tjh-2013-3

Volume 30

Issue 3

September 2013

40 TL

ISSN 1300-7777

Review Article Quality of Life and Supportive Care in Multiple Myeloma Melda Cömert et al.; İzmir, Turkey TURKISH JOURNAL OF HEMATOLOGY • VOL.: 30

Research Articles Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on GraftVersus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation Oral Nevruz, et al.; Ankara, Turkey

R e g u l ation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Expression in Primary Acute Leukemic Cells by Chemotherapeutics Shengmei Chen, et al.; Henan Province, P.R. China

Knowledge, Attitudes, and Practices of Hematologists Regarding Fertility Preservation in Turkey Mert Küçük, et al.; Aydın, Turkey

Prognostic Significance of Bcl-2 and p53 Protein Expressions and Ki67 Proliferative Index in Diffuse Large B-cell Lymphoma Betül Bolat et al.; İzmir, Kahramanmaraş, Turkey

Detection of Left Ventricular Regional Function in Asymptomatic Children with beta-Thalassemia Major by Longitudinal Strain and Strain Rate Imaging

ISSUE: 3

Ali Bay et al.; Gaziantep, Turkey

Endocrinological and Cardiological Late Effects Among Survivors of Childhood Acute Lymphoblastic Leukemia Pakize Karakaya, et al.; İzmir, Turkey

September 2013

Significance of Neuropilin-1 Expression in Acute Myeloid Leukemia Tarif H. Sallam, et al.; Cairo, Egypt; Sanaa, Yemen

Cover Picture: Dr. Ali Uğur Ural Macahel, Artvin

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Editor-in-Chief

International Review Board

Aytemiz Gürgey

Nejat Akar Görgün Akpek Serhan Alkan Çiğdem Altay Koen van Besien Ayhan Çavdar M.Sıraç Dilber Ahmet Doğan Peter Dreger Thierry Facon Jawed Fareed Gösta Gahrton Dieter Hoelzer Marilyn Manco-Johnson Andreas Josting Emin Kansu Winfried Kern Nigel Key Korgün Koral Abdullah Kutlar Luca Malcovati Robert Marcus Jean Pierre Marie Ghulam Mufti Gerassimos A. Pangalis Antonio Piga Ananda Prasad Jacob M. Rowe Jens-Ulrich Rüffer Norbert Schmitz Orhan Sezer Anna Sureda Ayalew Tefferi Nükhet Tüzüner Catherine Verfaillie Srdan Verstovsek Claudio Viscoli

TOBB Economy Technical University Hospital, Ankara, Turkey Maryland School of Medicine, Baltimore, USA Cedars-Sinai Medical Center, USA Ankara, Turkey Chicago Medical Center University, Chicago, USA Ankara, Turkey Karolinska University, Stockholm, Sweden Mayo Clinic Saint Marys Hospital, USA Heidelberg University, Heidelberg, Germany Lille University, Lille, France Loyola University, Maywood, USA Karolinska University Hospital, Stockholm, Sweden Frankfurt University, Frankfurt, Germany Colorado Health Sciences University, USA University Hospital Cologne, Cologne, Germany Hacettepe University, Ankara, Turkey Albert Ludwigs University, Germany University of North Carolina School of Medicine, NC, USA Southwestern Medical Center, Texas, USA Georgia Health Sciences University, Augusta, USA Pavia Medical School University, Pavia, Italy Kings College Hospital, London, UK Pierre et Marie Curie University, Paris, France King’s Hospital, London, UK Athens University, Athens, Greece Torino University, Torino, Italy Wayne State University School of Medicine, Detroit, USA Rambam Medical Center, Haifa, Israel University of Köln, Germany AK St Georg, Hamburg, Germany Charité University, Berlin, Germany Santa Creu i Sant Pau Hospital, Barcelona, Spain Mayo Clinic, Rochester, Minnesota, USA Istanbul Cerrahpaşa University, İstanbul, Turkey Minnesota University, Minnesota, USA The University of Texas MD Anderson Cancer Center, Houston, USA San Martino University, Genoa, Italy

Past Editors Erich Frank Orhan Ulutin Hamdi Akan

Language Editor Leslie Demir

Ankara, Turkey

Associate Editors Ayşegül Ünüvar İstanbul University, İstanbul, Turkey

Celalettin Üstün Minnesota University, Minnesota, USA

Cem Ar İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul, Turkey

İbrahim Haznedaroğlu Hacettepe University, Ankara, Turkey

İlknur Kozanoğlu Başkent University, Adana, Turkey

Hale Ören Dokuz Eylül University, İzmir, Turkey

Mehmet Ertem Ankara University, Ankara, Turkey

Muzaffer Demir Trakya University, Edirne, Turkey

Reyhan Diz Küçükkaya İstanbul Bilim University, İstanbul, Turkey

Assistant Editors A. Emre Eşkazan İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul, Turkey

Ali İrfan Emre Tekgündüz Dr. A. Yurtaslan Ankara Oncology Training and Research Hospital, Ankara, Turkey

Nil Güler On Dokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay Osmangazi University, Eskişehir, Turkey

Selami Koçak Toprak Başkent University, Ankara, Turkey

Şule Ünal Hacettepe University, Ankara, Turkey

Ümit Üre Bakırköy Dr. Sadi Konuk Training and Research Hospital, İstanbul, Turkey

Statistic Editor Hülya Ellidokuz

Senior Advisory Board Yücel Tangün Osman İlhan Muhit Özcan

Veysel Sabri Hançer İstanbul Bilim University, İstanbul, Turkey

Zühre Kaya Gazi University, Ankara, Turkey

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Publishing Services

Editorial Office İpek Durusu Bengü Timoçin

GALENOS PUBLISHER Molla Gürani Mah. Kaçamak Sk. No: 21, Fındıkzade-İstanbul Phone: +90 212 621 99 25 • Fax: +90 212 621 99 27 • www. galenos.com.tr

Contact Information Editorial Correspondence should be addressed to Dr. Aytemiz Gürgey Editor-in-Chief Address: 725. Sok. Görkem Sitesi Yıldızevler No: 39/2, 06550 Çankaya, Ankara / Turkey Phone : +90 312 438 14 60 E-mail : agurgey@hacettepe.edu.tr

All other inquiries should be adressed to TURKISH JOURNAL OF HEMATOLOGY Address: İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550 Çankaya, Ankara / Turkey Phone : +90 312 490 98 97 Fax : +90 312 490 98 68 E-mail : info@tjh.com.tr ISSN: 1300-7777

Turkish Society of Hematology

Publishing Manager Sorumlu Yazı İşleri Müdürü

Teoman Soysal, President Muzaffer Demir, General Secretary Hale Ören, Vice President İbrahim Haznedaroğlu, Research Secretary Fahir Özkalemkaş, Treasurer Zahit Bolaman, Member Mehmet Sönmez, Member

Muzaffer Demir

Management Address Yayın İdare Adresi

Türk Hematoloji Derneği İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550 Çankaya, Ankara / Turkey

Online Manuscript Submission

Publishing House / Yayınevi

http://mc.manuscriptcentral.com/tjh

Web page

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade-İstanbul Tel: +90 212 621 99 25 Faks: +90 212 621 99 27 E-posta: info@galenos.com.tr Baskı: Uniform Basım San. ve Turizm Ltd. Şti.

www.tjh.com.tr

Printing Date / Basım Tarihi

Owner on behalf of the Turkish Society of Hematology Türk Hematoloji Derneği adına yayın sahibi Teoman Soysal

Üç ayda bir yayımlanan İngilizce süreli yayındır.

International scientific journal published quarterly.

01.09.2013

Cover Picture

Ali Uğur Ural was born in 1960, Turkey. He is currently working in Bayındır Hospital, Department of Hematology, Ankara, Turkey.

Macahel is the name of a region at the border between Turkey and Georgia. It is connected to the county of Borçka of the province Artvin on the Turkish side of the border. It is also known as “Camili”.

With its friendly people, virgin nature, honey, and bridges, Macahel is a piece of heaven under the protection of UNESCO.

Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile Turkish Journal of Hematology’nin Türk Hematoloji Derneği İktisadi İşletmesi tarafından yayınlanmasına karar vermiştir.

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AIMS AND SCOPE The Turkish Journal of Hematology is published quarterly (March, June, September, and December) by the Turkish Society of Hematology. It is an independent, non-profit peer-reviewed international English-language periodical encompassing subjects relevant to hematology.

The Editorial Board of The Turkish Journal of Hematology adheres to the principles of the World Association of Medical Editors (WAME), International Council of Medical Journal Editors (ICMJE), Committee on Publication Ethics (COPE), Consolidated Standards of Reporting Trials (CONSORT) and Strengthening the Reporting of Observational Studies in Epidemiology (STROBE).

The aim of The Turkish Journal Hematology is to publish original hematological research of the highest scientific quality and clinical relevance. Additionally, educational material, reviews on basic developments, editorial short notes, case reports, images in hematology, and letters from hematology specialists and clinicians covering their experience and comments on hematology and related medical fields as well as social subjects are published.

General practitioners interested in hematology and internal medicine specialists are among our target audience, and The Turkish Journal of Hematology aims to publish according to their needs. The Turkish Journal of Hematology is indexed, as follows: - PUBMED Central - Science Citation Index Expanded - EMBASE - Scopus - CINAHL - Gale/Cengage Learning - EBSCO - DOAJ - ProQuest - Index Copernicus - Tübitak/Ulakbim Turkish Medical Database - Turk Medline

Impact Factor: 0.494 Subscription Information

The Turkish Journal of Hematology is sent free-of-charge to members of Turkish Society of Hematology and libraries in Turkey and abroad. Hematologists, other medical specialists that are interested in hematology, and academicians could subscribe for only 40 $ per printed issue. All published volumes are available in full text free-ofcharge online at www.tjh.com.tr.

Address: İlkbahar Mah., Turan Güneş Bulvarı, 613 Sok., No: 8, Çankaya, Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh Web page: www.tjh.com.tr E-mail: info@tjh.com.tr

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Permissions

Requests for permission to reproduce published material should be sent to the editorial office.

Editor: Professor Dr. Aytemiz Gürgey Adress: Ilkbahar Mah, Turan Günes Bulvarı, 613 Sok., No: 8, Çankaya, Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh Web page: www.tjh.com.tr E-mail: info@tjh.com.tr Publisher Galenos Yayinevi Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul Telefon : 0212 621 99 25 Fax : 0212 621 99 27 info@galenos.com.tr

Instructions for Authors Instructions for authors are published in the journal and at www. tjh.com.tr

Material Disclaimer Authors are responsible for the manuscripts they publish in The Turkish Journal of Hematology. The editor, editorial board, and publisher do not accept any responsibility for published manuscripts.

The journal is printed on acid-free paper.

Editorial Policy Following receipt of each manuscript, a checklist is completed by the Editorial Assistant. The Editorial Assistant checks that each manuscript contains all required components and adheres to the author guidelines, after which time it will be forwarded to the Editor in Chief. Following the Editor in Chief’s evaluation, each manuscript is forwarded to the Associate Editor, who in turn assigns reviewers.

Generally, all manuscripts will be reviewed by at least three reviewers selected by the Associate Editor, based on their relevant expertise.

Associate editor could be assigned as a reviewer along with the reviewers. After the reviewing process, all manuscripts are evaluated in the Editorial Board Meeting.

Turkish Journal of Hematology’s editor and Editorial Board members are active researchers. It is possible that they would desire to submit their manuscript to the Turkish Journal of Hematology.

This may be creating a conflict of interest. These manuscripts will not be evaluated by the submitting editor(s). The review process will be managed and decisions made by editor-in-chief who will act independently. In some situation, this process will be overseen by an outside independent expert in reviewing submissions from editors.

TURKISH JOURNAL OF HEMATOLOGY INSTRUCTIONS TO AUTHORS The Turkish Journal of Hematology accepts invited review articles, research articles, brief reports, case reports, letters to the editor, and hematological images that are relevant to the scope of hematology, on the condition that they have not been previously published elsewhere. Basic science manuscripts, such as randomized, cohort, cross-sectional, and case control studies, are given preference. All manuscripts are subject to editorial revision to ensure they conform to the style adopted by the journal. There is a single blind kind of reviewing system.

Manuscripts should be prepared according to ICMJE guidelines (http://www.icmje.org/). Original manuscripts require a structured abstract. Label each section of the structured abstract with the appropriate subheading (Objective, Materials and Methods, Results, and Conclusion). Case reports require short unstructured abstracts.

Letters to the editor do not require an abstract. Research or project support should be acknowledged as a footnote on the title page.

Technical and other assistance should be provided on the title page.

Conflict-of-Interest Statement: To prevent potential conflicts of interest from being overlooked, this statement must be included in each manuscript. In case there are conflicts of interest, every author should complete the ICMJE general declaration form, which can be obtained at: http://www.icmje.org/coi_disclose.pdf.

Abstract and Keywords: The second page should include an abstract that does not exceed 300 words. For manuscripts sent by authors in Turkey, a title and abstract in Turkish are also required. As most readers read the abstract first, it is critically important. Moreover, as various electronic databases integrate only abstracts into their index, important findings should be presented in the abstract.

Objective: The abstract should state the objective (the purpose of the study and hypothesis) and summarize the rationale for the study.

Materials and Methods: Important methods should be written respectively.

Results: Important findings and results should be provided here.

Conclusion: The study’s new and important findings should be highlighted and interpreted.

Original Manuscripts Title Page Title: The title should provide important information regarding the manuscript’s content. The title must specify that the study is a cohort study, cross-sectional study, case control study, or randomized study (i.e. Cao GY, Li KX, Jin PF, Yue XY, Yang C, Hu X. Comparative bioavailability of ferrous succinate tablet formulations without correction for baseline circadian changes in iron concentration in healthy Chinese male subjects: A single-dose, randomized, 2-period crossover study. Clin Ther. 2011; 33: 2054-2059).

The title page should include the authors’ names, degrees, and institutional/professional affiliations, a short title, abbreviations, keywords, financial disclosure statement, and conflict of interest statement. If a manuscript includes authors from more than one institution, each author’s name should be followed by a superscript number that corresponds to their institution, which is listed separately.

Please provide contact information for the corresponding author, including name, e-mail address, and telephone and fax numbers.

Running Head: The running head should not be more than 40 characters, including spaces, and should be located at the bottom of the title page.

Word Count: A word count for the manuscript, excluding abstract, acknowledgments, figure and table legends, and references, should be provided not exceed 2500 words. The word count for an abstract should be not exceed 300 words.

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Other types of manuscripts, such as case reports, reviews, perspectives, and editorials, will be published according to uniform requirements.

Provide 3-10 keywords below the abstract to assist indexers. Use terms from the Index Medicus Medical Subject Headings List (for randomized studies a CONSORT abstract should be provided (http://www.consort-statement.org).

Introduction: The introduction should include an overview of the relevant literature presented in summary form (one page), and what ever remains interesting, unique, problematic, relevant, or unknown about the topic must be specified. The introduction should conclude with the rationale for the study, its design, and its objective(s).

Materials and Methods: Clearly describe the selection of observational or experimental participants, such as patients, laboratory animals, and controls, including inclusion and exclusion criteria and a description of the source population. Identify the methods and procedures in sufficient detail to allow other researchers to reproduce your results.

Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for using them and an evaluation of their limitations.

Identify all drugs and chemicals used, including generic names, doses, and routes of administration. The section should include only information that was available at the time the plan or protocol for the study was devised (http://www.strobe-statement.org/fileadmin/ Strobe/uploads/checklists/STROBE_checklist_v4_combined.pdf).

Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify the reported results. Statistically important data should be given in the text, tables and figures. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used.

Results: Present your results in logical sequence in the text, tables, and figures. Do not present all the data provided in the tables and/or figures in the text; emphasize and/or summarize only important findings, results, and observations in the text. For clinical studies provide the number of samples, cases, and controls included in the study. Discrepancies between the planned number and obtained number of participants should be explained. Comparisons, and statistically important values (i.e. P value and confidence interval) should be provided.

Discussion: This section should include a discussion of the data.

New and important findings/results, and the conclusions they lead to should be emphasized. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by the data. Do not repeat the findings/results in detail; important findings/results should be compared with those of similar studies in the literature, along with a summarization. In other words, similarities or differences in the obtained findings/results with those previously reported should be discussed. Limitations of the study should be detailed. In addition, an evaluation of the implications of the obtained findings/results for future research should be outlined.

4. Book Chapter Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin: Genetics, Pathophysiology, Clinical Management. New York, Cambridge University Press, 2000.

5.Abstract Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract).

6.Letter to the Editor Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further definition of 20q deletion in myeloid leukemia using fluorescence in situ hybridization. Blood 1994;84:2821-2823.

7. Supplement Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr Transplant. 2005;9(Suppl 7):81-86

Brief Reports

Deeg HJ, O’Donnel M, Tolar J. Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure immunosuppressive therapy. Blood 2006;108:1485-1491.

Abstract length: Not to exceed 300 words.

Article length: Not to exceed 1200 words.

Introduction: State the purpose and summarize the rationale for the study.

Materials and Methods: Clearly describe the selection of the observational or experimental participants. Identify the methods and procedures in sufficient detail. Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for their use and an evaluation of their limitations. Identify all drugs and chemicals used, including generic names, doses, and routes of administration.

Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify the reported findings/results. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used.

Results: Present the findings/results in a logical sequence in the text, tables, and figures. Do not repeat all the findings/results in the tables and figures in the text; emphasize and/or summarize only those that are most important.

Discussion: Highlight the new and important findings/results of the study and the conclusions they lead to. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by your data.

2.Organization as author

Case Reports

Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in posthepatitis marrow aplasia. Lancet 1977;2:742-744.

Abstract length: Not to exceed 300 words.

Article length: Not to exceed 1200 words.

3.Book

Abstract

References Cite references in the text, tables, and figures with numbers in parentheses. Number references consecutively according to the order in which they first appear in the text. Journal titles should be abbreviated according to the style used in Index Medicus (consult List of Journals Indexed in Index Medicus). Include among the references any paper accepted, but not yet published, designating the journal and followed by, in press.

Examples of References: 1. List all authors.

Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger, 1961.

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Case reports should be structured as follows: An unstructured abstract that summarizes the case.

Introduction:A brief introduction (recommended length: 1-2 paragraphs).

Case Presentation: This section describes the case in detail, including the initial diagnosis and outcome.

Discussion:This section should include a brief review of the relevant literature and how the presented case furthers our understanding to the disease process.

Invited Review Articles Abstract length: Not to exceed 300 words.

Article length: Not to exceed 4000 words.

Review articles should not include more than 100 references. Reviews should include a conclusion, in which a new hypothesis or study about the subject may be posited. Do not publish methods for literature search or level of evidence. The study’s new and important findings should be highlighted and interpreted in the Conclusion section.

Images in Hematology Article length: Not exceed 400 words.

Authors can submit for consideration an illustration and photos(or when appropriate, two or more related images) that is interesting, instructive, and visually attractive, along with a few lines of explanatory text and references. Images in Hematology can include no more than 400 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title.

Letters to the Editor Article length: Not to exceed 500 words.

Letters can include no more than 500 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title.

Tables Supply each table on a separate file. Number tables according to the order in which they appear in the text, and supply a brief caption for each. Give each column a short or abbreviated heading. Write explanatory statistical measures of variation, such as standard deviation or standard error of mean. Be sure that each table is cited in the text.

Figures Figures should be professionally drawn and/or photographed.

Authors should number figures according to the order in which they appear in the text. Figures include graphs, charts, photographs, and illustrations. Each figure should be accompanied by a legend that does not exceed 50 words. Use abbreviations only if they have been introduced in the text. Authors are also required to provide the level of magnification for histological slides. Explain the internal scale and identify the staining method used. Figures should be submitted as separate files, not in the text file. High-resolution image files are not preferred for initial submission as the file sizes may be too large. The total file size of the PDF for peer review should not exceed 5 MB.

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Authorship Each author should have participated sufficiently in the work to assume public responsibility for the content. Any portion of a manuscript that is critical to its main conclusions must be the responsibility of at least 1 author.

Contributor’s Statement All submissions should contain a contributor’s statement page. Each manuscript should contain substantial contributions to idea and design, acquisition of data, or analysis and interpretation of findings.

All persons designated as an author should qualify for authorship, and all those that qualify should be listed. Each author should have participated sufficiently in the work to take responsibility for appropriate portions of the text.

Acknowledgments Acknowledge support received from individuals, organizations, grants, corporations, and any other source. For work involving a biomedical product or potential product partially or wholly supported by corporate funding, a note stating, “This study was financially supported (in part) with funds provided by (company name) to (authors’ initials)”, must be included. Grant support, if received, needs to be stated and the specific granting institutions’ names and grant numbers provided when applicable.

Authors are expected to disclose on the title page any commercial or other associations that might pose a conflict of interest in connection with the submitted manuscript. All funding sources that supported the work and the institutional and/or corporate affiliations of the authors should be acknowledged on the title page.

Ethics When reporting experiments conducted with humans indicate that the procedures were in accordance with ethical standards set forth by the committee that oversees human experimentation. Approval of research protocols by the relevant ethics committee, in accordance with international agreements (Helsinki Declaration of 1975, revised 2002 available at http://www.wma.net/e/policy/b3.htm, “Guide for the Care and use of Laboratory Animals” www.nap.edu/catalog/5140. html/), is required for all experimental, clinical, and drug studies.

Patient names, initials, and hospital identification numbers should not be used. Manuscripts reporting the results of experimental investigations conducted with humans must state that the study protocol received institutional review board approval and that the participants provided informed consent.

Non-compliance with scientific accuracy is not in accord with scientific ethics. Plagiarism: To re-publish-whole or in part-the contents of another author’s publication as one’s own without providing a reference. Fabrication: To publish data and findings/results that do not exist. Duplication: Use of data from another publication, which includes re-publishing a manuscript in different languages.

Salamisation: To create more than one publication by dividing the results of a study preternaturally.

We disapprove of such unethical practices as plagiarism, fabrication, duplication, and salamisation, as well as efforts to influence the review process with such practices as gifting authorship, inappropriate acknowledgements, and references. Additionally, authors must respect participant right to privacy.

On the other hand, short abstracts published in congress books that do not exceed 400 words and present data of preliminary research, and those that are presented in an electronic environment are not accepted pre-published work. Authors in such situation must declare this status on the first page of the manuscript and in the cover letter.

(The COPE flowchart is available at: http://publicationethics.org) Turkish Journal of Hematology uses plagiarism screening service to verify the originality of content submitted before publication.

Conditions of Publication All authors are required to affirm the following statements before their manuscript is considered: 1. The manuscript is being submitted only to The Turkish Journal of Hematology; 2. The manuscript will not be submitted elsewhere while under consideration by The Turkish Journal of Hematology; 3. The manuscript has not been published elsewhere, and should it be published in The Turkish Journal of Hematology it will not be published elsewhere without the permission of the editors (these restrictions do not apply to abstracts or to press reports for presentations at scientific meetings); 4. All authors are responsible for the manuscript’s content; 5. All authors participated in the study concept and design, analysis and interpretation of the data, drafting or revising of the manuscript, and have approved the manuscript as submitted. In addition, all authors are required to disclose any professional affiliation, financial agreement, or other involvement with any company whose product figures prominently in the submitted manuscript.

Copyright At the time of submission all authors will receive instructions for submitting an online copyright form. No manuscript will be considered for review until all authors have completed their copyright form. Please note, it is our practice not to accept copyright forms via fax, e-mail, or postal service unless there is a problem with the online author accounts that cannot be resolved. Every effort should be made to use the online copyright system. Corresponding authors can log in to the submission system at any time to check the status of any co-author’s copyright form. All accepted manuscripts become the permanent property of The Turkish Journal of Hematology and may not be published elsewhere-in whole or in part-without written permission.

Note: We cannot accept any copyright that has been altered, revised, amended, or otherwise changed. Our original copyright form must be used as is.

Units of Measurement Measurements should be reported using the metric system, according to the International System of Units (SI). Consult the SI Unit

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Conversion Guide, New England Journal of Medicine Books, 1992.

An extensive list of conversion factors can be found at http://www. unc.edu/~rowlett/units/scales/clinical_data.html. For more details, see http://www.amamanualofstyle.com/oso/public/jama/si_conversion_ table.html . Example for CBC.

Hematology component

SI units

RBC

6.7-11 x 1012/L

WBC

5.5-19.5 x109/L

Hemoglobin

116-168 g/L

PCV

0.31-0.46 L/L

MCV

39-53 fL

MCHC

300-360 g/L

MCH

19.5-25 pg

Platelets

300-700 x 109/L

Source: http://www.vetstream.com/felis/Corporate/993fhtm/ha-mat.htm

Abbreviations and Symbols Use only standard abbreviations. Avoid abbreviations in the title and abstract. The full term for an abbreviation should precede its first use in the text, unless it is a standard abbreviation. All acronyms used in the text should be expanded at first mention, followed by the abbreviation in parentheses; thereafter the acronym only should appear in the text.

Acronyms may be used in the abstract if they occur 3 or more times therein, but must be reintroduced in the body of the text. Generally, abbreviations should be limited to those defined in the AMA Manual of Style, current edition. A list of each abbreviation (and the corresponding full term) used in the manuscript must be provided on the title page.

Online Manuscript Submission Process The Turkish Journal of Hematology uses submission software powered

by ScholarOne Manuscripts. The website for submissions to The Turkish Journal of Hematology is http://mc.manuscriptcentral.com/tjh. This system is quick and convenient, both for authors and reviewers.

Setting up an account New users to the submission site will need to register and enter their account details before they can submit a manuscript. Log in, or click the “Create Account” button if you are a first-time user. To create a new account: After clicking the “Create Account” button, enter your name and e-mail address, and then click the “Next” button. Your e-mail address is very important. Enter your institution and address information, as appropriate, and then click the “Next” Button. Enter a user ID and password of your choice, select your area of expertise, and then click the “Finish” button.

If you have an account, but have forgotten your log-in details, go to “Password Help” on the journal’s online submission system and enter your e-mail address. The system will send you an automatic user ID and a new temporary password.

Full instructions and support are available on the site, and a user ID and password can be obtained during your first visit. Full support for authors is provided. Each page has a “Get Help Now” icon that connects directly to the online support system. Contact the journal administrator with any questions about submitting your manuscript to the journal (info@tjh.com.tr). For ScholarOne Manuscripts customer support, click on the “Get Help Now” link on the top right hand corner of every page on the site.

The Electronic Submission Process Log in to your author center. Once you have logged in, click the “Submit a Manuscript” link in the menu bar. Enter the appropriate data and answer the questions. You may copy and paste directly from your manuscript. Click the “Next” button on each screen to save your work and advance to the next screen.

Upload Files Click on the “Browse” button and locate the file on your computer.

Select the appropriate designation for each file in the drop-down menu next to the “Browse” button. When you have selected all the files you want to upload, click the “Upload Files” button. Review your submission before sending to the journal. Click the “Submit” button when you are finished reviewing. You can use ScholarOne Manuscripts at any time to check the status of your submission. The journal’s editorial office will inform you by e-mail once a decision has been made. After your manuscript has been submitted, a checklist will then be completed by the Editorial Assistant. The Editorial Assistant will check that the manuscript contains all required components and adheres to the author guidelines. Once the Editorial Assistant is satisfied with the manuscript it will be forwarded to the Senior Editor, who will assign an editor and reviewers.

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A-VIII

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CONTENTS A-XI

234

Obituary Review Article Quality of Life and Supportive Care in Multiple Myeloma Melda Cömert, Ajda Ersoy Güneş, Fahri Şahin, Güray Saydam

Commentary Current Management of Chronic Myeloid Leukemia with Tyrosine Kinase Inhibitors 247

İbrahim C. Haznedaroğlu

Research Articles

256

Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on Graft-Versus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation Oral Nevruz, Ferit Avcu, A. Uğur Ural1, Aysel Pekel, Bahar Dirican, Mükerrem Safalı, Elvin Akdağ, Murat Beyzadeoğlu, Tayfun İde, Ali Sengül

263

Regulation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Expression in Primary Acute Leukemic Cells by Chemotherapeutics Shengmei Chen, Yanfang Liu, Hui Sun, Ling Sun, Jie Ma, Dingming Wan, Zhongxing Jiang, Qiutang Zhang, Tao Li

269

Knowledge, Attitudes, and Practices of Hematologists Regarding Fertility Preservation in Turkey Mert Küçük, İrfan Yavaşoğlu, Ali Zahit Bolaman, Gürhan Kadıköylü

275

Prognostic Significance of Bcl-2 and p53 Protein Expressions and Ki67 Proliferative Index in Diffuse Large B-cell Lymphoma Betül Bolat Küçükzeybek, Sadi Bener, Aylin Orgen Çallı, Tuğba Doğruluk Paksoy, Bahriye Payzin

283

Detection of Left Ventricular Regional Function in Asymptomatic Children with beta-Thalassemia Major by Longitudinal Strain and Strain Rate Imaging Ali Bay, Osman Başpınar, Göksel Leblebisatan, Ali Seçkin Yalçın, Ahmet İrdem

290

Endocrinological and Cardiological Late Effects Among Survivors of Childhood Acute Lymphoblastic Leukemia Pakize Karakaya, Şebnem Yılmaz, Özlem Tüfekçi, Mustafa Kır, Ece Böber, Gülersu İrken, Hale Ören

300

Significance of Neuropilin-1 Expression in Acute Myeloid Leukemia Tarif H. Sallam, Manal A. Shams Eldin El Telbany, Hanan M. Mahmoud, Mutea A. Iskander

Brief Report

307

Effects of Omeprazole on Iron Absorption: Preliminary Study Mila Tempel, Anupama Chawla, Catherine Messina, Mahmut Yaşar Çeliker

Case Reports

311

A Case of Hypereosinophilic Syndrome Presenting with Multiorgan Thromboses Associated with Intestinal Obstruction Tao Sui, Qing Li1, Li Geng, Xinnv Xu, Yuming Li

A-IX

315

Late Onset Epstein Barr Virus Seropositive Posttransplant Lymphoproliferative Disorder in Two Renal Transplant Receivers Saime Paydaş, Semra Paydaş, Mustafa Balal, Arbil Açıkalın, Melek Ergin, Emel Gürkan, Fikri Başlamışlı

321

Ecthyma Gangrenosum-like Lesions in a Febrile Neutropenic Patient with Simultaneous Pseudomonas Sepsis and Disseminated Fusariosis Seven Uludokumacı, İlker İnanç Balkan, Bilgül Mete, Reşat Özaras, Neşe Saltoğlu, Teoman Soysal

325

A Neonatal Thrombosis Patient Treated Successfully With Recombinant Tissue Plasminogen Activator Kemal Erdinç, Serdar Ümit Sarıcı, Orçun Dabak, Orhan Gürsel, Adem Güler, Ahmet Emin Kürekçi, Fuat Emre Canpolat

328

Letters to the Editor

331

β-Thalassemia Mutation At Codon 37 (Tgg>>Tga) Detected In a Turkish Family Derya Güleç, İzmir, Turkey

333

Follicular Lymphoma Patient Relapsing with Paraneoplastic Sensory Neuronopathy (Ganglioneuropathy) Kadir Öztürk, Hakan Akgün, Çakar, Mustafa, Yusuf Emrah Eyi, Yakup Aksoy

336

Gynecomastia in a Boy with Chronic Myeloid Leukemia during Imatinib Therapy Betül Tavil, Sibel Kınık, Ahmet Gözen, Lale Olcay

338

Is There an Optimal Timing of Autologous Stem-Cell Transplantation for Multiple Myeloma in the Era of Novel Agents?

Selami Koçak Toprak

Images in Hematology

340

Mees’ Lines in an Acute Myeloid Leukemia Patient Soumaya Anoun, Meryem Qachouh, Mouna Lamchahab, Asmae Quessar, Said Benchekrou

341

The Side Effects of Imatinib Elmas Uzer, Ali Ünal, M. Yavuz Köker, Suat Ali Doğan

The Impact of Eltrombopag Administration on the Clinical Course of Severe Refractory Fatal Acquired Aplastic Anemia Ayşe Işık, Eylem Eliacık, İbrahim Haznedaroğlu, Salih Aksu, Nilgün Sayınalp, Yahya Büyükaşık, Hakan Göker, Osman Özcebe

A-X

Obituary

Professor Antonio Cao: A Remembrance

When I received Prof. Antonio Cao’s acceptance letter to his Centro Microcitemico at Cagliari University, I presumed that it was ushering in a new age in my life about the “molecular genetics of thalassemia in Turkish patients”.

I met Prof. Cao on a very hot August day in Cagliari/ Sardinia and the year was 1984. I was the first Turkish researcher accepted to the Thalassemia Center. He welcomed me and introduced me to Mario Pirastu, who had recently returned from Y.W. Kan’s laboratory in the United States and had created a new method for the identification of mutations in the beta-globin gene with usage for prenatal diagnosis [1].

In Sardinia, I had the chance to experience the establishment of a new molecular genetics laboratory and publish the first paper on the thalassemia mutations in a Turkish population [2].

Later on, I visited the center several times. Always I appreciated the sincerity of the Sardinians and I accepted Cagliari as my ‘second city’.

Prof. Cao’s center was an “International Thalassemia School”. Afterwards, several Turkish researchers visited the center and contributed to the molecular genetics of thalassemia in our population [3]. He created an algorithm to solve the thalassemia problem in Sardinia. This contribution helped in managing the thalassemia problem in different populations (Figure).

He had over 300 publications, mainly on different types of thalassemia and their molecular genetics. His group also contributed to the understanding of the molecular genetics of Wilson’s disease, especially in the Mediterranean area and including the Turkish population [4].

After his retirement, and even after his death, his scientific contributions continued [5].

Prof. Antonio Cao passed away last year, on 12 June 2012. His contribution to the thalassemia field and to Turkish researchers will always be remembered.

Nejat Akar MD, Prof.

TOBB-ETU Hospital, Pediatrics Department Ankara, Turkey A-XI 1

Figure 1: Prof. A. Cao’s handwriting, describing the algorithm for managing the thalassemia problem in Sardinia (Ankara, 1987) References 1. Pirastu M, Kan YW, Cao A, Conner BJ, Teplitz RL, Wallace RB. Prenatal diagnosis of beta-thalassemia. Detection of a single nucleotide mutation in DNA. N Engl J Med 1983;309:284287.

2. Akar N, Cavdar AO, Dessi E, Loi A, Pirastu M, Cao A. Beta thalassemia mutations in the Turkish population. J Med Genet 1987;24:378-379.

3. Sozuoz A, Berkalp A, Figus A, Loi A, Pirastu M, Cao A. Beta thalassemia mutations in Turkish Cypriots. J Med Genet 1988;25:766-768.

4. Loudianos G, Dessì V, Angius A, Lovicu M, Loi A, Deiana M, Akar N, Vajro P, Figus A, Cao A, Pirastu M. Wilson disease mutations associated with uncommon haplotypes in Mediterranean patients. Hum Genet 1996;98:640-642.

5. Cao A, Kan YW. The prevention of thalassemia. Cold Spring Harb Perspect Med 2013;3:a011775.

DOI: 10.4274/Tjh.2012.0192

Review

Quality of Life and Supportive Care in Multiple Myeloma Multiple Myelomada Yaşam Kalitesi ve Destek Tedavisi Melda Cömert, Ajda Ersoy Güneş, Fahri Şahin, Güray Saydam Ege University School of Medicine, Department of Hematology, İzmir, Turkey

Abstract: Multiple myeloma is the second most common haematological malignancy. Novel therapies have led to improvement in survival.

Current myeloma management is matching the progress made in improved survival through disease control while optimising quality of life with effective supportive care. Supportive treatment is an essential part of the therapeutic management of myeloma patients because it is directed towards improving the patient’s quality of life and also can improve survival. The aim of this review is to highlight the relationship among life of quality, supportive care, and improvement in survival.

Key Words: Multiple myeloma, Quality of life, Supportive care Özet: Multipl miyelom en sık görülen ikinci hematolojik malignitedir. Yeni tedaviler sağkalımda iyileşme sağlamıştır. Günümüzde uygulanan miyelom tedavi yönetimi, hastalığı kontrol altına alarak sağkalımda iyileşme sağlarken etkili destekleyici tedavi ile de yaşam kalitesini artırmaktadır. Destekleyici tedavi, hastaların yaşam kalitesini artırması ve sağkalımı iyileştirmesi nedeni ile miyelom hastalarının tedavi yönetiminin önemli bir parçasıdır. Bu derlemenin amacı yaşam kalitesi, destekleyici tedavi ve sağkalımdaki iyileşme arasındaki ilişkiyi vurgulamaktır.

Anahtar Sözcükler: Multipl miyelom, Yaşam kalitesi, Destekleyici tedavi Introduction

Multiple myeloma (MM) is the second most common haematological malignancy, comprising an estimated 1% of all cancers with an incidence of about 4.5 cases per 100,000 per year [1]. Significant progress in the treatment of MM has been achieved in the past 5 years [2,3,4]. Novel therapies led to improvement in survival, which resulted in an increasing symptom burden due not only to the disease itself, but also to the cumulative effects of treatments [5]. The significant challenge of

current myeloma management is matching the progress made in improved survival through disease control while optimising quality of life with effective supportive care from initial diagnosis to end-of-life care [5].

It is difficult to define clearly the meaning of the term “quality of life” and it carries different senses for everyone. It involves broad concepts that affect overall life contentment, including good health, adequate housing, employment, personal and family safety, interrelationships, education, and leisure pursuits.

Therefore, the life satisfaction most affected by health

Address for Correspondence: Melda CÖMERT, M.D., Hospital of Ege University Medicine Faculty, Hematology Hospital of Ege University Medicine Faculty, İzmir, 35100, Turkey Phone: +90 232 390 35 62 E-mail: meldacomert@hotmail.com Received/Geliş tarihi : December 4, 2012 Accepted/Kabul tarihi : April 1, 2013

234

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or illness is defined by the term “health-related quality of life” (HRQoL) [6,7]. Related to HRQoL, we can define supportive care as the treatment given to prevent, control, or relieve complications and side effects and to improve the patients’ and their families’ comfort and quality of life.

Quality of Life Questionnaires Karnofsky and Burchenal developed a clinical scale to quantify the functional performance of cancer patients in 1949 [8,9] (Table 1). Systematic assessment of HRQoL in cancer patients has received increasing interest over the past 2 decades. Cella et al. defined several advantages to including comprehensive HRQoL surveys in symptom trials in oncology. The most obvious is to test the hypothesis that HRQoL will be improved in addition to the symptom benefits [10]. Nevertheless, assessment of HRQoL has become an important focus of benefit for the treatment of patients with neoplastic diseases [11]. Wisloff reported that measurement of HRQoL before and during treatment contributes important prognostic information [12]. Therefore, it is very important to choose proper assessments and questionnaires. Effective and reliable questionnaires that include generic health status instruments, generic illness instruments, and disease-specific instruments are available for assessment of HRQoL [13]. Among the most widely used cancer questionnaires are the Functional Living Index-Cancer (FLIC), the European Organisation for Research and Treatment of Cancer

Table 1. Karnofsky Score [9] Score

Status

100%

Perfectly well

90%

Minor symptoms (can live a normal life)

80%

Normal activity with some effort

70%

Unable to carry on normal activity, but can care for oneself

60%

Requires occasional help with personal needs

50%

Disabled

40%

Nursing assistance and medical care, but is not hospitalised

30%

Severely disabled, in hospital

20%

Very sick (active support needed)

10%

Moribund

0%

Dead

Turk J Hematol 2013;30:234-246

(EORTC) QLQ-30, a specifically designed instrument for use among patients with MM known as the EORTC QLQ-MY20, and the Functional Assessment of Cancer Therapy (FACT) scale. Moreover, a disease-specific form of the FACT scale was developed through a structured iterative process for MM patients, called FACT-MM. This questionnaire incorporates open-ended questions with classic survey methods, detecting both known and new HRQoL issues for MM patients and the health care providers who treat them [4]. Etto et al. support the use of the EORTC QLQ-C30 as part of routine clinical care in MM patients in developing countries. Their results also suggest that the QLQ-C30 questionnaire for cancer patients seems to be more informative and easier to complete for the patients than the generic questionnaire [5]. The QLQ-C30 incorporates 9 multi-item scales: 5 functional scales (physical, role, cognitive, emotional, and social), 3 symptom scales (fatigue, pain, and nausea and vomiting), a global health and quality-of-life scale, and some single symptom measures. It is available in 16 languages. The EORTC QLQ-MY20 is meant for use among MM patients varying in disease stage and treatment modality (i.e. surgery, chemotherapy, radiotherapy, and hormonal treatment). It should always be complemented by the QLQ-C30. Therefore, it remains difficult to determine the best questionnaire for measurement purposes and patient groups [14].

In 2005, the Eastern Cooperative Oncology Group (ECOG) launched a clinical trial (E1A05) to evaluate a new treatment regimen for MM. HRQoL was determined to be a secondary goal for this trial, with the purpose of quantifying treatment-induced reduction of disease-specific symptoms as well as treatment-related symptoms and toxicities. Based on a comprehensive literature search, the authors concluded that no HRQoL instrument existed to adequately capture key MM symptoms and concerns from the patient’s perspective.

This led to collaboration among the ECOG Myeloma, Patient Outcomes and Survivorship, and Patient Representative committees to develop a patient-reported outcome (PRO) measure to assess MM symptoms and concerns [4]. The task force identified several types of measures that fall under the PRO umbrella, including HRQoL, functional status, symptom status, overall wellbeing, satisfaction with care, and treatment adherence.

What Affects the Quality of Life?

Previous studies have demonstrated that the most common physical symptoms indicated by MM patients 235

Turk J Hematol 2013;30:234-246

Cömert M: Quality of Life and Supportive Care in Multiple Myeloma

Table 2. Recommended regimens for bisphosphonates [32]

Pamidronate

Zoledronate

Clodronate

Dose

90 mg

4 mg

1600 mg

Application mode

3-h infusion

15-min infusion

2-h infusion or orally daily

Interval

Monthly

Monthly

Monthly

Table 3. Risk factors for infection and recommended form and dose of prophylaxis in multiple myeloma patients

Risk factors

Recommended form and dose of prophylaxis

Bacterial infections Active disease Poor performance Previous history of infections Hypogammaglobulinaemia Herpetic infections Patients treated with VAD Patients treated with high-dose dexamethasone Patients treated with bortezomib-based regimen

]]

-Trimethoprim/sulfamethoxazole, 160/800 mg, twice daily, orally - IVIG, 500 mg/kg, monthly, for up to 6 months

-Acyclovir, 400 mg, once daily, orally

Table 4. National Cancer Institute’s Common Terminology Criteria for Adverse Events and neuropathy grading

Neuropathy

Grade 0 Grade 1

Grade 2

Grade 3

Grade 4

Cranial

None

Asymptomatic, detected on examination or testing only

Symptomatic, not interfering with ADL*

Symptomatic, interfering with ADL

Life-threatening, disabling

Motor

None

Asymptomatic, weakness detected on exam or testing only

Symptomatic weakness, interfering with function but not interfering with ADL

Weakness Life-threatening, interfering with disabling ADL, bracing or assistance to walk indicated

Sensorial

None

Asymptomatic, loss of deep tendon reflexes, paresthesia (including tingling) but not interfering with function

Sensory alteration or paresthesia (including tingling) interfering with function but not interfering with ADL

Sensory alteration Disabling or paresthesia interfering with ADL

The National Cancer Institute’s Common Terminology Criteria for Adverse Events have a grade 5 toxicity category for all neuropathy items: death. *: Activities of daily living.

at presentation are especially skeletal pain and fatigue.

Despite significant improvements in the treatment of MM, it remains a chronic incurable disease that is associated with reduced HRQoL due to spontaneous fractures, spinal cord compression, osteolytic lesions, recurrent infections, renal failure, anaemia, mood disorders 236

accompanied by reduced physical functioning, and side effects of different types of treatment used to control this disease [15,16,17,18,19,20,21]. It is an important point that myeloma patients are typically in their sixth to seventh decade of life and have comorbidities.

Although the influence of comorbidities in MM patients

Turk J Hematol 2013;30:234-246

Cömert M: Quality of Life and Supportive Care in Multiple Myeloma

Table 5. World Health Organization (WHO) Pain Ladder [58]

Level 1 NSAIDs

Level 2 Weak opioids

Level 3 Strong opioids

Aspirin, ibuprofen, naproxen, COX-2 inhibitors

Codeine, dihydrocodeine, tramadol

Morphine, levomethadone, buprenorphine, fentanyl

NSAIDs: nonsteroidal anti-inflammatory drugs; COX-2: cyclooxygenase 2

Table 6. Risk factors of venous thromboembolism for the MM patients treated with lenalidomide or thalidomide [61]

Risk factors Myeloma-related Newly diagnosed myeloma Hyperviscosity Personal or family history of VTE Obesity (body mass index ≥30) Comorbidities: cardiac conditions, diabetes, renal impairment, chronic inflammatory disease Immobility Thrombophilias, myeloproliferative disorders, haemoglobinopathies Recent surgery (within 6 weeks): neurotrauma, orthopaedic, general, other Medications: ESAs, hormone replacement therapy, tamoxifen/stilboestrol

Treatment-related Combination chemotherapy High-dose steroid at ≥480 mg/month, dexamethasone or equivalent Doxorubicin

is yet unsettled, it has been demonstrated to affect progression-free survival and overall survival [22]. This can be considered as an important factor affecting the quality of life. Kleber et al. suggested that assessing the comorbidity status in MM, rather than considering specific age cut-offs alone, may allow better definition of patients’ status, more tolerability of treatment, and more knowledge about the best treatment allocations in upcoming patient cohorts [22]. Concern for the future and loss of labour are other contributing concerns that affect quality of life for younger patients. Blood tests raise anxiety and amplify the emotional effects of the disease. Wagner et al. reported that expert clinicians provided the highest HRQoL relevance ratings for bone pain, bodily pain, difficulty walking, tiring easily, feeling discouraged, interference with activities, and difficulty with self-care as a result of bone pain and fatigue.

Quantitative ratings by patients identified sexual function, uncertainty about health, fatigue, weight gain, and emotional concerns, such as worry about new symptoms and difficulty planning for the future, as most

1-2 Mild

5-6 3-4 Moderate

9-10 7-8 Severe

Figure 1. Wong-Baker facial grimace pain scale

relevant to HRQoL [4]. In a prospective populationbased study HRQoL and disease-specific complaints of patients with MM up to 10 years after diagnosis were described [23]. The findings of this study showed that patients with MM experience a much lower HRQoL compared to the general population, irrespective of the number of years since diagnosis. Patients with MM reported mean decreases between baseline and 1-year follow-up scores for quality of life (74%), fatigue (50%), 237

Turk J Hematol 2013;30:234-246

nausea and vomiting (71%), pain (59%), and dyspnea (66%). The most bothersome symptoms during the past week were tingling hands/feet (32%), back pain (28%), bone aches/pain (26%), pain in arm/shoulder (19%), and feeling drowsy (18%). Additionally, 37% worried about their future health, 34% thought about their disease, and 21% worried about dying [23].

Treatments and Quality of Life Some studies have been conducted about the relationship between myeloma treatments and quality of life. Alegre et al. specified that patients with relapsed or refractory MM treated with long-term lenalidomide reported clinically relevant improvements in certain quality-of-life and symptoms scores regardless of treatment response [24]. Transplant-setting studies have shown that patients’ treatment can have a transient adverse impact on HRQoL [25], but response and improved long-term outcomes are associated with an overall improvement in HRQoL [25,26]. Etto et al. reported that autologous stem cell transplantation (ASCT) improved the quality of life in Brazilian MM patients [5]. Khalafallah et al. reported that dosereduced tandem ASCT is well tolerated with low toxicity, although it has transient reduction in HRQoL during both transplants. Post-transplant follow-up showed significant improvement in overall HRQoL, reflecting positively on the overall disease outcome [27]. The activity of bortezomib was associated with improved HRQoL in a phase 3 APEX study [28]. Another phase 3 VISTA study showed that there were clinically expressive and statistically significant temporary reductions in HRQoL from baseline in patients receiving bortezomibmelphalan-prednisone (VMP) treatment and relatively lower HRQoL compared with patients treated with melphalan-prednisone (MP), associated with the toxicities arising from the addition of bortezomib to MP [29]. However, the results demonstrated that HRQoL is not compromised in the long term with VMP vs.

MP. Moreover, analyses of bortezomib dose intensity indicated better HRQoL in patients receiving lower dose intensity. Additionally, Delforge et al. suggested that clinically and statistically significant improvements in several aspects of HRQoL may occur following response onset in patients achieving an overall response to therapy and particularly complete remission (CR), the rate of which was significantly higher with VMP vs. MP [29]. A recent analysis of the HOVON49 phase 3 trial of 238

CĂśmert M: Quality of Life and Supportive Care in Multiple Myeloma

MP plus thalidomide (MPT) vs. MP alone in previously untreated elderly MM patients showed that the higher rates of toxicity associated with MPT, despite adversely affecting some HRQoL parameters during treatment, did not negatively affect global health scores vs. MP [29,30]. In a Nordic multicentre trial, 583 previously untreated MM patients were randomised to receive MP or MP + interferon Îą-2b at a dose of 5 million units subcutaneously, 3 days per week. During the first year of treatment the patients on interferon reported significantly more fever, chills, dry skin, fatigue, pain, nausea/vomiting, and appetite loss than the control patients. There was a moderate reduction of the global quality-of-life score and slight, nonsignificant, reductions of physical, emotional, cognitive, social, and role functioning scores. After the first year there were no statistically significant differences in any toxicity, symptoms, or quality-of-life scores, except for an increased frequency of dizziness in the interferon group [31].

Supportive Care The role of the physician in combining life quality and supportive care in MM patients is important because effective supportive treatment results in improved quality of life. Although much of the supportive care can be provided by haematologists, in some patients symptomatic management is achieved through the collaboration of colleagues in the fields of palliative medicine, pain management, clinical oncology, and orthopaedics [6]. A study by Wagner et al. revealed that patients may not discuss more personal aspects of their illness experience (e.g., anxiety, uncertainty, and sexual function) with their physicians. Physicians should educate patients about cognitive dysfunction associated with treatment and should assess them for cognitive decline [4].

In addition to chemotherapy, prophylaxis and supportive treatment of bone destruction, pain, anaemia, renal failure, fatigue, infections, hypercalcaemia, and emotional distress are essential parts of the therapeutic management of myeloma patients. The concerted action of supportive therapies can significantly help to maximise the benefits of treatment and to improve the wellbeing of myeloma patients in phases of disease progression as well as during phases of remission.

Management of symptoms in patients with myeloma at all stages should follow the principles of evidence-based

Cömert M: Quality of Life and Supportive Care in Multiple Myeloma

palliative medicine [6]. Thus, some guidelines were constituted. The aim of these guidelines is to summarise a national consensus of the haematological community and colleagues involved in the supportive care of patients with myeloma [6]. The supportive care definition is sufficiently broad to cover not only symptomatic treatment and palliative care but also the wide range of management options considered to be ‘haematological supportive care’, including anti-infectives, transfusion therapy, anticoagulation, and growth factors [6].

Supportive Care for Bone Disease Bone pain, particularly in the spine and chest, is the major symptom in MM, which presents at diagnosis in more than two-thirds of patients. Osteolytic lesions, fractures of long bones, vertebral collapse, and plasmacytomas, which directly affect neural tissues, are the most common causes of bone pain. Later in the course of the disease, pain often arises as a side effect of therapies, e.g., thalidomide or bortezomib neuropathy [32]. Long bone fractures usually require stabilisation by surgical fixation. Radiotherapy may be used as the sole treatment in selected cases, but it should be applied to all lesions prone to fracture. A single 8-10Gy fraction is recommended [32]. Vertebroplasty by percutaneous injection of low-viscosity liquid bone cement into the vertebral body has been used for pain relief in patients with spine involvement [33].

Kyphoplasty involves the creation of a cavity in the vertebral body and filling it with highly viscous cement, which will result in complete or partial restoration of the collapsed vertebral body. Analgesics, bed rest, and bracing are the other interventions, whose benefits are limited. Some patients may present with or develop instability of their spine or root compression because of primary disease or complications of vertebroplasty, requiring orthopaedic or neurosurgical interventions.

Bisphosphonates inhibit bone destruction by blocking the osteoclasts’ recruitment from progenitor cells, suppressing migration, proliferation, and differentiation of osteoclasts and inducing apoptosis of osteoclasts and myeloma cells. Bisphosphonates also inhibit the production of matrix metalloproteinase I and IL6, which is the most important growth hormone for myeloma cells [33,34,35,36,37]. The efficacy of clodronate, pamidronate, and zoledronate in preventing bone lesions has been investigated in several randomised trials, while for ibandronate limited data

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from randomised trials are available [32]. Additionally, a network meta-analysis showed superior overall survival with zoledronate compared with etidronate and a placebo. However, there was no difference between zoledronate and other bisphosphonates [38]. Recommended regimens of bisphosphonates are shown in Table 2. Pain improved and quality-oflife and performance statuses were better in patients who received bisphosphonates [33]. Bisphosponateinduced jaw osteonecrosis is an increasingly recognised complication of bisphosphonate therapy that was first described in 2003. Current evidence suggests that the risk is greater for jaw osteonecrosis with zoledronate than with other bisphosphonates. Cumulative dose and duration of treatment are important factors that play a role in this complication [39]. The risk increases in patients who have been taking bisphosphonates for more than 3 years [33]. Thus, bisphosphonates should be discontinued after 2 years of therapy in patients who have achieved CR and/or a plateau phase. For patients whose disease is active, who have not achieved a response, or who have threatening bone disease beyond 2 years, therapy can be tapered to 1 dose every 3 months [32].

Supportive Care for Anaemia Anaemia affects more than two-thirds of MM patients [33]. Anaemia is promoted by erythropoietin deficiency, shortened existence of red blood cells, death of immature erythroblasts due to the Fas ligand and TRAIL, decreased responsiveness of the erythron to proliferative signals of erythropoietin, and the myelosuppressive effect of the chemotherapy [32,33]. Anaemia may be managed by red blood cell transfusions in the shortterm in a symptomatic patient or by treatment with erythropoiesis-stimulating agents (ESAs) [6]. The improvement with transfusion is transient, and repeated transfusions will be required at intervals of 2-3 weeks.

Transfusions have some risks, such as immunological reactions, infections, volume and iron overload, and, in rare cases, even induction of graft-versus-host disease [40]. The high efficacy of erythropoietin in myeloma was documented as early as 1990 in a study on a small number of patients [41]. This pilot study’s findings have been confirmed by several phase 2 and 3 studies. ESAs are the preferred option as they decrease the frequency of transfusions, increase the mean haemoglobin levels, and improve quality of life and performance status 239

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[42,43]. It is recommended to start ESAs in patients with haemoglobin levels below 10 g/dL or with haemoglobin levels below 12 g/dL if symptoms due to anaemia are already present [44]. Patients with mild anaemia (Hb 12 g/dL) should be treated because a higher improvement in quality of life occurs when the haemoglobin level increases from 12 to 13 g/dL [33]. However, British guidelines suggest that the haemoglobin concentration should not rise above 120 g/L [45]. Treatment should be initiated with erythropoietin α or β at a dose of 10,000 U 3 times a week or at 30,000 U or 40,000 U once weekly, or with darbepoetin at 150 mg weekly or 500 mg every 3 weeks. The dose can be doubled after 4 weeks in patients with haemoglobin increases of <10 g/ dL. ESA treatment should be stopped after 6-8 weeks if there has been no haemoglobin response. ESA doses of <20,000 U/week may be adequate in patients where renal disease is the main cause of the anaemia [6].

American Society of Hematology and American Society of Clinical Oncology guidelines recommend ESAs to be administered at the lowest dose possible and the haemoglobin to be increased to the lowest concentration possible to avoid transfusions [46]. There is an increased risk of thrombotic events and hypertension in patients with cancer who are treated with ESAs [32]. There is a significant improvement in quality of life with a better sense of wellbeing, better exercise capacity, less fatigue, and abrogation of transfusion needs in patients responding to ESAs [47,48,49].

Supportive Care for Infections Infections, especially bacterial infections, are frequent complications of MM. Augustson et al. reported that up to 10% of myeloma patients die within 60 days after the diagnosis because of infective causes [50]. Increased predisposition to infections in myeloma is caused by deficits in humoral and cellular immunity, suppression of production of polyclonal immunoglobulins, and use of high-dose steroids in elderly patients or those with poor performance. Active disease is a risk factor for infections.

Administration of trimethoprim/sulfamethoxazole at 160 mg/800 mg, twice daily orally during the first 2 months of conventional induction chemotherapy, resulted in significantly decreased frequencies and severities of bacterial infections [51], but routine usage is not recommended because of antibiotic resistance and increased Clostridium difficile infection [6]. Although scientific data on antibiotic prophylaxis are insufficient, 240

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several studies suggested that prophylactic antimicrobial therapy should be based on the patient’s risk factors, such as previous history of infections and the type and dose of myeloma therapy [32]. Chapel et al. reported that replacement of intravenous immunoglobulin (IVIG) monthly for 1 year reduced the frequency and severity of infections in plateau-phase patients [52]. A dose of IVIG of 500 mg/kg administered every month for up to 6 months is recommended by guidelines for patients who suffer from recurrent infections and hypogammaglobulinaemia [6,32]. Patients treated with vincristine-adriamycin-dexamethasone (VAD), highdose dexamethasone, or a bortezomib-based regimens who are at high risk of reactivation or new acquisition of herpetic infections should receive antiviral prophylaxis with oral acyclovir, at 800 mg 4 times daily, or one of the newer antiviral drugs such as famciclovir or valacyclovir [32]. There are also current studies comparing antiviral prophylaxis with acyclovir at 400 mg orally, 3 times daily, and acyclovir at 400 mg once daily in patients treated with bortezomib. There was no statistically significant difference between the 2 groups in terms of herpetic infections [53]. Acyclovir should be given prior to starting to bortezomib treatment and discontinued 4 weeks after the last dose of treatment. Risk factors for infections and the recommended form and dose of prophylaxis are shown in Table 3. Granulocyte colony stimulating factor (G-CSF) may have a role in reducing treatment-associated neutropenia. It is routinely used after autologous and allogeneic transplantations.

Addition of G-CSF (5 µg/kg/day) to broad-spectrum antibiotics after high-dose chemotherapy decreases the mortality and morbidity rates, curbs superinfections, and prevents fungal infections [54], but there are also studies that do not support these data. Vaccination against influenza, Streptococcus pneumonia, and Haemophilus influenzae is recommended by guidelines, but the efficacy is not guaranteed [6].

Supportive Care for Peripheral Neuropathy Many patients with myeloma have subclinical or even clinical peripheral neuropathy at diagnosis, often due to comorbidities like diabetes mellitus, vitamin B12 deficiency, or carpal tunnel syndrome [6]. This will pose a risk for drug-induced neuropathy with bortezomib and thalidomide. The cause of the neuropathy in many cases of myeloma is not clear, but neurotoxic drugs, amyloidosis, and spinal cord or nerve root compression

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by plasmacytoma or lytic or extramedullary bone disease are the most common causes [6]. Bortezomibinduced neuropathy (BiPN) is characterised by pain and distal sensory neuropathy with suppression of reflexes, resulting in distal weakness in the lower limbs [55].

Grade 2 neuropathy requires 50% dose reduction of bortezomib and grades 3 and 4 neuropathies require drug discontinuation. Neuropathy grading is shown in Table 4.

anorexia, constipation, polydipsia, polyuria, fatigue, confusion, impairment of cognitive functions, coma) requires immediate supportive therapy with 3-6 L/day intravenous saline and high doses of loop diuretics (80-100 mg/day) with frequent evaluations of serum electrolytes [32]. Bisphosphonates can be used for the treatment of hypercalcaemia.

The symptoms of BiPN improve or completely resolve in the majority of patients after a median of 3 months following discontinuation of the drug, while in some patients maximum improvement may take 2 years [56]. Treatment for BiPN is symptomatic relief.

Prophylactic treatment is not effective. Thalidomideinduced neuropathy occurs in up to 75% of patients.

Daily drug dose, dose intensity, cumulative dose of â&#x2030;Ľ400 mg, and duration of therapy have been implicated in the pathogenesis. Symptomatic treatment for thalidomideand bortezomib-induced neuropathy usually comprises gabapentin, pregabalin, or tricyclic antidepressants [57].

Correction of vitamin B12 deficiency and treatment of comorbidities that cause neuropathy are important points for the management of neuropathy. Neuropathic pain scales can be used to define the degree of pain.

Guidelines recommend that superficial neuropathic pain should be treated with topical 5% lidocaine plaster and patients with chronic peripheral neuropathic pain should be considered for multimodal analgesic treatment, including an opioid, ion channel blocker, and serotonin-norepinephrine reuptake inhibitors (SNRIs) [6].

Pain is frequently the predominant symptom of myeloma at diagnosis. It is also a common indicator of relapse or progressive disease. Many myeloma patients suffer from pain, particularly in the skeleton [32]. Fractures, osteolytic bone lesions, spinal cord compression, and neuropathy are the most common causes of the pain in myeloma patients. Pain is a subjective experience, and for sufficient treatment, some pain scales must be used (Figure 1).

Supportive Care for Renal Failure Roughly 15%-25% of myeloma patients have a creatinine value of >2 mg/dL at diagnosis. Patients with reversed renal failure have better overall survival than those without improvement [58]. Dehydration, infections, analgesics, hypercalcaemia, and hyperuricaemia increase tubular cast formation. Fluid intake of at least 3 L per day, limitation of analgesic usage, prevention of infections, and oral or intravenous bicarbonate replacement can improve renal functions.

The most frequent metabolic complication of MM is hypercalcaemia, predominantly caused by tumourinduced bone resorption by osteoclast-activating factors such as various cytokines and prostaglandins [32]. Symptomatic hypercalcaemia (nausea, vomiting,

Supportive Care for Pain

Effective analgesia can be achieved by regular administration of oral medication in myeloma patients.

The WHO Pain Treatment Ladder (shown in Table 5) has been widely accepted for the treatment of tumourrelated pain.

NSAIDs must be given carefully because of renal toxicity and gastrointestinal effects. COX-2 inhibitors have less gastrointestinal and renal toxicity. Opioidsâ&#x20AC;&#x2122; long-term effect and tolerance are better, but they are more expensive. The adverse effects of opioids are dryness of mouth, nausea, and emesis. The combination of opioids and NSAIDs is more effective, but also more toxic. Adjuvant medications such as corticosteroids, anti-emetics, neuroleptics, antidepressants, and benzodiazepines should be given as required [32].

Supportive Care for Thromboembolism Myeloma and other plasma cell disorders have an association with venous thromboembolism (VTE) [59].

The incidence of VTE is highest during the first 3 to 4 months following the diagnosis [57]. Active disease, infections, previous VTE, and immobilisation are all known risk factors for VTE in myeloma patients.

Thalidomide and lenalidomide have been demonstrated to further increase this risk, particularly when combined with steroids or cytotoxic agents [6]. All myeloma patients starting thalidomide or lenalidomide should undergo a risk assessment for VTE. However, the optimal prophylaxis remains controversial. If the patient does not have a risk factor or has only one risk 241

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factor (risk factors are shown in Table 6), a standard dose of 325 mg/day or a low dose of 75-80 mg/day of aspirin is recommended, but in the case of 2 or more risk factors, low-molecular-weight heparin (LMWH) at a high-risk prophylactic dose, i.e. enoxaparin at 40 mg, or warfarin (target international normalised ratio [INR]: 2.5) is recommended, unless contraindicated [6]. Aspirin and warfarin showed similar efficacy in reducing thromboembolic events in patients with myeloma treated with thalidomide-based regimens compared with LMWH, but in elderly patients warfarin showed less efficacy than LMWH [60]. The duration of thromboprophylaxis remains unclear, but it is guided by risk factors such as active disease (e.g., for the first 4-6 months of treatment until disease control is achieved) and deescalated or discontinued unless there are ongoing significant risk factors [6].

Conclusion

risk factors, LMWH or warfarin (target INR: 2.5) is recommended, unless contraindicated.

• The duration of thromboprophylaxis remains unclear but is guided by risk factors such as active disease and is deescalated or discontinued unless there are ongoing significant risk factors.

• Prophylactic immunoglobulin is not routinely recommended but may be useful in patients with severe, recurrent bacterial infections and hypogammaglobulinaemia.

• Prophylactic acyclovir is recommended for patients receiving VAD, high-dose dexamethasone, or a bortezomib-based regimen who are at high risk of reactivation or new acquisition of herpetic infections and following autologous stem cell transplantation.

• Pain should be assessed regularly in myeloma patients at all stages of the disease and measured using a 0-10 or a verbal none-mild-moderate-severe scale.

Supportive treatment is an essential part of the therapeutic management of myeloma patients because while directed towards improving the patient’s quality of life, they also have significant effects against the disease and can improve survival [62]. Careful consideration of patients’ and caregivers’ reported symptoms and effective supportive care will result in improved quality of life and improved survival. Some recommendations are given below as clues for supportive treatment from the guidelines.

• Patients who repeatedly score pain as ≥5/10 should be referred to a palliative care or pain team.

Recommendations [45]

• Clinical evidence of a significant (e.g., above NCI grade 2) or progressive peripheral neuropathy at diagnosis should be appropriately investigated to identify treatable causes, and referral to a neurologist should be made so that appropriate neurological investigations can be performed.

• A therapeutic trial of ESA should be considered in a patient with persistent symptomatic anaemia (typically haemoglobin concentration of <100 g/L) in whom haematinic deficiency has been excluded.

• One of darbepoetin, epoetin alfa, or epoetin beta can be chosen. Dose-doubling after 4 weeks in patients with a haemoglobin increase of <10 g/dL can be considered. ESA treatment should be stopped after 6-8 weeks if there has been no haemoglobin response.

The haemoglobin concentration should not rise above 12 g/dL. • All patients who are due to start thalidomide- or lenalidomide-containing therapy should undergo a risk assessment for VTE. • In patients receiving thalidomide or lenalidomide, aspirin (75-325 mg) may be considered as VTE prophylaxis in low-risk patients, but in the case of ≥2 242

• Effective analgesia can be achieved by regular administration of oral medication. The WHO Pain Treatment Ladder (Table 5) has been widely accepted for the treatment of tumour-related pain.

• Local radiotherapy is helpful for pain control; a single-fraction dose of 8 Gy is recommended. The use of vertebroplasty or kyphoplasty may be considered in patients with persistent pain.

• Patients who develop a significant (e.g., above NCI grade 2) or progressive chemotherapy-induced peripheral neuropathy should be managed with graded dose reduction or drug withdrawal.

• All patients with chronic peripheral neuropathic pain should be considered for multimodal analgesic treatment including an opioid, ion channel blocker, and SNRI. • Dehydration, infections, analgesics, hypercalcaemia, and hyperuricaemia increase tubular cast formation.

Fluid intake of at least 3 L per day, limitation of analgesic usage, prevention of infections, and oral or intravenous bicarbonate replacement can improve renal functions.

Cömert M: Quality of Life and Supportive Care in Multiple Myeloma

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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60. Palumbo A, Cavo M, Bringhen S, Zamagni E, Romano A, Patriarca F, Rossi D, Gentilini F, Crippa C, Galli M, Nozzoli C, Ria R, Marasca R, Montefusco V, Baldini L, Elice F, Callea V, Pulini S, Carella AM, Zambello R, Benevolo G, Magarotto V, Tacchetti P, Pescosta N, Cellini C, Polloni C, Evangelista A, Caravita T, Morabito F, Offidani M, Tosi P, Boccadoro M. Aspirin, warfarin, or enoxaparin thromboprophylaxis in patients with multiple myeloma treated with thalidomide: a phase III, open-label, randomized trial. J Clin Oncol 2011;29:986993.

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DOI: 10.4274/Tjh.2013.0108

Commentary

Current Management of Chronic Myeloid Leukemia with Tyrosine Kinase Inhibitors Kronik Myeloid Lösemi Güncel Tedavisinde Tirozin Kinaz İnhibitörleri İbrahim C. Haznedaroğlu Hacettepe University Medical School, Department of Hematology, Ankara, Turkey

Abstract: The clinical outcomes and survival of tyrosine kinase inhibitor (TKI)-treated patients with chronic myeloid leukemia (CML) have been significantly improved. The aim of this editorial is to outline critical steps of TKI administration practices during the long-term clinical course of CML based on data obtained from randomized clinical trials and international recommendations.

The efficacy of TKI treatment, TKI side effects, off-target complications, and long-term morbidities due to both the disease and the drug are common arguments in the management of CML. Complete hematological response, early complete cytogenetic response, faster major molecular response, and deeper, more durable molecular responses (MR4, MR4.5, MR5) are the ultimate goals for TKI-receiving patients with CML. Key Words: Chronic myeloid leukemia, Tyrosine kinase inhibitor, Imatinib, Nilotinib, Dasatinib, Bosutinib, Ponatinib

Özet: Kronik myeloid lösemi (KML) tedavisinde tirozin kinaz inhibitörü (TKI) grubu ilaçların kullanımı klinik gidişi ve hastaların yaşam süresini son derece olumlu etkilemiştir. Bu yazının amacı, KML’nin uzun klinik seyri boyunca yapılan TKI uygulamalarının kritik basamaklarını randomize klinik çalışma verileri ve uluslararası rehberler ışığında irdelemektir. KML yönetiminde TKI tedavisinin etkinliği, TKI yan etkileri, ilaçların hedef-dışı komplikasyonları, ve hem hastalık seyrine hem de uygulanan ilaçlara bağlı uzun dönem morbiditeler önemli tartışma noktalarını teşkil etmektedir. Tam hematolojik yanıt, erken tam sitogenetik yanıt, daha çabuk gelişen, daha derin (MR4, MR4.5, MR5), ve dayanıklı major moleküler yanıtlar TKI tedavisi alan KML hastalarında klinik seyir boyunca ulaşılması gereken hedefler konumundadırlar.

Anahtar Kelimeler: Kronik myeloid lösemi, Tirozin kinaz inhibitörü, Imatinib, Nilotinib, Dasatinib, Bosutinib, Ponatinib Introduction Current initial frontline therapy for chronic myeloid leukemia (CML) is chronic oral administration of tyrosine kinase inhibitor (TKI) [1,2]. During the last decade, the introduction of TKI to the treatment regimen of CML has significantly affected the survival of patients. Imatinib mesylate was the first TKI in the clinic. The survival benefit

of imatinib in CML is excellent [3]. Next-generation TKIs, namely dasatinib [4,5], nilotinib [6], bosutinib [7], and ponatinib [8], were then developed for the management of CML patients. The clinical outcomes and survival of tyrosine kinase inhibitor (TKI)-treated patients with chronic myeloid leukemia (CML) have been significantly improved. The proper clinical and laboratory monitoring of CML patients

Address for Correspondence: İbrahim C. HAZNEDAROĞLU, M.D., Hacettepe University Medical School, Department of Hematology, Ankara, Turkey E-mail: ichaznedaroglu@gmail.com Received/Geliş tarihi : March 24, 2013 Accepted/Kabul tarihi : May 2, 2013

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Haznedaroğlu İC: Current Management of Chronic Myeloid Leukemia with Tyrosine Kinase Inhibitors

Table 1. European LeukemiaNet (ELN) 2013 recommendations for the clinical decision making in the chronic myeloid leukemia (CM)L patients receiving tyrosine kinase inhibitor (TKI) based on the response status during the follow-up and monitoring.

RESPONSE STATUS TO TKI BASED ON ELN 2013

MANAGEMENT IN THE PATIENT WITH CML

OPTIMAL RESPONSE TO THE TKI TREATMENT

THERE IS NO INDICATION THAT A CHANGE OF THERAPY MAY IMPROVE SIGNIFICANTLY THE OUTCOME (SURVIVAL)

FAILURE OF THE CURRENT TKI TREATMENT

THE RISK OF THE PROGRESSION AND DEATH FROM LEUKEMIA IS SIGNIFICANT. THE PATIENT SHOULD RECEIVE A DIFFERENT TREATMENT, WHENEVER AVAILABLE AND APPLICABLE

WARNING (PREVIOUSLY SUBOPTIMAL RESPONSE) FOR THE CURRENT TKI TREATMENT

THE CHARACTERISTICS OF THE DISEASE AND THE RESPONSE TO TREATMENT REQUIRE A MORE CAREFUL AND MORE FREQUENT MONITORING, THAT IS TO SAY A MOLECULAR AND A CYTOGENETIC TEST WITHIN LESS THAN 3 MONTHS, AND A MUTATIONAL ANALYSIS.

is absolutely necessary to reach those successful outcomes [9,10,11]. Complete hematological response (CHR), early complete cytogenetic response (CCyR), faster major molecular response (MMR), and deeper, more durable molecular responses (MR4, MR4.5, MR5) are the ultimate goals for TKI-receiving patients with CML [12]. During the era of 3rd generation TKIs, excellent molecular responses are the most important targets in CML. The surrogate markers of CML outcome (rate, depth, and time to cytogenetic and molecular response) are vital in the clinical management of the disease [1,13,14,15,16,17].

Critical evaluations of CML patients to hit those targets should be made at the baseline and at the 3rd, 6th, 12th, 18th, and 24th months after TKI administration. The treatment milestones are checked during the time-line of evaluation [12]. Therapeutic expectations have increased in the field of CML. The functional cure of the disease is now possible with TKIs. Likewise, the molecular responses of MR4 or MR4.5 could lead to the discontinuation of the drug with proper molecular monitoring (TFR; treatment-free remission) within the context of clinical trials [18]. On the other hand, disease progression (accelerated phase (AP) CML or blastic crisis (BC)) under TKI is a great disaster [19,20,21]. Survival after progression into AP/BC is still significantly shorter, even in the TKI era. However, the risk of progression has been decreased with the introduction of more powerful TKIs [22,23,24]. Major attention should be given for the prevention of disease progression, particularly for the treatment-nave CML or TKI-refractory diseases. Clinical response, the depth of response, and the impact of TKI use on the disease outcome should always be the focuses during the long-term management of CML [12].

Investigational efforts tried to improve the results of CML first-line therapy of imatinib obtained from the International 248

Randomized Study of Interferon and STI571 trials. Those attempts included imatinib dose increase, particularly in high-Sokal risk patients [25]; imatinib-based combinations [26]; and the setting of second-generation TKIs as first-line therapy [24,27]. Dose optimization studies of TKI, such as the German CML IV [28] and TIDEL [29], have been taken into account for increments in safety, efficacy, tolerability, adherence, and acceptably manageable drug toxicity. The aim of this review is to outline critical steps of TKI administration practices during the long-term clinical course of CML based on data obtained from randomized clinical trials (RCTs) and international recommendations. The efficacy of TKI treatment, TKI side effects, off-target complications, and long-term morbidities due to both the disease and the drug are common arguments in the management of CML. Standardized definitions of molecular response in CML under TKI have been given by the European LeukemiaNet (ELN). MR4 is achieved with a BCR-ABL expression of <0.01%, MR4.5 with <0.0032% BCR-ABLIS, and MR5 with <0.001% BCR-ABLIS [28].

Baseline Evaluation and Management of the Patient with CML Standard baseline evaluation of the de novo CML patient includes exact medical diagnosis of CML, basic laboratory evaluation covering complete blood count (CBC) [30] and peripheral blood smear (PBS), bone marrow histopathology, conventional cytogenetics and/or FISH analyses for the Ph* chromosome, and quantitative molecular analyses for BCRABL1. Tumor load and disease phase should be defined [12]. Newly diagnosed chronic-phase CML patients should be stratified based on the Sokal [31], Euro/Hasford [32], and EUTOS [33] CML prognostic scoring systems. Novel recent investigations for de novo CML patients have examined the validity of gene expression profiling, genetic polymorphisms,

HaznedaroÄ&#x;lu Ä°C: Current Management of Chronic Myeloid Leukemia with Tyrosine Kinase Inhibitors

next-generation genomics, multidrug resistance genes (MDR, OCT1), fusion transcripts, and preexisting BCR-ABL kinase domain mutations [34,35,36,37,38,39,40,41,42,43].

Current initial TKI treatment for chronic-phase CML is imatinib at 400 mg p.o. [12]. Second-generation TKIs, namely dasatinib at 100 mg p.o. [27] and nilotinib at 600 mg p.o. [24], have also been registered for the first-line therapy of CML. There is a tendency for the prescription of more powerful TKIs in high-Sokal risk CML patients and high-risk patients with complex karyotypic abnormalities at the beginning of the disease for the prevention of disease progression. Likewise, young and low prognostic risk CML patients are candidates for second-generation TKIs for the sake of drug discontinuation in the future. However, heterogeneous presentation and course of CML, individual characteristics, compliance and preferences of the patients, comorbidities, different toxicity profiles of the drugs, and the physician-clinical center experience must all be considered during the initial decision making for first-line TKI usage in newly diagnosed chronic-phase CML [12,24,27].

Evaluation and Management at the 3rd Month after the Initiation of TKI in the Patient with CML Standard disease assessments at the 3rd month of oral TKI administration for the chronic-phase CML patient include critical clinical evaluation and CBC/PBS to reveal CHR, cytogenetic analyses to evaluate the cytogenetic response, and quantitative molecular BCR-ABL analyses to identify molecular response [12]. Optimal response at the 3rd month of imatinib administration is CHR and minor cytogenetic response. However, particularly after the introduction of the powerful second-generation TKIs, namely nilotinib and dasatinib, to the first-line therapy of CML, the expectations in response become higher. Recent RCT studies [24,27,44,45,46,47,48,49,50] indicated that the critical BCR-ABL transcript level (10% cut-off value) the 3rd month following the start of TKI treatment may have prognostic significance in patients with CML. This scientific observation has been made with imatinib in GIMEMA [44], German CML IV [26], Hammersmith [51], DASISION [52], and ENESTnd [22] trials, and with dasatinib in DASISION [49] and with nilotinib in ENESTnd [22] trials. Challenges for the widespread routine use of the 10% BCR-ABL transcript cut-off at the 3rd month of TKI are present. First, the estimated ratio of BCR-ABL/ABL is highly technique-dependant. Many laboratories in the world are still not qualified for the international harmonization of scale (IS). High ratio values on the IS scale, house-keeping control gene problems, variations in samples, delays in the exact molecular assessment time after TKI administration, and early unexpected variation kinetics of response in indivi dual CML patients complicate the universal application of the 10% BCR-ABL transcript cut-off at the 3rd month of TKI. Furthermore, the tumor burden at diagnosis, prognostic

Turk J Hematol 2013;30:247-255

scoring, gene profile, cytoreduction with TKI dosage, treatment adherence, and numerous confounding effects may obscure the real-life picture at the 3rd month of TKI usage outside clinical trials. Nevertheless, any CML patient that does have a BCR-ABL over 10% after 3 months of TKI presents a strong warning, requiring more careful and more frequent monitoring based on the clear RCT data. If the CML patient exhibits no CHR and/or no minor cytogenetic response, the failure of the first-line TKI is evident. If the initial failed TKI treatment for CML was imatinib, then nilotinib or dasatinib should be given. If 1 of the 2 secondgeneration TKIs (nilotinib or dasatinib) was used as the firstline therapy and failed, the other (dasatinib or nilotinib) could be administered particularly based on the mutation data. During the treatment decision for 2nd line TKIs, a mutational analysis shall be performed. Increasing the dose of imatinib has been tried in the literature but seems to be a dying practice in the era of stronger TKIs. Drug tolerability and adherence to the treatment should always be sought.

Effective management of the treatment-related adverse effects is a vital part of the CML care [12].

Evaluation and Management at the 6th Month after the Initiation of TKI in the Patient with CML Standard disease assessments at the 6th month of oral TKI administration for the chronic-phase CML patient include critical clinical evaluation to establish CHR, cytogenetic analyses to evaluate the cytogenetic response, and quantitative molecular BCR-ABL analyses to identify molecular response. Optimal response at the 6th month of imatinib is at least partial cytogenetic response (Ph* chromosome lower than 35%) [12]. However, particularly after the introduction of the powerful second-generation TKIs, namely nilotinib and dasatinib, to the first-line therapy of CML, the expectations in response become higher. CCyR at 6 months and/or BCR-ABL below 1% following 6 months of second-generation TKIs are considered as optimal. Any CML patient that does have a BCR-ABL over 10% and/or Ph* chromosome over 35% after 6 months of TKI treatment (particularly nilotinib and dasatinib) may be accepted as a failed case and the treatment strategy may be changed. Those higher treatment milestones could be applied to the first-line imatinib-receiving CML patients and a switch to secondgeneration TKIs may be performed. Cumulative incidence of MMR is higher with both nilotinib and dasatinib. An early switch from imatinib to a second-generation TKI is rational since RCTs have indicated the higher probability of obtaining better responses as well as progression-free survival and overall survival [24,27]. Prevention of disease progression seems to be better achieved with more powerful second-generation TKIs. Specific long-term drug adverse effects (such as pleuropulmonary syndrome for dasatinib and metabolic syndrome for nilotinib), as well as increased treatment costs, should be considered. Drug tolerability and adherence to the treatment should always be sought [12].

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HaznedaroÄ&#x;lu Ä°C: Current Management of Chronic Myeloid Leukemia with Tyrosine Kinase Inhibitors

Evaluation and Management at the 12th Month after the

Evaluation and Management at the 24th Month and

Initiation of TKI in the Patient with CML

Thereafter Following the Initiation of TKI in the Patient

Standard disease assessments at the 12th month of oral TKI administration for the chronic-phase CML patient include critical clinical evaluation to establish CHR, cytogenetic analyses to examine the cytogenetic response, and quantitative molecular BCR-ABL analyses to identify molecular response [12]. Additional karyotypic abnormalities should be searched in the BM cytogenetics.

Optimal response at the 12th month of imatinib usage is at least CCyR. However, particularly after the introduction of the powerful second-generation TKIs, to the first-line therapy of CML, the expectations in response become higher [1,6,8,15,16,22,28,44,45,48,50,51,53,54,55,56, 57,58,59,60,61,62,63]. CCyR at 12 months and BCR-ABL below 0.1% following 6 months of second-generation TKIs are considered as optimal based on international guidelines.

Any CML patient that does have a BCR-ABL over 1% and/ or Ph* chromosome over 1% after 12 months of TKI usage (particularly nilotinib and dasatinib) may be accepted as a failed case and the treatment strategy may be changed.

Those higher treatment milestones could be applied to the first-line imatinib-receiving CML patients and a switch to second-generation TKIs may be performed. Drug tolerability and adherence to the treatment should always be sought [12].

Evaluation and Management at the

18th

Month after

the Initiation of TKI in the Patient with CML Standard disease assessments at the 18th month of oral TKI administration for the chronic-phase CML patient include critical clinical evaluation to establish CHR and CCyR, and quantitative molecular BCR-ABL analyses to identify molecular response [12]. Optimal response at the 18th month of imatinib is at least MMR. However, particularly after the introduction of the powerful secondgeneration TKIs, namely nilotinib and dasatinib, to the first-line therapy of CML, the expectations in response become higher [1,4,6,10,11,17,23,38,39,42,44,45,47,48,4 9,50,51,52,53,54,55,56,57]. CCyR at 18 months and BCRABL below 0.1% following 18 months of second-generation TKIs are considered as optimal. Any CML patient that does have a BCR-ABL over 1% and/or Ph* chromosome over 1% (absence of CCyR) after 18 months of TKI usage (particularly nilotinib and dasatinib) may be accepted as a failed case and the treatment strategy may be changed [15].

Those higher treatment milestones could be applied to the first-line imatinib-receiving CML patients and a switch to second-generation TKIs may be performed. Drug tolerability and adherence to the treatment should always be sought [12].

250

with CML Standard disease assessments at the 24th month of oral TKI administration for the chronic-phase CML patient include critical clinical evaluation to establish CHR and CCyR, and quantitative molecular BCR-ABL analyses to identify molecular response [12]. Optimal response at the 24th month of imatinib is at least the continuation of MMR. However, particularly after the introduction of the powerful second-generation TKIs, namely nilotinib and dasatinib, to the first-line therapy of CML, the expectations in response become higher. CCyR at 24 months and BCR-ABL below 0.1% following 24 months of second-generation TKIs are considered as optimal. Any CML patient that does have a BCR-ABL over 1% and/or Ph* chromosome over 1% after 24 months of TKI usage (particularly nilotinib and dasatinib) may be accepted as a failed case and the treatment strategy may be changed [15]. Those higher treatment milestones could be applied to the first-line imatinib-receiving CML patients and a switch to second-generation TKIs may be performed. Drug tolerability and adherence to the treatment should always be sought. Quality of life is especially a matter of concern in CML patients receiving long-term, maybe lifetime, TKI drugs [12].

In the case of intolerance to any TKIs and/or multi-TKIresistant CML cases with or without mutations, third-line treatment includes bosutinib, ponatinib, allogeneic stem cell transplantation, and experimental therapies [64-66]. ABL domain mutations leading to the increments in the BCRABL oncogenicity may be detected during the TKI therapy.

The TKI regimen may be altered with another TKI based on the type of the mutation. Sometimes the entire treatment strategy of CML has to be changed because of the presence of the ABL domain mutation. For instance, T315I is a unique mutation making the CML patient irresponsive to most TKIs (excluding ponatinib) and leads allografting to become an option in the case [15]. Combination treatments such as TKI plus interferon [67] are still a matter of research and are rarely used outside of clinical trials.

Evaluation for the discontinuation of TKIs in the superior TKI-responder patient with CML should be performed in the long term, for instance after 2 years. Patients with deeper molecular responses (MR4, MR4.5, MR5) are candidates for TKI discontinuation [68]. MR4 is achieved with a BCR-ABL expression of <0.01%, MR4.5 with <0.0032% BCR-ABLIS, and MR5 with <0.001% BCR-ABLIS [28,46]. Treatment-free remissions and re-induction of the remission with the same TKI seem to be possible based on the data from the STIM trial [68]. Pregnancy represents a cause for TKI discontinuation because of the negative impact of any TKI on organogenesis.

Haznedaroğlu İC: Current Management of Chronic Myeloid Leukemia with Tyrosine Kinase Inhibitors

Patients with AP/BC CML should be treated with the most powerful TKI available and multi-agent chemotherapy before allografting [19,20,21,65,69,70,71]. Since those patients with advanced-phase CML still do have a worse prognosis, prevention of disease progression is the most significant aspect of CML disease management.

Future Perspectives of CML ELN 2013 recommendations have established how to proceed in clinical decision making in the CML patients receiving TKIs based on the response status (Table 1). 72 The future of CML and TKI treatment will reveal better understandings of the disease pathobiology, leukemic stem cells, signal transduction, and their translations to the patient’s care [34,35,36,37,38,39,40,41,42,43]. Cure of CML and the eradication of minimal residual disease via the multi-hit drugs with distinct biological actions would be possible. The cessation of therapy with the aim of cure, stem cell depletion, stem cell exhaustion, and immunological control of the disease may be the future strategies in the management of CML. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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41. Schemionek M, Steidl U, Schwab A, Tenen DG, Mhairi C, Holyoake TL, Preisinger C, Mueller-Tidow C, Brümmendorf TH, Koschmieder S. Metastasis suppressor

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1 is downregulated in CML stem cells and overexpression impairs early leukemic cell propagation. ASH Annual Meeting Abstracts 2012;120:2776.

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44. Castagnetti F, Gugliotta G, Breccia M, Specchia G, Abruzzese E, Levato L, Cavazzini F, Iurlo A, Stagno F, Ferrero D, Porretto F, Martino B, Rupoli S, Intermesoli T, Fava C, Palandri F, Venturi C, Soverini S, Testoni N, Alimena G, Pane F, Saglio G, Martinelli G, Baccarani M, Rosti G. The BCR-ABL transcript levels at 3 and 6 months predict the long-term outcome of chronic myeloid leukemia patients treated frontline with imatinib mesylate: a Gimema CML WP analysis. ASH Annual Meeting Abstracts 2012;120:1678.

45. Gugliotta G, Castagnetti F, Palandri F, Breccia M, Levato L, Stagno F, Rege-Cambrin G, Zaccaria A, Specchia G, Usala E, Gozzini A, Martino B, Capucci A, Musto P, Tiribelli M, Abruzzese E, Soverini S, Bochicchio MT, Testoni N, Saglio G, Martinelli G, Pane F, Alimena G, Baccarani M, Rosti G. Cytogenetic and molecular responses at 3 months are associated with a better outcome in early chronic phase (ECP) chronic myeloid leukemia (CML) patients treated with nilotinib. ASH Annual Meeting Abstracts 2012;120:2797.

46. Hughes TP, Hochhaus A, Branford S, Müller MC, Kaeda JS, Foroni L, Druker BJ, Guilhot F, Larson RA, O’Brien SG, Rudoltz MS, Mone M, Wehrle E, Modur V, Goldman JM, Radich JP; IRIS investigators. Long-term prognostic significance of early molecular response to imatinib in newly diagnosed chronic myeloid leukemia: an analysis from the International Randomized Study of Interferon and STI571 (IRIS). Blood 2010;116:3758-3765.

47. Jain P, Kantarjian HM, Nazha A, Jabbour E, QuintásCardama A, Benjamini O, Pierce SA, Cardenas-Turanzas M, Verstovsek S, Borthakur G, Ravandi F, O’Brien S, Cortes JE. Early molecular and cytogenetic responses predicts for significantly longer event free survival (EFS) and overall survival (OS) in patients (pts) with newly diagnosed chronic myeloid leukemia (CML) in chronic phase (CP) - an analysis of 4 tyrosine kinase inhibitor (TKI) modalities (standard dose imatinib, high dose imatinib, dasatinib and nilotinib).

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48. Kim DDH, Lee HG, Kamel-Reid S, Lipton JH. BCR/ABL transcript at 3 months predicts long-term outcomes following second generation tyrosine kinase inhibitor therapy in the patients with CML in chronic phase who failed imatinib.

ASH Annual Meeting Abstracts 2012;120:2777.

49. Saglio G, Kantarjian HM, Shah N, Jabbour EJ, QuintasCardama A, Steegmann JL, Boqué C, Chuah C, Pavlovsky C, Mayer J, Ukropec J, Wildgust M, Hochhaus A. Early response (molecular and cytogenetic) and long-term outcomes in newly diagnosed chronic myeloid leukemia in chronic phase (CML-CP): exploratory analysis of DASISION 3-year data. ASH Annual Meeting Abstracts 2012;120:1675.

50. White DL, Saunders VA, Yeung DT, Grigg A, Hughes TP. Early molecular response to imatinib in CP-CML patients: the significance of early dose intensity and OCT-1 activity in responders and efficacy of dose escalation and switch to nilotinib in non-responders. ASH Annual Meeting Abstracts 2012;120:693.

51. Marin D, Hedgley C, Clark RE, Apperley J, Foroni L, Milojkovic D, Pocock C, Goldman JM, O’Brien S. Predictive value of early molecular response in patients with chronic myeloid leukemia treated with first-line dasatinib. Blood 2012;120:291-294.

52. Kantarjian H, O’Brien S, Jabbour E, Shan J, Ravandi F, Kadia T, Faderl S, Garcia-Manero G, Borthakur G, Cortes J. Impact of treatment end point definitions on perceived differences in long-term outcome with tyrosine kinase inhibitor therapy in chronic myeloid leukemia. J Clin Oncol 2011;29:3173-3178.

53. Choi SY, Lee SE, Kim SH, Jang EJ, Lee J, Oh YJ, Bang JH, Park JE, Byun JY, Jeon HL, Woodman RC, Szczudlo T, Jootar S, Kim HJ, Sohn SK, Park JS, Kim SH, Zang DY, Oh SJ, Kim DW. Nilotinib versus high-dose imatinib in early chronic phase CML patients who have suboptimal molecular responses to standard-dose imatinib: updated data from RE-Nice Study.

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54. Cortes J, Kantarjian H. How I treat newly diagnosed chronic phase CML. Blood 2012;120:1390-1397.

57. Falchi L, Kantarjian HM, Quintas-Cardama A, O’Brien S, Jabbour EJ, Ravandi F, Borthakur G, Garcia-Manero G, Verstovsek S, Burger JA, Luthra R, Cortes JE. Clinical significance of deeper molecular responses with four modalities of tyrosine kinase inhibitors as frontline therapy for chronic myeloid leukemia. ASH Annual Meeting Abstracts 2012;120:164.

58. Hughes TP, Lipton JH, Spector N, Leber B, Pasquini R, Clementino N, Schwarer AP, Etienne G, Guerci-Bresler A, Branford S, Purkayastha D, Collins L, Szczudlo T, Cervantes F. Switching to nilotinib is associated with continued deeper molecular responses in CML-CP patients with minimal residual disease after ≥ 2 years on imatinib: ENESTcmr 2-year follow-up results. ASH Annual Meeting Abstracts 2012;120:694.

59. Larson RA, Hochhaus A, Hughes TP, Clark RE, Etienne G, Kim DW, Flinn IW, Kurokawa M, Moiraghi B, Yu R, Blakesley RE, Gallagher NJ, Saglio G, Kantarjian HM. Leukemia 2012;26:2197-2203.

60. Rosti G, Castagnetti F, Gugliotta G, Palandri F, Baccarani M. Second-generation BCR-ABL inhibitors for frontline treatment of chronic myeloid leukemia in chronic phase.

Crit Rev Oncol Hematol 2012;82:159-170.

61. Rosti G, Gugliotta G, Castagnetti F, Breccia M, Levato L, Rege-Cambrin G, Capucci A, Tiribelli M, Zaccaria A, Bocchia M, Stagno F, Cavazzini F, Specchia G, Martino B, Cedrone M, Intermesoli T, Palandri F, Soverini S, Bochicchio MT, Testoni N, Alimena G, Pane F, Saglio G, Martinelli G, Baccarani M. Five-year results of nilotinib 400 mg BID in early chronic phase chronic myeloid leukemia (CML): high rate of deep molecular response - update of the GIMEMA CML WP Trial CML0307. ASH Annual Meeting Abstracts 2012;120:3784.

62. Sail KR, Chen L, Jackson J, Ericson SG, Haislip S, Ibison T, Gilmore J, Saleh MN. Treatment patterns among patients with Philadelphia chromosome positive chronic myeloid leukemia (Ph+ CML) treated with imatinib in a community setting. ASH Annual Meeting Abstracts 2012;120:3179.

55. Cortes JE, Brümmendorf TH, Khoury HJ, Hochhaus A, Apperley J, O’Brien S, Leip E, Kelly V, Ruffner K, Kantarjian HM. Assessment of early cytogenetic response as a predictor of long-term clinical outcomes in a phase 1/2 study of bosutinib in chronic phase CML. ASH Annual Meeting Abstracts 2012;120:2798.

63. Wu B, Zhong H, Saglio G, Chen F. Different strategies for firstline treatment of chronic myeloid leukaemia: an economic analysis. ASH Annual Meeting Abstracts 2012;120:4696.

56. Cortes JE, Pasquini R, Kantarjian HM, Joske D, Meillon LA, Shen Z, Piccolo C, Zernovak O, Sivarathinasami R, Eng DF, Kim DW, Hughes TP. First-line treatment and management of chronic myeloid leukemia (CML) in clinical practice: update of > 1800 patients (Pts) in the WORLD CML Registry.

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65. Cortes JE, Kim DW, Pinilla-Ibarz J, le Coutre P, Paquette R, Chuah C, Nicolini FE, Apperley J, Khoury HJ, Talpaz M, DiPersio JF, DeAngelo DJ, Abruzzese E, Rea D, Baccarani M, Muller MC, Gambacorti-Passerini C, Wong S, Lustgarten S, Rivera VM, Clackson T, Turner CD, Haluska FG, Guilhot F, Deininger MW, Hochhaus A, Hughes T, Goldman JM, Shah

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N, Kantarjian HG; The PACE Study Group. A pivotal phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) resistant or intolerant to dasatinib or nilotinib, or with the T315I BCR-ABL mutation: 12-month follow-up of the PACE Trial. ASH Annual Meeting Abstracts 2012;120:163.

66. Ahmed S, Saliba RM, De Lima M, Rondon G, Kebriaei P, Fisher TD, Jabbour E, Chen J, Kantarjian H, Anderlini P, Alousi AM, Hosing CM, Andersson B, Jones RB, Qazilbash MH, Shpall EJ, Giralt S, Champlin RE. Sequential treatment after allogeneic stem cell transplantation for chronic myelogenous leukemia. ASH Annual Meeting Abstracts 2012;120:3129.

67. Nicolini FE, Etienne G, Dubruille V, Roy L, Huguet F, Legros L, Giraudier S, Coiteux V, Guerci-Bresler A, Lenain P, Rea D, Ame S, Cony-Makhoul P, Gardembas M, Hermet E, Rousselot P, Gagnieu MC, Morisset S, Pivot C, Etienne M, Guilhot F, Dulucq S, Mahon FX. Pegylated interferon-{alpha} 2a in combination to nilotinib as first line therapy in newly diagnosed chronic phase chronic myelogenous leukemia provides high rates of MR4.5. Preliminary results of a phase II study. ASH Annual Meeting Abstracts 2012;120:166.

68. Mahon FX, Réa D, Guilhot J, Guilhot F, Huguet F, Nicolini F, Legros L, Charbonnier A, Guerci A, Varet B, Etienne G, Reiffers J, Rousselot P; Intergroupe Français des Leucémies Myéloïdes ChroniquesDiscontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 2010;11:1029-1035.

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69. Chae YK, Kantarjian HM, Romo CG, Whitaker C, Jabbour EJ, O’Brien S, Borthakur G, Ravandi F, Thomas DA, Pierce SA, Quintás-Cardama A, Cortes JE. Prognostic factors in the blastic and accelerated phase of patients with chronic myeloid leukemia (CML) in the tyrosine kinase inhibitor (TKI) era. ASH Annual Meeting Abstracts 2012;120:3786.

70. Ghanem H, Kantarjian HM, Jabbour EJ, Cortes JE, QuintasCardama A. Rare blast phase (BP) phenotypes of chronic myeloid leukemia (CML). ASH Annual Meeting Abstracts 2012;120:4434.

71. Strati P, Kantarjian HM, Thomas DA, O’Brien SM, Jabbour EJ, Quintas-Cardama A, Borthakur G, Faderl S, Ravandi F, Cortes JE. Hyper-CVAD plus imatinib or dasatinib for patients with lymphoid blastic phase of chronic myeloid leukemia. ASH Annual Meeting Abstracts 2012;120:3766.

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Research Article

DOI: 10.4274/Tjh.2013.0032

Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on Graft-Versus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation Ratlarda Kemik İliği Nakli sonrası gelişen Graft Versus Host Hastalığında Multipotent Mezenşimal Stromal Hücrelerin İmmünsupresif Etkileri Oral Nevruz1, Ferit Avcu1, A. Uğur Ural1, Aysel Pekel2, Bahar Dirican3, Mükerrem Safalı4, Elvin Akdağ5, Murat Beyzadeoğlu3, Tayfun İde5, Ali Sengül2 1Gülhane Medical Academy, Department of Hematology, Ankara, Turkey 2Gülhane Medical Academy, Department of Immunology, Ankara, Turkey 3Gülhane Medical Academy, Department of Radiation Oncology, Ankara, Turkey 4Gülhane Medical Academy, Department of Pathology, Ankara, Turkey 5Gülhane Medical Academy, Department of Medical Oncology Research Center, Ankara, Turkey

Abstract: Objective: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic bone marrow transplantation

(allo-BMT). While multipotent mesenchymal stromal cells (MSCs) demonstrate alloresponse in vitro and in vivo, they also have clinical applications toward prevention or treatment of GVHD. The aim of this study was to investigate the ability of MSCs to prevent or treat GVHD in a rat BMT model.

Materials and Methods: The GVHD model was established by transplantation of Sprague Dawley rats’ bone marrow and spleen cells into lethally irradiated (950 cGy) SDxWistar rat recipients. A total of 49 rats were randomly assigned to 4 study and 3 control groups administered different GVHD prophylactic regimens including MSCs. After transplantation, clinical GVHD scores and survival status were monitored.

Results: All irradiated and untreated control mice with GVHD died. MSCs inhibited lethal GVHD as efficiently as the standard GVHD prophylactic regimen. The gross and histopathological findings of GVHD and the ratio of CD4/CD8 expression decreased. The subgroup given MSCs displayed higher in vivo proportions of CD25+ T cells and plasma interleukin-2 levels as compared to conventional GVHD treatment after allo-BMT. Conclusion: Our results suggest that clinical use of MSCs in both prophylaxis against and treatment of established GVHD is effective. This study supports the use of MSCs in the prophylaxis and treatment of GVHD after allo-BMT; however, large scale studies are needed.

Key Words: Mezenchimal stromal cell, Bone Marrow Transplantation, İmmunsupresion Address for Correspondence: Oral NEVRUZ, MD Gülhane Medical Academy, Department of Hematology, Ankara, Turkey Phone: +90 312 304 41 04 E-mail: onevruz@hotmail.com Received/Geliş tarihi : January 28, 2013 Accepted/Kabul tarihi : April 29, 2013

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Nevruz O, et al: Effects of MSCs on GVHD

Özet: Amaç: Graft versus host hastalığı (GVHH) , başarılı bir kemik iliği nakli için önemli bir engel oluşturmaktadır. Multipotent mezenşimal stromal hücrelerin (MSH) immünsupresif etkileri, in vivo ve in vitro olarak gösterilmiş olmakla birlikte, GVHH’ nı önleme yönünde klinik uygulamalarda bulunmaktadır .

Gereç ve Yöntemler: Bu çalışmanın amacı ratlarda kemik iliği nakli sonrası oluşturulan GVHH’nı önleme ve tedavi etmede MSH nin etkinliğinin incelenmesidir. Bu amaçla 49 Sprague Dawley cinsi rat rastegele 4 çalışma, 3 kontrol grubuna ayrılmış ve gruplara MSH de içeren farklı GVHH önleyici tedaviler uygulanmıştır. Kemik iliği nakli sonrası GVHH skorlaması ve yaşama süreleri incelenmiştir.

Bulgular: Tüm ışınlanmış ve önleyici tedavi verilmemiş ratlar ölmüştür. MSH nin önleyici uygulamaları, standart GVHD önleyici tedavileri kadar etkin bulunmuştur. MSH uygulamaları, GVHH nın gözlemsel ve histolojik bulgularını ve CD4+/CD8+ oranını azaltmaktadır.Ayrıca MSH uygulanan gruplarda CD25+ T hücrelerinin in vivo oranıda daha yüksek olup, Allojeneik kemik iliği nakli sonrası standart GVHH tedavisi uygulananlara göre plazma İnterlökin-2 seviyesinin daha yüksek olarak saptanmıştır.

Sonuç: Bulgularımız MSH uygulamasının, GVHH nın hem önlenme hem de tedavi edilmesinde etkin olduğunu göstermiştir.

Ancak bu bulguların geniş ölçekli çalışmalarla desteklenmesi gerekmektedir.

Anahtar Sözcükler: Mezenkimal stromal hücre, Kemik iliği nakli, İmmünspresyon Introduction Allogeneic hematopoietic stem cell transplantation (alloHSCT) after high-dose marrow-ablative chemoradiotherapy is an effective treatment method in various hematologic, neoplastic, and congenital disorders. The major complication after allo-HSCT is the development of graft-versus-host disease (GVHD). GVHD is a life-threatening complication even when the major histocompatibility complex is matched [1,2]. Immunosuppressive therapy (i.e. cyclosporine and/ or steroids) is still the first-line treatment for established GVHD; however, the outcome for patients with steroidresistant acute GVHD is poor, as is overall survival [3].

Multipotent mesenchymal stromal cells (MSCs) are multipotent progenitor cells that can differentiate along multiple mesenchymal lineages including bone, cartilage, or fat and expand extensively in vitro [4,5]. The interest in MSC therapy has been raised by the observation that MSCs are able to modulate immune responses in vitro and in vivo [6]. MSCs display immunosuppressive properties that suppress the proliferation of T cells induced by alloantigens or mitogens [7]. Furthermore, MSCs have been reported to induce T cell division arrest, to inhibit the differentiation and maturation of dendritic cells, and to decrease the production of inflammatory cytokines by various immune cell populations [8,9,10]. These properties can be utilized in the context of allo-HSCT, particularly to modulate GVHD and graft rejection [6]. Therefore, MSCs can be thought of as promising agents for severe steroid-resistant acute GVHD and nonresponders can be treated with alternative methods, including MSCs (11).

The aim of this study was to evaluate the prophylactic and therapeutic potential of MSCs against GVHD using an established rat model of acute GVHD.

Materials and Methods Animals Female Wistar rats of 10-12 weeks old were used as recipients and male Sprague Dawley (SD) rats as donors. All procedures were performed according to the institutional guide for animal experimentation and the study protocol was approved by the institutional ethics committee.

Bone Marrow Preparation and Bone Marrow-Derived Rat MSC Generation Briefly, SD rats were sacrificed by decapitation and bone marrow (BM) was flushed with L-DMEM (Gibco, Grand Island, NY, USA) using a 23-gauge needle from femurs and tibias. The BM cells were then pelleted by centrifugation at 1000 rpm for 15 min. The BM cells were gently resuspended using an 18-gauge needle and filtered through a sterile nylon mesh. The viability was consistently >95% as determined by trypan blue exclusion.

For the MSC generation, BM cells were plated in 25cm2 polystyrene flasks in L-DMEM supplemented with 10% fetal bovine serum at 37 °C with 5% CO2 conditions (Gibco). Cells were allowed to adhere for 72 h followed by the removal of nonadherent cells and media were changed every 3 to 4 days. Adherent cells were detached using trypsin-EDTA solution-B (EDTA 0.05%, trypsin 0.25%, with phenol red; Biological Industries, Beit-Haemek, Israel) at 37 °C for 10 min and MSCs were expanded 3-4 times to achieve the desired cell numbers for use in in vitro and in vivo experiments.

Preparation of Viable Splenocytes SD rats weighing between 200 and 250 g were sacrificed by decapitation to be used as donors for splenocytes. After sterile splenectomy, spleens were collected and kept on ice with L-DMEM (Gibco) supplemented with penicillin (100 U/mL) 257

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and streptomycin (100 Âľg/mL). The spleens were disrupted in the medium by pressing spleen fragments between 2 glass slides. Spleen cell suspensions were filtered through a 100mesh nylon filter and washed 3 times with L-DMEM medium.

Splenocyte viability was consistently >95% as determined by trypan blue exclusion. Viable nucleated cells were counted and adjusted to 4.0x108/500 ÂľL. Allo-HSCT Procedure We generated a rat GVHD model in the context of alloHSCT with infusion of 4x108 donor splenocytes. Allo-HSCT was performed with isolation of 1x108 mononuclear cells from male SD rat donors, dilution in 0.5 mL of phosphatebuffered saline, and infusion to female Wistar rat recipients via the tail veins. The recipient animals were conditioned with a myeloablative regimen consisting of 950 cGy of total body irradiation prior to allo-HSCT. The GVHD prophylactic regimen was arranged as 3 mg/kg/day cyclosporine-A (CsA) and 0.25 mg/kg methotrexate (MTX) intraperitoneally at days +1, +3, and +6.

Experimental Design A total of 49 Wistar rats were enrolled in this study and they were randomly assigned to 4 study groups (SGs) and 3 control groups (CGs) (n=7 each). The GVHD model was generated in all SGs but only in one CG. 1. CG-I: Allo-HSCT and GVHD (enforced through donor splenocyte infusion).

2. CG-II: No allo-HSCT, myeloablative regimen only (total body irradiation).

3. CG-III: No allo-HSCT, no myeloablative regimen (no GVHD).

4. SG-I: Allo-HSCT, only standard GVHD prophylactic regimen (CsA+MTX) on day -1 of allo-HSCT. 5. SG-II: Allo-HSCT, given only MSCs (2x106 cells/kg) on day +1 after allo-HSCT. 6. SG-III: Allo-HSCT, given standard GVHD prophylactic regimen on day -1 of allo-HSCT plus MSCs (2x106 cells/kg) on day +1 of allo-HSCT. 7. SG-IV: Allo-HSCT, given MSCs (2x106 cells/kg) after observation of GVHD findings.

The rats were observed daily for clinical signs of GVHD, such as diffuse erythematous lesions (particularly of the ears and extremities), hyperkeratosis of the footpads, skin rash, diarrhea, anorexia, and weight loss (Figure 1).

Immunophenotypical (CD4, CD8, CD25, and plasma IL-2 levels) examinations and histopathological findings of GVHD (Table 1; Figure 2) following allo-HSCT were performed, and the survival of all groups was monitored.

Statistical Analysis To evaluate the effects of several variables on overall survival, Kaplan-Meier survival analysis (log-rank statistics) was performed. Differences among the treatment groups were assessed by nonparametric Mann-Whitney U tests.

Analyses were managed with SPSS 10.0 (SPSS Inc., Chicago, IL, USA) and the significance level was 5% (p<0.05).

Table 1. GVHD-related pathological findings of groups

Groups

Hepatic

(%)

Intestinal (n)

Dermatological (n)

BMT+GVHD+MSC

Damage of bile duct Periportal inflammation

(14.3) (42.9)

Cryptitis GI G II G III

(1) (2) (1)

G I: Focal/diffuse vacuolization of basal-layer (1) G II: Subepidermal infiltration of lymphocytes (1)

Mild portal inflammation

(42.9)

Cryptitis GI G II G III

(2) (2) (1)

Cryptitis GI G II

(1) (2)

Cryptitis GI

(3)

Cryptitis GI G II G III G IV

(1) (2) (3) (1)

CsA+MTX

MSC

CsA+MTX+MSC

BMT-CONTROL

258

Mild portal inflammation

(28.6)

Mild damage of hepatocytes Periportal inflammation

(28.6)

Damage of bile duct Cytoplasmic vacuolization Necrosis of hepatocytes Periportal inflammation

(28.6) (42.9) (14.3) (57.2)

G I: Focal/diffuse vacuolization of basal layer (3)

G I: Focal/diffuse vacuolization of basal layer (3) G I: Focal/diffuse vacuolization of basal layer (2) G I: Focal/diffuse vacuolization of basal-layer (4) G II: Spongiose+dyskeratotic keratinocyte, lymphocytes infiltration (3) G III: Subepidermal separation (1)

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70

Kaplan-Meier survival analyses 1.2 1.0

p<0.05*

8

GROUPS

p<0.05 and p<0.05*

6

RAD control H. control CsA+Mtx+MSC

4

MSC CsA+MSC

2 0.0

Figure 1. The weight changes of the study and control group rats during the experiment.

BMT+MSC 0

10

20

30

40

50

60

80

90 100

BMT+CONTROL

Survi

Figure 3. Kaplan-Meier survival curves of the study and control group rats (*: statistical significance in comparison with BMT-control). all groups following allo-HSCT. The expression of CD25 increased significantly (p<0.001) in all SGs and CG-I compared to CG-III. Intergroup comparisons of all SGs and CG-I in terms of CD25 expression were not statistically significant (Figure 5). The ratio of CD4/CD8 expression was significantly lower in SG-I (p=0.008) than in CG-III. In contrast, this expression was significantly higher in SG-II and SG-IV (p=0.014 for both) compared to CG-III (Figure 6). Plasma IL-2 levels were significantly increased in SG-I (p=0.032), SG-II (p=0.018), and SG-IV (p=0.032) compared to CG-I. This increase was much more prominent (p<0.001) in CG-I than in CG-III (Figure 7).

Figure 2. GVHD-related histopathological examination of groups. a, b, c: Hepatic pathology of GVHD (a: healthy control, 50x; b: BMT-control-GVHD, 100x; c: BMT-controlGVHD, 200x). d, e, f: Intestinal pathology of GVHD (d: healthy control, 50x; e: BMT-control-GVHD, 25x; f: BMTcontrol-GVHD, 200x); g, h, i: Dermatological pathology of GVHD (g: healthy control, 50x; h: BMT-control-GVHD, 100x; i: BMT-control-GVHD, 200x).

Results Wistar recipients from male SD donors were transplanted with allogeneic hematopoietic stem cells (allo-HSC) or MSCs as described above. The survival of the Wistar rats as shown in the Kaplan-Meier survival curve following allo-HSCT (Figure 3) was significantly longer in SG-I, SG-II, and SG-IV than in CG-I (p<0.05). However, the survival of SG-III was not significantly longer than in CG-I. The semiquantitative clinical scoring scale showed significant differences of the severity of GVHD (Figure 4). Clinical signs and symptoms of GVHD were significantly lower in SG-I and SG-II than in CG-I (p<0.05 and p<0.05, respectively). However, SGIII and SG-IV were not significantly different from CG-I (p>0.05 and p>0.05, respectively).

Statistical evaluation was performed on day 28.

Immunophenotypical (CD45, CD4, CD8, and CD25) examinations and plasma IL-2 levels were performed for

Discussion Allo-HSCT is an increasingly used treatment modality for hematological malignancies [2]. However, GVHD is a life-threatening complication of allo-HSCT caused by donor lymphocytes reacting against host tissues and is a major contributor to morbidity and mortality associated with this procedure [1,12]. Several studies have suggested that MSCs could exert immunomodulatory properties to reduce the incidence of GVHD after allo-HSCT. GVHD can be readily controlled by escalation of systemic immunosuppression (prophylactic regimens or systemic high-dose steroids) in up to 70% of patients [13,14]. However, patients whose GVHD is refractory to this therapy have a poor prognosis.

There is no standard second-line or salvage therapy for these patients and various therapeutic modalities have been administered. However, the results so far have demonstrated limited efficacy and low long-term survival due to toxicity [15,16,17,18,19].

Administration of MSCs is an alternative option in the treatment of steroid-refractory GVHD. Immunomodulatory properties of MSCs have been exploited to reduce the incidence of GVHD after allo-HSCT [10,11,21,22,23,24,25].

First, the effectiveness of MSCs on GVHD was supported by in vitro studies. Ning et al. first reported the effectiveness of human BM MSCs on allogeneic T lymphocyte phenotype in vitro (26). In vivo studies have also supported the effectiveness 259

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Nevruz O, et al: Effects of MSCs on GVHD

of MSCs on GVHD. Tian et al. reported the mechanisms a) Diarrhea Alopecia Weight loss responsible for GVHD in the setting of co-transplantation of No No created a0 model No of acute 0GVHD allo-HSC and0MSCs. They in rats transplanted with allo-HSC plus donor-derived T Yes 1 Yes 1 yes 1 cells, with or without additional donor-derived MSC coSevere 2 They Severe transplantation. suggested 2 that BM-derived MSCs

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4

b)

BMT CONTROL BMT + MSC

3

Scale of GVHD

2.5

CyS-Mtx MSC

2

0

7

14 21 28 35

42 49

56 63

70

77 84

91 100

Figure 4. a) Evaluation of the severity of GVHD with the semiquantitative clinical scoring scale utilizing diarrhea, alopecia, and weight changes over the experiment period; b) degree of GVHD in the study and control group rats.

120 100 80 60

CD45

40

CD25 CD4

20

T BM

OL TR N CO

T BM

+

SC M

tx M A+ s C

OL SC TR SC M N + M tx CO M H. A+ s C

CD8

Figure 5. Results of immunophenotypical analyses (CD45, CD4, CD8, CD25) in the study and control groups: CD25 was significantly increased (p<0.001) in all study groups and was higher in CG-I than in CG-III. *: p<0.001 in comparison with healthy control (SG-I).

can prevent lethal GVHD following allo-HSCT by means of homeostasis of T lymphocyte subsets in vivo. Their study demonstrated that the value of CD8+ and CD4+ T cells and the ratio of Th1/Th2 T cell subsets decreased at the same time, so the proportion of CD4+, CD25+ T cells increased both in spleen lymphocytes and thymocytes in vivo after allo-HSCT with MSC co-transplantation [27]. Our results, too, suggest that BM-derived MSCs can prevent GVHD after allo-HSCT by means of homeostasis of T subsets in vivo.

We also report a decreased ratio of CD4/CD8 expression, along with an increased proportion of CD25+ T cells and plasma interleukin-2 levels in vivo after allo-HSCT with prophylactic MSC administration. We also determined the highest level of CD8 in the CsA+MTX group. The highest level of CD8 in the CsA+MTX group may have an additional vrole in the treatment of GVHD. Recent studies particularly suggested inducible CD8 cells to be useful in suppressing autoimmune reactions, although their function in the alloSCT setting has not been fully explored [28].

260

BMT CONTROL

BMT + MSC

CsA + Mtx

MSC

CsA+Mtx+MSC

H. CONTROL

CONTROL

Days

0

0

CyS+Mtx+MSC

1.5 1 0.5 0

0.2

Figure 6. The ratio of CD4/CD8 expression in the study and control groups. The ratio of CD4/CD8 expression was significantly increased in the MSC groups, but rather decreased in the CsA+MTX group in comparison with the healthy control. * p=0.008 in comparison with CG-III (healthy control). ** p=0.014 in comparison with CG-III (healthy control).

MSCs may be used for hematopoiesis enhancement, GVHD prophylaxis, and treatment of established severe acute GVHD in allo-HSCT patients. Previous studies have supported the use of MSCs in steroid-refractory GVHD [11,21,22,23,24,27]. Intravenous administration of MSCs has been well tolerated [29]. In 1994, Le Blanc et al. reported a case with successful treatment of grade IV acute GVHD of the gut and liver with third-party haploidentical MSCs. They postulated that MSCs have a potent immunosuppressive Veffect in vivo [30]. In 1998, the same researchers published the MSC treatment of steroid-resistant, severe, acute, and chronic GVHD as a phase II study [22]. Herrmann et al. reported a phase I study that was applied to MSC therapy for steroid-refractory acute and chronic GVHD. They administered 2 infusions per patient and the overall response rates for acute GVHD were complete in 7 and partial in 4, with no response in 1 patient. Of the 7 patients who achieved a complete response, 6 were still alive [31].

The combination of cyclosporine and short course of methotrexate is currently considered the standard prophylaxis of GVHD [32]. There is sufficient in vitro evidence to support the use of MSCs in the prevention and treatment of GVHD. However, it has been rarely reported that MSCs were very effective for GVHD prevention in vivo but not in the treatment of GVHD in the xenogeneic model of NOD/SCID mice. In addition, these studies reported no adverse events following the infusion of MSCs, making it possible to use these cells for prevention of acute GVHD [33,34]. Tisato et al. designed a study to address these questions in a xenogeneic model testing the ability of umbilical cord blood-derived MSCs to prevent and/or treat GVHD. They reported that MSCs of cord-blood origin are effective in the prevention but not the treatment of GVHD [33]. Another study depicted that MSCs suppress the lymphocyte proliferation in vitro but fail to prevent GVHD in mice (34). However, our study evaluated the clinical potential of MSCs for controlling GVHD in rats. According to our results, prophylactic in vivo use of MSCs was as

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Turk J Hematol 2013;30:256-262

References

300

Levels of IL-2 (pg/ml)

250

1. Ferrara JL, Deeg HJ. Graft-versus-host disease. N Engl J Med 1991;324:667-674.

200 150

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100 50 0

BMT CONTROL

BMT - MSC

CsA+Mtx

MSC

CsA+Mtx+MSC H.CONTROL

Figure 7. Plasma IL-2 levels (pg/mL) in the study and control groups. Statistical significance in comparison with the healthy control is shown. * p=0.032 in comparison with CG-III (healthy control). ** p=0.018 in comparison with CG-III (healthy control). *** p<0.001 in comparison with CG-III (healthy control).

effective as the standard prophylactic regimen in preventing GVHD. Furthermore, we did not observe any adverse events following the infusion of MSCs. Until recently, there were no published data regarding the preferred dose, timing, and frequency of MSC infusion. However, a randomized controlled phase III trial on the use of MSCs in acute GVHD in humans is currently underway, and the preliminary results are promising [35]. Recently, Kuzmina et al. reported a phase II human study on the use of MSCs for prevention of acute GVHD. This prospective clinical trial was based on random patient allocation to two groups receiving either standard GVHD prophylaxis or standard GVHD prophylaxis combined with MSC infusion. They demonstrated the efficacy of MSCs in GVHD prophylaxis in a limited number of patients with no adverse events directly attributable to administration of MSCs (36). However, our in vivo rat study displayed similar efficacies for both the standard GVHD prophylactic regimen (CsA+MTX) and the prophylactic MSC regimen alone. The immunosuppressive effect of combination of standard GVHD prophylaxis (CsA+MTX) and MSC infusion was also more potent. Although MSC treatment was tolerated well, this potent immunosuppressive effect of the CsA+MTX+MSC combination was associated with increased mortality in our study. The potent immunosuppressive effect of CsA+MTX+MSC combination is to be proven by further studies.

To conclude, clinical use of MSCs in both prophylaxis against and treatment of established GVHD seems to be effective. However, MSC infusion combined with the standard GVHD prophylactic regimen causes stronger immunosuppression, with the potential of resultant early mortality. Clinical use of MSCs in prophylaxis and treatment of GVHD after allo-BMT requires further clinical trials.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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14. Van Lint MT, Milone G, Leotta S, Uderzo C, Scimè R, Dallorso S, Locasciulli A, Guidi S, Mordini N, Sica S, Cudillo L, Fagioli F, Selleri C, Bruno B, Arcese W, Bacigalupo A. Treatment of acute graft-versus-host disease with prednisolone: significant survival advantage for day +5 responders and no advantage for nonresponders receiving anti-thymocyte globulin. Blood 2006;107:4177-4181.

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Research Article

DOI: 10.4274/Tjh.2013.0027

Regulation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Expression in Primary Acute Leukemic Cells by Chemotherapeutics Primer Akut Lösemi Hücrelerinde, Kemoterapötiklerin Tümör Nekrozis Faktör Alakalı Apoptozis Indükleyici Ligand Ekspresyonunu Düzenlemesi Shengmei Chen, Yanfang Liu, Hui Sun, Ling Sun, Jie Ma, Dingming Wan, Zhongxing Jiang, Qiutang Zhang, Tao Li Key Observation Laboratory in Henan Medical University, Department of Hematology First Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan Province, P.R. China

Abstract: Objective: The expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein and its regulation by chemotherapeutics were analyzed in primary acute leukemic cells.

Materials and Methods: Peripheral blood was collected from 16 patients with acute leukemia on days 0, 1, 3, and 5 of chemotherapy. The mononuclear cells were separated from the peripheral blood, and TRAIL expression was assessed by flow cytometry. The bone marrow mononuclear cells of patients with acute leukemia were separated before chemotherapy and cultured in vitro with VP-16 and/or interferon (IFN). The TRAIL expression level was detected after the cell culture.

Results: TRAIL expression in the mononuclear cells of peripheral blood was significantly upregulated on day 1 (p<0.05) and then significantly decreased on day 5 after chemotherapy (p<0.05). Results from the in vitro culture revealed that VP-16 upregulated TRAIL expression in the bone marrow mononuclear cells of patients with acute leukemia, but the binding of VP-16 to IFN did not enhance TRAIL expression as compared with VP-16 alone (p>0.05).

Conclusion: OA single chemotherapy mechanism for leukemia may suffice to induce TRAIL expression and promote the apoptosis of leukemic cells.

Key Words: TRAIL, acute leukemia, chemotherapeutics

Özet: Amaç: Primer akut lösemi hücrelerinde, tümör nekrozis faktör alakalı apoptozis indükleyici ligand (TRAIL) protein ekspresyonu ve bunun kemoterapötikler yoluyla kontrolü analiz edildi.

Address for Correspondence: Shengmei CHEN, M.D., Department of Hematology, First Affiliated Hospital, Zhengzhou University, Key Observation Laboratory in Henan Medical University, Zhengzhou 450052, Henan Province, P.R. China Phone: +86-0371-66295132 E-mail: shengmeichen@163.com Received/Geliş tarihi : 29 March, 2013 Accepted/Kabul tarihi : 26 January, 2013

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Turk J Hematol 2013;30:263-268

Gereç ve Yöntemler: 16 akut lösemi hastasından, kemoterapinin 0, 1, 3 ve 5. günlerinde periferik kan örnekleri toplandı.

Mononükleer hücreler periferik kandan ayrılarak akım sitometrik olarak TRAIL ekspresyonu değerlendirildi. Akut lösemili hastaların kemik iliği mononükleer hücreleri kemoterapi öncesi ayrılarak, VP-16 ve/veya interferon (IFN) ile kültive edildi.

TRAIL ekspresyonu, hücre kültüründen sonra çalışıldı.

Bulgular: Periferik kandan elde edilen mononükleer hücrelerdeki TRAIL ekspresyonu 1. günde (p<0.05) anlamlı olarak yüksekti ve kemoterapi sonrası 5. gündeyse belirgin olarak düşük saptandı (p<0.05). In vitro kültür sonuçları; VP-16’nın akut lösemili hastaların kemik iliği mononükleer hücrelerindeki TRAIL ekspresyonunu arttırdığını ve fakat tek başına VP-16 ile karşılaştırıldığında IFN ile kombine VP-16’nın TRAIL ekspresyonunu arttırmadığını göstermiştir (p>0.05).

Sonuç: Lösemide tekli kemoterapi mekanizmasının TRAIL’i indüklemek ve lösemik hücreleri apoptozise yönlendirmek için yeterli olduğu görülmüştür.

Anahtar Sözcükler: TRAIL, akut lösemi, kemoterapötikler Introduction The process of programmed cell death or apoptosis has a key effect on the occurrence, development, and stability of an organism. This process also has an important role in the inhibition of tumor cell growth and immune surveillance.

Aside from the tumor necrosis factor (TNF) and the Fas ligand (FasL), the TNF-related apoptosis-inducing ligand (TRAIL), also known as the apoptosis 2 ligand or Apo2L [1], is a member of the TNF superfamily that induces apoptosis by binding to specific death receptors and activating multiple apoptotic signaling pathways [2]. TRAIL induces apoptosis in tumor cells but has low sensitivity to normal cells [3]. Various key cells of the immune system for tumor surveillance partially depend on TRAIL expression to perform cytotoxic effects and inhibit the occurrence and migration of tumors; these cells include the CD4+ T cells, natural killer (NK) cells, macrophages, and dendritic cells [4]. TRAIL can induce the apoptosis of malignant leukemia cells without harming normal cells [5]. TRAIL expression can also induce the differentiation of surviving leukemic cells and normal myeloid precursors into mature monocytoids and regulate normal hematopoiesis [6]. Liu et al. [7] detected the expression of TRAIL and its receptors in acute myeloid leukemic cells and discovered the high expression of TRAIL, DR4, and DR5 in patients. However, the effects of chemotherapeutics on TRAIL expression in primary acute leukemic cells are rarely reported. Our study used flow cytometry to detect TRAIL expression after incubating acute leukemic cells with chemotherapeutics in vivo and in vitro. We then investigated the significance of TRAIL expression in chemotherapy for acute leukemia. The effects of TRAIL-related drugs were likewise studied. Our study provides a scientific basis for the clinical application of TRAIL. Materials and Methods Subjects and Specimen Processing Blood samples were obtained from 16 patients diagnosed with acute leukemia based on their cytomorphology, 264

histochemistry, and cellular immunology. Peripheral blood was collected from the patients on days 0, 1, 3, and 5 of chemotherapy. Heparin was added to the samples for anticoagulation. Mononuclear cells were separated using a lymphocyte separation medium (Ficoll; density, 1.007) by a standard method, and the concentration of the cells was adjusted to 1x107 cells/L. Bone marrow (10 mL per patient) was collected from 12 patients before chemotherapy.

Mononuclear cells were then separated and cultured in a medium containing VP-16 and interferon (IFN). The cells were incubated for 0, 24, 48, and 72 h before they were collected. The final cell concentration was adjusted to 1x107 cells/L with phosphate-buffered saline (PBS; BD, USA). This study was conducted in accordance with the Declaration of Helsinki and with approval from the Ethics Committee of the First Affiliated Hospital, Zhengzhou University. Written informed consent was obtained from all participants.

Cell Culture The acute leukemic mononuclear cells cultured with VP16 and IFN in medium (cell concentration, 1x107 cells/L) were divided into 4 groups: the control group, VP-16 group (final VP-16 concentration, 50 µg/mL), IFN group (final IFN concentration, 2000 U/mL), and VP-16+IFN group (same final concentrations as in the VP-16 and IFN groups).

The cells were incubated under saturated humidity at 37 °C in an incubator with 5% CO2.

Detection of TRAIL Expression by Flow Cytometry The initial and cultured cells were divided into 2 tubes each. The first tube had CD45 monoclonal antibodies alone added, whereas the second one contained both CD45 and TRAIL monoclonal antibodies. The TRAIL antibodies were diluted 20-fold in PBS prior to use. Each tube then had 100 µL of cell suspension added. The tubes were incubated in the dark at room temperature for 30 min and washed with PBS 3 times. Afterwards, TRAIL expression was detected by flow cytometry (FACstar; BD, USA).

The detection results were presented as mean fluorescence intensity (MFI). The MFI of the total mononuclear cells and leukemic cells were recorded as T-MFI and L-MFI, respectively.

Chen S, et al: Regulation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Expression in Primary Acute Leukemic Cells by Chemotherapeutics

Statistical Analysis The statistical significance of the data was determined using SPSS 10.0. Data were presented as the mean ± standard deviation. The comparison between groups was performed using the paired Student’s t-test. Differences were considered statistically significant at p<0.05.

Results TRAIL Expression during Chemotherapy The T-MFI of TRAIL in patients with initial acute leukemia was significantly increased after chemotherapy, as compared with that before chemotherapy (p<0.01), and then it gradually decreased. The TRAIL expression on day 5 after chemotherapy was significantly decreased as compared with that on day 1 (p<0.05). The L-MFI of TRAIL in patients was distinctly higher on day 1 after chemotherapy than that before the chemotherapy (p<0.01). No significant differences in the TRAIL expression were found among the other days after chemotherapy (p>0.05; Table 1).

Effect of Chemotherapeutics on the TRAIL Expression in Acute Leukemic Bone Marrow Cells in Vitro The T-MFI of TRAIL was enhanced at 24 and 48 h after incubation with both VP-16 and IFN (p<0.05), as compared with that before incubation (p<0.05). L-MFI significantly increased at 24 h after incubation (p<0.01; Table 2).

Influence of Different Drugs on TRAIL Expression

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Treatment with VP-16 and the combination of VP-16 with IFN upregulated TRAIL expression in the bone marrow mononuclear cells of patients with acute leukemia at 24 h after the treatment, as compared with that in the control group (p<0.05). IFN induced TRAIL expression in leukemic cells, but its effect was not statistically significant (p>0.05).

The TRAIL expression level induced by IFN was distinctly different from that induced by the combination of IFN and VP-16. No pronounced changes in the TRAIL expression level were observed between the incubation with VP-16 alone and the drug combination (p>0.05; Table 3).

Discussion Plasilova et al. [5] reported that TRAIL can induce the apoptosis of leukemic cells such as K562, HL-60, and ML-1 in a dose-dependent manner. TRAIL reduces the formation of myeloid colony forming unit-granulocyte/macrophage (CFU-GM) colonies in patients with acute non-lymphocytic leukemia (ANLL), chronic myeloid leukemia (CML), and myelodysplastic syndromes, as well as inhibits cell growth.

However, the growth and proliferation of selected cells are not affected by TRAIL expression, which includes the cells in ANLL patients with completely remitted blood, cells in lymphoma patients with unaffected bone marrow, normal bone marrow precursors, and normal umbilical cord blood precursors. Lee et al. [8] treated a mixture of cord blood mononuclear cells and Jurkat cells with TRAIL. They found that Jurkat cells are specifically eliminated without

Table 1. The TRAIL expression in primary acute leukemic cells during chemotherapy.

Chemotherapy n

Day 0 16

Day 1 16

Day 3 15

Day 5 14

T-MFI

3.20±0.76

3.86±0.88*

3.91±1.22

3.29±0.88#

L-MFI

3.59±1.23

4.05±1.53*

4.34±1.77

3.91±1.31

*P < 0.05, compared with day 0; # P < 0.05, compared with day 1.

Table 2. The TRAIL expression in acute leukemic cells treated with VP-16 and INF for different time.

Culture with drug n

0h 7

24 h 7

48 h 6

72 h 4

T-MFI

3.44±1.12

5.12±1.20*

6.42±2.73

6.20±3.32

L-MFI

3.39±0.94

5.38±1.01*

5.26±1.97

4.04±1.36

*P < 0.05, compared with 0h

Table 3. The TRAIL expression in acute leukemic cells treated with different drugs.

Drug n

Control 12

VP-16 12

INF 12

VP-16 and INF 12

T-MFI

5.05±0.89

6.32±1.68*

5.35±1.28#

6.74±2.15*

L-MFI

5.02±1.13

6.06±1.73*

5.41±1.40#

6.36±1.55*

*P < 0.05, compared with control; # P < 0.05, compared with VP-16 and INF

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decreasing the number of normal CFU-GM colonies. A previous study showed that most Ph(+) leukemic cells are resistant to the apoptosis induced by FasL but highly sensitive to TRAIL [9]. The death receptors DR4 and DR5 are expressed on the surface of these TRAIL-sensitive cells.

However, these receptors are expressed in low levels or are absent in TRAIL-resistant cell lines. Therefore, the sensitivity of leukemic cells to TRAIL depends on the expression of death receptors on their surfaces. The induction of apoptosis has an important role in monitoring tumor immunity and chemotherapy by increasing the expression of TRAIL and its specific death receptors as well as their interaction. In this study, we utilized flow cytometry to investigate the effect of chemotherapy on TRAIL expression in the leukemic cells of patients with initial acute leukemia. The results showed that the T-MFIs of TRAIL on days 0, 1, 3, and 5 after chemotherapy were 3.20±0.76, 3.86±0.88, 3.91±1.22, and 3.29±0.88, respectively. T-MFI significantly increased on day 1 after chemotherapy as compared with that before chemotherapy (p<0.01). However, no obvious differences in TRAIL expression were found between day 0 and days 3 or 5. The TRAIL expression level on day 5 after chemotherapy was significantly decreased as compared with that on day 1 after chemotherapy (p<0.05). No significant differences in TRAIL expression were observed among the other days (p>0.05). The L-MFIs of TRAIL in acute leukemia patients were 3.59±1.23, 4.05±1.53, 4.34±1.77, and 3.91±1.31 on days 0, 1, 3, and 5, respectively. L-MFI was significantly higher on day 1 after chemotherapy than before the chemotherapy (p<0.01). No significant differences in TRAIL expression were found among the other days (p>0.05).

Chemotherapeutics function by inducing the apoptosis of leukemic cells. TRAIL is a type II transmembrane protein that induces apoptosis, which maintains cell homeostasis in normal cells and has cytotoxic effects on tumor cells.

This study reveals that chemotherapeutics could promote TRAIL expression in leukemic cells. TRAIL expression eventually decreased with prolonged chemotherapy. Thus, chemotherapy may have improved TRAIL expression, which induced the apoptosis of the acute leukemic cells.

Previous studies showed that IFN-α can upregulate TRAIL expression on the surfaces of active T cells [9]. Allogeneic stem-cell transplantation (Allo-SCT), as a curative treatment option for acute leukemia and chronic granulocytic leukemia, mainly eliminates leukemic cells via the immune-mediated graft-versus-leukemia (GVL) effect. Shlomchik and Pear [10] reported that the GVL effect is not reduced in Fasdeficient mice, which implies that the TRAIL/death receptor interaction might influence the elimination of immunemediated CML malignant cloning. Furthermore, FasL/Fas interaction is not the main signaling pathway for inducing apoptosis. Wen et al. [11] revealed that anti-leukemia drugs such as Ara-C and daunorubicin promote DR5 expression in leukemic cell lines and induce a cytotoxic effect. This study 266

investigated TRAIL expression in mononuclear cells of acute leukemia patients in complte remission. We discovered that TRAIL expression was significantly increased on day 1 after chemotherapy as compared with that before the chemotherapy (p<0.05). However, no obvious differences in TRAIL expression were found between day 0 and days 3 or 5. The TRAIL expression level on day 5 after chemotherapy was significantly decreased as compared with that on day 1 after chemotherapy (p<0.05). No significant difference in TRAIL expression was found among the other days studied (p>0.05). However, the TRAIL expression in leukemic cells and CD8+ T cells was not significantly changed (p>0.05).

These results demonstrated that TRAIL expression did not increase in leukemic cells and CD8+ T cells of patients in complete remission. The elimination of residual leukemic cells did not rely on TRAIL expression during the consolidation phase of treatment. The chemotherapy enhanced TRAIL expression in all mononuclear cells. We hypothesize that the increased TRAIL expression was not observed in the leukemic and CD8+ T cells, but it was the case in other mononuclear cells. The chemotherapy may have partially depended on the TRAIL expression of immune cells such as NK cells and macrophages to kill the residual leukemic cells. Subsequently, TRAIL binds to the death receptors to activate the apoptosis signaling pathway.

The combination of Ara-C and IFN-α remarkably increases the remission rate of CML patients and prolongs patient survival [12]. Kanako compared the anti-leukemia effect between TRAIL and STI-571 and found that malignant myeloid cells, as well as a type of malignant lymphocyte, are the most sensitive to STI-571 among the 8 types of malignant lymph cell lines. Four naturally STI-571-resistant cell lines are sensitive to TRAIL, whereas 5 STI-571-sensitive cell lines are resistant to TRAIL. However, all the tested cell lines are highly sensitive to the combination of TRAIL and STI-571. In our study, the bone marrow mononuclear cells of patients with acute leukemia were treated with VP16 and IFN. The T-MFIs of TRAIL were 3.44±1.12, 5.12±1.20, 6.42±2.73, and 6.20±3.32 at 0, 24, 48, and 72 h of treatment, respectively. The T-MFI of TRAIL was enhanced at 24 and 48 h after incubation with VP-16 and IFN (p<0.05), as compared with that before incubation (p<0.05). By contrast, L-MFI was 3.39±0.94, 5.38 ± 1.01, 5.26±1.98, and 4.04±1.36 at 0, 24, 48, and 72 h of incubation, respectively. L-MFI was significantly increased at 24 h after incubation (p<0.01). No significant differences in L-MFI were found among the other treatment times (p>0.05).

VP16 is a topoisomerase II inhibitor commonly used in chemotherapy protocols for acute leukemia to induce the apoptosis of leukemic cells. IFN is an antitumor cytokine involved in immune regulation by inhibiting cell hyperplasia.

The combination of these 2 drugs can enhance TRAIL expression in leukemic cells, which is consistent with the observation that chemotherapy promotes TRAIL production in leukemic cells. These drugs can kill leukemic cells and induce apoptosis by promoting TRAIL expression.

Chen S, et al: Regulation of Tumor Necrosis Factor-related Apoptosis-inducing Ligand Expression in Primary Acute Leukemic Cells by Chemotherapeutics

To further study the effects of these drugs, the cells were divided into 4 groups according to drug treatment. The results showed that the T-MFIs of TRAIL in the control, VP-16, IFN, and VP-16+IFN groups were 5.05±0.89, 6.32±1.68, 5.35±1.28, and 6.74±2.15, respectively, with the corresponding L-MFIs of 5.02±1.13, 6.06±1.73, 5.41±1.40, and 6.36±1.55. VP-16 as well as the combination of VP16 with IFN upregulated TRAIL expression in the bone marrow mononuclear cells of patients with acute leukemia compared with that in the control group (p<0.05). However, IFN alone did not cause any significant change (p>0.05).

The TRAIL expression level induced by IFN was distinctly different from that induced by the combination of IFN and VP-16 (p<0.05). No obvious changes in the TRAIL expression levels were observed between the incubation with VP-16 alone and the drug combination (p>0.05). This result strongly suggested that TRAIL expression is primarily regulated by V16 but not by IFN. This observation is not consistent with the published literature, which indicates that IFN-α can upregulate TRAIL expression on the T cell surface. This difference may be related to the different culture systems used in our experiments. Furthermore, the mononuclear cells cultured in our experiment were primary leukemic cells, with only a small proportion of immune cells. Thus, IFN alone had a weak effect on inducing TRAIL expression and promoting apoptosis.

It is reported that IFN and its therapeutic applications have serious side effects, such as the lethal graft-versushost disease effect of Allo-SCT and resistance to STI-571 in CML treatment. These side effects limit the effectiveness and persistence of these therapeutic option [13]. Only 2 out of 19 acute myeloid leukemia cases demonstrated leukemia cell apoptosis (>10%) after TRAIL treatment on mononuclear cells alone. However, the effect increases when TRAIL treatment is combined with fludarabine, cytarabine, or daunorubicin. Nearly half of the mononuclear cells underwent apoptosis under this treatment, and the proapoptotic effect was as strong as that of caspase-8.

Conticello et al. [14] confirmed that bortezomib with TRAIL induces the expression of TRAIL and its receptor to improve the sensitivity to the treatment. With the increased binding of TRAIL and the death receptors, more leukemia cells are subjected to apoptosis. TRAIL is characterized by a specific anti-leukemia effect, which makes it a promising therapy for leukemia. The role of TRAIL receptors in the TRAIL-induced apoptotic pathway still needs to be further explored. Given the limited number of reports to date, more cases are necessary to confirm the conclusions drawn from this study.

Various forms of recombinant TRAIL proteins have been successfully developed. Human Jurkat cells are more sensitive to the recombinant leucine zipper–TRAIL [15], which induces the apoptosis of human breast cancer MDA231 cells without harming normal breast epithelium cells

Turk J Hematol 2013;30:263-268

and other normal tissues. The continuous and systematic injection of excess TRAIL to non-human primates does not change the clinical and histopathological index. This finding demonstrates the safe application of TRAIL in primates, which indicates progress in the eventual application of TRAIL in humans. Ichikawa et al. [16] produced a specific DR5 monoclonal antibody, TRA-8, which is sensitive to primary and metastatic liver cells and specifically binds to DR5 without inducing the apoptosis of normal liver cells.

In vitro and in vivo animal experiments show the cytotoxic effect of other proteins, such as the recombinant isoleucine zipper–TRAIL (hFlex-TRAIL) [17] and the plasmid pRevTRE-TRAIL [18]. We think that the clinical application of recombinant human TRAIL to human malignant tumors, including leukemia, can enhance the sensitivity of chemotherapeutics and increase the GVL effect of Allo-SCT, as well as reduce the required drug dose and the side effects, thereby effectively improving the leukemia treatment and patient prognosis.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673682.

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3. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi AControl of TRAILinduced apoptosis by a family of signaling and decoy receptors. Science 1997;277:818-821.

4. Taniguchi T, Ogasawara K, Takaoka A, Tanaka N. IRF family of transcription factors as regulators of host defense. Annu Rev Immunol 2001;19:623-655.

5. Plasilova M, Zivny J, Jelinek J, Neuwirtova R, Cermak J, Necas E, Andera L, Stopka T. TRAIL (Apo2L) suppresses growth of primary human leukemia and myelodysplasia progenitors. Leukemia 2002;16:67-73.

6. Secchiero P, Gonelli A, Mirandola P, Melloni E, Zamai L, Celeghini C, Milani D, Zauli G. Tumor necrosis factor-related apoptosis-inducing ligand induces monocytic maturation of leukemic and normal myeloid precursors through a caspase-dependent pathway. Blood 2002;100:2421-2429.

7. Liu Z, Xu X, Hsu HC, Tousson A, Yang PA, Wu Q, Liu C, Yu S, Zhang HG, Mountz JD. CII-DC-AdTRAIL cell gene therapy inhibits infiltration of CII-reactive T cells and CIIinduced arthritis. J Clin Invest 2003;112:1332-1341.

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8. Lee NS, Cheong HJ, Kim SJ, Kim SE, Kim CK, Lee KT, Park SK, Baick SH, Hong DS, Park HS, Won JH. Ex vivo purging of leukemia cells using tumor-necrosis-factor-related apoptosis-inducing ligand in hematopoietic stem cell transplantation. Leukemia 2003;17:1375-1383.

9. Uno K, Inukai T, Kayagaki N, Goi K, Sato H, Nemoto A, Takahashi K, Kagami K, Yamaguchi N, Yagita H, Okumura K, Koyama-Okazaki T, Suzuki T, Sugita K, Nakazawa S. TNF-related apoptosis-inducing ligand (TRAIL) frequently induces apoptosis in Philadelphia chromosome-positive leukemia cells. Blood 2003;101:3658-3667.

10. Matte CC, Cormier J, Anderson BE, Athanasiadis I, Liu J, Emerson SG, Pear W, Shlomchik WD. Graft-versus-leukemia in a retrovirally induced murine CML model: mechanisms of T-cell killing. Blood 2004;103:4353-4361.

11. Wen J, Ramadevi N, Nguyen D, Perkins C, Worthington E, Bhalla K. Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute leukemia cells. Blood 2000;96:3900-3906.

12. Guilhot F, Chastang C, Michallet M, Guerci A, Harousseau JL, Maloisel F, Bouabdallah R, Guyotat D, Cheron N, Nicolini F, Abgrall JF, Tanzer J. Interferon alfa-2b combined with cytarabine versus interferon alone in chronic myelogenous leukemia. French Chronic Myeloid Leukemia Study Group.

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13. Jones DT, Ganeshaguru K, Mitchell WA, Foroni L, Baker RJ, Prentice HG, Mehta AB, Wickremasinghe RG. Cytotoxic drugs enhance the ex vivo sensitivity of malignant cells from a subset of acute myeloid leukaemia patients to apoptosis induction by tumour necrosis factor receptor-related apoptosis-inducing ligand. Br J Haematol 2003;121:713-720.

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14. Conticello C, Adamo L, Vicari L, Giuffrida R, Iannolo G, Anastasi G, Caruso L, Moschetti G, Cupri A, Palumbo GA, Gulisano M, De Maria R, Giustolisi R, Di Raimondo F. Antitumor activity of bortezomib alone and in combination with TRAIL in human acute myeloid leukemia. Acta Haemotol 2008;120:19-30.

15. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999;5:157-163.

16. Ichikawa K, Liu W, Zhao L, Wang Z, Liu D, Ohtsuka T, Zhang H, Mountz JD, Koopman WJ, Kimberly RP, Zhou T. Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity. Nat Med 2001;7:954-960.

17. Wu X, He Y, Falo LD Jr, Hui KM, Huang L. Regression of human mammary adenocarcinoma by systemic administration of a recombinant gene encoding the hFlexTRAIL fusion protein. Mol Ther 2001;3:368-374.

18. Wei XC, Wang XJ, Chen K, Zhang L, Liang Y, Lin XL. Killing effect of TNF-related apoptosis inducing ligand regulated by tetracycline on gastric cancer cell line NCI-N87. World J Gastroenterol 2001;7:559-562.

Research Article

DOI: 10.4274/Tjh.2012.0015

Knowledge, Attitudes, and Practices of Hematologists Regarding Fertility Preservation in Turkey Türkiye’de Hematologların Fertilite Prezervasyonu Konusunda Bilgi, Tutum ve Davranışları Mert Küçük1, İrfan Yavaşoğlu2, Ali Zahit Bolaman2, Gürhan Kadıköylü2 1Adnan Menderes University, Faculty of Medicine, Department of Obstetrics and Gynecology, Aydın, Turkey 2 Adnan Menderes University, Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Aydın, Turkey

Abstract: Objective: Fertility preservation stands before us as an issue of quality of life for cancer patients and their partners and

families. Therefore, the object of the present study was to determine the extent of the knowledge that hematologists have about fertility preservation and to understand their attitudes and practices regarding this matter.

Materials and Methods: A total of 25 hematologists participated in a survey. The questionnaire included questions on sociodemographic characteristics and awareness concerning the subject of fertility preservation, as well as questions designed to determine the extent of the knowledge that hematologists had on the subject and to understand their attitudes and practices in this context.

Results: Of the participants in the study, all expressed their awareness of the adverse effects that the various treatments they were prescribing could have on fertility; 2 (8%) revealed that they had never heard of the concept of fertility preservation. Of the participants, 19 (76%) indicated that they did not have adequate knowledge about fertility preservation, but 22 (88%) fortunately expressed a need for acquiring more knowledge about the subject. Of the respondents, 23 (92%) said that they did not have any brochures or published resources on this subject and stated their belief that if hematologists did have such documents, they would have more opportunity to discuss the various fertility preservation options with patients. All of the participants in the survey supported the idea of the Turkish Society of Hematology publishing a guidebook on this subject and organizing a session on fertility preservation in their regular congress.

Conclusion: Meeting the needs of hematologists for training and knowledge in the subject of fertility preservation and ensuring the development of appropriate attitudes and practices in this area is an important issue. The Turkish Society of Hematology may play a significant key role.

Key Words: Hematologists, Fertility preservation, Attitude

Özet: Amaç: Fertility prezervasyonu önemli bir hayat kalitesi konusu olarak kanserli hastalar, partnerleri ve aileleri için karşımıza çıkmaktadır. Bu nedenle bu çalışmada hematologların fertilite prezervasyonu konusunda ne ölçüde bilgi sahibi olduklarını belirlemek, ve hematologların bu konuda tutum ve davranışlarını anlamak amaçlanmıştır.

Address for Correspondence: Mert KÜÇÜK M.D., Adnan Menderes University, Faculty of Medicine, Department of Obstetrics and Gynecology, Aydın, Turkey Phone: +90 256 218 18 00 E-mail: dr.mertkucuk@gmail.com Received/Geliş tarihi : February 2, 2012 Accepted/Kabul tarihi : August 22, 2012

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Gereç ve Yöntemler: Ankete toplam 25 hematolog katıldı. Anket katılımcıların sosyodemografik özellikleri, fertilite

prezervasyonu konusunda farkındalıkları kadar hematologların bu konuda bilgilerinin seviyesini belirlemek ve bu bağlamda tutum ve davranışlarını anlamak için sorular içerdi.

Bulgular: Katılımcıların tamamı reçete ettikleri çeşitli tedavilerin fertilite üzerine olumsuz etkileri olabileceği konusunda farkındalıkları olduğunu belirtti; iki katılımcı (%8) ise fertilite prezervasyonu kavramını hiç duymadığını ifade etti.

Katılımcıların 19’u (%76) fertilite prezervasyonu konusunda yeterli bilgiye sahip olmadığını ifade etti ve şanslı olarak katılımcıların 22’si (%88) fertilite prezervasyonu konusunda daha fazla bilgi almak ihtiyacı olduğunu ifade etti. Katılımcıların 23’ü (%92) ellerinde bu konuda broşür veya basılı kaynak olmadığını ifade etti ve hematologların böyle dökümanları bulunması halinde çeşitli fertilite prezervasyon seçeneklerini tartışmak için daha fazla fırsatları olacağını belirttiler. Katılımcıların tamamı Türk Hematoloji Derneğinin bu konuda bir klavuz basması fikrini desteklediklerini ve düzenli kongrelerinde fertilite prezervasyonu konusunda bir oturum olmasını desteklediklerini belirtti.

Sonuç: Hematologların fertilite prezervasyonu konusunda eğitim ve bilgi ihtiyaçlarının karşılanması ve bu konuda uygun tutum ve davranışlarının geliştirilmesinin sağlanması önemli bir husustur. Bu konuda Türk Hematoloji Derneği önemli ve kilit bir rol oynayabilir.

Anahtar Sözcükler: Hematologlar, Fertilite prezervasyonu, Tutum

Introduction The advances made in providing effective treatment options and the steadily increasing percentage of cured patients or of patients with 5-year life expectancies in hematological malignancies have brought the subject of such patients’ quality of life into the foreground [1]. In this respect, the quality of life of patients of reproductive age demands particular attention. Studies on fertility or fertility preservation have highlighted the importance of these issues in terms of the quality of life of cancer patients [2,3].

Research indicates that loss of fertility or the fear of a loss of fertility among young cancer survivors is a significant trigger of psychological morbidity [4].

With the development of combination chemotherapies and steady improvements in more effective treatment modalities, today the 5-year survival rate of patients with Hodgkin’s lymphoma has reached a level of 90% [5]. It is known, however, that many types of treatment used in Hodgkin’s lymphoma or other hematological malignancies have a gonadotoxic effect [3]. Fertility problems thus appear before us as a major quality of life issue in the case of young patients with this disease [5].

Today, the subject of fertility preservation is steadily gaining more and more attention. An increasing number of clinicians are reporting that patients and their families and spouses are fearful about loss of fertility and reporting their will and desire to discuss fertility preservation options.

Major associations in the United States have published recommendations on this subject [6,7].

Today, sperm and embryo cryopreservation are the first options that are offered in fertility preservation [6,7,8]. Live pregnancies attained with oocyte and ovarian tissue cryopreservation represent the advanced fertility preservation options that are available today. There are many 270

more options that are still in their experimental stages at the moment.

There are only a limited number of studies in the literature today that have explored the matter of fertility preservation from the standpoint of hematologists. To the best of our knowledge, there has been no study conducted in Turkey on the knowledge, attitudes, and practices of hematologists regarding fertility preservation. This study therefore was carried out to understand how aware hematologists are about the subject of fertility preservation, the extent of their knowledge of this topic, their attitudes toward fertility preservation, and what their practices are in this area as a part of their daily work.

Materials and Methods The study was conducted as descriptive and crosssectional research. An attempt was made to reach members of the Turkish Society of Hematology. Hematologists who gave their consent and agreed to participate in the study were asked to fill out the questionnaire. The respondents answered questions about their sociodemographic characteristics and their awareness, knowledge, attitudes, and practices regarding the subject of fertility preservation.

The participants were informed that the questionnaire would be implemented with full confidentiality and anonymity.

The questions in the survey were prepared in light of previous studies. These questions probed into the participants’ sociodemographic characteristics and their awareness, knowledge, attitudes, and practices regarding the subject of fertility preservation. The questionnaire was initially administered to 3 hematologists in order to test the comprehensibility of the questions. The validity and reliability of the questionnaire was studied and the internal consistency coefficient of the questionnaire was found to be 0.85.

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The questions were revised on the basis of the feedback received. The study protocol was approved by the local ethics committee.

Statistics The statistical analysis was performed using version 11.5 of the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA). Descriptive characteristics such as frequency and summary characteristics were calculated for variables of interest.

Results A total of 16 male (64%) and 9 female (36%) hematologists responded to the questionnaire. In the present study, Cronbach’s alpha was found as 0.83. The mean age of the participating hematologists was 47.36±4.32 years. Of the responding hematologists, 4 (16%) worked at private hospitals, 2 (8%) at state hospitals and at trainingresearch hospitals, and 19 (76%) at university hospitals. The characteristics of the participants are presented in Table 1.

All respondents stated their awareness of the possible adverse effects on fertility of the treatment modalities that they were using. Twenty-three (92%) revealed that they had heard of the concept of fertility preservation while 2 (8%) asserted that they had not. The responses of the participants are given in Table 2.

Of the participants, 15 (60%) said that they did not inform patients about fertility preservation options as a routine procedure and 11 (44%) stated that they did not obtain informed consent from the patients or their legal guardians regarding the adverse effects of drugs and/or treatment procedures on fertility. Twenty-three (92%) expressed their feeling that patients and their families should be informed about the subject with the obtaining of written informed consent. On the other hand, 14 (56%) of the participants revealed that their patients or patients’ families knew about and asked whether they could discuss the topic of fertility preservation.

Of the participants, 23 (92%) said that they did not have a brochure or any other printed materials on the subject of fertility preservation, and they stated that if such documents could be made available, they would have more opportunity to discuss fertility preservation with patients and their partners and families.

Of the respondents, 19 (76%) said that they did not feel that they had enough information about fertility preservation and, fortunately, 22 (88%) expressed their desire to learn more.

Among the hematologists participating in the survey, 24 (96%) said that they should consider fertility preservation a priority topic when planning a modality of treatment for patients; only 15 (60%), however, did say that they considered the subject when planning the treatment.

Of the participants, 17 (68%) professed knowledge about fertility preservation options for male patients and 11 (44%) said that they knew about fertility preservation options for female patients.

Of the participants, 22 (88%) said that they did not have enough knowledge about oocyte cryopreservation and 13 (52%) reported the same about ovarian tissue cryopreservation. Fifteen (60%) of the participants revealed that they had never recommended sperm cryopreservation to any patients, 19 (76%) stated that they had not recommended ovarian tissue cryopreservation, 20 (80%) stated that there had been no instance where they had recommended oocyte cryopreservation, and, finally, all participants said that they had never recommended embryo cryopreservation.

Eighteen (72%) of the participants indicated that they had never read any publication on fertility preservation; 20 (80%) stated that they had not read any publication about the subject in the last 6 months.

Among the respondents, 20 (80%) expressed their approval of patients postponing their treatments for a short period (4 weeks, for example) to accommodate the fertility preservation process.

Fourteen (56%) of the participants said that the hospital in which their patients were treated had no clinic or assisted conception unit for fertility preservation and 17 (68%) stated that these should be established. Another 20 (80%) said that they had no knowledge about the costs of fertility preservation options.

Of the participants, 22 (88%) said that they were not familiar with the recommendations on fertility preservation published by the American Society of Clinical Oncology (ASCO) and American Society of Reproductive Medicine (ASRM). Another 24 (96%) admitted that they did not know whether the Turkish Society of Hematology had published a guidebook on fertility preservation, and all participants asserted that the Turkish Society of Hematology should have a guidebook and that they would support its publication and sessions about fertility preservation at congresses of the Turkish Society of Hematology.

Table 1. Physician characteristics, n=25.

n (%) Age (years)

47.36±4.32

Sex Male

16 (64)

Female

9 (36)

Institution where physician works University hospital

19 (76)

Private hospital

4 (16)

State hospital and Ministry of Health training and research hospital

2 (8)

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Discussion It was found that most of the hematologists in the study were aware that the treatment modalities they used had an adverse effect on fertility. A majority of the hematologists stated that they have heard of the concept of fertility preservation. It was also found that most of the hematologists did not routinely inform their patients about fertility preservation options. Most of the hematologists that participated in the survey stated their lack of adequate knowledge about fertility preservation but, fortunately, most of them expressed their desire to learn more about the topic.

As seen in the results obtained from the survey, a growing number of patients and their families are asking to be informed about the matter of fertility preservation and wish to discuss this with their doctors. It is our belief that it is very important that patients are informed about this subject. The same view has been expressed by ASCO and ASRM [7,8,9]. Patients should be informed and thus made ready to make their own informed decisions. The active participation of patients’ families should also be ensured in sessions where patients are provided information about fertility preservation. This is important because the subject is not only a source of anxiety for the patient but also creates intense feelings in close relatives such as the patient’s spouse.

Most of the hematologists in our study said that they did not routinely inform patients about fertility preservation before the start of treatment. In studies carried out abroad, half of male and female cancer patients were reported as not remembering being informed about fertility preservation [8,9,10]. Those who did remember said that they were not happy with the quality and amount of information that they were given [11,12]. Explaining the adverse effects on fertility of treatment modalities, informing patients about fertility preservation, or referring the patient to another physician for the purpose of obtaining this information is the responsibility of the patient’s attending physician [6].

Because there is no test today to indicate which patients’ fertility will be affected by prescribed treatments and what the impact will be, it is our belief that all patients should be informed about this subject [4]. Fertility preservation alternatives should be explained in detail to all patients and patients should be given the chance to decide. The matter of fertility is an important topic that affects quality of life following treatment. In a study by Schover et al., it was found that 76% of young cancer survivors with no children wished to eventually be parents and, for this reason, were interested in learning about the positive and negative effects of treatment on fertility. In this context, it was also reported that patients were concerned about the negative impact of treatment on a possible pregnancy and on the fetus, should a pregnancy occur [10].

As the majority of the hematologists participating in this study indicated in the questionnaire, there were no brochures or informative booklets available about fertility 272

preservation that could be given to patients to read. Such booklets are generally made available to patients as standard procedure at clinics in Western countries. As the majority of hematologists indicated in the questionnaire in the present study, the availability of this kind of informative printed booklets will enhance the dialog of clinicians and patients on this subject and patients will find increased opportunity to bring up the matter for a more productive discussion.

Organizations such as FertileHope [13] in the United States produce brochures of this kind and provide patients with support and guidance.

The majority of the hematologists stated that no clinics dealing with fertility preservation existed in the hospital in which they worked. It was also observed that hematologists were not knowledgeable about the costs of possible fertility preservation options. It may be that the reason a large majority of the hematologists had not referred patients to a fertility preservation center was because they did not know where such centers dealing with fertility preservation were located or the probable cost. It is our belief that associations of professionals in the areas of fertility, hematology, and oncology should collaborate and share information, perhaps forming working committees among themselves if necessary. An oncofertility consortium has been established in the United States [14]. We feel it would be useful for the Turkish Society of Hematology to announce on its website the locations of centers involved in fertility preservation in all regions of the country so that hematologists can easily find the center closest to them.

Another finding in our study was that some hematologists did not consider fertility preservation a priority topic at the time that a course of treatment was being planned for the patient. Other studies on this subject support this finding [15]. Clinicians are more liable to prioritize the discussion of the patient’s potential life-threatening complications. It is, however, true that as effective treatments cause 5-year survival and cure rates to rise, the topic of fertility will increasingly come to the fore as an issue of quality of life [10], and it is imperative that clinicians realize this. Patients and their families are often in a state of shock over lifethreatening complications and in such a situation, the subject of fertility options may be completely overlooked or there may be a lack of interest in discussing the matter.

It is very important that hematologists inform their patients in detail about fertility preservation before treatment begins [9]. The information presented to the patient should contain knowledge about the adverse effects of treatment on fertility. The hematologist should provide the patient and the patient’s family with the fertility preservation options and the success rates of fertility preservation modalities, also informing the patient that some options are still in their experimental stages. The knowledge that hematologists impart to their patients should also include information on

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Table 2. Knowledge, attitudes, and practices of hematologists regarding fertility preservation.

Question

Yes n (%)

No n (%)

1.Have you ever heard of the concept of fertility preservation?

23 (92)

2 (8)

2. Do you believe you know enough about fertility preservation?

6 (24)

19 (76)

3. Is fertility preservation a priority for you when you are planning a course of treatment for your patient?

15 (60)

10 (40)

4. Do you believe that fertility preservation should be a priority when planning a course of treatment for your patient?

24 (96)

1 (4)

5. Do you believe you need to have education/more knowledge about the topic of fertility preservation?

22 (88)

3 (12)

6. Do you offer your patients and their families information about fertility preservation as routine procedure?

10 (40)

15 (60)

7. Do your patients or their families ask you for information about fertility preservation?

14 (56)

11 (44)

8. Are you aware that the drugs you use in treatment have toxic effects on gonads and also have the capacity to affect your patient’s fertility in the future?

25 (100)

0 (0)

9. Do you give your patients and/or their families information about the gonadotoxic effects of drugs and treatment?

21 (84)

4 (16)

10. Are your patients aware of the effects the drugs they are taking have on fertility?

10 (40)

15 (60)

11. Do you inform your patients and receive informed consent from them regarding the adverse effects on fertility of drugs and/or treatment procedures?

14 (56)

11 (44)

12. Do you believe that your patients should be informed about this topic in writing?

23 (92)

2 (8)

13. Do you have booklets and/or brochures on fertility preservation at your clinic to inform patients about the topic?

2 (8)

23 (92)

14. If you had printed brochures or booklets on this subject, would you have more of an opportunity to discuss fertility preservation with your patients?

23 (92)

2 (8)

15. Do you know about fertility preservation options for male patients?

17 (68)

8 (32)

16. Do you know about fertility preservation options for female patients?

11 (44)

14 (56)

17. Have you ever recommended sperm cryopreservation to any of your patients?

10 (40)

15 (60)

18. Do you believe you know enough about ovarian tissue cryopreservation?

12 (48)

13 (52)

19. Have you ever recommended ovarian tissue cryopreservation to any of your patients?

6 (24)

19 (76)

20. Do you believe you know enough about oocyte cryopreservation?

3 (12)

22 (88)

21. Have you ever recommended oocyte cryopreservation to any of your patients?

5 (20)

20 (80)

22. Do you believe you know enough about embryo cryopreservation?

3 (12)

22 (88)

23. Have you ever recommended embryo cryopreservation to any of your patients?

0 (0)

25 (100)

24. Have you ever referred any of your patients to an assisted conception unit or a specialist on fertility preservation?

6 (24)

19 (76)

25. Would you look favorably on a patient’s wishing to postpone treatment for a short time (e.g., 4 weeks) for the purpose of fertility preservation?

20 (80)

5 (20)

26. Is there a clinic or an assisted conception unit at the hospital at which you treat your patients?

11 (44)

14 (56)

27. Do you believe the hospital at which you treat your patients should have a clinic or an assisted conception unit 17 (68) for fertility preservation?

8 (32)

28. Have you ever read any publications on fertility preservation?

7 (28)

18 (72)

29. Have you read any publications on fertility preservation in the last 6 months?

5 (20)

20 (80)

30. Concerning your approach to your patient on the subject of fertility preservation, do you know about the recommendations published by ASRM (American Society of Reproductive Medicine) and ASCO (American Society of Clinical Oncology)?

3 (12)

22 (88)

31. Do you think the Turkish Society of Hematology has a guidebook on fertility preservation?

1 (4)

24 (96)

32. Do you believe that the Turkish Society of Hematology should have a guidebook on fertility preservation?

25 (100)

0 (0)

33. Would you like a session on fertility preservation scheduled at hematology congresses?

25 (100)

0 (0)

34. Do you know anything about the costs of fertility preservation for the patient?

5 (20)

20 (80)

273

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Küçük M, et al: Fertility Preservation

the costs of fertility preservation options. It is important that the clinician give the patient the address and telephone number of a person or clinic that can be reached and from which information about fertility preservation can be acquired. We also feel that the hematologist’s own coordination with that person or clinic will be important in choosing a fertility preservation option that is suitable to the patient’s clinical status. It is also thought that obtaining written informed consent from the patient or their legal guardians will be important in terms of medicolegal matters.

Based on this study with a limited number of hematologists, it is not possible to specify to what extent these results are generalizable to all hematologists. Thus, we assume that the results of this study can be generalized to a limited degree. Further research with larger numbers of participants should be conducted on the topic.

It is our belief that publishing guidebooks to offer guidance to clinicians about fertility preservation options is important. The respondents to the questionnaire support the publication of such a guidebook by the Turkish Society of Hematology. ASCO and ASRM have made recommendations about fertility preservation in various publications [6,7,8].

The British Fertility Society [16] has also made similar recommendations. Reviewing these recommendations allows clinicians to be more comfortable about this topic and act on it quickly and in coordination. We feel that it is important that a subcommittee that may be formed under the Turkish Society of Hematology prepare a guidebook that will provide hematologists with guidance on this issue.

Most of the hematologists responding to the questionnaire believed that they did not know enough about fertility preservation, but fortunately they exhibited a desire to receive education on this topic. The respondents also supported the idea that the Turkish Society of Hematology should organize a session on fertility preservation at their regular congresses. An initiative of this kind by the Turkish Society of Hematology may be significant in terms of eliminating the lack of knowledge in this area. At the same time, we also think that making the topic of fertility preservation a standard part of the Society’s fellowship education program will be instrumental in enhancing the levels of knowledge.

Conclusion Meeting the needs of hematologists for more education and knowledge on the topic of fertility preservation and improving their knowledge, attitudes, and practices in this area is of great importance. The Turkish Society of Hematology may play a significant and key role in such efforts.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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References 1. Küçük M, Bolaman AZ, Yavaşoğlu İ, Kadıköylu G. Fertilitypreserving treatment options in patients with malignant hematological diseases. Turk J Hematol 2012;29:207-216.

2. Rieker PP, Fitzgerald EM, Kalish LA. Adaptive behavioral responses to potential infertility among survivors of testis cancer. J Clin Oncol 1990;8:347-355.

3. Tschudin S, Bitzer J. Psychological aspects of fertility preservation in men and women affected by cancer and other life-threatening diseases. Hum Reprod Update 2009;15:587-597.

4. Moynihan C. Testicular cancer: the psychosocial problems of patients and their relatives. Cancer Surv 1987;6:477-510.

5. van der Kaaij MA, van Echten-Arends J, Simons AH, KluinNelemans HC. Fertility preservation after chemotherapy for Hodgkin lymphoma. Hematol Oncol 2010;28:168-179.

6. Lee SJ, Schover LR, Partridge AH, Patrizio P, Wallace WH, Hagerty K, Beck LN, Brennan LV, Oktay K; American Society of Clinical Oncology. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. J Clin Oncol 2006;24:2917-2931.

7. Ethics Committee of the American Society for Reproductive Medicine. Fertility preservation and reproduction in cancer patients. Fertil Steril 2005;83:1622-1628.

8. Practice Committee of the American Society for Reproductive Medicine; Practice Committee of the Society for Assisted Reproductive Technology. Ovarian tissue and oocyte cryopreservation. Fertil Steril 2006;86(Suppl 1):142-147.

9. Schover LR, Brey K, Lichtin A, Lipshultz LI, Jeha S. Knowledge and experience regarding cancer, infertility, and sperm banking in younger male survivors. J Clin Oncol 2002;20:1880-1889.

10. Schover LR, Rybicki LA, Martin BA, Bringelsen KA. Having children after cancer. A pilot survey of survivors’ attitudes and experiences. Cancer 1999;86:697-709.

11. Thewes B, Meiser B, Rickard J, Friedlander M. The fertilityand menopause-related information needs of younger women with a diagnosis of breast cancer: a qualitative study.

Psychooncology 2003;12:500-511.

12. Partridge AH, Gelber S, Peppercorn J, Sampson E, Knudsen K, Laufer M, Rosenberg R, Przypyszny M, Rein A, Winer EP. Web-based survey of fertility issues in young women with breast cancer. J Clin Oncol 2004;22:4174-4183.

13. Beck LN. The gift of hope: my personal experience. J Natl Cancer Inst Monogr 2005;34:1-2.

14. Nagel K, Cassano J, Wizowski L, Neal MS. Collaborative multidisciplinary team approach to fertility issues among adolescent and young adult cancer patients. Int J Nurs Pract 2009;15:311-317.

15. Green D, Galvin H, Horne B. The psycho-social impact of infertility on young male cancer survivors: a qualitative investigation. Psychooncology 2003;12:141-152.

16. http://www.britishfertilitysociety.org.uk/practicepolicy/ documents/fccpaper.pdf (accessed November 10, 2011).

Research Article

DOI: 10.4274/Tjh.2011.0034

Prognostic Significance of Bcl-2 and p53 Protein Expressions and Ki67 Proliferative Index in Diffuse Large B-cell Lymphoma Diffüz Büyük B Hücreli Lenfomada Bcl-2 ve p53 Protein Ekspresyonu ve Ki67 Proliferasyon İndeksinin Prognostik Önemi Betül Bolat Küçükzeybek1, Sadi Bener1, Aylin Orgen Çallı1, Tuğba Doğruluk Paksoy2, Bahriye Payzin3 1Department of Pathology, İzmir Atatürk Training and Research Hospital, İzmir, Turkey 2Department of Pathology, Kahramanmaraş Yenişehir Government Hospital, Kahramanmaraş, Turkey 3Department of Hematology, İzmir Atatürk Training and Research Hospital, İzmir, Turkey

Abstract: Objective: Diffuse large B-cell lymphoma (DLBCL) is a high-grade neoplasm that has heterogeneous properties in clinical,

morphological, and immunophenotypic aspects. In the present study the effects of p53, Bcl-2, and Ki67 on prognosis and their relationships with clinical parameters were examined.

Materials and Methods: Thirty-five patients who had been diagnosed with nodally located DLBCL at İzmir Atatürk Training and Research Hospital between January 1999 and June 2006 were included in the study. The Ann Arbor classification system was used to determine the stage of the patients. The patients were evaluated according to age, sex, stage, B symptoms, extranodal involvement, and lactate dehydrogenase (LDH) level as well as immunohistochemically. P53 protein and Bcl-2 oncoprotein expressions and Ki67 proliferation index were assessed immunohistochemically.

Results: High Bcl-2 expression was found in 9 patients (25.7%), high p53 expression was found in 10 patients (28.6%), and high Ki67 was observed in 23 patients (65.7%). There was no significant correlation between p53 expression, Bcl-2 expression, or Ki67 proliferation index and age, sex, stage, B symptoms, extranodal involvement, LDH level, and overall survival (p>0.05).

We did not find a relationship among p53 expression, Bcl-2 expression, Ki67 proliferation index, and prognosis (p>0.05). There was no significant relationship between overall survival and age, sex, stage, B symptoms, extranodal involvement, or LDH level (p>0.05). Our results revealed that Bcl-2 and p53 protein expressions and Ki67 proliferation index have no effect on overall survival of patients with DLBCL. Conclusion: The prognostic importance of p53 and Bcl-2 protein expressions and Ki67 proliferation index in DLBCL, which has biological and clinical heterogeneity, can be understood in a large series of studies that have subclasses and immunohistochemical markers with optimal cut-off values.

Key Words: Diffuse large B-cell lymphoma, p53, Bcl-2, Ki67, Prognosis

Özet: Amaç: Diffuz büyük hücreli B lenfoma (DBBHL) klinik, morfolojik, immunofenotipik ve genetik özellikleri ile heterojenite

gösteren yüksek dereceli bir neoplazmdır. Çalışmamızda DBBHL’da hücre siklusu düzenleyicisi olan p53 (tümör supresör Address for Correspondence: Betül BOLAT KÜÇÜKZEYBEK, M.D., Department of Pathology, İzmir Atatürk Training and Research Hospital, İzmir, Turkey Phone: +90 232 243 43 43 E-mail: bbkzeybek@yahoo.com Received/Geliş tarihi : December 1, 2011 Accepted/Kabul tarihi : September 24, 2012

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gen), apoptozisi inhibe edici onkoprotein olan Bcl-2 ve hücre proliferasyon belirleyicisi olan Ki67 ekspresyonlarının klinik parametrelerle ilişkisini ve prognoz üzerindeki etkilerini araştırdık.

Gereç ve Yöntemler: Çalışmaya Ocak 1999 - Haziran 2006 tarihleri arasında, İzmir Atatürk Eğitim ve Araştırma Hastanesi Patoloji Bölümü’nde, nodal yerleşimli DBBHL tanısı alan 35 olgu alındı. Hastaların evrelemesinde Ann Arbor sınıflaması kullanıldı. Olgular yaş, cinsiyet, evre, B semptomları, ekstranodal tutulum, LDH düzeyi ve sağ kalımları yanı sıra immünohistokimyasal olarak; p53 protein ekspresyonu, Bcl-2 onkoprotein ekspresyonu ve Ki67 proliferasyon indeksi açısından değerlendirildi.

Bulgular: Yüksek Bcl-2 ekspresyonu 9 hastada (%25.7), yüksek p53 ekspresyonu 10 hastada (%28.6), yüksek Ki67 ekspresyonu 23 hastada (%65.7) saptandı. p53 ekspresyonu, Bcl-2 protein ekspresyonu ve Ki67 proliferasyon indeksi ile yaş, cinsiyet, evre, B semptomları, ekstranodal tutulum, LDH düzeyi ve sağ kalım arasında istatistiksel olarak anlamlı ilişki saptanmadı (p>0.05).

Tüm olgularda p53, Bcl-2 ve Ki67 ekspresyonları ile prognoz arasındaki ilişki istatistiksel olarak anlamlı değildi (p>0.05). Sağ kalım süresi ile yaş, cinsiyet, evre, B semptomları, ekstranodal tutulum ve LDH düzeyi arasında istatistiksel olarak anlamlı ilişki bulunmadı (p>0.05). Bu çalışmada Bcl-2 ve p53 protein ekspresyonları ile Ki67 proliferasyon indeksinin, DBBHL’lı hastaların yaşam süresi üzerinde etkili olmadığı bulunmuştur.

Sonuç: p53 ve Bcl-2 protein ekspresyonları ile Ki67 proliferasyon indeksinin, DBBHL’daki prognostik önemi, immunohistokimyasal markerların optimal cut-off değerlerinin belirlendiği, subgrup ayrımının yapıldığı daha geniş serili çalışmalarda, biyolojik ve klinik heterojenitesi olan bu hastalıkta, daha net anlaşılacaktır.

Anahtar Sözcükler: Diffüz büyük B hücreli lenfoma, p53, Bcl-2, Ki67, Prognoz

Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma, comprising 30%-40% of all nonHodgkin’s lymphoma (NHL). It has heterogeneous clinical features and varies markedly in response to treatment and in prognosis [1,2]. The response rate is 60%-80% in NHL after acceptable therapy. The 5-year overall survival rate is higher than 55%. Patients who responded to chemotherapy and were cured after therapy could be estimated using clinic and laboratory results. Therapy plans can be determined using these prognostic factors [3,4]. Although survival can be estimated on the basis of clinical parameters, molecular abnormalities in a panel of suppressor proteins and oncogenic proteins have also been reported related with prognosis [5,6,7]. In the present study we investigated the effects of p53 (tumor suppressor protein), which is a cell cycle regulator; Bcl-2 oncoprotein, which is an inhibitor of apoptosis; and Ki67, which is a cell proliferation marker on prognosis, and their relationships with clinical parameters.

Materials and Methods Patients: Thirty-five patients who had been diagnosed with nodally located DLBCL at İzmir Atatürk Training and Research Hospital between January 1999 and June 2006 were included in the study. The patients were evaluated according to age, sex, stage, B symptoms, extranodal involvement, and lactate dehydrogenase (LDH) level as well as immunohistochemically. p53 and Bcl-2 oncoprotein expressions and Ki67 proliferation index were assessed immunohistochemically. The clinical parameters and the outcome were reviewed retrospectively. All clinical and laboratory data, along with the follow-up data, were obtained from the hospital records or patients’ charts. Overall survival 276

(OS) was calculated from the date of diagnosis until death or last follow-up. Affected lymph nodes from all of the patients were examined by biopsy (Figure 1) and lymphoma was diagnosed according to WHO lymphoma classification.

Immunohistochemistry: Paraffin sections were immunostained by the labeled streptavidin-avidinbiotin method with the antibodies for Ki67 (clone SP6, Neomarkers), Bcl-2 (clone 124, Dako), and p53 (clone DO7, Dako). Positive staining of small reactive lymphocytes for Bcl-2 was provided as an internal control. A previously known positive case was used as an external control in order to evaluate p53 reactivity. A known positive control (normal tonsil) was used to evaluate Ki67 reactivity.

Three categories were defined for Bcl-2 [5,6,8,9] and p53 [6,7,8,10] expressions: negative when none or less than 10% of tumor cells showed staining, low expression when 10%50% of tumor cells showed staining, and high expression when >50% of tumor cells showed staining. The cases were divided into 2 groups: a high Ki67 expression (>40%) group and a low Ki67 expression (<40%) group [1,11].

Statistical Analysis: Survival curves were drawn according to the Kaplan-Meier method and compared by log-rank test. The relationship between p53, Bcl-2, and Ki67 expressions and clinicopathological parameters was evaluated by chi-square test for data qualification and Fisher’s exact test for data categorization. Differences were considered as significant if the p value was less than 0.05.

The study was approved by the ethics committee.

Results Patient characteristics are summarized in Table 1.

Follow-up duration ranged from 0.5 to 68 months, with an average of 17.6 months. Fifteen (42.9%) patients were followed until death, whereas 20 (57.1%) patients were still

Küçükzeybek BB, et al: Prognostic Significance of İmmunohistochemical Markers in Diffuse Large B-cell Lymphoma

alive at the end of the study. There were 21 (60%) men and 14 (40%) women in the study. The average age of the patients was 53.6 years. All patients were classified according to the Ann Arbor classification. As such, 12 patients (34.3%) had stage 1-2 and 23 patients (65.7%) had stage 3-4 disease.

Elevated serum LDH levels were observed in 26 patients (74.3%). Eighteen (51.4%) patients had B symptoms at the time of diagnosis. Thirteen (37.1%) patients presented with involvement of extranodal sites. p53 protein expression was high in 10 (28.6%) patients (Figure 2), low in 12 (34.3%) patients, and negative in 13 (37.1%) patients. Bcl-2 protein expression was high in 9 (25.7%), low in 4 (11.4%), and negative in 22 (62.9%) of the 35 cases (Figure 3). Ki67 expression was high in 23 (65.7%) patients and low in 12 (34.3%) patients (Figure 4).

Turk J Hematol 2013;30:275-282

significant relationships between p53, Bcl-2, and Ki67 expressions and prognosis (p>0.05) (Figures 5, 6, 7). There was no significant relationship between overall survival and age, sex, stage, B symptoms, extranodal involvement, or LDH level (p>0.05).

Treatment records of 27 patients were attained. Twentythree patients had been treated with 6-8 cycles of CHOP chemotherapy regimens including cyclophosphamide, doxorubicin, vincristine, and prednisolone. Only 4 patients had been treated with 6-8 cycles of a rituximab and CHOP chemotherapy regimen. Therefore, statistical analysis could not be done for the chemotherapy protocols.

Discussion

There was no significant correlation between p53 and Bcl-2 expressions or Ki67 proliferation index and age, sex, stage, B symptoms, extranodal involvement, LDH level, and overall survival (p>0.05) (Table 2). We did not find

DLBCL exhibits heterogeneous clinical features and varies markedly in response to treatment and prognosis [1,12]. Although survival can be estimated on the basis of clinical parameters, molecular abnormalities in a panel of suppressor proteins and oncogenic proteins have also been reported to be related to prognosis [5,6,7].

Figure 1. Morphologic features of DLBCL (hematoxylin and eosin, 440x).

Figure 3. Bcl-2 cytoplasmic positivity of >50% (440x).

Figure 2. p53 nuclear positivity of >50% (440x).

Figure 4. Ki67 nuclear positivity of >40% (220x).

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Survival Functions P53 score 1.0

%10-50 low exp <%10 negative >%50 high exp

0.8

No. (%) 22 (62.8) 13 (37.2)

Sex Male Female

21 (60) 14 (40)

Stage I+II III+IV

12 (34.3) 23 (65.7)

B symptoms (+) (-)

18 (51.4) 17 (48.6)

Extranodal involvement (+) (-)

13 (37.1) 22 (62.9)

LDH level High Normal

26 (74.3) 9 (25.7)

0.2

Deceased

15 (42.9)

0.0

Alive

15 (42.9)

p53 expression <10% 10%-50% >50%

13 (37.1) 12 (34.3) 10 (28.6)

Bcl-2 expression <10% 10%-50% >50%

22 (62.9) 4 (11.4) 9 (25.7)

Ki67 expression <40% >40%

12 (34.3) 23 (65.7)

Cum Survival

Age <60 >60

0.6

0.4

0.2

0.0 0

10

20

40

30

50

60

70

Time (m)

Figure 5. Overall survival related to p53 expression.

Survival Functions 1.0

Ki 67 score <%40 low exp

Cum Survival

0.8

>%40 high exp

0.6 0.4

0

10

20

30

40

50

60

70

Time (m)

Figure 6. Overall survival related to Ki67 expression.

Survival Functions 1.0

bcl score %10-50 low exp <%10 negative

0.8

Cum Survival

Table 1. Characteristics of the patients and p53, Bcl-2, and Ki67 expressions.

>%50 high exp

0.6 0.4 0.2

0.0 0

10

20

30

40

50

60

70

Time (m)

Figure 7. Overall survival related to Bcl-2 expression.

Several studies reported that age [7,12], serum LDH level [5,7,10,13], the involvement of extranodal sites [7,10], the stage of the disease [5,10,13,11,12,13,14,15], and B symptoms [10,15] were significant clinical predictors of survival of patients with DLBCL. In the present study, 278

similar to the other studies, there was no statistically significant relationship between OS and age [5,11,13,14], sex [5,10,14,15], stage [16], B symptoms [13], extranodal involvement [11,13,15], or LDH level [11,15]. p53 can be considered as a tumor suppressor protein. It is involved in the regulation of cell survival by interaction with G-S phase transition within the cell cycle and by induction of apoptotic cell death [17,18]. The incidence of p53 expression in DLBCL varies between 5.0% and 71.0% [5,6,7,10,14,19-22]. In the present study, p53 expression was identified in 62.9% of all cases and high p53 expression was identified in 10 patients (28.6%). Although Ichikawa

6 (50%)

0.721 9 (39.1%)

14 (60.9%)

6 (50%)

9 (75%)

4 (33.3%)

6 (60%)

4 (40%) 8 (66.7%) 3 (23.1%)

10 (76.9%) 5 (55.6%) 2 (50%)

9 (40.9%)

13 (59.1%) Alive

Outcome

Deceased

2 (50%)

4 (44.4%)

0.939

7 (70%) 9 (75%)

0.087

6 (26.1%)

17 (73.9%)

3 (25%) 3 (30%) 3 (25%) 3 (23.1%)

6 (66.7%) High

3 (75%)

5 (22.7%)

17 (77.3%)

Normal LDH level

1 (25%)

3 (33.3%)

0.828

10 (76.9%)

0.929

10 (43.5%) 3 (25%)

1

0.463 13 (56.5%) 9 (75%)

3 (30%) 6 (50%) 4 (30.8%)

7 (70%) 6 (50%) 9 (69.2%)

4 (44.4%) 0 (0%) 9 (40.9%) Present involvement

13 (59.1%) Absent Extranodal

4 (100%)

5 (55.6%)

0.259

5 (50%) 5 (41.7%)

5 (50%) 7 (58.3%)

8 (61.5%) 6 (66.7%) 1 (25%) 11 (50%) Present symptoms

11 (50%) Absent B

3 (75%)

3 (33.3%)

0.373

5 (38.5%)

0.524

13 (56.5%) 5 (41.7%)

0.489 10 (43.5%) 7 (58.3%)

8 (66.7%)

0.607

8 (34.8%)

15 (65.2%)

4 (33.3%)

6 (60%) 8 (66.7%)

4 (40%) 4 (33.3%)

9 (69.2%) 7 (77.8%)

2 (22.2%)

III+IV

2 (50%)

8 (36.4%)

14 (63.6%)

I+II Stage

2 (50%)

5 (55.6%)

0.588

4 (30.8%)

0.895

12 (52.2%) 2 (16.7%)

1

0.070 11 (47.8%) 10 (83.2%)

6 (50%)

1 (10%)

9 (90%) 6 (50%) 6 (6.2%)

7 (53.8%)

3 (75%)

1 (25%) 8 (36.4%)

14 (63.6%)

Female

Male

Sex

4 (44.4%)

0.496

6 (50%) 5 (38.5%)

0.071

11 (47.8%) 2 (16.7%)

0.139 12 (52.2%) 10 (83.3%) 0.347 2 (20%)

6 (50%) 8 (61.5%)

6 (66.7%) 6 (27.3%)

1 (25%)

16 (72.7%)

>60

Age

≤60

3 (75%)

3 (33.3%)

0.104

8 (80%)

Ki67 < 40%, low exp.

P53 10%50%, low exp.

P53 < %10, negative p Bcl-2 > 50%, high exp.

Bcl-2 10%-50%, low exp.

Bcl-2 < 10%, negative DLBCL (n = 35)

Table 2. Expressions of Bcl-2, p53, and Ki67 in relation to clinical characteristics.

P53 > 50%, high exp.

p

Ki67 > 40%, high exp.

P

Küçükzeybek BB, et al: Prognostic Significance of İmmunohistochemical Markers in Diffuse Large B-cell Lymphoma

Turk J Hematol 2013;30:275-282

et al. reported that patients who had high p53 expression had increased LDH levels and advanced-stage disease [21], Sanchez et al. [5], Kramer et al. [6], Wilson et al. [19], and Rujirojindakul et al. [23] did not demonstrate statistical correlation between p53 expression and age, sex, LDH level, and B symptoms in patients with DLBCL. The present study failed to show a relationship between p53 protein expression and any of the clinical variables studied. Several studies reported a relationship between p53 expression and OS of patients with DLBCL [8,19,21]. Zhang et al. reported an inverse relationship between p53 expression and diseasefree survival time [7]. Pervez et al. observed p53 nuclear expression in 52.13% of cases; it was concluded that p53 overexpression was associated with decreased OS (22).

Piris et al. found a correlation between p53 expression and OS in high-grade lymphomas, and patients with combined expression of Bcl-2 and p53 in tumors had poorer prognosis than those with p53 expression only, which was particularly significant in lymph nodes in DLBCL cases [24].

Kramer et al. showed that p53 expression was only related to a high tumor burden and was not an independent risk factor for survival in patients with DLBCL [6]. Similarly, in the present study, no significant correlation was found between p53 expression and OS or disease-free survival [5,10,23]. It has been found that p53 expression had no significant effect on OS. Bcl-2, an antiapoptotic protein, belongs to a large family of proteins involved in the regulation of programmed cell death [25]. The effects of Bcl-2 on clinical course have been widely studied with quite a few lymphoma types previously [5,6,7,10,13,19,20,26,27]. The incidence of Bcl2 expression in DLBCL varies between 24.0% and 77.0% [57,10,13,20,22,24,26,27,28]. In the present study, 13 (37.1%) cases out of 35 showed Bcl-2 protein expression in tumor cells and 9 (25.7%) cases out of 35 exhibited high levels (>50.0% of tumor cells staining) of Bcl-2 protein expression.

The relationship between Bcl-2 protein expression and clinical parameters has been evaluated previously. Bcl2 protein expression has been found positive in patients with advanced disease stage [6,8,10,28], high LDH levels [27,29], and advanced age [8,20]. This study, similar to the other studies, failed to show a relationship between Bcl-2 protein expression and any of the clinical variables studied [6,10,19,26,28]. The prognostic significance of Bcl-2 protein expression is controversial [5,6,7,8,9,10,13,19,20,26,27].

Some studies [8,9,10,20,27,28,30] showed that Bcl-2 protein overexpression was associated with poor OS, but some other studies [5,6,7,13,19,26] showed no difference in OS. Pervez et al. reported positive Bcl-2 protein expression in 75 of 117 (64.1%) patients in their study on 117 patients with DLBCL. However, there was no significant difference in OS between patients with negative or weakly positive Bcl-2 and high Bcl-2 expressions [22]. Similarly, in the present study, 279

Turk J Hematol 2013;30:275-282

Küçükzeybek BB, et al: Prognostic Significance of İmmunohistochemical Markers in Diffuse Large B-cell Lymphoma

we did not show any difference in the outcome of patients with Bcl-2-positive or Bcl-2-negative DLBCL. Iqbal et al. observed a relationship between Bcl-2 protein expression and survival in the ABC subgroup of DLBCL, which was not seen in the entire DLBCL group that was examined [29].

Different results were observed concerning the prognostic significance of Bcl-2 expression because of different cut-off values and methodological differences.

Ki67 is a proliferation marker for several human neoplasms. Ki67 detects a nuclear antigen associated with cell proliferation [31]. The prognostic value of Ki67 has been the subject of different studies in the past. Although most of the studies showed a high proliferation index to be an adverse prognostic marker, there were inconsistent results, as well [32,33,34]. High Ki67 expression was observed in 23 patients (65.7%) in the present study. There was no correlation between Ki67 expression level and any studied clinicopathological factors. Miller et al. [32] reported that tumor proliferation of >80.0% was associated with poorer survival in previously untreated patients with aggressive NHL, whereas Hall et al. [35] found that patients who responded well to chemotherapy were less likely to relapse if they had >80.0% tumor proliferation. Jovanovic et al. reported that the patients with a high proliferative fraction (Ki67 > 60.0%) had a worse OS rate with log-rank test analysis [30]. Conversely, Hasselblom et al. found that patients with low Ki67 expression (<49.0%) had significantly worse progression-free survival and OS as independent risk factors [36]. In this study, similar to the other studies, there was no statistically significant relationship between Ki67 expression and prognosis [7,10,11,15].

The effects of p53 and Bcl-2 protein expressions and Ki67 proliferation index on prognosis of DLBCL have been investigated. The results from prior studies are variable and controversial. Methodological differences, different cut-off levels of immunohistochemical markers, and differences in the treatment regimens and follow-up time may account for the variation in results.

The present study revealed that Bcl-2 and p53 protein expressions and Ki67 proliferation index have no effect on the OS of patients with DLBCL. The prognostic importance of p53 and Bcl-2 protein expressions and Ki67 proliferation index in DLBCL, which has biological and clinical heterogeneity, can be understood in a large series of studies that have subclasses and immunohistochemical markers with optimal cut-off values.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

280

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Research Article

DOI: 10.4274/Tjh.2012.0065

Detection of Left Ventricular Regional Function in Asymptomatic Children with beta-Thalassemia Major by Longitudinal Strain and Strain Rate Imaging Beta Talasemi Majör Tanılı Asemptomatik Çocuklarda Sol Ventrikül Bölgesel Fonksiyonlarının Longitidunal Strain ve Strain Rate Görüntüleme ile Değerlendirilmesi Ali Bay1, Osman Başpınar2, Göksel Leblebisatan3, Ali Seçkin Yalçın1, Ahmet İrdem2 1Gaziantep University Medical Faculty, Department of Pediatric Hematology, Gaziantep, Turkey 2Gaziantep University Medical Faculty, Department of Pediatric Cardiology, Gaziantep, Turkey 3Gaziantep Children’s Hospital, Department of Pediatric Hematology, Gaziantep, Turkey

Abstract: Objective: Cardiac failure due to iron overload remains the most common cause of death in patients with betathalassemia major. This study aimed to evaluate myocardial function in children with beta-thalassemia major using standard echocardiography technique and strain rate imaging.

Materials and Methods: Conventional echocardiographic analysis, tissue velocity imaging, and strain/strain rate imaging of the left ventricle were evaluated in 48 children with beta-thalassemia major (19 girls, 29 boys; 8.39±4.05 years) and 22 healthy children (11 girls, 11 boys; 8±3.72 years).

Results: Conventional echocardiographic examinations revealed that beta-thalassemia patients had larger left ventricular endsystolic diameter, end-diastolic and end-systolic volume, left ventricular mass index, and mitral early/late diastolic flow velocity ratio (p<0.05). Strain and strain rate imaging study of the basal lateral wall of the left ventricle was higher in patients than in controls, at p=0.035 and p=0.008, respectively.

Conclusion: We found that superior systolic strain and strain rate imaging of the left ventricle indicated the presence of regional systolic function in the left ventricular wall. We suggest that left ventricle volume and mass index parameters might be more sensitive than the other conventional and strain/strain rate imaging parameters during childhood. However, the adulthood strain and strain rate imaging values may be lower than controls, exceeding the critical level of iron overload.

Key Words: Childhood, Regional left ventricular cardiac function, Strain, Strain rate imaging, Thalassemia Özet: Amaç: Beta talasemi majörlü hastalarda demir yüklenmesine bağlı kalp yetmezliği en sık ölüm nedenidir. Bu çalışmada beta talasemi majörlü çocuklarda standart ekokardiyografi ve strain rate görüntüleme ile myokardiyal fonksiyonun değerlendirilmesi amaçlanmıştır.

Address for Correspondence: Osman BAŞPINAR, MD, Gaziantep University Medical Faculty, Department of Pediatric Cardiology, 27310 Gaziantep, Turkey Phone: ++ 90 342 360 39 28 E-mail: osmanbaspinar@hotmail.com Received/Geliş tarihi : May 20, 2012 Accepted/Kabul tarihi : October 22, 2012

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Turk J Hematol 2013;30:283-289

Bay A, et al: Left Ventricular Regional Function with Thalassemia

Gereç ve Yöntemler: Çalışmamızda rutin ekokardiyografik değerlendirme ile beraber sol ventrikülün doku velosite

görüntülenmesi, strain/strain rate görüntülemesi beta talasemi majörlü 48 çocukta (19 kız, 29 erkek; 8.39±4.05 yıl) ve 22 sağlıklı çocukta (11 kız, 11 erkek; 8±3.72 yıl) ölçülmüştür.

Bulgular: Rutin ekokardiyografik değerlendirmede beta talasemi majörlü hastalarda daha geniş sol ventrikül sistol sonu çap,

diyastol ve sistol sonu hacmi olduğu, kitlesinin artmış olduğu ve mitral kapak erken/geç diyastolik akım oranının artmış olduğu görüldü (p<0.05). Sol ventrikül bazal lateral duvarı strain ve strain rate görüntülemesi değerleri hasta grubunda kontrole göre anlamlı yüksek değerdeydi (sırası ile p=0.035 ve p=0.008).

Sonuç: Sol ventrikül superior strain ve strain rate görüntülemesinde sol ventrikül duvarının bölgesel sistolik fonksiyonunu koruduğunu bulduk. Çocukluk çağında sol ventrikül hacim ve kitle indeksi göstergelerinin diğer geleneksel ve strain/strain rate görüntüleme göstergelerine göre daha duyarlı olabileceğini düşünmekteyiz. Bununla beraber, erişkin döneminde demir yüklenmesinin kritik seviyeyi aşması ile strain ve strain rate görüntüleme değerleri kontrol olgularınınkinden daha düşük bulunabilir.

Anahtar Sözcükler: Çocukluk çağı, Bölgesel sol ventriküler kardiyak fonksiyonlar, Strain, Strain hız görüntüleme, Talasemi

Introduction Cardiac failure due to iron overload remains the most common cause of death in patients with beta-thalassemia major, accounting for up to 71% of all deaths from this disease [1,2]. Cardiac complications are related to left ventricle dysfunction leading to gradual cardiac failure and cardiogenic death. Standard echocardiographic measurements may remain normal until late stages during this disease process.

A number of cardiological parameters of left ventricular (LV) function have therefore been assessed to determine their efficacy in identifying early myocardial iron overload in order to prevent heart failure and avert its poor prognosis through increased chelation therapy. The stored iron in the heart is heterogeneous. Regional myocardial asynchrony characterizes diastolic abnormalities even in the absence of changes in systolic functions reported in thalassemia patients and, thus, LV diastolic function may be more sensitive as an early marker of myocardial iron overload. A number of techniques used in clinical practice have been utilized to assess diastolic function in thalassemia major [3,4,5].

Strain rate imaging (SRI) is a new noninvasive echocardiographic method for the analysis of local myocardial stress. SRI is potentially superior to tissue Doppler imaging (TDI) in regional myocardial function assessment [6,7,8]. This study aimed to evaluate myocardial function in children with beta-thalassemia major by using the standard echocardiography technique TDI and SRI and to compare them with healthy controls.

Material and Method Patient Population Two distinct groups were studied. The patient group comprised 48 beta-thalassemia major patients (19 girls, 29 boys; median 8, mean 8.39±4.05 years). Thalassemia major patients were selected from cases in follow-up in the 284

Gaziantep University Pediatric Hematology Department and Gaziantep Children’s Hospital. The diagnosis of thalassemia was based on hemogram, blood smear, hemoglobin electrophoresis, and clinical evaluation. All patients were under chelation therapy with an oral iron chelator (deferasirox, 30 mg/kg/d) or a parenteral iron chelator (deferoxamine, 40 mg/kg 5 days a week). Serum ferritin levels were noted as ng/mL. Inclusion criteria were diagnosis of beta-thalassemia major, normal renal function, normal left ventricular functions, normal estimated pulmonary pressures by echocardiographic Doppler evaluation, and absence of congenital or acquired structural heart or lung diseases. All thalassemia patients had asymptomatic heart failure and were in New York Heart Association functional class I. They had been receiving blood transfusions since the age of 6 months to 2 years. Twenty-three (47.9%) of the patients had been splenectomized. Severity of iron overload was defined by serum ferritin level. Twenty-two healthy children (11 girls, 11 boys; 8±3.72 years) without history of cardiac disease were included in the study as a control group. All children were in normal sinus rhythm and had normal resting 12-lead electrocardiographs. All studies were performed in accordance with the rules of the local ethics committee. Informed consent was obtained from all participants prior to the study.

Echocardiography Conventional echocardiography (Vivid 3, GE Vingmed Ultrasound, Horten, Norway) and strain and strain rate imaging were performed by the same experienced pediatric cardiologist. Echocardiographic images were obtained in the parasternal long-axis and short-axis, and apical 2-chamber and 4-chamber views were obtained with standard transducer positions. Conventional echocardiographic measurements were done according to the American Society of Echocardiography guidelines [9]. Left ventricle mass was

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Bay A, et al: Left Ventricular Regional Function with Thalassemia

calculated using the Devereux formula [10]. Left ventricle mass index was calculated by dividing the left ventricle mass by body surface area. All examinations were videotaped and contemporary electrocardiography traces were recorded.

From the parasternal long-axis view of the LV end-diastolic and end-systolic diameters, interventricular septal and posterior wall thicknesses were expressed in millimeters. We measured LV end-systolic and end-diastolic (EDV) volumes from the apical 4-chamber view. Left ventricle fractional shortening (FS) and ejection fraction (EF) were measured using the Teichholz formula. LV filling was evaluated by pulse wave Doppler from the apical 4-chamber view with the sample volume position at the tips of the mitral valve, and velocities in early (E) and late (A) diastole were recorded, in addition to the calculation of the E/A ratio. Furthermore, myocardial velocities of the left ventricle were evaluated by tissue velocity imaging (TVI), strain (S), and SRI. Digital data of color TDI were transferred for offline analysis with EchoPAC-PC software (GE Vingmed Ultrasound). Scanning was performed longitudinally from the apex to acquire apical 2-chamber and 4-chamber views with a 3-MHz transducer and a frame rate of 100 ± 20 frames/s, depending on the heart rate, to minimize the noise level. Myocardial velocities were measured for the local motion of a tissue by TVI and S, and the local rates of deformations were measured by SRI. Longitudinal strain and strain rate in the basal septal, basal lateral, mid-septal, and mid-lateral wall were estimated by measuring the spatial velocity gradient over a computational area of 3 mm × 5 mm. Tissue velocity, strain, and strain rate were obtained at each site from 3 consecutive b eats and average values were calculated. End-diastole was defined as the R peak in echocardiography, and end-systole was defined as the end of the T wave in echocardiography. Peak positive systolic values were calculated from the extracted curve. From the 4-chamber view, the TVI sample volume was located sequentially at septal and lateral sites of the valvular ring.

Mean values of early diastolic, late diastolic, and systolic myocardial velocities were calculated as cm/s.

Statistical Analysis Statistical analysis was performed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). All data were expressed as mean ± standard derivation. Comparison of measurements between 2 groups was analyzed with the unpaired Student t-test.

Correlation coefficients between various measurements were determined by the linear regression analysis. A p value of less than 0.05 was considered significantly different.

Results The demographic characteristics of the patients with beta-thalassemia major and the control group are presented in Table 1. The 2 groups were similar regarding age and body surface area. Conventional echocardiographic examinations revealed that beta-thalassemia patients had larger LV endsystolic diameter (cm), end-diastolic and end-systolic volume (mL), LV mass index (g/m2), and mitral early/late diastolic flow velocity ratio. Mitral early-to-late diastolic flow velocity ratio as determined by conventional echocardiography was significantly higher in thalassemia major patients compared to the control group (Table 2).

In the TVI technique, left ventricle lateral late diastolic myocardial velocity was lower in beta-thalassemia patients compared to the control group, and the lateral early-tolate diastolic myocardial velocity ratio was higher in betathalassemia patients compared to the control group (Table 3).

Strain and SRI study included the following measurements: basal lateral and septal segments and mid-lateral and septal segments of the LV walls. The basal lateral walls’ S and SRI measurements were higher in patients compared to controls: p=0.035 and p=0.008, respectively (Tables 4 and 5).

Pearson’s correlation coefficient showed significant correlations between the serum ferritin levels and the following echocardiographic variables: LV end-diastolic volumes (r=0.288, p=0.049), LV end-systolic volumes (r=0.354, p=0.015), EDV (r=0.288, p=0.049), EF (r=-0.380, p=0.008), FS (r=-0.378, p=0.009), E (r=-0.302, p=0.044), septal early TVI (r=-0.329, p=0.026), and lateral systolic TVI (r=-0.330, p=0.025).

Table 1. Demographic and hematologic characteristics of the patient and control groups.

Patient group (n=48)

Control group (n=22)

p

Sex

19 girls, 29 boys

11 girls, 11 boys

Age (years)

8.4±4.5 (2-16)

8.0±3.7 (2-14)

0.702

Height (cm)

119.77±19.67 (85-156)

126.59±23.03 (88-175)

0.242

Weight (kg)

23.28±8.69 (10-42)

30.09±14.54 (12-66)

0.052

Body surface area (m2)

0.87±0.23 (0.54-1.34)

1.01±0.32 (0.53-1.64)

0.092

Mean serum ferritin (µg/L)

2543.75±1790.65 (454-7669)

-

Splenectomy (%)

23 patients (47.9%)

285

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Bay A, et al: Left Ventricular Regional Function with Thalassemia

Table 2. Conventional and basic systolic and diastolic echocardiographic parameters of the patient and control groups.

Patients (n=48)

Controls (n=22)

p

Left atrial diameter (cm)

2.44±0.47

2.41±0.44

0.824

Aortic diameter (cm)

2.13±0.35

2.18±0.42

0.635

Left atrial/aortic diameter ratio

1.16±0.19

1.12±0.16

0.412

Left ventricular end-diastolic diameter (cm)

3.89±0.67

3.59±0.51

0.052

Left ventricular end-systolic diameter (cm)

2.46±0.54

2.10±0.45

0.006*

Diastolic interventricular septal thickness (cm)

0.74±0.15

0.75±0.11

0.767

Systolic interventricular septal thickness (cm)

1.01±0.18

1.06±0.19

0.317

Diastolic posterior wall thickness (cm)

0.64±0.14

0.63±0.15

0.818

Systolic posterior wall thickness (cm)

0.95±0.15

0.90±0.12

0.123

Left ventricular end-diastolic volume (mL)

68.78±27.45

55.90±19.20

0.030*

Left ventricular end-systolic volume (mL)

23.97±14.50

16.66±6.90

0.006*

Left ventricular stroke volume (mL)

46.25±17.81

39.33±13.03

0.078

Left ventricular ejection fraction (%)

67.85±7.54

70.66±4.53

0.062

Left ventricular fractional shortening (%)

37.61±5.74

39.38±3.66

0.133

Left ventricular mass (g)

80.92±39.49

67.48±33.67

0.159

Left ventricular mass index (g/m2)

86.14±25.47

64.28±17.50

0.000*

Mitral early diastolic flow velocity (m/s)

1.00±0.20

1.00±0.12

0.990

Mitral late diastolic flow velocity (m/s)

0.51±0.13

0.56±0.10

0.082

Mitral early/late diastolic flow velocity ratio

2.09±0.51

1.78±0.21

0.001*

Data expressed as means±standard deviations, *statistically significant.

Discussion The most common cause of morbidity and mortality in thalassemia major patients is cardiomyopathy due to iron overload. Heart disease is mainly expressed by a particular cardiomyopathy that progressively leads to heart failure and death [1,2,11]. Iron toxicity has been attributed to the production of free oxygen radicals, as a result of the Fenton and Haber-Weiss reactions, which take place in the presence of free iron, the most toxic form of iron.

Since cardiac function remains normal until late stages in the spectrum of iron cardiomyopathy, other tools are necessary to anticipate and prevent iron cardiomyopathy.

Cardiac magnetic resonance imaging, tissue Doppler echocardiography, radionuclide angiography, and stress echocardiography are useful imaging studies for the detection of early cardiac dysfunction. Recently, tissue Doppler echocardiography and radionuclide angiography (with exercise or low-dose dobutamine stimulation) have been shown to detect regional wall motion abnormality, even in early-stage thalassemic patients [11]. This finding may also reflect patchy, nonhomogeneous deposition of iron in cardiac muscle.

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Fitchett et al. demonstrated the deposition of iron within the myocytes rather than the interstitium, and this cardiac iron deposition was patchy [12]. Lattanzi et al. and Vogel et al. showed that regional wall motion abnormalities and regional iron overload were seen in patients with betathalassemia major [13,14]. These regional changes can be easily detected with an echocardiographic assessment including TVI and S/SRI analysis.

The novel contribution of our study is the demonstration of the superiority of SI over the conventional echocardiographic parameters, LVEF and LVFS, in the detection of regional myocardial function. Our patients had superior systolic S and SRI of the LV lateral wall, indicating the presence of regional systolic function in the left ventricular wall. We suggest that LV volume and mass index parameters might be more sensitive than the other conventional and S/SRI echocardiographic parameters. However, adulthood S/SRI sensitivity may be lower than in the controls.

Regular clinical follow-up is strongly recommended for precocious detection of symptoms and signs of myocardial dysfunction [15]. On the hypothesis that longitudinal ventricular function may give early information about

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Bay A, et al: Left Ventricular Regional Function with Thalassemia

Table 3. Left ventricular tissue velocity imaging (cm/s) parameters of the patient and control groups.

Patients (n=48)

Controls (n=22) p

Septal systolic myocardial velocity

0.8 ± 0.2

0.8 ± 0.1

0.264

Septal early diastolic myocardial velocity

1.5 ± .0.3

1.6 ± 0.2

0.104

Septal late diastolic myocardial velocity

0.7 ± 0.6

0.7 ± 0.2

0.989

Septal early/late diastolic myocardial velocity ratio

2.34 ± 0.76

2.17 ± 0.57

0.328

Lateral systolic myocardial velocity

0.7 ± 0.2

0.8 ±.0.1

0.091

Lateral early diastolic myocardial velocity

1.9 ± 0.5

1.9 ± 0.3

0.923

Lateral late diastolic myocardial velocity

0.6 ± 0.2

0.8 ± 0.1

0.001*

Lateral early/late diastolic myocardial velocity ratio

3.10 ± 0.84

2.42 ± 0.55

0.001*

Data expressed as means±standard deviations, *statistically significant.

Table 4. Left ventricular strain (%) parameters of the patient and control groups.

Patients (n=48)

Controls (n=22)

p

Basal lateral wall

13.99±11.08

9.13±7.36

0.035*

Mid-lateral wall

9.00±6.97

11.22±7.27

0.238

Basal septal wall

11.09±8.58

11.28±9.30

0.936

Mid-septal wall

13.84±10.22

16.11±7.65

0.310

Data expressed as means±standard deviations.

Table 5. Left ventricular strain rate imaging (1/s) parameters of the patient and control groups.

Patients (n=48)

Controls (n=22)

p

Basal lateral wall

1.27±0.9

0.82±0.46

0.008*

Mid-lateral wall

0.97±0.66

0.70±0.49

0.064

Basal septal wall

1.16±0.88

1.17±0.58

0.981

Mid-septal wall

1.34±0.88

1.07±0.40

0.083

Data expressed as means±standard deviations.

impending ventricular function, we used TVI and S/ SRI measurements [6,7]. Regional findings suggest the presence of differences in myocardial function between the asymptomatic patients with beta-thalassemia major in the early stage of disease and the healthy controls. These regional changes can be easily detected with an echocardiographic assessment including TVI and S/SRI analysis.

Previous studies have shown that beta-thalassemia major is frequently associated with progressive LV dysfunction, leading to congestive heart failure. LV diastolic function, measured by traditional measurements such as transmitral flow recordings, is preserved until the final stages of diseases [16,17,18]. Conventional Doppler indices frequently lead to incorrect diagnosis.

In our patients, chronic iron overload resulted in significant increase in serum ferritin and significantly increased LVMI. However, EF and FS were not altered significantly in patients compared with controls, suggesting preserved systolic functioning until late stages of the disease.

This finding is consistent with those of Parale et al. and Seliem et al. [18,19].

Magri et al. suggested TDI and strain imaging as a potential method for detecting early stages of abnormal iron deposition [20]. Olivieri et al. and Bosi et al. demonstrated that serum levels of ferritin below 2500 ng/mL were considered as the safe level [1,21]. Our mean value of serum ferritin was at this level. Fitchett et al. demonstrated iron deposition within the myocytes rather than the interstitium and this cardiac iron deposition was patchy [12]. Lattanzi et al. and Vogel et al. showed that regional wall motion abnormalities and regional iron overload were seen in patients with beta-thalassemia major [13,14]. We found wall motion abnormality located in the basal lateral segment of the LV wall. There is no clear explanation for this observation.

S/SRI offers a new way of measuring regional tissue deformation noninvasively with both good spatial and temporal resolution. Authors have demonstrated it to be a tool that would appear to offer reproducible and reliable data [8]. Our findings suggest that S/SRI parameters have the potential to detect early myocardial changes that precede abnormal LV filling.

This study demonstrates the ability of S/SRI to accurately depict regional myocardial function in asymptomatic betathalassemia patients with normal systolic and diastolic function. LV dilatation or increased LVMI was balanced with increased basal segment S/SRI measurements. During this period, increased LV dilatation may progress to lower S/SRI values and to LV diastolic and systolic function disorders.

Until the critical level of iron overload, peak systolic strain and SRI may accurately reflect local systolic function, and we may say that iron overload is not related to a specific area of LV myocardium and that cardiac changes are possibly related to a threshold level of iron overload.

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Bay A, et al: Left Ventricular Regional Function with Thalassemia

While serum ferritin levels remain above 2500 µg/L, echocardiographic measurements such as basal TVI, S, and SRI parameters might show compensatory increase although LV dilatation starts with normal systolic function.

This compensation would probably be inadequate at greater ferritin levels and systolic and diastolic functions would globally decrease eventually. Therefore, clinical manifestations usually occur in adulthood rather than childhood. According to the Frank-Starling law, if heart volume increases, this compensation might be inadequate, and these parameters worsen through adulthood.

Accurate assessments of cardiac dysfunction and cardiac iron status are currently based on imaging techniques. The T2-star magnetic resonance is currently the best noninvasive modality to estimate iron in the heart and other organs [22].

In addition, it can be used for monitoring myocardial iron levels during iron chelation therapy. Unfortunately this examination cannot be performed in our hospital; we could not correlate this technique with the results.

One of the limitations of our study is that we could not compare the patients within every ferritin level. In asymptomatic patients, normal systolic and diastolic functions might be related to good LV compensation.

Strengthening the chelation treatment in this period may normalize these measurements. Even when LVEF and LVFS are normal, increased LV mass and volume may be seen.

In this study we found compensatory increase in S and SRI measurements, and these parameters would probably worsen first to be followed by cardiac failure appearing due to decreased LVEF and LVFS. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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2. Leonardi B, Margossian R, Colan SD, Powell AJ. Relationship of magnetic resonance imaging estimation of myocardial iron to left ventricular systolic and diastolic function in thalassemia. JACC Cardiovasc Imaging 2008;1:572-578.

3. Chrysohoou C, Greenberg M, Pitsavos C, Panagiotakos DB, Ladis V, Barbetseas J, Brili S, Singh S, Stefanadis C. Diastolic function in young patients with beta-thalassemia major: an echocardiographic study. Echocardiography 2006;23:38-44.

4. Aypar E, Alehan D, Hazirolan T, Gümrük F. The efficacy of tissue Doppler imaging in predicting myocardial iron load in patients with beta-thalassemia major: correlation with T2* cardiovascular magnetic resonance. Int J Cardivasc Imaging 2010;26:413-421.

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5. Balci YI, Gurses D. Detection of early cardiac dysfunction in patients with β-thalassemia major and thalassemia trait by tissue doppler echocardiography. Pediatr Hematol Oncol 2011;28:486-496.

6. Nesbitt GC, Mankad S. Strain and strain rate imaging in cardiomyopathy. Echocardiography 2009;26:337-344.

7. Nesbitt GC, Mankad S, Oh JK. Strain imaging in echocardiography: methods and clinical applications. Int J Cardiovasc Imaging 2009;25:9-22.

8. Voigt JU, Lindenmeier G, Werner D, Flachskampf FA, Nixdorff U, Hatle L, Sutherland GR, Daniel WG. Strain rate imaging for the assessment of preload-dependent changes in regional left ventricular diastolic longitudinal function. J Am Soc Echocardiogr 2002;15:13-19.

9. Sahn DJ, DeMaria A, Kisslo J, Weyman A. Recommendations regarding quantitation in M-mode echocardiography: results of a survey of echocardiographic measurements.

Circulation 1978;58:1072-1083.

10. Devereux RB. Detection of left ventricular hypertrophy

by M-mode echocardiography. Anatomic validation, standardization, and comparison to other methods.

Hypertension 1987;9:19-26.

11. Cohen AR, Galanello R, Pennell DJ, Cunningham MJ, Vichinsky E. Thalassemia. Hematology Am Soc Hematol Educ Program 2004:14-34.

12. Fitchett DH, Coltart DJ, Littler WA, Leyland MJ, Trueman T, Gozzard DI, Peters TJ. Cardiac involvement in secondary haemochromatosis: a catheter biopsy study and analysis of myocardium. Cardiovasc Res 1980;14:719-724.

13. Lattanzi F, Bellotti P, Picano E, Chiarella F, Mazzarisi A, Melevendi C, Forni G, Landini L, Distante A, Vecchio C. Quantitative ultrasonic analysis of myocardium in patients with thalassemia major and iron overload. Circulation 1993;87:748-754.

14. Vogel M, Anderson LJ, Holden S, Deanfield JE, Pennell DJ, Walker JM. Tissue Doppler echocardiography in patients with thalassaemia detects early myocardial dysfunction related to myocardial iron overload. Eur Heart J 2003;24:113-119.

15. Marcí M, Pitrolo L, Lo Pinto C, Sanfilippo N, Malizia R. Detection of early cardiac dysfunction in patients with Beta thalassemia by tissue Doppler echocardiography.

Echocardiography 2011;28:175-180.

16. Koren A, Garty I, Antonelli D, Katzuni E. Right ventricular cardiac dysfunction in beta-thalassemia major. Am J Dis Child 1987;141:93-96.

17. Kremastinos DT, Tsiapras DP, Tsetsos GA, Rentoukas EI, Vretou HP, Toutouzas PK. Left ventricular diastolic Doppler characteristics in beta-thalassemia major. Circulation 1993;88:1127-1135.

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18. Parale GP, Pawar SS, Tapare VS. Assessment of LV diastolic function in patients with beta-thalassemia major with special reference to E/Eann ratio. J Pediatr Hematol Oncol 2009;31:69-73.

19. Seliem MA, Al-Saad HI, Bou-Holaigah IH, Khan MN, Palileo MR. Left ventricular diastolic dysfunction in congenital chronic anaemias during childhood as determined by comprehensive echocardiographic imaging including acoustic quantification.

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21. Bosi G, Crepaz R, Gamberini MR, Fortini M, Scarcia S, Bonsante E, Pitscheider W, Vaccari M. Left ventricular remodelling, and systolic and diastolic function in young adults with beta thalassaemia major: a Doppler echocardiographic assessment and correlation with haematological data. Heart 2003;89:762-766.

22. Anderson LJ. Assessment of iron overload with T2* magnetic resonance imaging. Prog Cardiovasc Dis 2011;54:287-294.

20. MagrĂŹ D, Sciomer S, Fedele F, Gualdi G, Casciani E, Pugliese P, Losardo A, Ferrazza G, Pasquazzi E, Schifano E, Mussino E, Quaglione R, Piccirillo G. Early impairment of myocardial function in young patients with beta-thalassemia major. Eur J Haematol 2008;80:515-522.

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Research Article

DOI: 10.4274/Tjh.2012.0094

Endocrinological and Cardiological Late Effects Among Survivors of Childhood Acute Lymphoblastic Leukemia Akut Lenfoblastik Lösemi Tedavisi Sonrası Yaşayan Çocuklarda Endokrinolojik ve Kardiyolojik Geç Yan Etkiler Pakize Karakaya1, Şebnem Yılmaz2, Özlem Tüfekçi2, Mustafa Kır3, Ece Böber4, Gülersu İrken2, Hale Ören2 1Department

of Pediatrics, Dokuz Eylül University Faculty of Medicine, İzmir, Turkey

2Department

of Pediatric Hematology, Dokuz Eylül University Faculty of Medicine, İzmir, Turkey

3Department

of Pediatric Cardiology, Dokuz Eylül University Faculty of Medicine, İzmir, Turkey

4Department

of Pediatric Endocrinology, Dokuz Eylül University Faculty of Medicine, İzmir, Turkey

Abstract: Objective: Survival rates for childhood acute lymphoblastic leukemia (ALL) have significantly improved and late effects of therapy have been important in the follow-up of survivors. The objective of this study is to identify the endocrinological and cardiological late effects of ALL patients treated in our pediatric hematology unit.

Materials and Methods: Patients treated for ALL with BFM protocols after at least 5 years of diagnosis and not relapsed were included in the study. Endocrinological late effects (growth failure, obesity, insulin resistance, dyslipidemia, thyroid gland disorders, osteopenia/osteoporosis, and pubertal disorders) and cardiological late effects were evaluated. The study group was evaluated with anthropometric measurements, body mass index, and laboratory testing of fasting glucose, insulin, serum lipids, thyroid functions, and bone mineral densities. Echocardiography and pulsed wave Doppler imaging were performed for analysis of cardiac functions.

Results: Of the 38 ALL survivors, at least 1 adverse event occurred in 23 (60%), with 8 of them (21%) having multiple problems. Six (16%) of the survivors were obese and 8 (21%) of them were overweight. Subjects who were overweight or obese at the time of diagnosis were more likely to be overweight or obese at last follow-up. Obesity was more frequently determined in patients who were younger than 6 years of age at the time of diagnosis. Insulin resistance was observed in 8 (21%) subjects.

Insulin resistance was more frequently seen in subjects who had family history of type 2 diabetes mellitus. Hyperlipidemia was detected in 8 (21%) patients. Hypothyroidism or premature thelarche were detected in 2 children. Two survivors had osteopenia. Cardiovascular abnormalities occurred in one of the subjects with hypertension and cardiac diastolic dysfunction.

Conclusion: We point out the necessity of follow-up of these patients for endocrinological and cardiological late effects, since at least one adverse event occurred in most of our cases.

Key Words: Cardiotoxicity, Chemotherapy, Childhood, Endocrinology, Leukemia, Late effects

Address for Correspondence: Hale ÖREN, M.D., Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, 35340 Balçova, İzmir, Turkey GSM: +90 532 666 90 50 E-mail:hale.oren@deu.edu.tr Received/Geliş tarihi : July 25, 2012 Accepted/Kabul tarihi : May 8, 2013

290

Karakaya P, et al: Endocrinological and Cardiological Late Effects Among Survivors of Childhood Acute Lymphoblastic Leukemia

Turk J Hematol 2013;30:290-299

Özet: Amaç: Akut lenfoblastik lösemili (ALL) çocuklarda sağkalım oranı belirgin düzelmiş ve izlemlerde geç yan etkiler açısından değerlendirilmeleri önemli hale gelmiştir. Bu çalışmanın amacı çocuk hematoloji kliniğimizde tedavisi tamamlanan akut lösemi olgularında uzun dönemde gelişebilecek endokrinolojik ve kardiyolojik geç etkilerin belirlenmesidir.

Gereç ve Yöntemler: Lösemi tanısı aldığı tarihten itibaren en az beş yıl geçmiş, relaps gelişmemiş ve BFM protokolleri ile tedavi edilmiş hastalar endokrinolojik geç etkiler (boy kısalığı, obezite, insülin direnci, dislipidemi, tiroid hastalığı, osteopeni/ osteoporoz, puberte sorunları) ve kardiyolojik geç etkiler açısından değerlendirildi. Çalışma grubunda antropometrik ölçümler, vücut kitle indeksi, açlık kan şekeri, insülin, serum lipid ve tiroid fonksiyon testleri ve kemik mineral dansiteleri araştırıldı.

Kardiyak fonksiyonların analizinde ekokardiyografi ve pulsed Doppler görüntüleme kullanıldı.

Bulgular: 38 olgudan 23’ünde (%60) en az bir, 8 olguda (%21) ise birden fazla geç etki olduğu görüldü. Altı olgunun (%16) fazla kilolu, 8’inin (%21) obez olduğu belirlendi. Tanı anında fazla kilolu ya da obez olanlarda ve tedavi bitiminde fazla kilolu ya da obez olanlarda son değerlendirmede obezite daha yüksek orandaydı. Tanı anında altı yaşın altında olanlarda obezite daha fazlaydı. Sekiz olguda (%21) insülin direnci vardı. Obez ya da fazla kilolu olanlarda ve ailede tip 2 DM öyküsü bulunanlarda insülin direnci daha yüksek oranda bulundu. Sekiz olguda (%21) hiperlipidemi saptandı. Hipotiroidi veya prematür telarş birer olguda, osteopeni 2 olguda görüldü. Kardiyolojik geç etki olarak bir olguda hipertansiyon ve hafif diastolik disfonksiyon olduğu belirlendi.

Sonuç: Olguların çoğunda en az bir geç yan etki gözlendiğinden, bu çocukların endokrinolojik ve kardiyolojik geç etkiler açısından takip edilmelerinin gerekli olduğu ortaya çıkmaktadır .

Anahtar Sözcükler: Kardiyotoksisite, Kemoterapi, Çocukluk çağı, Endokrinoloji, Lösemi, Geç etki

Introduction Survival rates of children treated for acute lymphoblastic leukemia (ALL) have improved significantly over the last 4 decades. More than 85% of children with ALL are expected to become long-term survivors [1,2]. Many long-term survivors of childhood cancer will develop chronic physical or psychosocial problems as a result of their cancer or its therapy [3,4,5]. The follow-up of late effects has assumed increasing importance as there are large numbers of childhood leukemia survivors [6]. In this study we aimed to determine the endocrinological and cardiological late effects in childhood ALL survivors treated with BFM protocols in our pediatric hematology unit.

Materials and Methods Patients The patients included in this single-center crosssectional study fulfilled the following criteria: diagnosis of ALL in our center between January 1993 and December 2007; age under 18 years at the time of diagnosis; treatment in our center according to the ALL BFM-90 and ALL BFM95 treatment protocols; no relapse, not including treatment with bone marrow transplantation; and a follow-up time of ≥5 years after the first diagnosis. In order to collect data the survivors over 18 years of age or the legal guardians of younger survivors were called by phone and informed about the study. Those who agreed to participate in the study were called to the outpatient clinic and informed consent

was obtained. A total of 38 patients were included in the study. Data including information about age, sex, age at diagnosis, anthropometric measures at diagnosis, initial echocardiographic measurements, leukemia type, subtype and risk group according to BFM protocols, administered treatment protocols, history of central nervous system disease and cranial irradiation, details about date and dosage of cranial irradiation, cumulative anthracycline and steroid doses, and the time after diagnosis were obtained from the medical records of the patients.

ALL BFM-90 and BFM-95 Protocols The ALL BFM-90 treatment protocol was administered between 1993 and 1995 and the ALL BFM-95 was administered thereafter. In both the ALL BFM-90 and BFM-95 protocols, patients were assigned to a standardrisk group (SRG), medium-risk group (MRG), or high-risk group (HRG). Both protocols consist of 4 phases: induction, consolidation, reinduction, and continuation. In ALL BFM90 and ALL BFM-95, the cumulative anthracycline doses were 180 mg/m2, 240 mg/m2, and 300 mg/m2 in the SRG, MRG, and HRG, respectively. In ALL BFM-90, preventive cranial radiotherapy was indicated for T-cell ALL and the MRG and HRG with a dose of 12 Gy. Therapeutic irradiation dose for patients with initial involvement of the central nervous system was 12 Gy for patients 1 to 2 years old and 24 Gy for patients of more than 2 years of age. In ALL BFM-95, preventive radiotherapy was only indicated for T-cell ALL and the HRG, and the central nervous systemdirected therapy dose was reduced to 18 Gy from 24 Gy. 291

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Turk J Hematol 2013;30:290-299

Infants younger than 1 year were neither preventively nor therapeutically irradiated. The duration of treatment was 24 months for all patients in ALL BFM-90, 36 months for male MRG patients, and 24 months for all patients in “ALL BFM 95” [7].

Evaluation of Endocrinological Late Effects The standing height of the survivors at the time of diagnosis and at the time of the study was noted. Height percentiles were defined using the Centers for Disease Control and Prevention (CDC) growth charts [8]. Height below the third percentile was accepted as short stature. Body mass index (BMI) of the patients at the time of diagnosis and at the time of the study was calculated as weight in kilograms divided by height in meters squared. BMI percentile for age and sex was determined using reference standards from the CDC [8]. Subjects with BMI at or above the 95th percentile for their age were defined as obese and those with BMI at or above the 85th percentile for their age were defined as overweight. For adult subjects, overweight was defined as a BMI of 25-29, whereas obesity was defined as a BMI of ≥30.

Fasting insulin, glucose, and lipid levels were measured.

The estimate of insulin resistance was calculated by the homeostasis model assessment (HOMA) index. A cut-off HOMA level of >2.5 in children and >4.0 in adolescents was used to identify insulin-resistance status [9]. Total Table 1. The characteristics of 38 leukemia survivors.

Features Median age (years) At diagnosis (min-max) At the time of study (min-max) Sex

4.5 (1-17) 12 (7-28) n=38

%

Male

18

47

Female

20

53

ALL BFM-95

34

89

ALL BFM-90

4

11

Standard/medium-risk group

25

66

High-risk group

13

34

Protocol

cholesterol of >200 mg/dL and low-density lipoprotein cholesterol of >130 mg/dL were defined as high [10].

Cumulative steroid dose, history of cranial irradiation, familial history of type 2 diabetes mellitus, hyperlipidemia, and insulin resistance were all noted and tested for relationships with obesity.

Thyroid function tests (free T4 and thyroid-stimulating hormone) were used for evaluation of thyroid gland disorders. Thyroid ultrasonography was performed when goiter was detected in physical examination or in the case of abnormal thyroid functions.

Bone mineral density (BMD) was measured from the lumbar spine by dual energy X-ray absorptiometry. Ageand sex-based z-scores of BMDs indicating osteopenia or osteoporosis risks were calculated. Z-score values above -1 standard deviation (SD) were considered as normal, between -1 SD and -2 SDs as osteopenia, and below -2 SDs with clinically significant fracture history as osteoporosis [11].

Pubertal stages of the patients were assessed according to the Tanner stages [12].

Bone ages were evaluated according to the atlas of Greulich and Pyle [13]. Precocious puberty was defined as development of pubertal changes before 8 years of age in girls and 9 years of age in boys [14]. Delayed puberty was defined as the lack of any secondary sexual characteristics by the age of 13 years in girls and 14 years in boys [15].

If precocious puberty or pubertal delay was detected, further evaluations with measurement of follicle-stimulating hormone, luteinizing hormone, estradiol, and testosterone levels were performed. Menarche ages of female survivors were noted.

Evaluation of Cardiological Late Effects Systolic and diastolic blood pressure measurements were evaluated according to age, sex, and height. Blood pressure readings were classified as normal, prehypertensive, or hypertensive [16].

Cardiomegaly was defined as measurement of the cardiothoracic ratio at >50% on telecardiography. A standard 12-lead electrocardiography (ECG) was recorded

Risk group Table 2. Late effects in acute lymphoblastic leukemia survivors.

Cranial irradiation

n

(%)

12 Gy

13

34

Overweight

8

21

18 Gy

3

8

Obesity

6

16

None

22

58

Hyperlipidemia

8

21

Insulin resistance

8

21

Cumulative anthracycline dose ≤300 mg/m2

38

100

Osteopenia

2

5

>300 mg/m

0

0

Premature thelarche

1

3

292

Karakaya P, et al: Endocrinological and Cardiological Late Effects Among Survivors of Childhood Acute Lymphoblastic Leukemia

Turk J Hematol 2013;30:290-299

and analyzed for rhythm disturbances and ventricular hypertrophy findings. The echocardiographic imaging was performed with a Philips IE 33 equipped with 1- and 5-MHz transducers. A standardized M-mode, 2-dimensional, Doppler echocardiogram examination was performed with multiple orthogonal parasternal, apical, and subcostal views with the patient in the left lateral decubitus position.

All echocardiogram examinations and blood pressure measurements were performed by a pediatric cardiologist when patients were in resting position. To assess the capacity of the left ventricle, the following parameters were measured: left ventricular end-diastolic and end-systolic dimension and the septal and posterior wall thickness in diastole. Systolic function of the left ventricle was defined by 2 parameters: the ejection fraction and the fractional shortening. Diastolic function of the left ventricle was assessed with Doppler echocardiogram. Parameters included peak early mitral blood flow velocity (E) and peak mitral velocity during atrial contraction (A); the value for the E/A ratio was calculated.

An E/A ratio of <1.00 was considered as abnormal with moderate diastolic dysfunction. Deceleration time of peak early blood flow velocity and isovolumic relaxation time were measured [17,18,19]. An ejection fraction of 60% or more was considered as normal [20].

Statistics All statistical analyses were performed using SPSS 15. Categorical data were expressed as frequencies and percents and continuous variables were expressed as mean ± SD. Differences between groups for categorical variables were compared by chi-square and Fisher exact tests. For continuous variables, the Mann-Whitney U test was used.

Values of p<0.05 were selected as reflecting statistical significance.

Results Characteristics Of the 38 ALL survivors, 20 (53%) were females and 18 (47%) were males. The median age was 12 years (range: 7-28 years). They had been off therapy for an average of 8 years (range: 5-17 years, SD: 3.4 years). Table 1 shows the characteristics of the 38 patients.

At least 1 adverse event occurred in 23 (60%) of the 38 survivors, with 8 of them (21%) having multiple problems (Table 2).

Endocrinological Late Effects Six (16%) of the survivors were obese and 8 (21%) of them were overweight. Subjects who were overweight or obese at the time of diagnosis were more likely to be overweight or obese at the last follow-up. Twenty-six percent of the patients with normal BMI and 86% of overweight or obese patients at the time of diagnosis were overweight or obese at the last follow-up. The difference among the groups was statistically significant (p=0.003). The comparisons of some factors for the obese, overweight, and normal-weight groups are shown in Table 3. Obesity was more frequently detected in patients who were younger than 6 years of age at diagnosis. While 6 (27%) of the survivors under 6 years of age at diagnosis were obese, none of the survivors ≥6 years of age at diagnosis were obese (p=0.023) at the last follow-up. Sex and receiving of cranial radiotherapy had no significant association with the development of overweight or obesity (p>0.005). Insulin resistance was higher in the obese group. Cumulative corticosteroid doses (as hydrocortisone equivalents) in the MRG and HRG were 14,900 mg/m2 and 22,000 mg/ m2, respectively, and there was no significant difference for

Table 3. The comparison of some factors for obese, overweight, and normal-weight groups.

Features

Obese n=6

Overweight n=8

Normal n=24

p

Mean age at diagnosis, years <6 years (%) ≥6 years (%)

6 (100) 0

4 (50) 4 (50)

12 (50) 12 (50)

>0.005

Sex Male, n (%) Female, n (%)

4 (67) 2 (33)

4 (50) 4 (50)

11 (46) 13 (54)

>0.005

Mean age at the time of study, years

12±2.68

18.28±7.84

13.9±5.54

>0.005

Mean follow-up time, years

8.33±1.5

9.14±3.93

7.73±3.6

>0.005

Mean cumulative steroid dose, mg/m2

17,266

18.450

17.887

>0.005

History of cranial radiotherapy, n (%)

2 (33)

4 (50)

10 (41.6)

>0.005

Family history of type 2 diabetes mellitus, n (%)

3 (50)

3 (50)

8 (33.3)

>0.005

Hyperlipidemia, n (%)

1 (16.6)

3 (37.5)

1 (16.6)

>0.005

Insulin resistance, n (%)

3 (50)

2 (25)

3 (12.5)

<0.005 293

Turk J Hematol 2013;30:290-299

overweight and obesity with respect to cumulative doses between the risk groups (p=0.880). The mean height SDs of all leukemia survivors at the time of diagnosis and at the time of study were 0.89 and 0.45, respectively. The difference in the mean height SDs before and after treatment was not statistically significant (p=0.056). On the other hand, the mean height SDs were statistically lower in the group that received cranial radiotherapy (p=0.037).

Fasting blood glucose levels of all subjects were in normal range (mean: 84.5±7 mg/dL, range: 70-98). Insulin resistance was observed in 8 (21%) subjects. Insulin resistance was more frequently seen in obese subjects and in those who had family history of type 2 diabetes. While 7 (47%) of the patients with family history of type 2 DM had insulin resistance, only 1 (4%) patient with no family history of diabetes had insulin resistance (p=0.002). Sex, age at diagnosis, treatment in the HRG, and receiving of cranial radiotherapy had no significant associations with insulin resistance (p>0.005). Hyperlipidemia was detected in 8 (21%) of the 38 survivors. Of these 8 survivors with hyperlipidemia, 4 of them had normal BMIs while the others were obese/overweight. Triglyceride and total cholesterol levels were not significantly different between subjects who were treated with cranial radiotherapy and those who received chemotherapy only. There was also no significant difference in total cholesterol and triglyceride levels between obese/overweight subjects and subjects with a normal BMI. The mean age at menarche among female survivors was 11.9±0.78 years (range: 10-13). Premature thelarche was detected in one female patient. None of the patients had short stature. Hypothyroidism was observed in one of the survivors with normal thyroid ultrasonography and negative thyroid autoantibodies. The patient with hypothyroidism was not in the HRG and did not receive cranial radiotherapy, and hypothyroidism was detected during laboratory examination without any symptoms. Two survivors had osteopenia. They were males and older than 6 years of age at the time of diagnosis. One of them was in the HRG and received 12-Gy cranial radiotherapy.

Cardiological Late Effects The cardiovascular examinations, including physical examination, ECG, and echocardiogram, were normal for all patients at the time of diagnosis of ALL. Cardiovascular abnormality at follow-up was detected in only one female survivor with hypertension and cardiac diastolic dysfunction.

She was 5 years old at the time of diagnosis and 13 years at the time of the study. Her blood pressure was measured as 135/85 mmHg, her E/A ratio was <1.00, and her ejection fraction was 62% at the time of the study. She was in the SRG/MRG and her cumulative anthracycline dose was 240 mg/m2. She also had obesity, hyperlipidemia, and insulin resistance.

The distribution of late effects did not differ between the SRG/MRG and the HRG (p=0.766). There was also no statistically significant difference for the distribution of late 294

Karakaya P, et al: Endocrinological and Cardiological Late Effects Among Survivors of Childhood Acute Lymphoblastic Leukemia

effects between subjects who did or did not receive cranial radiotherapy (p>0.005).

Discussion Among our childhood leukemia survivors, 60% had at least 1 adverse event and 21% had multiple problems.

Oeffinger et al. [3] found that 62.3% of survivors had at least 1 chronic condition. Geenen et al. [4] reported that almost 75% of survivors had 1 or more adverse events.

Therapeutic exposures, including cranial radiotherapy and certain chemotherapy agents, will place ALL survivors at risk of development of serious late effects from their therapy [21]. These data emphasize the need for regular screening programs for childhood ALL survivors. The most common late effect detected in our ALL survivors was overweight/ obesity. We found a high prevalence of overweight/obesity in our study, consistent with the other studies in the literature [22,23,24]. The prevalence of obese and overweight children among the general pediatric population was reported as 3.7%-6.1% and 10.3%-12.2%, respectively, in 2 different studies from Turkey [25,26]. According to these results, the prevalence of overweight/obesity detected in our population of leukemia survivors is higher than the prevalence detected in the general Turkish population.We observed that children younger than 6 years of age at diagnosis were more likely to be obese at last follow-up. In previous studies it was reported that younger children at diagnosis had a greater likelihood of becoming overweight or obese as adults than did older children at diagnosis [22,24,27]. One important risk factor for obesity in ALL is the rapid growth in adiposity and early ‘adiposity rebound’, which appears to be a typical consequence of the treatment and may explain why younger patients with ALL, particularly those with onset of the disease during the toddler and preschool years, are at the highest risk of obesity [28]. Regarding the risk factors for obesity, no significant difference was found in BMIs between patients having received cranial irradiation and those who received chemotherapy only. These findings were consistent with the findings of other studies in the literature [29,30].

There was also no significant association between high cumulative steroid dose and overweight/obesity. Further investigations with large numbers of participants are required to understand the impact of genetic background, environmental factors, and treatment components on the development of obesity in childhood cancer survivors.

Short stature and a decrease in growth velocity is a wellknown side effect of acute leukemia treatment in children, especially in those who receive cranial radiotherapy. Growthhormone deficiency is the most common endocrinopathy detected after cranial radiotherapy [21]. Consistent with these findings, in this study the mean height SD at the last follow-up was significantly lower than the mean height SD at the time of diagnosis in those who received cranial

Karakaya P, et al: Endocrinological and Cardiological Late Effects Among Survivors of Childhood Acute Lymphoblastic Leukemia

radiotherapy. On the other hand, the mean height SD of all leukemia survivors, including those who did not receive cranial radiotherapy, was also lower at the last follow-up, but the difference was not statistically significant. Although chemotherapy, and especially corticosteroids, can result in growth delay during treatment, it is known that this is normally followed by a period of catch-up growth after completion of therapy [21].

Insulin resistance was highly prevalent among our ALL survivors, which is in accordance with earlier studies [31,32].

Oeffinger et al. [33] reported increased prevalence of insulin resistance in young adult survivors of childhood ALL for both sexes treated with and without cranial radiotherapy. In this study, 21% of the subjects were found to have insulin resistance. Sex, treatment with cranial radiotherapy, and BMI were not significantly associated with insulin resistance. We observed that obese subjects and those with family history of type 2 diabetes were more likely to have insulin resistance.

The development of insulin resistance is multifactorial.

Increased rates of insulin resistance in leukemia survivors may be the result of interactions between familial background and environmental factors such as treatment components or nutritional status.

Hyperlipidemia was detected in 8 (21%) of the 38 survivors. Survivors of childhood ALL are at risk for visceral obesity, dyslipidemia, insulin resistance, and hypertension [32,34,35]. These features of metabolic syndrome are linked to growth-hormone deficiency and in turn to cranial radiotherapy [36,37]. However, obesity and metabolic syndrome components (as well as growth-hormone deficiency) have also been described in patients treated with chemotherapy only [38].

In our study, none of the survivors were found to have short stature. Growth-hormone deficiency is reported as the most common endocrinopathy detected after cranial radiotherapy [39]. It is more frequent with radiotherapy doses higher than 24 Gy, but it has been observed with doses as low as 18 or 10 Gy given as a single dose as part of total-body irradiation [39,40]. In addition to higher cranial radiotherapy doses, risk factors for growth-hormone deficiency and short stature include younger age at diagnosis and female sex [39,40,41]. The reason for not detecting short stature in our patients might be due to lower cranial radiotherapy doses (â&#x2030;¤18 Gy).

Precocious puberty is a well-described late effect of cranial radiotherapy at doses of 18 to 24 Gy, and is more common in girls [42,43,44]. However, most female ALL survivors experience menarche at a normal age [45,46].

Premature thelarche was detected in one survivor in our study. The small number of events in this study might be due to relatively lower cranial radiotherapy doses. The mean age at menarche among our ALL survivors was 11.9Âą0.78 years. Other studies from Turkey reported the mean age at menarche as 12.4 and 13.04, respectively [47,48]. The mean self-reported age of our leukemia survivors at menarche was similar to Turkish population norms.

Turk J Hematol 2013;30:290-299

Studies that have examined thyroid dysfunction following ALL therapy have either reported no significant thyroid function abnormalities [49,50] or more subtle compensated hypothyroidism [51,52,53]. The Childhood Cancer Survivor Study Group reported that cranial radiotherapy alone was insufficient to induce reportable hypothyroidism, whether central or primary in origin. On the other hand, they also noted that craniospinal radiotherapy used in ALL was associated with a higher amount of thyroid gland exposure and was strongly associated with subsequent hypothyroidism [54]. In our study, none of the patients had received craniospinal radiotherapy, and hypothyroidism was detected in only one patient of the MRG who had not received cranial radiotherapy.

Among our ALL survivors, 2 were found to have osteopenia. Osteonecrosis (avascular necrosis) and decreased BMD are both reported to be associated with treatment of ALL, specifically with prolonged corticosteroid exposure [21]. Decreased BMD is known to be caused by a number of factors, including the disease itself, concurrent serious infections, poor nutrition, decreased physical activity, and abnormalities in vitamin D metabolism, in addition to various components of treatment (especially glucocorticoids) and radiotherapy [55]. Halton et al. [56] found that BMD diminished during the first 2 years of chemotherapy in patients older than 11 years old at the time of diagnosis but remained stable in those who were younger.

Both Van der Sluis et al. [57] and Lequin et al. [58] also reported no significant long-term effects on height, BMD, or lean body mass in children with ALL treated with highdose dexamethasone and methotrexate but not with cranial irradiation. The small number of cases of osteopenia in this study might be related to the younger age of the subjects at diagnosis (84% of them were <10 years of age at diagnosis) and the relatively lower doses of cranial radiotherapy. Only 3 (8%) of the subjects received 18 Gy of cranial radiotherapy and 13 (34%) of them received 12 Gy. In this study, clinical cardiovascular abnormality was detected in one subject with hypertension and cardiac diastolic dysfunction. Through a variety of different mechanisms, it appears that survivors of childhood ALL have an increased prevalence of several cardiovascular risk factors and thus are at increased risk for developing cardiovascular disease. Cardiotoxicity is associated with exposure to anthracyclines and irradiation to the heart, particularly at a young age [59,60]. Doses of â&#x2030;Ľ300 mg/m2 of doxorubicin or daunorubicin are associated with the highest risk of cardiotoxicity, usually in the form of left ventricular dysfunction [61]. ALL survivors, particularly those who receive more than 24 Gy of cranial radiotherapy, may be at an even further increased risk for cardiac events due to additional cardiovascular risk factors like obesity, physical 295

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Turk J Hematol 2013;30:290-299

inactivity, and metabolic syndrome [62]. In a study from Turkey including 55 ALL survivors and 38 healthy controls with a mean follow-up time of 34.1Âą21 months, no overt cardiotoxicity was reported [63]. Rammeloo et al. [64] compared cardiac functions in ALL survivors who were treated with 100 mg/m2 of daunorubicin and in survivors who were not treated with anthracycline. They detected no difference in left ventricular functions between the 2 groups. In our study, HRG patients (34%) received 300 mg/ m2 anthracycline and the remaining (66%) had lower doses.

The small number of cardiovascular events in this study might be due to the moderate doses of anthracyclines and a relatively shorter follow-up time with an average of 8 years, since it is well established that the heart compensates for about 5-15 years following therapy in most survivors with anthracycline-induced cardiomyopathy [60].

There are some limitations of this study. One limitation is the relatively small size of the study group. Further investigations with large numbers of participants are needed for detection of the late effects in a wide spectrum in leukemia survivors. Another limitation is related to the adequacy of the methods used to evaluate cardiological late effects. The methods that we performed to evaluate cardiological late effects in this study were not sensitive enough to detect subclinical cases. There are some other methods reported in the literature to evaluate subclinical cardiological toxicity, including tissue Doppler imaging, myocardial performance index, and some biomarkers like B-type natriuretic peptide (BNP) and proBNP [63,65,66]. A more comprehensive, detailed detection of cardiological late effects including subclinical changes could have been performed if we had been able to use these mentioned methods.

In conclusion, 60% of ALL survivors had at least one endocrinological or cardiological late effect. Survivors of childhood leukemia are at risk of developing endocrinological and cardiological late effects as a result of exposure to chemotherapy and radiotherapy. Therefore, long-term, regular, and comprehensive follow-up of childhood ALL survivors is recommended for early recognition of these late effects and to prevent development of worse outcomes.

Conflicts of Interest Statement The authors have no conflicts of interest to declare.

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43. Ogilvy-Stuart AL, Clayton PE, Shalet SM. Cranial irradiation and early puberty. J Clin Endocrinol Metab 1994;78:12821286.

44. Quigley C, Cowell C, Jimenez M, Burger H, Kirk J, Bergin M, Stevens M, Simpson J, Silink M. Normal or early development of puberty despite gonadal damage in children treated for acute lymphoblastic leukemia. N Engl J Med 1989;321:143-151.

45. Chow EJ, Friedman DL, Yasui Y, Whitton JA, Stovall M, Robison LL, Sklar CA. Timing of menarche among survivors of childhood acute lymphoblastic leukemia: a report from the Childhood Cancer Survivor Study. Pediatr Blood Cancer 2008;50:854-858.

46. Mills JL, Fears TR, Robison LL, Nicholson HS, Sklar CA, Byrne J, Menarche in a cohort of 188 long-term survivors of acute lymphoblastic leukemia. J Pediatr 1997;131:598602.

47. Semiz S, Kurt F, Kurt DT, Zencir M, Sevinç O. Pubertal development of Turkish children. J Pediatr Endocrinol Metab 2008;21:951-961.

48. Ekerbicer HC, Celik M, Kiran H, Kiran G. Age at menarche in Turkish adolescents in Kahramanmaras, Eastern Mediterranean region of Turkey. Eur J Contracept Reprod Health Care 2007;12:289-293.

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acute leukemia. Pediatr Hematol Oncol 1988;5:35-38.

50. Lando A, Holm K, Nysom K, Rasmussen AK, FeldtRasmussen U, Petersen JH, Müller J. Thyroid function in survivors of childhood acute lymphoblastic leukaemia: the significance of prophylactic cranial irradiation. Clin Endocrinol (Oxf) 2001;55:21-25.

51. Shalet SM, Beardwell CG, Twomey JA, Jones PH, Pearson D. Endocrine function following the treatment of acute leukemia in childhood. J Pediatr 1977;90:920-923.

52. Pasqualini T, McCalla J, Berg S, Poplack DG, Rose SR, Nisula BC, Cassorla F. Subtle primary hypothyroidism in patients treated for acute lymphoblastic leukemia. Acta Endocrinol (Copenh) 1991;124:375-380.

53. Mohn A, Chiarelli F, Di Marzio A, Impicciatore P, Marsico S, Angrilli F. Thyroid function in children treated for acute lymphoblastic leukemia. J Endocrinol Invest 1997;20:215219.

54. Chow EJ, Friedman DL, Stovall M, Yasui Y, Whitton JA, Robison LL, Sklar CA. Risk of thyroid dysfunction and subsequent thyroid cancer among survivors of acute lymphoblastic leukemia: a report from the Childhood Cancer Survivor Study. Pediatr Blood Cancer 2009;53:432427.

55. Alikasifoglu A, Yetgin S, Cetin M, Tuncer M, Gumruk F, Gurgey A, Yordam N. Bone mineral density and serum bone turnover markers in survivors of childhood acute lymphoblastic leukemia: comparison of megadose methylprednisolone and conventional-dose prednisolone treatments. Am J Hematol 2005;80:113-118.

56. Halton JM, Atkinson SA, Fraher L, Webber C, Gill GJ, Dawson S, Barr RD. Altered mineral metabolism and bone mass in children during treatment for acute lymphoblastic leukemia. J Bone Miner Res 1996;11:1774-1783.

57. Lequin MH, van der Shuis IM, Van Rijn RR, Hop WC, van ven Huevel-Eibrink MM, MuinckKeizer-Schrama SM, van Kuijk C. Bone mineral assessment with tibial ultrasonometry and dual-energy X-ray absorptiometry in long-term survivors of acute lymphoblastic leukemia in childhood. J Clin Densitom 2002;5:167-173.

58. van der Sluis IM, van den Heuvel-Eibrink MM, Hählen K, Krenning EP, de Muinck Keizer-Schrama SM. Bone mineral density, body composition, and height in long-term survivors of acute lymphoblastic leukemia in childhood.

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60. Lipshultz SE, Lipsitz SR, Sallan SE, Dalton VM, Mone SM, Gelber RD, Colan SD. Chronic progressive cardiac dysfunction years after doxorubicin therapy for childhood acute lymphoblastic leukemia. J Clin Oncol 2005;23:26292936.

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SE. Relationship between cumulative anthracycline dose and late cardiotoxicity in childhood acute lymphoblastic leukemia. J Clin Oncol 1998;16:545-550.

62. Oeffinger KC. Are survivors of acute lymphoblastic leukemia (ALL) at increased risk of cardiovascular disease? Pediatr Blood Cancer 2008;50(Suppl 2):462-467.

63. Gulen H, Kazanci E, Mese T, Uzunkaya D, Erbay A, Tavli V, Vergin C. Cardiac functions by myocardial performance index and QT dispersion in survivors of childhood lymphoblastic leukaemia. Minerva Pediatr 2007;59:107113.

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66. Ruggiero A, De Rosa G, Rizzo D, Leo A, Maurizi P, De Nisco A, Vendittelli F, Zuppi C, Mordente A, Riccardi R. Myocardial performance index and biochemical markers for early detection of doxorubicin-induced cardiotoxicity in children with acute lymphoblastic leukaemia. Int J Clin Oncol 2012. Epub ahead of print 2012 Aug 22.

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Rammeloo LA, Postma A, Sobotka-Plojhar MA, BinkBoelkens MT, Berg A, Veerman AJ, Kamps WA. Low-dose daunorubicin in induction treatment of childhood acute lymphoblastic leukemia: no long-term cardiac damage in a randomized study of the Dutch Childhood Leukemia Study Group. Med Pediatr Oncol 2000;35:13-19.

299

Research Article

DOI: 10.4274/Tjh.2011.0005

Significance of Neuropilin-1 Expression in Acute Myeloid Leukemia Ciddi, Dirençli,Öldürücü, Kazanılmış Aplastik Aneminin Klinik Seyrine E Uygulamasının Katkısı Tarif H. Sallam1, Manal A. Shams Eldin El Telbany1, Hanan M. Mahmoud1, Mutea A. Iskander2 1Clinical

Pathology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt

2Clinical

and Chemical Pathology, Faculty of Medicine, Sanaa University, Sanaa, Yemen

Abstract: Objective: Neuropilin-1 is a vascular endothelial growth factor receptor that acts as a mediator of angiogenesis. Its importance in hematological malignancies such as acute myeloid leukemia (AML) remains to be elucidated. The aim of this study was to evaluate the significance of neuropilin-1 expression in AML patients by both flow cytometry and real-time polymerase chain reaction (PCR) in regard to its diagnostic and prognostic values.

Materials and Methods: Bone marrow aspirates of 44 patients with de novo AML and 12 relapsed AML patients were examined in this study. Ten subjects with nonhematological malignancy serving as the control group were also included.

Results: Neuropilin-1 expression by flow cytometry showed a highly significant increase in de novo and relapsed AML patients with a mean of 27.1±17.5% and 21.5±16.6%, respectively, compared to control group’s mean of 3.4±1.9%. A cut-off value of 6% was established as differentiating patients from the control group. By real-time PCR, no statistical significance was found in de novo and relapsed AML patients with a mean of 1.9±3.6 IU/L and 0.3±0.2 IU/L, respectively, compared to the control group’s mean of 0.3±0.1 IU/L. Neuropilin-1 surface expression by flow cytometry showed a significant correlation with total leukocyte count and a negative correlation with hemoglobin level in de novo AML patients. In relapsed AML patients, positive significant correlations were found with age, bone marrow blast percentage, and CD14. Neuropilin-1 mRNA level by real-time PCR showed a positive significant correlation with peripheral blood blast percentage and CD117 and a negative correlation with hemoglobin level in de novo AML patients. In relapsed patients, a positive correlation was found with lactate dehydrogenase.

Conclusion: Neuropilin-1 can be used as a tool for diagnosis and prognosis in AML patients.

Key Words: Neuropilin-1, Acute myeloid leukemia Özet: Amaç: Neuropilin-1 anjiyogenez mediatörü olarak rol oynayan vasküler endotelyal büyüme faktörü reseptörüdür. Akut miyeloid lösemi (AML) gibi hematolojik malignitelerdeki önemi tam aydınlatılamamıştır. Bu çalışmanın amacı; AML hastalarındaki hem akım sitometri hem de gerçek-zamanlı polimeraz zincir reaksiyonu (PZR) ile ile tanısal ve prognostik değerleri göz önünde bulundurularak değerlendirilmesidir.

Address for Correspondence: Tarif H. SALLAM, M.D, Ain Shams University Hospitals. Lotfy El-sayed Street, Abbaseya, Cairo, Egypt GSM: +002 02 24824073 E-mail: hananmm74@yahoo.comv Received/Geliş tarihi : October 16, 2011 Accepted/Kabul tarihi : May 16, 2013

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Sallam TH, et al: Significance of NRP-1 Expression in AML

Turk J Hematol 2013;30:300-306

Gereç ve Yöntemler: Bu çalışmada 44 de novo AML ve relaps olmuş 12 hastanın kemik iliği aspiratları incelendi. Hematoloji dışı malignitesi olan 10 kişinin örnekleri de kontrol grubu olarak dahil edildi.

Bulgular: Akım sitometri ile saptanan neuropilin-1 sunumu, de novo ve relaps AML hastalarında, sırasıyla, ortalama %27.1±17.5 ve %21.5±16.6 bulundu ve kontrol grubuna (ortalama %3.4±1.9) göre son derece anlamlı artış gösterdi.

Hastalardan kontrol grubunu ayırmada cut-off değeri %6 olarak belirlenmiştir. Gerçek-zamanlı PZR ile saptanan neuropilin-1 sunumu de novo ve relaps AML hastalarında, sırasıyla, ortalama 1.9 ±3.6 IU/L ve 0.3±0.2 IU/L bulundu ve bu değerler kontrol grubuna (ortalama 0.3±0.1 IU/L) göre istatistiksel olarak anlamlı değildi. De novo AML hastalarında akım sitometri ile saptanan neuropilin-1 yüzey sunumu, toplam lökosit sayısı ile anlamlı korelasyon ve hemoglobin düzeyi ile negatif korelasyon göstermekteydi. Relaps AML hastalarında ise yaş, kemik iliğindeki blast oranı ve CD14 ile aralarında anlamlı korelasyon bulunmuştur. De novo AML hastalarında gerçek- zamanlı PZR ile bakılan neuropilin-1 mRNA düzeyi, periferik kandaki blast yüzdesi ve CD117 ile pozitif anlamlı korelasyon, hemoglobin düzeyi ile de negatif korelasyon göstermiştir. Relaps hastalarda laktat dehidrogenaz ile pozitif korelasyon bulunmuştur.

Sonuç: Neuropilin-1, AML hastalarında tanı ve prognozda bir araç olarak kullanılabilir.

Anahtar Kelimeler: Neuropilin-1, Akut Miyeloid Lösemi Introduction Neuropilins are single-pass transmembrane glycoproteins that were found to be receptors for the vascular endothelial growth factor (VEGF) family of angiogenesis factors, suggesting that the receptor could be involved in blood vessel formation [1,2].

VEGF is an important cytokine that contributes to disease evolution in various myeloid neoplasms. In particular, VEGF has been described as a mediator of leukemia-associated angiogenesis as well as an autocrine growth regulator in leukemic cells [1].

In contrast to physiological angiogenesis, the number of studies suggesting a role for neuropilins in human tumor growth and angiogenesis is increasing rapidly [2].

Neuropilin-1 (NRP-1) was found to be a receptor or coreceptor for the specific isoform VEGF165 and expressed on endothelial cells (ECs) and several types of tumor cells.

VEGF, as a key factor for angiogenesis and tumor growth, exerts its functions mainly through activation of 2 tyrosine kinase receptors: VEGFR-1 (Flt 1) and VEGFR-2 (KDR).

Unlike Flt 1 and KDR, NRP-1 does not possess intrinsic tyrosine kinase activity and thus forms a complex with KDR to transmit signals of VEGF. When co-expressed with KDR on ECs, NRP-1 promotes the binding of VEGF165 to KDR and enhances VEGF-mediated mitogenic and chemotactic activity [3].

Acute myeloid leukemia (AML) is the most common acute leukemia affecting adults, and its incidence increases with age [4]. Much evidence indicates that bone marrow (BM) neoangiogenesis, orchestrated by different angiogenic growth factors, is implicated in the pathogenesis of AML. VEGF and its receptors are major regulators in neoangiogenesis in AML [5].

Experimental and clinical evidence indicates that BM cells participate in the process of new blood vessel formation.

However, the molecular mechanisms underlying their recruitment and their exact role are still elusive. BM cells

are recruited to the sites of neoangiogenesis through the NRP-1 receptor and they are essential for the maturation of the activated endothelium and the formation of arteries. The 165-aa isoform of VEGF, which activates the endothelium and recruits NRP-1 myeloid cells, is a powerful arteriogenic agent.

In contrast, neither the shortest VEGF121 isoform, which does not bind NRP-1 and thus does not recruit BM cells, nor semaphorin 3A, which attracts cells but inhibits endothelial activation, is capable of sustaining arterial formation [6].

Thus, this work was planned to study the diagnostic and prognostic significance of NRP-1 expression in AML patients by both flow cytometry and real-time polymerase chain reaction (PCR).

Materials and Methods Subjects This study was carried out on 56 diagnosed AML patients attending the Hematology/Oncology Clinic of the Ain Shams University Hospitals. Forty-four newly diagnosed AML patients (18 males and 26 females) ranged in age from 18 to 70 years with a mean of 39.5±13.1 years. These patients comprised 6 with M0, 10 with M1, 10 with M2, 8 with M3, 2 with M4, 2 with M5, 2 with M6, and 4 with M7 French-American-British (FAB) subtypes. Twelve relapsed AML patients (6 males and 6 females) were also included in this study; their ages ranged from 24 to 70 years with a mean of 44.6±17.2 years. These patients included 2 with M1, 4 with M2, 2 with M4, 2 with M5, and 2 with M6 FAB subtypes; none of them had M0 or M3 FAB subtypes. The diagnosis of AML was based on the standard morphological and immunophenotyping (IPT) criteria [5,6]. Ten subjects with nonhematological malignancy matching the patients in age and sex were recruited to serve as the control group.

The study design was approved by the Research Ethics Committee of the Ain Shams University Faculty of Medicine and was conducted according to the Helsinki Declaration of 1975. All samples were taken after obtaining written consent from the patients and the controls.

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All subjects and controls underwent the following procedures: A- Detailed taking of history and clinical examination: Stress was placed on lymphadenopathy and hepatosplenomegaly.

B- Laboratory investigations: 1- Complete blood count (CBC) using a Coulter Counter T660 (Coulter, Hialeah, FL, USA) with examination of Leishman-stained peripheral blood smears for differential leukocyte count and peripheral blood blast count (PB blast %).

2- BM aspiration with examination of Leishman-stained films for bone marrow blasts.

3- IPT analysis and NRP-1 expression of the BM aspirate using a Coulter EPICS XL flow cytometer. The acute leukemia panel of monoclonal antibodies used for IPT was either fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled. The monoclonal antibodies used were T-cell lymphoid markers (CD2, CD3, CD5, and CD7), B-cell lymphoid markers (CD10, CD19, and CD20), myeloid markers (CD13, CD33, CD14, CD61, CD117, and myeloperoxidase), and common progenitor markers (CD34 and HLA-DR).

4- Detection of NRP-1 by real-time PCR on BM using the SLAN real-time PCR system.

Sampling Two milliliters of PB was delivered into an EDTA vacutainer tube (1-2 mg/mL blood) for CBC. Four milliliters of BM aspirate was collected from each patient and control under completely aseptic conditions using sterile vacutainers and delivered into 2 EDTA vacutainer tubes. vSamples were divided as follows: 2 mL for IPT and NRP-1 surface expression by flow cytometry and the other 2 mL for RNA extraction, amplification, and NRP-1 gene detection by realtime PCR. Immunophenotyping For all of the monoclonal antibodies, including an antibody for the isotype-matched negative control monoclonal antibody, 50 µL of BM sample was pipetted into several tubes. Next, 5 µL of FITC- or PE-labeled MoAbs (provided by Coulter, except for the PE-labeled anti-NRP-1 from R&D Company, UK) was pipetted into the test tube and 5 µL of isotype-matched conjugated immunoglobulins was pipetted into the control tube to determine the nonspecific binding. Cells and monoclonal antibody mixtures were gently vortexed and both tubes were incubated for 15 min at room temperature in the dark. One milliliter of lysing solution was added to each tube for 2-5 min and mixed well.

At the end, 1 mL of phosphate-buffered saline was added.

The blasts were electronically gated using forward scatter against side scatter. At least 20% of cells should express the marker to be considered positive for the IPT panel, except 302

Sallam TH, et al: Significance of NRP-1 Expression in AML

for CD34, which is considered positive if only ≥10% of cells are positive. For NRP-1 expression, both the percentage and intensity of cells were determined.

Real-Time PCR of NRP-1 Total RNA was extracted from the blood specimens using the NucleoSpin RNA blood kit (Macherey-Nagel, Germany) following the manufacturer’s instructions. The total RNA was transcripted to cDNA and amplified using RNA-direct SYBR Green Real Time PCR master mix (Toyobo, Japan).

This was done by using a 2X Master Mix for 1-step real-time PCR using a thermostable DNA polymerase derived from Thermus thermophilus (Tth). Tth DNA polymerase exhibits reverse transcriptase activity in the presence of Mn2+ ions.

This system allows for 1-step real-time PCR, including reverse transcription and PCR steps. Two micrograms of total RNA was reverse-transcribed into cDNA and the PCR components of the 20 µL of total volume included 10 µL SYBR Green master mix, 2.5 mM Mn, and 0.25 µM of each primer. The sequences of the forward and reverse primers for NRP-1 were AAG ACC TTC TGC CAC TGG GAA CAT and AGT TGC CAT CTC CTG TGT GAT CCT, respectively [7], with a total amplicon of 103 base pairs.

The real-time PCR thermal cycler program was done using LG AdvanSure SLAN (LG Life Science, South Korea) at 90 °C for 30 s, reverse transcription at 61 °C for 20 min, and predenaturation at 95 °C for 60 s following 45 PCR cycles of 95 °C for 15 s, 60 °C for 15 s, and 74 °C for 45 s followed by melting curve analysis. The housekeeping β-actin gene was used for normalization. The primer sequences for β-actin were forward CCA AGC CCA ACC GTG AGA AGA T and reverse CAA CGT TCC GTG AGG ATC TTC A. Statistical Analysis Data were analyzed using SPSS 16 and are expressed as mean values ± standard deviations. The Kruskal-Wallis test was used for comparison between more than 2 groups and the Wilcoxon rank-sum test was used for comparisons between 2 nonparametric results. The Pearson correlation coefficient test was used for correlations. Receiver operating characteristic (ROC) curves were used to detect the best cut-off levels and Kaplan-Meier curves for survival were calculated.

Results The results of this study are summarized in Table 1 and Figures 1,2,3,4,5.

Results of NRP-1 by Flow Cytometry (Figures 1 and 2) A diagnostic cut-off level of NRP-1 percentage expression was detected in this study, discriminating AML patient groups (either comparing patients as a whole or after dividing patients into de novo and relapsed groups) from the healthy control group, and this level was found to be 6% of cells expressing NRP-1 surface antigen.

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Sallam TH, et al: Significance of NRP-1 Expression in AML

NRP-1 percentage expression for all AML cases was 25.9±17.2% taking all patients as a single group, 27.1±17.5% in the group of patients with de novo AML, and 21.5±16.6% in the group of patients with relapsed AML. Mean fluorescence intensity (MFI) levels of NRP-1 was 1.7±0.7 taking all patients as a single group, 1.7±0.8 in the group of patients with de novo AML, and 1.5±0.4 in the group of patients with relapsed AML. The mean percent positivity of NRP-1 detected by flow cytometry in the control group was 3.4±1.9%, while for MFI the mean level was 5.4±3.9.

There was a highly significant difference in NRP-1 surface expression, both as percent positivity or in its MFI, comparing the control group to patient groups after categorizing patients into de novo and relapsed groups (p<0.001 in all comparisons).

However, comparing the de novo and relapsed patient groups, no statistical significance was revealed in NRP-1 expression concerning both percentage and MFI (p=0.401 and p=0.954, respectively) (Table 1).

In de novo AML patients, a highly significant positive correlation was found between NRP-1 percentage expression and total leukocyte count (TLC) (r= 0.577, p=0.005), and a negative correlation was seen between NRP-1 percentage expression and hemoglobin (Hb) levels (r= -0.534, p=0.01), with no significant correlation found between NRP-1 percentage expression and any other parameters including age, platelet count, lactate dehydrogenase (LDH), PB blast %, CD33, CD13, CD14, CD117, CD61, myeloperoxidase, CD2, CD5, CD7, CD34, HLA-DR, CD10, and survival time.

Relapsed AML patients showed a positive and highly significant correlation between NRP-1 percentage expression and age (r= 0.986, p<0.001) and positive relationships between NRP-1 percentage and both BM blast % and CD14 (r= 0.882,0.899 respectively and p=0.02, 0.015 respectively). However, for other parameters such as TLC, Hb, platelet count, PB blast %, LDH, CD33, CD13, CD117, CD61, myeloperoxidase, CD2, CD5, CD7, CD34, HLA-DR, and CD10, no significant correlations were found.

Results of NRP-1 by Real-Time PCR (Figures 3 and 4) The mean value of the NRP-1 RNA level in de novo and relapsed AML patients was 1.9±3.6 IU/L and 0.3±0.2 IU/L, respectively, while the mean level was 0.3±0.1 IU/L in the control group.

No statistically significant difference was found comparing NRP-1 RNA levels between patients and the control group for the 2 groups of de novo patients and relapsed patients (p=0.745 and p=0.661, respectively) (Table 1).

In the de novo AML patient group, a positive and highly significant correlation was found between NRP-1 RNA level and TLC (r=0.551, p=0.008), as well as between NRP-1 RNA and both PB blast % and CD117 (r= 0.452, 0.451 respectively and p=0.035, 0.035 respectively), but a negative significant correlation was detected with Hb (r=-0.504, p=0.017). There was no significant correlation between NRP-1 RNA level and age, platelet count, LDH, BM blast %, CD33, CD13, CD14, CD61, myeloperoxidase, CD2, CD7, CD34, HLA-DR, CD10, or survival time.

A positive significant correlation between NRP-1 RNA and LDH (r=0.912, p=0.011) was detected in relapsed AML patients with no other significant correlation between NRP-1 RNA and any other parameters: age, TLC, Hb, platelet count, PB blast %, BM blast %, CD33, CD13, CD14, CD117, myeloperoxidase, CD2, CD5, CD7, CD34, HLA-DR, and CD10.

30 25 20 15 10 5 0

% MR

All patients AML

Denovo AML

Relapsed

Control

Control

AML

Figure 1. Mean value of NRP-1 (% and MFI) by flow cytometry in studied AML patients.

Table 1. Comparison between different patient groups and the control group as regards NRP-1 expression by flow cytometry and RT-PCR.

Parameters

FCM of NRP-1 %

RT-PCR of NRP-1 MFI

p

Sig.

p

Sig.

p

Sig.

All patients versus control

<0.001

HS

<0.001

HS

0.69

NS

De novo versus control

<0.001

HS

<0.001

HS

0.745

NS

Relapsed versus control

0.005

HS

0.002

HS

0.661

NS

De novo versus relapse

0.401

NS

0.954

NS

0.695

NS

FCM: Flow cytometry, RT-PCR: real-time polymerase chain reaction, Sig: significance, NS: nonsignificant at >0.05, HS: highly significant at <0.001.

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Sensitivity and Specificity ROC curves (Figure 5) were used to define the best cutoff values and to show the diagnostic performance of NRP-1 in discriminating de novo AML patients from the controls.

Survival Studies Kaplan-Meier curves for overall survival in de novo AML patients were calculated. Survival time for de novo AML patients ranged from 10 to 24 months with a mean of 11.20Âą0.84. No statistically significant association was

Figure 2. NRP-1 by flow cytometry of acute myeloid leukemia patient. Right: Gated blasts (60%); left: NRP-1 percentage expression (53.6%) and MFI (2.2).

NRP-1

2 1.5 1

NRP-1

0.5 0 AII AML patients

Denovo AML

Relapsed AML

found between either survival time or overall survival and NRP-1 expression by either flow cytometry or real-time PCR. Discussion AML is a heterogeneous group of diseases characterized by uncontrolled proliferation of clonal neoplastic hematopoietic precursor cells and impaired production of normal hematopoiesis [8]. Angiogenesis plays a significant role in the pathogenesis of AML and in the mechanism of disease progression [9].

NRP-1 is a VEGF co-receptor and is commonly overexpressed in regions of physiological and pathological angiogenesis, but neuropilin-mediated gene expression and the definitive role of neuropilins in angiogenic processes are not fully characterized [10].

The aim of this study was to evaluate the significance of NRP-1 expression in AML patients by both flow cytometry and real-time PCR. Fifty-six AML patients were studied for detection of NRP-1 levels both by flow cytometry (presented as percentage expression and MFI) and its RNA level as estimated by real-time PCR. Patients included in this work were classified for better evaluation into 2 groups: de novo and relapsed AML cases.

All patients expressed NRP-1 by flow cytometry and the cut-off level for AML diagnosis was determined to be 6% positivity. This study is one of the few that address the issue of NRP-1 in AML. Comparison of patientsâ&#x20AC;&#x2122; NRP-1 percent positivity or MFI to those of the control group showed a highly significant difference between groups. However, by real-time PCR, no statistical significance was found while comparing patients to controls.

Consistent with our results, a study done by Schuch et al. [7] found that NRP-1 was expressed in acute myeloid leukemic cells. Kreuter et al. [9] reported that the expression

Control

Figure 3. Mean value of NRP-1 by real-time PCR in studied AML patient groups.

100 90 80 70 60 50 40 30 20 10 0

0

20 NRP-1

40

60 Den. AML

80

100

Best cutoff

Area under the curve NRP-1 1.000

Figure 4. NRP-1 RNA level by real-time PCR in the de novo AML group.

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Figure 5. ROC curve analysis showing the diagnostic performance of NRP-1 for discriminating de novo AML from the control by flow cytometry.

Sallam TH, et al: Significance of NRP-1 Expression in AML

of NRP-1 was increased in the bone marrow of AML patients.

Lu et al. [3] examined mRNA expression of NRP-1 in leukemic cells and found that it was increased in AML patients compared with healthy controls. NRP-1 expression was directly correlated with myeloblast percentage in the bone marrow of patients with AML, suggesting that NRP-1 correlates with tumor load.

Staber et al. [11] demonstrated that recurrent AML is commonly associated with mRNA expression changes in a set of 58 genes, such as the angiogenic moleculesâ&#x20AC;&#x2122; fibroblast growth factor receptor-1 and thrombospondin-2. The discrepancy between our results regarding NRP-1 by realtime PCR and those of other researchers may be because the characterization of NRP-1 by real-time PCR is not standardized, leading to difference in results.

Similar to studies done by Lu et al. [3] and Kreuter et al. [9], no statistical correlation was found between NRP1 expression and sex, hepatomegaly, splenomegaly, or lymphadenopathy. However, in this study, NRP-1 percentage expression by flow cytometry showed a high positive correlation with age in relapsed patients.

Studying NRP-1 expression in relation to prognosis, there was a highly positive and significant correlation between it and TLC in de novo AML patients as measured either by flow cytometry or real-time PCR, which Lu et al. [3] failed to detect using PCR techniques.

A negative correlation was found here between both NRP1 expression by flow cytometry and NRP-1 RNA by realtime PCR and Hb concentration in de novo AML cases. In contrast, Lu et al. [3] reported no correlation between NRP1 RNA as measured by real-time PCR and Hb concentration.

A significant positive correlation was detected between NRP-1 percentage expression and BM blasts in relapsed cases. Another positive correlation was found between NRP1 RNA and PB blasts in de novo cases. This was in agreement with Lu et al. [3], who reported that increased NRP-1 expression was directly correlated with the blast percentage in the PB of AML patients.

In relapsed cases, a significant correlation was found between NRP-1 percentage expression and CD14. This was in agreement with the findings of Bradstok et al. [12], who reported that correlation with CD14 had a poorer outcome in AML patients There was a positive correlation between NRP-1 expression and LDH. Wimazal et al. [13] reported that a progression to AML was diagnosed in 60% of patients with increasing LDH. They also showed that an increase in LDH over time is associated with a higher probability of AML evolution and a reduced probability of survival, and so they recommended using LDH as a prognostic follow-up parameter in AML.

Turk J Hematol 2013;30:300-306

The current study showed a significant positive correlation between NRP-1 and CD10 percentage expression.

However, in a study by Webber et al. [14] excluding t(15;17) cases, they showed association between CD10 percentage expression and complete remission of AML patients.

In a study by Younan et al. [15] on Egyptian AML patients using real-time quantitative RT-PCR, it was revealed that NRP-1 was expressed in 95% of AML cases with levels higher in patients than controls, and there was a statistically significant difference in NRP-1 levels between patients who went into complete remission and those who did not. They concluded that NRP-1 is significantly associated with acute leukemia and that its level might serve as an indicator for disease severity and progression.

Anti-VEGF antibodies have received much attention lately for their ability to block tumor angiogenesis and prolong the life of cancer patients [16]. In 2004, bevacizumab (Avastin), a humanized monoclonal antibody against VEGF-A, became the first antiangiogenic drug approved by the US Food and Drug Administration as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Another anti-VEGF antibody, ranibizumab (Lucentis), a monoclonal antibody Fab, has been successful in the treatment of neovascularization associated with wet neovascular age-related macular degeneration, thereby alleviating blindness in patients [17]. However, in cancer patients, the anti-VEGFâ&#x20AC;&#x201C;chemotherapy combination has had adverse effects, including hypertension, impaired wound healing, and arterial thrombotic events [18]. Thus, a promising development surfaced, whereby it was reported that antibodies to NRP-1 in combination with anti-VEGF enhanced the ability of anti-VEGF to block tumor growth [19].

Silencing of the NRP-1 gene results in a significant decrease of VEGF- induced cell proliferation and migration in HEL cells, suggesting the role of VEGF in leukemia progression via its newly identified receptor, NRP-1.

Therefore, the blocking of NRP-1 signaling may represent a novel therapeutic approach for the treatment of a subset of AML [3].

In conclusion, NRP-1 by flow cytometry can be used as a diagnostic tool for AML diagnosis. It can also be used to detect prognosis by both flow cytometry and real-time PCR techniques.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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Sallam TH, et al: Significance of NRP-1 Expression in AML

References 1. Vales A, Kondo R, Aichberger KJ, Mayerhofer M, Kainz B, Sperr WR, Sillaber C, Jäger U, Valent P. Myeloid leukemias express a broad spectrum of VEGF receptors including neuropilin-1 (NRP-1) and NRP-2. Leuk Lymphoma 2007;48:1997-2007.

2. Latil A, Bièche I, Pesche S, Valéri A, Fournier G, Cussenot O, Lidereau R. VEGF overexpression in clinically localized prostate tumors and neuropilin-1 overexpression in metastatic forms. Int J Cancer 2000;89:167-171.

3. Lu L, Zhang L, Xiao Z, Lu S, Yang R, Han ZC. Neuropilin-1 in acute myeloid leukemia: expression and role in proliferation and migration of leukemia cells. Leuk Lymphoma 2008;49:331-338.

4. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin 2002;52:23-47.

5. Zhou J, Mauerer K, Farina L, Gribben JG. The role of the tumor microenvironment in hematological malignancies and implication for therapy. Front Biosci 2005;10:15811596.

6. Zacchigna S, Pattarini L, Zentilin L, Moimas S, Carrer A, Sinigaglia M, Arsic N, Tafuro S, Sinagra G, Giacca M. Bone marrow cells recruited through the neuropilin-1 receptor promote arterial formation at the sites of adult neoangiogenesis in mice. J Clin Invest 2008;118:20622075.

7. Schuch G, Machluf M, Bartsch G Jr, Nomi M, Richard H, Atala A, Soker S. In vivo administration of vascular endothelial growth factor (VEGF) and its antagonist, soluble neuropilin-1, predicts a role of VEGF in the progression of acute myeloid leukemia in vivo. Blood 2002;100:46224628.

8. Tallman MS, Gilliland DG, Rowe JM. Drug therapy for acute myeloid leukemia. Blood 2005;106:1154-1163.

9. Kreuter M, Steins M, Woelke K, Buechner T, Berdel WE, Mesters RM. Downregulation of neuropilin-1 in patients with acute myeloid leukemia treated with thalidomide. Eur J Haematol 2007;79:392-397.

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10. Staton CA, Kumar I, Reed MW, Brown NJ. Neuropilins in physiological and pathological angiogenesis. J Pathol 2007;212:237-248.

11. Staber PB, Linkesch W, Zauner D, Beham-Schmid C, Guelly C, Schauer S, Sill H, Hoefler G. Common alterations in gene expression and increased proliferation in recurrent acute myeloid leukemia. Oncogene 2004;23:894-904.

12. Bradstock K, Matthews J, Benson E, Page F, Bishop J. Prognostic value of immunophenotyping in acute myeloid leukemia. Australian Leukaemia Study Group. Blood 1994;84:1220-1225.

13.

Wimazal F, Sperr WR, Kundi M, Vales A, Fonatsch C, Thalhammer-Scherrer R, Schwarzinger I, Valent P. Prognostic significance of serial determinations of lactate dehydrogenase (LDH) in the follow-up of patients with myelodysplastic syndromes. Ann Oncol 2008;19:970-976.

14. Webber BA, Cushing MM, Li S. Prognostic significance of flow cytometric immunophenotyping in acute myeloid leukemia. Int J Clin Exp Pathol 2008;1:124-133.

15. Younan S, Elhoseiny S, Hammam A, Gawdat R, El-Wakil M, Fawzy M. Role of neuropilin-1 and its expression in Egyptian acute myeloid and acute lymphoid leukemia patients. Leuk Res 2012;36:169-173.

16. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target.

Nature 2005;438:967-974.

17. Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY, Kim RY; MARINA Study Group. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med 2006;355:1419-1431.

18. Hurwitz H, Saini S. Bevacizumab in the treatment of metastatic colorectal cancer: safety profile and management of adverse events. Semin Oncol 2006;33:26-34.

19. Pan Q, Chanthery Y, Liang WC, Stawicki S, Mak J, Rathore N, Tong RK, Kowalski J, Yee SF, Pacheco G, Ross S, Cheng Z, Le Couter J, Plowman G, Peale F, Koch AW, Wu Y, Bagri A, Tessier-Lavigne M, Watts RJ. Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth. Cancer Cell 2007;11:53-67.

Brief Report

DOI: 10.5152/tjh.2013.0042

Effects of Omeprazole on Iron Absorption: Preliminary Study Omeprazol’un Demir Absorpsiyonundaki Etkileri: Ön Çalışma Mila Tempel1, Anupama Chawla1, Catherine Messina2, Mahmut Yaşar Çeliker1 1Department

of Pediatrics, Stony Brook Long Island Children’s Hospital, Stony Brook, USA

2Department

of Preventive Medicine, Stony Brook University Medical Center, Stony Brook, USA

Abstract: Objective: Increasing numbers of pediatric and adult patients are being treated with proton pump inhibitors (PPIs). PPIs are known to inhibit gastric acid secretion. Nonheme iron requires gastric acid for conversion to the ferrous form for absorption.

Ninety percent of dietary and 100% of oral iron therapy is in the nonheme form. To the best of our knowledge, the effect of PPIs on iron absorption has not been studied in humans. Our study assessed the relationship between omeprazole therapy and iron absorption in healthy subjects.

Materials and Methods: We recruited 9 healthy volunteers between June 2010 and March 2011. Subjects with chronic illness, anemia, or use of PPI therapy were excluded. Serum iron concentrations were measured 1, 2, and 3 h after the ingestion of iron (control group). The measurements were repeated on a subsequent visit after 4 daily oral administrations of omeprazole at a dose of 40 mg (treatment group).

Results: One female and 8 male volunteers were enrolled in the study with a mean age of 33 years. There was no statistical difference detected between baseline, 1-h, 2-h, and 3-h iron levels between control and treatment groups.

Conclusion: Administration of omeprazole for a short duration does not affect absorption of orally administered iron in healthy individuals.

Key Words: Iron, Omeprazole, PPI, Proton pump inhibitors, Anemia Özet: Amaç: Giderek artan sayıda pediatrik ve yetişkin hastalar proton pompa inhibitörleri (PPI) ile tedavi edilmektedir. PPI’ların mide asidini inhibe ettikleri bilinmektedir. Heme’e bağlı olmayan demir ferroz şekline geçerek absorb edilebilmesi için mide asidi gerektirir. Diyetteki demirin yüzde doksanı ve tedavide kullanılan demirin yüzde yüzü heme’e bağlı olmayan durumdadır.

Bilgimize göre PPI’ların demir absobsiyonunda nasıl etki ettiği insanlarda araştırılmamıstır. Araştırmamız omeprazol tedavisi ile demir absorbsiyonu arasındaki ilişkiyi sağlıklı kişilerde incelemiştir.

Address for Correspondence: Mahmut YAŞAR ÇELIKER, M.D., Flushing Pediatric Medicine, PC 004 196th Street, Fresh Meadows, NY 11365, USA E-mail: myceliker@gmail.com Received/Geliş tarihi : February 6, 2013 Accepted/Kabul tarihi : April 15, 2013

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Tempel M, et al: Effects of Omeprazole on Iron Absorption: Preliminary Study

Gereç ve Yöntemler: Haziran 2010 ile Mart 2011 arasında 9 sağlıklı gönüllüyü çalışmamiz için davet ettik. Kronik hastalığı veya anemisi olan kişilerle PPI tedavisinde olan kişileri dışladık. Serum demir konsantrasyonunu demir alımından 1, 2, ve 3 saat sonra ölçtük (kontrol grubu). Bu ölçümleri bir sonraki ziyarette 4 günlük oral 40 mg dozunda omeprazol tedavisinden sonra tekrarladık (tedavi grubu).

Bulgular: Ortalama yaşları 33 yıl olan 8 erkek ve 1 kadın gönüllü çalışmamıza katıldı. Kontrol grubu ile tedavi grubu arasında bazal, 1 saat, 2 saat, ve 3 saat sonraki demir konsantrasyonları arasında istatistiksel anlamlı bir fark görülmedi.

Sonuç: Sağlıklı kişilerde kısa bir zaman sürecince verilen omeprazol oral olarak alınan demirin absorbsiyonunu etkilemez.

Anahtar Sözcükler: Demir, Omeprazol, PPI, Proton pompa inhibitörleri, Anemi Introduction Gastric acid plays an important role in the absorption of iron. Dietary iron can be divided into 2 types with respect to absorption: the heme type, derived from animal blood and muscle, which is well absorbed and comprises about 10%30% of the normal Western diet, and the more common nonheme type, derived from plants (fruits, vegetables, grains, nuts), which requires an acidic gastric environment for absorption [1,2]. Although the heme component is absorbed independent of gastric pH, the nonheme part requires an acidic pH for absorption [3].

At physiologic pH and in the presence of oxygen, iron exists predominantly in the highly insoluble ferric Fe(III) form and therefore is poorly bioavailable [4]. Ingested ferrous ions are readily oxidized to ferric ions by dissolved oxygen [5]. Stomach acid is important in releasing iron from ligands in food and in solubilizing ferric iron by converting it to ferrous form [6].

It has been speculated that inhibition of gastric acid secretion may lead to reduction in iron and vitamin B12 absorption [7]. The development of iron malabsorption has been shown in a variety of hypo- or achlorhydric conditions.

Heath and Patek observed that among patients with irondeficiency anemia, the rate of the hemoglobin response to iron therapy was lower in those who had achlorhydria than in those without [8]. Goldberg et al., using radiolabeled iron, showed that patients with achlorhydria have diminished absorption of iron [9]. These data clearly demonstrate the importance of gastric acid secretion in iron absorption.

Increasing numbers of pediatric and adult patients are being treated with proton pump inhibitors (PPIs), often for several months. There is a limited amount of research specifically looking at the effects of PPIs and absorption of orally administered iron. Our study is the first such human study to assess this relationship in healthy subjects.

Materials and Methods The protocol was approved by the institutional review board and written informed consent was obtained from all subjects. Subjects were recruited from a pool of healthy volunteers, 18 to 50 years old, who responded to our advertisement. Those with low ferritin levels <10 ng/mL 308

for females, <20 ng/mL for males) or anemia (hemoglobin <115 g/L for females, <130 g/L for males), pregnant, or with malabsorption disorders, major gastrointestinal surgery, malignancy or any chronic conditions involving the gastrointestinal system, renal disease, or hematological, cardiac, or pulmonary conditions were excluded during the screening visit. Menstruating females came to their study visits 10-13 days after their last day of menstrual bleeding in order to synchronize menstrual cycles at the time of testing.

All subjects were asked to continue their regular diet as we intended this study to be applicable to real-life situations.

At the second visit, baseline serum iron level and total iron-binding capacity were obtained after a minimum of 8 h of fasting. Ferrous sulfate (650 mg; 130 mg of elemental iron) was then administered orally as a tablet preparation, followed by determination of serum iron concentration 1, 2, and 3 h after the ingestion of iron. Male subjects returned 1 week and female subjects returned 1 month later for their last visit. Subjects were instructed to take omeprazole (40 mg) daily 30 min before breakfast for 4 days prior to their third visit (post-PPI), when after a minimum of 8 h of fasting, subjects were given 40 mg of omeprazole. During this visit, 650 mg of ferrous sulfate was administered orally.

Serum iron concentration was measured before and 1, 2, and 3 h after the ingestion of iron. Baseline and post-omeprazole serum iron level determination was done around the same time of the day at each visit.

Statistical Analysis Sample calculations were made using PASS (Power Analysis and Sample Size 2002, Number Crunch Statistical Systems, Kaysville, UT, USA). A sample size of 10 achieves 80% power to detect a difference of -50 (decrease by 50 µg/dL) between the baseline mean of 100 µg/dL and day 7 mean of 50 µg/dL with an estimated standard deviation of 50 µg/dL and with a significance level (alpha) of 0.05 using a 2-sided paired t-test.

Each subject served as her/his own control. Thus, serum iron concentrations were compared for the control vs. treatment conditions using the t-test for paired samples.

Tests of significance were 2-sided and evaluated at the p<0.05 level. Data analyses were conducted using SPSS (Version 19, IBM SPSS Statistics, Somers, NY, USA).

Turk J Hematol 2013;30:307-310

Tempel M, et al: Effects of Omeprazole on Iron Absorption: Preliminary Study

Results We recruited 9 healthy volunteers, 8 males and 1 female, who completed the study between June 2010 and March 2011. Mean age at the time of study was 33. A single blood sample in 2 subjects was hemolyzed and therefore excluded from the analysis.

Serum iron levels measured before taking omeprazole (control) and after 5 days of omeprazole therapy (treatment) were compared (Figure 1). When serum iron concentrations at different time points were compared between the control and treatment groups, there was no statistical difference (Table 1).

Discussion After oral administration of PPI, the onset of the antisecretory effect of omeprazole occurs within 1 h, with the maximum effect occurring within 2 h [10]. The inhibitory effect of omeprazole on acid secretion increases with repeated once-daily dosing, reaching a plateau after 4 days. Bastani et al. showed a significant rise in serum iron concentration of 164±32% µg/dL above baseline 2 h after oral ingestion, plateauing afterwards [11]. These studies

Serum iron concentration (µg/dL

Effects of Omeprazole on Iron Aborption Control Treatment

suggested that a 5-day course of PPI treatment would be sufficient length of treatment and 3 h of serum iron-level monitoring would be sufficient to detect serum changes in iron levels following the ingestion of iron preparation.

Our study was done in healthy subjects to minimize factors other than PPIs that may influence iron absorption.

We did not restrict the diet of the subjects to maintain real-life applicability. In rats that were on a normal diet, omeprazole did not alter iron absorption, in agreement with our results [12].

Previous human and animal studies suggested that omeprazole may inhibit iron absorption in iron-depleted states. Two adult patients with iron deficiency anemia from erosive gastritis had their anemia corrected only after discontinuation of omeprazole [13]. Omeprazole inhibits iron absorption in rats that were fed an iron-deficient diet [12]. Another study showed that treatment of patients with hereditary hemochromatosis with PPIs led to significant reduction in the volume of blood removed annually [14]. In that report, postprandial iron absorption studies in 14 subjects with hemochromatosis demonstrated decreased absorption of nonheme iron from a test meal by approximately 50% after 7 days of PPI therapy. These studies suggest that PPIs may inhibit iron absorption in iron-depleted as well as ironoverloaded individuals.

Although our sample size was small, it was calculated based on prior studies in which serum iron levels decreased by 50% after 7 days or more of PPI therapy in hemochromatosis patients and a case report by Sharma et al. [13,14]. Ad hoc sample size calculations using iron levels from the 3 h following treatment showed that we would have needed 117,268 subjects in each group for the observed difference to be statistically significant. This suggests that the small sample size is not likely to account for our observations.

Conclusions

0 hour

1 hour

2 hours

3 hours

Time after administration of ferrous sulfate

Figure 1. Changes in the serum iron levels at indicated time points after single parenteral administration of ferrous sulfate at 6 mg elemental iron/kg per dose (max. 130 mg of elemental iron) compared to baseline (hour 0).

Our study demonstrates that short duration of omeprazole use does not affect iron absorption in iron-replete healthy individuals who are on a normal diet. Since omeprazole’s effect was shown in iron-depletion or iron-overload states, it is plausible that omeprazole may have an effect on iron absorption only in abnormal iron metabolic states. Although short-term omeprazole therapy does not appear to affect

Table 1. Mean change in serum iron concentrations (µg/dL) in subjects before and after oral iron challenge (paired samples t-test). SD: standard deviation.

Time

Before PPI Mean (SD)

After PPI Mean (SD)

Before vs. after Mean difference (SD)

p

Baseline (n=7)

70.3 (19.9)

82.9 (29.8)

12.6 (24.2)

0.22

1 h (n = 7)

117.4 (33.3)

145.1 (53.3)

27.7 (52.4)

0.24

2 h (n=7)

163.9 (45.3)

180.6 (44.7)

16.7 (56.4)

0.46

3 h (n=7)

181.1 (52.5)

181.7 (55.6)

0.6 (59.6)

0.98 309

Tempel M, et al: Effects of Omeprazole on Iron Absorption: Preliminary Study

Turk J Hematol 2013;30:307-310

iron absorption, since PPIs are increasingly being used for longer durations, further studies are warranted to evaluate their effects on long-term use.

6. Miret S, Simpson RJ, McKie AT. Physiology and molecular biology of dietary iron absorption. Annu Rev Nutr 2003;23:283-301.

Competing Interests

7. Reynolds JC. The clinical importance of drug interactions with antiulcer therapy. J Clin Gastroenterol 1990;12(Suppl 2):54-63.

There are no financial or nonfinancial competing interests to disclose by any of the authors.

References 1. Khatib MA, Rahim O, Kania R, Molloy P. Iron deficiency anemia: induced by long-term ingestion of omeprazole. Dig Dis Sci 2002;47:2596-2597.

2. Massey AC. Microcytic anemia. Differential diagnosis and management of iron deficiency anemia. Med Clin North Am 1992;76:549-566.

3. Johnson LR. Physiology of the Gastrointestinal Tract, 2th ed. New York, Raven Press, 1987.

4. McKie AT, Barrow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly E, Mudaly M, Richardson C, Barlow D, Bomford A, Peters TJ, Raja KB, Shirali S, Hediger MA, Farzaneh F, Simpson RJ. An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 2001;291:17551759.

5. Goto K, Tamura H, Nagayama M. Mechanism of oxygenation of ferrous ion in neutral solution. Inorg Chem 1970;9:963964.

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8. Heath CW, Patek AJ. The anemia of iron deficiency. Medicine 1937;16:267-350.

9. Goldberg A, Lochhead AC, Dagg JH. Histamine-fast achlorhydria and iron absorption. Lancet 1963;1:848-850.

10.

Medical Economics. Physiciansâ&#x20AC;&#x2122; Desk Reference 2002.

Thomson PDR, Oradell, NJ, USA, 2001.

11. Bastani B, Islam S, Boroujerdi N. Iron absorption after single pharmacological oral iron loading test in patients on chronic peritoneal dialysis and in healthy volunteers. Perit Dial Int 2000;20:662-666.

12. Golubov J, Flanagan P, Adams P. Inhibition of iron absorption by omeprazole in rat model. Dig Dis Sci 1991;36:405-408.

13. Sharma VR, Brannon MA, Carloss EA. Effect of omeprazole on oral iron replacement in patients with iron deficiency anemia. South Med J 2004;97:887-889.

14. Hutchinson C, Geissler CA, Powell JJ, Bomford A. Proton pump inhibitors suppress absorption of dietary non-haem iron in hereditary haemochromatosis. Gut 2007;56:12911295.

DOI: 10.4274/Tjh.2012.0141

Case Report

A Case of Hypereosinophilic Syndrome Presenting with Multiorgan Thromboses Associated with Intestinal Obstruction İntestinal Tıkanıklık ile İlişkili Multiorgan Yetmezliği ile Başvuran Bir Hipereozinofilik Sendrom Olgusu Tao Sui1, Qing Li1*, Li Geng1, Xinnv Xu2, Yuming Li1 1Department

of Hematology, Tianjin First Center Hospital, Tianjin, China

2Key

Lab for Critical Care Medicine of the Ministry of Health, Tianjin First Center Hospital, Tianjin, China

*This

author contributed equally to this work and should be considered as co-first author

Abstract: Idiopathic hypereosinophilic syndrome (HES) is a disease characterized by persistent hypereosinophilia (>1.5×109/L) for more than 6 months in the absence of other causes of reactive eosinophilia. Patients with HES presenting with multiorgan thromboses are rare. Herein we report a 57-year-old man with HES who presented with deep venous thrombosis of the lower extremities, portal thrombosis, pulmonary embolism, and mesenteric venous thrombosis, which led to intestinal obstruction.

Key Words: Eosinophilia, Hypereosinophilic syndrome, Thrombosis, Intestinal obstruction

Özet: İdiyopatik hipereozinofilik sendrom (HES) reaktif eozinofilinin diğer nedenlerinin yokluğunda 6 aydan uzun süren persistan eozinofili (>1.5×109/L) ile karakterize bir hastalıktır. Multiorgan trombozları ile başvuran HES’li olgular nadirdir. Burada, alt ekstremite derin ven trombozu, portal tromboz, pulmoner embolizm, ve özellikle intestinal tıkanıklığa yol açan mezenterik venöz tromboz ile başvuran 57 yaşında bir erkek olgu sunulmuştur.

Anahtar Sözcükler: Eozinofili, Hipereozinofilik sendrom, Tromboz, Intestinal tıkanıklık Introduction Idiopathic hypereosinophilic syndromes (HESs) are rare disorders that comprise a heterogeneous group of diseases characterized by unexplained persistent, nonreactive overproduction of eosinophils (>1.5×109/L) persistent for more than 6 months. HES can cause multiple organ damage/dysfunction, mainly involving the skin, heart, lung, gastrointestinal tract, and nervous system [1].

The clinical manifestations of HES include fever, rash, fatigue, cough, shortness of breath, muscle aches, and diarrhea. The most common presenting manifestation is thrombosis, such as mural thrombus of the heart, inferior vena cava thrombosis, superficial venous thrombosis, portal thrombosis, deep venous thrombosis, cerebral arteriolar and venous thrombosis, and intracardiac thrombi [2, 3,4,5]. Simultaneous multiple organ thromboses associated with HES rare. Herein, we report a unique case of HES

Address for Correspondence: Tao SUI, M.D., Department of Hematology, Tianjin First Center Hospital, 24# Fukang Road, Nankai District, Tianjin 300192, China Phone: +86 22 236 265 19 E-mail: taosui71@yahoo.com.cn Received/Geliş tarihi : October 03, 2012 Accepted/Kabul tarihi : February 04, 2013

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Turk J Hematol 2013;30:311-314

presenting with deep venous thrombosis of the lower extremities, portal thrombosis, pulmonary embolism, and especially mesenteric venous thromboembolism, which led to intestinal obstruction.

Case Report A 57-year-old man was admitted to our department for pain and swelling of both lower extremities accompanied with fever for more than 10 days. His medical and family histories were unrevealing. His vital signs were within the normal range and the clinical examination was normal, except for hyperemia, warmth, slight tenderness, and edema of both lower legs, especially in the right leg. White blood cell count was high (23.9×109/L) with 66.5% eosinophils (15.9 ×109/L), hemoglobin of 141 g/L, and thrombocytopenia of 11×109/L. Liver and renal function tests were normal. A test for parasites in the stool and serum was negative. Tests for tumor markers were negative. The work-up for allergic and rheumatologic diseases were also negative (including anti-nuclear, anti-dsDNA, anti-neutrophilic cytoplasmic antibodies; anti-phospholipid antibodies; anti-SSA, antiSSB, anti-histone antibodies). A bone marrow aspiration and biopsy were performed and hematological malignancies were excluded. The bone marrow aspiration revealed a significant proliferation of trilineage cells and a prominent component of mature-appearing eosinophils comprising 68% of the total cellularity. The bone marrow biopsy showed normal cellularity with increased eosinophils.

Polymerase chain reaction indicated a negative BCR-ABL while fluorescence in situ hybridization indicated a negative FIP1L1-PDGFRA gene fusion. An ultrasound confirmed the arteriosclerosis of both lower limbs accompanied by multiple plaques and thrombosis of the right popliteal vein. An abdominal ultrasound revealed fatty liver and thrombosis of the portal vein. Echocardiography showed that the patient’s atrioventricular cavity size, ventricular wall thickness, range of motion, and valve morphology were all normal. Ejection fraction was 69%.

At this stage, HES with multiorgan thromboses was suspected. Methylprednisolone (40 mg/day) was commenced and piperacillin was initiated, empirically.

Hemorrhagic spots appeared on the skin of both lower limbs, and blood blisters of the oral mucosa were observed at a certain period. Thus, platelets were transfused.Pain and swelling of both lower extremities were alleviated using the aforementioned treatment. At day 10, abdominal distension and abdominal pain occurred and the abdominal pain could not be relieved by anisodamine hydrochloride. It gradually worsened and abdominal tenderness emerged with rebound tenderness as well as muscle tension. A plain abdominal radiograph revealed intestinal obstruction. No neurologic abnormalities were noted. White blood cell count was 8.07×109/L with 26.2% eosinophils (2.12×109/L), 107 g/L hemoglobin, and 18×109/L platelets. No evident presence of fragmented red blood cells was observed in the 312

peripheral smear. The renal function was normal and The prothrombin time (PT) was 18.2 s and the activated partial thromboplastin time (aPTT) was 40.9 s. Fibrinogen was 8.1 g/L and D-dimer was 9.6 µg/mL. Computed tomography (CT) results suggested that for the thrombosis of the superior mesenteric vein (Figure 1), emergency partial excision of the small intestine could be performed. The histopathological investigation confirmed the extensive small bowel hemorrhage, necrosis, and mesenteric vascular thrombosis. Postoperative recovery was good. Continued treatment with glucocorticoid and anticoagulant therapy was not performed due to the patient’s very low platelet counts with higher risk of bleeding. On day 29, the patient had dyspnea and breath-holding accompanied by chest pain and intermittent expectoration with bloody streaks. Oxygen saturation was 85% without oxygen support. Auscultation of the lungs revealed moist rales. Pulmonary CT angiography showed embolism in the bilateral pulmonary arteries and their branches, as well as bilateral pleural effusion (Figure 2). Meanwhile, protein C and S levels were normal.

An ultrasound of the abdomen and both lower limbs showed that the thrombosis of the right popliteal vein and portal vein had no significant alteration compared with

Figure 1: CT showing the thrombosis of the superior mesenteric vein (arrow).

Figure 2: Pulmonary CT angiography showing embolism in the pulmonary arteries and their branches (arrow).

Sui T, et al:A Case of Hypereosinophilic Syndrome Presenting with Multiorgan Thromboses Associated with Intestinal Obstruction

previous images. The white blood cell count was 8.69×109/L, with 31.3% eosinophils (2.72×109/L), 90 g/L hemoglobin, and 21×109/L platelets. The PT was 17.9 s and the APTT was 57.6 s. Fibrinogen was 2.43 g/L and D-dimer was 2.6 µg/mL. Heparin (2000 IU/day) was initiated. Platelets were transfused when the counts were lower than 10 × 109/L. On day 35, the patient’s eosinophil count returned to normal; the white blood cell count was 6.68 × 109/L with 0.2% eosinophils (0.02×109/L), hemoglobin of 90 g/L, and platelets of 19 × 109/L. The patient’s condition also gradually stabilized. CT showed that the pulmonary embolism was significantly improved in compared with the previous images. Prednisone (28 mg/day) and coumadin (2.5 mg/ day) were initiated. Thus far, follow-up has been consistent; the eosinophil and platelet counts were within normal limits and no recurrence of thrombosis was observed. Informed consent was obtained.

Discussion HES is a rare hematological disorder, and its diagnosis is set when patients have persistently elevated eosinophil counts of >1.5 × 109/L for more than 6 months and other causes of reactive eosinophilia have been excluded through histopathological evaluation and imaging studies for internal organ involvement.

Our patient was FIP1L1-PDGFRA-negative and did not show any evidence of T or B cell clonality. The diagnosis of idiopathic HES was considered after asthma, eczema, parasitic infestation, autoimmune disorders, and malignant neoplasm were ruled out. However, hereditary thrombophilia cannot be excluded due to the fact that hereditary factors such as factor V Leiden mutation, prothrombin G20210 mutation, and AT deficiency were not investigated. To the best of our knowledge, multiorgan thromboses, as a presentation of HES especially associated with intestinal obstruction, is rare and have only been reported by Kobayashi et al. [6].

When our patient presented with venous thrombosis of both legs, the portal vein, and the superior mesenteric vein, anticoagulation was not performed due to the severe thrombocytopenia accompanied with mucosal bleeding.

Glucocorticoids and platelet transfusions were preferred.

The eosinophil counts gradually decreased, suggesting that our patient exhibited response to glucocorticoids. However, unfortunately the patient developed pulmonary embolism.

Initially, we assumed that the embolism was related to thrombus shedding, and so an ultrasound was performed again. The images showed that the thrombosis of the right popliteal vein and the portal vein had no significant alteration compared to previous images. Pulmonary CT angiography showed multiple embolism in the pulmonary arteries.

Thus, the pulmonary embolism may have been due to local eosinophilic infiltration that resulted in hypercoagulability.

Therefore, heparin was used for anticoagulant therapy with intermittent platelet transfusions. To date, no other thrombosis has occurred and the platelets generally reached the normal range.

Turk J Hematol 2013;30:311-314

Glucocorticoids are the first-line therapy for FIP1L1PDGFRA-negative HES and are very effective for decreasing eosinophil counts [7]. INF-α, hydroxyurea, and antiIL-5 monoclonal antibodies are considered in case of glucocorticoid resistance. In our case, eosinophil counts decreased gradually with glucocorticoids. However, despite the decrease in the eosinophil counts, the risk for thrombosis recurrence did not disappear as reflected by an intestinal obstruction which may have been related to the thrombosis of the mesenteric vein. Although our patient’s eosinophil counts were significantly reduced by glucocorticoid administration, the patient needed emergency operation for the intestinal obstruction. Eosinophil counts do not always seem tomatch with organ damage.

The link between eosinophilia and the thrombotic event is still unclear. One possible reason is the infiltration of eosinophils in involved tissues [8,9,10]. A number of cytotoxic substances are then released, including highly cationic molecules such as eosinophil cationic protein, major basic protein, ribonuclease eosinophil-derived neurotoxin, oxidizing molecules such as eosinophil peroxidase and free oxygen radicals, and enzymes such as elastase and collagenase. Eosinophils can also produce lipid mediators such as leukotrienes and prostaglandins. Another reason is the presence of predispositions to thrombosis, such as immobility, genetic hypercoagulability, or advanced atherosclerosis [4,11,12].

In conclusion, HES with thromboembolic events in multiple organs is usually difficult to manage. Therefore, patients should be monitored carefully for the potential complications of the disease, and treatment should be performed according to the situation of each individual patient.

Acknowledgments This work was supported by the Tianjin Science and Technology Committee (11JCZDJC18600).

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Klion A. Hypereosinophilic syndrome: current approach to diagnosis and treatment. Annu Rev Med 2009;60:293-306.

2. Kocaturk H, Yilmaz M. Idiopathic hypereosinophilic syndrome associated with multiple intracardiac thrombi.

Echocardiography 2005;22:675-676.

3. Kikuchi K, Minami K, Miyakawa H, Ishibashi M. Portal vein thrombosis in hypereosinophilic syndrome. Am J Gastroenterol 2002;97:1274-1275.

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Sui T, et al:A Case of Hypereosinophilic Syndrome Presenting with Multiorgan Thromboses Associated with Intestinal Obstruction

4. Narayan S, Ezughah F, Standen GR, Pawade J, Kennedy CT. Idiopathic hypereosinophilic syndrome associated with cutaneous infarction and deep venous thrombosis. Br J Dermatol 2003;148:817-820.

9. Sakuta R, Tomita Y, Ohashi M, Nagai T, Murakami N. Idiopathic hypereosinophilic syndrome complicated by central sinovenous thrombosis. Brain Dev 2007;29:182184.

5. Grigoryan M, Geisler SD, St Louis EK, Baumbach GL, Davis PH. Cerebral arteriolar thromboembolism in idiopathic hypereosinophilic syndrome. Arch Neurol 2009;66:528531.

10.

Uemura K, Nakajima M, Yamauchi N, Fukayama M, Yoshida K. Sudden death of a patient with primary hypereosinophilia, colon tumours, and pulmonary emboli.

J Clin Pathol 2004;57:541-543.

6. Kobayashi M, Komatsu N, Kuwayama Y, Bandobashi K, Kubota T, Uemura Y, Taguchi H. Idiopathic hypereosinophilic syndrome presenting acute abdomen.

Intern Med 2007;46:675-678.

11. Gümrük F, Gürgey A, Altay C. A case of hypereosinophilic syndrome associated with factor V Leiden mutation and thrombosis. Br J Haematol 1998;101:208-209.

7. Park YM, Bochner BS. Eosinophil survival and apoptosis in health and disease. Allergy Asthma Immunol Res 2010;2:87-101.

8. Liapis H, Ho AK, Brown D, Mindel G, Gleich G. Thrombotic microangiopathy associated with the hypereosinophilic syndrome. Kidney Int 2005;67:1806-1811.

314

12. Johnston AM, Woodcock BE. Acute aortic thrombosis despite anticoagulant therapy in idiopathic hypereosinophilic syndrome. J R Soc Med 1998;91:492-493.

DOI: 10.4274/Tjh.2012.0017

Case Report

Late Onset Epstein Barr Virus Seropositive Posttransplant Lymphoproliferative Disorder in Two Renal Transplant Receivers Böbrek Nakli Alıcısı İki Hastada Geç Dönemde Gelişen Epstein Barr Virüs Seropozitif Lenfoproliferatif Hastalık Saime Paydaş2, Semra Paydaş3, Mustafa Balal2, Arbil Açıkalın4, Melek Ergin4, Emel Gürkan3, Fikri Başlamışlı3 1Çukurova

University Faculty of Medicine, Department of Nephrology, Adana, Turkey

2Çukurova

University Faculty of Medicine, Department of Oncology, Adana, Turkey

3Çukurova

University Faculty of Medicine, Department of Hematology, Adana, Turkey

4Çukurova

University Faculty of Medicine, Department of Pathology, Adana, Turkey

Abstract: Posttransplant malignancy is one of the most important complications of organ transplantation. Immunosuppressive drugs, viral infections such as human herpes virus 8 or Epstein-Barr virus, exposure to carcinogenic factors such as sun, and host factors can be etiologic factors in the development of malignant disease. In this paper we report 2 cases of late posttransplant lymphoproliferative disorder with malign behavior.

Key Words: Renal Transplantation, Lymphoproliferative disorder, Burkitt lymphoma

Özet: Nakil sonrası dönemde malign hastalık gelişmesi organ naklinin en önemli komplikasyonlarından biridir. Malign hastalık gelişmesinde; immünsüpressif ilaçlar, human herpes virüs 8, Ebstein Barr virüs gibi viral enfeksiyonlar, güneşe maruz kalma ve kişiye ait özellikler etyolojik faktörler olarak sayılabilir. Burada nakil sonrası geç dönemde gelişen ve malign gidiş gösteren lenfoproliferatif hastalıklı iki olgu rapor edilmişti Anahtar Kelimeler: Böbrek Nakli, Lenfoproliferatif hastalık, Burkitt lenfoma Introduction After cardiovascular complications and infections, malignancy is the third most common cause of death in renal transplant receivers (RTRs) [1,2,3]. Malignant disorders account for the 20% of deaths in RTRs every year and this rate increases to 30% in cases followed for more than 20 years [4]. Increase in some cytokines such as transforming growth factor beta, interleukin-10 and vascular endothelial

growth factor; immunodeficiency against viral infections; and DNA injury are etiologic factors in the development of posttransplant malignancy [5]. In transplant patients, Kaposi sarcoma, non-melanoma skin cancers, and nonHodgkin lymphoma are the most common cancers, and the risk of these cancers has been found to be increased by 20fold as compared with the normal population. The risk of renal cancer is reported to be increased 15-fold and a 5-fold

Address for Correspondence: Saime PAYDAŞ, M.D., Çukurova University Faculty of Medicine, Department of Nephrology, 01330 Adana, Turkey Phone: +90 322 338 73 20 E-mail: serkupeli@yahoo.com Received/Geliş tarihi : February 5, 2012 Accepted/Kabul tarihi : September 19, 2012

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increase risk has been found for melanoma, leukemia, and hepatobiliary, cervical, and vulvovaginal cancer. Two-to 3-fold increases have been reported for testicular, bladder, colorectal, lung, prostate, stomach, esophagus, pancreas, ovarian, and breast cancers [5].

Case Reports Case 1 Renal transplantation after a short course of hemodialysis (HD) had been performed in a 22-year-old woman from her father in 1997. There was no severe complication in the early posttransplant period and she did not receive antithymocyte globulin (ATG) or high-dose corticosteroids.

She had no hypertension and/or proteinuria and other complications. Drugs used in this case and the clinical outcome are summarized in Table 1. Calcineurin inhibitors (cyclosporin A [CysA] for 51 months and tacrolimus for 85 months), azathioprine at 100 mg/day, and corticosteroid at 4 mg daily for 4 years were used in the follow-up period.

Corticosteroid administration was ceased when she was found to be positive for hepatitis C virus (HCV). At that

time, her serum creatinine was 1.8 mg/dL and cyclosporine and azathioprine were substituted with tacrolimus and mycophenolate mofetil (MMF). In 2008 she wished to become pregnant and MMF was replaced by azathioprine. In the last year, she was receiving tacrolimus, and azathioprine.

In December 2009, she was hospitalized due to fever, anemia, and thrombocytopenia. At this time, physical exam was negative except for the forearm cellulitis. Chest X-ray, abdominal ultrasonography and bloodâ&#x20AC;&#x201C;urine cultures were found to be negative. Serologic tests for Salmonella, Brucella, human immunodeficiency virus, and cytomegalovirus (CMV) were found to be negative. Epstein-Barr virus (EBV) IgM was negative and EBV IgG was positive and HCV polymerase chain reaction was found to be positive.

Piperacillin-tazobactam plus linezolid were prescribed but the fever continued. Bone marrow aspiration showed dysplastic changes and periodic acid Schiff (PAS)-negative blasts, which were compatible with acute lymphoblastic leukemia L3 or Burkitt cells. Histopathological bone marrow biopsy revealed post-transplant lymphoproliferative disease (PTLD), monomorphic PTLD, and Burkitt lymphoma

Table 1. Clinical and laboratory findings of the first patient.

Baseline

Fourth year

Eleventh year

Twelfth year

May 1997

July 2001

September 2008

2009

CysA (Co) (ng/mL)

152

285 / ceased

-

-

Tacrolimus (Co) (ng/mL)

-

After CysA cessation, 6.1

6

2.2

Azathioprine

100 mg/day

-

50 mg/day

50 mg/day

MMF

-

1.5 g/day

-

-

Prednisolone

4 mg/day

Ceased

-

-

Hematocrit (%)

31.5

32.5

30.8

25

White blood cells (x109/L)

8000

7400

5400

5100

153,000

206,000

209,000

93,000

16/1.8

22/1.7

17/1.6

22/1.7

Platelets

(x109/L)

BUN/creatinine (mg/dL)

Table 2. Clinical outcome and therapy of the second patient.

1985

1995 HD

Edema, atrophic kidney

1997-2000 2000 TRx-

Chronic renal failure, stage 5

HD

2001

2005-2006

HD

HD

Nephrectomy

CysAazathioprineprednisolone

HD

TRx-

Burkitt lymphoma

R-CHOP

Trx-: Renal transplantation, HD: Hemodialysis, CysA: Cyclosporin A, MMF: mycophenolate mofetil.

316

2007-2009 2009-2010

Sirolimus MMF Prednisolone

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(Figures 1a and 1b). There was diffuse infiltration of the bone marrow by monotonous, medium-sized cells with multiple nucleoli, basophilic cytoplasm, and numerous mitoses.

Starry sky appearance was present. Immunohistochemically, Pax-5 was positive while terminal deoxynucleotidyl transferase (Tdt), CD3, and myeloperoxidase (MPO) were negative in tumor cells. Immunosuppressive drugs were ceased. Abdominopelvic magnetic resonance imaging (MRI) showed pelvic fluid and retroperitoneal lymph nodes.

Unconsciousness developed and there was no evidence of nuchal rigidity, papillary stasis or lateralized neurologic findings. Cerebral MRI showed diffuse thickening and contrast uptake in the meningeal structures. Cytology of the lumbar puncture showed blastic infiltration. CODOX-M

including cyclophosphamide, doxorubicin, vincristine, methotrexate, calcium folinate, granulocyte colonystimulating factor, Ara-C and rituximab was prescribed, but uremia developed and HD was performed. Nevertheless, fever and hypotension developed and she died.

a

a

b

b

c

Figure 1. Diffuse infiltration of the bone marrow by monotonous, medium-sized cells with multiple nucleoli, basophilic cytoplasm, and numerous mitoses. Starry sky appearance is present. Immunohistochemically, Pax-5 was positive; Tdt, CD3, and MPO were negative in tumor cells. a) Burkitt lymphoma tumor cells with numerous mitoses (hematoxylin, 400×); b) monotonous, medium-sized cells diffusely infiltrating bone marrow with starry sky appearance (hematoxylin, 100×).

Figure 2. Diffuse large B cell lymphoma showing atypical large lymphoid cells with multilobated nuclei: a) hematoxylin–eosin, 100x; b) hematoxylin–eosin, 400x; c) tumor specimen CD20 positivity (immunohistochemistry 400x).

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Case 2 A 17-year-old male was admitted to our unit with endstage renal failure. He had history of atrophic kidneysince he was 7 years old. The patient received renal transplantation from his father after 2 years of HD in 1997. There was no severe complication in the early post-transplant period and neither ATG nor high-dose corticosteroid was used.

During follow-up he received prednisolone and CysA plus azathioprine. At the end of 2 years, there was increase in blood urea nitrogen (BUN) and creatinine levels, and edema developed. Renal biopsy showed vascular rejection.

Prednisolone was given 500 mg/day for 5 days, but renal function did not improve and HD was initiated again.

The transplanted kidney was removed due to abscess formation 5 years after transplantation and renal biopsy showed chronic rejection. He developed pneumonia and imaging revealed ascites, cardiomegaly, pericardial effusion and pulmonary interstitial infiltrations. There was evidence of left ventricular hypertrophy, mitral annular calcification, and left atrial dilatation at echocardiography.

Peritoneal biopsy showed active chronic inflammation and mesothelial cell hyperplasia. Upper endoscopy showed gastroesophageal reflux disease, hiatal hernia and Barrettâ&#x20AC;&#x2122;s metaplasia. In 2005 the patient was hospitalized due to abdominal pain, nausea and vomiting. Paraaortic multiple conglomerate lymph nodes and splenomegaly were detected upon abdominopelvic CT. Biopsy taken by laparotomy was compatible with non-Hodgkin lymphomaâ&#x20AC;&#x201C;diffuse large B cell lymphoma. Diffuse proliferation of large lymphoid cells with vesicular nuclei containing fine chromatin and nucleoli were found. Some tumor cells had multilobated nuclei.

Immunohistochemically, leukocyte common antigen and CD20 were found to be positive while CD30 and CD3 were negative. EBV-encoded ribonucleic acid (EBER) was negative by in situ hybridization (ISH); see Figures 2a, 2b, and 2c.

Six cycles of rituximab-doxorubicin-cyclophosphamidevincristine-prednisone (R-CHOP) were administered. After chemotherapy, complete remission was achieved. In 2006, chemotherapy was completed. In the follow-up period, CT examination was negative for lymphoma (Table 2).

In 2009, cadaveric renal transplantation was performed.

Low-dose ATG (1 mg/kg daily) was given for 5 days and then maintenance sirolimus (target level: 3-12) and MMF (2 g daily) plus prednisolone were prescribed. In the last visit in 2010, BUN was 18 mg/dL, creatinine 0.7 mg/dL, hemoglobin 16.2 g/dL, hematocrit 49.2%, white blood cell count 7.7x10.9/L, and daily proteinuria 30 mg/day. EBV IgG was positive and IgM was negative.

[6]. EBV has a central role in the pathogenesis of PTLD [7,8,9], although not all PTLD is EBV-related. The most clearly defined risk factor for PTLD is primary EBV infection, which increases the risk for PTLD by 10- to 76fold [10,11]. However, EBV positivity is not the rule. EBVrelated viral disease and EBV-related malignant disease may develop with direct and indirect effects of the virus [12].

Fever, neutropenia, pneumonia, enteritis, meningitis, or encephalitis may develop secondary to the direct effects of the virus. Indirect effects of the immunomodulatory effect of the virus may cause increased risk of immune suppression and opportunistic infections via the secretion of cytokines, chemokines, or growth factors. Additionally, viral infections may change the surface antigen expression (for example, human leukocyte antigen) and provoke rejection reaction and/or contribute to oncogenesis with dysregulated cellular proliferation. Infection with one virus may stimulate the replication of other viruses (like CMV+HCV) or immunosuppression [13,14,15,16,17]. EBV positivity was present in both of our cases and HCV was present in one of them. The spectrum of disease ranges from benign polyclonal B cell infectious mononucleosis-like disease to malignant monoclonal lymphoma. The majority is of B cell origin, although T cell, NK-cell, and null cell tumors have been described. T cell PTLD has been demonstrated in 10% to 15% of cases, especially in the late transplant period; within allografts, it can be confused with graft rejection or other viral infections. Lymphomas comprise up to 15% of tumors among adult transplant recipients (51% in children), with mortality of up to 40% to 60%. Many deaths are associated with allograft failure after withdrawal of immune suppression during treatment of malignancy.

Compared with lymphoma in the general population, PTLD has increased extranodal involvement, bad response to conventional therapies, and poorer outcomes.

The use of muromonab-CD3 or antithymocyte globulin seems to be associated with an increased risk of PTLD, especially in the first year [18,19]. In RTRs belatacept was associated with an increased incidence of PTLD [20].

Tacrolimus is commonly associated with an increased risk of malignancy compared to cyclosporine [18,19]. mTOR inhibitors might protect against the development of PTLD. PTLD includes a wide range of histopathology.

Histopathological findings are important both for estimating the prognosis and treatment decision, and they have been classified in different ways. There are 4 groups [21]: 1) Early lesions: reactive and plasmacytic hyperplasia, infectious mononucleosis-like.

Discussion

2) PTLD polymorphic: polyclonal (rare), monoclonal.

PTLD is 20-fold more common in patients receiving organ transplantation as compared with normal populations [5]. PTLD is related to viral infections, especially EBV

3) PTLD monomorphic: B cell lymphoma (diffuse large B cell lymphoma, Burkitt/Burkitt-like lymphoma), plasma cell myeloma, T cell lymphoma (peripheral T cell lymphoma and other types).

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4) Other types (rare): Hodgkin disease-like lesions (associated with methotrexate therapy), plasmacytoma-like lesions.

According to this classification, cases 1 and 2 were compatible with the third group.

The cessation or decreasing of the dose of immunosuppressive drug is efficient in two-thirds of cases of EBV-related PTLD. This possibility is low in cases with EBVrelated conditions and develops in more than 1 year after transplantation. In these cases, there is a tendency for more malignant behavior. However, some cases may respond to the decreasing of immunosuppressive drug dosages, which should be considered in these cases [21].

In our first case, B cell lymphoma developed 12 years after transplantation and the patient presented with anemia and thrombocytopenia. Bone marrow aspiration/biopsy and cerebrospinal fluid were found to be positive for malignant cells. HCV and EBV IgG positivity were present in this case and there was no evidence of HCV-related liver disease. Two viral infections may be responsible as the risk factor for both the rejection and the development of malignant disease. In our second case, Burkitt lymphoma was localized in the abdomen and serum EBV IgG was found to be positive.

Tumor specimen was negative for EBER by ISH. In the first case, ISH for EBER was not done due to the acid exposure of the bone marrow specimen.

The case 1 of the current study patient both tacrolimus and CysA and the second case received only CysA. Additionally, our first patient was receiving immunosuppressives while the lymphoma developed, while in our second case lymphoma was detected 5 years after the cessation of immunosuppressive drugs. Azathioprine was used in both cases and MMF was used in the first case during the prelymphoma period. MMF was found to be relatively safe for development of PTLD in early and late periods [18,19].

In both of our cases, lymphoma developed in the post-transplant later period. For this reason, prognosis was poor and it was necessary to use anti-neoplastic drugs for the treatment of lymphoma. In the first case, lymphoma developed while the patient was receiving immunosuppressives, and in second case, PTLD developed 5 years after the cessation of immunosuppressive drugs.

The first patient was treated with immunosuppressives for 12 years, but the second patient received them for almost 2 years. In case 1, chemotherapy was started, but the patient died with complications. Patient 2 was treated by R-CHOP. After 2 years, complete remission was achieved and a second renal transplantation was performed. Calcineurin inhibitor was not used, while mTOR+MMF was given without any adverse event. In general, the longer the cancer-free interval before transplantation, the smaller the recurrence risk.

For most malignant neoplasms, a period of 2 to 5 years is recommended [22]. Informed consent was obtained.

In conclusion, although ATG is the main drug accused in the development of PTLD, both of our 2 patients had not been treated with ATG. Lymphoma develops generally while patients are receiving immunosuppressive drug(s) and regression of PTLD has been reported with the cessation of immunosuppressives. However, high-grade lymphoma developed in both of our patients and they were treated with aggressive combination chemotherapy.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1 Howard RJ, Patton PR, Reed AI, Hemming AW, Van der Werf WJ, Pfaff WW, Srinivas TR, Scornik JC. The changing causes of graft loss and death after kidney transplantation.

Transplantation 2002;73:1923-1928.

2. Briggs JD. Causes of death after renal transplantation.

Nephrol Dial Transplant 2001;16;1545-1549.

3. Collins AJ, Kasiske B, Herzog C, Chen SC, Everson S, Constantini E, Grimm R, McBean M, Xue J, Chavers B, Matas A, Manning W, Louis T, Pan W, Liu J, Li S, Roberts T, Dalleska F, Snyder J, Ebben J, Frazier E, Sheets D, Johnson R, Li S, Dunning S, Berrini D, Guo H, Solid C, Arko C, Daniels F, Wang X, Forrest B, Gilbertson D, St Peter W, Frederick P, Eggers P, Agodoa L. Excerpts from the United States Renal Data System 2003 Annual Data Report: atlas of end-stage renal disease in the United States. Am J Kidney Dis 2003;42( Suppl 5):1-230.

4. Mahony JF, Caterson RJ, Coulshed S, Stewart JH, Sheil AG. Twenty and 25 years survival after cadaveric renal transplantation. Transplant Proc 1995;27:2154-2155.

5. Wimmer CD, Rentsch M, Crispin A, Illner WD, Arbogast H, Graeb C, Jauch KW, Guba M. The janus face of immunosuppression - de novo malignancy after renal transplantation: the experience of the Transplantation Center Munich. Kidney Int 2007;71:1271-1278.

6. Liebowitz D. Epstein-Barr virus and a cellular signaling pathway in lymphomas from immunosuppressed patients.

N Engl J Med 1998;338:1413-1421.

7. Opelz G, Henderson R. Incidence of non-Hodgkin lymphoma in kidney and heart transplant recipients. Lancet 1993;342:1514-1516.

8. Paya CV, Fung JJ, Nalesnik MA, Kieff E, Green M, Gores G, Habermann TM, Wiesner PH, Swinnen JL, Woodle ES, Bromberg JS. Epstein-Barr virus-induced posttransplant lymphoproliferative disorders. ASTS/ASTP EBV-PTLD 319

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Task Force and The Mayo Clinic Organized International Consensus Development Meeting. Transplantation 1999;68:1517-1525.

9. Preiksaitis JK, Keay S. Diagnosis and management of posttransplant lymphoproliferative disorder in solid-organ transplant recipients. Clin Infect Dis 2001;33(Suppl 1):3846.

10. Walker RC, Marshall WF, Strickler JG, Wiesner RH, Velosa JA, Habermann TM, McGregor CG, Paya CV. Pretransplantation assessment of the risk of lymphoproliferative disorder. Clin Infect Dis 1995;20:1346-1353.

11. Walker RC, Paya CV, Marshall WF, Strickler JG, Wiesner RH, Velosa JA, Habermann TM, Daly RC, McGregor CG. Pretransplantation seronegative Epstein-Barr virus status is the primary risk factor for posttransplantation lymphoproliferative disorder in adult heart, lung, and other solid organ transplantations. J Heart Lung Transplant 1995;14:214-221.

12. Kotton CN, Fishman JA. Viral infection in the renal transplant recipient. J Am Soc Nephrol 2005;16:17581774.

13. Rubin RH. The direct and indirect effects of infection in liver transplantation: pathogenesis, impact, and clinical management. Curr Clin Top Infect Dis 2002;22:125-154.

14. Ljungman P. Beta-herpesvirus challenges in the transplant recipient. J Infect Dis 2002;186(Suppl1):99-109.

15. Boeckh M, Nichols WG. Immunosuppressive effects of betaherpesviruses. Herpes 2003;10:12-16.

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16. Reinke P, Prรถsch S, Kern F, Volk HD. Mechanisms of human cytomegalovirus (HCMV) (re)activation and its impact on organ transplant patients. Transpl Infect Dis 1999;1:157164.

17. Dockrell DH, Paya CV. Human herpesvirus-6 and -7 in transplantation. Rev Med Virol 2001;11:23-36.

18.

Opelz G, Dรถhler B. Lymphomas after solid organ transplantation: a collaborative transplant study report. Am J Transplant 2004;4:222-230.

19. Robson R, Cecka JM, Opelz G, Budde M, Sacks S. Prospective registry-based observational cohort study of the long-term risk of malignancies in renal transplant patients treated with mycophenolate mofetil. Am J Transplant 2005;5:29542960.

20. Durrbach A, Pestana JM, Pearson T, Vincenti F, Garcia VD, Campistol J, Rial Mdel C, Florman S, Block A, Di Russo G, Xing J, Garg P, Grinyรณ J. A phase III study of belatacept versus cyclosporine in kidney transplants from extended criteria donors (BENEFIT-EXT study). Am J Transplant 2010;10:547-557.

21. Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant 2009;9(Suppl 3):1-155.

22. Knoll G, Cockfield S, Blydt-Hansen T, Baran D, Kiberd B, Landsberg D, Rush D, Cole E; Kidney Transplant Working Group of the Canadian Society of Transplantation. Canadian Society of Transplantation consensus guidelines on eligibility for kidney transplantation. CMAJ 2005;173:1181-1184.

DOI: 10.4274/Tjh.2012.0030

Case Report

Ecthyma Gangrenosum-like Lesions in a Febrile Neutropenic Patient with Simultaneous Pseudomonas Sepsis and Disseminated Fusariosis Eşzamanlı Pseudomonas Sepsisi ve Dissemine Fusariozisi Olan Febril Nötropenik Hastada Ektima Gangrenozum Benzeri Lezyonlar Seven Uludokumacı1, İlker İnanç Balkan1, Bilgül Mete1, Reşat Özaras1, Neşe Saltoğlu1, Teoman Soysal2 1İstanbul 2İstanbul

University Cerrahpasa Medical Faculty, Infectious Diseases and Clinical Microbiology İstanbul, Turkey University Department of Infectious Diseases and Clinical Microbiology, Cerrahpaşa Faculty of Medicine İstanbul, Turkey

Abstract: Fusarium spp. is an opportunistic mold that causes disseminated infections in immunocompromised patients. It is important to make a definite diagnosis because of high mortality rates. We present the case of a 27-year-old pregnant woman diagnosed with acute myeloid leukemia with a prolonged febrile neutropenic period. She developed ecthyma gangrenosum-like lesions and simultaneously had Pseudomonas bacteremia and disseminated fusariosis. Histopathological and microbiological features of skin lesions had a critical role in differential diagnosis. Ecthyma gangrenosum-like lesions due to disseminated fusariosis might be easily misdiagnosed as lesions associated with Pseudomonas unless tissue cultures and histopathological examinations are performed.

Key Words: Disseminated fusariosis, Pseudomonas aeruginosa sepsis, Ecthyma gangrenosum-like lesions

Özet:

Fusarium spp. immündüşkün hastalarda dissemine enfeksiyonlara neden olan oportünistik bir küf mantarıdır. Yüksek mortalite oranları nedeniyle kesin tanıya gitmek önemlidir. Sunmakta olduğumuz olguda uzamış febril nötropenik dönemde bulunan AML tanılı 27 yaşında bir gebede Pseudomonas bakteremisi ile dissemine fusariozis eşzamanlı gelişmiş ve aynı dönemde ektima gangrenozum benzeri lezyonlar ortaya çıkmıştır. Ayırıcı tanıda deri lezyonlarının histopatolojik ve mikrobiyolojik özellikleri önemli bir yer tutmuştur. Dissemine fusariozise bağlı ektima gangrenozum benzeri lezyonlar doku kültürü ve histopatolojik incelemeler yapılmazsa kolaylıkla Pseudomonas enfeksiyonu ile ilişkili lezyonlarla karıştırılabilir.

Anahtar Sözcükler: Dissemine Fusariozis, P. aeruginosa sepsisi, Ektima gangrenozum-benzeri lezyonlar

Introduction Fusarium spp. is an opportunistic mold that causes disseminated infections in immunocompromised patients [1]. Cutaneous lesions like ecthyma gangrenosum are

seen in 70% of the cases [2,3]. We present a patient with acute myeloid leukemia in a febrile neutropenic period with ecthyma gangrenosum-like cutaneous lesions who had Pseudomonas bacteremia and disseminated fusariosis simultaneously. An Internet search of the English-language

Address for Correspondence: Seven ULUDOKUMACI, M.D., Istanbul University Cerrahpasa Medical Faculty, Infectious Diseases and Clinical Microbiology, Istanbul, Turkey GSM: +90 532 208 23 59 E-mail: seven_bilgen@yahoo.com Received/Geliş tarihi : February 19, 2012 Accepted/Kabul tarihi : November 14, 2012

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literature (Medline 1966-2011) revealed no reported cases of ecthyma gangrenosum-like lesions due to simultaneous Pseudomonas bacteremia and disseminated fusariosis.

We aim to draw attention to the cutaneous lesions in immunocompromised patients, as they may cause diagnostic challenges because of being associated with different invasive etiologic agents while having similar appearances.

Case Report A 27-year-old patient, 9 weeks pregnant and diagnosed with acute myeloid leukemia, was admitted with a febrile neutropenic attack and initially treated empirically with ceftazidime. On the fifth day of admission, conventional amphotericin B (Amp B) at 1.2 mg/kg/day was added to the treatment because of persistent fever. Computed tomography (CT) of the thorax revealed multiple nodules on the right lung. On day 8, medical abortion was performed.

After abortion, her general condition worsened, respiratory

distress developed, and the treatment was switched to meropenem (1 g, q8h) and levofloxacin (750 mg, q24h).

The patient was transferred to the intensive care unit (ICU) and underwent chemotherapy (ARA-C, 170 mg/day D1-7).

After 2 weeks of follow-up in the ICU, her general condition was partially improved and she was taken back to the hematology department. In the first month of antifungal therapy, follow-up thoracic CT demonstrated progression of acinar nodules and consolidations bilaterally in the lung parenchyma. Serum galactomannan test results were found positive 2 times consecutively. With preliminary diagnosis of probable invasive pulmonary aspergillosis, Amp B was switched to voriconazole (loading dose of 6 mg/kg, q12h, 2 doses; maintenance of 4 mg/kg, q12h).

After one month of this therapy, because of persisting fever, bronchoscopy was performed. In the bronchoalveolar lavage culture, Chryseobacterium indologenes and Candida glabrata (voriconazole MIC: 4 Âľg/mL) were isolated.

Figure 1: Ectyhma gangrenosum related to pseudomonas septicemia (center) and ecthyma gangrenosum-like lesions related to fusariosis (upper left corner).

Figure 3: Fusarium spp. colonies grown from skin biopsy specimen culture.

Figure 2: Axillary nodular skin lesion related with fusariosis.

Figure 4: Eye involvement of disseminated fusariosis.

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Although colonization of these microorganisms could not be excluded, because of the persisting fever and high levels of acute phase reactants, these agents were accepted as pathogenic microorganisms. Treatment was changed to caspofungin (50 mg/day) and piperacillin/tazobactam (PIP/ TAZ; 3 × 4.5 g/day) according to the susceptibility tests.

During the patient’s follow-up, clinical signs due to the progression of leukemia were detected. On days 83-88 of admission, fludarabine + ARA-C chemotherapy was given.

On day 17 of PIP/TAZ therapy, while fever and neutropenia were still persisting, an ulcerated erythematous lesion with a necrotic center was detected on the patient’s right hip, and there were smaller acneiform, erythematous nodular lesions on the left side of the abdominal wall, the left hip, the forearm, and the axilla (Figures 1 and 2). During the infection period, a total of 8 sets of hemocultures were obtained. Pseudomonas aeruginosa was isolated in 4 sets of hemocultures. Ciprofloxacin (2×400 mg/day) and amikacin (1×1 g/day) were added to PIP/TAZ according to the susceptibility tests. Histopathological analysis of the skin lesion on the right hip revealed ecthyma gangrenosum.

Fusarium spp. was isolated from the skin biopsy of the erythematous nodular lesions on the left hip (Figure 3).

Simultaneously, the patient complained of loss of vision in her right eye, and fundoscopic examination revealed fungal endophthalmitis (Figure 4). Liposomal Amp B was added to caspofungin, but because of severe allergic reaction, it was replaced by voriconazole. Informed consent was obtained.

Combined antibacterial therapy for Pseudomonas bacteremia was completed in 3 weeks. With daily granulocyte colony stimulating factor therapy, the patient became non-neutropenic and her follow-up bone marrow biopsy revealed signs of remission. A convulsion occurred on day 21 of combined antifungal therapy. Cranial magnetic resonance imaging showed disseminated lesions compatible with fungal infection. Antifungal treatment was continued and remission of lesions was observed 2 weeks later. After combination therapy for one month, she was discharged from the hospital with oral voriconazole. During one year of follow-up after discharge, the patient remained in remission hematologically and voriconazole was ceased at month 9 with resolution of radiological signs due to pulmonary fungal infection.

Discussion Co-infections of Fusarium spp. and P. aeruginosa are rarely reported [4]. Fusarium spp. is a mold that may cause superficial infections like keratitis and onychomycosis in immunocompetents, as well as severe disseminated or locally invasive (lung, sinuses, etc.) infections in immunocompromised patients [3]. In patients with hematological malignancies and prolonged fever and neutropenia, it is important to make a differential diagnosis between fusariosis and invasive pulmonary aspergillosis because of their similar clinical presentations. Negativity of

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galactomannan antigen and absence of typical radiological signs of aspergillosis can favor a diagnosis of fusariosis, whereas recent studies reported that galactomannan can also be positive in cases of fusariosis [5,6]. Disseminated fusariosis is described as the involvement of two or more non-contiguous regions [7]. Skin involvement is the first clue in most disseminated fusariosis cases and often occurs at an early stage of the disease [8]. Multiple erythematous macular or papular painful lesions are reported in 70% of cases. Lesions usually have a necrotic center resembling ecthyma gangrenosum and are described as ecthyma gangrenosum-like lesions [3]. In our case, there were two different types of cutaneous involvement. One of them was consistent with fusariosis, whereas other lesions were evaluated as ecthyma gangrenosum secondary to P. aeruginosa bacteremia. To our knowledge, this is the first case reported in the literature of ecthyma gangrenosumlike lesions with simultaneous Pseudomonas sepsis and disseminated fusariosis. In the management of fusariosis, conventional or liposomal Amp B may be used singly; successful results were achieved also by combining it with voriconazole or posaconazole [8,9,10]. In the present case, voriconazole was combined with caspofungin instead of liposomal Amp B and complete response to treatment was achieved, with total resolution of cranial and pulmonary lesions.

Conclusion It is crucial to carefully detect every single skin lesion in patients with hematological malignancies. These lesions could be incorrectly attributed to Pseudomonas aeruginosa bacteremia and disseminated fusariosis would easily be overlooked unless tissue cultures and histopathology were performed.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Musa MO, Al Eisa A, Halim M, Sahovic E, Gyger M, Chaudhri N, Al Mohareb F, Seth P, Aslam M, Aljurf M. The spectrum of Fusarium infection in immunocompromised patients with haematological malignancies and in nonimmunocompromised patients: a single institution experience over 10 years. Br J Hematol 2000;108:544-548.

2. Bodey GP, Boktour M, Mays S, Duvic M, Kontoyiannis D, Hachem R, Raad I. Skin lesions associated with Fusarium infection. J Am Acad Dermatol 2002;47:659-666.

3. Nucci M, Anaissie E. Cutaneous infection by Fusarium species in healthy and immunocompromised hosts: implications for diagnosis and management. Clin Infect Dis 2002;35:909-920.

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UludokumacÄą S, et al: Ecthyma Gangrenosum-like Lesions in a Febrile Neutropenic Patient with Simultaneous Pseudomonas Sepsis and Disseminated Fusariosis

4. Yang YS, Ahn JJ, Shin MK, Lee MH. Fusarium solani onychomycosis of the thumbnail coinfected with Pseudomonas aeruginosa: report of two cases. Mycoses 2011;54:168-171.

8. Jossi M, Ambrosioni J, Macedo-Vinas M, Garbino J Invasive fusariosis with prolonged fungemia in a patient with acute lymphoblastic leukemia: case report and review of the literature. Int J Infect Dis 2010;14:354-356.

5. Mikulska M, Furfaro E, Del Bono V, Gualandi F, Raiola AM, Molinari MP, Gritti P, Sanguinetti M, Posteraro B, Bacigalupo A, Viscoli C. Galactomannan testing might be useful for early diagnosis of fusariosis. Diagn Microbiol Infect Dis 2012;72:367-369.

9. Durand-Joly I, Alfandari S, Benchikh Z, Rodrigue M, Espinel-Ingroff A, Catteau B, Cordevant C, Camus D, Dei-Cas E, Bauters F, Delhaes L, De Botton S. Successful outcome of disseminated Fusarium infection with skin localization treated with voriconazole and amphotericin B-lipid complex in a patient with acute leukemia. J Clin Microbiol 2003;41:4898-4900.

6. Tortorano AM, Esposto MC, Prigitano A, Grancini A, Ossi C, Cavanna C, Cascio GL. Cross-reactivity of Fusarium spp. in the Aspergillus Galactomannan enzyme-linked immunosorbent assay. J Clin Microbiol 2012;50:10511053.

7. Boutati EI, Anaissie EJ. Fusarium, a significant emerging pathogen in patients with hematologic malignancy: ten yearsâ&#x20AC;&#x2122; experience at a cancer center and implications for management. Blood 1997;90:999-1008.

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10. Guzman-Cottrill JA, Zheng X, Chadwick EG. Fusarium solani endocarditis successfully treated with liposomal amphotericin B and voriconazole. Pediatr Infect Dis J 2004;23:1059-1061.

DOI: 10.4274/Tjh.07641

Case Report

A Neonatal Thrombosis Patient Treated Successfully with Recombinant Tissue Plasminogen Activator Rekombinan Doku Plazminojen Aktivatörü ile Başarılı Bir Şekilde Tedavi Edilmiş Bir Neonatal Tromboz Olgusu Kemal Erdinç1, Serdar Ümit Sarıcı2, Orçun Dabak1, Orhan Gürsel3, Adem Güler4, Ahmet Emin Kürekçi3, Fuat Emre Canpolat2 1Gülhane

Military Academy of Medicine, Department of Pediatrics, Ankara, Turkey Military Academy of Medicine, Departments of Pediatrics and Neonatology, Ankara, Turkey 3Gülhane Military Academy of Medicine, Departments of Pediatrics and Hematology, Ankara, Turkey 4Gülhane Military Academy of Medicine, Department of Cardiovascular Surgery, Ankara, Turkey 2Gülhane

Abstract: Herein we report an asphyctic preterm neonate with respiratory distress and prothrombotic risk factors that responded positively to rtPA treatment following 2 attacks of acute thrombosis.

Key Words: Thrombosis, Neonate, Tissue plasminogen activator, Preterm

Özet:

Bu yazıda solunum sıkıntısı ve asfiksi ile doğmuş, protrombotik risk faktörleri de olan bir prematüre bebekte iki tromboz atağının rekombinant doku plazminojen aktivatörü ile başarılı bir şekilde tedavi edildiği sunulmuş, ilacın etkinliği ve bu grup hastalarda dahi uygulanılabilirliği vurgulanmıştır.

Anahtar Sözcükler: Tromboz, Yenidoğan, Doku plazminojen aktivatörü, Prematüre

Introduction Thrombolytic treatment has increased in importance in recent years due to a significant increase in the number of neonatal cases diagnosed with thrombosis as a result of diagnostic and therapeutic interventions performed during the neonatal period [1]. Thrombolytic treatment is an alternative treatment for the elimination of thrombi, which may cause life-threatening organ and tissue destruction.

Recombinant tissue plasminogen activator (rtPA), which is commonly used for the treatment of myocardial infarction and stroke in adults, has also been used to treat cardiac

thrombi in patients with congenital heart disease and right-to-left shunting [2,3,4]. Research on the use of rtPA in neonates is limited [5]. Herein we report an asphyctic preterm neonate with respiratory distress and prothrombotic risk factors that responded positively to rtPA treatment following 2 attacks of acute thrombosis.

Case Report A preterm male neonate weighing 940 g that was born to a 25-year-old gravida 8, para 2 mother was transferred 3h after birth due to respiratory distress from another hospital to our neonatal intensive care unit in an intubated and

Address for Correspondence: Kemal ERDİNÇ, M.D., Gülhane Military Academy of Medicine, Department of Pediatrics, Ankara, Turkey Phone: +90 312 304 43 88 E-mail: kemalerdinc2005@yahoo.com Received/Geliş tarihi : September 25, 2010 Accepted/Kabul tarihi : March 05, 2011

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hand (bag)-ventilated manner in a transport incubator. The mother had not menstruated during the previous 2 years and, therefore, the neonate’s gestational age was estimated to be 27 weeks based on the Ballard score. The mother’s obstetric history included 5 abortions and 2 curettages, and an emergency cesarean section due to placenta previa the day she presented with vaginal bleeding to a hospital in a neighboring town.

Physical examination of the neonate showed hypothermia, hypotonia, cyanosis, intercostal and subcostal retractions, grunting, and tachypnea. The patient was diagnosed with hyaline membrane disease, and mechanical ventilation with intratracheal surfactant treatment, parenteral fluid, and empirical antibiotic treatment was initiated. Coldness, pallor, and cyanosis developed in the distal region of the right forearm soon after venipuncture of the right antecubital vein. Pulse oximetry showed no impulse (pulse and saturation) from this region, distal pulses could not be palpated, and Doppler ultrasonography showed positive brachial pulses, and negative radial and ulnar pulses.

The thrombosis was located at the distal end of the right brachial artery. There was no blood passing to the distal region. Hematochezia was observed 8h after birth.

Intravenous alteplase infusion (0.2 mg·kg·h–1) was initiated as treatment for the thrombosis in the right forearm. Prior to the initiation of this treatment basal hematologic factor levels and etiologic hematologic parameters were as follows: D-dimer: 12 ng mL–1; aPTT: 45 s; PT: 13 s; INR: 176 mg dL–1; protein C activity: 21%; protein S activity: 34%; AT III level: 22 mg dL–1; platelet count: 231,000 mm–3. Infusion was stopped after 30 min, following complete recovery.

Hematochezia was subsequently observed 3 times during the 1st d of life, but did not recur during the 2nd day of life.

The patient was extubated after 4 day of mechanical ventilation support. On the 13th d of life spontaneous and acute thrombosis recurred in the distal region of the left foot, and coldness, pallor, and cyanosis developed in the 1st, 2nd, and 4th phalanges of the left foot. Blood was withdrawn to analyze the coagulation profile, and heparin treatment with a 100 U kg–1 loading dose and 25 U·kg·h–1 maintenance dose, and intravenous alteplase infusion (0.2 mg·kg·h–1) were initiated and administered for 8 h. Fresh frozen plasma infusion (10 mL kg–1 b.i.d.) was also initiated. This treatment regimen resulted in complete recovery in 48 h.

Etiologic investigation of the recurring thrombus formation showed that the neonate had a heterozygous factor V Leiden mutation, a homozygous plasminogen activator inhibitor-1 (PAI-1) 5G/5G polymorphism, a heterozygous methyl tetrahydrofolate reductase (MTHFR) polymorphism, and a normal prothrombin 20210A gene structure. Maintenance treatment with subcutaneous low-molecular weight heparin (enoxaparin) (0.2 mg kg–1 b.i.d.) was initiated and a therapeutic anti-factor Xa level of 0.5-1 U mL–1 was targeted.

At the time this report was written, the patient was being followed-up as an outpatient with the diagnosis of chronic lung disease of prematurity.

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Discussion The incidence of symptomatic neonatal thrombosis, including that in the central nervous system, is reported to be 0.51 per 10,000 live births [1]. Along with advancements in diagnosis and treatment, and because of various invasive interventions performed in neonates, the annual incidence may increase to 2.4 per 1000 in neonatal intensive care units [6]. Thrombi may occur at arterial catheter entry regions and in the presence of prothrombotic risk factors, such as factor V Leiden mutation, and deficiencies in protein C and protein S. There is no consensus concerning the optimal mode of treatment for thrombosis, especially in neonates.

Thrombolytic treatment is being used with increasing frequency for thrombi in neonates. Thrombolytic treatment is an alternative treatment for the elimination of thrombi, which can cause life-threatening organ and tissue destruction [7].

A study that included 14 neonate with a diagnosis of thrombosis treated with rtPA reported that is was a safe treatment option [8]. rtPA has been effectively used to treat catheter-related intracardiac thrombus and vena cava inferior thrombus [9,10]. A study that included 20 children (age between 1 day and 16 years) with thrombosis reported that 11 patients treated with rtPA had a complete response [11]. In the presented case, the mother’s history of recurrent abortus, hematochezia during the first hours of life, and development of an acute thrombus in the region of the brachial artery suggested that the patient had an underlying prothrombotic disease. In addition to this clinical finding as the patient was born preterm (estimated gestational age: 27 weeks) and had respiratory distress syndrome we chose to treat the infant with rtPA in accordance with a previous report of a patient with a brachial artery thrombus that was successfully treated with rtPA [12]. rtPA is characterized by a locally potent effect, which helps minimize the occurrence of systemic side effects. rtPA has a rapid curative effect on thrombi, which is a great benefit as it can minimize the incidence of acute tissue and organ necrosis. In the presented case a rapid response to the patient’s first attack of brachial artery thrombus was achieved using rtPA. Although heparin was required in addition to rtPA for the patient’s second attack of thrombus (lower extremity), complete recovery was achieved, indicating that the combination of these 2 drugs is safe for the treatment of recurrent thrombi. The combination of heparin and thrombolytic treatment has previously been reported to be safe and effective [13]. Although data about the use of this drug combination in neonates, especially preterm neonates, are limited, it is an alternative choice of treatment in cases of life-threatening illness and for preventing acute necrosis in extremities due to thrombus [5]. The presented case had a complete response without any tissue loss, following rapid diagnosis and treatment. The literature contains few reports on the use of rtPA in preterm neonates, and fewer cases with

Erdinç K, et al: A Neonatal Thrombosis Patient Treated Successfully With Recombinant Tissue Plasminogen Activator

birth weights <1000 g, as in the presented case [14]. The presented case shows that use of rtPA in preterm neonates, even repeated doses, can be safe and effective; however, larger prospective, controlled clinical trials are needed to more fully demonstrate the safety and effectiveness of rtPA in neonates. We have written inform consent.

Acknowledgment We are grateful to Nejat Akar, MD and Yonca Eğin, PhD, of Ankara University, School of Medicine, Department of Molecular Hematology.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Nowak-Göttl U, Kosch A, Schlegel N. Neonatal thromboembolism. Semin Thromb Hemost 2003;29:227234.

2. Nakashima T, Minematsu K. Prospects of thrombolytic therapy for acute ischemic stroke. Brain Nerve 2009;61:1003-1012.

3. Aschermann M, Horák J, Reznícek V, Blohlávek J, Aschermann O. Fibrinolytic therapy in acute myocardial infarct. Cas Lek Cesk 2003;142:582-585.

4. Klinge J, Hofbeck M, Ries M, Schaf J, Singer H, von der Emde J. Thrombolysis of modified Blalock-Taussig shunts in childhood with recombinant tissue-type plasminogen activator. Z Kardiol 1995;84:476-480.

5. Monagle P, Michelson AD, Bovill E, Andrew M. Antithrombotic therapy in children. Chest 2001;119(Suppl 1):344-370.

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6. Schmidt B, Andrew M. Neonatal thrombosis: report of a prospective Canadian and international registry. Pediatrics 1995;96:939-943.

7. Raffini L. Thrombolysis for intravascular thrombosis in neonates and children. Curr Opin Pediatr 2009;21:9-14.

8. Hartmann J, Hussein A, Trowitzsch E, Becker J, Hennecke KH. Treatment of neonatal thrombus formation with recombinant tissue plasminogen activator: six years experience and review of the literature. Arch Dis Child Fetal Neonatal Ed 2001;85:18-22.

9. Torres-Valdivieso MJ, Cobas J, Barrio C, Muñoz C, Pascual M, Orbea C, Rodriguez E, Gutiérrez F. Successful use of tissue plasminogen activator in catheter-related intracardiac thrombus of a premature infant. Am J Perinatol 2003;20:9196.

10. Khan JU, Takemoto CM, Casella JF, Streiff MB, Nwankwo IJ, Kim HS. Catheter-directed thrombolysis of inferior vena cava thrombosis in a 13-day-old neonate and review of literature. Cardiovasc Intervent Radiol 2008;31(Suppl 2):153-160.

11. Knöfler R, Dinger J, Kabus M, Müller D, Lauterbach I, Rupprecht E, Taut-Sack H, Weissbach G. Thrombolytic therapy in children--clinical experiences with recombinant tissue-plasminogen activator. Semin Thromb Hemost 2001;27:169-174.

12.

Grieg A. Thrombolysis of a neonatal brachial artery thrombosis with tissue plasminogen activator. J Perinatol 1998;18:460-462.

13. Manco-Johnson M, Nuss R. Neonatal thrombotic disorders.

Neoreviews 2000;10:201-204.

14.

Anderson B, Urs P, Tudehope D, Ward C. The use of recombinant tissue plasminogen activator in the management of infective intracardiac thrombi in pre-term infants with thrombocytopaenia. J Paediatr Child Health 2009;45:598-601.

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DOI: 10.4274/Tjh-2013.0005

Letter to the Editor

The Impact of Eltrombopag Administration on the Clinical Course of Severe Refractory Fatal Acquired Aplastic Anemia Ciddi, Dirençli,Öldürücü, Kazanılmış Aplastik Aneminin Klinik Seyrine Eltrombopag Uygulamasının Katkısı Ayşe Işık, Eylem Eliaçık, İbrahim C. Haznedaroğlu, Salih Aksu, Nilgün Sayınalp, Yahya Büyükaşık, Hakan Göker, Osman Özcebe Hacettepe University Faculty of Medicine, Division of Hematology, Ankara, Turkey

Severe aplastic anemia (SAA) has an aggressive clinical course and represents a “difficult-to-treat” situation with current medications [1]. Eltrombopag is a c-mpl receptor agonist oral thrombopoietin-mimetic drug, mainly active in immune thrombocytopenic purpura (ITP) [2]. Single-agent oral eltrombopag produced hematological responses in 11 of 25 cases of aplastic pancytopenia, with trilineage responses observed in some, suggesting a stimulatory effect of early myeloid progenitors in a pilot clinical trial [3]. We would like to share our experience with eltrombopag in 2 patients with SAA refractory to conventional immunosuppressive treatment. Since the thrombopoietin/c-mpl receptor system is present in early hematopoiesis and in hematopoietic stem cells (HSCs) [4], SAA is an area of potential clinical application for thrombopoietin receptor agonists, including eltrombopag [5].

A 19-year-old male patient was admitted to our emergency room with the complaints of nasal bleeding and ecchymosis. His medical history was unremarkable. On physical examination, the patient was appropriately alert and oriented, and no abnormality was detected except for mucosal petechial hemorrhages and ecchymosis. Laboratory studies revealed pancytopenia with Hb of 6.4 g/dL,

Address for Correspondence: Ayşe IŞIK, M.D., Hacettepe University Faculty of Medicine, Division of Hematology, Ankara, Turkey Phone: +90 312 305 15 36 E-mail: isik_ayse@yahoo.com Received/Geliş tarihi : January 5, 2013 Accepted/Kabul tarihi : April 17, 2013

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leukocyte count of 1.5x109/L, platelet count of 4x109/L, and reticulocyte count of 0.2%. His absolute neutrophil count was 0.5x109/L. His coagulation profile was normal. The peripheral blood smear was consistent with the complete blood count, without any morphological abnormalities.

Additionally, immunologic and virology tests were carried out, revealing no abnormality. He had a negative DEB test.

We did not perform molecular genetic analysis tests for DKC1, TERC, or TINF2 because they are not available at our center.

After his diagnosis was confirmed with bone marrow aspiration and biopsy as aplastic anemia with bone marrow cellularity of <10% and without any evidence of dysplasia, 1 mg/kg steroid and 5 mg/kg cyclosporin were started.

During his follow-up, acute vision loss developed and retinal hemorrhage was detected; when his platelet count was below 20x109/L despite platelet transfusions. Daily platelet transfusion was started in an attempt to raise the platelet count since sufficient response was not obtained with immunosuppressive treatment. With the diagnosis we started to test for human leukocyte antigen (HLA) typing, but because the test results take time, we first started

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cyclosporin and steroid then when we added ATG to patients medication.

Following horse ATG at a dose of 40 mg/kg for 4 days, the patient became more cytopenic and needed more frequent transfusions, especially platelet suspensions.

Despite multiple platelet transfusions, his platelet counts remained below 50x109/L and so eltrombopag at 50 mg/day was begun (Figure 1). Although eltrombopag is not licensed for use in SAA cases, we provided the drug via application to the Ministry of Health for off-label use of eltrombopag.

Both patients signed an informed consent form. During the follow-up period, profound swelling developed in the left part of his face. Computed tomography (CT) scanning was consistent with mucormycosis. Antifungal treatment was begun and he underwent surgical debridement. One week after the eltrombopag initiation, the patient’s platelet counts remained below 50x109/L without any transfusion support, but we did not notice marked changes in Hb or neutrophil counts. However, septic shock complicated the clinical picture and the patient died.

A 44-year-old female patient with a history of bronchial asthma was admitted to our emergency service with the complaints of epistaxis, bleeding from the ears, and vision loss. Her complete blood count revealed Hb of 6.2 g/dL, leukocyte count of 2.2x109/L, platelet count of 4x109/L, and reticulocyte count of 1% with normal coagulation parameters.

Her neutrophil count was 0.7x109/L. Her MRI showed subdural hematoma, which was more prominent in the right parietal and left frontoparietal regions, also accompanied by subarachnoid hemorrhage. She was counseled that neurosurgery and surgical procedures were not needed, and

Işık, et al: The Impact of Eltrombopag Administration on the Clinical Course of Severe Refractory Fatal Acquired Aplastic Anemia

dexamethasone (16 mg/day) as an antiedema therapy and phenytoin were recommended. Her bone marrow aspiration and biopsy were consistent with aplastic anemia, with bone marrow cellularity of <10% and no evidence of dysplasia.

We did not perform a DEB test or molecular genetic analysis for DKC1, TERC, and TINF2, as those tests are not available at our center. We started cyclosporine A in addition to dexamethasone. Because of her intracranial hemorrhage, we did not plan ATG administration in order to avoid profound thrombocytopenia due to the drug itself after intracranial hemorrhage. During this period, she remained profoundly thrombocytopenic, requiring twice daily platelet transfusions to achieve a platelet count over 100x109/L, and we started eltrombopag at 50 mg/day (Figure 1). Although we obtained a relative response to platelet transfusion we could not achieve platelet count of 100x109/L, which is recommended for her intracranial hemorrhage, we started eltrombopag. Following the initiation of the drug, her liver function test results elevated and skin rashes appeared. Skinpunch biopsy was consistent with drug eruptions. Following the addition of oral and topical antihistaminic drugs, her skin rashes disappeared. Her liver function tests returned to normal with a dose reduction to 25 mg/day. After then increasing the dose again to 50 mg/day, her liver function values remained controlled. Two weeks after the initiation of the eltrombopag, her platelet transfusion requirement was reduced with platelet counts reaching the 80.000/µL range without transfusions. As enough time was not passed for occurence of response to immunsuppresive therapy, we attributed the relative recovery in platelet counts and decrease in platelet transfusion requirement to eltrombopag.

After eltrombopag administration we did not notice marked

Figure 1: Platelet counts (x109/L) of Patient 1 (top) and Patient 2 (bottom). Eltrombopag was started on the days marked with arrows.

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changes in Hb or neutrophil counts. Her follow-up MRI showed regression of the intracranial hemorrhage. Her donor screening tests revealed a related HLA-matched donor, and accordingly allogeneic HSC transplantation was planned. During her hospitalization, abdominal pain and fever developed. Her abdominal CT scan was consistent with typhlitis without perforation. Antimicrobial therapy was started. After 2 days, she became septic and died.

In summary, both cases of SAA presented here suggest that eltrombopag could reduce transfusion requirements in patients with platelet transfusion-dependent aplastic anemia. However, the drug had no impact on the morbidity or mortality in our patients with SAA. Informed consent was obtained.

Olnes et al. obtained clinical response in aplastic thrombocytopenia via the administration of eltrombopag, the c-mpl agonist oral thrombopoietin mimetic [3].

Likewise, Komatsu et al. previously disclosed that platelet counts were increased after intravenous PEG-reHuMGDF, a truncated recombinant version of c-mpl ligand (thrombopoietin), in aplastic anemia and myelodysplasia.

The peak platelet level has been observed within 5 to 6 weeks after the initiation of PEG-reHuMGDF treatment [5]. The efficacy of thrombopoietins for the reversal of thrombocytopenia requires the presence of hematopoietic and megakaryopoietic progenitors in the bone marrow (BM). Thus, thrombopoietins are active in diseases with BM megakaryocyte mass such as ITP and thrombocytopenia following nonmyeloablative chemotherapy. However, thrombopoietins are ineffective in thrombocytopenias due to myeloablation as a consequence of the inherent kinetics of thrombopoiesis [5]. Therefore, quantification of BM megakaryocyte mass before eltrombopag therapy can predict the response to thrombopoietins in thrombocytopenia.

The number of BM megakaryocytes was expressed as the number of GPIIb/IIIa-positive cells per cellular area, which was calculated by subtracting the fatty area from the total area in a previous study [6]. Similar techniques could be

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applied to the paraffin-embedded BM sections of patients with aplastic pancytopenia to search BM megakaryocyte masses for the prediction of eltrombopag-respondent versus nonrespondent cases [3].

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Scheinberg P, Young NS. How I treat acquired aplastic anemia. Blood 2012;120:1185-1196.

2. Saleh MN, Bussel JB, Cheng G, Meyer O, Bailey CK, Arning M, Brainsky A; EXTEND Study Group. Safety and efficacy of eltrombopag for treatment of chronic immune thrombocytopenia: results of the long-term, open-label EXTEND study. Blood 2013;121:537-545.

3. Olnes MJ, Scheinberg P, Calvo KR, Desmond R, Tang Y, Dumitriu B, Parikh AR, Soto S, Biancotto A, Feng X, Lozier J, Wu CO, Young NS, Dunbar CE. Eltrombopag and improved hematopoiesis in refractory aplastic anemia. N Engl J Med 2012;367:11-19.

4. Sitnicka E, Lin N, Priestley GV, Fox N, Broudy VC, Wolf NS, Kaushansky K. The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells. Blood 1996;87:4998-5005.

5. Haznedaroglu IC, Goker H, Turgut M, Buyukasik Y, Benekli M. Thrombopoietin as a drug: biologic expectations, clinical realities, and future directions. Clin Appl Thromb Hemost 2002;8:193-212.

6. Nagasawa T, Hasegawa Y, Shimizu S, Kawashima Y, Nishimura S, Suzukawa K, Mukai H, Hori M, Komeno T, Kojima H, Ninomiya H, Tahara T, Abe T. Serum thrombopoietin level is mainly regulated by megakaryocyte mass rather than platelet mass in human subjects. Br J Haematol 1998;101:242-244.

DOI: 10.4274/Tjh-2013.0073

Letter to the Editor

β-Thalassemia Mutation At Codon 37 (TGG>>TGA) Detected In A Turkish Family Bir Türk Ailesinde Gözlenen Kodon 37 β Talasemi Mutasyonu Derya Güleç, Sibel Bilgili, Nuriye Uzuncan, Bozkaya, Giray, Nur Soyer, Baysal Karaca İzmir Bozyaka Eductaion Research and Hospital, Biochemitry Department, İzmir

To the Editor, The β-globin gene mutation at codon 37 [TGG (Trp)→TGA (stop codon)] gives rise to a β0-thalassemia that was described first by Boehm et al. in 1986 in a Saudi Arabian family [1]. Thereafter, other nonsense codon 37 mutations have been reported [1,2,3,4]. Another mutation at codon 37 (TGG/TAG; tryptophan→stop codon) has also been reported previously [5,6].

Premature stop of translation results in a truncated protein and usually the phenotype of β-thalassemia major in homozygous individuals.

We have found an example of the nonsense codon (TGG→TGA; Trp→Stop) in a Turkish family. We report 3 cases with 1 homozygous and 2 heterozygous mutations at codon 37 causing a premature stop codon.

Human fetal hemoglobin is present in vivo as both an acetylated F1 (ααγγacetyl) form by the presence of acetyl groups at the NH2 termini of the γ chains and a nonacetylated F0 (ααγγ) form. The fraction of the total fetal hemoglobin in acetylated form (F1) is about 10%, a value similar to that reported previously for cord erythrocytes and mostly in newborns [7,8].

A 37-year-old female patient (case 1) was admitted to our hospital with symptoms of anemia and repeated blood transfusion dependence once a year. Her red blood cell count (RBC) was 4.34x1012/L, hemoglobin (Hb) was 97 g/L 9 g/L, mean corpuscular volume (MCV) was 69.1 fL (<80 fL), and mean corpuscular hemoglobin (MCH) was 22.4 pg (<27 pg ). Her hemoglobin subtypes were quantified by high-performance liquid chromatography and HbA was 0%

Table 1: Hematological and genetic data of the present study.

Parameters

Patient

Son

Sister

12

RBC (10 /L)

4.34

5.43

5.32

MCV( fL)

69.1

55

62.3

MCH (pg)

22.4

17.6

19.3

MCHC (g/L)

32.5

320

310

Hb (g/L)

97

96

103

HbA (%)

0.0

78.4

81.4

HbA2 (%)

1.0

4.8

5.2

HbF0+F1 (%)

99.0

11.6

6.2

β Genotype

β Codon 37 (TGG/TGA) β Codon 37 (TGG/TGA) homozygous heterozygous

β Codon 37 (TGG/ TGA) heterozygous

Address for Correspondence: Derya GÜLEÇ, M.D., Biyokimya İzmir Bozyaka Eğitim Araştırma Hastanesi İzmir, Turkey GSM: +90 505 525 16 42 E-mail: deryaglc@yahoo.com.tr Received/Geliş tarihi : February 26, 2013 Accepted/Kabul tarihi : May 02, 2013

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Turk J Hematol 2013;30:331-332

Güleç D: β-Thalassemia Mutation At Codon 37 (Tgg>>Tga) Detected In A Turkish Family

(70.0%-95%), HbF0 was 89.0% (<1.5.0%), HbF1 was 10.0%, and HbA2 was 1.0% (<3.5%). The blood smear showed microcytosis, hypochromia, teardrop cells, and target cells. The patient’s family was originally from the eastern region of Turkey and we were not able to take her parents’ blood samples. Consanguinity is not known to be the case in this family. Her 1-year-old son’s (case 2) and her sister’s (case 3) hematological parameters are given with the patient’s in Table 1.

The β-globin genomic DNA was analyzed after receiving informed consent. The β-globin regions of interest were amplified from isolated DNA in a single multiplex polymerase chain reaction and DNA sequencing analyses were done using an ABI 310 sequencer (Applied Biosystems, Foster City, CA, USA). Direct forward and reverse sequencing of the genes revealed that case 1 was homozygous and the other cases were heterozygous for the codon 37 (TGG→TGA) mutation. This mutation results in the production of a premature termination codon (tryptophan→stop codon) and gives rise to β0-thalassemia. Informed consent was obtained.

Prevention of β-thalassemia requires knowledge of the molecular spectrum occurring in the population at risk. This knowledge is particularly necessary when prevention control is applied to a multiethnic population. The frequency of this nonsense codon 37 mutation in the Turkish population is not known.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

332

References 1. Boehm CD, Dowling Kazazian HH Jr. Use in the characterization (beta 37 TGG----TGA) 1986;67:1185-1188.

CE, Waber PG, Giardina PJ, of oligonucleotide hybridization of a beta zero-thalassemia gene in a Saudi Arabian family. Blood

2. Bozdogan ST, Unsal C, Erkman H, Genc A, Yuregir OO, Muslumanoglu MH, Aslan H. Nonsense β-thalassemia mutation at codon 37 (TGG>TGA), detected for the first time in three Turkish cases. Hemoglobin 2012;36:283-288.

3. Gallano P, Girodon E, Ghanem N, Font LL, del Rio E, Martin J, Goossens M, Baiget M. High prevalence of the betathalassaemia nonsense 37 mutation in Catalonians from the Ebro delta. Br J Haematol 1992;81:126-127.

4. Sadiq MF, Huisman TH. Molecular characterization of betathalassemia in north Jordan. Hemoglobin 1994;18:325332.

5. Kornblit B, Taaning P, Birgens H. Beta-thalassemia due to a novel nonsense mutation at codon 37 (TGG-->TAG) found in an Afghanistani family. Hemoglobin 2005;29:209-213.

6. Li D, Liao C, Li J, Tang X. The codon 37 (TGG-->TAG) beta(0)-thalassemia mutation found in a Chinese family.

Hemoglobin 2006;30:171-173.

7. Abraham EC, Cope ND, Braziel NN, Huisman TH. On the chromatographic heterogeneity of human fetal hemoglobin.

Biochim Biophys Acta 1979;577:159-169.

8. Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory. Clin Chem 2004;50:1736-1747.

DOI: 10.4274/TJH-2013.0125

Letter to the Editor

Follicular Lymphoma Patient Relapsing with Paraneoplastic Sensory Neuronopathy (Ganglioneuropathy) Paraneoplastik Sensoryal Nöropati (Ganglionopati) ile Tekrarlayan Foliküler Lenfoma Olgusu Kadir Öztürk, Hakan Akgün, Çakar, Mustafa, Yusuf Emrah Eyi, Yakup Aksoy Gülhane Military Medical Academy, Department of Gastroenterology, Ankara, Turkey

To the Editor, Neurological symptoms can be seen in 5%-8% of lymphoma patients. The most frequent causes of neurological symptoms are herpes virus infection, neurotoxicity due to vincristine, Guillain–Barré syndrome, and the nerve involvement of the tumor [1]. Paraneoplastic polyneuropathy is a rare neurological complication of lymphoma [2]. Here we report a case of relapsed follicular lymphoma presenting with paraneoplastic sensory neuropathy (ganglioneuropathy).

A 45-year-old woman was admitted to the hospital 2 years ago with the complaints of numbness in the hands and feet, unsteadiness, weight loss, fever, and swellings in the neck, axilla, and inguinal region. She was diagnosed with follicular lymphoma through the excisional biopsy of the cervical lymph node. She was diagnosed with stage 3B disease based on the presence of lymphadenopathies in the paratracheal, precarinal, axillary, and inguinal regions and no infiltration by bone marrow examination. The treatment started with rituximab (500 mg/day), cyclophosphamide (1125 mg/day), adriablastine (80 mg/day), vincristine (2 mg/day), and prednisolone (80 mg/day). Therapy was

completed in 8 courses with 21-day intervals. After the therapy, all of the symptoms had regressed and the case was evaluated as in remission with a follow-up PET scan (Figure 1). Gradually increasing unsteadiness, numbness, and pain and burning sensation in the hands and feet developed 1 month following that PET study. She applied to the neurology service and it was found that she had normal muscle strength but mild ataxia, positive Romberg test, and prominent hypoesthesia in the distal regions of the extremities and bilateral impairment of the deep tendon reflexes and indifferent plantar responses. Informed consent was obtained.

An electromyography revealed sensorial gangliopathy.

On physical examination the positioning and the vibration sensations of the patient were found to be diminished.

Intravenous pulse steroid therapy (1000 mg/day) was administered for 5 days. Two days after this therapy, a severe and generalized pain developed in all of the extremities and joints. In neurological examination, distinct quadriparesis, ataxia, dysmetria, dysdiadochokinesia, hypoesthesia of the 4 extremities, and bilateral abolished deep tendon reflex with indifferent plantar responses were determined. There was

Address for Correspondence: Kadir ÖZTÜRK, M.D., Gülhane Military Medical Academy, Department of Gastroenterology, Ankara, Turkey GSM: +90 535 277 39 7? E-mail: kadirozturk3041@gmail.com Received/Geliş tarihi Accepted/Kabul tarihi

: April 10, 2013 : May 6, 2013

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Turk J Hematol 2013;30:333-335

Figure 1: Follow-up PET reveals the remission of follicular lymphoma.

Figure 2: PET reveals relapse of follicular lymphoma (green circle). neither vibration nor position sensation below the knees or elbows. Sensory-motor axonal neuropathy, which affected the motor component of the upper and lower extremities, was seen in repeated EMG. Plasmapheresis therapy was performed every other day for 7 days, but the patient derived no benefit from the therapy. Abdominal ultrasonography was then performed and a tumor located in the left upper anterior region and consistent with lymph node involvement was detected.

Pathological lymph nodes were seen in a the mesenteric, paraaortic, and aortocaval regions with abdominal computed tomography (CT). A tumor that was located in the left upper anterior region and consistent with lymph node involvement was also detected by PET scan (Figure 2). The levels of thyroid-stimulating hormone, vitamin B12, and 334

Öztürk K, et al: Follicular Lymphoma Patient Relapsing with Paraneoplastic Sensory Neuronopathy (Ganglioneuropathy)

folate were normal. Herpes simplex virus (HSV) antibody was negative. The levels of immunoglobulin and the lumbar puncture were inconclusive. There were no pathologic signs in the evaluation of cerebral, cervical, and thoracolumbar magnetic resonance imaging (MRI). Therefore, the patient was diagnosed with paraneoplastic polyneuropathy due to relapsed non-Hodgkin lymphoma. Dexamethasone (40 mg/day), cisplatin (50 mg/day), and cytosine arabinoside (3 g/day) were given in 3 courses. Progressive enlargement of the lymph nodes upon follow-up CT suggested tumor progression. High-dose chemotherapy (BCNU 300 mg/m2, etoposide 200 mg/m2, Ara-C 200 mg/m2, melphalan 140 mg/m2) was given and autologous stem cell transplantation (ASCT) was performed.

Following the ASCT and high-dose chemotherapy there was an improvement in motor functions, although mild numbness of the hands and feet continued. The sensorymotor axonal polyneuropathy of the upper and lower extremities was demonstrated in follow-up EMG. The pathogenesis of paraneoplastic neuropathy is not clear yet, but it is attributed to autoimmune mechanisms in most cases. Both axonal and demyelinating involvement of the peripheral nerves can be seen in paraneoplastic neuropathy patients [3]. In our case, the HSV antibody was negative and there was history of vincristine usage.

However, the symptoms appeared 9 months after the use of vincristine. Vincristine-induced neuropathy usually appears between weeks 2 and 18 [4]. For this reason, the neurological symptoms were thought not to occur due to use of vincristine. Guillain–Barré syndrome was eliminated as there was no albuminocytologic dissociation by the analysis of cerebrospinal fluid and there was no demyelinating disease upon EMG. As there were no pathologic signs upon cerebral, cervical, and thoracolumbar MRI evaluation, the patient was diagnosed with paraneoplastic polyneuropathy due to relapsed follicular lymphoma.

Paraneoplastic ganglioneuropathy is rare. Ganglioneuropathy is a syndrome characterized by a symmetric/ asymmetric involvement of sensorial components, diminishing or disappearance of reflexes, ataxia, glove- and stocking-type pain, and dysesthesia. These symptoms can be seen in any localization of the body, including the face. The potential of the sensory-motor nerve might be diminished while the motor transmission speed is normal or almost normal in electrophysiological studies [5]. In our case, a simultaneous axonal involvement was seen, associated with ganglioneuropathy symptoms such as clinical unsteadiness and numbness, and later on, additional motor involvement was seen. The level of vitamin B12 was normal and there was no history of alcoholism or diabetes.

In conclusion, paraneoplastic sensory neuropathy should be kept in mind in patients who present with neuropathy, particularly in the patients with malignancy. Neurotoxic drugs, infection, tumor invasion, and autoimmune disorders should be eliminated and the primary disease should be treated accordingly.

Öztürk K: Follicular Lymphoma Patient Relapsing with Paraneoplastic Sensory Neuronopathy (Ganglioneuropathy)

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Hughes RA, Britton T, Richards M. Effects of lymphoma on the peripheral nervous system. J R Soc Med 1994;94:526530.

Turk J Hematol 2013;30:333-335

3. Vallat JM, Leboutet MJ, Hugon J, Tabaraud F, Chartier JP, Boulesteix JM. Demyelinating polyneuropathy associated with cancer-a nerve biopsy study of eight cases. Clin Neuropathol 1988;7:217.

4. Legha SS. Vincristine neurotoxicity. Pathophysiology and management. Med Toxicol 1986;1:421-427.

5. Dalmau J, Rosenfeld MR. Paraneoplastic neurologic syndromes. In: Kasper DL, Braunwald E, Hauser S, Longo D, Jameson JL, Fauci AS, (eds). Harrison’s Principles of Internal Medicine. New York, McGraw-Hill, 2005.

2. Symonds RP, Hogg RB, Bone I. Paraneoplastic neurological syndromes associated with lymphomas. Leuk Lymphoma 1994;94:487-490.

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DOI: 10.4274/TJH-2013-0109

Letter to the Editor

Gynecomastia in a Boy with Chronic Myeloid Leukemia during Imatinib Therapy Kronik Miyeloid Lösemili Bir Erkek Çocukta, Imatinib Tedavisi Sırasında Gelişen Jinekomasti Betül Tavil1, Sibel Kınık2, Ahmet Gözen3, Lale Olcay1 1Ankara

Oncology Research and Training Hospital, Unit of Pediatric Hematology, Turkey

2Başkent

University School of Medicine, Department of Pediatrics, Unit of Pediatric Endocrinology, Turkey

3Ankara

Oncology Research and Training Hospital, Unit of Radiology, Turkey

4Ankara

Oncology Research and Training Hospital, Unit of Pediatric Hematology, Turkey

To the Editor, Chronic myeloid leukemia (CML) is a rare disease in children, accounting for 2%-3% of leukemias in this age group.

Current treatment options of CML include tyrosine kinase inhibitors (TKIs) and allogeneic hematopoietic stem cell transplantation (HSCT) for children. Allogeneic HSCT has been recommended following attainment of remission with TKIs. Since long-term outcome and adverse effects of TKIs are uncertain in children, some authors have recommended HSCT within 2 years of treatment with TKIs [1,2,3]. Imatinib mesylate is one of the TKIs; it inhibits all ABL tyrosine kinases and selectively suppresses the ATP-binding site of platelet-derived growth factor receptor (PDGF-R) and c-KIT, which are expressed in various types of neoplasms and also in normal cells. It is well tolerated with mild adverse effects.

Common and usually mild side effects of imatinib mesylate include edema, muscle cramps, skin rash, and conjunctival inflammation. An altered bone and mineral metabolism, a reduction of testosterone, and gynecomastia are the other defined side effects of imatinib mesylate, which were attributed to the PDGF-R and c-KIT inhibition in normal cells by imatinib mesylate. Gynecomastia development during the course of imatinib mesylate treatment has rarely been reported in the English literature [4,5,6,7]. Herein, we have described a 14-year-old boy with CML who developed gynecomastia while receiving imatinib mesylate for nearly 1.5 years.

A 14-year-old boy was admitted to hospital with complaints of malaise, cough, and pain in the lower extremities. His previous medical history was unremarkable. There was no consanguinity between parents and he had 2 healthy siblings.

On his physical examination, he had mild hepatomegaly (2 cm below the costal margin) and massive splenomegaly (18 cm below the costal margin). His laboratory studies revealed that

his hemoglobin was 100 g/L; hematocrit, 23%; white blood cell count, 365x109/L with 62% neutrophils, 14% stab cells, 6% metamyelocytes, 4% myelocytes, and 14% lymphocytes; mean corpuscular volume, 87 fL; and platelet count, 415x109/L. Biochemical parameters were all normal. Studies for Epstein– Barr virus, cytomegalovirus, herpes viruses, toxoplasmosis, measles, mumps, rubella, parvovirus, Salmonella, and Brucella did not show any ongoing infection with these agents. His abdominal ultrasonography revealed hepatosplenomegaly.

His echocardiography was normal. Bone marrow aspiration smears revealed hypercellularity and hyperactivity in the myeloid lineage with less than 5% blasts. Cytogenetic study of his bone marrow showed clonal positivity for t(9;22). Thus, the patient was diagnosed with CML in the accelerated phase.

After the diagnosis of CML, he received cytosine arabinoside (150 mg/m2/day) and mitoxantrone (10 mg/m2/day) 2 times at a 1-week interval. Hydration (3000 mL/m2/day and 20 mEq/L, NaHCO3) and allopurinol treatment were continued until his leukocyte count decreased to normal levels.

After learning that t(9;22) was positive, imatinib mesylate treatment (260 mg/m2) was started. Complete hematological response was achieved after 1 month of therapy. His t(9;22) was still positive after 3 months of imatinib mesylate.

Complete molecular response was achieved 9 months after the diagnosis. While he was receiving imatinib mesylate treatment, a human leukocyte antigen-identical sibling donor was found for HSCT. However, his father refused the HSCT and the patient and his father preferred continuation of drug therapy (imatinib mesylate). At month 17 of imatinib mesylate therapy, he was found to have developed bilateral gynecomastia. His pubertal development was not completed at diagnosis (Tanner grade 1), whereas he was in puberty when gynecomastia was diagnosed. His β-human chorionic gonadotropin and α-fetoprotein levels were normal. His free

Address for Correspondence: Lale OLCAY, M.D., Ankara Oncology Research and Training Hospital, Unit of Pediatric Hematology, Ankara, Turkey Gsm: +90 532 760 09 82 E-mail: laleolcay@hotmail.com.tr Received/Geliş tarihi : March 24, 2013 Accepted/Kabul tarihi : May 8, 2013

336

Olcay L, et al: Posttransplantation Hypothyroidism

testosterone level was decreased (3.48 pg/mL, normal range: 12-30) and his estradiol level was increased (25.69 pg/mL, normal range: 2-18). Bilateral breast ultrasonography also showed gynecomastia (Figures 1a and 1b). After he developed gynecomastia, he was irregularly admitted to the hospital for follow-up visits. When he presented to the hospital due to an accelerated phase of CML, it was learned that he had discontinued imatinib therapy for the last 8 months. It was found that the gynecomastia had resolved. Imatinib mesylate treatment was started again and complete hematological and molecular responses were achieved. During the consecutive 1-year follow-up, gynecomastia did not recur. However, later it was learned that he irregularly took the imatinib mesylate, and he was then lost to follow-up. Informed consent was obtained.

Imatinib is a signal transduction inhibitor; it inhibits the oncogenic tyrosine kinase BCR-ABL, c-KIT, and PDGF-R. c-KIT and PDGF-R are receptors of tyrosine kinases expressed in the testis and they are involved in testosterone production.

These receptors are inhibited by imatinib mesylate treatment.

This inhibition may be responsible for the development of gynecomastia. Gambacorti-Passerini et al. [7] reported that 7 of 38 men receiving imatinib for CML developed gynecomastia. They also measured hormone concentrations in 38 men receiving imatinib for CML at baseline and during

Turk J Hematol 2013;30:336-337

treatment. The mean follow-up duration was 23.6 months in that study. A comparison of hormone concentrations in 21 patients before and during treatment showed that patients who developed gynecomastia had a reduction in free testosterone concentrations [7]. Liu et al. [4] reported that among 57 male patients treated with imatinib mesylate due to advanced or recurrent gastrointestinal stromal tumor, 6 (10.5%) developed gynecomastia during the treatment. Their serum free testosterone levels were within the normal range, whereas 3 had increased serum estradiol level [4]. Thus, reduced free testosterone levels and increased serum estradiol levels may be seen in patients receiving imatinib mesylate.

Imbalance of sex hormones may be the cause of gynecomastia during treatment with imatinib mesylate. Free testosterone level was decreased and serum estradiol level was increased in our patient. At diagnosis, his pubertal development was not completed, whereas his puberty was completed during treatment and gynecomastia developed after puberty. We think that the gynecomastia resolved since the patient discontinued therapy first and then did not take it regularly during the consecutive follow-up period. In conclusion, boys who receive imatinib mesylate treatment should be examined from this aspect during follow-up visits. It should be kept in mind that gynecomastia can develop in a boy with CML following long-term imatinib mesylate treatment.

Conflict of Interest Statement The authors declare that they have no conflicts of interest that could be perceived as having influenced the impartiality of the materials presented.

Funding The present study received no grant from a funding agency in the public, commercial, or for-profit sectors.

Refenences

Figure 1: a) Breast tissue of 32x7.5 mm on the right side by ultrasonography. b) Breast tissue of 30x7 mm on the left side.

1. Suttorp M, Yaniv I, Schultz KR. Controversies in the treatment of CML in children and adolescents: TKIs versus BMT? Biol Blood Marrow Transplant 2011;17(Suppl 1):115-122.

2. Suttorp M Innovative approaches of targeted therapy for CML of childhood in combination with paediatric haematopoietic SCT. Bone Marrow Transplant 2008;42(Suppl 2):40-46.

3. Suttorp M, Millot F. Treatment of pediatric chronic myeloid leukemia in the year 2010: use of tyrosine kinase inhibitors and stem-cell transplantation. Hematology Am Soc Hematol Educ Program 2010;2010:368-376.

4. Liu H, Liao G, Yan Z. Gynecomastia during imatinib mesylate treatment for gastrointestinal stromal tumor: a rare adverse event. BMC Gastroenterol 2011;11:116.

5. Tanriverdi O, Unubol M, Taskin F, Meydan N, Sargin G, Guney E, Barutca S. Imatinib-associated bilateral gynecomastia and unilateral testicular hydrocele in male patient with metastatic gastrointestinal stromal tumor: a literature review. J Oncol Pharm Pract 2012;18:303-310.

6. Caocci G, Atzeni S, OrrĂš N, Azzena L, Martorana L, Littera R, Ledda A, La Nasa G. Gynecomastia in a male after dasatinib treatment for chronic myeloid leukemia. Leukemia 2008;22:2127-2128.

7. Gambacorti-Passerini C, Tornaghi L, Cavagnini F, Rossi P, Pecori-Giraldi F, Mariani L, Cambiaghi N, Pogliani E, Corneo G, Gnessi L. Gynaecomastia in men with chronic myeloid leukaemia after imatinib. Lancet 2003;361:1954-1956.

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DOI: 10.4274/Tjh.2013.0079

Letter to the Editor

Is There an Optimal Timing of Autologous Stem-Cell Transplantation for Multiple Myeloma in the Era of Novel Agents?

Yeni İlaçların Döneminde, Multiple Myelomada Kök Hücre Nakli İçin Uygun Bir Zamanlama Var mıdır?

Selami Koçak Toprak

Başkent University School of Medicine, Department of Hematology, Ankara, Turkey

To the Editor, In the last couple of years, parallel to the developments in our knowledge of disease pathogenesis, the inclusion of immunomodulatory drugs like thalidomide and lenalidomide and various protease inhibitors such as bortezomib to treatment protocols in clinical practice has led to dramatic changes in the treatment of multiple myeloma (MM). These agents particularly have survival advantages in relapse/refractory cases, while changing the classic first-line treatment approach with their recent good results in frontline treatment [1]. The performance of high-dose therapy plus autologous stem cell transplantation (HDT-ASCT) as soon as possible in eligible patients has been the standard treatment approach for almost 20 years [2]. In this period, in many regions, including Turkey, the performance of generally 3 to 6 cycles of an induction treatment consisting of vincristine– doxorubicin–dexamethasone (VAD) with the aim of improving hematopoietic cell collection by firstly decreasing plasma cell infiltration and reducing the tumor burden and increasing post-transplantation complete response rates has been widely done. However, the pre-transplantation results of this treatment have always been disappointing [1]. Nevertheless, the pre- and post-transplantation response rates of a treatment combination like thalidomide– bortezomib–dexamethasone (VTD) have been prominently superior, while the presence of bad prognosis markers, Address for Correspondence: Selami Koçak TOPRAK, M.D., Baskent University School of Medicine, Department of Hematology, Ankara, Turkey Phone: +90 532 656 02 06 E-mail: sktoprak@yahoo.com Received/Geliş tarihi : March 4, 2013 Accepted/Kabul tarihi : March 15, 2013

338

especially t(4;14), del(13q), and del(17p), requires the use of new agents [3]. Supporting this, a study by Palumbo et al. revealed that induction combinations including bortezomib and consequent post-transplantation consolidativemaintenance lenalidomide have led to a complete response (CR) in 66% of cases [4].

In the era of such good results in pre-transplantation treatment with new agents, should the HDT-ASCT approach be used in every patient as soon as possible? Or should it absolutely be performed? Depending on the increased rate of CR and prolonged progression-free survival (PFS) when compared with conventional chemotherapy in several randomized phase III studies, HDT-ASCT has been considered the standard of care for eligible patients with newly diagnosed MM [5]. However, HDT-ASCT is not curative and progression/relapses occur in most patients. Furthermore, according to a systematic review and meta-analysis published in 2007, single HDT-ASCT was not superior to conventional first-line treatments in terms of total survival; additionally, rates of treatmentrelated mortality were also higher [6]. Results of phase III studies have proven that combinations of new agents like VTD, bortezomib–doxorubicin–dexamethasone, lenalidomide with low-dose dexamethasone (Rd), bortezomib–dexamethasone, thalidomide–dexamethasone, and low-dose bortezomib–thalidomide–dexamethasone

Selami Koçak Toprak; Optimal timing of autologous transplantation for multiple myeloma

are superior to conventional approaches when used in first-line treatment [7]. The high overall and CR rates obtained with new agents have led to suspicions regarding the role and timing of transplantation in the minds of all clinicians, especially in the United States; in the US and many other countries, the idea has developed that first-line induction treatment with new agents should be prolonged until relapse/progress -with the condition that stem cells are collected in this period- and, thus, that transplantation should be left for relapse. In this regard, Rajkumar suggested the risk-adopted approach in the selection of first-line treatment for newly diagnosed MM patients and indicated that in standard-risk disease (about 75% of all cases), firstline treatment should consist of Rd × 4 cycles (stem cells would be collected during this period), followed by early or delayed HDT-ASCT performed in accordance with the patient’s choice [8]. Intermediate-risk patients, who are 10% of the population, should receive bortezomib-based firstline treatment followed by early HDT-ASCT and then again bortezomib-based maintenance treatment. In the remaining 15% who have high-risk disease, induction with bortezomib and lenalidomide-based combination should be followed

by HDT-ASCT and then bortezomib-based maintenance treatment; however, it was pointed out that in this patient group with a median survival of 2-3 years, clinical studies about more efficient treatments are required [8,9]. In a prior study, new agents were used and patients were evaluated in 2 groups according to HDT-ASCT performed in the first year or later; in the group with induction treatment consisting of lenalidomide–dexamethasone, 4-year survival was over 80%, independently of the timing of transplantation [10].

Nevertheless, in the phase III study of Boccadoro et al., new agents were compared with HDT-ASCT in 402 newly diagnosed MM patients and total survival was not different between 2 arms, while PFS was significantly higher in the arm with autologous stem cell transplantation [11]. As a result, despite prominently superior results of first-line treatment with combinations consisting of new agents, followed by HDT-ASCT and then by maintenance treatment with new agents, in order to decide to exclude HDT-ASCT and perform first-line induction treatment with new agents until relapse, results of multi-center randomized studies performed on large patient groups should be awaited.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Harousseau JL. How to select among available options for the treatment of multiple myeloma. Ann Oncol 2012;23(Suppl 10):334-338.

2. Harousseau JL, Moreau P. Autologous hematopoietic stemcell transplantation for multiple myeloma. N Engl J Med 2009;360:2645-2654.

Turk J Hematol 2013;30:338-339

3. Cavo M, Tacchetti P, Patriarca F, Petrucci MT, Pantani L, Galli M, Di Raimondo F, Crippa C, Zamagni E, Palumbo A, Offidani M, Corradini P, Narni F, Spadano A, Pescosta N, Deliliers GL, Ledda A, Cellini C, Caravita T, Tosi P, Baccarani M; GIMEMA Italian Myeloma Network. Bortezomib with thalidomide plus dexamethasone compared with thalidomide plus dexamethasone as induction therapy before, and consolidation therapy after, double autologous stem-cell transplantation in newly diagnosed multiple myeloma: a randomised phase 3 study. Lancet 2010;376:2075-2085.

4. Palumbo A, Gay F, Falco P, Crippa C, Montefusco V, Patriarca F, Rossini F, Caltagirone S, Benevolo G, Pescosta N, Guglielmelli T, Bringhen S, Offidani M, Giuliani N, Petrucci MT, Musto P, Liberati AM, Rossi G, Corradini P, Boccadoro M. Bortezomib as induction before autologous transplantation, followed by lenalidomide as consolidationmaintenance in untreated multiple myeloma patients. J Clin Oncol 2010;28:800-807.

5. Blanes M, de la Rubia J. Role of autologous bone marrow transplant in multiple myeloma. Curr Opin Oncol 2012;24:733-741.

6. Koreth J, Cutler CS, Djulbegovic B, Behl R, Schlossman RL, Munshi NC, Richardson PG, Anderson KC, Soiffer RJ, Alyea EP 3rd. High-dose therapy with single autologous transplantation versus chemotherapy for newly diagnosed multiple myeloma: A systematic review and meta-analysis of randomized controlled trials. Biol Blood Marrow Transplant 2007;13:183-196.

7. Lonial S, Miguel JF. Induction therapy for newly diagnosed multiple myeloma. J Natl Compr Canc Netw 2013;11:1928.

8. Moreau P, Rajkumar SV. Should all eligible patients with multiple myeloma receive autologous stem-cell transplant as part of initial treatment? Leuk Res 2012;36:677-681.

9. Rajkumar SV. Multiple myeloma: 2012 update on diagnosis, risk-stratification, and management. Am J Hematol 2012;87:78-88.

10. Kumar SK, Lacy MQ, Dispenzieri A, Buadi FK, Hayman SR, Dingli D, Gay F, Sinha S, Leung N, Hogan W, Rajkumar SV, Gertz MA. Early versus delayed autologous transplantation after immunomodulatory agents-based induction therapy in patients with newly diagnosed multiple myeloma. Cancer 2012;118:1585-1592.

11. Boccadoro M, Cavallo F, Nagler A, Ben Yehuda D, Omedè P, Cavalli M, Levi A, Crippa C, Siniscalchi A, Brasca P, Carella AM, Zanetti BA, Patriarca F, Pezzati S, Montefusco V, Stanevsky A, Lupo B, Caravita T, Di Raimondo F, Palumbo AP. Melphalan/prednisone/lenalidomide (MPR) versus highdose melphalan and autologous transplantation (MEL200) in newly diagnosed multiple myeloma (MM) patients: a phase III trial. J Clin Oncol 2011;29(Suppl):8020.

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Images in Hematology

Mees’ Lines in an Acute Myeloid Leukemia Patient Akut Miyeloid Lösemi Hastasında Mees Çizgileri

DOI: 10.4274/Tjh.2013.0048

A 26-year-old black male was treated for acute myeloid leukemia, subtype AML4. The karyotype showed trisomy of chromosome 8 and monosomy of chromosome 15. The patient received 5 courses of chemotherapy with complete remission after the first induction. Each aplasia was marked by deep infection, often treated with antibiotherapy and antifungal treatment. Chemotherapy courses included aracytine, daunorubicin, etoposide, and kidrolase. The patient developed multiple white lines on the fingernails, known as Mees’ lines [1,2]. The distance between the lines showed that the patient received the 2 last courses of chemotherapy with a large delay. Each line corresponds to the start of a chemotherapy course. Cytotoxic agents and infections can induce the temporary arrest of proliferation of the nail matrix, which is called Mees’ lines of the nail plate.

The patient remains in complete remission with a followup of 6 months and the Beau’s lines of his nail plate have disappeared completely. Informed consent was obtained.

Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Huang TC, Chao TY. Mees lines and Beau lines after chemotherapy. CMAJ 2010;182:149.

2. Hinds G, Thomas VD. Malignancy and cancer treatmentrelated hair and nail changes. Dermatol Clin 2008;26:5968.

Soumaya Anoun, Meryem Qachouh, Mouna Lamchahab, Asmae Quessar, Said Benchekroun Hopital du 20 Aout – Hematology & Pediatric Oncology Residence Fares 5 rue Abou Fares Bnou Hamdane, Casablanca, Morocco

Address for Correspondence: Soumaya ANOUN, M.D., Hopital du 20 Aout – Hematology & Pediatric Oncology Residence fares 5 rue abou fares bnou hamdane, Casablanca 20500 Morocco Phone: +90 212 662 391 10 E-mail: soumaya.anoun@gmail.com Received/Geliş tarihi : February 12, 2013 Accepted/Kabul tarihi : March 25, 2013

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Images in Hematology

DOI: TJH-2011.0018

The Side Effects of Imatinib Imatinib’in Yan Etkileri

Figure 3: Maculopapular rash on the back.

Figure 1: Maculopapular rash on the face, neck and trunk.

Figure 2: Maculopapular rash on the right arm and trunk.

Elmas Uzer, Ali Ünal, M. Yavuz Köker, Suat Ali Doğan* University of Erciyes - Division of Hematology, Kayseri, Turkey *University of Erciyes - Department of Infectious Diseases and Clinical Microbiology , Kayseri, Turkey Address for Correspondence: Elmas UZER, M.D., Erciyes University Mehmet Kemal Dedeman Hematology and Onkology Hospital Kayseri 38039 Turkey Phone: +90 352 437 49 37 - 27440 E-mail: mdelmasuzer@yahoo.com Received/Geliş tarihi : October 31, 2011 Accepted/Kabul tarihi : November 22, 2012

A 43-year-old male patient was diagnosed with chronic myelogenous leukemia (CML), and hydroxyurea treatment was begun 3 months before consultation. Physical examination revealed the spleen 2 cm below the costal margin. The Philadelphia chromosome was positive. After hydroxyurea treatment, imatinib (Gleevec) was started. On the 15th day of imatinib treatment, the patient was admitted because of fever, diarrhea, and generalized rash. Informed consent was obtained.

Whole blood count showed a hemoglobin concentration of 10.5 g/L, white cell count of 23,000/mm3, and platelet count of 240,000/mm3. Imatinib treatment was stopped to see if these symptoms were related to the drug. After cessation of imatinib, the fever resolved and the skin lesions disappeared. Imatinib was changed to dasatinib. The patient takes dasatinib at 100 mg and the treatment is on-going.

A wide spectrum of dermatologic toxicities has been associated with imatinib, among which a maculopapular rash is the most common event. Imatinib-induced skin rash is believed to be due to the blockade of the c-kit protein, which is present in skin. Severe toxicity of skin rash, fever, and diarrhea associated with imatinib has been reported.

The initial dose for chronic-phase disease, 400 mg/day, is very well tolerated. A higher risk for developing rash will occur with imatinib doses higher than 600 mg/day, advanced age, and female sex. In many patients who experience unacceptable adverse effects, transient dose reduction or treatment interruption allows for patients to resolve the adverse effects.

Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

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Advisory Board of This Issue (September 2013) Abdülkadir Babaoğlu, Turkey Ahmet Emre Eşkazan, Turkey Akif Yeşilipek, Turkey Aygül Doğan Çelik, Turkey Aytemiz Gürgey, Turkey Benjamin Bonavida, USA Berk Çağlar, USA Bülent Kantarcıoğlu, Turkey Burhan Ferhanoğlu, Turkey Burhan Turgut, Turkey Çağlar Berk, USA Can Boğa, Turkey Cem Akın, USA Cengiz Beyan, Turkey Davut Albayrak, Turkey Dina Fouad, Egypt Elif Ünal, Turkey Fatih Demirkan, Turkey Feride Duru, Turkey Feridun Acar, Turkey

Ferit Avcu, Turkey George Buchanan, USA Gökhan Erdem, Turkey Gülersu İrken, Turkey Gülsüm Emel Pamuk, Turkey Güray Saydam, Turkey Gürhan Kadıköylü, Turkey Hakan Özdoğu, Turkey Hamdi Akan, Turkey Handan Çipil, Turkey Luiz Martinez-Lostao, Spain Mahmut Bayık, Turkey Mehmet Ali Erkurt, Turkey Mehmet Ali Özcan, Turkey Mehmet Yılmaz, Turkey Meral Beksaç, Turkey Mine Hekimgil, Turkey Nazan Sarper, Turkey Nil Güler, Turkey Nilgün Sayınalp, Turkey

Nurcan Arat, Turkey Nuri Karadurmuş, Turkey Olga Meltem Akay, Turkey Orhan Ayyıldız, Turkey Owen J Mcginn, UK Rauf Haznedar, Turkey Reyhan Diz Küçükkaya, Turkey Roberto Stasi, UK Selami Koçak Toprak, Turkey Selim Çorbacıoğlu, Germany Selin Aytaç, Turkey Sermin Özkal, Turkey Tayfun Uçar, Turkey Tunç Fışgın, Turkey Türkan Patıroğlu, Turkey Veysel Sabri Hançer, Turkey Yi Le, USA Yunus Erdem, Turkey Yusuf Baran, Turkey Zeynep Şıklar, Turkey