Turkish Journal of Hematology Volume: 31 - Issue: 4 by LookUs Scientific

Issue 4

December 2014

40 TL

ISSN 1300-7777

Volume 31

Review Article Diagnosis of Invasive Fungal Diseases in Hematological Malignancies: A Critical Review of Evidence and Turkish Expert Opinion (TEO-2) Sevtap Arıkan Akdağlı, et al.; Ankara, Bursa, Kayseri, İstanbul, Turkey

Research Articles The Relationship between P-Selectin Polymorphisms and Thrombosis in Antiphospholipid Syndrome: A Pilot Case-Control Study Nilüfer Alpay, et al.; İstanbul, Turkey

Serum Bcl-2 Levels in Patients with β-Thalassemia Minor: A Pilot Study İrfan Yavaşoğlu, et al.; Aydın, Turkey

Duffy and Kidd Genotyping Facilitates Pretransfusion Testing in Patients Undergoing Long-Term Transfusion Therapy Diana Remeikiene, et al.; Kaunas, Lithuania

Bone-Specific Alkaline Phosphatase Levels among Patients with Multiple Myeloma Receiving Various Therapy Options Güven Çetin, et al.; İstanbul, Diyarbakır, Turkey

The Association of HLA Class 1 and Class 2 Antigens with Multiple Myeloma in Iranian Patients Arezou Sayad, et al.; Tehran, Iran

New Insights on Iron Study in Myelodysplasia Noha M. El Husseiny, et al.; Cairo, Egypt

Cover Picture: Işıl Erdoğan Winter’s Tale, Abant, Bolu

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  Memorial Şişli Hospital, İstanbul, Turkey  Santa Creu i Sant Pau Hospital, Barcelona, Spain  Mayo Clinic, Rochester, Minnesota, USA  Istanbul Cerrahpaşa University, İstanbul, Turkey  University of Minnesota, 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

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

Cengiz Beyan Gülhane Military Medical Academy, Ankara, Turkey

Hale Ören Dokuz Eylül University, İzmir, Turkey

İbrahim C. Haznedaroğlu Hacettepe University, Ankara, Turkey

İlknur Kozanoğlu Başkent University, Adana, Turkey

Mehmet Ertem Ankara University, Ankara, Turkey

A. 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

İnci Alacacıoğlu Dokuz Eylul University, Ankara, Turkey

Nil Güler On Dokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay Osmangazi University, Eskişehir, Turkey

Selami Koçak Toprak Ankara University, Ankara, Turkey

Şule Ünal Hacettepe University, Ankara, Turkey

Veysel Sabri Hançer İstanbul Bilim University, İstanbul, Turkey

Zühre Kaya

Statistic Editor Hülya Ellidokuz

Senior Advisory Board Yücel Tangün Osman İlhan Muhit Özcan

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

Publishing Manager Sorumlu Yazı İşleri Müdürü A. Muzaffer Demir

Management Address Yayın İdare Adresi

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

Publishing House / Yayınevi

Online Manuscript Submission

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul, Turkey Tel: +90 212 621 99 25 Faks: +90 212 621 99 27 E-posta: info@galenos.com.tr Baskı: Senk Ofset Matbaacılık Reklam Promosyon ve Tan. Hiz. San. Dış. Tic. Ltd. Şti. Tel.: +90 212 493 26 26 Topkapı Litros yolu, No: 24, Zeytinburnu, İstanbul, Turkey

http://mc.manuscriptcentral.com/tjh

Web page www.tjh.com.tr

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

Printing Date / Basım Tarihi 25.11.2014

Cover Picture

Işıl Erdoğan was born in 1982, Turkey. She is currently working at İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul, Turkey. Üç ayda bir yayımlanan İngilizce süreli yayındır. International scientific journal published quarterly. 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 - 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: 0.340 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 Telephone : 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.

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. Deeg HJ, Oâ&#x20AC;&#x2122;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. 2.Organization as author Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in posthepatitis marrow aplasia. Lancet 1977;2:742-744. 3.Book Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger, 1961.

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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. Fanconiâ&#x20AC;&#x2122;s anemia, transplantation, and cancer. Pediatr Transplant. 2005;9(Suppl 7):81-86

Brief Reports Abstract length: Not to exceed 150 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.

Case Reports Abstract length: Not to exceed 100 words. Article length: Not to exceed 1200 words. Case Reports can include maximum 1 figure and 1 table or 2 figures or 2 tables.

Case reports should be structured as follows:

Abstract 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. Authors who will prepare review articles should already have published research articles on therel evant subject. The study’s new and important findings should be highlighted and interpreted in the Conclusion section. There should be a maximum of two authors for review articles.

Images in Hematology Article length: Not exceed 200 words. Authors can submit for consideration an illustration and photos 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 200 words of text, 5 references, and 3 figure or table. No abstract, discussion or conclusion are 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

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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.

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) We use iThenticate to screen all submissions for plagiarism before publication. 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. Authors of accepted manuscripts will receive electronic page proofs and are responsible for proofreading and checking the entire article within two days. Failure to return the proof in two days will delay publication. If the authors cannot be reached by email or telephone within two weeks, the manuscript will be rejected and will not be published in the journal.

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.

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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 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

revisions can be recommended. Following initial peer-review, articles judged worthy of further consideration often require revision. Revised manuscripts generally must be received within 3 months of the date of the initial decision. Extensions must be requested from the Associate Editor at least 2 weeks before the 3-month revision deadline expires; The Turkish Journal of Hematology will reject manuscripts that are not received within the 3-month revision deadline. Manuscripts with extensive revision recommendations will be sent for further review (usually by the same reviewers) upon their re-submission. When a manuscript is finally accepted for publication, the Technical Editor undertakes a final edit and a marked-up copy will be e-mailed to the corresponding author for review and to make any final adjustments.

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CONTENTS 342

Review Article

357

Research Articles

Diagnosis of Invasive Fungal Diseases in Hematological Malignancies: A Critical Review of Evidence and Turkish Expert Opinion (TEO-2) Sevtap Arıkan Akdağlı, Alpay Azap, Figen Başaran Demirkazık, Beyza Ener, Sibel Aşcıoğlu Hayran, Özlem Özdemir Kumbasar, Gökhan Metan, Zekaver Odabaşı, Ömrüm Uzun, Hamdi Akan

The Relationship between P-Selectin Polymorphisms and Thrombosis in Antiphospholipid Syndrome: A Pilot Case-Control Study Nilüfer Alpay, Veysel Sabri Hançer, Burak Erer, Murat İnanç, Reyhan Diz-Küçükkaya

363

Serum Bcl-2 Levels in Patients with β-Thalassemia Minor: A Pilot Study İrfan Yavaşoğlu, Gökhan Sargın, Gürhan Kadıköylü, Aslıhan Karul, Zahit Bolaman

367

Duffy and Kidd Genotyping Facilitates Pretransfusion Testing in Patients Undergoing Long-Term Transfusion Therapy Diana Remeikiene, Rasa Ugenskiene, Arturas Inciura, Aiste Savukaityte, Danguole Raulinaityte, Erika Skrodeniene, Renata Simoliuniene, Elona Juozaityte

374

Bone-Specific Alkaline Phosphatase Levels among Patients with Multiple Myeloma Receiving Various Therapy Options Güven Çetin, Ahmet Emre Eşkazan, M. Cem Ar, Şeniz Öngören Aydın, Burhan Ferhanoğlu, Teoman Soysal, Zafer Başlar, Yıldız Aydın

381

The Relationship of T Helper-2 Pathway Components Interleukin-4, Interleukin-10, Immunoglobulin E, and Eosinophils with Prognostic Markers in Non-Hodgkin Lymphoma: A Case-Control Study Nil Güler, Engin Kelkitli, Hilmi Atay, Dilek Erdem, Hasan Alaçam, Yüksel Bek, Düzgün Özatlı, Mehmet Turgut, Levent Yıldız, İdris Yücel

388

The Association of HLA Class 1 and Class 2 Antigens with Multiple Myeloma in Iranian Patients Arezou Sayad, Mohammad Taghi Akbari, Mahshid Mehdizadeh, Elham Roshandel, Soheila Abedinpour, Abbas Hajifathali

394

New Insights on Iron Study in Myelodysplasia Noha M. El Husseiny, Dina Ahmed Mehaney, Mohamed Abd El Kader Morad

Case Reports

399

Severe Myelotoxicity Associated with Thiopurine S-methyltransferase*3A/*3C Polymorphisms in a Patient with Pediatric Leukemia and the Effect of Steroid Therapy Burcu Fatma Belen, Türkiz Gürsel, Nalan Akyürek, Meryem Albayrak, Zühre Kaya, Ülker Koçak

403

Intravascular Large B-Cell Lymphoma Diagnosed on Prostate Biopsy: A Case Report Nazan Özsan, Banu Sarsık, Asu Fergün Yılmaz, Adnan Şimşir, Ayhan Dönmez

408

Primary Splenic Angiosarcoma Revealed by Bone Marrow Metastasis Soumaya Anoun, Sofia Marouane, Asmae Quessar, Said Benchekroun

411

Aplastic Anemia Associated with Oral Terbinafine: A Case Report and Review of the Literature Bülent Kantarcıoğlu, Hüseyin Kemal Türköz, Güven Yılmaz, Funda Pepedil Tanrıkulu, Işık Kaygusuz Atagündüz, Cafer Adıgüzel, Tülin Fıratlı Tuğlular

A-IX

417

Letters to the Editor

420

c.761C>T Mutation Linked Hyper IgM Syndrome Presenting with Hypertransaminasemia and Arthritis Mehmet Halil Celiksoy, Stephan Borte, Aydan İkincioğulları, Meltem Ceyhan Bilgici, Filiz Karagöz, Ayhan Gazi Kalaycı, Alişan Yıldıran

422

Blastic Plasmacytoid Dendritic Cell Neoplasm: Single-Center Experience with Two Cases in One Year Alexandra Agapidou, Sophia Vakalopoulou, Dimitra Markala, Christina Chadjiaggelidou, Maria Tzimou, Theodosia Papadopoulou, Vasileia Garypidou

424

Mogamulizumab Treatment in a Hemodialysis Patient with Adult T-Cell Leukemia/Lymphoma Mari Yoshihara, Yasushi Kubota, Makoto Fukuda, Tomoya Kishi, Yuji Ikeda, Shinya Kimura

426

Chediak-Higashi Syndrome: A Case Report of a Girl without Silvery Hair and Oculocutaneous Albinism Presenting with Hemophagocytic Lymphohistiocytosis Murat Elevli, Halil Uğur Hatipoğlu, Mahmut Civilibal, Nilgün Selçuk Duru, Tiraje Celkan

428

Gaucher Cells or Pseudo-Gaucher Cells: That’s the Question Deniz Gören Şahin, Hava Üsküdar Teke, Mustafa Karagülle, Neslihan Andıç, Eren Gündüz, Serap Işıksoy, Olga Meltem Akay

430

Quilty Effect after Extracorporeal Photopheresis in a Patient with Severe Refractory Cardiac Allograft Rejection Özgür Ulaş Özcan, Tamer Sayın, Gürbey Soğut, Aylin Heper, Hüseyin Göksülük, Veysel Kutay Vurgun, Cansın Tulunay Kaya, Elif Ezgi Üstün, Osman İlhan, Çetin Erol

432

A Pediatric Patient with Intravenous Cyclosporine Anaphylaxis Who Tolerated the Oral Form Pamir Işık, Namik Özbek, Emine Dibek Mısırlıoğlu, Turan Bayhan, Suna Emir, Fatih Mehmet Azık, Bahattin Tunç

434

Acquired Hemophilia Şinasi Özsoylu

435

Images in Hematology

437

Aggressive Multiple Myeloma with Unusual Morphology Mehmet Sönmez, Hasan Mücahit Özbaş, Nilay Ermantaş, Ümit Çobanoğlu

2014 Index

2014 Subject Index

2014 Author Index

Significant Differences in Thymic Index of Thalassemia Major Patients Yeşim Oymak, Bülent Güzel, Hüseyin Gümüş, Erdem Dağlıoğlu, Ali Ayçiçek, Ahmet Koç, Derya Özyürük

Generalized Necrobiotic Xanthogranuloma in a Patient with Multiple Myeloma Maria Jimenez Esteso, Jose Verdu, Francisco de Paz, Fabian Tarin

A-X

NEWS ISH 2014 World Congress Report of the Chair of Council The XXXVth World Congress of the International Society of Hematology (ISH) was held on September 4-5, 2014, in Beijing, China, hosted by the Chinese Society of Hematology (CSH). The President of the Congress was Prof. Xiaojun Huang, the Co-Chairs were Prof. Changgeng Ruan and Prof. Saijuan Chen, and Prof. Kaiyan Liu served as the Secretary-General. The ISH 2014 Beijing World Congress was highly successful and well organized, with a balanced scientific program in basic and clinical hematology. The venue was beautifully located next to the Beijing Olympic Garden with the spectacular Bird’s Nest Stadium and Beijing National Aquatics Center. There were 420 international members with a total of 1450 attendees from 34 countries, and 410 abstracts were submitted to the meeting. ISH Travel Awards sponsored by the Congress Organizing Committee were presented to 22 junior hematologists from 20 countries. The scientific program started with Prof. Zhu Chen’s excellent Miwa Lecture entitled “Acute Promyelocytic Leukemia: Achievements, Challenges and Expectations”. Zhu Chen (Shanghai Institute of Hematology) described his original contributions in the role of ATRA and its implications in APL. He reviewed the mechanisms of ATRA in APL, the efficacy of arsenic trioxide (ATO), its synergy with ATRA, and lessons learned from leukemic genomics. Chen also focused on treatment algorithms in low-, intermediate-, and highrisk APL patients. His talk was followed by Jacob M. Rowe (Shaare Zedek Medical Center, Jerusalem, Israel), who reviewed the role of stem cell transplantation, its indications, and survival data in AML. Rowe addressed clear indications and eligibility of adult AML patients for auto- and allogeneic HSCT. He also explained current challenges as well as controversies in allo-HSCT and RIC for older patients in CR1, the safety of MUD, the rate of relapse, the ability to “cure” at relapse, and GVL effect. In the IAD-EADAPD joint session entitled “Genomic and Molecular Medicine in Hematology”, Seichi Ogawa (Kyoto, Japan) covered novel somatic mutations in AML and MDS. Ruben Mesa (Mayo Clinic, Scottsdale, Arizona, USA) clearly highlighted molecular targeting in myeloid malignancies and state-of-the art knowledge in myeloproliferative neoplasms. Mesa elegantly covered targeting issues from TKIs, i.e. imatinib, to novel therapeutics and their future implications. Tayfun Özçelik (Bilkent University, Ankara, Turkey) demonstrated very convincing data for careful interpretation of gene mutations in hematologic malignancies. Özçelik also cautioned us on “reading” and “translation” of genome sequencing data, giving excellent case examples from his original research. He explained his pioneering research in genomic sequencing and DNA-based diagnostics, including forensics in Turkey, as well as his recent discovery of the VLDR mutation in cerebellar hypoplasia associated with “quadrupedal gait in humans”. During the congress there were also sessions on case-based presentations. The meeting also included various presentations on acute leukemias, lymphomas, CLL, CML, multiple myeloma, and coagulation and platelet disorders. The oral and poster sessions were well attended. During the ISH business meetings, David Gómez-Almaguer (Mexico) was elected as the

new Secretary-General of the Inter-American Division (IAD), replacing Carlos Ponzinibbio (Argentina), who had completed his term. Sabri Kemahlı was reelected as the Secretary-General of the European-African Division (EAD). Future Perspectives The ISH is the only world hematology organization, established by the national hematology societies in 1946. The ISH has always had the strongest commitment to serve, educate, and provide up-to-date knowledge, as well as to collaborate with professional societies including the ASH, ICSH, ISTH, and ISEH. As the newly elected Chair of Council, I envision a future for the Society as a strong source of global education in hematology, embracing all countries. I am planning to serve the Society within the framework of the following major topics: (a) to have an interactive, regularly updated, and user-friendly website; (b) to establish scientific working groups dealing with specified issues; (c) to establish a ‘Code of Conduct’ for scientific ethics; (d) to make the biannual congresses a venue where all basic and clinical researchers are able to present their latest findings, and where hematologists in general practice can be informed and inspired by the very best courses and lecturers; (e) to establish training courses and educational meetings between the biannual congresses in joint activity with national hematology meetings, aimed at enhancing the training and professional development of junior members and particularly those in Africa, Asia, Eastern Europe, and Latin America; (f) to improve our fiscal responsibilities and our financial status; and (g) to improve communication among ISH members through an efficient website and to stimulate interactive bridging with other related professional hematological societies.

We look forward to seeing you all at the ISH 2016 World Congress, in Glasgow-UK, on April 18-21, 2016 (http://www.ish2016.com). Emin Kansu, M.D., FACP ISH Chair of Council eminkansu47@gmail.com A-XI

Review Article

DOI: 10.4274/tjh.2014.0218

Diagnosis of Invasive Fungal Diseases in Hematological Malignancies: A Critical Review of Evidence and Turkish Expert Opinion (TEO-2) Hematolojik Malignitelerde Görülen İnvazif Fungal İnfeksiyonların Tanısında Tanı Araçları: Kanıtlara Eleştirel Bakış ve Türk Uzman Görüşleri (TUG-2) Sevtap Arıkan Akdağlı1, Alpay Azap2, Figen Başaran Demirkazık3, Beyza Ener4, Sibel Aşcıoğlu Hayran5, Özlem Özdemir Kumbasar6, Gökhan Metan7, Zekaver Odabaşı8, Ömrüm Uzun5, Hamdi Akan9 1Hacettepe

University Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, Ankara, Turkey 3Hacettepe University Faculty of Medicine, Department of Radiology, Ankara, Turkey 4Uludağ University Faculty of Medicine, Department of Microbiology, Bursa, Turkey 5Hacettepe University Faculty of Medicine, Department of Infectious Diseases, Ankara, Turkey 6Ankara University Faculty of Medicine, Department of Pulmonary Diseases, Ankara, Turkey 7Erciyes University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, Kayseri, Turkey 8Marmara University Faculty of Medicine, Department of Infectious Diseases, İstanbul, Turkey 9Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey 2Ankara

Abstract: One of the most problematic issues in hematological malignancies is the diagnosis of invasive fungal diseases. Especially, the difficulty of mycological diagnosis and the necessity of immediate intervention in molds have led to the adoption of “surrogate markers” that do not verify but rather strongly suggest fungal infection. The markers commonly used are galactomannan (GM), beta-glucan, and imaging methods. Although there are numerous studies on these diagnostic approaches, none of these markers serve as a support for the clinician, as is the case in human immunodeficiency virus (HIV) or cytomegalovirus (CMV) infections. This paper has been prepared to explain the diagnostic tests. As molecular tests have not been standardized and are not used routinely in the clinics, they will not be mentioned here. Key Words: Diagnosis, Invasive fungal infection, Imaging, Serology

Özet: Hematolojik malignitelerin tedavisinde önemli sorunlardan birisi de invazif fungal infeksiyonların tanısıdır. Mikolojik tanıdaki zorluklar ve hızlı müdahele etme gerekliliği, küf mantarlarının tanısında dolaylı işaretleyicilerin geliştirilmesine yol açmıştır. En sık kullanılan tanı testleri galaktomannan, beta-glukan ve moleküler yöntemlerdir. Her ne kadar bu tanı yöntemleri ile ilgili

Address for Correspondence: Hamdi AKAN, M.D., Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone: +90 532 424 26 40 E-mail: hamdiakan@gmail.com Received/Geliş tarihi : May 30, 2014 Accepted/Kabul tarihi : August 14, 2014

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çok sayıda araştırma yapılmakta ise de, hiçbiri HIV ya da CMV’de olduğu gibi doğrudan yarar sağlamamaktadır. Tanı ile ilgili bu yazı klinisyene eldeki araçları kullanma konusunda yol göstermeyi amaçlamaktadır. Moleküler testlere henüz standardize edilmediği ve klinik kullanıma girmediği için bu yazıda değinilmeyecektir. Anahtar Sözcükler: Tanı, İnvazif fungal infeksiyon, Görüntüleme, Seroloji Introduction One of the most problematic issues in hematological malignancies is the diagnosis of invasive fungal diseases. In particular, the difficulty of mycological diagnosis and the necessity of immediate intervention in cases of molds have led to the adoption of “surrogate markers” that do not verify but rather strongly suggest fungal infection. The markers commonly used are galactomannan (GM), beta-glucan, and imaging methods. Although there are numerous studies on these diagnostic approaches, none of these markers serve as support for the clinician, as in human immunodeficiency virus (HIV) or cytomegalovirus (CMV) infections. This paper has been prepared to explain the diagnostic tests. As molecular tests have not been standardized and are not used routinely in clinics, they will not be mentioned here. Conventional Microbiological Diagnostic Methods Clinical manifestations of invasive fungal infections (IFIs) generally are not agent-specific. Therefore, radiological, histopathological, and microbiological features are important in diagnosis. Although the radiological and histopathological findings are very important and provide possible evidence, the causative agent should be grown in culture and preferably be identified to the species level for definitive diagnosis. Accordingly, conventional (classical) microbiological diagnostic methods remain the gold standard in the diagnosis of IFIs [1,2,3,4].

be acquired by histopathological evaluation of the biopsy samples. The staining methods used in microbiological and histopathological evaluation for the diagnosis of opportunistic mycoses and the goals of use are summarized in Table 1 [5,6]. Culture The isolation of fungi in culture is a conventional method, which remains the gold standard in providing the definitive diagnosis of IFIs. The collection of sufficient amounts of appropriate sample, rapid transport of the specimen to the laboratory, the informing of the laboratory of the tentative clinical diagnosis, and performance of the laboratory examinations without delay according to standard recommendations are among the main factors affecting the success of a culture [5,6,7]. The culture method carries some disadvantages along with important advantages; therefore, the use of additional serological diagnostic methods, particularly those aimed at early diagnosis, is recommended along with culture. The advantages and disadvantages related to the culture method [4,8,9] are presented in Table 2. Available Guidelines Related to the Microbiological Diagnosis of Invasive Fungal Infections In previous years, guidelines for or involving sections on fungal infection diagnosis have been published. The names and major content of these guidelines are summarized in Table 3.

Conventional Microbiological Diagnostic Methods and Current Guidelines

Recommendations for Direct Microscopic Examination and Culture Applications

Direct microscopic examination and culture of the clinical samples constitute the conventional microbiological diagnostic methods.

Direct microscopic examination and culture are the main indispensable methods for IFI diagnosis. Direct microscopic examination, due to its advantages such as being rapid and providing a possible pre-diagnosis, should necessarily be performed. The culture method, with advantages such as identification of the causative agent at species level and accordingly prediction of antifungal susceptibility profile at the species level further allowing performance of antifungal susceptibility tests for that strain, is the gold standard that should be performed as the fundamental diagnostic method whenever possible. The use of culture in diagnosis has also received the highest recommendation level as the main diagnostic method in the current guidelines.

Direct Microscopic Examination Direct microscopic examination of the sample is a rapid and practical method that provides a chance for a possible pre-diagnosis. The results of direct microscopy also allow the interpretation of culture contamination in specimens from which a mold is isolated later. The major factors affecting the sensitivity of direct microscopic examination include the collection of a sufficient volume of a suitable clinical sample, application of staining methods within the frame of related principles, and performance of microscopic examination by experienced people on a sample involving the whole specimen. The sensitivity of direct microscopy widely varies depending on these factors. In order to evaluate the microscopic structures of fungi, different stains and wet coating methods can be used. Furthermore, very important evidence for the diagnosis of fungal infections can

Diagnosis of Fungemia: Isolation of Fungi in Blood Cultures Principles of collecting samples for the isolation of fungi in blood culture are the same as the standard principles of blood culture [3]. Blood culture is an important method for the isolation of Candida, Fusarium, Scedosporium, 343

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thermally dimorphic fungi causing endemic mycosis, and the other yeasts with clinical importance [Blastoschizomyces (Saprochaete), Rhodotorula, Trichosporon, etc.] and for the diagnosis of infections caused by these fungi. If the fact that Turkey is not in an endemic region for thermally dimorphic fungi is considered, excluding possible travel-related infections, isolation of fungi causing endemic mycosis from blood does not carry primary importance. Fungi other than those causing endemic mycoses and expected to be isolated in blood culture can be isolated in blood culture at varying rates in our country, with Candida spp. being the

leading isolate. However, the sensitivity of blood culture in candidemia diagnosis is generally below the desired level. Autopsy studies show that candidemia can be diagnosed by blood culture in 50-70% of cases [10,11]. Different methods or parameters are being tested to enhance the sensitivity of blood culture in fungemia diagnosis, and the leading methods are lysis centrifugation method and use of fungal media. The lysis centrifugation method is especially recommended for the identification of dimorphic fungi in blood culture [12]. However, studies published in previous years, particularly those

Table 1. Direct microscopic examination methods used in the diagnosis of opportunistic mycoses [5,6].

Method

Aim of Use

Frequently Observed Structures

Wet-mount potassium hydroxide (KOH) preparation

By dissolution of organic matter in the sample, allows detection of a possible fungus in the sample more easily

Hyphae, arthroconidia

Wet-mount method using India ink

Visualization of the capsule

Capsule is seen as a “halo” that surrounds the yeast cell

Gram staining

Especially in detecting yeasts

Gram-positive, budding yeast cells (+pseudohyphae, -real hyphae)

Fluorescent staining with calcofluor-white

Performed to increase the probability of detecting microscopic fungal structures

Hyphae, arthroconidia

Immunofluorescence staining/Giemsa

Detection of Pneumocystis jirovecii

Cyst/trophozoite

Gomori’s methenamine silver (GMS) staining

Histopathological examination of tissue samples regarding all fungal structures

Hyphae/yeast cells

Table 2. The advantages and disadvantages of culture method in the diagnosis of invasive fungal infections [4,8,9].

Advantages

Disadvantages

It is a “gold standard” method allowing definitive diagnosis.

Sensitivity and specificity are variable and may be affected by several factors. Sensitivity rates are usually below the desired levels. Collection of a sufficient volume from the appropriate sample is among the most important factors affecting sensitivity.

Provides a chance for identification to the genus and species level. Helps to estimate the antifungal susceptibility profile (in accordance with the knowledge on primary resistance).

In the assessment of growth, cutoff of colony-forming units that can be used in the differentiation of contamination-colonization-infection is not available. False positive results due to contamination risk are possible (especially for molds).

Allows performance of antifungal susceptibility As the growth period is long (especially when molds are the caustesting for the causative strain and hence determina- ative agents), its benefit in early diagnosis is limited. tion of the differences in the susceptibility profile at strain level. 344

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involving open systems, reported data indicating that the use of the lysis centrifugation method might increase the risk of contamination [4,13]. Beyond that, as the lysis centrifugation method has been reported to be mainly useful in the diagnosis of dimorphic fungi, it does not carry primary importance in Turkey. On the other hand, use of fungal media has been observed to be important not only for the isolation of fungi causing endemic mycosis, but also for the growth duration of yeasts (especially for Candida glabrata); use of fungal media has been reported to shorten the isolation period [14,15]. This finding is important, as C. glabrata is isolated in varying rates in blood culture at different centers in our country. When aerobic culture bottles of different automated systems are used for the isolation of fungi, the isolation rate of C. glabrata has been demonstrated to differ according to the automated system used, and the use of fungal media is

particularly recommended with systems yielding low rates of isolation [16]. Other than that, study data are available showing that the use of fungal media can be beneficial in intensive care or hematology cases, where yeast growth in blood may be found together with resistant or multiple bacterial growth [14]. In light of these data, although current guidelines primarily stipulate the use of automated, validated blood culture systems, they emphasize that the sensitivity of the system may change depending on the species studied and the system itself [3]. The knowledge that the use of blood culture bottles containing fungal media may shorten the growth period and the recommendation that the decision related to the use of these media should be made by the related center also appear in the guidelines [12]. Additionally, the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM) guidelines offer the use of 2 aerobic blood culture bottles instead of a single

Table 3. The names and contents of new guidelines presenting recommendations related to the microbiological diagnosis of invasive fungal infections.

Reference No.

Guideline

Content

CLSI M54-A

Guidelines for implementing methods directed at identification of fungi by direct microscopic examination and culture in clinical samples

[12]

ECIL-3 - Guidelines for Classical Diagnostic Procedures

Classical diagnostic methods used for the diagnosis of IFIs in leukemia cases

[4]

ECIL - Guidelines for the Use of Biological Markers

Recommendations related to the use of biological markers for the diagnosis of IFIs in leukemia cases and hematopoietic stem cell donors

[22]

ECIL-3 - Guidelines for Mucormycosis

Recommendations for the diagnosis and treatment of mucormycosis in cases of hematological malignancies

[23]

ESCMID – Guidelines for the Diagnosis Recommendations related to the use of conventional and [3] of Candida Diseases other diagnostic methods in different clinical presentations of Candida IDSA-ASM Guidelines

Recommendations for the use of a microbiology [17] laboratory in the diagnosis of infectious diseases (presents recommendations on fungal infection agents along with other microorganism groups)

ESCMID-ECMM Guidelines for Phaeohyphomycosis

Recommendations related to the diagnosis and treatment of systemic phaeohyphomycosis caused by dematiaceous fungi

[24]

ESCMID-ECMM Guidelines for Hyalohyphomycosis

Recommendations for the diagnosis and treatment of fusariosis, scedosporiosis, and other hyalohyphomycoses

[25]

ESCMID-ECMM Guidelines for Mucormycosis

Recommendations related to the diagnosis and treatment of mucormycosis

[26]

ESCMID-ECMM Guidelines for Rare Invasive Yeast Infections

Recommendations for the diagnosis and treatment of rare invasive yeast infections

[27]

CLSI: Clinical and Laboratory Standards Institute, ECIL-3: European Conference on Infections in Leukaemia-3, ESCMID: European Society of Clinical Microbiology and Infectious Diseases, IDSA: Infectious Diseases Society of America, ASM: American Society for Microbiology, ECMM: European Confederation of Medical Mycology.

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bottle for inoculation as an alternative to inoculation into fungal media, if yeast fungemia is suspected [17]. Recommendation for the Isolation of Fungi from Blood Culture If the conditions of our country are taken into account, fungi likely to be isolated from blood include Candida spp., the other yeasts, and Fusarium spp. In accordance with the recommendations of the guidelines and the studies performed using Candida spp., it is recommended that the decision of using fungal media for inoculation should be made considering the properties of the automated system and the patient population of that center. Routine Antifungal Susceptibility Testing and Recommendations Antifungal susceptibility testing should be performed with certain indications to direct antifungal treatment, not for all fungal strains isolated in routine practice. The indications of antifungal susceptibility testing have gained accuracy mostly for Candida strains, and comprehensive studies on other fungi, especially Aspergillus, are also being carried out. The indications for antifungal susceptibility testing include strains isolated from normally sterile sites, unresponsiveness to antifungal treatment, history of previous antifungal treatment use, and decreased susceptibility to antifungal drug(s) or isolates belonging to a resistant strain. Antifungal susceptibility testing should be performed and interpreted using reference methods for antifungal susceptibility tests from the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antibiotic Susceptibility Testing (EUCAST) by staff and centers experienced in

antifungal susceptibility testing and educated in mycology [3,4,18,19,20,21]. Serological Tests Serological tests, developed to aid in early diagnosis and recommended to be used along with conventional methods, are another test group that carries importance in the microbiological diagnosis of IFIs. Currently, the important serological tests used in routine practice are the GM antigen test, beta-glucan test, cryptococcal antigen test, and combined mannan-anti-mannan testing. Galactomannan Test GM is a molecule composed of mannan and galactofuranose polymers found in the cell wall of Aspergillus spp. and some other molds (Penicillium and Fusarium spp.) [28,29]. It may be released into the environment during active growth of the fungus. It has been shown in several studies that determination of GM in serum, bronchoalveolar lavage (BAL) fluid, and other samples by a commercial kit using the sandwich enzyme immunoassay (EIA) method (Bio-Rad Laboratories, Marne-La-Coquette, France) is beneficial in the diagnosis of invasive aspergillosis (IA) [30,31,32,33]. The results are read by a spectrophotometer and expressed as optical density index. It is strongly (AII) recommended by the ECIL that it be performed as a screening test in bone marrow transplants and in patients with hematological malignancies in whom the incidence of IA is high (5-15%). Again, strong evidence (AII level) supports screening to be performed at 2- to 3-day intervals at least twice a week, and an optical density index of >0.7 in a single sample and an optical density index of >0.5 in 2 consecutive samples

Table 4. Factors affecting the performance of galactomannan testing.

Factor Affecting Interpretation Galactomannan Performance Patient population

Sensitivity is high in bone marrow transplants with neutropenia and in patients with hematological malignancies

Site of infection

Sensitivity is low in local infections

Sampling strategy

Sensitivity is high in frequent testing

IA incidence

Sensitivity is higher in cases of GM >7%

The causative Aspergillus spp.

Sensitivity is low in A. fumigatus infections

Molecular configuration of GM

Sensitivity is low if the specific epitope in the side chain of GM is <4

Exposure to antifungal agent

Sensitivity is low in patients receiving treatment or prophylaxis

Semi-synthetic β-lactam antibiotics

May lead to false positive results

Infection caused by rare molds

Cross-reaction in Fusarium infections

Nutritional factors

False positive results by GM-containing food

Laboratory experience

Pre-treatment of the samples is a critical step that may lead to false positive and false negative results

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suggests IA [22]. It is important to continue screening in patients diagnosed with IA by GM testing (BII) and it was demonstrated in studies that not observing a decrease in optical density may be related to poor prognosis [34]. Additionally, revised European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/ MSG) criteria emphasize that probable IA diagnosis can be made by GM positivity even if conventional microbiological evidence is not available [35]. There are several factors affecting the performance of the GM test. These factors are summarized in Table 4. It is emphasized that there may be false negative results in non-neutropenic patients receiving antifungal prophylaxis/ treatment and in local infections, and false positive results in patients with Fusarium infections and in those receiving semi-synthetic β-lactam antibiotics [28,36,37,38,39,40]. However, recent studies showed a very low rate of false positive GM related to piperacillin/tazobactam, which was considered as the most common antibiotic associated with false GM results [41,42,43]. Such kind of false positive results can be avoided by obtaining the serum sample just before the next dose of semi-synthetic β-lactam antibiotics [44].

(bone marrow transplants and patients with hematological malignancies) are followed, if a result can be achieved in 24 h and if computed tomography (CT) is available, it is recommended to be used as a screening test. In Turkey, GM testing twice a week for hospitalized patients is covered by reimbursement. Multidisciplinary follow-up is needed in high-risk patients and performance of bronchoscopy and BAL GM analysis after CT is important to exclude or support the diagnosis. There is a decrease in the sensitivity of GM testing, and especially serum GM, in patients receiving prophylaxis. It can be used as a diagnostic test when clinical findings develop in this kind of patient or at centers where scanning cannot be performed. However, it should not be forgotten that this is a supplementary test that should always be used together with other data. 1,3-beta-D-Glucan Test

There are also various factors affecting the performance of BAL GM testing. While the use of antifungal drugs active in molds leads to an apparent decrease in sensitivity, no significant difference could be found related to Aspergillus spp. Furthermore, there is no difference in the sensitivity of BAL GM in patients with or without neutropenia [45,50]. Similar to serum GM testing, the use of semi-synthetic β-lactam antibiotics may lead to false positive results [45]. The standardization of bronchoscopy and bronchoscopic material is the most urgent problem that should be solved regarding BAL GM testing.

The 1,3-beta-D-glucan (BG) test is one of the promising non-culture-based early diagnostic tests for the diagnosis of IFIs. BG is a cell wall component of many fungi, especially Candida and Aspergillus spp. [51,52]. This test is based on the reaction of BG with factor G of the horseshoe crab coagulation cascade. In a preliminary study of 30 candidemic patients and 30 healthy controls, a serum BG level of 60 pg/ mL was chosen as the cutoff. After determining this cutoff value, the BG test was evaluated in 283 patients with acute myeloid leukemia or myelodysplastic syndrome and the sensitivity, specificity, and negative predictive value of the test in proven and probable IFIs was 100%, 90%, and 100%, respectively [51]. Later, a cutoff value of 80 pg/mL, which is the currently accepted positive cutoff value for the test, was found to have better specificity in a multicenter study [53]. In an in vitro study, reactivity of 127 clinical fungal isolates belonging to 40 different genera was evaluated with the Glucatell assay [54]. Compared with the reactivity of Aspergillus spp. with the BG test, Bipolaris spicifera, Sporothrix schenckii, Wangiella dermatitidis, and Penicillium marneffei isolates showed stronger reactivity and Paecilomyces spp., Scopulariopsis spp., Fusarium spp., Phialophora verrucosa, and Exophiala jeanselmei showed some reactivity. Among the tested clinical yeast isolates, Saccharomyces spp., Rhodotorula rubra, and Trichosporon asahii were found to have similar test reactivity when compared to Candida spp. In a clinical study, sensitivity of the BG test for the diagnosis of Candida parapsilosis was found to be lower compared to other Candida spp. (78% vs. 90%, respectively) [53]. This test can also be positive in Pneumocystis jirovecii infections and the sensitivity of the test is very high: 96%, with a specificity of 84% [55]. The performance of the test in BAL fluid was also evaluated; however, the sensitivity of detecting an IFI using BAL specimens was not significantly increased over testing of serum alone [56].

Conclusively, GM is a biomarker that can be used along with clinical, radiological, and conventional microbiological tests in IA diagnosis. In centers where high-risk patients

The BG test is usually not positive in the case of fungal colonization, and detection of circulating BG levels may be used as a surrogate marker not only for diagnosing IFIs

Other than serum, GM testing can be performed in BAL fluid. It was reported in all meta-analyses that the sensitivity of BAL GM is higher and the specificity is lower than that of serum GM testing [45,46,47]. It is recommended to perform GM analysis in bronchoscopic samples of patients at high risk of IA as its negative predictive value is high. It was pointed out that a BAL GM optical index of <0.5 excludes IA diagnosis, whereas an optical index of >3 supports definitive diagnosis [48,49]. The values between these 2 limits are debatable. However, it is recommended in meta-analyses that the cutoff index value be taken as 1.5 in patients with hematological malignancies and 1 in mixed patient populations [45,46]. A study comparing culture and GM in BAL fluid samples emphasized that growth might be expected when the optical index is >1, and a correlation with clinical studies was also demonstrated [50].

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but also for assessing the effectiveness of therapy [51,57]. Diagnostic levels and serum kinetics of the BG test were found to be similar when compared to the GM antigen test for the diagnosis of IA in neutropenic patients [57]. In the same study, the combination of BG and GM antigen tests improved the specificity and positive predictive value to 100%. In another clinical study performed in hematologic malignancy patients with IA, the sensitivity of the BG test and GM antigen test was 67% and 38%, respectively [39]. Interestingly, diagnostic accuracy of the GM antigen test was 13% per serum sample in A. fumigatus-positive patients compared to 49% positivity in patients with non-fumigatus Aspergillus spp.; BG test positivity was similar between A. fumigatus and non-fumigatus Aspergillus spp. The overall pooled sensitivity and specificity of the test for the diagnosis of IFIs is 77% and 85% [58]. Those values seem promising, but there are no prospective studies evaluating the use of the BG test in the diagnosis and treatment of IFIs in hematology-oncology patients. Two consecutive positive antigenemia assays have very high specificity, positive predictive value, and negative predictive value but the sensitivity is not satisfactory, so the BG test needs to be combined with clinical, radiological, and microbiological findings [59]. Ideal cutoff values and timing of serum samples (like 2 or 3 times a week) should be determined. Currently, use of the test at least twice a week is moderately recommended in some guidelines for the

screening or diagnosis of invasive Aspergillus and Candida infections [3,22]. Table 5 summarizes the differences between the GM and BG tests. Cryptococcal Antigen Tests The polysaccharide capsule that surrounds Cryptococcus neoformans is the target structure in serological tests, due to its antigenic structure. LA and EIA are the methods accepted in the serological diagnosis of cryptococcosis [22]. The concordance of LA and EIA kits developed by different companies was found to be higher than 90% [62]. In a systematic review performed after January 1998 evaluating 7 studies, 6 of which were retrospective, it was reported that cerebrospinal fluid (CSF) antigen test sensitivity in cryptococcal meningitis was 97% [63]. Although the effect of CSF antigen titer on disease prognosis has not been clearly defined, a CSF or serum antigen titer higher than 1/512 was associated with disseminated infection and unresponsiveness to treatment [64,65]. Antigen titer, tested again at least 7 days after the first test, was reported to decrease by 4-fold in patients with treatment response [66]. The sensitivity of the serum antigen test was reported to be between 62% and 67% in pulmonary cryptococcosis and cases of other organ involvement. Antigen test specificity in both serum and CSF samples was determined to be 93-100% [64,65,66,67]. Serum antigen positivity has been reported in HIV-positive patients with disseminated disease. It should

Table 5. Basic characteristics of galactomannan and 1,3-beta-D-glucan tests including bronchoalveolar lavage [56,60,61].

Galactomannan

1,3-beta-D-glucan

Diagnostic spectrum

Aspergillusa

Panfungalb

Method

‘Sandwich’ ELISA Latex agglutination

Calorimetric

Commercial kit

Bio-Rad (France) Pastorex Aspergillus

Fungitell (USA) Fungitec-G MK (Japan) Wako (Japan) Maruha (Japan)

Cutoff (serum) BAL

0.5-1.5 1

Fungitell, 80 pg/mL Others, 11-20 pg/mL

Early diagnosis

5-8 days

3-10 days

Serum, plasma Sensitivity Specificity

29%-100% 20%-100%

47%-98% 86%-98%

BAL (sensitivity, specificity)

56%-100%, 76%-100%

50%-93%, 55%-73%

Use in monitoring treatment

Cost

aBe cautious in positivity to Penicillium, Histoplasma capsulatum, and Fusarium. bExcept Mucorales and Cryptococcus neoformans.

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be taken into account that serum antigen tests may give false negative results in cancer patients [22]. False positive test results at low titers were reported more frequently in LA tests than in EIA. False negative results are more likely with kits where clinical samples are not pre-treated with pronase [68]. In a study performed between January 1989 and December 1999, 3828 CSF cryptococcal antigen tests of cancer patients were evaluated, and positive results were reported in 12 patients (0.3%). Six of these patients were found to have other conditions involving the central nervous system and it was recommended that test results of such patients be interpreted carefully because of probable false positive results [69]. Cryptococcal antigen testing in serum and CSF samples of patients with cryptococcal meningitis and disseminated cryptococcosis is recommended with an AII evidence level, and the use of the test in following treatment response is recommended with a CIII evidence level by the ECIL [22]. Although cryptococcosis is not a common disease in Turkey, cryptococcal meningitis should be included in the differential diagnosis of central nervous system disorders of stem cell transplants or patients treated with intense chemotherapy regimens, and cryptococcal antigen testing should be performed on the CSF samples of such patients. Mannan/Anti-Mannan Tests Determination of mannan antigen (Mn) and antimannan antibody (A-Mn) by ELISA method has become one of the most frequently used serological methods in the diagnosis of invasive candidiasis [22]. A systematic review, evaluating 14 studies (13 retrospective, 1 prospective) including a total of 1220 patients (453 invasive candidiasis cases, 767 controls), reported that when performed separately the sensitivity of Mn and A-Mn tests was 58% and 59%, respectively, whereas the specificity was 93% for Mn and 83% for A-Mn. However, if the positivity of one of these tests (Mn/A-Mn combination) is considered to be sufficient for diagnosis, sensitivity reaches 83% without a significant decrease in specificity (86%) [70]. In 73% of 45 patients with candidemia, at least one of the tests yielded positive results 6-7 days before the culture results were available, and in 21 patients who developed hepatosplenic candidiasis, a positive result was achieved approximately 16 days before the culture results [71,72]. Among Candida spp., the highest sensitivity was achieved for Candida albicans [70]. Seven of the studies evaluated in this review were performed in cancer patients, while the other 7 studies were carried out in intensive care units and surgery clinics. The heterogeneity of the methods used in the studies is intriguing. When the hematology-oncology patients were evaluated as a subgroup, the sensitivity of the test was 71-100% and the specificity ranged between 53% and 92% [70]. It was reported that response to Mn was especially significant after resolution of neutropenia [73]. A study that compared the Mn, A-Mn, and

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BG test results in 56 candidemia patients, including 9 stem cell transplants or patients with hematological malignancies and 12 patients with solid tumors, reported the sensitivity of the tests as 58.9%, 62.5%, and 87.5%, respectively, and the specificity as 97.5%, 65%, and 85.5%, respectively. While the diagnostic sensitivity of the Mn/A-Mn combination increased to 89.3%, specificity remained at 63% [74]. ECIL guidelines recommend the use of Mn and A-Mn together with an evidence level of BII, and the use of Mn/A-Mn combination in the diagnosis of hepatosplenic candidiasis with an evidence level of BIII and in the diagnosis of candidemia in hematology-oncology patients with evidence level CII. In the same guidelines, the use of the BG test in the diagnosis of invasive fungal disease is recommended at the BII evidence level [22]. In the ESCMID guidelines for the diagnosis of Candida infections, the use of Mn/A-Mn combination is recommended with the same evidence level as the use of BG. Recommendation Considering the economic burden of performing 2 separate tests for the Mn/A-Mn combination, the similar clinical pictures in cancer patients with several other fungi besides Candida, and the panfungal character of the BG test contributing to the diagnosis of most of these conditions, the use of the BG test seems to be more suitable in neutropenic cancer patients in Turkey. Each center should decide which test they will use according to the incidence of invasive fungal disease, technical infrastructure, and target patient groups. Radiological Diagnosis of Invasive Fungal Infections CT is a more sensitive imaging method than chest X-ray in the diagnosis and differential diagnosis of pulmonary infections in immunosuppressed patients. CT is particularly indicated if chest X-ray is normal or near normal in patients suspected of pulmonary infections [75,76]. Currently, the entire lung can be scanned in a single breath-hold with multislice spiral CT (MSCT) systems. Continuously acquired slices of 5 mm in thickness can be used in routine evaluation. However, in order to identify the halo sign seen in fungal infections, thinner slices (1-2 mm) should be used in evaluation. The resolution of thin slices obtained with MSCT is sufficient to determine parenchymal lesions, and the use of classical high-resolution CT (HRCT) technique before MSCT is not necessary. MSCT especially provides better imaging than HRCT in patients who cannot hold their breath. However, while HRCT scans obtained in the prone position are helpful in the diagnosis of patients suspected of early fibrosis, HRCT scans obtained in expiration phase aid in the diagnosis of patients suspected of bronchiolitis obliterans [75]. There is no need to use intravenous contrast media for MSCT for detection and differential diagnosis of parenchymal infections in immunosuppressed patients. 349

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However, at the later stages, if there is suspicion of a rare vascular complication such as mycotic pulmonary artery aneurysm or aortic aneurysm, intravenous contrast media should be used. Intravenous contrast media are used for liver and spleen imaging of patients suspected of disseminated candidiasis [75]. Symptoms of IFIs are not specific in immunosuppressed patients and differential diagnosis from infections caused by other microorganisms and noninfectious causes should be made. Therefore, radiologists should be informed in detail about the history and clinical-laboratory findings (primary disease, history of chemotherapy or radiotherapy, fever, respiratory distress, neutropenia, etc.) of the patients when they are evaluating thoracic CT scans. Thorax CT Findings in Invasive Fungal Infections Chest X-ray findings in immunosuppressed neutropenic patients are nonspecific and the assessment should be done by CT. A nodule is detected in the thorax CT scans of 82%94% of patients with IFIs; the nodule is generally 1 cm or more in diameter. Nodules are more frequent in IFIs than in bacterial or viral infections. In particular, the “halo” sign at the periphery of the nodule or the “air crescent” sign within the nodule are findings in favor of IFIs [75,76,77].

Figure 1. Invasive Aspergillus infection showing a nodule with a halo sign at the periphery (arrows).

The halo sign is a ground-glass opacity surrounding a nodule or a mass in CT [78]. It was first described as a sign of hemorrhage around the foci of IA (Figure 1). Besides Aspergillus infections, it may be seen in Candida infections and mucormycosis. The halo sign is nonspecific, and it may be seen in other nodules with hemorrhage or as an in situ adenocarcinoma at the periphery of invasive adenocarcinoma. The halo sign is seen more frequently in patients with hematological malignancies and stem cell transplants than in solid organ transplants. While a halo sign is found in the majority of patients with invasive Aspergillus infection in the first days of infection (88-96%), its prevalence decreases with the progression of disease and it is present in 18-19% of patients at the end of 2 weeks [79,80]. The reversed halo sign, seen in approximately 4% of IFIs, is defined as a focal, rounded area of ground-glass opacity surrounded by a complete or nearly complete ring of consolidation. While it is seen in 19% of mucormycosis cases, its prevalence is lower than 1% in Aspergillus infections. Therefore, the reversed halo sign shows that mucormycosis should be considered, and it aids in selecting the appropriate antifungal agent in treatment [81].

Figure 2. Aspergillus infection showing a nodule with cavitation and air crescent sign (arrow).

At 2-3 weeks after commencement of treatment, along with the resolution of neutropenia, cavitation develops within the consolidation or within the nodule. Cavitation usually shows that prognosis is good. An air crescent sign may be present or absent. The air crescent sign is a crescent-like air space within the cavity, separating a mass from the cavity wall [78] (Figures 2 and 3). Characteristically, it shows that

the infarcted lung is separated from the wall in IA. However, it can be seen in conditions such as tuberculosis infection, Wegener granulomatosis, lung cancer, and hemorrhage within the cavity. Although the air crescent sign is a rare finding in the early stages of invasive Aspergillus infections, the prevalence increases with the progression of disease [75].

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The nodule may grow within 10 days of commencing antifungal treatment in IFIs. Some researchers explained this by the gathering of immune cells along with the improvement in bone marrow. Hence, even though the nodule enlarges in the first 10 days of treatment, if the GM level is low and the number of neutrophils is high, it is recommended not to change the antifungal treatment [79,80].

Figure 3. Invasive aspergillosis infection showing nodular densities, some with cavitation and air crescent signs.

Aspergillus bronchopneumonia develops in approximately 10% of invasive Aspergillus infections. This infection, also known as airway IA, is characterized by the presence of Aspergillus organisms deep in the airway basement membrane. Clinical manifestations include bronchopneumonia, tracheobronchitis, and bronchiolitis. There may be consolidation areas predominantly in the peribronchiolar regions. Lobar consolidations may be rarely seen. Generally, there are no radiological findings in acute tracheobronchitis. In rare cases, there may be thickening of tracheal or bronchial walls. “Tree-in-bud” signs, which indicate endobronchiolar or peribronchiolar disease, and centrilobular nodules may be seen in bronchiolitis. Centrilobular nodules may also be detected in endobronchial spread of tuberculosis, atypical tuberculosis infections, viral pneumonias, and mycoplasma pneumonias. Therefore, when bronchiolitis findings are detected in a CT scan, primarily infections related to bacterial or viral microorganisms are considered, as they are more frequent [76]. There is pulmonary involvement in 30% of cases of mucormycosis. Radiological findings are not specific; consolidation, nodule, mass, cavitation, or abscess development may be seen (Figure 4) [76].

Figure 4. Mucormycosis showing nodular densities, some with cavitation.

In Pneumocystis jiroveci pneumonia, although chest X-rays are normal, perihilar ground-glass opacities showing patchy or geographic distribution can be identified in HRCT (Figure 5). Frequently, there is interlobular septal thickening. Cystic changes can be seen in the lungs of acquired immunodeficiency syndrome (AIDS) patients receiving prophylaxis [76]. The Diagnosis of Extra-Pulmonary Invasive Fungal Infections CT is the method of choice in suspicion of IFIs in the paranasal sinuses, and magnetic resonance imaging (MRI) should be performed in cases of suspicion of central nervous system involvement. Ultrasound, CT, or MRI can be used for examining the abdomen in disseminated candidiasis; typically small abscesses with a target-like appearance are seen in the spleen and liver [82]. Interpretation

Figure 5. Pneumocystis pneumonia showing bilateral, perihilar ground-glass opacities; the lung periphery is spared.

Although currently there are various opportunities in the diagnosis of invasive fungal diseases, their implementation is somewhat difficult. None of these tests can be used to directly diagnose invasive fungal disease and they generally carry more meaning when they are used together. Radiological diagnosis seems to remain the most rapid and 351

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easy method to use; however, it carries a risk of false positive results. Serological tests such as GM, BG, and Mn/A-Mn are not available at all institutions and carry a risk of false positive or negative results, and delays in reporting make it difficult to use them in diagnosis. Molecular approaches are not yet recommended, as they are not standardized, carry a risk of false positive results, and do not have widespread use; however, in the very near future they have the potential to hold an important place in diagnosis. 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. Perfect JR. Fungal diagnosis: how do we do it and can we do better? Curr Med Res Opin 2013; 29(Suppl 4):3-11. 2. Rueping MJ, Vehreschild JJ, Cornely OA. Invasive candidiasis and candidemia: from current opinions to future perspectives. Expert Opin Investig Drugs 2009;18:735-748. 3. Cuenca-Estrella M, Verweij PE, Arendrup MC, ArikanAkdagli S, Bille J, Donnelly JP, Jensen HE, Lass-Flörl C, Richardson MD, Akova M, Bassetti M, Calandra T, Castagnola E, Cornely OA, Garbino J, Groll AH, Herbrecht R, Hope WW, Kullberg BJ, Lortholary O, Meersseman W, Petrikkos G, Roilides E, Viscoli C, Ullmann AJ; ESCMID Fungal Infection Study Group. ESCMID guideline for the diagnosis and management of Candida diseases 2012: diagnostic procedures. Clin Microbiol Infect 2012;18(Suppl 7):9-18. 4. Arendrup MC, Bille J, Dannaoui E, Ruhnke M, Heussel CP, Kibbler C. ECIL-3 classical diagnostic procedures for the diagnosis of invasive fungal diseases in patients with leukaemia. Bone Marrow Transplant 2012;47:1030-1045. 5. Larone DH. Medically Important Fungi: A Guide to Identification, 5th ed. Washington DC, ASM Press, 2011. 6. Murray PR, Rosenthal, KS, Pfaller MA. Medical Microbiology, 7th ed. Amsterdam, Elsevier, 2013. 7. Hazen KC, Howell SA. Mycology and antifungal susceptibility testing. In: Garcia LS (ed). Clinical Microbiology Procedures Handbook. Washington DC, ASM Press, 2007. 8. Perfect JR, Cox GM, Lee JY, Kauffman CA, de Repentigny L, Chapman SW, Morrison VA, Pappas P, Hiemenz JW, Stevens DA; Mycoses Study Group. The impact of culture isolation of Aspergillus species: a hospital-based survey of aspergillosis. Clin Infect Dis 2001;33:1824-1833. 9. Horvath JA, Dummer S. The use of respiratory-tract cultures in the diagnosis of invasive pulmonary aspergillosis. Am J Med 1996;100:171-178. 10. Clancy CJ, Nguyen MH. Finding the “missing 50%” of invasive candidiasis: how nonculture diagnostics will improve understanding of disease spectrum and transform patient care. Clin Infect Dis 2013;56:1284-1292. 352

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Research Article

DOI: 10.4274/tjh.2013.0091

The Relationship between P-Selectin Polymorphisms and Thrombosis in Antiphospholipid Syndrome: A Pilot Case-Control Study Antifosfolipid Sendromunda P-Selectin Polimorfizmi ile Tromboz arasındaki İlişki: Pilot Olgu Kontrol Çalışması Nilüfer Alpay1, Veysel Sabri Hançer2, Burak Erer1, Murat İnanç1, Reyhan Diz-Küçükkaya3 1İstanbul

University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Rheumatology, İstanbul, Turkey Bilim University Faculty of Medicine, Department of Medical Biology and Genetics, İstanbul, Turkey 3İstanbul Bilim University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 2İstanbul

Abstract: Objective: The selectins are cell adhesion molecules that mediate the interactions among leukocytes, activated platelets, and endothelial cells. We aimed to investigate whether P-selectin polymorphisms are associated with thrombosis in patients with antiphospholipid syndrome (APS).

Materials and Methods: The diagnosis and classification of APS were based on the report of an international workshop. Genomic DNA was extracted from citrated blood samples of all subjects. Three single nucleotide polymorphisms associated with the P-selectin coding region (S290N, c.1087G>A; N562D, c.1902G>A; T715P, c.2363A>C) were assessed.

Results: There were 26 APS (65%) patients with thrombosis. The number of patients without thrombosis was 14 (35%). The frequency of the N562D-DN genotype was significantly higher in patients with APS than in healthy controls (p=0.003). The frequency of this genotype was significantly higher in patients with APS with thrombosis compared with patients with no thrombosis (p=0.03). The N562D-NN genotype was found at a higher frequency in patients with APS than in healthy controls (p=0.004).

Conclusion: Our results suggest that the N562D polymorphism of the DN genotype of P-selectin is associated with an increased risk of thrombosis in patients with APS.

Key Words: P-selectin polymorphisms, Thrombosis, Antiphospholipid syndrome Özet: Amaç: Hücre adezyon molekülü olan selektinler, lökositlerle, aktive plateletler ya da endotel hücreleri arasındaki etkileşime aracılık eder. Bu çalışmada antifosfolipid sendromu (AFS) hastalarında tromboz riski ile P-selektin polimorfizmleri arasındaki ilişkinin araştırılması amaçlanmaktadır.

Address for Correspondence: Nilüfer ALPAY, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Rheumatology, İstanbul, Turkey Phone: +90 212 414 20 00 E-mail: nalpay@istanbul.edu.tr Received/Geliş tarihi : March 15, 2013 Accepted/Kabul tarihi : October 4, 2013

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Alpay N, et al: P-Selectin Polymorphisms and Thrombosis in Antiphospholipid Syndrome

Gereç ve Yöntemler: AFS tanısı “International Workshop” tanı ve sınıflandırma kriterleri ile konulmuştur. Hastaların periferik kan örneklerinden DNA elde edilmiştir. P-selektin gen bölgesiyle ilişkili üç tane tek nükleotid polimorfizmi (S290N, c.1087G>A; N562D, c.1902G>A; T715P, c.2363A>C) araştırılarak genotipleri belirlenmiştir. Bulgular: Hasta grubunda 26 trombozlu (%65) AFS olgusu ve 14 (%14) trombozu olmayan AFS olgusu yer almaktadır. Hasta ve kontrol grubu kıyaslandığında incelenen polimorfizmlerden N562D-DN genotipi hasta grubunda anlamlı olarak yüksek bulunmuştur (p=0,003). Yine hasta grubuna bakıldığında trombozlu AFS grubunda trombozu olmayan AFS grubuna kıyasla N562D-DN genotipine anlamlı olarak sık rastlanmıştır (p=0,03). N562D NN genotipi ise kontrol grubunda hasta grubuna oranla daha yüksek sıklıktadır (p=0,004). Sonuç: P-selektin N562D polimorfizmi DN genotipi primer AFS hastalarında tromboz riski ile ilişkili olduğu söylenebilir. Anahtar Sözcükler: P-selektin polimorfizmleri, Tromboz, Antifosfolipid sendrom Introduction Antiphospholipid syndrome (APS) is an autoimmune disease characterized by pregnancy morbidity and arterialvenous thrombosis in the presence of antiphospholipid antibodies (aPLAs) [1]. Although the relationship between aPLAs and thrombosis is known, the mechanisms of thrombosis in APS has not been fully elucidated. Selectins, which are cell adhesion molecules, mediate the interactions among leukocytes, activated platelets, and endothelial cells. P-selectin, which can be identified as a soluble form in plasma, intercedes in the attachment and rolling of leukocytes on activated endothelial cells and is involved in the recruitment of leukocytes to thrombi [2,3]. Novel data suggest that the levels of soluble P-selectin (sP-selectin) are increased in APS patients with thrombosis [4]. In this study, we aim to investigate whether P-selectin polymorphisms are associated with thrombosis in patients with APS. Materials and Methods Patients and Controls Forty adult patients with APS and 40 healthy subjects with no history of thrombosis or autoimmune disease were included in the study. The history of disease, physical examination, and screening of lupus anticoagulant (LA) and serum anticardiolipin IgG and IgM levels were assessed for all patients. The diagnosis and classification of APS were based on an international consensus statement [1]. The subjects participating in the study had no LA and/or serum anticardiolipin-related systemic diseases or risk factors such as hypertension or hyperlipidemia for thrombosis. LA was diagnosed using activated partial thromboplastin time, kaolin clotting time, and Russell’s viper venom test according to published criteria [5]. IgG and IgM anticardiolipin antibodies were determined by enzyme-linked immunosorbent assay [6], and levels equal to or greater than 4 standard deviations were regarded as positive. The

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anticardiolipin antibody and LA tests were repeated 3 months after the first determination. APS patients with thrombosis had arterial and/or venous thrombosis as well as aPLA positivity. aPLA-positive patients with no thrombosis suffered from either first trimester fetal losses or thrombocytopenia and had persistently positive aPLA test results but no thrombotic complications over at least 3 years of follow-up. The study protocol was approved by the local ethics committee, and written and signed informed consent was obtained from all participants. Genotyping Genomic DNA was extracted from citrated blood samples. Three single nucleotide polymorphisms associated with the P-selectin coding region (S290N, c.1087G>A; N562D, c.1902G>A; T715P, c.2363A>C) were assessed. Polymerase chain reaction (PCR) was done in a total volume of 25 µL containing 2 U of Taq DNA polymerase (Fermentas), 2 mmol/L MgCl2, 0.2 mmol/L of each dNTP, 2.5 µL of 10X PCR buffer, and 50 ng of genomic DNA. Allele specific primers were used in the following concentrations: 15 pmol of 290N-R (5’-TAAATGAATTCAGTCCATGGTTCCTACAT-3’), 5 pmol of 290S-R (5’-CACAGTCCATGGTTCCTTGAC-3’), 11 pmol of 290common (5’-TGTGTGGCTTTTCTCCTTTC-3’), 2 pmol of 562D-R (5’-ATTGCCCTACCAGCTTAAAGCCG TAGTC-3’), 7 pmol of 562N-R (5’-CTCCAGCTTAAAGCCGTTCTT-3’), 10.5 pmol of 562common (5’-TGAATATATAAGTGA ATGAACTTTGTG-3’), 3.5 pmol of 715P-R (5’-CCT GCT TGATAG GTT GCC ACG GAA GG-3’), 8 pmol of 715TR (5’-GCAGGT TGG CAC GGT TGT-3’), and 9 pmol of 715common (5’-CTGTGA AAT GCT CAG AAC TAC ATG3’). PCR amplification was carried out in a GeneAmp PCR System 9700 Thermo Cycler (Applied Biosystems, USA) using 36 cycles of 94 °C for 25 s, 57 °C for 25 s, and 72 °C for 25 s. PCR products were separated on agarose gels and stained with ethidium bromide. The PCR products were 115, 205, and 182 bp long for S290N, N562D, and T715P, respectively.

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Statistical Analysis Data are expressed as mean ± SD, number (%), or median (range). Test statistics were computed using the MannWhitney U test and the Kruskal-Wallis test. The chi-square test and odds ratio were used to calculate the 95% confidence intervals. Correlation coefficients and significance were calculated by Spearman’s test to assess the differences between groups. For all tests, a 2-tailed p-value of <0.05 was considered statistically significant. Statistical analyses were performed using the software package SPSS 15 running on Windows NT. Results There were 26 APS (65%) patients with thrombosis, 12 (46%) of which cases involved veins, 10 (38%) arteries, and 4 (15%) veins and arteries together. The number of APS patients without thrombosis was 14 (35%). The mean age of patients (80% female) was 39.4±9.5 years. The characteristics of patients are seen in Table 1. The frequency of the N562D-DN genotype was significantly higher in patients with APS than healthy controls (p=0.003). The frequency of this genotype was significantly higher in patients with APS with thrombosis compared to patients without thrombosis (p=0.03). The N562D-NN genotype was found at a higher frequency in patients with APS than in healthy controls (p=0.004). The frequency of the N562D-NN genotype was not different between

patients without thrombosis and control subjects (p=0.21). On the other hand, S290N and T715P polymorphisms were not different between patient and control groups (Tables 2 and 3). There was no relationship between aPLA, thrombocytopenia, or pregnancy loss and any polymorphism. Discussion P-selectin is expressed on activated platelets and endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) is found in neutrophils and monocytes, and these are from microparticles. Connecting P-selectin/PSGL-1 activated leukocytes for endothelium rolling, and providing the release of tissue factor initiates thrombosis [7]. Previous studies suggest that sP-selectin levels increased in patients with thrombosis after a finding of the association of P-selectin and thrombosis in an animal model [8,9,10]. Additionally, P-selectin polymorphisms were detected in patients with thrombosis, but not always together with high sP-selectin levels. High sP-selectin levels were detected in patients with systemic lupus erythematosus and APS [11,12]. According to other studies, P-selectin polymorphisms play a role in the pathogenesis of systemic lupus erythematosus [13,14]. On the other hand, the relationship between polymorphism of PSGL-1 and ischemic cerebrovascular disease was shown previously [15]. Roldan et al. demonstrated that short alleles of PSGL-1 protect against cardiovascular disease [16].

Table 1. Characteristics of the patients with antiphospholipid syndrome.

APS with Thrombosis

aPLA+ Patients All Patients without Thrombosis

p-Value

Number

Age (mean ± SD)

39.5±10.3

39.2±8.2

39.4±9.5

0.82

Female/male

17/7 (71%)

15/1 (93%)

32 (80%)

0.08

Fetal losses

9 (52%)

12 (80%)

21 (65%)

0.003*

Thrombocytopenia Mild Moderate Severe

9 (39%)

5 (31%)

14 (35%)

0.43

4 (16%)

2 (13%)

6 (15%)

0.35

2 (8%)

2 (13%)

4 (10%)

0.23

3 (12%)

1(6%)

3 (7%)

0.43

Lupus anticoagulant

13 (54%)

12 (75%)

25 (63%)

0.05*

Anticardiolipin antibodies IgG IgM

22 (92%)

14 (88%)

36 (92%)

0.36

19 (79%)

11 (69%)

30 (75%)

0.26

13 (54%)

10 (63%)

23 (57%)

0.48

*: statistically significant.

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Alpay N, et al: P-Selectin Polymorphisms and Thrombosis in Antiphospholipid Syndrome

Table 2. The frequency of S290N, N562D, and T715P polymorphisms in the patients and control groups.

SNP

Rs Number

Genotype All Patients (n=40)

Patients with Thrombosis (n=24)

Patients without Thrombosis (n=16)

Control (n=40)

S290N

rs6131 (G>A)

21 (52%)

15 (62%)

6 (38%)

25 (63%)

19 (48%)

9 (38%)

10 (62%)

13 (32%)

None

2 (5%)

6 (15%)

2 (8%)

4 (25%)

8 (20%)

30 (75%)

21 (88%)

9 (56%)

17 (42%)

4 (10%)

1 (4%)

3 (19%)

15 (38%)

31 (77%)

18 (75%)

13 (81%)

32 (80%)

7 (18%)

5 (21%)

2 (13%)

8 (20%)

2 (5%)

1 (4%)

1 (6%)

None

N562D

T715P

rs6127 (G>A)

rs6136 (A>C)

SNP: single nucleotide polymorphism.

vAdditionally, the relationship PSGL-1 VNRT polymorphisms

and risk of thrombosis in APS patients was shown by DizKucukkaya et al. [17]. The relationship between P-selectin polymorphism and thrombosis in APS patients was shown for the first time in our study. The c.1087G>A, c.1902G>A, and c.2363A>C polymorphisms lead to S290N, N562D, and T715P P-selectin gene variations, respectively. These variations are in the genetic region encoding the repeated part of the P-selectin gene and may be effective in binding P-selectin to PSGL-1. Therefore, our data are valuable in order to determine risk factors other than traditional ones for thrombosis in APS despite the fact that we did not estimate sP-selectin levels.

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In conclusion, our results suggest that the N562D polymorphism DN genotype of P-selectin is associated with an increased risk of thrombosis in patients with APS. The NN genotype of the same polymorphism might be protective against thrombosis in those patients. The effect of N562D polymorphism on sP-selectin levels will be studied in future work. 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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Table 3. The differences in S290N, N562D, and T715P polymorphisms among groups (group 1: all patients, group 1a: patients with thrombosis, group 1b: patients without thrombosis, group 2: control subjects).

SNP S290N

Between Groups Between Groups Between Groups Between Groups 1 and 2 1a and 2 1b and 2 1a and 1b SS; p-value OR CI

0.24 0.6 0.2-1.6

0.60 1.0 0.3-2.8

0.13 2.7 0.8-9.2

0.11 2.7 0.7-10.2

SN; p-value OR CI

0.12 1.8 0.7-4.6

0.78 0.8 0.2-2.3

0.07 0.2 0.09-0.9

0.11 0.3 0.09-1.3

0.24 -

0.38 -

0.50 -

DD; p-value OR CI

0.38 0.7 0.2-2.2

0.29 2.7 0.5-14.2

0.72 0.75 0.19-2.9

0.16 0.2 0.04-1.7

DN; p-value OR CI

0.003* 4.0 1.5-10.5

0.001* 0.1 0.02-0.4

0.38 0.5 1.1-1.8

0.03* 5.4 1.1-25.9

NN; p-value OR CI

0.004* 0.1 0.05-0.6

0.003* 13.8 1.6-112.9

0.21 2.6 0.6-10.6

0.16 0.1 0.01-2.0

TT; p-value OR CI

0.50 0.8 0.2-2.5

0.75 1.3 0.3-4.4

0.61 0.9 0.2-4.03

0.47 0.6 0.1-3.2

TP; p-value OR CI

0.50 0.8 0.2-2.6

0.58 0.9 0.2-3.3

0.70 1.7 0.3-9.3

0.40 1.8 0.3-10.9

PP; p-value OR CI

0.24 -

0.37 -

0.28 -

0.64 0.6 0.03-11.2

NN; p-value N562D

T715P

SNP: single nucleotide polymorphism, OR: odds ratio, CI: confidence interval, *: statistically significant.

References 1. Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, Brey R, Derksen R, Harris EN, Hughes GR, Triplett DA, Khamashta MA. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum 1999;42:13091311.

2. Ishiwata N, Takio K, Katayama M. Alternatively spliced isoform of P-selectin is present in vivo as a soluble molecule. J Biol Chem 1994;269:23708-23715. 3. Albert B, Johnson A, Lewis J, Raf M, Roberts K, Walter P. Molecular Biology of the Cell. 5th ed. New York, Garland Science, 2008.

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4. Devreese K, Peerlinck K, Hoylaerts MF. Thrombotic risk assessment in the antiphospholipid syndrome requires more than the quantification of lupus anticoagulants. Blood 2010;28:870-878.

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12. Joseph JE, Donohoe S, Harrison P, Mackie IJ, Machin SJ. Platelet activation and turnover in the primary antiphospholipid syndrome. Lupus 1998;7:333-340.

6. Harris EN. Antiphospholipid antibodies. Br J Haematol 1990;74:1-9.

13. Jacob CO, Reiff A, Armstrong DL, Myones BL, Silverman E, Klein-Gitelman M, McCurdy D, Wagner-Weiner L, Nocton JJ, Solomon A, Zidovetzki R. Identification of novel susceptibility genes in childhood-onset systemic lupus erythematosus using uniquely designed candidate gene pathway platform. Arthritis Rheum 2007;56:4164-4173.

7. Théorêt JF, Yacoub D, Hachem A, Gillis MA, Merhi Y. P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation. Thromb Res 2011;128:243-250.

14. Morris DL, Graham RR, Erwig LP, Gaffney PM, Moser KL, Behrens TW, Vyse TJ, Graham DS. Variation in the upstream region of P-Selectin (SELP) is a risk factor for SLE. Genes Immun 2009;10:404-413.

8. Furie B, Furie BC. Role of platelet P-selectin and microparticle PSGL-1 in thrombus formation. Trends Mol Med 2004;10:171178.

15. Lozano ML, González-Conejero R, Corral J, Rivera J, Iniesta JA, Martinez C, Vicente V. Polymorphisms of P-selectin glycoprotein ligand-1 are associated with neutrophil-platelet adhesion and with ischaemic cerebrovascular disease. Br J Haematol 2001;115:969-976.

5. Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. Thromb Haemost 1995;74:1185-1190.

9. Ay C, Jungbauer LV, Sailer T, Tengler T, Koder S, Kaider A, Panzer S, Quehenberger P, Pabinger I, Mannhalter C. High concentrations of soluble P-selectin are associated with risk of venous thromboembolism and the P-selectin Thr715 variant. Clin Chem 2007;53:1235-1243. 10. Ay C, Jungbauer LV, Kaider A, Koder S, Panzer S, Pabinger I, Mannhalter C. P-selectin gene haplotypes modulate soluble P-selectin concentrations and contribute to the risk of venous thromboembolism. Thromb Haemost 2008;99:899-904. 11. Joseph JE, Harrison P, Mackie IJ, Isenberg DA, Machin SJ. Increased circulating platelet-leucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis. Br J Haematol 2001;115:451-459.

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16. Roldan V, Gonzales-Conejero R, Marin F, Pineda J, Vicente V, Corral J. Short alleles of P-selectin glycoprotein ligand-1 protect against premature myocardial infarction. Am Heart J 2004;148:602-605. 17. Diz-Kucukkaya R, Inanc M, Afshar-Kharghan V, Zhang QE, López JA, Pekcelen Y. P-selectin glycoprotein ligand-1 VNTR polymorphisms and risk of thrombosis in the antiphospholipid syndrome. Ann Rheum Dis 2007;66:1378-1380.

Research Article

DOI: 10.4274/tjh.2013.0152

Serum Bcl-2 Levels in Patients with β-Thalassemia Minor: A Pilot Study β-Talasemi Minörlü Hastalarda Serum Bcl-2 Düzeyleri: Pilot Çalışma İrfan Yavaşoğlu1, Gökhan Sargın2, Gürhan Kadıköylü1, Aslıhan Karul3, Zahit Bolaman1 1Adnan

Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey Menderes University Faculty of Medicine, Department of Internal Medicine, Aydın, Turkey 3Adnan Menderes University Faculty of Medicine, Department of Biochemistry, Aydın, Turkey 2Adnan

Abstract: Objective: Anti-apoptotic proteins such as Bcl-2 and Bcl-xL may play a role in the survival of erythroid progenitor cells. Information about these proteins in patients with β-thalassemia minor is limited. We aimed to determine the levels of serum Bcl-2 in patients with β-thalassemia minor.

Materials and Methods: Ninety-seven patients (60 females and 37 males with mean age of 29±21 years) with β-thalassemia minor were enrolled in this study. The diagnosis of β-thalassemia minor was based on whole blood counts, family history, and HbA2 levels estimated by high-performance liquid chromatography. The control group comprised 23 healthy adults (17 females and 6 males with mean age of 58±9 years) without anemia. The levels of serum Bcl-2 were measured by enzyme-linked immunosorbent assay. Mann-Whitney U tests were used in statistical evaluation and p<0.05 was accepted as statistically significant.

Results: Although there was no statistically significant difference between patients with β-thalassemia minor and the control group for the level of serum Bcl-2 (p>0.05), these levels were higher in β-thalassemia minor patients than controls.

Conclusion: There are damaged beta chains in β-thalassemia minor. Therefore, it is expected that premature death of red blood cells may occur due to apoptosis. The mean age of the control group was higher than that of the β-thalassemia minor group; this may be why Bcl-2 levels were higher in the β-thalassemia minor group. It is known that older age constitutes a risk for increased apoptosis. Other proteins (Bad, Bax, etc.) and pathways [CD95 (Fas) ligand] associated with apoptosis should be evaluated in future studies including more patients.

Key Words: β-Thalassemia minor, Bcl-2, Apoptosis

Address for Correspondence: Gökhan SARGIN, M.D., Adnan Menderes University Faculty of Medicine, Department of Internal Medicine, Aydın, Turkey Phone: +90 553 424 10 97 E-mail: gokhan_sargin@hotmail.com Received/Geliş tarihi : April 29, 2013 Accepted/Kabul tarihi : July 19, 2013

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Özet: Amaç: Bcl-2 ve Bcl-xL gibi antiapoptotik proteinler eritroid progenitör hücrelerin yaşam süresinde rol oynayabilir. β-talasemi minör hastalarda bu proteinler ile ilgili bilgiler sınırlıdır. Biz, β-talasemi minörlü hastalarda serum Bcl-2 düzeylerinin belirlenmesi amaçladık. Gereç ve Yöntemler: β-talasemi minör tanılı doksan yedi hasta (60 kadın ve 37 erkek, yaş ortalamaları 29±21 yıl) bu çalışmaya dahil edildi. β-talasemi minör tanısı tam kan sayımı, aile öyküsü ve yüksek performanslı sıvı kromatografisine dayanan HbA2 düzeyleri ile konulmuştur. Kontrol grubu anemisi olmayan 23 sağlıklı yetişkin (17 kadın ve 6 erkek, yaş ortalaması 58±9 yıl) idi. Serum Bcl-2 düzeyleri ELISA yöntemi ile ölçüldü. İstatistiksel değerlendirmede Mann-Whitney U testi kullanıldı ve p<0.05 istatistiksel olarak anlamlı kabul edildi. Bulgular: Serum Bcl-2 düzeyinde β-talasemi minörlü hastalar ile kontrol grubu arasında istatistiksel olarak anlamlı farklılık olmamasına rağmen (p>0.05), β-talasemi minörlü hastalarda bu seviyeler kontrol grubuna göre daha yüksekti. Sonuç: β-talasemi minörde hasarlı beta zincirleri bulunmaktadır. Bu nedenle, kırmızı kan hücrelerinin erken ölümünün apoptoz nedeniyle olması beklenmektedir. Kontrol grubunun yaş ortalaması talasemi taşıyıcılarından yüksektir, bundan dolayı Bcl-2 düzeyleri β-talasemi minörde yüksek olabilir. İleri yaşın artmış apoptoz için bir risk oluşturtuğu bilinmektedir. Bu nedenle, 40 yaş üzerindeki talasemi minörlülerde, kontrole göre Apoptoz ile ilişkili diğer protein (Bad, Bax, vb.) ve yolaklar [CD95 (Fas) ligand] hasta sayısının daha fazla olduğu çalışmalarda değerlendirilmelidir.

Anahtar Sözcükler: β-Talasemi minor, Bcl-2, Apoptoz Introduction Translations or mutations of mRNA lead to a deficiency of globin synthesis and thus cause thalassemia syndromes [1]. Thalassemia syndromes are inherited disorders that occur as a result of abnormal synthesis of α/β-globin. Cytoplasmic inclusion bodies (including unpaired globin molecules) damage red blood cells. As a result, the life span of erythrocytes is shortened [1,2]. Cell death is regulated by many intra- and extracellular signals. The ratio of anti-apoptotic molecules (Bcl-xl, Mcl-1, Bcl-w, A1, etc.) to apoptotic molecules (Bax, Bak, Bik, Bid, etc.) determines the future of the cell [3]. The Bcl-2 protooncogene is located on chromosome 18 and inhibits the apoptotic pathway. Bcl-2 is an important molecule in the early period of cell transformation [3,4]. In recent years, some studies have been published about Bcl-2 genes to predict their role in the development of many solid tumors. However, there are few studies about the expression of Bcl-2 in patients with β-thalassemia minor [5]. In this study, we aimed to determine the levels of serum Bcl-2 in patients with β-thalassemia minor. Materials and Methods Ninety-seven patients (60 females and 37 males with mean age of 29±21 years) with β-thalassemia minor were enrolled in this study. The diagnosis of β-thalassemia minor was based on whole blood counts, family history, and HbA2 levels estimated by high-performance liquid chromatography (Agilent 1100 Series HPLC Value System, Waldbronn, Germany). The control group comprised 23 healthy adults (17 females and 6 males with mean age of 58±9 years) without anemia. The levels of 364

serum Bcl-2 were measured using a commercial enzyme-linked immunosorbent assay kit (Biosource, Cat. No. TMA 0311, Camarillo, CA, USA). Age, sex, hemoglobin, hematocrit, mean corpuscular volume (MCV), whole blood cell counts, and serum levels of Bcl-2 were recorded. Venous blood samples were taken under the supervision of medical personnel and were measured with an ADVIA 2120 instrument (Siemens, Erlangen, Germany). Signed informed consent was obtained from all participants. SPSS 15 (SPSS Inc., Chicago, IL, USA) and the Mann-Whitney U test were used in statistical evaluation of data and p<0.05 was accepted as statistically significant. Results In Table 1, age, hemoglobin, MCV, and serum Bcl-2 levels are given. Although there was no statistically significant difference between patients with β-thalassemia minor and the control group for the level of serum Bcl-2 (p>0.05), levels in β-thalassemia minor patients were higher than in the controls. Bcl-2 levels of the patients with β-thalassemia minor and the control group are shown by dot-plot distribution in Figure 1. The relationships between age, hemoglobin, and MCV values and serum Bcl-2 levels are shown in Table 2. We evaluated serum Bcl-2 levels of 22 controls and 27 patients with β-thalassemia minor of over 40 years. Bcl-2 levels were statistically significantly higher among patients than in the control group (p=0.045). Discussion In this study, we did not find any significant difference in Bcl-2 levels between patients with β-thalassemia minor and controls.

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Erythropoietin increases Bcl-xL expression in erythroidCFU colonies [5]. Significantly increased Bcl-xL and Mcl-1 protein levels and the suppression of Bax protein are observed in CD34 (+) cells, induced by erythropoietin or stem cell factor [9]. Myelodysplastic syndrome (MDS) is characterized with ineffective erythropoiesis and increased apoptosis of hematopoietic precursor cells. A correlation was observed with increased proteins associated with Bcl-2, but the mechanism is not yet fully understood [10]. Levels of Bad, Bax, and Bcl-xS were especially higher in patients with MDS-refractory anemia (RA) or RA with ring sideroblasts [10]. Many changes in oncogenes and tumor suppressor genes may have a role in the pathogenesis of many diseases. Bcl-2 is located in the nucleus or membranes of the endoplasmic reticulum, while mitochondria were shown to be in neoplastic cells in B-cell neoplasms with t(14;18). The mechanism of Bcl-2 activation is still unknown; however, increased levels of cytochrome-c as a center of apoptosis may play a role [11]. Hockenbery et al. reported that Bcl-2 inhibits apoptosis by changing mitochondrial functions [12].

were reported about these proteins and genetic mechanisms in patients with thalassemia [2,3,4,5]. Increased synthesis of fetal hemoglobin may improve the symptoms of β-thalassemia. Cytotoxic drugs such as hydroxyurea and cytarabine may affect the synthesis of fetal hemoglobin by stimulating precursor cells [1]. Castaneda et al. determined that short-chain fatty acids [arginine butyrate, sodium α-methylhydrocinnamate, sodium 2,2-dimethylbutyrate, sodium 3,4-(methylenedioxy) cinnamate, 2-(quinazolin-4-ylamino) butanoic acid, and 4-(trifluoromethyl) sulfanyl aniline acetic acid] increased the erythroid-BFU colonies and endogenous fetal globin gene expressions [5]. In addition, anti-apoptotic genes were specifically regulated and Bcl-2 levels were increased by short-chain fatty acids. It is known that increased apoptosis is observed with older age [14]. Cao et al. reported that neutrophils of patients with paroxysmal nocturnal hemoglobinuria expressed apoptosisrelated CD95, Bcl-2, and Bax without significant differences from the normal controls [15]. The results of a study by Ismail et al. suggest a more significant role for Bcl-x as an antiapoptotic regulator in CD34 (+) cells in aplastic anemia than Bcl-2 [16]. The limitations of our study were the existence of an age gap between the 2 groups and a limited number of

36.00 34.28 Bcl-2

Antiapoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) are necessary for the survival of erythroid precursor cells [6]. Proteins such as Bax and Bcl-xS trigger apoptosis of erythroid cells. According to some studies, Bcl-2 and Bax genes may be regulated by the p53 gene. If the ratio of anti-apoptotic proteins to pro-apoptotic proteins is more than 1, erythroid cells continue their life span [7]. Additionally, members of the Bcl-2 family may control apoptosis by acting as pro-oxidant agents [8].

Translations or mutations of mRNA lead to deficiency of globin synthesis and thus cause thalassemia syndromes. It was reported in many studies that some cancers may be treated by regulation genes on pre-RNA and mRNA regions. Changes in apoptotic or anti-apoptotic pathways may be a result of genetic modulation [13]. β-Thalassemia is characterized by accelerated apoptosis of erythroid precursor cells. In many studies, limited data

34.00

32.74

32.00

30.00

Controls

Thalessemia minor

Groups

Figure 1. Serum Bcl-2 levels with dot-plotdistribution.

Table 1. Hemoglobin, mean corpuscular volume, and serum Bcl-2 levels in patients with β-thalassemia minor and the control group.

Hemoglobin (g/dL)

β-Thalassemia Minor (n=97)

Control (n=23)

Female (n=60)

Male (n=37)

Female (n=17)

Male (n=6)

9.9±1.2

13.1±0.8

12.6±1.3

15.4±1.4

p-Value <0.001

Mean corpuscular volume (fL)

64±8

86±3.4

<0.001

Bcl-2 (ng/mL)

34.2±7.6

32.7±13

>0.05

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Yavaşoğlu İ, et al: Serum Bcl-2 Levels and β-Thalassemia Minor

Table 2. The comparison of serum Bcl-2 levels between age and mean corpuscular volume groups.

Bcl-2 p(ng/mL) Value Age of more than 40 years Control (n=22) β-Thalassemia minor (n=27) Hemoglobin levels ≥11.4 g/dL (n=41) <11.4 g/dL (n=56) Mean corpuscular volume (fL) <64 fL (n=55) ≥64 fL (n=64)

32.3±13.4 31.8±5.1

0.045

36±8.9 32.9±6.4

5. Castaneda S, Boosalis MS, Emery D, Thies A, Faller DV, Perrine SP. Enhancement of growth and survival and alterations in Bcl-family proteins in β-thalassemic erythroid progenitors by novel short-chain fatty acid derivatives. Blood Cells Mol Dis 2005;35:217-226.

0.047

6. Koury MJ, Sawyer ST, Brandt SJ. New insights into erythropoiesis. Curr Opin Hematol 2002;9:93-100.

35.4±8.3 32.8±6.5

7. Chao DT, Korsmeyer SJ. Bcl-2 family: regulators of cell death. Annu Rev Immunol 1998;16:395-419. 0.097

cases. Moreover, we could not evaluate other anti-apoptotic proteins (Bad, Bax, etc.) or pathways [CD 95 (Fas) ligand] in this study. The control group comprised a small number of patients and the control group comprised 23 healthy adults without anemia. Another limitation of this study is that we did not know the levels of annexin V, cleaved caspase-3, or mRNA regulation of Bcl-2. In conclusion, there are damaged beta chains in β-thalassemia minor. Therefore, it is expected that premature death of red blood cells may be due to apoptosis. The mean age of the control group was higher than that of the β-thalassemia minor patients, which may be the reason for Bcl-2 levels being higher in the β-thalassemia minor patients. It is known that older age constitutes a risk for increased apoptosis. Other proteins and pathways associated with apoptosis should be evaluated in future studies including larger populations. 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. Benz EJ. Disorders of hemoglobin. In: Fauci AS, Braunwald E, Kasper DL, Hauser SL, Longo DL, Jameson JL, Loscalzo J (eds). Harrison’s Principles of Internal Medicine, 18th ed. New York, McGraw-Hill, 2012. 2. Quek L, Thein SL. Molecular therapies in beta-thalassaemia. Br J Haematol 2007;136:353-365. 3. Yong J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997;275:1129-1132.

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4. Hacki J, Egger L, Monney L, Conus S, Rosse T, Fellay I, Borner C. Apoptotic crosstalk between the endoplasmic reticulum and mitochondria controlled by Bcl-2. Oncogene 2000;19:2286-2295.

8. Haddad JJ. On the antioxidant mechanisms of Bcl-2: a retrospective of NF-κB signaling and oxidative stress. Biochem Biophys Res Commun 2004;17;322:355-363. 9. Josefsen D, Myklebust JH, Lomo J, Sioud M, Blomhoff HK, Smeland EB. Differential expression of Bcl-2 homologs in human CD34(+) hematopoietic progenitor cells induced to differentiate into erythroid or granulocytic cells. Stem Cells 2000;18:261-272. 10. Boudard D, Vasselon C, Berthéas MF, Jaubert J, Mounier C, Reynaud J, Viallet A, Chautard S, Guyotat D, Campos L. Expression and prognostic significance of Bcl-2 family proteins in myelodysplastic syndromes. Am J Hematol 2002;70:115125. 11. Tsujimoto Y, Gorham J, Cossman J, Jaffe E, Croce CM. The t (14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining. Science 1985;229:1390-1393. 12. Hockenbery D, Nunez G, Milliman C. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990;348:334-336. 13. Bauma JA, Kole R. Modulation of RNA splicing as a potential treatment for cancer. Bioeng Bugs 2011;2:125-128. 14. Fulle S, Centurione L, Mancinelli R, Sancilio S, Manzoli FA, Di Pietro R. Stem cell ageing and apoptosis. Curr Pharm Des 2012;18:1694-1717. 15. Cao YR, Shao ZH, Liu H, Zhao MF, He GS, Shi J, Bai J, Fu R, Tu MF, Wang HQ, Xing LM, Cui ZZ, Sun J, Jia HR, Yang TY. Expression of apoptosis-related proteins in bone marrow neutrophils of patients with paroxysmal nocturnal hemoglobinuria. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2005;13:871-884. 16. Ismail M, Gibson FM, Gordon-Smith EC, Rutherford TR. Bcl-2 and Bcl-x expression in the CD34+ cells of aplastic anaemia patients: relationship with increased apoptosis and upregulation of Fas antigen. Br J Haematol 200;113:706-712.

Research Article

DOI: 10.4274/tjh.2013.0075

Duffy and Kidd Genotyping Facilitates Pretransfusion Testing in Patients Undergoing Long-Term Transfusion Therapy Uzun Süreli Transfüzyon Tedavisi Alan Hastalarda Duffy ve Kidd Genotiplendirme Transfüzyon Öncesi İncelemeleri Kolaylaştırıyor Diana Remeikiene1, Rasa Ugenskiene2, Arturas Inciura3, Aiste Savukaityte2, Danguole Raulinaityte2, Erika Skrodeniene4, Renata Simoliuniene5, Elona Juozaityte3 1Lithuanian

University of Health Sciences, Institute of Oncology, Department of Haematology, Kaunas, Lithuania University of Health Sciences, Institute of Oncology, Oncology Research Laboratory, Kaunas, Lithuania 3Lithuanian University of Health Sciences, Institute of Oncology, Department of Oncology, Kaunas, Lithuania 4Lithuanian University of Health Sciences, Department of Laboratory Medicine, Kaunas, Lithuania 5Lithuanian University of Health Sciences, Department of Physics, Mathematics, and Biophysics, Kaunas, Lithuania 2Lithuanian

Abstract: Objective: Conventional serologic typing of red blood cell systems other than ABO and RhD can be inaccurate and difficult

to interpret in patients who have recently undergone blood transfusion. While molecular-based assays are not used routinely, the usefulness of genotyping was investigated in order to determine patients who may benefit from this procedure. Materials and Methods: Blood samples were taken from 101 patients with haemato-oncological, chronic renal, or gastroenterological diseases and from 50 donor controls; the samples were tested for Fya and Fyb by applying serologic and genetic methods. All patients had received 3 or more units of RBCs during the last 3 months. An average of 6.1 RBC units were transfused per patient. The average length of time from transfusion until blood sampling was 24.4 days. The haemagglutination test was applied for serological analysis, and the restriction length polymorphism assay was used for genotyping. Results: In total, 33 (32.7%) patients showed positive reactions with anti-Fya or anti-Fyb while being negative genetically. Falsepositive Fya results were found in 23 samples, and false-positive Fyb in 10 specimens. During the last 3 months, significantly more RBC units were transfused to patients with discrepant results than to those with accurate phenotyping/genotyping results: median of 5 (mean ± SE: 6.85±0.69) versus median of 4 (mean: 5.71±0.51), respectively (p=0.025). The median length of time after the last transfusion was 25 days (mean: 28.72±2.23 days) in the group with accurate phenotyping/genotyping results versus a median of 14 days (mean: 15.52±1.95 days) in the group with discrepant results (p=0.001). Phenotypes and genotypes coincided in all donor samples. Conclusion: Genotyping assays for the Duffy system should be considered if the patient underwent blood transfusion less than 3 or 4 weeks before the sample collection. If the time frame from RBC transfusion exceeds 6 weeks, Duffy phenotyping can provide accurate results. Key Words: Duffy phenotyping, Kidd phenotyping, Genotyping, Multitransfused patients Address for Correspondence: Diana REMEIKIENE, M.D., Lithuanian University of Health Sciences, Institute of Oncology, Department of Haematology, Kaunas, Lithuania Phone: +370 37326303 E-mail: diana.remeikiene@kaunoklinikos.lt Received/Geliş tarihi : February 28, 2013 Accepted/Kabul tarihi : November 1, 2013

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Özet: Amaç: Eritrositlerin ABO ve RhD sistemleri dışındaki konvansiyonel serolojik tiplendirmesini son zamanlarda sık kan transfüzyonu yapılmış olan kişilerde yorumlamak yanlış ve zor olabilir. Bazı moleküler incelemeler henüz rutin olarak kullanılmamasına rağmen, genotiplendirmenin yararı bu işlemden fayda görecek hastaları belirlemek amacıyla incelenmiştir. Gereç ve Yöntemler: Hemato-onkoloijk, kronik böbrek hastalığı veya gastrointestinal hastalığı olan 101 hastadan ve kontrol grubu olan 50 kişiden karşılaştırmak üzere kan örnekleri alındı. Numuneler serolojik ve genetik yöntemler kullanılarak Fya ve Fyb için test edildi. Bütün hastalara son 3 ay içinde 3 ve daha fazla ünite eritrosit süspansiyonu transfüzyonu yapılmıştı. Her hasta için ortalama transfüzyon 6,1 ünite olarak hesaplandı. Transfüzyondan örneklerin alınmasına kadar geçen ortalama süre 24,4 gündü. Hemaglütinasyon testi serolojik analiz için uygulandı, ve uzunluğu kısıtlanmış polimorfizm testi genotipleme için kullanıldı. Bulgular: Genetik olarak negatif olan, toplam 33 (%32,7) hastada anti-Fya veya anti-Fyb ile pozitif reaksiyon elde edildi. Yirmi üç örnekte yanlış pozitif Fya sonucu, 10 örnekte yanlış pozitif Fyb sonucu elde edildi. Son 3 ayda fenotiplendirme/genotiplendirme sonuçları tutarsız olanlarda uyumlu olanlara göre anlamlı olarak daha fazla eritrosit süspansiyonu transfüzyon yapılmıştı: sırasıyla ortanca 5 (ortalama ± SE: 6,85±0,69) ve ortanca 4 (ortalama ± SE: 5,71±0,51) (p=0,025). Fenotiplendirme/genotiplendirmesi sonuçları uyumlu olan grupta transfüzyondan test aşamasına kadar geçen ortanca süre 25 gün (ortalama: 28,72±2,23) iken, sonuçları uyumsuz çıkan grupta bu süre ortanca 14 (ortalama: 15,52±1,95) gündü. Tüm donör örneklerinde fenotip ve genotipler tutarlıydı. Sonuç: Örnek alınmasından 3 veya 4 hafta öncesinde transfüzyon alan kişilerde Duffy sistemi için genotiplendirme yapılması uygun olabilir. Eğer eritrosit süspansiyonu transfüzyonundan sonra geçen süre 6 haftadan fazla ise, Duffy fenotiplendirmeyle uygun ve güvenilir sonuçlar sunabilir. Anahtar Sözcükler: Duffy fenotiplendirme, Kidd fenotiplendirme, Genotiplendirme, Çoklu transfüzyon alan hastalar Introduction Patients who require multiple transfusions of red blood cells (RBCs), such as those with sickle cell disease (SCD) or β-thalassaemia, have a higher potential risk of alloimmunisation and delayed haemolytic transfusion reactions (DHTRs). The widely described risk of developing antibodies, mostly to Rh, Kell, Duffy, Kidd, and MNS systems, ranges from 18% to 47% [1,2]. Programs to prevent alloimmunisation have been implemented in the centres treating patients with SCD and β-thalassaemia [1,3,4]. In addition to ABO and RhD matching, protocols range from providing limited antigen-matched RBCs for Rh and Kell to extended antigen-matched RBCs for Rh, Kell, Duffy, Kidd, and MNS systems prior to transfusion. Accurate phenotyping of multitransfused patients is often complicated, mostly due to the presence of circulating transfused donor RBCs in the recipient’s blood, leading to discrepancies in the assessment of tests results. The importance of the genotyping of clinically relevant antigens (such as C, c, E, K, Fya, Fyb, Jka, Jkb, and S) in addition to phenotyping is still being discussed for patients with SCD, β-thalassaemia, and other haemoglobinopathies. However, there is a lack of information about the need for molecular testing for other groups of patients who depend on long-term RBCs transfusions, such as those with myelodysplastic syndrome, myelofibrosis, or chronic renal failure [5,6,7]. Delayed haemolytic transfusion reactions are often an issue in these patients, affecting their quality of life

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and sometimes being fatal. The selection of antigen-negative RBCs in order to reduce alloimmunisation is often required for patients with long-term transfusions and for those with formed alloantibodies. The most common causes of DHTR include antibodies against Rh, Kell, Kidd, Duffy, and MNS systems [3,8,9,10]. Serological testing and evaluation of the antigens and antibodies of the Duffy and Kidd systems are among the main problems in multitransfused patients. The correlation between serological and molecular typing of Duffy and Kidd systems demonstrates the benefits of genotyping in patients who depend on chronic RBC transfusions. The aim of our study was to estimate the value of DNAbased typing of Duffy and Kidd systems in chronically transfused non-SCD or β-thalassaemia patients, and to establish the impact of the amount of transfused RBCs and the time from the last transfusion on the discrepancy of the results. Materials and Methods Patients Peripheral blood samples were obtained from 101 patients with haematological and oncological diseases, chronic renal failure, and gastrointestinal diseases. All patients received 3 or more units of RBCs during the last 3 months. The inclusion criteria were the time frame from the last RBC transfusion being shorter than 8 weeks and the need for further

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transfusions. None of the patients were tested for Fya, Fyb, Jka, or Jkb antigens before transfusions. Most of the RBC units received by the haematology patients and some of those received by the oncology patients were leucodepleted, while others were non-leucodepleted. Thus, 62 patients (61.4%) were transfused with leucoreduced RBCs, and 39 (38.6%) with entirely (or by more than half) non-leucoreduced RBCs. The median number of transfusion events per patient was 3 (mean: 3.33±0.21, range: 1-12). A median of 2 (mean: 1.85±0.04, range: 1-4) RBC units were administered per transfusion event and 4 (mean: 6.1±0.41, range: 3-24) units per patient. Donors In total, 49 blood donors served as controls in our study. Blood samples of donors were obtained by taking 1 or 2 segments of tubes from RBC bags used for transfusions. This control group and the patient group were used to determine frequencies of Duffy and Kidd phenotypes in a Lithuanian population. Blood samples of all patients and donors were tested for Fya, Fyb, Jka, and Jkb antigens by applying serological and molecular methods. Approval for the study was obtained from the Regional Bioethics Committee. Serotyping Peripheral blood samples were used for Duffy and Kidd phenotyping. Fya, Fyb, Jka, and Jkb antigens were determined by haemagglutination using anti-Fya, anti-Fyb, anti-Jka, and anti-Jkb Coombs reactive (polyclonal, human) reagents (Antitoxin GmbH, Germany) and DG Gel Coombs cards (Diagnostic Grifols, S.A., Spain). Each microtube of the card contained polymerised dextran in a buffered medium containing preservatives and low ionic strength solution, and was mixed with polyspecific anti-human globulin. The tests were performed according to the manufacturer’s recommendations. The results of the agglutination were expressed by using plus/minus values. Positive results were evaluated from 1+ to 4+.

Genotyping The DNA was extracted from peripheral blood leucocytes using a DNA extraction kit (GeneJet Genomic DNA Purification Kit, Thermo Fisher Scientific, USA), following the manufacturer’s instructions. Polymorphism was identified by polymerase chain reaction-restriction length polymorphism (PCR-RFLP) analysis according to Reid et al. [7]. Briefly, each PCR reaction was carried out in a total volume of 25 µL containing 1X DreamTaq standard buffer, template DNA, 50 pM of each primer, 2.0 mM MgCl2, 200 µM of each dNTP, and 1 U of DreamTaq DNA polymerase (Thermo Fisher Scientific) with annealing at 62 °C. The amplification products were then digested overnight by restriction endonuclease BanI (Thermo Fisher Scientific), following the manufacturer’s instructions. The fragments were separated electrophoretically using 2% agarose gel containing ethidium bromide. The Duffy antigen is present in 2 major allelic forms, FY*A and FY*B, differing in an amino acid at position 42 (Gly42Asp) of the Duffy antigen receptor. The amino acid Table 1. The results of Duffy and Kidd system phenotyping and genotyping in 101 multitransfused patients.

Genotype

Phenotype

Combination of FY alleles

Fy(a+b-)

Fy(a+b+)

Fy(a-b+)

FY*A/FY*A

FY*A/FY*B

FY*B/FY*B

JK*A/JK*A

JK*A/JK*B

JK*B/JK*B

Combination of JK alleles

Kappa 0.368, p<0.001 for Duffy system; kappa 0.404, p<0.001 for Kidd system.

Table 2. Duffy phenotyping and genotyping discrepancies and time from last red blood cells transfusion.

Time from the Last Transfusion <4 Weeks n=69

>4 Weeks n=32

Result

Number

Percent of Total

Number

Percent of Total

Genotype FY*A/FY*A - phenotype Fy(a+b+)

30.3

Genotype FY*B/FY*B -phenotype Fy(a+b+)

57.6

12.1

Total

87.9†

12.1†

†: statistically significant difference (z-test, p<0.05).

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dichotomous results of 2 tests was measured using Cohen’s kappa. Differences were considered significant at p<0.05. IBM SPSS Statistics 20 was used for the data analysis.

change occurs because of G125A polymorphism in the Duffy antigen receptor for the chemokine gene (DARC). The Kidd antigen system is known to comprise 2 major alleles, JK*A and JK*B, which result from a single nucleotide polymorphism (838G→A) in gene SLC14A1. The corresponding JK*A and JK*B antigens differ by a single amino acid (D280N).

Results Donors Coincidence between phenotype and genotype was observed in all 49 donor samples.

Statistical Analysis

Patients

The comparison of medians between the groups was performed by applying the nonparametric Mann-Whitney U test. We did not compare the means because the values of the studied variables were not normally distributed (KolmogorovSmirnov test, p<0.05). The 2-proportion z-test, chi-square test, and Fisher’s exact test (for small samples) were used for categorical data analysis. The level of agreement between

Disagreements of phenotype and genotype between the Duffy and Kidd systems were found in one-third of the samples of 101 patients who had recently undergone transfusion. The results of phenotyping and genotyping are presented in Table 1. To analyse the impact of time from the last transfusion on the discrepancies of the results, patients were distributed

Table 3. Kidd phenotyping and genotyping discrepancies and time from last red blood cells transfusion.

Time from the Last Transfusion <4 Weeks n=69

>4 Weeks n=32

Results

Number

Percent of Total

Number

Percent of Total

Genotype JK*A/JK*A - phenotype Jk(a+b+)

53.1

9.4

Genotype JK*B/JK*B -phenotype Jk(a+b+)

31.3

6.2

Total

84.4†

15.6†

†statistically significant difference (z-test, p<0.05).

Table 4. Distribution of Duffy and Kidd antigens and phenotypes.

Patients, Controls, n

Percent of Total

Literature, %

p-Value

Fya+

105

0.172*

Fyb+

124

82.7

0.5*

Fy(a+b+)

52.7

0.207*

Fy(a+b-)

17.3

0.5*

Fy(a-b+)

0.172*

Jka+

116

75.3

0.923*

Jkb+

105

Jk(a+b+)

47.3

0.568*

Jk(a+b-)

0.052*

Jk(a-b+)

22.7

0.201*

Duffy System

Kidd System

*: Frequencies of Duffy and Kidd antigens and phenotypes were compared with literature data (nonparametric binomial test).

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into 2 groups. The first group included those who received RBCs less than 4 weeks before and the second group more than 4 weeks before the sample collection. The influence of the number of transfused RBCs on the disagreement of the results was also analysed. Duffy System A total of 33 (32.7%) phenotype/genotype discrepancies were assessed. However, in samples that did not contain one of the Duffy antigens, discrepant results were found in 68.8% of cases. Significant differences in phenotype/genotype disagreements between the 2 aforementioned groups were found in 29 and 4 cases, respectively (Table 2). The comparison of the duration of time after the last transfusion between the 2 groups of patients with accurate and discrepant phenotype/genotype results also showed a significant difference. The median of days after the last transfusion was found to be 14 (mean: 15.52±1.95) in the group with discrepant results versus 25 (mean: 28.72±2.23) in the group with accurate results (p=0.001). No effect of transfused RBCs per year on Duffy phenotyping and genotyping discrepancies was detected: 605 units [median:5 (mean: 8.91±1.58, range: 3-46)] were transfused to patients with accurate phenotype/genotype results, and 294 units [median: 6 (mean: 8.91±1.4, range: 3-81] to those with discrepant results (p=0.207). Significantly different results were found when comparing the number of RBC units transfused during the last 3 months between the 2 aforementioned groups. A median of 5 (mean: 6.85±0.69) units were transfused during this period to the patients with discrepant phenotype/genotype results versus 4 (mean: 5.71±0.5) to those with accurate results (p=0.025). Kidd System Disagreements between genotyping and serologic typing for Jka/Jkb were found in 32 (31.7%) blood samples, while in samples that did not contain one of the Kidd antigens, discrepant results were found in 65.3% of cases. Significantly more discrepant phenotype/genotype results were found in the samples of patients of the first group than in those of the second group, at 27 and 5 cases, respectively (Table 3). The time after the last transfusion was shown to be significantly different between the 2 groups of patients. The median of days after the last transfusion was found to be 10.5 (mean: 16.09±2.45) in the group with discrepant results versus 25 (mean: 28.26±2.13) in the group with accurate results (p=0.01). No effect of the transfused RBCs per year on Kidd phenotyping and genotyping discrepancies was detected: 649 units [median: 5 (mean: 9.41±1.66, range: 3-81)] were transfused to patients with accurate phenotype/genotype

results, and 250 units [median: 8 (mean: 7.84±0.72, range: 3-18)] to those with discrepant results (p=0.188). There was no difference with regard to the number of RBC units transfused during the last 3 months between the 2 aforementioned groups, either. A median of 6 (mean: 6.28±0.48) units were transfused during this period to the patients with discrepant phenotype/genotype results versus a median of 4 (mean: 5.99±0.56) to those with accurate results (p=0.71). Phenotype Frequencies Genotyping results were used to determine the expected Duffy and Kidd phenotype frequencies. No differences were found when comparing these results with those of other authors [8,9]. The phenotypes of 101 patients and 49 controls are presented in Table 4. Discussion It is still disputable which situations require extended typing of transfused RBCs beyond routine matching for ABO, RhD, and existing antibodies. Although a number of studies found that SCD or β-thalassaemia patients may benefit from extended antigen matching, there is still a lack of recommendations in this context for other patients undergoing long-term RBC transfusions [1,11,12]. Additional antibodies are known to occur in 20% to 90% of previously alloimmunised patients [13,14,15]. Schonewille et al. evaluated the incidence of new antibody formation with subsequent transfusion in 22% of alloimmunised haematooncology patients and in 20%-25% of the non-haematology/ oncology cohort [14,15]. The transfusion of extended antigen-matched RBCs, including Duffy and Kidd systems, has been shown to lower alloimmunisation in about 70% of cases [16,17]. According to these studies as well as those investigating SCD patients, extended antigen matching could be recommended for haemato-oncology patients and other subjects who undergo long-term transfusions. We agree with the many authors who state that antigen typing of recently transfused patients is not always accurate, as their peripheral blood contains transfused cells [5,6,13,18,19]. Several reports describe this type of discrepancy in up to 10% of cases for the Duffy and Kidd systems by comparing phenotyping and genotyping results [6,18,20]. These findings differ from our data, which showed a much higher rate of discrepancies (32.7% and 31.7%), although the results cannot be accurately compared because other authors failed to provide information about the number of transfused RBC units or the time frame from the last transfusion. In contrast to a number of previous studies [6,18,20,21,22], our data do not suggest the presence of additional Duffy system alleles (which are known to be associated with reduced or abolished gene expression) either in patients or in

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donors. This is likely due to the fact that only genotypes of the Caucasian population were tested in our study. The phenotyping/genotyping results in a group of patients who received transfusions within a time period of 7 days before sample collection also differed from those reported earlier, particularly for the Kidd system [5,7]. In this group, a total of 24 samples in our study showed positive (2+ to 4+) results for Fya, Fyb, Jka, and Jkb, although genotypically, 14 of them were Fya- or Fyb-negative, and 14 were Jka- or Jkb-negative. Reid et al. described concordant phenotype/genotype results for 7 samples and mixed-field agglutination for 32 samples for Duffy antigens; they also described coincident results for 16 samples and mixed-field agglutination for 31 samples for the Kidd system [7]. Our data suggest the need for blood group genotyping (including the Duffy and Kidd systems), which was proposed by many authors [1,2,3,4,5,6,7,11,13,16,18,19,20,23,24,25]. Nevertheless, one should determine when genotyping should replace serology or be combined with it for patients undergoing long-lasting RBC transfusions. This is particularly important for institutions that do not perform blood group genotyping routinely. We found in our study that Duffy genotyping/ phenotyping disagreements depended on the number of transfused RBC units during the last 3 months. However, it would be difficult to predict how many units would cause these discrepancies or to make specific recommendations. The limitation of our study is the small number of subjects, which complicated their grouping according to the number of transfused RBC units. The results of our study regarding the effect of the length of time after the last transfusion on serology findings are useful in clinical practice. A significantly lower number of Duffy and Kidd phenotyping/genotyping discrepancies was found in patients who underwent their last transfusions more than 3 (p=0.01 and p=0.017, respectively) or 4 (p=0.003 and p=0.018, respectively) weeks before the sample collection. Only one disagreement for the Duffy system (compared to 3 for the Kidd system) was found when the time from the last transfusion was from 6 to 7 weeks. No discrepancies were found for either system when the last transfusion was performed more than 7 weeks before. Our data are also in agreement with previous reports indicating that the range of agglutination in mixed-field reactions does not predict the actual antigen typing [7]. For example, 10 patients in the discrepant group with 2+ reaction for Fya and 2+ or 3+ reaction for Fyb had FY*B/FY*B genotypes. In the group with accurate results, 7 genotypically FY*A/ FY*B samples also showed 2+ agglutination with anti-Fya. An exception could be made for serologic reactions with the expression of 1+. All 5 of the 1+ reactions in the Duffy system typing as well as 8 respective reactions in the Kidd typing 372

Remeikiene D, et al: Duffy and Kidd Genotyping for Transfusions

genetically showed no allele expression. Those reactions were classified as serologically negative. More research is needed to confirm this statement. Conclusions Our study showed that Duffy or Kidd phenotyping with agglutination tests is inappropriate for patients who have recently undergone blood transfusion. Genotyping should be considered when the patient underwent transfusion less than 4 weeks before the sample collection. The group of patients who recently (<7 weeks) received RBC transfusion and require genotyping in addition to phenotyping are those who are homozygous for either Duffy or Kidd, but this cannot be ascertained using serology. Agglutination tests can be reliably used for donors and patients who have been transfused more than 7 weeks before. Furthermore, the accurate typing of both the Kidd and Duffy systems before the first transfusion could be beneficial. 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. Wilkinson K, Harris S, Gaur P, Haile A, Armour R, Teramura G, Delaney M. Molecular typing augments serologic testing and allows for enhanced matching of red blood cells for transfusion in patients with sickle cell disease. Transfusion 2012;52:381-388. 2. Hillyer CD, Shaz BH, Winkler AM, Reid M. Integrating molecular technologies for red blood cell typing and compatibility testing into blood centers and transfusion services. Transfus Med Rev 2008;22:117-132. 3. Ribeiro KR, Guarnieri MH, Costa DC, Costa FF, Pelegrino J Jr, Castilho L. DNA array analysis for red blood cell antigens facilitates the transfusion support with antigen-matched blood in patients with sickle cell disease. Vox Sang 2009;97:147152. 4. Chou ST, Westhoff CM. The role of molecular immunohematology in sickle cell disease. Transfus Apher Sci 2011;44:73-79. 5. Rozman P, Dovc T, Gassner C. Differentiation of autologous ABO, RHD, RHCE, KEL, JK and FY blood group genotypes by analysis of peripheral blood samples of patients who have recently received multiple transfusions. Transfusion 2000;40:936-942. 6. Guelsin GA, Sell AM, Castilho L, Masaki VL, Melo FC, Hashimoto MN, Higa TT, Hirle LS, Visentainer EL. Benefits of blood group genotyping in multi-transfused patients from the south of Brazil. J Clin Lab Anal 2010;24:311-316.

Remeikiene D, et al: Duffy and Kidd Genotyping for Transfusions

7. Reid ME, Rios M, Powell VI, Charles-Pierre D, Malavade V. DNA from blood samples can be used to genotype patients who have recently received a transfusion. Transfusion 2000;40:48-53. 8. Meny GM. The Duffy blood group system: a review. Immunohematology 2010;26:51-56. 9. Westhoff CM, Reid ME. Review: the Kell, Kidd, Duffy and Kidd blood group systems. Immunohematology 2004;20:37-49.

Turk J Hematol 2014;31:367-373

17. Verduzco LA, Nathan DG. Sickle cell disease and stroke. Blood 2009;114:5117-5125. 18. Castilho L, Rios M, Bianco C, Pellegrino J Jr, Alberto FL, Saad ST, Costa FF. DNA-based typing of blood groups for the management of multiply-transfused sickle cell disease patients. Transfusion 2002;42:232-238. 19. Avent ND. Large-scale blood group genotyping: clinical implications. Br J Haematol 2008;144:3-13.

10. Schwickerath V, Kowalski M, Menitove JE. Regional registry of patient alloantibodies: first-year experience. Transfusion 2010;50:1465-1470.

20. Castilho L, Rios M, Pellegrino J Jr, Carvalho MHM, Alberto FL, Saad STO, Costa FF. Genotyping of Kell, Duffy, Kidd and RHD in patients with Î˛-thalassemia. Transfusion 2000;22:6976.

11. Castilho L, Rios M, Pellegrino J Jr, Saad STO, Costa FF. Blood group genotyping facilitates transfusion of Î˛-thalassemia patients. J Clin Lab Anal 2002;16:216-220.

21. Castilho L. The value of DNA analysis for antigens in the Duffy blood group system. Transfusion 2007;47:28-31.

12. Klapper E, Zhang Y, Figueroa P, Ness P, Stubbs J, Abumuhor I, Bailey J, Epperson L, Tauscher C, Enriques E, Hashmi G, Seul M. Toward extended phenotype matching: a new operational paradigm for the transfusion service. Transfusion 2010;50:536-546. 13. Denomme GA, Flegel WA. Applying molecular immunohematology discoveries to standards of practice in blood banks: now is the time. Transfusion 2008;48:24612475. 14. Schonewille H, de Vries R, Brand A. Alloimmune response after additional red blood cell antigen challenge in immunized hematooncology patients. Transfusion 2009;49:453-457.

22. Cotorruelo C, Biondi C, Racca L, Borras SG, Racca A. Duffy genotyping facilitates transfusion therapy. Clin Exp Med 2009;9:249-251. 23. Westhoff CM. Molecular testing for transfusion medicine. Curr Opin Hematol 2006;13:471-475. 24. Castro O, Sandler SG, Houston-Yu P, Rana S. Predicting the effect of transfusion only phenotype-matched RBCs to patients with sickle cell disease: theoretical and practical implications. Transfusion 2002;42:684-690. 25. Lomas-Francis C. The value of DNA analysis for antigens of the Kidd blood group system. Transfusion 2007;47:23-26.

15. Schonewille H, van de Watering L, Brand A. Additional red blood cell alloantibodies after blood transfusions in a nonhematologic alloimunized patient cohort: is it time to take precautionary measures? Transfusion 2006;46:630-655. 16. Anstee DJ. Red cell genotyping and the future of pretransfusion testing. Blood 2009;114:248-256.

373

Research Article

DOI: 10.4274/tjh.2013.0004

Bone-Specific Alkaline Phosphatase Levels among Patients with Multiple Myeloma Receiving Various Therapy Options Farklı Tedavi Rejimleri Alan Multipl Myelom Hastalarında Kemik Spesifik Alkalen Fosfataz Düzeyleri Güven Çetin1, Ahmet Emre Eşkazan2, M. Cem Ar3, Şeniz Öngören Aydın4, Burhan Ferhanoğlu4, Teoman Soysal4, Zafer Başlar4, Yıldız Aydın4 1Bezmialem

Vakıf University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey Training and Research Hospital, Clinic of Hematology, Diyarbakır, Turkey 3İstanbul Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 4İstanbul University Cerrahpaşa Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 2Diyarbakır

Abstract: Objective: This study aimed to investigate the impact of the different therapy regimens used in multiple myeloma (MM) on bone-specific alkaline phosphatase (BALP) levels.

Materials and Methods: One hundred and thirteen patients with MM were included in the study. Patients were grouped according to the regimens they received, as follows: group 1, melphalan and prednisolone (MP); group 2, vincristine, adriablastin, and dexamethasone (VAD); group 3, thalidomide plus dexamethasone; and group 4, bortezomib plus dexamethasone. BALP levels were measured before treatment and at the third and sixth months of treatment. A fifth group consisted of patients in the post-treatment remission period at study entry (no-treatment group). Results: The BALP levels at the third and sixth months of the treatment were significantly higher than the pre-treatment levels in the bortezomib and the no-treatment groups, whereas no significant difference was observed in the MP, VAD, and thalidomide groups.

Conclusion: Considering that BALP is a surrogate marker of bone formation, our study suggests that bortezomib more efficiently leads to the improvement of bone disease in myeloma than other treatment options.

Key Words: Multiple myeloma, Bone-specific alkaline phosphatase, Bortezomib, Thalidomide Özet: Amaç: Bu çalışmanın amacı; multipl myelomda (MM) kullanılan farklı tedavi rejimlerinin kemik-spesifik alkalen fosfataz (BALP) düzeyleri üzerine etkisini incelemektir.

Address for Correspondence: Güven ÇETİN, M.D., Bezmialem Vakıf University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey Phone: +90 532 551 47 98 E-mail: drgvn@mynet.com Received/Geliş tarihi : January 2, 2013 Accepted/Kabul tarihi : July 19, 2013

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Çetin G, et al: Multiple Myeloma and Bone-Specific Alkaline Phosphatase

Gereç ve Yöntemler: Çalışmaya 113 MM hastası dahil edildi. Hastalar aldıkları tedavi rejimlerine göre; 1. grup, melfalan ve prednizolon (MP); 2. grup, vinkristin, adriablastin ve deksametazon (VAD); 3. grup talidomid artı deksametazon; 4. grup, bortezomib artı deksametazon olarak gruplara ayrıldı. BALP düzeyleri tedaviden önce, tedavinin üçüncü ve altıncı aylarında ölçüldü. Çalışmanın başında, tedavi sonrası remisyon dönemindeki hastalardan beşinci bir grup oluşturuldu (tedavi almayan grup). Bulgular: Bortezomib grubu ve tedavi almayan grupta tedavinin üçüncü ve altıncı aylarında bakılan BALP düzeyleri, tedavi öncesine göre anlamlı olarak yüksek bulunurken; MP, VAD, ve talidomid gruplarında anlamlı fark saptanmadı. Sonuç: BALP’nin kemik formasyonunun bir belirteci olduğu göz önünde tutulduğunda, bu çalışma ile diğer tedavi seçeneklerine göre bortezomib tedavisinin miyelomda kemik hastalığının iyileşmesine daha etkin bir şekilde yol açtığı gösterilmiştir. Anahtar Sözcükler: Multipl Myelom, Kemik Spesifik Alkalen Fosfataz, Bortezomib, Talidomid Introduction Multiple myeloma (MM) is a malignant disease characterized by anemia, monoclonal protein in the serum and/or the urine, osteolytic bone lesions, hypercalcemia, and renal insufficiency resulting from uncontrollable clonal hyperproliferation of plasma cells in bone marrow [1,2,3]. The skeletal system problems that appear either at the time of the diagnosis or during the progression of MM cause a decrease in the quality of life and lead patients to present to health services. The rate of skeletal system-related complications including bone pain, osteolytic lesions, and pathological fractures reaches up to 80% in patients with MM [4,5]. In the studies concerning MM, it has been demonstrated that there is a significant relation between the bone-specific alkaline phosphatase (BALP) levels and bone pain, lytic lesions, and bone fractures [1,2]. Until now, there has been no large study showing the effect of various therapy options used in the treatment of MM on the changes in bone metabolism caused by myeloma. The aim of the present study was to investigate the relationship between pre-treatment and post-treatment BALP levels and the therapy options in patients with MM, and to expose the effect of these therapy options on bone metabolism. Materials and Methods Patients One hundred and thirteen patients diagnosed with MM in accordance with the criteria of the International Myeloma Working Group and followed in the Hematology Division of the Internal Medicine Department of the İstanbul University Cerrahpaşa Medical Faculty between November 2006 and July 2009 were included in the present study. The study cohort consists of patients with MM that have been followed and/ or treated for at least 6 months regardless of their age, sex, stage of the disease, and type of the therapy. Continuity of the out-patient clinic visits for follow-ups was confirmed, and the general status of the patients had not deteriorated. The study

was planned in accordance with the Helsinki Declaration, and the approval of the Ethics Committee of the İstanbul University Cerrahpaşa Medical Faculty was obtained. Written informed consent of the patients to participate in the study was received. Participants who chose to quit the study for any reason, cases lacking any results from the analyses done for the evaluations, cases of non-continuance of follow-up visits, and patients who died before completing the follow-up period were not included in the study. Methods The melphalan and prednisone (MP; group 1); vincristine, adriamycin (doxorubicin), and dexamethasone (VAD; group 2); thalidomide (group 3); and bortezomib (group 4) regimens were as defined before [6,7,8,9]. International Myeloma Working Group response criteria were used while evaluating the patients’ responses to these treatment modalities [10]. All patients had received monthly zoledronic acid treatment, and since the serum BALP values could be affected, the patients with renal insufficiency who required dialysis were excluded from the study population. The levels of BALP (initial, third month, and sixth month), serum protein electrophoresis, C-reactive protein, erythrocyte sedimentation rate, lactate dehydrogenase, total protein, albumin, hemoglobin, beta-2 microglobulin, creatinine, glomerular filtration rate, calcium, quantitative immunoglobulin and light chain, and immunofixation electrophoresis of all patients were analyzed in the Fikret Biyal Central Biochemistry Laboratory of the İstanbul University Cerrahpaşa Medical Faculty. Four milliliters of peripheral blood was taken from all patients into tubes containing EDTA both prior to the treatment and at the third and sixth months of treatment. BALP levels were measured by means of radioimmunoassay technique using the Ostease BALP Kit of International Diagnostic Systems Ltd. The normal ranges of BALP levels among pre-menopausal women, post-menopausal women, and men are 11.6-29.6 U/L, 14.2-42.7 U/L, and 15.0-41.3 U/L, respectively, as given by the manufacturer. 375

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Çetin G, et al: Multiple Myeloma and Bone-Specific Alkaline Phosphatase

Complete blood count was evaluated with a Beckman Coulter HMX auto-analyzer in the laboratory of the Hematology Division. Bone marrow aspiration and biopsies were evaluated in the Pathology Department of Cerrahpaşa Medical Faculty. Statistical Evaluation Statistical analyses were done with the NCSS 2007 package program. In addition to the descriptive methods (mean, standard deviation), Friedman’s test was used for the repetitive measurements of the multiple groups, the KruskalWallis test was used for intergroup comparisons, Dunn’s multiple comparison test was used for the comparison of sub-groups, and chi-square and Fisher’s exact tests were used for the comparison of qualitative data. The level of statistical significance was set at p<0.05. Results Among the 147 patients with MM, 113 patients were eligible for evaluation. Of these 113 patients, 52 (40%) were female and the average age was 60.5 years (range: 30-84 years). The patients included in the study were either newly diagnosed (n=45) or had active relapsed disease (n=42), or were receiving no treatment (n=26) [including patients with

both stage I disease (n=11) and with post-treatment remission (n=15)]. The patients who had received treatment were divided into groups receiving MP, VAD, thalidomide, and bortezomib. There were 45 newly diagnosed patients, of which 25 had received MP, whereas 20 had received VAD. The 42 active relapsed patients had received novel agents (thalidomide was given in 17 patients and bortezomib in 25 patients). The distribution of the groups was homogeneous, and there was no statistical difference between the groups according to age or sex at p=0.134 and p=0.183, respectively. IgG was the most common type of paraproteinemia. The distribution rates of M protein were as follows: 70 patients (61.9%) had IgG, 29 patients (25.66%) had IgA, 6 patients (5.30%) had kappa light chain, and 8 patients (7.07%) had lambda light chain type disease. The majority of the patients were in the advanced stage. According to the Durie-Salmon (DS) staging system, 11 (9.7%) patients were in stage 1, 27 (23.9%) patients were in stage 2, and 75 (66.4%) patients were in stage 3. According to the International Staging System, 50 patients (44.2%) were in stage 1, 41 patients (36.3%) were in stage 2, and 22 patients (19.5%) were in stage 3. The distributions of the patients according to the myeloma types and stages are displayed in Table 1.

ISS

DSSS

Myeloma type

Table 1. The distribution of the patients according to the myeloma types and stages (DSSS: Durie-Salmon Staging System, ISS: International Staging System, MP: melphalan and prednisone, VAD: vincristine, adriamycin, and dexamethasone).

376

MP group (n=25) n (%)

VAD group (n=20) n (%)

Thalidomide Bortezomib group (n=17) group n (%) (n=25) n (%)

No-treatment group (n=26) n (%)

IgA/Kappa

(12)

(25)

(11.8)

(16)

(15.3)

IgA/Lambda

(0)

(5)

(41.2)

(4)

(7.7)

IgG/Kappa

(52)

(50)

(17.6)

(44)

(30.8)

IgG/Lambda

(24)

(5)

(23.5)

(24)

(30.8)

Kappa

(4)

(10)

(0)

(4)

(7.7)

Lambda

(8)

(5)

(5.9)

(8)

(7.7)

(0)

(42.3)

(28)

(20)

(5.9)

(24)

(26.9)

(4)

(0)

(5.9)

(0)

(60)

(80)

(82.3)

(64)

(26.9)

(8)

(0)

(5.9)

(12)

(3.9)

(20)

(60)

(35.3)

(48)

(57.7)

(48)

(30)

(35.3)

(32)

(34.6)

(32)

(10)

(29.4)

(20)

(7.7)

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Çetin G, et al: Multiple Myeloma and Bone-Specific Alkaline Phosphatase

There was no significant difference according to the pretreatment, third month, and sixth month mean BALP values among the MP, VAD, and thalidomide groups (p=0.069, p=0.148, p=0.254; Table 2). There was a significant change between the pre-treatment, third month, and sixth month mean BALP values in the bortezomib group (p=0.002). While the sixth month mean BALP value was significantly higher than the pre-treatment and third month mean BALP values (p=0.003), no significant difference was found between pretreatment and third month values (p>0.05). Significant change was found between initial, third month, and sixth month mean BALP values of the no-treatment group (p=0.0001) (Table 2). The initial mean BALP values were found to be significantly lower than the third and sixth month mean BALP values (p=0.001, p=0.035). Since the patient numbers in the groups were relatively small, especially when divided according to DS stages, we did not compare BALP values among the patient groups according to the DS staging system. Discussion Myeloma-induced skeletal problems negatively affect the quality of life [11,12,13]. The activation of osteoclasts and the suppression of osteoblasts are the main events in MM. Bone formation is suppressed. Therefore, bone lesions are totally lytic in patients with MM [14]. Osteoclasts usually proliferate on the resorptive surfaces adjacent to the myeloma cells in the bone and do not proliferate in the regions that do not have tumoral involvement [15]. Until the new treatment options were developed, MP, high-dose dexamethasone, VAD, and multi-drug combination chemotherapies were the combinations most frequently used in MM [16,17,18,19]. The new drugs that have recently been included in the treatment of MM are molecular-targeted drugs that target the micro-environment of the bone. Among those drugs, good therapy responses have been obtained with the combination of thalidomide, bortezomib, or lenalidomide with high-dose dexamethasone [20]. The treatment of myeloma

bone disease has mainly been directed toward the inhibition of osteoclastic activity with the use of bisphosphonates [11]. Anti-myeloma therapies that cause remission are usually not accompanied by an increase in the osteoblast indicators or in bone mineral density [21,22]. When compared with patients with monoclonal gammopathy of undetermined significance or with the healthy controls, deterioration in osteoblastic activity in the patients with MM was observed with a decrease in osteocalcin or BALP levels [23]. In healthy subjects, BALP accounts for approximately 50% of the total alkaline phosphatase (TAP) in the circulation. BALP reflects both the bone formation and the bone degradation more sensitively than alkaline phosphatase [24]. Serum BALP shows a remarkable correlation with the dynamic parameters of bone formation [25]. The fact that there is a relationship between the increase in serum BALP and osteoblastic activity in MM has been demonstrated in another study, as well [26]. Some preclinical trials again raised the suggestion that the inhibition of proteasome might enhance osteoblastic activity [27]. From this perspective, proteasome inhibitors represent a group of new anti-tumor drugs [28] and have strong anti-myeloma effects [29]. Bortezomib, which is a proteasome inhibitor, is the first drug used in the treatment of MM that has similar effects on the osteoblastic function together with important anti-myeloma activity [11,14,15,30,31,32,33,34]. Bortezomib, a firstgeneration proteasome inhibitor that has been developed to be used as an anti-neoplastic agent, inhibits the 26S proteasome that is located on the chymotryptic region and inhibits the proliferation of chemotherapy-sensitive, chemotherapyresistant, and dexamethasone-resistant MM cells as well as the proliferation of MM cells that have been recently isolated from the bone marrow of the patients [35,36]. The first evidence concerning the effect of bortezomib on bone metabolism came from a 63-year-old female patient with κ-chain MM who had undergone sequential autologous transplantation and was then treated with

Table 2. The change in BALP values among different treatment groups (BALP: bone-specific alkaline phosphatase).

BALP value (U/L)

MP group (n=25)

VAD group (n=20)

Thalidomide group (n=17)

Bortezomib group (n=25)

No-treatment KW group (n=26)

Initial

32.73±32.19

34.54±20.89

21.64±8.54

21.87±14.11

24.26±14.99

9.79

0.069

Third month

27.42±12.69

34.94±20.61

26.17±17.29

23.2±12.89

29.96±16.18

6.78

0.148

Sixth month

22.96±10.69

33.16±19.78

27.63±17.68

28.75±13.12

28.43±13.64

5.34

0.254

0.7

4.507

12.48

15.431

0.368

0.705

0.105

0.002

0.0001

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1 mg/m2 bortezomib on days 1, 4, 8, and 11 of a 21-day cycle. The paraprotein response of myeloma was associated with a rapid increase in TAP. The effect continued for a few therapy cycles and the BALP values were normal during the relapses [9,37]. Sezer et al. reported a negative correlation between RANKL and osteoprotegerin [38]. Subsequently, a significant increase was determined in BALP levels in the group receiving bortezomib as compared to the control group, and this finding was interpreted as osteoblastic activity increasing the effect of bortezomib. Thereafter, the change in alkaline phosphatase levels was retrospectively analyzed and evaluated in 2 large studies (APEX and SUMMIT studies) in which bortezomib was used as a single agent in patients with recurrent/refractory MM [39,40]. Data obtained from all these studies show that bortezomib increases osteoblastic activity. In the present study, the initial, third month, and sixth month BALP levels were not significantly different in the MP, VAD, and thalidomide groups. Significant change was observed among the pre-treatment, third month, and sixth month mean BALP values of the bortezomib group. The sixth month mean BALP value was found statistically higher than the pre-treatment and third month mean BALP values. Significant change was observed between the initial, third month, and sixth month mean BALP values of the notreatment group. The initial mean BALP value was found statistically lower than the third and sixth month mean BALP values, whereas no statistically significant difference was observed between the other times. According to our findings, the high BALP values at the third and sixth months of therapy in the group receiving bortezomib can be interpreted as the osteoblastic activity being increased in this group. The results of our study are also in line with the clinical findings of Shimazaki et al., who defined high BALP levels following bortezomib combination therapy in resistant MM [41]. In addition, it was reported that there was no significant change in BALP levels in patients receiving dexamethasone, whereas there was a significant increase in BALP levels in the myeloma patients receiving bortezomib [39,42]. In the present study, we determined a significant difference between the pretreatment and post-treatment BALP values with bortezomib therapy. On the other hand, the degree of improvement obtained by chemotherapy not being in line with the healing degree of the bone disease may be related to the increased osteoclastic activity of MM even in the plateau period [21,22]. In the present study, the third and sixth month mean BALP values being high in the no-treatment group may be explained by decreased osteoclastic and increased osteoblastic activity, because the majority of the patients in this group were in remission (complete and/or almost complete). In some studies concerning MM, it was determined that BALP levels were associated with bone pain, lytic lesions, and bone fracture

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[43]. From this point of view, achieving complete remission and treating the bone disease appear to be important aims for decreasing the morbidity and mortality of the patients. In conclusion, the use of proteasome inhibitors such as bortezomib, together with bisphosphonates, will no doubt lead to much more positive outcomes in myeloma treatment. 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. Dispenzieri A, Lacy MQ, Greipp PR. Multiple myeloma. In: Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader B (eds). Wintrobe’s Clinical Hematology. Philadelphia, Lippincott Williams & Wilkins, 2004. 2. Kyle RA, Rajkumar SV. Epidemiology of the plasma-cell disorders. Best Pract Res Clin Haematol 2007;20:637-664. 3. Tricot G, Fassas A. Multiple myeloma and other plasma cell disorders. In: Hoffman R, Benz EJ Jr, Shattil SJ, Furie B, Cohen HJ, Silberstein LE, McGlave P (eds). Hematology Basic Principles and Practice. Philadelphia, Elsevier Churchill Livingstone, 2005. 4. Kyle RA, Rajkumar SV. Drug therapy: multiple myeloma. N Engl J Med 2004;351:1860-1873. 5. Melton LJ 3rd, Kyle RA, Achenbach SJ, Oberg AL, Rajkumar SV. Fracture risk with multiple myeloma: a population-based study. J Bone Miner Res 2005;20:487-493. 6. San Miguel JF, Schlag R, Khuageva NK, Dimopoulos MA, Shpilberg O, Kropff M, Spicka I, Petrucci MT, Palumbo A, Samoilova OS, Dmoszynska A, Abdulkadyrov KM, Schots R, Jiang B, Mateos MV, Anderson KC, Esseltine DL, Liu K, Cakana A, van de Velde H, Richardson PG. VISTA Trial Investigators. Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma. N Engl J Med 2008;359:906-917. 7. Segeren CM, Sonneveld P, van der Holt B, Baars JW, Biesma DH, Cornellissen JJ, Croockewit AJ, Dekker AW, Fibbe WE, Löwenberg B, van Marwijk Kooy M, van Oers MH, Richel DJ, Schouten HC, Vellenga E, Verhoef GE, Wijermans PW, Wittebol S, Lokhorst HM. Vincristine, doxorubicin and dexamethasone (VAD) administered as rapid intravenous infusion for firstline treatment in untreated multiple myeloma. Br J Haematol 1999;105:127-130. 8. Rajkumar SV, Hayman S, Gertz MA, Dispenzieri A, Lacy MQ, Greipp PR, Geyer S, Iturria N, Fonseca R, Lust JA, Kyle RA, Witzig TE. Combination therapy with thalidomide plus dexamethasone for newly diagnosed myeloma. J Clin Oncol 2002;20:4319-4323.

Çetin G, et al: Multiple Myeloma and Bone-Specific Alkaline Phosphatase

9. Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D, Rajkumar SV, Srkalovic G, Alsina M, Alexanian R, Siegel D, Orlowski RZ, Kuter D, Limentani SA, Lee S, Hideshima T, Esseltine DL, Kauffman M, Adams J, Schenkein DP, Anderson KC. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 2003;348:2609-2617. 10. Durie BG, Harousseau JL, Miguel JS, Bladé J, Barlogie B, Anderson K, Gertz M, Dimopoulos M, Westin J, Sonneveld P, Ludwig H, Gahrton G, Beksac M, Crowley J, Belch A, Boccadaro M, Cavo M, Turesson I, Joshua D, Vesole D, Kyle R, Alexanian R, Tricot G, Attal M, Merlini G, Powles R, Richardson P, Shimizu K, Tosi P, Morgan G, Rajkumar SV. International Myeloma Working Group. International uniform response criteria for multiple myeloma. Leukemia 2006;20:1467-1473. 11. Berenson JR. Myeloma bone disease. Best Pract Res Clin Haematol 2005;18:653-672. 12. Terpos E, Dimopoulos MA. Myeloma bone disease: pathophysiology and management. Ann Oncol 2005;16:12231231. 13. Jantunen E. Bisphosphonate therapy in multiple myeloma: past, present, future. Eur J Haematol 2002;69:257-264. 14. Taube T, Beneton MN, McCloskey EV, Rogers S, Greaves M, Kanis JA. Abnormal bone remodelling in patients with myelomatosis and normal biochemical indices of bone resorption. Eur J Haematol 1992;49:192-198. 15. Bataille R, Chappard D, Baste M. Excessive bone resorption in human plasmacytomas: direct induction by tumour cells in vivo. Br J Haematol 1995;90:721-724. 16. Alexanian R, Bonnet J, Gehan E, Haut A, Hewlett J, Lane M, Monto R, Wilson H. Combination chemotherapy for multiple myeloma. Cancer 1972;20:382-389. 17. Alexanian R, Yap BS, Bodey GP. Prednisone pulse therapy for refractory myeloma. Blood 1983;62:572-577. 18. Alexanian R, Dimopoulos MA, Delasalle K, Barlogie B. Primary dexamethasone treatment of multiple myeloma. Blood 1992;80:887-890. 19. Alexanian R, Barlogie B, Tucker S. VAD-based regimens as primary treatment for multiple myeloma. Am J Hematol 1990;33:86-89. 20. Dimopoulos MA, Zervas K, Kouvatseas G, Galani E, Grigoraki V, Kiamouris C, Vervessou E, Samantas E, Papadimitriou C, Economou O, Gika D, Panayiotidis P, Christakis I, Anagnostopoulos N. Thalidomide and dexamethasone combination for refractory multiple myeloma. Ann Oncol 2001;12:991-995. 21. Terpos E, Palermos J, Tsionos K, Anargyrou K, Viniou N, Papassavas P, Meletis J, Yataganas X. Effect of pamidronate administration on markers of bone turnover and disease activity in multiple myeloma. Eur J Haematol 2000;65:331336.

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22. Diamond T, Levy S, Day P, Barbagallo S, Manoharan A, Kwan YK. Biochemical, histomorphometric and densitometric changes in patients with multiple myeloma: effects of glucocorticoid therapy and disease activity. Br J Haematol 1997;97:641-648. 23. Corso A, Arcaini L, Mangiacavalli S, Astori C, Orlandi E, Lorenzi A, Passamonti F, Klersy C, Pascutto C, CanevariSciorati A, Lazzarino M. Biochemical markers of bone disease in asymptomatic early stage multiple myeloma. A study on their role in identifying high risk patients. Haematologica 2001;86:394-398. 24. Canterbury District Health Board. Endolab Quality Manual REC-26. Christchurch, New Zealand, Canterbury District Health Board, 2007. 25. Abildgaard N, Glerup H, Rungby J, Bendix-Hansen K, Kassem M, Brixen K, Heickendorff L, Nielsen JL, Eriksen EF. Biochemical markers of bone metabolism reflect osteoclastic and osteoblastic activity in multiple myeloma. Eur J Haematol 2000;64:121-129. 26. Zangari M, Yaccoby S, Cavallo F, Esseltine D, Tricot G. Response to bortezomib and activation of osteoblasts in multiple myeloma. Clin Lymphoma Myeloma 2006;7:109-114. 27. Roodman GD. Pathogenesis of myeloma bone disease. Blood Cells Mol Dis 2004;32:290-292. 28. Abroun S, Ishikawa H, Tsuyama N, Liu S, Li FJ, Otsuyama K, Zheng X, Obata M, Kawano MM. Receptor synergy of interleukin-6 (IL-6) and insulin-like growth factor-I that highly express IL-6 receptor α myeloma cells. Blood 2004;103:2291-2298. 29. Kyle RA. Multiple myeloma: review of 869 cases. Mayo Clin Proc 1975;50:29-40. 30. Tassone P, Forciniti S, Galea E, Morrone G, Turco MC, Martinelli V, Tagliaferri P, Venuta S. Growth inhibition and synergistic induction of apoptosis by zoledronate and dexamethasone in human myeloma cells. Leukemia 2000;14:841-844. 31. Ochiai N, Yamada N, Uchida R, Fuchida S, Okano A, Okamoto M, Ashihara E, Inaba T, Shimazaki C. Nitrogen-containing bisphosphonate incadronate augments the inhibitory effect of farnesyl transferase inhibitor tipifarnib on the growth of fresh and cloned myeloma cells in vitro. Leuk Lymphoma 2005;46:1619-1625. 32. Ochiai N, Yamada N, Uchida R, Fuchida S, Okano A, Hatsuse M, Okamoto M, Ashihara E, Shimazaki C. Combination therapy with thalidomide, incadronate and dexamethasone for relapsed or refractory multiple myeloma. Int J Hematol 2005;82:243-247. 33. Bladé J, Samson D, Reece D, Apperley J, Björkstrand B, Gahrton G, Gertz M, Giralt S, Jagannath S, Vesole D. Criteria for evaluating disease response and progression in patients

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with multiple myeloma treated by high-dose therapy and haemopoietic stem cell transplantation. Myeloma Subcommittee of the EBMT. European Group for Blood and Marrow Transplant. Br J Haematol 1998;102:1115-1123. 34. Garrett IR, Chen D, Gutierrez G, Zhao M, Escobedo A, Rossini G, Harris SE, Gallwitz W, Kim KB, Hu S, Crews CM, Mundy GR. Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro. J Clin Invest 2003;111:1771-1782. 35. Hideshima T, Richardson P, Chauhan D, Palombella VJ, Elliott PJ, Adams J, Anderson KC. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis and over comes drug resistance in human multiple myeloma cells. Cancer Res 2001;61:3071-3076. 36. Hideshima T, Chauhan D, Richardson P, Mitsiades C, Mitsiades N, Hayashi T, Munshi N, Dang L, Castro A, Palombella V, Adams J, Anderson KC. NF-kappa B as a therapeutic target in multiple myeloma. J Biol Chem 2002:277:16639-16647. 37. Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Richardson PG, Hideshima T, Munshi N, Treon SP, Anderson KC. Biologic sequelae of nuclear factor-kappa B blockade in multiple myeloma: therapeutic applications. Blood 2002;99:40794086. 38. Sezer O, Heider U, Zavrski I, Kühne CA, Hofbauer LC. RANK ligand and osteoprotegerin in myeloma bone disease. Blood 2003;101:2094-2098. 39. Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, Facon T, Harousseau JL, Ben-Yehuda D, Lonial S, Goldschmidt H, Reece D, San-Miguel JF, Bladé J, Boccadoro M, Cavenagh J, Dalton WS, Boral AL, Esseltine DL, Porter JB, Schenkein D, Anderson KC. Assessment of Proteasome Inhibition for Extending Remissions (APEX) Investigators. Assessment of proteasome inhibition for extending remissions (APEX) investigators. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med 2005;352:2487-2498.

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40. Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin D, Rajkumar SV, Hideshima T, Xiao H, Esseltine D, Schenkein D, Anderson KC; SUMMIT Investigators. Clinical factors predictive of outcome with bortezomib in patients with relapsed, refractory multiple myeloma. Blood 2005;1106:2977-2981. 41. Shimazaki C, Uchida R, Nakano S, Namura K, Fuchida SI, Okano A, Okamoto M, Inaba T. High serum bone-specific alkaline phosphotase level after bortezomib-combined therapy in refractory multiple myeloma: possible role of bortezomib on osteoblast differentiation. Leukemia 2005;19:1102-1103. 42. Zangari M, Esseltine D, Lee CK, Barlogie B, Elice F, Burns MJ, Kang SH, Yaccoby S, Najarian K, Richardson P, Sonneveld P, Tricot G. Response to bortezomib is associated to osteoblastic activation in patients with multiple myeloma. Br J Haematol 2005;131:71-73. 43. Terpos E. Biochemical markers of bone metabolism in multiple myeloma. Cancer Treat Rev 2006;32(Suppl 1):15-19.

Research Article

DOI: 10.4274/tjh.2013.0328

The Relationship of T Helper-2 Pathway Components Interleukin-4, Interleukin-10, Immunoglobulin E, and Eosinophils with Prognostic Markers in Non-Hodgkin Lymphoma: A Case-Control Study Yardımcı T Lenfosit-2 Yolağı Bileşenlerinden İnterlökin-4, İnterlökin-10, İmmünglobülin E ve Eozinofiller’in Hodgkin-Dışı Lenfoma’nın Prognostik Belirteçleri ile Olan İlişkisi-Kontrollü Klinik Çalışma Nil Güler1, Engin Kelkitli1, Hilmi Atay1, Dilek Erdem2, Hasan Alaçam3, Yüksel Bek4, Düzgün Özatlı1, Mehmet Turgut1, Levent Yıldız5, İdris Yücel2 119

Mayıs University Faculty of Medicine, Department of Hematology, Samsun, Turkey Mayıs University Faculty of Medicine, Department of Oncology, Samsun, Turkey 319 Mayıs University Faculty of Medicine, Department of Biochemistry, Samsun, Turkey 419 Mayıs University Faculty of Medicine, Department of Biostatistics, Samsun, Turkey 519 Mayıs University Faculty of Medicine, Department of Pathology, Samsun, Turkey 219

Abstract: Objective: Increased risk for non-Hodgkin lymphoma (NHL) is associated with infections and environmental agents. We hypothesized that these factors chronically trigger the T helper-2 (Th2) pathway and result in lymphoma. We investigated the role of the Th2 pathway by exploring the relationships between components of the Th2 pathway, interleukin (IL)-10, IL-4, immunoglobulin E (IgE), and eosinophils, and prognostic markers of NHL.

Materials and Methods: Thirty-one NHL patients and 27 healthy controls were enrolled. IL-10, IL-4, IgE, and eosinophils were measured. IL-4 and IL-10 were analyzed with the enzyme amplified sensitivity immunoassay method.

Results: High IL-10 levels were correlated with several poor prognostic features, short early survival, and lymphopenia. There was a positive correlation between albumin and IL-4 levels and a negative correlation between IL-10 and albumin. There was no relationship related with eosinophils and IgE. We found remnant increased IL-4, which could be a clue for the triggering of the Th2 pathway in the background.

Conclusion: There is a need for differently designed studies to detect the place of the Th2 pathway in NHL. Key Words: Chemokines, Cytokines, Lymphocytes, Non-Hodgkin lymphoma, Th2 pathway Address for Correspondence: Nil GÜLER, M.D., 19 Mayıs University Faculty of Medicine, Department of Hematology, Samsun, Turkey Phone: +362 312 19 19- 3521 E-mail: nilvecay@yahoo.com Received/Geliş tarihi : September 25, 2013 Accepted/Kabul tarihi : November 18, 2013

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Güler N, et al: The Place of T Helper-2 Pathway in NHL

Özet: Amaç: Non-Hodgkin lenfoma (NHL) riski enfeksiyonlar ve çevresel ajanlarla artmış olarak ilişkilendirilmiştir. Hipotezimize göre bu faktörlere kronik olarak maruz kalmak T helper-2 (Th2) yolağını aktifler ve lenfomaya sebep olur. Bu amaçla Th2 yolağı komponentleri olan IL-10, IL-4, IgE, ve eozinofille, NHL’nin prognostik belirteçleri arasındaki ilişkiye baktık. Gereç ve Yöntemler: Otuz bir NHL hastası ve 27 sağlıklı kontrolde IL-10, IL-4, IgE ve eozinofil değerlerine bakıldı. IL-4 ve IL-10 EASIA metodu ile ölçüldü. Bulgular: Yüksek IL-10 düzeyleri pek çok kötü prognostik özellikle, çok kısa yaşam süresi ve lenfopeni ile ilişkiliydi. Albümin açısından IL-10 ile negatif ilişki, IL-4 ile pozitif ilişki bulundu. Eozinofil ve IgE ile ilgili herhangi bir ilişki kurulamadı. Bazı hastalarda gözlenen artmış IL-4 geri planda kalmış Th2 yolağı aktivasyonunun bir ip ucu olabilir. Sonuç: Th2 yolağının NHL patogenezindeki yerini tespit etmek için farklı dizaynlarda çalışmalara ihtiyaç vardır.

Anahtar Sözcükler: Kemokinler, Sitokinler, Lenfositler, Hodgkin dışı lenfoma, Th2 yolağı Introduction Increased risk for Non-Hodgkin lymphoma (NHL) is associated with infections, environmental agents, and immune suppression. The T helper-2 (Th2) immune reaction is typically characterized by expression of interleukin (IL)-4, IL-5, IL-9, IL-10, and IL-13; the recruitment of eosinophils, basophils, and mast cells; and immunoglobulin (Ig) G-to-IgE antibody class switching [1]. Exposure to an antigen may cause an allergic reaction, but chronic exposure to the same allergen at a low dose can cause immune tolerance via T regulatory (T-reg) cell-associated Th2 suppression [2]. Anergy is defined as the inability of antigen-specific cells to generate an allergic reaction to an antigen [3]. IL-10 has a role in anergy as well as in immune suppression, whereas IL-4 plays a predominant role in allergy [3]. IL-10 is produced mainly by Th2 cells, T-reg cells, monocytes, and B lymphocytes and, in small amounts, by Th1 cells. IL-10 inhibits the proliferation of Th1 and Th2 cells in response to specific antigens. IL-10 also inhibits the production of gammainterferon (IFN-γ) and IL-2 by Th1 cells; the production of IL-4 and IL-5 by Th2 cells; the production of IL-6, IL-8, IL12, TNF-α, and IL-1β by mononuclear phagocytes; and the production of TNF-α and IFN-γ by natural killer cells. It further inhibits monocytes [3,4,5]. Therefore, IL-10 is known as a strongly inhibitory cytokine. IL-10 causes differentiation of T-reg cells from T helper cells. T-reg cells secrete IL-10, IFN-γ, TGF-β, and IL-5 [3]. T-reg cells also suppress functions of Th1 and Th2 cells [6]. Secretion of IL-4 by the Th2 pathway induces allergy. Desensitization treatments can be done if an allergic patient is chronically treated with a related allergen at a low dose. Allergic individuals develop anergy via IL-10. We hypothesized that chronic stimulation by environmental agents triggers the Th2 pathway and anergy. The Th2 pathway is shifted from IL-4 to IL-10 in anergy. It is well known that high IL-10 triggers the differentiation of T-reg cells from T

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helper cells. Once T-reg cells are increased, these cells will start to inhibit functions of Th2 and Th1 cells and start to secrete their own IL-10. In addition, if high levels of IL-10 persist, it causes serious immune suppression. Therefore, we examined the relationships between prognostic markers in NHL and components of the Th2 pathway, such as IL-10, IL4, IgE, and eosinophils. Figure 1 illustrates our hypothesis. Materials and Methods Thirty-one newly diagnosed NHL patients and 27 healthy controls were enrolled. The median ages of patients and controls were 64 (range: 19-85) and 26 (range: 23-60) years, respectively. All participants were free of history of allergy, dermatitis, or parasites in stool samples, because allergies and parasites can trigger the Th2 pathway and could cause misleading results. Twenty-seven patients had diffuse large B cell lymphoma (DLBCL) and 4 patients had T-cell lymphoma. The characteristics of the patients are given in Table 1. Initial blood samples were collected to test IL-4, IL-10, and IgE levels. Patient pretreatment values for eosinophils, hemoglobin, lymphocytes, lactate dehydrogenase (LDH), C-reactive protein (CRP), sedimentation, b2 microglobulin, and albumin were collected from the patient records. A second blood sample for IL-4 and IL-10 was collected from only the patients after completion of the 4 cycles of chemotherapy. The chemotherapy regimens were decided by their physicians. The blood samples for IL-10 and IL-4 were collected into tubes with no anticoagulant. The samples were centrifuged within 2 h at 4000 rpm for 10 min, and the serum was separated and kept at -80 °C. The IL-4 and IL-10 concentrations were analyzed from serum samples using the enzyme amplified sensitivity immunoassay (EASIA) method in accordance with the instructions of the kit’s manufacturer (DIAsource ImmunoAssays, Cat. No. KAP1281 and KAP1321, Belgium).

Güler N, et al: The Place of T Helper-2 Pathway in NHL

These IL-4 and IL-10 assays are specific for endogenous human IL-4 and IL-10, respectively. The samples were processed in duplicate. The detection limits of the kits for IL-4 and IL-10 are 1.2 pg/mL and 1.6 pg/mL, respectively. The intraassay and interassay variation of the IL-4 kit was 3.8% and 4.5%, respectively. The intraassay and interassay variation of the IL-10 kit was 2.8% and 2.8%, respectively. The results are presented in pg/mL. The Eastern Cooperative Oncology Group (ECOG) performance status system and the Ann Arbor staging system were used. International Prognostic Index (IPI) scores were categorized as low risk (scores 0-2) or high risk (scores 3-5). T-cell lymphoma patients were treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy. DLBCL patients were treated with rituximab plus CHOP. Overall survival (OS) was determined as the time between the diagnosis of lymphoma and the last evaluation in the hospital or death for any reason during the study. The university’s local ethics committee approved this study and all participants gave informed consent. This study is in accordance with the Helsinki Declaration of 1975. Statistical Analysis Statistical analyses were performed using SPSS 15.0. The normality of distribution was checked by KolmogorovSmirnov test. For variables not confirming to normal distribution comparisons were analyzed with the MannWhitney U-test. The Wilcoxon signed-rank test, KruskalWallis test, Spearman test, Pearson chi-square test, and Fisher exact test were also used. Statistical significance was accepted at p<0.05. The Kaplan-Meier test and log rank analysis were performed for survival analysis. Results IL-4 values were a median of 19.41 (min: 18.67, max: 43.05) and a mean of 20.72±4.8 in the control group. They were a median of 19.41 (min: 19.04, max: 2081.7) and a mean of 96.27±369.65 in the patient group. IL-10 values were a median of 7.1 (min: 6.6, max: 8.24) and a mean of 7.2±0.5 in the control group. They were a median of 8.2 (min: 6.1, max: 3155.4) and a mean of 163.93±574.5 in the patient group. The maximum values of IL-10 and IL-4 in the control group were used as cut-off values for determining increased IL-10 and IL-4 levels. Thus, the cut-off values were 8.24 pg/ mL and 43.05 pg/mL for IL-10 and IL-4, respectively. As mentioned above, the medians of IL-10 and IL-4 in the control group were 7.1 pg/mL and 19.4 pg/mL, respectively. Fifteen of the 31 lymphoma patients (48%) exhibited increased IL10 and 5 patients (16%) exhibited increased IL-4 levels. Four patients exhibited elevations of both IL-4 and IL-10.

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There was a positive correlation between IL-4 levels and albumin (r=0.44, p=0.013), whereas a negative correlation was observed between IL-10 and albumin (r=-0.46, p=0.01) using Spearman’s correlation test. IL-10 was positively correlated with CRP (r=0.58, p=0.001) and LDH (r=0.764, p<0.001). The pretreatment IL-10 values were significantly correlated with IPI [low and high: median of 6.65 (range: 6.12-28) and 29.13 (range: 6.65-3155), respectively; p=0.001], with lymphoid tissue size [≤5 and >5 cm: median of 7.18 (range: 6.12-687) and 43.06 (range: 6.65-3155), respectively; p=0.016], with ECOG performance [<2 and ≥2: median of 7.18 (range: 6.12-28) and 61.01 (range: 6.65-3155), respectively; p=0.007), and with stage (early and advanced; p=0.003). Because of these correlations, we hypothesized that high IL10 levels negatively affected prognosis, given their relationship with high LDH, high CRP, high IPI score, lymphoid tissue size of greater than 5 cm, poor ECOG performance, advanced stage, and albumin. By contrast, we hypothesized that high IL-4 levels positively affected prognosis because of their positive relationship with albumin in our study. To test this hypothesis, we classified the patients according to IL-10 and IL-4 values. We thought if IL-10 is a poor prognostic marker and IL-4 is a good prognostic marker, the group with the best prognostic profile for interleukins would have low IL-10 and high IL-4, and the group with the worst prognostik profile for interleukins would have high IL-10 and low IL-4. A group with relatively good prognostic profile for interleukins would have low IL-10 and low IL-4; a group with relatively poor prognostic profile for interleukins would have high IL-10 and high IL-4. Groups were established as follows: group 1, IL-10 of ≤8.24 and IL-4 of >43.05 (1 patient); group 2, IL-10 of ≤8.24 and IL-4 of ≤43.05 (15 patients); group 3, IL-10 of >8.24 and IL-4 of >43.05 (4 patients); group 4, IL-10 of >8.24 and IL-4 of ≤43.05 (11 patients). Because it only included a single patient, group 1 was not evaluated. We did not observe a significant difference between groups 2 and 3 with respect to albumin (p=0.548), while there was a difference between groups 2 and 4 (p=0.005) and between groups 3 and 4 (p=0.026). The significant difference between groups 3 and 4 was noteworthy because both of these groups consisted of patients with high IL-10. However, IL-4 was higher in group 3 and lower in group 4. The highest albumin values were observed in group 3. As mentioned above, there was a negative correlation between IL10 levels and albumin in the entire patient sample (r=-0.46, p=0.01). Despite the high IL-10 level, albumin levels were high in group 3. The lack of a difference between group 2 and group 3 was also important. IL-10 levels were low in group 2. The albumin values in group 3 did not differ from those of group 2 despite

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high IL-10 levels. These results suggest that IL-4 alleviates or restores the repressive effects of IL-10 on albumin (Figure 2). Seven of 15 patients with high IL-10 (46%) and 4 of 16 patients with low IL-10 (25%) died during the follow-up for a total number of 11 deceased patients. Ten of these 11 patients died before the completion of the fourth cycle of chemotherapy. Our mean observation period was 281.44 days (range: 19402). The minimum observation time in living patients was 112 days. Therefore, we relied on short-time surveying. The highest IL-10 value in living patients was 107.1 pg/ mL, 13-fold higher than the cut-off value. This value was found in patient number 21. This patient also exhibited a very high IL-4 value of 2081 pg/mL, 48-fold higher than the cut-off value.

Güler N, et al: The Place of T Helper-2 Pathway in NHL

Table 1. The characteristics of the patients.

Characteristics

Variables

Lymphoma type

DLBCL

T-cell lymphoma

≤60

>60

≥2

Normal

High

1-2

3-4

Low risk (score: 0-2)

High risk (score: 3-5)

Normal

High

Normal

Low

Normal

Low

Normal

High

Normal

High

Absent

Present

Normal

High

<5 cm

≥5 cm

Age ECOG

LDH Stage

IPI

CRP

Albumin

Figure 1. Presentation of the hypothesis. Dashed arrows indicate suppressive effect.

Hemoglobin Eosinophils IgE B symptoms

β2 microglobulin

Lymphoid tissue

Figure 2. Mean albumin values (g/dL) after removal of group 1 (with 1 patient only) from analysis. 384

NA: not available.

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For survival analysis, the patients were classified into 3 groups according to the cut-off value (8.24 pg/mL) and the highest value (107 pg/mL) in living patients: Group A, IL-10 of ≤8.24 (16 patients); group B, 8.24 < IL-10 ≤107 (11 patients); and group C, IL-10 of >107 (4 patients). The Kaplan-Meier test and log rank analysis were performed for the survival analysis.

and 4 (25%) of 16 patients with low IL-10 had lymphocyte counts of ≤1x109/L. We observed a difference between the participants with low IL-10 (≤8.24), consisting of 27 controls and 16 patients (43 individuals total), and the participants with high IL-10 (>8.24; 15 patients). Lymphocyte counts were decreased in the latter group (p=0.002).

The mean survival times were 323 days in group A, 285 days in group B, and 38.7 days in group C. The differences were statistically significant (p=0<0.001). Severe early survival differences were noted in group C (Figure 3). Groups A, B, and C contained 15 and 1, 9 and 2, and 3 and 1 DLBCL and T-cell lymphoma patients, respectively.

Differences in the relationships between IL-4 and age, CRP, hemoglobin, extranodal involvement, ECOG performance, stage, lymphopenia, LDH, lymphoid tissue size, IPI score, ≥2 microglobulin, or B symptoms were not observed.

IgE levels were high in 8 patients. There was no relationship between increased IgE levels and age, IPI score, LDH, ECOG performance, stage, IL-10, IL-4, albumin, or lymphocyte count. There were only 2 patients with high eosinophil count. The pretreatment lymphocyte count was ≤1x109/L in 11 (35.5%) patients. Seven (47%) of 15 patients with high IL-10

The characteristics of patients with T-cell lymphoma are shown in Table 2. Discussion In this study, high IL-10 levels were correlated with several poor prognostic features, including low albumin. However, IL-4 was positively correlated with albumin. In addition, IL-4 was able to overcome the negative effects of IL-10 on albumin. To our knowledge, this is the first study to detect a positive relationship between IL-4 and albumin and the ability of IL-4 to overcome the effects of IL-10 on albumin in NHL patients. IL-4 is an agent used in experimental treatment for NHL. The rationalization for this treatment is based on the observation of the inhibitory effect of IL-4 on NHL B cells and cancer cells in vitro [7,8]. In the literature, IL-10 is generally connected to the poor prognostic factors of NHL. One of the most noteworthy relevant studies was performed by Blay et al. They observed detectable IL-10 levels in 46% of patients with active disease. Detectable IL-10 levels were related to very short survival [9]. However, Cortes et al. could not observe a relationship with complete remission, failure-free survival, or OS. There was also no relationship between IL-10 and any prognostic factors except B symptoms [10].

Figure 3. Survival analysis as days related to IL-10 levels.

Lech-Maranda et al. observed a relationship between detectable IL-10 levels and age of >60, ECOG status of ≥2,

Table 2. The characteristic of patients with T-cell lymphoma.

Patient Type number

IL-10, pg/mL

IL-4, pg/mL

Lymphocytes, IgE x109/L

Eosinophil Exitus Follow-up Albumin count, (days) x109/L

PTCL

45.76 ↑

69.56 ↑

0.89↓

239↑

PTCL

687.2 ↑↑ 19.04 N

0.2↓

↓

PTCL

6.65 N

20.91 N

2.06

AITL

43.06↑

19.418 N

2.00

164↑

8.0 ↑↑

275

↓

PTCL: peripheral T-cell lymphoma, AITL: angioimmunoblastic T-cell lymphoma, ↓: low, N: normal, ↑: high, ↑↑: very high. The cut-off values used were 8.24 pg/mL and 43.05 pg/mL for IL-10 and IL-4, respectively.

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advanced stage, bulky tumor mass, high LDH, high IPI score, ≥2 microglobulin, anemia, existence of B symptoms, low albumin, low CR rate, and shorter progression-free survival and OS [11]. Nacinovic-Duletic et al. reported similar results. The patients with high IL-10 exhibited shorter survival [12]. However, Guney et al. observed only the relationship between IL-10 and high LDH and bone marrow involvement [13]. They detected significant decreases in IL10 levels after chemotherapy. Fabre-Guillevin et al. did not find any relationship between IL-4 or IL-10 and complete remission, failure-free survival, or OS [14]. As mentioned before, Lech-Maranda et al. observed a negative correlation between albumin and IL-10 [11]. Our finding was consistent with this result; in addition, we observed a positive effect of IL-4 on albumin. Lymphopenia is related to poor prognosis in many cancers [15,16]. In our study, the pretreatment lymphocyte count was negatively correlated with IL-10. To our knowledge, this is the first study in which lymphopenia was found to be related to high IL-10 levels in NHL. Some researchers have observed this relationship in sepsis [17,18]. We cannot define the source of high IL-10 levels in our study; it could be Th2, T-reg, or tumor cells. According to our hypothesis, NHL pathogenesis starts with Th2 pathway activation. We know that the increased secretion of IL-10 by Th2 cells can promote the development of T-reg cells from T helper cells, and newly developed T-reg cells begin to secrete their own IL-10 and inhibit both Th1 and Th2 functions [3,6]. As mentioned above, IL-10 is a strong inhibitor cytokine. In situations of very highly increased IL-10 levels, the inhibitor effect of IL-10 will be particularly enormous and will result in strong inhibition of Th1 and Th2 cell function. With the suppressive effect of IL-10, Th2 cannot produce IL-4. Our interpretation of the increase in IL-4 that was observed in some patients (5 patients with increased pretreatment IL-4 and 3 with increased posttreatment IL-4 values) is that there may be remnant Th2 pathway activation, which could have escaped the suppressive effect of IL-10. It is hard to show the initial activity of Th2 cells at the time of disease development because we are most likely catching patients after the switching of Th2 cell activity to increased T-reg cell activity and at a point when the disease is well established. To our knowledge, this is the first study to investigate the place of the Th2 pathway in NHL through the components of Th2 by detection of IgE, eosinophils, IL-10, and IL-4. IL-10 was related to very early death in this study. Therefore, we think that other treatment options may be more effective for patients with very high IL-10, such as antiIL-10 antibody in addition to CHOP. However, one could also make a different comment: if an increasing IL-10 level is accompanying NHL, the increased IL-10 may be a reaction of the immune system, and supporting this reaction may help to control the aggression of lymphoma. 386

Güler N, et al: The Place of T Helper-2 Pathway in NHL

Acknowledgment Special thanks to Timuçin Güler for interpretation of our results. 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. Authorship Contributions: Nil Güler: Proposed the hypothesis, designed the research, collected data, performed research, analyzed and interpreted data, wrote the manuscript. Engin Kelkitli: Collected data, performed research. Hilmi Atay: Collected data, performed research. Dilek Erdem: Collected data, performed research. Hasan Alaçam: Performed research, analyzed and interpreted data. Yüksel Bek: Performed statistical analysis, analyzed and interpreted data. Düzgün Özatlı: Collected data, performed research. Mehmet Turgut: Collected data, performed research. Levent Yıldız: Performed research. İdris Yücel: Collected data, analyzed and interpreted data.

References 1. Oliphant CJ, Barlow JL, McKenzie AN. Insights into the initiation of type 2 immune responses. Immunology 2011;134:378-385. 2. Chapoval S, Dasgupta P, Dorsey NJ, Keegan AD. Regulation of the T helper cell type 2 (Th2)/T regulatory cell (Treg) balance by IL-4 and STAT6. J Leukoc Biol 2010;87:10111018. 3. Roncarolo MG, Battaglia M, Gregori S. The role of interleukin 10 in the control of autoimmunity. J Autoimmun 2003;20:269-272. 4. Commins S, Steinke JW, Borish L. The extended IL-10 superfamily: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29. J Allergy Clin Immunol 2008;121:1108-1111. 5. Del Prete G, De Carli M, Almerigogna F, Giudizi MG, Biagiotti R, Romagnani S. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J Immunol 1993;150:353-360. 6. Askenasy N, Kaminitz A, Yarkoni S. Mechanisms of T regulatory cell function. Autoimmun Rev 2008;7:370-375. 7. Chang TL, Peng X, Fu XY. Interleukin-4 mediates cell growth inhibition through activation of Stat-1. J Biol Chem 2000;275:10212-10217. 8. Defrance T, Fluckiger AC, Rossi JF, Magaud JP, Sotto JJ, Banchereau J. Antiproliferative effects of interleukin-4 on freshly isolated non-Hodgkin malignant B-lymphoma cells. Blood 1992;79:990-996.

Güler N, et al: The Place of T Helper-2 Pathway in NHL

9. Blay JY, Burdin N, Rousset F, Lenoir G, Biron P, Philip T, Banchereau J, Favrot MC. Serum interleukin-10 in non-Hodgkin’s lymphoma: a prognostic factor. Blood 1993;82:2169-2174. 10. Cortes JE, Talpaz M, Cabanillas F, Seymour JF, Kurzrock R. Serum levels of interleukin-10 in patients with diffuse large cell lymphoma: lack of correlation with prognosis. Blood 1995;85:2516-2520. 11. Lech-Maranda E, Bienvenu J, Michallet AS, Houot R, Robak T, Coiffier B, Salles G. Elevated IL-10 plasma levels correlate with poor prognosis in diffuse large B-cell lymphoma. Eur Cytokine Netw 2006;17:60-66. 12. Nacinovic-Duletic A, Stifter S, Dvornik S, Skunca Z, Jonjic N. Correlation of serum IL-6, IL-8 and IL-10 levels with clinicopathological features and prognosis in patients with diffuse large B-cell lymphoma. Int J Lab Hematol 2008;30:230-239. 13. Guney N, Soydinc HO, Basaran M, Bavbek S, Derin D, Camlica H, Yasasever V, Topuz E. Serum levels of interleukin-6 and interleukin-10 in Turkish patients with aggressive non-Hodgkin’s lymphoma. Asian Pac J Cancer Prev 2009;10:669-674. 14. Fabre-Guillevin E, Tabrizi R, Coulon V, Monnereau A, Eghbali H, Soubeyran I, Soubeyran P. Aggressive non-Hodgkin’s lymphoma: concomitant evaluation of interleukin-2, soluble interleukin-2 receptor, interleukin 4, interleukin-6, interleukin-10 and correlation with outcome. Leuk Lymphoma 2006;47:603-611.

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15. Kim YR, Kim JS, Kim SJ, Jung HA, Kim SJ, Kim WS, Lee HW, Eom HS, Jeong SH, Park JS, Cheong JW, Min YH. Lymphopenia is an important prognostic factor in peripheral T-cell lymphoma (NOS) treated with anthracyclinecontaining chemotherapy. J Hematol Oncol 2011;4:34. 16. Ray-Coquard I, Cropet C, Van Glabbeke M, Sebban C, Le Cesne A, Judson I, Tredan O, Verweij J, Biron P, Labidi I, Guastalla JP, Bachelot T, Perol D, Chabaud S, Hogendoorn PC, Cassier P, Dufresne A, Blay JY; European Organization for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group. Lymphopenia as a prognostic factor for overall survival in advanced carcinomas, sarcomas, and lymphomas. Cancer Res 2009;69:5383-5391. 17. Roth G, Moser B, Krenn C, Brunner M, Haisjackl M, Almer G, Gerlitz S, Wolner E, Boltz-Nitulescu G, Ankersmit HJ. Susceptibility to programmed cell death in T-lymphocytes from septic patients: a mechanism for lymphopenia and Th2 predominance. Biochem Biophys Res Commun 2003;308:840-846. 18. Le Tulzo Y, Pangault C, Gacouin A, Guilloux V, Tribut O, Amiot L, Tattevin P, Thomas R, Fauchet R, Drénou B. Early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome. Shock 2002;18:487-494.

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Research Article

DOI: 10.4274/tjh.2013.0098

The Association of HLA Class 1 and Class 2 Antigens with Multiple Myeloma in Iranian Patients İranlı Multipl Miyelom Hastalarında HLA Sınıf 1 ve Sınıf 2 Antijenlerinin Birlikteliği Arezou Sayad1, Mohammad Taghi Akbari2, Mahshid Mehdizadeh3,4, Elham Roshandel3, Soheila Abedinpour3, Abbas Hajifathali3 1Shahid

Beheshti University of Medical Sciences, Department of Medical Genetics, Tehran, Iran Modares University Faculty of Medical Science, Department of Medical Genetics, Tehran, Iran 3Shahid Beheshti University of Medical Sciences, Taleghani Bone Marrow Transplantation Center, Tehran, Iran 4Shahid Beheshti University of Medical Sciences, Pediatric Congenital Hematologic Disorders Research Center, Tehran, Iran 2Tarbiat

Abstract: Objective: Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of malignant plasma cells. According to results of some studies, it has been suggested that the HLA class 1 and 2 genes have susceptibility effects on MM. Studies of different populations have reported different HLA class 1 and 2 alleles that affect MM. In this study, we assessed the association of HLA class 1 and class 2 antigens with MM in Iranian patients.

Materials and Methods: We performed a case-control genotyping study with 105 Iranian MM patients that were selected from the bone marrow transplantation department of Taleghani Hospital and 150 controls using single specific primerpolymerase chain reaction with the HLA-Ready Gene ABDR Kit. Results: Our results demonstrated that 21% of patients versus 12% of controls and 11% of patients versus 3% of controls carried HLA-A*03 and HLA-B*18, respectively. The MM patients had a significant increase in the frequency of HLA-A*03 and HLA-B*18 alleles in comparison to control subjects (p=0.039, OR=2.057 and p=0.013, OR=3.567, respectively).

Conclusion: Our findings suggested that the HLA-A*03 and HLA-B*18 alleles have significant susceptibility effects on MM in the Iranian population. However, compared to other populations, the above-mentioned alleles had different statuses. Since there are not many studies evaluating and calculating this association among ethnic groups, further studies among other populations are needed to explain the exact association of the HLA genes with MM.

Key Words: Multiple myeloma, HLA-A, HLA-B, HLA-DRB1, Genetic susceptibility Özet: Amaç: Multiple myeloma (MM), malign plazma hürelerinin klonal çoğalması ile karakterize bir B hücre neoplazisidir. Çeşitli çalışmaların sonuçlarına göre, bazı sınıf 1 ve 2 HLA genlerinin hastalığa yatkınlık sağladığına dair görüşler ortaya atılmıştır.

Address for Correspondence: Abbas HAJIFATHALI, M.D., Shahid Beheshti University of Medical Sciences, Taleghani Bone Marrow Transplantation Center, Tehran, Iran E-mail: ar.sayad@yahoo.com Received/Geliş tarihi : March 17, 2013 Accepted/Kabul tarihi : June 10, 2013

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Sayad A, et al: Association of HLA with Multiple Myeloma

Farklı popülasyonlarda yapılan çalışmalarda, farklı HLA sınıf 1 ve 2 allellerinin MM üzerine etkisi olduğu bildirilmiştir. Bu çalışmada, İranlı MM hastalarında HLA sınıf 1 ve sınıf 2 antijenlerinin birlikteliğini değerlendirdik. Gereç ve Yöntemler: HLA-Ready Gene ABDR kitleriyle tekli spesifik primer polimeraz zincir reaksiyonu yönteminin kullanıldığı bu olgu-kontrol genetiplendirme çalışmasında, hasta grubuna Taleghani Hastanesi kemik iliği nakli bölümünden seçilen 105 İranlı MM hastası ve 150 de kontrol olgusu dahil edilmiştir. Bulgular: Çalışma sonucunda, HLA-A*03 hasta grubunda %21 ve kontrol grubunda %12 bulunurken, HLA-B*18 ise hasta grubunda %11 ve kontrol grubunda %3 olarak saptanmıştır. MM hastalarının HLA-A*03 ve HLA-B*18 allele sahip olma oranı kontrol olgularıyla karşılaştırıldığında istatistiki olarak anlamlı olacak şekilde yüksek bulunmuştur (p=0,039, OR=2,057 ve p=0,013, OR=3,567, sırasıyla). Sonuç: Bizim bulgularımız, İran toplumunda HLA-A*03 ve HLA-B*18 allel varlığının istatistiki olarak anlamlı olacak şekilde MM’ye yatkınlık yarattığını ortaya koymaktadır. Bununla birlikte, diğer toplumlara bakıldığında adı geçen allellerin aynı sonucu doğurmadıkları görülmektedir. Farklı etnik gruplar arasındaki bu birlikteliği değerlendiren fazla sayıda çalışma olmadığı için, gelecek dönemlerde MM’li hastalarda HLA genlerinin birlikteliğinin sonuçlarını izah edebilecek daha ayrıntılı çalışmalara gereksinim vardır.

Anahtar Sözcükler: Multipl miyelom, HLA-A, HLA-B, HLA-DRB1, Genetik yatkınlık Introduction Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of malignant plasma cells, and evidence indicates that the bone marrow microenvironments of tumor cells have a crucial role in myeloma pathogenesis [1]. Neurological and impaired hemopoiesis symptoms, bone complications, renal failure, and infection are some of the heterogeneous clinical features [2]. MM is the second most prevalent blood cancer after nonHodgkin lymphoma [3]. MM represents approximately 1% of all cancers, 2% of all cancer deaths, and 10% of hematological malignancies. The prevalence of MM varies among different populations. Blacks have a 2-fold higher incidence than whites, while the Japanese, Chinese, and South Koreans have the lowest incidence [4,5]. Although the exact etiology of MM is unknown, the genetic factor has an important effect on it. Simply, MM is a condition characterized by the unlimited proliferation of plasma cells. Since the HLA genes are associated with a variety of immunologic diseases, they may be involved as a crucial factor in MM [6]. In 1970 for the first time, after recognition of HLA class 1 and prior to identification of HLA class 2, the susceptibility effects of HLA genes on MM were studied [7,8,9,10,11,12]. Not many studies have taken sufficient account of the effect of HLA genes on the susceptibility to MM in different populations. These studies reported different susceptible or protective alleles of HLA genes in association with MM. Some studies indicated no significant association between HLA-A and -B genes and MM, while other studies demonstrated that HLA-A3, -B18, -Bw65, and -DRw14 had associations with MM [13,14,15]. In the present study, for the first time, the associations of HLA class 1 and 2 genes with MM in Iranian patients were investigated.

Materials and Methods Patients and Controls One hundred and five Iranian patients were selected from the bone marrow transplantation department of Taleghani Hospital. The diagnosis of MM was made by an oncologist. Additionally, one hundred and twenty ethnically, age-, and sex-matched healthy individuals without personal or familial history of cancer or autoimmune disorders were included as controls. The subjects gave informed consent to participate. DNA Extraction and HLA Genotyping Genomic DNA from venous peripheral blood samples was extracted by the salting-out method [16]. HLA typing was performed at the Tehran Medical Genetics Laboratory. HLA-A, -B, and -DRB1 genotyping was carried out based on lowresolution single specific primer-polymerase chain reaction (SSP-PCR) HLA typing with the HLA-Ready Gene ABDR Kit (Inno-Train Diagnostik GmbH, Germany) according to the manufacturer’s recommendation. The PCR products were run on 2% agarose gel. Statistical Analysis By using chi-square and Fisher exact tests, comparisons between HLA-A, -B, and -DRB1 alleles of MM patients and the controls were performed. SPSS 18.0 for Windows was used for analysis and p<0.05 was considered to be statistically significant. The p-value was corrected with Bonferroni correction. In statistics, the Bonferroni correction is a method used to counteract the problem of multiple comparisons. Odds ratio (OR) and 95% confidence interval (CI) were determined. Results Distributions of sex and age of MM patients and the controls are shown in Table 1. The allele frequencies of HLA-A, -B, and

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Sayad A, et al: Association of HLA with Multiple Myeloma

-DRB1 in MM patients and controls are demonstrated in Table 2. As outlined in Table 2, no significant associations between HLA-DRB1 and MM were observed. Low-resolution HLA typing revealed that 21% of patients versus 12% of controls and 11% of patients versus 3% of controls carried HLA-A*03 and HLA-B*18, respectively. The MM group had a significant increase in the frequency of HLA-A*03 and HLA-B*18 alleles in comparison to control subjects (p=0.039, OR=2.057 and p=0.013, OR=3.567, respectively) (Table 2). Discussion After description of a serological technique for HLA typing, studies on the association of the HLA genes with susceptibility to or protection against different disease were begun. In our research, for the first time, we investigated the association of HLA class 1 and 2 genes with MM disease in Iranian patients. In our study, the HLA-A*03 and HLA-B*18 alleles had higher frequencies in MM patients than in control individuals and had significantly positive associations with MM. Therefore, the HLA-A*03 and HLA-B*18 alleles have a susceptibility effect in Iranian MM patients (p=0.039 and OR=2.057, p=0.013 and OR=3.567, respectively). Consistent with our results, in 2002, a study on 68 MM patients in southern Africa reported that the HLA-B*18 allele had an association (p<0.005, OR=6.3) and HLA-DRB1 had no association with MM significantly. In contrast to our results, however, that study found that there was no statistically significant association between HLA-B and MM [14]. Patel et al. demonstrated no significant association between antigens Table 1. Distributions of sex and age of multiple myelom (MM) patient and control groups. Variables

MM patients

Controls

Female [no. (%)]

47 (45)

69 (46)

Male [no. (%)]

58 (55)

81 (54)

Mean age ± SD, age range (years)

59±3.0, 39-74

60±2.9, 35-75

390

at either the A or the B locus in MM patients compared to controls [15]. Additionally, in a study on black and white men in 1992, it was shown that black MM patients had significantly higher HLA-Bw65 and HLA-DRw14 allele frequencies than black controls, while white MM patients had a higher A3 allele frequency than white controls [13]. A study on German, Dutch, American, and English subjects showed a weak association between HLA-B5 and MM [8,9,10,17]. No significant HLA-A and -B allele associations were demonstrated in French and Swiss MM patients, although the MM patients from another area of France showed a negative association between HLAAw32 and MM [6,7,12]. Ludwig and Mayr, in 1982, reported that comparisons between all available studies showed a significantly increased frequency of HLA-B5 [18]. HLA allele associations in our population demonstrated some differences from previous published studies on MM. Variations among ethnic groups may support these differences. Not many studies have taken sufficient account of the association between HLA and MM. Moreover, there are not many studies that have evaluated and calculated this association among ethnic groups. It is suggested that other populations be studied to find the correlation of HLA and MM to find out the exact association. Conclusion The HLA-A*03 and HLA-B*18 alleles have significant susceptibility effects on MM in the Iranian population. However, compared to other populations, the abovementioned alleles had different statuses. There are other reports that show both consistency and inconsistency with our results. Ethnic variations among different populations in part explain these controversies. Further studies with large sample sizes or family studies are needed to confirm the exact associations of HLA genes with MM. 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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Table 2. The allele frequencies of HLA-A, -B, and -DRB1 in multiple myelom (MM) patient and control groups.

Alleles

MM patients, Controls, n=210 [no. (%)] n=300 [no. (%)]

p-valuea

pc-valueb

OR (95% CI)

HLA-A*01

19 (9)

26 (9)

0.881

1.048 (0.564-1.948)

HLA-A*02

48 (23)

53 (27)

0.148

1.381 (0.891-2.140)

HLA-A*03

46 (21)

36 (12)

0.003

0.039

2.057 (1.276-3.316)

HLA-A*11

17 (8)

27 (10)

0.72

0.891 (0.472-1.679)

HLA-A*23

8 (4)

10 (3)

0.032

3.921 (1.028-14.957)

HLA-A*24

38 (18)

40 (18)

0.141

1.436 (0.885-2.330)

HLA-A*26

8 (4)

14 (5)

0.131

2.337 (0.754-7.245)

HLA-A*29

4 (2)

2 (1)

0.235c

2.893 (0.525-15.943)

HLA-A*30

4 (2)

10 (3)

0.454c

1.922 (0.426-8.680)

HLA-A*31

13 (6)

12 (5)

0.259

1.584 (0.708-3.543)

HLA-A*32

1 (1)

10(3)

0.647c

0.474 (0.049-4.585)

HLA-A*33

2 (1)

5 (2)

0.705c

0.567 (0.109-2.952)

HLA-A*68

2 (1)

5 (2)

0.705c

0.567 (0.109-2.952)

HLA-B*07

11 (5)

18 (6)

0.715

0.866 (0.400-1.874)

HLA-B*08

5 (2)

10 (3)

0.283c

2.415 (0.571-10.216)

HLA-B*09

2 (1)

1.000c

1.433 (0.200-10.252)

HLA-B*13

4 (2)

12 (4)

0.182

0.466 (0.148-1.465)

HLA-B*14

0 (0)

3 (1)

0.272

HLA-B*15

2 (1)

3 (1)

1.000c

0.952 (0.158-5.747)

HLA-B*18

23 (11)

10 (3)

0.001

0.013

3.567 (1.660-7.664)

HLA-B*27

6 (3)

10 (3)

0.761

0.853 (0.305-2.384)

HLA-B*35

52 (25)

84 (28)

0.416

0.846 (0.566-1.265)

HLA-B*37

0 (0)

3 (1)

0.272c

1.707 (1.587-1.837)

HLA-B*38

17 (8)

18 (6)

0.357

1.380 (0.694-2.745)

HLA-B*39

6 (3)

4 (1)

0.222

2.176 (0.607-7.810)

HLA-B*40

6 (3)

6 (2)

0.530

1.441 (0.458-4.531)

1.433 (0.200-10.252)

HLA-B*41

2 (1)

1.000c

HLA-B*44

17 (8)

21 (7)

0.643

1.170 (0.602-2.276)

HLA-B*48

2 (1)

3 (1)

1.000c

0.952 (0.158-5.747)

HLA-B*49

5 (2)

6 (2)

0.771

1.195 (0.360-3.969)

`HLA-B*50

2 (1)

7 (2)

0.244

0.402 (0.083-1.957)

51 (17)

0.330

0.782 (0.477-1.283)

HLA-B*51

(14)

HLA-B*52

6 (3)

7 (2)

0.712

1.231 (0.408-3.717)

HLA-B*53

0 (0)

2 (1)

0.515

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HLA-B*54

0 (0)

3 (1)

0.272

HLA-B*55

0 (0)

2 (1)

0.515

HLA-B*56

0 (0)

3 (1)

0.272

HLA-B*57

5 (2)

0.567

1.439 (0.411-5.034)

HLA-B*58

8 (4)

5 (2)

0.131

2.337 (0.754-7.245)

HLA-DRB1*01

6 (3)

15 (5)

0.231

0.559 (0.213-1.465)

HLA-DRB1*03

29 (14)

30 (10)

0.186

1.442 (0.837-2.484)

HLA-DRB1*04

15 (7)

24 (8)

0.720

0.885 (0.452-1.730)

HLA-DRB1*07

8 (4)

12 (4)

0.913

0.950 (0.382-2.367)

HLA-DRB1*08

1 (1)

3 (1)

0.647c

0.474 (0.049-4.585)

HLA-DRB1*09

5 (2)

3 (1)

0.217c

2.415 (0.571-10.216)

HLA-DRB1*10

11 (5)

12 (4)

0.507

1.327 (0.574-3.066)

HLA-DRB1*11

80 (38)

126 (42)

0.376

0.850 (0.592-1.219)

HLA-DRB1*12

5 (2)

2 (1)

0.130

3.634 (0.698-18.912)

HLA-DRB1*13

19 (9)

30 (10)

0.719

0.895 (0.489-1.638)

HLA-DRB1*14

20 (10)

21 (7)

0.302

1.398 (0.738-2.651)

HLA-DRB1*15

5 (2)

12 (4)

0.316

0.585 (0.203-1.687)

HLA-DRB1*16

6 (3)

10 (3)

0.761

0.853 (0.305-2.384)

a: Value of chi-square or Fisher exact test prior to Bonferroni correction. b: Value of chi-square or Fisher exact test with Bonferroni correction. c: Tested with Fisher exact test. n: number of alleles. NS: not significant.

References 1. Kyle RA, Rajkumar SV. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leukemia 2009;23:3-9. 2. Raab MS, Podar K, Breitkreutz I. Multiple myeloma. Lancet 2009;374:324-339. 3. Collins CD. Problems monitoring response in multiple myeloma. Cancer Imaging 2005;5:119-126. 4. McPhedran P, Heath CW Jr, Garcia J. Multiple myeloma incidence in metropolitan Atlanta, Georgia: racial and seasonal variations. Blood 1972;39:866-873. 5. Blattner WA. Epidemiology of multiple myeloma and related plasma cell disorders: an analytic review. In: Potter M (ed.). Progress in Myeloma: Biology of Myeloma. New York, Elsevier/North Holland, 1980. 6. Cassuto JP, Piereschi J, Maiolini R, Dujardin P, Ribeil R, Masseyeff R. Marqueurs HLA dans les myélomes et les dysglobulinémies monoclonales bénignes. Nouv Presse Med 1981;10:252-253 (article in French). 7. Jeannet M, Magnin C. HL-A antigens in haematological malignant diseases. Eur J Clin Invest 1971;2:39-42.

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8. Bertrams J, Kuwert E, Bohme U, Reis HE, Gallmeier WM, Wetter O, Schmidt CG. HL-A antigens in Hodgkin’s disease and multiple myeloma. Tissue Antigens 1972;2:41-46. 9. Smith G, Walford RL, Fishkin B, Carter PK, Tanaka K. HLA phenotypes, immunoglobulins and K and L chains in multiple myeloma. Tissue Antigens 1974;4:374-377. 10. Mason DY, Cullen P. HL-A antigen frequencies in myeloma. Tissue Antigens 1975;5:238-245. 11. Van Camp BGK, Cole J, Peetermans ME. HLA antigens and homogeneous immunoglobulins. Clin Immunol Immunopathol 1977;7:315-318. 12. Saleun IP, Youinou P, Le Goff P, Le Menn G, Morin JF. HLA antigens and monoclonal gammapathy. Tissue Antigens 1979;13:233-235. 13. Pottern LM, Gart JJ, Nam J, Dunston G, Wilson J, Greenberg R. HLA and multiple myeloma among black and white men: evidence of a genetic association. Cancer Epidemiol Biomarkers Prev 1992;1:177-182. 14. Patel M, Wadee AA, Galpin J, Gavalakis C, Fourie AM, Kuschke RH, Philip V. HLA class I and class II antigens associated with multiple myeloma in southern Africa. Clin Lab Haematol 2002;24:215-219.

Sayad A, et al: Association of HLA with Multiple Myelomaâ&#x20AC;&#x192;

15. Leech SH, Bryan CF, Elston RC, Rainey J, Bickers JN, Pelias MZ. Genetic studies in multiple myeloma. 1. Association with HLA-Cw5. Cancer 1983;51:1408-1411. 16. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1998;16:1215.

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17. Festen JJM, Marrink J, Sijpesteijn JAK, van Loghem E, Nijenhuis LE, Mandema E. A study on the association between myelomatosis and immunoglobulin allotypes, HLA, and blood groups. Immunogenetics 1976;3:201-203. 18. Ludwig H, Mayr W. Genetic aspects of susceptibility to multiple myeloma. Blood 1982;59:1286-1291.

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Research Article

DOI: 10.4274/tjh.2012.0154

New Insights on Iron Study in Myelodysplasia Miyelodisplazide Demir Testleri ile İlgili Yeni Görüşler Noha M. El Husseiny1, Dina Ahmed Mehaney2, Mohamed Abd El Kader Morad1 1Cairo 2Cairo

University Faculty of Medicine, Department of Clinical Hematology, Cairo, Egypt University Faculty of Medicine, Department of Chemical Pathology, Cairo, Egypt

Abstract: Objective: Hepcidin plays a pivotal role in iron homeostasis. It is predominantly produced by hepatocytes and inhibits iron release from macrophages and iron uptake by intestinal epithelial cells. Competitive ELISA is the current method of choice for the quantification of serum hepcidin because of its lower detection limit, low costs, and high throughput. This study aims to discuss the role of hepcidin in the pathogenesis of iron overload in recently diagnosed myelodysplasia (MDS) cases.

Materials and Methods: The study included 21 recently diagnosed MDS patients and 13 healthy controls. Ferritin, hepcidin, and soluble transferrin receptor (sTFR) were measured in all subjects.

Results: There were 7 cases of hypocellular MDS, 8 cases of refractory cytopenia with multilineage dysplasia, and 6 cases of refractory anemia with excess blasts. No difference was observed among the 3 MDS subtypes in terms of hepcidin, sTFR, and ferritin levels (p>0.05). Mean hepcidin levels in the MDS and control groups were 55.8±21.5 ng/mL and 19.9±2.6 ng/ mL, respectively. Mean sTFR was 45.7±8.8 nmol/L in MDS patients and 31.1±5.6 nmol/L in the controls. Mean ferritin levels were significantly higher in MDS patients than in controls (539.14±83.5 ng/mL vs. 104.6±42.9 ng/mL, p<0.005). There was a statistically significant correlation between hepcidin and sTFR (r=0.45, p=0.039). No difference in hepcidin levels between males and females was observed, although it was lower in males in comparison to females (47.9±27.6 vs. 66.7±35.7, p>0.05). Conclusion: Hepcidin may not be the main cause of iron overload in MDS. Further studies are required to test failure of production or peripheral unresponsiveness to hepcidin in MDS cases.

Key Words: Hepcidin, Myelodysplasia, Iron overload Özet: Amaç: Hepsidin demir dengesinde önemli bir rol oynar. Temel olarak hepatositler tarafından üretilir ve intestinal epitel hücrelerinden demir alımını ve makrofajlardan demir salınımını inhibe eder. Kompetitif ELİZA en düşük düzeyleri saptayabilmesi, düşük maliyeti ve yüksek test kapasitesi ile kantitatif serum hepsidin miktari ölçümü için tercih edilen mevcut bir yöntemdir. Bu çalışmanın amacı son zamanlarda miyelodisplazi (MDS) tanısı almış olgularda demir yükünün patogenezinde hepsidinin rolünü tartışmaktır.

Address for Correspondence: Noha M. EL HUSSEINY, M.D., Cairo University Faculty of Medicine, Department of Clinical Hematology, Cairo, Egypt E-mail: dr_noha2002@yahoo.com Received/Geliş tarihi : October 16, 2012 Accepted/Kabul tarihi : January 21, 2013

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Turk J Hematol 2014;31:394-398

Gereç ve Yöntemler: Bu çalışmaya 21 yeni tanı MDS hastası ve 13 sağlıklı kontrol alındı. Ferritin, hepsidin ve soluble

transferrin reseptörü (sTFR) tüm olgularda ölçüldü. Bulgular: Hiposellüler MDS’li 7 olgu, multilineage displazili refrakter sitopenili 8 olgu ve artmış blastlı refrakter anemili 6 olgu vardı. Bu 3 MDS alt tipleri arasında hepsidin, sTFR ve ferritin düzeyleri açısından fark gözlenmedi (p>0,05). Ortalama hepsidin seviyesi sırasıyla MDS grubunda 55,8±21,5 ng/mL ve kontrol grubunda ise 19,9±2,6 ng/mL idi. Ortalama sTFR, MDS grubunda 45,7±8,8 nmol/L ve kontrol grubunda ise 31,1±5,6 nmol/L idi. Ortalama ferritin seviyeleri ise MDS grubunda kontrolden anlamlı olarak yüksekti (539,14±83,5 ng/mL vs. 104,6±42,9 ng/mL, p<0,005). Hepsidin ve sTFR arasında istatistiksel anlamlı bir ilişki vardı (r=0,45, p=0,039). Hepsidin düzeyleri erkeklerde kadınlarla karşılaştırıldığında daha düşük değerlerde bulunmasına karşılık erkek ve kadınlar arasında istatistiksel anlamlı fark görülmedi (47,9±27,6 vs. 66,7±35,7, p>0,05). Sonuç: Hepsidin MDS’de aşırı demir yükünün ana nedeni olmayabilir. MDS’li olgularda hepsidine çevresel yanıtsızlık veya üretimi yetersizliğini test edecek başka çalışmalara ihtiyaç vardır.

Anahtar Sözcükler: Hepsidin, Miyelodisplazi, Demir yükü Introduction Myelodysplastic syndrome (MDS) is a group of clonal hemopoietic disorders characterized by ineffective hematopoiesis, bone marrow dysplasia, and an increased risk of transformation to acute myeloid leukemia. The large majority of patients with MDS are anemic and eventually up to 90% of them require regular transfusions. In 50% to 60% of patients, anemia is severe, with hemoglobin levels below 10 g/dL [1]. The liver polypeptide hepcidin plays a pivotal role in iron homeostasis. In macrophages, it accelerates the degradation of the trans-membrane iron exporter ferroportin mRNA. In intestinal epithelial cells, it is believed to down-regulate divalent metal transporter-1, which is involved in the transfer of iron across the intestinal wall [2]. Previous reports on hepcidin levels in MDS showed conflicting results. Murphy et al. found that urinary excretion of hepcidin was lower in MDS in comparison to the control group and interpreted this finding as evidence for hepcidin’s role in iron overload in MDS [3]. However, others reported contrary findings [4]. In an earlier study, we measured prohepcidin in MDS and found it to be significantly lower in comparison to levels in the control group [5]. However, the utility of the prohepcidin assay is controversial [6]. The development of quantification techniques based on mass spectrometry [matrix-assisted laser desorption ionization, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), or liquid chromatography tandem mass spectrometry] has shown promising results. However, some of these approaches did not use internal standards for the quantification of hepcidin and are considered semi-quantitative; moreover they require specialized equipment that is not widely accessible [7]. Regarding MDS, there are only a few conflicting data about urinary hepcidin measured by using first generation semiquantitative techniques [8]. The competitive enzyme-linked

immunosorbent assay (ELISA) is currently the method of choice for the quantification of serum hepcidin because of its lower detection limit, low costs, and high throughput [6]. Although prolonged red blood cell transfusion therapy appears to be the main contributor to iron overload in MDS, many patients appear to develop iron overload at an early stage of the disease, before the onset of transfusions. It has been postulated that an altered production of hepcidin, the recently discovered key hormone regulating iron homeostasis, may play a role in this regard [8]. The aim of this study was to find out whether hepcidin plays a key role in the pathogenesis of iron overload seen in recently diagnosed MDS, or whether hepcidin levels are just a consequence of the interaction between ineffective erythropoiesis and blood transfusion. Materials and Methods This study included 21 MDS patients recently diagnosed in the Clinical Hematology Unit of Cairo University. All patients who had an MDS diagnosis of less than 6 months entered the study if they were not on iron chelation, had no evidence of infection and no renal or liver impairment, and did not receive more than 10 units of blood. Ethical committee approval of the study was obtained. Each individual in the study signed a consent form. Five milliliters of venous blood was obtained from each individual in the study after at least 8 h of fasting. For MDS patients, samples were taken at least 5 days after the last transfusion to avoid acute alterations in the hepcidin level. The samples were centrifuged at 3000 rpm for 10 min to separate sera and stored at -80 °C until analyzed. Serum levels of hepcidin were determined using a commercially available ELISA kit (DRG Instruments GmbH, Marburg, Germany) according to the manufacturer’s protocol. The 5% to 95% range in apparently normal, healthy adults is between 13.3 and 54.4 ng/mL.

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Soluble transferrin receptor (sTFR) concentrations were measured in serum, stored at -80 °C, with the use of a commercially available ELISA kit (Quantikine IVD Soluble Transferrin Receptor ELISA; R&D Systems Europe Ltd., Abingdon, UK). The reference interval is 8.7-28.1 nmol/L. Serum ferritin was quantified using the DRG ferritin kit (ELISA) kit (EIA-1872; DRG International Inc., Mountainside, NJ, USA). The reference interval in healthy males and females is 20-250 ng/mL and 10-120 ng/mL, respectively. Statistics Analysis SPSS 17 was used for descriptive analysis and comparisons. Spearman’s test was used for correlation analysis and analysis of variance (ANOVA) was used for the comparison of multiple parameters among different groups. Differences and correlations were considered significant when p<0.05. The Kolmogorov-Smirnov test was performed for normal distributions. After suitability analysis, non-parametric or parametric tests were performed.

and ferritin levels were higher in patients with MDS than in controls, only ferritin significantly differed between the 2 groups (p<0.005). Correlation analysis between hepcidin and sTFR was statistically significant (r=0.45, p=0.039, Figure 1). The correlations between hepcidin and other parameters are given in Table 2. There was no statistically significant difference in hepcidin levels between males and females, although levels were lower in males in comparison to females (47.9±27.6 ng/mL vs. 66.7±35.7 ng/mL, p>0.05). MDS patients were divided according to the type of MDS into 3 groups: refractory cytopenia with multilineage dysplasia (RCMD), 8 patients; hypoplastic MDS, 7 patients; and refractory anemia with excess blasts (RAEB), 6 patients. No statistically significant difference among the 3 groups in terms of hepcidin, sTFR, or ferritin was found (p>0.05, Table 3). Mean hepcidin/ferritin ratio in patients with MDS was higher than in the controls (0.48±1.2 vs. 0.32±0.19), but this was not statistically significant (p=0.6). Discussion

Results This study included 21 MDS patients (10 females, 11 males) recently diagnosed in the Clinical Hematology Unit of Cairo University (average of 6 months after the onset of symptoms) and 13 (6 females, 7 males) age- and sex-matched controls. The mean age was 56±10.2 years. Mean hemoglobin level of MDS patients was 6.8±4.8 g/dL. Median number of blood transfusions was 6 units (range: 3-9 units). Mean hepcidin levels in the MDS and control groups were 55.8±21.5 ng/mL and 19.9±2.6 ng/mL, respectively. Mean sTFR level in MDS patients was 45.7±8.8, while in the control group it was 31.1±0.6. Mean ferritin levels in the MDS patients and the controls were 539.14±83.5 ng/mL and 104.6±42.9 ng/mL, respectively (Table 1). Although the mean hepcidin, sTFR,

It has been reported that ineffective erythropoiesis enhances iron absorption in MDS through down-regulation of hepcidin and its prohormone such that serum ferritin rises to 500-600 ng/mL but seldom exceeds these values before Table 1. Iron parameters.

Parameter

Patients with MDS

Healthy controls

Hepcidin (ng/mL, mean±SD)

55.8±21.5

19.9±2.6

0.06

Ferritin (ng/mL, mean±SD)

539.14±83.5

104.6±42.9

0.005

31.1±5.6

0.6

sTFR 45.7±8.8 (nmol/L, mean±SD)

DS: myelodysplastic syndrome, sTFR: soluble transferrin receptor.

Table 2. Correlation between hepcidin and other parameters in MDS.

Figure 1. Correlation between hepcidin and sTFR.

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Parameter

Correlation coefficient

p-value

Ferritin

-0.165

0.47

0.15

0.5

sTFR

0.45

0.03

Age

-0.04

0.84

No. of transfused blood units

0.31

0.16

sTFR: soluble transferrin receptor.

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El Husseiny MN, et al: Iron Status in Myelodysplasia

Table 3. Comparison of different parameters in the 3 main types of MDS.

Parameters

RCMD

RAEB

Hypoplastic MDS

Hepcidin (ng/mL)

66.6

80.1

22.57

0.13

sTFR (nmol/L)

47.5

66.01

26.2

0.5

Ferritin (ng/mL)

527.3

676.8

434.5

0.7

MDS: myelodysplastic syndrome, RAEB: refractory anemia with excess blasts, RCMD: refractory cytopenia with multilineage dysplasia, sTFR, soluble transferrin receptor.

transfusion begins [9,10]. Moreover, MDS is characterized by iron overload secondary to blood transfusion. However, its impact on stimulation of pro-hepcidin release to inhibit iron absorption is less than the erythroid drive suppressing its release, as shown from our earlier results [5]. In this work, we included newly diagnosed (within 6 months of diagnosis) MDS cases to limit the impact of overtransfusion on hepcidin levels. Furthermore, blood samples were drawn at least 5 days after the last transfusion to abolish the effect of acute transfusion on hepcidin expression. We found hepcidin levels higher in MDS cases in comparison to the control group, but this difference was not statistically significant, which could be attributed to the small number of patients studied. Our results are in line with those of Qin et al., who found that both hepcidin and serum ferritin levels in MDS patients, regardless of transfusion dependency or the number of blood transfusions, were higher than those of healthy controls [11]. Our findings suggest that iron overload is not related to defective hepcidin release but is rather associated with ineffective erythropoiesis and blood transfusion. In another study using SELDI-TOF MS for hepcidin assay, serum hepcidin levels were measured to be slightly higher in MDS patients than in controls, but this difference did not reach statistical significance. Nevertheless, the hepcidin/ferritin ratio was significantly lower for the whole MDS population as compared to the controls, which is not consistent with our study in which the ratio was higher in MDS cases [8]. This could be explained by the higher mean ferritin levels of the MDS population in that study, which also included heavily transfused cases. Ganz et al. reported that serum hepcidin was high in low-grade MDS patients in correlation with their iron and oxidative status, and that it was further increased by treatment with deferasirox. They concluded that the hepcidin level represented a balance between the stimulating effect of iron overload and the inhibitory effects of erythropoietic activity and oxidative stress. These preliminary findings favor the rationale for iron chelation therapy in such patients [12]. Our results revealed no correlation between hepcidin level and ferritin in patients with MDS in the context of non-heavily transfused patients.

Qin et al. worked on heavily transfused MDS and found that the increase of hepcidin was not in synchrony with the increase in serum ferritin levels secondary to blood transfusion when the number of blood transfusions exceeded 24 U. Hepcidin levels showed a negative relationship to serum ferritin, reflecting the decreased ability of hepcidin to inhibit body iron absorption during the increase of blood transfusion, which finally led to iron overload. Dynamic monitoring of the hepcidin concentration could help in predicting the occurrence of iron overload in transfusion-dependent MDS patients [11]. The current data may indicate that there is no correlation between hepcidin and serum ferritin in MDS cases, and thus hepcidin may not be a main player in iron overload in MDS. A possibility of peripheral unresponsiveness to hepcidin in MDS or failure of production may be the underlying cause, but further studies are required. When divided into RCMD, RAEB, and hypoplastic MDS groups, the highest levels of hepcidin were in the RAEB group, indicating marked activity in the bone marrow, followed by the RCMD and the hypocellular MDS groups. However, the difference among different subgroups in terms of hepcidin, ferritin, or sTFR level did not reach statistical significance. Santini et al. found that mean hepcidin levels were consistently heterogeneous across different MDS subtypes, with the lowest levels in refractory anemia with ringed sideroblasts (1.43 nmol/L) and the highest in the RAEB (11.3 nmol/L) (p=0.003) [8]. Conclusion Iron overload in MDS involves many players. Further studies are required to reveal the causes of failure in erythropoiesis or the peripheral unresponsiveness to hepcidin in MDS cases. Acknowledgments Special thanks to all members of the Hematology Clinic of Cairo University for their help in collection of data. The researchers were given funding from Cairo University to import ELISA kits for hepcidin assay. 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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El Husseiny MN, et al: Iron Status in Myelodysplasia

References 1. Dreyfus F. The deleterious effects of iron overload in patients with myelodysplastic syndromes. Blood Rev 2008;22:29-34. 2. Davis SL, Littlewood TJ. The investigation and treatment of secondary anaemia. Blood Rev 2012;26:65-71. 3. Murphy PT, Mitra S, Gleeson M, Desmond R, Swinkels DW. Urinary hepcidin excretion in patients with low grade myelodysplastic syndrome. Br J Haematol 2009;144:451452. 4. Winder A, Lefkowitz R, Ghoti H, Leiba M, Ganz T, Nemeth E, Rachmilewitz EA. Urinary hepcidin excretion in patients with myelodysplastic syndrome and myelofibrosis. Br J Haematol 2008;142:669-671. 5. El Husseiny NM, Matter MM, Sabry RM, Amin IS. Serum prohepcidin level in myelodysplasia. Scand J Clin Lab Invest 2010;70:343-346. 6. Kroot JJ, Laarakkers CM, Geurts-Moespot AJ, Grebenchtchikov N, Pickkers P, van Ede AE, Peters HP, van Dongen-Lases E, Wetzels JF, Sweep FC, Tjalsma H, Swinkels DW. Immunochemical and mass-spectrometryâ&#x20AC;&#x201C;based serum hepcidin assays for iron metabolism disorders. Clin Chem 2010;56:1570-1579.

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7. Koliaraki V, Marinou M, Vassilakopoulos TP, Vavourakis E, Tsochatzis E, Pangalis GA, Papatheodoridis G, Stamoulakatou A, Swinkels DW, Papanikolaou G, Mamalaki A. A novel immunological assay for hepcidin quantification in human serum. PLoS One 2009;4:4581. 8. Santini V, Girelli D, Sanna A, Martinelli N, Duca L, Campostrini N, Cortelezzi A, Corbella M, Bosi A, Reda G, Olivieri O, Cappellini MD. Hepcidin levels and their determinants in different types of myelodysplastic syndromes. PLoS One 2011;6:23109. 9. Fleming MD. The regulation of hepcidin and its effects on systemic and cellular iron metabolism. Hematology Am Soc Hematol Educ Program 2008;151-158. 10. Nemeth E. Iron regulation and erythropoiesis. Curr Opin Hematol 2008;15:169-175. 11. Qin Y, Liu H, Ruan S, Cai YF, You XF, Song GQ. Detection of hepcidin in transfusion dependent myelodysplastic syndrome patients and its clinical significance. Zhonghua Xue Ye Xue Za Zhi 2011;32:758-761 (article in Chinese with English abstract). 12. Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Immunoassay for human serum hepcidin. Blood 2008;112:4292-4297.

Case Report

DOI: 10.4274/tjh.2013.0082

Severe Myelotoxicity Associated with Thiopurine S-Methyltransferase*3A/*3C Polymorphisms in a Patient with Pediatric Leukemia and the Effect of Steroid Therapy Pediatrik Bir Lösemi Olgusunda Tiyopurin S-Metiltransferaz *3A/*3C Polimorfizmi ile İlişkili Ağır Miyelotoksisite-Steroid Tedavisinin Etkisi Burcu Fatma Belen1, Türkiz Gürsel1, Nalan Akyürek2, Meryem Albayrak3, Zühre Kaya1, Ülker Koçak1 1Gazi

University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey University Faculty of Medicine, Department of Pathology, Ankara, Turkey 3Kırıkkale University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey 2Gazi

Abstract: Myelosuppression is a serious complication during treatment of acute lymphoblastic leukemia and the duration of myelosuppression is affected by underlying bone marrow failure syndromes and drug pharmacogenetics caused by genetic polymorphisms. Mutations in the thiopurine S-methyltransferase (TPMT) gene causing excessive myelosuppression during 6-mercaptopurine (MP) therapy may cause excessive bone marrow toxicity. We report the case of a 15-year-old girl with T-ALL who developed severe pancytopenia during consolidation and maintenance therapy despite reduction of the dose of MP to 5% of the standard dose. Prednisolone therapy produced a remarkable but transient bone marrow recovery. Analysis of common TPMT polymorphisms revealed TPMT *3A/*3C. Key Words: Myelosuppression, Thiopurine S-methyl transferase, Acute leukemia

Özet: Miyelosupresyon, akut lenfoblastik lösemi tedavisinde görülen ciddi bir komplikasyon olup, miyelosupresyon süresi kemik iliği yetmezlik sendromlarından veya genetik polimorfizmin yol açtığı ilaç farmakogenetiğinden etkilenmektedir. Merkaptopürin metabolizasında yer alan Thiopurin s-metil transferaz (TPMT) enziminin azalmış aktivitesine neden olan polimorfizmleri artmış kemik iliği toksisitesine yol açar. Burada, 15 yaşında TPMT *3A/*3C ve MTHFR polimorfizmleri saptanan ve konsolidasyon/ idame tedavisi boyunca MP’nin standart dozun %5’inde yoğun miyelosupresyon görülen ve steroid tedavisi ile geçici düzelme saptanan bir T-ALL olgusu sunulmaktadır.

Anahtar Sözcükler: Miyelosupresyon, Thiopurin S-metil transferaz, Akut lösemi

Address for Correspondence: Burcu Fatma BELEN, M.D., Gazi University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey Gsm: +90 532 581 45 51 E-mail: draida@yahoo.com Received/Geliş tarihi : March 6, 2013 Accepted/Kabul tarihi : April 22, 2013

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Belen FB, et al: Myelotoxicity with TPMT *3A/*3C Polymorphisms

Introduction Myelosuppression is a serious complication of chemotherapy in children with acute lymphoblastic leukemia (ALL). The duration and the severity of myelosuppression vary with genetic polymorphisms affecting drug pharmacokinetics and bone marrow failure syndromes [1,2]. The thiopurine S-methyl transferase (TPMT) enzyme is involved in the metabolism of 6-mercaptopurine (MP), a widely used cytostatic agent in childhood ALL. The levels of active MP metabolites are elevated during MP therapy in patients who carry TPMT mutations with decreased enzyme activity such as TPMT *2 (G238C), TPMT *3A (G460A and A719G), and TPMT *3C (A719G), leading to excessive bone marrow toxicity and being more profound in those with 2 as compared to 1 nonfunctional allele [2,3,4,5,6]. The contribution of methylenetetrahydrofolate reductase (MTHFR) mutations in MP-induced myelosuppression has not been well established [7,8]. We here describe the case of a 15-year-old girl with ALL and TPMT *3A/*3C and MTHFR polymorphisms who suffered from severe bone marrow suppression persisting during the consolidation and maintenance therapy. Case Presentation A 15-year-old girl presented with a white blood cell (WBC) count of 150x109/L, anemia, thrombocytopenia, and mediastinal enlargement. A diagnosis of T-cell ALL was made based on peripheral blood morphology and immunophenotyping by flow cytometer. Karyotyping disclosed t(11;14), but polymerase chain reaction (PCR) tests for t(4;11) and t(9;21) were negative. She was treated with the Berlin-Frankfurt-Münster (BFM) ALL-95 chemotherapy protocol [9]. She received induction chemotherapy uneventfully and achieved complete remission on day 33 with full bone marrow recovery. Five days after receiving the first 5 days of Protocol Ib, which contained 6-MP at 60 mg/m2 per day orally, cytosine arabinoside (c-ARA) at 75 mg/m2/day for 4 doses, and a single dose of 1000 mg/m2 cyclophosphamide, her WBC count, absolute neutrophil count (ANC), platelet count, and hemoglobin (Hb) level began to decrease and reached a minimum at week 14 (WBC: 0.8x109/L, ANC :0x109/L; platelets: 3x109/L; Hb: 5 g/dL). Chemotherapy was stopped; granulocyte colony-stimulating factor (G-CSF) was started at 5 µg/kg/day and increased to 10 µg/kg/day. Six weeks later, WBC and ANC returned to normal but Hb level and platelet count remained low. Bone marrow aspirations yielded dry taps. TPMT genotyping for G238C, G460A, and A719G was reported as negative. As WBC and ANC were within normal limits, chemotherapy was restarted, but her counts rapidly dropped (Figure 1). She remained pancytopenic for a further 6-week period, during which chemotherapy was

400

stopped, and numerous platelet and erythrocyte transfusions were given. Bone marrow biopsy showed cellularity of 5% with no residual leukemia and/or fibrosis. PCR analysis for parvovirus B19, Epstein-Barr virus, and cytomegalovirus and the diepoxybutane (DEB) test were negative. Because of protracted pancytopenia, the rest of Protocol Ib and intensification blocks were omitted and maintenance therapy with oral 6-MP and methotrexate (MTX) was started at 25% of protocol doses. Absolute neutrophil count dropped and remained below 0.8x109/mm3 despite G-CSF therapy. Genotyping for MTHFR revealed heterozygousity for C677T and A1298C polymorphisms. We were informed that the patient had TPMT *3A/*3C polymorphisms; the previous report of wild-type TPMT was a transcription error. The doses of 6-MP and MTX were reduced to 10% of protocol doses, but the patient remained severely pancytopenic and transfusiondependent. Bone marrow biopsy showed profound hypocellularity with occasional hematopoietic hot points and abundant fat cells, resembling aplastic anemia. Chemotherapy was stopped and oral prednisolone was started at a dose of 60 mg/m2 per day. WBC and platelet counts increased gradually starting from the second week, and bone marrow aspirate became normocellular and free of leukemic blasts at the end of 30 days of steroid therapy. Bone marrow biopsy showed 40%

Figure 1. Absolute neutrophil counts, hemoglobin levels, and platelet counts during ALL BFM-95 chemotherapy. ANC: absolute neutrophil count, Hb: hemoglobin, Plt: platelet count, CCP: cytosine arabinoside + 6-mercaptopurine treatment, CP: cyclophosphamide, G-CSF: granulocyte colony-stimulating factor, GC: glucocorticoid (prednisolone), Pr M: Protocol M-BFM 95, Pr II: Protocol II-BFM-95. Blanks at the right end of the figure represent chemotherapy cessation due to hyperglycemia and hypertriglyceridemia during Protocol II.

Belen FB, et al: Myelotoxicity with TPMT *3A/*3C Polymorphisms

cellularity without residual blasts. As blood counts returned to normal limits, intensification therapy with 4 blocks of MTX at 5 g/m2/day and 6-MP at 2.5 mg/m2/day (5% of protocol dose) was given over the next 54 days. During this period, WBC count and ANC remained between 2 and 3x109/L and platelet counts could be kept above 10x109/L by weekly transfusions (Figure 1). MTX levels were calculated at 24, 36, 42, 48, and 54 h after infusion of MTX as below the highest upper limit to give additional folinic acid rescue. Reinduction therapy with vincristine, dexamethasone, L-asparaginase, and doxorubicine caused profound pancytopenia complicated with prolonged febrile episodes, severe hyperglycemia, and liver dysfunction. Therefore, the second part of the reinduction therapy was omitted, and maintenance therapy was restarted at 10% of standard doses. She completed the 5-week maintenance therapy with 6-MP and MTX doses ranging from 5% to 10% and from 8% to 16%, respectively. After termination of maintenance chemotherapy, blood counts gradually increased and reached normal range within 3 months. Informed consent was obtained. Discussion and Review of the Literature Following intensive chemotherapy, blood counts recover within 2 to 4 weeks in most cases of ALL. Longer periods of bone marrow suppression are rare and can be caused by abnormalities of drug disposition pathways or congenital bone marrow failure syndromes with myelodysplasia. A clear relation with excessive bone marrow toxicity has been established only for thiopurine drugs and TPMT polymorphisms [1,2,3,4,5,6,10]. Patients homozygous or compound heterozygous for a nonfunctional TPMT allele develop pancytopenia 2 to 4 weeks after starting oral 6-MP and recover within 2-6 weeks [11,12]. The longest duration of pancytopenia was 137 days in an 8-year-old boy with ALL who was homozygous for TPMT *3A/*3A [13]. Bone marrow suppression in the present case with TPMT *3A/*3C polymorphisms was more severe as compared to previously described patients with 2 nonfunctional TPMT alleles. Pancytopenia developed shortly after introduction of 6-MP and persisted during maintenance therapy, despite reducing both 6-MP and MTX doses to 5% to 10%. Interruption of chemotherapy for 6-8 weeks and G-CSF therapy resulted in ANC recovery initially without significant increase in Hb levels or platelet counts, but the duration of bone marrow suppression was prolonged as 6-MP exposures increased, suggesting that myelotoxicity is dose-dependent. However, we did not investigate additional single nucleotide polymorphisms involved in thiopurine metabolism, such as inosine triphosphate pyrophosphatase, that might have been responsible for extreme bone marrow suppression in our patient [14]. She also had MTHFR C677T and A1298C

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polymorphisms, which cause reduced MTHFR activity, leading to decreased production of S-adenosylmethionine, a protector of the TPMT enzyme [15]. Karas-Kuzelicki et al. reported increased myelotoxicity in children with ALL who were heterozygous for at least one low-activity TPMT allele and for C677T and/or A1298C polymorphisms in the MTHFR gene [7]. In contrast, another study found lower frequency of severe myelotoxicity in pediatric ALL patients having both TPMT and C677T polymorphisms [8]. Interestingly, we did not observe excessive hematological toxicity during intensification therapy with high-dose (5 g/m2) MTX combined with low-dose MP (2.5% of protocol dose) in our patient, suggesting that compound heterozygosity for C677T and A1298C is not an additional risk factor for hematotoxicity in children with TPMT polymorphisms. An interesting finding in the present case is a quick bone marrow recovery induced by glucocorticoid therapy. Bone marrow cellularity increased from 5% at the beginning to 45% at the end of 4 weeks of 2 mg/kg prednisolone administration. Although steroid therapy is effective in the treatment of immune-mediated bone marrow suppression, its role in chemotherapy-induced bone marrow hypoplasia has not been well studied. A short course of highdose methylprednisolone therapy was reported to increase CD34 (+) progenitor cells and shorten chemotherapy-induced neutropenia in children with leukemia [16,17]. The molecular mechanisms of steroid effects on bone marrow regeneration need to be illuminated. Children with Fanconi anemia (FA) may experience severe bone marrow toxicity with alkylating agents like cyclophosphamide and busulphan [2,18]. Cyclophosphamide causes chromosomal breaks in FA patients. A recent study showed that cyclophosphamide-specific interstrand DNA cross-links were increased 15-fold in FA patients compared to non-FA patients [19]. Our patient had received high-dose cyclophosphamide in addition to 6-MP one week before development of initial bone marrow hypoplasia. She had none of the main clinical or laboratory features of FA, such as congenital defects, developmental abnormalities, elevated HbF level, or increased chromosomal fragility. Patients with myelodysplasia also show delayed bone marrow recovery after chemotherapy. Several cases of T-cell ALL have been described in patients with myelodysplasia associated with germline RUNX-1 mutation or microdeletion of 21q22 resulting in RUNX-1 deficiency [20]. The conventional karyotyping performed for our patient is not able to detect such a small deletion, but absence of thrombocytopenia history before development of leukemia and rapid bone marrow regeneration without dysplastic changes and complete hematologic reconstitution at day 33 of remission induction therapy exclude this possibility. Patients who are compound heterozygous for TPMT *3A/3C may have severe

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bone marrow hypoplasia even with minimal amounts of MP. Further investigation of other genetic factors involved in MP metabolism and measurement of the intracellular levels of thioguanine nucleotides may help in better treatment of chemotherapy-induced myelosuppression. Conclusion Compound heterozygosity for TPMT *3A/3C may be associated with severe bone marrow hypoplasia, even with minimal amounts of MP, in children with ALL. The role of glucocorticoids on bone marrow regeneration after chemotherapy should be investigated further. 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. Meeker ND, Yang JJ, Schiffman JD. Pharmacogenomics of pediatric acute lymphoblastic leukemia. Expert Opin Pharmacother 2010;11:1621-1632. 2. Borriello A, Locasciulli A, Bianco AM, Criscuolo M, Conti V, Grammatico P, Cappellacci S, Zatterale A, Morgese F, Cucciolla V, Delia D, Della Ragione F, Savoia A. A novel Leu153Ser mutation of the Fanconi anemia FANCD2 gene is associated with severe chemotherapy toxicity in a pediatric T-cell acute lymphoblastic leukemia. Leukemia 2007;21:72-78. 3. Lennard L, Gibson BE, Nicole T, Lilleyman JS. Congenital thiopurine methyltransferase deficiency and 6-mercaptopurine toxicity during treatment for acute lymphoblastic leukaemia. Arch Dis Child 1993;69:577-579. 4. McLeod HL, Coulthard S, Thomas AE, Pritchard SC, King DJ, Richards SM, Eden OB, Hall AG, Gibson BE. Analysis of thiopurine methyltransferase variant alleles in childhood acute lymphoblastic leukaemia. Br J Haemol 1999;105:696700. 5. Krynetski, EY, Evans WE. Genetic polymorphism of thiopurine S-methyltransferase: molecular mechanisms and clinical importance. Pharmacology 2000;61:136-146. 6. Relling MV, Hancock ML, Rivera GK, Sandlund JT, Ribeiro RC, Krynetski EY, Pui CH, Evans WE. Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus. J Natl Cancer Inst 1999;91:2001-2008. 7. Karas-Kuzelicki N, Jazbec J, Milek M, Mlinaric-Rascan I. Heterozygosity at the TPMT gene locus, augmented by mutated MTHFR gene, predisposes to 6-MP related toxicities in childhood ALL patients. Leukemia 2009;23:971-974. 8. Costea I, Moghrabi A, Laverdiere C, Graziani A, Krajinovic M. Folate cycle gene variants and chemotherapy toxicity in pediatric patients with acute lymphoblastic leukemia. Haematologica 2006;91:1113-1116.

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9. Kaya Z, Gursel T, Bakkaloglu SA, Kocak U, Atasever T, Oktar SO. Evaluation of renal function in Turkish children receiving BFM-95 therapy for acute lymphoblastic leukemia. Pediatr Hematol Oncol 2007;24:257-267. 10. Albayrak M, Konyssova U, Kaya Z, Gursel T, Guntekin S, Percin EF, Kocak U. Thiopurine methyltransferase polymorphisms and mercaptopurine tolerance in Turkish children with acute lymphoblastic leukemia. Cancer Chemother Pharmacol 2011;68:1155-1159. 11. Evans WE, Horner M, Chu YQ, Kalwinsky D, Roberts WM. Altered mercaptopurine metabolism, toxic effects, and dosage requirement in a thiopurine methyltransferase-deficient child with acute lymphocytic leukemia. J Pediatr 1991;119:985989. 12. Andersen JB, Szumlanski C, Weinshilboum RM, Schmiegelow K. Pharmacokinetics, dose adjustments and 6-mercaptopurine /methotrexate drug interactions with thiopurine methyltransferase deficiency. Acta Paediatr 1998;87:108-111. 13. McBride KL, Gilchrist GS, Smithson WA, Weinshilboum RM, Szumlanski CL. Severe 6-thioguanine-induced marrow aplasia in a child with acute lymphoblastic leukemia and inherited thiopurine methyltransferase deficiency. J Pediatr Hematol Oncol 2000;22:441-445. 14. Adam de Beaumais T, Fakhoury M, Medard Y, Azougagh S, Zhang D, Yakouben K, Jacqz-Aigrain E. Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy. Br J Clin Pharmacol 2011;71:575-584. 15. Milek M, Karas Kuzelicki N, Smid A, Mlinaric-Rascan I. S-adenosylmethionine regulates thiopurine methyltransferase activity and decreases 6-mercaptopurine cytotoxicity in MOLT lymphoblasts. Biochem Pharmacol 2009;77:1845-1853. 16. Cetin M, Hiรงsรถnmez G, Tuncer AM, Kansu E, Canpynar H. The effect of short-course high-dose corticosteroid therapy on peripheral blood CD34+ progenitor cells in children with acute leukemia. Exp Hematol 1996;24:1191-1194. 17. Elmas SA, Cetin M, Tuncer M, Hicsonmez G. Myeloprotective effect of short course high dose methyl prednisolone treatment before consolidation therapy in children with acute myeloblastic leukemia. Am J Hematol 2005;80:1-5. 18. Goldsby RE, Perkins SL, Virshup DM, Brothman AR, Bruggers CS. Lymphoblastic lymphoma and excessive toxicity from chemotherapy: an unusual presentation for Fanconi anemia. J Pediatr Hematol Oncol 1999;21:240-243. 19. Johnson LA, Malayappan B, Tretyakova N, Campbell C, MacMillan ML, Wagner JE, Jacobson PA. Formation of cyclophosphamide specific DNA adducts in hematological diseases. Pediatr Blood Cancer 2012;58:708-714. 20. Kundu M, Compton S, Garrett-Beal L, Stacy T, Starost MF, Eckhaus M, Speck NA, Liu PP. Runx1 deficiency predisposes mice to T-lymphoblastic lymphoma. Blood 2005;106:36213624.

Case Report

DOI: 10.4274/tjh.2013.0090

Intravascular Large B-Cell Lymphoma Diagnosed on Prostate Biopsy: A Case Report Prostat Biopsisinde Tanı Konulan İntravasküler Büyük B Hücreli Lenfoma: Olgu Sunumu Nazan Özsan1, Banu Sarsık1, Asu Fergün Yılmaz2, Adnan Şimşir3, Ayhan Dönmez2 1Ege

University Faculty of Medicine, Department of Pathology, İzmir, Turkey University Faculty of Medicine, Department of Hematology, İzmir, Turkey 3Ege University Faculty of Medicine, Department of Urology, İzmir, Turkey 2Ege

Abstract: Intravascular large B-cell lymphoma (IVLBCL) is a very rare type of non-Hodgkin lymphoma, usually affecting elderly patients and characterized by selective infiltration of neoplastic cells within blood vessels’ lumina. IVLBCL diagnosed with prostatic involvement is extremely rare. We report a patient of 65 years old, having mostly neurological complaints but diagnosed with IVLBCL upon histopathological examination of transurethral prostate resection material, which revealed large neoplastic cell infiltration totally limited within the lumens of small vessels. By immunohistochemistry, neoplastic cell infiltration was positive with MUM1, bcl-6, and bcl-2 and negative with ALK1, CD10, and CD30, with a high Ki-67 proliferation index. CD34 and CD31 staining showed expression in endothelial cells, highlighting the intravascular nature of neoplastic infiltrate. The patient unfortunately refused to receive treatment and died of the disease 8 months after the diagnosis. IVLBCL, though very rare, should be considered in differential diagnosis of all organ biopsies with intravascular infiltration. Further improvements in the understanding of the pathogenesis and biology of this rare type of lymphoma are mandatory. Key Words: Non-Hodgkin lymphoma, Intravascular large B-cell lymphoma, Prostate, Non-germinal center B-cell

Özet: İntravasküler büyük B hücreli lenfoma (İVBBHL), non-Hodgkin lenfomaların nadir bir tipidir, genellikle ileri yaşta görülür, ve neoplastik hücrelerin damar lümeni içerisinde seçici infiltrasyonu ile karakterlidir. Prostat tutulumu ile tanı konan İVBBHL olguları ise çok nadirdir. Altmış beş yaşında, daha çok nörolojik şikayetleri olan, ancak transüretral prostat rezeksiyon materyalinin histopatolojik incelemesinde, tamamen küçük damar lümenleri içerisinde sınırlı neoplastik büyük hücre infiltrasyonunun görülmesi ile tanı konan bir olguyu sunuyoruz. Neoplastik hücre infiltrasyonu immunhistokimyasal incelemede MUM1, bcl-6 ve bcl-2 ile pozitif, ALK1, CD10, CD30 ile negatif saptandı, Ki67 proliferasyon indeksi yüksekti. CD34 ve CD31 endotelial hücrelerde pozitif olup, neoplastik infiltrasyonun damar içi yerleşimini belirgin olarak ortaya koydu. Hasta, ne yazık ki tedavi almayı kabul etmedi ve sekiz ay içerisinde hastalık nedeniyle kaybedildi. IVLBCL, az görülmekle birlikte, intravasküler infiltrasyon içeren tüm organ biopsilerinde ayırıcı tanı içerisinde yer almalıdır. Bu nadir lenfoma tipinin patogenez ve biyolojisinin aydınlatılmasına ihtiyaç duyulmaktadır.

Anahtar Sözcükler: Non-Hodgkin lymphoma, İntravasküler büyük B hücreli lenfoma, Prostat, Non-germinal merkez B hücre Address for Correspondence: Nazan ÖZSAN, M.D., Ege University Faculty of Medicine, Department of Pathology, İzmir, Turkey Phone: +90 232 388 10 25 E-mail: nazanozsan@yahoo.com Received/Geliş tarihi : March 14, 2013 Accepted/Kabul tarihi : April 2, 2013

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Özsan N, et al: Intravascular Large B Cell Lymphoma

Introduction Intravascular large B-cell lymphoma (IVLBCL) is a very rare type of non-Hodgkin lymphoma and is characterized by selective infiltration of neoplastic cells within blood vessels’ lumina. IVLBCL is an aggressive disease, usually affecting elderly patients with a poor prognosis [1]. Prostatic involvement in IVLBCL is extremely rare and has been described in only a few case reports. We hereby report a 65-year-old male patient who presented with neurological symptoms and was diagnosed with IVLBCL based on transurethral prostate resection (p-TUR) material. Case Presentation A 65-year-old man was admitted to the hospital with complaints of cervical and back pain. His past history revealed no remarkable illnesses, and he had no evidence of human immunodeficiency virus infection or other causes of immunodeficiency. Cervical spine magnetic resonance (MR) imaging showed signs consistent with degenerative discopathies at multiple levels and an increase in signals at the level of the conus medullaris, consistent with myelitis. Informed consent was obtained.

peroxidase detection system with diaminobenzidine as a chromogen was used to visualize bound secondary antibodies. The intravascular neoplastic cells were positive for CD20 and negative for CD3 and pan-cytokeratin. The following panel of antibody testing was then performed: CD10, bcl-6, bcl2, IRF4/MUM1, CD30, Ki-67, CD34, ALK, and CD31. The antibodies were scored as positive when >30% of tumor cells showed immunoexpression. Neoplastic cell infiltration was positive with MUM1, bcl-6, and bcl-2 and negative with ALK1, CD10, and CD30. The Ki-67 proliferation index was high, showing expression in about 90% of neoplastic large cells. CD34 and CD31 stainings showed expression in endothelial cells, highlighting the intravascular nature of the neoplastic infiltrate. In situ hybridization analysis was also performed using oligonucleotides complementary to Epstein–Barr early RNA transcripts in tissue sections of paraffin-embedded tissue in the same automated stainer (Ventana Medical Systems), which revealed negative results. The immunohistochemical expressions (IRF4/MUM1 and bcl-6 positivity, with CD10 negativity) revealed a non-germinal center B cell (non-GCB) phenotype when classified according to the Hans criteria [2]. The patient was diagnosed with IVLBCL from the p-TUR material.

The patient also had complaints indicating lower urinary tract symptoms, such as difficulty in urinating. Laboratory findings showed an increase in lactate dehydrogenase (LDH) levels (682 U/L; reference scale: 240480 U/L) and a mild decrease in platelet count (83,000/µL; reference range: 150,000-450,000/µL). The prostate-specific antigen (PSA) level was in normal limits (PSA: 2.37 ng/mL; reference range: <4 ng/mL; free PSA: 0.374 ng/mL; F.PSA/T. PSA: 0.16; reference: >0.15). Ultrasonography revealed enlargement in prostatic dimensions of 41x40x36 mm in size. p-TUR with spinal anesthesia was performed. After the surgery, the patient developed paraplegia and urinary and fecal incontinence. Lumbar spine MR imaging done after the surgery revealed signs consistent with lumbar spondylolysis, degenerative changes at multiple levels, and an increase of signals in the conus medullaris, which may reveal myelitis. He was then given prednisolone at 1 g/day for 10 days with the diagnosis of myelitis upon neurology consultation. Histopathological examination of the p-TUR specimen revealed large neoplastic cell infiltration totally limited within the lumens of small vessels in the stroma, while acinar and glandular structures were all benign. Immunohistochemical analysis was performed on paraffin-embedded tissue sections with an automated stainer (Ventana Benchmark XT, Ventana Medical Systems, Tucson, AZ, USA) according to protocol. A streptavidin-biotin-

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Figure 1. A) Infiltration of neoplastic large cells in the lumen of small vessels in transurethral prostate resection material (hematoxylin and eosin, original magnification: x200) by immunohistochemistry; B) CD31 staining with expression in endothelial cells highlighted the intravascular nature of neoplastic infiltrate (immunoperoxidase, original magnification: x400); C) Neoplastic infiltration is negative with cytokeratine, while benign glandular structures are positive (immunoperoxidase, original magnification: x200); D) Ki67 staining showed a high proliferation index in neoplastic cells (immunoperoxidase, original magnification: x200).

Ă&#x2013;zsan N, et al: Intravascular Large B Cell Lymphoma

The patient was hospitalized in the hematology department with a diagnosis of lymphoma. No enlarged superficial lymph nodes were found on physical examination. Positron emission tomography-computed tomography (PET-CT) showed an increase in fluorodeoxyglucose signals in both kidneys, which was interpreted as consistent with an inflammatory origin. Upper abdominal ultrasonography revealed mild splenomegaly (135 mm). The patient was given prednisolone at 1 g/day for 10 days, when bone marrow biopsy was performed. Bone marrow biopsy revealed no infiltration of lymphoma from either morphology or immunohistochemistry. R-CHOP chemotherapy was planned, but the patient refused to receive therapy and was discharged at his request. He received no therapy for IVLBCL and died from the disease 8 months after the diagnosis. Discussion and Review of the Literature In the current World Health Organization classification of hematopoietic neoplasms, IVLBCL is defined as a rare type of extranodal large B-cell lymphoma characterized by the selective infiltration of neoplastic cells in the lumina of vessels and capillaries, with the exception of large arteries and veins [3]. The disease is widely disseminated in extranodal sites: the bone marrow, central nervous system, skin, lungs, liver, and spleen are the most common sites of involvement [4]. Lymph node infiltration and lymphadenopathy are usually absent [5]. The clinical signs and symptoms are variable, related to the site of involvement. Neoplastic cells are rarely seen in bone marrow and peripheral blood smears and so IVLBCL is very difficult to diagnose; most of the cases reported have been confirmed by autopsy or cutaneous biopsies [6]. IVLBCL diagnosed in prostate specimens is extremely rare

Figure 2. Neoplastic large cells filling the vessels are strongly positive with CD20 (immunoperoxidase, original magnification: x200).

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in the literature, usually reported as single cases; to the best of our knowledge, this is the 10th such case reported [1,7,8,9,10,11,12]. Furthermore, none of the patients in the study by Murase et al., with a large series of 96 patients, were diagnosed with prostatic involvement, indicating the rarity of infiltration in this site [6]. All reported cases of primary IVLBCL of the prostate are in elderly patients aged above 60 years [10], and the median age defined for all IVLBCL cases is 67 years (range: 13-85 years) [4]. There are no specific laboratory findings indicating IVLBCL, but most patients were reported to display few pathologic findings in their complete blood counts, which should raise suspicion. Ferreri et al., in the study of a series of 38 patients, reported anemia in nearly 65% of patients, increased LDH and Î˛2 microglobulin levels in more than 80% of patients, and an elevated sedimentation component in 14% of patients [1]. Our patient had an increased LDH level and mild thrombocytopenia. He had complaints indicating lower urinary tract symptoms, which led to a urinary tract examination. No increase in PSA levels was found, but ultrasonography revealed enlargement in prostatic dimensions, leading to p-TUR surgery. The diagnosis of IVLBCL was established based on histopathological and immunohistochemical assessment of the biopsy. Neoplastic cell infiltration was totally limited within the lumens of small vessels in the stroma, within benign acinar and glandular structures. Neoplastic cell infiltration was positive with CD20, MUM1, bcl-6, and bcl-2 and negative with ALK1, CD10, CD30, and cytokeratin, with a high Ki-67 proliferation index. CD34 and CD31 staining with expression in endothelial cells highlighted the intravascular nature of the neoplastic infiltrate (Figures 1 and 2). The immunohistochemical profile of infiltration revealed a non-GCB cell phenotype (negative for CD10 and positive for IRF4/MUM1 and bcl-6) when classified according to the Hans criteria. Kanda et al. suggested that most IVLBCL cases might originate from post-germinal center cells, based on the presence of somatic mutation in variable regions of immunoglobulin heavy chain genes [13]. Subsequent studies supported their assertion as most IVLBCL cases have been reported to be of non-GCB cell origin [5,6]. Our patient had no enlarged lymph nodes; bone marrow biopsy showed no infiltration of lymphoma. Upper abdominal ultrasonography revealed mild splenomegaly, but PET-CT showed no findings consistent with extra sites of infiltration; according to these findings, he had stage 1E disease. In a study of 38 intravascular lymphoma patients, Ferreri et al. reported that 40% of the patients diagnosed in vivo had stage 1E disease according to the Ann Arbor staging system, but a disseminated infiltration was shown by autopsy in some stage I disease patients when death occurred a short time after diagnosis, highlighting the limitations of staging procedures 405

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in intravascular lymphoma patients [1,14]. The relatively high proportion of false negativity in classical staging procedures can be attributed to the fact that IVLBCL infiltration may usually be seen without tumor masses and apparent signs of involvement or lymphadenopathy [15]. The clinical manifestations of IVLBCL are extremely variable and symptoms are mostly related to the involved organ. Interestingly, clinical manifestations of the disease have been shown to differ between 2 distinct geographical areas. In Asian countries, hemophagocytic syndrome, bone marrow involvement, hepatosplenomegaly, fever, and thrombocytopenia have been documented at high frequencies, while central nervous system and skin involvements are predominantly seen in European countries [1,16]. Despite the defined geographical differences, neurological symptoms at initial diagnosis were reported in high incidences in both the Asian and Western types of the disease at 25% and 36%, respectively [1,17]. Among the few cases reported with prostatic involvement, 2 patients presented with neurological signs indicating spinal cord lesions [8,9]. Our patient had some neurological symptoms interpreted as myelitis according to MR findings, and following p-TUR surgery with spinal anesthesia, paraparesis developed. Whether these neurological symptoms were due to real neurological pathologies or to the invasion of tumor cells into the central nervous system or peripheral nerves remains unclear because an autopsy was not performed. The disease has an aggressive behavior, usually with a short outcome and fatal course [1]. The use of rituximab-containing chemotherapy regimens for the treatment of IVLBCL has been reported to improve outcomes [17,18]. Ferreri et al. reported overall survival at 3 years as 81% in 33 patients receiving immunochemotherapy [18]. Highdose chemotherapy with the support of autologous stem cell transplantation was shown to have efficacy in several reports [19,20]. Despite all treatment modalities, our patient refused to receive treatment for lymphoma and unfortunately died of the disease 8 months after the diagnosis. IVLBCL, though very rare, should be considered in differential diagnosis for elderly patients with elevated LDH levels, fever of unknown origin, and unexplained neurological symptoms. As for prostatic involvement, lower urinary tract obstruction symptoms can be seen, but an elevated level of PSA is not a common finding for lymphoma infiltration in the prostate, including rare IVLBCL cases [21]. Biopsy and immunohistochemical assessment are required for the diagnosis. Early diagnosis can result in a better outcome with the recent treatment options. Further improvements in the understanding of the pathogenesis and biology of this rare type of lymphoma are mandatory to achieve better outcomes for IVLBCL patients.

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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. Ferreri AJ, Campo E, Seymour JF, Willemze R, Ilariucci F, Ambrosetti A, Zucca E, Rossi G, López-Guillermo A, Pavlovsky MA, Geerts ML, Candoni A, Lestani M, Asioli S, Milani M, Piris MA, Pileri S, Facchetti F, Cavalli F, Ponzoni M. International Extranodal Lymphoma Study Group (IELSG). Intravascular lymphoma: clinical presentation, natural history, management and prognostic factors in a series of 38 cases, with special emphasis on the ‘cutaneous variant’. Br J Haematol 2004;127:173-183. 2. Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G, Müller-Hermelink HK, Campo E, Braziel RM, Jaffe ES, Pan Z, Farinha P, Smith LM, Falini B, Banham AH, Rosenwald A, Staudt LM, Connors JM, Armitage JO, Chan WC. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood 2004;103:275-282. 3. Nakamura S, Ponzoni M, Campo E. Intravascular large B-cell lymphoma. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC, 2008. 4. Ferreri AJ, Dognini GP, Campo E, Willemze R, Seymour JF, Bairey O, Martelli M, De Renz AO, Doglioni C, Montalbán C, Tedeschi A, Pavlovsky A, Morgan S, Uziel L, Ferracci M, Ascani S, Gianelli U, Patriarca C, Facchetti F, Dalla Libera A, Pertoldi B, Horváth B, Szomor A, Zucca E, Cavalli F, Ponzoni M. Variations in clinical presentation, frequency of hemophagocytosis and clinical behavior of intravascular lymphoma diagnosed in different geographical regions. Haematologica 2007;92:486-492. 5. Shimada K, Kinoshita T, Naoe T, Nakamura S. Presentation and management of intravascular large B-cell lymphoma. Lancet Oncol 2009;10:895-902. 6. Murase T, Yamaguchi M, Suzuki R, Okamoto M, Sato Y, Tamaru J, Kojima M, Miura I, Mori N, Yoshino T, Nakamura S. Intravascular large B-cell lymphoma (IVLBCL): a clinicopathologic study of 96 cases with special reference to the immunophenotypic heterogeneity of CD5. Blood 2007;109:478-485. 7. Alfaro J, Espinoza A, Manciquez M, Moyano L, González N, Larrondo M, Figueroa G. Intravascular lymphoma treated with anti CD20 monoclonal antibodies. Report of one case. Rev Med Chil 2004;132:1403-1406 (article in Spanish with English abstract).

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8. Pusch G, Feher G, Szomor A, Kover F, Gomori E, Illes Z. Intravascular lymphoma presenting with neurological signs but diagnosed by prostate biopsy: suspicion as a key to early diagnosis. Eur J Neurol 2009;16:39-41. 9. Quintini G, Barbera V, Franco V, Florena AM, Spadola V, Mariani G. Uncommon presentations of non-Hodgkin’s lymphoma: case 1. Intravascular large B-cell lymphoma: diagnosis on prostate biopsy. J Clin Oncol 2003;21:564-565. 10. Xu M, Yang Q, Li M, Geng W, Huang W, Chen Y. Prostate involvement by intravascular large B-cell lymphoma: a case report with literature review. Int J Surg Pathol 2011;19:544547. 11. Yin XR, Liu H, Chen HQ. Intravascular lymphomatosis of the prostate gland. Zhonghua Bing Li Xue Za Zhi 2005;34:189-90 (article in Chinese). 12. Csomor J, Kaszás I, Kollár B, Pajor L, Egyházi Z, Fekete S, Egyed M, Timár B. Prolonged survival using anti-CD20 combined chemotherapy in primary prostatic intravascular large B-cell lymphoma. Pathol Oncol Res 2008;14:281-284. 13. Kanda M, Suzumiya J, Ohshima K, Haraoka S, Nakamura N, Abe M, Tamura K, Kikuchi M. Analysis of the immunoglobulin heavy chain gene variable region of intravascular large B-cell lymphoma. Virchows Arch 2001;439:540-546. 14. Ponzoni M, Ferreri AJ. Intravascular lymphoma: a neoplasm of ‘homeless’ lymphocytes? Hematol Oncol 2006;24:105112. 15. Ponzoni M, Ferreri AJ, Campo E, Facchetti F, Mazzucchelli L, Yoshino T, Murase T, Pileri SA, Doglioni C, Zucca E, Cavalli F, Nakamura S. Definition, diagnosis, and management of intravascular large B-cell lymphoma: proposals and perspectives from an international consensus meeting. J Clin Oncol 2007;25:3168-3173.

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17. Shimada K, Matsue K, Yamamoto K, Murase T, Ichikawa N, Okamoto M, Niitsu N, Kosugi H, Tsukamoto N, Miwa H, Asaoku H, Kikuchi A, Matsumoto M, Saburi Y, Masaki Y, Yamaguchi M, Nakamura S, Naoe T, Kinoshita T. Retrospective analysis of intravascular large B-cell lymphoma treated with rituximab-containing chemotherapy as reported by the IVL study group in Japan. J Clin Oncol 2008;26:3189-3195. 18. Ferreri AJ, Dognini GP, Govi S, Crocchiolo R, Bouzani M, Bollinger CR, D’Incan M, Delaporte E, Hamadani M, Jardin F, Martusewicz-Boros M, Montanari M, Szomor A, Zucca E, Cavalli F, Ponzoni M. Can rituximab change the usually dismal prognosis of patients with intravascular large B-cell lymphoma? J Clin Oncol 2008;26:5134-5136; author reply 5136-5137. 19. Sawamoto A, Narimatsu H, Suzuki T, Kurahashi S, Sugimoto T, Sugiura I. Long-term remission after autologous peripheral blood stem cell transplantation for relapsed intravascular lymphoma. Bone Marrow Transplant 2006;37:233-234. 20. Bertz H, Zeiser R, Lange W, Fetscher S, Waller CF, Finke J. Longterm follow-up after high-dose chemotherapy and autologous stem-cell transplantation for high-grade B-cell lymphoma suggests an improved outcome for high-risk patients with respect to the age-adjusted International Prognostic Index. Ann Oncol 2004;15:1419-1424. 21. Kataja VV, Colleoni M, Bergh J. ESMO Minimum Clinical Recommendations for diagnosis, treatment and follow-up of locally recurrent or metastatic breast cancer (MBC). Ann Oncol 2005;16(Suppl.1):10-12.

16. Murase T, Nakamura S, Kawauchi K, Matsuzaki H, Sakai C, Inaba T, Nasu K, Tashiro K, Suchi T, Saito H. An Asian variant of intravascular large B-cell lymphoma: clinical, pathological and cytogenetic approaches to diffuse large B-cell lymphoma associated with haemophagocytic syndrome. Br J Haematol 2000;111:826-834.

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Case Report

DOI: 10.4274/tjh.2013.0049

Primary Splenic Angiosarcoma Revealed by Bone Marrow Metastasis Kemik İliği Metastazı ile Açığa Çıkan Primer Splenik Anjiosarkom Soumaya Anoun1, Sofia Marouane2, Asmae Quessar1, Said Benchekroun1 1Hopital 2Hopital

20 AOUT, Clinic of Hematology and Pediatric Oncology, Casablanca, Morocco Ibn Rochd, Clinic of Histopathology, Casablanca, Morocco

Abstract: Primary splenic angiosarcomas are the most common malignant non-hematopoietic tumors of the spleen. Metastatic diseases were found in 69% of patients in a reported series but the incidence of bone marrow involvement is unclear. We report a rare case of a 25-years-old Moroccan woman with unsuspected primary splenic angiosarcoma revealed by bone marrow metastasis. She presented with serious anemia and splenomegaly. Bone marrow biopsy revealed proliferating spindle cells. Computed tomography scanning showed an enlarged spleen with heterogeneous lesions. Splenectomy was performed and retrospective histological study of the spleen confirmed the diagnosis. She died 1 year after splenectomy.

Key Words: Angiosarcoma, Splenomegaly, Bone marrow infiltration Özet: Primer splenik anjiosarkom dalağın hematolojik olmayan tümörlerinden en sık karşılaşılanıdır. Yayınlanmış serilerde metastaz %69 olarak belirtilmişse de kemik iliği metastazının sıklığı tam bilinmemektedir. Biz Faslı bir kadın hastada kemik iliği metastazı ile açığa çıkan daha önceden şüphelenilmemiş bir primer splenik anjiosarkom vakasını rapor etmek istiyoruz. Yirmi beş yaşındaki hastanın ciddi anemi ve splenomegalisi mevcuttu. Kemik iliği biyopsisinde kemik iliği alanlarının yerini prolifere olmuş iğsi hücrelere bıraktığı gözlendi. Bilgisayarlı Tomografide büyümüş dalak içinde heterojen lezyonlar görüldü. Splenektomi yapıldı ve dalak üzerinde yapılan histolojik çalışma primer splenik anjiosarkom tanısını doğruladı. Hasta kemoterapi aldı fakat splenektomiden 1 yıl sonra metastazdan öldü.

Anahtar Sözcükler: Anjiosarkom, Splenomegali, Kemik iliği tutulumu

Address for Correspondence: Soumaya ANOUN, M.D., Hopital 20 AOUT, Clinic of Hematology and Pediatric Oncology, Casablanca, Morocco Phone: +212 662 39 11 04 E-mail: soumaya.anoun@gmail.com Received/Geliş tarihi : February 12, 2013 Accepted/Kabul tarihi : March 29, 2013

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Introduction Bone marrow is one of the common sites to be involved with solid tumors that metastasize via the bloodstream. Micrometastases can be demonstrated in the bone marrow of 30%-75% of patients with common malignancies [1]. Metastases involved in cortical bones often present with bony pain, pathologic fracture, and hypercalcemia [2]. Marrow involvement appears to be a prerequisite for the development, as bony metastases occur at the sites with hematopoietic marrow [2]. Extensive infiltration of the bone marrow may compromise hematopoietic functions. Hematologic abnormalities suggestive of marrow infiltration are peripheral cytopenia and leukoerythroblastic changes, and their occurrence is largely due to marrow replacement by tumor infiltration and reactive marrow fibrosis [2].

Turk J Hematol 2014;31:408-410

In January 2011, a second bone marrow biopsy was performed. Microscopic study highlighted, in the medullary compartments, a spindle proliferation with marked vascular differentiation. This was represented by slits or sometimes large virtual vascular cavities, surrounded and partitioned by turgescent endothelial cells. Islets of residual hematopoiesis were identified; they consisted of elements belonging to the 3 myeloid lineages. The new immunohistochemical study showed the same profile. The spindle cells did not express c-kit (CD117), with a positive internal control. The diagnosis of medullary location of vascular kaposiform proliferation was confirmed. Further immunostaining with HHV8 was performed and was negative (Figure 3). HIV serology and splenectomy were discussed.

Bone marrow aspirate and biopsy can be used to easily diagnose medullary metastases. The incidence of bone marrow involvement varies with types of primary tumors. The solid tumors most frequently detected in bone marrow in adults are carcinomas of the breast, prostate, lungs, and gastrointestinal tract [3,4]. Angiosarcomas are rare, comprising only about 2% of all soft tissue sarcomas. Primary angiosarcomas can occur at any site of the body. The most common sites are the skin and superficial soft tissues, followed by the breast, liver, spleen, and bone. Primary splenic angiosarcoma is an uncommon primary tumor. Bone marrow metastasis in splenic angiosarcoma, however, is exceedingly rare. We report here the pathologic findings of a patient with bone marrow metastasis of a primary unsuspected splenic angiosarcoma. Case Presentation In December 2010, a 25-year-old woman complained of anemic syndrome. She presented with serious normocytic normochromic anemia with nodular splenomegaly. Bone marrow biopsy was performed. Histopathological analysis found regular bone trabeculae and delimiting medullary compartments with normal cellular richness, where the 3 myeloid lineages were represented at different maturation stages. There was a spindle proliferation delineating vascularlike slits, which were sometimes empty or contained erythrocytes. The cells presented moderate nuclear atypia, often with a prominent acidophilic nucleolus.

Figure 1. Bone marrow biopsy: â&#x20AC;&#x153;bloodyâ&#x20AC;? appearance with spindle cells proliferation, some cytologic atypia, and mitotic activity.

The immunohistochemical study showed that spindle cells expressed CD34 and did not express CD31. Results for CD117 (c-kit) were difficult to interpret (Figures 1 and 2). The medullary location of a vascular-like spindle proliferation was the most evident clue for diagnosis. Another bone marrow biopsy was recommended to better pinpoint the diagnosis. Informed consent was obtained.

Figure 2. Immunohistochemestry: CD34 (+), CD31 (-), HHV8 (-). 409

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Anoun S, et al: Primary Splenic Angiosarcoma

angiosarcomas arising in the spleen have a unique propensity for bone marrow metastasis, but this is unfortunately not well documented in the literature [8]. We have found 3 cases in the literature in which primary splenic angiosarcoma was extended to the bone marrow. The KI-67 proliferation index determines prognosis. Because splenic angiosarcoma is a rare tumor, no specific regimen of chemotherapy has been employed in enough cases to enable the drawing of a conclusion as to effects on survival [6]. Despite best efforts, the prognosis for this diagnosis is poor, with mean survival ranging from 10.3 to 14.4 months [9]. Conclusion Figure 3. Microscopy: spindle proliferation with marked vascular differentiation. Nuclear atypia, with very few atypical cells and some calcifications. An abdominal computed tomography scan showed an enlarged spleen with heterogeneous lesions. Splenectomy was done in February 2011. The spleen weighed 706 g and measured 21x14x8 cm. The outer surface was smooth. When cut, multiple whitish nodules measuring between 0.4 and 1 cm in diameter were seen. The splenic parenchyma was the site of saffron-colored deposits. At the hilum, 15 lymph nodes were isolated, measuring between 4 and 12 mm in diameter. Histological examination showed that the nodules described above corresponded to a spindle proliferation with marked vascular differentiation. Cells were sometimes epithelioidlike, presenting moderate to marked nuclear atypia, with very few atypical cells and budding nuclei, estimated at 10 mitoses per 10 fields at high magnification. Vascular slits were engorged by erythrocytes Moreover, there were calcifications with giant cells and a small focus of necrosis. Fourteen lymph nodes among the 15 examined were metastatic. The final diagnosis was that of a well-differentiated angiosarcoma (grade II) of the spleen with lymph node metastases (14 N+ of 15). The patient received a chemotherapy course. Unfortunately, the patient died 1 year after the splenectomy from metastasis. Discussion and Review of the Literature Primary splenic angiosarcoma is the most common malignant non-hematopoietic tumor of the spleen [5]. These tumors are rare, highly aggressive, and lethal. Metastases tend to occur early and spread widely [6]. Metastatic diseases were found in 69% of the patients in a reported series [7]. Early metastasis from splenic angiosarcoma largely contributes to its poor prognosis. Up to 86% of patients have distant metastases at the time of presentation. Bone marrow metastasis, however, is exceptionally rare. Interestingly, Wang et al. suggested that 410

We believe that the true incidence of bone marrow metastasis is underreported. Bone marrow aspiration and biopsy should be systematically performed in angiosarcoma patients, especially when hematological abnormalities are found on blood count. 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. Pantel K, Cote RJ, Fodstad O. Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 1999;91:11131124. 2. Wang C, Rabah R, Blackstein M, Riddell RH. Bone marrow metastasis of angiosarcoma. Pathol Res Pract 2004;200:551555. 3. Anner RM, Drewinko B. Frequency and significance of bone marrow involvement by metastatic solid tumors. Cancer 1977;39:1337-1344. 4. Papac RJ. Bone marrow metastases: a review. Cancer 1994:74:2403-2413. 5. Rosai J. Spleen. In: Rosai J (ed). Rosai and Ackerman’s Surgical Pathology. Vol. 2. 9th ed. Philadelphia, Mosby, 2004. 6. Ekinci Ö, Okur Ö, Aksoy F, Demiral G, Evcimik T, Yalman H, Yiğitbaşı R. Diagnosis, treatment, radiologic and pathologic findings of splenic angiosarcoma: a case report. Marmara Medical Journal 2009;22;56-58. 7. Falk S, Krishnan J, Meis JM. Primary angiosarcoma of the spleen. A clinicopathological study of 40 cases. Am J Surg Pathol 1993;17:959-970. 8. Datta J, Toro TZ, Keedy VL, Merchant NB. Synchronous bone marrow metastasis from primary splenic angiosarcoma. Am Surg 2010;76:160-162. 9. Varma N, Vaiphei K, Varma S. Angiosarcoma presenting with leucoerythroblastic anaemia bone marrow fibrosis and massive splenomegaly. Br J Haematol 2000;110:503.

Case Report

DOI: 10.4274/tjh.2013.0119

Aplastic Anemia Associated with Oral Terbinafine: A Case Report and Review of the Literature Oral Terbinafin İlişkili Aplastik Anemi: Bir Olgu Sunumu ve Literatür Derlemesi Bülent Kantarcıoğlu1, Hüseyin Kemal Türköz2, Güven Yılmaz3, Funda Pepedil Tanrıkulu3, Işık Kaygusuz Atagündüz3, Cafer Adıgüzel3, Tülin Fıratlı Tuğlular3 1Okmeydanı

Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey University Faculty of Medicine, Department of Pathology, İstanbul, Turkey 3Marmara University Faculty of Medicine, Department of Hematology, İstanbul, Turkey 2Marmara

Abstract: Onychomycosis (OM) is a common fungal infection of the toenails and/or fingernails that is highly prevalent in the general population and also responsible for significant morbidity. OM is caused by dermatophytes, nondermatophytic molds, or yeast. Today systemic antifungal agents are considered as the gold standard for all types of OM. Here we report a case of aplastic anemia associated with oral terbinafine use and a review of the literature on hematological toxicities associated with terbinafine. Key Words: Onychomycosis, Terbinafine, Aplastic anemia, Hematological toxicity, Pancytopenia, Adverse events

Özet: Onikomikoz (OM) el ve ayak tırnaklarının sık görülen fungal enfeksiyonudur. Genel toplumda prevalansı yüksek bir hastalık olması nedeniyle önemli morbiditeye yol açmaktadır. OM dermatofitler, nondermatofitik küf mantarları veya mayalar ile ortaya çıkan hastalıklardır. Günümüzde onikomikozun tedavisinde sistemik antifungal ajanlar tüm OM tiplerinde altın standart tedavi olarak kabul edilmektedir. Biz burada, oral terbinafin kullanımı sırasında gelişen bir aplastik anemi olgumuzu ve literatürde terbinafine ile ilişkilendirilmiş olan hematolojik toksisitelerin derlemesini sunuyoruz. Anahtar Sözcükler: Onikomikoz, Aplastik anemi, Terbinafin, Hematolojik toksisite, Pansitopeni, Yan etkiler Introduction Onychomycosis is a very frequent fungal nail infection. The prevalence can be as high as 28%-40%, especially in elderly populations. Terbinafine is an antifungal agent with both fungicidal and fungistatic properties, which is highly effective and is the most frequently used agent in onychomycosis. Oral terbinafine is generally well tolerated with minimal reports of serious drug reactions. These rare

adverse events are mostly reported as case presentations, and it is important to be familiar with them in order to be able to evaluate the risk and inform patients accordingly [1,2,3]. Here we report a case of aplastic anemia (AA) associated with oral terbinafine use and a review of the literature on hematological toxicities associated with terbinafine. Written informed consent was obtained from the patient and her husband for publication of this manuscript and accompanying images.

Address for Correspondence: Bülent KANTARCIOĞLU, M.D., Okmeydanı Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey Phone: +90 532 547 62 08 E-mail: bulentkantarcioglu@gmail.com Received/Geliş tarihi : April 7, 2013 Accepted/Kabul tarihi : May 14, 2013

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Case Presentation A 41-year-old female presented with malaise, severe fatigue, nausea, and vaginal bleeding in April 2011. In her past history she was healthy, except that she reported taking terbinafine pills for 8 weeks for the treatment of longstanding recurrent toenail onychomycosis. She did not report any immune reactions or allergies to any drugs or substances. Her previous gynecological examination was normal, with a normal β-human chorionic gonadotropin level. Her complete blood count (CBC) revealed pancytopenia with white blood cell count of 3.2x109/L, absolute neutrophil count of 0.8x109/L, hemoglobin of 7.4 g/dL, and platelet count of 34x109/L. Her physical examination was unremarkable with no evidence of lymphadenopathy or organomegaly, except for a few petechiae and ecchymoses on bilateral legs. Peripheral blood smear was consistent with pancytopenia. Reticulocyte count was 0.7. Liver enzymes were elevated [AST: 61 U/L (N: 10-37 U/L), ALT: 117 U/L (N: 10-40 U/L), ALP: 434 U/L (N: 0-270 U/L), GGT: 471 U/L (N: 7-49 U/L)]. Renal function tests and lactate dehydrogenase were normal. Bone marrow aspiration and biopsy revealed severe reduction of all cell lineages without evidence of neoplastic infiltration, dysplasia, or fibrosis. The counted cellularity was 5% in bone marrow. Bone marrow karyotype analysis was normal. A gastroenterology consultation performed for the liver enzyme abnormalities did not provide an etiologic factor, pointing toward drug-induced hepatitis. Further work-ups, including levels of vitamin B12 and folate; neck, chest, and abdominopelvic computerized tomography; serology and polymerase chain reaction (PCR) tests for viral hepatitis, human immunodeficiency, Epstein-Barr virus, parvovirus B19, and cytomegalovirus; FLAER test for paroxysmal nocturnal hemoglobinuria; antinuclear antibody test; HLA-DRB1*15; and quantiferon test for tuberculosis, were all negative. The patient was diagnosed with AA, which was not severe at that time. Terbinafine treatment was stopped. Due to the use of a drug with probable hematologic toxicity, follow-up with supportive care was planned for the patient. During 3-4 weeks of follow-up time, blood values worsened with the need for erythrocyte and thrombocyte transfusions, in accordance with very severe AA (SAA). She did not have a matched related donor for transplantation. After confirming the diagnosis with a second bone marrow biopsy, she received rabbit antithymocyte globulin (ATG) plus cyclosporine-A (CYC). The clinical outcome after ATG + CYC was poor due to transient worsening of hematopoiesis and infectious complications. She spent 3 months in the hospital with perianal abscess, invasive aspergillosis, zoster virus reactivation, and several catheter infections. She required physical and psychological rehabilitation. Fortunately, the blood values began to recover at the end of the fourth month and full hematologic recovery was achieved at the end of the 412

Kantarcıoğlu B, et al: Aplastic Anemia and Terbinafine

sixth month. The patient is still in complete remission after 18 months of ATG + CYC treatment (Figures 1 and 2). Discussion and Review of the Literature Onychomycosis refers to all fungal infections of the nails. It is difficult to cure, has high recurrence rates, and can significantly affect a patient’s quality of life. Topical therapies are generally ineffective, and today treatment with systemic antifungal agents is accepted as the gold standard method for onychomycosis. In clinical trials, continuous terbinafine has repeatedly demonstrated higher efficacy when compared to other antifungal treatments. The recommended dosage for the treatment of onychomycosis is 250 mg/day orally for 12 weeks for toenails and 6 weeks for fingernails [1,2,3]. Oral terbinafine is generally well tolerated with minimal reports of serious drug reactions. Two large-scale postmarketing surveillance studies showed that the incidence of serious

Figure 1. Bone marrow trephine biopsy: low cellularity in the bone marrow consistent with aplastic anemia (H&E, 20x).

Figure 2. Bone marrow trephine biopsy: a few hematopoietic cells intermixed with lymphocytes and plasmocytes in interstitial areas (H&E, 100x).

35/not

78/M

69/F

44/F

74/F

79/F

68/F

63/F

61/F

66/F

73/F

reported

60/F

46 days

67 days

27 days

46 days

39 days

Not reported

32 days

35 days

31 days

27 days

34 days

32 days

Duration of Age (years)/ Terbinafine Treatment Sex

Reference Number

Neu 0.00

Neu 0.04

WBC 2.2

Neu 0.9

WBC 3.8

Neu 0.14

Neu 0.04

WBC 1.9

reported

agranulocytosis

Not given;

Neu 0.5

WBC 1.5

Recovered

Reported Outcome

Recovered

recovered antibiotics, amphotericin, nystatin anorexia, candidiasis

Recovered

recovered

Not yet

Recovered

ulceration, fever,

None reported

No treatment reported

G-CSF, one dose

No treatment reported

Hospitalized, i.v. antibiotics Died

G-CSF

antibiotics,

Hospitalized, i.v.

Hospitalized, i.v. antibiotics Recovered

No treatment reported

Cephalexin

Hospitalized, i.v. antibiotics, G-CSF

Management

Not yet 250 mg/day

250 mg/day

Terbinafine Dose

Hospitalized, i.v.

Mouth and tongue

Mouth ulceration, ageusia

Mouth ulceration

None reported

Septic shock

None reported

chills, headache

Mouth ulceration, fever,

anorexia

Neu 0.03

Mouth/tongue ulceration,

Neu 0.3

Flu-like illness

Mouth ulcers, cellulitis

Mouth/tongue ulceration, fever, myalgia, malaise

Reported Symptoms

WBC 2.18

Neu 0.9

WBC 2.4

Neu 0.3

WBC 2.9

WBC 1.2 Neu 0.00

Nadir of Cytopenia

Table 1. Reported cases of terbinafine-associated hematological toxicity in the literature.

Kantarcıoğlu B, et al: Aplastic Anemia and Terbinafine Turk J Hematol 2014;31:411-416

413

414

55/F

42/M

63/M

15/M

53/F

75/M

53/F

41/F

Presented

8 weeks

Not reported

63 days

Not reported

4 weeks

30 days

4 weeks

6 weeks

recovery obtained with ATG + CYC

Rtc 0.1%

Not recovered

Recovered

Plt 12,000

Supportive care initially

Observation

1.5 mg/kg/day

Hospitalized, prednisolone

spontaneously,

250 mg/day

Malaise, fatigue, nausea, vaginal bleeding

Recovered

Hospitalized, i.v. antibiotics Recovered

G-CSF

Oral penicillin, observation

Hb 6.5

250 mg/day

Epistaxis

250 mg/day

Recovered

Neu 0.2

WBC 1.5

Plt 63,000

Plt 8000

Ecchymosis, buccal hematoma

nal pain

Plt 99,000

Nausea, vomiting, diarrhea, fever, abdomi-

Hb 13.5

Fatigue

Fever, sore throat

WBC 500

Neu 0.01

WBC 1.9

Neu 0.00

WBC 2900

Plt 68,000

G-CSF

Hospitalized, i.v.

G-CSF

antibiotics,

Hospitalized, i.v.

Hct 24.6

Not reported

250 mg/day

G-CSF

antibiotics,

Hospitalized, i.v.

G-CSF

antibiotics,

Hospitalized, i.v.

antibiotics,

Fever, gum bleeding

Fever, tongue ulceration,

250 mg/day

Not reported

Neu not reported

WBC 1400

Neu 0.34

WBC 3.5

Neu 0.00

Fever, dehydration, sepsis syndrome

syndrome

Neu 0.11 WBC 1.6

Mouth ulceration, fever, cellulitis, sepsis

WBC 1.6

M: male, F: female, WBC: white blood cell, Neu: neutrophil, Hb: hemoglobin, Hct: hematocrit, Plt: platelet, Rtc: reticulocyte count.

case

60/M

Table 1. Reported cases of terbinafine-associated hematological toxicity in the literature.

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Kantarcıoğlu B, et al: Aplastic Anemia and Terbinafine

side effects was <1% [4,5]. In contrast, 2 studies of registry data from Austria and Denmark drew attention to blood dyscrasias associated with terbinafine [6,7]. Notable adverse events have been reported, including hepatitis requiring liver transplantation [8], drug-induced lupus reactions, severe skin reactions such as Stevens-Johnson syndrome, and, much less often, neutropenia/agranulocytosis and thrombocytopenia [9,10,11,12,13,14,15,16,17]. To our knowledge, ours is the first reported case of AA associated with terbinafine use. AA is usually diagnosed within the setting of pancytopenia and hypocellular bone marrow when other diseases are excluded. SAA is almost always fatal if untreated. Once SAA is established, therapy should not be delayed in the hope of spontaneous recovery [18,19]. Many drugs have been associated with AA. The vast majority of patients exposed to these drugs do not develop AA and the reason for idiosyncratic reactions is unknown. AA can develop as a direct response to exposure, but it can also develop indirectly through immune-mediated mechanisms. P-glycoprotein (P-gp), the MDR-1 gene product, and the multidrug resistance-associated protein are energy-dependent transmembrane efflux pumps for a variety of lipophilic drugs. Underexpression of P-gp in normal cells might allow cytoplasmic accumulation of drugs and enhance their toxic effects. Two studies found that P-gp activity was decreased in patients with AA; levels were lowest in a subgroup with druginduced AA [20,21]. In our case, the prolonged use of the highly lipophilic agent terbinafine might have caused direct toxicity, leading to AA. Additionally, in a recently published study, the release of IL-8 and TNFα was significantly increased by treatment with terbinafine, which can explain how terbinafine may also cause immune-mediated injury [22]. Lupus-like reactions reported with terbinafine use may be an additional evidence of immune-mediated injury as an underlying mechanism. However, in most cases, the trigger of the mechanism of AA remains unclear. Historically, drug-induced AA has not been easily distinguished from idiopathic forms of the disease since causality is difficult to establish [23]. A review of hematologic toxicities associated with the use of terbinafine showed that the duration of terbinafine exposure leading to hematological toxicity is almost 1 month; the degree of cytopenia can be severe and patients mostly presented with infectious complications requiring hospitalization. While the clinical outcome was reversible in most cases, our patient required additional treatment with a high burden of risk and complications (Table 1). In conclusion, keeping in mind the slow and persistent course of onychomycosis requiring long-term treatment, the high rate of success achieved with terbinafine, and the

wide range of the population receiving terbinafine treatment, we advise detailed information of patients in regard to adverse events and we recommend monitorization of CBC at baseline and every month during terbinafine treatment. To our knowledge, this is the first case report of irreversible SAA following treatment with terbinafine that required immunosuppressive treatment with ATG + CYC. This case highlights the need for routine blood count monitoring during treatment with terbinafine. In these patients, clinicians should consider the rare incidence of SAA when there is agranulocytosis or pancytopenia. 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. Shemer A. Update: medical treatment of onychomycosis. Dermatol Ther 2012;25:582-593. 2. Grover C, Khurana A. An update on treatment of onychomycosis. Mycoses 2012;55:541-551. 3. Van Duyn GL, Elewski BE. Recent updates in oral terbinafine: its use in onychomycosis and tinea capitis in the US. Mycoses 2011;54:679-685. 4. Hall M, Monka C, Krupp P, O’Sullivan D. Safety of oral terbinafine: results of a postmarketing surveillance study in 25,884 patients. Arch Dermatol 1997;133:1213-1219. 5. O’Sullivan DP, Needham CA, Bangs A, Atkin K, Kendall FD. Postmarketing surveillance of oral terbinafine in the UK: report of a large cohort study. Br J Clin Pharmacol 1996;42:559-565. 6. ADRAC. Terbinafine and blood dyscrasias. Aust Adverse Drug React Bull 2004;23:19. 7. Bangsgaard N, Saunte DM, Folkenberg M, Zachariae C. Serious adverse events reporting on systemic terbinafine: a Danish register-based study. Acta Derm Venereol 2011;91:358-359. 8. Perveze Z, Johnson MW, Rubin RA, Sellers M, Zayas C, Jones JL, Cross R, Thomas K, Butler B, Shrestha R. Terbinafineinduced hepatic failure requiring liver transplantation. Liver Transpl 2007;13:162-164. 9. Pillans PI, Boyd IW. Toenails and agranulocytosis. Intern Med J 2007;37:572-575. 10. Gupta AK, Soori GS, Del Rosso JQ, Bartos PB, Shear NH. Severe neutropenia associated with oral terbinafine therapy. J Am Acad Dermatol 1998;38:765-767. 11. Ornstein DL, Ely P. Reversible agranulocytosis associated with oral terbinafine for onychomycosis. J Am Acad Dermatol 1998;39:1023-1024. 12. Shapiro M, Li LJ, Miller J. Terbinafine-induced neutropenia. Br J Dermatol 1999;140:1196-1197.

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13. Conjeevaram G, Vongthavaravat V, Sumner R, Koff RS. Terbinafine-induced hepatitis and pancytopenia. Dig Dis Sci 2001;46:1714-1716. 14. Aguilar C, Mueller KK. Reversible agranulocytosis associated with oral terbinafine in a pediatric patient. J Am Acad Dermatol 2001;45:632-634. 15. Kovacs MJ, Alshammari S, Guenther L, Bourcier M. Neutropenia and pancytopenia associated with oral terbinafine. J Am Acad Dermatol 1994;31:806. 16. Tsai HH, Lee WR, Hu CH. Isolated thrombocytopenia associated with oral terbinafine. Br J Dermatol 2002;147:627628. 17. Grunwald MH. Thrombocytopenia associated with oral terbinafine. Int J Dermatol 1998;37:634. 18. Scheinberg P. Aplastic anemia: therapeutic updates in immunosuppression and transplantation. Hematology 2012;2012:292-300.

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Kantarcıoğlu B, et al: Aplastic Anemia and Terbinafine

19. Guinan EC. Diagnosis and management of aplastic anemia. Hematology 2011;2011:76-81. 20. Calado RT, Garcia AB, Falcão RP. Decreased activity of the multidrug resistance P-glycoprotein in acquired aplastic anaemia: possible pathophysiologic implications. Br J Haematol 1998;102:1157-1161. 21. Calado RT, Garcia AB, Gallo DA, Falcão RP. Reduced function of the multidrug resistance P-glycoprotein in CD34+ cells of patients with aplastic anaemia. Br J Haematol 2002;118:320326. 22. Mizuno K, Fukami T, Toyoda Y, Nakajima M, Yokoi T. Terbinafine stimulates the pro-inflammatory responses in human monocytic THP-1 cells through an ERK signaling pathway. Life Sci 2010;87:537-544. 23. Mintzer DM, Billet SN, Chmielewski L. Drug-induced hematologic syndromes. Adv Hematol 2009;2009:495863.

DOI: 10.4274/tjh.2014.0150

Letter to the Editor

Significant Differences in Thymic Index of Thalassemia Major Patients Talasemi Major Hastalarının Timik İndeksinde Anlamlı Farklılık Yeşim Oymak1, Bülent Güzel2, Hüseyin Gümüş2, Erdem Dağlıoğlu3, Ali Ayçiçek2, Ahmet Koç2, Derya Özyürük4 1Dr.

Behçet Uz Children’s Hospital, Clinic of Hematology, İzmir, Turkey University Faculty of Medicine, Department of Pediatric Hematology, Şanlıurfa, Turkey 3Harran University Faculty of Medicine, Department of Radiology, Şanlıurfa, Turkey 4Şanlıurfa Children’s Hospital, Clinic of Pediatric Hematology Oncology, Şanlıurfa, Turkey 2Harran

To the Editor, The thymus can be detected by ultrasonography until the pre-adolescent period. After the early teens, it begins to decrease in size [1]. In thalassemia patients, all organs have the risk of organ dysfunction because of iron overload. Only a few studies showed the size of the thymus in older children and there were no studies in thalassemia patients [2,3]. The side effects of iron overload in thalassemia major (TM) patients’ organs were examined through a literature search, but it appears that the thymus has not been emphasized enough to provide an adequate number of studies. The purpose of this study was to determine whether the size of the thymus in thalassemia patients differed from that in healthy children. Sixty-five children with TM, aged 1 to 19 years, who had been followed up in the pediatric hematology department at Harran University were enrolled in the study between 1 July and 31 July 2012. Fort-three TM patients and healthy siblings of patients with another diagnosis were enrolled as a control group because there was no literature related to the normal range of thymus size for children older than 8 years. The thymic size (thymic index) was calculated by multiplying the largest transverse diameter by the largest longitudinal diameter, measured in millimeters by ultrasonography (Toshiba Corporation Medical System Division, Tokyo, Japan, type SSA 240 A with a 7.5-MHz linear probe) [4]. Ultrasonography was

Figure 1. Dot plot of thymic index among children with TM (□—.) and healthy controls (○ ----) at various ages. performed by the same radiologist. The average ferritin levels for the last 6 months were recorded. The study was approved by the Ethics Committee of Harran University. The differences between groups were tested with Mann-Whitney U tests. Correlations were evaluated with Spearman’s test.

Address for Correspondence: Yeşim OYMAK, M.D., Dr. Behçet Uz Children’s Hospital, Clinic of Hematology, İzmir, Turkey Gsm: +90 532 355 42 28 E-mail: yesimoymak@hotmail.com Received/Geliş tarihi : April 11, 2014 Accepted/Kabul tarihi : June 24, 2014

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The characteristics of the TM patients and the control group are shown in Table 1. There were no differences in terms of sex, age, weight, and height. However, the thymic index of the TM patients was lower than that of the control group (Figure 1). The TM patients’ mean (± standard deviation) ferritin level was 2967 (±1842) µg/mL. Ferritin level and thymic index did not correlate (r=-0205, p=0.104). Seven (10.7%) TM patients were splenectomized. The median (min-max) thymic index of the splenectomized patients was 0.0 (0.0-304.0); it was 230.5 (0.0-903.0) in the non-splenectomized patients (p=0.001). Splenectomized patients were older than non-splenectomized patients (p=0.001). In TM patients, the thymic index has not been previously studied. Although corticosteroid effect was eliminated and there were no differences in the age, sex, weight, and height, the thymic index of the TM patients was lower than that of the control group. Iron overload is the best-known harmful effect of chronic transfusion on organs, and it might affect the thymus as well. The size of the thymus is affected by several factors like steroids, infections, and X-rays [5,6,7,8,9]. It has also been found that thymus activity is related to thymus size [10]. In this study, finding a difference in thymus size between TM patients and controls supported the hypothesis that chronic transfusions might affect the thymus; however, there were weaknesses in the groups’ histories in terms of infection, malnutrition, and having undergone X-rays. This is a preliminary study, and the only difference that we knew about was that the groups were receiving chronic blood transfusions, which might contribute to decreased thymus size in TM patients. Zinc level, which is known to be lower in TM patients, may affect the size of the thymus, but there were no data available about the zinc levels of our patients [8]. This study found no association between the thymic index and ferritin level; however, each organ may be affected to a different degree by the same ferritin level [11]. Smaller thymus sizes in splenectomized patients might depend on their older ages. Table 1. The characteristics of the TM patients and the control group.

TM patients Control group n=65 n=43 Age (years)a

6.91 (1.00-19.00)†

7.04 (1.00-15.40)†

Sex (male/female)

31/34†

21/22†

Weight (kg)a

22 (5-43)†

22 (9-56)†

Height (cm)a

126 (64.0-160)†

119 (80-170)†

208 (0-903)*‡

391 (0-1189)*‡

Thymic indexa (mmxmm)

aMedian (min-max), †p>0.05, *p=0.01. TM: Thalassemia major.

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It was also shown that stress and aging caused thymic involution, which might protect the organism from the danger of autoimmune diseases [12]. In one study it was shown that thymus size declined with age in both children with atopic dermatitis and healthy controls. However, the size of the thymus among children with active atopic dermatitis was higher compared to healthy controls [3]. Having a smaller thymus may be an advantage for TM patients, who are prone to alloimmunization related to transfusion. In conclusion, thymic involution occurred more rapidly in the TM group than in the normal controls. Further studies that include other parameters, such as T2 magnetic resonance imaging of the thymus for iron load and factors that may affect thymus size, with bigger sample sizes are required to objectively determine the effect of iron overload on thymic involution. 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. Key Words: Thymus, Blood transfusion, Beta-thalassemia, Iron overload, Tymic lndex Anahtar Sözcükler: Timüs, Kan Transfüzyonu, Beta talasemi, Demir yüklenmesi, Timik indeks References 1. Aspinall R, Andrew D, Pido-Lopez J. Age-associated changes in thymopoiesis. Springer Semin Immunopathol 2002;24:87-101. 2. Adam EJ, Ignotus PI. Sonography of the thymus in healthy children: frequency of visualization, size, and appearance. AJR Am J Roentgenol 1993;161:153-155. 3. Olesen AB, Andersen G, Jeppesen DL, Benn CS, Juul S, Thestrup-Pedersen K. Thymus is enlarged in children with current atopic dermatitis. A cross-sectional study. Acta Derm Venereol 2005;85:240-243. 4. Yekeler E, Tambag A, Tunaci A, Genchellac H, Dursun M, Gokcay G, Acunas G. Analysis of the thymus in 151 healthy infants from 0 to 2 years of age. J Ultrasound Med 2004;23:1321-1326. 5. Kolte L, Dreves AM, Ersbøll AK, Strandberg C, Jeppesen DL, Nielsen JO, Ryder LP, Nielsen SD. Association between larger thymic size and higher thymic output in human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy. J Infect Dis 2002;185:15781585. 6. Caffey J, Di Liberti C. Acute atrophy of the thymus induced by adrenocorticosteroids: observed roentgenographically in living infants: a preliminary report. Am J Roentgenol 1959;82:530-540. 7. Griffith SP, Levine QR, Kaber DH, Blumenthal S. Evaluation of enlarged cardiothymic image in infancy: thymolytic effect of steroid administration. Am J Cardiol 1961;8:311-318.

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8. Hasselbalch H, Jeppesen DL, ErsbĂ¸ll AK, Lisse IM, Nielsen MB. Sonographic measurement of thymic size in healthy neonates. Relation to clinical variables. Acta Radiol 1997;38:95-98. 9. Mahyar A, Ayazi P, Pahlevan AA, Mojabi H, Sehhat MR, Javadi A. Zinc and copper status in children with betathalassemia major. Iran J Pediatr 2010;20:297-302. 10. Auwaerter PG, Kaneshima H, McCune JM, Wiegand G, Griffin DE. Measles virus infection of thymic epithelium in the SCID-hu mouse leads to thymocyte apoptosis. J Virol 1996;70:3734-3740.

11. Kolnagou A, Natsiopoulos K, Kleanthous M, Ioannou A, Kontoghiorghes GJ. Liver iron and serum ferritin levels are misleading for estimating cardiac, pancreatic, splenic and total body iron load in thalassemia patients: factors influencing the heterogenic distribution of excess storage iron in organs as identified by MRI T2*. Toxicol Mech Methods 2013;23:48-56. 12. Aronson M. Hypothesis: involution of the thymus with aging--programmed and beneficial. Thymus 1991;18:7-13.

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Letter to the Editor

DOI: 10.4274/tjh.2014.0081

c.761C>T Mutation Linked Hyper IgM Syndrome Presenting with Hypertransaminasemia and Arthritis Hipertransaminazemi ve Artrit ile Kendini Gösteren c.761C>T Mutasyonuna Bağlı Hiper IgM Sendromu Mehmet Halil Celiksoy1, Stephan Borte2, Aydan İkincioğulları3, Meltem Ceyhan Bilgici4, Filiz Karagöz5, Ayhan Gazi Kalaycı6, Alişan Yıldıran3 1Ondokuz

Mayıs University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Samsun, Turkey University Faculty of Medicine, Department of Clinical Immunolgy, Leipzig, Germany 3Ankara University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Ankara, Turkey 4Ondokuz Mayıs University University Faculty of Medicine, Department of Pediatric Radiology, Samsun, Turkey 5Ondokuz Mayıs University Faculty of Medicine, Department of Pathology, Samsun, Turkey 6Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Gastroenterology, Hepatology and Nutrition, Samsun, Turkey 2Leipzig

To the Editor, Hyperimmunoglobulin M syndrome (HIGM) is a primary immunodeficiency characterized with low IgG, IgA, and IgE and normal or elevated IgM levels due to a defect in class switching recombination [1]. In this report, we present a patient with CD40 ligand (CD40L) deficiency who had arthritis and hypertransaminasemia with no history of serious infection until 8 years of age. An 8-year-old male patient presented because of a 2-year history of knee swelling and pain. The patient had no history of serious infection and he had been followed for asthma and elevated transaminase level by the Department of Pediatric Gastroenterology for the past 3 years. His parents were not relatives. Physical examination revealed the following: both knees had tenderness, swelling, redness, and limited range of motion. Other systems were normal. Laboratory analysis revealed the Figure 1. a) There is c.761C>T mutation in the CD40 ligand gene. b) following results: Hb: 11.7 g/dL, Hct: 37.7%, MCV: 71.7 MR cholangiography findings: dilated common choledochal (black fL, leukocytes: 8.0x109/L, platelets:405x109/L, CRP: 18 star) and intrahepatic bile ducts, narrowing of the bile ducts, and irregularities at multiple levels (white arrows). c) Bile duct with mg/L, ESR: 25 mm/h, ALT: 103 U/L (normal range: 0-55), increasing concentric connective tissue in one portal tract (trichrome AST: 63 U/L (5-40). The patient was negative for stain, 10x). HBsAg, anti-HBs, anti-HCV, anti-CMV IgG, and CMV immunoglobulins were as follows: IgG: 1.27 g/L (5.5-17), by polymerase chain reaction (PCR), while he was IgA: 0.06 g/L (0.6-3.3), IgM: 5.09 g/L (0.6-2.7), IgE: 16 IU/ positive for anti-CMV IgM (252.7%, normal range: 90100). Isohemagglutinins were 1/512 positive. The serum mL. Examinations for arthritis did not reveal remarkable Address for Correspondence: Mehmet Halil CELİKSOY, M.D., Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Samsun, Turkey Phone: +90 362 312 19 19 E-mail: drmhc@hotmail.com Received/Geliş tarihi : February 21, 2014 Accepted/Kabul tarihi : July 3, 2014

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Celiksoy MH, et al: CD40L Deficiency and Hypertransaminasemia

results and no pathogen could be isolated. Flow cytometry revealed CD40L deficiency, while analysis showed c.761C>T mutation in the same gene (Figure 1a). The patient was diagnosed with HIGM syndrome. A liver biopsy was done and magnetic resonance (MR) cholangiography was conducted for hypertransaminasemia. MR cholangiography and biopsy specimen findings were consistent with sclerosing cholangitis (Figures 1b and 1c). Informed consent was obtained. In HIGM syndrome, clinical findings occur at early ages and the mean age of diagnosis is less than 1 year. Clinically, HIGM has similarities with recurring respiratory tract infections causing bronchiectasis and other humoral immune deficiencies presenting with sinus and ear infections [2]. Recurrent respiratory infections were absent in our case. Additionally, our patient was diagnosed with asthma. Recurrent respiratory infections may be confused with asthma attacks by physicians. Therefore, our patient could have been diagnosed late. Although other markers were negative, anti-CMV IgM values were always high in this case. CMV-DNA PCR tests, applied at intervals, were also negative. In HIGM syndrome, the serum IgM level is normal or high but serum IgG and IgA levels are low, because of a defect in isotype class switching of B cells [3]. Furthermore, there is no response to protein antigens, though some IgM anti-polysaccharide antibodies, including isohemagglutinins, can be produced [2]. Therefore, our patient had CMV IgM probably due to CMV infection. However, he could not produce CMV IgG because of the nature of his disease. Our patient was being monitored due to hypertransaminasemia in the gastroenterology outpatient clinic. Abdominal ultrasonography showed no cholangiopathy, but MR cholangiography findings were consistent with sclerosing cholangitis. Portal fibrosis was seen in only 1 of 4 vena portae samples in his liver biopsy. The most common characteristic finding of liver biopsy for sclerosing cholangitis

is interlobular and septal fibrosis of bile ducts known as ‘onion skin’ [4]. Our findings of liver biopsy were not characteristic for sclerosing cholangitis. When clinical and radiological findings were evaluated together with liver biopsy results, the patient was diagnosed with sclerosing cholangitis in an initial state. In conclusion, idiopathic arthritis and persistent hypertransaminasemia should also be considered in the differential diagnosis of primary immune deficiencies. 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. Key Words: Humoral immune response, Immunodeficiency diseases, Immune response disorder, Immunoglobulins, Hyper IgM syndrome, CD40L Anahtar Sözcükler: Hümoral immün yanıt, İmmün yetmezlikler, İmmün yanıt bozukluğu, İmmünglobulinler, Hiper IgM sendromu, CD40L References 1. Lee WI, Torgerson TR, Schumacher MJ, Yel L, Zhu Q, Ochs HD. Molecular analysis of a large cohort of patients with the hyper immunoglobulin M (IgM) syndrome. Blood 2005;105:1881-1890. 2. Davies EG, Thrasher AJ. Update on the hyper immunoglobulin M syndromes. Br J Haematol 2010;149:167-180. 3. Montella S, Maglione M, Giardino G, Di Giorgio A, Palamaro L, Mirra V, Ursini MV, Salerno M, Pignata C, Caffarelli C, Santamaria F. Hyper IgM syndrome presenting as chronic suppurative lung disease. Ital J Pediatr 2012;38:45. 4. Farrell RJ, Kelly CP. Sclerosing cholangitis and recurrent pyogenic cholangitis. In: Feldman M, Friedman LS, Sleisenger MH (eds). Sleisenger & Fordtran’s Gastrointestinal and Liver Disease: Pathophysiology, Diagnosis, Management, Vol 2, 7th ed. Philadelphia, Saunders, 2002.

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DOI: 10.4274/tjh.2013.0404

Letter to the Editor

Blastic Plasmacytoid Dendritic Cell Neoplasm: Single-Center Experience with Two Cases in One Year Blastik Plazmasitoid Dendritik Hücreli Neoplazi: Bir Yılda İki Olguyu İçeren Tek Merkez Deneyimi Alexandra Agapidou1, Sophia Vakalopoulou1, Dimitra Markala2, Christina Chadjiaggelidou1, Maria Tzimou1, Theodosia Papadopoulou1, Vasileia Garypidou1 1Aristotle University of Thessaloniki Faculty of Medicine, Hippokratio General Hospital, Second Propaedeutic Department of Internal Medicine, Division of Hematology, Thessaloniki, Greece 2Theagenion Cancer Hospital, Thessaloniki, Greece

To the Editor, A 78-year-old Caucasian female patient presented to our department with a cutaneous lesion on her right shoulder (Figure 1a). Laboratory data disclosed mild anemia (hemoglobin: 11.3 g/dL) thrombocytopenia (139x109/L) and 30% morphologically immature atypical cells in the peripheral blood. Bone marrow aspiration showed 5% infiltration of immature blast cells with the following immunophenotype: CD45 (+), CD123 (+), CD85k (+), CD33 (-), CD14 (-), CD16 (-), CD19 (-), CD5 (-), CD10 (-), CD20 (-), CD56 (+) 20%, CD4 (+), NG2 (+). No chromosomal alterations were detected by cytogenetic analysis of the bone marrow (46,XX). Specific karyotypic aberrations were not found. She had axillary, jugular, submandibular, and supraclavicular lymphadenopathy. Cutaneous, lymph node, and bone marrow biopsy confirmed the diagnosis of blastic plasmacytoid dendritic cell neoplasm (BPDCN). She was treated with cyclophosphamide, vincristine, adriamycin, and dexamethasone (Cy-VAD) as part of an acute lymphoblastic leukemia treatment-protocol. She achieved first complete remission. Due to the highly aggressive type of leukemia, we decided to continue with induction 2 chemotherapy (etoposide-cytosine arabinoside), but she died 5 months after the first sign due to multiorgan failure. One year later, a 75-year-old Caucasian male patient presented with a generalized purplish skin rash from the head to the lower extremities that expanded very rapidly (Figure 1b and 1c). Laboratory data revealed anemia (hemoglobin: 10.9 g/dL), thrombocytopenia (100x109/L), and

Figure 1. a) Skin lesion of Caucasian female patient, before treatment. b) Skin lesions of Caucasian male patient before treatment and c) after treatment (abdominal-lower genital area). 42% morphologically immature atypical cells in the peripheral blood. Bone marrow aspiration showed 88% infiltration of immature blast cells with the following immunophenotype: CD45 (+) low, CD43 (+), CD123 (+), CD56 (+), CD4 (+), CD34 (-). Computed tomography scans did not disclose pathologic lymphadenopathy. Histopathology of skin lesions showed blast cell infiltrate. Immunohistochemical analysis confirmed the presence of cells with the aforementioned immunophenotypic features. A basic immunophenotype with CD4 (+), CD56 (+), CD123 (+), and negative T, B, and NK cells led to the diagnosis of BPDCN as per the current WHO classification [1]. In the cytogenetic analysis, a pathologic karyotype was found (46,XY, del (12), (p12), del (17), (p11) [17]/46,XY [13]). He began acute myeloid leukemia-type chemotherapy with idarubicin and arabinoside-c and he achieved complete remission after induction. We changed his treatment plans and continued with CHOP due to his poor performance status, and, after 4 cycles, he still remains

Address for Correspondence: Alexandra AGAPIDOU, M.D., Aristotle University of Thessaloniki Faculty of Medicine, Hippokratio General Hospital, Second Propaedeutic Department of Internal Medicine, Division of Hematology, Thessaloniki, Greece Phone: +30 694 880 97 42 E-mail: alekagapidou@yahoo.gr Received/Geliş tarihi : December 2, 2013 Accepted/Kabul tarihi : January 14, 2014

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Agapidou A, et al: Blastic Plasmacytoid Dendritic Cell Neoplasm

without clinical signs. Informed consent was obtained. Plasmacytoid dendritic cells were first identified 50 years ago by Lennert and his associates [2]. BPDCN is a rare, highly aggressive hematopoietic malignancy that is characterized by cutaneous infiltration with or without bone marrow involvement. Its overall incidence is extremely low. The leukemic form of the disease is very rare. BPDCN predominantly affects males, and generally the elderly [3]. The majority of patients present with asymptomatic solitary or multiple cutaneous reddish-brown nodules. Clinically, this malignancy generally presents in the skin, often followed by bone marrow and blood involvement. However, any organ can be affected. The disease follows a short course and fulminant leukemia is the common terminal stage. Diagnosis is based on the expression of CD4, CD56, and CD123 in the absence of T-cell, B-cell, or myeloid markers. Although identification of the immunophenotypic features of BPDCN has improved its recognition, this entity remains diagnostically challenging. Insufficient knowledge of this entity and inadequate immunophenotypic investigation can lead to the misdiagnosis of a different leukemia. The prognosis of patients with BPDCN is poor, with a median survival of 12 months regardless of treatment type. Acute lymphoblastic leukemia-type treatment regimens are advised and a promising initial response may occur, but this is followed by quick relapse [4]. There is also the option of bone marrow transplantation for young patients with an acceptable performance status. In conclusion, we encountered a rare type of leukemia. The rarity of this disease does not enable prospective clinical trials to identify a better therapeutic strategy, which, at present, is based on clinicians’ experience and on cooperation among them.

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. Key Words: Plasmacytoid dendritic cell, Leukemia, Cutaneous lesion, CD4 (+), CD56 (+) Anahtar Sözcükler: Plazmasitoid Dendritik Hücre, Lösemi, Deri Lezyonu, CD4 (+), CD56 (+) References 1. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon, France, International Agency for Research on Cancer, 2008. 2. Marafioti T, Paterson JC, Ballabio E, Reichard KK, Tedoldi S, Hollowood K, Dictor M, Hansmann ML, Pileri SA, Dyer MJ, Sozzani S, Dikic I, Shaw AS, Petrella T, Stein H, Isaacson PG, Facchetti F, Mason DY. Novel markers of normal and neoplastic human plasmacytoid dendritic cells. Blood 2008;111:3778-3792. 3. Pagano L, Valentini CG, Pulsoni A, Fisogni S, Carluccio P, Mannelli F, Lunghi M, Pica G, Onida F, Cattaneo C, Piccaluga PP, Di Bona E, Todisco E, Musto P, Spadea A, D’Arco A, Pileri S, Leone G, Amadori S, Facchetti F; GIMEMA-ALWP (Gruppo Italiano Malattie Ematologiche dell’Adulto, Acute Leukemia Working Party). Blastic plasmacytoid dendritic cell neoplasm with leukemic presentation: an Italian multicenter study. Haematologica 2013;98:239-246. 4. Pinto-Almeida T, Fernandes L, Sanches M, Lau C, Lima M, Alves R, Selores M. A case of blastic plasmacytoid dendritic cell neoplasm. Ann Dermatol 2012;24:235-237.

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DOI: 10.4274/tjh.2014.0166

Letter to the Editor

Mogamulizumab Treatment in a Hemodialysis Patient with Adult T-Cell Leukemia/Lymphoma Erişkin T-hücreli Lösemi/Lenfomalı Hemodiyaliz Hastasında Mogamulizumab Tedavisi Mari Yoshihara1, Yasushi Kubota1,2, Makoto Fukuda3, Tomoya Kishi3, Yuji Ikeda3, Shinya Kimura1 1Saga

University Faculty of Medicine, Department of Internal Medicine, Division of Hematology Respiratory Medicine, and Oncology, Saga, Japan University Faculty of Medicine Hospital, Department of Transfusion Medicine, Saga, Japan 3Saga University Faculty of Medicine, Department of Internal Medicine, Division of Nephrology, Saga, Japan 2Saga

To the Editor, Here we describe, for the first time, a hemodialysis patient suffering from adult T-cell leukemia/lymphoma (ATL) who was treated with mogamulizumab, a defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody [1]. An 83-year-old female was admitted to the hospital suffering from fatigue, leukocytosis, and hypercalcemia. On admission, laboratory tests revealed that her leukocyte count was elevated to 76x109/L (>80% abnormal lymphocytes). Blood chemistry analysis revealed elevated lactate dehydrogenase (LDH: 808 IU/L) and soluble interleukin-2 receptor (sIL-2R: 43.465 U/ mL). Seropositivity for human T-cell leukemia virus type-1 (HTLV-1) was confirmed and monoclonal integration of HTLV1 was detected by Southern blotting of DNA isolated from peripheral blood (PB). She was diagnosed with acute ATL. Flow cytometric analysis demonstrated that the abnormal lymphocytes were CD3+CD4+CD25+. Computed tomography (CT) revealed multiple lymphadenopathies, involving the supraclavicular fossae and the inguinal, mediastinum, and para-aortic regions. She was treated with systemic chemotherapy (THPCOP regimen: cyclophosphamide, pirarubicin, vincristine, and prednisolone) on day 8 post-admission. Although the number of ATL cells in the PB gradually decreased, they were persistent. On day 18 of THP-COP, she experienced a high fever and hypotension. Because serum β-D-glucan levels were elevated and a chest CT revealed a pulmonary infiltrate, she was administered cefepime and voriconazole. However, she developed anuric acute renal failure, probably induced by voriconazole, and so emergent hemodialysis was initiated. Subsequently, both the number of ATL cells

Figure 1. Clinical course: time-points of THP-COP (cyclophosphamide, pirarubicin, vincristine, and prednisolone) and mogamulizumab administration. WBC counts, ATL cell counts, and LDH and creatinine levels throughout the clinical course, and the concentration of mogamulizumab in the plasma pre- and post-dialysis, are shown. CHDF: Continuous hemodiafiltration, HD: hemodialysis. and her LDH levels increased, suggesting that the THP-COP regimen was not working. Because the ATL cells expressed high levels of CCR4, she received an intravenous infusion of mogamulizumab (1.0 mg/kg) once a week for 8 weeks. The concentration of mogamulizumab in the plasma was measured using an enzyme-linked immunosorbent assay [2,3]. The plasma concentrations of mogamulizumab before and after hemodialysis during the first mogamulizumab infusion and

Address for Correspondence: Yasushi Kubota, M.D., Saga University Faculty of Medicine, Department of Internal Medicine, Division of Hematology Respiratory Medicine, and Oncology, Saga, Japan Phone: +81-952-34-2366 E-mail: kubotay@cc.saga-u.ac.jp Received/Geliş tarihi : April 24, 2014 Accepted/Kabul tarihi : August 12, 2014

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Yoshihara M, et al: Mogamulizumab in a Hemodialysis Patient with ATL

just before the second infusion (Ctrough) were 14,104.8 ng/mL, 16,092.2 ng/mL, and 5901.2 ng/mL, respectively. The plasma levels of mogamulizumab before and after hemodialysis were comparable, and Ctrough was in the range of published data [2], suggesting that therapeutic levels of mogamulizumab may be maintained in patients undergoing dialysis (Figure 1). Five months after the mogamulizumab treatment the leukemia relapsed. However, the patient did not accept any further treatment or a rechallenge with mogamulizumab. She was managed with best supportive care, and she died 10 months after diagnosis with ATL. ATL is an aggressive peripheral T-cell neoplasm that cannot usually be cured by conventional chemotherapy [4,5]. Allogeneic hematopoietic stem cell transplantation is now considered the only curable treatment for young patients with ATL, but it is not applied to elderly patients because of higher toxicities. Currently, chemotherapy is the only therapeutic option for elderly ATL patients but there are concers about their safety and efficacy [6]. Mogamulizumab is a humanized anti-CCR4 antibody with a defucosylated Fc region and has been used to treat relapsed/refractory CCR4-positive ATL with 50% efficacy in a phase II study [3,7]. The fucose content in the oligosaccharide structure in the Fc region of mogamulizumab is reduced using Potelligent technology, which enhances the antibody-dependent cellular toxicity because of increased binding affinity to the Fcγ receptor on effector cells [8]. More recently, mogamulizumab was also approved for the treatment of relapsed CCR4-positive peripheral T-cell lymphoma and cutaneous T-cell lymphoma in Japan [9]. To date, limited data exist about the application of mogamulizumab in patients undergoing hemodialysis. Here, mogamulizumab treatment resulted in complete remission of an elderly ATL patient with no major adverse events such as infusion reaction or skin rash (including re-exacerbation of renal function). Thus, administration of mogamulizumab may be considered as a safe therapeutic option in this setting. The present case shows that therapeutic mogamulizumab levels can be achieved and maintained in patients undergoing hemodialysis as mogamulizumab is not eliminated by the procedure. 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. Key Words: Adult T-cell leukemia/lymphoma, Mogamulizumab, Hemodialysis, CCR4, HTLV-1 Anahtar Sözcükler: Erişkin T-hücreli lösemi/lenfoma, Mogamulizumab, Hemodiyaliz, CCR4, HTLV-1 References 1. Ishida T, Utsunomiya A, Inagaki A, Yano H, Komatsu H, Iida S, Imada K, Uchiyama T, Akinaga S, Shitara K, Ueda R. Defucosylated humanized anti-CCR4 monoclonal antibody KW-0761 as a novel immunotherapeutic agent for adult T-cell leukemia/lymphoma. Clin Cancer Res 2010;16:15201531.

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2. Yamamoto K, Utsunomiya A, Tobinai K, Tsukasaki K, Uike N, Uozumi K, Yamaguchi K, Yamada Y, Hanada S, Tamura K, Nakamura S, Inagaki H, Ohshima K, Kiyoi H, Ishida T, Matsushima K, Akinaga S, Ogura M, Tomonaga M, Ueda R. Phase I study of KW-0761, a defucosylated humanized anti-CCR4 antibody, in relapsed patients with adult T-cell leukemia-lymphoma and peripheral T-cell lymphoma. J Clin Oncol 2010;28:1591-1598. 3. Ishida T, Joh T, Uike N, Yamamoto K, Utsunomiya A, Yoshida S, Saburi Y, Miyamoto T, Takemoto S, Suzushima H, Tsukasaki K, Nosaka K, Fujiwara H, Ishitsuka K, Inagaki H, Ogura M, Akinaga S, Tomonaga M, Tobinai K, Ueda R. Defucosylated anti-CCR4 monoclonal antibody (KW-0761) for relapsed adult T-cell leukemia-lymphoma: a multicenter phase II study. J Clin Oncol 2012;30:837-842. 4. Uchiyama T, Yodoi J, Sagawa K, Takatsuki K, Uchino H. Adult T-cell leukemia: clinical and hematologic features of 16 cases. Blood 1977;50:481-492. 5. Tsukasaki K, Hermine O, Bazarbachi A, Ratner L, Ramos JC, Harrington W Jr, O’Mahony D, Janik JE, Bittencourt AL, Taylor GP, Yamaguchi K, Utsunomiya A, Tobinai K, Watanabe T. Definition, prognostic factors, treatment, and response criteria of adult T-cell leukemia-lymphoma: a proposal from an international consensus meeting. J Clin Oncol 2009;27:453-459. 6. Fukushima N, Itamura H, Urata C, Tanaka M, Hisatomi T, Kubota Y, Sueoka E, Kimura S. Clinical presentation and outcome in patients of over 75 years old with malignant lymphoma. International Journal of Clinical Medicine 2011;2:246-253. 7. Ito A, Ishida T, Yano H, Inagaki A, Suzuki S, Sato F, Takino H, Mori F, Ri M, Kusumoto S, Komatsu H, Iida S, Inagaki H, Ueda R. Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model. Cancer Immunol Immunother 2009;58:1195-1206. 8. Niwa R, Shoji-Hosaka E, Sakurada M, Shinkawa T, Uchida K, Nakamura K, Matsushima K, Ueda R, Hanai N, Shitara K. Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T-cell leukemia and lymphoma. Cancer Res 2004;64:2127-2133. 9. Ogura M, Ishida T, Hatake K, aniwaki M, Ando K, Tobinai K, Fujimoto K, Yamamoto K, Miyamoto T, Uike N, Tanimoto M, Tsukasaki K, Ishizawa K, Suzumiya J, Inagaki H, Tamura K, Akinaga S, Tomonaga M, Ueda R. Multicenter phase II study of mogamulizumab (KW-0761), a defucosylated anti-cc chemokine receptor 4 antibody, in patients with relapsed peripheral T-cell lymphoma and cutaneous T-cell lymphoma. J Clin Oncol 2014;32:1157-1163. 425

DOI: 10.4274/tjh.2014.0049

Letter to the Editor

Chediak-Higashi Syndrome: A Case Report of a Girl Without Silvery Hair and Oculocutaneous Albinism Presenting with Hemophagocytic Lymphohistiocytosis Chediak-Higashi Sendromu: Gri Saç ve Okülokütanöz Albinizm Olmaksızın Hemofagositik Lenfohistiositoz ile Başvuran Bir Kız Çocuğu Olgu Sunumu Murat Elevli1, Halil Uğur Hatipoğlu2, Mahmut Civilibal2, Nilgün Selçuk Duru2, Tiraje Celkan3 1Sakarya 2Haseki

University Faculty of Medicine, Clinic of Pediatrics, Sakarya, Turkey

Education and Research Hospital, Clinic of Pediatrics, İstanbul, Turkey University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey

3İstanbul

To the Editor, Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive inherited disorder characterized by variable degrees of oculocutaneous albinism, severe immune deficiency and unassociated lymphoproliferative syndrome, and intracytoplasmic giant granules in leukocytes, monocytes, platelets, melanocytes, and erythroid precursors [1,2,3,4,5]. CHS is caused by mutations in the lysosomal trafficking regulator gene (LYST) [3,6]. The role of the LYST gene in the trafficking of granules results in defective release of melanin or cytolytic enzymes, causing hypopigmentation of the skin and hair as well as cytotoxic defect [3]. There are 2 clinical periods of the disease: stable and accelerated. In the accelerated phase, fever, hepatosplenomegaly, hepatitis, lymphohistiocytic infiltration, pancytopenia, coagulopathy, hemorrhage, and peripheral neuropathy are seen [1]. Herein, we report a case of CHS presented with hemophagocytic lymphohistiocytosis (HLH). A 5-month-old girl presented with fever. She was febrile and pale, and she had splenomegaly with no hepatomegaly. There were no neurological symptoms, lymphadenopathy, or bleeding signs. Her body temperature was over 38.8 °C for 8 days in spite of ampicillin/sulbactam and netilmicin therapy. Hematological investigation revealed hemoglobin of 6.3 g/dL, white blood cell count of 3.8x109/L (70% lymphocytes, 10% neutrophils), and Address for Correspondence: Halil Uğur HATİPOĞLU, M.D., Haseki Education and Research Hospital, Clinic of Pediatrics, İstanbul, Turkey Gsm: +90 532 680 67 65 E-mail: huhatipoglu@gmail.com Received/Geliş tarihi : February 2, 2014 Accepted/Kabul tarihi : May 13, 2014

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Figure 1. A. Hemophagocytosis on bone marrow smear. B. An intracytoplasmic giant granule in a monocyte on peripheral blood smear. C. Light microscopic image of the hair shaft shows abnormal clumping of melanin in patient’s hair. Pigment clumbs are small and uniformly and regularly distributed along the hair shaft. This is consistant with Chediak-Higashi syndrome. D. Our patient does not have oculocutaneous albinism and she is black-haired.

Turk J Hematol 2014;31:426-427

Elevli M, et al: Chediak-Higashi Syndrome Presented with Hemophagocytic Lymphohistiocytosis

platelet count of 84x109/L. Erythrocyte sedimentation rate was 25 mm/h, C reactive protein was 6.75 mg/dL, and ferritin was 635 ng/mL, with a normal fibrinogen level. Serum triglyceride level was 584 mg/dL with a normal serum cholesterol level. Serum lactate dehydrogenase, serum glutamic oxaloacetic transaminase, and serum glutamic pyruvate transaminase levels were 882 U/L, 254 U/L, and 54 U/L, respectively. Her fever was controlled with piperacillin/tazobactam and amikacin combined with fluconazole. Serological markers were negative for cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, parvovirus, Toxoplasma, rubella, and hepatitis B virus. Salmonella and Brucella tube agglutination test results were also negative. Blood and urine cultures did not demonstrate any infectious agents. Tandem MS, blood amino acid levels, urinary organic acid profile, and urinary mucopolysaccharide screening tests were all normal. On bone marrow aspiration smear, hemophagocytosis was observed. The patient was diagnosed with HLH and the HLH-2004 treatment protocol, including dexamethasone and etoposide, was administrated. No gene defect was determined on exons 2 and 3 of the perforin 1 coding region. On both bone marrow aspiration and peripheral blood smears, intracytoplasmic giant granules in lymphocytes and monocytes were observed. Hair analysis for CHS was consistent with CHS. At the end of the initial therapy, the disease was in remission. Continuation therapy of the HLH regimen was then started. Stem cell transplantation (SCT) has been planned. As there was no matched related donor for SCT at that time, we will be treating her according to HLH-2004 continuation therapy until a matched or mismatched unrelated donor is found. Informed consent was obtained. Our patient was diagnosed with HLH because there was hemophagocytosis on bone marrow aspiration (Figure 1a) and she had splenomegaly, fever, pancytopenia, hypertriglyceridemia, and high ferritin levels. Sometimes the CHS diagnosis can be considered only after the observation of gray hair and giant intracellular granules on a peripheral blood smear, and light microscopy of a hair shaft can facilitate a quick diagnosis [2]. We thought that the diagnosis could be CHS because of the bone marrow aspiration and peripheral blood smears findings (Figure 1b). Light microscopic images of the patient’s hair showed abnormal clumping of melanin. Pigment clumps were small and uniformly and regularly distributed along the hair shaft (Figure 1c). Our patient was therefore diagnosed by hair analysis although she does not have the CHS phenotype (Figure 1d). Patients with CHS may have a variable clinical presentation due to different mutations in the LYST gene. Nonsense and frameshift mutations of the LYST gene are associated with severe early-onset childhood CHS and characterized by fatal infections and HLH. Meanwhile, missense mutations of the same gene are associated with milder, late-onset CHS with slowly progressive neurological impairment or an adolescent form with infections but no HLH [2,6,7]. In our case there is probably a mutation of the LYST gene that is associated with the normal phenotype and the specific findings of CHS on peripheral blood smear, bone marrow aspiration smear, and light microscopic image of the hair shaft but causes earlyonset HLH. Genetic analysis is needed to explain this exactly.

In our case there was no specific treatment for CHS. Intravenous antibiotherapy, erythrocyte suspension, thrombocyte suspension, and, in accordance with the HLH 2004 protocol, dexamethasone and etoposide were used. The patient has been positively responsive to chemotherapy and, as the main treatment of CHS, SCT is planned, but a donor has not yet been found [8]. In conclusion, we think that it is not necessary to find oculocutaneous albinism and silvery hair in all patients to diagnose CHS. Bone marrow aspiration and peripheral blood smears and light microscopic images of the hair shaft may be sufficient for diagnosis in some cases without genetic analysis. 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. Key Words: Chediak-Higashi syndrome, Hemophagocytic lymphohistiocytosis, Oculocutaneous albinism, Lyst gene, Immune deficiency Anahtar Sözcükler: Chediak-Higashi sendromu, Hemofagositik lenfohistiositoz, Okülokütanöz albinizm, Lyst geni, İmmün yetmezlik References 1. Akbayram S, Akgun C, Basaranoglu M, Kaya A, Balta G, Ustyol L, Yesilmen O, Deger I, Oner AF. A case of ChediakHigashi syndrome presented with hemophagocytic lymphohistiocytosis. International Journal of Hematology and Oncology 2011;21:196-199. 2. Spritz RA. Genetic defects in Chediak-Higashi syndrome and the beige mouse. J Clin Immunol 1998;18:97-105. 3. Janka GE. Familial and acquired hemophagocytic lymphohistiocytosis. Eur J Pediatr 2007;166:95-109. 4. Pujani M, Agarwal K, Bansal S, Ahmad I, Puri V, Verma D, Pujani M. Chediak-Higashi syndrome - a report of two cases with unusual hyperpigmentation of the face. Turk Patoloji Derg 2011;27:246-248. 5. Karalı Z, Kılıç SŞ. Chediak-Higashi sendromu. Güncel Pediatri Dergisi 2007;5:99-104 (article in Turkish). 6. Kaya Z, Ehl S, Albayrak M, Maul-Pavicic A, Schwarz K, Kocak U, Ergun MA, Gursel T. A novel single point mutation of the LYST gene in two siblings with different phenotypic features of Chediak-Higashi syndrome. Pediatr Blood Cancer 2011;56:1136-1139. 7. Westbroek W, Adams D, Huizing M, Koshoffer A, Dorward H, Tinloy B, Parkes J, Helip-Wooley A, Kleta R, Tsilou E, Duvernay P, Digre KB, Creel DJ, White JG, Boissy RE, Gahl WA. Cellular defects in Chediak-Higashi syndrome correlate with the molecular genotype and clinical phenotype. J Invest Dermatol 2007;127:2674-2677. 8. Haddad E, Deist FL, Blanche S, Benkerrou M, Rohrlich P, Vilmer E, Griscelli C, Fischer A. Treatment of Chediak-Higashi syndrome by allogeneic bone marrow transplantation: report of 10 cases. Blood 1995;85:3328-3333. 427

DOI: 10.4274/tjh.2014.0019

Letter to the Editor

Gaucher Cells or Pseudo-Gaucher Cells: That’s the Question Gaucher Hücreleri ya da Pseudo-Gaucher Hücreleri: İşte Soru Bu Deniz Gören Şahin1, Hava Üsküdar Teke1, Mustafa Karagülle1, Neslihan Andıç1, Eren Gündüz1, Serap Işıksoy2, Olga Meltem Akay1 1Eskişehir 2Eskişehir

Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey Osmangazi University Faculty of Medicine, Department of Pathology, Eskişehir, Turkey

To the Editor, Bone marrow cells with morphological characteristics similar to Gaucher cells and without cytoplasmic crystalline inclusions are rare. These Gaucher-like or pseudo-Gaucher cells can be seen in a variety of conditions such as acute lymphoblastic leukemia, multiple myeloma, myelodysplasia, Hodgkin’s disease, thalassemia, and disseminated mycobacterial infection [1,2,3,4,5,6,7,8]. Since the presence of these cells may obscure neoplastic cells in multiple myeloma and may lead to misdiagnosis, it is important for hematologists and hematopathologists to be aware of such a condition in order to make a prompt and accurate diagnosis. Herein we report a case of multiple myeloma in which the presence of plasma cells was missed on initial histological diagnosis. A 44-year-old female without history of any previous systemic disease presented with oliguria, easy fatigability, and breathlessness for 7 days. On examination she had crepitating rales, jugular venous congestion, abdominal distension, and pretibial edema. The complete blood count showed Hb of 67 g/L, WBC count of 3.4x109/L, and platelet count of 69x109/L. Erythrocyte sedimentation rate was 112 mm/h. Bone marrow aspirate and biopsy were performed. The bone marrow aspirate revealed numerous large cells with plentiful cytoplasm and a small eccentric nucleus. Scattered among these were plasma cells, which were obscured by sheets of Gaucher-like cells (Figure 1A). Immunohistochemical staining of bone marrow biopsy showed that plasma cells were positive for CD38 and kappa light chain (Figures 1B and 1C), and the large cells were positive for CD68 (Figure 1D). There were crystalline

Figure 1. (A) Bone marrow aspirate smear (Giemsa 400x) showing pseudo-Gaucher cells with abundant cytoplasm, dense round deposits, and an eccentric pyknotic nucleus; immunohistochemical staining of bone marrow biopsy showed that plasma cells were positive for (B) CD38 and (C) kappa light chain; (D) the large cells were positive for CD68. inclusion bodies within these cells, which were negative for smooth muscle actin, HHF-35, and keratin. The erythroid and myeloid series were normal. Serum electrophoresis revealed an M band. Skull X-ray showed lytic bone lesions. Taken together, a diagnosis of multiple myeloma associated with a prominent pseudo-Gaucher histiocytic response was made. Gaucher-like cells have been described in various hematological disorders [1,2,3,4,5,6,7,8]. These cells are considered to be marrow macrophages seen in circumstances

Address for Correspondence: Olga Meltem AKAY, M.D., Eskişehir Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey Phone: +90 222 239297 E-mail: olga.akay@hotmail.com Received/Geliş tarihi : January 13, 2014 Accepted/Kabul tarihi : May 13, 2014

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Gören Şahin D, et al: Pseudo-Gaucher Cells

related to high cell turnover [9]. One striking feature is that pseudo-Gaucher cells cannot be distinguished from true Gaucher cells by routine hematoxylin-eosin staining. In order to differentiate them, iron staining should be performed. Gaucher cells show diffuse iron staining whereas pseudoGaucher cells do not. Electron microscopical features may also help distinguish pseudo-Gaucher cells from true Gaucher cells. On electron microscopy, pseudo-Gaucher cells do not contain typical tubular cytoplasmic inclusions, which are present in Gaucher cells. In addition, crystal-storing histiocytosis and sea blue histiocytosis should be considered in differential diagnosis. Macrophages with cytoplasmic crystalline inclusions are better regarded as crystal-storing histiocytes and this rare entity could be confused with Gaucher or pseudoGaucher cells [10]. Moreover, sea blue histiocytes should be kept in mind. However, these cells are heavily granulated with prominent vacuolation. We are reporting this case to increase the awareness among hematologists and hematopathologists of this rare association to avoid misdiagnosis. We also would like to highlight that the presence of pseudo-Gaucher cells in bone marrow should not be overlooked as they might be obscuring an underlying pathology. Awareness of possible associations, appropriate immunohistochemistry, and relevant additional investigations based on clinical findings are necessary for final diagnosis. 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. Key Words: Crystalline inclusion bodies, Gaucher cells, Multiple myeloma Anahtar Sözcükler: Kristal inklüzyon cisimcikler, Gaucher hücreleri, Multiple miyeloma

References 1. Busarla SV, Sadruddin FA, Sohani AR. Pseudo-Gaucher cells in disseminated mycobacterial infection. Am J Hematol 2013;88:155. 2. Bain BJ, Lee L. Pseudo-Gaucher cells in sickle cell anemia. Am J Hematol 2010;85:435. 3. Ash Image Bank. Waldenström macroglobulinemia with pseudo-Gaucher cells. Blood 2010;116:3388. 4. Saroha V, Gupta P, Singh M, Singh T. Pseudogaucher cells obscuring multiple myeloma: a case report. Cases J 2009;2:9147. 5. Sharma P, Khurana N, Singh T. Pseudo-Gaucher cells in Hb E disease and thalassemia intermedia. Hematology 2007;12:457-459. 6. Saito T, Usui N, Asai O, Dobashi N, Ida H, Kawakami M, Yano S, Osawa H, Takei Y, Takahara S, Ogasawara Y, Yamaguchi Y, Minami J, Aiba K. Pseudo-Gaucher cell proliferation associated with myelodysplastic syndrome. Int J Hematol 2007;85:350-353. 7. Zidar BL, Hartsock RJ, Lee RE, Glew RH, LaMarco KL, Pugh RP, Raju RN, Shackney SE. Pseudo-Gaucher cells in the bone marrow of a patient with Hodgkin’s disease. Am J Clin Pathol 1987;87:533-536. 8. Scullin DC Jr, Shelburne JD, Cohen HJ. Pseudo-Gaucher cells in multiple myeloma. Am J Med 1979;67:347-352. 9. Carrington PA, Stevens RF, Lendon M. Pseudo-Gaucher cells. J Clin Pathol 1992;45:360. 10. Schaefer HE. Gammopathy-related crystal-storing histiocytosis, pseudo- and pseudo-pseudo-Gaucher cells. Critical commentary and mini-review. Pathol Res Pract 1996;11:1152-1162.

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DOI: 10.4274/tjh.2014.0052

Letter to the Editor

Quilty Effect after Extracorporeal Photopheresis in a Patient with Severe Refractory Cardiac Allograft Rejection Şiddetli Kardiyak Allogreft Rejeksiyonu Olan Bir Hastada Ekstrakorporeal Fotoferez Sonrası Quilty Etkisi Özgür Ulaş Özcan1, Tamer Sayın1, Gürbey Soğut1, Aylin Heper2, Hüseyin Göksülük1, Veysel Kutay Vurgun1, Cansın Tulunay Kaya1, Elif Ezgi Üstün1, Osman İlhan3, Çetin Erol1 1Ankara

University Faculty of Medicine, Department of Cardiology, Ankara, Turkey University Faculty of Medicine, Department of Pathology, Ankara, Turkey 3Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey 2Ankara

To the Editor, Solid organ transplant rejection is still a problem despite the use of immunosuppressive therapy. The currently available regimens for transplant rejection may predispose patients to malignancies such as nonmelanoma skin cancers and opportunistic infections [1]. Extracorporeal photopheresis (ECP) is a leukapheresis-based immunomodulatory therapy in which lymphocytes collected from the patient are incubated with 8-methoxypsoralen, a photosensitizing agent, in the presence of UV-A radiation and then reinfused into the patient [2]. Here we report a case of severe refractory cardiac allograft rejection that was successfully treated by ECP. A 47-year-old man presented with severe decompensated heart failure and hemodynamic compromise 13 months after heart transplantation. Ejection fraction was 25% with transthoracic echocardiography. After immediate therapy with positive inotropes and diuretics, diagnostic coronary angiography, right heart catheterization, and endomyocardial biopsy were performed. Coronary angiography revealed no obstructive coronary artery disease. The endomyocardial biopsy showed perivascular and interstitial lymphocytic inflammatory infiltrate with sparse eosinophils in 2 separate locations that were identified as moderate acute cellular rejection (ISHLT 2R). Cyclosporin A level was 150 ng/ mL. Methylprednisolone pulses (1 g/day) for 3 days and equine anti-thymocyte globulin (Atgam) at 15 mg/kg/ day were administered for induction therapy. Due to the deterioration of clinical status and intervening pneumonia, immunosuppressive therapy was ceased and ECP was planned. Address for Correspondence: Özgür Ulaş ÖZCAN, M.D., Ankara University Faculty of Medicine, Department of Cardiology, Ankara, Turkey Phone: +90 312 508 24 10 E-mail: ozgurulasozcan@yahoo.com.tr Received/Geliş tarihi : February 3, 2014 Accepted/Kabul tarihi : June 9, 2014

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Figure 1. Endomyocardial biopsy specimen demonstrated endocardial lymphocytic aggregate. ECP sessions were performed twice a week for 2 months. After therapy the patient became minimally symptomatic with an ejection fraction of 50%. Repeated endomyocardial biopsy demonstrated lymphocytic aggregation confined to the endocardium, which was interpreted as Quilty effect, and remission of acute cellular rejection (Figure 1). The patient was discharged asymptomatically. Informed consent was obtained. Transplant rejection of solid organs remains an issue despite modern immunosuppressive regimens. The rate of rejection is 25% during the first year after heart transplantation [1].

Özcan UÖ, et al: Extracorporeal Photopheresis in Acute Setting

Acute cell-mediated rejection is characterized by infiltration of T cells directed against the allograft [3]. Biopsy grades of >2 R warrant accentuation of immunosuppression [4]. ‘Quilty effect’ refers to lymphocytic infiltration in the endocardium of cardiac allografts. Although the clinical significance of the Quilty effect is not fully known, it is understood that the Quilty effect does not reflect transplant rejection. Hemodynamic compromise, persistence or recurrence of rejection, and side effects or complications associated with intensive immunosuppressive therapy necessitate alternative approaches for handling rejection. ECP is indicated for prevention of acute and chronic rejection of cardiac transplants [5,6,7]. Favorable effects were also demonstrated for secondary prevention among patients with a history of acute rejection [8]. ECP is a relatively safe procedure. Serious side effects are rarely reported, most of which are related to hypotension and anemia secondary to volume changes and blood loss during the procedure. Risks of opportunistic infections or secondary malignancies have not been increased because of ECP [9]. Small studies demonstrated the benefits of ECP for primary and secondary prevention of cardiac allograft rejection, but treatment for acute refractory cardiac allograft rejection is questionable because of the scarcity of data. We suggest the use of ECP for this compelling condition. 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. Key Words: Allograft, Extracorporeal photopheresis, Heart transplantation, Quilty effect, Rejection Anahtar Sözcükler: Allogreft, Ekstrakorporeal fotoferez, Kalp transplantasyonu, Quilty etkisi, Rejeksiyon References 1. Taylor DO, Stehlik J, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, Kucheryavaya AY, Rahmel AO, Hertz MI. Registry of the International Society for Heart and Lung Transplantation: Twenty-sixth Official Adult Heart Transplant Report-2009. J Heart Lung Transplant 2009;28:1007-1022. 2. Marques MB, Schwartz J. Update on extracorporeal photopheresis in heart and lung transplantation. J Clin Apher 2011;26:146-151. 3. Stewart S, Winters GL, Fishbein MC, Tazelaar HD, Kobashigawa J, Abrams J, Andersen CB, Angelini A, Berry GJ, Burke MM, Demetris AJ, Hammond E, Itescu S, Marboe CC, McManus B, Reed EF, Reinsmoen NL, Rodriguez ER, Rose AG, Rose M, Suciu-Focia N, Zeevi A, Billingham ME. Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant 2005;24:1710-1720.

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4. Costanzo MR, Dipchand A, Starling R, Anderson A, Chan M, Desai S, Fedson S, Fisher P, Gonzales-Stawinski G, Martinelli L, McGiffin D, Smith J, Taylor D, Meiser B, Webber S, Baran D, Carboni M, Dengler T, Feldman D, Frigerio M, Kfoury A, Kim D, Kobashigawa J, Shullo M, Stehlik J, Teuteberg J, Uber P, Zuckermann A, Hunt S, Burch M, Bhat G, Canter C, Chinnock R, Crespo-Leiro M, Delgado R, Dobbels F, Grady K, Kao W, Lamour J, Parry G, Patel J, Pini D, Towbin J, Wolfel G, Delgado D, Eisen H, Goldberg L, Hosenpud J, Johnson M, Keogh A, Lewis C, O’Connell J, Rogers J, Ross H, Russell S, Vanhaecke J. International Society of Heart and Lung Transplantation Guidelines. The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. J Heart Lung Transplant 2010;29:914-956. 5. Szczepiorkowski ZM, Winters JL, Bandarenko N, Kim HC, Linenberger ML, Marques MB, Sarode R, Schwartz J, Weinstein R, Shaz BH. Apheresis Applications Committee of the American Society for Apheresis. Guidelines on the use of therapeutic apheresis in clinical practice--evidence-based approach from the Apheresis Applications Committee of the American Society for Apheresis. J Clin Apher 2010;25:83177. 6. Barr ML, Meiser BM, Eisen HJ, Roberts RF, Livi U, Dall’Amico R, Dorent R, Rogers JG, Radovancevic B, Taylor DO, Jeevanandam V, Marboe CC. Photopheresis for the prevention of rejection in cardiac transplantation. Photopheresis Transplantation Study Group. N Engl J Med 1998;339:1744-1751. 7. Barr ML, Baker CJ, Schenkel FA, McLaughlin SN, Stouch BC, Starnes VA, Rose EA. Prophylactic photopheresis and chronic rejection: effects on graft intimal hyperplasia in cardiac transplantation. Clin Transplant 2000;14:162-166. 8. Kirklin JK, Brown RN, Huang ST, Naftel DC, Hubbard SM, Rayburn BK, McGiffin DC, Bourge RB, Benza RL, Tallaj JA, Pinderski LJ, Pamboukian SV, George JF, Marques M. Rejection with hemodynamic compromise: objective evidence for efficacy of photopheresis. J Heart Lung Transplant 2006;25:283-288. 9. Hart JW, Shiue LH, Shpall EJ, Alousi AM. Extracorporeal photopheresis in the treatment of graft-versus-host disease: evidence and opinion. Ther Adv Hematol 2013;4:320-334.

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DOI: 10.4274/tjh.2014.0206

Letter to the Editor

A Pediatric Patient with Intravenous Cyclosporine Anaphylaxis Who Tolerated the Oral Form İntravenöz Siklosporin ile Anafilaksiye Giren ve Oral Formu Tolere Eden Bir Pediatrik Olgu Pamir Işık1, Namik Özbek1, Emine Dibek Mısırlıoğlu2, Turan Bayhan3, Suna Emir1, Fatih Mehmet Azık1, Bahattin Tunç1 1Ankara

Children’s Hematology and Oncology Education and Research Hospital, Clinic of Pediatric Hematology, Ankara, Turkey Children’s Hematology and Oncology Education and Research Hospital, Clinic of Pediatric Allergy, Ankara, Turkey 3Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey 2Ankara

To the Editor, Cyclosporine is a potent immune suppressant and prevents T-cell activation and graft-versus-host disease by inhibiting calcineurin phosphatase [1,2]. Anaphylactic reaction due to intravenous infusion is a rare side effect, reported in 0.1% of patients [3,4]. Cremophor EL (polyoxyethylated castor oil), a solubilizing agent of the parenteral cyclosporine, has been blamed for hypersensitivity reactions [5]. Here we report a patient who developed anaphylaxis due to an intravenous form of cyclosporine. A 17-year-old boy, diagnosed with relapsed T-cell lymphoblastic lymphoma after achieving complete remission, underwent bone marrow transplantation from his fully matched brother. Intravenous cyclosporine (3 mg/kg/day) was started at day -1. At day +10, he developed a maculopapular rash on his trunk, fever, and weight gain, diagnosed as engraftment syndrome. His symptoms improved with intravenous methylprednisolone treatment (2 mg/kg/day). At day +14, at 10 min after the start of cyclosporine infusion, he developed disseminated erythematous rash, respiratory distress, severe chest pain, and facial edema. His systolic blood pressure was within the normal range, but his pulse rate decreased to 50/ min. Cyclosporine infusion was stopped and adrenaline, methylprednisolone, and pheniramine were intravenously administered. His complaints resolved within 30 min. We concluded that this reaction had occurred against the castor oil that exists in the intravenous form. Since cyclosporine treatment was crucial, we decided to give a capsule formulation that did not contain castor oil (Panosporin®). The capsule

was given in a 3-dose graded challenge (25 mg, 25 mg, and 50 mg with 30-min intervals) without significant reaction. In the following period he received scheduled doses without any problem. Skin tests performed with intravenous cyclosporine 5 months after anaphylaxis revealed positive results in our patient, but the test was negative for the donor. Informed consent was obtained. Polyoxyethylated castor oil has been associated with severe anaphylaxis [6]. A recent report concerning cyclosporineinduced anaphylaxis revealed that 11 patients had a reaction with the intravenous form. Seven of these patients tolerated an oral formulation of cyclosporine. It was claimed that hypersensitivity reaction to one formulation of cyclosporine does not preclude the use of a different formulation [5]. Some patients in the literature were reported to have allergic reactions to a parenteral form of both cyclosporine and tacrolimus attributed to castor oil, which is present in the formulation of both drugs [7,8]. Those patients had tolerated the oral formulation of cyclosporine without castor oil, as did our patient. Since the oral form of Panosporin® does not contain castor oil, we chose this drug for treatment for our patient without further reactions. Proposed mechanisms related to allergic reactions due to polyoxyethylated castor oil include IgE-mediated hypersensitivity, complement activation, and mast cell degranulation [5]. Our patient had a positive intradermal test for intravenous cyclosporine including polyoxyethylated castor oil. The positivity of the intradermal test suggested an IgE-mediated reaction. Interestingly, intensive immune

Address for Correspondence: Pamir IŞIK, M.D., Ankara Children’s Hematology and Oncology Education and Research Hospital, Clinic of Pediatric Hematology, Ankara, Turkey Phone: +90 312 596 98 74 E-mail: pamir1968@yahoo.com Received/Geliş tarihi : May 26, 2014 Accepted/Kabul tarihi : July 1, 2014

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Işık P, et al: Intravenous Cyclosporine Anaphylaxis

suppression in bone marrow recipients may facilitate the rare reaction to castor oil [7]. In conclusion, in the case of anaphylaxis with the parenteral form, oral products of cyclosporine that do not contain castor oil can be tried in patients who underwent bone marrow transplantation. 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. Key Words: transplantation

Cyclosporine,

Toxicity,

Stem

cell

Anahtar Sözcükler: Siklosporin, Yan etki, Kök hücre nakli References 1. Bickel M, Tsuda H, Amstad P, Evequoz V, Mergenhagen SE, Wahl SM, Pluznik DH. Differential regulation of colony-stimulating factors and interleukin 2 production by cyclosporin A. Proc Natl Acad Sci U S A 1987;84:32743277. 2. Ringden O. Cyclosporine in allogeneic bone marrow transplantation. Transplantation 1986;42:445-452.

3. Beauchesne PR, Chung NS, Wasan KM. Cyclosporine A: a review of current oral and intravenous delivery systems. Drug Dev Ind Pharm 2007;33:211-220. 4. Kuiper RA, Malingré MM, Beijnen JH, Schellens JH. Cyclosporine-induced anaphylaxis. Ann Pharmacother 2000;34:858-861. 5. Volcheck GW, Van Dellen RG. Anaphylaxis to intravenous cyclosporine and tolerance to oral cyclosporine: case report and review. Ann Allergy Asthma Immunol 1998;80:159163. 6. Gelderblom H, Verweij J, Nooter K, Sparreboom A. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer 2001;37:15901598. 7. Takamatsu Y, Ishizu M, Ichinose I, Ogata K, Onoue M, Kumagawa M, Suzumiya J, Tamura K. Intravenous cyclosporine and tacrolimus caused anaphylaxis but oral cyclosporine capsules were tolerated in an allogeneic bone marrow transplant recipient. Bone Marrow Transplant 2001;28:421-423. 8. Nicolai S, Bunyavanich S. Hypersensitivity reaction to intravenous but not oral tacrolimus. Transplantation 2012;94:61-63.

433

Letter to the Editor

DOI: 10.4274/tjh.2014.0252

Acquired Hemophilia Kazanılmış Hemofili Şinasi Özsoylu Retired Professor of Pediatrics, Hematology and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society

To the Editor, I would like to highlight 3 of our patients, a 14-year-old boy and females of 4 and 41 years old [1,2] with acquired hemophilia B seen at İhsan Doğramacı Children’s Hospital (previously Hacettepe) between 1963 and 1973 among 343 patients with hemophilia [3] on account of the case of acquired hemophilia A in a 78-year-old man who was successfully treated with a combined immunosuppressive and immunoadsorption approach by Bilgin et al. as reported in a recent issue of this journal [4]. Two of our 3 patients with acquired hemophilia B, which is rarer than acquired hemophilia A, improved most likely due to corticosteroid administration in at least one case and without any intervention in the others. We have shown antibodies against factor VIII in more than 20% of patients with X-linked hemophilia A and extremely rarely in normal people in low titers [3]; however, very severe bleeding due to the presence of AHG antibodies was not frequent. On this occasion I would rather use the term ‘hereditary’ instead of ‘congenital’ hemophilia, which was used by Bilgin et al., since inheritance is not involved in most congenital disorders, such as congenital tuberculosis, syphilis, CMV,

rubella infection, etc. May I also indicate that methyl prednisolone at 1 mg/kg/day may not be effective as an immunosuppressive, but 30 mg/kg/day seems to be more effective? 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. Key Words: Acquired hemophilia Anahtar Sözcükler: Kazanılmış hemofili References 1. Özsoylu S. Bir kız çocuğunda geçici hemofili B. Çocuk Sag ve Hast Dergisi 1964;7:101-107. 2. Özsoylu S, Özer F. Acquired factor IX deficiency: a report of two cases. Acta Haemat 1973;50:305-314. 3. Özsoylu S, Yamak B, Hiçsönmez G, Yetgin S, Pirnar A. Evaluation of 343 cases of hemophilia. Excerpta Med Int Congr Ser 1974;356:158-161. 4. Bilgin AU, Özcan M, Ayyıldız E, İlhan O. The treatment of acquired hemophilia with combination therapy of immunosuppressives and immunoadsorption. Turk J Hematol 2014;31:194-197.

Reply I would like to thank you for your comment and interest in my article. Firstly you are right about this subject; hemophilia is a genetic disorder. However, congenital hemophilia is a term that can be used in literature. Secondly high dose of steroid was not preferred for this patient because of his age and comorbid disease. Aynur UĞUR BİLGİN

Address for Correspondence: Şinasi ÖZSOYLU, M.D., Retired Professor of Pediatrics, Hematology and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society E-mail: sinasiozsoylu@hotmail.com Received/Geliş tarihi : June 21, 2014 Accepted/Kabul tarihi : June 23, 2014

434

Images in Hematology

DOI: 10.4274/tjh.2013.0364

Clinical Image in Hematology Generalized Necrobiotic Xanthogranuloma in a Patient with Multiple Myeloma Multipl Myeloma Tan覺l覺 Bir Hastada Yayg覺n Nekrobiyotik Ksantogran羹loma

Figure 1. Dermatological lesions.

435

Clinical Image in Hematology

Turk J Hematol 2014;31:435-436

Figure 2. Morphological analysis of the bone marrow aspirate demonstrated abnormal plasma cells.

Figure 3. Skin biopsy studies revealed 2 granulomas containing Touton giant cells and foamy histiocytes.

Analysis of a skin biopsy obtained from a 42-year-old female presenting with multiple yellowish to reddish-brown nodules and plaques on the arm, chest (Figure 1), and abdomen showed that she had generalized necrobiotic xanthogranuloma (Figure 3). Informed consent was obtained. A week later, she was diagnosed with IgG-kappa multiple myeloma (Figure 2). The laboratory workup showed the following: Hb: 109 g/L, Hct: 0.34 L/L, WBC: 9.5x109/L, Plt: 194x109/L, with normal differential white cell count. Other studied parameters included immunoglobulin IgG:1790 mg/dL (normal range: 750-1750), IgA: 279 mg/dL (normal range: 90-450), IgM: 40 mg/dL (normal range: 70-280); and kappa light chains: 1830 mg/dL (normal range: 629-1320 mg/dL). The coexistence of paraproteinemias and necrobiotic xanthogranuloma is well described (above all, monoclonal gammopathy of unknown significance), and it would seem reasonable to recommend performing at least serum electrophoresis for patients affected by these rare conditions [1]. 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. Key Words: Multiple, Myeloma, Skin, Necrobiotic, Xanthogranuloma Anahtar Sözcükler: Multipl, Myeloma, Deri, Nekrobiyotik, Ksantogranüloma Maria Jimenez Esteso, Jose Verdu, Francisco de Paz, Fabian Tarin University General Hospital of Alicante, Clinic of Hematology and Hemotherapy, Alicante, Spain Phone: +0096 591 38 71

E-mail: pepeverd@hotmail.com

Received/Geliş tarihi

: October 30, 2013

Accepted/Kabul tarihi

: November 11, 2013

Reference 1. Rose A, Robinson M, Kamino H, Latkowski JA. Necrobiotic xanthogranuloma. Dermatol Online J 2012;18:30.

436

DOI: 10.4274/tjh.2013.0104

Morphology in Hematology Aggressive Multiple Myeloma with Unusual Morphology Olağan Dışı Morfoloji ile Multipl Miyelom

Figure 1. Atypical and multinucleated plasma cell infiltration in the bone marrow aspirate (Wright’s stain, 100x). A 66-year-old male patient was admitted to the hospital with backache, fatigue, and paraesthesia and spasm in both legs. He had lower extremity numbness and bladder and bowel incontinence. Physical examination revealed the absence of bilateral lower extremity reflexes and lower extremity weakness. Magnetic resonance imaging showed a large mass extending from T8 to T9, fracture of T9, and compression of the spinal cord. Informed consent was obtained. Laboratory results at initial evaluation revealed the following: haemoglobin: 118 g/L, haematocrit: 33.5%, white blood cell count: 6.7x109/L, platelets: 192x109/L, blood urea nitrogen: 7.5 mmol/L, creatinine: 113.1 µmol/L, calcium: 1.9 mmol/L, total protein: 63 g/L; albumin: 31 g/L; and erythrocyte sedimentation rate: 59 mm/h. Protein studies by nephelometry revealed IgA of 4.25 g/L (reference range: 0.7-4 g/L) and lambda light chain of 3.98 g/L (reference range: 0.9-2.1 g/L). A small monoclonal spike was present upon protein electrophoresis. Urine immunoelectrophoresis documented no monoclonal light chain. Bone marrow aspirate and biopsy were performed and the patient underwent surgical decompression and stabilisation of the thoracic spine. The bone marrow aspirate and biopsy morphology showed infiltration with atypical, multilobated, cleaved, and monocytoid nuclei plasma cells (Figures 1 and 2). The biopsy material stained positive with lambda light chain and CD138. 437

Morphology in Hematology

Turk J Hematol 2014;31:437-438

Figure 2. Atypical and multinucleated plasma cell infiltration in the bone marrow biopsy (H&E, 20x). The neoplastic plasma cells exhibit a variety of morphologies. Poorly differentiated plasma cells may show atypical morphology, making it difficult to recognise their plasma cell nature. Plasma cell morphology correlates with disease course, prognosis, and resistance to conventional treatment [1,2,3]. 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. Key Words: Multiple myeloma, Unusual morphology Anahtar Sözcükler: Multipl miyelom, Olağan dışı morfoloji Mehmet Sönmez1, Hasan Mücahit Özbaş1, Nilay Ermantaş1, Ümit Çobanoğlu2 1Karadeniz

Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey

2Karadeniz

Technical University Faculty of Medicine, Department of Pathology, Trabzon, Turkey

Phone: +90 462 377 58 48 E-mail: mesonmez@yahoo.com Received/Geliş tarihi

: March 20, 2013

Accepted/Kabul tarihi

: April 25, 2013

References 1. Stifter S, Babarovic E, Valkovic T, Seili-Bekafigo I, Stemberger C, Nacinovic A, Lucin K, Jonjic N. Combined evaluation of bone marrow aspirate and biopsy is superior in the prognosis of multiple myeloma. Diagn Pathol 2010;5:30. 2. Kyle RA, Rajkumar SV. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leukemia 2009;23:3-9. 3. Erkut N, Çobanoğlu Ü, Sönmez M. Multiple myeloma with multilobated plasma cell nuclei. Turk J Hematol 2011;28:158159.

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31st Volume Index / 31. Cilt Dizini SUBJECT INDEX - KONU DİZİNİ 2014

Abnormal Hemoglobins Hemoglobin / Hemoglobin, 97 Variant / Varyant, 97 Hemoglobinopathy / Hemoglobinopati, 97 Hb Lansing / Hb Lansing, 317 Abnormal hemoglobin / Anormal hemoglobin, 317 Alpha-globin / Alfa globin, 317 Hemoglobinopathy / Hemoglobinopati, 319 Hb Jabalpur / Hb Jabalpur, 319 High performance liquid chromatography / Yüksek performanslı sıvı kromatografisi, 319

Acute Leukemia Acute myeloid leukemia / Akut miyeloid lösemi, 40,128,143,149 NPM1 gene type A mutation / NPM1 geni tip A mutasyonu, 40 FLT3-ITD / FLT3-ITD, 40 Allele-specific polymerase chain reaction/ Allel-özgül polimeraz zincir reaksiyonu, 40 T-cell prolymphocytic leukemia / T-hücreli prolenfositik lösemi, 75 Cerebral involvement / Serebral tutulum, 75 Central nervous system / Santral sinir sistemi, 75 Stereotactic biopsy / Stereotaktik biyopsi, 75 LYL1 gene / LYL1 geni, 128 Myelodysplastic syndrome / Miyelodisplastik sendrom, 128 Chronic myelogenous leukemia in blast and accelerated phases / Akselere ve blastik evre kronik miyeloid lösemi, 128 Real-time polymerase chain reaction / Gerçek-zamanlı polimeraz zincir reaksiyonu, 128 Cytogenetically normal / Sitogenetiği normal, 143 Mutations / Mutasyonlar, 143 Prognosis / Prognoz, 143 Wilms tumor 1 gene / Wilms tümör 1 geni, 143 Cytokines / Sitokinler, 149 Granulocyte colony-stimulating factor / Granülosit koloni büyüme etkeni, 149 RT-PCR / RT-PCR, 149 Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 203 Isolated breast relapse / İzole meme nüksü, 203 Leukemia cutis / Lösemi kutis, 213 Aleukemic leukemia / Aleukemic lösemi, 213 Persistent / Persistan, 213

Thalassemia intermedia / Talasemi intermedia, 311 Leukemia / Lösemi, 311,321 Cancer / Kanser, 311 Infection / İnfeksiyon, 321 Liver abscess / Karaciğer absesi, 321 Clofarabine / Clofarabine, 325 Multi-relapsed Leukemia / Çoklu relaps lösemi, 325 Children / Çocuk, 325 Relaps / Relaps 325 Myelosuppression / Miyelosupresyon, 399 Thiopurine S-methyl transferase / Thiopurin S-metil transferaz, 399 Acute leukemia / Akut lösemi, 399

Anemia Microcytosis / Mikrositoz, 339 Sideroblastic anemia / Sideroblastik anemi, 339 Zinc deficiency / Çinko eksikliği, 339

Aplastic Anemia Mesenchymal stem cell / Mezenkimal kök hücreler, 180 Immune recovery / İmmün yeniden yapılanma, 180 Aplastic anemia / Aplastik anemi, 180,411 Allogeneic stem cell transplantation / Allogenik kök hücre nakli, 180 Onychomycosis/ Onikomikoz, 411 Terbinafine / Terbinafin, 411 Hematological toxicity / Hematolojik toksisite, 411 Pancytopenia / Pansitopeni, 411 Adverse events / Yan etkiler, 411

Autoimmune Hemolytic Anemia Autoimmune hemolytic anemia / Otoimmün hemolitik anemi, 328

Bone Marrow Cystinosis / Sistinozis, 106 Bone marrow / Kemik iliği, 106 Examination / İnceleme, 106 Gelatinous transformation / Jelatinöz transformasyon, 175 Bone marrow / Kemik iliği, 175 Sarcoidosis / Sarkoidoz, 192 Bone / Kemik, 192

Bone marrow / Kemik iliği, 192 Hematologic manifestations of systemic diseases / Sistemik hastalıkların hematolojik belirtileri, 211 Lymphoid cells neoplasms / Lenfoid hücreler tümörleri, 211 Bone marrow failure / Kemik iliği yetmezliği, 211 Sensitized serum / Sensitize serum, 266 Bone marrow cells / Kemik iliği hücreleri, 266 Rejection / Rejeksiyon, 266 Transplantation / Transplantasyon, 266 Angiosarcoma / Anjiosarkom, 408 Splenomegaly / Splenomegali, 408 Bone marrow infiltration / Kemik iliği tutulumu, 408

Chediak-Higashi Syndrome Chediak-Higashi syndrome / Chediak-Higashi sendromu, 210,313,426 Giant granules / Dev granüller, 210 Erytrophagocytosis / Eritrofagositoz, 210 Child / Çocuk, 313 Oral lesions / Oral lezyonlar, 313 Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiositoz, 426 Oculocutaneous albinism / Okülokütanöz albinizm, 426 Lyst gene / Lyst geni, 426 Immune deficiency / İmmün yetmezlik, 426

Chemotherapy Doxorubicin / Doksorubisin, 68 Nitric oxide / Nitrik oksit, 68 Nitric oxide synthase inhibitors / Nitrik oksit sentaz inhibitörleri, 68

Chronic Leukemia PDGFRA / PDGFRA, 56 Chronic eosinophilic Leukemia / Kronik eozinofilik lösemi, 56 Imatinib / İmatinib, 56 India / Hindistan, 56 Lymphoma / Lenfoma, 92 Chronic myeloid leukemia / Kronik myeloid lösemi, 92 Secondary malignancy / Sekonder malignite, 92

Coagulation Blood coagulation / Kan koagülasyonu, 261 Colloids/ Kolloidler, 261 Genital neoplasms / Genital neoplaziler, 261 Thromboelastography / Trombelastografi, 261

Granulocytic Sarcoma Granulocytic sarcoma / Granülositik sarkom, 88 Breast / Meme, 88 Stem cell transplantation / Kök hücre nakli, 88 18FDG-PET/CT / 18FDG-PET/CT, 88

Hematological Malignancies Invasive fungal infection / İnvazif fungal infeksiyonlar, 111, 342 Antifungal treatment / Antifungal tedavi, 111 Evidence / Kanıt, 111 Hematological malignancies / Hematolojik malignansi, 111

Acute myeloblastic leukemia / Akut miyeloblastik lösemi, 201 Granulocytes / Granülositler, 201 Acute leukemia / Akut lösemi, 201 Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiositoz, 201 Extramedullary myeloid tumor / Extramedüller miyeloid tümör, 295 Pancreas / Pankreas, 295 Allogeneic stem cell transplantation / Allogeneik kök hücre nakli, 295 Diagnosis / Tanı, 342 Imaging / Görüntüleme, 342 Serology / Seroloji, 342 Plasmacytoid dendritic cell / Plazmasitoid Dendritik Hücre, 422 Leukemia / Lösemi, 422 Cutaneous lesion / Deri Lezyonu, 422 CD4 (+) / CD4 (+), 422 CD56 (+) / CD56 (+), 422

Hemophagocytic Lymphohistiocytosis Acquired hemophagocytic lymphohistiocytosis / Edinsel hemofagositik lenfohistiyositoz, 103 Intravenous immunoglobulin / İntravenöz immunoglobulin, 103 Adenovirus-associated hemophagocytic lymphohistiocytosis / Adenovirus ilişkili hemofagositik lenfohistiyositoz, 103 Gaucher disease / Gaucher hastalığı, 307 Hemophagocytic lymphohistiocytosis / Hemafagositik lenfohistiositozis, 307 Hepatosplenomegaly / Hepatosplenomegali, 307 Hemophagocytosis / Hemofagositoz, 309 Preterm / Prematüre, 309 Toxoplasma gondii / Toxoplasma gondii, 309

Hemophilia Hemophilia A / Hemofili A, 17,101 Inhibitor formation / İnhibitör oluşumu, 17 F8 gene mutation / F8 gen mutasyonu, 17 Single nucleotide gene polymorphisms / Tek Nükleotid Gen Polimorfizmleri, 17 Interleukins/cytokines/ İnterlökinler/sitokinler, 17 Association study / İlişkilendirme çalışması, 17 Newborn / Yenidoğan, 101 Liver / Karaciğer, 101 Hemorrhage / Hemoraji, 101 Postnatal hemorrhagic shock / Doğum sonrası hemorajik şok, 101 Bleeding / Kanama, 101 Factor VIII deficiency / Faktör VIII eksikliği, 194 Acquired / Kazanılmış, 194 Immunosuppressive / Immunsupresif, 194 Hemophilia B / Hemofili B, 226 Factor IX / Faktör IX, 226 Mutation / Mutasyon, 226 Intron 3 / İntron 3, 226 mRNA splice site / mRNA eklenme bölgesi, 226 Acquired hemophilia / Kazanılmış hemofili, 434

Immune Thrombocytopenic Purpura Thrombocytopenia / Trombositopeni, 32 Long-term survival / Uzun dönem takip, 32 Children / Çocuk, 32 Cerebral infarction / Serebral enfarkt, 184 Idiopathic thrombocytopenic purpura / İdyopatik trombositopenik purpura, 184 Triple X syndrome / Triple X sendromu, 184

Iron Disorder Anemia / Anemi, 61 Hepcidin / Hepsidin, 394 Insulin resistance / İnsülin direnci, 61 Iron overload / Demir yükü, 394 Inflammation / İnflamasyon, 61 Obesity / Obezite, 61

Leukocyte Leukocyte populations / Lökosit popülasyonu, 49 C-reactive protein / C-reaktif protein, 49 Febrile children / Ateşli çocuk, 49

Lymphoma Lymphoma / Lenfoma, 92,188 Chronic myeloid leukemia / Kronik myeloid lösemi, 92 Secondary malignancy / Sekonder malignite, 92 Adrenal glands / Adrenal bez, 188 Addisonian crisis / Addison krizi, 188 Non-Hodgkin lymphoma / Non-Hodgkin lenfoma, 205,207,403 Lymphoid cells neoplasms / Lenfoid hücreler tümörler, 205 B-Cell neoplasms / B-Hücre tümörleri, 205,207 Pharmacotherapeutics / Farmakoterapötikler, 207 Cytogenetics / Sitogenetik, 290 Non-Hodgkin’s lymphoma / Hodgkin dışı lenfoma, 290,381 Other lymphoproliferative diseases / Diğer lenfoproliferatif hastalıklar, 290 Other leukemias / Diğer lösemiler, 290 Primary renal lymphoma / Primer renal lenfoma, 315 Follicular lymphoma / Foliküler lenfoma, 315 Treatment option / Tedavi seçeneği, 315 Primary adrenal non-Hodgkin lymphoma / Primer adrenal non-Hodgkin lenfoma, 332 Central nervous system / Merkezi sinir sistemi, 332 Prognosis / Prognoz, 332 Therapy / Terapi, 332 Vincristine / Vinkristin, 337 Neuropathy / Nöropati, 337 Vocal cord paralysis / Vokal kord paralizi, 337 Paralytic ileus / Paralitik ileus, 337 Chemokines / Kemokinler, 381 Cytokines / Sitokinler, 381 Lymphocytes / Lenfositler, 381 Th2 pathway / Th2 yolağı, 381 Adult T-cell leukemia/lymphoma / Erişkin T-hücreli lösemi/lenfoma, 424 Mogamulizumab / Mogamulizumab, 424 Hemodialysis / Hemodiyaliz, 424

CCR4 / CCR4, 424 HTLV-1 / HTLV-1, 424 Intravascular large B-cell lymphoma / İntravasküler büyük B hücreli lenfoma, 403 Prostate / Prostat, 403 Non-germinal center B-cell / Non-germinal merkez B hücre, 403 Multiple Myeloma Multiple myeloma / Multiple myeloma, 84,136,231,374,388,428,435,437 LDH / LDH, 84 Prognosis / Prognoz, 84 Renin-angiotensin system / Renin-anjiotensin sistemi, 136 Progenitor cell / Progenitör hücre, 136 CD34+ / CD34+, 136 Drug resistance / İlaç direnci, 231 Cell cycle / Hücre döngüsü, 231 Apoptosis / Apoptoz, 231 HLA-A / HLA-A, 388 HLA-B / HLA-B, 388 HLA-DRB1 / HLA-DRB1, 388 Genetic susceptibility / Genetik yatkınlık, 388 Bone-specific alkaline phosphatase / Kemik Spesifik Alkalen Fosfataz, 374 Bortezomib / Bortezomib, 374 Thalidomide / Talidomid, 374 Unusual morphology / Olağan dışı morfoloji, 437 Skin / Deri, 435 Necrobiotic / Nekrobiyotik, 435 Xanthogranuloma / Ksantogranüloma, 435

Lymphoproliferative Disorders Renal transplantation / Böbrek nakli, 79 Post-transplant lymphoproliferative disease/ Posttransplant lenfoproliferatif hastalık, 79 Lymphoma / Lenfoma, 79 Immunosuppression / İmmünsupresyon, 79 Rituximab / Rituksimab, 79 Abnormal karyotype / Anormal karyotip, 79

Mutation Myeloproliferative disorders / Miyeloproliferatif hastalıklar, 239 Myeloproliferative neoplasm / Myeloproliferatif neoplasm, 239 Essential thrombocythemia / Esansiyel trombositemi, 239 Polycythemia vera / Polisitemia vera, 239 Primary myelofibrosis / Primer myelofibrozis, 239 JAK2V617F mutation / JAK2V617F mutasyonu, 239 Bone marrow histopathology / Kemik iliği histolojisi, 239 Red cell mass / Kırmızı kan hücresi kitlesi, 239 Erythrocyte count / Kırmızı kan hücresi sayısı, 239

Myelodysplasia Myelodysplasia / Miyelodisplazi, 394

Myeloproliferative Disorders Myeloproliferative disorders / Miyeloproliferatif hastalıklar, 121,239 Endothelial cell protein C receptor / Endotelyal protein C reseptörü, 121

Thrombosis / Tromboz, 121 Myeloproliferative neoplasm / Myeloproliferatif neoplasm, 239 Essential thrombocythemia / Esansiyel trombositemi, 239 Polycythemia vera / Polisitemia vera, 239 Primary myelofibrosis / Primer myelofibrozis, 239 JAK2V617F mutation / JAK2V617F mutasyonu, 239 Bone marrow histopathology / Kemik iliği histolojisi, 239 Red cell mass / Kırmızı kan hücresi kitlesi, 239 Erythrocyte count / Kırmızı kan hücresi sayısı, 239

Plasmacytoma 286-Solitary Bone Plasmacytoma Progressing into Retroperitoneal Plasma Cell Myeloma with No Related End Organ or Tissue Impairment: A Case Report and Review of the Literature Solitary bone plasmacytoma / Soliter kemik plazzmasitoması, 286 Retroperitoneal plasma cell myeloma / Retroperitoneal plazma hücreli myeloma, 286 Related end organ or tissue impairment / İlişkili organ ya da doku disfonksiyonu, 286 Bone marrow plasmacytosis / Kemik iliği plazmasitozu, 286 Extramedullary myeloid tumor / Extramedüller miyeloid tümör, 295 Pancreas / Pankreas, 295 Allogeneic stem cell transplantation / Allogeneik kök hücre nakli, 295

Platelet Disorders Blood cell count / Kan hücresi sayımı, 107 Blood platelets / Kan trombositleri, 107 Blood platelet disorders / Kan trombosit bozuklukları, 107 Peripheral blood smear / Periferik kan yayması, 107

Paroxysmal Nocturnal Hemoglobinuria SLE / SLE, 323 CD55 / CD55, 323 CD59 / CD59, 323 PNH / PNH, 323

Pure Red Cell Aplasia Lenalidomide / Lenalidomid, 99 PRCA / PRCA, 99 MSD/ MDS, 99

Radiotherapy Castleman’s disease / Castleman hastalığı, 197 Radiotherapy / Radyoterapi, 197 Unicentric / Unisentrik, 197

Red Cell Membrane Disorder RBC / Eritrosit, 25 Membrane disorders / Zar bozuklukları, 25 Band 3 / Band 3, 25 Flow cytometry / Akım sitometri, 25

Sickle Cell Sickle cell disease / Orak hücre hastalığı, 255 Inflammation / Enflamasyon, 255 Cytokine / Sitokin, 255

TNFSF15 gene / TNFSF15 geni, 255

Stem Cell Transplantation Granulocytic sarcoma / Granülositik sarkom, 88 Breast / Meme, 88 Stem cell transplantation / Kök hücre nakli, 88 18FDG-PET/CT / 18FDG-PET/CT, 88 Epstein-Barr virus / Epstein-Barr virüs, 155 Parvovirus B19 / Parvovirus B19, 155 Allogeneic stem cell transplantation / Allojenik kök hücre transplantasyonu, 155 Real-time PCR / Gerçek zamanlı PCR, 155 Cytomegalovirus reactivation / Sitomegalovirüs reaktivasyonu, 276 Human leukocyte antigens / İnsan lökosit antijeni, 276 Allogeneic stem cell transplantation / Allojeneik kök hücre nakli, 276 Graft-versus-host disease / Graft versus host hastalığı, 276 Prognosis / Prognoz, 276 CMV scoring index / CMV skorlama indeksi, 276

Thalassemia Thalassemia / Talasemi, 5 Thalassemia intermedia / Talasemi intermedya, 5 Iron chelation / Demir şelasyonu, 5 Ineffective erythropoiesis / İnefektif eritropoez, 5 Iron overload / Demir yükü, 5 Beta-thalassemia / Beta-talasemi, 272 C282Y mutation / C282Y mutasyonu, 272 Hemochromatosis / Hemokromatozis, 272 Thalassemia intermedia / Talasemi intermedia, 311 Leukemia / Lösemi, 311 Cancer / Kanser, 311 β-Thalassemia minor / β-Talasemi minor, 363 Bcl-2 / Bcl-2, 363 Apoptosis / Apoptoz, 363 Thymus / Timüs, 417 Blood transfusion / Kan Transfüzyonu, 417 Beta-thalassemia / Beta talasemi, 417 Iron overload / Demir yüklenmesi, 417 Tymic lndex / Timik indeks, 417

Thrombosis P-selectin polymorphisms / P-selektin polimorfizmleri, 357 Thrombosis / Tromboz, 357 Antiphospholipid syndrome / Antifosfolipid sendrom, 357

Thrombotic Thrombocytopenic Purpura 216-Atypical Hemolytic Uremic Syndrome: Differential Diagnosis from TTP / HUS and Management Atypical hemolytic uremic syndrome (aHUS) / Atipik hemolitik üremik sendrom (aHÜS), 216 Thrombotic thrombocytopenic purpura (TTP) / Trombotik trombositopenik purpura (TTP), 216

Eculizumab / Eculizumab, 216 TTP/HUS/TTP / HUS, 216 Thrombotic microangiopathy (TMA) / Trombotik mikroanjiopati (TMA), 216 ADAMTS13 / ADAMTS13, 216

Transfusion Duffy phenotyping / Duffy fenotiplendirme, 367 Kidd phenotyping / Kidd fenotiplendirme, 367 Genotyping / Genotiplendirme, 367 Multitransfused patients / Çoklu transfüzyon alan hastalar, 367

Treatment Myocardial infarction after rituximab / Rituximab’den sonra myokard infarktı, 95 Posterior reversible encephalopathy syndrome (PRES) / Arka geri dönüşümlü ensefalopati sendromu (PRES), 109 Methotrexate / Metotreksat, 109 Magnetic resonance imaging / Manyetik rezonans görüntüleme, 109 Fluidattenuated inversion recovery / Sıvı zayıflatılmış dönüşüm kazanımı (FLAIR), 109 Invasive fungal infection / İnvazif fungal infeksiyonlar, 111 Antifungal treatment / Antifungal tedavi, 111 Evidence / Kanıt, 111 Hematological malignancies / Hematolojik malignansi, 111

Other Turkish Journal of Hematology: From “Istanbul Contribution to Clinical Science” to “Pubmed Central”, 1 Posterior reversible encephalopathy syndrome (PRES) / Arka geri dönüşümlü ensefalopati sendromu (PRES), 109 Methotrexate / Metotreksat, 109 Magnetic resonance imaging / Manyetik rezonans görüntüleme, 109 Fluidattenuated inversion recovery / Sıvı zayıflatılmış dönüşüm kazanımı (FLAIR), 109 Blood / Kan, 161 Blood component / Kan ürünü, 161 Wastage / İsraf, 161 Transfusion / Nakil, 161 Blood bank / Kan bankası, 161 Erythrocyte deformability / Eritrosit deformabilitesi, 168 RhoA / RhoA, 168

Rho-kinase / Rho-kinaz, 168 Y-27632 / Y-27632, 168 Fasudil / Fasudil, 168 Lysophosphatidic acid / Lizofosfatidik asit, 168 C3 / C3, 168 Cerebral infarction / Serebral enfarkt, 184 Idiopathic thrombocytopenic purpura / İdyopatik trombositopenik purpura, 184 Triple X syndrome / Triple X sendromu, 184 Castleman’s disease / Castleman hastalığı, 197 Radiotherapy / Radyoterapi, 197 Unicentric / Unisentrik, 197 Thrombocytopenia / Trombositopeni, 199 Kawasaki disease / Kawasaki hastalığı, 199 Aneurysm / Anevrizma, 199 Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 203 Isolated breast relapse / İzole meme nüksü, 203 Hematologic manifestations of systemic diseases / Sistemik hastalıkların hematolojik belirtileri, 211 Lymphoid cells neoplasms / Lenfoid hücreler tümörleri, 211 Bone marrow failure / Kemik iliği yetmezliği, 211 Hypocalcemic seizure / Hipokalsemik nöbet, 336 Infantile malign osteopetrosis / Malign infantil osteopetrosis, 336 TCIRG1 gene mutation / TCIRG1 gen mutasyonu, 336 Cyclosporine / Siklosporin, 432 Toxicity / Yan etki, 432 Stem cell transplantation / Kök hücre nakli, 432 Allograft / Allogreft, 430 Extracorporeal photopheresis / Ekstrakorporeal fotoferez, 430 Heart transplantation / Kalp transplantasyonu, 430 Quilty effect / Quilty etkisi, 430 Rejection / Rejeksiyon, 430 Crystalline inclusion bodies / Kristal inklüzyon cisimcikler, 428 Gaucher cells / Gaucher hücreleri, 428 Multiple myeloma / Multiple miyeloma, 428 Humoral immune response / Hümoral immün yanıt, 420 Immunodeficiency diseases / İmmün yetmezlikler, 420 Immune response disorder / İmmün yanıt bozukluğu, 420 Immunoglobulins / İmmünglobulinler, 420 Hyper IgM syndrome / Hiper IgM sendromu, 420 CD40L / CD40L, 420

31st Volume Index / 31. Cilt Dizini AUTHOR INDEX - YAZAR DİZİNİ 2014

A. Muzaffer Demir.............. 111,321 A1i Taher.........................................5 Abbas Behzad- Behbahani.............25 Abbas Hajifathali.........................388 Abdülkerim Yıldız.......................323 Adalet Meral Güneş......................32 Adnan Şimşir...............................403 Ahmet Ata Özçimen....................255 Ahmet Emre Eşkazan..................374 Ahmet Koç..................................417 Ahmet Özet.................................207 Aiste Savukaityte.........................367 Ajay Gogia............................. 95,203 Akın Uysal.....................................79 Alev Erikçi.....................................75 Alexandra Agapidou...................422 Ali Ayçiçek..................................417 Ali Fettah.....................................336 Ali Uğur Ural...............................231 Alişan Yıldıran.............................420 Alpay Azap..................................342 Altay Atalay.................................155 Amirhossein Emami....................149 Amr Radwan...................................5 Ana Cvetanovic...........................315 Anıl Kumar N................................56 Anthony Haddad.............................5 Aptullah Haholu...........................75 Arbil Açıkalın...................... 205,295 Arezou Sayad...............................388 Arturas Inciura............................367 Arzu Akçay..................................311 Asit Mridha.................................286 Aslıhan Karul..............................363 Asmae Quessar............................408 Asu Fergün Yılmaz......................403 Atahan Çağatay...........................111 Aydan İkincioğulları...................420 Ayfer Çolak.................................319 Ayhan Dönmez............................403 Ayhan Gazi Kalaycı.....................420

Aylin Heper.................................430 Aynur Küçükcongar......................49 Aynur Uğur Bilgin.......................194 Ayşegül Ünüvar............ 212,311,325 Ayşegül Verim.............................301 Ayşegül Zenciroğlu.....................101 Ayşenur Bahadır............................68 Ayşenur Öztürk...........................317 Aytemiz Gürgey..............................1 Ayten Bilir...................................261 Bahattin Tunç...................... 336,432 Bahoush Gholamreza..................307 Balkis Meddeb.............................188 Banu Aydın..................................101 Banu Sarsık..................................403 Barış Malbora...................... 101,103 Barış Naiboğlu.............................301 Begüm Şirin Koç................. 212,325 Bengü Timoçin................................1 Berna Bozkurt Duman................205 Berna Totan Ateş.........................197 Beyza Ener...................................342 Birol Baytan...................................32 Branimir Spassov...........................40 Bülent Çetin................................207 Bülent Eser..................................155 Bülent Güzel...............................417 Bülent Kantarcıoğlu....................411 Bülent Orhan...............................207 Bülent Zülfikar..............................17 Burak Erer...................................357 Burak Toprak..............................319 Burak Uz.....................................136 Burçin Beken....................... 199,210 Burcu Fatma Belen......................399 Burhan Ferhanoğlu.....................374 Cafer Adıgüzel............................411 Can Boğa.....................................180 Cansın Tulunay Kaya..................430 Cem Atabey...................................75 Cengiz Beyan...............................107

Cengiz Ceylan.............................319 Çetin Erol....................................430 Chang-Kun Lin...........................226 Christina Chadjiaggelidou..........422 Chun-Lian Jin.............................226 Çiğdem Aliosmanoğlu................313 Çiğdem Tepe Karaca...................301 Çiğdem Usul Afşar.............. 197,205 Cüneyt Müderrisoğlu....................61 Dalia Abdel Raouf.......................143 Danguole Raulinaityte................367 Debdatta Basu.............................175 Deniz Gören Şahin......................428 Deniz Teke....................................84 Deniz Tuğcu................................311 Derya Özyürük............................417 Derya Sağlam..............................155 Diana Remeikiene.......................367 Didem Atay.................................215 Didem Torun...............................317 Dilek Dilli....................................101 Dilek Erdem................................381 Dimitra Markala..........................422 Dina Ahmed Mehaney................394 Doaa Shahin................................128 Doğuş Vurallı................................49 Dong-Hua Cao............................226 Duran Karabel.............................313 Düzgün Özatlı.............................381 Ebru Koca...................................121 Ebru Sarıbeyoğlu.........................311 Elham Jaberi................................149 Elham Roshandel........................388 Elif Çalış......................................197 Elif Ezgi Üstün............................430 Elif Gülsüm Ümit........................321 Elif Suyanı...................................323 Elona Juozaityte..........................367 Emad Azmy.................................143 Emine Bağır.................................197 Emine Dibek Mısırlıoğlu.............432

Emine Türkkan...........................215 Emre Tekgündüz...........................92 Ender Ülgen..................................61 Engin Kelkitli...................... 201,381 Engin Yenilmez.............................68 Enver Öte....................................290 Ercan Kaya..................................338 Erdem Dağlıoğlu.........................417 Eren Gündüz.................. 88,338,428 Erika Skrodeniene.......................367 Erkut Erkurt................................197 Erol Ayyıldız................................194 Esma Altunoğlu............................61 Ezgi Uysalol........................ 212,325 Fabian Tarin................................436 Farzaneh Aboualizadeh.................25 Fatemeh Samiee Rad...................161 Fatih Mehmet Azık.....................432 Fatma Akyol................................319 Fatma Gümrük............. 199,210,272 Fatma Nur Sarı............................309 Ferda özbay Hoşnut....................103 Feride İffet Şahin.........................290 Feryal Karaca...............................197 Fevzi Altuntaş...............................92 Fezan Şahin Mutlu......................261 Fibo Ten Kate..............................239 Figen Atalay................................121 Figen Başaran Demirkazık..........342 Figen Kuloğlu.............................321 Figen Özçay................................103 Filiz Karagöz...............................420 Francisco de Paz.........................436 Funda Pepedil Tanrıkulu............411 Füsun Erdenen..............................61 Gargi Tikku.................................286 Ghasem Miri-Aliabad..................106 Gökhan Kuş................................261 Gökhan Metan............................342 Gökhan Sargın.................... 192,363 Gueorgui Balatzenko.....................40 Gülendam Bozdayı......................276 Gülşah Kaygusuz..........................79 Gülsan Türköz Sucak.......... 276,323 Gülsüm Kadıoğlu Şimşek...........309 Gülyüz Öztürk............................325 Günay Balta.................................272 Gupta Shweta..............................341 Gürbey Soğut..............................430 Gürhan Kadıköylü.............. 192,363 Güven Çetin................................374 Güven Yılmaz..............................411 Habib Alah Golafshan...................25 Hakan Göker...............................136 Hakan Özdoğu.................... 180,295

Halil Uğur Hatipoğlu..................426 Halis Akalın.................................111 Hamdi Akan..................... 1,111,342 Hamdi Cihan Emeksiz..................49 Hanjun Kim................................184 Hasan Alaçam..............................381 Hasan Celalettin Ümit.................321 Hasan Mücahit Özbaş.................438 Hasan Suat Arslantaş...................197 Hasibe Gökçe çınar.....................101 Hava Üsküdar Teke............... 84,428 Hayam Fathi Ghazi.....................128 Hela Ben Abid.............................188 Hendrik De Raeve.......................239 Hilmi Atay...................................381 Hong-Gui Xu..............................266 Hüseyin Göksülük......................430 Hüseyin Gümüş..........................417 Hüseyin Kemal Türköz...............411 Hüseyin Kılıç...............................155 İbrahim C. Haznedaroğlu...........136 İdris Kandemir............................155 İdris Yücel...................................381 İlknur Kozanoğlu........................180 İlknur Sivrikoz Ak.........................88 İnanç Değer Fidancı......................17 İpek Durusu....................................1 İrfan Yavaşoğlu.................... 192,363 Işık Kaygusuz Atagündüz...........411 Işınsu Kuzu...................................79 Itır Şirinoğlu Demiriz....................92 Ivan Petkovic...............................315 Ivica Pejcic..................................315 Jain Prantesh...............................341 Jan Jacques Michiels....................239 Ji-Yong Lee..................................184 Jian-Pei Fang...............................266 Jin-biao Zhang.............................328 Jing-Li..........................................328 Jing-Li Sun..................................226 Jose Verdu...................................436 Juwon Kim..................................184 Kaan Kavaklı.................................17 Kadir Acar...................................276 Kai Mu.........................................226 Kanay Yararbaş............................319 Kansu Büyükafşar.......................168 Kap Jun Yoon..............................184 Karima Kacem.............................188 Kenan Keven.................................79 King H. Lam................................239 Kubilay Ekiz................................201 Kübra Bölük................................215 Kürşat Kaptan.............................107 Lakshmaiah K Chinnagiriyappa.... 56

Lamia Aissaoui............................188 Leili Moezzi...................................25 Levent Yıldız....................... 201,381 Leyla Ağaoğlu...................... 311,325 Leylagül Kaynar..........................155 Li-juan Jia....................................328 Lu-Hong Xu................................266 M. Cem Ar........................ 17,61,374 Mahmut Civilibal........................426 Mahmut Kebapcı.........................338 Mahmut Yeral..............................180 Mahshid Mehdizadeh..................388 Maitreyee Bhattacharyya...............99 Makoto Fukuda...........................424 Malina Romanova.........................40 Margarita Guenova.......................40 Mari Yoshihara............................424 Maria Jimenez Esteso..................436 Maria Tzimou..............................422 Mehmet Halil Celiksoy...............420 Mehmet Kanbay............................84 Mehmet Sönmez.........................438 Mehmet Turgut................... 201,381 Mehmet Yekta Öncel...................309 Mehran Karimi..............................25 Melek Ergin.................. 197,205,295 Melike Sezgin Evim.......................32 Meltem Ceyhan Bilgici................420 Meltem Olga Akay................ 88,261 Memiş Hilmi Atay.......................201 Meral Günaldı..................... 205,295 Merih Kızıl Çakar.......................323 Meryem Albayrak........................399 Miljan Krstic...............................315 Mina Bahrololoumi Shapourabadi..............................149 Mine Kadıoğlu...............................68 Miri-Aliabad Ghasem..................307 Mirjana Balic...............................315 Mirjana Todorovic.......................315 Mohamad Mehdi Daneshi Kohan............................161 Mohamed Abd El Kader Morad...........................394 Mohamed Sabry..........................143 Mohammad Mohammadi...........149 Mohammad Reza Khorramizadeh...........................149 Mohammad Taghi Akbari...........388 Mojtaba Tabatabaei Yazdi...........149 Monica Jain.................................286 Mualla Çetin...............................199 Müge Gökçe................................311 Müge Sayitoğlu...........................136 Muhit Özcan...............................194

Murat Elevli................................426 Murat İnanç.................................357 Murat Kutlay.................................75 Murat Söker................................313 Müsemma Karabel......................313 Mustafa Başak...............................84 Mustafa Çetin..............................155 Mustafa Karagülle................. 88,428 Mustafa N. Yenerel......................216 Mutlu Arat............................. 79,111 Nadia El-Menshawy............ 128,143 Naghmeh Rajaei..........................149 Naji Mallat.......................................5 Nalan Akyürek............................399 Namık Özbek...................... 103,432 Namratha N. Rajkumar.................56 Nazan Özsan...............................403 Necmiye Canacankatan..............255 Neda Setayesh.............................149 Nejat Akar............................. 97,317 Neşe Saltoğlu...............................111 Neşe Yaralı........................... 309,336 Neslihan Andıç............................428 Nikolay Stoyanov..........................40 Nil Güler.....................................381 Nilay Ermantaş............................438 Nilgün Kurucu..............................68 Nilgün Sayınalp...........................136 Nilgün Selçuk Duru....................426 Nilüfer Alpay...............................357 Noha M. El Husseiny..................394 Nurdan Uras................................309 Nurullah Okumuş.......................101 Oğuz K. Başkurt..........................168 Oğuz Kara...................................205 Olga Meltem Akay.............. 338,428 Ömer Bilaç..................................301 Ömer Devecioğlu......... 212,311,325 Ömrüm Uzun..............................342 Osman İ. Özcebe.........................136 Osman İlhan........................ 194,430 Özden Hatırnaz...........................136 Özden Yener Çakmak.................109 Özge Ağlamış..............................336 Özge Özer...................................290 Özgür Ulaş Özcan.......................430 Özkan Sayan.................................75 Özlem Özdemir Kumbasar.........342 Padhi Somanath..........................332 Pamir Işık....................................432 Paul Tyan........................................5 Pelin Mutlu.................................231 Penka Ganeva................................40 Pervin Topçuoğlu..........................79 Piltan Büyükkaya........................201

Prakas Kumar Mandal...................99 Prashant Mehta...........................203 Pritinanda Mishra.......................175 Quan-le Zhang............................328 R. Nalan Tiftik.............................168 Rafat Mohebbi Far.......................161 Raihane Ben Lakhal....................188 Raja Paramanik..................... 95,203 Rajesh Grover..............................286 Rajive Kumar...............................203 Rakhee Kar..................................175 Ramzi Ben Amor.........................188 Rasa Ugenskiene.........................367 Renata Simoliuniene...................367 Reyhan Diz-Küçükkaya..............357 Reza Ranjbaran.............................25 Rıdvan Ali...................................111 Sachin Khurana.............................95 Saeed Hashemi Bozchlou............149 Şahika Zeynep Akı......................276 Sahoo Jayaprakash......................332 Said Benchekroun.......................408 Salah Aref....................................143 Salih Aksu...................................136 Sami Zriba...................................188 Şamil Hızlı...................................103 Sandeep Saha.................................99 Sang Sun Hwang.........................184 Sara Erol......................................101 Sarjana Dutt..................................56 Seda Kibaroğlu............................109 Sedigheh Sharifzadeh....................25 Selami Koçak Toprak... 109,121,290 Selçuk Göçmen.............................75 Selma Gökahmetoğlu..................155 Selma Ünal.......................... 255,272 Selvi Kelekçi................................313 Sema Anak.......................... 212,325 Sema Arayıcı...............................309 Sema Karakuş..............................121 Semra Paydaş....................... 197,295 Şeniz Öngören Aydın.......... 111,374 Serap Işıksoy...............................428 Serap Karaman.................... 212,325 Serdar Beken...............................101 Şerife Efsun Antmen...................255 Server Hande Çağlayan.................17 Seval Kul.....................................168 Sevde Seçer..................................336 Sevgi Kalayoğlu-Beşışık...............111 Sevgin Taner................................336 Sevtap Arıkan Akdağlı................342 Şeyda Erdoğan.............................205 Seyyal Rota..................................276 Shahla Ansari..............................106

Shahrouz Sheidaii.......................301 Shinya Kimura............................424 Shyamali Dutta..............................99 Sibel Aşcıoğlu Hayran.................342 Şinasi Özsoylu.............................434 Sinem Civriz Bozdağ.....................92 Slavica Stojnev............................315 Sofia Marouane...........................408 Soheila Abedinpour....................388 Solafa El Sharawy........................143 Sophia Vakalopoulou..................422 Soumaya Anoun..........................408 Spiro Konstantinov.......................40 Sreeya Das...................................175 Stavri Toshkov..............................40 Stephan Borte..............................420 Sukhal Shashvat..........................341 Şule Mine Bakanay........................79 Şule Şengül....................................79 Şule Ünal............................. 199,210 Süleyman Durmaz.......................155 Suna Emir....................................432 Suzin Çatal Tatonyan..................136 Svetislav Vrbic.............................315 Tahereh Kalantari..........................25 Tamer Sayın................................430 Teoman Soysal............................374 Theodosia Papadopoulou...........422 Tihomit Dikov...............................40 Timur Tunçalı...............................79 Tiraje Celkan...............................426 Tomoya Kishi..............................424 Tufan Öge...................................261 Tülay Güler.................................109 Tülay Tecimer.............................207 Tülin Fıratlı Tuğlular..................411 Tuphan Kanti Dolai......................99 Turan Bayhan..............................432 Türkiz Gürsel........................ 49,399 Ufuk Can.....................................109 Ufuk Gündüz..............................231 Uğur Dilmen...............................309 Uğur Özbek.................................136 Ülker Koçak................................399 Umesh Das....................................56 Ümit Çobanoğlu.........................438 Vasil Hrischev...............................40 Vasileia Garypidou......................422 Vehbi Erçolak..............................205 Velat Şen......................................313 Veysel Haksöyler.........................295 Veysel Kutay Vurgun...................430 Veysel Sabri Hançer.....................357 Vishwanath Sathyanarayanan.......56 Visweswariah Lakshmi Devi.........56

Walid Bouteraa............................188 Wei-min Li..................................328 Wen-Jun Weng............................266 Wilfried Schroyens......................239 Xiang-Wei Ma.............................226 Xiao-Li Liu..................................226 Xiao-Zhong Bai...........................226 Yahya Büyükaşık.........................136 Yasushi Kubota...........................424 Yeşim Oymak..............................417 Yıldız Aydın.................................374

Yosr Ben Abdennebi....................188 Young Uh....................................184 Yousefian Saeed...........................106 Yuan-kai Bo.................................328 Yücel Erbilgin.............................136 Yuji Ikeda....................................424 Yüksel Bek...................................381 Yurdanur Kılınç............................17 Zafer Başlar............................ 17,374 Zafer Gülbaş................................338

Zaher Belhadj Ali........................188 Zahit Bolaman..................... 192,363 Zahra Abdolazimi........................161 Zargham Sepehrizadeh...............149 Zekaver Odabaşı..........................342 Zerrin Yılmaz..............................290 Zeynep Karakaş............ 212,311,325 Zübeyde Nur Özkurt..................276 Zühre Kaya............................ 49,399 Zwi Berneman.............................239

Advisory Board of This Issue (December 2014) Ahmet Emre Eşkazan, Turkey Ahmet Muzaffer Demir, Turkey Alexandra Agapidou, Greece Ayşegül Ünüvar, Turkey Ben E. de Pauw, the Netherlands Burhan Turgut, Turkey Canan Albayrak, Turkey Davut Albayrak, Turkey Deniz Yılmaz Karapınar, Turkey Duran Canatan, Turkey Erol Erduran, Turkey Fahir Özkalemkaş, Turkey Fahri Şahin, Turkey Fatma Gümrük, Turkey Ferit Avcu, Turkey Fevzi Altuntaş, Turkey

Harika Okutan, Turkey Ioannis Kotsianidis, Greece Işınsu Kuzu, Turkey Klara Dalva, Turkey Mahmut Bayık, Turkey Marek Sanak, Poland Maria Papaioannou, Greece Mehmet Yılmaz, Turkey Meletios Dimopoulos, Greece Meliha Nalçacı, Turkey Meral Beksaç, Turkey Mounia Bendari, France Mualla Çetin, Turkey Murat Akova, Turkey Mustafa Ünübol, Turkey Mutlu Arat, Turkey

Namık Özbek, Turkey Nazan Sarper, Turkey Nükhet Tüzüner, Turkey Olga Meltem Akay, Turkey Owen J McGinn, United Kingdom Özgür Mehtap, Turkey Reyhan Diz Küçükkaya, Turkey Rıdvan Ali, Turkey Selami Koçak Toprak, Turkey Semra Paydaş, Turkey Shigeki Takemoto, Japan Şule Ünal, Turkey Türkan Patıroğlu, Turkey Zühre Kaya, Turkey