Cancer Therapy Volume 5 Issue A by Cancer Therapy

CANCER THERAPY

Volume 5 Number 1 June, 2007

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 Devarajan, Prasad M.D., Cincinnati Children's Hospital, USA Der Channing, J. Ph.D, Lineberger Comprehensive Cancer Center, 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. UniversitĂ di Modena e Reggio Emilia, Italy Fiebig, Heiner H, Albert-Ludwigs-UniversitĂ¤t, 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 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

!!!!!!!!!!!!!!!!!!!!!!!! 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-

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Table of contents Cancer Therapy Vol 5 Number 1, June 2007 Pages

Type of Article

Article title

1-10

Research Article

Reversal of doxorubicin resistance by adenovirus-mediated transfer of cyclooxygenase-2 antisense in multidrug-resistant MCF-7 cells

11-18

Review Article

Development of farnesyl transferase inhibitors as anticancer agents: current status and future

Floriana Morgillo, Ho-Young Lee

19-28

Review Article

Treatment of vaginal intraepithelial neoplasia

Vedat Atay, Murat Muhcu, Ahmet Cantug Caliskan

29-34

Research Article

Thermo-chemo-radiotherapy for advanced pancreatobiliary cancer

35-42

Research Article

Retrospective study of the influence of anaemia in patients with advanced head and neck cancer received postoperative radiotherapy

43-54

Research Article

Concurrent chemoradiotherapy for locally advanced cervical cancer using Gemcitabine: non-randomized comparison of three sequential protocols

Terumi Kamisawa, Atsutake Okamoto, Katsuyuki Karasawa, Tsuneo Sasaki Sherif A. Abdel-Wahab, Mohamed M. El-Basiouny, Hatem M. Abdalla, Hany, M. Abdel-Aziz, Maha Margerges, Ali M. Azmy, Branislav Jeremic Kundan S. Chufal, Madhup Rastogi, Madhu Srivastava, M.C. Pant, M.L.B. Bhatt, Kirti Srivastava

55-66

Review Article

Merits of anti-cancer plants from the Arabian Gulf region

Amr Amin, Mohamed Mousa

67-76

Research Article

Overcoming K562Dox resistance to STI571 (Gleevec) by downregulation of P-gp expression using siRNAs

Raquel T. Lima, JosĂŠ Eduardo GuimarĂŁes, M. Helena Vasconcelos

77-88

Review Article

The role of chemotherapy in the treatment of advanced non-small cell lung cancer elderly patients

89-96

Research Article

Down-regulation of MDR1 by continuous exposure to cisplatin in LLC-PK1 cells

97-104

Research Article

Four cardiac hormones cause cell death in 81% of human ovarian adenocarcinoma cells

105-112

Research

Symptom clusters among cancer

Antonio Rossi, Paolo Maione, Giuseppe Colantuoni, Carmine Ferrara, Alba De Vita, Ciro Guerriero, Dario Nicolella, Filomena del Gaizo, Cesare Gridelli Kohji Takara, Masayuki Tsujimoto, Misato Kokufu, Noriaki Kitada, Toshiyuki Sakaeda, Noriaki Ohnishi, Teruyoshi Yokoyama Brian A. Vesely, Ehrentraud J. Eichelbaum, Abdel A. Alli, Ying Sun, William R. Gower, Jr., David L. Vesely Margot E. Kurtz, J Cleo Kurtz,

Authors (corresponding author is in boldface) Marwa W. Kamel, Salama A. Salama, Samira Saleh, Abdel-Moneim M. Osman, Mohamed M. Sayed-Ahmed, Ayman Al-Hendy

Article

patients and effects of an educational symptom control intervention

Charles W. Given, Barbara Given

113-116

Review Article

Neural stem cell-based cancer therapy and brain tumor stem cells

Philippe Taupin

117-124

Research Article

An intensive sequenced adjuvant chemotherapy regimen for breast cancer, a pilot study of the cancer and leukemia group b

Sushil Bhardwaj, David Duggan, Jeffrey J. Kirshner, Susan Woolf, James F. Holland, Donald A. Berry, Larry Norton, I. Craig Henderson

125-132

Research Article

Therapy of primary and metastatic murine tumors by intratumoral administration of interleukin-2 plasmid DNA with electroporation

Holly M. Horton, Rod R. Planchon, Michael Sawdey, Larry Smith, Alain Rolland, David C. Kaslow

133-136

Research Article

Nerve-sparing low semi-cystectomy in bladder: preliminary results

Zhong Wang, Yin-Tian Yuan, Guiting Lin, Benchun Liu

137-142

Research Article

Determination of epidermal growth factor receptor (EGFR) in patients with colorectal cancer (Institutional series)

143-150

Research Article

Bio-immunotherapy in the treatment of equine sarcoid, the commonest tumour of the horse

Arkom Cheirsilpa, Preecha Ruangvejvorachai, Anant Karalak, Soisuda Sangprakarn, Sangduan Pummai, Suleeporn Sangrajrang Raija E. Hallamaa

151-160

Review Article

Chemotherapy in AndrogenIndependent Prostate Cancer (AIPC): Whatâ&#x20AC;&#x2122;s next after taxane progression?

Jeanny B. Aragon-Ching, William L. Dahut

Cancer Therapy Vol 5, page 1 Cancer Therapy Vol 5, 1-10, 2007

Reversal of doxorubicin resistance by adenovirusmediated transfer of cyclooxygenase-2 antisense in multidrug-resistant MCF-7 cells Research Article

Marwa W. Kamel1,2, Salama A. Salama1, Samira Saleh3, Abdel-Moneim M. Osman2, Mohamed M. Sayed-Ahmed2, Ayman Al-Hendy1,* 1

Department of Obstetrics & Gynecology, University of Texas Medical Branch, Galveston, Texas, USA Department of Pharmacology and Experimental Oncology, Cancer Biology, National Cancer Institute, Cairo University, Egypt 3 Department of Pharmacology, Faculty of Pharmacy, Cairo University, Cairo, Egypt 2

__________________________________________________________________________________ *Correspondence: Ayman Al-Hendy, MD, PhD, FRCSC, FACOG, Department of Obstetrics & Gynecology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555, USA; Telephone: (409) 747-1668; Fax: (409) 747-1669; email: ayalhend@utmb.edu Key words: Gene therapy, drug resistance, breast cancer Abbreviations: Adenomatous Polyp Prevention on Vioxx, (APPROVe); adenovirus vector expressing COX-2 antisense, (Ad-COX-2 AS); cyclooxygenase-2, (COX-2); deoxynucleotide triphosphate mixture, (dNTP); doxorubicin-resistant breast cancer cell line, (MCF7/DOX); enzyme-linked chemiluminescence, (ECL); ethylenediaminetetraacetic adid, (EDTA); multidrug-resistance, (MDR); phosphate buffered saline, (PBS); phosphate buffered saline-Tween, (PBS-T); plaque forming units, (PFU); radioimmunoprecipitation, (RIPA); sodium dodecylsulfate, (SDS) Received: 22 November 2006; Accepted: 12 December 2006; electronically published: January 2007

Summary Increased expression of cyclooxygenase-2 (COX-2) has been implicated in many human malignancies, and it is associated with tumor progression, angiogenesis, inhibition of apoptosis, and development of multidrug-resistant (MDR) phenotype. Both selective and nonselective COX-2 inhibitors have been used in clinical trials as adjuvant therapy-too often at the expense of increased systemic toxicity. In the current study, we evaluated whether adenovirus vector expressing COX-2 antisense (Ad-COX-2 AS) has therapeutic potential to modulate the drug resistance and the expression of MDR gene MDR1, antiapoptotic gene Bcl-2, and cell cycle regulating gene cyclin D1 in doxorubicin-resistant breast cancer cell line (MCF-7/DOX). Our data show that MCF-7/DOX cells are more efficiently transduced by adenovirus compared with the parental MCF-7 cell line. Transduction of MCF-7/DOX cells with Ad-COX-2 AS (multiplicity of infection [MOI] of 10, 20, 50, plaque forming units [PFU]/cell) reduced the IC50 value of doxorubicin by 13-, 19-, and 20-fold, respectively, compared with Ad-LacZâ&#x20AC;&#x201C;transduced cells (P < 0.05). Furthermore, Ad-COX-2 AS downregulates the expression of the MDR1, the antiapoptic protein Bcl-2, and the cell cycle regulating protein cyclin D1 in MCF-7/DOX cells. In conclusion, MCF-7/DOX cells are more efficiently transduced with Ad-COX-2 AS compared with parental MCF-7 cells. Transduction of MCF-7/DOX cells with AdCOX-2 AS reverses their resistance to doxorubicin. The promising efficacy of Ad-COX-2 AS in reversing drug resistance deserves further investigation.

(Chow et al, 2005; Tari et al, 2005; Zatelli et al, 2005; Mazhar et al, 2006; Raspollini et al, 2006). The effects of COX-2 overexpression in cancer cells are mediated by interactions with downstream effectors of COX-2. Recent reports suggest that expression of the COX-2 enzyme upregulates expression of MDR1/P-glycoprotein (P-gp), an exponent of resistance to anticancer drugs (Ratnasinghe

I. Introduction There is considerable evidence to suggest that the expression of cyclooxygenase-2 (COX-2) plays an important role in the development of many human malignancies, and that it confers a survival advantage that eventually facilitates tumor development, progression, angiogenesis, and resistance to chemotherapeutic agents

Kamel et al: Cyclooxygenase-2 and gene therapy of drug resistance et al, 2001; Patel et al, 2002; Ziemann et al, 2002; Sorokin 2004; Surowiak et al, 2005), suggesting that COX-2 is involved in inducing the chemoresistance phenotype. Accordingly, numerous in vivo and in vitro studies indicate that COX-2 inhibitors (eg, coxibs) enhance the efficacy of various anticancer therapy methods (Gasparini et al, 2003). COX-2 plays a role not only in the growth of cancer cells but also in inhibiting the apoptosis pathway. Various experimental studies have shown a positive correlation between the expression of COX-2 and the inhibition of apoptosis (Meric et al, 2006). COX-2 overexpression is associated with increased amounts of the antiapoptotic protein Bcl-2 (Tsujii and DuBois, 1995). Furthermore, after a pre-exposure of tumor cells to prostaglandin E2 in vitro, a 4- to 5-fold increase in the levels of the antiapoptotic protein Bcl-2 has been observed, whereas the level of the Bax proapoptotic protein remained unchanged (Sheng et al, 1998). In addition to its antiapoptotic effect, COX-2 enhances tumor cell proliferation by inducing the expression of cyclin D1 (Wu et al, 2004; Patel et al, 2005; Zhang et al, 2006). Taken together, these data show a causal relationship between the expression of COX-2 and the inhibition of apoptosis, stimulation of cell progression, and development of drug resistance in tumor cells. Therefore, selective COX-2 inhibitors have emerged as potentially important chemopreventive and therapeutic agents in cancer treatment (Becerra et al, 2003; Chow et al, 2005; Mazhar et al, 2006). Despite considerable evidence suggesting a therapeutic role for COX-2 inhibitors in the chemoprevention and possible treatment of breast cancer, the available COX-2 inhibitors are not optimal compounds, and their use is associated with serious cardiovascular effects (Becerra et al, 2003; Dogne et al, 2006; Meric et al, 2006). In fact, the recent withdrawal of rofecoxib due to increased cardiovascular and thrombotic risk during the Adenomatous Polyp Prevention on Vioxx (APPROVe) trial has underscored new safety concerns about this class of drug. Thus, development of novel strategies that target COX-2 inhibitors selectively into tumor cells, while avoiding systemic toxicity, will be an invaluable addition in cancer management. The application of gene therapy approaches that can be adapted to transcriptional or transductional targeting of particular tissues or cell types, or certain cancer cells, represents a novel therapy that is distinct from traditional pharamaceutical treatment modalities. Antisense oligonucleotides to specific cellular RNA have shown great promise in both research and clinical studies as these sequence-specific agents are able to modulate the expression of targeted genes. In principle, the most important feature of antisense oligonucleotides is their ability to base-pair with the target RNA and either block its translation or facilitate its destruction by RNase H, an enzyme that destroys RNA in a DNA/RNA duplex (Tyson-Capper and Europe-Finner, 2006). In this study, we have examined the potential therapeutic effect of adenovirus vector expressing COX-2 antisense (Ad-COX2 AS) on modulating the expression of multidrugresistance (MDR) gene MDR1, antiapoptotic gene Bcl-2, and cell cycle regulating gene cyclin D1, as well as the

chemosensitivity to doxorubicin in a doxorubicin-resistant breast cancer cell line (MCF-7/DOX).

II. Materials and Methods A. Cell culture and cell lines The human breast cancer cell line MCF-7 was purchased from the American Type Culture Collection (Rockville, Md). MCF-7/DOX cells, a human breast adenocarcinoma cell line highly resistant to doxorubicin and overexpressing the MDR protein P-gp170 (Chen et al, 2002), was a kind gift from Dr. Kapil Mehta at the University of Texas M.D. Anderson Cancer Center, Houston, Texas. The cell lines were maintained at 37ºC in a 95% humidified, 5% CO2 atmosphere in RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, and 1% penicillin and streptomycin (Gibco BRL, Life Technologies, Long Island, NY). To maintain the resistance phenotype, the MCF-7/DOX cells were grown in the presence of 1 !g/mL doxorubicin (Chen et al, 2002).

B. Recombinant adenoviral vector and in vitro transduction The replication-deficient Ad-COX-2 AS under the control of the cytomegalovirus promoter was a generous gift from Dr. Chuan-Ming Hao (Vanderbilt University School of Medicine, Nashville, Tenn). An adenovirus expressing a marker gene coding for bacterial "-galactosidase (Ad-LacZ) was a kind gift from Dr. Savio Woo (Mount Sinai School of Medicine, New York, NY). Large-scale production of Ad-COX-2 AS and AdLacZ was performed as we described previously (Al-Hendy and Auersperg, 1997), with a typical batch yield of 2 # 1010 PFU/mL. For the in vitro transduction, MCF-7/DOX cells were grown as described above until they reached 70% to 80% confluence. Then, the cells were transduced with the Ad-LacZ reporter vector or Ad-COX-2 AS at the desired MOI for 4 hours in transduction media as we described previously (Al-Hendy and Auersperg, 1997). The transduction media were removed and replaced with maintenance media. The transduction efficiency was determined in Ad-LacZ–transduced cells by staining for Ad-LacZ expression with b-galactosidase staining kit (Stratagene, La Jolla, Calif), according to the manufacturer’s protocol. To study the effect of the Ad-COX-2 AS on MCF-7/DOX cells, the cells were transduced with different MOI (10, 20, 50, and 100 PFU/cell) of Ad-COX-2 AS or Ad-LacZ as a negative control.

C. Cytotoxicity assay MCF-7/DOX cells transduced with the indicated virus were plated in 6-well plates (5 # 105 cells/well) and allowed to attach for 12 hours. The growth media were replaced with fresh media containing different concentrations of doxorubicin (0.01– 20 !M). After 72 hours, the media were removed from the wells, and the cells were washed twice with phosphate buffered saline (PBS). The number of viable cells per well was determined in triplicate using a fluorometric assay with Hoechst 33258 (bisbenzimide), as described previously (Houston et al, 2003). A similar set of experiments was performed with the Trypan blue (Sigma Chemical Co, St. Louis, Mo) exclusion test. The concentrations of doxorubicin required to inhibit growth by 50% (IC50 values) were calculated from the cytotoxicity curves using GraphPad Prism Software (GraphPad Software Inc, San Diego, Calif). The fold-reversal of doxorubicin resistance was calculated by dividing the IC50 values in the cells transduced with Ad-LacZ vector by those in Ad-COX-2 AS–transduced cells.

D. Determination of MDR1 expression by RT-PCR 2

Cancer Therapy Vol 5, page 3 Forty-eight or seventy-two hours after transduction of MCF-7/DOX cells with the indicated virus, total RNA was extracted from cells with RNA STAT- 60 kit (Tel-Test Inc, Friendswood, Tex) and quantified by UV absorbance spectroscopy. Total RNA (2 !g) was reverse-transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, Calif) in a final volume of 50 !L. The reaction mixture contained 1# reverse transcriptase buffer (500 mM each deoxynucleotide triphosphate mixture [dNTP], 3 mM MgCl2, 75 mM KCl, 50 mM TRIS-HCl [pH 8.3]; 10 units RNase inhibitor; 10 mM dithiothreitol; 500 !M per dNTP; 50 units reverse transcriptase; and 1.5 mM random hexamers. The thermal cycling parameters for reverse transcription reactions were as follows: 25°C for 10 minutes, 48°C for 30 minutes, and 95°C for 5 minutes. In a total volume of 50 !L, the MDR1 and GAPDH were co-amplified. The reaction mixture contained the following ingredients: 2# Ready Mix REDTaq PCR reaction mix, MDR1 forward primer (10 pmol/!L) [5!-ATA TCA GCA GCC CAC ATC AT-3!], MDR1 reverse primer (10 pmol/!L) [5-GAA GCA CTG GGA TGT CCG GT-3!], GAPDH forward primer (10 pmol/!L) [5!-GCC AAA AGG GTC ATC ATC TC-3], GAPDH reverse primer (10 pmol/!L) [5!-GTA GAG GCA GGG ATG ATG TTC-3!]. The amplification cycles were as follows: initial denaturation step at 94°C for 3 minutes, then 33 cycles at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1.5 minutes, followed by 72°C for 15 minutes. The PCR products were electrophoresed on 1.5% agarose gel, and band intensities were quantified using Alpha Imager Imaging System (Alpha Innotech Corporation, San Leandro, Calif), and the ratio of the intensity from MDR1 to GAPDH from each lane was graphed and analyzed.

blocked with 5% nonfat milk in phosphate buffered saline-Tween (PBS-T) for 1 hour and then incubated overnight at 4°C with the corresponding primary antibodies. After three washes in 0.1% PBS-T, immunoreactive proteins were detected by using horseradish peroxidase-conjugated secondary antibodies (1:5000; Promega Corp, Madison, Wis). Detection by enzyme-linked chemiluminescence was performed according to the manufacturer’s protocol (ECL plus, Amersham Pharmacia Biotech). The intensity of each protein band was determined using a scanning densitometer (Epson 4870, Epson America, Long Beach, Calif). "-actin was used as internal control for normalization of the protein concentrations and loading precision.

F. Statistical analysis All assays were performed in triplicate. The results were expressed as mean ± standard error of the mean (SEM). Statistical analysis was determined by Student’s t test. Significance was reached when P < 0.05.

III. Results A. Adenovirus vector transduces MCF7/DOX cells with higher efficiency compared with the parental MCF-7 cells We first wanted to assess the ability of replicationincompetent adenovirus to transduce MCF-7/DOX cells versus the doxorubicin-sensitive parental MCF-7 cells. The transduction efficiency of the adenoviral vectors in MCF-7/DOX cells was tested using adenovirus LacZ (AdLacZ) vector. The adenovirus vector efficiently transduced MCF-7/DOX cells (Figure 1), and the transduction efficiency was proportional to the multiplicity of infections (MOI) used. The optimal transduction efficiency was obtained at an MOI of 100 plaque-forming units [PFU]/cell. At MOIs of 1, 10, 20, 50, and 100 PFU/cell, the percentages of !-galactosidase positive cells were 7 ± 1.16, 17 ± 3, 30 ± 5.3, 85 ± 1.5, and 95 ± 1.5, respectively. On the other hand, Ad-lacZ vector transduced the parental cells (doxorubicin-sensitive MCF7 cells) less efficiently compared with MCF-7/DOX cells. At similar MOIs (1, 10, 20, 50, and 100 PFU/cell), the percentages of !-galactosidase positive MCF-7 cells were 4 ± 1.2, 11 ± 1.8, 19 ± 2.6, 60 ± 3.8, and 85 ± 2.7, respectively (Table 1).

E. Western blotting The cells were harvested 72 hours after transduction with either of the indicated viruses for protein analysis. The cells were washed in PBS, lysed in radioimmunoprecipitation (RIPA) lysis buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.1% sodium dodecylsulfate (SDS), 1% nonidet-P40, 0.5% deoxycholate) containing 5 mM ethylenediaminetetraacetic adid (EDTA) and protease inhibitors (phenylmethyl sulfonyl fluoride, leupeptin, and aprotinin) at the ratio of 100 $L buffer for 3 # 106 cells. After 30 minutes on ice with some vortexing, the lysates were centrifuged at 13,000 # g for 20 minutes at 4°C. Protein concentration was determined with bicinchoninic acid protein assay kit (Pierce Technology Co, Rockford, Ill) according to the manufacturer’s protocol. Equal amounts of proteins (20 $g) were mixed with SDS reducing buffer. Protein samples were separated on acrylamide SDS-PAGE then transferred onto polyvinyldifluoride membrane (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were

Kamel et al: Cyclooxygenase-2 and gene therapy of drug resistance Figure 1. Adenovirus-infected MCF-7/DOX cells. The transduction efficiency was MOI-dependent and about 85% of the cells were transduced at an MOI of 50 PFU/cell.

Table 1. Percentage of "-Galactosidase Positive Cells After Transducing MCF-7/DOX Cells or the Parental MCF-7 Cells With Different MOIs of Adenovirus-expressing "-Galactosidase. Multiplicity of Infection (PFU/Cell) 1 10 20 50 100

MCF-7/DOX (%) 7 ± 1.16 17 ± 3 30 ± 5.3 85 ± 1.5 95 ± 1.5

Transduced Cells Parental MCF-7 Cells (%) 4 ± 1.2 11 ± 1.8 19 ± 2.6 60 ± 3.8 85 ± 2.7

B. Ad-COX-2 AS sensitizes MCF-7/DOX cells to doxorubicin

C. Ad-COX-2 AS inhibits MDR1 gene expression

Based on the mounting evidence suggesting that overexpression of COX-2 in tumor cells confers resistance to chemotherapeutic agents, we have evaluated whether Ad-COX-2 AS has the therapeutic potential to modulate drug resistance in the doxorubicin-resistant breast cancer cell line MCF-7/DOX. Our data show that Ad-COX-2 AS sensitizes MCF-7/DOX cells to the cytotoxic effects of doxorubicin. Sensitivity to doxorubicin was assessed by a cytotoxicity assay, which compared the IC50 values in MCF-7/DOX cells transduced with Ad-COX-2 AS with IC50 values in cells transduced with the same MOI as AdLacZ. Transduction with Ad-COX-2 AS (MOI: 10, 20, 50, and 100 PFU/cell) lowered the IC50 of doxorubicin in MCF-7/DOX cells by 2.7-, 16-, 20- and 20-fold, respectively, compared with cells transduced with a similar MOI of Ad-LacZ (Figure 2A). However, Ad-LacZ did not influence the sensitivity of MCF-7/DOX cells to doxorubicin (Figure 2B). These data clearly indicated that Ad-COX-2 AS reversed the doxorubicin-resistant phenotype of MCF-7/DOX cells.

To determine if the transduction of MCF-7/DOX cells with Ad-COX-2 AS affects the expression of the MDR1 gene, and consequently the sensitivity to doxorubicin, the expression of MDR1 mRNA was quantified by reverse transcription polymerase chain reaction (RT-PCR) in MCF-7/DOX cells transduced with the different MOI of Ad-LacZ or Ad-COX 2 AS. Transduction of MCF-7/DOX with Ad-LacZ (20–100 PFU/cell) did not alter the MDR1 expression (Figure 3A). However, Ad-COX-2 AS (MOI 10, 20, 50, and 100 PFU/cell) significantly downregulated MDR1 gene expression by 21%, 35%, 39%, and 48%, respectively, compared with the Ad-LacZ–transduced cells or untransduced control cells (Figure 3A, 3B). To confirm the effect of Ad-COX 2 AS on the MDR1 gene expression, Western blot analysis was used to evaluate the MDR1 protein level. Consistent with its effect on the mRNA level, Ad-COX 2 AS reduced the MDR1 protein expression in MCF-7/DOX cells (Figure 4A). Quantitatively, transduction of MCF-7/DOX cells with Ad-COX-2 AS at MOI of 10, 20, 50, or 100 PFU/cell significantly reduced MDR1 protein expression levels by 29%, 35%, 45%, and 47%, respectively, compared with untransduced or Ad-LacZ–transduced cells (Figure 4B).

Cancer Therapy Vol 5, page 5

Figure 2. Effect of different MOIs of (A) Ad-COX-2 AS or (B) Ad-LacZ on the sensitivity of MCF-7/DOX cells to doxorubicin. MCF7/DOX cells were transduced with Ad-COX-2 AS or Ad-LacZ at different MOIs. Twenty-four hours after transduction, the cells were treated with different concentrations of doxorubicin for 72 hours. Then, the cell proliferation was determined and the IC 50 was calculated. Values represent the mean ± SEM from three similar experiments. Figure 3. (A) Effect of Ad-LacZ or Ad-COX-2 AS on MDR1 mRNA expression in MCF-7/DOX cells. MCF-7/DOX cells were transduced with the different MOI of Ad-LacZ or Ad-COX-2 AS. Forty-eight hours later, RNA was extracted and the MDR1 mRNA was amplified using RT-PCR. GAPDH was co-amplified and used as internal control. The PCR products were electrophoresed on 1.5% agarose gel. Lane 1: DNA marker; Lane 2: normal MCF-7 cells; Lane 3: control MCF-7/Dox cells; Lane 4–6: MCF-7/DOX transduced with Ad-LacZ (20, 50, and 100 PFU/cell); Lane 7–10: MCF-7/DOX transduced with AdCOX-2 AS (10, 20, 50, and 100 PFU/cell). (B) The ratio of the band intensity of MDR1 to GAPDH from the RT-PCR. Values are expressed as mean ± SD (*P < 0.01). This figure shows the results of quantifying the density of PCR products by Alpha Imager Imaging System. *Significantly different from untransduced control cells and cells transduced with Ad-LacZ (P < 0.05). Data are presented as the mean ± SEM of three independent experiments.

Kamel et al: Cyclooxygenase-2 and gene therapy of drug resistance

Figure 4. (A) Effect of Ad-COX-2 AS on MDR1 protein expression in MCF-7/DOX cells. The cells were transduced with the different MOI of Ad-LacZ or Ad-COX-2 AS. Seventy-two hours later, cell lysates were prepared and an equal amount (50 $g) of protein was separated by 8% SDS-PAGE, and then transferred onto nitrocellulose membranes. The membranes were immunoblotted with monoclonal antiMDR1 antibody or antibody against "-actin. Lane 1: normal MCF-7 cells; Lane 2: untransduced MCF-7/DOX cells; Lane 3–5: MCF-7/DOX cells AdLacZ (10, 20, 50, and 100 PFU/cell); Lane 6–9, MCF-7/DOX transduced with Ad-COX-2 AS (10, 20, 50, and 100 PFU/cell). (B) Results of quantifying the density of protein bands by Alpha Imager Imaging System. *Significantly different (P < 0.05) compared with control MCF7/Dox cells and cells transduced with Ad-LacZ using two-sided distribution (Student’s t test). Results are the representative of three similar experiments (mean ± SE).

transduced cells, Ad-COX-2 AS at MOIs of 10, 20, 50, and 100 PFU/cells decreased the Bcl-2 expression by 17%, 25%, 32%, and 40%, respectively (Figure 6B). On the other hand, Ad-COX-2 AS had a minimal effect on Bax expression (Figure 7A, 7B).

D. Ad-COX-2 AS downregulates cyclin D1 expression in MCF-7/DOX cells Compelling evidence suggests a role for COX-2 in the growth of cancer cells via induction of the expression of cell cycle regulating genes. Therefore, we investigated the effect of Ad-COX-2 AS on cyclin D1 expression in MCF-7/DOX cells. As expected, Ad-COX-2 AS downregulates the expression of cyclin D1 in MCF7/DOX cells in an MOI-dependent fashion (Figure 5A). Transduction with Ad-COX-2 AS at MOI of 10, 20, 50, and 100 PFU/cells downregulates cyclin D1 expression in MCF-7/DOX cells by 22%, 35%, 42%, and 43%, respectively, compared with cells transduced with AdLacZ or untransduced control cells (Figure 5B).

IV. Discussion COX-2 overexpression is reported in many malignancies, including breast cancer (Ranger et al, 2004; Schmitz et al, 2006; Singh et al, 2006). Elevated tumor prostaglandin E2, a major COX-2 metabolite, has been implicated in angiogenesis, tumor growth, invasion, reduced apoptosis, and resistance to anticancer drugs (Raspollini et al, 2005; Surowiak et al, 2005; Zatelli et al, 2005; Krysan et al, 2006). A functional role of COX-2 in tumor development and progression has been demonstrated by both overexpression (Liu et al, 2001) and disruption (Chulada et al, 2000) of the COX-2 gene as well as application of drugs blocking either COX-1/-2 or COX-2 alone (Wang and DuBois, 2004; Harris et al, 2005; Harris et al, 2006). It has been reported that COX-2 upregulates MDR1/P-gp expression, which confers the resistance to anticancer drugs (Ratnasinghe et al, 2001; Patel et al, 2002; Ziemann et al, 2002; Sorokin 2004; Surowiak et al, 2005). In addition, COX-2 expression is

E. Effect of Ad-COX-2 AS on Bcl-2 and Bax expression in MCF-7/DOX cells Selective inhibitor of COX-2 has been reported to inhibit growth and induce apoptosis in several cancer cell lines (Sanborn and Blanke, 2005; Pyrko et al, 2006). Thus, we examined expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax in MCF-7/DOX cells after transduction with Ad-COX-2 AS. Western blotting analysis demonstrated that Ad-COX-2 AS significantly reduced the expression of Bcl-2 protein (Figure 6A). Compared with untransduced control cells and Ad-LacZ

Cancer Therapy Vol 5, page 7 Figure 5. Expression of cyclin D1 protein as measured with Western blot in MCF-7/DOX cells transduced with the different MOI of Ad-LacZ or Ad-COX-2 AS. MCF-7/DOX cells were transduced with Ad-LacZ or Ad-COX-2 AS. Seventy-two hours after transduction, cell lysates were prepared and equal amounts were separated by 12% SDS-PAGE, and then transferred onto nitrocellulose membranes. The membranes were immunoblotted with monoclonal anti-cyclin D1 antibody or antibody against "-actin. (A) Results of developing the x-ray film. Lane 1: untransduced MCF7/DOX cells; Lane 2–5: MCF7/DOX cells Ad-LacZ (10, 20, 50, and 100 PFU/cell); Lane 6–9: MCF7/DOX transduced with Ad-COX-2 AS (10, 20, 50, and 100 PFU/cell). (B) Results of quantifying protein expression by Alpha Imager Imaging System. *Significantly different (P < 0.05) compared with control MCF7/Dox cells and cells transduced with Ad-LacZ using two-sided distribution (Student’s t test). Results are the mean ± SEM for three different experiments.

Figure 6. Expression of Bcl-2 protein as measured with Western blot in MCF-7/DOX cells transduced with the different MOI of Ad-Lac Z or Ad-COX 2 AS. MCF-7/DOX cells were transduced with Ad-LacZ or Ad-COX-2 AS. Seventy-two hours after transduction, cell lysates were prepared and equal amounts were separated by 12% SDS-PAGE, and then transferred onto nitrocellulose membranes. The membranes were immunoblotted with monoclonal antiBcl-2 antibody or antibody against !-actin. (A) Results of developing the x-ray film. Lane 1: untransduced MCF-7/DOX cells; Lane 2–5: MCF-7/DOX cells Ad-LacZ (10, 20, 50, and 100 PFU/cell); and Lane 6–9: MCF7/DOX transduced with Ad-COX-2 AS (10, 20, 50, and 100 PFU/cell). (B) Results of quantifying protein expression by Alpha Imager Imaging System. *Significantly different (P < 0.05) compared with control MCF7/Dox cells and cells transduced with Ad-LacZ using two-sided distribution (Student’s t test). Results are the mean ± SEM for three different experiments.

Kamel et al: Cyclooxygenase-2 and gene therapy of drug resistance

Figure 7. Expression of Bax protein as measured with Western blot in MCF-7/DOX cells transduced with the different MOI of Ad-LacZ or Ad-COX-2 AS. MCF-7/DOX cells were transduced with Ad-LacZ or Ad-COX-2 AS. Seventy-two hours after transduction, cell lysates were prepared and equal amounts were separated by 12% SDS-PAGE, and then transferred onto nitrocellulose membranes. The membranes were immunoblotted with monoclonal antiBax antibody or antibody against "-actin. (A) Results of developing the x-ray film. Lane 1: untransduced MCF-7/DOX cells; Lane 2–5: MCF7/DOX cells Ad-LacZ (10, 20, 50, and 100 PFU/cell); Lane 6–9: MCF7/DOX transduced with Ad-COX-2 AS (10, 20, 50, and 100 PFU/cell). (B) Results of quantifying protein expression by Alpha Imager Imaging System. *Significantly different (P < 0.05) compared with control MCF7/Dox cells and cells transduced with Ad-LacZ using two-sided distribution (Student’s t test). Results are expressed as mean ± SEM for three different experiments.

associated with inhibition of the apoptotic pathway in tumor cells (Tsujii and DuBois 1995; Meric et al, 2006), and it induces cell proliferation by upregulation of cyclin D1 (Wu et al, 2004; Patel et al, 2005; Zhang et al, 2006). Therefore, COX-2 inhibitors have been used as chemopreventive and therapeutic agents in cancer treatment (Chow et al, 2005; Harris et al, 2006; Mazhar et al, 2006). Unfortunately, currently available orally administered COX-2 inhibitors are not optimal compounds, and their use is associated with serious cardiovascular effects (Becerra et al, 2003; Dogne et al, 2006; Meric et al, 2006). In fact, the US Food and Drug Administration has recently ordered rofecoxib to be withdrawn from the American market due to increased cardiovascular and thrombotic risks (Dogne et al, 2006; Schnitzer 2006). Others systemic COX-2 inhibitors have also been voluntarily withdrawn. Thus, development of strategies that target COX-2 selectively into tumor cells with minimal systemic toxicity will be a welcomed addition to cancer treatment. The effects of COX-2 in gene expression are generally mediated by formation of cAMP, the down stream effector of COX-2. Generally, genes regulated by COX-2 harbor cAMP response element (CRE) in their promoter regions. MDR1, Cyclin D1, and Bcl-2 genes have been reported to be up-regulated by cAMP, an effect mediated through cAMP response element (CRE) in their promoter regions (Chang et al, 2006; Xiang et al, 2006; Ziemannet al, 2006). Thus, abrogation of COX-2 expression using the Ad-COX-2 AS can cause a decrease in the cellular level of cAMP with subsequent down

regulation of MDR1, Cyclin D1, and Bcl-2 genes expression. Although the mechanism by which decrease cyclin D1 gene expression affect the sensitivity of tumor cells to doxorubicin is not known, it has been reported that the expression of genes that control the cell cycle are of critical importance in determining the sensitivity of cells and tumors to drugs (chemosensitivity) and radiation. It has been consistently reported tumor cell lines expressing higher of Cyclin D1 demonstrated resistance to cytotoxic drugs compared with cells expressing lower levels (Hochhauser et al, 1996; Henriksson et al, 2006). A gene therapy strategy could be the answer to this dilemma, since it provides effective localized delivery of a particular gene product while avoiding undesirable systemic side effects. In this study, we test the feasibility of using Ad-COX-2 AS as a gene therapy approach to selectively shut down COX-2 enzyme expression in tumor cells. The impact of transducing the MCF-7/DOX cells with Ad-COX-2 AS upon the expression of MDR1, Bcl-2, Bax, and cyclin D1 protein expression as well as the sensitivity to doxorubicin cytotoxicity was assessed. Our results demonstrated that the adenovirus vector transduces MCF-7/DOX cells more efficiently compared with doxorubicin-sensitive parental cells. This finding is consistent with several earlier reports suggesting that adenovirus vector preferentially transduces anticancerresistant tumor cells more efficiently compared with the parental cell line (Shirakawa et al, 2001; AmbriovicRistov et al, 2004; Holm et al, 2004). Our results also indicated that transduction of MCF7/DOX cells with Ad-COX-2 AS resulted in a dramatic

Cancer Therapy Vol 5, page 9 effectiveness of 5-flurouracil, epirubicin and cyclophosphamide with celecoxib treatment in breast cancer patients. Biomed Pharmacother 59 Suppl 2, S298-S301. Chulada PC, Thompson MB, Mahler JF, Doyle CM, Gaul BW, Lee C, Tiano HF, Morham SG, Smithies O, Langenbach R (2000) Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min mice. Cancer Res 60, 4705-4708. Dogne JM, Hanson J, Supuran C, Pratico D (2006) Coxibs and cardiovascular side-effects: from light to shadow. Curr Pharm Des 12, 971-975. Gasparini G, Longo R, Sarmiento R, Morabito A (2003) Inhibitors of cyclo-oxygenase 2: a new class of anticancer agents? Lancet Oncol 4, 605-615. Glasgow JN, Everts M, Curiel DT (2006) Transductional targeting of adenovirus vectors for gene therapy. Cancer Gene Ther 13, 830-844. Harris RE, Beebe-Donk J, Doss H, Burr DD (2005) Aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs in cancer prevention: a critical review of non-selective COX2 blockade (review). Oncol Rep 13, 559-583. Harris RE, Beebe-Donk J, Alshafie GA (2006) Reduction in the risk of human breast cancer by selective cyclooxygenase-2 (COX-2) inhibitors. BMC Cancer 6, 27. Holm PS, Lage H, Bergmann S, Jurchott K, Glockzin G, Bernshausen A, Mantwill K, Ladhoff A, Wichert A, Mymryk JS, Ritter T, Dietel M, Gansbacher B, Royer HD (2004) Multidrug-resistant cancer cells facilitate E1-independent adenoviral replication: impact for cancer gene therapy. Cancer Res 64, 322-328. Houston KD, Copland JA, Broaddus RR, Gottardis MM, Fischer SM, Walker CL (2003) Inhibition of proliferation and estrogen receptor signaling by peroxisome proliferatoractivated receptor gamma ligands in uterine leiomyoma. Cancer Res 63, 1221-1227. Krysan K, Reckamp KL, Sharma S, Dubinett SM (2006) The potential and rationale for COX-2 inhibitors in lung cancer. Anticancer Agents Med Chem 6, 209-220. Liu CH, Chang SH, Narko K, Trifan OC, Wu MT, Smith E, Haudenschild C, Lane TF, Hla T (2001) Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 276, 18563-18569. Mazhar D, Ang R, Waxman J (2006) COX inhibitors and breast cancer. Br J Cancer 94, 346-350. Meric JB, Rottey S, Olaussen K, Soria JC, Khayat D, Rixe O, Spano JP (2006) Cyclooxygenase-2 as a target for anticancer drug development. Crit Rev Oncol Hematol 59, 51-64. Patel MI, Subbaramaiah K, Du B, Chang M, Yang P, Newman RA, Cordon-Cardo C, Thaler HT, Dannenberg AJ (2005) Celecoxib inhibits prostate cancer growth: evidence of a cyclooxygenase-2-independent mechanism. Clin Cancer Res 11, 1999-2007. Patel VA, Dunn MJ, Sorokin A (2002) Regulation of MDR-1 (Pglycoprotein) by cyclooxygenase-2. J Biol Chem 277, 38915-38920. Puhlmann U, Ziemann C, Ruedell G, Vorwerk H, Schaefer D, Langebrake C, Schuermann P, Creutzig U, Reinhardt D (2005) Impact of the cyclooxygenase system on doxorubicininduced functional multidrug resistance 1 overexpression and doxorubicin sensitivity in acute myeloid leukemic HL-60 cells. J Pharmacol Exp Ther 312, 346-354. Pyrko P, Soriano N, Kardosh A, Liu YT, Uddin J, Petasis NA, Hofman FM, Chen CS, Chen TC, Schonthal AH (2006) Downregulation of survivin expression and concomitant induction of apoptosis by celecoxib and its non-

improvement in the sensitivity of these cells to the cytotoxic effect of doxorubicin. This reversal of the chemoresistance phenotypes is associated with downregulation of MDR1 gene expression. These observations are consistent with the well-documented effect of COX-2 inhibitors on sensitizing the tumor cells to chemotherapeutic agents and the regulation of MDR1 gene expression (Sorokin 2004; Puhlmann et al, 2005; Zatelli et al, 2005). Furthermore, Ad-COX-2 AS downregulated the expression of the antiapoptotic gene Bcl-2 and the cell cycle promoting gene cyclin D1. Similar effects have been observed with the conventional COX-2 inhibitors (Takada et al, 2004; Arellanes-Robledo et al, 2006). When the gene therapy era launched, it became evident that cancer would be an appropriate target for this developing field. Most importantly, the application of the gene therapy approach, which can be adapted to transcriptional targeting into a particular tissue or cell type, or certain cancer cells, represents a novel therapy that is distinct from traditional COX-2 inhibitors. The substantial advances in the adenovirus vector design raised expectations for this adenovirus-mediated gene therapy approach to be advantageous to other vectors (Al Hendy and Salama, 2006; Glasgow et al, 2006; Rein et al, 2006). Vector genomes can be manipulated to express therapeutic transgenes specifically into tumor cells utilizing transductional and transcriptional targeting strategies. Contrary to the conventional COX-2 inhibitors, the deployment of the Ad-COX-2 AS approach can avoid the undesirable systemic side effects on various organs. In conclusion, this â&#x20AC;&#x153;proof-of-conceptâ&#x20AC;? study suggests that gene therapy modalities using adenovirus vector expressing Ad-COX-2 AS are a promising treatment modality for breast cancer, especially in patients who have had unsuccessful traditional chemotherapeutic treatment.

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Kamel et al: Cyclooxygenase-2 and gene therapy of drug resistance cyclooxygenase-2-inhibitory analog, dimethyl-celecoxib (DMC), in tumor cells in vitro and in vivo. Mol Cancer 5, 19. Ranger GS, Jewell A, Thomas V, Mokbel K (2004) Elevated expression of cyclooxygenase-2 in breast cancer and ductal carcinoma in situ has no correlation with established prognostic markers. J Surg Oncol 88, 100-103. Raspollini MR, Amunni G, Villanucci A, Baroni G, Taddei GL (2006) Cyclooxygenase-2, angiogenesis, tumor cell proliferation, P-glycoprotein in advanced ovarian serous carcinoma. Am J Obstet Gynecol 194, 1203. Raspollini MR, Amunni G, Villanucci A, Boddi V, Taddei GL (2005) Increased cyclooxygenase-2 (COX-2) and Pglycoprotein-170 (MDR1) expression is associated with chemotherapy resistance and poor prognosis. Analysis in ovarian carcinoma patients with low and high survival. Int J Gynecol Cancer 15, 255-260. Ratnasinghe D, Daschner PJ, Anver MR, Kasprzak BH, Taylor PR, Yeh GC, Tangrea JA (2001) Cyclooxygenase-2, Pglycoprotein-170 and drug resistance; is chemoprevention against multidrug resistance possible? Anticancer Res 21, 2141-2147. Rein DT, Breidenbach M, Curiel DT (2006) Current developments in adenovirus-based cancer gene therapy. Future Oncol 2, 137-143. Sanborn R, Blanke CD (2005) Cyclooxygenase-2 inhibition in colorectal cancer: boom or bust? Semin Oncol 32, 69-75. Schmitz KJ, Callies R, Wohlschlager J, Kimmig R, Otterbach F, Bohr J, Lee HS, Takeda A, Schmid KW, Baba HA (2006) Overexpression of cyclooxygenase-2 is an independent predictor of unfavourable outcome in node-negative breast cancer but is not associated with protein kinase B (Akt)and mitogen-activated protein kinases (ERK1/2, p38) activation or with Her-2/neu signalling pathways. J Clin Pathol 59, 685-691. Schnitzer TJ (2006) Update on guidelines for the treatment of chronic musculoskeletal pain. Clin Rheumatol 25, 22-29. Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN (1998) Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 58, 362-366. Shirakawa T, Sasaki R, Gardner TA, Kao C, Zhang ZJ, Sugimura K, Matsuo M, Kamidono S, Gotoh A (2001) Drug-resistant human bladder-cancer cells are more sensitive to adenovirusmediated wild-type p53 gene therapy compared to drugsensitive cells. Int J Cancer 94, 282-289. Singh B, Berry JA, Shoher A, Lucci A (2006) COX-2 induces IL-11 production in human breast cancer cells. J Surg Res 131, 267-275.

Sorokin A (2004) Cyclooxygenase-2: potential role in regulation of drug efflux and multidrug resistance phenotype. Curr Pharm Des 10, 647-657. Surowiak P, Materna V, Matkowski R, Szczuraszek K, Kornafel J, Wojnar A, Pudelko M, Dietel M, Denkert C, Zabel M, Lage H (2005) Relationship between the expression of cyclooxygenase 2 and MDR1/P-glycoprotein in invasive breast cancers and their prognostic significance. Breast Cancer Res 7, R862-R870. Takada Y, Bhardwaj A, Potdar P, Aggarwal BB (2004) Nonsteroidal anti-inflammatory agents differ in their ability to suppress NF-kappaB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation. Oncogene 23, 9247-9258. Tari AM, Simeone AM, Li YJ, Gutierrez-Puente Y, Lai S, Symmans WF (2005) Cyclooxygenase-2 protein reduces tamoxifen and N-(4-hydroxyphenyl)retinamide inhibitory effects in breast cancer cells. Lab Invest 85, 1357-1367. Tsujii M, DuBois RN (1995) Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 83, 493-501. Tyson-Capper AJ, Europe-Finner GN (2006) Novel targeting of cyclooxygenase-2 (COX-2) pre-mRNA using antisense morpholino oligonucleotides directed to the 3' acceptor and 5' donor splice sites of exon 4: suppression of COX-2 activity in human amnion-derived WISH and myometrial cells. Mol Pharmacol 69, 796-804. Wang D, DuBois RN (2004) Cyclooxygenase-2: a potential target in breast cancer. Semin Oncol 31, 64-73. Wu CR, Zhang Y, Rose ME, Graham SH (2004) Cyclooxygenase-2 activity contributes to neuronal expression of cyclin D1 after anoxia/ischemia in vitro and in vivo. Brain Res Mol Brain Res 132, 31-37. Zatelli MC, Luchin A, Piccin D, Tagliati F, Bottoni A, Vignali C, Bondanelli M, degli Uberti EC (2005) Cyclooxygenase-2 inhibitors reverse chemoresistance phenotype in medullary thyroid carcinoma by a permeability glycoprotein-mediated mechanism. J Clin Endocrinol Metab 90, 5754-5760. Zhang GS, Liu DS, Dai CW, Li RJ (2006) Antitumor effects of celecoxib on K562 leukemia cells are mediated by cell-cycle arrest, caspase-3 activation, and downregulation of Cox-2 expression and are synergistic with hydroxyurea or imatinib. Am J Hematol 81, 242-255. Ziemann C, Schafer D, Rudell G, Kahl GF, Hirsch-Ernst KI (2002) The cyclooxygenase system participates in functional mdr1b overexpression in primary rat hepatocyte cultures. Hepatology 35, 579-588.

Cancer Therapy Vol 5, page 11 Cancer Therapy Vol 5, 11-18, 2007

Development of farnesyl transferase inhibitors as anticancer agents: current status and future Review Article

Floriana Morgillo and Ho-Young Lee* Departments of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX

__________________________________________________________________________________ *Correspondence: Ho-Young Lee, PhD, Department of Thoracic/Head and Neck Medical Oncology, Unit 432, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-6363; Fax: (713) 796-8655; e-mail: hlee@mdanderson.org Key words: farsenyl transferase inhibitors, anticancer agents, RAS signaling, FTIs; conventional chemotherapy, radiotherapy Abbreviations: colorectal cancer, (CRC); farnesyltransferase, (FTase); geranylgeranyltransferase, (GGTase); guanosine diphosphate, (GDP); guanosine triphosphate, (GTP); human head and neck squamous cell carcinoma, (HNSCC); mouse mammary tumor virus, (MMTV); non-small-cell lung cancer, (NSCLC); Partial Response, (PR) Received: 18 October 2005; Revised: 22 December 2006 Accepted: 02 January 2007 electronically published: January 2007

Summary Ras proteins belong to the GTPase family of proteins. They participate in the control of several signal transduction pathways, which include cell growth, differentiation, proliferation and survival. Ras mutation is one of the most frequent aberrations in cancer and plays a fundamental role in tumorigenesis. In order to be activated, Ras requires localization to the plasma membrane. This subcellular localization is dependent on a posttranslational modification consisting of protein farnesylation, which is a catalysed by the farnesyl transferase enzyme. Several different classes of agents that blocks FTase have been developed to inhibit Ras processing and consequently the growth of ras mutated tumor. Preclinical data revealed that although FTIs inhibit the growth of ras-transformed cells, they are also potent inhibitors of a wide range of cancer cell lines that contain wild-type ras and affect multiple signal transduction pathways independent of Ras. Phase I and II clinical trials confirmed a relevant antitumor activity and a low toxicity, although no improvement in overall survival has been reported in phase III trials. FTIs are a promising class of novel antineoplastic agents, but in solid tumors their activity seems to be modest. Therefore, studies on combination of these drugs with cytotoxic agents, ionizing radiation and other targeted therapeutic agents are warranted to develp better therapeutic strategies in cancer. of prenylation (Khosravi-Far et al, 1992; Gelb, 1997) by either farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase) (Zhang and Casey, 1996) (Figure 1). Upon activation, Ras triggers a series of downstream signaling pathways, including Raf/MEK/MAPK, which induce an increase in the levels of Cyclin D1 that promote the progression of cells through the G1 checkpoint and into S phase, leading to proliferation (Gille, 1999). Activated Ras also stimulates PI3K/Akt signaling pathways that stimulate cell proliferation and survival (Gille and Downward, 1999). In addition, Ras interacts with specific integrin-alpha cytoplasmic domains and small GTP-binding protein, such as Rac and the Rho proteins, which may lead to increase in the invasive capacity of neoplastic cells (Clark, 1996). Ras can be constitutively activated trough point mutations or indirectly through other genetic abnormalities

I. RAS signaling The Ras family of proteins are 21kDa membraneassociated guanine nucleotide binding proteins; there are currently three ras proto-oncogenes (H-, N- and K-ras) which encode four proteins, known as H-Ras, N-Ras and two K-Ras proteins, K-Ras 4A and K-Ras4B (Barbacid, 1987). Ras proteins has been known to participate in critical network of signal transduction pathways that lead to cellular proliferation, survival, differentiation and invasion/metastasis, all of which play a critical role in the process of malignant transformation. Ras proteins alternate between an inactive form bound to guanosine diphosphate (GDP) and an active form bound to guanosine triphosphate (GTP) (Jurnak, 1985), transfer information from receptors on the cell surface to signaling systems (McCormick, 1994). In order to be activated, Ras proteins need to be post-translationally modified through a process 11

Morgillo and Lee: FTIs as anticancer agents

Figure 1. In order to become activated, Ras proteins must be localized to the inner side of the plasma membrane through a process, which is called prenylation (Khosravi-Far et al, 1992), (Gelb, 1997). This process consists in the covalent addition of an isoprenoid moiety, farnesyl or geranylgeranyl, issued from the cholesterol biosynthetic pathway to carboxy-terminal cysteine of substrate proteins (Zhang and Casey, 1996). This reaction is catalyzed by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase) (Zhang and Casey, 1996) and occurs on the cysteine in a CAAX consensus sequence. In the CAAX sequence the “C” represents the cysteine, “A” is an aliphatic amino acid and “X” any aminoacid. Usually, the terminal X residue determines whether farnesylation or geranylgeranylation occurs (Casey et al, 1991), (Yokoyama et al, 1991). Once the prenyl group attaches to the CAAX moiety, the AAX part is cleaved. The C-terminal farnesyl-cysteine moiety is then carboxymethylated and a fatty acid palmitate residue is attached. This makes the Ras protein hydrophobic and facilitates its transfer to the cell membrane, where it becomes phosphorylated when activated by upstream tyrosine kinase signaling.

(Bos et al, 1987; Fujita et al, 1984; Sawyers et al., 1995). Ras mutation is one of the most frequent aberration in cancer and has been observed in about 30% of human cancer, with about 90 % in pancreatic cancer (Almoguera et al, 1988) and in 10 to 65% of hematologic malignancies (Bos, 1989; Rodenhuis, 1992). Tumor angiogenesis and metastases have been proposed to be associated with oncogenic ras mutations that mediate the increased expression of angiogenic growth factor and key metalloproteinase, such as VEGF, gelatinase and

stromelysin (Su et al, 1993; Zabrenetzky et al, 1994; Rak et al, 1995). Since farnesylation is the most important step in Ras activation (Kato et al, 1992) and subsequent downstream signaling, agents that blocks FTase have been developed to impact cancer cell survival and proliferation. Several different classes of FTIs can be summarized in four main classes: (1) Farnesyl Pyrophosphate analogues, (2) CAAX peptidomimetics, (3) bisubstrate inhibitors, (4) Non peptidomimetic inhibitors. Table 1.

Table 1. Different classes of FTIs Class of FTI Farnesyl Pyrophosphate analogues (FPP analogs) Synthetic agents: RPR-115135; J-104,871 (Yonemoto et al, 1998!"Russo et al, 2001)" CAAC Peptidomimetics Synthetic agents: FTI-276; B956; L731,735; L778,123 Bisustrate Inhibitors Synthetic agents: BMS-185878; BMS186511 (Manne et al, 1995) Nonpeptidomimetic Inhibitors Synthetic agents: R115777 (Tipifarnib) (Rowinsky et al, 1999); SCH66336 (Lonafarnib) (Liu et al, 1998); BMS214662 (Sarasar) (Rose et al, 2001)

Clinical development No antitumor activity in vivo (Leonard, 1997), (Rowinsky et al, 1999)

Side Effects

Phase I (Britten et al, 2001)

Grade IV thrombocytopenia, fatigue, cardiac conduction abnormalities (Britten et al, 2001)

Limited data in vivo

Phase I (Adjei et al, 2000; Zujewski et al, 2000) Phase II (Johnston et al, 2003; Kurzrock et al, 2003; Winquist et al, 2005) Phase III (Rao et al, 2004)

Myelosuppression, fatigue, neurotoxicity, cardiac conduction abnormalities, diarrhea, renal effects (Cortes et al, 2002)

Cancer Therapy Vol 5, page 13 SCH66336 appears to inhibit hypoxia- or growth factorâ&#x20AC;&#x201C; stimulated and constitutive secretion of angiogenic factors, such as HIF-1! and VEGF, in aerodigestive tract cancer cells. SCH66336 affects the level of HIF-1! at the posttranscriptional level by decreasing its stability and modifying the proteasome-dependent degradation pathway through inhibition of the interaction between HIF-1! and Hsp90 under hypoxic and normoxic IGF-stimulated conditions and therefore decreasing the VEGF production. The over-expression of Hsp90, but not constitutive Akt or constitutive MEK, restored HIF-1! expression in IGFstimulated or hypoxic cells but not in unstimulated cells. SCH66336 has also been demonstrated to induce the expression of IGF-BP3, a major IGF-binding proapoptotic protein in serum (Lee et al, 2004). Furthermore, SCH66336 inhibited activation of NF-"B and NF-"Bregulated gene expressions induced by carcinogens and inflammatory stimuli (Takada et al, 2004). In addition, SCH66336 has shown to inhibit microtubule cytoskeleton, resulting in microtubule stabilization and suppression of microtubule dynamics (Marcus et al, 2005). The microtubule stabilizing actions of SCH66336 on could be due to its effects on histone deacetylase 6 (HDAC6), the known tubulin deacetylase (Marcus et al, 2005), or on centromere-binding proteins (Ashar et al, 2000).

A. Preclinical and clinical activities of FTIs 1. Preclinical activities of FTIs FTIs have shown abilities to inhibit the tumor cell growth in vitro and in vivo. FTIs modulate cell cycle depending on cellular context. In human tumor cell lines, FTIs induce accumulation of cells in G0/G1 phase of the cell cycle, which is often correlated with a p53-dependent p21waf/cip1 induction in many cell types (Tamanoi et al, 2001). FTIs have been demonstrated to inhibit the growth of chemically induced lung tumor with K-Ras mutations in immunocompetent mice (Lantry et al, 2000) and of human bladder, colon and fibrosarcoma tumor xenografts (Nagasu et al, 1995). FTIs have been shown to inhibit the growth of mammary and salivary tumors developed in transgenic mice bearing oncogenic H-ras under the control of the mouse mammary tumor virus (MMTV) tissue-specific promoter (Kohl et al, 1995). Other convincing results have been obtained from mice harboring multiple genetic alterations. FTIs induce tumor regression in H-ras/c-myc mice as well as in oncogenic H-ras transgenic mice that lack p53 (Barrington et al, 1998). In addition to their antitumor efficacy in animal models, FTIs lack toxicity arguing for a high therapeutic index (Kohl et al, 1995). All of these collective findings indicate effective antitumor activities of FTIs. Although FTIs were originally designated to inhibit farnesylation and activation of Ras, accumulating evidence suggesting that FTIs affect multiple signal transduction pathways independent of Ras. FTIs have been demonstrated to inhibit anchorage-independent growth of many human cancer cell types, irrespective of whether they express wild-type or mutated ras (Liu et al, 1998; Feldkamp et al, 2001). RhoB, a protein which is prenylated has been suggested to mediate response to FTI treatment (Du et al., 1999); RhoB can be farnesylated or geranylgeranylated and the anti-tumor effects of FTIs is likely to depend on the accumulation of geranylgeranylated forms of RhoB (Du and Prendergast, 1999; Prendergast and Oliff, 2000). FTIs have also demonstrated the ability to inhibit the PI3K/AKTmediated growth and adhesion-dependent survival pathways and induce apoptosis via Ras-independent mechanism (Jiang et al, 2000); the nonpeptidomimetic FTI, SCH 66336 was shown to induce apoptosis in several human head and neck squamous cancer cell lines in association with a reduction in total and phosphorylated AKT, Bcl-2 and Bcl-XL, with no change in Bax (Chun et al, 2003). A recent study suggested an important role of anitangiogenic activity for the effects of FTIs in aerodigestive tract tumors, revealing a new role for this class of drugs in cancer management (Han et al., 2005). Treatment with SCH66336 alone led to regression of orthotopic tongue tumors of human head and neck squamous cell carcinoma (HNSCC) in mice by interfering with neoangiogenesis processes. Its action seems to be mediated by inhibition of growth and migration of endothelial cells demonstrated in three-dimensional cultures and in capillary tube formation assays of HUVECs on Matrigel-coated culture plates. Furthermore,

2. Combination with chemotherapy or radiotherapy

conventional

FTIs target different downstream effectors according to hostâ&#x20AC;&#x201C;tumor interactions, histological tumor type and stage of the tumor and their anti-tumor effects are quite heterogeneous from a prominent anti-angiogenic to an anti-proliferative and an apoptotic effect in different tumors (End et al, 2001). Moreover, resistance to FTIs has been reported probably by overexpression of antiapoptotic proteins. Thus, as a single agent, FTIs appear to have modest clinical effects that are not sufficient to induce a long-term tumor inhibition. Combination with other well-chosen targeted therapy might synergize with FTIs. Pre-clinical studies confirm that FTIs can be useful in combination therapy and have showed that combination with cisplatine, taxanes or gemcitabine can improve response (Sun et al, 1999). Combination of FTI and taxanes is sustained by the fact that FTIs sensitize tumor cells to paclitaxel-induced mitotic arrest (Moasser et al, 1998). Pre-clinical findings also support the combination of FTIs and GGTase-I inhibitors (GGTIs). Whereas FTIs inhibit the farnesylation of H-Ras, they do not completely inhibit the prenylation of Ki-Ras, which remains prenylated in FTI-treated cells because of its modification by the GGTase-I. Therefore, cells transformed with KiRas tend to be more resistant to FTIs than H-Rastransformed cells. Thus combination of FTIs and GGTIs is an interesting approach for tumors harboring Ki-Ras mutations. Some highly selective inhibitors of GGTase have been conceived but no GGTI are currently in clinical trials. In fact, in contrast to the FTIs, GGTIs seem to have substantial effects on cell signaling in normal cells,

Morgillo and Lee: FTIs as anticancer agents inducing cell cycle arrest and apoptosis (Sebti and Hamilton, 1997). The combinations of SCH66336 and IGFBP-3 (Lee et al, 2004), a major serum IGF-binding protein that regulates IGF-mediated cancer cell survival and growth, has been tested. Since Ras-mediated signaling has been suggested to block IGFBP-3 activity (Martin and Baxter, 1999), blockade of Ras-mediated signaling could increase the antitumor activity of IGFBP-3. SCH66336 has also shown enhanced antiproliferative effects on several cancer cell lines but not on normal cells, when combined with PI3K inhibitors, such as LY294002 and wortmannin (Edamatsu et al, 2000); inhibitors of PI3K/AKT pathway might have unmasked the proapoptotic effects of FTIs in malignantly transformed (Du et al, 1999). Antiproliferative effects of this combination were accompanied by increased apoptosis in vitro and in vivo.

Since transformation of cells by oncogenic Ras mutants has been shown to increase the expression of some metalloproteinases, such as gelatinase and stromelysin, which are involved in tumor metastasis (Su et al, 1993), FTIs and metalloproteinase inhibitors could be a potent anti-metastatic combination. In addition, combination of FTIs with anti-angiogenic agents needs to be studied as a synergic anti-metastatic strategy after surgery in tumor with a high-expected rate of metastastic spread.

3. FTIs in clinical trials The FTIs entered clinical development, so far, are R115777 (Zarnestra), SCH-66336 (Sarasar) (Table 2), L778, 123 and BMS-214662. BMS-214662 and L-778, 123

Table 2. Clinical development of FTIs Drug

Dose

Toxicities

Clinical Outcome

R115777 (Zujewski et al, 2000)

25-1300 BID oral 5d (q14d) I mg/mq

Neurophaty, fatigue, nausea, vomiting, headache, anaemia, hypotension

1/27 SD in CRC

(Karp et al, 2001) 100-1200 BID oral 21d I mg/mq (Adjei et al, 2003) 300 BID oral 3 /4 wks II mg/mq

Neurotoxicity

20% RR

Neuthropenia, anaemia, anorexia

7 SD

(Johnston et al, 2003) (Rao et al, 2004)

3/ 26 PR 9 /26 SD No statistical significant difference in overall survival as compared to best supportive care No statistical significant difference in overall survival

300-400 mg/mq 300 mg/mq

(Van Cutsem et al, 200 2004) mg/mq

Schedule

Phase

BID oral continuous BID oral 21d (q28d)

II III

Myelosuppression, paresthesia, diarrhoea, rash, fatigue Myelosuppression, prash, diarrhea

BID oral continuously in combination with Gemcitabine

III

Neutropenia, thrombocytopenia

SCH66336 (Adjei et al, 2000) 25-400 mg/mq

BID oral 7d (q21d) I

Nausea, vomiting, diarrhea, fatigue, renal insufficiency, anorexia

1 /20 PR in NSCLC 8 /20 SD

(Winquist et al, 2005)

200 mg/mq

BID oral continuously

1 /14 SD 6 /14 PD

(Sharma et al, 2002)

200 mg/mq

BID oral continuously

Anaemia,neuthropenia, thrombocytopenia, fatigue, anorexia, nausea Fatigue, diarrhoea, nausea Diarrhoea, hyperbilirubinemia, neuropathy, neuthropenia

8 /21 PR

Thrombocytopenia, neuthropenia

9/31 PR 1 /31 CR

(Khuri et al, 2004) 100-150 BID oral I mg/mq continuously with taxol 135mg/mq (Theodore et al, 100-150 BID oral with II 2005) mg/mq Gemcitabine 1000 mg/mq

3 SD

Cancer Therapy Vol 5, page 15 are administrated intravenously, whereas the two other agents, R115777 and SCH-66336, are given orally with different schedules. Dose-limiting toxicities have included myelosuppression, gastrointestinal disorders, peripheral neuropathy and fatigue. Because of cardiac conduction abnormalities, the clinical development of L-778, 123 has been discontinued. The results from Phase I studies are encouraging. R115777 has given evidence of clinical activity in a minority of patients including those with nonsmall-cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic cancer (Zujewski et al, 2000). In phase II study, it has been evaluated as single agent in advanced breast cancer achieving a response rate of 11% and disease stabilization in 35% of patients (Johnston et al, 2003). Other trials have been conducted in patients with malignant glioma and haematological malignancies and interesting results have been documented (Kurzrock et al, 2003). A phase III study has been conducted in patients with advanced refractory colorectal cancer who had failed two prior chemotherapy regimens. Unfortunately, median survival was comparable if R1115777 (Zarnestra) or placebo was administered (Rao et al, 2004). The activity of R1115777 has been tested also in hematologic malignancies; a Phase I trial of R1115777 in patients with refractory or relapsed Acute Myeloid Leukemia (AML) or Acute Lymphocytic Leukemia (ALL), blast phase Chronic myeloid Leukemia (CML), or high-risk previously untreated AML has been reported by Karp et al. (Karp et al., 2001). On a total of 34 patients, the response rate was of 29%, including two complete responses (CR). Interestly, eight of the 25 AML treated patients responded, including 3 of 6 with newly diagnosed AML, and 5 of 19 with refractory or relapsed disease, indicating a specific activity of the drug in this setting of patients. SCH66336 is orally active on a BID-dosing schedule. The MTD varied based on the schedule but seems to be included in the range 25-400 mg BID. In the first phase I study with SCH66336, of 20 patients treated, 1 NSCLC patient experienced a Partial Response (PR) and remained in the study for 14 months. Disease stabilization (8/20) were also described for 5-10 cycles. SCH66336 showed to be able to inhibit FTase at clinically relevant dose by using prelamin A as surrogate marker for inhibition of FTase (Adjei et al, 2000). In the phase II study in transitional cell carcinomas previously failing chemotherapy, myelosuppression was dose limiting with patients experiencing additional toxicities of fatigue, anorexia, nausea, vomiting, confusion, dyspnoea and dehydration. Despite significant toxicities, no responses were observed (Winquist et al, 2005). BMS-214662 has been investigated in phase I single agent studies as infusion over 1 h every 3 weeks and evidence of activity was observed by tumor regressions in patients with colorectal and breast cancer. In addition a reduction in tumor size greater than 40% occurred in 1 patient with NSCLC. Furthermore, several other patients had disease stabilisation for up to 10 cycles (Ryan et al, 2004). In a recent phase I study, preliminary evaluation of the efficacy demonstrated a minor response in one patient with nonâ&#x20AC;&#x201C; small cell lung cancer with bony metastases. Stable disease, as best protocol response, was reported in 16

(64%) of 25 patients. Of these, 15 experienced disease stabilization for a period longer than 12 weeks. A prolonged stabilization of disease was reported for seven patients with thyroid carcinoma (Papadimitrakopoulou et al, 2005). L-778, 123 is a peptidomimetic inhibitor of FT that is administered as a continuous infusion. Despite showing responses in head/neck and NSCLC, clinical investigations have been discontinued due to the evidence of cardiac conduction abnormalities, manifested as a prolongation of the QTc interval. At a clinical level, many studies of FTIs and standard cytotoxic agents are ongoing. Toxicities appear manageable with evidence of clinical activity in heavily pre-treated patients, some of whom had been resistant to the given cytotoxic as a single agent. Combination of SCH66336 with paclitaxel has been reported (Khuri et al, 2004). The most common toxicities were myelosuppression and diarrhea. Promising preliminary evidence of efficacy was documented with 8/21 patients demonstrating PR. Responses were encouraging also in another study of SCH66336 combined with gemcitabine in patients with advanced urothelial tract cancer: 9/31 patients had partial responses and 1/31 had a complete response achieving an overall response rate of 32.3% (Theodore et al, 2005). On the other side, no benefit is reported with the combination of gemcitabine and R115777, in pancreatic cancer (Van Cutsem et al, 2004). In parallel, combination of FTI with radiotherapy is under investigation. Ras oncogenes have been reported to confer resistance to ionizing radiation (McKenna et al, 1990; Kim et al, 2004; Cengel and McKenna, 2005). Pre-clinical data have demonstrated that FTIs are radiation sensitizers in selected cell lines. Tumors bearing wild-type (CohenJonathan et al, 1999) or mutated H-Ras (Bernhard et al, 1998) can be sensitized to !-radiation by FTIs. Based on the known radioresistance of hypoxic cells, the oxygenation of tumor xenografts in nude mice after treatment with another FTI, L744, 832, has been evaluated. Results indicated that FTI treatment markedly improved the oxygenation of xenografts from tumor cell lines with H-ras mutations. In contrast, xenografts from tumors without ras mutations had equivalent hypoxia regardless of the treatment. These results suggested that FTI treatment might be useful in the radiosensitisation of tumors with H-ras activation (Cohen-Jonathan et al, 2001). One phase I study combining gamma radiation with the FTI L778, 123 demonstrated that such a combination was feasible with minimal toxicity. More importantly, 2 CR in head and neck cancer and 1 CR in NSCLC were observed (Hahn et al, 2002). FTIs are a promising class of novel antineoplastic agents. As single agents have significant activity in myeloid leukemias, but in solid tumors their activity seems to be modest and these drugs probably need to be studied in combination with cytotoxic agents, ionizing radiation and other novels targeted drugs, such as antiangiogenic agents.

Morgillo and Lee: FTIs as anticancer agents not for cytoskeletal organization. J Biol Chem 21, 14814-14818.

Acknowledgements This work was supported by National Institutes of Health grants RO1 CA100816-01 and R01 CA109520-01 (to H-Y. Lee) and American Cancer Society grant RSG04-082-01-TBE 01 (to H-Y. Lee).

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Cancer Therapy Vol 5, page 19 Cancer Therapy Vol 5, 19-28, 2007

Treatment of vaginal ıntraepithelial neoplasia Review Article

Vedat Atay*, Murat Muhcu, Ahmet Cantu! Çalı"kan GATA Haydarpasa Hospital, Department of Obstetrics and Gynecology, Istanbul, Turkey

__________________________________________________________________________________ *Correspondence: Vedat Atay, MD, Associate Professor of Obstetrics & Gynecology, GATA Haydarpasa Hospital, Dept. of Obstetrics and Gynecology, Kadikoy, 34668, Istanbul, Turkey; Tel: +90 216 3459187; Fax: +90 216 3459177; E-mail: vedatatay@hotmail.com Key words: Vaginal intraepithelial neoplasia, Human papilloma virus, Cervical intraepithelial neoplasia (CIN), Partial vaginectomy, Therapeutic vaccine Abbreviations: Cervical intraepithelial neoplasia (CIN); Human papilloma virus (HPV); Loop electrosurgical excision procedure (LEEP); Vaginal intraepithelial neoplasia (VAIN) Received: 11 December 2006; Accepted: 23 Decemmber 2006; electronically published: January 2007

Summary Vaginal intraepithelial neoplasia (VAIN) is a rare disorder that, in most instances, will regress after initial treatment. Multifocality of VAIN is a significant risk factor for recurrence. Patients with VAIN require careful long-term monitoring after initial therapy, because risk of recurrence and even progression to invasion. Treatment should be individualized depending on the location, number of lesions, and previous treatments. Future therapies will be directly or indirectly antiviral, targeting HPV protein functions or enhancing the ability of the immun system to resolve infection or inducing apoptosis indirectly in HPV-infected cells.

Cure is defined as the cytologic and histologic eradication of VAIN. The term remission defined as no abnormal cytology or examination, with at least one negative cytologic evaluation and examination and at least 4 months of follow-up after treatment. While diagnosis of VAIN within 12 months after primary treatment is called as residuel disease, recurrent disease is defined as diagnoses at a later time after primary treatment. The term progression and regression are two ends of VAIN follow up while first one is used for the diagnoses of invasive cancer, the second one is used for resolving the lesion without any treatment during the follow up period. The cure rates for first-line, subsequent, and overall treatment methods are analyzed and the overall cure rate is used to compare their effectiveness. In the published literature on VAIN many studies have focused on single treatment modalities and have included small numbers of patients with short follow-up periods. So that they have little value according to evidence based medicine The purpose of this review is to evaluate the management of women with VAIN.

I. Introduction The first report of VAIN was reported by Graham and Meigs in 1952 (DiSaia and Creasman, 2002). VAIN is thought to be a precursor to malignant disease. High risk types of HPV (16,18 and 33 HPV types) are present in all patients affected by VAIN. This finding is to suggest the HPV impact in the etiology of this neoplasia (Minucci et al, 1995; Sugase and Matsukura, 1997). Histological grading of VAIN is done by the use of similar criteria to that used for cervical intraepithelial neoplasia (CIN). VAIN 1 is characterized by only slight nuclear and cellular atypia and preservation of stratification. Evident nuclear and cellular atypical chages are seen in the case of VAIN 2 and VAIN 3. VAIN 3 is demonstrated by complete loss of stratification. On the other hand, VAIN is categorized as low-grade (VAIN 2 and 3) and high-grade (VAIN 3). The histological classification of VAIN had been originated from that of CIN. The natural history of VAIN was supposed to be similar to that of CIN altough there were only a few limited information. Two reports addressed the pre-malignant potential of VAIN. The confirmation of epidemiological and molecular similarities between patients with VAIN and vaginal cancer leading authors conclude the malignant potential of the preceding lesion (Punnonen et al, 1989; Brinton et al, 1990). Therefore gynecologists manage abnormal cervical cytology i.e. colposcopy, biopsy and treatment similiar to that of their cervical counterpart.

II. Insidence VAIN is a rare clinical condition, accounts for approximately 0.4% of lower genital tract intraepithelial neoplasia (Cramer and Cutler, 1974). The incidence of VAIN is found to be 0.2-0.3 and 0.42 per 100.000 women, respectively, in the United States (Henson and Tarone, 1997). The incidence of VAIN is expected to rise due to 19

Atay et al: Treatment of vaginal 覺ntraepithelial neoplasia the more common use of cytologic screening and colposcopy and also increased knowledge about this unique entity. VAIN is more common amoung the patients aged more than 60 years (Hummer, 1970). The mean interval from hysterectomy to the diagnosis of VAIN is 8 years (range 60-120 years).

IV. Natural history of VAIN Progression rate of VAIN to invasive form is about 5%. This rate is significantly higher than the progression rate of patients treated for CIN. Progression to cervical cancer after conisation occurs in 0,3 % of patients with cancinoma in situ (Coppleson et al, 1992). The reason for this difference could be either orginates from different biologic behaviour or less effective treatment and followup for VAIN than for CIN. Little information exist on the natural history of VAIN. In a limited study of 23 patients with VAIN, 2 (9%) of the patiets showed progression to invasive cancer, 3 (13%) persisted in the same position, and 18 (78%) regressed spontaneously during a followed up period for 3 years without treatment. It must be emphasized that in this limited study only five of these women had a grade 3 lesion (Aho et al, 1991). Additionally, in a report on 32 patients undergoing upper vaginectomy for VAIN 3, 9 patients (28%) were found to have an underlying invasive cancer (Bornstein and Kaufman, 1988). On the other hand, spontaneous remissions after diagnostic biopsies or simple excision of the lesions have been reported. In the study reported by Rome and colleagues, the results of patients with low-grade VAIN showed 12% progression potential if left untreated (Rome and England, 2000). And also, VAIN has the pottential of spontaneous regression without any treatment. The use of of invasive treatment methods to treat low-grade VAIN is not appropriate but choosing treatment protocols which are non-invasive, having minimal side effects and low costing can be reasonable and worthwhile. For this reason, some medications reported such as topical application of 5fluorouracil, trichloroacetic acid 50%, or imiquimod 5% creme for treating low grade VAIN (Krebs, 1989; Buck and Guth, 2003; Hao Lin et al, 2005). It has been reported that untreated VAIN 3 lesions had the 20% risk of progression to invasive vaginal carcinoma within the 3 years (MacLeod et al, 1997; Aho et al, 1991) According to many reported studies the rate of development of invasive vaginal cancer varied between 3 and 12% among patients treated for VAIN (Sillman et al, 1997). Therefore, it is mandatory to eradicate of grade VAIN whenever it is diagnosed, inorder to reduce mortality from invasive cancer.

III. Risk factors for developing VAIN A number of risk factors were found by various studies for development of VAIN, including a history of previous abnormal Papanicolaou smear, genital warts, radiotherapy, immnosupression and history of or concominant CIN or cervical cancer (Gallup and Morley, 1975; Bowen-Simpkins and Hull, 1975; Muram et al, 1982; Brinton et al, 1990; Aho et al, 1991; Sillman et al, 1997). Radiotherapy is known to be one of the risk factors for VAIN. A shorter latency for VAIN in the patients with cervical malignancy treated by hysterectomy and subsequent radiation with respect to non-radiated patients was observed, but VAIN degree in the former group was higher than in the latter (Audet-Lapointe et al, 1990; Sillman et al, 1997). Having a treatment for a genital truct neoplasia is seen over half of patients with VAIN (Aho et al, 1991; Sherman and Paul, 1993). VAIN is frequently associated with invasiv or intraepithelial cervical neoplasia. Exposure of same carcinogenetic agents of cervix and vagina is a convincing reason for this association. Because of less susceptibility of vaginal epithelium to carcinogenesis than cervix, neoplasia develops in vaginal epithelium only in exceptional circumstances. Reports have suggested that 1% or 2% of patients will have vaginal recurrence after hysterectomy for a similar lesion of cervix. Although several studies suggested that between 20% to 40% patients with vaginal cancers have had hysterectomy for benign disease, (DiSaia and Creasman, 2002) routine cytological follow-up is not necessary for patients who have had a hysterectomy for benign disease (U.S. Preventive Services Task Force. Screening, 2003). Furthermore current guidelines suggest to continue with an indefinite follow-up with Pap smears after hysterectomy for an intraepithelial or an invasive lesion of the cervix and vulva. The need for screening after hysterectomy with the indication of CIN is a matter of debate because the likelihood of vaginal cancer is low but real. Careful inspection and colposcopic evaluation of the upper vagina before hysterectomy for cervical cancer and intraepithelial neoplasia should be done for coexisting VAIN because VAIN is an entity that could be preventable or detectable at an early stage. Some vaginal cancers might originate from occult foci of neoplastic epithelium buried in the vaginal vault. Therefore vaginal vault should not be closed in patients with cervical neoplasia. Additionally, three normal smears over a 10-year period is demanded for a satisfactory follow up in hysterectomized patients with an indication of CIN. On the other hand, HPV vaccines might eliminate persistent HPV infection and eliminating the need for screening guidelines of lower genital tract neoplasia, in the near future.

V. Diagnosis Even postcoital spotting or unusual vaginal discharge can be the symptoms, most of the patients are asymptomatic. The first sign of a possible neoplasia is usually an abnormal Pap smear from vagina in a posthysterectomized patient. Patients who on evaluation of the cervix for an abnormal Pap smear have no identifiable lesion either on the portio or the endocervix and on further evaluation will be found to have disease in the vagina. Additionally, vaginal neoplasia should be considered if postmenopausal posthysterectomized patient who is not sexually active, has the symptom of vaginal bleeding. After the diagnoses by an abnormal Pap smear, it is crucial to identify lesion or lesions in the vagina. It has been reported that most VAIN patients have no visible

Cancer Therapy Vol 5, page 21 lesions. The follow-up of abnormal Papanicolaou smear usually needs colposcopic evaluation in order to identify abnormal areas for issue biopsy. Colposcopy, which has been effective in identifying lesions on the cervix, can also be used in the vagina, with some handicaps due to the lenght, surface area, and redundancy of the vagina, so vaginal colposcopy is much more difficult and timeconsuming procedure. The most helpful tool before colposcopic evaluation is the use of local estrogen in postmenopausal patient with abnormal Pap smear. This examination is performed using an acetic acid difficult to visualize the angles of the vaginal apex owing to redundancy or tunneling, it is crucial to inspect the entire vaginal vault. After no aceto-white changes are identified, the use of Lugol’s staining is recommended by authors in the vaginal mucosa. Lugol’s staining of the vagina, which seems to be nonspesific for the cervix, can be very helpful in identifying nonstaining areas in the vagina. Biopsy specimen of aceto-white and hypostaining areas should be obtained during colposcopy. While aceto-white epithelial areas are seen in all of the VAIN cases, vascular changes can be seen in almost 50% of VAIN cases. Sometimes colposcopically a raised, white, well-defined area can be identified. Unfortunately, sometimes vaginal epithelium “buried” above the vaginal apex at the time of vault closure following hysterectomy, (Gallup and Morley, 1970; Hoffman et al, 1989) so that neoplastic lesion within the vault scar may remain unnoticed at the time of colposcopy. VAIN lesions may also be noted with the naked eye and increased vascularization such as punctation may also be seen. The identified lesions may be very small and restricted to the upper vagina, particulary at the vault area in posthysterectomized patients. The upper third of the vagina is the place of location for the majority of lesions (95,8 %) and also, lesions mainly locate at the apex with a multifocal manner (75%) in most of the cases (Lenehan et al, 1986).

multicentric low genital intraepithelial neoplasia (Ait Menguellet et al, 2006). The term multicentric disease refers to coexistence of CIN with VAIN and/or VIN. In multicentric disease mostly lesions involve two sites, only less than 10% of them involve the three sites. High recurrence rates are reported for multicentric lesions because of inadequancy of current treatments.

VII. Treatment of VAIN The management of women with VAIN remains contraversial because most studies about VAIN had relatively small and inadequate number of patients for a conclusion (Krebs, 1989; Petrilli et al, 1980; Caglar et al, 1981; Capen et al, 1982; Jobson and Homesley, 1983; Curtin et al, 1985; Lenehan et al, 1986; Brinton et al, 1990; Aho et al, 1991; Hoffman et al, 1991; Hoffman et al, 1992; Fanning et al, 1999; Robinson et al, 2000) , did not confirm the pathologic diagnosis, (Benedet and Sanders, 1984; Lenehan et al, 1986), and did not define the outcome measured for determining success of treatment clearly (Petrilli et al, 1980; Capen et al, 1982; Hoffman et al, 1991; Fanning et al, 1999). The overall recurrence rate was reported to be 10- 42% in the literature (Petrilli et al, 1980; Caglar et al, 1981; Capen et al, 1982; Jobson and Homesley 1983; Curtin et al, 1985; Lenehan et al, 1986; Krebs, 1989; Brinton et al, 1990; Aho et al, 1991; Hoffman et al, 1991; Hoffman et al, 1992; Fanning et al, 1999; Robinson et al, 2000). There are lots of treatment alternatives which have various success rates and associated complications. These treatment options are including local excision, partial or total vaginectomy, loop electrosurgical excision procedure (LEEP), chemosurgery including excisional therapies; laser vaporazation, topical 5-flourourocil, or topical trichloroacetic acid as an ablative therapy. There are various advantages and disadvantages of each treatment option. There were no randomized trials exist in the literature, and making a direct comparison of these studies is difficult owing to selection bias and different evaluation techniques. The treatment choice for a patient with VAIN is made by the light of various clinical factors such as number of the lesions, location, previous radiation therapy, previous VAIN treatment, sexual activity, in patients with recurret VAIN, previous treatment modality, and operator experience and patient preference. Even though most lesions regress after initial treatment, some recur, sometimes repeatedly, and may progress to invasiv cancer. Recurrence is more often for Grade 3 disease, multifocal lesions, and VAIN associated with other anogenital neoplasia. Many investigators have found partial vaginectomy, 5-FU, and CO2 laser to be associated with 0- 20%, 7- 20% and 0- 42% recurrence rates, respectively (Petrilli et al, 1980; Caglar et al, 1981; Capen et al, 1982; Jobson and Homesley, 1983; Curtin et al, 1985; Lenehan et al, 1986; Krebs, 1989; Brinton et al, 1990; Aho et al, 1991; Hoffman et al, 1991, 1992; Fanning et al, 1999; Robinson et al, 2000), did not confirm the pathologic diagnosis, (Benedet and Sanders, 1984; Lenehan et al, 1986). Attention must be paid on length of follow up periods while comparing the results of therapies among different studies because the length of follow up period

VI. Risk factors of recurrence and progression to carcinoma Dodge and colleagues reported multifocality and the method of treatment as to be statistically significant risk factors among the multiple risk factors assessed for their influence on recurrence (Dodge et al, 2001) Sillman et al also found that mulfifocality and multicentrality to be significant risk factors for persistence or recurrence (Sillman et al,1997). Multifocal lesions are more difficult to treat than unifocal lesions because some lesions could be missed during treatment (Benedet and Sanders, 1984; Hoffman et al, 1991). The increment in the rate of development of invasive vaginal cancer will be expected if the follow-up periods are lengthen. Factors associated with development of invasive vaginal cancer among women with VAIN are unknown. In literature, few studies have been published about multicentric intraepithelial neoplasia (Spitzer et al, 1989; Beckmann et al, 1991; Stanbridge and Butler, 1983; Planner and Hobbs, 1987). Ait Menguellet and colleagues reported that 4,4 % of the patients with CIN presented

Atay et al: Treatment of vaginal Äąntraepithelial neoplasia influence the success and recurrence rates. For example studies reporting high success rates may have relatively short follow-up periods. Beside the effect of time interval; size, number and diffusion of lesions are the other factors that must be kept in might while evaluating different studies. For example multiple or extensive lesions are more difficult to fully visualize and treat than small focal lesions.

were followed-up as an outpatient for evaluation with Papanicolaou smears every 3 to 4 months for at least 1 year, then every 6 months for 1 year, and then yearly thereafter unless recurrence. Recurrence was defined as VAIN on a colposcopic directed biopsy. Follow-up was available in 52 patients; 46 (88%) remain without recurrence at a mean follow-up of 25 months. In this study, upper vaginectomy was efficacious for the treatment of VAIN. Twenty-three (22%) patients had negative findings on final pathologic examination, and squamous cell carcinoma was found in 12% of patients in the region of the scar at the vaginal apex. Atrophy of the vagina may have played a role in negative pathology, as it is recognized that severe atrophy can be a complicating factor in diagnosing dysplasia. In addition, biopsy and/or regresion of VAIN can contribute to this finding. Some of these lesions were not grossly apparent, may be small, and therefore, may be easily missed by the pathologist. The cure rate of upper vaginectomy has varied from 68% to 88% which was the highest rate among all the treatment modal ities according to multipl studies (Hoffman et al, 1989,1992; Benedet and Sanders, 1984; Fanning et al, 1990; Hoffman et al, 1992; Diakomanolis et al, 2002a,b; Indermaur et al, 2005). A technique of upper vaginectomy accomplishes removal of the entire vault scar. Colposcopic evaluation of the entire vaginal vault should be performed before operation. The entire vaginal vault scar and angles must be included in the surgical specimen. After these landmarks are noted, the resection line is scrartched with the electrocautery, maintaining a margin of 5 to 10 mm.The posterior vaginal mucosa is grasped with clamps and separated from underlying rectum toward thje vaginal vault until the peritoneum is identified and enter the peritoneum, in an effort to ensure complete removal of the upper vaginal mucosa. After that vaginal mucosa anteriorly is placed on traction and separated from the bladder. If there is difficulty identifying this plane, the operatorâ&#x20AC;&#x2122;s finger can be placed around the specimen to improve traction and provide beter exposure. Once this space is development, gentle blunt separation will help isolation of the lateral attachments consist of uterosacral and cardinal ligament remnants and require serial division and ligation. Once the specimen is removed, peritoneal edge to the vaginal mucosa is closed with a running interlocked suture. If the vaginal epithelium is closed, vaginal lenght may be compromised, and recurrent VAIN may be missed in the buried scar. Upper vaginectomy has been considered to be the treatment of the choice for apical VAIN grade 3, mainly because it provides specimen for complete histopatologic review (Benedet and Sanders, 1984; Hoffman et al, 1989, 1992). A 28% prevalance of an occult invasive squamous cell carcinoma in the upper vaginectomy specimens was observed by Hoffman et al (Hoffman et al, 1992) Preoperatively undiagnosed superfically invasive carcinoma cases can all be adequately treated with upper vaginectomy without any further treatment. Some small studies support the treatment of superficially invasive squamous cell carcinoma with upper vaginectomy (Peters et al, 1985; Eddy et al, 1990; Indermaur et al, 2005).

A. Surgical treatment 1. Biopsy- local excision In some instance, small lesions can be removed entirely with biopsy or simple excision. Local excision of the involved area, which have 64-67% remission rates according to different studies, is the treatment of choice when there is a single well-located small lesion in many patients (Benedet and Sanders, 1984; Sillman et al, 1997). The authors suggested that subsequent inflammation and exfoliation of the abnormal epithelium after the excision or biopsy might have a contribution between the clearance of VAIN.

2. Partial upper vaginectomy Partial vaginectomy seems to be the best treatment option in unifocal lesions or multifocal lesions involving the upper third of the vagina by the advantage of providing a surgical specimen to assess the possibility of invasive vaginal cancer and to control the resection margins because invasive vaginal cancer can be found occacionally among women with VAIN III of the vaginal vault. (Hoffman et al, 1992). On a review of 124 smears and 70 corresponding biopsies in 45 women diagnosed to have VAIN, the authors reported a cyto-histologic discrepancy of more than 2 grades in 30% of cases (Sherman and Paul, 1993). This caused concern over the use of ablative therapy in the treatment of VAIN. Recently, more authors realized the impotance of exclusion of occult invasion and reverted to the use of excisional therapies. Partial upper vaginectomy which can be performed via transvaginally or transabdominally is associated with minimal postoperative pain and no vaginal irritation (Curtis et al, 1992; Ireland and Monaghan, 1989). Partial vaginectomy has been called the standart treatment. But it also involves complications like hemorrhage, bladder or rectum injury, and shortening or stenosis of vagina. Even tough topical chemotherapy and laser vaporization are less time consuming treatment modalities and can treat multifocal lesions, they have more and undesired complictions such as more vaginal irritation and they do not allow for a tissue diagnosis. Indermaur and colleagues, evaluated the use of upper vaginectomy for the treatment of VAIN, retrospectively (Indermaur et al, 2005). 60% of patients with VAIN had a history of previous hysterectomy for either CIN or carcinoma. Thirty-six patients had undergone an upper vaginectomy for grade 2 to 3 VAIN. Before the procedure, colposcopic-directed biopsy was performed to confirm that lesions were confined to the upper one third of the vagina. Ten percent had intraoperative complications. Four patients had postoperative complications. Patients

Cancer Therapy Vol 5, page 23 As a result, surgical extirpation as partial or total vaginectomy has been considered to be the most effective method of eradicating VAIN (Petrilli et al, 1980; Lenehan et al, 1986; Curtis et al, 1992). Furthermore, this method offers the opportunity for complete histological evaluation of the disease. However, there are disadvantages of vaginal shortening, stenosis and obliteration and morbidity associated with surgical procedures (Petrilli et al, 1980; Lenehan et al, 1986). Moreover, partial or even total vaginectomy does not guarantee against recurrence (Petrilli et al, 1980; Lenehan et al, 1986; Curtis et al, 1992; Sillman et al, 1997).

treated, 12 (34%) developed abnormal cytology, which of three had residual VAIN 3, five had recurrent VAIN 3, and four had invasive cancer diagnosed. Authors concluded that surgical complications of wide local excision could be severe, particularly in irradiated patients and should only be performed in experienced centers.

B. Ablative treatment of VAIN Multifocal lesions and lesions involving the lower one part of the vagina are more commonly treated with less invasive techniques than vaginectomy in reducing or avoiding operative morbidity, obtaining minimal anatomic and sexual function disruption. They may also preferable in patients who were previously irradited or have a shortened vagina.

3. Total vaginectomy Total vaginectomy is not a prefered procedure because vaginal intercourse becomes impossible without a graft and also it stil has the potential of recurrence and progression (Sillman et al, 1997).

1. Topical 5-FU Topical 5-FU has provided good results and prolonged remission periods, previously (Petrilli et al, 1980; Woodman et al, 1984). It is thought to be an ideal method for multifocal VAIN and recurrences. Vaginal pain and burning, piruritis, and ulcerations are major problems. Because of these complications it is difficult to obtain patient compliance (Petrilli et al, 1980; Lenehan et al, 1986; Krebs HB. 1989). Especially, it is difficult to tolarate for postmenopausal patients with atropic vaginas. Sillman et al reported a cure rate of 70% after a single initial treatment of chemosurgery, and an 82% cure rate in patients with recurrence (Sillman et al, 1985). Upper vaginectomy and chemosurgery which consists of topical 5-FU and upper vaginectomy, are comperative in cure rates therefore, addition of topical 5-FU to the upper vaginectomy may be unnecessary. The poor compliance, associated morbidities, and the potentially buried epithelium are the resons for low rate of success of treatment with 5-FU. Chemosurgery could be recommended for low-grade lesions at little risk of progression to invasiv cancer (Sillman et al, 1997).

4. LEEP excision procedure Disadvantages of partial upper vaginectomy is requirement of significant surgical expertise because of proximity of the bladder and rectum, time-consumption and significant hemorrhage.(Hoffman et al, 1992). Advantage of LEEP to partial upper vaginectomy over laser is the presence of a histologic specimen. LEEP partial upper vaginectomy can be performed as an outpatient surgical procedure. An electrical current is steam envelope in the tissue during the excision procedure. For this reason, 0. 5% lidocaine with epinephrine is injected directly beneath the vaginal mucosa producing a wheal to separate the subvaginal tissue from the bladder and rectum. The procedure results in minimal lateral tissue damage similar to the effects of the laser. Excision consist of the vaginal mucosa and a portion of the submucosal tissue. The distal circumference is sent separately for pathologic examination as the surgical margin. The coagulation ball is not used. The vaginal apex is closed anteriorly to posteriorly in many times because of postoperative hemorrhage. Fanning et al experienced LEEP excision procedure in 15 patients, and reported a 30 minute mean operative time, with minimal blood loss and a 7% complication rate (Fanning et al, 1999). In this study, although, none of the low-grade vaginal intraepithelial lesions received additional treatment, patients with histologic evidence of high-grade VAIN in the LEEP specimen received postoperative intravaginal 5-FU cream. The 5-FU was administrated as 1. 5 gr intravaginally once a week for 10 week begining 4 to 6 weks postoperatively. LEEP partial upper vaginectomy can be performed quicker than cold knife partial upper vaginectomy with less blood loss and fewer complications. However, this can be proven only by a prospective, randomized study.

2. CO2 laser vaporization The use of CO2 laser vaporization for the treatment of VAIN has been reported especially for lesions in other localization than apex and for the need of sexual function prevention in young sexually active women. Being an outpatient procedure, association with minimal blood loss and suitable usage for multifocal lesions are some advantages of laser. Potantial problems with laser therapy including bleeding and damage to the bowel or bladder. It is also likely that some of the VAIN lesions could be missed. VAIN localized in vaginal vault is a challenging indication for laser therapy. Many authors have cautioned against the use of laser in treatment of VAIN III lesions in the posthysterectomize vaginal vault because of the possibility of missing invasive cancer (Hoffman et al, 1991). Laser ablation for VAIN requires expensive equipment and expertise. Even tough the adequate depth of ablation with CO2 laser is 1. 5 mm, in a study of histologic evaluation of biopsies in patients with VAIN involved epithelium ranged from 0. 1 to 1.4 mm in

5. Wide local excision Danny et al, studied the usage of wide local excision of the vaginal vault in the treatment of VAIN 3, retrospectively (Chang et al, 1999). In this study, wide local excision is found to be efficious in treating high grade VAIN, with 66% of patients remeaning disease free at a median follow up of 44 months. Of the 35 patients 23

Atay et al: Treatment of vaginal 覺ntraepithelial neoplasia thickness. The success rate of laser vaporization various in the literature, with many studies reporting high recurrence rates. Hoffmann et al, reported a 42% of recurrence in 26 patients, 3 (11,5%) of whom had invasive carcinomas at the time of recurrence and they did not recommend CO2 laser alone for the treatment of high-grade VAIN, especially when it is located in the region of the vaginal vault scar (Hoffman et al, 1991). Lenehan et al, found laser ablation less effective then electrocautery and vaginectomy (Lenehan et al, 1986). Moreover, MacLeod et al, and Stuart et al demonstrated invasive disease at the time of recurrence in 7. 1 and 3. 7% of the patients respectively (Stuart et al, 1988; MacLeod et al, 1997). Laser vaporization of the vagina in 14 patient with VAIN resulted in a recurrence rate of 57%, possibly as a result of neoplastic epithelium buried above the vaginal vault or recessed in the vaginal angles (Petrilli et al, 1980). The inaccessible location of some VAIN lesions, extension into vault tunnels or buried in the vault scar after hysterectomy appeared to be the likely cause of therapeutic failure and also lesions in deep vaginal angles after hysterectomy are technically difficult to treat with laser. For this reason, an accurate appraisal of the extent and severity of the disease must be made before therapy. These findings discourage the use of CO2 laser for the treatment of VAIN (Woodman et al, 1984; Diakomanolis et al, 1996). Laser ablation should not be performed unless the full extent of abnormal vaginal epithelium can be visualized, there is no suspicion of an invasion, and there is no gross scarring or distortion of the vaginal vault. It can be suggested that in selected patients VAIN appeared to be adequately treated with CO2 laser, but after this therapy all patients should be closely follow-up for persistance, recurrence, or progression to invasion in long term. Ablative surgery may induce scarring that can compromise the diameter of the vagina and interfere with coital function.

D. Treatment modalities for eradicating etiologic factor 1. Topical Imiquimod 5% Cream All of the already mentioned therapies aim to treat the lesion itself instead of treating the etiologic factor which is underlying HPV. Imiquimod , an imidazoquinoline heterocyclic amine is an immune response modifier that regulates the immun system, fighting with the etiologic agent of VAIN, the HPV (Miller et al, 1999). The drug makes its action by mimicing the natural response of the organism to a viral infection, resulting thus in the production of cytokines (Sidky et al, 1992). Cytokines are known to stimulate the induction of natural killer cells, which exhibit a cytotoxic action to the HPV-infected and the carcinogenic cells (Gibson et al, 1995). According to the kinetics of imiquimod cytokines are detected in 1- 2 hours following application of the cream, and also the maximum production is detected approximately 8 hours later. The exact time that cytokines are maintained in high levels is not known although some authors belive that they may reach a 16-week period (Tyring et al, 1998). The purpose of Imiquimod cream therapy is to stimulate the immun system to resist HPV and not treat its results. Buck et al, repoted the use of imiquimod 5% cream therapy for treating low grade VAIN. According to this report a very low dosing regimen of imiquimod 5% cream (0.25 mg, once weekly for 3 weeks) seems to be an effective with well toleration by 85% of patients with low grade VAIN (Buck and Guth, 2003) Although biopsy which is performed before treatment could be a contributing factor of clearance rate, a very low dosing regimen of imiquimod 5% cream in the treatment of VAIN is the high rate of sustained clearance with its simplicity of usage and the relative absence of associated adverse events. Haidopoulos et al, evaluted the treatment of highgrade VAIN lesions with local application of imiquimod 5% cream in their study (Haidopoulos et al, 2005). The conclusion of this study emphasized that local imiquimod 5% cream therapy should not be considered as permanent; young, HPV-positive patients with multifocal high-grade VAIN not involving the vaginal vault or with previous treatment failures might be reserved for a rather radical therapy and gain some extra time of normal sexual life. However, after the imiquimod cream, closely follow-up should be necassary to make it certain so that no cases of invasive vaginal cancer are missed.

C. Irradiation therapy In irradiation therapy, success rate approaching from 86% to 100%, but a complication rate of nearly 36% including moderate-to-severe vaginal stenosis, rectal bleeding, and premature ovarian failure and subsequent neoplasia make it less acceptable for use in younger patients. Especially, it is contrindicated in previously irradiated patients because of its high local morbidity. Based on the associated toxicity, brachytherapy should be appropriate for patients who are no longer sexually active and for whom vaginal function is not a concern (Gallup and Morley, 1975; Ogino et al, 1998; HernanandezLinares et al, 1980). Moreover, persistent VAIN or progression to invasiv carcinoma is reported even after high-dose-rate brachytherapy. (MacLeod et al, 1997). It should be reserved for patients in whom other ablative methods are unsuccesful or not technically possible, or for patients unsuitable for surgical treatment (Lenehan et al, 1986; MacLeod et al, 1997).

2. Therapeutic vaccine The examination of biopsies from high-grade VAIN revealed the existance of HPV DNA with a rate of 80100% (Sugase and Matsukura, 1995; van Beurden et al, 1997). Therapeutic effect of vaccine against HPV is to induce systemic cell-mediated immune response in order to effect the local cytotoxicity against the virus-infected cell. The recombinant vaccinia for virus expressing HPV16 and HPV- 18 E6 and E8 (TA-HPV) has been described (Borysiewicz et al, 1996; Boursnell et al, 1996; Kaufmann et al, 2002). Boosting HPV-spesific cell-mediated immune responses may reduce progression to carcinoma and could

Cancer Therapy Vol 5, page 25 the lower genital tract. Eur J Obstet Gynecol Reprod Med Biol, in press. Audet-Lapointe P, Body G, Vauclair R, Drouin P, Ayoub J (1990) Vaginal intraepithelial neoplasia. Gynecol Oncol 36, 232-239. Baldwin PJ, van der Burg SH, Boswell CM, Offringa R, Hickling JK, Dobson J, Roberts JC, Latimer JA, Moseley RP, Coleman N, Stanley MA, Sterling J (2003) Vaccinia expressed human papillomavirus 16 and 18 E6 and E7 as a therapeutic vaccination for vulval and vaginal intraepithelial neoplasia. Clin Cancer Res 9, 5205-5213. Beckman AM, Acker R, Christiansen AE, Sherman KJ (1991) Human papillomavirus infection in women with multicentric neoplasia. Am J Obstet Gynecol 165, 1431-1437. Benedet JL, Sanders BH (1984) Carcinoma in situ of the vagina. Am J Obst Gynecol 148, 695-700. Bornstein J, Kaufman RH (1988) Combination of surgical excision and carbon dioxide laser vaporization for multifocal vulvar intraepithelial neoplasia. Am J Obstet Gynecol 158, 459-464. Borysiewicz LK, Fiander A, Nimako M, Man S, Wilkinson GWG, Westmoreland D, Evans AS, Adams M, Stacey SN, Boursnell MEG, Rutherford E, Hickling JK, Inglis SC (1996) A recombinant vaccinia virus encoding human papillomavirus types 16 and 18 E6 and E7 proteins as immunotherapy for cervical cancer. Lancet 347, 1523-1527. Boursnell ME, Rutherford E, Hickling JK, Rollinson EA, Munro AJ, Rolley N, McLean CS, Borysiwicz LK, Vousden K, Inglis SC (1996) Construction and characterisation of a recombinant vaccinia virus expressing human papillomavirus proteins for immunotherapy of cervical cancer. Vaccine 14, 1485-1494. Bowen-Simpkins P, Hull MG (1975) Intraepithelial vaginal neoplasia following immunosuppressive therapy treated with topical 5-Fu. Obstet Gynecol 46, 360-362. Brinton LA, Nasca PC, Mallin K, Schairer C, Rosenthal J, Rothenberg R, Yordan E Jr, Richart RM (1990) Case-control study of in situ and invasive carcinoma of the vagina. Gynecol Oncol 38, 49-54. Buck HW, Guth KJ (2003) Treatment of vaginal intraepithelial neoplasia (primarily low grade) with imiquimod 5% cream. J Low Genit Tract Dis 7, 290-293. Caglar H, Hertzog RW, Hreshchyshyn MM (1981) Topical 5fluorouracil treatment of vaginal intraepithelial neoplasia. Obstet Gynecol 58, 580-583. Capen CV, Masterson BJ, Magrina JF, Calkins JW (1982) Lase therapy of vaginal intraepithelial neoplasia. Am J Obstet Gynecol 142, 973-976. Cheng D, Ng TY, Ngan HY, Wong LC (1999) Wide local excision (WLE) for vaginal intraepithelial neoplasia (VAIN). Acta Obstet Gynecol Scand 78, 648-652. Coppleson M, Atkinson KH, Dalrymple JC (1992) Cervical squamous and glanduler intraepithelial neoplasia. Gynecol Oncol 20, 83-110. Cramer D, Cutler S (1974) Incidence and histopathology of malignancies of the female genital organs in the United States. Am J Obstet Gynecol 118, 443-460. Curtin JP, Twiggs LB, Julian TM (1985) Treatment of vaginal intraepithelial neoplasia with the CO2 laser. J Reprod Med 30, 942-944. Curtis P, Shepherd JH, Lowe DG, Jobling T (1992) The role of partial colpectomy in the management of persistent vaginal neoplasia after primary treatment. Br Obstet Gynaecol 99, 587-589. Di Saia PJ, Creasman WT (2002) Clinical Gynecologic Oncology. 6th ed. St Louis, Mo, Mosby. Diakomanolis E, Rodolakis A, Boulgaris Z, Blachos G, Michalas S (2002a) Treatment of vaginal intraepithelial neoplasia with

lead to disease clearance. Baldwin et al, suggested that the vaccine might have an effect on HPV-positive vulvar and VAIN. In their study, five of 12 (42%) patients showed at least a 50% reduction in total lesion diameter over 24 weeks with 1 patient showing complete regression of her lesion. Overall, 83% of women showed some improvement with an avarage decrease in lesion size of 40%. All cases showed an increase in IgG titer as a T-cell response to the vaccinia virus (Baldwin et al, 2003). Nowadays, the effect of vaccination on HPV infection and HPV associated neoplastic disease is less clear. Usage of vaccination as a therapy is limited to only small numbers of patients with high risk HPV dysplasia or carcinoma, so that dramatic clinical responses have not been occured. However, many authors sugget that stimulation of the immun response in high risk HPV-infected individuals may be to produce at least a partial effect on neoplastic disease.

VIII. Follow-up after treatment Long term follow-up is necassary because of the high risk of residual or recurrent disease. Follow-up included periodical clinical examinations, cytology and colposcopy at 3 months, at 6 months and after 12 months for the first year and once a year subsequently (Gemmell et al, 1990.) Frega et al reported that positivity to high-risk HPV was found significantly higher in the patients with relaps to VAIN (Frega et al, 2006, in press). They suggest to perform a (positive) high-risk HPV DNA test 6 months after treatment because they found it to be more predictive than abnormal cytology. Diagnostic accurancy for HPV DNA test in the prediction of VAIN persistence: Sensitivity at 90%, specificity at 78%, positive predictive value at 56%, and negative predictive value at 92%. Frega et al suggest that performing high-risk HPV DNA may improve the current guidelines on the follow-up of patients with VAIN in addition to cytological diagnosis since it can indicate VAIN persistance/ progression to vaginal carcinoma before cytology becomes abnormal.

IX. Conclusion Upper vaginectomy as a reasonable safe and efficacious procedure is the treatment of choice for women with VAIN in the region of the vaginal vault, especially when hysterectomy was done for CIN or carcinoma. To minimize complications and morbidities, it is useful to refer the occasional case of VAIN, particularly in an irradiated patient, to experienced centers for management. Future therapies will be directly or indirectly antiviral, targeting HPV protein functions or enhancing the ability of the immun system to resolve infection or inducing apoptosis indirectly in HPV-infected cells.

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From left to right: Ahmet Cantu! Çalı"kan, Vedat Atay, Murat Muhçu

Atay et al: Treatment of vaginal 覺ntraepithelial neoplasia

Cancer Therapy Vol 5, page 29 Cancer Therapy Vol 5, 29-34, 2007

Thermo-chemo-radiotherapy for advanced pancreatobiliary cancer Research Article

Terumi Kamisawa1,*, Atsutake Okamoto2, Katsuyuki Karasawa3, Tsuneo Sasaki4 1

Department of Internal Medicine, Department of Surgery 3 Department of Radiology 4 Department of Chemotherapy, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, Japan 2

__________________________________________________________________________________ *Correspondence: Dr. Terumi Kamisawa, Department of Internal Medicine, Tokyo Metropolitan Komagome Hospital, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo, Japan; Tel: +81-3-3823-2101; Fax: +81-3-3824-1552; E-Mail: kamisawa@cick.jp Key words: hyperthermia, chemotherapy, radiotherapy, pancreas cancer, gallbladder cancer, bile duct cancer Abbreviations: complete regression (CR); no change (NC); partial regression (PR); percutaneous transhepatic biliary drainage (PTBD); radiofrequency (RF); thermo-chemo-radiotherapy (TCRT) Received: 12 December 2006; Revised: 9 February 2007 Accepted: 26 February 2007; electronically published: March 2007

Summary The majority of gallbladder, bile duct, pancreas, and cholangiocellular cancers are unresectable at the time of clinical presentation because of involvement of major vessels near the tumor or because of distant metastases, and active treatment is sometimes overlooked for patients with unresectable tumors. To improve the treatment results, thermo-chemo-radiotherapy (TCRT) was performed for locally advanced pancreatobiliary cancers (Stage III or IV): gallbladder cancer (n=32), extrahepatic bile duct cancer (n=9), pancreas cancer (n=4), and cholangiocellular cancer (n=2). Radiofrequency capacitive hyperthermia was administered simultaneously with chemotherapeutic agents once weekly immediately following radiotherapy at 2 Gy. We administered heat to the patient for 40 minutes after the tumor temperature had risen to 42°C. The chemotherapeutic agents employed were cis-platinum (50 mg/m2) in combination with 5-fluorouracil (800 mg/m2) or methotrexate (30 mg/m2) in combination with 5fluorouracil (800 mg/m2). Number of heat treatments ranged from 2 to 11 sessions (mean±SD, 4.7±2.7). Total dose of radiotherapy ranged from 16 to 90 Gy (49.5±4.3). With respect to tumor regression, CR rate and CR+PR rate were 16% and 63% in gallbladder cancer, and 0% and 0% in pancreas cancer and cholangiocellular cancer. As for resolution of bile duct obstruction, CR rate and CR+PR rate were 30% and 75% in gallbladder cancer, and 33% and 67% in bile duct cancer. Mean survival and 1-year survival rate were 9.5±6.4 months (mean±SD) and 31% in gallbladder cancer; 13.0±10.7 and 67% in bile duct cancer; 4.7±1.4 and 0% in pancreas cancer; and 4.8±0.8 and 0% in cholangiocellular cancer. No major side effects occurred. At autopsy, marked hyalinization or fibrosis with necrosis extensively replaced bile duct tumor and wall, in which suppressed cohesiveness of cancer cells and degenerative cells were sparsely observed. We prefer TCRT as a new medical approach for patients with locally advanced gallbladder and bile duct cancer.

the tumor or because of distant metastases, and active treatment is sometimes overlooked for patients with unresectable tumors (Donohue et al, 1990; Okamoto et al, 2004; Miwa et al, 2006). Extrahepatic bile duct carcinomas are typically slowgrowing and locally invasive tumors which spread along nerves and invade adjacent vascular structures (Suzuki et al, 1989). Complete resection is sometimes difficult due to local extension which occurs perineurally, via lymphatic channels, by subepithelial spread in the duct wall, and by direct

I. Introduction Gallbladder cancer, pancreas cancer, and cholangiocellular cancer are the most difficult malignancies to detect at an early stage; at diagnosis, the tumor has generally already invaded adjacent organs or major vessels. Consequently, surgeons are generally involved with an admittedly limited patient group. The majority of tumors are unresectable at the time of clinical presentation because of involvement of major vessels near

Kamisawa et al: Thermo-chemo-radiotherapy for advanced pancreatobiliary cancer between those who underwent TCRT and chemo and/or radiation.

invasion of adjacent blood vessels (Sakamoto et al, 1998). If not resected, these pancreatobiliary cancers have a dismal prognosis (D’Anglica et al, 2004; Okamoto et al, 2004). Hyperthermia has been used in combination with radiation therapy and chemotherapy, and is considered to be effective for certain type of tumors (Arcangeli et al, 1983). We have conducted a clinical trial of regional hyperthermia in combination with chemoradiotherapy for advanced pancreatobiliary cancers including gallbladder, pancreas, extrahepatic bile duct, and intrahepatic cholangiocellular cancers (Okamoto et al, 1996; Kamisawa et al, 2005). In this study, clinical effectiveness of thermo-chemo-radiotherapy (TCRT) for these locally advanced pancreatobiliary cancers was evaluated.

B. Thermo-chemo-radiotherapy techniques

The heating equipment was a radiofrequency (RF) capacitive heating device, Thermotron RF-8 (Yamamoto Vinita Company, Osaka, Japan). With the patient in the prone position, the target was sand witched with upper and lower electrodes, and an 8 MHz RF wave was applied. We administered heat to the patient for 40 min after the tumor temperature had risen to 42°C. It took about 10-20 minutes to get 42°C in tumor. Tumor temperature was measured continuously using a needle thermosensor on each heating. The thermosensor was inserted beside the tumor from the skin surface through an 18G angiocatheter under ultrasonographic guidancy. Skin surface of the patient was cooled by circulating water. Whole treatment time was about 1 hour. The chemotherapeutic agents employed were cis-platinum (50 mg/m2) in combination with 5-fluorouracil (800 mg/m2) in 20 cases or methotrexate (30 mg/m2) in combination with 5-fluorouracil (800 mg/m 2) in 27 cases. The hyperthermia and chemotherapeutic agents were administered simultaneously once weekly immediately (generally within 15 minutes) following radiotherapy at 2 Gy (Figure 1). Number of heat treatments ranged from 2 to 11 sessions (mean±SD, 4.7±2.7). Total dose of radiotherapy ranged from 16 to 90 Gy (49.5±14.3). Three cases of gallbladder cancer and two cases of bile duct cancer were retreated.

II. Methods A. Study patients TCRT was performed in 32 patients with gallbladder cancer, 9 with extrahepatic bile duct cancer, 4 with pancreas cancer, and 2 with intrahepatic cholangiocellular cancer. According to the pTNM system proposed by the American Joint Committee on Cancer (AJCC, 2002), the cancers were radiologically confirmed as Stage III (n=38) or Stage IV (n=9) tumors (Table 1). In patients with unresectable gallbladder cancer, mean survival and 1-year survival rate were compared

Table 1. Patient characteristics and pTNM staging.

Gallbladder cancer Bile duct cancer Pancreas cancer Cholangiocellular cancer

No. of patients

Male/Female

32 9 4 2

9/23 8/1 2/2 2/0

Average Age (years) 63.4 69.4 62.3 66.7

T4, M0

T4, M1

23 9 4 2

9 0 0 0

Table 2. Effectiveness of thermo-chemo-radiotherapy for gallbladder, bile duct, and cholangiocellular cancer.

Gallbladder cancer (n=32) Gallbladder cancer (n=20) Bile duct cancer (n=9) Pancreas cancer (n=4) Cholangiocellular cancer (n=2)

Tumor regression CR PR NC 5 15 12

Resolution of the bile duct CR PR NC 6 3

0 0

(TCRT)

0 0

Figure 1. Schedule of thermo-chemo-radiotherapy.

4 2

9 3

5 3

Cancer Therapy Vol 5, page 31 Side effects were evaluated and graded according to National Cancer Institute Common Toxicity Criteria (NCI-CTC, 1989). Life table survival probabilities were calculated using the Kaplan-Meier method. The generalized Wilcoxon test was used to assess the difference in survival rates.

C. Response and toxicity criteria The effectiveness of TCRT on nonresectable gallbladder, pancreas, and cholangiocellular cancers was evaluated in terms of tumor regression on follow-up CT. Tumor regression was graded as complete regression (CR: more than 80% tumor volume reduction), partial regression (PR: 50%-80% regression), and no change (NC) (Figure 2A, B). The effectiveness of TCRT for resolution of biliary obstruction in bile duct cancer and gallbladder cancer with biliary involvement was evaluated by cholangiography. Response of obstructed bile ducts was graded as complete regression (CR: more than 80% resolution), partial regression (PR: 50%-80% resolution), and no change (NC) (Figure 3A, B). At autopsy, we histologically examined the gallbladder in 11 patients with gallbladder cancer and the bile duct in 3 patients with bile duct cancer treated with TCRT.

III. Results A. Effectiveness of TCRT With respect to tumor regression due to TCRT, among the 32 patients with gallbladder cancer, 5 achieved CR, 15 PR, and 12 NC, while NC was recorded in all 4 patients with pancreas cancer and all 2 patients with cholangiocellular cancer. CR rate and CR+PR rate were 16% and 63% in gallbladder cancer, and 0% and 0% in pancreas cancer and cholangiocellular cancer.

Figure 2. On CT scan, large gallbladder cancer with thickening of the gallbladder wall (A) was markedly reduced after thermo-chemoradiotherapy (B).

Figure 3. On cholangiography, completely obstructed bile duct due to bile duct cancer (A) was resolved after thermo-chemoradiotherapy (arrows) (B).

Kamisawa et al: Thermo-chemo-radiotherapy for advanced pancreatobiliary cancer As for resolution of bile duct obstruction, among the 20 patients with gallbladder cancer involving the bile duct, 6 achieved CR, 9 PR, and 5 NC; among the 9 patients with bile duct cancer, 3 achieved CR, 3 PR, and 3 NC. CR rate and CR+PR rate were 30% and 75% in gallbladder cancer and 33% and 67% in bile duct cancer (Table 2). The percutaneous transhepatic biliary drainage (PTBD) tube could be removed when placing a self-expandable metallic stent into the patency-restored bile duct after TCRT in 4 patients with gallbladder cancer and in 3 patients with bile duct cancer. Mean survival and 1-year survival rate were 9.5±6.4 months (mean±SD) and 31% in gallbladder cancer; 13.0±10.7 and 67% in bile duct cancer; 4.7±1.4 and 0% in pancreas cancer; and 4.8±0.8 and 0% in cholangiocellular cancer. Mean survival and 1-year survival rate of patients with unresectable gallbladder cancer treated with chemo and/or radiation were 5.7±4.2 and 11%. A significant improvement of long-term survival was exhibited in patients treated with TCRT compared with those treated with chemo and/or radiation (p<0.01).

in early death from cholangitis. Hence, one of the desirable strategies for these advanced cancers appears to be controlling the involvement of the hepatic hilar bile duct and the hepatoduodenal ligament. The effectiveness of TCRT in gallbladder and bile duct cancer in the present study was noted. In respect to regression of gallbladder cancer, CR rate and CR+PR rate were 16% and 63%. As for resolution of bile duct obstruction, CR rate and CR+PR rate were 30% and 75% in gallbladder cancer and 33% and 67% in bile duct cancer. Moreover, the PTBD tube could be removed when placing a self-expandable metallic stent into the patencyrestored bile duct after TCRT in 4 patients with gallbladder cancer and 3 patients with bile duct cancer. A significant improvement of long-term survival was exhibited in patients with gallbladder cancer treated with TCRT (9.5±6.4 months)compared with those treated with chemo and/or radiation. The mean survival months in the patients treated with TCRT was also better than 4.2 months of those treated with radiation reported in the literature (Todoroki, 1997). It therefore appears that involvement of the hepatoduodenal ligament can be controlled by TCRT in many patients with gallbladder cancer or bile duct cancer. Two biological interactions with radiation, a radiosensitizing effect (Kim et al, 1982; Arcangeli et al, 1991) and a direct cytotoxic effect on tumor cells (Overgaard, 1977) have been suggested to be involved in the mechanism of hyperthermia. When the target lesion is heated to around 42°C, the cancer killing effect of radiation or anticancer drug is enhanced (Song, 1984). Experimental studies strongly suggest that the extracellular pH of the tumor and a hypoxic environment sensitize tumor cells to direct heat killing (Lin et al, 1992). Hyperthermia in combination with radiotherapy for superficial malignancies has contributed to a better rate of complete response and reduction in the rate of recurrence when compared with radiotherapy alone (Perez et al, 1989). Data on hyperthermia for deep-seated tumors were still preliminary, because heating and thermal measurement techniques had not been established. However, the effectiveness of TCRT for deep-seated tumors has recently been revealed by prospective randomized studies (Harima Y et al, 2000; Van der Zee et al, 2000). Sugimachi and his colleagues (Sugimachi et al, 1988) established the treatment protocol of TCRT and demonstrated its effectiveness. They treated esophageal carcinoma with TCRT and showed significant improvement in the clinical effectiveness and 5-year survival ratio. Anatomically, the gallbladder and bile duct are located near the abdominal wall, which is advantageous to heating and thermal measurement. Thus, it seems that TCRT could be a favorable treatment strategy for advanced gallbladder and bile duct cancer. On the other hand, TCRT demonstrated little benefit in pancreas and cholangiocellular cancer. Heating of pancreas tumors is rather difficult due to the deep location of the pancreas, and continuous heating of hepatic tumors is also difficult as a result of circulation in the liver. In conclusion, this study established the TCRT treatment protocol for advanced gallbladder and bile duct

B. Histological findings at autopsy in patients treated with TCRT Marked hyalinization or fibrosis with necrosis extensively replaced gallbladder or bile duct tumor and wall, in which suppressed cohesiveness of cancer cells and degenerative cells were sparsely observed. Many lively cancer cells invaded adjacent organs outside the treatment field. However, most lively cancer cells were detected in the peripheral area inside the treatment field.

C. Complications of TCRT Complications of TCRT were nausea and vomiting (Grade 1-2, 25 cases), gastritis (Grade 2, 11 cases), leukocytopenia (Grade 2, 8 cases; Grade 3, 2 cases; Grade 3, 1 case), gastric or duodenal ulcer (Grade 2, 2 cases), fistula due to tumor necrosis (2 cases), thrombocytopenia (Grade 1, 1 case), and hemobilia from ruptured pseudoaneurysm of the hepatic artery (1 case). These complications were successfully treated conservatively.

IV. Discussion The majority of gallbladder, pancreas, and cholangiocellular cancers are unresectable at the time of clinical presentation because of involvement of major vessels near the tumor or because of distant metastases (Donohue et al, 1990; Okamoto et al, 2004; Miwa et al, 2006). In bile duct cancer particularly located near the hepatic bifurcation, and in gallbladder cancer involving the hepatoduodenal ligament, complete resection is sometimes difficult due to local extension. As deeply invasive bile duct cancers are apt to spread the intrahepatic bile duct or the connective tissues in the hepatoduodenal ligament with encasement of major vessels, cancer cells that spread to the ligament often cannot be removed completely, even when dissection of the tissue is performed (Suzuki et al, 1989). In nonresected cases, tumor involving the hepatic hilus frequently triggers obstructive jaundice which cannot be controlled, resulting

Cancer Therapy Vol 5, page 33 Overgaard J (1977) Effect of hyperthermia on malignant cells in vivo. A review and hypothesis. Cancer 37, 2637-2648. Perez CA, Gillespie B, Pajak T, Hornback NB, Emani B, Rubin P (1989) Quality assurance problems in clinical hyperthermia and their impact on therapeutic outcome: a report by the radiation therapy oncology group. Int J Radiat Oncol Biol Phys 16, 551-558. Sakamoto E, Nimura Y, Hayakawa N, Kamiya J, Kondo S, Nagino M, Kanai M (1998) The pattern of infiltration at the proximal border of hilar bile duct carcinoma: a histologic analysis of 62 resected cases. Ann Surg 227, 405-411. Song CW (1984) Effect of local hyperthermia and blood flow and microenvironment: a review. Cancer Res 44, 47214730. Sugimachi K, Matsuda H, Ohno S, Fukuda A, Matsuoka H, Mori M, Kuwano H (1988) Long term effects of hyperthermia combined with chemotherapy and irradiation of the treatment of patients with carcinoma of the esophagus. Surg Gynecol Obstet 167, 319-323. Suzuki M, Takashima T, Ouchi K, Matsuno S (1989) The development and extension of hepatohilar bile duct carcinoma. A three-dimensional tumor mapping in the intrahepatic biliary tree visualized with the aid of a graphics computer system. Cancer 64, 658-666. Todoroki T (1997) Radiation therapy for primary gallbladder cancer. Hepatogastroenterology 44, 1229-1239. Van der Zee, Gonzalez Gonzalez D, van Rhoon GC, van Dijk JD, van Putten WL, Hart AA (2000) Comparison of radiotherapy plus hyperthermia in locally advanced pelvic tumors: a prospective randomized multicenter trial. Lancet 335, 1119-1125.

cancer. We prefer TCRT as a new medical approach for patients with these tumors when they appear to be locally advanced.

References American Joint Committee on Cancer (2002) TNM classification of malignant tumors. 6th Edition. Philadelphia: Wiley-Liss, Inc. Arcangeli G, Cividalli A, Nervi C, Creton G, Lovisolo G, Mauro F (1983) Tumor control and therapeutic gain with different schedules of combined radiotherapy and local external hyperthermia in human cancer. Int J Radiat Oncol Biol Phys 9, 1125-1134. Arcangeli G, Casale C, Colistro F, Benassi M, Lovisolo G, Begnozzi L (1991) One versus four heat treatments in combination with radiotherapy in metastatic mammary carcinoma. Int J Radiat Oncol Biol Phys 21, 1569-1574. Dâ&#x20AC;&#x2122;Angelica MI, Jarnagin WR, Blumgart LH (2004) Resectable hilar cholangiocarcinoma: surgical treatment and long-term outcome. Surg Today 34, 885-890. Donohue JH, Nagorney DM, Grant CS, Tsushima K, Ilstrup DM, Adson MA (1990) Carcinoma of the gallbladder. Does radical resection improve outcome? Arch Surg 125, 237241. Harima Y, Nagata K, Harima K, Oka A, Ostapenko VV, Shikata N, Ohnishi T, Tanaka Y (2000) Bax and Bcl-2 protein expression following radiationtherapy versus radiation plus thermoradiotherapy in stage IIIB cervical carcinoma. Cancer 88,132-138. Kamisawa T, Tu Y, Egawa N, Karasawa K, Matsuda T, Tsuruta K, Okamoto A (2005) Thermo-chemo-radiotherapy for advanced bile duct carcinoma. World J Gastroenterol 11, 4206-4209. Kim JH, Hahn EW, Ahmed SA (1982) Combination hyperthermia and radiation therapy for malignant melanoma. Cancer 50, 478-482. Lin JC, Levitt SH, Song CW (1992) Relationship between vascular thermotolerance and intratumor pH. Int J Radiat Oncol Biol Phys 22, 123-129. Miwa S, Miyagawa S, Kobayashi A, Akahane Y, Nakata T, Mihara M, Kusama K, Soeda J, Ogawa S (2006) Predictive factors for intrahepatic cholangiocarcinoma recurrence in the liver following surgery. J Gastroenterol 41, 893-900. National Cancer Institute Common Toxicity Criteria (1989) Appendix A Grading of toxicity. Manual of oncologic therapeutics. Philadelphia: J.B. Lippincott. Okamoto A, Tsuruta K, Ishiwata J, Isawa T, Kamisawa T, Tanaka Y (1996) Treatment of T3 and T4 carcinomas of the gallbladder. Int Surg 81, 130-135. Okamoto A, Matsumoto G, Tsuruta K, Baba H, Karasawa K, Kamisawa T, Egawa N (2004) Intraoperative radiation therapy for pancreatic adenocarcinoma. The Komagome hospital experience. Pancreas 28, 296-300.

Terumi Kamisawa

Kamisawa et al: Thermo-chemo-radiotherapy for advanced pancreatobiliary cancer

Cancer Therapy Vol 5, page 35 Cancer Therapy Vol 5, 35-42, 2007

Retrospective study of the influence of anaemia in patients with advanced head and neck cancer received postoperative radiotherapy Research Article

Sherif A. Abdel-Wahab1,*, Mohamed M. El-Basiouny1, Hatem M. Abdalla1, Hany M. Abdel-Aziz1, Maha Margerges1, Ali M. Azmy1, Branislav Jeremic2 1 2

Department of Radiation Oncology, Ain Shams University, Cairo, Egypt Applied Radiation Biology and Radiotherapy Section, IAEA, Vienna, Austria

__________________________________________________________________________________ *Correspondence: Dr. Sherif Ahmed Abdel-wahab, M.D, 28 Elfareek Mohamed ibrahim street, Nasr City, Cairo, Egypt; Tel: 0020101551422; Fax: 00202 6870778; Email: sherifok69@hotmail.com Key words: Anaemia, Head and Neck cancer, Haemoglobin, Radiotherapy Abbreviations: cyclooxygenase-2, (COX-2); vascular endothelial growth factor, (VEGF) Received: 24 November 2006; Revised: 31 January 2006 Accepted: 4 March 2007; electronically published: March 2007

Summary This retrospective study was conducted to investigate the impact of pre-treatment haemoglobin level on the local recurrence and overall survival in patients received postoperative radiation therapy for locally advanced head and neck cancer. 331 patients with AJCC stage III, IVA or IVB head and neck cancers were treated with postoperative radiation therapy alone with doses ranging from 60-66 Gy, (2Gy/F). Baseline (pre-radiotherapy) haemoglobin values were recorded and the outcome was measured in terms of locoregional control rates and overall survival according to it. The median age was 52.2 years and 29% of patients were women. Mean pre-treatment haemoglobin was 10.7 g/dl (8.3-13.1g/dl). Median follow up of surviving patients was 42 months, and 27% of patients had local recurrence and 52% had died. Two-and five â&#x20AC;&#x201C;year loco-regional control for anaemic patients was 71% and 64% respectively, while patients with normal pre-treatment haemoglobin levels had 2- and 5- year locoregional control of 80% and 73% respectively (p= 0.042). Two-and five â&#x20AC;&#x201C;year overall survival for anaemic patients was 64% and 48% respectively, while patients with normal pre-treatment haemoglobin levels had 2- and 5- year survival rates of 72% and 57% respectively (p= 0.021). Multivariate analysis revealed risk group and low pre-treatment Hgb to be statistically significant predictors for increased locoregional recurrence and lower survival rates. The low pretreatment haemoglobin level below 11/dl was correlated with statistically significant adverse treatment outcomes in terms of locoregional control rates as well as survival rates in patients receiving postoperative radiation therapy for advanced head and neck cancers.

that render them an ideal model for evaluating the influence of anaemia and the associated tumour hypoxia on the outcomes of postoperative radiation therapy (Shasha et al, 2000). With the controversy over the optimal management of patients; there are still evidences for better locoregional control for the patients treated with postoperative radiation therapy (Vikram et al, 1984). However, both a recent metaanalysis and several subsequent large randomized trials have demonstrated the superiority of combined chemoradiotherapy over surgery alone or radiation therapy alone being used depending on the disease stage, performance status, and patient and physician preference in patients with advanced head and

I. Introduction Head and neck carcinoma is comprised of a heterogeneous group of tumours that arise from the epithelial lining of the oral cavity, pharynx, and larynx, and it is considered as a locoregional disease (Kumar, 2000). A number of treatment options are available for patients with head and neck cancer, with single modality (surgery or radiotherapy alone) and combined modality (surgery with pre or postoperative radiation therapy, or combined chemoradiotherapy) management strategies being used depending on the disease stage, performance status, and patient and physician preference. Despite their heterogeneity, head and neck cancers share many features 35

Abdel-Wahab et al: Influence of anaemia in patients advanced H&N cancer received postoperative RT neck cancer with respect to local-regional control, diseasefree survival, and overall survival. Combined chemoradiotherapy is rapidly becoming the standard of care for the non-surgical management of locally advanced disease (Adelstein et al 2000). Haemoglobin level has been reported to be a prognostic factor in a number of malignancies treated with radiation therapy, particularly carcinoma of the uterine cervix and head and neck carcinoma. Most studies have reported low pre-treatment haemoglobin levels to be a poor prognostic factor for tumour control and/or patient survival (Chua et al, 2004). The mechanism by which anaemia worsens outcome is a multifactorial process, and it may be in part explained by low haemoglobin levels resulting in the regions of low oxygen tension within the tumour, which leads to hypoxia induced radioresistance in the tumor cell population (Molls et al, 1998). In addition, anaemia results in altered expression of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), which may influence tumour behaviour (Dunst et al, 1999). A study including 114 patients with locally advanced cancer cervix patients had showed that although the association between anemia and poor response to treatment is not retained in multivariate analysis, in that series, basal and nadir haemoglobin status was strongly prognostic for disease free survival as well as overall survival, thus suggesting that the clinical impact of anemia is more likely to reflect tumour intrinsic aggressiveness rather than tumor susceptibility to treatment per se (Ferrandina et al, 2006). The cyclooxygenase-2 (COX-2) protein is highly expressed in a variety of human cancers and has been reported to promote tumor growth. Nonsteroidal anti-inflammatory drugs such as etodolac and celecoxib have been shown to inhibit COX-2 activity and may play a role in the chemoprevention of cancer. Etodolac and celecoxib do not alter the COX-2 protein levels but inhibit its enzyme activity to reduce prostaglandin E2 production. Survivin, an inhibitor protein of apoptosis, mediates anti-apoptosis and promotes cell division in cancer cells. COX-2 inhibitors can increase the reduction of survivin protein expression, growth inhibition, and death in different human cancer cells (Lin et al, 2005). Production of basic-fibroblast growth factor (bFGF, a pro-angiogenesis factor) was inhibited by etodolac in a dose-dependent manner.Furthermore, telomerase activity was inhibited and expression of hTERT mRNA was significantly inhibited with etodolac, leading to the conclusion that anti-tumor effects of etodolac on many cancer cells are due to inhibition of both angiogenesis and telomerase activity (Hasegawa et al, 2005). Dose of 50 Gy in 25 fractions over 5 weeks (conventional fractionation) has been reported to be sufficient to control subclinical disease in the neck in 90% of patients in whom the neck has not been dissected (Fletcher 1973 and 1974). While it was found that patients who underwent neck surgery, a higher radiation dose is required up to 57.6 Gy in 32 fractions over 6! weeks (Peters et al, 1993). A Hypothesis for increased dose requirement after surgery suggests that a dissected area would be less well vascularised and, therefore, less well

oxygenated than an undisturbed tumour bed. The impact of anaemia in cases of postoperative radiation therapy may therefore be as important as in the primary radiation therapy setting. A large retrospective study done by Frommhold and co-workers in 1998, including 889 patients with head and neck cancers showed a reduction in locoregional failure with increasing pretreatment haemoglobin. The aim of this retrospective study was to determine the impact of pre-treatment haemoglobin level on the outcomes of the postoperative radiation therapy for patients with advanced stages of head and neck cancers.

II. Materials and methods A. Patient population and eligibility criteria Eligible patients had to have previously untreated, histopathogically proven squamous cell carcinoma arising from head and neck (oropharynx, nasopharynx, oral cavity, larynx, hypopharynx or paranasal sinuses (with an advanced tumour according to AJCC III, IVA or IVB, and no distant metastasis, M0). Patients eligible for this study had to be at least 18 years of age and no older than75 years, with a performance status of 0, 1, or 2, according to the WHO scale; they had to have a determined pre-treatment haemoglobin level (within two weeks prior to radiation therapy) and all patients must be treated with radiation therapy (doses of 60-66 Gy) without chemotherapy. Patients were considered anaemic if the pre-treatment haemoglobin levels were <11g/dl. Patients had to have at least six months of regular follow up after finishing their treatment. The stage of the tumour was determined on the basis of the AJCC staging criteria 2002. Metastatic work up and pretreatment investigations were done including the laboratory (haematology and chemistry) as well as the radiological investigations (chest radiography, computed tomography of the site of the primary tumour and the neck). Three hundred and thirty one patients with advanced head and neck cancers in the period from January 1995 to June 2001 were identified, and the data were retrieved on host characteristics (age at diagnosis, gender, and pre-radiotherapy haemoglobin level), tumour characteristics (tumour site, T stage, N stage, pathological grade, and extra-capsular extension from lymph nodes) and treatment â&#x20AC;&#x201C;related factors (surgical margin status, total dose, fractionation).Patients were excluded from the study if they had received preoperative radiation therapy or combined chemoradiotherapy, if the intent of treatment was palliative, if the tumour histology was not squamous cell carcinoma or if the patient had lost more than 20% of his weight.

B. Surgery Eligible patients underwent primary surgery performed with curative intent. The criteria of surgical resection of the tumour and neck dissection followed the accepted criteria for adequate excision. Involved/positive surgical margins were identified if the tumour was within 1 mm of the surgical margins.

C. Radiation therapy All eligible patients received postoperative radiation therapy consisting of conventionally fractionated doses of 60-66 Gy, 2 Gy per day in five weekly fractions. Treatments were conducted on cobalt 60 machine or linear accelerator 6 MV. Radiation therapy technique was individualized for each patient; however in general, patients were treated supine; large treatment

Cancer Therapy Vol 5, page 37 volume including the primary tumour site as well as all draining lymph nodes at risk received a dose of up to 54 Gy in 27 fractions over an overall treatment time of 5! weeks. The dose to the spinal cord was limited to 45 Gy and areas at high risk for recurrence received a boost of 12Gy. Total tumour dose was 66 Gy in 33 fractions in 6.5 weeks.

more was found in 38% of total number of patients, while 62% of them had pre-treatment haemoglobin less than 11/dl. The median follow-up time was 42 months (6-97 months).

Table 1. Patient, tumour and treatment characteristics.

D. Follow up Follow up was undertaken during treatment where the patients were examined at least weekly, and then evaluation was done every two months for the first twelve months, every four months for the next 24 months, every six months for the next two years, and annually thereafter. Complications, performance status, weight and tumour response were evaluated at baseline, weekly for the first eight weeks, and at each follow up evaluation. The first evaluation included a clinical examination and computed tomography scan for the neck. Chest X-ray and abdominal ultrasonography were performed every 6-12 months.

Characteristic Gender Male Female Age(yr) Median Range Site of primary tumour Oropharynx Nasopharynx Oral cavity Larynx Hypopharynx Sinus Tumour stage Tx T1 T2 T3 T4 Nodal stage N0 N1 N2 N3 Tumour grade Grade I Grade II Grade III Unknown Extracapsular extension Positive Negative Unknown Resection margin Positive Negative Unknown Pre-treatment haemoglobin Mean Range

E. Definition of outcomes The study outcome measures were locoregional recurrence which was defined as a histologically confirmed tumour recurring within the previously irradiated field and it was considered as the primary endpoint of the study, while overall survival (OS) was defined as the percentage of patients in the study who had survived for a defined period of time and it was calculated from the date of diagnosis to the date of death of disease or the date last seen. Overall survival was considered as the secondary endpoint of the study.

F. Statistical analysis Survival analyses were carried out using SPSS statistical software package, version 12.0. Survival curves calculated using the Kaplan-Meier method (Kaplan and Meier, 1958), and survival curves were compared in univariate analyses using the Log-rank test (two-sided P values). For multivariate analyses, a Cox regression model was used. A difference between the anaemic and non anaemic groups is considered statistically significant with (p value<0.05). Regarding the different prognostic variables, we grouped the low, and intermediate risk factors in one category (one node affection , grade I, II or negative margins)versus high-risk group (stage III, IV ,more than one or L.N>3 cm, grade 3, close or positive surgical margins).

III. Results A. Patient, characteristics

tumor

and

treatment

The records of 331 patients with advanced head and neck cancers who presented and treated with postoperative radiation therapy at the Department of Radiation Oncology and Nuclear Medicine, Ain Shams University Hospitals in the period from January 1995 to June 2001 were reviewed. Baseline patient, tumour and treatment characteristics are presented in (Table 1). Median age for patients was 52.2 years (34-71 years), and 71 % of patients were men. Primary tumour site was 31% in oropharynx, 24% in nasopharynx, 20.5% in oral cavity, 19% in larynx, 4.5% in hypopharynx and only 1% of the patients were in the paranasal sinuses. Lymph node metastasis was discovered at time of presentation in 73% of patients with haemoglobin 11 g/dl or higher and in 70% of patients with haemoglobin less than11 g/dl. Pre-treatment haemoglobin values ranged from 8.3-13.1g/dl with the mean value of 10.7g/dl. Pre-treatment haemoglobin level of 11/dl or 37

Number and percentage

235(71%) 96(29%) 52.2 34-71 102 (31%) 79 (24%) 68 (20.5%) 63 (19%) 15(4.5%) 4 (1%) 67(20%) 16(5%) 33(10%) 99(30%) 116(35%) 139(42%) 73 (22%) 63(19%) 56(17%) 30(9%) 122(37%) 106(32%) 73(22%) 99(30%) 103(31%) 129(39%) 162(49%) 145(44%) 24(7%)

10.7 g/dl 8.3-13.1g/dl

Abdel-Wahab et al: Influence of anaemia in patients advanced H&N cancer received postoperative RT patients to non anaemic patients was 2.95 (95% CI: 1.01– 7.08). For the high risk group the hazard ratio was 8.12 (95% CI: 2.01–49) to the intermediate/low risk patients. While in the multivariate analysis, for the overall survival; risk-group and pre-treatment Hgb showed to be independent prognostic factors for overall survival. The hazard ratio for anaemic patients to non-anaemic patients was 2.43 (95% CI: 1.1–71.2). For the high risk group the hazard ratio was 2.01 (95% CI: 1.05-7.2) to the intermediate/low risk patients (Table 2).

B. Locoregional control After a median follow-up time of 42 months (6-86 months) 27% of the total number of the patients enrolled into this study had developed local recurrence. The Kaplan-Meier curve for locoregional recurrence free survival for all patients showed that the actuarial 2- and 5year locoregional control rates were 65% each. Pretreatment haemoglobin significantly influenced recurrence-free survival. Anaemic patients with a low preradiotherapy haemoglobin level showed a higher level of loco-regional failure. Two-and five –year loco-regional control for anaemic patients was 71% and 64% respectively, while patients with normal pre-treatment haemoglobin levels had 2- and 5- year locoregional control of 80% and 73% respectively (p= 0.042), (Figure 1).

IV. Discussion Anaemia is well known to be prevalent in the general population and is even more common among cancer patients. It is estimated that 27%–50% of patients undergoing cancer treatment have baseline anaemia (Nissenson, 2003). In the current study anaemia was defined in relation to the cutoff levels stated by the World Health Organization (11 g/dl) (Nowrousian, 2002). In this study the median age of the patients selected was 52.2 years and the male to female ratio were in accordance with the majority of studies (Peters et al, 1993). In comparison with other publications there was an evidence of higher incidence of anaemia among patients in our study and lesser mean haemoglobin level. In our retrospective study pretreatment anaemia in patients with advanced stages of head and neck cancers we had demonstrated a great influence on the outcome of treatment with postoperative radiation therapy in terms of locoregional control and overall survival rates. This is consistent with the findings of a large number of published trials that have identified a strong correlation between haemoglobin level and the efficacy of radiation therapy in different types of malignancies such as cancer cervix uteri (Dunst et al, 2003), squamous cell carcinoma

C. Overall survival A total of 52% of patients enrolled into this study had died. The 2- and 5- year overall survival rates for all patients are 57% and 42% respectively. Pre-treatment haemoglobin was noticed to significantly influence the overall survival; where anaemic patients with a low preradiotherapy haemoglobin level showed a lower survival rate. Two-and five –year overall survival for anaemic patients was 64% and 48% respectively, while patients with normal pre-treatment haemoglobin levels had 2- and 5- year survival rates of 72% and 57% respectively (p= 0.021), (Figure 2).

D. Multivariate analysis In the multivariate analysis for recurrence, risk-group and pre-treatment showed to be independent prognostic locoregional recurrence. The hazard ratio

locoregional haemoglobin factors for for anaemic

Figure 1. Kaplan-Meier curve of Locoregional Recurrence-free survival according to the pre-treatment Hgb level.

Figure 2. Kaplan-Meier curve of overall survival according to the pre-treatment Hgb level.

Cancer Therapy Vol 5, page 39 Table 2. Multivariate analysis of variables for locoregional control and survival rates. Variable Loco-regional control Risk group Pre-treatment Hgb Overall survival Risk group Pre-treatment Hgb

Regression coefficient

p-value

95% CI

2.13 1.02

0.031 0.042

8.12 2.95

2.01-49 1.01-7.08

0.998 0.925

0.033 0.021

2.01 2.43

1.05-7.2 1.1-71.2

of head and neck (Frommhold et al, 1997), ovarian cancer (Munstedt et al, 2003) and lung cancer (Wigren et al, 1997). On multivariate analysis, the risk group as well as the low pretreatment haemoglobin level was statistically significant predictors of locoregional recurrence as well as lower survival rates. A recent metaanalysis of 60 studies estimated an overall 65% increase in risk of death in cancer patients who were anaemic (Caro et al, 2001). However, the pathophysiology of anaemia and its relation to poorer outcomes in solid tumour patients remains controversial. Many authors had proposed several possible mechanisms by which a low haemoglobin concentration may impair survival: (1) impairment of tumour oxygenation would reduce the effectiveness of chemotherapy and radiotherapy; (2) indirect resultant of decreased quality of life; and (3) inadequate amount of treatment delivered to the patient (Littlewood 2001). In addition to previously mentioned mechanisms, anaemia results in altered expression of angiogenic cytokines, such as (VEGF, !FGF, IL-8, TNF, etc), responsible in the course of various signal transduction cascades for the proliferation, migration and differentiation of the endothelial cells responsible for the development of neovasculature in a tumour. As the tumour increases in size, so do the hypoxic areas and the angiogenic signals responsible for development and metastasis increase (Dunst et al, 1999). Additionally, there is some evidence that tumour cells respond to hypoxia with the expression of a variety of genes coding for oxygen regulated proteins such as c-jun or p53 (Molls et al, 1998). Unlike other published studies we estimated the impact of a threshold level of pretreatment haemoglobin of "11gm/dl, while other studies had estimated the treatment outcomes in association with haemoglobin level of " 13 gm/dl (Canaday et al, 1999). This raises the need for a more cooperation into a multicenter study in order to better define the term anaemia from the oncologic point of view. This difference could be attributed to the lower socioeconomic status of the population in our study. Importantly, findings of about half of previously published studies suggest that the haemoglobin threshold in the radiation oncology setting should be within the range of 12-14 g/dl. Although not investigated as extensively as pretreatment haemoglobin, a decrease in haemoglobin

levels during radiation therapy also appears to negatively affect postradiation outcomes of patients with head and neck or cervical cancer (Skladowski et al, 1999). The prognostic significance of anaemia is not merely a phenomenon of radiation therapy outcomes; it appears to apply to the surgical setting as well. In a recent study, preoperative anaemia (haemoglobin level <13 g/dl and <12 g/dl in men and women, respectively) was associated with a significantly worse 5-year prognosis (60% versus 85% in patients without anaemia; p = 0.002) and high risk of treatment failure (relative risk, 3.0) in patients with surgically treated glottic squamous cell carcinomas. Furthermore, subgroup analysis of 162 male patients with T1 tumours and clean margins revealed that each 1-g/dl haemoglobin decrease was associated with a relative risk of locoregional relapse of 1.4 in male patients with T1 tumours. This finding suggests that haemoglobin may be a continuous risk factor that retains clinical relevance within the accepted range of normal. This finding is particularly interesting given the fact that early glottic cancers are quite small and not generally considered hypoxic tumours (Guttenberger et al, 1999). Degner and Sutherland found in 1988 that early correction of mild-to-moderate anaemia in the radiation oncology setting has the potential to modify the hypoxic environment of solid tumours. An increase of haemoglobin by 20% produces a theoretical decrease in hypoxic tissue volume of approximately 30%. Strategies to increase the haemoglobin level prior to radiation therapy in patients with head and neck cancer may lead to improved survival and loco-regional control. Various methods have been used in an attempt to overcome hypoxia-induced radioresistance, including hyperbaric oxygen (Overgaard, 1995), nitroimidazoles (Overgaard, 1994), ARCON (accelerated radiotherapy carbogen and nicotinamide), blood transfusion (Bush et al, 1978) and erythropoietin therapy to combat anaemia during radiotherapy. Treating anaemia with blood transfusions can be associated with problems of allo-immunisation, allergic reactions, circulatory overload, immunosuppression (Groopman and Itri 1999), short-lived elevation in haemoglobin levels (Barrett-Lee et al, 2000) and a lack of blood supply in many countries. The use of rhEPO overcomes many of these drawbacks, and has been shown to reduce transfusion requirement (Cascinu et al, 1994) and increase 39

Abdel-Wahab et al: Influence of anaemia in patients advanced H&N cancer received postoperative RT Classification and characterization of anemia in cancer (2002) In: Nowrousian MR, ed. Recombinant human erythropoietin (rhEPO) in clinical oncology. Wien, New York, Springer, 23-38. Daly T, Poulsen MG, Denham JW, et al (2003) The effect of anaemia on efficacy and normal tissue toxicity following radiotherapy for locally advanced squamous cell carcinoma of the head and neck. Radiother Oncol 2, 113-22. Degner FL, Sutherland RM (1988) Mathematical modelling of oxygen supply and oxygenation in tumor tissues: prognostic, therapeutic, and experimental implications. Int J Radiat Oncol Biol Phys 15, 391-397. Dische S (1991) Radiotherapy and anaemia; the clinical experience. Radiother Oncol 20(suppl 1), 35-40. Dubray B, Mosseri V, Brunin F, Jaulerry C, Poncet P, Rodriguez J, Brugere J, Point D, Giraud P, Cosset JM (1996) Anemia is associated with lower local-regional control and survival after radiation therapy for head and neck cancer: a prospective study. Radiology 201, 553-558. Dunst J, Kuhnt T, Strauss HG, Krause U, Pelz T, Koelbl H, Haensgen G (2003) Anemia in cervical cancers: Impact on survival, patterns of relapse, and association with hypoxia and angiogenesis. Int J Radiat Oncol Biol Phys 56, 778787. Dunst J, Pigorsch S, Hansgen G, Hintner I, Lautenschlager C, Becker A (1999) Low hemoglobin is associated with increased serum levels of vascular endothelial growth factor (VEGF) in cancer patients. Does anaemia stimulate angiogenesis? Strahlenther Onkol 175, 93-96. Fein DA, Lee WR, Hanlon AL, Ridge JA, Langer CJ, Curran WJ Jr, Coia LR (1995) Pretreatment hemoglobin level influences local control and survival of T1-T2 squamous cell carcinoma of the glottic larynx. J Clin Oncol 13, 2077-2083. Ferrandina G, Distefano M, Smaniotto D (2006) Anemia in patients with locally advanced cervical carcinoma administered preoperative radiochemotherapy: Association with pathological response to treatment and clinical outcome. Gynecol Oncol 103, 500-505. Fletcher GH (1973) In: Fletcher GH, ed. Textbook of Radiotherapy, 2nd edn. Philadelphia:Lea& Febiger. Fletcher GH (1974) Clinical dose-response curve of subclinical aggregated of epithelial cells and its practical application in the management of human cancers. In, Friedman M, Editor, Biological and clinical basis of radiosensitivity, Thomas CC, Springfield, IL, 485-501. Frommhold H, Guttenberg R and Henke M (1998) The impact of blood haemoglobin content on the outcome of radiotherapy. The Freiburg Experience. Strahlenther Oncol 174(suppl 4), 31-34. Groopman JE, Itri LM (1999) Chemotherapy-induced anemia in adults: incidence and treatment. J Natl Cancer Inst 91, 1616-1634. Guttenberger R, Lutterbach J, Beck C (1999) Anemia is associated with decreased local control of surgically treated squamous cell carcinomas of the glottic larynx. Int J Radiat Oncol Biol Phys 45(suppl 1), 203. Harrison L, Blackwell K (2004) Hypoxia and Anemia: Factors in Decreased Sensitivity to Radiation Therapy and Chemotherapy? The Oncologist (9suppl), 31-40. Harrison L, Sasha D, Shiaova L, White C, Ramdeen B, Portenoy R (2001) Prevalence of anemia in cancer patients undergoing radiation therapy. Semin Oncol 2 suppl 8, 54-9. Harrison L, Sasha D, White C, Ramdeen B (2000) RadiotherapyAssociated Anemia: The Scope of the Problem. Oncologist (5suppl), 1-7. Hasegawa K,Ohashi Y,Ishikawa K, Yasue A, Kato R, Achiwa Y, Nishio E, Udagawa Y (2005) Expression of

quality of life in cancer patients with anaemia (Case et al, 1993). The National Comprehensive Cancer Network (NCCN) guidelines recommended transfusion or erythropoietin for symptomatic patients with haemoglobin of 10â&#x20AC;&#x201C;11 gm/dl and stated that erythropoietin should strongly be considered if haemoglobin falls to less than 10 g/dl. These recommendations were based on studies that revealed an improvement in the quality of life of cancer patients, but not patient survival with anaemia correction (Mock et al 2003). Phase III studies evaluating the correction of anaemia via erythropoietin have shown mixed results with some studies reporting a decrease in patient survival despite an improvement in haemoglobin levels. Diverse functions of erythropoietin are reviewed, including its potential to inhibit apoptosis via the JAK2/STAT5/BCL-X pathway. Correction of anaemia by the use of blood transfusions has also shown a decrement in patient survival, possibly through inflammatory and/or immunosuppressive pathways (Varlotto and Stevenson, 2005).

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cyclooxygenase-2 in uterine endometrial cancer and 40

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Patel SG and Shah JP (2005) TNM Staging of the Head and Neck: Striving for Uniformity Among Diversity. Cancer J Clin 55, 242-258. Peters LJ, Goepfert H, Ang KK, Byers RM, Maor MH, Guillamondegui O, Morrison WH, Weber RS, Garden AS, Frankenthaler RA, et al (1993) Evaluation of the dose for post-operative radiation therapy of head and neck cancer: first report of a prospective randomized trial. Int J Radiat Oncol Biol Phys 26, 3-11. Sch채fer U, Micke O, M체ller SB, Sch체ller P and Willich N (2003) Hemoglobin as an Independent Prognostic Factor in the Radiotherapy of Head and Neck Tumors. Strahlenther Onkol 8, 527-34. Shasha D, George MJ, Harrison LB (2000) Once-weekly dosing of epoetin alfa increases hemoglobin and improves quality of life in anemic cancer patients receiving radiation therapy either concomitantly or sequentially with chemotherapy. Blood 96, 434a. Skladowski K, Tarnawski R, Maciejewski B, Wygoda A, Slosarek K (1999) Clinical radiobiology of glottic T1 squamous cell carcinoma. Int J Radiat Oncol Biol Phys 43, 101-106. Varlotto J, Stevenson MA (2005) Anemia, tumour hypoxemia, and the cancer patient. Int J Radiat Oncol Biol Phys 63, 2536. Vikram B, Strong EW, Shah JP, Spiro R (1984) Failure at the primary site following multimodality treatment in advanced head and neck cancer. Head Neck Surg 6, 724-729. Warde P, O'Sullivan B, Bristow RG, Panzarella T, Keane TJ, Gullane PJ, Witterick IP, Payne D, Liu FF, McLean M, Waldron J, Cummings BJ (1998) T1/T2 glottic cancer managed by external beam radiotherapy: the influence of pretreatment hemoglobin on local control. Int J Radiat Oncol Biol Phys 41, 347-353. Wigren T, Oksanen H, Kellokumpu-Lehtinen P (1997) A practical prognostic index for inoperable non small-cell lung cancer. J Cancer Res Clin Oncol 123, 259-266.

Oncol 26, 1419-28. Hong JH, Tsai CS, Chang JT, Wang CC, Lai CH, Lee SP, Tseng CJ, Chang TC, Tang SG (1998) The prognostic significance of pre- and posttreatment hemoglobin levels in patients with squamous cell carcinoma of the cervix treated by radiotherapy. Int J Radiat Oncol Biol Phys 41, 823-830. Kaplan E and Meier P (1958) Nonparametric estimation from incomplete observations. J Am Stat Assoc 53, 457-481. Kramer S, Gelber RD, Snow JB, Marcial VA, Lowry LD, Davis LW, Chandler R (1987) Combined radiation therapy and surgery in the management of advanced head and neck cancer: final report of study 73-03 of the Radiation Therapy Oncology Group. Head Neck Surg 10, 19-30. Kumar P (2000) Impact of anemia in patients with head and neck cancer. Oncologist 5, 13-18. Lee WR, Berkey B, Marcial V et al (1998) Anemia is associated with decreased survival and increased locoregional failure in patients with locally advanced head and neck carcinoma: a secondary analysis of RTOG 85-27. Int J Radiat Oncol Biol Phys 42, 1069-1075. Lin J, Hsiao PW, Chiu TH, Chao JI (2005) Combination of cyclooxygenase-2 inhibitors and oxaliplatin increases the growth inhibition and death in human colon cancer cells. Biochem Pharmacol 70, 658-67. Littlewood TJ (2001) The impact of hemoglobin levels on treatment outcome in patients with cancer. Semin Oncol 28(2suppl8), 49-53. Macdonald G and Hurman DC (2004) Influence of anaemia in patients with head and neck cancer receiving adjuvant postoperative radiotherapy in the Grampian region. Clin Oncol (R Coll Radiol) 1, 63-70. Mock V, Atkinson A, Barsevick A, Cella D, Cimprich B, Cleeland C, Donnelly J, Eisenberger MA, Escalante C, Hinds P, Jacobsen PB, Kaldor P, Knight SJ, Peterman A, Piper BF, Rugo H, Sabbatini P, Stahl C; National Comprehensive Cancer Network (2003) Cancer-related fatigue. Oncology (Williston Park) 14, 151-161. Molls M, Stadler P, Becker A et al (1998) Relevance of oxygen in radiation therapy: mechanisms of action, correlation to lo haemoglobin levels. Strhlenther Onkol 174, 13-16. Munstedt K, Kovacic M, Zygmunt M, Von Georgi R (2003) Impact of hemoglobin levels before and during chemotherapy on survival of patients with ovarian cancer. Int J Oncol 23, 837- 843. Nissenson A (2003) Anemia: Not just an innocent bystander? Arch Intern Med 163, 1400-1405. Nordsmark M, Overgaard M, Overgaard J (1996) Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck. Radiother Oncol 41, 31-39. Overgaard J (1994) Clinical evaluation of nitroimidazoles as modifiers of hypoxia in solid tumors. Oncol Res 6, 509-518. Overgaard J (1995) Modification of hypoxia-from Gottwald Schwarz to nicotinamide: have we learned the lesson? In: Kogelnik HD, ed. Progress in Radio-Oncology 5, 469-475. Padersen D, Sogaard H, Overgaard J, Bentzen SM (1995) Prognostic value of pretreatment factors in patients with locally advanced carcinoma of the uterine cervix treated by radiotherapy alone. Acta Oncol 34, 787-795.

Sherif A. Abdel-Wahab

Abdel-Wahab et al: Influence of anaemia in patients advanced H&N cancer received postoperative RT

Cancer Therapy Vol 5, page 43 Cancer Therapy Vol 5, 43-54, 2007

Concurrent chemoradiotherapy for locally advanced cervical cancer using Gemcitabine: nonrandomized comparison of three sequential protocols Research Article

Kundan S. Chufal1,*, Madhup Rastogi2, Madhu Srivastava2, M.C. Pant2, M.L.B. Bhatt2, Kirti Srivastava2 1 2

Department of Oncology, Batra Hospital and Medical Research Centre, New Delhi, India Department of Radiotherapy, King Georgeâ&#x20AC;&#x2122;s Medical University, Lucknow, U.P, India

__________________________________________________________________________________ *Correspondence: Kundan Singh Chufal, Department of Oncology, Batra Hospital and Medical Research Centre, 1, Tughlakabad Institutional Area, New Delhi 110062, India; Tel: 911165681785; Fax: 911129957661; e-mail: kundan_25@rediffmail.com Key words: concurrent chemotherapy, gemcitabine, cervix, locally advanced, cisplatin, radiotherapy Abbreviations: Disease free survival, (DFS); External beam radiotherapy, (EBRT); Gemcitabine and radiation, (CGR); High Dose Rate, (HDR); International Federation of Gynecology and Obstetrics, (FIGO); Intracavitary radiotherapy, (ICRT); Locoregional relapse free survival, (LRFS); Low Dose Rate, (LDR); National Cancer Institute, (NCI); overall survival, (OS)

Received: 20 November 2006; Revised: 2 February 2007 Accepted: 20 February 2007; electronically published: March 2007

Summary Concurrent chemoradiotherapy approach using Gemcitabine group (CGR group) was retrospectively compared with other two sequential protocols, one was radiotherapy alone (RT group) and other was concurrent cisplatin (CCR group). Total 140 patients were enrolled into these three protocols and131 patients were evaluated. (CGR group = 44 patients, CCR group = 32 patients and RT group = 55 patients). Radiotherapy was delivered to a total dose of 50Gy in 25 fractions followed by Intracavitary radiotherapy. Concurrent chemotherapy was administered weekly with either cisplatin 40 mg/m2 or Gemcitabine 300 mg/m2. In CGR group 41/44 (93.2%) patients showed complete response (CR) while it was 44/55 (80%) and 22/32 (68.8%) in RT group and CCR group respectively (p = 0.004). Toxicity in terms of neutropenia, nausea and vomiting and diarrhea was significantly higher in CGR group. Survival functions revealed no significant differences among the groups. Concurrent chemoradiotherapy with Gemcitabine in this study showed excellent response rates but it could not be translated into better survival functions probably because of toxicity which resulted in prolongation of treatment time.

There were many issues like lacunae in the design of many of these trials, inadequate eligibility criteria, use of improper control arm, toxicity etc (Saibishkumar et al, 2005; Datta and Agrawal, 2006). In spite of these issues it is used as a routine clinical practice in majority of oncology centers in our country. Results of the trial by NCI of Canada clinical trial group did not reveal any benefit with concurrent chemoradiotherapy (Pearcey et al, 2002). Metanalysis by green et al revealed significantly better survival using concurrent chemoradiotherapy as compared to radiotherapy alone (Green et al, 2006). In their subgroup analysis survival advantage was more

I. Introduction Carcinoma cervix is one of the most common cancers among rural Indian women (Stewart and Kleihues, 2003). Usually cervical carcinoma presents as a locally advanced disease with parametrial infiltration. Treatment of locally advanced carcinoma of cervix had undergone paradigm shift over the last decade. Results of five randomized trials (Keys et al, 1999; Morris et al, 1999; Rose et al, 1999; Whitney et al, 1999; Peters et al, 2000) coerced National Cancer Institute (NCI) to flash an alert regarding the use of concurrent chemoradiotherapy using cisplatin.

Chufal et al: Concurrent gemcitabine with radiation for locally advanced carcinoma cervix radiotherapy (ICRT), irrespective of cause were excluded from the analysis to reduce the disparity among the groups. A complete physical examination with pelvic examination (PE) performed by a multidisciplinary team was required to determine the clinical stage according to the International Federation of Gynecology and Obstetrics (FIGO) classification. Patients underwent chest X-ray, intravenous pyelography, computed tomography scan or ultrasonography of the abdomen and pelvis, sigmoidoscopy (when needed) and cystoscopy.

pronounced among early stage disease. Although there was the heterogeneity among the trials included in the metanalysis, author recommended Cisplatin 40 mg/m2 weekly as a chemotherapy agent to be used along with radiation. Recently gemcitabine has shown promising results in some phase I and II trials as a radiosensitising agent (Shewach and Lawrence, 1996; Lawrence et al, 1997; Latz et al, 1998; Pattaranutaporn et al, 2001; Zarba et al, 2003; Umanzor et al, 2006). Gemcitabine is activated intracellulary by deoxycytidine kinase and is converted into two active metabolites gemcitabine diphosphate and triphosphate, which target RNA and DNA. It is considered to be an attractive compound to combine with ionizing radiation for several reasons. First, as DNA synthesis inhibitors, nucleoside analogues have the potential to inhibit the repair of genomic damage induced by ionizing radiation. Second, because they are preferentially cytotoxic to proliferative cells, these analogues may decrease the number of tumor clonogen and thus slow down cell repopulation during fractionated radiotherapy. Tumor shrinkage induced by these compounds may improve tumor oxygenation and thus counter the detrimental effect of tumor hypoxia on radiation response. Third, as this compound induces an S phase arrest which is the radioresistant phase while radiation acts mostly on G1/M phase of cell cycle so both these modalities complement each other and the result is synergistic effect. Considering this attractive hypothesis, we conducted a phase II trial of concurrent chemotherapy with gemcitabine along with radiation regarding its feasibility in our patient population. To understand the overall impact of this approach we retrospectively compared it with other two sequential protocols (one was radiotherapy alone and the other was concurrent chemoradiotherapy with cisplatin) used in our institution.

A. Radiotherapy External beam radiotherapy (EBRT), 50 Gy/25 fractions, was delivered using Cobalt-60 unit with 80 cm SSD. Upper border of the pelvic field was at L4-L5 junction; lower border was at lower most part of obturator foramen, which was modified according to the vaginal extent of disease. Lateral borders were kept 1.5 cm beyond the widest pelvic brim. When four field box technique was used, the anterior and posterior borders of the lateral portals were kept at anterior part of pubic symphysis and S2-S3 junction respectively. For ICRT patients were referred to other centers within 1 to 2 weeks. At one center they applied Cesium-137 Low Dose Rate (LDR) ICRT and a dose of 25 to 30 Gy at point A was given, while at other center they used Iridium198 High Dose Rate (HDR) ICRT to a total dose of 18 Gy in three sittings or 19 Gy in two sittings (with a gap of one week between each sittings) was given.

B. Cisplatin chemotherapy Cisplatin was administered weekly for 5 weeks during EBRT, beginning on the first day of radiation. Cisplatin infusion was administered prior to radiation. A dose of 40 mg/m2 was used and administered via a peripheral vein to patients in an outpatient setting as follows: 1,000 mL of normal saline for 4 hours followed by cisplatin diluted in 500 mL of normal saline containing 62.5 mL of 20% mannitol for 1 h, followed by 1000 mL of normal saline for 4 hours. Intravenously, 8 mg of Dexametasone, 50mg of Ranitidine and 8 mg of Ondansetron were employed as antiemetic prophylaxis.

C. Gemcitabine chemotherapy

II. Materials and methods

Treatment consisted of weekly gemcitabine at a dose of 300 mg/m2 diluted in 300 ml of normal saline administered intravenously over 30 minutes prior to radiation. A total of 5 cycles were planned. Chemotherapy was withheld in case of unacceptable toxicity or progression of disease.

Forty six patients with biopsy proven squamous cell carcinoma of the cervix with stage IIB and Stage III were entered into the protocol using concurrent Gemcitabine and radiation (CGR) from august 2003 to august 2004. Two other sequential protocols were included for retrospective comparison with CGR protocol. Fifty eight patients were treated by radiotherapy alone (RT) and 36 patients with concurrent cisplatin and radiation (CCR) with same eligibility criteria as for CGR protocol from august 2001 to July 2002 and august 2002 to July 2003 respectively. Before enrollment of patients, our institutional review board and clinical research committee approved the trial. All patients gave their written informed consent. Eligibility was restricted to the patients who had not been treated with prior radiation or chemotherapy and patients other than the stage I, Stage IIA and Stage IV. Patients with disease outside the pelvis and paraaortic lymph nodes metastasis were ineligible and this was determined by contrast enhanced computed tomography or ultrasonography of abdomen and pelvis. KPS of more than or equal to 70 and no previous history of cancer were required for eligibility. Other eligibility criteria were as follows: leukocyte count >3.0 ! 109/L; platelet count >100 ! 109/L; hemoglobin "10 g/dL (after transfusion if necessary); serum creatinine level <2.0 mg/dL (177#mol/L); serum bilirubin level <1.5 ! upper normal limit; and serum aspartate aminotransferase level <3 ! upper normal limit. Patients who did not received Intracavitary

D. Treatment toxicity For safety evaluations, acute toxicity was assessed according to the NCI CTCC v 2.0, and for acute and late radiotherapy toxicity RTOG classification of adverse events was used. When hematological toxicity was more than or equal to grade III, chemotherapy and radiation both were withheld. For grade II hematological toxicity only chemotherapy was withheld while radiation was continued. When non hematological acute radiation morbidity was more than or equal to grade III, radiation was interrupted.

E. Statistical methods Statistical analysis was done by Software, SPSS for windows 10.0.1 standard version. For a phase II trial, patient characteristics, safety profile of the concurrent modality treatment administration, and response rates were characterized by descriptive methods. Locoregional relapse free survival (LRFS), Disease free survival (DFS) and overall survival (OS) curves were calculated according to the Kaplan- Meier method.

Cancer Therapy Vol 5, page 45 For LRFS all local and /or regional recurrences and deaths due to disease were taken as events, for DFS all the deaths because of disease were taken as events, while for overall survival (OS) all deaths regardless of any cause were taken as events. Log rank test was used to compare the survival differences between the different sub groups.

CCR and CGR group while in RT group majority of patients were treated with conventional anterioposterior and posteroanterior two field technique. Gap of 5 days or more was considered significant and it was significantly higher in CGR group as compared to RT group. Main reason for gap among CGR group was neutropenia, diarrhea and nausea and vomiting (Table 4). Duration of hospitalization was also higher among patients in CGR group. Mean duration of hospitalization was 4.81(+ 2.1) days, 3.76(+ 1.4) days and 4.23(+ 1.5) days among patients in CGR group, CCR group and RT group respectively (Table 4) and this difference was statistically significant.

III. Results A. Patient’s characteristics From August 2001 to August 2004 three different protocols were initiated sequentially in Department of Radiation Oncology, King George’s Medical University, Lucknow, India. Total 140 patients were enrolled into these protocols (CGR = 46 patients, CCR = 36 patients and RT = 58 patients) and finally 131 patients completed treatment and were eligible for evaluation. Among CGR group one patient expired just after starting treatment because of cardiac cause and one patient refused further chemotherapy after initial two cycles of chemotherapy. Among CCR group one patient refused ICRT treatment while two patients refused chemotherapy after first cycle of cisplatin and one patient defaulted within one week of starting treatment because of personal family reasons. In RT group two patients refused ICRT while one patient lost to follow up during treatment. Details of patient’s characteristics and treatment related parameters are given in Table 1.There were no significant differences among all the three groups of patients except for follow up duration, external beam treatment time, overall treatment time and technique of external beam radiotherapy. Gap between EBRT and ICRT was not significantly different among all the three groups. Median duration of gap was 2.4 days (95% CI 1.9, 2.9) in CGR group while it was 2.3 days (95% CI 1.7, 2.6) and 2.1 days (95% CI 1.6, 2.3) in CCR group and RT group respectively. Mean follow up for CGR group was 23.1 months (95%CI 21.4, 24.8), for CCR group it was 26.1 months (95%CI 21.1, 31.0) and for RT group it was 32.4 months (95%CI 28.0, 36.8) (p = 0.002).

D. Survival Mean follow up time for all the patients was 27.7 months (95%CI 25.4, 30.1). Overall survival for all the patients was 68.7% (95%CI 67.6, 69.8) with median overall survival time of 38 months (95%CI 26.2, 49.8). DFS, LRFS, and DMFS were 66.3% (95%CI 65.2, 67.4), 69.6% (95%CI 68.5, 70.7) and 93.6% (95%CI 92.6, 94.7) respectively (Figures 1, 2 and 3). Median DFS time was 33 months (95%CI 31.7, 38.3) while Median LRFS time and DMFS time was not reached. DMFS was 100 % at 2 year for CCR group while it was 84.5% for CGR group. This difference was statistically significant with p value of 0.024. Details of survival functions are outlined in Table 5.

IV. Discussion Treatment of carcinoma cervix has evolved immensely over the last decade. For locally advanced carcinoma cervix overall prognosis has always been somber until the shift in the treatment paradigm came with concurrent chemoradiotherapy protocol. Although recently one phase III randomized trial (Pearcey et al, 2002) has shown results against this approach, still standard treatment for patients with carcinoma cervix undergoing radiotherapy is concurrent chemoradiotherapy. The major foundation for this approach came into existence with the results of 5 randomized Phase III trials (Keys et al, 1999; Morris et al, 1999; Rose et al, 1999; Whitney et al, 1999; Peters et al, 2000) after which NCI flashed the alert that all patients with carcinoma cervix undergoing radiotherapy should be considered for concurrent chemoradiotherapy approach. The most standard approach is the use of concurrent Cisplatin 40 mg/m2 weekly along with radiation. Despite of using concurrent cisplatin along with radiation locoregional failure rate is still in the range of 30-40% (Keys et al, 1999; Morris et al, 1999; Rose et al, 1999; Whitney et al, 1999; Peters et al, 2000). There is obvious need for other approaches which could have a positive impact on the locoregional failure rate. Gemcitabine has shown promising radiosensitising effect in preclinical as well as in clinical phase II trials (Shewach and Lawrence, 1996; Latz et al, 1998; Lawrence et al, 1997; Pattaranutaporn et al, 2001; Zarba et al, 2003; Umanzor et al, 2006). There are three Phase II trials (Pattaranutaporn et al, 2001; Zarba et al, 2003; Umanzor et al, 2006) using gemcitabine alone or in combination with cisplatin concurrently with radiation (Table 6).

B. Response At the end EBRT 92(70.2%) patients had complete response, 37(28.2%) patients had partial response and 2(1.6%) patients had no response to the treatment. When the response was assessed at 3 month after completion of ICRT there was 11.5% gain in complete response rate. Overall 107(81.7%) patients had complete response, 22(16.8%) patients had partial response and 2(1.5%) patients had no response to treatment. Responses when compared among CGR group versus CCR group, it revealed statistically significant differences in terms of complete response at the end of EBRT and end of 3 month after completion of ICRT (Table 2).

C. Toxicity and treatment compliance Grade III and IV toxicities within each group are described in Table 3. Neutropenia and Dermatitis was significantly higher in CGR group while diarrhea was significantly higher in RT group. Neutropenia and dermatitis may resulted from increased radiosenstivity while increase in diarrhea was mainly because of technique of EBRT which was Four field box technique in 45

Chufal et al: Concurrent gemcitabine with radiation for locally advanced carcinoma cervix

Table1. Patients and treatment related parameters RT (n = 55) Age in years (Mean) (95% CI) Range P value *BHL in gm% (mean) (95% CI) Range P value † AWHL in gm% (mean) (95% CI) Range P value ‡ EXTT in days (mean) (95% CI) Range P value § OTT in days (mean) (95% CI) Range P value Follow up in months (mean) (95% CI) Range P value Histopathology ! Well Differentiated SCC (n = 43) Moderately differentiated SCC (n = 42) Poorly differentiated SCC (n = 24) Unknown (n = 22) P value FIGO Group staging Stage IIB Stage IIIA Stage IIIB P value Radiotherapy Technique **CTFT (n = 63) †† FFBT (n = 68) P value

RT + Cisplatin (n = 32)

45.9 (43.5, 48.2) 26 to 61

RT + Gemcitabine (n = 44)

48 (45.1, 50.9) 33 to 61 0.531 10.4 (10.4, 11.3) 7.5 to 14 0.086 12.1 11.6, 12.6 8.3 to 15 0.516 37 35.8, 38.2 35 to 47 0.000 53.1 51, 55.1 42 to 66 0.731 26.1 21.1, 31.1 3 to 51 0.002

46.3 (43.6, 49) 32 to 63

10.7 (10.3, 10.9) 7.5 to 14 12.2 11.9, 12.6 8.3 to 15 36.9 36.2, 37.7 35 to 47 54.2 52.3, 56.2 42 to 92 32.4 28, 36.8 4 to 56

10.2 (9.8, 10.6) 6.4 to 13 11.9 11.5, 12.3 8.5 to 14 43.8 41.3, 46.4 36 to 58 58.3 56.0, 60.5 44 to 76 23.1 21.5, 24.8 10 to 32

8 0.840

30 5 20

18 2 12

27 3 14 0.786

37 18

18 14

8 36 0.000

*BHL: Baseline Hemoglobin level, †AWHL: Average weekly hemoglobin level, ‡EXTT: External beam radiotherapy treatment time, ! § OTT: Overall treatment time, SCC: Squamous cell carcinoma, **CTFT: Conventional two field technique, ††FFBT: Four field box technique.

Cancer Therapy Vol 5, page 47 Table 2. Response at the completion of EBRT and at the end of 3 months after completion of ICRT Post *EBRT response

†

RT (n = 55)

RT + Cisplatin (n = 32)

RT + Gemcitabine (n = 44)

38(69.1)

18(56.2)

36(81.8)

16(29.1)

13(40.6)

8(18.2)

No response

1(1.8)

1(3.2)

0(0)

Progression

Overall Response ‡ CR (%) §

PR (%)

P Value !

Post ICRT response

Overall Response CR (%)

0.011 (RT + Gemcitabine versus RT + Cisplatin) 0.122 (RT versus RT + Gemcitabine) 0.235 (RT versus RT + Cisplatin) RT RT + Cisplatin (n = 55) (n = 32)

RT + Gemcitabine (n = 44)

44(80)

22(68.8)

41(93.2)

10(18.2)

9(28.1)

3(6.8)

No response

1(1.8)

1(3.2)

0(0)

Progression

PR (%)

P Value

0.004 (RT + Gemcitabine versus RT + Cisplatin) 0.055 (RT versus RT + Gemcitabine) 0.255 (RT versus RT + Cisplatin)

*EBRT: External beam radiotherapy, †RT: Radiotherapy, ‡CR: Complete response, §PR: Partial response, Intracavitary radiotherapy.

ICRT:

Table 3. Incidence of Grade II or III toxicities during treatment

HEMATOLOGICAL Leucopenia (%) P value Neutropenia (%) P Value Thrombocytopenia (%) P Value Anemia (%) P Value NON HEMATOLOGICAL Nausea and Vomiting (%) P Value Dermatitis (%) P Value Proctitis (%) P Value Cystitis (%) P Value Diarrhea (%) P Value

*RT / (n = 55)

RT + Cisplatin / (n = 32)

RT + Gemcitabine / (n = 44)

3 (5.4)

5 (15.6)

8 (18.1) 0.264

2 (3.6)

6 (18.7)

16 (36.3) 0.000

8 (14.5)

5 (15.6)

12 (27.2) 0.176

8 (14.5)

9 (28.1)

14 (31.8) 0.167

4 (7.2)

10 (31.2)

6 (13.6) 0.236

17 (30.9)

6 (18.7)

24 (54.5) 0.049

29 (52.7)

13 (40.6)

33 (75) 0.865

31 (56.3)

24 (75)

25 (56.8) 0.246

10 (18.1)

5 (15.6)

5 (11.3) 0.048

*RT: Radiotherapy

Chufal et al: Concurrent gemcitabine with radiation for locally advanced carcinoma cervix Table 4. Treatment compliance

Treatment breaks (> 5 days) † P value Reason for breaks Personal reasons Nausea / Vomiting Bladder Toxicity Diarrhea Neutropenia Hospitalization required Duration of hospitalization (mean value in days) † (+ Standard Deviation) § P value

*RT (n = 55) 9 (16.3)

RT + Cisplatin RT + Gemcitabine (n = 32) (n = 44) 13 (40.6) 21 (47.2) 0.043 (Pearson Chi Square)

2 (3.6) 2 (3.6) 1 (1.8) 4 (7.2) 0 (0) 13 (23.6) 4.23 (+ 1.5)

1 (3.1) 2 (4.5) 3 (9.3) 4 (9) 1 (3.1) 2 (4.5) 5 (15.2) 5 (11.3) 3 (9.3) 8 (18.2) 17 (53.1) 27 (61.3) 3.76 4.81 (+ 1.4) (+ 2.1) 0.073 (Pearson Chi Square)

*RT: Radiotherapy †P Value was significant between RT + Gemcitabine versus RT alone. ‡Mean value is for those select patients who were hospitalized in each group. §Given P Value is between RT + Cisplatin versus RT + Gemcitabine. Figure 1. Comparison of Locoregional Relapse Free Survival curve among three different sequential protocol using concurrent gemcitabine with radiation, radiotherapy alone and concurrent cisplatin with radiation respectively. There was no significant difference among these protocols.

Figure 2. Comparison of Disease Free Survival curve among three different sequential protocol using concurrent gemcitabine with radiation, radiotherapy alone and concurrent cisplatin with radiation respectively. There was no significant difference among these protocols.

Cancer Therapy Vol 5, page 49 Figure 3. Comparison of Overall Survival curve among three different sequential protocol using concurrent gemcitabine with radiation, radiotherapy alone and concurrent cisplatin with radiation respectively. There was no significant difference among these protocols.

Table 5. Survival at the end of 2 year *RT (n = 55)

RT + Cisplatin (n = 32)

RT + Gemcitabine (n = 44)

Locoregional Relapse Free Survival (LRFS) â&#x20AC;

Median LRFS time in months (95%CI) LRFS at 2 year Log Rank test p value

69%

61.6% 0.276

79.9%

30 (18.9, 41.0) 59.4% 0.670

28 (26.2, 29.7) 68.3%

Disease Free Survival (DFS) Median DFS time in months (95% CI) DFS at 2 year Log Rank test p value

34 (24.9, 43) 68.3%

Distant Metastases Free Survival (DMFS) Median DMFS time in months (95%CI) DMFS at 2 year Log Rank test p value

97.6%

100% 0.024

NR 84.5%

Overall Survival (OS) Median OS time in months (95%CI) OS at 2 year Log Rank test p value

38 (21.3, 54.6) 68.3%

37 (25.3, 48.7) 62.3% 0.590

NR 74.2%

*RT: Radiotherapy, â&#x20AC; NR: Not reached.

If we compare our results in terms of response rate and survival functions there was not much difference, though out of 3 studies, 2 have used combination of gemcitabine and cisplatin. Pattaranutaporn et al, 2001 has used concurrent gemcitabine 300mg/m2 alone concurrently with radiation. Patient numbers were scanty to draw any definitive conclusion but overall response rate was very encouraging that may transform into good survival functions in future. There were 17 (89.5%) complete

responses, one partial response and one no response. After a median follow up of 19.9 months, one year DFS was 84.2% and overall survival was 100%. In our study for CGR group median follow up time was 23.5 months (+ 5.5). There were 41 (93.2%) complete responses and three partial responses. DFS and OS at 2 year in our study was 68.3% and 74.2% respectively. Our response rates were comparable to other studies but survival functions showed disparity on comparison.

Chufal et al: Concurrent gemcitabine with radiation for locally advanced carcinoma cervix Table 6. Summary of various studies using Gemcitabine as concurrent chemoradiotherapy for carcinoma cervix Study Pattaranutapar et al [14] January to April 1999 Phase II Number of patients = 19

Eligibility Stage III B. Age < 70 years. ECOG performance scale < 2 Abdominal CECT for paraaortic node assessment was done. Disease outside pelvis not eligible.

Treatment *EBRT: 50Gy in 25 fractions 2Gy per fraction (After 30Gy central shield) † ICRT: Low Dose Rate Cesium-137, 28 to 29Gy during EBRT period. Chemotherapy: Concurrent weekly Gemcitabine 300mg/m2 5 or 6 cycles.

Toxicity Only one case of Grade III diarrhea. Grade I and II Leucopenia, anemia and diarrhea was present in 50% patients. Late Toxicity: Grade III Cystitis in one patient.

EBRT: 50.8Gy in 28 fractions 1.8Gy per fraction. ICRT: Low Dose Rate Cesium-137, 30 to 35Gy after 1 to 2 weeks of EBRT. Chemotherapy: Concurrent weekly Gemcitabine 125 mg/m2 and cisplatin 40 mg/m2.

Results Response: Complete response = 17(89.5%), Partial response = 1(5.25%) and No response = 1(5.25%) Median follow up time was 19.9 months. At the end of one year 2 residual disease and one distant metastases was present. ‡ OS = 100% at one year § DFS = 84.2% at one year. Response: Complete response = 32(88.8%), Partial response = 3(8.3%) and No response = 1(2.7%) Median follow up time was 26 months. Three year DFS = 67% and OS = 72% Note: 50% of the patients were of stage IIIB.

Zabra et al [15] July 1999 to March 2001 Number of patients Phase I = 16 Phase II = 20

Stage IIB, III and IVA. ECOG performance scale < 2. Disease outside pelvis not eligible.

Umanzoretal et [16] Phase II April 2001 to June 2003 Number of patients = 23 Evaluable = 20

Stage IIB or IIIB KPS > 70. Positive aortic and Para aortic lymph node were ineligible.

EBRT: 50Gy in 25 fractions 2Gy per fraction ICRT: After a gap of 1 to 2 weeks Low Dose Rate Cesium-137, 30 to 35Gy. Chemotherapy: Concurrent weekly Gemcitabine 125 mg/m2 and cisplatin 40 mg/m2. EBRT: 50Gy in 25 fractions 2Gy per fraction ICRT: After a gap of 1 to 2 weeks Low Dose Rate Cesium-137, 25 to 30 Gy or High Dose Rate Iridium-198, 18Gy in 3 sittings (6Gy/sittings) or 19Gy in 2 sittings (9.5Gy/sittings). Chemotherapy: Concurrent weekly Gemcitabine 300mg/m2 five cycles.

Response: Complete response = 18(90%), Partial response = 2(10%) and No response = 0 Median Follow up = 22.2 months. Two year Progression free survival = 80% and OS = 85%. Response: Complete response = 41(93.2%), Partial response = 3(6.8%) and No response = 0 Median follow up time was 23.5 months At 2 year Locoregional relapse free survival = 79.9%, DFS = 68.3%, Distant metastasis free survival = 84.5% and OS = 74.2%.

Grade 3 Neutropenia = 1 and Grade 3 diarrhea = 1. Grade 2 diarrhea = 7 and Grade 2 neutropenia = 3

This Study Phase II August 2003 to August 2004 Number of patients = 44 in concurrent gemcitabine group

Stage IIB, IIIA and IIIB KPS > 70 Positive aortic and Para aortic lymph node and Distant metastases were ineligible.

Duenas et al, 2005 in his phase II randomized study compared two preoperative concurrent chemoradiotherapy protocol followed by type III radical hysterectomy for stage IB2 to IIB and showed better pathological response with Cisplatin 40mg/m2 and Gemcitabine 125mg/m2 as compared to Cisplatin 40mg/m2. Among Cisplatin group complete pathological response rate was 55% while it was 77.5% in Gemcitabine and Cisplatin group and the difference was statistically significance (p = 0.0201). Although author has not reported survival functions and presumed that better pathological responses will result in better survival functions. In their study after a median follow up of 20 months one patient relapsed in gemcitabine and cisplatin arm at distant site while in cisplatin arm 5 patients relapsed at locoregional and distant sites. Among adverse effects leucopenia was significantly higher in gemcitabine and cisplatin group.

Grade 4 neutropenia 1/26 patients at dose level 125 mg/m 2. Grade III Diarrhea mucositis, Nausea and vomiting was present in 3/26 patients.

27 patients required hospitalization with mean duration of hospitalization = 4.81 days. 16 patients had Grade II or III neutropenia.

Similarly time to deliver chemo and EBRT time both were significantly higher among cisplatin and gemcitabine group. The results of this study bear an important implication as responses mentioned above are pathological response and it could be presumed that these responses will transform into better survival rate. One important concern is delay in delivery of chemotherapy cycles, reduction in number of chemotherapy cycles and prolongation of EBRT treatment time in cisplatin and gemcitabine group. This difference was statistically significant. These delays can compromise the survival functions in further follow up. In our study when we compared our other two sequential protocols with CGR group, response rates were definitely better in CGR group as compared to CCR group after EBRT as well as after 3 months of completion of ICRT (Table 2). Interestingly there was no statistically

Cancer Therapy Vol 5, page 51 significant difference in response rates among CCR group and RT group (Table 2). Another important point in our study is when we compared 2 year survival functions than there were no differences among these protocols (Table 4) thus better response rate in CGR group was not converted into better survival functions. Overall treatment compliance was poor in CGR group (Table 4). Hospitalization was required in 27 patients in CGR group, 13 patients in RT group and 17 patients in CCR group. Treatment breaks were significantly higher in CGR group (Table 4). Prolonging the treatment duration among the CGR group because of toxicity may be the limitation which resulted in compromise of survival functions when compared to other two groups. Also incidence of distant metastases was highest in CGR group and was statistically significant (Table 4). Probably radiosensitising dose of gemcitabine used in our study was not enough to counter the micrometastasis and this needs consideration in future phase II and phase III studies. In an overview by Datta and Agrawal in 2006, where they critically analyzed the 5 randomized Phase III clinical trial responsible for NCI alert regarding use of concurrent chemoradiotherapy concluded that further exploration is required for the use of this approach prior to incorporating it into routine clinical care especially for patients from Indian subcontinent with locally advanced cervical cancer. In there final remark they qualify that chemoradiotherapy could be a standard form of treatment for early cancer of cervix. Non randomized comparison of three sequential protocols in our study clearly revealed disease, patients and treatment related variables were equally distributed among the groups (Table 1) with no significant differences in terms of OS and LRFS while there was increased toxicity and poor DMFS in CGR group.

efficacy of concurrent gemcitabine with radiotherapy in terms of survival functions. The doubt raised in this study could only be answered in the light of Phase III randomized trial. Till then concurrent chemoradiotherapy with Cisplatin 40 mg/m2 or radiotherapy alone with intracavitary brachytherapy should be the standard treatment. Though the role of concurrent chemoradiotherapy with cisplatin in developing countries especially in locally advanced cervical cancer still needs to be established and in our study comparison of CCR group with RT group revealed no significant differences in terms of response rates or survival function. This further needs observation in future phase III trials.

References Datta NR, Agrawal S (2006) Does the evidence support the use of concurrent chemoradiotherapy as a standard in the management of locally advanced cancer of the cervix, especially in developing countries? Clin Oncol (R Coll Radiol) 18, 306-12. Duenas-Gonzalez A, Cetina-Perez L, Lopez-Graniel C, Gonzalez-Enciso A, Gomez-Gonzalez E, Rivera-Rubi L, Montalvo-Esquivel G, Munoz-Gonzalez D, Robles-Flores J, Vazquez-Govea E, de La Garza J, Mohar A (2005) Pathologic response and toxicity assessment of chemoradiotherapy with cisplatin versus cisplatin plus gemcitabine in cervical cancer: a randomized Phase II study. Int J Radiat Oncol Biol Phys 61, 817-23. Green J, Kirwan J, Tierney J, Vale C, Symonds P, Fresco L, Williams C, Collingwood M (2006) Concomitant chemotherapy and radiation therapy for cancer of the uterine cervix. The Cochrane Database of Systematic Reviews Keys HM, Bundy BN, Stehman FB, Muderspach LI, Chafe WE, Suggs CL 3rd, Walker JL, Gersell D (1999) Cisplatin, radiation and adjuvant hysterectomy compared with radiation and adjuvant hysterectomy for bulky stage IB cervical carcinoma. N Engl J Med 340, 1154-1161. Latz D, Fleckenstein K, Eble M, Blatter J, Wannenmacher M, Weber KJ (1998) Radiosenzitizing potential of gemcitabine (29,29-difluoro-29-deoxycytidine) within the cell cycle in vitro. Int J Radiat Oncol Biol Phys 41, 875- 82. Lawrence TS, Eisbruch A, Shewach DS (1997) Gemcitabinemediated radiosensitizer. Semin Oncol 24(2suppl7), 24-8 (review). Morris M, Eifel PJ, Lu J, Grigsby PW, Levenback C, Stevens RE, Rotman M, Gershenson DM, Mutch DG (1999) Pelvic radiation with concurrent chemotherapy compared with pelvic and para-aortic radiation for high-risk cervical cancer. N Engl J Med 340, 1137-1143. Pattaranutaporn P, Thirapakawong C, Chansilpa Y, Therasakvichya S, Ieumwananontachai N, Thephamongkhol K (2001) Phase II study of concurrent gemcitabine and radiotherapy in locally advanced stage IIIB cervical carcinoma. Gynecol Oncol 81, 404-407. Pearcey R, Brundage M, Drouin P, Jeffrey J, Johnston D, Lukka H, MacLean G, Souhami L, Stuart G, Tu D (2002) Phase III trial comparing radical radiotherapy with and without cisplatin chemotherapy in patients with advanced squamous cell cancer of the cervix. J Clin Oncol 20, 966-72. Peters WA 3rd, Liu PY, Barrett RJ 2nd, Stock RJ, Monk BJ, Berek JS, Souhami L, Grigsby P, Gordon W Jr, Alberts DS (2000) Concurrent chemotherapy and pelvic radiation therapy compared with pelvic radiation therapy alone as adjuvant therapy after radical surgery in high-risk early-stage cancer of the cervix. J Clin Oncol 18, 1606-1613.

V. Conclusion CGR group was superior to CCR group in terms of complete response rate. OS and LRFS showed no significant difference among different groups with poor DMFS in CGR group. We have used gemcitabine alone as concurrent chemotherapy agent and we found it as a good radiosensitising agent, as it resulted in good response rates but this could not be translated into better OS and LRFS. The possible reason could be: 1. Limitation of nonrandomized retrospective comparison of sequential protocols which were initiated at different time interval. 2. Increased toxicity resulting in increase in overall treatment duration thus counter balancing any benefit achieved in CGR group in long run. 3. Dose of Gemcitabine used in this protocol was not effective in controlling micrometastatic disease thus increasing distant metastases. This resulted in compromise in overall survival. Our results had limitations some of which are mentioned above but still had enough ground to question efficacy of concurrent gemcitabine in locally advanced cervical cancer compared to other standard treatment. One cannot draw definitive conclusion regarding survival from this study but can foresee the trends which questions the 51

Chufal et al: Concurrent gemcitabine with radiation for locally advanced carcinoma cervix Rose PG, Bundy BN, Watkins EB, Thigpen JT, Deppe G, Maiman MA, Clarke-Pearson DL, Insalaco S (1999) Concurrent cisplatin-based radiotherapy and chemotherapy for locally advanced cervical cancer. N Engl J Med 340, 1144-1153. Saibishkumar EP, Patel FD, Sharma SC (2005) Results of a phase II trial of concurrent chemoradiation in the treatment of locally advanced carcinoma of uterine cervix: an experience from India. Bull Cancer 92, 7-12. Shewach DS, Lawrence TS (1996) Gemcitabine and radiosensitization in human tumor cells. Invest New Drugs 14, 257- 63. Stewart BW, Kleihues P, eds. (2003) World cancer report. Lyon, IARC Press, 215-224 Umanzor J, Aguiluz M, Pineda C, Andrade S, Erazo M, Flores C, Santillana S (2006) Concurrent cisplatin/gemcitabine chemotherapy along with radiotherapy in locally advanced cervical carcinoma: A phase II trial. Gynecol Oncol 100, 7075. Whitney CW, Sause W, Bundy BN, Malfetano JH, Hannigan EV, Fowler WC Jr, Clarke-Pearson DL, Liao SY (1999) Randomized comparison of fluorouracil plus cisplatin versus hydroxyurea as an adjunct to radiation therapy in stage IIBeIVA carcinoma of the cervix with negative para-aortic lymph nodes: a Gynecologic Oncology Group and Southwest Oncology Group study. J Clin Oncol 17, 1339-1348.

Zarba JJ, Jaremtchuk AV, Gonzalez Jazey P, Keropian M, Castagnino R, Mina C, Arroyo G; GETICS (Grupo de Estudio, Tratamiento e Investigacion del Cancer del Sur) Argentina (2003) A phase I-II study of weekly cisplatin and gemcitabine with concurrent radiotherapy in locally advanced cervical cancer. Ann Oncol 14, 1285-1290.

Kundan S. Chufal

Cancer Therapy Vol 5, page 55 Cancer Therapy Vol 5, 55-66, 2007

Merits of anti-cancer plants from the Arabian Gulf region Review Article

Amr Amin*, Mohamed Mousa Department of Biology, UAE University, Al-Ain, P.O. Box 17551, U.A.E.

__________________________________________________________________________________ *Correspondence: Amr Amin, Department of Biology, UAE University, Al-Ain, P.O. Box 17551, U.A.E; Tel: +9713-7134381; Fax: +97137671291; e-mail: a.amin@uaeu.ac.ae Key words: Flavonoids, Tannins, Alkaloids, Volatile oils, Saponins, Cardiac glycosides, Anthraquinones, Sterols/Triterpines, Coumarin, Glucosinolates Abbreviations: chamomile, (CO); complementary and alternative medicine , (CAM); phosphatidyl-serine, (PS); polyphyllin D, (PD); protodioscin, (PTN); sister chromatid exchanges, (SCEs); Solanum nigrum L., (SNL); United Arab Emirates, (UAE) Received: 28 November 2006; Revised: 21 January 2007 Accepted: 9 February 2007; electronically published: March 2007

Summary Modern cancer therapies have significantly prolonged the life of the average cancer patient, but still have not succeeded in reducing cancer mortality in certain types of cancer. Complementary and alternative medicine (CAM), which is not a mainstream therapy, has introduced a new element of hope. CAM encompasses a wide spectrum of modalities, practices, theories and beliefs and is based upon dietary supplements, herbal products, and other natural products as defined by The US National Center for Complementary and Alternative Medicine. Extensive research in the last few years has revealed that regular consumption of certain fruits and vegetables can reduce the risk of acquiring specific cancers. Phytochemicals derived from such fruits and vegetables, referred to as chemopreventive agents; include tannins, coumarins, lignans, quinones, stilbenes, curcuminoids, flavonoids and other groups of substances. Various edible and medicinal plants have been extensively studied for their ability to inhibit tumor development. The use of traditional medicines is therefore, quite common in developing countries and is spreading rapidly in developed countries. The Arabian Gulf region has a long tradition of herbal therapies. Herbal medicine constitutes a significant part of this regionâ&#x20AC;&#x2122;s heritage and until recently functioned as its main health-care system. Interestingly, medicinal plants from the Arab region have received little international attention. In the botanical literature there are 600 known plant species in UAE, 1204 in Oman, 2250 in Saudi Arabia, 2088 in Egypt and 2367 in Palestine. Less than 10% of these plants has been screened for their medicinal potential. In the Arabian region, medicinal use is well established for Plantago major, Plantago lanceolata and Commiphora myrrha as anti-tumor remedies. Here, we present an up-to-date review of Arabian medicinal plants known for their anticancer activities.

synthetic compounds can prevent, suppress, or reverse the progression of cancer. Although tumors have traditionally been treated with chemotherapeutic agents, the advent of compounds which prevent malignancies represent an emerging field and offer new options (Mann and DuBois, 2004). The first major meeting devoted to cancer prevention was organized by The American Association for Cancer Research in October 2002. With thousands of participants, this was a key event that brought cancer prevention to the forefront. In the past 5 years, the Food and Drug Administration approved tamoxifen for reducing the risk of breast cancer and celecoxib for the prevention of

I. Introduction Cancer, after cardiovascular disease, is the second leading cause of death (Kutluk and Kars, 1998; Odunsanya, 2001; Cetingoz et al, 2002; Turgay et al, 2005). Worldwide about 10 million people per year are diagnosed with cancer and more than 6 million die of the disease and over 22 million people in the world are cancer patients (Pinar, 1998; Steward and Kleihues, 2003). When cancer is diagnosed, therapists face a formidable range of challenges. Treatment usually consists of various combinations of surgery, radiation therapy, and chemotherapy but despite these therapeutic options, cancer remains associated with high mortality. Natural and some 55

Amin and Mousa: Merits of anti-cancer plants from the Arabian Gulf region familial adenomatous polyposis (Sabichi et al, 2003). The identification of these two drugs represents a paradigm shift in oncology from cancer treatment to cancer prevention. Cancer is a complex disease that is normally associated with a wide range of escalating effects both at the molecular and cellular levels. It therefore seems unlikely that chemoprevention follows simplistic rules and formulations. The old saying "Prevention is always better than cure" is particularly true in the case of cancer where a cure, if at all possible, is associated with high cytotoxic loads and/or invasive procedures. With our growing understanding of the molecular etiology of cancer, it has become apparent that strategies which limit DNA damage and/or increase the probability of DNA repair by inhibiting aberrant proliferation will decrease cancer incidence (Bertram, 2001). Primary cancer preventive strategies are those aimed at removing exposure to carcinogens, be they chemical in the case of tobacco, physical, in the case of UV exposure or mutifactorial in the case of diet and obesity. These preventive strategies have had mixed success. Some have been rather successful, such as the widespread elimination of asbestos, controls on aflatoxin contamination in foods and strict limitations on the amount of exposure to ionizing radiation. Success in primary prevention is reflected in the decreasing incidence of lung cancer, particularly in men, which parallel an earlier decrease in the consumption of tobacco (Bertram and Vine, 2005). Secondary cancer prevention inhibits the consequences of carcinogen exposure. Unfortunately, due to the inappropriate intervention, dosage or, in the case of retinoids, unacceptable toxicity, this strategy has had only limited success in clinical situations. Tertiary cancer prevention relies upon the identification and removal of pre-neoplastic lesions and the success of this approach can be readily observed with the dramatic decrease of uterine cancer resulting from the widespread screening for cervical cancer screening by the Pap test as from the early 1940â&#x20AC;&#x2122;s. At the present time, this approach is unfortunately limited to tissues that are easily accessed (Petignat et al, 2006). A variety of approaches have been employed in cancer chemoprevention. These include changes in diet, supplementation with specific vitamins and minerals, or administration of pharmacologic compounds. Investigators have identified approximately 400 drugs, vitamins, hormones and other agents that might help in preventing cancer. Clinical trials are underway to investigate an increasing number of agents. Most of these trials involve healthy people with a higher-than-average risk of cancer (Dunn and Ford, 2001; Amin and Buratovich, 2007). To sum up, adequate nutrition is a key element of a healthy lifestyle and is associated with a lowered risk for chronic illnesses. The consumption of five portions of fruits and vegetables per day is proposed to sustain optimal health and especially coloured food items are recommended. Data from epidemiological studies consistently show an inverse correlation between the intake of fruits and vegetables and the incidence of several diseases such as cardiovascular, ophthalmological,

gastrointestinal or neurodegenerative disorders and some types of cancer (Van Duyn and Pivonka, 2000). It has been postulated that among the different dietary components of fruits and vegetables, secondary plant constituents (such as phytochemicals) play a major role in disease prevention (Stahl and Seis, 2005; Al-Akhras et al, 2007). The Arabian Gulf region is one of the birthplaces of herbal therapy (Hasan et al, 2000). Herbal medicine occupies a significant part of this regionâ&#x20AC;&#x2122;s heritage and until recently functioned as the main health care system. Despite the wide variety of herbal species in the region with about 600 species in UAE (Jongbloed, 2003), 1204 in Oman (Ghaznafar, 1994), 2250 in Saudi Arabia (Migahid, 1990), 2088 in Egypt (Tackholm, 1974) and 2367 in Palestine (Zohary, 1973) (most of the species present in the two later countries are also recorded in the Gulf region), only less than 10% of these species have been screened for their medicinal uses. Initially, many medicinal plants and their applications were documented in the Middle East and the Arabian Gulf and in such countries as in the United Arab Emirates (UAE) (ElGhonemy, 1993), Qatar (Rizk and El-Ghazaly, 1995), Oman (Ghaznafar, 1994), Saudi (Rahman et al, 2004), Jordan and Egypt (Bolous, 1983). Most of the available literature is based upon information collected from local informants and as it lacks proper laboratory investigations, most of these reports are of dubious value. Since, medicinal plants represent an important health and economic component to Gulf region biodiversity, it is essential to furnish a complete inventory of the medicinal components of the flora of any country for purposes of conservation and sustainable use. At the present time, the high public demand for unconventional therapies has led many countries in the region to devote time and money to the exploration of the potential of their medicinal flora. There is a wide variation in the floral diversity of the Gulf region (Table 1). The highest number of species was recorded in Iran with about 8000 species followed by Iraq with 3000 species and the lowest was Kuwait with 282 species. Information about the diversity and distribution of medicinal plants in the Arabian Gulf region is still scant, although some reviews are available, e.g. Saudi Arabia and UAE. Medicinal plants from the Arabian Gulf area show a wide variety and range from 250 to more than 300 species (Rahman et al, 2004). Approximately 300 species in Saudi Arabia and 100 in the UAE are endemic to the region. For instance, UAE has recently established the "Zayed complex for traditional and herbal medicine," a state-ofthe-art center that specializes in the identification and characterization of native herbs. It also offers patient treatment with herbal remedies for some chronic diseases such as diabetes mellitus, gastric ulcer, psoriasis, and bronchial asthma. When compared to the medicinal plants of China and India where 5000 and 3000 species, respectively are recorded, it appears that the medicinal potential of North African flora has been clearly underestimated. Table 2 shows that only, about 64 species are used for skin lesions, 42 for eye diseases and 34 for wounds. In the Arabian

Cancer Therapy Vol 5, page 57 Gulf region, about 100 of the recorded medicinal plants are used as diuretics, 64 as stimulants, and 54 species as aphrodisiacs. Table 1. Floral diversity in the Middle East region. Region Country Total Medicinal species Bahrain 248

Endemic 0

Endemic (%) 0

Species/ 1000ha! 3.6

Iran

8000

1400

17.5

48.9

Iraq

3000

190

6.3

68.6

Kuwait

282

194.8

Oman

1200

6.1

56.6

Qatar

306

278.0

Saudi Arabia

2028

1.7

9.4

Socotra (Yemen) UAE

815

230

28.2

71.4

Yemen

1650

135

4.8

31.3

Algeria

3164

Egypt

2121

Libya

1825

Morocco

3675

Sinai (Egypt) Tunisia

984

Syria

3000

Palestine Jordan

2225 2100

Lebanon

2600

Turkey

8650

Middle East Mediterran ean Europe China India USA

15000

Arabian Gulf

600

300

13.3 54

7.2

21.3

North Africa

Mediterranean

Over all

2196

352

10.4 44.6 30

3.1

151

700

500

141.7

395

163.0

165 145

7.4 7.3

25.9

311

2549

2675

30.9

25000

1000

11000 32200 35000 19473

5000 3000

Source IUCN, 2002; UNEP, 2002 Riazi, 1997; IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002 Ghaznafar, 1994; IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002; Rizk and ElGhazaly, 1995 IUCN, 2002; UNEP, 2002; Rahman et al, 2004 Heywood, 1997; Majid, 1997 El-Ghonemy, 1993; Jongbloed, 2003 IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002 Bolous, 1983; T채ckholm, 1974 Hussien, 1985; UNEP, 2002 IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002 Zohary, 1973 IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002 IUCN, 2002; UNEP, 2002 FAO, 1997 Heywood, 1997 Heywood, 1997 Duke and Ayensu, 1985 Jain and Robert, 1991 IUCN, 2002; UNEP, 2002

Amin and Mousa: Merits of anti-cancer plants from the Arabian Gulf region Table 2. Medicinal uses of plants in Middle-East region Disease

Anthelmintic Aphrodisiac Asthma Bites and sting Boils (skin) Bone (fractures) Bruise Cataracts Childbirth Cold Colic Cosmetics Cough Cramps Demulcent Diabetes Diarrhea Disinfectant Diuretic Earache Emetic Emollient Epilepsy Expectorant

No. of species North Arabian Gulf Africa 18 29 18 54 8 24 25 6 8 15 14 1 7 4 6 3 19 11 11 24 33 5 11 2 30 39 9 1 9 2 9 10 30 22 4 8 30 100 2 2 11 29 7 41 6 11 11 34

Disease

Eye diseases Eczema Febrifuge Flatulence Gums Hair Jaundice Joints Laxative Menstruation Nosebleed Perfume Poultice Sedative Skin diseases Snuff Stimulant Swelling Toothache Ulcer Vernal diseases Warts Wounds

To date, little attention has been directed to the potential anti-cancer herbs in the region. The most highly represented active ingredients in medicinal plants from the Arabian Gulf area are mainly flavonoids and tannins, which are present in more than ninety percent of the recorded species. However, based on the representative

No. of species North Arabian Gulf Africa 42 26 6 8 24 37 10 2 7 5 23 11 14 22 21 1 23 39 14 1 3 3 8 1 17 1 9 25 64 2 3 2 11 64 24 7 17 12 23 20 6 2 4 1 34 34

species of each active ingredient, many active components are also represented among the flora of the Arab Gulf region (Al-Yahya et al, 1985; Rizk, 1986; Rizk and AlNowaihi, 1989) (Figure 1).

Figure 1. Representation of active ingredients in medicinal plants recorded in the Arabian Gulf region.

Cancer Therapy Vol 5, page 59 Many botanical species and different plant parts contain a diverse array of polyphenolic compounds that are synthesized as floral pigments and are mainly used to attract insect pollinators. They also play a role as antimicrobial/fungal defense mechanisms, and act as photoprotectants (Dixon and Steele, 1999). Based on their chemical structure, polyphenolic compounds are classified into isoflavonoids, flavonoids, stilbenes and lignans (Cos et al, 2003; Dijsselbloem et al, 2004).

is used in the formulations of a number of ayurvedic compounds (Kirtikar and Basu, 1993). Pharmacological studies carried out by several workers have also indicated that P. zeylanica L. extract possesses antiplasmodial (Simonsen et al, 2001), antimicrobial (Durga et al, 1990), antihyperglycemic (Olagunju et al, 1999), insecticidal (Kubo et al, 1983) and antiallergic (Dai et al, 2004) properties. P. zeylanica L. extract also stimulates the central nervous system (Bopaiah and Pradhan, 2001) and is cytotoxic to tumor cells (Lin et al, 2003). In the Palestinian and Israeli territories, extracts of Teucrium polium and Pistacia lentiscus, among others, are known to treat liver disease, jaundice, diabetes, fertility problems and cancer. Most recently, extracts of these two plants have been shown not to be toxic in addition to effectively suppress Fe2+-induced lipid peroxidation. As the aerial parts of Teucrium polium and Pistacia lentiscus are rich in flavonoids, it was concluded that the ability of these plants to suppress Fe2+-induced lipid peroxidation was mediated by flavonoids (Ljubuncic et al, 2005). In Saudi Arabia, aerial parts of Commiphora opobalsamum (L.) (Balessan) are commonly used to treat various diseases. However, its potential use in stomach problems and cancer has been reported only recently. Flavonoids, saponins, volatile oil, sterol and triterpenes have all been revealed in Balessan and thus might contribute to its anticancer activity (Howiriny et al, 2005). Among many other effects, Apium graveolens L. [celery, family: Umbelliferae], is particularly known for its anti-cancer (Sultana et al, 2005) and antioxidant effects (Momin and Nair, 2002). Phytochemical investigations of celery seeds revealed the presence of terpenes like limonene, flavonoids like apigenin and phthalide glycosides. Apigenin is an antioxidant that was documented as one of the major celery's active principals in Apium graveolens (Miean and and Mohammed, 2001). The efficacy of celery as an anti-cancer remedy may then be attributed to the presence of flavonoids, particularily apigenin in its extract (Hamza and Amin, 2007). Apigenin is a widely distributed plant flavonoid that was recently reported as an antitumor agent. Apigenin inhibits the growth of human cervical carcinoma cells by activating apoptosis, Confirmation of induction of apigenin-induced apotosis in HeLa cells was confirmed by DNA fragmentation assays and induction of sub-G1 phase by flow cytometry. Recent findings suggest that apigenin is a strong candidate for development as an anti-cervical cancer agent. Apigeninâ&#x20AC;&#x2122;s preventive effect is shown to be mediated through induction of p53 expression, which causes cell cycle arrest and apoptosis (Duthie and Crozier, 2000; Pei-wen et al, 2005). Butein is another polyphenolic compound, which can be extracted from Rhus verniciflua or the heartwood of Dalbergia odorifera. It induces apoptosis in HL-60 cells (Kim et al, 2001) and B16 melanoma cells (Iwashita et al, 2000) by regulating BCL-2 family proteins. Other properties, such as anti-inflammatory activities (Chan et al, 1998), antinephritic effects (Hayashi et al, 1996), antioxidant properties (Lee et al, 2002), have been documented as well. Only recently have butein's potential as a pharmacological agent been extended to the

II. Flavonoids Despite the tremendous advancements in the understanding and treatment of cancer, there is no sure-fire cure for a variety of cancers to date. Therefore, natural protection against cancer has recently been receiving a great deal of attention not only from cancer patients but, surprisingly, from physicians as well. Phytoestrogens, plant-derived secondary metabolites, are normally divided into three main classes: flavonoids, coumestans and lignans. Flavonoids are found in almost all plant families. Flavonoids are present in different plant parts including the leaves, stems, roots, flowers and seeds and are among the most popular anti-cancer candidates worldwide. Flavonoidic derivatives have a wide range of biological actions such as antibacterial, antiviral, anti-inflammatory, anticancer, and anti-allergic activities. Some of these benefits are attributed to the potent antioxidant effects of flavonoids, which include metal chelation and free-radical scavenging activities (Amin and Buratovich, 2007). Flavonoids are the most abundant active ingredients among the Arabian Gulf inhabitant species. A wide variety of plant-derived flavonoids are naturally present in the human diet or are normally consumed for medicinal reasons. Flavonoids are reported to inhibit specific enzymes, which include hydrolases, oxidoreductase, DNA synthases, RNA polymerases, lipoxygenase and gluthation S-transferase. They also block several digestive enzymes, including !-amylase, trypsin and lipase (Koshihara et al, 1984; Griffiths, 1986; Reddy and Aggarwal, 1994; Sadik et al, 2003). As a result, a rising number of authorized physicians are prescribing pure flavonoids to treat many important common diseases. Dragon's blood is the popular name for a dark-red viscous sap produced by Croton lechleri. This herb is used in folk medicine as an anti-inflammatory (Pieters et al, 1993), anti-microbial (Ubillas, 1994) and anticancer (Hartwell, 1969; Lopes et al, 2004). Similarly, crude extracts from plants like Colubrina macrocarpa, Hemiangium excelsum and Acacia pennatula have been shown to possess a selective cytotoxic activity against human tumor cells KB, HCT-15 COLADCAR and UISOSQC-1 (Popoca et al, 1998). Another member of the family Leguminosae has been shown to have significant anti-breast cancer potential (Amin et al, 2005). In that study, we have shown that Fenugreek can significantly protect rats against drug-induced breast cancer. Both the whole plant and the roots of Plumbago zeylanica L. are commonly used in the treatment of rheumatic pain, dysmenorrhea, carbuncles, contusion of the extremities, ulcers and elimination of intestinal parasites (Chiu and Chang, 2003). In India, P. zeylanica L. 59

Amin and Mousa: Merits of anti-cancer plants from the Arabian Gulf region modulation of estrogen metabolism. This effect could be essential in the prevention and treatment of estrogenresponsive breast cancers (Wang et al, 2005).

characterized into 179 isoprenoids. Individual isoprenoids suppressed the proliferation of B16 and HL-60 promyelocytic leukemia cells with varying degrees of potency. Cell cycle arrest at the G0-G1 phase and apoptosis account, at least in part, for this suppression. These findings suggest that the cancer-protective property of fruits, vegetables, and related products is partly conferred by the cumulative impact of volatile isoprenoid constituents (Smith-Warner et al, 2001; Tatman and Mo, 2002). For instance, geraniol, is an acyclic dietary monoterpene, found in aromatic herb oils. The anti-tumor efficacy of geraniol was evaluated on TC-118 human tumors, transplanted into Swiss nu/nu mice. Geraniol (150 "M) caused a two-fold reduction of thymidylate synthase and thymidine kinase expression in cancer cells. In nude mice, the combined administration of 5fluorouracil (20 mg/kg) and geraniol (150 mg/kg) caused a 53% reduction of the tumor volume, whereas a 26% reduction was obtained with geraniol alone (Carnesecchi et al, 2004). Geraniol exerts an anti-tumor activity against various cancer cells both in vitro and in vivo (Duncan et al, 2004). Geraniol has also been shown to inhibit Caco-2 cell growth by reducing DNA synthesis leading to a blockade of the cells in the S-phase of the cell cycle (Carnesecchi et al, 2001). In addition, geraniol increased the cytotoxicity of 5-FU and enhanced the uptake of the drug in human colon cancer cell lines (Carnesecchi et al, 2001). Different preparations of chamomile (CO) (Matricaria chamomilla) are used to treat various diseases, including inflammation and cancer. Recent studies have shown a dose-dependent inhibitory effect of CO essential oil on sister chromatid exchanges (SCEs) caused by different mutagens. Therefore, CO has been considered as an effective antimutagen (Berhow et al, 2000; HernandezCeruelos et al, 2002; Patel et al, 2007). Carvone, a monoterpene, has been shown to reduce fore-stomach tumor formation by about 60% and pulmonary adenoma formation by nearly 35%, due to its capacity to inhibit Nnitrosodiethylamine-induced carcinogenesis. Dietary monoterpenes were found to be helpful, both in prevention and therapy of cancer (Crowell, 1999). After being extracted and purified, carvone has been utilized throughout the centuries as a component of caraway (Carum carvi), dill (Anethum graveolens) and spearmint (Mentha spicata) seeds (DeCarvalho et al, 2005). Caraway is naturally found in Turkey, Iran and North Africa. Ancient Arabs were the first to use this herb for hysterical complaints and pale complexions. Another source of carvone is dill (seeds). Dill is an annual plant, native to the Mediterranean region. Dill seeds have been used for thousands of years: they were popular in Ancient Egypt (DeCarvalho et al, 2005).

III. Tannins Tannins, phenolic phytochemicals, which are natural constituents of green tea, are considered to have cancerpreventive properties (Lambert and Yang, 2003; Keil et al, 2004). Condensed tannins, isolated from black beans, did not affect the growth of normal cells, but induced cell death in cancer cells in a dose-dependent manner. This cell death was associated with a concentration-dependent decrease of ATP and a deterioration of cellular gross morphology (Swami et al, 2003; Bawadi et al, 2005). Sorghum is a rich source of various phytochemicals including tannins. Relative to other cereals or fruits, sorghum fractions possess high antioxidant activity. These fractions may offer similar health benefits commonly associated with fruits. Sorghum was adapted to grow in the U.A.E. environment, and was found to contain reasonable levels of dietary fiber, minerals and antioxidants to replace part of wheat flour in wheat-based food products (Ragaee et al, 2005). Available epidemiological evidence suggests that Sorghum consumption reduces the risk of certain types of cancer in humans compared to other cereals. The high concentration of phytochemicals in sorghum may be responsible for its protective effects (Rooney and Faubion, 1983; Awika and Rooney, 2004).

IV. Alkaloids Historic medicinal practice used Cat's Claw, also known Uncaria tomentosa, as an effective treatment for several health disorders, which include chronic inflammation, gastrointestinal dysfunction such as ulcers, tumors and infections. The efficacy of Cat's Claw was originally believed to be due to the presence of oxindole alkaloids. Water-soluble Cat's Claw extracts were shown not to contain significant amounts of alkaloids (<0.05%), and yet still were shown to be very effective. Most recently, the active ingredients of a water-soluble Cat's Claw extract were shown to inhibit cell growth without cell death, thus providing enhanced opportunities for DNA repair, immune stimulation, anti-inflammation and cancer prevention (Blumenthal, 2003; Sheng et al, 2005). These active ingredients were chemically defined as quinic acid esters and were also bioactive in vivo as quinic acid.

V. Volatile oils Cancer-preventive constituents of fruits and vegetables may inhibit carcinogen activation, enhance carcinogen detoxification, prevent carcinogens from interacting with critical target sites, or impede tumor progression. These activities, however, are achievable only when levels of individual bioactive constituents reach beyond those attainable from a normal balanced diet. Isoprenoids, a broad class of mevalonate-derived phytochemicals ubiquitous in the plant kingdom, suppress the proliferation of tumor cells and the growth of implanted tumors. Volatile isoprenoid constituents of food products spanning seven plant families have been

VI. Saponins An Iranian experimental study with mice indicated that saffron (Crocus sativus L.) stigma and petal extracts exhibited antinociceptive effects in chemical pain tests and acute and/or chronic anti-inflammatory activity. It was suggested that these effects of saffron extracts might be due to their content of flavonoids, tannins, anthocyanins, alkaloids, and saponins. Studies in animal models and with 60

Cancer Therapy Vol 5, page 61 cultured human malignant cell lines have demonstrated both the antitumor and cancer preventive activities of saffron and its main ingredients. Many possible mechanisms for these activities have been proposed. Ongoing clinical trials that use actual reduction of cancer incidence as the primary endpoint may soon provide a direct evidence of the anticancer effectiveness of saffron (Abdullaev and Espinosa-Aguirre, 2004). Hibiscus sabdariffa L., family: Malvaceae), an annual shrub (Migahid, 1990), is commonly used as a beverage. In folk medicine, H. sabdariffa has been commonly used for its anti-hypertension properties. Anthocyanin pigments confer its color and make it a valuable food product. Many biological activities of anthocyanin, such as antioxidative, hypocholesterole-mic and hepatoprotective activities have been reported. Administration of H. sabdariffa extract before chemotherapeutic treatments has been shown to reduce the testicular damage and ameliorate the drop in sperm quality associated with chemotherapy, in addition to retaining the control values of oxidative stress markers. The antioxidant activity of H. sabdariffa could be attributed to its phenolic contents; namely, anthocyanins (Amin and Hamza, 2006). The aqueous root extracts of some Astragalus species are used to treat leukemia and promote wound healing (Calis et al, 1997; Bedir et al, 2000). The roots of Astragalus species (Fabaceae) are known to be rich in polysaccharides and saponins (Yesilada et al, 2005). Astragalus L., the largest genus in the family Leguminosae is represented by thirty-two species in Egypt (Evans, 1998; Boulos, 1983). Some species of this genus have been reported as having immunostimulant, cardiovascular and antiviral activities (Rios and Waterman, 1997). Extracts of Astragalus kahiricus have been shown to have a reproducible cytotoxicity against the A 2780 ovarian cancer cell line, with an IC50 value of 25 "g/mL (Radwan et al, 2004). Tribulus terrestris, a member of the family Zygophyllaceae, is widely distributed in the entire Gulf region (El-Ghonemy, 1993). The Tribulus seeds are recommended for hemorrhages, kidney stone, and gout. Tribulus extract contains protodioscin (PTN), a steroidal saponin that has been extensively used for the treatment of various ailments, such as urinary, cardiovascular, and gastrointestinal disorders. Administration of Tribulus extracts to humans and animals improves libido and spermatogenesis. Saponin from Tribulus is also known for its hypoglycemic effect. In our recent study, ethanolic extract of Tribulus has shown a significant antioxidant activity against STZ-induced diabetes (Amin et al, 2006). In a search for new anticancer agents, a novel compound polyphyllin D (PD) (diosgenyl !-Lrhamnopyranosyl-(1!2)-(!-L-arabinofuranosyl)-(1!4)][#-D-glucopyranoside) has been identified was shown to induce DNA fragmentation in a hepatocellular carcinoma cell line (HepG2) derivative with drug resistance (RHepG2). PD is a saponin originally found in a tradition Chinese medicinal herb Paris polyphylla. It has been used to treat liver cancer in China for many years. PD has been reported as a potent anticancer agent that can overcome drug resistance in R-HepG2 cells and elicit programmed cell death via mitochondrial dysfunction (Henry et al,

2002; Cheung et al, 2005). Five saponins (diosgenin, hecogenin, tigogenin, sarsasapogenin, smilagenin) have been tested for their biological activities on human 1547 osteosarcoma cells. All examined saponins have shown a significant role on tested cell line in term of proliferation rate, cell cycle distribution and apoptosis induction (Swami et al, 2003). A bacterial metabolite of ginseng saponin (20-O-(-Glucopyranosyl)-20(S)-protopanaxadiol; IH901) is suggested to be a potential chemopreventive agent. IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of both procaspase-3 and caspase-8. Available data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Spragueâ&#x20AC;&#x201C; Dawley rats were not affected by IH901 (0â&#x20AC;&#x201C;60 M). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent (Bosch et al, 1999; Oh and Lee, 2004).

VII. Cardiac glycosides In addition to their cardiotonic function (Kazazoglou et al, 1983; Guh et al, 2000), cardiac glycosides have been suggested to regulate several cellular functions, such as cell proliferation, apoptosis and anti-apoptosisin different cell types (Al-Yahya et al, 1985; Orlov et al, 1999; Verheye-Dua and Bohm, 2000). A large number of plant extracts contain cardiac glycosides. Digoxin/digitoxin and ouabain are two well-known cardiac glycosides derived from Digitalis and Strophanthus gratus, respectively. Cardiac glycosides have recently been shown to possess a significant therapeutic potential against human prostate cancers. Ouabain, for example, was reported to induce cytostatic and cytotoxic effects in androgen-independent prostate cancer PC-3 cells in a time- and concentrationdependent manner (Huang et al, 2004). Salvia officinalis is the most popular herbal remedy in the Middle East to treat common health complications. Salvia species (Labiatae) are known for their antitumor (Liu et al, 2000) effects. Phytochemically, the whole plant contains several antioxidants that protect against cellular peroxidative damage such as rosmarinic acid (Huang and Zhang, 1992) and several phenolic glycosides (Wang et al, 1999). Salvia officinalis has been recently shown to restore normal levels of serum aminotransferases and to protect against drug-induced hepatotoxicity (Amin and Hamza, 2005).

VIII. Anthraquinones Daylilies (Hemerocallis spp.) are herbaceous, clumpforming, perennial monocots that are indigenous to Asia where they are utilized both as a food item and a medicinal agent (Uezu, 1997). Daylilies have been reportedly used throughout eastern Asia for treating a variety of diseases including depression, inflammation, and insomnia (Cichewicz and Nair, 2002). Many studies have demonstrated that Hemerocallis species possess antioxidant (Cichewicz and Nair, 2002) and antischistosome compounds (Cichewicz et al, 2002). It has 61

Amin and Mousa: Merits of anti-cancer plants from the Arabian Gulf region been previously reported that Hemerocallis extracts inhibit fibroblast proliferation (He, 1994) and induce cancer cells to undergo differentiation (Hata et al, 1998); however, the active constituents were never identified. Recently, the bioactive constituents of the flowers and roots of Hemerocallis have been isolated and characterized. A number of new anthraquinones that exhibit in vitro antitumor effects against four human cancer cell lines has been identified. The anti-proliferative activity, the topoisomerase inhibitory and the cytotoxicity of these compounds have also been documented (Cichewicz et al, 2004).

this protection remains unknown even though several different mechanisms have been proposed (Raicht et al, 1980; Rao and Janezic, 1992; Awad et al, 2000a, b). Prostatic 5!-reductase and prostatic aromatase activities were decreased in rats supplemented with phytosterols (Mettlin, 1997; Awad et al, 1998) indicating that they may suppress prostate metabolism and growth. In independent studies, sitosterol has been shown to alter tumor growth (Hannun and Linardic, 1994; Awad et al, 1996). The incorporation of sitosterol in the membranes of HT-29 cells resulted in a significant decrease in sphingomyelin and an increase in phosphatidylcholine (Hannun and Linardic, 1994). Thus, the inhibition of tumor growth could be explained by the effect of phytosterols on the sphingomyelin cycle and increased production of ceramide, which suggest an alteration of signal transduction pathways (Leikin and Brenner, 1989; Tapiero et al, 2003). Previous studies on the cancer chemopreventive effects of natural sources (Nakamura et al, 2002a) have shown gallic acid and methyl gallate, which were isolated from Juca fruits of Caesalpinia ferrea (Leguminosae), as the active constituents. These studies were conducted using the Epsteinâ&#x20AC;&#x201C;Barr virus early antigen activation assay (Ito et al, 1981; Nakamura et al, 2002b). In addition, Lantana camara L. possesses several medicinal properties and is commonly used in folk medicine for its antipyretic, antimicrobial and antimutagenic properties (Fernanda et al, 2005). Solanum nigrum L. (SNL) is a common herb that grows wildly and abundantly in open fields. It has been used in traditional folk medicine because of its diuretic and antipyretic effects. More specifically, it has been used for a long time in oriental medicine to cure inflammation, edema, mastitis and hepatic cancer (Perwaiz et al, 1995). Fruit extracts of SNL have been reported to induce significant DNA damage in human lymphocytes (Chen and Peng, 2001), and to effectively scavenge hydroxyl radicals. Furthermore, for the past three decades, many glycoproteins have been isolated from mushrooms, fungi, yeasts, algae, lichens and plants. The reported biological activities of these glycoproteins have been shown to include immunomodulatory, anticancer and antioxidant, hypolipidemic and hypocholesterolemic effects. However, the mechanisms of the biological functions of such glycoproteins are still not completely understood (Perwaiz et al, 1995). A glycoprotein, with an approximate molecular mass of 150 kDa, was recently extracted from Solanum nigrum L. (Lee and Lim, 2003; Heo et al, 2004). This glycoprotein has a strong scavenging effect against reactive oxygen radicals, and effectively inhibits cellular growth of JA221 and XL1Blue cells. Moreover, it has been reported that SNL glycoprotein has a cytotoxic effect against MCF-7 and HT-29 cells, even at low concentrations (Lim et al, 2002; Heo et al, 2004; Lee et al, 2005). Once a harsh area with limited resources, the Arabian Gulf region has undergone a considerable change over the past few decades after the discovery of petroleum reserves. Despite the significant economic and societal changes, scientific research still needs more attention to document and highlight the cultural heritage of this region

IX. Coumarin The diverse biological activities of natural and synthetic coumarin derivatives as anticoagulants and antithrombotics are well known (Murray et al, 1982). Some of the coumarin derivatives are also reported as antiHIV (Spino et al, 1998) agents, lipid-lowering (Madhavan et al, 2003) agents, and antioxidants (Kontogiorgis and Hadjipavlou-Litina, 2003). They have also been found to inhibit lipid peroxidation and to possess vasorelaxant (Hoult and Paya, 1996) and anticancer activity (Kempen et al, 2003). Many coumarin derivatives are also known as free radical scavengers (Mora et al, 1990) and two naturally occurring coumarins have been found to exhibit cytotoxicity against a panel of mammalian cancer cell lines (Reutrakul et al, 2003). Recently, a series of aryl sulfonamides, a synthesized series of novel compounds that possess both coumarin and sulfonamide entities in one molecule, has been reported as potent inhibitors of various cancer cells at very low concentrations (Rubenstein et al, 2001). Based on their diverse biological activities, both coumarins and aryl sulfonamides have been evaluated for their antitumor activity (Reddy et al, 2004).

X. Glucosinolates Glucosinolates are amino acid-derived natural plant products that coexist with thioglucosidases called myrosinases. When plant tissue is disrupted, glucosinolates come into contact with myrosinases and are hydrolyzed into unstable aglucones that rearrange into a number of products, primarily isothiocyanates. Isothiocyanates have a wide range of biological activities, which include repellents of generalist insects and microorganisms and attractants of specialist herbivorous insects (reviewed in Halkier, 1999). In addition, the isothiocyanate sulforaphane (4methylsulfinylbutylisothiocyanate) derived from glucoraphanine is a very potent cancer-preventing phytochemical as it induces a detoxifying system in mammalian livers ( Naur et al, 2003).

XI. Sterols/Triterpines Phytosterols, especially -sitosterol, are plant sterols that have been shown to exert protective effects against cardiovascular diseases (American Heart Association, 2001) and many types of cancer (Moghadasian, 2000; Awad et al, 2004). They have been reported to protect against cancer development, however, the mechanism of

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particularly in the field of alternative medicine. Pharmacological, toxicological and phytochemical studies should be carried out in this field in order to ascertain the effectiveness and toxicity of the plant-derived products. Therapeutic strategies can then be designed based on the most effective and least toxic of these products. Furthermore, special attention should be given to the promising plants in this part of the world and to protect them from extinction by excessive and radical exploitation. This can only be achieved through a solid collaboration between appropriate governmental authorities and research investigators.

Acknowledgements The authors thank Dr. Michael Buratovich (Spring Arbor University, USA) for proofreading the manuscript and Ms. Karima Al-Mansouri (Biology Department, UAE University) for her valuable assistance formatting the manuscript.

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Cancer Therapy Vol 5, page 67 Cancer Therapy Vol 5, 67-76, 2007

Overcoming K562Dox resistance to STI571 (Gleevec) by downregulation of P-gp expression using siRNAs Research Article

Raquel T. Lima1,2, José Eduardo Guimarães1,2,3, M. Helena Vasconcelos1,4* 1

Cancer Biology Group, IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal 2 Faculty of Medicine of the University of Porto, Porto, Portugal 3 Hospital São João, Porto, Portugal 4 Department of Microbiology, Faculty of Pharmacy of the University of Porto, Porto, Portugal

__________________________________________________________________________________ *Correspondence: Maria Helena Vasconcelos, IPATIMUP, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal. Tel: +351 22 5570700; Fax: +351 22 5570799; E-mail: hvasconcelos@ipatimup.pt Keywords: P-gp, MDR, STI571, Gleevec, Imatinib, RNAi, siRNA, CML Abbreviations: chronic myeloid leukemia, (CML); control siRNA, (CRNAi); multidrug resistance, (MDR); P-glycoprotein, (P-gp); RNA interference, (RNAi); short-hairpin RNAs, (shRNAs) Received: 23 June 2006; Revised: 22 December 2006 Accepted: 27 February 2007; electronically published: March 2007

Summary Resistance to STI571 is possibly due to several mechanisms, including the overexpression of P-glycoprotein (P-gp). The objective of the present study was to verify if downregulation of P-gp expression with specific siRNAs, in the K562Dox overexpressing P-gp cell line, would allow to overcome resistance of these cells to this drug. Uptake of fluorescently-labelled siRNAs was verified by fluorescence microscopy and confirmation of siRNAs efficiency in reducing P-gp protein expression was carried out by Western Blot. Transfection of the K562Dox cells with the siRNAs prior to treatment with STI571 enhanced the effects of this drug, as confirmed by counting the number of viable cells. This increase in the sensitivity was due to an increase in cellular apoptosis, as verified by the TUNEL assay. This data indicates that P-gp downregulation increased sensitivity of CML cells to STI571, by means of promoting apoptosis.

shown to specifically bind to the catalytic pocket of the BCR-ABL, avoiding the association of ATP to this protein and thereby blocking its tyrosine kinase activity (Savage and Antman 2002). This drug has been shown to be highly effective in the treatment of CML, especially in the chronic phase of the disease. However, several reports already state that this drug is not as effective in the more advanced phases of the disease, indicating that resistance to this drug is arising (Gorre et al, 2001; Kano et al, 2001; O'Dwyer et al, 2002; Weisberg and Griffin 2003). Resistance to STI571 has been attributed to several mechanisms, including amplification and mutations of the BCR-ABL gene (Gorre et al, 2001; Roche-Lestienne et al, 2002), increase in !"1-acid glycoprotein levels (Gambacorti-Passerini et al, 2000) and overexpression of multidrug resistance (MDR) genes, namely the MDR1 gene that codifies for P-gp (Mahon et al, 2000; Druker

I. Introduction More than 90% of chronic myeloid leukemia (CML) cases are associated with the presence of an acquired genetic abnormality, the Philadelphia chromosome (Deininger et al, 2000a; Kurzrock et al, 2003). This is a shortened chromosome 22, which results from a reciprocal translocation between chromosomes 9 and 22. As a result of the translocation, the Philadelphia chromosome has the BCR-ABL fusion gene, which codes for a 210KDa chimeric protein with the same name. This protein has a deregulated tyrosine kinase activity resulting in the activation of several transduction pathways in the cell, shown to be responsible for the CML phenotype (Deininger et al, 2000a,b; Shet et al, 2002; Calabretta and Perrotti 2004). Recently, a potent tyrosine kinase inhibitor named STI571 mesylate has been developed, which has been

Lima et al: Overcoming K562Dox resistance to (Gleevec) levels of P-gp expression throughout the experiments, all experiments with the K562Dox cells were carried out 6 days after this treatment with doxorubicin. Two different siRNAs targeting the MDR1 mRNA, previously designed by Wu et al, (2003) and Nieth et al, (2003), were used and named MDR-CR and MDR-FE siRNAs, respectively. Thus, the MDR-CR siRNA had the following sequences: sense – 5’GGA AAA GAA ACC AAC UGU CdTdT3’ and antisense –5’GAC AGU UGG UUU CUU UUC CdTdT3’. The MDR-FE siRNA had the following sequences: sense– 5’AAU GUU GUC UGG ACA AGC AdTdT3’ and antisense –5’UGC UUG UCC AGA CAA CAU UdTdT3’. A negative control siRNA was used with the following sequences (designed by Qiagen): sense – 5’UUC UCC GAA CGU GUC ACG UdTdT3’ and antisense –5’ACG UGA CAC GUU CGG AGA AdTdT3’. A negative control siRNA (with the same sequence) labelled with Alexa Fluor 488 fluorochrome (Qiagen) was used in some experiments. Treatments with the control siRNA are referred to as CRNAi. All siRNAs were purchased from Qiagen and resuspended in siRNA suspension buffer, according to the manufacturer’s instructions.

2002; Nimmanapalli and Bhalla 2002; Knight and McLellan 2004). P-gp is a 170KDa transmembrane glycoprotein involved in the cellular efflux of several drugs. It is known to confer MDR to cells and shown to have a high expression in cancers that are intrinsically resistant to therapy, such as blastic phase CML (Goldstein et al, 1989; Fardel et al, 1996). Several studies have shown that downregulation of P-gp expression results in the sensitization of cells to several chemotherapeutical drugs. Indeed, the use of antisense or rybozime technologies targeting the MDR1 gene has allowed to increase the sensitivity of human hepatocarcinoma cells (Wang et al, 2003), CML (Sola and Colombani 1996; Motomura et al, 1998), ovarian (Pan et al, 2001), colon and breast cancer cells to doxorubicin (Ramachandran and Wellham 2003) as well as to increase the sensitivity of chronic and acute myeloid leukemia cells to daunorubicin (Sola and Colombani 1996; Motomura et al, 1998). Furthermore, downregulation of P-gp expression by RNA interference (RNAi) has allowed the resistance of several cell lines to be overcome (Nieth et al, 2003; Wu et al, 2003; Celius et al, 2004), including of CML to several drugs (Yague et al, 2004). The involvement of P-gp in resistance to STI571 has been previously described by other authors. Indeed, P-gp overexpressing cell lines showed increased resistance to STI571 (Che et al, 2002; Kotaki et al, 2003; Mahon et al, 2003; Illmer et al, 2004). Furthermore, in the course of this work, by downregulating P-gp expression using shorthairpin RNAs (shRNAs), Rumpold and collaborators were able to overcome the resistance of CML cells to STI571 (Rumpold et al, 2005). However, other studies reported that the overexpression of P-gp in K562 cells did not confer resistance to STI571 (Ferrao et al, 2003; Zong et al, 2005). Furthermore, mdr1a/1b-null CML mice did not respond better to STI571 treatment (Zong et al, 2005). Therefore, these conflicting data illustrate the need to validate the relevance of P-gp in the resistance of CML to STI571. The purpose of the present study was to carry out such validation, by downregulating P-gp expression in CML cells that overexpress P-gp and that are resistant to STI571. Furthermore, the work intended to verify if downregulation of P-gp expression with siRNAs increased sensitivity of CML cells to STI571 by means of promoting apoptosis in these cells. This would allow to validate P-gp as a possible molecular therapeutic target, for adjuvant therapy in the treatment of CML with STI571.

B. Transfection of K562Dox cells with siRNAs and verification of uptake of the siRNAs and of downregulation of P-gp protein expression K562Dox cells (5x105 cells/well in 24-well plates) were transfected with siRNAs using jetSI# Reagent (Qbiogene). The manufacturer’s instructions were followed, using siRNA concentrations of 200nM and no FBS during the initial 4 hours of transfection. Following 4 hours of incubation, 400µl of medium containing 25% FBS was added to each well and cells were further incubated. The uptake of the siRNAs by the K562Dox cell line was verified by transfecting this cell line with 200nM control siRNA labelled with Alexa Fluor 488 and examining cells by fluorescence microscopy. To analyze P-gp and BCR-ABL protein expression, cells were lysed in Winman’s buffer (1% NP-40, 0.1M Tris-HCl pH 8.0, 0.15M NaCl and 5mM EDTA) with EDTA-free protease inhibitor cocktail (Boehringer Mannheim) and proteins were quantified and separated: i) in 4-20% Tris-Glycine gel (Novex) in the case of P-gp analysis or ii) in 10% Bis-Tris gels (Sambrook et al, 1989) in the case of BCR-ABL. Proteins were then transferred to a nitro-cellulose membrane (Amersham) with the Novex Electrophoresis System. The membranes were incubated with mouse anti-P-gp (F4 clone; 1:2500, Sigma) or with mouse anti-BCR-ABL (1:50; Santa Cruz Biotechnology) and then incubated with goat anti-mouse IgG -HRP (1:2000, Santa Cruz Biotechnology). The signal was detected with the ECL Amersham kit (Amersham), the Hyperfilm ECL (Amersham) and the Kodak GBX developer and fixer twin pack (Sigma) as previously described (Lima et al, 2004). The intensity of the bands obtained in each film was further analyzed using the software Quantity One – 1D Analysis (Bio-Rad, USA).

II. Materials and Methods

C. Response of K562 and K562Dox cells to treatments with STI571, in terms of viable cell number and programmed cell death

A. Cell lines and siRNAs Two CML cell lines in blast phase were used in this study, K562 (ECACC, European Collection of Cell Cultures, UK) and K562Dox (a kind gift from Professor J.P.Marie, Paris, France). Both these cell lines were routinely cultured in RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum (FBS) and maintained at 37ºC in a humidified atmosphere containing 5% CO2. The K562Dox cell line, which overexpresses P-gp, had previously been obtained by others, by selection of the K562 cell line after exposure to doxorubicin. To maintain P-gp expression in the K562Dox cell line, 1µM doxorubicin was added to the cells every two weeks. In order to maintain equal

In the experiments to determine the response of both cell lines to STI571 mesylate (Novartis), cells were plated in 24-well plates at 5x105 cells/well using serum free medium and incubated at 37ºC in a humidified atmosphere containing 5% CO 2. After 4 hours of incubation, 400 µl of medium containing 25% FBS was added to each well and cells were further incubated. Different concentrations of STI571 mesylate (0, 0.25, 0.5, 0.75, 1.0 and 1.25µM) were added to the cells 24 hours after plating. Viability was assessed 24 hours and 48 hours after drug exposure, with the Trypan Blue exclusion assay.

Cancer Therapy Vol 5, page 69 In the experiments to investigate sensitization to STI571 after transfection with siRNAs, STI571 was added only at the concentrations of 0.5µM or 1µM, 24 hours after transfecting the cells as previously described. As control, the equivalent volume of solvent of the drug (water) was also added to the cells at the same time point. Viability was assessed 48 hours after treatment with STI571, using the Trypan Blue exclusion assay. Apoptosis was also analysed in these cells, 48 hours after treatment with STI571, using the “in situ cell death detection kit” (Roche). In brief, cytospins were prepared and fixed in 4% paraformaldehyde solution. Cells were permeabilised (0.1% Triton X-100 in 0.1% sodium citrate) and incubated with TUNEL reaction mix, according to the optimized procedure recommended by the manufacturer (enzyme dilution 1:20). Cells were observed in a DM IRE 2 microscope (LEICA) and a semiquantitative evaluation was performed by counting a minimum of 500 cells per slide.

B. Cellular response to STI571 To verify the response of both K562 and K562Dox cells to STI571, both cell lines were treated with different concentrations of this drug. The effects of STI571 were analyzed 24h and 48h later, by counting the number of viable cells and analyzing the results as a percentage of control cells (cells that were not treated with STI571). In what concerns the K562 cell line, a decrease in the number of viable cells was verified with the increase in STI571 concentration, as expected (Figure 2 - A and B). The IC50 concentration was only achieved at 48h and with the highest drug concentration tested, 1.25µM (Figure 2B). On the other hand, and also as expected, the response of the K562Dox cell line was different from that observed in the K562 cell line. Indeed, treatment of K562Dox cells with STI571 concentrations up to 0.5µM did not affect their viability. A modest decrease in the number of viable cells was observed, but only with concentrations of STI571 greater than 0.5µM (Figure 2 – A and B). Furthermore, the 1.25µM STI571 concentration (determined to be the IC50 concentration in the K562 cell line) only caused a decrease in the number of the K562Dox viable cells to 76% of the control (Figure 2B). Since the IC50 was only achieved 48h following treatment with STI571, this length of treatment with this drug was chosen for the consecutive experiments. To further confirm that the effect of STI571 was different in the two cell lines, the levels of apoptosis were analysed by the TUNEL assay 48h following treatment of the cells with 1µM STI571. The results (Table 1) confirmed that the K562Dox cells are resistant to the apoptosis inducing effects of STI571.

D. Statistical analysis Results were expressed as mean ± SE. Differences between treatments with STI571 and the respective treatments with solvent were analyzed using a two-tailed paired Student’s t-test (Statview for PC), as appropriate. Significance was defined as P$ 0.05.

III. Results A. P-gp and BCR-ABL basal protein expression in K562 and K562Dox cells The K562Dox cell line was chosen to carry out the present study, since it had been shown to have an increased expression of P-gp, responsible for conferring the multiple drug resistance (MDR) phenotype. In order to confirm that the levels of P-gp expression were higher in K562Dox than in K562 cells, total protein extracts of these two cell lines were analyzed by Western Blot. It was observed that K562Dox cells express P-gp while K562 cells do not (Figure 1A). The levels of BCR-ABL protein expression in both K562 and K562Dox cell lines were also verified by Western Blot, since STI571 acts by blocking BCR-ABL protein function. Results show that there are similar levels of this protein in both cell lines (Figure 1B). Furthermore, since resistance to STI571 may be due to mutations in the BCR-ABL gene, the K562Dox cell line was analyzed (Centro Genética Clínica, Portugal) and no mutations were detected (data not shown).

C. Verification of the effect of the siRNAs: uptake of the siRNAs and downregulation of P-gp protein expression The optimum conditions for transfection using the jetSI# transfection reagent were previously established (data not shown). Uptake of the siRNAs was confirmed by using fluorescently-labelled siRNAs and by verifying the presence of small green fluorescent aggregates localized near the DAPI-stained nuclei, as visualized by fluorescence microscopy (Figure 3). Indeed, it was verified that 24 hours after transfection, 53% of the cells presented the green fluorescent aggregates, indicating a successful transfection of the siRNAs.

Figure 1. Basal levels of P-gp and BCR-ABL protein expression in K562 and K562Dox cell lines. (A) Western Blots (20 µg protein per lane) were probed for P-gp and reprobed for Actin. (B) Western Blots (30 µg protein per lane) were probed for BCR-ABL and reprobed for Actin.

Lima et al: Overcoming K562Dox resistance to (Gleevec)

Figure 2. Response of the K562 and K562Dox cell lines to different STI571 concentrations. This analysis was performed at 24 hours (A) and at 48 hours (B) after the treatment with the drug. The full line indicates the response of the K562 cell line and the dashed line indicates the response of the K562Dox cell line, both represented as % of viable cells in relation to control cells (cells without treatment with STI571).The graph represents the mean ± SE of 3 independent experiments for 0.25µM, 0.75µM and 1.25µM STI571 concentrations and of 4 independent experiments for the remaining concentrations (0µM, 0.5µM and 1µM). The vertical bars represent the standard errors.

Table 1. Induction of apoptosis in K562 and K562Dox cells by STI571. The values represent the mean ± SE of the % of apoptotic cells from 3 independent experiments. H2O

1µM STI571

K562

2%±0%

7%±0%

K562Dox

3%±0%

4%±0%

To confirm if the siRNAs were efficient, the expression of P-gp protein was analyzed 24 and 48 hours after transfection. It was verified that 24 hours after transfection with either of the siRNAs for P-gp (MDR-CR or MDR-FE), there was a specific downregulation of P-gp expression (Figure 4A-left panel). When carrying out semi-quantitative analysis of Western Blots from 3 independent experiments, it was possible to confirm that this downregulation was more pronounced with the MDRCR siRNA than with the MDR-FE siRNA, 24 hours after transfection (Figure 4B-left panel). Indeed, this semi-

quantitative analysis showed that, 24 hours after transfection, the levels of P-gp expression in the cells transfected with the MDR-FE or MDR-CR siRNAs decreased to 67% or 43% of the levels of the cells transfected with the control siRNAs (CRNAi), respectively (Figure 4B -left panel). When analyzing the results 48 hours after transfection, it was possible to verify that P-gp protein levels in the treatments with siRNAs for MDR1 had returned to levels similar to the treatments with the control siRNAs (Figures 4A,B-right panels).

Figure 3. Uptake of Alexa Fluor 488labelled control siRNA in the K562Dox cell line. Uptake was verified by fluorescence microscopy, 24 hours after transfection of the siRNA with jetSI# reagent. Nuclei were stained with Dapi and green fluorescence resulted from siRNA internalization. The mean ± SE of the percentage of internalization of the siRNA is represented in the bottom left corner (obtained from 3 independent experiments). The bar in the image represents 100µm.

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Figure 4. Analysis of P-gp protein levels after transfection with siRNAs. (A) Protein expression analysis by Western Blot. Proteins were extracted at 24h and 48h after transfection of K562Dox cells with control siRNA (CRNAi) or with the MDR-FE or MDR-CR siRNAs. Blots (10 µg protein per lane) were probed for P-gp and reprobed for actin. (B) Semi-quantitative analysis of the intensity of the bands obtained by Western Blot. Each bar represents the mean ± SE of the ratios P-gp/actin, obtained from the intensity of the bands from 3 independent experiments.

Figure 5. Effects of downregulation of P-gp on the sensitization to 0.5µM STI571. Results are represented as a % of the K562Dox Control cells treated with the solvent of STI571 (water), analysed 48 hours after this treatment, considering this as 100% in all experiments. For each group of columns, the filled bar represents the number of viable cells after treatment with solvent and the dashed bar represents the treatment with 0.5µM STI571. The results are the mean ± SE of 3 independent experiments. The first group of columns represents the effect of STI571 or its solvent in the number of viable control cells (Control). The remaining groups of columns represent the effects of the treatment with STI571 or its solvent in cells previously transfected with control siRNA (CRNAi) or siRNAs for P-gp (MDR-FE or MDR-CR). * Represents P! 0.05 between the treatment with STI571 and the respective treatment with solvent, analysed individually for each group of columns.

Lima et al: Overcoming K562Dox resistance to (Gleevec)

Figure 6. Effects of downregulation of P-gp on the sensitization to 1µM STI571. Results are represented as a % of the K562Dox control cells treated with the solvent of STI571 (water), analysed 48 hours after treatment, considering this as 100% in all experiments. For each group of columns, the filled bar represents the treatment with solvent and the dashed bar represents the number of viable cells after treatment with 1"M STI571. The results are the mean ± SE of 4 independent experiments. The first group of columns represents the effect of STI571 or its solvent in the number of viable control cells (Control). The remaining groups of columns represent the effects of the treatment with STI571 or its solvent in cells previously transfected with control siRNA (CRNAi) or siRNAs for P-gp (MDR-FE or MDR-CR). * Represents P! 0.05 between the treatment with STI571 and the respective treatment with solvent, analysed individually for each group of columns.

hand, when cells were transfected with the MDR-CR or MDR-FE siRNAs, there was an increase in the effects of the STI571. This was verified, in the case of the MDR-FE siRNA treatment, by the decrease in the viable cell number from 88% (after treatment with solvent) to 75% (after treatment with 0.5µM STI571). The effect of STI571 was even stronger in the cells previously transfected with the MDR-CR siRNA. In this case, there was a statistically significant decrease in the viable cell number from 94% (after treatment with solvent) to 71% (after treatment with 0.5µM STI571). Analysing the results from the treatment with 1µM STI571 (Figure 6), the addition of STI571 to the Control cells (not transfected) did not significantly affect their viability, as expected (Figure 6–first group of columns). Transfection of the cells with control siRNA (CRNAi) did also not affect their response to 1 µM STI571 (Figure 6– second group of columns). In both these cases a small decrease in the viable cell number was observed after treatment with STI571, but was not considered statistically significant. However, when cells treated with 1µM STI571 had previously been transfected with the MDR-FE or MDR-CR siRNAs, a significant decrease in the viable cell number was verified. In both cases there was an accentuated decrease from 86% (after solvent treatment) to 58% (after STI571 treatment), which was considered statistically significant (Figure 6– last 2 groups of columns). This sensitization effect was seen when cells were treated for 48h with STI571. No effect was seen when cells were treated for 24h only (results not shown) possibly due to the fact that the effect of STI571 was only

D. Effects of downregulation of P-gp expression in the cellular response to STI571: effects on the number of viable and apoptotic cells To investigate whether downregulation of P-gp expression in the K562Dox cells was sufficient to sensitize these resistant cells to treatment with STI571, cells previously transfected with siRNAs for P-gp were treated with 0.5 or 1µM STI571 and the number of viable cells was counted 48 hours after treatment. The choice of STI571 concentrations and length of treatment to be used was based on the above mentioned response of both cells lines (K562 and K562Dox) to this drug (Figure 2). Indeed, it had been observed that these concentrations affected the sensitive cell line (K562) reducing its viable cell number to 58% or to 54% of the control cells 48h after treatment with 0.5 or 1µM STI571 respectively, but did not strongly affect the viable cell number of the resistant cell line (K562Dox). Results were analyzed as a percentage of the viable Control cell number (treated only with water) and are presented in Figure 5 (for 0.5µM STI571) and Figure 6 (for 1 µM STI571). When analysing the results obtained from treating the cells with 0.5µM STI571 (Figure 5) it was observed that the addition of STI571 to the Control cells (not transfected) did not significantly affect their viability, as expected. Indeed, only a small reduction in the viable cell number was observed, to 88% of its control with solvent (Figure 5-first group of columns). It was also observed that transfection of the cells with the control siRNA (CRNAi) did not affect their response to STI571, since a similar viable cell number was obtained after the addition of solvent or STI571 (Figure 5-second group of columns). On the other 72

Cancer Therapy Vol 5, page 73 evident in these cell lines 48h after treatment (see Figure 2). In order to clarify if this reduction in the viable cell number, observed after transfection with the siRNAs for P-gp and treatment with STI571, was due to an increase in apoptosis, the TUNEL assay was carried out 48 hours after drug treatment. Results from this assay were analyzed by fluorescence microscopy and the percentage of apoptotic

cells in the different treatments was determined (Figure 7). Transfection of the cells with any of the siRNAs did not cause, on its own, a significant increase in the levels of apoptosis, since these levels only increased from 2% in the Control treatment to 3% in the transfected treatments (Figure 7-top panel). Furthermore, when 1 µM STI571 was

Figure 7. Apoptosis in K562Dox cells following treatment with 1µM STI571, detected by the TUNEL assay. Apoptosis was determined 48h after treatment with the STI571. The values represent the mean ± SE of the % of apoptotic cells, determined after counting at least 500 cells per experiment in 3 independent experiments. A typical apoptotic cell is indicated with the arrow A and a typical non-apoptotic cell is indicated with the arrow B. Nuclei are labelled with DAPI. * Represents P! 0.05 between the treatment with 1"M STI571 and the respective treatment with solvent. The bar represents 200µm.

added to the Control cells or to the CRNAi cells, there was no significant increase in the % of apoptotic cells. However, in the cells in which the P-gp expression had previously been downregulated by RNAi, the treatment with 1 µM STI571 led to a significant increase in the levels of apoptosis, in comparison to the respective treatment with its solvent. Indeed, in cells transfected with either the MDR-FE or the MDR-CR siRNAs, the % of apoptosis significantly increased from 3% (after treatment with solvent) to 7% (after treatment with 1 µM STI571).

responsible for resistance to STI571. Such results were already anticipated since they are in agreement with the results from previously published studies carried out by other authors, in which cells derived from the K562 cell line, selected for resistance to other drugs and overexpressing P-gp, were resistant to STI571 when compared to the parental cell line, K562 (Che et al, 2002; Kotaki et al, 2003; Mahon et al, 2003; Illmer et al, 2004). However, the results contradict those from other studies in which P-gp overexpression in the K562 cell line did not increase its resistance to STI571 (Ferrao et al, 2003; Zong et al, 2005). The reason for such contradicting data may be the difference in the experimental models used, the first two and the present work based on cell lines which had high levels of P-gp expression obtained by exposure to a drug, whereas in the last two studies the high levels of Pgp were obtained by transfection of cDNA. It is known that cells that are selected by resistance to drug treatment may have other alterations, apart from P-gp overexpression (Zong et al, 2005). Such alterations could justify the results observed in the present study and in the studies from the other authors (Che et al, 2002; Kotaki et al, 2003; Mahon et al, 2003; Illmer et al, 2004). On the other hand, studies in which cells are engineered to overexpress P-gp are based upon an “aberrant” model and therefore the lack of resistance to the STI571 observed in these studies (Ferrao et al, 2003; Zong et al, 2005) may be due to this unnatural model. These existent conflicting data deriving from a diversity of experimental models show that it is important

IV. Discussion In order to validate the importance of P-gp in the resistance of CML cells to STI571, two blastic-phase CML cell lines, one expressing and the other not expressing P-gp, were used as a model. It was important that the only difference between these cells, that could justify a difference in response to STI571, was at the level of P-gp expression. Therefore, it was imperative to confirm that one cell line had a high expression level of Pgp, whereas the other cell line did not express P-gp (at the limit of detection of the technique used-Western Blot). Following the same line of thought, it was confirmed that there were no differences in the levels of BCR-ABL expression between the two cell lines, and that there were no mutations of BCR-ABL in the resistant cell line, that could justify that resistance. The two cell lines responded differently to various concentrations of STI571 suggesting that P-gp was

Lima et al: Overcoming K562Dox resistance to (Gleevec) to complement these studies, in order to further validate the relevance of P-gp in the resistance to STI571. The experimental model used in this study was based on a recently discovered biotechnology, which relies on a natural mechanism existent in cells to silence gene expression, RNAi. We aimed at using one cell line that overexpressed P-gp, which had been selected by drug resistance and therefore could have other alterations that could justify resistance to STI571, and reducing P-gp expression in this cell line using the natural RNAi methodology, in order to determine if P-gp was the responsible alteration for such resistance. The RNAi technology with siRNAs relies on the uptake of the siRNAs by the cells. The optimized conditions showed relatively low levels of uptake of the siRNAs (53%). However, this was considered to be acceptable since these cells are known to be very difficult to transfect. Once the accepted mechanism for RNAi is based on specific degradation of the target mRNA and therefore reduction in the targeted protein expression, the analysis of the protein levels are necessary in order to confirm if and when the siRNAs are active. The data here presented indicate that the siRNAs were capable of reducing P-gp expression. The reduction was pronounced at 24h and was greater in the cells transfected with the MDR-CR siRNA than with the MDR-FE siRNA. The difference in the effect of these siRNAs is probably due to the fact that they target different regions of the mRNA. In fact, previous knowledge that some siRNAs hybridize with the target mRNA better than others, was the reason to work from the outset with two different siRNAs, targeting the same mRNA. Indeed, it is known that the efficacy of hybridization between siRNAs and its target mRNA depends on several factors, such as the thermodynamic structure of the siRNAs themselves and the structure of the target mRNA (Schubert et al, 2005). It was also verified that none of the siRNAs used was capable of totally silencing P-gp expression. This was already expected and is possibly due to the low percentage of transfection in this cell line, as verified by the uptake of the fluorescent siRNA, and also to the transient effect of the transfections, as verified by the Western Blot results. One possible way to overcome this transient RNAi effect is to create stable cell lines through the transfection of vectors cloned with shRNAs, allowing for the continuous expression of siRNAs in the cells and therefore a permanent RNAi effect. This approach has already been carried out by others in order to downregulate P-gp expression in several cell lines (Celius et al, 2004; Stege et al, 2004; Yague et al, 2004), including the one used in this study (Rumpold et al, 2005). However, the use of siRNAs has the advantage of yielding faster results. Furthermore, it is possible that siRNAs themselves could become therapeutic weapons in the future. In fact, there are already ongoing clinical trials with the use of siRNAs for other diseases (Hede 2005). The transfections with the control siRNA caused some toxicity in the K562Dox cell line (considered as nonspecific effects since there was no observed reduction of P-gp in the Western Blot, in the CRNAi treatment). This toxicity, observed in the cells transfected with the control

siRNA and treated with solvent (Figures 5 and 6–second groups of columns-filled bars) could justify the apparent lack of effect of 0.5µM STI571 in these cells, when comparing to the control treatment and analysing the results of STI571 in relation to its solvent. This toxicity could be possibly avoided if different control siRNA sequences had been designed. Some authors indicate that other controls should be used in RNAi experiments, such as siRNAs that have already been confirmed to participate in the RNAi machinery but that are not known to interfere with the protein expression of mammalian cells (Hannon and Rossi 2004). Pre-treatment of cells with siRNAs for P-gp enhanced their sensitivity to STI571. The fact that the increase in the sensitivity to 0.5µM STI571 was only considered statistically significant when cells were treated with the MDR-CR siRNA, but not when they were treated with the MDR-FE siRNA (Figure 5), is in agreement with the data shown above in which the MDR-CR siRNA decreased P-gp expression to a higher extent than the MDR-FE siRNA (Figure 4) and is therefore probably due to the different potency of the two siRNAs. Increasing STI571 concentration from 0.5µM to 1µM caused a more pronounced increase in sensitivity to STI571, considered statistically significant and similar for cells transfected with the MDR-FE or the MDR-CR siRNAs. Indeed, the increase in the sensitivity of these cells was of such an order that their response to 1µM STI571 (viable cell number = 56%) became similar to the response of the K562 cell line (sensitive cell line) to that same STI571 concentration (viable cell number = 54%). This proved that it is possible to revert STI571 drug resistance, in this cell line, by downregulating MDR1 gene expression. STI571 has been shown to induce apoptosis in the K562 cell line (Jacquel et al, 2003) and the results here presented prove that this is also the case for the K562Dox cell line. Indeed, results from the TUNEL assay with 1µM STI571 confirmed that once STI571 is able to reduce the viable cell number, it does so by means of increasing apoptosis. The data here presented allows to conclude that downregulation of P-gp protein expression with siRNAs permitted sensitization to STI571, of CML cells which overexpressed P-gp. This suggests that P-gp may be considered a good molecular therapeutic target, as an adjuvant to therapy with STI571. Furthermore, the enhancement of the effect of STI571 was due to an increase in apoptosis.

Acknowledgements The authors would like to thank Novartis Oncology Portugal for financial support and Novartis Pharma for the STI571 used in this study. We would also like to thank Prof. Clara Sambade and Prof. Paula Soares for general advice and finally Patrícia Pontes, Joana Figueiredo and Hugo Seca for technical assistance.

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Cancer Therapy Vol 5, page 77 Cancer Therapy Vol 5, 77-88, 2007

The role of chemotherapy in the treatment of advanced non-small cell lung cancer elderly patients Review Article

Antonio Rossi, Paolo Maione, Giuseppe Colantuoni, Carmine Ferrara, Alba De Vita, Ciro Guerriero, Dario Nicolella, Filomena del Gaizo, and Cesare Gridelli* Division of Medical Oncology, “S.G. Moscati” Hospital, Avellino – Italy

__________________________________________________________________________________ *Correspondence: Cesare Gridelli, M.D., Division of Medical Oncology, “S.G. Moscati” Hospital, Città Ospedaliera, Contrada Amoretta, 83100 Avellino, Italy; Tel: + 39 0825 203573; Fax: + 39 0825 203556; e-mail: cgridelli@libero.it Key words: NSCLC, elderly patients, chemotherapy Abbreviations: Eastern Cooperative Oncology Group, (ECOG); Elderly Lung cancer Vinorelbine Italian Study, (ELVIS); comprehensive geriatric assessment, (CGA); median survival time, (MST); Multicenter Italian Lung cancer in the Elderly Study, (MILES); non-small-cell lung cancer, (NSCLC); overall response rates, (ORR); performance status, (PS); progression-free survival, (PFS); quality of life, (QoL); small-cell lung cancer, (SCLC); Southwest Oncology Group, (SWOG); time to progression, (TTP) Received: 27 December 2006; Revised: 19 January 2007 Accepted: 30 January 2007; electronically published: March 2007

Summary More than 50% of non-small-cell lung cancer (NSCLC) patients are diagnosed over the age of 65 and approximately one-third of all patients are over the age of 70. Elderly patients tolerate chemotherapy poorly compared to their younger counterpart because of the progressive reduction of organ function and comorbidities related to age. This is why these patients are often not considered eligible for aggressive platinum-based chemotherapy, the standard of care for advanced NSCLC. With the current evidence, in clinical practice, singleagent chemotherapy with a third-generation drug should be the recommended option for non-selected elderly patients with advanced NSCLC. Subset analyses suggest that the efficacy of platinum-based combination chemotherapy is similar in fit older and younger patients, with an acceptable increase in toxicity for elderly patients. However, feasibility of platinum-based chemotherapy remains an open issue and has to be proven in prospective randomised trials. High priority should be also given to the evaluation of the role of new targeted therapies. Moreover, a comprehensive geriatric assessment for individualized treatment choice in NSCLC elderly patients is mandatory. The Authors review the currently-available evidences for the treatment of elderly patients affected by advanced NSCLC reporting their own experiences in the field. (Fentiman et al, 1990). Moreover, many of these patients have pre-existing co-morbid conditions, which, independent of cancer-related symptoms, may adversely affect organ function and impact on functional status (Yancik et al, 2001). Further, co-morbidities often require treatment with multiple drugs, which may interfere with chemotherapy or its toxicity profile. Moreover, the disparity between apparent decreased cancer aggressiveness in an individual patient and the high rate of cancer mortality in older age groups may be because the survival data are confounded by special problems common to geriatric populations (comorbidity, ‘poly-pharmacy’, physician or family bias regarding diagnosis and treatment of elderly, age-associated life stresses). These factors may increase death rates and counteract any primary influence that aging might have to reduce tumour aggressiveness (Ershler and Long, 1997).

I. Introduction More than 50% of lung cancers, the leading causes of cancer-related deaths in Europe and in other Western countries, are diagnosed in patients aged over 65 and about 30% in patients aged over 70 (Jemal et al, 2006). In the United States (US), approximately 13% of the patients have small-cell lung cancer (SCLC) and, consequently, more than 85% have non-small cell lung cancer (NSCLC) (Govindan et al, 2006). Clearly, lung cancer in elderly patients is an increasingly common problem which the practitioner of oncology must face. Since most patients with NSCLC have advanced disease at diagnosis, chemotherapy is the mainstay of management. Elderly patients often present with medical and physiological characteristics that make the selection of their optimal treatment more challenging. Unfortunately, as a result, these patients are at risk of being under-treated 77

Rossi et al: The role of chemotherapy in the treatment of advanced non-small cell lung cancer elderly patients In current practice, the elderly, often excluded from participation in clinical trials, receive untested or inadequate treatment, based on a long-held but completely undocumented notion that cancers in older people are less aggressive (Rossi and Gridelli 2006). Lung cancer is no exception to this observation; on the contrary, data show that it can be considered the paradigm of elderly underrepresentation in clinical research (Hutchins et al, 1999; Lewis et al, 2003; Yee et al, 2003). To confirm these data, a recent survey analyzing trials for cancer drug registration conducted over seven years (from 1995 to 2002), showed a great disparity between percentages of elderly patients in the general population and in those on clinical trials for lung cancer: 67% versus 35% (Talarico et al, 2004). Under-representation was particularly notable for patients older than 75 years. This phenomenon can significantly affect the generalizability of trial results that depend largely upon whether subjects enrolled in clinical trials are fully representative of the entire spectrum of patients suffering from that disease. We must consider that it is very difficult to establish a maximum age for chemotherapy in the elderly. Within epidemiological literature the age of 65 is usually considered as a cut-point to select elderly population. On the contrary, in clinical trials, the age of 70 is frequently used as lower limit for patient selection while a cut-off age of 75 years is less common. Obviously, indirect comparison of trials including or not patients aged 65 to 70 may be biased. In clinical practice biological instead of chronological age should be considered. Unfortunately, to date, laboratory tests and geriatric evaluation are inadequate to define ageing; therefore, at present, chronological age should be used as a frame of reference for clinical trials. A cut-off of 70 years of age seems to be the most appropriate. In fact, 70 years of age may be considered as the lower boundary of senescence, because the incidence of age-related changes starts to increase after the age of 70 years (Balducci, 2000). The evidence regarding the tolerability and efficacy of anticancer treatments for elderly patients affected by NSCLC comes from two different types of publications: prospective clinical trials specifically designed for the elderly and, retrospective analyses conducted on the subgroup of elderly patients enrolled onto clinical trials that did not have an upper age limit. For the latter type of studies, it is easy to argue that when a clinical trial is designed to test the efficacy of a treatment intended for younger patients, only a selected proportion of elderly patients will be considered for enrolment, and so the results may not necessarily extend to the general nonselected elderly population (Perrone et al, 2002). In fact, a recent report demonstrated that elderly specific trials are needed to define optimal cancer therapy in this subset of patients. This was a pooled analysis of elderly patients who participated in elderly-specific trials (age > 65 years) or in age-unspecified studies (age > 18 years). The median age of elderly patients was greater in elderly-specific trials and the toxicity reported in elderly patients was lower in elderly-specific trials than that reported in age-unspecified studies (Jatoi et al, 2005). It means that elderly-specific trials are providing quality care in the elderly.

In order to further individualize treatment choice in elderly patients, it is important to practice not only the patient’s basic medical history and the standard cancer staging, but also a comprehensive geriatric assessment (CGA). The CGA includes assessment of comorbidity, socio-economic issues, nutritional status, poly-pharmacy, functional dependence, emotional and cognitive conditions, an estimate of life expectancy and recognition of frailty. All these various facets of the patient’s health and environment may interfere with therapy. CGA has been demonstrated to add substantial information with respect to the functional assessment of elderly cancer patients (Repetto et al, 2002) allowing important prognostic discrimination even among patients characterized by a good performance status (PS). The CGA may be too lengthy for a busy clinical practice. Therefore, a number of screening instruments have been developed to select those older patients who may benefit from a full CGA. Of these, the evaluation proposed by the Cardiovascular Health Study, that allows the classification of elderly patients into three groups (fit, pre-frail, frail) according to five items (unintentional weight loss, selfreported exhaustion, weakness, walking speed and level of physical activity) (Fried et al, 2001) has gained particular prominence, because it is well correlated with mortality and risk of functional dependence. This classification has been proposed as a standard language for the classification of older individuals. Another simple screening instrument is the Vulnerable Elderly Survey 13 (VES-13), which includes 13 simple questions, the answers of which are scored; patients whose total score is four or higher may benefit from a full CGA (Saliba et al, 2001). Also patients’ self-reported quality of life (QoL) evaluation can add significant prognostic information. When the prognostic role of the baseline QoL, as measured by the EORTC C30 global QoL score, was evaluated in elderly patients diagnosed with NSCLC, QoL score was a strong and independent prognostic factor for survival in patients undergoing first-line treatment for their NSCLC (Maione et al, 2005). These results confirm also in elderly patients the strong prognostic role of self-assessed QoL in patients with advanced lung cancer (Ganz et al, 1991) and show that a simple, self-reported questionnaire may add useful information to baseline evaluation of the patient. This report is focused on the currently-available evidences for the treatment of elderly patients affected by advanced NSCLC and the Authors report their own experiences in this setting.

II. Single-agent chemotherapy The introduction into clinical practice of new, active and well-tolerated drugs has stimulated clinical research. The first randomized phase III trial in advanced NSCLC elderly patients was the Elderly Lung cancer Vinorelbine Italian Study (ELVIS). In this trial, 161 chemotherapynaïve patients (! 70) were randomized to receive vinorelbine (30 mg/m2 on days 1 and 8, every 3 weeks) or best supportive care (ELVIS Group 1999). A significant survival advantage with a better QoL was observed for vinorelbine compared with the control group (median survival time [MST]: 27 versus 21 weeks, P = 0.04). 78

Cancer Therapy Vol 5, page 79 Patients treated in the vinorelbine arm scored better than controls on many QoL subscales. The ELVIS trial represents a landmark regarding the feasibility and the palliative role of chemotherapy in elderly advanced NSCLC patients. Gemcitabine is one of the most widely used drugs for the treatment of NSCLC. Several phase II trials specifically designed for elderly patients with advanced NSCLC have confirmed the role of gemcitabine in this setting. In patients older than 70 years, gemcitabine yielded overall response rates (ORR) of 18% to 38% and MST of 6.8 to 9 months (Ricci et al, 2000; Altavilla et al, 2000; Martoni et al, 2001; Gridelli et al, 2001). Gemcitabine was generally well tolerated, with only two of these studies showing any grade 3 to 4 hematologic toxicities. The taxanes (paclitaxel and docetaxel) have demonstrated both activity and tolerability in the treatment of advanced NSCLC. A recent review of two phase II trials of paclitaxel 210 mg/m2 every 3 weeks compared outcome according to age (< 70 versus ! 70 years). This analysis showed no differences in ORR or MST between the age groups but the most frequent toxicity, neutropenia, tended to occur more frequently in the older patient group (89.3% versus 73.9% in younger patients) (Nakamura et al, 2000). In an effort to reduce toxicity, weekly regimens of singleagent paclitaxel have been investigated in three phase II studies resulting in ORR and MST ranging from 3% to 23% and from 6.8 to 10.3 months, respectively, with reasonable toxicity profiles (Fidias et al, 2001; Garbo et al, 2001; West et al, 2001). Regarding docetaxel, in a phase II trial, weekly doses of docetaxel were well tolerated (Hainsworth et al, 2000). A phase II randomized trial of the two different schedules of administration of docetaxel (weekly or every 3 weeks) in advanced NSCLC elderly patients or patients with PS 2 confirmed comparable activity and lower hematologic toxicity for the weekly schedule (Lilenbaum et al, 2004). Recently, a randomized phase III trial reported that docetaxel (60 mg/m" day 1 every 3 weeks) provided significantly longer progression-free survival (PFS) (5.5 months versus 3.1 months; p < 0.001), a significantly higher ORR (22.7% versus 9.9%; p = 0.019) a more favourable 1-year survival rate (58.6% versus 36.7%) and significantly better disease-related symptom improvement than vinorelbine (25 mg/m" days 1 and 8, every 3 weeks) in elderly patients with advanced NSCLC (Kudoh et al, 2006). These trial, enrolling 182 patients, fails to detect a significant difference in MST, the primary objective of the study, despite a longer median survival in favour of docetaxel-treated patients than vinorelbine-treated patients (14.3 versus 9.9 months, respectively; p = 0.065). Major toxicity was grade 3-4 neutropenia in both arms, 82.9% in docetaxel and 69.3% in vinorelbine group (p = 0.031). This is the first prospective, randomized phase III trial in which a single-agent treatment results superior, in several outcomes but the primary end-point, to another one in the treatment of advanced NSCLC elderly patients. Therefore, further larger phase III trials needed to confirm these results.

III. Non-platinum-based polichemotherapy In order to improve the results obtained with singleagent chemotherapy, some non-platinum-based combinations have been developed. The most studied nonplatinum-based regimen is gemcitabine plus vinorelbine. Two phase III trials have compared gemcitabine plus vinorelbine combination with a single-agent therapy (Frasci et al, 2000; Gridelli et al, 2003). A small study of gemcitabine 1200 mg/m2 plus vinorelbine 30 mg/m2 versus vinorelbine 30 mg/m2 alone (n = 120 patients) closed early when an interim analysis showed that the combination conferred a significant survival advantage over the single agent (MST: 29 versus 18 weeks, p < 0.01) and a better ORR (Frasci et al, 2000). However, the 18week survival for single-agent vinorelbine in this study is markedly lower than the 28- to 38-week survival times reported in six large randomised trials, including three studies dedicated entirely to elderly patients (Rossi et al, 2005; Kudoh et al, 2006). The Multicenter Italian Lung cancer in the Elderly Study (MILES) trial, enrolling 700 patients with NSCLC, is the largest phase III study of the elderly published to date (Gridelli et al, 2003). Patients were randomised to receive single-agent chemotherapy with vinorelbine 30 mg/m2 or gemcitabine 1200 mg/m2 or combination therapy with vinorelbine 25 mg/m2 plus gemcitabine 1000 mg/m2, all treatments recycled every 3 weeks. Combination treatment had no advantage in terms of ORR, time to progression (TTP), MST or QoL over single-agent therapy. Moreover, although toxicity was considered acceptable in all groups, it was higher with the combination than with the single agents. The authors concluded that single-agent therapy with vinorelbine or gemcitabine is preferable to the combination for treatment of advanced NSCLC in elderly patients. Table 1 summarises the randomized phase III trials performed in advanced NSCLC elderly patients.

IV. Platinum-based chemotherapy Cisplatin administration is associated with significant haematological and non-haematological toxicity (nephrotoxicity, ototoxicity, neurotoxicity) and the evaluation of the risk versus benefit ratio should be particularly rigorous in elderly patients. In this patient population, reduction of creatinine clearance and cisplatin renal excretion is expected to increase the potential for toxicity, and the presence of comorbidities and compromised PS may preclude cisplatin administration in a significant proportion of elderly patients (Oshita et al, 1995). Compared to cisplatin, carboplatin causes lower rates of emesis, nephrotoxicity and neurotoxicity, and it represents an appealing alternative for platinum-based chemotherapy, although safety remains a problem especially in terms of hematological toxicity, particularly if it is administered in combination with other myelotoxic agents. Platinum-based chemotherapy for elderly patients with advanced NSCLC has been evaluated both in retrospective and prospective trials.

Rossi et al: The role of chemotherapy in the treatment of advanced non-small cell lung cancer elderly patients

Table 1. Results from phase III trials of advanced non-small-cell lung cancer in elderly patients. Author ELVIS trial, 1999

Frasci, 2000

Gridelli, 2003

Kudoh, 2006

Regimen Vinorelbine vs Best Supportive Care Vinorelbine vs Vinorelbine + Gemcitabine

Age (years) ! 70

! 70

Vinorelbine or Gemcitabine vs Vinorelbine + Gemcitabine

No.pts 76

RR (%) 20

MST (months) 6.5

78 60

NA 15

4.8 4.2

6.7

233 233

18 16

8.3 6.5

232

6.9

9.9

22.7

14.3

! 70

Vinorelbine vs Docetaxel

> 70

ELVIS = Elderly Lung cancer Vinorelbine Italian Study; RR = response rate; MST = median survival time; NA = not applicable.

vinorelbine, and the SWOG 9308 trial that compared cisplatin plus vinorelbine with cisplatin alone (Nguyen et al, 1999). The results showed no statistically significant influence of age on MST (8.6 versus 6.9 months for < 70 versus ! 70 year old patients; p = 0.06), TTP (4.2 versus 3.9 months; p = 0.62) or toxicity. Moreover, these trials seem to support the higher tolerability of carboplatin versus cisplatin, at least at a dose of 100 mg/m2: 46% of elderly patients who received cisplatin and vinorelbine discontinued chemotherapy due to unacceptable toxicity, and this proportion was significantly higher than the discontinuation rate for toxicity in younger patients. In contrast, only 16% of elderly patients treated with carboplatin and paclitaxel stopped treatment for toxicity reasons, without a significant age-related trend (Nguyen et al, 1999). A third retrospective study of a trial of two different schedules of carboplatin plus paclitaxel confirmed these findings (Hensing et al, 2003). Other elderly subset analyses have also been reported recently (Lilenbaum et al, 2005; Belani and Fossella 2005). The Cancer and Leukemia Group B (CALGB) compared carboplatin plus paclitaxel with paclitaxel alone and found no differences in MST between patients aged < 70 and those aged > 70. A secondary analysis of the study showed that the survival advantage for the platinum-based doublet was also seen in the subgroup of elderly patients (8 versus 5.8 months), although this difference was not statistically significant, due to the limited number of elderly enrolled (Lilenbaum et al, 2005). First-line docetaxel plus either cisplatin or carboplatin was compared with vinorelbine plus cisplatin in the TAX 326 trial. A subset analysis of 401 patients aged ! 65 was conducted (Belani and Fossella 2005) and showed that docetaxel plus cisplatin provided substantial increases in survival (1 year: 52% versus 41%; 2 year: 24% versus 17%) compared with vinorelbine plus cisplatin. Both docetaxel regimens were well tolerated in the elderly

A. Retrospective analyses of platinumbased chemotherapy The issue of cisplatin- and carboplatin-based therapy for elderly patients with advanced NSCLC has been addressed in some retrospective analyses of large randomised trials without an upper age limit in the inclusion criteria (Nguyen et al, 1999; Kelly et al, 2001; Langer et al, 2002, 2003; Rocha Lima et al, 2002; Hensing et al, 2003; Belani and Fossella 2005; Belani et al, 2005; Lilenbaum et al, 2005). A retrospective analysis of Eastern Cooperative Oncology Group (ECOG) 5592 compared the effects of first-line cisplatin plus either etoposide or paclitaxel in 86 patients aged ! 70 years with those in 488 younger patients (Langer et al, 2002). There were no significant differences between the younger and older patients in any of the efficacy measures, with only minimal differences in toxicity between the age groups (the elderly had worse leukopenia and neuropsychiatric disorders). An additional analysis of the small subset of patients aged > 75 revealed no differences in outcomes compared with patients aged 70â&#x20AC;&#x201C;75, and there was only a borderline increase in leukopenia in the older age group (p = 0.06). Langer et al, also analysed data from ECOG 1594 in which four treatment combinations (cisplatin/paclitaxel, cisplatin/docetaxel, cisplatin/gemcitabine and carboplatin/paclitaxel) were evaluated as first-line chemotherapy for NSCLC (Langer et al, 2003). No significant differences were reported for RR (24.5% versus 22.1%) or MST (8.3 versus 8.2 months) for the 227 patients (20%) aged ! 70 compared with those aged < 70. Only a marginally significant (p = 0.04) increase in grade 4 toxicities was reported in the elderly population. A similar retrospective analysis was undertaken for the Southwest Oncology Group (SWOG) 9509 trial, which compared carboplatin plus paclitaxel with cisplatin plus

Cancer Therapy Vol 5, page 81 subgroup, although the lowest incidence of toxicity occurred in the docetaxel plus carboplatin arm. Belani et al, reported retrospective analyses related to elderly patients randomized to receive carboplatin plus weekly paclitaxel versus carboplatin plus standard paclitaxel. For the elderly subgroup, the efficacy of the weekly regimen appeared to be higher in terms of RR, TTP and MST with reduction in neuropathy as compared to the standard regimen of paclitaxel and carboplatin (Belani et al, 2005). Overall, these analyses show a similar outcome of platinum-based therapy for elderly patients compared to their younger counterparts, in terms of RR and MST, with a small but significant increase in toxicity in the elderly and no significant adverse effect on QoL. However, it must be noted that the aforementioned analyses suffer from selection bias. In fact, the percentage of elderly patients enrolled in these studies did not exceed 20% of the actual study population. So, elderly patients enrolled in these sort of trials are not representative of the whole elderly population but rather of a small subgroup thought by investigators to be eligible for aggressive treatments (Perrone et al, 2002).

Table 2 summarizes retrospective data analyses of elderly patients enrolled in phase III trials with platinumbased chemotherapy.

B. Prospective trials with platinum-based chemotherapy Prospective clinical trials of platinum-based chemotherapy with inclusion criteria limited to the elderly population are needed. Interest in reproducing cisplatin efficacy in elderly patients led to several prospective phase II trials testing the combination of third-generation cytotoxic agents with cisplatin in modified schedules or attenuated doses to obtain an active and well-tolerated treatment. These employed schemas are not the conventional ones (75-100 mg/m" on day 1, every 3 weeks) in fact, weekly regimens (Lippe et al, 2000; Mattioli et al, 2002; Berardi et al, 2003; Ohe et al, 2004), fractionated doses (Buffoni et al, 2006), low doses (Feliu et al, 2003) and 4 weeks recycled schedules (Perreira et al, 2004), were used in order to improve chemotherapy tolerability. Four main phase II trials tested the combination of cisplatin and gemcitabine reporting a ORR ranging between 32-44% with a MST of 9-10 months.

Table 2. Retrospective data analyses of elderly patients enrolled in phase III trials with cisplatin- or carboplatin-based chemotherapy Author Nguyen, 1999 Kelly, 2001 Langer, 2002 Rocha Lima, 2002 Langer, 2003

Hensing, 2003

Treatment

Age

No.pts

RR (%)

CDDP+GEM

> 70 < 70 > 70 < 70 > 70 < 70 > 70 < 70

53 207 117 491 86 488 31 222

15 29 n.r. n.r. 23.3 21.5 16 31

MST (months) 7.7 9.4 6.9 8.6 8.5 9.1 5.7 8.0

> 70 < 70

227 912

25 22

8.3 8.2

n.s.

> 70 < 70 > 65 all ages > 65 all ages > 65 all ages > 70 < 70 > 70 < 70 > 70 < 70

67 163 149 408 134 404 118 406 70 147 63 151 77 207

27 20 n.r. 32 n.r. 25 n.r. 24 25.7 28.6 19 19.2 36 30

7.1 7.8 12.6 11.3 9.9 10.1 9.0 9.4 9.2 9.6 7.7 11.4 8.0 8.5

n.s.

CBDCA+TAX CDDP+VNR CDDP+VP-16 CDDP+TAX CDDP+VBL CDDP+TAX CDDP+TXT CDDP+GEM CBDCA+TAX CBDCA+TAX CDDP+TXT

Belani, 2005

CDDP+VNR CBDCA+TXT CBDCA+TAXw

Belani, 2005 CBDCA+TAX Lilenbaum, 2005

CBDCA+TAX

P n.s. 0.06 n.s. n.s.

n.s. n.s. n.s. n.r. n.r. n.s.

CDDP = cisplatin; CBDCA = carboplatin; TAX = paclitaxel; TXT = docetaxel; VNR = vinorelbine, GEM = gemcitabine; VBL = vinblastine; VP-16 = etoposide; RR = response rate; MST = median survival time; S = survival; w = weekly; n.r. = not reported; n.s. = not significant.

Rossi et al: The role of chemotherapy in the treatment of advanced non-small cell lung cancer elderly patients The treatment was well tolerated (Lippe et al, 2002; Berardi et al, 2003; Feliu et al, 2003; Moscetti et al, 2005). The cisplatin plus vinorelbine schedule has been tested in three phase II trials reporting a ORR of 33-50% with a MST of 7.4-11 months. This combination was safe, too (Mattioli et al, 2002; Pereira et al, 2004; Buffoni et al, 2006). Ohe et al, treated 33 patients aged > 75 years with weekly cisplatin plus docetaxel reporting a ORR of 52% and a MST of 15.8 months with a modest toxicity (Ohe et al, 2004). The results of phase II trials employing third generation cisplatin-based chemotherapy are reported in Table 3. Among the new generation carboplatin-based regimens, two phase II studies were performed adding carboplatin to vinorelbine, but reported activity (14% and 27% RR) does not suggest any clinical improvement compared to single agent chemotherapy also in consideration of a significant rate and degree of mielotoxicity (Colleoni et al, 1996; Santomaggio et al, 1996). In contrast, the combination of low-dose carboplatin (AUC 4) and gemcitabine produced a RR of 37.5% and a MST of 9.0 months, with a favourable toxicity profile (Maestu et al, 2003). Recently, the results of a subset analysis of carboplatin plus gemcitabine from a randomized phase II study were reported. Patients aged 70

or more achieved similar benefit from this combination in terms of RR and MST with equally good tolerability of younger ones (Masters et al, 2005). Variable results have been observed with attenuated doses or modified weekly schedules of carboplatin plus paclitaxel; RRs have ranged from 14% to 60.9% and MSTs from 7.1 to 13.6 months (Jatoi et al, 2003; Choi et al, 2003; Okamoto et al, 2003; Marsland et al, 2005; Inoue et al, 2006; Pujol et al, 2006). Table 4 summarises the results of phase II trials with third-generation carboplatin-based chemotherapy in advanced NSCLC elderly patients employing attenuated doses or weekly administration of carboplatin.

V. Targeted therapies Gefitinib (ZD1839, Iressa) and erlotinib (OSI774, Tarceva), two orally available selective and reversible inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, have demonstrated activity as single-agent therapy in heavily pretreated patients with NSCLC (Gridelli et al, 2004). Subset analysis of several studies that have included patients aged ! 70 years suggests that gefitinib is active and generally well tolerated in this population (Gridelli et al, 2004). The combination of gefitinib with either

Table 3. Phase II trials of cisplatin-based chemotherapy with third-generation agents and modified schedules or attenuated doses of cisplatin Author Mattioli, 2002* Pereira, 2004 Buffoni, 2006 Lippe, 2002 Berardi, 2003 Feliu, 2003 Moscetti, 2005 Ohe, 2004

Regimen CDDP + VNR CDDP + VNR CDDP + VNR CDDP + GEM CDDP + GEM CDDP + GEM CDDP + GEM CDDP + TXT

CDDP dose 25 mg/m2, weekly 60-90 mg/m" 30 mg/m", day 1 and 8 35 mg/m2, weekly 35 mg/m2, weekly 50 mg/m2 75 mg/m", day 2 25 mg/m2, weekly

Age (years) > 65 > 70 > 70 ! 65 ! 70 ! 70 > 65 ! 75

No. Pts 36 44 30 29 48 46 46 33

RR (%) 36 50 33 48 31.8 35 45.6 52

MST (months) 11 7.5 7.4 10 9 10.2 15 15.8

*including 3 unfit patients; CDDP = cisplatin; VNR = vinorelbine; GEM = gemcitabine; TXT = docetaxel; RR = response rate; MST = median survival time; S = survival

Table 4. Phase II trials of modified administration of carboplatin-based chemotherapy with third-generation agents Author Colleoni, 1996 Santomaggio, 1996 Maestu, 2003 Masters, 2005째 Jatoi, 2003 Choi, 2003* Okamoto, 2005 Marsland, 2005* Inoue, 2006 Pujol, 2006

Regimen Carboplatin + vinorelbine Carboplatin + vinorelbine Carboplatin + gemcitabine Carboplatin + gemcitabine Carboplatin + paclitaxel Carboplatin + paclitaxel Carboplatin + paclitaxel Carboplatin ! paclitaxel Carboplatin + paclitaxel Carboplatin + paclitaxel Carboplatin + paclitaxel

Age (years) ! 70 ! 60 ! 65 > 70 > 65 ! 65 ! 70 ! 70 ! 70 > 70

No. 22 44 88 32 49 35 25 61 60 42 51

RR (%) 14 27 37.5 43.3 14 40 28 22.4 60.9 45 43

MST (months) n.r. 6.5 9.0 10.2 1-year S 31% 8.6 12.3 8.2 9.2 14 13.6

째Retrospective analysis from a phase II study; *Including unfit patients; RR = response rate; MST = median survival time; n.r. = not reported; S = survival

Cancer Therapy Vol 5, page 83 vinorelbine or gemcitabine was investigated in a prospective study of 60 patients aged ! 70 years with advanced NSCLC. Gefitinib combined with gemcitabine showed low activity, but was generally well tolerated. In contrast, toxicity was unacceptable in the vinorelbine arm in which 18 of 25 patients (72%) had grade 3/4 neutropenia, and there were 3 treatment-related deaths with no ORR reported (Scagliotti et al, 2004). Very recently, erlotinib has been tested prospectively in a phase II study in patients aged more than 70 years with previously untreated advanced NSCLC. Preliminary results on 80 patients, suggest that erlotinib is well tolerated with encouraging activity (ORR of 10.9%) as first-line treatment of advanced NSCLC of elderly patients. Adverse events were generally mild, although all responding patients developed a rash (which in 2 cases was severe) (Jackman et al, 2005). Recently, we concluded the accrual in a randomized phase II study, named CALC-1 (Cetuximab in Advanced Lung Cancer), in which cetuximab (IMC-C225), a humanmouse chimeric anti-EGFR monoclonal antibody, is administered in combination with gemcitabine or gemcitabine followed by cetuximab in advanced NSCLC patients unsuitable for combination chemotherapy. The humanized monoclonal antibody bevacizumab, directed against vascular endothelial growth factor (VEGF), is the first and, currently, the only pure antiangiogenic agent licensed for use in combination with fluorouracil-based chemotherapy for first-line treatment of patients with metastatic colorectal cancer in the US and Europe (Gridelli et al, 2006). The clinical development of bevacizumab combined with chemotherapy in the treatment of advanced NSCLC has produced exciting results (Johnson et al, 2004; Sandler et al, 2006). These studies represent the first evidence of superior efficacy of targeted therapy combined with chemotherapy over chemotherapy alone in the treatment of NSCLC. Despite squamous histology was excluded, because of risk of grade 5 hemoptysis reported in the phase II randomized study (Johnson et al, 2004), in the following phase III trial, there were 15 treatment-related deaths in bevacizumab group, including 5 from pulmonary haemorrhage (Sandler et al, 2006). No data about bevacizumab and elderly patients have been reported, but the toxicity profile of bevacizumab, does not appear particularly suitable to the elderly population, often characterised by cardiovascular comorbidities. In fact, a recent analysis from 1,745 patients included in 5 different clinical trials, with different tumours, showed that 7.1% of patients older than 65 years and treated with bevacizumab suffered thromboembolic complications, and that it went up to 17.9% in those with a previous history of atherosclerosis (Skillings et al, 2005). Moreover, bevacizumab is to be administered intravenously and in combination with chemotherapy, thus not being associated with the advantages of oral single agent targeted therapies. In fact oral adiministration and the possibility of some targeted agents to be administered without chemotherapy are factors encouraging their use in elderly patients.

VI. Octogenarians In general, little data exist regarding the outcome of chemotherapy in NSCLC patients aged 80 or more, a rapidly expanding, potentially vulnerable population cohort. In an age-specific subanalysis of trial ECOG 1594, investigators observed only 9 patients more than 80 years of age (less than 1% of all enrolees). It is notable that only 1 of these 9 patients was able to complete four cycles of chemotherapy. Efficacy in this group was very poor: 0% ORR; PFS of 2.2 months, and MST of only 4.2 months. These patients fared worse than patients aged 70-79 when treated with platinum-based combinations with time to progression and survival roughly half that observed in the much larger cohort of patients 70-79 years of age (Langer et al, 2003). A combined analysis of two separate trials performed in elderly patients provides additional information (Hesketh et al, 2005). One study compared weekly docetaxel to every 3-week docetaxel, and enrolled 26 patients aged 80 or older. The second trial, tested sequential vinorelbine-docetaxel and included 23 patients over age 80. Tolerance to treatment was similar between patients aged 70-79 and those over 80 years. For patients with performance status 0-1, median survival was actually shorter in the octogenarian group compared to patients aged 70-79 (7 versus 11 months). For PS 2 patients, survival was equally poor in both groups (4 versus 5 months). Clearly, more studies in this age group need to be conducted.

VII. Second-line chemotherapy Very recently, evidences regarding the feasibility of a second-line chemotherapy in advanced NSCLC elderly patients are emerging. A retrospective analysis of a randomized phase III trial of pemetrexed versus docetaxel in the second-line treatment of advanced NSCLC showed that elderly benefit of treatment as their younger counterparts. The reported results for elderly patients are as follows: 47 patients enrolled in the pemetrexed arm and 39 in the docetaxel group; TTP was 4.6 and 2.9 months, MST was 9.5 and 7.7 months, 12-months survival was 20.4% and 23.1%, 24-months survival was 6.1% and 10.6%, respectively. A smaller number of patients, 40 in the pemetrexed group and 36 in the docetaxel arm, were evaluable for ORR which was 5% and 5.6% with a stable disease of 60% and 41.7%, respectively. Overall, 40 patients in the pemetrexed group and 37 in the docetaxel arm were evaluable for grade 3-4 toxicity reported neutropenia in 12.5% and 29.7%, neutropenia febrile in 2.5% and 18.9%, anemia in 0% and 2.7%, muscle weakness in 5% and 0%, respectively (Weiss et al, 2006). The authors concluded that for elderly patients with advanced NSCLC and good PS, second-line cytotoxic therapy is appropriate, with pemetrexed producing a more favorable toxicity profile compared to docetaxel. These results may appear very attractive, but we strongly suggest to be careful in overemphasizing them, as they derived from a retrospective analysis which could be influenced by selection bias like that reported in the first-line setting.

Rossi et al: The role of chemotherapy in the treatment of advanced non-small cell lung cancer elderly patients In addition, a phase II trial reported good activity and toxicity profile for a modified schedule of docetaxel (37.5 mg/m2 on days 1 and 8 every 3 weeks) (Tibaldi et al, 2006).

of age. This group is a particularly attractive target for studies using molecular targeted and other potentially less toxic oral single agents. While we are still working hard to define the best first-line treatment to be generalized for this growing population, we are going into another minefield, such as second-line therapy. It may be very dangerous without a correct and pragmatic strategy. In our opinion, only second-line prospective studies, specifically addressed to elderly patients with advanced NSCLC, are welcome. Moreover, in order to better define elderly patients enrolled in clinical trials, every patient should receive a complete functional assessment at baseline allowing a clearer interpretation of trial results.

VIII. Conclusions NSCLC remains a major problem in the US, western civilization, and developing countries. Unfortunately, approximately 90% of all NSCLC patients will eventually die from their disease. SEER data in the U.S. suggest that 69 is the median age at diagnosis (Havlik et al, 1994). With the aging of the population one may expect that the prevalence of lung cancer among the elderly will increase. The use of chemotherapy in the elderly NSCLC population is complicated by a number of age-related issues. The reduced organ function and higher prevalence of comorbid disorders associated with the increasing of age affect treatment decisions. This, together with the lack of a clear definition of elderly, contributes to low numbers of elderly participants being included in clinical trials. The place of platinum-based treatment for elderly patients with NSCLC has traditionally been seen as questionable because of the perceived higher risk of toxicity. However, several retrospective subset analyses of platinum-based chemotherapy trials have demonstrated no or minimal differences between elderly patients who met the stringent eligibility criteria of the clinical trials and younger patients treated with platinum-based therapies. The results of subset analyses, however, should be interpreted with care due to the potential risk of selection bias. A new generation of clinical trials specifically designed for the elderly population are needed and should include the development and validation of new measures and tools to define ‘biological’ versus ‘chronological’ age. Moreover, several treatment options should be evaluated such as non-platinum-based combination chemotherapy; platinum-based combination chemotherapy; new biologic agents (Rossi et al, 2005). With the evidence currently available from large randomised trials, single-agent chemotherapy with a thirdgeneration agent, can be considered a recommended option for elderly advanced NSCLC patients (Pfister et al, 2004; Gridelli et al, 2005). However, which single-agent? Several factors should be considered by the clinician when choosing the drug to be administered. This choice should take into account the expected toxicity profile of the agent, pharmacokinetics, organ function and co-morbidities. Platinum-based chemotherapy may represent a valid option for fit elderly patients with adequate organ function. Obviously, a proper selection of these patients is mandatory and further experimental evidence on this topic appears essential. Randomized phase III trials with adequate power to address the main questions regarding the efficacy and tolerability of platinum-based chemotherapy in advanced NSCLC elderly patients are needed, especially in selected elderly good PS patients (Rossi et al, 2005). The lack of data on octogenarians remains troublesome. Based on retrospective analyses, clinicians should exercise caution when applying the existing data for patients aged 70 to 79 years to the those over 80 years

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From left to right Paolo Maione, Giuseppe Colantuoni, Anna Verrengia, Filomena Del Gaizo, Cesare Gridelli, Rosa Bruno, Carmine Ferrara, Costantina Mazza, Lucia Marra

Rossi et al: The role of chemotherapy in the treatment of advanced non-small cell lung cancer elderly patients

Cancer Therapy Vol 5, page 89 Cancer Therapy Vol 5, 89-96, 2007

Down-regulation of MDR1 by continuous exposure to cisplatin in LLC-PK1 cells Research Article

Kohji Takara1,*, Masayuki Tsujimoto1, Misato Kokufu1, Noriaki Kitada1, Toshiyuki Sakaeda2, Noriaki Ohnishi1, Teruyoshi Yokoyama1 1 2

Department of Hospital Pharmacy, Faculty of Pharmaceutical Sciences, Kyoto Pharmaceutical University, Kyoto, Japan Department of Hospital Pharmacy, School of Medicine, Kobe University, Kobe, Japan

__________________________________________________________________________________ *Correspondence: Kohji Takara, Ph.D., Department of Hospital Pharmacy, Faculty of Pharmaceutical Sciences, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan; Fax: +81-75-595-4752; E-mail: takara@mb.kyotophu.ac.jp Key words: MDR1/P-glycoprotein, down-regulation, cisplatin, LLC-PK1 cell Abbreviations: 50% growth inhibitory concentration, (IC50); !-actin, (BA); ciclosporin, (CsA); cisplatin, (CDDP); horseradish peroxidase, (HRP) Received: 3 December 2006; Revised: 17 January 2007 Accepted: 17 March 2007; electronically published: March 2007

Summary P-glycoprotein/MDR1 is considered to be a protection mechanism against various types of toxins or stress. MDR1dependent protection may be activated under the cytotoxic conditions induced by a nephrotoxic anticancer drug cisplatin. Previously, we clarified that the transient exposure (48 h) to cisplatin up-regulated MDR1. This study examined the effects of continuous exposure to cisplatin on MDR1 function and expression in a porcine kidney epithelial LLC-PK1 cell as a model of renal tubular cells. Two sublines that had acquired resistance to cisplatin were newly established from LLC-PK1 by continuous exposure to cisplatin, and designated as LLC/CDDP-1 and LLC/CDDP-2 cells. MDR1 function in LLC/CDDP cells was examined by assessing the growth inhibition by the MDR1 substrate paclitaxel and the uptake of the MDR1 substrate Rhodamine123. The level of MDR1 was evaluated by RT-PCR and immunoblotting. The IC50 values of paclitaxel in both LLC/CDDP cells were lower, but not significant, than that in LLC-PK1 cells, implying an increase in sensitivity to paclitaxel by continuous exposure to cisplatin. The level of MDR1 mRNA in both LLC/CDDP cells was significantly lower than that in LLC-PK1 cells, and MDR1 protein also decreased. These were confirmed by the fact that the inhibitory effects of an MDR1 substrate/inhibitor ciclosporin on Rhodamine123 uptake were remarkably low in LLC/CDDP cells. On the other hand, the uptake of Rhodamine123 in LLC/CDDP cells was significantly lower than that in LLC-PK1 cells, conflicting with the down-regulation of MDR1. This was presumably explained by the reduced Rhodamine123 uptake by the continuous exposure to cisplatin. In conclusion, continuous exposure to cisplatin was clarified to down-regulate MDR1 in a dissimilar way to the case of its transient exposure reported previously.

and/or toxic exogenous substances or metabolites out of the body. Thus, MDR1 is considered to be a protection mechanism against various types of toxins or stress. On the other hand, cisplatin is one of the effective anticancer drugs used in the treatment of various solid tumors at present (Christian, 1992; Kuhlmann et al, 1997). However, the chief limit to the efficacy of cisplatin is its severe nephrotoxicity (Hartmann and Lipp, 2003). Thus, we hypothesized that MDR1-dependent protection may be activated under the cytotoxic conditions induced by cisplatin. For this solution, we previously examined the effects of cisplatin on MDR1, and clarified that the

I. Introduction The kidney has developed a high-capacity transport system to prevent urinary loss of filtered nutrients, and simultaneously, to facilitate tubular secretion of a variety of xenobiotics (Inui et al, 2000). As one of these secretory transport systems, P-glycoprotein/MDR1, which belongs to the ATP Binding-Cassette (ABC) transporters, has been attracted as an efflux transporter (Takara et al, 2006). MDR1 is expressed in the apical membranes of normal tissues, including the kidneys, liver, intestine and brain (Schinkel and Jonker, 2003), and exports unnecessary

Takara et al: Down-regulation of MDR1 by continuous exposure to cisplatin in LLC-PK1 cells culture medium); 3 h later, the absorbance was determined at 450 nm with a reference wavelength of 630 nm. The 50% growth inhibitory concentrations (IC50) of anticancer drugs were calculated using the sigmoid inhibitory effect model: E = E max x [1-C" / (C" + IC50")]. The E and E max represent the surviving fraction (% of control) and its maximum, respectively; C and " represent the drug concentration in the medium and the sigmoidicity factor, respectively.

exposure for 48 h to cisplatin enhanced the activity of MDR1 function and increased MDR1 mRNA expression (about 70%) (Takara et al, 2003). In addition, it has been demonstrated that the single administration of cisplatin to rats increased the MDR1 level in the kidney by ca. 3-fold (Demeule et al, 1999). Huang and colleagues also demonstrated in 2001 the up-regulation of MDR1 mRNA in the rat kidney under conditions of cisplatin-induced nephrotoxicity. Gathering these findings, it was suggested that the transient exposure to cisplatin accelerated MDR1 in the kidneys. However, cisplatin is repetitively but not singly administered to patients in combination chemotherapy for cancer treatment. Thus, it is crucial to examine whether the repetitive administration of cisplatin affects MDR1, but no information is currently available. Herein, two sublines, with resistance to cisplatin, were newly established from a porcine kidney epithelial cell line, LLC-PK1, by continuous exposure to cisplatin, and designated as LLC/CDDP-1 and LLC/CDDP-2 cells. Using these cells as a model of tubular cells exposed continuously to cisplatin, the effects of continuous exposure to cisplatin on MDR1 and multidrug resistanceassociated protein 1 (MRP1) (Kruh and Belinsky, 2003; Haimeur et al, 2004), which is also a family of ABC transporter, were examined.

D. RT-PCR analysis of MDR1 and MRP1 mRNAs The extraction of total RNA from the cells and RT-PCR were carried out as previously reported (Takara et al, 2002b, 2003). The cells (2 x 106 cells/5 mL/dish) were seeded on a 60 mm diameter plastic culture dish and incubated at 37°C for 48 h. Total RNA was extracted using a GenEluteTM Mammalian Total RNA kit (Sigma-Aldrich), and the aliquots of RNA were used for reverse transcription (RT) and cDNA-PCR using a RNA PCR kit (AMV) ver. 2.1 (TakaraBio Inc., Shiga, Japan). The RT reaction was carried out at 30°C for 10 min and subsequently at 42°C for 30 min in a thermal cycler (iCyclerTM, Bio-Rad Laboratories, Inc., CA), and terminated by heating to 99°C for 5 min, followed by cooling at 5°C for 5 min. PCR primers for the amplification of MDR1, MRP1 and !-actin (BA) were previously reported (Kim et al, 1995; Takara et al, 2002b, 2003) and synthesized by Proligo Japan K. K. (Kyoto, Japan). PCR amplification was initiated by one cycle of 94°C for 2 min followed by 35 (for MDR1 and MRP1) or 25 (for BA) sequential cycles of denaturation at 94°C for 45 sec, annealing at 60°C for 45 sec, and extension at 72°C for 45 sec using a thermal cycler (BioRad). PCR products were separated on Tris-acetate-EDTA 3% agarose gels containing ethidium bromide, and visualized with ultraviolet illumination at 312 nm and photographed with a Polaroid camera. Densitometric analysis was performed on a Macintosh computer using NIH Image version 1.63 (National Institutes of Health, Betheda, MD, USA), and the band density ratio was calculated.

II. Materials and methods

A. Chemicals Cisplatin was purchased from Sigma-Aldrich Chemical Corp. (St Louis, MO, USA). Carboplatin and paclitaxel were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Nedaplatin (Lot. No. 1001) was a gift from Shionogi & Co., Ltd. (Osaka, Japan). WST-1 and 1-methoxy PMS were purchased from Dojindo Laboratories (Kumamoto, Japan). Rhodamine123 was purchased from Molecular Probes, Inc. (Eugene, OR, USA).

E. Immunoblotting of MDR1 and MRP1 Cells were cultured and treated with the protocol mentioned above. After treated, cells were harvested and lysed with CelLytic™-M (Sigma-Aldrich) and centrifuged at 10,000 rpm (9,100 g) at 4°C for 15 min. The supernatant was collected, and the total protein content was determined according to the Bradford method (Bradford, 1976) using bovine "-globulin as the standard. An aliquot of protein (20 or 10 µg) was loaded in each lane to detect the expression of MDR1, MRP1 or BA, respectively, electrophoresed on a 7.5% SDS-polyacrylamide gel by the method of Laemmli (Laemmli, 1970), and transferred to a polyvinylidene difluoride (PVDF) membrane Immun-Blot™ (pore size 0.2 µm, Bio-Rad). For immunoblotting (Takara et al, 2006a), the membranes were blocked with 5% skim milk (Wako) in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO 4, 137 mM NaCl and 0.1% Tween 20) at 37°C for 1 h. The blots were incubated with anti-MDR1 monoclonal antibody C219 (1:200, Zymed Laboratories Inc., South San Francisco, CA, USA), anti-MRP1 monoclonal antibody QCRL1 (1:100, Abcam plc., Cambridge, UK), or anti-BA monoclonal antibody (1:5,000, Sigma-Aldrich) for 2 h, then with horseradish peroxidase (HRP)-linked whole sheep antibody to mouse IgG (NA931, Amersham Biosciences) as a secondary antibody for 1 h, and washed five times with PBS-T. Except where stated specifically, all washing and incubation steps were performed at ambient temperature. MDR1, MRP1 and BA were detected with the HRP chemiluminescent reaction (Immobilon TM Western Chemiluminescent HRP Substrate, Millipore Corp., Billerica, MA, USA) according to the manufacturer’s instructions. Blots were then exposed to a

B. Establishment of cisplatin-resistant LLCPK1 subline LLC-PK1 cells were maintained in culture medium consisting of Medium199 (Invitrogen, Corp., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Lot. No. 41K2300, Sigma-Aldrich) without antibiotics. The cisplatinresistant LLC-PK1 cells were isolated by continuous exposure to cisplatin. The concentration of cisplatin to which the cells were exposed was 0.1 µM cisplatin for the first three months and then the cells were further exposed to 1 µM cisplatin for ca. six months. Then, two sublines possibly resistant to 1 µM cisplatin were isolated, and named LLC/CDDP-1 and LLC/CDDP-2. These sublines showed a microscopic morphology similar to LLC-PK1 cells, and also had in their ability to form domes, important indicators of the renal tubular origin of the LLC-PK1 cells. For maintaining the characteristics, these sublines were cultured in complete culture medium containing 1 µM cisplatin.

C. Growth inhibition assay The cytotoxicity of platinum derivatives or paclitaxel in LLC-PK1 and LLC/CDDP cells was evaluated by WST-1 assay (Takara et al, 2003, 2002a, b). Cells (5,000 cells/well) were seeded into 96-well plates in 100 µL culture medium without any drugs, and 24 h later the culture medium was exchanged to that containing the test drugs at various concentrations. After incubation for 72 h at 37°C, the culture medium was exchanged to 110 µL of that containing WST-1 (10 µL WST-1 + 100 µL

Cancer Therapy Vol 5, page 91 Polaroid film Biosciences).

using

ECL

Mini-Camera

(Amersham

B. Growth inhibitory effects of paclitaxel in LLC-PK1 and LLC/CDDP cells Figure 1 shows the growth inhibitory effects of paclitaxel, a representative MDR1 substrate, in LLC-PK1 and LLC/CDDP cells. The IC50 values of paclitaxel in both LLC/CDDP cells were lower, but not significantly so, than that in LLC-PK1 cells and the relative resistance in LLC/CDDP cells was less than 1.

F. Transport experiments of Rhodamine123 The transport activity of MDR1 was evaluated by the transport experiments using Rhodamine123, which was a substrate for MDR1 (Takara et al, 2002b, 2003). LLC-PK1 and LLC/CDDP cells (5 x 104 cells/mL/well) were seeded into a 24well plate, and incubated for 48 h in a humidified atmosphere of 5% CO2-95% air at 37°C. Then, the cells were washed three times with warmed Hanks’ balanced salt solution (HBSS), and the uptake reaction was started by the addition of fresh HBSS containing 10 µM Rhodamine123 with or without 10 µM ciclosporin (CsA), which was a representative substrate/inhibitor for MDR1. The reaction was stopped by the aspiration of HBSS from the wells, followed by washing quickly three times using ice-cold phosphate buffered saline (PBS). After finishing the uptake experiments, cells in the well were lysed with 0.3 M NaOH, and neutralized with 0.3 M HCl. The aliquots of the cell lysate were transferred into a 96-well black plate, and the fluorescence intensity of Rhodamine123 measured with an excitation wavelength of 485 nm and emission wavelength of 535 nm. The protein content in the sample was measured using the Lowry method (Lowry et al, 1951).

C. Expression profile of MDR1 and MRP1 expression in LLC-PK1 and LLC/CDDP cells Expression profiles of MDR1 or MRP1 in LLC-PK1 and LLC/CDDP cells were examined (Figures 2 and 3). The expression of MDR1 mRNA was observed in LLCPK1 and LLC/CDDP cells (Figure 2A). The levels of MDR1 mRNA in LLC/CDDP-1 and LLC/CDDP-2 cells were one-fifth and one-thirtieth of that in LLC-PK1 cells, respectively (p<0.01, Figure 2B). These were confirmed by immunoblotting (Figure 2C). On the other hand, MRP1 expression was also observed in all cells (Figure 3). The level of MRP1 expression in both LLC/CDDP cells was comparable to that in LLC-PK1 cells by RT-PCR and immunoblotting (Figure 3).

G. Statistical analysis Comparison among more than three groups was performed by repeated or non-repeated one-way analysis of variance (ANOVA) followed by Dunnett or Scheffe test, respectively. p values less than 0.05 (two-tailed) were considered significant.

D. Time courses of Rhodamine123 uptake by LLC-PK1 and LLC/CDDP cells

III. Results

The time course of Rhodamine123, a substrate for MDR1, uptake by LLC-PK1 and LLC/CDDP cells was examined (Figure 4). Rhodamine123 uptake was timedependent in LLC-PK1 and both LLC/CDDP cells. Unexpectedly, Rhodamine123 uptake in both LLC/CDDP cells was significantly lower than that in LLC-PK1 cells. To clarify this contradiction, the effects of 10 µM ciclosporin (CsA), a representative MDR1 substrate/inhibitor, on Rhodamine123 uptake for 120 min were examined (Table 2). In the presence of 10 µM CsA, Rhodamine123 uptake showed ca. 2-fold increase. In the case of LLC/CDDP cells, the addition of CsA led to the significant increase (ca. 1.2-fold) of Rhodamine123 uptake in LLC/CDDP-1 cells, but its increase was markedly small compared with that in LLC-PK1 cells. In addition, the effects of CsA on Rhodmaine123 uptake were not comprehensively observed in LLC/CDDP-2 cells.

A. Growth inhibitory effects of cisplatin, carboplatin and nedaplatin in LLC-PK1 and LLC/CDDP cells The 50% growth inhibitory concentration (IC50) for platinum derivatives, cisplatin, carboplatin and nedaplatin in LLC-PK1 and LLC/CDDP cells are summarized in Table 1. The IC50 values of cisplatin were significantly higher than LLC-PK1 cells in both LLC/CDDP cells. Both LLC/CDDP cells showed resistance to cisplatin, and their level of resistance was higher in LLC/CDDP-1 cells compared with LLC/CDDP-2 cells. In the case of carboplatin and nedaplatin, the IC50 values in both LLC/CDDP cells were significantly higher than that in LLC-PK1 cells. Both LLC/CDDP cells also showed resistance to carboplatin and nedaplatin.

Table 1. IC50 values for cisplatin, carboplatin and nedaplatin in LLC-PK1 and LLC/CDDP cells.

Drug Cisplatin Carboplatin Nedaplatin

LLC-PK1

LLC/CDDP-1

IC50 (µM) 2.56 ± 0.88 28.1 ± 8.18 1.19 ± 0.14

IC50 (µM) 16.2 ± 0.02** 251 ± 26.2** 53.6 ± 0.42**

LLC/CDDP-2 R.R.a) 6.3 8.9 45

IC50 (µM) 5.74 ± 0.78** 107 ± 6.51** 53.0 ± 0.16**

R.R. 2.2 3.8 45

Each IC50 value was determined by WST-1 assay, and represents the mean ± S.E (n=4). ** p<0.01 significantly different from the value of LLC-PK1 cells. a)Relative resistance was calculated by that the IC50 values for cisplatin in LLC/CDDP cells was divided by that in LLC-PK1 cells.

Takara et al: Down-regulation of MDR1 by continuous exposure to cisplatin in LLC-PK1 cells Table 2. Cellular accumulation of Rhodamine123 in LLC-PK1 and LLC/CDDP cells in the absence or presence of ciclosporin A (CsA). Cell LLC-PK1 LLC/CDDP-1 LLC/CDDP-2

Cellular accumulation (nmol/mg protein/120 min) - CsA + CsA 5.08 ± 0.19 9.89 ± 0.46** 3.64 ± 0.25 4.34 ± 0.10* 4.01 ± 0.27 4.30 ± 0.39

Fold-increase 1.95 1.19 1.07

Cells were incubated with 10 µM Rhodamine123 in the absence or presence of 10 µM CsA for 120 min. Each value represents the mean ± S.E (LLC-PK 1: n=8, LLC/CDDP: n=4). Fold-increase represents the ratio of the accumulation in the presence of CsA to that in the absence of CsA. * and ** p<0.05 and 0.01 significantly different from the accumulation in the absence of CsA in the corresponding cells, respectively. Figure 1. Growth inhibitory effects of paclitaxel and their IC 50 values in LLC-PK1 (open circles), LLC/CDDP-1 (closed triangles) and LLC/CDDP-2 (closed squares) cells. Each point and value represents the mean ± S.E (n=4). a) Relative resistance was calculated by that the IC 50 values for cisplatin in LLC/CDDP cells was divided by that in LLC-PK 1 cells.

Figure 2. Expression profile of MDR1 in LLC-PK1 and LLC/CDDP cells (A) Total RNA was extracted from cells, and MDR1 and bactin (BA; internal standard gene) mRNA expressions were evaluated by RT-PCR. A representative electrophoretogram is presented, and PCR products of MDR1 and BA were 167 bp and 731 bp, respectively (B) Results are expressed as percentages of the MDR1/BA mRNA levels in LLC-PK1 cells. Each bar represents the mean ± S.E (n=3) (C) Total protein was extracted from cells, and electrophoresed on a 7.5% SDS-polyacrylamide gel. For immunoblotting, the blots were incubated with anti-MDR1 (C219) or anti-BA antibodies. BA was used as the reference protein. A representative immunoblot is presented, and their sizes were 170 kDa and 42 kDa for MDR1 and BA, respectively.

Cancer Therapy Vol 5, page 93

Figure 3. Expression profile of MRP1 in LLC-PK1 and LLC/CDDP cells (A) Total RNA was extracted from cells, and MRP1 and bactin (BA; internal standard gene) mRNA expressions were evaluated by RT-PCR. A representative electrophoretogram is presented, and PCR products of MDR1 and BA were 184 bp and 731 bp, respectively (B) Total protein was extracted from cells, and electrophoresed on a 7.5% SDS-polyacrylamide gel. For immunoblotting, the blots were incubated with anti-MRP1 (QCRL1) or anti-BA antibodies. BA was used as the reference protein. A representative immunoblot is presented, and their sizes were 190 kDa and 42 kDa for MDR1 and BA, respectively.

Figure 4. Time courses of Rhodamine123 uptake in LLC-PK1 and LLC/CDDP cells LLC-PK1 (circles), LLC/CDDP-1 (triangles) and LLC/CDDP-2 (squares) cells were incubated with 10 ÂľM Rhodamine123 at the indicated times. Each point represents the mean Âą S.E (circle: n=8, other symbols: n=4). * and ** p<0.05 and 0.01 significantly different from LLC-PK 1 cells, respectively.

cisplatin-resistant LLC/CDDP cells were used as a model of kidney tubular cells exposed continuously to cisplatin. First, the sensitivity to paclitaxel, a representative MDR1 substrate, was compared between LLC-PK1 and LLC/CDDP cells (Figure 1). The IC50 values of paclitaxel in both LLC/CDDP cells were lower, but not significant, than that in LLC-PK1 cells, and the relative resistance in LLC/CDDP cells was less than 1 (Figure 1), implying an increase in the sensitivity to paclitaxel due to the continuous exposure to cisplatin. In addition, the level of MDR1 in LLC/CDDP-1 and LLC/CDDP-2 cells was significantly lower than that in LLC-PK1 cells (Figure 2).

IV. Discussion In the present study, the two sublines, which had acquired resistance to cisplatin, were newly isolated from LLC-PK1 cells by the continuous exposure to cisplatin, and named LLC/CDDP-1 and LLC/CDDP-2. LLC/CDDP1 and LLC/CDDP-2 cells had 6.3 and 2.2-fold resistances to cisplatin compared with LLC-PK1 cells (Table 1), respectively, and also resistance to carboplatin and nedaplatin (Table 1), indicating the cross-resistance to platinum derivatives. To evaluate whether the repetitive administration of cisplatin affects MDR1, the novel

Takara et al: Down-regulation of MDR1 by continuous exposure to cisplatin in LLC-PK1 cells Collectively, the continuous exposure to cisplatin was suggested to decrease the activity of MDR1 function, presumably via the down-regulation of MDR1. This was also confirmed by the fact that the inhibitory effects of CsA on Rhodamine123 uptake were remarkably low or zero in LLC/CDDP cells, compared with that in LLC-PK1 cells (Table 2). However, the present findings were dissimilar to a previous report where the exposure for 48 h to cisplatin accelerated MDR1 in LLC-PK1 cells (Takara et al, 2003). In addition, other reports demonstrated that the single administration of cisplatin to rats increased MDR1 levels in the kidney (Demeule et al, 1999; Huang et al, 2001), and that MDR1 was rapidly up-regulated when HeLa cells were exposed to cisplatin (Sakaeda et al, 2002). Although the exposure for 48 h to cisplatin might transitorily result in the acceleration of MDR1 as a protection mechanism against xenobiotics and/or stress, MDR1 was not considered to play a protective role against the cytotoxicity of cisplatin, which was not a substrate for MDR1 (Takara et al, 2002a). Thus, continuous exposure to cisplatin may result in the down-regulation of MDR1 in a manner dissimilar to the case of its transient exposure. In addition, cisplatin uptake from apical side in tubular cells have been reported to be mediated by organic cation transporter and copper influx transporter (Ban et al, 1994; Yonezawa et al, 2006; Zhang et al, 2006), and the resistance to cisplatin in tumor cells was one part due to reduced expression of uptake transporter (Song et al, 2004). Collectively, the continuous exposure to cisplatin may reduce the expression of their uptake transporters, and this might cause the reduced uptake of a substrate for MDR1, because cisplatin and some MDR1 substrate were substrates for organic cation transporter. As results, the expression of MDR1 might be decreased by the reduced accumulation of MDR1 substrate in cells. On the other hand, continuous exposure to cisplatin showed no remarkable alteration in the level of MRP1 expression (Figure 3). As cisplatin was not also a substrate for MRP1 (Haimeur et al, 2004), MRP1 did not presumably play a role in a protection mechanism against cellular toxicity caused by exposure to cisplatin. In the transport experiments with Rhodamine123, its uptake in LLC/CDDP cells was significantly lower than that in LLC-PK1 cells (Figure 4), conflicting with the down-regulation of MDR1. Recently, it was reported that some transporter might have participated in the entry process of Rhodamine123 into cells (Cho et al, 2000). Therefore, the decrease in Rhodamine123 uptake in LLC/CDDP cells was not considered to be caused by the acceleration of MDR1, but this may be explained by the reduced Rhodamine123 transport from an apical side by the continuous exposure to cisplatin. In clinical usage, the target maximum plasma concentration of cisplatin at the end of infusion is in the range of 1 to 2 mg/L as total platinum, that is, ca. 5-10 µM as cisplatin (Sasaki et al, 1989; O'Dwyer et al, 2000; Urien and Lokiec, 2004). In addition, cisplatin concentrations in proximal tubular epithelial cells were reported to exceed plasma concentrations 5-fold (Kuhlmann et al, 1997). As the cisplatin concentration used here was considered to be

achievable in the body, the present findings suggested the occurrence of a drug-drug interaction via the downregulation of MDR1 in the kidneys. Moreover, the downregulation of MDR1 by continuous exposure to cisplatin is considered to result in the decrease in MDR1-mediated protection against the xenobiotics and/or stress, and this may in part participate in the induction of nephrotoxicity due to cisplatin. In conclusion, continuous exposure to cisplatin was clarified to down-regulate MDR1 in a manner dissimilar to the case of its transient exposure. Therefore, the function of MDR1 in the kidneys may decrease in patients who receive the repetitive administration of cisplatin.

Acknowledgments The authors are grateful to Shionogi & Co., Ltd. for providing nedaplatin. We thank Dr. Giuliana Decorti and collaborators at the Department of Biomedical Sciences (University of Trieste, Italy) for their helpful advice regarding the sequences of the oligonucleotides as primers for !-actin. This work was supported in part by an “Open Research” Project for a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Cancer Therapy Vol 5, page 95 their clinical application. Nephrol Dial Transplant 12, 2478-2480. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259), 680-685. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-275. O'Dwyer PJ, Stevenson JP, and Johnson SW (2000) Clinical pharmacokinetics and administration of established platinum drugs. Drugs 59, 19-27. Sakaeda T, Nakamura T, Hirai M, Kimura T, Wada A, Yagami T, Kobayashi H, Nagata S, Okamura N, Yoshikawa T, Shirakawa T, Gotoh A, Matsuo M, and Okumura K (2002) MDR1 up-regulated by apoptotic stimuli suppresses apoptotic signaling. Pharm Res 19, 1323-1329. Sasaki Y, Tamura T, Eguchi K, Shinkai T, Fujiwara Y, Fukuda M, Ohe Y, Bungo M, Horichi N, Niimi S, Minato K, Nakagawa K, and Saijo N (1989) Pharmacokinetics of (glycolate-0,0')-diammine platinum (II), a new platinum derivative, in comparison with cisplatin and carboplatin. Cancer Chemother Pharmacol 23, 243-246. Schinkel AH, and Jonker JW (2003) Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 55, 3-29. Song IS, Savaraj N, Siddik ZH, Liu P, Wei Y, Wu CJ, and Kuo MT (2004) Role of human copper transporter Ctr1 in the transport of platinum-based antitumor agents in cisplatinsensitive and cisplatin-resistant cells. Mol Cancer Ther 3, 1543-1549. Takara K, Obata Y, Yoshikawa E, Kitada N, Sakaeda T, Ohnishi N, and Yokoyama T (2006a) Molecular changes to HeLa

cells on continuous exposure to cisplatin or paclitaxel. Cancer Chemother Pharmacol 58, 785-793. Takara K, Sakaeda T, and Okumura K (2006b) An update on overcoming MDR1-mediated multidrug resistance in cancer chemotherapy. Curr Pharm Des 12, 273-286. Takara K, Sakaeda T, Yagami T, Kobayashi H, Ohmoto N, Horinouchi M, Nishiguchi K, and Okumura K (2002a) Cytotoxic effects of 27 anticancer drugs in HeLa and MDR1overexpressing derivative cell lines. Biol Pharm Bull 25, 771-778. Takara K, Tsujimoto M, Kokufu M, Ohnishi N, and Yokoyama T (2003) Up-regulation of MDR1 function and expression by cisplatin in LLC-PK 1 cells. Biol Pharm Bull 26, 205-209. Takara K, Tsujimoto M, Ohnishi N, and Yokoyama T (2002b) Digoxin up-regulates MDR1 in human colon carcinoma Caco-2 cells. Biochem Biophys Res Commun 292, 190194. Urien S, and Lokiec F (2004) Population pharmacokinetics of total and unbound plasma cisplatin in adult patients. Br J Clin Pharmacol 57, 756-763. Yonezawa A, Masuda S, Yokoo S, Katsura T, and Inui K (2006) Cisplatin and oxaliplatin, but not carboplatin and nedaplatin, are substrates for human organic cation transporters (SLC22A1-3 and multidrug and toxin extrusion family). J Pharmacol Exp Ther 319, 879-886. Zhang S, Lovejoy KS, Shima JE, Lagpacan LL, Shu Y, Lapuk A, Chen Y, Komori T, Gray JW, Chen X, Lippard SJ, and Giacomini KM (2006) Organic cation transporters are determinants of oxaliplatin cytotoxicity. Cancer Res 66, 8847-8857.

Takara et al: Down-regulation of MDR1 by continuous exposure to cisplatin in LLC-PK1 cells

Cancer Therapy Vol 5, page 97 Cancer Therapy Vol 5, 97-104, 2007

Four cardiac hormones cause cell death in 81% of human ovarian adenocarcinoma cells Research Article

Brian A. Vesely, Ehrentraud J. Eichelbaum, Abdel A. Alli, Ying Sun, William R. Gower, Jr., David L. Vesely* Departments of Internal Medicine, Molecular Medicine, Molecular Pharmacology and Physiology James A. Haley Veterans Medical Center and University of South Florida Cardiac Hormone Center, Tampa, Florida 33612

__________________________________________________________________________________ *Correspondence: David L. Vesely, M.D., Ph.D., Professor of Medicine, Molecular Pharmacology and Physiology, Director, Cardiac Hormone Center, University of South Florida Medical School, 13000 Bruce B. Downs Blvd., Tampa, Florida 33612; Telephone: (813) 972-7624; Fax: (813) 972-7623; Email: david.vesely@med.va.gov Key words: Ovarian adenocarcinomas, therapeutics, cardiac hormones, DNA synthesis Abbreviations: analysis of variance (ANOVA);atrial natriuretic peptide (ANP); bromodeoxyuridine (BrdU); extracellular-signal regulated kinase (ERK); kaliuretic peptide (KP); long acting natriuretic peptide (LANP); mitogen activated protein (MAP); Natriuretic peptide receptors (NPR); vessel dilator (VDL) Received: 9 January 2007; Revised: 24 January 2007 Accepted: 16 March 2007; electronically published: March 2007

Summary Ovarian cancer has the highest mortality rate among all gynecological malignancies with an estimated 20,180 new cases and 15,310 deaths in the United States in 2006. Thus, for every four women diagnosed with ovarian cancer there were three deaths from ovarian cancer in 2006 in the United States. Four hormones synthesized by one gene in the heart i.e. atrial natriuretic peptide (ANP), vessel dilator, long acting natriuretic peptide (LANP), and kaliuretic peptide were evaluated with dose response (1 to 100 ÂľM) experiments for the ability to decrease the number of human ovarian carcinoma cells in vitro and their DNA synthesis. There was an 81%, 74%, 71%, 70% and 53% increase in cancer cell death of ovarian adenocarcinoma cells within 24 hours secondary to vessel dilator, kaliuretic peptide, ANP, LANP, and their intracellular mediator cyclic GMP, respectively (each at 100 Âľ M) (p<0.0001). These hormones decreased DNA synthesis within the ovarian carcinoma cells from 69% to 85% (p<0.0001). Natriuretic peptide receptors (NPR) -A, and -C were present in the human ovarian adenocarcinoma cells to mediate ANPâ&#x20AC;&#x2122;s effects. This study indicates that four cardiac hormones have potent anticancer effects in ovarian cancer cells by increasing cell death up to 81% of human ovarian cells within 24 hours of treatment mediated in part by a strong inhibition of DNA synthesis within these cancer cells.

(International Federation of Gynecology and Obstetrics) stage III disease and 25% for FIGO stage IV disease. As pointed out recently, it is time for new approaches for treating ovarian cancer (McGuire, 2006). A family of peptide hormones synthesized mainly in the heart have significant anticancer effects on human prostate, breast, colon, kidney and pancreatic adenocarcinoma cells (Vesely et al, 2003, 2005a,b, 2006a; Gower, 2005) as well as upon small-cell (Vesely et al, 2005c) and squamous lung carcinoma cells in vitro (Vesely et al, 2006b). Within this family of peptide hormones, one gene in the heart synthesizes a 126 amino acid (a.a.) prohormone which with proteolytic processing results in four peptide hormones consisting of 1) the first 30 a.a. of this prohormone (i.e., long acting natriuretic peptide, LANP), 2) a.a. 31-67 of this prohormone i.e.

I. Introduction Ovarian cancer has the highest mortality rate among all gynecological malignancies with an estimated 20,180 new cases and 15,310 deaths in the United States in 2006 (Jemal et al, 2006). This high mortality is in part due to at least 60% of women with this disease having advanced disease at the time of initial diagnosis (Naora H and Montell DJ, 2005). Presently ovarian cancer is treated with cytoreductive surgery and chemotherapy with a preferred combination of paclitaxil/carboplatin producing an initial response with improvement in progression-free and overall survival rates (McGuire et al, 1996, McGuire, 2006). However, the majority of patients eventually experience a recurrence of the disease (Alberts, 1999), with the five-year survival rate being only 37% for FIGO

Vesely: Four cardiac hormones cause cell death in 81% of human ovarian adenocarcinoma cells g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate and supplemented with 0.01 mg/ml bovine insulin, 80%; and 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) at a temperature of 37°C in a 5% CO2 environment as recommended by the ATCC. Cells were dispensed in new flasks with subculturing two to three times per week.

vessel dilator (VDL), 3) a.a. 79-98 i.e. kaliuretic peptide (KP), and 4) a.a. 99-126 of the 126 a.a. prohormone i.e. atrial natriuretic peptide (ANP) (Figure 1). Vessel dilator appears to have the strongest anticancer effects decreasing up to 97% the number of human prostate and colon cancer cells within 24 hours (Gower et al, 2005; Vesely et al, 2005b). In vivo, vessel dilator decreases the volume of human pancreatic adenocarcinomas growing in athymic mice the most (49% decrease in tumor volume in one week), while all four peptide hormones stop the growth of this cancer in vivo (Vesely et al, 2004). None of these cardiac hormones have ever been investigated on any gynecological cancer. The four cardiac hormones were evaluated for their ability to decrease the number (i.e. eliminate or cause cell death) of human ovarian cancer cells and to inhibit DNA synthesis within the human ovarian adenocarcinoma cells.

C. Ovarian cancer cell growth protocol After the ovarian carcinoma cells were subcultured for 24 hours they were then seeded to coverslips in 24-well plates (Nunclon TM, Roskilde, Denmark) with 1 mL of the above media. There were 65,000 cells seeded to each coverslip. After 24 hours, the well plates were washed twice with phosphate-buffered saline to remove the fetal bovine serum. Removal of serum was carried out to completely remove all variables in serum (EGF, insulin etc.) in order that interpretation of any data obtained would be straightforward. After 24 hours of serum deprivation for the control and treated cancer cells, media volume was reduced to 250 µL per well with, or without, the respective peptide hormones in dose-response curves with concentrations up to and including 100 µM (1% of this volume). Human ovarian carcinoma cells were then incubated for various periods of time (24, 48, 72, and 96 hours). The number of ovarian cancer cells were then counted with a cell counter (Thomas Scientific!, Swedesboro, NJ) evaluating ten fields of the microscope slide at x 40 along the X-axis with an Olympus BH-2 microscope (Atlanta, Georgia). This evaluation was repeated on six separate occasions with the number of ovarian adenocarcinoma cells reflecting 60 observations for each group, i.e., 60 observations for controls and 60 observations for each of the four groups with respective peptide hormones and for the group with intracellular mediator cyclic GMP. In the Results, ten fields were examined on each microscope slide. The results of the ten fields were pooled and the average of the ten fields is illustrated in the Results section.

II. Materials and Methods The peptide hormones used in this investigation were from Phoenix Pharmaceuticals, Inc., Belmont, CA.

A. Human ovarian carcinoma cells A cell line (ATCC number HTB-161; also designated NIH:OVCAR-3) of human ovarian adenocarcinoma cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). This ovarian carcinoma which is estrogen, progesterone and androgen receptor positive was isolated in 1982 by T. C. Hamilton and R. F. Ozols (Hamilton et al, 1983). This ovarian carcinoma cell line was established from a 60 year old Caucasian lady whose cancer was progressive after combination chemotherapy with cyclophosphamide, Adriamycin and cisplatin (Hamilton et al, 1983). This ovarian cancer is resistant in vitro to clinically relevant concentrations of Adriamycin (5 x 10-8 M), melphalan (5 x 10-6 M) and cisplatin (5 x 10-7 M) (Hamilton et al, 1983). These ovarian cancer cells form ovarian cancers in 100% of athymic mice within 21 days.

D. Determination of DNA synthesis To investigate whether these peptide hormones were inhibiting DNA synthesis, bromodeoxyuridine (BrdU) incorporation into the human ovarian carcinoma cells was utilized as previously described from our laboratory (Vesely et al, 2003, 2005a,b,c, 2006a,b). BrdU was from BD Bioscience, San Jose, California. After 24 hours in culture with 1 µM of LANP, vessel dilator, kaliuretic peptide, ANP, or cyclic GMP,

B. Culture of the human ovarian carcinoma cells Propagation of these human ovarian cancer cells was in Roswell Park Medical Institute (RPMI) 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5

Figure. 1. The atrial natriuretic peptide gene in the heart synthesizes a 126 amino acid (a.a.) prohormone with which proteolytic processing results in the formation of four cardiac hormones. These four cardiac hormones, i.e., 1) LANP consists of the first 30 amino acids of the 126 a.a. prohormone, 2) vessel dilator (VDL), a.a. 31-67 of the prohormone, 3) KP, a.a. 79-98 of this prohormone and 4) ANP, consisting of a.a. 99-126 of the 126 a.a. prohormone.

Cancer Therapy Vol 5, page 99 respectively, or with no peptide hormone (i.e., control), BrdU in a final concentration of 10 µM in the cell culture medium was added for 45 minutes, which is the time in which the cells are in the logarithmic phase of cell proliferation.

measures of analysis of variance (ANOVA) evaluated with statistical module of Excel software. To be considered statistically significant, we required a probability value to be < 0.05 (95% confidence limits).

E. Cyclic GMP effects on ovarian cancer cell number and DNA synthesis

III. Results A. Decreased number of human ovarian carcinoma cells by four peptide hormones synthesized by the cardiac ANP gene

Cyclic GMP is one of the known mediators of the previously described biologic effects of these peptide hormones (Waldman et al, 1984; Vesely, 1997). For the present investigation of the mechanism of these peptide hormones’ ability to decrease the number of ovarian cancer cells and DNA synthesis within the ovarian adenocarcinoma cells, 8bromoguanosine 3’,5’-cyclic monophosphate (i.e.,8-bromocyclic GMP, Sigma Chemical Company) was utilized. 8-Bromocyclic GMP is a cell-permeable analog of cyclic GMP.

F. ANP receptors carcinoma cells

human

The number of human ovarian carcinoma cells in culture for 24 hours decreased 81%, (down to 17 ± 1 cancer cells from 91 ± 2 cells), 67% and 56% secondary to vessel dilator at its 100 µM, 10 µM, and 1 µM concentrations, respectively (p<0.0001 for each) (Figure 2). Dose-response curves revealed that LANP in culture for 24 hours decreased the number of ovarian carcinoma cells 70% (decreased to 27 ± 2 cancer cells), 51% and 35% at its 100 µM, 10 µM, and 1 µM concentrations, respectively (p<0.001 for each) (Figure 2). Exposure of the human ovarian carcinoma cells to kaliuretic peptide resulted in a 74% (23 ± 2 cancer cells), 55% and 37% decrease at its 100 µM, 10 µM, and 1 µM concentrations, respectively (p<0.001 for each) (Figure 2). The addition of ANP decreased the number of ovarian cancer cells in 24 hours by 71%, 60% and 37% at its 100 M, 10 µM, and 1 µM concentrations (p<0.001 for each). Thus, with respect to their ability to eliminate human ovarian adenocarcinoma cells, when these cells were exposed to identical 100 µM concentrations of these peptide hormones for 24 hours, was vessel dilator > kaliuretic peptide > ANP > LANP. When the number of ovarian carcinoma cells was examined immediately after the incubation with the respective peptide hormones, there was no decrease in the number of ovarian cancer cells

ovarian

Ovarian carcinomas have never been examined to determine if they have natriuretic peptide hormone receptors. When it was found that these peptide hormones decreased the number of human ovarian carcinoma cells, it was then evaluated whether ovarian carcinoma cells have ANP receptors to mediate ANP’s effects. Western blots of the NPR -A, and -C were performed utilizing fifty micrograms of protein extract from the ovarian carcinoma cells loaded onto each lane of a discontinuous 7.5% Tris-HCl SDS-PAGE gel (Bio-Rad; Hercules, CA), separated by electrophoresis for 120 min at 100 volts, and then transblotted onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ) for 75 min at 100 volts in Towbin buffer as previously described from our laboratory (Vesely et al, 2005a,b,c, 2006a,b).

G. Statistical evaluation The data obtained in this investigation are illustrated as mean±SEM. Maximum changes in cell death (evaluated six times with ten areas of microscope slide evaluated each time) and DNA synthesis within groups were determined by repeated

Figure 2. Dose-response of KP, ANP, VDL, and LANP anticancer effects on human ovarian carcinoma cells revealed that at each increasing concentration of these four peptide hormones there was a significant (p<0.05) decrease in ovarian cancer cells (i.e. cancer cells killed) within 24 hours when evaluated by repeated measures of ANOVA. Vessel dilator caused the same decrease as the other peptide hormones at a 10-fold lower concentration, as observed in this figure (n=60 for each group).

Vesely: Four cardiac hormones cause cell death in 81% of human ovarian adenocarcinoma cells indicating that these peptide hormones’ anticancer effects were not due to their being toxic to the cells. In the wells with a decreased number of ovarian carcinoma cells secondary to the cardiac hormones at 24 hours there was evidence of cellular debris indicating that the cancer cells had become necrotic, i.e. no longer viable.

significant difference comparing these different concentrations at 24 hours and 48 hours). Two days after exposure to the respective peptide hormones (i.e., 72 hours) as illustrated in Figure 3 for 100 µM concentration, there was no increase in the percent of the remaining ovarian carcinoma cells. Thus, with vessel dilator there was an 83%, 68% and 57% decrease while with kaliuretic peptide there was a 76%, 54%, and 39% decrease at 72 hours in human ovarian carcinoma cells at their 100 µM, 10 µM, and 1 µM concentrations, respectively. LANP caused a 71%, 53%, and 37% decrease while with ANP there was a 73%, 62% and 38% decrease in ovarian cancer cells at their 100 µM, 10 µM, and 1 µM concentrations. Three days after exposure to the respective peptide hormones, the percent of ovarian cancer cells in the treated groups were the same at 48, 72 and 96 hours compared to 24 hours (Figure 3). Thus, the ovarian cancer cells that had not been exposed to vessel dilator for three days had a 83%, 68%, and 56% decrease in cancer cells compared to control and with kaliuretic peptide there was a 76%, 54%, and 38% decrease. The decrease in human ovarian cancer cells secondary to LANP and ANP at 96 hours was 71%, 53%, and 37% and 74%, 63% and 33% at their 100 µM, 10 µM, and 1 µM concentrations, respectively.

B. No increase in percent of ovarian cancer cells for three days after initial 24 hour exposure of these peptide hormones When the ovarian carcinoma cells were followed for three days after treatment with vessel dilator, LANP, kaliuretic peptide and ANP there was no increase in the percent of ovarian cancer cells at 45, 72, and 96 hours after the decrease in the number of the ovarian cancer cells at 24 hours by the four peptide hormones from the cardiac ANP prohormone gene (Figure 3). Thus, when exposed to vessel dilator for 24 hours but without exposure to vessel dilator for the next 24 hours, the decrease in number of human ovarian cancer cells at 48 hours was 82%, 68%, and 56% at 100 µM, 10 µM, and 1 µM of vessel dilator (non-significant difference from the amount of decrease at 24 hours). At 48 hours the decrease in ovarian cancer cells secondary to kaliuretic peptide was nearly identical to that observed at 24 hours with a 75%, 54%, and 38% decrease with 100 µM, 10 µM, and 1 µM of kaliuretic peptide (nonsignificant difference from 24 hours). At 48 hours, after an exposure to LANP for only 24 hours at its 100 µM, 10 µM and 1 µM concentrations, there was a 71%, 52%, and 36% decrease in ovarian cancer cells. Exposure to ANP for 24 hours but without exposure to ANP for the next 24 hours resulted in no proliferation of ovarian cancer cells as there was a 72%, 61%, and 38% decrease in ovarian carcinoma cells with 100 µM, 10 µM, and 1 µM of ANP (non-

C. Cyclic GMP decreases human ovarian adenocarcinoma cell number Cyclic GMP at 100 µM decreased ovarian cancer cells 56% (p<0.01) at 24 hours (Figure 2). Decreasing the concentration of cyclic GMP to 10 µM and 1 µM resulted in 45% and 29% decrease, respectively, in ovarian adenocarcinoma cells at 24 hours (Figure 2).

Figure 3. Time course in decrease of human ovarian cancer cell number with 100 µM concentration of atrial natriuretic peptide (!), kaliuretic peptide (!), vessel dilator ("), long acting natriuretic peptide (#), and cyclic GMP (") at 24, 48, 72, and 96 hours were significant at p<0.001 compared to placebo-treated ($) ovarian carcinoma cells when evaluated by repeated analysis of variance. These ovarian cancer cells were only treated with the cardiac hormones in the first 24 hours (n=60 for each group).

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Cancer Therapy Vol 5, page 101 There was no proliferation in ovarian carcinoma cells for three days after being exposed to cyclic GMP. Thus, 24 hours after exposure to 100 µM of cyclic GMP (i.e. at 48 hours in Figure 3) there was a 56% decrease in ovarian cancer cells while at 72 hours (i.e. 2 days after exposure to cyclic GMP) and at 96 hours (3 days after exposure to cyclic GMP) there was a 57% decrease in ovarian cancer cells (Figure 3).

chosen for this investigation as ovarian cancer has the worst 5-year survival of the gynecological cancers (Alberts, 1999). Ovarian cancer is in an advanced stage in 60% of women when first diagnosed (Naora H and Montell DJ, 2005) and the majority of patients experience a recurrence of this cancer (Alberts, 1999). This knowledge indicates that there is a strong need for adjunct anticancer therapy for this cancer. The present investigation suggests that the four cardiovascular hormones which eliminate up to 81% of the ovarian carcinoma cells in 24 hours, may be useful adjuncts to surgery to treat ovarian carcinomas. Vessel dilator decreased the number of human ovarian carcinoma cells the most at each of the respective concentrations of the peptide hormones (Figure 2). In the dose-response curves of the present investigation, when vessel dilator concentration was increased 10-fold and 100-fold (i.e., 10 µM and 100 µM), vessel dilator decreased the number of human ovarian carcinoma cells by 67% and 81%, respectively, within 24 hours compared to 56% decrease at its 1 µM concentration (Figure 2). Vessel dilator also decreases human pancreatic (Vesely et al, 2003), breast (Vesely et al, 2005a), colon (Gower et al, 2005), kidney (Vesely et al, 2006a) and prostate (Vesely et al, 2005b) adenocarcinomas as well as small-cell (Vesely et al, 2005c) and squamous lung cancer cells (Vesely et al, 2006b) in vitro the most. This information plus the knowledge that vessel dilator decreases the tumor volume of human pancreatic adenocarcinomas the most in vivo (Vesely et al, 2004) suggests that vessel dilator has the most significant anticancer properties of the four peptide hormones with anticancer effects in the present investigation. Vessel dilators’ effects are also stronger than clinically relevant concentrations of Adriamycin, melphalan and cisplatin (Hamilton et al, 1983) on this same ovarian cancer cell line. It is important to note that after 24 hours of incubation with the four peptide hormones that cellular debris was present, suggesting that cellular necrosis was occurring.

D. Inhibition of DNA synthesis in human ovarian carcinoma cells by LANP, vessel dilator, ANP, kaliuretic peptide and cyclic GMP To help determine the mechanism of the decrease in ovarian carcinoma cells and the inhibition of cellular proliferation of ovarian cancer cells by the above four hormones, the present study investigated if their effects were owing to an inhibition of DNA synthesis. Vessel dilator, LANP, kaliuretic peptide and ANP each at their 1 µM concentrations inhibited DNA synthesis when incubated with the human ovarian carcinoma cells for 24 hours by 85%, 69%, 76%, and 70%, respectively (p<0.001 for each) (Figure 4). Cyclic GMP (1µM) inhibited DNA synthesis in the ovarian carcinoma cells by 53% (Figure 4).

E. NPR-A and C-receptors are present in human ovarian carcinoma cells Ovarian carcinoma cells have never been evaluated to determine whether they have NPR-A and/or -C receptors. When the human ovarian carcinoma cells were evaluated by Western blots, the NPR-A and -C receptors were demonstrated to be present (Figure 5).

IV. Discussion This investigation is the first evidence of anticancer effects of four peptide hormones synthesized within the heart on any gynecological cancer. Ovarian carcinoma was

Figure 4. Decrease in DNA synthesis by atrial natriuretic peptide (ANP), kaliuretic peptide, vessel dilator, long acting natriuretic peptide (LANP), and cyclic 3’,5’ guanosine monophosphate (cGMP). The 69% to 85% decrease in DNA synthesis in the ovarian cancer cells secondary to the four cardiac hormones (each at 1 µM) was significant (p<0.001) while cyclic GMP (53% decrease) was significant at p<0.01 compared to control (i.e., untreated) cells when evaluated by repeated measures of analysis of variance (ANOVA) (n=30 for each group).

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Vesely: Four cardiac hormones cause cell death in 81% of human ovarian adenocarcinoma cells

Figure 5. Natriuretic peptide receptors (NPR) A-, and -C are present in human ovarian cancer cells as evidenced by western blot analysis with a 1:4000 dilution of R1214 polyclonal antibody directed against the COOH terminus of the natriuretic peptide A-receptor (kindly provided by Dr. David L. Garbers, University of Texas Southwestern, Dallas, Texas) and a 1:1000 dilution of Omori antibody to the NPR-C receptor (generously provided by Dr. Kenji Omori, Osaka, Japan). The left side blots are the positive (presence of signal in rat lung) controls for each of the receptors with the NPR-A receptor in human ovarian cancer cells being the upper panel at 130 Kilo Dalton (kDa) and NPR-C receptor at 66 kDa in the human ovarian carcinoma cells is shown in the lower panel. The negative controls for these experiments was absence of signal with normal rabbit serum, rabbit IgG, and after pre-absorption of NPR-A antibody with NPR-A protein or pre-absorption of NPR-C antibody with NPR-C protein. Albumin (bovine serum albumin, BSA) (70 kDa) was used in addition to Bio Rad Percision Plus Dual color standards to identify bands corresponding to the NPR-A and –C receptor, respectively. Reprobing with actin was used as a loading control. The ovarian carcinoma cells for receptor analysis were scraped from 100 mm dishes in ice cold mammalian protein extraction reagent (M-PER; Pierce, Rockford, IL, USA) containing HaltTM phosphatase inhibitor (Pierce) and HaltTM protease inhibitor (Pierce).

These peptide hormones’ anticancer effects are at concentrations above concentrations at which they normally circulate in the human body. The circulating concentrations in healthy humans of LANP, vessel dilator, kaliuretic peptide and ANP are 1528±158 pg/ml; 1595±157 pg/ml; 213±42 pg/ml, and 63±2 pg/ml, respectively (Daggubati et al, 1997). These peptide hormones do increase during the trimesters of a normal pregnancy (Merkouris et al, 1990) and increase further in the circulation in pre-eclampsia (Merkouris et al, 1991) but their concentrations in pre-eclampsia are not as high as the pharmacological concentrations needed for significant anticancer effects. This is similar to essentially all currently used anticancer chemotherapeutic agents, i.e. they are utilized at pharmacological rather than physiological concentrations to elicit anticancer effects. Each of the four peptide hormones from the cardiac ANP prohormone inhibited 69%-85% of DNA synthesis in the human ovarian carcinoma cells. We have previously demonstrated that the DNA synthesis-inhibiting properties of these peptide hormones synthesized by the cardiac ANP gene are directly due to the peptide hormones themselves

as when their specific antibodies are incubated with the peptide hormones the antibodies completely block these peptide hormones ability to decrease cancer cell number and their DNA synthesis (Vesely et al, 2005b). The antibodies by themselves did not block DNA synthesis (Vesely et al, 2005b). These findings suggest that one important mechanism of action of the four peptide hormones in the present investigation’s ability to inhibit cancer cell number is via their ability to inhibit DNA synthesis. These four peptide hormones have been demonstrated to localize to the nucleus of cancer cells where they can directly inhibit DNA synthesis (Saba et al, 2005). Their ability to inhibit DNA synthesis is specifically mediated by the intracellular messenger cyclic GMP, which inhibited DNA synthesis itself in the ovarian cancer cells, as utilizing a cyclic GMP antibody completely blocks each of these peptide hormones’ ability to inhibit DNA synthesis (Gower et al, 2005). The present investigation is the first evaluation of whether ovarian cancer cells contain cardiac hormone receptors. The NPRA and NPR-C receptors were present in these ovarian

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Cancer Therapy Vol 5, page 103 De Palo EF, Woloszczuk W, Meneghetti M, DePalo CB, Nielsen HB, and Secher NH (2000) Circulating immunoreactive proANP (1-30) and proANP (31-67) in sedentary subjects and athletes. Clin Chem 46, 843-847. Franz M, Woloszczuk W, and Horl WH (2000) N-terminal fragments of the proatrial natriuretic peptide in patients before and after hemodialysis treatment. Kidney Int 58, 374378. Franz M, Woloszczuk W, and Horl WH (2001) Plasma concentration and urinary excretion of N-terminal proatrial natriuretic peptides in patients with kidney diseases. Kidney Int 59, 1928-1934. Gower WR Jr, Vesely BA, Alli AA, and Vesely DL (2005) Four peptides decrease human colon adenocarcinoma cell number and DNA synthesis via guanosine 3’5’-cyclic monophosphate. Int J Gastrointestinal Cancer 36, 77-88. Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, Whang-Peng J, Rogan AM, Green WR, and Ozols RF (1983) Characterization of a human ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors. Cancer Res 43, 5379-5389. Hunter EFM, Kelly PA, Prowse C, Woods FJ, and Lowry PJ (1998) Analysis of peptides derived from pro atrial natriuretic peptide that circulate in man and increase in heart disease. Scan J Clin Lab Invest 58, 205-216. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, and Thun MJ (2006) Cancer statistics, 2006. CA Cancer J Clin 56, 106-130. McGuire WP (2006) Is it time for some new approaches for treating advanced ovarian cancer? J Natl Cancer Inst 98, 1024-1026. McGuire WP, Hoskins WJ, Brady MF, Kucera PR, Partridge EE, Look KY, Davidson M, and Clarke-Pearson DL (1996) Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N Eng J Med 334, 1-6. Merkouris RW, Miller FC, Catanzarite V, Quirk JG Jr, Rigg LA, and Vesely DL (1991) The N-terminal and C-terminal portions of the atrial natriuretic factor prohormone increase during preeclampsia. Am J Obstet Gynecol 164, 1197-1202. Merkouris RW, Miller FC, Catanzarite V, Rigg LA, Quirk JG Jr, and Vesely DL (1990) Increase in the plasma levels of the Nterminal and C-terminal portions of the prohormone of atrial natriuretic factor in normal pregnancy. Am J Obstet Gynecol 162, 859-864. Naora H, Montell DJ (2005) Ovarian cancer metastasis: integrating insight from disparate model organisms. Nat Rev Cancer 5, 355-366. Saba SR, Garces AH, Clark LC, Soto J, Gower WR Jr, and Vesely DL (2005) Immunocytochemical localization of atrial natriuretic peptide, vessel dilator, long acting natriuretic peptide, and kaliuretic peptide in human pancreatic adenocarcinomas. J Histochem Cytochem 53, 989-995. Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103, 211-225. Sun Y, Eichelbaum EJ, Wang H, and Vesely DL (2006a) Vessel dilator and kaliuretic peptide inhibit ERK 1/2 in human prostate cancer cells. Anticancer Res 26, 3217-3222. Sun Y, Eichelbaum EJ, Wang H, and Vesely DL (2006b) Atrial natriuretic peptide and long acting natriuretic peptide inhibit ERK 1/2 in prostate cancer cells. Anticancer Res 26, 41434148. Vesely BA, Alli AA, Song S, Gower WR Jr, Sanchez-Ramos J, and Vesely DL (2005b) Four peptide hormones specific decrease (up to 97%) of human prostate carcinoma cells. Eur J Clin Invest 35, 700-710. Vesely BA, Eichelbaum EJ, Alli AA, Sun Y, Gower WR Jr, and Vesely DL (2006a) Urodilatin and four cardiac hormones

carcinoma cells to help mediate ANP’s anticancer effects in the ovarian cancer cells. In addition to these four cardiac hormones inhibiting DNA synthesis within cancer cells, which appears to be their final step in their mechanism of action in cancer cells, these four peptide hormones also decrease the activation of extracellular-signal regulated kinase (ERK) 1/2 (Sun et al, 2006a,b). Extracellular signal-regulated kinase is a mitogen activated protein kinase (MAP kinase) important for the growth of cancers (Davis, 2000; Schlessenger, 2000). Mitogens such as epidermal growth factor which activate ERK 1/2 to cause proliferation and growth of cancer cells (Schlessenger, 2000) have up to 97% of their activation blocked by these four cardiac hormones (Sun et al, unpublished observation). These four cardiac hormones also directly block up to 97% of the phosphorylation (i.e. activation) of ERK 1/2 (Sun et al, 2006a,b). The ERK 1/2 cascade final step in proliferation of cancer cells is the stimulation of DNA synthesis within the cancer cells (Schlessenger, 2000). As demonstrated in the present investigation of human ovarian carcinoma cells, this step is also blocked by the four cardiac hormones. There was an estimated 15,310 ovarian cancer deaths in 2006 in the United States with surgery and current chemotherapy i.e. for every four new cases diagnosed there were three deaths from ovarian cancer in 2006 (Jemal et al, 2006). This knowledge plus the fact that up to 60% of ovarian carcinomas are in advanced stages at the time of diagnosis (Naora H and Montell DJ, 2005) indicates there is definite need to develop new approaches for the therapy of ovarian cancer. The present investigation details not only one but four new potential therapies, which kill up to 81% of human ovarian cancer cells within 24 hours. These four human cardiac hormones which circulate normally in the human body (Winters et al, 1989; Vesely et al, 1989; Daggubati et al, 1997; Hunter et al, 1998; DePalo et al, 2000; Frantz et al, 2000, 2001) have no known cytotoxic effects to normal cells (Vesely et al, 2003). It is also important to note that vessel dilator eliminates more of these ovarian adenocarcinoma cells in vitro than clinically relevant concentrations of Adriamycin, melphalan and cisplatin (Hamilton et al, 1983).

Acknowledgements We thank Charlene Pennington for excellent secretarial assistance. This investigation supported in part by Merit Awards from the United States Department of Veterans Affairs (DLV, WRG, Jr).

References Alberts DS (1999) Treatment of refractory and recurrent ovarian cancer. Semin Oncol 26, 8-14. Daggubati S, Parks JR, Overton RM, Cintron G, Schocken DD, and Vesely DL (1997) Adrenomedullin, endothelin, neuropeptide Y, atrial, brain, and C-natriuretic prohormone peptides compared as early heart failure indicators. Cardiovascular Res 36, 246-255. Davis RJ (2000) Signal transduction by the JNK group of MAP kinases. Cell 103, 211-225.

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Vesely: Four cardiac hormones cause cell death in 81% of human ovarian adenocarcinoma cells decrease human renal carcinoma cell number. Eur J Clin Invest 36, 810-819. Vesely BA, Fitz SR, Gower WR Jr, and Vesely DL (2006b) Vessel dilator: Most potent of the atrial natriuretic peptides in decreasing the number and DNA synthesis of squamous lung cancer cells. Cancer Lett 232, 226-231. Vesely BA, McAfee Q, Gower WR Jr, and Vesely DL (2003) Four peptides decrease the number of human pancreatic adenocarcinoma cells. Eur J Clin Invest 33, 998-1005. Vesely BA, Song S, Sanchez-Ramos J, Fitz SR, Alli A, Solivan SM, Gower WR Jr, and Vesely DL (2005c) Five cardiac hormones decrease the number of human small-cell cancer cells. Eur J Clin Invest 35, 388-398. Vesely BA, Song S, Sanchez-Ramos J, Fitz SR, Solivan SM, Gower WR Jr, and Vesely DL (2005a) Four peptide hormones decrease the number of human breast adenocarcinoma cells. Eur J Clin Invest 35, 60-69. Vesely DL (1997) Signal transduction: Activation of the guanylate cyclase-cyclic guanosine-3â&#x20AC;&#x2122;5â&#x20AC;&#x2122; monophosphate system by hormones and free radicals. Am J Med Sci 314, 311-323. Vesely DL, Clark LC, Garces AH, McAfee QW, Soto J, and Gower WR, Jr (2004) Novel therapeutic approach for cancer using four cardiovascular hormones. Eur J Clin Invest 34, 674-682. Vesely DL, Norsk P, Winters CJ, Rico DM, Sallman AL, and Epstein M (1989) Increased release of the N-terminal and Cterminal portions of the prohormone of atrial natriuretic

factor during immersion-induced central hypervolemia in normal humans. Proc Soc Exp Biol Med 192, 230-235. Waldman SA, Rapoport RM, and Murad F (1984) Atrial natriuretic factor selectively activates membranous guanylate cyclase and elevates cyclic GMP in rat tissues. J Biol Chem 259, 14332-14334. Winters CJ, Sallman AL, Baker BJ, Meadows J, Rico DM, and Vesely DL (1989) The N-terminus and a 4000 molecular weight peptide from the mid portion of the N-terminus of the atrial natriuretic factor prohormone each circulate in humans and increase in congestive heart failure. Circulation 80, 438449.

David L. Vesely

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Cancer Therapy Vol 5, page 105 Cancer Therapy Vol 5, 105-112, 2007

Symptom clusters among cancer patients and effects of an educational symptom control intervention 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 University Distinguished Professor, 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: symptom cluster; cancer; intervention; symptom control; utilization of services Abbreviations: American Joint Committee on Cancer, (AJCC) Received: 12 December 2006; Revised: 16 January 2007 Accepted: 28 January 2007; electronically published: March 2007

Summary An abundance of research has observed that patients with cancer experience multiple concurrent symptoms, and recent research suggests that certain clusters of symptoms may have a synergistic effect on one another and on future patient outcomes. In this study we first identified relevant symptom clusters for a sample of cancer patients, and then tested the effectiveness of an educational symptom control intervention in reducing the severities of these symptom clusters. Finally, we investigated whether the symptom control intervention had a carry-over effect on utilization of physician, hospital and emergency room services. Two hundred twenty-two patients diagnosed with a solid tumor (breast, lung, other) and undergoing the first two cycles of their first course of chemotherapy were recruited and randomly assigned to an intervention group (N=110) or a control group receiving usual care (N=112). From the fifteen cancer-related symptoms under consideration, a factor analysis identified three important symptom clusters: cluster A (nausea, vomiting, diarrhea, poor appetite), cluster D (insomnia, pain, fatigue) and cluster E (difficulty breathing, cough). The intervention proved to be effective in reducing severities for symptom cluster D, as well as reducing the number of hospitalizations. The results of this study provided valuable insight into symptom clusters associated with cancer and its treatment, and revealed that the well recognized pain, fatigue, insomnia cluster was favorably impacted by the educational intervention. On the clinical level, the results suggest that physicians and oncology nurses should think in terms of these naturally clustering symptoms when recommending plans for symptom management, in order to be more effective in the overall management of a larger spectrum of symptoms.

the presence of two or more symptoms, these symptoms may have a catalytic effect on one another. For example, pain could be perceived considerably worse in the presence of fatigue or nausea, or in the presence of both symptoms, pain could be proportionally more severe. Along the same line, Barsevick and colleagues have noted in 2006 that pain could result in sleep disturbances and fatigue. Thus, common elements among individual symptoms may link them as members of a cluster. Certain clusters of symptoms may have a synergistic effect on one another and on future patient outcomes. For example, patient quality of life as well as functional limitations have been accounted for by sets or groups of symptoms (Given et al, 1993). Miaskowski and colleagues have in 2004

I. Introduction An abundance of research has observed that patients with cancer experience multiple concurrent symptoms. Prevalent symptoms among patients undergoing a course of chemotherapy include pain, fatigue, insomnia, loss of appetite, nausea, diarrhea, constipation, weight loss, dry mouth, mouth sores and skin problems (Mor et al, 1992; Given et al, 1993,1994; Sarna 1993; Donnelly and Walsh, 1995; Berger, 1998; Chang et al, 1998; Morasso et al, 1999; McCorkle et al, 2000). The complexity and multi-dimensionality of symptoms have recently received attention in the literature. Lenz and colleagues suggested in 1997 that in

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Kurtz et al: Symptom clusters identified shared variance among several symptoms and correlation between symptom pairs such as fatigue-pain, fatigue-insomnia, pain-depression as an indication of symptom clustering. Dodd and colleagues suggested in 2001 that a symptom cluster should consist of a group of three or more concurrent symptoms that are related to each other that may or may not have a common etiology. According to Miaskowski and colleagues, 2004, the symptoms in a cluster may be related through a common mechanism or etiology, by sharing common variance, or producing different outcomes than individual symptoms. A clear scientific basis for the requirement of at least three symptoms in a cluster has not been established, and since there does not seem to be a consensus on this issue, we have adopted the point of view that a cluster should consist of two or more symptoms that are concurrent and related (conceptually related and all pairwise correlations are significant). In the past, an important component of symptom management has been reducing patient emotional distress and enhancing coping. In recent years, several studies have refocused symptom management by exploring interventions addressing multiple cancer-related physical symptoms such as nausea, pain and fatigue, and have established that patients can learn to incorporate strategies to reduce their symptom severity (Burish and Tope 1992; Smith et al, 1994; Breitbart and Payne 1998; Roscoe et al, 2000; Thomas and Weiss 2000; Hickock et al, 2001; Morrow et al, 2003; Given et al, 2004). However, a dearth of research has addressed the question of whether such educational interventions not only help to alleviate symptoms, but also result in reduced use of health care services. In past research this team examined aspects of utilization of services by cancer patients (Kurtz et al, 2005). The current work extends this investigation by inquiring how an educational intervention, working through clusters of physical symptoms, may influence utilization of services. Thus, we designed a study to investigate the following research questions: 1. What are relevant symptom clusters for cancer patients undergoing chemotherapy? 2. Does an educational symptom control intervention effectively reduce the severities of these symptom clusters? 3. Is there a carryover effect resulting in a reduction of physician visits, hospitalizations and emergency room visits? More specifically, we first identified relevant symptom clusters from 15 cancer-related symptoms (nausea, vomiting, trouble sleeping, difficulty breathing, diarrhea, coordination problems, poor appetite, fever, cough, dry mouth, constipation, mouth sores, inability to concentrate, pain and fatigue) for a sample of cancer patients, and then tested the effectiveness of an educational intervention in reducing the severities of these symptom clusters. Importantly, we have taken the research further by investigating the clustering of symptoms after twenty weeks rather than at the beginning of the intervention (baseline), in order to capture the full spectrum of symptoms produced by the cancer and its treatment. Most research on symptom clusters has been

cross-sectional in nature, and no study could be located that investigated symptom clusters further along the trajectory of the cancer experience in the context of a longitudinal intervention. Finally, we investigated whether the symptom control intervention had a carry-over effect on utilization of physician, hospital and emergency room services.

II. Materials and Methods A. Sample The present study used data from a larger longitudinal controlled intervention. To be eligible for the larger study, patients had to be 21 years of age or older, recently diagnosed with a solid tumor, have a designated family caregiver, and have completed no more than the first two cycles of their first chemotherapy prior to their baseline interview. Patients who had undergone chemotherapy for a previous disease were not eligible, nor were patients receiving radiation therapy at time of entry. Patients had to agree to telephone interviews at baseline, 10 and 20 weeks. In addition, patients randomly assigned to the intervention group had to commit to participate in a 10-contact, 20-week educational intervention administered by nurses and targeted to symptom management. Nurse recruiters from two comprehensive cancer centers and four community oncology settings were trained according to study protocol and approached 609 patients who were undergoing a first course of chemotherapy. Two hundred sixtythree patients agreed to participate and signed consent forms. Among the 346 patients who refused participation, 155 (45%) were not interested, 59 (17%) had no caregiver, 55 (16% were too overwhelmed by the disease and its treatment, and 48 (14%) indicated that they were too busy, or indicated other personal reasons. Of the 263 patients who signed the consent forms, 26 did not complete the intake interview because they were too ill, or had discontinued their chemotherapy. The remaining 237 patients were first stratified by cancer site and then randomized into either the 10-contact, 20-week intervention, which focused on managing symptoms, or to a conventional care control group. Conventional care was the usual practice for each setting. Of these 237 patients, 222 reported information on their service utilization, and these patients (110 in the intervention group, 112 in the control group) formed the sample for the study. The baseline interview was administered to the patients by telephone within two weeks of recruitment into the study. Patients assigned to the intervention group were contacted to begin the intervention. The 10 nursing intervention contacts occurred in alternating fashion, with the five in-person encounters coinciding with regular visits to the oncology center, and the five intervening telephone intervention contacts occurring approximately two weeks following each in-person contact. A letter informed patients assigned to the control group that they would receive standard care. All patients received follow-up data collection interviews at 10 weeks (midpoint) and 20 weeks (end of intervention). Forty-seven patients were lost to attrition between baseline and the 10-week interview, and an additional 39 patients were lost between the 10- and 20-week interviews, leaving 136 patients that completed all three interviews. Of these 136 patients, 127 provided data on their service utilization at 20 weeks. We analyzed the effects of attrition by comparing patients that were retained and lost to attrition at either 10 or 20 weeks, both by group and combined, according to gender, cancer stage, cancer site and recruitment location. In addition, we tested for differences in baseline values of physician visits, hospitalizations, emergency room visits, and comorbid conditions by group. Overall, there were no differences in attrition rates between the two groups, either at 10 weeks or 20

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Cancer Therapy Vol 5, page 107 weeks. Retention rates for the intervention group at 10 and 20 weeks were 82.7% and 58.2%, respectively, while the corresponding rates for the control group were 75.0% and 64.3%. There were however higher rates of attrition at 10 weeks for male patients in the intervention group (28.6% versus 12.3% for females, p=0.046), as well as for the entire sample (32.1% versus 16.5% for females, p=0.020).

problems, arthritis, rheumatism, fractured hip, liver disease, other major health problem), which they currently experienced. A comorbidity index was computed as a count of the number of comorbid conditions present. For this study, we employed the Tumor, Node, Metastasis (TNM) staging system 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) (Henson, 1985; 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). Data from audits of patient records were used to determine the patients’ treatment status (currently in treatment or not) at 10 weeks and 20 weeks. Service utilization was assessed in terms of the number of physician visits, hospitalizations and emergency room visits. Patients were asked at each interview (baseline, 10 weeks and 20 weeks) whether they had visited their primary care physician and/or medical oncologist due to their cancer treatment during the past three months, and if so, how many times. Thus the utilization of physician’s services was calculated as the total number of visits to a primary care physician or medical oncologist during the past three months. Similar measures were constructed for the number of hospitalizations (separate admissions) and emergency room visits during the past three months. Patients were queried on the frequency and severity of 15 cancer-related symptoms (nausea, vomiting, trouble sleeping, difficulty breathing, diarrhea, coordination problems, poor appetite, fever, cough, dry mouth, constipation, mouth sores, inability to concentrate, pain and fatigue). More precisely, at baseline, 10 weeks and 20 weeks, patients were asked how many days during the past two weeks they had experienced each of these 15 symptoms, and then to rate their severity on a scale of 010 (0=not present,…, 10=worst possible). Informed consent procedures for the intervention study were approved by the appropriate university committee on research involving human subjects as well as the institutional review boards of the participating recruitment sites.

B. Intervention The 10-contact (5 in person, 5 by telephone) 20-week nursing intervention was guided by a model drawing heavily on Bandura’s framework (Bandura, 1977,1997) which posits that self-management strategies are learned through practice and skills mastery based on verbal persuasion that a strategy will work, for example, to reduce symptom severity. However, as D’Zurrilla and Nezu have noted in 2001, solutions for specific problems require specific follow-up and evaluation. If a strategy is effective it can be continued; if not, then modifications must be made. For example, the nurse and patient identified problems, the nurse proposed interventions and together they evaluated the patient’s ability to undertake appropriate strategies that, if performed successfully, could address the symptom management problem. More specifically, patients who received the intervention were queried at each contact regarding the severity of 15 symptoms (nausea, vomiting, trouble sleeping, difficulty breathing, diarrhea, coordination problems, poor appetite, fever, cough, dry mouth, constipation, mouth sores, inability to concentrate, pain and fatigue). Severity of the symptoms was rated by patients on an 11-point scale, ranging from zero (not present) to ten (worst possible). When severity was rated as five or higher, that symptom was transferred to the plan of care to be addressed by the nurse for symptom management. As an example, if a patient reported severity of pain at level five, the nurse would determine what medications were being taken, and if they were being taken at appropriate intervals and at the prescribed dosage. The nurse might suggest a pain diary to capture the pattern and level of severity, would encourage the patient to communicate the level of pain to the oncologist, and might suggest other strategies for pain management to complement the pain medication. All recommended intervention strategies were recorded in a computer-guided documentation. At subsequent interviews, previously recommended strategies were assessed to determine if they had been tried, and if they were successful. If the strategy had not been tried, the nurse worked with the patient to determine why the strategy was not implemented, and might suggest revisions. If a strategy was effective, it was retained. Plans were adjusted based on intervention effectiveness and problem resolution. Quality assurance was completed for all nurses on a monthly basis to ensure that they followed protocol at all sites. Monthly audiotapes of nurses’ calls with their patients were completed by each nurse, and were audited for completeness and quality by the Quality Assurance Coordinator. In addition, monthly reviews with in person feedback sessions were held with all nurse interveners to review the fidelity of implementing the recommendations for all their telephone encounters.

D. Analysis All analyses were performed on data from the 20 week interview (N=127), except when controlling for baseline values of certain variables. As an initial step, basic descriptive statistics were computed for the socio-demographic variables as well as means, standard deviations and pairwise correlations for all scale variables employed in the study. In order to determine valid symptom clusters, the symptom frequency variables at 20 weeks were first submitted to a principal components factor analysis with varimax rotation. In this way we identified potential clusters of symptoms that tended to occur concurrently. Only factors with eigenvalue greater than one were accepted, and loading coefficients in the rotated component matrix less than 0.40 were suppressed. From the factors thus generated, only those were accepted that included conceptually related symptoms that were pairwise significantly correlated. Potential symptom clusters were also required to be non-overlapping. Once these natural symptom clusters were determined, a summated symptom severity subscale was computed for each of the symptom clusters. The effect of the intervention on each of the symptom clusters established at 20 weeks was assessed with a separate analysis of variance model that included group, cancer site, stage

C. Measures The baseline interview elicited patient demographic information such as age, gender and comorbid conditions. Information on cancer site and stage of disease was obtained through audits of patient records. Time dependent variables such as symptom severity and chemotherapy status were measured at baseline, ten weeks (midpoint), and 20 weeks (end of intervention). Patients were asked at baseline to identify from a list of 12 comorbid conditions (high blood pressure, diabetes, other cancer, chronic bronchitis/emphysema, heart problems, stroke, emotional

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Kurtz et al: Symptom clusters of disease and chemotherapy status as factors, while adjusting for comorbid conditions and baseline values of the corresponding symptom severity measure for the cluster. Finally, the effects of the intervention on utilization of services (physician visits, hospitalizations and emergency room visits) were tested with separate analysis of variance models, each having group, cancer site, stage of disease and chemotherapy status as factors, while including comorbid conditions, baseline values of the service utilization variable and the individual symptom cluster severity measures as covariates.

Means and standard deviations for the service utilization variables and symptom clusters are presented in Table 3. A preliminary examination of baseline and 20 week values for the intervention and control groups suggests that the intervention may have played a role in decreasing symptom severities for cluster D, and reducing the number of hospital and emergency room visits. These possible effects were analyzed more closely in the analysis of variance models. The correlation results (Table 4) suggest that symptom cluster D may play a role in physician visits, while clusters A, D and E are significantly correlated with emergency room visits. Also of note is the substantial correlation of comorbidity with physician visits as well as with symptom clusters A and D. Finally, none of the symptom clusters was significantly correlated with hospitalizations. The regression analyses for the symptom clusters (Table 5) revealed that the intervention was indeed successful in significantly reducing symptom severities for symptom cluster D. Baseline values of symptom severity for clusters D and E were also significant in the sense that patients with high symptom severity levels for these clusters at baseline were likely to also have elevated levels for these clusters at 20 weeks. Patients with more comorbid conditions tended to have higher symptom severity levels for symptom clusters A and D, confirming the correlation results. Patients with breast or lung cancer experienced higher severity levels for symptom cluster D than patients with other cancers, while lung cancer patients also experienced higher levels for cluster E than patients with other cancers.

III. Results Demographic data are presented in Table 1. The average patient age was 58.3 years, 81.7% were women, 62.9% suffered from late stage disease, and 65.1% were still in treatment. Breast cancer accounted for 45.7% of cases, lung cancer 26.8% and the remaining 27.6% suffered from colon, prostate, gynecological, pancreatic, lymphoma or uterine cancer. The factor analysis provided six potential factors, of which four were non-overlapping, conceptually related, and accounted for 11 of the 15 individual symptoms (Table 2). Three of these clusters (A, D and E) satisfied the additional criteria that all pairs in the cluster were significantly correlated and were retained in the model. Cluster C was rejected on the basis of its overlap with cluster D, while cluster F was rejected because of weak correlation between the items in the cluster. Finally, cluster B was rejected as the items in the cluster had no obvious conceptual relation. Thus the final symptom clusters were cluster A (nausea, vomiting, diarrhea, poor appetite), cluster D (insomnia, pain, fatigue) and cluster E (difficulty breathing, cough).

Table 1. Patient Sociodemographic Characteristics, Cancer Site, Stage, Treatment Status at 20 Weeks, by Group and Combined (N=127).

Characteristic Gender Female Male Age Mean SD Comorbidity Mean SD Cancer Site Breast Lung Other Stage of Disease Early Late Treatment Status Yes No

Intervention Group (N=58) N %

Control Group (N=69) N %

Combined (N=127) N

47 11

81.0 19.0

56 12

82.4 17.6

103 23

81.7 18.3

60.2 10.4

56.8 11.4

58.3 11.0

2.3 1.4

1.8 1.6

2.0 1.5

27 14 17

46.6 24.1 29.3

31 20 18

44.9 29.0 26.1

58 34 35

45.7 26.8 27.6

22 35

38.6 61.4

24 43

35.8 64.2

46 78

37.1 62.9

19 39

32.8 67.2

25 43

36.8 63.2

44 82

65.1 34.9

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Cancer Therapy Vol 5, page 109 Table 2. Rotated Factor Matrixâ&#x20AC; with Component Loading Factors (N=136)*. Symptom

Cluster A

Nausea Vomiting Insomnia Difficulty breathing Diarrhea Coordination problems Poor appetite Fever Cough Dry mouth Constipation Mouth sores Inability to concentrate Pain Fatigue

0.731 0.682

Percent variance explained

23.6

Cluster B (rejected)

Cluster C (rejected)

Cluster D

Cluster E

Cluster F (rejected)

0.830 0.763 0.560

0.486 0.766

0.536 0.463 0.842 0.825 0.620 0.642 0.753

10.9

0.495 0.624

0.582 0.426

8.6

8.2

6.9

6.7

â&#x20AC; Principal components analysis with varimax rotation *All available data at 20 weeks were used to determine symptom clusters.

Table 3. Means and Standard Deviations for Physician Visits, Hospitalizations, Emergency Room Visits and Symptom Clusters, at Baseline and 20 Weeks, by Group (N=127).

Service Utilization Variable Physician Visits Hospitalizations Emergency Room Visits Symptom Cluster Cluster Aa Cluster Dd Cluster Ee

Intervention Group (N=58) Baseline 20 Weeks Mean SD Mean SD

Control Group (N=69) Baseline 20 Weeks Mean SD Mean SD

Combined (N=127) Baseline 20 Weeks Mean SD Mean SD

0.61 0.07 0.33

1.36 0.32 0.81

0.79 0.04 0.18

1.24 0.19 0.63

0.74 0.20 0.46

1.32 0.47 1.98

0.57 0.26 0.57

1.13 0.68 1.97

0.68 0.14 0.40

1.33 0.41 1.54

0.67 0.16 0.39

1.18 0.53 1.52

7.33 9.33 1.14

7.56 6.20 2.65

2.03 5.53 1.50

5.48 5.79 2.96

8.21 10.15 2.29

8.20 6.10 4.26

2.16 8.43 2.45

4.41 7.05 3.94

7.80 9.77 1.76

7.79 6.13 3.64

2.10 7.11 2.02

4.91 6.64 3.55

Nausea, vomiting, diarrhea, poor appetite Pain, fatigue, insomnia e Difficulty breathing, cough d

Table 4. Correlations at 20 Weeks for All Study Variables (N=127).

Hospitalizations ER Visits Cluster Aa Cluster Dd Cluster Ee Comorbidity

Physician Visits 0.037 0.112 0.154 0.236** 0.100 0.315**

Hospitalizations 0.300** 0.143 0.003 0.129 0.071

Nausea, vomiting, diarrhea, poor appetite Pain, fatigue, insomnia e Difficulty breathing, cough d

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ER Visits

Cluster A

Cluster D

Cluster E

0.208* 0.267** 0.241** 0.209*

0.468** 0.319** 0.332**

0.420** 0.344**

0.178*

Kurtz et al: Symptom clusters Table 5. Regression Analysis for Symptom Clusters (N=127).

Baseline Value Group (1=Intervention, 2=Control) Cancer Site Breast versus Other Lung versus Other Stage of Disease (1=Early, 2=Late) Chemotherapy Status (1=Treatment, 0=No Treatment) Comorbid Conditions

Cluster Aa Coeff Sig 0.076 0.164 -0.499 0.564

Cluster Dd Coeff Sig 0.499 0.000 -3.105 0.001

Cluster Ee Coeff Sig 0.480 0.000 -0.556 0.297

0.550 0.872 -0.889

0.650 0.473 0.408

3.957 3.826 -0.032

0.003 0.005 0.979

0.520 2.108 0.046

0.480 0.006 0.944

-0.764

0.446

-1.237

0.260

-0.256

0.675

1.159 0.000 R2=0.160

1.126 0.002 R2=0.439

0.307 0.093 R2=0.398

Nausea, vomiting, diarrhea, poor appetite Pain, fatigue, insomnia e Difficulty breathing, cough d

The evidence for effectiveness of the intervention in reducing utilization of health care services was mixed. The intervention did significantly reduce the number of hospitalizations (Table 6), but not emergency room visits or physician visits. Patients reporting more physician visits at baseline tended to also report more physician visits at 20 weeks, and the same was true for hospitalizations. Patients with more comorbid conditions reported more physician visits at 20 weeks, while patients with lung cancer reported more emergency room visits than patients with other cancers. Higher levels of cluster E were

predictive of more emergency room visits. Cluster D presented an apparent anomaly, in that it was uncorrelated with hospitalizations, and surprisingly, higher levels of this cluster corresponded to fewer hospitalizations. The findings with respect to utilization of services, particularly hospital visits and emergency room visits, must be viewed with some caution, as the distributions of these variables were not normal, as required by the method of analysis. However, it is well known that the analysis of variance procedure used is generally quite tolerant of deviations from these normality assumptions.

Table 6. Regression Models for Physician Visits, Hospitalizations and Emergency Room Visits (N=127).

Baseline Value Group (1=Intervention,2=Control) Cancer Site Breast versus Other Lung versus Other Stage of Disease (1=Early, 2=Late) Chemotherapy Status (1=Treatment, 0=No Treatment) Comorbid Conditions Cluster Aa Cluster Dd Cluster Ee

Physician Visits Coefficient Significance 0.230 0.010 0.263 0.242

Hospitalizations Coefficient Significance 0.576 0.000 -0.220 0.020

Emergency Room Visits Coefficient Significance 0.168 0.068 -0.262 0.378

-0.173 -0.300 -0.385

0.579 0.354 0.174

0.006 -0.072 -0.022

0.961 0.584 0.841

-0.313 -0.848 -0.061

0.435 0.047 0.863

0.180

0.479

0.076

0.459

0.179

0.583

0.215 -0.017 0.024 0.024

0.008 0.492 0.247 0.495 R2=0.222

0.036 0.010 -0.022 0.026

0.277 0.351 0.009 0.072 R2=0.302

0.096 0.012 0.028 0.091

0.368 0.724 0.301 0.049 R2=0.185

Nausea, vomiting, diarrhea, poor appetite Pain, fatigue, insomnia e Difficulty breathing, cough d

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Cancer Therapy Vol 5, page 111 on pain relief, which is physician driven, as pain medication and its proper dosage and timing lies in the province of the physician, and can be implemented in the home setting. The results of this study provided valuable insight into symptom clusters associated with cancer and its treatment, and revealed that the well recognized pain, fatigue, insomnia cluster was favorably impacted by the educational intervention. On the clinical level, the results suggest that physicians and oncology nurses should think in terms of these naturally clustering symptoms when recommending plans for symptom management, in order to be more effective in the overall management of a larger spectrum of symptoms. There was some carryover effect of the intervention on patient hospitalizations, but as the average number of hospitalizations was so small, particularly for the intervention group (baseline mean = 0.07, 20-week mean = 0.03), this finding may have limited clinical relevance.

IV. Discussion Of the clusters generated by the factor analysis, cluster D (pain, fatigue and insomnia) is perhaps the most easily recognized, and has been studied by other authors. In their study of this cluster, Beck, Dudley and Barsevick (2005) found that pain influences fatigue directly as well as indirectly by its effect on sleep. Given et al, (2001) also found, in a large sample (826) of elderly patients with cancer, that this cluster of symptoms had a consistent and significant effect on losses in functioning unrelated to patients’ type of cancer treatments, stage of disease or comorbid conditions. In a similar vein, (Sarna, 1993), in a study of 69 women with lung cancer, observed the concurrence of pain, fatigue and insomnia, and found these symptoms to be strongly correlated with quality of life and disruptions in functional status. The symptoms in cluster A (nausea, vomiting, diarrhea, poor appetite) are all closely associated with chemotherapy, so it was certainly expected that this group of symptoms would form a natural cluster. Other authors have mentioned nausea, vomiting and poor appetite as a natural symptom cluster (Dodd et al, 2004). The addition of diarrhea to this cluster seems natural, and is validated both by the factor analysis and the inter-item correlations (minimum pairwise correlation = 0.181, p=0.041). The symptoms in cluster E (cough, dyspnea) are most frequently associated with lung cancer, however they would be expected to occur concurrently for other cancers as well, albeit with lower frequency. This is evidenced by their substantial correlation (r = 0.340, p < 0.001). The intervention was successful in decreasing severity levels for symptom cluster D. It was particularly gratifying to see that this cluster was positively impacted by the intervention, since pain, fatigue and insomnia are so pervasive and disruptive in the lives of cancer patients, with fatigue in particular generally considered to be the most frequent and distressing symptom experienced by cancer patients (Ahles et al, 1984; Portenoy et al, 1992; Richardson 1995; Visser and Smets, 1998; Dodd et al, 2001). A further investigation of the individual symptoms in this cluster revealed that the intervention was responsible for significant reductions in fatigue (p = 0.002) and insomnia (p < 0.001), but not pain. The intervention had a direct effect on the number of hospitalizations, with patients in the intervention group reporting fewer hospitalizations than patients in the control group. In contrast, the intervention showed no direct effects for either physician visits or emergency room visits. In fact, as seen in Table 2, physician visits for the intervention group increased slightly at 20 weeks over their baseline values, while patients in the control group experienced a slight decrease. A possible explanation for this phenomenon may lie in the fact that patients in the intervention group were encouraged and empowered, as one aspect of the intervention process, to communicate their concerns regarding symptom control issues with their physicians. With respect to symptom clusters, we have noted the apparent anomaly of more severe cluster D symptoms resulting in fewer hospitalizations. A partial explanation for this unexpected result could be that the intervention for cluster D symptoms would focus primarily

Acknowledgement This research was supported by a grant from the National Cancer Institute, Grant # RO1 CA79280, Family Homecare for Cancer: A Community-Based Model, and in affiliation with the Walther Cancer Institute, Indianapolis, Indiana.

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Cancer Therapy Vol 5, page 113 Cancer Therapy Vol 5, 113-116, 2007

Neural stem cell-based cancer therapy and brain tumor stem cells Research Article

Philippe Taupin National Neuroscience Institute, Singapore and National University of Singapore and Nanyang Technological University, Singapore

__________________________________________________________________________________ *Correspondence: Philippe Taupin, Ph.D., National Neuroscience Institute, Singapore, 11 Jalan Tan Tock Seng, Singapore 308433; Tel: (65) 6357 7533; Fax: (65) 6256 9178; e-mail: obgpjt@nus.edu.sg Key words: adult neurogenesis, cancer stem cells, cellular therapy, gene therapy, central nervous system Abbreviations: brain tumor stem cells (BTSCs); cancer stem cells (CSCs); neural stem cells (NSCs); subventricular zone (SVZ) Received: 3 December 2006; Revised: 16 January 2007 Accepted: 23 January 2007; electronically published: March 2007

Summary The confirmation that neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) offers new and promising opportunities to treat a broad range of neurological diseases and injuries, particularly neurodegenerative diseases like Alzheimerâ&#x20AC;&#x2122;s and Parkinsonâ&#x20AC;&#x2122;s diseases. Beside this potential, NSCs are being considered for the treatment of brain cancers, and in view of recent developments in stem cell biology that cancer may be a stem cell disease; researchers are revisiting the origin of brain tumors and attempting to isolate and characterize brain tumor stem cells (BTSCs). The existence of BTSCs would have significant consequences for our understanding and the treatment of brain tumors.

phenotypes of the nervous system, neurons, astrocytes and oligodendrocytes, whereas neural progenitor cells are multipotent cells with limited proliferative capacity (Potten and Loeffler, 1990). Neural progenitor cells and self-renewing multipotent NSCs have been isolated and characterized from adult mammalian brain, supporting the existence of NSCs in the adult CNS (Reynolds and Weiss, 1992; Gage et al, 1995; Taupin and Gage, 2002). Because of their potential to generate the main phenotypes of the nervous system, NSCs hold the promise to cure a broad range of neurological diseases and injuries. Neurogenesis also occurs in the olfactory epithelium that produces new olfactory sensory neurons throughout adulthood, including in human (Calof and Chikaraishi, 1989; Murrell et al, 1996). Neural progenitor and stem cells have been isolated and characterized from the adult olfactory epithelium, including human; the so-called olfactory stem cells (OSCs) (Roisen et al, 2001; Winstead et al, 2005). OSCs present several advantages over NSCs for therapy. OSCs can be isolated by simple biopsy from the nose, circumventing the need for invasive surgery to isolate them, and from the patient himself, providing candidate stem cells for autologous transplantation in the nervous system (Feron et al, 1998). Therefore, OSCs provide an

I. Introduction Contrary to the long held-belief, neurogenesis occurs in the adult mammalian brain, including in human (Eriksson et al, 1998; Gage, 2000; Altman, 2000). Neurogenesis occurs primarily in two regions of the adult brain, the subventricular zone (SVZ) along the ventricle, and the subgranular zone, a layer beneath the dentate gyrus of the hippocampus (Taupin and Gage, 2002). In the SVZ, newborn cells migrate to the olfactory bulb, through the rostro-migratory stream, where they differentiate into interneurons (Lois C and Alvarez-Buylla, 1994). In the subgranular zone, newborn cells migrate to the granular layer of the dentate gyrus, where they differentiate into granule-like cells (Cameron et al, 1993). In human, contrary to other mammals, like rodents and non-human primates, no evidence of new neuronal cells in the SVZ or in the pathway to the olfactory bulb were reported, suggesting that neurons in the adult human olfactory bulb are not replaced (Sanai et al, 2004). However, new neuronal cells may migrate through a different pathway yet to be identified (Taupin, 2006). It is hypothesized that new neuronal cells in the adult brain originate from NSCs (Gage, 2000; Taupin and Gage, 2002). NSCs are selfrenewing multipotent cells that generate the main 113

Taupin: NSCs and cancer alternative and promising source of adult stem cells for therapy (Marshall et al, 2006). Cancers result from genetic and epigenetic changes in normal growth-controlled cells; the cells become unable to terminally differentiate, to control their ability to proliferate, and acquire the ability to invade other tissues and spread through the body or metastasize (Cahill et al, 1998, 1999). With the advance in stem cell biology, a debate has emerged regarding the origin of tumors. Though the traditional view considers that tumors arise from the dedifferentiation of mature cells in response to genetic and epigenetic alterations, there are now evidences, particularly outside the CNS, that a small population of cells with stem cell properties may be the target for transformation leading to tumors; a theory known as the stem cell theory of carcinogenesis (Trosko and Chang, 1989). According to this theory, cancers would originate from populations of stem cells, termed cancer stem cells (CSCs). With the recent confirmation that neurogenesis occurs in the adult brain, and NSCs reside in the adult CNS, researchers are devising strategies to use neural progenitor and stem cells to treat brain tumors and are looking for evidence that brain tumors may be stem cell diseases.

III. Brain tumor stem cells With the development in stem cell biology, it is proposed that the carcinogenic process may start in a stem cell (Trosko and Chang, 1989). According to this theory, tumors would originate from the transformation of normal stem cells to cancer cells or CSCs (Reya et al, 2001). It is proposed that because of their property to live for long period of time and divide over time (self-renewal), mutations and epigenetic changes would accumulate in stem cells, leading to aberrant growth and tumor formation (Kondo, 1983). The basis of the stem cell theory of carcinogenesis lies on the following observations: each cancer appears to be "monoclonal" in origin, tumors, like normal tissues, are heterogeneous in nature, and stem cells and cancer/tumorigenic cells share common features; selfrenewal and asymmetrical division (Nowell, 1976; Pardal et al, 2003). Asymmetrical division is a process that gives rise to two daughter cells with different developmental potentials leading to the cellular heterogeneity. Therefore, not all cells in tumors have the same ability to proliferate and maintain the growth of tumors: only a relatively small fraction of cells in tumors, the CSCs, possess the ability to proliferate and self-renew abnormally. As CSCs go through the carcinogenic process, some of them differentiate and give rise to the various cells of the tumor. These tumor cells, as opposed to CSCs, lose the ability to proliferate and self-renew, make up most of the tumors, and become the phenotypic signature of the tumor. The stem cell theory of carcinogenesis is supported by recent data showing that a minority of tumorigenic cells from myeloid leukemia have the ability to form new tumors when transplanted in immune compromised mice. As few as 100 CSCs prospectively identified and isolated from the tumors by identification of cell surface markers were able to form tumors in mice, whereas tens of thousands cells with alternate phenotypes isolated from the tumors failed to form tumors upon transplantation. The tumorigenic subpopulation could be serially passaged; each time cells within this population generate new tumors composed of mixed populations of non-tumorigenic cells that resemble the patient’s tumor phenotype. The isolation and characterization of these minor populations of cells with the stem cell properties shows that neoplastic clones are maintained exclusively by a rare fraction of cells that would originate from the transformation of normal stem cells (Lapidot et al, 1994; Bonnet and Dick, 1997; Al-Hajj et al, 2003). The identification of CSCs has profound consequences for the treatment of tumors (Al-Hajj et al, 2004). Recently, CSCs have been isolated and characterized prospectively from brain tumors. The identification of CSCs from brain tumors or brain tumor stem cells (BTSCs) has been made by applying the principles of stem cell biology to brain tumor cell populations; tumor cell populations were isolated using specific cell surface markers, and characterized for their stem and cancer cell properties in vitro and ex vivo. Hemmati and colleagues isolated in 2003, tumorigenic cells with characteristics similar to NSCs from pediatric brain tumors, including medulloblastomas and gliomas. These cells are selfrenewing multipotent; they produce proliferating

II. Neural stem cell-based cancer therapy Neural progenitor and stem cells isolated from the adult brain provide a promising model for cellular therapy, not only because of their ability to differentiate into the various cell types of the nervous system and to integrate the host tissue upon grafting (Gage et al, 1995; Suhonen et al, 1996; Shihabuddin et al, 2000), but also for their ability to migrate to degenerated, injured and tumor sites in the nervous system, when transplanted in the CNS or administered by systemic injection or through the cerebrospinal fluid (Aboody et al, 2000; Brown et al, 2003; Glass et al, 2005; Macklis, 1993; Wu et al, 2002; Fujiwara et al, 2004). Further, neural progenitor and stem cells can be genetically engineered in vitro (Gage et al, 1995; Liu et al, 1999). These properties considerably broadened the spectrum of diseases and injuries that can be treated using neural progenitor and stem cells, like Alzheimer’s disease, Huntington’s disease and multiple sclerosis -where the degeneration is widespread and for which “classic” cell transplantation may not be suitable-, and to neurological diseases caused by genetic deficiencies, like Niemann-Pick's disease (Pluchino et al, 2003; Shihabuddin et al, 2004). The properties of neural progenitor and stem cells to be genetically engineered and to migrate to tumor sites have been proposed for the treatment of brain tumors. It is proposed to genetically engineer neural progenitor and stem cells with “suicide genes”, like genes coding for cytolytic activities or antitumor cytokines: transplanted or peripherally administered such genetically engineer neural progenitor and stem cells would then migrate to the tumor sites where they would attack and destroy tumor cells (Yip et al, 2003; Shah et al, 2005).

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Cancer Therapy Vol 5, page 115 neurospheres which differentiate into neurons and glia. These cells also differentiate into abnormal cells with multiple differentiation markers and express many genes characteristic of NSCs and other stem cells, like the cell surface marker CD133, transcription factor Sox2 and neural RNA binding protein musashi 1 (Hemmati et al, 2003). Singh et al (2003) identified a population of cells in human brain tumors, medulloblastomas, astrocytomas, ependymomas and gangliogliomas that expresses the cell surface marker CD133 and elicit CSCs characteristics; the CD133+ isolated cells correspond to a small fraction of the entire brain tumor cell population, express the NSC marker nestin, exhibit increased self-renewal capacity, generate clonal tumor spheres in culture, and are capable of tumor initiation upon transplantation in to the brains of immune compromised mice. Injections as few as 100 cells from the CD133-positive cell population produced tumors that could be serially transplanted and possess the characteristics of the patient's original tumor, whereas injections of 105 cells of the CD133-negative cell population engrafted but did not cause tumor (Singh et al, 2004a). These data show that the CD133+ cell population elicits the characteristics and corresponds to a population of BTSCs. In all, these data show that brain tumors may contain a population of BTSCs, acting as tumor-founding cells at the clonal level (Singh et al, 2004b; Vescovi et al, 2006). The identification of BTSCs has profound consequences for the treatment of brain tumors, as conventional strategies to treat brain tumors may not lead to the destruction of the source of the tumors. On the one hand, stem cells that are target for transformation are quiescent cells that would resist to radiation treatment. On the other hand, recent studies report that brain tumors may originate from the SVZ (Uchida et al, 2004), a neurogenic area in the adult mammalian brain. Tumor ablation may also not result in a cure for brain tumors, as an ectopic origin for BTSCs would signify recurrent formation of the tumors. Though there are accumulated evidences for a stem origin for cancers, the hypothesis about NSCs or BTSCs as a cell population highly susceptible for neoplastic transformation and responsible for tumor recurrence after local treatments in the CNS is still highly controversial and remains to be further confirmed.

and brain cancers may not be a disease originating purely in stem cells. The origin of brain tumors remains therefore to be further investigated and confirmed.

Acknowledgments P.T. is supported by grants from the NMRC, BMRC, and the Juvenile Diabetes Research Foundation.

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IV. Conclusion The confirmation that neurogenesis occurs in the adult brain and NSCs reside in the adult CNS has tremendous consequences not only for our understanding of brain development and cellular therapy, but also for the origin, treatment and cure of brain tumors. Properties of NSCs to migrate to tumors and to be genetically engineered provide a promising opportunity to treat brain tumors. The isolation and characterization of BTSCs suggest that brain tumors may be stem cell diseases, which may lead to the development of new strategies to cure brain tumors. The stem cell theory of carcinogenesis predicts that cancer cells and normal stem cells may share common mechanisms and pathways. Therefore, the identification of BTSCs may also lead to a better understanding of the mechanism leading to carcinogenesis. However, though the evidences are compelling, cancers 115

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Sanai N, Tramontin AD, Quinones-Hinojosa A, Barbaro NM, Gupta N, Kunwar S, Lawton MT, McDermott MW, Parsa AT, Garcia-Verdugo JM, Berger MS, Alvarez-Buylla A (2004) Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 427, 740-744. Shah K, Bureau E, Kim DE, Yang K, Tang Y, Weissleder R, Breakefield XO (2005) Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression. Ann Neurol 57, 34-41. Shihabuddin LS, Horner PJ, Ray J, Gage FH (2000) Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci 20, 8727-8735. Shihabuddin LS, Numan S, Huff MR, Dodge JC, Clarke J, Macauley SL, Yang W, Taksir TV, Parsons G, Passini MA, Gage FH, Stewart GR (2004) Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology. J Neurosci 24, 10642-10651. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63, 5821-5828. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB (2004a) Identification of human brain tumor initiating cells. Nature 432, 396-401. Singh SK, Clarke ID, Hide T, Dirks PB (2004b) Cancer stem cells in nervous system tumors. Oncogene 23, 7267-7273. Suhonen JO, Peterson DA, Ray J, Gage FH (1996) Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature 383, 624-627. Taupin P, Gage FH (2002) Adult neurogenesis and neural stem cells of the central nervous system in mammals. J Neurosci Res 69, 745-749. Taupin P (2006) Neurogenesis in the adult central nervous system. C R Biol 329, 465-475. Trosko JE, Chang CC (1989) Stem cell theory of carcinogenesis. Toxicol Lett 49, 283-295. Uchida K, Mukai M, Okano H, Kawase T (2004) Possible oncogenicity of subventricular zone neural stem cells: case report. Neurosurgery 55, 977-987. Vescovi AL, Galli R, and Reynolds BA (2006) Brain tumor stem cells. Nat Rev Cancer 6, 425-436. Winstead W, Marshall CT, Lu CL, Klueber KM, Roisen FJ (2005) Endoscopic biopsy of human olfactory epithelium as a source of progenitor cells. Am J Rhinol 19, 83-90. Wu S, Suzuki Y, Noda T, Bai H, Kitada M, Kataoka K, Nishimura Y, Ide C (2002) Immunohistochemical and electron microscopic study of invasion and differentiation in spinal cord lesion of neural stem cells grafted through cerebrospinal fluid in rat. J Neurosci Res 69, 940-945. Yip S, Aboody KS, Burns M, Imitola J, Boockvar JA, Allport J, Park KI, Teng YD, Lachyankar M, McIntosh T, O'Rourke DM, Khoury S, Weissleder R, Black PM, Weiss W, Snyder EY (2003) Neural stem cell biology may be well suited for improving brain tumor therapies. Cancer J 9, 189-204.

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Cancer Therapy Vol 5, page 117 Cancer Therapy Vol 5, 117-124, 2007

An intensive sequenced adjuvant chemotherapy regimen for breast cancer, a pilot study of the cancer and leukemia group b Research Article

Sushil Bhardwaj1,*, David Duggan2, Jeffrey J. Kirshner3, Susan Woolf4, James F. Holland1, Donald A. Berry5, Larry Norton6, I. Craig Henderson7 1

Division of Medical Oncology, Mount Sinai School of Medicine, New York NY Department of Medicine, SUNY Upstate Medical University 3 Syracuse Hematology-Oncology CCOP, Syracuse, NY 4 CALGB Statistical Center, Duke University, Durham NC 5 Department of Biostatistics, MD Anderson Cancer Center, Houston TX 6 Memorial Sloan-Kettering Cancer Center, New York NY 7 Medical Oncology, Moffitt-Long Hospital, University of California at San Francisco CA 2

__________________________________________________________________________________ *Correspondence: Sushil Bhardwaj, MD, FACP, Director, Cancer Program, Chief, Sub-section Hematology/Oncology, Good Samaritan Hospital, 255 Lafayette Avenue, Suffern, NY 10901, USA; Tel: 845-368-8500; Fax: 845-368-8460; Email: sushbhard@aol.com Key words: Breast Cancer, Adjuvant Chemotherapy, CMFVP, doxorubicin Abbreviations: Cancer and Leukemia Group B, (CALGB); Cyclophosphamide, doxorubicin and 5-fluorouracil), (CAF); cyclophosphamide, methotrexate, 5-fluorouracil, vincristine and prednisone, (CMFVP); Disease-free, (DFS); overall survival, (OS); State University of New York, (SUNY)

The research for CALGB 8743 was supported, in part, by grants from the National Cancer Institute (CA31946) to the Cancer and Leukemia Group B (Richard L. Schilsky, MD, Chairman). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. The following institutions participated in the study: Institution Name CALGB Statistical Office Mount Sinai School of Medicine SUNY Upstate Medical University Syracuse Hematology-Oncology Assoc. CCOP Washington University School of Medicine

Location Durham, NC New York, NY Syracuse, NY Syracuse, NY St. Louis, MO

Principal Investigator Stephen George, Ph.D. Lewis R. Silverman, MD Stephen L. Graziano, M.D. Jeffrey Kirshner, M.D. Nancy Bartlett, MD

supported by Grant # CA33601 CA04457 CA21060 CA45389 CA77440

Received: 8 March 2007; Accepted: 13 March 2007; electronically published: April 2007

Summary In order to establish the feasibility of adjuvant chemotherapy consisting of sixteen weeks of dose-dense CMFVP (cyclophosphamide, methotrexate, 5-fluorouracil, vincristine and prednisone) administered in the original dose and schedule of the "Cooper Regimen", followed by four monthly, 3-day cycles of escalating dose doxorubicin in patients with node-positive stage II and stage III A breast carcinoma. Forty-eight women (31 pre, 17 peri/postmenopausal) with node-positive, stage II and III A breast carcinoma were treated. Median number of involved axillary nodes was 3 (range:1-27). Toxicity was primarily myelosuppression associated with the doxorubicin component. All patients recovered from treatment without sequelae. With a median follow-up of 11.8 years, the 7year disease-free survival was 55% Âą 7%. Of 31 premenopausal patients, 15 relapsed; 13 of these subsequently died. Of 17 peri/postmenopausal patients, 7 relapsed; all 7 of these later died. This study establishes the feasibility of a dose-escalated regimen (doxorubicin) following a dose-dense regimen (CMFVP) in the adjuvant setting. However, after adjusting for differences in patientsâ&#x20AC;&#x2122; prognoses, survival and disease-free survival are not very different from those of other breast cancer adjuvant therapy trials.

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Bhardwaj et al: Sequenced adjuvant chemotherapy for breast cancer A third multi-institutional, randomized trial (CALGB 8541) of three different dose-schedules of CAF (Cyclophosphamide, doxorubicin and 5-fluorouracil) demonstrated a superior disease-free and overall survival for a high dose and dose-dense arm of CAF over a lower dose-intense schedule (Budman et al, 1998). Because the response rate to VATH in metastatic disease was only 45%8 and because doxorubicin was felt to be the most active drug in the program, another more intensive single agent doxorubicin regimen was evaluated to explore the maximum tolerated dose, with the expectation that it would be more effective. Women with metastatic breast carcinoma were treated with 3-day courses of doxorubicin in escalating doses. An overall response rate of 85%, a complete response rate of 38%, and median duration of unmaintained complete or partial remission of 11 months was observed (Jones et al, 1987). These data are superior to other reported doxorubicin results for metastatic breast cancer. Hence, to confirm the sequential concept established by the superiority of CMFVP-VATH in CALGB 8082 and to take advantage of any potential benefit that might be associated with an escalation of doxorubicin dose, an adjuvant regimen was designed using CMFVP for 16 weeks, given in the identical dose and schedule as reported by Cooper in 1988, followed by another non crossresistant intensive regimen: 3-day cycles of doxorubicin. A pilot feasibility study of this regimen led to a diseasefree survival of 80.5% at 60 months (Bhardwaj et al, 1993; Holland et al, 1994). A broader confirmatory pilot study was conducted (CALGB 8743) as a limited access protocol, the results of which are reported in this communication.

I. Introduction The presence of metastases in axillary lymph nodes at the initial presentation of breast carcinoma is an important prognostic determinant for the subsequent development of systemic metastatic disease (Fisher et al, 1969). Adjuvant chemotherapeutic regimens, whether of one or multiple drugs in combination, have 5-year failure rates for node positive Stage II breast cancer of 30% to 42% (Buzdar et al, 1981; Fisher et al, 1986; Bonnadonna, 1989). Thus new approaches to the strategy of adjuvant chemotherapy are worthy of study. Several regimens of adjuvant chemotherapy have been used in an effort to diminish the recurrence rate of breast carcinoma. The â&#x20AC;&#x153;Cooper regimenâ&#x20AC;? CMFVP (cyclophosphamide, methotrexate, 5-fluorouracil, vincristine and prednisone), was initially administered to 73 women with 4 or more metastatic axillary nodes. After a median of 5.5 years of follow-up, the estimated diseasefree survival was 68% at 8 years (Cooper et al, 1979). A more recent update with follow-up extending out to 12 years continues to demonstrate similar results (Cooper, 1988). In a large multi-institutional, randomized trial (CALGB 7581) conducted by the Cancer and Leukemia Group B (CALGB), a modification of the 5-drug combination, CMFVP, was shown to be superior to the 3drug combination CMF given by identical schedule in women with 4 or more nodes involved (Tormey et al, 1983). A subsequent trial (CALGB 8082) by the CALGB involved a random allocation after seven months of CMFVP to either continue CMFVP for six more months or to receive another non cross-resistant regimen, VATH (vinblastine, adriamycin, thiotepa and halotestin), which had been shown to be active in advanced disease (Hart et al, 1981). The CMFVP-VATH regimen was based on the concept that, because cancer cell populations are diverse, with preexistent or evoked genetic changes conveying resistance, utilizing two regimens of chemotherapy in sequence may more effectively eradicate the cells that are resistant to a single adjuvant chemotherapeutic regimen. With a median follow-up duration of 11.5 years, diseasefree survival is statistically significantly superior for VATH crossover as compared with continuation of CMFVP (P = .004, median: 8.7 vs. 5.0 years). There was improved overall survival with CMFVP-VATH over CMFVP-CMFVP (P = .046; median: >14 years vs. 10 years) (Perloff et al, 1996).

II. Patients and Methods A. Patient sample The trial was activated on June 10, 1987 and closed to patient accrual after 48 entries on July 11, 1989. As a limited access pilot study, only 3 main CALGB member institutions and their affiliates accrued patients. These main members were: State University of New York (SUNY) Health Science Center at Syracuse, Mount Sinai Medical Center, New York and Washington University/Barnes Hospital, St. Louis (Table 1). The treatment regimen included intensive CMFVP (the "Cooper Regimen") followed by intensive doxorubicin. Figure 1 details the chemotherapeutic agents, their respective doses and delivery schedule.

Table 1. CALGB 8743: Patient Accrual by Institution

Main Member Mount Sinai Hospital SUNY at Syracuse Washington Univ./Barnes Hosp TOTAL:

Accrual for: Main Member Affiliate 15 2 10 20 0

CALGB = Cancer and Leukemia Group B SUNY = State University of New York Univ. = University

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Total 17 30 1 48

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Figure 1. CALGB 8743 Treatment Schema. Eligibility requirements included: (a) histologically confirmed adenocarcinoma of the breast, (b) pathologically confirmed node-positive stage II and stage III, (c) radical/modified radical mastectomy or lumpectomy with axillary staging, (d) performance status 0-1, (e) ER known or pending, (f) no prior cytotoxic regimens, (g) no prior radiation therapy, (h) < 4 weeks since mastectomy or axillary staging and (i) written informed consent. Of the 48 patients accrued, two patients were ineligible, one due to N2 axillary nodal involvement and prior RT and one due to multifocal tumors. All 48 patients received treatment.

III. Results A. Patient characteristics Table 2 shows demographic and pretreatment characteristics for patients entered onto CALGB 8743. The median patient age was 42 years. Only 6% of patients were 60 years or older. Nearly two-thirds of the sample was premenopausal. About three quarters of the patients had primary tumors larger than 2 cm. Approximately half the patients had 1-3 involved axillary nodes; 13% of patients had 10 or more involved nodes. About 80% of patients had undergone mastectomy as their primary surgery. Most patients were estrogen receptor positive (56%) or progesterone receptor positive (48%). About 40% of patients received tamoxifen after completion of chemotherapy.

B. Variables examined We examined a series of demographic and pretreatment clinical variables. These included: patient age, menopausal status, tumor size, number of positive axillary nodes, estrogen and progesterone receptor status, and type of surgery. The outcome variables were disease-free survival and overall survival. Disease-free survival was the time from study entry to first relapse or death due to any cause, for patients who did not relapse; disease-free survivors were censored at date last known to be free from disease. Patients were generally examined every 3 months in the first year, every 4 months in the second year and every six months indefinitely thereafter. Films and scans were obtained when patients were symptomatic, or annually. Overall survival was the time from study entry to death due to any cause; survivors were censored at date of last followup.

B. Disease-free (DFS) and overall survival (OS) Disease-free survival and overall survival at selected time points are shown on Table 3. At the time of reporting, 24 patients have failed (relapsed or died without relapse). Of these 24 failures, 15 premenopausal and 7 postmenopausal patients relapsed; 1 premenopausal and 1 postmenopausal patient died without relapse. Twenty-two (22) patients have died; 2 of these died without recurrent disease.

C. Statistical methodology Survival curves were drawn using the Kaplan-Meier product limit method. We used the Cox proportional hazards model to relate disease-free survival and overall survival with possible prognostic variables. We used the Pearson correlation coefficient to assess the correlation between pairs of prognostic variables. All p-values are 2-sided. According to an intent-totreat approach, all 48 patients, regardless of eligibility status, are included in analyses. Data included herein were extracted from the CALGB database in February 2001. The median follow-up is 11.8 years (range from 5.1 to 13.5 years).

C. DFS and OS (CALGB 8743 vs. CALGB 8541) We have compared the Kaplan-Meier disease-free and overall survival curves observed in the current trial (8743) with those seen with the high-dose CAF arm of the previously reported CALGB trial 8541, n=519 (Figures 2, 3). Although treatment was not randomized, and recognizing that it is always dangerous to compare groups of patients across studies, the similarity of the survival curves for the two different treatment strategies, CMFVPDoxorubicin and high-dose CAF, is striking.

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Bhardwaj et al: Sequenced adjuvant chemotherapy for breast cancer Table 2. CALGB 8743: Patient Characteristics Race: White Hispanic Black Other Age at study entry: < 40 40-49 50-59 60-69 70+ Median Menopausal status: Premenopausal Perimenopausal Postmenopausal Tumor size: ! 2 cm > 2 cm Performance score: 0

Number of positive nodes: 1-3 4-9 10+ Unknown + Median Surgery: Lumpectomy Mastectomy ER status: Negative Positive Unknown PR status: Negative Positive Unknown Received RT: No Yes

40(83%) 3(6%) 3(6%) 2(4%) 19(40%) 15(31%) 11(23%) 3(6%) 0(0%) 42; range = 26-66 31(65%) 4(8%) 13(27%) 11(23%) 37(77%)

28(58%) 13(27%) 6(13%) 1(2%) 3; range = 1-27 8(17%) 40(83%) 17(35%) 27(56%) 4(8%) 20(42%) 23(48%) 5(10%) 43(90%) 5(10%)

CALGB = Cancer and Leukemia Group B, ER = estrogen receptor, PR = progesterone receptor, RT = radiotherapy

48(100%)

Table 3. CALGB 8743: Disease-Free (DFS) and Overall Survival (OS) Time 3 years 5 years 7 years

% Disease-Free ± Standard Error 81% ± 6% 60% ± 7% 55% ± 7%

CALGB: Cancer and Leukemia Group B

Figure 2. CALGB 8743 and 8541 (High dose arm): Disease-Free Survival

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% Surviving± Standard Error 92% ± 4% 79% ± 6% 64% ± 7%

Cancer Therapy Vol 5, page 121

Figure 3. CALGB 8743 And 8541 (High Dose Arm): Overall Survival

We performed multivariate Cox proportional hazards regressions on DFS and OS using both CALGB 8743 and the high dose arm of CALGB 8541. The standard clinical prognostic variables included: number of positive nodes, tumor size, receptor status, and menopausal status. We also included an indicator variable for treatment on either study. The results in Table 4 show that number of positive nodes and tumor size were significantly correlated with DFS and OS; however, the other clinical variables were not. In addition, treatment regimen did not correlate with DFS or OS.

During doxorubicin, the most frequent grade 3+ toxicities were: leukopenia (67%), granulocytopenia (56%), nausea & vomiting (33%), and thrombocytopenia (24%) (Table 5). No cardiac toxicity or extravasation was seen. Amenorrhea was seen in 44% of premenopausal patients after CMFVP, 68% of premenopausal patients after doxorubicin, and 86% of premenopausal patients during protocol treatment and active follow-up. There was no fatal toxicity. Few toxicities reached life-threatening severity (Grade 4). Of the Grade 4 toxicities, only one case each of peripheral neuropathy, skin toxicity and granulocytopenia occurred during the CMFVP part of the regimen. The other Grade 4 leukopenia, granulocytopenia and thrombocytopenia were reported during doxorubicin treatment.

D. Toxicity During CMFVP, the most frequent grade 3+ toxicities were: leukopenia (19%), granulocytopenia (18%), peripheral neuropathy (15%), and hepatic (10%). Table 4. CALGB 8743 vs. High Dose Arm of CALGB 8541

Variable # Positive Nodes Tumor Size Receptor Status Meno status Study

Comparison 10 vs. 1 5 vs. 2 Pos vs. Neg Pre vs. Post 8541 vs. 8743

Variable # Positive Nodes Tumor Size Receptor Status Meno status Study

Comparison 10 vs. 1 5 vs. 2 Pos vs. Neg Pre vs. Post 8541 vs. 8743

Disease-Free Survival RR 95% Lower 2.21 1.74 1.55 1.23 1.28 0.97 1.31 1.04 0.94 0.62 Overall Survival RR 95% Lower 2.32 1.79 1.86 1.09 1.07 0.79 1.23 0.94 1.11 0.71

95% Upper 2.80 1.95 1.71 1.66 1.44

P-value <0.0001 0.0002 0.084 0.025 0.78

95% Upper 2.98 1.83 1.45 1.60 1.74

P-value <0.0001 0.0082 0.66 0.13 0.64

CALGB: Cancer and Leukemia Group B, Meno status = menopausal status, Pos = positive, Neg = negative

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Bhardwaj et al: Sequenced adjuvant chemotherapy for breast cancer Table 5. Severe (Grade 3) or Life-threatening (Grade 4) toxicity events, by Regimen Type Toxicity granulocytopenia leukopenia thrombocytopenia anemia infection hepatic nausea & vomiting diarrhea pulmonary neuro (cns) neuro (pns) fever without infection stomatitis phlebothrombosis skin

Grade 3 16% 19% 0% 0% 4% 10% 6% 2% 2% 2% 13% 0% 2% 2% z

CMFVP Grade 4 2% 0% 0% 0% 0% 0% 0% 0% 0% 0% 2% 0% 0% 0% 2%

Grade 3 18% 47% 22% 4% 4% 0% 33% 4% 2% 0% 0% 2% 9% 0% 0%

Doxorubicin Grade 4 38% 20% 2% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%

neuro (cns) = central nervous system, neuro (pns) = peripheral nervous system

regimens in short courses based on a different concept (Goldie and Coldman, 1979). No important differences in outcome have been ascribed to this strategy. This is presumably attributable to inadequate suppression of a sensitive population of cells by a single course of the first regimen, leaving a large fraction of cells surviving, which could rapidly regrow during a second regimen to which they might not be susceptible. The current design continued the first regimen of CMFVP for 16 weeks in the original doses and schedule described by Cooper in 1988. This had not been done in the prior CALGB studies (Tormey et al, 1983; Perloff et al, 1996). The second regimen of doxorubicin (Jones et al, 1987) was introduced as soon as host tolerance permitted, theoretically near the nadir of residual tumor burden, following the chemotherapeutic impact of the first regimen. Four cycles of dose-escalated doxorubicin administration were then given (Jones et al, 1987). Including the time for recovery from toxicity the entire course was completed in 9 months. Although no definitive statement about efficacy can be made in the absence of a randomized control arm, this sequenced adjuvant chemotherapy for node-positive breast cancer has produced encouraging results. The data support the initial pilot study which was limited to Stage II node-positive patients (Bhardwaj et al, 1993; Holland et al, 1994). Bonnadonna et al have reported superior relapse-free and overall survival for patients with breast cancer with more than three metastatic axillary nodes treated with moderately intensive doxorubicin followed by CMF compared to those patients treated with an alternating doxorubicin/CMF schedule. Ten-year relapse free survival was better for the sequential rather than the alternating regimen, 42% vs. 28%, and 10-year overall survival was 58% vs. 44% (Bonnadonna et al, 1995). The concept of therapeutic symmetry (Day 1986) supports the administration of the more effective regimen following the lesser treatment. This concept favors our approach of

IV. Discussion Metastatic breast cancer is almost always fatal. In newly diagnosed breast cancer patients at high risk of recurrence, the application of intensive adjuvant therapy is based on the premise that treatment has a greater potential for being curative when administered at a time of low tumor burden. Dose intensity has been postulated as a determinant of the efficacy of adjuvant chemotherapy for breast cancer (Hryniuk et al, 1986). CALGB 8541 demonstrated that a critical threshold of drug dose must be achieved to effect clinically significant cell-kill (Budman et al, 1998). The role of high dose chemotherapy with stem cell support for the adjuvant therapy of breast cancer patients remains to be established (Peters et al, 1999, National Institutes of Health Consensus Development Conference Statement, 2000). The eradication of breast cancer by drugs is chiefly limited by the development of resistance. The phenomenon appears to be frequently attributable to selection of preexistent populations of cells insensitive to the concentration of the agent which kills sensitive cells. The experimental design advanced in the present work is the same concept as CMFVP-VATH: an approach to the problem which takes cognizance of the Gompertzian kinetics of breast cancer growth and regression (Norton, 1985, 1988). The model suggests that even for patients with small tumor burdens, intensive therapy (dose-dense and/or doseescalated) is necessary to drive tumors beneath a theoretical cure volume. The current program allows for the application of an initial dose-dense intensive chemotherapy regimen (CMFVP) at a time of low tumor volume following surgical reduction, followed by a second non cross-resistant dose-escalated intensive regimen (doxorubicin) at a time of significant tumor regression following the first regimen. Prior studies in other tumors where two different treatments have been used have often alternated the 122

Cancer Therapy Vol 5, page 123 Day RS (1986) Treatment sequencing, asymmetry, and uncertainty; protocol strategies for combination chemotherapy. Can Res 46, 3876-3885. Fisher B, Redmond C, Fisher ER, Wolmark N (1986) Systemic Adjuvant therapy in treatment of primary operable breast cancer: National surgical adjuvant breast and bowel project experience Proceedings of the NIH consensus development conference on adjuvant chemotherapy and endocrine therapy for breast cancer: NCI Monograph 1. 35-43. Fisher B, Slack NH, Bross IDJ (1969) Cancer of the breast: Size of neoplasms and prognosis. Cancer 24, 1071-1080. Goldie JH, Coldman AJ (1979) A mathematic model for relating the drug sensitivity of tumors to their spontaneous mutation rate. Cancer Treat Rep 63, 1727-1733. Hart RD, Perloff M, Holland JF (1981) One-day VATH (vinblastine, adriamycin, thiotepa and halotestin) Therapy for Advanced Breast Cancer Refractory to Chemotherapy. Cancer 48, 1522-1527. Henderson IC, Berry DA, Demetri GD, Cirrincione CT, Goldstein LJ, Martino S, Ingle JN, Cooper MR, Hayes DF, Tkaczuk KH, FLeming G, Holland JF, Duggan DB, Carpenter JT, Frei E III, Schilsky RL, Wood WC, Muss HB, Norton L 1998 Improved disease-free and overall survival from the addition of sequential paclitaxel but not from the escalation of doxorubicin dose level in the adjuvant chemotherapy of patients with node-positive primary breast cancer (BC). Proc Am Soc Clin Oncol 17, 101a. Holland JF, Bhardwaj S, Norton L (1994) Re: “We may be lost, but we’re sure makin’ good time”. Cancer Invest 12, 270272. Hryniuk W, Levine MN (1986) Analysis of dose intensity for adjuvant chemotherapy trials in stage II breast cancer. J Clin Oncol 4, 1162-1170. Hudis CA, Seidman AD, Baselga J, Raptis G, Lebwohl D, Gilewski T, Currie V, Moynahan ME, Sklarin N, Fennelly D, et al (1995) Sequential adjuvant therapy with doxorubicin/paclitaxel/cyclophosphamide for resectable breast cancer involving four or more axillary nodes. Semin Oncol 6(Suppl 15), 18-23. Jones RB, Holland JF, Bhardwaj S, Norton L, Wilfinger C, Strashun A (1987) A phase I-II study of intensive dose adriamycin for advanced breast cancer. J Clin Oncol 5. National Institutes of Health Consensus Development Conference Statement Adjuvant Therapy for Breast Cancer: Vol. 17, No. 4, November 1-3, 2000 (http //odp.od.nih.gov/consensus/cons/114/114_statement.htm). Norton L (1985) Implications of kinetic heterogeneity in clinical oncology. Semin Oncol 12, 231-249. Norton L (1988) A gompertzian model of human breast cancer growth. Cancer Research 48, 7067-7071. Perloff M, Norton L, Korzun AH, Wood WC, Carey RW, Gottlieb A, Aust JC, Bank A, Silver RT, Saleh F, Canellos GP, Perry MC, Weiss RB, Holland JF (1996) Postsurgical adjuvant chemotherapy of stage II breast carcinoma with or without crossover to a non-cross-resistant regimen: a cancer and leukemia group B study. J Clin Oncol 14, 1589-1598. Peters W, Rosner G, Vredenburgh J, Shpall E, Crump M, Richardson P, Marks L, Cirrincione C,. Wood W, Henderson I, Hurd D, Norton L (1999) A prospective randomized comparison of two doses of combination alkylating agents (AA) as consolidation after CAF in high-risk primary breast cancer involving ten or more axillary lymph nodes (LN); preliminary results of CALGB 9082/SWOG9114/NCIC MA13. Proc Am Soc Clin Oncol 18. Abstract 2a. Tormey DC, Weinberg VE, Holland JF, Weiss RB, Glidewell OJ, Perloff M, Falkson G, Falkson HC, Henry PH, Leone LA (1983) A Cancer and Leukemia Group B study. A randomized trial of five and three drug chemotherapy and

administering doxorubicin after the CMFVP. One school of new strategies for adjuvant chemotherapy of breast cancer places major emphasis on dose intensity and dose density, utilizing sequential short courses of single drugs in higher dose with cytokine support (Hudis et al, 1995). A pilot trial of dose-dense sequential administration of Doxorubicin, Taxol and Cyclophosphamide (A-T-C) with cytokine support applies concepts similar to dose-dense CMFVP followed by doseescalated doxorubicin. The event-free survival of the former approach utilizing Taxol in an adjuvant setting in patients with " 4 nodes, with median follow-up of 39 months, is 81% (95% C.I.: 65-90%) (Hudis et al, 1995).The administration of a combination of Doxorubicin and Cyclophosphamide (AC) followed by Taxol (AC-T) in the adjuvant treatment of breast cancer has resulted in improved recurrence-free survival and overall survival for sequential AC-T over dose escalated AC (Henderson et al, 1998). This is yet another application of the concept of sequential therapy that was first studied by the CALGB in CMFVP-VATH (Perloff et al, 1996) which led to CMFVP-Doxorubicin. A further application of this concept is being studied in the current intergroup study of A-T-C. The results of the present trial of CMFVPDoxorubicin at 11.8 years median observation time, are equivalent to the best arms of other reported programs prior to the use of Taxanes in the adjuvant treatment of breast cancer. CMFVP-Doxorubicin serves as a conceptual bridge between the older applications of the strategy of sequential dose-dense treatments, CMFVP-VATH, (Perloff et al, 1996) through the recently reported therapeutic advantage of AC-T (Henderson et al, 1998) over dose-escalated AC leading to the current trial of A-TC.

References Bhardwaj S, Holland JF, Norton L (1993) An intensive sequenced adjuvant chemotherapy for breast cancer. Cancer Invest 11, 6-9. Bonnadonna G (1989) Conceptual and practical advances in the management of breast cancer. J Clin Oncol 7, 1380-1397. Bonnadonna G, Zambetti M, Valgussa P (1995) Sequential or alternating doxorubicin and CMF regimens in breast cancer with more than three positive nodes: Ten year results. JAMA 273, 542-547. Budman DR, Berry DA, Cirrincione CT, Henderson IC, Wood WC, Weiss RB, Ferree CR, Muss HB, Green MR, Norton L, Frei E 3rd (1998) Dose and dose intensity are determinants of outcome in the adjuvant treatment of stage II nodepositive female breast cancer. J Natl Cancer Inst 90, 120512. Buzdar AU, Smith TL, Blumenschein GR, et al (1981) Adjuvant chemotherapy with fluorouracil, doxorubicin and cyclophosphamide (FAC) for stage II or III breast cancer: 5year results. In Adjuvant Therapy of cancer III (Salmon SE, Jones SE, eds). New York. Grune & Stratton, pp 419-426. Cooper RG (1988) Adjuvant chemotherapy for breast cancer: 20 years experience using CMFVP chemotherapy. Semin Oncol 15, 29-34. Cooper RG, Holland JF, Glidewell O (1979) Adjuvant chemotherapy of breast cancer. Cancer 44, 793-798.

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Sushil Bhardwaj

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Cancer Therapy Vol 5, page 125 Cancer Therapy Vol 5, 125-132, 2007

Therapy of primary and metastatic murine tumors by intratumoral administration of interleukin-2 plasmid DNA with electroporation Research Article

Holly M. Horton2,*, Rod R. Planchon1, Michael Sawdey1, Larry Smith1, Alain Rolland1, David C. Kaslow3 1

Vical Inc., 10390 Pacific Center Court, San Diego, CA 92121 Xencor, 111 W. Lemon Ave., Monrovia, CA 91016 3 Merck Research Laboratories, 351 N. Sumneytown Pike, North Wales, PA 19454 2

__________________________________________________________________________________ *Correspondence: Holly Horton, 111 W. Lemon Ave., Monrovia, CA 91016, USA; Phone: 626-737-8006; Fax: 626-256-3562; e-mail: hhorton@xencor.com Key words: IL-2, plasmid DNA, electroporation, immunotherapy Abbreviations: phosphate-buffered saline (PBS); electroporation (EP); Interleukin-2 (IL-2); intratumoral (i.t.); intradermally (i.d.); plasmid DNA (pDNA); subcutaneous (s.c.) Received: 26 December 2006; revised: 4 March 2007 Accepted: 11 April 2007; electronically published: April 2007

Summary Interleukin-2 (IL-2) is an approved therapy for metastatic melanoma and renal cell carcinoma but its widespread use is limited by serious side effects associated with systemic administration of recombinant IL-2 protein. In the present study, we evaluated whether intratumoral (i.t.) injection of a plasmid DNA (pDNA) encoding IL-2 followed by electroporation (EP) of the injected tumor to facilitate pDNA uptake by tumor cells could enhance antitumor efficacy. Mice bearing subcutaneous (s.c.) Cloudman melanoma tumors were injected i.t. with 30 !g of IL-2 pDNA with or without subsequent EP of the tumor. Mice receiving the IL-2 pDNA with EP had a significant reduction in tumor growth compared to mice receiving IL-2 pDNA alone. In addition, 60% of the mice treated with the IL-2 pDNA with EP became tumor-free. The pharmacokinetics of IL-2 in the injected tumor and serum were determined following a single i.t. injection of IL-2 pDNA with or without EP. Detectable IL-2 levels in the tumor persisted for at least a week and were 2-3 fold higher with EP. In addition, IL-2 levels were 20-200 fold higher in tumors compared to sera suggesting that intralesional IL-2 pDNA therapy results in mainly localized secretion, which could reduce the severe systemic toxicity associated with recombinant IL-2. The mechanism of action of IL-2 pDNA therapy was evaluated in mice depleted of either NK cells, CD4+, or CD8+ T cells. Depletion of CD8+ T cells resulted in a marked diminution of the antitumor efficacy of IL-2 pDNA suggesting that CD8+ T cells contribute to the IL-2 pDNA antitumor response. The ability of the IL-2 pDNA-electroporation therapy to affect distant metastases was evaluated in the metastatic M5076 sarcoma model. Mice bearing intradermal M5076 tumors and treated i.t. with 30 !g of IL-2 pDNA with EP had a significant reduction in liver metastases. These results suggest that i.t. administration of IL-2 pDNA with EP is able to enhance both local and systemic antitumor efficacy of IL-2 and supports an ongoing multicenter clinical trial evaluating IL-2 pDNA-EP therapy of human metastatic melanoma.

3 times per week reduced the severity of side effects. In the latter trial, patients developed adverse events of Grade 1 and 2 severity, rather than Grade 3 and 4 severities common with intravenous recombinant IL-2 therapy. An alternative way to deliver IL-2 which may require less frequent injection and which may also result in fewer side effects is intralesional injection of a plasmid DNA (pDNA) encoding IL-2. Previously, a pDNA encoding IL-

I. Introduction Recombinant interleukin-2 (IL-2, Proleukin 速) is an approved therapy for metastatic melanoma and renal carcinoma; however, treatment is limited by serious side effects often resulting in discontinuation of the therapy (Atkins et al, 1999; Tarhini and Agarwala, 2005). Recently, Radney and colleagues demonstrated in 2003, that local intratumoral (i.t.) injection of recombinant IL-2 125

Horton et al: Therapy of primary and metastatic murine tumors by Il-2 and pDNA with EP C57BL/6 mice were injected intraperitoneally (i.p.) with 104 M5076 cells. Two weeks later, the mice were euthanized and the ascites fluid was collected and re-injected into a larger group of mice. The second ascites collection was pooled, washed three times in RPMI medium (Invitrogen, Carlsbad, CA) and resuspended in freezing medium consisting of RPMI with 40% fetal bovine serum (Hyclone, Logan, UT) and 10% dimethyl sulfoxide (Sigma, St. Louis, MO). The cells were frozen in small aliquots in liquid nitrogen for future use.

2 complexed with cationic lipid was found to significantly reduce tumor growth in several rodent tumor models (Saffran et al, 1998; Horton et al, 1999) and Phase 1 and 2 trials of a human IL-2 pDNA complexed with cationic lipid for therapy of renal carcinoma demonstrated excellent tolerability of the therapy (Figlin et al, 1999). The present research explores the technique of pDNA electrotherapy to further enhance the antitumor efficacy of intralesional IL-2. The application of brief electric pulses to tissues to facilitate intracellular uptake of administered genes is a technique which has been used successfully in animal models to deliver a wide variety of genes (Heller, 2003). Aihara and Miyazaki, 1998 and Hartikka and colleagues demonstrated in 2001 that intramuscular EP could enhance the in vivo transfection efficiency of pDNA by up to 10-100-fold. EP has also been evaluated clinically with the chemotherapeutic agent bleomycin for localized ablation of head and neck tumors, with 57% of the patients developing a partial or complete local response to the therapy (Bloom and Goldfarb, 2005). Chi and colleagues demonstrated in 2002, that i.t. injection of a combination of pDNAs encoding IL-2 and GM-CSF followed by EP reduced tumor growth in a murine hepatoma model, while i.t. delivery of IL-2 pDNA with EP reduced the growth of B16F10 melanoma in C57BL/6 mice and human esophageal tumors in nude mice (Lohr et al, 2001; Matsubara et al, 2001). As a means of further evaluating whether EP could enhance both local and systemic antitumor efficacy of IL2 pDNA, we evaluated i.t. administration of IL-2 pDNA with or without EP in immunocompetent mouse models of primary and metastatic tumors. Our results in the primary Cloudman melanoma model demonstrated that IL-2 pDNA therapy with EP resulted in a sustained increase in IL-2 levels within the electroporated tumor tissue as well as significant reduction in primary tumor growth. Immune cell subset depletion studies suggested that CD8+ T cells play a major role in the IL-2 pDNA antitumor mechanism of action. In a metastatic sarcoma model, we demonstrated that treatment of a primary tumor with the IL-2 pDNA-EP therapy resulted in a significant inhibition of liver metastases. These results may represent an improved method for local delivery of IL-2 pDNA to affect both local and metastatic tumors.

C. Plasmids The IL-2 pDNA was constructed by cloning sequences encoding secreted murine IL-2 cDNA into the eukaryotic expression pDNA, VR1051, containing the CMV promoter/enhancer and a transcription terminator derived from the rabbit !-globin gene (Hartikka et al, 1996; Saffran et al, 1998). The backbone pDNA, VR1055, (lacking the IL-2 gene) was used as a control. All pDNA used in these studies was formulated in 1x phosphate-buffered saline (PBS).

D. EP Conditions Mice were anesthetized for all EP procedures using inhalant isoflurane. A square wave pulse generator (model T820, BTX, Holliston, MA) and modified caliper electrodes (model 384, BTX) were used to electroporate murine tumors. The calipers were modified by replacing the 15 x 15 x 3 mm stainless steel electrode plates with otherwise identical plates containing protruding extensions at the base. The extensions measured 7 x 6 x 3 mm and were used to allow for a better fit of the caliper electrode to a 20-80 mm3 tumor. Before EP, conductivity gel (Spectrode 360 electrode gel, Parker Labs, Fairfield, NJ) was applied to the surface of the plates. Immediately after pDNA injection, the plates were applied to the shaved skin on either side of the tumor and 8 consecutive square-wave electrical pulses of 1 msec duration were administered at an electric field strength of 800 V/cm.

E. Tumor efficacy studies For the melanoma model, DBA/2N mice were injected s.c. with 5 x 105 Cloudman S91 M3 melanoma cells. For the metastatic M5076 sarcoma model, C57BL/6 mice were injected intradermally (i.d.) with 5 x 105 M5076 sarcoma cells. Mice bearing palpable tumors of approximately 20-80 mm3 (Day 10 for the Cloudman model and Day 18 for the M5076 model) were randomly assigned to groups such that each group had similar mean tumor volume (n=10-20 mice per group). Mice were then anesthetized with inhalant isoflurane until negative toe pinch and injected i.t. with 30 "g of either control pDNA or IL-2 pDNA in 25 "l with or without EP every 3rd or 4th day for a total of 3 treatments (Days 10, 14 and 18 for the Cloudman model and Days 18, 21 and 25 for the M5076 model). Tumors were measured in 3 dimensions (length x width x height) using calipers. Tumor volume was determined from the formula for volume of a sphere: tumor volume (mm3) = 0.52 (length x width x height) (Tomayko and Reynolds, 1989). Animals with tumor size greater than 6000 mm3 were euthanized.

II. Materials and methods A. Mice Six to twelve week old DBA/2N and C57BL/6 mice were obtained from Jackson Labs (Bar Harbor, ME) and Taconic (Oxnard, CA). All animal studies were performed with approval of Vicalâ&#x20AC;&#x2122;s Institutional Animal Care and Use Committee in accordance with U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals.

F. Pharmacokinetics DBA/2N mice were injected s.c. with 5 x 105 Cloudman melanoma cells. Mice bearing tumors of approximately 20-80 mm3 were randomly assigned to groups such that each group had similar mean tumor volume. Mice received a single i.t. injection of 30 "g of either control pDNA or IL-2 pDNA followed by EP. On Days 1, 2 and 8 post pDNA injection, serum was collected by cardiac puncture and tumors were collected from euthanized mice (n=5 mice per timepoint). Tumors and sera were stored at -

B. Cells Cloudman melanoma (S91 M3) cells (American Type Culture Collection, Rockville, MD) were grown in Hamâ&#x20AC;&#x2122;s F-12K medium (Irvine Scientific, Santa Ana, CA) with 15% equine serum, 2.5% fetal bovine serum (Hyclone, Logan, UT), 0.1% 2mercaptoethanol and 1% GlutaMAX (Invitrogen, Carlsbad, CA). M5076 reticulum cell sarcoma cells were obtained as ascites from the DCTD tumor repository (NCI, Frederick, MD).

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Cancer Therapy Vol 5, page 127 80oC until processing. Tumor lysates were prepared from the frozen tumor tissue according to the procedure described by Hartikka and colleagues in 1996 for frozen muscle tissue. IL-2 levels in tumor lysates and sera were analyzed using a murine IL2 ELISA as described by the manufacturer (R & D Systems, Minneapolis, MN).

4), PE-conjugated anti-CD8 (clone 53-5.8) and biotin-conjugated anti-CD49b (cone DX5) (BD Pharmingen, Carlsbad, CA).

H. Statistical analysis Tumor growth expressed as tumor volume and the number of metastatic tumor nodules were analyzed using Wilcoxon Rank Sum Test. P values < 0.05 were considered significant.

G. In vivo depletion of NK and T cells

III. Results

Anti-CD4 antibody (clone GK1.5, American Type Culture Collection, Rockville, MD) was purified from mouse ascites fluid (Harlan Bioproducts for Science, Madison, WI). Purified anti-CD8 (clone 53-6.7) and anti-NK (anti asialo GM1) antibodies were purchased from Southern Biotech (Birmingham, AL) and Wako Chemicals (Richmond, VA), respectively. In vivo ablation of immune cell subsets was performed by i.p. injection of 200 "g of anti-CD4 or 500 "g of either anti-CD8 or anti-NK antibody on Days -1, +1 and +3 relative to the first pDNA injection and once per week thereafter. Control tumor-bearing mice were injected according to the same schedule with 500 Âľg of rat or rabbit IgG isotype control (Southern Biotech, Birmingham, AL). Immune cell depletion was verified by FACs analysis of splenocytes harvested once per week from sentinel mice treated according to the same schedule. Splenocytes were stained with one of the following antibodies to determine immune cell depletion: FITC-conjugated anti-CD4 (clone RM4-

A. Higher levels of IL-2 in tumor after EP In a pharmacokinetics study, mice bearing s.c. Cloudman melanoma received a single i.t. injection of 30 "g of IL-2 pDNA with or without EP. Sera and tumors were harvested on Days 1, 2 and 8 after the pDNA injection and analyzed for IL-2 levels. The levels of IL-2 in the tumor were 2-3 fold higher when the IL-2 pDNA was administered with EP (Figure 1A). Tumors treated with IL-2 pDNA with EP had mean IL-2 levels of 545, 190 and 90 pg/ml compared to tumors treated with IL-2 pDNA alone which had 200, 100 and 30 pg/ml on Days 1, 2, and 8, respectively. While tumor IL-2 levels were highest one day after IL-2 pDNA injection,

Figure 1. EP increases IL-2 levels in tumor and serum. DBA2/N mice bearing palpable tumors (20-80 mm 3) were injected once with 30 "g of either control pDNA (VR1055) or IL-2 pDNA (VR1111). Immediately after pDNA injection, the tumors were electroporated at 800V/cm for 8 pulses at 1 msec/pulse. On Days 1, 2 and 8 post i.t. pDNA injection, mice were euthanized, and tumors and sera collected. Tumor lysates and sera were analyzed using a murine IL-2 ELISA. Mean IL-2 levels in tumors (A) and sera (B) are shown (n = 5 mice per timepoint).

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Horton et al: Therapy of primary and metastatic murine tumors by Il-2 and pDNA with EP (545 pg/ml vs 200 pg/ml, electroporated vs. nonelectroporated tumors), tumor IL-2 was still readily detectable 8 days after a single i.t. injection of IL-2 pDNA. This is in marked contrast to the results of a previous pharmacokinetics study in which no IL-2 was detectable 24 hours after a single i.t. injection of recombinant IL-2 protein (Horton et al, 1999). In serum, 3-fold higher levels of IL-2 were found one day after pDNA injection of mice whose tumors were treated with IL-2 pDNA with EP compared to IL-2 pDNA alone (Figure 1B). Mice treated with IL-2 pDNA with EP had mean serum levels of 30 pg/ml while mice treated with IL-2 pDNA alone had mean serum levels of 10 pg/ml. IL-2 levels in serum were 20-200-fold lower than in the tumor, suggesting that the IL-2 expressed after in vivo EP is primarily localized to tumors. Mice injected i.t. with non-coding control pDNA with EP had only background levels of IL-2 in tumors and sera, suggesting that little, if any, endogenous murine IL-2 is induced by pDNA-EP treatment. These results suggest that EP may increase the in vivo transfection efficiency of pDNA resulting in greater transgene expression at the site of administration.

B. IL-2 pDNA-EP therapy reduces tumor growth and leads to tumor rejection Mice bearing s.c. Cloudman melanoma tumors were treated by i.t. injection of 30 "g of IL-2 or control pDNA with or without EP on Days 10, 14 and 18. Mice receiving the IL-2 pDNA with EP had a significant reduction in primary tumor growth compared to mice treated with IL-2 pDNA without EP (p<0.02 on Days 26 to 47) (Figure 2A). Moreover, mice treated with IL-2 pDNA without EP had the same kinetics of tumor growth as mice treated with control pDNA with or without EP. By Day 43, mice treated with IL-2 pDNA with EP had a mean tumor volume of 50 mm3, while mice treated with the IL-2 pDNA without EP had a mean tumor volume of 1600 mm3 (p=0.01 on Day 43). Mice injected with control pDNA with EP (backbone plasmid lacking the IL-2 gene) had a mean tumor volume of 1300 mm3 by Day 43. In addition to reducing tumor size, the IL-2 pDNAEP therapy resulted in complete responses in 60% of the mice (Figure 2B). In contrast only 20% of the mice receiving the IL-2 pDNA without EP became tumor-free. None of the mice treated with control pDNA (with or without EP) became tumor-free. Taken together, the efficacy and pharmacokinetics data shown in Figures 1

Figure 2. IL-2 pDNA administration with EP leads to tumor regression. DBA2/N mice bearing palpable tumors (20-80 mm3) were injected i.t. with 30 "g of either control pDNA (VR1055) or murine IL-2 pDNA (VR1111) (n=10 mice per group). Immediately after pDNA injection, the tumors were electroporated at 800 V/cm for 8 pulses at 1 msec/pulse. The mice received the i.t. pDNA therapy on days 10, 14 and 18 post-tumor cell injection. (A.) Mean tumor volume for mice treated with control pDNA or IL-2 pDNA with or without EP. (B.) Percent of tumor-free mice on day 54 of the study (n=10 mice per group). * p < 0.023 for IL-2 pDNA + EP compared to all groups. The dip in the curve for the control pDNA + EP group on Day 36 and for the IL-2 pDNA + EP group on day 40 was due to the death of one mouse with a large tumor burden.

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Cancer Therapy Vol 5, page 129 spontaneous metastases in the liver. Mice bearing i.d. M5076 tumors were injected i.t. with 30 "g of IL-2 pDNA with EP on Days 18, 21 and 25. On Day 35, the mice were euthanized, livers collected and metastases enumerated. Interestingly, mice whose primary tumor was treated by i.t. injection of IL-2 pDNA with EP had a significant reduction in the number of metastatic tumor nodules in the liver (Figure 3A, C). While 65% of the mice treated with IL-2 pDNA with EP had 2 or fewer liver tumor nodules on Day 35, none of the mice treated with control pDNA or saline with EP had 2 or fewer liver tumor nodules (Figure 3A, B). In fact, among the mice treated with control pDNA followed by EP, 55% had 3-85 liver tumor nodules and 45% had liver tumor nodules that were too numerous to count. Similarly, in the saline-treated group, 35% of the mice had 3-85 tumor nodules and 65% of the mice had tumor nodules that were too numerous to count. EP was required for the IL-2 anti-metastatic effect as delivery of IL-2 without EP resulted in development of a similar number of metastatic tumor nodules as the mice treated by i.t. administration of control pDNA with EP (data not shown). The results of this study suggest that treatment of a primary tumor with IL-2 pDNA with EP may be efficacious for preventing the spread of metastatic disease.

and 2 suggest that greater in vivo transfection efficiency and, hence greater antitumor efficacy, is obtained with IL2 pDNA administered with EP at the tumor site.

C. IL-2 pDNA-EP therapy requires CD8+ T cells The mechanism of action of the IL-2 pDNA therapy was investigated by antibody depletion of immune cell subsets in tumor-bearing mice. Mice bearing Cloudman melanoma tumors were injected i.p. with anti-CD4, antiCD8, anti-NK or control IgG on Days -1, +1, +3 relative to the first pDNA injection and once per week thereafter. Sentinel mice injected i.p with the same antibodies according to the same schedule were checked once per week to confirm depletion of the immune cell subsets. Mice were injected i.t. with 30 "g IL-2 pDNA with EP on Days 12, 16 and 20. 70 and 50% of the mice treated with either anti-CD4 or anti-NK antibodies followed by IL-2 pDNA-EP therapy became tumor-free by Day 42, respectively (Table 1). This result was similar to the mice treated with control IgG followed by IL-2 pDNA-EP therapy in which 50% of the mice became tumor-free, suggesting that CD4+ T cells and NK cells do not play a major role in the IL-2 pDNA-mediated immune response. In contrast, only 20% of the mice treated with anti-CD8 followed by i.t. IL-2 pDNA-EP therapy became tumor-free suggesting that CD8+ T cells are important effectors for the IL-2-mediated antitumor response. As a further demonstration of the role of a specific immune response involved in the IL-2 pDNA antitumor efficacy, eight mice that became tumor-free after IL-2 pDNA-EP therapy, were re-challenged two months later on the opposite flank with 5 x 106 Cloudman melanoma cells. None of the mice developed tumors upon rechallenge (0/8), in contrast to na誰ve mice challenged with the same dose of cells for which 100% (10/10) developed tumors (data not shown).

IV. Discussion In this study we demonstrate that i.t. administration of IL-2 pDNA followed by EP can have a potent antitumor effect in primary and metastatic tumor models. Mice treated with IL-2 pDNA-EP had a significant reduction in tumor growth leading to a complete response in the majority of mice. Furthermore, although previous studies have demonstrated antitumor efficacy of IL-2 pDNA for treatment of primary tumors, (Lohr et al, 2001; Matsubara et al, 2001; Chi et al, 2002) we have now demonstrated that EP delivery of IL-2 pDNA can significantly enhance its antitumor efficacy such that metastatic tumor growth is markedly reduced. In a metastatic model, 65% of the mice whose primary tumor was treated with IL-2 pDNA-EP had 2 or fewer metastatic tumor nodules in the liver. In contrast, none of the mice whose primary tumor was treated with control pDNA with EP had 2 or fewer liver metastatic nodules and 55% had liver metastases that were too numerous to count.

D. IL-2 pDNA-EP therapy prevents systemic metastases A metastatic sarcoma model (M5076) was employed to evaluate whether IL-2 pDNA therapy of a primary tumor could be efficacious against metastatic disease. In this model, mice bearing i.d. M5076 sarcoma develop

Table 1. Percentage of tumor-free mice after depletion of immune cell subsets and treatment with IL-2 pDNA. Antibody Treatmenta Anti-CD8 Anti-NK Anti-CD4 Control IgG

% Tumor-free Miceb 20 50 70 50

Tumor-bearing mice were depleted of immune cell subsets by i.p. injection of anti-CD8 (53-6.7), anti-CD4 (GK1.5), anti-NK (antiasialo GM1) or control IgG antibodies on Days -1, +1, +3 relative to pDNA injection and once per week thereafter. Immune cell depletion was confirmed by analysis of spleens from sentinel mice injected according to the same schedule. Antibody-treated mice were injected i.t. with 30 "g IL-2 pDNA with EP on days 12, 16 and 20. b Percent tumor-free mice determined on Day 42 post tumor cell injection (n=10 mice per group).

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Horton et al: Therapy of primary and metastatic murine tumors by Il-2 and pDNA with EP

Figure 3. Treatment of primary tumor with IL-2 pDNA-EP prevents establishment of systemic metastases. C57BL/6 mice bearing i.d. M5076 tumors (20-80 mm3) were injected i.t. with saline or with 30 "g of either control pDNA or IL-2 pDNA with EP on Days 18, 21 and 24 post tumor cell injection. On Day 35, mice were euthanized, livers collected and metastases enumerated. (A.) Number of individual tumor nodules in the livers of mice (n = 20 mice per group) are shown for each group. Mice treated with Il-2 pDNA with EP had a significant reduction in liver metastases [*p=0.0004, Wilcoxon Rank Sum Test, a value of 100 was used as a conservative estimate for liver nodules that were too numerous to count (TNTC)]. Liver sections from a mouse treated with (B.) control pDNA with EP or (C.) IL-2 pDNA with EP. TNTC = metastases that are too numerous to count.

The mechanism of action of the IL-2 pDNA-EP therapy appears to involve CD8+ T cells as the therapy was not as effective in mice depleted of this immune cell subset but was effective in mice depleted of CD4+ or NK cells. In addition, the local IL-2 pDNA-EP therapy appeared to generate a systemic tumor-specific immune response as demonstrated by the fact that mice that rejected primary tumors after the IL-2 pDNA-EP therapy were immune to rechallenge with tumor cells on the opposite flank. Based on our data, it is possible that treatment of the primary tumor with IL-2 pDNA-EP involves local IL-2 recruitment and activation of CD8+ T cells followed by circulation of tumor-specific T cells able to recognize and destroy disseminated cancer cells. In support of the latter hypothesis, we previously demonstrated that local delivery of IL-2 pDNA complexed with cationic lipid resulted in regression of renal carcinoma in mice and generation of tumor-specific cytotoxic T cells (CTL) that were able to lyse tumor cells (Saffran et al., 1998). Locally delivered IL-2 may also be helpful in reversing the tolerant state of T cells residing within the tumor. Zippelius et al. (2004) demonstrated that tumorspecific T cells within tumors are often functionally tolerant and unable to secrete interferon # (IFN#); however, when T cells are harvested from tumors and stimulated with IL-2 ex vivo, they are often able to secrete IFN# and lyse tumor cells in vitro (Overwijk et al., 2003).

Murine ovarian tumors treated by local injection of IL-2 pDNA complexed with lipid revealed a marked increase in IFN# within the tumor ascites (Horton et al., 1999). Thus, i.t. administration of IL-2 pDNA with EP may increase levels of the T helper 1 (TH1) cytokine, IFN#, within the tumor and this may be helpful in reversing the functional tolerance of T cells at the tumor site. One of the major advantages of local delivery of IL-2 by i.t. injection of IL-2 pDNA is the possibility of avoiding the serious side effects commonly associated with systemic IL-2 therapy. We demonstrate in mice that delivery of IL-2 pDNA with EP can result in 2-3 fold higher levels of IL-2 in tumors, while maintaining relatively low IL-2 levels in serum. In mouse models, the amount of recombinant IL-2 protein that must be administered to result in similar antitumor efficacy can, in some cases, cause systemic toxicity (Ettinghausen et al., 1986; Papa et al., 1986). In contrast, in the present study, no side effects were found for the mice treated by i.t. injection of IL-2 pDNA followed by EP and levels of IL-2 in the serum were very low. Since many of the side effects of recombinant IL-2 therapy are due to high serum levels of IL-2 (Schomburg et al., 1994), an alternative method of delivery such as local injection of IL-2 pDNA may be beneficial. In summary, we describe the antitumor efficacy and pharmacokinetics of locally delivered IL-2 pDNA in rodent tumor models. Our results suggest that by

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Cancer Therapy Vol 5, page 131 Horton HM, Dorigo O, Hernandez P, Anderson D, Berek JS, Parker SE (1999) IL-2 plasmid therapy of murine ovarian carcinoma inhibits the growth of tumor ascites and alters its cytokine profile. J Immunol 163, 6378-6385. Lohr F, Lo DY, Zaharoff DA, Hu K, Zhang X, Li Y, Zhao Y, Dewhirst MW, Yuan F, Li CY 2001 Effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation. Cancer Res 61, 3281-3284. Matsubara H, Gunji Y, Maeda T, Tasaki K, Koide Y, Asano T, Ochiai T, Sakiyama S, Tagawa M (2001) Electroporationmediated transfer of cytokine genes into human esophageal tumors produces anti-tumor effects in mice. Anticancer Res 21, 2501-2503. Overwijk WW, Theoret MR, Finkelstein SE, Surman DR, de Jong LA, Vyth-Dreese FA, Dellemijn TA, Antony PA, Spiess PJ, Palmer DC, Heimann DM, Klebanoff CA, Yu Z, Hwang LN, Feigenbaum L, Kruisbeek AM, Rosenberg SA, Restifo NP (2003) Tumor regression and autoimmunity after reversal of a functionally tolerant state of self reactive CD8+ T cells. J Exp Med 198, 569-580. Papa MZ, Vetto JT, Ettinghausen SE, Mule JJ, Rosenberg SA (1986) Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin-2 in mice. Cancer Res 46, 5618-5623. Radny P, Caroli UM, Bauer J, Paul T, Schlegel C, Eigentler TK, Weide B, Schwarz M, Garbe C (2003) Phase II trial of intralesional therapy with interleukin-2 in soft tissue metastases. Br J Cancer 89, 1620-1626. Saffran DC, Horton HM, Yankauckas MA, Anderson D, Barnhart KM, Abai AM, Hobart P, Manthorpe M, Norman JA, Parker SE (1998) Immunotherapy of established tumors in mice by intratumoral injection of interleukin-2 plasmid DNA: Induction of CD8+ T-cell immunity. Cancer Gene Ther 5, 321-330. Schomburg A, Kirchner H, Lopez-Hanninen E, Menzel T, Rudolph P, Korfer A, Fenner M, Poliwoda H, Atzpodien J (1994) Hepatic and serologic toxicity of systemic interleukin-2 and/or interferon-alpha. Evidence of a riskbenefit advantage of subcutaneous therapy. Am J Clin Oncol 17, 199-209. Schwartz RN, Stover L, Dutcher J (2002) Managing toxicities of high-dose interleukin-2. Oncology (Williston Park). 16, 1120. Tarhini AA, Agarwala SS (2005) Interleukin-2 for the treatment of melanoma. Curr Opin Investig Drugs 6, 1234-1239. Tomayko MM, Reynolds CP (1989) Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 24, 148-154. Zippelius A, Batard P, Rubio-Godoy V, Bioley G, Lienard D, Lejeune F, Rimoldi D, Guillaume P, Meidenbauer N, Mackensen A, Rufer N, Lubenow N, Speiser D, Cerottini JC, Romero P, Pittet MJ (2004) Effector function of human tumor-specific CD8+ T cells in melanoma lesions: A state of functional tolerance. Cancer Res. 64, 2865-2873.

increasing the in vivo transfection efficiency of IL-2 pDNA administered directly to tumors with EP, we may be able to enhance its antitumor efficacy while maintaining reduced systemic IL-2 levels. In addition, we demonstrate that treatment of a primary tumor with IL-2 pDNA-EP therapy can prevent the establishment of systemic metastases. These results suggest that local delivery of IL-2 pDNA with EP has promise for the treatment of cancer and support Vicalâ&#x20AC;&#x2122;s ongoing Phase 1 trial of IL-2 pDNA delivered by EP for therapy of melanoma.

Acknowledgements The authors would like to thank Mary Wloch and Luane Reyes for performing FACs analysis.

References Aihara H, Miyazaki J (1998) Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16, 867-870. Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, Abrams J, Sznol M, Parkinson D, Hawkins M, Paradise C, Kunkel L, Rosenberg SA (1999) High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: Analysis of 270 patients treated between 1985 and 1993. J Clin Oncol 17, 2105-2116. Bloom DC, Goldfarb PM (2005) The role of intratumor therapy with electroporation and bleomycin in the management of advanced squamous cell carcinoma of the head and neck. Eur J Surg Oncol 31, 1029-1035. Chi CH, Wang YS, Lai YS, Chi KH 2002 Anti-tumor effect of in vivo IL-2 and GM-CSF electrogene therapy in murine hepatoma model. Anticancer Res 22, 315-321. Ettinghausen SE, Rosenberg SA (1986) Immunotherapy of murine sarcomas using lymphokine-activated killer cells: Optimization of the schedule and route of administration of recombinant interleukin-2. Cancer Res 46, 2784-2792. Figlin RA, Parker SE, Horton HM (1999) Technology evaluation: Interleukin-2 gene therapy for the treatment of renal cell carcinoma. Curr Opin Mol Ther 1, 271-278. Hartikka J, Sawdey M, Cornefert-Jensen F, Margalith M, Barnhart K, Nolasco M, Vahlsing HL, Meek J, Marquet M, Hobart P, Norman J, Manthorpe M (1996) An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther 7, 1205-1217. Hartikka J, Sukhu L, Buchner C, Hazard D, Bozoukova V, Margalith M, Nishioka WK, Wheeler CJ, Manthorp M, Sawdey M (2001) Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: Plasmid dependence of muscle damage and effect of poloxamer 188. Mol Ther 4, 407-415. Heller R (2003) Delivery of plasmid DNA using in vivo electroporation. Preclinica 1, 198-208.

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Nerve-sparing low semi-cystectomy in bladder: preliminary results Research Article

Zhong Wang1, Yin-Tian Yuan2, Guiting Lin3, Benchun Liu3,* 1

Urology and Andrology Department, Ninth People's Hospital, Medical College of Shanghai Jiaotong University, Shanghai 200011, PR China 2 Department of Urology, Shouguang Municipal People’s Hospital, Shandong 262700, PR China 3 Department of Urology, University of California, San Francisco, CA94143, USA

__________________________________________________________________________________ *Correspondence: Benchun Liu, M.D., Ph.D. HSW1434, 513 Parnassus Avenue, San Francisco, CA94143, USA; Tel: 415-514-2017; Fax: 415-514-0756; e-mail: benchunliu@yahoo.com Key words: bladder neoplasms; cystectomy; quality of life; erectile dysfunction Abbreviations: erectile dysfunction, (ED); International Index of Erectile Function, (IIEF5) Received: 30 November 2006; Revised: 25 January 2007 Accepted: 8 March 2007; electronically published: April 2007

Summary In order to study the effect of nerve-sparing low semi-cystectomy and improve sexual function related to the quality of post-operative life for patient with multiple and infiltrated cancer within trigone or neck of bladder, six male patients enrolled into this study who were suffering with multiple and invasive cancers within trigone and/or neck of bladder. All cases underwent low semi-cystectomy and 5 of them received the technique for neurovascular bundle preservation successfully. Organ-confined bladder transitional cellular carcinomas with negative surgical margins were confirmed in all patients by post-operative histopathological examinations, which infiltrating to the muscle layer of bladder (T2a-bNx-0M0, TNM system). All patients were followed up to 9~36 months. With nerve-sparing surgery, sexual function has been preserved in 4 of these 5 patients (the IIEF5 score was 14 points or more). Radical cystectomy was performed on 1 case for tumor relapse. One has unilateral hydronephresis slightly. Qualities of life are better in these victims of bladder cancer than whom with traditional radical cystectomy. Our conclusion is that erectile function can be reserved by technique of nerve-sparing during the operation of low semi-cystectomy, but the patient should be selected strictly.

preservation in 1983 (Marshall 1998). Some studies have shown that nerve-sparing radical prostatectomy can decrease the incidence of erectile dysfunction (ED) from 90% to 17%~36%, but sexual function of patients’ can still be influenced indirectly by the urinary diversion to some extent, especially in young patients (Wang et al, 1998; Mirone et al, 2003; Nakashima et al, 2003). In order to preserve their sexual function, six young patients suffered with invasive bladder cancer within trigone and/or neck of bladder, who had the indication for the traditional radical cystectomy, were performed nerve-sparing low semicystectomy.

I. Introduction Radical cystectomy is an ideal therapy for invasive bladder cancer without metastasis. But usually little patient want to accept it because of higher risk of many kinds of complications, such as urinary diversion and sexual dysfunction (ejaculation dysfunction, erectile dysfunction, etc.), which influence the quality of life significantly (Wagner and Russo 2000; Mirone et al, 2003). As the patients’ demanding for a better life quality, such complications are becoming more and more important for health providers to think about before operation. So it is emphasized much more than before by urologist to improve patient’s quality of life, especially erectile function for young patient, with essential prerequisite of removing the malignant tumor radically. Dr. Walsh has demonstrated in 1998 that traditional surgery will injure the pelvic nerve bundle, and they also discussed the technique for neurovascular bundle

II. Patients and methods Six male patients enrolled into this study, aging from 29~52 years old (mean age at 38.7 ± 7.5 years old). All cases were diagnosed as multiple invasive bladder cancer within bladder trigone and/or neck before operation,

133

Wang et al: Nerve-sparing low semi-cystectomy in bladder: preliminary results without metastasis. They received low semi-cystectomy. Nervesparing surgery was given up in one case because of the bleeding and incapable of dissection during dissociating the prostate lateral ligament. Neurovascular bundles were preserved successfully in other 5 cases. One of these 5 cases, who was 29 years old, had the demanding of childbirth, his prostate was preserved during operation because healthy posterior urethra was shown through urethroscope. Bladder transitional cellular carcinomas with negative surgical margins were diagnosed during operation according frozen section pathology. Surgical manner of Walsh was improved (Marshall, 1998; Ali et al, 2004). The interspace between Denonvilliers fascia and rectum was separated bluntly. When dissociating the prostate, the neurovascular bundle of prostate can be found on the lateral of prostate capsule to seminal vesicle. After push away pelvic fascia of both sides from prostate capsule, the lateral branches should be cut off as nearly as possible to the prostate in order to avoid injuring the main trunk of neurovascular bundle. Some part of prostate capsule should be preserved while separating the apex of prostate. After cut off the urethra and pulling the apex of prostate, the space between posterior of prostate and anterior wall of rectum was dissociated retrograde. Prostate can be resected by incising prostate capsule from anterior. Surgical capsule of prostate can be preserved if itâ&#x20AC;&#x2122;s difficult to dissect around prostate. Then bladder and/or ureter can be cut over 2cm to the margin of tumor. If no cancer cell infiltrating in the incision edge and the apex of prostate were confirmed by frozen section examination, the ureter was pulled through a submucosal tunnel to create an anti-reflux mechanism. After Reshaping bladder neck individually according residue proximal urethra or capsule of prostate and with dragging Foley catheter for fixation, fashioned bladder neck was directly anastomosised with urethra (capsule of prostatic apex). A negative pressure drainage-tube was put into the interspace of post-pubis. Urine routine test, B-ultrasound investigation, and cystoscopic examinations were scheduled during follow up. International Index of Erectile Function (IIEF5) questionnaire was used for assessment of erectile function. Preventive intravesical instilled with thiotepa (60mg) or Bacillus CalmetteGuerin (BCG, 50mg) was performed after operation consecutively (once per week for 8 weeks, and then once per month for 10 months).

muscle layer of bladder (T2a-bNx-0M0, TNM system). Grade I~II, II, and III were 2, 3, and 1 case(s) respectively. After operation, there were two patients had stress urinary incontinence but got well at week 4 and 7 respectively. Temporary urine leakage around anastomosised orifice of bladder neck and urethra occurred in one patient. Drainage-tube was removed at week 4 for this case. All patients were followed up for 9~36 months (mean at 15 months). Sexual function was preserved in 4 cases among 6 according IIEF5 questionnaire (the IIEF5 score was 14 points or more). The other 2 cases arose ED, 1 of them was given up the nerve-sparing surgery because of the bleeding and incapable of dissection during operation. So the rate of sexual function preservation is 80% (4/5) in the cases received nerve-sparing surgery. One patient whose prostate was mostly preserved was found that operation had no influence on his sexual function by follow up, and his wife was labored a boy successfully two years later. The patient received radical cystectomy and urinary diversion eventually because of tumor relapse and hydronephresis at his left side. Another one case was found with slightly hydronephresis by Bultrasound, and he is still being followed up without any treatment. No other complications, such as enormous residual urine and urinary incontinence, were found in these patients (Table 1).

IV. Discussion Operation of genitourinary system is the main cause of nosocomial sexual dysfunction. Many scholars have made efforts on searching for the substitute of the bladder, such as Kock pouch, ileocecum conduit, bladder regeneration to avoid the inconvenience caused by the cystectomy and urinary diversion. Unfortunately these surgeries are too complex, with many kinds of complications, and also expensive (Wang and Xue 1994; Wang et al, 1997; Hart et al, 1999). To those young patients suffering with invasive cancer within bladder trigone or neck, radical cystectomy and urinary diversion not only affect normal function of urinary tract but also induce significant inconvenience to the daily life, especially the sexual behavior. Low semi-cystectomy

III. Results Organ-confined bladder transitional cellular carcinomas with negative surgical margins were confirmed in all patients by post-operative histopathological examinations, which infiltrating to the Table 1. Clinical features, therapy, and follow-up of patients Patient

Tumor stage

Surgery

I II III IV

T2bN0M0 T2aN0M0 T2aNxM0 T2bN0M0

Nerve-sparing Nerve-sparing Nerve-sparing Nerve-sparing

T2bN0M0

T2bNxM0

Routine technique Nerve-sparing

Follow-up Complications

Treatment

Sexual function

Stress urinary incontinence Unilateral hydronephresis Relapse, hydronephresis Anastomosised orifice urine leakage Stress urinary incontinence

Medication Self-cure Radical cystectomy Removed drainage tube late Self-cure

Preserved Loss Preserved Preserved

None

Preserved

134

Loss

Cancer Therapy Vol 5, page 135 can reserve not only physiological miction but also sexual function. Carefully selected patients are necessary for this kind of surgery. In our group, patients were young men with organ-confined bladder carcinoma in bladder trigone and/or near bladder neck. All of them strongly asked for preservation of their sexual and ejaculatory functions. Also, the indication must be restricted to selected patients with very low potential risk of local recurrence. Dr. Salem reported in 2005 that preservation of the vas deferens, whole prostate, and seminal vesicle for radical cystectomy is a good option in selected young men with bladder carcinoma and with petition of fertility. Sexual function-sparing cystoprostatectomy has been widely accepted recently. We have further demonstrated that the neurovascular bundle is derived from the celiac plexus by autopsy. It is formed by the vessels and fibers which control the bladder, prostate, seminal vesicle and corpus cavernosum, descending between the lateral posterior of prostate and the anterior wall of rectum (Marshall 1998; Ali et al, 2004). According to literature report, unilateral neurovascular bundle preservation is enough for patients to reserve sexual function (Mulcahy 2000). During operation procedure, we tried to preserve both neurovascular bundles. The result is similar to that of Walsh et al, and 80% of patient reserved sexual function in the present study (Walsh 1998; Martis et al, 2005). A device (Cavermap) has been invented by Klotz to prevent the nerve from being injured accident (Klotz, 2000). This kind device can be used to do real-time monitoring during cystectomy or prostatectomy because the coursor of the neurovascular bundle can be determined accurately by taking signal whether the nerve is stimulated or not. Thereby the aim of nerve preservation can be reached easily. It’s no doubt that the rate of nerve-sparing will be increased by using this device. Bladder preservation brings chances for tumor recurrence. To resect the tumor completely should be the original purpose of sexual nerve sparing low semicystectomy. Tumor recurrence can be effectively prevented by regular irrigation of bladder post operation. Meanwhile, intensive follow up is necessary and very important. Transurethral resection of a bladder tumor (TUR-Bt) should be performed in time on some patients suffering with tumor relapse. A few patients who have had childbirth with multiple relapse or progression of cancer should accept radical cystectomy. Prostatectomy won’t arise urinary retention commonly because the resistance of the bladder outlet is reduced. Incidence of postoperative urethral stricture can be greatly decreased by eversion of the bladder mucosa and anastomosised with the capsule of prostate apex. Unilateral hydronephresis occurred in one case of the present study. It may be caused by the tightness of the papilla. No reflux showed at micturation phase by cystography. Our result suggests that this alternative operation has clinical utility with no serious complications presented in other cases. Further studies with more cases and longer follow-up will be done.

Acknowledgements This study was supported by Shanghai Pujiang Project (05JC14035) and Shanghai Natural Scientific Foundation (04ZR14066), PR China. We show our grateful to the patients enrolled into this study.

References Ali M, Johnson IP, Hobsom J, Mohammadi B, Khan F (2004) Anatomy of the pelvic plexus and innervation of the prostate gland. Clin Anat 17, 123-129. Hart S, Skinner EC, Meyerowitz BE, Boyd S, Lieskovsky G, Skinner DG (1999) Quality of life after radical cystectomy for bladder cancer in patients with an ideal conduit, or cutaneous or urethral Kock pouch. J Urol 162, 77-81. Klotz L (2000) Neurostimulation during radical prostatectomy: improving nerve-sparing techniques. Semin Urol Oncol 18, 46-50. Marshall FF (1998) Surgery of the bladder. In Campbell’s Urology, 7th ed. Edited by Walsh PC, Retik AB, Vaughan ED and Wei AJ. Philadelphia: WB Saunders Co, Vol 3, chapt 105, pp3274-3297. Martis G, D’Elia G, Diana M, Ombres M, Mastrangeli B (2005) Prostatic Capsule- and Nerve-sparing Cystectomy in Organconfined Bladder Cancer: Preliminary Results. World J Surg 29, 1277-1281. Mirone V, Imbimbo C, Plamieri A, Longo N, Fusco F (2003) Erectile dysfunction after surgical treatment. Int J Androl 26,137-140. Mulcahy JJ (2000) Erectile function after radical prostatectomy. Semin Urol Oncol 18, 71-75. Nakashima M, Nishiyama H, Yagijashi Y, Shingo Y, Toshiyuki K, Tomonori H, Osamu O (2003) A case report of a young patient with invasive bladder cancer. Hinyokika Kiyo 49, 745-748. Salem HK (2005) Preservation of Ejaculatory and Erectile Function after Radical Cystectomy for Urothelial Malignancy. J Egypt Nat Cancer Inst 17, 239-244. Wagner JR, Russo P (2000) Urologic complications of major pelvic surgery. Semin Surg Oncol 10, 216-220. Walsh PC (1998) Nerve-sparing radical prostatectomy for early stage prostate cancer. Semin Oncol 15, 351. Wang Z, Xue ZY (1994) Complications and their treatments after bladder regeneration. Clin J Urol 9, 137-138. Wang Z, Zhang YF, Kong XG (1998) Low semi-cystectomy in treating trigone of bladder neck carcinoma. Chinese J Surg 36,141-1428. Wang Z, Zhang YF, Li CL (1997) Typing and significance of Xray on the venous erectile dysfunction. Chinese J Med 5, 212-214.

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Determination of epidermal growth factor receptor (EGFR) in patients with colorectal cancer (Institutional series) Research Article

Arkom Cheirsilpa1, Preecha Ruangvejvorachai1, Anant Karalak1, Soisuda Sangprakarn3, Sangduan Pummai1, Suleeporn Sangrajrang1,* 1

National Cancer Institute, Bangkok, Thailand Department of Pathology Faculty of Medicine Chulalungkorn University 3 Department of Pathology King Chulalungkorn Memorial Hospital 2

__________________________________________________________________________________ *Correspondence: Suleeporn Sangrajrang, Research Division, National Cancer Institute, 286/1 Rama VI road Ratchatavi, Bangkok, Thailand 10400; Tel: (66-2) 3547025 ext. 1405, 1414; Fax: (66-2) 3547037; Email: sulee@health.moph.go.th; suleesang@yahoo.com Key words: EGFR, colorectal cancer, prognosis Abbreviations: carbohydrate antigen, (CA19-9); carcinoembryonic antigen, (CEA); Epidermal growth factor receptor, (EGFR); Immunohistochemistry, (IHC) Received: 2 January 2007; Revised: 29 January 2007 Accepted: 20 April 2007; electronically published: May 2007

Summary EGFR is involved in the epidermal growth factors pathway that regulates cellular processes and is associated with the development of many types of cancer including colorectal cancer. EGFR-targeted could be an alternative option to improve clinical outcome in tumors with EGFR expression. This study aimed to examine the expression of EGFR and its prognostic value of EGFR in colorectal cancer. We investigated surgically resected tissue specimens of 99 colorectal cancer tissues for expression of EGFR by immunohistochemistry staining (IHC). Medical records were reviewed to set clinical information. Results: EGFR expression were detected in 34 (34.3%) patients of 99 patients with colorectal cancer. Neither age nor sex was correlated with the presence of EGFR. There was a statistically significant difference in the prevalence of EGFR expression between the early disease group (stage I-II) (25%) and the advanced disease group (stage III-IV) (75%) (p<0.01). A statistically significant relationship was also noted between the presence of EGFR and lymph node status (p=0.03). The presence of EGFR expression was related with higher grade of differentiation (p=0.05). Overexpression of EGFR was associated with high initial serum carcinoembryonic antigen (CEA) concentration (p=0.01), but no correlation between carbohydrate antigen (CA199) and EGFR expression was observed (p=0.25). In addition, patients with EGFR expression had a higher risk for disease recurrence compared with those EGFR negative (p=0.04). However, there was no relationship between EGFR expression and overall survival (p=0.40). Our findings indicate that EGFR expression correlate with histoclinical factors and may assist in predicting disease recurrence in colorectal cancer.

The beginning of EGF to EGFR produces a biological signal to the cell that initiates several functions that promote tumor growth, including cell invasion and metastasis, repair and new blood vessel formation (angiogenesis), all of which are essential components to the ongoing survival of the tumor (Schlessinger, 2000; Yarden, 2001). These discoveries have led to development of pharmacologic interventions that target the molecular and cellular consequences of these alterations (Ciardiello and Tortorn, 2003; Grunwald and Hidalgo, 2003). To our knowledge, data regarding EGFR expression in colorectal carcinoma are limited, particularly in Thailand. In a

I Introduction Colorectal cancer is one of the most common cancer in Thailand, the third in frequency among males and the fifth among females (Martin and Pongnikorn, 2003). The EGFR is also known as HER-1 or erb-B1 is a ubiquitous 170-kd membranous-spanning glycoprotein composed of an amino-terminal extra cellular ligand-binding domain, a hydrophobic transmembranous region, and a cytoplasmic domain that contains the tyrosine kinase domain and carboxy-terminal region that contains critical by tyrosine residues and receptors regulatory motifs (Arteaga, 2001).

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Cheirsilpa et al: Determination of EGFR in patients with colorectal cancer Kaplan-Meier method was used to estimate survival possibility as a function of time, and survival differences were analyzed by the log rank test. Two-tailed Ps of "0.05 were considered to be statistically significant. All data analysis were performed using a standard statistical program.

clinical study, EGFR expression has been demonstration to be associated with poor outcome in colorectal cancer patient with stage IV (Goldstein and Armin, 2001). EGFR expression has also been reported to be associated with poor response in patient with locally advanced colorectal cancer treated by radiation (Giralt et al, 2002). In this study, we evaluated EGFR expression in formaline-fixed paraffin embedded tissue sample of colorectal cancer patients. We also explore the relationship between the extent of its expression and histology clinical characteristics that might have an impact on prognosis and survival of the patients in order to access whether the apparent useful criteria in selection treatment.

III. Results A. Patient characteristics Clinical and histological characteristics of the 99 colorectal cancer patients were listed as in Table 1. There were 99 patients: 54 male and 45 female. Their age range 33-84 years old and the mean age was 60±10.94. Most of the adenocarcinoma (70.7%) were located in the colon and 29.3% in the rectum. Histopathological grade were: Well differentiated 49, moderated differentiated 46 and poorly differentiated only 4. Twelve patients had stage I, 34 patients had stage II, 38 patients had stage III, and 10 patients had stage VI disease.

II. Materials and Methods A. Patients and tumour specimens Formalin- fixed paraffin embedded tumor specimens of 99 colorectal cancer patients were recruited for determining EGFR by IHC. Medical records were reviewed to obtain clinical information then mapping both information in order to find any clinical value in term of prognostic and predicted factors. Characteristic studies included age, sex, tumor site , tumor size, degree of histological differentiation (well/moderate/poor) number of position lymph node counted during the slide review and classified as N 1 or N2 based on the American joint committee on cancer (AJCC) TNM classification (Geene, 2002). An institutional review board approval was obtained to conduct this retrospective study. Patient confidentially was maintained with the patients database kept in a data file with access limited to study authors.

B. Association between EGFR expression and clinicopathologic features EFGR antibody produced immunostaining labeling of variable intensity and extend in the membrane of tumour cells in 34 of 99 colorectal carcinomas (34.3%).

Table 1. Patient demographics Characteristic Age Mean Range Sex Male Female Site Colon Rectum Tumor site T1 T2 T3 T4 TX Node status N0 N1 N2 Nx TMN stage I II III IV Unknown Histopathological grade Well differentiated Moderated differentiated Poorly differentiated

B. Immunohistochemistry Immunohistochemiscal stains were performed on paraffinembedded tissue sections. Sections were deparaffinized and rehydrated in a series of alcohols and xylene according to established procedures. Sections for immunostaining were treated with 3% hydrogen peroxide for 10 min to quench myeloperoxidase and then cleared in running water followed by a 5 min rinse in distilled water. The section were incubated for 5 min with protinase K and non-specific binding site were blocked. Incubations with primary antibody were made for 1 h at room temperature (1:100). The peroxidase labeled polymer conjugated to goat antimouse method was used to detect antigen-antibody reaction (DAKO Envision + System, DAKO Corporation) for 30 min at room temperature. Section were then visualized with 3,3’diaminobenzidine as a chremogen for 5 min and conterstained with Mayer’s hematoxylin. Negative controls included replacement of the primary antibody with nonreacting antibodies. Positivity for EGFR expression is defined as any membrane staining above background level, whether or not completely circumferential. For EGFR staining intensity was classified as the following: negative 0;+1 if the circumferential membrane pattern was complete or incomplete with a waek intensity; +2 circumferential staining with a moderate intensity; and +3 complete strong circumferential staining.

C. ELISA for CEA and CA19-9 For the analysis of CEA and CA 19-9, we used commercially available kits (Roche, Mannheim, Germany). The assay was performed according to the manufacturer’s recommendations.

D. Statistical analysis Associations between categorical groups were evaluated using the !2 or the Fisher’s exact test when appropriate. The

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No. of Patients 60 ± 10.94 33 – 84 54 45 70 29 1 20 56 20 2 48 29 20 2 12 34 38 10 5 49 46 4

Cancer Therapy Vol 5, page 139 Twenty tumours had a week staining (1+), nine tumours had moderate staining (2+), and five tumours had a strong staining (3+) (Table 2, Figure 1). Neither age or sex was correlated with the presence of EGFR. There was a statistically significant difference in the prevalence of EGFR expression between the early disease group (stage I-II) (25%) and the advanced disease group (stage III-IV) (75%) (p<0.01). A statistically significant relationship was noted between the presence of EGFR and lymph node status (p=0.03) (Table 3). When EGFR expression in colonic carcinomas was compared with the grade of differentiation, the statistically significant difference was observed between moderately differentiated (58.8%) and well differentiated (35.3%). (p=0.05). In this study, we can not compare the histological grade between well and poorly differentiated since the number of poorly differentiated tumours are too small. The incidence of recurrent disease is also described in Table 3. Six patients with EGFR overexpression had a high risk for recurrent

disease (17.7%), while only 3 patients with negative for EGFR had disease recurrence (4.6%) (p=0.04).

C. Correlation biomarkers

EGFR

other

For colorectal cancer patients, CEA and CA19-9 are commonly used as clinical tool in the diagnosis and monitoring of metastasis in our institute. The cut-off value of CEA and CA19-9 among Thai population are 6.5 ng/ml and 37 U/ml, respectively. Thirty-two (35.2%) of 91 patients had higher initial CEA concentration. We observed that EGFR-positive tumours had 16 (51.6%) of high initial CEA concentration, while EGFR-negative tumours had 16 (26.7%) of high initial CEA concentration (Table 4). There was a statistical significance correlation between the presence of EGFR and high initial CEA concentration (p=0.02). No association was seen between EGFR expression and high initial concentration of CA19-9 (p=0.25).

Table 2. Contribution of adenocarcinoma cells with different EGFR intensities to the percentage of reactive Cells.

% stained cells " 5% > 5 â&#x20AC;&#x201C;25 % > 25 â&#x20AC;&#x201C; 50 %

No. cases 30 3 1

EGFR staining intensity 1+ 19 1 -

2+ 8 1 -

3+ 3 1 1

EGFR: epidermal growth factor receptor 1+: weak staining: 2+: moderate staining:3+: strong staining.

Figure 1. EGFR immunostaining in colorectal carcinoma (a) negative staining (b) 1+ staining intensity (c) 2+ staining intensity (d) 3+ staining intensity (Original magnification x400).

139

140 Table 3. Distribution of the clinico-pathological characteristics according to the EGFR expression EGFR Feature

Positive n (%)

Negative n (%)

p- value

" 50 >50

6 (17.65) 28 (82.35)

16 (24.61) 49 (75.38)

0.42

Male Female

18 (52.94) 16 (47.06)

37 (55.38) 28 (44.62)

0.70

22 (64.71) 12 (35.29)

48 (73.85) 17 (26.15)

0.34

8 (25.00) 24 (75.00)

38 (61.29) 24 (38.71)

< 0.01*

9 (28.13) 23 (71.87)

39 (60.00) 26 (40.00)

0.003*

12 (35.29) 20 (58.82) 2 (5.89)

37 (56.92) 26 (40.00) 2 (3.08)

0.05 *$

28 (82.35) 6 (17.65)

62 (95.38) 3 (4.62)

0.04*

Age

Sex

Site Colon Rectum Stage I and II III and IV Lymph node status NN+ Histophatological grade Well differentiated Moderated differentiated Poorly differentiated Disease recurrence No Yes $

p-value for well differentiated VS moderate differentiated

Table 4. Association between EGFR expression and other biomarkers EGFR Marker Carcinoembryonic antigen (ng/ml) CEA < 6.5 CEA # 6.5 Carbohydrate antigen (19-9) (U/ml) CA 19-9 < 37 CA 19-9 # 37

Positive n (%)

Negative n (%)

p-value

15 (48.38) 16 (51.62)

44 (73.33) 16 (26.67)

0.02*

21 (75.00) 7 (25.00)

46 (85.19) 8 (14.81)

0.25

have different outcomes. Obstruction or perforation, tumor grades, venous invasion, perineural invasion, age, gender, or allelic loss of chromosome 18q have been identified as factors possibly independent for survival (Takebayashi et al. 2004) but only a few of them have impact on colorectal cancer patientsâ&#x20AC;&#x2122; prognosis and survival. Recently there has been much discussion about potential prognosis markers for colorectal cancer such as Epidermal Growth Factor Receptor (EGFR) (Resnick et al, 2004; Nicholson et al, 2001). EGFR is highly expressed in a variety of solid tumors, colorectal cancer, lung cancer and head & neck cancer (Spano et a, 2005). This study was performed to retrospectively evaluate EGFR overexpression by immunohitochemical staining in formaline-fixed paraffin-embedded tissue samples collected from 99 consecutive patients who underwent surgical treatment at National Cancer Institute, Bangkok,

D. Correlation of EGFR expression and patient survival The primary endpoint in this study was overall survival, as measured from the time of diagnosis. We analyzed the association between EGFR expression and overall survival of 99 patients. The mean survival was 78 months (95% CI, 61-95) in the patients whose tumors expressed EGFR, which was not significant different from mean survival of 155 months (95% CI, 131-179) observed in patients with an absence of EGFR expression in tumors (p=0.40)(Figure 2).

IV Discussion TNM staging or classification is useful in selecting the colorectal cancer patients for specific treatment, but it is not sufficient as several patients in the same stage may 140

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Figure 2. Overall survival according to EGFR-status

Thailand during 2001 to 2004 from the department of pathology. In our study, EGFR is overexpression in 34 among 99 colorectal cancer patients. This number can be counted as 34.34% compare to 30% - 77% in the previous reports (Nakae et al, 1993; De-Jong et al, 1998; Yasui et al, 1988). This wide range of the EGFR overexpression is attributed to different detection techniques and time of storage of cut tissue sections (Atkins et al, 2004). We also found a significant association of EGFR with lymph node status (p=0.03), tumours stage (p<0.01), and differentiation grade (p=0.05). In addition, in our series EGFR status is a significant prognostic factor for disease recurrence (p=0.04). Elevated levels of serum CEA are commonly used as a clinical tool in the diagnosis, monitoring of metastasis (Ito et al, 2002), and determining response to treatment (Yamamoto et al, 2004). Overexpression of EGFR was associated with higher initial CEA concentration in our study (p=0.02). EGFR has been shown to be a strong prognostic indicator in certain cancer types (Nicholson et al, 2001); however, its role as an independent prognostic marker in colonic cancer has not been clearly defined yet. In certain studies, increased EGFR expression in colon cancer was associated with tumour stage, metastatic potential, relapse-free, and overall survival (Steele et al, 1990; Kluftinger et al, 1992; Mayer et al, 1993; Radinsky et al, 1995; De-Jong et al, 1998), although other studies have shown no such correlation (Komuta et al, 1995; Saeki et al, 1995; Barozzi et al, 2002; Lee et al, 2002; McKay et al, 2002). Determination of EGFR status may also identify patient populations that would benefit from anti-EGFR therapies. It also has the potential to provide an accurate means of monitoring the efficacy of anti-EGFR treatments and lead to significant improvements in cancer therapy. Currently, the data from many clinical studies have shown promising results (Mendelsohn and Baselga, 2003), although the

correlation between EGFR expression and response to therapy remains unclear (Ciardiello and Tortora, 2003). In conclusion, presents results indicate that EGFR expression is an indicator of poor prognosis and disease recurrence. Additional larger studies will be needed to determine EGFR expression and response to targeted antiEGFR therapy.

Acknowledgements This work was supported by the Terry Fox Foundation

References Arteaga CL (2001) The epidermal growth factor receptor: from mutant oncogene in nonhuman cancers to therapeutic target in human neoplasia. J Clin Oncol 15, 32-40. Atkins D, Reiffen KA, Tegtmeier CL, Winther H, Bonato MS, Storkel S (2004) Immunohistochemical detetion of EGFR paraffin embedded tumor tissues: variation in staining intensity are to choice of fixation and stronger time of tissues sections. J Histochem cytochem 52, 893-901. Barozzi C, Ravaioli M, Dâ&#x20AC;&#x2122; Errico A, Grazi GL, Poggioli G, Cavrini G, Mazziotti A, Grigioni WF (2002) Relevance of biologic markers in colorectal carcinoma: a comparative study of a broad panel. Cancer (Phila) 94, 647-57. Ciardiello F, Tortora G (2003) Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur J Cancer 39, 1348-54. De-Jong KP, Stellema R, Karrenbeld A, De Jong KP, Koudstaal J, Gouw AS, Sluiter WJ, Peeters PM, Sloolf MJ, De Vries EG (1998) Clinical relevance of transforming growth factor $, epidermal growth factor receptor, p53 and Ki67 in colorectal liver metastasis and corresponding primary tumors. Hepatology 284, 971-9. Geene FL (2002) American Joint Committee on Cancer,

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142 American Cancer Society. American Joint Committee on Cancer cancer staging manual. New York: Springer-Verlag. Giralt J, Eraso A, Armengol M, Rossello J, Majo J Ares C, Espin E, Benavente S, de Torres I (2002) EGFR is a predictor of tumor response in locally advanced rectal cancer patients treated with preoperative radication therapy. Int J Radical Oncol Biol Phys 54, 1400-1465. Goldstein NS, Armin M (2001) EGFR immunohistochemical reactivity in patients with AJC on cancer stage IV colon adenocarcinoma. Cancer 92,1331-1346. Grunwald V, Hidalgo M (2003) Development inhibitions of the epidermal growth factor receptor for cancer treatment. J Natl Cancer Inst 95, 851-867. Ito K, Hibi K, Ando H, Hidemura K, Yamazaki T, Akiyama S, Nakao A (2002) Usefulness of analytical CEA doubling time and half-life time for overlooked synchronous metastases in colorectal carcinoma. Jpn J Clin Oncol 32, 54-8. Kluftinger AM, Robinson BW, Ouenville NF, Finley RJ, Davis NL (1992) Correlation of epidermal growth factor receptor and c-erbB2 oncogene product to known prognostic indicators of colorectal cancer. Surg Oncol 1, 97-105. Komuta K, Koji T, Izumi S, Matsumoto T, Kohara N, Motojima K, Kanematsu T(1995) Expression of epidermal growth factor receptor for messenger RNA in human colorectal carcinomas assessed by non-radioactive in-situ hybridization. Eur J Surg Oncol 21, 269-75. Lee JC, Wang ST, Chow NW, Yang HB (2002) Investigation of the prognostic value of coexpressed erbB family members for the survival of colorectal cancer patients after curative surgery. Eur J Cancer 38, 1065-71. Martin N, Pongnikorn S (2003) colon and rectum. In: Cancer in Thailand Vol III (Sriplung H, Sontipong S, Martin N, Wiangnon S, Vootiprux V, Cheirsilpa A, Kanchanabat C, Khuhaprema T, eds). Bangkok, Bangkok Medical Publiher, pp 31-33. Mayer A, Takimoto M, Fritz E, Schellander G, Kofler K, Ludwig H (1993) The prognostic significance of proliferating cell nuclear antigen, epidermal growth factor receptor, and mdr gene expression in colorectal cancer. Cancer (Phila) 71, 2454-60.

Nakae S, Shimada E, Urakawa T (1993) Study of c-erbB-2 protein and epidermal growth factor receptor expression and DNA ploidy pattern in colorectal carcinoma. J Surg Oncol 54, 249-251. Nicholson RI, Gee JM, Harper ME (2001) EGFR and cancer prognosis. Eur J Cancer 37, S9-S15. Radinsky R, Risin S, Fan D, Dong Z, Bielenkerg D, Bucana CD, Fidler IJ (1995) Level and function of epidermal growth factor receptor predict the matastatic potential of human carcinoma cells. Clin Cancer Res 1, 19-31. Resnick M, Routhier J, Konkin T, Sabo E, Prico VE (2004) Epidermal growth factor receptor, c-MET, %-Cathenin, and p53 expression as prognostic indicators in stage II colon cancer: A tissue microarray study. Clin Cancer Res 10, 3069-3075. Saeki T, Salomaon DS, Johnson GR, Gullick WJ, Mandai K, Yamagami K, Moriwqki S (1995) Association of epidermal growth factor related peptides and type I receptor kinase receptors with prognosis of human colorectal carcinomas. Jpn J Clin Oncol 25, 240-9. Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103, 211-225. Spano JP, Lagorce C, Allan D, Milano G, Domont J, Benamouziq R, Attar A, Benichou J, Martin A, Morere JF, Raphael M, Penault-Llorca F, Breau JL, Faqard R, Khayat D, Wind P (2005) Impact of EGFR expression on colorectal cancer patient prognosis and survival. Ann Oncol 16, 102108. Steele RJ, Kelly P, Ellul B, Eremin O (1990) Epidermal growth factor receptor expression in colorectal cancer. Br J Surg 77, 1352-4. Takebayashi S, Hickson A, Ogawa T, Tung KY, Mireta H, Ueda Y, Grenman R, Fisher SG, Carey TE (2004) Loss of chromosome arm 18q with tumor progression in head and neck squamous cancer. Genes Chromosomes Cancer 41, 145-154. Yamamoto M, Maehara Y, Sakaguchi Y, Mine Y, Mamanaka T, Korenaga D, Okamura T (2004) Distributions in CEA doubling time differ in patients with recurrent colorectal carcinomas. Hepatogastroenterology 51, 147-51. Yarden Y (2001) The EGFR family and its ligands in human cancer: signaling mechanisms and therapeutic opportunities. Eur J Cancer 37, S3-S8. Yasui W, Sumiyoshi H, Hata J Kameda T, Ochiai A, Ito H, Tahara E (1988) Expression of epidermal growth factor receptor in human gastric and colonic carcinomas. Cancer Res 48, 137-141.

McKay JA, Murray LJ, Curran S, Ross VG, Clark C, Murray GI, Cassidy J, McLead HL (2002) Evaluation of the epidermal growth factor receptor (EGFR) in colorectal tumors and lymph node metastases. Eur J Cancer 38, 2258-64. Mendelsohn J, Baselga J (2003) Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 21, 2787-99.

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Cancer Therapy Vol 5, page 143 Cancer Therapy Vol 5, 143-150, 2007

Bio-immunotherapy in the treatment of equine sarcoid, the commonest tumour of the horse Research Article

Raija E. Hallamaa Veterinary Clinic, Pisteenkaari 4, 03100 Nummela Institute of Bio-Immunotherapy, Gylden Str. 2B, 00200 Helsinki, Finland

__________________________________________________________________________________ *Correspondence: Raija Hallamaa, DVM, Institute of Bio-immunotherapy, Gylden Str. 2B, 00200 Helsinki, Finland; e-mail: raija.hallamaa@elisanet.fi Key words: Horses, Clinical treatment, Microscopical examinations, Chromosomal examinations, Statistical analyses, regressing tumour tissue, Antibody findings Abbreviations: Bovine papillomavirus, (BPV); haematoxylin and eosin, (HE) Received: 4 December 2006; Revised: 7 February 2007 Accepted: 9 May 2007; electronically published: May 2007

Summary Equine sarcoids of 32 horses with either primary or recurrent tumours were treated by active specific bioimmunotherapy. The present treatment consisted of a partial surgical extirpation of tumours, repeated immunizations with autogenous polymerized tumour tissue and supportive dietary treatment with stannic chloride and folic acid. Healing of the unexcised tumour tissue was followed by serial biopsies studied under light and electron microscope. Disease-free intervals after bio-immunotherapy lengthened significantly as compared with the disease-free intervals after previous conventional surgery. Regression of the tumour tissue could not be explained by chromosomal alterations, classical immunological reactions or any form of cell death including apoptosis. Transformed mitochondria containing electron dense bodies were found in regressing tumour tissue following immunizations, but not before. These transformed mitochondria seem to be involved in the regulation of normal and malignant cell growth.

tissue and supportive dietary treatment. It has been shown to be an effective and practical method to treat equine sarcoid (Tallberg et al, 1994; Kinnunen et al, 1999). This treatment does not necessitate a total extirpation of tumour tissue. The chance to leave the base in the horse skin without endangering the clinical state, made it possible to follow the healing process both by inspections and serial biopsies. The aim of this article is to summarize the clinical results of the series of horses treated by bioimmunotherapy in 1991-2006 and to delineate the histopathological and chromosomal changes found in the regressing tumour tissue during the treatment.

I. Introduction Equine sarcoid is a benign, but locally aggressive, skin tumour of horses, donkeys and mules (Jackson 1936). This fibroblastic tumour (Figure 1a) is found mainly in young horses, and the typical predilection sites are the head, limbs and ventral abdomen (Ragland et al, 1970; Torrontegui and Reid 1994). Equine sarcoid may occur as a single tumour or as multiple tumours. Bovine papillomavirus (BPV) or a closely related virus is suggested to play a role in the aetiology and pathogenesis (Trenfield et al, 1985; Angelos et al, 1991; Carr et al, 2001a). Equine sarcoid has a marked tendency to recur despite various treatments, especially after surgery, if tumour tissue has not been skilfully and radically removed (Jackson 1936; Ragland et al, 1970; Marti et al, 1993; Brostrรถm 1995; Knottenbelt and Matthews 2001). Unfortunately, equine sarcoid commonly affects sites, where complete extirpation is difficult or impossible to perform. Bio-immunotherapy consists of repeated immunizations with autogenous polymerized tumour

II. Materials and methods A. Horses A total of 32 horses with histologically confirmed equine sarcoid were entered into the present study, which started in 1991. Horses had either primary, previously untreated sarcoids (18 horses) or recurrent, previously once or several times surgically treated sarcoids (14 horses). The horses were not selected; the only prerequisite for a horse to be included was the

143

Hallamaa: Bio-immunotherapy in the treatment of sarcoid size of a tumour or tumours. The minimum amount of tissue for autovaccine was 10 g corresponding to a tumour approximately 2 to 3 cm in diameter.

homogenised between the second and third wash and finally washed with 0.6% sodium azide. The supernatant was decanted. The polymer particles with addition of 0.6% sodium azide were further homogenised, ten times by using a ceramic plunger in glass tubes. The polymer particles with sodium azide as a preservative were bottled and stored at +4°C. Before each vaccination, sodium azide was washed off by centrifuging the dose of polymer particles with 0.9% NaCl. More detailed instructions have been described earlier (Tallberg et al, 1991). A modification of bio-immunotherapy as used on human cancer patients (Tallberg et al, 1979) was used on these horses. Immunizations were given as bilateral intradermal injections in the neck and also in the loins if tumours were situated in the caudal half of the horse. Horses were vaccinated at 2- to 4-week intervals until no tumour tissue could visibly be found in the debulked base. Tuberculin (purified protein derivative PPD 10 TU/ml, Statens Seruminstitut, Copenhagen) as adjuvant was mixed 1:1, using 0.5 or 1.0 ml of the autovaccine immediately before vaccinations. A detailed immunization schedule has been described earlier (Tallberg et al, 1994; Kinnunen et al, 1999). The supportive dietary treatment consisted of oral administration of stannic chloride (20 mg) and folic acid (5-10 mg) mixed in the daily fodder during the immunization period. If the normal vitamin supplements in horse fodder included enough folic acid, no supplementary folic acid was given. Six horses were left without stannic chloride supplements. The efficacy of bio-immunotherapy was followed by inspections and photography (Figure 1a-1d) and was assessed according to the length of the disease-free interval.

B. Clinical treatment Tumours were partially removed by leaving the base in the skin. Of the horses with multiple tumours, only the largest (diameter >2 cm) and tumours of fibroblastic type were excised. The autovaccine was made from the debulked part of the tumour according to the polymerization technique devised by Avrameas and Ternynck, 1967. The removed part was cleaned of necrotic tissue, cut into small pieces and homogenised at +4°C into a suspension with distilled water by using a high-speed homogeniser. The homogenate was frozen overnight at !20°C for the further disruption of the tumour cells. Next day the homogenate was ultrasonicated and centrifuged at +4°C. The supernatant was harvested and the precipitate discarded. The volume and protein concentration of the supernatant was measured. If the protein concentration was under 1.2%, patient’s own serum was added until a concentration of 1.2% was achieved. The supernatant was polymerized with ethyl chloroformate and sodium acetate (pH 4.8) as a buffer into insoluble polymer particles, by continuously mixing with a magnet for 60 min. The pH was kept between 4.5 and 4.8 by adding NaOH (1 mol/l). After 30 minutes, sodium acetate (0.2 mol/l) was added as equal volume to that of the original supernatant. After polymerization, the supernatant was decanted saving the polymer particles. The polymer was washed three times by centrifugation with sterile physiological saline at +4°C,

Figure 1. Equine sarcoid tumour in the ventral abdomen at the beginning of bio-immunotherapy (a). The same tumour five weeks after the partial excision combined with active specific bio-immunotherapy. Regression has started at the margin of the tumour, while the centre still shows fibroblastic tumour tissue (b). A small scar tissue is found at the site of the previous sarcoid 13 months later (c). The tumour has not recurred during the 12-year follow-up. All scar tissue has reverted to normal skin (d).

144

Cancer Therapy Vol 5, page 145 The disease-free interval was regarded as the time between the bulk excision and the first regrowth found at the site of surgery, at any new sites or in the small multiple tumours that were not excised at all.

chromosomal changes Wilcoxon’s matched pair test was used to analyse the difference of polyploidism before and during the regression. The Mann-Whitney test for quantitative and Fisher’s exact test for qualitative variables were used when differences between the features of the primary and initially recurrent sarcoids were evaluated. Two sided p-values < 0.05 were considered significant in all tests.

C. Microscopic examinations The samples for light and electron microscopy were collected from 18 horses at the time of the excision and the follow-up biopsies when visibly normal epithelisation was observed at the base of the debulked tumour. The specimens were taken as longitudinal sections from the healthy skin to the centre of the tumour base. Changes were followed by successive sections obtained from these longitudinal biopsies. For light microscopy samples were fixed in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, embedded in paraffin, sectioned at 6 "m and stained routinely with haematoxylin and eosin (HE). For electron microscopy samples were fixed in 2% formaldehyde with 1.25% glutaraldehyde solution and with 0.1 M phosphate buffer added immediately before biopsy, at pH 7.2. Samples were washed in the same buffer as was used in the fixation, postfixed in phosphate buffered 1% OsO4, dehydrated and embedded in Epon. Samples were sectioned at 60 nm and post-stained with lead citrate and uranyl acetate. Three fields from each sample were scanned and the number of normal and transformed mitochondria was calculated. The values were expressed as the percentage of transformed mitochondria of the total number of mitochondria found.

III. Results A. Clinical findings Of the horses, 50% had a single tumour and the other half had multiple tumours. The mean number of tumours in the horses with multiple sarcoids was 5.3+6.9 (range 230 tumours). The size of the tumours ranged from 1 to 8 cm, the mean diameter of the largest sarcoids being 3.4+1.7 cm. Tumours were situated on the trunk, head and limbs; the trunk was affected most commonly. Of the horses, 12 had either one or several tumours at sites where conventional surgery would have been difficult or impossible to perform. Clinical results of the 32 horses treated by active specific bio-immunotherapy are presented in Table 1. The mean disease-free interval after conventional surgery had been 5.2+3.9 months (range 0.5-12) in the horses (n=14, marked by bolt type in Table 1) which had been surgically treated once or several times before bio-immunotherapy started. After bio-immunotherapy, the disease-free intervals of these horses lengthened significantly (p=0.0015), approximately more than eight times, as compared with the previous conventional surgery (Table 2). The horses with primary tumours had significantly longer disease-free intervals following bio-immunotherapy than the horses with initially recurrent sarcoids when the horses had been followed for at least one year (p=0.0164, the Mann-Whitney test). A total of seven horses (26%) got a recurrence after bio-immunotherapy when the horses had been followed for at least 2-3 years (Table 1); 12% of the horses with a primary and 45% of the horses with initially a recurrent tumour. However, the difference between the recurrence rates of the horses with a primary and with initially a recurrent tumour, was not significant (p=0.0837). Most of the recurrences were found in 12 months. Four of the seven horses with relapse following bio-immunotherapy were treated anew with autogenous tumour vaccine made from this recurrent tumour. Of the four horses, three did not develop recurrences after repeated bio-immunotherapy. A total of 26 horses became disease-free after initial or repeated bio-immunotherapy. Most of the horses got 2 to 5 immunizations, the mean being 5.1+3.9 (range 2-23). Horses with recurrent sarcoids in the beginning of bio-immunotherapy usually required more immunizations to cause tumour regression than the horses with primary tumours. No side-effects relating to bio-immunotherapy could be found during the treatment or the long follow-up from 1991 to 2006.

D. Chromosomal examinations Chromosomal aberrations were studied on the sarcoids of 11 horses with primary or recurrent sarcoids and on the healthy skin samples of the 8 control horses with or without sarcoids. Of the 8 control horses, four were healthy horses, three recovered from sarcoids, while one horse still had sarcoids. The studies on chromosomal polyploidism were performed according to standard technique by Medix Company, Clinical Laboratory, Espoo, Finland, with the exception that sarcoids’ cells were cultured as normal human fibroblasts and not as human tumour cells, as usually. The samples were incubated for 5-15 days at 37°C in a CO 2 atmosphere until obvious cell growth was found. The presence of polyploidy was expressed as the percentage of polyploidy mitoses among the 100 mitoses detected. The value of 10% or lower, normally found in human tissues, was regarded as a guide.

E. Examinations for autoantibodies Agglutinating antibodies were titrated by slide agglutination with the antigenic polymer particles of the extirpated tumour tissue and with serum dilutions of 1:1, 1:2, 1:4, 1:8, 1:16 and 1:100. Precipitating autoantibodies were analysed by double diffusion in agar gel. Tumour homogenates were diluted 1:1, 1:10 and 1:100 and the sera 1:1 and 2-fold dilutions from 1:2 to 1:32.

F. Statistical analyses The efficacy of bio-immunotherapy was evaluated according to the disease-free intervals. Wilcoxon’s matched pair test was used to analyse the difference between the disease-free intervals after previous conventional surgery and following bioimmunotherapy in the horses with initially recurrent tumours. The difference between the disease-free intervals after bioimmunotherapy in the horses with primary sarcoids and in the horses previously treated by conventional surgery was compared using the Mann-Whitney test. The difference between the histopathological changes before and during the tumour regression was evaluated using the exact McNemar’s test. For

B. Macroscopic findings in the regressing tumour tissue Visibly normal epithelisation started at the margin of the incised tumour and progressed gradually to the centre (Figure 1b). The mean rate of epithelisation was

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Hallamaa: Bio-immunotherapy in the treatment of sarcoid 0.25+0.15 mm/day, i.e. a tumour base 3 cm in diameter had completely regressed and was covered by normal skin in approximately 9 weeks. Neither exuberant granulation tissue nor harmful scar tissue developed at the site of incision (Figures 1c and 1d). Hair growth was total in 64% of the horses, partial in 11%, while the site was left hairless in 25%. The small multiple tumours that were left without partial excision decreased in size or dropped off during a 2-year follow-up. If the debulked tumour recurred, the unoperated tumours also became active.

i.e. approximately in 9.2+3.1 weeks. The histopathological picture in the biopsies taken at the beginning of bioimmunotherapy was typical of equine sarcoid (Figure 2). Both the epidermal and dermal components of sarcoid were found in all biopsies. Light microscopic studies showed that the epidermal part became normal earlier than the dermal (Hallamaa et al, 2005). In the dermal component, healing seemed to proceed from the deeper part of the tumour to the superficial part and finally the last tumoural fibroblasts were found near the dermoepidermal junction. Neither lymphocyte nor natural killer cell infiltration were found during regression (Figure 3). Neutrophils were found only in the tumours that were ulcerated and inflamed in the beginning of bioimmunotherapy, but not later on when the surface of the epidermis had healed fully. In electron microscopy, transformed mitochondria (Figure 4) were found in all biopsies collected after

C. Microscopic findings in the regressing tumour tissue The first biopsies were taken at time of the bulk excision and the follow-up biopsies when the base of the tumour began visibly to heal (Figure 1b). In most of the horses, visibly normal epithelisation was observed at the margin of the base after the second or third immunization,

Table 1. Clinical results after active specific bio-immunotherapy in equine sarcoid. Horses with recurrent tumour at the beginning of bio-immunotherapy are marked by bolt type. Horses without recurrence after bio-immunotherapy A C D E F G H I J K L M N O* P R W Y Z BB CC DD* EE FF GG Total 25 Horses with recurrence after bio-immunotherapy

Follow-up periods/ months (years) 72 (6) 72 (6) 120 (10) 132 (11) 27 (2.2) 102 (8.5) 102 (8.5) 84 (7) 72 (6) 66 (5.5) 56 (4.7) 60 (5) 60 (5) 148 (12.4) 48 (4) 64 (5.3) 71 (5.9) 36 (3) 54 (4.5) 58 (4.8) 4 (0.3) 3 (0.3) 1 (0.1) mean 65.7+38.3 (5.5+3.2) Disease-free intervals before recurrence/months (years)

10 (0.8)

5 (0.4)

17 (1.4)

9 (0.8)

8 (0.7)

30 (2.5)

5 (0.4)

Total 7

mean 12.0+8.9 (1.0+0.7)

Total 32

mean 53.2+40.8 (4.4+3.4)

*2 horses underwent euthanasia during treatment due to diseases not related to equine sarcoid.

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Cancer Therapy Vol 5, page 147

Table 2. The disease-free intervals after previous surgery and after bioimmunotherapy in the horses with recurrent tumours at the beginning of bio-immunotherapy. Horses

Disease-free intervals after previous surgery / months

Disease-free intervals after first bioimmunotherapy / months

148

0,5

EE Total 12

mean 4.7+3.8

mean 39.7+41.9

Difference between the disease-free intervals is significant (p=0.0015) Figure 2. Histologically equine sarcoid consists of an epidermal and dermal part. Hyperplasia and epidermal projections protruding into the dermis are typical for the transformed epidermis, while the dermal part shows numerous whorlforming fibroblasts and collagen (HE).

Figure 3. Equine sarcoid tissue two weeks after the second immunization with autogenous tumour vaccine. No leucocyte infiltration could be found in the regressing tumour tissue (HE).

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Hallamaa: Bio-immunotherapy in the treatment of sarcoid Figure 4. Transformed mitochondria with internal electron dense bodies were found in the regressing tumour tissue following active specific bioimmunotherapy (EM 4000x).

immunization and while the tumour was regressing, but not before (p=0.002, the exact McNemar’s test). These transformed mitochondria contained electron dense bodies, the size of which varied from 0.1 to 3 "m and number from one to several in separate mitochondria (Tallberg et al, 1995). No traces of apoptosis or necrotic cell death could be found during regression.

the most suitable tumours for wide excision were selected (Martens et al, 2001) or when the horses were operated in recumbency under general anaesthesia (Broström 1995), respectively. Most of the recurrences after bioimmunotherapy developed within 12 months, while following conventional surgery recurrences are usually found within six months (Ragland et al, 1970; Broström 1995). In the present study, no recurrences were found after two and half years; the time after which recurrences usually have not been found (Theon and Pascoe 1994). The present treatment was practical. Tumours at delicate sites could be removed without difficulty and no deforming scar tissue was formed, despite the partial extirpation. Horses with either single tumour or multiple tumours could be treated, and it was not necessary to manage all tumours in horses with multiple sarcoids as has to be done by conventional surgery or most of the usual sarcoid therapies (Vanselow et al, 1988; Marti et al, 1993; Knottenbelt and Walker 1994; Theon and Pascoe 1994; Spoormakers et al, 2003). No side-effects relating to the therapy could be found during repeated immunizations or the long follow-up. Water insoluble polymerized protein particles do not induce autoimmune reactions, which has been confirmed earlier in animal experiments and studies on human cancer patients (Tallberg et al, 1979; Tykkä 1981). The supportive dietary treatment may not play an important role in horses, as it has been shown to have in human patients (Tallberg et al, 1996; Tallberg 2003). Most of the horses had got folic acid in their normal daily vitamin supplements before bio-immunotherapy started. Of the six horses left without stannic chloride supplementation, only one developed a recurrence after bio-immunotherapy. Bovine papillomavirus (BPV) is closely associated with the aetiology of equine sarcoid (Trenfield et al, 1985; Angelos et al, 1991; Carr et al, 2001a, b). BPV-DNA has been found generally in the sarcoids but also in the healthy skin of sarcoid affected horses (Carr et al, 2001a; Bogaert et al, 2005). However, the role of BPV in the progression of equine sarcoid is still unclear, as is the possibility of viral latency (Bogaert et al, 2005). In the present study, there were horses both with and without close contact with cattle. An interesting case was the mare in Figures 1a-1d. This mare and two of her foals, which spent several years

D. Chromosomal findings Chromosomal aberrations, in the form of polyploidism, were found in tumour cells both before and during immunizations, as described previously (Tallberg et al, 1995). However, the mean number of polyploidy mitoses before the immunization was not significantly higher than the number in regressing tumour tissue, 13.0+10.9% (range 1-33) and 8.1+6.2% (range 1-18), respectively. Moreover, the mean number of polyploidism, 16.6+11.8% (range 6-43), found in the cells of the healthy horse skin with and without sarcoids did not significantly differentiate from the value of sarcoid cells.

E. Antibody findings No marked elevation was found in the autoimmune titre of immunized horses during the healing; only a low slide agglutinating titre was detected before immunizations and these titres decreased or disappeared after immunizations and tumour regression (Tallberg et al, 1994). A precipitating antibody titre was not found in any sera.

IV. Discussion Bio-immunotherapy consisting of active specific immunotherapy combined with supportive dietary treatment was shown to be a promising method to treat equine sarcoid. Disease-free intervals following bioimmunotherapy lengthened significantly in the horses with relapse after previous conventional surgery. However, these horses tended to develop a recurrence more often than the horses that received bio-immunotherapy as the first treatment. The total success rate was 74%, thus being competitive with the results of the usual treatments in equine sarcoid (Broström 1995; Martens et al, 2001; Spoormakers et al, 2003). Exceptionally high success rates after skilful surgery, 82% and 80%, were achieved, when

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Cancer Therapy Vol 5, page 149 in the birth stable in contact with cattle, got equine sarcoids, while the other three foals of the mare sold before the age of one and half year have not got sarcoids. Age about 1.5 years seems to be a critical point in the development of the disease; before that age sarcoids have not been reported (Marti et al, 1993; Reid et al, 1994; Torrontegui and Reid 1994; Broström 1995) and were not found in the present study either. At an age of about two years the horse has finished the period of fast growth. It may be that horses have some protective factors as yearlings, or after that age they are more susceptible to viral infection and development of equine sarcoid, or latent viral infection with a risk of future disease. If a virus is a causative agent, systemic immunological treatments rather than only local therapies would be better in the treatment of equine sarcoid. The antigenic marker seems to change when the sarcoid recurs. Horses which developed a recurrent tumour following bio-immunotherapy were revaccinated with the autovaccine prepared from the initial sarcoid, but no positive effect was seen (Kinnunen et al, 1999). When the horses were retreated with the autovaccine made from the recurrence, most of the horses reacted positively. It is possible that the horse is susceptible to re-infection with another type of bovine papillomaviruses. Equine sarcoid was an ideal target to study tumour tissue before and during regression, since bioimmunotherapy did not entail a radical excision. Serial biopsies could be collected from the tumour tissue left in the skin. This incomplete surgery is not ethically relevant for human patients. However, the disease of the horses was not aggravated after partial excision combined with bio-immunotherapy. Macroscopically the regression of the debulked tumour resembled normal wound healing with an epithelisation rate typical of lacerations in the healthy horse skin (Stashak 1990). The regression of tumour tissue without classical immunological reactions or any form of cell death was an interesting finding, since after nonspecific immunotherapy and chemotherapy both leucocyte infiltrations and necrotic or apoptotic cell death have been found (Vanselow et al, 1988; Spoormakers et al, 2003). The only novel and different finding in the regressing tumour as compared with the tumour before bioimmunotherapy was the numerous intramitochondrial electron dense bodies that appeared after immunization. Similar electron dense bodies have not been earlier described to be colonized in sarcoid cells or mitochondria (Madewell and Munn 1989; Scheffler 1999). However, electron dense bodies had been found previously in chloroma leukaemia cells of rats treated by bioimmunotherapy, but these findings were then erroneously regarded as artefacts (Tallberg et al, 1986; Tallberg 2000). Not until these transformed mitochondria were found in the regressing horse tumours following bioimmunotherapy, were they linked to the healing process (Tallberg et al, 1994; Tallberg 2000). Later intramitochondrial dark bodies have been found also in the human malignant histiocytoma cells when the tumours are in regression after dietary biotherapy (Tallberg et al, 1996; Tallberg 2003). The fact that similar mitochondrial changes have been detected in three different mammalian

species implies that mitochondria play an important role not only in energy transformation, but also in the intracytoplasmic regulation systems. In sarcoids, fibroblasts are normally differentiated and they usually do not show a marked mitotic rate (Jackson 1936; Tarwid et al, 1985). However, they may divide rapidly, especially after mechanical irritation (Ragland et al, 1970; Tarwid et al, 1985; Broström 1995; Knottenbelt and Matthews 2001; Carr et al, 2001a). Following bio-immunotherapy these normally differentiated fibroblasts seem to be returned to a healthy state by transformed mitochondria. It has been suggested that cell cycle progression is dependent on mitochondrial ATP production and there could exist an energetic checkpoint at which the cell evaluates its energy stocks and decides to pause or proceed towards the cell cycle (Alirol and Martinou 2006). In the present study, healing could not be explained by classical immunological reactions, chromosomal alterations related to polyploidism or any form of cell death. However, regression simulated a spontaneous healing process. It seems that mitochondria in mammals are involved in restoring cells back to normal healthy function.

Acknowledgements I thank Docent Thomas Tallberg, DVM Elise SaarioPaunio and Dr. Jan Dabek for their advice and helpful suggestions. I am grateful to Professor Seppo Sarna for his advice with statistical analyses. This study was supported by the Finnish Society of Sciences and Letters, Ulla Hamberg’s Fund, and the Suoma Viiala Foundation, Finland.

References Alirol E and Martinou JC (2006) Mitochondria and cancer: is there a morphological connection? Oncogene 25, 4706-4716. Angelos JA, Marti E, Lazary S and Carmichael LE (1991) Characterization of BPV-like DNA in equine sarcoids. Arch Virol 119, 95-109. Avrameas S and Ternynck T (1967) Biologically active waterinsoluble proteinpolymers. Their use for isolation of antigens and antibodies. J Biol Chem 242, 1651-1659. Bogaert L, Martens A, De Baere C and Gasthuys F (2005) Detection of bovine papillomavirus DNA on the normal skin and in the habitual surroundings of horses with and without equine sarcoids. Res Vet Sci 79, 253-258. Broström H (1995) Equine Sarcoids. A Clinical and Epidemiological Study in Relation to Equine Leucocyte Antigens (ELA). Acta vet scand 36, 223-236. Carr EA, Theon AP, Madewell BR, Griffey SM and Hitchcock ME (2001a) Bovine papillomavirus DNA in neoplastic and nonneoplastic tissues obtained from horses with and without sarcoids in the western United States. Am J Vet Res 62, 741-744. Carr EA, Theon AP, Madewell BR, Hitchcock ME, Schlegel R and Schiller JT (2001b) Expression of a transforming gene (E5) of bovine papillomavirus in sarcoids obtained from horses. Am J Vet Res 62, 1212-1217. Hallamaa RE, Saario E and Tallberg Th (2005) Macroscopical and Histopathological Changes in Regressing Primary and Recurrent Equine Sarcoids During Active Specific Bioimmunotherapy. In Vivo 19, 761-768.

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Hallamaa: Bio-immunotherapy in the treatment of sarcoid Jackson C (1936) The incidence and pathology of tumours of domestic animals in South Africa. J Vet Sci Anim Indust 6, 378-385. Kinnunen RE, Tallberg Th, Stenbäck H and Sarna S (1999) Equine Sarcoid Tumour Treated by Autogenous Tumour Vaccine. Anticancer Res 19, 3367-3374. Knottenbelt DC and Matthews JB (2001) A Positive Step Forwards in the Diagnosis of Equine Sarcoid. Vet J 161, 224-226. Knottenbelt DC and Walker JA (1994) Topical treatment of the equine sarcoid. Equine Vet Educ 6, 72-75. Madewell BR and Munn RJ (1989) Tubuloreticular Inclusions in Equine Connective Tissue Neoplasms. J Comp Path 100, 449-452. Martens A, De Moor A, Vlaminck L, Pille F and Steenhaut M (2001) Evaluation of excision, cryosurgery and local BCG vaccination for the treatment of equine sarcoids. Vet Rec 149, 665-669. Marti E, Lazary S, Antczak DF and Gerber H (1993) Report of the first international workshop on equine sarcoid. Equine Vet J 25, 397-407. Ragland WL, Keown GH and Spencer GR (1970) Equine Sarcoid. Equine Vet J 2, 2-11. Reid SWJ, Smith KT and Jarrett WFH (1994) Detection, cloning and characterisation of papillomaviral DNA present in sarcoid tumours of Equus asinus. Veterinary Record 135, 430-432. Scheffler IE (1999) Mitochondria New York, Wiley-Liss, Inc. , Publication. . Spoormakers TJP, Klein WR, Jacobs JJL, Van Den Ingh ThSGAM, Koten JW and Den Otter W (2003) Comparison of the efficacy of local treatment of equine sarcoids with IL-2 or cisplatin/IL-2. Cancer Immunol Immunother 52, 179184. Stashak TS (1990) Advances in Wound Management. In: White NA (eds) Current practice of equine surgery JB Lippincott Comp Philadelphia, . 34-37. Tallberg T, Tykkä H, Halttunen P, Mahlberg KL, Uusitalo R, Carlsson O, Sandstedt B, Oravisto K, Lehtonen T, Sarna S and Strandström H (1979) Cancer immunity. The effect in cancer-immunotherapy of polymerised autologous tumour tissue and supportive measures. Scand J clin Lab Invest 39, 1-35. Tallberg Th (2000) Mitochondria seem to regulate the genome, in the chromosomes they have phylogenetically created. Annual Meeting of Finnish Physiological Society 24th and 25th Nov 2000, 36-38. Tallberg Th (2003) Development of a Combined Biological and Immunological Cancer Therapy Modality a review of bioimmunotherapy. J Australian Coll Nutr and Env Med 22, 321. Tallberg Th, Kinnunen RE, Palkama A, Saario EMK and Borgström G (1994) Equine Sarcoid Successfully Treated by Bio-Immunotherapy. Dtsch Zschr Onkol 26, 34-40. Tallberg Th, Kinnunen RE, Stenbäck H, Virtanen I and Palkama A (1995) Bio-Immunotherapy of Equine Sarcoid with

Special Reference to Intracellular Histopathological Reactions. Dtsch Zschr Onkol 27, 157-163. Tallberg Th, Mahlberg K and Kalima T (1986) Experimental and Clinical Studies on Cancer Immunotherapy. In: Eds. Klippel KF and Macher E Present Status of Non-Toxic Concepts in Cancer Proc Int Symp , Nonnweiler/Trier, April, pp. 237263. Tallberg Th, Matous B, Tykkä H, Vosmik Fr, Sarna S, Bauer J, Krajsova I and Sundell B (1991) Malignant Melanoma. Effect of Post-operative Specific Immunotherapy and Dietary Supplementation on Progress and Prognosis. Tumor Diag and Therap 12, 139-146. Tallberg Th, Stenbäck H, Dabek J and Palkama A (1996) Complete disappearance of human malignant histiocytoma cells following dietary biotherapy, leading to activation of inductional control mediated by mitochondria. J Aust Coll Nutr and Env Med 15, 5-10. Tarwid JN, Fretz PB and Clark EG (1985) Equine Sarcoids: A Study with Emphasis on Pathologic Diagnosis. Compend Contin Educ Pract Vet 7, 293-301. Theon AP and Pascoe JR (1994) Iridium-192. interstitial brachytherapy for equine periocular tumours: treatment results and prognostic factors in 115 horses. Equine Vet J 27, 117-121. Torrontegui BO and Reid SWJ (1994) Clinical and pathological epidemiology of the equine sarcoid in a referral population. Equine Vet Educ 6, 85-88. Trenfield K, Spradbrow PB and Vanselow B (1985) Sequences of papillomavirus DNA in equine sarcoids. Equine Vet J 17, 449-452. Tykkä H (1981) Active specific immunotherapy with supportive measures in the treatment of advanced palliatively nephrectomised renal adenocarcinoma. A controlled clinical study. Scand J Urol and Nephrol, Suppl. 63. Vanselow BA, Abetz I and Jackson ARB (1988) BCG emulsion immunotherapy of equine sarcoid. Equine Vet J 20, 444447.

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Cancer Therapy Vol 5, page 151 Cancer Therapy Vol 5, 151-160, 2007

Chemotherapy in Androgen-Independent Prostate Cancer (AIPC): Whatâ&#x20AC;&#x2122;s next after taxane progression? Review Article

Jeanny B. Aragon-Ching, William L. Dahut* Medical Oncology Branch, National Cancer Institute, National Institutes of Health

__________________________________________________________________________________ *Correspondence: William L. Dahut, M.D., Medical Oncology Branch, National Cancer Institute, Building 10, Rm 12N226, 9000 Rockville Pike, Bethesda, MD 20892, USA; Phone: 301-435-8143; Fax: 301-435-3854; e-mail: dahutw@mail.nih.gov Key words: Chemotherapy, prostate cancer, mitoxantrone, docetaxel, epothilones, satraplatin Abbreviations: androgen-independent prostate cancer, (AIPC); National Cancer Institute, (NCI), overall survival, (OS); progressionfree survival, (PFS); prostate-specific antigen, (PSA) Received: 19 April 2007; Accepted: 9 May 2007; electronically published: May 2007

Summary Prostate cancer is the most common non-cutaneous cancer in the United States. Although most are diagnosed at earlier stages of disease, a significant number of patients will eventually progress to metastatic androgenindependent prostate cancer (AIPC) and will receive chemotherapy. The benefit of chemotherapy in overall survival has been demonstrated in studies utilizing docetaxel. However, duration of response is short and therapeutic options are limited after taxane failure. There is a need for effective chemotherapeutic agents in the second-line setting, either alone or in combination. Some of these regimens may also ultimately translate to the front-line chemotherapeutic setting as an alternative or perhaps in combination with a taxane.

docetaxel based chemotherapy. Several second-line hormonal treatment have been utilized in this setting, but responses had been short and non-durable (Goktas and Crawford, 1999; Klotz, 2000). In this population of patients where hormone-refractory state emerges, palliation with chemotherapy has been utilized. The use of chemotherapy especially during earlier analysis of single chemotherapeutic agents have been disappointing, with response rates of 8.7% and median survival of 10-12 months (Yagoda and Petrylak, 1993), until the recent introduction of taxanes (Gulley and Dahut, 2004). Among the first systemic agents studied was mitoxantrone, which was approved based on symptomatic improvement of quality of life (Tannock et al, 1996). Subsequently, treatment with docetaxel and prednisone was FDA-approved for the treatment of AIPC because of the demonstration of improved overall survival (OS) of 18.9 months versus mitoxantrone and prednisone with OS of 16.5 months (Tannock et al, 2004). The use of the docetaxel and estramustine showed similar survival advantage to docetaxel and prednisone, but with more toxicity (Gulley and Dahut, 2004; Petrylak et al, 2004). Despite the first clear advance in the treatment of metastatic prostate cancer, the median time to PSA

I. Introduction Prostate cancer is the leading non-cutaneous cause of malignancy in American men and it is estimated that 218,890 men will be diagnosed with prostate cancer and 27,050 would die from the disease in 2007 (Jemal et al, 2007). Since the advent of prostate specific antigen (PSA) screening, the majority of patients are diagnosed with localized disease and about 5% are diagnosed after the cancer has metastasized (Ries et al, 2006). Primary therapy for localized prostate cancer typically includes radical prostatectomy, external beam radiation therapy, brachytherapy, or active surveillance, but 30-40% of patients will eventually develop recurrent or metastatic disease (Dillioglugil et al, 1997). Androgen deprivation therapy achieved through medical or surgical castration has been the cornerstone of treatment for patients with metastatic disease (Huggins and Hodges, 1941; Figg et al, 1997; Sharifi et al, 2005). However, almost all patients progress to androgen-independent phenotype after a median of 18 - 36 months (Figg et al, 1997; Sharifi et al, 2005). Once metastatic androgen-independent prostate cancer (AIPC) develops, responses to alternative hormonal therapy or chemotherapy are not durable, with a median overall survival of approximately 18 months with 151

Aragon-Ching and Dahut: Chemotherapy in Androgen-Independent Prostate Cancer (AIPC) using mitoxantrone or ixabepilone as 2nd line regimens after taxane failure, respectively. Furthermore, taxanes retain activity whether used before or after mitoxantrone in a retrospective analysis, with equivalent progressionfree survival and overall survival in either sequence (Oh et al, 2006). Thus, although the use of mitoxantrone and prednisone after docetaxel has some activity, response is modest, and other agents with better potency are needed after taxane-based chemotherapy.

progression with taxane therapy remains limited to about 6-8 months, with many patients progressing thereafter (Savarese et al, 2001). Therefore, there is a clear need for new therapeutic strategies for patients with advanced AIPC who have failed previous taxane chemotherapy. This review will focus on several potential secondline chemotherapeutic agents that have shown promising results in the treatment of metastatic prostate cancer.

II. Chemotherapy in the second-line setting

B. Satraplatin and other platinum agents Satraplatin, formerly known as JM-216, is a thirdgeneration orally available platinum analogue that has similar but improved properties compared to other platinum agents like cisplatin, carboplatin, and oxaliplatin (Kelland, 2000). Similar to other platinum agents, satraplatin exerts its biological activity via reactive biotransformation products that bind to DNA , forming DNA adducts that cause the inhibition of DNA replication, cell cycle arrest, and induction of apoptosis, and repaired by the nucleotide excision repair (NER) mechanism, but not recognized by the DNA mismatch repair system that acts upon cisplatin and carboplatin adducts (Kelland, 2000). Unlike the older platinum generations, satraplatin is more lipophilic and more chemically stable, offering improved oral bioavailability which enables satraplatin to be administered orally. Pre-clinical studies with satraplatin demonstrated cytotoxic and anti-tumor activities comparable to cisplatin or carboplatin, as well as improved toxicity profiles with nephrotoxicity (as compared with cisplatin) and neurotoxicity (as compared with oxaliplatin), with myelosuppression as the major dose-limiting toxicity (Sternberg, 2005). Platinum agents, in general, has been studied in AIPC, but single-agent cisplatin response has been in the range of 0% - 19% (Rossof et al, 1979; Merrin, 1980; Qazi and Khandekar, 1983; Moore et al, 1986). However, single-agent carboplatin has shown some activity with 50% disease stabilization (Canobbio et al, 1993), and carboplatin in combination with paclitaxel and estramustine (TEC) has shown some antitumor activity with response rates up to 45% in patients with measurable disease (Kelly et al, 2001; Solit et al, 2003), or in previous taxane failures (Tay et al, 2004). Recently, carboplatin has been used for second-line treatment in patients who have failed prior docetaxel therapy (Oh et al, 2006). Updated results from a phase II trial that evaluated 34 patients using a combination regimen of docetaxel 60 mg/m2 and a carboplatin area under the curve of 4 (AUC 4 mg/ml/min) every 21 days showed PSA declines of >50% in 6 of 34 patients (18%, 95% C.I. 7-35%), with a median duration of PSA response of 7.4 months (95% C.I. 2.8-7.4 months) (Oh et al, 2007). There was also an observed partial response in 3 out of 21 patients who had measurable disease at baseline (14%; 95% C.I. 3-36%). These findings suggest that there may be synergism between carboplatin and docetaxel that warrants further investigation of its use in patients who have failed taxanes. These promising results using older generation platinum agents led to studies using the third generation satraplatin. Preclinical studies in human AIPC cell lines exhibited sensitivity to satraplatin. Satraplatin entered

Chemotherapy has recently been considered active in the treatment of prostate cancer. Although the use of taxanes (specifically docetaxel), has been shown to confer a survival benefit in AIPC, progressive disease after use of taxanes remain a vexing problem and present a hindrance to long-term survival in these patients. Several chemotherapeutic agents have been investigated in the 2nd line setting. With reported success of some of these agents, investigation with upfront use in first line treatment in AIPC will be in the foreseeable future. The following section will discuss the most commonly used or most promising chemotherapeutic regimens for AIPC in the second-line setting.

A. Mitoxantrone The overall survival benefit demonstrated with the use of docetaxel in metastatic prostate cancer has become accepted standard of care in this population of patients. However, there are still a proportion of patients who may not tolerate the adverse effects and the use of the anthracenedione mitoxantrone, and prednisone may be considered appropriate initial regimen for these patients (Berthold et al, 2005). Although cross-over studies conducted using mitoxantrone and docetaxel are few in number, there is a suggestion that number of PSA declines of at least 50% achieved with mitoxantrone administered after first-line docetaxel may be inferior to first-line mitoxantrone (Michels et al, 2005). The percentage of patients who experienced a 50% or greater decline in PSA levels after therapy has been shown to occur from 6% (for 2nd line mitoxantrone) to 12% for first-line mitoxantrone use (Michels et al, 2005; Oh et al, 2005). However, the median total duration of PFS for both chemotherapy courses together, from the start of the first to progression after the second type of chemotherapy, was no different whether mitoxantrone or a taxane was used first (39.9 weeks versus 38.7 weeks, respectively, P = 0.67). The median OS also did not differ significantly between the two groups: 15.2 months for the mitoxantrone-first group versus 17.1 months for the taxane-first group. Therefore, mitoxantrone may still offer some benefit when used as 2nd line treatment after primary taxane therapy. Recently, Lin et al reported a trial using ixabepilone (discussed more extensively at later sections of this review) or mitoxantrone after primary taxane failure (Lin et al, 2006). PSA decline rates of 50 % were as high as 20% with mitoxantrone as 2nd line after taxane therapy, and 30% as 3rd line (after taxane and ixabepilone failures), with equivalent overall survival of 13 months or 12.5 months,

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Cancer Therapy Vol 5, page 153 clinical trials in 1992. Phase I trials of single-agent satraplatin have explored different dosing schedules with the recommended dosing schedule in chemo-naĂŻve patients of 80 - 120 mg/m2 for 5 consecutive days every 4 - 5 weeks (McKeage et al, 1994; McKeage et al, 1995; McKeage et al, 1997; Beale et al, 1998; Sessa et al, 1998; Kurata et al, 2000). Based on promising preclinical and clinical efficacy in prostate cancer, several phase II and III trials have been conducted in AIPC (Table 1). Satraplatin for first-line treatment of AIPC has been conducted in a Phase II trial CA142-013 and two Phase III trials (CA142029 and EORTC 30972), while satraplatin for second-line treatment of AIPC has been studied in one Phase II trial, CA142-026 and a Phase III trial (SPARC). However, much of these studies, except the Phase II CA142-013 and recently concluded SPARC trial, were prematurely terminated as part of a commercial decision of the original pharmaceutical sponsor. Analyses of the data are discussed herein. CA142-013 was a phase II trial conducted at multiple US sites which accrued 39 patients with AIPC. The starting dose was 120 mg/m2/day for 5 days, but most patients had the dose reduced to 100 mg/m2/day for 5 days because of excessive toxicity (Latif et al, 2005). Patients received a total of 155 courses (median 2, range 1-16) of satraplatin. Dose delays (77% of courses) and dose reductions (31% of courses) were common and were mainly due to myelosupression. Response was assessed in 32 patients, 10 (26%) had partial response (PSA decline of at least 50 % without disease progression during or before response period), 14 (36%) had stable disease while PSA progression was seen in 8 (21%) patients. Of 20 (54%) patients with measurable disease two patients had a documented partial response. The median survival for the whole cohort is 16.7 months (95% CI, 9.3 - 19.2 months) and the median PSA response duration was 3.8 months

with a median progression-free survival of 7.7 months in 32 assessable patients. The first phase III trial using satraplatin and prednisone for first-line treatment of AIPC was led by the European Organization for Research and Treatment of Cancer (EORTC) trial 30972 (Sternberg et al, 2005). Although the target accrual was 380 patients, only 50 patients were enrolled when the study was terminated early by the sponsoring company. Patients with symptomatic AIPC were randomized to treatment with satraplatin (100 mg/m2/day for 5 days every 5 weeks) plus prednisone (10 mg bid daily) (N = 27) or prednisone alone (N=23). All patients have been followed until progression or death. Forty-two patients have died, most due to prostate cancer. A > 50% decrease in PSA was observed in 2/23 (8.7%) on the prednisone alone arm versus 9/27 (33.3%) in the satraplatin + prednisone arm (P=0.046). Toxicity was minimal in both arms; one patient on each arm died due to stomach perforation, most likely related to prednisone. Compliance to treatment was excellent. The median progression-free survival (PFS) was twice as long in the satraplatin + prednisone arm versus the prednisone alone arm (5.2 versus 2.6 months, p=0.023). Median overall survival (OS) also favored the satraplatin arm, 14.9 versus 11.9 months. This difference was not statistically significant, probably due to the small patient numbers. The second trial (CA142-029) was a randomized, double blind, placebocontrolled study initiated in December 1998. Fourteen patients with symptomatic AIPC were enrolled and randomized to treatment with either 100 mg/m2/day satraplatin for 5 days plus BID administration of 10 mg prednisone for 5 days (N = 7), or placebo plus BID administration of 10 mg prednisone alone for 5 days (N = 7) every 5 weeks. The primary end-point was pain response and at the time of study termination, only 14 patients were enrolled, and therefore, no formal analysis was conducted.

Table 1. Satraplatin studies in androgen-independent prostate cancer Trial

Phase

Number patients

CA142-013 (Latif et al, 2005)

CA142-029 EORTC 30972 (Sternberg et al, 2005)

III

CA142-026

SPARC (Petrylak 2007)

Regimen

Results PR: 26%; SD:36%; OS:16.7 mos (95% CI, 9.3 â&#x20AC;&#x201C; 19.2 mos)

S 120 mg/m2/day x 5 d q 4 wks S 100 mg/m2/day x 5 d q 5 wks + P 10 mg BID (n=7 pts) x 5 d q 5 wks vs P 10 mg BID (n=7) x 5 d q 5 wks S 100 mg/m2/day x 5 d q 5 wks + P 10 mg BID (n=27 pts) vs. P 10 mg BID (n=23) S 80-100 mg/m2/day x 5 d q 3 wks + P 10 mg BID

950

S 80 mg/m2/day x 5 d q 5 wks + P 5 mg BID vs. P 5mg BID

al, III

No formal analysis; terminated prematurely PSA response: 33.3% (S+P) vs. 8.7% (P); PFS: 5.2 vs. 2.6 mos, p=0.023; OS:, 14.9 versus 11.9 months (NS) No formal analysis; terminated prematurely 40% RR; PFS: 11.7 (S+P) vs. 9 wks (P); at 12 mos, 16% (S+P) and 7% (P) had not progressed

Legends: SPARC: SatraPlatin Against Refractory Cancers; PFS: Progression-free survival; OS: overall survival; RR: risk reduction; SD: stable disease; CI: confidence interval; S: Satraplatin; P: Prednisone;mos: months; wks: weeks; d:days; PR: Partial response; NS: Not significant

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Aragon-Ching and Dahut: Chemotherapy in Androgen-Independent Prostate Cancer (AIPC) CA142-026 was the first trial using satraplatin and prednisone for second-line treatment of AIPC, using a regimen of 80-100mg/m2/day for 5 days, every 3 weeks. Again, the study was terminated with only 10 subjects enrolled. SPARC (SatraPlatin Against Refractory Cancers) is a Phase III pivotal trial that opened in 2003. It is a multicenter, double-blind, placebo-controlled, randomized Phase III trial assessing satraplatin plus prednisone versus placebo plus prednisone as 2nd line chemotherapy treatment of AIPC. Satraplatin 80 mg/m2 or placebo was administered daily on days 1-5 of a 35-day cycle, and prednisone 5 mg was given twice daily on days 1-35 (Petrylak et al, 2007). A total of 950 patients were accrued at more than 200 clinical sites in fifteen countries on four continents. Results from this study showed a 40% reduction in the risk of progression, p<0.00001, Hazard Ratio of 0.6 (95% Confidence Interval: 0.5-0.7). The improvement seen in progression-free survival by patients treated with satraplatin increased over time. Progressionfree survival at the median (50th percentile) demonstrated a 13% improvement in patients who received satraplatin plus prednisone (11 weeks) compared to patients who received prednisone plus placebo (9.7 weeks). At 6 months, 30% of patients in the satraplatin arm had not progressed, compared to 17% of patients in the control arm. At 12 months, 16% of patients who received satraplatin had not progressed, compared to 7% of patients in the control arm. Pain response was 24.2% for satraplatin and prednisone versus 13.8% for prednisone and placebo (p<0.005) and PSA response was 25.4% for the combination versus 12.4% for prednisone (p<0.01). In summary, satraplatin with prednisone is emerging as an active treatment regimen for AIPC patients in the second-line setting, especially for those patients who have

progressed on taxane-based regimens. Final results of the SPARC trial are being awaited to determine whether satraplatin and prednisone would have an impact in overall survival. For the first line setting, current available studies on satraplatin have insufficient statistical power to conclude equivalence with standard therapy. Therefore, additional studies are needed to confirm the promising results seen thus far with the use of satraplatin and prednisone in the upfront treatment of metastatic AIPC.

C. Epothilones The class of antineoplastic agents called epothilones function in a manner similar to taxanes in microtubule stabilization. Microtubules are essential for normal mitosis and cell division. Polymerization of heterodimeric !/" tubulin subunits, with multiple isoforms of both ! and " tubulin present in proliferating human cells, is regulated by several microtubule-associated proteins (Jordan et al, 1993). Differences in the individual binding result in differences in tubulin function between epothilones and taxanes (Bode et al, 2002). Preclinical studies suggest that tumor cells resistant to taxanes will retain sensitivity to epothilones and hence provide a role for these class of compounds in the setting of clinical progression after taxane therapy (Bhandari and Hussain, 2005). Of the four known drugs in the epothilone class forms A-D, Azaepothilone B (BMS-247550; Ixabepilone) and epothilone B (EPO906; patupilone) have been most widely studied for AIPC. Ixabepilone has been used predominantly in chemotherapy-na誰ve metastatic patients (Table 2). Initial phase I study in solid tumors showed promising anti-tumor activities (Goodin et al, 2004), which led to several phase

Table 2. Selected Ixabepilone studies in prostate cancer Investigator

Smaletz et al (2003)

Hussain et al (2005)

Kelly et al (2004)

Phase of study

Number patients

Regimen

Results

Ixabepilone at 2 dose levels: 35 mg/m2 and 40 mg/m2 and oral EMP (280 mg TID x 5 days) every 3 weeks

11/12 (92%) with PSA decline of # 50%; Objective response in soft tissue (57%) and bone metastasis (40%)

Ixabepilone at 40 mg/m2 every 3 weeks

33% PSA response; PFS of 6 mos (95% CI, 4 - 8 mos), OS of 18 mos (95% CI, 13 - 24 mos)

Arm 1: 45 pts on ixabepilone 35 mg/m2 and EMP 280 mg TID x 5 days q 3 wks vs. Arm 2: 47 pts treated with ixabepilone 35 mg/m2

# 50% PSA decline: 31/45 (69%) pts in combination arm vs. 21/44 (48%) pts in ixabepilone alone; days to progression: 141 in combination arm vs. 145 in ixabepilone arm

Legends: EMP: Estramustine phosphate; pts: patients; mos: months; TID: Three times a day; PFS: progression-free survival; OS: Overall survival;

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Cancer Therapy Vol 5, page 155 II studies that was used in AIPC. Single agent phase II trial was conducted by South-West Oncology Group (SWOG, 0111) using ixabepilone 40 mg/m2 intravenously (IV) over 3 hours every 3 weeks (Hussain et al, 2004). The primary objective of this study was PSA response and patients were given upfront first-line chemotherapy with ixabepilone. Of the 41 patients enrolled, 16 patients (39%) had a # 50% PSA decline, and 14 of the responding patients (34%) had a confirmed PSA decrease. The median progression-free survival (PFS) was 6 months. These results were published in 2005 with 42 eligible patients, 14/42 (33%; 95% CI, 20% to 50%) PSA responses, with 72% of patients achieving # 80% declines in PSA. The PFS was 6 months (95% CI, 4 to 8 months), and the median survival is 18 months (95% CI, 13 to 24 months) (Hussain et al, 2005). Adverse effects from ixabepilone were mainly hematological and neurological with 17% occurrence of grade 3 or 4 neutropenia, while 12% of grade 3 sensory neuropathy occurred. Ixabepilone in combination with estramustine (EMP), a nornitrogen mustard linked to estradiol via a carbamate bond, was also studied in another phase II trial (Smaletz et al, 2003). Thirteen patients were treated at 2 dose levels of 35 mg/m2 and 40 mg/m2 in combination with EMP 280 mg three times daily for 5 days. The phase II dose of ixabepilone combined with EMP was determined to be 35 mg/m2 every 3 weeks, and a decline in PSA of # 50% was found in 11 of 12 patients (92%). A subsequent study enrolling a total of 92 patients with 45 patients treated with ixabepilone and EMP versus 47 patients treated with ixabepilone alone was done (Kelly et al, 2004). Objective response was seen in 8 of 25 patients (32%) in the ixabepilone alone arm and 11 of 23 patients (48%) in the combination arm, but days to PSA progression was similar in both arms. Despite low-dose warfarin prophylaxis, the combination arm had a 9% incidence of grade 3 or 4 thrombotic event. Since preclinical studies have shown no crossresistance between epothilones and taxanes, the secondline use of epothilones after taxanes may hold some promise. Although most of the studies that have been reported for ixabepilone have been for front-line treatment of AIPC, the activity of second-line ixabepilone after initial taxane treatment has been described in a 2-arm, non-comparative randomized phase II study (Lin et al, 2006). Forty-one evaluable patients were assigned to receive either: Mitoxantrone 14 mg/m2 IV every 3 weeks with 5 mg twice daily of prednisone 5 mg twice daily (MP) or Ixabepilone 35 mg/m2 IV every 3 weeks. The study's primary endpoint was to detect a > 50% PSA decline by Consensus Criteria in at least 25% of 2nd-line patients for each arm. The median follow-up was 5.0 months at the time of data presentation with a median number of 3 cycles administered to each 2nd-line arm. Median survival from protocol entry was equivalent, with 13.0 months for the ixabepilone arm and 12.5 months with MP. Confirmed 2nd-line post-therapy response of >50% PSA declines were observed in 17% of ixabepilone patients (95% CI = 7-32) and 20% of MP patients (95% CI = 9-35). Partial responses in patients with measurable disease were observed in only 1 out of 18 patients on 2nd-

line ixabepilone (6%; 95% CI = 0.1-27.3) and in 1 out of 15 patients on 2nd-line MP (7%; 95% CI = 0.2-31.9). The median duration on 2nd-line treatment was similar in both ixabepilone and MP arms, at 2.2 months and 2.3 months, respectively. Crossover to 3rd-line treatment seemed to occur more with MP, in 68% of MP patients versus 39% of ixabepilone patients. Again, the confirmed 3rd-line posttreatment >50% PSA declines were similar in both arms, occurring in 3 out of 24 ixabepilone treated patients and in 4 out of 13 MP patients. The most common grade 3/4 toxicity associated with 2nd-line treatment was neutropenia with occurrence of 41% for ixabepilone patients and 54% of MP patients. Conversely, there is some activity with second-line taxane therapy in patients who were previously treated with ixabepilone with a median time to PSA progression of 4.6 months (Rosenberg et al, 2005). Patupilone (EPO906; Epothilone B), a more potent microtubule stabilizer than paclitaxel formulated in polyethylene glycol-300 (Wartmann et al, 2000), has been studied in previous taxane-failure patients. In a phase II study of 3 out of 4 weekly 2.5 mg/m2 patupilone in AIPC patients, 7 of 28 patients (25%) had a response of 50% PSA decline (Hussain et al, 2004). Three of these 7 patients had received previous taxane-based chemotherapy, although the median duration of the PSA response was short, at 2.2 months.

D. Other agents Vinorelbine is a semi-synthetic vinca alkaloid that has shown some activity in AIPC. The vinca alkaloids, similar to taxanes and epothilones, work by perturbing the dynamic equilibrium of microtubule polymerization and depolymerization (Horwitz, 1992). Single-agent studies showed a 50% decrease in PSA levels sustained for 3-4 weeks in 13% to 17% of evaluable patients (Fields-Jones et al, 1999; Morant et al, 2002). Vinorelbine and hydrocortisone has been used for palliative benefit in a phase III study compared to hydrocortisone alone (Abratt et al, 2004), although this study excluded patients who have had prior chemotherapy. This study included 414 patients, a regimen using vinorelbine given 30 mg/m2 on days 1 and 8 every 3 weeks and hydrocortisone 40 mg/day versus hydrocortisone alone was used. The PSA response rate ( 50% decline of PSA sustained for at least 6 weeks) was 30.1% (95% CI 24% to 36%) in the combination arm and was 19.2% (95% CI 14% to 25%) in the hydrocortisone alone arm. The 6-month progression-free survival (PFS) rates were 33.2% versus 22.8%, and the median durations of PFS were 3.7 versus 2.8 months. The combination regimen was relatively well tolerated with the majority of patients receiving a median relative dose intensity of 90%. Another vinca alkaloid, vincristine, has shown antitumor activity in conjunction with cyclophosphamide and dexamethesone (CVD regimen) in AIPC (Daliani et al, 2003). The patients received oral cyclophosphamide, 250 mg daily on Days 1-14; intravenous vincristine, 1 mg daily on Days 1, 8, and 15; and oral dexamethasone, 0.75 mg twice daily on Days 1-14. Cycles were repeated every 28 days. Fifteen of 52 patients (29%; 95% CI 18-42%) had a 50% decrease in serum PSA level, the median overall 155

Aragon-Ching and Dahut: Chemotherapy in Androgen-Independent Prostate Cancer (AIPC) PFS duration was 10.11 weeks (95% CI 8.91-14.87), and median OS duration was 10.6 months (95% CI 7.24-14.1). Toxicity, which was mainly hematologic, was also acceptable in this study. Capecitabine has also been investigated in AIPC. In a small study of 25 patients, response was observed in 25% of patients, but because of toxicity (3 deaths in the study), further investigation in phase III trials was not pursued (Morant et al, 2004). This trial utilized capecitabine at 1250 mg/m2 BID on days 1-14, every 3 weeks. However, a recent phase II study using a combination of 3 out of 4 weekly docetaxel (35 mg/m2/week) and capecitabine (625 mg/m2 twice daily on days 5 - 18) in 46 patients was shown to be well tolerated and showed a 68.2% response defined as PSA reduction of # 50% (Ferrero et al, 2006). The median overall survival was 17.7 months (95% CI, 15.8 months to not reached).

benefit from chemotherapy agents. For instance, patients with DNA repair gene polymorphisms have been shown to exhibit platinum sensitivity in a variety of cancers (Kang et al, 2006; Olaussen et al, 2006). Presence of variant gene polymorphisms may help predict response to platinum agents in prostate cancer and forms the basis of a planned clinical trial at NCI using satraplatin in patients with different nucleotide excision repair (NER) gene polymorphisms.

IV. Conclusions Chemotherapy currently has a well defined role in the treatment of prostate cancer. Although improvements in OS have been demonstrated using taxanes, responses are short with current standard therapy, and improvements in clinical endpoints by using combination chemotherapy, along with the use of synergistic cytostatic agents such as angiogenic inhibitors (i.e., bevacizumab, thalidomide), can be obtained. Second-line chemotherapeutic agents for prostate cancer patients who have progressed after taxanes remain very limited. Perhaps a better understanding of the mechanisms of drug resistance, discovery of new agents, and targeting of new pathways in the emergence of AIPC, would ultimately lend better survival with the use of standard, and evolving combination therapies.

III. Future Directions There is an urgent need for newer agents or varying combinations of chemotherapeutic drugs that will improve upon the responses seen with docetaxel and prednisone. However, most of these studies are conducted as first-line regimens for the treatment of AIPC. Previous studies using varying combinations of docetaxel with agents using different mechanisms of action show promise in synergistic combinations (Dahut et al, 2004; Beer et al, 2007). Synergism with other agents including antiangiogenic drugs like thalidomide and bevacizumab is quickly emerging as one of the most promising therapies. In a phase II trial of bevacizumab, thalidomide, docetaxel, and prednisone (Ning et al, 2007), treatment consisted of docetaxel 75 mg/m2 plus bevacizumab 15 mg/kg on day 1, every 21 days as a cycle, plus thalidomide 200 mg and prednisone 10 mg daily. Enoxaparin was used for thrombosis prevention and pegfilgrastim initiated after detection of grade >3 neutropenia. Twenty-three of 33 patients (70%) had >80% PSA declines and objective response rates (ORR) of 64%, and was well tolerated with significant toxicities involving the following: febrile neutropenia (4/33 patients), syncope (3/33 patients), colon perforation or fistula (2/33 patients), grade 3 bleeding (2/33 patients), thrombosis (2/33 patients). This trial is the first study to combine antiangiogenic agents of different mechanisms with docetaxel in metastatic AIPC. Most of the accrued patients have unfavorable characteristics as evidenced by a high Gleason score (median Gleason score of 8) and a rapid PSA doubling time (median of 1.6 months). This trial is currently ongoing at the NCI and may pave the way for a future CALGB trial to determine the benefits of combined anti-angiogenic therapy with standard chemotherapeutic agents. Other agents in combination with docetaxel have shown promising activity, including calcitriol (Beer et al, 2007), estramustine and bevacizumab (Picus et al, 2003), lenalidomide (Moss et al, 2007), and several of these combinations are currently being investigated in cooperative trials. Apart from strategies combining multi-agent chemotherapy, understanding of pharmacogenomics will also help determine which patients would ultimately

Acknowledgements This project has been supported by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research, National Institutes of Health. The content of this publication does not reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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