Cancer Therapy Volume 6 Issue B

CANCER THERAPY

Volume 6 Number 2 December, 2008

CANCER THERAPY FREE ACCESS www.cancer-therapy.org

!!!!!!!!!!!!!!!!!!!!!!!! Editor

Teni Boulikas Ph. D., CEO Regulon Inc. 715 North Shoreline Blvd. Mountain View, California, 94043 USA Tel: 650-968-1129 Fax: 650-567-9082 E-mail: teni@regulon.org

Teni Boulikas Ph. D., CEO, Regulon AE. Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9853849 Fax: +30-210-9858453 E-mail: teni@regulon.org

!!!!!!!!!!!!!!!!!!!!!!!! Assistant to the Editor Maria Vougiouka B.Sc., Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9858454 Fax: +30-210-9858453 E-mail: maria@cancer-therapy.org

!!!!!!!!!!!!!!!!!!!!!!!! Editorial Board

Ablin, Richard J., Ph.D., Arizona Cancer Center, University of Arizona, USA Armand, Jean Pierre, M.D. Ph.D., European Organization for Research and Treatment of Cancer (EORTC), Belgium Aurelian, Laure, Ph.D., University of Maryland School of Medicine, USA Berdel, Wolfgang E, M.D., University Hospitals, Germany Bertino, Joseph R., M.D., Cancer Institute of New Jersey, USA Beyan Cengiz, M.D., Gulhane Military Medical Academy, Turkey Bottomley, Andrew, Ph.D., European Organization for Research and Treatment of Cancer Data Center (EORTC), Belgium Bouros, Demosthenes, M.D., University Hospital of Alexandroupolis. Greece Cabanillas, Fernando, M.D, The University of Texas M. D. Anderson Cancer Center, USA Castiglione, Monica, MHA, SIAK/IBCSG Coordinating Center, Switzerland Chou, Kuo-Chen, Ph.D., D.Sc., Pharmacia Upjohn, USA Chu, Kent-Man, M.D., University of Hong Kong Medical Center, Queen Mary Hospital, Hong Kong, China Chung, Leland W.K, Ph.D., Winship Cancer Institute, USA Coukos, George, M.D., Ph.D., Hospital of the University of Pennsylvania, USA Darzynkiewicz, Zbigniew, M.D., Ph.D., New York

Medical College, USA Der Channing, J. Ph.D, Lineberger Comprehensive Cancer Center, USA Devarajan, Prasad M.D., Cincinnati Children's Hospital, USA Dritschilo, Anatoly, M.D., Georgetown University Hospital, USA Duesberg, Peter H., Ph.D, University of California at Berkeley, USA El-Deiry, Wafik S. M.D., Ph.D., Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, USA Federico, Massimo, M.D. Università di Modena e Reggio Emilia, Italy Fiebig, Heiner H, Albert-Ludwigs-Universität, Germany Fine, Howard A., M.D., National Cancer Institute, USA Frustaci, Sergio, M.D., Centro di Riferimento Oncologico di Aviano, Italy Georgoulias, Vassilis, M.D., Ph.D., University General Hospital of Heraklion, Greece Giordano, Antonio, M.D., Ph.D., Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, USA Greene, Frederick Leslie, M.D., Carolinas Medical Center, USA Gridelli, Cesare M.D., Azienda Ospedaliera, "S.G.Moscati", Italy Hengge, Ulrich, M.D., Heinrich-Heine-University Duesseldorf, Germany Huber, Christian M.D., Johannes-Gutenberg-

University, Germany Hunt, Kelly, M.D., The University of Texas M. D. Anderson Cancer Center, USA Kamen, Barton A., M.D. Ph.D, Cancer Institute of New Jersey, USA Kaptan, Kürsat, M.D., Gülhane Military Medicine Academy, Turkey Kazuma, Ohyashiki, M.D., Ph.D., Tokyo Medical University, Japan Kinsella, Timothy J. M.D., The research Institute of University Hospitals in Cleveland, USA Kmiec, Eric B, Ph.D., University of Delaware, USA Kosmidis Paris, M.D., "Hygeia" Hospital, Athens, Greece Koukourakis Michael, M.D., Democritus University of Thrace, Greece Kroemer, Guido, M.D. Ph.D., Institut Gustave Roussy, France Kurzrock, Razelle, M.D., F.A.C.P., M. D. Anderson Cancer Center, USA Leung, Thomas Wai-Tong M.D., Chinese University of Hong Kong, China Levin, Mark M.D., Sister Regina Lynch Regional Cancer Center, Holy Name Hospital, USA Lichtor, Terry M.D., Ph.D., Rush Medical College, USA Liebermann, Dan A., Ph.D., Temple Univ. School of Medicine, USA Lipps, Hans J, Ph.D., Universität Witten/Herdecke, Germany Lokeshwar, Balakrishna L., Ph.D., University of Miami School of Medicine, USA Mackiewicz, Andrzej, M.D., Ph.D., University School of Medical Sciences (USOMS) at Great Poland Cancer Center, Poland Marin, Jose J. G., Ph.D., University of Salamanca, Spain McMasters, Kelly M., M.D., Ph.D., University of Louisville, J. Graham Brown Cancer Center, USA Morishita, Ryuichi, M.D., Ph.D., Osaka University, Japan Mukhtar, Hasan Ph.D., University of Wisconsin, USA Ng, Eddie YK, Ph.D., Nanyang Technological University, Singapore Norris, James Scott, Ph.D., Medical University of South Carolina, USA Palu, Giorgio, M.D., University of Padova, Medical School, Italy Park, Jae-Gahb, M.D., Ph.D., Seoul National University College of Medicine, Korea Perez-Soler, Roman M.D., The Albert Einstein Cancer Center, USA Peters, Godefridus J., Ph.D., VU University Medical Center (VUMC), The Netherlands Poon, Ronnie Tung-Ping, M.D., Queen Mary Hospital, Hong Kong, China Possinger, Kurt-Werner, M.D., Humboldt University, Germany Rainov G Nikolai M.D., D.Sc., The University of Liverpool. UK Randall, E Harris, M.D., Ph.D., The Ohio State University, USA Ravaioli Alberto, M.D. Ospedale Infermi, Italy

Remick, Scot, C. M.D., University Hospitals of Cleveland, USA Rhim, Johng S M.D., Uniformed Services University of Health Sciences, USA Schadendorf, Dirk, M.D., Universitäts-Hautklinik Mannheim, Germany Schmitt, Manfred, Ph.D., Universität München, Klinikum rechts der Isar, Germany Schuller, Hildegard M., D.V.M., Ph.D., University of Tennessee, USA Slaga, Thomas J., Ph.D., AMC Cancer Research Center (UICC International Directory of Cancer Institutes and Organisations), USA Soloway, Mark S., M.D., University of Miami School of Medicine, USA Srivastava, Sudhir, Ph.D., MPH, MS, Division of Cancer Prevention, National Cancer Institute, USA Stefanadis, Christodoulos, M.D., University of Athens, Medical School, Greece, Stein, Gary S Ph.D., University Of Massachusetts, USA Tirelli, Umberto, National Cancer Institute, Italy Todo, Tomoki, M.D., Ph.D., The University of Tokyo, Japan van der Burg, Sjoerd H, Leiden University Medical Center, The Netherlands Wadhwa Renu, Ph. D., Nat. Inst. of Advan. Indust. Sci. and Technol. (AIST), Japan Waldman, Scott A. M.D., Ph.D., USA Walker, Todd Ph.D., Charles Sturt University, Australia Watson, Dennis K. Ph.D., Medical University of South Carolina, Hollings Cancer Center, USA Waxman, David J., Ph.D., Boston University, USA Weinstein, Bernard I., M.D., D.Sci (Hon.), Columbia University, USA Werner, Jochen Alfred M.D., Philipps-University of Marburg, Germany Wieand, Harry Samuel Ph.D., National Surgical Adjuvant Breast and Bowel Project (NSABP), USA Yamada, Akira Ph.D., Kurume University Research Center for Innovative, Japan Yu, Dihua M.D., Ph.D., The Univ. Texas M. D. Anderson Cancer Center, USA Zagon, Ian, Ph.D., The Pennsylvania State University, USA

!!!!!!!!!!!!!!!!!!!!!!!! Associate Board Members

Chen, Jiguo, Ph.D, The University of Texas Health Science Center at San Antonio, USA Chen, Zhong, M.D, Ph.D, National Institute of Deafness and other Communication Disorders, National Institutes of Health, USA Dietrich Pierre Yves, Hopitaux Universitaires de GenFve Switzerland Jeschke Marc G, M.D., Ph.D. Universität Erlangen-Nürnberg. Germany Limacher Jean-Marc, MD Hôpitaux Universitaires de Strasbourg, France Los Marek J, M.D., Ph.D. University of Manitoba, USA Mazda Osam, M.D., Ph.D. Kyoto Prefectural University of Medicine, Japan Merlin Jean-Louis, Ph.D Centre Alexis Vautrin, National Cancer Institute University Henri Poincaré France Okada Takashi, M.D., Ph.D. Jichi Medical School Japan Pisa Pavel, M.D, Ph.D. Karolinska Hospital, Sweden Squiban Patrick, MD Transgene SA France Taupin, Philippe, Ph.D., National University of Singapore, Singapore Tsuchida Masanori, M.D, Ph.D Niigata University Graduate School of Medical and Dental Sciences Japan Ulutin, Cuneyt, M.D., Gulhane Military Medicine Academy, Turkey Xu Ruian, Ph.D., The University of Hong Kong, Hong Kong

!!!!!!!!!!!!!!!!!!!!!!!! For submission of manuscripts and inquiries: Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org

Instructions to authors: Cancer Therapy FREE ACCESS www.cancer-therapy.org

Scope This journal, bridging various fields is one of the most rapid with free access at www.cancertherapy.org. The scope of Cancer Therapy is to rapidly publish original and in-depth review articles on cancer embracing all fields from molecular mechanisms to results on clinical trials. Articles (both invited and submitted) review or report novel findings of importance to a general audience in cancer therapy, molecular medicine, gene discovery, and molecular biology with emphasis to molecular mechanisms and clinical applications. The journal will accept papers on all aspects of cancer, at the clinical, preclinical or cell culture stage on chemotherapy and new experimental drugs, gene discovery, cancer immunotherapy, DNA vaccines, use of DNA regulatory elements in gene transfer, cell therapy and drug discovery related to cancer therapy. The authors are encouraged to elaborate on the molecular mechanisms that govern a cancer therapy approach. To make the publication attractive authors are encouraged to include color figures. Type of articles Both review articles and original research articles will be considered. Original research articles should contain a generous introduction in addition to experimental data. The articles contain information important to a general audience as the volume is addressed to researches outside the field. There is no limit on the length of the articles provided that the subject is interesting to a general audience and covers exhaustively a field. The typical length of each manuscript is 12-60 manuscript pages (approximately 4-20 printed pages) plus Figures and Tables. Free of Charge publication, Complimentary reprints & Subscriptions There are no charges for color figures or page numbers. Corresponding authors get a one-year free subscription (hard copy) plus 25 reprints free of charge. The free subscription can be renewed for additional years by having one paper per year accepted for publication. Sections of the manuscript Each manuscript should have a Title, Authors, Affiliation, Corresponding Author (with Tel, Fax, and E-mail), Summary, and Introduction; review articles are subdivided into headings I, II, III, etc. (starting with I. Introduction) and subdivided into A, B, C, etc. You can further subdivide into 1, 2, 3, etc. Research articles are divided into Summary; I. Introduction; II. Results; III Discussion; Acknowledgments IV. Materials and Methods and References. Please include in your text citations the name of authors and year in parenthesis; for three or more authors use: (name of first author et al, with year); for two authors please use both names. Please delete hidden text for references. In the reference list, please, type references with year and Journal in boldface and provide full title of the article such as: Buschle M, Schmidt W, Berger M, Schaffner G, Kurzbauer R, Killisch I, Tiedemann J-K, Trska B, Kirlappos H, Mechtler K, Schilcher F, Gabler C, and Birnstiel ML (1998) Chemically defined, cell-

free cancer vaccines: use of tumor antigen-derived peptides or polyepitope proteins for vaccination. Gene Ther Mol Biol 1, 309-321. Please use Microsoft Word, font “Times” (Mac users) or “Times New Roman” (PC users) and insert Greek or other characters using the “Insert/Symbol” function in the Microsoft Word rather than simple conversion to font “Symbol”. Please boldface Figure 1, 2, 3 etc. as well as Table 1, 2, etc. throughout the text. Please provide the highest quality of prints of your Figures; whenever possible, please provide in addition an electronic version of your figures (optional). Corresponding authors are kindly requested to provide a color (or black/white) head photo of themselves (preferably 4x5 cm or any size), as we shall include these in the publication. Submission and reviewing Peer reviewing is by members of the Editorial Board and external referees. Please suggest 2-3 reviewers providing their electronic addresses, mailing addresses and telephone/fax numbers. Authors are being sent page proofs. Cancer Therapy (Volume 1, 2003) is published on high quality paper with excellent reproduction of color figures and electronically. Reviewing is completed within 5-15 days from receiving the manuscript. Articles accepted without revisions (i.e., review articles) will be published online (www.cancertherapy.org) in approximately 1 month following submission. Please submit an electronic version of full text and figures preferably in jpeg format. The electronic version of the figures will be used for the rapid reviewing process. High quality prints or photograph of the figures and the original with one copy should be sent via express mail to the Editorial Office. Citation in MedLine Articles accepted for publication by GTMB or Cancer Therapy can be included in MedLine (PubMed) as full articles upon the request of authors provided that the authors have completed their published work under a government grant by NIH (or EU/Japan government grant). If this is you case, please consult the NIH Manuscript Submission System http://www.nihms.nih.gov/. Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org The free electronic access to articles published in "Cancer Therapy" to a big general audience, the attractive journal title, the speed of the reviewing process, the no-charges for page numbers or color figure reproduction, the 25 complimentary reprints, the rapid electronic publication, the embracing of many fields in cancer, the anticipated high quality in depth reviews and first rate research articles and most important, the eminent members of the Editorial Board being assembled are prognostic factors of a big success for the newly established journal.

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Table of contents Cancer Therapy Vol 6 Number 2, December 2008

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

367-376

Research Article

Margot E. Kurtz, J Cleo Kurtz, Charles W. Given, Barbara Given

377-388

Review Article

Age and depressive symptomatology: impact on the relative effectiveness of two pain and fatigue control interventions among cancer patients Drug resistance in lung cancer

389-394,

Case report

Carcinosarcoma of endometrium: A case study with eleven DNA fluorescent in situ hybridization probes

Shamim A. Faruqi, Holly Prescott, Christopher Harsch, Harvey Spector, Joel S. Noumoff1

395-408

Research Article

William C. Dooley, Hernan I. Vargas, Alan J. Fenn, Mary Beth Tomaselli, Jay K. Harness

409-412

Case Report

Randomized Study of Preoperative Focused Microwave Phased Array Thermotherapy for Early-Stage Invasive Breast Cancer Metachronous thyroid metastasis of primary rectal adenocarcinoma

413-420

Research Article

421-424

Research Article

425-438

Review Article

439-444

Review Article

445-456,

Review Article

457-462

Review Article

463-476

Review Article

477-490

Review

Outcome of Hispanics with colorectal cancer residing in Puerto Rico with access to adequate health care facilities: results are not inferior to USA or European caucasians Morphoeic type basal cell carcinoma of the perianal region: rare manifestation of a common disease Highlights from ASCO and ECCO 2007: Update on targeted treatment of metastatic renal cell carcinoma Functional and structural analysis of androgen receptors for anti-cancer drug discovery Actual role of multitargeted therapy in renal cell carcinoma (RCC)

Canine hemangiosarcoma: A tumor of contemporary interest Early breast cancer: A review The role of panitumumab in metastatic

Jozef !karda, Marian HajdĂşc, VĂ­t!zslav Kolek

Raffaele Longo, Francesco Torino, Roberta Sarmiento, Domenico Gattuso, Cinzia Bernardi, Giampietro Gasparini Luis Ignacio Echenique, Fernando Cabanillas, Viviana Freire, Ignacio A. Echenique

Sai Krishna Balakrishnan, Pankaj K Singh, Lucy Jane Smith, Vinay K Singh Robert J. Motzer

Zeina Nahleh

Alessandro Sciarra, Giovanni B. Di Pierro, Andrea Alfarone, Francesco Minisola, Alessandro Gentilucci, Stefano Salciccia, Luca Iannotta, Francesca Antonini, Cristiano Cristini, Franco Di Silverio, Vincenzo Gentile Stuart C. Helfand Kyriakos Kalogerakos, Chrisostomos Sofoudis, Nikolaos Baltayiannis Maria Anna Bareschino, Clorinda

Article

colorectal cancer

491-494

Case Report

Extrathyroidal anaplastic transformation

495-510

Review Article

511-536

Research Article

537-544

Review Article

545-552

Research Article

553-562

Research Article

563-570

Research Article

571-576,

Research Article

From here to eternity - the secret of Pharaohs: Therapeutic potential of black cumin seeds and beyond Analysis of gene expression profiles in the programmed cell death rat retinal ganglion cells (RGC-5) Mouse mammary tumor virus: a cause of breast cancer in humans? Techniques for intratumoral chemotherapy of lung cancer by bronchoscopic drug delivery Dosimetric comparison of tomotherapy versus 4-field pelvic box altered fractionation radiotherapy treatment plans for invasive squamous cell carcinoma of the cervix Alterations in antioxidant enzyme activities and increased oxidative stress in cyclophosphamide- induced hemorrhagic cystitis in the rat Dosimetric evaluation of helical tomotherapy treatment planning for non-small cell lung cancer

577-596

Research Article

597-602

Review Article

603-610

Research Article

611-616

Research Article

617-622

Research Article

623-628

Research Article

Gastrointestinal stromal tumor of the pancreatoduodenal complex: A detailed review and development of new prognostic scoring system Contemporary management of adrenocortical carcinoma Disruption of Survivin in K562 cells elevates telomerase activity and protects cells against apoptosis induced by the Bcr-abl kinase inhibitor STI571 Efficacy of methotrexate as primary single agent therapy for non metastatic and low risk metastatic gestational trophoblastic neoplasia at the University of the Philippines-Philippine General Hospital (UP-PGH) A Meta analysis assessment of bevacizumab in colorectal cancer inpatient to decide therapy with limited data Inhibition of endothelial cell adhesion and in vitro angiogenesis by Taurolidine

Schettino, Paolo Maione, Antonio Rossi, Dario Nicolella, Fortunato Ciardiello, Cesare Gridelli Todd Swanson, Senthilselvi Nanthakumar, Samuel Bugis, Sam M. Wiseman Subhash Padhye, Sanjeev Banerjee, Aamir Ahmad, Ramzi Mohammad, Fazlul H Sarkar Abdelnaby Khalyfa, Mohamed O. Buazza, Muad A. Maya, Nigel Cooper, Bryant A. Minnis Ish Ahmed, James R. Harvey, Simi Ali, John A. Kirby, Thomas WJ Lennard Firuz Celikoglu, Seyhan I Celikoglu, Eugene P Goldberg Don Yee, Robert Pearcey, George Dundas, John Hanson, Marc Mackenzie, Don Robinson, Colin Field, Lori Underwood, Raul Urtasun, Nadeem Pervez, B. Gino Fallone

Premila Abraham, Indirani Kanakasabapathy, Preethi Kulothungan Karen Chu, George Rodrigues, Slav Yartsev, A. Rashid Dar1, Edward Yu1,3, Robert Ash1, Brian Yaremko1, Marc MacKenzie, Harvey Quon, Glenn Bauman, Wilson Roa Raja R Gopaldas, Lori J Toedter, Sanford Dorman, Chand Rohatgi

Troy Guthrie Jr, Nikesh Jasani Zhanxiang Wang, Louis M. Pelus

Maria Stephanie Fay SamadanCagayan

Fabiana Rubba, Salvatore Panico, Patrizia Cuccaro, Antonio Della Vecchia, Maria Triassi Thomas Mรถhler, Martina WillhauckFleckenstein, Reinhard Schwartz-Albiez, Annette Merling, Hanns Mรถhler

629-646

Research Article

Diagnostic and Therapeutic Efficacy of Imaging Modalities in Non-Small Cell Lung Cancer (NSCLC): experience from a Phase III clinical study using tumor targeted Lipoplatin nanoparticles The role of percutaneous ultrasound guided radiofrequency ablation (RFA) in patients with breast cancer hepatic metastases

647-654

Research Article

655-664

Review Article

Antiestrogen therapy for Breast Cancer: An overview

665-672

Research Article

Detection of Epstein-Barr virus in Pediatric Hodgkin’s Lymphoma in Iran by immunohistochemistry and in situ hybridization

673-682

Research Article

683-686

Research Article

Conventional versus hyperfractionated radiotherapy in locally advanced head and neck cancer Efficacy of cancer treatment in Armenia: where is it going?

687-698

Review Article

Paraneoplastic syndromes in lung cancer

Stefanie Heinemann, Peter Zabel, HansPeter Hauber

699-706

Research Article

Luigi Vigorè, Fernando Brivio, Luca Fumagalli, Roberto Vezzo, Giusy Messina, Franco Rovelli, Massimo Colciago, Giovanna D’Amico, Giuseppe Di Fede, Paolo Lissoni

707-710

Case Report

711-728

Review Article

A clinical study of T-regulatory lymphocyte function in cancer patients in relation to tumor histotype, disease extention, lymphocyte subtypes and cortisol secretion An atypical tongue metastasis of renal cell carcinoma in a patient with metachronous hepatocellular carcinoma Chemotherapy in the treatment of primary liver tumours

Faye Lazarioti, Teni Boulikas

Gianpaolo Carrafiello, Domenico Laganà, Chiara Recaldini, Monica Mangini, Federico Fontana, Elisa Cotta, Maria Petullà, Raffaele Novario2, Salvatore Cuffari, Gianlorenzo Dionigi, Carlo Fugazzola Kishor S Kumar, Mahesh M.J Kumar Farzaneh Jadali, Abdollah Karimi, Fatemeh Fallah, Golareh Habibi, Ahmadreza Shamshiri, Atoosa Gharib, Mostafa Sharifian, Shahnaz Armin, Soraya Tabatabaii, Masumeh Mohkam, Zahra Chavoshizadeh, Reza Shiari, Saiid Fahimzad, Saiid Maham, Elham Mazaheri-Tehrani Azza Abd El-Naby, Aly Tawfek, Hala M. El-Shenshawy, Amal Halim, Rasha Hamdy Armen K. Nersesyan

Raffaele Longo, Daniela Baldini, Giampietro Gasparini

Jose J G Marin, Beatriz Castaño, Pablo Martinez-Becerra, Ruben Rosales, Maria J Monte

Cancer Therapy Vol 6, page 367! Cancer Therapy Vol 6, 367-376, 2008

Age and depressive symptomatology: impact on the relative effectiveness of two pain and fatigue control interventions among cancer patients Research Article

Margot E. Kurtz1,*, J Cleo Kurtz2, Charles W. Given3, Barbara Given4 1

Department of Family and Community Medicine Department of Mathematics 3 Department of Family Medicine 4 College of Nursing, Michigan State University, East Lansing, Michigan 48824, USA 2

__________________________________________________________________________________! *Correspondence: Margot E. Kurtz, PhD, Professor, Department of Family and Community Medicine, Michigan State University, B 211 West Fee Hall, East Lansing, MI 48824, USA; Tel: 517.353.4732; Fax: 517.353.6613; e-mail: kurtzm@msu.edu Key words: pain; fatigue; cancer; intervention; depression; age Abbreviations: American Joint Committee on Cancer, (AJCC); automated telephone symptom management intervention, (ATSM); Center for Epidemiological Studies Depression Scale, (CES-D); nurse administered symptom management intervention, (NASM); Tumor, Node, Metastasis, (TNM) Received: 6 May 2008; Revised: 5 June 2008 Accepted: 11 June 2008; electronically published: June 2008

Summary Pain and fatigue are among the most frequently occurring symptoms experienced by patients with cancer. Numerous studies have described the impact these symptoms can have on patients’ physical and psychological functioning as well as quality of life throughout the trajectory of the cancer experience. Recent research has focused on different types of interventions to control cancer symptoms. Prominent among these are nurse administered interventions and automated telephone interventions. In this study we investigated whether, in the presence of high (low) depression at intake, a nurse administered symptom management intervention (NASM) was more effective than an automated telephone symptom management intervention (ATSM) in the reduction of cancer pain and fatigue. Secondly, we investigated whether NASM was more effective than ATSM in the reduction of cancer pain and fatigue among older versus younger patients. Three hundred fifty-one patients were recruited who had been diagnosed with a solid tumor cancer or non-Hodgkin’s lymphoma. These patients were randomly assigned to NASM (180) and ATSM (171). Separate ANCOVA models for predicting pain and fatigue showed that there was no difference in the effectiveness of NASM and ATSM in controlling either pain or fatigue, based on level of depression at intake. However, the ATSM intervention outperformed the NASM intervention in controlling severity of fatigue among younger patients, while NASM outperformed ATSM in controlling pain among older patients. These findings point in the direction that oncologists, when identifying the appropriate style of intervention for an individual patient, may need to determine, in consultation with the patient, which symptoms should receive the highest priority, and what approach would feel most comfortable to them, so that optimal symptom control can be achieved.

I. Introduction

1999; Given et al, 2001; Escalante 2003). Studies have indicated that during the six months following diagnosis, pain and fatigue seem to be the most problematic symptoms (Longman et al, 1997; Maquart-Moulin et al, 1999). The severity of these symptoms may be a function of the site and stage of the cancer, comorbidity, treatment regimen, or patient characteristics such as age and gender, and may change over time (Sarna 1993; Degner and Sloan

Pain and fatigue are among the most frequently occurring symptoms experienced by patients with cancer. Numerous studies have described the impact these symptoms can have on patients’ physical and psychological functioning as well as quality of life throughout the trajectory of the cancer experience (Portenoy et al, 1995; Groopman 1998; Jacobsen and Stein 367

Kurtz et al: Pain and Fatigue Control among Cancer Patients! symptom management intervention (ATSM) in the reduction of cancer pain and fatigue? 2. Is NASM more effective than ATSM in the reduction of cancer pain and fatigue among older versus younger patients?

1995; Pater et al, 1997; Stone et al, 1999; Redeker et al, 2000; Given et al, 2001, Kroenke 2001; Engel et al, 2003; Miaskowski 2004; Bower et al, 2006; Doorenbos et al, 2006a; Yoon et al, 2008). Pain and fatigue are prevalent particularly during chemotherapy, and some researchers suggest that their presence may add to the severity of other symptoms and increase the complexity of symptom management (Irvine et al, 1994; Miaskowski and Lee 1999; Given et al, 2001). The picture is further complicated, as patients with cancer experience considerable distress, and many manifest depressive symptoms in response to their illness (Dugan et al, 1998). As many as half of these patients may experience depressive symptomatology at a level that would qualify for clinical diagnosis (Hann et al, 1999), and this adds to the burden of treatment and may lead to more difficulty with general management and symptom control (Montazeri et al, 1998; Stommel et al, 2002, 2004). As observed by Maxwell in 2000, existing studies indicate a high prevalence rate and poor management of cancer pain in the elderly. In a recent retrospective study of cancer patients admitted to nursing homes, between 25% and 40% of these elderly cancer patients were found to experience daily pain, however 26% of those experiencing pain received no analgesia, and only 26% received morphine or its equivalent. Some of the barriers to good pain management in the elderly seem rooted in ageism. For example, elderly patients often underreport their pain because they expect pain to be a normal part of aging, or they do not wish to trouble their physician and be considered a “bad� patient. Bedeau and colleagues noted in 2004 that among cancer patients with pain, there were tendencies for younger persons to report more intense pain, and the elderly reported using more passive pain coping strategies. They indicated the need to develop interventions tailored to the unique characteristics of each age group. Recent research has focused on different types of interventions to control cancer symptoms (Given et al, 2002; Yates 2004; Doorenbos et al, 2006B; Godino et al, 2006; Sikorskii et al, 2006). Prominent among these are nurse administered interventions (Yates 2004; Sikorskii et al, 2006) and automated telephone interventions (Sikorskii et al, 2007). Sikorskii and colleagues compared in 2007 the effectiveness of a nurse administered symptom management intervention compared to an automated telephone symptom management intervention in controlling a composite of 17 cancer-related symptoms. The results suggested that while both interventions achieved a clinically significant reduction in symptom severity, neither was shown to be conclusively more effective overall than the other. This begs the question as to how these two types of interventions behave with prominent symptoms such as pain and fatigue, and with subgroups of patients (older versus younger, depressed versus not depressed). To address these questions we investigated the following: 1. In the presence of high (low) depression at intake, is a nurse administered symptom management intervention (NASM) more effective than an automated telephone

II. Materials and Methods A. Sample The study we are reporting, which focuses on control of the important symptoms pain and fatigue, uses data from a larger multifaceted longitudinal symptom control intervention involving cancer patients and their caregivers. This larger study included a random controlled trial focusing on a composite measure of a broad spectrum of cancer related symptoms, comparing a NASM intervention with an ATSM intervention, as well as a separate patient/caregiver intervention. Patients were recruited simultaneously for these studies from two comprehensive cancer centers, one community oncology program, and six hospital affiliated community oncology centers. Nurses were subcontracted from the participating institutions and trained to implement the recruitment and intervention protocols. In order to be eligible for the study, patients had to be: (1) 21 years of age or older, (2) have a diagnosis of a solid tumor cancer or non-Hodgkins lymphoma, (3) be currently undergoing chemotherapy (patients concurrently undergoing radiation therapy or surgery were excluded), (4) have a touchtone telephone, and (5) be able to speak and read English. Based on information in their medical records, 1605 cancer patients were eligible for the combined studies and were approached by nurse recruiters. Eight hundred fifteen expressed their willingness to participate by signing an informed consent form. Their socio-demographic information was entered into a web-based tracking system. An automated voice response version of the MD Anderson Symptom Inventory was used during the recruitment phase to screen patients for symptom severity (Cleeland et al, 2000). According to established guidelines, a score of two or higher suggests a need for monitoring. Patients scoring two or higher on severity of at least one symptom (range 0 -10) were accepted into the study. Patients not scoring two or higher on any of the symptoms were called twice weekly for up to six weeks. Those failing to report a severity score of two or higher on any symptom were sent a letter thanking them for participation and were not entered into any of the studies. Of the 806 patients remaining, 257 had designated family caregivers and thus entered the patient/caregiver trial, 78 dropped out prior to entry and another 34 dropped out prior to the baseline interview, leaving 437 patients who completed the baseline interview. Of these, 219 were randomly assigned to the ATSM and the remaining 218 to the NASM. Figure 1 shows the accrual process and attrition in more detail. Informed consent procedures for the clinical intervention were approved by the appropriate university committee on research involving human subjects, as well as the institutional review boards of the individual recruitment sites.

368

Cancer Therapy Vol 6, page 369! skipped week five, were called in week six, skipped week seven, and received a final call in week eight. At 10 weeks all patients received a second interview to assess their status and measure the effectiveness of the interventions. A total of 75 patients dropped out between baseline and 10 weeks (34 from NASM and 41 from ATSM).

B. Intervention Patients accepted into both interventions underwent an intake interview and received a copy of a corresponding Symptom Management Guide (SMG) designed to accompany their intervention. Patients in both groups received a telephone call in each of the first four weeks,

Figure 1. Flowchart of Study Accrual and Attrition.

conditions (p=0.020), were more depressed (p=0.003) and were more likely to be suffering from late stage disease (p=0.007). !"# $%&"# "$'()# *+,'"-"# +.# $%-# ")/0$+/"# 01&.# 1.(# *1$&2'-3# 4-# (-",5&6-# $%-# &.$-57-.$&+."# 1"# $%-)# 5-81$-# $+# $%-"-# $4+# ")/0$+/"9# :%-# 01$&-.$"# &.# $%-# ;!<=#

The 351 patients who completed the 10 week intervention (either ATSM or NASM) formed the sample for this two-wave longitudinal study. A comparison of baseline information for those who dropped out, compared to those continuing in the study, revealed that those patients who dropped out reported more comorbid

369

Kurtz et al: Pain and Fatigue Control among Cancer Patients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n the ATSM intervention, the patients were queried on the severity of their pain and fatigue by a prerecorded pleasant female voice. Patients rated the severity of their pain and fatigue by pressing the appropriate numbers on their telephone keypads. For symptoms above threshold, patients were referred to the appropriate section of the SMG for information on strategies to manage that symptom. The ATSM was programmed so that on the subsequent contact, patients were asked whether they had tried the strategies suggested in the SMG, and if so, were they helpful in alleviating the symptoms. Patients could respond with numerical prompts using their telephone keypad. When the symptoms above threshold at the last contact had been reviewed, the system then elicited the current severity of pain and fatigue. Figure 2 contains examples of strategies available for moderate pain (severity level 4-6). In the NASM, the nurse intervened in the areas of teaching, prescribing, communication with the health care provider (HCP) and counseling and support. The right-hand column summarizes information on strategies for pain contained in the SMG used in the ATSM.

interview. Comorbid conditions were assessed at baseline as a count of comorbid conditions present, selected from a list of 15 comorbid conditions (diabetes, hypertension, cardiovascular disease, etc.) (Katz et al, 1996). For this study, we employed the !"#$%&' ($)*&' +*,-.,-./.' 0!(+1' .,-2/32' .4.,*# promulgated by the American Joint Committee on Cancer (AJCC) in the United States. Determination of the stage involves consideration of a number of variables which are important for prognosis (e.g., extent of the tumor, histological type, differentiation, metastasis, etc.), and classifies tumors on a scale of 0-IV (0=localized…IV=distant metastasis) (Beahrs et al, 1992; Hermanek and Sobin 1992). To minimize the problem of small or empty cells in the analysis, stage was dichotomized into two groups: “early” (stages 0, I, II) and “late” (stages III, IV). Severity of the symptoms pain and fatigue was scored by patients on a scale ranging from 0 (not present) to 10 (worst possible) at the baseline interview, each of the six telephone contacts and the 10 week interview. Such 10-point scales are recommended in the National Cancer Care Network (NCCN) for measuring symptom severity (NCCN 2008), and are commonly used by physicians in the practice setting. Depressive symptomatology was measured by the Center for Epidemiological Studies Depression Scale (CES-D) (Radloff 1977; Roberts and Vernon 1983). This is a well-established instrument used in population-based studies of depression, and has been used in many studies with cancer patients (Berry et al, 1984). The scale consists of 20 items, each scored on a scale of 0-3 (0=rarely/none of the time,..., 3=almost all of the time). The usual composite score was computed by summing the scores for the 20 items on the scale, with higher scores corresponding to greater depressive symptomatology. Respondents with a CES-D score of 16 or higher are commonly classified as depressed (Fechner-Bates et al, 1994). To establish a baseline for depressive symptomatology, patients were categorized at intake as either depressed (CES-D score 16 or higher), or not depressed (CES-D score less than 16).

D. Analysis As an initial step, basic descriptive statistics were computed for the socio-demographic variables as well as means and standard deviations for baseline values of all scale variables employed in the study. To investigate our research questions we used separate analysis of covariance models for predicting pain (respectively fatigue) based on intervention type, age group, baseline depression status, gender and stage of disease, as well as the interactions of intervention with depression status and intervention with age group, while adjusting for differences in the number of comorbid conditions and baseline severity of pain (respectively fatigue). Since the interaction of intervention with depression status did not approach significance for either pain or fatigue, this term was dropped from the models.

C. Measures Age, gender, cancer site and stage of disease were obtained from an audit of patients’ records, entered into the tracking system, and confirmed during the baseline

370

Cancer Therapy Vol 6, page 371! NASM Teach: !! Knowledge of pain management: prescriptive (administration, long vs. short-acting, break through pain treatment, OTC medications) !! Knowledge of pain management: non-prescriptive (ability to anticipate needs, barriers to pain control, alternative therapies) !! Problem solving strategies !! Coping strategies !! Stress importance of break through pain and HCP titrating medications for pain control Prescribe: !! Initiate prescriptive pain management !! Antiemetics !! Bowel management !! Other symptom management (fatigue, insomnia) Communicate with HCP: !! Bowel obstruction !! Inability to function/get out of bed !! Need for narcotic !! Pain uncontrolled with current medications !! Side effects of medication uncontrolled !! Rapidly progressing pain intensity Counsel and Support: !! Cognitive reframing !! Communicate and express feelings !! Coping strategies !! Optimistic thinking !! Uncertainty reduction

ATSM Tips to help manage your pain: !! Take your pain medication around the clock !! Try using heat or cold to your area of pain. Wrap your heating blanket or ice pack in a towel to protect your skin. !! Try using a journal or diary to keep track of your pain; this may help your doctor or nurse manage your pain too. What can family members and friends do to help me with my pain? !! Ask family members and friends to call your doctor if your pain medication is not helping to decrease your pain, or if your pain level is above 5. !! Ask them to remind you to take your pain medication as prescribed and stay on an even schedule, even through the night. What should I report to my doctor or nurse? !! How many hours per day you have pain, and how long the pain lasts after taking something for relief. !! Pain that is new or different from your last doctor’s visit. !! Pain along with fatigue, nausea, vomiting, or abdominal bloating. Where can I get more information? !! Contact a support group through local ACS office or cancer center. !! Call the American Cancer Society at: 1-800-ACS2345 or visit on the internet at: http://www.cancer.org. Click on Patients, Family and Friends, then Coping with Treatment.

Figure 2. Typical Strategies Proposed (NASM) and Information Provided (ATSM) to Address Moderate Pain (Severity Levels 4-6).

Means and standard deviations for pain and fatigue at baseline and 10 weeks according to age group and baseline depression group are presented in Table 2. It appears that the NASM may have some advantage over the ATSM in controlling pain among older patients (age 63 and above), as mean severity scores for pain decreased between baseline and 10 weeks for patients in NASM, while mean scores increased for patients in ATSM. No obvious differences were apparent for the two depression groups. Looking at fatigue, we see indications that the ATSM outperformed the NASM for younger patients (age < 53), as there is a substantial drop in these patients’ mean fatigue severity scores between baseline and 10 weeks for patients in ATSM, with a much smaller improvement in scores for patients in NASM. As was mentioned earlier, the interaction effect of intervention group and depression status was not a significant predictor of severity of either pain or fatigue, which suggested that the two interventions did not behave differently with respect to either pain or fatigue for individuals who were depressed versus those who were not depressed. To further buttress this conclusion we implemented the models for pain and fatigue again for

We also investigated briefly whether the inclusion of a composite measure of the severity of other symptoms aside from pain and fatigue in the models would appreciably alter the results of interest, and found that this was not the case. Similarly, we experimented with the inclusion of a dichotomous variable separating out those patients experiencing pain and fatigue concurrently versus those where this was not the case, and again found no appreciable change in our results. Accordingly, these variables were not included in the models. All computations were carried out with SPSS 14.0.

III. Results Descriptive statistics are presented in Table 1. The average patient was 60 years old and suffered from 1.96 comorbid conditions. The patients were predominately female (75.6 %) and most were diagnosed with late stage disease (85.4%). Breast cancer accounted for 42.8% of cases, 15% were diagnosed with lung cancer, 13.3% with colon cancer, and the remaining 28.9% suffered from genital/urinary cancer, gastrointestinal cancer, gynecological cancer, pancreatic cancer, non-Hodgkins lymphoma, mesothelioma or other cancers. 371

Kurtz et al: Pain and Fatigue Control among Cancer Patients! significant predictors of severity of fatigue at 10 weeks. In this case, patients who were more depressed at baseline reported greater severity of fatigue at 10 weeks than those who were less depressed at baseline. In order to further clarify the interaction effect of intervention group and age group, we repeated the analysis of covariance for pain severity and fatigue severity for each of the three age groups separately, removing the interaction term. The result was that for patients less than 53 years of age, the ATSM intervention was more effective in reducing fatigue severity at 10 weeks than the NASM intervention (adjusted difference in means=1.435, p=0.002). For patients aged at least 53 but less than 63 there were no significant differences between the two interventions for either pain severity or fatigue severity. Finally, for individuals age 63 and above, the NASM intervention was marginally more effective than the ATSM intervention in reducing severity of pain at 10 weeks (adjusted difference in means=0.731, p=0.056).

each depression group separately, removing the interaction term. The results showed no effect of the intervention group for either pain or fatigue, for either group of patients. The interaction of intervention group and depression status was accordingly dropped from both models. In the case of the interaction of intervention group and age group however the situation was very different. The analysis of covariance model for pain severity revealed that the interaction of intervention group and age group was a significant predictor of pain severity, along with comorbidity and baseline pain severity, with more comorbid conditions or more severe pain at baseline corresponding to more severe pain at 10 weeks. (See Table 3) Patients who were more depressed at baseline (CESD ! 16) tended to experience marginally more severe pain at 10 weeks (p=0.06) than patients were less depressed at baseline (CESD<16). The analysis of covariance model for severity of fatigue similarly showed the interaction of intervention group and age group as well as baseline fatigue to be

Table 1. Patient sociodemographic characteristics, comorbidity, cancer site and stage of disease (N=351).

Characteristic Gender Male Female Stage of Disease Early Late Age Group Age < 53 53 <= Age < 63 Age >= 63 Depression Status CESD ! 16 CESD < 16 Cancer Site Breast Colon Lung Genital/Urinary Gastrointestinal Gynecological Pancreatic Non-Hodgkins Lymphoma Mesothelioma Other Age Comorbidity†â€

NASM N Percent

ATSM N Percent

Combined N Percent

44 136

24.4 75.6

42 129

24.6 75.4

86 265

24.5 75.5

26 152

14.6 85.4

43 128

25.1 74.9

69 280

19.8 80.2

67 54 59

37.2 30.0 32.8

65 60 45

38.2 35.3 26.5

132 114 104

37.7 32.6 29.7

52 126

29.2 70.8

52 118

30.4 69.0

104 244

29.9 70.1

77 24 27 11 6 14 6 9 3 3 Mean 59.97 1.96

42.8 13.3 15.0 6.1 3.3 7.8 3.3 5.0 1.7 1.7 Std Dev 11.49 1.52

67 24 25 14 6 16 6 10 2 1 Mean 56.78 1.96

39.2 14.0 14.6 8.2 3.5 9.4 3.5 5.8 1.2 0.6 Std Dev 12.11 1.59

144 48 52 25 12 30 12 19 5 4 Mean 56.88 1.96

41.0 13.7 14.9 7.1 3.4 8.5 3.4 5.4 1.4 1.1 Std Dev 11.78 1.55

Scale 0-15

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Cancer Therapy Vol 6, page 373! Table 2. Means and standard deviations for pain and fatigue by age group and baseline depression, at baseline and ten weeks (N=351).

Overall Age Group Age < 53 53 <= Age < 63 Age >= 63 Baseline Depression Group CESD < 16 CESD >= 16

Overall Age Group Age < 53 53 <= Age < 63 Age >= 63 Baseline Depression Group CESD < 16 CESD >= 16 †

Pain† Baseline NASM Mean

SD

Ten Weeks NASM Mean SD

ATSM Mean

SD

1.97

2.70

1.46

2.25

1.58

2.40

2.80 2.35 2.50

2.18 2.27 1.31

2.80 2.93 2.15

2.21 1.19 0.85

2.69 2.00 1.64

1.40 1.75 1.64

2.32 2.45 2.48

1.37 3.23 Fatigue† Baseline NASM Mean

2.28 2.95

1.76 2.48

2.52 3.05

0.99 2.63

1.87 2.69

1.44 1.92

2.20 2.80

SD

ATSM Mean

SD

Ten Weeks NASM Mean SD

ATSM Mean

SD

4.39

2.66

4.33

2.79

3.45

2.76

3.01

2.66

4.52 4.63 4.03

2.55 2.47 2.95

4.55 4.57 3.76

2.74 2.44 3.25

4.09 3.04 3.10

2.88 2.42 2.83

2.65 3.50 2.89

2.58 2.83 2.53

3.85 5.83

2.59 2.28

3.72 5.81

2.67 2.49

2.98 4.67

2.65 2.70

2.67 3.81

2.41 3.05

SD

ATSM Mean

1.91

2.62

2.63 1.69 1.31

Scale 0-10

Table 3. Analysis of variance for pain and fatigue (N=351).

Intervention Age Group Intervention by Age Group Baseline Depression Group Stage of Disease Gender Comorbidity Baseline Pain Baseline Fatigue

Pain F Value 0.441 1.666 3.555 3.413 0.007 0.002 7.020 41.641

Significance 0.522 0.191 0.030 0.066 0.933 0.964 0.008 0.000

Fatigue F Value 2.970 0.319 3.678 6.062 1.526 0.843 2.267 18.683 R2=0.154

R2=0.203

Significance 0.086 0.727 0.026 0.014 0.218 0.359 0.133 0.000

an ideal study population to compare the effectiveness of two competing symptom management interventions. For research question 1 the results indicate clearly that, although depression played a role in predicting both severity of pain and severity of fatigue at 10 weeks for both interventions, there was no detectable difference between the effectiveness of the two interventions in controlling the severity of either pain or fatigue. A closer inspection of Table 2 reveals that for each of the

IV. Discussion The patients in our study were quite ill with 80.2% having been diagnosed with late stage disease, were diverse in age, and approximately one-third of the patients in each intervention group were severely depressed (CESD at least 16) at baseline. As such, these patients could potentially benefit from assistance in managing their symptomatology, particularly pain and fatigue, and were

373

Kurtz et al: Pain and Fatigue Control among Cancer Patients! symptom distress in cancer patients. Cancer 89, 16341646. Degner L, Sloan J (1995) Symptom distress in newly diagnosed ambulatory cancer patients and as a predictor of survival in lung cancer. J Pain Symptom Manage 10, 423-431. Doorenbos AZ, Given CW, Given B, Verbitsky N (2006a) Symptom experience in the last year of life among individuals with cancer. J Pain Symptom Manage 32, 403-412. Doorenbos A, Given B, Given C, Verbitsky N (2006b) Physical functioning: effect of behavioral intervention for symptoms among individuals with cancer. Nurs Res 55, 161-171. Dugan W, McDonald MV, Passik SD, Rosenfeld BD, Theobald D, Edgerton S (1998) Use of the Zung selfrating depression scale in cancer patients: feasibility as a screening tool. Psycho-Oncol 7, 483-493. Engel J, Kerr J, Schlesinger-Raab A, Eckel R, Sauer H, Hölzel D (2003) Comparison of breast and rectal cancer patients’ quality of life: results of a four year prospective field study. Eur J Cancer Care 12, 215223. Escalante CP (2003) Treatment of cancer-related fatigue: an update. !"##$%&'Care Cancer 11, 79-83. Fechner-Bates S, Coyne JC, Schwenk TL (1994) The relationship of self-reported distress to depressive disorders and other psychopathology. J Consult Clin Psych 63, 350-359. Given CW, Given B, Azzouz F, Kozacik S, Stommel M (2001) Predictors of pain and fatigue in the year following diagnosis among elderly cancer patients. J Pain Symptom Manage 21, 456-466. Given B, Given C, McCorkle R, Kozachik S, Cimpich B, rahbar MH, Wijcik C (2002) Pain and fatigue management: results of a nursing randomized clinical trial. Oncol Nurs Forum 29, 949-956. Godino C, Jodar L, Duran A, Martinez I (2006) Nursing education as an intervention to decrease fatigue perception in oncology patients. Eur J Oncol Nurs 10, 150-155 Groopman JE (1998) Fatigue in cancer and HIV/AIDS. Oncology 12, 345-346. Hann D, Winter K, Jacobsen P (1999) Measurement of depressive symptoms in cancer patients: evaluation of the Center for Epidemiological Studies Depression Scale (CES-D). J Psychosom Res 46, 437-443. Hermanek P, Sobin LH, editors (1992) Classification of malignant tumors, 4th edition, 2nd rev., Berlin, SpringerVerlag. Irvine D, Vincent L, Graydon J, Bubella N, Thompson L (1994) The prevalence and correlates of fatigue in patients receiving treatment with chemotherapy and radiotherapy. Cancer Nursing 17, 367-378. Jacobsen PB, Stein KD (1999) Is fatigue a long-term side effect of breast cancer treatment? J Moffett Cancer Center 6, 256-263. Katz JN, Chang LC, Sangha O, Fossel A, Bates D (1996) Can comorbidity be measured by questionnaire rather than medical record review? Med Care 34, 73-84.

interventions there is an apparent improvement in both severity of pain and severity of fatigue between baseline and 10 weeks, the situation being that more depressed patients begin and end the interventions at higher levels than those who are less depressed. Thus, although both intervention types proved to be efficacious, it is clear that more depressed patients tend to suffer worse pain and fatigue. This finding reinforces the importance of oncologists, physicians and health care professionals caring for cancer patients staying attuned to their patients’ depressive symptomatology, particularly when assisting patients in managing pain and fatigue. Patient age appears to be an important factor in determining the appropriate style of intervention for the management of pain and/or fatigue. Clearly, the study revealed that for younger patients, the automated telephone intervention outperformed the nurse administered intervention in controlling the patients’ severity of fatigue. We conjecture that this may be due to the fact that younger patients are more comfortable with and accustomed to the use of technology, and may have a preference for this intervention approach as they can utilize it whenever they feel they need it. On the other hand, the fact that the nurse assisted intervention outperformed the automated telephone intervention in controlling pain among older patients may have a similar explanation. Older patients may be less experienced with automated systems, and may have a preference for the personal contact offered by the nurse patient interaction. These findings point in the direction that oncologists, when identifying the appropriate style of intervention for an individual patient, may need to determine, in consultation with the patient, which symptoms should receive the highest priority, and what approach would feel most comfortable to them, so that optimal symptom control can be achieved.

Acknowledgement This research was supported in part by National Cancer Institute Grant # RO1 CA030724 Automated Telephone Monitoring for Symptom Management, Charles Given, PI, Barbara Given, Co-PI, and in affiliation with the Walther Cancer Institute, Indianapolis, Indiana.

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Cancer Therapy Vol 6, page 377! Cancer Therapy Vol 6, 377-388, 2008

Drug resistance in lung cancer Review Article

Jozef ! karda1,*, Marian HajdĂşch2,*, VĂ­t"zslav Kolek3 1

Department of Pathology Laboratory of Experimental Medicine, Pediatric Department 3 Department of Tuberculosis and Respiratory diseases Faculty of Medicine and Dentistry, Palacky University, Olomouc 2

__________________________________________________________________________________! *Correspondence: Joseph Skarda MD DVM, LF Palacky University, Olomouc LF Palacky University, Department of Pathology and Laboratory of Molecular Pathology, Olomouc, Czeck Republic; Tel./Fax: 004-2060-3436129; e-mail address: jojos@email.cz Key words: Drug resistance, lung cancer, resistance-related proteins, protooncogenes, DNA microarray technology Abbreviations: 50% inhibitory concentration, (IC50); antioxidant enzymes, (AOE); called multidrug resistance, (MDR); cyclindependent kinases (cdks); glutathione peroxidase, (GPX); glutathione S-transferase, (GST); glutathione, (GSH); hypoxia-inducible factor-1, (HIF-1); lung resistance protein, (LRP); Metallothioneins, (MTs); multidrug resistance-associated protein, (MRP); non-small cell lung cancer, (NSCLC); O6-alkylguanine-DNA alkyltransferase, (ATase); P-glycoprotein, (P-gp); small cell lung cancer, (SCLC); surface enhanced laser desorption/ionisation, (SELDI); Thymidylate synthase, (TS); topoisomerase II, (topo II); World Health Organization, (WHO) Received: 7 May 20038; Revised: 5 June 2008 Accepted: 13 June 2008; electronically published: June 2008

Summary Drug resistance is an important problem in the treatment of patients with lung cancer. Tumours become resistant not only to the drugs used initially, but also to those to which they have not yet been exposed. Data obtained from various sources indicate that multiple mechanisms contribute to drug resistance. Many of them are inter-related or independent of each other, but may exist simultaneously in cancer cells or subpopulation of cells, producing an overall drug-resistant phenotype. The resistance-related proteins P-glycoprotein, glutathione-dependent enzymes, topoisomerases, metallothioneins, thymidylate synthase and O6-alkyl-DNA alkyltransferase have been found in various human lung tumours, but these alone cannot explain the drug-resistant phenotype. Cell-cycle-related proteins, angiogenic factors, protooncogenes, and tumour suppressor genes also play a role in the resistant phenotype. In future, a key challenge will be to determine the relative quantitative contributions of each of these mechanisms to overall resistance. The use of DNA microarray technology in drug resistance in lung cancer will yield insight into the mechanisms of drug resistance and the rational design of more effective strategies to circumvent resistance.

frequently in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Most patients with SCLC have an initial response to chemotherapy, but the majority relapses and their tumours will be largely refractory to further treatment. NSCLC are inherent resistant and are generally no responsive to initial chemotherapy.

I. Introduction Lung cancer is a global problem. It is the most frequent cancer in the world, and the epidemic of lung cancer is still continuing. The global incidence of lung cancer is increasing at 0.5% per year. Consequently, lung cancer will remain a major cause of world wide cancer death in the 21st century (Haugen, 2000). The prognosis of lung cancer is very poor: nearly 80% of patients die within 1 year of diagnosis. Despite major advances in patient management, chemotherapy and radiotherapy made over the past decades, long-term survival is obtained in only 5-10% of the patients (Richardson and Johnson, 1993; Mattern et al, 2002). The major problem in lung cancer chemotherapy is the emergence of inherent and acquired drug resistance of the cancer cells. Resistance to anticancer agents is observed

II. Definition of drug resistance in vivo and in vitro Clinical drug resistance criteria are defined on the basis of tumour response criteria documented by the World Health Organization (WHO) or other such organizations. When a patient does not show more than a partial response after chemotherapy treatment, the tumour is considered to be resistant to the anticancer drugs used.

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karda et al: Drug resistance in lung cancer (MDR) which describes the broad pattern of crossresistance that is seen following the development of resistance to certain drugs. MDR cells consistently over express a 170 kDa membrane-associated glycoprotein (Pglycoprotein) which is encoded by the ()&*#gene (Gros et al, 1986). Based on the homology of the ()&*#gene to a variety of genes encoding membrane transport proteins and on the ability of P-glycoprotein (P-gp) to bind drugs, it is believed that the multidrug resistance results from the function of P-gp as an energy-dependent drug efflux pump (Bradley et al, 1988). Relatively high levels of P-gp/mdr1 expression have been shown in many intrinsically drug-resistant tumours derived from tissues which normally express P-gp, including adrenal gland, kidney, liver, colon and rectum (Fojo et al, 1987). Generally low, but detectable levels of ()&*# gene expression on performance of RNA slot blot analysis or RT-PCR have been found in lung cancer as well as in normal lung tissue despite the intrinsic chemoresistance of lung cancer (Lai et al, 1989; Abe et al, 1994). Other studies using similar techniques also demonstrated that lung tumours contain low or undetectable levels of mdr1 mRNA (Goldstein et al, 1989; Shin et al, 1992). In contrast, using various monoclonal antibodies reactive with P-gp, some studies have revealed a relatively high expression of this protein in lung cancer (Radosevich et al, 1989). The inconsistency with the very low mdr1 levels found in lung tumours may be attributed to the varying sensitivity of the different methods to detect mdr1/P-gp expression (Herzog et al, 1992). Several SCLC and NSCLC cell lines made resistant to some MDR related drugs or derived from tumours’ relapsing after chemotherapy have been shown to express mdr1 mRNA and P-gp (Lai et al, 1989; Twentyman et al, 1986). In NSCLC, there appears to be a relationship between P-gp expression and drug resistance !"#$!%&' (Volm et al, 1991). The significance of mdr1/P-gp expression as a predictor for response to therapy in lung cancer is not yet clear. Studies including larger numbers of patients are necessary to answer this question. In conclusion, there is no evidence to suggest a major role of P-gp in the MDR of lung tumours. The over expression of mdr1/P-gp alone does not completely explain intrinsic MDR, and thus other drug resistance mechanisms are thought to exist in lung cancers (Table 1).

Criteria for !"# $!%&' drug resistance are not clear. In most studies, the 50% inhibitory concentration (IC50) values are used. Various !"# $!%&' test procedures have been used to test the sensitivity or resistance of lung tumour cells to cytotoxic drugs prior to therapy. The soft agar clonogenic assay (Hamburger and Salmon, 1977), the most widely used test system for !"#$!%&' drug selection, has proven to be of limited practical use in lung cancer since drug testing can be performed in only a minority of cases because of technical problems. Renewed interest in !"# $!%&' testing has been generated by the use of dye exclusion assays which are simple colorimetric tests for cell proliferation and survival (Weisenthal et al, 1983). The use of xenografts for testing drugs !"# $!$' has also been used (Mattern et al, 1984; 1987; 1988). In most instances good agreement was found between the !"#$!%&' and the !"#$!$' results. However, it is common to all test systems used that tumours which demonstrate resistance during testing will not, with a high degree of probability, respond to therapy in the clinic (Mattern et al, 1982). Thus, lung cancers exhibit a drug resistance profile !"#$!%&' paralleling that observed in clinical practice. Cell lines established from untreated SCLC are often sensitive !"# $!%&' to cytotoxic drugs whereas cell lines from NSCLC or relapsed SCLC are resistant to multiple agents (Carmichael et al, 1985; Ruckdeschel et al, 1987). In an effort to understand better the mechanisms of drug resistance, many !"# $!%&' selected lines have been generated by continuous or pulsed exposure to drugs. Such induced drug resistance has been associated very often with changes in expression of certain intracellular and plasma proteins which are partly characteristic of resistance to certain drugs. Tc-99m MIBI single photon emission computed tomography imaging may be a functional probe of overexpression of Pgp in patients with lung carcinoma. However, Tc-99m MIBI single photon emission computed tomography imaging cannot be used to identify the MDR involved in the MRP1 or LRP in these patients (Zhou et al, 2001).

III. Specific mechanisms of drug resistance A.! Multidrug resistance One mechanism of resistance which has now been well characterized is the so-called multidrug resistance

Table 1: Resistance-related proteins and influenced anticancer agents/Resistance-related proteins Increased or decreased. Resistance to drugs: P-glycoprotein Increased Topoisomerase II Decreased Glutathione-S transferase- Increased Metallthionein Increased

alkyltransferase Increased Thymidylate synthase Increased Cell cycle-related proteins Decreased

DOX, ACT, VCR, VP16, VM26, COL, VDS, VBL, MTC DOX, ACT, VP16, VM26, MITOX, m-AMSA DOX, ACT, VP16, VM26, L-PAM, DDP, CTX, CHL, BCNU DOX, DDP, L-PAM, CHL 06-alkylguanine DNA BCNU, CCNU, ACNU, PC, CTX 5-FU, MTX, DOX, DDP DOX, MTX, VCR, ARA-C, HU, 5-FU, CTX

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!""#$%&'(&)*+,- 5-fluorouracil, (5-FU); actinomycin D, (ACT); amsacrine)m-AMSA); carmustine, (BCNU); chlorambucil, (CHL); cisplatin, (DDP); colchicine, (COL); cyclophosphamide, (CTX); cytosine arabinoside, (ARA-C); doxorubicin, (DOX); etoposide, (VP16); hydroxyurea, (HU); lomustine, (CCNU); melphalan, (L-PAM); methotrexate, (MTX); mitomycin C, (MTC); mitoxantrone, (MITOX); procarbacin, (PC); tenoposide, (VM26); vinblastine, (VBL); vincristine, (VCR); vindesine, (VDS)

dependent enzymes have been detected in cell lines derived from NSCLC compared to SCLC cell lines (D. Arpa and Liu, 1989). It is speculated that these alterations could account for the differences in drug sensitivity between these tumours types. Evidence that GST may be involved in drug resistance has come from the generation of drug-resistant cell lines &*- %&(#) and from transfection studies with GST cDNAs (Nakagawa et al, 1990). Changes in GST expression are most marked in cell lines made resistant to nitrogen mustard compounds and nitrosoureas and redox cycling drugs such as doxorubicin (Whelan et al, 1989). GST-! isoenzyme is also over expressed in lung tumours of smokers compared to non smokers (Volm et al, 1991). It is suggested that GST over expression may be a part of an adaptive response to environmental stress to protect against toxic injury. Frequently, a co expression of P-gp and GST-! is found (Volm et al, 1991; Linsenmeyer et al, 1992). But also a coordinated induction of MRP1 and GSH-related enzymes is reported in malignant cells after exposure to cytostatic agents (Van der Kolk et al, 1999). These observations led to the suggestions that these genes share common regulatory mechanisms, and that perhaps a single transcription factor or regulating protein may be involved in their regulation. However, it is unlikely that alterations in the GST are causally related to the development of drug resistance in lung tumours, but rather that co-modification along with other resistance related enzymes could mediate drug resistance. Besides these detoxifying enzymes, normal lung is also efficiently protected against exogenous free radicals by antioxidant enzymes (AOEs). Major human AOE include superoxide dismutases, catalase and enzymes associated with GSH metabolism, all of which are expressed in human lung. In addition, human lung also expresses several thiol-containing proteins including the families of thioredoxins, thioredoxin reductases and peroxiredoxins. Their expression in human lung is located mainly to alveolar macrophages, bronchial epithelial cells and alveolar epithelium, critical areas in the oxidant protection of human lung (Kinnula et al, 2004). These proteins not only have effects on cell proliferation and cell death, but also protect both non-malignant and malignant cells against radiation and chemotherapy. The redoxregulating proteins are highly expressed in lung tumours (Soini et al, 2001) and are associated with lymph node status and prognosis in NSCLC (Kakolyris et al, 2001).Peroxiredoxins also have effects on the progression and prognosis of lung cancer (Lehtonen et al, 2004).

Within the past few years a variety of further MDR associated genes was identified: mainly the discovery of the MDR- associated protein MRP1 going along with its family members MRP2 to MRP6 of this transporters family (Cole et al, 1992; Borst et al, 1999) and the lung resistance-related protein (LRP) acting as the major vault protein in the nucleocytoplasmic transport (Scheper et al, 1993). They confer the MDR phenotype that is distinct in pattern from mdr1-related resistance, but includes many of the same drugs. MRP belongs to the ABC transporter gene super family and operates as an ATP-dependent primary active transporter for substrates conjugated with glucuronide or glutathione (Ishikawa et al, 2000). Over expression of MRP and LRP is frequently observed in primary NSCLC, especially in the well-differentiated squamous cell carcinomas (Nooter et al, 1996) and expression is significantly higher in NSCLC samples when compared to SCLC samples (Dingemanns et al, 1996).There is also evidence that expression of MRP1 and LRP can be up regulated by sub lethal exposure of lung cancer cells to some MDR-related drugs (Berger et al, 2000; Yoshida et al, 2001). Ota and colleagues examined in 1995 the levels of expression of MRP in 104 NSCLC and found that MRP over expression was associated with a worse prognosis in patients that received postoperative chemotherapy with MRP-related anticancer drugs (vindesine/etoposide). However, the concomitant operation of several resistance mechanisms may be often necessary to cause the MDR phenotype. Experimental data indicate that the genes coding for P-gp, MRP and LRP are differentially regulated by extra cellular stimuli.

B. Glutathione-dependent enzymes Another important arsenal that cells utilize to detoxify the cytotoxic effects of anticancer drugs are glutathione (GSH) and the GSH-related enzymes glutathione S-transferase (GST) and glutathione peroxidase (GPX). GST catalyzes the conjugation of electrophilic metabolites to GSH to facilitate their excretion. GPX utilizes GSH to remove reactive oxygen intermediates. GSH and its related enzymes are ubiquitously distributed in many normal tissues as well as tumors and are involved in resistance to a wide variety of drugs such as alkylating agents, anthracyclines and vinca alkaloids (Tew, 1994). There is increasing evidence that these enzymes are a determinant factor in the sensitivity of lung tumors to anticancer drugs (Carmichael et al, 1988; Sharma et al, 1993). GST isoenzymes are found in significant amounts in bronchioles and alveoli of normal lung (Awasthi et al, 1987) and most intensely in the bronchial epithelium (Anttila et al, 1993). A number of studies have shown that the amount of GST isoenzymes is even higher in tumors of the lung relative to the surrounding normal tissue (Di Ilio et al, 1988; Clapper et al, 1991). In particular, high levels of glutathione

C. Topoisomerases In addition to P-gp and non-P-gp-mediated MDR, other mechanisms for resistance to multiple drugs have been described including frequent alterations of topoisomerase II (topo II) activity (Eijdems et al, 1985;

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karda et al: Drug resistance in lung cancer growth factors, tumours promotors and many other chemicals. Some stressful environmental conditions such as heat, cold and starvation also induce MT (Hamer, 1986).Recently, the synthesis of MT by tumour cells has been proposed as a possible mechanism for the intracellular inactivation of metal-containing chemotherapeutic agents such as cisplatin. MT content and MT mRNA levels correlated well with the sensitivity of SCLC cell lines to cisplatin (Kasahara et al, 1991). A transfected cell line that over expresses MT proved not only resistant to cisplatin but also resistant to chlorambucil, melphalan and doxorubicin (Kelley et al, 1988). However, cells of various origins selected for cisplatin resistance often, but not always show increased MT expression, suggesting that an increased MT expression alone may not be the sole mediator of cisplatin resistance. Matsumoto and colleagues found in 1997 that the proportion of MT-positive tumours was significantly higher in treated NSCLC compared with untreated NSCLC and treated SCLC, whereas Joseph and colleagues demonstrated in 2001 that MT over expression was predictive of short-term survival in patients with SCLC undergoing chemotherapy. In a study with human NSCLC, we found a significant relationship between MT expression and doxorubicin resistance !"#$!%&' (Mattern et al, 1992). Thus, a number of factors may be involved in the development of drug resistance in lung tumour cells and expression of MT may be one of them.

Cole et al, 1991). Topo II is an ambiguous nuclear enzyme that is essential for many aspects of DNA function, including replication, recombination and transcription. There is evidence that this enzyme is the target of many clinically important antineoplastic drugs such as anthracyclines, ellipticines, amsacrines and epipodophyllotoxins (D’Arpa and Liu, 1989; Zijlstra et al, 1990). These drugs stabilize the cleavable complex formed between topo II and DNA, resulting in increased DNA excision, detectable as DNA single-strand or doublestrand breaks, and DNA-protein cross-links. Drug-induced cell destruction is proportional to the level of topo II, the more enzyme the greater the toxicity. This explains how a reduction in topo II could be a major mechanism of resistance to many antineoplastic drugs. Topo II has also been reported to play a role in cell proliferation. High levels of this enzyme are found in proliferating cells, and very low levels in quiescent cells (Zijlstra et al, 1990). In contrast, there are some reports that increased topo II is associated with resistance to certain DNA-damaging agents (Dingemans et al, 1999). It is also speculated that the increased affinity of topo II for cross-linked DNA in alkylating agents-resistant cells contribute to alkylator resistance by changing DNA topology, thereby facilitating DNA repair (Eder et al, 1995; Pu and Bezwoda, 1999). In surgical tissue samples of primary untreated lung tutors, significant intra- and intertumour variation in topo II expression has been observed. Topo II activity is higher in NSCLC as compared to breast cancer (McLeod et al, 1994). On the other hand, topo II activities of SCLC cell lines have been reported to be 2-fold higher than those for NSCLC cell lines, corresponding to their sensitivities to doxorubicin and etoposide (Kasahara et al, 1992). A correlation between topo II gene expression and sensitivity to doxorubicin, etoposide and cisplatin was also found in lung cancer cell lines not selected !"# $!%&' for drug resistance (Giaccone et al, 1992). Most lung cancer cell lines selected for resistance to doxorubicin demonstrate decreased levels of topo II expression in addition to P-gp over expression (Eijdems et al, 1985). Therefore, low levels of topo II expression may predict reduced sensitivity of human lung cancer to several drugs. However, as with mdr1/P-gp expression, this cannot solely explain the drug-resistant phenotype of NSCLC.

E.O6-alkylguanine-DNA alkyltransferase A number of DNA-damaging anticancer agents attack the O6 position on guanine, forming the potent cytotoxic DNA adduct. The DNA repair enzyme O6alkylguanine-DNA alkyltransferase (ATase), encoded by the gene ()(*, repairs alkylation at this site and is responsible for protecting tumour and normal cells from these agents. ATase activity varies widely among different organs, with lung tissue on average lower than others (Citron et al, 1991). However, ATase activity in normal peripheral lung tissue of smokers is significantly higher than that of nonsmokers (Drin et al, 1994; Mattern et al, 1998). Most human lung tumours contain amounts of ATase similar or greater than the tissue from which they originate (Kelley et al, 1988). In SCLC, ATase was found to be significantly lower than in NSCLC, but wide interindividual variations were observed (OberliSchrämmli et al, 1994). However, approximately 12% of human lung tumours are deficient in this enzyme (Citron et al, 1993). These ATase-deficient tumours are very sensitive to the cytotoxic effects of agents that alkylate the O6-position of guanine, such as nitrosoureas (Pegg, 1990). Thus, evidence suggesting a possible role for ATase in drug resistance of lung tumours comes from the following observations: (1) the level of ATase in tumour cells correlates well with the sensitivity to nitrosoureas (Brent et al, 1985), (2) transfection of the gene for alkyltransferase to ATase-deficient cells decreases the sensitivity to alkylating agents (Kaina et al, 1991), and (3) depletion of the activity of this enzyme by addition of O6benzylguanine significantly enhances toxicity (Dolan et al, 1993). Whether the ATase gene is a member of and

D. Metallothioneins Metallothioneins (MTs) are intracellular proteins of low molecular weight (6-7 kDa) that are present in a wide variety of eukaryotes. MT are characterized by a high content of cysteine and the ability to bind heavy metal ions including zinc, copper, cadmium and platinum. The physiological function of MT is not well understood. Most mammalian tissues contain a basal level of MT, which may vary with the type of tissue. MT has also been demonstrated in a variety of malignancies including colorectal tumours (Ă–fner et al, 1994), testicular germ cell tumours (Chin et al, 1993) and ovarian tumours (Murphy et al, 1991). MT in lung cancer tissue is significantly elevated when compared to non-malignant lung tissue (Hartet al, 1993). The synthesis of MT is easily inducible in lung or other organs by certain hormones, cytokines, 380

Cancer Therapy Vol 6, page 381! 1974; Arai et al, 1994). Moreover, patients whose lung tumours have a high proportion of cells in the S-phase generally die earlier than patients whose tumours have a low proportion of these cells (Volm et al, 1985; 1988; 1995; Alama et al, 1990). Thus, the determination of cell proliferation in clinical material provides a potentially useful marker to estimate sensitivity or resistance to anticancer drugs. Cell proliferation is regulated by both growth stimulatory and growth-inhibitory proteins (Sherr, 1993). Protein complexes that are composed of cyclins and cyclin-dependent kinases (cdks) are important factors for cellular proliferation. Cyclins are regulatory proteins for cdks and are differentially synthesized and degraded at specific points during the cell cycle (Cordon-Cardo, 1995). Five major classes of mammalian cyclins have been described (cyclin A-E). Cyclin C, D and E reach their peak of synthesis and activity during the G1 phase and regulate the transition from G1 to S-phase. Cyclins A and B achieve their maximum peaks during S- and G2-phases. We and others could demonstrate that cyclin A expression closely correlates with the proportion of Sphase cells measured by flow cytometry (Volm et al, 1997). Furthermore, patients with cyclin A-positive lung carcinomas had significantly shorter median survival times than patients with cyclin A-negative carcinomas. A significant correlation between expression of cyclin A and response of NSCLC to doxorubicin "#! $"%&' was also detected (Volm et al, 1997).

coregulated with other stress-responsive genes and controlled by a common set of transcription factors remains to be elucidated. !

F. Thymidylate synthase Thymidylate synthase (TS) plays a central role in DNA biosynthesis and is the target of many chemotherapeutic agents, such as 5-fluorouracil, methotrexate and fluorodeoxyuridine (Washtien, 1982). Moreover, tumour cells resistant to cisplatin and doxorubicin display increased levels of this enzyme (Scanlon et al, 1988; Chu et al, 1991). Human NSCLC strongly express TS in a high percentage of cases (Volm and Mattern, 1992a; 1992b). This expression of TS is significantly related to doxorubicin resistance "#!$"%&' and associated with cross-resistance to 5-fluorouracil (Volm et al, 1979). Moreover, TS-positive lung tumours have been noted to be clinically progressive, the affected patients living a significantly shorter time than those with TS negative tumours (Volm and Mattern, 1992a). In addition, evaluation of intratumoral TS activity accurately predicts responsiveness to 5-FU-based chemotherapy in NSCLC patients (Huang et al, 2000; Shintani et al, 2004). It has often been reported that the higher the TS level, the more resistant is the cell to antineoplastic drugs, in particular to 5-fluorouracil (Johnston et al, 1995). However, the relevance of TS relates not only to the importance of this enzyme as a chemotherapeutic target, but also as a DNA synthetic enzyme associated with cell division and proliferation (Stammler et al, 1995; Nakagawa et al, 2004). Nevertheless, high intrinsic levels of TS do not necessarily lead to higher proliferation rates than in cases with low levels of TS (Pestalozzi et al, 1995). A recent study has shown that TS protein binds to ()*+( mRNA suggesting an involvement in the coordinate regulation of a number of other genes (Chu et al, 1994).

H. Hypoxia Because the rate of neovascularisation frequently fails to keep pace with tumour growth, tumour vasculature is often inadequate for the tumour mass. Therefore, many solid tumours contain subpopulations of cells that are hypoxic and are relatively resistant to certain drugs (Teicher, 1994) and irradiation (HÜckel et al, 1993). This is partly caused to poor vascularisation that reduces the influx of cytostatic agents and lowers the levels of oxygen and nutrients. A growing body of evidence indicates that cells respond to hypoxic stress by altering the expression of specific genes or proteins (Wilson and Sutherland, 1989; Sutherland et al, 1996). Hypoxia is known to induce one or more transcription factors, the best characterized of which is hypoxia-inducible factor-1 (HIF-1), which in turn stimulates expression of several genes including those involved in drug resistance and endothelial cell growth. Hypoxia-induced resistance to doxorubicin and to methotrexate has been attributed to an amplification of the P-glycoprotein gene and the dihydrofolate reductase gene (Rice et al, 1986; 1987; Luk et et al, 1990; Kalra et al, 1993). Murphy and colleagues have recently shown in 1994 that metallothionein IIA mRNA levels were significantly increased during hypoxia and during reoxygenation. O’Dwyer and colleagues investigated in 1994 the effects of hypoxia on the expression of a group of enzymes involved in drug metabolism. Exposing colon carcinoma cells to hypoxia resulted in a notably increased glutathione content. In a clinical study with NSCLC, it has been shown that poor vascularisation, as measured by

G. Cell cycle-related proteins There exists general agreement that cancer chemotherapy is most successful when used on rapidly growing malignant cells (Valeriote and Van Putten, 1975). Experimental data obtained in a variety of systems ranging from mammalian cell cultures to transplanted rodent tumours show that proliferating cells are more sensitive to most cytotoxic agents than are resting cells (Drewinko et al, 1981). These experimental data are supported by the clinical observations that fast-growing tumours usually respond to treatment, whereas tumours with a low rate of proliferation very often show no response. To estimate the proliferative activity of cancer, various techniques including 3H-thymidine labeling (Alama et al, 1990) or flow-cytometric analysis (Volm et al, 1985; 1988) have been used. Several antibodies have also been produced that label preferentially the nuclei of proliferating (Volm et al, 1995a) and nonproliferating cells (Volm et al, 1995b). Although human lung tumours show a wide variation in proliferative activity and tumour doubling times, NSCLC have on average lower labelling indices and longer doubling times than SCLC, perhaps partly accounting for their resistance to cytotoxic drugs (Muggia,

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! karda et al: Drug resistance in lung cancer Overproduction of bcl-2, a blocker of apoptosis, prevents cell death induced by nearly most anticancer drugs and radiation, thus contributing to treatment failures in patients with cancer (Miyashita and Reed, 1993; Sartorius and Krammer, 2002). However, several homologs of bcl-2 have been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the bcl-2 protein. Thus, the role of bcl-2 as a clinically significant prognostic factor of drug resistance remained open (Martin et al, 2003). In a study with 85 human squamous cell lung carcinomas, we found a positive correlation between expression of bcl-2 and expression of the resistance related proteins P-gp and GST-! . Moreover, all bcl-2- positive carcinomas were resistant to doxorubicin in an !"# $!%&' predictive test (Volm and Mattern, 1995). These results indicate that bcl-2 may contribute to drug resistance in NSCLC.

vessel density, correlates with an upregulation of glutathione Stransferase-! , metallothionein and thymidylate synthase (Koomägi et al, 1995). In another study involving rectal cancer, poor angiogenesis is also linked to an expression of glutathione S-transferase and metallothionein (Mattern et al, 1996). Moreover, lung tumours with low microvessel density and low VEGF expression were more frequently to doxorubicin !"# $!%&' than tumours with high microvessel density and high expression of VEGF (Volm et al, 1996). These studies show that hypoxia or poor vascularisation result in over expression of certain detoxicating enzymes which provides an additional insight into cell resistance. Some studies have demonstrated the presence of drug resistance mechanisms in endothelial cells of normal and tumour tissue (Cordon-Cardo et al, 1990; Terrier et al, 1990). The MDR-associated P-glycoprotein and glutathione Stransferase have been localized in normal human endothelial cells and in the stroma of some tumours. Furthermore, Huang and Wright found in 1994 that some members of the fibroblast growth factor family, which are potent angiogenic peptides, may mediate resistance to some cytotoxic agents and modify gene amplification properties of tumour cells. Furthermore, there is experimental evidence that tumour cells and vascular endothelial cells within a solid tumour may stimulate each other by paracrine factors (Rak and Kerbel, 1996). On the basis of these studies, it seems reasonable to hypothesize that a highly vascularised tumour may produce elevated levels of angiogenic peptides that induce proliferation of chemo resistant endothelial cells which may confer tumour cell resistance to conventional anticancer therapy.

IV. Co-expression of resistance mechanisms and their putative regulation During the past few years it has become apparent that multiple mechanisms of resistance play a role in the clinical manifestation of drug resistance. The study of drug resistance in lung cancer has not identified one single, specific mechanism as a major cause of the resistance observed in a clinical setting. There are now various reports that cell populations exist in human lung tumours which have several resistance mechanisms at once. The parallel assessment of drug resistance parameters in human tumours has shown that individual tumours exhibit different patterns: none, several or all of the monitored resistance markers are elevated. This indicates that each tumour has its own unique resistance factor profile. In lung tumours, Oberli-Schrämmli and colleagues observed in 1994, in a majority of tumours, the concomitant over expression of ATase and GSH-related parameters. In contrast, over expression of ATase together with P-gp was never observed. There was no correlation between ATase and GSH or its enzymes in colorectal tumours (Redmond et al, 1991), however, ATase was frequently co-expressed with other drug resistance parameters in ovarian tumours (Joncourt et al, 1998). An increased expression of P-gp was detected not only concomitant to an over expression of GST, but also accompanied by a coordinate over expression of metallothionein and thymidylate synthase in human lung tumour (Volm and Mattern, 1992). A relationship exists between the extent of resistance measured !"# $!%&' and the number of detected resistance mechanisms. With an increasing extent of resistance, the number of resistance mechanisms increases (Volm et al, 1992). The reasons for the concomitant expression of different resistant mechanisms in human lung tumours are unknown. The increased expression of several resistance markers might be the result of induction of a cascade ofrelated gene products triggered by chemotherapy or environmental factors. It was found from !"# $!%&' studies that NSCLC of smokers are more frequently resistant and express a higher degree of P-gp and GST-! than tumours of non-smokers (Volm et al, 1991). Thus, smoking may up regulate different

I. Programmed cell death The recent progress in the field of biology has indicated that programmed cell death (apoptosis) plays an important role in the chemotherapy-induced tumour cell killing. Since the different antineoplastic agents induce a similar pattern of cell death, it was suggested that a common pathway of apoptosis could exist in the drug induced apoptosis and the defect in the signaling pathway of apoptosis could cause a new form of multidrug resistance in tumour cells. Recent studies in human leukemia cells have demonstrated that chemo sensitivity also depends on activation of caspases that are an integral part of the CD95 signalling pathway (Los et al, 1997). Inhibition of caspases not only retarded the apoptotic process but also provided protection from drug-induced death. In a study with NSCLC, caspase-3 expression correlated with a lower incidence of lymph node involvement and the median survival time was longer for patients with caspase-3-positive tumours than for those with caspase-3-negative tumours (Koomägi and Volm, 2000). Thus, impairment in the protease effectors’ phase of apoptosis may lead to chemo resistance against several anticancer drugs that is not due to other well-characterized resistance mechanisms such as over expression of anti apoptotic bcl-2-related proteins or increased expression of P-gp (Friesen at al, 1997).

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Cancer Therapy Vol 6, page 383! (Chu et al, 1994). Whether the ATase gene is a member of and co-regulated with other stress-responsive genes and controlled by a common set of transcription factors remains to be elucidated.

detoxifying enzymes, depending on histopathological and clinicopathological variables, to protect the cells from carcinogens but as a consequence render them resistant to drugs. The coordinate expression of different resistance mechanisms in the same tumour may explain why tumours are also resistant to drugs not involved in therapy and why a single marker, e.g. GST-! , may serve as a general marker for resistance and prognosis, irrespective of whether it is itself involved in the resistance mechanism (Mulder et al, 1995).Another explanation for the presence of different resistance mechanisms in human tumours is that tumours are mostly detected at a relatively late stage when they are already large and have metastasized. These tumours are for the most part hypoxic and the vascular networks for supply of oxygen and nutrients is substantially lower (Mattern et al, 1996). In fact, it has been shown that various resistance parameters are up regulated in tumours with poor vascularisation (Koom채gi et al, 1995) and that the reduced vascularisation of tumours together with up regulated resistance-related proteins may represent an import contributing factor to the poor response to chemotherapy and irradiation. There are several hints that detoxifying systems may share common regulatory elements. One possibility is that the resistance factors present in human tumours belong to a set of genes that can be co-ordinately expressed to protect cells from injury and against different xenobiotics. Many oncogene products are implicated in the regulation of cellular proliferation and, because the growth rate of tumours is an important determinant for the response of tumours to chemotherapy, oncogenes might influence drug resistance by regulation of proliferative activity. It has been reported that c-fos is involved in growth control and cellular differentiation (Verma, 1986). The cfos protein is associated with the gene product of the proto-oncogene cjun. The c-fos/c-jun protein complex binds specifically to a DNA sequence referred to as the AP-1 binding site and thereby affects the transcriptional expression of cellular genes (Sassone-Corsi et al, 1988). It has been also demonstrated that the promoter region of the Chinese hamster P-gp gene contains the AP-1 binding site and that this latter is essential for full promoter activity (Teeter et al, 1991). The promoter region of the genomic GST-! also contains an AP-1 motif, which suggests that this gene may be regulated by the cellular oncogenes c-fos and c-jun (Morrow et al, 1989). In a clinical study, surgical specimens of NSCLC of untreated patients were analyzed for expression of c-fos, c-jun and for resistance to doxorubicin. A significant association between drug resistance and expression of c-fos and c-jun proteins was found (Volm, 1993). With a c-fos-transfected cell line it was demonstrated that a ribozyme-mediated decrease in cfos expression was associated with reduced levels of thymidylate synthase, DNA polymerase " and metallothionein IIA (Scanlon et al, 1991). These results suggest that Fos may mediate DNA replication and repair processes through transcriptional activation of the aforementioned genes. A recent study has shown that thymidylate synthase protein binds to c-myc mRNA suggesting an involvement in the coordinate regulation of a number of other genes

V. Future directions The recent development of DNA microarray technology for large-scale analyses of gene expression has had a profound impact on biomedical research. Microarrays allow the simultaneous analysis of thousands of genes or proteins in a single experiment. Thus, it is not surprising that the old concept of prediction of drug response and individualized therapy is experiencing a revival. Staunton and colleagues determined in 2001 whether the gene expression signatures of untreated cells are sufficient for the prediction of drug sensitivity. Using a panel of 60 human cancer cell lines, gene expressionbased classifiers of sensitivity or resistance of 232 compounds were generated. They found that the accuracy of chemo sensitivity prediction was considerably better than would be expected by chance. Kudoh and colleagues used in 2000 the cDNA microarray to monitor the expression profiles of MCF-7 cells that are selected for resistance to doxorubicin. They found that a subset of genes was constitutively over expressed in cells selected for resistance to doxorubicin. Ikehara and colleagues conducted in 2004 a study with 47 human lung tumors (using cDNA microarray analysis) to determine whether expression levels of genes were correlated with survival after chemotherapy. They analyzed the expression levels of 1176 genes and found that three genes, G1/S-specific cyclin D2, type II cGMPdependent protein kinase and hepatocyte growth factor like protein, were significantly correlated with survival. Wigle and colleagues performed in 2002 expression profiling on tumour specimens from 39 NSCLC patients and could identify distinct profiles of gene expression correlating with disease-free survival. Significant technological advances in protein chemistry in the last decades have established mass spectrometry as a tool for protein study. The recently developed ProteinChip technology using surface enhanced laser desorption/ionisation (SELDI) mass spectrometry facilitate protein profiling of complex biological mixtures and could be used to discriminate e.g. normal vs. tumor tissues or treated vs. untreated cells. Preliminary results with this technology show that this method could be used to classify and predict histological subgroups as well as nodal involvement and survival in resected NSCLC (Yanagisawa et al, 2003; Zhukov et al, 2003). The promising aspect of all these new methods is the hope that it will be improve the ability to identify those patients who are at high risk of failing therapy.

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Cancer Therapy Vol 6, page 389! Cancer Therapy Vol 6, 389-394, 2008

Carcinosarcoma of endometrium: A case study with eleven DNA fluorescent in situ hybridization probes Case report

Shamim A. Faruqi1,*, Holly Prescott1, Christopher Harsch1, Harvey Spector2, Joel S. Noumoff1 1 2

Department of OB/GYN Department of Pathology, Crozer-Chester Medical Center, Upland, PA, USA.

__________________________________________________________________________________! *Correspondence: Shamim A. Faruqi, Ph.D., Director, Division of Gynecologic Oncology Research, Department of OB/GYN, CrozerChester Medical Center, One Medical Center Blvd., Upland, PA 19013, USA; Tel: 610-447-2775; Fax: 610-447-2939; e-mail: gynoncob@aol.com Key words: Mixed mullerian tumor, carcinosarcoma, endometrium, DNA probes, fluorescent !"# $!%& hybridization; ZNF217, PML, RARA, 5p15, 5q31, 7q22, inv(16), Her-2/Neu Abbreviations: promyelocytic leukemia, (PML); retinoic acid receptor alpha, (RARA) Received: 15 January 2007; Revised: 26 October 2007 Accepted: 13 April 2008; electronically published: June 2008

Summary Carcinosarcomas (previously identified as Mixed Mullerian Tumors, or MMT) of the uterus are uncommon and complex malignancies. Cytogenetic studies of these tumors are few and molecular investigations of their oncogenes are rare. We present a case including a study of this tumor with a panel of eleven fluorescent DNA probes of PML/RARA, RB-13q, c-myc, 20q13.2 (ZNF217), inv 16, Cyclin-D, LAVysion (to determine EGFR counts), Her2/Neu, 19p13.3, 7q22/7q35, and 5p15/5q31 by means of fluorescence in situ hybridization technique. In this investigation we identified the deletion of both 5q31 and one 5p15 loci, partial deletion of the 7q22 region, fusion of the PML gene at chromosome 15q22 to the RARA gene at 17q21.1, inversion of chromosome 16 and low level amplification of Her-2/neu. More importantly, the probe for the oncogene ZNF217 at 20q13.2 revealed significant amplification. This is the first known finding of this oncogene in a carcinosarcoma. ZNF217 has recently been implicated in certain ovarian and breast cancers, suggesting a potential primary role in the etiological onset of these conditions.

gene abnormality. For example, use of the drug Herceptin to treat Her-2/Neu-positive breast cancers or the use of RNA-i to stop the abnormal activity of a particular gene (Nakata et al, 2005). These approaches to contain malignancy require knowledge as to which specific cancer genes are abnormal in a given case. With these points in mind, we present this case study which includes an investigation using a panel of eleven FISH probes covering fifteen loci, chosen for their potential roles in the initiation or progression of the tumor.

I. Introduction Carcinosarcomas are rare malignancies where two different tissue types, namely carcinomatous element and sarcomatous element are present, contribute towards the formation of this complex tumor. Although only a small number of cases have been studied, so far the clones of this malignancy could be fairly complex as seen in their genomic makeup (Mitelman 1994; Halbwedl et al, 2005). A comprehensive plan for this type of tumor requires understanding of its genetic makeup for effective treatment and follow-up care of patients. This has become more important as there is now a radical change in the strategy behind finding treatments for cancer. Identifying genetic abnormalities, especially at the molecular level, would ideally enhance our understanding of the tumor biology and ultimately optimize treatment strategies. Today the focus is not only to find a single miracle drug for all the cancers; rather, the effort is highly diversified, such as finding a specific drug (or drugs) for a specific

II. Case report The patient initially presented as a 69 year old African American female gravida 8, para 8, some 20 years postmenopausal with no history of hormone use. When seen on routine annual examination, she complained of new onset of postmenopausal spotting and on physical examination what was felt to represent a new pelvic mass noted. The patient’s review of systems was essentially negative. Past medical history was significant for hypertension, cardiac arrhythmia and surgical

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!"#$%&'()'"*+',"#-&./0"#-/1"'/2'(.3/1()#&$1' ' history consistent with that of inguinal hernia repair. Physical examination revealed a mass palpable abdominally, apparently arising from the pelvis and extending above the symphysis which she herself had noted. Pelvic examination showed the vagina to be of normal mucosa and cervix without lesion. The uterus was palpable at approximately 12 cm in size with no obvious adnexal pathology appreciated. Ultrasound examination revealed an endometrial mass which was expanding the uterus. CT and MRI scans were also consistent with a similar uterine enlargement, describing a dilated endometrial canal containing enhancing material and myometrial thickening. A suggestion of right pelvic sidewall adenopathy was noted as was a ventral abdominal hernia. Exploratory laparotomy revealed a 15 cm uterus with an enlarged lower uterine segment. Total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, pelvic and paraaortic lymph node dissection and repair of ventral wall hernia were performed. The final pathology report revealed a carcinosarcoma invading the superficial myometrium with no evidence of angiolymphatic involvement. All lymph nodes were benign as were the adnexa and omentum. Immunohistochemical analysis favored a carcinosarcoma of the uterus with the epithelial component focally expressing neuroendocrine activity.

min to 2XSSC at ambient temperature. Slides were then treated with a pepsin solution for 10 min at 37oC (pepsin stock solution was made by dissolving 0.5g of pepsin in 5 ml d.H2O, 20ul of this solution was diluted in 40ml of 0.1N HCl and preheated to 37oC), again washed in 2XSSC for 5 min at ambient temperature and fixed in 4% formalin for 5 min. A final pass is made through 2XSSC for 5 min and slides were dehydrated in an ethanol series (75%, 85%, and 100% for 2, 2 and 5 minutes, respectively). Slides were then examined for individual cells under the microscope and 2-3ul of probe was added to the area on each slide with the most individual cells. Slides were placed in Hybrite programmed at 80oC for 1 min and 37oC for 48 hours. After this time, cover slips were removed and the slides were washed in 0.1% NP-40 in 2XSSC at 73oC for one min, followed by a wash in similar solution for 10 sec at ambient temperature and finally mounted in DAPI-1. Microscopic examination was for signal number and color under UV light; responsive cells were scored and photographed.

IV. Results Eleven DNA FISH probes covering fifteen oncogenes were used in this study, and consolidated results for the two abnormal monochromatic probes – ZNF217 (Figure 1) and Her-2/Neu (Figure 2) - are given (Table 1). Averaged results of the dichromatic (red/green) probes 7q22/7q35, 5p15/5q31 and PML/RARA are also presented (Table 2, Figures 3-5). Inversion 16, due to its three possible colors, is presented by itself (Table 3). Probes for RB-13q, cyclin D1, LAVysion, 19p13.3 and cmyc were normal. The cells stained with the PML/RARA fusion probe (Figure 5), on examination, had 106 cells where a single red and single green signal were seen together, which typically appeared yellow. Four clear signals - two pairs of independent genes - were seen only in one single cell out of the 125 cells scored. The averaging of probes per cell suggests that there is one copy of each gene missing from almost all cells counted, and the remaining copy may be a fusion product.

III. Materials and Methods Tumor sample was accessioned by the histopathologist while following the IRB protocol. For oncogenic evaluation, directly labeled, fluorescent DNA probes of RB13q, c-myc, ZNF217, cyclin-d, LAVysion, Her-2/Neu, 19p13.3, dual-color probes 5p15/5q31 and 7q22/7q35, the fusion probe for inversion 16, as well as the linkage probe PML/RARA (monochromatic when PML and RARA genes are linked together, and dichromatic when the linkage is normal) were procured from Vysis of Abbott Molecular (Downers Grove, Illinois, USA) for fluorescence insitu hybridization staining. 5-6 um thick sections of the tumor in paraffin block were cut onto individual slides and exposed overnight at 56oC, deparaffinated with three ten minute changes in warmed (56oC) xylene and placed in acetone for five minutes. Slides were passed through ethanol twice. They were then treated twice for 5 min each in a citric acid solution (0.2g citric acid, Sigma Cat# C0759; 2.7g Sodium Citrate, Sigma Cat# S-4641; 1 liter d.H2O) on the high setting of a microwave oven, cooled, and exposed for 5

Table 1. Signal Counts for Monochromatic FISH Probes Used in the Oncogenic Study of Carcinosarcoma. Probe Name! ZNF217! Her-2/Neu!

0 signals!

1 signal! 2 signals!

3 signals!

4 signals!

>4 signals!

Cells!

Avg. Sig./Cell! % Abnorm!

0! 0!

0! 12!

4! 18!

32! 7!

59! 10!

100! 99!

4.45! 2.51!

5! 52!

95.0%! 47.5%!

Table 2. Mean Counts for Dichromatic FISH Probes Used in the Oncogenic Study of Carcinosarcoma. Probe Name*! 7q35/7q22! 5q31/5p15! PML/RARA!

Mean Cell! 1.80! 0.08! 0.91!

Red

Probes/! Mean Cell! 1.39! 0.90! 1.01!

Green

Probes/

Total Cells Scored!

% Abnormal Cells!

51! 49! 125!

94.1%! 100%! 99.2%!

*Probes are listed in Red/Green order with relation to the target sites. This is not necessarily how the probes are identified by Vysis, but is a simplification in order to make presentation of the data simpler. PML/RARA has had yellow (fusion) responses distributed as 1 red and 1 green. There was a fusion rate of 84.8% (106/125).

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Cancer Therapy Vol 6, page 391! Table 3. Mean Counts for Trichromatic Probe Inversion (16) in Carcinosarcoma. Probe Color*! Yellow! Red! Green!

Average Probes/Cell, 59 cells! 0.51! 1.31! 0.46!

*Probe Combinations Identified are: 1Y, 2Y, 1R, 2R, 3R, 4R, 1G, 2G, 1Y+1R, 1Y+2R, 1Y+3R, 2Y+1R, 1R+1G, 2R+1G, 3R+1G, 4R+1G, 1Y+1R+1G (Y being yellow, R being red, and G being green). The following probes resulted in principally normal signal patterns, that is, two (+/- 0.2) red signals per cell: c-myc, RB-13q, cyclin-D1, LAVysion and 19p13.3

Figure 2. Single cell image of Her-2/Neu, red fluorophore, showing a single cell with three highly positive responses. Note that the response in the lower left-hand corner is outside of the cell.

Figure 1. Single cell image of ZNF217, red fluorophore, showing a single cell with five probe responses.

Figure 4. Single cell showing the single green fluorescence of 5p15. This cell is representative of the average appearance of cells stained with this probe – a single copy of 5p15 is all that appears in most of these cells.

Figure 3. Single cell showing the yellow result for 7q22/7q35. The genetic proximity of these two loci leads normal chromosomes to fluoresce yellow. One chromosome appears to be deleted in this image.

Figure 5. Two cells showing yellow fusion products for PML/RARA. Each cell only provided one fusion gene, suggesting deletion of the complementary gene.

Figure 6. Two cell image of Inversion (16), each cell containing a single red fluorophore and one yellow fusion fluorophore.

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!"#$%&'()'"*+',"#-&./0"#-/1"'/2'(.3/1()#&$1' ' in cancers of the breast (Kumaravel and Briston, 2005; Shimada et al, 2005) and ovary (Marwah et al, 2007) and uterus (Raspollini et al, 2005; Vilella et al, 2006). Hyperactivity of this gene is correlated to poor prognosis in patients with cancers of these tissues (Borst et al, 1990; Berchuck et al, 1991). Amant and colleagues in a study in 2004 of uterine carcinosarcomas reported five cases out of 21 examined with positive staining for Her-2/Neu in the carcinomatous portion of the tumor, where all sarcomatous portions were negative for said gene; this pattern is similar to the results for ZNF217, c-myc and PML/RARA. Her2/Neu is also an oncogene which plays a role in the epithelial cells. 36.8% of our cells are found to have amplified signals of Her-2/Neu. These results may indicate that Her-2/Neu mutation is a chronologically later occurrence in this case. Translocation between chromosomes 15 and 17 yields a hybrid protein, the fusion product of the PML (promyelocytic leukemia) and RARA (retinoic acid receptor alpha) genes. This translocation is commonly associated with acute promyelocytic leukemia, APL (Caprodossi et al, 2005). In a normal cell, two red and two green signals are observed, while any abnormalities may have the appearance of a yellow signal, due to the adjacency of the red and green signals for each gene. Evidence of translocation in this tumor is quite high with only one cell out of 125 showing a normal pattern of two red and two green signals. 106 of 125 cells analyzed (84.8%) showed a fusion pattern in one chromosome set and a deletion in the other (1Y, or 1 R/G proximity). The dual-color probe 7q22/7q35 provided normal results for the 7q35 probe and suggested a deletion for 7q22. Research has suggested that deletions in the 7q22 region are associated with uterine fibroids (Vanharanta et al, 2005), hairy cell leukemia (Andersen et al, 2004) and with an increased susceptibility of Hodgkin’s lymphoma patients towards a secondary neoplasia (Lillington et al, 2002). Our other dual-color probe, 5q15/5q31, suggested single and double deletions, respectively. 5q15 deletion has been shown to be a factor in oral squamous cell carcinomas (Noutomi et al, 2006) and cervical carcinomas (Arias-Pulido et al, 2002). Jenkins and colleagues reported in 1993 an ovarian carcinoma with an add 5(q31), suggesting that abnormality of this gene may play a role in carcinomatous tissues. Deletion of 5q or 7q and loss of either chromosome 5 or 7 have been found in hematologic malignancies, principally myelodysplastic syndrome (MDS) and acute myelocytic leukemia (Beyer et al, 2004). RB-13q, or RB-1, is located at 13q14 and its deletion is associated with the onset of retinoblastoma (Knudson, 1971). Since its discovery as a retinoblastoma initiator and the discovery of other complementary genes linked to retinoblastoma causation, RB-13q has been implicated in a number of different cancers, including epithelial ovarian cancer (Villeneuve et al, 1999). However, there has been no evidence of amplification of this gene with regard to carcinosarcoma analyses in the current literature, and our findings do not differ in this respect. Cyclin D1, located at 11q13, has been noted as abnormal in other gynecologic cancers (Zhuang et al,

Inversion 16 is a three color probe (Yellow = Y, Red =R, Green =G) covering two loci within the CBFB gene, which produced multiple combinations of visualized probes (Figure 6). Data are presented in terms of the average number of probes per cell of a given color.

V. Discussion Carcinosarcomas of the uterus are neoplasms with carcinomatous as well as sarcomatous elements. Emoto and colleagues inferred in 1997 that the sarcomatous portion of the tumor originated from the carcinomatous element. On the basis of chromosomal analysis, we had reached the same conclusion in our laboratory (Faruqi et al, 1997). Prior studies on carcinosarcoma oncogenes have investigated multiple composite probes, such as in the present investigation. Those studies which were published previously have yielded conflicting or varied results. To date, studies have implicated amplification of plateletderived growth factor, beta chain, PGDF-B (Kacinski et al, 1989), the oncogenes Her-2/Neu, HRAS, KRAS, and tumor suppressor p53 (Lynch et al, 1998). More commonly, c-myc oncogene amplification in carcinosarcomas has been connected with significant range differentials from normal expression (Monk et al, 1994; Jeffers et al, 1995). C-myc is one of the common oncogenes amplified in a variety of malignancies, including both hematologic cancers and solid tumors, such as breast, cervix and ovary. N-myc, while sporadically found in these cancers, has not been shown to have the significance as an indicator that c-myc has been attributed (Emoto, et al, 1997). As noted previously, our c-myc value averaged that of a normal cell (2.15 probes/cell versus an expected 2.00 in a normal cell). We therefore find it unlikely that c-myc was the initial oncogene activated in this case. We have instead found interest in the location of the ZNF217 oncogene at 20q13.2. The probe targets this specific locus of the chromosome, and it is a gene whose amplification has been identified in other malignancies, including ovarian (Dimova et al, 2005) breast (Collins et al, 1998), and non-endocrine cancers such as colorectal carcinoma (Rooney et al, 2004) and urothelial tumors (Toncheva and Zaharieva, 2005). The high percentage of cells showing amplification in our sample (95%) suggests that ZNF217 is a potential etiological factor in this carcinosarcoma. Most of the reports of oncogenic activity of this region are from ovarian carcinomas (Collins et al, 1998; Tanner et al, 2000; Dimova et al, 2005) and as best as we can determine, there is no evidence of its presence in sarcomas. As pointed out earlier, ZNF217 is a gene which is frequently amplified in carcinomas. This gene therefore is potentially of significance with regard to the etiology of this tumor, and hence supporting Emoto’s inference regarding the sarcomatous element’s origin. To the best of our knowledge, this is the first study to have definitively shown ZNF217 as a significant amplification source and a possible oncogenic initiator in this uterine malignancy. Similarly, the proto-oncogene Her-2/Neu (also called erbB-2) at 17q21.1 was also amplified, though not to the extreme level as ZNF217. Her-2/Neu is expressed mainly 392

Cancer Therapy Vol 6, page 393! Dimova I, Yosifova A, Zaharieva B, Raitcheva S, Doganov N, Toncheva D (2005) Association of 20q13.2 copy number changes with the advanced stage of ovarian cancer-tissue microarray analysis. Eur J Obstet Gynecol Reprod Biol. 118, 81-5. Emoto M, Iwasaki H, Oshima K, Kikuchi M, Kaneko Y, Kawarabayashi T (1997) Characteristics of rhabdomyosarcoma cell lines derived from uterine carcinosarcomas. Virchows Arch 431, 249-56. Faruqi SA, Deger RB, Noumoff JS (1997) Non-random chromosomal aberrations in two Mixed Mullerian sarcomas of the endometrium. Amer J Hum Genet A362, Abstr. 2118 Halbwedl I, Ullmann R, Kremser ML, Man YG, Isadi-Moud N, Lax S, Denk H, Popper HH, Tavassoli FA, Moinfar F (2005) Chromosomal alterations in low-grade endometrial stromal sarcoma and undifferentiated endometrial sarcoma as detected by comparative genomic hybridization. Gynecol Oncol 97, 582-7. Jeffers MD, Richmond JA, Macaulay EM (1995) Overexpression of the c-myc proto-oncogene occurs frequently in uterine sarcomas. Mod Pathol 8, 701-4. 8"-E6-*% ]!'% !)$+"2+% 4% 8$'% B+)2D<"$,"$% 5'% 5"$*<-*% 4'% 4)12% ]8'% 5<A$)+G% ;'% ;""-"#% 7'% 9)$+3)--% ^J% K0112L% (#+<,"-"+6>% *+UA6"*% <M% "V6+1"26)2% <=)$6)-% >)$>6-<3)J% $%&'()* +(&(,* $-,./(&(,%SRKRL'%S_@T_J% Kacinski BM, Carter D, Kohorn EI, Mittal K, Bloodgood RS, Donahue J, Kramer CA, Fischer D, Edwards R, Chambers SK, et al, (1989) Oncogene expression in vivo by ovarian adenocarcinomas and mixed-mullerian tumors. Yale J Biol Med 62, 379-92. Knudson AG Jr (1971) Mutation and cancer, statistical study of retinoblastoma. Proc Natl Acad Sci USA 68, 820-3. Kumaravel TS, Bristow RG (2005) Detection of genetic instability at HER-2/neu and p53 loci in breast cancer cells using Comet-FISH. Breast Cancer Res Treat 91, 89-93. ^6% 5'% /)6-"*@!)-A6"$)% B'% ;U<% I^'% 7U)-% `'% BU-% `'% 9622*% /'% 9U)-,% 7'% (<226-*% (('% ^"U-,% 5('% 7$)#% 8I'% CU"$*V"$,% aJ% KZWWSL/U2+6V2"% $<2"*% <M% +1"% >)-A6A)+"% <-><,"-"% bacZRS% 6-% <=)$6)-% "V6+1"26)2% -"<V2)*+6>% V$<,$"**6<-J% 3&,* 4* $%&'()% RZWK[L'%RT_Q@SQJ% Lillington DM, Micallef IN, Carpenter E, Neat MJ, Amess JA, Matthews J, Foot NJ, Lister TA, Young BD, Rohatiner AZ (2002) Genetic susceptibility to Hodgkin’s disease and secondary neoplasias, FISH analysis reveals patients at high risk of developing secondary neoplasia. Ann Oncol 13 Suppl 1, 40-3. Lynch HT, Casey MJ, Lynch J, White TE, Godwin AK (1998) Genetics and ovarian carcinoma. Semin Oncol 25, 265-80. Marwah N, Bansal C, Gupta S, Singh S, Sapna , Arora B. (2007) Immunohistochemical study of the expression of Her-2/Neu oncogene in ovarian lesions. Indian J Pathol Microbiol 50(3), 482-92. Mitelman F (1994) Catalogue of Chromosome Aberrations in Cancer. Wiley and Sons Publishing. New York, NY. Monk BJ, Chapman JA, Johnson GA, Brightman BK, Wilczynski SP, Schell MJ, Fan H (1994) Correlation of Cmyc and HER-2/neu amplification and expression with histopathologic variables in uterine corpus cancer. Am J Obstet Gynecol 171, 1193-8. Noutomi Y, Oga A, Uchida K, Okafuji M, Ita M, Kawauchi S, Furuya T, Ueyama Y, Sasaki K (2006) Comparative genomic hybridization reveals genetic progression of oral squamous cell carcinoma from dysplasia via two different pathways. J Pathol 210, 67-74. ])*V<226-6% /]'% BU*6-6% ?'% C3U--6% 7'% 5),26"$)-6% /'% ?)AA"6% C'% /)$>16<--6% /'% B>)$*"226% 7'% ?)AA"6% 7^J% K!""5L% (de@Z'% >@ ;f?% )-A% 9F]@ZO-"U% "gV$"**6<-% 6-% U+"$6-"% >)$>6-<*)$><3)*X%

2001), but not noted in carcinosarcomas; our results do not differ. The gene targets that are the most likely initiators of this tumor - ZNF217, PML/RARA, 7q22, and 5p15/5q31 deserve further exploration to determine their specific roles. The latter three targets are being researched in terms of hematologic cancers, but ZNF217 seems to play a particularly strong role in gynecologic cancers. ZNF217’s function is poorly understood in normal cells, but its overexpression is associated with a reduction in the p53 and pRB proteins in ovarian cells (Li et al, 2007). Given that our FISH results for RB suggested a normal quantification of genes, further study would require us to determine if the expression level is also normal. An ideal next step will be an immunohistochemical analysis of both RB1 and p53 activity to determine if the same principle is at work in uterine malignancies as well. If this proves to be the case, additional study of ZNF217 will be needed to identify its role in the initiation and progression of carcinosarcomas.

References Amant F, Vloeberghs V, Woestenborghs H, Debiec-Rychter M, Verbist L, Moerman P, Vergote I (2004) ERBB-2 gene overexpression and amplification in uterine sarcomas. Gynecol Oncol 95, 583-7. Andersen CL, Gruszka-Westwood A, Ostergaard M, Koch J, Jacobsen E, Kjeldsen E, Nielsen B (2004) A narrow deletion of 7q is common to HCL, and SMZL, but not CLL. Eur J Haematol 72, 390-402. Arias-Pulido H, Narayan G, Vargas H, Mansukhani M, Murty VV (2002) Mapping common deleted regions on 5p15 in cervical carcinoma and their occurrence in precancerous lesions. Mol Cancer 1, 1-3. Berchuck A, Rodriguez G, Kinney RB, Soper JT, Dodge RK, Clarke-Pearson DL, Bast RC Jr (1991) Overexpression of HER-2/neu in endometrial cancer is associated with advanced stage disease. Am J Obstet Gynecol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orst MP, Baker VV, Dixon D, Hatch KD, Shingleton HM, Miller DM (1990) Oncogene alterations in endometrial carcinoma. Gynecol Oncol 38, 364-6. Caprodossi S, Pedinotti M, Amantini C, Santoni G, Minucci S, Pelicci PG, Fanelli M (2005) Differentiation Response of Acute Promyelocytic Leukemia Cells and PML/RARa Leukemogenic Activity Studies by Real-Time RT-PCR. Mol Biotechnol 30, 231-8. Collins C, Rommens JM, Kowbel D, Godfrey T, Tanner M, Hwang SI, Polikoff D, Nonet G, Cochran J, Myambo K, Jay KE, Froula J, Cloutier T, Kuo WL, Yaswen P, Dairkee S, Giovanola J, Hutchinson GB, Isola J, Kallioniemi OP, Palazzolo M, Martin C, Ericsson C, Pinkel D, Albertson D, Li WB, Gray JW (1998) Positional cloning of ZNF217 and NABC1, genes amplified at 20q13.2 and overexpressed in breast carcinoma. Proc Natl Acad Sci USA 95, 8703-8.

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!"#$%&'()'"*+',"#-&./0"#-/1"'/2'(.3/1()#&$1' ' !"#$%#&'()* +,)'#"&* #"* !#'-%'(,.* /,"0-"&* +#"* ',"$-'-1* '2-",!(-&3*!"#$%&'()#%&'*4567897:4;5<=* >##%-?*@A9*B##%&#%$*C9*D)E,1?-%*DF9*D)G-#1*AG9*F,&&(1?* H9* FI"",%* J9* DI"",?* KL=* 6*++,8* M2-* ),%1(1,'-* #%)#$-%-* NOEP7<* (&* +"-QI-%'.?* ,/!.(+(-1* (%* )#.#%* ),%)-"=* -( ./01&'* PRS6T89*PUP;U=* Shimada M, Imura J, Kozaki T, Fujimori T, Asakawa S, Shimizu N, Kawaguchi R (2005) Detection of Her2/neu, c-MYC and ZNF217 gene amplification during breast cancer progression using fluorescence in situ hybridization. Oncol Rep 13, 63341. M,%%-"* DD9* K"-%/,%* J9* V#I.* C9* H#2,%%&&#%* W9* D-.'X-"* @9* @-Y#Z()*M9*B#"$*C9*L&#.,*HH=*6*+++8*E"-QI-%'*,/!.(+(),'(#%*#+* )2"#/#&#/,.* "-$(#%* PRQ7P;Q7T* (%* #Z,"(,%* ),%)-"=* 2'3#( 2/#%(4$5*56:89*7UTT;4=* Toncheva D, Zaharieva B. (2005) F#-[(&'-%)-* #+* )#!?* %I/\-"* )2,%$-&*#+*1(++-"-%'*$-%-&*6LOVSC9*-"\B;79*-"\B;P9*FD]F9* FFO^7* ,%1* NOEP7<8* (%* I"#'2-.(,.* 'I/#"&=* 678&79( :3&'* P56P89*UT;4T=

Vanharanta S, Wortham NC, Laiho P, Sjoberg J, Aittomaki K, Arola J, Tomlinson IP, Karhu A, Arango D, Aaltonen LA (2005) 7q deletion mapping and expression profiling in uterine fibroids. Oncogene 24, 6545-54. _(..-..,* HC9* F#2-%* J9* J/('2* ^A9* A(\&2##&2* A9* A-"&2/,%* ^=* 6*++;8*A`>;Pa%-I*#Z-"-[!"-&&(#%*(%*I'-"(%-*!,!(..,"?*&-"#I&* ),%)-"&* ,%1* ('&* !#&&(\.-* '2-",!-I'()* (/!.(),'(#%&=* <#0( -( !"#$%&'(2/#%$9*756:89*7U4<;4RP=* _(..-%-IZ-* HB9* J(.Z-"/,%* DB9* C.1-"-'-* B9* F.(\?* bC9* G(* A9* F"#$2,%* KC9* @#1",'X* VF9* H-%0(%&* >B=* 6=>>>8* G#&&* #+* /,"0-"&* .(%0-1* '#* B>FC7* !"-)-1-&* .#&&* ,'* (/!#"',%'* )-..* )?).-* "-$I.,'#"?* $-%-&* (%* -!('2-.(,.* #Z,"(,%* ),%)-"=* !$#$5( 219&8&5&8$5(2/#%$9*P:6789*5:;4=* Zhuang YH, Sarca D, Weisz A, Altucci L, Cicatiello L, Rollerova E, Tuohimaa P, Ylikomi T (2001) Cell typespecific induction of cyclin D and cyclin-dependent kinase inhibitor p27(kip1) expression by estrogen in rat endometrium. J Steroid Biochem Mol Biol 78, 193-9.

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Cancer Therapy Vol 6, page 395! Cancer Therapy Vol 6, 395-408, 2008

Randomized Study of Preoperative Focused Microwave Phased Array Thermotherapy for EarlyStage Invasive Breast Cancer Research Article

William C. Dooley1,*, Hernan I. Vargas2, Alan J. Fenn3, Mary Beth Tomaselli4, Jay K. Harness5 1

The University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center Torrance, California 3 Massachusetts Institute of Technology, Lexington, Massachusetts 4 Comprehensive Breast Center, Coral Springs, Florida 5 St. Joseph’s Hospital, Orange, California 2

__________________________________________________________________________________! *Correspondence: William C. Dooley, MD, Department of Surgery, Breast Institute, University of Oklahoma, 920 S.L. Young Boulevard, Suite 2290, Oklahoma City, Oklahoma, USA, 73104; Fax: (405) 271-4443; E-mail: William-dooley@ouhsc.edu Key words: breast cancer, ablation, minimally invasive, focused microwave thermotherapy, cancer treatment Abbreviations: cumulative equivalent minutes, (CEM); ductal carcinoma in situ, (DCIS); focused microwave thermotherapy, (FMT); hematoxylin and eosin, (H&E)

This manuscript is original and has not been published or under consideration by any other peer-reviewed journal. A portion of the results presented in this manuscript is taken in part with permission from Chapter 8 of the book: Fenn AJ, Breast Cancer Treatment by Focused Microwave Thermotherapy, Jones and Bartlett Publishers, Sudbury, MA 2007. Received: 31 October 2007; Revised: 2 June 2008 Accepted: 6 June 2008; electronically published: July 2008

Summary Microwave energy is a promising method of tumor ablation, because it can preferentially heat and kill high-water high-ion content breast carcinomas, compared to adipose and glandular tissues. The aim of this study was to investigate the hypothesis that preoperative focused microwave thermotherapy (FMT) kills breast carcinomas prior to surgery and reduces the incidence of positive margins. This is a prospective, randomized multi-center study of preoperative focused microwave phased array thermotherapy for patients with T1, T2 invasive breast cancer receiving breast conservation therapy. Tumor dose was measured as cumulative thermal equivalent minutes (CEM) of treatment relative to 43oC. Outcomes measured were pathologic margin status, surgical reexcision rates including second incisions, excised tissue volume, pathologic tumor necrosis, focused microwave thermotherapy related side effects. Interim statistical analysis was performed on a study group of 75 patients, consisting of 34 patients (mean age, 59.4 years) treated with thermotherapy prior to surgery and 41 patients (mean age, 58.0 years) that received surgery alone. At enrollment based on ultrasound measurements, mean tumor diameter was 1.7 cm in the thermotherapy arm versus 1.6 cm in the control arm (p=0.49). After treatments were completed, in the thermotherapy arm 0 of 34 (0%) patients had positive margins and in the surgery-alone arm 4 of 41 (9.8%) patients had positive margins (p=0.13). Preoperative focused microwave thermotherapy can be performed safely with minimal morbidity. Confirmatory data from a larger study should be considered to determine whether focused microwave thermotherapy can reduce the rate of positive margins compared with breast conservation surgery alone. implementation of screening mammography have contributed to a decrease in the size of breast cancer (Cady et al, 1996). Therefore, less invasive procedures such as breast conservation instead of mastectomy have been increasingly used for the treatment of breast cancer (Fisher

I. Introduction Breast cancer treatment has changed considerably in the last three decades. Changes in therapy have followed changes in clinical presentation (Edney, 2002). Increased patient education and awareness, public advocacy and the 395

Dooley et al: Preoperative Focused Microwave Phased Array Thermotherapy for Early-Stage Invasive Breast Cancer studies of adaptively focused microwave thermotherapy for breast cancer used a phased array of 8 radiating antenna elements at 915 MHz for a tightly focused microwave beam in phantom breast tissue with a maximum dimension on the order of 2 cm (Fenn et al, 1994) which might be useful for treatment of small breast cancers such as T1a (>0.1 to !0.5 cm) or T1b (>0.5 to !1 cm) tumors. Although this tightly-focused beam could be electronically steered to treat a larger region in the breast (Fenn, 1994), for general treatment of T1 tumors (!2 cm) the resulting treatment time would then be excessive when taking account of the additional time needed to steer the beam and heat and kill microscopic cancer cells in the tumor margins. Thus, because of margin considerations this tighter focus is not desirable for T1 or T2 breast cancers. Transcutaneous wide-aperture waveguide applicators opposing the breast are used to produce a wide-field focused microwave beam with a maximum heating dimension of 8 to 10 cm to irradiate and heat the high-water, high-ion content primary breast lesion as well as tumor cells in the margins (Gardner et al, 2002; Vargas et al, 2004, 2007; Fenn, 2007). Preoperative focused microwave thermotherapy has been explored in combination with preoperative anthracycline-based chemotherapy for improved tumor response compared to preoperative anthracycline-based chemotherapy alone in a small randomized study for patients with large T2, T3 breast carcinomas (Vargas et al, 2007) " the desired tumor thermal dose during active microwave heating for this study was in the range of 80 to 100 CEM at tumor temperatures in the range of 44 to 46ºC in each of two treatments administered during the first two of four cycles of chemotherapy. The primary aims of the present study were to determine whether focused microwave thermotherapy prior to surgery could 1) reduce the rate of close or positive margins and/or 2) reduce the rate of second incisions compared to surgery alone in a multi-center randomized setting. Tumor temperatures desired in this study were in the range of 48 to 52ºC with the equivalent tumor thermal dose delivered during active microwave heating in the range of 140 to 180 minutes relative to 43ºC, while avoiding skin damage and other morbidity. To reach the desired minimum thermal dose of 210 minutes (210 CEM43), additional thermal equivalent minutes were accumulated during a cool-down phase after microwaves were powered off.

et al, 2002). The impact of breast cancer tumor margins (negative, close, or positive) on locoregional recurrence has been reviewed in detail (Singletary, 2002). For example, from three studies involving 912 patients (Singletary, 2002), after breast conservation therapy when negative margin width for invasive ductal carcinoma was defined as 1 mm, the mean local recurrence rates with 45 to 123-month follow-up were 4.3%, 6.6%, 19% for negative, close (tumor cells 1 mm or less from the cut edge of the surgical specimen), positive margins, respectively. Similarly, after breast conservation, when negative margin width for ductal carcinoma in situ (DCIS) is defined as 1 mm, local recurrence rates tend to be significantly higher when positive margins for DCIS occur compared to negative margins (Singletary, 2002). The goal of any improved breast conservation procedure is to conserve as much breast tissue as possible, while achieving negative margins for carcinomas and to reduce recurrence rates without significant added side effects. Breast cancer tumor ablation as part of a multimodality approach in the treatment of breast cancer is the subject of recent interest as discussed in a number of review articles (Hall-Craggs, 2000; Singletary, 2001; Noguchi, 2003; Agnese and Burak, 2005; Huston and Simmons, 2005; Esser et al, 2007). Clinical rationale for exploring thermal ablation of primary breast carcinomas prior to surgery for improving breast conservation includes (1) reducing the rate of positive margins to reduce recurrence and (2) reducing the rate of second incisions to improve cosmesis (Fenn, 2007). Furthermore, if thermal ablation can reliably kill all breast cancer cells prior to surgery and can provide a recurrence rate equal to or less than that provided by surgery, then thermal ablation could potentially be used instead of breast conservation surgery (Singletary, 2001; Noguchi, 2003; Agnese and Burak, 2005; Huston and Simmons, 2005; Esser et al, 2007; Fenn, 2007). The use of thermal energy with radiofrequency (Jeffrey et al, 1999; Izzo et al, 2001; Singletary et al, 2002; Burak et al, 2003; Fornage et al, 2004), interstitial laser photocoagulation (Dowlatshashi et al, 2002; Dowlatshashi et al, 2004), focused ultrasound (Huber et al, 2001; Wu 2005), cryotherapy (Pfleiderer et al, 2002; Sabel et al, 2004), or focused microwaves (Gardner et al, 2002; Vargas et al, 2004; Fenn, 2007; Vargas et al, 2007) has demonstrated some success in achieving ablation of breast tumors. A previous dose-escalation study of externally applied focused microwave thermotherapy (FMT) for treatment of primary breast cancer in 25 patients demonstrated that a cumulative equivalent minutes (CEM) tumor thermal dose of 210 minutes or greater (relative to 43oC) is predictive of 100% necrosis for invasive breast carcinomas (Vargas et al, 2004). Microwave energy is promising because it can preferentially heat and damage high-water high-ion content breast carcinomas, compared to lesser degrees of heating that occurs in lower-water lower-ion content adipose and glandular tissues (Fenn, 2007; Joines et al, 1994; Campbell and Land, 1992). Adaptive microwave phased arrays, commonly used for radar applications (Fenn, 2008), can be used to concentrate (focus) the microwave energy at a tumor site in tissue (Fenn, 1991; Fenn et al, 1999). Initial preclinical

II. Patients and Methods A. Patient Selection Between November 2002 and May 2004, patients with primary invasive breast carcinomas seen at (1) University of Oklahoma, Oklahoma City; (2) Harbor-UCLA Medical Center, Torrance, California; (3) Comprehensive Breast Center, Coral Springs, Florida; (4) Mroz-Baier Breast Care Center, Memphis, Tennessee; (5) Pearl Place, Tacoma, Washington; (6) St. Joseph’s Hospital, Orange, California; (7) Royal Bolton Hospital, Bolton, UK; (8) Breast Care Specialists, Norfolk, Virginia; (9) Breast Care, Las Vegas, Nevada; and (10) Carolina Surgery, Gastonia, North Carolina were invited to participate in this FDAapproved thermal dose safety and efficacy clinical trial. This study was approved and monitored by the Human Subjects

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Cancer Therapy Vol 6, page 397! Committee at each participating institution. Other eligibility criteria included: (1) Karnofski performance status >70%, (2) core needle biopsy-proven invasive breast cancer with clinical classification T1 (0 to 2 cm clinical diameter, with the further designations T1a (>0.1 cm to 0.5 cm), T1b (>0.5 cm to 1.0 cm), T1c (>1.0 cm to 2.0 cm)) or T2 (>2 cm to 5 cm clinical diameter), (3) planned breast-conservation treatment by partial mastectomy or lumpectomy followed by radiation therapy, (4) visible tumor measurable by ultrasound, (5) absence of involvement of the skin or pectoralis muscle. All patients were required to undergo counseling and to sign written, informed consent. Specific exclusion criteria were (1) pregnancy, (2) breastfeeding, (3) presence of breast implants, (4) clinically significant heart disease, (5) pacemakers or defibrillators, (6) unable to tolerate prone position or breast compression, or (7) diagnosis of cancer made by lumpectomy or incisional biopsy. Other exclusion criteria were: (8) known bleeding diathesis, (9) laboratory evidence of coagulopathy (PT, INR > 1.5; PTT > 1.5), (10) thrombocytopenia (platelet count < 100,000/mm3), (11) anticoagulant therapy, (12) evidence of chronic liver disease or renal failure, (13) presence of any factor or condition, other than tumor size, which would preclude lumpectomy including multicentric (multifocal) disease or prior history of collagen vascular disease, or (14) breast cancer with a high probability of extensive intra-ductal in situ disease, Additional exclusions were (15) clinical tumor fixation to the pectoralis major muscle or skin, (16) involvement of the nipple or inflammatory breast cancer, or (17) skin metastases.

B. Study Treatment Plan

Design,

Treatment

treated toward the midpoint of the applicator apertures, leaving an air gap of about 1.0 to 2.0 cm from the breast tissue to allow the desired airflow and surface cooling. During treatment, the amount of breast compression, focused microwave power, aircooling of the skin are adjusted to reduce or avoid discomfort for the patient-the microwave phase focusing is verified and adjusted under computer control as necessary after any change in breast compression. For treatment of small or large tumors, a single focal position is used for the entire therapy by means of the single microwave focusing probe positioned within the tumor throughout the treatment. During some treatments, the microwave applicators are repositioned closer or farther from the breast tissue, the relative phase of the applicators is adjusted under computer control to maintain the focus at the microwave focusing probe. Figure 4 shows a photograph of a breast cancer patient receiving a focused microwave thermotherapy treatment. Subject to patient tolerance and the success in heating the tumor to the target temperature, the maximum allowed thermotherapy treatment time was sixty minutes in this study. The clinical rationale and technology for this approach of wide-field focused microwave phased array thermotherapy for tumor ablation was previously described (Fenn et al, 1999; Gardner et al, 2002; Vargas et al, 2003, 2004, 2007; Fenn, 2007). Patients were monitored for toxicity following treatment. Breast conservation surgery was to be performed within 60 days of thermotherapy.

System,

This study was designed as a prospective, multi-center randomized study with two treatment arms (thermotherapy plus surgery, surgery alone as the control arm). The planned study population was 222 patients allowing for 10% patient attrition. An interim analysis was planned to be performed when approximately 50% of the patients had all of the necessary data collected. Microwave treatment was performed on an outpatient basis using local anesthesia with patients in the prone position. A Food and Drug Administration IDE-approved two-channel 915 MHz focused microwave adaptive phased array thermotherapy system (Microfocus APA-1000 APA; Celsion (Canada) Limited) was used in this study. A schematic diagram of this system is shown in Figure 1, a photograph of the clinical system is shown in Figure 2. This minimally invasive treatment system uses opposing microwave waveguide applicators and produces a phase-focused microwave field in the compressed breast to heat and destroy high-water high-ion content tumor tissue. A sensor catheter is used to monitor parameters in the tumor during the focused microwave treatment. Prior to inserting the catheter, a local anesthetic (1% lidocaine) is infused at the skin entry point, then the skin is nicked with an 11 blade. A 16-gauge (1.65 mm OD, 1.22 mm ID) closed-end plastic catheter is inserted into the tumor under ultrasound guidance, a single-use disposable combination E-field focusing sensor (1.12 mm OD) and fiber optic temperature sensor (Figure 3) is inserted in the catheter to focus the microwaves and measure the tumor temperature during thermotherapy. The combination sensor has the fiber optic temperature sensor at the tip and the E-field sensor is 1.5 cm from the tip. The patient is positioned prone with the breast pendulant through a hole in the treatment table and the breast is compressed to a thickness of between about 4 and 8 cm depending both on size of the breast and on patient comfort. Seven temperature sensors are taped using thin sterile elastic skin closures to the skin and nipple to monitor the skin temperature during thermotherapy. Two microwave applicators are positioned on opposite sides of the compression plates with the tumor to be

Figure 1. Schematic diagram for a focused microwave adaptive phased array thermotherapy system for heating a breast tumor in the compressed breast. Reproduced from Fenn et al, 1999 with kind permission from Taylor & Francis.

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Figure 2. Focused microwave adaptive phased array thermotherapy clinical system for treating breast cancer. Photo courtesy of Celsion (Canada) Limited.

thermal dose is more rapidly accumulated in the tumor. The thermal treatment time required for a 210-minute equivalent treatment at 43ºC can be reduced, for example, to about 6.4 minutes at 48ºC, 3.2 minutes at 49ºC, or 1.6 minutes at 50ºC. The cumulative equivalent minutes (CEM) thermal dose was calculated from the measured temperatures recorded by the sensor in the tumor and also for seven sensors on the skin and nipple-the desired tumor thermal dose during active microwave heating for this study was in the range of 140 to 180 CEM43ºC at tumor temperatures in the range of 48 to 52ºC. To reach therapeutic temperature from the initial tumor temperature, the desired heating rate of the tumor was in the range of 1!C/minute to 2!C/minute. Once the desired temperature range and thermal dose range is achieved during active microwave heating (140 to 180 equivalent minutes) the microwave power is reduced to zero and breast compression is maintained during a 5-minute cool down period. During the cool-down period, as a result of reduced blood flow from the breast compression and the thermal insulation of the surrounding breast tissues, the thermal dose continues to accumulate in the tumor toward the goal of a minimum of 210 thermal equivalent minutes. As an example of the gross tumor necrosis induced by focused microwave thermotherapy, Figure 7 shows a photograph of a gross section from a case in the prior Phase II doseescalation study (Vargas, 2004). In this case, the peak tumor temperature was 48.4!C and the equivalent thermal dose was 206 minutes, which produced an induced tumor necrosis of 85% by volume with a rim of viable tumor.

Figure 3. Photograph of a combination E-field and fiber optic temperature probe for focused microwave thermotherapy treatments. The specific absorption rate (SAR) is a quantitive measure of the rise in temperature during the time period in which heat energy is applied to tissue (Field and Hand, 1990). The focused microwave thermotherapy system shown in Figure 2 has been used to heat breast phantoms with a 6 cm thickness and with simulated tumors of various sizes (Fenn, 2007). Figures 5 and 6 show measured specific absorption rate profiles in the simulated 6-cm thick breast phantom with simulated tumors of dimensions 2 ! 2 ! 2 cm and a 2 ! 2 ! 6 cm, respectively. The x axis is parallel to the breast compression dimension and spans the central 4 cm region. The z axis is the lateral dimension and is parallel to the long dimension of the simulated tumor. Due to the difference in water and ion contents in the simulated breast tissue and in the simulated breast tumor in these two examples, the microwave SAR profile matches closely the dimensions of the tumor.

D. Outcomes Measured In this study, outcomes measured were pathologic margin status, surgical reexcision (intraoperative and second incision) rates, excised tissue volume, pathologic tumor necrosis, focused microwave thermotherapy related side effects. Margins for the primary excision were assessed relative to multi-color inking to identify the medial, lateral, superior, inferior, anterior, posterior aspects of the breast tissue. In this study, tumor margins were assessed as follows: A positive margin was used to refer to breast tumor cells located at the surgical margin, close margin referred to breast tumor cells located at a distance 1 mm or less from the surgical margin, negative margin referred to breast tumor cells located at a distance greater than 1 mm from the surgical margin.

C. Thermal Dose Experimental studies support the concept that tumor cell heating for 60 minutes at 43ºC is tumoricidal, the period of time to kill tumor cells decreases by a factor of two for each degree increase in temperature above about 43ºC (Sapareto and Dewey, 1984). During the tumor temperature increase above 43ºC,

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Figure 4. Photograph showing a breast cancer patient receiving focused microwave thermotherapy treatment. Photo courtesy of Celsion (Canada) Limited.

Figure 5. Measured relative specific absorption rate (SAR) profile for a simulated compressed breast phantom with a 2 x 2 x 2 cm simulated breast tumor. Reproduced from Fenn, 2007 with kind permission from Jones and Bartlett Publishers, Sudbury, MA.

Figure 6. Measured relative specific absorption rate (SAR) profile for a simulated compressed breast phantom with a 2 x 2 x 6 cm simulated breast tumor. Reproduced from Fenn, 2007 with kind permission from Jones and Bartlett Publishers, Sudbury, MA.

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Figure 7. Photograph of gross section in which focused microwave phased array thermotherapy induced 85% tumor necrosis. Reproduced from Vargas et al, 2004 with kind permission from Springer Science and Business Media.

Tumor cell kill was based on tumor necrosis and was estimated from hematoxylin and eosin (H&E) histological sections (performed at each participating site) from wide local excision of the primary breast tumor. Necrosis was estimated and expressed as a percentage of necrotic tumor areas in relation to necrotic and viable tumor areas. If viable tumor cells were found either close (1 mm or less from the inked margin) or at the inked margin, intraoperative reexcision was recommended by the pathologist. Adverse events, vital signs, laboratory measurements (complete blood count, blood urea nitrogen and creatinine, bilirubin, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, alkaline phosphatase, routine chemistries) were monitored to evaluate the safety and tolerability of the thermal dose. Performance status was recorded after thermal therapy.

from the study analysis were as follows: 1 patient had an excisional biopsy (prior to enrollment there was no tumor remaining) who was enrolled by error, 1 patient had a T3 tumor (the tumor clinical size was 9 cm, the patient received a double mastectomy instead of breast conservation), 5 patients did not receive thermotherapy and were withdrawn from the study, 2 patients went on chemotherapy prior to surgery, 3 patients had multifocal tumors. In the surgery-alone arm, 5 patients excluded were as follows: 1 patient signed informed consent but did not participate, 1 patient developed metastatic pancreatic cancer and was withdrawn from the study, 2 patients had multifocal tumors, 1 patient had extensive DCIS. Demographics for the 75 patients included in the interim statistical analysis are summarized in Table 1. Thirty-four patients (mean age: 59.4 years) were treated with thermotherapy prior to surgery and 41 patients (mean age 58.0 years) received surgery alone. At enrollment, mean tumor maximum diameter based on ultrasound was 1.7 cm (range 0.7 to 3.6 cm) in the thermotherapy arm versus 1.6 cm (range 0.7 to 2.7 cm) in the surgery alone arm. Clinically in the thermotherapy arm there were 64.7% T1 and 35.3% T2 tumors, in the surgery alone arm there were 75.6% T1 and 24.4% T2 tumors. Clinically, at enrollment in the thermotherapy arm 93.9% of patients were node negative and 6.1% were node positive, in the surgery alone arm 87.8% of patients were node negative and 12.2% were node positive. Based on pathologic final diagnosis, 32 of 34 (94%) patients in the thermotherapy arm had invasive ductal carcinomas compared with 37 of 41 (90%) patients in the surgery alone arm. DCIS was present in 12 of 34 (35.3%) patients in the thermotherapy arm compared to 25 of 41 (61%) in the surgery alone arm (p=0.04) at final diagnosis.

E. Statistical Analysis Statistical differences between thermotherapy and surgery alone groups were quantified using Student t test and Fisher’s exact test (InStat, GraphPad Software, Inc.), as appropriate (Armitage, 1994). All tests were two-sided and p values !0.05 were considered statistically significant. Parameters were quantified by mean, range, standard deviation (SD), 95% confidence interval (CI) as appropriate. Cases excluded from the statistical analysis included patients that did not receive treatment, patients with multifocal tumors, patients with extensive DCIS determined by pathological evaluation of the excised breast tissue, patients discontinued or withdrawn from the study, patients that received an excisional biopsy, or patients with tumors not T1 or T2.

III. Results A. Patient Characteristics A total of 92 patients were enrolled (46 in each arm), 17 cases were excluded from the interim statistical analysis, based on the criteria discussed in the previous section, providing a study group of 75 patients for analysis. In the thermotherapy arm, 12 patients excluded 400

Cancer Therapy Vol 6, page 401! Table 1. Demographic and tumor characteristics of the study population of 75 patients for the interim analysis. Reproduced from Fenn, 2007 with kind permission from Jones and Bartlett Publishers, Sudbury, MA

N Age, Years Tumor Size Based on Ultrasound Measurements at Enrollment

Clinical Tumor Classification at Enrollment

Clinical Nodal Status at Enrollment Tumor Histology (final diagnosis)

Tumor Grade (final diagnosis)

Mean Range

Thermotherapy 34 59.4 42-89

Surgery Alone 41 58.0 41-89

Mean, cm

1.7

1.6

Range, cm 95% Confidence Interval, cm

0.74-3.64

0.70-2.73

1.47-1.94

1.44-1.77

T1a

0%

2.4%

T1b T1c T2

20.6% 44.1% 35.3%

19.5% 53.6% 24.4%

Negative / Positive

93.9% / 6.1%

87.8% / 12.2%

Invasive Ductal Carcinoma Invasive Lobular Carcinoma Colloid DCIS Component Present High Grade Intermediate Grade Low Grade

94%

90%

3%

7%

3% 35% 40% 43% 17%

3% 61% 36% 33% 31%

B. Thermotherapy Characteristics and Tumor Necrosis

In the thermotherapy arm, 16 of 34 (47.0%) tumors were located laterally, 9 of 34 (26.5%) medially, 9 of 34 (26.5%) at either the 6 o’clock or 12 o’clock position. In the surgery-alone arm, 19 of 41 (46.3%) tumors were located laterally, 12 of 41 (29.3%) medially, 10 of 41 (24.4%) at either the 6 o’clock or 12 o’clock position. In the thermotherapy arm, 79.4% of patients were postmenopausal compared to 73.2% in the surgery-alone arm. In the thermotherapy arm, 76.5% of patients were estrogen receptor (ER) positive, 50% progesterone receptor (PR) positive, 21.2% HER-2/neu positive. In the surgery-alone arm, 82.8% of patients were ER positive, 75.6% were PR positive, 34.1% were HER-2/neu positive.

The relevant thermal parameters for the 34 thermotherapy-treated patients are summarized in Table 2. Breast compression at the start of thermotherapy had a mean value of 5.3 cm (range 3.0 to 9.2 cm). Microwave treatment time had a mean value of 26.6 minutes (range 5.0 to 60 minutes). The cumulative equivalent thermal dose had mean value182.0 minutes (range 0 to 645.0 minutes). Microwave treatment energy dose had mean value 150.7 kilojoules (range 28.8 to 350.3 kilojoules). Tumoricidal temperatures (> 43oC) were reached in 31 of 34 (91.2%) patients. The desired target tumor temperature of (48oC or greater) was achieved in 15 of 34 (44.1%)

Table 2. Thermal parameters achieved in the thermotherapy study arm of 34 patients in the interim analysis. Reproduced from Fenn, 2007 with kind permission from Jones and Bartlett Publishers, Sudbury, MA

Breast Compression During Microwave Treatment (cm) Microwave Treatment Time (minutes) Microwave Energy Dose (kilojoules) Peak Tumor Temperature (!C) Time at Tumor Temperature !48!C Cumulative Thermal Dose CEM43 (minutes) Number of Days Between Thermotherapy and Surgery

Mean

Range

Standard Deviation

95% Confidence Interval

5.3

3.0-9.2

1.4

4.8-5.9

26.6 160.7 46.7 1.2 182.0

5.0-60.0 28.8-350.3 34.6-51.4 0.0-5.2 0-645.0

14.9 88.5 3.2 1.66 152.6

21.4-31.8 129.3-192.1 45.5-47.8 0.6-1.8 127.9-236.1

19.6

7-60

14.1

14.2-25.1

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Dooley et al: Preoperative Focused Microwave Phased Array Thermotherapy for Early-Stage Invasive Breast Cancer surgery-alone arm (p=0.07). The mean volume of excised breast carcinoma (residual) from gross pathology in the first surgery was 3.24 cc in the thermotherapy arm and 3.05 cc in the surgery-alone arm (p=0.82). The mean volume of excised breast tissue including first excision and all reexcisions was 117.8 cc in the thermotherapy arm and 94.7 cc in the surgery-alone arm (p=0.13). The pathologic margins and second incision results are given in Table 4. In the thermotherapy arm 29 of 34 (85.3%) patients had negative margins, in the surgeryalone arm 30 of 41 (73.2%) had negative margins (p=0.26). In the thermotherapy arm, 5 of 34 (14.7%) patients had close margins, in the surgery-alone arm 7 of 41 (17.1%) had close margins (p=0.81). In the thermotherapy arm 0 of 34 (0%) patients had positive margins and in the surgery-alone arm 4 of 41 (9.8%) patients had positive margins (p=0.13). In the thermotherapy arm, 2 of 34 (5.9%) patients received a second incision, in the surgery-alone arm 4 of 41 (9.8%) patients received a second incision (p=0.68).

patients. The targeted thermal dose of 140 to 180 equivalent minutes during active microwave heating was achieved in 20 of 34 (58.8%) patients. With the additional tumor heating that occurred during the cool-down phase, the minimum desired thermal dose of 210 minutes was achieved in 17 of 34 (50%) patients. Patients underwent breast-conserving surgery on average 19.6 days after thermotherapy (range: 7-60 days, SD=14.1 days, 95% CI 14.2 to 25.1 days). Mean pathologic tumor necrosis by volume was 29.8% (range 0 to 100%) in the thermotherapy arm and 0.1% in the surgery-alone arm (p=0.0001). In the group of 17 patients that received the minimum desired thermal dose of 210 equivalent minutes, the minimum targeted temperature of 48!C was achieved in 15 of 17 (88.2%) cases and the mean tumor necrosis by volume for these 17 patients was 38.0% with two of the patients having 100% tumor necrosis. The desired target tumor temperature of (48oC or greater) was achieved in 15 of 34 (44.1%) patients. In the group of 15 patients that received the minimum desired target temperature of 48oC, the minimum thermal dose of 210 equivalent minutes was achieved in 15 of 15 (100%) cases and the mean tumor necrosis by volume for these 15 patients was 36.3% with two of the patients having 100% tumor necrosis. Therefore, the cases receiving the target temperature and target thermal dose were highly overlapping.The most consistent high degree of tumor necrosis occurred when all of the following parameters occurred together: 1) a cumulative thermal dose greater than 210 minutes was achieved in the tumor, 2) the tumor temperature was maintained above 48!C for greater than 2.0 minutes, 3) the microwave treatment time was greater than 10 minutes, 4) the microwave energy dose was greater than 50 kilojoules.

D. Side Effects Side effects for the 34 patients in the interim-analysis group receiving thermotherapy prior to surgery are listed in Table 5. In this study, 3 of 34 (8.5%) patients receiving thermotherapy had a skin burn less than 3 cm in size that, in each case was excised during the subsequent breast conserving surgery procedure. These three patients received peak skin temperatures of 44.2!C, 42.4!C, 41.7!C and skin thermal doses of 5.7, 1.5, 2.7 equivalent minutes, respectively. One of the patients (case 2004) in this study received a skin burn at the site of scar tissue on the skin. This skin burn is likely associated with reduced blood flow in the scar tissue, which can increase the scar tissue temperature relative to surrounding normal skin tissue that has normal blood flow which can remove heat.With respect to the overall thermotherapy treatment, 15 of 34 (44.1%) of patients had no discomfort during thermotherapy, 11 of 34 (32.4%) had mild discomfort, 3 of 34 (8.8%) had moderate discomfort, 5 of 34 (14.7%) had intolerable discomfort. During thermotherapy treatment 3 of 34 (8.8%) patients experienced nausea. None of the patients (0 of 34 (0%)) in the thermotherapy arm developed a fever, hypotension, dizziness, or ulceration as a result of thermotherapy. Skin reddening (erythema) occurred in 4 of 34 (11.8%) patients as a result of mechanical compression of the skin or from microwave treatment. Edema was reported in 3 of 34 (8.8%) cases. Moderate ecchymosis possibly related to thermotherapy occurred in 1 of 34 (2.9%) patients-the event resolved within 9 days. Mild nipple retraction possibly related to thermotherapy occurred in 1 of 34 (2.9%) patients. Mild subcutaneous fibrosis occurred in 1 of 34 (2.9%) patients. Moderate abscess caused by necrotic tissue surrounding the tumor occurred in 1 of 34 (2.9%) patients-the patient was given antibiotics and the wound was irrigated, the event was resolved within 29 days. Hematoma / seroma was reported in 6 of 34 (17.6%) thermotherapy patients-3 cases were mild, 2 cases were mild/moderate, 1 case was moderate severity.

C. Excised Tissue and Margin Status Predicted and actual excised breast tissue volumes for the thermotherapy arm and control arm are given in Table 3. At enrollment, based on ultrasound tumor measurements in three dimensions, the calculated mean tumor volume (Ellipsoidal volume = Length x Width x Depth x 0.524) was 2.5 cc in the thermotherapy arm and 1.5 cc in the surgery-alone arm (p=0.14). Assuming a 2 cm surgical margin surrounding the ellipsoidal tumor, the predicted mean volume of excised breast tissue was 88.7 cc in the thermotherapy arm and 80.4 cc in the surgeryalone arm (p=0.18). The mean volume of excised breast tissue in the first surgery, including any intraoperative reexcisions, was 115.0 cc (range 24.3 to 363.1 cc, 95% CI 91.2 to 139.0 cc) in the thermotherapy arm and 90.7 cc (range 4.4 to 282.0 cc, 95% CI 71.4 to 110.0 cc) in the surgery alone arm (p=0.11). The rate of intraoperative reexcisions during the first surgery was 0.74 per patient in the thermotherapy arm and 0.71 per patient in the surgeryalone arm (p=0.88). Based on gross pathology, the maximum dimension of excised breast tumor had mean value 1.87 cm in the thermotherapy arm and 1.85 cm in the surgery-alone arm (p=0.94). The gross maximum dimension of the initial excised breast tissue (prior to any re-excisions) had mean value 8.3 cm in the thermotherapy arm and 7.4 cm in the 402

Cancer Therapy Vol 6, page 403! Table 3. Summary of excised tissue volumes in both arms of the study for the interim analysis. Reproduced from Fenn, 2007 with kind permission from Jones and Bartlett Publishers, Sudbury, MA

Number of patients per arm Ultrasound measured maximum diameter at enrollment (cm)

Ultrasound measured ellipsoidal volume at enrollment (cc)

Thermotherapy 34

Surgery Alone 41

P

tumor

Mean

1.7

1.6

0.61

tumor

Range 95% Confidence Interval Mean

0.74-3.64 1.49-1.97 2.5

0.7-2.73 1.44-1.77 1.5

0.14

Range

0.13-14.12

0.1-6.7

95% Confidence Interval Mean

1.33-3.58 88.7

1.05-2.03 80.4

Range

52.4-182.6

50.2-133.8

95% Confidence Interval

77.9-99.5

73.9-86.8

Mean

0.74

0.71

0.88

Mean

1.87

1.85

0.94

Range

0.0-4.5

0.8-3.3

95% Confidence Interval Mean Range

1.5-2.2 8.3 4.5-14.0

1.6-2.1 7.4 3.0-11.7

95% Confidence Interval

7.6-9.0

6.7-8.1

Mean

3.24

3.05

Range

0-13.95

0.15-14.15

95% Confidence Interval

2.0-4.47

1.98-4.12

Mean Range 95% Confidence Interval Mean Range

117.8 24.3-363.1 93.7-141.9 41.7 -65 to 253

94.7 4.4-282.0 75.1-114.2 16.5 -94 to 240.9

95% Confidence Interval

12.0 to 71.5

-6.5 to 39.4

Predicted breast tissue excision volume based on 2 cm margin surrounding ellipsoidal tumor measured by ultrasound in three dimensions (cc) Number of interoperative reexcisions per patient Pathology measured maximum tumor diameter from initial excised specimen (cm)

Pathology measured maximum tissue diameter of initial excised specimen (cm)

Pathology measured ellipsoidal tumor volume from excised specimens in first surgery (cc) Actual breast tissue excision ellipsoidal volume including first excision and all reexcisions including second incisions (cc) % Excess breast tissue (by volume) excised compared to predicted

0.18

0.07

0.82

0.13

0.18

DCIS = ductal carcinoma in situ P>0.05 not significant

Table 4. Summary of margin status and second incision rates in both arms of the study in the interim analysis. Reproduced from Fenn, 2007 with kind permission from Jones and Bartlett Publishers, Sudbury, MA. Thermotherapy 34

Surgery Alone 41

P

Rate of negative margins

29/34 (85.3%)

30/41 (73.2%)

0.26

Rate of close margins

5/34 (14.7%)

7/41 (17.1%)

0.81

Rate of positive margins

0/34 (0%)

4 of 41 (9.8%)

0.13

Rate of close or positive margins Invasive only

5/34 (14.7%)

11/41 (26.8%)

0.26

3/34 (8.8%)

7/41 (17.1%)

0.33

DCIS only

0/34 (0%)

4/41 (9.8%)

0.13

Both invasive and DCIS

2/34 (5.9%)

0/41 (0%)

0.20

2/34 (5.9%)

4/41 (9.8%)

0.68

Number of patients per arm Margin status at completion of first surgery

Incidence rate for type of tumor cells involved in close or positive final margins (Invasive only, DCIS only or both invasive and DCIS) for first surgery Rate of second incisions DCIS = ductal carcinoma in situ P>0.05 not significant

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Dooley et al: Preoperative Focused Microwave Phased Array Thermotherapy for Early-Stage Invasive Breast Cancer Table 5. Summary of side effects for the group of 34 patients in the interim analysis receiving focused microwave thermotherapy prior to surgery. Side Effect Abscess Hematoma/Seroma Subcutaneous Fibrosis Nipple retraction Ecchymosis Fever Hypotension Dizziness Ulceration Nausea Edema Erythema Discomfort during thermotherapy None Mild Moderate Intolerable

No. of Patients 1 6 1 1 1 0 0 0 0 3 3 4

% 2.9 17.6 2.9 2.9 2.9 0 0 0 0 8.8 8.8 11.8

15 11 3 5

44.1 32.4 8.8 14.7

et al, 1996; Vernon et al, 1996; van der Zee et al, 2000; Falk and Issels, 2001). The main objective of this study was to determine whether focused microwave phased array thermotherapy could decrease the rate of positive margins, without clinically significant toxicity in a multi-center randomized setting. The target cumulative thermal dose of 210 equivalent minutes was achieved in 17 of 34 (50%) patients and the target tumor temperature of 48oC or greater was achieved in 15 of 34 (44.1%) patients. Based on H&E staining, complete (100%) necrosis of breast cancer was achieved in 2 of 17 (11.8%) cases receiving the targeted cumulative thermal dose-in both of these cases there was no pathologic evidence of viable invasive carcinoma or DCIS cells. To achieve a high degree of necrosis of breast cancer, in addition to the target cumulative thermal dose of 210 equivalent minutes, this study indicates that the target tumor temperature should also be maintained above 48!C for greater than 2.0 minutes. Tumor cell kill measured by other pathologic testing such as nicotinamide adenine dinucleotidediaphorase (NADH) and immunohistochemistry, as used in other breast tumor ablation studies (Gardner et al, 2002; Huston and Simmons, 2005), were not evaluated in this study. An apparent reduction in the incidence of positive margins was suggested (but did not reach statistical significance) in the thermotherapy arm; however, both consistent delivery of the targeted tumor thermal dose and consistent tumor necrosis were not achieved in this study. The desired target tumor temperature of (48oC or greater) was achieved in 15 of 34 (44.1%) patients. The targeted thermal dose of 140 to 180 equivalent minutes during active microwave heating was achieved in 20 of 34 (58.8%) patients. With the additional tumor heating that occurred during the cool-down phase, the minimum desired thermal dose of 210 minutes was achieved in 17 of 34 (50%) patients. The maximum allowed treatment time (60 minutes) was reached in only one case. The tumor

IV. Discussion There is an increasing interest in the use of minimally invasive ablative techniques in the treatment of breast cancer (Singletary, 2001). A new treatment paradigm in which systemic therapy may be followed by minimally invasive local therapy to eradicate any residual local tumor has been proposed and is the subject of research studies. This paradigm is seen as the next step in an evolution of therapy that is designed to offer lessinvasive means of therapy to patients presenting with small cancers that are detected as a result of screening practices (Singletary, 2001). The objective of using minimally invasive treatment techniques is to cause complete local tumor regression and long-term local control of breast cancer with minimal damage to the surrounding normal breast tissue and the skin. Other characteristics of the ideal treatment are that it must be an outpatient procedure using a percutaneous or transcutaneous application without the need of sedation or general anesthesia. The morbidity and local complications must be minimal. Thermal ablation has taken a prominent role in minimally invasive approaches to treat neoplasms (Singletary, 2001). Freezing can be achieved through cryoablation (Pfleiderer et al, 2002), whereas heat energy can be generated by the use of interstitial laser photocoagulation (Dowlatshashi et al, 2002), radiofrequency induced coagulation (Izzo et al, 2001), focused ultrasound (Huber et al, 2001),or focused microwave ablation (Vargas et al, 2004, 2007; Fenn, 2007). The cytotoxic effects of thermotherapy on cancer cells using temperatures in the range of at least 45 to 53ยบC have been demonstrated on cancer cells in vitro (Gerhard, 1978). Clinical trials have demonstrated improved efficacy of hyperthermia with temperatures in the range of about 42 to 46ยบC when used with radiation therapy and chemotherapy (Valdagni and Amichetti, 1993; Overgaard

404

Cancer Therapy Vol 6, page 405! (Ryoo et al, 1989; Smitt et al, 1995; Recht et al, 1996; Park et al, 2000). However, this difference in positive margins did not reach statistical significance and might be the result of a type 2 statistical error requiring a larger clinical trial to demonstrate a statistical difference. Therefore, the hypothesis that focused microwave phased array thermotherapy treatment prior to breast conservation surgery provides significant tumor cell kill in the primary tumor and in the margins and is responsible for a low incidence of positive margins requires further critical study. Two other factors might be responsible for this finding regarding positive margins: 1) A smaller volume of breast tissue on average was excised in the surgeryalone arm and might have contributed to a higher rate of positive margins in the surgery-alone arm compared to preoperative thermotherapy; however, this volume difference was not statistically significant. Possible explanations for the larger volume of breast tissue excised in the thermotherapy arm might include increased firmness (induration) of the breast cancer mass as a result of thermotherapy effects leading to better resection, or investigator bias because this was not a blinded study. Future studies of focused microwave thermotherapy could investigate whether induration of the breast tumor occurs more frequently in the thermotherapy arm compared to surgery alone. Future studies should investigate whether thermal effects on tissue such as induration and coagulative necrosis have any impact on hemostasis in surgery. 2) At final pathologic diagnosis, a higher rate of ductal carcinoma in-situ (61.0% in the surgery-alone arm versus 35.3% in the thermotherapy arm (p=0.04), based on final pathology) might have been a contributing factor to the higher rate of positive margins in the surgery-alone arm compared to thermotherapy-two of the four cases in the surgery-alone arm had positive margins for DCIS. Elements of intraductal carcinoma might extend outside the tumor mass and can be difficult to detect preoperatively with mammography (Holland, 1990). This characteristic of DCIS is a potential pitfall of minimally invasive ablative approaches. In addition, microwaveheating properties of DCIS lesions need to be evaluated in future studies to determine whether DCIS is high-water high-ion content similar to invasive breast carcinomas. If DCIS cells are high-water high-ion content similar to invasive breast carcinoma, they might be successfully treated with wide-field focused microwaves and more global heating of the breast while sparing the normal healthy breast tissue. Studies demonstrate that the differentiation of DCIS is correlated with the grade of the associated invasive ductal carcinoma (Douglas-Jones et al, 1996; Cadman et al, 1997) indicating a pathological similarity between DCIS and invasive ductal carcinoma cells. If microwave thermotherapy does not preferentially heat DCIS compared to normal breast tissues, then DCIS might be a potential exclusion criterion. In this study, patients were determined to have DCIS at the final pathohistological assessment (post surgery) from the excised tissue. Future studies of thermal ablation must consider selecting patients with invasive carcinoma with lower risk of an intraductal carcinoma component based on pre-treatment mammography and percutaneous biopsy

thermal dose achieved for this case was 81.5 minutes and the peak tumor temperature achieved was 45.2!C. For this case, skin temperatures were elevated in the range of 40 to 42!C, but were not too high to cause any skin burn, erythema, or edema, the patient tolerated the treatment. In this study, the mean microwave treatment time was 26.6 minutes, suggesting that longer treatment times could have been used, which could have increased the percentage of patients receiving the desired thermal dose. The decision to stop a treatment prior to completing the targeted thermal dose depended primarily on the temperature achieved in the tumor and on the skin, as well as on patient comfort. In a typical stopped treatment, skin temperatures were in the range of about 40!C to 42!C and the tumor temperature was in the range of about 43!C to 47!C. Additional breast compression, elevated microwave power levels, adjustment of the skin cooling air-flow rate, applicator spacing to the breast tissue might have improved the heating rate of the tumors. As each patient can tolerate a different amount of breast compression for different periods of time during focused microwave thermotherapy, the investigator or technician operating the equipment must make decisions regarding the treatment parameters, which are subject to experience. For externally applied focused microwave heating of the breast for thermotherapy-alone treatments at target tumor temperatures in the range of 48!C to 50!C, additional skin cooling might be desirable. In terms of possible skin burns, air cooling in the present clinical system appears to be sufficient based on many treatments that have been conducted in patients; however, increased air cooling could be considered to decrease the possibility of skin burns. In this study, 3 of 34 (8.5%) patients receiving thermotherapy had a skin burn less than 3 cm in size that, in each case was excised during the subsequent breast conserving surgery procedure. During treatments, the microwave power can be lowered based on skin temperature measurements to decrease the skin temperatures and decrease the possibility of skin burns. Water cooling of the skin is not an option for the current focused microwave thermotherapy system which uses non-contact air-cooled applicators. Some of the cases in which the desired thermal treatment was not achieved might be a result of the learning curve associated with using a new treatment technology in a multi-institutional study setting. At this interim analysis, although an apparent (potential) reduced incidence of positive margins in the thermotherapy arm was suggested (p=0.13, but not statistically significant), due to the observed inconsistent delivery of the desired thermal dose and inconsistent tumor necrosis, it was decided to close the study early. The intent then was to document the data collected in this study to be used in developing a future, larger, clinical study of focused microwave thermotherapy (with improvements in the treatment delivery protocol) for preoperative treatment of breast carcinomas. An observation arising from this study is the 0% rate of positive margins in the thermotherapy arm in comparison to 9.8% positive margins in the surgery alone arm and reported incidences of positive margins after breast conserving surgery in the literature of 4% to 44% 405

Dooley et al: Preoperative Focused Microwave Phased Array Thermotherapy for Early-Stage Invasive Breast Cancer this possibility was not investigated in this study. A future study could consider investigating any such correlation. The local anesthetic used in this study is 1% lidocaine and it is injected into the breast in the region of the skin nick, prior to the skin nick, for insertion of the catheter that is placed into the tumor. The lidocaine injectate is ionic which could tend to be heated by microwave energy more rapidly in comparison to low ion content tissue. In this study, there did not appear to be any significant heating effects from infusion of lidocaine such as skin burn in the vicinity of the entry point for the tumor catheter. Approaches to measure the success of thermal ablation are essential if a non-surgical management is contemplated. Imaging with mammography is unlikely to provide additional valuable information. Ultrasound is most valuable in providing easy targeting and placement of the sensors within the mass, but may not provide information about the viability of the tumor. On the other hand, delayed contrast-enhanced magnetic resonance imaging has the potential to show the effects of treatment as areas of devascularization (non-enhancement) within the treated area (Hall-Craggs, 2000). Alternatively, more extensive tissue sampling in follow up with core-biopsy or vacuum-assisted core biopsy may provide essential information of residual tumor. In the current study, FMT was conducted by the placement of a percutaneous sensor catheter for focusing of the microwaves (E-field sensor) and measurement of tumor temperatures (temperature sensor). In the future, focused microwave thermotherapy has the potential of being a transcutaneous procedure that does not require the insertion of any needle-type devices and targeting may be conducted based on imaging alone. Regarding the possibility of skin burns, it is relatively easy to heat a superficial tumor with a single microwave applicator facing the tumor, it tends to be more difficult for a second applicator on the opposite side of the breast to contribute significant power to this distal superficial tumor without burning the proximal skin. In this study, the protocol excluded patients with clinical tumor fixation to the skin, involvement of the nipple, or skin metastases. In this study the primary aims were to improve pathologic margins and reduce the rate of second incisions. Radiological quantitative assessment by mammography, MRI, ultrasound (pretreatment and presurgery) is planned to be used and documented in a future study. The ultimate goal of thermal ablation with focused microwaves would be to develop a protocol for ablationonly treatment and eliminate surgery. A clinical study to attempt to demonstrate this goal could be considered, once a consistent 100% tumor ablation with focused microwave thermotherapy is demonstrated pathologically in a randomized multi-center setting in which patients receive thermotherapy prior to surgery. It is worth noting that a portion of the patient population is unable to tolerate general anesthesia. Ablative procedures such as focused microwave thermotherapy or other heat-delivery techniques may be the only recourse for some patients.

results (Cadman et al, 1997; Bagnall et al, 2001). Future studies could determine whether wide-field focused microwave thermotherapy has an ablative effect on DCIS (Fenn, 2007). In the group of five patients with multifocal tumors that were excluded from the main analysis, each patient had positive margins after the first surgery was completed. Future studies of focused microwave thermal ablation should consider using magnetic resonance imaging (Huber, 2001; Saslow et al, 2007) to better identify patients with multifocal tumors or DCIS to possibly exclude these patients prior to enrollment. If focused microwave thermal ablation for multifocal tumors is to be studied in a clinical trial setting, to achieve negative margins multiple ablation treatments (one for each focal tumor site) should be considered. It is worth noting that in addition to sparing lowwater, low-ion content normal fatty breast tissues, focused microwave therapy should spare the chest wall and pleura. The opposing microwave applicator apertures are oriented, approximately, at right angles with respect to the chest wall and pleura and would tend not to heat these regions. The ability of focused microwave phased array thermotherapy to consistently deliver a specified therapeutic thermal dose and minimum temperature to breast cancer tumors and achieve consistent 100% pathologic tumor cell kill might depend on a number of factors including (1) breast compression thickness which impacts blood flow in the tumor and in surrounding tissues, the required penetration depth for the microwaves, (2) tumor size, (3) tumor histology, (4) tumor location, (5) accuracy, number and positioning of tumor temperature probes, (6) initial temperature of the tumor, (7) magnitude of skin surface cooling, (8) patient tolerance, (9) length of time between thermotherapy and surgery, (10) method of pathologic evaluation of tumor cell kill. It would be desirable in future clinical studies of pre-surgical heatalone focused microwave phased array breast thermotherapy to explore a more consistent delivery of a minimum 210-minute thermal dose (with a corresponding tumor temperature in the range of about 48 to 50ยบC) and higher thermal doses, potentially in the range of 240 to 360 thermal equivalent minutes, for more effective and consistent tumor cell kill (Fenn, 2007). In this study, the wide-field focused microwave thermotherapy system uses single sensor temperature measurements which provides limited monitoring and control of tumor thermal dose. Future studies of FMT could explore measuring the tumor blood perfusion rate with Doppler ultrasound when the breast is compressed prior to the start of thermotherapy, to assess the ability to overcome perfusion effects and more effectively heat the tumor-these perfusion measurements could be used as a guide to determine the desired breast compression thickness prior to the start of thermotherapy. Future studies conducted at more consistent and/or higher therapeutic doses of FMT will provide meaningful information regarding the success of this therapy for heatalone treatment of breast cancer based on pathological response. Comparison with other means of percutaneous thermal ablation of breast cancer will then be possible. It is possible that breast hydration due to the menstrual cycle can correlate with treatment effects, but

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Cancer Therapy Vol 6, page 407! Century Perspective, Volume I: Congress Abstracts, (Chapman JD, Dewey WC, Whitmore GF, Editors), Academic Press, San Diego, California, 290. Fenn AJ (2007) 60*#%"(7#.&*0(20*#"8*."(9:(;,&4%*-()$&0,<#5*( 2+*08,"+*0#=:1(Sudbury, MA,(Jones and Bartlett Publishers. Fenn AJ (2008) >-#="$5*(>."*..#%(#.-(?+#%*-(>00#:%(@,0(/#-#0( #.-( 7,884.$&#"$,.%, Norwood, MA, Artech House Publishers, 133-160. Fenn AJ (1994) Minimally Invasive Monopole Phased Arrays for Hyperthermia Treatment of Breast Cancer, Proc 1994 Int Symp Antennas, Nice, France, November 8-10, 1994, pp. 418-421. Fenn AJ, Cheung AY, Cao H (1994) Adaptive focusing experiments for minimally invasive monopole phased arrays in hyperthermia treatment of breast cancer, 16th Annual IEEE Engineering in Medicine and Biology Society International Conference, Baltimore, Maryland, November 36, 1994, 766-767. Fenn AJ, Wolf GL, Fogle RM (1999) An adaptive phased array for targeted heating of deep tumors in intact breast: animal study results. Int J Hyperthermia 15, 45-61. Field SB and Hand JW, editors (1990) >.( A."0,-4&"$,.( ",( "+*( ?0#&"$&#'(>%=*&"%(,@(7'$.$&#'(B:=*0"+*08$#, London, Taylor & Francis, 242-274. Fisher B, Anderson S, Bryant J, Margolese RG, Deutsch M, Fisher ER, Jeong JH, Wolmark N (2002) Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpectomy, lumpectomy plus irradiation for the treatment of invasive breast cancer. N Engl J Med 347, 1233-1241. Fornage BD, Nour SN, Ross MI, Mirza AN, Kuerer HM, Edeiken BS, Ames FC, Newman LA, Babiera GV, Singletary SE (2004) Small (!2-cm) breast cancer treated with USguided radiofrequency ablation: feasibility study, Radiology 231, 215-226. Gardner RA, Vargas HI, Block JB, Vogel CL, Fenn AJ, Kuehl GV, Doval M (2002) Focused microwave phased array thermotherapy for primary breast cancer. Ann Surg Oncol 9, 326-332. Gerhard H, Klinger HG, Gabriel E (1978) Short term hyperthermia: In vitro survival of different human cell lines after short exposure to extreme temperatures. in 7#.&*0( 2+*0#=:( 9:( B:=*0"+*08$#( #.-( /#-$#"$,., Streffer C, editor, Baltimore-Munich: Urban & Schwarzenberg. 201-203. Hall-Craggs MA, (2000) Interventional MRI of the breast: minimally invasive therapy, Eur Radiol 10, 59-62. Holland R, Hendriks JH, Vebeek AL, Mravunac M, Schuurmans Stekhoven JH (1990) Extent, distribution, mammographic/histological correlations of breast ductal carcinoma in situ. Lancet 335, 519-522. Huber PE, Jenne JW, Rastert R, Simiantonakis I, Sinn HP, Strittmatter HJ, von Fournier D, Wannenmacher MF, Debus J (2001) A new noninvasive approach in breast cancer therapy using magnetic resonance imaging-guided focused ultrasound surgery. Cancer Res 61, 8441-8447. Huston TL and Simmons RM (2005) Ablative therapies for the treatment of malignant diseases of the breast. Am J Surg( 189, 694-701. Izzo F, Thomas R, Delrio P, Rinaldo M, Vallone P, DeChiara A, Botti G, D'Aiuto G, Cortino P, Curley SA (2001) Radiofrequency ablation in patients with primary breast carcinoma. A pilot study of 26 patients. Cancer 92, 20362044. Jeffrey SS, Birdwell RL, Ikeda DM, et al. (1999) Radiofrequency ablation of breast cancer. First report of an emerging technology. Arch Surg 134, 1064-1068. Joines WT, Zhang Y, Li C, Jirtle RL (1994) The measured electrical properties of normal and malignant human tissues from 50 to 900 MHz. Med Phys 21, 547-50.

In the present study, thermal ablation with focused microwave thermotherapy was tolerated in a majority of cases and no significant complications were recorded. A higher thermal dose than used in this study would be required for increased tumor necrosis. The results of this study are suggestive of a reduction in positive margins in the preoperative thermotherapy plus breast-conserving surgery arm. A larger randomized study is required to verify this conclusion.

Acknowledgements Other investigators participating in this clinical study were: Christine T. Mroz, MD (Mroz-Baier Breast Care Center, Memphis, Tennessee), Lynne P. Clark, MD (Pearl Place, Tacoma, Washington), Claire M. Carman, MD (Breast Care Specialists, Norfolk, Virginia), John Winstanley, MD (Royal Bolton Hospital, Bolton, United Kingdom), Sandra B. Schultz, MD (Carolina Surgery, Gastonia, North Carolina), Theodore M. Potruch, MD (Breast Care, Las Vegas, Nevada). Study sponsored by Celsion (Canada) Limited.

References Agnese DM, Burak WE (2005) Ablative approaches to the minimally invasive treatment of breast cancer. Cancer J 11, 77-82. Armitage P and Berry G (1994) !"#"$%"$&#'( )*"+,-%( $.( )*-$&#'( /*%*#0&+1( 2+$0-( 3-$"$,., Oxford: Blackwell Science. 156163, 283-311. Bagnall MJ, Evans AJ, Wilson AR, Pinder SE, Denley H, Geraghty JG, Ellis IO (2001) Predicting invasion in mammographically detected microcalcification. Clin Radiol 56, 828-32. Burak WE Jr, Agnese DM, Povoski SP, Yanssens TL, Bloom KJ, Wakely PE, Spigos DG (2003) Radiofrequency ablation of invasive breast carcinoma followed by delayed surgical excision, Cancer 98, 1369-1376. Cadman B, Ostrowski J, Quinn C (1997) Invasive ductal carcinoma accompanied by ductal carcinoma $.(%$"4((DCIS): comparison of DCIS grade with grade of invasive component. The Breast 6, 132-137. Cady B, Stone MD, Schuler JG, Thakur R, Wanner MA, Lavin PT (1996) The new era in breast cancer: invasion, size, nodal involvement dramatically decreasing as a result of mammographic screening. Arch Surg 131, 301-308. Campbell AM and Land DV (1992) Dielectric properties of female human breast tissue measured $.( 5$"0, at 3.2 GHz. Phys Med Biol 37, 193-210. Douglas-Jones AG, Gupta SK, Attanoos RL, Morgan JM, Mansel RE (1996) A critical appraisal of six modern classifications of ductal carcinoma in situ of the breast (DCIS): correlation with grade of associated invasive carcinoma. Histopathology 29, 397-409. Dowlatshashi K, Dieschbourg JJ, Bloom KJ (2004) Laser therapy of breast cancer with 3-year follow-up, The Breast J 10, 240-243. Dowlatshashi K, Francescatti DS, Bloom KJ (2002) Laser therapy for small breast cancers. Am J Surg 184, 359-363. Edney JA (2002) Breast cancer treatment for the future based on lessons from the past. Am J Surg 184, 477-483. Falk MH, Issels RD (2001) Hyperthermia and Oncology, Int J Hyperthermia1 17, 1-18. Fenn AJ (1991) Adaptive hyperthermia for improved thermal dose distribution, Radiation Research: A Twentieth-

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Dooley et al: Preoperative Focused Microwave Phased Array Thermotherapy for Early-Stage Invasive Breast Cancer Noguchi M (2003) Minimally invasive surgery for small breast cancer, J Surg Oncol 84,94-101. Overgaard J, Gonzalez Gonzalez D, Hulshof MCCH, Arcangeli G, Dahl O, Mella O, Bentzen SM (1996) Hyperthermia as an adjuvant to radiation therapy of recurrent or metastatic malignant melanoma. A multicentre randomized trial by the European Society for Hyperthermic Oncology. Int J Hyperthermia 12, 3-20. Park CC, Mitsumori M, Nixon A, Recht A, Connolly J, Gelman R, Silver B, Hetelekidis S, Abner A, Harris JR, Schnitt SJ (2000) Outcome at 8 years after breast-conserving surgery and radiation therapy for invasive breast cancer: influence of margin status and systemic therapy on local recurrence. J Clin Oncol 18, 1668-1675. Pfleiderer SO, Freesmeyer MG, Marx C, Kuhne-Heid R, Schneider A, Kaiser WA (2002) Cryotherapy of breast cancer under ultrasound guidance: Initial results and limitations. Eur Radiol 12, 3009-3014. Recht A, Come SE, Henderson IC, Gelman RS, Silver B, Hayes DF, Shulman LN, Harris JR (1996) The sequencing of chemotherapy and radiation therapy after conservative surgery for early stage breast cancer. N Engl J Med 334, 1356-1361. Ryoo MC, Kagan AT, Wollin M, Tome MA, Tedeschi MA, Rao AR, Hintz BL, Kuruvilla AM, Nussbaum H, Streeter OE Jr, et al (1989) Prognostic factors for recurrence and cosmesis in 393 patients after radiation therapy for early mammary carcinoma. Radiology 172, 555-559. Sabel MS, Kaufman CS, Whitworth P, Chang H, Stocks LH, Simmons R, Schultz M (2004) Cryoablation of early-stage breast cancer: Work-in-progress report of a multiinstitutional trial, Ann Surg Oncol 11, 542-549. Sapareto SA, Dewey WC (1984) Thermal dose determination in cancer therapy. Int J Radiat Oncol Biol Phys 10, 787-800. Saslow D, Boetes C, Burke W, Harms S, Leach MO, Lehman CD, Morris E, Pisano E, Schnall M, Sener S, Smith RA, Warner E, Yaffe M, rews KS, Russell CA; American Cancer Society Breast Cancer Advisory Group (2007) American cancer society guidelines for breast screening with MRI as an adjunct to mammography. CA Cancer J Clin 57, 75-89. Singletary SE (2001) Minimally invasive techniques in breast cancer treatment. Seminars in Surg Onc 20, 246-50. Singletary SE (2002) Surgical margins in patients with earlystage breast cancer treated with breast conservation therapy. Am J Surgery, 184, 383-93. Singletary SE, Fornage BD, Sneige N, Ross MI, Simmons R, Giuliano A, Hansen N, Kuerer HM, Newman LA, Ames FC, Babiera G, Meric F, Hunt KK, Edeiken B, Mirza AN (2002) Radiofrequency ablation of early-stage invasive breast tumors: an overview, Cancer J 8, 177-180. Smitt MC, Nowels KW, Zdeblick MJ (1995) The importance of lumpectomy surgical margin status in long term results of breast conservation. Cancer 76, 259-267. Valdagni R, Amichetti M (1993) Report of long-term follow-up in a randomized trial comparing radiation therapy and radiation therapy plus hyperthermia to metastatic lymphnodes in stage IV head and neck patients. Int J Radiat Oncol Biol Phys 28, 163-169.

van der Zee, J, Gonzalez Gonzalez D, van Rhoon GC, van Dijk JDP, van Putten WLJ, Hart AAM (2000) Comparison of radiotherapy alone with radiotherapy plus hyperthermia in locally advanced pelvic tumors: a prospective, randomised, multicentre trial. Lancet 355, 1119-25. van Esser S, Maurice van den Bosch AAJ, van Diest PJ, Mali WTM, Borel Rinkes IHM, van Hillegersberg R (2007) Minimally invasive ablative therapies for invasive breast carcinomas: An overview of current literature. World J Surg 31, 2284-92. Vargas HI, Dooley WC, Fenn AJ, Tomaselli MB, Harness JK (2007) Study of preoperative focused microwave phased array thermotherapy in combination with neoadjuvant anthracycline-based chemotherapy for large breast carcinomas. Cancer Ther 5 401-408. Vargas HI, Dooley WC, Gardner RA, Gonzalez KD, HeywangKobrunner SH, Fenn AJ (2003) Success of sentinel lymph node mapping after breast cancer ablation with focused microwave phased array thermotherapy. Am J Surg 186, 330-332. Vargas HI, Dooley WC, Gardner RA, Gonzalez KD, Venegas R, Heywang-Kobrunner SH, Fenn AJ (2004) Focused microwave phased array thermotherapy for ablation of earlystage breast cancer: Results of thermal dose escalation. Ann Surg Oncol 11, 139-46. Vernon CC, Hand JW, Field SB, Machin D, Whaley JB, van der Zee J, van Putten WL, van Rhoon GC, van Dijk JD, Gonzalez Gonzalez D, Liu FF, Goodman P, Sherar M (1996) Radiotherapy with or without hyperthermia in the treatment of superficial localized breast cancer: results from five randomized controlled trials, Int J Radiat Oncol Biol Phys 35, 731-744. Wu F, Zhi-Wang ZB, Zhu H, Chen WZ, Zou JZ, Bai J, Li KQ, Jin CB, Xie FL, Su HB (2005) Extracorporeal high intensity focused ultrasound treatment for patients with breast cancer. Breast Cancer Res Treat 92, 51-60.

William C. Dooley

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Metachronous thyroid metastasis of primary rectal adenocarcinoma Case Report

Raffaele Longo1,*, Francesco Torino1, Roberta Sarmiento1, Domenico Gattuso1, Cinzia Bernardi2, Giampietro Gasparini1 1 2

Division of Medical Oncology, “San Filippo Neri” Hospital Rome, Italy Division of Anathomy & Pathology, “San Filippo Neri” Hospital Rome, Italy

__________________________________________________________________________________! *Correspondence: Raffaele Longo, MD, Division of Medical Oncology ‘San Filippo Neri’ Hospital, Via Martinotti 20, 00135 Rome, Italy; Tel: +39-06-33062272; Fax: +39-06-33062414; E-mail: raflongo@libero.it Key words: Colorectal cancer, Thyroid metastasis, chemotherapy Abbreviations: 5-fluorouracil, (5-FU); leucovorin, (LV) Received: 17 June 2008; Revised: 23 June 2008 Accepted: 26 June 2008; electronically published: July 2008

Summary Thyroid metastases occur frequently from lung, breast and renal cancer, with an overall incidence of 1.25% to 24.2% reported in autopsy studies (Shimaoka et al, 1961; Czech et al, 1982). Only a few cases of colorectal cancer metastatic to the thyroid have been reported to date. We describe the case of a 60-year old man who underwent right hemithyroidectomy with laterocervical lymph node dissection for a thyroid metastasis from a previous rectal adenocarcinoma surgically treated 11 years before.

II. Case report

I. Introduction

In September 1996, a 60 year-old Caucasian man underwent radical resection of a moderately differentiated low rectal adenocarcinoma (TNM stage pT3, pN0, M0; AJCC, 1997; Dukes B2) followed by 6 cycles of adjuvant chemotherapy with 5-fluorouracil (5-FU: 425 mg/mq/d, d1-5, every 28 days) and leucovorin (LV: 20 mg/mq/d, d 1-5, every 28 days) (Mayo Clinic regimen) and standard loco-regional radiotherapy (50.4 Gy). In June 2001, a chest X-ray and then a whole body CT scan documented 2 lung metastases in the upper left lobe. No other systemic lesions were observed. Tumor marker levels (CEA and CA 19.9) were also in the normal ranges. The patient underwent lung metastasectomy. No other systemic treatments were started. In May 2004, the patient was submitted to lobectomy of the upper left lung for a new single metastasis, documented by a whole body CT scan. In June 2007,the patient noticed a swelling in the right side of his neck associated with dysphonia. Physical examination revealed a nodule in the right thyroid lobe along with an enlarged right, lower, laterocervical lymph nodes. Ultrasonography showed a mass, 3.0 x2.0 cm in size, with a non-uniform internal echo pattern and associated tracheal compression (Figure 1A, red arrow). A total body CT confirmed these data (Figure 1B, red arrow). No other concomitant diseases were documented. Blood chemistry, thyroid hormones, CEA, and CA 19.9 levels were all in the normal ranges. The patient underwent fine-needle aspiration (FNA) of the thyroid lesion. FNA cytology documented the presence of neoplastic cells, characteristic of

The occurrence of clinically-relevant thyroid metastases from colorectal cancer is rare compared to that from other tumors such as renal cell, breast, and lung cancer (Shimaoka et al, 1961; Czech et al, 1982). In contrast to clinical evidence, autopsy data show an incidence of 4% of thyroid metastases in patients with primary colon cancer (Czech et al, 1982). This discrepancy might be explained by the following: 1. only a few thyroid metastases become clinically symptomatic, 2. the frequent concomitant presence of metastases in other organs, such as liver, lung or brain, that usually determine the worse prognosis of these patients potentially masking thyroid metastases, 3. thyroid metastases could be mistaken for other thyroid diseases (Ivy, 1984). Based on these considerations, in a patient with a history of a previous cancer, the presence of a thyroid lesion requires careful evaluation typically with ultrasound scan, thyroid hormone tests, and fine-needle aspiration and/or biopsy. Treatment of thyroid metastases is not well established and depends on the presence of clinicallyrelevant symptoms, other concomitant systemic metastases, pre-existing co-morbidities and the patient’s overall prognosis.

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Longo et al: Metachronous thyroid metastasis of primary rectal adenocarcinoma rectal adenocarcinoma (Figure 1C, E!E, 20x, red arrow) and whith a pattern similar to that found in the primary tumor and in lung metastases. Due to the lack of other systemic metastases, the patient’s good performance status (ECOG: 0), the patient’s symptoms and the radiological tracheal compression, a right hemithyroidectomy plus isthmectomy with laterocervical lymph nodes dissection was performed in August 2007. Histology showed tall columnar cells in acinar structures infiltrating normal tissues (Figure 1D, E!E, 10x, red arrows). At the immunohistochemical analysis, tumor cells were negative for both TTF1 (Figure 1E, 10x, red arrows) and thyroglobulin, and positive for cytokeratin 20 (Figure 1F, 20x, red arrows). In contrast to this pattern, normal follicular cells were positive for both TTF1 (Figure 1E, 10x, green arrows) and thyroglobulin, and negative for cytokeratin 20 (Figure 1F, 20x, green arrows). Immunohistochemistry was consistent with metastasis from rectal cancer (Ivy, 1984; Kumamoto et al, 2006; Mattavelli et al, 2006; Iguchi et al, 2007). Metastases in 2 lymph nodes were also reported. Thyroid hormone replacement therapy was started. The patient was submitted to 12 cycles of postoperative systemic chemotherapy with 5-FU (400 mg/mq/d, d1-2, bolus; 600 mg/mq/d, d1-2, continuous infusion, every 14 days) and LV (100 mg/mq/d, d 1-2, every 14 days) (De Gramont regimen). He is

now alive, in good general condition and disease-free.

III. Discussion Thyroid metastases are rare, with an overall incidence of 1.25% to 24.2% reported in autopsy studies, the most frequent primary sites being breast and lung cancers (Shimaoka et al, 1961). In clinical series, renal cell carcinoma is the most common primary tumor associated to thyroid metastases (Czech et al, 1982). Despite an incidence of 4% of thyroid metastatic involvement secondary to colorectal cancer described in a large autopsy series, only a few clinical cases are reported in the literature to date (Shimaoka et al, 1961). Metastatic tumor involving the thyroid gland may pose diagnostic problems (Ivy, 1984; Iguchi et al, 2007). As metastatic tumors do not clinically differ from primary thyroid cancers and other thyroid benign diseases, it is crucial to consider this possibility in patients with a history of previous primary cancers in order to avoid misdiagnosis.

Figure 1. (A) Ultrasonography demonstrated a thyroid lesion in the right lobe of 3.0 x 2.0 cm in size with a non-uniform internal echo pattern (red arrow) and initial signs of tracheal displacement and compression (green arrow). (B) Chest CT scan confirmed the presence of a non-homogeneous thyroid lesion in the right lobe with tracheal displacement and initial aspects of compression (red arrow). (C) Fine-needle aspiration (FNA) cytology of the thyroid lesion documented a cluster of neoplastic cells, characteristic of rectal adenocarcinoma (red arrow). (D) Histology showed tall columnar cells in acinar structures (red arrows) infiltrating normal thyroid tissue. (E) At immunohistochemistry, tumor cells resulted negative for TTF1 (red arrows) whereas normal follicular cells were positive (green arrows). (F) In contrast to the immunohistochemical pattern shown in Fig. 1E, tumor cells were positive for cytokeratin 20 (red arrows) whereas normal thyroid cells were negative for this marker (green arrows).

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Cancer Therapy Vol 6, page 411! that passes laterally along the middle hemorrhoidal artery and the iliac nodes and the obturator fossa. These anatomical characteristics might explain the presence of systemic metastases, particularly in the lung, without an hepatic secondary involvement. Both these two considerations could make the diagnosis very difficult as this clinical and radiological pattern might be confounded with that of other thyroid diseases that are usually treatedin a different way.

Three hypotheses could explain the rarity of thyroid metastases: first, mechanical stress associated with the abundant blood supply to the thyroid and its fast blood flow could make adhesion and implantation of metastatic tumor cells difficult (Iguchi et al, 2007); second, sex hormones effect at thyroid tissue may alter the occurrence of thyroid metastases that are more frequent in the women (Kumamoto et al, 2006; Mattavelli et al, 2006); third, the high oxygen saturation index and high iodine content of the thyroid gland could inhibit the growth of cancer cells (Willis, 1931). This last hypothesis is supported by the observation that patients with benign thryoid diseases, such as adenomatous goiter, that are characterized by lowoxygen/iodine states, have an increased risk of developing thyroid metastases (Smith et al, 1987). Though controversial, the treatment of choice is surgery aimed at reducing potential airway compression and difficulty with swallowing (Rosen et al, 1995). The extent of surgery can vary from partial lobectomy to total thyroidectomy and lymph node dissection depending on patient and disease factors. The affected lobe and isthmus are usually removed. The extent of the surgical procedure does not seem to influence long-term prognosis, which is understandably worse in the case of concurrent systemic spread (Rosen et al, 1995). As is always the case, the final treatment recommendation should take into account the presence of synchronous metastases in other organs, the patient’s performance status and co-morbidities, as well as the clinical relevance of thyroid metastases-related symptoms (Poon et al, 2004). Our case illustrates two interesting points. First, the patient had a long cancer history of 11 years from the diagnosis of primary rectal cancer and the development of thyroid metastases. In clinical practice, it is not very common to observe a similar long history in colorectal cancer patients, particularly when metastases in other organs, such as in the lung, like in this case, are previously reported. Tumor genetic variables might explain this behaviour. The second aspect is related to the lack of other synchronous lesions. This latter aspect is more frequently observed in low rectal adenocarcinoma and depends on the particular venous lymphatic drainage of the lower rectum

References Czech JM, Lichtor TR, Carney JA, van Heerden JA (1982) Neoplasms metastatic to the thyroid gland. Surg Gynecol Obstet 155, 503-505. Iguchi T, Matsuoka J, Sato S, Okumura Y, Omori M, Mifune H, Akaki S, Kanazawa S (2007) F-18 FDG PET demonstration of a thyroid metastasis in a patient with colon cancer. Clin Nucl Med 32, 361-362. Ivy HK (1984) Cancer metastatic to the thyroid gland: a diagnostic problem. Mayo Clin Proc 59, 856-859. Kumamoto K, Utsumi Y, Sugano K, Hoshino M, Suzuki S, Takenoshita S (2006) Colon carcinoma metastasis to the thyroid gland: report of a case with a review of the literature. Tumori 92, 252-256. Mattavelli F, Collini P, Gervasoni C, Pizzi N, Pallotti F, Pastorino U (2006) Double, metachronous thyroid metastases of colon cancer. Tumori 92, 249-251. Poon D, Toh HC, Sim CS (2004) Two case reports of metastases from colon carcinoma to the thyroid. Ann Acad Med Singapore 33, 100-102. Rosen IB, Walfish PG, Bain J, Bedard YC (1995) Secondary malignancy of the thyroid gland and its management. Ann Surg Oncol 2, 252-256. Shimaoka K, Sokal JE, Pickren JW (1961) Metastatic neoplasmas in the thyroid gland: pathological and clinical findings. Cancer 15, 557-565. Smith SA, Gharib H, Goellner JR (1987) Fine-needle aspiration: usefulness for diagnosis and management of metastatic carcinoma to the thyroid gland. Arch Intern Med 147, 3112. Willis RA (1931) Metastatic tumor in the thyroid gland. Am J Path 7, 187-211.

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Outcome of Hispanics with colorectal cancer residing in Puerto Rico with access to adequate health care facilities: results are not inferior to USA or European caucasians Research Article

Luis Ignacio Echenique1,*, Fernando Cabanillas2, Viviana Freire1, Ignacio A. Echenique1 1 2

Hospital Auxilio Mutuo, San Juan, Puerto Rico Auxilio Centro de Cancer, Hospital Auxilio Mutuo, San Juan, Puerto Rico

__________________________________________________________________________________! *Correspondence: Luis Ignacio Echenique, Hospital Auxilio Mutuo, Torre Medica Suite 218, Ave. Ponce de Leon # 735, San Juan, Puerto Rico, 00918; Tel/Fax:787-296-0949/7872-296-0958; e-mail: liegio@yahoo.com Key words: Hispanics, colorectal cancer, health care facilities, Demographics, Outcomes, rectal carcinomas Abbreviations: carcinoembryonic antigen, (CEA); sphincter preservation rate, (SPR); Surveillance, Epidemiology and End Results, (SEER); Total mesorectal excision, (TME) Received: 20 November 2008; Revised: 18 March 2008 Accepted: 5 June 2008; electronically published: July 2008

Summary Colorectal cancer cure rates vary significantly according to ethnicity and race. No data regarding this are available for Puerto Ricans. We examined the outcome of 202 Puerto Ricans with colorectal cancer managed by one surgeon. We evaluated survival rates and sphincter preservation rate (SPR). Most presented with localized disease (69% stage I- II), 18% stage III and 13% stage IV. Median age was 66. Five year overall survival and cause specific survival were 77% and 85% respectively. SPR varied according to the location: 56/57 (98%) for those in the high/middle rectum (6-15 cm from the anal verge) and 12/24 (50%) for those in the low rectum. Age distribution was: <55 y/o=19.7%, 55-64= 25%, 65-74=28.9%, >75=23%. This age distribution reveals a younger population than USA. Data from the Island Cancer Registry confirmed this. Our survival data compares favorably with that of American Whites; this suggests that Puerto Ricans residing in the island don’t have an adverse prognosis provided adequate medical care is available. The unfavorable outcome of Hispanics might be related to disparities in health care access. In view of the younger age distribution, colorectal cancer screening for Puerto Ricans might have to be modified to start at 45 years.

the Veteran’s Administration system, where all patients have access to medical care, the results of AfricanAmericans with the same stage of disease are equal to those of Caucasians, thus suggesting that the difference in survival in the USA is mostly attributable to disparities in health care. Recent data suggest that the clinical outcome for Hispanics with colorectal cancer is superior to that of African-Americans but inferior to non-Hispanic whites (Clegg et al, 2002). It is not clear whether certain genetic differences might account at least in part for this difference. However, it is difficult to define accurately what a Hispanic is. For example there are huge differences between the genetic stock of immigrant Mexicans, whose

I. Introduction Colorectal cancer is a potentially curable disorder but cure rates vary significantly according to race (Howe et al, 2006), disease stage (Steele et al, 1994), surgical modality used (Wibe et al, 2002), hospital surgical volume (McGrath, 2005) as well as the surgeon’s volume (McGrath et al, 2005), and more recently whether adjuvant chemotherapy is used for stage II-III cases (Andre et al, 2004). In the USA, African-Americans have fared poorly when compared with Caucasians (Clegg et al, 2002). This has been attributed both to racial disparities in the delivery of adequate health care as well to potential biological differences in their colorectal cancer genes. However, in

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Echenique et al: Outcome of Hispanics with colorectal cancer with access to adequate health care facilities! cm and high rectum as 11-15 cm. Clinical stage was determined with the use of CT scans of abdomen and pelvis as well as chest x-rays or CT of chest. For clinical staging of rectal primaries, rectal sonograms were performed in 36 (44%) of the 81 cases and 57% of low and mid-rectal lesions; in cases which didn’t have a sonogram staging was performed with a CT scan. Total mesorectal excision (TME) was the surgical approach we used in patients with rectal cancer. Chemoradiation was utilized preoperatively usually for all T3-T4 lesions and for all low rectal cases except those who were managed with a local transanal excision. Those in the mid rectum with T3-T4 were also treated preoperatively. Usually cases with high rectal presentation didn’t receive preoperative radiation except selected cases who either had unfavorable prognostic features such as evidence of lymph node involvement or T3-T4 on CT or mucinous histology. Statistical analysis for survival and disease free survival was performed by Kaplan-Meier technique and log rank test was used to determine significance. All other statistical significance analysis was performed by chi square test.

genes are mostly of native Aztec and Mayan Indian extraction versus Puerto Ricans whose genetic makeup is more complex, being derived from a prominent mixture of Africans, European Caucasians and native Taino Indians (Fernandez-Cobo et al, 2001). On the other extreme are Hispanics from Buenos Aires, Argentina whose genes are mostly of European Caucasian derivation. Unfortunately there are not much data available regarding the outcome of Puerto Rican Hispanics with colorectal cancer who reside in Puerto Rico. This malignancy is the second most common tumor in Puerto Rico. For this reason we have examined the clinical features and outcome of 202 patients with colorectal cancer, all operated by the same colorectal surgeon, in a private practice setting. Although this sample might not be representative of the whole island, it does provide a profile of the experience with a group of patients whose socioeconomic level, although higher than average for the island, is uniformly composed of Puerto Rican “Hispanics”. Another advantage of this study is that we were able to obtain detailed and reliable data on stage, treatment delivered and outcome. This is frequently a limitation with data from tumor registries which rely on information derived from various sources.

III. Results A. Demographics Table 1 summarizes the demographic features of our population. Sixty nine percent of the patients presented with localized disease (stage I-II) and only 13% presented with metastatic stage IV disease. The median age was 66. Close to 45% of the patients were younger than 65 at the time of presentation. Figure 1 shows the age distribution in graphic form.

II. Methods We retrospectively reviewed the charts of 202 consecutive patients with colorectal cancer operated from January 2000 through January 2007. Median follow up of patients who are alive is 31 months; mean is 39 months and range 1-88 months. This study includes all patients operated during that time period by a single surgeon, Dr. Ignacio Echenique, who is a colorectal surgeon based at Auxilio Mutuo Hospital. The Auxilio Mutuo Hospital is a private non-profit institution. Although it accepts patients from essentially all health insurance plans including Medicare, it doesn’t contract with “Reforma” health insurance, which is the local government’s insurance plan for indigent patients. Consequently the patient mix is of a higher socioeconomic level than most hospitals in the island. The following data were collected: age, gender, preoperative carcinoembryonic antigen (CEA) level, clinical and pathological stage, site of primary tumor (when rectal in origin, the distance from the anal verge was also identified), type of surgery performed, whether sphincter preservation was performed, whether preoperative chemotherapy and radiation was given and whether postoperative adjuvant chemotherapy was delivered. Overall survival, cause-specific survival and failure-free survival information was also examined. Overall survival was defined as the interval from the date of surgery to the date of death irrespective of the cause of death. Failure free survival was defined as the interval from the date of surgery until the first evidence of relapse or lethal toxicity from treatment. Causespecific survival (in this case, cancer related mortality) was calculated from the date of surgery to the date of death from cancer. For the latter analysis, five patients who died of unrelated causes such as cardiovascular or others not directly linked to the tumor or to lethal toxicity from treatment, were censored at the time of death. All survival curves were plotted using the KaplanMeier technique (Kaplan and Meier, 1958). For rectal cases, the level in the rectum was calculated usually by rigid proctoscopy and/or digital rectal examination and expressed as the distance from the anal verge. Low rectum was defined as 0-5 cm from the anal verge, mid-rectum as 6-10

Table 1. Demographic Features. Characteristic Median Age Gender Female Male Stage I II III IV Histology Well differentiated Moderately well differentiated Poorly differentiated Adenocarcinoma NOS* Primary Tumor Site Rectum Right Colon Sigmoid Left Colon Transverse Colon Level in Rectum** High (11-15 cm) Mid (6-10 cm) Low (0-5 cm) Median CEA preop * NOS=not otherwise specified **measured from the anal verge

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N 202 202 103 99 202 71 68 37 26 202 105 78

Distribution 66 (23-88) 100% 51% 49% 100% 35% 34% 18% 13% 100% 41% 49%

7 12 202 81 59 37 14 11 81 23 34 24 156

4% 6% 100% 40% 29% 18% 7% 5% 100% 28% 42% 30% 2.1 (0.1-5,000)

Cancer Therapy Vol 6, page 415!

Figure 1. Histogram Age Distribution Echenique Series. All Cases, All Stages

B. Outcome of colorectal cases

Figure 4, the postoperative pathological stage was used except for those cases who were clinical stage IV before surgery. The cancer specific survival for the same patients is shown in Figure 4. For stage I, II, III and IV the projected 5 year cancer specific survivals respectively were 96%, 92%, 87% and 28%. There was no significant difference in cause specific survival among stages I-III but the lower survival of stage IV cases was statistically significant. Figure 5 shows the failure free survival of the same cases shown in Figure 3 and 4.

The overall survival and the cause specific survival curves are shown in Figure 2. These curves include all stages of colon and rectal cancer. Five year overall survival and cause specific survival were 77% and 85% respectively. Five deaths considered not to be related to cancer were censored in the cause specific survival curve in this figure. Figure 3 depicts the overall survival of all colorectal cases according to stage. For this analysis as well as for

Figure 2. Survival of All Colon and Rectal Cases (N=202)

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Figure 3. Overall Survival of All Cases According to Stage

Figure 4. Cancer Specific Survival According to Stage

C. Management of colon cases excluding rectum

preoperatively and 16 of these also received chemotherapy concurrently. When all 24 cases with low rectal lesions are considered, 12 (50%) had sphincter preserving surgery. All patients who underwent sphincter saving procedures routinely had a temporary diverting colostomy.

For the 121 cases in whom the primary was located outside of the rectal area, information on the use of chemotherapy is shown in Table 2 which summarizes the use of chemotherapy according to stage. Most patients with stage II-III received adjuvant chemotherapy while 90% of stage IV received palliative chemotherapy.

E. Outcome of colon cases compared with rectal Figure 6 compares the cause specific survival of colon versus rectal carcinomas excluding those with stage IV presentation. No significant differences were observed, although the survival and failure free survival trends favor colon over rectum. When this same analysis was carried out focusing exclusively on stage I-III cases, the same pattern was observed (data not shown).

D. Management of rectal carcinomas Of the 81 cases of rectal primaries, 42 received preoperative radiation therapy, of which 41 also received chemotherapy concurrently. Of the 24 cases with low rectal primaries 17 received radiation therapy

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Figure 5. Failure Free Survival According to Stage

Table 2. Use of chemotherapy in colon cases. Stage I II III IV

N 30 43 25 20

# treated with chemotherapy 1 (3%) 29 (67%) 22 (88%) 18 (90%)

Figure 6. Cancer Specific Survival of Colon vs Rectum Excluding Stage IV

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F. Outcome of rectal cases

highly curable. However a closer look at Table 3 reveals that the age distribution in our series is very different from the USA, with a much higher proportion of cases diagnosed at a younger age. In order to determine if the young age of onset is an isolated event limited to the practice of one surgeon, we reviewed the island-wide statistics by contacting the Puerto Rico Cancer Registry at the Dept. of Health. The results confirmed that the proportion of patients younger than 55 years is also higher for the whole island as compared with USA (P<.00001) and similar to the results observed in our series (P=0.54). In a sense, our findings regarding the younger age of colorectal cancer patients in our series from Puerto Rico parallel those observed in African-Americans in USA (Agrawal et al, 2005). The colorectal screening recommendations for African Americans have recently been modified. The first colonoscopy for this minority group should be performed at the age of 45 years instead of 50 according to the American Gastroenterology Association (Agrawal et al, 2005). We propose that the current screening recommendations for Puerto Ricans should be changed so that it would start earlier. In order to capture that same ~80% of cases in their pre-malignant phase, we would have to start screening at 45 years old. This still would miss 10 cases who were younger than 45 years old when they were diagnosed but it would capture in the premalignant phase 82% of patients who were diagnosed after age 55 and in addition would have detected at an earlier stage those 7% diagnosed between 45-55 years old. A possible explanation for this earlier age of onset of colorectal cancer in Puerto Rico could be that the life expectancy in the island might be lower than USA. This could result in patients dying at an early age and thus not being at risk for developing colon cancer at an older age. In order to rule out this possibility as an explanation for the earlier age of onset, we compared the life expectancy in Puerto Rico with that of USA. In USA the life expectancy for females in 2001 was 79.8 and for males 74.4, as compared with 80.0 and 71.0 respectively in Puerto Rico (National Center for Health Statistics Health, United States 2005 With Chartbook on Trends in the Health of Americans, 2005). Consequently, we don’t feel this is the explanation for the younger age of onset in Puerto Rico.

In the 81 cases of rectal primaries, it was possible to preserve the anal sphincter in 68 (84%). Of 57 with mid or high rectal presentations, 56 (98%) had sphincter preserving surgery as compared with 12/24 (50%) of low rectal cases. The failure free survival of patients whose sphincters were saved was not inferior to those ten requiring an abdominoperineal resection. In fact, the failure free survival tended to be superior for those who underwent sphincter saving surgery with 75% being failure free at five years versus 57% for those whose sphincter had to be sacrificed (P=.09). The same trend for a superior failure free survival was observed when the low rectal cases were analyzed separately. Of our cases with rectal cancer six were able to be treated with local excision and one recurred locally. This was managed with radiation and an abdominoperineal resection and the patient has remained continuously free of disease since then. Of our cases with rectal cancer, six were stage IV leaving 75 patients with stage I-III treated by radical resection, of which 11 experienced a relapse. Of these 11, only 2 were local recurrences (2.6 % of the 75 with stage I-III). Both local recurrences occurred in low rectal cases with ultrasound documented T3 and T4 lesions. The remaining 9 relapses were 8 distant and 1 distant plus local.

IV. Discussion The current colorectal screening recommendations in Puerto Rico are based on the age distribution of this disorder in the USA (Smith et al, 2002). Colon cancer in most instances is known to arise from a benign adenomatous polyp which with time develops molecular genetic events that lead to dysplasia and eventually to its transformation into an adenocarcinoma (Vogelstein et al, 1988). These events on the average take 10 years to fully develop from a polyp to an invasive adenocarcinoma. Table 3 summarizes the (Surveillance, Epidemiology and End Results) SEER data on the age distribution of cases of colorectal carcinoma in the USA (Clegg et al, 2002). Seventy one percent of the USA cases are diagnosed after the age of 65 years and approximately 20% before 60 years. This means that the vast majority of colorectal cancers would be detected in their premalignant phase if screening colonoscopy starts at 50 years which is the current recommendation. A large fraction of the other 20% would be detected in their early stages when they are

Table 3. Comparison of age distribution in USA versus Puerto Rico. Country USA (SEER) Puerto Rico (our series) Puerto Rico Cancer Registry

N 91,850 202

<55 12.2% 18.8%*

55-64 16.8% 24.3%

65-74 27% 32.2%

>75 41.9% 24.8%

<65 29% 43.1%

1,538

17.9%**

N.A.

N.A.

N.A.

N.A.

*P value USA SEER vs Puerto Rico (our series) =.0046 **P value USA SEER vs Puerto Rico Cancer Registry <.00001 *** P value for our series vs Puerto Rico Cancer Registry = .54

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Cancer Therapy Vol 6, page 419! cumulative incidence at 5 yrs in patients with stage I-III tumors. If the T1 tumors are excluded from the denominator, the cumulative local relapse rate at 5 yrs was 7.5%. This is an acceptable rate and is in keeping with the published literature (Martling et al, 2000; Sauer et al, 2004). Most important is the fact that the high sphincter preservation rate was achieved without sacrificing the effectiveness of the surgical procedure as determined by the fact that the failure free survival of those whose sphincter was preserved was excellent. An inspection of the overall survival, cause specific survival and failure free survival curves shown in Figures 3-5 reveals the surprising finding that there were no major differences between stages I, II and III. Only for stage IV was there a significant difference. Although unexpected, part of the explanation could be that the vast majority of patients with stage III and most with stage II, received adjuvant therapy. Adjuvant therapy, particularly for stage III, has led to a major improvement in the outcome of these cases. In the seminal article where Andre et al describe the results of their randomized adjuvant trial, the Folfox chemotherapy arm had a 3 year failure free survival of 72.2% for stage III cases and 87% for stage II (Andre et al, 2004). Since the number of patients with stage III in our series is relatively small (N=28), our 3 year failure free survival of 85% for stage III, has a standard deviation of 26% which is compatible with Andre’s results of 72.2% survival. Our 3 year failure free survival of 89% for stage II was very similar to Andre’s results of 87%. In summary, our data suggest that the current recommendations for screening colonoscopy for the Puerto Rican population residing in the island might have to be reconsidered. In view of the younger age of Puerto Ricans with colorectal cancer, screening colonoscopy might have to start at 45 years of age. This is a theoretical proposal which should be confirmed with facts. Since our data regarding overall survival, failure free survival and sphincter preservation rates are in the range of that described for American Whites, this suggests that being of Puerto Rican extraction doesn’t carry adverse prognostic implications provided adequate medical care is available. Perhaps the slightly less favorable outcome of Hispanics with colorectal cancer in the USA might be related to disparities in health care access (Howe !"#$%, 2006). Finally our data suggest that when an experienced high volume colorectal surgeon performs colon cancer surgery with adequate facilities and a suitable infrastructure, the results are comparable with those obtained in North America and Europe.

A review of the 1973-2002 SEER survival data for colorectal cancer from the USA National Cancer Institute (Howe et al, 2006) reveals some similarities and some differences from our data. In first place, there was no significant difference in the survival rate for colon versus rectal cancer in the USA. Such is our experience also; we didn’t observe any statistically significant difference in the survival of colon versus rectal primaries (Figure 6). However, a notable difference is our 5 year overall survival which appears superior: 78% for our population versus 65% for USA. This difference can probably be explained by various factors. In first place, our patients were predominantly early stage with 69% in the category of stage I-II, however, our stage III cases also had an excellent outcome when compared with earlier stages. Another possible explanation is that our data are more recent since they were generated from 2000-07 which covers a 7 year period instead of 29 years (from 19732002). Recent advances in management are more prominently reflected in our series. Another contributing factor could be that our patient population is significantly younger. Younger age is known to correlate with a longer survival (Mitry et al, 2001), although Rodriguez-Bigas has identified very young age, defined as <30 years, as an adverse prognostic factor (Rodriguez-Bigas et al, 1996). In addition, our data pertain to a single surgeon who is highly experienced in colorectal cancer surgery. Finally, there could be a selection bias in favor of a higher socioeconomic status for our patient population. It is a well known fact that African-Americans with colorectal cancer have a significantly lower 5 year survival. In the 1983-90 SEER data, the 5-year survival rate for blacks with colorectal cancer was 49.5% versus 60.1% for whites (Clegg et al, 2002). This lower survival results in a lower average survival for the whole USA population. The sphincter preservation rate in rectal carcinoma cases, particularly for cases with low rectal primary tumors, is a good measure of the suitability of the surgery performed. In the past the sphincter had to be frequently sacrificed in most patients with rectal cancers. In 34 studies that included 6,570 patients, the average sphincter preservation rate was 50% for all cases with rectal cancer and 32% for the lower third of the rectum (Di Betta et al, 2003). Our sphincter preservation rate of 84% for all rectal cancers and 50% for the lower third of the rectum compares favorably with the rates recorded in those 6,570 cases. Advances in preoperative staging through accurate staging with transrectal ultrasound, CT scans and MRI and the recognition of the importance of invasion of the tumor into the mesorectal plane has allowed for a more accurate selection of patients for preoperative chemo-radiation (Rifkin and Marks, 1985; Zmora et al, 2004). This, coupled with the introduction of TME in the surgical treatment of low and mid rectal cancers, allows resection of tumors closer to the dentate line without a decrease in the survival rates thus allowing a higher number of patients to undergo sphincter saving surgery (Guillem et al, 2007). With TME, usually in combination with preoperative chemoradiation therapy, we achieved a very low local recurrence rate of 2/75 (2.6%) or 5.5%

References Agrawal S, Bhupinderjit A, Bhutani MS, Boardman L, Nguyen C, Romero Y, Srinivasan R, Figueroa-Moseley C; Committee of Minority Affairs and Cultural Diversity, American College of Gastroenterology (2005) Colorectal cancer in African Americans. Am J Gastroenterol 100, 51523, discussion 514. André T, Boni C, Mounedji-Boudiaf L, Navarro M, Tabernero J, Hickish T, Topham C, Zaninelli M, Clingan P, Bridgewater J, Tabah-Fisch I, de Gramont A; Multicenter International

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Echenique et al: Outcome of Hispanics with colorectal cancer with access to adequate health care facilities! Study of Oxaliplatin/5-Fluorouracil/Leucovorin in the Adjuvant Treatment of Colon Cancer (MOSAIC) Investigators (2004) Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med 350, 2343-51. Clegg LX, Li FP, Hankey BF, Chu K, Edwards BK (2002) Cancer survival among US whites and minorities: a SEER (Surveillance, Epidemiology, and End Results) Program population-based study. Arch Intern Med 162, 1985-93. Di Betta E, D'Hoore A, Filez L, Penninckx F (2003) Sphincter saving rectum resection is the standard procedure for low rectal cancer. Int J Colorectal Dis 18, 463-9. Fernandez-Cobo M, Jobes DV, Yanagihara R, Nerurkar VR, Yamamura Y, Ryschkewitsch CF, Stoner GL (2001) Reconstructing population history using JC virus: Amerinds, Spanish, and Africans in the ancestry of modern Puerto Ricans. Hum Biol 73, 385-402. Guillem JG, Chessin DB, Shia J, Suriawinata A, Riedel E, Moore HG, Minsky BD, Wong WD (2007) A prospective pathologic analysis using whole-mount sections of rectal cancer following preoperative combined modality therapy: implications for sphincter preservation. Ann Surg 245, 8893. Howe HL, Wu X, Ries LA, Cokkinides V, Ahmed F, Jemal A, Miller B, Williams M, Ward E, Wingo PA, Ramirez A, Edwards BK (2006) Annual report to the nation on the status of cancer, 1975-2003, featuring cancer among U.S. Hispanic/Latino populations. Cancer 107, 1711-42. Kaplan EL, Meier P (1958) Non-parametric estimation from incomplete observations. J Am Stat Assoc 53, 457-481. Martling AL, Holm T, Rutqvist LE, Moran BJ, Heald RJ, Cedemark B (2000) Effect of a surgical training programme on outcome of rectal cancer in the County of Stockholm. Stockholm Colorectal Cancer Study Group, Basingstoke Bowel Cancer Research Project. Lancet 356, 93-6. McGrath DR, Leong DC, Gibberd R, Armstrong B, Spigelman AD (2005) Surgeon and hospital volume and the management of colorectal cancer patients in Australia. ANZ J Surg 75, 901-10. Mitry E, Benhamiche AM, Jouve JL, Clinard F, Finn-Faivre C, Faivre J (2001) Colorectal adenocarcinoma in patients under 45 years of age: comparison with older patients in a welldefined French population. Dis Colon Rectum 44, 380-7. National Center for Health Statistics Health, United States 2005 With Chartbook on Trends in the Health of Americans (2005) Hyattsville, Maryland. ! Rifkin MD, Marks GJ (1985) Transrectal US as an adjunct in the diagnosis of rectal and extrarectal tumors. Radiology 157, 499-502. Rodriguez-Bigas MA, Mahoney MC, Weber TK, Petrelli NJ (1996) Colorectal cancer in patients aged 30 years or younger. Surg Oncol 5, 189-94.

Sauer R, Becker H, Hohenberger W, Rödel C, Wittekind C, Fietkau R, Martus P, Tschmelitsch J, Hager E, Hess CF, Karstens JH, Liersch T, Schmidberger H, Raab R; German Rectal Cancer Study Group (2004) Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med 351, 1731-40. Smith RA, Cokkinides V, von Eschenbach AC, Levin B, Cohen C, Runowicz CD, Sener S, Saslow D, Eyre HJ; American Cancer Society (2002) American Cancer Society guidelines for the early detection of cancer. CA Cancer J Clin 52, 822. Steele GD, Jr. (1994) The National Cancer Data Base report on colorectal cancer. Cancer 74, 1979-89. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL (1988) Genetic alterations during colorectal-tumor development. N Engl J Med 319, 525-32. Wibe A, Møller B, Norstein J, Carlsen E, Wiig JN, Heald RJ, Langmark F, Myrvold HE, Søreide O; Norwegian Rectal Cancer Group (2002) A national strategic change in treatment policy for rectal cancer--implementation of total mesorectal excision as routine treatment in Norway. A national audit. Dis Colon Rectum 45, 857-66. Zmora O, Dasilva GM, Gurland B, Pfeffer R, Koller M, Nogueras JJ, Wexner SD (2004) Does rectal wall tumor eradication with preoperative chemoradiation permit a change in the operative strategy? Dis Colon Rectum 47, 1607-12.

Luis Ignacio Echenique

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Cancer Therapy Vol 6, page 421! Cancer Therapy Vol 6, 421-424, 2008

!"#$%"&'()*+$&),-.-/)(&//)(-#('0"1-)"2)*%&)$&#'-0-/) #&3'"04)#-#&)1-0'2&.*-*'"0)"2)-)("11"0)5'.&-.&) Research Article

Sai Krishna Balakrishnan678, Pankaj K Singh97)Lucy Jane Smith3, Vinay K Singh4) 1

ST1 Core Surgical Trainee, Luton & Dunstable Hospital NHS Trust, Lewsey road, Luton LU4 0DZ, UK Clinical Research Fellow, Neurovascular Surgery, Royal Hallamshire Hospital, Sheffield S10 2JF, UK 3 FY 2, Dept of General Surgery, Leicester Royal Infirmary, Infirmary Square, Leicester LE1 5WW, UK 4 Registrar Trauma and Orthopaedics Surgery, Luton & Dunstable Hospital NHS Trust, Lewsey Road, Luton LU4 0DZ, UK 2

__________________________________________________________________________________! *Correspondence: SK Balakrishnan, ST1 Core Surgical Trainee, Luton & Dunstable Hospital NHS Trust, Lewsey road, Luton LU4 0DZ, UK; Phone: 00-44-7757669448; Email: we.publish@googlemail.com Key words: Basal cell carcinoma, perianal region, morphoeic type Abbreviations: Basal cell carcinoma, (BCC); basal cell nevus syndrome, (BCNS); basaloid carcinoma, (BC); carcinoembrionic antigen, (CEA); epithelial membrane antigen, (EMA) Received: 14 April 2008; revised: 6 June Accepted: 10 June 2008; electronically published: July 2008

Summary Basal cell carcinoma (BCC) is the most common dermatological malignancy. Sunlight being the most important aetiological factor, it remains an exceptional rarity in non-sun exposed parts, including the perianal area. We report an atypical case of BCC of the perianal area in an elderly man, who presented with itching and haemorrhoids. The macroscopic appearance of the lesion was typical of the morphoeic variety. The case was successfully treated by wide local excision and the patient remained symptom-free at three years. The case remains unique, both in terms of the perianal location being unusual, as well as the rare macroscopic presentation. Due to a striking histopathological resemblance, perianal basaloid carcinoma (BC) makes an important differential diagnosis. Histopathologists must be able to distinguish between the two entities, as both exhibit entirely different biological and clinical behaviours, thus requiring dramatically different treatments.

specific history of past skin cancers, exposure to carcinogens like arsenic or other chemicals, nude sunbathing or tanning. In addition, the patient denied any history of burns or trauma to the perianal region, homosexuality. The patient sought consultation mainly due to concerns regarding the increasing size of a ‘mole’ near the anus. On examination an erythematous, small, raised plaque, 3 mm in diameter was present in the perianal region at the 4 o'clock position (Figure 1). The lesion was non-tender and firm on palpation without evidence of active bleeding, and was confined to the perianal skin in its entirety without extension into the anal canal. Proctoscopy revealed the presence of co-existing grade-1 haemorrhoids. Due to the growing size and suspicious nature of the lesion, it was decided excision should be performed to establish a histopathological diagnosis. The patient was admitted for further investigations and the lesion was excised with a 5 mm margin under general anaesthetic. The haemorrhoids were banded at the same sitting. A surgical specimen of 12x8 mm size, preserved in 10% buffered formalin, was sent for histopathological examination. On staining with haematoxylin and eosin, typical basophilic cells were seen with dominant peripheral palisading nuclei. There were no signs of necrosis, and

I. Introduction Basal cell carcinoma (BCC) is the most common type of human dermatological malignancy accounting for more than 75% of all non-melanomatous skin cancers (Miller, 1991). It is mainly seen in sun-exposed sites with ultraviolet rays playing an important role in etiopathogenesis (Gloster, 1996). BCC of non-sun exposed sites like the perianal area remains a rarity, constituting less than 0.1 per cent of all anorectal tumors (Espana et al, 1992; Gibson et al, 2001), and prompts the need for exploring some alternative etiological factors other than ultraviolet rays.

I. Case report A 60-year-old white Caucasian male attended our outpatient clinic with a two year history of itching and a slowly growing ‘mole’ in his perianal area. The patient had smoked cigarettes for the past 40 years and had no significant past medical history and was not immunosuppressed. There was no

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Balakrishnan et al: Perianal basal cell carcinoma! there was a clear plane of demarcation between the tumour and surrounding normal tissue (Figure 2). Further immunohistochemical evaluation showed positive staining for Ki-67 (Figure 3) and bcl-2 (Figure 4). After establishing the margins were tumour free, the patient was discharged. He remained symptom-free at three years with no signs of recurrence.

A. Epidemiology In spite of being the commonest skin cancer, perianal occurrence of BCC is rare. In a large series, Gibson and colleagues, scrutinized in 2001 18,943 cases of BCC diagnosed at their institute between 1985 and 1996 and retrieved only 20 cases of perianal BCC, constituting 0.1 per cent of the total. A slight male preponderance has been noted by some authors! (Armitage et al, 1955) but the fact is not agreed upon universally (Nielsen et al, 1981). BCC of the perianal region primarily affects the elderly population, with most of the patients presenting in the sixth and seventh decades (Armitage et al, 1955; Nielsen et al, 1981).

II. Discussion Basal cell carcinoma is by far the commonest form of skin malignancy in humans and accounts for more than 75% of all non-melanomatous skin cancers (Miller, 1991). Ultraviolet rays and exposure to sun remain the two most important aetiological factors with the face being the most preferred site (Gloster, 1996). Not surprisingly, BCC of non-sun exposed sites like the perianal area remains a rarity,!accounting for less than 0.1 per cent of all anorectal tumours (Espana et al, 1992; Gibson et al, 2001).

B. Risk factors Whereas sunlight and ultraviolet rays are well accepted risk factors for the common photo-distributed variety, the exact etiology for perianal BCC is a matter of debate. A number of theories have been postulated by different authors. Ultraviolet radiation itself has been

!

! Figure 2. Haematoxylin and Eosin x200; to show typical peripheral nuclear palisading of a basal cell carcinoma. A clear plane of demarcation can be seen between the tumour and normal tissue (arrows).

!

! Figure 4. !"#$% staining x200; shows strong cytoplasmic staining of the tumour cells.

Figure 1. A diagrammatic representation of superficial lesion measuring about 3 mm situated between 3 and 4 O’ clock position (arrow).

Figure 3. Ki67 is a nuclear proliferation marker. This illustrates the relatively high percentage of positively staining tumour nuclei within the lesion.

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Cancer Therapy Vol 6, page 423! carries a far graver prognosis due to an aggressive course and early metastases (Alvarez-Canas et al, 1996) BC has a completely different treatment protocol to BCC. Basaloid carcinoma, also known as cloacogenic carcinoma or transitional carcinoma is considered to be a variant of squamous cell carcinoma by most authors. It originates from the transitional cell epithelium of anus recapitulating features of this zone in both light and electron-microscopy (Gillespie et al, 1978). On the other hand, the histological features that support the diagnosis of BCC are prominent nuclear palisading, absence of necrosis, low invasiveness, minimal nuclear pleomorphism and an absence of atypical mitosis (Alvarez-Canas et al, 1996). Alvarez-Canas and colleagues discussed in 1996 the different ways of differentiating between these two closely related entities. The different immunoreactivity patterns for keratin are shown by two neoplasms. Whereas BC can be readily labelled, BCC remain consistently negative, except for the squamous foci.! Levy and colleagues suggested in 1991 the value cytokeratin polypeptide expression in distinguishing between BC and BCC. Cytokeratin 19 labelled BC extensively, but was almost absent in BCC. Quite the contrary, cytokeratin 10/11 immunoreactivity was observed in BCC, but not in BC. Immunohistochemical markers such as carcinoembrionic antigen (CEA), epithelial membrane antigen (EMA), and the lectin Ulex europaens agglutinin I (UEA-I), shows the similar results. All these show a positive reactivity with BC, while failed to stain BCC. Monoclonal antibodies Ber-EP4 is another tool to establish the diagnosis. BCC shows an intensive labelling with BC being uniformly negative. At last, the S-phase fraction analysed by flowcytometry could also prove helpful in distinguishing perianal BCC from BC with S-phase fraction being remarkably greater in BC than BCC.

proposed to be involved in the pathogenesis of non-sun exposed BCC by depressing the immune surveillance, even at a distant site (Strickland et al, 1985). Gibson et al, and others (Gorlin et al, 1987;! Kraemer et al, 1987) suggested the possibility of underlying genetic factors. A particular association has been noted with xeroderma pigmentosa, acanthosis nigricans and, Haily-Haily disease. Basal cell nevus syndrome (BCNS) may be especially important, with 25% (n=5) of perianal BCC diagnosed by Gibson and colleagues !"# $%%&, occurring secondary to BCNS. Immunosuppression and ionizing radiation may also play a role. Gibson and colleagues reported in 2001 a case of perianal BCC who had received radiotherapy (dose not known) for prostate carcinoma. They also implicate previous trauma and burn scars as possible etiological factors in the development of perianal BCC. In spite of doubtful suggested significance of HPV 2 and 16 (Eliezri et al, 1990; Pierceall et al, 1991; Gibson et al, 2001), investigators have failed to detect the presence of HPV from the perianal variety (Nahass et al, 1992; Nehal et al, 1998; Kort et al, 1995). None of the above mentioned factors were noted in our patient; however pruritus ani and chronic perianal irritation, as suggested by Gibson and colleagues in 2001 may be a possible underlying cause.

C. Histopathological considerations Perianal BCC shares a number of macroscopic and microscopic characteristics with its counterpart, photodistributed variety. The usual location is perianal skin, but can extend to the anal canal (Alvarez-Canas et al, 1996). Macroscopically, the ulcerated type is the most common presentation. In the Gibson and colleagues in 2001, series 29.4% cases have showed a tendency of central ulceration. It has been suggested that the common occurrence of ulceration in perianal BCC has a possible correlation with delay in diagnosis. The patient may consider the initial symptoms of itching and irritation as trivial, or the physician himself may mistake the condition with inflammatory dermatoses (Collins et al, 1981). The absence of ulceration and small size of tumour in our patient indicated an early diagnosis and treatment. Our case remains a unique in terms of the macroscopic presentation of the morphoeic type, as described above. After a thorough search of literature we could not find any other case of a morphoeic type BCC in perianal region. To author’s knowledge this is the first reported case of morphoeic type of perianal BCC. Contrary to our case, where the size was 3 mm, the average size of lesion at the time of diagnosis was 1.95 cm in Gibson at all series. Perianal BCC shows a huge variety of microscopic patterns with solid being most common (Alvarez-Canas et al, 1996).

E. Prognosis and treatment Most of the authors think that similar to photodistributed variety perianal BCC carries a good prognosis if diagnosed and treated early. In spite of few reports, metastasis (Nielson et al, 1981) and local recurrence (Gibson et al, 2001) remains an exception. Wide local excision with a 5 mm tumour free margin is considered to be curative. Due to the low propensity for nodal involvement, no further screening is required, if there is 5 mm tumour free margin (Gibson et al, 2001). Kharfi and colleagues advocated in 1992 Moh’s micrographic surgery yielding the best results, especially when the tumour is large, or the histology is aggressivee.g., morphaeform, metatypical and micrnodular. The aim is to preserve the maximum possible normal tissue. Carbon-dioxide laser and electrodesiccation with curettage are other modalities tried successfully. Nielsen and colleagues reported in 1981 a crude five year survival rate of 72.6%, with no deaths attributed to the tumour itself.

D. Differential diagnoses- clinical and histopathological Squamous cell carcinoma and condyloma accuminata are the two most important differential diagnoses for a clinician. On the other hand, the histopathologist must distinguish BCC from the basaloid carcinoma (BC) of anus, which it histopathologically resembles closely, but

III. Conclusions The relative rarity of perianal BCC requires a high index of suspicion and dictates the need for the 423

Balakrishnan et al: Perianal basal cell carcinoma! Gloster GM, Broadland DG (1996) The epidemiology of skin cancer. Dermatol Surg 22, 217-22. Gorlin RJ (1987) Nevoid basal cell carcinoma syndrome. Medicine 66, 98-113. Kharfi M, Mokhtar I, Fazaa B, Pierard-Franchimont C, Rustom KAl, Nikkels AF, et al, (1992) Vulvar basal cell carcinoma. Eur J Dermatol 2, 81-84. Kort R, Fazaa B, Bouden S (1995) Perianal basal cell carcinoma. Int J Dermatol 34, 427-428. Kraemer KH, Lee MM, Scotto J (1987) Xeroderma pigmentosum: cutaneous, ocular, and neurologic abnormalities in 830 published cases. Arch Dermatol 123, 241-250. Levy R, Czernobilsky B, Geiger B (1991) Cytokeratin polypeptide expression in a cloacogenic carcinoma and in the normal anal canal epithelium. Virchows Arch A Pathol Anat 418, 447-455. Miller SJ (1991) Biology of basal cell carcinoma (part 1). J Am Acad Dermatol 24, 1-13. Nahass GT, Blauvelt A, Leonardi CL, Penneys NS (1992) Basal cell carcinoma of the scrotum. J Am Acad Dermatol 26, 574-578. Nehal KS, Levine VJ, Ashinoff R (1998) Basal cell carcinoma of the genitalia. Dermatol Surg 24, 1361-1363. Neilson OV, Jenson SL (1981) Basal cell carcinoma of the anusa clinical study of 34 cases. Br J Surg 68, 856-857. Pierceall WE, Goldberg LH, Ananthaswamy HN (1991) Presence of human papillomavirus type 16 DNA sequences in human nonmelanoma skin cancers. J Invest Dermatol 23, 880-884. Strickland PT, Creasia D, Kripke ML (1985) Enhancement of two-stage skin carcinogenesis by exposure of distant skin to UV radiation. J Natl Cancer Inst 74, 1129-1134.

enthusiastic efforts of biopsying every unusual lesion in the perianal area. In the absence of sunlight as a causative agent, other aetiological factors need to be considered. Due to high cure rates the importance of early diagnosis and treatment cannot be overemphasised. Owing to the striking histological resemblance and entirely different biological and clinical behaviours, the histopathologist needs to be aware of differentiating between BCC and BC, and reporting whether there is a 5 mm tumour free margin.

References Alvarez-Canas MC, Fernandez FA, Rodilla IG, Val-Bernal JF (1996) Perianal basal cell carcinoma: a comparative histologic, immunohistochemical, and flow cystometric study with basaloid carcinoma of the anus. Am J Dermaopathol 18, 371-379. Collins PS, Farber GA, Hegre AM (1981) Basal cell carcinoma of the vulva. J Dermatol Surg Oncol 7,711-713. Eliezri YD, Silverstein SJ, Nuovo GJ (1990) Occurrence of human papilloma virus type 16 DNA in cutaneous squamous and basal cell neoplasms. J Am Acad Dermatol 23,836-842. Espana A, Redondo P, Idoate MA, Serna MJ, Quintanilla E (1992) Perianal basal cell carcinoma. Clin Exp Dermatol 17, 360-362. Gibson GE, Ahmed I (2001) Perianal and genital basal cell carcinoma: A clinicopathologic review of 51 Carcinoma of the anal canal. J Am Acad Dermatol 45, 68-71. Gillespie JJ, MacKay B (1978) Histogenesis of cloacogenic carcinoma. Fine structure of anal transitional epithelium and cloacogenic carcinoma. Hum Pathol 9, 579-587.

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Cancer Therapy Vol 6, page 425! Cancer Therapy Vol 6, 425-438, 2008

Highlights from ASCO and ECCO 2007: Update on targeted treatment of metastatic renal cell carcinoma Review Article

Robert J. Motzer Memorial Sloan-Kettering Cancer Center, New York, NY

__________________________________________________________________________________! *Correspondence: Robert J Motzer, MD, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA; Tel.: 646 422 4312; Fax: 212 988 0806; E-mail: motzerr@mskcc.org Key words: Metastatic renal cell carcinoma, Receptor tyrosine kinase inhibitors, Targeted therapy, Combination therapy, First-line treatment, Second-line treatment, Cytokine-refractory, Sunitinib, Sorafenib, Temsirolimus, Bevacizumab, Novel agents, Interferon-alfa, Tumor response, Overall survival, Prognostic factors, Quality of life, Cost-efficacy Abbreviations: Advanced Renal Cell Carcinoma Sorafenib Expanded Access Program-North America, (ARCCS); Adverse event, (AE); American Society of Clinical Oncology, (ASCO); confidence interval, (CI); continuous daily dosing, (CDD); dose-limiting toxicity, (DLT); Eastern Cooperative Oncology Group, (ECOG); epidermal growth factor receptor, (EGFR); European Cancer Conference, (ECCO); Functional Assessment of Cancer Therapy - Biological Response Modifiers, (FACT-BRM); glial cell line-derived neurotrophic factor receptor, (RET); hazard ratio, (HR); imatinib mesylate, (IM); interferon-alfa, (IFN-! ); intravenously, (IV); loss of function, (LOF); mammalian target of rapamycin, (mTOR); Memorial Sloan-Kettering Cancer Center, (MSKCC); metastatic renal cell carcinoma, (mRCC); million units, (MU); objective response rate, (ORR); orally, (PO); overall survival, (OS); partial response, (PR); performance status, (PS); platelet-derived growth factor, (PDGF); platelet-derived growth factor receptor, (PDGFR); progression-free survival, (PFS); progressive disease, (PD); quality-adjusted life-year, (QALY); quality of life, (QoL); receptor tyrosine kinase, (RTK); renal cell carcinoma, (RCC); selective endothelin-A receptor antagonist, (SERA); stable disease, (SD); stem-cell factor receptor, (KIT); time to tumor progression, (TTP); time without symptoms and toxicity, (TWiST); twice daily, (BID); vascular endothelial growth factor, (VEGF); vascular endothelial growth factor receptor, (VEGFR); Von Hippel-Lindau, (VHL) Received: 10 June 2008; Revised: 20 July 2008 Accepted: 21 July 2008; electronically published: July 2008

Summary Metastatic renal cell carcinoma (mRCC) is highly resistant to conventional forms of treatment and is associated with a poor prognosis in most patients; only approximately 10% of patients with metastatic disease remain alive 5 years after diagnosis. However, recent years have witnessed the successful development of a number of targeted agents and combination therapies that have shown efficacy and tolerability in the treatment of mRCC in first-line and second-line settings, including sunitinib, sorafenib, temsirolimus, and bevacizumab plus interferon-alfa. This report reviews current reference standards for the first- and second-line treatment of mRCC, and discusses efficacy and safety data for the targeted agents in this setting based on presentations given at the 2007 American Society of Clinical Oncology and European Cancer Conference meetings. Clinical trial data presented at the meetings concerning novel targeted agents, including everolimus, axitinib, and volociximab, are also reviewed, and new combination therapies, sequential treatments, and dosing schedules with targeted agents are discussed. As targeted therapies are beginning to improve prognoses in patients with mRCC, and quality of life (QoL) is becoming an increasing focus, so the importance of prognostic and predictive factors for treatment response and survival is gaining increasing recognition. Recent trial evidence concerning prognostic and predictive factors that may assist in tailoring treatments and optimizing patient management are reviewed. Finally, key data presented at the meetings concerning the cost-efficacy of targeted agents and their effects on QoL in mRCC patients are evaluated.

al, 2006). RCC is estimated to account for about 2% of all cancers worldwide, with the highest rates observed in North America, Australia, and Europe (Parkin et al, 2005). Until recently, patients with metastatic RCC (mRCC) had

I. Introduction In the USA, approximately 51,000 new cases of renal cell carcinoma (RCC) and approximately 12,900 deaths from this malignancy were expected in 2007 (Schรถffski et 425

Motzer: Highlights from ASCO and ECCO 2007: Update on targeted treatment of metastatic renal cell carcinoma Barcelona, Spain). The report reviews current reference standards for the first- and second-line treatment of mRCC, and summarizes clinical trial data concerning novel targeted agents, new combination therapies and dosing schedules with targeted agents, and prognostic and predictive factors for treatment response and survival with RTK inhibitors. Through this report, I aim to translate findings from recent trials into the clinical practice setting.

extremely poor prognoses, as advanced disease is highly resistant to conventional forms of chemotherapy, radiotherapy, and hormonal therapy (Lilleby and Fosså, 2005; Rohrmann et al, 2005). As a result, the 5-year survival rate for patients with metastatic disease is approximately 10% (Godley and Taylor, 2001; Ravaud, 2007). Within the last 3 years, advances in our understanding of mRCC have resulted in the successful development of a number of novel targeted anticancer agents that have shown efficacy and tolerability in the clinical setting. This has resulted in a move away from conventional cytokine-based therapies, which are associated with low response rates and high risks of toxicity (Négrier et al, 1998; Yang et al, 2003a; McDermott et al, 2005; Schöffski et al, 2006). Several targeted agents are currently indicated for the treatment of advanced RCC, including sunitinib malate (SUTENT®; Pfizer Inc., New York, NY, USA), sorafenib (Nexavar®; Bayer Healthcare AG, Leverkusen, Germany), and temsirolimus (Torisel™; Wyeth Pharmaceuticals Inc., Philadelphia, PA, USA). Sunitinib is an oral multitargeted inhibitor of several receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptors (VEGFR-1, -2, and -3), platelet-derived growth factor receptors (PDGFR-! and -" ), stem-cell factor receptor (KIT), FMS-like tyrosine kinase 3, colony-stimulating factor 1 receptor, and glial cell line-derived neurotrophic factor receptor (RET) (Abrams et al, 2003; Mendel et al, 2003; Murray et al, 2003; O’Farrell et al, 2003; Kim et al, 2006). RTKs play a key role in tumor growth and angiogenesis (Krause and Van Etten, 2005), and VEGF and PDGF are viable targets in the RCC setting. Sunitinib is approved multinationally for the treatment of advanced RCC and imatinib-resistant/-intolerant gastrointestinal stromal tumors (SUTENT® Prescribing Information, 2007). Sorafenib, also a multikinase inhibitor, targets a range of RTKs (VEGFR-1, -2, and -3, PDGFR-" , c-KIT, and RET) and downstream Raf kinase isoforms (Raf1, BRaf, and mutant !"#$% V600E) in tumor cells and the tumor vasculature (Wilhelm et al, 2006; Nexavar® Prescribing Information, 2007). Sorafenib is indicated for the treatment of patients with advanced RCC who are unsuitable for interferon-alfa (IFN-! ) or interleukin-2 therapy or in whom such treatment has failed, and for the treatment of patients with hepatocellular carcinoma (Nexavar® Summary of Product Characteristics, 2007). Temsirolimus is an inhibitor of mammalian target of rapamycin (mTOR) that reduces levels of hypoxiainducible factor-1 and -2! and VEGF, and arrests tumor cell growth; temsirolimus is indicated for the treatment of advanced RCC (Torisel™ Prescribing Information, 2007). This report discusses the use of targeted agents for the treatment of mRCC, including sunitinib, temsirolimus, sorafenib, and the anti-VEGF monoclonal antibody, bevacizumab (Avastin®; F. Hoffmann-La Roche Ltd, Basel, Switzerland). The data are based on presentations given at the American Society of Clinical Oncology (ASCO) 43rd annual meeting (1-5 June 2007; Chicago, Illinois, USA), and include updates from the 14th European Cancer Conference (ECCO; 23-27 September 2007;

II. First-line treatment of mRCC Current international treatment guidelines (Ljungberg et al, 2007; National Comprehensive Cancer Network, 2008) recommend angiogenesis inhibitors, including sunitinib, bevacizumab plus IFN-! , temsirolimus (in poorrisk patients), and sorafenib (in selected patient populations), as first-line treatments for mRCC. Efficacy and safety data for a number of targeted agents, including sunitinib, temsirolimus, sorafenib, and bevacizumab, in the first-line setting in mRCC were presented at the ASCO and ECCO 2007 meetings. &

A. Sunitinib Sunitinib is indicated for the first-line treatment of clear-cell mRCC, when administered as an oral dose of 50 mg/day for 4 weeks, followed by a 2-week rest period in a 6-week cycle (Schedule 4/2); this agent continues to be the standard of care in the first-line setting (Motzer et al, 2007a). Updated data from the phase III clinical trial support the significant efficacy advantage of sunitinib over IFN-! in patients with mRCC, regardless of their prognostic risk, according to Memorial Sloan-Kettering Cancer Center (MSKCC) risk status (Motzer et al, 2007b,c). Independent assessment showed an objective response in 39% of sunitinib-treated patients (n=365) compared with 8% of patients treated with IFN-! (n=346) and a median progression-free survival (PFS) of 11.0 months (95% confidence interval [CI], 10.7-13.4) with sunitinib versus 5.1 months (95% CI, 3.9-5.6) with IFN-! (Motzer et al, 2007c). When assessed by MSKCC risk group, median PFS was longer in each prognostic group treated with sunitinib compared with IFN-! therapy (Motzer et al, 2007b,c). Sunitinib 37.5 mg in combination with IFN-! 3 million units (MU) may have clinical utility as a first-line combination therapy for patients with mRCC, as shown in a recent phase I dose-finding study (Kondagunta et al, 2007a,b). Three patients showed partial responses (PR) in this study, resulting in an objective response rate (ORR) of 12% (95% CI: 2.5-31.2) in 25 evaluable patients; median time to tumor progression (TTP) was 11.9 months (95% CI: 5.5-12.3). Dose-limiting toxicities (DLTs) with this combination treatment included myelosuppression and fatigue. The investigators concluded that sunitinib 37.5 mg in combination with IFN-! 3 MU is tolerable in this patient population, while higher dose combinations (sunitinib 50 mg/IFN-! 9 MU, sunitinib 50 mg/ IFN-! 6 MU, and sunitinib 37.5 mg/ IFN-! 6 MU) were poorly tolerated (Kondagunta et al, 2007b).

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Cancer Therapy Vol 6, page 427! insufficient number of patients with intermediate prognostic risk was enrolled to enable any meaningful assessment of this subgroup (Dutcher et al, 2007). These data suggest that, in line with international treatment guidelines, temsirolimus may be an effective treatment for poor-risk RCC patients who exhibit three or more predictors of short survival (Figure 1).

B. Temsirolimus Phase III clinical trial data in patients with mRCC receiving temsirolimus, the latest agent indicated for the treatment of advanced RCC, were released immediately prior to the ASCO 2007 meeting and updated at ECCO 2007 (De Souza et al, 2007; Hudes et al, 2007). In patients with untreated, advanced RCC and poor prognoses (defined as exhibiting ! 3 of 6 modified MSKCC risk criteria predictive of short survival), weekly single-agent intravenous temsirolimus 25 mg significantly improved overall survival (OS; 10.9 versus 7.3 months; hazard ratio [HR], 0.78; 95% CI, 0.63-0.97; p=0.0252) and PFS (3.8 versus 1.9 months; HR, 0.74; 95% CI, 0.60-0.90; p=0.003) compared with IFN-" monotherapy. However, OS in the temsirolimus/IFN-" combination arm of the trial did not differ significantly from that in the IFN-" monotherapy group (8.4 versus 7.3 months; HR, 0.93; 95% CI, 0.751.15; p=0.4902). Further supporting the benefits of temsirolimus in poor-risk mRCC, fewer patients receiving single-agent temsirolimus reported severe (grade 3-4) adverse events (AEs) compared with IFN-" monotherapy (69% versus 79%; p=0.024) (De Souza et al, 2007). Based on the data published by Hudes and colleagues in 2007, the US Food and Drug Administration approved temsirolimus for the treatment of advanced RCC. Further analyses of data from this trial showed that temsirolimus was superior to IFN-" , regardless of tumor histology, with improved OS and PFS in both clear-cell and non-clear-cell RCC with temsirolimus versus IFN-" (clear-cell RCC: OS, 10.6 versus 8.2 months; PFS, 5.5 versus 3.8 months; other RCC subtypes: OS, 11.6 versus 4.3 months; PFS, 7.0 versus 1.8 months) (Dutcher et al, 2007). The data also revealed improvements in OS and PFS with temsirolimus versus IFN-" therapy regardless of age (<65 versus ! 65 years), and showed no apparent agerelated differences in terms of reported AEs. In the trial, temsirolimus was shown to be superior to IFN-" in patients with poor prognostic features (OS, 10.2 versus 6.0 months; median PFS, 5.1 versus 2.3 months). However, an

C. Sorafenib Data from a phase II trial of first-line sorafenib in 189 patients with advanced RCC found no significant improvement in the primary endpoint of PFS compared with the active comparator IFN-" (5.7 versus 5.6 months, respectively, p=0.504), suggesting that sorafenib may be of limited benefit as a first-line treatment for advanced RCC (Szczylik et al, 2007). The ORR, determined by independent assessment, was 5% with sorafenib 400 mg twice daily (BID) versus 9% with IFN-" . Of interest, however, was the finding that tumor regression occurred in 68% of patients receiving sorafenib 400 mg orally (PO) BID compared with 39% of patients receiving IFN-" , and sorafenib was associated with a longer time to health status deterioration than IFN-" , as determined using the Functional Assessment of Cancer Therapy - Biological Response Modifiers scale (HR, 0.50; 95% CI, 1.43-2.85; p=0.0001) (Figure 2) (Szczylik et al, 2007). The effects of sorafenib dose-escalation and switching from IFN-" to sorafenib were also assessed in patients with disease progression in this trial. No objective responses were noted in 44 patients who were doseescalated from sorafenib 400 mg BID to 600 mg BID, but there was an additional PFS benefit of 4.1 months following escalation from the 400 mg to the 600 mg dose, and the higher dose was well tolerated. Among the 41 patients who switched from IFN-" to sorafenib therapy, 22% demonstrated objective responses by independent assessment, with a median PFS of 5.7 months (Szczylik et al, 2007).

Figure 1. Hazard ratios for overall survival by patient subgroup in patients with metastatic renal cell carcinoma receiving temsirolimus or interferon-alpha. Reproduced from Dutcher et al, 2007 with kind permission from Journal of Clinical Oncology.

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Motzer: Highlights from ASCO and ECCO 2007: Update on targeted treatment of metastatic renal cell carcinoma

Figure 2. Time to health status deterioration in patients with metastatic renal cell carcinoma treated with sorafenib versus interferon-alfa (IFN-! ), as determined using the Functional Assessment of Cancer Therapy - Biological Response Modifiers (FACT-BRM) scale. Reproduced from Szczylik et al, 2007 with kind permission from Journal of Clinical Oncology.

D. Bevacizumab

with favorable (11.1 versus 5.7 months, respectively; p=0.0012) and intermediate (8.4 versus 5.3 months, respectively; p=0.0017) MSKCC risk status, while nonsignificant improvements were observed in the poor-risk subgroup (3.3 versus 2.6 months, respectively; p=0.25). ORR was 25.5% with combination therapy versus 13.1% with IFN-! monotherapy (p<0.0001). The most common serious AEs in both arms of the trial were fatigue (37% versus 30%) and anorexia (17% versus 8%), with combination or single-agent therapy, respectively. Findings from the large AVOREN and CALBG 90206 trials indicate that bevacizumab, in combination with IFN-! , is associated with a statistically significant improvement in PFS and tumor response when administered in the first-line setting. In a smaller randomized phase II trial, the addition of erlotinib to bevacizumab was well tolerated but did not provide additional clinical benefit compared with bevacizumab alone (Bukowski et al, 2007a). However, data from the trial support the clinical benefit of bevacizumab in patients with previously untreated mRCC.

Randomized clinical trials have reported beneficial effects with bevacizumab in patients with advanced RCC (Yang et al, 2003b; Bracarda et al, 2007; Bukowski et al, 2007a; Escudier et al, 2007a; Melichar et al, 2007; Rini et al, 2008). Data on the efficacy and safety of bevacizumab (10 mg/kg intravenously [IV] every 2 weeks) in combination with IFN-! versus IFN-! alone in the firstline setting were reported in the randomized, controlled, multicenter, phase III AVOREN trial in 649 patients with advanced RCC (Bracarda et al, 2007; Escudier et al, 2007a; Melichar et al, 2007). Bevacizumab plus IFN-! significantly increased PFS compared with IFN-! plus placebo, as determined by investigator assessment (10.2 versus 5.4 months; HR, 0.63, p<0.0001) (Escudier et al, 2007a). PFS benefits with bevacizumab plus IFN-! were achieved regardless of baseline prognostic factors (Bracarda et al, 2007) or IFN-! dose reductions (Melichar et al, 2007). Furthermore, ORR was significantly higher in the bevacizumab/IFN-! group than in the IFN-! plus placebo group (31% versus 13%; p<0.0001); however, OS could not be determined due to a lack of mature data. The most common serious (grade 3-4) AEs associated with bevacizumab/IFN-! versus IFN-! plus placebo were fatigue and hypertension (23% versus 15%, and 3.9% versus 0.7%, respectively). Hemorrhage was reported in 11 patients (3.3%) receiving bevacizumab plus IFN-! versus 1 patient (0.3%) receiving IFN-! plus placebo. More recently, interim data from the similar CALBG 90206 study were reported at the ASCO 2008 Genitourinary Cancers Symposium. In this study, patients with clear-cell mRCC were randomized to receive firstline bevacizumab and IFN-! (n=369) or IFN-! alone (n=363) at the same doses as in the AVOREN trial (Rini et al, 2008). Overall, patients achieved a median PFS of 8.5 months (95% CI, 7.5-9.7) with bevacizumab plus IFN-! versus 5.2 months (95% CI, 3.1-5.6) with IFN-! alone (HR, 0.71; 95% CI, 0.61-0.83; p<0.0001). Compared with monotherapy, significant improvements in PFS were also achieved with combination therapy in patient subgroups

E. Atrasentan A phase II trial of the selective endothelin-A receptor antagonist (SERA), atrasentan (Xinlay™; Abbott Laboratories, IL, USA), has recently been completed in patients with mRCC (Manola et al, 2007). Atrasentan inhibits the activity of endothelin-1, a protein implicated in stimulating the spread of cancer cells. The phase II trial showed that atrasentan (10 mg/day PO) was well tolerated but findings from the trial did not support its use as a firstline monotherapy in patients with mRCC (Manola et al, 2007). The data revealed a median PFS with atrasentan of 2.3 months (95% CI, 2.0-3.5 months), and a percentage of patients remaining progression-free at 6 months, stratified according to disease status and prior immunotherapy, of between 0% and 17%.

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Cancer Therapy Vol 6, page 429! use of this agent in the second-line setting (Gore et al, 2007a,b). In the program, antitumor activity with sunitinib was reported across a diverse patient population, and safety and tolerability outcomes were similar to those observed in previous clinical trials with sunitinib (Motzer et al, 2006a,b). In 3997 evaluable patients, the ORR was 11.4%, with a further 44.6% of patients exhibiting stable disease (SD) at ! 3 months. Sunitinib was associated with acceptable tolerability in specific patient subgroups, including patients with poor performance status (PS ! 2), older patients (aged >65 years), and patients with brain metastases (Gore et al, 2007b). Sunitinib also demonstrated substantial antitumor activity in 61 bevacizumab-refractory patients in a phase II, single-arm, open-label study (George et al, 2007a; Hutson et al, 2007). Partial responses (PRs) were observed in 23% of patients (n=14) and SD in 59% of patients (n=36), with a median PFS of 30.4 weeks (95% CI, 18.336.7). The positive findings from this study in patients who had received previous treatment with bevacizumab indicate an absence of cross-resistance between these two agents that target the VEGF pathway (George et al, 2007a; Hutson et al, 2007). This suggests that the therapeutic activity of sunitinib in RCC may be mediated via multiple pathways, and sunitinib may even inhibit signaling pathways involved in bevacizumab resistance.

III. Recent advances in second-line treatment of RCC National and international treatment guidelines support several targeted agents that have been associated with improved patient outcomes compared with historical therapies as second-line treatments in mRCC (Ljungberg et al, 2007; National Comprehensive Cancer Network, 2008). Currently recommended targeted therapies include sorafenib, sunitinib, temsirolimus, and bevacizumab in patients with cytokine-refractory mRCC. Data presented at the 2007 ASCO and ECCO congresses concerning the use of targeted agents in the second-line treatment of mRCC are discussed below.

A. Sunitinib Clinical benefit with sunitinib in the second-line setting was confirmed in an updated, pooled efficacy analysis of two phase II trials that reported substantial antitumor activity in patients with cytokine-refractory mRCC (Rosenberg et al, 2007). In 168 evaluable patients, investigator-assessed ORR was 45% (95% CI, 39-54) and PFS was 8.4 months (95% CI, 7.9-10.7). Median OS was 19.9 months for the pooled population, which compared favorably with historical experience of other agents in the second-line setting, in which median OS of 12.7 months was reported (Motzer et al, 2004). Prognostic factors for survival were similar to those previously identified in association with cytokine therapy, including performance status, time from diagnosis to treatment, and serum hemoglobin level (Table 1) (Motzer et al, 2004; Rosenberg et al, 2007). Updated data from the larger of these two trials in 105 evaluable patients, showed an ORR of 33% (95% CI, 24-43%) with a median duration of response of 14.0 months. Median TTP and PFS were 10.7 and 8.8 months, respectively, and median survival was 23.9 months; 43 patients remained alive at a median follow-up of 29.7 months (Motzer et al, 2007d). Efficacy and safety data from an international expanded access program of sunitinib in mRCC were presented at both the 2007 ASCO and ECCO meetings; these data provide a practical clinical perspective on the

B. Sorafenib Final data from the phase III TARGET trial (Target Approaches in Renal Cancer Global Evaluation Trial) of second-line sorafenib versus placebo with best supportive care in advanced RCC showed no statistically significant difference in OS between sorafenib and placebo (median OS, 17.8 versus 15.2 months; HR, 0.88; p=0.146) (Escudier et al, 2007b; Bukowski et al 2007b). However, some clinicians consider that sorafenib may offer clinically important benefits, and it seems likely that the crossover of placebo patients to the sorafenib treatment arm, due to significant improvements in PFS with sorafenib, may have confounded the final analysis.

Table 1. Pretreatment predictors of overall survival as determined by multivariate analysis from a pooled analysis of 168 patients with metastatic renal cell carcinoma treated with sunitinib as second-line therapy in two single-arm phase II clinical trials. Reproduced from Rosenberg et al, 2007 with kind permission from Journal of Clinical Oncology. Variable Time interval from diagnosis to treatment ! 1 year vs <1 year Hemoglobin* ! 13/11.5 g/dL vs <13/11.5 g/dL Baseline ECOG PS 0 vs 1 or 2 Number of metastatic sites 0 or 1 vs 2 or ! 3

Hazard ratio 0.384

95% CI 0.255-0.580

P-value <0.0001

0.427

0.280-0.651

<0.0001

0.552

0.363-0.840

0.0056

0.445

0.218-0.906

0.0257

*Cut point is 13 g/dL for males and 11.5 g/dL for females CI = confidence interval, ECOG PS = Eastern Cooperative Oncology Group performance status

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Motzer: Highlights from ASCO and ECCO 2007: Update on targeted treatment of metastatic renal cell carcinoma phase, and targeting VEGF, thus inhibiting angiogenesis (Jac et al, 2007). In a phase II, single-arm trial, administration of everolimus at an oral dose of 10 mg/day by continuous daily dosing (CDD) in a 28-day cycle to 37 evaluable patients with progressive mRCC in the secondline setting resulted in a PR in 12 patients (32.4%) and SD for >3 months in 19 patients (51.4%). Median OS in the trial was >11.5 months (range 1->20) (Jac et al, 2007). Treatment-related AEs included mucositis, skin rash, pneumonitis, thrombocytopenia, anemia, laboratory abnormalities, and decreased metabolic activity (Jac et al, 2007). Following positive early results, studies are ongoing in patients in whom prior sunitinib or sorafenib therapy failed.

In the open-label ARCCS (Advanced Renal Cell Carcinoma Sorafenib Expanded Access Program-North America) trial, sorafenib treatment was evaluated in 2502 patients with advanced RCC who would not otherwise have been eligible for inclusion in clinical trials (Knox et al, 2007). Fifty percent (n=1255) of these patients received sorafenib as a second-line treatment. Of 936/1255 secondline patients, one patient had a PR (3%) and 161 patients (17%) showed unconfirmed responses. SD was observed in 759 patients (81%), and clinical benefit was achieved in 84% of patients. Similar results were observed in the 1247 patients in the program who received sorafenib as a firstline treatment. More than 1150 patients have been recruited into EU-ARCCS, the European arm of this openlabel study, and analyses are ongoing in these patients (Beck et al, 2007). Despite the positive results achieved in this program to date, the trial is limited by its non-rigorous design and short duration of follow-up (~12 weeks).

B. Axitinib Axitinib is a potent small molecule RTK inhibitor of VEGFRs, PDGFR-! , and c-KIT (Rugo et al, 2005) that has been shown to be active in cytokine-refractory mRCC (Rini, 2005). Activity of axitinib as a second-line therapy in patients with mRCC that is refractory to tyrosine kinase inhibitors, such as sunitinib and sorafenib, is of clinical interest (Rini et al, 2007a). An open-label, multicenter, phase II trial was recently undertaken to investigate oral axitinib administered twice daily at a dose of 5 mg in this setting (Rini et al, 2007a,b). In 62 evaluable patients who had received prior treatment with sorafenib or sorafenib and sunitinib, axitinib therapy was associated with PR in 15% of patients (n=9) and SD in 37% of patients (n=23), with some level of tumor shrinkage in 51% of patients (Figure 3). Median PFS was not reached, but preliminary data indicated an overall median PFS of >7.7 months (Rini et al, 2007a,b). Based on these data, axitinib appears to exhibit substantial antitumor activity in patients with sorafenib-refractory mRCC, and a tolerability profile that is manageable and characteristic of this class of agents.

IV. Novel targeted agents in the second-line treatment of RCC Promising data with several newer targeted agents in the second-line setting in mRCC were presented at the 2007 ASCO and ECCO meetings, including data from trials of the mTOR inhibitor, everolimus (RAD001; Certican, Novartis International AG, Basel, Switzerland), and the novel agents axitinib (AG-013736; Pfizer Inc., New York, NY, USA) and volociximab (M200; Biogen Idec, Zug, Switzerland; PDL Biopharma Inc., Redwood City, CA, USA).

A. Everolimus Like temsirolimus, everolimus is an oral mTOR inhibitor that is believed to exert anti-tumor activity by shutting down tumor cell responses to nutrients and growth factors, arresting the cell cycle in the late G1 ! !

Figure 3. Tumor regression, measured as maximum percent reduction in target lesions, in 42 evaluable patients with TKI-refractory metastatic renal cell carcinoma receiving axitinib monotherapy in the second-line setting. Reproduced from Rini et al, 2007 with kind permission from Journal of Clinical Oncology.

! !

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C. Volociximab

with an overall median PFS of 11.3 months. The most common grade 3 treatment-related AEs were diarrhea, which occurred in 14% of patients (n=6), and nausea and rash, each in 5% of patients (n=2 each); no grade 4 treatment-related AEs were observed (Redman et al, 2007). Combined inhibition of mTOR and PDGFR may prevent tumor growth and angiogenesis through vertical blockade of the PI3K/AKT pathway and its stimulatory effects on VEGF (Arsham et al, 2004; Chan et al, 2007). The therapeutic potential of such blockade provided the rationale for a phase II study of combination therapy with everolimus (2.5 mg/day PO) and imatinib mesylate (IM; Glivec速; Novartis, Basel, Switzerland; 600 mg/day PO) in patients with previously treated advanced RCC (Chan et al, 2007). Among the 10 patients who were evaluable for the primary endpoint (PFS), three patients were progression-free at 3 months. Nine patients were evaluable for best response by RECIST, among whom SD was the best response in seven patients and PD occurred in two patients. The combination of everolimus and IM was associated with moderate toxicity. Grade 3 AEs included fatigue, thrombocytopenia, peripheral edema, rash, and abdominal pain. In addition, two cases of grade 3 pneumonitis and angioedema (n=1 each) were considered as potential everolimus-related toxicities (Chan et al, 2007). In a similar phase I-II, single-arm study, PTK/ZK (PTK787/ZK222584; Novartis, Basel Switzerland; Schering, Berlin, Germany; 1000 or 1250 mg/day PO), a novel orally active multitargeted kinase inhibitor against VEGFR and PDGFR, was evaluated in combination with everolimus (5 or 10 mg/day PO) in 27 patients with advanced solid tumors who may have received prior VEGF inhibitor therapy (Speca et al, 2007). Preliminary data from 13 mRCC patients who were evaluable for response showed PR in two patients (15%) and SD for >3 months in eight patients (62%); median TTP in these patients was 6 months. DLTs included grade 3 asthenia and mucositis, and grade 4 hypertension (Speca et al, 2007). The investigators concluded that the combination of PTK/ZK 1,000 mg plus everolimus 5 mg was well tolerated and was associated with clinical activity in patients with advanced RCC, despite prior treatment with VEGF pathway inhibitors in some cases (Speca et al, 2007).

Volociximab is a chimeric monoclonal antibody that blocks the binding of fibronectin in the extracellular matrix to integrin a5! 1, and thus induces apoptosis in proliferating endothelial cells (Yazji et al, 2007). The efficacy and safety of volociximab was assessed in a multicenter, open-label, phase II study in 40 evaluable patients with refractory or relapsed clear-cell mRCC. Patients received intravenous volociximab at a dose of 10 mg/kg every 2 weeks until disease progression. PR was confirmed in one patient, with SD in 32 patients (80%). Median TTP was 4 months, and median OS had not been reached at 22 months, with 68% of patients remaining alive at 22 months (Yazji et al, 2007). Most frequently reported side effects included grade 1-2 fatigue, nausea, dyspnea, and arthralgia. The investigators concluded that volociximab was well tolerated and was associated with anti-tumor effects in a large percentage of patients. Further studies of this novel agent are planned.

V. Combination therapy with targeted agents in RCC Combination therapy with targeted agents is a logical next step in optimizing the treatment of patients with mRCC. Combinations of targeted agents may offer more complete inhibition of a single pathway (vertical blockade) or may inhibit several pathways to mediate a range of therapeutic effects (horizontal blockade); however, this type of treatment has raised concerns regarding increased risks of toxicities compared with single-agent treatments. Data from several phase I/II trials of combination therapies with targeted agents were presented at the 2007 ASCO and ECCO meetings (Chan et al, 2007; Feldman et al, 2007; Merchan et al, 2007; Patel et al, 2007; Patnaik et al, 2007; Redman et al, 2007; Speca et al, 2007). Temsirolimus in combination with bevacizumab was evaluated in a phase I study in 12 patients with stage 4 mRCC, in which patients could receive full doses of each agent in monthly cycles (temsirolimus 25 mg/week IV; bevacizumab 10 mg/kg once every 2 weeks) (Merchan et al, 2007). This combination demonstrated clinical activity, including PR in 7 patients, without enhanced toxicity (Merchan et al, 2007). In contrast, however, data from a similar phase I study of temsirolimus (15 or 25 mg/week IV) in combination with sorafenib (200 or 400 mg BID) in patients with advanced solid tumors revealed significant mucocutaneous toxicity when full doses of sorafenib were given. PR was observed in 2/24 patients and prolonged SD was noted in one patient. Evaluations of this combination at revised dose levels are ongoing (Patnaik et al, 2007). The combination of oral sunitinib (37.5 or 50 mg/day on Schedule 4/2) and gefitinib (Iressa; AstraZeneca, London, UK; 250 mg/day), an orally active selective epidermal growth factor receptor (EGFR) inhibitor, was assessed in a phase I/II trial based on preclinical data, which suggested that simultaneous inhibition of VEGFR and EGFR may act synergistically to inhibit tumor growth (Patel et al, 2007; Redman et al, 2007). Among 42 patients with mRCC who were evaluable for a response, PR was noted in 17 patients (40%) and SD in 19 patients (45%),

VI. Sequential treatment of targeted agents in RCC Until recently, few data have been available on the use of sequential targeted therapies in patients with mRCC in whom antiangiogenic treatments have failed. The 2007 ASCO and ECCO meetings highlighted several studies that have begun to address this important clinical question. Anecdotal reports have suggested that patients respond to sunitinib treatment following sorafenib failure and vice versa (Dham and Dudek, 2007; Sablin et al, 2007). A study presented at ASCO 2007 evaluated the efficacy of sequential therapy with sorafenib or sunitinib in 37 patients who had switched treatment due to disease progression (n=32) or unacceptable toxicity (n=5) 431

Motzer: Highlights from ASCO and ECCO 2007: Update on targeted treatment of metastatic renal cell carcinoma the use of alternate doses and schedules with sunitinib and sorafenib in mRCC. Following favorable phase III trials with second-line sorafenib (400 mg BID) showing efficacy in mRCC patients, a single-arm, phase II, dose-escalation study in 44 evaluable patients with mRCC demonstrated that the majority of patients were able to tolerate much higher doses of sorafenib (Amato et al, 2007). Patients underwent monthly dose-escalation from 400 mg BID to 600 and then 800 mg BID, and received treatment for a median duration of ! 6 months. Doses were escalated to 600 mg BID in 41 of 44 patients (93%) and to 800 mg BID in 32 of 44 patients (73%). The investigators reported that treatment was well tolerated, with AEs including hand-foot syndrome, skin rash, diarrhea, alopecia, fatigue, hypertension, hypophosphatemia, and elevated amylase and lipase levels, as might be expected with this regimen. ORR was a considerable 55%, with complete response noted in 16% of patients and PR in 39% of patients; 20% of patients exhibited SD for ! 6 months (Amato et al, 2007). In addition to the wealth of available data regarding the use of sunitinib 50 mg/day PO administered on Schedule 4/2, a recent study showed that CDD with sunitinib 37.5 mg PO had comparable efficacy to intermittent dosing with sunitinib 50 mg PO in 107 patients with cytokine-refractory mRCC (Srinivas et al, 2007; Escudier et al, 2007c). The study investigated the efficacy and tolerability of CDD and compared morning and evening dosing regimens. In total, a PR was confirmed in 22% of patients, while 48% of patients exhibited SD for >3 months; median PFS was 8.3 months (95% CI, 6.58.8). The study showed no differences between morning and evening dosing. The investigators concluded that administration of sunitinib by CDD is associated with a manageable tolerability profile and additional flexibility in terms of dose scheduling (Escudier et al, 2007c). This dosing regimen may offer a useful alternative to existing intermittent treatment schedules and may be explored in future combination studies. Findings from a pharmacokinetic/pharmacodynamic analysis of the exposure-response profile of sunitinib in mRCC, based on data from three phase II trials in 237 patients, supported the benefits of CDD with sunitinib (Houk et al, 2007a,b). The meta-analysis showed greater efficacy in patients with the greatest exposure to the drug, and revealed that increased exposure was associated with a higher probability of PR, greater OS, and longer TTP. Higher exposure was also associated with greater changes in tumor volume. Comparable efficacy was observed when sunitinib was administered as an oral dose of 37.5 mg by CDD and when it was given at a dose of 50 mg/day PO on Schedule 4/2 (Figure 4) (Houk et al, 2007a,b).

following first-line sunitinib or sorafenib (Dham and Dudek, 2007). The study showed that disease control was achieved with both sequential treatments; however, median duration of SD was longer in patients who received sorafenib followed by sunitinib rather than vice versa (sorafenib followed by sunitinib, 32 weeks [range 637]; sunitinib followed by sorafenib, 8 weeks [range 410]) (Dham and Dudek, 2007). A retrospective analysis in France assessed sequential therapy in 90 patients who received sorafenib followed by sunitinib (n=68) or sunitinib followed by sorafenib (n=22) over a 3-year period in clinical studies or extended-access programs (Sablin et al, 2007). Overall, PR rates were 17.6% for sorafenib followed by sunitinib and 22.7% for sunitinib followed by sorafenib. PD occurred in only six patients receiving both drugs, all of whom were of intermediate or poor risk, with 3 or more metastatic sites. These data support the sequential use of sorafenib and sunitinib and suggest no cross-resistance between the two therapies (Sablin et al, 2007). A subsequent study, presented at ECCO 2007, assessed the comparative toxicity of sunitinib and sorafenib by self-reported questionnaire in patients who received the two treatments sequentially, 17 patients receiving sorafenib first and 10 patients receiving sunitinib first (Plantade et al, 2007). Patients reported fatigue as a more important side-effect of sunitinib than sorafenib, irrespective of the order of administration. Stomatitis was more common with sunitinib, regardless of treatment sequence, while diarrhea and hand-foot syndrome were more common with sorafenib. Anorexia occurred with similar frequency with both drugs, and patients reported similar effects on quality of life (QoL) with both agents. Overall, treatment with sunitinib was more acceptable to patients than sorafenib therapy (Plantade et al, 2007). Data from studies such as these highlight the potential of sequential targeted therapies, indicating that responses to treatment can be achieved in mRCC patients who receive targeted agents following the failure of an initial RTK inhibitor. Findings from the studies suggest that improved responses are achieved in patients who receive sunitinib following sorafenib failure, and that sunitinib is more acceptable to patients (Dham and Dudek, 2007; Plantade et al, 2007; Sablin et al, 2007).

VII. Refining existing therapies using alternate schedules and doses As our clinical understanding of existing RTKs and other targeted agents increases, so also does our interest in optimizing currently available treatment regimens in order to improve patient outcomes. Several research groups presented data at the ASCO and ECCO 2007 meetings on

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Figure 4. Comparison of time to progression and overall survival in patients with metastatic renal cell carcinoma receiving sunitinib at 50 mg/day following a 4/2 Schedule (4 weeks on treatment followed by 2 weeks off treatment in a 6-week cycle; n=188) or at a dose of 37.5 mg/day by continuous daily dosing (CDD) (n=49). Reproduced from Houk et al, 2007 with kind permission from Journal of Clinical Oncology.

Choueiri et al, 2007); however, variation in treatment outcomes occurs and the association remains unclear (Choueiri et al, 2007). To address this issue, Choueiri and colleagues used polymerase chain reaction analysis to assess VHL mutational status in 123 mRCC patients who had received prior treatment with anti-VEGF therapy (sunitinib, sorafenib, axitinib, or bevacizumab); findings were correlated with clinical and laboratory features and treatment outcomes. Loss of function (LOF) mutations (p=0.03), normal hemoglobin levels (p=0.01), and the absence of prior radiotherapy (p=0.04) were identified as independent predictors of response in a multivariate analysis. The presence of a LOF mutation was associated with an ORR of 52% versus 31% for those without the mutation. Of the four treatments, sunitinib was associated with the greatest ORR in patients with VHL mutations or promoter hypermethylation, while axitinib conferred the greatest ORR in patients with wild-type VHL status. However, PFS and OS were not affected by VHL status. The researchers concluded that certain types of VHL mutation - such as LOF mutations, which have been correlated with poor patient prognosis (Kim et al, 2005) may be associated with greater responses to VEGFtargeted therapies (Choueiri et al, 2007).

VIII. Prognostic and predictive factors The identification of prognostic or predictive factors for individual patient outcomes is important in order to develop tailored treatments that reduce the risk of relapse and enhance the chance of successful management. Prognostic and predictive factors for survival were the focus of a number of publications and presentations at the 2007 ASCO and ECCO meetings. Motzer and colleagues undertook an analysis of prognostic risk factors based on data from the phase III trial of first-line sunitinib versus IFN- in mRCC (Motzer et al, 2007a). They observed improvements in PFS with sunitinib versus IFN- , irrespective of MSKCC prognostic risk factor group (favorable, intermediate, or poor), indicating that sunitinib is an effective treatment for a range of mRCC patient populations (Motzer et al, 2007b,c). Using a multivariate analysis, the researchers identified three baseline prognostic factors predictive of prolonged PFS with sunitinib (Eastern Cooperative Oncology Group PS 0 versus 1 [p=0.007], time from diagnosis to treatment ! 1 year versus <1 year [p<0.001], and corrected calcium level " 10 versus >10 mg/dL [p=0.0084]). Median PFS was 14.9 months (95% CI, 13.417.4) with sunitinib versus 8.4 months (95% CI, 7.8-11.0) with IFN- in patients with favorable risk; 10.7 months (95% CI, 8.3-11.4) versus 3.8 months (95% CI, 3.7-5.1), respectively, in patients with intermediate risk; and 3.9 months (95% CI, 2.5-13.5) versus 1.2 months (95% CI, 1.0-5.1), respectively, in patients with poor-risk mRCC (Motzer et al, 2007c). Von Hippel-Lindau (VHL) tumor suppressor gene inactivation through mutation is strongly associated with clear-cell RCC in a majority of cases (Kaelin, 2002; Rini and Small, 2005). VHL inactivation is thought to be associated with a greater objective response to VEGFtargeted therapy (Kaelin, 2002; Rini and Small, 2005;

IX. QoL and cost-effectiveness As newer targeted therapies in oncology offer patients longer disease-free periods, there is an increasing need to understand the effects of these therapies on patient QoL and management costs. At both the ASCO and ECCO 2007 meetings, Cella and colleagues (Cella et al, 2007a,b) examined the correlation between baseline QoL variables and PFS in the previously reported phase III trial comparing sunitinib with IFN- as first-line therapy in patients with mRCC (Motzer et al, 2007). The analyses showed that baseline

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Motzer: Highlights from ASCO and ECCO 2007: Update on targeted treatment of metastatic renal cell carcinoma cytokines and/or targeted agents. Sunitinib and sorafenib have shown efficacy in this setting, while novel agents, such as axitinib, have yielded positive data in patients with RTK-refractory disease. As in first-line treatment, solid clinical evidence supports the efficacy of sunitinib in this setting, including poor-prognosis patients and those who would not normally be eligible for enrolment in clinical trials. Recent studies have demonstrated clinical efficacy with sunitinib in patients aged over 65 years, patients with brain metastases, and those with poor PS. Improved outcomes in a range of patient populations have lead to investigations of sunitinib, sorafenib, and other targeted agents using alternative treatment strategies, including CDD, and as a part of combination and sequential therapies, in order to optimize treatment efficacy. The publication of long-term survival data for sunitinib in the mRCC setting is anticipated at ASCO 2008, and these data and the subsequent release of mature survival data for other targeted agents is likely to strengthen the robust body of evidence supporting their use in this setting. Historically, mRCC is a highly treatment-resistant malignancy for which few therapeutic options have been available until recently. The ongoing development of novel targeted therapies, greater understanding of prognostic factors, and the investigation of alternative dosing schedules and treatment combinations are continuing to improve our ability to treat patients with mRCC. Such advances may assist clinicians in tailoring treatment strategies to individual patients and combating problems of treatment resistance and treatment-related toxicities in order to prolong PFS and improve QoL.

QoL score was predictive of PFS, regardless of treatments and other demographic and clinical characteristics at baseline (Cella et al, 2007a). The superior treatment effect of sunitinib on PFS in comparison with IFN-! remained when QoL and other baseline variables were controlled (Cella et al, 2007a). However, treatment advantages with sunitinib versus IFN-! were greater in European, Australian, and Canadian patients than in US patients (Cella et al, 2007b). In another phase III study, QoL was compared in poor-prognosis mRCC patients receiving first-line temsirolimus and IFN-! versus either agent alone (Parasuraman et al, 2007). The study investigated QoL in terms of quality-adjusted time without symptoms of progression or toxicity (TWiST), and found that survival benefits with temsirolimus were correlated with improvements in QoL. Patients receiving temsirolimus alone exhibited 38% greater TWiST than those receiving IFN-! alone (6.5 versus 4.7 months, p=0.0005, respectively). However, there was no significant difference in TWiST values between the combination and IFN-! monotherapy treatment arms (P=0.1288) (Parasuraman et al, 2007). Data from the international phase III trial of first-line sunitinib in the mRCC setting (Motzer et al, 2007a) have also been used to evaluate the cost-effectiveness of sunitinib in comparison with IFN-! in mRCC patients from a US societal perspective (Rémak et al, 2007; Négrier et al, 2007). Markov models were used to project survival and costs over 5- and 10-year time horizons based on data from the trial. The evaluation showed that the probability of sunitinib providing a cost-effective alternative to IFN-! was 45.9% with a threshold of $50,000 per quality-adjusted life-year (QALY), and 64.9% with a threshold of $100,000 per QALY. Furthermore, when compared with multiple therapies, sunitinib was the optimal treatment above a threshold of $55,000 per QALY. These data suggest that sunitinib represents a costeffective first-line treatment for mRCC, with costeffectiveness ratios within the established cost thresholds that society is willing to pay for health benefits (Rémak et al, 2007; Négrier et al, 2007).

Acknowledgements Editorial support was provided by ACUMED® (Tytherington, UK) with funding from Pfizer Inc.

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X. Conclusions In recent years, greater understanding of the mechanisms involved in mRCC growth and angiogenesis have prompted the development and evaluation of a number of targeted antitumor agents that have been associated with improved clinical outcomes in this setting. Randomized clinical trials have shown that sunitinib, temsirolimus, and bevacizumab are effective in the firstline treatment of mRCC. Robust clinical trial data support sunitinib as the current standard of care in this patient population, while clinical findings suggest a potential role for temsirolimus in poor-risk patients. Bevacizumab, in combination with IFN-! , has been shown to confer clinical benefit in patients with previously untreated mRCC. However, there is little evidence to support sorafenib as a first-line treatment for mRCC. A host of agents is available for use in the secondline setting in patients with mRCC that is refractory to 434

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Cancer Therapy Vol 6, page 439! Cancer Therapy Vol 6, 439-444, 2008

Functional and structural analysis of androgen receptors for anti-cancer drug discovery Review Article

Zeina Nahleh Karmanos Cancer Institute, Division of Hematology-Oncology, Wayne State University, 41 4HWCRC, Detroit, USA

__________________________________________________________________________________! *Correspondence: Zeina Nahleh, M.D., Karmanos Cancer Institute, Division of Hematology-Oncology, Wayne State University, 4100 John R, 4HWCRC, Detroit, MI 48201, USA; Tel: 313-576-8722; fax: 313-576-8767; e-mail: nahlehz@karmanos.org Key words: Androgen receptor, cancer, nuclear, structural Abbreviations: Androgen Receptor, (AR); Androgen Response Element, (ARE); binding protein, (CBP); cyclic AMP responsive element binding protein, (CREB); DNA-binding domain, (DBD); epidermal growth factor receptor, (EGFR); estrogen receptor, (ER); glucocorticoid receptor, (GR); Hormone Response Elements, (HREs); insulin-like growth factor-I( IGF-I); ligand binding domain, (LBD); Nuclear Receptor, (NR); peroxisome proliferator-activated receptor (PPAR); progesterone receptor, (PR); prostate-specific antigen, (PSA); retinoic acid receptor, (RAR); Selective Estrogen Receptor Modulators, (SERMs); steroid receptor coactivator 1, (SRC1); steroidogenic receptor 1, (SF1); thyroid receptor, (TR); vitamin D receptor, (VDR)

Received: 1 July 2008; Revised: 18 July 2008 Accepted: 21 July 2008; electronically published: August 2008

Summary Understanding the molecular mechanism(s) by which Nuclear Receptors (NRs) regulate developmental processes is an essential step toward the utilization of one or many of these mechanisms for anticancer drug research. This manuscript discusses the structures, functions and molecular mechanisms that regulate the interactions of the Androgen Receptors (AR) as a model for potential drug targeting in hormonally-driven solid tumors. ARs ‘nuclear function, transcriptional factors and their cytoplasmic interaction with growth factor-initiated signaling pathways and coregulatory proteins are reviewed. Potential mechanisms for AR antagonism, inhibition, and modulation, as well as newer strategies are discussed.

1 (SF1).

I. Introduction to Nuclear Receptors Super family

II. The Androgen structural analysis

The nucleus is a cellular compartment that is crucial for coordinating the responses to diverse signals. It plays a critical role in cell differentiation, proliferation, apoptosis, and metabolism. This is mostly established through the regulatory effects of lipophilic ligands such as steroidal and non-steroidal hormones and the interaction with their specific nuclear receptors (NRs) (Thompson et al, 1966). NRs can be classified into three classes(2): Class 1 is the steroid receptor family, and includes the progesterone receptor (PR), the estrogen receptor (ER), the glucocorticoid receptor (GR), the androgen receptor (AR) and the mineralocorticoid receptor; Class 2 or the thyroid/ retinoid family, includes the thyroid receptor (TR), vitamin D receptor (VDR), the retinoic acid receptor (RAR) and the peroxisome proliferator-activated receptor (PPAR; and Class 3 of nuclear receptors, known as the orphan receptor family and includes steroidogenic receptor

Receptor:

a

Understanding the molecular mechanism(s) by which NRs regulate developmental processes would be an essential step toward the utilization of one or many of these mechanisms for anticancer drug development (Suzuki et al, 1993; Ribeiro et al, 1995; Aranda and Pascual, 2001; Tata, 2002; Berns et al, 2003; RobinsonRechavi et al, 2003; Xu and Li, 2003; Ring and Dowsett, 2004; Perissi and Rosenfeld, 2005; Pooley et al, 2006; Bain et al, 2007; Lange et al, 2007; Rody et al, 2007; Pearce et al, 2008). A typical nuclear receptor is a single polypeptide chain with three major domains: a variable amino-terminal domain (the A/B domain), a highly conserved DNAbinding domain (DBD or C domain), and a less conserved carboxyl-terminal ligand binding domain (LBD or E 439

Nahleh: Functional and structural analysis of androgen receptors for anti-cancer drug discovery Guo et al, 2006). These factors may serve as the basis for studying AR function and role as a drug target in other endocrine driven tumors like breast cancer.

domain) (Tata, 2002; Robinson-Rechavi et al, 2003; Bain et al, 2007). All nuclear receptors modulate gene transcription, although amongst the three classes there are differences in the mechanisms through which this is achieved (Ribeiro et al, 1995; Aranda and Pascual, 2001; Bain et al, 2007). The steroid receptors (class 1) which include AR are bound to Heat Shock Proteins (Hsp), such as Hsp90 and Hsp70, in the cytoplasm. Upon binding to their hormonal ligand, the activated receptors undergo restructuring, release Hsps, translocate into the nucleus, and the receptor then binds directly to specific Hormone Response Elements (HREs) in the promoter regions of target genes. ER recognizes a consensus sequence of AGGTCA, and the remaining steroid receptors recognize a consensus AGAACA sequence. DNA binding is coupled to the recruitment of co-activator proteins such as the p160 family (Xu and Li, 2003), and subsequent transcriptional activation. AR classically targets the Androgen Response Element (ARE) -containing prostate-specific antigen (PSA), maspin, p21, and fibroblast growth factor 1 promoters (Lange et al, 2007). Additional mechanisms have been described; for example treatment with steroid hormones also leads to an upregulation of regulatory molecules that lack a classical ARE in their proximal promoter regions, such as androgen-regulated epidermal growth factor receptor (Brass et al, 1995; Lange et al, 1998), c-fos (Richer et al, 1998; Church et al, 2005) and cyclin D1 (Groshong et al, 1997; Gregory et al, 2001). Without canonical HREs, NRs regulation of these genes can occur through indirect DNA-binding mechanisms, as in the case of AR regulating genes via interaction with the SMAD family of intracellular proteins. In the following sections, we will focus the discussion on AR as a model for drug targeting in the treatment of endocrine driven cancers, based on the experience learned in prostate cancer.

A. AR Mutations Structural mutations in the nuclear hormone receptors gave insight into many inherited disease processes and further elucidated the role of NRs in various pathologies (Suzuki et al, 1993; Ribeiro et al, 1995; Berns et al, 2003; Ring and Dowsett, 2004; Pooley et al, 2006; Rody et al, 2007; Pearce et al, 2008). Mutations in Xlinked ARs result in testicular feminization syndrome with androgen unresponsiveness or hypo-responsiveness. Furthermore, earlier studies have suggested that genetic variants in some of the nuclear hormone receptors may be associated with increased cancer risk, although results have been inconsistent (Rody et al, 2007; Pearce et al, 2008). The loss of certain nuclear hormone receptors in human tumors may also have significant implications on outcome. For example, the loss of the AR expression together with the observed loss of other steroid hormone receptors in BRCA1-mutated breast tumors may lead to a hormone-independent growth or to anti-hormone resistant growth of these tumors (Berns et al, 2003). In turn, the increased AR expression level was shown to be associated with the development of resistance to antiandrogen therapy (Chen et al, 2004). The extent of this increased expression and its frequency need to be further elucidated. Also, the development of certain nuclear receptors gene mutations may confer endocrine therapy-resistance, as seen in breast cancer (Ring and Dowsett, 2004) and prostate cancer (Suzuki et al, 1993).

B. AR interaction with growth factors The frequency of AR mutation is generally low and probably only accounts for less than 10% of the cases surveyed (Taplin et al, 2003). On the other hand, the interaction between growth factor and AR-signaling pathways in prostate cancer cells has been well documented. It is believed that cross talk with other signaling pathways may allow AR to continue to function even in the absence of androgens or in the presence of very low levels of androgens. Exploration and understanding of these other signaling pathways may be a potential venue for targeting AR for cancer therapeutics. Most research on cross talk between ARs and androgen-independent signaling pathways has occurred in the context of prostate cancer and therefore may reflect dysfunctional cellular processes. Current data support at least three distinct ways in which ARs engage in cross talk with other signaling pathways. First, kinases from other signal transduction pathways directly phosphorylate AR and modulate its transcriptional activity. Second, kinases from other signal transduction pathways phosphorylate dedicated steroid receptor coactivator/corepressor proteins and regulate their activity. Third, AR participates directly in cross talk via protein-protein interactions with signal transduction intermediates from other signaling pathways (Lange et al, 2007). Thus, AR would be a model for drug target as it functions not only as a transcription factor but also as a node that integrates multiple extracellular signals.

III. The Androgen Receptor: drug targeting and endocrine resistance In adult males, AR is essential for maintaining many male-specific organs, including the prostate gland. Blocking the activity of AR results in loss of the secretory activity and epithelial apoptosis in the prostate (Staack et al, 2003). This normal function of AR has been exploited for more than 65 years as the basis for androgen ablation therapy in prostate cancer (Denis and Griffiths, 2000). However, despite an initial response to the treatment, the therapy eventually fails in virtually all patients, resulting in androgen-independent prostate cancer. However, the majority of androgen-independent prostate cancers continue to express AR and to transactivate AR-responsive genes (Staack et al, 1995; Chen et al, 2004). Therefore, AR signaling appears to play a central role in androgendependent treatment response, but may continue to be the basis of antiandrogen resistance mechanisms (Guo et al, 2006). Mutations and amplification of AR, alterations in protein kinases, growth factors, and nuclear receptor coactivators have all been proposed to modulate AR signaling and may, therefore, play key roles in the development of androgen independence of prostate cancer as detailed below (Taplin et al, 2003; Chen et al, 2004; 440

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D. AR and coregulatory proteins

Some of these interactions are listed below: increases in autocrine and paracrine growth factor loops are among the most commonly reported changes correlated with the progression of prostate cancer from a localized and androgen-dependent disease to a disseminated and androgen-independent disease. Similar observations have been made in experimental models of prostate cancer in which androgen-dependent prostate cancer cell lines require exogenous growth factors to efficiently form tumors in athymic mice, whereas androgen-independent prostate cancer cell lines do not (Gleave et al, 1992; Pietrzkowski et al, 1993; Thalmann et al, 1994). Moreover, forced overexpression of HER2/neu in androgen-dependent prostate cancer cells drives androgenindependent growth (Craft et al, 1999). Also, inhibition of epidermal growth factor receptor (EGFR)/HER2 signaling can inhibit prostate cancer cell growth !"#$!%&' and !"#$!$' (Agus et al, 2002; Mellinghoff et al, 2002) as well as AR transcriptional activity, protein stability, DNA binding, and phosphorylation on serine 81 (Mellinghoff et al, 2004). Previous work by Hobisch et al. has suggested that AR is transcriptionally activated in a ligand-independent manner by insulin-like growth factor-I (IGF-I), EGF, and keratinocyte growth factor in human prostate tumor cell lines (Culig et al, 1994). HER-2/neu activates the AR pathway in the absence of ligand and increases AR responses in the presence of low levels of androgen in prostate cancer cells (Craft et al, 1999).

Other studies have implicated a direct role for AR coregulatory proteins in prostate cancer. Transcriptional coregulators are frequently overexpressed in advanced prostate cancer, facilitating AR activity. These include steroid receptor coactivator 1 (SRC-1), cyclic AMP responsive element binding protein (CREB), binding protein (CBP), p300, amplified in breast cancer 1 (AIB1). All contribute to prostate cancer progression to androgen independence and have been identified as targets of signaling pathways (He et al, 2000, 2002, 2004, 2006; Lange et al, 2007). Their role in other tumor types need to be further elucidated. #

E. AR modulators

antagonists,

inhibitors

and

Flutamide, nilutamide and bicalutamide are nonsteroidal androgen antagonists currently used in the treatment of prostate cancer. The mechanism of action of bicalutamide has been further elucidated by studying the crystal structure of a mutant form of AR, the ARW741L mutant, when bound to bicalutamide (Bohl et al, 2005a). This naturally occurring mutation confers agonistic activity to bicalutamide and is thought to contribute to the bicalutamide withdrawal syndrome (resistance of prostate tumor cells to antiandrogen therapy). The mutation generates additional space such that the sulfonyl-linked phenyl ring of bicalutamide is accommodated at the location of the missing indole ring of W741. In wild-type AR, the presence of the W741 side chain probably forces bicalutamide to protrude out thus precluding the active position of H12. In contrast, due to their modest size, flutamide and nilutamide probably antagonize AR through the mechanism of “passive antagonism”.

C. AR in” other “tumor types The interaction between AR and other growth factor signaling pathways was further elucidated recently in both prostate and other tumor types (Migliaccio et al, 2005). A novel interplay between the EGF-activated EGFR, its partner HER 2/neu, and ARs was suggested using experiments on MCF-7 and LNCaP cells. These are hormone-responsive cells derived from human mammary and prostate cancers, respectively; they express AR and either ER! (MCF-7) or ER" (LNCaP) and are widely used to analyze the effect of sex-steroid hormones and the cross-talk between growth factors and steroid hormones. The EGF-activated Src, which is associated with ER/AR dimer, acts strongly on EGFR phosphorylation. Conversely, when ER and/or AR are locked in an inactive conformation (i.e., by hormone antagonists or when the steroid receptor levels are down-regulated), their action on Src and EGFR is missing or heavily impaired and EGFinduced EGFR tyrosine phosphorylation is minimal. Interestingly, in MCF-7 cells, steroid antagonists and silencing of steroid receptor genes abolished the EGFelicited DNA synthesis, thus indicating that such an effect requires steroid receptors. A new model of cross-talk between steroid receptors and EGFR with a central role of the physical and functional interactions between EGFR, steroidal receptors, and Src has been suggested (Migliaccio et al, 2005). From these and many other reports, it seems that cross-talk between growth factors and ARs, which occur at multiple levels, is involved in cancer progression and in endocrine resistance.

F. AR: Novel therapeutic approaches Current treatments of prostate cancer with AR antagonists are facing problems of adverse effects and resistance similar to those observed with the Selective Estrogen Receptor Modulators (SERMs). The lack of AR structures in the antagonist conformation obviously limits the structural information that could aid in the development of selective androgen receptor modulators (SARMs) on a rational basis. Studies have shown that resistant AR mutants that convert antagonists into agonists adopt an active conformation when bound to these ligands (Bohl et al, 2005a,b; Salvati et al, 2005; Baek et al, 2006). Mutations alleviate steric hindrance in the hormone binding pocket and permit accommodation of antagonists in the active AR conformation. Potential solutions to approach this problem are : 1) Use the current knowledge of the structural basis for binding of nonsteroidal ligands to these AR mutants in order to allow for the rational modification of existing drugs to obtain effective antagonists which will retain an antagonist profile against the wild-type and mutant ARs. 2) Conduct further studies to visualize further how antagonist-bound AR interacts with corepressors, since the recruitment of corepressors by AR appears to be required for the therapeutic effectiveness of antiandrogens (Baek et al, 2006). 3) Pursue a detailed

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Nahleh: Functional and structural analysis of androgen receptors for anti-cancer drug discovery enzymatic components of coactivator and corepressor complexes function at transcriptional and posttranscriptional levels? Clearly, the many secrets of nuclear receptor regulation need to be further unraveled. We considered AR as a study model. It appears that every component of the AR signaling pathway (growth factors, growth factor receptors, AREs, coactivators that include kinase substrates and AR itself) may be a potential target for anti-cancer therapeutics. This constellation of mutually reinforcing mechanisms however underscores the continuous challenge ahead to develop effective therapies for cancer Many studies as detailed above have given us enormous insight into the properties of nuclear receptor, yet it is still not fully clear how the subunits of the receptors interact to generate a holoprotein function. AR is an exciting target for cancer research and treatment. Most research on ARs and androgen- dependent and independent signaling pathways has occurred in the context of prostate cancer. However, we believe that AR as a therapeutic target should be explored in other tumors (Nahleh, 2008). For example, 70% of breast cancers express AR which has been implicated in the pathogenesis of this disease (Sola, 1993; Moinfar et al 2003; Nahleh, 2008), rendering AR an ideal target for further exploration in breast cancer. In addition, newer approaches to designing inhibitors should be explored in order to overcome the current inescapable resistance in cancer treatment that targets ARs. The understanding of the AR structural and functional domains, the transcriptional regulation, and the coordination with numerous coactivators and corepressors is the beginning. A detailed understanding of the rules of regulation applied on a genome-wide basis should be pursued including the exploration of chromatin remodeling complexes that are recruited to their target genes and can be potential cancer therapeutic targets that can activate, nullify or switch their function. A combination of these potential targets with other known agents such as selective AR modulators (SARMS) and SERMs can potentially achieve additional activity. Clearly, the stage is set for an exciting new era in which the nuclear receptor regulation exemplified by AR as an anti cancer strategy will be further unraveled.

understanding of the rules of regulation applied on a genome-wide basis. For example, a particular feature of AR is that it prefers the aromatic-rich motif FxxLF which can be found in a subset of AR-specific coactivators (He et al, 2002) and in the A/B domain of AR itself where it mediates an interaction with the LBD that has been shown to be important for androgen-regulated gene expression (He et al, 2000). It has been suggested that an evolutionary decline in LxxLL motif binding that favors interdomain interaction through the N-terminal FxxLF sequence could account for the functional predominance of AF-1 in AR (He et al, 2004). However, the ARV730M mutant that displays increased affinity for LxxLL fragments and increases AR activation has been identified as a somatic mutation in prostate cancer (He et al, 2006). This information can be, for example, the basis of the possibility of developing specific peptidomimetics that mimic the binding face of LxxLL coactivator motifs and block the interaction of the ARs with required cofactors in target cells (Leduc et al, 2003; Hall and McDonnell, 2005). It is anticipated that drugs of this type could be used in combination with selective AR modulators (SARMS) and SERMs to achieve a complete blockage of AF-2 activity. Other potential features of AR that could be pursued for therapeutic purposes involve tyrosine phosphorylation which modulates AR transcriptional activity, as suggested by the work of Brodie A. and her colleagues (Guo et al, 2006). They have observed that the overall level of tyrosine phosphorylation is significantly increased in hormone-refractory human prostate tumor xenografts derived from castrated male mice, suggesting that tumor cells may utilize the autocrine/paracrine factors activating tyrosine kinases to compensate for loss of androgens. Furthermore, AR is tyrosine phosphorylated in response to several growth factors, including EGF, heregulin, and IL6. An interesting observation on growth factor-induced androgen-independent activation of AR transcriptional activity is that growth factor (e.g., EGF or IL-6) alone in general can only induce a few-fold increase of AR transcriptional activity. This seems to be negligible in comparison to the magnitude of the optimal induction by androgens. Thus, it is possible that a few-fold increase of AR transcriptional activity may be sufficient for regulating a subset of AR-regulated genes that are required for promoting the survival/growth of prostate cancer cells (Guo et al, 2006). EGF, heregulin, and IL-6 are known to be either highly expressed in prostate tissues or elevated in prostate tumors, and could be pursued as a potential target for modulation

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Actual role of multitargeted therapy in renal cell carcinoma (RCC) Review Article

Alessandro Sciarra*, Giovanni B. Di Pierro, Andrea Alfarone, Francesco Minisola, Alessandro Gentilucci, Stefano Salciccia, Luca Iannotta, Francesca Antonini, Cristiano Cristini, Franco Di Silverio, Vincenzo Gentile Urology, Department ‘’U.Bracci’’, University ‘’Sapienza’’, Rome, Italy

__________________________________________________________________________________! *Correspondence: Alessandro Sciarra, Department of Urology ‘’U. Bracci’’, University ‘’Sapienza’’, Viale Policlinico 155, 00161 Rome, Italy; e-mail: a.sciarra@lycos.it Key words: renal cell carcinoma (RCC), angiogenesis, VEGF, PDGF, VHL, tyrosine kinase inhibitor, sunitinib, sorafenib, targeted therapy, temsirolimus Abbreviations: basic fibroblast growth factor (bFGF); complete response, (CR); Eastern Cooperative Oncology Group (ECOG); Epidermal Growth Factor Receptor (EGFR); European Organization For Research and Treatment of Cancer (EORTC); European urological association, (EAU); FMS-like tyrosine kinase-3 (FLT3); hypoxia inducible factor, (HIF); interferone, (IFN); interleukine, (IL); mammalian-target of rapamycin, (mTOR); National Comprehensive Cancer Network (NCCN); objective response rate (ORR); overall survival (OR); plateled-derived growth factor, (PDGF); progression-free survival (PFS); renal cell carcinoma, (RCC); Response Evaluation Criteria in Solid Tumors (RECIST); Southwest Oncology Group (SWOG); tyrosine kinase, (TK); University of California Integrated Staging System (UISS); vascular endothelial growth factor, (VEGF); Von Hippel Lindau (VHL) Received: 27 May 2008; Revised: 24 June 2008 Accepted: 3 July 2008; electronically published: August 2008

Summary This paper reviews the recent progress in understanding the molecular basis of RCC tumourigenesis, and the development and application of new therapies targeted at key molecules involved in angiogenesis, a key process in tumour growth and progression, in order to address the treatment approaches with multitargeted therapies in advanced RCC. We reviewed the international recent literature using Pubmed search. RCC is genetically linked to factors regulating angiogenesis, in particular vascular endothelial growth factor (VEGF). Sunitinib is a multitarget receptor tyrosine-kinase (TK) inhibitor, acting on VEGF receptor (VEGFR) and platelet-derived growth factor receptors (PDGFR). Sorafenib is an oral multikinase inhibitor (VEGFR and PDGFR) showing also inhibitors effect on the Raf system. Similar compound, axitinib, is now in clinical development and directly inhibit the VEGF and PDGF receptors (VEGFR and PDGFR). Bevacizumab is a recombinant human antibody against VEGF binds and it neutralizes all biologically active isoforms of VEGF. Phase I trials showed a consistent safety profile with these new therapies. Phase II and phase III trials showed that these antiangiogenic agents are effective in the treatment of advanced RCC. Survival benefits exist in particular when advanced RCC patients undergo cytoreductive nephrectomies before the initiation of the systemic therapy. Although patients with mRCC are now offered a better prognosis, several questions remain: how to optimise the clinical use of new agents; differences among different compounds; the identification of patients most likely to benefit from multitargeted therapy; advantages of combination or sequential therapies. The new targeted therapies have showed therapeutic feasibility and efficacy, leading to dramatically improvement in survival, both progression-free and overall, and with acceptable toxicity. Multitargeted therapy with Sunitinib and Sorafenib has been approved to FDA and is revolutioning how we clinically approach advanced RCC.

lives each year, and > 200000 new cases are diagnosed annually (Ferlay et al, 2000). Although prognosis is predominantly for the 20% of patients presenting with distant metastases, 5-yr survival is < 10% (SEER Cancer Statistics Review 1975-2004, 2007). Such statistics have

I. Introduction The incidence of renal cell carcinoma (RCC) has been increasing over the past decades. Overall, cancer of the kidney and renal pelvis, of which RCC is the most common type (Kovacs et al, 1997) claims about 100000 445

Sciarpa et al: Actual role of multitargeted therapy in renal cell carcinoma In the last years new advances have begun to revolutionize the management of advanced RCC and offer hope for the future. First, a proven role for aggressive surgical resection of the primary lesion and metastasectomy has been underlined. Second, a series of exciting new approaches, so called “targeted therapy�, are revolutionizing the management of advanced RCC. In particular, the recent progress in understanding the molecular basis of RCC has led to the identification of angiogenesis, PDGF (platelet-derived growth factor) and VEGF (vascular endothelial growth factor) as a significant therapeutic target in patients with advanced RCC (Ferrara, 2004). Both PDGF and VEGF promote angiogenesis (Iliopoulos et al, 1996; Wiesener et al, 2001). Several studies using strategies to inhibit VEGF and PDGF activity have demonstrated significant antitumour effects in the management of RCC. In addition, studies have showed that these cytokines are frequently over-expressed in clear-cell RCC, likely contributing to the hypervascularity (Takahashi et al, 1994; Tsuchiya et al, 2001). This article reviews the present and future perspectives of advanced RCC treatment and will address, in particular results from clinical trials and strategies to maximize the potential of these agents in advanced RCC. We will try to highlight how far we have come, where we are now and how far we are going. Several reviews related to this topic have been published; here we will focus on new findings and suggestions. In particular we reviewed the international recent literature using PUBMED search (advanced RCC, targeted therapy), in order to analyze new findings on the role of anti-angiogenic targeted therapies in advanced RCC.

prompted an intensive search for newer more effective therapies for metastatic RCC advanced. The diagnostic trend is mainly due to the use of non-invasive abdominal imaging procedures, which show incidental renal tumours. The majority of incidentally detected RCC are at low stage. The management of clinically localized RCC is represented by surgical treatment and it represents a consistent approach to offer a cure for patients with this localized disease. However, a stable proportion of 20%30% of patients still present with metastatic disease and one quarter with clinical localized tumour at the time of nephrectomy will later develop metastatic disease (Rabinovitch et al, 1994; Zisman et al, 2002). These patients would clearly benefit from effective adjuvant therapy, but there is no clinically proven adjuvant treatment for patients at high risk of relapse following surgery (Jacobsohn and Wood, 2006). Previous therapies for RCC advanced, including chemotherapy, hormonal therapy, immunotherapy and radiotherapy have generally had only limited success in a small proportion of patients. The RCC is consistently resistant to cytotoxic chemotherapy with response rates for single agent and combination therapies generally < 15% and often with important or unacceptable toxicity (Motzer and Russo, 2000). Investigation into newer therapies alone or combination of drugs has led to little results. In recent years, basic research into the molecular basis of RCC tumourigenesis has resulted in developement of agents blocking key proteins of angiogenesis and able to improve the prognosis and survival in patients with RCC, providing response rates of > 40%. Previous therapies for advanced RCC including chemotherapies, hormonal therapy, immunotherapy and radiotherapy have only had limited success, leading to response rates generally < 10% (Bukowski, 1997; No author listed, 1999; Atzpodienet al, 2001; Brouwerset al, 2005). The immunogenity of RCC has represented the basis supporting the use of immunotherapy in advanced RCC. As immunotherapeutic agents either interleukin-2 (IL-2) and interferon-! (IFN-! ) have been used. High dose IL-2 was approved by US FDA, as it leads to occasional durable complete responses (Fyfe et al, 1995). However, due to its toxicity, a minority of patients are eligible for high-dose IL-2 therapy and only 5% experience a durable complete remission (Fyfe et al, 1995). However, improved remission rate did not always correlate with survival benefit, but it seems that IFN therapy leads to modest improvements in survival (No author listed, 1999). Combination of IL-2 and IFN has not significative improved clinical results (Mcdermott et al, 2005). Several combination of cytokines, with or without other systemic therapies, have been trialled, but no regimen seems to be consistently superior. In addition, only few patients would likely benefit from cytokine therapy, therefore these therapeutics are indicated only for selected patients with intermediate prognosis and good risk profile (Motzer and Bukowski, 2006; Mcdermott et al, 2005; Ljungberg et al, 2007). Based on this, new therapies for metastatic RCC patients have been long sought.

II. Clinical rationale for a targeted therapy: the concept of cytoreductive surgery An important consideration in the management of advanced RCC is the role of removing the primary tumour, the cytoreductive nephrectomy. Generally, surgeons prefer to remove the whole tumour, which sometimes involves considerable morbidity and mortality (Kirkali and Van Poppel, 2007). Indeed, patients with advanced disease have a high risk of recurrence and require, in many cases, multidisciplinary approaches to increase survival and outcomes (Rigaud et al, 2006; Ljungberg et al, 2007). Different retrospective studies on metastatic RCC cases identified nephrectomy as a favourable treatment if entire tumour is removed and surgery is combined with systemic therapy (Wood, 2003). Moreover, previous studies showed that locally advanced or metastatic RCC cases treated with primary nephrectomy and cytokines improved survival and treatment outcomes than cases without cytoreductive surgery (Zisman et al, 2003; Haferkamp et al, 2007). Based on this, 2 prospective randomized trials were initiated on T-any, N-any, M1 RCC cases and ECOG performance status 0 or 1. The Southwest Oncology Group (SWOG) randomized 241 metastatic RCC cases to IFN-! alone or nephrectomy followed by IFN-! therapy (Wood,

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Cancer Therapy Vol 6, page 447! necrosis) based on the Mayo Clinic experience on RCC cases (Frank et al, 2002). These systems may be useful for the selection of high risk patients most suitable for adjuvant treatment trials and the assignation of patients with metastatic RCC to different treatment strategies and different targeted therapies.

2003). Patients submitted to cytoreductive nephrectomy had a survival advantage over those receiving IFN-! alone (11.1 versus 8.1 months respectively: p=0.012) (Flanigan et al, 2001). The second trial, the European Organization For Research and Treatment of Cancer (EORTC) trial randomized 85 advanced RCC patients to IFN-! alone versus nephrectomy plus IFN-! (Mickisch et al, 2001). Also in this trial the median survival was significantly better for the surgery arm (17 versus 7 months: p=0.010). The benefit of cytoreductive surgery was more evident in ECOG score 0 cases, but not dependent on site of metastasis. The development of new targeted therapies, with promising activity (Escudier et al, 2007; Motzer et al, 2007) and greater results than those with immunotherapy, more set the problem of whether and when surgery is required, in which patients it is useful combining these therapies and surgical approach, and whether or not multitargeted therapy might obviate the nephrectomy in certain patients, even if now there are no data to support this proposal (Hudes et al, 2007). The benefit with targeted therapies in phase 3 clinical trials has been so that these are now recommended in EAU guidelines for treatment of metastatic RCC (Ljungberg et al, 2007) and United States NCCN guidelines (Motzer et al, 2006). All these data strongly suggest that an overall survival benefit exists when advanced RCC patients undergo cytoreductive nephrectomy before the starting of a systemic therapy (Flanigan et al, 2004). This concept has been proved using immunotherapy as a systemic therapy but it should be proved also for targeted therapy as systemic treatment. EAU guidelines raccomend nephrectomy for patients with metastatic disease and good performance status when combined with INF-! (Ljungberg et al, 2007). Proper patients selection is essential to maximize the benefits of cytoreductive nephrectomy (Haferkamp et al, 2007). Prognostic algorithms and nomograms are useful tools in order to identify those patients who are more likely to benefit from combination therapy. For example, Garcia and Rini considered in 2007 optimal candidates those with (Kovacs et al, 1997) good performance status, (Ferlay et al, 2000) a resectable primary tumour representing the majority of tumour borden, (SEER Cancer Statistics Review 1975-2004, 2007) no evidence of central nervous system metastases, (Zisman et al, 2002) no prohibitive medical conditions, (Rabinovitch et al, 1994) no evidence of rapidly progressive extrarenal disease. Resection of solitary (or limited in number) metastases can be associated to significant results in 30% of such patients. Good results have been obtained in cases with pulmonary metastases or also after excision of hepatic, adrenal, brain and pancreatic metastases. At now, there is an increasing interest in using integrated staging system to select RCC cases in different prognostic categories. TNM stage, Furhman grade and ECOG score, compose the University of California Integrated Staging System (UISS) which now stratifies 6 categorie, 3 for non-metastatic and 3 for metastatic RCC. A second validated score is the SSIGN (stage, size, grade,

III. Action mechanism multitargeted therapies

of

The main targeted therapy in RCC recently analysed is that against angiogenesis, VEGF and PDGF signalling pathway. This targeted therapy appears to be associated with more favourable clinical results. In the present review, we considered the analysis of the mechanism of action and the results from clinical trials related to specific anti-VEGF and anti-PDGF therapies.

A. Experimental rationale for targeted therapy 1. Angiogenesis The clinical knowledge that RCC is a highly vascular cancer and that the Von Hippel Lindau (VHL) protein has an important role in sporadic RCC, have made antiangiogenic strategies an attractive approach (15-21). RCC is genetically linked to factors regulating angiogenesis, such as VEGF, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) (Iliopoulos et al, 1996; Tsuchiya et al, 2001; Wiesener et al, 2001; Ferrara, 2004; Motzer and Bukowski, 2006; Negrier, 2006; Ljungberg et al, 2007). VEGF is the strongest proangiogenic protein in RCC and there is a strong rationale for blocking VEGF in this tumor. Approximately 60% of clear RCC have an inactivated VHL tumor suppressor gene (50% through somatic mutation and 10% through promoter methylation). A normal VHL protein indirectly blocks transcription of hypoxic-inducible genes, such as VEGF (Iliopoulos et al, 1996; Tsuchiya et al, 2001; Wiesener et al, 2001; Ferrara, 2004; Motzer and Bukowski, 2006; Negrier, 2006; Ljungberg et al, 2007). Therefore, under hypoxic conditions or VHL gene inactivation, there is an induction of VEGF transcription, over-expression of VEGF protein and angiogenesis (Iliopoulos et al, 1996; Wiesener et al, 2001). Different studies showed that nearly all RCC express high levels of VEGF and that this over-expression correlates with tumor progression and prognosis (Ljungberg et al, 2007). VEGF has several isoforms: VEGF-A is involved in angiogenesis, whereas VEGF-C and VEGF-D are more related to lymphoangiogenesis (Motzer and Bukowski, 2006; Negrier, 2006). VEGF plays major roles in different steps of tumor development with increase of microvascular permeability, permeabilization of blood vessels, extravasation of plasma proteins, introduction of endothelial cell division and migration, promotion of endothelial cell survival (Iliopoulos et al, 1996). Hypoxia in RCC plays an important role in the VEGF pathway. When tumors are exposed to hypoxia as a result of a rapid growth beyond existing blood supplies, the tumor become necrotic. Larger tumors have an inadequate blood supply and thus, hypoxia

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Sciarpa et al: Actual role of multitargeted therapy in renal cell carcinoma angiogenesis, which is disregulated in cancer. In solid tumours Bevacizumab is not currently approved for use in RCC. This drug acts through 3 potential mechanisms: 1) repression of microvasculature, 2) normalization of mature vasculature, 3) inhibition of the production of new vasculature. In RCC the direct effect of Bevacizumab on microvasculature, leads to tumour regression and slowing the tumour growth (Figure 1).

occurs, causing up-regulation of VEFG (Iliopoulos et al, 1996). For this reason in the peri-necrotic areas the hypoxic conditions are a major stimulus for the production of VEGF. There are two possible approaches for targeting VEGF signaling: ligand blockade through a monoclonal antibody (Bevacizumab) and blocking signalling by targeting the receptors with either a monoclonal antibody or a small-molecule tyrosine kinase inhibitor (Sorafenib and Sunitinib).

C. Temsirolimus Temsirolimus is intravenously administered and inhibits the mTOR kinase (Hu-Lowe et al, 2002). Stimulation of mTOR results in the expression of several proteins involved in cell cycle progression, and also controls protein synthesis in response to oxygen starvation (including synthesis of HIF-1! in RCC cells) (Hudson et al, 2002; Patel et al, 2006) (Figure 1). It has been incorporated into the recently revised EAU Guidelines on RCC (Ljungberg et al, 2007) and the United States National Comprehensive Cancer Network (NCCN) Guidelines (Motzer et al, 2006).

2. mTOR pathway The mammalian target of rapamycin (mTOR) pathway has a central role in the regulation of cell growth, and it appears to be dysregulated in cancer (Hudes et al, 2007). mTOR receives stimuli from growth factors, hormones, nutrients and it stimulates proteins syntesis. The mTOR pathway also contributes to angiogenesis (Hudes et al, 2007). A UCLA study showed that mTOR pathways is affected in RCC patients with poor prognostic factors (Hudes et al, 2007). As inhibitors of this pathway, at present there are 3 mTOR inhibitors that are in clinical development as anticancer agents: Temsirolimus, AP23572 and RAD001.

D. Sunitinib Sunitinib is a recent small molecule, acting as multitargeted receptor tyrosine kinase inhibitor, implicated in tumour angiogenesis, growth, and progression (Tsuchiya et al, 2001; Bergers et al, 2003; Sulzbacher et al, 2003). It showed antitumour activity against RCC cells dependent upon signalling through PDGF receptor, VEGF

B. Bevacizumab Bevacizumab is a recombinant human antibody against VEGF, that it is binds and it neutralizes all biologically active isoforms of VEGF (Presta et al, 1997). It has a central role in the regulation of growth and

! Figure 1. Targeted therapies on angiogenesis in RCC. Reproduced from European Urology Supplements, 2008 with kind permission from Elsevier B.V. HIF = hypoxia inducible factor; mTOR = mammalian target of rapamycin; PDGF = platelet-derived growth factor; PDGFR = PDGF receptor; VEGF = vascular endothelial growth factor; VEGFR = VEGF receptor

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Cancer Therapy Vol 6, page 449! and epistaxis more commonly in the high dose Bevacizumab arm. All toxicity episodes were reversible after cessation of the therapies. The study showed that time to progression was significantly longer in patients treated with high dose Bevacizumab (4.8 months) than in those with placebo (2.5 months; p=0.001; HR=2.55). No differences between placebo and low dose Bevacizumab arms in terms of progression-free survival (PFS) were found. In this trial the tumour burden reduction observed in Bevaciumab arms, did not meet criteria for objective response (Response Evaluation Criteria in Solid Tumors (RECIST) as ! 30% tumor burden reduction). More recently the effect of Bevacizumab monotherapy in untreated RCC was defined in a randomized phase II trial of Bevacizumab (10mg/kg every 2 weeks) Âą Erlotinib, an oral Epidermal Growth Factor Receptor (EGFR) tyrosine kinases inhibitor (150 mg /day) (Bukowsy et al, 2006). The objective response rate (ORR) was 13% and median PFS with Bevacizumab monotherapy was 8.5 months. The addition of Erlotinib did not improve clinical results. Two different randomized phase III trials using Bevacizumab in combination with IF versus IF alone have completed accrual (CALGB-90206 and B017705) (Sonpavde et al, 2007). Recently, promisig results have also been reported in combination with INF-" during a phase 3 trial in patients with metastatic RCC: PFS was 10.2 month with Bevacizumab plus INF-" compared with 5.4 month for INF-" alone (Escudier et al, 2007).

receptor, FMS-like tyrosine kinase-3 (FLT3) and stem cell factor receptor (KIT) for proliferation and progression (Mendel et al, 2003). In addition, sunitinib showed to block angiogenesis through inhibition of PDGF receptor, and VEGFR signalling in VEGFR-expressing vascular endothelial cells, and PDGFR-expressing pericytes, stromal cells, or fibroblast. Therefore, Sunitinib may block the tumour growth and progression directly inhibiting its cells as well as blocking angiogenesis through inhibition of vascular endothelial cells and associated supporting cells (Figure 1).

E. Sorafenib Sorafenib is a multikinase inhibitor that was originally developed because of its inhibition on the Raf system (regulatory system on the transcription of relevant proteins), and on several tyrosine kinases that regulates cell proliferation and angiogenesis (Yang et al, 2003). In particular Sorafenib targets the VEGFR and PDGFR and the kinase activity of both C-Raf and B-Raf . All these data suggest that Sorafenib in RCC may inhibit tumour growth by a duel mechanism of action: either directly on the tumor (Raf signalling) and/or indirectly on tumour angiogenesis (VEGFR and PDGFR signalling).

IV. Clinical results from phase I-III trials Different phase I-III clinical trials using Bevacizumab, Sorafenib, Sunitinib and Temsirolimus have been developed and all show that each of these antiangiogenic agents is effective in the treatment of metastatic RCC (Patard, 2008). In most of these studies, the population analyzed was represented by metastatic RCC in progression after cycles of cytokine therapies. Nowadays, sunitinib is recommended as first-line therapy for patients with metastatic RCC and low or intermediate risk; this follows the initial approval for only use in cytokine-refractory patients (Motzer et al, 2006a,b). Recent studies have also demonstrated the efficacy of sorafenib in second-line treatment of metastatic RCC (Cancer Pain Assessment and Treatment Curriculum Guidelines, 1992; Motzer and Bukowski, 2006; Motzer et al, 2006b; Ratain et al, 2006; Escudier et al, 2007; Hudes et al, 2007).

B. Temsirolimus Temsirolimus is considered as first-line treatment in poor-risk patients, as defined by modified MSKCC criteria, and has shown a survival benefit compared with IFN-" in these patients (Houk et al, 2002). In a multicentre phase III study, 626 previously untreated and with poor prognosis were randomised to receive either 25 mg temsirolimus weekly, 3 MU INF-" (with an increase to 18 MU) subcutaneously three times week, or combination therapy with 15 mg temsirolimus weekly plus 6 MU INF-" three times a week. Results showed better response in patients receiving temsirolimus alone: median survival was 7.3 months in INF-" goup, 10.9 months in temsirolimus group and 8.4 in temsirolimus plus INF-" . There was no significant difference in OS between temsirolimus plus INF-" compared with INF-" alone. Termsirolimus as a single agent was better tolerated than IFN-a alone and combination therapy (Houk et al, 2002).

A. Bevacizumab Bevacizumab is indicated in US for treatment of advanced RCC, instead EAU recommends this drug as first-line therapy in poor risk patients with metastatic RCC (Ljungberg et al, 2007). The clinical efficacy of Bevacizumab in metastatic RCC was first investigated in a randomized phase II trial in which 116 cases received placebo versus low-dose (3mg/kg) Bevacizumab, versus high dose (10mg/kg) Bavacizumab (intravenously every 2 weeks) (Yang et al, 2003). All patients showed disease progression after at least one cycle of systemic cytokines treatment (95% IL-2). No life-threatening toxicities or deaths related to Bevacizumab were reported. Toxicity episodes included hypertension (36% of cases), proteinuria

C. Sunitinib Sunitinb is indicated for treatment of advanced RCC and/or metastatic RCC (Sutinib malate: summary of product characteristics, 2006). Either as first-line therapy or as second-line therapy in disease refractory to cytokine therapy (Motzer et al, 2006a,b, 2007). The recommended dose for Phase II trials using Sunitinib, was defined in phase I trials as 50 mg/day orally for 4 weeks, followed by 2 weeks off, in repeated 6 weeks cycles (Faivre et al, 449

Sciarpa et al: Actual role of multitargeted therapy in renal cell carcinoma Sunitinib as first-line therapy in patients with good and intermediate risk (Ljungberg et al, 2007). Sunitinib was also evaluated in patients who have failed prior Bevacizumab therapy in a phase II trial (61 cases). An ORR of 16% was demonstrated with 61% of cases with stable disease.

2006). A recent meta-analysis of the pharmacocynetic of Sunitinib from 10 phase I studies, determined that hepatic and renal function had no effect (Houk et al, 2002). In phase I studies, fatigue was the most commonly reported adverse event (70% of cases included) (Pawson, 2007). This symptom was generally associated with lethargy, asthenia, weakness. Hypothyroidsm was also observed in approximately 65 % of treated cases who underwent thyroid testing in clinical trials.The results of phase I trials suggest that thyroid disfunction should be considered in the differential diagnosis of fatigue during Sunitinib therapy. The most common grade 3-4 toxicities reported included fatigue (10%), neutropenia (8%), thrombocytopenia (7%) (Chow et al, 2007). Two phase II trials have been conducted in patients with metastatic RCC in progression after cytokine therapies. The two trials had similar elegibility criteria (age ! 18 yr, histologic comfirmation of RCC, measurable disease with evidence of metastases, failure of one cytokine therapy because of disease progression ECOG performance status " 1, and adeguate organ function) and treatment plans. Treatment was continued until disease progression, unacceptable toxicity or withdrawal of consent., with primary end point the ORR and secondary end points PFs, overall survival (OR) and safety. In a population of 168 evaluable patients, Sunitinib induced an ORR of 40% with a median time to progression of 8.7 months and a median survival of 16.4 months. A longer PFS was observed in patients with favourable ECOG score and normal serum haemoglobin. The Sunitinib was well tolerated in the two phase 2 trials, with manageable toxicity and the most common adverse events were fatigue and diarrhoea (Motzer et al, 2006a,b). These results induced US Food and Drug Administration (FDA) approval of Sunitinib for the treatment of advanced RCC. A large, randomized, phase 3 study demonstrated that Sunitinib significantly increased PFS and ORR compared with INF- in previous untreated patients with mRCC and its superiority as first-line therapy (Motzer et al, 2006b, 2007). In this multicentre study, 750 patients were randomized in 1:1 ratio to receive either Sunitinib or IFN, and Sunitinib (50 mg) was administered orally once daily in 6 weeks cycles consisting of 4 weeks of treatment followed by 2 weeks without treatment. After a median treatment duration of 11 months for Sunitib and 4 months for IFN- the median PFS was 11 months for the Sunitinib arm and 4 months for the IFN- arm (HR=0.415, p<0.0001) . The ORR was 31% versus 6% (p<0.00001). Four patients were considered to have had CR (complete response) with Sunitinib and two patients with IFN- ., and median duration of response was 12 months for Sunitinib and 10 months for IFN- . Therefore, Sunitinib showed acceptable safety profile, although most general adverse events of all grades occurred more frequently in the Sunitinib group, adverse events leading to withdrawal from the study occurred in 8% of cases with Sunitinib and 13% with IFN- . Data are not yet mature for the assessment of overall survival, but the investigators concluded that Sunitinib should be considered the new standard for the first line treatment of metastatic RCC and, based on these outcomes, the EAU guidelines recommend

D. Sorafenib In December 2005, the FDA approved Sorafenib for the treatment of patients with advanced RCC (Goodman et al, 2007). In European Union it is indicated as therapy in patients with advanced RCC refractory to cytokine therapy or who are unsuitable for such therapy; instead, in US it is indicated for patients with advanced RCC (Nexavar summary of product characteristics, 2006; Nexavar US prescribing information, 2007). The EAU guidelines recommend the use of Sorafenib as second-line therapy in mRCC. The approval was based on randomized trials evaluating Sorafenib, in particular in cases in progression after cytokine therapies. Sorafenib has been evaluated as monotherapy in four, phase I, trials with distinct schedules of administration: interrupted dosing and continuous administration (Strumberg et al, 2007). In these trials 173 patients with advanced solid tumours were enclosed. Sorafenib was generally well tolerated at dosing " 400 mg bid. The most frequently reported adverse events related to the drug were fatigue (40%), anorexia (35%), diarrhoea (34%) and rush handfoot skin reaction (25%). These adverse events were mainly < grade 3 (90%) and were resolved either reducing the dose or discontinuing Sorafenib. A treatment related hypertension was observed in 5-11% of cases. The maximal tolerated dose for Sorafenib was defined in these trials as 400 mg bid. The hypothesis is that reactions are related to EGFR and Raf inhibition, whereas hypertension to VEGF inhibition (Flaherty et al, 2006). A first phase II randomized discontinuation trial evaluated the effect of Sorafenib in 202 patients with advanced RCC (all with ECOG score 0 or 1; 75% clear RCC; 89% with prior nephrectomy; 76% with prior IL-2 or IF therapy) (Ratain et al, 2006). Patients received Sorafenib 400 mg bid during an initial run-in period and thereafter (12 weeks) cases with changes in tumour measurement < 25% were randomized to Sorafenib versus placebo for other 12 weeks. Patients with 25% tumour growth discontinued treatment whereas patients with 25% tumour shrinkage continued open-label Sorafenib. At 24 weeks, 50% of Sorafenib treated patients were progression-free versus 18% of the placebo group (p=0.007). Median PFS was 24 weeks in Sorafenib versus 6 weeks in placebo group (p=0.008). A randomized phase II trial of Sorafenib versus IFN in untreated metastatic RCC is ongoing to define the role of this drug in this setting (Rini, 2006). The clinical advantage of Sorafenib was confirmed in a multicentre, randomized, phase III trial, that compared Sorafenib with placebo as second-line therapy in patients with advanced metastatic RCC and, subsequently, randomized in a double-blind fashion to receive either Sorafenib or placebo.The PFS was longer in the Sorafenib arm (5.5 mo vs 2.8 mo) and the analysis demonstrated that Sorafenib reduce the risk of death 450

Cancer Therapy Vol 6, page 451! In a phase I trial, 24 patients with metastatic RCC received Bevacizumab plus Sorafenib. The combination showed preliminary evidence of antitumor activity but a dose-limiting toxicity related to hand-foot syndrome, anorexia, and fatigue, was reported (Sosman et al, 2008). Combining of anti-VEGF strategies with agents targeting other relevant mechanisms in advanced RCC have been also planned. A phase II trial analyzed the combination of Bevacizumab with Erlotinib (EGFR inhibitor). The trial showed that Erlotinib does not significantly add to the clinical efficacy of Bevacizumab (Bukowsy et al, 2006). A promising combination strategy considered antiVEGF drugs with Temsirolimus, a mammalian target of rapamycin inhibitor (PFS of 3.7 months versus 1.9 months, p=0.0001 in a phase III trial comparing Temsirolimus with IF in advanced RCC) (Hudes et al, 2007).

compared to placebo. Sorafenib was superior than placebo also in relation to CR (1 patient vs none), PR (10% vs 2%), SD (74% vs 53%) and PD (12% vs 37%). The most common adverse events were diarrhoea, fatigue, rash, hand-foot skin reactions, alopecia, nausea, and about 10% of patients discontinued the treatment because of these (Escudier et al, 2007).

E. Combination Sorafenib or Sunitinib

therapies

using

The rationale for targeting VEGF in advanced RCC is important, but also other factors, including PDGF, are able to influence the angiogenenetic process in this tumour. Therefore, enhanced antitumours activity, could be obtained by a simultaneous targeting of different factors involved in this process. Different preclinical studies suggest that IF has antiangiogenic properties and that combinating 2 or more agents with antiangiogenic properties may result in additive or synergistic antitumour activity (Rini, 2006). Clinical trials have been started to examine the possibility of combination targeted therapies in advanced RCC. Combination of Bevacizumab and IF versus IF monothrerapy has been evaluated in two randomized phase III trials as first line therapy in metastatic RCC (Rini et al, 2006). However these trials did not fully define the additive or synergic effects of this combination regimen due to the absence of a Bevacizumab monotherapy control arm. Two phase II trials combining sorafenib 400 mg bid with IF 3-times week have been reported (Gollob et al, 2006; Ryan et al, 2007). These trials suggest that this combination regimen can be safely used in untreated advanced RCC patients. Among 24 cases evaluable for response, the ORR by RECIST criteria was 42% (38% partial and 4% complete response); moreover 46% of cases showed stable disease.

V. Paradigmatic cases report Several case reports on extreme cases of advanced RCC treated by targeted therapies have been recently reported in the literature. These cases help also to suggest new possible indications of targeted therapies in RCC cases. Here, we present, as example, a case that shows the efficacy of anti-angiogenic targeted therapies as neoadjuvant treatment to surgery in advanced RCC. In March 2007, a 71 year old white male was diagnosed at CT scan as having a voluminous (8.0 cm in diameter) solid mass at left kidney level (Figure 2a). Moreover, the CT scan (chest, abdomen and pelvis) evidenced lymph-nodes involvement (maximal diameter 3 cm, at para-aortic and left renal vein level) and a thrombus at left renal vein and inferior vena cava level (Figure 3a). No other distant metastases were evidenced (also a bone scan was negative). ECOG performance status was 3. Family history for kidney or other neoplasm was negative.

Figure 2. (A) Pre-treatment CT scan showing a 9.0 cm solid mass at left kidney level. (B) Post Sorafenib treatment CT scan, showing a 9.0 cm solid mass at left kidney level with modification of its pathway

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s Figure 3.. (A) Pre-treatment CT scan showing a thrombus at left renal vein and inferior vena cava level. (B) Post Sorafenib treatment CT scan showing a reduction in thrombus extension and vena cava involvement.

In March 2007 the patient started a medical treatment with Sorafenib given orally at a dose of 400 mg daily. Treatment with sorafenib was well tolerated without side effects. There was also an improvement in ECOG performance status (score 2), starting after 3 months. In August 2007 a new CT scan confirmed: the presence of a 9.0 cm in diameter solid mass at left kidney level (Figure 2b); no modifications at lymph-nodes level; a reduction in thrombus extension, limited at left renal vein level without involvement of the inferior vena cava (Figure 3b). After 6 months of neoadjuvant treatment with sorafenib, in September 2007 the patient was submitted to an open left radical nephrectomy. No intra-operative and post operative complications were developed. Macroscopic examination at left kidney level showed a 9 x 7 cm solid mass involving the upper pole and the medial aspect of the kidney. The mass was well delimited by a pseudo-capsule. The renal capsule, the ureter and the renal vessels resulted no involved by the mass and, in particular, the renal vein resulted free from thrombus. Histological examination revealed that over 90% of the solid mass was substituted by necrotic tissue: the residual neoplastic component was diagnosed as a clear cell Renal Cell Carcinoma (RCC), Furhman grade 2. At pathology, all lymph-nodes removed resulted negative for neoplastic involvement. At now (February 2008) the patient is in good physical conditions; there is a normalization in ECOG performance status (score 1) and the quality of life is good. Treatment with sorafenib was interrupted after surgery. At 5 months from radical nephrectomy (February 2008) a new CT scan was negative for local progression or distant metastases. It is important to underline that the patient did not develop embolic events.

VI. Future points to be addressed Guidelines on how to treat advanced RCC as first line treatment or at failure as second line treatment, using targeted agents are emerging. The information that we presented supports the clinical rationale for targeting angiogenesis and the VEGF pathways and they show that this strategy is feasible and it can result in tumor shrinkage and PFS advantages. To better use this kind of targeted therapy, different points should be developed in the future: 1) the identification of the clinical characteristics of RCC patients that can be used to predict outcomes and responses to therapy; 2) the definition of the differences among different compounds; 3) the advantage of combination therapies; 4) the possibility of sequential treatments. The selection of patients who most likely to respond to targeted therapies, should be crucial to optimize the use of these agents in the clinical setting. Advanced RCC, prior nephrectomy, a clear cell histology were inclusion criteria in almost trials. Clear cell histology criterion was based on the biology of Von Hippel Lindau inactivation and subsequent VEGF hyperexpression, which is confirmed to this histologic type of RCC. In some trials, however, there have been reports of activity of anti-VEGF agents also in cases with non clear cell histology. This point should be better defined in future trials. The median PFS of 13 months obtained with VEGF targeted agents in metastatic RCC is significantly superior to that reported in other trials involving earlier therapies (Choueiri et al, 2008). The real value of PFS, however, may change in relation of the selected group of patients who is considered for the treatment. Predictive biomarkers of response should also be largely investigated and VEGF expression is an obvious candidate. Another important point is the statistically similar PFS of patients receiving VEGF targeted therapy for

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Cancer Therapy Vol 6, page 453! Moreover, considering that Sorafenib can target endothelial cells also through Raf pathways rather than through VEGFR pathways, it could be possible that Sorafenib may overcome a resistence of the tumour to VEGF inhibitors such as Sunitinib. All these suggestions strongly support the need for a direct comparison of targeted therapies. An Eastern Cooperative Oncology Group (ECOG) phase III trial, with patients randomized to Sorafenib versus Sunitinib versus placebo is ongoing. To understand the role and the advantage of a combination therapy in this setting, different questions should be addressed: 1) which target is most important? 2) which agent better inhibits each target? 3) Is there a synergic effect using combination therapy? 4) Which is the advantage to be reached (improvement in the rate and duration of clinical response, prevention of clinical resistence)? 5) Which population may better respond (first line therapy, cytokine resistant tumors, second or third line targeted therapy)? There are different concepts of combination targeted therapy for RCC. “Horizontal blockade” is when different target molecules are contemporaneally and individually inhibited: tumour cells (EGFR), endothelial cells (VEGFR) and pericyte (PDGF). To obtain this kind of blockade, specific single inhibitors or multitargeted agents (Sorafenib and Sunitinib) could be used. “Vertical blockade” is when a specific pathway is targeted and than inhibited at 2 or more different levels (for example VEGF and TK).

metastatic disease as first line or second line treatment (Choueiri et al, 2008). This finding supports the hypothesis that the biology of RCC that mediates response to anti VEGF therapies may not be influenced by prior exposure to cytokine therapies. This represent another important point that should be better defined. Recently, a possible treatment algorithm incorporating the rationale for use of various agents in advanced RCC has been proposed (Figure 4). The recent approval of different multitargeted Tirosyn Kinases inhibitors that both modify the natural history of RCC must induce to compare these drugs together. Also this approach helps to determine how best to use them in metastatic RCC. In this kind of analysis, Sunitinib bound 73 kinases in addition to VEGFR, whereas Sorafenib bound 40 additional kinases. The clinical relevance of inhibiting “off target” kinases has not been yet defined in terms of clinical advantage. Sorafenib, unlike Sunitinib, is also an inhibitor of Raf kinase. Also in this case, the relevance of EGFR and Raf system in RCC is controversial and therefore it remains uncertain whether Raf activity inhibition has a clinical role in the treatment of advanced RCC. It should be interesting to define a possible different role of Sorafenib, Sunitinib and Bevacizumab as first line therapy or after cytokine treatment resistance. The drugs should also be extensively examined and compared in the treatment of RCC patients with non clear tumours. It is possible that one drug over the other may have a potential role in the treatment of papillary and chromophobe RCC.

Figure 4. Treatment algorithm for advanced RCC. Reproduced from Porta et al, 2007 with kind permission from Kidney Cancer Journal.

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Sciarpa et al: Actual role of multitargeted therapy in renal cell carcinoma cG250 in patients with metastasized clear cell renal cell carcinoma. J Clin Oncol 23, 6540-6548. Bukowski RM (1997) Natural history and therapy of metastatic renal cell carcinoma: the role of interleukin-2. Cancer 80, 1198-1200. Bukowski RM, Kabbinavar F, Figlin RA, Flaherty K, Srinivas S, Vaishampayan U, Drabkin H, Dutcher J, Scappaticci F, McDermott D (2006) Bevacizumab with or without Erlotinib in metastatic renal cell carcinoma (RCC). J Clin Oncol 24, 222s. Porta C, Paglino C, Imarisio I (2007) Algorithm for Systemic Therapies in Metastatic Kidney Cancer in the Era of Targeted Therapies. Kidney Cancer Journal 5, 57-60. Cancer Pain Assessment and Treatment Curriculum Guidelines (1992) The Ad Hoc Committee on Cancer Pain of the American Society of Clinical Oncology [see comments]. J Clin Oncol 10, 1976-1982. Choueiri TK, Garcia JA, Elson P et al (2007) Clinical factors associate with outcome in patients with metastatic clear-cell renal cell carcinoma treated with vascular endothelial growth factor targeted therapy. Cancer 110, 543-50. Chow L, Eckhardt G (2007) Sunitinib:from rational design to clinical efficacy. J ClinOncol 7, 884-96. Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, Negrier S, Chevreau C, Solska E, Desai AA, Rolland F, Demkow T, Hutson TE, Gore M, Freeman S, Schwartz B, Shan M, Simantov R, Bukowski RM; TARGET Study Group (2007) Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 356, 125-134. Escudier B, Koralewski P, Pluzanska A, Ravaud A, Bracarda S, Szczylik C, Chevreau C, Filipek M, Melichar B, Moore N (2007) A randomized, controlled, double-blind phase III study (AVOREN) of bevacizumab/interferon-! 2a vs placebo/interferon-! 2a as first-line therapy in metastatic renal cell carcinoma (Abst 3). J Clin Oncol 25, No 18S (June 20 Supplement). European Urology Supplements (2008) Vol 7. Faivre S, Delbaldo C, Vera K, Robert C, Lozahic S, Lassau N, Bello C, Deprimo S, Brega N, Massimini G, Armand JP, Scigalla P, Raymond E (2006) Safety, pharmacokinetic, an antitumor activity of SU11248, a novel oral multitarget tyrosine kinase inhibitor, in patients with cancer. J Clin Oncol 24, 25-35. Ferlay J, Bray F, Pisani P (2000) Cancer incidence, mortality and prevalence worldwide. 2001;Version 1.0. GLOBOCAN. Ferrara N (2004) Vascular endothelial growth factor as a target for anticancer therapy. The Oncologist 9, 2-10. Flaherty KT, Sorafenib delivering a targeted drug to the right targets. Expert Rev Anticancer Ther 7, 617-626. Flanigan RC, Mickisch G, Sylvester R, Tangen C, Van Poppel H, Crawford ED (2004) Cytoreductive nephrectomy in patients with metastatic renal cancer: a combined analysis. J Urol 171, 1071-1076. Flanigan RC, Salmon SE, Blumenstein BA, Bearman SI, Roy V, McGrath PC, Caton JR Jr, Munshi N, Crawford ED (2001) Nephrectomy followed by interferon alfa-2b compared with interferon alfa-2b alone for metastatic renal cell cancer. N Engl J Med 345, 1655-59. Frank I, Blute ML, Cheville JC, Lohse CM, Weaver AL, Zincke H (2002) An outcome prediction model for patients with clear renal cell carcinoma treated with radical nephrectomy based on tumor stage, size, grade and necrosis: the SSIGN score. J Urol 168, 2395-2400. Fyfe G, Fisher RI, Rosenberg SA, Snzol M, Parkinson DR, Louie AC (1995) Results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interlukin-2 therapy. J ClinOncol 13, 688-696.

In RCC the horizontal blockade is intended to block tumour progression acting at different levels, either directly on tumour cells or indirectly on angiogenesis. The vertical blockade helps to resolve the problem of resistance that may develop during targeted therapy. In terms of resistance to targeted therapy for advanced RCC, there are no guidelines at present on how sequencing drugs or how to treat RCC after failure of first or second line agents. The hypothesis of no cross-reactivity among the different targeted therapies has set the basis for a strategy based on a sequential therapy. The determination of the correct sequence in using targeted therapy in RCC is a very important point that also support the rationale for continuing targeting the angiogenic pathways throughout progression and resistance of the RCC tumour.

VII. Final comment The large number of scientific articles published on the role of targeted therapy (in particular antiangiogenic/anti-VEGF) in advanced RCC, strongly underlines the actual relevance and the innovative role of this therapeutic approach in a setting in which other previous systemic treatments (chemiotherapy, radiotherapy and partially also cytokine inhibitors) failed. These new therapeutic approaches sustain the role of surgery, either in terms of cytoreductive nephrectomy or metastatectomy, as first treatment for advanced RCC. On the contrary the activity of these agents without prior nephrectomy remains undefined. It is possible to predict a future extension for the indications of these targeted therapies in RCC, for example as adjuvant to nephrectomy in high risk non metastatic cases, or as neoadjuvant, before surgery. We must also underline limits of these therapies. The novel agents are more likely to be cytostatic than cytotoxic and therefore we still fall to achieve complete and durable responses, but only advantages in terms of PFS or as disease stabilization can be expected.

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Tsuchiya N, Sato K, Akao T, Kakinuma H, Sasaki R, Shimoda N, Satoh S, Habuchi T, Ogawa O, Kato T (2001) Quantitative analysis of gene expressions of vascular endothelial growth factor-related factors and their receptors in renal cell carcinoma. Tohoku J Exp Med 195, 101-113. Wiesener MS, Münchenhagen PM, Berger I, Morgan NV, Roigas J, Schwiertz A, Jürgensen JS, Gruber G, Maxwell PH, Löning SA, Frei U, Maher ER, Gröne HJ, Eckardt KU (2001) Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1alpha in clear cell renal carcinomas. Cancer Res 61, 5215-5222. Wood CG (2003) The role of cytoreductive nephrectomy in the management of metastatic renal cell carcinoma. Urol Clin North Am 30, 581-88. Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL, Steinberg SM, Chen HX, Rosenberg SA (2003) A randomized trial with Bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N EnglJ Med 349, 427-434. Zisman A, Pantuck AJ, Wieder J, Chao DH, Dorey F, Said JW, deKernion JB, Figlin RA, Belldegrun AS (2002) Risk group assessment and clinical outcome algorithm to predict the natural history of patients with surgically resected renal cell carcinoma..J Clin Oncol 20, 4559-4566. Zisman A, Wieder JA, Pantuck AJ, Chao DH, Dorey F, Said JW, Gitlitz BJ, deKernion JB, Figlin RA, Belldegrun AS (2003) Renal cell carcinoma with tumor thrombus extension: biology, role of nephrectomy and response to immunotherapy. J Urol 169, 909-916.

Alessandro Sciarra

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Cancer Therapy Vol 6, page 457! Cancer Therapy Vol 6, 457-462, 2008

Canine hemangiosarcoma: A tumor of contemporary interest Review Article

Stuart C. Helfand Oncology, Oregon State University, Magruder Hall, Corvallis, Oregon, USA

__________________________________________________________________________________! *Correspondence: Stuart C. Helfand, D.V.M., Professor, Diplomate ACVIM (Oncology and Internal Medicine), Oregon State University, Magruder Hall, Corvallis, OR 97331, USA; Tel: 541-737-4830; Fax: 541-737-4818; e-mail: Stuart.Helfand@oregonstate.edu Key Words: Canine hemangiosarcoma, tumor, adjuvant chemotherapy, immunotherapy, receptor tyrosine kinase Abbreviations: galectin-3, (Gal-3); hemangiosarcoma, (HSA); histone deacetylase, (HDAC); inducible protein-10, (IP-10); interleukin12, (IL-12); liposome-encapsulated muramyl tripeptide phosphotidylethanolamine, (L-MTP-PE); receptor tyrosine kinase, (RTK); suberoylanilide hydroxamic acid, (SAHA) Received: 9 June 2008; electronically published: August 2008

Presented in the Theilen Tribute Symposium at UC Davis 31st May- 1st June 2008.

Summary Hemangiosarcoma is an endothelial cell-derived malignancy that continues to be a fatal cancer in dogs. Death is almost always due to local and systemic metastases despite numerous attempts to alter the clinical course with adjuvant chemotherapy. A benefit from doxorubicin-based chemotherapy, albeit modest, has been established unambiguously and represents the current standard of adjuvant care following surgical removal of hemangiosarcoma. Survival times, however, typically are not appreciably prolonged beyond six months. A small number of studies employing immunotherapy approaches indicate the potential to develop this modality for better tumor control. As more information detailing cellular and molecular aspects of hemangiosarcoma cell function is revealed, new opportunities for development of novel treatments, with implications for antiangiogenic approaches for cancer, are emerging. Targeting hemangiosarcoma cell surface adhesion molecules, differentiation molecules, and growth factor receptor pathways may provide the opportunities needed to impact on hemangiosarcoma growth and improve clinical outcome.

I. Introduction

II. Lessons learned from adjuvant chemotherapy

Dogs develop hemangiosarcoma (HSA) more frequently than any other species. Hemangiosarcoma originates from transformed endothelial cells and while endothelial malignancies such as angiosarcoma and Kaposi sarcoma are seen in humans, their incidence is low compared to canine HSA. At least 7% of all canine cancer is due to HSA and its profoundly aggressive behavior almost always portends an unfavorable outcome in affected dogs. The veterinary profession has singularly dealt with the challenge of this deadly cancer but despite the plethora of novel treatment strategies, HSA has refused to be tamed and remains a formidable clinical problem. Despite the frustrations associated with numerous unrewarding, yet novel treatment efforts for HSA, the quest to improve survival time has provided a unique opportunity to learn about endothelial cell biology including subcellular pathways and the process of angiogenesis, the formation of new blood vessels.

It has been more than 20 years since the first reports of adjuvant chemotherapy for canine HSA were published. Since then, numerous studies have appeared, all of which confirm several apparent truths. These include: 1) adjuvant chemotherapy extends the disease free interval and survival time following removal of the primary (splenic) HSA; 2) the benefit obtained by adjuvant chemotherapy is short lived; 3) adjuvant chemotherapy protocols should include doxorubicin. There is a consensus of opinion that chemotherapy given during the micrometastasis phase of HSA extends survival, but close inspection of various reports fails to show much difference in outcome regardless of the novel question studied. Doxorubicin-containing protocols rarely extend life much beyond six months compared to the two or three month survival time of dogs not receiving chemotherapy (Hammer et al, 1991; Sorenmo et al, 1993; Ogilvie et al,

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Helfand: Canine hemangiosarcoma: A tumor of contemporary interest 1996). More recent efforts to improve survival employed a variety of strategies including the destruction of intraabdominal metastasis by intraperitoneal administration of liposome-doxorubicin (Sorenmo et al, 2007), continuous low-dose oral chemotherapy (i.e., cyclophosphamide, etoposide, piroxicam) (Lana et al, 2007) and immunotherapy using a potentiated allogeneic HSA antigen vaccine combined with doxorubicin (U'Ren et al, 2007), all failed to improve survival time compared to that observed with systemically administered doxorubicin containing protocols. Survival times of canine HSA patients were similarly unaltered by modified adjuvant doxorubicin protocols testing an accelerated (dose intensified) protocol (Sorenmo et al, 2004) or the addition of a putatively antiangiogenic antibiotic minocycline (Sorenmo et al, 2000). Table 1 summarizes the median survival times reported in these studies. Death is always due to widespread metastasis, often affecting the lungs (Figure 1). Taken together, it is readily apparent that major breakthroughs in controlling canine HSA will not come from adjuvant chemotherapy alone.

III. Potential of immunotherapy Several lines of evidence indicate immunotherapy may play a role in suppressing HSA micrometastases. These include approaches that elicited both cell-mediated and humoral responses. In fact, the most impressive survival times reported for canine HSA patients were those described by Vail and colleagues in 1995 in which dogs were treated with the macrophage activator liposome-encapsulated muramyl tripeptide phosphotidylethanolamine (L-MTP-PE) concurrently with multiple chemotherapy cycles consisting of doxorubicin and cyclophosphamide. The median survival time for all 18 dogs receiving chemoimmunotherapy was 277 days, but of note were the eight dogs with stage I disease that had a median survival time of 425 days, although not significant in comparison to the placebo-treated dogs due to the small numbers. The recent report by U’ren and colleagues in 2007 provides evidence of the potential to elicit humoral immune responses to unknown HSA

Table 1. Median survival times of dogs with hemangiosarcoma treated with splenectomy and chemotherapy. Study

Year

Treatment

Wood and colleagues Hammer and colleagues

1998 1991

Sorenmo and colleagues Ogilvie and colleagues Lana and colleagues* Sorenmo and colleagues Sorenmo and colleagues Sorenmo and colleagues Lana and colleagues*

1993 1996 2007 2000 2004 2007 2007

splenectomy, no chemotherapy vincristine, doxorubicin, cyclophosphamide doxorubicin, cyclophosphamide doxorubicin doxorubicin doxorubicin, minocycline doxorubicin every 2 weeks pegylated doxorubicin intraperitoneal daily low dose etoposide, cyclophosphamide, piroxicam

Median survival time (days) 86 145 202 172 133 170 257(I)#, 210 (II), 107 (III) 131 178

*Same study comparing standard doxorubicin every three weeks vs. daily low dose etoposide, cyclophosphamide, and piroxicam. # Results reported by clinical stage in parenthesis.

Figure 1. Canine hemangiosarcoma pulmonary metastasis. Diffuse pulmonary metastatic hemangiosarcoma nodules are readily visible (numerous small white densities) in the thoracic radiograph of a dog presented for a penile hemangiosarcoma (left). At postmortem (right), the lungs are filled with large numbers of round maroon nodules comprised of metastatic hemangiosarcoma. A section of lung has been excised (center) to establish a hemangiosarcoma cell line from metastatic lesions (see Figure 3).

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Cancer Therapy Vol 6, page 459! normally triggered by the malignant cells (Akhtar et al, 2004; Dickerson et al, 2004). Figure 2 shows an example of canine HSA-induced neovascularization in a corneal angiogenesis assay. Furthermore, using a canine HSA xenograft model in which HSA was transplanted into immunoincompetent mice, we demonstrated unambiguously the potential for IL-12 to suppress growth of canine HSA (Akhtar et al, 2004). Although there are, as yet, not many studies exploring a role for immune modulation in controlling HSA, it appears that such strategies may offer promise, especially when combined with other modalities.

antigenic determinants following vaccination with allogeneic canine HSA cell lysates in combination with liposome containing (non-coding) DNA complexes (LDC). The inclusion of LDC with the tumor cell lysate was intended as an adjuvant as LDC reportedly trigger enhanced immune responses in a variety of vaccine protocols (U'Ren et al, 2007). While the survival time of the treated dogs was not much different than animals given chemotherapy alone, the development of specific humoral immune responses to the HSA vaccine is a promising step in overcoming immune tolerance. Our laboratory has investigated the potential to develop interleukin-12 (IL-12) as an adjuvant therapy for canine HSA. This line of research evolved from our interest in exploiting the immunotherapeutic potential of IL-12 coupled with the discovery that IL-12 also mediates antiangiogenic activity. Interleukin-12 is a potent inducer of cell-mediated immune responses making it attractive as part of immunotherapeutic strategies for cancer. It is a potent inducer of interferon-!, which in turn stimulates production of interferon inducible protein-10 (IP-10) and the chemokine Mig (monokine induced by interferon-!), both of which mediate angiostatic activity. Since HSA cells likely share functional properties in common with neoangiogenic endothelium within tumors, i.e., mitotically active endothelial cells that form new blood vessels within the tumor microenvironment, agents that inhibit angiogenesis may also be of value in suppressing HSA growth. Using a combination of in vitro and in vivo methods, we demonstrated that IL-12 could be targeted to adhesion molecules (i.e., "v#3 integrin) expressed by dividing endothelial cells comprising the neovasculature induced by canine HSA (and other tumor) cells. This in turn, resulted in marked suppression of vascular ingrowth

IV. Form provides clues to function We have just begun to scratch the surface in understanding the characteristics of the transformed cells that give rise to canine HSA. Learning more about them on a cellular and subcellular level will ultimately reveal clues as to where they may be vulnerable which in turn, will facilitate design of strategies that can lead to improved therapeutic outcomes. Information is gradually accruing relating to HSA cell dependency on external growth factors, expression of surface receptors that interact with the extracellular environment, signaling pathways, stem cells, and genetic abnormalities. A variety of contemporary research tools are helping with these discoveries, including cellular, molecular, protein, and genomic interrogation techniques. Table 2 shows a partial list of surface molecules reportedly expressed by canine HSA cells. Assuredly, more features of HSA cells will come to light as research progresses. Several of these have been the subjects of studies exploring unique therapeutic strategies.

Figure 2. Canine hemangiosarcoma cells are inducers of angiogenesis. In this corneal angiogenesis assay, a polyvinyl sponge containing canine hemangiosarcoma cells surgically placed into a corneal pocket of a BALB/c mouse elicited ingrowth of new blood vessels arising from the normal limbic vessel (bottom). After binding to receptors expressed on mature endothelial cells comprising the limbic vessel, proangiogenic proteins released by the hemangiosarcoma cells induce angiogenesis with arborized neovessels clearly homing towards the hemangiosarcoma cells, the source of the angiogenic stimulus. Details of this assay can be found in Dickerson et al (Dickerson et al, 2004).

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Helfand: Canine hemangiosarcoma: A tumor of contemporary interest Table 2. Surface molecules of potential interest in canine hemangiosarcoma. Molecule !v"3 integrin (adhesion molecule)! (Akhtar et al, 2004; Fosmire et al, 2004)! ICAM-1 (Thamm et al, 2006) c-kit (CD117) (Akhtar et al, 2004; Fosmire et al, 2004) VEGFRs (Akhtar et al, 2004) PDGFR (Helfand, unpublished) Galectin-3 (Johnson et al, 2007) CD34, CD133, c-kit (Lamerato-Kozicki et al, 2006) PTEN (cytoplasmic) (Dickerson et al, 2005)

Importance RGD targeting (cytokines, chemotherapy) Adhesion molecule Tyrosine kinase inhibition Tyrosine kinase inhibition, mAb target Tyrosine kinase inhibition Gal-3 inhibition (modified citrus pectin, lactulosyl-L-leucine) HSA stem cells Tumor suppressor protein

Another recent report by Lamerato-Kozicki and colleagues in 2006 was the first to clarify that a small subset (i.e., <1%) of canine HSA cells coexpress surface markers of hematopoietic stem cells (CD133, CD34, c-kit) with commitment to endothelial differentiation. This study lends credence to the idea that HSA is really a bone marrow derived malignancy with progenitor cells arising from lineage committed stem cells that circulate and eventually give rise to HSA in specific organs. The significance of this finding is not trivial because it implies that complete eradication of HSA in affected dogs will require elimination of these apparent HSA stem cells. Cancer stem cells have proven to be the most resilient and resistant to therapy and are capable of regenerating a tumor even from a residual microscopic tumor population. This may help to explain, in part, the poor results obtained with chemotherapy of HSA in the microscopic disease setting that is routine after splenectomy for a primary splenic HSA. Information regarding the HSA genome is gradually emerging as well, which may facilitate identification of numerous genes in tumor cells that are either over- or under-expressed compared to non-cancerous endothelial cells. The advent of genomics, sequencing of the entire canine genome, and commercially available canine gene chips that allow vast numbers of genes to be examined in microarrays, is beginning to reveal the complexity of canine HSA at the gene level. To this end, splenic HSAs from Golden retrievers, a breed at above average risk for developing this cancer, have recently been found to have a unique pattern of gene under expression (Dr. Jaime Modiano, personal communication 2008). It is tempting to wonder if some of these genes may actually code for tumor suppressor proteins that may have been silenced either as a consequence or as a cause of malignant transformation. The single case report of a dog with splenic HSA that was treated with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), that lived >1000 days post splenectomy, more

As mentioned earlier, the author’s laboratory investigated a novel approach targeting IL-12 to the adhesion molecule !v"3 integrin by molecular engineering of a fusion protein consisting of the peptide ligand for !v"3 integrin (RGD4C) combined with IL-12 (Dickerson et al, 2004). This strategy resulted in a vastly superior anti-antiangiogenic effect against neovascularization induced by canine HSA (and other tumor histologies) compared to that observed with untargeted IL-12. More importantly, IL-12 suppressed growth of canine HSA in a xenograft model, but the !v"3 integrin-targeted IL-12 was vastly more potent than untargeted IL-12 in suppressing the growth of tumors in a murine neuroblastoma model (Dickerson et al, 2004). Taken together, it would seem that there is merit in pursuing novel therapeutic constructs that target !v"3 integrin. An informative review of this concept was recently published describing benefits of targeting a variety of molecules capable of arresting tumor cell growth to !v"3 integrin including radioisotopes, TNF-!, doxorubicin, IL-12, and others (Temming et al, 2005). We are continuing to investigate this approach in our laboratory for canine HSA. Johnson and colleagues chose to explore targeting galectin-3 (Gal-3) they believed to be expressed on the surface of canine HSA cells (Johnson et al, 2007). Gal-3 is a member of a group of surface carbohydrate binding proteins, overexpressed in numerous human malignancies, and participates in a variety of cellular processes important to cancer including cell differentiation, cell-cell and extracellular matrix adhesions, metastasis, and apoptosis (Johnson et al, 2007). It also participates in angiogenesis providing a rationale to examine it as a target in canine HSA. The authors identified Gal-3 expression on naturally occurring canine HSA biopsies and showed that proliferation of a Gal-3 positive murine HSA cell line could be inhibited in vitro by Gal-3 inhibitors. Clearly, more needs to be done to determine if Gal-3 blockade can be developed as a meaningful therapy for canine HSA, but the novelty of this approach bears obvious consideration.

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Cancer Therapy Vol 6, page 461! strategies will continue to come forth. Most likely, progress will be the result of multimodality interventions as canine HSA has proven itself to be a stubborn foe.

than four times longer than most dogs with this malignancy, may possibly be explained on the basis of upregulation of silenced tumor suppressor genes by the SAHA treatment (Cohen et al, 2004). HDAC inhibitors are a class of new anticancer agents capable of inhibiting promiscuous histone deacetylation in cancer cells that results in gene silencing. There are alternative explanations for the favorable results in this case report, such as the potential for a good outcome regardless of treatment as this dog’s tumor was reported to be low grade (Thamm, 2005). There is continuing interest in developing HDAC inhibitors for canine HSA and the example of genomic investigation of canine HSA in Golden retrievers will be instructional in exploring the effects of HDAC inhibitors on HSA from a mechanistic perspective. Since we discovered transcripts in canine HSA cells that encode c-kit and VEGFR-1 and -2 as well as their respective ligands, stem cell factor and VEGF (Akhtar et al, 2004; Fosmire et al, 2004), we have been interested in examining the possibility that these cell surface receptors, members of the receptor tyrosine kinase (RTK) family, may be important players in this malignancy. Much has been written about RTKs and their role in various malignancies. Briefly, RTKs are at the interface of the cell and its extracellular environment and respond to external stimuli by triggering intracellular growth promoting pathways (London, 2004). In health, they are protooncogenes in which their activity is tightly regulated, but numerous transforming mutations have been reported in a variety of cancers in which they function as oncogenes. Indeed, many malignancies appear to be addicted to mutated tyrosine kinases as a means to promote their immortality. Activated RTKs (as well as cytoplasmic tyrosine kinases), rely on phosphorylation of tyrosine residues for their activity (London, 2004). We are continuing to explore the role of certain RTKs in canine HSA and have found that PDGFR-!, an RTK important in endothelial differentiation and vascular development (Zhu, 2006), is expressed by canine HSA cells. Development of PDGFR-! antagonists is an active area of cancer research because of the potential to arrest neoangiogenesis in a broad range of cancers. In regards to HSA, this concept is entirely compatible with the idea that canine HSA is a malignancy of primitive endothelial cells (Fosmire et al, 2004). The ligand for PDGFR-! is PDGFBB and when stimulated by PDGF-BB, PDGFR-! is activated through phosphorylation of various tyrosine residues. Figure 3 shows a western blot illustrating activation (i.e., phosphorylation) of PDGFR-! in response to stimulation by exogenous PDGF-BB in a canine HSA cell line developed in the author’s laboratory. We are continuing to examine the importance of this pathway in canine HSA. It would seem that we are now finally able to begin to develop potentially meaningful therapeutic approaches based on specific information about the composition of HSA cells. The stage is set through the processes of discovery and innovation, for advancements that are likely to improve the prognosis for dogs with HSA. As more is revealed about the dysregulated processes driving canine HSA, it is likely that previously unimaginable treatment

Acknowledgments The author would like to thank numerous collaborators and colleagues for their contributions to various aspects of the hemangiosarcoma research summarized here including Drs. Erin Dickerson, Jaime Modiano (and his laboratory team), Nasim Akhtar, Robert Auerbach, Matthew Breen, John Wojcieszyn, Valerie MacDonald, and Michelle Turek. The author would also like to thank Marcia Padilla, Wade Edris, and Kevin Marley for their excellent contributions to some of the studies mentioned in this review. Support for studies from the Helfand laboratory came from the National Institutes of Health (R01 CA86264), Morris Animal Foundation (D03CA-71), AKC Canine Health Foundation (2025), Midwest Athletes Against Childhood Cancer, and the University of Wisconsin School of Veterinary Medicine Companion Animal Fund.

Figure 3. Response of PDGFR-! to stimulation by PDGF-BB in a canine hemangiosarcoma cell line (Rio-HSA). Cell lysates from hemangiosarcoma cells (Rio-HSA) derived from a pulmonary hemangiosarcoma metastatic lesion in a dog (Figure 1) were examined by western blot analysis following overnight culture in serum starved medium without (0 min) or with PDGFBB (30 ng/ml) after 10 (middle lane) and 60 minutes (right lane) and probed with an antibody specific for canine PDGFR-! phosphotyrosine position 857 located in the kinase domain. The cellular response is normal in this line in that the cells were not autophosphorylated (time 0), but responded rapidly to PDGF-BB stimulation by phosphorylating tyrosine after 10 minutes with down regulation of the phosphotyrosine response by 60 minutes despite further stimulation. As a loading control, the blot was stripped and reprobed with an antibody (Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes total PDGFR-! regardless of phosphorylation status (bottom).

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Helfand: Canine hemangiosarcoma: A tumor of contemporary interest Efficacy and toxicity of a dose-intensified doxorubicin protocol in canine hemangiosarcoma. J Vet Intern Med 18, 209-213. Sorenmo KU, Jeglum KA, Helfand SC (1993) Chemotherapy of canine hemangiosarcoma with doxorubicin and cyclophosphamide. J Vet Intern Med 7, 370-376. Temming K, Schiffelers RM, Molema G, Kok RJ (2005) RGDbased strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Resist Updat 8, 381-402. Thamm DH (2005) SAHA and hemangiosarcoma: another view. Vet Comp Oncol 3, 101-101. Thamm DH, Dickerson EB, Akhtar N, Lewis R, Auerbach R, Helfand SC, MacEwen EG (2006) Biological and molecular characterization of a canine hemangiosarcoma-derived cell line. Res Vet Sci 81, 76-86. U'Ren LW, Biller BJ, Elmslie RE, Thamm DH, Dow SW (2007) Evaluation of a novel tumor vaccine in dogs with hemangiosarcoma. J Vet Intern Med 21, 113-120. Vail DM, MacEwen EG, Kurzman ID, Dubielzig RR, Helfand SC, Kisseberth WC, London CA, Obradovich JE, Madewell BR, Rodriguez C, Fidel J, Susaneck S, Rosenberg M (1995) Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) adjuvant immunotherapy for splenic hemangiosarcoma in the dog: a randomized multi-institutional clinical trial. Clin Cancer Res 1, 1165-1170. Wood CA, Moore AS, M GJ, Ablin LA, Berg RJ, Rand WM (1998) Prognosis for dogs with stage I or II splenic hemangiosarcoma treated by splenectomy alone: 32 cases (1991-1993). J Am Anim Hosp Assoc 34, 417-421. Zhu Z (2006) PDGFR!: a multifaceted player in vascular and hematopoietic development. Blood 108, 1788-1789.

References Akhtar N, Padilla ML, Dickerson EB, Steinberg H, Breen M, Auerbach R, Helfand SC (2004) Interleukin-12 inhibits tumor growth in a novel angiogenesis canine hemangiosarcoma xenograft model. Neoplasia 6, 106-116. Cohen LA, Powers B, Amin S, Desai D (2004) Treatment of canine haemangiosarcoma with suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. Vet Comp Oncol 2, 243-248. Dickerson EB, Akhtar N, Steinberg H, Wang Z-Y, Lindstrom MJ, Padilla ML, Auerbach R, Helfand SC (2004) Enhancement of the antiangiogenic activity of interleukin-12 by peptide targeted delivery of the cytokine to avb3 integrin. Mol Cancer Res 2, 663-673. Dickerson EB, Thomas R, Fosmire SP, Lamerato-Kozicki AR, Bianco SR, Wojcieszyn JW, Breen M, Helfand SC, Modiano JF (2005) Mutations of phosphatase and tensin homolog deleted from chromosome 10 in canine hemangiosarcoma. Vet Pathol 42, 618-632. Fosmire SP, Dickerson EB, Scott AM, Bianco SR, Pettengill MJ, Meylemans H, Padilla M, Frazer-Abel AA, Akhtar N, Getzy DM, Wojcieszyn J, Breen M, Helfand SC, Modiano JF (2004) Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium. Lab Invest 84, 562-572. Hammer AS, Couto CG, Filppi J, Getzy D, Shank K (1991) Efficacy and toxicity of VAC chemotherapy (vincristine, doxorubicin, cyclophosphamide) in dogs with hemangiosarcoma. J Vet Intern Med 5, 160-166. Johnson KD, Glinskii OV, Mossine VV, Turk JR, Mawhinney TP, Anthony DC, Henry CJ, Huxley VH, Glinsky GV, Pienta KJ, Raz A, Glinsky VV (2007) Galectin-3 as a potential therapeutic target in tumors arising from malignant endothelia. Neoplasia 9, 662-670. Lamerato-Kozicki A, R, Helm KM, Jubala CM, Cutter GC, Modiano JF (2006) Canine hemangiosarcoma originates from hematopoietic precursors with potential for endothelial differentiation. Exp Hematol 34, 870-878. Lana S, U'ren L, Plaza S, Elmslie R, Gustafson D, Morley P, Dow S (2007) Continuous low-dose oral chemotherapy for adjuvant therapy of splenic hemangiosarcoma in dogs. J Vet Intern Med 21, 764-769. London C (2004) Kinase inhibitors in cancer therapy. Vet Comp Oncol 2, 177-193. Ogilvie GK, Powers BE, Mallinckrodt CH, Withrow SJ (1996) Surgery and doxorubicin in dogs with hemangiosarcoma. J Vet Intern Med 10, 379-384. Sorenmo K, Duda L, Barber L, Cronin K, Sammarco C, Usborne A, Goldschmidt M, Shofer F (2000) Canine hemangiosarcoma treated with standard chemotherapy and minocycline. J Vet Intern Med 14, 395-398. Sorenmo K, Samluk M, Clifford C, Baez J, Barrett JS, Poppenga R, Overley B, Skorupski K, Oberthaler K, Van Winkle T, Seiler G, Shofer F (2007) Clinical and pharmacokinetic characteristics of intracavitary administration of pegylated liposomal encapsulated doxorubicin in dogs with splenic hemangiosarcoma. J Vet Intern Med 21, 1347-1354. Sorenmo KU, Baez JL, Clifford CA, Mauldin E, Overley B, Skorupski K, Bachman R, Samluk M, Shofer F (2004)

Stuart C. Helfand

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Cancer Therapy Vol 6, page 463! Cancer Therapy Vol 6, 463-476, 2008

Early breast cancer: A review Review Article

Kyriakos Kalogerakos1,*, Chrisostomos Sofoudis1, Nikolaos Baltayiannis2 1 2

Breast Unit Metaxa Cancer Hospital, Piraeus Greece Department of Thoracic Surgery Metaxa Cancer Hospital, Piraeus, Greece.

__________________________________________________________________________________! *Correspondence: Kyriakos Kalogerakos, Metaxa Cancer Hospital, 51 Botasi, Piraeus, Greece; e-mail: ageliki@diagoras-travel.gr Key words: Early breast cancer, early breast cancer diagnosis, early breast cancer treatment, minimally invasive procedures Abbreviations: 5-fluorouracil, adriamycine, cyclophosphamide, (FAC); 5-fluorouracil, epirubicin, cyclophosphamide, (FEC); Advanced Breast Biopsy Instrumentation system, (ABBI); American Joint Committee on Cancer, (AJCC); American Society of Clinical Oncology, (ASCO); Aromatase inhibitors, (AIs) axillary lymph node dissection, (ALND); Axillary lymph node dissection, (ALND); breast conserving surgery, (BCS); breast conserving therapy, (BCT); clinical breast examination, (CBE); cyclophosphamide, methotrexate, 5fluorouracil , (CMF); ductal carcinoma in situ, (DCIS); Early Breast Cancer Trialists' Collaborative Group, (EBCTCG); estrogen receptor, (ER); extensive intraductal carcinoma, (EIC); fine needle aspiration, (FNA); Focused Ultrasound Ablation, (FUA); Food and Drug Administration, (FDA); Magnetic resonance imaging, (MRI); Minimally Invasive Breast Biopsy, (MIBB); progesterone receptor, (PR); radiationtherapy, (RT); Radiofrequency Ablation, (RFA); selective estrogen receptor modulator, (SERM); sentinel lymph node biopsy, (SLNB); sentinellymph node, (SLN); tumor, nodal, metastasis, (TNM) Received: 2 July 2008; Revised: 4 August 2008 Accepted: 21 August 2008; electronically published: September 2008

Summary Breast cancer remains a common disease throughout the world. The prognosis of early breast cancer is generally favorable. Especially, ductal carcinoma in situ has been regarded as a non-life-threatening disease. Therefore, early diagnosis and early onset of the treatment has been important. Early age at menarche, late age at first birth and late age at menopause are related to breast cancer risk. Examination by mammography and ultrasonography is still the most effective means of detection for premenopausal and postmenopausal women, respectively. Additionally, there have been important advances in MRI, sentinel lymph node biopsy, breast-conserving surgery, partial breast irradiation, neoadjuvant systemic therapy and adjuvant systemic therapy. Another approach is to treat primary tumors without surgery. For this purpose, several new minimally invasive procedures, including radiofrequency ablation, interstitial laser ablation, focused ultrasound ablation and cryotherapy, are currently under development and may offer effective tumor management and provide treatment options that are psychologically and cosmetically more acceptable to the patients than are traditional surgical therapies. Here we review new knowledge about early breast cancer the last years.

(Welsh Cancer Intelligence and Surveillance Unit (WCISU), 2004; Office for National Statistics, 2005). One-third of new breast cancer cases are aged 70 years or over. The likelihood of diagnosis increases with age, doubling about every 10 years until the menopause, when the rate of increase slows dramatically (Quinn et al, 2001, Mcpherson et al, 2000). In Greece the incidence is more than 3000 per year and the annual increase over the last 20 years has been 1.3 %. The disease is unusual before the age of 40, but increases rapidly thereafter. Although the incidence of breast cancer has been increasing in Greece its mortality rate has been decreasing. This decline in mortality could be due to widespread use of mammography, advances in evaluation techniques and

I. Introduction Breast cancer continues to be the most commonly diagnosed cancer in women in the United States, accounting for 26% of all female cancers (Jemal et al, 2006). In 2007, approximately 178, 480 women and 2, 030 men will be diagnosed with invasive breast cancer and 40, 460 women and 480 men will die from the disease (Ries et al, 1975-2000; American Cancer Society. Cancer Facts and Figures 2004 and 2005). Additional breast cancer is the most common cancer amongst women in England and Wales: 38, 651 women were diagnosed as having breast cancer in 2003. The most common age at diagnosis was between 55 and 59 years, although the median age was between 60 and 64 years

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Kalogerakos et al: Early breast cancer: A review With stage 0, the cancer is non-invasive, meaning it has not spread to surrounding normal tissue (sometimes called carcinoma !"#$!%&). In stage I cancer, the tumor is two centimeters in size or smaller and has not spread outside the breast. And, in stage II, either: !!There is no tumor in the breast, but cancer is found in the axillary lymph nodes (nodes under the arms); or, !!The tumor is two centimeters or smaller and has spread to the axillary lymph nodes; or, !!The tumor is two-to-five centimeters and has spread to the axillary lymph nodes; or, !!The tumor is larger than five centimeters and has not spread to the axillary lymph nodes or, !!The number of lymph nodes involved with cancer is not more than three (Table 1).

effective adjuvant treatment. Despite this, approximately 1600 patients die from breast cancer every year in Greece. TNM stages I, II and IIIA are the "early" stages of invasive carcinoma and most of these tumors are traditionally considered operable. More than 90% of breast cancer diagnoses are made early in the disease. Early-stage breast cancer is potentially curable with surgery, radiation therapy and systemic therapy (Mirshahidi and Abraham, 2004). In patients with early breast cancer who receive appropriate treatment, 5-year survival rates are in excess of 75%. This article is a review for early-stage breast cancer.

II. Definition The term "early breast cancer" refers to breast cancer in stages 0, I and II at the time of diagnosis (AJCC, 2002). Table 1. TNM classification Primary tumor (T).

TX-Primary tumor cannot be assessed T0-No evidence of primary tumor Tis-Carcinoma in situ !! Tis (DCIS)-Intraductal carcinoma in situ !! Tis (LCIS)-Lobular carcinoma in situ !! Tis (Paget's)-Paget's disease of the nipple with no tumor; tumor-associated Paget's disease is classified according to the size of the primary tumor T1-Tumor 2 cm or less in greatest dimension !! T1mic-Microinvasion 0.1 cm or less in greatest dimension !! T1a-Tumor more than 0.1 but not more than 0.5 cm in greatest dimension !! T1b-Tumor more than 0.5 cm but not more than 1 cm in greatest dimension !! T1c-Tumor more than 1 cm but not more than 2 cm in greatest dimension T2-Tumor more than 2 cm but not more than 5 cm in greatest dimension T3-Tumor more than 5 cm in greatest dimension T4-Tumor of any size with direct extension to (a) chest wall or (b) skin, only as described below: !! T4a-Extension to chest wall !! T4b-Edema (including peau d'orange) or ulceration of the breast skin, or satellite skin nodules confined to the same breast !! T4c-Both (T4a and T4b) !! T4d-Inflammatory carcinoma Note: Dimpling of the skin, nipple retraction, or any other skin change except those described for T4b and T4d may occur in T1-3 tumors without changing the classification. Regional lymph nodes (N): Clinical classification NX-Regional lymph nodes cannot be assessed (eg, previously removed) N0-No regional lymph node metastases N1-Metastasis to movable ipsilateral axillary lymph nodes(s) N2-Metastasis to ipsilateral axillary lymph node(s) fixed or matted, or in clinically apparent ipsilateral internal mammary nodes in the absence of evident axillary node metastases !! N2a-Metastasis to ipsilateral axillary lymph node(s) fixed to one another (matted) or to other structures !! N2b-Metastasis only in clinically apparent (as detected by imaging studies [excluding lymphoscintigraphy] or by clinical examination or grossly visible pathologically) ipsilateral internal mammary nodes in the absence of evident axillary node metastases N3-Metastasis to ipsilateral infraclavicular lymph node(s) with or without clinically evident axillary lymph nodes, or in clinically apparent ipsilateral internal mammary lymph node(s) and in the presence of clinically evident axillary lymph node metastases, or metastasis in ipsilateral supraclavicular lymph nodes with or without axillary or internal mammary nodal involvement !! N3a-Metastasis to ipsilateral infraclavicular lymph node(s) !! N3b-Metastasis to ipsilateral internal mammary lymph node(s) and clinically apparent axillary lymph nodes !! N3c-Metastasis in ipsilateral supraclavicular lymph nodes with or without axillary or internal 464

Cancer Therapy Vol 6, page 465! mammary nodal involvement Regional lymph nodes: Pathologic classification (pN)-Classification is based upon axillary lymph node dissection (ALND) with or without sentinel lymph node dissection (SLND). Classification based solely on SLND without ALND should be designated (sn) [eg, pN0 (i +) (sn)). pNX-Regional lymph nodes cannot be assessed (eg, previously removed, or not removed for pathologic study) pN0-No regional lymph node metastasis; no additional examination for isolated tumor cells (ITCs, defined as single tumor cells or small clusters not greater than 0.2 mm, usually detected only by immunohistochemical or molecular methods but which may be verified on hematoxylin and eosin (H&E) stains. ITCs do not usually show evidence of malignant activity [eg, proliferation or stromal reaction]) !! pN0 (i -)-No histologic nodal metastases and negative by immunohistochemistry (IHC) !! pN0 (i +)-No histologic nodal metastases but positive by IHC, with no cluster greater than 0.2 mm in diameter !! pN0 (mol -)-No histologic nodal metastases and negative molecular findings (by reverse transcriptase polymerase chain reaction, RT-PCR) !! pN0 (mol +)-No histologic nodal metastases, but positive molecular findings (by RT-PCR) pN1-Metastasis in 1 to 3 ipsilateral axillary lymph node(s) and/or in internal mammary nodes with microscopic disease detected by SLND but not clinically apparent !! pN1mi-Micrometastasis (greater than 0.2 mm, none greater than 2.0 mm) !! pN1a-Metastasis in 1 to 3 axillary lymph nodes !! pN1b-Metastasis to internal mammary lymph nodes with microscopic disease detected by SLND but not clinically apparent !! pN1c-Metastasis in 1 to 3 ipsilateral axillary lymph node(s) and in internal mammary nodes with microscopic disease detected by SLND but not clinically apparent. If associated with more than 3 positive axillary nodes, the internal mammary nodes are classified as N3b to reflect increased tumor burden. pN2-Metastasis in 4 to 9 axillary lymph nodes or in clinically apparent internal mammary lymph nodes in the absence of axillary lymph nodes !! pN2a-Metastases in 4 to 9 axillary lymph nodes (at least one tumor deposit >2 mm) !! pN2b-Metastasis in clinically apparent internal mammary lymph nodes in the absence of axillary lymph nodes pN3-Metastasis in 10 or more axillary lymph nodes, or in infraclavicular lymph nodes, or in clinically apparent ipsilateral internal mammary lymph nodes in the presence of one or more positive axillary nodes; or in more than three axillary lymph nodes with clinically negative microscopic metastasis in internal mammary lymph nodes; or in ipsilateral supraclavicular lymph node(s) !! pN3a-Metastasis in 10 or more axillary lymph nodes (at least one tumor deposit greater than 2.0 mm), or metastasis to the infraclavicular lymph nodes !! pN3b-Metastasis in clinically apparent ipsilateral internal mammary lymph nodes in the presence of one or more positive axillary nodes; or in more than three axillary lymph nodes with microscopic metastasis in internal mammary lymph nodes detected by SLND but not clinically apparent !! pN3c-Metastasis in ipsilateral supraclavicular lymph node(s) Distant metastasis (M) MX-Distant metastasis cannot be assessed M0-No distant metastasis M1-Distant metastasis STAGE GROUPINGS Stage 0-Tis N0 M0 Stage I-T1 N0 M0 (including T1mic) Stage IIA-T0 N1 M0; T1 N1 M0 (including T1mic); T2 N0 M0 Stage IIB-T2 N1 M0; T3 N0 M0 Stage IIIA-T0 N2 M0; T1 N2 M0 (including T1mic); T2 N2 M0; T3 N1 M0; T3 N2 M0 Stage IIIB-T4 Any N M0 Stage IIIC-Any T N3 M0 Stage IV-Any T Any N M1

diagnosis, the more likely that a genetic component may be involved. About 5-10% of breast cancer is thought to be linked to changes (mutations)in certain genes. The most common are those of the BRCA 1 and BRCA 2 genes. Women with mutations in BRCA 1 or BRCA 2 have a high risk of

III. Risk factors Women with a family history of breast cancer should obtain as much information as possible about those relatives, including age at onset and type of cancer. The risk of breast cancer development related to family history increases with the number of affected relatives, specific lineage and age at diagnosis. The younger the age at 465

Kalogerakos et al: Early breast cancer: A review is an additional tool to further define abnormalities detected on CBE or mammography (Figure 1). Several studies have reported that mammographic screening reduces breast cancer mortality by 23% (Vachon et al, 2007). Digital mammography employs detection software that can highlight suspicious lesions in the breast not initially seen by a radiologist. Magnetic resonance imaging (MRI) is recommended as a screening tool for women who have a 20%-25% or greater increased lifetime risk of breast cancer. That includes women with a strong family history of breast cancer and women who are survivors of a previous malignancy that was treated with chest radiation therapy (Kaiser et al, 2008). MRI is not routinely indicated for women with a personal history of breast cancer, despite a 5%-10% increase in risk of a second primary cancer in the first 10 years after diagnosis, as the use of adjuvant chemotherapy and/or hormonal therapy significantly decreases overall risk to less than 5% (Hazard and Hansen, 2007).

developing breast cancer, ovarian cancer and several other types of cancer during their lifetimes. However, most cases of breast cancer occur “by chance”. The causes are still unknown, but there is probably a combination of factors including lifestyle factors, environmental factors and hormone factors. A list of several risk factors for breast cancer are shown in Table 2 (Mcpherson et al, 2000; Ceschi et al, 2007; Evans and Howell, 2007; Kiley and Hammond, 2007; Pruthi et al, 2007; Vitiello et al, 2007).

IV. Diagnosis Early breast cancer does not usually cause pain. When the cancer grows, it causes changes in the size or shape of the breast: a lump or thickening may be noticeable. In advanced cases the tumour can show signs of ulceration of the skin and fixation to the chest wall and in the worst cases large lymph nodes may be present (Reeder, 2007; Albrand and Terret, 2008; Rolz-Cruz and Kim, 2008). If any of these symptoms appears a proper investigation should be initiated. The “triple diagnosis” includes clinical examination, mammography and/or ultrasonography and fine-needle aspiration for cytology or coreneedle biopsy for histopathological examination (Soares and Johnson, 2007). Mammography provides radiographic images of the breasts with at least two sets of images, the mediolateral oblique and cranial-caudal views. It remains the most reliable and widely used method of breast cancer screening. Radiation exposure to the breast and surrounding structures is limited to one rad per breast when performed with a modern mammography unit. Ultrasonography, another imaging tool, uses sound waves that pass through a gel-covered skin probe to determine whether nodules or densities found on a mammogram or physical examination are solid or cystic. The benefit of total breast ultrasound continues to be studied and it is not considered a replacement for screening mammography but

Table 2. Risk factors for breast cancer Elderly Developed country Age at menarche before 11years Age at menopause after 54 years Age at first full pregnancy in early 40s Family history Previous benign disease (atypical hyperplasia) Cancer in the other breast Diet with high intake of saturated fat Body mass index >35 Alcohol consumption (excessive intake) Exposure to ionising radiation Oral contraceptives Hormone replacement therapy Diethylstilbestrol (during pregnancy)

Figure 1. Mammography: Early cancer of the left breast.

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Cancer Therapy Vol 6, page 467! Today a growing number of breast lesions, visible on MRI only, are being detected, posing diagnostic difficulties. Since the development of so-called “breast biopsy coils” MRI-guided percutaneous large-core or vacuum-assisted needle biopsy has become available in some selected centers with success (Perlet et al, 2006).

V. Minimal invasive diagnosis For a long time, open surgical breast biopsy after needlewire localization was considered to be the standard diagnostic procedure for nonpalpable lesions. But now the international guidelines state that at least 90% of breast cancer patients should have received a diagnosis of malignancy before entering the operating room (Mastology EESo. EUSOMA Guidelines. 2005, 2006). Several different percutaneous biopsy techniques are applied to obtain material of nonpalpable lesions: fine needle aspiration (FNA), large-core needle biopsy and vacuumassisted needle biopsy. FNA is a well-established tool for the evaluation of palpable breast lumps but it can’t to distinguish between invasive and in situ cancer and frequently we take inadequate sampling and we have false-negative rates (Wells, 1995). These problems with the application of FNA have led to the introduction of large-core needle biopsy for the diagnosis of nonpalpable breast lesions. Large-core needle biopsy is less operator-dependent than FNA. It allows identification of an invasive component additional it facilitates the assessment of tumor grade and provides sufficient material for additional immunochemistry staining. Diagnostic accuracy of large-core needle biopsy is high 93-99%, whereas falsepositive results are extremely rare (Verkooijen, 2002). However, in some cases, the severity of the disease is underestimated. In up to 40%-50% of needle biopsies containing high-risk lesions these are underestimated. In an attempt to reduce disease underestimate rates, vacuum-assisted breast biopsy was introduced in 1995. With this technique, tissue samples are acquired by using a single insertion of a probe (11-gauge) and vacuum suction to retrieve core specimens. Several studies have showed that vacuum-assisted needle biopsy can reduce the high-risk and some advocate vacuum-assisted needle biopsy (Kettritz et al, 2004). Ultrasound guidance is the technique of first choice for percutaneous biopsy and can be applied for image guidance of FNA, large-core needle biopsy and vacuumassisted needle biopsy. But some nonpalpable lesions cannot be identified by ultrasound. For these types of lesions, stereotaxis is used. With stereotactic imaging, two digital images of the targeted lesion are taken at +15o and -15o from the central axis. This allows precise calculation of the coordinates of the lesion. With this information, a biopsy needle can be inserted into the lesion and while the biopsies are being harvested, repeat stereotactic images can be taken to confirm the position of the needle. Stereotactic image guidance can be provided either by add-on devices, which are attached to standard mammography units, or dedicated prone biopsy tables. With the latter, the patient is positioned in the prone position on a biopsy table while her affected breast passes through an opening in the table (Vlastos and Verkooijen, 2007).

VI. Staging The TNM staging system was designed to be a useful instrument in determining the prognosis of cancer patients and in planning their treatment. The system is derived from tumour size (T), lymph node status (N) and distant metastasis (M). Clinical stage is based on all information, including physical examination and imaging before surgery. Pathological staging (pTNM) adds additional information gained by examination of the tumour microscopically by a pathologist.

A. Definition of pTNM 1. Primary tumour (T) Tx, primary tumour cannot be assessed; T0, no evidence of primary tumour; Tis, carcinoma in situ or Paget disease of the nipple; T1, tumour 20 mm or less; T2, tumour more than 20 mm but nor more than 50 mm; T3, tumour more than 50 mm; T4, tumour of any size with direct extension to chest wall or skin, or inflammatory breast cancer.

2. Regional lymph nodes N0, no node metastasis (includes cases with only isolated tumour cells, or small clusters of cells, not more than 0.2 mm); N1mi, micrometastasis (larger than 0.2 mm, but none larger than 2 mm); N1, metastasis in 1-3 ipsilateral axillary node(s) and/or in ipsilateral internal mammary nodes with microscopic metastasis detected by sentinel lymph node dissection but not clinically apparent; N2 metastasis in 4-9 ipsilateral axillary lymph nodes or in clinically apparent internal mammary lymph node(s); N3, metastasis in 10 or more ipsilateral axillary lymph nodes, or in infra- or supraclavicular lymph nodes, or in both ipsilateral axillary lymph nodes and clinically apparent ipsilateral internal mammary lymph nodes. 13.

3. Distant metastasis (M) M0, no distant metastasis; M1, presence of distant metastasis (AJCC, 2002; Singletary et al, 2002, Woodward et al, 2003) (Table 1).

B. Treatment 1. Surgical therapy Breast cancer surgery has changed dramatically over the past 20 years. With the emergence of breast conserving therapy (BCT), many women now have the option of preserving a cosmetically acceptable breast without sacrificing survival (Veronesi et al, 2002). BCT refers to surgical removal of the tumor without removing excessive amounts of normal breast tissue. The aim of BCT are to provide a cancer operation equivalent to mastectomy and a cosmetically acceptable breast, with a low rate of recurrence in the treated breast (Veronesi et al, 467

Kalogerakos et al: Early breast cancer: A review Absolute contraindications include pregnancy (first or second trimester), diffuse suspicious calcifications, previous radiation to the region and inability to achieve negative margins (particularly with EIC-extensive intraductal carcinoma). Relative contraindications include two or more gross tumors (multicentric disease) in different quadrants, tumor greater than 5 cm initially or after neoadjuvant chemotherapy, large tumorbreast ratio for cosmesis and collagen vascular disease (Daniel et al, 2008). It’s truth that breast conserving surgery is not an option for all women. If the tumour is ! 4cm, multifocal or if radiotherapy has to be avoided, mastectomy is the method of choice. Regardless of the method used, an axillary lymph node dissection is always mandatory. The reason for this is that we know from several studies that the axillary lymph node status is the most important prognostic factor for recurrence and survival (Moore and Kinne, 1997; Orr, 1999). Two different operations of the axilla can be preformed. Traditional axillary lymph node dissection or sentinel lymph node biopsy (Figure 2). The former has been the standard procedure for a long time with additional side effects such as sensory disturbances, lymphedema, pain, seroma formation, poorer cosmetics and infections (Sener et al, 2001; Blanchard et al, 2003; Reitsamer et al, 2003). The sentinel node biopsy is by definition the first lymph node to receive lymphatic drainage from a tumour. Today, the technique is considered to be standard procedure (Bergqvist et al, 2008).

1990; Fisher et al, 2002). All of the available data, including six randomized trials directly comparing BCT with mastectomy and an overview of completed trials, show equivalent survival with BCT as compared to mastectomy (Early Breast Cancer Trialists’ Collaborative Group, 2000). The critical obstacle to widespread acceptance and utilization of BCT is the risk of in-breast recurrence. Most doctors advise against BCT and instead recommend mastectomy if they estimate the risk of in breast recurrence to be >10 to 15 percent over the succeeding 5 to 10 years, even after surgery and radiation. BCT provides an acceptable alternative to mastectomy for many, but is applicable to only 60 to 75 % of newly diagnosed women. The last years a growing number of women with early-stage breast cancer seem to be choosing to have the whole breast removed instead of just the cancerous lump, doctors are reporting. Now, a study of about 5, 500 women at the Mayo Clinic in Rochester, Minn., shows that mastectomies are on the rise (The Associated press, 2008). The study was released Thursday 05.15.2008 by the American Society of Clinical Oncology and will be presented at the group's annual meeting later this month. In the Mayo Clinic study, about 45 percent of breastcancer patients chose mastectomies in 1997. That declined to 30 percent in 2003, then started to rise. By 2006, 43 percent were opting for the more radical treatment (www.azstarnet.com, 05.16.2008). There are very few contraindications to BCT. For most women, the choice of BCT versus mastectomy can be a matter of personal preference.

Figure 2. Sentinel lymph node biopsy (SLNB) is standard care for patients with early-stage breast cancer.

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Cancer Therapy Vol 6, page 469! The procedure was well-tolerated under local anesthesia and sedation but the investigators don’t proposed the RAF as an alternative to open surgery because the patients have residual disease after application of the intervention (Jeffrey et al, 1999; Singletary et al, 2002; Hayashi et al, 2003; Fornage et al, 2004).

2. Minimally invasive procedures Today breast conservation therapy has become the treatment standard for early-stage breast cancer patients and sentinel lymph node biopsy allows prediction of axillary lymph node status without the need for axillary lymph node dissection (Sener et al, 2001; Blanchard et al, 2003; Reitsamer et al, 2003; Albrand and Terret, 2008; Bergqvist et al, 2008; Doughty, 2008). The next challenge is to treat the primary tumor without open surgery but with minimally invasive procedures. Percutaneous tumor excision, radiofrequency ablation (RFA), interstitial laser ablation, focused ultrasound ablation (FUS) and cryotherapy provide interesting alternatives to open breast surgery.

5. Focused Ultrasound Ablation Thermal tumor ablation has also been evaluated using FUS. After localization of the tumor within the breast, ultrasound can be focused and rapidly generate a substantial increase in local temperatures of up to 90°C by converting acoustic energy into heat. FUS ablation heats the tumor and causes cell damage and tumor death (Chen et al, 1999). FUS is based on a 1.5-MHz ultrasound source. Tumor ablation is monitored through temperature probes and skin monitors. Duration of FUS ablation is usually 10 minutes. The major advantage of FUS over other ablative techniques is that no skin incisions are needed. However, tumors close to the skin may be treated with less success and with such adverse effects as skin burns.

3. Percutaneous Stereotactic Excision Percutaneous stereotactic biopsy techniques have been used as a treatment option for excision of benign and malignant breast lesions (Fine et al, 2003). Stereotactic biopsy systems, including the Advanced Breast Biopsy Instrumentation (ABBI) system (U.S. Surgical, Norwalk, CT, http://www. ussurgical.com), other vacuum-assisted core-sampling devices such as the Mammotome (Ethicon, Cornelia, GA, http://www.ethicon.com) and the Minimally Invasive Breast Biopsy (MIBB; U.S. Surgical Corporation), were developed and subsequently used in a percutaneous excisional purpose; although the patients who treated with these approaches were highly selected and conclusions cannot be applied to all breast cancer patients.

6. Laser Ablation Another technique currently being investigated for local treatment of breast cancer is laser ablation. Laser ablation is a technique that generates heat and subsequently causes cell death and tumor destruction. Laser energy is delivered directly to the target tumor through a fiberoptic probe inserted under imaging guidance. Several laser types have been evaluated and used for thermal ablation: the Nd:YAG laser (1064 d, 1, 320 nm), semiconductor diode laser (805 nID) and argon laser (488 and 514 nID). Laser type 805 nID was used more because it is a portable device and may be applied in tumors through special needles. Laser ablation consists in delivering 2-2.5 W in 500 s (>1, 000 J for each fiber) on the tumor. The size of tumor destruction can be increased with the use of several fibers. Laser treatments may be performed under imaging guidance (mammography, ultrasound, or MRI). A target temperature of 80°C-100°C is generated during 1520 minutes to obtain tumor ablation. Laser ablation for the treatment of early-stage breast cancer has not been studied extensively, but some have shown that small tumors can be ablated with negative margins (Mumtaz et al, 1996). After technical improvements, the success rate for complete tumor ablation rose to 93% (Harms, 2001).

4. Radiofrequency Ablation Radiofrequency ablation has been used successfully for the treatment of primary or metastatic tumors of numerous organs, such as liver, lungs, bones, central nervous system, pancreas, kidneys, or prostate Radiofrequency Ablation destroys the tumor with heat (Arciero and Sigurdson, 2008, Lehman and Landman, 2008; Steinke, 2008; White and D'Amico, 2008). A radiofrequency probe (15-gauge) with RFA electrodes is inserted in the tumor and an alternating highfrequency electric current (400-500 kHz) is administered. The heat that is generated affects the cell membrane’s fluidity and the cytoskeleton proteins and finally acts on the nuclear structure, resulting in the interruption of cell replication. This finally leads to irreversible tumor destruction, as tumor cells are more susceptible to heat than are normal cells. The RFAtargeted tumor volume depends on applied tension (up to 200 W). Under imaging guidance, the RFA probe is inserted into the center of the lesion and a star-like array of electrodes is deployed from the tip of the probe. At least 5 minutes are necessary to gradually reach the target temperature (95°C). This temperature is maintained for 15 minutes to achieve complete ablation and is followed by a 1-minute cool-down period. Temperature is monitored during the entire procedure by sensors (van Esser et al, 2007). Several studies evaluated the use of RFA ablation in the treatment of breast cancer.

7. Cryotherapy Cryotherapy was initially developed and used in the treatment of nonoperable liver metastases from colorectal cancers. Cryotherapy uses coldness to achieve tumor destruction (Whitworth and Rewcastle, 2005). Energy is produced by an external generator composed of an argon or nitrogen freezing system and a helium heating system. Cryosurgery involves the use of a freezing probe linked to the generator. Several probes (up to seven) can be used simultaneously to treat larger tumors, as thermal conduction increases the volume of cooled tissue. The 469

Kalogerakos et al: Early breast cancer: A review to others and need not take special precautions with clothes, urine, or stool. Typical side effects include skin reactions ranging from mild redness, dryness and itching to less frequent moist desquamation, ulceration and infection. All usually heal well after treatment. The patient may also experience occasional mild shooting pains in the treated breast and axilla, as well as some fatigue, which is not usually related to low blood counts. There is a low but long-term risk of scarring or fibrosis (eg, fat necrosis) of the treated breast and tumor bed, alteration of breast symmetry and hyperpigmentation, telangiectasias, or altered skin texture (Kissinet al, 1986, Meric et al, 2002). Adding radiotherapy after surgery increases the risk of lymphedema-especially following axillary lymph node dissection (18%) as opposed to sentinel lymph node dissection (10%)-and decreased range of motion of the ipsilateral upper extremity (Meric et al, 2002). Other potential side effects, such as neuropathy, plexopathy, radiation pneumonitis, rib fracture, cardiac events and mortality in women with left breast cancer and risk of secondary primary malignancies, all average less than 1%. There is no correlation to risk of contralateral breast cancer (Bartelink et al, 2001; Recht et al, 2001). In spite of the data and excellent tolerability of treatment, more than 40% of women with early-stage breast cancer still opt for mastectomy, despite long-term local recurrence and survival rates comparable with those for BCT. Up to 25% of women who undergo lumpectomy do not proceed to radiation therapy (Recht et al, 2001). According to the American Society of Clinical Oncology (ASCO) guidelines postoperative radiotherapy after mastectomy, is recommended to patients with tumours >5cm regardless of lymph node status and to patients with four or more positive lymph nodes (Recht et al, 2001). This recommendation is some what controversial, as two Danish randomised studies have shown a survival benefit from radiotherapy in patients with tumours <5cm and one-three positive lymph nodes (Overgaard et al, 1999).

probe is inserted in the center of the tumor under imaging guidance (ultrasound or MRI) through a tiny incision. Once the probe is positioned correctly, an iceball is created at the needle tip. This iceball destroys the tumor as well as 5-10 mm of additional breast tissue surrounding the lesion. During each freeze cycle, temperatures from -185째C to -70째C are obtained and constantly monitored (Staren et al, 1997; Sabel et al, 2004; Vlastos et al. 2004). Currently, the U.S. Food and Drug Administration has approved cryotherapy without resection as a treatment option for core biopsyproven fibroadenomas. For early-stage breast cancer (tumors less than 10-15 mm), cryotherapy is promising, as this technique can be realized under local anesthesia (Bouchardy et al, 2003; Caleffi et al, 2004).

8. Radiotherapy after surgery Postoperative radiotherapy is known to substantially reduce the risk of locoregional recurrence and improve breast cancer mortality, both when given after mastectomy and after breast-conserving surgery (EBTCG, 2005). The meta-analysis by the EBCTCG included a total of 7300 patients who underwent breast-conserving surgery +/- postoperative radiotherapy towards the remaining breast. The locoregional recurrence rate after 5 years was 7% versus 26% (reduction 19%) and 15 years breast cancer mortality risks 30.5% versus 35.9% (reduction 5.4%) (EBTCG, 2005). Patients have many fears in anticipating the radiation experience. Fortunately for patients having BCT, the treatment course is usually well tolerated and produces limited side effects. Of note, if systemic chemotherapy is indicated, it will usually be completed before the initiation of radiotherapy because there is no negative impact on local control or disease-free survival and the chemotherapy may benefit overall survival (Whelan et al, 2002). Doses of 45 to 50 Gray (Gy) are typically given to the whole breast, in daily, Monday-to-Friday fractions of 180 to 200 centiGray (cGy), over a 5-week period, followed by a tumor bed boost of an additional 10 to 16 Gy over 1 to 2 weeks (Shelley et al, 2000; Owenet al, 2006). Pathologic nodal status will determine whether regional nodal groups also require concomitant adjuvant radiotherapy in doses that are similar to those given to the whole breast. In discussing treatment with the patient, the clinician should explain that treatments are typically given on a linear accelerator and that there will be a treatment planning session or simulation of about an hour before the start, which will define the treatment fields and mark the skin. Total daily treatment time usually averages 15 to 20 minutes. The treatments are not painful and the radiation cannot be seen or felt. Radiotherapy is a local treatment that works only where the beams are pointed. Breast irradiation will not cause hair loss of the scalp, nausea, or lowered immunity and it should not harm the heart, lungs, or spinal cord. Reassure the patient that she will not be radioactive, does not need to monitor physical proximity

9. Adjuvant chemotherapy Adjuvant systemic therapy refers to the administration of chemotherapy, hormone therapy and/or trastuzumab (a monoclonal antibody directed against HER2) following primary surgery for early breast cancer. The purpose is to eliminate or delay the subsequent appearance of clinically occult micrometastases, thought to account for distant treatment failures in women undergoing local therapy alone. A major determinant of the choice of adjuvant therapy is whether an individual breast cancer expresses estrogen (ER) or progesterone (PR) receptors. Hormone therapy benefits patients with hormone receptor-positive breast cancer, but not those with hormone receptornegative disease (Berry et al, 2005). The use of adjuvant chemotherapy was first introduced in the mid-1970s.Two prospective randomised trials in patients with node positive patients showed

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Cancer Therapy Vol 6, page 471! et al, 2006). Compared to women who did not receive chemotherapy, those who received it were significantly more likely to visit emergency rooms for all causes (61 versus 42 percent) and for chemotherapy-related serious adverse effects (16 versus 5 percent). The percentages of chemotherapy recipients who were hospitalized or visited the emergency room during the year after their breast cancer diagnosis were: fever or infection 8.4 %, neutropenia or thrombocytopenia 5.5 %, dehydration or electrolyte disorders 2.5 %, nausea, emesis, or diarrhea 2.4 %, anemia 2.2 %, constitutional symptoms 2 %, deep venous thrombosis or pulmonary embolus 1.2 % and malnutrition 0.9 %. These data suggest that the risks of chemotherapyrelated side effects in populations of breast cancer patients may be higher than those reported from large clinical trials, due in part to the intense monitoring and supportive care that patients receive in clinical trials.

promising results in terms of delayed tumour recurrence (Fisher et al, 1975). Despite the fact that both studies had very short follow-up times (18 and 27 months, respectively) adjuvant chemotherapy was considered the treatment of choice for many women in most developed countries. The original regimen used was cyclophosphamide, methotrexate, 5-fluorouracil (CMF). Thereafter, many other regimes have been used. According to the meta-analysis by EBCTCG, adjuvant poly chemotherapy, consisting of either CMF, 5fluorouracil, adriamycin, cyclophosphamide (FAC) or 5fluorouracil, epirubicin, cyclophosphamide (FEC), reduces both recurrence and mortality from breast cancer (Bonadonna et al, 1976). The absolute reduction in breast cancer mortality for women <50 years was 10% and for women aged 50-69 3%. When CMF-based poly chemotherapy was compared with anthracycline-based there was a moderate but significant advantage for anthracyclines, especially in women <50 years (Singal and Iliskovic, 1998). Adjuvant chemotherapy reduces the annual breast cancer death in both node negative and node positive patients. The absolute improvement in 15-years survival is 5% (intention to treat 20) for node negative and 15% for node positive patients. Hence, overtreatment with systemic chemotherapy is a common problem in node negative patients, especially as the side effects generally increase as the drugs get more effective. For example, the FEC regimen induces cardiotoxicity and the incidence of secondary leukaemia (Martin et al, 2005; Praga et al, 2005). The role of taxanes (docetaxel and paclitaxel) has been investigated in several studies (Henderson et al, 2003; Roche et al, 2004). A recent meta-analysis, including 15.500 patients treated with either docetaxel or paclitaxel, shows an absolute survival gain for node positive patients of 3% compared to anthracyclines (Bria et al, 2006). Due to increased toxicity, taxanes are only recommended for patients at moder16 ate/high risk of recurrence (node positive, human epidermal growth factor receptor 2 (Her2) positive, young age). Nausea, vomiting, stomatitis (mucositis) and bone marrow suppression are acute and reversible side effects of systemic chemotherapy. Alopecia is nearly universal with regimens containing an anthracycline or a taxane, while the extent of alopecia varies with the dose and schedule of CMF. Approximately 40 percent of women treated with oral CMF develop alopecia compared to 67 percent of those receiving IV CMF (Fisher et al, 1990; Zambetti et al, 1992). The number, nature and costs of severe adverse effects experience by younger (ie, nonelderly) women receiving chemotherapy for breast cancer is relatively unknown and may be more common than suspected from the results of large clinical trials. This was illustrated in a series of 12, 239 women aged 63 or younger with newly diagnosed breast cancer (4075 of whom received chemotherapy during the 12 months after the initial diagnosis), derived from a database of medical claims made by individuals with employerprovided health insurance between 1998 and 2002 (Hassett

10. Adjuvant therapy antibody Trastuzumab

with

monoclocal

Around 20% of breast cancers overexpress HER2 and this is associated with an adverse prognosis. Trastuzumab is a humanised monoclonal antibody directed against the external domain of the receptor with clinical activity as a single agent in patients whose cancers overexpress HER2. Approximately 25-30% of breast cancer tumours have an amplification of the Her2 gene or overexpression of its protein product (Vlastos and Verkooijen, 2007). Overexpression of the receptor is associated with increased disease recurrence and worse prognosis. Adjuvant trastuzumab in combination with or followed chemotherapy has been investigated in five randomised studies (Piccart-Gebhart et al, 2005; Romond et al, 2005; Slamon et al, 2005; Joensuu et al, 2006). Pooled results from the four major trials and the smaller FinHer study have recently been analysed in a metaanalysis by Viani and colleagues in 2007. It concluded that trastuzumab showed a significant reduction of mortality (p<0.00001), recurrence (p<0.00001) and metastases rates (p<0.00001) compared to patients never treated with adjuvant trastuzumab (Viani et al, 2007). Due to these results, all Her2 positive patients treated with adjuvant chemotherapyn should be considered for one year treatment with trastuzumab. So trastuzumab is becoming a common component of adjuvant treatment programmes for most women whose tumours overexpress HER2. Side effects such as cardiac toxicity grade III or IV was reported in all five studies especially after treatment with antracyclines. The risk for cardiac toxicity was 2.45 fold higher (95% CI 1.89-3.16) in the group of patients treated with trastuzumab (Viani et al, 2007). However, patients that developed cardiac heart failure generally improved on removal of the agent. Despite this, regularly monitoring of the heart is recommended throughout treatment. In Sweden but and in others countries of the Europe an echocardiogram is preformed prior to starting the treatment and thereafter every third month (Andersson et al, 2002; Sengupta et al, 2008). 471

Kalogerakos et al: Early breast cancer: A review Aromatase inhibitors (AIs) markedly suppress plasma estrogen levels in postmenopausal women by inhibiting or inactivating aromatase, the enzyme responsible for synthesizing estrogens from androgenic substrates (Smith and Dowsett, 2003) (Table 3). In contrast to tamoxifen, these compounds lack partial agonist activity. Several trials have investigated the effectiveness of aromatase inhibitors in postmenopausal women with ERpositive, early breast cancer. Regardless of whether it is given “up front� or sequentially after tamoxifen an improvement in treatment outcomes have been noted (Coombes et al, 2004; Jakesz et al, 2005; Howell et al, 2005; Thurlimann et al, 2005). The MA-17 trial compared letrozole versus placebo following five years of tamoxifen (Goss et al, 2003). The MA17 trial showed that the aromatase inhibitor letrozole further decreased the risk of recurrence and improved overall survival (OS) for node positive patients when given as extended treatment after five years of tamoxifen (Jemal et al, 2006). AIs are associated with an increased incidence of musculoskeletal complaints, although the prevalence of these symptoms is unclear. Most published trials as well as data derived from patient surveys suggest that as many as 44 to 47 percent of women experience joint pain or stiffness while taking an AI in the adjuvant setting (Crew et al, 2006). In contrast to tamoxifen, which has estrogenic (ie, protective) effects in the bones of postmenopausal women, all AIs cause bone loss by lowering endogenous estrogen levels. The best way to prevent bone loss associated with AIs is unclear. Guidelines from ASCO and others suggest that women with T-scores lower than -2.5 should exercise and receive calcium, vitamin D and a bisphosphonate; use of a bisphosphonate is "optional" for women with bone densities between -1.5 and -2.5 (Hillner et al, 2003; Perez and Weilbaecher, 2006). However, most endocrinologists recommend pharmacologic therapy for postmenopausal women with T-scores less than -2.0, regardless of risk factors for fracture and with T-scores less than -1.5 if risk factors are present. In all of the trials, compared to tamoxifen alone, AIs have been associated with a lower risk of venous thromboembolic and ischemic cerebrovascular events. In some but not all trials, AIs have also been associated with an increase in the risk of ischemic cardiovascular disease compared to tamoxifen, although the magnitude of the excess risk appears to be small (Mouridsen, 2006; Mouridsen et al, 2007).

11. Endocrine therapy Tamoxifen, a selective estrogen receptor modulator (SERM), inhibits the growth of breast cancer cells by competitive antagonism of estrogen at the ER. However, its actions are complex and it also has partial estrogen agonist activity. These agonist effects can be both beneficial (eg, prevention of bone demineralization) and detrimental (increased risk of uterine cancer and thromboembolic events) (Lee et al, 2008). Several overviews of randomised trials have shown reduced mortality in the adjuvant setting. The latest Oxford overview (15 years follow-up) confirmed a 31% reduction in mortality in women with ER-positive disease who received tamoxifen for five years, regardless of age, menopausal status or nodal status and a 39% reduction in the incidence of contralateral breast cancer (EBTCG, 2005; Albrand and Terret, 2008). A number of potential adverse effects are associated with the administration of tamoxifen. These include hot flashes and vaginal discharge in the short-term and a longterm increase in the risk of thromboembolic events, as well as a two- to three-fold higher risk of endometrial cancer and uterine sarcomas (Rutqvist et al, 1995; Cosman and Lindsay, 1999; Benson and Pitsinis, 2003; Riggs and Hartmann, 2003). Several factors may contribute to tamoxifen resistance in breast cancer, including variable expression of estrogen receptor alpha and beta isoforms, interference with binding of co-activators and co-repressors, alternatively spliced ER mRNA variants and modulators of ER expression such as epidermal growth factor (EGF) and its receptor (EGFR1, also called HER1) as well as the type 2 EGFR, also called HER2 (Lipton et al, 2005). Emerging studies also suggest that relative resistance to tamoxifen may be related to inheritance of certain drug metabolizing CYP2D6 genotypes that are associated with a reduced activation of tamoxifen to its active metabolite endoxifen. However, at present, routine assay to identify the CYP2D6 genotype as a means of selecting patients for tamoxifen is not considered standard practice (Jin et al, 2005). Tamoxifen 20 mg daily is a standard adjuvant treatment option for both premenopausal and postmenopausal women with ER+ early breast cancer. Until more data become available, the recommended duration of therapy is five years. Aromatase is an enzyme that naturally converts oestrogen from androgen. In premenopausal women, most of the oestrogen is produced in the ovaries, but in postmenopausal women, most oestrogen is synthesised in peripheral tissue from conversion of androgens (Simpson, 2003). Table 3. Aromatase inhibitors Generation First (nonselective) Second (selective) Third (superselective)

Steroidal (type 1) Formestane Exemestane (Aromasin)

472

Nonsteroidal (type 2) Aminoglutethimide Fadrozole Anastrozole (Arimidex) Letrozole (Femara)

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VII. Conclusion Increased awareness among women and improvement in diagnostic procedures have enabled earlier and better detection of breast cancer. Improvement in breast cancer treatment has undoubtedly also increased the long-term survival of patients as reflected by the improved overall survival across all breast cancer stages. The prognosis of breast cancer has become relatively good, with current 10-year relative survival about 70% in most western populations.

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The role of panitumumab in metastatic colorectal cancer Review Article

Maria Anna Bareschino1,2,^, Clorinda Schettino1,2,^, Paolo Maione2, Antonio Rossi2, Dario Nicolella2, Fortunato Ciardiello1, Cesare Gridelli2,* 1

Division of Medical Oncology, Department of Clinical and Experimental Medicine and Surgery ‘F. Magrassi and A. Lanzara’, Second University of Naples, School of Medicine Via S. Pansini 5, 80131 Naples, Italy. 2 Division of Medical Oncology, “S.G. Moscati” Hospital, Avellino, Italy.

__________________________________________________________________________________! *Correspondence: Cesare Gridelli, M.D., Division of Medical Oncology, “S.G. Moscati” Hospital, Contrada Amoretta, 83100 Avellino, Italy; Telephone: 39-0825-203573; Fax: 39-0825-203556; e-mail: cgridelli@libero.it Key words: EGFR, mCRC, Panitumumab, K-RAS, FISH, Monoclonal Antibodies Abbreviations: antibody-dependent cellular cytotoxicity and complement-dependent cellular, (ADCC); area under the concentrationtime curve, (AUC); best supportive care, (BSC); chromogenic in situ hybridization, (CISH); disease control rate, (DCR); eastern cooperative oncology group, (ECOG); epidermal growth factor, (EGF); epidermal growth factor receptor, (EGFR); food and drugs administration, (FDA); gene copy number, (GCN); health related quality of life, (HRQoL); immunohistochemistry, (IHC); monoclonal antibody, (mAbs); metastatic colorectal cancer, (mCRC); median survival time, (MST); maximum tolerated dose, (MTD); progression of disease, (PD); platelet derived growth factor, (PDGF); progression free survival, (PFS); partial responses, (PR); quality of life, (QoL); response rate, (RR); stable disease, (SD); transforming growth factor ! , (TGF-! ); time to progression, (TTP); vascular endothelial growth factor, (VEGF)

^This author have equally contribute to this manuscript

Received: 3 July 2008; Revised: 1 August 2008 Accepted: 4 August 2008; electronically published: September 2008

Summary Colorectal cancer constitutes one of the most common malignancies and the second leading cause of death from cancer in the western world representing one million new cases and half a million deaths annually worldwide. Even if therapeutic options and survival times for patients with metastatic colorectal cancer are improved considerably over the last decade, due to availability of modern chemotherapy agents such as irinotecan and oxaliplatin, there is a need for new active treatment options in this setting. Two classes of biologic agents have recently approved for the treatment of mCRC: monoclonal antibodies direct against vascular endothelial growth factor and monoclonal antibodies direct against the epidermal growth factor receptor. Panitumumab is the first fully monoclonal antibody that binds EGFR approved by Food and Drugs Administration for the treatment of EGFR expressing CRC patients with disease progression on or following fluoropyrimidine, oxaliplatin, and irinotecan containing chemotherapy regimens based on improvement in progression free survival and response rate.

response rate (RR) of approximately 20% and reaching median survival of 10-12 months (Simmonds et al, 2000). Several options are currently available for the mCRC treatment including different regimens of chemotherapeutic compounds. The addition of irinotecan to 5-FU in first line therapy increased RR to 40-50%, prolongs median time to progression (TTP) by approximately three months and resulted in an improvement in survival of 2-3 months approximately

I. Introduction Colorectal cancer (CRC) belongs to the leading causes of death from cancer. Approximately 20% of patients with CRC present metastatic disease, and it is estimated that ~ 50% of CRC patients develop metastases and eventually die of disease. The treatment of metastatic CRC (mCRC) was based on fluoropyrimidine chemotherapy for over 50 years producing objective tumor

477

Bareschino et al: The role of panitumumab in metastatic colorectal cancer phase II randomized clinical trial demonstrating a statistically significant improvement of RR (22% vs 10.8%; P=0.007) and progression free survival (PFS) (4.1 vs 1.5 months; P<0.001) when cetuximab was given in combination with irinotecan compared to cetuximab alone in irinotecan refractory mCRC. Survival, however did not differ significantly between two groups, although its was numerically longer for patients receiving cetuximab and irinotecan (8.6 vs 6.9 months P=0.48) (Cunningham et al, 2004). The efficacy of single agent cetuximab in heavily pre-treated mCRC patients has been recently confirmed by a phase III randomized clinical trial, in witch patients randomized to receive cetuximab achieved a statistically significant improvement in OS compared with those receiving best supportive care (BSC) alone [median OS: 6.1 versus 4.6 months; hazard ratio (HR)=0.766; P=0.0048, stratified log-rank test] (Jonker et al, 2007). Based on this finding, in October 2007 regular approval for single-agent cetuximab was granted for use in this patient population. The most important phase II/III trials investigating the role of cetuximab in combination with chemotherapy in first line setting and second and further line treatment of mCRC are summarized in the tables 1 and 2 (Table 1, 2). Bevacizumab, a recombinant humanized monoclonal IgG1 antibody directed against the VEGF, was approved in the US in 2004 for use in combination with irinotecan containing regimens for the first-line treatment of mCRC. This approval was based on the demonstration that the addition of bevacizumab to chemotherapy provides significant clinical benefit to patients by increasing OS, PFS, and overall RR (Kabbinavar et al, 2003; Hurwitz et al, 2004) (Table 3).

compared to 5-FU/LV alone (Douillard et al, 2000; Saltz et al, 2000) and also in pretreated patients irinotecan monotherapy increased the median overall survival (OS) by 2-3 months from the 6-8 months (Rougier et al, 1998; Cunningham et al, 1998). Similar results were obtained adding oxaliplatin to infused 5-FU/LV (De Gramont et al, 2000, Giacchetti et al, 2000). The comparison between the irinotecan/infused 5FU/LV (FOLFIRI) and oxaliplatin/infused 5-FU/LV (FOLFOX) regimens sequentially used, regardless of order, show that these regimens are equally effective resulting in median survival time (MST) reaching 21 months approximately (Turnigand et al, 2004). Data from 11 phase III clinical trials has showed that median OS is significantly correlated with the percentage of patients who received all three drugs (5-FU, irinotecan and oxaliplatin) in the course of their disease (P =0.0001) independently of sequence in which they were administered (Grothey et al, 2004, 2005). Therefore, several novel targeted agents are being investigated in combination with chemotherapy and as single agents. Two classes of biologic drugs has recently been approved for the treatment of mCRC: Epidermal Growth Factor Receptor (EGFR) inhibitors (cetuximab and panitutumab) and Vascular Endothelial Growth Factor (VEGF) inhibitor (bevacizumab). Cetuximab is a chimeric IgG1 monoclonal antibody (mAb) that binds to the extracellular domain of the EGFR, and was the first biologic agent to receive approval in 2004 by Food and Drugs Administration (FDA) in the United States (US) for use in combination with irinotecan for the treatment of EGFR expressing irinotecan refractory mCRC patients and as single agent in those intolerant to irinotecan-based chemotherapy. This approval was based on results of a

Table 1. Cetuxuximab in combination with chemotherapy in second and third line mCRC treatment. Treatment

N째pts

Study details

Second/third Line

218

Cetuximab + irinotecano vs Cetuximab Cetuximab+ irinotecano vs irinotecan Cetuximab+ irinotecano Cetuximab+ irinotecano Cetuximab;

Second/third Line

111 648 650

Second/third Line Second/third line Second/third line

79 121 2

14 Second/third line

40

Second/third Line

123

Cetuximab+ irinotecano Cetuximab/ capecitabine/ oxaliplatin Cetuximab+ irinotecan

RR (%) 22.9

TTP (months) 4.1

PFS (months)

OS (months) 8.6

-

Study Phase II randomized, Cunningham and colleagues Phase III, Sobrero and colleagues

10.8 16.4

1.5 -

4.0

6.9 10.7

4.2

-

2.6

10.0

17.7 *

9.7

-

-

-

-

-

-

Phase II open-label, Buzaid and colleagues Phase II open-label, Saltz and colleagues Phase II retrospective, Chung and colleagues

20

3.0

-

10.7

Phase II open-label, Souglakos and colleagues

13.8

-

12.1

9.5

Phase II open-label, Lim and colleagues

26.6 17 25

25

Pts= patients; RR= response rate; PFS= progression free survival; TTP= time to progression; OS= overall survival.

478

Cancer Therapy Vol 6, page 479! Table 2. Cetuxuximab in combination with chemotherapy in mCRC first line treatment.

Treatment

N째pts

Study details

Second/third Line

218

Cetuximab + irinotecano vs Cetuximab

Second/third Line

111 648

RR (%) 22.9

79

Second/third line

121

Second/third line

PFS (months)

OS (months) 8.6

-

Cetuximab+ irinotecano vs irinotecan

650 Second/third Line

TTP (months) 4.1

10.8 16.4

1.5 -

4.0

6.9 10.7

4.2

-

2.6

10.0

17.7 *

9.7

-

-

-

-

-

-

20

3.0

-

10.7

13.8

-

12.1

9.5

Cetuximab+ irinotecano 26.6

Cetuximab+ irinotecano

2

14 Second/third line

40

Second/third Line

123

17 25

Cetuximab; Cetuximab+ irinotecano Cetuximab/ capecitabine/ oxaliplatin

25

Cetuximab+ irinotecan

Study Phase II randomized, Cunningham and colleagues Phase III, Sobrero and colleagues Phase II openlabel, Buzaid and colleagues Phase II openlabel, Saltz and colleagues Phase II retrospective, Chung and colleagues Phase II openlabel, Souglakos and colleagues Phase II openlabel, Lim and colleagues

* 1-year rate of PFS Pts= patients; RR= response rate; PFS= progression free survival; TTP= time to progression; OS= overall survival;

Table 3. Registration trials with biologic agents in mCRC.

Trial

Treatment

Cunningham et al. 2004 (phase II)

Cetuximab vs Cetuximab + Irinotecan 5FU/LV vs 5FU/LV+Bev 5mg/kg vs 5FU/LV+Bev 10mg/kg IFL+ Placebo vs IFL+ Bev

Kabbinavar et al. 2003 (phase II)

Hurwitz et al. 2004 (phase III) Van Cutsem et al. 2007 (phase III)

Pmab + BSC vs BSC

N째 Pts RR TTP (%) (months) 10.8 1.5 111

PFS (months)

OS (months)

_

6.9

218

22.9 4.1

_

8.6

36 35

17 40

5.2 9.0

_ _

13.8 21.5

33

24

7.2

_

16.1

411 402

34.8 44.8

6.2 10.6

15.6 20.3

231 232

10 0

8* 7.3*

NR** NR**

*Weeks; ** No significant differences was observed (HR= 1.00). Pts= patients; RR= response rate; PFS= progression free survival; TTP= time to progression; Bev= bevacizumab; pmab= panitumumab;

479

Bareschino et al: The role of panitumumab in metastatic colorectal cancer BSC= best supportive care; NR= no reported.

production of fully humanized antibodies that in contrast with chimeric antibodies results to be less immunogenic and to have reduced Fc domain based effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cellular (ADCC). The result is the non-immunogenic mAbs generation, with reduction of the risk of hypersensitivity reactions during treatment administration and of the formation of human antimouse antibodies which can reduce the efficacy of the agents (Chua et al, 2006). Panitumumb was generated by immunizing the XenoMouse strain of mice with human cervical epidermal carcinoma cell line A431 known for EGFR iperexpression (Yang et al, 2001). This drug binds specially and selectively to the EGFR and blocks binding of EGF and TGF-! reducing EGFR signalling causing cell cycle arrest at G0/G1, apoptosis, decrease production of growth factors such as VEGF and pro-inflammatory cytokine as interleukin-8. The panitumumab-coated receptor is rapidly internalized resulting in receptor down-regulation although is not clear if the receptor is next degraded or recycled to the plasma membrane. Panitumumab was evaluated for pharmacologic activity in human tumor cell lines in vitro and in vivo in human xenograft tumors in immunodeficient mice; and for toxicity and pharmacokinetics profile in immunodeficient mice and cynomolgus monkeys. Tissue binding studies showed that panitumumab bound with moderate to strong intensity to surface EGFR in tissue samples of epithelial origin from both human and cynomolgus monkey. Treatment of tumor-bearing immunodeficient mice with panitumumab alone or in combination with several different biological agents or chemotherapy regimens resulted in delayed growth of human colon, breast, or pancreatic cancers (Yang et al, 1999). The preclinical activity of this agent is more pronounced in cells expressing the EGFR at levels of " 15000 per cell and inactive in EGFR negative tumors suggesting that a threshold level of EGFR expression is required for tumor response to panitumumab (Ranson et al, 2003). Panitumumab administered as a single agent exhibits non-linear pharmacokinetics. Following a single 1-hour infusion the area under the concentration-time curve (AUC) increased in a greater than dose-proportional manner, and clearance of panitumumab decreased from 30.6 ml/kg per day to 4.6 ml/kg per day as the dose increased from 0.75 mg/kg to 9 mg/kg. However, at doses above 2 mg/kg, the AUC of panitumumab increased in an approximately dose-proportional manner. Following the recommended dose regimen (6 mg/kg given once every 2 weeks as a 1-h infusion), panitumumab concentrations reached steady-state levels by the third infusion peak and trough concentrations of 213 ± 59 and 39 ± 14 µg/ml, respectively. The area under the concentration-time curve (AUC0-tau) and clearance were 1,306 ± 374 µg · day/ml and 4.9 ± 1.4 ml/kg/day, respectively. The elimination half-life was approximately 7.5 days range, 3.6-10.9 days. Selected covariates such as age, gender, race, mild-to-moderate renal and hepatic

Panitumumab, a fully human monoclonal antibody that targets EGFR is approved in the US in September 2006 by FDA for the treatment of mCRC EGFR positive patients and is approved in Europe in 2007 by EMEA only in wild-type K-RAS mCRC patients. In this review we will focus on the clinical results available with panitumumab in the treatment of colorectal cancer and the molecular mechanisms involved in resistance and response to this drug.

II. Biology of EGFR The EGFR is a member of the ErbB family cell membrane receptors which also includes ErbB2/Neu/HER2, ErbB-3/HER3, and ErbB-4/HER4 that are important mediators of cell growth, differentiation and survival. The EGFR is a transmembrane glycoprotein, that consists of an extracellular domain that recognizes and binds to specific ligands, a hydrophobic trans-membrane domain, involved in interactions between receptors within the cell membrane, and an intracellular domain that contains the tyrosine kinase enzymatic activity. The are several ligands binding EGFR including Epidermal Growth Factor (EGF), Transforming Growth Factor ! (TGF-! ), neuregulin family and some others. Once the ligand binds to the extracellular domain, EGFR undergoes homo-dimerization or hetero-dimerization. Dimerization induces the activation of the tyrosine kinase domain, which leads to autophosphorylation of critical tyrosine residues on the cytoplasmic terminal. These tyrosine residues serve as attachment sites for a range of cellular docking proteins, activating a variety of downstream signalling cascades to affect gene transcription (Yarden et al, 2001). Three pathways downstream of EGFR have been identified: the Ras/Raf mitogen-activated protein (MAP) kinase, PI3K/Akt, and Jak2/STAT3 pathways. Activation of these pathways starts a cascade of complex cell biochemistry that result in different cellular responses such as proliferation, migration, differentiation or apoptosis. Elevated levels of EGFR expression have been found in a variety of epithelial tumors: colorectal, head and neck, breast, renal cell cancer (Salomon et al, 1995). The blockade of EGFR signalling in cancer cells causes not only inhibition of cell proliferation but also other effects that could be relevant in the clinical setting including: induction of apoptosis; anti-angiogenesis through inhibition of angiogenic growth factors production; inhibition of invasion and metastasis; potentiation of anti-tumor activity of cytotoxic drugs and radiotherapy (Normanno et al, 2003).

III. Panitumumab: mechanism action and pharmacokinetics

of

Panitumumab (Vectibix, ABX-EGF; Amgen Inc, Thousand Oaks, CA) is a fully human IgG2 monoclonal antibody targeting the EGFR. Panitumumab was initially developed using XenoMouse transgenic technology based on inactivation of the endogenous mouse immunoglobulin genes that are replaced by genes containing the human heavy and K chains. This technology permits the

480

Cancer Therapy Vol 6, page 481! dysfunction, had no apparent effect on pharmacokinetics of this drug (Giusti et al, 2007).

fluoropyrimidine and either irinotecan or oxaliplatin, were tested for EGFR expression using immunohistochemistry (IHC). The patients were also prospectively divided in two cohorts: patients with EGFR staining of 2+ and 3+ ! in 10% of cells (cohort A), and patients EGFR staining 2+ and 3+ " 10% of cells, but 1+, 2+ or 3+ ! 10% of tumor cells (cohort B). Panitumumab was administrated at dose of 2.5 mg/kg weekly. This trial has reported a RR of 9% and a 29 % of SD resulted similar between two groups; PFS and MST was 14 weeks and 9.0 months respectively. The major toxicities reported were rush and fatigue; skin rash was reported in 95% of patients, (5% grade 3 and 0% grade 4 toxicity) (Hecth et al, 2007). Another phase II trial have evaluated panitumumab activity in 150 refractory or pretreated mCRC patients with low (1%-9%) or negative (<1%) tumor EGFR levels detected by IHC. Patients received panitumumab at dose of 6 mg/Kg every two weeks. Disease Control Rate (DCR) was 35% in patients with low EGFR levels, and 28% in patients with negative EGFR tumors; PFS was 7.8 and 8.3 weeks respectively. Median OS was 8.7 months for patients with low EGFR levels and 10.1 months patients with negative EGFR tumors. Results confirmed earlier findings that panitumumab has anti-tumor activity in patients with low or undetectable EGFR tumor membrane levels (Hecht et al, 2008a). However these data are in contrast to preclinical data suggesting that a threshold level of EGFR expression is required for tumor response to panitumumab (Cohenuram et al, 2007). The predictive value of EGFR positivity is therefore more questioned. At moment several bias characterize IHC methodology such as a significant variability in EGFR immunoreactivity depending on fixation method; an important reduction in EGFR staining intensity occurred with increased storage time of the tissue samples; and different IHC EGFR expression between an individual patients primary tumor and metastatic tissue specimens. Another possible explanation may be the presence of two biologically distinct EGFRs (high and low affinity for EGF) and may be hypothesized that a small number of high-affinity EGFR might be responsible for susceptibility to mAbs treatment even if its absolute number is below the threshold of IHC detection (Francoual et al, 2006). Panitumumab have also a good safety and tolerability profile in combination with chemotherapy as showed in a phase II clinical trial in which panitumumab at dose of 2.5 mg/kg/week was combined with irinotecan- and 5-FU based treatment in previously untreated mCRC patients. Initially, nineteen patients were treated with panitumumab plus the bolus 5-FU (IFL regimen), and twenty-four with infusional 5-FU (FOLFIRI) regimen. Has been reported an overall RR of 46% in IFL and 42% in FOLFIRI regimen, PFS was 5.6 and 10.9 months and OS was 17 and 22.5 months for IFL and FOLFIRI group, respectively. Skin toxicity was the predominant toxicity observed, occurring at any grade in 84% and 75% of patients treated in the IFL and FOLFIRI cohorts, respectively; another common toxicity reported was grade 3/4 diarrhea occurred specially in IFL arm (58%) compared to FOLFIRI regimen (25%). In general, the incidence of toxicities was lower in the

the

IV. Clinical efficacy A. Phase I clinical trials Forty-three patients with several cancer types such as colorectal, esophageal, and prostate cancer, enrolled in a phase I trial have received up to 4 weekly doses of panitumumab monotherapy, from 0.01 to 2.5 mg/kg with no loading dose. Panitumumab has been well tolerated, no allergic reactions infusion-related were reported and no human antihuman antibodies were detected. The rash, characteristic of EGFR blocking antibodies, was observed in all patient receiving a dose of 2.0 mg/kg/week or more of panitumumab. One patient with colorectal cancer had for 4 months stable disease (SD) at the 1.5 mg/kg dose and SD was achieved for 7 months in one patient with esophageal cancer at the 0.1 mg/kg dose; one patient with prostate cancer experienced a minor response for 6 months at dose 0.75 mg/kg (Figlin et al, 2002). The exposure and tolerability profile is comparable between different dose levels and schedules of panitumumab monotherapy ranging from 0.01 to 5.0 mg/kg once per week (QW), 6.0 mg/kg once every 2 weeks (Q2W) or 9.0 mg/kg once every 3 weeks (Q3W), and no maximum tolerated dose (MTD) was reached as reported in a phase I trial. In this trial ninety-six EGFR positive solid tumor patients received panitumumab; among 39 CRC patients has been reported 5 (12.8%) partial responses (PR), and 9 SD were observed (Weiner et al, 2008). In other studies in which panitumumab was administered at 6 mg/kg every two weeks or 9 mg/kg every three weeks has been showed that both schedules were generally well tolerated resulting in exposure and tolerability profiles comparable to dose of 2.5 mg/kg weekly (Arends et al, 2005; Weiner et al, 2005). Panitumumab is also being studied in combination with chemotherapy (FOLFIRI or FOLFOX) and motesanib diphosphate (AMG 706), an oral drug with activity against multiple tyrosine kinases including VEGF, Platelet Derived Growth Factor (PDGF) and Kit receptors in a phase Ib clinical trial. Among forty-five patients enrolled were reported 6 dose-limiting toxicities: FOLFIRI n=4, all grade 3 (diarrhea n=2; deep vein thrombosis n=1; high GI output n=1); FOLFOX n=2 (all fatigue, grade 3). Treatment-related adverse events for both regimens (FOLFIRI/FOLFOX) included: fatigue 55/58%, anorexia 24/50% diarrhea 24/33% epistaxis 27/0% and hypertension 15/8%. The overall RR was 11/22 (50%) for the combination of biologic agents with FOLFIRI chemotherapy and 5/10 (50%) for the same combination of biologics plus FOLFOX; these preliminary data show that the combination of panitumumab and AMG 706 plus chemotherapy was well tolerated with little effect on AMG 706 pharmacokinetics (Schwartzberg et al, 2007).

B. Phase II clinical trials In a phase II clinical trial tumor specimens from 148 mCRC patients, who had failed treatment with a 481

Bareschino et al: The role of panitumumab in metastatic colorectal cancer FOLFIRI treated patients cohort (Berlin et al, 2007a). In

the table 4 were summarized phase II studies (Table 4).

Table 4. Panitumumab phase II-III studies in mCRC. Pts (n째) 148 EGFR low (43) EGFR high (105) 150 EGFR negative (71) EGFR low (79)

RR (%)

PFS (weeks)

Naive

Phase

Authors

5 8

8 8

NO

II

Hecht (2007)

8.3 7.8

NO

II

Hecht (2008)

46 42

5.6 10.9

YES

II

Berlin (2007)

4.2 5.1

43 IFL + pmab (19) FOLFIRI + pmab (24) 463 pmab (231) BSC (232) 230 Iri/Bev+pmab (115) Iri/Bev (115)

10 0

8 7.3

YES

III

Van Cutsem (2008)

55 46

10.6* 10.7*

YES

III

Hecht (2008)

823 Ox/Bev+pmab (413) Ox/Bev (410)

45 46

9.5* 11.0*

YES

III

Hecht (2008)

* months Pts= patients; RR= response rate; PFS= progression free survival; EGFR= epidermal growth factor receptor; pmab= panitumumab; BSC= best supportive care; Iri= irinotecan; Bev=bevacizumab; Ox= oxaliplatin

C. Phase III clinical trials

following treatment with a fluoropyrimidine, irinotecan, and oxaliplatin were randomized to receive panitumumab 6 mg/kg every 2 weeks plus BSC (n = 231) or BSC alone (control arm; n = 232). Patients were required to have 1% or more EGFR tumor cell membrane staining by IHC.

The efficacy and safety of panitumumab has been evaluated in a randomized, controlled, open label, phase III study (Figure 1). In this trial, a total of 463 mCRC patients with documented progression of disease during or

Figure 1. Panitumumab + Best Supportive Care (BSC) vs BSC alone in mCRC, design of the registered phase III clinical trial.

Randomization stratification: !! Easter Cooperative Oncology Group (ECOG) score: 0-1vs 2 Geografic region : Western EU vs central vs Eastern EU vs rest of the world 482

Cancer Therapy Vol 6, page 483!

on PFS favored panitumumab vs BSC in spite of age or ECOG status. Similar PFS and OS times and ORR were seen between elderly and younger pts and within both ECOG status groups, among patients receiving panitumumab. This evidence suggest that the efficacy and tolerability of panitumumab in mCRC patients was similar regardless of age and ECOG status (Van Cutsem et al, 2007b). Another analysis of this study, characterises the association of PFS with health related quality of life (HRQoL), symptoms related to disease, and OS. At week 8 lack of disease progression was associated with significantly and clinically important lower CRC related symptoms for both treatment groups and higher HRQoL only for panitumumab treated patients. OS favoured patients alive at week 8 without progression of disease (PD) compared to patients reporting PD (Siena et al, 2007). A further analysis of this trial has evaluated patients randomized to BSC arm who received panitumumab in the separate crossover study. Among 232 patients receiving BSC, 176 with PD has been enrolled in the crossover study. The objective RR was 11.6% (1 CR, 19 PR), and 33% (58/176) of patients achieved SD; median PFS and MST were 9.4 (95% CI= 8.0 to 13.4) weeks and 6.3 months (95% CI 5.1-6.8) respectively. The most frequent reported adverse event was skin toxicity. These findings were consistent with that seen in patients receiving panitumumab plus BSC in the phase III study as well as in previous panitumumab monotherapy studies and also in these patients panitumumab appear well tolerated with a manageable toxicities profile (Van Cutsem et al, 2008a). The preliminary safety data of the first phase III clinical trial investigating the combination of panitumumab with FOLFIRI chemotherapy as second line treatment for mCRC patients has been recently reported. Seven hundred one patients were randomly assigned to receive panitumumab plus FOLFIRI at standard dose or FOLFIRI alone. The most common adverse events observed were skin toxicity (61%), diarrhea (55%), nausea (43%), fatigue (28%) (Peeters et al, 2008). Another randomized, multicenter phase III clinical trial have tested the panitumumab in combination with FOLFOX chemotherapy in untreated mCRC patients. After a median follow up time of 15 weeks a safety interim analysis including 601 patients have showed that panitumumab is well tolerated and most common toxicities were: skin reactions reported in approximately half of patients, diarrhea (43%), nausea (39%), neutropenia and fatigue (Siena et al, 2008). A phase III study to assess whether the addition of panitumumab to first-line chemotherapy (either oxaliplatin or irinotecan-based) plus bevacizumab would have improved PFS compared to treatment with chemotherapy plus bevacizumab alone (PACCE trial) has been conducted. Of this complex and intricate trial has been examined separate strata of patients receiving either oxaliplatin or irinotecan based chemotherapy/bevacizumab Âą panitumumab. Based on the first interim efficacy analysis of oxaliplatin stratum demonstrating a shorter PFS and an increased incidence of toxicities for the

A significant improvement of PFS was observed in patients who received panitumumab plus BSC compared with those who received BSC alone; median PFS time was 8 weeks (95% CI = 7.9 to 8.4) for panitumumab and 7.3 weeks (95% CI=7.1 to 7.7) for BSC arm. Patients receiving panitumumab achieved a decrease of 46% in the rate of tumor progression compared with those receiving BSC alone (HR=0.54; 95% CI= 0.44 to 0.66). After a 12-month minimum follow-up, 10% of RR (all PR) among patients receiving panitumumab and no response in the BSC group were observed (P<0.0001); median duration of response was 17.0 weeks (95% CI= 7.9 to 76.7); however no difference in OS (HR= 1.00; 95% CI= 0.82 to 1.22 P=0.81) among two groups was reported. Among patients receiving BSC alone 176 (76%) received panitumumab in a cross-over protocol after progression disease and the median time to cross-over was 7 weeks. The high rate and rapidity of cross over of the patients in the BSC group and the similar activity of panitumumab observed in the cross-over population could confounded survival data. Panitumumab was well tolerated and skinrelated toxicities including dermatitis acneiform, pruritus, erytema, rash, skin exfoliation, paronychia, dry skin and skin fissures occurred in 90% of patients in the panitumumab group and in 9% of patients in the BSC group respectively. Among patients in the panitumumab arm, a better PFS was reported for patients with a worst severity of grade 2 to 4 versus grade 1 skin toxicity (HR=0.62; 95% CI= 0.44 to 0.88); 19/22 (86%) of responders patients had a maximum skin toxicity severity of either grade 2 or 3, and only 3 (14%) responders patients had a maximum skin-toxicity severity of grade 1. Other common adverse events were hypomagnesemia and diarrhea occurred in 36% and 21 % of patients in the panitumumab arm (Van Cutsem et al, 2007a). Based on results of this trial on September 2006 the FDA granted approval to panitumumab (Vectibix, Amgen, Thousand Oaks, CA) for the treatment of patients with EGFR-expressing m- CRC with disease progression on or following fluoropyrimidine, oxaliplatin, and irinotecan containing chemotherapy regimens. This is the first clinical trial to demonstrate the superiority of a targeted agent used as monotherapy in mCRC compared to BSC alone. However a phase III randomized clinical study has showed that cetuximab as a single agent compared to BSC significantly improves survival in mCRC patients refractory to approved chemotherapy agents including irinotecan, oxaliplatin and fluoropyrimidines. In this study the median survival was 6.1 months for patients treated with cetuximab vs 4.6 months for patients receiving BSC alone (P=0.005; HR=0.77), futhermore cetuximab improves PFS and preserve quality of life (QoL) (Jonker et al, 2007). An exploratory analysis of registrative trial including elderly (<65 vs >65 years) and poor performance status [Eastern Cooperative Oncology Group (ECOG) score 0-1 vs 2-3] patients has been conducted. The treatment effect

483

Bareschino et al: The role of panitumumab in metastatic colorectal cancer efficacy has been evaluated in mCRC patients enrolled in several clinical trials. Data emerging from five clinical trials (4 phase II and 1 phase III studies) including a total of 612 patients show that the most common skin toxicities (any grade) were erythema 54%, pruritus 53%, dermatitis acneiform 52%, and rush 39%. The RR was 3.3% in patients with grade 0- 1 vs 12.6% in patients with grade 2-4 skin toxicity, also PFS, and OS appeared to favor patients with grade 2-4 skin toxicity (PFS 8.0 vs 13.1 weeks respectively; OS 4.5 vs 8.5 months respectively P<0.0001). This large combined analysis demonstrates that the severity of skin rash was correlated with increased efficacy of panitumumab in terms of overall clinical outcome (Berlin et al, 2007b). Considering that the skin toxicities could be assemble only after the start of treatment it may not be considered as a predictive marker but can serve as a surrogate marker of efficacy.

combination oxaliplatin/bevacizumab plus panitumumab (Ox/bev + pmab) in March 2007 Amgen discontinued this study. An recent updated analysis of 823 patients randomized in the oxaliplatin stratum have reported a PFS of 9.5 months in Ox/bev + pmab arm compared to 11 months in the Ox/bev alone and an unfavorable benefit/risk report for this combination in the treatment of mCRC patients (Hecht et al, 2008b). These data suggest that a negative interaction between the combination of VEGF and EGFR antibodies can not be excluded. In fact similar results were recently reported in a randomized phase III study (CAIRO II) in which has been investigated the effect of adding cetuximab to capecitabine and oxaliplatin (CapOx) and bevacizumab in untreated mCRC. The combination of both antibodies to CapOx regimen results in a significant decrease of PFS compared to bevacizumab plus CapOx chemotherapy (Punt et al, 2008). However, early studies have suggested that combining EGFR, VEGF inhibitors and chemotherapy improve efficacy (Shaheen et al 2001; Saltz et al, 2007). In a phase II clinical trial (BOND2) the combination of cetuximab, bevacizumab and irinotecan vs cetuximab and bevacizumab alone in irinotecan-refractory mCRC patients has been compared. The results of this trial suggest a preliminary evidence of the clinical benefit combining cetuximab and bevacizumab and the activity seen with the addition of bevacizumab seems to be favorable when compared to historical controls treated with cetuximab/irinotecan or cetuximab alone in this setting (Saltz et al, 2007). Preliminary data of irinotecan stratum (PACCE Trial) including 230 patients show that the RR were higher in the irinotecan/bevacizumab (iri/bev) plus panitumumab compared to iri/bev alone (55% vs 46%) such as toxicities related to treatment, while PFS was similar between two arms, (10.6 vs 10.7 months) and no difference in OS was seen. Most patients withdrew due to non progressive events (59% on panitumumab + iri/bev arm, 71% on iri/bev) similar to the Oxaliplatin chemotherapy cohort, limiting the utility of PFS as a valid endpoint in this trial (Hecht et al, 2008c). In the table 4 were summarized the phase III studies (Table 4).

B. The predictive role of EGFR gene copy number The identification of patients who are likely to benefit from EGFR targeted mAbs is increasingly crucial for improving therapeutic strategies; the first step is to identify the genetic alterations associated with the clinical response, the second is the elucidation of molecular basis for primary or acquired resistance to these drugs. A first retrospective trial to evaluate the association between EGFR gene copy number (GCN) and response to mAbs (cetuximab/panitumumab) has been conducted by Moroni. Tumor specimens from 31 mCRC patients receiving panitumimab or cetuximab were analysed for EGFR GCN by fluorescent in situ hybridization (FISH). Eight of nine patients with objective responses had an increased EGFR GCN, while among non responders only one patients had an increased EGFR GCN (P<0.0001). An interesting aspect of this trial is the demonstration of heterogeneity across tumor areas with regard to EGFR amplification with tumor areas showing normal genoma vs other areas showing clear amplification (Moroni et al, 2005). The predictive role of EGFR GCN has been evaluated in other subsequent clinical trials including mCRC patients treated with cetuximab (Lenz et al, 2006; Lievre et al, 2006). An increased EGFR GCN assessed by chromogenic in situ hybridization (CISH) was significantly associated with an objective tumor response to cetuximab (P=0.04) (Lievre et al, 2006). In another study conducted by Lenz et al the evaluation of EGFR GCN by polymerase chain reaction (PCR) was performed on 34 mCRC patients receiving cetuximab. However this trial have failed to show an association between GCN and response and PFS, but a significant positive correlation with OS was seen and it could reflect the role of EGRF GCN as an independent prognostic variable (Lenz et al, 2006). The discrepancies between these studies could be a result of methodologic differences for this reason prospective designed trials are needed to address this question. The association between EGFR GCN and clinical

V. Clinical and molecular predictors of response to Panitumumab A. Skin toxicity as a potential surrogate marker of panitumumab efficacy The major challenge for an optimal use of EGFR targeting drugs is to define which patients are more likely to have a therapeutic advantage from the treatment. Inhibition of EGFR by Mabs or tyrosine kinase inhibitors (TKIs) leads to the development of a characteristic papulopustular rush also related to as acneiform rush that is histologically characterized by a neutrophilic infiltrate in the dermal tissue particularly in the infundibular part of hair follicle (Busam et al, 2001). Data from several clinical trials with cetuximab show a positive correlation between rash and response and/or survival (Saif et al, 2007). The predictive value of skin toxicity severity for panitumumab 484

Cancer Therapy Vol 6, page 485! 33%), and a longer PFS 7.7 vs 5.5 months, respectively (Bokemeyer et al, 2008). KRAS mutation status has been also retrospective evaluated in tumor specimens from the phase I/II EVEREST trial, in which irinotecan-refractory mCRC patients were treated with irinotecan and escalating doses of cetuximab. Patients treated with cetuximab dose escalation have reported an higher RR compared to those receiving cetuximab at standard dose (46.4 % vs 21.1%, respectively), KRAS wild type patients achieved considerable benefit from irinotecan plus cetuximab treatment, while KRAS mutated patients did not profit from irinotecan plus cetuximab treatment and cetuximab dose escalation did not increase responses in these patients (Tejpar et al, 2008). In a series of 48 mCRC patients treated with cetuximab or panitumumab, it has been demonstrated that the presence of KRAS and/or of B-RAF mutations is negatively associated with the possibility of a PR (P=0.005), and the median TTP was significantly worse in patients bearing a mutated KRAS and/or B-RAF gene in their tumours (P=0.0259) as compared with those carrying wild-type genes (Benvenuti et al, 2007). KRAS status was retrospectively analyzed in metastatic CRC patients enrolled in the registratory panitumumab phase III trial. K-RAS mutations were found in 43% of 427 patients. In this trial PFS results significantly greater among wild type KRAS patients receiving panitumumab (12.3 weeks for panitumumab vs 7.3 weeks for BSC). While in KRAS mutated patients, in whom no panitumumab benefit was reported (HR=0.99), PFS was 7.4 vs 7.3 weeks in panitumumab and BSC group respectively. Wild type KRAS patients receiving panitumumab have also a better RR (17% vs 0%) and longer OS (HR=0.67, 95% CI 0.55 to 0.82) compared with mutant group (Amado et al, 2008). Another phase II trial (PRECEPT trial) has been designed to evaluate the effect of KRAS mutation status on the combination of panitumumab with FOLFIRI chemotherapy following first-line FOLFOX and bevacizumab. At moment preliminary data of first 51 patients suggest that this combination appear well tolerated and active (14% PR) as just observed in unselected population; however data of RR, OS and PFS according to KRAS status will be presented (Cohn et al, 2008).

outcome has been evaluated in a larger and more homogeneous patients cohort from the registratory phase III clinical trial. In patients treated with panitumumab (58 pts), a mean EGFR GCN <2.5/nucleus or <40% of tumor cells displaying chromosome 7 polysomy within the tumor predict for shorter PFS (P=0.039 and P=0.029, respectively) and OS (P=0.015 and P=0.014, respectively). None of treated patients had tumor response when the EGFR GNC was less than the value of <2.47/nucleus whereas 6/20 obtained objective response when EGFR GNC was superior than this cutoff value (P=0.0009). Six of 19 patients with chromosome 7 polysomy ! 43% obtained objective tumor response compared to none of 39 patients with chromosome 7 polysomy less then this value (P=0.0007). Evaluation of BSC-treated patients (34 pts) showed no correlation between EGFR GCN or chromosome 7 polysomy status and PFS, suggesting a predictive, rather than prognostic value of this genetic feature. These data confirm that non increased EGFR GCN is associated to failure of response to mAbs, conversely seem to emerge that only a fraction of tumors with increased EGFR GNC achieves objective response (Sartore-Bianchi et al, 2007).

C. RAS/RAF mutation status as a selection marker for panitumumab treatment EGFR signalling pathway includes the activation of downstream GTPases encoded by RAS genes. Mutations in the RAS family members results in EGFR-independent MAPK pathway activation and they are found in approximately 30% to 50% of CRC tumors, and are also common in other tumor types (Esteller et al, 2001; Malumbres et al, 2003). Several trials indicates that the presence of mutant K-RAS in lung and CRC tumors correlates with poor prognosis (Andreyev et al, 2001; Bazan et al, 2002) and is associated with lack of response to TKIs and mAbs (Pao et al, 2005; Lievre et al, 2006; Benvenuti et al, 2007; De Roock et al, 2007; Di Fiore et al, 2007). A retrospective molecular analyses to evaluate the influence of KRAS mutation status in first line patients treated with FOLFIRI with or without cetuximab enrolled in CRYSTAL study, has been recently reported. KRAS mutations were detected in 35.6% (192/540) of pts with evaluable samples. A statistically significant difference in favor of cetuximab was seen in KRAS wild type pts for PFS (P=0.0167) and best overall response [59.3% (cetuximab + FOLFIRI) vs 43.2% (FOLFIRI), P=0.0025]. Subgroup analyses in KRAS mutated patients show no significant differences between treatment groups for PFS (P=0.75; HR= 1.07) and best overall response (P=0.46) (Van Cutsem et al, 2008b). Efficacy analyses of the randomized phase II OPUS trial have previously failed to show a significant improvements in PFS or overall response when cetuximab was added to FOLFOX versus FOLFOX alone in the firstline mCRC treatment; however a following molecular analysis of KRAS status have show a higher RR in wild type KRAS patients compared to mutated patients (61% vs

VI. Conclusion The panitumumab have increased our repertoire of biological agents and is the first fully human mAbs approved for the treatment of mCRC. The advantage of this drug compared to other mAbs such as cetuximab is the less antigenic potential with less hypersensitivity reactions and no antibody formation. Panitumumab have demonstrated safety and promising activity in several phase I and II clinical trials, however it’s approval is based on a clinically modest but highly statistically significant improvement of PFS as showed in a single, open-label, randomized registratory phase III trial. PFS is a surrogate end point that is probably to predict effect on survival. However no difference in survival in favor to panitumumab were reported in this study and it may be 485

Bareschino et al: The role of panitumumab in metastatic colorectal cancer Arends R, Yang B, Schwab G, Lockbaum P, Funelas C, Roskos L (2005) Flexible dosing schedules of panitumumab (ABXEGF) in cancer patients (Abst 3089). J Clin Oncol 23, No 16S (June 1 Supplement). Arnold D, Höhler T, Dittrich C, Lordick F, Seufferlein T, Riemann J, Wöll E, Herrmann T, Zubel A, Schmoll HJ (2008) Cetuximab in combination with weekly 5fluorouracil/folinic acid and oxaliplatin (FUFOX) in untreated patients with advanced colorectal cancer: a phase Ib/II study of the AIO GI Group. Ann Oncol in press. Bazan V, Migliavacca M, Zanna I, Tubiolo C, Grassi N, Latteri MA, La Farina M, Albanese I, Dardanoni G, Salerno S, Tomasino RM, Labianca R, Gebbia N, Russo A (2002) Specific codon 13 K-ras mutations are predictive of clinical outcome in colorectal cancer patients, whereas codon 12 Kras mutations are associated with mucinous histotype. Ann Oncol 13, 1438-46. Bendell JC, Uronis H, Morse M, Blobe G, Aklilu M, Nixon A, Niedzweicki D, Ashton J, Howard L, Hurwitz H (2008) Initial results of a phase II study of oxaliplatin (OX), capecitabine (CAP), bevacizumab (BV), and cetuximab (CET) in the treatment of metastatic colorectal cancer (mCRC). 2008 Gastrointestinal Cancers Symposium. Benvenuti S, Sartore-Bianchi A, Di Nicolantonio F, Zanon C, Moroni M, Veronese S, Siena S, Bardelli A (2007) Oncogenic activation of the RAS/RAF signalling pathway impairs the response of metastatic colorectal cancer to antiepidermal growth factor receptor antibody therapies. Cancer Res 15, 2643-48. Berlin J, Posey J, Tchekmedyian S, Hu E, Chan D, Malik I, Yang L, Amado RG, Hecht JR (2007) Panitumumab with irinotecan/leucovorin/5-fluorouracil for first-line treatment of metastatic colorectal cancer. Clin Colorectal Cancer 6, 42732. Berlin J, Van Cutsem E, Peeters M, (2007) Predictive value of skin toxicity severity for response to panitumumab in patients with metastatic colorectal cancer (mCRC): A pooled analysis of five clinical trials (Abst. 4134). J Clin Oncol 25, No 18S (June 20 Supplement). Busam KJ, Capodieci P, Motzer R, Kiehn T, Phelan D, Halpern AC (2001) Cutaneous side-effects in cancer patients treated with factor receptor antibody c225. Br J Dermatol 144, 1169-76. Chua YJ, Cunningham D (2006) Panitumumab. Drugs today 42, 711-19. Chung KY, Shia J, Kemeny NE, Shah M, Schwartz GK, Tse A, Hamilton A, Pan D, Schrag D, Schwartz L, Klimstra DS, Fridman D, Kelsen DP, Saltz LB (2005)Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol. 23, 1803-10. Ciuleanu TE, Kurteva G, Ocvirk J, Beslija S, Koza I, Papamichael D, Vrbanec D, T. Brodowicz T, Scheithauer W, Zielinski CC (2008) A randomized, open-label CECOG phase II study evaluating the efficacy and safety of FOLFOX6 + cetuximab versus FOLFIRI + cetuximab as first-line therapy in patients (pts) with metastatic colorectal cancer (mCRC) (Abstr 4032). J Clin Oncol 26, No 15S (May 20 Supplement). Cohenuram M, Saif MW (2007) Epidermal growth factor receptor inhibition strategies in pancreatic cancer: past, present and the future. JOP 8, 4-15. Cohn AL, Smith DA, Neubauer MA, Houston G, Khandelwal P, Wiggans RG, Suzuki S, Yassine , Deeter R, Sikorski R (2008) Panitumumab (pmab) regimen evaluation in colorectal cancer to estimate primary response to treatment (PRECEPT): Effect of KRAS mutation status on second-line

due to several reasons as the rapid crossover study before the first tumor assessment and the high rate of patients in the panitumumab arm who had an unscheduled tumor evaluation before the first planned analysis. The addition of panitumumab to combination of irinotecan or oxaliplatin-based chemotherapy plus bevacizumab, as first line treatment (PACCE trial) have demonstrated no additive effect on PFS, even a shorter PFS in oxaliplatinum stratum, and an increased toxicity profiles for both combinations. However panitumumab efficacy in mCRC seem to be confined to wild type KRAS patients, indicating that KRAS status must be considered when selecting mCRC patients as candidates for this treatment. Finally confirmation of clinical benefit will be further required, and future research areas to investigate may be: the combination with other targeted agents, to test this drug in other setting of disease such as in curative settings, and to asses prospectively whether KRAS mutations may also influence response to panitumumab when combined to chemotherapeutic agents.

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Morse M, Chen HX (2007) Randomized phase II trial of cetuximab, bevacizumab, and irinotecan compared with cetuximab and bevacizumab alone in irinotecan-refractory colorectal cancer: the BOND-2 study. J Clin Oncol 25, 4557-61. Sartore-Bianchi A, Moroni M, Veronese S, Carnaghi C, Bajetta E, Luppi G, Sobrero A, Barone C, Cascinu S, Colucci G, Cortesi E, Nichelatti M, Gambacorta M, Siena S (2007) Epidermal growth factor receptor gene copy number and clinical outcome of metastatic colorectal cancer treated with panitumumab. J Clin Oncol 25, 3238-45. Schwartzberg LS, Hurwitz H, Stephenson J, Kotasek D, Goldstein D, Tebbutt N, McGreivy J, Y. Sun Y, Yang L, Burris H (2007) Safety and pharmacokinetics (PK) of AMG 706 with panitumumab plus FOLFIRI or FOLFOX for the treatment of patients (pts) with metastatic colorectal cancer (mCRC) (Abst 4081). J Clin Oncol 25, No 18S (June 20 Supplement). Shaheen RM, Ahmad SA, Liu W, Reinmuth N, Jung YD, Tseng WW, Drazan KE, Bucana CD, Hicklin DJ, Ellis LM (2001) Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors. Br J Cancer. 85, 584-89. Siena S, Peeters M, Van Cutsem E, Humblet Y, Conte P, Bajetta E, Comandini D, Bodoky G, Van Hazel G, Salek T, Wolf M, Devercelli G, Woolley M, Amado RG (2007) Association of progression-free survival with patient-reported outcomes and survival: results from a randomised phase 3 trial of panitumumab. Br J Cancer 97, 1469-74. Siena S, Tabernero J, Burkes RL, Cassidy J, Cunningham D, Barugel ME, Humblet Y, McPhie C, Shing M, Douillard J (2008) Phase III study (PRIME/20050203) of panitumumab (pmab) with FOLFOX compared with FOLFOX alone in patients (pts) with previously untreated metastatic colorectal cancer (mCRC): Pooled safety data. (Abst 4034).! J Clin Oncol 26, No 15S (May 20 Supplement). Simmonds PC (2000) Palliative chemotherapy for advanced colorectal cancer: systematic review and meta-analysis. Colorectal Cancer Collaborative Group. BMJ 321, 531-35. Sobrero AF, Maurel J, Fehrenbacher L, Scheithauer W, Abubakr YA, Lutz MP, Vega-Villegas ME, Eng C, Steinhauer EU, Prausova J, Lenz HJ, Borg C, Middleton G, Kröning H, Luppi G, Kisker O, Zubel A, Langer C, Kopit J, Burris HA 3rd (2008)EPIC: phase III trial of cetuximab plus irinotecan after fluoropyrimidine and oxaliplatin failure in patients with metastatic colorectal cancer. J Clin Oncol. 26, 2311-19. Souglakos J, Kalykaki A, Vamvakas L, Androulakis N, Kalbakis K, Agelaki S, Vardakis N, Tzardi M, Kotsakis AP, Gioulbasanis J, Tsetis D, Sfakiotaki G, Chatzidaki D, Mavroudis D, Georgoulias V (2007) Phase II trial of capecitabine and oxaliplatin (CAPOX) plus cetuximab in patients with metastatic colorectal cancer who progressed after oxaliplatin-based chemotherapy. Ann Oncol 2007 18, 305-10. Tabernero J, Van Cutsem E, Díaz-Rubio E, Cervantes A, Humblet Y, André T, Van Laethem JL, Soulié P, Casado E, Verslype C, Valera JS, Tortora G, Ciardiello F, Kisker O, de Gramont A (2007) Phase II trial of cetuximab in combination with fluorouracil, leucovorin, and oxaliplatin in the first-line treatment of metastatic colorectal cancer. J Clin Oncol 25, 5225-32 Tejpar S, Peeters M, Humblet Y, Vermorken JB, De Hertogh G, De Roock W, Nippgen J, von Heydebreck A, Stroh C, Van Cutsem E (2008) Relationship of efficacy with "#$% status (wild type versus mutant) in patients with irinotecanrefractory metastatic colorectal cancer (mCRC), treated with irinotecan (q2w) and escalating doses of cetuximab (q1w):

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Cancer Therapy Vol 6, page 489! The EVEREST experience (preliminary data) (Abstr 4001). J Clin Oncol 26, No 15S (May 20 Supplement). Tournigand C, AndrĂŠ T, Achille E, Lledo G, Flesh M, MeryMignard D, Quinaux E, Couteau C, Buyse M, Ganem G, Landi B, Colin P, Louvet C, de Gramont A (2004) FOLFIRI followed by FOLFOX6 or the reverse sequence in advanced colorectal cancer: A randomized GERCOR study. J Clin Oncol 22, 229-37. Van Cutsem E, Lang I, D'haens G, Moiseyenko V, Zaluski J, Folprecht G, Tejpar S, Kisker O, Stroh C, Rougier P (2008) KRAS status and efficacy in the first-line treatment of patients with metastatic colorectal cancer (mCRC) treated with FOLFIRI with or without cetuximab: The CRYSTAL experience (Abst 2). J Clin Oncol 26, No 15S (May 20 Supplement). Van Cutsem E, Nowacki M, Lang I, Cascinu S, Shchepotin I, Maurel J, Rougier P, Cunningham D, Nippgen J, KĂśhne C (2007) Randomized phase III study of irinotecan and 5FU/FA with or without cetuximab in the first-line treatment of patients with metastatic colorectal cancer (mCRC): The CRYSTAL trial (Abst 4000). J Clin Oncol 25, No 18S (June 20 Supplement). Van Cutsem E, Peeters M, Siena S, Humblet Y, Hendlisz A, Neyns B, Canon JL, Van Laethem JL, Maurel J, Richardson G, Wolf M, Amado RG (2007) Open-label phase III trial of panitumumab plus best supportive care compared with best supportive care alone in patients with chemotherapyrefractory metastatic colorectal cancer. J Clin Oncol 25, 1658-64. Van Cutsem E, Peeters M, Siena S, Humblet Y, Hendlisz A, Neyns B, Canon JL, Van Laethem JL, Wolf M, Amado RG (2007) A phase III randomized controlled trial of panitumumab (Pmab) in patients (pts) with metastatic colorectal cancer (mCRC): Subset analyses in elderly pts and in pts with poor performance status (Abst 349). Proceedings of the 2007 Gastrointestinal Cancers Symposium, Orlando. Van Cutsem E, Siena S, Humblet Y, Canon JL, Maurel J, Bajetta E, Neyns B, Kotasek D, Santoro A, Scheithauer W, Spadafora S, Amado RG, Hogan N, Peeters M (2008) An open-label, single-arm study assessing safety and efficacy of

panitumumab in patients with metastatic colorectal cancer refractory to standard chemotherapy. Annals Oncol 19, 9298. Weiner LM, Belldegrun AS, Crawford J, Tolcher AW, Lockbaum P, Arends RH, Navale L, Amado RG, Schwab G, Figlin RA (2008) Dose and schedule study of panitumumab monotherapy in patients with advanced solid malignancies. Clin Cancer Res 14, 502-08. Yang XD, Jia XC, Corvalan JR,Wang P, Davis CG (2001) Development of ABX-EGF, a fully human anti-EGF receptor monoclonal antibody, for cancer therapy. Crit Rev Oncol Hematol 38, 17-23. Yang XD, Jia XC, Corvalan JR,Wang P, Davis CG, Jakobovits A (1999) Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. Cancer Res 59,1236-43. Yarden Y, Sliwkowski MX (2001) Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2, 127-37.

Cesare Gridelli

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Extrathyroidal anaplastic transformation Case Report

Todd Swanson1, Senthilselvi Nanthakumar2, Samuel Bugis1, Sam M. Wiseman1,* 1

Department of Surgery, St. Paul’s Hospital & University of British Columbia, Vancouver, British Columbia,Canada Department of Pathology & Laboratory Medicine, St. Paul’s Hospital & University of British Columbia, Vancouver, British Columbia,Canada 2

__________________________________________________________________________________! *Correspondence: Sam Wiseman, MD, FRCSC Department of Surgery, St. Paul’s Hospital, Burrard Bldg., Room C-303, 1081 Burrard St., Vancouver, BC, V6Z 1Y6. Canada; Tel: (604) 806-9108; Fax: (604) 806-9957; E-mail: smwiseman@providencehealth.bc.ca Key words: Extrathyroidal anaplastic transformation Abbreviations: Anaplastic thyroid carcinoma, (ATC); differentiated thyroid cancer, (DTC); papillary thyroid cancer, (PTC) Received: 29 April 2008; Revised: 14 July 2008 Accepted: 16 July 2008; electronically published: September 2008

Summary Anaplastic thyroid carcinoma (ATC) is an uncommon and highly lethal endocrine malignancy. Accumulating clinical, pathological, and molecular evidence suggests that ATC represents the final end-point in the malignant progression of differentiated thyroid cancer (DTC). Previous reports have described ATC identified in neck lymph nodes only at the time of recurrence of differentiated thyroid cancers (papillary, follicular, and Hürthle cell carcinoma). We report a case of ATC that presented as disease identified within cervical lymph nodes of an individual diagnosed with regionally metastatic papillary thyroid cancer (PTC). This individual’s treatment included thyroidectomy and unilateral selective neck dissection followed by postoperative radioactive Iodine-131 therapy and external beam radiation treatment. Four years following treatment this individual has remained disease free. This unique case of the synchronous presentation of a PTC primary tumor and ATC in neck nodal metastasis provides further clinical evidence to support the concept that ATC transforms from pre-existing DTC. It also illustrates a good outcome for ATC detected early and treated aggressively.

thyroid cancer. Papillary, follicular, and Hürthle cell thyroid carcinomas have all been identified in association with ATC. A review of the world literature reported that from 23% to 90% of ATC may have an associated DTC component (Wiseman et al, 2003). Japanese investigators have reported recurrences of DTC in cervical lymph nodes leads to the development of progressively more aggressive and undifferentiated thyroid tumor characteristics (Ozaki et al, 1999). In a cohort of 14 patients these authors demonstrated that 15% of nodal recurrence eventually exhibited anaplastic characteristics. Several other investigators have also reported ATC as being diagnosed in cervical lymph node recurrences following a previously treated DTC (Kapp et al, 1982; Mooradian et al, 1983). Follicular thyroid cancer resection, followed by a diagnosis of ATC in lung metastasis, has also been reported (Moore et al, 1985). Papillary thyroid cancer (PTC) with associated ATC has also been described in extra-thyroidal tissues including brachial cleft cysts (Togashi et al, 2004) and medial ectopic thyroid tissue (Nussbaum et al, 1981). Hürthle cell thyroid cancer with associated mediastinal ATC has also been reported (Mai et al, 2000). We were unable to

I. Introduction Anaplastic thyroid cancer (ATC) represents one of the deadliest and most aggressive human malignancies. With widespread adoption of immunohistochemistry, ATC was histopathologically separated from medullary cancer and lymphoma of the thyroid in the 1980s (Holting et al, 1990). Historically, an ATC diagnosis implied a mean survival that ranged from 4 to 12 months and a dismal 5 year survival that ranged from 1.0% to 7.1% (Wiseman et al, 2003). Multimodal therapy, which includes surgery, chemotherapy, and radiation therapy, has recently improved ATC 3 year disease free survival (Crevoisier et al, 2004) in those individuals who can tolerate these aggressive treatment regimens. Cure is uncommon and with multimodal therapy ATC patients generally succumb to metastatic disease (Venkatesh and Ordonez, 1990). Traditionally mortality from ATC was secondary to locally advanced disease despite palliation with tracheostomy and/or tracheal stenting (Green and Mack, 2006). Accumulating clinical, pathological, and molecular evidence over the last two decades has supported the concept that ATC arises from pre-existing differentiated 491

Swanson et al: Extrathyroidal anaplastic transformation contained metastatic PTC. Two weeks later, the patient underwent a planned left selective left neck dissection for the palpable nodal metastases. Due to gross cancer involvement, the sternocleidomastoid muscle and internal jugular vein were resected, but the left accessory nerve was preserved. Pathologic evaluation revealed multiple cancer containing nodes. A matted mass of nodes replaced by metastatic PTC measured 4 cm x 2.5 cm x 2 cm in size. Within these level 2 neck nodes an associated focus of ATC was identified. The ATC was a spindle cell subtype (Figure 1). The metastatic PTC was positive for pancytokeratin and cytokeratin 19 immunostaining and the ATC focus was positive for pankeratin and vascular antigen CD31 and negative for cytokeratin19 and vascular markers Factor VIII and CD34 immunostaining. Postoperatively, the patient received 1850 MBq of Iodine-131 and 30 fractions of external beam radiation to his neck for a total dose of 60 Gy. A follow up radioactive iodine-123 scan carried out 1 month after completion of treatment did not demonstrate any evidence of residual disease. The patient was alive with no evidence of disease recurrence when followed up 4 years after treatment.

identify any prior reports of ATC being diagnosed in association with the nodal metastasis of a primary DTC at disease presentation.

II. Case Presentation A 68 year old male presented with a one year history of an enlarging left neck mass. His medical history was significant for a 10 year smoking history, coronary artery disease, WolffParkinson-White syndrome, and hypertension. He had no prior history of head and neck radiation exposure or family history of thyroid disease or cancer. Clinical examination revealed a firm 2.5 cm level 2 left neck mass and flexible nasopharyngoscopy revealed no mucosal abnormalities and normal vocal cord appearance and function. The patient subsequently underwent a fine needle aspiration of the left neck mass, which was diagnosed as metastatic PTC. A total thyroidectomy and central neck dissection was subsequently carried out and pathologic evaluation identified PTC (2 cm x 2 cm x 1.5 cm, follicular variant) arising from the left lobe of the thyroid and two of three lymph nodes removed

Figure 1. (A) Low power to contrast PTC (left) and adjacent anaplastic carcinoma (right) from lymph node metastasis. Scale for magnification at top for upper panel. (B) PTC (power 5 x "A"). (C) Anaplastic carcinoma (power 5x "A").

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Cancer Therapy Vol 6, page 493! development of more effective treatments for individuals diagnosed with this fatal thyroid malignancy.

III. Discussion Currently, the incidence of ATC has been decreasing while the incidence of DTC is on the rise (Agrawal et al, 1996). There are several possible explanations for this observed decline which include: the decrease in incidence of endemic goiters due to adoption of iodized salt into the diets of developing countries, and earlier and more aggressive treatment of DTC (Are and Shaha, 2006). The incidence of DTC has been steadily increasing for greater than 10 years due to an increase in the number of newly diagnosed small PTC (Davies and Welsh, 2006). Survival rates of individuals diagnosed with ATC in association with a DTC may be better than for individuals diagnosed with ATC without a DTC component (Rodriguez and Pinero, 2000). Generally most long-term ATC survivors have their tumor incidentally noted in the specimen that was either resected for DTC or benign disease (Wiseman et al, 2003). This observation suggests that early initiation of therapy at a more critical junction in the malignant progression of the ATC may impact patient outcome. Accumulating molecular evidence has provided insight into the progression of ATC from DTC. Intrachromosomal changes identified utilizing inter-simplesequence-repeat polymerase chain reaction has provided evidence for anaplastic transformation (Wiseman et al, 2003). There is a higher incidence of mutations in the p53 gene in ATC when compared to DTC (Fagin et al, 1993). Wild type p53 antigen is undetectable in normal thyroid cells as well as most DTC (Donghi et al, 1993). Shingu and colleagues, reported in 2000 a DTC that transformed into ATC in a nodal recurrence and found that the DTC over expressed the p53 protein as did the ATC. Apart from p53, many genes and their products may be involved in the transformation of DTC into ATC. A recent study from our center evaluating 12 ATC cases, with associated DTC components, for a panel of 63 molecular markers; identified thyroglobulin, Bcl-2, MIB-1, E-cadherin, p53, ! -catenin, topoisomerase II-" , and vascular endothelial growth factor as being altered during the transformation process (Wiseman et al, 2007). Other markers that have been reported to be altered, and possibly involved in anaplastic transformation include: Met, c-myc, Nm23, ras, Cyclin D1, Cyclin E, Aurora A, Aurora C, uPA-R, and HER4 (Wiseman et al, 2003, 2007). Currently in-vivo and in-vitro studies evaluating molecular targeted therapy for ATC treatment show exciting potential for future clinical application (Kim and Prichard, 2006).

References Agrawal S, Rao R, Parikh E (1996) Histological trends in thyroid cancer 1969-1993: a clinico-pathologic analysis of the relative proportion of anaplastic carcinoma of the thyroid. J Surg Onc 63, 251-255. Are C, Shaha A (2006) Anaplastic thyroid carcinoma: biology, pathogenesis, prognostic factors, and treatment approaches. Ann Surg Onc 13, 453-464. Crevoisier R, Baudin E, Bachelot A (2004) Combined treatment of anaplastic thyroid carcinoma with surgery, chemotherapy and hyperfractionated accelerated external radiotherapy. Int J Radiat Oncol Biol Phys 60, 1137-1143. Davies L, Welsh G (2006) Increasing incidence of thyroid cancer in the United States1973-2002. JAMA 295, 2164- 2167. Donghi R, Longoni A, Pilotti S, Michieli P, Della Porta G, Pierotti MA (1993) Gene p53 mutations are restricted to poorly differentiated and undifferentiated carcinomas of the thyroid gland. J Clin Invest!91, 1753-1760. Fagin JA, Matsuo K, Karmakar A, Chen DL, Tang SH, Koeffler HP (1993) High Prevalence of Mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J Clin Invest!91, 179-184. Green L, Mack L (2006) Anaplastic thyroid cancer and primary thyroid lymphoma: a review of these rare thyroid malignancies. J Surg Onc 94, 725-736. Hölting T, Möller P, Tschahargane C, Meybier H, Buhr H, Herfarth C (1990) Immunohistochemical reclassification of anaplastic carcinoma reveals small and giant cell lymphoma. World J Surg14, 291-295. Kapp D, Livolsi V, Sanders M (1982) Anaplastic carcinoma following well-differentiated thyroid cancer: etiological considerations. Yale J Biol Med 55, 521-528. Kim S, Prichard C (2006) Cetuximab and irinotecan interact synergistically to inhibit the growth of orthotic anaplastic thyroid carcinoma xenografts in nude mice. Clin Cancer Res 12, 600-607. Mai D, Mai K, Shamji F (2000) Fine needle aspiration biopsy of anaplastic thyroid carcinoma developing from a Hürthle cell tumor: a case report. Acta Cytol 45, 761-764. Mooradian AD, Allam CK, Khalil MF, Salti I, Salem PA (1983) Anaplastic transformation of thyroid cancer: report of two cases and review of the literature. J Surg Oncol 23, 95-98. Moore J, Bacharach B, Choi H (1985) Anaplastic transformation of metastatic follicular carcinoma of the thyroid. J Surg Onc 29, 216-221. Nussbaum M, Buchwald RP, Ribner A, Mori K, Litwins J (1981) Anaplastic carcinoma arising from median ectopic thyoid (thyroglossal duct remnant). Cancer!48, 2724-2728. Ozaki O, Ito K, Mimura T, Sugino K, Ito K (1999) Anaplastic transformation of papillary thyroid carcinoma in recurrent disease in regional lymph nodes: A histological and immunohistochemical Study. J Surg Onc 70, 45-48. Rodriguez J, Pinero A (2000) Clinical and histological differences in anaplastic thyroid carcinoma. Euro J Surg 166, 34-38. Shingu K, Kobayashi S, Yokoyama S, Fujimori M, Asanuma K, Ito KI, Hama Y, Maruyama M, Kusama R, Amano J (2000) Likely transformation of papillary thyroid carcinoma into anaplastic carcinoma during postoperative radioactive iodine131 therapy: report of a case. Surg Today 30, 910-913. Togashi S, Oka K, Kanayama R, Koyamatsu S, Tobita T, Yatabe Y, Matsumoto T, Hakozaki H (2004) Thyroid anaplastic

IV. Conclusions Ongoing molecular study may allow for identification of DTCs, which are at greatest risk of undergoing anaplastic transformation. More aggressive initial therapy of these ‘transformation prone’ tumors may further decrease the mortality due to ATC by preventing it from ever developing. Targeted therapeutic drugs are currently being applied to ATC and could build on the treatment benefits recently demonstrated using multimodal therapy. Our case illustrates how a good outcome in ATC can be accomplished with aggressive multimodal therapy. Overall, recent study of the transformation of DTC into ATC has offered insights that will be important for the 493

Swanson et al: Extrathyroidal anaplastic transformation carcinoma transformed from papillary carcinoma in extrathryoid areas!"Auris Nasus Larynx 31, 287-292. Venkatesh Y, Ordonez N (1990) Anaplastic carcinoma of the thyroid. Cancer 66, 321-330. Wiseman S, Loree TL, Hicks WL (2003) Anaplastic thyroid cancer evolved from papillary carcinoma: demonstration of anaplastic transformation by means of the inter-simple sequence repeat polymerase chain reaction. Arch Otolaryngol Head Neck Surg 129, 96-100. Wiseman SM, Griffith OL, Deen S, Rajput A, Masoudi H, Gilks B, Goldstein L, Gown A, Jones SJ (2007) Identification of Molecular Markers Altered During Transformation of Differentiated Into Anaplastic Thyroid Carcinoma. Arch Surg 142, 717-727. Wiseman SM, Loree TR, Rigual NR, Hicks WL Jr, Douglas WG, Anderson GR, Stoler DL (2003) Anaplastic transformation of thyroid cancer: review of clinical, pathological, and molecular evidence provides new insights into disease biology and future therapy. Head Neck 25, 662-670.

Sam M. Wiseman

Wiseman SM, Masoudi H, Niblock P, Turbin D, Rajput A, Hay J, Bugis S, Filipenko D, Huntsman D, Gilks B (2007) Anaplastic Thyroid Carcinoma: Expression Profile of Targets for Therapy Offers New Insights for Disease Treatment. Ann Surg Onc14, 719-729.

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From here to eternity - the secret of Pharaohs: Therapeutic potential of black cumin seeds and beyond Review Article

Subhash Padhye, Sanjeev Banerjee, Aamir Ahmad, Ramzi Mohammad, Fazlul H Sarkar* Department of Pathology and Division of Internal Medicine, Barbara Ann Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI- 48201, USA

__________________________________________________________________________________! *Correspondence: Fazlul H Sarkar, Department of Pathology and Division of Internal Medicine, Barbara Ann Karmanos Cancer Institute, Wayne State University, School of Medicine, Room-740 HWCRC Bldg., 110 E Warren, Detroit, MI 48201, USA; Tel: 313576-8327; Fax: 313-576-8389; E-mail: fsarkar@med.wayne.edu Key words: Thymoquinone, Chemoprevention, Chemotherapy Abbreviations: 1, 2-dimethyl hydrazine, (DMH); 2,2/-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), (ABTS); 2,2'-diphenyl-ppicrylhydrazyl, (DPPH); 20-methylcholanthrene, (MC); 5-fluorouracil, (5-FU); 5-lipooxynenase, (5-LOX); Advanced Glycation End Products, (AGEs); angiotensin II, (AT II); buthionine sulfoximine, (BSO); butylated hydroxytoluene, (BHT); carbon tetrachloride, (CCl4); chromosomal aberrations, (CAs); diabetes mellitus, (DM); dihydrothymoquinone, (DHTQ); dithymoquinone, (DTQ); Dithymoquinone, (TQ2, III); doxorubicin, (DOX); Epigallocatechin-3-gallate, (EGCG); Experimental Allergic Encephalitis, (EAE); fibrosarcoma, (FsaR); Gentamicin, (GM); glutathione peroxidase, (GPx); glutathione, (GSH); High Performance Liquid Chromatography, (HPLC); hyperhomocysteinemia, (HHcy); interleukin-6, (IL-6); Leucotiriene-C4-synthase, (LT4synthase); leukotrienes, (LT); levo-dopa, (L-dopa); lipopolysaccharide, (LPS); Long-Evans Tokushima Otsuka, (LETO); malondialdehyde (MDA); median inhibitory concentration, (IC50); mitogen-activated protein kinases, (MAPKs); multi-drug resistant, (MDR); Multiple Sclerosis, (MS); N(omega)-nitro-l-arginine methyl esters, (l-NAME); N-Acetyl Glucosamine, (NAG); nitrate/nitrite, (NOx); nitric oxide, (NO); nuclear factor kappa B, (NF-! B); Otsuka Long-Evans Tokushima Fatty, (OLETF); ovalbumin, (OVA); polobox domain, (PBD); Pololike kinase 1, (Plk1); proximal tubular epithelial cells, (pTECs); proximal tubular epithelial cells, (pTECs); quinone-reductase, (QR); reactive nitrogen species, (RNS); squamous cell carcinoma, (SCC VII); streptozotocin, (STZ); superoxide dismutase, (SOD); tert-butyl hydroperoxide, (TBHP); tert-butylhydroquinone, (TBHQ); Thin Layer Chromatography, (TLC); thiobarbituric acid-reactive substances, (TBARS); Thymohydroquinone, (THQ); Thymoquinone, (I); thymoquinone, (TQ) Received: 23 July 2008; Revised: 22 August 2008 Accepted: 25 August 2008; electronically published: September 2008

Summary Over many centuries humans have been mining the bounties of nature for discovering substances that have been used for the treatment of all human diseases; many such remedies are useful even today as modern day medicine. Emerging evidence also suggests that the search is still continuing for harnessing active compounds from nature in combating human illnesses although pharmaceutical industries are equally active for synthesizing small molecule compounds as novel therapeutics. The lesson learned over many centuries clearly suggests that further sophisticated search for finding compounds from natural resources together with robust characterization and chemical synthesis will lead to the discovery of novel drugs that may have high therapeutic efficacy against all human diseases including cancer. Black cumin seed (Nigella sativa) oil extracts have been used for many centuries for the treatment of many human illnesses, and more recently the active compound found in black seed oil, viz. thymoquinone (TQ) has been tested for its efficacy against several diseases including cancer. However, further research is needed in order to assess the full potential of TQ as a chemopreventive and/or therapeutic agent against cancers. Here, we have summarized what is known regarding the value of black seed oil and its active compound TQ, and how this knowledge will help us to advance further research in this field by creating awareness among scientists and health professionals in order to appreciate the medicinal value of TQ and beyond.

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Padhye et al: Chemopreventive and chemotherapeutic potential of Thymoquinone! yellow, pink, pale blue or pale purple, with 5-10 petals. The fruit is a capsule composed of several united follicles, each containing numerous seeds while in some species (e.g. !"#$%%&'4&-&(/$.&), the capsule is large and inflated. The parts of the plant most commonly used for the therapeutic purposes in the “Alternative Medicinal” systems are the seeds (Figure 1B) which are contained in an inflated capsule formed from the united follicles containing considerable amount of oil having pungent and bitter taste. Commonly the seeds are used primarily as a spice and food preservative. In folk medicinal practices they are ingested with food or mixed with honey and are primarily used as lactogogues, carminitative and antihelmnthic agents. The seeds have also been used as diuretics, anti-hypertensive, muscle relaxants and as immunity enhancers in immune-compromised people. Importantly, the seeds have been reported to be safe when used orally in moderate amount in food (DerMarderosian. et al, 2005). They have been shown to stimulate uterine contractions when used in large amounts, leading to abortion (Aqel and Shaheen, 1996). There are reports that the oil from the seeds can be used to treat dermatitis topically (Zedlitz et al, 2002). Several beneficial pharmacological effects have been attributed to various crude or purified components of these seeds including antihistaminic (Chakravorty, 1993), antihypertensive (Zaoui et al, 2000), hypoglycemic (Al-Hader et al, 1993), antifungal (Khan et al, 2003), anti-inflammatory (AlGhamdi, 2001) along with significant anti-neoplastic (Worthen et al, 1998) activities. These studies collectively provide early indication that further development of agents derived from black cumin seeds could be useful in modern medicine.

I. Introduction Out of the several accompanying articles found in the tomb of Egyptian Pharaoh Tutankhamen were the seeds of Black cumin [!"#$%%&' (&)"*&; (Zohary and Hopf, 2001)] not to be mistaken with common cumin seed (+,-".,-' /0-".,-). It is a spice that grows in the Mediterranean region and in Western Asian countries including India, Pakistan and Afghanistan. The historical references to these seeds are also found in some of the oldest religious and medical texts. For example, it is referred to as ‘Melanthion’ by Hippocrates and Dioscorides, while the Bible describes it as the ‘curative black cumin’ (Isaiah 28:25, 27 NKJV). It is, therefore, no wonder that they were thought to be worthy accompaniments in the ‘From Life here to Eternity’ by the pharaoh as described earlier. The black cumin herb goes by many different names. For example, in old Latin it is called as ‘Panacea’ meaning ‘cure all’ while in Arabic it is termed as ‘Habbah Sawda’ or ‘Habbat el Baraka’ translated as ‘Seeds of blessing’. In India it is called as Kalonji while in China it is referred as Hak Jung Chou (Aggarwal et al, 2008). The plant belongs to the 1&.,./,%&/$&$' family of flowering plants and genus of about 14 species including !"#$%%&' &2*$.("(3 !"#$%%&' /"%"&2"(3' !"#$%%&' 4&-&(/$.$3' !"#$%%&' 5"(6&."/&3' !"#$%%&' ".)$#2"78%"&3' !"#$%%&' ."#$%%&()2,-3' !"#$%%&' 82"$.)&%"(' &.4' !"#$%%&' (&)"*&3' 2$(6$/)"*$%0. Among these, !"#$%%&' (&)"*& is the species most exhaustively investigated for therapeutic purposes although other species have also been implicated for therapeutic uses (Aggarwal et al, 2008). The species grow to 20-30 cm tall, with finely divided leaves wherein the leaf segments are narrowly linear to threadlike (Figure 1A). The flowers are white,

Figure-1. (A) Morphological features of !"#$%%&'(&)"*&'plant, and (B) black cumin seeds containing oil having Thymoquinone (TQ) as the active principle.

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Cancer Therapy Vol 6, page 497!

A. Chemistry of the active compounds found in black seed extracts

constituents in the oil including TQ and Thymohydroquinone (THQ, termed ‘Nigellone’ by earlier workers) depends upon storage conditions. This is an important reminder to the researchers using oil as a source of test substance in various biological assays because such results may include a factor of variability depending upon the source and storage conditions of the Black seed oil. A HPLC method for quantifying the putative pharmacologically active constituents: TQ, DTQ (dithymoquinone), THQ, and thymol (THY), in the oil of !"#$%%&'(&)"*& seed has been described earlier (Ghosheh et al, 1999) employing a reversed-phase C18 analytical column and by using an isocratic mobile phase of water:methanol:2-propanol (50:45:5% v/v) at a flow rate of 2 ml min-1 and UV detection at 254 nm for TQ, DTQ, and THY, and at 294 nm for THQ. This method provides a good quality control methodology for the pharmacologically active components in these widely used !"#$%%&' (&)"*& extracts as a natural remedy for many human illnesses. Although TQ has been investigated presently for determining its therapeutic potential, the other constituents of !"#$%%&' (&)"*& seed oil also deserve further investigation for assessing their therapeutic value. Chemically, TQ belongs to 2, 5-di-substituted benzoquinone class of compounds having methyl and isopropyl groups at C-2 and C-5 positions, respectively. The compound can be readily prepared in gram quantities by oxidation of thymol as shown previously (Dockal et al, 1955). Since many of the biological activities of TQ largely originate from its antioxidant properties, the electrochemical characteristics are important. Michelitsch and Rittmannsberger (Michelitsch and Rittmannsberger, 2003) have described the polarographic behavior of TQ in Sörensen buffer: methanol (3:7, v/v; pH 8.5) and have found that the compound exhibits a single, reversible peak at dropping mercury electrode at -0.095 V vs. Ag/AgCl electrode. This perhaps can explain its facile interconversion under biological environment leading to its antioxidant nature. The polarographic method can be applied to determine TQ in black seed oil preparations where the limit of detection has been calculated to be 0.05µg/ml.

Most of the earlier studies on this plant involved use of either the seeds or the oil extracted from it. For example, the ethanol extract of !"#$%%&' (&)"*& seeds has been shown to possess antitumor activity as well as lifespan expanding activity in mice bearing Ehrlich ascites tumor cells by Musa and co-workers (Musa et al, 2004). Benkaci-Ali and co-workers (Benkaci-Ali et al, 2006) investigated different methods of extracting oil from !"#$%%&' (&)"*& and found that microwave extraction gave the best results in terms of reduction of extraction time and better yield in obtaining higher amounts of oil, and this is particularly significant since microwave extraction is a green technique reducing environmental burden. The chemical composition of the black !"#$%%&'(&)"*& seed is diverse and contains amino acids, proteins, carbohydrates, fixed and volatile oils, alkaloids, saponins and many other compounds. Thin Layer Chromatography (TLC) screening of the oil samples showed the presence of four main components, viz. thymoquinone, carvacrol, tanethole and 4-terpineol, which demonstrated respectable radical scavenging property. These four constituents and the essential oil possessed variable antioxidant activity when tested in the 2,2'-diphenyl-p-picrylhydrazyl (DPPH) assay for non-specific hydrogen atom or electron donation. The oil samples showed variable antioxidant activity which was ascribed mainly to the variable composition of these constituents (Abou Basha et al, 1995). According to the common practices of ‘evidencebased herbal medicine’, the bioactive constituents of the volatile oil of black seed (54%) were identified by ElDakhakhany in 1963 showing that Thymoquinone (I) or, in short, TQ was the main active constituent of volatile oil of the black seed although it is accompanied by other analogous compounds such as Thymol (II) and Thymoquinone dimer named as Dithymoquinone (TQ2, III) (Figure 2). Ghosheh et al. (Ghosheh et al, 1999) carried out High Performance Liquid Chromatography (HPLC) analysis of the oil of !"#$%%&' (&)"*& using the isocratic mobile phase of water-methanol-2-propanol (50:45:5 %v/v) which revealed that the concentration of different

Figure-2. Chemical structures of Thymoquinone, Thymol and Dithymoquinone.

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Padhye et al: Chemopreventive and chemotherapeutic potential of Thymoquinone! Additionally, it was able to produce significant reductions in hepatic antioxidant enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase. It has been shown that TQ could inhibit iron-dependent microsomal lipid peroxidation efficiently in rats with doxorubicin-induced hyperlipidemic nephropathy (Badary et al, 2000). The compound was observed to decrease cellular oxidative stress by inducing glutathione in experimental allergic encephalomyelitis in female Lewis rats (Mohamed et al, 2003). Multiple epidemiological studies have shown that a high intake of antioxidant rich foods is inversely related to cancer risk (Borek, 2004). Recent experimental and clinical studies have implicated oxidative stress in the development and progression of different cancers (Kim et al, 2004; Pathak et al, 2005). Present findings suggest that TQ has a potent chemopreventive potential of inhibiting the process of carcinogenesis by modulating lipid peroxidation and cellular antioxidant milieu (Badary, et al, 1999; Badary et al, 2007). Wilson and co-workers (Wilson-Simpson et al, 2007) have analyzed the effect of low and high doses of Epigallocatechin-3-gallate (EGCG), Selenium, and TQ on ES-2 ovarian cells at 24, 48, and 72 h in terms of morphology, cell count, and biochemical markers. During this phase I study, experimental groups were administered physiological doses of specific antioxidants, EGCG, selenium, and TQ. Selenium exhibited the largest effect on biochemical assays. Analysis of collected data showed that the antioxidants suppress metabolic activity, alter behavioral responses, and cause molecular damage. However, it has not been shown that the use of antioxidants results in total destruction of ES-2 ovarian cancer cells. Norwood et al. (Norwood et al, 2007) further confirmed the effects of sustained drug delivery of TQ, EGCG and 5-fluorouracil (5-FU) on the metabolic activity as well as structural changes in the SW-626 human colon cancer cells. Their results indicate that the sustained drug delivery of EGCG and TQ induces significant cellular destruction and interference of cellular metabolic functions which are comparable with damages caused by sustained drug delivery of 5-FU. Morphological cellular changes occurring at 24 h after exposure to these two agents are also comparable to those caused by 5-FU. Whether these results are indicative of a safer alternative to 5-FU using natural photochemicals remains to be seen and thus further in-depth investigations are needed. Sayed-Ahmed and co-worker (Sayed-Ahmed and Nagi, 2007) have investigated the possible protective effects of TQ against Gentamicin (GM)-induced nephrotoxicity. Supplementation with TQ resulted in significant decrease in reduced glutathione (GSH), and increased levels of glutathione peroxidase (GPx), catalase and ATP, and a complete reversal of the GM-induced increase in blood urea nitrogen, creatinine, thiobarbituric acid-reactive substances (TBARS) and total nitrate/nitrite (NOx), and decrease in GSH, GPx, CAT and ATP to control values. Histopathological examination of kidney tissues confirmed the biochemical data wherein TQ supplementation prevents GM-induced degenerative changes in kidney tissues, suggesting that these effects, at

Khalife and Lupidi (Khalife and Lupidi, 2007) have found that TQ reacts with GSH, NADH and NADPH chemically. Such reactions occurring under the physiological conditions clearly indicate the formation of two products, viz. glutathione dihydrothymoquinone after rapid reaction with GSH and dihydrothymoquinone (DHTQ) after slow reaction with NADH and NADPH, respectively. Measurement of antioxidant activity of the reduced compounds against organic radicals such as 2,2/azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and DPPH revealed a potential scavenging activity for glutathionyl-dihydrothymoquinone similar to that of DHTQ while TQ showed lower scavenging activities. It is interesting to note that these compounds exhibit antioxidant capacity equivalent to Trolox which is considered as a standard antioxidant compound. These results indicate a possible intracellular non-enzymatic activation of TQ dependent upon GSH, NADH and NADPH representing perhaps the ‘cellular switch’ for modulating cellular antioxidant defenses. The crystal structure of TQ was determined by Pagola and colleagues in 2004 using high-resolution powder diffraction which indicated that it belongs to the triclinic system with Z = 2 and space group P . The thermal analysis performed on the crystals indicated that weak van der Walls forces are present in the molecules in the solid state and that only one crystalline system exists in the compound. Availability of this crystal structure opens up the possibility of using it for molecular modeling in various protein ligands.

B. Biological Activities of TQ Amongst the various bioactivities examined for TQ or the extracts of black seed oil, most are concerned with the anticancer or the antioxidant potential although only few have shown a direct correlation between the two. Several possible targets have been suggested for the observed activities and yet there seems to be no unique target. In this connection recent studies reported by Kaseb et al. have indicated effects of TQ on cell cycle regulatory and proapoptotic proteins in prostate cancer cells (Kaseb et al, 2007), and more recent studies have documented the cancer cell specific effects of TQ affecting multiple targets (Sethi et al, 2008; Yi et al, 2008; Aggarwal et al, 2008), suggesting that TQ deserves further in-depth investigations for delineating its role as an anticancer agent. Moreover, a few studies have been devoted to antiinflammatory properties and they obviously have connotations to the anti-cancer activities of TQ and thus prompt further in-depth investigation as indicated below.

1. Antioxidant Activity TQ has been shown to exhibit antioxidant property through different mechanisms in several recent reports. For example, it inhibits the production of 5-hydroxyeicosa-tetraenaoic as well as 5-lipoxygenase products (ElDakhakhny et al, 2002), both of which are required for the viability of colon cancer cells. It was shown to work as a scavenger of various reactive oxygen species including superoxide radical anion and hydroxyl radicals (Kruk et al, 2000; Mansour et al, 2002; Badary et al, 2003). 498

Cancer Therapy Vol 6, page 499! mg/kg). The smaller dose of TQ (5 mg/kg) produced partial protection, which clearly suggests that the effects of TQ could be partly due to its antioxidant activity. Mansour and co-workers have studied in 2002 the effects of TQ on antioxidant enzymes, lipid peroxidation and DT-diaphorase in hepatic, cardiac and kidney tissues of normal mice. Treatment with the different doses of TQ produced significant reductions in hepatic SOD, CAT and GSH-Px activities although neither produced any change in GST activity nor influenced reduced glutathione content in any of the tissues studied. These differences were attributed to varying concentrations of DT-diphorase enzyme present in different tissues; highest being in the hepatic tissues which was responsible for the reduction of TQ to DHTQ. These results revealed that TQ and DHTQ acted not only as superoxide anion scavengers but as general free radical scavengers. The median inhibitory concentration (IC50) values for TQ and DHTQ in biochemical and photochemical assays were in the nanomolar and micromolar ranges, respectively. These authors have suggested that the reported beneficial !"#$!$' protective effects of TQ are likely to be through the combined antioxidant properties of both compounds, which needs further confirmatory studies. Badary and Gamal have studied in 2001 the inhibitory effects of TQ against 20-methylcholanthrene (MC)-induced fibrosarcoma tumorigenesis where the compound delayed the onset of MC-induced fibrosarcoma tumors that appeared at 12 weeks and produced less MC-induced mortality. Compound alone showed a significant induction in the enzyme activities of hepatic GST and quinonereductase (QR). Mice treated with TQ along with MC showed reduction in hepatic lipid peroxides and increased GSH content and increased enzyme activities of GST and QR compared to the control group. The IC50 value for TQ against fibrosarcoma cells was found to be 15 ÂľM, indicating its potential as a powerful chemopreventive and/or therapeutic agent. The effects of TQ on carbon tetrachloride (CCl4)induced hepatotoxicity have been investigated in male Swiss albino mice by Nagi and colleagues in 1999 (Nagi et al, 1999). Oral administration of TQ in a single dose (100 mg/Kg) resulted in a significant protection against the hepatotoxic effects of CCl4. When tested as a substrate for mice hepatic DT-diaphorase in the presence of NADH, TQ was found to undergo reduction to DHTQ which turned out to be more potent than TQ and butylated hydroxytoluene (BHT). The IC50 values for DHTQ, TQ and BHT were found to be 0.34, 0.87 and 0.58 ÂľM, respectively. The data suggests that the protective action of TQ against CCl4-induced hepatotoxicity may be mediated through the combined antioxidant properties of TQ and its metabolite DHTQ. The influence of TQ on doxorubicin (DOX)-induced hyperlipidemic nephropathy and oxidative stress in rats has been examined by al-Shabanah and colleagues in 1998. Treatment with TQ significantly suppressed DOXinduced proteinuria, albuminuria, and urinary excretion of N-Acetyl Glucosamine (NAG) which confirmed the involvement of free radicals in the pathogenesis of nephropathy induced by DOX. The study suggested that

least in part, may be related to the ability of TQ to modulate cellular oxidative stress. The protective effects of TQ have been assessed by Khattab and Nagi in 2007 in rats after chronic inhibition of nitric oxide (NO) synthesis with N (omega)-nitro-l-arginine methyl esters (l-NAME). Treatment with TQ decreased the elevated creatinine and increased GSH levels compared to normal levels and inhibited the !"# $!%&' production of superoxide radicals in enzymatic and non-enzymatic systems, thus offering protection against l-NAME-induced hypertension and renal damage possibly via antioxidant activity of TQ. Both (!)*++,# -,%!$, oil and TQ can partly protect gastric mucosa from acute alcohol-induced mucosal injury which is partly ascribed to their radical scavenging activity (Kanter et al, 2005). The gastroprotective activity of (!)*++,# -,%!$, oil and TQ against gastric mucosal injury induced by ischemia/reperfusion in rats was also investigated by El-Abhar and co-workers (El-Abhar et al, 2003) who found that both agents offer protection against the gastric lesions and these might be related to the conversion of the gastric mucosal redox state. Interestingly, Farah and colleagues in 2005 have compared the effects of the water soluble and lipid soluble fractions of Black seed and pure TQ on A549 cells in culture for 24, 48 and 72 h wherein the cell number was found to decrease and remained so for the duration of the study. The water soluble fraction showed a trend similar to TQ but the ethanol fraction showed a negative shift in cell number at 48 h when compared with the control. This is contrary to the expectation that lipid soluble TQ shall accompany the ethanol fraction and hence needs further corroboration from the HPLC profiles of both the fractions, which clearly suggests further in-depth investigation. The state of hyperhomocysteinemia (HHcy) appears to be associated with higher risks of coronary, cerebral and peripheral vascular diseases as well as a number of other clinical conditions and is thought to be capable of inducing a pathogenic state of oxidative stress although its underlying molecular mechanisms are not fully elucidated. El-Saleh and co-workers (El-Saleh et al, 2004) have shown that active antioxidant components of black seeds of (!)*++,#-,%!$, plants are capable of rendering protection against the development of methionine-induced HHcy and its associated state of oxidative stress. Pre-treatment of rats with an oral dose of 100 mg/kg of TQ for 30 min and for one week provided complete protection against induced HHcy after methionine load (100 mg/kg). Under the state of induced HHcy, there were significant increases in the plasma levels of triglycerides, lipid peroxidation, and cholesterol as well as in the activities of glutathione peroxidase and SOD although catalase activity was not affected. The total antioxidant status was significantly depressed. All of these effects were almost totally blocked by the prior treatment with TQ. Mahgoub has investigated in 2003 the effects of TQ on acetic acid-induced (intracolonic injection of 3% acetic acid) colitis in rats. The study showed that pre-treatment of rats for 3 days with TQ (10 mg/kg) was able to give complete protection against acetic acid-induced colitis as against the control group treated with significantly higher dose of sulfasalazine (500

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Padhye et al: Chemopreventive and chemotherapeutic potential of Thymoquinone! lead to a delay in cancer progression and may improve patient’s morbidity and mortality as well. Arachidonic acid is another example of a major precursor of several classes of signal transduction molecules whose metabolism is altered significantly in human carcinogenesis. For example, it has been well established that 5-lipooxynenase (5-LOX) converts arachidonic acid to hydroxyl-eicosaterraenoic acids or leukotrienes (LT) which, in turn, enhance proliferation and survival and suppress apoptosis of human cells. It may, therefore, be expected that inhibition of 5-LOX protein may lead to apoptosis (Hoque et al, 2005). As a result, potential of TQ in suppressing inflammation through inhibition of leukotrienes constitutes a very active area of research at the present time. The compound is reported to be a potent inhibitor of leukotrienes formation in human blood cells (Mansour and Tornhamre, 2004). The inhibitory effect was found to be dose as well as time-dependent, and the effect was exerted on both 5-lipooxgenase and Leucotiriene-C4-synthase (LT4synthase) activity (Mansour and Tornhamre, 2004). In another study (Mahgoub, 2003), the rats pre-treated with oral TQ doses showed complete protection against acetic acid-induced colitis compared to sulfasalazine (500 mg/kg) control group wherein TQ was found to suppress the production of NO by macrophages which is useful in ameliorating the inflammatory and autoimmune conditions (El-Mahmoudy et al, 2002). El-Dakhakhny and co-workers have evaluated in 2002 the effects of (!)*++,# -,%!$, oil, TQ and polythymoquinone (Nigellone) on the synthesis of 5-LOX products from polymorphonuclear leukocytes from rats. TQ inhibited the production of 5-LOX products (IC50= 0.26 mg/ml) and 5-HETE production (IC50=0.36 mg/ml) which may be ascribed to its antioxidant potential. These observations explain the traditional use of (!)*++,# -,%!$, oil for ameliorating inflammatory conditions in various folk medicinal systems. El-Gazzar et al, reported in 2007 that TQ inhibits LPS-induced pro-inflammatory cytokine production in RBL-2H3 cells by blocking GATA transcription factor expression and promoter binding, which demonstrates its anti-inflammatory effect (ElGazzar et al, 2007). The compound inhibits LPS-induced IL-5 and IL-13 mRNA expression as well as protein production but not the production of IL-10. Since LTs are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells by Mansour and his coworker in 2004 found that it provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 µM, respectively, at 15 min. Their major inhibitory effect was on the 5-lipoxygenase activity (IC50=3µM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-HETE in sonicated polymorphonuclear cell suspensions. Staurosporine, which is an unselective protein kinase inhibitor, failed to prevent inhibition of LTC4 synthase activity induced by TQ which clearly indicates that the compound indeed inhibits the

TQ might be applicable as a protective agent against proteinuria and hyperlipidemia associated with nephrotic syndrome. The cardio toxicity of the widely used antitumor agent, Doxorubicin, has been suggested to result from the generation of oxygen free radicals. These workers observed that TQ offers protection against doxorubicin-induced cardio toxicity without compromising its antitumor activity. This finding was based on significant reductions in serum lactate dehydrogenase and creatine kinase elevated levels, and further supplemented by histopathological examination of cardiac tissue. The compound did not alter the plasma and heart DOX levels as monitored by fluorometric analysis. TQ has also been tested in isolated rat hepatocytes as a hepatoprotective agent against the toxicity inflicted by tert-butyl hydroperoxide (TBHP) by Daba and AbdelRahman in 1998 and compared against the protection offered by known hepatoprotective agent, silybin. Although both compounds prevented TBHP-induced depletion of GSH to the same extent, degree of protection by TQ against the liver enzyme leakage was less than that by silybin. The antioxidant and pro-oxidant effects of TQ and a synthetic structurally-related compound, viz. tertbutylhydroquinone (TBHQ), were examined !"# $!%&' by Badary et al. (Badary et al, 2003). Both compounds efficiently inhibited iron-dependent microsomal lipid peroxidation in a concentration-dependent manner with IC50 values of 16.8 and 14.9 µM, respectively. TBHQ was stronger than TQ as a scavenger of DPPH and hydroxyl radicals whereas TQ was more active than TBHQ as a superoxide anion scavenger. Only TBHQ significantly promoted DNA damage in the bleomycin-Fe (III) system. These results suggest that both compounds have strong antioxidant potentials although TQ acts mainly as a potent superoxide anion scavenger. Moreover, Al-Majed and co-workers (Al-Majed et al, 2006) have evaluated the neuroprotective effect of TQ against transient forebrain ischemia-induced neuronal damage in the rat hippocampus. The pre-treatment of ischemic rats with the compound decreased the elevated levels of MDA and increased GSH, catalase and SOD activities to normal levels. TQ and its reduced product, THQ, inhibited the !"# $!%&' non-enzymatic lipid peroxidation in hippocampal homogenate induced by ironascorbate. The IC50 for TQ and THQ were found to be 12 and 3 µM respectively. This spectacular protection makes TQ a promising agent in pathologies implicating neurodegenaration such as cerebral ischemia.

2. Anti-inflammatory Chemopreventive Activity of TQ

and

Inflammation has been known to produce proinflammatory cytokines and diverse reactive oxygen species (ROS) and reactive nitrogen species (RNS) creating pre-disposition to various patho-physiological disorders such as Cohn’s disease or ulcerative colitis (Greenstein et al, 1979; Ekbom et al, 1990; D'Haens et al, 1993; Brentnall et al, 1996; Krok and Lichtenstein, 2004), gastric helicobacter pylori infection (Crowe, 2005), and colorectal adenocarcinoma (Ekbom et al, 1990; Nielsen et al, 2005). Intervention in the inflammatory cascade can 500

Cancer Therapy Vol 6, page 501! neurodegeneration in hippocampus after chronic toluene exposure in rats. Treatment with TQ caused morphologic improvement on neurodegeneration indicating necessity of further preclinical research in this area. The author has also studied the effects of same compounds on histopathological changes of sciatic nerves in streptozotocin (STZ)-induced diabetic rats (Kanter, 2008a). The histopathological evaluation of the tissues in diabetic animals treated with these compounds showed fewer morphological alterations. Myelin breakdown decreased significantly after treatment with both. The ultra-structural features of axons also showed remarkable improvement. These results suggest that further preclinical research may be able to highlight the advantages of using these agents as a potential treatment on peripheral neuropathy in STZ-induced diabetic rats. McDermott and co-workers have assessed in 2008 the chemoprotective potential of two antioxidants, EGCG and TQ, against n-hexane toxicity in terms of increase in ROS formation with a corresponding decrease in Jurkat Tcell proliferation since n-Hexane is an important industrial solvent and ambient air pollutant. Treatment of cells with EGCG, at a concentration reached in plasma, reduced the ROS formation caused by exposure to n-hexane and inhibited the decrease in cell proliferation. Similar effects were obtained with TQ. Both the compounds were able to significantly reduced n-hexane-induced LDH leakage to the control levels. As indicated earlier, NF-! B is a molecular target of TQ (Sethi et al, 2008), and further studies by Mohamed and colleagues in 2005 showed the effects of TQ on the inhibition of activation of NF-! B in an experimental autoimmune encephalomyelitis in the rat model of multiple sclerosis. The encephalomyelitis was induced in Lewis rats by injecting myelin basic protein emulsified in complete Freund’s adjuvant. Several clinical and biochemical parameters including activation of NF-! B were determined to assess the degree of protection. TQ was able to counter peri-vascular cuffing and infiltration of mononuclear cells in the brain and spinal cord, increase the red blood cell glutathione, and inhibit the activation of NF-! B in the brain and spinal cord. These results clearly provide some early indication that many of the biological activity of TQ could in part be due to inactivation of NF! B and its downstream genes. Collectively, these results suggest that NF-! B is a molecular target of TQ among many other legitimate targets. Moreover, El-Gazzar and colleagues have investigated in 2007 the effect of TQ on LPS-induced TNF-" production in the rat basophil cell line, RBL-2H3 (El Gazzar et al, 2007). The administration of TQ to LPSstimulated cells did not noticeably alter NF-! B cytosolic activation or nuclear expression as demonstrated by western blot analysis. Instead, the compound significantly increased the amount of the repressive NF-! B p50 homodimer and simultaneously decreased the amount of transactivating NF-! B p65:p50 heterodimer, bound to the TNF-" promoter as revealed by electrophoretic mobility shift and chromatin immunoprecipitation assays. These results suggest that TQ attenuates the pro-inflammatory response in LPS-stimulated mast cells by modulating

formation of leukotrienes in human blood cells (Mansour and Tornhamre, 2004). The anti-inflammatory activity of Black cumin seed oil has also been evaluated using carrageenan-induced paw edema in rats and croton oil-induced ear edema in mice by Hajhashemi and colleagues in 2004. Although oral administration of the oil at doses of 100, 200 and 400 Âľl/kg did not exert a significant anti-inflammatory effect in the carrageenan test, the intraperitoneal injection of the same doses significantly inhibited carrageenan-induced paw edema (Hajhashemi et al, 2004). The oil could also reduce croton oil-induced edema at smaller doses and was found to produce a significant analgesic effect in acetic acid-induced writhing, formalin and light tail flick tests. It seems that mechanism other than opioid receptors is involved in the analgesic effect of the oil since naloxone, an opioid antagonist, could not reverse this effect. Being one of the major components of the oil (13.5%), TQ obviously has an important role in these pharmacological effects. Experimental Allergic Encephalitis (EAE) is a Tcell mediated autoimmune disease, which resembles the human disease of Multiple Sclerosis (MS) in rodents. The infiltration of inflammatory cells and the induction of astrocyte proliferation correlate with the severity of the disease. Since oxidative stress has been postulated as the causative factor of initiation and progression of MS, the amelioration of the inflammation by TQ was examined by El-Gouhary and colleagues in 2005 who showed potent effects, which were thought to occur via induction of glutathione (El-Gouhary et al, 2005). El-Gazzar and colleagues has investigated in 2005 whether TQ affects Th2 cytokine response !"# $!%&' in lipopolysaccharide (LPS)-activated rat mast cells. TQ significantly inhibited LPS-induced IL-5 and IL-13 mRNA expression and protein production but did not affect IL-10 production probably by blocking GATA transcription factor expression (El-Gazzar et al, 2005). The inhibitory effects of TQ on activation of the redoxsensitive transcription factor nuclear factor kappa B (NF! B) and interleukin-6 (IL-6) were studied !"# $!%&' (Sayed and Morcos 2007). Human proximal tubular epithelial cells (pTECs) in vitro were cultivated and stimulated with Advanced Glycation End Products (AGEs) and the effects of TQ were studied. A significant reduction of AGEinduced NF-! B-activation and IL-6 expression was observed in Human proximal tubular epithelial cells (pTECs) cultivated and stimulated with AGEs. Sayed studied in 2008 the effect of angiotensin II (AT II) on proximal tubular epithelial cells (pTECs) !"# $!%&'. AT II has been found to activate NF-! B and its controlled genes, IL-6, in a time-dependent manner wherein the first point of maximum NF-! B activation occurs after 12 h and the second after 3.5 days, respectively (Sayed 2008). TQ was found to decrease NF! B activation in a dose-dependent manner with maximum inhibitory effect at a concentration of 500nM. Preincubation of pTECs with TQ leads to disappearance of the second peak of NF-! B. These data suggest the therapeutic value of TQ in delaying end stage renal diseases in diabetics. Kanter has investigated in 2008 the possible beneficial effects of Black seed oil and TQ on

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Padhye et al: Chemopreventive and chemotherapeutic potential of Thymoquinone! documented by multiple observations. For example, the p53 knockout-mice invariably develop spontaneous tumors within first 6 months of life highlighting the protective role played by p53 against cancer (el-Deiry et al, 1993). Although virtually all human tumors deregulate either the pRB or p53 pathway or sometimes both, the unique effects of TQ on p53 protein clearly warrant further studies in determining the precise molecular targets of TQ (Yamasaki, 2003). Moreover, TQ induced growth inhibition in spindle carcinoma cells by inducing G2/M cell-cycle arrest which was associated with an increase in p53 expression and down-regulation of cyclin B1 protein. It is worth mentioning that p53 can regulate the G2/M transition through either induction of p21 or 14-3-3 , a protein that normally sequesters cyclin B1-Cdc2 complexes in the cytoplasm, ultimately leading to the induction of apoptosis (Yonish-Rouach et al, 1991; Shaw et al, 1992; Hermeking et al, 1997; Bunz et al, 1998). These limited studies further suggest that it would be highly desirable to investigate the effects of TQ on other proteins that are involved in G2-M transition in order to delineate the molecular mechanism(s) by which TQ may function as an inhibitor of cell cycle progression and thus as an anti-tumor agent. Having shown that TQ is an anti-cellular and antineoplastic drug that induces p53-dependent apoptosis in human colon cancer cells, Roepke and colleagues have evaluated in 2007 the anti-proliferative and pro-apoptotic effects of TQ in two human osteosarcoma cell lines with different p53 mutation status. Cell viability was reduced more selectively in MG63 tumor cells than in normal human osteoblasts. Flow cytometric analysis showed that TQ induced a much greater increase in the Pre-G1 (apoptotic) cell population, but no cell cycle arrest in MG63 cells. G2/M arrest in MNNG/HOS cells was associated with p21WAF1 up-regulation. Using three DNA damage assays, the compound was confirmed to induce greater extent of apoptosis in p53 null MG63 cells. Although the Bax/Bcl-2 ratios were not differentially modulated in both cell lines, the mitochondrial pathway appeared to be involved in apoptosis induced by TQ in MG63 by showing the cleavage of caspases-9 and -3, respectively. Since TQ was found to induce p53independent apoptosis in human osteosarcoma cells, it suggests the potential clinical usefulness of TQ for the treatment of these malignancies. The serine/threonine kinase Polo-like kinase 1 (Plk1) is over-expressed in many types of human cancers, and has been implicated as an adverse prognostic marker. Plk1 localizes to its intracellular anchoring sites via its polobox domain (PBD). Reindl and colleagues have reported in 2008 that TQ and its synthetic C-1 imino analog, Poloxin, are good inhibitors of Plk 1 PBD !"# $!%&' and cause deregulation of its cellular localization, chromosomal defects, mitotic arrest and apoptosis in HeLa cells (Reindl et al, 2008). These results provide early indication as to the value of further research into the development of synthetic analogs of TQ as anticancer agents. Ivankovic and colleagues have investigated in 2006 the anti-tumor activity of TQ and THQ in L929 mouse fibroblasts and two other tumor cell lines, viz. squamous cell carcinoma

nuclear transactivation of NF-! B and TNF-" production. The above results collectively suggest that further in-depth research in this area is warranted.

3. Anti-proliferative (anti-tumor) and Cell Cycle Regulatory Activity Shoieb and colleagues have investigated in 2003 the anti-proliferative effects of TQ in cancer and normal cell lines, viz. canine osteocarcinoma (COS31) and its cisplatin-resistant variant (COS31/rCDDP), human breast adenocarcinoma (MCF-7), Human ovarian adenocarcinoma (BG-1) and Mandin-Darby canine (MDCK) cells (Shoieb et al, 2003). The compound was found to inhibit proliferation in a concentration-dependent manner as assessed by MTT assay. MDCK cells (normal kidney cells) were the most resistant cells to the inhibitory effects of TQ (IC50 = 101 µM). Ait and colleagues have evaluated in 2007 the anti-tumor properties of the black seed oil and its ethyl extract against P815 cell line and both were found to be cytotoxic. The extracts were also tested on a variety of cell lines such as ICO1, Vero cells and BSR cell line which showed that the extent of cytotoxicity depends upon the tumor cell type. In animal model employing DBA2/P815 (H2d) mouse model it was observed that the injection of the essential oil into the tumor site significantly inhibited solid tumor development as well as the incidence of liver metastasis, thus improving mouse survival. These results indicate that the anti-tumor activity or cell growth inhibition could in part be due to the effect of TQ on cell cycle. The cell cycle checkpoints allow the cells to correct possible defects and avoid progression to cancer (Hartwell and Weinert, 1989; Hartwell and Kastan, 1994). There are two major checkpoints to detect DNA damage: one at the G1-S transition that prevents the cell from replicating damaged DNA and one at the G2-M transition that prevents chromosome segregation, if the chromosome is not intact. The principle activity of TQ was found to be due to its effects on the expression of cell cycle regulatory proteins. The treatment of cells with 30 µM concentration for 48 h induced G1 cell-cycle arrest in papilloma cells, which correlated with a sharp increase in the expression of the cyclin-dependent kinase inhibitor p16 and downregulation of cyclin D1 protein expression (Gali-Muhtasib et al, 2004b). It would be helpful to define further targets of TQ by studying other cell cycle regulating proteins in future studies. In Flow cytometric studies of DNA content by propidium iodide staining it has been revealed that TQ induces G1 cell-cycle arrest of osteosarcoma cancer cells (COS31) as well as human colon cancer cells (HCT-116), at 100µM concentration treated for 48 h (Gali-Muhtasib et al, 2004a). The effect was observed starting after 24 h at a concentration of 50 µM for COS31 cells while for HCT116 cells it started at 60 µM, respectively. The G1 arrest was associated with up-regulation of p21WAF1 in HCT-116 cells which was suggested as the principal transcriptional target of p53 in the context of the G1 checkpoint. The resulting high levels of p21WAF1 blocks cdk2 activity and possibly cdk4 and cdk6 activities leading to G1 arrest. There was also an up-regulation of p53 expression which plays important roles in cancer development as 502

Cancer Therapy Vol 6, page 503! application of TQ alone or in combination with other agents as novel therapeutics. Although 5-FU continues to be the chemotherapeutic gold-standard for the treatment of colon cancer, the side effects of 5-FU are numerous due to its ability to attack both healthy and cancerous cells. Hence, Norwood and colleagues have compared in 2007 the effects of 5-FU and natural chemotherapeutic agents, EGCG and TQ on the metabolic activity as well as structural changes in the SW626 human colon cancer cell line. These studies indicate a significant cellular destruction and interference of cellular metabolic functions which opens up new possibility for sustained drug delivery of other natural agents for the safe alternative treatment of colon cancer. Consequently the studies were extended by these workers (Richards et al, 2007) to the ceramic drug delivery system of TQ which demonstrated the greatest reduction in cell count as well as the most cell membrane damage according to malondialdehyde (MDA) levels. This supports the hypothesis that sustained delivery of antioxidants may be a means of treating cancers, both safely as well as effectively. Obviously further studies are needed to test the mechanisms behind these reactions. Badary and colleagues have shown in 2007 that TQ protects the mice against benzo(a)pyrene-induced forestomach carcinogenesis and chromosomal aberrations (CAs) in mouse bone marrow cells when supplemented in the drinking water (Badary et al, 2007). It was observed that daily intake of the compound before and after or during exposure to benzo(a)pyrene significantly reduced the frequencies of CAs and damaged cells compared to the highly clastogenic activity of B(a)P alone. Womack and colleagues have evaluated in 2006 the effects of a single dose of 5 ÂľM of TQ which showed a 50% reduction in Hep-2 cell numbers after 24 h. After 48 h the cells exhibited a four-fold decrease in total cell number which indicates that TQ given in a sub-therapeutic dose could alter cellular viability. Tan and colleagues have also examined in 2006 the effects of TQ on the proliferation and viability of PANC-1 cell line. The PANC-1 cells were treated with three pre-determined doses of TQ (5, 25, and 50 Âľg/ml) and medium viability and morphology were examined microscopically after each 24 h interval. The compound was found to be the potent inhibitor of human pancreatic carcinoma, reducing their propagation activities. Recently, Richards and co-workers have used the androgen-dependent LNCaP human prostate cancer cell line as a cell model to evaluate the physiological effects to conventional treatments with both low doses and high doses of TQ (Richards et al, 2006). All treated cells showed a reduction in cell growth, and high doses of the compound seemed to be the most potent. The group treated with high doses of TQ also demonstrated the greatest decrease in total protein levels in comparison to the control. Morphologically, the cells demonstrated significant changes, such as swelling and irregularity in appearance upon TQ exposure. These results seem to suggest that TQ could serve as a chemopreventive agent for prostate cancer. However, further experiments are needed to understand the mechanism involved. Because

(SCC VII) and fibrosarcoma (FsaR), respectively. Both compounds showed dose dependent potent cytotoxicities, which was more pronounced in tumor cells compared to L929 normal fibroblasts. The growth inhibitory (antitumor) effects of TQ were further studied by Badary and colleagues in 2001 studied fibrosarcoma induced by 20methylcholanthrene (MC) in male Swiss albino mice. Administration of TQ (0.01% in drinking water) one week before and after MC treatment significantly inhibited the tumor incidence and tumor burden compared with the results in the group receiving MC alone (Badary et al, 2001). Moreover, TQ also delayed the onset of MCinduced fibrosarcoma tumors indicating that it could be a powerful chemopreventive agent against MC-induced fibrosarcomas (Badary et al, 2001). Gali-Muhtasib and colleagues have also evaluated in 2008 the therapeutic potential of TQ in two different murine colon cancer models, viz.1, 2-dimethyl hydrazine (DMH) and xenografts. They examined the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor invasion !"#$!%&' and found that the tumor multiplicity was reduced from 17.8 in the DMH group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long lasting since tumors did not re-grow even when TQ injection was discontinued for 10 weeks. In a xenograft model of HCT116 colon cancer cells, TQ significantly delayed the growth of the tumor cells. In addition to TQ, a recent review article has shown the beneficial effects of many chemical compounds isolated from natural resources (Aggarwal et al, 2008), suggesting that exploitation of natural compounds for therapeutic application is an active area of research. Moreover, recent studies have also shown that NF-! B is a legitimate target of TQ which was associated with cell growth inhibition and induction of apoptosis in cancer cells (Sethi et al, 2008). As indicated earlier, Kaseb and colleagues have observed in 2007 the biological effects of TQ in the inhibition of DNA synthesis, cell proliferation, and viability of prostate cancer cells (LNCaP, C4-2B, DU145, and PC-3) but not of the non-cancerous (BPH-1) prostate epithelial cells which was mechanistically linked with the down-regulation of AR and E2F-1. In LNCaP cells, this was associated with a dramatic increase in p21WAF1 (Cip1), p27 (Kip1), and Bax proteins. It also blocked the progression of synchronized LNCaP cells from G1 to S phase, with a concomitant decrease in AR and E2F-1 as well as the E2F-1-regulated proteins necessary for cell cycle progression. In a xenograft prostate tumor model, TQ inhibited growth of C4-2Bderived tumors in nude mice which was associated with a dramatic decrease in AR, E2F-1, and cyclin A as determined by Western blot analysis (Kaseb et al, 2007). These results collectively suggest that TQ may prove to be an effective agent in treating hormone-sensitive as well as hormone-refractory prostate cancers with reasonable degree of selectivity and other cancers (Kaseb et al, 2007; Aggarwal et al, 2008; Sethi et al, 2008). In the following section we will catalogue several other studies that are relevant to cancer prevention and/or therapy although further scientific studies are needed in order to justify the

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Padhye et al: Chemopreventive and chemotherapeutic potential of Thymoquinone! TQ has been shown to initiate apoptosis even via p53-independent pathways through activation of caspase3, 8 and 9 in p53-null myeloblastic leukemia HL-60 cells (El-Mahdy et al, 2005). It was observed that caspase-8 activity was highest after 1 h following the treatment of TQ, while caspase-3 activity was highest after 6 h respectively. These observations were explained on the basis of up-regulation of pro-apoptotic Bax protein along with down-regulation of anti-apoptotic Bcl-2 proteins resulting in enhance Bax/Bcl-2 ratio. It is thus apparent that TQ induces apoptosis through modulation of multiple targets and hence is a promising phytochemical that could be useful for the killing of many types of cancer cells. These results are also supported by reports in prostate and other cancer cells (Kaseb et al, 2007; Aggarwal et al, 2008; Sethi et al, 2008). A very recent report has identified checkpoint kinase 1 homolog, CHEK1, a serine/threonine kinase, as the target of TQ, leading to apoptosis in p53+/+ colon cancer cells (Gali-Muhtasib et al, 2008b). The study compared the effect of TQ on p53+/+ as well as p53-/- HCT116 colon cancer cells and p53+/+ cells were found to be more sensitive to TQ in terms of DNA damage and apoptosisinduction. As a possible explanation for such sensitivity, it was observed that CHEK1 was up-regulated upto 9 folds in p53-null HCT116 cells. Further, transfection of p53 cDNA and CHEK1 siRNA in p53 null cells resulted in restoration of apoptosis to the levels of p53+/+ cells. The results were also corroborated "+' *"*- and it was demonstrated that tumors lacking p53 had higher levels of CHEK1 which was associated with poorer apoptosis, advance tumor stages and worse prognosis. Despite the potential interest in TQ as a skin antineoplastic agent, its mechanism of action was not examined for a long time. Gali-Muhtasib and colleagues studied in 2004 the cellular and molecular events involved in antineoplastic activity of TQ using primary mouse keratinocytes, papilloma (SP-1) and spindle carcinoma cells. The non-cytotoxic concentrations of TQ reduce the proliferation of neoplastic keratinocytes by 50%. The sensitivity of cells to TQ treatment appears to be stage dependent. For example, papilloma cells are twice as sensitive to the growth inhibitory effects of TQ as the spindle cancer cells. At longer times of incubation the compound induced apoptosis in both cell lines by increasing the ratio of Bax/Bcl-2 protein expression and decreasing Bcl-xL protein. Collectively, these findings support a potential role for TQ as a chemopreventive agent, particularly at the early stages of skin tumorigenesis. Rooney and Ryan sought to understand in 2005 the mechanisms involved in the action of ! -hederin and TQ which are the active constituents of !"#$%%&'(&)"*&, against human laryngeal carcinoma (HEp-2) cancer cells by using buthionine sulfoximine (BSO), a selective inhibitor of GSH synthesis, to determine the importance of GSH in the apoptosis elicited and using cisplatin as internal standard. Both compounds elicited necrosis and apoptosis with a higher incidence of the latter induced by TQ. BSO significantly enhanced ! -hederin- and cisplatin- mediated toxicity without changes in apoptosis or necrosis levels.

TQ is a potent agent, it has been explored further for combination with other known chemopreventive agents; one such study reported by Brewer and colleagues showed in 2006 that selenomethione, lycopene and thymoquinone could be effective on SiHa cells in the presence or absence of estrogen (Brewer et al, 2006). Their results indicate that selenomethione alone appeared to be chemo-protective, however, when used in combination with estrogen, lycopene and TQ, it caused cellular damage as evidenced by decreased proliferation rate, increased glutathione levels, and increased MDA levels (Brewer et al, 2006). It appears that TQ is a pleiotropic agent targeting multiple signaling pathways in many patho-physiological conditions as documented earlier and thus TQ has also been tested for its optimal delivery. Martin et al. have determined the effects of TQ on the viability and metabolic activity of SH-SY5Y human neuroblastoma cells alone or challenged with levo-dopa (L-dopa) using conventional and sustained drug delivery routes (Martin et al, 2006). These results suggest that the compound is able to offer some protection although the exact mechanism for this is not yet known. It is suggested that the compound may be preventing the quinone formation, which has been implicated in the pathogenesis of Parkinson's disease as a result of L-dopa auto-oxidation and thus it could be further investigated as a neuroprotective agent. Further studies using a crude gum, a fixed oil, and two purified components of !"#$%%&' (&)"*& seed, viz. TQ and DTQ were assessed for their cytotoxicity on several parental and multi-drug resistant (MDR) human tumor cell lines (Worthen et al, 1998). Although as much as 1% w/v of the gum or oil was devoid of cytotoxicity, both TQ and DTQ were cytotoxic for all of the tested cell lines. The parental cell lines and their corresponding MDR variants, viz. Doxorubicin-resistant and etopside-resistant cell lines, were equally sensitive to TQ and DTQ. The MDR modulator, viz. quinine, reversed MDR Dx-5 cell resistance to DOX and ETP by 6- to 16-fold, but had no effect on the cytotoxicity of TQ or DTQ These results suggest further pre-clinical "+' *"),- and "+' *"*- animal experiments to advance our knowledge on the subject.

4. Apoptosis Induction Activity It has been shown that TQ triggers apoptosis in HCT116 cells in a dose and time-dependent manner, starting at a concentration of 100 ÂľM after 12 h of incubation which is associated with a 2.5 to 4.5 fold increase in p53 and p21WAF1 mRNA expression and a significant decrease in Bcl-2 protein levels (Yamasaki, 2003). Co-incubation with pifithrin-! , a p53 inhibitor, restored the Bcl-2, p53 and p21WAF1 levels to the untreated control levels and absolved the effects of TQ. These results suggest role of TQ in influencing cell cycle regulators involved in apoptosis as well as in down-regulating the anti-apoptotic proteins. This was supported by similar effects on primary mouse keratinocytes, papilloma (SP-1), and spindle carcinoma cells respectively. At longer incubation times (48 h) the compound induced apoptosis in both cell lines by increasing the ratio of Bax/Bcl-2 protein expression and down-regulating the Bcl-xL protein.

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Cancer Therapy Vol 6, page 505! upon LPS stimulation. In absence of stimulation (IL-1" ) insignificant increase in nitrite concentration was observed which turned significant upon LPS stimulation. Sera of these animals, however, showed significant increase in TNF- ! level. The compound TQ normalized the elevated nitrite and cytokine profiles both !"# $!%&' and !"# $!$' but had no significant effect on the already decreased parameters in chronically affected OLETF rats. These data suggest that there is a tendency for macrophage inflammatory products to increase in acute type I Diabetes and to decrease in chronic type II Diabetes respectively. Collectively, TQ has been suggested to have the potential to normalize the elevated levels of these macrophagederived inflammatory mediators. It has been established that NO is involved in the destruction of " -cells during the development of type I DM. El-Mahmoudy and colleagues demonstrated in 2005 the possibility of rescuing " -cells by intervention with TQ using STZ rat diabetic model. The hyperglycemic and hypoinsulinemic responses to STZ were significantly abrogated in rats treated with TQ, and this abrogation persisted for 1 month after termination of the treatment. TQ was found to have no effect on either I B degradation or NF- B activation, although it significantly inhibited both p44/42 and p38 mitogen-activated protein kinases (MAPKs) which contribute to the transcriptional machinery of inducible nitric oxide synthase and NO production, respectively. These data emphasize the protective role of TQ against development of type I diabetes via NO inhibitory pathway. The protective effect of black seed extract and TQ has been studied on mouse cells infected with schistosomiasis by Aboul-Ela in 2002. Bone marrow cells and spleen cells in both !"# $!%&'# (")# !"# $!$' experiments were used to evaluate the potentially protective effects on the induction of chromosomal aberrations. Karyotyping of the mice cells illustrated that the main abnormalities were gaps, fragments and deletions especially in chromosomes 2, 6 and some in chromosomes 13 and 14. The seed extract as well as TQ both offered protection against the chromosomal aberrations induced by schistosomiasis. Nigellone is the carbonyl polymer of TQ, isolated from *!+,--(#.(%!$(#L. seeds. The polymer is far less toxic but retains much of the pharmacologic properties of TQ, which is the active principle of the plant. Chakravorty’s investigations (Chakravorty, 1993), carried out on rat peritoneal mast cells !"#$!%&', have shown that nigellone, in relatively low concentrations, is very effective in inhibiting histamine release induced by the secretagogues antigen in sensitized cells, viz. compound 48/80, and the calcium ionophore,viz.A23187 respectively. The mechanism of action seems to involve decreasing intracellular calcium by inhibiting its uptake and stimulating the efflux as well as by an inhibition of protein kinase C. El-Gazzar and colleagues have examined in 2006 the effect of TQ on airway inflammation in a mouse model of allergic asthma. Intraperitoneal injection of TQ before airway challenge of ovalbumin (OVA)-sensitized mice resulted in a marked decrease in lung eosinophilia and the elevated Th2 cytokines; both !"#$!$' and !"#$!%&', following stimulation of lung cells with OVA. TQ also

The compound TQ and cisplatin significantly decreased GSH levels in a dose-dependent manner, with BSO pretreatment synergistically depleting GSH levels in only cells treated with TQ. Since the caspase-3 inhibitor significantly decreased apoptosis induced by the latter combination, it was concluded that GSH depletion and caspase 3-activation mediate apoptosis induced by TQ in this cell line, suggesting that exploitation of cause and effect relationship between the biological activities of TQ with many cellular targets are warranted. Tumor growth and metastasis are known to be angiogenesis-dependent and several clinical trials have suggested that anti-angiogenic therapy might provide an attractive target for therapeutic intervention. Alteration in the balance between pro-angiogenic and anti-angiogenic molecules in the local tissue microenvironment may provide the starting point for such strategy using the active constituent of black seed oil. The anti-aniogenic effects of TQ have been assessed by cell proliferation and migration assays (Bawadi et al, 2004). The TQ significantly decreased the proliferation of human breast (MCF-7), colon (Caco-2) and prostate (DU-145) cancer cells at concentration of 100 µM and also prevented their metastasis. It inhibited HIF-1! expression and decreased HIF-1! DNA binding activity in all cancer cells in addition to reducing VEGF and cathepsin D secretion in normal human lung fibroblast cells. It, however, did not affect normal cell proliferation even at 200µM concentration. Presently, there are no studies reported, dealing with effects of TQ on angiogenesis.

5. Other Activities The immunomodulatory and immunotherapeutic potentials of black seed oil and its active ingredients have been discussed by Salem in 2005. The oil and some of its active ingredients showed beneficial immunomodulatory properties, augmenting the T cell- and natural killer cellmediated immune responses. Further studies are urgently required to explore bystander effects of TQ on the professional antigen presenting cells, including macrophages and dendritic cells, as well as its modulatory effects upon Th1- and Th2-mediated inflammatory immune diseases which is likely to substantially improve the immunotherapeutic application of TQ in clinical settings. El-Mahmoudy and co-workers (El-Mahmoudy et al, 2005b) have compared the macrophage-derived cytokine and NO profiles in type I and type II diabetes mellitus (DM) in order to determine whether TQ has any modulatory effects. Peritoneal macrophages were collected from Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a model for type II DM and its control, Long-Evans Tokushima Otsuka (LETO) rats, as well as from STZ-injected LETO ones as a model for type I DM. The cells were cultured and incubated with or without TQ (10 µM) in the absence or presence of lipopolysaccharide. Nitrite, IL-1" and TNF-! are significantly higher in macrophage supernatants and sera of the acutely affected STZ-LETO rats either with or without LPS stimulation compared to corresponding controls. On the other hand, chronically diabetic OLETF rats' macrophage supernatants showed significant decreases of IL-1" and TNF-! level

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Padhye et al: Chemopreventive and chemotherapeutic potential of Thymoquinone! Al-Majed AA, Al-Omar FA, Nagi MN (2006) Neuroprotective effects of thymoquinone against transient forebrain ischemia in the rat hippocampus. Eur J Pharmacol 543, 40-47. al-Shabanah OA, Badary OA, Nagi MN, al-Gharably NM, alRikabi AC, Al-Bekairi AM (1998) Thymoquinone protects against doxorubicin-induced cardiotoxicity without compromising its antitumor activity. J Exp Clin Cancer Res 17, 193-198. Aqel M, Shaheen R (1996) Effects of the volatile oil of Nigella sativa seeds on the uterine smooth muscle of rat and guinea pig. J Ethnopharmacol 52, 23-26. Badary,O.A., al-Shabanah,O.A., Nagi,M.N., al-Rikabi,A.C., and Elmazar,M.M. (1999). Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone. Eur. J. Cancer Prev. +, 435-440. Badary OA, bd-Ellah MF, El-Mahdy MA, Salama SA, Hamada FM (2007) Anticlastogenic activity of thymoquinone against benzo(a)pyrene in mice. Food Chem. Toxicol 45, 88-92. Badary OA, bdel-Naim AB, bdel-Wahab MH, Hamada FM (2000) The influence of thymoquinone on doxorubicininduced hyperlipidemic nephropathy in rats. Toxicology 143, 219-226. Badary OA, Gamal el-Din AM (2001) Inhibitory effects of thymoquinone against 20-methylcholanthrene-induced fibrosarcoma tumorigenesis. Cancer Detect Prev 25, 362368. Badary OA, Taha RA, Gamal el-Din AM, bdel-Wahab MH (2003) Thymoquinone is a potent superoxide anion scavenger. Drug Chem Toxicol 26, 87-98. Bawadi HA, Bansode RR, Losso JN (2004) Thymoquinone in the control of hypoxia-induced angiogenic disease biomarkers: Insight into the mechanism of action in vitro 2004 IFT Annual Meeting 2004 Las Vegas NV USA 7-122004. Benkaci-Ali F, Baaliouamer A, Meklati BY (2006) Kinetic study of microwave extraction of essential oil of !"#$%%&' (&)"*& L.seeds. Chromatographia 64, 227-231. Borek C (2004) Dietary antioxidants and human cancer. Integr Cancer Ther 3, 333-341. Brentnall TA, Haggitt RC, Rabinovitch PS, Kimmey MB, Bronner MP, Levine DS, Kowdley KV, Stevens AC, Crispin DA, Emond M, Rubin CE (1996) Risk and natural history of colonic neoplasia in patients with primary sclerosing cholangitis and ulcerative colitis. Gastroenterology 110, 331-338. Brewer J, Benghuzzi H, Tucci M (2006) Effects of thymoquinone, lycopene, and selenomethione in the presence of estrogen on the viability of SiHa cells in vitro. Biomed Sci Instrum 42, 37-41. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B (1998) Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497-1501. Chakravorty N (1993) Inhibition of histamine release from mast cells by Nigellone. Ann Allergy 70, 237-242. Crowe SE (2005) Helicobacter infection, chronic inflammation, and the development of malignancy. Curr Opin Gastroenterol 21, 32-38. Daba MH, Abdel-Rahman MS (1998) Hepatoprotective activity of thymoquinone in isolated rat hepatocytes. Toxicol Lett 95, 23-29. DerMarderosian A, Lawrence L, Beutler J, Grauds C, Tatro DS, Cirigliano, DeSilva D (2005) The Review of Natural Products, 4th Edn. , Facts and Comparisions. Lipincott Williams & Wilkins). D'Haens GR, Lashner BA, Hanauer SB (1993) Pericholangitis and sclerosing cholangitis are risk factors for dysplasia and

decreased the elevated serum levels of OVA-specific IgE and IgG1. Histological examination of lung tissue demonstrated that the compound significantly inhibited allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells. It was concluded that TQ attenuates allergic airway inflammation by inhibiting Th2 cytokines and eosinophil infiltration into the airways and thus demonstrating its potential anti-inflammatory role during the allergic response in the lung.

III. Conclusions and Future Directions We have attempted in summarizing the state-ofknowledge on the biological activity of TQ; however in this short article we were not able to include all the published results and thus we hope that the authors whose results have not been included in this article will forgive us. It is however tantalizing to speculate that because TQ has attracted significant scientific attention in recent years, further pre-clinical and clinical research to assess the health benefits of TQ is urgently needed. Moreover, emerging evidence suggests that synthetic analogues of treasures from natural resources could indeed be useful for making more effective compounds than TQ which should also be an active area of research. It is hoped that this article will promote such research and thus novel agents could be discovered based on the chemical structure of TQ for the prevention and/or treatment of human diseases in the near future.

Acknowledgement The authors’ work cited in this review was partly funded by grants from the National Cancer Institute, NIH (5R01CA083695, 5R01CA101870, and 5R01CA108535) awarded to FHS, a sub-contract award to FHS from the University of Texas MD Anderson Cancer Center through a SPORE grant (5P20-CA101936) on pancreatic cancer awarded to James Abbruzzese, and a grant from the Department of Defense (DOD Prostate Cancer Research Program DAMD17-03-1-0042) awarded to FHS.

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Fazlul H Sarkar

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Analysis of gene expression profiles in the programmed cell death rat retinal ganglion cells (RGC-5) Research Article

Abdelnaby Khalyfa1,2,*, Mohamed O. Buazza1, Muad A. Maya1, Nigel Cooper2, Bryant A. Minnis1 1 2

Department of Pediatrics, Kosair Children’s Hospital Research Institute, Department of Anatomical Sciences and Neurobiology, University of Louisville, Louisville, KY

__________________________________________________________________________________! *Correspondence: Abdelnaby Khalyfa, Ph.D., Department of Pediatrics, Kosair Children’s Hospital Research Institute, University of Louisville, School of Medicine, 570 S. Preston St., Room 204, Louisville, KY, 40202, USA; Tel: 502-852-7524; Fax: 502-852-2215; Email: a.khalyfa@louisville.edu Key words: Genome microarray, gene expression, agilent microarray, RGC, RGC-5 Abbreviations: ciliary neurotrophic factor, (CNTF); death agonist, (BAD); false discover rate, (FDR); fetal bovine serum, (FBS); Gene Ontology, (GO); glycogen synthase kinase 3, (GSK-3); intraocular pressure, (IOP); ischemic preconditioning, (IPC); nitric oxide, (NO); photo-multiplier tube, (PMT); retinal ganglion cells, (RGCs) Received: 11 March 2008; Revised: 24 March 2008 Accepted: 11 April 2008; electronically published: September 2008

Summary The death of retinal ganglion cells (RGCs) is the primary cause of losing vision in glaucoma, and the death of these cells may be due to the loss of neurotrophic factors. Molecular networks underlying RGCs response to serum deprivation are not fully understood. We hypothesized that there would be change in the gene expression levels associated with the biological pathways over the period of time for RGC-5 serum deprivation. The aim of this study was to further identify and characterize biological pathways for apoptotic RGC-5 time dependent programmed cell death. Agilent technology (genome-scale, long oligonucleotide microarrays) was used to characterize candidate genes involved in apoptotic signal transduction pathways in rat retinal ganglion cells. We identified the most highly up-and down-regulated genes for each time point. In addition, we identified 80 genes across all time points using one-way ANOVA (p-value <0.05). The most highly sinfigicant differentially expressed genes were imported into PathwayStudio software to build biological pathways of RGC cell death mechanisms. Gene Ontology (GO) and network analyses were employed to identify significant biological processes and regulatory networks critical for RGC-5s response to serum deprivation time courses. Microarray data was validated using Real Time PCR. For the differentially expressed genes, only the annotated genes were considered. We demonstrated that complement component 3 (C3), the major complement protein of the alternative complement pathway, was up-regulated in serum-deprived RGC-5. Immunocytochemistry and confocal microscopy showed that C3 protein was detected in isolated RGC-5, including the rat retina itself. Our results provide a detailed view of the distinct temporal changes in gene expression induced by time courses of RGC-5 serum deprivation, and demonstrates the potential for discovery in applying systems biology approaches to understanding the mechanisms underlying RGC-5s response to apoptotic signals. An understanding of the apoptotic pathway in RGCs may lead to the development of new treatments that could block the apoptotic cell process during periods of stress.

pressure (IOP) is one of the highest risk factors of this potentially blinding disease (Armaly et al, 1980; Quigley, 1999; Sommer et al, 1991). The precise mechanisms involved in glaucoma have yet to be determined; however, a better appreciation of the factors involved in ganglion cell death is crucial to future development of an overall

I. Introduction Glaucoma is one of the leading causes of vision loss characterized by progressive retinal ganglion cell (RGC) death (Quigley, 1996). In the eye, elevated pressure is associated with glaucoma and leads to apoptosis of neuronal RGCs, (Charles et al, 2005). This intraocular 511

Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5 RGC-5 cells induced apoptosis associated with the activation of caspases cascade, and also loss of mitochondrial function revealed by cytosolic release of cytochrome c (Charles et al, 2005). Analysis of large-scale gene expression has become a fundamental approach to functional genomics for identification of clinical diagnostic and potential drug targets. Microarray gene expression measures the expression levels for thousands of genes or the whole genome at the same time and allowing the analysis of differential gene expression patterns under different conditions (Schena et al, 1995; Lockhart and Winzeler, 2000). The advent of microarray technology along with exponential accumulation of biological data have recently led to a massive search for new knowledge that can be applied to improve our quality of life, with focus on human diseases (Ring and Ross, 2002). Many applications of microarrays have been reported, including behavioral neuroscience (Karssen et al, 2006), disease classification (Golub et al, 1999), and discovery of new disease subtypes (Bhattacharjee et al, 2001); ischemic preconditioning (IPC) in the rat retina (Kamphuis et al, 2007), in the optic nerve head after exposure to elevated intraocular pressure using a rat glaucoma model (Johnson et al, 2007) purified at retinal ganglion cells (Farkas et al, 2004). In addition, Ivanov and colleague in 2006 (Ivanov et al, 2006) compared a purified primary culture of RGCs with adult retinal ganglion cells. To identify groups of genes that are similarly regulated across biological samples, a variety of mathematical methods were developed that partitioned genes (samples) into groups, or clusters. Each gene product may be involved in one or more functions. These functions are accurately described in the Gene Ontology database. The GO consortium1 produces three independent ontologies for the gene products. The three ontologies form the basis to describe the molecular function, the biological process, and the cellular component of the gene products. The genetic and phenotypic heterogeneity of retinal dystrophies poses a problem with respect to rational development of therapeutic strategies. Identification of pathways that lead to pathogenesis, and delineation of the physiological functions of disease related genes, can pilot development of a systematic and global approach to genebased treatments (Yu et al, 2004). Therefore we attempted to characterize genome expression and biological pathways that RGCs use when they undergo apoptosis during serum deprivation. Our previous studies highlighted common and unique genomic changes after serum deprivation using basic statistical analysis (Khalyfa et al, 2007); whereas this study focuses on signal transduction pathways for each individual time point using additional detailed statistical analysis. In this study, we compared the genomic response of serum deprivation after induction of apoptosis with control non-serum deprived samples. A number of genes were regulated in the serum deprived and non-serum deprived samples; the majority of these modifications were previously unreported. We hypothesized that there would be changes in the gene expression levels associated with the biological pathways

strategy for treatment (Garcia-Valenzuela et al, 1995; Quigley et al, 1995). Recent studies have suggested that RGC may directly be involved in glaucoma pathogenesis instead of an inadvertent response to increased IOP (Wang et al, 2007). The first apoptotic RGCs were described in a rat optic nerve axotomy model (Berkelaar et al, 1994; Garcia-Valenzuela et al, 1995). Apoptosis of RGCs related to glaucoma has been demonstrated in monkey models (Quigley et al, 1995), in human models (GarciaValenzuela et al, 1995; Tatton et al, 2001), and in several other animal models. This type of cell death contains distinct morphological features involving a programmed series of molecular events genetically regulated by the inflicted cells, and can be induced by various stimuli (Wyllie et al, 1980). The RGCs are sustained by neurotrophic factors that are transported in a retrograde orientation to the ganglion cells (Pease et al, 2000). Neurotrophic factors withdrawal has been hypothesized as a primary cause of RGC death in glaucoma (Wadia et al, 1998; Charles et al, 2005). Charles and colleagues in 2005 reported that molecular pathways are implicated in RGC-5 cell death through the intrinsic apoptotic pathway upon the release of cytochrome c from mitochondria. Apoptosis is essential for normal tissue development, and contributes to certain forms of pathological cell loss. RGCs death is a common feature of many ophthalmic disorders such as glaucoma, optic neuropathies, and retinovascular diseases, including diabetic retinopathy and retinal vein occlusions. RGC death has been reported to occur via a variety of mechanisms, such as oxidative stress (Bonne et al, 1998), excitatory amino acids (Dreyer, 1998), nitric oxide (NO) (Neufeld, 1999), and apoptosis (McKinnon, 1997). Gupta and Yucel indicated in 2007 that there are similarities between glaucoma and neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease (which include the selective loss of neuron populations), trans-synaptic degeneration (in which the disease spreads from injured neurons to connected neurons), and common mechanisms of cell injury and death. RGCs are widely used as a model system for neurobiological studies of development, trauma, neuronal degeneration and regeneration (Farkas et al, 2004). Yuan and Yankner in 2000 reported that neuronal apoptosis plays a role construed as significant in neurodegenerative diseases; and elucidation of the cell death machinery in neurons promises to provide multiple points of therapeutic intervention. Agarwal and colleagues indicated in 2007 that neurotrophins are a family of proteins which promote development, survival, and differentiation of neurons, and deprivation of neurotrophic factors potentially ensues RGC apoptosis in glaucoma. Agarwal and colleagues have also determined in 2007 the expression profile of neurotrophins and their receptors in cultured primary rat retinal ganglion cells (RGCs) and the transformed RGC-5 cells. Apoptosis-related signaling pathways were investigated in a RGC-5 cell line deprived of growth factors after serum withdrawal from the culture medium (Charles et al, 2005). Ganglion cells die in several retinal diseases, including glaucoma and diabetic retinopathy (Gardner et al, 2002; Levin and Gordon, 2002). Previously, we have show that serum deprivation of

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Cancer Therapy Vol 6, page 513! 25 X fragmentation buffer were incubated at 60ºC for 30 minutes. A hybridization mixture of 490 !L was incubated at 60°C for 17 hrs in a hybridization chamber. The microarray slides were washed for approximately 30 seconds each in 6x SSPE, 0.005% sarcosine and 0.006x SSPE. Hybridization and washing of arrays was processed in an aseptic environment for protection from contamination by dust and dirt.

over a period of time for RGC-5 serum deprivation. Our aim was to provide an overview of the discrepancies in genomic expression levels occurring in serum deprived RGC-5; and to identify, in time- dependent biological pathways, individuals and groups of genes potentially involved in differential gene expression. Furthermore, the genomic response to serum deprivation after induction of apoptotic cell death was examined to construct biological pathways conceivably participating in apoptotic cell death. The application of Agilent oligonucleotide-based microarray technology was utilized when RGC-5s were induced to apoptosis by serum deprivation for 0, 8, 24, 48, and 96 hrs. We have investigated the molecular and cellular pathways of serum deprivation in a time dependent manner through combining large-scale gene expression approaches, data analysis, and careful selection of the differentially expressed genes. We also identified several of the neuronal apoptosis genes, and are in the process of up-loading the data into GEO databases. Our understanding of how deprivation of neurotrophic agents causes neuronal cell death relates to the signal transduction cascades by which these factors normally maintain variability.

C. Data analysis Microarray slides were scanned immediately following hybridization using a laser Typhoon 9410 scanner of 10 µm resolutions (GE HealthCare, Piscataway, NJ) exciting the Cy3 (Datta et al, 1999). Microarrays were scanned at 600 photomultiplier tube (PMT) voltages, and images were captured by ImageQuant software v. 5.2 (GE HeathCare, Piscataway, NJ). ArrayVision v.8.0 software (Imaging Research Inc., Ontario, Canada) was used to calculate the median intensities for each spot of Cy3 fluorescent signals. The net signal intensity was calculated by subtracting the background median intensities from the median of the spot intensities. The background corrected intensities were then normalized to the total signal intensities of each spot by the median. The differentially expressed genes were identified by the net intensity ratios between the two test groups. The Student %-test )-values (in which variances between the groups are not assumed to be equal) were used to compare intensities between the two groups. Log ratios for individual genes were calculated for each serum-deprived (test)/nondeprived (control) sample, from which the mean log ratio for each sample group was determined.

II. Materials and Methods A. Cell Cultures and RNA isolation Transformed retinal ganglion cells (Krishnamoorthy et al, 2001), RGC-5 (previously obtained from Dr. Neeraj Agarwal), were routinely cultured in DMEM containing 1 g/L glucose, supplemented with 10% fetal bovine serum (FBS), 100 ! /g/mL penicillin, and 100 ! g/mL streptomycin at 37°C. To induce apoptosis of RGC-5 cells, growth medium was withdrawn (serum starvation), and cells were maintained in DMEM free serum for 0, 8, 24, 48 and 96 hours (Khalyfa et al, 2007). The total RNA was extracted using RNase mini columns (RNeasy; Qiagen, Valencia, CA), followed by DNase treatment as described in the manufacturer's protocols. Total RNA was extracted from each time point and maintained at -800C until used for analysis. The RNA integrity was verified with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and RNA Integrity Numbers (Khan-Farooqi et al, 2005).

D. Statistical analysis Student’s t-test was used to identify differentially expressed genes in each time point. This test was carried out on each gene in the data set. To test appropriate statistical thresholds for each time point, accounting for the multiplicity of testing thousands of genes in each data set, we used ANOVA and the false discover rate (FDR). For desired FDR, a threshold p-value of 0.05 was used (Benjamini and Hochberg, 1995).

E. Identification of signaling pathways Pathway Studio software was used to examine the gene list obtained from our microarray analysis to classify any specific signaling pathways, gene regulation, and protein interaction maps. Biological pathway networks of differentially expressed genes (RefSeq accession numbers) were only modeled onto the software. This software extracts functional information on specific genes from the RetNet database using a natural language processing algorithm named MedScan. Data analyzed with this program could be resolved into cogent models of the specific biological pathways activated under the experimental conditions used in the microarray analyses.

B. Microarray analysis Equal quantities of total RNA samples were labeled using Agilent low RNA input fluorescent linear amplification kit (Agilent), and hybridized to three independent identical arrays of Agilent oligonucleotide probe sequences. Three independent biological samples from each time point were used for cDNA synthesis. Four hundred !g of total RNA was reverse transcribed into cDNA briefly using Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT) with oligo promoter primer, and incubated at 40oC for 2 hours. cRNAs were synthesized by !"# $!%&' transcription using T7 RNA polymerase, and labeled with cyanine 3-labeled dCTP (PerkinElmer, Boston MA). The labeled products were further purified using RNeasy mini purification columns (Qiagen). Twelve picomoles of the fluorescently labeled cRNAs were used for each of the microarray hybridizations. Preparation of hybridization solution was accomplished using Agilent’s !" s!%( Hybridization Kit Plus. The Agilent 60mer array was comprised of 22,775 rat probe sequences, with each array containing one probe sequence (60-mer) per transcript that was spotted using a non-contact spotter onto glass slides. Hybridizations were performed using Agilent’s oligo processing protocols. Approximately 0.75 !g of labeled cRNA and 5 !l of

F. Gene ontology and clustering Functional classification of the differentially expressed genes, based on their cellular component, biological process, and molecular functions, were conducted using GO. To further clarify the functional roles of the differentially expressed genes, GO analysis was conducted using GeneSifter ("#$%!&'()*!&&+*! Seattle, WA) and GeneSpring software (Silicon Genetics, Agilent Technologies, Palo Alto, CA). The Ontology report provided information on how many genes in the list posses a particular ontology, and how many genes on the array have that same ontology. A z-score was calculated for each ontology, and if the z-score was greater than two or less than negative two, this indicated ontology was significantly over or under-represented in the list. The results from GeneSifter and GeneSpring were linked

513

Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5 to KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.ad.jp/kegg) databases for bioinformatics integration. To examine expression profiles of differentially expressed genes, clustering analysis was used with aid of GeneSifter software. This allowed genes to be grouped together based on the degree of similarity between their temporal expression profiles. Hierarchical dendrograms were drawn with the gene tree algorithm, and genes sharing similar expression profiles tended to be clustered together. The location of a branch containing the genes can be considered a benchmark for assessing resemblance of genetic expression. This measure is usually a correlation coefficient that compares the regulation of one gene to another at various experimental conditions. Genes were selected for the construction of a gene tree if the expression of the gene was twofold greater or less in the serum deprived samples, relative to that in the control (non-serum deprived).

PBS and incubated with the secondary goat antibody coupled to Alexa green 488 (Molecular Probes, Inc), and diluted to 1:200 in buffer for 1hr at room temperature. The samples were then rinsed in PBS. To visualize the nuclei, the sections and cells were stained with Hoechst nuclear stain (Invitrogen) for 5-10 minutes at room temperature in the dark. The sections and cells were again rinsed in PBS and double distilled water and mounted with an anti-fade mounting media. Next, the sections were examined with a Zeiss LSM410 confocal microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) using a 40X water immersion objective (numerical aperture 1.2). Samples were excited at 488 nm using an argon ion laser (Melles Girot/Omnichrome, Carlsbad, CA), and fluorescence was collected using a 510-525nm emission filter. The Hoechst nuclear stain was excited at 364 nm with a UV laser (Coherent Innova Technology, Santa Clara, CA), and fluorescence emission was collected using a 400-440nm emission filter.

G. Real-time PCR

III. Results

Quantitative Real-Time PCR (Applied Biosystems, Foster City, CA; 7300 instrument) of the differentially expressed genes detected in both serum-deprived (8, 24, 48 and 96 hrs) and nondeprived samples (zero time point), were used to validate microarray results. The same total RNA from apoptotic and nonapoptotic RGC-5 samples, used for microarray hybridization, was used for quantitative real-time PCR. One micrograms of the same total RNA used for hybridization was reverse transcribed using a High-Capacity cDNA Archive Kit, and TaqMan® Master Mix Reagent Kit (Applied Biosystems, Foster City, CA). The total volume of PCR reactions was carried out for each transcript of interest in 40 !l. The primer pairs for each cDNA were obtained from Applied Biosystem’s demand-assay (Applied Biosystems, Foster City, CA). The sequences for each selected gene from the microarray results were BLASTed against !"##$%& '()*+,-.$% on the NCBI database. Sequences of the primers designed were selected to be within the same region of the gene used to develop the microarray sequence probes. To ensure specific amplification, various negative controls were included in the PCR reaction. Triplicate PCR reactions were performed in 96-well plates for each gene in parallel with the 18S rRNA. All reactions were performed in triplicates, and statistical significance was evaluated by the Student t-test. The RT-PCR reaction program included the following: the initial step of 2 minutes at 50 C, denaturation at 95 C for 10 minutes, followed by 40 thermal cycles of denaturation (15 seconds at 95oC) and elongation (1 min at 60oC). The relative expression of the gene of interest was analyzed using the 2-!!CT method.

A. Differentially expressed genes RGC-5 Agilent rat microarray was used to generate a genome-wide expression profile for RGC-5 serum deprivation in order to characterize the molecular mechanisms, and identify apoptosis-related genes possibly involved in signal transduction pathways. Of the 22,775 transcripts, we identified two categories of genes (known and unknown) during the four separate time points. For the known genes, 168 were differentially expressed at 8hrs, 417 at 24hrs, 218 at 48hrs and 247 at 96 hrs. In addition 545 unknown genes were differentially expressed at 8hrs, 1550 at 24 hrs, 793 at 48 hrs, and 915 at 96hrs. Table 1 shows the top 10 up-and down-regulated differentially expressed genes in RGC-5 serum deprived time course 8, 24, 48 and 96 hrs, including their RefSeq accession number, UniGene number, chromosome location, tissue or cells of expression, sub-cellular location, fold change and p-value. The differentially expressed transcripts were identified in each individual RGC-5 serum deprived time point using statistical analysis and Student’s #-test p-values (p<0.05). In addition, the data presented in Table 2 shows the differentially expressed genes involved in neuronal cell death at 8, 24, 48 and 96 hrs.

B. Biological pathways To better understand the biological significance of the differentially expressed genes in RGC-5 serum deprived time course samples, the top 5 up and the top 5 down-regulated genes were used to build biological pathways utilizing the PathwayStudio software (Figure 1). The differentially expressed genes obtained from the microarray data was imported into PathwayStudio (Ariadne Genomics Inc.). Biological pathways were constructed using the differentially expressed genes in 8, 24, 48, and 96 hrs. The data (Figure 1, Panel A) exhibits the signaling pathways for the genes that were differentially expressed in 8 hrs serum deprivation. We found 7 candidate genes associated with the 10 genes (5 up-regulated, highlighted in /0$+, and 5 down-regulated, highlighted in&*-(0+#) identified by the microarray analysis using PathwayStudio. A total of 17 genes led to the further identification of three cellular processes which included regulation of signal transduction, proliferation, and

H. Confocal microscopy Adult Sprague-Dawley rats (Whole eyes) were isolated, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at a thickness of 5 !m, and deparaffinized prior to immunocytochemical treatment. RGC-5 cells were also fixed in 4% paraformaldehyde, and rinsed in phosphate buffered saline (PBS; pH 7.4). The samples were immersed in a 0.1 Triton X100 solution for 30 min, and rinsed three times in PBS for 5 minutes. Samples were then treated with a 0.05M solution of glycine for 15 min. Subsequently, the sections from tissues and cells were incubated in a blocking solution of 5% BSA and 10% normal serum of a second antibody host animal in PBS for 15 min. This was followed by an overnight incubation at 4ºC with primary antibodies for complement component C3 (Santa Cruz Biotechnology, Santa Cruz, CA), diluted to 1 !g/ml in a solution of 1% BSA and 1% normal serum of secondary antibody host animal within PBS. For controls, the primary antibody was omitted and replaced by goat IgG in the same concentration. After overnight incubation, the samples were rinsed four times in

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Cancer Therapy Vol 6, page 515! We also found 30 candidate genes associated with the 10 genes identified by the microarray analysis, and five major biological processes were identified: 1) protein degradation, 2) differentiation, 3) cell survival, 4) proliferation, and 5) apoptosis. The data in Figure 1, Panel D displays 31 candidate genes associated with the 10 genes identified by the microarray analysis. Six major biological processes were identified: 1) apoptosis, 2) proteolysis, 3) differentiation, 4) proliferation, 5) cell survival, and 6) secretion.

apoptosis (!"##$%). The data (Figure 1, Panel B) shows the signaling pathways for the genes that were differentially expressed (top 5 up-regulated and top 5 down-regulated) in the 24 hrs. We identified 15 genes associated with the top 10 regulated genes identified by the microarray analysis, making a total of 25 genes leading to further identification of four cellular processes: 1) secretion, 2) proteolysis, 3) DNA protection, and 4) differentiation (Figure 1 Panel B). The biological pathway for the differentially expressed genes in 48 hr serum-deprived RGC-5 is shown in Figure 2, Panel C.

Table 1. List of most highly 10 down and up-differentially expressed genes in each time point of RGC-5 serum deprived. Symbol

Description

RefSeq ID

UniGene ID

chrom. Location

Expression Area

Subcellar Location

Fold change

p-value

Rn.22129 !

2q44!

brain; eye; other !

secreted!

0.344!

0.0003!

Rn.3272!

6q16!

brain; eye; other!

nuclear!

0.363!

0.0287!

Rn.22609 !

8q21!

other!

0.398!

0.0172!

Rn.3723 !

6q16!

0.407!

0.0221!

Rn.22258 !

5q36!

brain; other! brain; eye; other !

membrane! plasma membrane! NA!

0.422!

0.0419!

Rn.950 !

1q22!

brain; other!

NA!

0.425!

0.0265!

Rn.44298 !

2q11!

brain; other!

NA!

0.445!

0.0212!

Rn.24539 ! Rn.9828 !

13q26! 8q31!

secreted ! cyctoplasmic!

0.450! 0.456!

0.0488! 0.0321!

Rn.10957 !

1q22!

brain; other! eye; other! brain; eye; other !

membrane!

0.520!

0.0045!

Rn.1658 !

8q21!

brain; eye; other !

mitochondrial!

2.310!

0.0293!

Rn.14526 !

7q34!

membrane!

2.418!

0.0182!

Rn.54554 ! Rn.9942 !

8q23! 3q42!

NA! extracellular!

2.433! 2.506!

0.0334! 0.0118!

Rn.6302 !

2q45!

mitochondrial!

2.525!

0.0332!

Rn.18592 ! Rn.38451 !

4q34! 7q31!

NA! NA!

2.984! 3.152!

0.0407! 0.0456!

Rn.10211 !

20p12!

NA!

3.686!

0.0238!

Rn.764 !

15p14!

nuclear!

3.829!

0.0042!

membrane!

4.620!

0.0256!

NA!

0.270!

0.0005!

NA!

0.338!

0.0232!

NA!

0.349!

0.0269!

8hrs cysteine rich protein 61 ! Cyr61!

Id2!

Kcnj1! Ifrd1! Srm! Pace4! Rasa1! Tgfb2! Rbp2! Igf1r!

Atpi! Slc16a8! Cryab! Svs4!

Acadm! Mgll! A5D3! Lss! Lgals3! Fdft1! Col1a2! Anx3! Odc1!

NM_031327! Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein ! NM_013060! Potassium inwardlyrectifying channel, subfamily J, member 1 ! NM_017023! interferon-related developmental regulator 1 ! NM_019242! spermidine synthase ! NM_053464! subtilisin - like endoprotease ! NM_012999! RAS p21 protein activator NM_013135! 1! transforming growth factor, beta 2 ! NM_031131! retinol-binding protein 2 ! NM_012640! iInsulin-like growth factor 1 receptor ! NM_052807! ATPase inhibitor (rat mitochondrial IF1 protein) ! NM_012915! solute carrier family 16, member 8 ! NM_031744! crystallin, alpha polypeptide 2 ! NM_012935! seminal vesicle protein-4 ! NM_012662! acyl-Coenzyme A dehydrogenase, C-4 to C12 straight-chain ! NM_016986! monoglyceride lipase ! NM_138502! A5D3 protein ! NM_145093! 2,3-oxidosqualene: lanosterol cyclase ! NM_031049! lectin, galactose binding, NM_031832! soluble 3 ! farnesyl diphosphate farnesyl transferase 1! NM_019238! procollagen, type I, alpha 2! annexin III (Lipocortin III) ! arnitine decarboxylase !

NM_053356!

Rn.3252 ! 15p12! 24hrs Rn.16629 ! 4q13!

NM_012823!

Rn.6589 !

14q22!

NM_012615!

Rn.874 !

6q16!

515

other! brain; eye; other! other! brain; eye; other! brain; eye; other! other; brain! brain; eye; other! brain; eye; other! brain; eye; other! brain; eye; other! brain; eye; other! brain; eye; other!

Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5 Hsp60!

NM_022229!

Rn.44658 !

9q22!

NM_022929!

Rn.54458 !

10q12!

NM_032070!

Rn.29981 !

7q22!

NM_053827!

Rn.4445 !

5q36!

Cyslt2!

heat shock protein 60 (liver) ! Kv channel-interacting protein 1 ! non-histone chromosomal architectural protein HMGI-C ! procollagen-lysine, 2oxoglutarate 5dioxygenase 1 ! cysteinyl leukotriene CysLT2 receptor ! profilin II x!

Cyr61!

cysteine rich protein 61 !

NM_031327!

Rn.22129 !

2q44!

Dpp7!

dipeptidylpeptidase 7 !

NM_031973!

Rn.3363 !

3q13!

Cds1!

NM_031242!

Rn.18983 !

14q22!

Fbln5! Prdx4! Stmn1!

CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 1 ! eucine rich repeat protein 3, neuronal ! fibulin 5 ! peroxiredoxin 4 ! stathmin 1 !

Mgp!

matrix gla protein !

P2rx4!

Kcnip1! Hmgic!

Plod1!

Cyslt2!

Lrrn3!

Fcgrt!

C3!

NM_133413!

Rn.74252 !

15p11!

NM_030873!

Rn.3515 !

2q31!

NM_030856!

Rn.39124 !

6q21!

NM_019153! NM_053512! NM_017166!

Rn.1699 ! Rn.17958 ! Rn.555 !

6q32! NA! 5q36!

NM_012862!

Rn.2379 !

4q43!

purinergic receptor P2X, NM_031594! ligand-gated ion channel, 4! Fc fragment NM_033351! immunoglobulin G receptor ! complement component 3 ! NM_016994!

Rn.7176 !

12q16!

Rn.2306 !

1q22!

Rn.11378 !

9q11!

procollagen, type I, alpha 2! ornitine decarboxylase !

NM_053356! NM_012615!

Rn.874 !

6q16!

NM_031716!

Rn.63486 !

7q33!

Cyr61!

WNT1 inducible signaling pathway protein 1! cysteine rich protein 61 !

NM_031327!

Rn.22129 !

2q44!

Coq3!

coenzyme Q (ubiquinone) ! NM_019187!

Rn.3824 !

5q21!

Tgfb2!

transforming growth factor, beta 2 ! follistatin-related protein !

NM_031131!

Rn.24539 !

13q26!

NM_024369!

Rn.2979 !

11q21!

annexin III (Lipocortin III) ! biglycan !

NM_012823!

Rn.6589 !

14q22!

NM_017087!

Rn.783 !

Xq37!

solute carrier family 6, member 6 ! regulator of G-protein signaling protein 2 ! alanine and arginine rich domain containing protein! lectin, galactosidebinding, soluble, 2 (galectin 2)! stathmin 1 !

NM_017206!

Rn.9968 !

4q34!

NM_053453!

Rn.1892 !

13q21!

NM_145093!

Rn.38451 !

7q31!

NM_133599!

Rn.2982 !

7q34!

NM_017166!

Rn.555 !

5q36!

Odc1! Wisp1!

Frp! Anx3! Bgn! Slc6a6! Rgs2! Aard! Lgals2!

Stmn1!

0.0255!

0.362!

0.0236!

other!

NA!

0.364!

0.0422!

brain; eye; other!

membrane!

0.375!

0.0047!

membrane!

0.376!

0.0063!

NA!

0.392!

0.0032!

secreted!

0.410!

0.0198!

lysosomal!

4.037!

0.0144!

membrane!

4.067!

0.0012!

NA! secreted! NA!

4.243! 4.645! 4.719!

0.0045! 0.0138! 0.0094!

cyctoplasmic!

4.920!

0.0017!

secreted!

5.345!

0.0226!

membrane!

6.005!

0.0348!

membrane!

6.411!

0.0122!

NA!

6.457!

0.0116!

NA!

0.106!

0.0015!

NA!

0.252!

0.0291!

secreted!

0.256!

0.0401!

secreted!

0.281!

0.0004!

mitochondrial!

0.360!

0.0356!

secreted!

0.360!

0.0138!

secreted!

0.367!

0.0212!

NA!

0.375!

0.0275!

secreted!

0.377!

0.0392!

membrane!

0.379!

0.0022!

NA!

3.081!

0.0223!

NA!

3.273!

0.0438!

NA!

3.456!

0.0102!

cyctoplasmic!

3.626!

0.0121!

other! brain; eye; other! brain; eye; other! brain; eye; other! brain; eye; other! brain; eye; other! other! other! brain; eye; other! brain; eye; other!

brain; other!

48 hrs Rn.16629 ! 4q13!

Col1a2!

0.355!

other!

mitochondrial! plasma membrane!

brain; other!

516

brain; eye; other! brain; eye; other! brain; eye; other! brain; eye; other!

eye; other! brain; eye; other! brain; eye; other! brain; other! brain; eye; othre! brain; eye; othre! brain; eye; othre! brain; eye; othre! brain; other! braina; other!

brain; other! brain; eye; othre!

Cancer Therapy Vol 6, page 517! Ccl5! Lamp2! Bhlhb3!

Lamp1!

Lcn2! C3!

chemokine (C-C motif) ligand 5 ! lysosomal-associated membrane protein 2 ! basic helix-loop-helix domain containing, class B3 ! lysosomal associated membrane protein 1 (120 kDa) ! lipocalin 2 ! complement component 3 !

Col1a2!

procollagen, type I, alpha 2! Cstn2! calsyntenin 2 ! (Epn2)! EH domain binding protein epsin 2 ! Tgfb2! transforming growth factor, beta 2 ! Lu! lutheran blood group (Auberger b antigen included) ! Hmgic! non-histone chromosomal architectural protein HMGI-C ! Prkwnk1! protein kinase, lysine deficient 1 ! Lmnb1! lamin B1 ! Col5a3! collagen, type V, alpha 3 ! Gabra4! gamma-aminobutyric acid (GABA-A) receptor, subunit alpha 4 ! Rgs2! regulator of G-protein signaling protein 2 ! Epha7! Eph receptor A7 ! Fth1! ferritin subunit H ! Cebpd!

Dnase1! C3!

CCAAT/enhancerbinding, protein (C/EBP) delta ! lectin, galactosidebinding, soluble, 2 (galectin 2) ! complement component 1, s subcomponent ! chemokine (C-C motif) ligand 5 ! deoxyribonuclease I ! complement component 3 !

Mgp!

matrix gla protein !

Lgals2!

C1s! Ccl5!

NM_031116!

Rn.8019 !

10q26!

NM_017068!

Rn.1722 !

Xq11!

NM_133303!

Rn.10784 !

4q43!

brain; eye; othre! brain; eye; othre!

secreted!

4.262!

0.0202!

membrane!

4.378!

0.0403!

membrane!

4.406!

0.0261!

nuclear! secreted!

4.811! 5.535!

0.0318! 0.0431!

NA!

7.264!

0.0151!

NA! membrane!

0.113! 0.320!

0.0005! 0.0045!

cyctoplasmic!

0.374!

0.0088!

brain; other!

secreted!

0.390!

0.0159!

brain; other!

NA!

0.394!

0.0479!

other! brain; eye; other! brain; other! brain; other!

NA!

0.406!

0.0377!

cyctoplasmic! nucleoplasmic! NA!

0.406! 0.430! 0.431!

0.0427! 0.0234! 0.0408!

brain; other!

NA!

0.439!

0.0077!

brain; other! brain; other! brain; eye; other! brain; eye; other!

NA! membrane!

5.827! 5.832!

0.0127! 0.0258!

NA!

6.143!

0.0027!

cyctoplasmic!

7.259!

0.0223!

NA!

8.427!

0.0326!

NA!

9.599!

0.0413!

secreted! secreted!

18.160! 18.922!

0.0287! 0.0282!

NA!

31.580!

0.0015!

secreted!

66.370!

0.0363!

brain; eye; other! NM_012857!

Rn.40177 !

16q12!

NM_130741! NM_016994!

Rn.11303 ! Rn.11378 !

NM_053356!

96hrs Rn.16629 ! 4q13!

NM_134377! NM_021852!

Rn.81212 ! Rn.44273 !

8q31! 10q23!

NM_031131!

Rn.24539 !

13q26!

NM_031752!

Rn.7003 !

1q21!

NM_032070!

Rn.29981 !

7q22!

NM_053794!

Rn.18103 !

4q42!

NM_053905! NM_021760! NM_080587!

Rn.11362 ! Rn.38654 ! Rn.81205 !

18q12! 8q13! 14q11!

NM_053453!

Rn.1892 !

13q21!

NM_134331! NM_012848!

Rn.10181! Rn.54447 !

5q21! 1q43!

NM_013154!

Rn.6975 !

NA!

NM_133599!

Rn.2982 !

7q34!

NM_138900!

Rn.4037 !

4q42!

NM_031116!

Rn.8019 !

10q26!

NM_013097! NM_016994!

Rn.6077 ! Rn.11378 !

10q12! 9q11!

NM_012862!

Rn.2379 !

4q43!

C. Networks analyses

3q11! 9q11!

brain; eye; other! eye; other! brain; eye; other! brain; eye; other! NA! brain; eye; other!

brain; other! brain; eye; other! brain; eye; other! other! brain; eye; other! brain; eye; other!

1.5 fold (p-value <0.05). Furthermore, by applying more stringent statistical methods (Benjamini and Hochberg, 1995), 80 genes were identified (57 up-regulated, and 23 down-regulated) using a 2-fold cutoff and p-value (<0.05).! The highly significant differentially expressed genes (80) identified in all serum deprived RGC-5 were classified based on their involvement in specific biological processes using the GO database (http://www.geneontology.org). These genes were classified into three functional categories: 1) biological

A large number of genes were identified in each time point of serum deprived cells using a threshold cutoff of a 1.5-fold change and a !-value <0.05. We identified 137 transcripts down-regulated at 8hrs, 409 at 24 hrs, 228 at 48hrs, and 244 at 96hrs; we also identified 257 transcripts up-regulated at 8 hrs, 875 at 24 hrs, 352 at 48 hrs and 588 at 96hrs. In addition, using one-way ANOVA among all serum deprived samples (0, 8, 24, 48, and 96 hrs), there were 786 transcripts identified; among those 216 upregulated above 1.5 fold, and 483 down-regulated below

517

Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5

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Cancer Therapy Vol 6, page 519!

Figure 1. Schematic representation of direct biological association of differentially expressed genes involved in RGC-5 serum deprived time course (8, 24, 48, and 96 hrs). The biological pathways for the differentially expressed genes were conducted using PathwayStudio software (Ariadne Genomics Inc., Rockville, MD). Biologically linked proteins indicated by nodes, and biological processes are shown in the diagram. The top 5 down-regulated genes are in !"#$% &'"'(, and the top 5 up-regulated genes are in )*'"$+% &'"'(,% The colors highlighted in !"#$ and )*'"$+ are the genes identified by microarray analysis, and the colors in ($-% are% the potential target genes identified by the aid of PathwayStudio. In 8 hrs, three major biologic processes were identified (regulation of signal transduction, proliferation, and apoptosis) by .$""'/ color (Panel A). In 24 hrs, four major biologic processes were identified (secretion, proteolysis, DNA protection, and differentiation) and are represented by yellow color squares (Panel B). In 48 hrs, five major biologic processes were identified (protein degradation, differentiation, cell survival, proliferation and apoptosis), and are represented by .$""'/ color (Panel C). In 96 hrs, six major biologic processes were identified (apoptosis, proteolysis, differentiation, proliferation, cell survival, and secretion) (Panel D).

signaling pathway) for the 80 genes were identified. The genes involved in the cell cycle pathway were cyclindependent kinase inhibitor 2A, cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4), growth arrest and DNA-damage-inducible 45 alpha; genes involved in complement and coagulation cascades were complement component 1, s subcomponent, and complement component 3; genes involved in MAPK signaling pathway were calcium channel, voltage-dependent, T type, ! 1G subunit, and growth arrest and DNA-damage-inducible 45! . It should be understood that a gene may be involved in more than one pathway. Subcategories of the molecular function of these 80 genes were indicated for further investigation. These two sources of annotation provided an overview of the biological process, molecular function, cellular localization and cellular pathway information associated with a particular gene product.

process, 2) cellular component, and 3) molecular function. Table 3 summarizes the GO for the 80 most frequently differentially expressed genes, under serum deprivation conditions. The ontology of the genes in the biological process was classified as follows: physiological process (34%), cellular process (33%), regulation of biological process (13%), development (8%), response to stimulus (8%), and growth (2%). For the cellular components, genes were classified as cell (24%), cell part (24%), organelle (16%), extracellular region (12%), extracellular region part (9%), organelle part (6%), protein complex (5%), membrane-enclosed lumen (3%), and extracellular matrix (1%) (Table 3). For the molecular functions, genes were classified as binding (45%), catalytic activity (23%), signal transducer activity (11%), transporter activity (7%), transcription regulator activity (5%), enzyme regulator activity (4%), and structural molecule activity (4%) (Table 3). This annotation provided an overview of the biological process, molecular function, cellular localization and pathway information associated with a particular gene product. To gain insight into the biological significance of those 80 differentially expressed genes, several biological pathways using standard KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified. For example, three major KEGG pathways (cell cycle, complement and coagulation cascades, and MAPK

D. Biological regulated genes

characterization

of

To understand the biological meaning of our investigation, we annotated the 80 significantly differentiated expressed genes {71% up-regulated (57 genes), and 29% down-regulated (23 genes)} identified by our microarray assesments with the controlled vocabulary provided by the Gene Ontology Consortium. Multifunctional proteins contain several annotations

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Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5

Figure 2. Hierarchical clustering for the significant differentially expressed genes in RGC-5 serum deprived time dependent manner. Panel A shows a heat map of the clustered genes and data presented in the matrix. The row coordinate is assigned to represent the level of single gene expression, and the column coordinate is assigned to the sample (0, 8, 24, 48 and 96 hrs), with red corresponding to high expression, green corresponding to low expression and black corresponding to an intermediate level of expression. Genes significantly changed above 2-fold across the time course following serum deprivation were only included in clustering. The averaged normalized intensity from triplicate arrays is represented by the color of the corresponding cell in the matrix. Color intensity reflects the magnitude of the signal intensity. Panel B shows hierarchical cluster of the genes arranged in the dendrogram in which the patterns and length of the branches reflect the relationship of the expression level. The ordering of the rows and columns was determined using hierarchical clustering and the associated dendrogram for the samples shown at the top of the figure.

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Cancer Therapy Vol 6, page 521! Table 2. List of neuronal differentially expressed genes detected in serum deprived RGC-5 time course p-value <0.05. 8 hrs Symbol! Cyr61 Tgfb2 Nfil3 Bdnf Bcl2l10 Ctf1 Dnase1 Mmp9 Ucn Sod1 Ngfr Prdx2 Dap Mapk8ip Cyr61 Prkg2 Tgfb2 Kcnb1 Gzma Bmf Madh7 Bok Tfpt Dnase1 Sod1 Gch Bad Prdx2 Bnip3l Gchfr Ngfrap1 Mapk8ip Pdcd4 Cyr61 Tgfb2 Kcnb1 Nrg1 Bok Sp1 Bard1 Bnip3l Rcl Gchfr Dnase1 Gch Gadd45a Cyr61 Tgfb2 Bcl2l Sp1

RefSeq number!

Gene Description! cysteine rich protein 61 transforming growth factor, beta 2 nuclear factor, interleukin 3, regulated Brain derived neurothrophic factor BCL2-like 10 (apoptosis facilitator) cardiotrophin 1 Deoxyribonuclease I matrix metalloproteinase 9 urocortin Superoxide dimutase 1, soluble Nerve growth factor receptor, fast peroxiredoxin 2 death-associated protein mitogen activated protein kinase 8 interacting protein 24 hrs cysteine rich protein 61 protein kinase, cGMP- dependent, type 2 transforming growth factor, beta 2 Potassium voltage gated channel, member 1 granzyme A Bcl-2 modifying factor MAD homolog 7 (Drosophila) Bcl-2-related ovarian killer protein TCF3 (E2A) fusion partner (in childhood leukemia) Deoxyribonuclease I Superoxide dimutase 1, soluble GTP cyclohydrolase 1 bcl-2 associated death agonist peroxiredoxin 2 BCL2/adenovirus E1B 19 kDa-interacting protein 3-like GTP cyclohydrolase I feedback regulatory protein nerve growth factor receptor (TNFRSF16) mitogen activated protein kinase 8 interacting protein programmed cell death 4 48 hrs cysteine rich protein 61 transforming growth factor, beta 2 Potassium voltage gated channel, member 1 neuregulin 1 Bcl-2-related ovarian killer protein Sp1 transcription factor BRCA1-associated RING domain protein 1 BCL2/adenovirus E1B 19 kDa-interacting protein 3-like putative c-Myc-responsive GTP cyclohydrolase I feedback regulatory protein Deoxyribonuclease I GTP cyclohydrolase 1 growth arrest and DNA-damage-inducible 45 alpha 96 hrs cysteine rich protein 61 transforming growth factor, beta 2 B cell lymphoma 2 like Sp1 transcription factor

521

Fold change!

p-value!

NM_031327 NM_031131 NM_053727 NM_012513 NM_053733 NM_017129 NM_013097 NM_031055 NM_019150 NM_017050 NM_012610 NM_017169 NM_022526 NM_053777

0.344 0.450 0.611 0.828 1.102 1.174 1.257 1.296 1.552 1.577 1.610 1.787 1.900 1.925

0.000 0.049 0.003 0.019 0.035 0.012 0.027 0.046 0.046 0.044 0.022 0.041 0.006 0.011

NM_031327 NM_013012 NM_031131 NM_013186 NM_153468 NM_139258 NM_030858 NM_017312 NM_138870 NM_013097 NM_017050 NM_024356 NM_022698 NM_017169 NM_080888 NM_133595 NM_053401 NM_053777 NM_022265

0.410 0.506 0.558 0.578 0.578 0.597 0.631 0.686 1.349 1.464 1.638 1.713 1.986 2.080 2.262 2.317 1.806 2.838 2.317

0.020 0.012 0.020 0.036 0.043 0.020 0.024 0.021 0.039 0.023 0.033 0.022 0.040 0.010 0.020 0.003 0.021 0.005 0.011

NM_031327 NM_031131 NM_013186 NM_031588 NM_017312 NM_012655 NM_022622 NM_080888 NM_133525 NM_133595 NM_013097 NM_024356 NM_024127

0.281 0.360 0.468 0.555 0.560 0.753 1.754 2.087 2.169 2.326 2.343 2.550 3.076

0.000 0.014 0.028 0.020 0.027 0.029 0.050 0.031 0.013 0.000 0.006 0.008 0.043

NM_031327 NM_031131 NM_031535 NM_012655

0.338 0.390 0.479 0.536

0.037 0.016 0.045 0.017

Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5 Ghr Gzma Bok Lfg Dnase1l3 Mmp9 P2rx1 Nradd Atf3 Prdx2 Bad Trib3 Bnip3l Tpt1 Aqp1 Gch Nr4a1 Gadd45a Dnase1

growth hormone receptor granzyme A Bcl-2-related ovarian killer protein lifeguard deoxyribonuclease I-like 3 matrix metalloproteinase 9 Purinergic receptor P2X, ligand-gated ion channel, 1 neurotrophin receptor associated death domain Activating transcription factor 3 peroxiredoxin 2 bcl-2 associated death agonist tribbles homolog 3 (Drosophila) BCL2/adenovirus E1B 19 kDa-interacting protein 3-like tumor protein, translationally-controlled 1 aquaporin 1 GTP cyclohydrolase 1 immediate early gene transcription factor NGFI-B growth arrest and DNA-damage-inducible 45 alpha Deoxyribonuclease I

NM_017094 NM_153468 NM_017312 NM_144756 NM_053907 NM_031055 NM_012997 NM_139259 NM_012912 NM_017169 NM_022698 NM_144755 NM_080888 NM_053867 NM_012778 NM_024356 NM_024388 NM_024127 NM_013097

0.614 0.615 0.650 1.172 1.223 1.347 1.585 1.679 1.690 2.206 2.219 2.269 2.428 2.559 3.598 3.863 4.165 4.653 18.922

0.015 0.028 0.035 0.021 0.032 0.040 0.021 0.007 0.050 0.042 0.043 0.046 0.048 0.018 0.044 0.027 0.025 0.023 0.028

Table 3. Gene Ontology for the highly significance differentially expressed genes detected in all time courses above 2-fold change. Category Biological Process

Ontology physiological process cellular process regulation of biological process development response to stimulus growth

Gene percentage (%) 34 33 13 8 8 2

z-score 2.36 1.29 0.93 0.03 0.62 0.79

Cellular Components

cell cell part organelle extracellular region extracellular region part organelle part protein complex membrane-enclosed lumen extracellular matrix

24 24 16 12 9 6 5 3 1

-3.87 -3.87 -0.42 1.92 1.02 -0.54

binding catalytic activity signal transducer activity transporter activity transcription regulator activity enzyme regulator activity structural molecule activity

45 23 11 7 5 4 4

0.22 0.05 -0.29 -0.35 -0.35 0.34 0.77

Molecular Function

0.76 -0.36

for each gene. In addition, the molecular function for down-regulated genes were also classified as binding (44%), catalytic activity (39%), signal transducer activity (6%), structure molecule activity (6%), and transporter activity (6%), as shown in Table 5.

corresponding to their different functions. Several molecular functions were categorized for the up-regulated genes, which included binding (45%), catalytic activity (19%), signal transducer activity (10%), transporter activity (8%), enzyme regulator activity (6%), transcription regulator activity (6%), and structure molecule activity (4%), as shown in Table 4. The data presented in Table 4 displays the following: the total number of genes involved in each molecular function category, gene name, accession number and the function

E. Clusters of gene expression profiles The 80 highly significant genes were clustered, as shown in Figure 2. The selected genes contained in the

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Cancer Therapy Vol 6, page 523! B, shows that genes segregating into seven major branches of the dendrogram were assigned seven cluster similarities based on their expression profiles across multiple conditions. Out of the 80 significantly differentiated expressed genes, 57 were up-regulated and 23 were down-regulated (Figure 3 and 4). The diagram in Figure 3, Panel A, shows a partition cluster for the up-regulated genes using silhouette widths. The heat map for the up-regulated genes is depicted in Figure 3, Panel B, and the hierarchical cluster analysis in Figure 3, Panel C. The partition cluster is shown in Figure 4, Panel A, the heat map in Figure 4, Panel B, and the hierarchical cluster in Figure 4, Panel C, using the same type of clustering indicated above for the down regulated genes.

clusters have expression values above 2-fold changes: either up or down-regulated, and p-values <0.05. GeneSifter software utilized clustering analysis to identify discrepancies in gene expression, and association of these changes to sets of genes with similar profiles. Figure 2, Panel A, shows the heat map cluster analysis of gene expression alterations across a serum deprived time course; each color represents a single gene expression value of the sample. The intersection of a gene and sample is colored according to its expression value: red indicates high expression and green indicates low expression. A hierarchical clustering method was used to group genes differentially expressed in a time dependent manner after induction of apoptosis on the basis of similar expression patterns (Figure 2, Panel B). The data in Figure 2, Panel

Figure 3. Hierarchical clustering of the significant up-regulated genes detected in RGC-5 serum deprived time course. Panel A can be construed as gene patterns dramatically changed above 2-fold, across time course following serum deprivation. Panel B shows the heat map of the hierarchically clustered genes. Each row represents a single gene and each column an experimental conditions (0, 8, 24, 48 and 96 hrs). The averaged normalized intensity from triplicate arrays is represented by the color of the corresponding cell in the matrix. Color intensity is proportional to the magnitude of signal intensity. Panel C displays a hierarchical cluster of genes arranged in the dendrogram, where the patterns and length of the branches reflect the relationship of expression level in 5 different experimental conditions. Genes were arranged in a dendrogam, where the patterns and length of the branches reflect the relationship of the expression level in 5 different experimental conditions.

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Figure 4. Hierarchical clustering of the significant down-regulated genes detected in RGC-5 serum deprived time course. Panel A displays partitioning methods k-medoids (18) algorithms. Only genes displaying significant 2-fold change across time course after serum deprivation were included in the hierarchical analysis, as evident from the graph. Panel B shows a heat map of hierarchically clustered genes. Data are presented in a matrix; each row represents a single gene, and each column represents experimental conditions (0, 8, 24, 48 and 96 hrs). The averaged normalized intensity from triplicate arrays is represented by the color of the corresponding cell in the matrix. Color intensity is proportional to the magnitude of signal intensity. Panel C shows a hierarchical cluster of the genes arranged in the dendrogram where the patterns and length of the branches reflect the relationship of the expression level in 5 different experimental conditions. In this cluster, genes are connected iteratively based on their similarity; genes with similar expression patterns were grouped together and connected by a series of branches, called dendrogram (or clustering tree).

Table 4. List of the differentially up-regulated genes found to be associated in the molecular function using Gene Ontology. Category !

Gene Name!

Binding!

AT rich interactive domain 5, (Mrf1 like)! ATPase, H transporting, lysosomal V0 subunit c, (Atp6v0c)!

!

Accession Number! NM_0010349 34! NM_130823!

NM_030845! NM_022214!

chemokine activity

Calcium channel, voltage-dependent, T type, alpha 1G subunit, (Cacna1g) !

NM_031601!

!

CCAAT/enhancer binding protein (C/EBP), delta! Chemokine (C-X-C motif) ligand 1, (Cxcl1) ! Chemokine (C-X-C motif) ligand 5, (Cxcl5) !

NM_013154!

!

DNA binding! ATPase activity, coupled to transmembrane movement of ions; hydrogen-transporting ATPase activity; metal ion binding; protein binding calcium ion binding; cation channel activity; ion channel activity; low voltage-gated calcium channel activity; voltage-gated calcium channel activity DNA binding; protein binding; protein homodimerization activity growth factor activity

!

!

Function!

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Cancer Therapy Vol 6, page 525! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Chromatin assembly factor 1, subunit B p60, (Chaf1b) ! Complement component 3, (C3)! Cyclin-dependent kinase inhibitor 2A, (Cdkn2a) !

NM_0010247 41! NM_016994! NM_031550!

Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4), (Cdkn2c) ! Deoxyribonuclease I, (Dnase1) !

NM_131902!

Fc receptor, IgG, low affinity III, (Fcgr3) ! Ferritin, heavy polypeptide 1, (Fth1) !

NM_053843!

Golgi associated, gamma adaptin ear containing ARF binding protein 2, (Gga2) ! Growth arrest and DNA-damageinducible 45 alpha, Gadd45a! Growth factor, erv1 homolog (S. cerevisiae), Gfer ! Lectin, galactoside-binding, soluble 2, Lgals2 ! Mitochondrial ribosomal protein L23, Mrpl23 ! Nuclear factor, erythroid derived 2, like 2, Nfe2l2 ! RAB13, member RAS oncogene family, Rab13 ! response gene to complement 32, Rgc32! Solute carrier family 25 (mitochondrial carrier, phosphate carrier), member 25, Slc25a25 !

NM_013097!

NM_012848! BQ191223!

ferroxidase activity; kinase binding metal ion binding; oxidoreductase activity; protein binding ADP-ribosylation factor binding; protein binding

NM_024127!

Protein binding!

NM_013222!

protein binding

NM_133599!

galactoside binding!

NM_022529!

RNA binding; structural constituent of ribosome

NM_031789!

NM_253722!

DNA binding; protein binding; transcription factor activity; transcription regulator activity; transcriptional activator activity GTPase activity; nucleotide binding

NM_054008!

protein binding

NM_145677!

calcium ion binding; transporter activity

ATPase activity, coupled to transmembrane movement of ions; hydrogen ion transporter activity; hydrogentransporting ATP synthase activity, hydrogentransporting ATPase activity, hydrolase activity; metal ion binding; protein binding complement component C1r activity

Catalytic activity!

ATPase, H transporting, lysosomal V0 subunit c, Atp6v0c!

NM_130823!

!

NM_0010028 04! NM_138900!

!

Complement component 1, r subcomponent, C1r ! Complement component 1, s subcomponent, C1s ! Deoxyribonuclease I, Dnase1 !

!

Ferritin, heavy polypeptide 1, Fth1 !

NM_012848!

!

Flavin containing monooxygenase 1, Fmo1 ! GTP cyclohydrolase 1; Legumain, Gch ! RAB13, member RAS oncogene family, Rab13 !

NM_012792!

!

chromatin binding; histone binding; unfolded protein binding protein binding; receptor binding cyclin-dependent protein kinase inhibitor activity; DNA binding; protein binding; transcription factor activity cyclin-dependent protein kinase inhibitor activity; DNA binding; transcription factor activity actin binding; calcium ion binding; deoxyribonuclease I activity; hydrolase activity; protein binding IgG binding; IgG receptor activity; receptor activity

NM_024356!

complement component C1s activity; peptidase activity actin binding; calcium ion binding deoxyribonuclease I activity; endonuclease activity; hydrolase activity; protein binding ferroxidase activity; kinase binding; metal ion binding; oxidoreductase activity; protein binding dimethylaniline monooxygenase (N-oxide-forming) activity; monooxygenase activity; transporter activity hydrolase activity

NM_253722!

GTPase activity; nucleotide binding

Chemokine (C-X-C motif) ligand 1, Cxcl1 !

NM_030845!

Growth factor activity!

NM_022214!

chemokine activity!

NM_016994! NM_053843!

protein binding; receptor binding IgG binding; IgG receptor activity; receptor activity

!

Chemokine (C-X-C motif) ligand 5, Cxcl5 ! Complement component 3, C3! Fc receptor, IgG, low affinity III, Fcgr3 ! Toll-like receptor 2, Tlr2 !

NM_198769!

receptor activity

Transporter activity!

ATPase, H transporting, lysosomal V0 subunit c, Atp6v0c !

NM_130823!

ATPase activity, coupled to transmembrane movement of ions; hydrogen ion transporter activity; hydrogen-

! ! Signal transducer activity! ! ! !

NM_013097!

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Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5 transporting ATP synthase activity, rotational mechanism; hydrogen-transporting ATPase activity, rotational mechanism; hydrolase activity; metal ion binding; protein binding calcium ion binding; cation channel activity; ion channel activity; low voltage-gated calcium channel activity; voltage-gated calcium channel activity dimethylaniline monooxygenase (N-oxide-forming) activity; monooxygenase activity; transporter activity ion binding; transporter activity

!

Calcium channel, voltage-dependent, T type, alpha 1G subunit; Cacna1g !

NM_031601!

!

Flavin containing monooxygenase 1, Fmo1 ! and Solute carrier family 25 (mitochondrial carrier, phosphate carrier), member 25, Slc25a25 !

NM_012792!

Enzyme regulator activity!

Cyclin-dependent kinase inhibitor 2A, Cdkn2a !

NM_031550!

cyclin-dependent protein kinase inhibitor activity; DNA binding; protein binding; transcription factor activity

!

Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4), Cdkn2c !

NM_131902!

cyclin-dependent protein kinase inhibitor activity; DNA binding; transcription factor activity

Transcription regulator activity! !

Cyclin-dependent kinase inhibitor 2A, Cdkn2a !

NM_031550!

Cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4), Cdkn2c ! Nuclear factor, erythroid derived 2, like 2, Nfe2l2 !

NM_131902!

cyclin-dependent protein kinase inhibitor activity; DNA binding; protein binding; transcription factor activity cyclin-dependent protein kinase inhibitor activity; DNA binding; transcription factor activity DNA binding, protein binding; transcription factor activity; transcription regulator activity; transcriptional activator activity extracellular matrix structural constituent; structural constituent of bone

!

!

Structural molecule activity! !

NM_145677!

NM_031789!

Matrix Gla protein, Mgp !

NM_012862!

Mitochondrial ribosomal protein L23, Mrpl23 !

NM_022529!

F. Confirmation expressed genes

of

differentially

RNA binding; structural constituent of ribosome

PCR results indicated that C3 is appreciably up-regulated in RGC-5 serum deprived cells.

To confirm the expression data from our microarray results, we validated 10 differentially expressed genes, including the following: BAD, AKT1, AKT2, AKT3, GSK3a GSK3b, Pik3r1, Pik3cb, Pik3c2g, and SGK; all for quantitative RT-PCR analysis. These genes were construed as having invariable trends of change by microarray analysis and real-time quantitative PCR (Table 6). Expression levels of 18S were used as an internal housekeeping gene control to normalize all selected genes observed from the microarray data (Table 6). Mean fold changes from microarray vs. RT-PCR experiments were calculated. Expression ratios obtained by quantitative RTPCR are in accordance with the ratios obtained from using microarrays. In general, relative expression level changes conveyed by microarray analysis were greater than those revealed by real-time RT-PCR; especially for genes that were clearly over-expressed. This may be explained by the increased sensitivity, and perhaps, the better reliability of microarray compared to the RT-PCR method. RT-PCR was performed in parallel for genes differentially expressed at each time point to confirm or support the detected changes of expression levels. A gene expression ratio greater than 1 is considered up-regulated, while a gene expression ratio less than 1 is down-regulated. The information gathered from our microarray data and RT-

H. Confocal microscopy Confocal microscopy was used to determine the expression pattern of complement protein (C3), using antibody against C3 in RGC-5 cells and rat retina. For the adult rat, the immunofluorescence labeling of C3 in the retina (Figure 5, Panel C) was observed predominantly in the RGC layer (white arrows in Figure 5, Panel C) and nerve fiber layer (fluorescent green arrow in Figure 5, Panel C), with positive immunolabeling in the photoreceptor cells (blue arrow in Figure 5, Panel C); RPE (light blue arrow in Figure 5, Panel C); choroids (red arrow in Figure 5, C); and an inner plexiform layer (purple arrow in Figure 5, Panel C). Diffuse labeling was observed throughout the thickness of the retina. The DIC image of the retina is shown in Figure 5, Panel A. The retina was also stained with Hoechst nuclear stain (Figure 5, Panel B), and the merged image for C3 and Hoechst nuclear stain is shown in (Figure 5, Panel D). As expected, there was no a positive immunoreactivity in IgG control retinas (data not shown). For the RGC-5 cells, the data in Figure 6, Panel C displayed intense C3 immunoreactivity in the cytosol with no reactivity in the nucleus. Merged images of C3 and Hoechst nuclear stain immunofluorescence clearly showed that C3 was expressed in the cytosol of the transformed RGC-5 cells (Figure 6, Panel D). As expected, there was no positive immunoreactivity with goat IgG controls (data not shown).

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Cancer Therapy Vol 6, page 527! Table 5. List of the differentially down-regulated genes found to be associated in the molecular function using Gene Ontology. ! Category ! Binding! !

Gene Name ! Adenylate kinase 1, Ak1 Carbonic anhydrase VB, mitochondrial , Ca5b Crystallin, alpha B, Cryab

! !

Accession Number! NM_024349!

Function!

NM_001005551! NM_012935! NM_138548!

structural constituent of eye lens; unfolded protein binding ATP binding; deoxyribonuclease activity; DNA binding; kinase activity; magnesium ion binding; nucleoside diphosphate kinase activity; nucleotide binding; transferase activity poly(U) binding; protein binding; RNA binding

Expressed in non-metastatic cells 1, Nme1 !

Heterogeneous nuclear ribonucleoprotein H1, Hnrph1 Insulin-like growth factor 1, Igf1

!

Serum/glucocorticoid regulated kinase, Sgk

!

WNT1 inducible signaling pathway protein 1, Wisp1

Catalytic activity! ! !

Adenylate kinase 1, Ak1 Carbonic anhydrase VB, mitochondrial, Ca5b Expressed in non-metastatic cells 1, Nme1

! !

Lanosterol synthase, Lss Lysyl oxidase-like 1, Loxl1

! !

Signal transducer activity! Structural molecule activity ! Transporter activity!

Serum/glucocorticoid regulated kinase, Sgk Transmembrane 7 superfamily member 2, Tm7sf2 Insulin-like growth factor 1, Igf1

adenylate kinase activity; kinase activity; nucleotide binding; transferase activity lyase activity; metal ion binding

NM_080896!

NM_001082477 NM_019232!

growth factor activity; hormone activity; insulin-like growth factor ; receptor binding; protein binding; steroid binding nucleotide binding; protein kinase activity; protein serine/threonine kinasemactivity; transferase activity

NM_031716!

insulin-like growth factor binding; protein binding

NM_024349!

adenylate kinase activity; kinase activity; nucleotide binding; transferase activity lyase activity; metal ion binding

NM_001005551! NM_138548!

NM_031049! NM_001012125! NM_019232! NM_001013071!

ATP binding; deoxyribonuclease activity; DNA binding; kinase activity; magnesium ion binding; nucleotide binding; transferase activity isomerase activity; lanosterol synthase activity electron carrier activity; oxidoreductase activity, acting on the CH-NH2 group of donors, oxygen as acceptor nucleotide binding; protein kinase activity; protein serine/threonine kinase activity; transferase activity delta14-sterol reductase activity, oxidoreductase activity

NM_001082477!

growth factor activity; hormone activity; insulin-like growth factor receptor binding; protein binding; steroid binding

Crystallin, alpha B, Cryab

NM_012935!

structural constituent of eye lens; unfolded protein binding

Potassium channel, subfamily K, member 2, Kcnk2

NM_172041!

outward rectifier potassium channel activity; potassium channel activity; voltage-gated potassium channel activity

neurodegenerative, autoimmune, cardiovascular and cancer diseases. As our understanding of apoptosis increases, further opportunities will arise for tailor-made therapies that will result in improvements in the clinical setting. Retinal ganglion cells play a key role in integrating visual information and relaying it to the visual centers of the brain via the optic nerve, and the health of these cells depends on the availability of various neurotrophic factors. A number of studies have shown that neurotrophins promote survival of injured RGCs and other retinal neurons (Mittag and Schmidt, 2004; Thanos and Emerich, 2005). It has been shown that optic neuropathies or retinopathies that disrupt the retrograde transport of trophic factors to the RGCs induce stress to these cells and

IV. Discussion In this study, we further identified and characterized differentially expressed genes, potentially involved in the apoptotic cell death of RGC-5 serum deprived with time courses 0, 8, 24, 48 and 96 hrs. This was performed using Agilent’s oligonucleotide-based microarrays. The serum deprivation time course was chosen based on DNA fragmentation, and cell death to survival ratios (Charles et al, 2005; Khalyfa et al, 2007). There has been limited progress in the past in identifying apoptosis-related genes in many models of neuronal cell death. Apoptosis is a process of cellular suicide, consisting of precise transcriptional alterations of a multitude of genes involved in a wide range of pathologic conditions that include:

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Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5 Table 6. Comparative analysis of microarray data and Real Time RT-PCR. Comparison of fold changes in expression for selected genes by microarray and real-time PCR. The data shown are means Âą SD from three replicates. Microarray p-value <0.05 Gene Name Phosphoinositide 3kinase, regulatory subunit, polypeptide 1 (p85 alpha) phosphatidylinositol 3-kinase, catalytic subunit, beta isoform phosphatidylinositol 3-kinase, C2 domain containing, gamma polypeptide v-akt murine thymoma viral oncogene homolog 1 Murine thymoma viral (v-akt) oncogene homolog 2 thymoma viral proto-oncogene 3 (Akt3), mRNA glycogen synthase kinase 3 alph glycogen synthase kinase 3 beta bcl-2 associated death agonist Complement component 3 complement component 1, s subcomponent growth arrest and DNA-damageinducible 45 alpha

RT-PCR p-value <0.05

Symbol

RefSeq number

Pik3r1

NM_013005

1.10

0.83

0.98

0.85

0.98

0.75

0.42

0.76

Pik3cb

NM_053481

0.89

0.68

0.70

0.83

0.98

0.74

0.42

0.76

Pik3c2g

NM_053923

0.69

0.92

0.59

0.70

0.24

0.37

0.07

0.11

Ak1

NM_033230

0.80

0.88

0.59

0.70

0.9

0.82

0.44

0.21

Akt2

NM_017093

1.01

0.97

0.90

0.56

1.13

1.05

0.9

0.61

Akt3

NM_031575

0.93

0.47

1.12

0.55

0.75

0.37

0.33

0.26

Gsk3a

NM_017344

1.16

0.93

0.62

1.03

1.16

0.84

0.49

0.61

Gsk3b

NM_032080

0.96

0.75

0.68

0.69

0.91

0.84

0.53

0.46

Bad

NM_022698

1.66

1.99

1.24

2.22

1.28

2.28

2.13

1.95

C3

NM_016994

1.15

6.46

7.26

31.58

0.26

11.25

14.89

24.61

C1s

NM_138900

0.88

2.23

2.86

9.60

0.81

4.39

20.41

24.15

Gadd45a

NM_024127

1.37

2.04

3.08

4.65

1.51

2.03

2.23

2.73

8 hrs

24 hrs

48 hrs

96 hrs

8 hrs

24 hrs

48 hrs

96 hrs

response. Our data provides insight into how gene expression in RGC-5 responds to treatment of serum deprivation with several time points, and how gene expression data is useful in identifying genes involved in different biochemical pathways. Differential gene expression analysis is one of the most widely used applications of microarray technology. To categorize the overall global changes of gene expression profiling in RGC-5 serum deprivation using a time-dependent manner (8, 24, 48 and 96 hrs), which includes a wide range of apoptotic cell death, we identified the significantly differentiated expressed genes between cell survival and cell death phenotype in each time point, and we applied multiple comparisons between all samples using different statistical analyses. In microarray data anlyses, genes are eventually divided into two groups, those that demonstrate differential expression and those that do not, often based on essentially arbitrary statistical or ad hoc criteria. We used a test to identify differentially expressed genes between two time points (i.e. 8hrs vs. 0 hr, 24 hrs vs. 0 hrs,

eventually their death. For example, in glaucoma, elevated intraocular pressure was shown to obstruct axonal transport at the optic nerve head, causing blockage of retrograde transport of neurotrophins (Anderson and Hendrickson, 1974; Nickells, 1996; Pease et al, 2000). Neurotrophins are a family of regulatory factors that mediate the differentiation and survival of neurons. It has been shown that the deprivation of these trophic factors is associated with vision loss in glaucoma resulting from apoptosis of the RGCs (Rabacchi et al, 1994; Quigley et al, 1995). It is clear that neurotrophins play an important role in the health and pathology of the RGCs (Agarwal et al, 2007). The cross-talk among various apoptotic pathways is complex. To understand the intricacies of cell death machinery, microarray analysis can facilitate a comprehensive evaluation of the role of individual signaling pathways in mediating apoptosis, and it can also provide insights into how apparently distinct pathways network with each other to coordinate a balanced 528

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Figure 5. Confocal immunocytochemical localization of complement components (C3) in the adult rat retina. A DIC image of retina is showing various layers of rat retina Panel A. The nuclear staining by Hoechst stain is shown in Panel B. Intense C3 immunolabeling Panel C was detected in retinal ganglion cell (RGC) layer (white arrow) including the nerve fiber layer (fluorescent blue arrow). The C3 labeling was also detected in the inner plexiform layer (purple arrow), photoreceptors (blue arrow), and the choroids (red arrow). The merged image of B and C is shown in D. INL indicates inner nuclear layer; ONL indicates outer nuclear layer; RPE indicates retinal pigment epithelium layer; and CH indicates choroids.

Figure 6. Confocal immunocytochemical localization of complement components (C3) in RGC-5 cells. The DIC image is shown in Panel (A) and the nuclear staining by Hoechst stain is shown in Panel B. Arrows in Panels A and B depict the cells and the nuclei of the RGC-5 cells, respectively. Complement components (C3) in green (arrows in Panel C). The merged images of these RGC-5 cells are shown in Panel D (arrows). CFH was expressed in the cytoplasm (arrows in Panels B and D).

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Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5 involved in cell survival and apoptotic pathways. We identified a series of interacting genes that constitute a potentially important gene-regulatory network related to cell death and survival pathways (Figure 7). We believe that gene expression profiling and identification of novel genes would provide a significant increase in our knowledge to the mechanisms and pathways regulating the transition from cell survival to cell death. We also believe that the pathway presented in Figure 7 is important in the apoptotic gene networks because those genes were confirmed using QRT-PCR in RGC-5 cells. In Figure 7, we included C3 and C1s which had previously validated by Khalyfa and colleagues in 2007, in addition to the genes that validated in this study by RT-PCR in order to build a gene-network interactions and possible link between complement components and PI3K/Akt gene members. Many apoptotic pathways have been reported in other literature. The decision between cell survival and death is believed to depend on the balance of pro- and antiapoptotic members of the Bcl-2 family, whom dimerize in the cell. Bad, a pro-apoptotic BH3-only containing protein, is one of the pro-apoptotic members of the Bcl-2 family, and is the target of a network of upstream survival signals. It has been indicated that PI3K/Akt/Bad signal transduction pathway is engaged in the control of apoptosis in many different cell types, particularly through phosphorylation of the Bcl-2 family protein Bad (Campos et al, 2003). Here, we show the involvement of this pathway in the programmed cell death of RGC-5 response to time dependent serum deprivation. In our microarray data, we identified the PI3K, Akt, GSK isoforms, and Bad. The validated genes, which are known to be involved in the PI3K pathway, were also used to build biological pathways. We believe PI3K is important because it is involved in both cell death and cell survival. To the best of our knowledge this study is the first to demonstrate the gene network interactions between C3, C1s and PI3K/Akt members in RGC-5 serum deprivation. Based on our data, we suggest that may be there is a link among those genes based on their gene interactions and connectivity.! Previously, it has been shown that complement activation occurs in the retina, that has been subjected to elevated IOP, which may have implications in the pathophysiology of glaucoma (Kuehn et al, 2006). P13K directly regulates certain cytoplasmic apoptotic pathway. Several studies have shown that PI3K and its downstream effectors, protein serine/threonine kinases (Akt), is a major signaling pathway by which survival factors prevent apoptosis (Yao and Cooper, 1995). PI3K plays a crucial role in implementing alterations in a broad range of cellular functions in response to extracellular signals. Several targets of the PI3K/Akt signaling pathway have been identified, which may underlie the ability of this regulatory cascade to promote survival (Datta et al, 1999). Our microarray data shows three isoforms of PI3K were found to be consistently changed over time of serum deprivation. PI3K has three isoforms: Pik3r1, Pik3cb, and Pik3c2g, encoded by three different genes located on chromosomes 2q12, 8q31, and 4q44, respectively. Activation of PI3K pathway promotes proliferation and

48 hrs vs. 0 hr, and 96 hrs vs. 0 hr), then we used AVOVA for multiple comparisons to identify genes either up-or-down regulated utilizing GeneSifter software FDR <0.05. We are not expecting to have the same number genes for each time point to be the same even we used the same p-value cutoff (<0.05) for all time points. Based on the DNA fragmentation and cell death numbers for all the time points, 24 hrs was less apoptotic comparing to 48 and 96 hrs. We noticed that at 24 hrs the number of differentially expressed shows the highest number of genes (412) comparing to other time points. The highest number of genes in 24 hrs was surprise. We identified 80 genes that were differentially expressed at least two-fold following serum deprivation in all time points. Of these 80 genes, there were 57 genes up-regulated and 23 genes down-regulated. These particular gene expression discrepancies demonstrate a common retinal response to cell death, plausibly implicated in retinal cell death. Furthermore, besides monitoring changes in the expression patterns of individual genes at each time point, we also used the differentially expressed genes to design biological pathways. Our results provide a comprehensive view of the distinct temporal changes in gene expression induced by RGC-5 serum deprivation using GO and network analyses to identify significant biological processes and regulatory networks. We showed how gene expression data is useful in identifying genes involved in different biochemical pathways (Figure 1, Panels, A-D, and Table 1). Our data show that ontology analysis revealed that genes related to apoptosis as a group were significantly regulated in response to serum deprivation. Many of these genes are involved in proliferation, cell survival, differentiation, and apoptosis, as documented (Figure 1). Of the 80 genes there were three KEGG pathways significantly enriched in the genes associated with cell death, cell cycle, complement and coagulation cascades, and MAPK signaling pathway) in all time points of serum deprivation. For example, MAPK represents a critical link between cell surface signal transduction and nuclear processes, including regulation of cell proliferation, differentiation, and survival (Nozaki et al, 2001; Huffman et al, 2004). Our results also revealed that serum deprivation induced distinct sets of RGC-5 genes, which exhibited different temporal expression patterns (Tables 3, 4 and 5). The clustering process aided in distinguishing frequent expression patterns and highly specialized patterns, which probably required the participation of specialized sets of transcription factors. The up-regulated genes formed the largest clusters, followed by the down-regulated genes. All clusters showed the relative consistency of up-regulated and down-regulated genes (Figure 3). As indicated in Figure 3, the genes that are classified into the same clusters may have shared biological functions or may have been part of the same biological pathways. The comparative gene expression profile of RGC-5 serum deprivation conveyed resemblance of microarray data to quantitative RT-PCR. The microarray data were confirmed using 12 differentially expressed genes by realtime PCR (Table 6). Such validated genes were used to formulate and assemble biological pathways, conceivably

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Figure 7. Biological pathway for selected genes used for validation via QRT-PCR. The biological pathways for the selected genes were conducted using PathwayStudio software. The blue color represents the selected genes identified in microarray data and used for validation. Biologically linked proteins are indicated by nodes, and biological processes are shown in the diagram.

survival in several different cell types of the immune system. For example, (Homma et al, 2007) demonstrated that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stages of rat retinal ganglion cells after optic nerve injury. Yu and colleagues also suggested in 2006 that insulin protects retinal neurons from oxidative stressinduced apoptosis. The ciliary neurotrophic factor (CNTF) promotes cell survival via the PI3K signaling pathway in !"#$% and in !"!%& (Ikeda et al, 2004). PI3K activates, phosphorylates and regulates the activity of several targets, including kinases, transcription factors and other regulatory molecules. The complement component has been implicated in the pathogenesis of neurodegenerative diseases (Stasi et al, 2006), and posses the potential to activate intracellular signaling pathways, such as TNF cascade, which can lead to the activation of downstream mediators (Alexander et al, 2005). Complement component 3 (C3) is the most abundant complement protein, playing a key role in the complement cascades that consist of the classical,

alternative, and lectin pathways (Sekine et al, 2001). Activation of C3 via the alternative pathway can induce TNF release (Levy et al, 2003). The complement expression was up-regulated within the retina of two glaucoma models (DBA/2 mouse and monkey) and some human glaucomatous eyes (Stasi et al, 2006). C3 is the converging point for activation of all three complement pathways, and is critical in biological processes mediated by complement activation (Sekine et al, 2001). In spite of the pathway where the complement cascade becomes activated, all pathways share the same downstream events that start with the generation of different C3 convertases (Sekine et al, 2001). The up-regulation of C3 and C1s, both part of the complement system, demonstrate an apoptotic immune response to the serum deprived RGC. The consistent up-regulation of complement proteins along with the simultaneous, consecutive down-regulation of Akt suggests that these systems may be interrelated. The complement cascade is initiated to help clear pathogens. C3 is activated proteolytically by enzyme complexes (the C3 convertases) yielding the 531

Khalyfa et al: Analysis of gene expression profiles in the programmed cell death in RGC-5 apoptotic response in tumor cells. Overexpression of Akt3 results in contractile dysfunction and increased susceptibility to cardiac injury (Taniyama et al, 2005). Akt may also promote cell survival in an indreict fashion by regulating another major signaling enzyme-glycogen synthase kinase 3". The glycogen synthase kinase 3 (GSK-3) is another gene was validated by qRT-PCR and highlighted in this study. The GSK-3 is a serine-threonine kinase encoded by two isoforms, termed GSK-3" and GSK-3! , that are inactivated upon phosphorylation by PKB (Woodgett, 1990; Cross et al, 1995). The two isoforms are encoded by different genes and share nearly identical sequences in their kinase domains. Inhibition of GSK-3! results in the protection against apoptosis (Pap and Cooper, 1998). It has been implicated in several major neurological disorders, and has been originally identified as a regulator of glycogen metabolism (Embi et al, 1980). GSK3" mediates an interaction between two major forms of synaptic plasticity in the brain (Peineau et al, 2007). GSK3" is involved in neurodevelopment, regulation of neuronal plasticity, cell survival, and potentially, it is a key component of several psychiatric and neurodegenerative diseases (Grimes and Jope, 2001). In our data, GSK isoforms were down-regulated throughout each time point of serum deprivation (Table 6). GSK3 inhibition occurs when the p110 of PI3K/Akt pathway is activated by growth factors. Activated Akt then phosphorylates GSK3!, inhibiting GSK3 kinase activity. This activation of the p110-PI 3-kinase/Akt pathway, and inhibition of GSK3, delivers a strong anti-apoptotic signal to the cell (Turenne and Price, 2001). Serum withdrawal from cultured cortical neurons resulted in apoptosis along with decreased Akt activity and increased GSK3!!"#$%&%$'! (Grimes and Jope, 2001)(!The ability of trophic factors to promote cell survival has been attributed in part to the PI3K/Akt kinase cascade. Several targets of the PI3K/Akt signaling pathway have been recently identified that may explain the ability of this regulatory cascade to promote survival (Datta et al, 1999). It is obvious that studying gene expression for thousands of genes at once allows us to examine biological pathway’s analyses rather than looking at a single gene marker. Genes with common functions often exhibit correlated expression levels, which can be used to identify sets of interacting genes from microarray data (Voy et al, 2006). Gene Ontology (Ashburner et al, 2000) and clustering are the most commonly used methods for identifying a large group of genes detected by microarray data. Microarrays gene expression profiling have been used extensively in many human diseases which provided new possibilities for gaining superior insights into the pathogenesis of various diseases; to define biomarkers and possibly new candidate targets for novel treatments (Lindberg and Kappos, 2006). During the apoptotic process, the molecular players interact closely with each other to accelerate or interrupt the cellular process, some of these either up-regulated or down-regulated based on the signal of stimuli to the cells. The up-regulated genes are the candidates for the apoptotic signals and the down-regulated genes are the

anaphylatoxin C3a [which (Sekine et al, 2001) mediates inflammation (Bokisch et al, 1969)], and the major fragment C3b [which can covalently attach to target surfaces via its reactive thioester (Law et al, 1979)]. C3 plays a central role in the activation of the complement system, and C1s are involved specifically in the classical pathway. One significant effect of complement activation can be apoptosis, which can occur directly through cellular events stimulated by C5b-9, C5a (Sato et al, 1999; Nauta et al, 2002). The up-regulation of complement components (C1S and C3) in our data was also supported by the recent studies using microarray data published by (Yang et al, 2007) who suggested that the up-regulation of those genes in experimental glaucoma and optic nerve transaction rats injury. The pharmacological blockade of the PI3K/Akt pathway has been shown to reduce cell death in PC12 cells (Hillion et al, 2006). Here we show Akt was down regulated in both microarrays and QRT-PCR. Our data show that down-regulation of Akt members (Table 6). The down-regulation of Akt expression levels is an indication of apoptoic cell death in RGC-5. This Akt protein kinase has been identified as an important mediator of cell survival (Hillion et al, 2006). Akts are primary downstream effectors of PI3K. There are three closely related isoforms, which include: Akt1, (PKB!), Akt2 (PKB") and Akt3 (PKB#), encoded by three different genes located on chromosomes 14q32, 19q13 and 1q43, respectively. The Akt signaling pathway is recognized as one of the most imperative pathways in regulating cell survival. The activation of Akt pathway provides cells with a survival signal that allows them to withstand apoptotic stimuli (Yao and Cooper, 1995). Hillon and colleagues found that the pharmacological blockade of the PI3K/Akt pathway abated cell death (Hillion et al, 2006). In the nerve system, Akt expression is substantially upregulated during cellular stress, suggesting Akt’s involvement in cellular protection. For example, Akt targets several key proteins needed for keeping cells alive, including apoptosis regulators and transcription factors (Yuan and Yankner, 2000). Activated Akt protein supports the survival of neurons in the absence of trophic factors, whereas a dominant-negative mutant of Akt inhibits neuronal survival even in the presence of survival factors. Activated Akt has been shown to mediate cell survival by phosphorylation of several targets including Bad (Thompson and Thompson, 2004). Phosphorylated Bad and Bax proteins ensues release of anti-apoptotic Bcl-xL and Bcl-2 proteins and promotes survival (Masters et al, 2002). Bcl-2 associated death agonist (BAD), which is continually up- regulated throughout each time-point, as proven by microarray and QRT-PCR (Table 6), is known to be a pro-apoptotic gene (Campos et al, 2003). Dephosphorylated Bad translocates to the mitochondria where it binds to either the death antagonists Bcl-2 or BclxL, and prevents them from performing their per-survival activity, thus leading to cell death (Yang et al, 1995). Akt phosphorylates and inactivates GSK-3! (Cross et al, 1995), and (Koseoglu et al, 2007) suggested that inhibiting all three AKT isoforms is necessary to elicit maximal

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Cancer Therapy Vol 6, page 533! prevention of diseases like glaucoma. Apoptosis is involved in a wide range of pathologic conditions, including neurodegenerative disease. The ability to understand apoptosis processes will arise for tailor-made therapies that will result in improved clinics. We believe identification of the potential target genes are and the biological pathways leading to apoptosis will be of great benefit therapeutically in the future. Only functional studies of individual genes can establish their actual role and mechanisms of action within apoptotic pathways. Our microarray data, highlighted by GO annotations and statistical analyses, have generated new hypotheses that could not be generated by any other approach. These hypotheses provide directions to investigations for elucidating the molecular mechanisms that lead to neuronal apoptosis. Theses studies may reveal unsuspected molecular pathways controlling the survival/death of neurons, and ultimately contribute to the identification of new target genes for the treatment of neurodegenerative diseases including glaucoma.

candidates for cell survival. Many of these genes and their products can activate several pathways including PI3K/Akt as a cell survival pathway. This enzyme inhibits apoptosis by many downstream effects. Most traditional research into neurodegenerative disease including glaucoma is focused on developing drugs that inhibit neuronal dysfunction and death early in the disease process. One approach is to block the apoptotic signal trigger and activation of anti-apoptotic pathways by treatment with neurotrophic factors is another approach. Our data, together with previously published (Khalyfa et al, 2007) suggest that the induction of RGC-5 cell death lead to complex changes in gene expression and these changes involve modulation of positive and negative regulatory pathways. We believe better understanding of the molecular and cellular targets will led to the identification of specific drug targets. Genotyping for those candidate genes using the single nucleotide polymorphisms is another direction of research. As we mentioned above, glaucoma is the second leading cause of blindness in the world-wide, and successful development of a product capable of directly protecting both retina and optic nerve from glaucomatous damage would represent a significant advance in the treatment of the disease. Cell culture and animal studies support the concept of neuroprotective therapies may prevent RGC cell death after diverse kinds of injuries. Apoptosis is consequently of profound significance in physiology, pathology, and therapeutic medicine. Apoptosis of RGCs is central glaucomatous progression in human and experimental models. The analysis of the molecular mechanisms involved in apoptosis is therefore of great importance in developing gene and drug therapies for many disease where the control of apoptosis is perturbed including glaucoma. The control of apoptosis is complex processes and involves many genes. Some of these genes are relatively well characterized, but there many genes have yet to be identified. Many experiments in the past have demonstrated that the requirement of de novo gene expression during neuronal apoptosis. We have chosen to use RGC-5 cell line to mimic certain events seen in RGC death. Although previous studies implicated individual genes or genetic pathways during apoptosis, the complete spectrum of genes involved in the distinct temporal domains is mostly unknown. To begin a comprehensive survey of the gene based mechanisms that underline neuronal apoptosis, we have used the unprecedented experimental opportunities that genome sequences and the DNA microarray technology to perform genome-scale expression analysis in RGC-5 in cell survival and cell death using RGC-5 cell line. In this study, we highlighted the use of this technology and identifying differentially expressed genes in time dependent serum deprivation which might be helpful for identifying specific pathway or targeting molecules in particular pathway. In conclusion, we have identified a large number of differentially expressed genes in RGC-5 time dependent manor of apoptotic cell death. These alterations of gene expression may help to provide essential support to develop unique markers and strategies for efficient

Aknowledgement This work was partially supported by NIH:NCRR P2016481. The authors would like to thank Dr. Neerj Agarwal for helping in confocal microscope data.

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networks for low-dose radiation using graph theoretical algorithms. PLoS Comput Biol 2, e89. Wadia JS, Chalmers-Redman RM, Ju WJ, Carlile GW, Phillips JL, Fraser AD, Tatton WG (1998) Mitochondrial membrane potential and nuclear changes in apoptosis caused by serum and nerve growth factor withdrawal: time course and modification by (-)-deprenyl. J Neurosci 18, 932-947. Wang JT, Kunzevitzky NJ, Dugas JC, Cameron M, Barres BA, Goldberg JL (2007) Disease gene candidates revealed by expression profiling of retinal ganglion cell development. J Neurosci 27, 8593-8603. Woodgett JR (1990) Molecular cloning and expression of glycogen synthase kinase-3/factor A. The EMBO journal 9, 2431-2438. Wyllie AH, Kerr JF, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68, 251-306. Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ (1995) Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 80, 285-291. Yang Z, Quigley HA, Pease ME, Yang Y, Qian J, Valenta D, Zack DJ (2007) Changes in gene expression in experimental glaucoma and optic nerve transection: the equilibrium between protective and detrimental mechanisms. Invest Ophthalmol Vis Sci 48, 5539-5548. Yao R, Cooper GM (1995) Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267, 2003-2006. Yu J, Mears AJ, Yoshida S, Farjo R, Carter TA, Ghosh D, Hero A, Barlow C, Swaroop A (2004) From disease genes to cellular pathways: a progress report. Novartis Found Symp 255, 147-160; discussion 160-144, 177-148. Yu XR, Jia GR, Gao GD, Wang SH, Han Y, Cao W (2006) Neuroprotection of insulin against oxidative stress-induced apoptosis in cultured retinal neurons: involvement of phosphoinositide 3-kinase/Akt signal pathway. Acta Biochim Biophys Sin (Shanghai) 38, 241-248. Yuan J, Yankner BA (2000) Apoptosis in the nervous system. Nature 407, 802-809.

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Mouse mammary tumor virus: a cause of breast cancer in humans? Review Article

Ish Ahmed, James R. Harvey, Simi Ali, John A. Kirby, Thomas WJ Lennard* Applied Immunobiology and Breast Research Group, Institute of Cellular Medicine, Newcastle University, UK

__________________________________________________________________________________! *Correspondence: Professor Thomas Lennard, School Of Surgical & Reproductive Sciences3rd Floor, William Leech Building Faculty Of Medical Sciences Framlington Place Newcastle Upon Tyne Ne2 4hh, UK; Tel: 0191 222 7067; Fax: 0191 222 8514; E-mail: T.W.J.Lennard@ncl.ac.uk Key words: Mouse mammary tumour virus (MMTV), breast cancer Abbreviations: 3' long terminal repeat, (LTR); ductal carcinoma in situ, (DCIS); fluorescence nested PCR, (FN-PCR); human endogenous retrovirus, (HERV); infiltrating ductal carcinoma, (IDC); lobulo-alveolar, (LA); mouse mammary tumour virus, (MMTV); open reading frame, (ORF); superantigen, (Sag); terminal ductal lobular unit, (TLDU) Received: 13 February 2008; Revised: 20 June 2008 Accepted: 30 June 2008; electronically published: September 2008

Summary The mouse mammary tumour virus (MMTV), a B-type retrovirus, is known to be the most common cause of breast cancer in mice. However, its role in human breast cancer is very controversial. Here we review the evidence that supports the role of this virus in causing human breast cancer. It’s historical background, its possible transmission from mice to human and its mechanism of actions have also been reviewed.

newborn young of an inbred high-cancer mouse line (tumour incidence >80%) were fostered by females of a low tumour line. The fostered mice and their progeny lived considerably longer and had a tumour incidence of only 7.4%. With this and some other similar experiments, he concluded that an agent in milk influences the development of breast tumours. Though initially isolated in the milk, this agent was also obtained from both spontaneous and transplanted mammary carcinomas, lactating mammary tissue and whole blood. Bittner also showed that this agent could be carried through several passages in association with tumour transplants in mice that did not carry the milk influence themselves. The agent was smaller than a bacterium as it could pass through bacterial and seitz filters. By 1945, Andorvent went on to describe this milk factor as a virus (Gross, 1970), and gradually, this became widely known as the ‘Bittner Virus’. Today, the term MMTV includes Bittner’s virus and other closely related viruses (Labat, 1998). It is now well established that MMTV causes breast tumours in laboratory mice (Callahan, 1996), but its role in causing breast cancer in humans is a subject of significant controversy.

I. Introduction Breast cancer is the most common cancer in women worldwide, accounting for about 25% of all malignancies in women; the proportion is higher in western, developed, countries. In England, there were around 36,500 new cases diagnosed in 2003, representing 32 per cent of all cancers in women and a rate of 120 cases per 100,000 women. (Quinn et al, 2003). Several risk factors have been implicated in breast cancer including age, diet, nulliparity, obesity and family history (Kelsey, 1993). With genetic predisposition (mutations in BRCA1 and BRCA2) only accounting for approximately 5% of breast cancers (Szabo and King, 1997), extensive efforts have been made to account for the remainder. The mouse mammary tumour virus (MMTV) has been implicated as one of the unrecognized factors that might account for remainder of sporadic cases. The geographical variation in incidence of breast cancer provides additional grounds for this theory.

II. The origin of mouse mammary tumour virus The interest in MMTV as a cancer causing agent was generated several decades ago when Bittner demonstrated in 1939 an extra chromosomal factor in mice that contributes to breast cancer incidence. In his experiments,

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Ahmed et al: Mouse mammary tumor virus: a cause of breast cancer in humans? Identification of the MMTV envelope proteins in human breast cancer by using antibody against them was one of the earlier methods used. In one of these series, positive reactions were seen in 51 of 131 (39%) breast carcinomas with negative reactions in all normal breast tissue (Mesa-Tejada et al, 1978). However, the use of this antibody against the MMTV .-2elope protein has been disputed and places an uncertainty on the validity of these results (Hareuveni and Lathe, 1990). One of the major problems encountered at that time was the identification of human endogenous retrovirus (HERV) in the human genome. The significant homology between HERV and MMTV made it extremely difficult to differentiate between the two. As a consequence, earlier results were disputed with claims that the identification of MMTV may actually be HERV. This problem was overcome when, among others, Wang and colleagues isolated a 660-bp sequence in human breast cancer (Wang et al, 1995). This sequence was highly homologous to the .-2 gene of MMTV and had a very low homology to HERVs. Isolating DNA from fresh and frozen breast samples and using the polymerase chain reaction, they detected this sequence in 38% of the 314 breast cancer samples and in none of the normal breast tissues. A similar 250-bp sequence homologous to MMTV were detected in 39.7% of the 151 human breast cancers and in 1 of 27 normal breast samples assayed from paraffin-embedded sections. Wang and colleagues suggested in 1998 that their findings may represent exogenous sequences from an agent similar to MMTV. They next investigated whether these .-2-positive tumours produced mRNA by assessing the expression of the 660-bp sequence in the human breast samples using reverse transcription PCR. They demonstrated the expression of this sequence in 66% of the .-2-positive tumours and in none of the .-2-negative tumours. Subsequent sequence analysis confirmed that the expressed sequence was 98% homologous to the .-2 gene of the MMTV with only one small stretch of 46 bp that was homologous to the endogenous viral sequences. The fact that not all .-2-positive tumours produced mRNA was slightly intriguing, and possible explanations suggested were degradation of the mRNA or that not all the cells within the tumour express the viral sequences (Wang et al, 1998). The use of MMTV .-2 sequences by Wang and colleagues in 1998 and his interesting results rekindled further interests on the role of MMTV in human breast cancer. Several groups undertook similar strategies in attempts to define such a role. Etkind and colleagues used in 2000 a similar approach and analysed the presence of MMTV-like .-2 sequences in breast tumours by analysing them for the presence of a 250bp DNA fragment after nested PCR reactions. The sequence was present in 37% of the 73 samples of human breast cancer. None of the normal breast tissues had these sequences. DNA sequencing confirmed these sequences to be 99-100% homologous to the MMTV .-2 gene sequences. Interestingly, they also identified these sequences in 3 of 19 non-Hodgkin’s lymphoma (Etkind et al, 2000). Identifying the source of this sequence in human breast cancers is of paramount importance. To this end,

III. MMTV may explain the geographical variation in human breast cancer The incidence of human breast cancer varies significantly worldwide. The incidence is very low in Asia and Africa, very high in north America and northern Europe with an intermediate incidence belt in southern Europe and Latin America (Ahearne et al, 1999). People from lower incidence areas are known to develop an increase in its incidence when they move to an area of higher incidence (Winter et al, 1999). Several theories have been hypothesized to explain this increase in breast cancer incidence with migration. Andreeva and colleagues have reviewed 79 studies identifying risks and protective factors for the acquired risk. In their review, they have described that several studies (15 of the 79) have highlighted the importance of environmental and behavioural factors after migration. These include modification in diet, delay in childbirth, avoidance of breastfeeding and increasing socioeconomic status (Andreeva et al, 2007). Several groups have tried to explain the geographical variation in breast cancer incidence. Stewart and colleagues have described an explanation for this difference in incidence based on the distribution of MMTV in house mice of the genus !"#$%$!&$'"#(")"#*$!&$ ("#+,-."# and !&$ /0'.#+1("# (Stewart et al, 2000). !&$ '"#(")"# is present mainly in eastern Europe and !&$ /0'.#+1("# in western Europe (Sage et al, 1993). MMTV occurs both as exogenous infectious particles and as endogenous proviruses. !&$ /0'.#+1("# mice carried more exogenous virus and had more endogenous proviral loci than !&$'"#(")"#. Based on the incidence of human breast cancers described by Parkins and colleagues in 1997, he showed that the incidence of human breast cancer is higher in western Europe, inhabited by !&$ /0'.#+1("#, than eastern European lands of !&$ '"#(")"#. Interestingly, MMTV has been isolated from human breast cancer samples in other regions inhabited by this mouse specie. These include North America, Australia and Tunisia (Forsyth, 1985; Wang et al, 1995; Pogo et al, 1999; Etkind et al, 2000; Ford et al, 2004b; Levine et al, 2004). Regions not inhabited by this mice, including Vietnam, Sweden and Austria, are areas from where MMTV has not been isolated from human breast cancersamples (Ford et al, 2003; Witt et al, 2003; Bindra et al, 2007). It thus appears that human breast cancer may have a zoonotic connection with humans acquiring the virus from mice.

IV. Isolation of MMTV in human samples MMTV is known to cause breast tumours in laboratory mice. Numerous attempts have been made to identify a human analogue of MMTV that may cause cancers in human breasts. Most of the available data and its conclusion may point towards the presence of such an analogue in human breast cancer. Whether this plays any role in human breast cancer progression remains to be identified.

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Cancer Therapy Vol 6, page 539! show any significant difference between tumour grade and the presence of the MMTV !"# sequence (Zammarchi et al, 2006).

Melana and colleagues have tried to establish an exogenous source by examining the 250-bp sequence in archived cancer and normal tissues of the same patient. Of paired breast samples (normal and cancer) from 106 patients, they found 30% of 106 human breast cancers and only 1 of 106 normal breast tissues to be !"# positive (Melana et al, 2001). These results strongly indicated that the normal breasts of patients with !"# positive cancers do not contain the sequence, and led the authors to conclude that these sequences are of exogenous origin. However, they could not explain the finding of this sequence in the normal breast of one individual. The corresponding breast cancer from this patient was !"# negative. After several tests, they concluded that this may have been due to potential contamination with cancer tissue or DNA. The presence of the MMTV sequences in human breast cancers appears to have a geographical variation with areas of high and low positivity. In one study, MMTV-like gene sequences were amplified in only 1 of 120 and 0 of 40 breast cancer biopsy tissues from Vietnamese and Australian Vietnamese women, respectively. The same study showed the sequence in 19 of 45 breast cancers from Caucasian-Australian women (Ford et al, 2003). Earlier, this sequence was detected in 37.7% of 70 paraffin sections of human breast cancers in Italian women (Pogo et al, 1999). Recently, this sequence was isolated in 5 of 119 (4.2%) breast cancer biopsies in Mexican women (Zapata-Benavides et al, 2007). Most of the claims on the presence of MMTV in human breast cancers were initially based upon the amplification of MMTV-like sequences. However, in 2001, Liu and colleagues Isolated the complete virus from two human breast cancer samples that were !"# positive. This was done by amplifying three overlapping fragments and using the sequences to construct the complete virus by nested PCR. This 9.9-kb provirus was 95% homologous to MMTV and had a low homology to HERV. These proviral sequences were virtually absent in normal breast tissues (2%) and again suggest an exogenous origin (Liu et al, 2001). Primary culture of cells isolated from ascites or pleural effusion of patients with metastatic breast cancer have shown to contain viral sequences in their DNA, expressed env protein and retroviral particles on electron microscopy (Melana et al, 2007). A fluorescence nested PCR (FN-PCR) has recently been used to detect copies of the MMTV viral genome. Of the 45 frozen human breast cancers obtained by laser micro-dissection, the MMTV !"# sequence was found in 15 (33%). The sequence was amplified from none of the normal breast tissues and other cancers. Sequence analysis confirmed sequences amplified from breast cancer was highly homologous to the MMTV genome (Zammarchi et al, 2006). MMTV has also been shown to correlate with tumour grade in human breast cancers. In one study, MMTV-like !"# sequences were present in 26.3% of ductal carcinoma in situ and 53.8% of infiltrating ductal carcinoma (IDC) (Ford et al, 2003). In a similar study, these sequences were present in 23% of IDC grade I, 34% of IDC grade II and 38% of IDC grade III (Ford et al, 2004b). However, Zammarchi and colleagues failed to

V. Transmission of MMTV in mice and humans MMTV, a B-type retrovirus, exists both as endogenous proviruses, which are transmitted vertically following Mendelian patterns of inheritance, and exogenous infectious viruses which are transmitted horizontally. The Mouse Mammary Tumour Virus originally isolated by Bittner was an exogenous MMTV. Both endogenous and exogenous forms of the virus may be present in a mouse; several mouse strains are known to carry variable copies of endogenous MMTV regardless of whether they shed MMTV particles (Varmus et al, 1972). Although all inbred mice strains contain MMTV proviral integrants ($%#&), very few produce infectious MMTV particles. Examples of endogenous MMTVs include $%#1, $%#'(, $%#'7)* $%#'17)* $%#'43* and* $%#'50. $%#-1 and $%#-2, in some mice strains, are known to be expressed in mammary glands and leads to the productions and secretion of the exogenous virus in milk. Significant differences have been described in the biology of the two strains. $%#-1 (in mice strain C3H) has a long latency period and a low incidence of tumours (50%). In contrast, $%#-2 (in mice strain GR) induces tumours at an early age and at a high incidence (>90%). New virus strains are produced from the recombination between endogenous and exogenous virus with a broader host range (Golovkina et al, 1994). Sarkar et al have described detailed characterization of the exogenous forms of MMTV in the mice strain RIII/Sa (Sarkar et al, 2004). These mice express MMTV-1 and MMTV-2 in their milk and mammary tumours. None of these viruses resemble another known exogenous MMTV; MMTV-4, the exogenous virus of BR6 mice (BR6 MMTV). MMTV-3 was another virus isolated in R111/Sa. In contrast to MMTV-1 and MMTV-2, this was an endogenous form of the virus originating from $%#-17. The authors concluded, based on expression pattern of the viruses in mice tumours and mutation analyses, that MMTV-2 may be the major determinant for tumorigenesis in these mice. In mice, transmission of exogenous viruses following ingestion of infected milk by the pups from a viremic mother has been described (Ross, 1998). The virions pass through the gut wall to the spleen and infect lymphoid cells. It is presumed that the passage to the spleen is via the gastric vein as neonatal mice with short gastric veins have an increased infective susceptibility (Roubinian and Blair, 1980). Thereafter, MMTV infects B cells in lymphoid tissue of the gut such as the peyer’s patches. It appears that, for efficient infection, MMTV requires the expression of a protein called superantigen (Sag). These bind to major histocompatibility complex (MHC) class II molecules and stimulate T cells by interacting with the V beta domain of the T cell receptor (Acha-Orbea and MacDonald, 1995). An open reading frame (ORF), located at the 3' long terminal repeat (LTR) of the provirus, encodes for this protein (Held et al, 1993). Upon MMTV infection, Sag is presented to the appropriate T cell subset 539

Ahmed et al: Mouse mammary tumor virus: a cause of breast cancer in humans? do not exclude the possibility of a zoonotic origin of MMTV in humans. It remains unclear how humans may acquire MMTV. This may be through contact with mice faeces or dust. This theory has been supported by Stewart and colleagues who have cited ancient documents indicating the presence of mouse faecal pellets in stored grains (Stewart et al, 2000). However, this does not account for the increase in breast cancer is urban areas where human contact with mice is reduced compared to rural areas. An alternate suggestion has been made by Szabo and colleagues They suggest that a close homologue of MMTV may be present in cats and that cats may then transmit the virus to humans (Szabo et al, 2005). This is supported by the isolation of MMTV-like viral particles from feline cells following serial passages of MMTV from mice strains. These variants had been shown to be able to infect cells of a number of hosts (Howard et al, 1977). Prior to this, viruslike particles had been isolated from spontaneous cat mammary tumours, though these have not been characterized any further (Feldman and Gross, 1971). Theoretically, MMTV from mice may be acquired by cats through feeding and this may be transferred to their offspring via milk or licking. Humans, according to this hypothesis, may acquire the virus though contact with mice or cats (Szabo et al, 2005).

by B cells. The resulting immune reactions lead to preferential clonal expansion of infected B cells including memory cells. A stable MMTV infection is hence accomplished resulting in the spread to a variety of epithelial surfaces, of which the mammary epithelial is the most common (Smith, 1966; Callahan and Smith, 2000). There is a complex interplay of the T cell repertoire as a response to MMTV infection. Whilst Sags have been considered to lead to T-cell dependent expansion of infected B cells, Sags of some MMTVs result in deletion of V ! T cells. It has been demonstrated that the exogenous form of MMTV in RIII/Sa mice interacts with T cell subsets; V ! -2 T cells and V ! -8 T cells. Infection with these MMTVs lead to the deletion of V ! -2 T cells (>95%) in mice free of wild borne MMTV (BALB/c). Similarly, V ! -8 cells were also deleted, though to a much lesser degree (56%) (Uz-Zaman et al, 2003). Deletion of V ! -2 cells was much faster than deletion of V ! -8 cells. Moreover, MMTV infection induced clonal deletion of the V ! -2 and V ! -8 expressing immature thymocytes. It has been suggested that the two strains of RIII/Sa derived exogenous viruses, MMTV-1 and MMTV-2 have specificities for the V ! T cells; MMTV-1 specific for V ! -2 T cells and MMTV-2 specific for V ! -8 T cells. There have been suggestions that the virus may have a similar mechanism in humans. Labrecque and colleagues have shown that human T cells proliferate efficiently in response to minor lymphocyte stimulatory (Mls) antigens (Sags encoded by endogenous MMTV proviruses). An interesting observation they made was that this response was limited to T cells expressing a restricted set of T cell receptor V beta chains, and these are highly homologous to the mouse V beta chains that interact with Mls (Labrecque et al, 1993). Wang and colleagues have reported a 630-bp MMTV-like LTR sequence in 41.5% of human breast cancers (Wang et al, 2001). They have shown that these human isolates are highly homologous to the MMTV LTR SAg gene, and can stimulate a similar human T-cell response in vitro (Wang et al, 2004). It is not clear how the virus is acquired by humans. A similar transmission in humans through milk has been considered, which could explain the earlier observed increased risk of breast cancer in women whose mothers had breast cancer (Bucalossi and Veronesi, 1957; Tokuhata, 1969). However, these observations did not provide evidence of an association, and did not assess breast feeding status of the mothers. Moreover, the distribution of human breast cancers is low in areas where breast feeding is more common (MacMahon et al, 1970; Fraumeni and Miller, 1971). Titus-Ernstoff and colleagues specifically addressed this hypothesis by evaluating the risk of breast cancer in women who were breast fed by mothers who subsequently developed the disease. In this large population based study, they did not find any evidence of a positive association between having been breast fed and risk of breast cancer. They also showed that breast cancer was not increased by having been breast fed by a mother who subsequently developed the disease. These findings make a strong argument against the presence of such a transmissible agent in human breast milk (Titus-Ernstoff et al, 1998). However, these results

VI. Mechanism of action of MMTV MMTV is an insertional mutagen and inserts its DNA into the host DNA. One of the consequences of MMTV integration into the host genome is the genetic alteration of different genes in mammary tumours. Wint-1, Wnt-3, Wnt-10b, Fgf-3, Fgf-4, Fgf-8, int-3, int-6 were initially implicated [reviewed in (Callahan, 1996)]. More recently, 33 common insertion sites were identified in a high-throughput retroviral insertional mutagenesis screen in MMTV-induced human breast cancers (Szabo et al, 2005). It appears that the cells expressing MMTV sequences in humans may have an enriched transcriptional profile of genes involved in inflammation. This follows from the comparison made by Fernandez-Cobo and colleagues on the expression profiles of two sub-lines of the human breast cancer cell line MCF-7, one containing and one lacking the MMTV-like !"# sequence (Fernandez-Cobo et al, 2006). Of the genes upregulated in the MCF-7 containing MMTV-like sequences, there were 5 TNF and 2 TGF-! connected genes. They suggest that this enhanced expression of interferon-related genes in the !"# positive MCF-7 cells may suggest an increased potential for cell growth. Mutations in p53, a tumour suppressor gene, are known to be associated with human breast cancer. Estimates suggest the prevalence of p53 mutations in human breast cancer to be around 16-40% (Soong et al, 1997; Meng et al, 1999). Faedo and colleagues have demonstrated its increased prevalence in archival human breast cancers where MMTV-like sequences are present compared to tumours without these sequences (Faedo et al, 2004). The significance of this observation clearly warrants further clarity. 540

Cancer Therapy Vol 6, page 541! similarity was seen in lower grade human tumour. However, they could not demonstrate a significant correlation between the degree of similarity and the presence MMTV in the specimen (by PCR).

The cellular receptor that MMTV reacts with to initiate infection has recently been identified. Transferring receptor 1 (TfR1), a single membrane-spanning glycoprotein, binds the MMTV at the cell surface in mice. The virus is then trafficked to the endosomal compartment where fusion with the cell membrane occurs. Its human orthologue, however, was not able to mediate infection of human cells (Ross et al, 2002). Hence, identification of a receptor that mediates viral entry into the human cells remains to be identified. Even though such a receptor has not been identified yet, Indik and colleagues have confirmed the ability of MMTV to infect a range of human cells (Indik et al, 2005). Infection required the MMTV !"#elop protein to be intact. Heat inactivation of the virus and specific neutralising anti-MMTV serum could block the infection.

VII. MMTV

Pathology

of

tumours

VIII. Does MMTV cause human breast cancers? One of the strongest arguments in support of this notion has been made by Ford and colleagues who have based their arguments around some of the postulates (Ford et al, 2004c) suggested by Evans and Mueller that establish a causal link between an agent and the development of tumour (Evans and Mueller, 1990). According to Evans et al, the putative agent must be shown to be consistently associated with the disease and the agent must precede the onset of disease. In their arguments, Ford and colleagues describe several groups to have demonstrated the presence of MMTV-like sequences in a large proportion of human breast cancers, and in very few normal breast tissues. They also describe their group’s longitudinal study in which all non-malignant breast tissues excised up to 24 months after surgery for breast cancer were negative for MMTV-like sequences (Ford et al, 2004b). Even though many research groups have detected the MMTV-like sequences in human breast cancers using different approaches, several groups have not been able to do so. One of the recent was from a group in Sweden. Using real time PCR with a very high sensitivity, they did not detect the MMTV-like sequence in 18 human breast cancers they examined (Bindra et al, 2007). Yin and colleagues detected the sequence in only 1 of 60 human breast cancers by PCR (Yin et al, 1997). Witt and colleagues examined 50 Austrian human breast cancer samples and did not detect the MMTV !"# sequence in any of them. Even the breast cancer cell lines they examined did not reveal the sequence (Witt et al, 2003). Mant and colleagues detected the sequence in 16% of 44 human breast cancer samples, but all turned up to be falsepositives on subsequent DNA sequencing comprising host genomic DNA (Mant et al, 2004). Demonstrating the presence of MMTV prior to the onset of human breast cancer has not met much success. One of the earlier evidence in support of this has been the seroconversion of a woman working with MMTV-infected mice. Remaining seronegative over a 28 month period, she became seropositive at the 32nd month. She developed a breast mass 9 month later which was later confirmed to be an infiltrating ductal carcinoma (Poon et al, 1983). More recently, Ford and colleagues has reported a control subject who had !"#-positive benign breast tissue and went on to develop breast cancer (Ford et al, 2004a). Strong favour for this association has also been made by Holland and colleagues in 2004. In their review, they write, “ taken together, the presence of MMTV-like gene in a large proportion of breast cancers, which has been confirmed in many laboratories; the presence of the entire virus in breast cancers; reproduction of the virus from breast cancer cells $"%#$&'(; the demonstrated infectivity of !"#-positive breast cancer cells $"% #$&'( for normal breast cells; and variable penetrance in diverse populations present a plausible basis for considering causality (Wang

with

The mammary gland of mice and humans differs in structure and hormonal stimulation, although their basic biology and histology are quite similar. Most mice strains have 5 pairs of mammary glands in contrast to humans with one pair. The functional unit of the mammary gland, the terminal ductal lobular unit (TLDU) in humans and lobulo-alveolar units (LA) in mice, are hormone sensitive. Both have similar epithelial cells, produce milk and are the site of origin of most mammary cancers [reviewed in Cardiff and Wellings, 1999; Cardiff and Kenney, 2007]. Metastatic patterns of mouse mammary tumour are different from those observed in human breast tumours. Systemic dissemination in humans is seen following initial spread to the draining lymph nodes. In contrast, mouse mammary tumours rarely metastasize to regional lymph nodes; most metastases are seen in pulmonary vessels (Vaage and Harlos, 1987). Classification of spontaneous mouse mammary tumours has been based historically on the original description of Dr Thelma Dunn (Dunn, 1959). These fall into Dunn Types A (microacinar), B (ductal) or C. Type A tumours comprise small rounded regular sized and shaped cells with loose connective tissue. Type B tumours have a solid appearance with some glands and cysts. Types A and B are the predominant forms accounting for almost 90% of spontaneous mouse mammary tumours. In the past, mouse mammary tumours have been considered to be morphologically different from human breast cancers (Hamilton, 1974). Wellings observed that the histological characteristics of mouse mammary tumours were seen in some human breast cancer specimens (Wellings, 1980). Recent observations have demonstrated more similarities between human and mouse mammary tumours. Lawson et al have compared the morphology of human breast tumours and mouse mammary tumours (Lawson et al, 2006). They compared the morphology of specimens of human IDC and ductal carcinoma in situ (DCIS). They demonstrated that 39.9% of IDC (17 of 43 specimens) had ‘some’ histological characteristics similar to those associated with mouse mammary tumours. This similarity was inversely correlated with tumour grade; greater

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Ahmed et al: Mouse mammary tumor virus: a cause of breast cancer in humans? samples. J Gen Virol. 88, 1806-1809. Bittner JJ (1939) Relation of nursing to the extra-chromosomal theory of breast cancer in mice. Am J Cancer 35, 90-97. Bucalossi P, Veronesi U (1957) Some observations on cancer of the breast in mothers and daughters. Br J Cancer 11, 337347. Callahan R (1996) MMTV-induced mutations in mouse mammary tumors: their potential relevance to human breast cancer. Breast Cancer Res Treat 39, 33-44. Callahan R, Smith GH (2000) MMTV-induced mammary tumorigenesis: gene discovery, progression to malignancy and cellular pathways. Oncogene 19, 992-1001. Cardiff RD, Kenney N (2007) Mouse mammary tumor biology: a short history. Adv Cancer Res 98, 53-116. Cardiff RD, Wellings SR (1999) The comparative pathology of human and mouse mammary glands. J Mammary Gland Biol Neoplasia 4, 105-122. Dunn T (1959) Morphology of mammary tumors in mice. In: Homburger F (ed). Physiopathology of cancer. A.J. Phiebig: New York, 38-83. Etkind P, Du J, Khan A, Pillitteri J, Wiernik PH (2000) Mouse mammary tumor virus-like ENV gene sequences in human breast tumors and in a lymphoma of a breast cancer patient. Clin Cancer Res 6, 1273-1278. Evans AS, Mueller NE (1990) Viruses and cancer. Causal associations. Ann Epidemiol 1, 71-92. Faedo M, Ford CE, Mehta R, Blazek K, Rawlinson WD (2004) Mouse mammary tumor-like virus is associated with p53 nuclear accumulation and progesterone receptor positivity but not estrogen positivity in human female breast cancer. Clin Cancer Res 10, 4417-4419. Feldman DG, Gross L (1971) Electron microscopic study of spontaneous mammary carcinomas in cats and dogs: viruslike particles in cat mammary carcinomas. Cancer Res 31, 1261-1267. Fernandez-Cobo M, Melana SM, Holland JF, Pogo BG (2006) Transcription profile of a human breast cancer cell line expressing MMTV-like sequences. Infect Agent Cancer 1, 7. Ford C, Faedo M, Delprado W, Rawlinson W (2004a) Mouse mammary tumor virus-like gene sequences in breast tumors of Australian and Vietnamese women. Clin Cancer Res 10, 802. Ford CE, Faedo M, Crouch R, Lawson JS, Rawlinson WD (2004b) Progression from normal breast pathology to breast cancer is associated with increasing prevalence of mouse mammary tumor virus-like sequences in men and women. Cancer Res 64, 4755-4759. Ford CE, Faedo M, Rawlinson WD (2004c) Mouse mammary tumor virus-like RNA transcripts and DNA are found in affected cells of human breast cancer. Clin Cancer Res 10, 7284-7289. Ford CE, Tran D, Deng Y, Ta VT, Rawlinson WD, Lawson JS (2003) Mouse mammary tumor virus-like gene sequences in breast tumors of Australian and Vietnamese women. Clin Cancer Res 9, 1118-1120. Forsyth A (1985) Mammals of the Canadian Wild. Camden House Printing: London. Fraumeni JF, Jr., Miller RW (1971) Breast cancer from breastfeeding. Lancet 2, 1196-1197. Golovkina TV, Jaffe AB, Ross SR (1994) Coexpression of exogenous and endogenous mouse mammary tumor virus RNA in vivo results in viral recombination and broadens the virus host range. J Virol 68, 5019-5026. Gross L (1970) Oncogenic Viruses. Pergamon Press: Coford, New York. Hamilton JM (1974) Comparative aspects of mammary tumors. Adv Cancer Res 19, 1-45.

et al, 1995; Etkind et al, 2000; Liu et al, 2001; Ford et al, 2003; Holland et al, 2003; Levine et al, 2004). The discovery of an antibody response that indicated infection occurred before breast cancer became apparent should add to the mounting evidence that a major proportion of human breast cancer is horizontally acquired”. Several other factors need careful consideration in linking MMTV with human breast cancer. There seems to be an unambiguous belief in MMTV’s role in causing breast cancer in mice. Comparing the mice and human cancer models points out several similarities and differences. The virus acts as an insertional mutagen in mice, and this may be the case in humans as well [reviewed in (Stewart et al, 2000). Stewart and colleagues have shown that immunosuppression decreases breast cancer incidence in mice. He has also shown a reduction in human breast cancers in patients who are completely immunosuppressed (Stewart and Heppner, 1997). While this may support MMTV’s role in human breast cancer, this is in marked contrast to the observed effect of most of the established oncogenic viruses where immunosuppression predisposes to malignant progression. TfR1, the receptor that MMTV reacts with to initiate infection in mice, has not been shown to be the receptor that mediates infection in human cells (Indik et al, 2005). However, they have confirmed the ability of MMTV to infect human cells. More recently, they have also shown that human cells infected by the virus can rapidly spread between human cells in culture, express MMTV structural proteins and release spike B-type virions. The group was able to block the infectivity of these by anti-MMTV antibody and the replication of the virus by an inhibitor of reverse transcription. They argue that the demonstration of their replication in human cells lends more weight to the link between MMTV and human breast cancer (Indik et al, 2007). Clearly, there remains a lot of controversy on the role of MMTV in human breast cancers. There certainly are many observations that draws one to believe a strong association between the two, but a lot more needs to be proven before such causation may be established.

Acknowledgements The Royal College of Surgeons of Edinburgh, the Seahouse & District Cancer Research and Relief Fund, the Cancer Research Prevention Trust and Northumbria Healthcare NHS Trust for their financial support.

References Acha-Orbea H, MacDonald HR (1995) Superantigens of mouse mammary tumor virus. Annu Rev Immunol. 13, 459-486. Ahearne PM, Leach SD, BW F (1999) Invasive breast cancer. In: Feig BW, Berger DH, Fuhrman GM (eds). The MD Anderson surgical oncology handbook. Lippinscott Williams & Williams: Philadelphia, 13-37. Andreeva VA, Unger JB, Pentz MA (2007) Breast cancer among immigrants: a systematic review and new research directions. J Immigr Minor Health 9, 307-322. Bindra A, Muradrasoli S, Kisekka R, Nordgren H, Warnberg F, Blomberg J (2007) Search for DNA of exogenous mouse mammary tumor virus-related virus in human breast cancer

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Cancer Therapy Vol 6, page 543! Hareuveni M, Lathe R (1990) Breast cancer sequences identified by mouse mammary tumor virus (MMTV) antiserum are unrelated to MMTV. Int J Cancer 46, 1134-1135. Held W, Waanders GA, Shakhov AN, Scarpellino L, AchaOrbea H, MacDonald HR (1993) Superantigen-induced immune stimulation amplifies mouse mammary tumor virus infection and allows virus transmission. Cell 74, 529-540. Holland JF, Melana S, Wang Y, Fernandez-Cobo M, Jiang J-D, Pogo BG-T (2003) Human mammary tumor virus (HMTV) is horizontally, not vertically transmitted. Proc Am Soc Clin Oncol 22, 873.. Holland JF, Pogo BG (2004) Mouse mammary tumor virus-like viral infection and human breast cancer. Clin Cancer Res 10, 5647-5649. Howard DK, Colcher D, Teramoto YA, Young JM, Schlom J (1977) Characterization of mouse mammary tumor viruses propagated in heterologous cells. Cancer Res 37, 26962704. Indik S, Gunzburg WH, Kulich P, Salmons B, Rouault F (2007) Rapid spread of mouse mammary tumor virus in cultured human breast cells. Retrovirology 4, 73. Indik S, Gunzburg WH, Salmons B, Rouault F (2005) Mouse mammary tumor virus infects human cells. Cancer Res 65, 6651-6659. Kelsey JL (1993) Breast cancer epidemiology: summary and future directions. Epidemiol Rev. 15, 256-263. Labat ML (1998) Possible retroviral etiology of human breast cancer. Biomed Pharmacother 52, 6-12. Labrecque N, McGrath H, Subramanyam M, Huber BT, Sekaly RP (1993) Human T cells respond to mouse mammary tumor virus-encoded superantigen: V beta restriction and conserved evolutionary features. J Exp Med 177, 1735-1743. Lawson JS, Tran DD, Carpenter E, Ford CE, Rawlinson WD, Whitaker NJ, Delprado W (2006) Presence of mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast cancer. J Clin Pathol 59, 1287-1292. Levine PH, Pogo BG, Klouj A, Coronel S, Woodson K, Melana SM, Mourali N, Holland JF (2004) Increasing evidence for a human breast carcinoma virus with geographic differences. Cancer 101, 721-726. Liu B, Wang Y, Melana SM, Pelisson I, Najfeld V, Holland JF, Pogo BG (2001) Identification of a proviral structure in human breast cancer. Cancer Res 61, 1754-1759. MacMahon B, Lin TM, Lowe CR, Mirra AP, Ravnihar B, Salber EJ, Trichopoulos D, Valaoras VG, Yuasa S (1970) Lactation and cancer of the breast. A summary of an international study. Bull World Health Organ 42, 185-194. Mant C, Gillett C, D'Arrigo C, Cason J (2004) Human murine mammary tumour virus-like agents are genetically distinct from endogenous retroviruses and are not detectable in breast cancer cell lines or biopsies. Virology 318, 393-404. Melana SM, Holland JF, Pogo BG (2001) Search for mouse mammary tumor virus-like env sequences in cancer and normal breast from the same individuals. Clin Cancer Res 7, 283-284. Melana SM, Nepomnaschy I, Sakalian M, Abbott A, Hasa J, Holland JF, Pogo BG (2007) Characterization of viral particles isolated from primary cultures of human breast cancer cells. Cancer Res 67, 8960-8965. Meng L, Lin L, Zhang H, Nassiri M, Morales AR, Nadji M (1999) Multiple mutations of the p53 gene in human mammary carcinoma. Mutat Res 435, 263-269. Mesa-Tejada R, Keydar I, Ramanarayanan M, Ohno T, Fenoglio C, Spiegelman S (1978) Detection in human breast carcinomas of an antigen immunologically related to a group-specific antigen of mouse mammary tumor virus. Proc Natl Acad Sci U S A 75, 1529-1533.

Parkin D, Whelan S, Raymond L, Young J (1997) Cancer Incidence in Five Continents. IARC Scientific Publications: Lyon. Pogo BG, Melana SM, Holland JF, Mandeli JF, Pilotti S, Casalini P, Menard S (1999) Sequences homologous to the mouse mammary tumor virus env gene in human breast carcinoma correlate with overexpression of laminin receptor. Clin Cancer Res 5, 2108-2111. Poon MC, Tomana M, Niedermeier W (1983) Serum antibodies against mouse mammary tumor-virus-associated antigen detected nine months before appearance of a breast carcinoma. Ann Intern Med 98, 937-938. Quinn M, Babb P, Brock A, Kirby L, J J (2003) Cancer Trends in England and Wales 1950-1999. Office for National Statistics. Ross SR (1998) Mouse mammary tumor virus and its interaction with the immune system. Immunol Res 17, 209-216. Ross SR, Schofield JJ, Farr CJ, Bucan M (2002) Mouse transferrin receptor 1 is the cell entry receptor for mouse mammary tumor virus. Proc Natl Acad Sci U S A 99, 12386-12390. Roubinian JR, Blair PB (1980) Short gastric veins as the major portal of entry for milk-borne murine mammary tumor virus. J Natl Cancer Inst 65, 795-800. Sage R, Atchley W, Capanna E (1993) House mice as models in systematic biology. Syst Biol 42, 523-561. Sarkar NH, Golovkina T, Uz-Zaman T (2004) RIII/Sa mice with a high incidence of mammary tumors express two exogenous strains and one potential endogenous strain of mouse mammary tumor virus. J Virol 78, 1055-1062. Smith GH (1966) Role of the milk agent in disappearance of mammary cancer in C3H/StWi mice. J Natl Cancer Inst. 36, 685-701. Soong R, Iacopetta BJ, Harvey JM, Sterrett GF, Dawkins HJ, Hahnel R, Robbins PD (1997) Detection of p53 gene mutation by rapid PCR-SSCP and its association with poor survival in breast cancer. Int J Cancer 74, 642-647. Stewart TH, Heppner GH (1997) Immunological enhancement of breast cancer. Parasitology 115 Suppl, S141-153. Stewart TH, Sage RD, Stewart AF, Cameron DW (2000) Breast cancer incidence highest in the range of one species of house mouse, Mus domesticus. Br J Cancer 82, 446-451. Szabo CI, King MC (1997) Population genetics of BRCA1 and BRCA2. Am J Hum Genet 60, 1013-1020. Szabo S, Haislip AM, Garry RF (2005) Of mice, cats, men: is human breast cancer a zoonosis? Microsc Res Tech 68, 197208. Titus-Ernstoff L, Egan KM, Newcomb PA, Baron JA, Stampfer M, Greenberg ER, Cole BF, Ding J, Willett W, Trichopoulos D (1998) Exposure to breast milk in infancy and adult breast cancer risk. J Natl Cancer Inst 90, 921-924. Tokuhata GK (1969) Morbidity and mortality among offspring of breast cancer mothers. Am J Epidemiol 89, 139-153. Uz-Zaman T, Ignatowicz L, Sarkar NH (2003) Mouse mammary tumor viruses expressed by RIII/Sa mice with a high incidence of mammary tumors interact with the V beta-2and V beta-8-specific T cells during viral infection. Virology 314, 294-304. Vaage J, Harlos JP (1987) Spontaneous metastasis from primary C3H mouse mammary tumors. Cancer Res 47, 547-550. Varmus HE, Bishop JM, Nowinski RC, Sarker NH (1972) Mammary tumour virus specific nucleotide sequences in mouse DNA. Nat New Biol 238, 189-191. Wang Y, Go V, Holland JF, Melana SM, Pogo BG (1998) Expression of mouse mammary tumor virus-like env gene sequences in human breast cancer. Clin Cancer Res 4, 25652568. Wang Y, Holland JF, Bleiweiss IJ, Melana S, Liu X, Pelisson I, Cantarella A, Stellrecht K, Mani S, Pogo BG (1995)

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microdissection approach. J Pathol 209, 436-444. Zapata-Benavides P, Saavedra-Alonso S, Zamora-Avila D, Vargas-Rodarte C, Barrera-Rodriguez R, Salinas-Silva J, Rodriguez-Padilla C, Tamez-Guerra R, Trejo-Avila L (2007) Mouse Mammary Tumor Virus-Like Gene Sequences in Breast Cancer Samples of Mexican Women. Intervirology 50, 402-407.

Thomas WJ Lennard

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Cancer Therapy Vol 6, page 537! Cancer Therapy Vol 6, 537-544, 2008

Mouse mammary tumor virus: a cause of breast cancer in humans? Review Article

Ish Ahmed, James R. Harvey, Simi Ali, John A. Kirby, Thomas WJ Lennard* Applied Immunobiology and Breast Research Group, Institute of Cellular Medicine, Newcastle University, UK

__________________________________________________________________________________! *Correspondence: Professor Thomas Lennard, School Of Surgical & Reproductive Sciences3rd Floor, William Leech Building Faculty Of Medical Sciences Framlington Place Newcastle Upon Tyne Ne2 4hh, UK; Tel: 0191 222 7067; Fax: 0191 222 8514; E-mail: T.W.J.Lennard@ncl.ac.uk Key words: Mouse mammary tumour virus (MMTV), breast cancer Abbreviations: 3' long terminal repeat, (LTR); ductal carcinoma in situ, (DCIS); fluorescence nested PCR, (FN-PCR); human endogenous retrovirus, (HERV); infiltrating ductal carcinoma, (IDC); lobulo-alveolar, (LA); mouse mammary tumour virus, (MMTV); open reading frame, (ORF); superantigen, (Sag); terminal ductal lobular unit, (TLDU) Received: 13 February 2008; Revised: 20 June 2008 Accepted: 30 June 2008; electronically published: September 2008

Summary The mouse mammary tumour virus (MMTV), a B-type retrovirus, is known to be the most common cause of breast cancer in mice. However, its role in human breast cancer is very controversial. Here we review the evidence that supports the role of this virus in causing human breast cancer. It’s historical background, its possible transmission from mice to human and its mechanism of actions have also been reviewed.

newborn young of an inbred high-cancer mouse line (tumour incidence >80%) were fostered by females of a low tumour line. The fostered mice and their progeny lived considerably longer and had a tumour incidence of only 7.4%. With this and some other similar experiments, he concluded that an agent in milk influences the development of breast tumours. Though initially isolated in the milk, this agent was also obtained from both spontaneous and transplanted mammary carcinomas, lactating mammary tissue and whole blood. Bittner also showed that this agent could be carried through several passages in association with tumour transplants in mice that did not carry the milk influence themselves. The agent was smaller than a bacterium as it could pass through bacterial and seitz filters. By 1945, Andorvent went on to describe this milk factor as a virus (Gross, 1970), and gradually, this became widely known as the ‘Bittner Virus’. Today, the term MMTV includes Bittner’s virus and other closely related viruses (Labat, 1998). It is now well established that MMTV causes breast tumours in laboratory mice (Callahan, 1996), but its role in causing breast cancer in humans is a subject of significant controversy.

I. Introduction Breast cancer is the most common cancer in women worldwide, accounting for about 25% of all malignancies in women; the proportion is higher in western, developed, countries. In England, there were around 36,500 new cases diagnosed in 2003, representing 32 per cent of all cancers in women and a rate of 120 cases per 100,000 women. (Quinn et al, 2003). Several risk factors have been implicated in breast cancer including age, diet, nulliparity, obesity and family history (Kelsey, 1993). With genetic predisposition (mutations in BRCA1 and BRCA2) only accounting for approximately 5% of breast cancers (Szabo and King, 1997), extensive efforts have been made to account for the remainder. The mouse mammary tumour virus (MMTV) has been implicated as one of the unrecognized factors that might account for remainder of sporadic cases. The geographical variation in incidence of breast cancer provides additional grounds for this theory.

II. The origin of mouse mammary tumour virus The interest in MMTV as a cancer causing agent was generated several decades ago when Bittner demonstrated in 1939 an extra chromosomal factor in mice that contributes to breast cancer incidence. In his experiments,

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Ahmed et al: Mouse mammary tumor virus: a cause of breast cancer in humans? Identification of the MMTV envelope proteins in human breast cancer by using antibody against them was one of the earlier methods used. In one of these series, positive reactions were seen in 51 of 131 (39%) breast carcinomas with negative reactions in all normal breast tissue (Mesa-Tejada et al, 1978). However, the use of this antibody against the MMTV .-2elope protein has been disputed and places an uncertainty on the validity of these results (Hareuveni and Lathe, 1990). One of the major problems encountered at that time was the identification of human endogenous retrovirus (HERV) in the human genome. The significant homology between HERV and MMTV made it extremely difficult to differentiate between the two. As a consequence, earlier results were disputed with claims that the identification of MMTV may actually be HERV. This problem was overcome when, among others, Wang and colleagues isolated a 660-bp sequence in human breast cancer (Wang et al, 1995). This sequence was highly homologous to the .-2 gene of MMTV and had a very low homology to HERVs. Isolating DNA from fresh and frozen breast samples and using the polymerase chain reaction, they detected this sequence in 38% of the 314 breast cancer samples and in none of the normal breast tissues. A similar 250-bp sequence homologous to MMTV were detected in 39.7% of the 151 human breast cancers and in 1 of 27 normal breast samples assayed from paraffin-embedded sections. Wang and colleagues suggested in 1998 that their findings may represent exogenous sequences from an agent similar to MMTV. They next investigated whether these .-2-positive tumours produced mRNA by assessing the expression of the 660-bp sequence in the human breast samples using reverse transcription PCR. They demonstrated the expression of this sequence in 66% of the .-2-positive tumours and in none of the .-2-negative tumours. Subsequent sequence analysis confirmed that the expressed sequence was 98% homologous to the .-2 gene of the MMTV with only one small stretch of 46 bp that was homologous to the endogenous viral sequences. The fact that not all .-2-positive tumours produced mRNA was slightly intriguing, and possible explanations suggested were degradation of the mRNA or that not all the cells within the tumour express the viral sequences (Wang et al, 1998). The use of MMTV .-2 sequences by Wang and colleagues in 1998 and his interesting results rekindled further interests on the role of MMTV in human breast cancer. Several groups undertook similar strategies in attempts to define such a role. Etkind and colleagues used in 2000 a similar approach and analysed the presence of MMTV-like .-2 sequences in breast tumours by analysing them for the presence of a 250bp DNA fragment after nested PCR reactions. The sequence was present in 37% of the 73 samples of human breast cancer. None of the normal breast tissues had these sequences. DNA sequencing confirmed these sequences to be 99-100% homologous to the MMTV .-2 gene sequences. Interestingly, they also identified these sequences in 3 of 19 non-Hodgkin’s lymphoma (Etkind et al, 2000). Identifying the source of this sequence in human breast cancers is of paramount importance. To this end,

III. MMTV may explain the geographical variation in human breast cancer The incidence of human breast cancer varies significantly worldwide. The incidence is very low in Asia and Africa, very high in north America and northern Europe with an intermediate incidence belt in southern Europe and Latin America (Ahearne et al, 1999). People from lower incidence areas are known to develop an increase in its incidence when they move to an area of higher incidence (Winter et al, 1999). Several theories have been hypothesized to explain this increase in breast cancer incidence with migration. Andreeva and colleagues have reviewed 79 studies identifying risks and protective factors for the acquired risk. In their review, they have described that several studies (15 of the 79) have highlighted the importance of environmental and behavioural factors after migration. These include modification in diet, delay in childbirth, avoidance of breastfeeding and increasing socioeconomic status (Andreeva et al, 2007). Several groups have tried to explain the geographical variation in breast cancer incidence. Stewart and colleagues have described an explanation for this difference in incidence based on the distribution of MMTV in house mice of the genus !"#$%$!&$'"#(")"#*$!&$ ("#+,-."# and !&$ /0'.#+1("# (Stewart et al, 2000). !&$ '"#(")"# is present mainly in eastern Europe and !&$ /0'.#+1("# in western Europe (Sage et al, 1993). MMTV occurs both as exogenous infectious particles and as endogenous proviruses. !&$ /0'.#+1("# mice carried more exogenous virus and had more endogenous proviral loci than !&$'"#(")"#. Based on the incidence of human breast cancers described by Parkins and colleagues in 1997, he showed that the incidence of human breast cancer is higher in western Europe, inhabited by !&$ /0'.#+1("#, than eastern European lands of !&$ '"#(")"#. Interestingly, MMTV has been isolated from human breast cancer samples in other regions inhabited by this mouse specie. These include North America, Australia and Tunisia (Forsyth, 1985; Wang et al, 1995; Pogo et al, 1999; Etkind et al, 2000; Ford et al, 2004b; Levine et al, 2004). Regions not inhabited by this mice, including Vietnam, Sweden and Austria, are areas from where MMTV has not been isolated from human breast cancersamples (Ford et al, 2003; Witt et al, 2003; Bindra et al, 2007). It thus appears that human breast cancer may have a zoonotic connection with humans acquiring the virus from mice.

IV. Isolation of MMTV in human samples MMTV is known to cause breast tumours in laboratory mice. Numerous attempts have been made to identify a human analogue of MMTV that may cause cancers in human breasts. Most of the available data and its conclusion may point towards the presence of such an analogue in human breast cancer. Whether this plays any role in human breast cancer progression remains to be identified.

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Cancer Therapy Vol 6, page 539! show any significant difference between tumour grade and the presence of the MMTV !"# sequence (Zammarchi et al, 2006).

Melana and colleagues have tried to establish an exogenous source by examining the 250-bp sequence in archived cancer and normal tissues of the same patient. Of paired breast samples (normal and cancer) from 106 patients, they found 30% of 106 human breast cancers and only 1 of 106 normal breast tissues to be !"# positive (Melana et al, 2001). These results strongly indicated that the normal breasts of patients with !"# positive cancers do not contain the sequence, and led the authors to conclude that these sequences are of exogenous origin. However, they could not explain the finding of this sequence in the normal breast of one individual. The corresponding breast cancer from this patient was !"# negative. After several tests, they concluded that this may have been due to potential contamination with cancer tissue or DNA. The presence of the MMTV sequences in human breast cancers appears to have a geographical variation with areas of high and low positivity. In one study, MMTV-like gene sequences were amplified in only 1 of 120 and 0 of 40 breast cancer biopsy tissues from Vietnamese and Australian Vietnamese women, respectively. The same study showed the sequence in 19 of 45 breast cancers from Caucasian-Australian women (Ford et al, 2003). Earlier, this sequence was detected in 37.7% of 70 paraffin sections of human breast cancers in Italian women (Pogo et al, 1999). Recently, this sequence was isolated in 5 of 119 (4.2%) breast cancer biopsies in Mexican women (Zapata-Benavides et al, 2007). Most of the claims on the presence of MMTV in human breast cancers were initially based upon the amplification of MMTV-like sequences. However, in 2001, Liu and colleagues Isolated the complete virus from two human breast cancer samples that were !"# positive. This was done by amplifying three overlapping fragments and using the sequences to construct the complete virus by nested PCR. This 9.9-kb provirus was 95% homologous to MMTV and had a low homology to HERV. These proviral sequences were virtually absent in normal breast tissues (2%) and again suggest an exogenous origin (Liu et al, 2001). Primary culture of cells isolated from ascites or pleural effusion of patients with metastatic breast cancer have shown to contain viral sequences in their DNA, expressed env protein and retroviral particles on electron microscopy (Melana et al, 2007). A fluorescence nested PCR (FN-PCR) has recently been used to detect copies of the MMTV viral genome. Of the 45 frozen human breast cancers obtained by laser micro-dissection, the MMTV !"# sequence was found in 15 (33%). The sequence was amplified from none of the normal breast tissues and other cancers. Sequence analysis confirmed sequences amplified from breast cancer was highly homologous to the MMTV genome (Zammarchi et al, 2006). MMTV has also been shown to correlate with tumour grade in human breast cancers. In one study, MMTV-like !"# sequences were present in 26.3% of ductal carcinoma in situ and 53.8% of infiltrating ductal carcinoma (IDC) (Ford et al, 2003). In a similar study, these sequences were present in 23% of IDC grade I, 34% of IDC grade II and 38% of IDC grade III (Ford et al, 2004b). However, Zammarchi and colleagues failed to

V. Transmission of MMTV in mice and humans MMTV, a B-type retrovirus, exists both as endogenous proviruses, which are transmitted vertically following Mendelian patterns of inheritance, and exogenous infectious viruses which are transmitted horizontally. The Mouse Mammary Tumour Virus originally isolated by Bittner was an exogenous MMTV. Both endogenous and exogenous forms of the virus may be present in a mouse; several mouse strains are known to carry variable copies of endogenous MMTV regardless of whether they shed MMTV particles (Varmus et al, 1972). Although all inbred mice strains contain MMTV proviral integrants ($%#&), very few produce infectious MMTV particles. Examples of endogenous MMTVs include $%#1, $%#'(, $%#'7)* $%#'17)* $%#'43* and* $%#'50. $%#-1 and $%#-2, in some mice strains, are known to be expressed in mammary glands and leads to the productions and secretion of the exogenous virus in milk. Significant differences have been described in the biology of the two strains. $%#-1 (in mice strain C3H) has a long latency period and a low incidence of tumours (50%). In contrast, $%#-2 (in mice strain GR) induces tumours at an early age and at a high incidence (>90%). New virus strains are produced from the recombination between endogenous and exogenous virus with a broader host range (Golovkina et al, 1994). Sarkar et al have described detailed characterization of the exogenous forms of MMTV in the mice strain RIII/Sa (Sarkar et al, 2004). These mice express MMTV-1 and MMTV-2 in their milk and mammary tumours. None of these viruses resemble another known exogenous MMTV; MMTV-4, the exogenous virus of BR6 mice (BR6 MMTV). MMTV-3 was another virus isolated in R111/Sa. In contrast to MMTV-1 and MMTV-2, this was an endogenous form of the virus originating from $%#-17. The authors concluded, based on expression pattern of the viruses in mice tumours and mutation analyses, that MMTV-2 may be the major determinant for tumorigenesis in these mice. In mice, transmission of exogenous viruses following ingestion of infected milk by the pups from a viremic mother has been described (Ross, 1998). The virions pass through the gut wall to the spleen and infect lymphoid cells. It is presumed that the passage to the spleen is via the gastric vein as neonatal mice with short gastric veins have an increased infective susceptibility (Roubinian and Blair, 1980). Thereafter, MMTV infects B cells in lymphoid tissue of the gut such as the peyer’s patches. It appears that, for efficient infection, MMTV requires the expression of a protein called superantigen (Sag). These bind to major histocompatibility complex (MHC) class II molecules and stimulate T cells by interacting with the V beta domain of the T cell receptor (Acha-Orbea and MacDonald, 1995). An open reading frame (ORF), located at the 3' long terminal repeat (LTR) of the provirus, encodes for this protein (Held et al, 1993). Upon MMTV infection, Sag is presented to the appropriate T cell subset 539

Ahmed et al: Mouse mammary tumor virus: a cause of breast cancer in humans? do not exclude the possibility of a zoonotic origin of MMTV in humans. It remains unclear how humans may acquire MMTV. This may be through contact with mice faeces or dust. This theory has been supported by Stewart and colleagues who have cited ancient documents indicating the presence of mouse faecal pellets in stored grains (Stewart et al, 2000). However, this does not account for the increase in breast cancer is urban areas where human contact with mice is reduced compared to rural areas. An alternate suggestion has been made by Szabo and colleagues They suggest that a close homologue of MMTV may be present in cats and that cats may then transmit the virus to humans (Szabo et al, 2005). This is supported by the isolation of MMTV-like viral particles from feline cells following serial passages of MMTV from mice strains. These variants had been shown to be able to infect cells of a number of hosts (Howard et al, 1977). Prior to this, viruslike particles had been isolated from spontaneous cat mammary tumours, though these have not been characterized any further (Feldman and Gross, 1971). Theoretically, MMTV from mice may be acquired by cats through feeding and this may be transferred to their offspring via milk or licking. Humans, according to this hypothesis, may acquire the virus though contact with mice or cats (Szabo et al, 2005).

by B cells. The resulting immune reactions lead to preferential clonal expansion of infected B cells including memory cells. A stable MMTV infection is hence accomplished resulting in the spread to a variety of epithelial surfaces, of which the mammary epithelial is the most common (Smith, 1966; Callahan and Smith, 2000). There is a complex interplay of the T cell repertoire as a response to MMTV infection. Whilst Sags have been considered to lead to T-cell dependent expansion of infected B cells, Sags of some MMTVs result in deletion of V ! T cells. It has been demonstrated that the exogenous form of MMTV in RIII/Sa mice interacts with T cell subsets; V ! -2 T cells and V ! -8 T cells. Infection with these MMTVs lead to the deletion of V ! -2 T cells (>95%) in mice free of wild borne MMTV (BALB/c). Similarly, V ! -8 cells were also deleted, though to a much lesser degree (56%) (Uz-Zaman et al, 2003). Deletion of V ! -2 cells was much faster than deletion of V ! -8 cells. Moreover, MMTV infection induced clonal deletion of the V ! -2 and V ! -8 expressing immature thymocytes. It has been suggested that the two strains of RIII/Sa derived exogenous viruses, MMTV-1 and MMTV-2 have specificities for the V ! T cells; MMTV-1 specific for V ! -2 T cells and MMTV-2 specific for V ! -8 T cells. There have been suggestions that the virus may have a similar mechanism in humans. Labrecque and colleagues have shown that human T cells proliferate efficiently in response to minor lymphocyte stimulatory (Mls) antigens (Sags encoded by endogenous MMTV proviruses). An interesting observation they made was that this response was limited to T cells expressing a restricted set of T cell receptor V beta chains, and these are highly homologous to the mouse V beta chains that interact with Mls (Labrecque et al, 1993). Wang and colleagues have reported a 630-bp MMTV-like LTR sequence in 41.5% of human breast cancers (Wang et al, 2001). They have shown that these human isolates are highly homologous to the MMTV LTR SAg gene, and can stimulate a similar human T-cell response in vitro (Wang et al, 2004). It is not clear how the virus is acquired by humans. A similar transmission in humans through milk has been considered, which could explain the earlier observed increased risk of breast cancer in women whose mothers had breast cancer (Bucalossi and Veronesi, 1957; Tokuhata, 1969). However, these observations did not provide evidence of an association, and did not assess breast feeding status of the mothers. Moreover, the distribution of human breast cancers is low in areas where breast feeding is more common (MacMahon et al, 1970; Fraumeni and Miller, 1971). Titus-Ernstoff and colleagues specifically addressed this hypothesis by evaluating the risk of breast cancer in women who were breast fed by mothers who subsequently developed the disease. In this large population based study, they did not find any evidence of a positive association between having been breast fed and risk of breast cancer. They also showed that breast cancer was not increased by having been breast fed by a mother who subsequently developed the disease. These findings make a strong argument against the presence of such a transmissible agent in human breast milk (Titus-Ernstoff et al, 1998). However, these results

VI. Mechanism of action of MMTV MMTV is an insertional mutagen and inserts its DNA into the host DNA. One of the consequences of MMTV integration into the host genome is the genetic alteration of different genes in mammary tumours. Wint-1, Wnt-3, Wnt-10b, Fgf-3, Fgf-4, Fgf-8, int-3, int-6 were initially implicated [reviewed in (Callahan, 1996)]. More recently, 33 common insertion sites were identified in a high-throughput retroviral insertional mutagenesis screen in MMTV-induced human breast cancers (Szabo et al, 2005). It appears that the cells expressing MMTV sequences in humans may have an enriched transcriptional profile of genes involved in inflammation. This follows from the comparison made by Fernandez-Cobo and colleagues on the expression profiles of two sub-lines of the human breast cancer cell line MCF-7, one containing and one lacking the MMTV-like !"# sequence (Fernandez-Cobo et al, 2006). Of the genes upregulated in the MCF-7 containing MMTV-like sequences, there were 5 TNF and 2 TGF-! connected genes. They suggest that this enhanced expression of interferon-related genes in the !"# positive MCF-7 cells may suggest an increased potential for cell growth. Mutations in p53, a tumour suppressor gene, are known to be associated with human breast cancer. Estimates suggest the prevalence of p53 mutations in human breast cancer to be around 16-40% (Soong et al, 1997; Meng et al, 1999). Faedo and colleagues have demonstrated its increased prevalence in archival human breast cancers where MMTV-like sequences are present compared to tumours without these sequences (Faedo et al, 2004). The significance of this observation clearly warrants further clarity. 540

Cancer Therapy Vol 6, page 541! similarity was seen in lower grade human tumour. However, they could not demonstrate a significant correlation between the degree of similarity and the presence MMTV in the specimen (by PCR).

The cellular receptor that MMTV reacts with to initiate infection has recently been identified. Transferring receptor 1 (TfR1), a single membrane-spanning glycoprotein, binds the MMTV at the cell surface in mice. The virus is then trafficked to the endosomal compartment where fusion with the cell membrane occurs. Its human orthologue, however, was not able to mediate infection of human cells (Ross et al, 2002). Hence, identification of a receptor that mediates viral entry into the human cells remains to be identified. Even though such a receptor has not been identified yet, Indik and colleagues have confirmed the ability of MMTV to infect a range of human cells (Indik et al, 2005). Infection required the MMTV !"#elop protein to be intact. Heat inactivation of the virus and specific neutralising anti-MMTV serum could block the infection.

VII. MMTV

Pathology

of

tumours

VIII. Does MMTV cause human breast cancers? One of the strongest arguments in support of this notion has been made by Ford and colleagues who have based their arguments around some of the postulates (Ford et al, 2004c) suggested by Evans and Mueller that establish a causal link between an agent and the development of tumour (Evans and Mueller, 1990). According to Evans et al, the putative agent must be shown to be consistently associated with the disease and the agent must precede the onset of disease. In their arguments, Ford and colleagues describe several groups to have demonstrated the presence of MMTV-like sequences in a large proportion of human breast cancers, and in very few normal breast tissues. They also describe their group’s longitudinal study in which all non-malignant breast tissues excised up to 24 months after surgery for breast cancer were negative for MMTV-like sequences (Ford et al, 2004b). Even though many research groups have detected the MMTV-like sequences in human breast cancers using different approaches, several groups have not been able to do so. One of the recent was from a group in Sweden. Using real time PCR with a very high sensitivity, they did not detect the MMTV-like sequence in 18 human breast cancers they examined (Bindra et al, 2007). Yin and colleagues detected the sequence in only 1 of 60 human breast cancers by PCR (Yin et al, 1997). Witt and colleagues examined 50 Austrian human breast cancer samples and did not detect the MMTV !"# sequence in any of them. Even the breast cancer cell lines they examined did not reveal the sequence (Witt et al, 2003). Mant and colleagues detected the sequence in 16% of 44 human breast cancer samples, but all turned up to be falsepositives on subsequent DNA sequencing comprising host genomic DNA (Mant et al, 2004). Demonstrating the presence of MMTV prior to the onset of human breast cancer has not met much success. One of the earlier evidence in support of this has been the seroconversion of a woman working with MMTV-infected mice. Remaining seronegative over a 28 month period, she became seropositive at the 32nd month. She developed a breast mass 9 month later which was later confirmed to be an infiltrating ductal carcinoma (Poon et al, 1983). More recently, Ford and colleagues has reported a control subject who had !"#-positive benign breast tissue and went on to develop breast cancer (Ford et al, 2004a). Strong favour for this association has also been made by Holland and colleagues in 2004. In their review, they write, “ taken together, the presence of MMTV-like gene in a large proportion of breast cancers, which has been confirmed in many laboratories; the presence of the entire virus in breast cancers; reproduction of the virus from breast cancer cells $"%#$&'(; the demonstrated infectivity of !"#-positive breast cancer cells $"% #$&'( for normal breast cells; and variable penetrance in diverse populations present a plausible basis for considering causality (Wang

with

The mammary gland of mice and humans differs in structure and hormonal stimulation, although their basic biology and histology are quite similar. Most mice strains have 5 pairs of mammary glands in contrast to humans with one pair. The functional unit of the mammary gland, the terminal ductal lobular unit (TLDU) in humans and lobulo-alveolar units (LA) in mice, are hormone sensitive. Both have similar epithelial cells, produce milk and are the site of origin of most mammary cancers [reviewed in Cardiff and Wellings, 1999; Cardiff and Kenney, 2007]. Metastatic patterns of mouse mammary tumour are different from those observed in human breast tumours. Systemic dissemination in humans is seen following initial spread to the draining lymph nodes. In contrast, mouse mammary tumours rarely metastasize to regional lymph nodes; most metastases are seen in pulmonary vessels (Vaage and Harlos, 1987). Classification of spontaneous mouse mammary tumours has been based historically on the original description of Dr Thelma Dunn (Dunn, 1959). These fall into Dunn Types A (microacinar), B (ductal) or C. Type A tumours comprise small rounded regular sized and shaped cells with loose connective tissue. Type B tumours have a solid appearance with some glands and cysts. Types A and B are the predominant forms accounting for almost 90% of spontaneous mouse mammary tumours. In the past, mouse mammary tumours have been considered to be morphologically different from human breast cancers (Hamilton, 1974). Wellings observed that the histological characteristics of mouse mammary tumours were seen in some human breast cancer specimens (Wellings, 1980). Recent observations have demonstrated more similarities between human and mouse mammary tumours. Lawson et al have compared the morphology of human breast tumours and mouse mammary tumours (Lawson et al, 2006). They compared the morphology of specimens of human IDC and ductal carcinoma in situ (DCIS). They demonstrated that 39.9% of IDC (17 of 43 specimens) had ‘some’ histological characteristics similar to those associated with mouse mammary tumours. This similarity was inversely correlated with tumour grade; greater

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Ahmed et al: Mouse mammary tumor virus: a cause of breast cancer in humans? samples. J Gen Virol. 88, 1806-1809. Bittner JJ (1939) Relation of nursing to the extra-chromosomal theory of breast cancer in mice. Am J Cancer 35, 90-97. Bucalossi P, Veronesi U (1957) Some observations on cancer of the breast in mothers and daughters. Br J Cancer 11, 337347. Callahan R (1996) MMTV-induced mutations in mouse mammary tumors: their potential relevance to human breast cancer. Breast Cancer Res Treat 39, 33-44. Callahan R, Smith GH (2000) MMTV-induced mammary tumorigenesis: gene discovery, progression to malignancy and cellular pathways. Oncogene 19, 992-1001. Cardiff RD, Kenney N (2007) Mouse mammary tumor biology: a short history. Adv Cancer Res 98, 53-116. Cardiff RD, Wellings SR (1999) The comparative pathology of human and mouse mammary glands. J Mammary Gland Biol Neoplasia 4, 105-122. Dunn T (1959) Morphology of mammary tumors in mice. In: Homburger F (ed). Physiopathology of cancer. A.J. Phiebig: New York, 38-83. Etkind P, Du J, Khan A, Pillitteri J, Wiernik PH (2000) Mouse mammary tumor virus-like ENV gene sequences in human breast tumors and in a lymphoma of a breast cancer patient. Clin Cancer Res 6, 1273-1278. Evans AS, Mueller NE (1990) Viruses and cancer. Causal associations. Ann Epidemiol 1, 71-92. Faedo M, Ford CE, Mehta R, Blazek K, Rawlinson WD (2004) Mouse mammary tumor-like virus is associated with p53 nuclear accumulation and progesterone receptor positivity but not estrogen positivity in human female breast cancer. Clin Cancer Res 10, 4417-4419. Feldman DG, Gross L (1971) Electron microscopic study of spontaneous mammary carcinomas in cats and dogs: viruslike particles in cat mammary carcinomas. Cancer Res 31, 1261-1267. Fernandez-Cobo M, Melana SM, Holland JF, Pogo BG (2006) Transcription profile of a human breast cancer cell line expressing MMTV-like sequences. Infect Agent Cancer 1, 7. Ford C, Faedo M, Delprado W, Rawlinson W (2004a) Mouse mammary tumor virus-like gene sequences in breast tumors of Australian and Vietnamese women. Clin Cancer Res 10, 802. Ford CE, Faedo M, Crouch R, Lawson JS, Rawlinson WD (2004b) Progression from normal breast pathology to breast cancer is associated with increasing prevalence of mouse mammary tumor virus-like sequences in men and women. Cancer Res 64, 4755-4759. Ford CE, Faedo M, Rawlinson WD (2004c) Mouse mammary tumor virus-like RNA transcripts and DNA are found in affected cells of human breast cancer. Clin Cancer Res 10, 7284-7289. Ford CE, Tran D, Deng Y, Ta VT, Rawlinson WD, Lawson JS (2003) Mouse mammary tumor virus-like gene sequences in breast tumors of Australian and Vietnamese women. Clin Cancer Res 9, 1118-1120. Forsyth A (1985) Mammals of the Canadian Wild. Camden House Printing: London. Fraumeni JF, Jr., Miller RW (1971) Breast cancer from breastfeeding. Lancet 2, 1196-1197. Golovkina TV, Jaffe AB, Ross SR (1994) Coexpression of exogenous and endogenous mouse mammary tumor virus RNA in vivo results in viral recombination and broadens the virus host range. J Virol 68, 5019-5026. Gross L (1970) Oncogenic Viruses. Pergamon Press: Coford, New York. Hamilton JM (1974) Comparative aspects of mammary tumors. Adv Cancer Res 19, 1-45.

et al, 1995; Etkind et al, 2000; Liu et al, 2001; Ford et al, 2003; Holland et al, 2003; Levine et al, 2004). The discovery of an antibody response that indicated infection occurred before breast cancer became apparent should add to the mounting evidence that a major proportion of human breast cancer is horizontally acquired”. Several other factors need careful consideration in linking MMTV with human breast cancer. There seems to be an unambiguous belief in MMTV’s role in causing breast cancer in mice. Comparing the mice and human cancer models points out several similarities and differences. The virus acts as an insertional mutagen in mice, and this may be the case in humans as well [reviewed in (Stewart et al, 2000). Stewart and colleagues have shown that immunosuppression decreases breast cancer incidence in mice. He has also shown a reduction in human breast cancers in patients who are completely immunosuppressed (Stewart and Heppner, 1997). While this may support MMTV’s role in human breast cancer, this is in marked contrast to the observed effect of most of the established oncogenic viruses where immunosuppression predisposes to malignant progression. TfR1, the receptor that MMTV reacts with to initiate infection in mice, has not been shown to be the receptor that mediates infection in human cells (Indik et al, 2005). However, they have confirmed the ability of MMTV to infect human cells. More recently, they have also shown that human cells infected by the virus can rapidly spread between human cells in culture, express MMTV structural proteins and release spike B-type virions. The group was able to block the infectivity of these by anti-MMTV antibody and the replication of the virus by an inhibitor of reverse transcription. They argue that the demonstration of their replication in human cells lends more weight to the link between MMTV and human breast cancer (Indik et al, 2007). Clearly, there remains a lot of controversy on the role of MMTV in human breast cancers. There certainly are many observations that draws one to believe a strong association between the two, but a lot more needs to be proven before such causation may be established.

Acknowledgements The Royal College of Surgeons of Edinburgh, the Seahouse & District Cancer Research and Relief Fund, the Cancer Research Prevention Trust and Northumbria Healthcare NHS Trust for their financial support.

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Thomas WJ Lennard

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Cancer Therapy Vol 6, page 545! Cancer Therapy Vol 6, 545-552, 2008

Techniques for intratumoral chemotherapy of lung cancer by bronchoscopic drug delivery Research Article

Firuz Celikoglu1, Seyhan I Celikoglu1, Eugene P Goldberg2 1 2

University of Istanbul, Cerrahpasa Medical Faculty and Institute of Lung Diseases and Tuberculosis, Istanbul, Turkey University of Florida, Biomaterials Center, Department Materials Science & Engineering, Gainesville, FL, USA

__________________________________________________________________________________! *Correspondence: Seyhan I Celikoglu Ph.D., Urguplu Caddesi, No. 16, Yesilyurt, 34800 Istanbul, Turkey; Tel.:+90 (542) 433 02 88; Fax: +90 (212) 663 96 38; E-mail addresses: seyhan@celikoglu.biz Eugene P Goldberg, Ph.D., University of Florida, Biomaterials Center, Department Materials Science & Engineering, P.O. Box 116400, Gainesville, FL, USA; Tel.: +352 392 4907; fax: +352 383 7416; E-mail: egold@mse.ufl.edu Key words: Direct intratumoral injection; Endobronchial chemotherapy; Interventional therapeutic bronchoscopy; Lung cancer therapy Abbreviations: 5-fluorouracil, (5-FU); computerized axial tomography, (CAT); endo-bronchial intratumoral chemotherapy, (EITC); endoscopic ultrasound bronchoscope, (EBUS); intratumoral, (IT); Sentinel lymph nodes, (SLN) Received: 11 March 2008; Revised: 8 June 2008 Accepted: 20 June 2008; electronically published: September 2008

Summary The objective of this review is to provide a detailed description of the technique for a new non-systemic therapeutic modality for NSC lung cancer chemotherapy. Described here is the technique for intratumoral injection of one or more cytotoxic drugs directly into tumor tissue through a flexible bronchoscope by means of a needle-catheter. The procedure is termed “endo-bronchial intratumoral chemotherapy (EITC)”. EITC is a loco-regional form of chemotherapy as well as an ablative cytotoxic procedure for removal of endobronchial tumor bulk for NSC lung cancer that presents with bronchial obstruction. In addition to the advantage of tumor burden reduction inside the airway lumen, intratumoral delivery of cytotoxic drugs may be regarded as an improved neoadjuvant therapy for use prior to irradiation and/or surgery. EITC differs significantly from conventional intravenous chemotherapy by virtue of the localized non-systemic route of drug delivery. The advantages include: precise delivery of a drug superdose directly to the tumor mass (a dose impossible to deliver safely by normal systemic chemotherapy), and little systemic drug toxicity (in contrast to systemic intravenous drug delivery which is severely dose-limited due to general toxicity). Future multi-center randomized clinical trials will be essential to confirm the short-term and longterm beneficial effects of EITC as a therapeutic strategy for more effective NSCLC treatment. Clinical studies should address the potential benefits for neoadjuvant intratumoral therapy of early NSCLC in combination with other treatments such as brachytherapy and/or photodynamic therapy, and surgery.

obstruction. IT injection is readily achieved through a flexible bronchoscope by means of an ordinary needlecatheter. This procedure, termed “endobronchial intratumoral chemotherapy (EITC)” is described here and is important for consideration as a more effective approach to debulking endobronchial tumor obstruction and neoadjuvant chemotherapy of lung cancer (Wagai et al, 1982; Celikoglu et al, 1991, 1997, 2004, 2006a,b; Liu et al, 2000; Celikoglu and Celikoglu, 2003; Seymour et al, 2006). Our objective is to update information concerning clinical techniques for the direct injection of cytotoxic drugs into endobronchial malignant tumors in order to instruct bronchoscopists and interventional pulmonologists and to facilitate the use of this treatment modality.

I. Introduction Intravenous chemotherapy often leads to severe complications and abandonment of therapy due to systemic toxicity. To overcome this problem, many recent studies have been devoted to alternative modes of delivery for anticancer drugs to decrease systemic toxicity and improve the therapeutic index for approved cancer drugs. Several studies have shown that intratumoral delivery of cytotoxic drugs by direct injection into the solid tumor mass can provide extremely high doses of drug throughout the tumor with minimal systemic toxicity. Direct intratumoral (IT) injection of cancer drugs therefore represents a new treatment paradigm which may be especially valuable for treating lung cancer, especially when complicated by endobronchial tumor mass 545

Celikoglu et al: Endobronchial intra tumoral chemotherapy! axial tomography (CAT) scan of the chest and upper abdomen should be carefully reviewed to locate with precision the extent of the extraluminal localization of the tumor. If it is considered necessary, a PET scan and a mediastinoscopy should be done. If available, though not mandatory, the use of an endoscopic ultrasound bronchoscope (EBUS) may be helpful in some cases to locate any extra luminal malignant growth IT treatment. Autofluorescence bronchoscopy, if available, may also be useful to define the margins of malignant infiltration on the bronchial mucous membrane.! An intravenous access site is established, and normal saline is infused during the procedure. Standard monitoring includes an electrocardiogram, non-invasive blood pressure recording and oxygen saturation. Supplemental oxygen is administered nasally as deemed necessary. Topical anesthesia of the oral-nasalpharynx is achieved with 4% lidocaine. Conscious sedation is achieved with 3-5 mg intravenous midasolam and meperidine.

II. Method For bronchoscopic intratumoral injection, patients undergo routine bronchoscopy through any type of standard flexible bronchoscope under local anesthesia in a fully equipped endoscopy suite; at either hospital or outpatient clinics. In addition to a bronchoscopist, the procedure requires 2 dedicated assistants. The IT injection technique consists of the following steps: 1. Bronchoscopy, to visualize the lesion to be treated. 2. Introduction of a needle-device through the working channel of the bronchoscope. 3. Insertion of the needle into the lesion and injection of drug into the tumor tissue. 4. Repeated needle insertions, as many as considered appropriate to completely perfuse the tumor mass. 5. IT drug injections usually performed weekly. In patients previously treated by standard cancer therapies, EITC can continue up to one to two months as deemed necessary. In newly diagnosed patients, after 4 weekly injection sessions standard cancer treatments may be administered. 6. At end of each bronchoscopic injection session, removal of drug induced necrotic debris may be appropriate using mechanical dissection or other ablation techniques accompanied by irrigation and aspiration.

D. Bronchoscopic complications Bronchoscopy may be generally regarded as quite safe and well tolerated if performed by an experienced bronchoscopist. Infrequent serious complications are usually associated with patients who have predisposing health factors: 1.! a history of asthma may result in bronchospasm 2.! infection may occur in immunocompromised patients 3.! superior vena cava obstruction predisposes to laryngeal edema and bleeding 4.! ischemic heart disease and dysrithmias are contraindications for bronchoscopy 5.! bleeding diatheses may give excessive bleeding when biopsy specimens are taken 6.! drug allergies should be noted and anticoagulants and/or cardiorespiratory drugs may need to be stopped A retrospective study by Credle and colleagues indicated a complication incidence of 0.45% and 0.01% mortality for bronchoscopy (Credle et al, 1974). However, as might be expected, patients with preexisting infections often exhibit a significant incidence of post-bronchoscopy infection and pneumonia. Antibiotic treatment is therefore needed for patients at risk for infectious disease complications. Some degree of airways obstruction and hypoxemia is inherent in bronchoscopy. We therefore recommend use of supplementary oxygen through the bronchoscope during EITC. This is mandatory for all patients with oxygen tension of 70 mm Hg or less. A single nasal oxygen delivery catheter is convenient for this purpose. Supplementary oxygen should be continued in these patients for at least two hours after the EITC procedure.

A. Criteria for patient selection and eligibility Patients treated for non-small call lung carcinoma are eligible for endobronchial intratumoral chemotherapy by bronchoscopy based on the following criteria: 1.! Patients present with inoperable lung carcinoma and/or recurrent disease 2.! Patients present with symptomatic airways obstruction with symptoms such as coughing, shortness of breath, postobstructive pneumonia or atelectasis 3.! Occlusion or partial occlusion of bronchus 4.! Prior surgery is not a contraindication 5.! Disease verified by bronchoscopy 6.! Patients may be receiving systemic chemotherapy and/or radiation 7.! No age limitation 8.! Metastatic disease may be present 9.! Require informed consent 10.!Not eligible if patient has predisposing factors for diagnostic bronchoscopy It is important to note that EITC is intended to serve as a loco-regional neoadjuvant treatment.

B. Staging of NSCLC and EITC Treatment EITC may be used for palliative debulking at all stages of NSCLC (from Stage I to Stage IV) including all patients who had been previously treated by other conventional modalities and who have relapsed with endobronchial obstructive tumor growth. In cases of advanced and previously treated NSCLC, the use of EITC usually benefits the quality of life but no evidence is available for prolonged survival. For newly diagnosed NSCLC at all stages, EITC can be useful as a neoadjuvant therapy or a sensitizer (with intratumoral Cisplatin) for radiotherapy. In these cases, prolonged survival may be achieved. Here too, EITC may be effective for down-staging NSCLC by removal of tumor bulk, clearing the airways and bronchus, and enabling surgery for patients previously indicated as inoperable.

E. Bronchoscopic EITC Procedure The flexible bronchoscope is passed trans-nasally or orally and introduced into the trachea in the usual manner. A full inspection of the tracheal-bronchial tree with conventional white light and auto-fluorescence, if available, is completed. Biopsy specimens would have been taken previously from the tumor to confirm the diagnosis. The biopsies from the sites proximal and distal to the lesion should also have been obtained to define tumor margins histologically. The lesions to be treated are inspected to determine whether they are projecting or bulging into the airway lumen or infiltrating the mucous membrane, and to define the position and extent of stenos or extrinsic compression that may exist. Tumor volume is estimated visually from visible length-width-height measurements over the bronchial surface. The volume of extra-luminal localization of the tumor is estimated from CAT scan or EBUS.

C. Preparation of patients Before carrying out intratumoral chemotherapy through a bronchoscope, routine clinical data is of course obtained from patients, including arterial blood gases, coagulation parameters, and blood biochemistry. The chest radiography and computerized

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Cancer Therapy Vol 6, page 547! remains exposed. This maneuver allows the bronchoscope body to support the needle, so preventing the tubing from kinking and giving the operator greater control over the exact placement and advancement of needle. At the moment when the needle is uncovered, an assistant fixes the needle device manually at the proximal aperture of the bronchoscope working channel, so that both hands of the operator are free. This way, one hand of the operator can direct the tip of bronchoscope with the lever and the other hand can push the body of the bronchoscope with the needle uncovered at its distal end. The entire bronchoscope body is then pushed until the tip of needle contacts the targeted tumor area. The needle is then embedded into the tumor tissue to a depth appropriate for injection of drug into the tissue.

F. Handling of the needle device in the bronchoscope working channel 1. Characteristics of “the needle-device” used for intratumoral injection. The needle-device consists of a needle-catheter placed in an outer jacket or sheath (steel or plastic). The needle-catheter consists of a metallic needle attached to the end of a plastic catheter. The placement of the needle-catheter within a sheath is to prevent damage to the working channel by the sharp pointed needle during advancement into the working channel of the bronchoscope. During advancement in the working channel, the needle must be retracted into the sheath or outer jacket. Conventional needle devices designed for “transbronchial needle aspiration biopsy” or an “esophageal sclerotherapy catheter”, which are normally available in all endoscopy departments are quite suitable for the procedure. !

G. Injection of the drug 1. Mode of insertion of the needle into the lesion Injections of drugs are made directly into the targeted tumor tissues: endoluminal mass, mucosal, sub-mucosal, intramural, peribronchial infiltrative lesions or extra-bronchial locations of the tumor growth. The mode of insertion of the needle may be varied depending on the location, form, and appearance of the lesion to be treated. If the bronchoscopic appearance of the tumor is a polypoid or exophitic mass protruding in the bronchial lumen then the needle is inserted vertically into the tumor and adjacent bronchial mucous membrane (Figure 1). When a malignant lesion is seen as a slightly protruding infiltration above the surface of bronchial mucous membrane or as an infiltration in the sub-mucosal area or elsewhere within the airway wall, the needle is inserted into the lesion at an oblique angle of approximately 45 degrees (Figure 3). In the case of extramural disease causing an extramural compressive obstruction of the bronchial lumen (either by a tumor mass or by a conglomerate of metastatic lymph nodes adjacent to the tracheal-bronchial tree), then the needle is inserted at the wall of the airway at an angle of about 60-90 degrees (Figure 4). Once the needle is embedded into the tumor tissue; a syringe loaded previously with the drug is placed at the proximal end of the needle-catheter and the assistant starts the injection. To obtain the best therapeutic advantage for this IT drug delivery, it is desired that the drug fully perfuse the entire tumor mass. For protection of the maintenance personnel from the any probable adverse effects, the syringes are loaded with cytotoxic drugs beforehand in the oncology department under special hoods.

2. Selection of the size and length of the needle according to the morphology of lesion The needle size and the length will vary according the type and extent of the tumor growth. For endo-luminal exophytic, polypoid and bulgy tumors inside the airway lumen, 19-21 gauge and 10mm length needles are required. Mucosal, sub-mucosal or mural tumor growths usually require 23-25 gauge and 5mm length needles. Longer needles are best for trans-bronchial injection in extra-mural compressive obstructions because of the long distance from lesions. Therefore, for injecting peribronchial masses or extramural hilar and subcarinal lymph nodes adjacent to the bronchial wall, 21 gauge and 15mm or longer needles are most useful with a stiffer catheter. !

3. Plastic needle A unique needle made entirely of plastic without any metallic ending appears to be particularly useful for peripheral applications. It consists of an outer sheath and inner plastic catheter with a sharpened, beveled tip. The inner catheter can be withdrawn into the sheath during passage through the bronchoscope. The advantage of this needle lies in its lack of the rigid section that is characteristic of metal needles. As a result, plastic needles can generally be passed around sharper bends inside the working channel of bronchoscope than metal needles. This property is particularly useful in injecting upper lobe apical segment and lower lobe superior segment lesions and is also useful for other distally localized tumors.

4. Placement of the needle device in the bronchoscope working channel

2. Procedures to achieve complete tumor perfusion of drug during injection into tumor tissue

Initially, the bronchoscope is introduced into the bronchial tree in the conventional manner to visualize the lesion. Then, the needle-device is put into the entrance hole at the proximal aperture of the bronchoscope working channel with needlecatheter retracted in the sheath. The needle device is then advanced slowly into the working channel. As a precaution, it is advisable to pass the needle device through the bronchoscope channel with little or no distal tip deflection of the bronchoscope. The bronchoscope can then be flexed as needed once the distal end of the sheath is visible and out of the working channel. When the tip of the sheath is visualized out of the distal end of the bronchoscope working channel, it is forwarded further until the tip of the sheath is approximately 20 mm above the area to be injected. The needlecatheter itself can then be forwarded from its sheath until the needle is seen to be entirely uncovered. We emphasize that the metal needle should be forwarded from its sheath only after the distal end of the plastic sheath is visible through the bronchoscope. At this point, it is helpful to withdraw the sheath into the bronchoscope working channel so that only the needle

For complete dispersion of the drug solution throughout the tumor mass, the following technique may be useful: 1. The total volume of the drug, calculated according the volume of tumor, should be injected by repeated injections at several insertion points on the tumor surface until the total volume of drug is administered. Approximately, 0.50 ml of the drug solution should be injected at each insertion site. 2. After injecting a 0.50 ml aliquot of the drug solution at one insertion site on the tumor surface, the injection is stopped, the needle is withdrawn entirely from the tumor; and is then embedded at another location. After injecting another 0.50 ml aliquot, injection is stopped again; the needle is removed, and is again embedded at another site. These repetitive injection maneuvers are repeated several times (3-6x) until the entire desired total dose is delivered. 3. In order to obtain the most complete tumor perfusion of the drug, the following technique may also be useful: after embedding the needle into the tumor with the assistant pushing

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Celikoglu et al: Endobronchial intra tumoral chemotherapy! the piston of the syringe to inject drug, the needle is moved up and down by the operator in a fanning manner in the tumor mass. 4. To avoid spillage and leakage of drug into the airway lumen during removal of the needle from the tumor mass by the

operator, the pressure applied to the piston of the syringe by the assistant is halted and the piston is pulled back slightly to create a small negative pressure.

Figure 1. Shows the needle inserted vertically into an endoluminal polypoid or exophitic mass.

Figure 3. Shows the injection of the drug into tumor infiltrating the bronchial wall after removal of the endo-luminal component of the tumor mass.

Figure 4. Shows the injection of cytotoxic drug into a tumor that causes a compressive airway obstruction with intact bronchial mucous membrane. No bronchial fistula develops after intratumoral chemotherapy.

Figure 2. Shows the removal of tumor residues (debridements) by mechanical resection with forceps. The locally injected drug kills the malignant cells but does not remove them. The necrotic residues are therefore removed by mechanic debridement with forceps and facilitates delivery of additional cytotoxic drugs further into the tumoral mass if necessary.

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Cancer Therapy Vol 6, page 549! This procedure of repeated injections at different tumor sites, accompanied by up and down needle movements in a fanning manner provides for reasonably uniform dispersion of drug throughout tumor mass. After completing the delivery of the total volume of the drug, the needle is retracted into its sheath to prevent damage to the operating channel of the bronchoscope and then the entire needle-device is pulled back from the operating channel of the bronchoscope. After removal of the needle device from the bronchoscope working channel, irrigation and aspiration may be appropriate to avoid any local adverse effects of the drug due to any drug leakage.

chemotherapy with favorable results (Liu et al, 2000). Long acting formulas of cisplatin, mitoxantrone, carboplatin and paclitaxel are under investigation for intratumoral chemotherapy (Harper et al, 1999; Goldberg et al, 2006). !

J. Intratumoral Cisplatin injection for local neoadjuvant chemotherapy (prior to irradiation or surgery) in patients with NSC lung cancer presenting with bronchial obstruction Recent clinical trials with systemic cisplatin chemotherapy have shown cisplatin to be one of the most active agents against NSCLC. It is therefore the most frequently used cytotoxic drug in conventional systemic combination chemotherapy (Sandler et al, 2000; Gatzemeier et al, 2001). It has also been demonstrated that intravenous cisplatin can sensitize malignant cells to irradiation (Shaake-Konig et al, 1994; Yapp et al, 1998). Moreover, cisplatin in a collagen gel has been reported to successfully treat a variety of localized malignant tumors. Cisplatin has been administered by direct endoscopic injection for palliation of obstructive cancer of the esophagus (Monga et al, 2000), by IT injection for head and neck cancers (Burris et al, 1998), by CAT-scan guided IT injection into malignant liver tumors (Engelmann et al, 2002) and by endoscopic IT injection into gastric tumors (Monga et al, 1998). Endobronchial intratumoral chemotherapy with cisplatin was initiated by Celikoglu and colleagues for lung cancer patients presenting with bronchial obstruction. In previously untreated inoperable lung cancer patients, satisfactory results were obtained using bronchoscopic IT injections as a neoadjuvant chemotherapy prior to irradiation. Significant relief of bronchial obstruction was seen for 75% of treated patients (Celikoglu et al, 2006a). Celikoglu and colleagues have also shown that intratumoral chemotherapy with cisplatin can result in down-staging NSCLC and enable surgical intervention (Celikoglu et al, 2006b).!

3. Post procedure clean-up of bronchoscope working channel At the completion of the bronchoscopic drug injection procedure, it is advisable to aspirate 1 liter of saline through the working channel of the bronchoscope in order to remove any remaining drug. The entire bronchoscope is then cleaned in a automated cleaner.

H. Volume of cytotoxic drug solution to be injected into tumor tissue The volume of the solution to be delivered by intratumoral injection and the dose of the drug to be administered are calculated from the estimated volume of the tumor (not according to the body weight of the patient). A volume of 0.5 ml of drug solution is usually readily injected for each cubic centimeter of the tumor volume. Exophytic or polypoid endobronchial tumor volume may be approximated from the bronchoscopic view. The volume of any existing extramural or extra bronchial component of the tumor may be estimated from previous CAT scan or EBUS by the equation (0.5xLxWxH) where “L” is the greatest length; “W” is the greatest width, and “ H” is the greatest depth or height of the tumor (Monga et al, 2000). A total solution volume of about half the estimated tumor volume is usually safely injected into the tumor tissue (at several injection sites) without leakage.

A. Cisplatin dosage for intratumoral injection at each IT injection session Cisplatin has been used in the solution form which is available for intravenous administration (Celikoglu et al, 2006a,b). Cisplatin may be utilized at a concentration of 2 or 4 mg/mL. That means, if a 0.5 mL aliquot is injected for each cc of tumor volume; 1 or 2 mg cisplatin is administered at each treatment session. Although the total dose of cisplatin delivered by intratumoral injection is based on the estimated total volume of the tumor mass, it is preferable that a total dose of not more than 40 mg of cisplatin be injected at each IT injection session (Celikoglu et al, 2006a,b).

I. Characteristics of cancer drugs used for intratumoral chemotherapy Preferred cytotoxic drugs for IT injection should not induce significant local necrotic changes in the normal mucosa, should have a normal pH of approximately 7.4, and should exhibit direct anti-neoplastic activity. Drugs that require hepatic activation (i.e. cyclophosphamide) are not suitable for intratumoral injection. For intratumoral injection, standard approved anticancer drugs in solutions for intravenous administration are used. Current research is also being devoted to the preparation of polymer-drug compositions that can enhance drug stability and prolong intratumoral activity with more limited diffusion away from the injection tumor. Injectable drug-loaded nano-meso-microspheres, liposomes, and polymer gels (i.e. collagen or alginate gels) promise to offer improved therapeutic IT efficacy in the future (Goldberg et al, 2002, 2006; Almond et al, 2003). Until such modified drugs are available for clinical use, currently approved aqueous I.V. drug solutions are useful for IT chemotherapy. Celikoglu and colleagues have studied in 1997 mixed drug regimens composed of methotrexate, bleomycin, mitoxantrone, mitomycin C, and 5-fluorouracil (5-FU) for inratumoral injection (Celikoglu et al, 1997). Celikoglu and colleagues also studied in 2003 5-FU alone at higher doses for IT treatments with satisfactory debulking results (Celikoglu and Celikoglu, 2003. Liu, has reported the use of carboplatin for intratumoral

K. Scheduling of injection sessions and duration of intratumoral chemotherapy treatment 1. For previously untreated patients For previously untreated patients, cisplatin may be administered four times at weekly intervals (on days 1, 8, 15 and 22). Three to seven days after the last session of intratumoral chemotherapy, irradiation may be initiated if deemed appropriate (Celikoglu et al, 2006ba5). In operable cases or in cases where down-staging was achieved after EITC, surgery may be carried out 3-7 days after termination of IT treatment (Celikoglu et al, 2006b). !

2. For patients previously treated by surgery, by extra-beam irradiation, or by systemic chemotherapy For such patients, IT chemotherapy is carried out at weekly intervals. If a response is obtained, EITC therapy can continue for 6-8 weeks or longer until the disappearance of the

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Celikoglu et al: Endobronchial intra tumoral chemotherapy! endobronchial tumor growth. If there is no positive response after 3 weeks of weekly EITC injections, the IT therapy may be stopped. Other endobronchial ablation techniques such as laser photoresection, electrocautery, cryotherapy may then be tried. In cases of poor response to other interventional bronchoscopic procedures, it may be appropriate to evaluate EITC.

It is reasonable to regard the following factors as contributory to the haemostasis: 1.! Vasoconstriction of small vessels in or around the tumor growth due to reaction with a very high concentrations of the cytotoxic drug; 2.! Oligemia caused by the compression of small vessels in the tumor due to the volume of drug solution injected into the restricted intratumoral volume; 3.! Occlusive intravascular thromboses in the tumor vessels. The haemostasis is advantageous in that it facilitates resection of the necrotic tumor mass with forceps and removal of tumor tissue debris (Celikoglu and Celikoglu, 2003; Celikoglu et al, 2006a,b)

3. Patient follow-up For those patients previously treated by other standard cancer therapies, if bronchial obstruction has been relieved by IT chemotherapy, follow-up bronchoscopic examinations should be performed at least every three months. Biopsy specimens of the treated areas are taken and examined by the same pathologist who examined the initial specimens. It is advisable to use autofluorescence to detect any tumor recurrence. If tumor recurrence is noted, patients may be indicated to receive additional IT treatment. The same follow-up protocol is advised after radiotherapy.

L. Effects of intratumoral cytotoxic drugs on tumor tissue

delivery

N. Removal of residual tumor debris by ablation techniques after EITC of obstructive endo-luminal tumors Although a significant reduction in tumor size may be affected by IT chemotherapy and may thereby be sufficient to alleviate symptoms, tumor size reduction in some cases may not adequately restore the airway passage as quickly as desired. Cytotoxic drug injection into the tumor kills the cancerous cells but does not immediately result in elimination of necrotic residues. Thus, in urgent life threatening situations with severe dyspnea where immediate obstruction relief is obligatory, tumor cell debris, necrotic residues, and fibrin plugs should be removed by other means. In such cases, endobronchial ablation procedures such as mechanical resection, laser photoresection, electocautery or cryotherapy should be utilized. The limited haemostatic effect provided by IT drug injection often facilitates the removal of tumor residues by mechanical resection with forceps without serious bleeding (Figure 2). Piece by piece resection with forceps can therefore be useful for debulking of tissue residues after IT endo-bronchial chemotherapy (Celikoglu and Celikoglu, 2003; Celikoglu et al, 2006a,b).

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1. Early effects The tumor mass is usually seen through the bronchoscope as an endo-luminal protruding polypoid mass. While injecting the drug solution, the tumor around the needle becomes swollen, pale and whiter. This is probably due to edema of the tumor tissue and compression of blood vessels inside the tumor by the volume of injected solution.

2. Delayed effects Three to seven days after intratumoral drug injection, bronchoscopic examination normally reveals some reduction of the tumor mass. Tumor surfaces appear to be covered with a white-yellow gel-like substance. Histopathology indicates the presence of necrotic tissue, devitalized tumor cell residues, fibrin coagulum, dissipated secretions and white blood cells. In some cases, weeks to months later, the tumor may disappear completely leaving a grey colored scar tissue in place of the tumor (Celikoglu and Celikoglu, 2003; Celikoglu et al, 2006a,b). This necrotizing effect of locally administered cytotoxic drug is presumably due to contact and interaction of the malignant cells with the extremely high concentration of drug to which the tumor is exposed by direct drug injection (Goldberg et al, 2002, 2006). A few days after IT drug delivery the texture of the malignant tissue may appear to soften and become brittle and is then easily separated into small fragments. This is likely due to an early necrobiosis or necrosis. As a result of the early necrosis and reduction in size of the tumor mass, in some cases the diameter of the airway passage may be amplified and symptoms alleviated even after the first session of treatment. In such cases, it is also possible to observe rather rapid recovery from accompanying pathology, i.e. atelectasis or obstructive pneumonia. When the tumor growth is present as an infiltrative process with slight protrusion on the surface of the bronchial mucous membrane, the infiltrative processes improve markedly after drug injection. This infiltrative tumor growth is usually converted to a white-yellow “membrane-like� substance (Celikoglu and Celikoglu, 2003; Celikoglu et al, 2006a,b).

M. Haemostatic effect injection of cytotoxic drugs

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O. Adverse effects of intratumoral chemotherapy During injection of the cytotoxic drug, patients report no pain or other disturbing sensations. Injection of the drug into normal mucous membranes around the malignant tissue also does not cause any important symptoms. No adverse effects such as irritation or necrosis have been noted due to contact of the drug solution with the surface of normal bronchial mucous membrane. After IT chemotherapy, transient moderate chest pain, a slight sensation of nausea, and/or a moderate fever of one day duration may be noted. No other important systemic or local side effects are observed. No systemic toxicity such as interstitial pneumonia, bone marrow suppression, worsening of inflammatory findings or hair loss has been observed. Nephrotoxicity, neurotoxicity, and myocarditis, which are commonly associated with systemic intravenous administration of drugs such as cisplatin, are also not seen (Liu et al, 2000; Celikoglu et al, 2006a,b).

P. Dispersion of drugs in the tumor mass 1. Drug distribution by diffusion The success of intratumoral chemotherapy is dependent upon relatively complete permeation of the injected malignant lesion with a very high concentration of drug with minimal rapid diffusion from the injected tumor tissue into the systemic circulation. Protocols involving repetitive injections and weekly treatments for several weeks are designed to maximize the probability that all tumor cells in the injected lesion are subjected to lethal drug concentrations. Fortunately, although small drug molecules may associate with tumor tissues by inherent tissue

intratumoral

Clinical observations indicate that intratumoral injection of cytotoxic drugs may also provide a haemostatic effect on the tumor bulk. The bleeding tendency of tumor tissues is thereby often reduced after IT drug injection. In such situations, piecemeal resection of tumor debris with forceps does not provoke further bleeding (Figure 2).

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Cancer Therapy Vol 6, page 551! affinity and by DNA binding, some diffusion away from the injection sites via the extra-cellular tumor tissue fluid can also occur to facilitate tumor perfusion by most drugs. The extent of such diffusion is proportional to ratio of the injection site drug concentration and the drug concentration in the surrounding tumor tissue. When there is a relatively uniform drug concentration within the tumor tissue, drug diffusion out of the tumor will be dependent on the rate of drug passage into the lymphatic and vascular systems. Repeated IT injections facilitate complete permeation of drug into the entire tumor mass. The drug transport process is also affected by the relative porosity of the tumor tissue with some variation in such porosity according to tumor type and size. Clinical CT scan observations have demonstrated that even when only a small part of the tumor is visible by bronchoscopy, the injection of cytotoxic drugs into an endoluminal portion of the tumor is effective in also decreasing the size of the extramural part of the tumor. This suggests that anticancer drugs injected into one portion of the tumor may diffuse throughout the tumor mass (Goldberg et al, 2002, 2006).

drug and the rate of transport into the systemic circulation is thereby so limited that no adverse systemic effects are observed.

IV. Discussion and Conclusions Reviewed here are the techniques and clinical indications for EITC. It is noted that EITC may be used as a preoperative or neoadjuvant procedure or used concomitantly or sequentially with other standard lung cancer treatments and interventional therapeutic bronchoscopic procedures. EITC is a novel therapeutic paradigm for lung cancer treatment, particularly for widespread application to malignant endobronchial tumor growths. By providing local delivery of cancer drugs, EITC achieves a significant reduction in systemic toxicity with few of the toxic side effects of systemic chemotherapy. The EITC procedure is not yet used to its full potential. Increased training for interventional pulmonologists in the techniques for bronchoscopic EITC is needed to help broaden clinical perspectives and to make clearer the therapeutic scope and opportunities for EITC. Increased attention should be focused on such less invasive and less toxic treatment strategies for lung cancer. In a recent study, Nader reported that intratumoral administration of cisplatin, as an adjunct to brachytherapy, was a safe and potentially useful modality to assist in the management of endobronchial tumor obstruction and possibly for long term efficacy. It was concluded that further studies are needed to more fully assess the benefits of this treatment (Nader, 2007). In addition to the obvious advantage of tumor burden reduction inside the airway lumen, intratumoral bronchoscopic delivery of cytotoxic drugs provides a unique loco-regional neo-adjuvant therapy for use prior to irradiation or surgery (Celikoglu et al, 2006a,b). It is chemotherapy which differs from intravenous chemotherapy only by its local route of delivery. Although the number of clinical studies to date are limited, intratumoral chemotherapy has been found to be advantageous compared to intravenous chemotherapy for effecting efficient tumor regression with minimal sideeffects. One example is a comparative clinical study by Liu and colleagues where intratumoral chemotherapy with carboplatin was combined with systemic carboplatin and found to be superior to systemic chemotherapy alone for prolonged survival of lung cancer patients (Liu et al, 2000). In addition to the development of statistically significant survival data, future clinical studies should also encompass: different drug combinations, IT drug dose response, timing of EITC injections, evaluation of drug serum levels vs. time, and mechanistic studies designed to measure T-cell/killer cell activity and tumor-specific immune response.

2. Sentinel lymph node drug perfusion (drainage of drug molecules via afferent lymphatic vessels) Sentinel lymph nodes (SLN) are the first lymphatic drainage site of a tumor and the likely site of initial metastatic tumor cell dissemination (Tiffet et al, 2005). Although the overall prognostic significance SLN micro-metastases in early lung cancer remains unclear, recent studies suggest that for larger and potentially resectable lung cancers there is a significant 5year survival advantage in patients with adenocarcinoma who do not have SLN micro-metastases when compared to patients with SLN micro-metastasis (62% with metastasis vs. 86% with no metastasis) (Wu et al, 2001). IT injected drug molecules can be transported by afferent lymphatic vessels into the sentinel and draining lymph nodes. Such drug transport to the lymph nodes may be expected to have a valuable therapeutic effect by eradication of lymphatic micrometastases. Clinical studies performed using pre-surgical radioisotopes, blue dye techniques, and even the blue cancer drug mitoxantrone, support this view of drug molecule transport from an IT injected site to sentinel and draining lymph nodes (Izbicki et al, 1996; Baitchev et al, 2001; Wu et al, 2001; Lardinois et al, 2003; Tiffet et al, 2005). As also suggested by Lardinois et al, drug injected through the bronchoscope into normal tissues around the tumor is also transported to sentinel lymph nodes. We deduce from this that EITC may have the added advantage of inhibiting metastasis by cytotoxic action on tumor cells which are migrating into the lymph nodes that drain the tumor area (Lardinois et al, 2003).

3. Low systemic concentration of IT injected drugs Although we have no human clinical data, animal studies have shown that very high doses of cytotoxic drugs (>10-30 times normal intravenous doses) are achieved within tumors by intratumoral injection. However, only minute amounts of IT drug pass into the systemic circulation (Goldberg et al, 2002, 2006). Minimal systemic drug concentrations occur because of several unique aspects of IT injection. Depending on the molecular structures of specific cancer drugs, factors such as drug-tissue affinity, drug instability, rapid drug metabolism, low plasma solubility, and low tissue diffusion rates limit the extent of drug transport into surrounding tissues and vasculature. Furthermore, a major drug transport pathway for IT injected drug molecules is via the afferent lymphatics to the sentinel lymph nodes and then leave lymph glands by efferent lymphatic vessels which ultimately empty into the thoracic duct and right lymphatic duct which then empty into the venous circulation. The amount of

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lung cancer tumor nodules in mice. Clin Cancer Research 5, 4242-4248. Izbicki JR, Passlick B, Hosch SB, Kubuschock B, Schneider C, Busch C, Knoefel WT, Thetter O, Pantel K (1996) Mode of spread in the early phase of lymphatic metastasis in nonsmall cell lung cancer: significance of nodal metastasis. J Thorac Cardiovasc Surg 112, 623-630. Lardinois D, Brack T, Gaspert A, Spahr T, Schneiter D, Steinert HC, Weder W (2003) Bronchoscopic radioisotope injection for sentinel lymph-node mapping in potentially resectable nonsmall-cell-lung-cancer. Eur J Cardio-Thorac Surg 23, 824-827. Liu M, Ma P, Lu Z (2000) Local chemotherapy by fibrobroncoscopy for advanced broncogenic carcinoma. Chin J Tuberc Respir Dis 23, 550-551. Monga SP, Wadleigh R, Adib H, Harmon JW, Berlin M, Mishra L (1998) Endoscopic treatment of gastric cancer with intratumoral cisplatin/epinephrine injectable gel: a case report. Gasrointest Endosc 48, 415-417. Monga SP, Wadleigh R, Sharma A, Adib H, Strader D, Singh G, Harmon JW, Berlin M, Monga DK, Mishra L (2000) Intratumoral therapy of cisplatin/epinephrine injectable gel for palliation in patients with obstructive esophageal cancer. Am J Clin Oncol (CCT) 23, 386-392. Nader AD (2007) Intratumoral chemotherapy as an adjuct to endobronch覺al brachytherapy. Chest 32, (Suppl) 459s. Sandler AB, Nemunaitis J, Denham C, von Pawel J, Cormier Y, Gatzemeier U, Mattson K, Manegold C, Palmer MC, Gregor A, Nguyen B, Niyikiza C, Einhorn LH (2000) Phase III trial gemcitabine plus cisplatin versus cisplatin alone with patients with locally advanced or metastatic non-small lung cancer. J Clin Oncol 18, 122-130. Seymour CW, Krimsky WS, Sager J, Kruklitis RJ, Lund ME, Musani AI, Sterman DH (2006) Transbronchial needle injection: A systemic review of a new diagnostic and therapeutic paradigm. Respiration 73, 78-89. Shaake-Konig C, van den Bogaert W, Daleiso O, Festen J, Hoogenhout J (1994) Radiosensitization by cytotoxic drugs. The EORTC experience by radiotherapy and lung cancer cooperative groups. Lung Cancer 10, S263-270. Tiffet O, Nicholson AG, Khaddag, Ladas G, Dubois F, Goldstraw P (2005) Feasibility of detection of sentinel lymph node in peripheral non-small lung cancer with radio isotopic and blue dye techniques. Chest 127, 443-448. Wagai F, Kinoshita M, Watanabe RSH, Kitamura S (1982) The direct injection of several anti-cancer drugs into the primary lung cancer through a fiberoptic bronchoscope. Jpn J Thorac Dis 20, 170. Wu J, Ohta Y, Minato H (2001) Nodal occult metastasis in patients with peripheral lung adenocarcinoma of 2.0 cm or less in diameter. Ann Thorac Surg 71, 1772-1778. Yapp DT, Lloyd DK, Zhu E, Lehnert SM (1998) The potentiation of the effect of radiation treatment by intratumoral delivery of cisplatin. Int J Radiat Oncol Rio Phys 42, 413-420.

From left to right: Firuz Celikoglu, Seyhan I Celikoglu, Eugene P Goldberg

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Cancer Therapy Vol 6, page 545! Cancer Therapy Vol 6, 545-552, 2008

Techniques for intratumoral chemotherapy of lung cancer by bronchoscopic drug delivery Research Article

Firuz Celikoglu1, Seyhan I Celikoglu1, Eugene P Goldberg2 1 2

University of Istanbul, Cerrahpasa Medical Faculty and Institute of Lung Diseases and Tuberculosis, Istanbul, Turkey University of Florida, Biomaterials Center, Department Materials Science & Engineering, Gainesville, FL, USA

__________________________________________________________________________________! *Correspondence: Seyhan I Celikoglu Ph.D., Urguplu Caddesi, No. 16, Yesilyurt, 34800 Istanbul, Turkey; Tel.:+90 (542) 433 02 88; Fax: +90 (212) 663 96 38; E-mail addresses: seyhan@celikoglu.biz Eugene P Goldberg, Ph.D., University of Florida, Biomaterials Center, Department Materials Science & Engineering, P.O. Box 116400, Gainesville, FL, USA; Tel.: +352 392 4907; fax: +352 383 7416; E-mail: egold@mse.ufl.edu Key words: Direct intratumoral injection; Endobronchial chemotherapy; Interventional therapeutic bronchoscopy; Lung cancer therapy Abbreviations: 5-fluorouracil, (5-FU); computerized axial tomography, (CAT); endo-bronchial intratumoral chemotherapy, (EITC); endoscopic ultrasound bronchoscope, (EBUS); intratumoral, (IT); Sentinel lymph nodes, (SLN) Received: 11 March 2008; Revised: 8 June 2008 Accepted: 20 June 2008; electronically published: September 2008

Summary The objective of this review is to provide a detailed description of the technique for a new non-systemic therapeutic modality for NSC lung cancer chemotherapy. Described here is the technique for intratumoral injection of one or more cytotoxic drugs directly into tumor tissue through a flexible bronchoscope by means of a needle-catheter. The procedure is termed “endo-bronchial intratumoral chemotherapy (EITC)”. EITC is a loco-regional form of chemotherapy as well as an ablative cytotoxic procedure for removal of endobronchial tumor bulk for NSC lung cancer that presents with bronchial obstruction. In addition to the advantage of tumor burden reduction inside the airway lumen, intratumoral delivery of cytotoxic drugs may be regarded as an improved neoadjuvant therapy for use prior to irradiation and/or surgery. EITC differs significantly from conventional intravenous chemotherapy by virtue of the localized non-systemic route of drug delivery. The advantages include: precise delivery of a drug superdose directly to the tumor mass (a dose impossible to deliver safely by normal systemic chemotherapy), and little systemic drug toxicity (in contrast to systemic intravenous drug delivery which is severely dose-limited due to general toxicity). Future multi-center randomized clinical trials will be essential to confirm the short-term and longterm beneficial effects of EITC as a therapeutic strategy for more effective NSCLC treatment. Clinical studies should address the potential benefits for neoadjuvant intratumoral therapy of early NSCLC in combination with other treatments such as brachytherapy and/or photodynamic therapy, and surgery.

obstruction. IT injection is readily achieved through a flexible bronchoscope by means of an ordinary needlecatheter. This procedure, termed “endobronchial intratumoral chemotherapy (EITC)” is described here and is important for consideration as a more effective approach to debulking endobronchial tumor obstruction and neoadjuvant chemotherapy of lung cancer (Wagai et al, 1982; Celikoglu et al, 1991, 1997, 2004, 2006a,b; Liu et al, 2000; Celikoglu and Celikoglu, 2003; Seymour et al, 2006). Our objective is to update information concerning clinical techniques for the direct injection of cytotoxic drugs into endobronchial malignant tumors in order to instruct bronchoscopists and interventional pulmonologists and to facilitate the use of this treatment modality.

I. Introduction Intravenous chemotherapy often leads to severe complications and abandonment of therapy due to systemic toxicity. To overcome this problem, many recent studies have been devoted to alternative modes of delivery for anticancer drugs to decrease systemic toxicity and improve the therapeutic index for approved cancer drugs. Several studies have shown that intratumoral delivery of cytotoxic drugs by direct injection into the solid tumor mass can provide extremely high doses of drug throughout the tumor with minimal systemic toxicity. Direct intratumoral (IT) injection of cancer drugs therefore represents a new treatment paradigm which may be especially valuable for treating lung cancer, especially when complicated by endobronchial tumor mass 545

Celikoglu et al: Endobronchial intra tumoral chemotherapy! axial tomography (CAT) scan of the chest and upper abdomen should be carefully reviewed to locate with precision the extent of the extraluminal localization of the tumor. If it is considered necessary, a PET scan and a mediastinoscopy should be done. If available, though not mandatory, the use of an endoscopic ultrasound bronchoscope (EBUS) may be helpful in some cases to locate any extra luminal malignant growth IT treatment. Autofluorescence bronchoscopy, if available, may also be useful to define the margins of malignant infiltration on the bronchial mucous membrane.! An intravenous access site is established, and normal saline is infused during the procedure. Standard monitoring includes an electrocardiogram, non-invasive blood pressure recording and oxygen saturation. Supplemental oxygen is administered nasally as deemed necessary. Topical anesthesia of the oral-nasalpharynx is achieved with 4% lidocaine. Conscious sedation is achieved with 3-5 mg intravenous midasolam and meperidine.

II. Method For bronchoscopic intratumoral injection, patients undergo routine bronchoscopy through any type of standard flexible bronchoscope under local anesthesia in a fully equipped endoscopy suite; at either hospital or outpatient clinics. In addition to a bronchoscopist, the procedure requires 2 dedicated assistants. The IT injection technique consists of the following steps: 1. Bronchoscopy, to visualize the lesion to be treated. 2. Introduction of a needle-device through the working channel of the bronchoscope. 3. Insertion of the needle into the lesion and injection of drug into the tumor tissue. 4. Repeated needle insertions, as many as considered appropriate to completely perfuse the tumor mass. 5. IT drug injections usually performed weekly. In patients previously treated by standard cancer therapies, EITC can continue up to one to two months as deemed necessary. In newly diagnosed patients, after 4 weekly injection sessions standard cancer treatments may be administered. 6. At end of each bronchoscopic injection session, removal of drug induced necrotic debris may be appropriate using mechanical dissection or other ablation techniques accompanied by irrigation and aspiration.

D. Bronchoscopic complications Bronchoscopy may be generally regarded as quite safe and well tolerated if performed by an experienced bronchoscopist. Infrequent serious complications are usually associated with patients who have predisposing health factors: 1.! a history of asthma may result in bronchospasm 2.! infection may occur in immunocompromised patients 3.! superior vena cava obstruction predisposes to laryngeal edema and bleeding 4.! ischemic heart disease and dysrithmias are contraindications for bronchoscopy 5.! bleeding diatheses may give excessive bleeding when biopsy specimens are taken 6.! drug allergies should be noted and anticoagulants and/or cardiorespiratory drugs may need to be stopped A retrospective study by Credle and colleagues indicated a complication incidence of 0.45% and 0.01% mortality for bronchoscopy (Credle et al, 1974). However, as might be expected, patients with preexisting infections often exhibit a significant incidence of post-bronchoscopy infection and pneumonia. Antibiotic treatment is therefore needed for patients at risk for infectious disease complications. Some degree of airways obstruction and hypoxemia is inherent in bronchoscopy. We therefore recommend use of supplementary oxygen through the bronchoscope during EITC. This is mandatory for all patients with oxygen tension of 70 mm Hg or less. A single nasal oxygen delivery catheter is convenient for this purpose. Supplementary oxygen should be continued in these patients for at least two hours after the EITC procedure.

A. Criteria for patient selection and eligibility Patients treated for non-small call lung carcinoma are eligible for endobronchial intratumoral chemotherapy by bronchoscopy based on the following criteria: 1.! Patients present with inoperable lung carcinoma and/or recurrent disease 2.! Patients present with symptomatic airways obstruction with symptoms such as coughing, shortness of breath, postobstructive pneumonia or atelectasis 3.! Occlusion or partial occlusion of bronchus 4.! Prior surgery is not a contraindication 5.! Disease verified by bronchoscopy 6.! Patients may be receiving systemic chemotherapy and/or radiation 7.! No age limitation 8.! Metastatic disease may be present 9.! Require informed consent 10.!Not eligible if patient has predisposing factors for diagnostic bronchoscopy It is important to note that EITC is intended to serve as a loco-regional neoadjuvant treatment.

B. Staging of NSCLC and EITC Treatment EITC may be used for palliative debulking at all stages of NSCLC (from Stage I to Stage IV) including all patients who had been previously treated by other conventional modalities and who have relapsed with endobronchial obstructive tumor growth. In cases of advanced and previously treated NSCLC, the use of EITC usually benefits the quality of life but no evidence is available for prolonged survival. For newly diagnosed NSCLC at all stages, EITC can be useful as a neoadjuvant therapy or a sensitizer (with intratumoral Cisplatin) for radiotherapy. In these cases, prolonged survival may be achieved. Here too, EITC may be effective for down-staging NSCLC by removal of tumor bulk, clearing the airways and bronchus, and enabling surgery for patients previously indicated as inoperable.

E. Bronchoscopic EITC Procedure The flexible bronchoscope is passed trans-nasally or orally and introduced into the trachea in the usual manner. A full inspection of the tracheal-bronchial tree with conventional white light and auto-fluorescence, if available, is completed. Biopsy specimens would have been taken previously from the tumor to confirm the diagnosis. The biopsies from the sites proximal and distal to the lesion should also have been obtained to define tumor margins histologically. The lesions to be treated are inspected to determine whether they are projecting or bulging into the airway lumen or infiltrating the mucous membrane, and to define the position and extent of stenos or extrinsic compression that may exist. Tumor volume is estimated visually from visible length-width-height measurements over the bronchial surface. The volume of extra-luminal localization of the tumor is estimated from CAT scan or EBUS.

C. Preparation of patients Before carrying out intratumoral chemotherapy through a bronchoscope, routine clinical data is of course obtained from patients, including arterial blood gases, coagulation parameters, and blood biochemistry. The chest radiography and computerized

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Cancer Therapy Vol 6, page 547! remains exposed. This maneuver allows the bronchoscope body to support the needle, so preventing the tubing from kinking and giving the operator greater control over the exact placement and advancement of needle. At the moment when the needle is uncovered, an assistant fixes the needle device manually at the proximal aperture of the bronchoscope working channel, so that both hands of the operator are free. This way, one hand of the operator can direct the tip of bronchoscope with the lever and the other hand can push the body of the bronchoscope with the needle uncovered at its distal end. The entire bronchoscope body is then pushed until the tip of needle contacts the targeted tumor area. The needle is then embedded into the tumor tissue to a depth appropriate for injection of drug into the tissue.

F. Handling of the needle device in the bronchoscope working channel 1. Characteristics of “the needle-device” used for intratumoral injection. The needle-device consists of a needle-catheter placed in an outer jacket or sheath (steel or plastic). The needle-catheter consists of a metallic needle attached to the end of a plastic catheter. The placement of the needle-catheter within a sheath is to prevent damage to the working channel by the sharp pointed needle during advancement into the working channel of the bronchoscope. During advancement in the working channel, the needle must be retracted into the sheath or outer jacket. Conventional needle devices designed for “transbronchial needle aspiration biopsy” or an “esophageal sclerotherapy catheter”, which are normally available in all endoscopy departments are quite suitable for the procedure. !

G. Injection of the drug 1. Mode of insertion of the needle into the lesion Injections of drugs are made directly into the targeted tumor tissues: endoluminal mass, mucosal, sub-mucosal, intramural, peribronchial infiltrative lesions or extra-bronchial locations of the tumor growth. The mode of insertion of the needle may be varied depending on the location, form, and appearance of the lesion to be treated. If the bronchoscopic appearance of the tumor is a polypoid or exophitic mass protruding in the bronchial lumen then the needle is inserted vertically into the tumor and adjacent bronchial mucous membrane (Figure 1). When a malignant lesion is seen as a slightly protruding infiltration above the surface of bronchial mucous membrane or as an infiltration in the sub-mucosal area or elsewhere within the airway wall, the needle is inserted into the lesion at an oblique angle of approximately 45 degrees (Figure 3). In the case of extramural disease causing an extramural compressive obstruction of the bronchial lumen (either by a tumor mass or by a conglomerate of metastatic lymph nodes adjacent to the tracheal-bronchial tree), then the needle is inserted at the wall of the airway at an angle of about 60-90 degrees (Figure 4). Once the needle is embedded into the tumor tissue; a syringe loaded previously with the drug is placed at the proximal end of the needle-catheter and the assistant starts the injection. To obtain the best therapeutic advantage for this IT drug delivery, it is desired that the drug fully perfuse the entire tumor mass. For protection of the maintenance personnel from the any probable adverse effects, the syringes are loaded with cytotoxic drugs beforehand in the oncology department under special hoods.

2. Selection of the size and length of the needle according to the morphology of lesion The needle size and the length will vary according the type and extent of the tumor growth. For endo-luminal exophytic, polypoid and bulgy tumors inside the airway lumen, 19-21 gauge and 10mm length needles are required. Mucosal, sub-mucosal or mural tumor growths usually require 23-25 gauge and 5mm length needles. Longer needles are best for trans-bronchial injection in extra-mural compressive obstructions because of the long distance from lesions. Therefore, for injecting peribronchial masses or extramural hilar and subcarinal lymph nodes adjacent to the bronchial wall, 21 gauge and 15mm or longer needles are most useful with a stiffer catheter. !

3. Plastic needle A unique needle made entirely of plastic without any metallic ending appears to be particularly useful for peripheral applications. It consists of an outer sheath and inner plastic catheter with a sharpened, beveled tip. The inner catheter can be withdrawn into the sheath during passage through the bronchoscope. The advantage of this needle lies in its lack of the rigid section that is characteristic of metal needles. As a result, plastic needles can generally be passed around sharper bends inside the working channel of bronchoscope than metal needles. This property is particularly useful in injecting upper lobe apical segment and lower lobe superior segment lesions and is also useful for other distally localized tumors.

4. Placement of the needle device in the bronchoscope working channel

2. Procedures to achieve complete tumor perfusion of drug during injection into tumor tissue

Initially, the bronchoscope is introduced into the bronchial tree in the conventional manner to visualize the lesion. Then, the needle-device is put into the entrance hole at the proximal aperture of the bronchoscope working channel with needlecatheter retracted in the sheath. The needle device is then advanced slowly into the working channel. As a precaution, it is advisable to pass the needle device through the bronchoscope channel with little or no distal tip deflection of the bronchoscope. The bronchoscope can then be flexed as needed once the distal end of the sheath is visible and out of the working channel. When the tip of the sheath is visualized out of the distal end of the bronchoscope working channel, it is forwarded further until the tip of the sheath is approximately 20 mm above the area to be injected. The needlecatheter itself can then be forwarded from its sheath until the needle is seen to be entirely uncovered. We emphasize that the metal needle should be forwarded from its sheath only after the distal end of the plastic sheath is visible through the bronchoscope. At this point, it is helpful to withdraw the sheath into the bronchoscope working channel so that only the needle

For complete dispersion of the drug solution throughout the tumor mass, the following technique may be useful: 1. The total volume of the drug, calculated according the volume of tumor, should be injected by repeated injections at several insertion points on the tumor surface until the total volume of drug is administered. Approximately, 0.50 ml of the drug solution should be injected at each insertion site. 2. After injecting a 0.50 ml aliquot of the drug solution at one insertion site on the tumor surface, the injection is stopped, the needle is withdrawn entirely from the tumor; and is then embedded at another location. After injecting another 0.50 ml aliquot, injection is stopped again; the needle is removed, and is again embedded at another site. These repetitive injection maneuvers are repeated several times (3-6x) until the entire desired total dose is delivered. 3. In order to obtain the most complete tumor perfusion of the drug, the following technique may also be useful: after embedding the needle into the tumor with the assistant pushing

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Celikoglu et al: Endobronchial intra tumoral chemotherapy! the piston of the syringe to inject drug, the needle is moved up and down by the operator in a fanning manner in the tumor mass. 4. To avoid spillage and leakage of drug into the airway lumen during removal of the needle from the tumor mass by the

operator, the pressure applied to the piston of the syringe by the assistant is halted and the piston is pulled back slightly to create a small negative pressure.

Figure 1. Shows the needle inserted vertically into an endoluminal polypoid or exophitic mass.

Figure 3. Shows the injection of the drug into tumor infiltrating the bronchial wall after removal of the endo-luminal component of the tumor mass.

Figure 4. Shows the injection of cytotoxic drug into a tumor that causes a compressive airway obstruction with intact bronchial mucous membrane. No bronchial fistula develops after intratumoral chemotherapy.

Figure 2. Shows the removal of tumor residues (debridements) by mechanical resection with forceps. The locally injected drug kills the malignant cells but does not remove them. The necrotic residues are therefore removed by mechanic debridement with forceps and facilitates delivery of additional cytotoxic drugs further into the tumoral mass if necessary.

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Cancer Therapy Vol 6, page 549! This procedure of repeated injections at different tumor sites, accompanied by up and down needle movements in a fanning manner provides for reasonably uniform dispersion of drug throughout tumor mass. After completing the delivery of the total volume of the drug, the needle is retracted into its sheath to prevent damage to the operating channel of the bronchoscope and then the entire needle-device is pulled back from the operating channel of the bronchoscope. After removal of the needle device from the bronchoscope working channel, irrigation and aspiration may be appropriate to avoid any local adverse effects of the drug due to any drug leakage.

chemotherapy with favorable results (Liu et al, 2000). Long acting formulas of cisplatin, mitoxantrone, carboplatin and paclitaxel are under investigation for intratumoral chemotherapy (Harper et al, 1999; Goldberg et al, 2006). !

J. Intratumoral Cisplatin injection for local neoadjuvant chemotherapy (prior to irradiation or surgery) in patients with NSC lung cancer presenting with bronchial obstruction Recent clinical trials with systemic cisplatin chemotherapy have shown cisplatin to be one of the most active agents against NSCLC. It is therefore the most frequently used cytotoxic drug in conventional systemic combination chemotherapy (Sandler et al, 2000; Gatzemeier et al, 2001). It has also been demonstrated that intravenous cisplatin can sensitize malignant cells to irradiation (Shaake-Konig et al, 1994; Yapp et al, 1998). Moreover, cisplatin in a collagen gel has been reported to successfully treat a variety of localized malignant tumors. Cisplatin has been administered by direct endoscopic injection for palliation of obstructive cancer of the esophagus (Monga et al, 2000), by IT injection for head and neck cancers (Burris et al, 1998), by CAT-scan guided IT injection into malignant liver tumors (Engelmann et al, 2002) and by endoscopic IT injection into gastric tumors (Monga et al, 1998). Endobronchial intratumoral chemotherapy with cisplatin was initiated by Celikoglu and colleagues for lung cancer patients presenting with bronchial obstruction. In previously untreated inoperable lung cancer patients, satisfactory results were obtained using bronchoscopic IT injections as a neoadjuvant chemotherapy prior to irradiation. Significant relief of bronchial obstruction was seen for 75% of treated patients (Celikoglu et al, 2006a). Celikoglu and colleagues have also shown that intratumoral chemotherapy with cisplatin can result in down-staging NSCLC and enable surgical intervention (Celikoglu et al, 2006b).!

3. Post procedure clean-up of bronchoscope working channel At the completion of the bronchoscopic drug injection procedure, it is advisable to aspirate 1 liter of saline through the working channel of the bronchoscope in order to remove any remaining drug. The entire bronchoscope is then cleaned in a automated cleaner.

H. Volume of cytotoxic drug solution to be injected into tumor tissue The volume of the solution to be delivered by intratumoral injection and the dose of the drug to be administered are calculated from the estimated volume of the tumor (not according to the body weight of the patient). A volume of 0.5 ml of drug solution is usually readily injected for each cubic centimeter of the tumor volume. Exophytic or polypoid endobronchial tumor volume may be approximated from the bronchoscopic view. The volume of any existing extramural or extra bronchial component of the tumor may be estimated from previous CAT scan or EBUS by the equation (0.5xLxWxH) where “L” is the greatest length; “W” is the greatest width, and “ H” is the greatest depth or height of the tumor (Monga et al, 2000). A total solution volume of about half the estimated tumor volume is usually safely injected into the tumor tissue (at several injection sites) without leakage.

A. Cisplatin dosage for intratumoral injection at each IT injection session Cisplatin has been used in the solution form which is available for intravenous administration (Celikoglu et al, 2006a,b). Cisplatin may be utilized at a concentration of 2 or 4 mg/mL. That means, if a 0.5 mL aliquot is injected for each cc of tumor volume; 1 or 2 mg cisplatin is administered at each treatment session. Although the total dose of cisplatin delivered by intratumoral injection is based on the estimated total volume of the tumor mass, it is preferable that a total dose of not more than 40 mg of cisplatin be injected at each IT injection session (Celikoglu et al, 2006a,b).

I. Characteristics of cancer drugs used for intratumoral chemotherapy Preferred cytotoxic drugs for IT injection should not induce significant local necrotic changes in the normal mucosa, should have a normal pH of approximately 7.4, and should exhibit direct anti-neoplastic activity. Drugs that require hepatic activation (i.e. cyclophosphamide) are not suitable for intratumoral injection. For intratumoral injection, standard approved anticancer drugs in solutions for intravenous administration are used. Current research is also being devoted to the preparation of polymer-drug compositions that can enhance drug stability and prolong intratumoral activity with more limited diffusion away from the injection tumor. Injectable drug-loaded nano-meso-microspheres, liposomes, and polymer gels (i.e. collagen or alginate gels) promise to offer improved therapeutic IT efficacy in the future (Goldberg et al, 2002, 2006; Almond et al, 2003). Until such modified drugs are available for clinical use, currently approved aqueous I.V. drug solutions are useful for IT chemotherapy. Celikoglu and colleagues have studied in 1997 mixed drug regimens composed of methotrexate, bleomycin, mitoxantrone, mitomycin C, and 5-fluorouracil (5-FU) for inratumoral injection (Celikoglu et al, 1997). Celikoglu and colleagues also studied in 2003 5-FU alone at higher doses for IT treatments with satisfactory debulking results (Celikoglu and Celikoglu, 2003. Liu, has reported the use of carboplatin for intratumoral

K. Scheduling of injection sessions and duration of intratumoral chemotherapy treatment 1. For previously untreated patients For previously untreated patients, cisplatin may be administered four times at weekly intervals (on days 1, 8, 15 and 22). Three to seven days after the last session of intratumoral chemotherapy, irradiation may be initiated if deemed appropriate (Celikoglu et al, 2006ba5). In operable cases or in cases where down-staging was achieved after EITC, surgery may be carried out 3-7 days after termination of IT treatment (Celikoglu et al, 2006b). !

2. For patients previously treated by surgery, by extra-beam irradiation, or by systemic chemotherapy For such patients, IT chemotherapy is carried out at weekly intervals. If a response is obtained, EITC therapy can continue for 6-8 weeks or longer until the disappearance of the

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Celikoglu et al: Endobronchial intra tumoral chemotherapy! endobronchial tumor growth. If there is no positive response after 3 weeks of weekly EITC injections, the IT therapy may be stopped. Other endobronchial ablation techniques such as laser photoresection, electrocautery, cryotherapy may then be tried. In cases of poor response to other interventional bronchoscopic procedures, it may be appropriate to evaluate EITC.

It is reasonable to regard the following factors as contributory to the haemostasis: 1.! Vasoconstriction of small vessels in or around the tumor growth due to reaction with a very high concentrations of the cytotoxic drug; 2.! Oligemia caused by the compression of small vessels in the tumor due to the volume of drug solution injected into the restricted intratumoral volume; 3.! Occlusive intravascular thromboses in the tumor vessels. The haemostasis is advantageous in that it facilitates resection of the necrotic tumor mass with forceps and removal of tumor tissue debris (Celikoglu and Celikoglu, 2003; Celikoglu et al, 2006a,b)

3. Patient follow-up For those patients previously treated by other standard cancer therapies, if bronchial obstruction has been relieved by IT chemotherapy, follow-up bronchoscopic examinations should be performed at least every three months. Biopsy specimens of the treated areas are taken and examined by the same pathologist who examined the initial specimens. It is advisable to use autofluorescence to detect any tumor recurrence. If tumor recurrence is noted, patients may be indicated to receive additional IT treatment. The same follow-up protocol is advised after radiotherapy.

L. Effects of intratumoral cytotoxic drugs on tumor tissue

delivery

N. Removal of residual tumor debris by ablation techniques after EITC of obstructive endo-luminal tumors Although a significant reduction in tumor size may be affected by IT chemotherapy and may thereby be sufficient to alleviate symptoms, tumor size reduction in some cases may not adequately restore the airway passage as quickly as desired. Cytotoxic drug injection into the tumor kills the cancerous cells but does not immediately result in elimination of necrotic residues. Thus, in urgent life threatening situations with severe dyspnea where immediate obstruction relief is obligatory, tumor cell debris, necrotic residues, and fibrin plugs should be removed by other means. In such cases, endobronchial ablation procedures such as mechanical resection, laser photoresection, electocautery or cryotherapy should be utilized. The limited haemostatic effect provided by IT drug injection often facilitates the removal of tumor residues by mechanical resection with forceps without serious bleeding (Figure 2). Piece by piece resection with forceps can therefore be useful for debulking of tissue residues after IT endo-bronchial chemotherapy (Celikoglu and Celikoglu, 2003; Celikoglu et al, 2006a,b).

of

1. Early effects The tumor mass is usually seen through the bronchoscope as an endo-luminal protruding polypoid mass. While injecting the drug solution, the tumor around the needle becomes swollen, pale and whiter. This is probably due to edema of the tumor tissue and compression of blood vessels inside the tumor by the volume of injected solution.

2. Delayed effects Three to seven days after intratumoral drug injection, bronchoscopic examination normally reveals some reduction of the tumor mass. Tumor surfaces appear to be covered with a white-yellow gel-like substance. Histopathology indicates the presence of necrotic tissue, devitalized tumor cell residues, fibrin coagulum, dissipated secretions and white blood cells. In some cases, weeks to months later, the tumor may disappear completely leaving a grey colored scar tissue in place of the tumor (Celikoglu and Celikoglu, 2003; Celikoglu et al, 2006a,b). This necrotizing effect of locally administered cytotoxic drug is presumably due to contact and interaction of the malignant cells with the extremely high concentration of drug to which the tumor is exposed by direct drug injection (Goldberg et al, 2002, 2006). A few days after IT drug delivery the texture of the malignant tissue may appear to soften and become brittle and is then easily separated into small fragments. This is likely due to an early necrobiosis or necrosis. As a result of the early necrosis and reduction in size of the tumor mass, in some cases the diameter of the airway passage may be amplified and symptoms alleviated even after the first session of treatment. In such cases, it is also possible to observe rather rapid recovery from accompanying pathology, i.e. atelectasis or obstructive pneumonia. When the tumor growth is present as an infiltrative process with slight protrusion on the surface of the bronchial mucous membrane, the infiltrative processes improve markedly after drug injection. This infiltrative tumor growth is usually converted to a white-yellow “membrane-like� substance (Celikoglu and Celikoglu, 2003; Celikoglu et al, 2006a,b).

M. Haemostatic effect injection of cytotoxic drugs

of

O. Adverse effects of intratumoral chemotherapy During injection of the cytotoxic drug, patients report no pain or other disturbing sensations. Injection of the drug into normal mucous membranes around the malignant tissue also does not cause any important symptoms. No adverse effects such as irritation or necrosis have been noted due to contact of the drug solution with the surface of normal bronchial mucous membrane. After IT chemotherapy, transient moderate chest pain, a slight sensation of nausea, and/or a moderate fever of one day duration may be noted. No other important systemic or local side effects are observed. No systemic toxicity such as interstitial pneumonia, bone marrow suppression, worsening of inflammatory findings or hair loss has been observed. Nephrotoxicity, neurotoxicity, and myocarditis, which are commonly associated with systemic intravenous administration of drugs such as cisplatin, are also not seen (Liu et al, 2000; Celikoglu et al, 2006a,b).

P. Dispersion of drugs in the tumor mass 1. Drug distribution by diffusion The success of intratumoral chemotherapy is dependent upon relatively complete permeation of the injected malignant lesion with a very high concentration of drug with minimal rapid diffusion from the injected tumor tissue into the systemic circulation. Protocols involving repetitive injections and weekly treatments for several weeks are designed to maximize the probability that all tumor cells in the injected lesion are subjected to lethal drug concentrations. Fortunately, although small drug molecules may associate with tumor tissues by inherent tissue

intratumoral

Clinical observations indicate that intratumoral injection of cytotoxic drugs may also provide a haemostatic effect on the tumor bulk. The bleeding tendency of tumor tissues is thereby often reduced after IT drug injection. In such situations, piecemeal resection of tumor debris with forceps does not provoke further bleeding (Figure 2).

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Cancer Therapy Vol 6, page 551! affinity and by DNA binding, some diffusion away from the injection sites via the extra-cellular tumor tissue fluid can also occur to facilitate tumor perfusion by most drugs. The extent of such diffusion is proportional to ratio of the injection site drug concentration and the drug concentration in the surrounding tumor tissue. When there is a relatively uniform drug concentration within the tumor tissue, drug diffusion out of the tumor will be dependent on the rate of drug passage into the lymphatic and vascular systems. Repeated IT injections facilitate complete permeation of drug into the entire tumor mass. The drug transport process is also affected by the relative porosity of the tumor tissue with some variation in such porosity according to tumor type and size. Clinical CT scan observations have demonstrated that even when only a small part of the tumor is visible by bronchoscopy, the injection of cytotoxic drugs into an endoluminal portion of the tumor is effective in also decreasing the size of the extramural part of the tumor. This suggests that anticancer drugs injected into one portion of the tumor may diffuse throughout the tumor mass (Goldberg et al, 2002, 2006).

drug and the rate of transport into the systemic circulation is thereby so limited that no adverse systemic effects are observed.

IV. Discussion and Conclusions Reviewed here are the techniques and clinical indications for EITC. It is noted that EITC may be used as a preoperative or neoadjuvant procedure or used concomitantly or sequentially with other standard lung cancer treatments and interventional therapeutic bronchoscopic procedures. EITC is a novel therapeutic paradigm for lung cancer treatment, particularly for widespread application to malignant endobronchial tumor growths. By providing local delivery of cancer drugs, EITC achieves a significant reduction in systemic toxicity with few of the toxic side effects of systemic chemotherapy. The EITC procedure is not yet used to its full potential. Increased training for interventional pulmonologists in the techniques for bronchoscopic EITC is needed to help broaden clinical perspectives and to make clearer the therapeutic scope and opportunities for EITC. Increased attention should be focused on such less invasive and less toxic treatment strategies for lung cancer. In a recent study, Nader reported that intratumoral administration of cisplatin, as an adjunct to brachytherapy, was a safe and potentially useful modality to assist in the management of endobronchial tumor obstruction and possibly for long term efficacy. It was concluded that further studies are needed to more fully assess the benefits of this treatment (Nader, 2007). In addition to the obvious advantage of tumor burden reduction inside the airway lumen, intratumoral bronchoscopic delivery of cytotoxic drugs provides a unique loco-regional neo-adjuvant therapy for use prior to irradiation or surgery (Celikoglu et al, 2006a,b). It is chemotherapy which differs from intravenous chemotherapy only by its local route of delivery. Although the number of clinical studies to date are limited, intratumoral chemotherapy has been found to be advantageous compared to intravenous chemotherapy for effecting efficient tumor regression with minimal sideeffects. One example is a comparative clinical study by Liu and colleagues where intratumoral chemotherapy with carboplatin was combined with systemic carboplatin and found to be superior to systemic chemotherapy alone for prolonged survival of lung cancer patients (Liu et al, 2000). In addition to the development of statistically significant survival data, future clinical studies should also encompass: different drug combinations, IT drug dose response, timing of EITC injections, evaluation of drug serum levels vs. time, and mechanistic studies designed to measure T-cell/killer cell activity and tumor-specific immune response.

2. Sentinel lymph node drug perfusion (drainage of drug molecules via afferent lymphatic vessels) Sentinel lymph nodes (SLN) are the first lymphatic drainage site of a tumor and the likely site of initial metastatic tumor cell dissemination (Tiffet et al, 2005). Although the overall prognostic significance SLN micro-metastases in early lung cancer remains unclear, recent studies suggest that for larger and potentially resectable lung cancers there is a significant 5year survival advantage in patients with adenocarcinoma who do not have SLN micro-metastases when compared to patients with SLN micro-metastasis (62% with metastasis vs. 86% with no metastasis) (Wu et al, 2001). IT injected drug molecules can be transported by afferent lymphatic vessels into the sentinel and draining lymph nodes. Such drug transport to the lymph nodes may be expected to have a valuable therapeutic effect by eradication of lymphatic micrometastases. Clinical studies performed using pre-surgical radioisotopes, blue dye techniques, and even the blue cancer drug mitoxantrone, support this view of drug molecule transport from an IT injected site to sentinel and draining lymph nodes (Izbicki et al, 1996; Baitchev et al, 2001; Wu et al, 2001; Lardinois et al, 2003; Tiffet et al, 2005). As also suggested by Lardinois et al, drug injected through the bronchoscope into normal tissues around the tumor is also transported to sentinel lymph nodes. We deduce from this that EITC may have the added advantage of inhibiting metastasis by cytotoxic action on tumor cells which are migrating into the lymph nodes that drain the tumor area (Lardinois et al, 2003).

3. Low systemic concentration of IT injected drugs Although we have no human clinical data, animal studies have shown that very high doses of cytotoxic drugs (>10-30 times normal intravenous doses) are achieved within tumors by intratumoral injection. However, only minute amounts of IT drug pass into the systemic circulation (Goldberg et al, 2002, 2006). Minimal systemic drug concentrations occur because of several unique aspects of IT injection. Depending on the molecular structures of specific cancer drugs, factors such as drug-tissue affinity, drug instability, rapid drug metabolism, low plasma solubility, and low tissue diffusion rates limit the extent of drug transport into surrounding tissues and vasculature. Furthermore, a major drug transport pathway for IT injected drug molecules is via the afferent lymphatics to the sentinel lymph nodes and then leave lymph glands by efferent lymphatic vessels which ultimately empty into the thoracic duct and right lymphatic duct which then empty into the venous circulation. The amount of

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Celikoglu et al: Endobronchial intra tumoral chemotherapy! in patients with early breast carcinoma. J Chemother 13, 440-443. Burris HA 3rd, Vogel CL, Castro D, Mishra L, Schwarz M, Spencer S, Oakes DD, Korey A, Orenberg EK (1998) Intratumoral cisplatin/epinephrine-injectable gel as a palliative treatment for accessible solid tumors: a multicentric pilot study. Otolaryngol Head Neck Surg 118, 496-503. Celikoglu F, Celikoglu SI (2003) Intratumoral chemotherapy with 5-fluorouracil for palliation of bronchial cancer in patients with severe airway obstruction. J Farmacy Pharmacol 55, 1441-1448. Celikoglu F, Celikoglu SI, York AM, Goldberg EP (2006a) Intratumoral administration of cisplatin through a bronchoscope followed by irradiation for treatment of inoperable non-small cell obstructive lung cancer. Lung Cancer 51, 225-236. Celikoglu SI, Celikoglu F, Goldberg EP (2004) Intratumoral chemotherapy through a flexible bronchoscope. J Bronchol 11, 260-265. Celikoglu SI, Celikoglu F, Goldberg EP (2006b) Endobronchial intratumoral chemotherapy (EITC) followed by surgery in early non-small-cell lung cancer with polypoid growth causing erroneous impression of advanced disease. Lung Cancer 54, 339-346. Celikoglu SI, Celikoglu F, Karayel T, Demirci S, Cagatay T (1997) Direct injection of anticancer drugs into endobronchial tumors for major airway obstruction. Post Grad Med J 73, 159-162. Celikoglu SI, Karayel T, Demirci S, Celikoglu F, Serdengecti S, Gulbaran Z, Yanardag H (1991) Direct injection of several anticancer drugs into endobronchial malignant tumor in the palliation of major airway obstruction (abstract). Eur Respir J 4 (Suppl 14), 303 S. Credle WF, Smiddy JF, Elliott C (1974) Complications of fiberoptic bronchoscopy, Am Rev Resp Disease 109, 67-72. Engelmann K, Mack MG, Straub R, Eichler K, Zangos S, Orenberg E, Vogl TJ (2002) CT-guided intratumoral administration of cisplatin/epinephrine gel for treatment of malignant liver tumors. Brit J Cancer 86, 524-529. Gatzemeier U, von Pawel J, Gottfried M, ten Velde GP, Mattson K, DeMarinis F, Harper P, Salvati F, Robinet G, Lucenti A, Bogaerts J, Gallant G (2001) Phase III comperative study of high dose cisplatin versus a combination of paclitaxel and cisplatin in patients with advanced non-small lung cancer. J Clin Oncol 19, 2108-2109 Goldberg EP, Almond BA, Hadba AR, Cuevas BJ, York A, Enriquez I (2006) Nano-mesosphere drug carriers for localized cancer chemotherapy. Proc NSTI Nanotech 2, 1-4. Goldberg EP, Hadba AR, Almond BA, Marotta JS (2002) Intratumoral cancer chemotherapy and immunotherapy: opportunities for non-systemic preoperative drug delivery. J pharm Pharmacol 54, 159-180. Harper E, Dang W, Lapidus RG, Garver RI Jr (1999) Enhanced efficacy of a novel controlled release paclitaxel formulation (paclimer delivery system) for local-regional threreapy of

lung cancer tumor nodules in mice. Clin Cancer Research 5, 4242-4248. Izbicki JR, Passlick B, Hosch SB, Kubuschock B, Schneider C, Busch C, Knoefel WT, Thetter O, Pantel K (1996) Mode of spread in the early phase of lymphatic metastasis in nonsmall cell lung cancer: significance of nodal metastasis. J Thorac Cardiovasc Surg 112, 623-630. Lardinois D, Brack T, Gaspert A, Spahr T, Schneiter D, Steinert HC, Weder W (2003) Bronchoscopic radioisotope injection for sentinel lymph-node mapping in potentially resectable nonsmall-cell-lung-cancer. Eur J Cardio-Thorac Surg 23, 824-827. Liu M, Ma P, Lu Z (2000) Local chemotherapy by fibrobroncoscopy for advanced broncogenic carcinoma. Chin J Tuberc Respir Dis 23, 550-551. Monga SP, Wadleigh R, Adib H, Harmon JW, Berlin M, Mishra L (1998) Endoscopic treatment of gastric cancer with intratumoral cisplatin/epinephrine injectable gel: a case report. Gasrointest Endosc 48, 415-417. Monga SP, Wadleigh R, Sharma A, Adib H, Strader D, Singh G, Harmon JW, Berlin M, Monga DK, Mishra L (2000) Intratumoral therapy of cisplatin/epinephrine injectable gel for palliation in patients with obstructive esophageal cancer. Am J Clin Oncol (CCT) 23, 386-392. Nader AD (2007) Intratumoral chemotherapy as an adjuct to endobronch覺al brachytherapy. Chest 32, (Suppl) 459s. Sandler AB, Nemunaitis J, Denham C, von Pawel J, Cormier Y, Gatzemeier U, Mattson K, Manegold C, Palmer MC, Gregor A, Nguyen B, Niyikiza C, Einhorn LH (2000) Phase III trial gemcitabine plus cisplatin versus cisplatin alone with patients with locally advanced or metastatic non-small lung cancer. J Clin Oncol 18, 122-130. Seymour CW, Krimsky WS, Sager J, Kruklitis RJ, Lund ME, Musani AI, Sterman DH (2006) Transbronchial needle injection: A systemic review of a new diagnostic and therapeutic paradigm. Respiration 73, 78-89. Shaake-Konig C, van den Bogaert W, Daleiso O, Festen J, Hoogenhout J (1994) Radiosensitization by cytotoxic drugs. The EORTC experience by radiotherapy and lung cancer cooperative groups. Lung Cancer 10, S263-270. Tiffet O, Nicholson AG, Khaddag, Ladas G, Dubois F, Goldstraw P (2005) Feasibility of detection of sentinel lymph node in peripheral non-small lung cancer with radio isotopic and blue dye techniques. Chest 127, 443-448. Wagai F, Kinoshita M, Watanabe RSH, Kitamura S (1982) The direct injection of several anti-cancer drugs into the primary lung cancer through a fiberoptic bronchoscope. Jpn J Thorac Dis 20, 170. Wu J, Ohta Y, Minato H (2001) Nodal occult metastasis in patients with peripheral lung adenocarcinoma of 2.0 cm or less in diameter. Ann Thorac Surg 71, 1772-1778. Yapp DT, Lloyd DK, Zhu E, Lehnert SM (1998) The potentiation of the effect of radiation treatment by intratumoral delivery of cisplatin. Int J Radiat Oncol Rio Phys 42, 413-420.

From left to right: Firuz Celikoglu, Seyhan I Celikoglu, Eugene P Goldberg

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Cancer Therapy Vol 6, page 553! Cancer Therapy Vol 6, 553-562, 2008

Dosimetric comparison of tomotherapy versus 4field pelvic box altered fractionation radiotherapy treatment plans for invasive squamous cell carcinoma of the cervix Research Article

Don Yee1,*, Robert Pearcey1, George Dundas1, John Hanson2, Marc Mackenzie3, Don Robinson3, Colin Field3, Lori Underwood3, Raul Urtasun1, Nadeem Pervez1, B. Gino Fallone3 1

Department of Radiation Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada Preventive Oncology and Epidemiology, Cross Cancer Institute, Edmonton Alberta, Canada 3 Department of Medical Physics, Cross Cancer Institute, Edmonton, Alberta, Canada 2

__________________________________________________________________________________! *Correspondence: Dr. Don Yee, Department of Radiation Oncology, Cross Cancer Institute, 11560 University Avenue, Edmonton Alberta CANADA T6G 1Z2,; Tel: +1 (780) 432 8783; Fax: +1 (780) 432 8380; E-mail: donyee@cancerboard.ab.ca Key words: Tomotherapy, radiotherapy, squamous cell carcinoma of the cervix, tumour control, histogram comparison Abbreviations: computed tomography, (CT); dose volume histogram, (DVH); gross tumour volume, (GTV); Intensity modulated RT, (IMRT); megavoltage CT, (MVCT); Planning target volume one, (PTV1); radiotherapy, (RT); simultaneous integrated boost, (SIB);! tumour control probability, (TCP) Received: 9 June 2008; Revised: 29 July 2008; Accepted: 31 July 2008; electronically published: September 2008

Summary Altered fractionation radiotherapy features variations in frequency of radiotherapy delivery and/or amount of radiotherapy delivered at each treatment in an attempt to improve radiotherapy efficacy, depending on a given tumour’s biologic nature. Several altered fractionation radiotherapy clinical trials for cervix cancer have been completed, but despite promising local control rates, these trials have been unsuccessful due to excessive radiotherapy toxicities. Radiotherapy in these trials was planned using traditional 4-field box techniques. Advanced radiotherapy planning and delivery systems such as helical tomotherapy offer the potential of planning and delivering altered fractionation regimens with improved critical structure avoidance. The purpose of this project is to perform a dosimetric comparison between tomotherapy and 4-field altered fractionation radiotherapy plans for cervix cancer cases. Two planning target volumes (PTVs) were defined for ten cervix cancer cases: PTV 1 contained pelvic lymph nodes, gross tumour, uterus and parametria and PTV 2 contained gross tumour, uterus and parametria. A tomotherapy and 4-field plan was created for each case. A dose of 45 Gy/25 fractions was prescribed to PTV1 with a concomitant boost of 10 Gy to PTV2. Plans were normalized such that 95% of PTV 2 received the prescribed dose. Plans were compared based on dose volume histogram criteria, target volume conformity indices and normal tissue complication and tumour control probability estimates. Tomotherapy plans had significantly better conformity indices for PTV2 coverage. Four-field plans had significantly higher minimum, median and mean PTV1 doses. Rectal, bladder and small bowel doses were significantly lower in tomotherapy plans. PTV2 tumour control probabilities were not significantly different between planning modalities. Bladder, rectal, and small bowel normal tissue complication estimates were significantly lower for tomotherapy plans. This data indicates tomotherapy plans confer superior boost volume coverage and critical structure avoidance compared with traditional 4-field plans for locally advanced cervical cancer. Direct clinical comparisons between tomotherapy and 4-field pelvic box radiotherapy plans for cervix cancer are needed to quantify tomotherapy’s clinical benefits in terms of tumour control, radiotherapy toxicity rates and patient quality of life.

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Yee et al: Tomotherapy versus 4-field box radiotherapy treatment plans for carcinoma of the cervix toxicities compared with a similar cohort of patients treated with conventional RT (Mundt et al, 2001, 2003). More recent trials involving small numbers of cervix cancer patients treated definitively with varying IMRT techniques and concurrent chemotherapy reported promising early toxicity and locoregional control rates (Gerszten et al, 2006; Salama et al, 2006; Beriwal et al, 2007). No reports of the use of helical tomotherapy planning and delivery technology for definitive locoregional RT treatment of locally-advanced cervix cancer exist however. In previously published altered fractionation RT trials for cervix cancer, RT was planned using traditional “4-field box” techniques. No altered fractionation trials using advanced high-precision IMRT or tomotherapy for cervix cancer have been reported. Tomotherapy’s ability to deliver highly conformal RT to target structures while minimizing RT dose to adjacent normal structures may allow for planning of altered fractionation RT regimens with the potential of avoiding the excessive RT toxicities reported in previous altered fractionation trials for cervix cancer. The purpose of this study is to perform a dosimetric comparison between helical tomotherapy versus traditional 4-field box radiotherapy plans for cervix cancer cases.

I. Introduction With the incorporation of concurrent chemotherapy into radiotherapy (RT) regimens for cervix cancer, local control and overall survival rates have significantly improved (Green et al, 2001). Challenges in cervix cancer treatment remain, however. Concurrent chemoradiotherapy regimens may result in higher rates of treatment-related toxicity (Kirwan et al, 2003) and RT treatment outcomes are still limited by factors of total RT dose and overall RT treatment time (Fyles et al, 1992; Girinsky et al, 1993; Lanciano et al, 1993; Perez et al, 1995, 1998; Petereit et al, 1995). Data from these various patient cohorts indicate that higher RT doses delivered without prolonging overall treatment time improve tumour control rates for cervix cancer. Attempts to improve RT outcomes for cervix cancer with altered fractionation RT regimens designed to deliver higher total RT doses without prolonging overall treatment time have been hampered by unacceptable rates of RT toxicities (Varghese et al, 1992; Grigsby et al, 1998). Advanced RT planning and delivery systems now offer improved RT delivery accuracy. Intensity modulated RT (IMRT) technology enables the variation of RT intensity across a beam, resulting in the ability to deliver more conformal RT doses to target volumes and lower doses to surrounding normal tissues (Purdy, 1999). IMRT has been used to treat different tumours including head and neck and prostate with encouraging early results (Zelefsky et al, 2002; Lauve et al, 2004). The improved accuracy afforded by current-day IMRT technology may enable planning and delivery of altered fractionation RT regimens with less severe treatment-related toxicity profiles than those associated with altered fractionation schemes planned and delivered with more traditional RT planning systems. Helical tomotherapy represents a next-generation IMRT planning and delivery system. Helical tomotherapy features an intensity-modulated beam, which rotates around the patient as the patient is translated longitudinally through the gantry. Helical tomotherapy units also feature the capability of acquiring megavoltage CT (MVCT) images of target and normal structures at each treatment (Beavis, 2004). The capability of acquiring MVCT images with the patient on the treatment bed just prior to the actual RT treatment is felt to provide the ability to deliver extremely accurate daily RT treatments based on the daily CT image (Welsh, et al 2006). Since tomotherapy units have only recently become commercially available, published clinical experience with this technology is limited. Compared with other tumours, experience in treating cervix cancer patients with IMRT techniques is sparse. A specific commercial IMRT planning system has been shown to provide adequate coverage of pelvic node volumes with improved normal tissue sparing in cervix cancer patients compared with conventional planning techniques (Portelance et al, 2001). Early clinical reports of using IMRT to treat small numbers of selected cervix cancer patients in the post-operative setting with adjuvant doses of RT suggests that the treatments are tolerable and associated with decreased incidences of acute and late RT

II. Materials and methods The study was initiated after receiving institutional Research Ethics Committee approval. Ten patient cases with the following eligibility criteria were identified: biopsy-proven cervix cancer, treated with concurrent chemoradiotherapy, having undergone a computed tomography (CT) scan for RT planning. At the time this work was conducted, all patients with invasive cervix cancer assessed in our department underwent an examination under anesthesia, CT scan of the abdomen and pelvis and chest x-ray to stage their disease. Planning CT images were anonymized. Rectum, bladder, small bowel and gross tumour volume (GTV) [cervical tumour, uterus and involved pelvic lymph nodes] were contoured on each patient data set at an AcQ-Sim Workstation. The nodal clinical target volume (pelvic nodes at risk but not grossly involved) was defined by contouring bilateral internal and external iliac vessels with a 1 cm margin. Planning target volume one (PTV1) contained the nodal clinical target volume, parametria and GTV with a margin of 1.5 cm. PTV2 consisted of the GTV with a 1.5 cm margin. A margin of 5mm was used to define PTV1 and PTV2 at any direct interface with rectum, bladder or small bowel. All CT image sets with volumes were transferred to the department’s treatment planning system (Helax version 6.02) and Tomotherapy Hi-Art 2.0 workstation. The external beam component of a radical course of RT for each case was the focus of comparison for this study. We assume that the dosimetry from the brachytherapy component of treatment usually offered to cervix cancer patients would be identical regardless of external beam RT planning modality and therefore did not use any brachytherapy dose calculations in our comparison. A tomotherapy plan and a traditional 4-field plan were created for each patient case. Traditional 4-field plans were created using the department’s Helax version 6.02 treatment planning system. Helical tomotherapy plans were created with Tomotherapy Hi-Art 2.0 treatment planning software. For tomotherapy plans, 45 Gy in 25 daily fractions was prescribed to PTV1 while at the same time delivering a simultaneous integrated boost to PTV2 of 10 Gy (i.e. total PTV2 dose of 55 Gy in 25 fractions). Normal tissue constraints defined for tomotherapy

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Cancer Therapy Vol 6, page 555! plans included: <50% of bladder, rectum and small bowel to receive 40, 35 and 35 Gy, respectively. For 4-field plans, 45 Gy in 25 daily fractions was prescribed to PTV1 with a concomitant boost of 10 Gy in 5 fractions to PTV2. Small bowel, rectal and bladder shielding was defined by multi-leaf collimation in the 4field plans. Tomotherapy and 4-field plans for each case were normalized such that 95% of PTV2 received the prescribed dose. Plans used 6 MV photons. Radiation plans were compared using dose volume histogram (DVH) criteria and calculations of tumour control probability (TCP) for target structures and normal tissue complication probability (NTCP) for critical normal structures. Data extracted from target and normal structure DVH’s for comparison included: minimum, maximum and mean dose, along with doses to 10%, 90% and 95% (D10, D90 and D95) of a given volume. PTV1 and PTV2 conformity indices were calculated to describe the slope of the shoulder of the target volume DVH. This index is defined as:

III. Results A. Test Cases Ten cases meeting the inclusion criteria were identified from Gynecologic Oncology Clinic records. Table 1 outlines their clinical features and staging information.

B. Dose-volume histogram comparison Table 2 summarizes DVH comparison criteria for target and critical structure volumes in all treatment plans. Four-field plans had significantly higher minimal, median, mean and D10 PTV1 doses and significantly higher minimal and D10 PTV2 doses. Tomotherapy plans had significantly lower minimum, maximum, median, mean, D10, D90 and D95 bladder doses. Median, mean and D10 rectal doses were significantly lower in the tomotherapy plans. Tomotherapy plans had significantly lower maximum, median and D10 small bowel doses. PTV2 conformity indices were significantly better in tomotherapy plans. Figures 1-5 present average DVH’s for target and critical structures for both 4-field (created with Helax system) and tomotherapy plans.

"!D90 ! D10 %! &! 1! #! $! median '! where values closest to 1 indicate increased conformity. TCP and NTCP estimates were calculated using Warkentin’s module (Warkentin et al, 2004). This module calculates NTCP and TCP based on two NTCP models (Lyman’s dose response model and individual and population-based variants of the critical volume model) and two TCP models (a Poisson-based and a linear-quadratic-based model).

C. Normal tissue complication tumour control probability

! A. Statistics

Table 3 summarizes mean NTCP and TCP estimates for target and critical structure volumes in 4-field and tomotherapy plans. The mean TCP estimate for PTV1 was significantly higher for 4-field plans. The mean TCP estimate for PTV2 was not significantly different between tomotherapy and 4-field plans. The mean bladder NTCP value calculated using the CV (pop) model was significantly lower for tomotherapy plans. Mean rectal NTCP calculated using the Lyman model and small bowel NTCP calculated with both Lyman and CV (pop) models were significantly lower for tomotherapy plans.

Descriptive statistics were used to describe clinical and staging features of the 10 test cases and criteria means with 95% confidence intervals calculated for the 4-field and tomotherapy plans. Mean differences between DVH criteria were analyzed using two-tailed paired t-tests with a p-value less than 0.05 considered significant. SAS software (version 9.1, SAS Institute, Cary, NC) was used for the analysis.

! Table 1. Staging and clinical features of test cases. Case ID Test Case 1 Test Case 2

FIGO Stage IB2 IIIB

Test Case 3 Test Case 4

IB2 IIB

Test Case 5

IIB

Test Case 6 Test Case 7

IIIB IIB

Test Case 8 Test Case 9

IIA IIB

Test Case 10

IB2

and

Clinical Details Barrel-shaped cervix Circumferential involvement of cervix with bilateral extension to pelvic sidewall, bilateral hydronephrosis, right external iliac lymphadenopathy 4 cm lesion confined to cervix 5x4 cm tumor with extension to bilateral parametrial tissues, proximal vagina with bilateral pelvic lymphadenopathy Lesion on posterior lip of cervix with extension to left parametrial tissues 6 cm cervical mass with extension to left pelvic sidewall Bilateral parametrial extension with left pelvic lymphadenopathy 5 cm cervix mass with extension to right lateral fornix Tumor replaces entire cervix with extension to left parametrium 4.5 cm lesion confined to cervix

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Yee et al: Tomotherapy versus 4-field box radiotherapy treatment plans for carcinoma of the cervix Table 2. Averaged dose-volume histogram criteria for target and normal tissue volumes in conventional and tomotherapy plans. Structure

DVH Criteria

Conventional Mean (95% CI)

PTV1

Minimum Maximum Median Mean D10 D90 D95 Conformity Index Minimum Maximum Median Mean D10 D90 D95 Conformity Index Minimum Maximum Median Mean D10 D90 D95 Minimum Maximum Median Mean D10 D90 D95 Minimum Maximum Median Mean D10 D90 D95

43.8 (43.2 - 44.4) 58.2 (57.7 - 58.7) 54.1 (53.1 - 55.2) 53.2 (52.4 - 54.0) 56.7 (56.5 - 57.0) 47.7 (46.0 - 49.3) 46.5 (45.0 - 48.1) 1.168 (1.136-1.200) 53.9 (53.4 - 54.5) 58.1 (57.7 - 58.6) 56.2 (55.9 - 56.5) 56.2 (55.9 - 56.5) 57.0 (56.7 - 57.3) 55.3 (55.0 - 55.6) 55.0 (54.7 - 55.4) 1.030(1.026-1.035) 32.6 (27.2 - 38.1) 57.1 (56.5 - 57.7) 52.9 (51.6 - 54.1) 51.0 (49.0 - 53.1) 56.0 (55.6 - 56.5) 43.2 (38.1 - 48.2) 39.0 (34.4 - 43.6) 11.8 (1.4 - 22.3) 57.4 (57.0 - 57.9) 54.8 (53.5 - 56.1) 46.2 (41.1 - 51.2) 56.9 (56.5 - 57.4) 21.1 (6.7 - 35.5) 15.4 (1.8 - 28.9) 3.1 (2.0 - 4.2) 57.7 (57.2 - 58.1) 42.0 (36.4 - 47.6) 39.4 (35.8 - 43.1) 55.5 (54.6 - 56.3) 19.8 (14.1 - 25.5) 14.3 (8.8 - 19.8)

PTV2

Bladder

Rectum

Small Bowel

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Tomotherapy Mean (95% CI) 39.2 (37.3 - 41.2) 57.8 (57.0 - 58.6) 47.5 (46.8 - 48.2) 48.6 (48.0 - 49.2) 53.2 (52.4 - 54.0) 46.0 (45.7 - 46.3) 45.7 (45.4 - 46.0) 1.152 (1.136-1.168) 48.6 (46.5 - 50.7) 57.7 (57.1 - 58.3) 56 (55.7 - 56.2) 55.9 (55.7 - 56.1) 56.3 (56.0 - 56.6) 55.3 (55.1 - 55.4) 55.0 (55.0 - 55.0) 1.019(1.015-1.023) 19.6 (15.3 - 23.8) 56.5 (56 - 57.1) 38.5 (34.1 - 43.0) 38.9 (35.2 - 42.6) 53.3 (52 - 54.7) 25.3 (20.7 - 29.8) 23.3 (18.9 - 27.7) 7.4 (3.6 - 11.3) 56.8 (56.2 - 57.4) 41.7 (38.9 - 44.5) 37.5 (34.3 - 40.7) 53.6 (52.5 - 54.6) 14.5 (7.1 - 21.9) 11.6 (4.9 - 18.2) 11.8 (8.5 - 15.1) 56.8 (56.1 - 57.5) 36.2 (34.3 - 38) 36.6 (35.1 - 38.2) 52.2 (50.6 - 53.8) 21.0 (17.9 - 24.0) 18.3 (14.9 - 21.8)

p value 0.0008 0.3422 <0.0001 <0.0001 <0.0001 0.0413 0.2271 0.4040 <0.0001 0.2399 0.2565 0.1160 0.0019 0.7175 0.5871 <0.0001 <0.0001 0.0364 <0.0001 <0.0001 0.0015 <0.0001 <0.0001 0.2915 0.1284 <0.0001 0.0002 0.0002 0.1155 0.2951 <0.0001 0.0478 0.0463 0.1086 0.0007 0.5562 0.0697

Cancer Therapy Vol 6, page 557!

Figure 1. Averaged PTV1 DVH’s. (tomo=tomotherapy)

Figure 2. Averaged PTV2 DVH’s. Figure DVH’s.

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3.

Averaged

bladder

Yee et al: Tomotherapy versus 4-field box radiotherapy treatment plans for carcinoma of the cervix Figure 4. Averaged rectal DVH’s.

Figure 5. Averaged small bowel DVH’s.

techniques offer significant dosimetric advantages in terms of dose conformity and critical structure avoidance when compared to traditional 4-field techniques. Though one would expect that tomotherapy technology would be able to plan more conformal treatments with improved normal structure avoidance compared with traditional 4-field techniques, this project quantifies these dosimetric improvements for a simulated accelerated course of RT for cervix cancer. The feasibility of using other intensity-modulated techniques to plan simultaneous integrated boost (SIB) RT for gynecologic tumours has been demonstrated (Guerrero et al, 2005). The dosimetric superiority of helical tomotherapy techniques over a dynamic IMRT technique for head and neck cancer cases has been demonstrated (van Vulpen et al, 2005). Whether or not other intensity-modulated techniques would provide the same magnitude of dosimetric benefits conferred by tomotherapy for the clinical situations used for this study is unknown but is under investigation by our group. Tomotherapy allows for delivery of differential daily doses of RT to target volumes that lie within larger target volumes. This in effect delivers a true SIB to areas of

IV. Discussion Guided by the radiobiologic principles of shortening overall treatment time and RT dose escalation to increase tumour control probability and decrease tumour cell repopulation, several altered fractionation RT trials for cervix cancer have been conducted in which RT was planned with traditional 4-field techniques (Faria and Ferrigno, 1997; Kavanagh et al, 1997; Grigsby et al, 1998; 2002; Macleod et al, 1999; Viswanathan et al, 1999; Chun et al, 2000). The promising local control rates reported in each of these trials support the radiobiologic rationale behind such regimens, but the reported local control gains were unfortunately offset by unacceptably high RT toxicity rates. Advanced RT planning and delivery technology may provide a means to achieve dose escalation and altered fractionation objectives for cervix cancer without causing excessive RT toxicities observed in previous altered fractionation RT clinical trials which used traditional RT planning and delivery technologies. This study reports the first dosimetric comparison between helical tomotherapy and traditional 4-field box altered fractionation RT plans for cervix cancer. Our data indicate tomotherapy 558

Cancer Therapy Vol 6, page 559! the relative differences seen in the normal structure NTCP estimates between the two planning modalities suggests that tomotherapy plans may provide improved therapeutic ratios compared with their traditional 4-field counterparts in this study. This hypothesis will ultimately need testing in future clinical trials. Our data quantifies the benefits of tomotherapy over 4-field box radiotherapy plans for cervix cancer in terms of anticipated radiotherapy doses to defined target volumes and adjacent critical structures and relative estimates of tumour control and normal tissue complication probabilities. Future direct clinical comparisons of these two treatment planning modalities in appropriately-powered randomized clinical trials are required to assess whether or not the dosimetric and theoretical radiobiologic advantages we report here translate into true clinical benefits for patients in terms of improved local tumour control and cure rates, lower radiotherapy-related toxicity rates, and maintenance of patient-related quality of life. These future direct clinical comparisons will require proper stratification for patientand tumour-related prognosticators such as FIGO stage to assess whether tomotherapy technology confers varying magnitudes of clinical benefits for different patient subgroups. While our data supplies the dosimetric support to proceed with using tomotherapy for cervix cancer patients, several issues need addressing prior to clinical implementation. These issues include: 1) Uterine and cervix movement and cervix tumour regression during a course of external beam RT (Huh et al, 2004; Lee et al, 2004). The effect of this movement and regression on very conformal tomotherapy dose distributions is likely to be significant but has not been rigorously studied. With tomotherapy’s ability to acquire daily MVCT images of target volume location/size, treatment portals may be adjusted during a course of RT based on tumour regression rates provided by MVCT imaging. Ideal methodology for the timing and magnitude of such adjustments is yet to be defined. 2) Target volume definition. Our study was done using target volumes defined on CT image sets. The resolution of CT scans may not enable complete delineation of all gross disease in the cervix or parametria. While most authors investigating advanced RT delivery techniques to treat pelvic nodal targets use the pelvic vessels plus a uniform margin to define the pelvic nodal volume, no consensus regarding the definition of this volume exists. Improved definition of pelvic nodal locations may allow for smaller margins to be placed around pelvic vessels (Taylor et al, 2005), reducing the treatment volume. Our group is actively investigating the feasibility of using MRI for cervix cancer treatment planning, as future clinical assessments of tomotherapy for cervix cancer will require accurate target volume definition to ensure that the highly-conformal radiotherapy dose distributions provided by tomotherapy are delivered to all actual sites of disease. 3) The ideal altered dose/fractionation regimen for cervix cancer is still unknown. Our study does show that modest external beam dose escalation with acceptable

gross disease that lie within a larger volume at risk for harbouring micrometastases without having to deliver two separate fractions of RT per day. The SIB technique decreases the overall treatment time, which theoretically reduces the effects of accelerated tumour cell repopulation. Delivering higher doses per fraction to areas of gross disease would also theoretically increase the radiobiologic enhancement effects of concurrent cisplatin chemotherapy (Kavanagh et al, 2002). The concomitant boost technique has been shown in a randomized setting to improve locoregional control for head and neck squamous cell carcinoma patients (Fu et al, 2000). Dose escalation using a SIB technique may enhance tumour response and shrinkage to provide better geometry for the intracavitary brachytherapy portion of cervix cancer treatments. This may be advantageous for bulky locally advanced tumours which decrease brachytherapy insert quality due to suboptimal geometry. This may translate into more precise dose delivery to gross tumour involving the cervix and parametria and clinically improved local control, disease-free and overall survival. Four-field plans created on this study had significantly higher PTV1 doses and TCP estimates. The significantly higher PTV1 doses in the 4-field plans may be a reflection of the greater dose heterogeneity throughout PTV1 in these plans as demonstrated in Figure 1. This increased dose heterogeneity is likely a result of the inferior boost dose conformity around PTV2 in the 4-field plans. The significantly higher PTV1 TCP values should be interpreted cautiously, as the parameters incorporated into the NTCP/TCP module we used were derived from empiric data describing probability of controlling macroscopic disease (Okunieff et al, 1995). If criteria from this same empiric database were used, the tomotherapy mean PTV1 dose of 48.6 Gy would satisfy the requirement to control microscopic disease. Though small bowel NTCP estimates favoured tomotherapy plans, some small bowel DVH criteria only demonstrated non-significant trends favouring tomotherapy plans. The lack of statistical significance in some of the small bowel DVH values may be due to the low case numbers but may also be a result of the anatomic relationship between small bowel and pelvic node volumes. A feature of tomotherapy delivery is that small regions immediately superior and inferior to the treated region receive radiation as the treatment starts and finishes. The extra length treated is (1-pitch)* jaw width (15 mm in our plans) and may have contributed to our small bowel DVHs. Realistically, the small bowel likely shifts position during a course of RT which in effect would spread a lower dose of RT over a larger volume of small bowel than is depicted in a DVH. Daily MVCT imaging and dose re-construction capabilities available on tomotherapy software would produce DVHs which are more representative of the actual doses delivered to a given volume. The ability of biologic models to accurately and reliably select more clinically effective RT plans has not been established. While our absolute TCP and NTCP values may not be directly applicable to clinical situations,

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Yee et al: Tomotherapy versus 4-field box radiotherapy treatment plans for carcinoma of the cervix Stevens R, Rotman M, Gaffney D (2002) Long-term followup of RTOG 88-05: twice-daily external irradiation with brachytherapy for carcinoma of the cervix. Int J Radiat Oncol Biol Phys 54, 51-57. Grigsby PW, Lu JD, Mutch DG, Kim RY, Eifel PJ (1998) Twice-daily fractionation of external irradiation with brachytherapy and chemotherapy in carcinoma of the cervix with positive para-aortic lymph nodes: Phase II study of the Radiation Therapy Oncology Group 92-10. Int J Radiat Oncol Biol Phys 41, 817-822. Guerrero M, Li XA, Ma L, Linder J, Deyoung C, Erickson B (2005) Simultaneous integrated intensity-modulated radiotherapy boost for locally advanced gynecological cancer: radiobiological and dosimetric considerations. Int J Radiat Oncol Biol Phys 62, 933-939. Huh SJ, Park W, Han Y (2004) Interfractional variation in position of the uterus during radical radiotherapy for cervical cancer. Radiother Oncol 71, 73-79. Kavanagh BD, Gieschen HL, Schmidt-Ullrich RK, Arthur D, Zwicker R, Kaufman N, Goplerud DR, Segreti EM, West RJ (1997) A pilot study of concomitant boost accelerated superfractionated radiotherapy for stage III cancer of the uterine cervix. Int J Radiat Oncol Biol Phys 38, 561-568. Kavanagh BD, Schefter TE, Wu Q, Tong S, Newman F, Arnfield M, Benedict SH, McCourt S, Mohan R (2002) Clinical application of intensity-modulated radiotherapy for locally advanced cervical cancer. Semin Radiat Oncol 12, 260-271. Kirwan JM, Symonds P, Green JA, Tierney J, Collingwood M, Williams CJ (2003) A systematic review of acute and late toxicity of concomitant chemoradiation for cervical cancer. Radiother Oncol 68, 217-226. Lanciano RM, Pajak TF, Martz K, Hanks GE (1993) The influence of treatment time on outcome for squamous cell cancer of the uterine cervix treated with radiation: a patternsof-care study. Int J Radiat Oncol Biol Phys 25, 391-397. Lauve A, Morris M, Schmidt-Ullrich R, Wu Q, Mohan R, Abayomi O, Buck D, Holdford D, Dawson K, Dinardo L, Reiter E (2004) Simultaneous integrated boost intensitymodulated radiotherapy for locally advanced head-and-neck squamous cell carcinomas: II--clinical results. Int J Radiat Oncol Biol Phys 60, 374-387. Lee CM, Shrieve DC, Gaffney DK (2004) Rapid involution and mobility of carcinoma of the cervix. Int J Radiat Oncol Biol Phys 58, 625-630. MacLeod C, Bernshaw D, Leung S, Narayan K, Firth I (1999) Accelerated hyperfractionated radiotherapy for locally advanced cervix cancer. Int J Radiat Oncol Biol Phys 44, 519-524. Mundt AJ, Mell LK, Roeske JC (2003) Preliminary analysis of chronic gastrointestinal toxicity in gynecology patients treated with intensity-modulated whole pelvic radiation therapy. Int J Radiat Oncol Biol Phys 56, 1354-1360. Mundt AJ, Roeske JC, Lujan AE, Yamada SD, Waggoner SE, Fleming G, Rotmensch J (2001) Initial clinical experience with intensity-modulated whole-pelvis radiation therapy in women with gynecologic malignancies. Gynecol Oncol 82, 456-463. Okunieff P, Morgan D, Niemierko A, Suit HD (1995) Radiation dose-response of human tumors. Int J Radiat Oncol Biol Phys 32, 1227-1237. Perez CA, Grigsby PW, Castro-Vita H, Lockett MA (1995) Carcinoma of the uterine cervix. I. Impact of prolongation of overall treatment time and timing of brachytherapy on outcome of radiation therapy. Int J Radiat Oncol Biol Phys 32, 1275-1288. Perez CA, Grigsby PW, Chao KS, Mutch DG, Lockett MA (1998) Tumor size, irradiation dose, long-term outcome of carcinoma of uterine cervix. Int J Radiat Oncol Biol Phys

normal structure DVH’s is achievable with tomotherapy, but was not designed to define the optimal altered dose/fractionation regimen. Different altered dose/fractionation regimens designed and delivered with innovative high-precision RT planning and delivery technologies warrant clinical evaluation. We conclude that our study supplies dosimetric rationale for the use of tomotherapy to plan radiobiologically advantageous RT plans for cervix cancer. Clinical implementation of tomotherapy will help determine whether or not the dosimetric and estimated radiobiologic advantages conferred by tomotherapy plans we report in this preliminary work translate into improved local control and cure rates, lower treatment-related toxicity rates and better quality of life for cervix cancer patients.

Acknowledgements This work was supported by a grant from the Alberta Cancer Board.

References Beavis AW (2004) Is tomotherapy the future of IMRT? Br J Radiol 77, 285-295. Beriwal S, Gan GN, Heron DE, Selvaraj RN, Kim H, Lalonde R, Kelley JL, Edwards RP (2007) Early clinical outcome with concurrent chemotherapy and extended-field, intensitymodulated radiotherapy for cervical cancer. Int J Radiat Oncol Biol Phys 68, 166-171. Chun M, Kang S, Ryu H, Chang K, Oh Y, Ju H, Lee E (2000) Modified partial hyperfractionation in radiotherapy for bulky uterine cervical cancer: reduction of overall treatment time. Int J Radiat Oncol Biol Phys 47, 973-977. Faria SL, Ferrigno R (1997) Hyperfractionated external radiation therapy in stage IIIB carcinoma of uterine cervix: a prospective pilot study. Int J Radiat Oncol Biol Phys 38, 137-142. Fu KK, Pajak TF, Trotti A, Jones CU, Spencer SA, Phillips TL, Garden AS, Ridge JA, Cooper JS, Ang KK (2000) A Radiation Therapy Oncology Group (RTOG) phase III randomized study to compare hyperfractionation and two variants of accelerated fractionation to standard fractionation radiotherapy for head and neck squamous cell carcinomas: first report of RTOG 9003. Int J Radiat Oncol Biol Phys 48, 7-16. Fyles A, Keane TJ, Barton M, Simm J (1992) The effect of treatment duration in the local control of cervix cancer. Radiother Oncol 25, 273-279. Gerszten K, Colonello K, Heron DE, Lalonde RJ, Fitian ID, Comerci JT, Selvaraj RN, Varlotto JM (2006) Feasibility of concurrent cisplatin and extended field radiation therapy (EFRT) using intensity-modulated radiotherapy (IMRT) for carcinoma of the cervix. Gynecol Oncol 102, 182-188. Girinsky T, Rey A, Roche B, Haie C, Gerbaulet A, Randrianarivello H, Chassagne D (1993) Overall treatment time in advanced cervical carcinomas: a critical parameter in treatment outcome. Int J Radiat Oncol Biol Phys 27, 10511056. Green JA, Kirwan JM, Tierney JF, Symonds P, Fresco L, Collingwood M, Williams CJ (2001) Survival and recurrence after concomitant chemotherapy and radiotherapy for cancer of the uterine cervix: a systematic review and meta-analysis. Lancet 358, 781-786. Grigsby P, Winter K, Komaki R, Marcial V, Eifel P, Doncals D,

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Cancer Therapy Vol 6, page 561! 41, 307-317. Petereit DG, Sarkaria JN, Chappell R, Fowler JF, Hartmann TJ, Kinsella TJ, Stitt JA, Thomadsen BR, Buchler DA (1995) The adverse effect of treatment prolongation in cervical carcinoma. Int J Radiat Oncol Biol Phys 32, 1301-1307. Portelance L, Chao KS, Grigsby PW, Bennet H, Low D (2001) Intensity-modulated radiation therapy (IMRT) reduces small bowel, rectum, bladder doses in patients with cervical cancer receiving pelvic and para-aortic irradiation. Int J Radiat Oncol Biol Phys 51, 261-266. Purdy JA (1999) 3D treatment planning and intensity-modulated radiation therapy. Oncology (Williston Park) 13, 155-168. Salama JK, Mundt AJ, Roeske J, Mehta N (2006) Preliminary outcome and toxicity report of extended-field, intensitymodulated radiation therapy for gynecologic malignancies. Int J Radiat Oncol Biol Phys 65, 1170-1176. Taylor A, Rockall AG, Reznek RH, Powell ME (2005) Mapping pelvic lymph nodes: guidelines for delineation in intensitymodulated radiotherapy. Int J Radiat Oncol Biol Phys 63, 1604-1612. van Vulpen M, Field C, Raaijmakers CP, Parliament MB, Terhaard CH, MacKenzie MA, Scrimger R, Lagendijk JJ, Fallone BG (2005) Comparing step-and-shoot IMRT with dynamic helical tomotherapy IMRT plans for head-and-neck cancer. Int J Radiat Oncol Biol Phys 62, 1535-1539.

Varghese C, Rangad F, Jose CC, Raveendran P, Subhashini J, Ramadas K, Idicula J, Pais A, Roul RK (1992) Hyperfractionation in advanced carcinoma of the uterine cervix: a preliminary report. Int J Radiat Oncol Biol Phys 23, 393-396. Viswanathan FR, Varghese C, Peedicayil A, Lakshmanan J, Narayan VP (1999) Hyperfractionation in carcinoma of the cervix: tumor control and late bowel complications. Int J Radiat Oncol Biol Phys 45, 653-656. Warkentin B, Stavrev P, Stavreva N, Field C, Fallone BG (2004) A TCP-NTCP estimation module using DVHs and known radiobiological models and parameter sets. J Appl Clin Med Phys 5, 50-63. Welsh JS, Lock M, Harari PM, Tome WA, Fowler J, Mackie TR, Ritter M, Kapatoes J, Forrest L, Chappell R, Paliwal B, Mehta MP (2006) Clinical implementation of adaptive helical tomotherapy: a unique approach to image-guided intensity modulated radiotherapy. Technol Cancer Res Treat 5, 465-479. Zelefsky MJ, Fuks Z, Leibel SA (2002) Intensity-modulated radiation therapy for prostate cancer. Semin Radiat Oncol 12, 229-237.

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Cancer Therapy Vol 6, page 563! Cancer Therapy Vol 6, 563-570, 2008

Alterations in antioxidant enzyme activities and increased oxidative stress in cyclophosphamide- induced hemorrhagic cystitis in the rat Research Article

Premila Abraham1,*, Indirani Kanakasabapathy 2, Preethi kulothungan1 1 2

Department of Biochemistry Departments of Anatomy, Christian Medical College, Bagayam, Vellore 632002, Tamil Nadu, India

__________________________________________________________________________________! *Correspondence: Dr Premila Abraham, Associate Professor, Department of Biochemistry; Christian Medical College, Bagayam, Vellore 632002, Tamil Nadu, India Tel: +91-416-2284267, +91-416-2284458; Fax: +91-416-2262788; E-mail: premilaabraham@yahoo.com, premilaabraham@cmcvellore.ac.in Key words: cyclophosphamide; hemorrhagic cystitis; Oxidative stress; Myeloperoxidase; Antioxidant enzymes Abbreviations: 1-chloro-2, 4-dinitrobenzene, (CDNB); cyclophosphamide, (CP); glutathione peroxidase, (GPO); Glutathione S transferase, (GSTase); haematoxylin and eosin, (H & E); hemorrhagic cystitis, (HC); malondialdehye, (MDA); Protein carbonyl, (Pco); Reactive Oxygen species, (ROS); Superoxide dismutase, (SOD) Received: 16 July 2008; Revised: 4 August 2008 Accepted: 7 August 2008; electronically published: September 2008

Summary The mechanism of cyclophosphamide (CP) induced hemorrhagic cystitis (HC) is not clear although the production of acrolein is suggested to be involved. It has been demonstrated that detoxifying acrolein does not prevent HC symptoms completely. This suggests that acrolein production may not be the sole cause of HC; other mechanisms may be involved. It is important to verify the mechanism of cyclophosphamide induced bladder damage in order to perform cancer chemotherapy effectively by minimising the side effect. Our aim is to verify whether there is any alteration in the activities of the antioxidant enzymes and oxidative stress in cyclophosphamide induced HC, using a rat model. Adult male rats were administered a single injection of CP at the dose of 150/ kg body wt intraperitoneally and sacrificed 6 hours or 16 hours after the dose of CP. Vehicle treated rats served as control. The bladder was used for light microscopic studies and biochemical studies. Myeloperoxidase activity, a marker of neutrophil infiltration was measured in bladder homogenates. The markers of oxidative damage including protein carbonyl content, protein thiol, malondialdehyde and conjugated dienes were assayed in the homogenates. The activities of the antioxidant enzymes, superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase and glutathione S transferase were assayed in the bladder . Six-hour after treatment with CP the mucosa became edematous and the cells of the urothelium were not compact. Cellular exudates were observed in the lumen. The condition became worse in sixteen hours, wherein edema of lamina propria with epithelial and sub-epithelial hemorrhage was seen. Myeloperoxidase activity was increased significantly 6 hours after treatment with CP and increased further at 16 hours. All the parameters of oxidative stress that were studied were significantly elevated. The activities of the antioxidant enzymes were significantly lowered. The results of the present study suggest that alteration in the activities of antioxidant enzymes and oxidative stress is responsible, at least in part, for CP induced bladder damage. It is suggested that CP treatment of rats induces oxidative stress in the urinary bladder, depletion of antioxidant enzymes, and the oxidative stress contributes to neutrophil infiltration into bladder and hence inflammation.

receiving a high intravenous dose. The urological side effects vary from transient irritative voiding symptoms to life-threatening HC. The urotoxicity of these nitrogen mustard cytostatics is believed to be based on the formation of 4-hydroxy metabolites, in particular, renal excretion of acrolein, which is formed from hepatic microsomal enzymatic hydroxylation. Mesna, an acrolein binding and detoxifying compound within the urinary collecting system, has been widely used as an effective

I. Introduction Cyclophosphamide is widely used in the treatment of solid tumors and B cell malignant disease, such as lymphoma, myeloma, chronic lymphocytic leukemia and Waldenstrom’s macroglobulinemia. Hemorrhagic cystitis (HC) is a major dose-limiting side effect of cyclophosphamide (CP) and ifosfamide (Levine and Richie, 1989). The incidence of this side effect is related to the dosage and can be as high as 75% in patients 563

Abraham et al: Alterations in antioxidant enzyme activities and increased oxidative stress in cyclophosphamide bladder was used for biochemical assays and another part for histological assessment.

agent against CP induced cystitis, but significant HC is still being encountered clinically (Brock et al, 1981; West, 1997). Since detoxifying acrolein does not remove symptoms of HC completely, it is proposed that mechanisms other than direct contact of acrolein with bladder mucosa may also be involved in CP induced HC. It is important to elucidate the mechanism of CP induced HC in order minimize the toxic and dose limiting side effect of CP. Elimination of the side effects of CP can lead to better tolerance of the drug and a more efficient and comfortable therapy can be achieved for patients in need of CP treatment. Reactive oxygen species (ROS) production in inflammatory states is considered to play an important role in the initiation and progression of tissue injury. Inflammatory cells such as macrophages, neutrophils and monocytes produce ROS which react with macromolecules such as lipids, proteins and nucleic acids because of their high reactivity, causing tissue injury. The cellular antioxidant systems helps to minimize ROS induced tissue injury. These include enzymes such as superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase and glutathione S transferase as well as non-enzymatic anti-oxidants as glutathione, protein thiol, ascorbic acid and tocopherol (Babiak et al, 1998). The present study focused on oxidative stress and alteration in antioxidant enzymes and neutrophil infiltration in order to elucidate the mechanism of CP induced cystitis. To this end, a time course study was carried out in order to study the effect of CP on the histology of the urinary bladder and markers of oxidative stress, antioxidant enzyme activities and neutrophil infiltration. The results of the present study demonstrate that CP induced hemorrhagic cystitis is associated with depletion of antioxidant enzymes, increased oxidative stress, and neutrophil infiltration.

D. Histology The tissues were fixed overnight in 10 % buffered neutral formalin, processed to paraffin wax, sectioned at 5 !m, and stained with haematoxylin and eosin (H & E) for examination by light microscopy.

E. Biochemical assays Bladder tissue was weighed and homogenized appropriate buffers and used for the following assays.

in

1. Myeloperoxidase (Wallace et al, 1989) Myeloperoxidase activity was measured with Odianisidine- H O assay. The rate of decomposition of H2 O2by myeloperoxidase was determined by measuring the rate of color development at 460 nm. To 10!l of sample, 11!l of H O, 17!l of O- dianisidine and 962 !l of phosphate buffer were added and the color read at 460nm at an interval of 30 seconds for 4 minutes and the rate of change/minute was determined. Extinction coefficient of 1.13 X 104 cm -1 was used for the calculation. One unit is the amount of enzyme decomposing 1!mole of peroxide per minute.

2. Malondialdehyde Malonaldehyde content was measured as described by Ohkawa and colleagues in 1979.The mixture consisted of 0.8 ml of sample (1mg), 0.2 ml of 8.1 % SDS, 1.5 ml of 20 % glacial acetic acid adjusted to pH 3.5, and 1.5 ml of 0.8 % aqueous solution of TBA. The mixture was made up to 4ml with distilled water and heated at 95 C for 60 min using a glass ball as condenser. After cooling with tap water, 1ml distilled water and 5ml n-butanol and pyridine mixture (15:1) were added and the solution was shaken vigorously. After centrifugation at 2000g for 10 minutes the absorbance of the organic layer was measured at 532nm. Amount of thiobarbituric reacting substances formed is calculated from standard curve prepared using 1, 1’, 3, 3’ tetramethoxy propane and the values expressed as nmoles per mg protein.

II. Materials and methods A. Animals

3. Conjugated diene

Adult male Wistar rats (200-250g) were used for the experiments. The study was approved by animal ethics Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA), Government of India. The guidelines were followed. Dosage and route of administration of cyclophosphamide (CP) were determined from that described in literature (Ahluwalia et al, 1994).

Total lipids were extracted as described by Chan and Levett, in 1977 and evaporated to dryness under nitrogen. This was dissolved in 1 ml heptane and the absorbance was measured at 233nm. The amount of conjugated diene formed in the sample is calculated using a molar absorption co-efficient of 2.52 x104.

4. Protein carbonyl content Protein carbonyl content was measured using DNPH as described by Sohal and colleagues in 1993.To 0.5 ml of sample (1-2mg), an equal volume of 10 mM DNPH in 2 N HCl was added and incubated for 1 hr shaking intermittently at room temperature. Corresponding blank was carried out by adding only 2N HCL to the sample. After incubation, the mixture was precipitated with 10 % TCA (final concentration) and centrifuged. The precipitate was washed twice with ethanol:ethylacetate (1:1) and finally dissolved in 1 ml of 6 M guanidine HCl, centrifuged at low speed and the supernatant was read at 366nm. The difference in absorbance between the DNPH treated and HCl treated sample is determined and expressed as nmoles of carbonyl groups per mg of protein, using extinction co-efficient of 22 mM-1cm1.

B. Animal treatment The rats were divided into three groups and were treated as follows. The rats in group I (n = 8) received a single intraperitoneal injection of CP in saline at the dose of 150 mg/kg body weight .The rats in group II (n=8) received a single intraperitoneal injection of CP in saline at the dose of 150 mg/kg body weight. The rats (n=6) in group III received saline alone as a vehicle control. The rats in group I were killed 6 hours after the dose of CP and the rats in group II were killed 16 hour after the dose of CP.

C. Tissue procurement Rats were killed by exsanguination. The urinary bladder was removed and blotted dry before weighing. A part of the

5. Protein thiol groups Thiol groups were measured as described by Sedlak and Lindsay in 1968. To 1 ml of the sample suspension (1 mg

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Cancer Therapy Vol 6, page 565! protein/ml), 1 ml of 10 % TCA containing 1 mM EDTA was added. The protein precipitate was separated by high speed centrifugation for 10 min. For total thiol estimation the sample was taken directly with out precipitation. To this, 1 ml of solution I and 0.5 % SDS were added followed by 2 ml of solution II and 30 !l of DTNB. The tubes were mixed well and kept in the dark for 15 min at room temperature. The intense yellow colour of the nitromercapto benzoate anion formed from the DTNB reaction with the thiol was read at 412 nm which -1 1has a molar absorption of 13,600 mM-1cm1.

ml with water. Reaction was started by adding 0.2 ml of H2O2 and change in OD at 340 nm was followed. Extinction coefficient of 6.1 mm-1 was used for the calculation. One unit is the amount needed to oxidize 1 nmole of NADPH/min.

F. Statistical Analysis The results are expressed as mean Âą S.D. Comparison between groups was done using ANOVA. P value of < 0.05 was considered as statistically significant.

III. Results

6. Assay of anti-oxidant enzyme activities

A. Histology (Figure 1)

i. Superoxide dismutase Superoxide dismutase was measured as described by Ohkuma and colleagues in 1982. The assay mixture consisted of 100!l ofphosphate buffer, 10!l of BSC, 50!lof Triton X-100, 5!l of EDTA, 5!l of xanthine oxidase, 50!l ofxanthine is added. To this finally 150 !l MTT and sample (50-150 !g protein) were added and, the volume is made up to 1 ml with water. The mixture was incubated for 5 minutes at room temperature (30°C) and the reaction was terminated with the addition of 1ml of stop buffer. This was read at 540nm. Amount of superoxide formed is calculated using the molar extinction coefficient of MTT formazan -of 17,000 M-1cm-1 at pH 7.4 to 10.5. The percentage of inhibition by the presence of SOD is calculated 540from the reduction of the MTT colour formation as compared to the MTT formazan formed in the absence of SOD, which is taken as 100 %. One unit of SOD is defined as the amount of protein required to inhibit MTT reduction by 50%.

In control animal, the urinary bladder had the urothelium formed by tightly packed cells with little intercellular space. The basement membrane that separates the epithelium from the underlying lamina propria was intact. There was no breach in it. The connective tissue that constituted the lamina propria was dense with normal vascular supply. The mucosa was thrown into folds and there were sub-epithelial crypts. The smooth muscle coat underlying the lamina propria displayed circular and longitudinal muscle coat with minimal connective tissue packing. There were no exudates in the lumen (Figure 1A). Contrary to the normal picture presented above, the bladder wall in treated animals showed damages, which became severe with increased time after treatment with the drug. In six-hour case the mucosa became edematous and the cells of the urothelium were not compact. There seemed to be cellular exudates in the lumen. Mucosal content formed follicular cystitis (Figure 1B). However hemorrhage was not seen in this group. The condition became worse in sixteen hours, where edema of lamina propria with epithelial and sub-epithelial hemorrhage was seen (arrows in Figure 1C).

ii. Catalase Catalase activity is estimated by measuring the change in absorption at 240 nm using H2O2 as substrate (Aebi, 1984). To 1 ml of 30 mM buffered H2O2, the enzyme (sample) was added to start the reaction. The final volume was made up to 2 ml with 0.05 M phosphate buffer pH 7.0. Change in OD was observed for 2 min at 240 nm. One unit is the activity that disproportionates H2O2at the rate of 10-3 absorbance/sec. iii. Glutathione-S-transferase (GSTase) The activity of GSTase is measured spectrophotometrically using the substrate 1-chloro-2, 4-dinitrobenzene (CDNB) (Awasthi et al, 1980). To 0.1 ml of 1M potassium phosphate buffer pH 6.5, following reagents were added: 0.1 ml of 10 mM GSH, 0.05 ml 20 mM CDNB and water and made up the volume to 1 ml. The reaction was started by adding the enzyme and change in OD at 340 nm is measured for 1-2 min. One unit of enzyme is the amount required to conjugate 1 !mole of substrate with glutathione in one minute.

B. Biochemical findings The biochemical results obtained for the bladder are shown in figure 2. Myeloperoxidase activity, a marker of neutrophil infiltration was increased by 72 % and by 122% in the urinary bladder, six hour and 16 hour respectively after treatment with CP as compared with control (Figure 2A). Protein carbonyl (Pco) content, an early and sensitive marker of oxidative damage to proteins was increased by 113% at 6 hour and by 50 % sixteen hour after treatment with CP (Figure 2B). Protein thiol, one of the important antioxidants and indicators of oxidative damage to proteins was decreased by 62 % and by 42 % 6 hours and 16 hours after treatment with CP respectively (Figure 2C). The markers of lipid peroxidation, namely malondialdehye (MDA) and conjugated diene were increased significantly in bladders of CP treated rats 16 hours after treatment as compared with control (Figure 2D,E). Conjugated diene was elevated by 367% and MDA by 131 %. With regard to the antioxidant enzymes, a significant decrease in glutathione peroxidase (GPO) activity (50 %) was observed 16 hours after treatment with CP (Figure 2F). With respect to Superoxide dismutase (SOD) activity, a 54 % decrease was observed 16 hour after treatment with CP (Figure 2G). Theactivity of the drug detoxifying enzyme, Glutathione S transferase (GSTase) was

iv. Glutathione reductase In the presence of enzyme, hydrogen is transferred from NADPH to GSSG and the reaction can be measured at 340 nm (Racker, 1955). To the reaction mixture containing 0.05 ml of 1 M phosphate buffer pH 7.6, 0.15 ml of 10 mM EDTA, 0.1 ml of 1 mM NADPH, and 0.1 ml 10 mM GSSG, the enzyme was added. The volume was made up to 1 ml and the decrease in OD at 340 nm was measured for 2-3 min. One unit is the amount of enzyme needed to oxidise 1 !mole of NADPH/min. v. Glutathione peroxidase Total peroxidase is determined by following the oxidation of NADPH at 340 nm using hydrogen peroxide (Nakamura and Hosada, 1974). To 0.25 ml of 0.4 M phosphate buffer, 0.2 ml of 4 mM EDTA, 0.2 ml of 10 mM GSH, 0.2 ml of NaN3, 0.2 ml of 1.6 mM NADPH, 0.03 ml glutathione reductase (one unit) and the enzyme (sample) was added. Total volume was made up to 2

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Abraham et al: Alterations in antioxidant enzyme activities and increased oxidative stress in cyclophosphamide with CP respectively as compared with control as shown in Figure 2I. No significant alteration in the activity of catalase was observed in the bladders of CP treated rats (Figure 2J).

decreased by 52 % and by 66 % 6 hour and 16 hour after treatment with CP respectively as compared with control (Figure 2H). Glutathione reductase activity was increased by 53 % and 25 % six hour and 16 hour after treatment

Figure 1. (A). Control animal. Arrow points to folded mucosa with epithelium showing tight packing of cells and dense connective tissue in lamina propria. Sub-epithelial crypts are also seen in this figure. There is no hemorrhage. Magnification ! 100. (B). Six hours after treatment with cyclophosphamide. Arrows point to edematous lamina propria and cellular and fluid exudates in the lumen. Magnification ! 100. (C). Sixteen hours after treatment with cyclophosphamide. Arrows point to hemorrhage in the epitheliumand in the lamina propria. The lamina propria is edematous. Magnification ! 100.

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Figure 2. (A) Myeloperoxidase activity in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, * P < 0.05 as compared with control, # P< 0.05 as compared with 6 hour. (B) Protein carbonyl content in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, * P < 0.05, **P<0.02 as compared with control. (C) Protein thiol levels in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, * P < 0.05, **P <0.01 as compared with control, # P< as compared with 6 hour (D) Malondialdehyde levels in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, *P<0.01 as compared with control, P < 0.01 as compared with 6 hour. (E) Conjugated diene levels in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, *P<0.02 as compared with control, # P < 0.02 as compared with 6 hour. (F) Glutathione peroxidase activity in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, *P<0.02 as compared with control, # P < 0.02 as compared with 6 hour. (G) Superoxide dismutase activity in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, * P < 0.01 as compared with control, # P < 0.01 as compared with 6 hour. (H) Glutathione S transferase activity in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, * P < 0.05, ** P<0.02 as compared with control. (I) Glutathione reductase activity in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group, *P < 0.05, **P<0.02 as compared with control, # P<0.05 as compared with 6 hour. (J) Catalase activity in the bladder of control rats and experimental rats 6 hour and 16 hours following treatment with CP. Data represent mean ± SD, n= 5-7 in each group.

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Abraham et al: Alterations in antioxidant enzyme activities and increased oxidative stress in cyclophosphamide ROS production in inflammatory diseases is considered to play an important role in the initiation and progression of tissue injury. Inflammatory cells such as macrophages, neutrophils and monocytes produce ROS which can react with biological macromolecules such as lipids, proteins and nucleic acids because of their high reactivity, causing tissue injury. On the other hand, the ROS produced are thought to contribute to the subsequent neutrophil infiltration to tissue (Petrone et al, 1980; Hotter et al, 1997). It is known that ROS and cellular redox status regulate expressions of proinflammatory cytokines in inflammatory or non-inflammatory cells at gene level (DeForge et al, 1993; Shi et al, 1999; Haddad 2000; Haddad et al, 2001). Increased ROS production has been shown to initiate and facilitate neutrophil infiltration leading to the inflammation (Bradley et al, 1982). In the present study, the early and sensitive indicator of oxidative stress namely Pco was significantly increased 6 hr after treatment with CP and began to decrease thereafter, but still remained elevated as compared with control. Besides, protein thiol content in the bladder was reduced 6 hour after treatment with CP. Increase in MPO activity was observed 6 hour after treatment with CP. The MPO activity increased further at 16 hours. Therefore it is suggested that oxidative stress initiates and facilitates neutrophil infiltration. The activated neutrophils generate reactive oxygen species leading to inflammation. ROS not only regulate cytokine expression through nuclear transcriptional factors but also affect adhesion molecules (Suzuki et al, 1997; Droge 2002). ROS contribute towards increased transendothelial and transepithelial permeability (Rao et al, 2000; Meyer et al, 2001). The increase of transepithelial permeability allows toxins to permeate through the barrier, which leads to inflammation. ROS produced by the CP treatment may possibly cause the above events, leading to the CP-induced hemorrhagic cystitis. Comparing the histological findings with that of the biochemical findings, it was observed that oxidative stress and neutrophil infiltration was evident 6 hours after treatment with CP, when the mucosa became edematous and cellular exudates were seen in the lumen. Sixteen hours after treatment with CP the activity of MPO increased further, the parameters of oxidative stress were elevated and histologically a more severe damage was observed. Increase in oxidant stress and decrease in the antioxidant status was accompanied by neutrophil infiltration and bladder damage. This study clearly shows that increased oxidative stress, depletion of antioxidant enzymes, and neutrophil infiltration contribute to CP induced hemorrhagic cystitis. In the light of the current data, is suggested that oxidative stress contributes at least in part, to CP induced bladder damage. CP treatment of rats induces oxidative stress, depletes cellular antioxidants in the urinary bladder which contribute to neutrophil infiltration into bladder. In the future, investigation of the effect of administration of MPO inhibitors and natural antioxidants such as vitamin C in the prevention of hemorrhagic cystitis may be planned. It is believed that the proposed study will increase the

IV. Discussion Urotoxicity is one of the major dose limiting side effects of CP. The urotoxicity of CP is thought to be due to the formation of acrolein that damages the urothelium. Therefore, Mesna, an acrolein binding and detoxifying compound within the urinary collecting system, has been widely used as an agent against CP induced cystitis, but significant HC is still being encountered clinically (Brock et al, 1981). Since detoxifying acrolein does not remove symptoms of HC completely, it is speculated that mechanisms other than direct contact of acrolein with bladder mucosa may be involved in CP induced HC. Oxidative stress is known to be involved in several diseases and to be the cause of toxicity of many drugs. Therefore, in the present study the parameters of oxidative stress and the activities of antioxidant enzymes were studied and compared with histological findings. The parameters of oxidative stress i.e. conjugated dienes, malondialdehyde and protein carbonyl content were found to be markedly increased in the bladders of CP treated rats suggesting that CP treatment caused oxidative damage to the lipids and proteins of the bladder. Protein carbonyl content was increased significantly 6 hour after treatment with CP and, MDA and Conjugated diene were elevated significantly 16 hours after treatment with CP. In parallel with their production, protein thiol content, an important antioxidant in the bladder was lowered. Recently, Topal and colleagues in 2005 and Sadir colleagues in 2007 have demonstrated that MDA levels increase in the bladder of rats after the administration of CP. Antioxidant enzyme activities were measured to evaluate the antioxidant capacity. Antioxidant enzyme activities are reported to be altered in drug induced inflammatory conditions. In NSAIDs induced intestinal toxicity, SOD and GPO activities were shown to be decreased in parallel with the severity of damage (de la Lastra et al, 2000; Villegas et al, 2001). In the present study a decrease in the activities of the important antioxidant enzymes namely SOD, GPO, GSTase was observed in the bladders of CP treated rats. Since oxidative stress preceded the decrease in the activities of antioxidant enzymes it is suggested that the decrease in the activities of antioxidant enzymes is a consequence of increased oxidative stress in the bladder. It is proposed that the decrease in the activities of the antioxidant enzymes contribute to the urotoxicity of CP. An increase in the activity of glutathione reductase is considered to be protective response of the body to the oxidative stress induced by CP. It has been demonstrated that activated neutrophils secrete enzymes such as myeloperoxidase, elastase and other proteases. MPO plays a fundamental role in the oxidant production by the neutrophils and has been used as an effective index of inflammation due to the correlation between MPO activity and the histological analysis of neutrophil infiltration (Sekizuka and Grisham, 1988). In the present study, MPO levels were elevated indicating that neutrophil infiltration contributes to CP induced cystitis.

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Cancer Therapy Vol 6, page 569! Meyer TN, Schwesinger C, Ye J, Denker BM, Nigam SK (2001) Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity. J Biol Chem 276, 22048-22055. Nakamura W, Hosada S (1974) Purification and properties of rat liver glutathione peroxidase. Biochim Biophys Acta 358, 251-261. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95, 351-8. Ohkuma N, Matsuo S, Tutsui M, Ohkawara A. [Superoxide dismutase in the epidermis (author's transl)] (1982) Nippon Hifuka Gakkai Zasshi 92, 583-50. Petrone WF, English DK, Wong K, McCord JM (1980) Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc Natl Acad Sci USA 77, 1159-1163. Racker E (1955) Glutathione reductase from bakers' yeast and beef liver. J Biol Chem 217, 855-865. Rao RK, Li L, Baker RD, Baker SS, Gupta A (2000) Glutathione oxidation and ATPase inhibition by hydrogen peroxide in Caco-2 cell monolayer. Am J Physiol Gastrointest Liver Physiol 279, G332-340. Sair S, Deveci S, Korkmaz A (2007) Alpha tocopherol, beta carotene, and melatonin administration protects cyclophosphamide induced oxidative damage to bladder tissue in rats. Cell Biochem Funct 25, 521-52 Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 25, 192-205. Sekizuka E, Grisham MB, Li M (1988) Inflammation induced intestinal hyperemia in the rat: role of neutrophils. Gastroenterology 95, 1528-1534. Shi MM, Chong I, Godleski JJ, Paulauskis JD (1999) Regulation of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages. Immunology 97, 309-315. Sohal RS, Agarwal S, Dubey A , Orr WC (1993) Protein oxidative damage is associated with life expectancy of houseflies. Proc Natl Acad Sci U S A 90, 7255-7259. Suzuki YJ, Forman HJ, Sevanian A (1997) Oxidants as stimulators of signal transduction. Free Radic Biol Med 22, 269-285. Topal P, Oztas Y, Korkmaz A (2005) Melatonin ameliorates bladder damage induced by cyclophosphamide in rats. J Pineal Research 38, 272-277. Villegas I, Alarcon de la Lastra C, La Casa C, Motilva V, Martin MJ (2001)Effects of food intake and oxidative stress on intestinal lesions caused by meloxicam and piroxicam in rats. Eur J Pharmacol 414, 79-86. Wallace JL, MacNaughton W K, Morris GP, Beck PL (1989) Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 96, 29-36. West NJ (1997) Prevention and treatment of hemorrhagic cystitis. Pharmacotherapy 4, 696-706.

clinical utility of the drug by overcoming cystitis, the most limiting side effect of cyclophosphamide.

Acknowledgements The study was supported by Department of Science and Technology (DST) New Delhi.

References Aebi IH (1984) Catalase in vitro. Methods Enzymol 105, 121-6. Ahluwalia A, Maggi CA, Santicioli P, Lecci A, Giuliani S (1994) Characterization of the capsaicin-sensitive component of cyclophosphamide-induced inflammation in the rat urinary bladder. Br J Pharmacol 111, 1017-22. Awasthi YC, Dao DD, Saneto RP (1980) Interrelationship between anionic and cationic forms of glutathione Stransferases of human liver. Biochem J 191, 1-10. Babiak RMV, Campello AP, Carnieri EGS (1998) Methotrexate: pentose cycle and oxidative stress. Cell Biochem Funct 16, 283-293. Bradley PP, Priebat DA, Christensen RD, Rothstein G (1982) Measurement of cutaneous Inflammation: estimation of neutrophil content with an enzyme marker. J Inves Dermatol 78, 206-209. Brock N, Pohl J, Stekar J (1981) Studies on the urotoxicity of oxazaphosphorine cytostatics and its prevention. 1. Experimental studies on the urotoxicity of alkylating compounds. Eur J Cancer 17, 595-601. Chan HW, Levett G (1977) Autoxidation of methyl linoleate. Separation and analysis of isomeric mixtures of methyl linoleate hydroperoxides and methyl hydroxylinoleates. Lipids 12, 99-104. de la Lastra CA, Nieto A, Motilva V, Martin MJ, Herrerias JM, Cabre F (2000) Intestinal toxicity of ketoprofen-trometamol vs its enantiomers in rat. Role of oxidative stress. Inflamm Res 49, 627-632. DeForge LE, Preston AM, Takeuchi E, Kenney J, Boxer LA, Remick DG (1993) Regulation of interleukin 8 gene expression by oxidant stress. J Biol Chem 268, 2556825576. Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82, 47-95 Haddad JJ (2000) Glutathione depletion is associated with augmenting a proinflammatory signal: evidence for an antioxidant/prooxidant mechanism regulating cytokines in the alveolar epithelium. Cytokines Cell Mol Ther 6, 177187. Haddad JJ, Safieh-Garabedian B, Saade NE, Land SC (2001) Thiol regulation of pro-inflammatory cytokines reveals a novel immunopharmacological potential of glutathione in the alveolar epithelium. J Pharmacol Exp Ther 296, 996-1005. Hotter G, Closa D, Prats N, Pi F, Gelpi E, Rosello-Catafau J (1997) Free radical enhancement promotes leucocyte recruitment through a PAF and LTB4 dependent mechanism. Free Radic Biol Med 22, 947-954. Levine AL, Richie PJ (1989) Urological complications of cyclophosphamide. J Urol 141, 1063-1069.

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Dosimetric evaluation of helical tomotherapy treatment planning for non-small cell lung cancer Research Article

Karen Chu1, George Rodrigues1,2, Slav Yartsev3, A. Rashid Dar1, Edward Yu1,3, Robert Ash1, Brian Yaremko1, Marc MacKenzie4, Harvey Quon5, Glenn Bauman1,3, Wilson Roa5 1

Department of Radiation Oncology, London Regional Cancer Program, London Health Sciences, London, ON, Canada Department of Epidemiology and Biostatistics, University of Western Ontario, London, ON, Canada 3 Department of Medical Biophysics, University of Western Ontario, London, ON, Canada 4 Department of Medical Biophysics, Cross Cancer Institute, Edmonton, AB, Canada 5 Department of Radiation Oncology, Cross Cancer Institute, Edmonton, AB, Canada 2

__________________________________________________________________________________! *Correspondence: Dr. George Rodrigues, Department of Radiation Oncology, London Regional Cancer Program, London, Ontario, N6A 4L6, Canada; Phone: 519 685 8600 ext. 53347; Fax: 519 685 8736; e-mail: george.rodrigues@lhsc.on.ca Key words: Helical Tomotherapy; Three-dimensional Conformal Radiotherapy; Dosimetric Comparison; Lung Cancer Abbreviations: 3D Conformal Radiotherapy, (3DCRT); Computer tomography, (CT); Cross Cancer Institute, (CCI); DICOM RT; Dose, (D); Dose-volume histograms, (DVH); Gross tumor volume, (GTV); Gy, (Gray); Helical tomotherapy, (HT); Intensity Modulate Radiotherapy, (IMRT); London Regional Cancer Program, (LRCP); Megavoltage CT, (MVCT); Non-small cell lung cancer, (NSCLC); Ontario, (ON); Planning target volume, (PTV); Volume, (V) Received: 7 April 2008; Revised: 25 may 2008 Accepted: 5 June 2008; electronically published: September 2008

Summary Helical tomotherapy (HT) is a novel technique to deliver intensity modulated radiation therapy guided by 3D megavoltage CT imaging. The purpose of our study is to assess the dosimetric parameters related to HT and 3DCRT in advanced non-small cell lung cancer (NSCLC). Eleven patients from the London Regional Cancer Centre and the Cross Cancer Institute with NSCLC underwent individualized treatment planning on both HT and 3DCRT. Corresponding HT and 3DCRT plans for each patient were analyzed using dose-volume histograms for GTV, PTV (median dose 60Gy/30 fractions), and critical structures (lung V5-30, esophageal V50-60, and spinal cord D1). Observed differences in tumor and normal tissue dosimetry were assessed for statistical significance using paired t-tests. A statistically significant improvement on GTV homogeneity but not PTV homogeneity was found in relation with HT. 3DCRT was associated with improved V5 (14%, p = 0.02), V10 (9%, p = 0.04) and V15 (6%, p = 0.04). However, there was no difference in V20 (2%, p = NS); while HT demonstrated superior V30 (5%, p = 0.002). HT achieved excellent tumor coverage relative to 3DCRT in the setting of routinely clinically planned radiation therapy with improvements in the V30 lung parameter. This was at the expense of a modest increase in V5-V15 total lung dose.

utilized to improve the therapeutic ratio between tumor control and early and late critical structure effects. Helical tomotherapy (HT) is a novel technique that combines intensity-modulated radiation therapy (with a binary multileaf collimator) with a helical CT (Mackie et al, 1993, 1999). A megavoltage linear accelerator is mounted on a ring gantry, and dose is delivered via a continuous helical beam as the patient progresses through the ring. HT has the ability to produce daily megavoltage CT (MVCT) images, enabling image-guided adjustment of

I. Introduction Lung cancer is the second most common malignancy and accounts for 29% of cancer deaths in Canada (Canadian Cancer Society, 2007). Despite aggressive multi-modality treatment of locally-advanced non-small cell lung cancer (NSCLC), 5 year overall survival still remains low at 15-20%. Advancements in tumor imaging, 4-dimensional computed tomography (CT), respiratory gated therapy, image guided therapy, and intensitymodulated radiation therapy delivery are currently being 571

Chu et al: Evaluation of helical tomotherapy in NSCLC! Table 1. Patient demographics.

interfraction setup errors (Ruchala et al, 1999; Ruchala et al, 2000; Welsh 2004) and dose delivery verification (Kapatoes, 2001). HT has been studied in the context of both traditional (Mehta 2001; Scrimger et al, 2003; Kron et al, 2004) and stereotactic body (Fuss et al, 2006, Hodge et al, 2006) NSCLC treatment. Preliminary investigations of tumor motion effects (Kanagaki et al, 2007), adaptive therapy (Ramsay et al, 2006) and assessment of tumor response by serial MVCT (Kupelian et al, 2005, Seibert et al, 2007) have been reported in the literature. In evaluating stage III locally-advanced NSCLC, mean normalized dose to total lung and the V20 were decreased by 30% and 22% respectively using HT when compared to 3DCRT plans (Scrimger et al, 2003). HT also spared both the spinal cord and esophagus. In the comparison of HT to IMRT plans in locally-advanced lung cases including microscopic elective nodal coverage excellent dose homogeneity was found with HT, though this was attained at the expense of higher mean lung dose, esophageal dose, and spinal cord dose (Kron et al, 2004). However, once the elective nodal volume was excluded, the mean lung dose was significantly improved from an average of 28 to 16Gy. HT has been implemented clinically at each of our institutions since 2003, and prospective evaluation has demonstrated that HT can generate dosimetrically superior equivalent radiotherapy treatment plans compared to 3dimensional conformal radiotherapy (3DCRT), and that such plans can be delivered safely (Bauman et al, 2007). Concerns regarding low doses of radiotherapy to large volumes of lung tissue have been described in the medical literature (Yorke et al, 2002). With the increasing adoption of arc-based tomotherapy with the potential for delivery of this low dose lung radiation, a dosimetric analysis of this phenomenon is appropriate. Therefore, the objective of our study was to compare HT and 3DCRT NSCLC plans dosimetrically in terms of target and normal tissue parameters in a series of clinically treated patients.

Variable Age Gender Staging

Tumor Location

Male Female T1N3M0 T2N0M0 T3N2M0 T4N2M0 T4N3M0 pT2N3M0 Sub aortic fossa Lingula Left Lower Lobe Left Upper Lobe Right Middle Lobe Right Lower Lobe Right Upper Lobe

Median 70 years, Range 54-85 years n=3 (27%) n=8 (73%) n=1 (9%) n=1 (9%) n=4 (36%) n=3 (27%) n=1 (9%) n=1 (9%) n=1 (9%) n=1 (9%) n=2 (18%) n=1 (9%) n=1 (9%) n=1 (9%) n=4 (36%)

B. Radiotherapy prescription A minimum dose of 50 Gy to the PTV was required for entry into the study cohort. The treating radiation oncologist approved the final prescription dose to the PTV and reviewed all DVH information prior to treatment. The median prescription dose was 60Gy in 30 fractions and varied (1 patient at 51.5Gy, 1 patient at 55Gy, and 9 of 11 patients at 60Gy) depending upon the particular clinical scenario. The HT and 3DCRT plans were prescribed to the same total dose, and to the same prescription point. All patients were treated at a standard fractionation of 2 Gy per day. DVH optimization was primarily based on achieving acceptable homogeneity of target dose (GTV and PTV), the reduction of the V20Gy bilateral lung parameter, and clinically acceptable maximum spinal cord dosage (see Helical Tomotherapy and Conventional 3CDRT planning below).

C. Helical tomotherapy planning CT datasets and all contoured volumes were transferred to the tomotherapy planning workstation (Tomotherapy Inc. Madison, WI, USA) using the DICOM RT protocol. A uniform slice thickness and separation of 3 mm was used after 256x256 voxel CT dataset resampling. The planning system used an inverse planning process based on least-squares minimization of an objective function guided by precedence, importance and penalty factors. The LRCP and the CCI used similar inverse treatment planning parameters, and these have previously been published in the literature (8). The initial lung class solution prioritized GTV coverage and avoidance of contralateral lung, ipsilateral lung and spinal cord. Maximum allowable GTV/PTV target heterogeneity was from -5% to +7% of the prescription dose. The volume of total lung treated to ! 20 Gy was limited to a maximum of 35%. The maximum dose to spinal cord was limited to 48Gy. HT planning used pitch of 0.286, fan beam width of 5cm, and initial modulation factor of 3. Dose was calculated using a superposition/convolution algorithm on the normal cell grid setting (256x256). The helical dose delivery was emulated in 51 projections per rotation and the dose calculation used a total of 24 different angles for the dose-spread array of the incident 6 MV beam.

II. Materials and Methods A. Patients and target design Eleven patients with NSCLC were included in this study (Table 1). Nine patients were treated at the London Regional Cancer Program (LRCP) in London, Ontario, and 2 patients were treated at the Cross Cancer Institute (CCI) in Edmonton, Alberta. All patients had 3-dimensional, multislice CT simulation with 3 mm slice thickness. The gross tumor volume (GTV) was contoured by a thoracic radiation oncologist based on diagnostic CT and pathological information. Positron Emission Tomography was not employed in this treatment cohort. A margin of 1.0 to 2.0 cm was applied around the GTV to define the planning target volume (PTV). Full elective nodal irradiation was not utilized in this study. Lung, esophagus, and spinal cord were contoured for all patients, and these structures were used as avoidance volumes of interest. The total lung was defined as the bilateral external lung contour excluding GTV. The external surface of the esophagus was either contoured from the thoracic inlet to the gastroesophageal junction or for the length of the treatment field depending on institutional/radiation oncologist practice. The external surface of the spinal cord was contoured from the lung apex to the level of L1.

D. Conventional 3DCRT planning Matched conventional treatment plans were created for

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Cancer Therapy Vol 6, page 573! each patient using standard multifield 3DCRT treatment principles. All 3DCRT plans were generated on a commercial treatment planning system (Theraplan Plus 3.8 with Dose Calculation Module 2.0, Nucleotron, Kanata, ON). Coplanar multifield (3-5 fields) 3DCRT planning was performed to optimize target homogeneity, while meeting the same normal tissue dosimetric constraints specified for HT. Field shaping with 120-leaf multileaf collimation was utilized to shape the radiation ports.

IIIB disease. Mean GTV was 127.9 cc (range 4.1-282.1 cc), and mean PTV was 391.3 cc, (range 76.6-874.7 cc). Mean spinal cord volume (56.24 cc, range 33.0-96.7 cc), mean total lung volume (4034.3cc, range 1673.6-6611.0 cc), and mean esophageal volume (37.6 cc, range 7.5667.4 cc) were also calculated. Patients were treated to a median dose of 60Gy (range 51.5-60 Gy) in 30 fractions (range 22-30 fractions) prescribed to the 95% (range 90100%) isodose line. Ultimately, seven patients were treated with HT and 4 patients were treated with 3CDRT at the discretion of the treating radiation oncologist based on dosimetric analysis and treatment unit availability. The calculated raw 1%, 5%, 95% and 99% point DVH doses to the GTV and PTV for each of HT and 3DCRT are presented in Table 2. The calculated differences between these parameters are presented in this same table. Calculated mean differences (D1-D99) and (D5D95) for the GTV and PTV are presented in this same table. Both (D1-D99) and (D5-D95) demonstrated statistically significantly improved homogeneity about the GTV when HT is used. There were no statistically significant differences in PTV homogeneity found between HT and 3DCRT. The resultant normal tissue dosimetry is presented in Table 3. 3DCRT was associated with significantly improved normal lung dosimetry in the range of lowest doses: V5 (14%, p = 0.02), V10 (9%, p = 0.04) and V15 (6%, p = 0.04). However, there was no observed difference in V20 (2%, p = NS), while HT created a better lung sparing effect at a dose of 30 Gy, V30 (5%, p = 0.002). Esophageal V50 and V55 were not significantly different when comparing 3DCRT to HT; however a small (3%) borderline statistically significant difference (p=0.05) in esophageal V60 was observed. The maximal spinal cord dose, D1 showed a benefit towards 3DCRT with a mean difference of 12.7 Gy (p = 0.02). A categorical analysis of target and normal tissue DVH parameters was also performed (Table 4) further emphasizing the differences between the two treatment delivery systems.

E. Statistical Methodology Dose-volume histograms (DVH) were generated for the GTV and PTV, for both HT and 3DCRT. From these results, PTV and GTV homogeneity indices were calculated for each patient as differences between observed doses: D5-D95 and D1-D99 (where “Dx” represents dose (D, in Gy) received by “x” % of the PTV or GTV). Considering normal tissue dosimetry, DVHs were generated for the total lung, esophagus and spinal cord, for both HT and 3DCRT, for all patients. From these DVHs, the corresponding fractional organ volumes treated to a set of specified relevant dose levels were identified for the total lung (V5, V10, V15, V20, V30) and for the esophagus (V50, V55, V60), where “V” represents the fractional volume (%) of an organ treated to a dose of “x” (Gy). For the spinal cord, we calculated the dose to 1% of the organ volume (D1), the dose to 50% of the organ volume (D50) and the maximum dose to the organ, again for all patients, for both HT and 3DCRT. All generated dosimetric parameters were compared directly between HT and 3DCRT, and any observed differences were assessed for statistical significance using a two-sided, paired Student’s t-test. The !" #$%&$%" null hypothesis for all comparisons was that there was no difference in any dosimetric parameter between HT and 3DCRT. The alternative hypotheses were that the difference in DVH parameter between HT and 3DCRT was non-zero.

III. Results A total of 11 patients with NSCLC were analyzed (Table 1). The median age was 70 years old with 3 male and 8 female patients. One patient had a Stage IB tumor, 4 patients had Stage IIIA disease and 6 patients had Stage Table 2. Target DVH parameters paired T-Test comparison.

Point Target Dose GTV D1 (Gy) GTV D5 GTV D95 GTV D99 PTV D1 PTV D5 PTV D95 PTV D99 Homogeneity GTV D1-D99 (Gy) GTV D5-D95 PTV D1-D99 PTV D5-D95

3DCRT Mean SD

HT Mean

3DCRT-HT Mean SD

Paired T-Test

SD

64.1 63.4 58.7 57.7 64.5 63.5 56.8 54.2

2.6 2.7 2.3 2.4 1.9 1.7 2.8 3.6

63.9 63.3 60.8 60.2 63.7 62.8 57.4 54.2

3.5 3.3 2.9 2.4 3.6 3.3 3.8 5.5

0.2 0.1 -2.1 -2.5 0.8 0.7 0.6 0.0

4.5 4.4 2.6 2.7 4.1 3.7 2.5 2.7

NS NS p=0.03 p=0.01 NS NS NS NS

11.2 4.1 12.5 6.7

17.5 1.6 8.6 2.3

7.9 2.5 13.0 5.4

13.4 1.5 10.9 3.7

3.3 1.6 -0.5 1.3

5.0 2.1 4.9 3.7

p=0.05 p=0.03 NS NS

" 3DCRT= Three-dimensional Conformal Radiation Therapy; HT=Helical Tomotherapy; SD=Standard Deviation; D=Dose; NS=Not Significant

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Chu et al: Evaluation of helical tomotherapy in NSCLC!

Table 3. Normal tissue DVH parameter paired T-Test comparison. 3DCRT Mean

HT Mean

SD

3DCRT-HT Mean SD

Paired T-Test

SD

Spinal Cord D50 (Gy) D1 (max dose)

11.0 29.1

14.5 15.6

13.5 41.8

10.8 7.0

-2.5 -12.7

16.3 14.8

NS p=0.02

Total Lung Volume V5 V10 V15 V20 V30

0.50 0.37 0.28 0.23 0.19

0.13 0.11 0.10 0.10 0.08

0.64 0.46 0.34 0.25 0.14

0.17 0.19 0.15 0.11 0.06

-0.14 -0.09 -0.06 -0.02 0.05

0.19 0.14 0.09 0.06 0.04

p=0.02 p=0.04 p=0.04 NS p=0.002

Esophagus V50 V55 V60

0.29 0.26 0.13

0.21 0.20 0.11

0.32 0.27 0.16

0.18 0.17 0.15

-0.03 -0.01 -0.03

0.20 0.17 0.15

NS NS p=0.05

V=Volume

Table 4. Categorical target/normal tissue comparison. 3DCRT>HT

3DCRT=HT

3DCRT<HT

Point Target Dose GTV D1 (Gy) GTV D5 GTV D95 GTV D99 PTV D1 PTV D5 PTV D95 PTV D99

n=8 (72%) n=7 (64%) n=1 (9%) n=2 (18%) n=9 (82%) n=9 (82%) n=4 (36%) n=5 (45%)

Homogeneity GTV D1-D99 (Gy) GTV D5-D95 PTV D1-D99 PTV D5-D95

n=9 (82%) n=9 (82%) n=5 (45%) n=6 (55%)

n=2 (18%) n=2 (18%) n=6 (55%) n=5 (45%)

Spinal Cord D50 D1

n=4 (36%) n=2 (18%)

n=7 (64%) n=9 (82%)

Total Lung Volume V5 V10 V15 V20 V30

n=1 (9%) n=3 (27%) n=3 (27%) n=3 (27%) n=10 (91%)

n=10 (91%) n=8 (73%) n=8 (73%) n=7 (64%) n=1 (9%)

Esophagus V50 V55 V60

n=4 (40%) n=6 (60%) n=4 (40%)

n=1 (9%)

n=1 (9%)

n=1 (10%) n=1 (10%) n=1 (10%)

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n=3 (27%) n=4 (36%) n=10 (91%) n=9 (82%) n=2 (18%) n=1 (9%) n=7 (64%) n=6 (55%)

n=5 (50%) n=3 (30%) n=5 (50%)

Cancer Therapy Vol 6, page 575! conformal avoidance IMRT capabilities of HT. However, these scenarios were not investigated as part of this report. Patients with large treatment volumes or that are receiving exquisite radiosensitizers may not be the ideal tomotherapy candidates due to the risk of generalized radiation pneumonitis. Clinical studies are currently ongoing at our respective institutions to address and catalogue such therapeutic ratio issues more fully.

IV. Discussion This study was designed to assess the dosimetric benefits of HT versus 3DCRT as it applies to the routine clinical planning and treatment of NSCLC. This report describes the initial clinical implementation experience of helical tomotherapy at two Canadian institutions. Helical tomotherapy plans (and their matched 3DCRT backup plans) were optimized to the level required to achieve safe and clinically appropriate radiation treatment. Other studies assessing non-clinical highly optimized dosimetric comparisons of HT with 3DCRT (10) and IMRT (8) have been previously published in the literature. These investigations were not limited by clinical timeframes and potentially reflect levels of higher optimization that may or may not be clinically achievable in routine practice. Consequently, our results are certainly relevant clinically, with each patient in this study actually received treatment according to one of the two treatment plans (HT or 3DCRT) that was generated. Although the treatment delivered to each patient could potentially have been optimized further (for example to enable delivery of a higher dose to the PTV) such optimization was not performed because the plans already met the standard clinical acceptability criteria in place at each institution at the time of treatment and the added benefits of additional optimization was deemed to be minimal. With respect to tumor coverage, the HT treatment plans were found to have the advantage of more homogeneous GTV coverage and reduction of total lung V30. This can be appreciated immediately by examining the 30 Gy to 60 Gy isodose lines on a typical treatment plan, as such lines begin to approximate the shape of the PTV very well at this dose level. However, with respect to the total lung volume, HT did not improve the dosimetry to the normal lung in the lowest dose range (20 Gy and less). Although in principle, lung dosimetry in this lowdose region could have been improved slightly through optimization, i.e. by increasing the weighting of the imposed normal lung constraints in the inverse treatment planning optimization algorithm. The dominant factor contributing to the comparatively worse lung dosimetry in the low-dose region is the inherent design of tomotherapy itself. Because of the ring gantry design in tomotherapy, the dose from the arc-based radiation creates a “dose-bath� phenomenon of low dose to normal lung tissue. For lung cancer (as well as for other thoracic malignancies) the tendency of tomotherapy to increase dose in the low-dose region could have significant negative implications in terms of late toxicity, as there is mounting evidence that the amount of normal lung exposure in the low-dose range is predictive of late toxicity than the amount exposed to higher doses (Yorke et al, 2002). In this regard, further studies are required to establish the potential clinical gain of using HT in routine clinical practice. Therefore, each clinical case should be judged individually, in terms of the dosimetric and imageguidance benefits of HT balanced against the increases in V5-V15 lung DVH parameters. Lung tumors near critical structures such as spinal cord, esophagus, liver, or heart probably provide the best clinical scenarios that can potentially take advantage of the image-guidance and

Acknowledgements The authors would like to acknowledge Larry Stitt for statistical consultation services and Dr. Belal Ahmad for manuscript comments.

References Bauman G, Yartsev S, Rodrigues G, Lewis C, Venkatesan VM, Yu E, Hammond A, Perera F, Ash R, Dar AR, Lock M, Baily L, Coad T, Trenka K, Warr B, Kron T, Battista J, Van Dyk J (2007) A prospective evaluation of helical tomotherapy. Int J Radiat Oncol Biol Phys!68, 632-41. Canadian Cancer Society/National Cancer Institute of Canada (2007) Canadian Cancer Statistics 2007, Toronto, Canada. Fuss M, Shi C, Papanikolaou N (2006) Tomotherapeutic stereotactic body radiation therapy: Techniques and comparison between modalities. Acta Oncol!45, 953-60. Hodge W, Tome WA, Jaradat HA, Orton NP, Khuntia D, Traynor A, Weigel T, Mehta MP (2006) Feasibility report of image guided stereotactic body radiotherapy (IG-SBRT) with tomotherapy for early stage medically inoperable lung cancer using extreme hypofractionation. Acta Oncol!45, 890-6. Kanagaki B, Read PW, Molloy JA, Larner JM, Sheng K (2007) A motion phantom study on helical tomotherapy: the dosimetric impacts of delivery technique and motion. Phys Med Biol 52, 243-55. Kapatoes JM, Olivera GH, Balog JP, Keller H, Reckwerdt PJ, Mackie TR (2001) On the accuracy and effectiveness of dose reconstruction for tomotherapy. Phys Med Biol!46, 943-66. Kron T, Grigorov G, Yu E, Yartsev S, Chen JZ, Wong E, Rodrigues G, Trenka K, Coad T, Bauman G, Van Dyk J (2004) Planning evaluation of radiotherapy for complex lung cancer cases using helical tomotherapy. Phys Med Biol! 21, 3675-90. Kupelian PA, Ramsey C, Meeks SL, Willoughby TR, Forbes A, Wagner TH, Langen KM (2005) Serial megavoltage CT imaging during external beam radiotherapy for non-smallcell lung cancer: observations on tumor regression during treatment. Int J Radiat Oncol Biol Phys!63, 1024-8. Mackie TR, Balog J, Ruchala K, Shepard D, Aldridge S, Fitchard E, Reckwerdt P, Olivera G, McNutt T, Mehta M (1999) Tomotherapy. Semin Radiat Oncol 9, 108-17. Mackie TR, Holmes T, Swerdloff S, Reckwerdt P, Deasy JO, Yang J, Paliwal B, Kinsella T (1993) Tomotherapy: a new concept for the delivery of dynamic conformal radiotherapy. Med Phys 20, 1709-19. Mehta M, Scrimger R, Mackie R, Paliwal B, Chappell R, Fowler J (2001) A new approach to dose escalation in non-small-cell lung cancer. Int J Radiat Oncol Biol Phys!49, 23-33. Ramsey CR, Langen KM, Kupelian PA, Scaperoth DD, Meeks SL, Mahan SL, Seibert RM (2006) A technique for adaptive image-guided helical tomotherapy for lung cancer. Int J Radiat Oncol Biol Phys!15, 1237-44. Ruchala KJ, Olivera GH, Kapatoes JM, Schloesser EA, Reckwerdt PJ, Mackie TR (2000) Megavoltage CT image reconstruction during tomotherapy treatments. Phys Med Biol!45, 3545-62.

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Chu et al: Evaluation of helical tomotherapy in NSCLC! Ruchala KJ, Olivera GH, Schloesser EA, Mackie TR (1999) Megavoltage CT on a tomotherapy system. Phys Med Biol! 44, 2597-621. Scrimger RA, Tome WA, Olivera GH, Reckwerdt PJ, Mehta MP, Fowler JF (2003) Reduction in radiation dose to lung and other normal tissues using helical tomotherapy to treat lung cancer, in comparison to conventional field arrangements. Am J Clin Oncol!26, 70-8. Seibert RM, Ramsey CR, Hines JW, Kupelian PA, Langen KM, Meeks SL, Scaperoth DD (2007) A model for predicting lung cancer response to therapy. Int J Radiat Oncol Biol Phys! 67, 601-9.

Welsh JS, Bradley K, Ruchala KJ, Mackie TR, Manon R, Patel R, Wiederholt P, Lock M, Hui S, Mehta MP (2004) Megavoltage computed tomography imaging: a potential tool to guide and improve the delivery of thoracic radiation therapy. Clin Lung Cancer!5, 303-6. Yorke ED, Jackson A, Rosenzweig KE, Merrick SA, Gabrys D, Venkatraman ES, Burman CM, Leibel SA, Ling CC (2002) Dose-volume factors contributing to the incidence of radiation pneumonitis in non-small-cell lung cancer patients treated with three-dimensional conformal radiation therapy. Int J Radiat Oncol Biol Phys 54, 329-39.

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Cancer Therapy Vol 6, page 577! Cancer Therapy Vol 6, 577-596, 2008

Gastrointestinal stromal tumor of the pancreatoduodenal complex: A detailed review and development of new prognostic scoring system Research Article

Raja R Gopaldas1,*, Lori J Toedter2, Sanford Dorman3, Chand Rohatgi4 1

Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston TX Department of Surgery, Easton Hospital, Easton PA 3 Department of Pathology, Easton Hospital, Easton PA 4 Associate in-charge, Division of Surgical Oncology, Easton Hospital; Clinical Associate, Drexel University College of Medicine 2

__________________________________________________________________________________! *Correspondence: Raja R. Gopaldas, MD, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston TX 77030, USA; Email: doctorgopaldas@yahoo.com Key words: GIST, stromal, imatinib mesylate, sunitinib, pancreatoduodenectomy, gastrointestinal stromal tumor, neoadjuvant, !"#$%&' ()*'gene Abbreviations: 18-Fluodeoxy-glucose Positron emission tomography, (18 FDG-PET); Endoscopic Retrograde Cholangiopancreatography, (ERCP); Endoscopic Ultrasound guided Fine-needle aspiration, (EUS-FNA); esophagogastroduodenoscopy, (EGD); fine needle aspiration biopsy, (FNAB); gastric autonomic nerve tumors, (GANT); gastrointestinal pacemaker cell tumor, (GIPACT); Gastrointestinal stromal tumors, (GISTs); high power fields, (HPF); Interstitial Cells of Cajal, (ICC); Muscle Specific Actin, (MSA); Smooth Muscle Actin, (SMA); smooth muscle tumors of uncertain malignant potential, (STUMP); stem cell factor, (SCF) Received: 22 May 2008; Revised: 20 June 2008 Accepted: 30 June 2008; electronically published: September 2008

Summary Gastrointestinal stromal tumor (GIST) is currently the most common gastrointestinal sarcoma of mesenchymal origin, with highest incidence in the 40 to 60 year age group. It has recently been identified as a distinct clinical and histopathologic entity. GIST's were previously misclassified as leiomyosarcoma/other spindle cell cancers, but currently constitute a whole spectrum of tumors that share several characteristics with interstitial cells of Cajal. Until its definitive molecular characterization, the management of GIST has been a topic of controversy and renewed interest in the field of surgical oncology. The management of GIST of the pancreatoduodenal complex has been an area of controversy from a surgical perspective as well. With delineation in the modalities of surgical management, a variety of options have been in vogue during the last decade. The article discusses the nature of GIST’s, their unusual presentation, their association with upper GI bleed, and their neural resemblance. We have encountered three gastrointestinal stromal tumors in the pancreatoduodenal complex that were similarly managed. Our cases clearly demonstrate the challenges associated in surgical and multimodal therapies for this entity. Two patients underwent “en bloc” Whipple’s procedure while the third underwent a Billroth II reconstruction. Gastrointestinal stromal tumors are comprised of a mixture of neural and myogenous features in varying proportions by electron microscopy, ranging from pure neural forms (plexosarcomas) to the purely spindle forms (leimyomas). The majority are mixed forms and differentiating them from paraganglioma, gangliocytic paraganglioma and neural endocrine tumors is important. Expression of c-kit (CD117) is noted in 95% of the cases. Management depends on the mitotic activity, grade and extent of the neoplasm, with the aim of achieving primary surgical cure with negative margins. Advanced or higher grade disease may require adjuvant chemotherapy with imatinab mesylate. GIST of the pancreatoduodenal complex is rare and management requires radical approaches. Prognosis is primarily dependent upon the ability to achieve negative margins without tumor spillage, thus favoring more radical procedures while avoiding unnecessary proximate intraoperative tumor handling (e.g. biopsies). Currently there are no definite protocols/guidelines for surgical management of GIST of the pancreatoduodenal complex due to the limited number of cases. We review all reported cases of GIST of the pancreatoduodenal region and propose management and intervention algorithms based on the known prognostic factors. We have designed a novel algorithm, the Gopaldas-Rohatgi score, that could function as a unique stratifying system for the management

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Gopaldas et al: Gastrointestinal stromal tumor of the pancreatoduodenal complex of GIST specific to the pancreatico-duodenal complex. The role of repeated resections, and new generation drugs (imatinib mesylate and sunitinib malate) in the management of pancreatoduodenal GIST are also discussed.

mechanisms of resistance and utilization of drugs with multi-targed mechanisms have only added to the complexity of management of these tumors (ContrerasHernรกndez et al, 2008). Over a period of fifteen years, we have encountered about 38 patients with gastrointestinal stromal tumors in a single institution. Of these, three patients presented with a mass in proximity to the pancreato-duodenal region requiring resection of some component of the duodenum. Two patients underwent pancreatoduodenectomy with primary tumors located in the second or third part of the duodenum. The third patient had a tumor located in the first part of the duodenum and was managed by a distal gastrectomy with duodenectomy followed by a Billroth II reconstruction. Each case demonstrated unique challenges in management both from an operative and multimodal approach. We first review briefly the biology of gastrointestinal stromal tumors and subsequently discuss in detail the multimodal approach to managing this complex entity, highlighting the essential factors that need to be taken into consideration before commencing treatment. We then discuss the role of targeted therapy utilizing tyrosine kinase inhibitors for this specific group of patients.

I. Introduction Gastrointestinal stromal tumors (GISTs) have been a subject of controversy for the last decade. Not only has their classification undergone a dramatic change from a pathologic and immunohistochemical standpoint, but their surgical management has been challenging both with respect to the operative modalities offered and the adjuvant therapies available. The advent of imatinib mesylate and sunitinib malate and their role in the management of gastrointestinal tumors has redirected management protocols for some of these tumors (Manley et al, 2002; Joensuu, 2006). Traditionally, surgical excision with attainment of negative margins has been the treatment modality of choice for most GISTs arising in the small bowel and stomach. A major challenge lies in the ability to achieve negative margins. In situations where the tumors arise in the vicinity of the pancreatoduodenal complex, the location of nearby anatomic structures often does not allow the surgeon to easily perform a marginnegative resection as in the case of small bowel or gastric tumors (Pierie et al, 2001). Tumors located in the vicinity of, adjacent to, or arising from the duodenum itself are characterized as poorly positioned GISTs. A radical approach in these cases would require a pancreatoduodenectomy, which is a major procedure associated with significant morbidities. Conservative approaches might be considered in certain cases. Controversy still surrounds the surgical approach, since the indications for different modalities are not well defined (Eisenberg, 2003). The recent introduction of imatinib mesylate (Gleevec: a tyrosine kinase inhibitor) as a drug for the treatment of GIST has gained strong momentum and has spearheaded the multimodal approach for treatment of GIST. Interesting developments in the

II. Case description A. Case 1 A 32-year-old male presented with severe malaise and weight loss. Initial evaluation revealed severe anemia (hemoglobin < 4 grams) which required multiple transfusions. Despite a normal appetite, he complained of a 4 lb weight loss over the previous 2 months. He also complained of occasional episodes of melanotic stools with similar episodes about a year prior when his hemoglobin was extremely low. He had refused an esophagogastroduodenoscopy (EGD) at that time.

Figure 1. Esophagogastroduodenoscopy. A fungating irregular friable mass is noted in the second part of the duodenum. The mass easily bled on contact. Initial biopsy was consistent with paraganglioma.

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Cancer Therapy Vol 6, page 579!

Figure 2. Computerized Tomogram. Heterogenous lobulated mass in the right upper quadrant measuring 6 X 5 X 5.2 cm. the masses are hypodense centrally suggesting the possibility of associated necrosis.

During the current admission he received a total of 6 units of packed red blood cells and an EGD was subsequently performed. A large (3 by 4cm) ulcerated mass was clearly visualized in the second part of the duodenum (Figure 1). Biopsies were obtained and initial analysis was consistent with paraganglioma. A CT scan was subsequently obtained which revealed a heterogenous mass in the right upper quadrant (Figure 2). Hypodensities within the mass suggested the possibility of central necrosis.

The patient subsequently underwent celiotomy, which revealed an 8 cm spherical, lobulated mass arising from the mesentery of the transverse colon and extending into the confluence of the duodenum and the pancreas. The mass derived a predominant part of its blood supply from the middle-colic artery. A decision was then made to proceed with an en-bloc resection consisting of an extended right hemicolectomy, a Whipple’s procedure and cholecystectomy (Figure 3).

Figure 3. Resected specimen. Demonstrating the location of the mass in the pancreato-duodenal complex. A.! Tumor mass B.! Duodenum C.! Pancreas D.! Transverse colon E.! Stomach

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colon and the posterior aspect of the head of the pancreas (Figure 4). Histologically the tumor was cellular in the non-necrotic areas, demonstrating two cellular types: there was a spindle cell component and an epithelial cell component (Figure 5). The epithelioid cells had large irregular nuclei with clearly visible eosinophilic nuclei. Mitotic figures were seen at about 1 per 50 high power fields. The epithelioid cells contained abundant eosinophilic cytoplasm. In the spindled areas of the tumor, cells had elongated nuclei and wispier eosinophilic cytoplasm. Nucleoli were not prominent in these cells. The staining pattern is indicated in Table 1. Although the initial endoscopic biopsy specimens were indicative of a paraganglioma, the final evaluations, especially with a positive CD 117 staining, were consistent with GIST.

i. Pathology The mass measured 8.0 by 5.0 by 4.0 centimeters. Although the mass was multinodular, the surface revealed no papillary excrescences. Upon opening the duodenum, there was a large ulcer measuring 1.5 by 3.5 cm. The mass appeared to be mostly extrinsic to the duodenum, and the cut surface showed patchy necrotic areas. The tumor was surrounded by a fibrous external capsule abutting the duodenum, the right and transverse

Figure 4. Low power image demonstrating smooth margin with no evidence of infiltrative pattern (100x).

Figure 5. High power demonstrating spindle shaped cells typically seen in GIST (400x).

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Cancer Therapy Vol 6, page 581! Table 1. Summary of data on our cases. Age at presentation(yrs) Gender Mode of presentation Hemoglobin ( g/dL) Procedure Duodenum involved Follow up Recurrence Imatinib(adjuvant) Pathology Tumor Size Chromogranin A Synaptophysin CD 117 CD 34 Neuron specific enolase CAM 5.2/AE-1 Keratins 39, 40, 43, 48, 50, 50.6 kD S-100 Protein MART-1 Melanoma associated marker Vimentin Mitoses /50HPF

Case I 32 Male Anemia <4 Whipple

Case II 49 Male Abdominal pain 8.1 Whipple

D2 3 yrs No No

D3 6 yrs No No

Case III 52 Male Abdominal pain 7.4 Antrectomy/DI resection/Billroth II D1 2 yrs No Yes

8 X 5 X 4 cm Negative Positive Positive Positive Positive Negative Positive Negative Positive 1

4 X 3 X 3 cm Negative Negative Positive Positive Negative Negative Negative Negative Positive <1

5 X 4 X 3 cm Negative Positive Positive Negative Positive Negative Positive Negative Positive 7

B. Case 2

C. Case 3

49 year old male with history of chronic anemia (Hb = 8.1 g/dL)and recurrent diverticulitis, presented with generalized abdominal pain. Previous episode of diverticulitis was about a year prior to current presentation. CT scan was obtained on initial work up and only mild inflammatory changes were noted around the sigmoid. However a mass was noted inferior to the head of the pancreas behind the colonic mesentery. Endoscopic Retrograde Cholangiopancreatography (ERCP) and Upper GI endoscopy was unremarkable. Small bowel follow through series revealed no luminal compression, although mucosal irregularity was noted in the distal duodenum. Positron emission tomographic scan was used to exclude any extra-abdominal disease. Since the mass was well defined and not present on the previous years tomographic imaging, surgical resection was contemplated. The mass measured 3 cm in largest dimension. Laparotomy revealed the mass to be arising form the third part of the duodenum and adherent to the pancreas. The ligament of Trietz, proximal jejunum, and fourth part of the duodenum was completely mobilized to gain adequate exposure. Complete resection en bloc could be accomplished only by Whipple’s procedure. There was no residual disease elsewhere in the abdomen. The patient was discharged form the hospital on post operative day 12. At 6 month follow up no further therapy was recommended. He has remained disease free 6 years post surgical resection.

52 year gentle man presented with a tumor early satiety, worsening regurgitation ad water brash for about 2 months. Medical history was significant for non insulin dependent diabetes. He had been treated with PPI for about a year for reflux disease. During work up, the hemoglobin was measure to be 7.4 g/dL with a hematocrit of 22%. An EGD was performed which revealed complete narrowing of the first part of the duodenum and the scope could not be passed beyond the pylorus. The pylorus itself appeared to be normal. A CT san revealed a mass about 4 cm in diameter arising from the first part of the duodenum extending into the lesser curvature of the stomach. Staging work-up was negative with no evidence of metastatic disease. The patient underwent an elective celiotomy. The tumor was pedunculated and arising form the serosal surface of the first part of the duodenum. Resection was successfully accomplished by performing an antrectomy and resection of the first part of the duodenum. The duodenal stump was closed and continuity was re-established via a Billroth II reconstruction. The patient developed ileus which resolved spontaneously with naso-gastric decompression. Oral intake was commenced on post operative day 10. He was discharged form the hospital on Day 14. Follow up at 15 days and 2 months after surgery was unremarkable.

i. Pathology Gross dimensions of the tumor were 4 by 5 by 3 cm, mostly arising from the serosal aspect of the first part of the duodenum. The entire luminal aspect of the first part of the duodenum was compress with 0.5cm ulceration in the duodenal bulb. Staining pattern is indicated in Table 1. Both spindle cell and epithelial cell components were observed. Mitotic figures were 7 per 50 HPF. The patient underwent concurrent treatment with Imatinib for a period of six months. There was no evidence of recurrent disease at 8, 12 and 24 months after follow up and he been doing well since.

i. Pathology On gross inspection, the tumor appeared to be arising from the superior aspect of the third part of the duodenum extending behind the head of the pancreas. Final tumor dimension was 3 by 3 by 4 cm and was well encapsulated. Entire capsule was intact, although adherent to the pancreas. Cross section of the tumor revealed areas of patchy necrosis. The tumor had extended up to the mucosa without evidence of any breach in continuity. The mitotic figures were about < 1 per 50 HPF. Histology was consistent with leiomyosarcoma with CD34 staining pattern consistent with GIST. All resected margins were negative.

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Gopaldas et al: Gastrointestinal stromal tumor of the pancreatoduodenal complex as a subdivision of GIST and managed similarly (Segal et al, 1994). GIST’s lack the distinct features of classic leiomyomas, schwannomas and their malignant counterparts (Kerr et al, 1999). Although the terms leiomyoblastoma and plexosarcoma (Appelman and Helwig, 1977; Dalaker and Harket, 1980) were used depending on the predominant cellular pattern, the inclusive term GIST was introduced to collectively* refer all these tumors (Saul et al, 1987). The discovery of the CD 34 stem cell antigen expression in these tumors was an important landmark and used as a major diagnostic criterion for GIST (van de Rijn et al, 1994; Miettinen et al, 1995). The observation of a more consistent expression of the '()*gene in a series of GIST (Ernst et al, 1998) led to a completely different approach to these tumors. Gain of function mutations in the juxtamembranous domain of the '()*gene results in a ligand independent activation of the CD 117 receptor (Hirota et al, 1995). Studies have correlated '()*gene mutations with the malignant potential of CD 117 expressing tumors (Lasota et al, 1999; Taniguchi et al, 1999).

III. Discussion Gastrointestinal stromal tumors have been recognized more frequently in the last decade as a clinicopathologic distinct group of tumors with a varying spectrum but characterized by distinct immmunohistochemical patters. GIST are characterized by a vast heterogeneity in cellular origin, differentiation, clinical behavior, anatomic distribution and prognosis most of which had been poorly understood until the past 5 years (Lee JS et al, 1995; Graadt van Roggen et al, 2001). Initially confused with smooth muscle tumors (Lavin et al, 1972; Miettinen, 1988), they were subsequently individualized based on ultrastructural and immunohistopathologic patterns. Our aim is to review in detail the management of GIST that arises from the duodenum or its vicinity. We define any tumor in contact with the duodenum, based on any imaging, as a tumor of the pancreatoduodenal complex. GISTs have been referred to by various acronyms, including GANT (gastric autonomic nerve tumors), STUMP (smooth muscle tumors of uncertain malignant potential) and GIPACT (gastrointestinal pacemaker cell tumor). The many acronyms only serve to highlight the controversy associated with the cellular origin of these tumors (Dierkes-Globisch et al, 2001).

B. Interstitial cells of Cajal and CD 117 mutation Torihashi and colleagues demonstrated in 1999 that blockade of '()* gene* signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype, indicating the plasticity between ICC and smooth muscle cells and the common origin based on in vitro studies. The same mechanism is proposed to be responsible for the etiopathogenesis of GIST (Pauwels et al, 2005). Huizinga and colleagues showed in 1995 that the '() gene variants were responsible for both ICC and intestinal pacemaker activity. CD 34 expression is noted in many cell types and hence CD 34 expression in GIST does not define lineage but merely suggests the primitive nature of the mesenchymal cells (Miettinen et al, 1995). Although their functional aspect remains controversial, some authors still believe the ICC to be an integral part of gut motor physiology, controlling peristalsis and muscular activity by acting as pacemaker cells and possibly as neurotransmitter mediators (Huizinga et al, 1997; Sircar et al, 1999). ICC and smooth muscle cells have been shown to originate from common precursors (Kluppel et al, 1998). Other features identifying GIST include ultra structural identification of complex interdigitating cell processes with rudimentary cell junctions (Rumessen, 1996). The ICC do not have unique identifying characteristics: identification is dependent on ultra structural features and their spatial relationship to surrounding cells, which is unfortunately distorted or lost in neoplatic cells (Sircar et al, 1999). Although they have close contact with nerve bundles and smooth muscles, their contractile apparatus is less well organized than in smooth muscles (Faussone-Pellegrini et al, 1990). The ligand for the membrane-bound tyrosine kinase receptor CD 117 encoded by the '()*gene happens to be stem cell factor (SCF). The interaction between SCF-CD 117 is

A. Defining GIST GISTs are best defined as a group of neoplasms of non-epithelial origin. Located along the entire length of the gastrointestinal tract, they arise either from epithelioid or spindle cells whose embryologic origins are shared with the Interstitial Cells of Cajal (ICC). They manifest along a spectrum ranging from those involving predominantly neural components to those with predominantly myoid components (Neuhaus et al, 2005). The term GIST was introduced in 1983 (Mazur and Clark, 1983) and for many years remained a vaguely defined type of tumor. In 1984, Herrera et al. described a subgroup of gastrointestinal stromal tumors termed GANT (gastric autonomic nerve tumors) or plexosarcomas (Herrera et al, 1984) based on ultra structural properties. Consequent to their similarity to autonomic nerve structures, they were presumed to originate from the intramural autonomic plexus. Kindblom and colleagues subsequently demonstrated in 1998 that the ICC of the gastrointestinal tract form a complex cellular neural network, which is proposed to play a vital role in intestinal motility. These cells exhibit both myoid and neural features, and were presumed to be candidates for the histogensis of the tumor. However, in detailed reviews that followed, GANT’s were reclassified as GIST’s. The discovery of the CD 117 !"#$%&"& by the '()* gene) gain of function mutation by Hirota and colleagues in 1998 was an important landmark responsible for the reclassification of these tumors into a distinct group. The previous GANT type of gastrointestinal stromal tumor were presumed to be a more aggressive variant of GIST (Tornoczky et al, 1999). Although immunohistochemistry may be helpful for classifying GIST, electron microscopy is considered the gold standard for making a diagnosis of GANT, which is now classified

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Cancer Therapy Vol 6, page 583! exon mutation has been defined for duodenal GIST due to the rarity of cases indicating their potentially different behavior. "#$! gene mutations leading to ligandindependent activation of the receptor, resulting in uncontrolled intracellular phosphorylation, are seen in 50 % of GIST’s (Hirota et al, 1998). Specific treatment of GIST relies on the utilization of tyrosine kinase inhibitors to block the over activity of the mutated CD 117 receptor and is the basis for current drugs utilized in adjuvant therapy (Lee et al, 2001).

important elsewhere for the maturation of germ cells, bone marrow stem cells, melanocytes and mast cells, and is not restricted to the ICC family (Huizinga et al, 1995). In normal gut, the ICC are the only cells which demonstrate positivity for CD 117, CD 34 and vimentin. ICC are also MSA negative, desmin negative, PGP negative, S100 negative and tryptase negative (Sircar et al, 1999). This is essential in differentiating GIST from other confounding tumor types. The close differential diagnosis would be ganglia and mast cells which are vimentin positive, CD 117! positive and yet CD 34 negative. Ganglion cells are NSE and S-100 positive. Schwannomas are PGP positive, S-100 positive, vimentin positive and negative for CD117, CD 34 and MSA. Leiomyomas on the other hand are MSA, desmin and vimentin positive and CD- 117/CD 34/PGP negative (Rumessen et al, 1992). Gain of function mutation in "#$! gene results in activation of the CD 117 without the necessity of ligand interaction. Clone analysis has shown the CD 117 receptor to be only immunoreactive on half of the cells (Hirota et al, 1998). This indicates that only one allele is mutated and responsible for the tumor phenotype, while the normal allele, which is not mutated in the remaining clones, is responsible for the staining characteristics. The mutant allele responsible for the neoplatic phenotype loses immunoreactivity to the CD 117antibodies. Loss of the second allele may result in a more aggressive tumor and increased metastasizing potential. Hence, a tumor arising out of a "#$!gene mutation may by virtue of mutation lose reactivity to CD -117and yet result in a gain of function mutation, thereby presenting as a clinically aggressive CD 117 negative stromal tumor (Hirota et al, 1998). A similar analogy to CD 34 reactivity has been proposed. Loss of %&'(() and CD 34 immunoreactivity within the same tumor is responsible for the variation in stromal characteristics of these immunophenotypic markers. Hence GIST’s, although most commonly CD 34 positive and CD 117 positive, could still present as a variant form, with either one or both markers staining negative. An aggressive form of GIST could thus be missed on immunohistochemical staining if all the alleles of CD 117 or CD 34 are mutated although this happens to be a very rare variant. CD 34 negativity without loss of reactivity to CD 117 happens to be the more common aggressive variant (Hirota et al, 2006). Clinically this is important when dealing with very aggressive types of GIST which lack consistent staining patterns, making diagnosis and treatment difficult. Platelet-derived growth factor receptor ! (PDGFR! ) has been shown to be mutated in certain types of GIST without the evidence of a "#$!gene mutation (Bernet et al, 2003). Tumor cells could be non-reactive to CD-117 antibodies due either to non-expression or mutation of "#$! gene resulting in expression of different epitopes of CD 117 which are not recognized by the CD-117antibodies. The latter case could be a gain of function mutation resulting in over expression of "#$! gene in the setting of non-reactive immunohistochemistry (Miettinen and Lasota, 2006). Mutations in the juxtamembranous domain of CD-117 (exon 11 and 9) are the most common in GIST’s of all sites (Buchdunger et al, 2000). No specific

C. Sub classification of gastrointestinal mesenchymal tumors In a series of 244 cases (Rudolph et al, 2002), antigen expression varied from tumor to tumor except for vimentin, which was consistently positive. CD 117 positive tumors were classified as GIST regardless of the co-expression of the other antigens. CD 117 negative tumors with strong expression of myogenic markers, and with no significant neural or CD 34 expressions, were considered GI leiomyogenic tumors. CD117 negative tumors with strong S-100 or GFAP or PGP positivity in the absence of myogenic tumor markers were considered GIGT (gastrointestinal glial/schwannian tumors). Concurrent expression of synaptophysin in CD 117 negative tumors was characterized as GINT. GIFT’s were a special category of tumors with null phenotype lacking any antigen expression except for vimentin. Strong CD 34 positive tumors that were CD 117 negative and did not fit any of the above criteria were classified as GINST (gastrointestinal negative stromal tumors) (Table 2). However, if the previous hypothesis that mutation of both "#$! *+,+! alleles results in absence of staining but in a more aggressive form of tumor, then GINSTs should indeed be a more aggressive form of GIST. The data presented by Rudolph and colleagues in 2002 and DeMatteo and colleagues in 2000 is consistent with that hypothesis. The percentage of tumor phenotypes varied according to the anatomic distribution of the tumor with GIST accounting for 100% of esophageal cases, 80 % of jejunal tumors and 54% of gastric tumors. No specific data is available for duodenal tumors. On an overall basis, 9% of all GINST’s occurred in the duodenum. GIFT’s were almost exclusively noted in the duodenum. Overall, 9.8 % occurred in the duodenum, with GIST’s and GIFT’s being the most common forms (Rudolph et al, 2002). Tumors categorized as GIFT’s, if assumed to be dual mutant forms of CD 34 and CD 117 would technically have a worse prognosis. The cumulative survival rate for GIST’s has been reported as around 0.6, GINST’s as 0.4 and GIFT’s as 0.2. Hence, CD-117 negative tumors, although classified as a separate group, could in fact be more aggressive forms of GIST, involving mutations of both the "#$! gene! alleles. Genetic analysis for "#$! mutations would be more helpful than immunohistochemistry in characterizing the nature of GIST’s. Of note is the fact that CD 117 negative stromal tumors exhibit more epithelioid features (Tortella et al, 1987).

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Gopaldas et al: Gastrointestinal stromal tumor of the pancreatoduodenal complex Table 2. Sub-classification of gastrointestinal mesenchymal tumors. GIST (Gastrointestinal stromal tumors) Leiomyoblastoma Plexosarcoma (GANT) GINST (Gastrointestinal negative stromal tumors) GILT (Gastrointestinal leiomyogenic tumors) GIGT (Gatrointestinal glial/schwannian tumors) GIFT (Gastrointestinal fibrous tumors) GINT (Gastrointestinal neuronal /glial tumors)

D. Malignant figures

potential

and

mitotic

CD -117 ( c-kit) positive Predominant spindle myoid cells Predominant epithelioid cells CD -117 ( c-kit) negative CD - 34 positive CD -117 ( c-kit) negative ! - Smooth muscle actin or desmin CD -117 ( c-kit) negative S-100 or glial fibrillary acidic protein CD -117 ( c-kit) negative vimentin CD -117 ( c-kit) negative Neuronal /glial markers

E. Anatomic distribution GIST accounts for most mesenchymal tumors within the gastrointestinal tract (Evans et al, 1985), highlighting the non-epithelial nature of the tumor. Tumors of true smooth muscle, neural (schwannoma), fibroblastic and vascular origin are thus excluded. Differentiating from pure smooth muscle cell tumors is an important aspect in the diagnosis of GIST. Smooth muscle tumors show variably spindled cells with cigar shaped nuclei and bipolar perinucler location of the cytoplasmic glycogen. Smooth Muscle Actin (SMA) and Muscle Specific Actin (MSA) are generally positive in smooth muscle tumors but patchy or absent in GIST (Bagnolo et al, 1998). GIST’s can arise in any part of the GI tract, but more commonly occur in the stomach (65 to 70%) and small bowel (30 to 45%), and less frequently in the esophagus, colon and rectum where true myogenic tumors predominate (Antonioli et al, 1989). Pure leiomyomas are less common than GIST’s in the duodenum, and the esophagus is the only location in the GI tract where leiomyomas predominate compared to GIST (Emory et al, 1999). The majority of GIST’s are located in the stomach and small intestine with only 4% located in the duodenum (Meesters et al, 1998). A recent review paper indicated a higher percentage in the duodenum but only in a single institution (Winfield et al 2006). Also, some of the retroperitoneal GIST tumors were found in a juxtaduodenal location, further indicating that these, in fact, might be arising from the duodenum itself (Lee et a; 2001).

Traditionally a mitotic index of more than 4 mitotic figures per 10 high power fields (HPF) was considered malignant. However, recent reports have proposed a revised mitotic index of more than 10 mitotic figures / 50 HPF to be more appropriate (Meesters et al, 1998). In a classification proposed by Ranchod and Kempson in 1977 for grading GIST, a tumor more than 4 cm in size was classified as a gastrointestinal sarcoma if it was hypercellular and associated with a high mitotic index. These tumors were considered low grade if the mitotic index was less than 10 per 50 HPF, and high grade if more than 10 per 50 HPF. Recent work by Fletcher and colleagues in 2002 has reclassified the malignant potential of GIST tumors based on size and is currently held as the standard. Cut off values for tumor sizes are 5cm and 10cm, while mitotic figures are 5 and 10 per 50 HPF. Tumors which have a mitotic index of more than 10 /50 HPF or a size of more than 10 cm or those which are intermediate in size (5 to 10 cm) with a mitotic index between 5 and 10 /50 HPF were all classified as high risk tumors. Tumors with a size of less than 5cm and a mitotic index of less than 5 per 50 HPF were considered low risk. A subset of this group with size less than 2 cm was classified as very low risk. All other combinations were classified as intermediate risk tumors. This classification, although useful in directing the indication for adjuvant therapy, does not help in identifying metastatic potential. Even tumors of low risk can potentially metastasize, thereby highlighting the point that all GIST's should be treated with caution (Schubert et al, 2006). The other problem associated with this risk stratification is that the mitotic grade will be known only after the final specimen is resected, and hence will not be helpful in guiding the extent of surgical resection preoperatively. Surgical resection has to be decided primarily on size and feasibility of resection based on the anatomic location of the tumor. A fine needle aspiration biopsy (FNAB) could be very helpful in confirming the diagnosis, but may be very difficult to use in stratifying low or intermediate grade tumors. The presence of mitotic figures in a FNAB is more likely consistent with a high risk tumor (Elliott, 2006).

F. Clinical presentation Overall, the peak incidence of GIST is in the fifth and sixth decades, being infrequent before the age of 40 (Hinz et al, 2006). Based on a review of the cases reported in the literature (Sakamoto et al, 2003; Hughes et al, 2004; Winfield et al, 2006) GIST’s of the pancreatoduodenal complex typically present with bleeding. Most small GIST’s are asymptomatic and go unnoticed until an esophagogastroduodenoscopy is performed for some other reason. Symptomatology is dependent on the size and the location of the tumor (Hompes et al, 2004). The most common complaint for patients with GIST is usually

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Cancer Therapy Vol 6, page 585! CT scan with reconstruction, if feasible. The relation of the tumor to the surrounding structures has to be delineated. CT scanning is considered the imaging modality of choice for anatomic evaluation of GIST (Lee et al, 2004). Numerous characteristics are assessed, but the most important features indicating a need for further workup are the sharpness of the tumor margin and its proximity to the luminal aspect of adjacent duodenum. Evaluating the extent of the mass, detecting possible metastasis, and assessing the resectability of disease during staging are easily accomplished utilizing a CT scan. A triphasic scan should be obtained for baseline evaluation (Blay et al, 2005). Nodal evaluation is unnecessary as these tumors very rarely involve lymph nodes. If tumors are juxtaduodenal, then an endoscopic ultrasound would be necessary to evaluate the interface between the tumor and the duodenum. If the tumor is abutting the duodenum, then a transluminal EUS-guided biopsy should be considered without the risk of contaminating the peritoneal cavity. A sharp interface noted on the CT scan indicates that the tumor may not be infiltrating the surrounding structures, allowing enucleation as a possibility if the tumor is small. Tumors that do not have a sharp delineation are most likely to require a radical approach. 18-Fluodeoxy-glucose Positron emission tomography (18 FDG-PET) may be used to evaluate the extent of the disease and to screen for metastatic disease. However, a PET scan should not be used for initial detection as it is very non-specific: it should be utilized only after previous evaluation with a CT scan. PET has a specific role only in staging a GIST. Since PET and CT scans have different uses, both should be obtained in all cases of duodenal GIST even if the primary tumor is very small. Even tumors with very low risk have a metastatic potential so PET is indicated in these cases also. In circumstances where small GIST’s are missed by CT scan, a PET scan would raise the index of suspicion by highlighting the area of metabolic activity. In addition, a PET scan also delineates the presence of surrounding inflammation due to hemorrhage or myxoid degeneration from that of tumor growth and infiltration (Goerres et al, 2005). PET and CT scan in combination precisely delineate lesions and allow intricate planning of the surgical procedure. The assessment of operability is a crucial step in the preoperative workup of a GIST, as the prognosis is excellent after complete resection (Emory et al, 2005). PET and CT are important in the current era of imatinib mesylate neoadjuvant treatments, as they help to categorize the disease as responsive, stable or progressive. However, absence of PET activity does not necessarily correlate with absence of tumor cells on pathology. It is also important to analyze the nature and appearance of these lesions after imatinib therapy. Appearance of new lesions may be difficult to identify since liver metastases that are initially occult, could present as new cystic changes due to tumor destruction consequent to adjuvant therapy on a follow up CT scan (Linton et al, 2006). The appearance of a nodule within a mass pattern is an ominous sign and consistent with recurrent disease or new onset resistance. Hence attention to detail and

vague abdominal pain or discomfort (De Marco et al, 2005). This is unlike the presentation noted in GIST of the duodenum, which typically manifests with bleeding and associated microcytic anemia (Carvajal et al, 2006). All three cases encountered in our institution presented initially with anemia. We attributed the bleeding to pressure erosion of the duodenum. As opposed to other areas of the GI tract, GIST’s arising from the duodenum do not have surrounding free space to grow, making the pliable duodenum the least resistant pathway for tumor expansion. Other presentations include anemia, anorexia, weight loss, nausea, fatigue, and acute intraperitoneal bleeding or perforation (Goh et al, 2005). The tumor may or may not arise from the duodenum per se, but rather from the retroperitoneum or any adjacent structure. As with most primary mesenchymal tumors, GIST’s of the pancreatoduodenal complex tend to be typically of submucosal origin, indicating that they have to be associated with the duodenum in most circumstances with extension possible into any of the surrounding structures. GIST’s tend not to infiltrate but to push surrounding structures away. That being said, the desmoplastic reaction in large tumors could make it technically difficult to identify the exact tissue plane. Pressure necrosis associated with the tumor pushing onto the surrounding structures could result in obliteration of the capsule due to inflammatory response. Based on classification by Yamada and Ichikawa in 1974, most tumors are Type 1 tumors, i.e. sessile or slightly raised. Rare reports of Type IV - polypoid lesions with long stalks arising from one region of the GI tract and causing a distal obstruction down stream could be found in the literature (Kim et al, 1999).

G. Diagnostic and staging workup With the initial presentation usually being an upper GI bleed, EGD is the first modality of evaluation. If the EGD reveals an obvious lesion, then a luminal biopsy should be considered. In most circumstances, GISTs are not diagnosed until there is a final pathologic examination of the resected specimen. GIST tumors with erosion into the lumen of the duodenum are easily accessible for tissue biopsy and a preoperative diagnosis can be established in some cases. In most circumstances, the tumors can be however easily approached via an Endoscopic Ultrasound guided Fine-needle aspiration (EUS-FNA). EUS-FNA has advantages in that intraperitoneal seeding of the tumor is avoided and a diagnosis can be established with reasonable certainty (Chatzipantelis et al, 2008). Adequate specimen sufficient enough to perform the appropriate immmunohistochemical stains could be easily obtained. It has been shown that that when combining cytologic and immunocytochemical studies, EUS-FNA is accurate and efficient in the diagnosis of GIST (Fu et al, 2002). However, in those cases where the tumor is not easily accessible for a transgastrointestinal luminal biopsy, a high index of suspicion is necessary. In such circumstances, transperitoneal biopsy is best avoided due to tumor spillage associated with the procedure. Instead a detailed non-invasive staging work up must be considered. Initial diagnostic work up consists of a contrast-enhanced 585

Gopaldas et al: Gastrointestinal stromal tumor of the pancreatoduodenal complex margins did not. However, subsequent data by Gold and colleagues in 2007, have demonstrated that R0 resection for GIST was associated with a statistically significant improvement in survival compared to either R1 or R2 resection. We strongly favor negative microscopic margins to be accomplished in the very first surgical specimen. The essence here is to avoid traveling though tumor cells at all possibilities and operative dissection should be carried out well away from the vicinity of the tumor. In certain cases the tumor may be easily lifted off surrounding structures. However, we would like to specify distinctly that only if the tumor is merely sitting on a surrounding structure should an enucleation be considered. This still leaves the pedicle of the tumor to be dealt with. We propose that a 2 cm margin should be obtained at the pedicle. The aim is to achieve negative margins, although it is not clear what the margin should be. Given the small number of cases and the high recurrence rate we feel that a 2 cm margin should be adequate for tumors less than 2 cm in size, or for those with a very low risk potential. If the tumor shows even the slightest evidence of inflammatory adherence to any surrounding structure, one could make a fitting argument not to dissect on the tumor in an attempt to separate it. The friability of the tumor is associated with a high risk of rupture. We prefer a no touch technique by staying away from the tumor even if it means resecting the surrounding structures. In essence, only very low risk tumors in the free wall of the duodenum can be technically resected by a wide local excision. A tumor in the duodenum of more than 2 cm in size would require more than 6cm of the duodenal wall to be resected (allowing for 2 cm on either side). Reconstruction of such a large defect could be done, but a resection of such magnitude would be associated with unnecessary proximate handling of the tumor. A pancreaticoduodenectomy in such cases would be a better modality as it keeps the dissection well away from the tumor and also provides the highest chance of achieving a negative margin. There are a few additional factors to be considered before a pancreaticoduodenectomy is performed. The performance status of the patient is important, especially when an operation of such magnitude is considered. A patient with good physiologic reserve, younger age, and no major comorbidity will better tolerate the operation. Such a patient is also more likely to benefit and have the highest chance of surgical cure offered. Tumors confined to the bulb of the duodenum (more than 2 cm proximal to the junction of the first part and second part of the duodenum) could technically be considered for a gastroduodenectomy with a Billroth II reconstruction provided the tumor is on the convex side of the duodenal sweep and well away from the pancreas. Resection of tumors in the fourth part of the duodenum could be accomplished by an intestinal derotation (Valdoni-Strong) procedure (De Nicola et al, 2005). However, if the tumor is located at the junction of the third and fourth part of the duodenum, we strongly suggest that a Whipple’s procedure be performed instead. As with any major cancer operation, the entire abdominal cavity should be assessed for metastatic

homogeneity of the lesions and not the size alone is crucial in determining response and early identification of resistance to treatment on follow up CT imaging (Shankar et al, 2005).

H. Surgical management The aim of surgery in any type GIST is to achieve negative microscopic margins. Surgery is the principal modality of treatment in patients with resectable GIST (Pierie et al, 2001; Rossi et al, 2003). Grossly negative margins are not prognostic indicators in GIST. Tumors of the pancreatoduodenal complex pose many technical challenges because of the surrounding anatomy. Unlike tumors of the small intestine or stomach where wide margins can be achieved without a significant technical challenge, tumors in the duodenum do not lend themselves to a simple method of operative resection. The issue to be addressed is the necessity of a pancreaticoduodenectomy for these tumors. The morbidity associated with pancreatoduodenectomy is quite high, so the indications for this procedure in GIST tumors must be specific. There have been reports of even large tumors being treated with wedge excisions especially if the tumor is predominantly extra luminal (Sawaki et al, 2003). In the case described by Sawaki and colleagues 2003, recurrent metastatic disease was noted within two years for a tumor that was resected from the duodenal bulb. In a case series described by Winfield and colleagues 2006, 5 out of 8 cases were treated by pancreaticoduodenectomy, but none of the tumors were located in the first portion of the duodenum. The question remains: Is pancreaticoduodenectomy the ideal procedure or could less radical approaches be entertained? To answer this we need to better understand the tumor biology. Based on a review of literature, there have been no consistent long term studies in answer to this question. There have been anecdotal cases where the surgical modality has varied from a simple enucleation to a radical pancreaticoduodenectomy. Based on a review by Piere and colleagues in 2001, complete resection is the only modality of cure. Even with complete resection fiveyear survival was only 42 % and incomplete resection was associated with a 9% survival. In addition, complete resection was achieved in only 59% of cases. The objective of surgery for primary GIST is the resection of tumor with an intact pseudocapsule. This is critical, as damaging the pseudocapsule may allow for bleeding/hemorrhage and/or dissemination. One might favor an argument that enucleation could be utilized as an option as these tumors do not invade the surrounding structures. GIST’s typically are very friable and enucleation is associated with the risk that the pseudocapsule could be violated. Violation of the pseudocapsule could potentially result in spillage of tumor cells and henceforth upstage the disease. With the potential of “cure” only achievable through surgery, complicated by the fact that chemoradiation is not very helpful in GIST, we feel that enucleation should not be attempted at all in GIST. Initial studies by De Matteo and colleagues in 2000 have indicated that although size correlated with poorer prognosis, positive microscopic

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Cancer Therapy Vol 6, page 587! Whipple’s should be avoided: if the patient has a tumor of less than 5 cm in size then one should probably perform a radical resection if the performance status of the patient is excellent. However, this approach remains controversial until more data are available. If the tumor is more than 5 cm then any procedure short of a Whipple’s, which can accomplish a resection of most of the tumor, should be considered. We propose this based on the tumor biology and by extrapolating data from studies that have evaluated options for GIST located in other areas of the GI tract. Another important factor influencing the prognosis of GIST tumors is the tumor size at presentation. Tumor size also influences the nature of resection to be accomplished, especially if the tumor is located in critical regions such as at the pancreatoduodenal interface. Tumor size at presentation predicts recurrence-free survival: patients with tumors >10 cm have a 5-year survival rate of 27%, while those with tumors <5 cm have 5-year survival of 82% (Raut et al, 2006). The possibility of discovering advanced disease should be clearly addressed with a pre-operative PET scan. Surgical cures are much more common when incidentally discovered GIST’s are included in the data. These asymptomatic tumors are often small, with benign histologic features, and may be evident only on pathologic examination of a specimen removed for another reason.

disease. The presence of any metastatic disease precludes any major resective procedure, especially a pancreaticoduodenectomy, as the risks undertaken with the operation in these cases are not worth taking since the disease is already advanced. However, palliative procedures may be indicated and biopsy of the suspected metastatic lesions with frozen section confirmation should be done. The role of debulking alone in GIST has not been clearly established. However, as a part of multimodal treatment for advanced disease, debulking has shown to improve survival (Gold et al, 2006). Raut and colleagues in 2006 reported their data on debulking GIST tumors for cases that were managed with adjuvant imatinib mesylate therapy. Patients were categorized into three groups based on response to tyrosine kinase inhibitor neoadjuvant therapy, namely stable disease, limited progression or generalized progression. After surgery, there was no evidence of disease in 78%, 25%, and 7% of patients with stable disease, limited progression, and generalized progression, respectively. Bulky residual disease remained after surgery in 4%, 16%, and 43% of the patients with stable disease, limited progression, and generalized progression. Twelvemonth progression-free survival was 80%, 33%, and 0% for patients with stable disease, limited progression, and generalized progression. Twelve-month overall survival was 95%, 86%, and 0% for patients with stable disease, limited progression, and generalized progression. They concluded that patients with advanced GIST’s exhibiting stable disease or limited progression on kinase inhibitor therapy have prolonged overall survival after debulking procedures. Even though partially successful treatment may render initially inoperable tumors potentially resectable from a technical standpoint, the biologic behavior of the tumor simulates metastatic disease and surgery in these cases is primarily cytoreductive (Neuhaus et al, 2005). It has been proposed (Rutkowski et al, 2006) that minimizing the tumor burden will decrease the likelihood of imatinib resistant clones from developing within the disease in situ. Surgery has little to offer in the setting of generalized progression. Surgical removal of residual disease after adjuvant therapy may allow complete remission in selected patients responsive to imatinib, theoretically prolonging survival provided imatinib is continued. Simultaneous metastasectomy with resection of the primary in pancreatoduodenal GIST at initial presentation has been reported (Stratopoulos et al, 2006) without neoadjuvant treatment. However, follow-up in this case was not of adequate duration to justify this approach until more data are available on the benefits of neoadjuvant imatinib mesylate. The paper by Raut and colleagues in 2006 does not specifically address the patient with duodenal GIST or cases where the surgeon discovers advanced disease for the first time at celiotomy. A surprise discovery puts the surgeon in a dilemma as to whether a radical resection should be performed or not. As data are limited, we suggest that if the tumor is of intermediate or low risk, despite the presence of metastatic disease, a debulking operation should be considered. As far as possible a

I. Laparoscopy In general, laparoscopy is not indicated once the location of the tumor and the stage has been established. However in doubtful cases where there is a high suspicion of peritoneal disease or liver metastasis, we suggest a laparoscopy and biopsy with intraoperative laparoscopic ultrasound guidance.

! "#!$%&!'()&!(*!+,-.+/+0!,&12)-.&! The phenylaminopyrimidine imatinib mesylate was the product of multiple rounds of selection for a compound capable of inhibiting the Bcr-Abl kinase that is prevalent in CML. The effects of the drug were discovered in 1996 by Druker et al. and have since been utilized in managing many solid tumors (Druker at el, 1996). Imatinib mesylate is a selective tyrosine kinase inhibitor. In addition to BcrAbl, imatinib mesylate inhibits KIT and PDGFR. Receptor tyrosine kinases that are not inhibited include EGFR, VEGFR-2, and IGFR (Tuveson et al, 2001; Singer et al, 2002). The mechanism of imatinib mesylate inhibition involves occupying the target kinase’s ATP binding site, denying both the substrate and energy source for tyrosine phosphorylation (Manley et al, 2002). As the majority of GIST’s are dependent on constitutive !"#$gene$signaling, experiments performed in GIST tissue culture demonstrated that imatinib mesylate was able to inhibit tissue growth. Until the work by Tuveson and colleagues 2001 GIST was considered a chemo resistant tumor with not many options available for advanced or aggressive disease. The use of imatinib mesylate as a tyrosine kinase inhibitor was primarily restricted to treatment for chronic myeloid leukemia until the work by Tuveson and colleagues where a specific

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Gopaldas et al: Gastrointestinal stromal tumor of the pancreatoduodenal complex PET clearly has an earlier predictive benefit compared to standard CT ((Demetri et al, 2002). In addition CT scanning has a tendency to falsely indicate therapeutic failure or imatinib resistance in cases where myxoid degeneration secondary to anti tyrosine kinase therapy may be observed as an increase in the size of lesions on CT scans. PET scans, on the contrary, show a response in these cases due to decreased metabolic activity (Goldstein et al, 2005). Tumor resistance to therapy is defined as progression of tumor burden noticed on imaging studies. Response does not always manifest as an immediate decrease in size of tumor lesions but rather as an initial inhibition of growth (Verweij at al, 2004). Absence of any tumor progression after 8-12 weeks of therapy is considered a response to treatment and labeled as stable disease. These patients who respond to the initial therapy should complete of a full 6 month course of treatment of up before surgery is contemplated. Some patients manifest a clear decrease in tumor burden after initiation of therapy, and are labeled as having responsive disease, while an increase in tumor burden (not size alone) despite therapy is defined as resistant disease. According to the Response Evaluation Criteria in Solid tumors (RECIST), initial resistance is defined as objective disease progression within 3 months of randomization (Therasse et al, 2000). This cut off point was selected to include progression documented at first response evaluation after 2 months of therapy but to exclude progression documented at second disease evaluation at 4 months after initiation of therapy. Late resistance was defined as tumor progression noticed after 3 months of initiation of imatinib therapy. The major drawback of the RECIST criteria is the fact that tumor response is judged primarily by unidimensional tumor size. This problem more pronounced in GIST treated with molecularly targeted agents such as imatinib where the tumor can increase in size despite good response. Also, focal progression within a responding GIST can be overlooked with the RECIST criteria (Choi et al, 2007). The Choi criteria use a combination of tumor size and density is better suited in early response evaluation and in predicting long-term prognosis (Benjamin et al, 2007). Initial resistance was noted in 12% of all cases analyzed by Van Glabbeke and colleagues in 2005. The presence of lung metastasis, absence of liver metastasis and baseline low hemoglobin of less than 8 gm/dl were highly significant adverse prognostic factors, while a high baseline granulocyte count showed borderline significance. A multivariate analysis of patients with late resistance revealed that tumor size, high baseline granulocyte count (> 5 x 109/L), non gastric primary tumor (which would technically include all duodenal GIST’s) and randomization into the low dose group (400 mg /day of imatinib) were independent factors of adverse prognosis. We thus infer that the highest possible tolerable dose of imatinib should be administered to all patients with pancreatoduodenal GIST who require neoadjuvant treatment. Late resistance to imatinib is independently predicted by the size of the lesions present at the commencement of neoadjuvant therapy. Thus, tumor size by itself forms an important prognostic factor for primary

effect was noted on cell lines with oncogenic allele mutations in !"#$ gene. This correlated with induction of apoptosis and decrease of cellular proliferation. With the first clinical anecdote of imatinib demonstrating significant promise in the treatment of metastatic GIST, GIST began to be included in many randomization trials for various advanced cases and now has been selectively indicated for use in patients with bulky, high grade or advanced disease (Joensuu et al, 2001). Studies have shown up to 90% of patients with metastatic disease stabilizing or responding to imatinib therapy (Demetri et al, 2002). Withdrawal from high dose imatinib therapy has in some cases been associated with a rebound effect leading to tumor flares. This is explained by the high concentration of stem cell factor (SCF) ligand accumulating in the serum (Bono et al, 2004). After therapy is initiated, careful surveillance is necessary to assess the adequacy of drug dosage and tumor response. Currently there are several trials underway by the EORTC and ACOSOG evaluating the efficiency of standard dose imatinib mesylate (400mg a day) with the high dose regimen (800mg per day) (Goldstein et al, 2005). A phase I EORTC study (Demetri et al, 2006), identified the highest feasible dose of imatinib to be 400mg bid and indicated extensive activity against tumor burden. Some studies have shown a small but statistically significant benefit in progression-free survival with the high dose regimen (Verweij at al, 2004).

K. The role of Sunitinib for resistant cases In patients who demonstrate a disease progression despite imatinib therapy, an alternate drug, sunitinib, which is a selective inhibitor of tyrosine kinase, may be helpful (Demetri et al, 2006). Sunitinib malate is a novel oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities. Sunitinib has been recently approved for the treatment of patients with GIST after disease progression or intolerance to imatinib mesylate therapy. Oral sunitinib has been shown to possess a high level of efficacy with the 50 mg daily dose for 4 weeks (Rutkowski et al, 2008). Currently, sunitinib malate is approved only as a second-line drug. Unlike imatinib mesylate, sunitinib is a multitargeted agent inhibiting tyrosine kinase, PDGFR A & B, vascular endothelial growth factor receptors and colony stimulating factor 1 receptor. Resistance to imatinib has been proposed based on mutation of KIT mediated tyrosine kinase receptor ligands and PDGFR ! receptors. Sunitinib's efficacy in these circumstances has been attributed to the multitargeted nature of the drug (Le Tourneau et al, 2007). Although this drug is still being evaluated, initial studies have shown a fourfold increase in median time to tumor progression compared to placebo (Joensuu, 2006).

L. Assessment of response to therapy: RECIST versus Choi criteria CT and PET are both necessary for the monitoring of tumor response. PET changes are seen much earlier then detectable CT changes consistent with imatinib response.

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Cancer Therapy Vol 6, page 589! with a combined score of 5 or more (potential scores ranging from 2 to 8) require neoadjuvant imatinib therapy. Imatinib mesylate is indicated in most patients with high grade GIST and grading is usually not possible unless the specimen in resected. In the setting of neoadjuvant therapy, assessment of mitotic index by preoperative biopsy techniques is technically difficult unless an adequate core biopsy specimen is available. At least 50 HPF need to be examined to definitely conclude a low or intermediate grade GIST. We prefer to avoid core needle biopsies due to potential intraperitoneal tumor spillage unless the tumor can be biopsied through an endoscopic approach. In other circumstances, we would proceed with only an FNA, which would complement the CT findings and help in identifying certain high grade GISTs. Although less tissue is sampled with an FNA, the presence of necrosis or higher mitotic figures may help in clinching the diagnosis. The presence of many mitotic indices in a smaller sample is indicative of a high-grade tumor. FNA specimens are preferred from a pre-operative perspective as they cause less tumor spillage, but they are more challenging with respect to grading the tumor. However, the very presence of mitotic cells and the Ki-67 labeling indexing in FNA specimens have been shown to be significant predictive factors for high grade GIST (Ando et al, 2002). After the score is calculated, the patients either undergo neoadjuvant therapy or operative exploration. Neoadjuvant therapy is initiated at full dose and patients are reevaluated with a CT and a PET scan at 6 to 8 weeks and again at 16 weeks. Patients with responding or stable disease after the follow up imaging studies undergo the full course of imatinib mesylate. Patients who demonstrate relapse or late resistance are either considered for alternative modalities of treatment with sunitinib or for definitive operative management or debulking procedures depending on the extent of the disease. Patients who respond to imatinib mesylate therapy are reassessed using the scoring system: if their score is less than 5 they are managed surgically. Those with a score of more than 5 are considered for definitive surgery or debulking procedures depending on the extent of the disease. The aim of operative exploration is to achieve negative margins without tumor spillage and, as indicated in the algorithm, the type of procedure varies depending on tumor location and the size. Caution has to be exercised when the surgical plan is modified based on therapeutic response to neoadjuvant treatment. Specifically in GIST, although tumor sizes may have decreased, there often is evidence of microscopic disease in portions of the specimen that previously had tumor-related PET activity but are currently PET inactive secondary to the neoadjuvant treatment. Utilization of the Choi criteria helps to assess tumor response in a systematic fashion and is more thorough compared to the classical RECIST criteria (Choi et al, 2004). Based on a study of 36 patients by Goh et al (2006), pathologic response of GIST to imatinib mesylate is usually incomplete and does not correlate with the complete response seen on PET scan. Their findings suggested that

disease. Treatment with imatinib is generally safe and well tolerated with the most common adverse effects being anemia, edema, nausea, diarrhea, myalgia, fatigue and skin rash (Fletcher et al, 2002). There are certain circumstances in which imatinib mesylate may not be useful as a therapeutic agent. Approximately 30 % of GIST’s have been noted to lack !"#$ gene mutations but instead possess an activating mutation of Platelet Derived Growth Factor Receptor-! (PDGFR! ) (Heinrich et al, 2003). Familial GIST with germ line mutations of PDGFRA genes and Neurofibromatosis (NF) genes has been reported. These cases are most likely to present with imatinib resistance due to different tumorigenic mechanisms (Nishida et al, 1998).

!"# $%&'('()# *+,-'*./0,# -1%20345# 6(# 0,).2'-1*'7#0332.071# Imatinib mesylate has a role both as neoadjuvant and adjuvant treatment. Based on the literature reviewed so far, the exact indication for imatinib as neoadjuvant therapy in pancreatoduodenal GIST is still controversial. In order to clearly identify the role of imatinib mesylate for treatment of patients with GIST of the duodenum, we created an algorithm based on the salient factors highlighted from limited literature enumerated above and our own experience. Our algorithm (Figure 6) begins at initial presentation, which can be characterized as involving either vague abdominal complaints or upper gastrointestinal bleeding. Each is evaluated as indicated in the algorithm until a diagnosis is established and the staging work-up completed. Most of these cases, as noted, are complex and require a thorough discussion at every institutions Tumor board before a definitive regimen is implemented for each patient. We designed a scoring system based on evaluation of published articles and have incorporated this into the algorithm which would determine those patients who are expected to require neoadjuvant treatment based on tumor size and surgical resectability (Table 3). We quantify tumor size on a scale of 1 to 4, with 1 representing tumors of less than 2 cm in size and 4 assigned to tumors 10 cm or greater in size. A similar ordinal scale is used to quantify surgical resectability, which is determined by the surgeon’s evaluation of the imaging studies and extent of disease. Surgical resectability is typically categorized into four groups based purely on technical aspects: resectable with no functional deficit; resectable with associated functional deficit; potentially respectable; and unresectable or metastatic disease. Procedures which could potentially result in functional disability include any form of reconstructive procedure other than a wide excision with a primary closure (Billroth II, Whipple’s procedure or a Valdoni-Strong procedure). All such complex procedures receive a score of 2. If the tumor is of questionable resectability based on diagnostic imaging, or if the surgeon is not sure of the surgical plan, we categorize it as potentially resectable and assign a score of 3. Patients with metastatic disease or unresectable disease or those with preoperative biopsy-proven evidence of high grade tumor are assigned a score of 4. We hypothesize that patients 589

Gopaldas et al: Gastrointestinal stromal tumor of the pancreatoduodenal complex surgical plan should be decided based on the baseline triphasic CT scan obtained on initial evaluation, and we

surgical resection will continue to play a vital role in the treatment of patients with advanced disease despite a response to imatinib mesylate. Hence we feel that the

Figure 6. Algorithm for management of GIST of the pancreatoduodenal complex.

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Cancer Therapy Vol 6, page 591! Table 3. Gopaldas- Rohatgi pre surgical scoring to determine neoadjuvant therapy for GIST of the pancreato-duodenal complex. Tumor Size cm Score

Surgical resectability Score Clearly Resectable with 1 no functional deficit Clearly Resectable with associated functional 2 deficit

! 2

1

2< tumor ! 5

2

5< tumor ! 10

3

Potentially resectable

3

4

Unresectable Or High Grade on preoperative biopsy

4

Tumor > 10

Total score !! Score is a summative score obtained by adding the scores. !! Score of less than 5 does not require neoadjuvant therapy !! Score equal to or more than 5 requires neoadjuvant therapy with imatinib mesylate !! Neoadjuvant therapy may convert unresectable tumors to resectable ones, but may not necessarily assure negative surgical margin !! Any surgery involving a reconstruction of the duodenum other than a wide excision with primary closure is considered as functional deficit

number of cases reported in literature, although there are ongoing trials, it will be a while before solid data for statistical analysis would be available; however, these guidelines may help in stratifying patients and, once enough data has been accumulated, a retrospective analysis or a prospective randomized controlled trial could provide us with more insight into the issue.

prefer to stick to the initial plan unless a significant morbidity is associated with it or unless the procedure is technically not feasible. Even though the tumor burden may have decreased, we feel that an aggressive approach based on the initial CT scan offers the best chance of obtaining negative margins. Necrosis of large and bulky tumors after imatinib mesylate therapy has been the cause of gastrointestinal hemorrhage, enterocutaneous fistula or even free perforation in certain cases of tumors elsewhere in the GI tract requiring emergent surgical intervention (Yen et al, 2006). The usual response to imatinib is a global effect: in cases of disseminated disease the response, irrespective of magnitude, will be seen at all sites. Patients who develop resistance to imatinib after initial successful response would benefit from surgery depending on the nature of the resistance, relapse, and extent of disease and feasibility of surgery. The scenario in which a patient is initially responsive to disease but subsequently develops stable disease is a subject of controversy (Neuhaus et al, 2005), and the issue remains as to whether these should be considered cases of treatment resistance due to development of a new mutation or be considered as maximal response with stabilization of disease. In our algorithm we propose that patients with stabilization of extent of disease after an initial response are responders and we recommend that they complete the full course of therapy before intervening surgically. However, we consider repeating CT and PET scans at 20 weeks from the initiation of therapy to ascertain the stability of the disease before completing the full course of treatment. There are no reliable data available yet for this particular scenario. Late resistance is still a controversial issue and we do not highlight it in our algorithm. The algorithm we have designed only serves as a guide that has made the decision making process easier in the patients we have handled. Due to the miniscule

!"#$%&'()*+#+,-.)/0# In general, any patient who has received preoperative imatinib will require post-operative treatment. The duration of treatment appears to be life long. We have developed a post-surgical scoring system for those patients who did not receive neoadjuvant imatinib mesylate based on initial tumor size, residual disease and histologic grade. The initial tumor size and histologic grade is summated, while the residual disease component is a multiplier to the summated score. A final score of 4 or more indicates a requirement for adjuvant imatinib therapy. The score is calculated based on final tumor size and grade, which are summative components, and the presence of residual disease which is a multiplier component. (Table 4) This mathematic model serves as an easy method of streamlining the decision making process for the need of post-operative imatinib, but as previously mentioned more data are required to determine the effectiveness of this scoring system. Patients with stable or responsive disease should remain on treatment. There has been enough data in the literature to support the finding that cessation of imatinib therapy in responders is associated with disease flare-up, thus requiring life long high-level suppressive doses (Blay et al, 2005). Pathology specimens resected after neoadjuvant imatinib may occasionally pose a diagnostic pitfall as GIST specimens from such patients have demonstrated complete loss of CD 117 immunoreactivity due to a change in phenotype (Pauwels et al, 2005). 591

Gopaldas et al: Gastrointestinal stromal tumor of the pancreatoduodenal complex Table 4. Gopaldas- Rohatgi post surgical scoring to determine adjuvant therapy for GIST of the pancreato-duodenal complex. Tumor Size cm

Score

! 2

1

Tumor Grade cm Score Low

Surgical outcome Multiplier Score

Total score {Size score + Grade score} x { multiplier}

No residual disease X1

Score less than 4 does not require adjuvant therapy

1

2< tumor ! 5

2

Intermediate

2

5< tumor ! 10

3

High

3

Score equal to 4 or more requires post operative adjuvant therapy Residual disease X2

Tumor > 10

4

All patients who received neoadjuvant therapy must receive post op therapy irrespective of score.

this might require major reconstructive operations in certain cases. The scoring system we have designed is based on extrapolating data available in the literature from GIST occurring elsewhere in the GI tract, as the amount of data available on pancreatoduodenal GIST is limited and will take time to accrue. The cut off points in our scoring system are based on clinical decisions pertaining to the cases we have reviewed and experienced. Although arbitrary to a certain degrees, we feel it would serve as a good tool until more data is available which would prompt revision of the scoring system. Despite limitations due to the paucity of data available, the algorithm we propose could serve as a reasonable guide for managing these aggressive tumors in critical locations such as the pancreatoduodenal complex. Ideally this could serve to streamline the treatment of these patients, and future data when available could be more easily analyzed and outcomes predicted within clinical trials.

Isoform analysis for specific exon 11 point mutation would be helpful in making the diagnosis in such circumstances. Those patients who demonstrate rapidly progressive disease should be considered for sunitinib, and imatinib should be discontinued (Blay et al, 2005). These patients should be considered for any of the ongoing clinical trials, as clear data are still not available for the management of patients with advanced progressive disease, which is uniformly fatal. The role of re-resection is not yet clearly defined. Focal resistance to imatinib mesylate therapy can develop in specific lesions, in which case limited resection of progressing lesions should be considered.

IV. Conclusion Gastrointestinal stromal tumors might well be called the tumors of the decade due to the tremendous growth in our scientific understanding of them and the controversies still surrounding them. The role of tyrosine kinase inhibitors has been clearly established in halting the progress of the disease in a certain number of patients, and has definitely enhanced the options available for managing these patients. Imatinib has been proven to be a valuable adjunct to surgery. Despite this, surgery remains the gold standard, primary modality for managing these tumors as the option offering the highest chance of cure. Caution has to exercised when dealing with stromal tumors that are CD-117 negative but clinically aggressive. These might be variant forms of GIST. Genetic analysis may be helpful in determining if a !"#$ gene mutation indeed exists. Endoluminal approaches are safer in establishing preoperative diagnosis and avoid peritoneal seeding. The essence of surgical management is to avoid violating the tumor capsule and unnecessary manipulation of the tumor itself. We also strongly advocate the principle of obtaining negative margins at the very first specimen. Positive margins at the first specimen are indicative of violation of tumor territory/capsule and negatively impacts prognosis. Hence, we favor a very aggressive surgical approach for managing patients with pancreatoduodenal GIST and dissect well away from the vicinity of the tumor, although

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Cancer Therapy Vol 6, page 597! Cancer Therapy Vol 6, 597-602, 2008

Contemporary management of adrenocortical carcinoma Review Article

Troy Guthrie Jr, Nikesh Jasani* Research and Education Baptist Cancer Institute, Jacksonville, FL, USA

__________________________________________________________________________________! *Correspondence: Nikesh Jasani MD Fellow University of Florida/Shands Jacksonville, FL, USA; Tel: 904-891-7065; Fax: 904-2441681; e-mail: nikjasani@gmail.com Key words: adrenocortical carcinoma, Epidemiology, molecular pathogenesis, Clinical presentation, Diagnosis, Treatment, Prognosis Abbreviations: Adrenocortical carcinoma, (ACC); cisplatin, doxorubicin, etoposide, (EDP); computerized tomography, (CT); dehydroepiandrosterone sulfate, (DHEAS); F-fluorodeoxyglucose positron emission tomography, (FDG-PET); First International Randomized Trial in Locally Advanced and Metastatic Adrenocortical Carcinoma Treatment, (FIRM-ACT) Received: 7 April 2008; Revised: 12 June 2008 Approved: 15 June 2008; electronically published: September 2008

Summary Adrenocortical carcinoma is a rare malignancy with a high mortality rate. Management of adrenocortical carcinoma has evolved with most advances occuring in the past decade. This review will touch on the pathogenesis, epidemiology and diagnosis of adrenocortical carcinoma. The article will focus on treatment and review surgical, radiation and chemotherapeutic indications in the managment of this lethal disease.

adrenal tissue and leads to cell line proliferation in ACC (Libe and Bertherat, 2005). Despite progress in genetics of ACC there are no conclusive pathways that have been identified that lead to ACC and much remains to be done.

I. Introduction Adrenocortical carcinoma (ACC) is a rare and heterogeneous malignancy with generally poor outcomes. Increasing use of imaging techniques have led to the discovery of silent adrenal tumors that may have malignant potential. Molecular pathogenesis still remains a mystery and is relatively poorly understood. Treatment options for advanced ACC are limited.

II. Epidemiology pathogenesis

and

III. Clinical presentation Patients present with either adrenal steroid hormone excess symptoms or symptoms from local tumor invasion. Rapidly progressing Cushing syndrome with or without virilization is often an initial presentation. A review of the German ACC registry showed 60% of cases had cortisol secretion based on pre-surgery hormone levels (Koschker et al, 2006). ACC can also lead to release of androgens which in females can cause hirsutism and virilization. In males estrogen secreting tumors can cause gynecomastia and testicular atrophy (Fassnacht et al, 2004). Rarely aldosterone producing tumors cause hypertension with hypokalemia. Hormonally inactive tumors present usually with abdominal discomfort or back pain from mass effect. Hemorrhage within tumor can also happen resulting in acute abdominal pain.

molecular

Benign Adrenocortical masses are common with a prevalence of 3% in people above age of 50 (National Institutes of Health, 2002; Grumbach et al, 2003). Actual adrenocortical carcinoma, however is quite rare having an incidence of 1-2 per million population. The age distribution is bimodal with a peak in childhood and second higher peak in fourth and fifth decade (Wajchenberg et al, 2000; NG and Libertino, 2003). Women are more commonly affected than men. TP53 is a tumor-suppressor gene located at 17p13, and controls cell proliferation (Allolio and Fassnacht, 2006). This acquired mutation is involved in pathogenesis of many types of cancer. TP53 mutations can be found in only 25% of sporadic ACC cases (Libe and Bertherat, 2005). The IGFII gene located at 11p15 encodes for a fetal growth factor. IGF-II is dependant on IGF-I which are both present in

IV. Diagnosis A. Imaging Imaging using computerized tomography (CT), magnetic resonance imaging (MRI) and Ffluorodeoxyglucose positron emission tomography (FDG597

Guthrie: Contemporary management of adrenocortical carcinoma PET) are being used to asses adrenal tumors. ACC have irregular borders and irregular areas of enhancement after intravenous contrast when using CT scanning. Imaging of the chest and abdomen is essential since liver and lung are the two most common sites of metastases. Recently a new imaging process using C-metomidate-PET has been used. This incorporates metomidate which binds to 11Bhydoxylase and can help distinguish lesions of adrenocortical origin from others (Khan et al, 2003). Benign adrenal tumors found on imaging present a diagnostic challenge to clinicians. Tumors larger than 6cm are usually resected due to a higher chance for malignancy in these larger size masses. Tumors between 3 and 6 cm are more difficult to separate benign versus malignant and should be observed with follow-up imaging every 3-12 months.

V. Treatment A. Early stage disease (stage I-III) Surgery is the mainstay of treatment and complete resection by skilled surgeon is the best chance for cure (Icard et al, 1992; Dackiw et al, 2001). R0 (no residual microscopic or macroscopic disease left behind) resection is goal of surgery involving en bloc resection of involved organs and lymphadenectomy. It is important to leave the tumor capsule in place thus reducing the risk of local recurrences (Dackiw et al, 2001). The use of laparoscopic adrenalectomy for ACC has been evaluated and found inferior to an open adrenalectomy approach in ACC that invades adjacent organs, having clinically enlarged lymph nodes or tumors greater than 10cm (Saunders and Doherty, 2004; Schteingart et al, 2005; Shen et al, 2005). Adjuvant medical therapy with Mitotane an oral adrenal-specific drug has been studied in ACC. Mitotane specifically has cytotoxic effect on adrenalcortical cells mainly in the reticular zone. Impairment of steroidgenesis is due to direct inhibition of steroidogenic enzymes (Hahner and Fassnacht, 2005). Mitotane causes adrenal insufficiency due to adrenolytic effect and increased clearance of glucocorticoids (Hague et al, 1989). Glucocorticoid replacement (in the range of 50mg hydrocortisone) is needed. Mitotane as adjuvant therapy was initially based on two retrospective studies which suggested improved survival compared to historical survival rates (Venkatesh et al, 1989). A recent publication by Terzolo and colleagues was a retrospective study looking at 177 patients with ACC who completed radical surgery which demonstrated that mitotane may prolong recurrence-free survival (Terzolo et al, 2007).

B. Laboratory work up It is necessary to obtain a complete blood count and serum chemistries. If signs of excess sex hormone secretion exist, then a hormonal laboratory evaluation should be completed. Hormonal evaluation includes random plasma rennin activity, plasma aldosterone concentration, ACTH, serum cortisol and 24 hour urine for creatinine, free cortisol, catecholamines, metanephrines and VMA (Kantarjian et al, 2006). Testosterone levels should also be measured as dehydroepiandrosterone sulfate (DHEAS) and estradiol.

C. Staging Staging for adrenocortical carcinoma is most often based on the Sullivan modification of the Macfarlane system (Macfarlane, 1958; Sullivan et al, 1978) (Figure 1).

Figure 1. Staging adrenocortical carcinoma.

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Cancer Therapy Vol 6, page 599! 2005). Another prospective trial by Khan and colleagues demonstrated the combination of streptozotocin and mitotane had improved disease free survival in metastatic ACC compared to historical controls (Khan et al, 2000). Based on the above prospective trials and these promising results a large phase III trial is underway FIRM-ACT (First International Randomized Trial in Locally Advanced and Metastatic Adrenocortical Carcinoma Treatment) and will compare EDP and mitotane versus streptozocin and mitotane chemotherapy in terms of survival and response rate. Until the results from FIRMACT trial are available, locally advanced or metastatic ACC patients should be treated with either EDP plus mitotane or streptozotocin and mitotane (Table 1). Radiation therapy as a single treatment modality has shown response rates of up to 42% (Percarpio and Knowlton, 1976; Magee et al, 1987; Markoe et al, 1991). Radiation therapy can be used in locally advanced disease not amenable to surgery. Surgery for recurrent metastatic disease has been incorporated in the management of ACC. There are small retrospective studies showing that surgical resection for recurrent disease had survival rates that equal those patients without recurrent disease who were just observed (Jensen et al, 1991; Bellantone et al, 1997; Schulick and Brennan, 1999).

Although this retrospective literature suggest a role for mitotane in the adjuvant setting may exist firm recommendations await randomized trial. No role for cytotoxic chemotherapy in the adjuvant setting has been defined. There is limited data in the use of radiation therapy in the adjuvant setting. Based on data from small retrospective trials no role for adjuvant radiation therapy to a adequately excised postoperative tumor bed exists and anectdotal evidence supports offering adjuvant radiation in stage III or high risk stage II disease (King and Lack, 1979; Magee et al, 1987; Markoe et al, 1991; Allolio and Fassnacht, 2006).

B. Unresectable locally advanced disease and metastatic disease When a complete surgical resection is not attainable, the mainstay of treatment involves the use of mitotane, cytotoxic chemotherapy and radiation in various combinations. One large study by Lubitz and colleagues showed response rate of 61% in patients with measurable disease treated with mitotane (Lubitz et al, 1973). However Numerous retrospective and prospective studies involving mitotane monotherapy have shown average response rates of 25% in advanced ACC. Complete responses have been reported with the use of mitotane in advanced ACC (Becker and Schumacher, 1975; Boven et al, 1984; Krzisnik et al, 1988; Remond et al, 1992; Ilias et al, 2001). Mitotane is associated with severe toxicities at adequate therapeutic doses. There are no large randomized trials to date that have incorporated mitotane therapy. Cytotoxic chemotherapy using cisplatin in combination with etoposide and/or doxorubicin has shown activity in advanced ACC. The largest prospective trial to date by Berruti and colleagues involved 72 patients with advanced ACC and showed a 49% response rate with 5 complete remissions using cisplatin, doxorubicin, etoposide (EDP) and mitotane (Berruti et al, 2005). Median overall survival was 28 months (Berruti et al,

C. Management of hormone excess ACC is frequently associated with hormone excess syndromes such as Cushing’s syndrome, excess androgen secretion and hyperaldosteronemia. This may result in hypokalemia, hypertension, osteoporosis, and infectous complications. Drugs such as ketoconazole, metypraone, aminoglutethimide and etomidate have been used to suppress steroid formation within the adrenal gland (Feldman, 1986; Miller and Crapo, 1993). The management of hormone excess in ACC should be monitored by experienced endocrinologist.

Table 1. Role of chemotherapy in adrenalcortical carcinoma Series

Year

Institution

Regimen

Number

Response

Berrutti(31)

2005

Italy

MEDP

72

35 PR, 5CR

Khan(32)

2000

EATSMRC

"#!

40

4 PR, 1CR

Williamson(33)

2000

SWOG

EPM

45

5 PR

Abraham(34)

1999

NCI

MEDV

28

4 PR, 1CR

Zidan(35)

1996

Israel

EP

1

1 PR

Bukowski(36)

1993

SWOG

MP

37

11 PR

Berruti(37)

1992

Italy

EDP

2

2 PR

cisplatin, (P); complete response, (CR); doxorubicin, (D); Endocrine Abdominal Tumors of the Swedish Medical Research Council, (EATSMRC); etoposide, (E); mitotane, (M); National Cancer Institute, (NCI); partial response, (PR); Southwest Oncology Group, (SWOG); streptozocin, (S); vincristine, (V)

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Guthrie: Contemporary management of adrenocortical carcinoma Bellantone R, Ferrante A, Boscherini M, Lombardi CP, Crucitti P, Crucitti F, Favia G, Borrelli D, Boffi L, Capussotti L, Carbone G, Casaccia M, Cavallaro A, Del Gaudio A, Dettori G, Di Giovanni V, Mazziotti A, Marrano D, Masenti E, Miccoli P, Mosca F, Mussa A, Petronio R, Piat G, Marazano L, et al (1997) Role of reoperation in recurrence of adrenal cortical carcinoma: results from 188 cases collected in the Italian National Registry for Adrenal Cortical carcinoma Surgery 122, 1212-1218. Berruti A, Terzolo M, Paccotti P, Veglio F, Pia A, Dogliotti L, Angeli A (1992) Favorable response of metastatic adrenocortical carcinoma to etoposide, adriamycin and cisplatin (EAP) chemotherapy Report of two cases Tumori 78, 345-8. Berruti A, Terzolo M, Sperone P, Pia A, Casa SD, Gross DJ, Carnaghi C, Casali P, Porpiglia F, Mantero F, Reimondo G, Angeli A, Dogliotti L (2005) Etoposide, doxorubicin and cisplatin plus mitotane in the treatment of advanced adrenocortical carcinoma: a large phase II prospective trial Endocr Relat Cancer 12, 657-666. Boven E, Vermorken JB, van Slooten H, Pinedo HM (1984) Complete response of metastasized adrenal cortical carcinoma with o,p’-DDD Cancer 53, 26-29. Bukowski RM, Wolfe M, Levine HS, Crawford DE, Stephens RL, Gaynor E, Harker WG (1993) Phase II trial of mitotane and cisplatin in patients with adrenal carcinoma: a southwest Oncology group Study L Clin Oncol 11, 161-5. Dackiw AP, Lee JE, Gagel RF, Evans DB (2001) Adenal cortical carcinoma World J Surg 25, 914-926. Fassnacht M, Kenn W, ALlolio B (2004) Adrenal tumors: how to establish malignancy? J Endocrinol Invest 27, 387-399. Feldman D (1986) Ketoconazole and other imidazole derivatives as inhibitors of steroidogenesis Endocr Rev 7, 409-420. Grumbach MM, Biller BM, Braunstein GD, Campbell KK, Carney JA, Godley PA, Harris EL, Lee JK, Oertel YC, Posner MC, Schlechte JA, Wieand HS (2003) Management of the clinically inapparent adrenal mass Ann Intern Med 138, 424-429. Hague RV, May W, Cullen DR (1989) Hepatic microsomal enzyme induction and adrenal crisis due to o,p”DDD therapy for metastatic adrenocortical carcinoma Clin Endocrinol (Oxf) 31, 51-57. Hahner S, Fassnacht M (2005) Mitotane for adrenocortical carcinoma treatment Curr Opin Investig Drugs 6, 386-394. Harrison LE, Gaudin PB, Brennan MF (1999) Pathologic features of prognostic significance for adrenocortical carcinoma after curative resection Arch Surg 134, 181-185. Icard P, Chapuis Y, Andreassian B, Bernard A, Proye C (1992) Adrenocortical carcinoma in surgically treated patients: a retrospective study on 156 cases by the French Association of Endocrine Surgery Surgery 112, 972-979. Ilias I, Alevizaki M, Philippou G, Anastasiou E, Souvatzoglou A (2001) Sustained remission of metastatic adrenal carcinoma during long-term administration of low dose mitotane J Endocrinol Invest 24, 532-535. Jensen JC, Pass HI, Sindelar WF, Norton JA (1991) Recurrent or metastatic disease in select patients with adrenalcortical carcinoma Aggressive resection vs chemotherapy Arch Surg 126, 457-461. Kantarjian MH, Wolff AR, Koller AC (2006) Adrenocortical Carcinoma MD Anderson Manual of Medical Oncology 4, 841-847. Khan TS, Imam H, Juhlin C, Skogseid B, Gröndal S, Tibblin S, Wilander E, Oberg K, Eriksson B (2000) Streptozocin and o,p’DDD in the treatment of adrenal cortical cancer patients: long-term survival in its adjuvant use Ann Oncol 11, 12811287.

VI. Prognosis Various prognostic markers have been associated with poor survival outcomes. Large tumors greater than 12 cm, high mitotic rates, high Ki67 immunohistochemical staining and mutated P53 are some factors associated with poor prognosis in ACC (Harrison et al, 1999; Stojadinovic et al, 2002). The two major predictors of survival in metastatic ACC were found to be number of tumoral organs at the time of first metastasis and mitotic index (Assié et al, 2007). Median 5 yr survival for stage I disease has been reported around 60%, stage II 58%, stage III 24%. Median survival for stage IV disease is less than 12 months (Allolio and Fassnacht, 2006).

VII. Conclusions and future The mangement of ACC has evolved through retrospective and prospective trials resulting in a multimodality approach to treatment of this rare malignancy. Surgical resection remains the cornerstone to curative treatment for ACC. Adjuvant therapy with mitotane is still not conclusive and this should be individually discussed with high risk patients until prospective clinical trial are available. Adjuvant radiation therapy to postoperative tumor bed can be used in high risk stage II or stage III disease. Unresectable locally advanced disease should be treated with cytotoxic chemotherapy consisting of EDP plus mitotane or streptozotocin and mitotane with radiation. Metastatic or recurrent disease should be treated with either above chemotherapy regimen and surgical resection of solitary metastasis should be pursued. Hormonal excess should be managed by experienced endocrinologist. Future advances in the pathogenesis and therapeutic management of ACC will depend on clinical trials. The FIRM-ACT trial is a multinational prospective trial and will be the first ever phase III trial in ACC. It will determine the standard cytotoxic chemotherapy for ACC. This trial randomizes patients to either the Berruti protocol (EDP plus mitotane) or the one by Khan (streptozotocin and mitotane) and intends to enroll 300 patients. This trial as well as other phase II trials will not only lead to advancement in the treatment of ACC but also stimulate research into the pathogenesis of the disease.

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Cancer Therapy Vol 6, page 603 ! Cancer Therapy Vol 6, 603-610, 2008

Disruption of Survivin in K562 cells elevates telomerase activity and protects cells against apoptosis induced by the Bcr-abl kinase inhibitor STI571 Research Article

Zhanxiang Wang, Louis M. Pelus* Department of Microbiology & Immunology, and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202

__________________________________________________________________________________! *Correspondence: Louis M. Pelus, Ph.D., Department of Microbiology & Immunology, Indiana University School of Medicine, 950 West Walnut Street, Indianapolis, IN 46202, USA; Tel: 317-2747565; Fax: 317-2747592; E-mail: lpelus@iupui.edu Key words: Survivin, Telomerase, Apoptosis, Leukemia Abbreviations: Ras GTPase-activating protein, (RasGAP); Chronic Myeloid Leukemia, (CML); Cisplatin, (CDDP); heat-inactivated fetal bovine serum, (HI-FBS); human telomerase reverse transcriptase, (hTERT); SDS-polyacrylamide gels, (SDS-PAGE); sodium dodecyl sulfate, (SDS)

Supported by grants HL079654 and HL69669 (to LMP) from the National Institute of Health.

Received: 7 May 2008; Revised: 25 August 2008 Accepted: 27 August 2008; electronically published: September 2008

Summary The Bcr-abl kinase inhibitor STI571 produces clinical responses in most patients with Chronic Myeloid Leukemia (CML); however, development of resistance limits utility. One strategy to overcome STI571 resistance is to decrease the level/activity of Bcr-abl. We reported that disruption of the anti-apoptotic protein Survivin promoted STI571induced apoptosis in Bcr-abl+ K562 cells, through caspase-dependent Bcr-abl degradation. To investigate the utility of Survivin disruption in drug-resistant CML cells, we generated STI571-resistant K562 cells by long-term culture with STI571. In contrast to parental cells, where Survivin disruption enhances STI571-induced apoptosis, Survivin disruption in STI571-resistant cells failed to promote STI571-induced apoptosis; rather it protected cells from STI571 and other apoptosis-inducing compounds. Even though Survivin levels were similar in parental and STI571resistant K562 cells, Survivin disruption in STI571-resistant cells increased telomerase activity, likely due to Bcrabl/c-abl degradation. Our results indicate that emergence of STI571 resistance in Bcr-abl+ K562 cells results from induction of additional pathways that circumvent STI571-responsiveness.

domain that interfere with STI571 binding (Shah et al, 2002). One possible strategy to overcome STI571 resistance would be to decrease Bcr-abl levels. Survivin blocks apoptosis by inhibiting caspases 3, 7 and 9 (Ambrosini et al, 1997; Shin et al, 2001; Pennati et al, 2004). We recently reported that Bcr-abl up regulates Survivin transcription and that Survivin disruption promotes STI571-induced apoptosis by disrupting mitochondrial membrane potential resulting in cytochrome-! release and caspase-dependent Bcr-abl degradation (Wang et al, 2005). Caspase-dependent degradation of Bcr-abl oncoprotein in cells in which

I. Introduction The Bcr-abl oncogene, encodes a cytoplasmic protein with constitutive tyrosine kinase activity and is found in cells of ~95% of CML patients (Kurzrock et al, 1988; Sawyers, 1999). The specific Bcr-abl inhibitor, STI571 (Gleevec速, imatinib mesylate), produces clinical responses in most patients with CML (Druker et al, 2001); however, the development of resistance (Hochhaus et al, 2001) has prompted the search for alternative treatments. The mechanisms of clinical resistance to STI571 can involve increased Bcr-abl through gene amplification (Gorre and Sawyers, 2002) or mutations in the Bcr-abl catalytic 603

Wang and Pelus: Disruption of Survivin elevates telomerase activity! membranes. The filters were incubated with specific antibodies followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin and developed by electrogenerated chemiluminescence (Amersham Biosciences, Piscataway, NJ). !

Survivin expression is disrupted may represent a pathway to enforce apoptosis in STI571-resistant cells. Since enhanced degradation of Bcr-abl should reduce its antiapoptotic effects, we investigated Survivin knockdown as a strategy to treat drug resistance to STI571. Using STI571-resistant K562 cells, we found that Survivin disruption did not promote STI571-induced apoptosis as seen in parental K562 cells, rather, it produced the opposite effect. Reduced apoptosis upon Survivin disruption in STI571-resistant K562 cells was accompanied by elevated telomerase activity associated with increased Bcr-abl/c-abl degradation. Our results suggest that development of STI571 resistance in K562 cells occurs through effects on multiple pathways, indicating that combined drug interventions will likely be necessary to overcome STI571 resistance.

D. Flow cytometry analysis for apoptosis Apoptosis was quantitated based on hypodiploid DNA content or Annexin-V binding as previously described (Wang et al, 2004). Cell apoptosis was analyzed using a FACScan and CellQuest software (BD Bioseinces). !

E. Telomerase activity and telomere length assay Telomerase activity and telomere length were analyzed using the Telo TAGGG Telomerase PCR ELISA and Telo TAGGG Telomere Length Assay kits (Roche Diagnostics, Indianapolis, IN) following the manufacturer’s instructions.

II. Material and Methods

III. Results

A. Reagents and antibodies

A. Disruption of Survivin in STI571resistant K562 cells fails to promote STI571induced apoptosis

Recombinant IL-3 and anti-human Survivin (AF886) were purchased from R&D Systems (Minneapolis, MN). Anti-c-abl (Ab-3) was from Oncogene (LaJolla, CA). Hemin and CDDP (Cisplatin) were purchased from Sigma Chemical Company (St. Louis, MO). Anti-hTERT and anti-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). STI571 (Gleevec) was obtained from Novartis Inc (Basel, Switzerland). The caspase 3-specific inhibitor Z-DEVD-FMK was from BD Biosciences (San Jose, CA).

We previously showed that Survivin disruption in K562 cells enhances STI571-induced apoptosis, accompanied by cytochrome-# release, disruption of mitochondrial membrane potential, and caspase-mediated Bcr-abl protein cleavage, suggesting that Survivin knockdown might overcome drug resistance to STI571 (Wang et al, 2005). STI571-resistant K562 cells were generated by culture of cells with increasing concentrations of STI571 (0.05 to 0.5 !M) over 6 months. Resistant K562 cells were significantly less sensitive to STI571 than parental cells (Figures 1A and 1B). To knockdown Survivin protein, parental and STI571-resistant K562 cells were transfected with retrovirus containing an antisense Survivin construct. Cells were also transfected with empty vector and wildtype Survivin as controls. Western blot analysis validated Survivin knockdown and over expression, respectively (Figure 1A and 1B, Western inserts). Westerns also demonstrated that STI571-resistant cells contained higher Survivin levels than parental cells, consistent with the facts that these cells contain more Bcr-abl as a consequence of STI571 resistance (Figure 2C) and that Bcr-abl regulates Survivin transcription (Wang et al, 2005). In the parental K562 cells, ectopic Survivin protected cells from STI571-induced apoptosis, while Survivin disruption promoted apoptosis as expected (Figure 1A). In STI571-resistant cells (Figure 1B), ectopic Survivin still protected the cells from STI571induced apoptosis although to a lesser degree than in parental cells, which is not unexpected, since these cells are drug resistant. However, Survivin disruption did not promote cell apoptosis, but rather protected cells to an equivalent degree as ectopic Survivin.

B. Cell lines, plasmids and transfection Human K562 cells, which express Bcr-abl, and MCF7 breast cancer cells were maintained in RPMI-1640 with 10% heat-inactivated fetal bovine serum (HI-FBS) (Hyclone Sterile Systems, Logan, UT) and 1% penicillin-streptomycin. STI571resistant cells were generated by stepwise culture with increasing concentration of STI571 (0.05-0.5 !M) over 6 months. Survivin and antisense-Survivin (reverse orientation) constructs were cloned in the IRES-EGFP-MIEG3 vector as described (Wang et al, 2004). Transfection of K562 cells was carried out as reported previously (Wang et al, 2005). Briefly, log phase K562 cells plus 30 !g of Survivin plasmid DNAs were mixed in a Bio-Rad gene Pulser Cuvette and incubated at 37°C for 15 minutes.!Following one pulse electroporation (360V, 960 F), cells were incubated at 37°C for 15 minutes and resuspended in RPMI-1640 + 10% HIFBS. After 48 hours, GFP+ cells were isolated by FACS (Becton Dickinson, San Jose, CA), expanded for one week and resorted for GFP+ cells. IL-3-dependent murine Ba/F3 cells were maintained in RPMI-1640 with 10% HI-FBS, 1% penicillinstreptomycin and 0.1 ng/mL rmIL-3. Construction of Ba/F3 cells stably expressing bicistronic retrovirus MIEG3 plasmids containing full-length or antisense mouse Survivin and transient transfection of MCF7 with full-length or antisense human Survivin were described previously (Wang et al, 2004).

C. Protein extraction and Western analysis Whole cell lysates for Western analysis were prepared in 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 !g/ml phenylmethyl sulfonyl fluoride, and 1 mM sodium orthovanadate. Aliquots were kept on ice for 30 minutes and centrifuged at 14,000! " for 30 minutes. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Fifty micrograms of protein was denatured in 2X SDS buffer at 95°C for 5 minutes, separated by electrophoresis on 10% SDS-polyacrylamide gels (SDS-PAGE) (Invitrogen, Carlsbad, CA) and transferred onto nitrocellulose

B. Survivin disruption decreases c-abl in MCF7 and BaF3 cells and Bcr-abl/c-abl in STI571- resistant K562 cells Survivin blocks caspase activity while Survivin disruption increases caspase activity, favoring apoptosis. 604

Cancer Therapy Vol 6, page 605 ! In both MCF7 cells transiently transfected with antisense Survivin and murine BaF3 cells stably transduced with antisense Survivin, Survivin disruption was associated with significantly lower levels of c-abl compared to vector control cells (Figures 2A and 2B). In parental and STI571-resistant K562 cells, Survivin disruption resulted in lower levels of both Bcr-abl and c-abl (Figure 2C). The demonstration of Bcr-abl/c-abl degradation suggests that alteration in caspase activity alone is not responsible for drug resistance.

The finding of protection from apoptosis in STI571resistant K562 cells led us to investigate caspase-3 activity. We previously showed that Survivin blocks caspase activity and caspase-dependent cleavage of Mdm2 in MCF7 and BaF3 cells (Wang et al, 2004) and several recent studies have described caspase-dependent cleavage of c-abl and Bcr-abl in K562 cells (Di Bacco and Cotter, 2002; Jacquel et al, 2003). Since Survivin inhibits caspase activity, and c-abl is a caspase substrate, we hypothesized that Survivin disruption could decrease c-abl and Bcr-abl.

Figure 1. Disruption of Survivin fails to promote STI571-induced apoptosis in STI571-resistant K562 cells. Parental (A) or STI571resistant (B) K562 cells transfected with wild-type Survivin (S), antisense (AS) Survivin or control vector (V) were cultured with STI571 for 48 hours. Apoptosis was determined by quantitating hypodiploid DNA content. Representative western blots (insert) show Survivin levels after transfection.!All data are expressed as Mean ÂąSEM from 3 experiments.!"#$%&%'&

Figure 2. Survivin knockdown promotes Bcr-abl and c-abl degradation and reduces hTERT levels. Human MCF7 breast cancer cells (A) and murine hematopoietic BaF3 proB cells (B) were transiently transfected with antisense (AS) Survivin or control vector (V). After 24 hours, cell lysates were subjected to Western analysis using an anti-c-abl antibody. (C). Parental and STI571resistant K562 cells! stably transfected with antisense (AS) Survivin and control vector (V) were harvested and whole cell lysates analyzed by western analysis using anti-c-abl, anti-Bcr-abl and anti-Survivin antibodies. Data are representative from 3 experiments with similar results. (D). hTERT protein was determined by western analysis in parental and STI571-resistant cells transiently transfected with antisense Survivin or control vector. Data are representative of 2 experiments with similar results.

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C. High telomerase activity was found in STI571-resistant K562 cells after disruption of Survivin Telomerase is a cellular RNA-dependent DNA polymerase that maintains the tandem arrays of telomeric TTAGGG repeats at eukaryotic chromosome ends (Greider and Blackburn, 1996; van Steensel et al, 1998). Telomerase activity is elevated in many cancers and immortalized cells (Engelhardt et al, 2000; Ohyashiki et al, 2002). It has been reported that c-abl directly associates with the catalytic protein subunit of human telomerase reverse transcriptase (hTERT) resulting in tyrosine phosphorylation and inhibition of telomerase activity (Kharbanda et al, 2000). Since Survivin disruption results in decreased Bcr-abl/c-abl levels, we reasoned that telomerase activity might increase as a consequence of reduced phosphorylation. In parental K562 cells, Survivin knockdown resulted in a marginal decrease in hTERT protein (Figure 2C; left panel); however, in drug-resistant K562 cells, hTERT protein was more dramatically reduced (Figure 2D; right panel). Interestingly, regardless of the hTERT protein level, telomerase activity was significantly increased in both parental and STI571resistant cells transfected with antisense Survivin (Table 1). In STI571-resistant K562 cells, basal telomerase activity was significantly higher than in parental cells (for both vector control and AS Survivin cells), indicating that long-term inhibition of Bcr-abl by STI571 increases telomerase activity, which is consistent with published reports (Bakalova et al, 2003; Drummond et al, 2004). In addition, STI571-resistant K562 cells, in which Survivin was disrupted, had much longer telomere lengths compared to STI571-resistant K562 cells transfected with control vector (not shown).

Figure 3. Survivin knockdown attenuates hemin-induced apoptosis in K562 cells. (A). Parental K562 cells transfected with an antisense Survivin construct or vector alone were cultured with 30 !M hemin for 72 hours and hypodiploid DNA content quantitated by flow cytometry. Data are Mean ± SEM apoptosis from 6 experiments. *P<0.01. (B) STI571-resistant K562 cells transfected with an antisense Survivin or vector were cultured with 30 !M hemin for 72 hours and hypodiploid DNA content and Annexin-V expression quantitated by flow cytometry. Data are Mean ± SEM from three experiments. *P<0.05.

D. Survivin knockdown attenuates Hemin-induced apoptosis in K562 cells Since Survivin knockdown leads to increased telomerase activity in both parental and STI571-resistant cells, we evaluated whether increased telomerase activity would result in reduced sensitivity to apoptosis induced by hemin, which induces apoptosis specifically by inhibiting telomerase activity (Benito et al, 1996; Yamada et al, 1998). In parental (Figure 3A) and resistant (Figure 3B) K562 cells, Survivin knockdown protected cells from hemin-induced apoptosis, although Survivin-mediated knockdown protected parental cells to a slightly greater degree than resistant cells. Inhibition of caspase-3 by the

Table 1. Effect of Survivin knockdown on Telomerase activity in parental and STI571-resistant K562 cells Telomerase Activity (OD 490 nm) V

*

AS

Parental K562

1.51 ± 0.04

1.79 ± 0.05 *

STI571 Resistant K562

1.89 ± 0.06 †

2.17 ± 0.09 *, †

P <0.05 compared to vector control; † P <0.05 compared to parental K562 cells.

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Cancer Therapy Vol 6, page 607 ! reduced apoptosis in resistant K562 cells upon Survivin disruption is a consequence of decreased c-abl levels or alternatively results from increased telomerase activity due to Bcr-abl/c-abl cleavage. Treatment with CDDP effectively induced apoptosis in control and K562 cells transfected with antisense Survivin (Figure 4C). Equivalent induction of apoptosis in both groups despite the fact that the antisense-transfected cells had significantly lower levels of c-abl indicates that c-abl cleavage does not play a significant role in apoptosis, and that Bcr-abl is the dominant mediator of apoptosis in these cells.

selective caspase inhibitor Z-VAD-FMK blocked apoptosis induced by STI571 in both control vector and antisense Survivin transfected K562 cells (Figure 4A). In contrast, caspase-3 inhibition was without effect on apoptosis induced by hemin either in the presence or absence of Survivin (Figure 4B), indicating that caspase-3 is not involved in hemin-induced apoptosis. While Bcr-abl and c-abl levels decrease in parental and drug-resistant cells after Survivin knockdown (Figure 2), likely resulting from increased caspase activity due to reduced Survivin protein, the reduction in c-abl is more dramatic. Since c-abl is required for CDDP-induced apoptosis (Gong et al, 1999) we questioned whether

Figure 4. Caspase-3 inhibition attenuates hemin-induced apoptosis in K562 cells. (A). Parental K562 cells transfected with an antisense Survivin construct or vector were cultured in 0.5 !M STI571 with or without Z-DEVD-FMK for 48 hours. Apoptosis was quantitated based upon hypodiploid DNA content. Data are from 3 separate experiments. *P<0.01. (B). Transfected parental K562 cells were cultured in 30 !M hemin with or without Z-DEVD-FK for 72 hours and hypodiploid DNA content quantitated by flow cytometry. Data are from 2 experiments. *P<0.05. (C). Transfected parental K562 cells were cultured in 30 !M CDDP for 48 hours. Apoptosis was quantitated based upon hypodiploid DNA content. Data are from 3 separate experiments. *P<0.01.

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Wang and Pelus: Disruption of Survivin elevates telomerase activity! and accumulating evidences indicates that c-abl may suppress telomerase activity (Kharbanda et al, 2000; Ohyashiki et al, 2002). Enhanced telomerase activity is a prognostic indicator for shorter survival in CML patients. It seems likely that both Bcr-abl and telomerase abnormalities are responsible for disease progression, since Bcr-abl strongly activates c-abl kinase activity, thereby potentiating telomerase suppression. Consistent with this hypothesis, extension of telomeres in CML patients after long-term treatment with Gleevec in both chronic phase and blast crisis has been reported (Drummond et al, 2004). Long-term treatment with antiBcr-abl/c-abl antisense oligonucleotides also significantly elongates telomeres and enhances hTERT activity, accompanied by increased cell proliferation (Bakalova et al, 2003, 2004; Brummendorf et al, 2003). In this context, combination treatment with drugs targeting the Bcr-abl kinase and compounds influencing telomere length may be a promising strategy in treating CML. The demonstration that inhibition of human telomerase enhances the effect of Gleevec supports this assumption (Tauchi et al, 2002). Survivin disruption elevated telomerase activity in both parental and drug-resistant K562 cells; however the consequence of apoptosis induction on these cells was opposite, raising the question of divergent versus convergent mechanisms. A possible explanation might be that in parental K562 cells, Bcr-abl cleavage by Survivin disruption produces only a moderate increase in telomerase activity that does not play a significant antiapoptotic role. However, after STI571 selection, Bcr-abl inhibition leads to reduced hTERT phosphorylation and more significantly, enhanced telomerase activity. Furthermore, additional cleavage of Bcr-abl by Survivin disruption imparts a more predominant role for telomerase in blocking apoptosis. Indeed, the highest telomerase activity was observed in STI571-resistant cells in which Survivin was disrupted. A recent report indicates that Survivin enhances telomerase activity in colon cancer through up regulation of hTERT transcription (Endoh et al, 2005). Consistent with this report, hTERT protein levels were decreased in K562 cells when Survivin protein levels are knocked down, however, telomerase activity did not parallel hTERT levels in K562 cells, pointing to potential differences between solid tumor cells and leukemia cells. This discrepancy might be explained by the fact that K562 cells like primary CML cells are distinct from most cancer and leukemia cells, in that they possess high levels of constitutively active Bcr-abl that phosphorylates hTERT and inhibits telomerase (Engelhardt et al, 2000; Ohyashiki et al, 2002, 1997). Blocking this inhibition either through chemical or biological means results in enhanced telomerase activity. Our results that Survivin disruption fails to promote apoptosis in resistant cells implies that drug resistance to STI571 affects additional signaling pathways in addition to increased gene amplification (Gorre and Sawyers, 2002) or mutations in the Bcr-abl catalytic domain (Shah et al, 2002) that have already been implicated in development of clinical resistance to STI571. However, although a Bcr-abl/telomerase axis pathway might play an important role it may only be

IV. Discussion Bcr-abl kinase renders cells insensitive to apoptosis induced by diverse stimuli, including most cytotoxic drugs (McGahon et al, 1994; Bedi et al, 1995). Since we demonstrated previously that Survivin blocks caspase activity and caspase-dependent cleavage of Mdm2 (Wang et al, 2004) and Bcr-abl and c-abl are caspase substrates (Di Bacco and Cotter, 2002; Jacquel et al, 2003), we expected that Survivin knockdown might attenuate STI571-resistance by increasing caspase-mediated Bcr-abl cleavage. However, although Bcr-abl protein was decreased by Survivin knockdown, Survivin disruption did not enhance apoptosis; rather it blocked apoptosis induced by STI571. Data presented herein, suggests that this apoptosis enhancing effect is linked to the telomerase pathway. Survivin blocks caspase 3 and 7 activity (O'Connor et al, 2000; Riedl et al, 2001) and promotes apoptosis. However, caspases mediate other biological processes besides apoptosis, such as cell differentiation (Gurbuxani et al, 2005) and can cleave a large number of cellular substrates, making it difficult to define a specific mechanism. Recently, it was reported that the p120 Ras GTPase-activating protein (RasGAP) can be cleaved into an N-terminal fragment by low levels of caspase, and that the cleavage products trigger antiapoptotic signals via activation of the Ras/PI-3 kinase/Akt pathway (Bartling et al, 2004). A similar mechanism might also occur in STI571-resistant K562 cells or Bcr-abl cleavage may trigger another signal pathway that increases telomerase activity. Alternatively, since c-abl associates with and directly phosphorylates hTERT and inhibits telomerase activity (Kharbanda et al, 2000), decreased Bcr-abl level resulting from Survivin disruption might interfere in this pathway and alter the apoptosis response. Telomerase maintains the tandem repeats of TTAGGG at the telomere of eukaryotic chromosomes that protects them from end-to-end fusions (Greider and Blackburn, 1996; van Steensel et al, 1998). It is upregulated in the vast majority of human tumors, and reconstitution of telomerase activity in cultured primary cells allows immortal growth (Bodnar et al, 1998; Kiyono et al, 1998; Jiang et al, 1999; Morales et al, 1999). The telomerase reverse transcriptase is a critical catalytic protein subunit within the telomerase complex (Feng et al, 1995; Weinrich et al, 1997). It has been reported recently that the c-abl tyrosine kinase directly associates with and phosphorylates hTERT and inhibits hTERT activity (Kharbanda et al, 2000). Therefore, c-abl protein and activity are potentially responsible for telomerase suppression. Moreover, chromosomal translocations that generate fusion proteins such as Tel-Abl and Tel-PDGFR, also correspond with low telomerase activity (Ohyashiki et al, 2002). Approximately 80% of CML patients show short telomere length and low telomerase activity in chronic phase (Ohyashiki et al, 1997), in contrasts to most leukemias, tumors and immortalized cell lines, where telomerase is usually overexpressed (Engelhardt et al, 2000; Ohyashiki et al, 2002). A link between the Bcr-abl with strong c-abl kinase activity and low telomerase activity during chronic phase CML has been suggested 608

Cancer Therapy Vol 6, page 609 ! Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, Adams RR, Chang E, Allsopp RC, Yu J, et al (1995) The RNA component of human telomerase. Science 269, 12361241. Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin WG Jr, Levrero M, Wang JY (1999) The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399, 806-809. Gorre ME, Sawyers CL (2002) Molecular mechanisms of resistance to STI571 in chronic myeloid leukemia. Curr Opin Hematol 9, 303-307. Greider CW, Blackburn EH (1996) Telomeres, telomerase and cancer. Sci Am 274, 92-97. Gurbuxani S, Xu Y, Keerthivasan G, Wickrema A, Crispino JD (2005) Differential requirements for survivin in hematopoietic cell development. Proc Natl Acad Sci U S A 102, 11480-11485. Hochhaus A, Kreil S, Corbin A, La Rosée P, Lahaye T, Berger U, Cross NC, Linkesch W, Druker BJ, Hehlmann R, Gambacorti- Passerini C, Corneo G, D'Incalci M (2001) Roots of clinical resistance to STI-571 cancer therapy. Science 293, 2163. Jacquel A, Herrant M, Legros L, Belhacene N, Luciano F, Pages G, Hofman P, Auberger P (2003) Imatinib induces mitochondria-dependent apoptosis of the Bcr-Abl-positive K562 cell line and its differentiation toward the erythroid lineage. FASEB J 17, 2160-2162. Jiang XR, Jimenez G, Chang E, Frolkis M, Kusler B, Sage M, Beeche M, Bodnar AG, Wahl GM, Tlsty TD, Chiu CP (1999) Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet 21, 111-114. Kharbanda S, Kumar V, Dhar S, Pandey P, Chen C, Majumder P, Yuan ZM, Whang Y, Strauss W, Pandita TK, Weaver D, Kufe D (2000) Regulation of the hTERT telomerase catalytic subunit by the c-Abl tyrosine kinase. Curr Biol 10, 568-575. Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, Klingelhutz AJ (1998) Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396, 84-88. Kurzrock R, Gutterman JU, Talpaz M (1988) The molecular genetics of Philadelphia chromosome-positive leukemias. N Engl J Med 319, 990-998. McGahon A, Bissonnette R, Schmitt M, Cotter KM, Green DR, Cotter TG (1994) BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood 83, 1179-1187. Morales CP, Holt SE, Ouellette M, Kaur KJ, Yan Y, Wilson KS, White MA, Wright WE, Shay JW (1999) Absence of cancerassociated changes in human fibroblasts immortalized with telomerase. Nat Genet 21, 115-118. O'Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio PC, Altieri DC (2000) Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc Natl Acad Sci U S A 97, 13103-13107. Ohyashiki JH, Sashida G, Tauchi T, Ohyashiki K (2002) Telomeres and telomerase in hematologic neoplasia. Oncogene 21, 680-687. Ohyashiki K, Ohyashiki JH, Iwama H, Hayashi S, Shay JW, Toyama K (1997) Telomerase activity and cytogenetic changes in chronic myeloid leukemia with disease progression. Leukemia 11, 190-194. Pennati M, Binda M, Colella G, Zoppe' M, Folini M, Vignati S, Valentini A, Citti L, De Cesare M, Pratesi G, Giacca M, Daidone MG, Zaffaroni N (2004) Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-induced apoptosis and decreases the tumorigenic

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Cancer Therapy Vol 6, page 611! Cancer Therapy Vol 6, 611-616, 2008

Efficacy of methotrexate as primary single agent therapy for non metastatic and low risk metastatic gestational trophoblastic neoplasia at the University of the Philippines-Philippine General Hospital (UPPGH) Research Article

Maria Stephanie Fay Samadan-Cagayan Department of Obstetrics and Gynecology, Section of Trophoblastic Diseases, College of Medicine, University of the Philippines- Philippine General Hospital, Manila, Philippines

__________________________________________________________________________________! *Correspondence: Maria Stephanie Fay Samadan-Cagayan, MD Associate Professor, Department of Pharmacology and Toxicology and and OB-GYNE, College of Medicine, University of the Philippines Manila, 547 Pedro Gil St. Ermita, Manila, Philippines 1000; Tel: (632) 526-4248; Fax: (632) 521-8251; E-mail: faye_cagayan@hotmail.com, cagayanmsfs@druginfo.ph Key words: Gestational trophoblastic neoplasia, Choriocarcinoma, Methotrexate, Chemotherapy Abbreviations: gestational trophoblastic disease, (GTD); Gestational trophoblastic neoplasia, (GTN); low risk metastatic gestational trophoblastic neoplasia, (LRMGTN); methotrexate, (MTX); nonmetastatic gestational trophoblastic neoplasia, (NMGTN); nonmetastatic trophoblastic disease, (NMTD) Received: 27 March 2008; Revised: 7 July 2008 Accepted: 3 September 2008; electronically published: September 2008

Summary To evaluate the efficacy and toxicity of methotrexate (MTX) as single agent chemotherapy in patients with nonmetastatic gestational trophoblastic neoplasia (NMGTN) and low risk metastatic gestational trophoblastic neoplasia (LRMGTN). Between 1988 and 2004, a total of 203 patients with NMGTN and LRMGTN received complete treatment at the UP-PGH Medical Center. These patients were initially treated with methotrexate 0.3-0.4 mg/kg/day intramuscularly (IM) for 5 days. Treatment courses were repeated every 7-10 days. Clinical characteristics and outcomes were analyzed. Of 203 patients initially treated with low dose IM MTX, 178 (88%) achieved complete remission after 4-5 cycles. Sixty per cent of patients also had hysterectomy. 25 patients (12.31%) were shifted to actinomycin due to toxicity and resistance with complete remission in 18 patients. Most common adverse effects were mucositis and gastrointestinal symptoms. Sustained remission rate was 98.5%. Patients who did not have complete response to single agent chemotherapy were given multiple agent therapy and subsequently attained remission. Methotrexate chemotherapy is effective and well-tolerated in treating patients with nonmetastatic and low risk metastatic gestational trophoblastic neoplasia.

prognosis when managed by single agent chemotherapy with the introduction of methotrexate in 1956 for the treatment of gestational trophoblastic disease (GTD) (Smith et al, 1982; Barter et al, 1987; Homesley et al, 1990; Homesly, 2002; Srisomboon et al, 2005; Sekharan et al, 2006). Most women with non-metastatic gestational trophoblastic neoplasia (NMGTN) and low risk metastatic GTN (LRMGTN) can be successfully diagnosed and treated with numerous single agent treatment protocols such as oral methotrexate, low dose intramuscular methotrexate for 5 days given every 1-2 weeks,

I. Introduction Gestational trophoblastic neoplasia (GTN) usually arises from 15-50% of molar pregnancies (Morrow et al, 1977; Berkowitz and Goldstein, 1981; Kohorn, 1982; Lurain et al, 1983; Barter et al, 1987; Homesly, 2002; Sekharan et al, 2006). The prognosis of gestational trophoblastic neoplasia (GTN) is determined by several risk factors such as human chorionic gonadotropin level, antecedent pregnancy, presence and number of metastasis and previous history of chemotherapy. Non-metastatic disease and low risk metastatic disease has an excellent

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Samadan-Cagayan: Efficacy of methotrexate as primary single agent therapy for NMGTN and LRMGTN were serially monitored. Patients were advised against conception for at least 1 year after completion of chemotherapy. Patients who refused treatment and/or quit treatment were all lost to follow up.

methotrexate with folinic acid rescue, actinomycin D and etoposide, all with good outcomes and with preservation of normal reproductive function (Smith et al, 1982; Barter et al, 1987; Petrilli et al, 1987; Berkowitz et al, 1990; Homesley et al, 1990; Lurain and Elfstrad, 1995; Matsui et al, 1998; Homesly, 2002; Srisomboon et al, 2005; Sekharan et al, 2006). Prompt diagnosis in women presenting with vaginal bleeding, appropriate staging and identification of risk score, timely management of disease and its complications and complications of chemotherapy as well as close monitoring of serum BhCG after remission allow for a prolonged life expectancy and good quality of life of GTN patients. Meticulous search for metastases should be done in order to properly classify patients as non-metastatic or metastatic and low or high risk so that appropriate therapy can be instituted. The diagnosis of NMGTN after mole evacuation is usually based on a rising or plateauing hCG titer and metastatic workups show no evidence of extrauterine trophoblastic disease. This study aims to review the efficacy of low dose intramuscular methotrexate as primary chemotherapy for patients diagnosed with NMGTN and LRMGTN at the UP-PGH Medical Center. Overall cure rate, chemoresistance rate and toxicity were also determined.

III. Results Charts of 411 patients diagnosed with GTN were reviewed during the study period, with 210 (51%), 47 (11.4%), 154 (37.4%]) patients diagnosed as NMGTN, low-risk metastatic GTN, and high-risk metastatic GTN, respectively (Table 1). Out of 257 patients eligible for single agent therapy, 35 patients went home against medical advice prior to treatment. Only 222 were actually started on the treatment with 19 patients going home against advice after 1-2 courses of chemotherapy. These patients were lost to follow up. Table 2 shows the clinicopathologic profile of the 203 patients who were able to complete therapy. Patient ages ranged from 18 to 56 (mean= 31.2 years). Majority of cases fell in the 25-34y/o group (44%), and the least number of cases, in the extremes of age: 15-19 (3%) and >50 (4%). Age was not an independent prognostic factor with regards to response to MTX. Over 87% of cases were preceded by a molar pregnancy, with a minority of cases anteceded by an abortion (9.5%) or term gestation (5.5%). All of the patients who had antecedent molar pregnancy achieved remission with methotrexate alone except for 5 patients who were shifted to actinomycin due to hepatotoxicity. These patients subsequently responded to actinomycin. Five patients with term antecedent pregnancy were subsequently shifted to actinomycin due to chemoresistance with complete remission in three. The remaining patients were subsequently shifted to multiple agent therapy with complete remission. Of the 203 patients who completed treatment, 15% (30/203) were diagnosed < 4 months from index pregnancy, 23% (47/203) at 4-6 months from pregnancy, 56% (114/203) at 7-12 months from pregnancy and 6% (12/203) had their index pregnancies more than 12 months. No significant difference with regard to response to MTX treatment was seen based on duration of disease. 64% of patients had parity of 2-4. Twenty three per cent (47/203) of patients had vaginal and/or lung metastases but site and number of metastases had no significant effect on response to treatment. One hundred twenty-two patients (60%) underwent adjuvant hysterectomy (Table 3). The major indication was completed family size (64%). Other indications for hysterectomy were uterine rupture (20.5%) and intractable vaginal bleeding (11.5%). Only 5 patients underwent hysterectomy primarily for chemoresistance (4%). Comparing the overall ! -HCG values of preoperative and post-operatively, a statistically significant reduction in the mean level was noted. (mean preoperative =98,863 versus post -operative=16,973.4, p<.001). One hundred thirty-five of the 203 patients (67%) evaluated experienced adverse drug reactions to the treatment regimen, the most common being mucositis (26%). Other adverse reactions noted were deranged liver function tests (18%), pruritus and rash (12%), hyperpigmentation (7%) and diarrhea (5%).

II. Materials and Method The medical records of 257 patients with nonmetastatic trophoblastic disease (NMTD) and LRMGTN who were treated at the UP- PGH Medical Center between January 1988 and December 2004 were reviewed. Patients were classified accordingly using the International Federation of Gynecology and Obstetrics anatomic staging and prognostic scoring. Low risk metastatic GTN was defined as FIGO stage II/III with risk score ! 7. (FIGO Oncology Committee Report, 2002). The patients socio-demographic profile including age, antecedent pregnancy, pretreatment hCG level, pathologic diagnosis if known, type and number of course of chemotherapy, toxicity and treatment outcome were analyzed. Baseline metastatic work up were carried out, including physical examination, chest x-ray, pelvic ultrasonography, computed tomography of the pelvis, abdomen, chest and brain when needed. Complete blood count, serum hCG level, liver function and renal function tests were evaluated. A diagnosis of post molar NMGTN was based on 2 consecutive risings (> 10%) or 3 consecutive plateauings (< 10%) of weekly hCG titers, and no metastasis was detected elsewhere. Patients with NMGTN and LRMGTN were treated with methotrexate 0.3-0.4 mg/kg/day intramuscularly (IM) for 5 days. Treatment courses were repeated every 7-10 days. Chemotherapy was postponed in cases of grade 3 or 4 toxicity. Grading of toxicity was based on the Gynecologic Oncology Group Common Toxicity Criteria (DiSaia and Creasman, 2002). Patients with deranged liver function or showed resistance to methotrexate were treated with actinomycin D 12 ug/kg intravenously daily for 5 days every 1-2 weeks. Combination chemotherapy were given to patients after failure of 2 sequential single agents. Patients underwent adjuvant hysterectomy after a course of chemotherapy for the following indications: completed family size and chemoresistance. Patients with uterine rupture and intractable vaginal bleeding on admission usually had hysterectomies prior to chemotherapy. Serial serum hCG levels were determined before each course of chemotherapy. Complete remission was diagnosed after 3 consecutive weekly hCG levels were within normal range. Additional 2 courses of chemotherapy were given after the first normal hCG level. After remission, hCG levels

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Cancer Therapy Vol 6, page 613! Table 1. Distribution of Patients according to FIGO stage and Risk Score. FIGO Stage I II III IV Total

Risk Score Low Risk 128 (73%) 14 (8%) 33 (19%) 0 (0%) 175 (43%)

High Risk 82 (35%) 24 (10%) 100 (42%) 30 (13%) 236 (57%)

Total 210 (52%) 38 (9%) 133 (32%) 30 (7%) 411 (100%)

Table 2. Clinicopathologic Profile of Patients with GTN. Age 15-24 25-34 35-49 >50 Antecedent Pregnancy Molar Pregnancy Term Pregnancy Abortion Pretreatment hCG level < 1,000 1,000- <10,000 10,000-< 100,000 >100,000 Duration of Disease < 4 months 4-6 months 7-12 months >12 months NO metastases With Metastases Vagina Lungs +/- vagina

41 114 40 8

20% 56% 20% 4%

173 11 19

85% 5.5% 9.5%

38 26 80 59

19% 13% 39% 29%

30 47 114 12 156 47 14 33

15% 23% 56% 6% 77% 23% 7% 16%

Table 3. Indication for Surgery Indications for Surgery Uterine rupture Completed family size/adjuvant Vaginal bleeding Chemoresistance

N= 122 25 78 14 5

20.5% 64% 11.5% 4%

risk metastatic GTN. It has been associated with high rate of remission with low morbidity in majority of cases (Smith et al, 1982; Homesley et al, 1990; Homesly, 2002; Srisomboon et al, 2005; Sekharan et al, 2006). In this study, patients with NMGTN and LRGTN were initially given low dose IM methotrexate. Best responses are seen in cases where patients present with an antecedent molar pregnancy with deranged bHCG following molar evacuation. High complete remission rates (90.2%) have been demonstrated with the traditional regimen (single agent MTX 20-25 mg IM daily for five days every two weeks), and usually just after one course (81.5%) (Homesly, 2002; Srisomboon et al, 2005; Sekharan et al, 2006). In our study, remission rate was comparable at 88%

Of the 203 patients started primarily on single agent methotrexate chemotherapy, 178 patients (88%) achieved remission after a mean of 4.3 cycles. 25 patients were shifted to actinomycin due to adverse drug reaction (44%), resistance (40%) and progression (16%). Eighteen patients achieved remission with actinomycin after 2-4 courses. Four patients had chemoresistance to actinomycin but were cured with combination chemotherapy. Three patients went home against advice. Sustained remission rate for these patients was 98.5%.

IV. Discussion Single agent chemotherapy appears reasonable as initial therapy for patients with non metastatic and low-

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Samadan-Cagayan: Efficacy of methotrexate as primary single agent therapy for NMGTN and LRMGTN adjuvant hysterectomy offers some benefit to lessen the chemotherapeutic cycles of a patient and to hasten cure. In this study, patients with NMGTN and LRGTN were given methotrexate and actinomycin. The results of this study verify previous reports of complete cure in all patients with NMGTN and LRMGTN treated with chemotherapy with or without adjuvant hysterectomy. The 178 patients treated initially treated with methotrexate in this study had primary complete remission rate of 88%, comparable to that of other studies (Smith et al, 1982; Homesley et al, 1990; Homesly, 2002). In conclusion, single agent low dose IM methotrexate chemotherapy appeared to be highly effective and welltolerated in treating NMGTN and LRMGTN. Virtually all patients with non-metastatic and low-risk disease can be cured using single agent therapy. The excellent cure rate with chemotherapy in gestational trophoblastic tumors allows the patients to attain complete remission while retaining their fertility.

and a sustained remission rate of 98.5% but usually after 4-5 courses of single agent therapy. The dose-limiting toxicities of MTX are bone marrow depression and gastrointestinal toxicities with nadir often appearing 7 to 10 days after treatment. Hematologic effects of MTX include leukopenia, thrombocytopenia and anemia. An early sign of GI toxicity is mucositis while severe toxicity may be manifested as diarrhea, ulceration and bleeding. To avoid toxicities, it is important to have alkalinization of the urine (pH>5.0), hydration and administration of leucoverin, a tetrahydrofolic acid derivative used to treat potential toxic effects of MTX overdoses. Leucoverin is also used as part of high-dose MTX regimens as planned rescue. There were very few serious or dose limiting toxicities noted in the present study and most of the toxicities were mild and manageable. Toxicity is minimized with leucovorin rescue, while still achieving comparable efficacy. In our institution, MTX is given daily at a dose of 0.3-0.4 mg/kg intramuscularly for 5 days at 7-10 day interval until normal hCG titer is reached. Most patients tolerate MTX at this dose with no need to give folinic acid and because of this, MTX is usually administered on an out-patient basis. However, folinic acid is given when a patient manifests hypersensitivity or toxicity to MTX and then changed to another treatment regimen, usually single agent actinomycin D. Actinomycin D is a highly effective chemotherapeutic agent in the treatment of NMGTN (Berkowitz et al, 1990; Homesley et al, 1990). This agent was used as secondary treatment in the present study. Out of 25 patients who were shifted to actinomycin due to toxicity and resistance, 18 patients responded very well resulting in complete remission. It is also an appropriate regimen for patients with liver or renal disease contraindicating methotrexate use. Adjuvant hysterectomy is considered unnecessary in treating NMGTN, since nearly all patients are cured with chemotherapy (Srisomboon et al, 2005). Although adjuvant hysterectomy in patients with NMGTN has been reported to decrease the amount of chemotherapy courses required to achieve remission, surgical intervention is usually restricted to removal of chemotherapy resistant foci and control of hemorrhage and infection in emergency cases (Berkowitz et al, 1986; DiSaia and Creasman, 2002). In the present series, adjuvant surgery remains an integral part of patient management. More than half of our patients (60%) underwent surgery for various reasons such as completed family size, tumor bleed, intractable vaginal bleeding and chemoresistance. There was a significant decrease of post operative BHCG levels among patients who underwent surgery. By this, it would mean faster time to achieve remission since beta hCG remains to be a very sensitive tumor marker for response to treatment of GTN. As was reported by Lurain and coleagues “ prompt regression of BHCG within one to two weeks of surgical resection predicts a favorable outcome” (Lurain, 2006). Although chemotherapy remains to be the cornerstone of management in GTD, surgery still plays a major role in improving a patient ‘s chance for cure with or without chemotherapy. In a tertiary institution such as PGH,

References Bagshawe KD, Dent J, Newlands ES, Begent RHJ, Rustin GJS (1989) The role of low-dose methotrexate and folinic acid in gestational trophoblastic tumours. Br J Obstet Gynaecol 96, 795-802. Bagshawe KD, Wilde CE (1964) Infusion therapy for pelvic trophoblastic fumors. Br J Obstet Gynaecol 71, 565-70. Barter JF, Soong SJ, Hatch KD, Orr JW Jr, Partridge EC, Austin JM Jr, Shingleton HM (1987) Treatment of nonmetastatic gestational trophoblastic disease with oral methotrexate. Am J Obstet Gynecol 157, 1166-8. Berkowitz RS, Goldstein DP (1981) Pathogenesis of gestational trophoblastic neoplasms. Pathobiol Ann 11, 391-411. Berkowitz RS, Goldstein DP, Bernstein MR (1986) Ten years’ experience with methotrexate and folinic acid as primary therapy for gestational trophoblastic disease. Gynecol Oncol 23, 111-8. Berkowitz RS, Goldstein DP, Bernstein MR (1990) Methotrexate infusion and folinic acid in primary therapy of nonmetastatic gestational trophoblastic tumors. Gynecol Oncol 36, 56-9. DiSaia PJ, Creasman WT (2002) Clinical gynecologic oncology. 6th ed. St. Louis, Mosby, pp 621-4. FIGO Oncology Committee Report (2002) FIGO Staging for gestational trophoblastic neoplasia 2000.! Int J Gynaecol Obstet 77:285-87. Hammond CB, Weed JC Jr, Currie JL (1980) The role of operation in the current therapy of gestational trophoblastic disease. Am J Obstet Gynecol 136, 844-56. Homesley HD, Blessing J, Rettenmaier M, Capizzi RL, Major F, Twiggs L (1990) Weekly intramuscular methotrexate for nonmetastatic gestational trophoblastic disease. Obstet Gynecol 36, 56-9. Homesly HD (2002) Treatment of non-metastatic gestational trophoblastic disease. edited by BW Hancock. Sheffield, pp. 293-304. Kohorn EI (1982) Hydatidiform mole and gestational trophoblastic disease in Southern Connecticut. Obstet Gynecol 59, 78-84. Lurain JR, Brewer JI, Torok EE, Halpern B (1982) Gestational trophoblastic disease, treatment results at the Brewer Trophoblastic Disease Center. Obstet Gynecol 60, 354-60. Lurain JR, Brewer JI, Torok EE, Halpern B (1983) Natural history of hydatidiform mole after primary evacuation. Am J Obstet Gynecol 145, 591-5.

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Cancer Therapy Vol 6, page 615! Lurain JR, Elfstrad EP (1995) Single-agent methotrexate chemotherapy for the treatment of nonmetastatic gestational trophoblastic tumors. Am J Obstet Gynecol 172, 574-9. Lurain JR (2006) The role of surgery in the management of high risk gestational trophoblastic neoplasia. J Reprod Med, 773776. Matsui H, Iitsuka Y, Seki K, Sekiya S (1998) Comparison of chemotherapies with methotrexate, VP-16 and actinomycinD in low-risk gestational trophoblastic disease, remission rates and drug toxicities. Gynecol Obstet Invest 46, 5-8. Morrow CP, Kletzsky OA, DiSaia PJ, Townsend DE, Mishell DR, Nakamura RM (1977) Clinical and laboratory correlates of molar pregnancy and trophoblastic disease. Am J Obstet Gynecol 128, 424-30. Mutch DG, Soper JT, Babevek CJ, Clarke-Pearson DL, Hammond CB (1990) Recurrent gestational trophoblastic disease, experience of the Southeastern Regional Trophoblastic Disease Center. Cancer 66, 978-82. Petrilli ES, Twiggs LB, Blessing JA, Teng NN, Curry S (1987) Single-dose actinomycin D treatment for nonmetastatic gestational trophoblastic disease. Cancer 60, 2173-6.

Sekharan PK, Sreedevi NS, Radhadevi VP, Beegam R, Raghavan J, Guhan B (2006) Management of postmolar gestational trophoblastic disease with methotrexate and folinic acid 15 years of experience. J Reprod Med 51, 835-840. Smith EB, Weed JC Jr, Tyrey L, Hammond CB (1982) Treatment of nonmetastatic gestational trophoblastic disease, results of methotrexate alone versus methotrexate-folinic acid. Am J Obstet Gynecol 72, 413-8. Srisomboon J, Suprasert P, Phongnarisorn C, Charoenkwan K, Siriaree S, Cheewakriangkrai C, Tantipalakorn C, Kietpeerakool C (2005) Treatment Results of Methotrexate and Folinic Acid as Primary Chemotherapy for Nonmetastatic Gestational Trophoblastic Neoplasia. J Med Assoc Thai 88, 886-888. Suzuka K, Matsui H, Iitsuka Y, Yamazawa K, SekiK, Sekiya S (2001) Adjuvant hysterectomy in low-risk gestational trophoblastic disease. Obstet Gynecol 97, 431-4.

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Cancer Therapy Vol 6, page 617! Cancer Therapy Vol 6, 617-622, 2008

A Meta analysis assessment of bevacizumab in colorectal cancer in-patient to decide therapy with limited data Research Article

Fabiana Rubba1,*, Salvatore Panico2, Patrizia Cuccaro1, Antonio Della Vecchia1, Maria Triassi1 1 2

Preventive Health Services Department, Federico II University Hospital, Naples Clinical and experimental medicine Department, Federico II University Hospital, Naples

__________________________________________________________________________________! *Correspondence: Fabiana Rubba, MD, PhD, Public, Preventive an Social medicine school, University "Federico II" Medical School, Ed 12-D, Via Pansini, 5 80131, Naples, Italy; Tel/Fax: 00397462675; e-mail: fabiana.rubba@unina.it Key words: EBM, meta-analysis, bevacizumab Abbreviations: 5-fluorouracil/leucovorin, (5-FU/LV); Colorectal cancer, (CRC); fluorouracil, (5-FU); irinotecan, bolus 5-FU, leucovorin, (IFL); leucovorin, 5-FU, irinotecan, (FOLFIRI); leucovorin, 5-FU, oxaliplatin, (FOLFOX); metastatic Colorectal cancer, (mCRC); Odds Ratios, (OR); Time to progression, (TTP); vascular endothelial growth factor, (VEGF)

Received: 30 July 2008; Revised: 1 September 2008 Accepted: 3 September 2008 electronically published: September 2008

Summary We evaluated the introduction of a new targeting antineoplastic drug, Bevacizumab, for a young woman and for almost 30 in - patients of Federico II University Hospital, Naples, the year when this drug was approved for treatment in Europe, by summoning systematic reviews to a multidisciplinary committee (with explicit declaration of no conflicting interest), in order to assess clinical efficacy and tolerance and to control hospital costs. The literature search was done using the keywords from the medical search engine PubMed (National Library of Medicine, Bethesda, MD, USA) (No authors listed, 1992; Rose, 1992; Davidoff et al, 1995; No authors listed, 1995; Gigerenzer and Edwards, 2003; Ioannidis, 2005). The quality of all studies was assessed by two independent reviewers using standard EPOC quality criteria (www.epoc.cochrane.org). We established as clinical efficacy outcome the 18 months survival and the 1 year Time to progression (TTP). 95% CI for ORs were the follow: 18 months Survival: bevacizumab/fluorouracil/leucovorin vs fluorouracil/leucovorin / (95% CI OR 0,7-3,9) and 1 Year Disease Free Time bevacizumab/fluorouracil/leucovorin vs fluorouracil/leucovorin / (95% CI OR 0,16-0,9). After 3 years the evidence produced consistent results and this finding support the decision of carry on bevacizumab therapy.

For nearly 35 years, the standard chemotherapeutic regimen for treating metastatic colorectal carcinoma consisted only of fluorouracil (5-FU), with an overall response rate of 10% and a median survival of 10 months. Significant progress in the treatment of colon cancer has been made in recent years. In mCRC, with the development of systematic chemotherapy, including 5fluorouracil, oxaliplatin, and irinotecan, disease progression has been slowed and survival time has increased. However, until ~2000, combination 5fluorouracil/leucovorin (5-FU/LV) was the only regimen used worldwide. Then, Targeted monoclonal antibody therapy has developed. Bevacizumab- an angiogenesis

I. Introduction New technologies progress to the continuous introduction of new drugs and increasing trend of new costs jeopardize the welfare prospective of health care systems. Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. With an estimated 153,760 newly diagnosed cases and 52,180 cancer-related deaths in the United States for 2007, it will remain the second leading cause of cancer-related death (Shiroiwa et al, 2007). Almost 20% of these patients will present at the time of diagnosis with metastatic colorectal cancer (mCRC).

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Rubba et al: Assessment of bevacizumab in colorectal cancer in-patient were carried out for principal outcome measures 18-months survival and 1 year TTP, in relation to available data. After three years, evidences were reviewed and systematic review was repeated in order to verify the therapeutic scenario and access if the decision to use the drug was correct (Kabbinavar et al, 2003, 2005; Hurwitz et al, 2004; Rothman and Greenland, 2005; Bir et al, 2007; Golfinopoulos et al, 2007; Kwon et al, 2007; Shiroiwa et al, 2007; Tappenden et al, 2007a,b; Saltz et al, 2008; Sato et al, 2008).

inhibitor and a recombinant humanized monoclonal antibody-blocks the activity of vascular endothelial growth factor (VEGF). VEGF is a growth factor ligand that binds to specific cells and stimulates new blood vessel formation. Bevacizumab has been approved for the treatment of mCRC in the United States (in 2004) and the European Union (in 2005). The American National Comprehensive Cancer Network has recommended the use of bevacizumab. The objective of the presented report is to use EBM methodology and meta analysis in a crucial Health choice context , as expensive new drug against colorectal cancer, in order to decide appropriately, despite the boundaries of limited and preliminary data.

III. Results The 2005 systematic review data are summarized in Table 1. The two Kabbinavar’s (Kabbinavar et al, 2003; 2005) studies were eligible for Meta –analysis. Meta analytical evaluation suggested that bevacizumab have a significant effect on time to progression and a consequent increase in the healthy life duration (Kanhemann Nobel lecture; Royal Swedish Academy of Sciences. www.nobel.se; No authors listed, 1998; Glasziu et al, 2003; Rothman and Greenland, 2005). Data concerning improving survival were not significant. 95% CI for Odds ratios ORs were the follow: !! Survival: bevacizumab/fluorouracil/leucovorin vs fluorouracil/leucovorin (95% CI OR 0,33-1,2) !! Disease -free time bevacizumab /fluorouracil/leucovorin vs fluorouracil/leucovorin (95% CI OR 0,22-0,96) Therefore, it was decided to use the new drug in patient despite the high costs of the therapy. Three year later an audit was performed in order to confirm the drug- approval decision. The 2008 systematic review data are summarized in Table 2.

II. Methods We evaluated the introduction of a new targeting antineoplastic drug, Bevacizumab, for 31 in - patients of Federico II University Hospital, Naples, the year when this drug was approved for treatment in Europe, by summoning systematic reviews to a multidisciplinary committee (with explicit declaration of no conflicting interest), in order to assess clinical efficacy and tolerance and to control hospital costs. The literature search was done using the keywords from the medical search engine PubMed (National Library of Medicine, Bethesda, MD, USA) (No authors listed, 1992; Rose, 1992; Davidoff et al, 1995; No authors listed, 1995; Gigerenzer and Edwards, 2003; Ioannidis, 2005). The quality of all studies was assessed by two independent reviewers using standard EPOC quality criteria (www.epoc.cochrane.org). We established as clinical efficacy outcome the 18 months survival and the 1 year TTP. Pooled Odds Ratios (OR) of comparable studies were calculated as log OR*w/w were “w” was study weight calculated as 1/variance (Rothman and Greenland, 2005). Meta-analysis

Table 1. 2005 systematic review. Kabbinavar et al, 2005

Hurwitz et al, 2004

Prospective randomized double controlled trial, sample (N 209.); periodical endpoints blind-assessment. 1 endpoint: median survival (months) 2 endpoint: disease free survival 3 endpoint: response rate age (>18ys); Prospective randomized controlled trial,

bind

median surv ival : 16,6 vs 12,9 months (p=0,16) progression free survival : 9,2 vs 5,5 months (p=0,0002) response rate:26% vs 15 % (p=0,055 )

double

bind

median survival : 20,3 vs 15,6 months (p<0,001) progression free survival : 10,6 vs 6,2 months (p<0,001) response rate:44,8% vs 34% (p<0,004)

sample (N 813 ). 1 endpoint: median survival (months) 2 endpoint: disease free survival 3 endpoint: response rate age (>18ys); Kabbinavar et al, 2003

Prospective randomized controlled trial; sample (104 pz.) age (>18 ys)

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median survival : 21.; 16.1 vs 13.8 progression free survival : 5.8; 7.2 vs 5,2 months response rate: 44 %; 24% vs 17%)

Cancer Therapy Vol 6, page 619! Table 2. 2008 systematic review. Study Sato et al, 2008

Saltz et al, 2008

Design To explain the background of CPT-11 and to discuss its role in the treatment of advanced or recurrent colorectal cancer in Japan and elsewhere together with the authors' own experience.

Outcome Dramatic prolongation of the survival of patients with advanced or recurrent colorectal cancer has been achieved by multi-drug chemotherapy using agents such as continuous 5-fluorouracil and leucovorin, irinotecan (CPT-11), L-OHP, bevacizumab and cetuximab

To evaluate the efficacy and safety of bevacizumab when added to first-line oxaliplatin-based chemotherapy (either capecitabine plus oxaliplatin [XELOX] or fluorouracil/folinic acid plus oxaliplatin [FOLFOX-4]) in patients with metastatic colorectal cancer (MCRC).

median survival : 21,3 vs 19,6 months (p<0,07) progression free survival : 9,4 vs 8 months (p<0,002)

sample (N 813 ). 1 endpoint: median survival (months) 2 endpoint: disease free survival age (>18ys); To evaluate the combination of bevacizumab with infusional 5-fluorouracil (5-FU), leucovorin (LV) and irinotecan (FOLFIRI) in patients with advanced colorectal cancer (CRC) pretreated with combination regimens including irinotecan and oxaliplatin sample (104 pz.)

Kwon et al, 2007.

1 endpoint: median survival (months) 2 endpoint: disease free survival age (>18ys); We conducted a retrospective study of first-line FOLFOX plus bevacizumab to better characterize the safety and efficacy of this regimen in the first-line treatment of MCRC

Bir et al, 2007

progression free survival: 3,9 months median survival: 10,9 months

median survival : 23,2 months progression free survival: 9,9 months

1 endpoint: median survival (months) 2 endpoint: disease free survival Shiroiwa et al, 2007

The purpose of this cost-effectiveness analysis was to examine the economic efficiency of treating mCRC with bevacizumab plus chemotherapy versus chemotherapy alone in Japan

Tappenden 2007.

et

al,

The aim of this study is to estimate the cost-effectiveness of adding bevacizumab to chemotherapy for patients with untreated metastatic colorectal cancer.

Tappenden 2007

et

al,

To assess the clinical effectiveness and cost-effectiveness of bevacizumab and cetuximab in the treatment of individuals with metastatic colorectal cancer (CRC).

This review highlights a number of areas for further research, including clarifying the true impact of first-line bevacizumab in combination with irinotecan and/or infusional 5-FU/FA, without subsequent bevacizumab treatment following disease progression, on OS in patients with metastatic CRC who are representative of the typical population of CRC patients in England and Wales.

Golfinopoulos et al, 2007

The aim of our study was to estimate the magnitude of survival and disease progression benefits with the use of different regimens in patients with advanced colorectal cancer

The disease progression benefits were even more prominent for the addition of irinotecan plus bevacizumab (HR 0.41 [0.28-0.60]); irinotecan plus oxaliplatin

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In this cost-effectiveness analysis in Japan, the ICERs of bevacizumab + FU/LV combination treatment, IFL, and secondline FOLFOX4 were high compared with other chemotherapies for mCRC. Given high acquisition costs in relation to clinical benefits, bevacizumab is unlikely to represent a cost-effective use of NHS resources

Rubba et al: Assessment of bevacizumab in colorectal cancer in-patient However, the therapeutic scenario is not significantly changed: a recent met analysis confirmed the Kabbinavar’s results and almost 1 year life prolongation was demonstrated using also irinotecan. To assess more reliable data concerning survival and to assess the best first -line therapy is a current challenge. Our report show that to assess met-analytic value of risk measures also in urgent decision-making context support a consistent, evidence-based judgement in a managerial issues as Hospital drug policy in severe and life-threatening diseases as Cancer.

No different conclusions have been derived concerning survival neither different consideration affecting time to progression of disease. There is no consensus on Cost-effectiveness of bevacizumab in first-time therapy. Consequently, drug-approval decision was confirmed.

IV. Discussion Bevacizumab was the first angiogenesis inhibitor to demonstrate improving health in patients with metastatic colorectal cancer by inhibition of all active isoforms of VEGF-A. A randomized phase III trial (AVF2107) with 813 previously untreated metastatic colorectal cancer patients showed that the addition of bevacizumab to IFL (irinotecan, bolus 5-FU, leucovorin) increased response rates as well as progression-free and overall survival. The toxicity profile revealed an increase in (bevacizumabdependent) hypertension, but this was manageable with standard oral antihypertensive agents (Shiroiwa et al, 2007). Today, bolus administration of 5-FU, as evaluated in 2005, is no longer considered standard treatment. Recently, bevacizumab has been combined with the infusional regimens FOLFOX (leucovorin, 5-FU, oxaliplatin) and FOLFIRI (leucovorin, 5-FU, irinotecan). First review yet suggested an ameliorating effect in Irinotecan combination therapy, despite first data were not pooled with Kabbinavar’s (Kabbinavar et al, 2003). New drugs are often introduced without long standing evaluation of clinical efficacy and tolerance assessment. This short track of drug evaluation favours the new drugs, even equivalent, but also may produce a burden of drug choices without consistent and long term effectiveness’ assessment Nowadays to measure clinical effectiveness and tolerance profile in hospital pathways together with costs is becoming a crucial and urgent step in new drugs surveillance and prescriptive habits monitoring. In our analysis, the “Affect Heuristic”, personal and moral perception of health context, could have influenced the judgement and favoured the bevacizumab statement. However, statistical and pooled analysis also demonstrated a good result in 1 year healthy life gain consistent with heuristic perception, strengthening the results of each single study. To conclude it may be pointed out that the response rate in bevacizumab users was associated to gain longer or at least a better life. Three years later cost-effectiveness analysis stressed the high cost of targeting drugs as bevacizumab in first line-therapy. In January 2007, NICE issued a statement in which bevacizumab and cetuximab were not recommended as treatment options for patients with mCRC.30. The basecase costs per QALY gained were £62,857 for bevacizumab combined with IFL compared with IFL alone, and £88,436 for bevacizumab combined with 5FU/LV compared with 5-FU/LV alone. Similar data were found from a Japanese analysis (Golfinopoulos et al, 2007; Shiroiwa et al, 2007).

References Bir A, Tan W, Wilding GE, Lombardo J, Fakih MG (2007) 5fluorouracil, leucovorin and oxaliplatin plus bevacizumab in the first-line treatment of metastatic colorectal cancer: a single-institute study. Oncology 72, 4-9. Davidoff F, Case K, Fried PW (1995) Evidence Based-Medicine: why all the fuss? Ann Int Med 122, 727. Gigerenzer G, Edwards A (2003) Simple tools for understanding risks: from innumeracy to insight. BMJ 327, 741-744. Glasziu P, Del Mar C, Salisbury J (2003) Evidence Based Work book.London: BMJ Book. Golfinopoulos V, Salanti G, Pavlidis N, Ioannidis JP (2007) Survival and disease-progression benefits with treatment regimens for advanced colorectal cancer: a meta-analysis. Lancet Oncol 8, 898-911. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F (2004) Bevacizumab plus Irinotecan, Fluorouracil, and Leucovorin for Metastatic Colorectal Cancer". N Engl J Med 350, 2335-42. Ioannidis JP (2005) Why most published research findings are false. PLoS Med 8, 696-701. Kabbinavar F, Hurwitz HI, Fehrenbacher L, Meropol NJ, Novotny WF, Lieberman G, Griffing S, Bergsland E (2003) Phase II, Randomized Trial Comparing Bevacizumab Plus Fluorouracil (FU)/Leucovorin (LV) With FU/LV Alone in Patients With Metastatic Colorectal Cancer. J Clin Oncol 21, 60-5. Kabbinavar FF, Schulz J, McCleod M, Patel T, Hamm JT, Hecht JR, Mass R, Perrou B, Nelson B, Novotny WF (2005) Addition of Bevacizumab to Bolus Fluorouracil and Leucovorin in First-Line Metastatic Colorectal Cancer: Results of a Randomized Phase II Trial. J Clin Oncol 23, 3697-705. Kanhemann Nobel lecture; Royal Swedish Academy of Sciences. www.nobel.se. Kwon HC, Oh SY, Lee S, Kim SH, Kim HJ (2007) Bevacizumab plus infusional 5-fluorouracil, leucovorin and irinotecan for advanced colorectal cancer that progressed after oxaliplatin and irinotecan chemotherapy: a pilot study. World J Gastroenterol 13, 6231-5. No authors listed (1992) Evidence-based Medicine Working Group. Evidence-based medicine: a new approach to the teaching of medicine. JAMA 268, 2420-5. No authors listed (1995) Evidence-based Medicine, in its place. Lancet 346, 785. No authors listed (1998) Users’guide to the medical literature. JAMA 4, 10. Rose G (1992) The strategy of Preventive Medicine. Oxford: Oxford University Press. Rothman K, Greenland K (2005) Modern Epidemiology. Oxford: Lippincott.

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Cancer Therapy Vol 6, page 621! Saltz LB, Clarke S, DĂ­az-Rubio E, Scheithauer W, Figer A, Wong R, Koski S, Lichinitser M, Yang TS, Rivera F, Couture F, SirzĂŠn F, Cassidy J (2008) Bevacizumab in combination with oxaliplatin-based chemotherapy as firstline therapy in metastatic colorectal cancer: a randomized phase III study. J Clin Oncol 26, 2013-9. Sato T, Hatate K, Ikeda A, Yamanashi T, Ozawa H, Onosato W, Nakamura T, Ihara A, Watanabe M (2008) Treatment of advanced or recurrent colorectal cancer with irinotecan in Japan and elsewhere. Expert Opin Pharmacother 9, 12238. Shiroiwa T, Fukuda T, Tsutani K (2007) Cost-effectiveness analysis of bevacizumab combined with chemotherapy for the treatment of metastatic colorectal cancer in Japan. Clin Ther 29, 2256-67. Tappenden P, Jones R, Paisley S, Carroll C (2007a) Systematic review and economic evaluation of bevacizumab and cetuximab for the treatment of metastatic colorectal cancer. Health Technol Assess 11, 1-128. Tappenden P, Jones R, Paisley S, Carroll C (2007b) The costeffectiveness of bevacizumab in the first-line treatment of

metastatic colorectal cancer in England and Wales. Eur J Cancer 43, 2487-94.

! From left to right: Salvatore Panico and Fabiana Rubba

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Cancer Therapy Vol 6, page 623! Cancer Therapy Vol 6, 623-628, 2008

Inhibition of endothelial cell adhesion and in vitro angiogenesis by Taurolidine Research Article

Thomas Möhler1,*, Martina Willhauck-Fleckenstein1,2, Reinhard SchwartzAlbiez2, Annette Merling2, Hanns Möhler3 1

University of Heidelberg, Department of Medicine V, Im Neuenheimer Feld 410, 69120 Heidelberg, German Cancer Research Center, Im Neuenheimer Feld 210, 69120 Heidelberg, 3 Institute of Pharmacology, ETH Zurich and University of Zurich, Winterthurerstr. 190, CH-8057 Zurich 2

__________________________________________________________________________________! *Correspondence: Dr. Thomas Möhler, MD, Department of Medicine V, (Haematology/Oncology/Rheumatology), University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany; Tel: 0049-163-5456620; e-mail: thomas.moehler@med.uniheidelberg.de Key words: Taurolidine, Endothelial cell proliferation, Endothelial adhesion, angiogenesis, Statistical analysis Abbreviations:! Human Bone Marrow Endothelial Cell line, (HBMEC); human Epidermal Growth Factor, (hEGF); human umbilical vein endothelial cells, (HUVECS); Phosphate buffered saline, (PBS)! Received: 27 May 2008; "#$%&#'(!)*!+,-,&.!)//0! Accepted: 8 September 2008; electronically published: September 2008

Summary Taurolidine is a clinically active tumor suppressing agent. To gather experimental evidence whether an antiangiogenic mechanism might contribute to the observed anti-tumor effects in vivo, we investigated the ability of this compound to inhibit endothelial proliferation and in vitro angiogenesis. Taurolidine inhibited proliferation of microvascular endothelial cells derived from skin, bone marrow and placenta origin in concentrations of 70 ! M and lower. Taurultam, a congener of taurolidine, inhibited proliferation less effectively. Both Taurolidine and Taurultam selectively inhibited adhesion of endothelial cells to laminin but not to collagen I and fibronectin. In the in vitro angiogenesis assay, Taurolidine induced inhibition starting at concentrations of 4.4 ! M. In conclusion, our data indicate an antiangiogenic mechanism of action of Taurolidine based on inhibition of endothelial proliferation and adhesion to laminin. We propose that the antiangiogenic effect of Taurolidine contributes to its therapeutic activity in cancer patients.

which the median life expectancy of patients is only 6-12 months despite some recent improvements in therapeutics (Stupp et al, 2007). Previously, it was demonstrated that Taurolidine, a derivative of the aminoacid taurin, and its congener Taurultam have the ability to inhibit proliferation of multiple tumor cell lines !"# $!%&' (Rodak et al, 2005). The inhibition of proliferation can be detected starting at concentrations of approximately 70 ! M !"# $!%&' corresponding to 20 ! g/ml. These initial observations led to the use of Taurolidine in two patients with recurrent glioblastoma on a compassionate use basis, resulting in a partial response in both patients (Stendel et al, 2004) . In addition, clinical improvement was observed in patients with gastric carcinoma (Braumann et al, 2006). Mechanistically, Taurolidine had previously been shown to induce an apoptotic cell death of tumor cells involving the induction of reactive oxygen species, the activation of

I. Introduction Antiangiogenesis was recognized in the recent decade as an important new strategy for the treatment of cancer (Carmeliet, 2005; Folkman, 2007). Several novel compounds with antiangiogenic activity have been approved for treatment of different cancer types or are recognized by international medical associations for the respective oncologic indications as Avastin®, Thalidomide and Thalidomide derivatives, sorafenib® and others (Kabbinavar et al, 2003; Moehler et al, 2004). Glioblastoma and visceral adenocarcinomas including colorectal, pancreatic and gastric carcinomas are highly vascularized tumors with a corresponding high expression of the potent angiogenic factor VEGF (Yamamoto et al, 1998; Konno et al, 2000; Jain et al, 2007). There is an urgent need for the development of novel therapeutic agents for such tumors, in particular for glioblastoma for 623

Möhler et al: Inhibition of endothelial cell adhesion and in vitro angiogenesis by Taurolidine

C. TCS®angiogenesis assay

apoptosis inducing factor and -depending on cell type- a cytochrom c pathway or enhancement of Fas ligand expression (Han et al, 2002; Stendel et al, 2003; Rodak et al, 2005). Using repeated intravenous infusions of Taurolidine it was shown that concentrations effective !"# $!%&' can be achieved !"# $!$' in the clinical setting (Stendel et al, 2007). Up to now, there are no reports describing the direct effect of Taurolidine on endothelial proliferation or angiogenesis. Therefore, in the present study, it was tested, whether Taurolidine and Taurultam would affect the proliferation of endothelial cells and the VEGF-induced !"#$!%&' angiogenesis.

To assess the effect of Taurolidine and Taurultam on angiogenesis we used an !"#$!%&' angiogenesis assay of TCS Cell Works (TCS Cell Works, Buckingham, UK). The assay was performed according to the manufacturers instruction. In brief, endothelial cells were incubated under endothelial culture conditions in 24-well-plates in the presence or absence of vascular endothelial growth factor VEGF (20 ng/ml) (Promocell, FRG) or VEGF plus Taurolidine in concentrations of 70.3 ! M to 1.1 ! M Medium was changed in 3 days intervals for a total culture duration of 10 days. VEGF and Taurolidine were added after each change of the medium in the same concentrations as in the beginning of the culture to ensure continued exposure of cultures with VEGF and/or Taurolidine. All experimental conditions were performed in triplicates. All experiments were repeated at least twice. At the end of the 10-day culture period, cultures were fixed with 2 % paraformaldehyde and stained with anti-CD31 antibodies followed by APAAP visualization (Figures 2,3) as previously described (Vacca et al, 2003). Formation of tubules was manually counted using a Zeiss inverted microscope with 400x magnification (high power field).

II. Materials and Methods A. Endothelial cell proliferation assay Primary human umbilical vein endothelial cells [HUVECS, Promocell, Germany], Human Bone Marrow Endothelial Cell line [HBMEC] (Rood et al, 2000) and the human dermal endothelial cell line [HMEC- 1] (Xu et al, 1994) were cultured in endothelial specific cell culture medium using ECGM (Endothelial Cell Growth Medium, Promocell, Germany) supplemented with hydrocortisone 1 ! g/ml, hEGF (human Epidermal Growth Factor) 0.5ng/ml and hbFGF (Fibroblast Growth Factor) 0.1 ! g/ml and Endothelial growth supplement as recommended by the manufacturer (Promocell Germany). Endothelial proliferation was performed according to Oliver et al. using colorimetric detection of cell number (Oliver et al, 1989). In each proliferation assay 1x104 ECs were used per well in a 96-well-plate (day 0). Cells were placed into 96-well plates 24 hrs before addition of compounds to exclude an effect on adhesion. All wells were coated with fibronectin in a concentration of 10 ! g/ml for 2 hrs at 37 C before addition of endothelial cells. Taurolidine (MW: 284.37) and Taurultam (MW: 136.18) were added to the culture medium on day 1 in concentrations between 20 ! g/ml and 0.3 ! g/ml (for Taurolidine: 70 ! M to 1.1 ! M, for Taurultam 146.8 ! M to 2.3! M). There was no change of medium or readdition of compounds. Three days after addition of the compounds, cells were fixed with 4 % Formalin/PBS and air dried. Cells were then stained with 1 % methylenblue solution in 1 M borate buffer for 10 min at room temperature. After washing, 0.1 N HCL was added and absorption was detected using an ELISA reader at 650 nm. In reference experiments it was shown that the absorption was strictly correlated with the cell number using our cell culture conditions. Each condition was performed parallel in 8 wells. Each experiment was repeated at least twice.

D. Compounds Taurolidine and Taurultam were kindly provided by Geistlich Pharma AG, 6110 Wolhusen, Switzerland.

E. Statistical analysis Statistical comparisons were performed using the Wilcoxan log rank test.

III. Results A. Endothelial cell proliferation Endothelial cell proliferation was strongly inhibited by Taurolidine and also by Taurultam. The results of a representative quantitative determination of endothelial cell numbers after 4 days of cultivation in the presence of Taurolidine or Taurultam (0.3-20 ! g/ml, corresponding to 1.1-70 ! M for Taurolidine and 2.3-146.8 ! M for Taurultam) are given in Figures 1A-C for the three endothelial cell lines tested (HBMEC [bone marrow origin], HMEC-1 [dermal origin], HUVECs [umbilical vein origin]) as compared to the PBS controls. For each cell line, the inhibition of proliferation achieved by Taurolidine exceeded the degree of inhibition by Taurultam by more than 50 %. The level of inhibition of proliferation by 70 ! M (20 ! g/ml) Taurolidine was statistically significant for each cell type in comparison to that by Taurultam (p<0.001). Thus, the sensitivity of all three endothelial cell lines was greater for Taurolidine than for Taurultam as shown most clearly by the magnitude of their response to 20 ! g/ml (70 ! M for Taurolidine) of either agent. The difference in potency between the two compounds appears even more pronounced when compared on a molar basis as 20 ! g/ml corresponds to 146.8 ! M of Taurultam and 70 ! M of Taurolidine. The effects of inhibition of proliferation for both compounds was more pronounced for the primary endothelial cells (HUVECs) in comparison to the two endothelial cell lines (HMEC-1 and HBMEC) as shown in Figure 1. With HUVECS, Taurolidine achieved a 92 % inhibition at 70 ! M whereas the same concentration resulted only in a 81 % and 51 % inhibition for HMEC-1 and HBMEC, respectively.

B. Endothelial adhesion assay For preparation of adhesion assay flat bottom 96-wellplates were coated with sterile fibronectin (Gibco), collagen I (Sigma) or laminin (Sigma) each at a concentration of 10 ! g/ml in Phosphate buffered saline (PBS) for 1 hr at 37° C in humidified atmosphere. Before stimulation with VEGF (20 ng/ml) in supplemented ECGM (10% FCS) for 14h, HBMECs were grown for 24h in starvation medium (ECGM 5% FCS). Cells were harvested and incubated for 30 min on ice in serum free ECGM with or without test compounds in concentrations from 1.1 ! M to 350 ! M for Taurolidine and 2.3 ! M to 734 ! M for Taurultam. Subsequently 4 x 104 HBMECs were placed into individual wells for the 96well-plates precoated with extracellular matrix compounds as described above. After 30 minutes at room temperature nonadherent cells were gently removed by a careful washing step using PBS. Adherent cells were detected using the methylen-blue assay described for the proliferation assay (s. above). Adhesion assays were repeated 3 times with at least 6 wells per condition.

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Figure 1 A-C Inhibition of endothelial cell proliferation. The proliferation of three human endothelial cell lines was tested ( HBMEC, HMEC-1and HUVEC) in the absence and presence of increasing concentrations of Taurolidine (TI) (1.1 ! M to 70 ! M) and Taurultam (TA) (2.3 ! M to 146.8 ! M). The values given in the figure are concentration in ! M. Cell density was determined using a colorimetric assay by an ELISA reader with linear correlation of optical density [OD] and cell density after 3 days of culture in the presence or absence of compounds. Significant inhibition with p-value <0.001 (for TI 17.5 !M/HUVECS: p-value = 0.04) is indicated by asterisk.

B. Inhibition of endothelial adhesion

C. Inhibition of angiogenesis

Adhesion of HBMEC to laminin, collagen and fibronectin was investigated using decreasing concentrations of Taurolidine and Taurultam (Figure 2). We observed a striking difference in the ability to inhibit HBMEC adhesion dependent on the different ECM molecules used for coating of the 96-well-plates. Taurolidine induced a significant dose dependent inhibition of endothelial adhesion to laminin starting at a Taurolidine concentration of 17 ! M. Similar to Taurolidine, Taurultam induced significant inhibition of adhesion to laminin (Figure 2). Neither Taurultam nor Taurolidine were able to inhibit adhesion to collagen I or fibronectin under our conditions.

We employed a dual cell type/complex angiogenesis !"#$!%&' assay to determine the effect of Taurolidine on !"# $!%&' angiogenesis. Taurolidine was chosen for these experiments as it was more efficient in inhibiting endothelial proliferation than Taurultam. Using this assay, VEGF in a concentration of 20 ng/ml induced a three-fold increase in the number of endothelial tubules as compared to PBS control (Figure 3 and Figure 4). Taurolidine in concentrations between 4.4 ! M (1.25 ! g/ml) and 70 ! M (20 ! g/ml) was able to significantly (p<0.005) inhibit !"# $!%&' tubule formation. At 70 ! M the angiogenesis index was suppressed to the control level corresponding to a 100 % inhibition. With 4.4 and 17.5 ! M approximately 50 % of inhibition was achieved.

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MĂśhler et al: Inhibition of endothelial cell adhesion and in vitro angiogenesis by Taurolidine

Figure 2. Inhibition of endothelial adhesion by Taurolidine and Taurultam. Adhesion of HBMEC was investigated in 96-well plates pre-coated with extracellular matrix protein as indicated. Number of adherent cells per well was determined using a colorimetric assay (OD with linear correlation to number of adherent cells). Mean OD per condition and standard deviation of one out of three independent experiments in the presence or absence of a range of concentrations of Taurolidine (A-C) and Taurultam (D-F) are shown for collagen I (Col I), laminin (Ln), fibronectin (Fn) (*p-value = 0.03; **p-value</= 0.01)

Figure 3 Inhibition of in-vitro angiogenesis by Taurolidin. Utilizing the in vitro TCSÂŽ angiogenesis assay, the formation of new blood vessels and tubules was investigated for HBMEC cells under control conditions (cell culture medium only), the addition of VEGF 20 ng/ml or VEGF 20 ng/ml in the presence of Taurolidine in concentrations from 1.1 ! M to 70 ! M. Formation of APAAP-stained tubules was counted using a Zeiss inverted microscope with 400x magnification (high power field). For examples see Figure 4. The VEGF-induced generation of tubules was significantly inhibited in the presence of Taurolidine (TI). Note the high potency of Taurolidine, being effective already at 4.4 ! M (*p-value < 0.04; for 70 !M p-value = 0.0001).

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Figure 4. Representative examples for Taurolidine induced inhibition of angiogenesis. The !"# $!%&'# TCS速 angiogenesis assay was used to assess the formation of capillary-like structures and new blood vessels and tubules from HBMEC cells under control conditions (cell culture medium only), the addition of VEGF 20 ng/ml or VEGF 20 ng/ml in the presence of Taurolidine in concentrations from 1.1 ! M to 70 ! M. After 10 days, cultures were fixed and stained with a monoclonal antibody to CD31 using the APAAP technique (s. Figure 3 for quantitative results). (A) control: cell medium only. (B) VEGFinduced tubule formation as well as VEGF-induced increased branching of tubules and formation of in-vitro capillaries. (C) inhibition of VEGF-induced tubule formation to control level by Taurolidine 70 ! M.

endothelial cells we employed proliferation and adhesion assays. We now provide clear evidence for an antiangiogenic effect of Taurolidine based on a direct effect on angiogenic endothelial cells. To further investigate the effect of Taurolidinde on endothelial cells we employed proliferation and adhesion assays. Our results demonstrate that Taurolidine downmodulates endothelial proliferation in a concentration between 17.5 ! M (for HUVECS) and 70 ! M (for HBMECs and HMECs) (Figure 1). This is in the concentration range that is known to induce inhibition of tumor cell proliferation (10-40 ! M) as well (Stendel et al, 2002, 2003). Intravenous infusion of Taurolidine in Glioblastoma patients in clinical trials resulted in serum concentrations of approx 245 ! M (Stendel et al, 2007). Thus, concentrations that led to antiangiogenic effects in vitro in our experimental systems are achieved in patients. As a second effect on endothelial cells we describe the ability of Taurolidine to inhibit endothelial adhesion (Figure 2). We observed that the antiadhesive effect of both compounds was dependent on ECM proteins with significant effects on laminin and under our experimental

We did not observe a significant antiangiogenic effect by Taurolidine at 1.1 ! M using our culture conditions. Thus, Taurolidine was found to be highly effective in inhibiting VEGF-induced angiogenesis.

IV. Discussion The proliferation of human endothelial cells was strongly inhibited by Taurolidine and Taurultam as shown for three different human endothelial cell lines (Figure 1). Taurolidine specifically inhibited adhesion of endothelial cells to laminin, but we did not observe inhibition of adhesion to collagen I or fibronectin (Figure 2). Moreover, Taurolidine displayed significant antiangiogenic effects in a complex dual cell/!"# $!%&' angiogenesis assay starting at concentrations of 4.4 ! M (Figure 3-4). An antiangiogenic effect of Taurolidine had previously been postulated based on its ability to suppress tumor cell VEGF production. The production of VEGF by glioblastoma cells was inhibited by a minimal concentration of 70 ! M (Rodak et al, 2005; Thornton et al, 2006). To further investigate the effect of Taurolidinde on

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Möhler et al: Inhibition of endothelial cell adhesion and in vitro angiogenesis by Taurolidine Konno H, Baba M, Tanaka T, Kamiya K, Ota M, Oba K, Shoji A, Kaneko T, Nakamura S (2000) Overexpression of vascular endothelial growth factor is responsible for the hematogenous recurrence of early-stage gastric carcinoma. Eur Surg Res 32, 177-181. Ljubimova JY, Fujita M, Khazenzon NM, Ljubimov AV, Black KL (2006) Changes in laminin isoforms associated with brain tumor invasion and angiogenesis. Front Biosci 11, 8188. Moehler TM, Hillengass J, Goldschmidt H, Ho AD (2004) Antiangiogenic therapy in hematologic malignancies. Curr Pharm Des 10, 1221-1234. Oliver MH, Harrison NK, Bishop JE, Cole PJ, Laurent GJ (1989) A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors. J Cell Sci 92, 513-518. Rodak R, Kubota H, Ishihara H, Eugster HP, Könü D, Möhler H, Yonekawa Y, Frei K (2005) Induction of reactive oxygen intermediates-dependent programmed cell death in human malignant ex vivo glioma cells and inhibition of the vascular endothelial growth factor production by taurolidine. J Neurosurg 102, 1055-1068. Rood PM, Calafat J, von dem Borne AE, Gerritsen WR, van der Schoot CE (2000) Immortalisation of human bone marrow endothelial cells: characterisation of new cell lines. Eur J Clin Invest 30, 618-629. Stendel R, Picht T, Schilling A, Heidenreich J, Loddenkemper C, Jänisch W, Brock M (2004) Treatment of glioblastoma with intravenous taurolidine. First clinical experience. Anticancer Res 24, 1143-1147. Stendel R, Scheurer L, Schlatterer K, Stalder U, Pfirrmann RW, Fiss I, Möhler H, Bigler L (2007) Pharmacokinetics of taurolidine following repeated intravenous infusions measured by HPLC-ESI-MS/MS of the derivatives taurultame and taurinamide in glioblastoma patients. Clin Pharmacokinet 46, 513-524. Stendel R, Scheurer L, Stoltenburg-Didinger G, Brock M, Mohler H (2003) Enhancement of Fas-ligand-mediated programmed cell death by taurolidine. Anticancer Res 23, 2309-2314. Stendel R, Stoltenburg-Didinger G, Al Keikh CL, Wattrodt M, Brock M (2002) The effect of taurolidine on brain tumor cells. Anticancer Res 22, 809-814. Stupp R, Hegi ME, Gilbert MR, Chakravarti A (2007) Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 25, 4127-4136. Thornton AD, Ravn P, Winslet M, Chester K (2006) Angiogenesis inhibition with bevacizumab and the surgical management of colorectal cancer. Br J Surg 93, 1456-1463. Vacca A, Ria R, Semeraro F, Merchionne F, Coluccia M, Boccarelli A, Scavelli C, Nico B, Gernone A, Battelli F, Tabilio A, Guidolin D, Petrucci MT, Ribatti D, Dammacco F (2003) Endothelial cells in the bone marrow of multiple myeloma. Blood 102, 3340-8. Xu Y, Swerlick RA, Sepp N, Bosse D, Ades EW, Lawley TJ (1994) Characterization of expression and modulation of cell adhesion molecules on an immortalized human dermal microvascular endothelial cell line (HMEC-1). J Invest Dermatol 102, 833-837. Yamamoto S, Yasui W, Kitadai Y, Yokozaki H, Haruma K, Kajiyama G et al (1998) Expression of vascular endothelial growth factor in human gastric carcinomas. Pathol Int 48, 499-506.

conditions lack of significant effect on collagen and fibronectin. The selectivity for laminin points to a specific interference of Taurolidine with binding of lamininspecific integrin receptors. Whether Taurolidine interferes with integrin binding sites or other integrin-related mechanisms e.g. signaling, remains a matter of further investigation (Ljubimova et al, 2006). Although we have not determined apoptosis of endothelial cells experimentally, we assume that the antiadhesive effect of Taurolidine leads to anoikis (apoptosis by inhibition of adhesion). The inhibition of angiogenesis by Taurolidine is not directly mediated by the antiadhesive effect as, in our in-vitro angiogenesis assay, Taurolidine was added to the adherent ECs. Whether Taurolidine affects adhesion of circulating endothelial progenitor cells needs to be evaluated in the future. Furthermore, studies on tumor cell adhesion to laminin are proposed to determine whether inhibition of adhesion is a further mechanism of the antitumor activity of Taurolidine. The suppression of angiogenesis based on inhibition of endothelial proliferation and adhesion can be considered as an important mechanism for the antitumor activity of Taurolidine. This is of pivotal importance since inhibition of angiogenesis is an important mechanism of tumor suppression in highly vascularized tumors such as glioblastoma. In summary, our data on the antiangiogenic effects of Taurolidine have important implications for the further clincial development of Taurolidine, in particular for the treatment of glioblastoma and other vascularized tumors.

Acknowledgement We thank Mrs. Ute Braun and Annette Merling for excellent technical assistance. Taurolidine and Taurultam were kindly provided by Geistlich Pharma AG, CH-6110 Wolhusen, Switzerland.

References Braumann C, Winkler G, Rogalla P, Menenakos C, Jacobi CA (2006) Prevention of disease progression in a patient with a gastric cancer-re-recurrence. Outcome after intravenous treatment with the novel antineoplastic agent taurolidine. Report of a case. World J Surg Oncol 4, 34. Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature, 438, 932-936. Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 6, 273-286. Han Z, Ribbizi I, Pantazis P, Wyche J, Darnowski J, Calabresi P (2002) The antibacterial drug taurolidine induces apoptosis by a mitochondrial cytochrome c-dependent mechanism. Anticancer Res 22, 1959-1964. Jain RK, di TE, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT (2007) Angiogenesis in brain tumours. Nat Rev Neurosci 8, 610-622. Kabbinavar F, Hurwitz HI, Fehrenbacher L, Meropol NJ, Novotny WF, Lieberman G et al (2003) Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 21, 60-65.

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Cancer Therapy Vol 6, page 629! Cancer Therapy Vol 6, 629-646, 2008

Diagnostic and Therapeutic Efficacy of Imaging Modalities in Non-Small Cell Lung Cancer (NSCLC): experience from a Phase III clinical study using tumor targeted Lipoplatin nanoparticles Review Article

Faye Lazarioti1, Teni Boulikas1,2,* 1

Regulon AE, Afxentiou 7, Alimos, Athens 17455, Hellas Regulon, Inc. 715 North Shoreline Blvd, Mountain View, California 94043, USA

2

__________________________________________________________________________________! *Correspondence: Teni Boulikas Ph.D., Regulon AE., Gregoriou Afxentiou 7, Alimos, Athens, 17455, Greece; Tel: +30-210-9853849; Fax: +30-210-9858453 ; E-mail: teni@regulon.org Key words: NSCLC, CT imaging, PET-CT, MRI, Lipoplatin, antiangiogenesis, tumor targeting Abbreviations: Bronchioalveolar Cell Carcinoma, (BAC); Computed Tomography, (CT); Computer Aided Diagnosis, (CAD); Fluorine18 fluorodeoxyglucose, (FDG); Low-Dose Computed Tomography, (LDCT); Magnetic Resonance Angiogram, (MRA); Magnetic Resonance Imaging, (MRI); Non-Small Cell Lung Cancer, (NSCLC); Partial response, (PR); Positron Emission Tomography, (PET); Progressive disease, (PD); Response Evaluation Criteria In Solid Tumors, (RECIST) ; Solitary Pulmonary Nodules, (SPNs); Stable disease, (SD); Standardized Uptake Value, (SUV); World Health Organization, (WHO) Received: 12 May 2008; Revised: 12 June 2008 Accepted: 23 June 2008; electronically published: September 2008

Summary Chest radiography, computed tomography (CT), positron emission tomography (PET), and PET-CT are powerful imaging tools used worldwide for the diagnosis and treatment strategy of NSCLC. Furthermore, we present examples of CT imaging using an exciting new anticancer drug, Lipoplatin, a liposomal nanoparticle formulation of cisplatin. The ability of Lipoplatin™ to target primary tumors and metastases, using the permeability of the vasculature and the growing tumor for its preferential extravasation, and to cause a greater damage to tumor tissue as compared to normal tissue has been demonstrated in animal and human studies. It was demonstrated that Lipoplatin™ can target and kill tumor endothelial cells and, thus, it has the properties of a chemotherapeutic and of an antiangiogenesis drug, combined together. CT scans from patients participating in a multicenter Phase III clinical study demonstrate appraisal of response to Lipoplatin plus paclitaxel as first line treatment in NSCLC. Our results and literature review suggest that key factors for effective chemotherapy treatment and response of NSCLC relate to histological type and early diagnosis. We further suggest that liposomes endowed with tumor targeting properties can be used as carriers of radioactive material in cancer imaging.

subtypes, adenocarcinoma, squamous cell carcinoma and undifferentiated large cell carcinoma, with frequencies 50%, 30% and 5%, respectively (Michelle et al, 2007). In the current era where we witness breakthrough technological advancements, radiology acquired a pivotal role in diagnosis and screening of various diseases, especially cancer. In the last twenty years, diagnostic methods were multiplied and became significantly sophisticated, influencing the clinical algorithms and modulating the therapeutic strategies of patients, particularly those suffering from cancer. However, the abundance of current imaging armamentarium, created difficulties and questions on the appropriateness and role

I. Introduction Lung cancer is the most prevalent malignancy worldwide causing 17.8% of all cancer deaths (Spiro et al, 2002) and the overall 5-year survival rate does not exceed 15%. Its incidence ranks second in the US, with 213,380 new cases and 160,390 deaths recorded in 2007 (US National Cancer Institute). Non small cell lung cancer, (NSCLC) is the most frequent occurring that accounts for 80% of all lung cancers (Ettinger et al, 2004), while the more aggressive and rapidly spreading small cell lung cancer, accounts for merely 20% of all cases. NSCLC is a slow spreading malignancy that consists of three major

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Lazarioti and Boulikas Diagnostic and Therapeutic Efficacy of Imaging Modalities in NSCLC need to do a CT-scan. In most circumstances, where previously no chest radiographs were undergone, these patients must also proceed with CT-scanning (Patz et al, 2000). Finally, through protocol dictation, chest radiography is a prerequisite for lung cancer patients considered before undergoing surgery. This is also accompanied with a CT scan of thorax, liver and adrenal glands (British Thoracic Society guidelines). Although various studies have attempted to stress the potential of chest radiography alone as a screening tool for lung cancer in terms of sensitivity, diagnostic efficacy, timing and cost-effectiveness, the analysis of the results is not encouraging. Currently, there is a an ongoing randomized trial from the US National Cancer Institute and the American College of Radiology Imaging Network, addressing the issue of low dose CT versus chest radiography as screening tools. It includes more than 50,000 individual cases and there is much anticipation of its results, which are to be announced in 2009; this study will probably resolve the ongoing issue of lung cancer surveillance.

of each radiology diagnostic method in the management of NSCLC patients. Furthermore, for crucial indications such as lung cancer, it is more meaningful and relevant to establish an improved diagnostic or therapeutic efficacy of these diagnostic tools in contemporary clinical practice. The purpose of this article is to review the present state of the art diagnostic tools, but also to present examples of CT imaging using a new anticancer drug, Lipoplatin, endowed with tumor targeting and antiangiogenic properties.

II. Chest radiography Many studies have assessed the sensitivity of conventional chest radiographs in detection of NSCLC. However, the general consensus is that a significant number of pulmonary nodules may escape detection, especially in regions of anatomical overlap, such as the ribs and clavicles. Furthermore, particularly for small pulmonary nodules, it is generally considered that lesions below 2 cm are hardly visible in chest radiography, (although one study reported that the lowest size limit of visible nodule was 9 mm, Kundel et al, 1981). Apart from these factors, the degree of sedulous interpretation of chest films, as well as the precise and optimal technique employed in their acquisition, seems to play a crucial role in visualizing new solitary pulmonary nodules. It was shown that nearly 90% of these nodules where visible in retrospect in prior radiographs (Muhm et al, 1983). In a recent study, it was reported that simple radiography has 71.6%Âą4.8 sensitivity (Tsubamoto et al, 2002) in the detection of lung cancer, with the lowest rates recorded in bronchioloalveolar carcinoma (the subtype of adenocarcinoma), which in at least 60% of cases is found in the periphery (Patz et al, 2000). A different study stated that 85% of undetected lung cancers are peripheral (Woodring et al, 1990), whilst other research studies exhibited only 40% of patients with stage I disease, were diagnosed solely by chest radiography. The results rendered no impact in mortality from the disease (Fontana et al, 1988). Common signs of lung cancer in chest films, although non-specific, are: (i) unilateral hilar enlargement if a central tumor is involved; (ii) peripheral pulmonary opacity, which usually is irregular but can be well circumscribed when bronchioloalveolar carcinoma is involved; (iii) total, lobar or segmental collapse if the tumor grows within the bronchus, as is the case with squamous cell carcinoma; (iv) pleural effusion when pleura is invaded; (v) broadening of mediastinum when paratracheal lymphadenopathy occurs; (vi) rising hemidiaphragm in phrenic nerve palsy; (vii) osteolytic lesions of the ribs when direct invasion of the chest wall occurs, (viii) and finally, the radiographic “Sâ€? sign of Golden pertains to the characteristic shape of the bulging interlobar right fissure as is deviated around a central tumor mass (Armstrong et al, 2000). Once a lesion is identified as suspicious in chest radiography, it is important to compare it with previous radiographs. More explicitly, if the lesion was present and remained unchanged over a 2-year period, it can be presumed benign (Good et al, 1958; Yankelevitz et al, 1997). Although this might be true, the patient will still

III. Computed Tomography A. Overview Computed tomography has evolved ever since the pioneering studies of the English engineers, Hounsfield and McCormack, but also since 1973, where the method of application to patients was determined. Currently, the more sophisticated technology, the multi-detector row CT (MDCT) along with advances in software applications, allows, finer image resolution, detection of subtle contrast differences and minimizes image acquisition time, decreasing in this way the motion and partial volume effects. On the other hand, during the last decade, data acquired mainly through large cancer screening studies worldwide (Henschke et al, 1999; Benjamin et al, 2003; Midthun et al, 2003; Swensen et al, 2003) have added invaluable knowledge to our understanding of the morphology patterns, growth rates and biological characteristics of lung cancer. The above features provided a higher level of confidence, particularly in diagnostic dilemmas, as in the case of lung cancer imaging, which is of uppermost importance to determination of the benign or malignant nature of solitary pulmonary nodules (SPN). Even though the differential diagnosis of SPN is vast and not limited to neoplastic etiology, the tumoral origin bears the highest probability in certain patient populations (Helen et al, 2006). Furthermore, it possesses such a high variability of appearance, in terms of size, location, morphology, enhancement pattern, and growth rate that in several cases it represents a difficult challenge even for the most competent radiologist. All these features will be analyzed in this review in more detail, but at this point one must briefly outline the major applications of computed tomography in lung cancer: !! detection and characterization of pulmonary nodules or bigger lesions !! reliable method (though not the most accurate) in staging

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Cancer Therapy Vol 6, page 631! Trial (Swensen et al, 1999) states that nodules in the range of 4-7 mm in diameter are malignant in 0.9%, and for those less than 3 mm the respective percentage is 0.2%. Except for the prognostic value of size in the characterization of the nodule, another important application is the relevant time of screening for the disease, a subject that will be detailed later in the review.

!! accurate guiding for fine needle biopsy of pulmonary lesions !! screening tool, through low dose alternative techniques. As discussed earlier, there are various features, characteristics and parameters that need special attention by the radiologist in order to reach a safe conclusion about the malignant or benign nature of a nodule. Before their description however, it is important to point out the exact definition of a pulmonary nodule, since it is the most common form of manifestation of NSCLC followed by a consolidation-like process and less frequently, multiple small subcentimeter irregularly edged nodules. A SPN is a round or oval opacity, smaller than 3 cm in diameter that is completely surrounded by pulmonary parenchyma, which is not associated with lymphadenopathy, atelectasis or pneumonia (Midthun et al, 1993).

3. Distribution Although, nodule distribution is not a safe predictor of its benign or malignant nature (Swensen et al, 1999), several studies have concluded that most malignant nodules tend to be located in the right lung and more specifically in the upper lobe (Swensen et al, 2000; Winner-Muram et al, 2002). This however, does not imply that the left lung and/or the lower lobes are spared, as the latter is usually affected in patients with idiopathic pulmonary fibrosis, particularly the periphery (Lee et al, 1996). In these cases, the left lung is affected 1.5 times less commonly than the right. (Garland et al, 1961) Furthermore, adenocarcinoma, as well as BCA and undifferentiated large cell carcinoma, tend to occur in the periphery, while the squamous cell carcinoma subtype, occurs centrally, producing obstructive symptoms (Quinn et al, 1996).

B. Lesion Characterization 1. Radiological pattern The major classification adapted from most studies consists of 3 types of nodules: !! Solid, which is the most frequent type of nodule appearance, but the less likely to be malignant compared to other attenuation types. Paradoxically however, most cases of NSCLC are detected in solid nodules. On the other hand, isolated solid nodules usually represent granulomas (Libby et al, 2004). !! Partly solid or mixed nodules are considered to have a higher probability of being malignant (Helen et al, 2006). This is usually seen in invasive adenocarcinoma, when the solid part of the nodule is located centrally and the size is increased (Henschke et al, 2002; Li et al, 2004). !! Non-solid or ground glass nodules are potentially malignant, but less than that of partly solid ones. In most cases, it may represent a benign condition, which is mainly due to inflammation. The malignant potential of such nodules is increased when they contain malignant precursors, as in a atypical adenomatous hyperplasia (Kerr et al, 2001; Vazquez et al, 2003). Nodules that contain mixed tissue and ground glass densities are more commonly associated with bronchioalveolar cell carcinoma (BAC), with the ground glass part usually lying peripherally. Although 60% of BAC cases are present as a solitary nodule (Matthews et al, 1975), it may also be present in a multifocal fashion. Mucinous subtype is an example of a multifocal lesion, that bears well defined nodules of varying sizes or groundglass opacities that coalesce and resemble consolidation or reticulonodular infiltration (Travis et al, 2005). However, solid tumors without air-bronchograms are more commonly found in poorly differentiated adenocarcinomas (Erasmus et al, 1997).

4. Morphological and constitutive characteristics The edge of the lesion, which has been studied more thoroughly, can provide the radiologist with an assumption rather than a definite impression for differentiating between a malignant or benign lesion. Despite this fact, there have been some features concerning the margin of the nodule that are strong predictors of malignancy and these are irregularity, spiculation, and lobulation (Gurney et al, 1993). The lobulated margin is due to the dynamic process of uneven growth rates of the tumor (Swensen et al, 1997), while the irregularity and spiculation result from the extension pattern of the cancer cells in the pulmonary structures (Heitzman et al, 1982). In the same way that an irregular margin does not necessarily indicate malignancy (Huston et al, 1987), the reverse is also true as up to one third of malignant lesions the margin observed is smooth (Bateson et al, 1965; Siegelman et al, 1980; Seemann et al, 2000). The same stands for most case of adenocarcinomas, particularly the poorly differentiated ones (Erasmus et al, 1994). Probably the most important feature for malignancy exclusion of a nodule is the presence of calcification, its pattern and locus of distribution correlated with the size of the nodule. We distinguish 4 patterns of calcification: central nidus, laminated, popcorn and diffuse. In the presence of one of the patterns, it has been stated that the likelihood of benignity approaches 100% (Siegelman et al, 1986; Zerhouni et al, 1986). However, up to 45% of benign nodules are not calcified and CT studies have shown that up to 13% of lung cancers have some calcification, particularly when it is located eccentrically in the nodule (Zerhouni et al, 1986). The pattern of distribution in malignant lesions may be amorphous, stippled or diffuse (Mahoney et al, 1990).

2. Size Although there is no formative size guideline to determine the nature of a nodule, it can generally be stated that the larger the nodule the higher the probability of malignancy, with more than 90% of the nodules (<2 cm) in a benign state (Gurney et al, 1993; Swensen et al, 2005). Data obtained from a Mayo Clinic CT Screening 631

Lazarioti and Boulikas Diagnostic and Therapeutic Efficacy of Imaging Modalities in NSCLC al, 2002), worthy of remark is the relatively low specificity and PPV for the detection of malignant lymph nodes, an issue that determines the important N status, that will be analyzed under the comparison of the various methods later in this review. Lymph node involvement and enlargement is of paramount importance in staging NSCLC and certainly requires particular attention. Earlier data demonstrated that the most important predictor of malignancy in a lymph node was a short axis diameter of more than 10 mm for mediastinal lymph nodes and 7 mm for hilar nodes (Glazer et al, 1985; Quint et al, 1986; Deslauriers et al, 2000). Although this indication bears some truth, it cannot be used always as a safe guide for distinguishing malignant from benign enlarged lymph nodes, since, particularly in adenocarcinoma, 25% of resected normal-sized lymph nodes were found to be metastatic (Kerr et al, 1992). Additionally, in a correlation study of clinical and pathologic staging, the 10 mm limit was found to have sensitivity and specificity of 64% and 62% respectively, for detection of malignancy (McLoud et al, 1992). Furthermore, there are still some factors that weaken the 10mm limit, such as the fact that mediastinum normally contains lymph nodes of variable size, orientation of the scan plane that affects measurement of the node, and comorbid conditions such as infection, inflammation or reactive hyperplasia, which provoke the same state. CT in general, can reliably determine assessment of the N status in NSCLC, but there are more sensitive methods for establishing the presence of metastases. Lung cancer, especially the small cell type, is commonly associated with hematogenous spread at the time of diagnosis. Concerning NSCLC, squamous cell carcinoma is the subtype less commonly associated with early metastasis. Routine CT examination for staging in NSCLC, includes liver and adrenals, common target metastatic sites, especially the latter being present in nearly 7% of NSCLC patients (Silvestri et al, 1995). MRI however, is slightly better in detection of secondary lesions in these organs, and is the method of choice for brain metastases, though uncommon for NSCLC to occur in isolation (Hooper et al, 1984; Klein et al, 1991) and can exist without creating symptoms, particularly with adenocarcinomas (Newman et al, 1974; Kormas et al, 1992). There is no doubt that CT accurately demonstrates bone involvement, usually occurring in vertabral column, but in symptomatic patients, bone scintigraphy in correlation with radiographs, seems to be a better approach for differentiation between metastasis and coexisting degenerative disease.

The presence of fat in a lesion or nodule is also considered a marker indicating benignity, a common constituent of hamartomas or lipomas. However, malignancy does not exclude its presence, especially metastatic lesions of liposarcomas and renal cell carcinomas (Muram et al, 2003). In up to 55% of BAC, air bronchograms resemble pseudocavitation. Although, the percentage seems quite high, it is a more common feature in small cell carcinomas (Zwirewich et al, 1991).

5. Contrast enhancement The administration of iodinated contrast material during CT examination and monitoring of the nodule behavior concerning its uptake is another rough guide for the benign or malignant nature of the lesion. Enhancement of the lesion is analogous to its vascularity and although non-specific, increased enhancement is usually related to malignancy (Swensen et al, 1995; Swensen et al, 1996). It was shown that after a standard dose of intravenous contrast, nodules that failed to enhance ! 15HU were found to be benign with a positive predictive value of nearly 99%, (Swensen et al, 2000) while the minimal enhancing rim sign with a density of <15HU surrounding a homogeneously low density centered nodule has been associated with increased probability of a benign lesion (Muhm et al, 2003). However, again this does not represent a clear-cut guide since the percentage of enhancement of malignant nodules with more than 15HU is only 58%, the remaining represents benign lesions or active inflammation.

C. Staging solely by computed tomography There are two actual methods for lung cancer staging, especially for NSCLC, as small cell carcinoma is staged in a different way. Initially, clinical staging is done simultaneously or at least shortly after diagnosis, but before the initiation of a specific treatment plan and the pathologic staging that is performed on patients that have undergone surgery (in this way the true extent of the disease is more accurately expressed). It is not so infrequently that the latter upgrades the initial clinical stage while the reverse only exceptionally may occur. Although nowadays, other imaging modalities with better accuracy may be used for staging NSCLC, CT was and remains the initial and routine method applied to patients. The staging of NSCLC follows the TNM system as defined by the American Joint Committee on Cancer (AJCC) (Greene et al, 2002). In a recent comparison study of the available techniques used for staging, De Wever and colleagues reported in 2004 that CT alone can assess globally the TNM status of a patient in 46% of cases compared to 70% for integrated PET-CT units. Furthermore, the authors report sensitivity, specificity, PPV, NPV and accuracy values of 83%, 68%, 60%, 88% and 74% respectively, for the detection of malignant lymph nodes which is the primary concern for correct staging of the cancer. From the listed values, particularly sensitivity, which in general are higher than those reported in previous studies (McLoud et al, 1992; Winer-Muram et

D. CT-guided pulmonary biopsy The more invasive biopsy techniques which, however, provide the most accurate characterization of a nodule or lesion, are video-assisted thoracoscopic and open surgical biopsy. However, the advances in CT technology, the small-sized nodules, the fact that some patients are not candidates for surgery and the avoidance of purely invasive procedures in suspicious benign nodules, has made the fine needle or core biopsy

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Cancer Therapy Vol 6, page 633! ground-glass opacities, 457 days for solid nodules with ground-glass component, and 149 days for purely solid nodule size less than 5 mm, they proposed that longer follow-up intervals are appropriate for non solid nodules. Various other studies (Henschke et al, 2004, 2006) concluded that safe criteria for probable exclusion of malignancy are nodular size less than 5 mm (Midthun et al, 2003) and presence of small foci of clustered nodules, which usually indicate inflammatory or post-inflammatory origin. Finally randomly distributed nodules have many differential diagnosis including cancer, and annual monitoring is adequate for avoiding delayed diagnosis in nodules of initial size less than 5mm (Henschke et al, 2004). As it was stated initially in this section, there is a large dispute concerning NSCLC screening by CT and there are several arguments supporting it. Firstly and most importantly, it has not been proven that screening produces an actual or statistically significant reduction in mortality rate (Flehinger et al, 1992; Chirikos et al, 2002). Secondly, certain limitations such as length bias i.e. loss of tumors that progress rapidly, and over diagnosis bias i.e. detection of tumors that would not have resulted in patient’ s death due to an extremely slow rate of growth. Thirdly, issues of cost-effectiveness of CT screening versus anti-smoking campaigns or alternative techniques such as efficient molecular sputum markers (Tockman et al, 1997) since it was shown among others, that CT surveillance requires further diagnostic evaluation by other methods in at least 23% of cases (Henschke et al, 1999; Chirikos et al, 2002). In conclusion, we can state that CT screening certainly has a place in surveillance of NSCLC. Valuable information regarding this issue are expected from the large randomized study sponsored by the US Cancer Institute and the American College of Radiology Imaging Network that has enrolled more than 50,000 individuals and compares the efficiency of screening by chest radiology and CT (Hillman et al, 2003).

techniques under CT guidance reliable alternatives for this purpose.

E. CT screening for NSCLC Screening for lung cancer is a matter of dispute since early 1980s. The facts that disease is usually diagnosed at an advanced stage (in US only 12% of patients present with stage I and 15% with stage II disease) (Wagner et al, 1996), there is less than 15% 5-year survival (Van Klaveren et al, 2001) and 7% 10-year survival, and that other efforts of screening such as sputum cytology were shown ineffective (Melamed et al, 1984; Kubik et al, 1986; Fontana et al, 1988), turned the surveillance issue towards imaging. Earlier studies tested the efficacy of chest radiology as a screening tool but the results were inconclusive as chest X-ray could detect stage I lung cancer in only 40% of patients, with no impact on disease mortality (Fontana et al, 1988). With the emergence and development of CT, and especially low-dose spiral MDCT, screening for NSCLC appeared more feasible. Several screening studies have reported an early stage lung cancer detection in 55-80% of cases. CT was introduced as a screening tool by Japanese investigators in early 1990s (Kaneko et al, 1996; Sone et al, 1998) with encouraging results. Recently, a large international screening study with 25.000 participants demonstrated that stage I disease can be detected in 80% of cases (Henschke et al, 1999). Toward this, the development of software applications and emergence of Computer Aided Diagnosis (CAD) (Kim et al, 2005; Li et al, 2005) revealed some other parameters to exploit and use as potential adjuncts in screening and diagnosis, such as volumetry, growth rate assessments and 3D reconstructions. The growth rate of a nodule is itself a prognostic factor in patients with lung cancer (Usuda et al, 1994), and volumetry is important for establishing the doubling time Td. This is calculated by the equation: Td=Tixlog2/3xlog(Di/Do) where Ti=interval time, Di=initial diameter and Do= final diameter. A 25% increase in diameter is approximately equivalent to volume doubling (Armstrong et al, 2000). Doubling time of lung cancer ranges from 30 to 490 days, with 20% of tumors exhibiting doubling time of more than 465 days (Winer-Muram et al, 2002). Although the majority of lung cancers have rapid or moderately rapid growth rates, benign lesions may also grow in this rate (Winer-Muram et al, 2005). This, combined with the fact that there are limitations in precise measurement such as the necrotic, hemorrhagic or ground glass components of many tumors, indicate that doubling time can not be used as an accurate screening criterion in practice, but probably as means of differentiation of neoplastic from infective nodules, especially if Td is extremely short. Recently however, the Fleischner Society (MacMahon et al, 2005) issued recommendations, according to which it is safe to characterize a nodule as benign if no growth has been observed in two years. Furthermore, based on the observations of Hasegawa et al, 2000, that mean volume doubling times were 813 days for

IV. Positron Emission Tomography (PET) Positron emission tomography (PET) allows imaging of physiological molecules labeled with radioisotopes as an expression of metabolism. PET has greatly contributed to oncology, and particularly in lung cancer diagnosis and management. In 1998, Medicare in USA justified its usage for certain clinical indications, after it was shown that it changed the treatment plan in 40% of cancer patients and more specifically 18% of lung cancer patients (Weber et al, 2003), and despite being expensive, in the long run, reduced the healthcare costs. Furthermore, Van Tinteren et al, demonstrated that PET reduced futile surgery by 51% and prevented unnecessary surgery in 1 of 5 patients in a prospective study of 188 patients suspected to have NSCLC (Van Tinteren et al, 2002). Although, a thorough description of the method is beyond the scope of this review, the basic principle is that fluorine-18 (18F) attaches to deoxyglucose to produce fluorodeoxyglucose (FDG), as a marker of cellular glucose metabolism. Tumor and inflammatory cells have higher 633

Lazarioti and Boulikas Diagnostic and Therapeutic Efficacy of Imaging Modalities in NSCLC treatment plan in 15% of patients (Cerfolio et al, 2004). Moreover, the more striking superiority of integrated PETCT is in assessment of N status, particularly the precise verification of N0 status (Scott et al, 1996; Vansteenkiste et al, 1998; Magnani et al, 1999; Hany et al, 2002; Antoch et al, 2003; Lardinois et al, 2003), the important differentiation of N1 and N2 (Asamura et al, 2000), and finally, the identification of N3 with supraclavicular involvement (Antoch et al, 2003; Aquino et al, 2003; Lardinois et al, 2003; Cerfolio et al, 2004).

rates of glucose metabolism due to higher glucose uptake. The amount of FDG trapped within the lesion can be identified with the use of PET camera, and quantitatively be assessed by the standardized uptake value (SUV), an objective measurement of positron activity in the region of interest. There is evidence (Brown et al, 1999; Higashi et al, 2000) for a correlation of the FDG uptake to the degree of differentiation of adenocarcinoma, and generally the subtype of NSCLC, with expression of Glut-1 glucose transporter. FDG-PET has a sensitivity range of 90-100% and specificity range 69-95% in detecting malignancy of a solitary nodule (Lowe et al, 1998; Herder et al, 2000; Gould et al, 2004). However, there is the size threshold of 10 mm, below which the above values drop significantly, since one study showed that FDG-PET missed the verified malignancy of 8 out of 20 nodules below 1 cm in size (Nomori et al, 2004). Apart from the nodular size limitation, FDG-PET can provide false positive results in well-differentiated tumors such as BAC (Abouzied et al, 2004; Verschakelen et al, 2004; Steinert et al, 2005) in granulomatous diseases such as tuberculosis and sarcoidosis and fungal pulmonary infections (Roberts et al, 2000). Furthermore, the serum glucose level of the patient, which if raised causes reduced FDG uptake into tumor cells, can influence imaging and subsequently the diagnostic efficacy of PET.

VI. MRI of NSCLC Cost, being time-consuming, and limited access are the main reasons that MRI is not widely used in NSCLC diagnosis and staging, apart from the fact that in general terms, superiority compared to CT has not been proved. There are instances however, that MRI may be employed for better results, namely detection of brain metastases which are more common with adenocarcinoma than squamous cell carcinoma, clarification of tumor invasion in local structures such as chest wall, pleura, heart and pericardium, and extension of superior sulcus tumors through the lung apex in the lower neck (Heelan et al, 1989). However, the increasing application of MDCT has obviated some of these indications for MRI, which is therefore not routinely employed in lung cancer patients. There have been some MRI techniques, such as MRA, which show sensitivity up to 90%, specificity up to 87% and accuracy up to 88% (Ohno et al, 2001) in mediastinal and hilar invasion, but even so, application of MRI is reserved for selected cases, including liver and adrenal metastases that have not been clarified by CT.

V. Staging with PET and comparison with other modalities The available PET scanners, though offering valuable metabolic information, leave a lot to be desired on the anatomical details and localization of lesions. This limitation however, was overcome initially by the visualfusion of CT and PET images and recently by the introduction of the dual-modality units or integrated PETCT scanners. The first approach, though more rewarding anatomically-wise, proved unsatisfactory and complex due to differences in patient positioning and motion-induced data misregistration (Townsend et al, 2001; Beyer et al, 2002). Integrated PET-CT, on the other hand, improved diagnostic accuracy when compared with the separately acquired approach, even from preliminary studies (Antoch et al, 2003; Aquino et al, 2003; Cerfolio et al, 2004). A recent study (Tsubamoto et al, 2002) that compared the efficacy of integrated PET-CT with CT alone, PET alone, and visually correlated PET/CT, demonstrated superiority in prediction of TNM status, both globally and individually for T, N and M parameters. Moreover, it demonstrated an accuracy of 84% in detection of malignant lymph nodes for integrated PETCT, with sensitivity, specificity, PPV and NPV of 83%, 84%, 75% and 90% respectively, versus 83%, 81%, 71% and 89% for PET alone, and 83%, 78%, 68% and 89% for visually correlated PET/CT. Although subtle differences, these findings were verified by other studies (Antoch et al, 2003, Aquino et al, 2003; Lardinois et al, 2003; Cerfolio et al, 2004; Shim et al, 2005) especially in stage I and II disease, leading to a change in tumor stage in 26% of patients compared to PET alone, and modulation of

VII. Radiological evaluation of the response to treatment The introduction of a standard evaluation of the response of neoplastic lesions to therapy is important in clinical practice, not only for the standardization of clinical protocols, but also for the management of the cancer patient. The international criteria were introduced in the 1980s by WHO, and have been recently updated and correlated with the advances in imaging technologies. WHO criteria used the bi-dimensional measurement approach and were defined as following: Complete Response (CR) as disappearance of all known lesions confirmed at 4 weeks. Partial response (PR) as >50% reduction in the sum of the products of the perpendicular diameters of all measurable lesions, lasting for at least 4 weeks, during which time no new lesions appeared and no existing lesions enlarged. Stable disease (SD) as 50% reduction to 25% increase in the sum of the products of the two perpendicular diameters of all measurable lesions and the appearance of no new lesions for 8 weeks. Progressive disease (PD) as a >25% increase in the product of the two perpendicular diameters of any measurable lesion over the size at study entry, or, for patients who responded, the size at the time of maximum regression and the appearance of new areas of malignant disease. In 1994, the European Organization for Research and Treatment in Oncology, the National Cancer Institute of

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Cancer Therapy Vol 6, page 635! RECIST criteria’s validity in precise and accurate tumor response evaluation, substantiating the need for revision, addition and modification more imperative (Suzuki et al, 2008).

the United States, and the National Cancer Institute of Canada Clinical Trials Group, set up a task force with the objective to review tumor measurement techniques. The resulting guidelines, an outcome of retrospectively analyzing more than 4000 patients from fourteen trials, based on the model proposed by James et al, 1999 were published in the Journal of National Cancer Institute in February 2000. According to these RECIST criteria (Response Evaluation Criteria In Solid Tumors), a single measurement, more specifically the largest diameter in the transverse plane, suffices for quantifying tumor burden. Furthermore, RECIST proposed that measurements should arbitrarily include and limited to five lesions per organ and ten lesions per patient in those with tumors in multiple organs. As far as the unidimensional measurement approach is concerned, the criteria for treatment response were modified accordingly, with partial response being defined as greater than 30% reduction in tumor diameter, stable disease being less than 30% reduction or less than 20% increase of lesion diameter, and disease progression being greater than 20% increase in tumor size (Therasse et al, 2000). Computed tomography and MRI are the most reliable and reproducible imaging modalities for measuring target lesions. The type of CT scanner is important regarding the slice thickness and minimum size lesion. For spiral CT scanners, the minimum lesion at baseline may be 10 mm, provided that images are reconstructed contiguously at 5 mm intervals. For conventional CT, there is a minimum lesion size limit of 20 mm at baseline, by use of a slice contiguous slice thickness of 10 mm. As a rule of thumb, the minimum size of the lesion should be no less than double the slice thickness (Therasse et al, 2000). In terms of radiological criteria, RECIS! criteria have limitations that every radiologist should be familiar with. More specifically, the cystic lesions by definition are non-measurable, although many lesions that may have a central cystic or necrotic component, which modifies during treatment, should be taken under consideration in evaluating the response. Furthermore, RECIST criteria ignore the morphological changes that occur within neoplastic lesions, such as calcification, hemorrhage or necrosis. Another drawback pertains to lymph node involvement. These should be measured in the short axis dimension, since it is the best predictor of metastatic disease presence. Apart from that, it can be hypothesized that a metastatic lymph node in complete remission will return to its normal dimensions, which on individual basis are not specified and can therefore not be defined (Husband et al, 2004). According to the RECIST criteria bone metastasis is defined as “non measurable”, but the efficacy of MRI in evaluating the size of bone lesion has been demonstrated, and is widely used in clinical practice (Ciray et al, 2001). Finally, a crucial matter that RECIST criteria “ignore”, is the application of maximum “axial diameter” as the sole measurement criterion of tumor response, without exploiting the new developments and tools of three-dimensional reconstruction imaging provided by MRI and MDCT. In conclusion, the gradually increasing use of the latter modalities supplemented by PET and integrated PET/CT scanning, tend to outdate

VIII. Lipoplatin A. Lipoplatin as a tumor targeting nanoparticle and as an anti-angiogenesis drug Cisplatin continues to be one of the cornerstone drugs in the treatment of epithelial malignancies among over 700 FDA-approved drugs. However, cisplatin damages, indiscriminately, cancerous and normal tissue. Its severe side effects arise from induction of apoptosis in normal tissue in treated patients especially in peripheral nerves, renal tubules, bone marrow and gastrointestinal tract. The side effects of chemotherapy have prompted academic institutions, as well as biotechnology and pharmaceutical companies to invent new ways of delivering drugs. One fruit of the effort evolved from the genesis of the field of liposomes, pioneered by Gregoriadis and Papahadjopoulos and in the creation of the field of nanotechnology aimed at wrapping up old drugs or new molecules into nanoparticles composed of a variety of peptides, polymers, dendrimers, and hyperbranched polymers (Boulikas et al, 2007). Patented platform technologies have been used for the liposomal encapsulation of cisplatin into tumor targeted 110-nm in diameter Lipoplatin™ nanoparticles (also termed Oncoplatin™ and Nanoplatin™). NonPEGylated liposomes are taken up by liver macrophages and destroyed with a half-life in body fluids of 20 min. On the contrary, PEGylated liposomes, such as those of Lipoplatin, display a half-life of 5 days in body fluids (Boulikas et al, 2004). The advantage of Lipoplatin™ over cisplatin is suggested to result from the ability of Lipoplatin™ to target primary tumors and metastases using the permeability of the vasculature of the growing tumor for its preferential extravasation and to cause a greater damage to tumor tissue compared to normal tissue as demonstrated in human studies (Figure 1). The Lipoplatin formulation uses the anionic lipid DPPG that gives Lipoplatin its fusogenic properties presumably acting at the level of entry of the drug through the cell membrane after reaching the target tissue (Figure 2). Animal studies suggested that genes wrapped up in Lipoplatin™ shells target not only the tumors after systemic delivery but also their vasculature and result in the expression of a functional gene product after crossing the cell membrane barrier (Figure 3). It is being inferred that Lipoplatin™ is endowed with the properties of cisplatin plus the ability of its nanoparticles to target and kill endothelial cells of tumor vasculature suggesting that this drug has two properties, that of a chemotherapy drug and that of an antiangiogenesis agent, combined together. Figure 3 shows a SCID mouse implanted with MCF7 human breast tumor cells that were allowed to develop into large measurable solid tumors at about 30 days postinoculation. The animal was injected i.p. with a 635

Lazarioti and Boulikas Diagnostic and Therapeutic Efficacy of Imaging Modalities in NSCLC that Lipoplatin causes apoptotic death to endothelial cells of tumor vasculature in addition to the apoptosis it induces to the tumor cells as deduced from previous studies in xenografts (Boulikas et al, 2004). All these observations lead to the conclusion that Lipoplatin not only kills tumor cells but also cells of the tumor vasculature. It can therefore be classified also as an anti-angiogenesis agent.

liposomally encapsulated plasmid carrying the betagalactosidase gene under control of the CMV promoter. The encapsulated plasmid had the same shell as Lipoplatin. Following systemic injection with the reporter beta-galactosidase gene, and at 24 h postinjection the carcass was stained with X-Gal to reveal the sites of transgene expression. It can be concluded that the liposomally encapsulated gene was dramatically concentrated into tumors, it crossed successfully the cell membrane, survived any lysosomal, endosomal or cytoplasmic nucleases, was imported into nuclei, was successfully expressed into RNA and translated into protein responsible for the blue staining 24-h from injection. It is evident that the sites of gene transfer and expression in vivo are primarily the tumor and the subcutaneous vasculature developed to supply the tumor with nutrients indicating that cells (endothelial cells) of tumor vasculature are the targets for entry of the liposome and expression of the foreign gene. A control experiment with the same amount of naked plasmid did not reveal gene expression in the tumor and most other tissues presumably as a result of plasmid degradation in the peritoneal cavity. Since the LipoGene vehicle mediates delivery and expression of the gene, both in tumor cell mass and in tumor vasculature, it is concluded that LipoGenes (and by extension Lipoplatin) enter through the cell membrane in both cell types. It is known that plasmid DNA is poorly taken across the nuclear membrane barrier. It is concluded

B. Tumor targeting in human studies Lipoplatin is preferentially concentrated in the primary tumor and the metastases in human patients undergoing chemotherapy. High tumor levels are seen at about 20 h from infusion of the drug under conditions where blood levels of Lipoplatin have dropped. Targeting is done at two levels: (i) after intravenous injection Lipoplatin is preferentially (40-times) concentrated into tumors by extravasation through the leaky tumor vasculature; (ii) once inside the tumor Lipoplatin is taken up more avidly by the cell membrane of the tumor cell compared to normal cell (5 times more). These two mechanisms together contribute to a 200-fold higher damage to cancer tissue compared to normal tissue and contribute to the low side effects of the drug. This was shown by intravenous infusion of Lipoplatin in four independent patient cases (one with hepatocellular adenocarcinoma, two with gastric cancer, and one with colon cancer) who underwent Lipoplatin infusion followed by a prescheduled surgery ~20h later (Table 1).

Figure 1 Left. Cross section of a nanoparticle of Lipoplatin. The model shows the lipid bilayer and the cisplatin molecules in its lumen (yellow spheres) with the PEG molecules on its surface (red hair-like structures) coating the particle with a hydrophilic inert polymer giving the ability to escape detection from macrophages and evade immune surveillance. Right. Lipoplatin nanoparticles evade immune surveillance because of their PEG coating and extravasate preferentially into primary tumors and metastases through the compromised endothelium of their vasculature due to their small size (110 nm) and long circulation. Even tiny tumors of a size of a pinhead, often invisible in CT scans of x-rays, sprout new vasculature in a process known as neoangiogenesis; these lesions are accessible to Lipoplatin. Thus, Lipoplatin nanoparticles can target primary tumors and micro metastases, which was shown in animal and human studies. In addition, Lipoplatin nanoparticles were proposed to be able to target the endothelium of the tumor vasculature causing apoptosis, thus, endowed with antiangiogenesis properties (Boulikas et al, 2007). The Figure depicts the extravasation process (right) through the compromised endothelium of the vasculature of the tumor. Reproduced from Boulikas et al, 2007 with kind permission from Therapy Press, Mountain View, California, USA.

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Figure 2. Activation of signalling pathways by cisplatin and Lipoplatin including the mitochondrial, DNA damage, ERK, PI3K/AKT1 and death receptor leading to caspase activation and apoptosis is shown. Ctr1, the major copper influx transporter, imports Cisplatin. Two copper efflux transporters, ATP7A and ATP7B, situated at the periphery of the cell membrane regulate the efflux of cisplatin. Instead, Lipoplatin bypasses Ctr1 thanks to the fusogenic DPPG lipid which commands direct fusion with the cell membrane and cisplatin deliver across the membrane barrier. In addition, because of its 110-nm particle size (compared to cisplatin with a molecular dimension of less than 1 nm) Lipoplatin is taken up by phagocytosis. Tumor cells are known to be more actively engaged in phagocytosis than normal tissue. Thus, Lipoplatin acts as a Dorian Horse for tumor cells. Lipoplatin is proposed to be able to bypass cisplatin resistance. Reproduced from Boulikas et al, 2007 with kind permission from Therapy Press, Mountain View, California, USA.

Figure 3. Targeting of the vasculature of the primary tumor and the metastases after systemic delivery of “Lipogenes� using our proprietary liposomal encapsulation technology. The photo shows a SCID mouse implanted with MCF-7 human breast tumor cells. Following systemic injection with the reporter !-galactosidase gene, the carcass was stained with X-Gal. Preferential staining of the tumors, especially of the vascular system around the tumors is evident. Reproduced from Boulikas et al, 2007 with kind permission from Therapy Press, Mountain View, California, USA.

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Lazarioti and Boulikas Diagnostic and Therapeutic Efficacy of Imaging Modalities in NSCLC Table 1. Summary of human targeting by Lipoplatin. Patient # and specimen #1 Liver tumor #1 Normal liver tissue #1 Colon metastasis #1 Normal colon tissue #2 Liver metastasis #2 Normal liver tissue #3 Stomach tumor 1 #3 Stomach tumor 2 #3 Normal stomach tissue #4 Colon tumor 1 #4 Colon tumor 2 #4 Normal Colon tissue

Trapped 5.18 16.45 4.44 0.06 34.51 16.94 44.17 28.46 2.62 4.42 1.86 0.02

Tumor/ normal 0.31 74.00 2.04 16.86 10.86 221.00 93.00

Reacted 33.18 3.16 2.17 0.08 96.64 4.00 220.45 37.92 3.97 6.85 5.83 0.04

Tumor/ normal 10.50 27.12 24.16 55.53 9.55 171.25 145.75

advanced stage pretreated pancreatic cancer patients. Myelotoxicity of grades 3 and 4 was observed at 125 mg/m2 of Lipoplatin™ and 1000 mg/m2 of gemcitabine and therefore this dose scheme was considered as DLT whereas the dose of 100 mg/m2 of Lipoplatin™ and 1000 mg/m2 of gemcitabine on days 1,15 in a 28-day cycle for 3 cycles as the MTD (Stathopoulos et al, 2005a). There are several ongoing Phase II studies the results of which will be reported including: Lipoplatin monotherapy against NSCLC, Lipoplatin plus gemcitabine against NSCLC, Lipoplatin plus intravenous navelbine against metastatic breast cancer and others. There are three ongoing Phase III studies. The first Phase III (LipoGEM), is a randomized multicenter clinical study that compares 120 mg/m2 Lipoplatin on days 1,8,15 plus 1g/m2 gemcitabine on days 1,8 (Arm A) with 100 mg/m2 cisplatin on day 1 plus 1g/m2 gemcitabine on days 1,8 (Arm B). The cycle in each arm is 21 days and treatment is given for six cycles or until disease progression as first line treatment in patients with nonsmall cell lung cancer (NSCLC). Overall, this Phase III study shows that Lipoplatin™ appears to have a better safety profile and equivalent or slightly improved therapeutic profile than cisplatin, when combined with gemcitabine, in patients with advanced NSCLC as first line treatment. Particularly important might be the significantly lower neuro- and nephro-toxicity of the Lipoplatin™ arm and its administration on an outpatient basis (Boulikas et al, 2007). The second Phase III (LipoTaxol), was initiated in April 2006 in Greece. This randomized Phase III uses 200 mg/m2 Lipoplatin plus 135 mg/m2 paclitaxel administered on day 1 repeated every 2 weeks (Arm A). Lipoplatin was infused for 8 hours in 1 Lit 5% dextrose. Arm B is 75 mg/m2 cisplatin (hydration of 2 Lit) and 135 mg/m2 paclitaxel, administered every two weeks. One cycle is 14 days and the plan was to give 9 cycles (treatments) per patient unless disease progression was detected before the 9th cycle. As of December 2006, 61 chemo naive patients were recruited with a median age of 65 (42-80). 54 were evaluable for response and toxicity, 27 in each arm. Response: Arm A: PR: 48.15% SD: 37.03% PD: 3.7% and clinical benefit 11.11%. Arm B: PR 44.44% SD 44.44% PD: 3.7% and clinical benefit 7.41%. Thus, both arms

Tumor specimens were first extracted in saline and the platinum that was solubilized was related to platinum trapped in tissues («Trapped» in Table 1) that presumambly did not cross the cell membrane. Salineinsoluble material from tumor specimens was then extracted in SDS (sodium dodecyl sulfate) that disolved membranes, proteins, RNA and DNA thus revealing the amount of platinum that was bound to macromolecules («Reacted» in Table 1). Platinum levels in tumor specimens were compared with levels in the adjacent normal tissue and the ratio was recorded (Table 1). Direct measurement of platinum levels in extracts from specimens from the excised tumor and the adjacent normal tissue as well as metastases (colon metastasis from a liver tumor, liver metastasis from a gastric cancer) showed that total platinum levels that reacted with macromolecules and caused damage to tissue were on the average 10 to 171 times higher in malignant tissue compared to the adjacent normal tissue specimens; most effective targeting was observed in colon cancer with an accumulation up to 200-fold higher in colon tumors compared to normal colon tissue. Gastric tumor specimens had the highest levels of drug than any other tissue (Boulikas et al, 2005, Table 1). In conclusion, Lipoplatin has the ability to preferentially concentrate in malignant tissue both of primary and metastatic origin following intravenous infusion to patients. In this respect, Lipoplatin emerges as a very promising drug in the arsenal of chemotherapeutics.

C. Clinical development of Lipoplatin A phase I study on Lipoplatin™ as second- or thirdline treatment has been completed on 27 patients (19 pancreatic carcinoma, 6 renal cell carcinoma, 1 with gastric cancer and 1 with squamous cell carcinoma of the head and neck) and with dose escalation from 25 mg/m 2 to 125 mg/m2. The highlights of this study were that Lipoplatin had a mild hematological and gastrointestinal toxicity and did not show any nephro-, neuro-, ototoxicity, did not cause hair loss and was void of most other side effects (Stathopoulos et al, 2005). This drug received the orphan drug status by EMEA in 2007 against pancreatic cancer. A phase I/II dose escalation study of Lipoplatin and gemcitabine has been completed in 638

Cancer Therapy Vol 6, page 639! according to RECIST criteria, in patients with stage IIIb/IV NSCLC who received 200mg/m2 Lipoplatin plus 135mg/m2 paclitaxel on day 1, dosage repeated every 2 weeks for 9 cycles. All baseline examinations were performed as closely as possible to the commencement of treatment. Follow-up examinations were normally performed after 4 cycles (8 weeks) and 9 cycles (18 weeks) whereas any response should be documented after 4 weeks. Confirmation of chemotherapy response is used to prevent any overestimation of the response rate observed. Figure 4 shows CT slices of a 77-year-old man following chemotherapy treatment with Lipoplatinpaclitaxel for low differentiated adenocarcinoma. The decrease of the primary lesion in the right lobe is remarkable after 4 cycles of the specific chemotherapy. Figure 5 demonstrates another case of stage IV adenocarcinoma with lymph node metastases, which after 5 cycles of Lipoplatin-paclitaxel regressed substantially. Although, these examples do not display the full capabilities of a modern CT scanner with various software enhancements, it is obvious to the naked eye that the volume reduction of the tumor is substantial, after several cycles of Lipoplatin-paclitaxel chemotherapy. Figure 6 shows a CT scan of a NSCLC patient with a large tumor mass in the right lobe that is almost absent in the CT scan at the same section after 9 cycles of Lipoplatin-paclitaxel. These examples exhibit equivalent morphologic tumor changes that validate the drug’s efficiency, which, compared to the alike chemotherapeutic agents, provides increased survival with better tolerance. Figure 7 shows a CT scan of a NSCLC patient with a soft tissue mass in the left lobe which regressed substantially after 9 cycles of Lipoplatin-paclitaxel.

show about the same response rate with a slight superiority in the Lipoplatin arm. Toxicity: Arm A: Renal toxicity in 1 patient (3.70%) neurotoxicity grade I-II in 7 patients (25.92%) nausea-vomiting in 5 patients (18.52%) myelotoxicity Grade I-II in 10 patients (37.04%). Arm B: Renal toxicity in 7 patients (25.92%), neurotoxicity Grade I-III in 12 patients (44.44%) nausea-vomit in 7 patients (25.92%) myelotoxicity Grade I-III in 17 patients (62.96%). Thus, the toxicity differences are very important between the two arms. In particular, the renal toxicity appears to be 7-fold lower (700% less) in the Lipoplatin arm. Also significantly lower are the neurotoxicity and myelotoxicity of Grade III (totally absent in the Lipoplatin arm). It was concluded that the response rate was similar but toxicity and in particular nephrotoxicity, neurotoxicity, and myelotoxicity was significantly lower in the Lipoplatin arm (Stathopoulos et al, 2007). The third Phase III (LipoFU), is a randomized, multicenter phase III trial against squamous cell carcinoma of the head and neck (SCCHN). The study is comparing 100 mg/m2/day Lipoplatin (days 1,8,15) plus 1,000 mg/m2/day 5-FU (days 1 to 5) every 21 days (one cycle) for 6 cycles (Arm A). The comparative arm (Arm B) uses 100mg/m2/day cisplatin (day 1) plus plus 1,000 mg/m2/day 5-FU (days 1 to 5) every 21 days (one cycle) for 6 cycles (Jehn et al, 2007).

IX. CT scans from the Lipoplatin plus paclitaxel phase III study As has been mentioned already, CT is an excellent tool for chemotherapy response evaluation while Lipoplatin, being a powerful anti-tumor agent serves an ideal candidate whose efficiency could be tested. Figures 4 through 7 display examples of CT-based therapy appraisal

Figure 4. CT slices (with mediastinal window settings) A. A low differentiated adenocarcinoma in the right upper lobe and B. After 4 cycles of Lipoplatin-Paclitaxel chemotherapy, the patient shows partial response (PR).

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Lazarioti and Boulikas Diagnostic and Therapeutic Efficacy of Imaging Modalities in NSCLC

Figure 5. (A) A large adenocarcinoma cell tumor (arrows) in the left lower lobe. (B) Follow-up scan taken after 5 cycles of LipoplatinPaclitaxel treatment show reduction of volume of the lesion.

Figure 6. Left: CT section demonstrating a large tumor mass in the right lobe. Right: Follow-up scan after 9 cycles of Lipoplatinpaclitaxel treatment, demonstrates a remarkable mass reduction.

Figure 7. Left: CT section demonstrating a large tumor mass in the left lobe. Right: Follow-up scan after 9 cycles of Lipoplatinpaclitaxel treatment, demonstrates mass reduction.

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Cancer Therapy Vol 6, page 641! detection at digital chest radiography (dCXR) in a fashion similar to that of conventional film-screen techniques (Wu et al, 2008). Functional imaging using 2-18 fluorodeoxyglucose positron emission tomography is increasingly used and when combined with anatomic imaging provides better staging information for both local disease and the extent of metastases (Berghmans et al, 2008). Fluorine-18 fluorodeoxyglucose (FDG) uptake of the primary lesions in patients with a new diagnosis of advanced-stage NSCLC does not have a significant relationship with survival (Hoang et al, 2008). Integrated fluorodeoxyglucose-positron emission tomography/ computed tomography has been proposed to be a predictor of malignancy in mediastinal lymph nodes; some patients with non-small cell lung cancer with a maximum standardized uptake value less than 5.3 in their N2 lymph nodes might be able to forego mediastinoscopy and proceed directly to thoracotomy (Lee et al, 2008). Virtual bronchoscopy using data sets from positron emission tomography (PET) and computed tomography (CT) offers a useful alternative to fiberoptic bronchoscopy, and is particularly promising for patients for whom fiberoptic bronchoscopy is not feasible, contraindicated or refused (Englmeier et al, 2008).

X. Discussion A. NSCLC imaging The overall mortality rate for lung cancer is high, and early diagnosis provides the best chance for survival. CT, MRI, and PET play an important role in the detection, diagnosis, and staging of NSCLC as well as in assessing response to therapy and monitoring for tumor recurrence after treatment (Erasmus et al, 2008). CT and MRI are used in staging and provide anatomical information but have well known limitations in differentiating reactive from malignant nodes, and fibrosis from active disease. PET (using in the vast majority of cases 18 fluorodeoxyglucose) appears to have high sensitivity and reasonable specificity for differentiating benign from malignant lesions as small as 1 cm. PET appears superior to CT imaging for mediastinal staging in NSCLC (Ung et al, 2007). CT is considered the standard technique for assessing morphologic findings and intrathoracic spread of a solitary pulmonary nodule (SPNs). Although the clinical role of MRI for SPNs remains limited, dynamic MRI and dynamic CT are useful for differentiating between malignant and benign SPNs (Fugimoto et al, 2008). A PET/CT fusion scan is the most sensitive and accurate method of non-invasive mediastinal nodal staging; this tool was suggested to be a component of clinical staging of all NSCLC patients (Whitson et al, 2008). PET/CT has provided an incremental dimension to the management of cancer patients by allowing the incorporation of important molecular images in radiotherapy treatment planning, ie, direct evaluation of tumor metabolism, cell proliferation, apoptosis, hypoxia, and angiogenesis. Low-dose computed tomography (LDCT) identifies small, early-stage, resectable lung cancer in a high-risk population (Macapinlac et al, 2008). Positron emission tomography (PET) is currently accepted as an important tool in oncology, mostly for diagnosis, staging and restaging purposes; It can also be used for target volume definition in radiotherapy treatment planning (Rembielak et al, 2008). Radiofrequency ablation (RFA) for thoracic tumours has emerged as a minimally invasive therapy option for primary and secondary lung tumours and has gained increasing acceptance for pain palliation. The procedure is well tolerated and the complication rates are low (Steinke et al, 2008). Approximately 20 to 40% of patients with surgically resected stage I non-small cell lung cancer (NSCLC) will develop recurrent disease. The 2-[18F]-fluoro-2-deoxy-dglucose positron emission tomography is an imaging tool for assessing clinical tumor, node, metastasis in NSCLC; the primary tumor standardized uptake value (SUV) is a prognostic factor for survival as suggested from metaanalysis (Berghmans et al, 2008). Positron emission tomography (PET) with 2-[18F] fluoro-2-deoxy-D-glucose (FDG) is also used often in staging NSCLC (Goodgame et al, 2008). 18FDG-PET and multislice computerized axial tomography (CT) scan are used for diagnosis, staging and response evaluation in NSCLC patients (Decoster et al, 2008). The lesion size, location, conspicuity, and histopathology impact the likelihood of lung carcinoma

B. Trends and prospects Visual analysis of tumour uptake of 99mTc-HYNICrh-annexin-V and scintigraphy in patients with lymphoma, NSCLC and head and neck squamous cell carcinoma gave a statistically significant correlation between changes in annexin tumour uptake and therapy outcome (Kartachova et al, 2008). Acquired resistance to chemotherapy is a major hurdle in previously treated patients and the reason for the low response in second-line treatment. The major factor of resistance appears to be linked with transport of the chemotherapy drug across the cell membrane barrier. In this capacity, Lipoplatin, suggested to enter by direct fusion with the cell membrane of the tumor cell rather than the Ctr1 transporter as cisplatin, was proposed to have applications in cisplatin resistant tumors (Boulikas et al, 2007). Human studies have shown an astonishing targeting of tumors and metastases from an accumulation in total platinum in cancer tissue compared to adjacent normal tissue; in this study (Matthews et al, 1974) four patients received an infusion of 100mg/m2 Lipoplatin one day before prescheduled surgery and blinded specimens obtained during surgery were analyzed for levels of platinum. The method was designed to differentiate between platinum trapped in tissue and platinum that had entered the tissue cells and reacted with macromolecules (proteins, DNA, RNA and others). The study showed that the highest platinum levels were attained in gastric cancer specimens, presumably due to their high vascularization, suggesting very successful targeting of this cancer using our liposome nanoparticle technology. This observation along with the mechanism of targeting that involves extravasation through the endothelium of the tumors that bears tiny “holes� and allows the passage of the long641

Lazarioti and Boulikas Diagnostic and Therapeutic Efficacy of Imaging Modalities in NSCLC circulating Lipoplatin nanoparticles led to the proposal that Lipoplatin is a chemotherapy and an antiangiogenesis drug (Boulikas et al, 2007). This proposal was further supported from animal studies showing that systemic injection of a beta-galactosidase “blue gene” plasmid wrapped up in liposomes having the same shell structure as Lipoplatin to SCID mice bearing MCF-7 human breast cancer resulted in the preferential staining of the tumors and of the endothelium of tumor vasculature (Boulikas et al, 2007). The mechanism(s) for the higher accumulation of Lipoplatin™ in tumor tissue, compared to normal tissue results in an overall 10 to 400-fold higher tumor cell uptake. The ability of Lipoplatin™ to target primary tumors and metastases and to cause a greater damage to tumor tissue compared to normal tissue contributes to its therapeutic efficacy. In this respect, Lipoplatin emerges as a very promising drug in the arsenal of chemotherapeutics. In the same capacity, liposomes having a similar outer shell as Lipoplatin nanoparticles are proposed as carriers of radioactive material in NSCLC and other cancer imaging. Their ability to concentrate into tumors and metastases, including micrometastases such as those in bones from a primary gastric cancer using Lipoxal (liposomal oxaliplatin) (Figure 8) appears to be a general property of these particles regardless of the active substance in their lumen. Even tiny micrometastases that

cannot be visualized by CT scans or chest x-rays need to sprout their vasculature and in doing so are potential targets of our nanoparticles. Thus the liposomes proposed here as carriers of radioactive imaging substances could detect tumors otherwise invisible by conventional techniques.

Acknowledgements Supported by the European grants RIGHT, Nanopharmaceutics and Myoaid, to Regulon AE as well as by two grants from the Hellenic government (General Secretariat of Research and Development) for the clinical development of Lipoplatin, Lipoxal and LipoVIL12. We are grateful to Dr. George Stathopoulos and Ioannis Stathopoulos (Errikos Dunant Hospital, Athens) and to Drs Kostas Marosis, Antonios Bastas, Pepi Mihalopoulou, Aspasia Provata, Ioannis Dimitroulis, Pantelis Giamboudakis, Dimosthenis Antoniou and Kostas Lolis (from Sotiria Pulmonary Disease Hospital, Athens), and to Drs Eva Kalatzi, Maria Karabatzaki, Evi Balamoti, Nassos Alevizopoulos, Panagiotis Kourtesis, Nikos Anyfantis, Dimitris Patestos, Stella Doukelli and Christos Karanikas (Regulon) for stimulating discussions.

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Cancer Therapy Vol 6, page 647! Cancer Therapy Vol 6, 647-654, 2008

The role of percutaneous ultrasound guided radiofrequency ablation (RFA) in patients with breast cancer hepatic metastases Research Article

Gianpaolo Carrafiello1, Domenico Laganà1, Chiara Recaldini1, Monica Mangini1, Federico Fontana1, Elisa Cotta1, Maria Petullà1, Raffaele Novario2, Salvatore Cuffari3, Gianlorenzo Dionigi4, Carlo Fugazzola1 1

Department of Radiology, University of Insubria, Department of Physical Health, University of Insubria, 3 Department of Anesthesia, University of Insubria, 4 Department of Surgical Sciences, University of Insubria, Varese, Italy 2

__________________________________________________________________________________! *Correspondence: Professor Gianpaolo Carrafiello, Department of Radiology, University of Insubria, Viale Borri 57, 21100 Varese, Italy ; e-mail: gcarraf@tin.it Key words: Radiofrequecy Thermal Ablation (RFA), breast cancer metastasis Abbreviations: international normalized ratio, (INR); Radiofrequency Thermal Ablation, (RFA) Received: 15 July 2008; Revised: 30 July 2008 Approved: 1 August 2008; electronically published: September 2008

Summary The aim of our paper is to evaluate the effectiveness of Radiofrequency Thermal Ablation (RFA) of liver metastasis from breast cancer and its impact on survival. Thirteen female patients (range: 36-82 years, media 54.5 years) underwent RFA for the treatment of 21 liver metastases from breast cancer. The procedures were performed under US guidance using a RF 2000 or RF 3000 generator system and Le Veen monopolar needle electrodes. Follow up was carried-out by CT after 1,3,6,12 months. Technical success was 100%. No major or minor complications occurred at the end of the procedure. One month technique effectiveness was 95% (20 /21 lack of enhancement in the ablated tumor was observed). The mean overall survival after RFA was 10.9 months. Mean survival was calculated after the initial diagnosis of breast cancer and was 90.92 months. In conclusion, RFA appears to be a useful adjunct to systemic chemotherapy and/or ormonotherapy, in the loco-regional treatment of hepatic metastases from breast cancer. RFA may also be a less invasive alternative to surgery in the loco-regional treatment of liver metastases from breast cancer.

metastases involving the liver (Scheneebaum et al, 1994). A small group of patients with metastasis confined to the liver (5-12%) appear to have an improved prognosis after hepatic resection and association with hormonal therapy or chemotherapy can be more effective (Hoe et al, 1991; Zinser et al, 1987). In the last ten years radiofrequency thermal ablation (RFA) has gained wide acceptance for the treatment of patients with non resectable primary and metastatic liver tumors (Curley et al, 1999; Machi, 2001). RFA has been demonstrated to be an effective cytoreductive strategy for a variety of metastatic liver malignancies, with few side effects and without worsening quality of life of the patients (DeMatteo et al, 2001; Di Carlo et al, 2003).

I. Introduction Liver metastases have been identified at autopsy in as many as 55–75% of patients with breast cancer (Smith, 1987). Of all the patients with metastatic breast cancer, 5% will developed hepatic metastasis only (Hoe et al, 1991). Typically, this is a late finding and metastases are already present at other sites (Wyld et al, 2003). Patients with metastatic breast cancer have a poor prognosis and are usually considered to have disseminated disease even if the disease appears to be localised to a single organ (Kemeny, 1983). Although chemotherapy still remains the treatment of choice for patients with metastatic breast cancer, response rates are low in patients with disseminated breast

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Carrafiello et al: The role of percutaneous ultrasound guided RFA in breast cancer hepatic metastases under endocrine therapy and 1 patient was under chemotherapy and endocrine therapy before RFA treatment. The average diameter of the treated lesions was 3.5 cm (range 0.5-7 cm). We calculated the interval between the diagnosis of initial breast cancer and evidence of liver metastases (range 0 - 240 months; mean 87 months). At the moment of diagnosis of primary tumor, hepatic metastases were already present in 2 patients. The characteristics of patients are listed in Table 1 and in Table 2.

The aim of our paper is to evaluate the effectiveness of RFA in patients with liver metastasis from breast cancer and its impact on survival.

II. Materials and Methods In our Institution, between November 2003 and October 2007, 13 female patients (age: 36-82 years, mean 54.5 years) underwent RFA for the treatment of 21 liver metastases from breast cancer. Six patients were under chemotherapy, six patients were

Table 1. Characteristics of patients Patients Age 1

82

2

45

3

55

4

56

5

57

6

70

7

76

8

46

9

42

10

54

11

36

12

43

13

49

Primary carcinoma Infiltrating ductal carcinoma Infiltrating ductal carcinoma Infiltrating ductal carcinoma Infiltrating ductal carcinoma Infiltrating ductal carcinoma Infiltrating lobular carcinoma Infiltrating ductal carcinoma Infiltrating lobular carcinoma Infiltrating ductal carcinoma Infiltrating ductal carcinoma Infiltrating ductal carcinoma Infiltrating lobular carcinoma Infiltrating ductal carcinoma

TNM pT3 pN1 M0 pT2 pN1 M0 pT1 pN0 M0 pT1 pN0 M0 pT3 pN2 M1 pT1 pN0 M0 pT2 pN0 M0 pT1 pN1 M0 pT2 pN3 M1 pT1 pN0 M0

pT3 pN1 M0 pT1 pN0 M0

TM (months) 7

Breast Surgery Yes

Systemic therapy ET

Hepatic metastases 2

Extrahepatic metastases -

24

Yes

CH

1

brain

51

Yes

ET

2

bone,axillaryly mphnodes

234

Yes

ET

1

-

0

Yes

ET

3

-

100

Yes

ET

1

adrenal

42

Yes

CH

1

-

250

Yes

ET CH

1

0

Yes

CH

3

axillarylymphn odeslung, pleura -

52

Yes

CH

1

-

58

Yes

CH

3

-

139

Yes

CH

1

lung, bone

17

Yes

ET

1

pleura

ET: endocrino therapy; TM: time to metastases; CH: chemotherapy

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Cancer Therapy Vol 6, page 649! Table 2. Patients survival characteristics. Patients 1 2 3 4 5 6 7 8 9 10 11 12 13

TR (months) 18 5 1 7 38 1 19 1

Survival after RFA (months) 21 5 8 2 8 1 5 10 6 44 4 26 2

Total survival (month) 42 31 72 238 11 106 50 262 17 105 62 166 20

TR: time to recurrence

Diagnostic workup included contrast-enhanced abdominal CT scan and a biopsy under US guidance. Treatment decision was based on biopsy-proven breast metastases. Decision for RFA was made upon consensus of the surgeon, oncologist and radiologist and proposed to patients. An informed written consent was obtained by all patients. All patients were treated with prophylactic antibiotics therapy consisted with cefazolin 1 g IV every 8 hours, three days before the procedure till 2 days afterwards. RFA was performed under deep sedation according to the principles of “monitored anaesthesia care�: the patients were administered propofol (50-120mg), alfetanyl (1-3mg) and mydazolam (1-3mg); the skin at the site of the planned needle puncture(s) was anesthetized with 1% lidocaine. All patients received oxygen during the procedures; blood pressure and heart rate were monitored. The procedures were performed by two interventional radiologists skilled in liver RFA, under US guidance (Philips IU22, Netherlands; ATL HDI 3500 Advanced Technology Laboratories, Bothell, Washington, USA). Ablations were performed using a RF 2000 or RF 3000 generator system (Boston Scientific Corp., Natick, MA, USA) and Le Veen monopolar needle electrodes (Boston Scientific Corp, Natick, MA, USA). The choice of electrode size was decided in relation with tumor dimensions and the safety margin was 5mm. The lesions with diameter ! 3 cm were treated with two or more overlapping ablation treatments with a maximum of 4 session for the largest lesion. All patients were asked for post-radiofrequency syndrome: postablation syndrome is a common phenomenon after RFA of solid abdominal tumors and consisted of transient flu-like symptoms(fever,malaise,pain, myalgia, nausea and vomiting) (Carrafiello et al, 2007). Follow up was carried-out by CT after 1, 3 6, 12 months. Technical success was evaluated as the correct placement of the needle in the lesion during the procedure. Technique effectiveness was evaluated by contrast enhncement CT carried out at 1 month: complete ablation was considered when lack of enhancement in the ablated tumor was observed; irregular or nodular peripheral contrast enhancement was considered as residual tumor (Choi et al, 2001). The Kaplan-Meier method was used to evaluate first metastasis occurrence rate and overall survival since the initial diagnosis and after RFA. MedCalc version 9.4.2.0 (MedCalc Software Mariakerke, Belgium) was used for the analyses.

III. Results Technical success was 100%. Inclusion criteria were: number of metastases up to three with the largest diameter " 7 cm; no evidence of extrahepatic disease or stable extrahepatic disease for at least 3 months; platelet count > 35 x10 3 / L; international normalized ratio (INR) < 1.5. No major or minor complications occurred at the end of the procedure. Post-radiofrequency syndrome was observed only in 3 patients, which were treated with acetaminophen. Mean follow-up was 12,9 months (range 4-35 months); at the last follow-up at 12 months, 3 patients died of extrahepatic dissemination of disease while 10 patients are still alive. Complete response at one month follow-up (technique effectiveness) was 95% since in 20/21 lesions lack of enhancement in the ablated tumor was observed. Repeat treatment was performed to treat local residual tumor in 1 patient (7 cm lesion). In our series, 7/21 lesions in 7/13 patients progressed in dimension at 7, 18, 19, and 38 months in chronologic order. This resulted in a mean primary local progressionfree interval of 16.6 months. The mean overall survival after RFA was 10.9 months. Mean survival was also calculated after the initial diagnosis of breast cancer and it was 90.92 months. The mean value of first metastasis occurrence rate was found approximately at 52 months, and the curve drop to zero at 250 months (Figure 1). The mean value of recurrence rate after RFA procedure was found approximately at 18 months and the curve drop to zero at 38 months (Figure 2). The percentage survival rate after the RFA procedure never goes below 67% and the maximum follow-up time is 35 months (Figure 3). The overall percentage survival rate never goes below 64% and the maximum follow-up time is 262 months (Figure 4).

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Carrafiello et al: The role of percutaneous ultrasound guided RFA in breast cancer hepatic metastases Figure 1. Occurrence rate of first metastasis. The occurrence rate of first metastasis vs time is shown. The 50% value was found approximately at 52 months, and the curve drop to zero at 250 months. The 95% confidence limits of the curve is also shown (thinner lines).

Figure 2. Recurrence rate after RFA. The recurrence rate after RFA vs time is shown in Figure 2. The 50% value was found approximately at 18 months, and the curve drop to zero at 38 months.

Figure 3. Survival after RFA. The percentage survival rate vs time after the RF procedure is shown. The curve never goes below 67% and the maximum follow-up time is 44 months.

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Cancer Therapy Vol 6, page 651! Figure 4. Overall survival. The overall percentage survival rate is shown in Figure 4. The curve never goes below 64% and the maximum follow-up time is 262 months.

which complete necrosis was obtained in 87% lesions (Lencioni, 2004). In a recent study, in 43 patients with no more than five hepatic metastases and no metastases larger than 5 cm diameter, was obtained a technique effectiveness of 96% (Jakobs et al, 2008). Technique effectiveness based on the 6-month contrast-enhanced CT follow-up was 88% in a recent small series of 14 patients having treated lesions with a mean largest diameter of 1.90 cm (Gunabushanam et al, 2007). In a more recent study Sofocleous and colleagues observed a technique effectiveness of 79% (Sofocleous et al, 2007). In determining technique effectiveness, also characteristic tumor and proximity to large branches of hepatic and portal veins are believed to be factors of greater importance than the site of origin of metastases (Lencioni et al 1998; Rossi et al, 1998; Livraghi et al, 2001; Gunabushanam et al, 2007; Sofocleous et al, 2007). Probably our high rate of technique effectiveness is due to multiple overlapping ablation treatments in the lesions with diameter ! 3 cm. The best results in terms of local tumor control were noted - according to literature- in patients with lesions smaller than 4 cm in diameter. As Lowes evidenced in his study, RFA is used to reduce tumor bulk in conjunction with systemic therapy and is a relatively simple percutaneous technique that has been demonstrated to be an effective cytoreductive strategy (Lawes et al, 2006). Livraghi and colleagues reported 63% of the patients were disease-free and were followed up for 4-44 months (Livraghi et al, 2001). Unfortunately, progression-free interval and overall survival curves were not calculated in that study and more direct comparisons and discussion are not possible. Our dates are similar to those reported in recent study of Gunabushanam and colleagues who demonstrated a 1 year survival rate of 64% and Sofocleous and colleagues that reported overall survival rate of 70%

IV. Discussion Approximately 40,000 women in Italy have breast cancer each year. More than 50% of patients with breast cancer will develop liver metastases. The presence of liver metastases implies a poor prognosis, signifying wide dissemination of disease and mean survival times ranging from 4 to 12 months. Five years survival of patients with metastatic breast cancer is 22% (Livraghi et al, 2001; Giordano et al, 2004; Lawes et al, 2006; Gunabushanam et al, 2007). Surgical resection with or without chemotherapy is considered the best treatment in selected patients with solitary liver metastases and a low surgical risk. However, solitary liver metastases in breast cancer patients are rare, occurring in only 4-5% of all cases. Most of the patients are not suitable for surgery because of their poor general condition or the stage of disease. Medical therapy for metastatic breast cancer consists of chemotherapy or endocrine therapy because even in the case of isolated metastases in one organ, diffuse tumor cell dissemination exists. In this case, survival ranged between 3 and 6 months, but rarely exceeded 2 years (Diaz et al, 2004; Gillams et al, 2005). Less invasive alternatives to surgical resection have become available. These include percutaneous thermal ablation using methods such RFA. Actually, RFA is a good alternative treatment in the management of high-risk surgical patients and in those with stable extra hepatic disease who are not candidates for resection. RFA is the preferred adjunct treatment (instead of surgery) to systemic chemotherapy in hepatic metastases and in patients with hepatic disease after chemotherapy (Lawes et al, 2006). There are limited data on the use of RFA in treating hepatic metastases from breast cancer. In the current study, technique effectiveness was 95%, which is similar to a prior study concerning 64 liver metastases from breast cancer in 24 patients, in which a 92% complete necrosis was noted (Livraghi et al, 2001) and to another study in

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Carrafiello et al: The role of percutaneous ultrasound guided RFA in breast cancer hepatic metastases standard treatment? Breast 13, 254-258. Frezza EE, Wachtel MS, Barragan B, Chiriva-Internati M, Cobos E (2007) The role of radiofrequency ablation in multiple liver metastases to debulk the tumor, a pilot study before alternative therapies. J Laparoendosc Adv Surg Tech A 17, 282-284. Gillams AR (2005) The use of radiofrequency in cancer. Br J Cancer 92, 1925-1929. Giordano SH, Buzdar AU, Smith TL, Kau SW, Yang Y, Hortobagyi GN (2004) Is breast cancer survival improving? Trends in survival for patients with recurrent breast cancer diagnosed from 1974 through 2000. Cancer 100, 44-52. Gunabushanam G, Sharma S, Thulkar S, Srivastava DN, Rath GK, Julka PK, Bhatnagar S (2007) Radiofrequency ablation of liver metastases from breast cancer, results in 14 patients. J Vasc Interv Radiol 18, 64-72. Hoe AL, Royle GT, Taylor I (1991) Breast liver metastasesincidence diagnosis and outcome. J R Soc Med 84, 714-16. Jakobs TF, Hoffmann RT, Schrader A, Stemmler HJ, Trumm C, Lubienski A, Murthy R, Helmberger TK, Reiser MF (2008) CT-Guided radiofrequency ablation in patients with hepatic metastases from breast cancer. Cardiovasc Intervent Radiol, in press. Kemeny N (1983) The systemic chemotherapy of hepatic metastases. In, Seminars in oncology. New York, NY, Grune & Stratton, 148-155. Lawes D, Chopada A, Gillams A, Lees W, Taylor I (2006) Radiofrequency ablation (RFA) as a cytoreductive strategy for hepatic metastasis from breast cancer. Ann R Coll Surg Engl 88, 639-642. Lencioni R (2004) Tumor radiofrequency ablation Italian network (TRAIN), survival analysis in breast cancer patients with isolated hepatic metastases. Paper presented at, 2004 Scientific Assembly of the Radiological Society of North America December 1, Chicago,IL. Lencioni R, Goletti O, Armillotta N (1998) Radiofrequency thermal ablation of liver metastases with cooled-tip electrode needle, results of a pilot clinical trial. Eur Radiol 8, 12051211. Livraghi T, Goldberg SN, Solbiati L, Meloni F, Ierace T, Gazelle GS (2001) Percutaneus radio-frequency ablation of liver metastases from breast cancer, initial experience in 24 patients. Radiology 220 145-149. Machi J (2001) Radiofrequency ablation for multiple hepatic metastases. Ann Surg Oncol 8, 379-380. Pocard M, Pouillart P, Asselain B, Salmon R (2000) Hepatic resection in metastatic breast cancer, results and prognostic factors. Eur J Surg Oncol 26, 155-159. Rossi S, Buscarini E, Garbagnati F, Di Stasi M, Quaretti P, Rago M, Zangrandi A, Andreola S, Silverman D, Buscarini L (1998) Percutaneous RF interstitial thermal ablation in the treatment of hepatic cancer. AJR Am J Roentgenol 170, 1015-1022. Schneebaum S, Walker MJ, Young D, Farrar WB, Minton JP (1994) The regional treatment of liver metastases from breast cancer. J Surg Onc 55, 26-32. Smith IE (1987) Recurrent disease. In, Harris JR, Hellman S, Henderson IC, Kinne DW,ed. Breast diseases. Philadelfia, PA, Lippincott, 369-384. Sofocleous CT, Nascimento RG, Gonen M, Theodoulou M, Covey AM, Brody LA, Solomon SM, Thornton R, Fong Y, Getrajdman GI, Brown KT (2007) Radiofrequency ablation in the management of liver metastases from breast cancer. AJR Am J Roentgenol 189, 883-889. Solbiati L, Goldberg SN, Ierace T, Livraghi T, Meloni F, Dellanoce M, Sironi S, Gazelle GS (1997) Hepatic metastases, percutaneous radiofrequency ablation with cooled tip electrodes. Radiology 205, 367-373.

and 30% respectively at 3 and 5 years; while Lowes and colleagues reported 30 month survival rate of 41,6% (Gunabushanam et al, 2007; Sofocleous et al, 2007). Compared to surgery, RFA offers the advantages of being less expensive and considerably less invasive (Solbiati et al, 1997; Frezza et al, 2007). Our results appear to be similar to those reported in a resection series by Pocard et al. in which the 3-year survival rate was 71%.! In more recent study Vlastos et al, in a series of 31 patients with isolated breast cancer metastases to the liver, treated with major liver resections and minor resections with or without RFA, demonstrated a mean survival of 63 months and overall 2 and 5 year survival rates of 86% and 61%, respectively, whereas 2 and 5 year disease – free survival rates 39% and 31% respectively. It may thus be reasonable to use RFA rather than surgery as a first-choice local therapy, particularly given the natural history of this disease, which suggests that local therapies (eg, surgery and in situ tumor ablation) will be curative in only a minority of patients. The use of RFA does not prevent the simultaneous or subsequent use of other, potentially complementary treatments.

V. Conclusion In our experience and according to literature, RFA appears to be a useful adjunct to systemic chemotherapy and/or ormonoterapy, in the loco-regional treatment of hepatic metastases from breast cancer. RFA may also be a less invasive alternative to surgery in the loco-regional treatment of liver metastases from breast cancer. A prospective randomized trial comparing systemic treatment alone and systemic treatment with RFA for selected breast cancer liver metastases is absolutely needed to show any advantage of the RFA in local and overall disease control when compared with systemic treatment alone.

References Carrafiello G, Laganà D, Ianniello A, Dionigi G, Novario R, Recaldini C, Mangini M, Cuffari S, Fugazzola C (2007) Post-radiofrequency ablation syndrome after percutaneous radiofrequency of abdominal tumours, one centre experience and review of published works. Australas Radiol 51, 550554. Choi H, Loyer EM, DuBrow RA, Kaur H, David CL, Huang S, Curley S, Charnsangavej C (2001) Radio-frequency ablation of liver tumors, assessment of therapeutic response and complications. RadioGraphics 21, S41-S54. Curley SA, Izzo F, Delrio P, Ellis LM, Granchi J, Vallone P, Fiore F, Pignata S, Daniele B, Cremona F (1999) Radiofrequency ablation of unresectable primary and metastatic hepatic malignancies, results in 123 patients. Ann Surg 230, 1-8. DeMatteo RP, Shah A, Fong Y, Jarnagin WR, Blumgart LH, Brennan MF (2001) Results of hepatic resection for sarcoma metastatic to liver. Ann Surg 234, 540-547. Di Carlo I, Grasso G, Patane' D, Russello D, Latteri F (2003) Liver metastases from lung cancer, is surgical resection justified? Ann Thorac Surg 76, 291-293. Díaz R, Santaballa A, Munárriz B, Calderero V (2004) Hepatic resection in breast cancer metastases, should it be considered

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Cancer Therapy Vol 6, page 653! Vlastos G, Smith DL, Singletary SE, Mirza NQ, Tuttle TM, Popat RJ, Curley SA, Ellis LM, Roh MS, Vauthey JN (2004) Long-term survival after an aggressive surgical approach in patients with breast cancer hepatic metastases. Ann Surg Oncol 11, 869-874. Wyld L, Gutteridge E, Pinder SE, James JJ, Chan SY, Cheung KL, Robertson JF, Evans AJ (2003) Prognostic factors for

patient with hepatic metastases from breast cancer. Br J Cancer 89, 284-90. Zinser JW, Hortobagyi GN, Buzdar AU, Smith TL, Fraschini G (1987) Clinical course of breast cancer patients with liver metastases. J Clin Oncol 5, 773-782.

From left to right upper row: Gianpaolo Carrafiello, Elisa Cotta, Salvatore Cuffari, Gianlorenzo Dionigi, Domenico LaganĂ From left to right lower row: Federico Fontana, Carlo Fugazzola, Monica Mangini, Chiara Recaldini, Maria PetullĂ

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Cancer Therapy Vol 6, page 655! Cancer Therapy Vol 6, 655-664, 2008

Antiestrogen therapy for Breast Cancer: An overview Review Article

Kishor S Kumar, Mahesh M.J Kumar* Centre for Cellular and Molecular Biology, Hyderabad, India

__________________________________________________________________________________ *Correspondence: Mahesh Kumar M.J, Scientist, Center for Cellular and Molecular Biology, Uppal Road, Hyderabad -500 007, India; Tel:+ 91-40-27192872; Fax : +91-40-27160591; Email: mahesh73@ccmb.res.in Key words: anti estrogenic therapy, blocking of estrogen receptor, estrogen synthesis inhibitor, estrogen receptor down regulator Abbreviations: bone mineral density (BMD); estrogen receptor (ER); estrone (E1); estradiol (E2); estriol (E3); estrogen responsible elements (ERS); multiple outcome of raloxifene evaluation (MORE); selective estrogen receptor modulator (SERM); selective estrogen receptor down regulator (SERD) Received: 17 June 2008; Revised: 21 August 2008 Accepted: 4 September 2008; electronically published: September 2008

Summary Breast cancer is the leading malignancy worldwide. Estrogen and estrogen receptors (ER) play a major role in breast cancer progression and development. In this review, our discussion is mechanism and action of estrogen and its receptors in breast cancer. Also, this paper will summarize the therapeutic methods available for anti hormonal therapy, molecular mechanism of action of anti hormonal agents, basic science of estrogen and estrogen receptors and current status of chemoprevention with selective estrogen receptor modulators (SERM).

receptor by selective down regulators (fluvestrant) and inhibiting the estrogen synthesis (aromatase inhibitors anastozole, leterozol) -result in substantial decreases of tumour progression and growth of patients (Wolmark and Dunn, 2001). Recent genetic, biochemical and pharmacological dissection of the estrogen signal transduction pathway has led to the identification of numerous proteins and processes that impinge on ER function. This paper will review the therapeutic methods available for anti hormonal therapy, estrogen synthesis, function, and metabolism and molecular mechanism of action of anti hormonal agents

I. Introduction Breast cancer is the leading malignancy in women, accounting for more than 3, 50,000 deaths per year worldwide (Ferlay et al, 2000). Estrogens play role in luminal mammary epithelial cell proliferation and development of mammary cancers. About 70% of human breast cancer is expressing estrogen receptors (Lacroxia and Leclercq, 2004). The mammary cancers are classified on the basis of growth requirement as estrogen dependent and estrogen independent tumor (Stoll, 1969; Furth, 1975). The relationship between estrogen receptor expression and cellular responsiveness to estrogen and antiestrogen has been extensively studied in cell lines (Katzenellen et al, 1987), animal models (Milholland et al, 1979) and humans (Rose, 1980) and its makes ER is one of the most important therapeutic targets in breast cancer (Sunderland and Osborne, 1991). ER is a transcription factor that regulates the genetic program of cell cycle progression and growth in mammary glands. ER alpha is considered as the primary receptor for mammary tumor development. Antiestrogenic therapy, blocks the ability of the estrogen and control the growth of breast cancer cells. It is very effective treatment against ER dependent breast cancers. Antiestrogenic treatments are blocking estrogen receptor by selective modulators (tamoxifen and raloxifene), destabilization and degradation of estrogen

II. Estrogen and Breast cancer Estrogen is one of the major risk factor of breast cancer progression and development. Over, 85% of the mammary cancers in women originated from the luminal mammary epithelial cells. Adenocarcinogenesis are classified according to their growth requirements for proliferation has been either hormone dependent or hormone independent tumors (Nandi et al, 1995). Various studies of breast cancer have reported to an increased risk associated with elevated blood levels of endogenous estrogen. The influence of serum estrogen concentration is associated with the menstrual cycle in premenopausal women and more stable concentration in postmenopausal

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Kumar and Kumar: Antiestrogen therapy for Breast Cancer: An overview esters of estrogens that comprise of a separate class of steroid hormones and they are synthesized in blood, circulated and bind to lipoproteins. Overall, less than 10 percent of serum estradiol is associated with lipoproteins, mainly high-density lipoproteins, but serum estradiol can be transferred to low-density lipoproteins (Tang et al, 1997). Lipoidal estrogens are more resistant to catabolism than free estrogens are therefore it takes lifetime to clean in the circulation. Lipoidal estrogens lower the concentration of estradiol to inhibit the oxidation of lowdensity lipoproteins reported in invitro system (Shwaery et al, 1997).

women. (Mark Clemons and Goss, 2001). In animal experiment, estrogen is lead to development of mammary tumors (Mahesh K et al, 2007) and the mechanisms of estrogen are contributed to each phase of the adenocarcinogenesis process of ignition, promotion and progression.

III. Synthesis Estrogen derivatives of estrone (E1), estradiol (E2), and estriol (E3) (Figure 1), the C18 steroids are derived from cholesterol. Cholesterol is taken up by steroidogenic cells and stored and moved in to the site of steroid synthesis (Scallen et al, 1985). The different steroids are formed by reduction in the number of carbon atoms from 27 to 18 (Kallen et al, 1998). Both cytochrome CYP17 (P450 17 hyderoxylase) and cytochrome CYP19 (P450 aromatase) are involved in estrogen biosynthesis (Haimen et al, 1999). Aromatization is the last step in estrogen formation and the P450 aromatare monoxygenase enzyme complex is present in the smooth endoplasmic reticulum and functions as a demethylase catalyst in this reaction. In three consecutive hydroxylation reactions, estrone (E1) and estradiol (E2) are formed from their obligatory precursor’s androstenedione and testosterone respectively.

V. Functions Estrogen stimulates growth, blood flow and water retention in sexual organs and is also involved in breast lipoprotein receptors, resulting in a decrease in serum concentrations of low-density lipoprotein cholesterol in the liver (Paganin et al, 1996). Estrogen is increasing the potential for blood coagulation. In gastrointestinal tract, estrogens may play role in protecting against colon cancer (Calle et al, 1995) and it increases the turgor and collagen production and reduces the depth of wrinkles on aged (Schmidt et al, 1996). In breast tissue, estrogen stimulates the growth and differentiation of the ductal epithelium, induces mitotic activity of ductal cylindric cells and stimulates the growth of connective tissue (Porter, 1974). The lobular units of terminal ducts of breast tissue of young women are highly responsive to estrogen and it exerts histamine-like effects on the microcirculation of the breast. The density of estrogen receptors in breast tissue is highest in the follicular phase of the menstrual cycle and falls after ovulation. In postmenopausal women with breast cancer, the tumor concentration of estradiol is high because of in situ aromatization and low serum estradiol concentrations. The ovarian estrogen hormone has a primary role in the establishment and maintenance of reproductive function. Additional functions of estrogen are in the skeleton development, cardiovascular system and brain (Mcdonnell et al, 2001).

IV. Metabolisms Estrogens are meeflora and subsequent reabsorption of the estrogen results in an enterohepatic circulation. Estrogens are also metabolized by hydroxylation and subsequent methylation to form catechol and methoxylated estrogens (Osewa et al, 1993). Hydroxylation of estrogens yields 2-hydroxyestrogens, 4hydroxyestrogens and 16 a-hydroxy estrogens (catechol estrogens), among which 4-hydroxyestrones and 16a hydroxy estradiols are considered as carcinogenic. Methylation of the 2- and 4-hydroxyestrogens by catechol O-methyltransferase yields methoxylated estrogen metabolites (Grubber and Hubber, 2001). Catechol estrogens are bound to estrogen receptors and have weak estrogenic activity in animals. Lipoidal estrogens are fatty

Figure 1. Three different types of estrogen.

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Cancer Therapy Vol 6, page 657! nuclear-receptor co-activators or repressors. Estrogen regulates the transcription of genes that lack functional estrogen-response elements by modulating the activity of other transcription factors and also it can enhance breast cancer cell survival and tumor growth (Donald et al, 2002).

VI. Molecular actions of estrogen The molecular mechanism of estrogen on nuclear DNA involves (Figure 2) estrogen and its nuclear receptors complex are bound to the regulatory regions of estrogenresponsive genes as dimers and also these binding is associated with basal transcription factors, co activators and co repressors to alter the gene expression (Levin, 2002). The ER is a ligand-dependent transcription factor which accounts for the latency of most estrogenic responses in target tissues (Means et al, 1972). Estrogen diffuses into the cell and it binds to the ligand-binding domain of the receptor. The complex of estrogen and estrogen receptor diffuses into the cell nucleus and these estrogens-estrogen receptor complexes bind to specific sequences of DNA called estrogen-response elements (EREs). The estrogen-estrogen receptor complexes are bound not only to the response elements but also to

VII. Estrogen receptors There are two subtypes of estrogen receptor and several isoforms and splice variants of each subtype was reported. The first subtype estrogen receptor ! , was cloned in 1986 and the second subtype of estrogen receptor ! is present in several nonclassic target tissues including the kidney, intestinal mucosa, lung parenchyma, bone marrow, bone, brain, endothelial cells and prostate gland (Enmark et al, 1997). In contrast, estrogen receptor " is

Figure 2. Molecular action of estrogen. E: Estrogen Receptor, ERE: estrogen responsible elements, P450 Scc: P450 side chain cleavage enzyme, CYP17: 17! -hydroxylase, 3! HDS: denotes 3! -hydroxysteroid dehydrogonase, DHEA: dehydroepiandrosterone, 17-KSR: 17-ketosteroid reductase, 17! -HDS: 17! hydroxysteroid dehydrogonase.

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Kumar and Kumar: Antiestrogen therapy for Breast Cancer: An overview loosely bound to its cytoplasmic or nuclear location, but they have attached to receptor-associated proteins. These proteins have served as chaperones and they stabilize the receptor in an un-activated state or mask the DNA binding domain of the receptor. Other receptor-associated proteins may contribute to cross talk between different signal pathways. The specific domain within ER ! is required for ligand binding, dimerization, DNA binding, and transactivation (McDonnell, 1999). ER ! also plays a role in negative regulation of ER " . In response to estrodial, ER " can activate the same genes as regulated by ER ! . However, ER " function is to inhibiting ER ! transcriptional activity in cells in which both receptors are expressed (Hall and McDonnell, 1999). The BRCA1 gene account for 40% to 50% of hereditary breast cancer. BRCA1 inhibit the signaling pathway by ligand activated estrogen receptor (ER ! ) through the estrogen responsive element and also block the transcription activation function of AF-2 of ER a (Fan et al, 1999).

found mostly in endometrium, breast-cancer cells and ovarian stroma. The development and growth of breast cancer is mediated through a balance of ER alpha and ER beta molecules. Estrogen receptor ! and " are members of nuclear hormone receptor super family, which has approximately 150 recognized members. Estrogen receptors have several functional domains. ER ! and ER " have 96% amino-acid identity in their DNA-binding domains, whereas there is only 53% homology in their ligand-binding domains.These receptors differ in their activation domains suggesting that, they may recruit different proteins to the transcription complexes and thereby altering the specificity of their genomic transcriptional effects (Pettersson and Gustafsson, 2001). The ligand-binding domain contains different sets of amino acids that bind to different ligands and this domain also interacts with co-regulatory proteins. The N-terminal domain a high degree of variability and normally it contains a transactivation domain that can interact directly with factors of the transcriptional machinery. The Cterminal domain has contributes to the transactivation capacity of the receptor. The DNA-binding domains of estrogen receptors ! and " are very similar and overall degrees of homology of the receptors are low in cells. The estrogen signal transduction pathway has led to identification of numerous proteins that impinge on ER function (Kuiper et al, 1996; Donald et al, 2002). ER ! is sequestered into target cell nuclei of large inhibitory heat shock protein complex. which enables the displacement of heat shock proteins and also facilitates the interaction of a receptor dimer within the regulatory regions of target genes. The interaction of ER ! with target gene promoters can occur either directly through specific estrogen response elements (EREs) or indirectly through contacts with other DNA bound transcription factors such as AP1, SP1 or NF- B. Once bound to DNA, the receptor can either positively or negatively regulate the target gene transcription (Donald et al, 2002). Cross talk between the genomic and second-messenger pathways probably has important roles in estrogenic control of cell proliferation and inhibition of apoptosis and may have implications for therapy (Levin, 2003). Catechol estrogen metabolites may participate in the regulation of pathways of gene expression or signaling or both through the estrogen receptor. The receptor is modulated to enhance or suppress gene activation in different target tissues (Anstead et al, 1997). One proposed mechanism is the balance of corepressor or co-activator proteins that can bind to the SERM-ER protein complex to switch off (co repressor) or switch on (co activator) the transcriptional potential of the complex. However, a thorough investigation is required for all the interacting signaling pathways mediated through the estrogen receptor and growth factors such as epidermal growth factor and insulin- like growth factor 1, that function in human breast tissue normal and tumor cells. This could facilitate the goal of achieving a comprehensive understanding of control of proliferation and apoptosis by estrogen and its metabolites on cancer cells and it is perhaps a new targets for combined therapeutic intervention. Ligand-dependent activation of estrogen receptors have unattached to its ligand and

VIII. Antiestrogen therapy In 1882, Thomas William Nunn recognized importance of ovarian functions in breast cancer growth. In 1989, Albert Schizinger proposed surgical oophorectomy to treat breast cancer. 37% of patients had a tumor response with oophorectomy was reported earlier (Beatson, 1896). “Ovarian hormone of estrus� and the hormones from the ovarian tissues were investigated and named as estrogens (Allen and Doisy, 1923). Estradiol have synthesized and injected into immature female rats to determine the site of actions (Jennsen and Jacbson, 1962). The ER was isolated from ovary and characterized in 1967. Mechanism of Antiestrogenic therapy exists in three pathways. 1. Blocking the estrogen receptors (Tamoxifen & Raloxifene) 2. Estrogen synthesis inhibitors (Aromatase) 3. Estrogen receptors down regulators (Fulvestrant)

IX. Blocking of estrogen receptors (Tamoxifen & Raloxifene) Tamoxifen is acting an antiestrogen by inhibiting the binding of estrogen-to-estrogen receptors. The Food and Drug Administration has approved Toamoxifen in 1977 for the treatment of women with advanced breast cancer (Jordan, 1999, 1995). The compound was administered to patients as trans-tamoxifen (as the citrate salt) because this isomer has higher affinity for estrogen receptors than cisisomer (Langan et al, 1994). Drugs such as tamoxifen are more properly referred to as selective estrogen-receptor modulators (SERM) because of their multiple activities. It has estrogen-antagonist activity in breast tissue accounting for its ability to inhibit tumor growth (Horwitz et al, 1996) (Figure 3). Tamoxifen is absorbed readily after oral administration and the usual dosage is 20 mg per day and the serum half-lives of tamoxifen and its major metabolites range from 7 to 14 days (Buckley et al, 1989; Sunderland and Osbrne, 1991; Langan et al, 1994). In long-term treatment, the concentrations of tamoxifen and its metabolites in serum remain constant for as long as 10 years and its reduced bioavailability is not a cause for

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Figure 3. Blocking of estrogen receptors (Tamoxifen & Raloxifene). SERM: Selective estrogen receptor modulators.

35%) but it does not reduce the non-vertebral fractures. However, it would reduce thromboembolism (44%), fetal stroke (49%), leg cramps, peripheral edema and gallbladder disease in breast cancer patients (Fisher et al, 1998). The influence of raloxifene on breast cancer incidence has been reported from several ongoing trials. The largest randomized trial to address the issue of breast cancer risk is with raloxifene therapy. In the Multiple Outcomes of Raloxifene Evaluation (MORE) study, 7,705 postmenopausal women existing with osteoporosis were randomized to receive either raloxifene at 60 or 120 mg/d or placebo. Breast cancer was monitored in the MORE study and initial reports showed 70% reduction in the incidence of invasive breast cancer with raloxifene treatment compared with placebo. Raloxifene reduced the incidence of estrogen receptor-positive breast cancers and did not reduce the estrogen receptor-negative cancers (Cummings et al, 1999; Ettinger et al, 1999). Raloxifene increases the BMD and it may lower the fracture risk in human. Raloxifene is not associated with endometrial proliferation, but clear information on raloxifene’s effect on endometrial cancer development is not yet available (Barrett et al, 2006).

acquired resistance. Tamoxifen can be detected in serum for several weeks and in tumor tissue for several months after the treatment is discontinued (Lien et al, 1991). The anti tumor effects of tamoxifen are thought to be due to its anti estrogenic activity mediated by competitive inhibition of estrogen binding to estrogen receptors (Osborne et al, 1996). As a consequence, tamoxifen inhibits the expression of estrogen-regulated genes including growth factors and angiogenic factors secreted by the tumor that may stimulate the growth by autocrine or paracrine mechanisms. The net result is blocking the G1 phase of the cell cycle and a slowing of cell proliferation. Tumors may regress because of this altered balance between cell proliferation and ongoing cell loss. Tamoxifen directly induces programmed cell death in tumour tissues (Ellis et al, 1997) and it reduces the risk of recurrence and death from breast cancer and also it provides effective palliation for patients with metastatic breast cancer. Therefore it is useful to treat the women who have estrogen-receptorpositive invasive breast cancer. An exception might be on the basis of prognostic factors such as an invasive or tubular mucinous or papillary tumor with negative axillary nodes - to have a very low risk of disseminated disease. Tamoxifen is an initial hormonal therapy of choice for postmenopausal women with metastatic breast cancer and it is used as first- or second-line therapy in younger women. It is safe and well tolerated, but the risk of endometrial cancer is high.

XI. Estrogen (Aromatase)

Synthesis

Inhibitors

Aromatase inhibitors are markedly suppressing the plasma estrogen levels in postmenopausal women by inhibiting or inactivating aromatase, the enzyme responsible for synthesis of estrogens from androgenic substrates. Aromatase is an enzyme of cytochrome P-450 super family and the product of the CYP19 gene (Evans et al, 1986) and it is present at lower levels in several non glandular tissues including subcutaneous fat, liver, muscle, brain, normal breast, and breast-cancer tissue (Miller et al, 1982). Peripheral aromatase activity and plasma estrogen levels are correlated with body-mass index in postmenopausal women. The concentration of estradiol in breast-carcinoma tissue is approximately 10 times higher than the concentration in plasma (Longcope et al, 1986). The level of estrogen is high in breast cancer patient and estrogen sends a growth signal to ER following which uncontrolled proliferation of cells occur. Aromatase inhibitors block the estrogen signaling that leads to fewer

X. Raloxifene Raloxifene is a second generation of selective estrogen-receptor modulators (SERM) and it was initially developed for breast cancer. Later, it was treated as an alternative to hormone replacement therapy for treatment of osteoporosis. Raloxifene is a benzothiophene derivative with a distinctly different structure from tamoxifen (Buzdar et al, 1998). Although, raloxifene has similar activity to tamoxifen in breast and bone it is devoid of agonist activity in the endometrium (Riggs et al, 2003). Raloxifene is reduces the incidence of breast cancer by 62%, and it does not increase the endometrial proliferation or neoplasm (Cummings et al, 1999). Raloxifene significantly reduces the risk of invasive breast cancer in human with positive estrogen receptor status and reduces the risk of clinical vertebral fractures (reactive reduction 659

Kumar and Kumar: Antiestrogen therapy for Breast Cancer: An overview estrone sulfate (Geisler et al, 2002). Aromatase has intra tumoral activity in the majority of breast carcinomas and isotopic assays shown that such activity contributes to decrease the plasma estrogen levels. Anastrozole, letrozole and exemestane are markedly inhibiting the breast cancer (Miller and Dixon, 2001). Overall trials shown that aromatase inhibitors are favorable to tamoxifen in post menopausal ER positive patients, secondary to decreased recurrence and a decrease in undesirable side effects of endometrial cancer and venous thrombosis. The disadvantage with aromatase inhibitors is the higher rate of osteoporosis and subsequent fracture as well as higher cholesterol levels.

growth signals to ER and hence cancer growth can be slowed down or stopped. Aromatase inhibitors inhibit several cytochrome P450 enzymes involved in adrenal steroidogenesis and these are redeveloped for use as “medical adrenalectomy� against advanced breast cancer (Santen et al, 1977). They are described as first-, second- and third-generation inhibitors according to the chronological order of their clinical development and they are further classified as type 1 type 2 inhibitors (Figure 4) according to their mechanism of action. Type 1 inhibitors are steroidal analogues of androstenedione and they bind to the same site on the aromatase molecule. Type I inhibitors, unlike androstenedione, bind irreversibly to aromatase and gets converted to reaction intermediates. Therefore, they are commonly known as enzyme in activators. Type 2 inhibitors are non steroidal and bind reversibly to the heme group of the enzyme by basic nitrogen atom. Anastrozole and letrozole are third-generation inhibitors and they bind at their triazole groups. Anastrozole, letrozole and exemestane are administered orally to the breast cancer patients (Steele et al, 1987). Type-I compound and a typeII compound have clinical efficacy, but the disadvantage of intramuscular injections are required to deliver the molecules. (Lonning et al, 1991; Wisemen and Adkins, 1998). Aminoglutethimide induces cytochrome P-450 activity, but it reduces tamoxifen levels (Lien et al, 1990). The third-generation aromatase inhibitors have been found in pre clinical studies to be more potent than aminoglutethimide. All of them markedly suppress the plasma estrogen levels in postmenopausal women. Clinical doses of anastrozole, exemestane and letrozole are greater than 97 percent (Geisler et al, 1998) as compared with aminoglutethimide (MacNeill, 1992). Letrozole was associated with greater aromatase inhibition than anastrozole and lower the plasma levels of estrone and

XII. Estrogen receptor regulators (Fulvestrant)

down

Fulvestrant is the selective estrogen receptor down regulator and FDA has recently approved it for use in advanced breast cancer and metastatic cancer (Howell, 2001). The agonist properties of tamoxifen and other SERMs can contribute to an increased risk of endometrial carcinoma and the development of resistance in tumor cells. Fulvestrant is a 7-alkyl analog of estradiol having a unique mechanism of action when compared with tamoxifen and other SERMs. Tamoxifen binds to the ER results in dissociation of heat shock proteins, homodimerization and inhibition of transactivation in breast tissue. Fulvestrant acts quite differently binding to ER and causes dissociation of heat shock proteins. However, the bulky side chain of fulvestrant prevents dimerization and results in degradation of ER. The result is not only inhibition of ER activity but also a dramatic decline in ER expression levels (Howell, 2001). As a result of down regulatory activity, fulvestrant has been termed a SERD (Figure 5).

Figure 4. Estrogen Synthesis Inhibitors (Aromatase).

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Cancer Therapy Vol 6, page 661!

Figure 5. Estrogen receptor down regulators or selective estrogen receptor down regulator (SERD).

more rapidly in the circulation (Howell et al, 2002). The main adverse effects of fulvestrant are nausea, vomition, constipation and diarrhea. Interestingly, fulvestrant does not cross the blood-brain barrier and it seems to cause less menopausal symptoms than other antiestrogens (Howell et al, 1995). It does not induce endometrial thickening, but longer follow-up of the larger clinical trials would be required to assess its effects on endometrial cancer risk (Figure 6).

Fulvestrant has notable efficacy in tamoxifenrefractory disease and it has demonstrated an equivalent or superior activity than anastrozole in the metastatic cancers. There has been recent debate existing in the dosing of fulvestrant in humans. The published pharmacokinetic data suggested that, once in a month of intra muscular injection of fulvestrant at the dose rate of 250 mg provides an adequate dose in the circulation, but it does not reach the steady-state level quickly. Further exploration of dosing of250 mg once in 2 weeks will reach a steady state

Figure 6. Molecular mechanism of action of anti estrogenic therapy.

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Kumar and Kumar: Antiestrogen therapy for Breast Cancer: An overview Calle EE, Miracle-McMahill HL, Thun MJ, Heath CW Jr (1995) Estrogen replacement therapy and risk of fatal colon cancer in a prospective cohort of postmenopausal women. J Natl Cancer Inst 87, 517-523. Cummings SR, Eckert S, Krueger KA (1999) The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. Multiple Outcomes of Raloxifene Evaluation. JAMA 281, 2189-2197. Donald P, McDonnell DP, John D Norris (2002) Connection and regulation of the human estrogen receptor. Science 296, 1642-1644. Enmark E, Pelto-Huikko M, Grandien K (1997) Human estrogen receptor b gene structure chromosomal localization and expression patterns. J Clin Endocrinol Metab 82, 42584265. Ellis PA, Saccani-Jotti G, Clarke R (1997) Induction of apoptosis by tamoxifen and ICI 182780 in primary breast cancer. Int J Cancer 72, 608-613. Ettinger B, Black DM, Mitlak BH (1999) Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. JAMA 282, 637-645. Evans CT, Ledesma DB, Schulz TZ, Simpson ER, Mendelson CR (1986) Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P450 mRNA. Proc Natl Acad Sci U S A. 3, 6387-6391. Fan J A Wand, Yuan R, Ma Y, Mena Q, Erodos MR, Pestell RG, Fand Yuan, Auborn KJ, Goldberg ID, Rosen EM (1999) BRCA1 inhibitor of estrogen receptor signaling in transfected cells. Science 284, 1354-1356. Ferlay, Bray, Pisani, P, Parkin, MD GLOBOCAN (2000) Cancer incidence, mortality and prevalence wordwide IARC press, Lyon. Fisher B, Costantino JP, Wickerham DL (1998) Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 90, 1371-1388. Furth J (1975) A comprehensive treatise, ed. Becker GF (Plenum New York) 1, 75-120. Geisler J, King N, Anker G (1998) In vivo inhibition of aromatization by exemestane, a novel irreversible aromatase inhibitor, in postmenopausal breast cancer patients. Clin Cancer Res 4, 2089-2093. Geisler J, Haynes B, Anker G, Dowsett M, Lonning PE (2002) Influence of letrozole and anastrozole on total body aromatization and plasma estrogen levels in postmenopausal breast cancer patients evaluated in a randomized, cross-over study. J Clin Oncol 20, 751-758. Grubber DM, Huber JG (2001) Tissue specificity: the clinical importance of steroid metabolites in hormone replacement therapy. Meturitas 37, 151-157. Haimen CA, Hankinson SE, Spiegelman D (1999) the relationship between a polymorphism in CYP17 with plasma hormone level and breast cancer. Cancer Res 59, 10151020. Hall JM, McDonnell DP (1999) The Estrogen Receptor ! isoform (ER! ) of the human estrogen receptor modulates ER transcriptional activity and is a key regulator of the cellular response to estrogen and antiestrogens. Endocrinology 140, 5566-5578. Horwitz KB, Jackson TA, Bain DC, Richer JK, Jakimoto GS, Tung L (1996) Nucleus receptor coactivations and corepressors. Mol Endocrinol 101167-77. Howell A (2001) Preliminary experience with pure antiestrogens. Clin Cancer Res 7, 4369-75s; discussion 4411s-2s. Howell A, Robertson JF, Quaresma Albano J, Aschermannova A, Mauriac L, Kleeberg UR, Vergote I, Erikstein B, Webster A, Morris C (2002) Fulvestrant, formerly ICI 182,780, is as

XIII. Conclusion Current anti estrogen receptor strategies include blocking by selective modulators (Tamoxifen and Raloxifene), destabilization and degradation of estrogen receptor by selective down regulator (fulvestrant) and disruption of estrogen synthesis (aromatase inhibitors such as anastrole, letrozole or exemestane), any of which alone can result in a substantial decrease of tumor growth of estrogen receptor positive patients. Resistance to therapy is most often observed during the treatment of advanced breast cancer. Currently, several clinical trials indicated that aromatase inhibitors should be used for hormonal therapy and also it is superior for prevention of recurrence. Treatment for recurrent cancer could be tailored for individual patient based on disease characteristics and other side effects such as osteopenia, cardiac risk, venous thrombosis and uterine cancer. The third generation aromatase inhibitor (anastrole, letrozole) is a new development in the endocrine treatment of estrogen receptor positive breast cancer in post menopausal woman. In advanced disease, letrozole is convincingly better than tamoxifen, but long-term effects of profound estrogen suppression in postmenopausal woman are unknown.Role of estrogen in breast cancer led to development of new preventive and therapeutic intervention that block estrogen receptor function or drastically reduce the levels of endogenous estrogen through inhibition of its synthesis. The development of additional strategies on the basis of the inhibition of estrogen metabolism, inactivation of reactive quinolones, silencing of estrogen receptors genes and specific inhibition of membrane estrogen-receptor activated second messenger pathways will probably lead to the availability of additional effective approaches. However, thorough investigation is required for all the interacting signaling pathways mediated through the estrogen receptor and growth factors, such as epidermal growth factor and insulin- like growth factor 1, that function in human breast tissue, normal and tumor cells.

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Mark Clemons MB, Paul Goss (2001) Estrogen and risk of breast cancer (A review) N Engl J Med 344, 4-276-285. McDonnell DP, Chang CY, Norris JD (2001) Capitalizing on the Complexities of Estrogen Receptor Pharmacology in the Quest for the Perfect SERM. Ann N Y Acad Sci 949, 16-35. McDonnell DP (1999) The Molecular Pharmacology of SERMs. Trends Endocrinol Metab 10, 301-311. Means AR, Comstock JP, Rosenfeld GC,, O'Malley BW (1972) Ovalbumin Messenger RNA of Chick Oviduct: Partial Characterization, Estrogen Dependent, and Translation in vitro. Proc Nalt Acad Sci USA 69, 5, 1146-11. Milholland MJ, Rosen F,, Kim U (1979) Mammary cancer: Selective action of estrogen receptors complex. Science 203, 361-363. Miller WR, Hawkins RA, Forrest AP (1982) Significance of aromatase activity in human breast cancer. Cancer Res 42, 3365-3368. Miller WR, Dixon JM (2001)! Local endocrine effects of aromatase inhibitors within the breast"! J Steroid Biochem Mol Biol!79#!93-102" Nunn TW (1882) On cancer of the breast. London: J & A Churchill. P. 71. Osborne CK, Elledge RM, Fuqua SAW (1996) Estrogen receptors in breast cancer therapy. Science Med 3, 32-41. Osewa Y, Higashiyama T, Shirizu Y, Yarborough C (1993) Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase. J Steroid Biochem Mol Biol 44, 469-80. Pettersson K, Gustafsson JA (2001) Role of estrogen receptor beta in estrogen action. Annu Rev Physiology 63,165-192. Riggs BL, Hartmann LC (2003) Selective estrogen-receptor modulators- mechanisms of action and application to clinical practice. N Engl J Med 348, 618-629. Porter JC (1974) Hormonal regulation of breast development and activity. J Invest Dermatol 63, 85-92. Rose C (1980) Therapeutic effect of tamoxifen related to estrogen receptor level. Recent result. Cancer Res 71, 134141. Nandi S, Raphael C Guzman, Janson Yang (1995) Hormone and mammary carcinogensis in mice, rat and humens : A unifying hypothesis. Proc Nalt Acad Sci USA 92, 36503657. Santen RJ, Samojlik E, Lipton A, Harvey H, Ruby EB, Wells SA, Kendall J (1977) Kinetic, hormonal and clinical studies with aminoglutethimide in breast cancer. Cancer 39, 29482958. Scallen TJ, Noland BJ, Gavey KL (1985) Sterol carrier protein 2 and fatty acid binding protein: separate and physiological function. J Bio Chem 260, 4733-4739. Schmidt JB, Binder M, Demschik G, Bieglmayer C, Reiner A (1996). Treatment of skin aging with topical estrogens. Int J Dermatol 35, 669-674. Shwaery GT, Vita JA, Keaney JF (1997) Antioxidant protersfer of LDL by Physiological concentration of 17 ! estradiol: requirements for estradiol modification. Circulation 95, 1378-3786. Stoll BA (1969) Hormonal management in breast cancer lippincott, Philadelphia. 3-5. Steele RE, Mellor LB, Sawyer WK, Wasvary JM, Browne LJ (1987) In vitro and in vivo studies demonstrating potent and selective oestrogen inhibition with the nonsteroidal aromatase inhibitor CGS 16949A. Steroids 50.147-161. Sunderland MA, Osborne CK (1991) Tamoxifen in premenopausal patients with metastatic breast cancer. A review. J Clin Oncol 9, 1283-1297. Tang M, Abplanalp W, Subbiab MT (1997) Association of estrogens with human plascma lipoproteins: Studies using

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Kumar and Kumar: Antiestrogen therapy for Breast Cancer: An overview estradiol 17 beta and its hydrophobic derivatives. J Lab Clin Med 129,447-452. Wolmark N, Dunn K (2001) The role of Tamoxifen in breast cancer prevention: issues sparked by the NSABP Breast cancer prevention Trial (P-1). Ann NY Acad Sci 949, 99108. Wiseman LR, Adkins JC (1998) Anastrozole: a review of its use in the management of postmenopausal women with advanced breast cancer. Drugs Aging 13, 321-332.

From left to right: Kishor S Kumar, Mahesh M.J Kumar

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Cancer Therapy Vol 6, page 665! Cancer Therapy Vol 6, 665-672, 2008

Detection of Epstein-Barr virus in Pediatric Hodgkin’s Lymphoma in Iran by immunohistochemistry and in situ hybridization Research Article

Farzaneh Jadali*, Abdollah Karimi, Fatemeh Fallah, Golareh Habibi, Ahmadreza Shamshiri, Atoosa Gharib, Mostafa Sharifian, Shahnaz Armin, Soraya Tabatabaii, Masumeh Mohkam, Zahra Chavoshizadeh, Reza Shiari, Saiid Fahimzad, Saiid Maham, Elham Mazaheri-Tehrani Pediatric Infectious Research Center, Mofid Children's Hospital, Shahid Beheshti Medical Center, Tehran, Iran

__________________________________________________________________________________! *Correspondence: Jadali Farzaneh MD, Associate Professor, Pediatric Pathologist, Head of Department of Pathology and PIRC Virology Committee, Mofid Children's Hospital Shahid Beheshti Medical University, Tehran, Iran; e-mail: fjadali@hotmail.com Key words: Epstein-Barr virus; Hodgkin’s Lymphoma, immunohistochemistry, !"# $!%& hybridization, Statistical analysis, gene expression Abbreviations: classical HL, (CHL); diaminobenzidine, (DAB); EBV encoded RNA -1, (EBER); Epstein-Barr virus, (EBV); Hodgkin's lymphoma, (HL); Immunohistochemistry, (IHC); In Situ Hybridisation, (ISH); latent membrane protein-1, (LMP1); lymphocyte depletion, (LD); lymphocyte rich, (LRC); mixed cellularity, (MC); nodular lymphocyte predominant HL; nodular sclerosis, (NS); phosphate buffered saline, (PBS); Statistical Package for Social Sciences, (SPSS) Received: 21 July 2008; Revised: 21 August 2008 Accepted: 3 September 2008; electronically published: September 2008

Summary Frequency of EBV detected in patients with Hodgkin's lymphoma (HL) is geographically diverse. In this study, we investigated the frequency of EBV in HL patients in Mofid Children’s Hospital, one of the major pediatric medical centers in Tehran, Iran. The presence of EBV was determined by immunohistochemistry with anti LMP-1(latent membrane protein-1) antibody and by In situ hybridization method for EBERs (EBV encoded RNA -1). We studied 36 pediatric cases with HL. The male to female ratio was 3:1 (males 72% and females 28%) and the patients were between 5-12 years old (with a median of 9 years). The presence of EBV was not independently correlated with histologic subtype (80.0% in Mixed cellularity, 63.6% in nodular sclerosis and 75.0% in Lymphocyte predominant; p-value=0.76), sex (76.9% in males and 50.0% in females; p-value=0.22), and age (76.2% in patients aged 5-9 years and 60.0% in age group 10-12 years; p-value=0.47)). The findings of this study provided further evidence that HL is etiologically heterogeneous. On the other hand, the distribution of EBV in Iranian children with HL follows a pattern intermediate to that of industrialised and non industrialised countries.

Although the pathogenesis of HL is unknown, its complex epidemiology, in particular the diversity of incidence observed in different age, gender, ethnic, geographic, and socioeconomic groups suggests that different environmental etiologic agents may play an important role in the pathogenesis of HL(5) (MacMahn, 1966; Gutensohn and Cole,1981). Serological studies in the 1970s indicated that Epstein-Barr virus (EBV) infection was associated with HL (Engel et al, 2000). Afterwards, hybridization blotting techniques confirmed presence of this virus in HL (Weiss et al, 1987). Localisation of EBV to Hodgkin ReedSternberg (HRS) cells was subsequently shown by In Situ

I. Introduction Hodgkin’s lymphoma (HL), a neoplasm of germinal center related B cells in nearly all cases (Marafioti et al, 2000; Stein et al, 2001) occurs as two clearly delineated entities, nodular lymphocyte predominant HL (NLPHL) and classical HL (CHL) (Stein et al, 2001). These two differ in clinical presentation and behavior, morphology, phenotype and molecular features (Stein et al, 2001). CHL is further classified into four subtypes: nodular sclerosis (NS), mixed cellularity (MC), lymphocyte rich (LRC), and lymphocyte depletion (LD) (Harris, 1998; Flavell and Murray, 2000).

665

Jadali et al: Detection of Epstein-Barr virus in Pediatric Hodgkin’s Lymphoma in a microwave superheating oven. Antigen retrieval step was carried out by autoclaving at 121ºC, 15 PSI for 20 minutes. The sections were then allowed to cool down to room temperature in the sodium citrate solution for at least 30 minutes, followed by three 5-minute washes in phosphate buffered saline (PBS). Endogenous peroxidase activity was blocked by placing sections in 3% hydrogen peroxide for 5 minutes and washed 3 times in distilled water. To minimize nonspecific background staining, sections were blocked for 30 minutes with normal rabbit or goat serum diluted 1:1 in PBS. After removing the excess serum, sections were incubated with primary antibodies against LMP1(clones CS 1-4, Dako), followed by two 5-minute washes of PBS. The sections were incubated for 30 minutes with biotinylated rabbit anti-mouse antibody, washed with PBS twice, followed by 30 minutes incubation with a streptavidin- biotinhorseradish peroxidase complex. Sections were washed in PBS for 45 minutes and visualized using diaminobenzidine (DAB). Finally, the sections were lightly counterstained with hematoxyline, dehydrated and mounted (Aster, 2004). The slides were reviewed by light microscopy by two pathologists blindly for any positivity stained in RS cells Figure 1.

Hybridisation (ISH) (Weiss et al,! 1989). There is increasing evidence confirming the involvement of EBV in the development of HL (Brousset et al, 1994). It has also been shown that a product of the EBV BNLF-1 gene, latent membrane protein-1 (LMP1), and an integral membrane protein of known transforming ability also affecting apoptosis "#! $"%&' is strongly expressed in all neoplastic cells in EBV positive HL (Wang et al, 1985). The sensitive and specific techniques In situ hybridization (ISH) for EBV-encoded RNA (EBER1) and Immunohistochemistry (IHC) for EBV latent membrane protein 1 (LMP1) can detect EBV latent gene products in HRS cells in approximately 40-50% of HL cases. These two methods are the best and known as Gold standard. EBV appears to be associated particularly with HL in certain special epidemiologic groups, such as patients in developing countries and those with HIV infection. Similarly, several groups have reported high frequency of EBV infection in HL in children from various geographic and ethnic backgrounds (Ambinder et al, 1993; Jarrett et al, 1993; Kusuda et al, 1998). These studies have also suggested that EBV infection may be linked more closely to cases of pediatric HL that arise in developing countries (Kusuda et al, 1998). There are very few studies regarding the frequency of EBV in childhood HL. Studies in developing countries reported a strong correlation between pediatric HL and EBV infection. Examples include South Africa (68%), Peru (96%), China (72%), India (96%), Malaysian (93%), Thailand (92.8%), Jordan (47%) and Kuwait (79%). (Chang et al,! 1993; Hiraiwa et al, 1997; Peh et al, 1997; Staratschek-Jox et al, 2000; Zhou et al, 2001; Rrmakar et al, 2003; Almasri and Khalidi; 2004; Dinand et al, 2007). The aims of this study were to analyze the frequency of EBV infection in HL in Iranian children and to compare this with reported frequencies from other countries.

C. In Situ hybridization EBV-encoded small RNAs (EBERs) were detected using single-stranded digoxigenin-labeled riboprobes complementary to EBER-1 transcripts. Paraffin sections were stained with fluoresceinated EBER-specific oligonucleotide probe (Novocastra, Newcastle, UK) as recommended by the manufacturer. In brief, after dewaxing and hydrating poly l lysine slides associated tissues in xylene and ethanol, they were placed in an incubation tray and covers with 100µl of proteinase K, 15 !g/ml for 30 minutes at 37ºC. These slides were then dehydrated in ethanol and dried. Twenty !l of probe hybridization solution, a fluorescent labeled random oligonucleotide cocktail (NCL-EBVK, Novocastra kit) were added to slides for 2 hours at 37 ºC. Slides were washed in TBS containing 0.1% v/v triton X-100 for 3 minutes. Hybridized probe was detected with peroxidase conjugated anti-FITC antibody diluted 1:150 in TBS, 3% w/v BSA, 0.1% v/v triton x-100 for 30 minutes in room temperature after incubating slides in an incubation tray and covering with 100!l blocking solution for 10 minutes. Subsequently, sections were washed in TBS solution for 3 minutes and alkalin phosphatase substrate buffer pH 9 for 5 minutes. Slides were again placed in an incubation tray and demonstrated alkalin phosphatase activity by covering sections with Diluet Enzyme Substrate 1:50 in 100mM Tris/Cl, 50mM MgCl2, 100mM NaCl pH 9.0 and 1!l of inhibitor (Levamisole, 1000X) to each ml of diluted enzyme substrate for overnight at room temperature. The next day, the sections were washed by running water for 5 minutes and counterstained in Mayer's haematoxylin for 4-6 seconds and mounted. The stained slides were reviewed under microscope by two pathologists blindly and the positivity of hybridization stain has been detected as a positive result (Figure 2) (Zhou et al, 2001).

II. Material and Methods A. Patient selection Iranian HL patients between 5-12 years of age (with a median of 9 years) were selected from the archives of departments of pathology at Mofid Children’s Hospital, Shaheed Beheshti Medical University in the period of 1996 to 2006. In this study, cases in this age group were classified as pediatric HL. All lesions were reviewed histologically by pathologists in our team (F.J). The diagnosis and subtyping of HL were based on widely accepted morphologic and immunophenotypic criteria previously (Rrmakar et al, 2003). After revision, 36 pediatric HL cases were available for this study. The cases were formalin fixed and paraffin embedded. Among these 36 cases, the frequency of male and female cases were 26 (72%) and 10 (28%). Basic clinical and paraclinic data were extracted from the patients’ records and paraffin embedded tissues were collected and transported to PIRC to perform IHC and ISH tests. Paraffin sections of selected 36 HL infants were included: 4 LP, 10 MC, 22 NS.

D. Statistical analysis For statistical analysis, Chi-square and Fisher's exact tests were perfomed using Statistical Package for Social Sciences (SPSS) software. Histologic subtypes were compared with patients’ age group and sex. EBV expression was compared to the histologic subtypes, as well as to the available demographic data. The two methods used to study EBV expression were also compared with each other by McNemar test. P-values <0.05 were considered to be significant.

B. Immunohistochemistry Immunoperoxidase staining for LMP-1 was performed on all the 36 cases in this study. Four micron sections were cut from each selected paraffin block on to Poly L lysine coated slides; sections were then dewaxed, rehydrated to distilled water and placed in 0.01 M sodium citrate solution at pH 6.0 for 5 minutes

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Cancer Therapy Vol 6, page 667! negative by IHC showed EBER positively by ISH and 9 (25.0%) cases which were EBER negative by ISH showed LMP1 positive by IHC. The results obtained by IHC was not in concordance with ISH results (! statistics=0.29, pvalue=0.07). Because two different Ags are detected Sex distribution of expression of the virus between males (20 out of 26) and females (5 out of 10) were 76.9% and 50.0% (p-value=0.22). With regard to the subtypes, there was a trend for the MC male 20 out of 26, female 5 out of 10cases to be EBV positive: 8 of 10 (80.0%), compared with 14/22 (63.6%) of the NS and 3/4 (75.0%) of the LP subtypes which was not statistically significant (pvalue=0.76). So in our study we found more incidence of EBV positivity in MC subtype than NS subtype. EBV positive rates were 76.2% (16/21) for the age group 5-9 years and 60.0% (9/15) for the age group 10-12 years. The difference in EBV expression among these two age groups was not statistically significant (p-value=0.47) (Table 2).

III. Results A. Histological Subtype The median age of the patients was 9 years (ranged 5 to 15 years). The peak incidence of HL was seen between the ages of 6 and 11 years. There was an overall male predominance, with 72% male and 28% female patients (3:1). Twenty two patients (61.1%) were classified as NS, while only ten (27.8%) were of the MC subtype. Fifteen of the 22 NS Patients (68.2%), eight of the 10 MC cases (80.0%) were male, giving a male to female ratio in NS and MC of 2:1 and 4:1, respectively Table 1.

B. EBV gene expression Results of the EBV investigation are given in Tables 1-2. EBV expression was considered positive if any ReedStenberg cells was positive by either method (IHC or ISH). EBV products were detected in the HRS cells of 36 HL cases by ISH and IHC are 16 (44.4%) and 21 (58.3%), respectively. Four (11.1%) cases which were LMP1

Figure 2. In situ hybridization using oligonucleotide probe EBER-l positive multinucleated Hodjkin's cell (x400).

Figure 1. EBV-LMPI positve cytoplasmic staining in Hodjkin's cell (x400).

Table 1. The frequency of EBV positive cases of Hodgkin's disease (1996-2006) by sex and age

Sex Male Female Age (Yrs) 5-9 10-12 Total

Hodgkin Cases (%)

EBV positive by IHC (%)

EBV positive by ISH (%)

EBV positive by either ISH or IHC (%)

26( 72.2) 14( 53.8)

17(65.4) 4(40)

14( 53.8) 2(20)

20(76.9) 5(50)

21(58.3) 15(41.7) 36(100)

13( 61.9) 8(53.3) 21(58.3)

10(47.6) 6( 40) 16(44.4)

16( 76.2) 9(60) 25(69.4)

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Jadali et al: Detection of Epstein-Barr virus in Pediatric Hodgkin’s Lymphoma Table 2. The frequency of EBV positive cases of HL by histological subtype. ! Subtype Hodgkin EBV positive EBV positive Cases (%)! by ISH (%)! by ICH (%)! Mixed Cellularity ! 10 (27.8) 5 (50) 7 (70.0) Nodular Sclerosis! 22 (61.1)! 9 (40.9) 12 (54.5) Lymphocyte 4 (11.1) 2 (50.0) 2 (50.0) Predominant Total! 36 (100) 16 (44.4) 21 (58.3)

EBV positive by either ISH or IHC (%) 8 (80.0)! 14 (63.6) 3 (75.0) 25 (69.4)

cellularity subtype is the most common type in developing countries (Zhou et al, 2001). In most developed countries 30-40% of EBV cases are associated with higher rates; however this association may reach to 90% level in some developing countries (Staratschek-Jox et al, 2000). A detailed review of the literature further confirms this view. We identified 18 published series of pediatric HL, which included data on the presence of EBV in HRS cells as defined by EBV-specific IHC and/or ISH which appeared to relate to separate patient populations (Table 3). These cases came from Europe, North and South America, Africa, and the Middle and Far East. The results from these published cases showed similar findings to this study. EBV infection of HRS cells was associated strongly with pediatric HL, particularly in some developing countries, where it was reported in approximately 50-60% of cases in series from Middle East containing UAE, Jordan and Egypt (Table 3) (Armstrong et al, 1993; Macfarlane et al, 1995; Staratschek-Jox et al, 2000).

IV. Discussion Our results showed that HL in Iranian children is linked with EBV infection of HRS cells. Most studies on HL have shown epidemiological differences between developed and developing countries. HL in the USA and Europe countries tend to have a bimodal age distribution with a young adult peak occurring between 20 and 34 years of age and a second peak occurring in the elderly patients between 50 and 74 years of age (MacMahn, 1966; Aster, 2004) .On the other hand, HL in developing countries lacks the young adult peak, but there is an increase in the incidence of HL in children <15 years of age (Correa and O' Conor, 1971; Zhou et al, 2001). This association is striking particularly in young children, regardless of histological type, as EBV is present in tumor cells in 50% of the children who are 10 years old or younger. Therefore pediatric HL should be regarded as a distinctive, EBV-related lymphoma. It has also been shown that the NS subtype predominates in developed countries, whereas the mixed

Table 3. EBV Gene Expression in Hodgkin and Reed-Sternberg Cells in Pediatric HL: Published Case Series. Studies

country

Age 0-10

Age >10

MC

NS

Total

!"

Razzouk et al, 1997

Study year 1997

USA

13/17

2/9

12/17

2/6

15/26

58

Weinreb, 1996

1996

UK

14/32

23/42

17/20

14/36

37/74

50

Armstrong et al, 1998 Brousset et al, 1993 Claviez et al, 1994 Leoncini et al, 1996 Weinreb et al, 1997 Chang et al, 1993 Armstrong et al, 1993 Precido, 1995

1998 1993 1994 1996 1996 1993 1993 1995

UK France Germany Italy Greece Peru Brazil Argentina

4/5

1/11 6/8 8/9

1/3 2/11

6/7 14/14 10/12 19/25

11/12 4/4 7/10 1/1

5/16 7/13 10/21 3/9 19/21 19/19 18/25 22/41

31 54 48 33 90 100 72 54

Monterroso et al, 1998

1996

10/10

19/24

34/42

81

Zhou et al, 2001

2001

Costa Rica China

7/13

82/89

5/7

89

Liu et al, 1998 Huh et al, 1996 Kusuda et al, 1997 Peh et al, 1997 Kusuda et al, 1998 Current study

1998 1996 1998 1997 1998 2007

Taiwan Korea Japan Malaysia Kenya Iran

5/5 4/6 4/9

7/8 2/2 8/9 12/13 6/10

1/4 2/2 . 6/6 2/3 10/22

93/10 4 4/6 12/16 4/4 14/15 16/18 18/36

7/9

3/12

18/18

1/1

86/91

9/10 12/12 13/25

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67 75 100 93 89 51

Cancer Therapy Vol 6, page 669! very young adult is associated with NS subtype (Andriko et al, 1997). The results in this study showed NS subtype with EBV positive samples is higher than MC subtype. Moreover, Glaser and colleagues analyzed in 1997 association of EBV and HL in international study from 14 former published studies in Europe and United states and showed that incidence of HL is low in childhood. They reported a marked young adult peak dominated by NS disease followed by plateau through later life with equal contributions from both NS and MC subtypes. Patterns vary in other parts of the world, but generally in lessdeveloped countries, childhood HL is more common. This disease then shows a steady age-related increase with no young adult peak. Therefore, overall contribution of MC disease is higher (Glaser et al, 1997). In concordance with previous reports (Delsol et al, 1992) the present findings suggest that LMP1 expression may be adequate for identifying EBV-associated CHL Moreover, in these reports using ISH methods is strongly suggested in cases with negative results (Pinto et al, 2006).

Pediatric HL series from Western Europe and the United States also reported high rates of EBV infection in tumors from children younger than 10 years old. Although these rates were generally lower than corresponding case series from developing countries, this trend was not significant. Epidemiologically, frequency of pediatric HL varies with geography and a different epidemiologic curve is seen with pediatric HL. The first peak in the bimodal ageincidence curve of HL, seen in the third decade in developed countries whereas it occurs earlier in developing countries (Evans et al, 1978; Weinreb et al, 1996; Staratschek-Jox et al, 2000) Furthermore, cases of HL in children 10 years of age or younger are particularly more common. In this study, EBV was found in 25 out of 36 (69.4%) children between 5-12 years. As in our cases, EBV was associated with pediatric HL, regardless of histologic subtypes. Similar findings with regard to the relative frequency of pediatric and adult HL in developing countries (particularly in young children) have been reported by other groups where cases of pediatric and adult HL differ in their epidemiology (Riyat, 1992, Leoncini et al, 1996; Kusuda et al, 1998). In developing communities, EBV infection usually occurs at an early age. The epidemiologic and socioeconomic features associated with childhood HL predispose to such early infection. In contrast, a significant proportion of children remain uninfected by the virus up to age of 15 in western countries (Henle and Henle, 1979). Although early infection is usually mild or asymptomatic, late infection is frequently associated with a more severe host response in the form of infectious mononucleosis, a rare disease in most economically developing communities (Rosdahl et al, 1974). The biologic factors that determine the development of HL in these children remain to be determined. We found a clear male predominance among our pediatric cases (male to female ratio, 3:1), and similar findings have been reported by other groups (Armstrong et al, 1993, Andriko et al, 1997). These observations remain unexplained, but they suggest that there might be some unknown gender-linked genetic or environmental factors are involved. A male predominance in the incidence of symptomatic disease has been reported with some common infectious agents (Green, 1992). However, the exact mechanism or the possibility that it is connected to a general or specific immune deficiency in males relative to females, remain to be proven.! Previous studies have shown that pediatric HL cases with EBV infection differ from adult HL cases in their subtypes (Harris, 1998; Engel et al, 2000; Flavell and Murray, 2000). Also, the epidemiology of their morphology appeared to vary with geographical origin. We compared pediatric HL cases from Europe and the United States with cases from Asia, Africa, Central America and South America; in the current study, EBV antigens were present in 63.6% NS subtype and 80.0% of MC cases. Flavel and colleagues reported in 2000 that there is a variation in the frequency of EBV associated HL with age; however, reports from above mentioned countries concluded that HL of childhood and older adult is commonly EBV associated with MC subtype, whereas

IV. Conclusion Our study provides compelling evidence that HL in children 12 years of age or younger is a specific EBVrelated disease with characteristic morphologic features that represent a rare complication of early EBV infection. The role of EBV in CHL still remains to be clarified. Moreover, the evaluation of the clinical implications of the presence of EBV for evolution, prognosis and pathogenesis of the disease requires further studies. The techniques of ISH (EBER1 RNA) and IHC applied to tissues fixed in formaldehyde and embedded in paraffin, as employed in our studies, may turn out to be quite valuable for future epidemiological studies on the association of EBV virus with CHL in other regions of Iran.

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Cancer Therapy Vol 6, page 671! Khalek ER, Mangoud A, El-Safy UR, Madanat F, Al Sheyyab M, Mpofu C, Revesz T, Rafii R, Tiedemann K, Waters KD, Barrantes JC, Nyongo A, Riyat MS, Mann JR (1996) The role of Epstein-Barr virus in Hodgkin’s disease from different geographical areas. Arch Dis Child 74, 27-31. Weiss LM, Movahed LA, Warnke RA, Sklar J (1989) Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin’s disease. N Engl J Med 320, 502-6.

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Left to right upper row: Atoosa Gharib, Abdollah Karimi, Saiid Maham, Golareh Habibi, Fatemeh Fallah Left to right lower row: Shahnaz Armin, Farzaneh Jadali, Saiid Fahimzad

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Cancer Therapy Vol 6, page 673! Cancer Therapy Vol 6, 673-682, 2008

Conventional versus hyperfractionated radiotherapy in locally advanced head and neck cancer Research Article

Azza Abd El-Naby1, Aly Tawfek2, Hala M. El-Shenshawy1,*, Amal Halim1, Rasha Hamdy1 1 2

Department of Clinical Oncology and Nuclear Medicine, Faculty of Medicine, Mansoura University, Dakhlia, Egypt Department of ENT, Faculty of Medicine, Mansoura University, Dakhlia, Egypt

__________________________________________________________________________________! *Correspondence: Hala Mohamed El- Shenshawy, Department of Clinical Oncology and Nuclear Medicine, Faculty of Medicine, Mansoura University, Dakhlia, Egypt; Tel: 0020123810407; Fax: 0096672501305; e-mail: kandeel36@yahoo.com Key words: conventional, hyperfractionated radiotherapy, head and neck, locally advanced Abbreviations: 5-fluorouracil, (5-FU); chemotherapy, (CT); complete response, (CR); conventional fractionated radiotherapy, (CFRT); European Organization for the Research and Treatment of Cancer, (EORTC); hyperfractionated radiotherapy, (HFRT); karnofsky performance status, (KPS); partial response, (PR); progressive disease, (PD); radiotherapy, (RT); stationary disease, (SD)

Accepted at the Annual Scientific Meeting of the Radiological Society of North America, 2007 (RSNA-2007), Radiation oncology section, November 25-November 30, 2007, Chicago-USA. Received: 19 November 2007; Revised: 14 January 2008 Accepted: 17 January 2008; electronically published: September 2008

Summary Radiotherapy is often the primary treatment of locally advanced squamous cell head and neck cancer, but the optimal fractionation schedule has been controversial. The aim of this study was to examine whether, after preceeding induction chemotherapy, hyperfractionated radiotherapy (HFRT) is superior to conventional fractionated radiotherapy (CFRT). Patients with locally advanced squamous cell head and neck cancer were treated with 3 cycles of cisplatin (100 mg/m2 D1) and 5-fluorouracil (1000 mg/m2 D1-4), repeated every 3 weeks. Then patients were randomized to receive either CFRT at 1.8-2 Gy / fraction / day, 5 day / week to 65- 70 Gy / 33- 35 fractions / 7 weeks or HFRT at 1.2 Gy / fraction, twice daily with a 6-h interfraction interval, 5 days / week to 76.8 Gy / 64 fractions / 7 weeks. All patients in both treatment arms received concomitant chemotherapy in the form of weekly bolus injection of cisplatin (20mg/m2). Of the 60 patients entered, only 53 patients were evaluable for outcomes. The primary end points were local control and progression- free survival. Chemotherapy was well tolerated, the overall response rate after induction chemotherapy was 73.6%, including 13.2% complete response rate. After completion of radiotherapy, patients treated with HFRT had an overall response rate of 96.2% vs 77.8% in CFRT (P= 0.037) and complete response rate of 65.4% in HFRT vs 40.7% in CFRT (P=0.013). After a median follow- up of 28 months, overall survival was 57.7% in HFRT vs 44.4% in CFRT (P= 0.068). The 2-year progression- free survival was 44% in HFRT vs 23.8% in CFRT (P=0.028). The 2- year locoregional control was significantly higher in HFRT (58.8%) than those with CFRT (36.4%) (P=0.021). The incidence of local recurrence rate was 41.2% in HFRT vs 63.6% in CFRT (P=0.019). However, the incidence of distance metastases was 7.7% in HFRT vs 11.1% in CFRT (P=0.439). Patients treated with HFRT had significantly greater acute side effects compared to CFRT. However, there was no significant increase of late effects. After induction chemotherapy, hyperfractionated radiotherapy is more efficaceous than conventional fractionated radiotherapy in locally advanced squamous cell head and neck cancer. Acute but not late effects are increased, but it is tolerable and manageable.

mainstay for this group of patients because chemotherapy alone has only palliative means. Apart from intrinsic radioresistance and sublethal damage repair of tumor cell

I. Introduction The prognosis of locally advanced head and neck cancer is still dismal. Radiation therapy (RT) is the 673

El-Naby et al: Conventional versus hyperfractionated radiotherapy in locally advanced head and neck cancer used and easily given. It results in a high response rate as it reduces the tumor mass facilitating its elimination by radiation. Synchronous or concurrent chemotherapy results in the delivery of RT and CT on the same days, typically CT is given for one or more days at the initiation of RT and then repeated in the same fashion several weeks later (Ang, 2007). By adding systemic drugs to radiation, both the tumor lesions within the irradiated field and micrometastases outside the irradiated area are exposed to the drugs' cytotoxic action. In addition, chemotherapeutic drugs may make tumor cell clonogens undergoing irradiation more susceptable to killing by ionizing radiation. This radiosensitizing property of chemotherapeutic agents constitutes the major rationale for concurrent chemoradiotherapy (Perez et al, 2004). The activity of cisplatin in head and neck squamous cell carcinoma was already documented in an early period (Knox et al, 1986). Some experimental studies showed synergism between RT and the drug in vitro (Zamboglou et al, 1989). The simultaneous administration of RT and CT with cisplatin and 5-fluorouracil (5-FU) proved feasible and highly effective in terms of objective response. The combination of 5-FU and cisplatin remains the best-studied and most active drug regimen. One advantage of this drug combination is that both agents are radiosensitizers (Douple, 1988). 5-FU is an S-phase specific agent with a short half- life (<10 min), making it particularly suitable for administration as continuous infusion, which enhances cell kill by increasing the exposure of dividing cells (Lockich et al, 1981). Addition of concomitant chemotherapy to either hyperfractionated or conventional fractionated RT could be a good option, since such a method of combining two treatment modalities seems to be the most promising approach, as demonstrated in meta-analyses and recent randomized studies (Lockich et al, 1981; Brizel et al, 1998; Pignon et al, 2000; Domenge et al, 2000). The aim of this study was as follows: (a) to analyse and compare locoregional control, progression-free survival and overall survival in both conventional fractionated radiotherapy arm and hyperfractionated radiotherapy arm. (b) to determine and compare the acute and late adverse effects in both treatment arms and (c) to determine variations in treatment outcome per arm in a variety of head neck sub-sites and T and N stages.

clonogens, hypoxia and repopulation are also known to be poor , with median 3-year overall survival and locoregional control rates of approximately 30% and 40% respectively (Hoeckel et al, 1996; Withers et al, 1988). Survival in patients with locally advanced head and neck cancer is determined by locoregional outcome, with a probability of less than 30% long- term tumor control despite optimal treatment with surgery and/ or conventional radiotherapy. In less advanced tumor stages, the probability of survival is up to 60%. In these cases, the results are influenced by locoregional recurrences and second tumors. The accumulated knowledge on tumor biology has resulted the investigators to modify conventional treatment modalities with the intention of achieving better local control bettering order to improve the survival rates (Galiana et al, 2002). A number of strategies have been explored during the last two decades to improve the outcome in locally advanced head and neck cancer (Stuschike et al, 1997; Fu et al, 2000). New fractionations in radiotherapy on head and neck cancer have emerged as a treatment of choice to increase local control of this disease. Altered fractionation is predicted to improve the therapeutic ratio through a differential response between tumors and normal tissues to fractionated radiotherapy. Hyperfractionated radiotherapy is probably the best studied form of altered classical fractionation of 2 Gy as a single daily fraction, and consists of administering two or more small fractions every day to reduce the repopulation of tumor cells between two consecutive fractions. The overall treatment time is not increased. Hyperfractionation has been considered as the most promising and tempting alternative to standard fractionation (Horiot et al, 1992; Hall, 1994; Stuschike et al, 1997). Retrospective studies of patients treated with accelerated fractionation and hyperfractionation have demonstrated an improvement in disease control of about 20%, as compared with historical controls treated with conventional daily radiation, without an increase in longterm toxicity. A prospective randomized trial by the European Organization for the Research and Treatment of Cancer (EORTC) showed an increase of 20% in the rate of locoregional control of oropharyngeal carcinoma at five years and an improvement of 14% in survival after treatment with 8050cGy of radiation in hyperfractionated doses as compared with rates achieved with once-daily radiotherapy (total doses, 7000 cGy), without any increase in acute or chronic toxic effects (Horiot et al, 1992; ElSayed and Nelson,1996). Even the most effective radiotherapy regimens result in local control rates not exceeding 50-70% and diseasefree survival rates not more than 30-40%. This circumstance has stimulated the investigation of treatments combining radiotherapy and chemotherapy. Several review articles have explored the different chemotherapeutic agents and RT schemes of these treatment programs (El-Sayed and Nelson, 1996). The combination of RT and chemotherapy (CT) is another promising approach investigated to improve results in advanced head and neck cancer. Induction or neoadjuvant chemotherapy performed before definitive RT was widely

II. Materials & Methods Between June 2004 and June 2006, 60 patients with locally advanced squamous cell carcinoma of the head and neck who attended to Clinical Oncology and Nuclear Medicine Department, Mansoura University Hospital, were randomly assigned in this prospective study.

A. Eligibility criteria Eligibility criteria included patients of either sex older than 18 years, hisitologically proved squamous cell carcinoma of the head and neck, karnofsky performance status (KPS) ! 60, locally advanced non metastatic stage III, IV (according to UICC/ AJCC stage classification for head and neck cancer) and no evidence of coexistent synchronous or previous malignant disease, previous head and neck radiotherapy, previous chemotherapy or previous surgery except biopsy only.

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Cancer Therapy Vol 6, page 675! Treatment fields were verified by simulator before starting treatment and weekly during treatment to ensure reproducibility. All patients were instructed to meticulous oral hygiene and change their eating habits. During RT, the patients were examined for acute toxicity every week. Toxicity of RT developing within 90 days from the beginning of RT (acute toxicity) was assessed according to RTOG and EORTC criteria (Perez et al, 1992). RT toxicity developing after 90 days (chronic/ late toxicity) was graded with the same scale for late sequelae and evaluated every 6 months. Mucositis was treated by saline mouth washes, pain management, antimicrobials (antibiotics, antivirals and antifungal) and maintaining nutritional intake.

B. Pretreatment evaluation Pretreatment evaluation included complete history, physical examination, head and neck examination including mirror and panendoscopic examination, histopathologic examination of the primary tumor or cervical lymph nodes, complete blood count (WBC count ! 3.5 109/L, platelets ! 100 10 9/L, hemoglobin ! 10g/dL), blood chemistry including liver function tests (serum bilirubin " 1.5 mg/dL), and kidney function test (serum creatinine " 1.5mg/dL), computed tomography and or magnetic resonance imaging of the head and neck to define the extent of the disease and metastatic workup including chest x-ray and imaging of liver by ultrasound or computed tomography in all patients. Bone scan was not routinely performed and was restricted to those with bone pain or elevated serum alkaline phosphatase. Dental care was applied to each eligible patients before therapy.

D. Post-treatment evaluation Response was assessed six weeks after completion of radiotherapy by clinical examination, endoscopic examination, and CT and/or MRI of head and neck. Criteria for response were as follows: complete response (CR) was defined as complete regression of all evidence of tumor. Partial response (PR) was defined as an estimated decrease in tumor size of 50% or more. Stationary disease (SD) was defined as < 50% decrease in tumor size or < 25% increase in pretreatment tumor size. Progressive disease (PD) was defined as > 25% increase in pretreatment tumor size. Re-evaluation was done at 3 months interval during the first two years of follow- up unless any manifestations of progression were developed. Chest radiography and ultrasonography of the liver were performed every year.

C. Treatment schedule Induction chemotherapy consisting of 3 cycles of cisplatin 100mg/m2 IV on day 1 and 5-fluorouracil 1000mg/m2 on days 1 through 4 by continous infusion, to be repeated every 3 weeks. All patients had prehydration with intravenous fluids for one day prior to chemotherapy. Cisplatin was administered as an infusion over one to two hours and 5- fluorouracil was administered as continous infusion. Antiemetics prophylaxis was routinely given, IV dexamethasone 10-20mg and diphenylhydramine 50mg IV were given prior to chemotherapy. The dose of cisplatin was decreased by 25% if serum creatinine level was elevated 1.5 to 2 times above the baseline values and by 50% if serum creatinine level were elevated 2 to 2.5 times above baseline. If creatinine levels were elevated more than 2.5 times the baseline value, no further cisplatin was given to that patient. Reassessment of response was performed at the end of the 3 cycles of chemotherapy. Radiotherapy began within 3 weeks of completion of the last cycle of induction chemotherapy and after re-assessment of response (primary tumor as well as lymph nodes) by clinical examination, endoscopic examination and CT and/or MRI. Out of 60 patients, only 56 patients completed 3 cycles of induction chemotherapy. All 56 patients were randomly assigned into two treatment arms. Arm I (n=28 patients) treated by conventional fractionated radiotherapy (CFRT) to total dose of 65-70 Gy, 1.8-2 Gy / fraction, one fraction / day, 5 days / week. Arm II (n= 28 patients) treated by hyperfractionated radiotherapy (HFRT) to total dose of 76.8 Gy, 1.2 Gy / fraction, 2 fractions / day with 6- h interval between fractions, 5 days / week. All patients in the two treatment arms received concomitant chemotherapy in the form of weekly bolus injection of cisplatin (20mg/m2). The primary tumor and draining lymphatic system were irradiated in most patients by two parallel- opposed lateral fields. In the initial lateral fields: arm I received 45 Gy (1.8 Gy / fraction / day, 5 days / week) , however, arm II received 43.2 Gy (1.2 Gy / fraction, 2 fractions / day, 5 days / week) after that these initial fields were reduced for spinal cord shielding. Further reductions were made whenever possible to limit the volume of tissues receiving high- dose RT. Patients were treated by 6 MV photons of a linear accelerator or Cobalt-60 teletherapy device with the centres of the fields marked on the shell. Electron beam irradiation was used to boost the dose to the posterior cervical lymph node chains and to the selected lymph node region as indicated. However, the supraclavicular nodes and nodes in the lower part of the neck were treated with the use of a single anterior field with midline blocking to prevent spinal cord overlap. Anterior lower neck field doses were prescribed at depth of 3 cm with a total dose of 50 Gy (2 Gy / fraction / day, 5 days /week). The inferior border of the lateral fields and the superior border of anterior lower neck field coincided on the skin.

E. Endpoints The primary endpoints were to analyse and compare locoregional control and acute and late adverse effects in both treatment arms. The secondary endpoints were to analyse and compare progression-free survival and overall survival in both treatment arms.

F. Statistical methods Pretreatment characteristics of both treatment arms were compared using the Chi- square test. Overall survival and progression- free survival were calculated using the KaplanMeier method. Responses were compared using the Chi- square test. Mann- Whitney U test used to compare the median responses, survival and progression- free survival in both treatment groups. Confidence intervals (CIs) were calculated using Cox's proportional hazard model. Prognostic factors related to response, survival and progression- free survival were assessed using Cox proportional hazards regression model. Informed consent was obtained from all patients, and ethical committee approval was received by our participating center. The randomization scheme was a permuted block design with an equal probability of assignment to either treatment arms. Patients were stratified by primary site of disease and stage of disease and were then randomized to receive one of the two treatments planned in the trial.

III. Results A. Patient's characteristics All sixty patients with locally advanced squamous cell carcinoma of the head and neck received induction chemotherapy but only 56 patients completed their 3 cycles of chemotherapy. These patients were randomly assigned into two treatment arms, either CFRT arm or HFRT arm with 28 patients in each treatment arm. In 675

El-Naby et al: Conventional versus hyperfractionated radiotherapy in locally advanced head and neck cancer IV (56.6%). The median age was 57 years, ranging from 20 to 80 years.

CFRT arm, only 27 patients completed their treatment as one patient died from tumor 2 weeks from beginning of RT. While in HFRT arm, only 26 patients completed their protocol as the remaining 2 patients died during the course of RT (one patient died from hepatitis and the other died from local disease). Only 53 patients out of 60 patients were analyzable for outcome. The median follow-up of all eligible patients were 28 months (range 12-34 months). Table 1 shows the pre-treatment patients characteristics. They were well balanced among the both treatment groups. The nasopharynx was the most common primary site. All patients were stage III (43.4%) and stage

B. Response The overall response rate after induction chemotherapy was 73.6% (95% CI: 0.617 to 0.855), including 13.2% complete response rate, 60.4% partial response rate and 26.4% stable disease. However, the overall response rate in all evaluable patients 6 weeks after the completion of concomitant chemoradiotherapy were

Table 1. Patients Characteristics Character Age(years): <60 ! 60 Sex: Male Female Smoking: Smoker Non smoker KPS: 60 70 80 90 Site: Oral cavity Maxillary sinus Nasopharynx Oropharynx Hypopharynx Larynx Grade I II III Undifferentiated T-stage T2 T3 T4 N-stage: N0 N1 N2 N3 AJC stage: III IV Hemoglobin concentration: ! 12 g/dl < 12 g/dl

Total No.

%

CFRT No.

%

HFRT No.

%

31 22

61.5 38.5

15 12

55.6 44.4

16 10

61.5 38.5

0.76 0.87

40 13

75.5 24.5

19 8

70.4 29.6

21 5

80.8 19.2

0.56 0.34

32 21

60.4 39.6

16 11

59.3 40.7

16 10

61.5 38.5

0.93 0.81

2 1 26 24

3.8 1.9 49.1 45.3

0 0 14 13

0 0 51.9 48.1

2 1 12 11

7.7 3.8 46.2 42.3

0.34 0.64 0.81 0.79

7 3 20 3 10 10

13.2 5.7 37.7 5.7 18.9 18.9

4 1 11 2 3 6

14.8 3.7 40.7 7.4 11.1 22.2

3 2 9 1 7 4

11.5 7.7 34.6 3.8 26.9 15.4

0.75 0.53 0.72 0.94 0.23 0.51

14 11 10 18

26.4 20.8 18.9 34

9 3 6 9

33.3 11.1 22.2 34.6

5 8 4 9

19.2 30.8 15.4 33.3

0.46 0.17 0.52 0.96

8 35 10

15.1 66 18.9

8 16 3

29.6 59.3 11.1

0 19 7

0 73.1 26.9

0.14 0.31 0.22

13 13 21 6

24.5 24.5 39.6 11.3

7 7 9 4

25.9 25.9 33.3 14.8

6 6 12 2

23.1 23.1 46.2 7.7

0.96 0.96 0.73 0.41

23 30

43.4 56.6

13 14

48.1 51.9

10 16

38.5 61.5

0.51 0.59

15 38

28.3 71.7

9 18

33.3 66.7

6 20

23.1 76.9

0.49 0.52

676

P-Value

Cancer Therapy Vol 6, page 677! grade 3 or worse late side effects as mucositis (P=0.026), xerostomia (P=0.032) and dysphagia (P=0.045), according to the RTOG that defines late effects as toxicity noted > 90 days from the start of radiotherapy. However, these late effects were actually prolonged acute effects. Table 5 had shown that globally there was no significant difference in frequency of grade 3 or worse late effects reported at 6-24 months after start of radiotherapy among the two treatment groups.

77.8% (95% CI: 0.621 to 0.935) in CFRT arm versus 96.2% (95% CI: 0.889 to 1.035) in HFRT arm (P= 0.037), including 40.7% complete response rate in CFRT vs 65.4% in HFRT (P= 0.013) and 37% partial response rate in CFRT vs 30.8% in HFRT (P= 0.433). However, 11.1% had stable disease in CFRT vs 3.8% in HFRT and 11.1% had progressive disease in CFRT vs 0% in HFRT (Table 2).

C. Toxicity and treatment compliance

D. Survival

Tables 3 and 4 show the site and grade of acute and late adverse effects by treatment groups. The most common sites of grade 3 or worse acute side effects were the mucous membranes and the pharynx. However, the most common sites of grade 3 or worse late effects were the mucous membranes, the pharynx and the salivary gland. Compared to conventional fractionated radiotherapy, the hyperfractionated radiotherapy had significantly increased grade 3 or worse acute side effects as mucositis (P=0.034), dysphagia (P=0.042) and neck edema (P=0.049). However, it had significantly increased

The median local recurrence free survival was 15 months (ranging from 4-34 months) in the CFRT group versus 25 months (ranging from 7-34 months) in the HFRT group. Hyperfractionated radiotherapy arm had significantly increased locoregional control rate at two years (58.9%) compared with CFRT arm (36.4%) (P=0.021). Results of Kaplan- Meier estimates of localregional control in both treatment groups are shown in Figure 1.

Table 2. Response after radiation therapy in both treatment groups

Response Complete response

CFRT Arm No. % 11 40.7

HFRT Arm No. % 17 65.4

P Value 0.013

Partial response

10

37

8

30.8

0.433

Stationary disease

3

11.1

1

3.8

0.159

Progressive disease

3

11.1

0

0

0.081

Overall response

21

77.7

25

96.2

0.037

Table 3. Acute adverse effects of radiation therapy reported within 90 days after start of radiotherapy in both treatment groups.

Organ /Tissue Skin toxicity (dermatitis)

Mucous membrane (mucositis)

Salivary gland (xerostomia) Pharynx /Eosphagus (dysphagia) Subcutaneous tissue (neck edema) Taste sensation (dysgeusia) Weight loss

Grade 1 2 3 4 1 2 3 4 1 2 1 2 3 1 2 1 2 1 2

CFRT Arm No. % 66.7 18 33.3 9 0 0 0 0 22.2 6 55.6 15 22.2 6 0 0 7 25.9 16 59.3 29.6 8 48.1 13 14.8 4 10 37 0 0 23 85.2 2 7.4 20 74 4 14.8

677

HFRT Arm No. % 42.3 11 50 13 3.8 1 3.8 1 7.6 2 46.2 12 42.3 11 3.8 1 6 23.1 17 65.4 3.8 1 65.4 17 26.9 7 12 46.2 1 3.8 20 76.9 5 19.2 13 50 10 38.5

P Value

0.221

0.034

0.89

0.042 0.049 0.14 0.05

El-Naby et al: Conventional versus hyperfractionated radiotherapy in locally advanced head and neck cancer Table 4. Late adverse effects of radiation therapy reported after 90 days after start of radiotherapy in both treatment groups.

Organ / Tissue Mucous membrane

Grade 1 2 3 4 1 2 1 2 3 4

Salivary gland Pharynx /Esophagus

Subcutaneous tissue Pharyngo-tracheal fistula

1 2 3 4 1 2 3 4

Laryngeal edema

Temporal lobe necrosis

CFRT Arm No. % 7.4 2 33.3 9 0 0 0 0 3 11.1 4 14.8 22.2 6 14.8 4 3.7 1 0 0 8 29.6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

HFRT Arm No. % 26.9 7 34.6 9 11.5 3 0 0 6 23.1 10 38.4 11.5 3 34.6 9 7.7 2 0 0 7 26.9 0 0 0 0 0 0 3.8 1 0 0 0 0 0 0 3.8 1 1 3.8

P Value

0.026

0.032 0.045

0.81 0.39

0.39

0.39

Table 5. Frequency of Grade 3 or worse late effects at various times after start of radiotherapy in both treatment groups. Time after start of radiotherapy(months) 6

CFRT Arm No. % 3 11.1

HFRT Arm No. % 4 15.4

P Value

12

2

7.4

4

15.4

0.085

18

2

7.4

3

11.5

0.238

24

1

3.7

2

7.7

0.481

Figure 1. Cumulative probability of local-regional free survival in both treatment groups.

678

0.572

Cancer Therapy Vol 6, page 679! months, ranging from 5-34 months vs 25 months, ranging from 7-34 months in HFRT, with no statistically significant difference (P=0.061). The 2-year overall survival in CFRT arm was 44.4% vs 57.7% in HFRT, with no statistically significant difference (P=0.068), (Figure 3). Table 6 shows 2-year local-regional control rate, progression-free survival and overall survival for the two treatment groups.

Progression includes the following events: local, regional, loco-regional and distant failure. The median progression-free survival was 9 months, ranging from 2-34 months in CFRT vs 19 months, ranging from 3-34 months in HFRT (P=0.031). In addition, there was improved 2year progression-free survival in HFRT 44 % vs 23.8% in CFRT, with statistically significant difference (P=0.028), (Figure 2). At a median follow-up of 28 months of all analyzed patients, the median overall survival in CFRT was 19

Figure 2. Cumulative probability of progression free survival in both treatment groups.

Figure 3. Cumulative probability of overall survival in both treatment groups.

Table 6. 2-year locoregional control, progression-free survival and overall survival in both treatment groups.

2-Year endpoints Locoregional Control

CFRT Arm No. % 4/11 36.4

HFRT Arm No. % 10/17 58.9

0.021

Progression-free survival

5/21

23.8

11/25

44

0.028

Overall survival

12/27

44.4

15/26

57.7

0.068

679

P Value

El-Naby et al: Conventional versus hyperfractionated radiotherapy in locally advanced head and neck cancer

E. Pattern of treatment failure

2003). It is possible that induction chemotherapy may have a role in increasing survival rates for squamous cell carcinoma of the head and neck patients and appropriate randomized trials testing this hypothesis need to be conducted (Cohen et al, 2004). Our results show activity and relatively low toxicity of a combined therapy consisting of cisplatin and 5- FU followed by concomitant chemoradiotherapy in the form of either CFRT or HFRT with cisplatin as radiosensitizer in both treatment groups. It was noted, however, that 6-8 weeks after treatment completion, a significant advantage of the HFRT arm over the CFRT arm was already present. The overall response at the completion of radiotherapy was 96.2 % in HFRT vs 77.7 % in CFRT with 65.4% CR in HFRT vs 40.7% in CFRT. Such a response rate has been seen in previous studies including induction cisplatin- fluorouracil regimens (Paccagnella et al, 1994; Lefebvre et al, 1996; Posner et al, 2000). Locoregional control represents the major endpoint of any curative radiotherapy. This randomized trial demonstrates a significant 2-year locoregional control rate benefit of a hyperfractionated radiotherapy regimen over a conventional regimen in locally advanced head and neck cancers. These findings had been seen in many large randomized trials that had compared hyperfractionated RT with conventional fractionated RT (Pinto et al, 1991; Horiot et al, 1992; Cummings et al, 1996; El-Sayed and Nelson, 1996; Fu et al, 2000; Jeremic et al, 2000; Galiana et al, 2002; Orecchia et al, 2002). In the EORTC trial (Horiot et al, 1997), 356 patients were randomized to receive 70 Gy in 35 fractions of 2 Gy/fraction in the CFRT arm versus 80.5 Gy in 70 fractions of 1.15 Gy delivered twice-a-day (6-h interfraction interval) in the HFRT arm, both over 7 weeks. The locoregional control was significantly higher in the twice-a-day arm (56% vs 38%; P=0.01). However, it was a trend in favour of hyperfractionated radiotherapy, especially for the most unfavourable T and N combinations. In our study we found that the locoregional control benefit of HFRT is of a larger magnitude in patients with unfavourable prognosis factors. Similar conclusions were reached with the EORTC hyperfractionation trial (Horiot et al, 1997). However, standard fractionation already reaches rather high control rates in the more favourable clinical presentations. Conversely, patients with large primaries and / or involved nodes do recur more often and sooner with the conventional regimen. Such conclusions were reported with EORTC trial (Horiot et al, 1997). Neoadjuvant chemotherapy followed by concomitant chemoradiotherapy resulted in reduction of tumor lesions as well as eradication of micrometastases outside the irradiated field, this resulted in high local control and low incidence of both local failure and distant metastases. Moreover, in hyperfractionated irradiation, the overall treatment time is reduced with respect to the increased total dose (76.8 Gy /7 weeks). This lead to increase in locoregional control, as the hypothesis that tumor repopulation during therapy is a major cause of treatment failure. Our findings go ahead with EORTC trial (Horiot et al, 1997), in which patients with locally advanced head

The primary site was the most common location of treatment failure. The 2-year locoregional failure rates were 63.6% in CFRT vs 41.1% in HFRT, (P=0.019). However, the incidence of distance metastases at 2-years was 11.1% in CFRT vs 7.7% in HFRT (P=0.439).

F. Prognostic factors with treatment outcomes On multivariate analysis for locoregional control, T category (T4 vs T2, T3; P=0.003) and tumor site (oral cavity or oropharynx vs all other sites; P=0.001) were significant independent adverse prognostic factors for locoregional control. On other hand, the HFRT arm was confirmed as an independent factor of good prognosis for locoregional control (P=0.03). However, on multivariate analysis for progression- free survival, T category (T4 vs T2, T3; P=0.001) and N–category (N2-N3 vs N0,N1; P=0.045), sex (male vs female; P=0.024) and smoker patients (P=0.033) had independent adverse prognostic impact on progression-free survival. Whilst, HFRT was associated with good prognosis for progression- free survival (P=0.04). In addition, on multivariate analysis for survival, sex (P=0.03), poor performance status (P=0.03), T4 (P=0.01), N2-N3 category (P=0.004) and stage IV (P=0.008) were independent factors associated with poor prognosis for survival.

IV. Discussion In most patients with advanced head and neck cancer, conventional radiotherapy does not result in long- term locoregional control of the tumor, and this failure ultimately proves fatal (Jeremic et al 2000). One of the ways to improve the therapeutic ratio is through modification of dose fractionation. Two types of altered fractionation regimens were predicted to offer therapeutic advantage: hyperfractionated and accelerated fractionation schedules. The main rationale of hyperfractionation is to increase the total doses through the use of multiple smaller dose fractions (1.1-1.2 Gy/ fraction) without increasing the overall treatment time, that allows to increase the probability of tumor control within the tolerance of late – responding normal tissues. The rationale for accelerated fractionation is that reduction in overall treatment time reduces the opportunity for tumor cell regeneration during treatment and therefore increases the probability of tumor control for a similar total dose. This improved tumor control would lead to a therapeutic gain because overall treatment time has little influence on the probability of late normal tissue injury, when the fraction size is not increased and the interval between dose fractions is sufficient for cellular repair to approach completion (Galiana et al, 2002). The combination of radiotherapy and chemotherapy is another promising approach investigated to improve results in advanced head and neck cancer. Induction chemotherapy and concomitant chemoradiotherapy might have complementary effects on overall disease control, with the former leading to a reduction of distant disease and the latter enhancing locoregional control (Haraf et al

680

Cancer Therapy Vol 6, page 681! conformal and intensity- modulated radiotherapy, which have the capability of the high-dose tumor target coverage while minimizing the dose to the volume of the surrounding normal tissues irradiated

and neck cancer recur more often and sooner with conventional regimen than those with hyperfractionated one. The improvement in locoregional control was responsible for better progression- free survival in HFRT arm than those in CFRT arm. These findings are in agreement with the finding of Adelstein and colleagues in 2002; Galiana and colleagues in 2002 and Olmi and colleagues in 2003. The improvement in locoregional control was responsible for improved 2-year overall survival in the HFRT arm than those of CFRT arm, however it did not reach to a statistically significant level. Our findings coincide with many studies (Horiot et al, 1997; Fu et al, 2000; Galiana et al, 2002). In our study, hyperfractionated schedules resulted in increased acute toxicity more than standard fractionation which go ahead with RTOG trial (Fu et al, 2000). The most common sites of grade 3 acute reactions were the mucous membrane and the pharynx, which was in accordance with other studies (Pinto et al, 1991; Antognoni et al, 1996; Horiot et al, 1997; Fu et al, 2000). In this study, grade 2 acute reactions were observed in the subcutaneous tissues and weight loss of patients in the HFRT group compared with those in the CFRT group. However Antognoni et al, 1996 reported greater proportion of mild complication of skin and salivary glands in patients treated with hyperfractionated radiotherapy but found no difference in other normal tissues. In Krstevska and Crvenkova, 2006, grade 2 acute reactions were observed in the skin and the larynx of patients in the hyperfractionated group compared with those in the conventional group. As regard late adverse effects, the most common sites of grade 3 late side effects were observed in the mucous membrane, the pharynx and the salivary gland, which go ahead with many studies (Pinto et al, 1991; Antognoni et al, 1996; Horiot et al, 1997; Fu et al, 2000), who reported that the most common sites of grade 3 late side effects were in pharynx and salivary gland. In addittion Krstevska and Crvenkova, 2006 reported that the most common sites of grade 3 late effects were in the mucous membrane. In our study, we used 90 days from treatment start as a cutoff date for scoring acute and late effects. Any acute adverse effects lasting > 90 days would have been reported as late effects. Some of these late effects were actually prolonged acute effects. In our study, most of the late effects (reported >90 days) resolved with time and there was no significant difference in the frequency of reported late effects at 6-24 months after treatment start among the two treatment groups. These findings coincide with many studies (Horiot et al, 1997; Fu et al, 2000; Olmi etal, 2003; Hehr et al, 2004). At present time, the major limitation of hyperfractionated radiotherapy or combined radiotherapy and chemotherapy for head and neck is increased acute reaction primarily acute mucositis (Kaanders et al, 1992; Brizel, 1998). Several toxicity antagonists are under active investigation (Trotti, 2001). In the future, some of these agents may decrease the acute and late effects of cancer therapy. The therapeutic ratio may also be improved by

V. Conclusion After induction chemotherapy, hyperfractionated radiotherapy seems to be more efficacious than conventional fractionated radiotherapy in locally advanced squamous cell carcinoma of the head and neck, by increasing significantly the progression-free survival and locoregional control. This improvement did not translate into a statistically significant overall survival improvement. However, HFRT was associated with significantly increased acute toxicity, but late toxicity was not significantly increased and it was tolerable and manageable.

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El-Naby et al: Conventional versus hyperfractionated radiotherapy in locally advanced head and neck cancer El-Sayed S, Nelson N (1996) Adjuvant and adjunctive chemotherapy in the management of squamous cell carcinoma of the head and neck region: A meta-analysis of prospective and randomized trials. J Clin Oncol 14, 838847. Fu KK, Pajak TF, Trotti A, Jones CU, Spencer SA, Phillips TL, Garden AS, Ridge JA, Cooper JS, Ang KK (2000) A radiation therapy oncology group(RTOG)phase III randomized study to compare hyperfractionation and two variants of accelerated fractionation to standard fractionation radiotherapy for head and neck squamous cell carcinomas: First report of RTOG 9003. Int J Radiat Oncol Biol Phys 48, 7-16. Galiana R, Boladeras A, Mesía R, Gómez J, de Juan A, Mañós M, Nogués J, Navarro V, Guedea F (2002) Twice-a- day radiotherapy for head and neck cancer: the Catalan Institute of Oncology experience. Radiother Oncol 64, 19-27. Hall EJ (1994) Radiobiology for the radiologist (4th ed.), Lippincott, Philadelphia, PA. Haraf DJ, Rosen FR, Stenson K, Argiris A, Mittal BB, Witt ME, Brockstein BE, List MA, Portugal L, Pelzer H, Weichselbaum RR, Vokes EE (2003) Induction chemotherapy followed by concomitant TFHX chemotherapy with reduced dose radiation in advanced head and neck cancer. Clin Cancer Res 9, 5936. Hehr T, Classen J, Schreck U, Glocker S, Bamberg M, Budach W (2004) Hyperfractionated accelerated radiotherapy alone and with concomitant chemotherapy to the head and neck: treated within and outside of randomized clinical trials. Int J Radiat Oncol Biol Phys 58, 1424-1430. Horiot JC, Le Fur R, N'Guyen T (1992) Hyperfraction versus conventional fraction in oropharyngeal carcinoma: final analysis of a randomized trial of the EORTC cooperative group of radiotherapy. Radiother Oncol 25, 231-241. Horiot JC, Bontemps P, van den Bogaert W, Le Fur R, van den Weijngaert D, Bolla M, Bernier J, Lusinchi A, Stuschke M, Lopez-Torrecilla J, Begg AC, Pierart M, Collette L (1997) Accelerated fractionation (AF)compared to conventional fractionation (CF)improves loco-regional control in the radiotherapy of advanced head and neck cancers: results of the EORTC 22851 randomized trial. Radiother Oncol 44, 111-121. Höckel M, Schlenger K, Mitze M, Schäffer U, Vaupel P (1996) Hypoxia and radiation response in human tumors. Semin Radiat Oncol 6, 3-9. Jeremic B, Shibamoto Y, Milicic B, Nikolic N, Dagovic A, Aleksandrovic J, Vaskovic Z, Tadic L (2000) Hyperfractionated radiotherapy with or without concurrent low- dose daily cisplatin in locally advanced squamous cell carcinoma of the head and neck: A prospective randomizrd trial. J Clin Oncol 18, 1455-1464. Kaanders JH, van Daal WA, Hoogenraad WJ, van der Kogel AJ (1992) Accelerated fractionation radiotherapy for laryngeal cancer, acute, and late toxicity. Int J Radiat Oncol Biol Phys 24, 497-503. Knox RJ, Friedlos F, Lydall DA, Roberts JJ (1986) Mechanism of cytotoxicity of anticancer platinum drugs: Evidence that cis-diamminedichloroplatinum(II)and cis-diammine-(1,1cyclobutanedicarboxylato)platinum(II)differ only in the kinetics of their interaction with DNA. Cancer Res 46, 1972-1979. Krstevska V, Crvenkova S (2006) Altered and conventional fractionated radiotherapy in locoregional control and survival

of patients with squamous cell carcinoma of the larynx, oropharynx, and hypopharynx. Croat Med J 47, 42-52. Lefebvre JL, Chevalier D, Luboinski B, Kirkpatrick A, Collette L, Sahmoud T (1996) Larynx preservation in pyriform sinus cancer: Preliminary results of a European Organization for Research and Treatment of Cancer phase III trial. J natl Cancer Inst 88, 890-898. Lokich J, Bothe A, Fine N, Perri J (1981) Phase I study of protracted venous infusion of 5- fluorouracil. Cancer 48, 2565-2568. Olmi P, Crispino S, Fallai C, Torri V, Rossi F, Bolner A, Amichetti M, Signor M, Taino R, Squadrelli M, Colombo A, Ardizzoia A, Ponticelli P, Franchin G, Minatel E, Gobitti C, Atzeni G, Gava A, Flann M, Marsoni S (2003) Locoregionally advanced carcinoma of the oropharynx: Concentional radiotherapy vs acceleratad hyperfractionated radiotherapy vs concomitant radiotherapy and chemotherapyA multicenter randomized trial. Int J Radiat Oncol Biol Phys 55, 78-92. Orecchia R, Jereczek-Fossa BA, Catalano G, Chiesa F, De Pas T, Masci G, Krengli M, Vavassori A, De Paoli F, Robertson C, Marrocco E, De Braud F (2002) Phase II trial of vinorelbine, cisplatin and continuous infusion of 5- fluorouracil followed by hyperfractionated radiotherapy in locally advanced head and neck cancer. Oncology 63, 115-123. Paccagnella A, Orlando A, Marchiori C, et al (1994) Phase III trial of initial chemotherapy in stage III or IV head and neck cancer: A study by the Gruppo di Studio sui Tumoti della Testa e del Collo. J Natl Cancer Inst 86, 265-272. Perez CA, Brady LW (1992) Overview. In: Perez CA, Brady LW, editors. Principles and practice of radiation oncology,2nd ed. Philadelphia: Lippincott-Raven; pp. 1-63. Perez CA, Brady LW, Roti JL (2004) Overview. In: Perez CA, Brady LW, editors. Principles and practice of radiation oncology,4th ed. Philadelphia: Lippincott-Raven; 33, 63. Pignon JP, Bourhis J, Domenge C, Designé L (2000) MACH-NC Collaborative Group: Chemotherapy added to locoregional treatment for head and neck squamous- cell carcinoma: Three meta-analysis of up-dated individual data. Lancet 355, 949-955. Pinto LH, Canary PC, Araújo CM, Bacelar SC, Souhami L Prospective randomized trial comparing (1991) hyperfractionated versus conventional radiotherapy in stages III and IV oropharyngeal cacinoma. Int J Radiat Oncol Biol Phys 21, 557-562. Posner MR, Colevas AD, Tishler RB (2000) The role of induction chemotherapy in the curative treatment of squamous cell cancer of the head and neck. Semin Oncol 27(suppl8), 13-24. Stuschike M,Thames HD (1997) Hyperfractionated radiotherapy of human tumors: Overview of the randomized clinical trials. Int J Radiat Oncol Biol Phys 37, 259-267. Trotti A (2001) Toxicity antagonists in cancer therapy. Curr Oncol 9, 569-578. Withers HR, Taylor JM, Maciejewski B (1988) The hazard of accelerated tumor clonogen repopulation during radiotherapy. Acta Oncol 27, 131-145. Zamboglou N, Pape H, Schnabel T, Wurm R, Bannach B, Fürst G, Schmitt G (1989) Combined radiotherapy with cis-or carboplatin in advanced head and neck tumors. Strahlenther Onkol 165, 647-651.

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Cancer Therapy Vol 6, page 683 ! Cancer Therapy Vol 6, 683-686, 2008

Efficacy of cancer treatment in Armenia: where is it going? Research Article

Armen K. Nersesyan Institute of Cancer Research, Medical University of Vienna, Vienna A-1090, Austria

__________________________________________________________________________________ *Correspondence: Armen K. Nersesyan (PhD, DSc), Environmental Toxicology Group, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8A, A-1090, Vienna, Austria; Tel: +431 4277 65146; Fax: +431 4277 9651; E-mail: armen.nersesyan@meduniwien.ac.at, armenn@freenet.am Key words: cancer treatment, cancer mortality Abbreviations: coefficient of efficacy of cancer treatment (CECT) Received: 15 May 2008; Revised: 28 July 2008 Accepted: 5 September 2008; electronically published: September 2008

Summary The efficacy of cancer treatment in Armenia was evaluated based on the data of cancer incidence and mortality from 1987 till 2004 and this parameter was compared with the data of some countries (both developing and developed). It has been shown that, unlike in some randomly chosen countries, mentioned parameter substantially decreased in Armenia during 17 years although it was relatively high in 1987. The reason(s) of this substantial decline is(are) unknown. Further more detailed investigations in this area are certainly warranted.

II. Materials and methods

I. Introduction

Because of absence of relevant data from cancer registry, a lot of sources were used to analyse cancer mortality. The sources include published scientific articles and also some websites which contain relevant information concerning Armenia (WHO, CIA of the USA). The data concerning the incidence of cancer and mortality were obtained from the thesis of Bazikyan (2006) which is based ob the official data of Ministry of Health of Armenia. The approach to evaluate the efficacy of cancer treatment based on the ratio mortality/incidence, or incidence/mortality was applied earlier (Galstyan et al. 2003), and this approach can give a possibility to estimate the efficacy of cancer treatment in a certain year very approximately. {The coefficient of efficacy (CECT) is equal to [1 – (number of died from cancer subjects/number of registered cancer patients) x 100%] or {[1 – (ratio mortality : incidence)] x 100%}}. Simply to say, the ratio mortality : incidence (x 100%) means the percentage of subjects diseased because of cancer, and one minus this ratio could show the approximate effectiveness of the treatment (based on assumption that all the patients were treated, and it is the case because almost all cancer patients in Armenia are treated). In these calculations both sexes and all localizations of cancer are considered. It is a little more precise in developing counties because in all of them (including Armenia) the incidence of cancer increases. As for the developed countries, an increased CECT can be due to both improved treatment and decline of cancer incidence.

Cancer is a major public health problem in any country of the world. It is noteworthy that in many developed European countries (Levi et al, 2007) and the USA (Jemal et al, 2008) cancer mortality declines which is mostly due to improved treatment of cancer and to a lesser extent to decline of cancer incidence of some localizations. This is very pronounced for the USA where the incidences of cancer deaths have decreased by 18.4 per cent among men and 10.5 per cent among women since the early nineties. In the EU the decline was 11.1% in males and 9.8% in females. Currently many countries publish annual statistics concerning cancer incidence and mortality. At present Armenia has no national cancer registry, and hence, such data are not available, except of the total number of registered cancer patients and deaths because of cancer. Based on very scant data, the incidences of cancer of some localizations in Armenian population, including male and female breast cancers and childhood leukosis were estimated (Barsegyan and Nersesyan, 1999; Galstyan et al, 2002 and 2003; Nersesyan et al, 2003a,b). It would be of interest to know if there are changes in cancer incidence and mortality during the last 17 years (1987-2004) in Armenia.

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Nersesyan: Efficacy of cancer treatment in Armenia: where is it going? ! years in Armenia because the mortality increased much more faster that the incidence of cancer. To test if mentioned calculations are true, the CECT in randomly chosen countries of Europe and also in the USA was calculated in the same way (Black et al, 1997; Wingo et al, 1998; Jemal et al, 2008; American cancer Society; Table 1). It can be noted that the CECT increases during 16 years in mentioned countries (from 2.0% in Estonia to 18.1% in Greece) indicating improved cancer treatment and to lesser extent, the decrease of cancer incidence (if any). Only Armenia was an exception where substantial decline of the CECT was observed. It is noteworthy than in 1987-1990 the CECT in Armenia was comparable with this parameter in Austria, Denmark, France, Germany and Estonia, and even was higher than in Greece, Latvia and Lithuania. But, unlike these countries, no improvement of cancer treatment was observed along with increased cancer incidence. The CECT in 2004-2006 in seven Republics of former the USSR was compared with the CECT of Armenia (Table 2). In all these countries the CECT was higher than in Armenia (from 7.8 in Latvia to 18.6 in Lithuania) which means better treatment of cancer in these countries. The data presented here are supported by the paper by Davydov and Aksel’ (2007). They analyzed officially declared data and also found that cancer mortality in Armenia grew significantly during the last years (by 37.5% and 24.7% for males and females, respectively). This shows one more time the correctness of the calculations presented in this paper. Cancer investigations in Armenia are of particular interest because this Republic is now almost mono-ethnic country "#$%!&'!(&()*+,-&.!+/0!1/20.-+.34!55567-+68&9:6! ;,! -3! 50**! <.&5.! ,=+,! ,=0/0! +/0! /+7-+*! +.> ethnic peculiarities in cancer incidence (Jemal et al, 2008). Another important peculiarity of current Armenia is that the number of medical doctors per 1000 inhabitants is one of the highest in the world (3 per 1000 inhabitants) and it is higher than in many developing and even in some developed counties (Denmark, for example-2.93 per 1000 inhabitants, according the WHO data).

III. Results and discussion In 1987 in Armenia died because of cancer (both sexes, all localizations) 3008 subjects (Barsegyan and Nersesyan, 1999 The numbers of deaths increased every year, and 4395 subjects died in 2004 (Bazikyan, 2006). At first glance it should be like this because Armenia is developing country, and in all developing countries the incidence of cancer increases at present. But the problem is that in Armenia were registered 3.166 million inhabitants in 1987, and in 2001, according to the common census only 3.002 million inhabitants. Earlier the data concerning population of Armenia were analysed and substantial discrepancies between officially declared and real data were found (Barsegyan and Nersesyan, 1999). According to the data of the CIA of the USA (https://www.cia.gov/), population of Armenia to July of 2008 is estimated as less than 3.0 million which is more realistic. This is supported by other independent source (http://worldfacts.us/Armenia.htm). Crude rate of cancer deaths in Armenia in 1987 was 95.1, and it was in 2004 either 140.7 per 100,000 subjects (the numbers were calculated based on the official data) or 154.3 (based on the of the CIA data). In both cases it is obvious that the cancer mortality increased substantially during 17 years (1.48-1.62-fold). Calculation of the CECT based on the data presented by Bazikian in 2006, shows that mentioned parameter was 41.2% in 1987 (when Armenia was a part of the USSR). After the independence of Armenia the CECT began to decline, and it was 33.1% in 1992, 28.5% in 1996, 33.5% in 2000, and 28.8% in 2004. Hence, the CECT declined from 41.2% in 1987 to 28.8% in 2004 (1.43-fold!). This is, of course, the indication of sufficiently decreased the CECT. It is interesting that mentioned number (1.43) is very close to the numbers which were obtained from the calculation of proportions of crude rates of mortality 1.481.62. The numbers of cancer deaths increased 1.46-fold from 1987 to 2004 (3008 and 4395 persons, respectively). It is noteworthy that the number of persons diagnosed with cancer increased only 1.21-fold (5119 and 6174, respectively). Hence, this circumstance really shows that the efficacy of cancer treatment declines during the last

Table 1. Coefficient of efficacy of cancer treatment in various countries Country Armenia Austria Denmark France Germany Greece Estonia Latvia Lithuania USA

Year 1990 41.2* 46.2 39.4 45.3 43.8 33.4 38.1 31.0 31.2 49.6*

All cancers, both sexes; data of * 1987; ** 2004; *** 2008

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Difference 2006 28.8** 51.4 52.5 56.8 55.9 51.5 40.1 36.6 47.4 60.6***

-12.4 5.2 13.1 11.5 12.1 18.1 2.0 5.6 16.2 11.0

Cancer Therapy Vol 6, page 685 ! Table 2. Efficacy of cancer treatment in some former Republics of the USSR in 2006. Country

Treatment efficacy

Armenia Belarus Estonia Latvia Lithuania Moldova Russia Ukraine

28.8* 39.6 40.1 36.6 47.4 38.9 37.4 38.6

Difference of treatment efficacy with Armenia 10.8 11.3 7.8 18.6 10.7 8.6 9.8

* data of 2004 Black RJ, Bray F, Ferlay J, Parkin DM (1997) Cancer incidence and mortality in the European Union: cancer registry data and estimates of national incidence for 1990. Eur J Cancer 33, 1075-107. Cancer Facts and Figures. American Cancer Society, 2001. Davydov MI, Aksel' EM (2007) The incidence of malignant tumors and mortality caused by them in Commonwealth of Independent States in 2005. Gerald Russian Akad Med Sci 11, 45-9. Galstyan AM, Khachatouryan IA, Nersesyan AK (2003) Breast cancer in males in Armenia (1980-2002). Archive of Oncology 11, 31-33. Galstyan AM, Ovanesbekova TG, Nersesyan AK (2002) Female breast cancer in Armenia (1980-2000). Archive of Oncology 11, 281-282. http://worldfacts.us/Armenia.htm http://www.armstat.am/en/ http://www.euro.who.int/document/obs/armsum2002r.pdf https://www.cia.gov/ http://nv.am/kurer.htm Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer statistics. CA Cancer J Clin 58, 71-96. Karim-Kos HE, de Vries E, Soerjomataram I, Lemmens V, Siesling S, Coebergh JW (2008) Recent trends of cancer in Europe: A combined approach of incidence, survival and mortality for 17 cancer sites since the 1990s. Eur J Cancer [Epub ahead of print]. Levi F, Lucchini F, Negri E, La Vecchia C (2007) Continuing declines in cancer mortality in the European Union. Ann Oncol 18, 593-5. Nersesyan AK, Daghbashyan SS, Danielyan SH, Arutyunyan RM (2003a) The incidence of childhood leukemia in Armenia (1991-2002). Archive of Oncology 11, 277-279. Nersesyan AK, Daghbashyan SS, Danielyan SH, Arutyunyan RM (2003b) The incidence of various types of childhood leukemia in Armenia (1991-2002). J BUON 8, 257-9. Wingo PA, Ries LA, Rosenberg HM, Miller DS, Edwards BK (1998) Cancer incidence and mortality, 1973-1995: a report card for the U.S. Cancer 82, 1197-207.

Also, the number of medical doctors-oncologists possessing PhD degree and even Doctor of science (the highest scientific degree in Armenia and all post-soviet countries which is almost the equivalent of habilitated PhD in some western countries) in Armenia is one of highest in the world (http://nv.am/kurer.htm). Taking these circumstances into consideration, such high difference in the CECT of ex-socialist countries and Armenia (to say nothing about the comparison with developed countries) is completely unclear. The reason of this strange phenomenon should be discussed carefully by the Union of Oncologists of Armenia and the Ministry of Health of this Republic. Bazikyan in his thesis (2006) stressed that optimization of anticancer struggle should include the creation of National Cancer Registry and some screening programs which can improve the diagnostics of cancer. It looks reasonable but along with these programs the causes of poor cancer treatment in Armenia must be found and then eliminated. The real goal of “optimization� of the anticancer struggle should be improvement of cancer treatment, to reach at least the efficacy registered almost twenty years ago (1987).

Acknowledgements The author would like to thank Dr. S. Bonassi (NCRI, Genoa, Italy) for some important suggestions.

References Barsegyan VS, Nersesyan AK (2000) Cancer incidence in Armenia (1970-1998). Archive of Oncology 8, 187. Bazikyan GK (2006) Epidemiology of cancer and some ways of optimization of cancer struggle in Armenia. DSc thesis. National Oncology Center, Yerevan, Armenia.

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Cancer Therapy Vol 6, page 687! Cancer Therapy Vol 6, 687-698, 2008

Paraneoplastic syndromes in lung cancer Review Article

Stefanie Heinemann, Peter Zabel, Hans-Peter Hauber* Medical Clinic, Research Center Borstel, Borstel, Germany

__________________________________________________________________________________! *Correspondence: Hans-Peter Hauber, MD, Medical Clinic, Research Center Borstel, Parkallee 35, 23845 Borstel, Germany; Phone: +49 (0)4537 188 364; Fax: +49 (0)4537 188 313; E-mail: hphauber@fz-borstel.de Key words: Anemia, antidiuretic hormone, autoantibody, cachexia, cancer, chemotherapy, clubbing, Cushing, dermatomyositis, hypertrophic pulmonary osteoarthropathy, hypercalcaemia, Lambert-Eaton, lung, neurologic, paraneoplastic, syndrome Abbreviations: Adrenocorticotrophic hormone, (ACTH); antidiuretic hormone, (ADH); atrial natriuretic peptide, (ANP); cancer procoagulant, (CP); ciliary neutropic factor, (CNTF); cyclic adenosine monophosphate, (cAMP); granulocyte colony stimulating factor, (G-CSF); Granulocyte macrophage- colony stimulating factor, (GM-CSF); hypertrophic pulmonary osteoarthropathy, (HPO); hypothalamic-pituitary-adrenal, (HPA); interferon, (IFN); interleukin, (IL); Lambert-Eaton myasthenic syndrome, (LEMS); lipid mobilizing factor, (LMF); low molecular weight heparin, (LMWH); non-small-cell lung cancer, (NSCLC); Palmo-plantar hyperkeratosis, (PPH); parathyroid hormone, (PTH); parathyroid hormone-related protein, (PTHrP); polyneuropathy, (PNP); proopiomelanocortin, (POMC); prostaglandins of the E series, (PGE); proteolysis-inducing factor, (PIF); small-cell lung cancer, (SCLC); syndrome of inappropriate antidiuretic hormone secretion, (SIADH); tissue factor, (TF); transforming growth factor alpha, (TGF)! ; tumor necrosis factor alpha, (TNF)! ; unfractionated heparin, (UFH); vascular endothelial growth factor, (VEGF); venous thromboembolism, (VTE); voltage-gated P/Q calcium channels, (VGCC)

Received: 21 August 2008; Revised: 11 September 2008 Accepted: 12 September 2008; electronically published: October 2008

Summary Paraneoplastic syndromes are frequently found in lung cancer. They can be the first manifestation of disease or recurrence. Neuromuscular, vascular, haematological and metabolic syndromes, as well as syndromes involving the connective and skeletal tissues and skin can be distinguished. While some of the paraneoplastic syndromes are commonly observed (eg. Anemia, cachexia) others are rare disorders (eg. Opsoclonus-myoclonus, tylosis). This review focuses on the epidemiology, pathogenesis, clinical symptoms and treatement options of frequent and clinical important paraneoplastic syndromes.

syndromes is broadened to include conditions such as anemia, cachexia, and hypercalcemia then the incidence and prevalence of paraneoplastic syndromes is much higher. Lung cancer and small-cell lung cancer (SCLC) in particular is the most common cancer to be associated with paraneoplastic syndromes. However, some paraneoplastic syndromes are more often found in non-small-cell lung cancer (NSCLC). For example hypertrophic pulmonary osteoarthropathy has most often been described in association NSCLC. The extent of paraneoplastic syndromes is unrelated to the size of the primary tumour. In some cases it may precede diagnosis of malignancy while in other cases it may occur late in the course of disease or may show up as the first symptom of recurrence. If lung cancer patients have paraneoplastic syndromes normally they suffer from only one syndrome. Literature data on multiple paraneoplastic syndromes in the same patient are sparse and are mostly described in case reports (Monsieur et al, 1995).

I. Introduction Paraneoplastic syndromes are common in lung cancer, and may be the first manifestation of the disease or its recurrence (Bunn and Ridgway, 1993). Paraneoplastic phenomena are not related to the direct invasion, obstruction, or metastasis (Patel et al, 1993; Spiro, 1995). Neuromuscular, vascular, haematological and metabolic syndromes, as well as syndromes involving the connective and skeletal tissues and skin can be distinguished. Table 1 gives an overview of the paraneoplastic syndromes that have been described in lung cancer patients. In the following epidemiology, pathogenesis, clinical findings, and treatment options of the most common paraneoplastic syndromes in lung cancer will be reviewed.

II. Epidemiology Paraneoplastic syndromes are estimated to occur in 7% to 15% of all patients with cancer (Richardson and Johnson, 1992). If the definition of paraneoplastic 687

Heinemann et al: Paraneoplastic syndromes in lung cancer Table 1. Paraneoplastic syndromes in lung cancer.

III. Hypercalcaemia Hypercalcaemia is frequently found in patients with lung cancer. Its incidence ranges from 2 to 6% at presentation to 8 to 12% throughout the course of disease (Spiro et al, 2007). It may arise from bone metastasis but can also be induced in a paraneoplastic manner by secretion of parathyroid hormone-related protein (PTHrP), calcitriol or other cytokines, including osteoclast activating factors. Hiraki and colleagues examined 1149 patients with lung cancer and found 6% to have hypercalcemia (Hiraki et al, 2004). Among those with hypercalcemia 51% had squamous cell carcinoma, 22% had adenocarcinoma, and 15% had SCLC. Most of those patients had advanced disease (stage III or IV). Median survival was only 3.8 months (Hiraki et al, 2004). Hypercalcaemia in lung cancer patients can be caused by either bony metastases or less commonly by paraneoplastic syndromes. Tumours can secrete PTHrP, calcitriol or other cytokines including osteoclast activating factors. Moseley and colleagues identified PTHrP expression in lung cancer cells (Moseley et al, 1987). PTHrP shares 70% sequence homology with PTH over the first 13 amino acids at the N-terminus. Both parathyroid hormone (PTH) and PTHrP bind to a common PTH/PTHrP receptor (Abou-Samra et al, 1992) and share similar biological activities (Horiuchi et al, 1987). However, expression of PTHrP has also been reported in normal tissue (Danks et al, 1989; Asa et al, 1990). This implicates that PTHrP has also a physiological effect. It is now clear that PTHrP has several other functions in addition to its PTH-like effects (Clines and Guise, 2005). PTH stimulates osteoclastic bone resorption and calcium reabsorption and inhibition of phosphate reabsorption from renal tubules. It also stimulates renal 1! -hydroxylase resulting in production of 1,25-(OH)2D3, which increases intestinal absorption of calcium and phosphate. These actions result in increased serum calcium. PTH actions are mediated through binding of the amino terminus of the PTH molecule to the PTH receptor, a member of the family of G protein-coupled receptors that contain seven transmembrane- spanning domains (Juppner et al, 1991). The ligand-bound PTH receptor activates adenylate cyclase, through the activation of G protein G! s, producing cyclic adenosine monophosphate (cAMP) while activating protein kinase A. In addition, the phospholipase C/protein kinase C system also contributes to PTH signal transduction (Mahon et al, 2002; Swarthout et al, 2002). Approximately 80% of cancer patients with hypercalcemia have detectable or increased plasma concentrations of PTHrP (Burtis et al, 1990). PTHrP has a multifunctional role in cancer. It mediates hypercalcaemia but also aids development and progression of osteolytic bone metastasis, regulates growth of cancer cells and acts as a cell survival factor (Luparello et al, 1993; Luparello et al, 1995; Li et al, 1996; Chen et al, 2002). Although PTHrP is responsible for he majority of cases of paraneoplastic hypercalcaemia there have been rare cases with ectopic PTH production in the tumour. This has been reported in SCLC as well as in squamous

Endocrine syndromes SIADH Nonmetastatic hypercalcemia Cushing syndrome Gynecomastia Hypercalcitonemia Elevated levels of LSH and FSH Hypoglycemia Hyperthyroidism Carcinoid syndrome Neurologic syndromes Subacute sensory neuropathy Mononeuritis multiplex Lambert-Eaton myasthenic syndrome Encephalomyelitis Necrotizing myelopathy Cancer associated retinopathy Opsoclonus-myoclonus Skeletal syndromes Hypertrophic osteoarthropathy Clubbing Renal syndromes Glomerulonephritis Nephrotic syndrome Metabolic syndromes Lactic acidosis Hypouricemia Systemic syndromes Cachexia Fatigue Fever Collagen-vascular syndromes Dermatomyositis and polymyositis Vasculitis Systemic lupus erythematosus Cutaneous syndromes Acquired hypertrichosis languinosa !"#$%&'()*#"($+')"&,&-. Erythema multiforme Tylosis Erythroderma Exfoliative dermatitis /0(-$%1.2.)-2*"20(-. Sweet syndrome Pruritus and urticaria Hematologic syndromes Anemia Leucocytosis and eosinophilia Thrombocytosis and thrombocytopenic purpura Coagulopathies Thrombophlebitis Thromboembolism Disseminated intravascular coagulation Reproduced from Scagliotti, 2001 and Carbone et al, 1970 with kind permission from European Respiratory Monthly and Annals of Internal Medicine respectively.! .

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Cancer Therapy Vol 6, page 689! have been implicated as possibly contributing to the hyponatremia found in lung cancer patients. However, only elevated plasma ADH levels were consistently found in patients with lung cancer and may explain the impaired ability to excrete the water load (Vorherr, 1974). Physiologically ADH is released from the posterior pituitary gland whereas in paraneoplastic SIADH cancer cells secrete ADH (Moses and Scheinman, 1991). Another possible mechanism is inappropriate peripheral baroreceptor stimulation of ADH release from the hypothalamus (Vorherr, 1974). ADH causes hyponatremia and hypoosmolality that interfere with urinary dilution, thereby preventing the excretion of ingested water. An interesting observation in the SIADH is the development of partial escape from ADH which tends to protect against progressive water retention (Jaenike and Waterhouse, 1961). Although ADH secretion or effect is inappropriately increased in the SIADH, up to one-third of patients have a downward resetting of the osmostat in which the plasma sodium concentration is normally regulated (and therefore stable) at a new lower level, typically between 125 and 135 mmol/L. Establishing the presence of this condition is clinically important because correcting the hyponatremia is both unnecessary and likely to be ineffective, since raising the plasma osmolality will stimulate both ADH release and thirst. Hyponatremia, plasma hypoosmolality and urine hyperosmolality with continuing sodium excretion are biochemical findings in SIADH. Water retention is typically only 2 to3 liters and does not lead to edema or anasarka. The severity of symptoms in SIADH is related to the degree of hyponatremia and the rapidity of fall in serum sodium. Anorexia, nausea, and vomiting are common symptoms. A rapid onset of hyponatremia can cause cerebral edema. This may lead to irritability, restlessness, personality changes, confusion, coma, seizures, and respiratory arrest. In patients with SCLC, SIADH resolves in up to 80% after administration of chemotherapy (List et al, 1986). Adjuvant management of SIADH includes fluid restriction to 800-1,000 ml/d to increase serum sodium. In patients with severe symptoms (severe confusion, convulsions, or coma) intravenous hypertonic saline (5%) solution (eg. 200-300 ml in 3-4 h) should be given.

cell carcinoma of the lung (Yoshimoto et al, 1989; Nielsen et al, 1996). While in haematological malignancies extra-renal production of 1,25-(OH)2D3 appears to be a major mediator of hypercalcaemia (Seymour et al, 1994) in lung cancer calcitriol does not seem to be the major mediator of PTH-like activity even if SCLC cell lines can synthesize 1,25-(OH)2D3 (Mawer et al, 1994). Other factors that can stimulate osteocalstic bone resorption and cause hypercalcaemia include interleukin (IL)-1, IL-6, transforming growth factor alpha (TGF)! , tumor necrosis factor alpha (TNF)! , and granulocyte colony stimulating factor (G-CSF) (Clines and Guise, 2005). Human TGF! and TNF! stimulate osteoclastic bone resorption !"# $!%&' and result in hypercalcaemia !"# $!$' (Bertolini et al, 1986; Yates et al, 1992; Ibbotson et al, 1995). Factors such as TGF! , IL-1, IL-6 and TNF! can also enhance the hypercalcaemic effects of PTHrP. Although prostaglandins of the E series (PGE) are powerful stimulators of bone resorption (Klein and Raisz, 1970) there role in tumour-associated bone destruction remains unclear (Mundy, 1995). They may be mediators of cytokine effects on the bone. Moreover, PGE expression has been described in the lung cancer tissue of normocalcaemic patients (Kukrja et al, 1982). This finding implicates that PGE may not be necessary to induce hypercalcaemia in cancer patients. Symptoms of hypercalcaemia include anorexia, nausea, vomiting, constipation, lethargy, polyuria, polydipsia, and dehydration. If untreated hypercalcaemia my lead eventually to confusion and coma. Renal failure and nephrocalcinosis are late manifestations, too. Symptomatic patients with a serum calcium of " 3 mmol/L required treatment that includes hydration and biphosphonate (Thomas et al, 2004).

IV. Syndrome of inappropriate antidiuretic hormone secretion Elevated levels of antidiuretic hormone (ADH) and impaired water handling can be observed in 30 to 70% of lung cancer patients (Patel et al, 1993). However, excess production of ADH does not always produce symptoms (Maurer et al, 1983; Bliss et al, 1990; Moses and Scheinman, 1991). Only 1 to 5% of all patients with lung cancer have symptoms attributable to the syndrome of inappropriate antidiuretic hormone secretion (SIADH). SIDAH is frequently caused by SCLC. In a study by List and co-workers approximately 10% of patients with SCLC had SIADH (List et al, 1986). In that study development of SIADH did not correlate with clinical stage or metastatic sites. SIADH occurred most often with initial presentation and promptly resolved with initiation of combined chemotherapy in 80% of the patients. Response to chemotherapy and survival was not influenced by the presence of SIADH (List et al, 1986). Recurrence of SIADH was associated with tumour progression. Biochemically, the SIADH production is defined as low serum sodium and a dilute plasma osmolality along with a higher, or "inappropriate," urine osmolality, in the presence of continued urinary sodium excretion. A variety of hormones including atrial natriuretic peptide (ANP)

V. Cushing syndrome Adrenocorticotrophic hormone (ACTH) is the most commonly produced hormone in lung cancer patients. Increased levels of ACTH may be detectable in up to 50% of patients with lung cancer (Mendelsohn and Baylin, 1984). ACTH secretion is almost always associated with SCLC (Hansen, 1990). About 30% of all cases of SCLC are associated with hypersecretion of ACTH. However, a clinical apparent Cushing syndrome is rare (Mennecier et al, 1999). Cushing syndrome has been described in 1 to 5% of patients with SCLC (Odell et al, 1979; Ilias et al, 2005) but this may overestimate the real rate. In 2005 Hansen and Bork reported only 3 out of 90 cases of Cushing syndrome to be attributable to SCLC (Hansen and Bork, 1985). Most commonly, Cushing syndrome occurred in patients with pulmonary carcinoid (35 of 90 689

Heinemann et al: Paraneoplastic syndromes in lung cancer chemotherapy in patients with SCLC and Cushing syndrome is only moderate (Shepherd et al, 1992). Severe symptoms should be treated symptomatically.

patients). While ectopic ACTH syndrome typically presents as Cushing syndrome in patients with SCLC bronchial carcinoid tumors are the most common occult sources of ACTH (Terzolo et al, 2001). SCLC associated with the ectopic ACTH syndrome is more resistant to chemotherapy and the severe hypercortisolism is responsible for a high rate of lifethreatening complications during treatment which worsens prognosis (Terzolo et al, 2001). Shepherd and collegues retrospectively analyzed the charts of 545 patients with SCLC. They identified 23 patiens (4.5%) with Cushing syndrome and ectopic ACTH production. These patients had a response rate to chemotherapy of only 46%, and their median survival was only 3.57 months (Shepherd et al, 1992). In pituitary cells ACTH is derived by cleavage from the precursor, pro-opiomelanocortin (POMC). It is unlikely that processing of POMC is as efficient in nonpituitary cells. ACTH precursors and ACTH-related peptides can be secreted by POMC expressing cells. ACTH precursors can be detected in the serum of patients with ectopic ACTH syndrome (Oliver et al, 2003). Stewart and co-workers reported grossly elevated levels of ACTH precursors in patients with ectopic ACTH syndrome and increased concentrations of ACTH precursors in patients with SCLC without evidence of ectopic ACTH syndrome (Stewart et al, 1994). SCLC cells have been shown to express POMC and secrete ACTH precursors (Stewart et al, 1989; White et al, 1989). POMC expression has also been observed in bronchial carcinoid tumour cells (Crosby et al, 1990). NSCLC is rarely associated with ectopic ACTH syndrome. In a case report of a patient with poorly differentiated squamous cell lung carcinoma and ectopic ACTH syndrome ACTH secretion of cancer cells was confirmed by immunohistochemical staining (Noorlander et al, 2006). Another case reported described paraneoplastic Cushing syndrome in a patient with adenocarinoma of the lung (Yoh et al, 2003). ACTH and ACTH precursors stimulate the adrenal glands to secrete glucocorticosteroids. The symptoms and signs of paraneoplastic Cushing syndrome result directly from chronic exposure to excess glucocorticoid because of ectopic ACTH-production. There is a large spectrum of manifestations from subclinical to overt syndrome. Diagnosis is often difficult because there is no pathognomonic symptom. An important clinical hint to the presence of Cushing syndrome is the simultaneous development and increasing severity of several of the following symptoms: Centripetal obesity, facial plethora, glucose intolerance, weakness, proximal myopathy, hypertension, psychological changes, easy bruisability, hirsutism, oligomenorrhea or amenorrhea, impotence, acne, oily skin, abdominal striae, ankle edema, osteoporosis, polydipsia, polyuria, hyperpigmentation, headache, fungal infection specially oral thrush and hypokalimea. To treat the paraneoplastic Cushing syndrome, treatment of the underlying disease is essential. In most cases treatment of the tumour will also improve the paraneoplastic syndrom. However, responses to

VI. Hematological abnormalities Hematologic abnormalities including anemia, leukocytosis, thrombocytosis, and eosinophilia are frequently observed in lung cancer patients. Anemia is common in lung cancer patients. In one series 38% of untreated patients had a haemoglobin ! 12 g/dL. In contrast 80% of patients that were treated with chemotherapy were anaemic at one time (Kosmidis and Krzakowski, 2005). Leukocytosis is often found in patients with lung cancer either at time of diagnosis or during the course of the disease (Thomson et al, 1986). In one study leukocytosis had been described in 15% of all patients with lung cancer (n = 227). Nearly all patients had NSCLC. Leukocytosis was thought to be due to overproduction of granulocyte-colony stimulating factor (Kasuga et al, 2001). Tumor-related leukocytosis was associated with a poorer prognosis compared to patients without leukocytosis (median survival: 4.6 months vs 20.8 months) (Kasuga et al, 2001). Leukocytosis has also been associated with hypercalcaemia (Kasuga et al, 2001; Hiraki et al, 2004). Thrombocytosis is observed in 16% to 32% of all lung cancer patients (Moller Pedersen and Milman, 1996; Aoe et al, 2004). It has been identified as an independent predictor of shorten survival (Moller Pedersen and Milman, 1996; Aoe et al, 2004). Eosinophilia in tissue or blood is rare. While tumourassociated tissue eosinophilia appear to have a better prognosis tumour-associated blood eosinophilia seem to be associated with a worse prognosis (Lowe, et al 1981). The cause of anemia associated with cancer is multifactorial. Bleeding, hemolysis, bone marrow infiltration, and nutritional deficiencies may all contribute to the development of anemia in patients with cancer. In addition, inflammatory cytokines, such as TNF-" , IL-1, IL-6, and IFN- , inhibit erythropoiesis, which leads to decreased production of erythrocytes, resulting in anemia. It has to be taken into account that anemia is a common complication of myelosuppressive chemotherapy (Groopman and Itri, 1999). On average, over one third of patients become anemic after three cycles of chemotherapy (Glaspy et al, 2002). Lung cancer has been shown to produce G-CSF which leads to leukocytosis (Asano et al, 1977). Granulocyte macrophage- colony stimulating factor (GMCSF) and interleukin-6 production of lung carcinomas has also been reported to be associated with leukocytosis (Sawyers et al, 1992; Matsuguchi et al, 1991). Anemia should be treated to improve quality of life. Erythropoiesis-stimulating agents such as epoetin and darbepoetin should be used with caution in cancer patients with anemia who are not receiving chemotherapy (Rizzo et al, 2008).

VII. Hypercoagulable disorders A variety of hypercoagulable disorders including Trousseau´s syndrome (migratory superficial thrombophlebitis), deep venous thrombosis and 690

Cancer Therapy Vol 6, page 691! The exact pathogenesis of digital clubbing and HPO is not known. In digital clubbing proliferation of connective tissue beneath the nail matrix is observed. Histochemical features of HPO include vascular hyperplasia, edema, and excessive fibroblast and osteoblast proliferation (Myers and Farquhar, 2001). In the past, neurogenic, hormonal, and vascular mechanisms have been discussed (Shneerson, 1981). More recently, the overexpression of vascular endothelial growth factor (VEGF) has been implicated as contributing to the pathogenesis of clubbing and HPO. Olan and collegues reported the case of a young woman with lung cancer and HPO. Serum levels of VEGF were elevated. After resection of the cancer VEGF levels fell and HPO remitted. Histochemical studies of the resected tumor showed increased VEGF messenger RNA expression, suggesting ectopic production by the lung cancer cells (Olan et al, 2004). Dermatomyositis is characterized by infarcts, perifascicular atrophy, endothelial cell swelling and necrosis, vessel wall membrane attack complex deposition, and myocyte-specific MHC I upregulation in the muscle. Histopathological findings in the skin include hyperkeratosis, epidermal basal cell vacuolar degeneration and apoptosis, increased dermal mucin deposition, and a cell-poor interface dermatitis. The precise link between malignancy and inflammatory myopathy remains incompletely understood (Casciola-Rosen et al, 2005). Although dermatomyositis has been classically considered as humorally mediated disease newer evidence suggest that cell-mediated mechanisms and innate immune system dysfunction play a more important role in the pathogenesis (Krathen et al, 2008). Myositis-specific autoantigens are expressed at high levels in regenerating cell in myositic muscles and in several cancer cells (Levine, 2006). This may provide a link between cancer and paraneoplastic myositis syndromes. Digital clubbing is an enlargement of the terminal segments of the fingers and/or toes due to proliferation of connective tissue beneath the nail matrix. HPO is a systemic disorder, which involves both a painful symmetrical arthropathy, usually of the ankles, wrists, and knees, and periosteal new bone formation in the distal long bones of the limbs. Myositis is characterized by muscle weakness and muscle pain. Typically proximal muscles are involved. Dermatomyositis also shows characteristic cutaneous findings of heliotrope eruption, Gottron's papules and a photodistributed eruption with poikiloderma. Raynaud phenomenon, interstitial lung disease and inflammatory arthritis can also be found. Symptoms of HPO may resolve after tumour resection. If a patient is not operable the usual treatment includes nonsteroidal anti-inflammatory agents or a bisphophonate (Amital et al, 2004). The mainstay of therapy for dermatomyositis is corticosteroids (Iorizzo and Jorizzo, 2008).

thromboembolism, dissiminated intravascular coagulopathy, thrombotic microangiopathy, and nonthrombotic microangiopathy can be found in lung cancer. The incidence of venous thromboembolism (VTE) in lung cancer patients is around 40-100 cases per 1000 person-years compared to an estimated 1-2 cases per 1000 person-years in the general population (Tesselaar and Osanto, 2007). Chew and collegues analyzed the data from 91.933 patients with newly diagnosed lung cancer and found that approximately 3% developed VTE within two years (Chew et al, 2008). Venous thromboembolism was associated with a higher risk of death within two years for NSCLC and SCLC. Lung cancer is one of the greatest purveyors of VTE (Girard et al, 2008). Tumour cells can directly activate the clotting through two procoagulants: tissue factor (TF) and cancer procoagulant (CP) (Molnar et al, 2007). Human TF is the physiological initiator of blood coagulation. Goldin-Lang and co-workers found increased expression of full length human TF and alternatively spliced human tissue factor in NSCLC tissue compared to healthy controls (Goldin-Lang et al, 2008). Moreover, in that study expression of tissue factor was correlated with tumour stage and prognosis. Active TF-bearing microparticles, which may originate from the tumour cells themselves, have been found in the circulation of cancer patients. Microparticle-associated TF activity may provide a link between cancer and thrombosis and play a decisive role in the pathogenesis of the prothrombotic state in cancer patients (Tesselaar et al, 2007). VTE in lung cancer should be treated the same was as in non-cancer patients. Data from the literature suggest that low molecular weight heparin (LMWH) is likely to be superior to unfractionated heparin (UFH) in the initial treatment of VTE (Akl et al, 2008). For the long term treatment of VTE in cancer patients LMWH reduce VTE but not dead compared to oral anticoagulant therapy (Akl et al, 2008). In cancer patients without previous thrombotic incidents heparin has been shown to have a survival benefit especially in patients with limited SCLC (Akl et al, 2007). In contrast oral anticoagulation may not prolong survival. Only in patients with extensive SCLC a survival benefit of six months from warfarin is suggested according to the data from the literature (Akl et al, 2007).

VIII. Skeletal and collagen-vascular syndromes Digital clubbing and hypertrophic pulmonary osteoarthropathy (HPO) is observed in approximately 12% of patients with adenocarcinoma of the lung and less frequently in other cell types (Stenseth et al, 1967). Inflammatory symptoms and pain may disappear with successful treatment of the tumour. Dermatomyositis and polymyositis are associated with neoplasms in 40% of all cases. Besides ovarian cancer SCLC is the most frequent type of cancer (Hill et al, 2001). Gomm and colleagues studied 100 patients with lung cancer (35% had SCLC, 65% had NSCLC). In that study one patient presented with dermatomysositis and 33 patients had polymyopathie (Gomm et al, 1990).

IX. Neurologic syndromes Paraneoplastic neurological syndromes are observed in only 0.01% of cancer patients chiefly those affected by 691

Heinemann et al: Paraneoplastic syndromes in lung cancer can be found (Lennon et al, 1995). Low titers of antibodies against the P/Q and the N type calcium channel were also found in approximately 50% of patients with paraneoplastic encephalomyeloneuropathic complications (Lennon et al, 1995). Paraneoplastic encephalomyelitis is characterized by neuronal loss and inflammatory infiltrates in particular areas of the nervous system (Henson and Urich, 1982). It usually causes a severe neurological dysfuction, and antedates the diagnosis of SCLC in >70% of cases. In the majority of patients with paraneoplastic encephalomyelitis an antineuronal antibody, anti-Hu, can be found (Graus et al, 1985, 1986). This antibody recognizes a family of RNA-binding proteins (HuD, HuC, Hel-N1 and Hel-N2) expressed in the nuclei of neurones and SCLC cells (Szabo et al, 1991; King and Dropcho, 1996). Despite their crucial role in the development and maintenance of the neuronal phenotype the function of Hu antigens in the tumour cells is unknown. There is even no evidence that the anti-Hu antibodies are the cause of the neuronal damage (Sillevis Smitt et al, 1995; Carpentier et al, 1998). Nevertheless anti-Hu antibodies represent a useful diagnostic marker (Molinuevo et al, 1998). The antibodies probably are part of a more complex immune response against Hu antigens that is initially driven to control tumour growth but misdirected to cause neurological dysfunction (Posner and Dalmau, 1997). At autopsy of patients with paraneoplastic neurologic syndromes lymphocytic infiltration is found in the areas of the central nervous system that correspond to neurologic deficits. This finding supports the hypothesis that autoantibodies play a key role in the pathogenesis of the neurologic syndromes. Clinical features of paraneoplastic syndromes correspond to the underlying neurologic deficits. LEMS is characterized by muscle weakness with a predominance of the hip girdle. The motor weakness progresses in a craniocaudal direction. Patients with limbic encephalopathy usually present with rapidly progressive short-term memory deficits, psychiatric symptoms, and seizures. Paraneoplastic PNP leads to distal symmetric sensorimotor deficits. Cerebellar degeneration is characterized by ataxia. Retinopathy leads to visus loss. Opsoclonus is characterized by irregular, continual and conjugated chaotic saccades of the eyes. Opsoclonus when accompanied by other symptoms of central nervous system involvement (head, appendicular myoclonus and truncal ataxia) constitutes the opsoclonus-myoclonus syndrome. Paraneoplastic autonomic neuropathy is associated with a variety of symptoms including hypothermia, hypoventilation, sleep apnea, intestinal pseudo-obstruction, and cardiac arrhythmias. Two general approaches have been tried to treat paraneoplastic neurologic syndromes based on the assumption that these syndromes are immune-mediated: removal of the antigen source by treatment of the tumour and suppression of the immune response. LEMS can be treated 3,4-diaminopyridine or intravenous immunoglobulin that have been shown to improve muscle strength. However, evidence from studies is limited (Maddison and Newsom-Davis, 2005). In patients with

lung, breast, ovarian or stomach cancer. However, these syndromes frequently cause major disability and limitation in patients´ daily activities (Mollina-Garrido et al, 2006). Neurologic syndromes in lung cancer include the LambertEaton myasthenic syndrome (LEMS), limbic encephalopathy, polyneuropathy (PNP), cerebellar degeneration, retinopathy, opsoclonus-myoclonus, and autonomic neuropathy (Swash and Schwartz, 1990; Martina and Clay, 2005). Paraneoplastic neurologic syndromes may occur almost exclusively with SCLC. Incidence in lung cancer patients has been reported to range between 4 and 5% but may probably be lower (Swash and Schwartz, 1990). In a 1991 survey of 150 consecutive SCLC patients only two patients had LEMS (1%) and one patient suffered from PNP (< 1%) (Elrington et al, 1991). In 2005 similar results were obtained in a study of 432 consecutive patients with SCLC (LEMS: 1.6%, PNP: < 1%, subacute cerebellar degeneration: < 1%, limbic encephalitis: < 1%) (Seute et al, 2004). In a study of 200 patients with paraneoplastic encephalomyelitis and anti-Hu antibodies pathological or X-ray evidence of a tumour was obtained in 83%. Diagnosis of SCLC was made in 74% of those with histological diagnosis (Graus et al, 2001). The prognosis of patients with SCLC and paraneoplastic encephalomyelitis is poor (Spiegelman et al, 1989; Graus et al, 2001). Opsoclonus-myoclonus is a rare paraneoplastic neurologic disorder that is most often associated with SCLC (Anderson et al, 1988; Bataller et al, 2001). The presence of the anti-neuronal antibodies in the serum indicate a poor prognosis (Margery et al, 2003). In a study by Hassan and co-workes SCLC patients with opsoclonusmyoclonus died within 3 months without treatment. In contrast, in that same study with appropriate chemotherapy about half of the patients reported improvement of neurologic symptoms and several became long-term survivors (6 to 84 months) (Hassan et al, 2008). Autoimmune mechanisms seem to be responsible for the development of neurologic syndromes in cancer. Autoantibodies are commonly found in neurologic syndromes associated with cancer. Autoantibodies that are directed against ligand- or voltage-gated channels have been identified in several neuromuscular syndromes. These include antibodies against voltage-gated calcium channels (Lambert-Eaton syndrome), antibodies against voltage-gated potassium channel (acquired neuromyotonia), and antibodies against the neuronal AChR in autonomic ganglia (autoimmune autonomic ganglionopathy). There is good evidence that antibodies in these disorders cause changes in synaptic function or neuronal excitability by directly inhibiting ion channel function (Vernino, 2007). In Lambert-Eaton syndrome antibodies against the presynaptic voltage-gated calcium channels can be found. This decreases calcium entry into the presynaptic terminal which prevents binding of vesicles to the presynaptic membrane and acetylcholine release (Mareska and Gutmann, 2004). Antibodies are most often directed against voltage-gated P/Q calcium channels (VGCC) but also antibodies against voltage-gated N calcium channels

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Cancer Therapy Vol 6, page 693! free tryptophan, a precursor of serotonin, are found (Brink et al, 2002). Amounts of tryptophan are closely related with reduced food intake. Changes in hormone levels and target-organ sensitivity have also been described in tumour patients. Elevated levels of cortisol and glucagons may amplify the acute-phase protein response in cancer patients (Schaur et al, 1979; Knapp et al, 1991). In addition, several proinflammatory cytokines including (TNF)-! , (IL)-1, IL-6, interferon (IFN)-" , and ciliary neutropic factor (CNTF) have been implicated in cachexia (Lelbach et al, 2007). Apart from factors produced by the host tumour derived mediators have been described that may play an important role in the pathogenesis of cachexia. These mediators include proteolysis-inducing factor (PIF) and lipid mobilizing factor (LMF) (Cariuk et al, 1997; Hiraki et al, 1997). At present therapy for cancer cachexia is difficult. Reversing malnutrition by traditional food intake is hampered by the protein-energy deficit and the associated wasting as well as the anorexia and the early satiety. Several drugs have been used to “repair� the altered metabolism. Corticosteroids are most widely used with a short benefit. Ibuprofen has been used with some effect due to its anti-inflammatory properties. Medroxyprogesteron acetate may improve appetite and stabilize weight. Eicosapentaenoic acid can lower the production of proinflammatory cytokines. Cancer-related fatigue is also extremely common. Up to 90% of cancer patients report fatigue symptoms while in most studies prevalence rates are 60% (Cella et al, 2001). Fatigue is a highly subjective multidimensional experience. Individuals may perceive fatigue as physical tiredness or exhaustion, a need for reduced activity, reduced motivation, and/or mental fatigue (Ahlberg et al, 2003). Fatigue is experienced due to cancer and to treatment. The basic mechanisms of fatigue are broadly characterized into two main components: peripheral and central. Peripheral fatigue occurs in the neuromuscular junctions and muscle tissues. This results in the inability of the peripheral neuromuscular apparatus to perform a task in response to central stimulation. Central fatigue arises from the progressive failure to transmit motor neuron impulses. This leads to difficulties in the initiation or maintenance of voluntary activities (Chaudhuri and Behan, 2004; Ronnback and Hansson, 2004). The etiology of fatigue is poorly understood. Several underlying mechanisms have been proposed based on studies in normal (exercise) conditions and in the context of chronic diseases, including chronic fatigue syndrome and rheumatoid arthritis (Ryan et al, 2007). Several mechanisms have been shown to be involved in cancer-related fatigue. Studies in patients with chronic fatigue syndrome have demonstrated raised plasma levels of free tryptophan, which could potentially lead to high central serotonin levels (Castell et al, 1999; Badawy et al, 2005). An increase in brain serotonin (5-HT) levels and/or upregulation of a population of 5-HT receptors, may lead to reduced somatomotor drive, modified hypothalamicpituitary-adrenal (HPA) axis function, and a sensation of reduced capacity to perform physical work (Andrews et al, 2004). Low levels of circulating cortisol have been

underlying cancer chemotherapy has been successful (Verschuuren et al, 2006). For other paraneoplastic neurologic syndromes there is evidence that prompt oncologic treatment and immunotherapy (eg. Immunosuppression) can be beneficial (Keime-Guibert et al, 2000; Rosenfeld and Dalmau, 2003; Vernino et al, 2004).

X. Cutaneous syndromes A variety of paraneoplastic cutaneous syndromes has been described in lung cancer patients (refer to Table 1). However, these syndromes are mostly non-specific and can be observed in both malignant and benign disease. Palmo-plantar hyperkeratosis (PPH) also called tylosis is a rare paraneoplastic syndrome in lung cancer. In the literature it is mostly described in case reports (Schwindt et al, 1970; Nomori et al, 1996; Engin et al, 2002). It may typically precede the diagnosis of lung cancer for months or years (Burgdorf and Goltz, 1987). Prognosis of patients with lung cancer and PPH is poor (Nomori et al, 1996). Akanthosis nigricans has also been described in several case reports to be associated with adenocarcinoma, with squamous cell lung carcinoma or also with alveolar cell carcinoma (Horiuchi et al, 1986; Menzies et al, 1988; Onai et al, 1989).

XI. Cachexia and fatigue Cancer cachexia is perhaps the most common manifestation of advanced malignant disease (50%) and is responsible for approximately 25% of deaths from cancer (Lelbach et al, 2007). The degree of cachexia is inversely correlated with survival time and always implies a poor prognosis. Symptoms of cachexia include anorexia, weight loss, muscle loss, anemia, and alterations in carbohydrate, lipid and protein metabolism. Cachexia is mainly driven by anorexia and metabolic alterations. Cancer patients frequently exhibit a relative glucose intolerance and insulin resistance. While significant loss of adipose tissue is observed in cancer cachexia lipolytic rates are not significantly increased. Lipogenesis seem to be reduced. Loss of skeletal muscle protein and decreased muscle protein synthesis is commonly observed in cachexia (Dworzak et al, 1998; Giordano et al, 2003). Changes in liver protein synthesis refect aspects of the so-called acute-phase protein response. This is part of the general adaptation of the body often seen in trauma, inflammation, infection, and cancer. An acute-phase protein response can be observed in a significant proportion of patients with lung cancer. The presence of an acute-phase protein response has been related to accelerated weight loss in patients with lung cancer (Harvie et al, 2003). A variety of mediators has been described to be involved in development of cachexia (Lelbach et al, 2007). The tumour behaves like a new metabolically active organ. However, it is not clear whether the metabolic changes result from mediators released by the cancer or the host response (Argiles et al, 2003, 2008; Lelbach et al, 2004). In general, serotinergic activity in the hypothalamus decreases appetite. In cancer patients increased levels of 693

Heinemann et al: Paraneoplastic syndromes in lung cancer treatment of venous thromboembolism in patients with cancer. Cochrane Database Syst Rev 16, CD006650. Akl EA, van Doormaal FF, Barba M, Kamath G, Kim SY, Kuipers S, Middeldorp S, Yosuico V, Dickinson HO, Sch端nemann HJ (2007) Parenteral anticoagulation for prolonging survival in patients with cancer who have no other indication for anticoagulation. Cochrane Database Syst Rev 18, CD006652. Amital H, Applbaum YH, Vasiliev L, Rubinow A (2004) Hypertrophic pulmonary osteoarthropathy: control of pain and symptoms with pamidronate. Clin Rheumatol 23, 330332. Anderson NE, Budde-Steffen C, Rosenblum MK, Graus F, Ford D, Synek BJ, Posner JB (1988) Opsoclonus, myoclonus, ataxia, and encephalopathy in adults with cancer: a distinct paraneoplastic syndrome. Medicine (Baltimore) 67, 100109. Andrews PLR, Morrow GR, Hickok JT (2004) In: Armes J, Krishnasamy M, Higginson I, eds. Fatigue in Cancer. Mechanisms and models of fatigue associated with cancer and its treatment: Evidence of pre-clinical and clinical studies. Oxford, Oxford University Press, pp 51-87. Aoe K, Hiraki A, Ueka H, Kiura K, Tabata M, Tanaka M, Tanimoto M (2004) Thrombocytosis is a useful prognostic indicator in patients with lung cancer. Respiration 71, 170173. Argiles JM, Moore-Carrasco R, Busquets S, Lopez-Soriano FJ (2008) Catabolic mediators as targets for cancer cachexia. Drug Discov Today 8, 838-844. Argiles JM, Moore-Carrasco R, Fuster G, Busquets S, LopezSoriano FJ (2003) Cancer cachexia: the molecular mechanism. Int J Biochem Cell Biol 35, 405-409. Asa SL, Henderson J, Goltzman D, Drucker DJ (1990) Parathyroid hormone-like peptide in normal and neoplastic human endocrine tissues. J Clin Endocrinol Metab 71, 1112-1118. Asano S, Urabe A, Okabe T, Sato N, Kondo Y (1977) Demonstration of granulopoietic factors in the plasma of nude mice transplanted with a human lung cancer and in the tumor tissue. Blood 49, 845-52. Badawy AA, Morgan CJ, Llewelyn MB, Albuquerque SR, Farmer A (2005) Heterogeneity of serum tryptophan concentration and availability to the brain in patients with the chronic fatigue syndrome. J Psychopharmacol 19, 385-391. Bataller L, Graus F, Saiz A, Vilchez JJ and for the Spanish Opsolconus-Myoclonus Study Group (2001) Clinical outcome in adult onset idiopathic or paraneoplastic opsoclonus-myoclonus. Brain 124, 437-443. Bertolini DR, Nedwin GE, Bringman TS, Smith DD, Mundy GR (1986) Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors. Nature 319, 516-518. Bliss DP Jr, Battey JF, Linnoila RI, Birrer, MJ, Gazdar AF, Johnson BE (1990) Expression of the atrial natriuretic factor gene in small cell lung cancer tumors and tumor cell lines. J Natl Cancer Inst 82:, 305-310. Brink M, Anwar D, Delafontaine P (2002) Neurohormonal factors in the development of catabolic/anabolic imbalance and cachexia. Int J Cardiol 85, 111-121. Bunn PA, Ridgway EC (1993) Paraneoplastic syndromes. In: DeVita VT, et al, eds. Cancer: Principles and Practice of Oncology. 4th Edn. JB Lippincott Co, pp 2026-2071. Burgdorf WHC, Goltz RW (1987) Figurate erythemas. In: Fitzpatrick TB, Eisen AZ. Wolff K, et al. eds. Dermatology in General Medicine. New York, McGraw-Hill, pp 10101018. Burtis WJ, Brady TG, Orloff JJ, Ersbak JB, Warrell RP Jr, Olson BR, Wu TL, Mitnick ME, Broadus AE, Stewart AF (1990)

observed in patients with chronic fatigue syndrome (Cleare, 2003). Cancer, and/or cancer treatment may also alter the function of the HPA axis, resulting in endocrine changes that cause or contribute to fatigue. Cancer may cause fatigue by circadian rhythm disruption. Several alterations in circadian function have been demonstrated in patients with cancer. These include changes in endocrine rhythms (e.g., cortisol, melatonin, and prolactin secretion), metabolic processes (e.g., temperature and circulating protein levels), the immune system (e.g., levels of circulating leukocytes and neutrophils), and rest-activity patterns (Focan et al, 1986; Mormont and Levi, 1997; Vgontzas and Chrousos, 2002; Sephton and Spiegel, 2003; Levin et al, 2005). Cancer can lead to a defect in the mechanism for regenerating ATP in skeletal muscle can compromise the ability to perform mechanical tasks (Andrews et al, 2004). Reduced oxidative muscle metabolism, epleted cellular ATP associated with a dysregulated 2',5'-oligoadenylate synthetase/RNase L pathway, and impaired synthesis of ATP have been reported (McCully et al., 1996; Lane et al, 1998; Forsyth et al, 1999). Proinflammatory cytokines, such as TNF-! and IL-1" , are implicated in many of the mechanisms proposed for the etiology of fatigue associated with cancer and various illnesses (Konsman et al, 2002). Treatment of cancer-related fatigue should be individualized according to the underlying pathology when a specific cause has been identified. In addition to older therapies, such as hematopoietics, antidepressants, corticosteroids, and psychostimulants, the new wakepromoting agent modafinil may offer an alternative therapeutic approach.

XII. Conclusions Paraneoplastic syndromes are common in lung cancer patients. Some paraneoplastic syndromes can severely affect organ function and quality of life. Treating the underlying cancer is the first step. However, specific therapy may also be necessary.

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Paraneoplastic syndromes in lung cancer Review Article

Stefanie Heinemann, Peter Zabel, Hans-Peter Hauber* Medical Clinic, Research Center Borstel, Borstel, Germany

__________________________________________________________________________________! *Correspondence: Hans-Peter Hauber, MD, Medical Clinic, Research Center Borstel, Parkallee 35, 23845 Borstel, Germany; Phone: +49 (0)4537 188 364; Fax: +49 (0)4537 188 313; E-mail: hphauber@fz-borstel.de Key words: Anemia, antidiuretic hormone, autoantibody, cachexia, cancer, chemotherapy, clubbing, Cushing, dermatomyositis, hypertrophic pulmonary osteoarthropathy, hypercalcaemia, Lambert-Eaton, lung, neurologic, paraneoplastic, syndrome Abbreviations: Adrenocorticotrophic hormone, (ACTH); antidiuretic hormone, (ADH); atrial natriuretic peptide, (ANP); cancer procoagulant, (CP); ciliary neutropic factor, (CNTF); cyclic adenosine monophosphate, (cAMP); granulocyte colony stimulating factor, (G-CSF); Granulocyte macrophage- colony stimulating factor, (GM-CSF); hypertrophic pulmonary osteoarthropathy, (HPO); hypothalamic-pituitary-adrenal, (HPA); interferon, (IFN); interleukin, (IL); Lambert-Eaton myasthenic syndrome, (LEMS); lipid mobilizing factor, (LMF); low molecular weight heparin, (LMWH); non-small-cell lung cancer, (NSCLC); Palmo-plantar hyperkeratosis, (PPH); parathyroid hormone, (PTH); parathyroid hormone-related protein, (PTHrP); polyneuropathy, (PNP); proopiomelanocortin, (POMC); prostaglandins of the E series, (PGE); proteolysis-inducing factor, (PIF); small-cell lung cancer, (SCLC); syndrome of inappropriate antidiuretic hormone secretion, (SIADH); tissue factor, (TF); transforming growth factor alpha, (TGF)! ; tumor necrosis factor alpha, (TNF)! ; unfractionated heparin, (UFH); vascular endothelial growth factor, (VEGF); venous thromboembolism, (VTE); voltage-gated P/Q calcium channels, (VGCC)

Received: 21 August 2008; Revised: 11 September 2008 Accepted: 12 September 2008; electronically published: October 2008

Summary Paraneoplastic syndromes are frequently found in lung cancer. They can be the first manifestation of disease or recurrence. Neuromuscular, vascular, haematological and metabolic syndromes, as well as syndromes involving the connective and skeletal tissues and skin can be distinguished. While some of the paraneoplastic syndromes are commonly observed (eg. Anemia, cachexia) others are rare disorders (eg. Opsoclonus-myoclonus, tylosis). This review focuses on the epidemiology, pathogenesis, clinical symptoms and treatement options of frequent and clinical important paraneoplastic syndromes.

syndromes is broadened to include conditions such as anemia, cachexia, and hypercalcemia then the incidence and prevalence of paraneoplastic syndromes is much higher. Lung cancer and small-cell lung cancer (SCLC) in particular is the most common cancer to be associated with paraneoplastic syndromes. However, some paraneoplastic syndromes are more often found in non-small-cell lung cancer (NSCLC). For example hypertrophic pulmonary osteoarthropathy has most often been described in association NSCLC. The extent of paraneoplastic syndromes is unrelated to the size of the primary tumour. In some cases it may precede diagnosis of malignancy while in other cases it may occur late in the course of disease or may show up as the first symptom of recurrence. If lung cancer patients have paraneoplastic syndromes normally they suffer from only one syndrome. Literature data on multiple paraneoplastic syndromes in the same patient are sparse and are mostly described in case reports (Monsieur et al, 1995).

I. Introduction Paraneoplastic syndromes are common in lung cancer, and may be the first manifestation of the disease or its recurrence (Bunn and Ridgway, 1993). Paraneoplastic phenomena are not related to the direct invasion, obstruction, or metastasis (Patel et al, 1993; Spiro, 1995). Neuromuscular, vascular, haematological and metabolic syndromes, as well as syndromes involving the connective and skeletal tissues and skin can be distinguished. Table 1 gives an overview of the paraneoplastic syndromes that have been described in lung cancer patients. In the following epidemiology, pathogenesis, clinical findings, and treatment options of the most common paraneoplastic syndromes in lung cancer will be reviewed.

II. Epidemiology Paraneoplastic syndromes are estimated to occur in 7% to 15% of all patients with cancer (Richardson and Johnson, 1992). If the definition of paraneoplastic 687

Heinemann et al: Paraneoplastic syndromes in lung cancer Table 1. Paraneoplastic syndromes in lung cancer.

III. Hypercalcaemia Hypercalcaemia is frequently found in patients with lung cancer. Its incidence ranges from 2 to 6% at presentation to 8 to 12% throughout the course of disease (Spiro et al, 2007). It may arise from bone metastasis but can also be induced in a paraneoplastic manner by secretion of parathyroid hormone-related protein (PTHrP), calcitriol or other cytokines, including osteoclast activating factors. Hiraki and colleagues examined 1149 patients with lung cancer and found 6% to have hypercalcemia (Hiraki et al, 2004). Among those with hypercalcemia 51% had squamous cell carcinoma, 22% had adenocarcinoma, and 15% had SCLC. Most of those patients had advanced disease (stage III or IV). Median survival was only 3.8 months (Hiraki et al, 2004). Hypercalcaemia in lung cancer patients can be caused by either bony metastases or less commonly by paraneoplastic syndromes. Tumours can secrete PTHrP, calcitriol or other cytokines including osteoclast activating factors. Moseley and colleagues identified PTHrP expression in lung cancer cells (Moseley et al, 1987). PTHrP shares 70% sequence homology with PTH over the first 13 amino acids at the N-terminus. Both parathyroid hormone (PTH) and PTHrP bind to a common PTH/PTHrP receptor (Abou-Samra et al, 1992) and share similar biological activities (Horiuchi et al, 1987). However, expression of PTHrP has also been reported in normal tissue (Danks et al, 1989; Asa et al, 1990). This implicates that PTHrP has also a physiological effect. It is now clear that PTHrP has several other functions in addition to its PTH-like effects (Clines and Guise, 2005). PTH stimulates osteoclastic bone resorption and calcium reabsorption and inhibition of phosphate reabsorption from renal tubules. It also stimulates renal 1! -hydroxylase resulting in production of 1,25-(OH)2D3, which increases intestinal absorption of calcium and phosphate. These actions result in increased serum calcium. PTH actions are mediated through binding of the amino terminus of the PTH molecule to the PTH receptor, a member of the family of G protein-coupled receptors that contain seven transmembrane- spanning domains (Juppner et al, 1991). The ligand-bound PTH receptor activates adenylate cyclase, through the activation of G protein G! s, producing cyclic adenosine monophosphate (cAMP) while activating protein kinase A. In addition, the phospholipase C/protein kinase C system also contributes to PTH signal transduction (Mahon et al, 2002; Swarthout et al, 2002). Approximately 80% of cancer patients with hypercalcemia have detectable or increased plasma concentrations of PTHrP (Burtis et al, 1990). PTHrP has a multifunctional role in cancer. It mediates hypercalcaemia but also aids development and progression of osteolytic bone metastasis, regulates growth of cancer cells and acts as a cell survival factor (Luparello et al, 1993; Luparello et al, 1995; Li et al, 1996; Chen et al, 2002). Although PTHrP is responsible for he majority of cases of paraneoplastic hypercalcaemia there have been rare cases with ectopic PTH production in the tumour. This has been reported in SCLC as well as in squamous

Endocrine syndromes SIADH Nonmetastatic hypercalcemia Cushing syndrome Gynecomastia Hypercalcitonemia Elevated levels of LSH and FSH Hypoglycemia Hyperthyroidism Carcinoid syndrome Neurologic syndromes Subacute sensory neuropathy Mononeuritis multiplex Lambert-Eaton myasthenic syndrome Encephalomyelitis Necrotizing myelopathy Cancer associated retinopathy Opsoclonus-myoclonus Skeletal syndromes Hypertrophic osteoarthropathy Clubbing Renal syndromes Glomerulonephritis Nephrotic syndrome Metabolic syndromes Lactic acidosis Hypouricemia Systemic syndromes Cachexia Fatigue Fever Collagen-vascular syndromes Dermatomyositis and polymyositis Vasculitis Systemic lupus erythematosus Cutaneous syndromes Acquired hypertrichosis languinosa !"#$%&'()*#"($+')"&,&-. Erythema multiforme Tylosis Erythroderma Exfoliative dermatitis /0(-$%1.2.)-2*"20(-. Sweet syndrome Pruritus and urticaria Hematologic syndromes Anemia Leucocytosis and eosinophilia Thrombocytosis and thrombocytopenic purpura Coagulopathies Thrombophlebitis Thromboembolism Disseminated intravascular coagulation Reproduced from Scagliotti, 2001 and Carbone et al, 1970 with kind permission from European Respiratory Monthly and Annals of Internal Medicine respectively.! .

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Cancer Therapy Vol 6, page 689! have been implicated as possibly contributing to the hyponatremia found in lung cancer patients. However, only elevated plasma ADH levels were consistently found in patients with lung cancer and may explain the impaired ability to excrete the water load (Vorherr, 1974). Physiologically ADH is released from the posterior pituitary gland whereas in paraneoplastic SIADH cancer cells secrete ADH (Moses and Scheinman, 1991). Another possible mechanism is inappropriate peripheral baroreceptor stimulation of ADH release from the hypothalamus (Vorherr, 1974). ADH causes hyponatremia and hypoosmolality that interfere with urinary dilution, thereby preventing the excretion of ingested water. An interesting observation in the SIADH is the development of partial escape from ADH which tends to protect against progressive water retention (Jaenike and Waterhouse, 1961). Although ADH secretion or effect is inappropriately increased in the SIADH, up to one-third of patients have a downward resetting of the osmostat in which the plasma sodium concentration is normally regulated (and therefore stable) at a new lower level, typically between 125 and 135 mmol/L. Establishing the presence of this condition is clinically important because correcting the hyponatremia is both unnecessary and likely to be ineffective, since raising the plasma osmolality will stimulate both ADH release and thirst. Hyponatremia, plasma hypoosmolality and urine hyperosmolality with continuing sodium excretion are biochemical findings in SIADH. Water retention is typically only 2 to3 liters and does not lead to edema or anasarka. The severity of symptoms in SIADH is related to the degree of hyponatremia and the rapidity of fall in serum sodium. Anorexia, nausea, and vomiting are common symptoms. A rapid onset of hyponatremia can cause cerebral edema. This may lead to irritability, restlessness, personality changes, confusion, coma, seizures, and respiratory arrest. In patients with SCLC, SIADH resolves in up to 80% after administration of chemotherapy (List et al, 1986). Adjuvant management of SIADH includes fluid restriction to 800-1,000 ml/d to increase serum sodium. In patients with severe symptoms (severe confusion, convulsions, or coma) intravenous hypertonic saline (5%) solution (eg. 200-300 ml in 3-4 h) should be given.

cell carcinoma of the lung (Yoshimoto et al, 1989; Nielsen et al, 1996). While in haematological malignancies extra-renal production of 1,25-(OH)2D3 appears to be a major mediator of hypercalcaemia (Seymour et al, 1994) in lung cancer calcitriol does not seem to be the major mediator of PTH-like activity even if SCLC cell lines can synthesize 1,25-(OH)2D3 (Mawer et al, 1994). Other factors that can stimulate osteocalstic bone resorption and cause hypercalcaemia include interleukin (IL)-1, IL-6, transforming growth factor alpha (TGF)! , tumor necrosis factor alpha (TNF)! , and granulocyte colony stimulating factor (G-CSF) (Clines and Guise, 2005). Human TGF! and TNF! stimulate osteoclastic bone resorption !"# $!%&' and result in hypercalcaemia !"# $!$' (Bertolini et al, 1986; Yates et al, 1992; Ibbotson et al, 1995). Factors such as TGF! , IL-1, IL-6 and TNF! can also enhance the hypercalcaemic effects of PTHrP. Although prostaglandins of the E series (PGE) are powerful stimulators of bone resorption (Klein and Raisz, 1970) there role in tumour-associated bone destruction remains unclear (Mundy, 1995). They may be mediators of cytokine effects on the bone. Moreover, PGE expression has been described in the lung cancer tissue of normocalcaemic patients (Kukrja et al, 1982). This finding implicates that PGE may not be necessary to induce hypercalcaemia in cancer patients. Symptoms of hypercalcaemia include anorexia, nausea, vomiting, constipation, lethargy, polyuria, polydipsia, and dehydration. If untreated hypercalcaemia my lead eventually to confusion and coma. Renal failure and nephrocalcinosis are late manifestations, too. Symptomatic patients with a serum calcium of " 3 mmol/L required treatment that includes hydration and biphosphonate (Thomas et al, 2004).

IV. Syndrome of inappropriate antidiuretic hormone secretion Elevated levels of antidiuretic hormone (ADH) and impaired water handling can be observed in 30 to 70% of lung cancer patients (Patel et al, 1993). However, excess production of ADH does not always produce symptoms (Maurer et al, 1983; Bliss et al, 1990; Moses and Scheinman, 1991). Only 1 to 5% of all patients with lung cancer have symptoms attributable to the syndrome of inappropriate antidiuretic hormone secretion (SIADH). SIDAH is frequently caused by SCLC. In a study by List and co-workers approximately 10% of patients with SCLC had SIADH (List et al, 1986). In that study development of SIADH did not correlate with clinical stage or metastatic sites. SIADH occurred most often with initial presentation and promptly resolved with initiation of combined chemotherapy in 80% of the patients. Response to chemotherapy and survival was not influenced by the presence of SIADH (List et al, 1986). Recurrence of SIADH was associated with tumour progression. Biochemically, the SIADH production is defined as low serum sodium and a dilute plasma osmolality along with a higher, or "inappropriate," urine osmolality, in the presence of continued urinary sodium excretion. A variety of hormones including atrial natriuretic peptide (ANP)

V. Cushing syndrome Adrenocorticotrophic hormone (ACTH) is the most commonly produced hormone in lung cancer patients. Increased levels of ACTH may be detectable in up to 50% of patients with lung cancer (Mendelsohn and Baylin, 1984). ACTH secretion is almost always associated with SCLC (Hansen, 1990). About 30% of all cases of SCLC are associated with hypersecretion of ACTH. However, a clinical apparent Cushing syndrome is rare (Mennecier et al, 1999). Cushing syndrome has been described in 1 to 5% of patients with SCLC (Odell et al, 1979; Ilias et al, 2005) but this may overestimate the real rate. In 2005 Hansen and Bork reported only 3 out of 90 cases of Cushing syndrome to be attributable to SCLC (Hansen and Bork, 1985). Most commonly, Cushing syndrome occurred in patients with pulmonary carcinoid (35 of 90 689

Heinemann et al: Paraneoplastic syndromes in lung cancer chemotherapy in patients with SCLC and Cushing syndrome is only moderate (Shepherd et al, 1992). Severe symptoms should be treated symptomatically.

patients). While ectopic ACTH syndrome typically presents as Cushing syndrome in patients with SCLC bronchial carcinoid tumors are the most common occult sources of ACTH (Terzolo et al, 2001). SCLC associated with the ectopic ACTH syndrome is more resistant to chemotherapy and the severe hypercortisolism is responsible for a high rate of lifethreatening complications during treatment which worsens prognosis (Terzolo et al, 2001). Shepherd and collegues retrospectively analyzed the charts of 545 patients with SCLC. They identified 23 patiens (4.5%) with Cushing syndrome and ectopic ACTH production. These patients had a response rate to chemotherapy of only 46%, and their median survival was only 3.57 months (Shepherd et al, 1992). In pituitary cells ACTH is derived by cleavage from the precursor, pro-opiomelanocortin (POMC). It is unlikely that processing of POMC is as efficient in nonpituitary cells. ACTH precursors and ACTH-related peptides can be secreted by POMC expressing cells. ACTH precursors can be detected in the serum of patients with ectopic ACTH syndrome (Oliver et al, 2003). Stewart and co-workers reported grossly elevated levels of ACTH precursors in patients with ectopic ACTH syndrome and increased concentrations of ACTH precursors in patients with SCLC without evidence of ectopic ACTH syndrome (Stewart et al, 1994). SCLC cells have been shown to express POMC and secrete ACTH precursors (Stewart et al, 1989; White et al, 1989). POMC expression has also been observed in bronchial carcinoid tumour cells (Crosby et al, 1990). NSCLC is rarely associated with ectopic ACTH syndrome. In a case report of a patient with poorly differentiated squamous cell lung carcinoma and ectopic ACTH syndrome ACTH secretion of cancer cells was confirmed by immunohistochemical staining (Noorlander et al, 2006). Another case reported described paraneoplastic Cushing syndrome in a patient with adenocarinoma of the lung (Yoh et al, 2003). ACTH and ACTH precursors stimulate the adrenal glands to secrete glucocorticosteroids. The symptoms and signs of paraneoplastic Cushing syndrome result directly from chronic exposure to excess glucocorticoid because of ectopic ACTH-production. There is a large spectrum of manifestations from subclinical to overt syndrome. Diagnosis is often difficult because there is no pathognomonic symptom. An important clinical hint to the presence of Cushing syndrome is the simultaneous development and increasing severity of several of the following symptoms: Centripetal obesity, facial plethora, glucose intolerance, weakness, proximal myopathy, hypertension, psychological changes, easy bruisability, hirsutism, oligomenorrhea or amenorrhea, impotence, acne, oily skin, abdominal striae, ankle edema, osteoporosis, polydipsia, polyuria, hyperpigmentation, headache, fungal infection specially oral thrush and hypokalimea. To treat the paraneoplastic Cushing syndrome, treatment of the underlying disease is essential. In most cases treatment of the tumour will also improve the paraneoplastic syndrom. However, responses to

VI. Hematological abnormalities Hematologic abnormalities including anemia, leukocytosis, thrombocytosis, and eosinophilia are frequently observed in lung cancer patients. Anemia is common in lung cancer patients. In one series 38% of untreated patients had a haemoglobin ! 12 g/dL. In contrast 80% of patients that were treated with chemotherapy were anaemic at one time (Kosmidis and Krzakowski, 2005). Leukocytosis is often found in patients with lung cancer either at time of diagnosis or during the course of the disease (Thomson et al, 1986). In one study leukocytosis had been described in 15% of all patients with lung cancer (n = 227). Nearly all patients had NSCLC. Leukocytosis was thought to be due to overproduction of granulocyte-colony stimulating factor (Kasuga et al, 2001). Tumor-related leukocytosis was associated with a poorer prognosis compared to patients without leukocytosis (median survival: 4.6 months vs 20.8 months) (Kasuga et al, 2001). Leukocytosis has also been associated with hypercalcaemia (Kasuga et al, 2001; Hiraki et al, 2004). Thrombocytosis is observed in 16% to 32% of all lung cancer patients (Moller Pedersen and Milman, 1996; Aoe et al, 2004). It has been identified as an independent predictor of shorten survival (Moller Pedersen and Milman, 1996; Aoe et al, 2004). Eosinophilia in tissue or blood is rare. While tumourassociated tissue eosinophilia appear to have a better prognosis tumour-associated blood eosinophilia seem to be associated with a worse prognosis (Lowe, et al 1981). The cause of anemia associated with cancer is multifactorial. Bleeding, hemolysis, bone marrow infiltration, and nutritional deficiencies may all contribute to the development of anemia in patients with cancer. In addition, inflammatory cytokines, such as TNF-" , IL-1, IL-6, and IFN- , inhibit erythropoiesis, which leads to decreased production of erythrocytes, resulting in anemia. It has to be taken into account that anemia is a common complication of myelosuppressive chemotherapy (Groopman and Itri, 1999). On average, over one third of patients become anemic after three cycles of chemotherapy (Glaspy et al, 2002). Lung cancer has been shown to produce G-CSF which leads to leukocytosis (Asano et al, 1977). Granulocyte macrophage- colony stimulating factor (GMCSF) and interleukin-6 production of lung carcinomas has also been reported to be associated with leukocytosis (Sawyers et al, 1992; Matsuguchi et al, 1991). Anemia should be treated to improve quality of life. Erythropoiesis-stimulating agents such as epoetin and darbepoetin should be used with caution in cancer patients with anemia who are not receiving chemotherapy (Rizzo et al, 2008).

VII. Hypercoagulable disorders A variety of hypercoagulable disorders including Trousseau´s syndrome (migratory superficial thrombophlebitis), deep venous thrombosis and 690

Cancer Therapy Vol 6, page 691! The exact pathogenesis of digital clubbing and HPO is not known. In digital clubbing proliferation of connective tissue beneath the nail matrix is observed. Histochemical features of HPO include vascular hyperplasia, edema, and excessive fibroblast and osteoblast proliferation (Myers and Farquhar, 2001). In the past, neurogenic, hormonal, and vascular mechanisms have been discussed (Shneerson, 1981). More recently, the overexpression of vascular endothelial growth factor (VEGF) has been implicated as contributing to the pathogenesis of clubbing and HPO. Olan and collegues reported the case of a young woman with lung cancer and HPO. Serum levels of VEGF were elevated. After resection of the cancer VEGF levels fell and HPO remitted. Histochemical studies of the resected tumor showed increased VEGF messenger RNA expression, suggesting ectopic production by the lung cancer cells (Olan et al, 2004). Dermatomyositis is characterized by infarcts, perifascicular atrophy, endothelial cell swelling and necrosis, vessel wall membrane attack complex deposition, and myocyte-specific MHC I upregulation in the muscle. Histopathological findings in the skin include hyperkeratosis, epidermal basal cell vacuolar degeneration and apoptosis, increased dermal mucin deposition, and a cell-poor interface dermatitis. The precise link between malignancy and inflammatory myopathy remains incompletely understood (Casciola-Rosen et al, 2005). Although dermatomyositis has been classically considered as humorally mediated disease newer evidence suggest that cell-mediated mechanisms and innate immune system dysfunction play a more important role in the pathogenesis (Krathen et al, 2008). Myositis-specific autoantigens are expressed at high levels in regenerating cell in myositic muscles and in several cancer cells (Levine, 2006). This may provide a link between cancer and paraneoplastic myositis syndromes. Digital clubbing is an enlargement of the terminal segments of the fingers and/or toes due to proliferation of connective tissue beneath the nail matrix. HPO is a systemic disorder, which involves both a painful symmetrical arthropathy, usually of the ankles, wrists, and knees, and periosteal new bone formation in the distal long bones of the limbs. Myositis is characterized by muscle weakness and muscle pain. Typically proximal muscles are involved. Dermatomyositis also shows characteristic cutaneous findings of heliotrope eruption, Gottron's papules and a photodistributed eruption with poikiloderma. Raynaud phenomenon, interstitial lung disease and inflammatory arthritis can also be found. Symptoms of HPO may resolve after tumour resection. If a patient is not operable the usual treatment includes nonsteroidal anti-inflammatory agents or a bisphophonate (Amital et al, 2004). The mainstay of therapy for dermatomyositis is corticosteroids (Iorizzo and Jorizzo, 2008).

thromboembolism, dissiminated intravascular coagulopathy, thrombotic microangiopathy, and nonthrombotic microangiopathy can be found in lung cancer. The incidence of venous thromboembolism (VTE) in lung cancer patients is around 40-100 cases per 1000 person-years compared to an estimated 1-2 cases per 1000 person-years in the general population (Tesselaar and Osanto, 2007). Chew and collegues analyzed the data from 91.933 patients with newly diagnosed lung cancer and found that approximately 3% developed VTE within two years (Chew et al, 2008). Venous thromboembolism was associated with a higher risk of death within two years for NSCLC and SCLC. Lung cancer is one of the greatest purveyors of VTE (Girard et al, 2008). Tumour cells can directly activate the clotting through two procoagulants: tissue factor (TF) and cancer procoagulant (CP) (Molnar et al, 2007). Human TF is the physiological initiator of blood coagulation. Goldin-Lang and co-workers found increased expression of full length human TF and alternatively spliced human tissue factor in NSCLC tissue compared to healthy controls (Goldin-Lang et al, 2008). Moreover, in that study expression of tissue factor was correlated with tumour stage and prognosis. Active TF-bearing microparticles, which may originate from the tumour cells themselves, have been found in the circulation of cancer patients. Microparticle-associated TF activity may provide a link between cancer and thrombosis and play a decisive role in the pathogenesis of the prothrombotic state in cancer patients (Tesselaar et al, 2007). VTE in lung cancer should be treated the same was as in non-cancer patients. Data from the literature suggest that low molecular weight heparin (LMWH) is likely to be superior to unfractionated heparin (UFH) in the initial treatment of VTE (Akl et al, 2008). For the long term treatment of VTE in cancer patients LMWH reduce VTE but not dead compared to oral anticoagulant therapy (Akl et al, 2008). In cancer patients without previous thrombotic incidents heparin has been shown to have a survival benefit especially in patients with limited SCLC (Akl et al, 2007). In contrast oral anticoagulation may not prolong survival. Only in patients with extensive SCLC a survival benefit of six months from warfarin is suggested according to the data from the literature (Akl et al, 2007).

VIII. Skeletal and collagen-vascular syndromes Digital clubbing and hypertrophic pulmonary osteoarthropathy (HPO) is observed in approximately 12% of patients with adenocarcinoma of the lung and less frequently in other cell types (Stenseth et al, 1967). Inflammatory symptoms and pain may disappear with successful treatment of the tumour. Dermatomyositis and polymyositis are associated with neoplasms in 40% of all cases. Besides ovarian cancer SCLC is the most frequent type of cancer (Hill et al, 2001). Gomm and colleagues studied 100 patients with lung cancer (35% had SCLC, 65% had NSCLC). In that study one patient presented with dermatomysositis and 33 patients had polymyopathie (Gomm et al, 1990).

IX. Neurologic syndromes Paraneoplastic neurological syndromes are observed in only 0.01% of cancer patients chiefly those affected by 691

Heinemann et al: Paraneoplastic syndromes in lung cancer can be found (Lennon et al, 1995). Low titers of antibodies against the P/Q and the N type calcium channel were also found in approximately 50% of patients with paraneoplastic encephalomyeloneuropathic complications (Lennon et al, 1995). Paraneoplastic encephalomyelitis is characterized by neuronal loss and inflammatory infiltrates in particular areas of the nervous system (Henson and Urich, 1982). It usually causes a severe neurological dysfuction, and antedates the diagnosis of SCLC in >70% of cases. In the majority of patients with paraneoplastic encephalomyelitis an antineuronal antibody, anti-Hu, can be found (Graus et al, 1985, 1986). This antibody recognizes a family of RNA-binding proteins (HuD, HuC, Hel-N1 and Hel-N2) expressed in the nuclei of neurones and SCLC cells (Szabo et al, 1991; King and Dropcho, 1996). Despite their crucial role in the development and maintenance of the neuronal phenotype the function of Hu antigens in the tumour cells is unknown. There is even no evidence that the anti-Hu antibodies are the cause of the neuronal damage (Sillevis Smitt et al, 1995; Carpentier et al, 1998). Nevertheless anti-Hu antibodies represent a useful diagnostic marker (Molinuevo et al, 1998). The antibodies probably are part of a more complex immune response against Hu antigens that is initially driven to control tumour growth but misdirected to cause neurological dysfunction (Posner and Dalmau, 1997). At autopsy of patients with paraneoplastic neurologic syndromes lymphocytic infiltration is found in the areas of the central nervous system that correspond to neurologic deficits. This finding supports the hypothesis that autoantibodies play a key role in the pathogenesis of the neurologic syndromes. Clinical features of paraneoplastic syndromes correspond to the underlying neurologic deficits. LEMS is characterized by muscle weakness with a predominance of the hip girdle. The motor weakness progresses in a craniocaudal direction. Patients with limbic encephalopathy usually present with rapidly progressive short-term memory deficits, psychiatric symptoms, and seizures. Paraneoplastic PNP leads to distal symmetric sensorimotor deficits. Cerebellar degeneration is characterized by ataxia. Retinopathy leads to visus loss. Opsoclonus is characterized by irregular, continual and conjugated chaotic saccades of the eyes. Opsoclonus when accompanied by other symptoms of central nervous system involvement (head, appendicular myoclonus and truncal ataxia) constitutes the opsoclonus-myoclonus syndrome. Paraneoplastic autonomic neuropathy is associated with a variety of symptoms including hypothermia, hypoventilation, sleep apnea, intestinal pseudo-obstruction, and cardiac arrhythmias. Two general approaches have been tried to treat paraneoplastic neurologic syndromes based on the assumption that these syndromes are immune-mediated: removal of the antigen source by treatment of the tumour and suppression of the immune response. LEMS can be treated 3,4-diaminopyridine or intravenous immunoglobulin that have been shown to improve muscle strength. However, evidence from studies is limited (Maddison and Newsom-Davis, 2005). In patients with

lung, breast, ovarian or stomach cancer. However, these syndromes frequently cause major disability and limitation in patients´ daily activities (Mollina-Garrido et al, 2006). Neurologic syndromes in lung cancer include the LambertEaton myasthenic syndrome (LEMS), limbic encephalopathy, polyneuropathy (PNP), cerebellar degeneration, retinopathy, opsoclonus-myoclonus, and autonomic neuropathy (Swash and Schwartz, 1990; Martina and Clay, 2005). Paraneoplastic neurologic syndromes may occur almost exclusively with SCLC. Incidence in lung cancer patients has been reported to range between 4 and 5% but may probably be lower (Swash and Schwartz, 1990). In a 1991 survey of 150 consecutive SCLC patients only two patients had LEMS (1%) and one patient suffered from PNP (< 1%) (Elrington et al, 1991). In 2005 similar results were obtained in a study of 432 consecutive patients with SCLC (LEMS: 1.6%, PNP: < 1%, subacute cerebellar degeneration: < 1%, limbic encephalitis: < 1%) (Seute et al, 2004). In a study of 200 patients with paraneoplastic encephalomyelitis and anti-Hu antibodies pathological or X-ray evidence of a tumour was obtained in 83%. Diagnosis of SCLC was made in 74% of those with histological diagnosis (Graus et al, 2001). The prognosis of patients with SCLC and paraneoplastic encephalomyelitis is poor (Spiegelman et al, 1989; Graus et al, 2001). Opsoclonus-myoclonus is a rare paraneoplastic neurologic disorder that is most often associated with SCLC (Anderson et al, 1988; Bataller et al, 2001). The presence of the anti-neuronal antibodies in the serum indicate a poor prognosis (Margery et al, 2003). In a study by Hassan and co-workes SCLC patients with opsoclonusmyoclonus died within 3 months without treatment. In contrast, in that same study with appropriate chemotherapy about half of the patients reported improvement of neurologic symptoms and several became long-term survivors (6 to 84 months) (Hassan et al, 2008). Autoimmune mechanisms seem to be responsible for the development of neurologic syndromes in cancer. Autoantibodies are commonly found in neurologic syndromes associated with cancer. Autoantibodies that are directed against ligand- or voltage-gated channels have been identified in several neuromuscular syndromes. These include antibodies against voltage-gated calcium channels (Lambert-Eaton syndrome), antibodies against voltage-gated potassium channel (acquired neuromyotonia), and antibodies against the neuronal AChR in autonomic ganglia (autoimmune autonomic ganglionopathy). There is good evidence that antibodies in these disorders cause changes in synaptic function or neuronal excitability by directly inhibiting ion channel function (Vernino, 2007). In Lambert-Eaton syndrome antibodies against the presynaptic voltage-gated calcium channels can be found. This decreases calcium entry into the presynaptic terminal which prevents binding of vesicles to the presynaptic membrane and acetylcholine release (Mareska and Gutmann, 2004). Antibodies are most often directed against voltage-gated P/Q calcium channels (VGCC) but also antibodies against voltage-gated N calcium channels

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Cancer Therapy Vol 6, page 693! free tryptophan, a precursor of serotonin, are found (Brink et al, 2002). Amounts of tryptophan are closely related with reduced food intake. Changes in hormone levels and target-organ sensitivity have also been described in tumour patients. Elevated levels of cortisol and glucagons may amplify the acute-phase protein response in cancer patients (Schaur et al, 1979; Knapp et al, 1991). In addition, several proinflammatory cytokines including (TNF)-! , (IL)-1, IL-6, interferon (IFN)-" , and ciliary neutropic factor (CNTF) have been implicated in cachexia (Lelbach et al, 2007). Apart from factors produced by the host tumour derived mediators have been described that may play an important role in the pathogenesis of cachexia. These mediators include proteolysis-inducing factor (PIF) and lipid mobilizing factor (LMF) (Cariuk et al, 1997; Hiraki et al, 1997). At present therapy for cancer cachexia is difficult. Reversing malnutrition by traditional food intake is hampered by the protein-energy deficit and the associated wasting as well as the anorexia and the early satiety. Several drugs have been used to “repair� the altered metabolism. Corticosteroids are most widely used with a short benefit. Ibuprofen has been used with some effect due to its anti-inflammatory properties. Medroxyprogesteron acetate may improve appetite and stabilize weight. Eicosapentaenoic acid can lower the production of proinflammatory cytokines. Cancer-related fatigue is also extremely common. Up to 90% of cancer patients report fatigue symptoms while in most studies prevalence rates are 60% (Cella et al, 2001). Fatigue is a highly subjective multidimensional experience. Individuals may perceive fatigue as physical tiredness or exhaustion, a need for reduced activity, reduced motivation, and/or mental fatigue (Ahlberg et al, 2003). Fatigue is experienced due to cancer and to treatment. The basic mechanisms of fatigue are broadly characterized into two main components: peripheral and central. Peripheral fatigue occurs in the neuromuscular junctions and muscle tissues. This results in the inability of the peripheral neuromuscular apparatus to perform a task in response to central stimulation. Central fatigue arises from the progressive failure to transmit motor neuron impulses. This leads to difficulties in the initiation or maintenance of voluntary activities (Chaudhuri and Behan, 2004; Ronnback and Hansson, 2004). The etiology of fatigue is poorly understood. Several underlying mechanisms have been proposed based on studies in normal (exercise) conditions and in the context of chronic diseases, including chronic fatigue syndrome and rheumatoid arthritis (Ryan et al, 2007). Several mechanisms have been shown to be involved in cancer-related fatigue. Studies in patients with chronic fatigue syndrome have demonstrated raised plasma levels of free tryptophan, which could potentially lead to high central serotonin levels (Castell et al, 1999; Badawy et al, 2005). An increase in brain serotonin (5-HT) levels and/or upregulation of a population of 5-HT receptors, may lead to reduced somatomotor drive, modified hypothalamicpituitary-adrenal (HPA) axis function, and a sensation of reduced capacity to perform physical work (Andrews et al, 2004). Low levels of circulating cortisol have been

underlying cancer chemotherapy has been successful (Verschuuren et al, 2006). For other paraneoplastic neurologic syndromes there is evidence that prompt oncologic treatment and immunotherapy (eg. Immunosuppression) can be beneficial (Keime-Guibert et al, 2000; Rosenfeld and Dalmau, 2003; Vernino et al, 2004).

X. Cutaneous syndromes A variety of paraneoplastic cutaneous syndromes has been described in lung cancer patients (refer to Table 1). However, these syndromes are mostly non-specific and can be observed in both malignant and benign disease. Palmo-plantar hyperkeratosis (PPH) also called tylosis is a rare paraneoplastic syndrome in lung cancer. In the literature it is mostly described in case reports (Schwindt et al, 1970; Nomori et al, 1996; Engin et al, 2002). It may typically precede the diagnosis of lung cancer for months or years (Burgdorf and Goltz, 1987). Prognosis of patients with lung cancer and PPH is poor (Nomori et al, 1996). Akanthosis nigricans has also been described in several case reports to be associated with adenocarcinoma, with squamous cell lung carcinoma or also with alveolar cell carcinoma (Horiuchi et al, 1986; Menzies et al, 1988; Onai et al, 1989).

XI. Cachexia and fatigue Cancer cachexia is perhaps the most common manifestation of advanced malignant disease (50%) and is responsible for approximately 25% of deaths from cancer (Lelbach et al, 2007). The degree of cachexia is inversely correlated with survival time and always implies a poor prognosis. Symptoms of cachexia include anorexia, weight loss, muscle loss, anemia, and alterations in carbohydrate, lipid and protein metabolism. Cachexia is mainly driven by anorexia and metabolic alterations. Cancer patients frequently exhibit a relative glucose intolerance and insulin resistance. While significant loss of adipose tissue is observed in cancer cachexia lipolytic rates are not significantly increased. Lipogenesis seem to be reduced. Loss of skeletal muscle protein and decreased muscle protein synthesis is commonly observed in cachexia (Dworzak et al, 1998; Giordano et al, 2003). Changes in liver protein synthesis refect aspects of the so-called acute-phase protein response. This is part of the general adaptation of the body often seen in trauma, inflammation, infection, and cancer. An acute-phase protein response can be observed in a significant proportion of patients with lung cancer. The presence of an acute-phase protein response has been related to accelerated weight loss in patients with lung cancer (Harvie et al, 2003). A variety of mediators has been described to be involved in development of cachexia (Lelbach et al, 2007). The tumour behaves like a new metabolically active organ. However, it is not clear whether the metabolic changes result from mediators released by the cancer or the host response (Argiles et al, 2003, 2008; Lelbach et al, 2004). In general, serotinergic activity in the hypothalamus decreases appetite. In cancer patients increased levels of 693

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observed in patients with chronic fatigue syndrome (Cleare, 2003). Cancer, and/or cancer treatment may also alter the function of the HPA axis, resulting in endocrine changes that cause or contribute to fatigue. Cancer may cause fatigue by circadian rhythm disruption. Several alterations in circadian function have been demonstrated in patients with cancer. These include changes in endocrine rhythms (e.g., cortisol, melatonin, and prolactin secretion), metabolic processes (e.g., temperature and circulating protein levels), the immune system (e.g., levels of circulating leukocytes and neutrophils), and rest-activity patterns (Focan et al, 1986; Mormont and Levi, 1997; Vgontzas and Chrousos, 2002; Sephton and Spiegel, 2003; Levin et al, 2005). Cancer can lead to a defect in the mechanism for regenerating ATP in skeletal muscle can compromise the ability to perform mechanical tasks (Andrews et al, 2004). Reduced oxidative muscle metabolism, epleted cellular ATP associated with a dysregulated 2',5'-oligoadenylate synthetase/RNase L pathway, and impaired synthesis of ATP have been reported (McCully et al., 1996; Lane et al, 1998; Forsyth et al, 1999). Proinflammatory cytokines, such as TNF-! and IL-1" , are implicated in many of the mechanisms proposed for the etiology of fatigue associated with cancer and various illnesses (Konsman et al, 2002). Treatment of cancer-related fatigue should be individualized according to the underlying pathology when a specific cause has been identified. In addition to older therapies, such as hematopoietics, antidepressants, corticosteroids, and psychostimulants, the new wakepromoting agent modafinil may offer an alternative therapeutic approach.

XII. Conclusions Paraneoplastic syndromes are common in lung cancer patients. Some paraneoplastic syndromes can severely affect organ function and quality of life. Treating the underlying cancer is the first step. However, specific therapy may also be necessary.

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Cancer Therapy Vol 6, page 699! Cancer Therapy Vol 6, 699-706, 2008

A clinical study of T-regulatory lymphocyte function in cancer patients in relation to tumor histotype, disease extention, lymphocyte subtypes and cortisol secretion Research Article

Luigi Vigorè1, Fernando Brivio2, Luca Fumagalli2, Roberto Vezzo1, Giusy Messina6, Franco Rovelli6, Massimo Colciago3, Giovanna!D’Amico4, Giuseppe Di Fede5, Paolo Lissoni6 1

Laboratory of Immunomicrobiology, St.Gerardo Hospital, Monza, Milan, Italy Division of Surgery, St.Gerardo Hospital, Monza, Milan, Italy 3 I.N.R.C.A Laboratory of Analyses, Casatenovo, Lecco, Italy 4 Research Center “Fondazione Tettamanti” Clinica Pediatrica Università Milano-Bicocca, Italy 5 Institute of Biological Medicine, Milan, Italy 6 Division of Radiation Oncology, St.Gerardo Hospital, Monza, Milan, Italy 2

__________________________________________________________________________________! *Correspondence: Dr. Paolo Lissoni, Divisione di Radioterapia Oncologica, Ospedale S.Gerardo, 20052 Monza, Milano, Italia; Fax: +390392332284, e-mail: p.lissoni@hsgerardo.org Key words: Anticancer immunity, immunosuppression, T regulatory lymphocytes Abbreviations: cytotoxic T lymphocyte-associated antigen-4, (CTLA-4); glucocorticoid-induced TNF-! receptor, (GITR); myeloidderived suppressor cells, (MDSC); NK cells, (CD16CD56); T cytotoxic lymphocytes, (CD8); T helper lymphocytes, (CD4); Tregulatory lymphocytes, (T-reg) Received: 24 July 2008; Revised: 11 September 2008 Accepted: 12 September 2008; electronically published: October 2008

Summary Several clinical investigations showed that the immune status is a prognostic variable in cancer patients, even tough the evaluation of the anticancer immunity is not generally considered in the medical oncology. Several immune parameters, including lymphocyte subsets and cytokine blood concentration, had been proposed to quantify the functional status of the anticancer immunity, but recent discoveries would suggest that the end-result of the various immune interactions is represented by a subtype of CD4 lymphocytes capable of suppressing the antitumor immune reaction, the so called T-regulatory lymphocytes (T-reg). This study was performed to detect T-reg count and percentage in solid tumor patients, in relation to tumor histotype, disease extension, lymphocyte sub-populations and cortisol circadian secretion. The study included 114 consecutive cancer patients affected by the most frequent tumor histotypes, 69 of whom showed a metastatic disease. In each patient we evaluated T-reg cells, identified as CD4+CD25+, in relation to T helper (CD4), T cytotoxic (CD8) and NK (CD16CD56) cells. Abnormally high values of T-reg cells were seen in 52/114 (46%) patients, and the percentage of high values of T-reg was significantly higher in metastatic patients than in non-metastatic ones. In contrast, no significant difference was seen in relation to tumor histotype. Patients with increased T-reg count had a significantly lower NK cell number. Finally no significant difference in T-reg number was seen between patients with altered or normal rithm of cortisol. The study confirmed that, irrespectively of tumor histotype the metastatic disease is associated with a progressive and increased T-reg generation, with a following suppression of anticancer immunity.

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Vigorè et al: T regulator lymphocytes in cancer patients ! cortisol (Sthephens et al, 2004), that in contrast may inhibit the activity of the most other T lymphocytes, namely that of T helper lymphocytes, with a following diminished production of IL-2 (Claman, 1998). As far as the mechanisms responsible for T-reg-induced suppression of the anticancer immunity are concerned, several experimental observations have shown that T-reg cells may suppress the antitumor immune response through the release of immunosuppressive cytokines, namely IL-10 and TGF-" (Dieckmann et al, 2002), even though other authors would suggest that the suppressive activity of Treg cells on CD4+ and CD8+ lymphocyte activation may be relatively independent from the action of cytokines, by mainly requiring cell surface contact (Birebent et al, 2004). IL-2 has been proven to be essential for T-reg generation and some authors consider IL-2 as the main growth factor of T-reg lymphocytes (Antony and Restito, 2005), but more adequate studies have demonstrated that IL-2 may induce both stimulation and inhibition of T-reg generation and activation (Malek and Bayer, 2004). In fact, IL-2 has appeared to induce and promote T-reg differentiation only in the presence of TGF-" (Chen et al, 2003). Therefore, IL-2 would constitute the main human cytokine in influencing the characteristics of the anticancer immunity, since it may be responsible for both activation and suppression of an effective immune response against cancer cell proliferation and dissemination (Wang et al, 2001), namely depending on the whole status of the cytokine network, in particular on the presence or in the absence of adequate concentrations of TGF-" . In the absence of TGF-" , IL-2 stimulates the anticancer immunity, whereas it counteracts the generation of an effective antitumor immunity in the presence of TGF-" . In other words, IL-2 would physiologically control both tolerance and immunity, depending on the presence of TGF-" and other less known factors (Annunziato et al, 2002). In fact, under cancer immunotherapy with IL-2 the percent of T-reg cells has been shown to decrease in responding patients and to enhance in those with disease progression (Cesana et al, 2006). However, the regulation of T-reg functions does not depend only on immune factors, since it is also under a neuroendocrine control (Ji et al, 2004). In particular, cortisol has appeared to stimulate T-reg cell generation (Ji et al, 2004), with a following enhanced release of IL-10, by representing the main mechanism responsible for cortisol-induced immunosuppression. From a clinical oncological point of view, preliminary observations showed an enhanced percent of circulating CD4+CD25+ lymphocytes in cancer patients, namely in those with advanced disease (Sasada et al, 2003). The present study was performed to better establish which is T-reg behaviour in cancer patients in relation to both tumor histotype and disease extension.

I. Introduction At present, there is no doubt about the existence of a sub-type of T lymphocytes, the so-called T regulatory lymphocytes (T-reg), capable of suppressing the cellular immune responses,including the anticancer immunity (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006). However, the exact definition of T-reg cells in terms of cell surface marker expression still remains controversial, particularly from a clinical point of view. All authors are in agreement to consider Treg lymphocytes as CD4+CD25+ cells, but at present it is still unknown whether the expression of CD4 and CD25 antigens may be sufficient to identify T-reg cells (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006), since several authors retain that the intracytoplasmatic expression of the FOX p3 protein is essential for the differentiation into T-reg cells (Ziegler, 2006; Zou, 2006).Recently, however, some preliminary observations would suggest that the cytoplasmatic expression of FOX p3 by CD4+CD25+ cells may be associated at least in some experimental conditions with a diminished, rather than with an enhanced immunosuppressive activity of T-reg cells (Siddiqui et al, 2007). In contrast, all authors agree that the expression of CD152 antigen, also called cytotoxic T lymphocyteassociated antigen-4 (CTLA-4) (Vasu et al, 2004), is fundamental for the immunosuppressive activity of T-reg cells (Takahashi et al, 2000), since the block of its expression by using anti-CTLA-4 monoclonal antibodies may abolish the suppressive activity of T-reg cells, with a following stimulation of the anticancer immunity in cancer patients (Knutson and Disis, 2007) and an enhanced incidence of autoimmune diseases in the healthy subjects (Lan et al, 2005). Therefore, the addition of a third marker, such as CD152 antigen, may allow to define a more homogeneous cell population provided by a regulatory activity with respect to the simple CD4+CD25+ expression (Dieckmann and Plottner, 2001). In fact, the suppressive regulatory action of CD4+CD25+CD152+ has appeared to be clearly higher than that played by the simple CD4+CD25+ T lymphocytes (Leong et al, 2006).This finding is not surprising, since the simple expression of CD25 marker, corresponding to the ! -chain of IL-2 receptor, is not an exclusive characteristic of T-reg lymphocytes, but it is a non-specific property of the overall activated T lymphocytes (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006). At present, preliminary clinical studies would show that the percent of circulating CD4+CD25+ cells may be about 10% of the all CD4+ lymphocytes, and that of CD4+CD25+CD152+ cells may be about 40% of the total CD4+CD25+ cells, then the expected percent of CD4+CD25+CD152+ in the healthy subjects would be less than 5% of the total circulating CD4+ lymphocytes (Jago et al, 2004). Finally, the expression of glucocorticoidinduced TNF-! receptor (GITR) is also associated with an evident suppressive activity by T-reg lymphocytes (Kanamaru et al, 2004), which in fact are stimulated by

II. Materials and methods The study included 114 consecutive solid tumor patients with locally limited or metastatic disease, whose clinical characteristics are shown in Table 1. Lung cancer and gastrointestinal tumors were the most frequent neoplasms in our patients. For the immune detections, venous blood samples were collected in the morning after an overnight fast. Operable patients and metastatic patients were investigated before the

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Cancer Therapy Vol 6, page 701! surgical operation and before the onset of chemotherapy, respectively, in an attempt to exclude the possible influence of the various anticancer therapies on the immune status of patients. In each sample, we have evaluated total lymphocyte count and the various lymphocyte subpopulations by a flow cytometric assay and monoclonal antibodies, including T helper lymphocytes (CD4), T cytotoxic lymphocytes (CD8), NK cells (CD16CD56), and T regulatory (T-reg) lymphocytes (CD4CD25). Normal values (95% confidence limits) of T-reg observed in our laboratory were below 240/mm3. Moreover, because of its importance in regulating lymphocyte functions and proliferation (Claman, 1998; Sthephens et al, 2004), the circadian rhythm of cortisol was also investigated by collecting blood samples at 8.00 A.M. and at 4.00 P.M., and cortisol serum concentrations were measured in duplicate by using the ECLA method. Data were reported as mean +/- SE, and statistically analyzed by the Student’s t test, the analysis of variance and the chi-square test, as appropriate.

(64%) vs 8/45(18%), ! < 0.01). Table 3 shows the mean number of T-reg and the mean percentages of T-reg with respect to both total lymphocytes and T helper (CD4+) lymphocytes observed in cancer patients in relation to their disease extension. The mean number of T-reg observed in metastatic patients was higher with respect to that found in patients with locally limited disease, without, however statistically significant differences. In contrast, the mean percentages of T-reg with respect to that of both lymphocytes and CD4 cells were significantly higher in metastatic patients than in the non-metastatic ones (!< 0.05 and !< 0.001,respectively). Moreover, within the metastatic group, patients with a normal lymphocyte count greater than 1500/mm3 showed a significantly higher mean number of T-reg with respect to the non-metastatic patients, whereas no difference was seen between nonmetastatic patients and metastatic patients with lymphocytopenia, consisting of lymphocyte count lower than 1500/mm3. In contrast, the mean percentages of T-reg with respect to total lymphocytes and CD4+ cells observed in both groups of metastatic patients with normal or low total lymphocyte count were significantly higher than in non-metastatic patients (lymphocytes: !< 0.025, CD4+

III. Results As reported in Table 2, an abnormally high number of T-reg was seen in 52/114 (46%) patients. Moreover, the percentage of cases with elevated number of T-reg observed in metastatic patients was significantly higher with respect to that found in non-metastatic patients (44/69 Table 1. Clinical characteristics of 114 solid tumor patients.

Characteristics M/F Median Age (years) Median performance status (Karnofsky’s score) Tumor histotypes Non-small cell lung cancer Breast cancer Colorectal cancer Gastric cancer Pancreatic cancer Prostate cancer Gynaecologic neoplasms Disease extension locally limited disease metastatic disease Dominant metastasis sites Soft tissues Bone Lung Liver Lung + liver Serouses

n 53/61 59 (35-76) 90 (70-100)

28 25 19 12 11 10 9 45 69 6 11 18 16 9 9

Table 2. Percentages of abnormally high values of CD4+CD25+ lymphocytes. Patients Overall patients Non-metastatic patients Metastatic patients

n (%) 52 / 114 (46%) 8 / 45 (18%) 44 / 69 (64%)*

"#$ < 0.01 %& non-metastatic patients.

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Vigorè et al: T regulator lymphocytes in cancer patients ! Table 3. Mean number of CD4+CD25+ lymphocytes and their mean percentages with respect to total lymphocytes and CD4+ lymphocytes in metastatic and non-metastatic patients. Patients

n

CD4+CD25+Cells (X ± SE)

% Lymphocytes (X ± SE)

% CD4+ Cells (X ± SE)

Non-metastatic patients Metastatic patients Low lymphocytes number Normal lymphocytes number

45 69 18 51

185 ± 38 304 ± 49 219 ± 54 457 ± 61***

9±2 20 ± 3 * 22 ± 3 19 ± 2**

22 ± 4 48 ± 5**** 51 ± 6**** 47 ± 4****

* !<0.05 "# non-metastatic patients; ** !<0.025 "# non-metastatic patients; *** !<0.01 "# non-metastatic patients; **** !<0.001 "# nonmetastatic patients

patients, without however significant differences with respect to the overall other histotypes. The metastatic disease was associated with a higher number of T-reg with respect to the non-metastatic group in all tumor histotypes, even though a statistically significant differences occurred for the only breast cancer (!<0.05) and colorectal cancer (!< 0.01).

cells: !< 0.001). The mean counts of NK and CD8 cells in relation to that of T-reg are reported in Table 4. As shown, no significant difference in the mean number of CD8 lymphocytes was found between patients with normal or abnormally elevated number of T-reg. On the contrary, patients with elevated number of T-reg showed a significantly lower number of NK cells with respect to that found in those with normal T-reg count. Finally, Table 5 shows the circadian rhythm of cortisol in relation to total lymphocytes, CD4+ cells and T-reg mean number. A normal cortisol rhythm, with morning values greater at least than 50% with respect to the values occurring during the afternoon, was found in 85/114 (75%). Total lymphocyte and CD4+ cell mean numbers observed in patients with altered cortisol rhythm were significantly lower than those found in patients with normal cortisol circadianicity (!<0.01), whereas no significant difference was seen in the mean number of T reg. Figure 1 and Figure 2 illustrate T-reg mean numbers in relation to tumor histoptypes in the overall patients and with respect to their disease extension, respectively. No significant difference was seen in relation to tumor histotype. The highest values of T-reg were observed in pancreatic cancer

IV. Discussion According to previous preliminary clinical investigations (Sasada et al, 2003; Cesana et al, 2006), this study confirms in a greater number of cancer patients that the metastatic disease is characterized by the evidence of an abnormally increased percentage of T-reg lymphocytes with respect to both total circulating lymphocytes and CD4+ lymphocytes. This finding does not seem to represent a specific characteristic of some tumor histotypes, then it could constitute a general alteration occurring during the progression of the neoplastic disease, by representing a fundamental immune parameter of cancer-related immunosuppression.

Table 4. Mean values of NK cells and CD8+ lymphocytes in cancer patients with normal or abnormally high values of CD4+CD25+ lymphocytes. Patients

n

Normal values of CD4+CD25+ cells High values of CD4+CD25+ cells

62 52

NK cells (n/mm3) (X ± SE) 339 ± 34 204 ± 22 *

CD8+ lymphocytes (n/mm3) (X ± SE) 439 ± 55 506 ± 57

* !<0.05 "# normal values of CD4+CD25+ lymphocytes.

Table 5. Mean numbers of total lymphocytes, T helper (CD4+) lymphocytes and T regulator lymphocytes (CD4+CD25+) in relation to cortisol circadian secretion in cancer patients. Cortisol circadian secretion Normal cortisol rhythm Altered cortisol rhythm

n 85 29

Lymphocytes 1978 ± 69 * 1537 ± 81

* $<0.01 "# patients with altered cortisol rhythm

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N/mm3 (X ± SE) CD4+ cells CD4+CD25+ cells 944 ± 65 * 209 ± 48 623 ± 87 244 ± 55

Cancer Therapy Vol 6, page 703!

Figure 1. CD4+CD25+ lymphocyte mean number in relation to tumor histotype.

Figure 2. CD4+CD25+ lymphocytes in relation to tumor histotype in metastatic and non-metastatic cancer patients.

their mechanisms of action may be represented by the stimulation of T-reg generation, with a consequent inhibition of the activation of an effective anticancer

Several immune molecules have appeared to suppress the anticancer immunity, namely IL-6, IL-10, IL-1, TNF-! and TGF-" , but it seems that the common end result of 703

Vigorè et al: T regulator lymphocytes in cancer patients ! cells are dependent on CTLA-4 expression. Eur J Immunol 34, 3485-96. Cesana GC, DeRaffele G, Cohen S, Moroziewicz D, Mitcham J, Stoutenburg J, Cheung K, Hesdorffer C, Kim-Schulze S, Kaufman HL (2006) Characterization of CD4+CD25+ regulatory T cells in patients treated with high-dose interkleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol 24, 1169-77. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM (2003) Conversion of peripheral CD4+CD25naïve T cells to CD4+CD25+ regulatory T cells by TGF-! induction of transcription factor Foxp 3. J Exp Med 198, 1875-86. Claman HN (1998) Corticosteroids and the immune system. Adv Exp Med Biol 245, 203-10. Dieckmann D, Bruett H, Ploettner H, Lutz MB, Schuler G (2002) Human CD4+CD25+ regulatory contact-dependent T cell induce IL-10 producing,contact-independent type-1regulatory T cells. J Exp Med 196, 247-53. Dieckmann D, Plottner H (2001) Ex vivo isolation and characterizationof CD4+CD25+ T cells with regulatory properties from human blood. J Exp Med 193, 1303-10. Ehrke MJ, Mihich E, Berd D, Mastrangelo MJ (1989) Effects of anticancer drugs on the immune system. Semin Oncol 16, 230-9. Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, Chauffert B, Solary E, Bonnotte B, Martin F (2004) CD4+ CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows Immunotherapy of established tumors to be curative. Eur J Immunol 34, 336-44. Ikemoto S, Yoshida N, Narita K, Wada S, Kishimoto T, Sugimura K, Nakatani T (2003) Role of tumor-associated macrophages in renal cell carcinoma. Oncol Rep 10, 1843-9. Jago CB, Yates J, Camara NOS, Lechler RI, Lombardi AG (2004) Differential expression of CTLA-4 among T cell subsets. Clin Exp Immunol 136, 463-71. Ji HB, Liao G, Faubion WA, Abadía-Molina AC, Cozzo C, Laroux FS, Caton A, Terhorst C (2004) Cutting edge, the natural ligand for glucocorticoid-induced TNF receptorrelated protein abrogates regulatory T cell suppression. J Immunol 172, 5823-7. Kanamaru F, Youngnak P, Hashiguchi M, Nishioka T, Takahashi T, Sakaguchi S, Ishikawa I, Azuma M (2004) Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. Immunol 172, 7306-14. Knutson KL, Disis M, Salazar L (2007) CD4 regulatory T cells in human cancer pathogenesis. Cancer Immunol Immunother 556, 271-85. Kusmartsev S, Gabrilovich DI (2005) STAT1 signaling regulates tumor-assolciated macrophage-mediate T cell deletion. J Immunol 174, 4880-91. Lan RY, Ansari AA, Lian ZX, Gershwin ME (2005) Regulatory T cells, development,function,and role in autoimmunity. Autoimmun Rev 4, 351-63. Leong PP, Mohammad R, Ibrahim N, Ithnin H, Abdullah M, Davis WC, Seow HF (2006) Phenotyping of lymphocytes expressing regulatory and effector markers in infiltrating ductal carcinoma of the breast. Immunol Lett 102, 229-36. Malek TR, Bayer AL (2004) Tolerance not immunity crucially depends on IL-2. Nat Rev Immunol 4, 665-74. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25, 677-86. Sasada T, Kimura M, Yoshida Y, Kanai M, Takabayashi A (2003) CD4+CD25+ regulatory T cells in patients with

immune reaction. On the same way, several immune cells are able to suppress the anticancer immunity, including macrophages, T helper-2 lymphocytes and some myeloidderived suppressor cells, but also in this case they would act in a suppressive way by promoting the generation of Treg. Then, the detection of T-reg amounts in terms of both absolute number and percentages with respect to total lymphocytes and CD4+ cells could constitute a simple and adequate clinical immune parameter to quantify the whole status of the anticancer immunity in the single cancer patient. Moreover, future clinical studies will be required to establish the possible prognostic significance of changes in T-reg percentage and number in relation to the anticancer efficacy of the various standard antitumor therapies. Moreover, it has to be remarked that T-reg lymphocytes would not represent the only immune cells involved in the suppression of the anticancer immunity. In fact, there is at least another fundamental immunosuppressive system, consisting of the monocytemacrophage cell lineage (Sica and Bronte, 2007). In more detail, it has been observed that the bone marrow may release myeloid precursors provided by suppressive activity on the antitumor immune response and defined as myeloid-derived suppressor cells (MDSC) (Kusmartsev and Gabrilovich, 2005). These cells have appeared to be characterized by the cell surface expression of GR-1, CD11b and CD80 antigens (Anderson et al, 2002; van Ginderachter et al, 2006). The myeloid suppressor cells would promote the generation and activation of T-reg lymphocytes, which at the other side would stimulate MDSC release from the bone marrow and M2 macrophage differentiation (Terabe et al, 2003; Wie et al, 2006). Moreover, the myeloid suppressive cells would inhibit the anticancer immunity by promoting macrophage differentiation into the M2 sub-type (Ikemoto et al, 2003), which plays a clear inhibitory effect on the anticancer immunity, namely through the release of IL-6 (Ueno et al, 2000), whereas the M1 macrophage sub-type may either stimulate or suppress the antitumor immunity (Mantovani et al, 2004). M1 and M2 macrophage sub-types have appeared to be characterized by a high production of IL-12 or IL-10, respectively (Ueno et al, 2000). Then, further studies by concomitantly evaluating T reg and MDSC count, will contribute to better define the immune mechanism responsible for the suppression of the anticancer immunity.

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Cancer Therapy Vol 6, page 705! gastrointestinal malignancies, possible involvement of regulatory T cells in disease progression. Cancer 98, 108993. Schwartz RH (2005) Natural regulatory T cells and selftolerance. Nat Immunol 6, 327-30. Shevach EM (2002) CD4+CD25+ suppressor T cells, more questions than answers. Nat Rev Immunol 2, 389-400. Sica A, Bronte V (2007) Altered macrophage differentiation and immune dysfunction in tumor development. J Clin Invest 117, 1155-66. Siddiqui SA, Frigola X, Bonne-Annee S, et al (2007) Tumorinfiltrating Foxp3 CD4+CD25+ T cells predict poor survuival in renal cell carcinoma. Clin Cancer Res 13, 2075-81. Stephens GL, McHugh RS, Whitters MJ, Young DA, Luxenberg D, Carreno BM, Collins M, Shevach EM (2004) Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J Immunol 173, 500820. Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, Mak TW, Sakaguchi S (2000) Immunologic and tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen. J Exp Med 192, 1285-94. Terabe M, Matsui S, Park JM, Mamura M, Noben-Trauth N, Donaldson DD, Chen W, Wahl SM, Ledbetter S, Pratt B, Letterio JJ, Paul WE, Berzofsky JA (2003) Transforming growth factor-! production and myeloid cells are an eggector mechanism trhough which CD1d-restricted T cells block cytotoxic T lymphocyte-mediated tumor immunosurveillance, abrogation prevents tumor recurrence. J Exp Med 198, 1741-52. Thomton AM, Shevach EM (2000) Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J Immunol 164, 183-90. Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, Koike M, Inadera H, Matsushima K (2000) Significance of macrophage chemo-attractant protein-1 in macrophage recruitment,angiogenesis and survival in human breast cancer. Clin Cancer Res 6, 3282-9. Van Ginderachter JA, Movahedi K, Hassanzadeh Ghassabeh G, Meerschaut S, Beschin A, Raes G, De Baetselier P (2006) Classical and alternative activation of mononuclear phagocytes, picking the best of both worlds for tumor promotion. Immunobiology 211, 487-501.

Vasu C, Prabhakar BS, Holterman MJ (2004) Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4 high T regulatory cells with target alloantigens specificity. J Immunol 173, 2866-76. von Boehmer H (2005) Mechanisms of suppression by suppressor T cells. Nat Immunol 6, 338-44. von Herrath MG, Harrison LC (2003) Regulatory lymphocytes, antigen-induced regulatory T cells in autoimmunity. Nat Rev Immunol 3, 223-32. Wang XB, Zheng CY, Giscombe R, Lefvert AK (2001) Regulation of surface and intracellularexpression of CTLA-4 on human peripheral T cells. Scand J Immunol 54, 453-8. Wie S, Kryczeck I, Zou W (2006) Regulatory T-cell compartmentalization and trafficking. Blood 108, 426-31. Yang R, Cai Z, Zhang Y, Yutzy WH 4th, Roby KF, Roden RB (2006) CD80 in immune suppression by mouse ovarian carcinoma-associated Gr-1+CD11b+ myeloid cells. Cancer Res 66, 6807-15. Ziegler SF (2006) FOXP3 of mice and men. Annu Rev Immunol 24, 209-26. Zou W (2006) Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 6, 295-307.

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Cancer Therapy Vol 6, page 699! Cancer Therapy Vol 6, 699-706, 2008

A clinical study of T-regulatory lymphocyte function in cancer patients in relation to tumor histotype, disease extention, lymphocyte subtypes and cortisol secretion Research Article

Luigi Vigorè1, Fernando Brivio2, Luca Fumagalli2, Roberto Vezzo1, Giusy Messina6, Franco Rovelli6, Massimo Colciago3, Giovanna!D’Amico4, Giuseppe Di Fede5, Paolo Lissoni6 1

Laboratory of Immunomicrobiology, St.Gerardo Hospital, Monza, Milan, Italy Division of Surgery, St.Gerardo Hospital, Monza, Milan, Italy 3 I.N.R.C.A Laboratory of Analyses, Casatenovo, Lecco, Italy 4 Research Center “Fondazione Tettamanti” Clinica Pediatrica Università Milano-Bicocca, Italy 5 Institute of Biological Medicine, Milan, Italy 6 Division of Radiation Oncology, St.Gerardo Hospital, Monza, Milan, Italy 2

__________________________________________________________________________________! *Correspondence: Dr. Paolo Lissoni, Divisione di Radioterapia Oncologica, Ospedale S.Gerardo, 20052 Monza, Milano, Italia; Fax: +390392332284, e-mail: p.lissoni@hsgerardo.org Key words: Anticancer immunity, immunosuppression, T regulatory lymphocytes Abbreviations: cytotoxic T lymphocyte-associated antigen-4, (CTLA-4); glucocorticoid-induced TNF-! receptor, (GITR); myeloidderived suppressor cells, (MDSC); NK cells, (CD16CD56); T cytotoxic lymphocytes, (CD8); T helper lymphocytes, (CD4); Tregulatory lymphocytes, (T-reg) Received: 24 July 2008; Revised: 11 September 2008 Accepted: 12 September 2008; electronically published: October 2008

Summary Several clinical investigations showed that the immune status is a prognostic variable in cancer patients, even tough the evaluation of the anticancer immunity is not generally considered in the medical oncology. Several immune parameters, including lymphocyte subsets and cytokine blood concentration, had been proposed to quantify the functional status of the anticancer immunity, but recent discoveries would suggest that the end-result of the various immune interactions is represented by a subtype of CD4 lymphocytes capable of suppressing the antitumor immune reaction, the so called T-regulatory lymphocytes (T-reg). This study was performed to detect T-reg count and percentage in solid tumor patients, in relation to tumor histotype, disease extension, lymphocyte sub-populations and cortisol circadian secretion. The study included 114 consecutive cancer patients affected by the most frequent tumor histotypes, 69 of whom showed a metastatic disease. In each patient we evaluated T-reg cells, identified as CD4+CD25+, in relation to T helper (CD4), T cytotoxic (CD8) and NK (CD16CD56) cells. Abnormally high values of T-reg cells were seen in 52/114 (46%) patients, and the percentage of high values of T-reg was significantly higher in metastatic patients than in non-metastatic ones. In contrast, no significant difference was seen in relation to tumor histotype. Patients with increased T-reg count had a significantly lower NK cell number. Finally no significant difference in T-reg number was seen between patients with altered or normal rithm of cortisol. The study confirmed that, irrespectively of tumor histotype the metastatic disease is associated with a progressive and increased T-reg generation, with a following suppression of anticancer immunity.

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Vigorè et al: T regulator lymphocytes in cancer patients ! cortisol (Sthephens et al, 2004), that in contrast may inhibit the activity of the most other T lymphocytes, namely that of T helper lymphocytes, with a following diminished production of IL-2 (Claman, 1998). As far as the mechanisms responsible for T-reg-induced suppression of the anticancer immunity are concerned, several experimental observations have shown that T-reg cells may suppress the antitumor immune response through the release of immunosuppressive cytokines, namely IL-10 and TGF-" (Dieckmann et al, 2002), even though other authors would suggest that the suppressive activity of Treg cells on CD4+ and CD8+ lymphocyte activation may be relatively independent from the action of cytokines, by mainly requiring cell surface contact (Birebent et al, 2004). IL-2 has been proven to be essential for T-reg generation and some authors consider IL-2 as the main growth factor of T-reg lymphocytes (Antony and Restito, 2005), but more adequate studies have demonstrated that IL-2 may induce both stimulation and inhibition of T-reg generation and activation (Malek and Bayer, 2004). In fact, IL-2 has appeared to induce and promote T-reg differentiation only in the presence of TGF-" (Chen et al, 2003). Therefore, IL-2 would constitute the main human cytokine in influencing the characteristics of the anticancer immunity, since it may be responsible for both activation and suppression of an effective immune response against cancer cell proliferation and dissemination (Wang et al, 2001), namely depending on the whole status of the cytokine network, in particular on the presence or in the absence of adequate concentrations of TGF-" . In the absence of TGF-" , IL-2 stimulates the anticancer immunity, whereas it counteracts the generation of an effective antitumor immunity in the presence of TGF-" . In other words, IL-2 would physiologically control both tolerance and immunity, depending on the presence of TGF-" and other less known factors (Annunziato et al, 2002). In fact, under cancer immunotherapy with IL-2 the percent of T-reg cells has been shown to decrease in responding patients and to enhance in those with disease progression (Cesana et al, 2006). However, the regulation of T-reg functions does not depend only on immune factors, since it is also under a neuroendocrine control (Ji et al, 2004). In particular, cortisol has appeared to stimulate T-reg cell generation (Ji et al, 2004), with a following enhanced release of IL-10, by representing the main mechanism responsible for cortisol-induced immunosuppression. From a clinical oncological point of view, preliminary observations showed an enhanced percent of circulating CD4+CD25+ lymphocytes in cancer patients, namely in those with advanced disease (Sasada et al, 2003). The present study was performed to better establish which is T-reg behaviour in cancer patients in relation to both tumor histotype and disease extension.

I. Introduction At present, there is no doubt about the existence of a sub-type of T lymphocytes, the so-called T regulatory lymphocytes (T-reg), capable of suppressing the cellular immune responses,including the anticancer immunity (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006). However, the exact definition of T-reg cells in terms of cell surface marker expression still remains controversial, particularly from a clinical point of view. All authors are in agreement to consider Treg lymphocytes as CD4+CD25+ cells, but at present it is still unknown whether the expression of CD4 and CD25 antigens may be sufficient to identify T-reg cells (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006), since several authors retain that the intracytoplasmatic expression of the FOX p3 protein is essential for the differentiation into T-reg cells (Ziegler, 2006; Zou, 2006).Recently, however, some preliminary observations would suggest that the cytoplasmatic expression of FOX p3 by CD4+CD25+ cells may be associated at least in some experimental conditions with a diminished, rather than with an enhanced immunosuppressive activity of T-reg cells (Siddiqui et al, 2007). In contrast, all authors agree that the expression of CD152 antigen, also called cytotoxic T lymphocyteassociated antigen-4 (CTLA-4) (Vasu et al, 2004), is fundamental for the immunosuppressive activity of T-reg cells (Takahashi et al, 2000), since the block of its expression by using anti-CTLA-4 monoclonal antibodies may abolish the suppressive activity of T-reg cells, with a following stimulation of the anticancer immunity in cancer patients (Knutson and Disis, 2007) and an enhanced incidence of autoimmune diseases in the healthy subjects (Lan et al, 2005). Therefore, the addition of a third marker, such as CD152 antigen, may allow to define a more homogeneous cell population provided by a regulatory activity with respect to the simple CD4+CD25+ expression (Dieckmann and Plottner, 2001). In fact, the suppressive regulatory action of CD4+CD25+CD152+ has appeared to be clearly higher than that played by the simple CD4+CD25+ T lymphocytes (Leong et al, 2006).This finding is not surprising, since the simple expression of CD25 marker, corresponding to the ! -chain of IL-2 receptor, is not an exclusive characteristic of T-reg lymphocytes, but it is a non-specific property of the overall activated T lymphocytes (Thomton and Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006). At present, preliminary clinical studies would show that the percent of circulating CD4+CD25+ cells may be about 10% of the all CD4+ lymphocytes, and that of CD4+CD25+CD152+ cells may be about 40% of the total CD4+CD25+ cells, then the expected percent of CD4+CD25+CD152+ in the healthy subjects would be less than 5% of the total circulating CD4+ lymphocytes (Jago et al, 2004). Finally, the expression of glucocorticoidinduced TNF-! receptor (GITR) is also associated with an evident suppressive activity by T-reg lymphocytes (Kanamaru et al, 2004), which in fact are stimulated by

II. Materials and methods The study included 114 consecutive solid tumor patients with locally limited or metastatic disease, whose clinical characteristics are shown in Table 1. Lung cancer and gastrointestinal tumors were the most frequent neoplasms in our patients. For the immune detections, venous blood samples were collected in the morning after an overnight fast. Operable patients and metastatic patients were investigated before the

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Cancer Therapy Vol 6, page 701! surgical operation and before the onset of chemotherapy, respectively, in an attempt to exclude the possible influence of the various anticancer therapies on the immune status of patients. In each sample, we have evaluated total lymphocyte count and the various lymphocyte subpopulations by a flow cytometric assay and monoclonal antibodies, including T helper lymphocytes (CD4), T cytotoxic lymphocytes (CD8), NK cells (CD16CD56), and T regulatory (T-reg) lymphocytes (CD4CD25). Normal values (95% confidence limits) of T-reg observed in our laboratory were below 240/mm3. Moreover, because of its importance in regulating lymphocyte functions and proliferation (Claman, 1998; Sthephens et al, 2004), the circadian rhythm of cortisol was also investigated by collecting blood samples at 8.00 A.M. and at 4.00 P.M., and cortisol serum concentrations were measured in duplicate by using the ECLA method. Data were reported as mean +/- SE, and statistically analyzed by the Student’s t test, the analysis of variance and the chi-square test, as appropriate.

(64%) vs 8/45(18%), ! < 0.01). Table 3 shows the mean number of T-reg and the mean percentages of T-reg with respect to both total lymphocytes and T helper (CD4+) lymphocytes observed in cancer patients in relation to their disease extension. The mean number of T-reg observed in metastatic patients was higher with respect to that found in patients with locally limited disease, without, however statistically significant differences. In contrast, the mean percentages of T-reg with respect to that of both lymphocytes and CD4 cells were significantly higher in metastatic patients than in the non-metastatic ones (!< 0.05 and !< 0.001,respectively). Moreover, within the metastatic group, patients with a normal lymphocyte count greater than 1500/mm3 showed a significantly higher mean number of T-reg with respect to the non-metastatic patients, whereas no difference was seen between nonmetastatic patients and metastatic patients with lymphocytopenia, consisting of lymphocyte count lower than 1500/mm3. In contrast, the mean percentages of T-reg with respect to total lymphocytes and CD4+ cells observed in both groups of metastatic patients with normal or low total lymphocyte count were significantly higher than in non-metastatic patients (lymphocytes: !< 0.025, CD4+

III. Results As reported in Table 2, an abnormally high number of T-reg was seen in 52/114 (46%) patients. Moreover, the percentage of cases with elevated number of T-reg observed in metastatic patients was significantly higher with respect to that found in non-metastatic patients (44/69 Table 1. Clinical characteristics of 114 solid tumor patients.

Characteristics M/F Median Age (years) Median performance status (Karnofsky’s score) Tumor histotypes Non-small cell lung cancer Breast cancer Colorectal cancer Gastric cancer Pancreatic cancer Prostate cancer Gynaecologic neoplasms Disease extension locally limited disease metastatic disease Dominant metastasis sites Soft tissues Bone Lung Liver Lung + liver Serouses

n 53/61 59 (35-76) 90 (70-100)

28 25 19 12 11 10 9 45 69 6 11 18 16 9 9

Table 2. Percentages of abnormally high values of CD4+CD25+ lymphocytes. Patients Overall patients Non-metastatic patients Metastatic patients

n (%) 52 / 114 (46%) 8 / 45 (18%) 44 / 69 (64%)*

"#$ < 0.01 %& non-metastatic patients.

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Vigorè et al: T regulator lymphocytes in cancer patients ! Table 3. Mean number of CD4+CD25+ lymphocytes and their mean percentages with respect to total lymphocytes and CD4+ lymphocytes in metastatic and non-metastatic patients. Patients

n

CD4+CD25+Cells (X ± SE)

% Lymphocytes (X ± SE)

% CD4+ Cells (X ± SE)

Non-metastatic patients Metastatic patients Low lymphocytes number Normal lymphocytes number

45 69 18 51

185 ± 38 304 ± 49 219 ± 54 457 ± 61***

9±2 20 ± 3 * 22 ± 3 19 ± 2**

22 ± 4 48 ± 5**** 51 ± 6**** 47 ± 4****

* !<0.05 "# non-metastatic patients; ** !<0.025 "# non-metastatic patients; *** !<0.01 "# non-metastatic patients; **** !<0.001 "# nonmetastatic patients

patients, without however significant differences with respect to the overall other histotypes. The metastatic disease was associated with a higher number of T-reg with respect to the non-metastatic group in all tumor histotypes, even though a statistically significant differences occurred for the only breast cancer (!<0.05) and colorectal cancer (!< 0.01).

cells: !< 0.001). The mean counts of NK and CD8 cells in relation to that of T-reg are reported in Table 4. As shown, no significant difference in the mean number of CD8 lymphocytes was found between patients with normal or abnormally elevated number of T-reg. On the contrary, patients with elevated number of T-reg showed a significantly lower number of NK cells with respect to that found in those with normal T-reg count. Finally, Table 5 shows the circadian rhythm of cortisol in relation to total lymphocytes, CD4+ cells and T-reg mean number. A normal cortisol rhythm, with morning values greater at least than 50% with respect to the values occurring during the afternoon, was found in 85/114 (75%). Total lymphocyte and CD4+ cell mean numbers observed in patients with altered cortisol rhythm were significantly lower than those found in patients with normal cortisol circadianicity (!<0.01), whereas no significant difference was seen in the mean number of T reg. Figure 1 and Figure 2 illustrate T-reg mean numbers in relation to tumor histoptypes in the overall patients and with respect to their disease extension, respectively. No significant difference was seen in relation to tumor histotype. The highest values of T-reg were observed in pancreatic cancer

IV. Discussion According to previous preliminary clinical investigations (Sasada et al, 2003; Cesana et al, 2006), this study confirms in a greater number of cancer patients that the metastatic disease is characterized by the evidence of an abnormally increased percentage of T-reg lymphocytes with respect to both total circulating lymphocytes and CD4+ lymphocytes. This finding does not seem to represent a specific characteristic of some tumor histotypes, then it could constitute a general alteration occurring during the progression of the neoplastic disease, by representing a fundamental immune parameter of cancer-related immunosuppression.

Table 4. Mean values of NK cells and CD8+ lymphocytes in cancer patients with normal or abnormally high values of CD4+CD25+ lymphocytes. Patients

n

Normal values of CD4+CD25+ cells High values of CD4+CD25+ cells

62 52

NK cells (n/mm3) (X ± SE) 339 ± 34 204 ± 22 *

CD8+ lymphocytes (n/mm3) (X ± SE) 439 ± 55 506 ± 57

* !<0.05 "# normal values of CD4+CD25+ lymphocytes.

Table 5. Mean numbers of total lymphocytes, T helper (CD4+) lymphocytes and T regulator lymphocytes (CD4+CD25+) in relation to cortisol circadian secretion in cancer patients. Cortisol circadian secretion Normal cortisol rhythm Altered cortisol rhythm

n 85 29

Lymphocytes 1978 ± 69 * 1537 ± 81

* $<0.01 "# patients with altered cortisol rhythm

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N/mm3 (X ± SE) CD4+ cells CD4+CD25+ cells 944 ± 65 * 209 ± 48 623 ± 87 244 ± 55

Cancer Therapy Vol 6, page 703!

Figure 1. CD4+CD25+ lymphocyte mean number in relation to tumor histotype.

Figure 2. CD4+CD25+ lymphocytes in relation to tumor histotype in metastatic and non-metastatic cancer patients.

their mechanisms of action may be represented by the stimulation of T-reg generation, with a consequent inhibition of the activation of an effective anticancer

Several immune molecules have appeared to suppress the anticancer immunity, namely IL-6, IL-10, IL-1, TNF-! and TGF-" , but it seems that the common end result of 703

Vigorè et al: T regulator lymphocytes in cancer patients ! cells are dependent on CTLA-4 expression. Eur J Immunol 34, 3485-96. Cesana GC, DeRaffele G, Cohen S, Moroziewicz D, Mitcham J, Stoutenburg J, Cheung K, Hesdorffer C, Kim-Schulze S, Kaufman HL (2006) Characterization of CD4+CD25+ regulatory T cells in patients treated with high-dose interkleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol 24, 1169-77. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM (2003) Conversion of peripheral CD4+CD25naïve T cells to CD4+CD25+ regulatory T cells by TGF-! induction of transcription factor Foxp 3. J Exp Med 198, 1875-86. Claman HN (1998) Corticosteroids and the immune system. Adv Exp Med Biol 245, 203-10. Dieckmann D, Bruett H, Ploettner H, Lutz MB, Schuler G (2002) Human CD4+CD25+ regulatory contact-dependent T cell induce IL-10 producing,contact-independent type-1regulatory T cells. J Exp Med 196, 247-53. Dieckmann D, Plottner H (2001) Ex vivo isolation and characterizationof CD4+CD25+ T cells with regulatory properties from human blood. J Exp Med 193, 1303-10. Ehrke MJ, Mihich E, Berd D, Mastrangelo MJ (1989) Effects of anticancer drugs on the immune system. Semin Oncol 16, 230-9. Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, Chauffert B, Solary E, Bonnotte B, Martin F (2004) CD4+ CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows Immunotherapy of established tumors to be curative. Eur J Immunol 34, 336-44. Ikemoto S, Yoshida N, Narita K, Wada S, Kishimoto T, Sugimura K, Nakatani T (2003) Role of tumor-associated macrophages in renal cell carcinoma. Oncol Rep 10, 1843-9. Jago CB, Yates J, Camara NOS, Lechler RI, Lombardi AG (2004) Differential expression of CTLA-4 among T cell subsets. Clin Exp Immunol 136, 463-71. Ji HB, Liao G, Faubion WA, Abadía-Molina AC, Cozzo C, Laroux FS, Caton A, Terhorst C (2004) Cutting edge, the natural ligand for glucocorticoid-induced TNF receptorrelated protein abrogates regulatory T cell suppression. J Immunol 172, 5823-7. Kanamaru F, Youngnak P, Hashiguchi M, Nishioka T, Takahashi T, Sakaguchi S, Ishikawa I, Azuma M (2004) Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. Immunol 172, 7306-14. Knutson KL, Disis M, Salazar L (2007) CD4 regulatory T cells in human cancer pathogenesis. Cancer Immunol Immunother 556, 271-85. Kusmartsev S, Gabrilovich DI (2005) STAT1 signaling regulates tumor-assolciated macrophage-mediate T cell deletion. J Immunol 174, 4880-91. Lan RY, Ansari AA, Lian ZX, Gershwin ME (2005) Regulatory T cells, development,function,and role in autoimmunity. Autoimmun Rev 4, 351-63. Leong PP, Mohammad R, Ibrahim N, Ithnin H, Abdullah M, Davis WC, Seow HF (2006) Phenotyping of lymphocytes expressing regulatory and effector markers in infiltrating ductal carcinoma of the breast. Immunol Lett 102, 229-36. Malek TR, Bayer AL (2004) Tolerance not immunity crucially depends on IL-2. Nat Rev Immunol 4, 665-74. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25, 677-86. Sasada T, Kimura M, Yoshida Y, Kanai M, Takabayashi A (2003) CD4+CD25+ regulatory T cells in patients with

immune reaction. On the same way, several immune cells are able to suppress the anticancer immunity, including macrophages, T helper-2 lymphocytes and some myeloidderived suppressor cells, but also in this case they would act in a suppressive way by promoting the generation of Treg. Then, the detection of T-reg amounts in terms of both absolute number and percentages with respect to total lymphocytes and CD4+ cells could constitute a simple and adequate clinical immune parameter to quantify the whole status of the anticancer immunity in the single cancer patient. Moreover, future clinical studies will be required to establish the possible prognostic significance of changes in T-reg percentage and number in relation to the anticancer efficacy of the various standard antitumor therapies. Moreover, it has to be remarked that T-reg lymphocytes would not represent the only immune cells involved in the suppression of the anticancer immunity. In fact, there is at least another fundamental immunosuppressive system, consisting of the monocytemacrophage cell lineage (Sica and Bronte, 2007). In more detail, it has been observed that the bone marrow may release myeloid precursors provided by suppressive activity on the antitumor immune response and defined as myeloid-derived suppressor cells (MDSC) (Kusmartsev and Gabrilovich, 2005). These cells have appeared to be characterized by the cell surface expression of GR-1, CD11b and CD80 antigens (Anderson et al, 2002; van Ginderachter et al, 2006). The myeloid suppressor cells would promote the generation and activation of T-reg lymphocytes, which at the other side would stimulate MDSC release from the bone marrow and M2 macrophage differentiation (Terabe et al, 2003; Wie et al, 2006). Moreover, the myeloid suppressive cells would inhibit the anticancer immunity by promoting macrophage differentiation into the M2 sub-type (Ikemoto et al, 2003), which plays a clear inhibitory effect on the anticancer immunity, namely through the release of IL-6 (Ueno et al, 2000), whereas the M1 macrophage sub-type may either stimulate or suppress the antitumor immunity (Mantovani et al, 2004). M1 and M2 macrophage sub-types have appeared to be characterized by a high production of IL-12 or IL-10, respectively (Ueno et al, 2000). Then, further studies by concomitantly evaluating T reg and MDSC count, will contribute to better define the immune mechanism responsible for the suppression of the anticancer immunity.

References Anderson CF, Gerber JS, Mosser DM (2002) Modulating macrophage function with IgG immune complexes. J Endotoxin Res 8, 477-81. Annunziato F, Cosmi L, Liotta F, Lazzeri E, Manetti R, Vanini V, Romagnani P, Maggi E, Romagnani S (2002) Phenotype,localization and mechanism of suppression of CD4+CD25+ human thymocytes. J Exp Med 196, 379-87. Antony PA, Restito NP (2005) CD4+CD25+ T regulatory cells,immunotherapy of cancer,and interleukin-2. J Immunother 28, 120-8. Birebent B, Lorho R, Lechartier H, de Guibert S, Alizadeh M, Vu N, Beauplet A, Robillard N, Semana G (2004) Suppressive properties of human CD4+CD25+regulatory T

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Cancer Therapy Vol 6, page 705! gastrointestinal malignancies, possible involvement of regulatory T cells in disease progression. Cancer 98, 108993. Schwartz RH (2005) Natural regulatory T cells and selftolerance. Nat Immunol 6, 327-30. Shevach EM (2002) CD4+CD25+ suppressor T cells, more questions than answers. Nat Rev Immunol 2, 389-400. Sica A, Bronte V (2007) Altered macrophage differentiation and immune dysfunction in tumor development. J Clin Invest 117, 1155-66. Siddiqui SA, Frigola X, Bonne-Annee S, et al (2007) Tumorinfiltrating Foxp3 CD4+CD25+ T cells predict poor survuival in renal cell carcinoma. Clin Cancer Res 13, 2075-81. Stephens GL, McHugh RS, Whitters MJ, Young DA, Luxenberg D, Carreno BM, Collins M, Shevach EM (2004) Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J Immunol 173, 500820. Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, Mak TW, Sakaguchi S (2000) Immunologic and tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen. J Exp Med 192, 1285-94. Terabe M, Matsui S, Park JM, Mamura M, Noben-Trauth N, Donaldson DD, Chen W, Wahl SM, Ledbetter S, Pratt B, Letterio JJ, Paul WE, Berzofsky JA (2003) Transforming growth factor-! production and myeloid cells are an eggector mechanism trhough which CD1d-restricted T cells block cytotoxic T lymphocyte-mediated tumor immunosurveillance, abrogation prevents tumor recurrence. J Exp Med 198, 1741-52. Thomton AM, Shevach EM (2000) Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J Immunol 164, 183-90. Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, Koike M, Inadera H, Matsushima K (2000) Significance of macrophage chemo-attractant protein-1 in macrophage recruitment,angiogenesis and survival in human breast cancer. Clin Cancer Res 6, 3282-9. Van Ginderachter JA, Movahedi K, Hassanzadeh Ghassabeh G, Meerschaut S, Beschin A, Raes G, De Baetselier P (2006) Classical and alternative activation of mononuclear phagocytes, picking the best of both worlds for tumor promotion. Immunobiology 211, 487-501.

Vasu C, Prabhakar BS, Holterman MJ (2004) Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4 high T regulatory cells with target alloantigens specificity. J Immunol 173, 2866-76. von Boehmer H (2005) Mechanisms of suppression by suppressor T cells. Nat Immunol 6, 338-44. von Herrath MG, Harrison LC (2003) Regulatory lymphocytes, antigen-induced regulatory T cells in autoimmunity. Nat Rev Immunol 3, 223-32. Wang XB, Zheng CY, Giscombe R, Lefvert AK (2001) Regulation of surface and intracellularexpression of CTLA-4 on human peripheral T cells. Scand J Immunol 54, 453-8. Wie S, Kryczeck I, Zou W (2006) Regulatory T-cell compartmentalization and trafficking. Blood 108, 426-31. Yang R, Cai Z, Zhang Y, Yutzy WH 4th, Roby KF, Roden RB (2006) CD80 in immune suppression by mouse ovarian carcinoma-associated Gr-1+CD11b+ myeloid cells. Cancer Res 66, 6807-15. Ziegler SF (2006) FOXP3 of mice and men. Annu Rev Immunol 24, 209-26. Zou W (2006) Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 6, 295-307.

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An atypical tongue metastasis of renal cell carcinoma in a patient with metachronous hepatocellular carcinoma Case Report

Raffaele Longo1,*, Daniela Baldini2, Giampietro Gasparini1 1 2

Division of Medical Oncology, “San Filippo Neri” Hospital, Rome Department of Anathomy ! Pathology, “San Filippo Neri” Hospital, Rome

__________________________________________________________________________________! *Correspondence: Raffaele Longo, MD, Division of Medical Oncology ‘San Filippo Neri’ Hospital, Via Martinotti 20, 00135 Rome, Italy; Tel: +39-06-33062272; Fax: +39-06-33062414; E-mail: raflongo@libero.it Key words: tongue metastasis, renal cell carcinoma, metachronous hepatocellular carcinoma Abbreviations: hepatocellular carcinoma, (HCC); Magnetic Resonance Imaging, (MRI); renal “clear cell” carcinoma, (RCC) Received: 21 August 2008; Revised: 3 September 2008 Accepted: 15 September 2008; electronically published: October 2008

Summary Primary tumors metastasizing to the tongue are very unusual and only anecdotal cases have been reported. There is an about 1% incidence rate of metastases to the oral cavity from other primary tumors, particularly lung, breast, skin, gastrointestinal tract, and liver (Azam et al, 2008). We describe the case of a 68-year-old caucasian man with two metachronous tumors, an hepatocellular (HCC) and a renal cell carcinoma (RCC), who presented an unusual tongue metastasis from this latter cancer 9 years after nephrectomy. Tongue metastasis of RCC is usually a manifestation of widespread disease and is associated with a poor prognosis, being the mean interval from diagnosis of tongue metastasis to death of 5.8 months (Goel et al, 2003; Azam et al, 2008). Treatment of tongue metastasis is often palliative and aims to provide an improvement of quality of life by means of pain relief and prevention of bleeding, infection, airway obstruction, and difficulty in swallowing. Surgical excision is recommended as primary treatment, with emphasis on preservation of tongue structure and function. However, new molecular targeted agents, such as sorafenib, sunitinib, bevacizumab, and temsirolimus, have shown promising results in the treatment of metastatic RCC.

II. Case Report

I. Introduction

In June 1997, a 68-year-old caucasian man, affected by chronic hepatitis C, was submitted to VIII liver segmentectomy for an hepatocellular carcinoma (HCC) (pT2, pN0, M0, G2 stage II, UICC, 1997). Four months later, during the follow-up, a whole body CT-scan and a Magnetic Resonance Imaging (MRI) documented the presence of a solid lesion in the left kidney, 3.5 x 2.5 cm in size. A fine-needle biopsy was consistent with the hypothesis of a primary renal cancer. For this reason, the patient underwent left nephrectomy. Histology confirmed the diagnosis of a metachronous renal “clear cell” carcinoma (RCC) (pT1, pN0, M0, G1-stage I, UICC, 1997). Because of liver recurrence of HCC, histologically proven by core needle biopsy, he received several sequential chemoembolizations for 2 consecutive years. Three months after the last loco-regional treatment, metastases of HCC in mediastinal lymph nodes were documented by CT-scan and mediastinoscopy, and a systemic chemotherapy was started with a combined regimen of weekly 4-epidoxorubicin and thalidomide, obtaining a stable disease. Due to progression of

Primary RCC metastasizing to the tongue is very unusual and only a few cases have been reported in the literature to date. Out of the 29 published cases, only 4 initially presented with tongue metastases before the diagnosis of primary tumor (Azam et al, 2008). The prognosis for patients with lingual RCC metastasis is poor (Goel et al, 2003; Azam et al, 2008). The treatment of tongue metastasis is usually palliative and aims to provide patient comfort by means of pain relief and prevention of bleeding, infection, airway obstruction, and difficulty in swallowing. Surgical excision is recommended as primary treatment with emphasis on preservation of tongue structure and function (Goel et al, 2003; Azam et al, 2008).

707

Longo et al: An atypical tongue metastasis of renal cell carcinoma in a patient with metachronous hepatocellular carcinoma disease in the mediastinal lymph nodes, in the early 2006, a 2ndline chemotherapy with a combination of oxaliplatin and gemcitabine was administered. After 4 months of treatment, during the last hospitalization, the patient presented mild pain in his oral cavity without any difficulty in swallowing. Oral examination showed an asymptomatic, irregular, hard, and protruding lesion of 3 centimeters at the largest diameter, localized on the left postero-lateral side of the tongue (Figure 1A and 1B, black arrows). The tongue mobility was normal and no

palpable cervical lymphadenopathy was found. Histological examination by core biopsy documented a metastasis of RCC. Tumor cells (black arrows) infiltrated muscular tissue (red arrows) without affecting the overlying mucosa (bleu arrows). (Figure 1C). We planned a debulking surgery of this lesion, but the patient refused any surgical procedures and other systemic treatments. He died 4 months later for an acute mediastinal syndrome.

Figure1. (AB) Oral examination showed an asymptomatic, irregular, hard, and protruding lesion with the largest diameter of 3 cm, localized on the left postero-lateral side of the tongue (black arrows).(C) Histology documented a metastasis of RCC. Tumor cells (black arrows) infiltrated muscular tissue (red arrows) without affecting the overlying mucosa (bleu arrows).

708

Cancer Therapy Vol 6, page 709! metastasis after interferon-! therapy (Shibayama et al, 1993). Finally, considering that metastatic tumors do not clinically differ from primary tongue cancers, it is crucial to consider this possibility in patients with history of previous primary tumors in order to avoid misdiagnosis and to provide the right treatment. In fact, in contrast with primary lingual tumors, secondary neoplasms are often managed with palliative intent, being their prognosis very poor.

III. Discussion RCC is a neoplasm with unpredictable spread (Azam et al, 2008). Approximately one-third of RCC patients presents metastasis at primary diagnosis; moreover, 3050% of patients with initially localized RCC will develop metastases. Overall, tongue metastasis is uncommon and only a few cases have been reported in the literature to date (Inai et al, 1987; Ishikawa et al, 1991; Okabe et al, 1992; Shibayama et al, 1993; Aguirre et al, 1996; Goel et al, 2003). Tongue metastases are very rare, being about 1% the incidence rate of metastases to the oral cavity from other primary tumors, most commonly: lung, breast, skin, gastrointestinal tract, and liver (Zegarelli et al, 1973; Kyan and Kato, 2004; Azam et al, 2008). Metastases are usually found months after the diagnosis of the primary tumor or at autopsy (Zegarelli et al, 1973; Kim et al, 1979; Kyan and Kato, 2004). Like primary tongue lesions, lingual metastases can be ulcerated or polypoid and tumor cells can infiltrate the muscle without involving the overlying mucosa (Zegarelli et al, 1973). Zegarelli and colleagues reported 12 cases of lingual metastases at autopsy out of 6881 patients with various malignancies and about 66% of metastases were localized at the base of the tongue (Zegarelli et al, 1973). Possible routes of metastatic spread to the tongue are the arterial, venous and lymphatic circulation. In a review of the literature, the base of tongue was the most common site involved, probably because of the rich network of lymphatic and blood vessels, through the dorsal lingual artery, and its immobility as compared to other parts of the tongue (Kim et al, 1979). Management of tongue metastasis is surgical excision followed by adjuvant radiotherapy to achieve local control of disease. Systemic treatments, such as interferon-! and new molecular targeted drugs, including sorafenib, sunitinib, bevacizumab, and temsirolimus, can offer a palliative benefit in some patients. Shibayama and colleagues reported a complete response of a tongue

References Aguirre A, Rinaggio J, Diaz-Ordaz E (1996) Lingual metastasis of renal cell carcinoma. J Oral Maxillofac Surg 54, 344-6. Azam F, Abubakerr M, Gollins S (2008) Tongue metastasis as an initial presentation of renal cell carcinoma: a case report and literature review. J Med Case Reports 2, 249. Goel MC, Williams DW, Evans H, Roberts JG (2003) Lingual metastasis from renal cell carcinoma management and review of the literature. Urol Int 71, 418-21. Inai T, Kagawa S, Aga Y, Akiyama K (1987) A renal cell carcinoma with metastasis to the tongue. Acta Urol Jpn 33, 1240-3. Ishikawa J, Morisue K, Imanishi O, Kamidono S (1991) Renal cell carcinoma metastatic to the tongue: a case report. Acta Urol Jpn 37, 263-5. Kim RY, Perry SR, Levy DS (1979) Metastatic carcinoma to the tongue: a report of two cases and a review of the literature. Cancer 43, 386-9. Kyan A, Kato SN (2004) Renal cell carcinoma metastatic to the base of tongue: a case report. Acta Urol Jpn 50, 791-3. Okabe Y, Ohoka H, Miwa T, Nagayama I, Furukawa M (1992) View from beneath: pathology in focus. Renal cell carcinoma metastasis to the tongue. J Laryngol Otol 106, 282-4. Shibayama T, Hasegawa S, Nakamura S, Tachibana M, Jitsukawa S, Shitani A, Morinaga S (1993) Disappearance of metastatic renal cell carcinoma in the base of the tongue after systemic administration of interferon-alpha. Eur Urol 24, 297-9. Zegarelli DJ, Tsukada Y, Pickren JW, Greene GW jr (1973) Metastatic tumor to the tongue: report of twelve cases. Oral Surg Oral Med Oral Pathol 35, 202-11.

709

Longo et al: An atypical tongue metastasis of renal cell carcinoma in a patient with metachronous hepatocellular carcinoma

710

Cancer Therapy Vol 6, page 707! Cancer Therapy Vol 6, 707-710, 2008

An atypical tongue metastasis of renal cell carcinoma in a patient with metachronous hepatocellular carcinoma Case Report

Raffaele Longo1,*, Daniela Baldini2, Giampietro Gasparini1 1 2

Division of Medical Oncology, “San Filippo Neri” Hospital, Rome Department of Anathomy ! Pathology, “San Filippo Neri” Hospital, Rome

__________________________________________________________________________________! *Correspondence: Raffaele Longo, MD, Division of Medical Oncology ‘San Filippo Neri’ Hospital, Via Martinotti 20, 00135 Rome, Italy; Tel: +39-06-33062272; Fax: +39-06-33062414; E-mail: raflongo@libero.it Key words: tongue metastasis, renal cell carcinoma, metachronous hepatocellular carcinoma Abbreviations: hepatocellular carcinoma, (HCC); Magnetic Resonance Imaging, (MRI); renal “clear cell” carcinoma, (RCC) Received: 21 August 2008; Revised: 3 September 2008 Accepted: 15 September 2008; electronically published: October 2008

Summary Primary tumors metastasizing to the tongue are very unusual and only anecdotal cases have been reported. There is an about 1% incidence rate of metastases to the oral cavity from other primary tumors, particularly lung, breast, skin, gastrointestinal tract, and liver (Azam et al, 2008). We describe the case of a 68-year-old caucasian man with two metachronous tumors, an hepatocellular (HCC) and a renal cell carcinoma (RCC), who presented an unusual tongue metastasis from this latter cancer 9 years after nephrectomy. Tongue metastasis of RCC is usually a manifestation of widespread disease and is associated with a poor prognosis, being the mean interval from diagnosis of tongue metastasis to death of 5.8 months (Goel et al, 2003; Azam et al, 2008). Treatment of tongue metastasis is often palliative and aims to provide an improvement of quality of life by means of pain relief and prevention of bleeding, infection, airway obstruction, and difficulty in swallowing. Surgical excision is recommended as primary treatment, with emphasis on preservation of tongue structure and function. However, new molecular targeted agents, such as sorafenib, sunitinib, bevacizumab, and temsirolimus, have shown promising results in the treatment of metastatic RCC.

II. Case Report

I. Introduction

In June 1997, a 68-year-old caucasian man, affected by chronic hepatitis C, was submitted to VIII liver segmentectomy for an hepatocellular carcinoma (HCC) (pT2, pN0, M0, G2 stage II, UICC, 1997). Four months later, during the follow-up, a whole body CT-scan and a Magnetic Resonance Imaging (MRI) documented the presence of a solid lesion in the left kidney, 3.5 x 2.5 cm in size. A fine-needle biopsy was consistent with the hypothesis of a primary renal cancer. For this reason, the patient underwent left nephrectomy. Histology confirmed the diagnosis of a metachronous renal “clear cell” carcinoma (RCC) (pT1, pN0, M0, G1-stage I, UICC, 1997). Because of liver recurrence of HCC, histologically proven by core needle biopsy, he received several sequential chemoembolizations for 2 consecutive years. Three months after the last loco-regional treatment, metastases of HCC in mediastinal lymph nodes were documented by CT-scan and mediastinoscopy, and a systemic chemotherapy was started with a combined regimen of weekly 4-epidoxorubicin and thalidomide, obtaining a stable disease. Due to progression of

Primary RCC metastasizing to the tongue is very unusual and only a few cases have been reported in the literature to date. Out of the 29 published cases, only 4 initially presented with tongue metastases before the diagnosis of primary tumor (Azam et al, 2008). The prognosis for patients with lingual RCC metastasis is poor (Goel et al, 2003; Azam et al, 2008). The treatment of tongue metastasis is usually palliative and aims to provide patient comfort by means of pain relief and prevention of bleeding, infection, airway obstruction, and difficulty in swallowing. Surgical excision is recommended as primary treatment with emphasis on preservation of tongue structure and function (Goel et al, 2003; Azam et al, 2008).

707

Cancer Therapy Vol 6, page 709! metastasis after interferon-! therapy (Shibayama et al, 1993). Finally, considering that metastatic tumors do not clinically differ from primary tongue cancers, it is crucial to consider this possibility in patients with history of previous primary tumors in order to avoid misdiagnosis and to provide the right treatment. In fact, in contrast with primary lingual tumors, secondary neoplasms are often managed with palliative intent, being their prognosis very poor.

III. Discussion RCC is a neoplasm with unpredictable spread (Azam et al, 2008). Approximately one-third of RCC patients presents metastasis at primary diagnosis; moreover, 3050% of patients with initially localized RCC will develop metastases. Overall, tongue metastasis is uncommon and only a few cases have been reported in the literature to date (Inai et al, 1987; Ishikawa et al, 1991; Okabe et al, 1992; Shibayama et al, 1993; Aguirre et al, 1996; Goel et al, 2003). Tongue metastases are very rare, being about 1% the incidence rate of metastases to the oral cavity from other primary tumors, most commonly: lung, breast, skin, gastrointestinal tract, and liver (Zegarelli et al, 1973; Kyan and Kato, 2004; Azam et al, 2008). Metastases are usually found months after the diagnosis of the primary tumor or at autopsy (Zegarelli et al, 1973; Kim et al, 1979; Kyan and Kato, 2004). Like primary tongue lesions, lingual metastases can be ulcerated or polypoid and tumor cells can infiltrate the muscle without involving the overlying mucosa (Zegarelli et al, 1973). Zegarelli and colleagues reported 12 cases of lingual metastases at autopsy out of 6881 patients with various malignancies and about 66% of metastases were localized at the base of the tongue (Zegarelli et al, 1973). Possible routes of metastatic spread to the tongue are the arterial, venous and lymphatic circulation. In a review of the literature, the base of tongue was the most common site involved, probably because of the rich network of lymphatic and blood vessels, through the dorsal lingual artery, and its immobility as compared to other parts of the tongue (Kim et al, 1979). Management of tongue metastasis is surgical excision followed by adjuvant radiotherapy to achieve local control of disease. Systemic treatments, such as interferon-! and new molecular targeted drugs, including sorafenib, sunitinib, bevacizumab, and temsirolimus, can offer a palliative benefit in some patients. Shibayama and colleagues reported a complete response of a tongue

References Aguirre A, Rinaggio J, Diaz-Ordaz E (1996) Lingual metastasis of renal cell carcinoma. J Oral Maxillofac Surg 54, 344-6. Azam F, Abubakerr M, Gollins S (2008) Tongue metastasis as an initial presentation of renal cell carcinoma: a case report and literature review. J Med Case Reports 2, 249. Goel MC, Williams DW, Evans H, Roberts JG (2003) Lingual metastasis from renal cell carcinoma management and review of the literature. Urol Int 71, 418-21. Inai T, Kagawa S, Aga Y, Akiyama K (1987) A renal cell carcinoma with metastasis to the tongue. Acta Urol Jpn 33, 1240-3. Ishikawa J, Morisue K, Imanishi O, Kamidono S (1991) Renal cell carcinoma metastatic to the tongue: a case report. Acta Urol Jpn 37, 263-5. Kim RY, Perry SR, Levy DS (1979) Metastatic carcinoma to the tongue: a report of two cases and a review of the literature. Cancer 43, 386-9. Kyan A, Kato SN (2004) Renal cell carcinoma metastatic to the base of tongue: a case report. Acta Urol Jpn 50, 791-3. Okabe Y, Ohoka H, Miwa T, Nagayama I, Furukawa M (1992) View from beneath: pathology in focus. Renal cell carcinoma metastasis to the tongue. J Laryngol Otol 106, 282-4. Shibayama T, Hasegawa S, Nakamura S, Tachibana M, Jitsukawa S, Shitani A, Morinaga S (1993) Disappearance of metastatic renal cell carcinoma in the base of the tongue after systemic administration of interferon-alpha. Eur Urol 24, 297-9. Zegarelli DJ, Tsukada Y, Pickren JW, Greene GW jr (1973) Metastatic tumor to the tongue: report of twelve cases. Oral Surg Oral Med Oral Pathol 35, 202-11.

709

Longo et al: An atypical tongue metastasis of renal cell carcinoma in a patient with metachronous hepatocellular carcinoma disease in the mediastinal lymph nodes, in the early 2006, a 2ndline chemotherapy with a combination of oxaliplatin and gemcitabine was administered. After 4 months of treatment, during the last hospitalization, the patient presented mild pain in his oral cavity without any difficulty in swallowing. Oral examination showed an asymptomatic, irregular, hard, and protruding lesion of 3 centimeters at the largest diameter, localized on the left postero-lateral side of the tongue (Figure 1A and 1B, black arrows). The tongue mobility was normal and no

palpable cervical lymphadenopathy was found. Histological examination by core biopsy documented a metastasis of RCC. Tumor cells (black arrows) infiltrated muscular tissue (red arrows) without affecting the overlying mucosa (bleu arrows). (Figure 1C). We planned a debulking surgery of this lesion, but the patient refused any surgical procedures and other systemic treatments. He died 4 months later for an acute mediastinal syndrome.

Figure1. (AB) Oral examination showed an asymptomatic, irregular, hard, and protruding lesion with the largest diameter of 3 cm, localized on the left postero-lateral side of the tongue (black arrows).(C) Histology documented a metastasis of RCC. Tumor cells (black arrows) infiltrated muscular tissue (red arrows) without affecting the overlying mucosa (bleu arrows).

708

Longo et al: An atypical tongue metastasis of renal cell carcinoma in a patient with metachronous hepatocellular carcinoma

710

Cancer Therapy Vol 6, page 711! Cancer Therapy Vol 6, 711-728, 2008

Chemotherapy in the treatment of primary liver tumours Review Article

Jose J G Marin*, Beatriz Casta単o, Pablo Martinez-Becerra, Ruben Rosales, Maria J Monte Laboratory of Experimental Hepatology and Drug Targeting, Biomedical Research Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Instituto de Salud Carlos III, University of Salamanca, Spain

__________________________________________________________________________________! *Correspondence: Jose J. G. Marin, Department of Physiology and Pharmacology, Campus Miguel de Unamuno E.I.D. S-09, 37007Salamanca, Spain; Tel: 34-923-294674; Fax: 34-923-294669; E-mail: jjgmarin@usal.es Key words: Chemotherapy, Primary liver tumours, Non-malignant primary liver lesions, Hepatocellular carcinoma, Hormonotherapy, Curative treatment, Chemotherapy, Palliative treatment, Immunotherapy, Systemic chemotherapy, Chemotherapy resistance Abbreviations: 5-fluorouracil, (5-FU); ATP-binding cassette, (ABC); Best supportive care, (BSC); Cisplatin + adriamycin + 5-FU + INF!, (PIAF); cholangiocarcinoma, (CGC); epidermal growth factor receptor, (EGFR); European Regional Development Fund, (ERDF); excision repair cross-complementing protein 1, (ERCC1); Gemcitabine + oxaliplatinum (GEMOX); Glutathione-S-transferases, (GSTs); Hepatocellular carcinoma, (HCC); ifosfamide, cisplatin and doxorubicin, (IPA); Interferon alpha, (INF!); Iridium-192, (192Ir); Luteinizing hormone-releasing hormone, (LHRH); Metallothioneins, (MTs); Multidrug resistance phenotype, (MDR); Multidrug resistance-associated proteins, (MRPs); Nucleotide excision repair, (NER); Organic anion-transporting polypeptide (OATP); Research on Membrane Transport Proteins, (REIT); Trans-arterial chemoembolization, (TACE); Tyrosine kinase inhibitors, (TKIs); UDPglucuronosyl transferases, (UGTs); Vascular endothelial growth factor, (VEGF); Xeroderma pigmentosum group A, (XPA); Xeroderma pigmentosum group C, (XPC) Received: 21 July 2008; Revised: 12 September 2008 Accepted: 16 September 2008; electronically published: October 2008

Summary Primary liver tumours, mainly hepatocellular carcinoma and cholangiocarcinoma, constitute the fifth most frequent types of cancer, whereas in the rank of mortality they are the third. The clinical relevance of liver tumours increases further if the number of deaths due to liver metastasis of cancers of extrahepatic origin is included. One important limitation in the clinical battle against these tumours is their marked refractivity to the available chemotherapy and the development of resistance to drugs during treatment. Owing to the existence of several types of primary liver tumours and the wide range of tissues from which metastatic cells can be released, the biology of tumours affecting the liver is diverse, and to this must be added inter-individual variability plus the fact that sensitivity/refractivity to drugs may change during the evolution of the tumour. In the context of the current most commonly used curative and palliative treatments, the present article reviews classic and newly available anticancer drugs that have been assayed to treat liver tumours, either alone or, more frequently, combined with other therapeutic options as adjuvant (pre- and post-surgery) chemotherapy. The main mechanisms responsible for the lack of efficacy of these drugs in primary liver tumours are also briefly described.

come third in the rank of mortality due to cancer, even though they constitute the fifth most frequent type of cancer. The clinical relevance of liver tumours is further exacerbated if the number of deaths due to liver metastasis of cancer of extrahepatic origin is taken into consideration. One important limitation in the clinical battle against these tumours is their marked refractivity to the available chemotherapy and the development of resistance during treatment. The complexity of the situation can be understood if it is considered that owing to the existence of several types of primary liver tumours and the broad

I. Introduction Liver tumours can be classified according to their origin as primary tumours, derived from liver cells, and secondary, i.e., due to metastasis of primary tumours located in other territories, frequently colorectal tumours (Benson, 2007). Depending on the severity of their prognosis, they can be classified as benign or malignant tumours (Table 1). Non-malignant lesions are commonly asymptomatic and, when detected incidentally, the recommendation may be surgical resection or simply follow up. In contrast, primary malignant liver tumours 711

Marin et al: Liver Cancer Chemotherapy! the biliary tree, the stomach or the duodenum, in which case they cause abdominal pain, fever or cholestasis. Some of these tumours, such as infantile hemangioendothelioma and mesenchymal hamartoma, are almost exclusively found in children. In adults, benign liver tumours include, in order of frequency (Table 1): hemangiomas (CaseiroAlves et al, 2007), focal nodular hyperplasia (LizardiCervera et al, 2006) and hepatocellular adenomas (Kalra et al, 1976; Lizardi-Cervera et al, 2006) in the liver parenchyma, and cholangiocellular adenomas and biliary cystadenomas, which are derived from biliary duct cells (Kim et al, 1998). The remaining benign liver tumours, such as biliary hamartoma, mesenchymal hamartoma, lipoma, myelolipoma, angiomyolipoma, angiomyelolipoma and others are very infrequent. Complete identification of the type of lesion is usually based on the clinical presentation, biochemical data, imaging techniques and histological examination of biopsies. The patient’s background regarding the use of oral anticonceptives, cirrhosis, etc. may be valuable information for orienting the diagnosis. The best therapeutic option for non-malignant primary liver tumours is usually surgical resection, which is recommended based on the size and predicted consequences of the tumour and on the risk of spontaneous rupture with intraperitoneal bleeding, which is frequently the case of hepatic adenomas. Moreover, the risk of transformation into malignant lesions should be also considered. This risk is higher in hepatic adenomas, cholangiocellular adenomas and biliary cystadenomas. Recent investigations have suggested that analysis of the genotype-phenotype correlations in benign liver tumours and the identification of novel markers of a higher risk of malignant transformation may modify the current protocols of diagnosis and follow-up of these patients (Rebouissou et al, 2008). Trans-arterial embolization, as an alternative to surgical resection, has been recommended only in infantile hemangioendothelioma, when resection is not possible. The need for an orthotopic liver transplant due to nonmalignant primary liver tumours is rare.

range of tissues from which metastatic cells reaching the liver may be released, the biology of tumours affecting the liver is quite diverse. Moreover, inter-individual variability, together with the fact that the sensitivity/refractivity to drugs may change during the evolution of the tumour, must also be taken into account. Liver tumours can be treated according to five different types of strategies. These include: i) surgical resection or liver transplant; ii) percutaneous intervention by ethanol injection or tumour ablation by radiofrequency; iii) trans-arterial intervention by tumour embolization, chemoperfusion or chemoembolization (TACE); iv) systemic chemotherapy and v) gene therapy. In the present article we shall review the relevance of classic and novel chemotherap"#$ic options in the context of these possible strategies to treat the different types of liver tumours. Although with some peculiarities and different therapeutic recommendations -in part due to the type of tumour, but also to the stage of development at which they are diagnosed- the basic mechanical procedures used to treat these tumours, including surgical resection, percutaneous ablation, arterial embolization and orthotopic liver transplant are similar for different types of tumours. In contrast, regarding their response to chemotherapy, this is very diverse, although in general low, owing to their very different biological characteristics. When located in the liver, secondary tumours usually maintain the same sensitivity to chemotherapy as the primary tumour. Thus, here we shall focus on the recommended treatments and the sensitivity/resistance to commonly used drugs only regarding primary liver tumours.

II. Treatment of primary liver lesions

non-malignant

Benign primary tumours form a heterogeneous group of hepatic lesions that share the common characteristic of the absence of uncontrolled growth or metastasis. They are usually detected incidentally during abdominal exploration using ultrasound or computerized tomography prescribed to diagnose other diseases, because they are frequently asymptomatic unless they are large enough to compress Table 1. Types of primary liver tumours. Localization Liver Parenchyma

Benign Hemangioma Focal nodular hyperplasia Hepatocellular adenoma Infantile hemangioendothelioma Mesenchymal hamartoma Others

Malignant Hepatocellular carcinoma Hepatoblastoma Hemangiosarcoma Fibrosarcoma Lymphosarcoma Others

Intrahepatic Biliary Tree

Cholangiocellular Adenoma Biliary Cystadenoma Biliary hamartoma Others

Cholangiocarcinoma Others

712

Cancer Therapy Vol 6, page 713! discovered in initial stages when it is still feasible to apply radical treatment. As in the case of other types of liver cancer to be reviewed below, complete tumour resection is the best therapeutic option for long-term survival in patients with HCC (Ringe et al, 1991). However, in addition to the technical feasibility of the surgical procedure other factors, such as the general health status of the patient, the degree of remnant liver function and the stage of the tumour, may limit the possibility of success of this therapeutic approach (Bruix and Sherman, 2005). Resection of HCC may follow similar guidelines to those used in the surgical removal of other liver malignancies. These include routine ultrasonography during the procedure to define tumour number, size and exact localization (Takayama et al, 1998). Dual-phase spiral computed tomography is the primary imaging technique in the diagnosis of focal liver lesions. The diagnostic confidence in the detection and characterization of small liver lesions can be improved by contrastenhanced ultrasonography (Janica et al, 2007). It is important to elucidate the relationship of the tumour with neighbouring vascular structures with ultrasonography, because in addition to avoiding tumour manipulation, the tumour must be rapidly and efficiently isolated from the vascular inflow and outflow to prevent iatrogenic metastasis during the surgical procedure (Jarufe, 2006). In patients with HCC, but in the absence of liver cirrhosis, partial hepatectomy is relatively safe and postsurgery mortality is lower than 5% (Bismuth et al, 1993; Fuster et al, 1996), even if the resection includes a large liver mass (Bismuth and Majno, 2000). The post-surgery survival of these patients reaches 80-92% in the first year; 61-86% three years after hepatectomy, and 41-74% after five years (Fong et al, 1999). Patients reaching the highest values in this range of survival are those with a single tumour, with a well preserved hepatobiliary function, without portal hypertension, and with normal serum bilirubin values (Llovet et al, 1999). Unfortunately, the proportion of patients with HCC but without liver cirrhosis is low. This is less than 40% in Asia and less than 10% in Western countries. This is important because in patients with liver cirrhosis the risk of post-surgery complications is higher, which accounts for mortality rates after tumour resection of between 10 and 20% (Ringe et al, 1991). This and other factors explain why the 5-year survival rate is only of 37% in this type of patients (Fong et al, 1999). In cases where surgical resection is not recommended, percutaneous intervention may represent the best choice, provided the tumour is in an early stage (Livraghi et al, 1995). In these cases, cancer cell death is induced by intratumour administration of ethanol or acetic acid or by physical means aimed at strongly increasing or reducing the temperature of the tumour cells by exposing them to radiofrequency, laser beams or cryoablation (Okada, 1999). Percutaneous ethanol injection is the method most used because it is cheap and easy, and the appearance of serious side effects is rare. Using this therapeutic approach, the success rate regarding tumour removal reaches 90-100% in HCC tumours smaller than 2

Chemotherapy is not commonly used to treat these tumours, because the efficacy of the drugs used to treat them has not been demonstrated, except in the case of infantile hemangioendothelioma, which has been reported to be sensitive to treatment with corticoids (Jackson et al, 1977).

III. Curative treatment hepatocellular carcinoma (HCC)

of

In adults, the most frequent type of malignant liver tumour is HCC, which accounts for 90% of malignant primary hepatic lesions (Anderson et al, 1992). Moreover, since it accompanies (and is probably closely related to) the growing frequency of cirrhosis, the incidence of HCC is increasing worldwide. Indeed, approximately three out of four cases of HCC are associated with cirrhosis of the liver. Furthermore, patients with viral hepatitis B and C infection have an enhanced risk of developing this malignancy. HCC may appear as a single tumour, in the form of several nodules, or as a diffuse lesion in the liver parenchyma. Histologically, HCC is often formed by highly vascularized trabecular groups of cells with a varying (from well differentiated to undifferentiated tumours) degree of similarity to hepatocytes. The neoplastic tissue often invades branches of the portal vein and even the hepatic vein. Expansion of the HCC occurs via four different mechanisms: i) centrifugal growth, which results in compression of the surrounding healthy tissue; ii) parasinusoidal dissemination by tumour invasion of the neighbouring tissue, either through the sinusoids or the parasinusoidal spaces; iii) venous dissemination involving the use of the branches of the portal vein to propagate the tumour in the retrograde direction. Invasion of the hepatic vein is less common, but when it occurs it can reach the cava vein or even the right auricle; iv) as a consequence of dissemination through the lymph vessels and the vascular system, HCC metastases can reach other organs, most frequently regional lymph nodes (48%) and the lung (73%). Common clinical manifestations of HCC include loss of body weight (80% of cases), fatigue and abdominal pain. The latter is usually vague and persistent but may become suddenly very intense due to intraperitoneal haemorrhage associated with the rupture of a necrotic nodule or the erosion of a vessel. Liver size is almost always increased. Splenomegalia occurs in one third of cases. The appearance of jaundice varies between 20% and 58%. Ascites accumulates in 50%-75% of patients. The prognosis of patients with HCC depends on the time when the diagnosis is established. Patients with good remnant liver function and asymptomatic HCC can survive for several years, even without treatment. In contrast, when patients are diagnosed in a very advanced stage of tumour progression and/or associated impaired liver function they may die in weeks. Fibrolamellar HCC constitutes a special sub-type of HCC that mainly affects young women without cirrhosis. This tumour is characterized by the presence of a dense fibrous infiltration and a lower invasiveness, which accounts for a better prognosis, especially if the tumour is 713

Marin et al: Liver Cancer Chemotherapy! (Wang et al, 2008). This and incomplete embolization usually result in the absence of full success in inducing tumour necrosis. Studies showing that tegafur/uracil inhibits tumor angiogenesis in several cancer types have suggested the use of chemotherapy based on tegafur/uracil after TACE in advanced HCC. The use of this strategy seems to have been effective in treating these tumours and no severe adverse events have been reported (Ueda et al, 2008). In the absence of metastasis, when the tumour cannot be surgically removed and/or the remnant liver function is very poor liver transplant may be the only choice, but only if the selection criteria are appropriate because of the low availability of donors, particularly in Asia. The development of techniques for donations from living donors has permitted this problem to be partially overcome (Fan et al, 2000; de Villa et al, 2003). A careful selection of patients to be treated in this way is crucial. Several selection criteria have been proposed, adapted, and improved over the last two decades, and this has contributed to enhancing the rate of success in patients with HCC undergoing liver transplant to 5-year survival rates without recurrence in up to 85%. In 1996, the socalled criteria of Milan were proposed, which considered as candidates for liver transplant patients with a single tumour of ! 5 cm or 2-3 tumours of ! 3 cm (Mazzaferro et al, 1996). These criteria were slightly relaxed later (Yao et al, 2001; Broelsch et al, 2005; Hiatt et al, 2005; Majno and Mazzaferro, 2006).

cm, 70% in lesions smaller than 3 cm, and 50% when the tumours are 5 cm or more in diameter (Lencioni et al, 1997). Thus, in many cases of HCC with a single tumour of ! 3 cm, percutaneous ethanol injection may be considered the treatment of choice (Bruix and Llovet, 2002). In the case of larger tumours, radiofrequency may offer a good alternative to surgical resection (Buscarini et al, 2001). This technique can be used either percutaneously or during laparotomy. The aim is to reach at least the same response rate as when using percutaneous ethanol injection but with fewer sessions. The advantage over percutaneous ethanol injection is more evident in the treatment of tumours larger than 3 cm. For these patients, the 5-year survival rate is 33-40% (Livraghi et al, 1999). Other approaches, such as laser beam irradiation, cryoablation and microwave-induced coagulation involve more complications, are still in the experimental phase, and have not yet been widely accepted (Okada, 1999). Trans-arterial embolization with lipiodol alone or using chemoembolization (TACE), in which the embolizing agent is mixed with anticancer drugs (e.g., doxorubicin, mitomycin or cisplatin), are the therapies of choice in the treatment of liver tumours that cannot be resected surgically or efficiently eliminated by percutaneous interventions (Lo et al, 2002). These procedures reach response rates (generally partial responses) of between 15% and 55%. Despite this, they may be useful to slow down tumour growth and delay vascular invasion in comparison with patients receiving only palliative treatment (Blum, 2005). However, it is very important to consider patient selection in these cases. Thus, patients must have a preserved liver function (generally Child A cirrhosis) and must be free of vascular invasion and extrahepatic dissemination (Llovet et al, 2002). This is because in patients with poor liver function (Child B or C) these therapies may result in acute liver failure, which abolishes the potential benefit of using this therapeutic approach. When appropriately used, chemoembolization, for instance using doxorubicin-loaded beads, has been shown to be an efficient therapy in the treatment of non-surgically resectable HCC, as demonstrated by the low complication rate, marked tumour response, and sustained reduction of alphafetoprotein levels (Malagari et al, 2008). TACE has been also proposed as a useful palliative strategy in patients with HCC on the waiting list for orthotopic liver transplantation (Alba et al, 2008). However, there is some controversy about whether preoperative TACE significantly improves the disease-free survival or the pattern of recurrence after curative resection of HCC. Studies based on retrospective analyses of preoperative TACE do not recommend this technique as a routine procedure before hepatectomy for a resectable HCC (Choi et al, 2007). In spite of the beneficial effects of TACE in some patients with HCC, as happens with other mechanisms of resistance to be reviewed below the tumour tends to adapt its biology to the new situation. Thus, the residual surviving cancerous tissue in HCC after TACE has a rich vascularity, in part due to increased vascular endothelial growth factor (VEGF) expression in the residual tumour

IV. Chemotherapy in the palliative treatment of HCC Many different drugs have been assayed for the treatment of HCC. However, success has been achieved in only 15% of patients, and then with only a partial response, usually associated with a high rate of side effects. These have been reduced in part by local administration through the hepatic artery. Since the liver is an organ sensitive to the action of sex hormones, and since hormone receptors are expressed in HCC, the usefulness of hormonotherapies, both anti-androgenic and antiestrogenic, has been investigated (Boix et al, 1993; Bl채ker et al, 2004). However, none of them has demonstrated a consistent rate of response or the ability to slow down tumour growth or an unequivocal modification of the survival time. Other options of systemic treatment have included the use of immunomodulators. However, the administration of interferon has afforded minimal antitumour activity, with no significant effect on survival time. Although other types of immunotherapy have not been effective either, promising results have been obtained with several new agents that should be investigated in adequately designed clinical trials. The most significant results obtained using these alternatives are reviewed below and are summarized in Figure 1.

A. Hormonotherapy Anti-androgenic agents have shown no efficacy even when combined with agonists of luteinizing hormonereleasing hormone (LHRH), such as flutamide (an anti-

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Cancer Therapy Vol 6, page 715! octreotide (sandostatin LAR) were carried out, no positive impact was found (Yuen et al, 2002; Becker et al, 2007). When lanreotide, another long-acting somatostatin analogue, was tested, a limited degree of antitumour activity was observed (Raderer et al, 2000; Cebon et al, 2006). In conclusion, although some patients occasionally appear to have a stable response to somatostatin analogues, the overall beneficial response regarding the inhibition of tumour progression and survival seems to be poor.

androgenic drug) and triptorelin (an LHRH agonist) (Grimaldi et al, 1998). Regarding anti-estrogenic agents, the usefulness of tamoxifen in the treatment of advanced HCC has long been discussed (Farinati, 1996), but it is now clear from the results of several randomized controlled trials that tamoxifen is not an efficient drug in the treatment of this disease (Castells et al, 1995; CLIP Group, 1998; Riestra et al, 1998; Liu et al, 2000; Chow et al, 2002; Barbare et al, 2005). In a meta-analysis of seven randomized controlled trials in which tamoxifen was compared with conservative management, this drug showed no significant antitumour effect or benefit for survival (Llovet and Bruix, 2003). Tamoxifen has also been suggested to inhibit P-glycoprotein-mediated drug resistance and this has led to assays of tamoxifen in combination with other chemotherapeutic agents. Unfortunately, such studies have revealed no benefit of the addition of tamoxifen to the chemotherapeutic regimen (Melia et al, 1987; Cheng et al, 1996, Raderer et al, 1996; Cheng et al, 1998). Although other anti-estrogen agent, megestrol, has shown modest activity in at least three trials (Chao et al, 1997; Farinati et al, 2001; Villa et al, 2001), to date no robust survival advantage has been shown either. Since somatostatin has a well known antitumour effect in neuroendocrine tumours, and because somatostatin receptors have been identified in HCC cells (Bl채ker et al, 2004; Cebon et al, 2006), several analogues of this hormone, such as octreotide, have been assayed to treat HCC. When this substance was first studied, in short series of patients, the results suggested a benefit of this approach (Kouroumalis et al, 1998). However, when studies in larger trials using a long-acting form of !

B. Immunotherapy Interferon alpha (INF! ), an immunomodulatory cytokine widely used in the treatment of chronic hepatitis, has demonstrated activity in preclinical models against several tumour types, including HCC (Ikeda et al, 1999; Wada et al, 2007). In addition, antiangiogenic and antiproliferative properties have been proposed (Lindner, 2002; von Marschall et al, 2003; Fujioka et al, 2006; Zhu et al, 2008). Thus, INF! monotherapy has been evaluated in patients with advanced HCC, but unfortunately, conflicting results were obtained. Two randomized controlled trials suggested better response rates and good tolerance when compared to doxorubicin (Lai et al, 1989) or best supportive care (BSC) (Lai et al, 1993). A more recent study failed to show benefits of INF! regarding the tumour progression rate and survival time. In addition, INF! was poorly tolerated in patients with cirrhosis and advanced HCC, leading to a high rate of dropout (Llovet et al, 2000).

Figure 1. Schematic representation of most interesting chemotherapeutic agents assayed in clinical practice to treat the main types of primary liver malignancies.

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Marin et al: Liver Cancer Chemotherapy! When the results from trials with different chemotherapeutic options are analyzed it should be recalled that patient populations and cancer biology are very heterogeneous, which may result in confusing data and the masking of positive responses in certain subgroups of patients (Nowak et al, 2004). Thus, the heterogeneity of the populations included in clinical trials, together with the differences in the design of such studies, make it very difficult to compare the results concerning response rates between different trials, as well as to extrapolate the conclusions to a general patient population. This problem, together with the poor response obtained with most regimens assayed until now, does not allow a standard chemotherapy regime to be established for all patients with advanced HCC. Almost all the available chemotherapeutic agents have been tested in monotherapy, mainly in phase-I and -II studies, to treat HCC (Table 2). However, in general the rate of objective responses has been very low (O´Reilly et al, 2001; Fuchs et al, 2002). Moreover, the response is commonly limited and transient and there is no consistent result indicating that patient survival is actually increased (Johnson, 2002; Yeo et al, 2005). This has restricted the

However, combinations of INF! with chemotherapy appear to be more efficient, as will be described below. Novel strategies based on immunotherapeutic agents are currently being developed, with promising results. These include the administration of other cytokines and the transfer of T-lymphocytes (Butterfield, 2004; Avila et al, 2006; Sun et al, 2006; Zerbini et al, 2006; Butterfield, 2007).

C. Systemic chemotherapy Several reasons account for the lack of substantial success in treating HCC with chemotherapeutic agents in systemic regimens: i) Most of these tumours are highly resistant to chemotherapy owing to mechanisms that will be briefly described below; ii) Tolerance to the treatment is limited due to the reduced remnant liver function; iii) The existence of cirrhosis may determine thrombocytopenia due to portal hypertension and hypersplenism, leading to delayed treatment cycles, decreased chemotherapy dose-intensity and impaired treatment efficacy (Taieb et al, 2006); iv) Finally, the use of immunosuppressive chemotherapy may reactivate chronic HBV infection, which is often present in these patients (Taieb et al, 2006).

Table 2. Response rate in mono- and poly-chemotherapy of hepatocellular carcinoma. Chemotherapy Regimen Mono-chemotherapy Doxorubicin 5-FU Epirubicin Mitoxantrone Capecitabine Etoposide Cisplatin Gemcitabine Irinotecan Poly-chemotherapy 5-FU + leucovorin Cisplatin-based regimens: Cisplatin + epirubicin + infusional 5-FU ECF (etoposide + cisplatin + 5-FU) Cisplatin + doxorubicin + capecitabine Cisplatin + mitoxantrone + infusional 5-FU Cisplatin + doxorubicin Infusional cisplatin + infusional 5-FU PIAF (cisplatin + doxorubicin + infusional 5-FU + INF!) Gemcitabine-based regimens: Gemcitabine + cisplatin GEMOX (Gemcitabine + oxaliplatin)

Response Rate 10-20% 6-10% 10-25% 10-25% 13%-25% 0-10% 0-15% Modest anti-tumour activity Minimal anti-tumour activity 10-28% 15% 15-20% 24% 24-27% 18-49% 47% 15-26% (*) 17-21% 17-40%

(*) Better results have been reported in patients with normal liver function as compared to those with cirrhosis and high serum bilirubin levels (response rate 50% vs 6%) 5- Fluorouracil, 5-FU; Interferon-alpha, INF!.

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Cancer Therapy Vol 6, page 717! as jaundice, choluria and pruritus (Shimoda and Kubota, 2007). As mentioned above regarding other liver tumours, surgery is also at present the best curative therapeutic option for patients with CGC (Yeh et al, 2004; Alberts et al, 2007). However, this is not always feasible or recommended, as happens in patients with multiple tumours (approximately 5% of them) and/or with subjacent parenchymal or biliary disease. Moreover, the majority (approximately two thirds) of these patients are diagnosed at advanced stages of the disease, approximately 50% of patients already have an invasion of lymph nodules and 10-20% have metastasis or lesions involving the portal structure or main hepatic artery (Pichlmayr et al, 1996). This implies that surgical resection is recommended in only a small number of patients with CGC (Khan et al, 2002). When resection is not possible, in principle liver transplantation might provide a good alternative (Iwatsuki et al, 1998). However, in contrast to HCC, patients with CGC have classically been excluded from liver transplant programs. This has been due in part to the high probability of recurrence of this type of tumour after resection. Nevertheless, improvements in survival due to novel adjuvant therapies suggest that this therapeutic option for CGC should be reappraised (Knight et al, 2007). In patients with unresectable CGC, the prognosis is poor and palliative measures should aim at increasing the quality of life and the relief of symptoms (pain, pruritus, jaundice) and, if possible, at extending patient survival (Anderson et al, 2004). Radiation therapy, chemotherapy or a combination of both approaches may constitute the only possible treatment for these patients (Shimoda and Kubota, 2007). Even in some cases in which surgical removal of the tumour is possible, complete elimination of the disease is not achieved, and hence a high rate of recurrence (60-90%) occurs (Malhi and Gores, 2006). This justifies the use of adjuvant therapies, radiotherapy and/or chemotherapy in combination with surgery (Chari et al, 2003a). However, the actual usefulness of these treatments is controversial (Hejna et al, 1998). In some series, radiotherapy after surgery has been found to be useful (Todoroki et al, 2000), whereas poorer results were obtained in other series (Pitt et al, 1995). In some cases, even an impairment of liver function has been reported (Cherqui et al, 1995). This justifies the restriction of radiotherapy to patients with unresectable CGC due to locally advanced disease but without evidence of distant metastases. These patients may benefit from palliative radiotherapy using a combination of external beam radiation and intraluminal brachytherapy with iridium-192 (192Ir) (Chari et al, 2003b). While the survival benefit of palliative radiotherapy is not clear, a role in the control of local disease seems more evident. The use of radiotherapy with or without concomitant chemotherapy may contribute to biliary decompression and the relief of pain (Onishi et al, 1995), permitting a better management of local biliary disease (Alden and Mohiuddin, 1994; Kuvshinoff et al, 1995; Vallis et al, 1996). In addition, a combination of regional chemotherapy using intra-arterial hepatic

use of chemotherapy in HCC to the aim of trying to slow down tumour growth and to minimize cancer-related symptoms. The drugs with the highest activity against HCC are 5-fluorouracil (5-FU), cisplatin and doxorubicin. However the best results are obtained when they are used in combined regimens (Table 2). Nevertheless, the response rates of patients with HCC to these drugs are relatively low (Chlebowski et al, 1984; Porta et al, 1995). Epirubicin, mitoxantrone and capecitabine have no better proven antitumour efficacy than doxorubicin (Dunk et al, 1985; Hochster et al, 1985; Colleoni et al, 1993; Pohl et al, 2001). Other chemotherapeutic drugs with modest activity against HCC are etoposide, vincristine, gemcitabine and irinotecan (Yang et al, 2000; Fuchs et al, 2002). Multiple combinations of antitumour drugs have been tested in patients with advanced HCC (Table 2). Cisplatin-based regimens appear to result in higher objective response rates than others not including cisplatin (Stuart, 2007). However, it is not clear that any of these regimens may confer survival benefit to treated patients. The response rates to regimens including two- and threedrug combinations are approximately the same. Among the cisplatin-based regimens, the best response rates have been obtained with the treatment known as PIAF (cisplatin + adriamycin + 5-FU + INF! ) (Leung et al, 1999, 2002; Lau et al, 2004). However, due to its high toxicity profile, it should be considered only for patients with a general good health status and minimal hepatic dysfunction (Leung et al, 2002; Yeo et al, 2005). More recently, the GEMOX regimen (gemcitabine + oxaliplatin) has been evaluated in a phase-II study, with promising results. In addition, this regimen lacks substantial renal and hepatic toxicity (Taieb et al, 2003; Louafi et al, 2007). !

V. Chemotherapy in the treatment of tumours of the biliary system Primary malignant tumours that originate in the biliary system include cholangiocarcinoma (CGC), gallbladder cancer and carcinoma of the ampulla of Vater. The latter two types of tumour show different clinical characteristics and will not be considered here. Although CGC is less frequent than HCC - the incidence of CGC is between 0.01% and 0.8%; this tumour represents 5%-10% of all hepatic primary malignancies-, CGC is more aggressive than HCC (Yeo et al, 1990). The typical age of presentation is between 50 and 70 years, but it may appear up to 20 years earlier in persons with an enhanced risk of developing this tumour. Among the factors favouring the development of CGC are primary sclerosing cholangitis, whose risk of appearance is in turn enhanced in patients by smoking and alcohol consumption, congenital disease (e.g., choledochal cyst, congenital hepatic fibrosis and Caroli disease), intrahepatic lithiasis, and several infections by parasites (e.g., "#$%$&'()* and +,)*-$&'()*) or viruses (e.g., HIV, HBV and HCV). Regarding anatomical localization, these tumours can be classified as intrahepatic (approximately 15%), perihiliar (60%), and distal (25%) cholangiocarcinomas. The clinical manifestations are abdominal pain, weight loss and, usually, signs related to biliary obstruction, such 717

Marin et al: Liver Cancer Chemotherapy! chemotherapy cannot be always used. Thus, systemic chemotherapy is recommended in most cases.

fluorodeoxyuridine and conformational radiotherapy has been reported to improve results in controlling local disease (Robertson et al, 1993). Regarding the use of chemotherapy alone as an adjuvant to surgery, controversial results have also been obtained (Goldstein et al, 1993; Todoroki, 2000; Yoshida et al, 2002; Kelley et al, 2004). The general consensus is that there is no clear benefit of chemotherapy for biliary tumours except in the case of certain gallbladder carcinomas (Takada et al, 2002). Moreover, the combination of radiotherapy plus chemotherapy has not afforded improved results (Figueras et al, 2000). Nevertheless, it is important to consider that no randomized clinical trials exist for any of these adjuvant therapies. The available data are from retrospective studies carried out in small groups of patients (Hejna et al, 1998). Thus, at present no adjuvant treatment is recommended outside clinical investigation (Hejna et al, 1998; Anderson et al, 2004). The use of neoadjuvant chemotherapy (or presurgery chemotherapy) is rarely recommended because of the presence of reduced liver function and jaundice at the moment of diagnosis in most patients with CGC (Anderson et al, 2004). In patients with metastatic CGC, systemic chemotherapy has been used as palliative treatment. For this purpose 5-FU was used first, with a poor response rate (0-10%) and a short median survival time (6.5 months). Subsequent use of gemcitabine has permitted better results to be obtained in several dosage schedules (8-60% and 6.3-16 months, respectively). Other regimens based on the use of single agents, such as paclitaxel, docetaxel and irinotecan have failed to attain response rates higher than 10%. In contrast, combined regimens have in general been more effective. The best results have been obtained for regimens based on 5-FU in combination with leucovorin, doxorubicin, mitomycin C, methyl-CCNU, streptozotocin, INF! and cisplatin plus epirubicin (a 10-40% response rate and a 2-12-month median overall survival) or gemcitabine including leucovorin/5-FU, cisplatin, oxaliplatin, docetaxel, irinotecan and capecitabine (a 950% response rate and a 5-15.4-month median overall survival), with a tolerable toxicity profile (Thongprasert, 2005). Data from several studies suggest that gemcitabine in monotherapy or combined with cisplatin or oxaliplatin represents one of the most active and well tolerated drugs. Another regimen with promising results is 5-FU and leucovorin with cisplatin. Nevertheless, a phase-III comparison between these different alternatives is needed (Thongprasert, 2005). Intra-arterial administration of chemotherapeutic agents permits response rates of up to 44% to be obtained (O´Grady, 2000; Khan et al, 2002). This may be useful in some cases both to reduce tumour size before surgery and to prolong survival in patients with non-resectable tumours (Burger et al, 2005; Wu et al, 2007). The best regimens assayed in this approach have been those based on gemcitabine including either cisplatin or oxaliplatin (Gusani et al, 2008). Unfortunately, owing to the high incidence of metastasis in patients with CGC, and hence the high frequency of recurrence, intra-arterial

VI. Chemotherapy in the treatment of hepatoblastoma, hemangiosarcoma and other liver malignancies Two malignant primary liver tumours that are uncommon in adults, also less frequent than HCC, are hepatoblastoma and hemangiosarcoma (Table 1). Other primary malignancies, such as lymphosarcoma, fibrosarcoma and leiomyosarcoma are even more infrequent. Among the liver metastases, the most frequent ones have their origin in primary tumours located in the gastrointestinal system (tumours of pancreas, stomach, small intestine, colorectal region, extrahepatic biliary tree and gallbladder), or lung cancer, breast cancer, melanoma, and endocrine carcinoid tumours. Whereas HCC is derived from differentiated liver parenchyma, hepatoblastoma develops in foetal liver tissue. Thus, HCC is rarely found in patients younger than 6 years, where this tumour is often associated with cirrhosis or congenital metabolic storage disease (Isaacs, 2007). Although hepatoblastoma is a very rare tumour, accounting for less than 1% of all paediatric cancers, it is the most frequent hepatic tumour in children (Miller et al, 1995). Hepatoblastoma is commonly found in otherwise healthy livers. The tumour is usually discovered as a single large mass, which is detected by antenatal ultrasonography or during childhood, sparked by the precocious onset of puberty. Approximately 50% of hepatoblastomas are encapsulated at the moment of the diagnosis. The prognosis of this malignancy depends on the possibilities of implementing surgical treatment (resection or transplant), but it is usually better than that of HCC. A group of paediatric liver tumours with intermediate characteristics has recently been classified as transitional liver cell tumours (Zimmermann, 2005). As in the case of other liver malignancies, complete surgical resection of the tumour also constitutes the treatment of choice and the only one with curative potential for hepatoblastoma. Nevertheless, in the last three decades, chemotherapy, both as adjuvant and neoadjuvant therapy, has become a useful tool to improve the outcome of these patients, with rates of survival at 5 years of approximately 75% !"#$%$ the value of 35% described 30 years ago (Pritchard et al, 2000; Schnater et al, 2002). The reason for the success is that hepatoblastoma is more sensitive than other liver malignancies to several cytotoxic agents, such as vincristine, doxorubicin, cyclophosphamide, ifosfamide, 5-FU, and cisplatin. Neoadjuvant chemotherapy has proved to be effective in increasing the number of hepatoblastomas that become resectable only after chemotherapy in up to 75%, and in decreasing the morbidity associated with surgery (Stringer et al, 1995; Seo et al, 1998). This strategy is aimed at shrinking the tumour, rendering it more solid, less prone to bleeding, and better delimited with respect to the surrounding healthy liver tissue. Thus, a safer and

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Cancer Therapy Vol 6, page 719! radiotherapy (Habrand et al, 1992) and intra-arterial chemoembolization (Malogolowkin et al, 2000; Xianliang et al, 2004). The outcome for hepatoblastomas in stage IV at diagnosis is not good, but cure is still possible in 25% to 30% of patients (Douglass et al, 1993; Ortega et al, 2000; Perilongo et al, 2000). Patients that have undergone primary tumour resection should also be considered for surgical removal of pulmonary metastases. In these advanced hepatoblastomas, the same chemotherapy regimens used in stage III tumours are recommended. The standard regimen is 4 courses of cisplatin/vincristine/5-FU (Ortega et al, 2000) or doxorubicin/cisplatin followed by an attempt at tumour resection. If the surgical intervention is successful, 2 postoperative courses of the same chemotherapy should be given. If the tumour is not resectable after the 4 courses mentioned above, alternative options should be considered. These include irinotecan (Katzenstein et al, 2002) or high-dose cisplatin with etoposide. Radiotherapy alone has been reported as being useless to treat hepatoblastoma, because the active dose required is higher than that tolerated by the liver. However, radiotherapy has been suggested to be potentially useful as adjuvant therapy in cases of noncomplete surgical removal of these tumours (Habrand et al, 1992; Douglass et al, 1993). Alternatively, intra-arterial liver administration of chemotherapeutic agents (Oue et al, 1998; Malogolowkin et al, 2000) or, if the metastatic disease is controlled, liver transplant could be considered (Superina and Bilik, 1996; Perilongo et al, 2000; Otte et al, 2004; Otte et al, 2005; Austin et al, 2006). Indeed, a high rate of survival (82%) after liver transplant in children with non-resectable hepatoblastomas has been reported (Otte et al, 2004). Owing to the promising role of chemotherapy in the treatment of hepatoblastomas, new clinical trials have been carried out by stratifying the high- and low-risk patients and consequently adapting the therapy in order to enhance the success rate and reduce the toxic side effects (Roebuck and Perilongo, 2006). Hemangiosarcomas as well as other primary sarcomas of the liver, such as leiomyosarcoma, epithelioid hemangioendothelioma, fibrosarcoma, malignant fibrous histiocytoma and embryonic sarcoma are very uncommon liver lesions. They constitute less than 1% of all hepatic malignancies (Weitz et al, 2007). The outcome of patients with primary hepatic sarcoma depends primarily on the tumour histology and the ability to achieve complete tumour resection. In general, the prognosis is poor, the patients usually dying within a year, and few reaching a 3year survival if the tumour cannot be resected. Diseasespecific survival is increased to 5 years in up to 80% of patients undergoing tumour resection. Unfortunately, in many cases (15%), as for other vascular tumours of the liver, intraperitoneal hemorrhage due to primary tumour rupture is the first clinical presentation, which can be lethal if not diagnosed and treated immediately. Once hemangiosarcoma is discovered, it is often too late for surgical intervention, with a life expectancy of less than 6 months. In these cases palliative TACE incorporating the

more complete resection with fewer complications can be performed (Schnater et al, 2003). In some cases, chemotherapy has been described to be able to completely eradicate pulmonary metastases and to eliminate liver foci in multinodular hepatoblastoma (Perilongo et al, 2000). Surgical resection of distant disease makes the cure of patients with hepatoblastoma possible. Resection of pulmonary metastases, which is often performed at the same time as the resection of the primary tumour, is recommended when the number of metastases is limited (Feusner et al, 1993). When possible, the resection of areas of locally invasive disease, such as diaphragm metastases and isolated brain metastases, is recommended (Robertson et al, 1997). The choice of the chemotherapeutic regimen to treat hepatoblastomas depends on the stage of the disease, which can be established with post-surgical or pre-surgical criteria. In hepatoblastomas diagnosed in stages I and II (post-surgical classification) or 1, 2 and 3 of PRETEX (pre-surgical classification), regimens based on cisplatin result in a survival rate of more than 90% (Douglass et al, 1993; Ortega et al, 2000; Perilongo et al, 2004). The therapeutic options include complete surgical resection followed by four courses of chemotherapy with cisplatin, vincristine and 5-FU or any comparable combination (Douglass et al, 1993). When the tumour has been completely resected and presents a purely foetal histology the patient can be treated with doxorubicin alone (Ortega et al, 2000) or carefully followed without further therapy (Douglass et al, 1993). Tumours in PRETEX stage 1 can be resected without neoadjuvant chemotherapy and the patients can be treated with chemotherapy after surgery (Pritchard et al, 2000). To treat hepatoblastomas in PRETEX stages 2 and 3, it has been suggested to initially administer 4-6 courses of single-agent cisplatin (Perilongo et al, 2004) or cisplatin in combination with doxorubicin (Pritchard et al, 2000) before attempting surgical resection. In hepatoblastomas diagnosed in stage III (postsurgical classification) or PRETEX stage 4, neoadjuvant chemotherapy plays an important role among the therapeutic options. The international SIOPEL-1 trial compared two preoperative regimens (cisplatin + vincristine + 5-FU !"#$%$ cisplatin and continuous infusion of doxorubicin). Overall survival and disease-free survival were not significantly different in the two arms of this study. However, toxicity was more severe for the second regimen (Ortega et al, 2000). Another regimen including doxorubicin and cisplatin at different doses and schedules has afforded comparable results with fewer secondary effects (Pritchard et al, 2000). The combination of ifosfamide, cisplatin and doxorubicin (IPA) has also shown efficacy (von Schweinitz et al, 1995). Encouraging results were obtained when patients who did not respond to this regimen were treated with carboplatin and etoposide (Haberle et al, 2003). When the resection of stage III hepatoblastomas is not possible, other alternatives in addition to chemotherapy should be considered. These include orthotopic liver transplant (Dower and Smith, 2000; Reyes et al, 2000; Otte et al, 2004; Czauderna et al, 2005; Otte and de Ville de Goyet, 2005; Austin et al, 2006),

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Marin et al: Liver Cancer Chemotherapy! reduced accumulation of drugs in the tumour, which suggests that they may play an important role in the refractivity of HCC to chemotherapy (Nies et al, 2001; Zollner et al, 2005). In some cases chemoresistance in HCC cells may be accounted for by the conjoint enhanced expression of both MDR1 and MRP2 (Minemura et al, 1999). Regarding the role of other members of the ABCC family in the chemoresistance of liver tumours, it is noteworthy that a relationship between MRP3 expression in CGC and resistance to etoposide, doxorubicin and epirubicin has been reported (Tepsiri et al, 2005). The ABCG family includes an important member as regards drug resistance: ABCG2 or BCRP (breast cancer resistance protein). There is experimental evidence that this transporter, which is able to export many cytostatic drugs, may play a role in the chemoresistance of HCC (Li et al, 2007; Takahata et al, 2008). Among the intracellular mechanisms involved in chemoresistance are those that modify drug metabolism, either by enhancing the production of an inactive metabolite, or, if the compound administered is a pro-drug that must be activated, by altering its biotransformation so that it decreases the formation of the active compound. Thus, tumour cells can inactivate anticancer drugs by over-expressing detoxifying enzymes, including phase-I enzymes, such as cytochrome P450 enzymes, or phase-II enzymes, such as those involved in conjugation with glucuronic acid or with glutathione. The expression of some of these enzymes has been found to be altered in HCC (Fritz et al, 1993; Murray el al, 1993). GlutathioneS-transferases (GSTs) and metallothioneins (MTs) are involved in cellular defence against many toxic compounds, including drugs. There is evidence that this is the case of the detoxification mechanism of cisplatin (Akiyama et al, 1999). Moreover, in chemoresistant cells derived from HCC enhanced glutathione levels and GST activity have been found (Jin et al, 2002). UDPglucuronosyl transferases (UGTs) may inactivate drugs through conjugation with glucuronic acid. Such is the case of SN-38, the active form of irinotecan, which is inactivated by the isoform UGT1A1 (Xu and VillalonaCalero, 2002), whose over-expression has been associated with reduced sensitivity to irinotecan in HCC cell lines (Takahata et al, 2008). Other mechanisms leading to chemotherapy resistance through reductions in the efficiency of the antitumour drug are characterized by changes in molecular targets. The following two examples illustrate this concept: i) FdUMP, the active metabolite of 5-FU, is a strong inhibitor of thymidylate synthase. Consequently, the expression level of this enzyme is very important for determining sensitivity to 5-FU. In this sense, resistance to 5-FU has been described in HCC cells with high expression of thymidylate synthase (Jin et al, 2002); ii) The mechanism of action of several anticancer drugs is based on their ability to interact with DNA topoisomerases involved in replication, recombination, chromosome segregation and transcription. Thus, in HCC cells with enhanced resistance to doxorubicin an increased expression of topoisomerase II has been found (Pang et al, 2005).

newer embolization agent Embospheres has been assayed to locally target and treat this aggressive tumour (Stambo and Guiney, 2007). Regarding systemic chemotherapy, the accumulated experience is promising, although very poor. Only few patients have been reported to have been treated with systemic chemotherapy (doxorubicin, cisplatin) alone, and only a few more patients have received chemotherapy after liver transplantation (Weiss and Enzinger, 1982).

VII. Chemotherapy resistance in liver tumours The lack of efficiency of anticancer agents against liver tumours may be due to complex mechanisms also involved in the resistance to chemotherapy of other tumours. Here we shall briefly review some examples specifically applied to liver tumours. One of these mechanisms is the reduction in the active concentration of the drug inside the tumour cells. Two mechanisms can account for this effect: low uptake (Zollner et al, 2005; Huang and SadĂŠe, 2006) and enhanced efflux (Gottesman et al, 2002). Regarding reduced drug uptake, the organic anion transporter OATP1B1, which is highly expressed in human liver and located in the basolateral membrane of normal hepatocytes, is able to transport antitumour drugs (Hagenbuch and Meier, 2003). This carrier is downregulated in HCC, which may result in a lower accumulation of antitumour drugs (Zollner et al, 2005). Changes in the expression in HCC of another member of the OATP family also expressed in normal hepatocytes, OATP1B3 (formerly OATP8), have been described (Vavricka et al, 2004). Moreover, the concentrative nucleoside transporter hCNT1, which is involved in the uptake of nucleoside analogues such as gemcitabine, shows a tendency to be down-regulated in HCC (Zollner et al, 2005). At the same time, an important role in the multidrug resistance phenotype (MDR) is played by plasma membrane proteins involved in the efflux of cytostatic drugs, hence causing a reduction in the intracellular levels of the active compound. The most relevant of these transporters in the chemoresistance of liver tumours are MDR1, MRP1, MRP2, MRP3 and ABCG2, which belong to the superfamily of ATP-binding cassette (ABC) proteins. MDR1 (ABCB1 or P-glycoprotein) plays an important role in drug efflux in several tissues. Among the long list of substrates that ABCB1 is able to transport are included many non-related antitumour drugs, which may account for the association of high ABCB1 expression in HCC with a poor response to chemotherapy (Chou et al, 1997; Ng et al, 2000). However, when inhibitors of ABCB1 have been administered in combination with systemic doxorubicin to treat HCC in clinical practice no improvement in the response was found (Lai et al, 1990). The ABCC family of multidrug resistance-associated proteins (MRPs) includes several isoforms expressed in HCC, such as MRP1 (Bonin et al, 2002) and MRP2 (Nies et al, 2001; Zollner et al, 2005), which are able to transport several cytostatic drugs. The presence of these pumps in the membrane of HCC cells has been associated with a 720

Cancer Therapy Vol 6, page 721! erlotinib and gefitinib (tyrosine kinase inhibitors, TKIs) or cetuximab (an anti-EGFR monoclonal antibody), able to bind to EGFR and inhibit its activation by its natural ligands, have been evaluated in patients with advanced HCC (Thomas and Abbruzzese, 2005). In addition, these TKIs include antiangiogenic activity, resulting from the induction of a decrease in the levels of VEGF (Pore et al, 2006). In a phase II study, erlotinib has shown an acceptable tolerance profile and limited clinical activity, as manifested by disease control in patients with advanced HCC (Philip et al, 2005). Gefitinib has been shown to inhibit cell proliferation and metastatic spread in HCC cell lines and $%&'$'( mouse models, respectively (Matsuo et al, 2003). Cetuximab has also been tested in patients with advanced HCC, showing a good safety profile but poor response rates and survival (Zhu et al, 2007b). However, early results suggest activity for cetuximab in combination with GEMOX (Louafi et al, 2007). In a phase II study, bevacizumab (an anti-VEGF monoclonal antibody), has proved to be safe and potentially effective in localized HCC in patients with well-preserved liver function. A reduction in disease progression in patients with previously diagnosed rapid tumour growth has been reported (Siegel et al, 2008). In patients with nonresectable or metastatic HCC, a moderate antitumour activity of bevacizumab in combination with the GEMOX regimen has been also found (Zhu et al, 2006). Sunitinib targets a variety of TKs in addition to VEGFR, including PDGFRs, KIT, RET, and FLT3, which are involved in tumour angiogenesis in HCC. The results of two phase-II studies in advanced HCC suggest a modest clinical efficacy for this agent (Faivre et al, 2007; Zhu et al, 2007a). Sorafenib blocks Raf kinases, which are key elements of the Raf/MEK/ERK signalling pathway that regulate many cellular functions such as proliferation, differentiation, transformation and apoptosis (Gollob et al, 2006). In addition, sorafenib inhibits tumour angiogenesis by blocking the activation of the tyrosine kinase receptors involved in neovascularization and tumour progression, including VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-!, cKIT, Flt3, and RET (Wilhelm et al, 2004). In a recent international phase-III study (SHARP) comparing sorafenib to placebo in advanced HCC, sorafenib has been shown to significantly increase both progression-free survival and overall survival, without any difference in the appearance of serious adverse events (Llovet et al, 2007).

Resistance to chemotherapy may be also due to an enhanced DNA repair capacity in tumour cells (Lage and Dietel, 1999). HCC is considered to be a tumour highly resistant to agents attacking DNA. However, the reason for this refractivity is not known. Nucleotide excision repair (NER) is one of the major mechanisms accounting for the repair of DNA adduct formation resulting from the use of alkylating agents, such as cisplatin (Reardon et al, 1999). In cisplatin-resistant cells, the expression of some proteins involved in NER has been found to be elevated, such as excision repair cross-complementing protein 1 (ERCC1) (Youn et al, 2004) and xeroderma pigmentosum group A (XPA) (Dabholkar et al, 1994). In colon cancer cells at least, a relationship between the expression levels of both proteins and the resistance of these cells to oxaliplatin has been found (Arnould et al, 2003, Boyer et al, 2004). The role of NER in chemoresistance of HCC is unknown, although several genes involved in NER, such as ERCC1, XPA and xeroderma pigmentosum group C (XPC) have been found to be more highly expressed in the tumour than in the surrounding healthy liver tissue (Fautrel et al, 2005). Impairments of the apoptosis machinery constitute an important group of processes accounting for refractivity to anticancer drugs such as cisplatin, cytarabine, etoposide and doxorubicin, whose mechanism of action involves the activation of apoptosis in tumour cells (Ormerod et al, 1996). The presence of abnormally functioning p53 is a common finding in drug-resistant tumour cells (Anthoney et al, 1996). This is also the case in many HCC, in which the apparent contradiction of high expression levels of p53, higher than in the surrounding liver tissue, is frequently observed (Fautrel et al, 2005). However, this protein is not functional, owing to accumulated mutations in the !"# gene (Hussain et al, 2007). This leads to the resistance of HCC cells to doxorubicin and paclitaxel (Chan and Lung, 2004). Moreover, over-expression of the anti-apoptotic factors Bcl-2 and Bcl-xL has been found in HCC cell lines and this has been associated with resistance to paclitaxel (Chun and Lee, 2004). Another way in which the survival of tumour cells may be increased is through the activation of protein tyrosine-kinases involved in the signalling of growth factors. This is the case of epidermal growth factor receptor (EGFR), which is over-expressed in most HCC (Buckley et al, 2008). When tumour cells are exposed to selective EGFR inhibitors, such as gefitinib, the survival and proliferation signalling pathways are blocked (Okano et al, 2006). Moreover, the resistance to SN-38 in HCC cells has been suggested to be mediated in part by activation of the PI3-K/Akt survival pathway. Thus, the inhibition of elements of this pathway may result in enhanced sensitivity to SN-38 (Koizumi et al, 2005).

IX. Conclusions and perspectives At present, surgical approaches, either by partial hepatectomy or transplantation, remain the treatments of choice for malignant liver tumours, including HCC, CGC and hepatoblastoma. However, because of the frequent existence of underlying liver disease and the limitation in organ availability, only a minority of patients affected are candidates for resection or transplantation. In the early and intermediate stages of liver tumours, local tumour ablation and chemoembolization, respectively, have afforded some benefits. No consistent positive results have been obtained using hormonotherapy, immunotherapy, or systemic chemotherapy, except in the case of hepatoblastomas,

VIII. Development of novel drugs The understanding of the mechanisms of chemotherapy resistance commented above has been the base for the development of novel therapeutic strategies aimed at interacting with molecular targets located in the apoptosis/survival pathways (anti-EGFR, anti-VEGF or anti-RAF therapies). Thus, several agents, such as 721

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which in general are more sensitive to chemotherapy. The lack of response of liver tumours to anticancer drugs is due to complex mechanisms, involving changes in the expression and/or function of the proteins involved in drug uptake/efflux, intracellular processes of signalling, DNA repair and death/survival control. Thus, current research efforts are focused on gaining a better understanding of the mechanisms of chemotherapy resistance in order to be able to better predict it before starting treatment and to develop novel strategies to overcome it.

Acknowledgements This study was supported in part by the Junta de Castilla y Leon (Grants GR75-2008, SA033A08, SA03508 and SA03608), Spain; Ministerio de Ciencia y Tecnologia, Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (Grant BFU2006-12577), Spain; The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), cofunded by the Ministerio de Educacion y Ciencia, Spain and the European Regional Development Fund (ERDF) (Grants BFU2005-24983-E/BFI and BFU2007-30688E/BFI) and belongs to the CIBERehd (Centro de Investigacion Biomedica en Red) for Hepatology and Gastroenterology Research (Instituto de Salud Carlos III, Spain). The authors thank N. Skinner for revision of the English text of the manuscript.

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Jose J G Marin

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Cancer Therapy Vol 6, page 711! Cancer Therapy Vol 6, 711-728, 2008

Chemotherapy in the treatment of primary liver tumours Review Article

Jose J G Marin*, Beatriz Casta単o, Pablo Martinez-Becerra, Ruben Rosales, Maria J Monte Laboratory of Experimental Hepatology and Drug Targeting, Biomedical Research Center for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Instituto de Salud Carlos III, University of Salamanca, Spain

__________________________________________________________________________________! *Correspondence: Jose J. G. Marin, Department of Physiology and Pharmacology, Campus Miguel de Unamuno E.I.D. S-09, 37007Salamanca, Spain; Tel: 34-923-294674; Fax: 34-923-294669; E-mail: jjgmarin@usal.es Key words: Chemotherapy, Primary liver tumours, Non-malignant primary liver lesions, Hepatocellular carcinoma, Hormonotherapy, Curative treatment, Chemotherapy, Palliative treatment, Immunotherapy, Systemic chemotherapy, Chemotherapy resistance Abbreviations: 5-fluorouracil, (5-FU); ATP-binding cassette, (ABC); Best supportive care, (BSC); Cisplatin + adriamycin + 5-FU + INF!, (PIAF); cholangiocarcinoma, (CGC); epidermal growth factor receptor, (EGFR); European Regional Development Fund, (ERDF); excision repair cross-complementing protein 1, (ERCC1); Gemcitabine + oxaliplatinum (GEMOX); Glutathione-S-transferases, (GSTs); Hepatocellular carcinoma, (HCC); ifosfamide, cisplatin and doxorubicin, (IPA); Interferon alpha, (INF!); Iridium-192, (192Ir); Luteinizing hormone-releasing hormone, (LHRH); Metallothioneins, (MTs); Multidrug resistance phenotype, (MDR); Multidrug resistance-associated proteins, (MRPs); Nucleotide excision repair, (NER); Organic anion-transporting polypeptide (OATP); Research on Membrane Transport Proteins, (REIT); Trans-arterial chemoembolization, (TACE); Tyrosine kinase inhibitors, (TKIs); UDPglucuronosyl transferases, (UGTs); Vascular endothelial growth factor, (VEGF); Xeroderma pigmentosum group A, (XPA); Xeroderma pigmentosum group C, (XPC) Received: 21 July 2008; Revised: 12 September 2008 Accepted: 16 September 2008; electronically published: October 2008

Summary Primary liver tumours, mainly hepatocellular carcinoma and cholangiocarcinoma, constitute the fifth most frequent types of cancer, whereas in the rank of mortality they are the third. The clinical relevance of liver tumours increases further if the number of deaths due to liver metastasis of cancers of extrahepatic origin is included. One important limitation in the clinical battle against these tumours is their marked refractivity to the available chemotherapy and the development of resistance to drugs during treatment. Owing to the existence of several types of primary liver tumours and the wide range of tissues from which metastatic cells can be released, the biology of tumours affecting the liver is diverse, and to this must be added inter-individual variability plus the fact that sensitivity/refractivity to drugs may change during the evolution of the tumour. In the context of the current most commonly used curative and palliative treatments, the present article reviews classic and newly available anticancer drugs that have been assayed to treat liver tumours, either alone or, more frequently, combined with other therapeutic options as adjuvant (pre- and post-surgery) chemotherapy. The main mechanisms responsible for the lack of efficacy of these drugs in primary liver tumours are also briefly described.

come third in the rank of mortality due to cancer, even though they constitute the fifth most frequent type of cancer. The clinical relevance of liver tumours is further exacerbated if the number of deaths due to liver metastasis of cancer of extrahepatic origin is taken into consideration. One important limitation in the clinical battle against these tumours is their marked refractivity to the available chemotherapy and the development of resistance during treatment. The complexity of the situation can be understood if it is considered that owing to the existence of several types of primary liver tumours and the broad

I. Introduction Liver tumours can be classified according to their origin as primary tumours, derived from liver cells, and secondary, i.e., due to metastasis of primary tumours located in other territories, frequently colorectal tumours (Benson, 2007). Depending on the severity of their prognosis, they can be classified as benign or malignant tumours (Table 1). Non-malignant lesions are commonly asymptomatic and, when detected incidentally, the recommendation may be surgical resection or simply follow up. In contrast, primary malignant liver tumours 711

Marin et al: Liver Cancer Chemotherapy! the biliary tree, the stomach or the duodenum, in which case they cause abdominal pain, fever or cholestasis. Some of these tumours, such as infantile hemangioendothelioma and mesenchymal hamartoma, are almost exclusively found in children. In adults, benign liver tumours include, in order of frequency (Table 1): hemangiomas (CaseiroAlves et al, 2007), focal nodular hyperplasia (LizardiCervera et al, 2006) and hepatocellular adenomas (Kalra et al, 1976; Lizardi-Cervera et al, 2006) in the liver parenchyma, and cholangiocellular adenomas and biliary cystadenomas, which are derived from biliary duct cells (Kim et al, 1998). The remaining benign liver tumours, such as biliary hamartoma, mesenchymal hamartoma, lipoma, myelolipoma, angiomyolipoma, angiomyelolipoma and others are very infrequent. Complete identification of the type of lesion is usually based on the clinical presentation, biochemical data, imaging techniques and histological examination of biopsies. The patient’s background regarding the use of oral anticonceptives, cirrhosis, etc. may be valuable information for orienting the diagnosis. The best therapeutic option for non-malignant primary liver tumours is usually surgical resection, which is recommended based on the size and predicted consequences of the tumour and on the risk of spontaneous rupture with intraperitoneal bleeding, which is frequently the case of hepatic adenomas. Moreover, the risk of transformation into malignant lesions should be also considered. This risk is higher in hepatic adenomas, cholangiocellular adenomas and biliary cystadenomas. Recent investigations have suggested that analysis of the genotype-phenotype correlations in benign liver tumours and the identification of novel markers of a higher risk of malignant transformation may modify the current protocols of diagnosis and follow-up of these patients (Rebouissou et al, 2008). Trans-arterial embolization, as an alternative to surgical resection, has been recommended only in infantile hemangioendothelioma, when resection is not possible. The need for an orthotopic liver transplant due to nonmalignant primary liver tumours is rare.

range of tissues from which metastatic cells reaching the liver may be released, the biology of tumours affecting the liver is quite diverse. Moreover, inter-individual variability, together with the fact that the sensitivity/refractivity to drugs may change during the evolution of the tumour, must also be taken into account. Liver tumours can be treated according to five different types of strategies. These include: i) surgical resection or liver transplant; ii) percutaneous intervention by ethanol injection or tumour ablation by radiofrequency; iii) trans-arterial intervention by tumour embolization, chemoperfusion or chemoembolization (TACE); iv) systemic chemotherapy and v) gene therapy. In the present article we shall review the relevance of classic and novel chemotherap"#$ic options in the context of these possible strategies to treat the different types of liver tumours. Although with some peculiarities and different therapeutic recommendations -in part due to the type of tumour, but also to the stage of development at which they are diagnosed- the basic mechanical procedures used to treat these tumours, including surgical resection, percutaneous ablation, arterial embolization and orthotopic liver transplant are similar for different types of tumours. In contrast, regarding their response to chemotherapy, this is very diverse, although in general low, owing to their very different biological characteristics. When located in the liver, secondary tumours usually maintain the same sensitivity to chemotherapy as the primary tumour. Thus, here we shall focus on the recommended treatments and the sensitivity/resistance to commonly used drugs only regarding primary liver tumours.

II. Treatment of primary liver lesions

non-malignant

Benign primary tumours form a heterogeneous group of hepatic lesions that share the common characteristic of the absence of uncontrolled growth or metastasis. They are usually detected incidentally during abdominal exploration using ultrasound or computerized tomography prescribed to diagnose other diseases, because they are frequently asymptomatic unless they are large enough to compress Table 1. Types of primary liver tumours. Localization Liver Parenchyma

Benign Hemangioma Focal nodular hyperplasia Hepatocellular adenoma Infantile hemangioendothelioma Mesenchymal hamartoma Others

Malignant Hepatocellular carcinoma Hepatoblastoma Hemangiosarcoma Fibrosarcoma Lymphosarcoma Others

Intrahepatic Biliary Tree

Cholangiocellular Adenoma Biliary Cystadenoma Biliary hamartoma Others

Cholangiocarcinoma Others

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Cancer Therapy Vol 6, page 713! discovered in initial stages when it is still feasible to apply radical treatment. As in the case of other types of liver cancer to be reviewed below, complete tumour resection is the best therapeutic option for long-term survival in patients with HCC (Ringe et al, 1991). However, in addition to the technical feasibility of the surgical procedure other factors, such as the general health status of the patient, the degree of remnant liver function and the stage of the tumour, may limit the possibility of success of this therapeutic approach (Bruix and Sherman, 2005). Resection of HCC may follow similar guidelines to those used in the surgical removal of other liver malignancies. These include routine ultrasonography during the procedure to define tumour number, size and exact localization (Takayama et al, 1998). Dual-phase spiral computed tomography is the primary imaging technique in the diagnosis of focal liver lesions. The diagnostic confidence in the detection and characterization of small liver lesions can be improved by contrastenhanced ultrasonography (Janica et al, 2007). It is important to elucidate the relationship of the tumour with neighbouring vascular structures with ultrasonography, because in addition to avoiding tumour manipulation, the tumour must be rapidly and efficiently isolated from the vascular inflow and outflow to prevent iatrogenic metastasis during the surgical procedure (Jarufe, 2006). In patients with HCC, but in the absence of liver cirrhosis, partial hepatectomy is relatively safe and postsurgery mortality is lower than 5% (Bismuth et al, 1993; Fuster et al, 1996), even if the resection includes a large liver mass (Bismuth and Majno, 2000). The post-surgery survival of these patients reaches 80-92% in the first year; 61-86% three years after hepatectomy, and 41-74% after five years (Fong et al, 1999). Patients reaching the highest values in this range of survival are those with a single tumour, with a well preserved hepatobiliary function, without portal hypertension, and with normal serum bilirubin values (Llovet et al, 1999). Unfortunately, the proportion of patients with HCC but without liver cirrhosis is low. This is less than 40% in Asia and less than 10% in Western countries. This is important because in patients with liver cirrhosis the risk of post-surgery complications is higher, which accounts for mortality rates after tumour resection of between 10 and 20% (Ringe et al, 1991). This and other factors explain why the 5-year survival rate is only of 37% in this type of patients (Fong et al, 1999). In cases where surgical resection is not recommended, percutaneous intervention may represent the best choice, provided the tumour is in an early stage (Livraghi et al, 1995). In these cases, cancer cell death is induced by intratumour administration of ethanol or acetic acid or by physical means aimed at strongly increasing or reducing the temperature of the tumour cells by exposing them to radiofrequency, laser beams or cryoablation (Okada, 1999). Percutaneous ethanol injection is the method most used because it is cheap and easy, and the appearance of serious side effects is rare. Using this therapeutic approach, the success rate regarding tumour removal reaches 90-100% in HCC tumours smaller than 2

Chemotherapy is not commonly used to treat these tumours, because the efficacy of the drugs used to treat them has not been demonstrated, except in the case of infantile hemangioendothelioma, which has been reported to be sensitive to treatment with corticoids (Jackson et al, 1977).

III. Curative treatment hepatocellular carcinoma (HCC)

of

In adults, the most frequent type of malignant liver tumour is HCC, which accounts for 90% of malignant primary hepatic lesions (Anderson et al, 1992). Moreover, since it accompanies (and is probably closely related to) the growing frequency of cirrhosis, the incidence of HCC is increasing worldwide. Indeed, approximately three out of four cases of HCC are associated with cirrhosis of the liver. Furthermore, patients with viral hepatitis B and C infection have an enhanced risk of developing this malignancy. HCC may appear as a single tumour, in the form of several nodules, or as a diffuse lesion in the liver parenchyma. Histologically, HCC is often formed by highly vascularized trabecular groups of cells with a varying (from well differentiated to undifferentiated tumours) degree of similarity to hepatocytes. The neoplastic tissue often invades branches of the portal vein and even the hepatic vein. Expansion of the HCC occurs via four different mechanisms: i) centrifugal growth, which results in compression of the surrounding healthy tissue; ii) parasinusoidal dissemination by tumour invasion of the neighbouring tissue, either through the sinusoids or the parasinusoidal spaces; iii) venous dissemination involving the use of the branches of the portal vein to propagate the tumour in the retrograde direction. Invasion of the hepatic vein is less common, but when it occurs it can reach the cava vein or even the right auricle; iv) as a consequence of dissemination through the lymph vessels and the vascular system, HCC metastases can reach other organs, most frequently regional lymph nodes (48%) and the lung (73%). Common clinical manifestations of HCC include loss of body weight (80% of cases), fatigue and abdominal pain. The latter is usually vague and persistent but may become suddenly very intense due to intraperitoneal haemorrhage associated with the rupture of a necrotic nodule or the erosion of a vessel. Liver size is almost always increased. Splenomegalia occurs in one third of cases. The appearance of jaundice varies between 20% and 58%. Ascites accumulates in 50%-75% of patients. The prognosis of patients with HCC depends on the time when the diagnosis is established. Patients with good remnant liver function and asymptomatic HCC can survive for several years, even without treatment. In contrast, when patients are diagnosed in a very advanced stage of tumour progression and/or associated impaired liver function they may die in weeks. Fibrolamellar HCC constitutes a special sub-type of HCC that mainly affects young women without cirrhosis. This tumour is characterized by the presence of a dense fibrous infiltration and a lower invasiveness, which accounts for a better prognosis, especially if the tumour is 713

Marin et al: Liver Cancer Chemotherapy! (Wang et al, 2008). This and incomplete embolization usually result in the absence of full success in inducing tumour necrosis. Studies showing that tegafur/uracil inhibits tumor angiogenesis in several cancer types have suggested the use of chemotherapy based on tegafur/uracil after TACE in advanced HCC. The use of this strategy seems to have been effective in treating these tumours and no severe adverse events have been reported (Ueda et al, 2008). In the absence of metastasis, when the tumour cannot be surgically removed and/or the remnant liver function is very poor liver transplant may be the only choice, but only if the selection criteria are appropriate because of the low availability of donors, particularly in Asia. The development of techniques for donations from living donors has permitted this problem to be partially overcome (Fan et al, 2000; de Villa et al, 2003). A careful selection of patients to be treated in this way is crucial. Several selection criteria have been proposed, adapted, and improved over the last two decades, and this has contributed to enhancing the rate of success in patients with HCC undergoing liver transplant to 5-year survival rates without recurrence in up to 85%. In 1996, the socalled criteria of Milan were proposed, which considered as candidates for liver transplant patients with a single tumour of ! 5 cm or 2-3 tumours of ! 3 cm (Mazzaferro et al, 1996). These criteria were slightly relaxed later (Yao et al, 2001; Broelsch et al, 2005; Hiatt et al, 2005; Majno and Mazzaferro, 2006).

cm, 70% in lesions smaller than 3 cm, and 50% when the tumours are 5 cm or more in diameter (Lencioni et al, 1997). Thus, in many cases of HCC with a single tumour of ! 3 cm, percutaneous ethanol injection may be considered the treatment of choice (Bruix and Llovet, 2002). In the case of larger tumours, radiofrequency may offer a good alternative to surgical resection (Buscarini et al, 2001). This technique can be used either percutaneously or during laparotomy. The aim is to reach at least the same response rate as when using percutaneous ethanol injection but with fewer sessions. The advantage over percutaneous ethanol injection is more evident in the treatment of tumours larger than 3 cm. For these patients, the 5-year survival rate is 33-40% (Livraghi et al, 1999). Other approaches, such as laser beam irradiation, cryoablation and microwave-induced coagulation involve more complications, are still in the experimental phase, and have not yet been widely accepted (Okada, 1999). Trans-arterial embolization with lipiodol alone or using chemoembolization (TACE), in which the embolizing agent is mixed with anticancer drugs (e.g., doxorubicin, mitomycin or cisplatin), are the therapies of choice in the treatment of liver tumours that cannot be resected surgically or efficiently eliminated by percutaneous interventions (Lo et al, 2002). These procedures reach response rates (generally partial responses) of between 15% and 55%. Despite this, they may be useful to slow down tumour growth and delay vascular invasion in comparison with patients receiving only palliative treatment (Blum, 2005). However, it is very important to consider patient selection in these cases. Thus, patients must have a preserved liver function (generally Child A cirrhosis) and must be free of vascular invasion and extrahepatic dissemination (Llovet et al, 2002). This is because in patients with poor liver function (Child B or C) these therapies may result in acute liver failure, which abolishes the potential benefit of using this therapeutic approach. When appropriately used, chemoembolization, for instance using doxorubicin-loaded beads, has been shown to be an efficient therapy in the treatment of non-surgically resectable HCC, as demonstrated by the low complication rate, marked tumour response, and sustained reduction of alphafetoprotein levels (Malagari et al, 2008). TACE has been also proposed as a useful palliative strategy in patients with HCC on the waiting list for orthotopic liver transplantation (Alba et al, 2008). However, there is some controversy about whether preoperative TACE significantly improves the disease-free survival or the pattern of recurrence after curative resection of HCC. Studies based on retrospective analyses of preoperative TACE do not recommend this technique as a routine procedure before hepatectomy for a resectable HCC (Choi et al, 2007). In spite of the beneficial effects of TACE in some patients with HCC, as happens with other mechanisms of resistance to be reviewed below the tumour tends to adapt its biology to the new situation. Thus, the residual surviving cancerous tissue in HCC after TACE has a rich vascularity, in part due to increased vascular endothelial growth factor (VEGF) expression in the residual tumour

IV. Chemotherapy in the palliative treatment of HCC Many different drugs have been assayed for the treatment of HCC. However, success has been achieved in only 15% of patients, and then with only a partial response, usually associated with a high rate of side effects. These have been reduced in part by local administration through the hepatic artery. Since the liver is an organ sensitive to the action of sex hormones, and since hormone receptors are expressed in HCC, the usefulness of hormonotherapies, both anti-androgenic and antiestrogenic, has been investigated (Boix et al, 1993; Bl채ker et al, 2004). However, none of them has demonstrated a consistent rate of response or the ability to slow down tumour growth or an unequivocal modification of the survival time. Other options of systemic treatment have included the use of immunomodulators. However, the administration of interferon has afforded minimal antitumour activity, with no significant effect on survival time. Although other types of immunotherapy have not been effective either, promising results have been obtained with several new agents that should be investigated in adequately designed clinical trials. The most significant results obtained using these alternatives are reviewed below and are summarized in Figure 1.

A. Hormonotherapy Anti-androgenic agents have shown no efficacy even when combined with agonists of luteinizing hormonereleasing hormone (LHRH), such as flutamide (an anti-

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Cancer Therapy Vol 6, page 715! octreotide (sandostatin LAR) were carried out, no positive impact was found (Yuen et al, 2002; Becker et al, 2007). When lanreotide, another long-acting somatostatin analogue, was tested, a limited degree of antitumour activity was observed (Raderer et al, 2000; Cebon et al, 2006). In conclusion, although some patients occasionally appear to have a stable response to somatostatin analogues, the overall beneficial response regarding the inhibition of tumour progression and survival seems to be poor.

androgenic drug) and triptorelin (an LHRH agonist) (Grimaldi et al, 1998). Regarding anti-estrogenic agents, the usefulness of tamoxifen in the treatment of advanced HCC has long been discussed (Farinati, 1996), but it is now clear from the results of several randomized controlled trials that tamoxifen is not an efficient drug in the treatment of this disease (Castells et al, 1995; CLIP Group, 1998; Riestra et al, 1998; Liu et al, 2000; Chow et al, 2002; Barbare et al, 2005). In a meta-analysis of seven randomized controlled trials in which tamoxifen was compared with conservative management, this drug showed no significant antitumour effect or benefit for survival (Llovet and Bruix, 2003). Tamoxifen has also been suggested to inhibit P-glycoprotein-mediated drug resistance and this has led to assays of tamoxifen in combination with other chemotherapeutic agents. Unfortunately, such studies have revealed no benefit of the addition of tamoxifen to the chemotherapeutic regimen (Melia et al, 1987; Cheng et al, 1996, Raderer et al, 1996; Cheng et al, 1998). Although other anti-estrogen agent, megestrol, has shown modest activity in at least three trials (Chao et al, 1997; Farinati et al, 2001; Villa et al, 2001), to date no robust survival advantage has been shown either. Since somatostatin has a well known antitumour effect in neuroendocrine tumours, and because somatostatin receptors have been identified in HCC cells (Bl채ker et al, 2004; Cebon et al, 2006), several analogues of this hormone, such as octreotide, have been assayed to treat HCC. When this substance was first studied, in short series of patients, the results suggested a benefit of this approach (Kouroumalis et al, 1998). However, when studies in larger trials using a long-acting form of !

B. Immunotherapy Interferon alpha (INF! ), an immunomodulatory cytokine widely used in the treatment of chronic hepatitis, has demonstrated activity in preclinical models against several tumour types, including HCC (Ikeda et al, 1999; Wada et al, 2007). In addition, antiangiogenic and antiproliferative properties have been proposed (Lindner, 2002; von Marschall et al, 2003; Fujioka et al, 2006; Zhu et al, 2008). Thus, INF! monotherapy has been evaluated in patients with advanced HCC, but unfortunately, conflicting results were obtained. Two randomized controlled trials suggested better response rates and good tolerance when compared to doxorubicin (Lai et al, 1989) or best supportive care (BSC) (Lai et al, 1993). A more recent study failed to show benefits of INF! regarding the tumour progression rate and survival time. In addition, INF! was poorly tolerated in patients with cirrhosis and advanced HCC, leading to a high rate of dropout (Llovet et al, 2000).

Figure 1. Schematic representation of most interesting chemotherapeutic agents assayed in clinical practice to treat the main types of primary liver malignancies.

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Marin et al: Liver Cancer Chemotherapy! When the results from trials with different chemotherapeutic options are analyzed it should be recalled that patient populations and cancer biology are very heterogeneous, which may result in confusing data and the masking of positive responses in certain subgroups of patients (Nowak et al, 2004). Thus, the heterogeneity of the populations included in clinical trials, together with the differences in the design of such studies, make it very difficult to compare the results concerning response rates between different trials, as well as to extrapolate the conclusions to a general patient population. This problem, together with the poor response obtained with most regimens assayed until now, does not allow a standard chemotherapy regime to be established for all patients with advanced HCC. Almost all the available chemotherapeutic agents have been tested in monotherapy, mainly in phase-I and -II studies, to treat HCC (Table 2). However, in general the rate of objective responses has been very low (O´Reilly et al, 2001; Fuchs et al, 2002). Moreover, the response is commonly limited and transient and there is no consistent result indicating that patient survival is actually increased (Johnson, 2002; Yeo et al, 2005). This has restricted the

However, combinations of INF! with chemotherapy appear to be more efficient, as will be described below. Novel strategies based on immunotherapeutic agents are currently being developed, with promising results. These include the administration of other cytokines and the transfer of T-lymphocytes (Butterfield, 2004; Avila et al, 2006; Sun et al, 2006; Zerbini et al, 2006; Butterfield, 2007).

C. Systemic chemotherapy Several reasons account for the lack of substantial success in treating HCC with chemotherapeutic agents in systemic regimens: i) Most of these tumours are highly resistant to chemotherapy owing to mechanisms that will be briefly described below; ii) Tolerance to the treatment is limited due to the reduced remnant liver function; iii) The existence of cirrhosis may determine thrombocytopenia due to portal hypertension and hypersplenism, leading to delayed treatment cycles, decreased chemotherapy dose-intensity and impaired treatment efficacy (Taieb et al, 2006); iv) Finally, the use of immunosuppressive chemotherapy may reactivate chronic HBV infection, which is often present in these patients (Taieb et al, 2006).

Table 2. Response rate in mono- and poly-chemotherapy of hepatocellular carcinoma. Chemotherapy Regimen Mono-chemotherapy Doxorubicin 5-FU Epirubicin Mitoxantrone Capecitabine Etoposide Cisplatin Gemcitabine Irinotecan Poly-chemotherapy 5-FU + leucovorin Cisplatin-based regimens: Cisplatin + epirubicin + infusional 5-FU ECF (etoposide + cisplatin + 5-FU) Cisplatin + doxorubicin + capecitabine Cisplatin + mitoxantrone + infusional 5-FU Cisplatin + doxorubicin Infusional cisplatin + infusional 5-FU PIAF (cisplatin + doxorubicin + infusional 5-FU + INF!) Gemcitabine-based regimens: Gemcitabine + cisplatin GEMOX (Gemcitabine + oxaliplatin)

Response Rate 10-20% 6-10% 10-25% 10-25% 13%-25% 0-10% 0-15% Modest anti-tumour activity Minimal anti-tumour activity 10-28% 15% 15-20% 24% 24-27% 18-49% 47% 15-26% (*) 17-21% 17-40%

(*) Better results have been reported in patients with normal liver function as compared to those with cirrhosis and high serum bilirubin levels (response rate 50% vs 6%) 5- Fluorouracil, 5-FU; Interferon-alpha, INF!.

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Cancer Therapy Vol 6, page 717! as jaundice, choluria and pruritus (Shimoda and Kubota, 2007). As mentioned above regarding other liver tumours, surgery is also at present the best curative therapeutic option for patients with CGC (Yeh et al, 2004; Alberts et al, 2007). However, this is not always feasible or recommended, as happens in patients with multiple tumours (approximately 5% of them) and/or with subjacent parenchymal or biliary disease. Moreover, the majority (approximately two thirds) of these patients are diagnosed at advanced stages of the disease, approximately 50% of patients already have an invasion of lymph nodules and 10-20% have metastasis or lesions involving the portal structure or main hepatic artery (Pichlmayr et al, 1996). This implies that surgical resection is recommended in only a small number of patients with CGC (Khan et al, 2002). When resection is not possible, in principle liver transplantation might provide a good alternative (Iwatsuki et al, 1998). However, in contrast to HCC, patients with CGC have classically been excluded from liver transplant programs. This has been due in part to the high probability of recurrence of this type of tumour after resection. Nevertheless, improvements in survival due to novel adjuvant therapies suggest that this therapeutic option for CGC should be reappraised (Knight et al, 2007). In patients with unresectable CGC, the prognosis is poor and palliative measures should aim at increasing the quality of life and the relief of symptoms (pain, pruritus, jaundice) and, if possible, at extending patient survival (Anderson et al, 2004). Radiation therapy, chemotherapy or a combination of both approaches may constitute the only possible treatment for these patients (Shimoda and Kubota, 2007). Even in some cases in which surgical removal of the tumour is possible, complete elimination of the disease is not achieved, and hence a high rate of recurrence (60-90%) occurs (Malhi and Gores, 2006). This justifies the use of adjuvant therapies, radiotherapy and/or chemotherapy in combination with surgery (Chari et al, 2003a). However, the actual usefulness of these treatments is controversial (Hejna et al, 1998). In some series, radiotherapy after surgery has been found to be useful (Todoroki et al, 2000), whereas poorer results were obtained in other series (Pitt et al, 1995). In some cases, even an impairment of liver function has been reported (Cherqui et al, 1995). This justifies the restriction of radiotherapy to patients with unresectable CGC due to locally advanced disease but without evidence of distant metastases. These patients may benefit from palliative radiotherapy using a combination of external beam radiation and intraluminal brachytherapy with iridium-192 (192Ir) (Chari et al, 2003b). While the survival benefit of palliative radiotherapy is not clear, a role in the control of local disease seems more evident. The use of radiotherapy with or without concomitant chemotherapy may contribute to biliary decompression and the relief of pain (Onishi et al, 1995), permitting a better management of local biliary disease (Alden and Mohiuddin, 1994; Kuvshinoff et al, 1995; Vallis et al, 1996). In addition, a combination of regional chemotherapy using intra-arterial hepatic

use of chemotherapy in HCC to the aim of trying to slow down tumour growth and to minimize cancer-related symptoms. The drugs with the highest activity against HCC are 5-fluorouracil (5-FU), cisplatin and doxorubicin. However the best results are obtained when they are used in combined regimens (Table 2). Nevertheless, the response rates of patients with HCC to these drugs are relatively low (Chlebowski et al, 1984; Porta et al, 1995). Epirubicin, mitoxantrone and capecitabine have no better proven antitumour efficacy than doxorubicin (Dunk et al, 1985; Hochster et al, 1985; Colleoni et al, 1993; Pohl et al, 2001). Other chemotherapeutic drugs with modest activity against HCC are etoposide, vincristine, gemcitabine and irinotecan (Yang et al, 2000; Fuchs et al, 2002). Multiple combinations of antitumour drugs have been tested in patients with advanced HCC (Table 2). Cisplatin-based regimens appear to result in higher objective response rates than others not including cisplatin (Stuart, 2007). However, it is not clear that any of these regimens may confer survival benefit to treated patients. The response rates to regimens including two- and threedrug combinations are approximately the same. Among the cisplatin-based regimens, the best response rates have been obtained with the treatment known as PIAF (cisplatin + adriamycin + 5-FU + INF! ) (Leung et al, 1999, 2002; Lau et al, 2004). However, due to its high toxicity profile, it should be considered only for patients with a general good health status and minimal hepatic dysfunction (Leung et al, 2002; Yeo et al, 2005). More recently, the GEMOX regimen (gemcitabine + oxaliplatin) has been evaluated in a phase-II study, with promising results. In addition, this regimen lacks substantial renal and hepatic toxicity (Taieb et al, 2003; Louafi et al, 2007). !

V. Chemotherapy in the treatment of tumours of the biliary system Primary malignant tumours that originate in the biliary system include cholangiocarcinoma (CGC), gallbladder cancer and carcinoma of the ampulla of Vater. The latter two types of tumour show different clinical characteristics and will not be considered here. Although CGC is less frequent than HCC - the incidence of CGC is between 0.01% and 0.8%; this tumour represents 5%-10% of all hepatic primary malignancies-, CGC is more aggressive than HCC (Yeo et al, 1990). The typical age of presentation is between 50 and 70 years, but it may appear up to 20 years earlier in persons with an enhanced risk of developing this tumour. Among the factors favouring the development of CGC are primary sclerosing cholangitis, whose risk of appearance is in turn enhanced in patients by smoking and alcohol consumption, congenital disease (e.g., choledochal cyst, congenital hepatic fibrosis and Caroli disease), intrahepatic lithiasis, and several infections by parasites (e.g., "#$%$&'()* and +,)*-$&'()*) or viruses (e.g., HIV, HBV and HCV). Regarding anatomical localization, these tumours can be classified as intrahepatic (approximately 15%), perihiliar (60%), and distal (25%) cholangiocarcinomas. The clinical manifestations are abdominal pain, weight loss and, usually, signs related to biliary obstruction, such 717

Marin et al: Liver Cancer Chemotherapy! chemotherapy cannot be always used. Thus, systemic chemotherapy is recommended in most cases.

fluorodeoxyuridine and conformational radiotherapy has been reported to improve results in controlling local disease (Robertson et al, 1993). Regarding the use of chemotherapy alone as an adjuvant to surgery, controversial results have also been obtained (Goldstein et al, 1993; Todoroki, 2000; Yoshida et al, 2002; Kelley et al, 2004). The general consensus is that there is no clear benefit of chemotherapy for biliary tumours except in the case of certain gallbladder carcinomas (Takada et al, 2002). Moreover, the combination of radiotherapy plus chemotherapy has not afforded improved results (Figueras et al, 2000). Nevertheless, it is important to consider that no randomized clinical trials exist for any of these adjuvant therapies. The available data are from retrospective studies carried out in small groups of patients (Hejna et al, 1998). Thus, at present no adjuvant treatment is recommended outside clinical investigation (Hejna et al, 1998; Anderson et al, 2004). The use of neoadjuvant chemotherapy (or presurgery chemotherapy) is rarely recommended because of the presence of reduced liver function and jaundice at the moment of diagnosis in most patients with CGC (Anderson et al, 2004). In patients with metastatic CGC, systemic chemotherapy has been used as palliative treatment. For this purpose 5-FU was used first, with a poor response rate (0-10%) and a short median survival time (6.5 months). Subsequent use of gemcitabine has permitted better results to be obtained in several dosage schedules (8-60% and 6.3-16 months, respectively). Other regimens based on the use of single agents, such as paclitaxel, docetaxel and irinotecan have failed to attain response rates higher than 10%. In contrast, combined regimens have in general been more effective. The best results have been obtained for regimens based on 5-FU in combination with leucovorin, doxorubicin, mitomycin C, methyl-CCNU, streptozotocin, INF! and cisplatin plus epirubicin (a 10-40% response rate and a 2-12-month median overall survival) or gemcitabine including leucovorin/5-FU, cisplatin, oxaliplatin, docetaxel, irinotecan and capecitabine (a 950% response rate and a 5-15.4-month median overall survival), with a tolerable toxicity profile (Thongprasert, 2005). Data from several studies suggest that gemcitabine in monotherapy or combined with cisplatin or oxaliplatin represents one of the most active and well tolerated drugs. Another regimen with promising results is 5-FU and leucovorin with cisplatin. Nevertheless, a phase-III comparison between these different alternatives is needed (Thongprasert, 2005). Intra-arterial administration of chemotherapeutic agents permits response rates of up to 44% to be obtained (O´Grady, 2000; Khan et al, 2002). This may be useful in some cases both to reduce tumour size before surgery and to prolong survival in patients with non-resectable tumours (Burger et al, 2005; Wu et al, 2007). The best regimens assayed in this approach have been those based on gemcitabine including either cisplatin or oxaliplatin (Gusani et al, 2008). Unfortunately, owing to the high incidence of metastasis in patients with CGC, and hence the high frequency of recurrence, intra-arterial

VI. Chemotherapy in the treatment of hepatoblastoma, hemangiosarcoma and other liver malignancies Two malignant primary liver tumours that are uncommon in adults, also less frequent than HCC, are hepatoblastoma and hemangiosarcoma (Table 1). Other primary malignancies, such as lymphosarcoma, fibrosarcoma and leiomyosarcoma are even more infrequent. Among the liver metastases, the most frequent ones have their origin in primary tumours located in the gastrointestinal system (tumours of pancreas, stomach, small intestine, colorectal region, extrahepatic biliary tree and gallbladder), or lung cancer, breast cancer, melanoma, and endocrine carcinoid tumours. Whereas HCC is derived from differentiated liver parenchyma, hepatoblastoma develops in foetal liver tissue. Thus, HCC is rarely found in patients younger than 6 years, where this tumour is often associated with cirrhosis or congenital metabolic storage disease (Isaacs, 2007). Although hepatoblastoma is a very rare tumour, accounting for less than 1% of all paediatric cancers, it is the most frequent hepatic tumour in children (Miller et al, 1995). Hepatoblastoma is commonly found in otherwise healthy livers. The tumour is usually discovered as a single large mass, which is detected by antenatal ultrasonography or during childhood, sparked by the precocious onset of puberty. Approximately 50% of hepatoblastomas are encapsulated at the moment of the diagnosis. The prognosis of this malignancy depends on the possibilities of implementing surgical treatment (resection or transplant), but it is usually better than that of HCC. A group of paediatric liver tumours with intermediate characteristics has recently been classified as transitional liver cell tumours (Zimmermann, 2005). As in the case of other liver malignancies, complete surgical resection of the tumour also constitutes the treatment of choice and the only one with curative potential for hepatoblastoma. Nevertheless, in the last three decades, chemotherapy, both as adjuvant and neoadjuvant therapy, has become a useful tool to improve the outcome of these patients, with rates of survival at 5 years of approximately 75% !"#$%$ the value of 35% described 30 years ago (Pritchard et al, 2000; Schnater et al, 2002). The reason for the success is that hepatoblastoma is more sensitive than other liver malignancies to several cytotoxic agents, such as vincristine, doxorubicin, cyclophosphamide, ifosfamide, 5-FU, and cisplatin. Neoadjuvant chemotherapy has proved to be effective in increasing the number of hepatoblastomas that become resectable only after chemotherapy in up to 75%, and in decreasing the morbidity associated with surgery (Stringer et al, 1995; Seo et al, 1998). This strategy is aimed at shrinking the tumour, rendering it more solid, less prone to bleeding, and better delimited with respect to the surrounding healthy liver tissue. Thus, a safer and

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Cancer Therapy Vol 6, page 719! radiotherapy (Habrand et al, 1992) and intra-arterial chemoembolization (Malogolowkin et al, 2000; Xianliang et al, 2004). The outcome for hepatoblastomas in stage IV at diagnosis is not good, but cure is still possible in 25% to 30% of patients (Douglass et al, 1993; Ortega et al, 2000; Perilongo et al, 2000). Patients that have undergone primary tumour resection should also be considered for surgical removal of pulmonary metastases. In these advanced hepatoblastomas, the same chemotherapy regimens used in stage III tumours are recommended. The standard regimen is 4 courses of cisplatin/vincristine/5-FU (Ortega et al, 2000) or doxorubicin/cisplatin followed by an attempt at tumour resection. If the surgical intervention is successful, 2 postoperative courses of the same chemotherapy should be given. If the tumour is not resectable after the 4 courses mentioned above, alternative options should be considered. These include irinotecan (Katzenstein et al, 2002) or high-dose cisplatin with etoposide. Radiotherapy alone has been reported as being useless to treat hepatoblastoma, because the active dose required is higher than that tolerated by the liver. However, radiotherapy has been suggested to be potentially useful as adjuvant therapy in cases of noncomplete surgical removal of these tumours (Habrand et al, 1992; Douglass et al, 1993). Alternatively, intra-arterial liver administration of chemotherapeutic agents (Oue et al, 1998; Malogolowkin et al, 2000) or, if the metastatic disease is controlled, liver transplant could be considered (Superina and Bilik, 1996; Perilongo et al, 2000; Otte et al, 2004; Otte et al, 2005; Austin et al, 2006). Indeed, a high rate of survival (82%) after liver transplant in children with non-resectable hepatoblastomas has been reported (Otte et al, 2004). Owing to the promising role of chemotherapy in the treatment of hepatoblastomas, new clinical trials have been carried out by stratifying the high- and low-risk patients and consequently adapting the therapy in order to enhance the success rate and reduce the toxic side effects (Roebuck and Perilongo, 2006). Hemangiosarcomas as well as other primary sarcomas of the liver, such as leiomyosarcoma, epithelioid hemangioendothelioma, fibrosarcoma, malignant fibrous histiocytoma and embryonic sarcoma are very uncommon liver lesions. They constitute less than 1% of all hepatic malignancies (Weitz et al, 2007). The outcome of patients with primary hepatic sarcoma depends primarily on the tumour histology and the ability to achieve complete tumour resection. In general, the prognosis is poor, the patients usually dying within a year, and few reaching a 3year survival if the tumour cannot be resected. Diseasespecific survival is increased to 5 years in up to 80% of patients undergoing tumour resection. Unfortunately, in many cases (15%), as for other vascular tumours of the liver, intraperitoneal hemorrhage due to primary tumour rupture is the first clinical presentation, which can be lethal if not diagnosed and treated immediately. Once hemangiosarcoma is discovered, it is often too late for surgical intervention, with a life expectancy of less than 6 months. In these cases palliative TACE incorporating the

more complete resection with fewer complications can be performed (Schnater et al, 2003). In some cases, chemotherapy has been described to be able to completely eradicate pulmonary metastases and to eliminate liver foci in multinodular hepatoblastoma (Perilongo et al, 2000). Surgical resection of distant disease makes the cure of patients with hepatoblastoma possible. Resection of pulmonary metastases, which is often performed at the same time as the resection of the primary tumour, is recommended when the number of metastases is limited (Feusner et al, 1993). When possible, the resection of areas of locally invasive disease, such as diaphragm metastases and isolated brain metastases, is recommended (Robertson et al, 1997). The choice of the chemotherapeutic regimen to treat hepatoblastomas depends on the stage of the disease, which can be established with post-surgical or pre-surgical criteria. In hepatoblastomas diagnosed in stages I and II (post-surgical classification) or 1, 2 and 3 of PRETEX (pre-surgical classification), regimens based on cisplatin result in a survival rate of more than 90% (Douglass et al, 1993; Ortega et al, 2000; Perilongo et al, 2004). The therapeutic options include complete surgical resection followed by four courses of chemotherapy with cisplatin, vincristine and 5-FU or any comparable combination (Douglass et al, 1993). When the tumour has been completely resected and presents a purely foetal histology the patient can be treated with doxorubicin alone (Ortega et al, 2000) or carefully followed without further therapy (Douglass et al, 1993). Tumours in PRETEX stage 1 can be resected without neoadjuvant chemotherapy and the patients can be treated with chemotherapy after surgery (Pritchard et al, 2000). To treat hepatoblastomas in PRETEX stages 2 and 3, it has been suggested to initially administer 4-6 courses of single-agent cisplatin (Perilongo et al, 2004) or cisplatin in combination with doxorubicin (Pritchard et al, 2000) before attempting surgical resection. In hepatoblastomas diagnosed in stage III (postsurgical classification) or PRETEX stage 4, neoadjuvant chemotherapy plays an important role among the therapeutic options. The international SIOPEL-1 trial compared two preoperative regimens (cisplatin + vincristine + 5-FU !"#$%$ cisplatin and continuous infusion of doxorubicin). Overall survival and disease-free survival were not significantly different in the two arms of this study. However, toxicity was more severe for the second regimen (Ortega et al, 2000). Another regimen including doxorubicin and cisplatin at different doses and schedules has afforded comparable results with fewer secondary effects (Pritchard et al, 2000). The combination of ifosfamide, cisplatin and doxorubicin (IPA) has also shown efficacy (von Schweinitz et al, 1995). Encouraging results were obtained when patients who did not respond to this regimen were treated with carboplatin and etoposide (Haberle et al, 2003). When the resection of stage III hepatoblastomas is not possible, other alternatives in addition to chemotherapy should be considered. These include orthotopic liver transplant (Dower and Smith, 2000; Reyes et al, 2000; Otte et al, 2004; Czauderna et al, 2005; Otte and de Ville de Goyet, 2005; Austin et al, 2006),

719

Marin et al: Liver Cancer Chemotherapy! reduced accumulation of drugs in the tumour, which suggests that they may play an important role in the refractivity of HCC to chemotherapy (Nies et al, 2001; Zollner et al, 2005). In some cases chemoresistance in HCC cells may be accounted for by the conjoint enhanced expression of both MDR1 and MRP2 (Minemura et al, 1999). Regarding the role of other members of the ABCC family in the chemoresistance of liver tumours, it is noteworthy that a relationship between MRP3 expression in CGC and resistance to etoposide, doxorubicin and epirubicin has been reported (Tepsiri et al, 2005). The ABCG family includes an important member as regards drug resistance: ABCG2 or BCRP (breast cancer resistance protein). There is experimental evidence that this transporter, which is able to export many cytostatic drugs, may play a role in the chemoresistance of HCC (Li et al, 2007; Takahata et al, 2008). Among the intracellular mechanisms involved in chemoresistance are those that modify drug metabolism, either by enhancing the production of an inactive metabolite, or, if the compound administered is a pro-drug that must be activated, by altering its biotransformation so that it decreases the formation of the active compound. Thus, tumour cells can inactivate anticancer drugs by over-expressing detoxifying enzymes, including phase-I enzymes, such as cytochrome P450 enzymes, or phase-II enzymes, such as those involved in conjugation with glucuronic acid or with glutathione. The expression of some of these enzymes has been found to be altered in HCC (Fritz et al, 1993; Murray el al, 1993). GlutathioneS-transferases (GSTs) and metallothioneins (MTs) are involved in cellular defence against many toxic compounds, including drugs. There is evidence that this is the case of the detoxification mechanism of cisplatin (Akiyama et al, 1999). Moreover, in chemoresistant cells derived from HCC enhanced glutathione levels and GST activity have been found (Jin et al, 2002). UDPglucuronosyl transferases (UGTs) may inactivate drugs through conjugation with glucuronic acid. Such is the case of SN-38, the active form of irinotecan, which is inactivated by the isoform UGT1A1 (Xu and VillalonaCalero, 2002), whose over-expression has been associated with reduced sensitivity to irinotecan in HCC cell lines (Takahata et al, 2008). Other mechanisms leading to chemotherapy resistance through reductions in the efficiency of the antitumour drug are characterized by changes in molecular targets. The following two examples illustrate this concept: i) FdUMP, the active metabolite of 5-FU, is a strong inhibitor of thymidylate synthase. Consequently, the expression level of this enzyme is very important for determining sensitivity to 5-FU. In this sense, resistance to 5-FU has been described in HCC cells with high expression of thymidylate synthase (Jin et al, 2002); ii) The mechanism of action of several anticancer drugs is based on their ability to interact with DNA topoisomerases involved in replication, recombination, chromosome segregation and transcription. Thus, in HCC cells with enhanced resistance to doxorubicin an increased expression of topoisomerase II has been found (Pang et al, 2005).

newer embolization agent Embospheres has been assayed to locally target and treat this aggressive tumour (Stambo and Guiney, 2007). Regarding systemic chemotherapy, the accumulated experience is promising, although very poor. Only few patients have been reported to have been treated with systemic chemotherapy (doxorubicin, cisplatin) alone, and only a few more patients have received chemotherapy after liver transplantation (Weiss and Enzinger, 1982).

VII. Chemotherapy resistance in liver tumours The lack of efficiency of anticancer agents against liver tumours may be due to complex mechanisms also involved in the resistance to chemotherapy of other tumours. Here we shall briefly review some examples specifically applied to liver tumours. One of these mechanisms is the reduction in the active concentration of the drug inside the tumour cells. Two mechanisms can account for this effect: low uptake (Zollner et al, 2005; Huang and SadĂŠe, 2006) and enhanced efflux (Gottesman et al, 2002). Regarding reduced drug uptake, the organic anion transporter OATP1B1, which is highly expressed in human liver and located in the basolateral membrane of normal hepatocytes, is able to transport antitumour drugs (Hagenbuch and Meier, 2003). This carrier is downregulated in HCC, which may result in a lower accumulation of antitumour drugs (Zollner et al, 2005). Changes in the expression in HCC of another member of the OATP family also expressed in normal hepatocytes, OATP1B3 (formerly OATP8), have been described (Vavricka et al, 2004). Moreover, the concentrative nucleoside transporter hCNT1, which is involved in the uptake of nucleoside analogues such as gemcitabine, shows a tendency to be down-regulated in HCC (Zollner et al, 2005). At the same time, an important role in the multidrug resistance phenotype (MDR) is played by plasma membrane proteins involved in the efflux of cytostatic drugs, hence causing a reduction in the intracellular levels of the active compound. The most relevant of these transporters in the chemoresistance of liver tumours are MDR1, MRP1, MRP2, MRP3 and ABCG2, which belong to the superfamily of ATP-binding cassette (ABC) proteins. MDR1 (ABCB1 or P-glycoprotein) plays an important role in drug efflux in several tissues. Among the long list of substrates that ABCB1 is able to transport are included many non-related antitumour drugs, which may account for the association of high ABCB1 expression in HCC with a poor response to chemotherapy (Chou et al, 1997; Ng et al, 2000). However, when inhibitors of ABCB1 have been administered in combination with systemic doxorubicin to treat HCC in clinical practice no improvement in the response was found (Lai et al, 1990). The ABCC family of multidrug resistance-associated proteins (MRPs) includes several isoforms expressed in HCC, such as MRP1 (Bonin et al, 2002) and MRP2 (Nies et al, 2001; Zollner et al, 2005), which are able to transport several cytostatic drugs. The presence of these pumps in the membrane of HCC cells has been associated with a 720

Cancer Therapy Vol 6, page 721! erlotinib and gefitinib (tyrosine kinase inhibitors, TKIs) or cetuximab (an anti-EGFR monoclonal antibody), able to bind to EGFR and inhibit its activation by its natural ligands, have been evaluated in patients with advanced HCC (Thomas and Abbruzzese, 2005). In addition, these TKIs include antiangiogenic activity, resulting from the induction of a decrease in the levels of VEGF (Pore et al, 2006). In a phase II study, erlotinib has shown an acceptable tolerance profile and limited clinical activity, as manifested by disease control in patients with advanced HCC (Philip et al, 2005). Gefitinib has been shown to inhibit cell proliferation and metastatic spread in HCC cell lines and $%&'$'( mouse models, respectively (Matsuo et al, 2003). Cetuximab has also been tested in patients with advanced HCC, showing a good safety profile but poor response rates and survival (Zhu et al, 2007b). However, early results suggest activity for cetuximab in combination with GEMOX (Louafi et al, 2007). In a phase II study, bevacizumab (an anti-VEGF monoclonal antibody), has proved to be safe and potentially effective in localized HCC in patients with well-preserved liver function. A reduction in disease progression in patients with previously diagnosed rapid tumour growth has been reported (Siegel et al, 2008). In patients with nonresectable or metastatic HCC, a moderate antitumour activity of bevacizumab in combination with the GEMOX regimen has been also found (Zhu et al, 2006). Sunitinib targets a variety of TKs in addition to VEGFR, including PDGFRs, KIT, RET, and FLT3, which are involved in tumour angiogenesis in HCC. The results of two phase-II studies in advanced HCC suggest a modest clinical efficacy for this agent (Faivre et al, 2007; Zhu et al, 2007a). Sorafenib blocks Raf kinases, which are key elements of the Raf/MEK/ERK signalling pathway that regulate many cellular functions such as proliferation, differentiation, transformation and apoptosis (Gollob et al, 2006). In addition, sorafenib inhibits tumour angiogenesis by blocking the activation of the tyrosine kinase receptors involved in neovascularization and tumour progression, including VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-!, cKIT, Flt3, and RET (Wilhelm et al, 2004). In a recent international phase-III study (SHARP) comparing sorafenib to placebo in advanced HCC, sorafenib has been shown to significantly increase both progression-free survival and overall survival, without any difference in the appearance of serious adverse events (Llovet et al, 2007).

Resistance to chemotherapy may be also due to an enhanced DNA repair capacity in tumour cells (Lage and Dietel, 1999). HCC is considered to be a tumour highly resistant to agents attacking DNA. However, the reason for this refractivity is not known. Nucleotide excision repair (NER) is one of the major mechanisms accounting for the repair of DNA adduct formation resulting from the use of alkylating agents, such as cisplatin (Reardon et al, 1999). In cisplatin-resistant cells, the expression of some proteins involved in NER has been found to be elevated, such as excision repair cross-complementing protein 1 (ERCC1) (Youn et al, 2004) and xeroderma pigmentosum group A (XPA) (Dabholkar et al, 1994). In colon cancer cells at least, a relationship between the expression levels of both proteins and the resistance of these cells to oxaliplatin has been found (Arnould et al, 2003, Boyer et al, 2004). The role of NER in chemoresistance of HCC is unknown, although several genes involved in NER, such as ERCC1, XPA and xeroderma pigmentosum group C (XPC) have been found to be more highly expressed in the tumour than in the surrounding healthy liver tissue (Fautrel et al, 2005). Impairments of the apoptosis machinery constitute an important group of processes accounting for refractivity to anticancer drugs such as cisplatin, cytarabine, etoposide and doxorubicin, whose mechanism of action involves the activation of apoptosis in tumour cells (Ormerod et al, 1996). The presence of abnormally functioning p53 is a common finding in drug-resistant tumour cells (Anthoney et al, 1996). This is also the case in many HCC, in which the apparent contradiction of high expression levels of p53, higher than in the surrounding liver tissue, is frequently observed (Fautrel et al, 2005). However, this protein is not functional, owing to accumulated mutations in the !"# gene (Hussain et al, 2007). This leads to the resistance of HCC cells to doxorubicin and paclitaxel (Chan and Lung, 2004). Moreover, over-expression of the anti-apoptotic factors Bcl-2 and Bcl-xL has been found in HCC cell lines and this has been associated with resistance to paclitaxel (Chun and Lee, 2004). Another way in which the survival of tumour cells may be increased is through the activation of protein tyrosine-kinases involved in the signalling of growth factors. This is the case of epidermal growth factor receptor (EGFR), which is over-expressed in most HCC (Buckley et al, 2008). When tumour cells are exposed to selective EGFR inhibitors, such as gefitinib, the survival and proliferation signalling pathways are blocked (Okano et al, 2006). Moreover, the resistance to SN-38 in HCC cells has been suggested to be mediated in part by activation of the PI3-K/Akt survival pathway. Thus, the inhibition of elements of this pathway may result in enhanced sensitivity to SN-38 (Koizumi et al, 2005).

IX. Conclusions and perspectives At present, surgical approaches, either by partial hepatectomy or transplantation, remain the treatments of choice for malignant liver tumours, including HCC, CGC and hepatoblastoma. However, because of the frequent existence of underlying liver disease and the limitation in organ availability, only a minority of patients affected are candidates for resection or transplantation. In the early and intermediate stages of liver tumours, local tumour ablation and chemoembolization, respectively, have afforded some benefits. No consistent positive results have been obtained using hormonotherapy, immunotherapy, or systemic chemotherapy, except in the case of hepatoblastomas,

VIII. Development of novel drugs The understanding of the mechanisms of chemotherapy resistance commented above has been the base for the development of novel therapeutic strategies aimed at interacting with molecular targets located in the apoptosis/survival pathways (anti-EGFR, anti-VEGF or anti-RAF therapies). Thus, several agents, such as 721

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which in general are more sensitive to chemotherapy. The lack of response of liver tumours to anticancer drugs is due to complex mechanisms, involving changes in the expression and/or function of the proteins involved in drug uptake/efflux, intracellular processes of signalling, DNA repair and death/survival control. Thus, current research efforts are focused on gaining a better understanding of the mechanisms of chemotherapy resistance in order to be able to better predict it before starting treatment and to develop novel strategies to overcome it.

Acknowledgements This study was supported in part by the Junta de Castilla y Leon (Grants GR75-2008, SA033A08, SA03508 and SA03608), Spain; Ministerio de Ciencia y Tecnologia, Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (Grant BFU2006-12577), Spain; The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), cofunded by the Ministerio de Educacion y Ciencia, Spain and the European Regional Development Fund (ERDF) (Grants BFU2005-24983-E/BFI and BFU2007-30688E/BFI) and belongs to the CIBERehd (Centro de Investigacion Biomedica en Red) for Hepatology and Gastroenterology Research (Instituto de Salud Carlos III, Spain). The authors thank N. Skinner for revision of the English text of the manuscript.

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Jose J G Marin

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Cancer Therapy Vol 6, page 729! Cancer Therapy Vol 6, 729-732, 2008

A successful veno-occlusive disease (VOD) prophylaxis strategy for patients undergoing stem cell transplantation Research Article

David C Gammon1,*, Kristi Holt3, Traci Dutton3, Janet Rogers2, Samer Al-Homsi2 1

Clinical Oncology Pharmacist, University of Massachusetts Memorial Medical Center, University Campus; Instructor UMass Medical School of Graduate Nursing 2 Blood and Marrow Transplant Program, University of Massachusetts Memorial Medical Center, University Campus 3 Pharm.D. Candidate, Massachusetts College of Pharmacy and Health Sciences, Worcester

__________________________________________________________________________________! *Correspondence: David C Gammon, B.S. PHARM.,! is a Clinical Oncology Pharmacist at the Massachusetts Memorial Medical Center, University Campus; Worcester, MA 01655, USA; e-mail: gammond@ummhc.org Key words: Veno-occlusive disease, bone marrow transplantation Abbreviations: bone marrow transplant, (BMT); Confidence Interval, (CI); deep venous thrombosis, (DVT); stem cell transplantation, (SCT); Veno-occlusive disease, (VOD)

Received: 26 August 2008; Revised: 25 September 2008 Accepted: 26 September 2008; electronically published: October 2008

Summary Veno-occlusive disease (VOD) is a common and serious complication associated with stem cell transplant regimens. Risk factors for VOD include chemotherapy and irradiation used in pre-transplant conditioning. Certain transplant conditioning regimens including drugs such as cytarabine, cyclophosphamide, and busulfan, as well as total body irradiation increase the risk of VOD. Because of the serious, often fatal, outcomes associated with VOD in transplant patients, strategies have been proposed to prevent it without consensus, some including the use of heparin. Ursodiol, studied in the past for the prevention or treatment of veno-occlusive disease, is thought to increase biliary flow, decrease biliary viscosity, and protect hepatocytes from harmful hydrophobic bile acids. Methods: Twenty-five patients admitted between February and December 2007 were identified as having received ursodiol as part of their transplant regimens. Seventeen of the twenty-five patients received at least one of the treatment regimens including cytarabine, cyclophosphamide, busulfan, and/or total body irradiation. None of the patients received prophylactic heparin for VOD. The records of each patient were screened retrospectively for documentation of veno-occlusive disease. Results: None of the twenty-five patients who received Ursodiol as part of their stem cell transplant regimen developed VOD. Conclusion: Stem cell transplant patients are at risk for VOD, based on their conditioning regimens. Ursodiol was used as VOD prophylaxis in twenty-five patients, none of whom developed VOD. Ursodiol should be further studied as a prophylactic part of stem cell transplant regimens.

Common causes of VOD are total body irradiation and drugs such as busulphan, carmustine, cyclophosphamide, cytarabine, actinomycin-d and dacarbazine (Pegram and Kennedy, 2001; Reddy and Faust, 2006). Characteristics of VOD are hyperbilirubinemia, ascites, hepatomegaly, and weight gain (Weisdorf, 2005; Reddy and Faust, 2006) No association has been found with VOD and the nature of the disease for transplant or the type of engraftment (Pegram and Kennedy, 2001). Ursodiol is a hydrophilic, nontoxic bile acid commonly found in small doses, approximately one

I. Introduction As stem cell transplants are preformed on a regular basis, the complications such as veno-occlusive disease (VOD) need to be prevented if possible. VOD most commonly occurs within a month after stem cell transplantation (SCT) but can develop during the administration of the preparative regimen approximately 50% of the time (Weisdorf, 2005). This complication is often a regimen-related toxicity that results in an injury to the sinusoidal wall of the liver causing an obstruction of the sinusoids and central veins (Reddy and Faust, 2006). 729

Gammon et al: VOD prophylaxis strategy for patients undergoing stem cell transplantation DRUGDEX® System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). In light of the recent controversy with heparin’s safety and supply we retrospectively reviewed the use of ursodiol, with no concomitant heparin, for the prevention of VOD in bone marrow transplant (BMT) patients with a variety of malignancies, therefore dissimilar conditioning regimens, that underwent either autologous or allogeneic stem cell transplantation.

percent of the total bile acid, within the body (Essell et al, 1998). Given exogenously, at a dose of 600 to 900mg daily, allows for the potential to replace the toxic hydrophobic bile acids from the conditioning regimens, thus, preventing damage to hepatic cells (Pegram and Kennedy, 2001; Reddy and Faust, 2006). As a VOD preventative agent, ursodiol has relatively no adverse effects associated with it, unlike other products that are used to prevent VOD such as heparin and pentoxifylline (Ursodiol. In: DRUGDEX® System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically; Park et al, 2002). FDA-approved uses for ursodiol include primary biliary cirrhosis, dissolution of gallstones and chronic cholestatic liver disease (Ursodiol. In: DRUGDEX® System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). Oral ursodiol undergoes significant firstpass metabolism therefore, with an intact gallbladder, is able to concentrate in the bile to reduce hydrophobic bile acids as well as decrease the detergent properties of the bile salts decreasing their hepatotoxicity (Ursodiol. In:

II. Patients and Study Design A. Patients This retrospective study included twenty-five patients admitted between February and December 2007 who were identified as having received ursodiol as part of their transplant regimen. All patients regardless of malignancy, conditioning regimen, or type of stem cell transplant (autologous or allogeneic) received ursodiol. Three patients were given antithrombolytics such as heparin or fondaparinux, to prevent deep venous thrombosis (DVT) during their transplant (Table 1).

Table 1. Ursodiol Patient Data. Name 1 2 3 4 5 6 7

Date of BMT 2/21/2007 2/23/2007 3/14/2007 3/24/2007 4/27/2007 5/1/2007 5/21/2007

Graft auto PB allo PB auto PB allo unrelated PB allo unrelated PB auto PB allo unrelated B

Conditioning Regimen VP/Carb/Ifos Flu/ATG/TBI-mini allo Melphalan CyTBI FAB-mini mud CBV FAB-mini mud

VOD no no no no no no no

8 9 10 11 12 13

6/7/2007 6/19/2007 6/27/2007 7/10/2007 7/16/2007 7/18/2007

allo PB allo unrelated PB auto PB allo unrelated PB allo PB auto PB

FAM-mini allo FAB-mini mud Melphalan Flu/ATG/TBI-mini mud Flu/TBI-mini allo Melphalan

no no no no no no

14 15

7/20/2007 7/27/2007

auto PB auto PB

Melphalan Melphalan

no no

16 17 18 19

8/21/2007 9/17/2007 9/21/2007 9/25/2007

allo cord allo PB allo unrelated PB allo unrelated PB

Flu/Cy/TBI Flu/TBI-mini allo FATBI-mini mud FAB-mini mud

no no no no

20 21 22 23 24 25

9/26/2007 10/29/2007 11/6/2007 11/28/2007 12/11/2007 12/26/2007

allo PB allo unrelated PB allo unrelated PB auto PB allo unrelated PB auto PB & BM

Flu/Bu-mini allo FAB-mini mud Cy/TBI/ATG Melphalan FAB-mini mud CBV

no no no no no no

Hep/LMWH none none none none none none Fondaparinux (DVT prophylaxis) none none none none none Enoxaparin (DVT prophylaxis) none Heparin (DVT prophylaxis) none none none Allergy to Heparin none none none none none none

auto PB= autologous peripheral blood; allo PB= allogeneic peripheral blood; allo cord= allogeneic umbilical cord blood; BM= bone marrow; mini mud= mini matched unrelated donor; mini allo= mini allogeneic; VP=etoposide; Carb=carboplatin; Ifos=ifosfamide; Flu=fludarabine; ATG= antithymocyte globulin (rabbit); TBI=total body irradiation; CyTBI=cylophosphamide + TBI; FAB=Fludarabine, antithymocyte globulin, busulfan; CBV= cyclophosphamide, carmustine, etoposide FAM= fluorouracil, doxorubicin, mitomycin;

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Cancer Therapy Vol 6, page 731! the most recent news about its safety and availability, makes it an inferior choice to ursodiol. Essell and colleagues showed that prophylactic ursodiol was more effective at preventing VOD than placebo in patients conditioned with busulfan and cyclophosphamide who underwent allogeneic BMT (P = 0.03) (Essell et al, 1998). Unfortunately, there was no statistical difference in overall 100-day survival, which is frequently associated with the identification of VOD, between the placebo and ursodiol groups (P=0.19) (Essell et al, 1998). A trial done by Park and colleagues showed that heparin plus ursodiol versus heparin alone produced no difference in outcome for preventing VOD (P = 0.348) (Park et al, 2002). When looking at the 100-day survival the ursodiol plus heparin group did not show a statistical difference from heparin alone (P = 0.298) (Park et al, 2002). Ohashi and colleagues did a recent randomized study which looked at a variety of conditioning regimens along with the different types of stem cell transplants (allogeneic and autologous) that incorporated either ursodiol or placebo to prevent VOD (Ohashi et al, 2000). Overall, the incidence of VOD occurred most often in the placebo arm (P=0.004) (Ohashi et al, 2000). Zirakzadeb and colleagues reviewed several articles on ursodiol and its use as a prophylactic agent in the prevention of VOD which showed that using ursodiol will decrease the incidence of VOD (pooled OR 0.33, 95% Confidence Interval (CI) 0.12, 0.90) (Zirakzadeh et al, 2000). This study also demonstrated that there was no statistical difference in survival in regards to trial type (P=0.89), conditioning regimen (P=0.64) or diagnosis criteria (P=0.36) (Zirakzadeh et al, 2000). Our study, while refraining from using prophylactic heparin, also showed that ursodiol is efficient in preventing veno-occlusive disease in patients with different conditioning regimens and stem cell transplantations. Each patient evaluated completed therapy with ursodiol, was conditioned with regimens that have the propensity to cause VOD, and underwent a SCT with no patient developing VOD symptoms. We plan a future analysis to verify 100-day survival. Park and colleagues demonstrated that the chief risk factor for developing VOD was an allogeneic stem cell transplant (P= 0.018), as previous reports show allogeneic transplant patients are at a higher risk for developing VOD (Park et al, 2000). Ohashi and colleagues also showed that the majority of their patients whom developed VOD were patients who underwent allogeneic STC; only one patient had an autologous SCT and developed VOD (Ohashi et al, 2000).Zirakzadeh and colleagues reported that allogenic transplants increase the risk of VOD (OR 0.33, 95% CI 0.11, 0.99) (Zirakzadeh et al, 2000). Our study, although small, included more patients that received allogeneic stem cells than autologous and none developed VOD.

B. Study design All autologous and allogeneic stem cell transplant patients were placed on ursodiol during their conditioning regimen. Several different types of conditioning regimens were used with busulfan, cyclophosphamide and/or total body irradiation being the most common. These three regimens have the highest potential for developing veno-occlusive disease, which is of great concern during stem cell transplantation. Every patient received oral ursodiol either 300mg twice a day or 300mg every morning and 600mg every evening for patients >90kg. Monitoring for ursodiol included: liver enzymes as well as watching for adverse symptoms such as nausea, vomiting, diarrhea and backache (Ursodiol. In: DRUGDEX速 System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). VOD monitoring included physical evaluation as well as computable assessment. The diagnosis was made by the physician and the criteria for diagnosis were an increase in total bilirubin >2mg/dl plus two of the following: hepatomegaly, ascites, and/or weight gain greater than five percent of their admission body weight. No difference in outcome between the patients was found in regards to age, sex and primary disease. For each patient their conditioning medications, type of SCT, and use of ursodiol and development of VOD was analyzed. The primary endpoint was the prevention of VOD with ursodiol while using no heparin.

III. Results Overall nine patients underwent autologous SCT and sixteen underwent allogeneic. Of those that underwent allogeneic SCT ten had an unrelated donor and one was given umbilical cord blood. While the patients varied in their conditioning regimens, with seventeen receiving conditioning regimens that have a high propensity to cause VOD, and different types of engraftment, their commonality was the fact that ursodiol was used to prevent VOD and heparin was not. Throughout the study period three patients needed DVT prophylaxis with each receiving one of the following enoxaparin, fondaparinux or heparin in doses appropriate for DVT. The one patient given heparin had a history of DVTs. The criteria that was used for the diagnosis of VOD, an increase in total bilirubin >2mg/dl plus two of the following: hepatomegaly, ascites, and/or weight gain greater than five percent of their admission body weight, is similar to the criteria that other trials, Essel and colleagues in 1998, Park and colleagues in 2002, and Ohashi and colleagues in 2000, used to evaluate and diagnose VOD. None of the SCT patients assessed, allogeneic and autologous SCT, developed VOD during the study period.

IV. Discussion This retrospective analysis showed that prophylactic ursodiol prevented the development of the complications associated with veno-occlusive disease after a bone marrow transplantation. As a drug, ursodiol has no serious side-effects associated with it, monitoring appropriate labs is routine, and it is dosed orally. Heparin, which is administered intravenously, has numerous adverse reactions associated with it such as bleeding, pain at the insertion site, increased liver enzymes, heparin-induced thrombocytopenia, and anaphylactoid reactions, mandatory monitoring during administration, along with

V. Conclusion Our results indicate that prophylactic ursodiol may be a valuable and an effective treatment option for the prevention of veno-occlusive disease. As no drug is universally accepted for the prevention of VOD, the use of a non-toxic drug such as ursodiol, which has been 731

Gammon et al: VOD prophylaxis strategy for patients undergoing stem cell transplantation Reddy K, Faust T (2006) Clinician’s guide to liver disease. 1st ed. Thorofare, NJ: Slack Inc. Ursodiol. In: DRUGDEXŽ System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically. Weisdorf DJ (2005) Hoffman: Hematology: Basic Principles and Practice. 4th ed. Philadelphia, Pennsylvania: Elsevier. Zirakzadeh A, Montori V, Imran H, Litzow M, Kumar S (2006) Ursodiol prophylaxis against hepatic veno-occlusive disease in hematopoietic stem cell transplant recipients: a systematic review and meta-analysis. Biol Blood Marrow Transplant! 12(suppl 1), 137-38.

effective at preventing VOD in several trials, could be considered as a prophylactic medication for stem cell transplant patients versus heparin. Larger trials with various types of bone marrow transplant patients and different conditioning regimens that use ursodiol as their primary prevention are needed to augment these findings. As we better understand the complete pathology of VOD we should be able to find a more effective preventative measure and treatment.

References Essell JH, Schroeder MT, Harman GS, Halvorson R, Lew V, Callander N, Snyder M, Lewis SK, Allerton JP, Thompson JM (1998) Ursodiol prophylaxis against hepatic complications of allogeneic bone marrow transplantation: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 128, 975-81. Ohashi K, Tanabe J, Watanabe R, Tanaka T, Sakamaki H, Maruta A, Okamoto S, Aotsuka N, Saito K, Nishimura M, Oh H, Matsuzaki M, Takahashi S, Yonekura S (2000) The Japanese multicenter open randomized trial of ursodeoxycholic acid prophylaxis for hepatic veno-occlusive disease after stem cell transplantation. Am J Hematol 64, 32-8. Park SH, Lee MH, Lee H, Kim HS, Kim K, Kim WS, Jung C, Im Y, Yoon S, Kang W, Park K, Park C, Kim S (2002) A randomized trial of heparin plus ursodiol versus heparin alone to prevent hepatic veno-occlusive disease after hematopoietic stem cell transplantation. Bone Marrow Transplant 29, 137-43. Pegram AA, Kennedy LD (2001) Prevention and treatment of veno-occlusive disease. Ann Pharmacother 35, 935-941.

David C Gammon

732

Cancer Therapy Vol 6, page 729! Cancer Therapy Vol 6, 729-732, 2008

A successful veno-occlusive disease (VOD) prophylaxis strategy for patients undergoing stem cell transplantation Research Article

David C Gammon1,*, Kristi Holt3, Traci Dutton3, Janet Rogers2, Samer Al-Homsi2 1

Clinical Oncology Pharmacist, University of Massachusetts Memorial Medical Center, University Campus; Instructor UMass Medical School of Graduate Nursing 2 Blood and Marrow Transplant Program, University of Massachusetts Memorial Medical Center, University Campus 3 Pharm.D. Candidate, Massachusetts College of Pharmacy and Health Sciences, Worcester

__________________________________________________________________________________! *Correspondence: David C Gammon, B.S. PHARM.,! is a Clinical Oncology Pharmacist at the Massachusetts Memorial Medical Center, University Campus; Worcester, MA 01655, USA; e-mail: gammond@ummhc.org Key words: Veno-occlusive disease, bone marrow transplantation Abbreviations: bone marrow transplant, (BMT); Confidence Interval, (CI); deep venous thrombosis, (DVT); stem cell transplantation, (SCT); Veno-occlusive disease, (VOD)

Received: 26 August 2008; Revised: 25 September 2008 Accepted: 26 September 2008; electronically published: October 2008

Summary Veno-occlusive disease (VOD) is a common and serious complication associated with stem cell transplant regimens. Risk factors for VOD include chemotherapy and irradiation used in pre-transplant conditioning. Certain transplant conditioning regimens including drugs such as cytarabine, cyclophosphamide, and busulfan, as well as total body irradiation increase the risk of VOD. Because of the serious, often fatal, outcomes associated with VOD in transplant patients, strategies have been proposed to prevent it without consensus, some including the use of heparin. Ursodiol, studied in the past for the prevention or treatment of veno-occlusive disease, is thought to increase biliary flow, decrease biliary viscosity, and protect hepatocytes from harmful hydrophobic bile acids. Methods: Twenty-five patients admitted between February and December 2007 were identified as having received ursodiol as part of their transplant regimens. Seventeen of the twenty-five patients received at least one of the treatment regimens including cytarabine, cyclophosphamide, busulfan, and/or total body irradiation. None of the patients received prophylactic heparin for VOD. The records of each patient were screened retrospectively for documentation of veno-occlusive disease. Results: None of the twenty-five patients who received Ursodiol as part of their stem cell transplant regimen developed VOD. Conclusion: Stem cell transplant patients are at risk for VOD, based on their conditioning regimens. Ursodiol was used as VOD prophylaxis in twenty-five patients, none of whom developed VOD. Ursodiol should be further studied as a prophylactic part of stem cell transplant regimens.

Common causes of VOD are total body irradiation and drugs such as busulphan, carmustine, cyclophosphamide, cytarabine, actinomycin-d and dacarbazine (Pegram and Kennedy, 2001; Reddy and Faust, 2006). Characteristics of VOD are hyperbilirubinemia, ascites, hepatomegaly, and weight gain (Weisdorf, 2005; Reddy and Faust, 2006) No association has been found with VOD and the nature of the disease for transplant or the type of engraftment (Pegram and Kennedy, 2001). Ursodiol is a hydrophilic, nontoxic bile acid commonly found in small doses, approximately one

I. Introduction As stem cell transplants are preformed on a regular basis, the complications such as veno-occlusive disease (VOD) need to be prevented if possible. VOD most commonly occurs within a month after stem cell transplantation (SCT) but can develop during the administration of the preparative regimen approximately 50% of the time (Weisdorf, 2005). This complication is often a regimen-related toxicity that results in an injury to the sinusoidal wall of the liver causing an obstruction of the sinusoids and central veins (Reddy and Faust, 2006). 729

Gammon et al: VOD prophylaxis strategy for patients undergoing stem cell transplantation DRUGDEX® System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). In light of the recent controversy with heparin’s safety and supply we retrospectively reviewed the use of ursodiol, with no concomitant heparin, for the prevention of VOD in bone marrow transplant (BMT) patients with a variety of malignancies, therefore dissimilar conditioning regimens, that underwent either autologous or allogeneic stem cell transplantation.

percent of the total bile acid, within the body (Essell et al, 1998). Given exogenously, at a dose of 600 to 900mg daily, allows for the potential to replace the toxic hydrophobic bile acids from the conditioning regimens, thus, preventing damage to hepatic cells (Pegram and Kennedy, 2001; Reddy and Faust, 2006). As a VOD preventative agent, ursodiol has relatively no adverse effects associated with it, unlike other products that are used to prevent VOD such as heparin and pentoxifylline (Ursodiol. In: DRUGDEX® System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically; Park et al, 2002). FDA-approved uses for ursodiol include primary biliary cirrhosis, dissolution of gallstones and chronic cholestatic liver disease (Ursodiol. In: DRUGDEX® System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). Oral ursodiol undergoes significant firstpass metabolism therefore, with an intact gallbladder, is able to concentrate in the bile to reduce hydrophobic bile acids as well as decrease the detergent properties of the bile salts decreasing their hepatotoxicity (Ursodiol. In:

II. Patients and Study Design A. Patients This retrospective study included twenty-five patients admitted between February and December 2007 who were identified as having received ursodiol as part of their transplant regimen. All patients regardless of malignancy, conditioning regimen, or type of stem cell transplant (autologous or allogeneic) received ursodiol. Three patients were given antithrombolytics such as heparin or fondaparinux, to prevent deep venous thrombosis (DVT) during their transplant (Table 1).

Table 1. Ursodiol Patient Data. Name 1 2 3 4 5 6 7

Date of BMT 2/21/2007 2/23/2007 3/14/2007 3/24/2007 4/27/2007 5/1/2007 5/21/2007

Graft auto PB allo PB auto PB allo unrelated PB allo unrelated PB auto PB allo unrelated B

Conditioning Regimen VP/Carb/Ifos Flu/ATG/TBI-mini allo Melphalan CyTBI FAB-mini mud CBV FAB-mini mud

VOD no no no no no no no

8 9 10 11 12 13

6/7/2007 6/19/2007 6/27/2007 7/10/2007 7/16/2007 7/18/2007

allo PB allo unrelated PB auto PB allo unrelated PB allo PB auto PB

FAM-mini allo FAB-mini mud Melphalan Flu/ATG/TBI-mini mud Flu/TBI-mini allo Melphalan

no no no no no no

14 15

7/20/2007 7/27/2007

auto PB auto PB

Melphalan Melphalan

no no

16 17 18 19

8/21/2007 9/17/2007 9/21/2007 9/25/2007

allo cord allo PB allo unrelated PB allo unrelated PB

Flu/Cy/TBI Flu/TBI-mini allo FATBI-mini mud FAB-mini mud

no no no no

20 21 22 23 24 25

9/26/2007 10/29/2007 11/6/2007 11/28/2007 12/11/2007 12/26/2007

allo PB allo unrelated PB allo unrelated PB auto PB allo unrelated PB auto PB & BM

Flu/Bu-mini allo FAB-mini mud Cy/TBI/ATG Melphalan FAB-mini mud CBV

no no no no no no

Hep/LMWH none none none none none none Fondaparinux (DVT prophylaxis) none none none none none Enoxaparin (DVT prophylaxis) none Heparin (DVT prophylaxis) none none none Allergy to Heparin none none none none none none

auto PB= autologous peripheral blood; allo PB= allogeneic peripheral blood; allo cord= allogeneic umbilical cord blood; BM= bone marrow; mini mud= mini matched unrelated donor; mini allo= mini allogeneic; VP=etoposide; Carb=carboplatin; Ifos=ifosfamide; Flu=fludarabine; ATG= antithymocyte globulin (rabbit); TBI=total body irradiation; CyTBI=cylophosphamide + TBI; FAB=Fludarabine, antithymocyte globulin, busulfan; CBV= cyclophosphamide, carmustine, etoposide FAM= fluorouracil, doxorubicin, mitomycin;

730

Cancer Therapy Vol 6, page 731! the most recent news about its safety and availability, makes it an inferior choice to ursodiol. Essell and colleagues showed that prophylactic ursodiol was more effective at preventing VOD than placebo in patients conditioned with busulfan and cyclophosphamide who underwent allogeneic BMT (P = 0.03) (Essell et al, 1998). Unfortunately, there was no statistical difference in overall 100-day survival, which is frequently associated with the identification of VOD, between the placebo and ursodiol groups (P=0.19) (Essell et al, 1998). A trial done by Park and colleagues showed that heparin plus ursodiol versus heparin alone produced no difference in outcome for preventing VOD (P = 0.348) (Park et al, 2002). When looking at the 100-day survival the ursodiol plus heparin group did not show a statistical difference from heparin alone (P = 0.298) (Park et al, 2002). Ohashi and colleagues did a recent randomized study which looked at a variety of conditioning regimens along with the different types of stem cell transplants (allogeneic and autologous) that incorporated either ursodiol or placebo to prevent VOD (Ohashi et al, 2000). Overall, the incidence of VOD occurred most often in the placebo arm (P=0.004) (Ohashi et al, 2000). Zirakzadeb and colleagues reviewed several articles on ursodiol and its use as a prophylactic agent in the prevention of VOD which showed that using ursodiol will decrease the incidence of VOD (pooled OR 0.33, 95% Confidence Interval (CI) 0.12, 0.90) (Zirakzadeh et al, 2000). This study also demonstrated that there was no statistical difference in survival in regards to trial type (P=0.89), conditioning regimen (P=0.64) or diagnosis criteria (P=0.36) (Zirakzadeh et al, 2000). Our study, while refraining from using prophylactic heparin, also showed that ursodiol is efficient in preventing veno-occlusive disease in patients with different conditioning regimens and stem cell transplantations. Each patient evaluated completed therapy with ursodiol, was conditioned with regimens that have the propensity to cause VOD, and underwent a SCT with no patient developing VOD symptoms. We plan a future analysis to verify 100-day survival. Park and colleagues demonstrated that the chief risk factor for developing VOD was an allogeneic stem cell transplant (P= 0.018), as previous reports show allogeneic transplant patients are at a higher risk for developing VOD (Park et al, 2000). Ohashi and colleagues also showed that the majority of their patients whom developed VOD were patients who underwent allogeneic STC; only one patient had an autologous SCT and developed VOD (Ohashi et al, 2000).Zirakzadeh and colleagues reported that allogenic transplants increase the risk of VOD (OR 0.33, 95% CI 0.11, 0.99) (Zirakzadeh et al, 2000). Our study, although small, included more patients that received allogeneic stem cells than autologous and none developed VOD.

B. Study design All autologous and allogeneic stem cell transplant patients were placed on ursodiol during their conditioning regimen. Several different types of conditioning regimens were used with busulfan, cyclophosphamide and/or total body irradiation being the most common. These three regimens have the highest potential for developing veno-occlusive disease, which is of great concern during stem cell transplantation. Every patient received oral ursodiol either 300mg twice a day or 300mg every morning and 600mg every evening for patients >90kg. Monitoring for ursodiol included: liver enzymes as well as watching for adverse symptoms such as nausea, vomiting, diarrhea and backache (Ursodiol. In: DRUGDEX速 System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). VOD monitoring included physical evaluation as well as computable assessment. The diagnosis was made by the physician and the criteria for diagnosis were an increase in total bilirubin >2mg/dl plus two of the following: hepatomegaly, ascites, and/or weight gain greater than five percent of their admission body weight. No difference in outcome between the patients was found in regards to age, sex and primary disease. For each patient their conditioning medications, type of SCT, and use of ursodiol and development of VOD was analyzed. The primary endpoint was the prevention of VOD with ursodiol while using no heparin.

III. Results Overall nine patients underwent autologous SCT and sixteen underwent allogeneic. Of those that underwent allogeneic SCT ten had an unrelated donor and one was given umbilical cord blood. While the patients varied in their conditioning regimens, with seventeen receiving conditioning regimens that have a high propensity to cause VOD, and different types of engraftment, their commonality was the fact that ursodiol was used to prevent VOD and heparin was not. Throughout the study period three patients needed DVT prophylaxis with each receiving one of the following enoxaparin, fondaparinux or heparin in doses appropriate for DVT. The one patient given heparin had a history of DVTs. The criteria that was used for the diagnosis of VOD, an increase in total bilirubin >2mg/dl plus two of the following: hepatomegaly, ascites, and/or weight gain greater than five percent of their admission body weight, is similar to the criteria that other trials, Essel and colleagues in 1998, Park and colleagues in 2002, and Ohashi and colleagues in 2000, used to evaluate and diagnose VOD. None of the SCT patients assessed, allogeneic and autologous SCT, developed VOD during the study period.

IV. Discussion This retrospective analysis showed that prophylactic ursodiol prevented the development of the complications associated with veno-occlusive disease after a bone marrow transplantation. As a drug, ursodiol has no serious side-effects associated with it, monitoring appropriate labs is routine, and it is dosed orally. Heparin, which is administered intravenously, has numerous adverse reactions associated with it such as bleeding, pain at the insertion site, increased liver enzymes, heparin-induced thrombocytopenia, and anaphylactoid reactions, mandatory monitoring during administration, along with

V. Conclusion Our results indicate that prophylactic ursodiol may be a valuable and an effective treatment option for the prevention of veno-occlusive disease. As no drug is universally accepted for the prevention of VOD, the use of a non-toxic drug such as ursodiol, which has been 731

Gammon et al: VOD prophylaxis strategy for patients undergoing stem cell transplantation Reddy K, Faust T (2006) Clinician’s guide to liver disease. 1st ed. Thorofare, NJ: Slack Inc. Ursodiol. In: DRUGDEXŽ System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically. Weisdorf DJ (2005) Hoffman: Hematology: Basic Principles and Practice. 4th ed. Philadelphia, Pennsylvania: Elsevier. Zirakzadeh A, Montori V, Imran H, Litzow M, Kumar S (2006) Ursodiol prophylaxis against hepatic veno-occlusive disease in hematopoietic stem cell transplant recipients: a systematic review and meta-analysis. Biol Blood Marrow Transplant! 12(suppl 1), 137-38.

effective at preventing VOD in several trials, could be considered as a prophylactic medication for stem cell transplant patients versus heparin. Larger trials with various types of bone marrow transplant patients and different conditioning regimens that use ursodiol as their primary prevention are needed to augment these findings. As we better understand the complete pathology of VOD we should be able to find a more effective preventative measure and treatment.

References Essell JH, Schroeder MT, Harman GS, Halvorson R, Lew V, Callander N, Snyder M, Lewis SK, Allerton JP, Thompson JM (1998) Ursodiol prophylaxis against hepatic complications of allogeneic bone marrow transplantation: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 128, 975-81. Ohashi K, Tanabe J, Watanabe R, Tanaka T, Sakamaki H, Maruta A, Okamoto S, Aotsuka N, Saito K, Nishimura M, Oh H, Matsuzaki M, Takahashi S, Yonekura S (2000) The Japanese multicenter open randomized trial of ursodeoxycholic acid prophylaxis for hepatic veno-occlusive disease after stem cell transplantation. Am J Hematol 64, 32-8. Park SH, Lee MH, Lee H, Kim HS, Kim K, Kim WS, Jung C, Im Y, Yoon S, Kang W, Park K, Park C, Kim S (2002) A randomized trial of heparin plus ursodiol versus heparin alone to prevent hepatic veno-occlusive disease after hematopoietic stem cell transplantation. Bone Marrow Transplant 29, 137-43. Pegram AA, Kennedy LD (2001) Prevention and treatment of veno-occlusive disease. Ann Pharmacother 35, 935-941.

David C Gammon

732

Cancer Therapy Vol 6, 729-732, 2008!

!

A successful veno-occlusive disease (VOD) prophylaxis strategy for patients undergoing stem cell transplantation Research Article!

David C Gammon1,*, Kristi Holt3, Traci Dutton3, Janet Rogers2, Samer Al-Homsi2 1

Clinical Oncology Pharmacist, University of Massachusetts Memorial Medical Center, University Campus; Instructor UMass Medical School of Graduate Nursing 2 Blood and Marrow Transplant Program, University of Massachusetts Memorial Medical Center, University Campus 3 Pharm.D. Candidate, Massachusetts College of Pharmacy and Health Sciences, Worcester

__________________________________________________________________________________ *Correspondence: David C Gammon, B.S. PHARM.,! is a Clinical Oncology Pharmacist at the Massachusetts Memorial Medical Center, University Campus; Worcester, MA 01655, USA; e-mail: gammond@ummhc.org Key words: Veno-occlusive disease, bone marrow transplantation! Abbreviations: bone marrow transplant, (BMT); Confidence Interval, (CI); deep venous thrombosis, (DVT); stem cell transplantation, (SCT); Veno-occlusive disease, (VOD)

!

!

Received: 26 August 2008; Revised: 25 September 2008! Accepted: 26 September 2008; electronically published: October 2008!

Summary Veno-occlusive disease (VOD) is a common and serious complication associated with stem cell transplant regimens. Risk factors for VOD include chemotherapy and irradiation used in pre-transplant conditioning. Certain transplant conditioning regimens including drugs such as cytarabine, cyclophosphamide, and busulfan, as well as total body irradiation increase the risk of VOD. Because of the serious, often fatal, outcomes associated with VOD in transplant patients, strategies have been proposed to prevent it without consensus, some including the use of heparin. Ursodiol, studied in the past for the prevention or treatment of veno-occlusive disease, is thought to increase biliary flow, decrease biliary viscosity, and protect hepatocytes from harmful hydrophobic bile acids. Methods: Twenty-five patients admitted between February and December 2007 were identified as having received ursodiol as part of their transplant regimens. Seventeen of the twenty-five patients received at least one of the treatment regimens including cytarabine, cyclophosphamide, busulfan, and/or total body irradiation. None of the patients received prophylactic heparin for VOD. The records of each patient were screened retrospectively for documentation of veno-occlusive disease. Results: None of the twenty-five patients who received Ursodiol as part of their stem cell transplant regimen developed VOD. Conclusion: Stem cell transplant patients are at risk for VOD, based on their conditioning regimens. Ursodiol was used as VOD prophylaxis in twenty-five patients, none of whom developed VOD. Ursodiol should be further studied as a prophylactic part of stem cell transplant regimens.

I. Introduction! As stem cell transplants are preformed on a regular basis, the complications such as veno-occlusive disease (VOD) need to be prevented if possible. VOD most commonly occurs within a month after stem cell transplantation (SCT) but can develop during the administration of the preparative regimen approximately 50% of the time (Weisdorf, 2005). This complication is often a regimen-related toxicity that results in an injury to the sinusoidal wall of the liver causing an obstruction of the sinusoids and central veins (Reddy and Faust, 2006). Common causes of VOD are total body irradiation and drugs such as busulphan, carmustine, cyclophosphamide, cytarabine, actinomycin-d and dacarbazine (Pegram and Kennedy, 2001; Reddy and Faust, 2006). Characteristics of VOD are hyperbilirubinemia, ascites, hepatomegaly, and weight gain (Weisdorf, 2005; Reddy and Faust, 2006) No association has been found with VOD and the nature of the disease for transplant or the type of engraftment (Pegram and Kennedy, 2001). Ursodiol is a hydrophilic, nontoxic bile acid commonly found in small doses, approximately one percent of the total bile acid, within the body (Essell et al, 1998). Given exogenously, at a dose of 600 to 900mg daily, allows for the potential to replace the toxic hydrophobic bile acids from the conditioning regimens, thus, preventing damage to hepatic cells (Pegram and Kennedy, 2001; Reddy and Faust, 2006). As a VOD preventative agent, ursodiol has relatively no adverse

effects associated with it, unlike other products that are used to prevent VOD such as heparin and pentoxifylline (Ursodiol. In: DRUGDEXÆ System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically; Park et al, 2002). FDA-approved uses for ursodiol include primary biliary cirrhosis, dissolution of gallstones and chronic cholestatic liver disease (Ursodiol. In: DRUGDEXÆ System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). Oral ursodiol undergoes significant first-pass metabolism therefore, with an intact gallbladder, is able to concentrate in the bile to reduce hydrophobic bile acids as well as decrease the detergent properties of the bile salts decreasing their hepatotoxicity (Ursodiol. In: DRUGDEXÆ System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). In light of the recent controversy with heparin's safety and supply we retrospectively reviewed the use of ursodiol, with no concomitant heparin, for the prevention of VOD in bone marrow transplant (BMT) patients with a variety of malignancies, therefore dissimilar conditioning regimens, that underwent either autologous or allogeneic stem cell transplantation.

II. Patients and Study Design A. Patients This retrospective study included twenty-five patients admitted between February and December 2007 who were identified as having received ursodiol as part of their transplant regimen. All patients regardless of malignancy, conditioning regimen, or type of stem cell transplant (autologous or allogeneic) received ursodiol. Three patients were given antithrombolytics such as heparin or fondaparinux, to prevent deep venous thrombosis (DVT) during their transplant (Table 1).

Table 1. Ursodiol Patient Data. Name 1 2 3 4 5 6 7

Date of BMT 2/21/2007 2/23/2007 3/14/2007 3/24/2007 4/27/2007 5/1/2007 5/21/2007

Graft auto PB allo PB auto PB allo unrelated PB allo unrelated PB auto PB allo unrelated B

Conditioning Regimen VP/Carb/Ifos Flu/ATG/TBI-mini allo Melphalan CyTBI FAB-mini mud CBV FAB-mini mud

VOD no no no no no no no

8 9 10 11 12 13

6/7/2007 6/19/2007 6/27/2007 7/10/2007 7/16/2007 7/18/2007

allo PB allo unrelated PB auto PB allo unrelated PB allo PB auto PB

FAM-mini allo FAB-mini mud Melphalan Flu/ATG/TBI-mini mud Flu/TBI-mini allo Melphalan

no no no no no no

14 15

7/20/2007 7/27/2007

auto PB auto PB

Melphalan Melphalan

no no

16 17 18 19

8/21/2007 9/17/2007 9/21/2007 9/25/2007

allo cord allo PB allo unrelated PB allo unrelated PB

Flu/Cy/TBI Flu/TBI-mini allo FATBI-mini mud FAB-mini mud

no no no no

20 21 22 23 24 25

9/26/2007 10/29/2007 11/6/2007 11/28/2007 12/11/2007 12/26/2007

allo PB allo unrelated PB allo unrelated PB auto PB allo unrelated PB auto PB & BM

Flu/Bu-mini allo FAB-mini mud Cy/TBI/ATG Melphalan FAB-mini mud CBV

no no no no no no

Hep/LMWH none none none none none none Fondaparinux (DVT prophylaxis) none none none none none Enoxaparin (DVT prophylaxis) none Heparin (DVT prophylaxis) none none none Allergy to Heparin none none none none none none

auto PB= autologous peripheral blood; allo PB= allogeneic peripheral blood; allo cord= allogeneic umbilical cord blood; BM= bone marrow; mini mud= mini matched unrelated donor; mini allo= mini allogeneic; VP=etoposide; Carb=carboplatin; Ifos=ifosfamide; Flu=fludarabine; ATG= antithymocyte globulin (rabbit); TBI=total body irradiation; CyTBI=cylophosphamide + TBI; FAB=Fludarabine, antithymocyte globulin, busulfan; CBV= cyclophosphamide, carmustine, etoposide FAM= fluorouracil, doxorubicin, mitomycin;

B. Study design

All autologous and allogeneic stem cell transplant patients were placed on ursodiol during their conditioning regimen. Several different types of conditioning regimens were used with busulfan, cyclophosphamide and/or total body irradiation being the most common. These three regimens have the highest potential for developing veno-occlusive disease, which is of great concern during stem cell transplantation. Every patient received oral ursodiol either 300mg twice a day or 300mg every morning and 600mg every evening for patients >90kg. Monitoring for ursodiol included: liver enzymes as well as watching for adverse symptoms such as nausea, vomiting, diarrhea and backache (Ursodiol. In: DRUGDEXÆ System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically). VOD monitoring included physical evaluation as well as computable assessment. The diagnosis was made by the physician and the criteria for diagnosis were an increase in total bilirubin >2mg/dl plus two of the following: hepatomegaly, ascites, and/or weight gain greater than five percent of their admission body weight. No difference in outcome between the patients was found in regards to age, sex and primary disease. For each patient their conditioning medications, type of SCT, and use of ursodiol and development of VOD was analyzed. The primary endpoint was the prevention of VOD with ursodiol while using no heparin.

III. Results Overall nine patients underwent autologous SCT and sixteen underwent allogeneic. Of those that underwent allogeneic SCT ten had an unrelated donor and one was given umbilical cord blood. While the patients varied in their conditioning regimens, with seventeen receiving conditioning regimens that have a high propensity to cause VOD, and different types of engraftment, their commonality was the fact that ursodiol was used to prevent VOD and heparin was not. Throughout the study period three patients needed DVT prophylaxis with each receiving one of the following enoxaparin, fondaparinux or heparin in doses appropriate for DVT. The one patient given heparin had a history of DVTs. The criteria that was used for the diagnosis of VOD, an increase in total bilirubin >2mg/dl plus two of the following: hepatomegaly, ascites, and/or weight gain greater than five percent of their admission body weight, is similar to the criteria that other trials, Essel and colleagues in 1998, Park and colleagues in 2002, and Ohashi and colleagues in 2000, used to evaluate and diagnose VOD. None of the SCT patients assessed, allogeneic and autologous SCT, developed VOD during the study period.

IV. Discussion This retrospective analysis showed that prophylactic ursodiol prevented the development of the complications associated with veno-occlusive disease after a bone marrow transplantation. As a drug, ursodiol has no serious side-effects associated with it, monitoring appropriate labs is routine, and it is dosed orally. Heparin, which is administered intravenously, has numerous adverse reactions associated with it such as bleeding, pain at the insertion site, increased liver enzymes, heparin-induced thrombocytopenia, and anaphylactoid reactions, mandatory monitoring during administration, along with the most recent news about its safety and availability, makes it an inferior choice to ursodiol. Essell and colleagues showed that prophylactic ursodiol was more effective at preventing VOD than placebo in patients conditioned with busulfan and cyclophosphamide who underwent allogeneic BMT (P = 0.03) (Essell et al, 1998). Unfortunately, there was no statistical difference in overall 100-day survival, which is frequently associated with the identification of VOD, between the placebo and ursodiol groups (P=0.19) (Essell et al, 1998). A trial done by Park and colleagues showed that heparin plus ursodiol versus heparin alone produced no difference in outcome for preventing VOD (P = 0.348) (Park et al, 2002). When looking at the 100-day survival the ursodiol plus heparin group did not show a statistical difference from heparin alone (P = 0.298) (Park et al, 2002). Ohashi and colleagues did a recent randomized study which looked at a variety of conditioning regimens along with the different types of stem cell transplants (allogeneic and autologous) that incorporated either ursodiol or placebo to prevent VOD (Ohashi et al, 2000). Overall, the incidence of VOD occurred most often in the placebo arm (P=0.004) (Ohashi et al, 2000). Zirakzadeb and colleagues reviewed several articles on ursodiol and its use as a prophylactic agent in the prevention of VOD which showed that using ursodiol will decrease the incidence of VOD (pooled OR 0.33, 95% Confidence Interval (CI) 0.12, 0.90) (Zirakzadeh et al, 2000). This study also demonstrated that there was no statistical difference in survival in regards to trial type (P=0.89), conditioning regimen (P=0.64) or diagnosis criteria (P=0.36) (Zirakzadeh et al, 2000). Our study, while refraining from using prophylactic heparin, also showed that ursodiol is efficient in preventing veno-occlusive disease in patients with different conditioning regimens and stem cell transplantations. Each patient evaluated completed therapy with ursodiol, was conditioned with regimens that have the propensity to cause VOD, and underwent a SCT with no patient developing VOD symptoms. We plan a future analysis to verify 100-day survival. Park and colleagues demonstrated that the chief risk factor for developing VOD was an allogeneic stem cell transplant (P= 0.018), as previous reports show allogeneic transplant patients are at a higher risk for developing VOD (Park et al, 2000). Ohashi and colleagues also showed that the majority of their patients whom developed VOD were patients who underwent allogeneic STC; only one patient had an autologous SCT and developed VOD (Ohashi et al, 2000).Zirakzadeh and colleagues reported that allogenic transplants increase the risk of VOD (OR 0.33, 95% CI 0.11, 0.99) (Zirakzadeh et al, 2000). Our study, although small, included more patients that received allogeneic stem cells than autologous and none developed VOD.

V. Conclusion

Our results indicate that prophylactic ursodiol may be a valuable and an effective treatment option for the prevention of veno-occlusive disease. As no drug is universally accepted for the prevention of VOD, the use of a non-toxic drug such as ursodiol, which has been effective at preventing VOD in several trials, could be considered as a prophylactic medication for stem cell transplant patients versus heparin. Larger trials with various types of bone marrow transplant patients and different conditioning regimens that use ursodiol as their primary prevention are needed to augment these findings. As we better understand the complete pathology of VOD we should be able to find a more effective preventative measure and treatment. !

References! Essell JH, Schroeder MT, Harman GS, Halvorson R, Lew V, Callander N, Snyder M, Lewis SK, Allerton JP, Thompson JM (1998) Ursodiol prophylaxis against hepatic complications of allogeneic bone marrow transplantation: a randomized, double-blind, placebocontrolled trial. Ann Intern Med 128, 975-81. Ohashi K, Tanabe J, Watanabe R, Tanaka T, Sakamaki H, Maruta A, Okamoto S, Aotsuka N, Saito K, Nishimura M, Oh H, Matsuzaki M, Takahashi S, Yonekura S (2000) The Japanese multicenter open randomized trial of ursodeoxycholic acid prophylaxis for hepatic veno-occlusive disease after stem cell transplantation. Am J Hematol 64, 32-8. Park SH, Lee MH, Lee H, Kim HS, Kim K, Kim WS, Jung C, Im Y, Yoon S, Kang W, Park K, Park C, Kim S (2002) A randomized trial of heparin plus ursodiol versus heparin alone to prevent hepatic veno-occlusive disease after hematopoietic stem cell transplantation. Bone Marrow Transplant 29, 137-43. Pegram AA, Kennedy LD (2001) Prevention and treatment of veno-occlusive disease. Ann Pharmacother 35, 935-941. Reddy K, Faust T (2006) Clinician's guide to liver disease. 1st ed. Thorofare, NJ: Slack Inc. Ursodiol. In: DRUGDEXÆ System [Internet database]. Greenwood Village, Colo: Thomson Healthcare. Updated periodically. Weisdorf DJ (2005) Hoffman: Hematology: Basic Principles and Practice. 4th ed. Philadelphia, Pennsylvania: Elsevier. Zirakzadeh A, Montori V, Imran H, Litzow M, Kumar S (2006) Ursodiol prophylaxis against hepatic veno-occlusive disease in hematopoietic stem cell transplant recipients: a systematic review and meta-analysis. Biol Blood Marrow Transplant!12(suppl 1), 137-38. !

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David C Gammon! !

Cancer Therapy Vol 6, page 733! Cancer Therapy Vol 6, 733-740, 2008

Cytotoxic effects of a combination of three natural compounds to leukemia cells in vitro Research Article

Markus Düchler*, Maciej Stepnik Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, 8 Sw. Teresy Street, 91-348 ód! , Poland

__________________________________________________________________________________! *Correspondence: Dr Markus Düchler, Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, 8 Sw. Teresy St., 91-348 ód! , Poland; e-mail: markusd@imp.lodz.pl; markus.duechler@univie.ac.at Key words: Caffeic acid phenethyl ester, methylglyoxal, parthenolide, leukemia cell line, natural compounds Abbreviations:!2',7'-dichlorodihydro-fluorescein diacetate, (DCF); 5-sulfosalicylic acid, (SSA); Caffeic acid phenethyl ester, (CAPE); epigallocatechin-3-gallate, (EGCG); fetal bovine serum, (FBS); green tea extract, (GTE); L-N-acetylcysteine, (NAC); mean fluorescence intensity, (MFI); Methylglyoxal, (MGO); Parthenolide, (PRT); Peripheral blood mononuclear cells, (HDL); phosphate-buffered saline, (PBS); propidium iodide, (PI); Resveratrol, (RES); room temperature, (RT)! Received: 8 September 2008; "#$%&#'(!19 September 2008! Accepted: 25 September 2008; electronically published: October 2008

Summary Many studies revealed anti-cancer activity of plant-derived compounds and suggest their potential application in prevention of carcinogenesis. Five drugs with previousely described anti-cancer activity were applied in double and triple combinations to identify those combinations with the highest potential to specifically decrease viability of leukemia cell lines. Caffeic acid phenethyl ester, parthenolide, methylglyoxal, green tea extract and resveratrol were applied to EL-4 mouse thymoma cells, human Jurkat T cells, K562 erythroleukemia cells, and HL-60 promyelocytic cells. Cell viability, production of reactive oxygen species and glutathione levels were determined. The combination consisting of caffeic acid phenethyl ester, methylglyoxal, and parthenolide showed the highest toxicity towards leukemic cells while sparing lymphocytes isolated from healthy donors. Cell death induced by this combination could be inhibited by N-acetylcysteine. Oxidative stress was deleterious only for cells with reduced glutathione levels. These findings indicate, that leukemia cell death could be induced efficiently and selectively by the selected drug combination, presumably in a redox-dependent manner.

apoptotic transcription factor NF-!B (Watabe et al, 2004). In hepatoma cells and a xenograft model of hepatoma, Chung et al. demonstrated anti-cancer activity of CAPE by inhibition of NF-!B activity and reduced expression of matrix metalloproteases, MMP-2 and MMP-9 (Chung et al, 2004). Parthenolide (PRT), a sesquiterpene lactone derived from a plant named ‘fewerfew’ (!"#"$%&'() *"+&,%#-'(), is a well-known inhibitor of NF-" B, and induces reactive oxygen species (ROS) production (Zhang et al, 2004; Wang et al, 2006). Its anti-cancer activity was demonstrated in multiple myeloma cells and B-cell chronic lymphocytic leukemia cells (Wang et al, 2006; Steele et al, 2006). Importantly, PRT also exerts specific cytotoxic activity against cancer stem cells (Guzman et al, 2005). Methylglyoxal (MGO) is formed as a side product during normal cellular glucose metabolism from dihydroxyacetone- and glyceraldehyde-3-phosphates. It is

I. Introduction Molecularly targeted anti-cancer drugs are characterized by their clearly defined mode of action and a high specificity to the malignant cells. In contrast, socalled natural compounds with anti-cancer activity often modify many intracellular signaling pathways simultaneously. Fortunately, many of them also show specificity towards transformed cells. In the current study, five different natural drugs known to induce cancer cell death were tested in all possible double and triple combinations for their cytostatic/cytotoxic potential against leukemia cells. Caffeic acid phenethyl ester (CAPE) is one of the active substances in honeybee propolis. CAPE treatment of transformed human osteogenic sarcoma induced cell cycle G2/M arrest, and triggered apoptosis (Yang et al, 2005). In breast cancer cells, CAPE induced apoptosis by Fas death receptor activation and inhibition of the mainly anti-

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Düchler and Stepnik: Natural drugs reduce leukemia cell viability! complemented with 10% FBS and antibiotics. Peripheral blood mononuclear cells (HDL) were isolated from healthy volunteers by Ficoll centrifugation and cultured in RPMI medium containing glutamine, 20% FBS, and antibiotics. EL-4 cell line was from ATCC (kindly provided by J. Dastych), all other cell lines used were purchased from DSMZ (Braunschweig, Germany).

also present in varying amounts in honey, reaching very high concentrations up to more than 700 mg/kg in New Zealand Manuka (!"#$%&#"'()(* &+%#,'-)() honey (Mavric et al, 2008). MGO is able to form protein adducts resulting in advanced glycation end products which in turn might induce ROS accumulation (Li et al, 2007). Because MGO is thought to cause complications in patients with diabetes mellitus, it is not generally well accepted as anticancer drug. However, a methylglyoxal-based anticancer formulation was demonstrated to have reasonable efficacy against a variety of cancers in humans and to lack any toxic effect (Talukdar et al, 2006, 2008). The major active polyphenol in green tea extract (GTE) is epigallocatechin-3-gallate (EGCG). EGCG modulates multiple signal transduction pathways to control the unwanted proliferation of malignant cells (Chen et al, 2008). EGCG inhibits NF-!B, PI3-K/Akt, Ras/Raf/MAPK and AP-1 signaling pathways (Khan and Mukhtar, 2008). In addition, EGCG inhibited the chymotrypsin-like activity of the proteasome (Nam et al, 2001). Resveratrol (RES) is found in several vegetal sources including the skin of grapes. RES exerts anti-carcinogenic, anti-inflammatory, and growth-modulatory effects and that may be partially ascribed to the inhibition of activation of NF-!B and AP-1 (Sunil et al, 2000; Kundu et al, 2006). Modulation of mitochondrial membrane potential and generation of ROS constitute early events of the RESinduced apoptotic pathway (Tinhofer et al, 2001). Combining two or more drugs is a common strategy to increase anti-cancer efficacy. Due to their multiple activities, the outcome of combining natural anti-cancer drugs is almost impossible to predict. Therefore, an empirical approach was used to find effective collaborations between those five compounds for specifically eradicating leukemic cells in vitro. The sought-after drug combination should show toxicity with high specificity to malignant cells and exert its activity to differend kinds of leukemia. The best combination identified in this screening rapidly reduced intracellular gluthatione levels and increased reactive oxygen species suggesting that cell death was induced based on a redoxdependent mechanism.

C. Assessment of cell viability Cell death was assessed based on propidium iodide (PI) staining. After incubation with drugs in complete culture medium for 24 hours at a cell density of 0.25x106 cells/ml, cells were washed once in phosphate-buffered saline (PBS) and resuspended in 0.25ml of 2!g/ml PBS/PI solution. After 10 min of staining (room temperature (RT), in the dark) cell fluorescence was measured by flow cytometry. PI-negative cells were defined as viable. Flow cytometric analysis was performed on a Cytomics FC500 MPL cytometer from Beckman Coulter (Fullerton, California). Data were analysed using the CPX software package. Cell viability and proliferation was determined by MTT (thiazolyl blue) assay (Sigma, Steinheim, Germany) as was described earlier (Mosmann, 1983).

E. Measurement of reactive oxygen species production Intracellular ROS levels were visualized after incubation with 2',7'-dichlorodihydro-fluorescein diacetate (DCF) at a final concentration of 10!M. The fluorescent dye was added for the last 30 min of the drug exposure period. After the incubation, cells were washed once in PBS, resuspended in PBS and examined immediately by flow cytometry.

F. Measurement of intracellular GSH content Changes in total cellular gluthathione levels were determined using a “Glutathione Assay Kit” following the included instructions. 1-5x107 cells were used for each sample. Briefly, cells were lysed by freeze/thawing, and protein was precipitated with 5% 5-sulfosalicylic acid (SSA) solution. After centrifugation, GSH in the supernatant was quantified by its ability to convert 5,5’-dithiobis(2-nitrobenzoic acid) into the yellow product 2-nitrobenzoic acid which was measured spectrophotometrically.

III. Results A. Identification of combinations with high cytotoxic/cytostatic efficacy to leukemic cells

II. Materials and Methods A. Drugs and chemicals

All possible double and triple combinations of the five drugs under investigation were tested in cell cuture against one murine and three human leukemia cell lines. Drug concentrations were chosen which did not cause more than 10% cell death in HL-60 as assessed by PI staining: CAPE 5µM, TGE 22.5 µg/ml (about 25µM EGCG), MGO 300µM, RES 2,5µM, and PRT 10µM. Cell proliferation/viability was measured after 24 hour incubations by MTT assay, and by FACS analysis after propidium iodide staining. The results of MTT assay for Jurkat and K562 cells (Figure 1a), and FACS results for EL-4 and HL-60 cells (Figure 1b) are shown. The ‘CMP’ combination of CAPE, MGO and PRT showed the highest cytotoxicity in three cell lines. When measured by MTT assay, CMP reduced viability by almost 100% in Jurkat and EL-4 cells, and by about 50% in HL-

Green tea extract (Pharmanex, Provo, Utah) was purchased as capsules containing 97% pure polyphenols extracted from green tea, half of which is EGCG. The contence of one capsule was dissolved in water at 45mg/ml resulting in a solution with about 50mM EGCG. Propidium iodide and DCF were obtained from Fluka (Sigma-Aldrich, St. Louis, Missouri, USA). CAPE, methylglyoxal, resveratrol, L-N-acetylcysteine (NAC), z-VADfmk, and the Glutathione Assay Kit were from from Sigma Chemical Company (Sigma-Aldrich, St. Louis, Missouri, USA).

B. Cell culture EL-4 mouse thymoma cells were cultured in DMEM, 10% fetal bovine serum (FBS), 2mM glutamine and antibiotics (penicillin 100U/ml and streptomycin 100 !g/ml). Jurkat T cells, K562 erythroleukemia cell line, and HL-60 promyelocytic cells were grown in RPMI 1640 medium containing glutamine

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Cancer Therapy Vol 6, page 735! 60. Determination of cell death by PI staining revealed

about 75% of dead cells in EL-4 cells, about 35% in HL-

Figure 1. Cytotoxic and cytostatic activity of five natural compounds alone or in combination. (A) K562 and Jurkat cells were incubated for 24 hours with drugs as indicated. Cell viability was assessed by MTT assay and is shown relative to untreated controls. One representative experiment done in triplicates is shown. (B) Viability of HL-60 and EL-4 cells after 24 hours incubations with drugs as

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DĂźchler and Stepnik: Natural drugs reduce leukemia cell viability! indicated was measured by PI staining and FACS analysis. The percentages of PI-negative, viable cells from one representative experiment done in triplicates are shown.

CMP incubation reduced cell viability of HDL by less than 10% in all cases.

60, and 80% in Jurkat. K562 cells were relatively resistant to any drug combination, showing only 10-15% reduction in cell viability with CAPE alone and CAPE containing combinations.

C. The influence of NAC and caspase inhibition on the cytotoxic activity of CMP

B. Mononuclear blood cells from healthy donors are resistant to the same treatment which efficiently kills leukemia cells.

To investigate the importance of ROS for cell death induction, the influence of N-acetylcysteine was evaluated. Addition of 1mM NAC completely protected Jurkat cells from the cytotoxic effects of CMP (Figure 3). To determine the involvement of caspases in cell death, the general caspase inhibitor z-VAD-fmk was added together with the CMP combination. z-VAD-fmk at 20ÂľM reduced cell death only by about 40% (Figure 3).

Peripheral blood mononuclear cells were isolated from healthy donors (n=4) and subjected to the some drug combinations at the same concentrations as the leukemic cell lines. HDL were highly resistant to the same treatment which showed high toxicity in leukemia cells (Figure 2).

Figure 2. Healthy donor lymphocytes are resistant to CMP treatment. Peripheral blood mononuclear cells were isolated from healthy donors and exposed to drugs for 24 hours. Cell viability was determined by PI staining and FACS analysis. The percentage of dead cells is shown. CP, CAPE and PRT.

Figure 3. Influence of N-acetylcysteine and caspase inhibitor on the cytotoxic effects of CMP drug treatment. Jurkat cells were incubated for 24 hours with the combination of CAPE, MGO and PRT (CMP). In parallel experiments either 1mM N-acetylcysteine (NAC) or 20!M caspase inhibitor z-VAD-fmk (CI) was added. Cell viability was measured after propidium iodide staining by flow cytometry, and the percentage of dead cells is shown.

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Cancer Therapy Vol 6, page 737!

D. Influence of drug treatment on intracellular levels of reactive oxygen species

during the first 4 hours of incubation. When measured after 24 hours, ROS production was at control levels in all combinations tested (not shown).

Increased ROS levels are thought to constitute an essential step in cell death induction by many different cytotoxic drugs. ROS levels were assessed after 1, 2 and 4 hours of incubation with CAPE, MGO and PRT, as well as their combinations. DCF fluorescence was then measured by flow cytometry. Changes in the mean fluorescence intensity (MFI) relative to untreated control cultures was interpreted as increase or decrease of the amount of internal ROS. PRT caused a rapid transient increase of intracellular ROS in Jurkat cells with a maximum during the first hour followed by a decrease during the following three hours (Figure 4). In all combinations containing PRT a similar pattern was observed although peak levels were lowered. CAPE and MGO did not induce ROS

E. Changes in intracellular GSH content upon drug treatment Finally, intracellular GSH levels were measured in drug treated cells after 6 hours and compared to control cultures (Figure 5). The combination of CAPE with PRT showed significant reduction of GSH only in Jurkat cells. CAPE combined with MGO reduced GSH levels in all cell lines to varying degrees. The triple combination CMP showed the strongest effect in all cell lines reducing the intracellular GSH level by 45% in EL-4, 65% in Jurkat, 60% in K562 and more than 90% in HL-60 cells.

Figure 4. Relative reactive oxygen levels levels in Jurkat cells after 1, 2, and 4 hours. DCF at a concentration of 10!M was added to cell cultures during the last 30 minutes of incubation with the tested drugs. Cells were washed in PBS and green fluorescence was measured immediately by flow cytometry. The mean fluorescence intensity is shown in arbitrary units. CP, CAPE and PRT; MP, MGO and PRT; CM, CAPE and MGO.

Figure 5. Glutathione content in different cell lines after drug treatment. 1-2x107 cells from for each condition were treated with drug combinations as indicated. After 6 hours cells were lysed by freeze/thawing in 5% sulfosalicylic acid and GSH content was measured spectrophotometrically.

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Düchler and Stepnik: Natural drugs reduce leukemia cell viability! defensive capacity of the cells and made them susceptible to cell death induction by ROS.

IV. Discussion Several plant derived compounds have raised considerable interest because of their anti-cancer activity towards a variety of malignancies including leukemia. Two or more drugs in combination might potentiate anticancer effects, but they could also impede each others activity. Due to their complex pharmacological action the outcome of combining natural drugs is not easy to predict. Therefore, all possible double and triple combinations of 5 different plant-derived compounds with known anti-cancer activity were screened for their combined cytotoxic activity against in 4 different leukemia cell lines. To easily identify synergistic cooperation between drugs, concentrations were chosen which showed only low toxicity for each single compound. One triple combination consisting of CAPE, MGO and PRT showed high specific toxicity to leukemia cells, killing about 50% to 90% of HL-60, EL-4 and Jurkat cells during 24 hours. K562 cells, which are well known to be highly resistant to apoptosis induction, were not affected by CMP. Importantly, lymphocytes isolated from normal healthy donors were completely resistant to the same drug exposure which efficiently killed leukemic cells. The cytotoxic effect of the CMP combination was partially independent from caspases and could be totally inhibited by N-acetylcysteine indicating that cell death was induced in a redox-dependent manner. An influence of CAPE, MGO and PRT (as single drugs) on cell viability and changes in the cellular redox status in leukemia cell lines has been described earlier. MGO induced apoptosis in HL-60 cells through inhibition of DNA synthesis (Kang et al, 1996). In Jurkat cells, MGO induced apoptosis by sustained activation of c-Jun N-terminal kinase (Du et al, 2000). The mechanism included production of superoxide anion and activation of apoptosis signaling kinase-1 (Du et al, 2001). MGO damaged mitochondria, and induced a pro-oxidant state through the generation of nitric oxide and superoxide anion. These two molecules combine to the highly toxic peroxynitrite radical, which oxidizes and deplets glutathione (Chang et al, 2005). PRT treated HL60 and Jurkat cells were described to become arrested in G2/M phase of the cell cycle and to undergo apoptosis (Pozarowski et al, 2003). In HL-60 cells, CAPE induced apoptosis was associated with GSH depletion and scavenging of hydrogen peroxide (Chen et al, 2001). Also in the current study, less ROS was produced by PRT in the presence of CAPE. Exposure to PRT alone showed the highest relative increase of ROS levels during the first hours, but this did not induce cell death. Solely increasing ROS seems not sufficient to kill the leukemic cells. Determination of intracellular GSH levels suggested that a concomittant reduction of GSH was necessary to allow ROS-mediated killing of malignant cells. A rapid decrease of GSH can happen by non-oxidative loss of glutathione in stress-induced apoptosis via GSH extrusion and might trigger the downstream events of apoptosis by leaving cells unprotected against thiol oxidation and free radical production (Ghibelli et al, 1995; Düchler et al, 2008). When PRT was combined with CAPE and MGO, GSH content was markedly reduced. In conclusion, these results suggest that the reduction of GSH levels lowered the

Acknowledgements This work was supported by the Nofer Institute of Occupational Medicine, grant Nr. IMP1.7/2006, and by the Marie Curie Transfer of Knowledge Program “EpiTok” Nr. 6PR/2004/509829.

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