tjh-2010-2 by LookUs Scientific

Issue 2

June 2010

40 TL

ISSN 1300-7777

Volume 27

TURKISH JOURNAL OF HEMATOLOGY • VOL.: 27 ISSUE: 2 June 2010

Solitary plasmacytoma

Esengül Koçak et al.; Lausanne, Switzerland, ûstanbul, Turkey

Mitoxantrone-induced acute cardiotoxicity and amifostine Vefki Gürhan KadÕköylü et al.; AydÕn, Denizli, Turkey

Ankaferd® Blood Stopper Duygu Özel Demiralp et al.; Ankara, Turkey

A49G polymorphism and autoimmune blood Faruk Aktürk et al.; ûstanbul, Turkey

Enhanced platelet adhesion in essential thrombocythemia Andreas C. Eriksson et al.; Sweden

Clinical aspects of sclerodermatous type GVHD Bengu Nisa Akay et al.; Ankara, Turkey

New effects of clopidogrel on platelets Derya ÖzsavcÕ et al.; ûstanbul, Turkey

Cover Picture : Dilek Argon ûstanbul and Shaft of Light

36.

8/86A/ +(MA72/2-Ä K21G5(6Ä

.DVĂąP Gloria Kongre Merkezi

Belek - Antalya

Bilimsel Sekreterya

Kongre SekreteryasÄą

TĂźrk Hematoloji DerneÄ&#x;i Turan GĂźneĹ&#x; Bulv. Sancak Mah.613 Sok. No: 8 Ă&#x2021;ankaya - Ankara 5FM t 'BLT & QPTUB QSFTT!UIE PSH US t 8FC XXX UIE PSH US

Serenas Turizm Kongre Org. Otelcilik A.Ĺ&#x17E;. Turan GĂźneĹ&#x; BulvarÄą 5. Cadde No:13 06550 YÄąldÄąz, Ă&#x2021;ankaya-Ankara 5FM t 'BY & QPTUB HPLDF POBZ!TFSFOBT DPN US t 8FC XXX TFSFOBT DPN US

ZZZ XlXValKePatoloMi org

Ǧ » Ú Ǧ Ǥ Ǥ ǡ ǡ

» Ǧ

ǡ ȋ Ȍǡ ǡ Cologne, Germany

» Ǧ

ǡ ǡ

» Ǧ

ǡ ǡ ǡ ǡ

» Ǧ

ǡ ǡ Germany

» Ǧ

ǡ ǡ ǡ ǡ

» Ǧ ǡ ǡ Ǥ Ǥǡ ǡ

» Ǧ Ǧ Ȁ ǡ

» Ǥ Ǧ ǯ ǡ ǡ

» Ǧ

ǡ Ǧ ǡ ǡ

» Ǧ

ǡ ǡ

» Ǥ Ǧ ǡ ǡ ǡ

Ǧ Ǧ Ǥ ǯ ǡ ǡ

» Ǧ Ǧ ǡ ǯ Muenster, Germany

3 International Congress rd

Leukemia Lymphoma Myeloma

ǣ ǡ ò ç Ç ͸ͳ͵Ǥ Ǥ ǣͺ Ͳ͸ͷͷͲ . Ǧ Ȁ o G ǣ Ϊ ͻͲ ͵ͳʹ ͶͻͲ ͻͺ ͻ͹ Ȉ ǣ Ϊ ͻͲ ͵ͳʹ ͶͻͲ ͻͺ ͸ͺ Ȉ ǣ Ǥ Ǥ Ǥ Ǧ ǣ ̷ Ǥ Ǥ ǡ ϐ ̷ Ǥ Ǥ

ǣ ò ç Ǥ ͷǤ ǣ ͳ͵ Ͳ͸ͷͷͲ Ç Ç ǡ . Ǧ Ȁ o G ǣ ΪͻͲ ͵ͳʹ ͶͶͲ ͷͲ ͳͳ Ȉ ǣ ΪͻͲ ͵ͳʹ ͶͶͳ Ͷͷ ͸ʹ ǣ Ǥ Ǥ Ǥ Ȉ Ǧ ǣ Ǥ ̷ Ǥ Ǥ

Turkish Society of Hematology

Serenas Tourism Congress & Organisation

Editor-in-Chief

International Review Board Nejat Akar (Turkey) Görgün Akpek (USA) Serhan Alkan (USA) Çiúdem Altay (Turkey) Ayhan Çavdar (Turkey) M.SÕraç Dilber (Sweden) Ahmet Doúan (USA) Jawed Fareed (USA) Gösta Gahrton (Sweden) Dieter Hoelzer (Germany) Marilyn Manco-Johnson (USA) Andreas Josting (Germany) Emin Kansu (Turkey)

Aytemiz Gürgey Associate Editors

Mutlu Arat Muzaffer Demir Reyhan Diz Küçükkaya Mehmet Ertem Hale Ören Mehmet Ali Özcan Teoman Soysal Ayýegül Ünüvar Celalettin Üstün Neýe YaralÕ Past Editors

Erich Frank Orhan Ulutin Hamdi Akan

Contact Information

Editorial Correspondence should be addressed to Prof.Dr.Aytemiz Gürgey Editor-in-Chief Hacettepe University Faculty of Medicine Pediatric Hematology Department 06100 SÕhhiye, Ankara/TURKEY Tel No : + 90 312 305 41 17 Fax No : + 90 312 305 41 17 E-mail : agurgey@hacettepe.edu.tr

Senior Advisory Board

Orhan Ulutin Yücel Tangün Osman ûlhan

Language Editor

Corinne Can

Statistic Editor

Mutlu Hayran

Turkish Society of Hematology

Editorial Secretary

ûpek Durusu Bengü Timoçin Cover Picture:

Dilek Argon was born in 1964, Ordu,Turkey. She is retired from Department of Hematology, Sisli Etfal Education and Research Hospital. She is the third prize winner of the "35. Turkish National Hematology Congress Competition of Photography" in October 2009.

Muhit Özcan, President Mutlu Arat, General Secretary Hale Ören, Vice President Muzaffer Demir, Research Secretary Teoman Soysal, Treasurer Fahir Özkalemkaý, Member Mehmet Sönmez, Member Türk Hematoloji Derneúi, 07.10.2008 tarihli 6 no’lu kararÕ ile Turkish Journal of Hematology’nin Türk Hematoloji Derneúi ûkdisadi ûýletmesi tarafÕndan yayÕnlanmasÕna karar vermiýtir.

Winfried Kern (Germany) Nigel Key (USA) Korgün Koral (USA) Abdullah Kutlar (USA) Ghulam Mufti (UK) Gerassimos A. Pangalis (Greece) Santiago Pavlovsky (Argentina) Antonio Piga (Italy) Ananda Prasad (USA) Orhan Sezer (Germany) Nüket Tüzüner (Turkey) Catherine Verfaillie (USA) Claudio Viscoli (Italy) All other inquiries should be adressed to

TURKISH JOURNAL OF HEMATOLOGY Sancak Mahallesi , Turan Güneý BulvarÕ 613. Sk. No:8 06550 Çankaya, Ankara/Turkey Tel. : +90 312 490 98 97 Fax : +90 312 490 98 68 E-mail: info@tjh.com.tr ISSN: 1300-7777

Web page

www.tjh.com.tr

Sahibi

Türk Hematoloji Derneúi adÕna Muhit Özcan

Sorumlu YazÕ ûýleri Müdürü Aytemiz Gürgey

YayÕn ve Yönetim Yeri Türk Hematoloji Derneúi Türk OcaúÕ Cad. 17/6 Caúaloúlu-Eminönü-ûstanbul Üç ayda bir yayÕnlanan yerel bilimsel dergidir.

Publisher: AVES YayÕncÕlÕk Address: KÕzÕlelma cad. 5/3 34096 FÕndÕkzade-ûstanbul-Turkey Phone: +90 212 589 00 53 Fax: +90 212 589 00 94 E-mail: info@avesyayincilik.com Place of printing: Görsel Dizayn Ofset MatbaacÕlÕk Tic. Ltd. üti. - +90 212 671 91 00 Date of printing: June 2010 Broadcast as: Local periodical

A-I

AIMS AND SCOPE The Turkish Journal of Hematology is the regular publishing organ of the Turkish Society of Hematology. This periodical journal covers subjects on hematology. The journal is an independent, peer-reviewed international periodical, published quarterly (March, June, September and December) in English language. The Turkish Journal of Hematology is a nonprofit scientific peer reviewed journal. The aim of the Turkish Journal Hematology is to publish original research papers of highest scientific and clinical value on hematology. Additionally, educational material, reviews on basic developments, editorial short notes, case reports, original views and letters from specialists on hematology, and hematology medicine covering their experience and comments as well as social subjects are published. General Practitioners interested in hematology, and internal medicine specialists, are also our target audience, and we will arrange the Turkish Journal of Hematology according to their needs. The Turkish Journal of Hematology is indexed in - Science Citation Index Expanded - Embase - Scopus - Cinahl - Index Copernicus - Gale Cengage Learning - EBSCO - DOAJ - ProQuest - Tübitak Ulakbim Türk TÕp Dizini Subscription Information The Turkish Journal of Hematology is sent free of charge to hematologists and academicians in our country as well as to other specialists interested in hematology. All published volumes in full text can be reached free of charge through the web site www.tjh.com.tr Adress: Sancak mah. Turan Güne¿ BulvarÕ 613. sok. No: 8 Çankaya-Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 Web page: www.tjh.com.tr E-mail: press@thd.org.tr Permissions Requests for permission to reproduce published material should be sent to the editorial office. Editor: Prof.Dr. Aytemiz Gürgey Adress: Sancak mah. Turan Güne¿ Bulvar› 613. sok. No: 8 Çankaya-Ankara, Turkey Telephone: +90 312 490 98 97 Fax: +90 312 490 98 68 E-mail: info@tjh.com.tr Instructions for Authors Instructions for authors are published in the journal and on the web page www.tjh.com.tr Material Disclaimer The author(s) is (are) responsible from the articles published in the Turkish Journal of Hematology. The editor, editorial board and publisher do not accept any responsibility for the articles. The journal is printed on acid-free paper.

A-II

INSTRUCTION TO AUTHORS The Turkish Journal of Hematology accepts invited review articles, research articles, brief reports, case reports, letters to the editor, and images in Hematology on subjects within the scope of hematology, on the condition that they have not been previously published elsewhere. All papers are subject to editorial revision for purpose of conformity to the style adopted by the Journal. Evaluation is a double blind kind of evaluation. Original research articles Regular Articles Maximum length for a Regular Article is 4,000 words of text. Abstracts must not exceed 150 words and should be a single psaragraph with no subheadings. Submissions are limited to a total of 7 figures/tables. References should be limited to 50. The sections of a Regular Article should include Abstract, Introduction, Material and Methods, Results, Discussion, References, Figure Legends. The format of the articles should comply with the Uniform Requirements for Manuscripts Submitted to Biomedical Journals: Writing and Editing for Biomedical Publication (http://www.icmje.org) Brief Reports Short manuscripts definitively documenting either experimental results or informative clinical observations will be considered as brief report. Brief Reports should not exceed 1,000 words of text not counting the abstract, figure legends, and references; abstracts must not exceed 150 words. Review Articles Review articles should not exceed 4,000 words in length, must include an abstract of 150 words or fewer, and may not have more than 100 references. Letters to the Editor Letters can include no more than 400 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title. Images in Haematology Authors can submit for consideration an illustration (or, where appropriate, two or more related images) which is interesting, instructive and visually attractive, with a few lines of explanatory text and references. The images (e.g. a clinical photograph, radiology, cytology, histology, a laboratory test) should be submitted in a digital format. Preparation of Manuscript Each of the following sections of the manuscript should be typed on separate pages. Title Page should include (in Turkish when possible): (a) title of the article in a concise but informative style, (b) first name, middle initial, last name of each author, (c) name of department(s) and institution(s) to which the work should be attributed, (d) name and address of author responsible for correspondence for the manuscript, (e) name and address of author to whom requests for reprints should

A-III

be addressed, (f) source(s) of support in the form of grants, equipments, drugs, etc., and (h) short running title of no more than 40 characters. Authorship Each author should have participated sufficiently in the work to take public responsibility for the content. Any part of an article critical to its main conclusions must be the responsibility of at least one author. All authors’ signatures should be included in the title page. The signed statement on absence of conflict of interests between authors is required. Acknowledgments Acknowledge support received from individuals, organizations, grants, corporations, or any other sources. For work involving a biomedical product or potential product partially or wholly supported by corporate funding, a note must be included stating: This study was supported (in part) by research funding from (company name) to (authors’ initials). Grant support, if received, needs to be stated and the specific granting institution(s) name(s) and grant numbers provided when applicable. Authors are expected to disclose, on the title page of their manuscripts, any commercial or other associations that might pose a conflict of interest in connection with the submitted article. All funding sources supporting the work, and institutional or corporate affiliations of the authors, should be acknowledged on the title page. Ethics When reporting experiments on human subjects indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation. An approval of research protocols by ethic 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 experimental, clinical and drug studies. Do not use patient names, initials, or hospital numbers, especially in any illustrative material. Manuscripts reporting the results of experimental investigations on human subjects must include a statement to the effect that procedures had received official institutional approval. The statement on the informed consent of patients is required. We frown upon unethical practices such as plagiarism, duplicate publication, ‘salami’ publication, and efforts to influence the review process with practices such as gifting authorship, inappropriate acknowledgements and references. Also, authors must respect patients’ right to privacy. Abstract and key words: The second page should include an Abstract which does not exceed 150 words. For manuscripts sent from Turkey, a title and abstract

in Turkish are required. The abstract should state the purpose of the study or investigation, basic procedures, methods, main findings, specific data, statistical significance and the principal conclusions. Provide 3 to 10 key words below the abstract to assist indexers. Use terms from the Medical Subject Headings List of Index Medicus. The text should be divided into sections with headings as follows: Objective, Methods, Results and Conclusion. Other types of articles such as case reports, reviews, perspectives and editorials will be published according to uniform requirements. Introduction: State the purpose of the article and summarize the rationale for the study. Materials and Methods: Describe your selection of the observational or experimental subjects clearly. Identify the methods and procedures in sufficient detail to allow other workers to reproduce the results. Give references to established methods (including statistical methods), provide references and brief modified methods, give reasons for using them and evaluate their limitations. Identify all drugs and chemicals used, including generic name(s), dose(s) and route(s) of administration. Statistics: Describe statistical methods in enough detail to enable a knowledgeable reader with access to the original data to verify the reported results. Give details about randomization, describe treatment complications, give number of observations, and specify any computer program used. Results: Present your results in logical sequence in the text, tables and illustrations. Do not repeat in the text all the data in the tables or illustrations; emphasize or summarize only important observations. Discussion: Emphasize the new and important aspects of the study and the conclusions that follow them. Link the conclusions with the goals of the study but avoid unqualified statements and conclusions not completely supported by your data. References: Identify references in text, tables and legends by Arabic numerals in parentheses. Number references consecutively in the order in which they are first mentioned in the text. The titles of the journals should be abbreviated according to the style used in Index Medicus; consult List of Journals Indexed in Index Medicus. Include among the references any papers accepted but not yet published, designating the journal and followed by “in press”. Articles in Journals 1. List all authors Williams RL, Hilton DJ, Pease S, Wilson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukemia inhibitory factor (LIF) maintains the developmental potential of embryonic stem cells. Nature 1988;336:684-687.

2. Organization as author Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in post-hepatitis marrow aplasia. Lancet 1977;2:742-744. 3. Complete book Adams DO, Edelson PJ, Koren HS. Methods for studying mononuclear phagocytes. San Diego: Academic Press, 1981. 4. Chapter of book. Smolen JE, Boxer LA. Functions of Neutrophils. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. 4th ed. New York: McGraw-Hill, 1991: 780-794. 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. Tables Type each table on a separate sheet. Number tables consecutively in the order of appearance in the text and supply a brief title for each. Give each column a short or abbreviated heading. Place explanatory statistical measures of variations such as standard deviation or standard error of mean. Be sure that each table is cited in the text. Illustrations Figures should be professionally drawn and photographed. Please send sharp, glossy, black and white photographic prints, usually 9 x 13 cm. Affix a label to the back of each figure indicating the number of the figure, first author’s name and top of the figure. Type legends for illustrations double-spaced, starting on a separate page with Arabic numerals corresponding to the illustrations. Explain the internal scale and identify method of staining. Units of Measurement Measurements should be reported in the metric system in terms of the International System of Units (SI). Consult SI Unit Conversion Guide, New England Journal of Medicine Books 1992, when necessary. Abbreviations and Symbols Use only standard abbreviations. Avoid abbreviations in the title and abstract. The full term for which an abbreviation stands should precede its first use in the text unless it is a standard abbreviation.

A-IV

ONLINE MANUSCRIPT SUBMISSION PROCESS Manuscripts can be submitted online at www.journalagent.com/tjh/ The online system consists of four main parts: manuscript submission module (MSM), editorial module, admin module and referee module. The editorial module, admin module and referee module work on the background and will not be open to the end user. The term module used in this document refers only to the MSM. As part of the peer-review system, authors will also receive the referee reports and can observe he current status of their manuscript(s) online. An online help is also available during the submission process. The module accepts the body of the manuscript as a whole document; thus, documents should be completed as a .doc or .rtf file before submission. The supported file extensions, fonts and other formats are given in Table 1. Table 1.

Supported formats .doc (MS Office for Windows or Macintosh) .rtf (rich text format)

Supported fonts Arial Times Helvetica Times New Roman Courier

Tables, figures and pictures Tables should be created in your original wordprocessing software or inserted in the original file from Excel or another compatible software. Please ensure the table or figure created complies with the limitations mentioned in Table 1. Tables created as a picture file are problematic and are not advised. Figures should be embedded in the original file, but the system also requires that they be sent separately. The supported image files are given in Table 1. Symbols Special characters not available on the keyboard can be accessed either from the insert menu (select symbol) or by selecting symbol as a font from the font window of the Formatting toolbar. Please check these characters in your original file and proofs as the softwares tend to replace these characters with others if they are unreadable. Submission online To submit a manuscript, you must first establish a login name and password, which can then be used indefinitely. After you login to the module, the first page accessed allows you to track the status of your current or previous manuscripts. To submit a new manuscript, login with your user name and password, then click on “Author” on your main menu. From this page, select “new article” pull-down menus. The next page is for the details of the institutions of the authors. On the next page where the authors are listed, the instructions and menus allow you to select the appropriate institution for each author from the list. Please remember to check the box next to the corresponding author. The title page

requires only the title; special characters listed on the bottom of the title input window can be used when needed. On the summary page which follows, please write the abstract of your manuscript in the appropriate window. This is followed by a keyword input page, which allows up to 10 keywords. Any special notes to the editor can be recorded on the next page. The body and figures of the manuscript are submitted on the next page. Locate the manuscript in your PC, then write the type of the file you are sending and give a description. Use this page to also send your images. Send your manuscript using the designated button on the next page. All pages are supported with help menus; if you require additional help or experience a problem, please send an e-mail to info@tjh.com.tr Manuscripts that have passed an initial screening by the. Editors are reviewed by members of the Editorial Board and/or other experts in the field. The Editors select the reviewers and make the final decision on the manuscript. Supported images .bmp .jpg .jpeg .gif .tif

Referees who review a manuscript remain unknown to the authors. Every manuscript is treated by the Editors and reviewers as privileged information, and they are instructed to exclude themselves from review of any manuscript that may involve a conflict of interest or the appearance of such. Following initial peer-review, articles judged worthy of further consideration often require revisions. Revised manuscript generally must be received within 3 months of the date on the initial decision. Extensions must be requested from the Associate Editor at least 2 weeks before the 3-month revision deadline expires. Otherwise Turkish Journal of Hematology will reject manuscripts which do not received within 3 months of the date on the initial revesion decision. A Copyright transfer and conflict of interest form signed by all authors, must also be submitted by fax to +90 3124909868. Both forms can be found at the web site www.tjh.com.tr Authors of accepted manuscripts will receive electronic page proofs directly from the printer and are responsible for proofreading and checking the entire article, including tables, figures, and references. Page proofs must be returned within 48 hours to avoid delays in publication. English-language editing All manuscripts are professionally edited by English language editor before publication. Online Early Turkish Journal of Hematology published abstracts of accepted articles online in advance of their publication in a printed issue.

CONTENTS Review Article

Radiation therapy for the solitary plasmacytoma

Esengül Koçak, Giorgio Ballerini, Abderrahim Zouhair, Mahmut Özýahin, Lausanne, Switzerland, ûstanbul, Turkey

Research Articles

The protection of the myocardium by amifostine against mitoxantrone-induced acute cardiotoxicity in rats

Vefki Gürhan KadÕköylü, ûbrahim Meteoúlu, Süleyman Demir, Hülya Aybek, Mete Kalak, Muharrem Balkaya, Çiúdem Yenisey, Zahit Bolaman, AydÕn, Denizli, Turkey

Functional proteomic analysis of Ankaferd® Blood Stopper

Cytotoxic T lymphocyte antigen-4 (CTLA-4) A49G polymorphism and autoimmune blood diseases

Enhanced platelet adhesion in essential thrombocythemia after in vitro activation

Clinical aspects of sclerodermatous type graft-versus-host disease after allogeneic hematopoietic cell transplantation

New in vitro effects of clopidogrel on platelets in hyperlipidemic and healthy subjects

Duygu Özel Demiralp, ûbrahim C. Haznedaroúlu, Nejat Akar, Ankara, Turkey Faruk Aktürk, Veysel Sabri Hançer, Reyhan Küçükkaya, ûstanbul, Turkey Andreas C. Eriksson, Kourosh Lotfi, Per A. Whiss, Linköping, Sweden

Hatice üanlÕ, Bengü Nisa Akay, Ender Soydan, Pelin Koçyiúit, Mutlu Arat, Osman ûlhan, Ankara, Turkey

Derya ÖzsavcÕ, Azize üener, Rabia Oba, Gülderen YanÕkkaya Demirel, Fikriye Uras, Turay Kevser YardÕmcÕ, ûstanbul, Turkey

Case Reports

109

A rare extramedullary involvement in myeloma: lung parenchyma and association with unfavorable chromosomal abnormalities

113

Complex cytogenetic findings in the bone marrow of a chronic idiopathic myelofibrosis patient

117

An unusual presentation of pediatric acute lymphoblastic leukemia with parotid gland involvement and dactylitis

120

First observation of Hb Tunis [beta124(H2)Pro>Ser] in Turkey

123

Özlem üahin BalçÕk, Murat Albayrak, Simten Daúdaý, Funda Ceran, Gülsüm Özet, Funda Demiraú, Osman Yokuý, Ankara, Kayseri, Turkey Tuúçe BulakbaýÕ BalcÕ, Meltem Yüksel, Zerrin YÕlmaz, Feride ûffet üahin, Ankara, Turkey

üule Ünal, BarÕý Kuýkonmaz, Yasemin IýÕk BalcÕ, Bülent Cengiz, Murat Tuncer, Aytemiz Gürgey, Erman Cilsal, Ayse Gültekingil, Fatma Gümrük, Ankara, Turkey Aylin Köseler, Hasan Koyuncu, Onur Öztürk, Anzel BahadÕr, Sanem Demirtepe, Ayfer Atalay, Erol Ömer Atalay, Denizli, Turkey

Letters to the Editor Ribosomal protein S19 -631 insertion is an African-originated mutation

Özge CumaoúullarÕ, Ayýenur Öztürk, Nejat Akar, Solaf Elsayed, Ezzat Elsobky, Bakhouche Houcher, Ankara, Turkey, Cairo, Egypt, Setif, Algeria

125

Lower FXII activity and thrombosis: a comment

126

IgA lambda oligoclonal gammopathy in multiple myeloma

128

Blood transfusion services in Iraq; an unfortunate field

130

Mega-dose methylprednisolone in hematologic and non-hematological disorders

132

Oseltamivir and G6PD deficiency

133

FLT3 – ITD positive acute lymphocytic leukemia, does it impact on disease´s course?

Viroj Wiwanitkit, Bangkok, Thailand

ûbrahim Tek, Dilsa MÕzrak, Güngör Utkan, Selami Koçak Toprak, Hüseyin Tutkak, Abdullah Büyükçelik, Bülent YalçÕn, Hakan Akbulut, Fikri ûçli, Ankara, Turkey Abbas Hashim Abdulsalam, Baghdad, Iraq üinasi Özsoylu, Ankara, Turkey üinasi Özsoylu, Ankara, Turkey

Sebastian Kobold, Nerbil KÕlÕç, John Scharlau, Carsten Bokemeyer, Walter Fiedler, Hamburg, Bonn, Germany, Basel, Switzerland

Images in Hematology 135

Pleural fluid plasmacytosis in a patient with plasma cell leukemia

Raihan Sajid, Bushra Moiz, Nausheen Kamran, Salman Naseem Adil, Karachi, Pakistan

A-V

Review

Radiation therapy for the solitary plasmacytoma Soliter plazmasitom'un radyoterapi ile tedavisi

Esengül Koçak1,2, Giorgio Ballerini1, Abderrahim Zouhair1, Mahmut Özýahin1 1Departments 2Cerrahpaýa

of Radiation Oncology, University of Lausanne Medical Center, Lausanne, Switzerland School of Medicine, University of ûstanbul, ûstanbul, Turkey

Abstract Plasma-cell neoplasms are classically categorized into four groups as: multiple myeloma (MM), plasma-cell leukemias, solitary plasmacytomas (SP) of the bone (SPB), and extramedullary plasmacytomas (EMP). These tumors may be described as localized or diffuse in presentation. Localized plasma-cell neoplasms are rare, and include SP of the skeletal system, accounting for 2-5% of all plasma-cell neoplasms, and EMP of soft tissue, accounting for approximately 3% of all such neoplasms. SP is defined as a solitary mass of neoplastic plasma cells either in the bone marrow or in various soft tissue sites. There appears to be a continuum in which SP often progresses to MM. The main treatment modality for SP is radiation therapy (RT). However, there are no conclusive data in the literature on the optimal RT dose for SP. This review describes the interrelationship of plasma-cell neoplasms, and attempts to determine the minimal RT dose required to obtain local control. (Turk J Hematol 2010; 27: 57-61) Key words: Solitary plasmacytoma, radiotherapy, multiple myeloma, management Received: September 17, 2009

Accepted: March 3, 2010

Özet Plazma hücre malignitesi klasik olarak dört gruptur: multiple myelom (MM), plazma hücre lösemisi, kemik yerleýimli soliter plazmasitom (SPB) ve ekstrameduller yerleýimli plazmasitom (EMP). Lokalize plazma hücre malignitesini iskelet sistemi yerleýimli SP ve EMP oluýturur. Plazmasitoma birçok farklÕ ýekilde tanÕmlanabilir. Çoúunlukla kemik iliúinde veya yumuýak dokuda plazma hücrelerinden oluýan bir kitle ýeklinde görülür. Henüz, soliter plazmositomlarÕn MM’a dönüýümü arasindaki iliýki tam olarak tanÕmlanmamÕýtÕr. Soliter plazmositomlar, hem SPB hem de EMP için ana tedavi radyoterapidir. Ancak, SP için uygun tedavi dozu için ortak bir data yoktur. Lokal kontrol için optimal doz hastalÕúÕn yerine ve boyutuna baúlÕ olarak 30 ve 50 Gy arasÕnda deúiýmektedir. Bu derlemede lokal kontrol için minimal RT dozu tanÕmlanmaya çalÕýÕlmÕýtÕr. (Turk J Hematol 2010; 27: 57-61) Anahtar kelimeler: Soliter plazmasitom, radyoterapi, multipl miyelom, tedavi yaklaýÕmÕ Geliý tarihi: 17 Eylül 2009

Kabul tarihi: 3 Mart 2010

Address for Correspondence: Dr. Esengül Koçak, MD, Department of Radiation Oncology, Cerrahpaýa School of Medicine, 34303 Cerrahpaýa, ûstanbul, Turkey Phone: +90 212 414 31 02 E-mail: dresengulkocak@gmail.com doi:10.5152/tjh.2010.01

Koçak et al. Solitary plasmacytoma

Introduction Plasma-cell neoplasms account for approximately 1-2% of human malignancies, and occur at a rate of 3.5/100,000 per year. Plasmacytoma, a clonal neoplastic disorder of bone marrow originating from plasma cells, the last maturation stage of B lymphocytes, may appear as four different diseases: multiple myeloma (MM, systemic disease), plasma-cell leukemias, extramedullary plasmacytoma (EMP), and solitary plasmacytoma (SP) of bone (SPB). This review article assesses the management of SPs, as the treatment of MM is mainly with systemic chemotherapy, and the role of local radiotherapy (RT) is restricted for palliation of symptoms. A diagnosis of SP is made when all the following criteria are satisfied [1]: a histologically confirmed single lesion with negative skeletal imaging outside the primary site, normal bone marrow biopsy (less than 10% monoclonal plasma cells), and no myeloma-related organ dysfunction. Men are affected more than women (male-to-female ratio 2:1) [2]. The median age at the diagnosis of SP ranges between 55 and 65 years, on average about 10 years younger than patients with MM. EMPs are less common than SPBs [2-4]. Laboratory signs of SP are usually related to the immunoglobulin production, if the secretory component is present. Solitary plasmacytoma most commonly presents in bone (80%). SPBs are most often found in the axial skeleton, usually the vertebrae and skull [2]. Patients usually present with bone pain, sometimes associated with neurologic compromise and/or pathologic fracture. Less commonly, SP presents in an EMP site (20%), usually found in the head and neck, and most often in the nasal cavity and nasopharynx [2,3,5,6]. The histologic diagnosis of EMP can be difficult, and the main differential diagnosis is extranodal marginal zone lymphoma [5,7]. Diagnostic Work-Up Diagnostic work-up at the presentation includes history, physical examination, complete blood count, bone marrow biopsy, serum protein electrophoresis, evaluation of the urine for myeloma protein, and skeletal survey. The incidence of a monoclonal gammopathy (M-protein) is present in 30-75% of cases (especially for bone presentation, usually minimally elevated [IgG <5 g/L, IgA <20 g/L], and urine monoclonal kappa or lambda <1.0 g/24 h) [8-9]. Tsang et al. [4] observed the following myeloma protein levels in the blood of 46 patients with SP: normal 59%, minimally elevated 28% and elevated 13%. Skeletal survey is preferred over bone scintigraphy because the latter is only 40-60% sensitive for detecting bone lesions since the osteoblastic response to bone destruction by myeloma is negligible. Approximately 25-50% of bone trabeculae must be destroyed with a bone defect to be visible on a plain radiography. Thus, computed tomography (CT) is more sensitive for detecting the extent of bone destruction. Some patients with suspected SPB will be upstaged following magnetic resonance imaging (MRI) with the detection of multiple vertebral lesions or bone marrow disease, with lesions being darker or isointense on T1-weighted images and hyperintense on T2-weighted images and enhanced with contrast [10-11] or positivity on 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) [12,13].

Turk J Hematol 2010; 27: 57-61

Management of Solitary Plasmacytoma Radiation therapy (RT) is the standard treatment for SP. Surgery is considered for bone instability, fracture, or when there is rapidly progressive neurologic deterioration such as spinal cord compression [1,14]. For patients treated with surgical excision, RT is still indicated to eradicate microscopic residual disease. Surgery alone without RT leads to an unacceptably high local recurrence rate [2]. A review of the recent literature for SPB indicated high local control rates with RT (7991%) but a modest overall survival of approximately 50% at 10 years. Özýahin et al. [2] reported local control was better with small tumors (<4 cm) in patients treated with RT; however, no difference was observed between bone and extramedullary SP. This is because of a high progression rate to MM, a finding consistently reported by many investigators [2,4,15-18]. Over 60% of patients with SPB will progress to myeloma, usually within two to four years after treatment. Özýahin et al. [2] showed that SP seemed to develop MM in two peaks. A first peak during the first three years post-treatment is probably due to undetected existing MM by an asymptomatic gross disease becoming more obvious; a second peak, corresponding to a real MM development of previously occult disease, is observed after 6-9 years. Therefore, it appears SPB may be an early form of MM. Vertebral disease has been reported to be a poor prognostic factor compared to other bony localization [2]. The presence of low level M-protein pre-radiation is very common, and by itself is not associated with a higher risk of progression to MM. Patients showing persistent M-protein levels for more than one year after RT are prone to progress to MM [16,19]. The addition of adjuvant chemotherapy to RT is, theoretically, attractive in an attempt to eradicate subclinical disease to prevent the disease relapsing as myeloma. Only one randomized trial suggested a benefit with adjuvant melphalan and prednisone given for three years after RT compared to RT alone [20]. However, this was a small study and the concerns regarding prolonged use of alkylating agents on the bone marrow do not justify its routine use. This study concluded that combined radiochemotherapy seemed to increase remission and survival duration. Targeting the mechanisms that control angiogenesis, which have an integral role in the pathophysiology of hematologic malignancies, could be an innovative therapeutic approach in the treatment of SP. Kumar et al. [19] reported high-grade angiogenesis in 64% of tumors in their series of 25 SPB patients, and found that angiogenesis is highly correlated with progression to MM. Therefore, antiangiogenic compounds such as thalidomide, vascular-endothelial growth factor or proteasome inhibitors may be promising in this disease. For EMP, complete surgical excision may be curative for small lesions. However, most patients with larger lesions or with tumor location not amenable to complete excision should receive local RT, and postoperative RT is indicated for incompletely excised lesions. In contrast to bone localization, EMPs are well controlled with local radiation. Of all plasma-cell tumors, EMPs have the best prognosis [5]. Evidence of local involvement of adjacent bone and/or lymph nodes does not necessarily indicate systemic disease, or signify a worse prognosis [21]. Conversion to MM is less likely than in patients with SPB with the best result

Koçak et al. Solitary plasmacytoma

Turk J Hematol 2010; 27: 57-61

reported in patients receiving surgery and/or RT [2,5,21-24]. Moreover, a substantial proportion of patients are cured of their disease. Although the 10-year survival varies widely in the recent literature (range: 50-78%), the two largest series reported 10-year survival rates of 72% [2] and 78% [21] (Tables 1, 2). Radiation Therapy The optimal treatment of most patients with SP is moderate-dose RT. Approximately 40-50 Gy are administered once daily at 1.8-2.0 Gy per fraction in a continuous course. For RT planning, MRI is extremely useful to delineate the gross tumor volume (GTV), both within and beyond the bone. This is important for the head and neck areas (e.g., paranasal sinuses), where inflammatory changes may be difficult to distinguish from tumor on CT scan. The FDG-PET could be used to delineate the tumor and response to treatment; however, the role of FDG-PET in defining tumor extent to assist in RT planning requires further investigation [12]. Clinical target volume (CTV) should encompass probable routes of microscopic spread, recognizing that barriers to the extension of local disease will vary according to the anatomical location, as will the morbidity of treating adjacent normal tissues. Jyothirmayi et al. [25] reported that, among 30 patients with SP treated with RT that encompassed only the tumor with a margin, no marginal recurrence was seen. Prophylactic regional lymph node coverage is not necessary in SPB, as the regional nodal failure rate is low after local RT without intentional coverage of adjacent nodes [4,23,25,26]. For EMP, nodal involvement at presentation is observed in only 10-20% of the patients, and planning target volume (PTV) does not justify covering the regional lymph nodes [2]. Strojan et al. [3], in a series of 26 patients, concluded that prophylactic nodal radiation is probably unnecessary.

Planning target volume (PTV) should account for setup variation from day to day and organ motion issues, typically adding 5-10 mm to the CTV depending on the immobilization technique employed. Although parallel-opposed fields are commonly adequate to encompass disease, CT-based planning and the use of conformal or intensity-modulated RT (IMRT) techniques should be used when needed to treat the PTV adjacent to critical structures. This is especially important in extramedullary disease involving the head and neck areas where avoidance of the eyes, optic nerves, and salivary glands is desirable to minimize morbidity. Solitary plasmacytomas are radiation-sensitive tumors. Most studies documented response rates of 85% or more for patients treated with 35 Gy or more. Some investigators reported better local control following doses of 45 Gy or more [6,27], while others have found no indication of improved outcome with higher doses [2,26]. A dose-response analysis of 81 patients by Mendenhall et al. [2,25] led to a recommendation of a minimum dose of 40 Gy for both bone and extramedullary SP. In their study, a total dose of 40 Gy or more gave a local failure rate of 6%, compared with a suboptimal 31% for lower doses [24]. Therefore, the standard practice is to administer a dose of 40-45 Gy or even higher for bulky tumors. However, in one of the largest studies (n=258), there was no evidence of improved local control with RT doses increasing from 30-50 Gy, even for the subset of patients with tumors larger than 4 cm [2]. In fact, there was a worse local control rate for the group receiving a total dose of 50 Gy or more, although this was not statistically significant [2]. These findings should be interpreted carefully because in these retrospective studies, dose-response rates are typically confounded by selection bias, as high doses are to be prescribed to larger tumors with worse prognosis.

Table 1. Solitary plasmacytoma of bone Author

MMFS

DFS

Tsang RW [4]

78% (8 yrs)

36% (8 yrs)

Knobel D [15]

206

79% (10 yrs)

51% (10 yrs)

46% (10 yrs)

50% (10 yrs)

Wilder RB [16]

90% (10 yrs)

38% (10 yrs)

57% (10 yrs)

59% (10 yrs)

KÕlçÕksÕz S [17]

94% (10 yrs)

Median 4.1 yrs

Median 3.2 yrs

68% (10 yrs)

Bolek TW [18]

96%

0% (15 yrs)

23% (15 yrs)

Note: Time interval (in parentheses) indicates follow-up point at which the outcome was observed LC: Local control; DFS: Disease-free survival; MMFS: Multiple myeloma-free survival; OS: Overall survival

Table 2. Extramedullary plasmacytoma Author

MMFS

DFS

Özýahin M [2]

74%

36% (10 yrs)

55% (10 yrs)

72% (10 yrs)

Strojan P [3]

87% (10 yrs)

92% (10 yrs)

87% (10 yrs)

61% (10 yrs)

Tsang RW [4]

93% (8 yrs)

84% (8 yrs)

Tournier-Rangeard L [6]

88.2% (10 yrs)

63.8% (10 yrs)

53.5% (10 yrs)

63.4% (10 yrs)

Liebross RH [8]

95%

56% (5 yrs)

78% (5 yrs)

KÕlçÕksÕz S [17]

95% (10 yrs)

Median 7.4 yrs

89% (10 yrs)

Bolek TW [18]

100%

67% (15 yrs)

80% (15 yrs)

Koçak et al. Solitary plasmacytoma

Durable local control is obtained in 85% of small tumors (<5 cm) with 35-40 Gy, and there is little evidence that higher doses are necessary, regardless of bone or extramedullary locations. However, SP of >5 cm have worse local control [2,4], and doses of 45-50 Gy are recommended in these larger tumors. Despite this, one should be aware that the quality of evidence supporting the use of higher RT doses is limited, and local recurrences are still infrequently observed even after doses exceeding 50 Gy [2,4]. Özýahin et al. [2] recommended a minimum dose of 45 Gy should be prescribed even for patients who undergo surgery. Conversely, in the event of a bulky tumor and no surgery, a dose to the CTV could be even higher than 45 Gy, possibly 50 Gy, to improve local control. Prognostic Factors Affecting the Outcome Advanced age is clearly associated with poor survival [2]. Similarly, higher risk of progression to MM was observed in the elderly in some studies [4,19,28] but not confirmed by others [2,29-32]. A bony presentation was consistently demonstrated to have a higher risk of MM progression with a 10-year rate of 76%, compared with an extramedullary presentation, in which the 10-year MM rate was 36% [2]. Subclinical osteopenia [33] or abnormal MRI scan of the spine [10,34,35] reflects presence of occult disease, and predicts rapid progression to systemic myeloma. A suppression of the normal immunoglobulin classes, also known as immunoparesis, correlates with a higher risk of progressing to MM [33,36]. M-protein levels are reported to be a predictive factor of occult disseminated disease, and patients showing persistent M-protein levels for more than one year after RT are prone to progress to MM [8,16,37]. Anaplastic plasmacytomas (those with a higher histologic grade) [26] and tumors with a high level of angiogenesis [9] are associated with poorer outcomes. Tumor size is reported to be an important prognostic factor in terms of local control. According to Tsang et al. [4], in tumors smaller than 5 cm, a high level of local control was achieved with 35 Gy, whereas those 5 cm or larger had a local failure rate of 58%. The importance of tumor bulk is also supported by others [2,30,31]. Follow-Up Since there is a significant risk of recurrence of disease as MM, repeat measurements of M-protein may detect the onset of systemic disease prior to the development of symptoms [16]. Complete blood counts should be taken periodically to evaluate bone marrow function. The occurrence of new bone pain requires further investigations, including imaging as appropriate. Conclusion Solitary plasmacytoma is a relatively uncommon malignancy that frequently presents in the vertebrae (SPB) or head and neck (EMP). Most patients are treated with moderate-dose RT. Some investigators reported better local control following doses of 45 Gy or more [6,27], while others have found no indication of improved outcome using higher doses [2,26]; a subset of patients may require surgical intervention. At the

Turk J Hematol 2010; 27: 57-61

present time, adjuvant chemotherapy is not indicated. The likelihood of local control after treatment is high. The most common pattern of relapse is systemic myeloma progression, which is more likely to occur after treatment for SPB compared with EMP. Strategies to improve outcome will be related to improved management of MM in the subset of patients who develop systemic relapse. Future prospective studies should focus on the use of adjuvant chemotherapy and/or novel therapeutic agents, i.e., proteosome inhibitors. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

Durie BG, Kyle RA, Belch A, Bensinger W, Blade J, Boccadoro M, Child JA, Comenzo R, Djulbegovic B, Fantl D, Gahrton G, Harousseau JL, Hungria V, Joshua D, Ludwig H, Mehta J, Morales AR, Morgan G, Nouel A, Oken M, Powles R, Roodman D, San Miguel J, Shimizu K, Singhal S, Sirohi B, Sonneveld P, Tricot G, Van Ness B. Myeloma management guidelines: a consensus report from the Scientific Advisors of the International Myeloma Foundation. Hematol J 2003;4:379-98. Ozsahin M, Tsang RW, Poortmans P, Belkacemi Y, Bolla M, Oner Dincbas F, Landmann C, Castelain B, Buijsen J, Curschmann J, Kadish SP, Kowalczyk A, Anacak Y, Hammer J, Nguyen TD, Studer G, Cooper R, Sengöz M, Scandolaro L, Zouhair A. Outcomes and patterns of failure in solitary plasmacytoma: A multicenter Rare Cancer Network study of 258 patients. Int J Radiat Oncol Biol Phys 2006;64:210-7. Strojan P, Soba E, Lamovec J, Munda A. Extramedullary plasmacytoma: clinical and histopathologic study. Int J Radiat Oncol Biol Phys 2002;53:692-701. Tsang RW, Gospodarowicz MK, Pintilie M, Bezjak A, Wells W, Hodgson DC, Stewart AK. Solitary plasmacytoma treated with radiotherapy: impact of tumor size on outcome. Int J Radiat Oncol Biol Phys 2001;50:113-20. Alexiou C, Kau RJ, Dietzfelbinger H, Kremer M, Spiess JC, Schratzenstaller B, Arnold W. Extramedullary plasmacytoma: tumor occurrence and therapeutic concepts. Cancer 1999;85:2305-14. Tournier-Rangeard L, Lapeyre M, Graff-Caillaud P, Mege A, Dolivet G, Toussaint B, Charra-Brunaud C, Hofstetter S, Marchal C, Peifert D. Radiotherapy for solitary extramedullary plasmacytoma in the head-and-neck region: A dose greater than 45 Gy to the target volume improves the local control. Int J Radiat Oncol Biol Phys 2006;64:1013-7. Hussong JW, Perkins SL, Schnitzer B, Hargreaves H, Frizzera G. Extramedullary plasmacytoma: a form of marginal zone cell lymphoma? Am J Clin Pathol 1999;111:111-6. Van de Berg BC, Lecouvet FE, Michaux L, Labaisse M, Malghem J, Jamart J, Maldague BE, Ferrant A, Michaux JL. Stage I multiple myeloma: value of MR imaging of the bone marrow in the determination of prognosis. Radiology 1996;201:243-6. Kumar S, Fonseca R, Dispenzieri A, Lacy MQ, Lust JA, Wellik L, Witzig TE, Gertz MA, Kyle RA, Greipp PR, Rajkumar SV. Prognostic value of angiogenesis in solitary bone plasmacytoma. Blood 2003;101:1715-7.

Koรงak et al. Solitary plasmacytoma

Turk J Hematol 2010; 27: 57-61

10.

11.

12.

13.

14.

15.

16.

17.

18. 19. 20.

21.

22.

23.

Liebross RH, Ha CS, Cox JD, Weber D, Delasalle K, Alexanian R. Solitary bone plasmacytoma: outcome and prognostic factors following radiotherapy. Int J Radiat Oncol Biol Phys 1998;41:1063-7.mination of prognosis. Radiology 1996;201:243-6. He Mx, Zhu MH, Zhang YM, Fu qg, Wu LL. Solitary plasmacytoma of spine: a clinical, radiologic and pathologic study of 13 cases. Zhonghua Bing Li Xue Za Zhi 2009;38:307-11. Kim PJ, Hicks RJ, Wirth A, Ryan G, Seymour JF, Prince HM, Mac Manus MP.Impact of 18F-fluorodeoxyglucose positron emission tomography before and after definitive radiation therapy in patients with apparently solitary plasmacytoma. Int J Radiat Oncol Biol Phys 2009;74:740-6. Schirrmeister H, Buck AK, Bergmann L, Reske SN, Bommer M. Positron emission tomography (PET) for staging of solitary plasmacytoma. Cancer Biother Radiopharm 2003;18:841-5. Soutar R, Lucraft H, Jackson G, Reece A, Bird J, Low E, Samson D. Guidelines on the diagnosis and management of solitary plasmacytoma of bone and solitary extramedullary plasmacytoma. Clin Oncol 2004;16:405-13. Knobel D, Zouhair A, Tsang RW, Poortmans P, Belkacemi Y, Bolla M, Oner FD, Landmann C, Castelain B, Ozsahin Ml. Prognostic factors in solitary plasmacytoma of the bone: a multicenter Rare Cancer Network study. BMC Cancer 2006;6:118. Wilder RB, Ha CS, Cox JD, Weber D, Delasalle K, Alexanian R. Persistence of myeloma protein for more than one year after radiotherapy is an adverse prognostic factor in solitary plasmacytoma of bone. Cancer 2002;94:1532-7. Kilciksiz S, Celik OK, Pak Y, Demiral AN, Pehlivan M, Orhan O, Tokatli F, Agaoglu F, Zincircioglu B, Atasoy BM, Ozseker N, Yersal O, Niang U, Haydaroglu A. Clinical and prognostic features of plasmacytomas: a multicenter study of Turkish Oncology Group-Sarcoma Working Party. Am J Hematol 2008;83:702-7. Bolek TW, Marcus RB, Mendenhall NP. Solitary plasmacytoma of bone and soft tissue. Int J Radiat Oncol Biol Phys 1996;36:329-33. Bataille R, Sany J. Solitary myeloma: clinical and prognostic features of a review of 114 cases. Cancer 1981;48:845-51. Aviles A, Huerta-Guzman J, Delgado S, Fernandez A, DiazMaqueo JC. Improved outcome in solitary bone plasmacytomata with combined therapy. Hematol Oncol 1996;14:111-7. Galieni P, Cavo M, Pulsoni A, Avvisati G, Bigazzi C, Neri S, Caliceti U, Benni M, Ronconi S, Lauria F. Clinical outcome of extramedullary plasmacytoma. Haematologica 2000;85:47-51. Chao MW, Gibbs P, Wirth A, Quong G, Guiney MJ, Liew KH. Radiotherapy in the management of solitary extramedullary plasmacytoma. Intern Med J 2005;35:211-5. Liebross RH, Ha CS, Cox JD, Weber D, Delasalle K, Alexanian R. Clinical course of solitary extramedullary plasmacytoma. Radiother Oncol 1999;52:245-9.

24.

25.

26.

27.

28.

29. 30.

31.

32.

33.

34.

35.

36.

37.

Mendenhall CM, Thar TL, Million RR. Solitary plasmacytoma of bone and soft tissue. Int J Radiat Oncol Biol Phys 1980;6:1497-501. Susnerwala SS, Shanks JH, Banerjee SS, Scarffe JH, Farrington WT, Slevin NJ. Extramedullary plasmacytoma of the head and neck region: clinicopathological correlation in 25 cases. Br J Cancer 1997;75:921-7. Jyothirmayi R, Gangadharan VP, Nair MK, Rajan B. Radiotherapy in the treatment of solitary plasmacytoma. Br J Radiol 1997;70:511-6. Frassica DA, Frassica FJ, Schray MF, Sim FH, Kyle RA. Solitary plasmacytoma of bone: Mayo Clinic experience. Int J Radiat Oncol Biol Phys 1989;16:43-8. Chak LY, Cox RS, Bostwick DG, Hoppe RT. Solitary plasmacytoma of bone: treatment, progression, and survival. J Clin Oncol 1987;5:1811-5. Bolek TW, Marcus RB, Mendenhall NP. Solitary plasmacytoma of bone and soft tissue. Int J Radiat Oncol Biol Phys 1996;36:329-33. Holland J, Trenkner DA, Wasserman TH, Fineberg B. Plasmacytoma: treatment results and conversion to myeloma. Cancer 1992;69:1513-7. Mayr NA, Wen BC, Hussey DH, Burns CP, Staples JJ, Doornbos JF, Vigliotti AP. The role of radiation therapy in the treatment of solitary plasmacytomas. Radiother Oncol 1990;17:293-303. Shih LY, Dunn P, Leung WM, Chen WJ, Wang PN. Localised plasmacytomas in Taiwan: comparison between extramedullary plasmacytoma and solitary plasmacytoma of bone. Br J Cancer 1995 71:128-33. Galieni P, Cavo M, Avvisati G, Pulsoni A, Falbo R, Bonelli MA, Russo D, Petrucci MT, Bucalossi A, Tura S. Solitary plasmacytoma of bone and extramedullary plasmacytoma: two different entities? Ann Oncol 1995;6:687-91. Moulopoulos LA, Dimopoulos MA, Smith TL, Weber DM, Delasalle KB, Libshitz HI, Alexanian R. Prognostic significance of magnetic resonance imaging in patients with asymptomatic multiple myeloma. J Clin Oncol 1995;13:251-6. Mariette X, Zagdanski AM, Guermazi A, Bergot C, Arnould A, Frija J, Brouet JC, Fermand JP. Prognostic value of vertebral lesions detected by magnetic resonance imaging in patients with stage I multiple myeloma. Br J Haematol 1999;104:723-9. Jackson A, Scarffe JH. Prognostic significance of osteopenia and immunoparesis at presentation in patients with solitary myeloma of bone. Eur J Cancer 1999;26:363-71. Dingli D, Kyle RA, Rajkumar SV, Nowakowski GS, Larson DR, Bida JP, Gertz MA, Therneau TM, Melton LJ 3rd, Dispenzieri A, Katzmann JA. Immunoglobulin free light chains and solitary plasmacytoma of bone. Blood 2006;108:1979-83.

Research Article

The protection of the myocardium by amifostine against mitoxantrone-induced acute cardiotoxicity in rats SÕçanlarda mitoksantronun yol açtÕúÕ akut kardiyotoksisteye karýÕ amifostin ile miyokardÕn korunmasÕ Vefki Gürhan KadÕköylü1, ûbrahim Meteoúlu2, Süleyman Demir3, Hülya Aybek3, Mete Kalak1, Muharrem Balkaya4, Çiúdem Yenisey5, Zahit Bolaman1 1Division

of Hematology, Adnan Menderes University Medical Faculty, AydÕn, Turkey of Pathology, Adnan Menderes University Medical Faculty, AydÕn, Turkey 3Division of Biochemistry, Pamukkale University Medical Faculty, Denizli, Turkey 4Adnan Menderes University, Faculty of Veterinary Medicine, AydÕn, Turkey 5Division of Biochemistry, Adnan Menderes University Medical Faculty, AydÕn, Turkey 2Division

Abstract Objective: Amifostine (AMI) has been used for the prevention of doxorubicin-induced cardiotoxicity in several experimental and a few clinical studies. The aim of this study was to investigate the effects of AMI on lipid peroxidation, protective enzymes, and mitoxantrone (MITO)-induced acute cardiotoxicity in the rat heart using biochemical tests and histopathological examinations. Materials and Methods: Thirty-six rats were divided into six groups (n=6 in each). Control rats were given intraperitoneal (i.p.) serum saline and AMI group rats were given 200 mg/kg AMI i.p. Rats received MITO-2.5 and 5 mg/kg i.p. in the MITO-2.5 and MITO-5 groups. AMI 200 mg/kg i.p. was administered 30 min. before the same doses of MITO in the MITO2.5+AMI and MITO-5+AMI groups. Results: The levels of cardiac enzymes such as creatinine phosphokinase-myocardial band and cardiac troponin T did not change. Malondialdehyde (MDA) levels increased in MITO groups compared to controls. Catalase and glutathione (GSH) levels in the MITO and MITO+AMI groups were higher than in controls. Superoxide dismutase and glutathione peroxidase levels were not different between MITO groups and controls. There was no difference in MDA levels between MITO+AMI groups and controls. Calcium deposition was not detected. The scores of fibrosis, apoptosis, inflammation, and degeneration in MITO groups were higher than in controls. The scores of fibrosis, degeneration and inflammation in MITO+AMI groups were lower. Conclusion: MITO caused lipid peroxidation and myocardial damage, and the myocardium increased catalase and GSH levels to prevent this damage. AMI can protect against MITO-induced acute cardiotoxicity, decreasing myocardial damage and lipid peroxidation. (Turk J Hematol 2010; 27: 62-9) Key words: Amifostine, acute cardiotoxicity, mitoxantrone, lipid peroxidation Received: September 17, 2009

Accepted: March 12, 2010

Address for Correspondence: Prof. Vefki Gürhan KadÕköylü, Adnan Menderes Üniversitesi TÕp Fakültesi Hematoloji Bilim DalÕ, 09100 AydÕn, Turkey Phone: +90 256 444 12 56 E-mail: gurhan@prontomail.com doi:10.5152/tjh.2010.02

KadÕköylü et al. Mitoxantrone-induced acute cardiotoxicity and amifostine

Turk J Hematol 2010; 27: 62-9

Özet Amaç: Amifostin (AMI) doksorubisinin yol açtÕúÕ kardiyotoksisiteden korunmada çeýitli deneysel ve bir kaç klinik çalÕýmada kullanÕlmÕýtÕr. Bu çalÕýmanÕn amacÕ SÕçan kalbindeki lipid peroksidasyonu, koruyucu enzimler ve mitoksantronun (MITO) yol açtÕúÕ akut kardiyotoksisite üzerinde AMI’nin etkilerini biyokimyasal ve histopatolojik incelemeler ile araýtÕrmaktÕ. Yöntem ve Gereçler: Her bir grupta 6 sÕçan olmak üzere 36 sÕçan 6 gruba bölündü. ûntraperitoneal (ip) olarak kontrol grubuna serum fizyolojik ve AMI grubuna 200 mg/kg AMI verildi. MITO 2.5 ve 5 gruplarÕndaki sÕçanlar ip MITO 2.5 ve 5 mg/kg aldÕ. MITO 2.5+AMI ve MITO 5+AMI gruplarÕnda aynÕ dozlarda MITO’dan 30 dk önce 200 mg/kg AMI uygulandÕ. Bulgular: Kretainin fosfokinaz-miyokardial bant ve kardiyak troponin T gibi kardiyak enzimlerin düzeyi deúiýiklik göstermedi. MITO gruplarÕndaki malondialdehid (MDA) düzeyleri kontrollere kÕyasla yüksekti. MITO ve MITO+AMI gruplarÕndaki katalaz ve glutatyon düzeyleri kontrollerden yüksekti. MITO+AMI ve kontroller arasÕnda süperoksit dismutaz ve glutatyon peroksidaz düzeyleri bakÕmÕndan fark yoktu. Kalsiyum birikimi saptanmadÕ. Fibrozis, dejenerasyon ve inflamasyon skorlarÕ MITO+AMI gruplarÕnda daha düýüktü. Sonuç: MITO lipid peroksidasyonu ve miyokardiyal zaralanmaya neden olurken miyokardiyum bu zaralanmadan korunmak için katalaz ve GSH düzeylerini arttÕrmaktadÕr. AMI miyokardiyal zararlanma ve lipid peroksidasyonu azaltarak MITO’nun yol açtÕúÕ akut kardiyotoksisiteye karýÕ koruyucu olabilmektedir. (Turk J Hematol 2010; 27: 62-9) Anahtar kelimeler: Amifostin, akut kardiyotoksisite, mitoksantron, lipid peroksidasyonu Geliý tarihi: 17 Eylül 2009

Kabul tarihi: 12 Mart 2010

Introduction Anthracyclines are antineoplastic drugs used in the treatment of hematological malignancies and solid tumors [1]. The mechanisms of the antitumor effect of these drugs are the inhibition of topoisomerase II, the intercalation between DNA base pairs, inhibiting synthesis of macromolecules, and the generation of free oxygen radicals, causing DNA damage and lipid peroxidation. The most important toxic effects of anthracyclines are myelosuppression and cardiotoxicity. These toxicities limit both their doses and effectiveness [1,2]. Anthracycline-induced chronic cardiotoxicity is dosedependent and cumulative. The cumulative cardiotoxicity doses are 500-550 mg/m2 for doxorubicin, 150 mg/m2 for idarubicin, and 40-100 mg/m2 for mitoxantrone (MITO), respectively. In these patients, left ventricular dysfunction and congestive heart failure occurred because of dilated cardiomyopathy [2-5]. Although myocardial adrenergic dysfunction including down-regulation of myocardial `-adrenergic receptors, intracellular calcium overload, induction of apoptosis, and release of cardiotoxic cytokines such as tumor necrosis factor-alpha and interleukin-2 may play a role in the pathogenesis of anthracycline-induced cardiotoxicity, focus in recent years has been on free oxygen radicals and lipid peroxidation [2,3,6-10]. One electron addition to quinine moiety in the tetracyclic ring of anthracyclines results in the formation of semiquinone and free oxygen radicals such as superoxide anion (O2-•), hydrogen peroxide (H2O2), and hydroxyl radicals (OH•), by NADPH (nicotinamide-adenine dinucleotide phosphate)-cytochrome c-reductase. O2-• converts to H2O2 by superoxide dismutase (SOD), and then H2O2 is metabolized to H2O by catalase. Ferrous (Fe2+) ions catalyze the conversion of H2O2 to OH•. The OH• is capable of abstracting a hydrogen atom from polyunsaturated fatty acids in membrane lipids to initiate lipid peroxidation. As a result of the interaction of the anthracycline-iron (Fe3+) complex, these radicals can cause both extensive tissue and DNA damage and decrease in peptides con-

taining sulfhydryl, reacting with macromolecules, such as membrane lipids, proteins and nucleic acids in myocardial tissue. Moreover, free oxygen radicals can decrease the levels of glutathione (GSH) and protective enzymes such as catalase, SOD, glutathione peroxidase (Gpx), and glutathione reductase [2,6,8,10-12]. Amifostine (AMI) is a cytoprotective prodrug that is dephosphorylated by alkaline phosphatase in tissues to active free thiol metabolite [11,12]. AMI has been used for the prevention of doxorubicin-induced cardiotoxicity in several experimental and a few clinical studies [9,11-16]. AMI protects the myocardium, inhibiting lipid peroxidation, apoptosis and the production of free oxygen radicals [9,11,12,15,17]. Mitoxantrone (MITO) is an anthracenedione antineoplastic drug, but its cardiotoxicity is less than that of doxorubicin. The mechanism of MITO-induced cardiotoxicity is probably similar to that of other anthracyclines [18-20]. There are limited experimental studies on MITO-induced cardiotoxicity [6,7,19,21,22], but we did not find any experimental study in the literature on the cardioprotective effects of AMI against MITO-induced acute cardiotoxicity. We thus investigated both the effects of AMI on lipid peroxidation and protective enzymes and the cardioprotective effects of AMI against MITO-induced acute cardiotoxicity in the rat heart using biochemical tests and histopathological examinations.

Materials and Methods Chemicals MITO (Mitoxantron Ebewe 20 mg/10 ml, Liba/Turkey), AMI (Ethyol 500 mg/10 ml, Er-Kim/Turkey), and serum saline were used as drugs in rats. Animals and Treatment Protocols Thirty-six male Wistar rats, 4 months old, weighing 156±30 g (88-242 g) were obtained from the Experimental Research Center of Ege University. Rats were kept in ventilated rooms at

KadÕköylü et al. Mitoxantrone-induced acute cardiotoxicity and amifostine

23±2°C with 10 hour (h) darkness/14 h light cycle and relative humidity of 60-75%. They were fed on standard feed (Best Feed-Turkey) and water ad libitum. Rats were divided into six groups of six rats each. There was no difference between groups with respect to weight (p>0.05). In the control group, rats received 1 ml/100 g serum saline intraperitoneally (i.p.). In the AMI group, 200 mg/kg AMI i.p. was administered. MITO-2.5 mg/kg and 5 mg/kg i.p. were injected in rats in the MITO-2.5 and MITO-5 groups, respectively. AMI 200 mg/kg i.p. was administered to rats 30 minutes before the same doses of MITO in the MITO-2.5+AMI and MITO-5+AMI groups, respectively. In previous experimental studies on MITO-induced chronic cardiotoxicity, 0.2-0.6 mg/kg MITO for 12-13 weeks was used. The cumulative doses were 2.4-7.2 mg/kg [20-23]. However, there is no study with the exception of cell culture on MITOinduced acute cardiotoxicity in rats. We thus performed this study with a single dose of 2.5 or 5 mg/kg MITO. Moreover, acute cardiotoxicity was evaluated 7 days after MITO administration, because anthracycline-induced cardiotoxicity is usually detected on the 7th day of administration [18,24]. Blood samples were obtained from the tail vein for cardiac enzymes before administration, one day after drug administration, and before sacrifice. Sera were separated and stored until analysis. The rats were sacrificed 7 days after administration by cervical dislocation and their hearts were removed. The hearts were washed with cold serum saline. Half of the heart was stored at -80°C for biochemical tests until analysis. The other half was fixed in 4% formaldehyde solution, and then embedded into paraffin for histopathological evaluations. An expert pathologist examined 4 m-thick tissue sections, hematoxylin and eosin-stained, without prior knowledge of the drugs given to the rats. The Institutional Animal Care and Use Committee and National Institutes of Health Guidelines for Animal Care were followed throughout the study [25]. The Local Animal Ethical Committee of the Veterinary Faculty of Adnan Menderes University approved our study. The Project Fund of Adnan Menderes University supported this study financially (Project Number: 6022). Biochemical Assays The serum levels of cardiac enzymes, such as creatinine phosphokinase-myocardial band (CK-MB) and cardiac troponin T (cTnT) were measured by electrochemiluminescence immunoassay method (Elecsys 2010 instruments, cTnT STAT and CK-MB STAT kits, Roche Diagnostics, Indianapolis, Indiana, USA). The heart tissues were thawed and homogenized with appropriate buffer solutions. All analyses including malondialdehyde (MDA), catalase, SOD, total GSH, and Gpx in the heart tissues were performed with Oxis Research products (Foster City, California, USA) using spectrophotometric/colorimetric methods. MDA, a lipid peroxidation product, was measured with a method using the commercial MDA-586 kit. This method is

Turk J Hematol 2010; 27: 62-9

based on the reaction of a chromogenic reagent, N-methyl-2phenylidone (NMPI) at 45°C [26]. Catalase was analyzed using the commercial catalase-520 kit. When incubated with H2O2, the tissue containing catalase is quenched with sodium azide. The amount of remaining H2O2 is determined by oxidative coupling reaction of 4-aminophenazone and 3,5-dichloro2-hydroxybenzenesulfonic acid and then catalyzed by horseradish peroxidase (HRP). The resulting quinone imine dye is measured at 520 nm [27]. SOD was measured with the method of Nebot et al. [28] using the commercial SOD-525 kit. This method is based on the SOD-mediated increase in the rate of autoxidation of 5,6,6,6 a,11b tetrahydro - 3,9,10 - trihydroxybenzo [c] fluorine (TTF) in aqueous alkaline solution to yield a chromophore with maximum absorbance at 525 nm. Total GSH was assayed with the method using the commercial GSH-420 kit. This method is based on the formation of a chromophoric thione [29]. Gpx was measured using the commercial Gpx-340 kit. Gpx catalyzes the reduction of H2O2 to H2O and organic peroxides to alcohols using GSH. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm for indirect measuring of Gpx activity [30]. Histopathological Evaluation Myocardial fibrosis, degeneration, apoptosis, inflammation, and calcium deposition were evaluated using grading described previously [31,32]. The grading for fibrosis was 1: minimal fibrosis in ventricles, septum or papillary muscles, 2: small foci of fibrosis involving small foci at multiple locations, 3: multiple foci of fibrosis involving more than one area, and 4: large diffuse fibrosis area involving ventricular septum and left ventricular papillary muscles. The grading for inflammation was 1: few scattered inflammatory cells, 2: minimal inflammatory infiltrates, 3: small localized multiple foci of inflammatory cells involving more than one area, and 4: diffuse severe inflammatory infiltrates. The grading for apoptosis was 1: single myocytes randomly distributed in ventricles, septum or papillary muscles, 2: single foci consisting of a few myocytes involving more than previously described locations, and 3: small localized, multiple foci of myocytic apoptosis involving more than one area. Billingham’s score for degeneration was 1: <5%, 1.5: 5-15%, 2: 16-25%, 2.5: 26-35%, and 3: >35%. The grading for calcium deposition was 1: occasional calcium deposits in ventricles, septum or papillary muscles, 2: apparent calcium deposits in previously described area, 3: apparent calcium deposits involving more than one area, and 4: large diffuse area of calcium deposition involving ventricular septum and left ventricular papillary muscles. Statistical Analysis All values were given as mean ± standard deviation. Oneway ANOVA, Tukey, and Bonferroni tests of post-hoc analysis were used for the comparison of multiple groups. Cardiac enzymes were compared with two-paired t test. SPSS 13.0 for Windows was used for all tests. P value <0.05 was as considered to be significant.

KadÕköylü et al. Mitoxantrone-induced acute cardiotoxicity and amifostine

Turk J Hematol 2010; 27: 62-9

Results Rats In the MITO-5 group, one rat died on the 7th day of the study. The heart was removed and washed with cold serum saline. The same procedures were done until analysis. Cardiac Enzymes The levels of cTnT and CK-MB were not different between the six groups and treatment days in all groups (p>0.05). The results of cardiac enzymes are shown in Table 1. Lipid Peroxidation, GSH and Protective Enzymes MDA levels in MITO groups were significantly higher than in control and AMI groups (p<0.05 for MITO-2.5 group and p<0.001 for MITO-5 group). There was no difference in MDA levels between MITO+AMI, control, and AMI groups (p>0.05). MDA levels of MITO-5+AMI group were significantly lower than of the MITO-5 group (p<0.005). Total GSH levels in MITO-2.5 (p<0.001 for both groups) and MITO-5 groups (p<0.001 and p<0.01) were higher than control and AMI groups. Moreover, these levels in MITO2.5+AMI (p<0.001) and MITO-5+AMI groups (p<0.001 and p<0.005) were still higher than in controls. Catalase levels in the MITO-2.5 (p<0.001 and p<0.005), MITO-5 (p<0.001), and MITO+AMI groups (p<0.001) were higher than in control and AMI groups. SOD levels of the MITO-5+AMI group were higher than in controls (p<0.005), but there was no difference between the other groups (p>0.05).

Gpx levels of the MITO-5+AMI group were lower than in the MITO-2.5 group (p<0.05), but these levels were not different between the other groups (p>0.05). The results of all biochemical analyses are shown in Table 1. Histopathological Changes Fibrosis score in the MITO-2.5 group was higher than in the control (p<0.005), AMI (p<0.05), and MITO-5 groups (p<0.005). Fibrosis decreased in the MITO-5+AMI group compared to the MITO-2.5 group (p<0.05). There was no difference between control and MITO-5+AMI groups (p>0.05). Inflammation scores in the AMI and MITO-2.5 (p<0.001 for both groups) and MITO- 5 groups (p<0.05) were higher than in the control group. Inflammation decreased in the MITO+AMI groups compared to the MITO-2.5 group (p<0.05 and p<0.001). There was no difference between control and MITO+AMI groups (p>0.05). Apoptosis score in the MITO-2.5 group was higher than in control, AMI (p<0.001 for both groups), and MITO-5 groups (p<0.05). While the score in the MITO-2.5+AMI group was higher than in controls (p<0.005), there was no difference between control and MITO-5+AMI groups (p>0.05). Billingham’s scores in the AMI, MITO-2.5, MITO-2.5+AMI (p<0.001 for all), and MITO-5 groups (p<0.01) were higher than in the control group. Degeneration in MITO-5+AMI group was decreased when compared to the MITO-2.5 group (p<0.005). There was no difference between controls and the MITO-5+AMI groups (p>0.05). There was no calcium deposition in any rat. Histopathological changes in all groups are given in Table 2. The figures of Grade 3 fibrosis, inflammation, apoptosis, and degeneration are seen in Figures 1-4, respectively.

Table 1. The results of biochemical analysis in the groups Control

Amifostine

Mitoxantrone-2.5

Mitoxantrone-5

Mitoxantrone-2.5 +

Mitoxantrone-5 +

(n=6)

Amifostine (n=6)

163±18

150±29

149±25

144±39

150±32

165±14

MDA (nmol/g tissue)

45.9±10.1

43±12.2

69.2±10.1a,b

82.7±14.8c,d

63.5±7.2

52.3±12.8e

Gpx (mU/g protein)

14.2±7

20.7±10.5

23±1.8

18±7.3

15.1±5.3

6±2.3g

Catalase (U/g protein)

1.15±0.15

1.3±0.25

2.61±0.43d,f

3.32±1.2c,d

3±0.47c,d

3.27±0.55c,d

SOD (U/g protein)

19.1±2.7

27.8±18.3

29.1±9.9

26.6±13.5

29.4±7.8

45.2±21f

GSH ( mol/g protein)

85±27.4

120.3±30.3

258±60.5c,d

196.4±29.5c,h

215.6±36c,d

198.1±30.8c,i

Day 0

0.01±0

0.03±0.04

Day 1

0.01±0

Day 8

0.01±0

0.1±0.15

0.23±0.33

0.09±0.1

0.04±0.05

0.83±1.8

Day 0

0.12±0.02

0.15±0.05

0.19±0.05

0.2±0.07

0.18±0.07

0.14±0.06

Day 1

0.1±0

0.13±0.07

0.1±0

0.18±0.18

0.12±0.04

0.01±0.05

Day 8

0.1±0

0.17±0.12

Weight (g)

Troponin-T (ng/ml)

CK-MB (ng/ml)

a:p < 0.05 as compared to AMI group, b:p < 0.05 as compared to control group, c:p < 0.001 as compared to control group, d:p < 0.001 as compared to AMI group, e:p < 0.005 as compared to MITO-5 group, f:p < 0.005 as compared to control group, g:p< 0.05 as compared to MITO-2.5 group, h:p < 0.01 as compared to AMI group, i:p < 0.005 as compared to AMI group

KadÕköylü et al. Mitoxantrone-induced acute cardiotoxicity and amifostine

Turk J Hematol 2010; 27: 62-9

Table 2. The results of histopathological evaluation in the groups Control

Amifostine

Mitoxantrone-2.5

Mitoxantrone-5

Mitoxantrone-2.5 +

Mitoxantrone-5 +

(n=6)

Amifostine (n=6)

Inflammation

1±0

2.3±0.5a

2.8±0.4a

2±0.6b

1.8±0.4c

1.5±0.5d

Degeneration

1±0

2.3±0.5a

2.8±0.4a

2±0.6e

2.3±0.3a

1.7±0.4f

1.2±0.4

1.3±0.5c

2.3±0.5g

1.2±0.4f

1.7±0.5

1.3±0.5c

Calcium deposition

1±0

Apoptosis

1±0

1.3±0.5

2.7±0.5a,h

1.7±0.5c

2.2±0.4g

1.8±0.8

Fibrosis

a:p < 0.001 as compared to control group, b:p < 0.05 as compared to control group, c:p < 0.05 as compared to MITO-2.5 group, d:p < 0.001 as compared to MITO-2.5 group, e:p < 0.01 as compared to control group, f:p < 0.005 as compared to MITO-2.5 group, g:p < 0.005 as compared to control group, h:p < 0.001 as compared to AMI group

Figure 1. Grade 3 fibrosis in heart tissue

(multiple foci of fibrosis involving more than one area; 4 m thick paraffin section, hematoxylin and eosin, original magnification x200)

Figure 2. Grade 3 inflammation in heart tissue

(small localized multiple foci of inflammatory cells involving more than one area; 4 m thick paraffin section, hematoxylin and eosin, original magnification x400)

Figure 3. Grade 3 apoptosis in heart tissue

(small localized, multiple foci of myocytic apoptosis involving more than one area; 4 m thick paraffin section, hematoxylin and eosin, original magnification x200)

Figure 4. Grade 3 degeneration in heart tissue

(more than 35% of degeneration; 4 m thick paraffin section, hematoxylin and eosin, original magnification x200)

Discussion In this study, MITO caused inflammation, degeneration, fibrosis, and apoptosis in heart tissue. Moreover MITO induced lipid peroxidation and increased catalase and total GSH levels.

It was shown in previous experimental studies that doxorubicin causes the generation of free oxygen radicals and lipid peroxidation. The effects of doxorubicin on GSH and protective enzymes such as catalase, Gpx and SOD are controversial

Turk J Hematol 2010; 27: 62-9

[9,11,12,33,34]. Although MITO induced lipid peroxidation and the generation of free oxygen radicals in liver microsomes [3,8,18], there are only a few experimental studies in which these effects in the heart are demonstrated [22,23]. In these studies, 0.5-0.6 mg/kg MITO for 12-13 weeks was administrated to investigate MITO-induced chronic cardiotoxicity. Moreover, histopathological changes for chronic cardiotoxicity due to the cumulative effects of MITO’s repeated administration were detected. In our study, we investigated the acute cardiotoxicity of single-dose MITO. Lipid peroxidation played a role in MITO-induced acute cardiotoxicity in our study. Lipid peroxidation was especially more evident in the MITO-5 group. Catalase, which dismutases H2O2 to H2O and O2-•, is an antioxidant enzyme. While catalase activities are highest in liver and erythrocytes, they are lowest in the heart and brain. Catalase activity in the heart is about 2% of that in the liver of humans, mice, and rats [33]. SOD, which catalyzes the dismutation of O2-• to H2O2 and O2, is a metalloenzyme. Fe-SOD in prokaryotes, Cu/Zn-SOD in cytosol and nuclei of eukaryotes and Mn-SOD in mitochondrial matrix of prokaryotes and eukaryotes are found [35]. Gpx catalyzes the conversion of H2O2 and reduced-GSH to H20 and oxidized GSH [36]. Catalase, SOD and Gpx play critical roles in protecting the myocardium from lipid peroxidation and free oxygen radicals under oxidative damage. It was detected in some experimental studies that doxorubicin and idarubicin decreased GSH, catalase, Gpx, and SOD levels in the heart tissue [9,11,37,38]. However, over-expression of catalase and Mn-SOD activities were detected after doxorubicin administration. Expression of catalase activity 60 to 100-fold higher than normal can exhibit protection from doxorubicin-induced lipid peroxidation in the heart of transgenic mice. But more than 200-fold increase in catalase activity can not provide this protection [2,33,39-41]. In some studies, no increase in the levels of GSH and these protective enzymes was found after MITO administration [20,39,41]. In our study, while Gpx and SOD levels did not change, GSH and catalase levels increased. Although it was not previously reported, this condition may be related to myocardial protection from MITO-induced cardiotoxicity. Doxorubicin can cause edema, cytoplasmic vacuolization, degeneration, myofibrillar loss, inflammation, apoptosis, and fibrosis in cardiomyocytes [13,14,21,22]. Billingham’s score was higher than in controls in the evaluation of chronic doxorubicin cardiotoxicity in two studies [14,21]. MITO may reduce cell viability acutely and accumulate in cardiomyocytes more than other anthracyclines such as epirubicin, idarubicin and carminomycin [6]. Moreover, MITO can induce apoptosis in the rat heart [2,42]. MITO-induced chronic cardiotoxicity was compared with doxorubicin in two studies. Koutinos et al. [22] administered 2 mg/kg doxorubicin and 0.5 mg/kg MITO once a week for 12 weeks. At the end of 12 weeks, Grade 1 and 2 degenerations were observed in 33% and 50% of rats treated with doxorubicin and in 83.3% and 16.6% of rats treated with MITO, respectively. Herman et al. [21] gave 1 mg/kg doxorubicin and 0.5 mg/kg MITO weekly for 12 weeks. Degeneration Grade 2 was detected in 100% of the doxorubicin group and

KadÕköylü et al. Mitoxantrone-induced acute cardiotoxicity and amifostine

91% of the MITO group. In both studies, histopathological findings in the doxorubicin group were more abundant than those of the MITO group. In our study, the scores of inflammation, degeneration, fibrosis, and apoptosis were higher in the MITO groups. Billingham’s score was more than Grade 1.5 in the MITO groups. Interestingly, histopathological changes were more apparent in the MITO-2.5 group. In spite of histopathological and biochemical changes in heart tissue, we did not detect a significant increase in serum cardiac enzymes in our study. cTnT and CK-MB have been used for the detection of anthracycline-induced cardiotoxicity in experimental studies. These enzymes increased in both acute and chronic doxorubicin cardiotoxicity [21,40,43]. However, Nazeyrollas et al. [13] found these enzyme levels as normal. cTnT levels were high in rats with chronic MITOinduced cardiotoxicity in only one study [21]. AMI decreased MITO-induced lipid peroxidation in our study. This decrease was especially evident in the MITO5+AMI group. While catalase and total GSH levels in all MITO+AMI groups, and SOD levels in only the MITO-5+AMI group increased, Gpx levels decreased in the MITO-5+AMI group compared to controls. However, AMI did not have any favorable effect on these parameters compared to the MITO groups. AMI did not change cardiac enzymes. Although AMI inhibits lipid peroxidation and the production of free oxygen radicals, and it increases the protective enzymes such as SOD, catalase, and GSH against doxorubicin-induced cardiotoxicity in some experimental studies [10,13-15]. we did not find any study in which the effects of AMI on MITO-induced cardiotoxicity were investigated. Thus, we could not compare our results with other studies. In our study, AMI caused degeneration and inflammation when compared to controls. AMI administration together with MITO decreased degeneration, apoptosis, inflammation, and fibrosis especially in the MITO-5+AMI group. Alone, AMI administration resulted in degeneration and inflammation in a few experimental studies, although these effects were less than those of doxorubicin [13,14,21]. AMI can decrease the histopathological findings of doxorubicin-induced cardiotoxicity including edema, vacuolization, myofibrillar loss, necrosis, and degeneration [11,13,14,44]. Dragojevic-Simic et al. [14] and Herman et al. [45] detected that AMI significantly decreased these cardiac damage scores. Dexrazoxane reduces the formation of free oxygen radicals induced by the doxorubicin-iron complex and the chronic cardiotoxic effects of doxorubicin in experimental and clinical studies [11,45]. Bjelogrlic et al. [44] and Herman et al. [45] compared the protective effects of dexrazoxane with AMI on doxorubicin-induced cardiotoxicity in two studies. Dexrazoxane was more cardioprotective than AMI. In one study, dexrazoxane attenuated the histopathological and biochemical changes of both doxorubicin- and MITO-induced cardiotoxicity [21]. In conclusion, MITO induced lipid peroxidation and caused myocardial damage. The myocardium increased catalase and GSH to prevent MITO-induced acute cardiotoxicity. AMI decreased both myocardial damage and MITO-induced lipid peroxidation. Thus, AMI can protect against MITO-induced acute cardiotoxicity.

KadÕköylü et al. Mitoxantrone-induced acute cardiotoxicity and amifostine

Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

Turk J Hematol 2010; 27: 62-9

17.

18.

References 1. 2.

3. 4.

8. 9.

10.

11. 12. 13.

14.

15.

16.

Hortobagyi GN. Anthracyclines in the treatment of cancer. An overview. Drugs 1997;54:1-7. Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracycline: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 2004;56:185-229. Shan K, Lincoff AM, Young JB. Anthracycline-induced cardiotoxicity. Ann Intern Med 1996;125:47-58. van Dalen EC, van der Pal HJH, Bakker PJ, Caron HN, Kremer LC. Cumulative incidence and risk factors of mitoxantroneinduced carditoxicity: a systematic review. Eur J Cancer 2004;40:643-52. Ghalie RG, Edan G, Laurent M, Mauch E, Eisenman S, Hartung HP, Gonsette RE, Butine MD, Goodkin DE. Cardiac adverse effects associated with mitoxantrone (Novantrone) therapy in patients with MS. Neurology 2002;59:909-13. Andersson BS, Eksborg S, Vidal RF, Sundberg M, Carlberg M. Anthraquinone–induced cell injury: acute toxicity of carminomycin, epirubicin, idarubicin and mitoxantrone in isolated cardiomyocytes. Toxicology 1999;135:11-20. Kim E, Giri SN, Pessah IN. Antithetical actions of mitoxantrone and doxorubicin on ryanodine-sensitive Ca++ release channels of rat cardiac sarcoplasmic reticulum: evidence for a competitive mechanism. J Pharmacol Exp Ther 1994;268:1212-21. Doroshow JH. Effect of anthracycline antibiotics on oxygen radical formation in rat heart. Cancer Res 1983:43:460-72. Bolaman Z, Cicek C, Kadikoylu G, Barutca S, Serter M, Yenisey C, Alper G. The protective effects of amifostine on adriamycininduced acute cardiotoxicity in rats. Tohoku J Exp Med 2005:207:249-53. Ehrke MJ, Maccubbin D, Ryoyama K, Cohen SA, Mihich E. Correlation between adriamycin-induced augmentation of interleukin 2 production and of cell-mediated cytotoxicity in mice. Cancer Res 1986;46:54-60. Dorr RT. Cytoprotective agents for anthracyclines. Semin Oncol 1996; 23: 23-34. Minotti G, Cairo G, Monti E. Role of iron in anthracycline cardiotoxicity: new tunes for and old song. FASEB J 1999;13:199-212. Nazeyrollas P, Frances C, Prevost A, Costa B, Lorenzato M, Kantelip JP, Elaerts J, Millart H. Efficiency of amifostine as a protection against doxorubicin toxicity in rats during a 12-day treatment. Anticancer Res 2003;23:405-9. Dragojevic-Simic VM, Dobric SL, Bokonjic DR, Vucinic ZM, Jacevic VM, Dogovic NP. Amifostine protection against doxorubucin cardiotoxicity in rats. Anti-Cancer Drugs 2004;15:169-78. Rigatos S, Stathopoulos G, Dontas I, Perrea-Kotsarelis D, Couris E, Karayannacos PE, Deliconstantinos G. Investigation of doxorubicin tissue toxicity: Does amifostine provide chemoprotection? An experimental study. Anticancer Res 2002;22:129-34. Catino A, Crucitta E, Latorre A, Sambiasi D, Calabrese P, Lorusso V. Amifostine as chemoprotectant in metastatic breast cancer patients treated with doxorubicin. Oncol Rep 2003;10:163-7.

19.

20.

21.

22.

23.

24.

25. 26.

27.

28.

29. 30.

31.

32.

33.

Provinciali M, Clavattini A, Di Stefano G, Argentati K, Garzetti GG. In vivo amifostine (WR-2721) prevents chemotherapyinduced apoptosis of peripheral blood lymphocytes from cancer patients. Life Sci 1999;64:1525-32. Herman EH, Zhang J, Hasinoff BB, Clark JR Jr, Ferrans VJ. Comparison of the structural changes induced by doxorubicin and mitoxantrone in the heart, kidney and intestine and characterization of the Fe(III)-mitoxantrone complex. J Mol Cell Cardiol 1997;29:2415-30. Faulds D, Balfour JA, Chrisp P, Langtry HD. Mitoxantrone: a review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the chemotherapy of cancer. Drugs 1991;41:400-49. Alderton PM, Gross J, Green MD. Comparative study of doxorubicin, mitoxantrone, and epirubicin in combination with ICRF-187 (ADR-529) in a chronic cardiotoxicity animal model. Cancer Res 1992;52:194-201. Herman EH, Zhang J, Rifai N, Lipshultz SE, Hasinoff BB, Chadwick DP, Knapton A, Ferrans VJ. The use of serum levels of cardiac troponin T to compare the protective activity of dexrazoxane against doxorubicin- and mitoxantrone-induced cardiotoxicity. Cancer Chemother Pharmacol 2001;48:297-304. Koutinos G, Stathopoulos GP, Dontas I, Perrea-Kotsarelis D, Couris E, Karayannacos PE, Deliconstantinos G. The effect of doxorubicin and its analogue mitoxantrone on cardiac muscle and on serum lipids: an experimental study. Anticancer Res 2002; 22: 815-20. Bouhour JB, Chiffoleau S, Delajartre AY. Experimental study of the subacute toxicity of mitoxantrone and doxorubicin in rats (subcutaneous and/or intraperitoneal route). Arch Mal Coeur Vaiss 1986;79:1238-44 (abstract). Viglione PN, Praprotnik A, Pinto JE. In vitro evaluation of acute effects of mitoxantrone (Novantrone) in rat and guinea pig atria. Pharmacol Toxicol 1993;72:208-12. Pitts M. A guide to the new ARENA/OLAW IACUC guidebook. Lab Anim 2002;31:40-2. Gerard-Monnier D, Erdelmeier I, Regnard K, Moze-Henry N, Yadan JC, Chaudiere J. Reactions of 1-methyl- 2-phenilodine with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation. Chem Res Toxicol 1998;11:1176-83. Fossati P, Perencipe L, Berti G. Use of 3,5-dichloro-2hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. Clin Chem 1980;26:227-31. Nebot C, Moutet M, Huet P, Xu JZ, Yadan JC, Chaudiere J. Spectrophotometric assay of superoxide dismutase activity based on the activated autoxidation of a tetracyclic catechol. Anal Biochem 1993;214:442-51. Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983;52:711-60. Ursini F, Maiorino M, Brigellius-Flohe R, Aumann KD, Roveri A, Schomburg D, Flohe L. Diversity of glutathione peroxidases. Methods Enzymol 1995;252:38-53. Herman EH, Zhang J, Chadwick DP, Ferrans VJ. Age dependence of the cardiac lesions induced by minoxidil in the rat. Toxicology 1996;110:71-83. Billingham ME, Mason JW, Bristow MR, Daniels JR. Anthracycline cardiomyopathy monitored by morphological changes. Cancer Treat Rep 1978;62:865-72. Kang YJ, Chen Y, Epstein PN. Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice. J Biol Chem 1996;271:12610-6.

KadÕköylü et al. Mitoxantrone-induced acute cardiotoxicity and amifostine

Turk J Hematol 2010; 27: 62-9

34.

35.

36.

37.

38.

39.

40.

Abd-Allah AR, Al-Majed AA, Mostafa AM, Al-Shabanah OA, DinAG, Nagi MN. Protective effect of Arabic gum against cardiotoxicity induced by doxorubicin in mice: a possible mechanism of protection. J Biochem Mol Toxicol 2002;16:254-9. Hegstad A, Ytrehus K, Myklebust R, Jorgensen l. Ultrastructural changes in the myocardial myocytic mitochondria: crucial step in the development of oxygen radical induced damage in isolated rat hearts? Basic Res Cardiol 1994;89:128–38. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 2006;160:1-40. Kalender S, Kalender Y, Ates A, Yel M, Olcay E, Candan S. Protective role of antioxidant vitamin E and catechin on idarubicin-induced cardiotoxicity in rats. Braz J Med Biol Res 2002;35:1379-87. Cigremis Y, Parlakpinar H, Polat A, Colak C, Ozturk F, Sahna E, Ermis N, Acet A. Beneficial role of aminoguanidine on acute cardiomyopathy related to doxorubicin-treatment. Moll Cell Biochem 2006;31:147-53. Doroshow JH, Davies KJ. Redox cycling of anthracyclines by cardiac mitochondria. Formation of superoxide anion, hydrogen peroxide, and hydroxyl radical. J Biol Chem 1996;261:3068-74. Yen HC, Oberley TD, Vichitbandha S, Ho YS, St Clair DK. The protective role of manganese superoxide dismutase against

41.

42.

43.

44.

45.

adriamycin-induced acute cardiac toxicity in transgenic mice. J Clin Invest 1996;98:1253-60. Crescimanno M, Flandina C, Rausa L, Sanquedolce R, D’Alessandro N. Morphological changes and catalase activity in the hearts of CD 1 mice following acute starvation or single doses of doxorubicin, epirubicin or mitoxantrone. Chemioterapia 1988;7:53-9. Kluza J, Marchetti P, Gallego MA, Fournier C, Loyens A, Beauvillain JC, Bailly C. Mitochondrial proliferation during apoptosis induced by anticancer agents: effects of doxorubicin and mitoxantrone on cancer and cardiac cells. Oncogene 2004;23:7018-30. Kojima S, Icho T, Hayashi M, Kajiwara Y, Kitabatake K, Kubota K. Inhibitory effect of 5, 6, 7, 8-tetrahydroneopterin on adriamycininduced cardiotoxicity. J Pharmol Exp Ther 1993;266:1699-704. Bjelogrlic SK, Radic J, Radulovic S, Jokanovic M, Jovic V. Effects of dexrazoxane and amifostine on evolution of doxorubicin cardiomyopathy in vivo. Exp Biol Med 2007;232:1414-24. Herman EH, Zhang J, Chadwick DP, Ferrans VJ. Comparison of the protective effects of amifostine and dexrazoxane against the toxicity of doxorubicin in spontaneously hypertensive rats. Cancer Chemother Pharmacol 2000;45:329-34.

Research Article

Functional proteomic analysis of Ankaferd® Blood Stopper Kanama durdurucu Ankaferd® ve etki mekanizmasÕnÕn proteomik analizi Duygu Özel Demiralp1, ûbrahim C. Haznedaroúlu2, Nejat Akar3 1Proteomics

Unit, Ankara University Biotechnology Institute, Ankara, Turkey of Hematology, Hacettepe University Faculty of Medicine, Ankara, Turkey 3Department of Pediatric Molecular Genetics, Ankara University Faculty of Medicine, Ankara, Turkey 2Department

Abstract Objective: Ankaferd® Blood Stopper (ABS) comprises a standardized mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. The basic mechanism of action for ABS is the formation of an encapsulated protein network that provides focal points for vital erythrocyte aggregation. ABS– induced protein network formation with blood cells, particularly erythrocytes, covers the primary and secondary hemostatic system without disturbing individual coagulation factors. Materials and Methods: To understand the effect mechanisms of ABS on hemostasis, a proteomic analysis using 2D gel electrophoresis and mass spectrometer was performed. Results: Proteins of plant origin in Ankaferd® were NADP-dependent-malic enzyme, ribulose bisphosphatecarboxylase-large chain, maturase K, ATP synthase subunit-beta, ATP synthase subunit-alpha, chalcone-flavanone isomerase-1, chalcone-flavanone isomerase-2, and actin-depolymerizing factor. Furthermore, functional proteomic studies revealed that proteins resembling human peptides have been detected within Ankaferd®, including ATP synthase, mucin-16 (CD164 sialomucin-like 2 protein), coiled-coil domain containing 141 hypothetical protein LOC283638 isoform 1, hypothetical protein LOC283638 isoform 2, dynactin 5, complex I intermediate-associated protein 30, mitochondrial, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, TP synthase, H+ transporting, mitochondrial actin binding 1 isoform, LIM domain and actin binding 1 isoform a, LIM domain and actin binding 1 isoform b, spectrin alpha non erythrocytic 1, prolactin releasing hormone receptor, utrophin, tet oncogene family member 2 isoform b, protein phosphatase 1 regulatory subunit 12A, NIMA (never in mitosis gene a)-related kinase, ATP-binding cassette protein C12, Homo sapiens malic enzyme 1, mitochondrial NADP(+)-dependent malic enzyme 3, ME2 protein, nuclear factor 1 B-type, abhydrolase domain-containing protein 12B, E3 SUMO-protein ligase PIAS2, alpha-1, 2-glucosyltransferase ALG10-A, cofilin, non-muscle isoform, 18 kDa phosphoprotein, p18, actin-depolymerizing factor (ADF), twinfilin-1, ankyrin repeat and FYVE domain-containing protein 1, usherin precursor, urotensin II receptor, interleukin 4, and midkine. Conclusion: Proteomic analysis of Ankaferd® represents a true basis for the upcoming Ankaferd® studies focusing on its wound healing, hemostatic, anti-infective, antineoplastic, and preservative biological actions. (Turk J Hematol 2010; 27: 70-7) Key words: Ankaferd, proteomics, hemostasis, bleeding Received: June 23, 2009

Accepted: November 19, 2009

Address for Correspondence: Asst. Prof. Duygu Özel Demiralp, Proteomics Unit, Ankara University Biotechnology Institute, 06100 Ankara, Turkey Phone: +90 312 222 58 17 - +90 312 222 58 26 E-mail: ozel@medicine.ankara.edu.tr doi:10.5152/tjh.2010.03

Demiralp et al. Ankaferd® Blood Stopper

Turk J Hematol 2010; 27: 70-7

Özet Amaç: Kanama durdurucu olarak bilinen Ankaferd (AKD); Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum ve Urtica dioica bitki özlerinin özel bir karÕýÕmÕdÕr. AKD nin temel etki mekanizmasÕ, hayati eritrosit agregasyon odaklarÕ olan enkapsüle protein aúÕ formasyonunu saúlayarak gerçekleýmektedir. AKD kan hücreleri ve özellikle eritrositler ile oluýumunu indüklediúi protein aúÕ sayesinde birincil ve ikincil haemostatik sistem üzerine etkisini koagülasyon faktörlerini birebir hasarlamadan gerçekleýtirir. Yöntem ve Gereçler: ABS nin hemostaz üzerindeki etki mekanizmasÕnÕ anlamak için 2D jel elektroforez ve kütle spektrometre yöntemleri kullanÕlarak proteomik analizleri yapÕldÕ. Bulgular: Ankaferd kanama durdurucu içeriúinde tanÕmlanan bitkisel proteinler: NADP-baúÕmlÕ malik enzim, Ribuloz bisfosfatkarboksilaz büyük zinciri, MturazK, ATPsentaz altünitesi beta, ATPsentaz altünitesi-alfa, Chalcon flavonon isomeraz-1, Chalcon-flavonon izomeraz 2 ve Aktin-depolimerizasyon faktördür. AyrÕca Ankaferd kapsamÕnda koagülasyon için oldukça önemli farklÕ insan proteinleri benzerleri de tanÕmlanmÕýtÕr, bu proteinler arasÕnda; ATP sentaz, musin16 (CD164sialomucin-benzer-2 protein), helezonal kangal taýÕyan protein-141, hypotetik protein LOC283638 izoform 1, hypothetik protein LOC283638 izoform 2, dinaktin 5, Kompleks 1 intermadia iliýikli protein 30, mitokondrial protein, NADH dehidrogenaz (Ubiquinone) 1 alpha altkompleks, TP sentaz H+ taýÕyÕcÕ protein, mitokondrial aktin baúlayÕcÕ protein 1, LIM kangal ve aktin baúlayÕcÕ alt ünite 1 izoform a, LIM kangal ve aktin baúlayÕcÕ alt ünite 1 izoform b, Spectrin alpha non eritrotik 1, Prolactin releasing hormone reseptör, Utrophin, tet onkogen aile üyesi 2 izoform b, Protein fosfotaz 1 regulatory altünit 12A, NIMA -iliýkili kinaz, ATP-baúlayÕcÕ protein C12, malik enzim 1, Mitochondrial NADP(+) baúÕmlÕ malik enzim 3, ME2 protein, Nuclear faktör 1B tipi , Abihidrolaz kangal taýÕyÕcÕ protein 12B, E3 SUMO-protein ligaz PIAS2, Alpha-1,2glucosyltransferase ALG10-A, Cofilin, 18 kDa fosfoprotein, p18, Aktin-depolymerizing faktör, ADF, Twinfilin-1, Ankirin tekrarlayan ve FYVE kangalÕ içeren protein 1, Usherin öncü proteini, Urotensin II reseptör yer almaktadÕr. Sonuç: Proteomik analizler sonucu elde edilen proteinler Ankaferdin hemostatik, yara iyileýtirme ve anti-inflamatuvar etkilerinin araýtÕrÕlmasÕna ÕýÕk tutacak ve açÕklayÕcÕ olacak niteliktedir. (Turk J Hematol 2010; 27: 70-7) Anahtar kelimeler: Ankaferd, proteomiks, hemostaz, kanama Geliý tarihi: 23 Haziran 2009

Kabul tarihi: 19 KasÕm 2009

Introduction Ankaferd® Blood Stopper (ABS) is a standardized herbal extract obtained from five different plants: Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica [1]. Topical safety of ABS in normal healthy human volunteers was shown in a randomized double-blind cross-over Phase I clinical trial after preclinical animal studies [2-6]. ABS has been approved for local topical applications in external post-surgical and post-dental surgery bleedings in Turkey (www.Ankaferd.com). Ankaferd has also been used topically for the management of hemorrhages uncontrolled by standard measures in a wide variety of difficult clinical conditions [7-13]. Ankaferd represents its unique local hemostatic effect by promoting the very rapid (<1 s) formation of a protein network, which acts as an anchor for vital physiological erythrocyte aggregation, covering the classical cascade model of the clotting system without independently acting on coagulation factors and platelets [1,12]. Unique effects of critical proteins inside the ABS on critical transcription factors [14] and in vitro anti-infectious [15] and anti-cancer [16,17] effects suggested that Ankaferd may also affect pathobiological courses of tissues in addition to its unique action on hemostasis [18]. Functional proteomics is the essential step to identify the protein library of a given product, such as ABS hemostatic agent. The key characteristics of mass analyzers for proteomics are sensitivity, resolution and accuracy. Furthermore, demonstrated anti-infectious [15,19] and anti-neoplastic

[16,17] actions of ABS in vitro represent the basis to search the in vivo efficacy and safety of this herbal product for rational phytotherapy in upcoming researches. Therefore, the protein content of ABS should be searched to proceed with those investigations. The aim of this study was to assess functional proteomics of ABS via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses.

Materials and Methods Two-Dimensional (2D) Sample Preparation The protein samples for 2D gel electrophoresis were prepared as follows: 200 ml of Ankaferd solution was precipitated with trichloroacetic acid (TCA). 100 l of 100% TCA was added for each 1 ml of sample after the vortex and ice bath incubation for 15 min and centrifuge at 14,000 g for 10 min at room temperature. The pellet was washed with 1 ml of ice-cold 85% acetone, vortexed to disperse pellet, then spun at top speed for 5 min at room temperature. The pellet was dried in a Speed Vac for 10-20 min to remove residual solvent. The pellet was re-suspended in 300 l of 2D sample rehydration solution containing 7 M urea (Sigma, USA), 2M ThioUrea (Sigma, USA), 0.2% pH 3-10 linear IPG Ampholyte (Bio-Rad Laboratories, USA), 4% CHAPS (Sigma, USA), 1% HED (2-hydroxyethyldisulfide, Sigma, USA), and 1% DTT (Dithiothreitol, Sigma, USA). The total protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, USA).

Demiralp et al. AnkaferdÂŽ Blood Stopper

1 (0,172) 1296 6335 2240

100

Turk J Hematol 2010; 27: 70-7

1 TOF LD+ 2.24e3

1297 6206 2201

1298 6575 1722 1347 6903 1601 1570 7443 1511 1348 6584 1430

1571 7222 1157

% 1349 6631 903

1672 7548 698

1759 8162 884

1562 7098 388 1350 6700 301

1252 7061 224

0 1000

1100

1200

1300

1400

1760.7361 375

2465 1414 336

1573 7876 267

2464 1882 247

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

2486 2307 279

2500

2600

2700

2800

2900

Figure 1. Spectra acquired from external calibration peptide mix, which includes sub-P, angiotensin, renin, ACTH and glu fib 5857771 1177.1566

663.9321

688.1873

943.9321

1209.7771

0 500

1000

Figure 2. Spectra acquired from AnkaferdÂŽ Blood Stopper via MALDI-TOF

2000

m/z

Demiralp et al. Ankaferd® Blood Stopper

Turk J Hematol 2010; 27: 70-7

Mass Spectrometry and Database Search The tryptic peptides on the MALDI target plate were analyzed with MasLynx 4.0, MALDI-time of flight mass spectrometer (Waters, UK). Mass spectra were recorded in the positive-ion mode. All spectra were acquired with external calibration of sub-P, angiotensin, renin, ACTH and glu fib mix (Figure 1). PLGS (Waters, UK) was obtained with SwissProt database with 50 ppm sensitivity. Proteins were evaluated by considering the number of matched tryptic peptides, the percentage coverage of the entire protein sequence, the apparent molecular weight (MW), and the pI of the protein (Figures 2, 3).

Two-Dimensional (2D) Gel Electrophoresis of Proteins During the process, 150 g of total proteins were rehydrated in 300 l of rehydration buffer overnight (Bio-Rad Laboratories, USA). Samples were loaded on first dimension strips (17 cm, pI 3-10 Linear IPG). The first dimension of isoelectric focusing was performed using a Protein IEF cell (Bio-Rad Laboratories, USA). After running, first dimension gels were equilibrated for 10 min in equilibration buffer I (6 M urea, 0.375 M Tris pH 8.8, 2% SDS, 20% glycerol, 2% (W/V) DTT (Sigma, USA)), followed by 10 min in equilibration buffer II (6 M urea, 0.375 M Tris pH 8.8, 2% SDS, 20% glycerol, 2.5% (W/V) iodoacetamide). Samples were then separated by second dimension on 4-12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Gels were stained with Sypro Ruby protein stain, and images were acquired and analyzed using PDQuest software 8.0 (BioRad Laboratories, USA).

Results To identify the hemostatic protein contents of ABS herbal extract, functional proteomic analysis was performed to shed light on the effect mechanisms of ABS on hemostasis. Dissolution of proteins was achieved by 2D gel electrophoresis. ABS, 1st dimension according to separation pI point and 2nd dimension separations according to MW, was evaluated by making a protein profile map with the help of fluorescent staining (Sypro-Ruby). PDQuest program 8.0 (Bio-Rad, USA) was used to analyze the protein profiles, and with the 3-D view analysis of the separated protein spots, the artifacts were eliminated and with the help of Proteome works, the spots were resumed from the gels (Figure 4). Proteins were digested

Gel Digestion of Proteins Selected spots from the gel were excised using a Proteome Works Spot Cutter (Bio-Rad Laboratories, USA) and transferred to a 96-well plate. The proteins were enzymatically digested and the tryptic peptides ZipTip (Millipore, France) purified. After ZipTip purification, the tryptic peptides were eluted from the ZipTip with 3 mg/100 l cyano-4-hydroxycinnamic acid (CHCA) solution in 50% acetonitrile (ACN)/0.1% TriFlora acetic acid and spotted directly onto wax-coated MALDI target plates. 100

1432.1871

1432.7771

1063.9321

1537.1871

1538.1873

1216.7771 1569.1425 1421.0839 1195.7712

2012.1566

1594.1566 1697.2821

1420.1478 1364.9240

2439.1425 1754.2665 1938.1987

0 1000

1100

1200

1300

1400

1500

1600

1700

Figure 3. Spectra of MATK-VITVI acquired via MALDI-TOF

1800

1900

2000

2100

2200

2300

2400

2500

2600

2700

2800

2900

m/z

Demiralp et al. Ankaferd® Blood Stopper

Turk J Hematol 2010; 27: 70-7

to peptides for mass spectrometer reflectron mode analysis in the range of 500-10.000 daltons. In the protein description, MALDI-TOF mass spectrometry, which describes m/z values by calculating flight time by matrixbased laser-dependent ionization, was preferred. The ABS protein peptides were analyzed in positive ion mode with external calibration and with 50ppm sensitivity by MassLynx4.0 (Waters, UK). Moreover, the spectra data were identified with PLGS (Waters, UK) program using UniProt/ Swiss-Prot data banks. Peptide equalizations were re-checked and evaluated in accordance with MW and pI with 2D gel electrophoresis analyses. Furthermore, the herbal proteins and human-proteinlike proteins were determined in ABS with the PLGS 5-7 PLGS score and 89-207 Mascot score. Regarding the herbal analyses, the herbal proteins identified in ABS (Table 1) via 2D gel electrophoresis and mass spectrometer analysis with MALDI-TOF are: • NADP-dependent malic enzyme • Ribulose bisphosphate-carboxylase large chain • Maturase K • ATP synthase beta subunit • ATP synthase alpha subunit • Chalcone flavanone isomerase-1 • Chalcone flavanone isomerase 2 • Actin-depolymerization factor (ADF) In addition, various human-protein-like proteins that are considerably important for hemostasis were also identified within ABS (Table 2). It was interesting to see that in addition to the herbal proteins, some human-like proteins that could represent many crossroads of hemostasis, infection, and neoplasia were found within ABS with the proteomic analysis.

Discussion In this study, the protein library of Ankaferd has been evaluated via MALDI-TOF analyses. Topical hemostatic efficacy of ABS has been previously tested in animals with normal [2,5] and defective hemostasis [3,6]. Short-term hematological and biochemical safety of the oral systemic administration of Ankaferd to rabbits has been shown [20]. No acute mucosal toxicity, hematotoxicity, hepatotoxicity, nephrotoxicity, and biochemical toxicity was observed during the short-term follow-up of the animals [20]. Those preclinical results reflect a starting point to search any possible systemic confounding effect of ABS when applied to internal topical surfaces. Physiological cell-based coagulation could be clinically managed via topical ABS application to prevent and treat bleeding in many distinct clinicopathological states [7-13]. Ankaferd-induced formation of the protein network with vital erythroid aggregation covers the entire physiological hemostatic process. Mainly, there are distinct important components of the Ankaferd-induced hemostatic network. Vital erythroid aggregation takes place with the spectrin ankyrin and actin proteins on the membrane of red blood cells. Based on the results of our study, essential erythroid proteins (ankyrin recurrent and FYVE bundle containing protein 1, spectrin alpha, actin-depolymerization factor, actin-depolymerizing factor, LIM bundle and actin binding subunit 1 isoform a, LIM bundle and actin binding subunit 1 isoform b, NADP-dependent malic enzyme, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, mitochondrial NADP (+) dependent malic enzyme 3, ribulose bisphosphatecarboxylase large chain, maturase K) and the required ATP bioenergy (ATP synthase, ATP synthase beta subunit, ATP synthase alpha subunit, ATP-binding protein C12, TP synthase H+ transporter protein, ADF, and alpha-1,2-

Table 1. Plant proteins inside the Ankaferd® Description

Accession No

Theoretical

Measured

Uni ProtKB/Swiss-Prot

pl/Mr

NADP-dependent malic enzyme

P51615

6/65.18

6.5/75

Rebulose bisphosphatem carboxylase large chain

Q37010

Undef/undef

9.0/100

Mt urase K

Q8HV93

9/62

7.0/55

ATP synthase subunit beta

Q0ZJ13

5.5/53

6.5/50

ATP synthase subunit alpha

Q0ZJ35

5.26/55,5

6.7/53

Chalcone-flavonone isomerase 1

P51117

5.25/25

6.5/35

Actin-depolymerzing factor

Q8SAG3

6.91/16.5

7/12

Figure 4. 3 Dimensional view of protein spots

Demiralp et al. AnkaferdÂŽ Blood Stopper

Turk J Hematol 2010; 27: 70-7

Table 2. Proteins resembling human peptides in AnkaferdÂŽ protein library TP synthase, H+ Transporting mitachondrial

A8K0K3

pl: 9.65/11400

8.00/25.000

acting binding 1 isoform

Q9UPN3

pl: 5.27/620.000

5.5/220.000

LIM domain and actin binding 1 isoform a

6.5/50.000

LIM domain and actin binding 1 isoform b

6.5/50.000

Spactirin alpha non erythrocytic 1

Q13813

pl: 5.22/28.400

6.5/40.000

Prolactin releasing hormone receptor

A5JUU5

pl: 9.43/41.100

9.00/40.000

Utroptin

P46939

pl: 5.21/40.000

6.5/40.000

tet oncogene family member 2 isoform b Protein phosphatase 1 regulatory subunit 12A

6.5/50.000 O14974

pl: 5.31/115.200

6.5/120.000

ATP binding cassette protein C12

Q96J65

pl: 8.64/152.000

7.00/150.000

highly similar to Homo sapiens malic enzyme 1

A8K168

pl: 5.79/64.100

6.5/75.000

Mitachondrial NADP (+) - dependent malic enzyme 3

Q6TCH8

pl: 6.31/29.700

6.5/30.000

Me2 protein

Q9BWL6

pl: 8.80/53.500

8.00/50.000

O00712

pl: 9.01/47.400

9.00/50.000

Abhydrolase domain-containing protein 12 B

Q7Z5M8

pl: 8.57/40.000

8.5/50.000

E3 SUMO-protein ligase PIAS2

O75928

pl: 7.47/68.200

7.5/75.000

Alpha- 1,2-glucosyltransferase ALG10-A

Q5BKT4

pl: 9.40/55.600

9.5/70.000

P23528

pl: 8.26/18.500

8.5/35.000

P60981

pl: 8.12/18.500

8.00/30.000

Q12792

pl: 5.7/128.200

6,5/120.000

Ankyrin repeat and repeat and FYVE domain containing protein 1

Q9P2R3

pl: 6.47/42.201

7.00/50.000

Usherin [Precursor]

O75445

pl: 6.42/572.200

6.5/220.000

Urotensin II receptor

Q9UKP6

pl: 10.6/42.100

9.57/50.000

NIMA never in mitosis gene a)- relatet kinas

Nuclear factor 1 B-type

Cofilin, non-muscle isoform 18 kDa phosphoprotein p18 Actin-depolymerizing factor ADF Twinfilin-1

IL-4 receptor IL-4ligand Midkine

glycosyltransferase ALG10-A) are included in the protein library of Ankaferd. Ankaferd also upregulates GATA/FOG transcription system affecting erythroid functions and urotensin II [14,18]. Urotensin II is also an essential component of Ankaferd and represents the link between injured vascular endothelium, adhesive proteins and active erythroid cells. Those concepts could be developed via MALDI-TOF proteomic molecular analyses, cytometric arrays, transcription analysis, and Scanning electron microscope (SEM) ultrastructural examinations as well as upcoming investigations interacting with basic and clinical research facilities. Ankaferd, in addition to its hemostatic activity, may also inhibit the growth of bacteria [19]. The anti-infectious activity of Ankaferd may represent an advantage over its current clinical use, since it inhibits the growth of bacteria in the area

used mainly for its hemostatic activity, such as in traumatic infected wounds. The antimicrobial activity of Ankaferd was tested against many pathogens [15]. The isolates included Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter spp., Stenotrophomonas maltophilia, methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase negative Staphylococcus, vancomycin-susceptible Enterococcus, and vancomycin-resistant enterococci (VRE). Antibacterial activities of Ankaferd against several gram-positive and gram-negative food and human pathogens were also reported in another study [19]. The mechanism of action regarding the anti-infective activity of ABS is currently unknown. Several proteins (Homo sapiens malic enzyme 1, dynactin 5, cofilin, utrophin, mucin 16 (CD164-sialomucin-

Demiralp et al. Ankaferd® Blood Stopper

like-2 protein), chalcone flavanone isomerase-1, chalcone flavanone isomerase 2, helezonal bundle transporter protein-141, hypothetical protein LOC283638 isoform 1, hypothetical protein LOC283638 isoform 2, complex I intermediate-related protein 30) in our proteomic analyses represent an important step to elucidate how Ankaferd biologically affects the components of numerous pathogens. Several hypotheses will need to be raised to understand the mechanism-of-action of Ankaferd on tumor tissue [16,17]. ABS is a hemostatic agent with pleiotropic effects [14,18,21,22]. The unique protein library of Ankaferd upregulates critical transcription factors including regulators of neoplasia such as p53 [14,18]. There is a close relationship between coagulation factor expressions and solid tumor progression, via mechanisms other than angiogenesis. Several proteins (midkine, interleukin 4, p18, 18 kDa phosphoprotein, SUMO-protein ligase PIAS2, abhydrolase bundle transporter protein, the precursor of usher, tet oncogene family member 2 isoform b, twinfilin-1, SUMOprotein ligase PIAS2, prolactin secretor hormone receptor, protein phosphatase 1 regulatory subunit 12A, never in mitosis gene a (NIMA)-related kinase, mitochondrial protein, mitochondrial actin binding protein 1) in our proteomic analyses represent an important step to elucidate how Ankaferd regulates cell cycle and other biological actions of the tumor tissue. The pleiotropic effects of ABS on vascular endothelium, blood cells, angiogenesis, cellular proliferation, vascular dynamics, and cellular mediators should be investigated to determine its potential role in many pathological states, including neoplastic disorders, infectious diseases and inflammation. Our proteomic results in this report within many crossroads of hemostasis, infection and neoplasia may shed further light and represent a novel starting point on that perspective for the new avenues of ABS.

Acknowledgements There are no conflicts of interest to report. Ankaferd Blood Stopper vials were supplied from Ankaferd Drug Inc., İstanbul, Turkey. We would like to thank Cagri Gumustekin from Ankara University Biotechnology Institute Proteomics Unit for technical support. This study was supported in part by Ankara University Biotechnology Institute, Proteomics project.

References 1.

Goker H, Haznedaroglu IC, Ercetin S, Kirazli S, Akman U, Ozturk Y, Firat HC: Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper. J Int Med Res 2008;36:163-70. A double-blinded, randomized, placebo-controlled, cross-over phase I clinical study of Ankaferd® Blood Stopper. In: Firat HC, Ozdemir O, Kosar A, Goker H, Haznedaroglu IC, editors. Annual Review of Ankaferd 08-09. Istanbul: Naviga Publications, 2009:64-72. Bilgili H, Kosar A, Kurt M, Onal IK, Goker H, Captug O, Shorbagi A, Turgut M, Kekilli M, Kurt OK, Kirazli S, Aksu S, Haznedaroglu

Turk J Hematol 2010; 27: 70-7

10.

11.

12.

13.

14.

15.

16.

17.

IC: Hemostatic efficacy of Ankaferd Blood Stopper (r) in a swine bleeding model. Med Prin Pract 2009;18:165-9. Cipil H, Kosar A, Kaya A, Uz B, Haznedaroglu IC, Goker H, Ozdemir O, Koroglu M, Kirazli S, Firat H. In vivo hemostatic effect of the medicinal plant extract Ankaferd Blood Stopper in rats pretreated with warfarin. Clin Appl Thromb Hemost 2009;15:270-6. Huri E, Akgul T, Ayyildiz A, Ustun H, Germiyanoglu C. Hemostatic role of a folkloric medicinal plant extract in a rat partial nephrectomy model: controlled experimental trial. J Urol 2009;181:2349-54. Karakaya K, Ucan HB, Tascilar O, Emre AU, Cakmak GK, Irkorucu O, Ankarali H, Comert M. Evaluation of a new hemostatic agent Ankaferd Blood Stopper in experimental liver laceration. J Invest Surg 2009;22:201-6. Kosar A, Cipil HS, Kaya A, Uz B, Haznedaroglu IC, Goker H, Ozdemir O, Ercetin S, Kirazli S, Firat HC. The efficacy of Ankaferd Blood Stopper in antithrombotic drug-induced primary and secondary hemostatic abnormalities of a rat-bleeding model. Blood Coagul Fibrin 2009;20:185-90 Arslan S, Haznedaroglu IC, Oz B, Goker H. Endobronchial application of Ankaferd Blood Stopper to control profuse lung bleeding leading to hypoxemia and hemodynamic instability. Resp Med 2009;2:144-6. Dogan OF, Ozyurda U, Uymaz OK, Ercetin S, Haznedaroglu I. New anticoagulant agent for CABG surgery. Eur J Clin Invest 2008;38:341. Ibis M, Kurt M, Onal IK, Haznedaroglu IC. Successful management of bleeding due to solitary rectal ulcer via topical application of Ankaferd Blood Stopper. J Altern Complement Med 2008;14:1073-4. Kurt M, Disibeyaz S, Akdogan M, Sasmaz N, Aksu S, Haznedaroglu IC. Endoscopic application of Ankaferd Blood Stopper as a novel experimental treatment modality for upper gastrointestinal bleeding: a case report. Am J Gastroenterol 2008;103:2156-8. Kurt M, Kacar S, Onal IK, Akdogan M, Haznedaroglu IC. Ankaferd blood stopper as an effective adjunctive hemostatic agent for the management of life-threatening arterial bleeding of the digestive tract. Endoscopy 2008;40:E262. Ucar Albayrak C, Caliskan U, Haznedaroglu IC, Goker H. Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper [letter and response]. J Int Med Res 2008;36:1447-9. Koray M, Ergun S, Saruhanoglu A, Tanyeri H. Use of a new local haemostatic agent Ankaferd Blood Stopper after surgical excision of eruption cyst: a case report. Int J Oral Maxillofacial Surg 2009;38:558. YÕlmaz E, Gulec S, Haznedaroglu IC, Akar N. Effects of Ankaferd on huvec transcription factors and erythrocyte protein profile. In: Haznedaroglu IC, Goker H, Ozdemir O, Kosar A, Firat H, editors. Ankaferd: Scientific Perspectives and Basic-Clinical Data. Istanbul: Naviga Publications, 2008:60. Fisgin NT, Cayci YT, Coban AY, Ozatli D, Tanyel E, Durupinar B, Tulek. Antimicrobial activity of plant extract Ankaferd Blood Stopper (r). Fitoterapia 2009;80:48-50. Goker H, Cetinkaya D, Kilic E, Haznedaroglu IC, Kirazli S, Firat H. Anti-cancer activity of Ankaferd Blood Stopper on osteosarcoma (SAOS-2) cell lines in vitro. In: Haznedaroglu IC,

Demiralp et al. Ankaferd® Blood Stopper

Turk J Hematol 2010; 27: 70-7

18.

19.

Goker H, Ozdemir O, Kosar A, Firat H, editors. Ankaferd: Scientific Perspectives and Basic Clinical Data. Istanbul: Naviga Publications, 2008:109. Goker H, Kilic E, Cetinkaya D, Buyukasik Y, Aksu S, Turgut M, Haznedaroglu I. Anti-cancer activity of Ankaferd on human colon cancer (CACO-2) in vitro. In: Haznedaroglu IC, Goker H, Ozdemir O, Kosar A, Firat H, editors. Ankaferd: Scientific Perspectives and Basic Clinical Data. Istanbul: Naviga Publications, 2008:108. Haznedaroglu IC. Molecular basis of the pleiotropic effects of Ankaferd Blood Stopper. IUBMB Life 2009; 61: 290.

20.

21.

22.

Akkoc N, Akcelik M, Haznedaroglu IC, Goker H, Turgut M, Aksu S, Kirazli S, Firat HC. In vitro anti-bacterial activities of Ankaferd medicinal plant extract. Turkiye Klinikleri Tip Bilimleri Dergisi 2009; 29: 410-5. Bilgili H, Captug O, Kosar A, Kurt M, Kekilli M, Shorbagi A, Kurt OK, Ozdemir O, Goker H, Haznedaroglu I: Oral systemic administration of Ankaferd Blood Stopper has no short-term toxicity in an “in vivo” rabbit experimental model. Clin Appl Thromb Hemost 2009 [in press]. Aydin S. Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper (r). J Int Med Res 2009; 37: 279.

Research Article

Cytotoxic T lymphocyte antigen-4 (CTLA-4) A49G polymorphism and autoimmune blood diseases Sitotoksik T lenfosit antijen-4 (CTLA-4) A49G polimorfizmi ve otoimmün kan hastalÕklarÕ Faruk Aktürk1, Veysel Sabri Hançer2, Reyhan Küçükkaya3 1Division

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

Abstract Objective: The cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is expressed on T lymphocytes, and inhibits the T-cell responses. In animal models, it has been shown that complete CTLA-4 deficiency was lethal due to massive infiltration of tissues by polyclonally proliferating lymphocytes. CTLA-4 A49G polymorphism, which has been suggested to reduce the inhibitory function of the CTLA-4 molecule, was found to be associated with various autoimmune diseases in recent studies. Material and Methods: In this study, we evaluated the frequency of CTLA-4 A49G polymorphism in 46 patients with autoimmune hemolytic anemia (AIHA), 62 patients with immune thrombocytopenic purpura (ITP), and 150 healthy individuals. Results: Allele frequencies and genotype distributions were similar in both ITP and AIHA patients compared to healthy individuals. In subgroup analysis, however, we found that in chronic lymphocytic leukemia (CLL) patients with AIHA (n=4), all patients had CTLA-4 A49G polymorphism (3 had AG, 1 had GG). There was no significant statistical association between G allele and systemic lupus erythematosus (SLE) or AIHA. Conclusion: These data suggest that CTLA-4 A49G polymorphism does not contribute to the pathogenesis of lymphoproliferative diseases itself, nor does it increase the risk of autoimmune complications in patients with lymphoproliferative disease. (Turk J Hematol 2010; 27: 78-81) Key words: Cytotoxic T lymphocyte antigen-4 (CTLA-4), A49G polymorphism, autoimmunity, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, chronic lymphocytic leukemia Received: December 26, 2008

Accepted: February 22, 2010

Özet Amaç: Sitotoksik T lenfosit antijen-4 (CTLA-4) ifadesi T lenfositlerde gerçekleýir ve T hücre cevabÕnÕ engeller. Hayvan modellerinde CTLA-4 yokluúunun, dokularÕn poliklonal çoúalan lenfositler tarafÕndan yoúun infiltrasyonu nedeni ile, ölümcül olduúu gösterilmiýtir. FarklÕ otoimmün hastalÕklar ile iliýkilendirilmiý CTLA-4 A49G polimorfizminin, CTLA-4 molekülünün inhibitör fonksiyonunu azalttÕúÕ düýünülmektedir. Yöntem ve Gereçler: Bu çalÕýmada otoimmun hemolitik anemi (OIHA) hastasÕ 46 kiýi, immun trombositopenik purpura (ITP) hastasÕ 62 kiýi ve 150 saúlÕklÕ kontrol bireyinde CTLA-4 A49G polimorfizmi çalÕýÕlmÕýtÕr. Address for Correspondence: Asst. Prof. Dr. Veysel Sabri Hançer, Istanbul Bilim University, Faculty of Medicine, Department of Medical Biology and Genetics, Esentepe, Istanbul, Turkey Phone: +90 533 634 30 14 E-mail: vshancer@istanbul.edu.tr doi:10.5152/tjh.2010.04

Aktürk et al. A49G polymorphism and autoimmune blood diseases

Turk J Hematol 2010; 27: 78-81

Bulgular: ITP ve OIHA hastalarÕndan oluýan iki grupta da saúlÕklÕ kontrol bireyleri ile karýÕlaýtÕrÕldÕúÕnda benzer allel frekanslarÕ ve genotip daúÕlÕmlarÕ saptanmÕýtÕr. Alt grup analizi gerçekleýtirildiúinde ise hem OIHA hem de KLL hastalÕúÕna sahip 4 bireyin hepsinin polimorfizme sahip olduúu gösterilmiýtir (3 AG, 1 GG). Risk alleli olan G OIHA, ûTP ve kontrol grubunda incelendiúinde istatistik olarak anlamlÕ bir farklÕlÕk saptanmamÕýtÕr. Sonuç: Bu verilerin ÕýÕúÕnda CTLA-4 A49G polimorfizminin lenfoproliferatif hastalÕklarÕn patogenezine bir katkÕsÕnÕn olmadÕúÕ veya lenfoproliferatif hastalÕúa sahip bireylerde otoimmün komplikasyonlarÕn geliýmesi açÕsÕndan risk oluýturmadÕúÕ sonucuna varÕlmÕýtÕr. (Turk J Hematol 2009; 27: 78-81) Anahtar kelimeler: Sitotoksik T lenfosit antijen-4 (CTLA-4), A49G polimorfizmi, otoimmünite, idiopatik trombositopenik purpura, otoimmün hemolitik anemi, kronik lenfositik lösemi Geliý tarihi: 26 AralÕk 2008

Kabul tarihi: 22 üubat 2010

Introduction Autoimmune disorders are the result of disturbed immune tolerance to self-antigens. After the presentation of an antigen in conjunction with HLA molecules on the surface of an antigen-presenting cell to a specific T-cell receptor (TCR), costimulatory signals are required for the T-cell responses [1]. A number of co-stimulatory molecules are present on the T-cell membrane, which may have stimulatory or inhibitory effects on T lymphocytes. The cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is expressed on T lymphocytes and inhibits the T-cell responses [2]. Mechanisms of down-regulation of T-cell immunogenic response by CTLA-4 may be due to competitive antagonism with CD28, increment in TCR stimulation threshold, and limitation of T-cell division capacity and activation of cell cycle arrest of T-cells after antigenic stimulation [2]. In animal models, it has been shown that complete CTLA-4 deficiency was lethal due to severe lymphoproliferative disorders with massive infiltration of tissues by polyclonally proliferating lymphocytes [3]. Blocking the CTLA-4/B7 interaction by monoclonal antibodies results in an increase in antigen-specific T-cell proliferation, whereas enhancing the inhibitory function of CTLA-4 suppresses T-cell responses [1,2]. These findings provide strong evidence that CTLA-4 has a crucial role in the negative regulation of T-cell responses. The CTLA-4 gene is located on chromosome 2 (2q33). Several polymorphisms were identified on the CTLA-4 gene [4]. The A49G polymorphism in exon 1 of the CTLA-4 gene is especially important because it alters the structure of the CTLA-4 protein by causing Thr17Ala amino acid substitution [5]. It has been suggested that this polymorphism reduces the inhibitory function of CTLA-4 [2]. In recent studies, the CTLA-4 A49G polymorphism was found to be associated with autoimmune diseases such as insulin-dependent diabetes, rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, primary biliary cirrhosis, Hashimoto’s thyroiditis, and Graves’ disease [1,2,4,6]. In this study, we evaluated the frequency of the CTLA-4 A49G polymorphism in patients with autoimmune hemolytic anemia (AIHA) and autoimmune thrombocytopenic purpura (ITP).

Materials and Methods Participants: The present study was approved by the local ethics committee (Istanbul Faculty of Medicine) and all

participants gave informed consent. Patients were selected from cases admitted to the Hematology Division of the Istanbul Faculty of Medicine from May 2003 to June 2006. One hundred and fifty healthy Turkish subjects, 46 patients with AIHA and 62 patients with ITP were included in our study. ITP and AIHA patients were diagnosed by standard methods and were being followed by the Hematology Department. In the AIHA group, median age was 53 (28-66) and male to female ratio was 13/33. Fourteen AIHA patients also had SLE, 4 had chronic lymphocytic leukemia (CLL), 2 had Evans’s syndrome, and 2 had autoimmune diseases other than SLE (autoimmune thyroiditis and autoimmune hepatitis). In the ITP group (n=62), median age was 41 (18-56) and male to female ratio was 17/45. Thirty-six patients had primary ITP. Twenty-six patients in the ITP group had antiphospholipid antibodies (aPLA), and 12 of those patients were diagnosed as definite antiphospholipid syndrome (with thrombosis and/or pregnancy morbidity). Fourteen ITP patients had only persistent aPLA-positivity. In the healthy control group (n=150), median age was 38 years (18-55), and male to female ratio was 67/83. Methods: Genomic DNA was isolated from peripheral venous blood samples using high pure polymerase chain reaction (PCR) template preparation kit (Roche Diagnostics). For the amplification of exon 1 to show CTLA-4 A49G polymorphism, 5’-GCTCTACTCCTGAAGACCT-3’ and 5’-AGTCTCACTCACTCACTTTGCAG-3’ primers were used. Amplified DNA samples were digested by Bbv I restriction endonuclease enzyme (New England Biolabs, Beverly, MA). Amplified fragment (162 bp) analyzed on 3.5% agarose gel was stained by ethidium bromide. The allele A was not cut by the enzyme and so left 162 bp; on the contrary, G allele was cut into two DNA fragments 88 and 74 bp long [4]. Statistical Analysis: Statistical analysis was performed using chi-square and Fisher’s exact tests at ‘Graphpad for Windows Version 3.0’. P values <0.05 were accepted as statistically significant. Results of the statistical analysis of comparisons of the allele frequencies are shown in Table 1.

Results Healthy Controls: One hundred and fifty healthy subjects were investigated in terms of CTLA-4 A49G polymorphism.

AktĂźrk et al. A49G polymorphism and autoimmune blood diseases

Turk J Hematol 2010; 27: 78-81

Table 1. Patient and control genotype and allele frequencies for the CTLA-4 A49G polymorphism n

Allele A

Allele G

p (vs *)

95% CI

Total AIHA

0.643

0.85

0.517-1.425

Primary AIHA

0.874

1.08

0.568-2.055

SLE+AIHA

0.660

0.83

0.363-1.918

CLL+AIHA

0.049

0.23

0.055-1.015

Total ITP

0.183

1.42

0.865-2.333

Primary ITP

0,1004

1.79

0.935-3.441

aPLA+ ITP

1.000

1.07

0.553-2.081

Healthy individuals*

150

215

AIHA: Autoimmune hemolytic anemia; SLE: Systemic lupus erythematosus; CLL: Chronic lymphocytic leukemia; ITP: Immune thrombocytopenic purpura; aPLA: Antiphospholipid antibodies; OR: Odds ratio; CI: Confidence interval

Genetic variation was distributed according to Hardy-Weinberg equilibrium. Genotype frequencies were 48% for AA, 47.3% for AG and 4.7% for GG. A-allele frequency was 71.6% and G-allele frequency was 28.4% (Table 1). There was no statistical difference between genders and allele frequencies in the healthy control group. AIHA Group: Twenty-one patients had AA (45.6%), 21 had AG (45.6%), and 4 had GG (8.8%) genotype in this group. A-allele frequency was 68.4% and G-allele frequency was 31.6%, which was similar to healthy controls. In sub-group analysis, in a small group of patients with CLL and AIHA (n=4), all patients were found to have CTLA-4 A49G polymorphism (3 had AG, 1 had GG) (Table 1). When we statistically compared the risk allele G in the AIHA and control groups, there was no statistical difference (p=0.643; odds ratio [OR]: 0.858; 95% confidence interval [CI]: 0.8658-2.33). In sub-group analysis, we found no difference between patients and controls for the risk allele G (Table 1). ITP Group: Thirty-eight had AA (61.3%), 21 had AG (33.9%), and 3 had GG (4.8%) genotype in this group. A-allele frequency was 78.4% and G-allele frequency was 21.6%. There was no statistical difference between the ITP group and healthy control group for the risk allele. In sub-group analysis, we similarly found no difference between ITP patients and controls for the risk allele G.

Discussion The role of the CTLA-4 A49G polymorphism in the development of autoimmune diseases is not well understood. Since CTLA-4 controls the amplitude of immune response and peripheral tolerance, this polymorphism could have an impact on autoimmunity in some way. The CTLA-4 A49G polymorphism may result in susceptibility to autoimmune disease [1,2,6,7]. The role of the CTLA-4 A49G polymorphism in the development of autoimmune blood diseases such as ITP and AIHA is not clear. In the only published study, which was done by Pavkovic et al. [5], it was shown that the CTLA-4 A49G polymorphism was significantly higher in patients with AIHA, but no association was found in patients with ITP. They also showed

that the G-allele frequency was highest among CLL patients who had developed AIHA. In that study, 60% of the AIHA cases were patients with CLL. Our study is the second study investigating the role of the CTLA-4 A49G polymorphism in patients with ITP and AIHA. Although we could not demonstrate an association between autoimmune blood diseases and the CTLA-4 A49G polymorphism, we found very interesting results in the CLL+AIHA subgroup, as in Pavkovicâ&#x20AC;&#x2122;s study. In our study, all CLL patients who had AIHA (n=4) were found to have G allele (3 had AG, 1 had GG). With the knowledge of the development of fatal lymphoproliferative disease in CTLA-4-deficient mice, these data suggest that the CTLA-4 A49G polymorphism may contribute to the pathogenesis of the lymphoproliferative diseases itself, or may increase the risk of autoimmune complications in patients with lymphoproliferative diseases. In a metaanalysis study, Lee et al. [6] demonstrated a significant statistical association between the A49G polymorphism and SLE susceptibility. Their meta-analysis did not show a significant association of other polymorphisms of CTLA-4 with SLE. Though the estimated ORs from the race-specific analysis were very similar, a statistically significant SLE association was found with Asians only (n=522). Similarly, in our study, there was no association between the A49G polymorphism and SLE in the Caucasian population based on this meta-analysis. When we statistically compared groups, there was no association between the AIHA and control group. In contrast to Lee et al. [6], in the present study, our findings demonstrated that there was no significant statistical association between G allele and SLE. Similar findings were seen for ITP (Table 1). In conclusion, our results showed that there is no association between the A49G polymorphism of the CTLA-4 gene and AIHA or ITP disease. Further genetic and clinical studies are required to understand the exact role of the CTLA-4 A49G polymorphism in the development of autoimmune and lymphoproliferative diseases. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

AktĂźrk et al. A49G polymorphism and autoimmune blood diseases

Turk J Hematol 2010; 27: 78-81

References 1.

2. 3.

Chambers CA, Kuhns MS, Egen JG, Allison JP. CTLA-4 mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu Rev Immunol 2001;19:565-94. Teft WA, Kirchhof MG, Madrenas J. A molecular perspective of CTLA-4 function. Annu Rev Immunol 2006;24:65-97. Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H, Mak TW. Lymphoproliferative disorders with early lethality in mice deficient in CTLA-4. Science 1995;270:985-8.

Sahin M, Erdogan MF, Erdogan G. Cytotoxic T lymphocyteassociated molecule-4 polymorphisms in Turkish Gravesâ&#x20AC;&#x2122; disease patients and association with probability of remission after antithyroid therapy. Eur J Intern Med 2005;16:352-5. Pavkovic M, Georgievski B, Cevreska L, Spiroski M, Efremov DG. CTLA-4 exon 1 polymorphism in patients with autoimmune blood disorders. Am J Hematol 2003;72:147-9. Lee YH, Harley JB, Nath SK. CTLA-4 polymorphisms and systemic lupus erythematosus (SLE): a meta-analysis. Hum Genet 2005;116:361-7. Dallos T, Kovacs L. CTLA-4 and the genetic predisposition on autoimmunity. Bratisl Lek Listy 2005;106:55-62.

Research Article

Enhanced platelet adhesion in essential thrombocythemia after in vitro activation In vitro aktivasyon sonrasÕ esansiyel trombositemide yüksek platelet adezyonu Andreas C. Eriksson1, Kourosh Lotfi2, Per A. Whiss1 1Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Linköping University, Sweden and Department of Hematology, University Hospital, Linköping, Sweden 2Division of Drug Research/Clinical Pharmacology, Department of Medical and Health Sciences, Linköping University, Sweden

Abstract Objective: Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by elevated platelet counts and increased risk of thrombosis. Ex vivo data suggest increased platelet reactivity in agreement with the increased thrombosis risk, while in vitro tests often detect decreased platelet activity. The present study aimed to investigate adhesion of ET-platelets in vitro, which is an aspect of platelet function that has been addressed in only a few studies on ET patients. Material and Methods: The study included 30 ET patients and 14 healthy controls. Platelet adhesion was measured with a static platelet adhesion assay. Results: The main finding was that ET-platelets were more readily activated by adhesion-inducing stimuli in vitro than control platelets. This was particularly evident in elderly patients and when using multiple stimuli, such as surfaces of collagen or fibrinogen combined with addition of adenosine 5’-diphosphate or ristocetin. Such multiple stimuli resulted in adhesion above the control mean +2 standard deviations for approximately 50% of the patients. Conclusion: The results are in accordance with the concept of increased platelet activity in ET, but opposite to most other in vitro studies. We suggest that the conditions in the adhesion assay might mimic the in vivo situation regarding the presence of chronic platelet activation. (Turk J Hematol 2010; 27: 82-90) Key words: Essential thrombocythemia, platelet activation, adhesion, thrombosis, platelet assay Received: December 4, 2009

Accepted: February 22, 2010

Özet Amaç: Esansiyel trombositemi (ET) platelet sayÕsÕnÕn artmasÕ ve yüksek tromboz riski ile karakterize kronik bir myeloproliferatif bozukluktur. Ex vivo veriler tromboz riskine uygun olarak artan platelet reaktivitesini öne sürerken in vitro testler sÕklÕkla platelet aktivitesinde azalma tespit etmektedir. Bu çalÕýmanÕn amacÕ ET-hastalarÕnda az sayÕda çalÕýmaya dahil edilmiý bir platelet fonksiyonu konusu olan ET-plateletleri adezyonunun in vitro incelenmesidir. Yöntem ve Gereçler: ÇalÕýmaya 30 ET hastasÕ ile 14 saúlÕklÕ kontrol dahil edilmiýtir. Statik platelet adezyonu tayini ile platelet adezyonu ölçülmüýtür. Bulgular: Temel bulgu ET plateletlerinin, in vitro adezyon indüklenmiý uyaranlar ile kontrol plateletlerinden daha kolay aktive olduúu olmuýtur. Bu durum özellikle yaýlÕ hastalarda ve adenosin 5-difosfat ya da ristosetin eklenerek kombine edilmiý kolajen ya da fibrinojen yüzeyler gibi çoklu uyaran kullanÕldÕúÕnda barizdir. Bu gibi çoklu uyaran hastalarÕn yaklaýÕk %50’sinde kontrol deúeri + 2 standart sapmanÕn üzerinde adezyon sonucunu vermiýtir.

Address for Correspondence: Andreas C. Eriksson, Division of Drug Research/Pharmacology, Department of Medical and Health Sciences, Linköping University, SE-581 85 Linköping, Sweden Phone: +46 10 1038071 Fax: +46 13 149106 E-mail: andreas.eriksson@liu.se doi:10.5152/tjh.2010.05

Eriksson et al. Enhanced platelet adhesion in essential thrombocythemia

Turk J Hematol 2010; 27: 82-90

Sonuç: Bulgular ET’de artan platelet aktivitesi konseptine uygun olsa da diúer in vitro çalÕýmalarÕn tersinedir. Adezyon tayininde koýullarÕn kronik platelet aktivasyonu varlÕúÕ ile ilgili olarak in vitro durumu taklit edebileceúini ileri sürüyoruz. (Turk J Hematol 2010; 27: 82-90) Anahtar kelimeler: Esansiyel trombositemi, platelet aktivasyonu, adezyon, tromboz, platelet tayini Geliý tarihi: 4 AralÕk 2009

Kabul tarihi: 12 üubat 2010

Financial support: The study was financed by grants from the County Council of Östergötland (2004/049 and LIO-5311). During the course of the research underlying this study, Andreas C. Eriksson was enrolled in Forum Scientium, a multidisciplinary doctoral program at Linköping University, Sweden.

Introduction Essential thrombocythemia (ET) is classified as a myeloproliferative neoplasm (MPN) [1] and affects about 1.5 middle aged to elderly individuals per 100,000 annually [2]. The disease is characterized by increased platelet counts and the occurrence of both thrombosis and bleeding [3]. Bleeding events are more common at very high platelet counts [4], probably as a consequence of the development of an acquired von Willebrand disease [5-7]. The most common complication is, however, thrombosis [3], which most often occurs in patients older than 60 years of age and in those with previous thrombosis [8]. Furthermore, a single point mutation in the JAK2 gene (Val617Phe) has been described [9-11], and is estimated to be present in about 60% of patients with ET [12]. The impact

of this mutation on the risk of thrombotic complications is still controversial [13,14]. Treatment alternatives in ET include the use of cytotoxic drugs to lower platelet number and/or use of the platelet inhibitor acetylsalicylic acid (ASA) [15]. Guidelines for the use of these drugs were recently prepared after reviewing 438 papers published from 1980 to August 2002 [16]. It was concluded that treatment with ASA should be highly recommended for secondary thrombotic prophylaxis and for patients suffering from microcirculatory disturbances. However, the authors also discussed the lack of larger studies for this uncommon disease leading to difficulties in retrieving good scientific evidence for the best treatment in ET. Consequently, no consensus could be reached regarding ASA treatment as primary thrombotic prophylaxis. One way of approaching this problem is to conduct further research on platelet function and on the thrombotic process in ET. Knowledge from such studies might be used to detect individual patients and/or define novel high-risk patient groups that would benefit from ASA. Several studies investigating platelet function in ET have already been performed. However, interpretations of the results are complicated since, depending

Table 1. Demographics of the included patients Gender (Male / Female) - n

8 / 22

Median age at blood sampling – years (range)

67 (30-87)

Median age at diagnosisa – years (range)

61 (31-84)

Median time from diagnosis to blood

samplinga

– years (range)

7 (1-27)

Treatment at blood sampling – n (%) - ASA

10 (33.3)

- ASA + Hydroxyurea

6 (20.0)

- No treatment

5 (16.7)

- Hydroxyurea

4 (13.3)

- Warfarin + Hydroxyurea

2 (6.7)

- Warfarin + ASA

1 (3.3)

- Warfarin

1 (3.3)

- Interferon

1 (3.3)

Disease historyb – n (%) - Thrombosis

9 (30.0)

- No complications

11 (36.7)

- Bleeding

6 (20.0)

- Thrombosis + Bleeding

4 (13.3)

aDetails

are missing for one patient

bDisease history includes events occuring before the time of blood sampling. Thrombosis events include deep vein thrombosis, thrombophlebitis, stroke, angina/myocardial infarction and transitory ischaemic attack. Bleeding events include haematoma, excessive menstruation, gum bleedings, haematuria, excessive bleedings from wounds, epistaxis and spontaneous bleedings

Eriksson et al. Enhanced platelet adhesion in essential thrombocythemia

on the assay used, both increased and decreased activity of ET-platelets have been reported [17]. In the present study, we used a novel assay of static platelet adhesion [18] to investigate platelet function in ET patients. The study used a screening approach and investigated adhesion to albumin, collagen, fibrinogen, and fibronectin in the presence of soluble activators such as adenosine 5’-diphosphate (ADP), adrenaline, ristocetin and L-_-lysophosphatidic acid (oleoyl-sn-glycero-3-phosphate, LPA). All of these surfaces and soluble activators, with the exception of albumin and ristocetin, are of obvious physiological importance. The inclusion of bacteria-derived ristocetin is motivated since it stimulates the otherwise flow-dependent interaction between von Willebrand factor (vWf) and glycoprotein (GP)-Ib-IX-V on platelets [19]. Albumin was included both as a negative control for basal adhesion and because it allows the detection of LPA-induced adhesion [20]. This experimental setup also allows investigation of both _2`1-dependent adhesion to collagen and _IIb`3-dependent adhesion to fibrinogen and albumin [21]. Since platelet adhesion is scarcely studied in ET, our aim was to investigate if this novel assay for measurement of static platelet adhesion could be used to detect platelet abnormalities in patients with ET.

Material and Methods Patients and Control Subjects The study included 30 ET patients (Table 1), diagnosed according to the World Health Organization (WHO) classification of tumors for 2008. Inclusion was made consecutively during a one-year period. Fourteen healthy controls (4 female, 10 male) with a median age of 49 years (range: 31-66 years) were also included. All controls were free from non-steroidal anti-inflammatory drugs or other drugs known to affect platelet function and were included consecutively during the same time period as the patients. The study conforms to the principles outlined in the Declaration of Helsinki (1975) and later revisions, and was approved by the ethical committee at the Faculty of Health Sciences in Linköping, Sweden (Dnr 03-043). All subjects gave their informed consent to be included in the study. Coating of Microplates The wells of 96-well microplates (Nunc Maxisorp, Roskilde, Denmark) were coated with different proteins by addition of 100 L of coating solution followed by incubation at 4°C at least overnight but for a maximum of 7 days. The different protein coating solutions consisted of 2 mg/ml human albumin (Pharmacia & Upjohn AB, Stockholm, Sweden), 2 mg/ml human fibrinogen (American Diagnostica Inc., Greenwich, Connecticut, USA), 10 g/ml human fibronectin (Roche Diagnostics, Mannheim, Germany) or 0.1 mg/ml collagen S from calf skin (Roche Diagnostics) combined with 2 g/ml horse tendon collagen (Biopool International, Ventura, California, USA). Preparation of Platelet-Rich Plasma (PRP) Blood was drawn from antecubital veins into Na-heparin tubes and centrifuged at 205×g for 20 min at room temperature (RT). The upper layer consisting of PRP was transferred to

Turk J Hematol 2010; 27: 82-90

a new tube and diluted 1:4 in a solution of 0.9% NaCl containing MgCl2. The final concentration of MgCl2 when performing the platelet adhesion assay was 5 mmol/L. Platelet Adhesion Assay Platelet adhesion was measured as previously described [18,21]. Briefly, protein-coated microplates were washed twice in 0.9% NaCl by plate inversion followed by addition of 50 L diluted PRP and 50 L platelet activator or solvent. The platelet activators used were ADP and LPA (Sigma-Aldrich, St Louis, Missouri, USA), adrenaline (NM Pharma, Stockholm, Sweden) and ristocetin (Diagnostica Stago, Asnières-sur-Seine, France). The microplates were incubated for 1 h at RT to allow platelet attachment and, thereafter, washed twice in 0.9% NaCl by plate inversion. To all wells, 140 L of a substrate buffer solution (pH 5.4) consisting of 0.1 mol/L sodium citrate, 0.1 mol/L citric acid, 0.1% (w/v) Triton X-100 and 1 mg/ml p-nitrophenyl phosphate (Sigma-Aldrich) was added. A separate microplate was used for estimation of 100% and 0% platelet adhesion. This was achieved by mixing 140 L substrate solution with 50 L diluted PRP or 50 L 0.9% NaCl, respectively. Background absorbance was then measured for all wells at 405 nm using a Spectramax microplate reader (Molecular Devices, Sunnyvale, California, USA) and the microplates were incubated for 40 min during constant shaking at RT. After incubation, 100 L of 2 mol/L NaOH was added to all wells followed by absorbance measurements at 405 nm. Percentage platelet adhesion was calculated from the absorbance values. It has previously been shown that for absorbance values between 0 and 1, the platelet count correlates well with the amount of product developed [18]. When performing the same type of analysis on one single patient with high platelet count we found that linear relationships between absorbance and platelet amount can be expected for absorbance values as high as 1.9 (r2 = 0.98). Allele-Specific Polymerase Chain Reaction (PCR) Blood samples from ET patients were analyzed for the JAK2 mutation (Val617Phe) as described earlier [9]. The only difference compared to the original protocol was the use of 35 PCR-cycles instead of 36. Statistics The effect of externally added platelet activators was analyzed with one-way Repeated Measures ANOVA followed by Bonferroni’s multiple comparison test. Two-way ANOVA and Bonferroni’s post test were used for comparisons between controls and patients. For the two-way ANOVA, all effects induced by externally added activators were analyzed as the effect measured reduced by the basal adhesion. The age distributions for controls and ET patients were analyzed with t-test and one-way ANOVA. The effect of JAK2 status on platelet adhesion was analyzed with t-test. Linear regression was used when investigating the influence of platelet amount on platelet adhesion and when analyzing the relationship between platelet amount and measured absorbance. All statistical calculations were performed using the software GraphPad Prism®, version 4 (GraphPad Software Inc., San Diego, California, USA).

Eriksson et al. Enhanced platelet adhesion in essential thrombocythemia

Turk J Hematol 2010; 27: 82-90

Fiăgure 1a

Fiăgure 1b

*** ***

30 25 20

***

*** ***

Platelet adhesion to collagen (%)

Platelet adhesion to albumin (%)

***

*** ***

*** ** ***

***

20 10

5 0

0 lve

P P AD AD

lve

P P AD AD

Fiăgure 1c

Platelet adhesion to fibrinogen (%)

*** ***

***

lve

P P AD AD

*** *** ***

***

20 10

1 10 1 10 nt nt nt P 1 10 lve DP P lve DP P P AD AD So A AD So A AD

lve

Fiăgure 1d 30

Platelet adhesion to fibronectin (%)

*** ***

***

1 10 1 10 1 10 nt nt nt lve DP DP lve DP DP lve DP DP o o o A A A A S S S A A

1 10 1 10 nt nt nt P 1 10 lve DP DP lve DP DP P o o A A A AD AD S S A

l So

Figure 1. Platelet adhesion to albumin (a), collagen (b), fibrinogen (c), and fibronectin (d) was significantly increased by ADP (1 and 10 mol/L) for both controls and ET-platelets. A dose-response relationship was seen on all surfaces for ET-platelets, but only on albumin and fibronectin for controls. Differences in basal adhesion (solvent) between the three groups were not significant. Data are presented as mean+SD. Filled bars = Controls (n = 11 except for fibronectin where n = 10, age range: 31-63 years), open bars = Age-matched patients (n = 14, age range: 30-66 years), patterned bars = Patients 67 years (n = 16, age range: 67-87 years). ns = not significant, **p<0.01, ***p<0.001

*** *** ***

*** ** ***

Platelet adhesion to albumin (%)

20 15

10 5

*** *** ***

ns ns

nt 0.1 10 10 nt 0.1 10 10 nt 0.1 10 10 lve dr PA PA lve dr PA PA lve dr PA PA o o o L L S A L 1+ S A L 1+ S A L 1+L . . . 0 0 r r r0 Ad Ad Ad

Figure 2. The combination of 0.1 mol/L adrenaline (Adr) and 10 mol/L LPA increased platelet adhesion synergistically to albumin for both ETplatelets and controls. Data are presented as mean+SD. Filled bars = Controls (n = 11, age range: 31-63 years), open bars = Age-matched patients (n = 14, age range: 30-66 years), patterned bars = Patients 67 years (n = 16, age range: 67-87 years). ns = not significant, **p<0.01, ***p<0.001

Results The aim of this study was to investigate whether platelets from ET patients show abnormal adhesion patterns compared to controls. Several patients were older than 70 years, which resulted in difficulties in finding age-matched healthy controls. Consequently, the control group was significantly younger than the patient group (p<0.01), and since age is considered a risk factor for thrombosis in ET [8], there is a potential risk that age might influence the results. To be able to compare the control group with the ET patients, we therefore divided the ET group in two, with one group being age-matched with the controls and the other group consisting of patients * 67 years. The ages of the controls (n = 14, median age: 49, range: 31-66) and the age-matched ET patients (n = 14, median age: 49, range: 30-66) were similar. However, the ages of the patients * 67 years (n = 16, median age: 77, range: 67-87) were significantly different from the other two groups (p<0.001) The prevalence of ASA-treatment was similar for the age-matched ET patients (8 of 14) and the patients * 67 years (9 of 16).

Eriksson et al. Enhanced platelet adhesion in essential thrombocythemia

Turk J Hematol 2010; 27: 82-90

Collagen Albumin

Fibrinogen

***

Figure 3b

Figure 3a

** ns

40 ns

Platelet adhesion (%)

40 30

20 10

ch ed at

AD P

P AD s ro l

nt Co

6 7

ye ar s

1 AD P

ye ar s

t. pa

Pa t.

AD P

P AD

ch ed

ro l

ye ar s

AD P

6 7

ch ed

AD P

AD s

ro l

ye ar s

t. pa

ch ed at

AD P

10 P AD s ro l nt Co

m eAg

0 0

Albumin Collagen Figure 3c

Fibrinogen

*** ** ns

ns 30 20

Platelet adhesion (%)

ns 20

Ad rs ye a Pa

ed at ch em Ag

r+ Ad t. pa

Co nt ro ls

Ri st Co o 1 n tro em l s at R ch ist o ed 1 pa Pa t. t. Ri st 67 o 1 ye ar s Ri st o 1

nt ro

Ad r

LP A

+L PA

Platelet adhesion (%)

Figure 3d

Figure 3. Platelet adhesion to albumin (filled circles), collagen (triangles) and fibrinogen (squares) induced by ADP (1 and 10 mol/L), ristocetin (Risto 1 mg/ml) or the combination of adrenaline (Adr 0.1 mol/L) and LPA (10 mol/L) with basal adhesion subtracted. The increase in platelet adhesion induced by the soluble platelet activators was generally higher for ET patients compared to controls (n = 12-14, age range: 31-66 years except in D where age range: 31-63 years) and there were no differences between age-matched patients (n = 14, age range: 30-66 years) and patients 67 years (n = 16, age range: 67-87 years). Horizontal lines represent mean values. ns = not significant, *p<0.05, **p<0.01, ***p<0.001

Effect of Externally Added Platelet Activators on Platelet Adhesion Our approach was to measure platelet adhesion to protein surfaces in the presence or absence of different externally added platelet activators. Basal adhesion to the different proteins in the absence of external activators (solvent) was not significantly different between the three groups (Figure 1). Addition of 1 and 10 mol/L ADP significantly increased platelet adhesion to all surfaces for controls as well as for both

patient groups (Figures 1a-d). Furthermore, there was a significant dose-response relationship on all surfaces when activating ET-platelets with ADP. For control platelets, this was only seen on albumin and fibronectin, i.e. the surfaces that induced the lowest adhesion. The only significant effects observed for adrenaline at 0.1 mol/L and ristocetin at 1 mg/ ml were increased adhesion to collagen and fibrinogen for both ET-platelets and controls as well as increased ristocetininduced adhesion to albumin for ET-platelets (not shown). When combining 0.1 mol/L adrenaline and 10 mol/L LPA,

Eriksson et al. Enhanced platelet adhesion in essential thrombocythemia

Turk J Hematol 2010; 27: 82-90

Proportion of ET-patients exceeding the mean+2SD of controls (%)

100

10 A

Ad r0

10 b Al

Co l

1 lA Co l

10 P

lR ist o

Co l

Fib rg

Ri st o

Figure 4. The relative proportion of all ET patients presenting with platelet adhesion to albumin (Alb), collagen (Coll) and fibrinogen (Fibrg) exceeding the mean + 2SD of the control group for different activating stimuli with basal adhesion subtracted (n = 30, age range: 30-87 years). Numerals on the x-axis represent concentrations of soluble platelet activators in mol/L except for ristocetin where the unit of concentration is mg/ml. Adr = adrenaline, Risto = ristocetin

adhesion of control- and ET-platelets to albumin was significantly increased compared to basal adhesion (solvent) and compared to adrenaline and LPA when added alone (Figure 2). This pattern of increased adhesion indicating synergistic effects was neither detected for controls nor ET-platelets on collagen and fibrinogen, but was present on fibronectin for both ET-groups but not for controls (not shown). Comparisons of Platelet Adhesion Between Controls and ET Patients When analyzing differences between the groups, we focused on those activators that produced significantly increased platelet adhesion for controls and both patient groups. Comparisons were made on the increase relative to basal adhesion induced by the externally added activators. For 10 mol/L ADP on fibrinogen, the increase was significantly higher for age-matched patients compared to controls, while there was no difference between controls and patients * 67 years (Figure 3a). Inversely, adding 10 mol/L ADP or the combination of adrenaline and LPA to albumin as well as 1 mol/L ADP to collagen or 1 mg/ml ristocetin to fibrinogen resulted in significantly greater increase in adhesion for patients * 67 years but not for age-matched ET patients compared to controls (Figures 3a-d). The increased adhesion to collagen induced by 10 mol/L ADP and 1 mg/ml ristocetin was significantly higher for both age-matched patients and patients * 67 years compared to controls (Figures 3b, c). None of the activating stimuli induced significant differences between the patient groups (Figures 3a-d). Influence of Platelet Amount, History of Thrombohemorrhagic Events and JAK2 Status on Platelet Adhesion Since ET patients have more platelets than healthy individuals, we investigated if the increased platelet activity found in this study could be explained by this quantitative difference. In

the cases where we observed a significant difference in the increase in platelet adhesion (1 mol/L ADP to collagen; 10 mol/L ADP to albumin, collagen and fibrinogen; 1 mg/ml ristocetin to collagen and fibrinogen; 0.1 mol/L adrenaline + 10 mol/L LPA to albumin), we performed linear regression analyses for the amount of ET-platelets added to wells versus platelet adhesion. Platelet amount was estimated by measurements of acid phosphatase activity, which correlates well with the actual platelet count [18]. However, there were no significant correlations between platelet amount and adhesion since r2-values ranged from 0.0099 to 0.094 for the seven different stimuli. Even though we found significant differences in platelet adhesion for ET patients compared to controls, we also found considerable variations within groups and consequently overlaps between the groups. We therefore further analyzed the results with specific attention to adhesion values for ET-platelets being higher than the mean + 2 standard deviations (SDs) calculated for controls (Figure 4) as described earlier [22]. Adhesion above mean + 2 SD was found for approximately 50% of the patients when investigating ristocetin and ADPinduced adhesion to collagen and fibrinogen. The prevalence of such high adhesion values was considerably lower on the albumin surface. Furthermore, this pattern remained in both the age-matched patient group and in patients * 67 years (not shown). In order to investigate whether a high adhesion phenotype could be connected to a history of thrombosis, we studied the disease characteristics of patients having adhesion above mean + 2 SD for five or more of the seven stimuli. However, this small group of nine patients was heterogeneous, containing patients suffering from bleeding and thrombosis as well as no events at all. At the time of blood sampling, five of those patients received a combination of anti-proliferative and anti-platelet treatment (hydroxyurea + ASA), one received hydroxyurea combined with warfarin, and three patients were untreated. Among the remaining 21 patients, three were treated with both anti-proliferative and anti-platelet drugs (ASA combined with hydroxyurea or with a history of P32-treatment). Also, for one of the three untreated patients in the high adhesion phenotype group, management of the disease was later changed to treatment with both hydroxyurea and ASA. A change from monotherapy into combined anti-platelet and anti-proliferative treatment (ASA+hydroxyurea or interferon) occurred in three patients in the larger group of 21 patients. We next investigated the influence on platelet adhesion of the recently discovered mutation in the JAK2 gene (Val617Phe), which is present in about 60% of all ET patients [12]. JAK2 status was available for eight of the nine patients described above who had highly adhesive platelets (one patient died during the course of the study). Four of those patients were JAK2positive and four were JAK2-negative. Among all patients analyzed for JAK2 status in this study (25 patients), 15 were negative and 10 were positive. When investigating the whole patient group, there were no differences between JAK2positive and JAK2-negative patients regarding platelet adhesion induced by the stimuli that caused differences between controls and patients.

Eriksson et al. Enhanced platelet adhesion in essential thrombocythemia

Discussion Different types of ex vivo platelet function measures have reported that ET-platelets are highly active. Flow cytometric studies in whole blood show increased platelet surface expression of P-selectin, increased binding of thrombospondin to platelets and increased amounts of platelet-leukocyte aggregates in MPN patients compared to controls [22,23]. Urine from ET patients has been reported to contain increased amounts of TXA2-metabolites [24]. Compared to normal subjects, an increased amount of platelet aggregates has been observed in blood from ET patients, and there is also an increased spontaneous aggregation of ET-platelets in plasma [25]. All those studies could potentially be used to explain the preponderance of thrombosis compared to bleeding in ET. However, interpretations get complicated when taking into account results obtained from analyses that activate platelets in vitro. In a study including 120 MPN patients, platelet aggregation was measured both in whole blood and in PRP after in vitro stimulation [26]. Platelets were hyperreactive when measuring whole blood aggregometry and using ADP and collagen as activators. In contrast, aggregation in PRP was decreased. Decreased platelet aggregation response in PRP to different platelet agonists for MPN patients has been reported by a number of other studies [27-30]. Decreased platelet function has also been observed with whole blood flow cytometry after in vitro activation [22] and when using the Platelet Function Analyzer (PFA-100) [30]. In accordance with this, we found a non-significant trend towards decreased basal adhesion to collagen and fibrinogen for ET-platelets in the present study. In contrast to these results, ET-platelets produce increased amounts of thromboxane B2 as compared to controls after in vitro stimulation with thrombin [31]. Regarding platelet aggregation in PRP, the most pronounced decrease in platelet function is often seen with adrenaline as activator [27,29,30]. This has been proposed to be the result of decreased amounts of _-adrenergic receptors [32]. However, this is opposite to our results, which show that ET-platelets have normal response to adrenaline alone and increased reactivity compared to controls when combined with LPA. Further, we observed a considerable individual variability both in controls and in ET patients regarding the combination of adrenaline and LPA. The appearance of individual variability in the response to adrenaline has been studied in the healthy population, and when including 140 individuals, 16% were classified as non-responders to adrenaline-induced platelet aggregation in plasma [33]. This variability in the adrenaline response in healthy subjects has been confirmed by other investigations also studying platelet function in plasma [20,34-36]. No study has been designed with the purpose of investigating individual variability of adrenaline-response in ET patients. However, when looking at individual variations obtained from studies investigating platelet aggregation in PRP for patients with MPNs, we find it possible that the variability exists in this group as well [30,32,37]. Particularly interesting is a study that investigated 76 MPN patients, and characterized 21% of patients as non-responders to adrenaline measured by aggregometry in PRP [27].

Turk J Hematol 2010; 27: 82-90

These results are very similar to the study on healthy donors described above, which reported 16% of healthy individuals as non-responders to adrenaline [33]. Our study, showing interindividual variation in the response to adrenaline combined with LPA, further supports the claim that the response to adrenaline in the ET population is highly heterogeneous. Furthermore, the increased response to adrenaline observed by us and not by others might be a result of the methodology used (described below). Age is a well-known risk factor for thrombosis in ET [8,38]. In agreement with this, our results show higher adhesion values for patients * 67 years versus controls, which correlate well with the increased thrombosis risk in this group. We also found that some stimuli were significantly more effective in inducing platelet adhesion on age-matched patients compared to controls, while differences between the two patient groups were lacking. A plausible explanation is that part of the increase in adhesion is connected to old age while the other part is connected to the disease itself. Our general finding in this study was that ET-platelets are more readily activated compared to controls. However, there was an overlap between the groups with several patients having responses in the same range as the control group. Since the JAK2 mutation occurs in only approximately 60% of ET patients [12], we decided to investigate if patients with high adhesion values were JAK2-positive. However, JAK2 status did not influence platelet adhesion. Furthermore, there were no clear connections between platelet adhesion and disease history. On the other hand, this study included eight patients treated with ASA combined with anti-proliferative treatment, and five of those were classified as high adhesion phenotype. Later, one untreated patient that we classified as high adhesion phenotype was, independent of our results, prescribed ASA combined with hydroxyurea. From a clinical point of view, it might be suggested that these patients, being treated with both anti-proliferative and anti-platelet drugs, were considered to have a more severe disease and therefore needed more extensive treatment. The other patients not classified as high adhesion phenotype were less likely to have combined antiproliferative and anti-platelet treatment. In this way, a high adhesion phenotype seems to be connected to clinical status. However, further studies are needed before any definite conclusions can be drawn regarding the predictive value of the adhesion assay for thrombosis. We aim to follow these patients in the future, not only for the purpose of detecting thrombosisprone individuals but also for investigating the abilities of the assay to guide anti-thrombotic treatment. It might be claimed that our use of PRP without the commonly performed adjustment of platelet count with autologous platelet-poor plasma (PPP) limits this study. However, recent studies have shown that adjustment of platelet count with PPP might induce decreased platelet activity, possibly through an inhibiting effect from PPP [39,40]. This suggests that comparisons between control platelets and platelets from ET patients might be misleading if adjusting the platelet count since the higher platelet count for ET patients demands more PPP for dilution [40]. Consequently, the increased adhesion for ET-platelets observed in this study might be the result of avoidance of PPP-induced artificial inhibition.

Eriksson et al. Enhanced platelet adhesion in essential thrombocythemia

Turk J Hematol 2010; 27: 82-90

It has been proposed that increased activity of ET-platelets in vivo results in circulating exhausted platelets that are not able to respond in vitro [41]. This explanation might also be of interest for the interpretation of the current results. We suggest that the increased reactivity in vitro observed by us, which is uncommon for other in vitro tests, might be a consequence of the relatively long one hour incubation period in the presence of both an activating surface and soluble agonists. This is rather different from e.g. flow cytometry and platelet aggregometry, which often measure platelet response within minutes after stimulation with a single agonist. We also noticed that strong stimuli such as high concentrations of ADP and ristocetin combined with the activating surfaces collagen and fibrinogen resulted in a relatively high proportion of adhesion values being higher than the mean + 2SD for controls. In contrast, weaker stimuli represented by adhesion measured on albumin resulted in comparatively few adhesion values that were higher than the control mean + 2SD. In addition, earlier studies show that secretion of ADP is important in this assay [20,21]. Thus, we propose that multiple stimuli, which most likely are of importance in vivo [42], and a long incubation time are both needed to induce secretion and activation of exhausted ET-platelets. In conclusion, we suggest that the present platelet assay mimics the in vivo situation regarding the presence of chronic platelet-activating stimuli resulting in increased risk of thrombosis. It must be remembered, however, that additional factors, such as vascular cells other than platelets and shear stress, are absent in our assay but affect thrombus formation in vivo. Nevertheless, the assay is reproducible over time [43] and it might be useful to predict thrombotic risk and monitor antiplatelet treatment in ET. Further prospective follow-up studies and larger patient material are needed to strengthen this hypothesis. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hellstrom-Lindberg E, Tefferi A, Bloomfield CD. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009;114:937-51. Johansson P. Epidemiology of the myeloproliferative disorders polycythemia vera and essential thrombocythemia. Semin Thromb Hemost 2006;32:171-3. Elliott MA, Tefferi A. Thrombosis and haemorrhage in polycythaemia vera and essential thrombocythaemia. Br J Haematol 2005;128:275-90. van Genderen PJ, Michiels JJ. Erythromelalgic, thrombotic and haemorrhagic manifestations of thrombocythaemia. Presse Med 1994;23:73-7.

10.

11.

12.

13.

14.

15. 16.

17.

18.

19.

Budde U, Schaefer G, Mueller N, Egli H, Dent J, Ruggeri Z, Zimmerman T. Acquired von Willebrand's disease in the myeloproliferative syndrome. Blood 1984;64:981-5. Budde U, Scharf RE, Franke P, Hartmann-Budde K, Dent J, Ruggeri ZM. Elevated platelet count as a cause of abnormal von Willebrand factor multimer distribution in plasma. Blood 1993;82:1749-57. van Genderen PJ, Budde U, Michiels JJ, van Strik R, van Vliet HH. The reduction of large von Willebrand factor multimers in plasma in essential thrombocythaemia is related to the platelet count. Br J Haematol 1996;93:962-5. Cortelazzo S, Viero P, Finazzi G, D'Emilio A, Rodeghiero F, Barbui T. Incidence and risk factors for thrombotic complications in a historical cohort of 100 patients with essential thrombocythemia. J Clin Oncol 1990;8:556-62. Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, Scott MA, Erber WN, Green AR. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005;365:1054-61. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, Garcon L, Raslova H, Berger R, Bennaceur-Griscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005;434:1144-8. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I, Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D'Andrea A, Frohling S, Dohner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ, Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7:387-97. Vannucchi AM, Guglielmelli P, Tefferi A. Advances in understanding and management of myeloproliferative neoplasms. CA Cancer J Clin 2009;59:171-91. Tefferi A, Elliott M. Thrombosis in myeloproliferative disorders: prevalence, prognostic factors, and the role of leukocytes and JAK2V617F. Semin Thromb Hemost 2007;33:313-20. Finazzi G, Barbui T. Evidence and expertise in the management of polycythemia vera and essential thrombocythemia. Leukemia 2008;22:1494-502. Harrison CN, Green AR. Essential thrombocythaemia. Best Pract Res Clin Haematol 2006;19:439-53. Barbui T, Barosi G, Grossi A, Gugliotta L, Liberato LN, Marchetti M, Mazzucconi MG, Rodeghiero F, Tura S. Practice guidelines for the therapy of essential thrombocythemia. A statement from the Italian Society of Hematology, the Italian Society of Experimental Hematology and the Italian Group for Bone Marrow Transplantation. Haematologica 2004;89:215-32. Wehmeier A, Sudhoff T, Meierkord F. Relation of platelet abnormalities to thrombosis and hemorrhage in chronic myeloproliferative disorders. Semin Thromb Hemost 1997;23:391-402. Eriksson AC, Whiss PA. Measurement of adhesion of human platelets in plasma to protein surfaces in microplates. J Pharmacol Toxicol Methods 2005;52:356-65. Berndt MC, Shen Y, Dopheide SM, Gardiner EE, Andrews RK. The vascular biology of the glycoprotein Ib-IX-V complex. Thromb Haemost 2001;86:178-88.

90 20.

21.

22.

23.

24.

25. 26.

27.

28. 29.

30.

31.

32.

33.

Eriksson et al. Enhanced platelet adhesion in essential thrombocythemia

Eriksson AC, Whiss PA, Nilsson UK. Adhesion of human platelets to albumin is synergistically increased by lysophosphatidic acid and adrenaline in a donor-dependent fashion. Blood Coagul Fibrinolysis 2006;17:359-68. Eriksson AC, Whiss PA. Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates. Blood Coagul Fibrinolysis 2009;20:197-206. Jensen MK, de Nully Brown P, Lund BV, Nielsen OJ, Hasselbalch HC. Increased platelet activation and abnormal membrane glycoprotein content and redistribution in myeloproliferative disorders. Br J Haematol 2000;110:116-24. Jensen MK, de Nully Brown P, Lund BV, Nielsen OJ, Hasselbalch HC. Increased circulating platelet-leukocyte aggregates in myeloproliferative disorders is correlated to previous thrombosis, platelet activation and platelet count. Eur J Haematol 2001;66:143-51. Rocca B, Ciabattoni G, Tartaglione R, Cortelazzo S, Barbui T, Patrono C, Landolfi R. Increased thromboxane biosynthesis in essential thrombocythemia. Thromb Haemost 1995;74:1225-30. Wu KK. Platelet hyperaggregability and thrombosis in patients with thrombocythemia. Ann Intern Med 1978;88:7-11. Balduini CL, Bertolino G, Noris P, Piletta GC. Platelet aggregation in platelet-rich plasma and whole blood in 120 patients with myeloproliferative disorders. Am J Clin Pathol 1991;95:82-6. Avram S, Lupu A, Angelescu S, Olteanu N, Mut-Popescu D. Abnormalities of platelet aggregation in chronic myeloproliferative disorders. J Cell Mol Med 2001;5:79-87. Ginsburg AD. Platelet function in patients with high platelet counts. Ann Intern Med 1975;82:506-11. Yamamoto K, Sekiguchi E, Takatani O. Abnormalities of epinephrine-induced platelet aggregation and adenine nucleotides in myeloproliferative disorders. Thromb Haemost 1984;52:292-6. Cesar JM, de Miguel D, Garcia Avello A, Burgaleta C. Platelet dysfunction in primary thrombocythemia using the platelet function analyzer, PFA-100. Am J Clin Pathol 2005;123:772-7. Mayordomo O, Carcamo C, Vecino AM, Navarro JL, Cesar JM. Arachidonic acid metabolism in platelets of patients with essential thrombocythaemia. Thromb Res 1995;78:315-21. Kaywin P, McDonough M, Insel PA, Shattil SJ. Platelet function in essential thrombocythemia. Decreased epinephrine responsiveness associated with a deficiency of platelet alphaadrenergic receptors. N Engl J Med 1978;299:505-9. Kambayashi J, Shinoki N, Nakamura T, Ariyoshi H, Kawasaki T, Sakon M, Monden M. Prevalence of impaired responsiveness to

Turk J Hematol 2010; 27: 82-90

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

epinephrine in platelets among Japanese. Thromb Res 1996;81:85-90. Nakahashi TK, Kambayashi J, Nakamura T, Le SN, Yoshitake M, Tandon NN, Sun B. Platelets in nonresponders to epinephrine stimulation showed reduced response to ADP. Thromb Res 2001;104:127-35. Pyo MK, Yun-Choi HS, Hong YJ. Apparent heterogeneous responsiveness of human platelet rich plasma to catecholamines. Platelets 2003;14:171-8. Theodoropoulos I, Christopoulos C, Metcalfe P, Dimitriadou E, Economopoulos P, Loucopoulos D. The effect of human platelet alloantigen polymorphisms on the in vitro responsiveness to adrenaline and collagen. Br J Haematol 2001;114:387-93. Raszeja-Specht A, Skibowska A, Bieniaszewska M, Szutowicz A. Relationships between thrombohemorrhagic complications and platelet function in patients with essential thrombocythaemia. Am J Hematol 2001;68:32-6. Landolfi R, Cipriani MC, Novarese L. Thrombosis and bleeding in polycythemia vera and essential thrombocythemia: pathogenetic mechanisms and prevention. Best Pract Res Clin Haematol 2006;19:617-33. Cattaneo M, Lecchi A, Zighetti ML, Lussana F. Platelet aggregation studies: autologous platelet-poor plasma inhibits platelet aggregation when added to platelet-rich plasma to normalize platelet count. Haematologica 2007;92:694-7. Grignani C, Noris P, Tinelli C, Barosi G, Balduini CL. In vitro platelet aggregation defects in patients with myeloproliferative disorders and high platelet counts: are they laboratory artefacts? Platelets 2009;20:131-4. Michiels JJ, Berneman Z, Schroyens W, Finazzi G, Budde U, van Vliet HH. The paradox of platelet activation and impaired function: platelet-von Willebrand factor interactions, and the etiology of thrombotic and hemorrhagic manifestations in essential thrombocythemia and polycythemia vera. Semin Thromb Hemost 2006;32:589-604. Graff J, Klinkhardt U, Harder S. Pharmacodynamic profile of antiplatelet agents: marked differences between single versus costimulation with platelet activators. Thromb Res 2004;113:295-302. Eriksson AC, Jonasson L, Lindahl TL, Hedback B, Whiss PA. Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study. J Transl Med 2009;7:42.

Research Article

Clinical aspects of sclerodermatous type graft-versushost disease after allogeneic hematopoietic cell transplantation Allogeneik hematopoietik hücre nakli sonrasÕ sklerodermatoz tip graft-versushost hastalÕúÕnÕn klinik yönleri Hatice üanlÕ1, Bengü Nisa Akay1, Ender Soydan2, Pelin Koçyiúit1, Mutlu Arat2, Osman ûlhan2 1University 2University

of Ankara School of Medicine Department of Dermatology, Ankara, Turkey of Ankara School of Medicine Department of Hematology, Ankara, Turkey

Abstract Objective: We aimed to evaluate the clinical features of sclerodermatous chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (AHSCT). Materials and Methods: We retrospectively analyzed 423 patients who underwent AHSCT. We assessed age, sex, pretransplant diagnosis, conditioning regimen, GVHD prophylaxis, and occurrence of acute GVHD (aGVHD), chronic lichenoid and chronic systemic GVHD, and clinical properties of sclerodermatous GVHD. Results: Sclerotic skin lesions developed in 22 patients after a mean of 752±647 days (median 480). aGVHD appeared in 17 patients, with hepatic involvement in 2, gastrointestinal tract involvement in 2 and skin involvement in 13 of these patients. Extensive chronic GVHD (liver, pulmonary, skin and oral mucosa) developed in 12 patients. Sclerosis was generalized in 19 patients (86.4%) and localized in 3 patients (13.6%). Leopard skin eruption appeared in 8 (36.4%) of the 19 patients with generalized sclerodermatous changes. In most cases, sclerotic lesions appeared on the trunk, and distal parts of the extremities were spared. Eight patients (36.4%) progressed from lichenoid to sclerodermatous lesions, 2 (9.1%) with lichenoid and sclerodermatous phases together and 12 (55.5%) with de novo sclerodermatous lesions. Five patients died because of late transplant-related complications. Conclusion: Sclerodermatous GVHD has a late onset and may be quite disabling. Unlike scleroderma, acral involvement is seen rarely. Although most lesions do not disappear in the course of the disease, most patients have a good prognosis. (Turk J Hematol 2010; 27: 91-8) Key words: Allogeneic hematopoietic stem cell transplantation, sclerodermatous graft-versus-host disease Received: September 1, 2009

Accepted: April 27, 2010

Özet Amaç: Allogeneik hematopoietik kök hücre nakli (Allo-HKHN) sonrasÕ geliýen sklerodermatoz graft-versus-host hastalÕúÕnÕn (GVHH) klinik özelliklerinin deúerlendirilmesi amaçlanmÕýtÕr. Yöntem ve Gereçler: Allo-HKHN yapÕlan 423 hasta retrospektif olarak analiz edilmiýtir. OlgularÕn yaý, cinsiyet, transplantasyon öncesi tanÕlarÕ, hazÕrlÕk rejimleri, GVHH proflaksileri, akut GVHH ve/veya kronik likenoid ve kronik sistemik GVHH mevcudiyeti ve geliýen sklerodermatoz GVHH’nÕn klinik özellikleri deúerlendirilmiýtir. Bulgular: Sklerotik lezyonlar 22 hastada ortalama 752±647 gün (ortanca 480) sonra geliýmiýtir. Akut GVHH 17 hastada geliýirken, bunlarÕn 2’sinde karaciúer, 2’sinde gastrointestinal sistem ve 14’ünde deri tutulumu gözlenmiýtir. YaygÕn kronik GVHH (karaciúer, akciúer, deri ve oral mukoza) 12 hastada geliýmiýtir. Skleroz 19 hastada (%86.4) jeneralize, 3 hastada (%13.6) lokalizeydi. Jeneralize sklerodermatoz deúiýiklikler izlenen 19 hastanÕn 8’inde (%36.4) leopar derisi görünümü

Address for Correspondence: Bengu Nisa Akay, MD, Department of Dermatology, Ankara University Faculty of medicine Ibni Sina Hospital, 06100 Ankara, Turkey Phone: +90 312 508 25 58 E-mail: nisaakay15@yahoo.com doi:10.5152/tjh.2010.06

üanlÕ et al. Clinical aspects of sclerodermatous type GVHD

Turk J Hematol 2010; 27: 91-8

mevcuttu. OlgularÕn çoúunda sklerotik lezyonlar gövdede yerleýirken, ekstremitelerin distali etkilenmemiýtir. OlgularÕn 8’i likenoid GVHH’dan sklerodermatoz GVHH’ye dönüýürken, 2’sinde her iki faz bir arada ve 12’sinde sklerodermatoz GVHH denovo olarak geliýmiýtir. Beý olgu transplantasyonla iliýkili geç komplikasyonlar nedeniyle vefat etmiýtir Sonuç: Sklerodermatoz GVHH geç baýlangÕçlÕ ve hastalar için oldukça sÕkÕntÕ oluýturabilen bir tablodur. SklerodermanÕn aksine akral tutulum nadiren görülür. Lezyonlar hastalÕk seyri boyunca kaybolmamakla birlikte pek çok olguda prognoz iyidir. (Turk J Hematol 2010; 27: 91-8) Anahtar kelimeler: Allogeneik hematopoietik kök hücre nakli, Sklerodermatoz Graft-Versus-Host HastalÕúÕ Geliý tarihi: 1 Eylül 2009

Kabul tarihi: 27 Nisan 2010

Introduction The development of acute and chronic graft-versus-host disease (aGVHD, cGVHD) after allogeneic hematopoietic stem cell transplantation (AHSCT) remains a major clinical problem associated with significant morbidity and mortality [1,2]. The skin, gastrointestinal tract and liver are the organs primarily affected [3]. Chronic GVHD (cGVHD) remains a major complication of AHSCT, and it affects more than 50% of long-term survivors of AHSCT [4]. cGVHD can occur anytime from months to years after AHSCT and may or may not be preceded by episodes of aGVHD. Lichenoid and sclerodermatous changes have been described [2-6]. Sclerodermatous cGVHD is distinguished by plaques of dermal sclerosis resembling morphea, and eventually by generalized scleroderma, often resulting in joint contractures [1-3,5-7]. Nevertheless, few data are available regarding the late sclerodermatous phase of cGVHD. Most authors do not separate lichenoid and sclerodermatous cGVHD in their reports. In this study, we describe the clinical features of 22 patients with sclerodermatous cGVHD who had received AHSCT.

Materials and Methods We retrospectively analyzed 423 patients who underwent AHSCT. The study was conducted by the Departments of Dermatology and Hematology in one of the largest university hospitals in Turkey. We reviewed the clinical characteristics after the appearance of sclerodermatous cGVHD. We assessed age, sex, pre-transplant diagnosis, conditioning regimen, GVHD prophylaxis, preceding aGVHD and/or lichenoid cGVHD, and clinical properties of sclerodermatous cGVHD. The patient data are summarized in Table 1. The diagnosis and grading of aGVHD and cGVHD were made according to the recent National Institutes of Health (NIH) consensus conference [8]. Mild cGVHD involves only one or two organs or sites (except lungs, with no clinically significant impairment). Moderate cGVHD involves at least one organ or site with clinically significant impairment but no major disability, or three or more organs or sites with no clinically significant functional impairment. Severe cGVHD indicates major disability caused by cGVHD [8]. The skin lesions in cGVHD were classified as: (1) lichenoid lesions or (2) sclerodermatous

lesions. Patients were classified as having generalized sclerodermatous cGVHD if more than two anatomic sites were involved and as localized in the remaining cases. The clinical diagnosis of cGVHD was established by a dermatologist and the diagnosis of cutaneous sclerodermatous GVHD was established based on both clinical and dermatopathological findings. Pigmentation changes like widespread, well-demarcated, hyperpigmented macules (leopard skin-like pigmentary changes) [2], areas of hypopigmentation, depigmentation, poikiloderma (atrophic and pigmentary changes), lichen sclerosus-like lesions (discrete to coalescent gray to white movable papules and plaques), keratosis pilaris, and ichthyosis were also evaluated. Presence of Raynaud phenomenon, sclerodactyly, and esophageal, joint and/or lung involvement was noted. Autoimmune markers such as anti-nuclear antibodies (ANA), anti-double-stranded DNA (anti-DsDNA), SCL70 and anti-centromere antibodies (ACA) were screened after the diagnosis of sclerodermatous GVHD. Moderate or severe GVHD was treated with 3-5 mg/kg/ day cyclosporine (CsA) and 1 mg/kg/day prednisone in patients already receiving CsA therapy (Seattle regimen). In non-responders, 15 mg/kg/dose twice daily mycophenolate mofetil (MMF) and/or extracorporeal photopheresis (ECP) were added to CsA therapy. UVAR XTS system (Therakos, Exton, PA, USA) was used during ECP. Each cycle of ECP consisted of two consecutive days at two-week intervals for the first three months and thereafter every four weeks until a maximum period of one year. Psoralen-UV-A (PUVA) therapy was given using oral 8-methoxypsoralen and a UV-A dosimetry regimen. PUVA was delivered three times weekly starting at a dose of 0.5 J/cm2 and increasing the dose by 0.5 J/ cm2 increments at each treatment to a maximum dose of 6 J/cm2. Patients were clinically examined twice weekly during the first three months and monthly afterwards. Response to therapy was defined as complete if less than 2% of the skin surface showed tightness and all other signs associated with cGVHD had disappeared [5]. Patients who did not show any improvement in the sclerotic changes were defined as nonresponsive. The response was defined as partial in the remaining cases. A statistical analysis was performed using SPSS 15.0. Simple descriptive statistics were tabulated. The chi-square test and Fisher’s exact probability test were used to analyze

üanlÕ et al. Clinical aspects of sclerodermatous type GVHD

Turk J Hematol 2010; 27: 91-8

Table 1. Patient data Age Recipient Donor Diagnosis

Type of

Conditioning Prophylaxis aGVHD cGVHD Skin

Skin pigmentation Therapy Survival Response

Sex

Transplantation

regimen

AML

ALLO

BU+CY

CSA+MTX

Moderate

pattern G

Hyperpigmentation Prednisone

disorders alive

ALL

ALLO

CY-TBI

CSA+MTX

Mild

Hypoand hyperpigmentation, poikiloderma

CsA MMF

exitus

CML

ALLO

BU+CY

CSA+MTX

Absent

Mild

CsA

alive

CML

ALLO

BU+CY

CSA+MTX

Mild

CsA

alive

CML

ALLO

BU+CY

CSA+MTX

Mild

Hyperpigmentation

CsA

alive

AML

ALLO

BU+CY

CSA+MTX

Mild

CsA

alive

AML

ALLO

BU+CY

CSA+MTX

Severe

HypoCsA+ exitus and prednisone hyperpigmentation

MDS

ALLO

BU+CY

CSA+MTX

Severe

Hyperpigmentation CsA +ECP

alive

CML

ALLO

BU+CY

CSA+MTX

Absent

Mild

Hypoand hyperpigmentation, poikiloderma

exitus

AML

ALLO

BU+CY

CSA+MTX

Severe

Hyperpigmentation CsA+ECP

alive

CML

ALLO

FLU-BU(iv)ATG

CSA+MMF

Absent

Mild

HypoMMF+ECP and hyperpigmentation

alive

AML

ALLO

BU+CY

CSA+MTX

Severe

HypoMMFand CsA+ECP hyperpigmentation, poikiloderma

alive

AML

ALLO

BU+CY

CSA+MTX

Moderate

Hyperpigmentation

MMF

alive

AML

ALLO

BU+CY

CSA+MTX

Severe

Hyperpigmentation

MMF CsA+ECP

alive

AML

ALLO

BU+CY

CSA+MTX

Moderate

HypoCsA+ prednisone alive and hyperpigmentation

ALLO

FLU+TBI

CSA+MMF

Mild

Hypoand hyperpigmentation

ECP

alive

MDS

ALLO

BU+CY

CSA+MTX

Moderate

Hyperpigmentation

MMF PUVA

alive

AML

ALLO

FLU+Mel

CSA+MMF

Mild

Hypoand hyperpigmentation,

CsA

exitus

CML

ALLO

BU+CY

CSA+MTX

Absent

Severe

Hyperpigmentation

ECP

alive

CML

ALLO

FLU+BU

CSA+MMF

Absent Moderate

Hypoand hyperpigmentation

MMF

alive

AML

ALLO

BU+CY

CSA+MTX

Mild

Hypoand hyperpigmentation

CsA

exitus

MDS

ALLO

BU+CY

CSA+MTX

Severe

Hyperpigmentation

MMF

alive

CsA

Prednisone+CsA+ECP ALL: Acute lymphoblastic leukemia; AML: Acute myeloblastic leukemia; CML: Chronic myelogenous leukemia; MDS: Myelodysplastic syndrome; Allo: Allogeneic; BM: Bone marrow; PB: Peripheral blood; BU: Busulphan; MTX: Methotrexate; CY: Cyclophosphamide; FLU: Fludarabine; ATG: Antithymocyte globulin; TBI: Total body irradiation; Mel: Melphalan; CSA: Cyclosporine; MMF: Mycophenolate mofetil; ECP: Extracorporeal photochemotherapy; PUVA: Psoralen-UV-A; G: Generalized; L: Localized; NR: Non-responder; PR: Partial response

üanlÕ et al. Clinical aspects of sclerodermatous type GVHD

differences between groups. Values of p<0.05 were considered statistically significant.

Results The development of sclerodermatous GVHD was observed in 22 (5.2%) out of 423 patients. Thirteen patients were male and 9 were female, with a mean age of 32±9 years. All the patients were transplanted from HLA-identical sibling donor. Sex mismatch was found to be statistically significant in the development of sclerodermatous GVHD (p=0.02). CsA plus short-term methotrexate (n=18) or MMF (n=4) was used for prophylaxis of GVHD (Table 1). History of aGVHD (> Grade II) was present in 17 patients (77%), with hepatic involvement in 2, gastrointestinal tract involvement in 2 and skin involvement in 13 patients. Eight patients (36.4%) progressed from lichenoid to sclerodermatous lesions, 2 (9.1%) with lichenoid and

Turk J Hematol 2010; 27: 91-8

sclerodermatous phases together and 12 (55.5%) with de novo sclerodermatous lesions. Clinical features of chronic sclerodermatous GVHD: Moderate (7 patients) or severe (5 patients) cGVHD developed in 12 patients, while it was mild in 10 patients. Five patients (23%) developed de novo chronic cutaneous GVHD without a previous aGVHD. Sclerotic lesions developed after a mean of 752±647 days (median 480). Immunosuppressive therapy was interrupted in 16 (72%) patients before sclerodermatous lesions had developed. Sclerosis was generalized in 19 patients (86.4%) and localized in 3 patients (13.6%). There was no statistically significant difference between the extensiveness of sclerodermatous GVHD and presence of previous aGVHD (p=1.00) or cGVHD (p=0.21). Widespread, welldemarcated, hyperpigmented macules and hypo-hyperpigmentation appeared in 8 (36.4%) and 11 (50%) patients, respectively (Figure 1). In most of the patients, sclerotic lesions

Figure 1. Multiple hyperpigmented macules that resemble leopard skin on the neck and cicatricial alopecia on the left temporal region

Figure 2. Sclerodermatous changes (multiple hyper-hypopigmented macules) of cGVHD presented with trunk involvement unlike acral involvement of scleroderma

Figure 3. Sclerodermatous cGVHD causing severe contractures of the wrists and fingers

Figure 4. Mucosal erosion and atrophy on the dorsal surface of the tongue

Turk J Hematol 2010; 27: 91-8

üanlÕ et al. Clinical aspects of sclerodermatous type GVHD

Discussion

Figure 5. Sclerodermatous poikilodermatous changes of cGVHD with ulceration on the upper arm

appeared on the trunk, and distal parts of the extremities were spared (Figure 2). Joint retractions and dysphagia developed in 2 (9%) patients (Figure 3). None of the patients had Raynaud phenomenon. Autoimmune markers like SCL-70, ACA, ANA, and anti-DsDNA were negative in all patients. Thirteen patients (59%) presented with accompanying oral mucosal involvement. Oral manifestations include reticular whitish plaques, erosions and ulcerations (Figure 4). The localizations of the lesions were buccal mucosa, tongue and gingiva. One patient presented with lichen sclerosis-et-atrophicus and one with septal panniculitis. Other associated lesions were poikiloderma (n=3) (Figure 5), pyogenic granuloma-like lesions (n=1), bullous lesions and erosions (n=1), ulcers (n=2) (Figure 5), eccrine hydrocystoma (n=1), acquired ichthyosis (n=2), cicatricial alopecia (n=3) (Figure 1), vitiligo (n=2), sicca syndrome (n=2), and salivary abnormalities (n=5). There was no standard therapy. None of the patients with localized scleroderma responded to CsA therapy. Partial response was achieved in five patients with extensive sclerodermatous GVHD. Three of them received ECP, MMF and CsA combination, one received only ECP, and one was given MMF and PUVA. The number of PUVA therapies in this patient was 48, and the total UVA dose was 135 J/cm2. Duration of ECP was one year in 4 patients showing partial response and six months in 4 patients without any response. Patients treated with ECP alone or ECP plus MMF, CsA or prednisone showed statistically significant improvement compared to patients treated with other treatment regimens (p=0.03). The effect of treatment appeared after three months and was maximal after seven or eight months. Five patients died because of late transplant-related complications. All these patients had extensive disease (multi-organ involvement).

The incidence of cGVHD is 30-50%. Of these, 90-100% develops cutaneous disease. cGVHD can occur de novo, but it is seen more often following aGVHD, which is the most important risk factor for the development of cGVHD. In our study, 17 of the 22 patients (77%) developed secondary chronic cutaneous GVHD following acute cutaneous GVHD, and in 5 patients (23%), de novo chronic cutaneous GVHD occurred without previous aGVHD. The mucocutaneous manifestations of cGVHD clinically resemble a wide variety of skin diseases, including lichen planus, lichenoid eruptions, sicca syndrome, morphea, scleroderma, and lichen sclerosus. Chronic cutaneous GVHD is categorized according to the type of lesions into lichenoid and sclerodermatous variants. Both types may occur in a single patient. While the literature precedent invariably associates the lichenoid manifestations of cGVHD as a part of the cutaneous manifestations of cGVHD, Magro et al. [9] suggested that the early-onset lichenoid GVHD is a unique form of aGVHD. Sclerodermatous cGVHD has the most severe skin involvement and appears late in the course of the disease [1]. It has been described in small series of patients [1-3,5]. In our study, the rate of sclerodermatous cGVHD among all surviving patients was similar (5.2%) to previously reported (3.4-3.6%) studies [2,3] but lower than in the study of Skert et al. (10.5%) [5]. Sclerotic lesions of our patients developed after a mean of 752±647 days (median 480, range: 3 months - 5 years). The length of time between AHSCT and the onset of sclerodermatous cGVHD has been reported within a wide range (292-2190 days, mean 730) [1-3,5]. Chosidow et al. [3] found that lichenoid GVHD always preceded the sclerodermatous phase. Shulman et al. [4] suggested that patients with generalized sclerodermatous GVHD followed a biphasic course, with first a generalized erythematous or violaceous rash, and then poikiloderma with sclerotic skin. In our study, 8 of our patients (36.4%) progressed from lichenoid to sclerodermatous lesions, 2 (9.1%) with lichenoid and sclerodermatous phases together and 12 (55.5%) with de novo sclerodermatous lesions. Sclerodermatous lesions begin with indurated plaques with loss of skin markings and appendages causing pain and chronic ulceration, predisposing to generalized wasting and pyogenic infections of the skin. Sometimes subcutaneous fat and fascia are also involved, resulting in eosinophilic fasciitislike appearance. Chosidow et al. [3] reported that 4 of their 7 patients showed fibrosis in the dermis extending to the subcutaneous fat, and this association has been suggested in two recent reviews [6,7]. Penas et al. [2] suggested that septal panniculitis should be described as a histological type of scleroder-

ĂźanlĂ&#x2022; et al. Clinical aspects of sclerodermatous type GVHD

matous GVHD, and they found septal panniculitis in 6 (50%) of the 12 patients with biopsy specimens available for evaluation. In the present study, we observed septal panniculitis and clinical fasciitis in only 1 patient. In our study, the rate of lichen sclerosus-like lesions was lower (4.4%) than in the previously reported (29-47%) studies [2,3]. These lesions progressed to sclerotic areas in the late phases of the disease. Considering the retrospective nature of the present study, it is possible that those patients with early lichenoid lesions might have been missed, and the actual incidence might not have been lower than the reported studies. The presence of both lichen sclerosus-like lesions and the histological findings of septal panniculitis in the disease process suggest that the sclerosis in sclerodermatous GVHD can start and affect any level of the skin and can extend to involve the complete dermis, the subcutis, and even the fascia. Despite the fact that sclerodermatous GVHD and scleroderma have some similarities in cutaneous fibrosis, a recent study comparing the dermal microvasculature in sclerodermatous GVHD to scleroderma suggested that sclerodermatous GVHD is a suitable model for studying dermal sclerosis but may not be applicable for studying the microvascular alterations characteristic of scleroderma. Focal capillary proliferation occurs in early sclerodermatous GVHD; however, loss of endothelial markers and dermal capillaries is seen in scleroderma but not in sclerodermatous GVHD [10]. Authors have stated that leopard skin eruption-like pigmentary changes (widespread, well-demarcated, hyperpigmented macules) precede, almost constantly, the development of evident sclerosis and are very distinctive [2,11]. We observed these changes preceding the sclerotic lesions in 8 (36.4%) patients. Poikiloderma was described as a frequent finding in the first reports of cGVHD [1]. Since then, it has been infrequently described [3]. In our study, we found poikiloderma in 3 patients (13.6%). Eccrine hydrocystoma has not yet been described in association with cutaneous GVHD. We found eccrine hydrocystoma in 1 patient. Here, the obstruction of the eccrine duct via sclerosis may be responsible for the development of eccrine hydrocystoma. Sclerodermatous cGVHD can give rise to reduced range of motion and secondary effects including loss of strength, endurance and functional capabilities. In our set, joint retractions and dysphagia were found in 2 (9%) patients. Acrosclerosis and Raynaud phenomenon, which are commonly seen in progressive scleroderma, are not frequent in sclerodermatous cGVHD [12]. None of our patients with sclerodermatous GVHD showed this phenomenon or underwent an edematous phase of systemic scleroderma. No female predominance was found. Sclerodermatous lesions tend to affect the trunk and proximal

Turk J Hematol 2010; 27: 91-8

extremities while distal parts of the extremities were spared. Autoimmune markers like SCL-70 and ACA were negative. All of these data suggest that, although patients fulfill some criteria for systemic sclerosis, both diseases could have different etiopathogeneses. In cGVHD, oral lesions are seen in approximately 80% of the patients. Oral manifestations may include xerostomia, lichen planus-like changes, reticular whitish plaques, erosions and ulcerations, and submucosal fibrosis [6,7,13]. Sicca syndrome of the eyes and the mouth can be seen and pyogenic granuloma formation has been reported as a rare finding [6,7,14]. Oral mucosa involvement was present in 13 (59%) of our patients. The most frequent findings were mucosal ulcerations and erosions. Survival rates for patients with cGVHD are approximately equal to rates for patients without cGVHD, regardless of treatment. Spontaneous resolution of sclerodermatous GVHD may occur. However, none of our patients showed spontaneous resolution of the lesions. cGVHD has direct influence on both mortality and morbidity. The most important causes of mortality are infections, liver dysfunction and cachexia [7]. Of the 22 patients that we studied, 5 patients died due to late transplantrelated complications. Numerous treatments, including prednisone, azathioprine, penicillamine, CsA, methotrexate, MMF, thalidomide, clofazimine, anti-CD20 monoclonal antibody, ECP, phototherapy with bath PUVA, UVA1 or UVB, etretinate or various combinations, have been tried with varying success in sclerodermatous GVHD [2,3,5,15-22]. The best therapeutic response has been achieved with etretinate, ECP, high doses of steroid and azathioprine, and methotrexate [2,5,15,16]. Anti-CD20 monoclonal antibody has been found to have significant activity in the treatment of refractory sclerodermatous GVHD [22]. Imatinib mesylate 400 mg/day, which enables inhibition of fibroblast growth and decreased collagen production via inhibition of the transforming growth factor beta (TGFbeta) and platelet-derived growth factor (PDGF) pathways, is found to be effective especially in patients with refractory sclerodermatous cGVHD [23]. We achieved partial response in 5 patients showing extensive sclerodermatous changes. Patients treated with ECP alone or ECP plus MMF, CsA or prednisone showed statistically significant improvement compared to patients treated with other treatment regimens (p=0.03). Several retrospective and prospective studies have shown the efficacy of ECP in the management of cGVHD [24]. These reports included patients who were not responsive to at least one line of therapy, in most cases steroids and CsA, and ECP had been used as adjunctive treatment. Similar to our patients, the effect of treatment

üanlÕ et al. Clinical aspects of sclerodermatous type GVHD

Turk J Hematol 2010; 27: 91-8

appeared after two or three months and was generally maximal after six months. The mechanism of action of ECP in GVHD is still not entirely understood. However, several studies seem to support the hypothesis that ECP operates as an immunological response modifier [24]. In addition, ECP causes PUVA damage to T cells and stimulates the differentiation of monocytes into active dendritic antigenpresenting cells. We achieved partial response in 1 patient receiving MMF and PUVA combination. Response was seen at the third month of the therapy. The number of PUVA therapies in this patient was 48 and the total UVA dose was 135 J/cm2. MMF alone was not found to be effective in 2 patients. In conclusion, sclerodermatous GVHD has a late onset and clinically may be quite disabling. Unlike scleroderma, acral involvement is seen rarely and most patients have trunk involvement with widespread, well-demarcated, hyperpigmented macules. Moderate or severe cGVHD precedes generalized sclerodermatous involvement in most patients. Although most lesions do not disappear in the course of the disease and are therapy-resistant, most patients have a good prognosis.

10.

11.

12. 13.

14.

15.

References 1. 2.

6. 7.

Graham Brown RAC, Sarkany I. Scleroderma like changes due to chronic graft versus host disease. Clin Exp Dermatol 1983;8:531-8. Peñas PF, Jones-Caballero M, Aragüés M, Fernández-Herrera J, Fraga J, García-Díez A. Arch Dermatol. 2002 Jul;138(7):924-34. PMID: 12071820 Chosidow O, Bagot M, Vernant JP, Roujeau JC, Cordonnier C, Kuentz M, Wechsler J, André C, Touraine R, Revuz J. Sclerodermatous chronic graft-versus-host disease: analysis of seven cases. J Am Acad Dermatol 1992;26:49-55. Shulman HM, Sale GE, Lerner KG, Barker EA, Weiden PL, Sullivan K, Gallucci B, Thomas ED, Storb R. Chronic cutaneous graft-versus-host disease in man. Am J Pathol 1978;91:545-70. Skert C, Patriarca F, Sperotto A, Cerno M, Filì C, Zaja F, Stocchi R, Geromin A, Damiani D, Fanin R. Sclerodermatous chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation: incidence, predictors and outcome. Haematologica 2006;91:258-61. Aractingi S, Chosidow O. Cutaneous graft-versus-host disease. Arch Dermatol 1998;134:602-12. Johnson ML, Farmer ER. Graft versus host reactions in dermatology. J Am Acad Dermatol 1998;38:369-92.

16.

17.

18.

19.

20.

Filipovich AH, Weisdorf D, Pavletic S, Socie G, Wingard JR, Lee SJ, Martin P, Chien J, Przepiorka D, Couriel D, Cowen EW, Dinndorf P, Farrell A, Hartzman R, Henslee-Downey J, Jacobsohn D, McDonald G, Mittleman B, Rizzo JD, Robinson M, Schubert M, Schultz K, Shulman H, Turner M, Vogelsang G, Flowers ME. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versushost disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 2005;11:945-56. Magro CM, Kerns MJ, Votava H, Vasil KE, Dyrsen ME, Morrison CD. Early-onset lichenoid graft-vs.-host disease: a unique variant of acute graft-vs.-host disease occurring in peripheral blood stem cell transplant recipients. J Cutan Pathol. 2009 Oct 15. [Epub ahead of print] Fleming JN, Shulman HM, Nash RA, Johnson PY, Wight TN, Gown A, Schwartz SM. Cutaneous chronic graft-versus-host disease does not have the abnormal endothelial phenotype or vascular rarefaction characteristic of systemic sclerosis. PLoS One. 2009;4:e6203. Roujeau JC, Revuz J, Touraine R. Graft versus host reactions. In: Rook A, Savin J, editors. Recent Advances in Dermatology. Vol 5. New York: Churchill Livingstone Inc, 1980: 131-57. Belch J. Raynoud’s phenomenon: its relevance to scleroderma. Ann Rheum Dis 1991;50:839-45. Atkinson K. Chronic graft versus host disease. Bone Marrow Transplant. 1990;5:69-82. Lee L, Miller PA, Maxymiw WG, Messner HA, Rotstein LE. Intraoral pyogenic granuloma after allogeneic bone marrow transplantation. Report of three cases. Oral Surg Oral Med Oral Pathol 1994;78:607-10. Marcellus DC, Altomonte VL, Farmer ER, Horn TD, Freemer CS, Grant J, Vogelsang GB. Etretinate therapy for refractory sclerodermatous chronic graft-versus-host disease. Blood 1999;93:66-70. Greinix HT, Volc-Platzer B, Rabitsch W, Gmeinhart B, GuevaraPineda C, Kalhs P, Krutmann J, Hönigsmann H, Ciovica M, Knobler RM. Successful use of extracorporeal photochemotherapy in the treatment of severe acute and chronic graft-versus-host disease. Blood 1998;92:3098-104. Vogelsang GB, Wolff D, Altomonte V, Farmer E, Morison WL, Corio R, Horn T. Treatment of chronic graft-versus-host disease with ultraviolet irradiation and psoralen (PUVA). Bone Marrow Transplant 1996;17:1061-7. Ghoreschi K, Thomas P, Penovici M, Ullmann J, Sander CA, Ledderose G, Plewig G, Kolb HJ, Röcken M. PUVA-bath photochemotherapy and isotretinoin in sclerodermatous graft-versus-host disease. Eur J Dermatol 2008;18:667-70. Wetzig T, Sticherling M, Simon JC, Hegenbart U, Niederwieser D, Al-Ali HK. Medium dose long-wavelength ultraviolet A (UVA1) phototherapy for the treatment of acute and chronic graft-versus-host disease of the skin. Bone Marrow Transplant 2005;35:515-9. Parker PM, Chao N, Nademanee A, O'Donnell MR, Schmidt GM, Snyder DS, Stein AS, Smith EP, Molina A, Stepan DE,

21.

22.

üanlÕ et al. Clinical aspects of sclerodermatous type GVHD

Kashyap A, Planas I, Spielberger R, Somlo G, Margolin K, Zwingenberger K, Wilsman K, Negrin RS, Long GD, Niland JC, Blume KG, Forman SJ. Thalidomide as salvage therapy for chronic graft-versus-host disease. Blood 1995;86:3604-9. Lee SJ, Wgner SA, McGarigle CJ, Bierer BE, Antin JH. Treatment of chronic graft-versus-host disease with clofazimine. Blood 1997;89:2298-302. Ratanatharathorn V, Ayash L, Reynolds C, Silver S, Reddy P, Becker M, Ferrara JL, Uberti JP. Treatment of chronic graft-

Turk J Hematol 2010; 27: 91-8

23.

24.

versus-host disease with anti-CD20 chimeric monoclonal antibody. Biol Blood Marrow Transplant 2003;9:505-11. Magro L, Catteau B, Coiteux V, Bruno B, Jouet JP, Yakoub-Agha I. Efficacy of imatinib mesylate in the treatment of refractory sclerodermatous chronic GVHD. Bone Marrow Transplant 2008;42:757-60. Carcagnì MR, De Aloe G, D'Ascenzo G, Rubegni P, Fimiani M. Extracorporeal photopheresis in graft-versus-host disease. J Dtsch Dermatol Ges 2008;6:451-7.

Research Article

New in vitro effects of clopidogrel on platelets in hyperlipidemic and healthy subjects Hiperlipidemili ve saúlÕklÕ olgularda trombositler üzerinde klopidogrelin yeni in vitro etkileri Derya ÖzsavcÕ1, Azize üener1, Rabia Oba1, Gülderen YanÕkkaya Demirel2, Fikriye Uras1, Turay Kevser YardÕmcÕ1 1Department 2Centro

of Biochemistry, Marmara University, Faculty of Pharmacy, ûstanbul, Turkey Laboratary, ûstanbul, Turkey

Abstract Objective: We aimed to detect novel in vitro effects of clopidogrel on platelets by assessment of the following parameters: malondialdehyde, glutathione, nitrite, aggregation response, and expressions of P-selectin, fibrinogen, apolipoprotein A1, apolipoprotein B, and phosphatidylserine. Materials and Methods: Platelets were obtained from healthy (n: 9) and hyperlipidemic (n: 9) volunteers. Expressions of P-selectin, fibrinogen, apolipoproteins A1/B and phosphatidylserine with and without clopidogrel were assayed by flow cytometry. Malondialdehyde, glutathione, aggregation and nitrite levels were also assayed. Results: Without clopidogrel, the baseline values of platelet aggregation, malondialdehyde, and expressions of P-selectin, fibrinogen and phosphatidylserine were significantly higher, whereas nitrite and expression of apolipoproteins A1/B were significantly lower in hyperlipidemics than in the healthy group. In both groups, clopidogrel significantly reduced aggregation and expression of fibrinogen, but it elevated nitrite levels. Clopidogrel significantly decreased P-selectin and phosphatidylserine expression and malondialdehyde but increased expressions of apolipoproteins A1/B only in hyperlipidemics. Conclusion: It seems that clopidogrel has some new in vitro antiplatelet effects. The present study is a basic in vitro study to suggest new insights into the effects of clopidogrel on platelet functions. (Turk J Hematol 2010; 27: 99-108) Key words: Platelets, hyperlipidemia, clopidogrel, apolipoprotein A1, apolipoprotein B, oxidative stress, nitrites, phosphatidylserine, P-selectin, glycoproteins IIb/IIIa Received: July 19,2009

Accepted: January 25, 2010

Özet Amaç: Klopidogrelin trombositler üzerinde yeni in vitro etkilerini tayin etmek: Malondialdehit, glutatyon, nitrit, aggregasyon cevabÕ, P-selektin, fibrinojen, apolipoprotein A1, apolipoprotein B ve fosfatidilserin ekspresyonlarÕ. Yöntem ve Gereçler: SaúlÕklÕ (n: 9) ve hiperlipidemik (n: 9) olgulardan trombositler elde edildi. Klopidogrelli ve klopidogrelsiz trombositlerde P-selektin, fibrinojen, apolipoprotein A1, apolipoprotein B ve fosfatidilserin ekspresyonlarÕ flow sitometre ile tayin edildi. Malondialdehit, glutatyon, aggregasyon ve nitrit seviyeleri de tayin edildi. Bulgular: Klopidogrel yokluúunda, hiperlipidemililerde kontrollere göre trombosit agregasyonu, malondialdehit, P-Selektin, fibrinojen ve fosfatidilserin ekspresyonunun baýlangÕç deúerleri yüksek; bununla birlikte nitrit, apolipoprotein A1 ve apolipoprotein B ekspresyonlarÕnÕnki ise daha düýüktü. Her iki grupta, klopidogrel anlamlÕ düzeyde aggregasyonu ve fibrinojen ekspresyonunu azalttÕ, fakat nitrit seviyelerini artÕrdÕ. Klopidogrel sadece hiperlipidemililerde P-selektin ve fosfatidilserin ekspresyonunu ve malondialdehiti azalttÕ ancak apolipoprotein A1 ve apolipoprotein B ekspresyonlarÕnÕ artÕrdÕ.

Address for Correspondence: Asst. Prof. Derya ÖzsavcÕ, Tibbiye Cad. No: 49, Haydarpasa, Kadiköy, ûstanbul,Turkey Phone: +90 216 450 27 73 E-mail: deryaozsavci@hotmail.com doi:10.5152/tjh.2010.07

100

ÖzsavcÕ et al. New effects of clopidogrel on platelets

Turk J Hematol 2010; 27: 99-108

Sonuç: Görüldüúü üzere klopidogrel bazÕ yeni in vitro antiplatelet etkilere sahiptir. Bu çalÕýma, trombosit fonksiyonlarÕ üzerinde klopidogrelin etkilerine yeni bir bakÕý açÕsÕ saúlayan temel in vitro bir çalÕýmadÕr. (Turk J Hematol 2010; 27: 99-108) Anahtar kelimeler: Trombositler, hiperlipidemi, klopidogrel, Apolipoprotein A-I, Apolipoprotein B, oksidatif stres, nitrite, fosfatidilserin, P-selektin, Glikoprotein IIb/IIIa Geliý tarihi: 19 Temmuz 2009

Kabul tarihi: 25 Ocak 2010

Introduction Atherothrombotic events due to vasoactivity, inflammation and also lipids may affect platelet function. Antiplatelet therapy is one of the most effective therapies for treatment of atherothrombotic and other associated diseases [1-4]. Platelet composition and function abnormalities in patients with hyperlipoproteinemia suggest that the circulating levels of lipids influence the platelet markers such as P-selectin [5]. In fact, it has been reported that increased low-density lipoprotein (LDL), especially oxidized-LDL, reduces platelet and macrophage nitric oxide (NO) synthase expression, and thus platelet activation increases [6]. There is some evidence of these lipoproteins interacting with platelets via specific receptors. It has been reported that there are high-density lipoprotein (HDL) and LDL binding sites on platelets, and those binding sites are reduced in familial hyperlipidemia [7,8]. In our previous study, we had detected that apolipoprotein-A1 (anti-apo-A1) was able to bind to the platelet surface indicating the presence of apo-A1 binding sites on platelets by a flow cytometric method [9]. On the other hand, the expression of P-selectin, fibrinogen, glycoprotein (Gp) IIb/IIIa, and circulating platelet-derived microparticles (PDMPs) on platelets are used as clinical markers for platelet activation status in several disorders including hyperlipidemia [10-13]. Additionally, occurrence of apoptosislike events in platelets has been confirmed by in vivo and in vitro studies. Phosphatidylserine (PS) appears on the outer cell membranes during the early stages of apoptosis. PS exposure serves as a procoagulant stimulus and a signal for phagocytic clearance of apoptotic cells including platelets as well as being an activation marker [14-16]. Reactive oxygen species have a number of important pro-atherogenic effects such as oxidation of LDL and activation of platelets [17]. It has been reported that in vivo platelet “release reaction” and malondialdehyde (MDA) formation are increased in hyperlipidemic patients [18]. The target of most of the antiplatelet treatments is either platelet agonist receptors or platelet fibrinogen receptor GpIIb/IIIa [19]. Several antiplatelet drugs have been developed to inhibit platelet activity in acute thrombotic events [20]. Clopidogrel, which is a thienopyridine derivative, blocks platelet aggregation irreversibly by binding to P2Y12 , one of the adenosine diphosphate (ADP) receptors on platelets. Clopidogrel affects GpIIb/IIIa complex activation. Several studies have reported that the essential action of thienopyri-

dine derivatives occurs upon their derivatization in the liver [21-23]. However, various other studies have reported that clopidogrel had different in vitro antiplatelet effects on platelets [24-26]. Although those studies have been carried out in different mediums such as whole blood, isolated platelets or platelet-rich plasma (PRP), the consensus of those studies is that hepatic biotransformation is not required for platelet inhibition by clopidogrel, which seems contrary to most of the in vivo studies. Considering those studies, our aim was to investigate some new effects of clopidogrel on platelets, which were obtained from healthy and hyperlipidemic volunteers under in vitro conditions. The novel in vitro effects of clopidogrel on the following parameters were investigated: apo A and apolipoprotein B (apo B) binding sites, expression of P-selectin and fibrinogen, PS exposure on the surface of platelets, aggregation response, MDA, glutathione (GSH) and nitrite levels in platelets. We think that our findings obtained under in vitro conditions will be useful in future studies to determine new targets for the drug.

Materials and Methods Subjects and Blood Sampling The study group consisted of 9 hyperlipidemic (7 males and 2 females) patients and 9 normolipidemic, age- and sexmatched control subjects (6 males and 3 females). This study was performed in accordance with the Declaration of Helsinki. Written evidence of informed consent was obtained from each participant. Table 1 shows some characteristics of the Table 1. Some characteristics of the healthy and hyperlipidemic groups Healthy Group Hyperlipidemic Group n Age, years

46 ±4.65

49±6.96 (NS)

Male/Female

6/3

7/2 (NS)

BMI, kg/m2

24.11±3.21

28.55±2.92*

T-CHO, mmol/L

4.66±0.20

7.70±0.36***

LDL-C, mmol/L

2.98±0.24

5.50±0.31***

VLDL-C, mmol/L

0.57±0.22

1.13±0.13***

TG, mmol/L

1.32±0.18

2.44±0.23***

HDL-C, mmol/L

1.51±0.25

0.94±0.14**

BMI: Body mass index; T-CHO: Total cholesterol; LDL-C: Low density lipoprotein cholesterol; VLDL-C: Very low density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; TG: Triglyceride; * p<0.05, ** p<0.01, *** p<0.001, NS: not significant

Turk J Hematol 2010; 27: 99-108

dyslipidemic group and healthy volunteers. Apo A and apo B levels were within the normal ranges. Exclusion criteria were: secondary hyperlipidemia (renal, liver, thyroid and cardiovascular disease), fasting glucose > 6.1 mmol/L, HbA1c (%) > 6, alcohol consumption, smoking, and use of anti-platelet, anticoagulant or lipid-lowering drugs within the previous eight weeks. All subjects were normotensive (<140/90 mmHg) and fasting glucose levels were in the normal range. The levels of total cholesterol (T-CHO) and triglyceride (TG) were determined by enzymatic methods using test kits with a Hitachi 917 analyzer. HDL-cholesterol (C) was measured by the dextran sulfate-Mg+2 precipitation method. LDL-C and very lowdensity lipoprotein (VLDL)-C were calculated by the formula of Friedewald. Fasting venous blood samples were obtained with a 21-gauge butterfly needle from healthy and hyperlipidemic volunteers. Whole blood was collected into tubes containing 3.2% sodium citrate and used for flow cytometric analysis. PRP was used for aggregation and other analysis. Drug Clopidogrel was obtained from Sanofi Synthelabo, France. In the reactions, 10 M clopidogrel was used at a final concentration, which is close to the therapeutic concentration in human plasma reported by Cruz et al. [24]. Flow Cytometric Analysis Expressions of P-selectin and fibrinogen on the surface of platelets were assayed according to the modified flow cytometric analysis method of Shattil et al. [27] as previously described. Briefly, citrated whole blood was diluted (1:10) in phosphate-buffered saline (PBS) (8 mM NaH2PO4, 5 mM KCl, 125 mM NaCl, 5 mM glucose and 0.5 g/L albumin) and mixed gently. Whole blood (50 L) was incubated with clopidogrel (10 M) for 5 min at 37oC. The following steps were not used so that platelet activation during the assay could be minimized: washing, centrifugation, gel filtration, and mixing vigorously. All blood samples were stimulated with ADP (10 M) for 5 min. Then, they were added to polypropylene tubes containing the appropriate amounts of fluorescein isothiocyanate (FITC)-labeled antibodies such as anti-CD62P-FITC (to detect platelet activation marker, P-selectin) (Immunotech, Coulter) or anti-fibrinogen-FITC (to detect platelet activation marker, platelet bound fibrinogen) (polyclonal rabbit antihuman fibrinogen-FITC, Dako Cytomation). The samples were incubated for 15 min at room temperature without stirring, and then an equal volume of 0.02 g/ml para-formaldehyde was added to stop the reaction. In parallel experiments, the same procedure was performed without addition of the drug. The samples were further diluted with PBS and kept in the dark at 4°C until flow cytometric measurement.

ÖzsavcÕ et al. New effects of clopidogrel on platelets

101

Diluted whole blood samples (with or without the drug) were stimulated with ADP (10 M) for 10 min to detect PS exposure (platelet early apoptosis) on the surface of the platelets. It has been reported that PS exposure on the surface of cells is commonly measured by flow cytometry of fluorescently labeled annexin V binding to these procoagulant phospholipids. Annexin V-FITC (25 g/ml) (Immunotech Coulter) was added to the samples and the tubes were kept on ice for 10 min. Then, 400 L of ice-cold binding buffer (10 mM HEPES/NaOH (pH 7.4) 140 mM NaCl, 2.5 mM CaCl2) was added to the blood samples, and flow cytometric analysis was performed. We used the same method in our previous study with a small modification for flow cytometric assay for apo-A1 and apo B, which were bound to the surface of platelets [9]. Diluted whole blood samples (with or without clopidogrel) were stimulated with ADP (10 M) for 5 min. Monoclonal antibodies (MoAbs) against apo-A1 or apo B were added (1/100 diluted as optimal Moab concentration). After 15 min incubation at room temperature in the dark, 5 L of FITC-labeled secondary antibody was added to the tubes. Then, the tubes were mixed and incubated for 15 min at room temperature in the dark. The tubes were centrifuged and the cells were washed with PBS two or three times by centrifugation to remove unbound FITClabeled secondary antibody. Progression of the reaction was stopped by adding an equal volume of 0.02 g/ml para-formaldehyde. The samples were diluted with PBS and kept at 4°C prior to flow cytometric analysis. All the samples, which were prepared as described above, were analyzed on a Beckman Coulter, EPICS XL-MCL flow cytometer. The flow cytometer was equipped with a 488 nm argon ion laser. The platelet population was identified by forward scatter for cell size and by side scatter for cell granularity. Alignment of the instrument was checked by calibration beads daily. An electronic bitmap was placed around the platelet population and CD41a-FITC was used to form a gate. The results were expressed as the percentage of antibodypositive platelets. Nonspecific and background fluorescence was determined by the use of FITC conjugated immunoglobulin G (IgG). Fifty thousand platelets were counted in each tube. Platelet Aggregation Platelet-rich plasma and platelet-poor plasma (PPP) were prepared by differential centrifugation. PRP was obtained by centrifugation of citrated whole blood for 8 min at 1500 rpm. PPP was obtained by centrifugation of PRP for 10 min at 3500 rpm. Clopidogrel was incubated with PRP (500 L) for 10 min. ADP (Chronolog, final concentration 5 M) was used to stimulate platelet aggregation. The aggregation responses were assayed on Chronolog-Lumi aggregometer and recorded as the mean percentage of maximum platelet aggregation both with and without clopidogrel.

102

ÖzsavcÕ et al. New effects of clopidogrel on platelets

Turk J Hematol 2010; 27: 99-108

before clopidogrel before clopidogrel

after clopidogrel

100

101

102 103 APO A1 FITC

after clopidogrel

100

104

101 102 APO B FITC

103

100

104

101

before clopidogrel

102 CD62P-FITC

103

104

after clopidogrel after clopidogrel before clopidogrel

before clopidogrel

100 100

101

102

103

104

101

102

103

104

Annexin V-FITC

antifibrinogen-FITC

Figure 1. Flow cytometric histograms of antibodies (ApoA, Apo B, CD62P-, antifibrinogen, annexinV) in a hyperlipidemic patient with or without clopidogrel. Mean channel values were used for the evaluation of negative and positive results. An unstained sample and another sample combined with related monoclonal antibodies (without clopidogrel) were used as negative controls. More than 20% shift in channel numbers is regarded as a cut off point for the negative/positive results

GSH and MDA Analyses Platelet-rich plasma (1.5 ml) was incubated with clopidogrel (10 M) at 37°C for 15 min. After centrifugation at 10,000 rpm for 15 min, the platelet pellet was obtained. After washing twice with Tris-NaCl EDTA buffer, the platelet pellet, which was treated either with or without clopidogrel, was suspended in distilled water. It was frozen and thawed four times and then centrifuged at 10,000 rpm for 15 min. The supernatant was used for GSH assay and 15% metaphosphoric acid was added according to the Mergel and Anderman method [28]. The results were expressed as microgram per 109 platelets. The precipitate was solubilized with Tris-NaCl buffer containing 1% Triton X-100 for 8 h and then centrifuged. After solubilization, platelet crude membrane MDA levels were assayed as a product of lipid peroxidation in supernatants with thiobarbituric acid reactive substances (TBARS) method [29]. The results were expressed as nmoL/mg crude protein. The protein concentration was determined according to the Bradford method and bovine serum albumin was used as the standard [30]. The sensitivities of GSH and MDA are as follows: GSH: 0.4-100 M,and MDA: 0.1 M. Nitrite Assay After centrifugation of PRP at 10,000 rpm for 15 min, the platelet pellet was obtained and washed twice with PBS buffer. The washed platelets were incubated with clopidogrel (10 M) and 1.44 mmol/L NADPH for 1 h at 37°C. Then, each sample, treated with or without clopidogrel, was incubated for 1 h at

37°C after the addition of 20 mU nitrate reductase, which reduced nitrate to nitrite. The platelets were frozen and thawed four times. After centrifugation, the supernatant was allowed to react with Griess reagent to form a chromophore; its absorption was measured subsequently at 546 nm. Sodium nitrite (0.2 to 4 M) was used as the standard [6]. The sensitivity of nitrite was 2 M. Statistical Analysis The results were presented as mean ± SD. Mean differences between the healthy and patient groups were calculated with the nonparametric Mann-Whitney U test. Analyses before and after the drug addition were compared using the Wilcoxon signed-rank test. Statistical analyses were performed with SPSS software. P values <0.05 were considered significant.

Results Figure 1 shows Flow cytometric histograms of antibodies (ApoA, Apo B, CD62P-, antifibrinogen, annexinV) in a hyperlipidemic patient with or without clopidogrel. Figure 2A shows the expression of fibrinogen and Figure 2B shows the expression of P-selectin on the ADP-stimulated platelet surface. Before clopidogrel was added, there were significant differences in the expression of both P-selectin (%) and fibrinogen (%) between the hyperlipidemic and the healthy groups. In both groups, the expression of fibrinogen (%) was significantly decreased after

Ă&#x2013;zsavcĂ&#x2022; et al. New effects of clopidogrel on platelets

Turk J Hematol 2010; 27: 99-108

clopidogrel; however, clopidogrel significantly decreased P-selectin expression on platelets only in hyperlipidemics. Phosphatidylserine exposure with ADP-stimulated platelets is shown in Figure 3. Before clopidogrel, there were significant differences in PS exposure (annexin V) between the hyperlipidemic and the healthy groups. After clopidogrel, platelet PS exposure (annexin V) was significantly decreased in the hyperlipidemic group, but not in the healthy group. We measured the levels of MDA and GSH in platelets. Figure 4A shows the levels of MDA and Figure 4B shows the levels of GSH before and after clopidogrel in the healthy and hyperlipidemic groups. Before clopidogrel, there were significant differences in the levels of MDA between the hyperlipidemic group and the healthy group, but not in GSH levels. MDA levels significantly decreased after clopidogrel in the hyperlipidemic group. On the other hand, GSH levels remained unchanged after clopidogrel in both groups Platelet aggregation responses before and after clopidogrel are shown in Figure 5. Before clopidogrel, there were significant differences in the aggregation (%) status between the hyperlipidemic patients and the healthy volunteers. After clopidogrel, platelet aggregation response significantly decreased in both healthy and hyperlipidemic groups (Figure 5A). Figure 5B shows the aggregation responses before and after clopidogrel in the hyperlipidemic group. Nitrite levels in platelets are shown in Figure 6. Before clopidogrel, there were significant differences in the levels of nitrite between the hyperlipidemic and the healthy groups. In both groups, platelet nitrite levels significantly increased after clopidogrel. Figure 7A shows the expression of platelet apo-A1 before and after clopidogrel in the hyperlipidemic group and healthy volunteers. Before clopidogrel, there were significant differences in the expressions of apo-A1 and apo B on the platelet surface between the hyperlipidemic patients and the healthy group. The expression of platelet apo-A1 significantly increased after clopidogrel in the hyperlipidemic group, but did not change in the healthy group. Figure 7B shows the expression of platelet apo B before and after clopidogrel in the hyperlipidemic and healthy groups. The apo B expression on the platelet surface significantly increased after clopidogrel in the hyperlipidemic group, but did not change in the healthy group.

metric method, we found that both P-selectin and fibrinogen expressions on the surface of platelets were significantly higher in the hyperlipidemic group compared to the healthy group. In our previous study, we had detected that the receptor numbers of GpIIb/IIIa, GpIIIa and P-selectin were significantly higher in hyperlipidemic patients [12]. In our present study, we confirmed our previous findings indicating that platelets are activated in the circulation in hyperlipidemic patients. In this study, we also observed that PS exposure, lipid peroxidation and aggregation response of platelets were all higher, whereas platelet nitrite levels were lower in hyperlipidemics than in the healthy group. It has been shown that increased PS exposure is an important signal of early apoptosis, platelet activation and procoagulant stimulus [14-16,35,36]. The increase of PS exposure, which we observed in this study, may be related to increased oxidative stress (high MDA levels), because oxidative stress is one of the mediators of apoptosis [37]. Due to the increasing platelet aggregation response and PS exposure, we can speculate that platelets from hyperlipidA HL+Clopidogrel

***

Normal+Clopidogrel

Normal 0

It has been reported that circulating activated platelets and increased thrombotic risk are related to many cardiovascular events such as angioplasty, stroke, diabetes mellitus, and hyperlipidemia [31-34]. In the present study, using a flow cyto-

100

Fibrinogen expression (%)

B +

HL+Clopidogrel

***

Normal+Clopidogrel

Normal

Discussion

103

P-selection expression (%)

Figure 2. Mean values for expressions of fibrinogen (A) and P-selectin (B) in platelets of different study groups. Normal: Platelets from the healthy group; HL: Platelets from the hyperlipidemic group *** P <0.001 vs normal a, P <0.01 vs normal +, P <0.01 vs HL

Ă&#x2013;zsavcĂ&#x2022; et al. New effects of clopidogrel on platelets

104

Turk J Hematol 2010; 27: 99-108

emic subjects are more susceptible to ADP and the apoptosisactivation process. In fact, it has been reported that elevated LDL, especially oxidized-LDL, may reduce NO synthase expression in platelets and macrophages. Due to the decrease in NO, platelet activation and cellular production of oxygen radicals *** 50

Annexin-V (PS exposure (%)

Normal Normal+Clopidogrel

HL HL+Clopidogrel

may increase [38]. The measurement of nitrite levels instead of NO has been used in several studies since it is an indirect indication of NO production. As it is known, when LDL is at high concentrations and when it is together with an agonist such as ADP (as in our study), it triggers platelet hyperactivity and vascular damage as an independent stimulant [39,40]. Therefore, in this study, both elevated plasma LDL levels and platelet low nitrite levels observed in the hyperlipidemic subjects may be some of the factors responsible for the increase in platelet activation, platelet lipid peroxidation and apoptosis. One of the platelet abnormalities, observed in the hyperlipidemic group, is the change in membrane receptor responses. It has been shown that patients with

20 10

Aggregation (%)

Figure 3. Mean values of annexin-V (PS exposure) in platelets of different study groups. Normal: Platelets from the healthy group; HL: Platelets from the hyperlipidemic group ***, P <0.001 vs normal +, P <0.01 vs HL

90 80 70

60 50 40

Normal Normal+Clopidogrel HL HL+Clopidogrel

30 20 10 0

A 0.15

Normal

ADP

MDA (nmol/mg protein)

Normal+Clopidogrel 0.10

HL HL+Clopidogrel Clopidogrel+ADP

0.05

Figure 5. (A) Platelet aggregation responses (%) of the study groups. Normal: Platelets from the healthy group; HL: Platelets from the hyperlipidemic group * P <0.05 vs normal a, P <0.01 vs normal +, P <0.001 vs HL (B) Platelet aggregation plots from the hyperlipidemic group

0.0

7 Normal Normal+Clopidogrel

HL HL+Clopidogrel

Figure 4. Mean MDA (A) and GSH levels (B) in platelets of different study groups. Normal: Platelets from the healthy group; HL: Platelets from the hyperlipidemic group ** P <0.01 vs normal +, P <0.01 vs HL

Nitrite (nmol/mg protein)

GSM (+g/109 platelets)

Normal Normal+Clopidogrel

HL HL+Clopidogrel

4 3 2

1 0

Figure 6. Mean values of nitrite levels in platelets of different study groups Normal: Platelets from the healthy group; HL: Platelets from the hyperlipidemic group * P <0.05 vs normal a, P <0.01 vs normal +, P <0.001 vs HL

Ă&#x2013;zsavcĂ&#x2022; et al. New effects of clopidogrel on platelets

Turk J Hematol 2010; 27: 99-108

atherosclerosis, diabetes and hyperlipidemia have significantly lower platelet LDL and HDL binding sites [7,8,41]. In our study, expressions of both apo B (also LDL) and apo A (also HDL) on platelets were lower in the hyperlipidemic patients. This is possibly related to the reactivity potential of platelets in the hyperlipidemic group. Based on our findings, the increase in platelet activation, apoptosis, oxidative status (MDA), aggregation, altered platelet membrane binding features, and decreased platelet nitrite levels in hyperlipidemia might elevate thrombotic risk causing atherogenesis to progress. New risk assessment criteria for thrombosis and cardiovascular diseases [42,43] and new drugs [44,45] for inhibition of coagulation are very important research areas. Clopidogrel is a drug that has been used for the secondary prevention of atherothrombotic events related to ischemia [46]. Although there are many in vivo reports related to the effects of clopidogrel on platelet functions, there are also various reports about its in vitro effects on platelets [24-26]. In those in vitro studies, it was shown that hepatic biotransformation is not required for platelet inhibition by clopidogrel. According to the findings of the study of De La Cruz et

A 90

Anti-Apo A Binding (%)

80 70

Normal Normal+Clopidogrel HL HL+Clopidogrel

***

60 50 40 30 20 10 0

B 90 80

Anti-Apo B Binding (%)

70 60 50 40

***

Normal Normal+Clopidogrel HL HL+Clopidogrel

30 20 10 0

Figure 7. Mean values for expressions of apoA1 (A) and apoB (B) in platelets of different study groups. Normal: Platelets from the healthy group; HL: Platelets from the hyperlipidemic group *** P <0.001 vs normal +, P <0.001 vs HL

105

al. [24], clopidogrel in vitro reduced ADP-induced platelet aggregation and increased endothelial NO production depending on dose. Arrebola et al. [25] showed that thromboxane B2 production and collagen-induced platelet aggregation were inhibited, but prostacyclin synthesis did not change following in vitro incubation with clopidogrel. Weber et al. [26] reported that the in vitro inhibition of platelet aggregation by clopidogrel is selective for ADP and does not require hepatic bioactivation. Regarding their findings, the inhibitory effects of clopidogrel were selective for ADP because no inhibition of platelet aggregation was seen with collagen, thrombin or thromboxane A2. Additionally, they have shown that the antiaggregant activity of clopidogrel was associated with the platelets and was not dependent on the presence of the compound in the test buffer. They also discussed that platelets are capable of generating the putative active metabolite of clopidogrel. This possibility is supported by the time-dependence of platelet inhibitory actions of clopidogrel in their study. In rats, Savi et al. [47] reported that clopidogrel (40 mg kg<1) was less effective in hepatectomized rats as compared to normal control rats. In addition, clopidogrel did inhibit platelet aggregation in isolated, blood-perfused rat livers. It seems that our results support some of their findings, but we have also observed some novel in vitro effects of clopidogrel. We found that in vitro clopidogrel significantly increased platelet nitrite levels in both hyperlipidemic and healthy groups. In vitro and in vivo studies show that clopidogrel may stimulate NO production via a Ca-dependent way in different cells. Thienopyridines enhance endothelial NO production, which influences vascular wall endothelial function, platelet function and inflammation [48]. In our study, the same mechanism may be responsible for the effects of clopidogrel on nitrite production. It was reported in a study that there is a significant decrease in fibrinogen binding to GpIIb/IIIa by an aspirin and clopidogrel combination and a reduction in platelet P-selectin expression by clopidogrel alone [49]. Similarly, in our study, in the presence of ADP, clopidogrel reduced the fibrinogen expression on the platelet surface, and thereby, presumably decreased the platelet activation in both groups. On the other hand, in vitro addition of clopidogrel to platelets reduced increased P-selectin expression only in hyperlipidemic patients. Storey et al. [50] showed that clopidogrel and P2Y12 receptor antagonist suppressed platelet aggregation, P-selectin expression and platelet-leukocyte conjugate formation, whereas aspirin had no such inhibitory effect. In our study, we also observed that clopidogrel has reducing effects on PS exposure, MDA levels and platelet aggregation

106

ÖzsavcÕ et al. New effects of clopidogrel on platelets

response in hyperlipidemic patients. It has been reported that the antiaggregant effect of thienopyridines is more potent when aggregation is induced with ADP than with collagen. Although in this study we have not enlightened the exact mechanism of clopidogrel, we have speculated on the possible mechanisms inspired from in vitro and in vivo studies. Clopidogrel may block ADP-induced platelet aggregation via G protein activation and inhibition of adenyl cyclase or it may be totally independent from these factors. At the same time, clopidogrel may impair the transmembrane migration of PS, and thus PS exposure reduces. In conclusion, the present study is a basic in vitro study to suggest new insights into the effects of clopidogrel on platelet functions. We observed that platelets from hyperlipidemic subjects may be more susceptible to agonists, activation and apoptosis. Disturbed lipid metabolism in those subjects may lead to physiochemical changes in platelet response and consequently may result in altered expression of surface membrane proteins, apoA and apoB. Those events may contribute to the atherosclerotic process by elevating thrombotic risk in hyperlipidemics. We have shown some novel effects (for example apo A and apo B binding sites on platelets) of clopidogrel under in vitro conditions. To our knowledge, there is no in vivo study related especially with the effects of clopidogrel on those platelet binding sites. It seems that clopidogrel, which is not subjected to biotransformation, especially affects hyperlipidemic platelets by reducing platelet thrombotic response and by altering membrane specifications. We are hopeful that our findings obtained under in vitro conditions can be used in further studies to determine new benefits or side effects of clopidogrel. Acknowledgment: This research was supported in part by Marmara University Research Unit Grants SAG 033/131102 and SAG 070/060904. We are grateful to Mr. Nazmi Uzunosmanoglu for his contribution to the preparation of this manuscript.

Turk J Hematol 2010; 27: 99-108

2. 3.

10.

11.

12.

13.

14.

References 15. 1.

Waltermann A, Fritsch P, Kyrle PA, Schoenauer V, Heinze G, Wojta J, Christ G, Huber K. Effects of pretreatment with clopidogrel and platelet and coagulation activation in patients undergoing elective coronary stenting. Thromb Res 2003;112:19-24.

Meyer BJ. Antithrombotic drugs: insights from cardiology. Cerebrovasc Dis 1998;8 5:19-27. Patrono C, Coller B, FitzGerald GA, Hirsh J, Roth G. Plateletactive drugs: the relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126(Suppl):234S-264S. Cheng JW. Pharmacoeconomic analysis of clopidogrel in secondary prevention of coronary artery disease. J Manag Care Pharm 2007;13:326-36. Bröijersen A, Karpe F, Hamsten A, Goodall AH, Hjemdahl P. Alimentary lipemia enhances the membrane expression of platelet P-selectin without affecting other markers of platelet activation. Atherosclerosis 1998;137:107-13. Chen LY, Mehta P, Mehta JL. Oxidized LDL decreases L-arginine uptake and nitric oxide synthase protein expression on human platelets. Circulation 1996;93:1740-6. Virgolini I, Li S, Yang Q, Banyai M, Koller E, Angelberger P, Sinzinger H. Binding of 111In-labeled HDL to platelets from normolipidemic volunteers and patients with heterozygous familial hyperlipidemia. Arterioscler Thromb 1992;12:849-61. Virgolini I, Koller E, Li S, Yang Q, Banyai M, Rauscha F, Pidlich J, Pirker W, Sinzinger H. Etofibrate increases binding of low and high density lipoprotein to human platelets of patients with type II hyperlipoproteinemia. Atherosclerosis 1993;102:217-26. Ozsavci D, Yardimci T, Demirel GY, Uras F, Hekim N, Ulutin ON. Apo A1 binding to platelets detected by flow cytometry. Thromb Res 2001;103:117-22. Sener A, Ozsavci D, Oba R, Demirel GY, Uras F, Yardimci KT. Do platelet apoptosis, activation, aggregation, lipid peroxidation and platelet-leukocyte aggregate formation occur simultaneously in hyperlipidemia? Clin Biochem 2005;38:1081-7. Tetik S, Uras F, Ekýioúlu-Demiralp E, Turay Yardimci K. Lowdensity lipoprotein specifically binds glycoprotein IIb/IIIa: a flow cytometric method for ligand-receptor interaction. Clin Appl Thromb Hemost 2008;14:210-9 Ozsavci D, Yardimci T, Demirel GY, Demiralp E, Uras F, Onder E. Flow cytometric assay of platelet glycoprotein receptor numbers in hyperlipidemia. Platelets 2002;13:223-9. Choudhury A, Chung I, Blann A, Lip G. Elevated platelet microparticle levels in nonvalvular atrial fibrillation: relationship to p-selectin and antithrombotic therapy. Chest 2007;131:809-15. Tait JF, Smith C, Wood BL. Measurement of phosphatidylserine exposure in leukocytes and platelets by whole blood flow cytometry with annexin V. Blood Cells Mol Dis 1999;15:271-8. Kingston JK, Bayly WM, Sellon DC, Meyers KM, Wardrop KJ. Measurement of the activation of equine platelets by use of fluorescent-labeled annexin-V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody. Am J Vet Res 2002;63:513-9.

ÖzsavcÕ et al. New effects of clopidogrel on platelets

Turk J Hematol 2010; 27: 99-108

16.

17. 18.

19. 20. 21.

22. 23.

24.

25.

26.

27.

28.

29. 30.

31.

32.

Rand ML, Wang H, Bang KW, Poon KSV, Packham MA, Freedman J. Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steadystate senescence. J Thromb Haemost 2004;2:651-9. Olas B, Wachowicz B. Resveratrol and vitamin C as antioxidant in blood platelets. Thromb Res 2002;106:143-8. Zahavi J, Betteridge JD, Jones NA, Galton DJ, Kakkar VV. Enhanced in vivo platelet release reaction and malondialdehyde formation in patients with hyperlipidemia. Am J Med 1981;70:59-64. Harker LA. Therapeutic inhibition of platelet function in stroke. Cerebrovasc Dis 1998;8:8-18. Chen WH. Antiplatelet resistance with aspirin and clopidogrel: is it real and does it matter? Curr Cardiol Rep 2006;8:301-6. Patrono C, Coller B, Fitzgerald GA, Hirsh J, Roth G. Plateletactive drugs: the relationships among dose, effectiveness, and side effects. Chest 2004;126:234-64. Herbert JM, Savi P. P2Y12, a new platelet ADP receptor, target of clopidogrel. Semin Vasc Med 2003;3:113-22. Savi P, Herbert JM. Clopidogrel and ticlopidine: P2Y12 adenosine diphosphate receptor antagonists for the prevention of atherothrombosis. Semin Thromb Hemost 2005;31:174-83. De La Cruz JP, Arrebola MM, Villalobos MA, Pinacho A, Guerrero A, Gonzalez-Correa JA, Sanches de la Cuesta F. Influence of glucose concentration on the effects of aspirin, ticlopidine and clopidogrel on platelet function and platelet subendothelium interaction. Eur J Pharmacol 2004;484:19-27. Arrebola MM, De la Cruz JP, Villalobos MA, Pinacho A, Guerrero A, Sánchez de la Cuesta F. In vitro effects of clopidogrel on platelet-subendothelium interaction, platelet thromboxane and endothelial prostacyclin production, and nitric oxide synthesis. J Cardiovasc Pharm 2004;43:74-82. Weber A, Reimann S, Schrör K. Specific inhibition of ADPinduced platelet aggregation by clopidogrel in vitro. Br J Pharmacol 1999;126:415-20. Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987;70:307-15. Mergel DC, Anderman G, Andermann C. Simultaneous determination of oxidized and reduced glutathione in human rabbit red cells. Meth Find Exp Clin Pharmacol 1979;1:277-82. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302-10. Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248-54. Schwartz MB, Hawiger J, Timmons S, Friesinger GC. Platelet aggregates in ischemic heart disease. Thromb Haemost 1980;43:185-8. Omoto S, Nomura S, Shouzu A, Nishikawa M, Fukuhara S, Iwasaka T. Detection of monocyte-derived microparticles in patients with Type II diabetes mellitus. Diabetologia 2002;45:550-5.

33.

34.

35.

36. 37. 38.

39.

40.

41.

42.

43.

44.

45.

46. 47.

107

de Man HF, Nieuwland R, van der Laarse A, Romijn F, Smelt AH, Gevers Leuven JA, Sturk A. Activated platelets in patients with severe hypertriglyceridemia: effect of triglyceride-lowering therapy. Atherosclerosis 2000;152:407-14. Nimpf J, Wurm H, Kostner GM, Kenner T. Platelet activation in normo- and hyperlipoproteinemias. Basic Res Cardiol 1986;81:437-53. Li J, Xia Y, Bertino AM, Coburn JP, Kuter DJ. The mechanism of apoptosis in human platelets during storage. Transfusion 2000;40:1320-9. Perrotta PL, Perrotta CL, Snyder EL. Apoptotic activity in stored human platelets. Transfusion 2003;43:526-35. Buttke TM, Sandstrom PA. Oxidative stress as a mediator of apoptosis. Immunol Today 1994;15:7-10. Liu SX, Chen Y, Zhou M, Wan J. Oxidized cholesterol in ox-LDL may be responsible for the inhibition of LPS-induced NO production in macrophages. Atherosclerosis 1998;136:43-9. Zhang Y, Shen D, Zou P, Wei W, Wang A, Yang L. Activity of platelet in patients with high level of LDL and the effect of LDL on platelet glycoproteins. Chin Med J (Eng) 1998;111:910-2. van Willigen G, Gorter G, Akkerman JW. LDLs increase the exposure of fibrinogen binding sites on platelets and secretion of dense granules. Arterioscler Thromb 1994;14:41-6. Virgolini I, Li S, Qiong Y, Koller E, Banyai M, Angelberger P, Sinzinger H. Binding of 111In-labeled LDL to platelets of normolipidemic volunteers and patients with heterozygous familial hypercholesterolemia. Arterioscler Thromb 1993;13:536-47. Akar N, Akar E, Deda C, Sipahi T, Orsal A. Factor V1691 G-A, prothrombin 20210 G-A, and methylenetetrahydrofolate reductase 677 C-T variants in Turkish children with cerebral infarct. J Child Neurol 1999;14:749-51. Unal S, Gumruk F, Aytac S, Yalnzoglu D, Gurgey A. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) levels and IL-6, TNF-polymorphisms in children with thrombosis. J Pediatr Hematol Oncol 2008;30:26-31. Iqbal O, Tobu M, Aziz S, Gerdisch M, Da Valle M, Demir M, Hoppensteadt DA, Ahmad S, Walenga JM, Fareed J. Successful use of recombinant hirudin and its monitoring by ecarin clotting time in patients with heparin-induced thrombocytopenia undergoing off pump coronary artery revascularization. J Card Surg 2005;20:42-51. Gül C, Kürüm T, Demir M, Ozbay G, Vural O, Igbal O, Fareed J. Acute myocardial infarction in a patient with essential thrombocythemia treated with glycoprotein IIb/IIIa inhibitor. Clin Appl Thromb Hemost 2004;10:77-9. Davis SM, Donnan GA. Secondary prevention for stroke after CAPRIE and ESPS-2. Cerebrovasc Dis 1998;8:73-7. Savi P, Herbert JM, Pfliegera AM, Dol F, Delebassee D, Combalbert J, Defreyn G, Maffrand JP. Importance of hepatic metabolism in the antiaggregating activity of the thienopyridine clopidogrel. Biochemical Pharmacol 1992;44:527-32.

108 48.

Ă&#x2013;zsavcĂ&#x2022; et al. New effects of clopidogrel on platelets

Turk J Hematol 2010; 27: 99-108

rin, clopidogrel, and the glycoprotein IIb/IIIa inhibitors abciximab

Antonino MJ, Mahla E, Bliden KP, Tantry US, Gurbel PA. Effect

and SR121566A. Clin Pharmacol Ther 2000;67:305-13.

of long-term clopidogrel treatment on platelet function and inflammation in patients undergoing coronary arterial stenting. 49.

50.

Storey RF, Judge HM, Wilcox RG, Heptinstall S. Inhibition of ADP

Am J Cardiol 2009;103:1546-50.

induced P-selectin expression and platelet-leukocyte conjugate for-

Klinkhadt U, Kirchmaier CM, Westrup D, Graff J, Mahnel R,

mation by clopidogrel and the P2Y12 receptor antagonist

Breddin HK, Harder S. Ex vivo-in vitro interaction between aspi-

AR-C69931MX but not aspirin. Thromb Haemost 2002;88:488-94.

Case Report

109

A rare extramedullary involvement in myeloma: lung parenchyma and association with unfavorable chromosomal abnormalities Miyelom’da nadir bir ekstramedüller tutulum: Akciúer parankimi ve olumsuz kromozomal anormalliklerle iliýkisi Özlem üahin BalçÕk1, Murat Albayrak1, Simten Daúdaý2, Funda Ceran2, Gülsüm Özet2, Funda Demiraú3, Osman Yokuý4 1Department

of Hematology, Oncology Education and Research Hospital, Ankara, Turkey of Hematology, Numune Education and Research Hospital, Ankara, Turkey 3Department of Pathology, Atatürk Chest Diseases and Chest Surgery Education and Research Hospital, Ankara, Turkey 4Department of Hematology, Kayseri Education and Research Hospital, Kayseri, Turkey 2Department

Abstract Although pulmonary complications developing secondary to lung infections and involvement in ribs occur frequently in multiple myeloma (MM), involvement of the lung parenchyma is quite rare. In clinical studies, the involvement of lung parenchyma has been found to be associated with unfavorable prognosis. Here, a MM case in whom involvement of lung parenchyma was accompanied by unfavorable prognostic cytogenetic markers is presented. A 62-year-old male presented with complaint of cough, and heterogeneous hypodense mass was detected in thorax computerized tomography. The patient underwent bronchoscopic biopsy. Pathological examination revealed diffuse plasma cell infiltration staining with kappa immunohistochemically. In bone marrow biopsy, plasma cell infiltration was observed. In conventional cytogenetic examination, hypodiploidy was established. In cytogenetic examination carried out with fluorescence in situ hybridization, deletion (13q) was determined. In conclusion, in patients diagnosed with MM and presenting with pulmonary mass lesion, lung involvement associated with plasma cell infiltration should also be considered in the differential diagnosis. As overall survival is low in these cases, more aggressive treatment approaches such as high-dose treatment should be immediately considered. (Turk J Hematol 2010; 27: 109-12) Key words: Myeloma, pulmonary involvement, prognostication, cytogenetics, molecular genetics, del(13q), hypodiploidy Received: May 7, 2008

Accepted: January 7 2009

Özet Multipl miyelomda (MM) akciúer enfeksiyonlarÕ ve göúüs kafesi kemiklerinde tutuluma baúlÕ geliýen pulmoner komplikasyonlar sÕkça görülmesine karýÕn, akciúer parankim tutulumu oldukça nadirdir. YapÕlan klinik çalÕýmalarda akciúer parankim tutulumunun kötü prognoz ile iliýkisi bulunmuýtur. Burada akciúer parankim tutulumuna olumsuz prognostik sitogenetik belirleyicilerin eýlik ettiúi bir MM olgusu sunulmaktadÕr. Address for Correspondence: Özlem üahin BalçÕk, M.D., Fatih University Medical School Department of Hematology, Ankara, Turkey Phone: +90 312 321 66 97 Office: +90 312 336 09 09 E-mail: drozlembalcik@yahoo.com doi:10.5152/tjh.2010.08

110

BalçÕk et al. Lung parenchyma involvement in myeloma

Turk J Hematol 2010; 27: 109-12

Öksürük ýikayeti ile baývuran 62 yaýÕnda erkek olguya çekilen toraks tomografisinde; saú orta lob düzeyinde heterojen hipodens kitle görünümü saptandÕ. Bronkoskopik biyopsi yapÕldÕ. Patolojik incelemede immünhistokimyasal olarak kappa ile diffüz boyanma gösteren plazma hücre infiltrasyonu saptandÕ. Kemik iliúi biyopsisinde plazma hücre infiltrasyonu görüldü. Konvansiyonel sitogenetik incelemede hipodiploidi saptandÕ. Fluorescence in situ hybridization ile yapÕlan sitogenetik incelemede delesyon (13q) tespit edildi. Sonuç olarak, akciúerde kitle lezyonu ile baývuran ve MM tanÕsÕ alan hastalarda ayÕrÕcÕ tanÕda MM a baúlÕ plazma hücre infiltrasyonu da düýünülmelidir. Bu olgular toplam saú kalÕmlarÕ düýük olduúundan, yüksek doz tedavi gibi daha agresif tedavi yaklaýÕmlarÕna hÕzla yönlendirilmelidir. (Turk J Hematol 2010; 27: 109-12) Anahtar kelimeler: Miyelom, akciúer tutulumu, prognoz, sitogenetik, moleküler genetik, del(13q), hipodiploidi Geliý tarihi: 7 MayÕs 2008

Kabul tarihi: 7 Ocak 2009

Introduction Multiple myeloma (MM) is a disease characterized by the proliferation of malignant plasma cells in bone marrow [1]. Recently, parallel to the developments in cytogenetics, it has been demonstrated that genetic abnormalities play an important part in the prognosis of MM as in other hematological and solid malignancies [2,3]. Conventional cytogenetics and fluorescence in situ hybridization (FISH) analysis contribute substantially to clinical follow-up at the stages of selection among treatment options and determination of prognosis. It has been shown that hypodiploidy and deletion (del) 13q are independent unfavorable prognostic factors [4,5]. MM may progress with extramedullary involvement. It may occur in all lymph nodes, skin, liver and spleen, and less frequently in the kidney and dura mater [1,6]. More rarely, involvements of the orbita, muscle, skin, and pericardium are seen [7-9]. Diagnosis is made with the establishment of monoclonal plasma cells in the involved organ. The response of these cases even to quite aggressive treatment approaches is not satisfactory [1,10]. Plasma cell diseases presenting with pleural effusion and plasmacytoma in the lung have been described [11,12]. However, the involvement of lung parenchyma occurs rarely in MM [13-17].

Table 1. Patient characteristics Age

Sex

Male

Hemoglobin (g/dl)

9.1 (14-17.5)

Leukocyte (x109/L)

7.1 (4.4-11.3)

Platelet (x109/L)

179 (150-450)

Creatinine* (mg/dl)

3.27 (0.5-1.4)

Calcium* (mg/dl)

15.1 (8.4-10.0)

Albumin* (g/dl)

3.2 (3.4-5)

LDH* (U/L)

154 (125-243)

CRP* (mg/L)

49 (0-5)

Beta-2 microglobulin* (mg/L)

16.9 (0.6-2.5)

*: Serum LDH: Lactate dehydrogenase; CRP: C-reactive protein.

Case Report A 62-year-old male patient presented with the complaints of fever, cough, sputum, and nonhomogeneous density on chest radiography. The patient’s characteristics are detailed in Table 1. Thoracic computed tomography (CT) revealed a heterogeneous hypodense mass appearance without air at the level of the right middle lobe (Figure 1). Bronchoscopic biopsy was carried out with the presumptive diagnosis of lung carcinoma. Pathological examination revealed strong and diffuse plasma cell infiltration staining with kappa immunohistochemically (Figures 2, 3). Chromogranin, neuron specific enolase, keratin and CD56 were applied to the bronchoscopic biopsy material; however, no staining was observed and carcinoid tumor was ruled out. With the establishment of plasma cell infiltration in the lung, the case was evaluated thoroughly for MM. Plasma cell infiltration staining diffusely and strongly with kappa immunohistochemically was seen in bone marrow trephine biopsy as well. In serum immunofixation electrophoresis, immunoglobulin (Ig) A kappa, and in urine immunofixation electrophoresis, kappa light chain, were established. Serum IgA level was found to be 8.04 g/L (0.70-4.00 g/L). Hypodiploidy was detected with conventional genetical examination. In FISH analysis, del(13q) was established in 16% of the cells examined. The case was regarded as stage IIIB according to prog-

Figure 1. At the level of right middle lobe, heterogenous hypodense mass lesion in thorax computerized tomography

Turk J Hematol 2010; 27: 109-12

Figure 2. Plasma cell infiltration in the bronchial wall (HE X200)

Figure 3. Plasma cells are positive with kappa light chain (Kappa X400)

nostic Durie Salmon staging system and stage III according to the International Staging System (ISS) for MM [18,19]. Chemotherapy including vincristine, adriablastin and dexamethasone was commenced. The patient did not respond to chemotherapy and died six months later due to progressive disease. All samples were drawn after obtaining formal written consent from the patient, and the case presentation was carried out in accordance with the Helsinki Declaration.

Discussion In MM, bone involvement in the thorax region and lung infections secondary to infections developing due to humoral immune deficiency related to hypogammaglobulinemia occur most frequently. Although pleural effusion and plasmacytoma in the lung are reported in association with MM, lung parenchyma is a rare site of extramedullary involvement [12,17]. Pulmonary involvement in MM is associated with rapid progression of the disease. In the study of Oymak et al. [16] of 38 patients referring to the Chest Diseases

BalĂ§Ă&#x2022;k et al. Lung parenchyma involvement in myeloma

111

Department and being followed with the diagnosis of MM, thoracal involvement was established in 19 (50%), lung involvement in 13 (35%) and thoracal bone invasion in 9 (24%). When the cases were evaluated according to their clinical and radiological findings, pneumonic infiltration was observed in 6, mass lesion in 2, multiple nodular lesions in 2, and interstitial infiltration in 3. In two patients with nodular lung infiltration and mass lesion, malignant plasma cell infiltration was demonstrated with percutaneous lung biopsy, and in the patient with reticulonodular infiltration, it was shown with bronchoscopic lung biopsy. In cases with pulmonary involvement, rapidly progressive disease was established, especially with renal involvement and pathological bone fractures. Similarly, in our case, plasma cell infiltration was detected with the biopsy obtained from the endobronchial lesion with bronchoscopic approach. The fact that our patient with renal involvement did not respond to chemotherapy and subsequently died is compatible with the reports in the literature. In the study of Damaj et al. [10], extramedullary involvement was established in 19 of 432 MM cases and these cases were evaluated. Of the cases recalcitrant to standard dose chemotherapy and thalidomide treatment, 8 responded to high-dose treatment supplemented with stem cell. Disease progressed in the other cases even under treatment. After a follow-up period of 13 months, 6 of these 11 patients died; partial remission was achieved in 4 and progressive disease developed in 2. It has been concluded that extramedullary involvement is rare in MM and that if it is present, the response to treatment is limited and the disease has an unfavorable prognosis. MM is quite a heterogeneous disease in terms of prognosis. There are cases in which survival is as short as a few months, while other cases have been described as surviving for more than 10 years. Although the disease has a similar presentation in all cases, prognosis is quite variable. Therefore, various prognostic parameters have been described for use in patient monitoring. While mostly clinical and biochemical parameters were used previously, cytogenetic methods are also used at present. Our case, with involvement of lung parenchyma and unfavorable prognosis, was evaluated with respect to biochemical and cytogenetic characteristics. The disease was determined as stage IIIB according to prognostic Durie Salmon staging system and as stage III according to ISS, with unfavorable prognostic characteristics. In our case, hypodiploidy was found with conventional cytogenetic examination and del(13q) with FISH analysis [20-22]. However, to our knowledge, there is no study aiming to determine the cytogenetic properties of cases with lung parenchyma involvement. Del(13q) and hypodiploidy are each independent negative prognostic parameters irrespective of other parameters. These cases should be evaluated in terms of cytogenetic characteristics. Thus, accompanying cytogenetic abnormalities can be identified and the necessary steps may be taken for targeting treatment. In the literature, there are reports on the association of lung parenchyma involvement with unfavorable prognosis [10,16]. In our case, identification of del(13q) and hypodiploidy as well

112

BalçÕk et al. Lung parenchyma involvement in myeloma

as the other negative prognostic parameters and rapid progression of the disease with no treatment response are consistent with the literature. In conclusion, in patients diagnosed with MM and presenting with pulmonary mass lesion, lung involvement associated with plasma cell infiltration should be kept in mind in the differential diagnosis. As the overall survival rate is low in these cases, more aggressive treatment approaches, such as highdose treatment, should be promptly considered. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

Tricot G. Multiple myeloma and other plasma cell disorders. In: Hoffman R, Benz EJ, Shattil SJ, Furie B, Cohen HJ, Silberstein LE et al., editors. Hematology Basic Principles and Practice. 4th ed. New York: Elsevier Churchill Livingstone, 2005:1501-21. Dewald GW, Kyle RA, Hicks GA, Greipp PR. The clinical significance of cytogenetic studies in 100 patients with multiple myeloma, plasma cell leukemia or amyloidosis. Blood 1985;66:380-90. Sawyer JR, Waldron JA, Jagannath S, Barlogie B. Cytogenetic findings in 200 patients with multiple myeloma. Cancer Genet Cytogenet 1995;82:41-9. Smadja NV, Bastard C, Brigaudeau C, Leroux D, Fruchart C; Groupe Français de Cytogénétique Hématologique. Hypodiploidy is a major prognostic factor in multiple myeloma. Blood 2001;98:2229-38. Fassas AB, Spencer T, Sawyer J, Zangari M, Lee CK, Anaissie E, Muwalla F, Morris C, Barlogie B, Tricot G. Both hypodiploidy and deletion of chromosome 13 independently confer poor prognosis in multiple myeloma. Br J Haematol 2002;118:1041-7. Haegelen C, Riffaud L, Bernard M, Carsin-Nicol B, Morandi X. Dural plasmacytoma revealing multiple myeloma. Case report. J Neurosurg 2006;104:608-10. Thoumazet F, Donnio A, Ayeboua L, Brebion A, Diedhou A, Merle H. Orbital and muscle involvement in multiple myeloma. Can J Ophthalmol 2006;41:733-6. Alexandrescu DT, Koulova L, Wiernik PH. Unusual cutaneous involvement during plasma cell leukaemia phase in a multiple myeloma patient after treatment with thalidomide: a case report and review of the literature. Clin Exp Dermatol 2005;30:391-4. Abelman W, Virchis A, Yong K. Extramedullary myeloma representing as a pericardial effusion with tamponade: two case reports and a further review of 19 cases in the literature. Mod Pathol 1995;8:257-9.

Turk J Hematol 2010; 27: 109-12

10.

11. 12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Damaj G, Mohty M, Vey N, Dincan E, Bouabdallah R, Faucher C, Stoppa AM, Gastaut JA. Features of extramedullary and extraosseous multiple myeloma: a report of 19 patients from a single center. Eur J Haematol 2004;73:402-6. Rodríguez JN, Pereira A, Martínez JC, Conde J, Pujol E. Pleural effusion in multiple myeloma. Chest 1994;105:622-4. Nonomura A, Mizukami Y, Shimizu J, Oda M, Watanabe Y, Kamimura R, Takashima T, Kitagawa M. Primary extramedullary plasmacytoma of the lung. Intern Med 1992;31:1396-400. Kamble R, Rosenzweig T. Diffuse pulmonary parenchymal involvement in multiple myeloma: antemortem diagnosis. Int J Hematol 2006;83:259-61. Marmor DB, Farber JL, Gottlieb JE. Acute respiratory distress syndrome due to pulmonary involvement by neoplastic plasma cells in multiple myeloma. Thorax 2006;61:455-6. Yokote T, Akioka T, Miyamoto H, Oka S, Hara S, Yamano T, Takasu T, Tsuji M, Hanafusa T.l. Pulmonary parenchymal infiltrates in a patient with CD20-positive multiple myeloma. Eur J Haematol 2005;74:61-5. Oymak FS, Karaman A, Soyuer I, Karaman H, Gülmez I, Demir R, Unal A, Ozesmi M. Pulmonary and chest wall involvement in multiple myeloma. Tuberk Toraks 2003;51:27-32. Luh SP, Lai YS, Tsai CH, Tsao TC. Extramedullary plasmacytoma (EMP): report of a case manifested as a mediastinal mass and multiple pulmonary nodules and review of literature. World J Surg Oncol 2007;5:123. Durie BG, Salmon SE. A clinical staging system for multiple myeloma. Correlation of measured myeloma cell mass with presenting clinical features, response to treatment, and survival. Cancer 1975;36:842-54. Greipp PR, San Miguel J, Durie BG, Crowley JJ, Barlogie B, Bladé J, Boccadoro M, Child JA, Avet-Loiseau H, Kyle RA, Lahuerta JJ, Ludwig H, Morgan G, Powles R, Shimizu K, Shustik C, Sonneveld P, Tosi P, Turesson I, Westin J. International staging system for multiple myeloma. J Clin Oncol 2005;23:3412-20. Fassas AB, Ward S, Muwalla F, Van Hemert R, Schluterman K, Harik S, Tricot G. Myeloma of the central nervous system: strong association with unfavorable chromosomal abnormalities and other high-risk disease features. Leuk Lymphoma 2004;45:291-300. Fassas AB, Muwalla F, Berryman T, Benramdane R, Joseph L, Anaissie E, Sethi R, Desikan R, Siegel D, Badros A, Toor A, Zangari M, Morris C, Angtuaco E, Mathew S, Wilson C, Hough A, Harik S, Barlogie B, Tricot G. Myeloma of the central nervous system: association with high-risk chromosomal abnormalities, plasmablastic morphology and extramedullary manifestations. Br J Haematol 2002;117:103-8. Chang H, Sloan S, Li D, Keith Stewart A. Multiple myeloma involving central nervous system: high frequency of chromosome 17p13.1 (p53) deletions. Br J Haematol 2004;127:280-4.

Case Report

113

Complex cytogenetic findings in the bone marrow of a chronic idiopathic myelofibrosis patient Kronik idiopatik myelofibrozis olgusunun kemik iliúinde kompleks sitogenetik bulgular Tuúçe BulakbaýÕ BalcÕ1, Meltem Yüksel2, Zerrin YÕlmaz1, Feride ûffet üahin1 1Department 2Dr.

of Medical Genetics, Baskent University Faculty of Medicine, Ankara, Turkey Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Ankara, Turkey

Abstract Chronic idiopathic myelofibrosis is a myeloproliferative disorder characterized by splenomegaly, myeloid metaplasia and reactive bone marrow fibrosis. Karyotype analysis of the bone marrow is an integral part of the diagnosis, especially as a discriminative tool in ruling out reactive conditions. The frequency of clonal cytogenetic anomalies in this disease is the highest among its group, varying between 30 and 75%. Among these, trisomy 1q, 20q-, 13q- and +8 are the most common aberrations. Here we report a 66-year-old male patient whose bone marrow biopsy revealed signs of chronic myeloproliferative changes and dysmegakaryopoiesis. He was administered hydroxyurea treatment, splenic radiotherapy and multiple transfusions. The patient worsened in the following months and the second bone marrow biopsy revealed myelofibrosis. Cytogenetic analysis of this bone marrow sample revealed a complex karyotype reported to be 46,XY,del(9) (q22q34),t(8;17;21)(q22;q21;q22)[23]/46,XY[2], with a previously undefined three-way translocation and deletion in chromosome 9. The patient died shortly thereafter. (Turk J Hematol 2010; 27: 113-6) Key words: Chronic idiopathic myelofibrosis, cytogenetics Received: August 15, 2008

Accepted: April 8, 2009

Özet Kronik idiopatik myelofibrozis, kronik myeloproliferatif hastalÕklardandÕr ve splenomegali, myeloid metaplazi ve reaktif kemik iliúi fibrozisi ile karakterizedir. Bu hastalÕklarÕn tanÕsÕnda, özellikle de reaktif durumlarÕ ekarte etmek için kemik iliúinden kromozom analizi oldukça önemli bir araçtÕr. ûdiopatik myelofibrozis, bu grup içerisinde en yüksek klonal sitogenetik anomali sÕklÕúÕna sahiptir (%30-75). Trizomi 1q, 20q-, 13q- ve +8 en sÕk gözlenen anomalilerdendir. Bu yazÕmÕzda masif splenomegali ile baývuran 66 yaýÕnda erkek olgunun bulgularÕ sunulmaktadÕr. Olgunun kemik iliúi biyopsisinde kronik myeloproliferatif deúiýiklikler ve dismegakaryopoez saptandÕ. Hidroksiüre tedavisi, splenik radyoterapi ve çok sayÕda transfüzyon alan hastanÕn kliniúi ilerleyen aylarda kötüleýti. YapÕlan ikinci kemik iliúi biyopsisisinde myelofibrozis gözlendi. Bu örneúin sitogenetik incelemesi sonucu daha önce tanÕmlanmamÕý üç yollu bir translokasyon ve 9. kromozomda delesyon saptandÕ. Karyotipi 46,XY,del(9)(q22q34),t(8;17;21)(q22;q21;q22)[23]/46,XY[2] olarak belirlendi. Olgu kÕsa süre sonra kaybedildi. (Turk J Hematol 2010; 27: 113-6) Anahtar kelimeler: Kronik idiopatik myelofibrozis, sitogenetik Geliý tarihi: 15 Aúustos 2008

Kabul tarihi: 8 Nisan 2009

Address for Correspondence: Prof. Dr. Feride ûffet üahin, Baýkent Üniversitesi TÕp Fakültesi TÕbbi Genetik Anabilim DalÕ, Kubilay Sokak No: 36 Maltepe 06570 Ankara, Turkey Phone: +90 312 232 44 00 E-mail: feridesahin@hotmail.com doi:10.5152/tjh.2010.09

114

BalcĂ&#x2022; et al. Complex karyotype in chronic idiopathic myelofibrosis

Turk J Hematol 2010; 27: 113-6

Introduction

myeloproliferative changes and dysmegakaryopoiesis. He received hydroxyurea treatment (20 mg/kg/day) and transfusion support for two years. The splenomegaly regressed with therapy, and then reappeared, when the patient had a poorer clinical status and needed weekly transfusions, in November 2006. The leukocyte count at that time was 9.000/mm3, hemoglobin was 8.8 g/dl and platelet count was 469.000/mm3. A second bone marrow biopsy was performed and showed myelofibrosis. Despite the hydroxyurea therapy, his spleen was enlarging and there was progressive decrease in hemoglobin and platelet counts. Palliative radiotherapy was started in February 2007 when his leukocyte count was 11.800 /mm3, hemoglobin 7.6 g/dl and platelet count 25.000 /mm3. At the end of radiotherapy, his spleen had been reduced to almost normal volume and the patient became transfusion-independent. He was re-admitted to the hospital in October 2007 due to fatigue, palpitation and left upper abdominal pain. On physical examination, he had massive splenomegaly; his leukocyte count was 3.200 /mm3, hemoglobin 7.4 g/dl and platelets 354.000 /mm3. A new bone marrow biopsy was performed to rule out leukemia. The results were compatible with the earlier diagnosis of myelofibrosis. Dexamethasone therapy was initiated and thalidomide therapy was planned. His bone marrow sample was sent to our laboratory for cytogenetic studies. Fluorescence in situ hybridization (FISH) with LSI BCR-ABL ES Dual Color Translocation Probe (Vysis, USA) was performed. Fusion signal was detected, though below the cut-off value of our laboratory, and was thus reported as negative. After routine cultures were set up using Bone Marrow Karyotyping Medium (Biological Industries, Israel) and Hematopoietic Cell Karyotyping Medium (Biological Industries, Israel), G-banding of chromosomes revealed a complex karyotype, including rearrangements in chromosomes 8, 9, 17 and 21. Following

Chronic myeloproliferative disorders (CMPDs) are a group of heterogeneous clonal stem cell disorders that can be defined by specific cytomorphological phenotypes and sometimes genetic features [1]. The frequency of clonal cytogenetic anomalies is variable in these disorders. Chronic idiopathic myelofibrosis (CIMF) is characterized by splenomegaly, myeloid metaplasia and reactive bone marrow fibrosis, and it has the highest karyotype aberration rate [1-3]. Approximately 50% of patients diagnosed with CIMF harbor cytogenetic anomalies, and among these, trisomy 1q, 20q-, 13q- and +8 are the most common aberrations [3-5]. Abnormalities of chromosomes 5, 6, 7, 9 and 12 have also been reported. If leukemic transformation occurs, clonal evolution to a more complex karyotype is common [1]. Thus, cytogenetic analysis is a valuable tool in CIMF, both at diagnosis to distinguish from secondary myelofibrosis and chronic myeloid leukemia (CML) and during the follow-up [1]. Here we report a CIMF case with multiple complex cytogenetic abnormalities and poor outcome to emphasize the significance of cytogenetic studies in patients with this disease. Case Report A 66-year-old male patient with congestive heart failure was referred to the hematology clinic because of low hemoglobin levels and thrombocytosis. He had dyspnea and fatigue and on physical examination had massive splenomegaly (spleen size 20 cm on abdominal ultrasound). The leukocyte count was 10.900/mm3, hemoglobin was 9.8 g/dl and platelet count was 1.699.000/mm3. The first bone marrow biopsy, which was performed in April 2004, revealed signs of chronic

Figure 1. Karyotype of the patient: 46,XY,del(9)(q22q34),t(8;17;21)(q22;q21;q22)

BalcĂ&#x2022; et al. Complex karyotype in chronic idiopathic myelofibrosis

Turk J Hematol 2010; 27: 113-6

analysis of 25 metaphases, the karyotype was designated according to ISCN 2005 and reported as: 46,XY,del(9) (q22q34),t(8;17;21)(q22;q21;q22)[23]/46,XY[2] (Figure 1). To define the rearrangements, FISH was performed with Whole Chromosome Paint probes 8, 9, 17 and 21 (Vysis, USA) on selected metaphases (Figures 2, 3). Based on the results, an interstitial deletion was found in chromosome 9 and a threeway translocation was verified between chromosomes 8, 17 and 21. Written informed consent was obtained from the patient. The patient died shortly after his bone marrow aspirate was sampled. Discussion Karyotype analysis of the bone marrow is an integral part of the diagnosis in myeloid disorders, and in recent years, it has

115

gained more importance in the diagnosis of CMPDs, especially as a discriminative tool in ruling out reactive conditions [5]. In CMPD patients, cytogenetic analysis is not the primary test as a diagnostic tool. The first-line diagnostic tools are clinical and hematological parameters. Both conventional cytogenetics and FISH are performed as the second-line tests. Chromosome banding visualizes the complete karyotype of the patients. FISH analyses can be used to detect suspected or known chromosome aberrations [1]. It may be difficult to obtain a representative sample from patients with CIMF because the bone marrow is fibrotic in this disease and the cells that are obtained have low proliferative capacity in vitro [4,6]. Thus, the overall frequency of cytogenetic abnormalities may not be determined with certainty. The reported frequencies range between 30 to 75% [4]. As mentioned above, the most common cytogenetic aberrations

17 17

der (17) 21

der (17)

der (21)

Figure 2. a: Metaphase spread of the patient; relevant chromosomes are marked with arrows b: The same metaphase after fluorescence in situ hybridization with Whole Chromosome Probe 8 Spectrum Orange (Vysis, USA); relevant chromosomes are marked with arrows

der (8)

der (8) der (17) der (17) 8

Figure 3. a: Metaphase spread of the patient; relevant chromosomes are marked with arrows b: The same metaphase after fluorescence in situ hybridization with Whole Chromosome Probe 17 Spectrum Orange (Vysis, USA); relevant chromosomes are marked with arrows

116

BalcĂ&#x2022; et al. Complex karyotype in chronic idiopathic myelofibrosis

found in CIMF include +8, 20q-, 13q-, +1q, +9 and +21. There is no specific aberration, and unlike myeloid leukemias, balanced translocations are rare [2,4]. Some of these anomalies (involving chromosomes 1, 5, 7, 9) are almost always associated with additional changes, and thus are suspected to be a result of genetic instability, while others (20q-, 13q-, +8) appear as sole anomalies and are thought to be pathogenetically relevant [5]. Of these, 13q- and 20q- are considered as favorable abnormalities while all other abnormalities are regarded as unfavorable, but the findings are not consistent in all series [5,6]. Regarding the current patient, the three-way translocation together with the deletion in the long arm of chromosome 9, resulting in such a complex karyotype, could have contributed unfavorably to the prognosis and could be associated with the progression of the disease. On the other hand, the fact that the patient received radiotherapy along with hydroxyurea treatment may raise the question of chromosome breaks due to radiation and myelotoxic drugs. Hydroxyurea treatment has not yet been associated with a significant increase in myeloblastic transformation in myeloproliferative disorders, but its myelotoxic effects could have contributed to the worsening hematological status and myelofibrosis in this patient [7,8]. Splenic irradiation is considered as a local palliative therapy to reduce the spleen size in elderly patients with CMPDs. The fact that CIMF is mostly seen in the older patient population, in whom surgical treatment would bring about higher rates of mortality and morbidity, has led clinicians to use this nonsurgical alternative in this group of patients. Complex karyotypic rearrangements in CIMF have not been reported to appear as a result of radiotherapy. However, the most expected lifethreatening complication of splenic irradiation is myelosuppression, and the potential outcome of this therapy has not yet been fully optimized for CIMF patients [9]. It has been known for many years that radiation exposure could cause single or double strand breaks in the chromosomes, leading to dicentric chromosomes, translocations and more complex rearrangements [10,11]. The hematological effect of radiotherapy has also been studied, and along with chemotherapy, it is known to cause different kinds of neoplasms [11,12]. Therefore, there is a theoretical possibility that the three-way complex translocation and the deletion in our patient may have been caused by the therapy he received during the course of his disease. Our patient worsened about eight months after receiving radiotherapy. Unfortunately, we do not have information about the cytogenetic status of the bone marrow in this patient before therapy, so it is not possible to decide whether the complex karyotype had been present from the beginning, or whether it was due to disease progression or to the radio- and/or chemotherapy.

Turk J Hematol 2010; 27: 113-6

It is obvious that cytogenetic analysis of bone marrow is the first-line analysis in both determining the diagnosis and foreseeing the prognosis in myeloid disorders. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1.

9. 10.

11. 12.

Haferlach T, Bacher U, Kern W, Schnittger S, Haferlach C. The diagnosis of BCR/ABL-negative chronic myeloproliferative diseases (CMPD): a comprehensive approach based on morphology, cytogenetics, and molecular markers. Ann Hematol 2008;87:1-10. Demory JL, Dupriez B, Fenaux P, LaĂŻ JL, Beuscart R, Jouet JP, Deminatti M, Bauters F. Cytogenetic studies and their prognostic significance in agnogenic myeloid metaplasia: a report on 47 cases. Blood 1988;72:855-9. Ahmed A, Chang CC. Chronic idiopathic myelofibrosis: clinicopathologic features, pathogenesis, and prognosis. Arch Pathol Lab Med 2006;130:1133-43. Djordjevic V, Dencic-Fekete M, Jovanovic J, Bizic S, Jankovic G, Bogdanovic A, Cemerikic-Martinovic V, Gotic M. Cytogenetics of agnogenic myeloid metaplasia: a study of 61 patients. Cancer Genet Cytogenet 2007;173:57-62. Tefferi A, Mesa RA, Schroeder G, Hanson CA, Li CY, Dewald GW. Cytogenetic findings and their clinical relevance in myelofibrosis with myeloid metaplasia. Br J Haematol 2001;113:763-71. Strasser-Weippl K, Steurer M, Kees M, Augustin F, Tzankov A, Dirnhofer S, Fiegl M, Simonitsch-Klupp I, Gisslinger H, Zojer N, Ludwig H. Prognostic relevance of cytogenetics determined by fluorescent in situ hybridization in patients having myelofibrosis with myeloid metaplasia. Cancer 2006;107:2801-6. Nand S, Stock W, Godwin J, Fisher SG. Leukemogenic risk of hydroxyurea therapy in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Am J Hematol 1996;52:42-6. Burkitt MJ, Raafat A. Nitric oxide generation from hydroxyurea: significance and implications for leukemogenesis in the management of myeloproliferative disorders. Blood 2006;107:2219-22. McFarland JT, Kuzma C, Millard FE, Johnstone PA. Palliative irradiation of the spleen. Am J Clin Oncol 2003;26:178-83. Tucker JD. Low-dose ionizing radiation and chromosome translocations: a review of the major considerations for human biological dosimetry. Mutat Res 2008;659:211-20. Cornforth MN. Perspectives on the formation of radiation-induced exchange aberrations. DNA Repair (Amst) 2006;5:1182-91. Leone G, Pagano L, Ben-Yehuda D, Voso MT. Therapy-related leukemia and myelodysplasia: susceptibility and incidence. Haematologica 2007;92:1389-98.

Case Report

117

An unusual presentation of pediatric acute lymphoblastic leukemia with parotid gland involvement and dactylitis Parotid bezi tutulumu ve daktilit ile baývuran alÕýÕlmadÕk bir pediatrik akut lenfoblastik lösemi vakasÕ üule Ünal1, BarÕý Kuýkonmaz1, Yasemin IýÕk BalcÕ1, Bülent Cengiz2, Murat Tuncer1, Aytemiz Gürgey1, Erman Cilsal3, Ayýe Gültekingil3, Fatma Gümrük1 1Division

of Pediatric Hematology, Hacettepe University Faculty of Medicine, Ankara, Turkey of Pediatric Infectious Diseases, Hacettepe University Faculty of Medicine, Ankara, Turkey 3Department of Pediatrics, Hacettepe University Faculty of Medicine, Ankara, Turkey 2Division

Abstract

Mumps infection during the course of childhood acute lymphoblastic leukemia (ALL) treatment has been reported to have a mild course and this was related to the intrinsic low cytopathological effect of the virus, contrasting with the severe course of measles and Varicella zoster virus infections in immunocompromised patients. Herein, we present a three-yearold girl, who was previously vaccinated against mumps infection, admitted with bilateral parotid swelling, dactylitis and serum immunoglobulin M positivity for mumps infection and diagnosed to have ALL with bilateral persistent parotid involvement, inconsistent with mumps infection. Acute leukemia should be suspected during the atypical course of any disease during childhood. Besides, mumps infection at presentation of ALL, as similar to infection emerging during the period of the leukemia treatment, has a mild course. (Turk J Hematol 2010; 27: 117-9) Key words: Mumps, parotitits, dactylitis, childhood, leukemia Received: March 12, 2008

Accepted: April 8, 2009

Özet

Çocukluk çaúÕ akut lenfoblastik lösemi (ALL) tedavisi boyunca geçirilebilecek kabakulak enfeksiyonunun, virusun düýük sitopatolojik etkileri nedeniyle, kÕzamÕk ve su çiçeúi enfeksiyonlarÕnÕn aksine daha hafif seyrettiúine dair raporlar bulunmaktadÕr. Daha önce kabakulak aýÕsÕ yapÕlmÕý olan üç yaýÕndaki kÕz hasta iki taraflÕ parotid bezinde ýiýlik ve daktilit nedeniyle baývurduúunda kabakulak IgM pozitifliúi saptanmÕý ve ileri incelemesinde bilateral parotid infiltrasyonunun eýlik ettiúi ALL tanÕsÕ konulmuýtur. Çocukluk çaúÕnÕn atipik seyirli tüm hastalÕklarÕnda akut lösemi akÕlda tutulmalÕdÕr. AynÕ zamanda tedavi sÕrasÕnda geçirilen kabakulak enfeksyonlarÕna benzer ýekilde, ALL tanÕsÕ anÕnda eýlik eden kabakulak enfeksiyonu da hafif seyretmektedir. (Turk J Hematol 2010; 27: 117-9) Anahtar kelimeler: Kabakulak, parotit, daktilit, çocukluk çaúÕ, lösemi Geliý tarihi: 12 Mart 2008

Kabul tarihi: 8 Nisan 2009

Address for Correspondence: üule Ünal, MD, Hacettepe University, Division of Pediatric Hematology 06100 Ankara, Türkiye Phone: + 90 312 305 11 70 E-mail: suleunal@hacettepe.edu.tr doi:10.5152/tjh.2010.10

118

Unal et al. An unusual presentation of pediatric ALL

Introduction Acute parotitis is common in childhood, mostly associated with mumps infection and among the other causes there exist Staphylococcus aureus, human immunodeficiency virus (HIV), Mycobacterium tuberculosis, echovirus, parainfluenza (types 1 and 3), coxsackievirus A, adenovirus or cytomegalovirus [1]. Leukemia and lymphoma may cause enlargement in the salivary glands with or without the lacrimal gland involvement [2]. Primary benign and malign neoplasms of the parotitis gland are rare in childhood. Herein, we present a three-year-old girl, who was previously vaccinated against mumps infection, admitted with bilateral parotid swelling with immunoglobulin (Ig) M positivity for mumps infection and diagnosed to have acute lymphoblastic leukemia (ALL) with bilateral parotid involvement. Previous data suggest that in malignant disease, the course of mumps is rarely severe and that the infection often remains subclinical, as in healthy children, contrasting with the severe course of measles and varicella [3]. However, data related to the mumps infection coinciding the presentation of childhood ALL is limited.

Case report A three-year old girl presented with a seven day history of painful, fusiform swelling in digits of the hands and feet followed by bilateral swelling of the parotids. The personal history revealed vaccination for measles-mumps-rubella at 12-month of age, in an otherwise healthy children. Body weight and height were at 50th percentile. The physical examination revealed bilateral submandibular, posterior auricular and occipital lymphadenopathies, bilateral painful parotid swelling and dacytylitis. No abdominal tenderness and hepatosplenomegaly were detected. Menengeal irritation signs were absent. Enzyme immunoassay for mumps revealed IgM positivity and the dac-

Figure 1. Dactylitis and parotid swelling prior to steroid treatment

Turk J Hematol 2010; 27: 117-9

tylitis was evaluated as reactive arthritis. Erythrocyte sedimentation rate was 32 mm/hour, anti-nuclear antibody was negative and serum complement 3 and 4 levels were within normal ranges. However since parotid swelling and dactylitis persisted by the 10th day of the onset of symptoms, she was referred to Hacettepe University, Department of Pediatrics, Division of Pediatric Hematology. Informed consent was obtained from the patient. The parotid glands were painless, whereas digits were still painfully swollen at presentation to Division of Hematology (Figure 1). The hemogram was unremarkable except a normocytic anemia (hemoglobin: 9.2 g/dl, MCV: 82 fl; white blood cell count 7.9x109/L, and platelet 124x109/L) and peripheral blood smear exhibited 72% lymphocyte, 12% neutrophil, 7% blast, 7% monocyte and 2% eosinophil. Serum lactate dehydrogenase (LDH) and uric acid were 631 IU/L and 5.6 mg/dl, respectively. The hemoglobin electrophoresis ordered for investigation of dactylitis etiology and excluded HbSS and AS status. The bone marrow aspiration exhibited 100% blasts with L1 morphology according to FAB classification and immunophenotyping revealed a diagnosis of CALLA positive B-cell ALL. The ultrasonographic evaluation of parotids revealed bilaterally swollen gland with hypoechoic areas and hyperechoic septations. Apart from the 4x4 mm and 6x4 mm of size lymphadenopathies within both glands there was another one adjacent to the left parotid gland with 23x9 mm of size. Abdominal ultrasonography revealed a paraaortic lymphadenopathy of 9x6 mm size. Cytologic evaluation of the cerebrospinal fluid showed leukemic involvement of the central nervous system. By the 11th day of onset of symptoms the mumps serology was reordered and seroconversion to IgG was demonstrated and specific treatment for ALL with modified St: Jude Total XIII protocol was initiated [4]. Within 24 2 hours of the onset of 600 mg/m /day methylprednisolone treatment, the parotitis and swelling dactylitis resolved dramatically (Figure 2).

Unal et al. An unusual presentation of pediatric ALL

Turk J Hematol 2010; 27: 117-9

119

Figure 2. Disapperance of dactylitis and parotid swelling by the 24th hour of steroid treatment

Discussion Mumps is a systemic illness caused by the paramyxovirus and the incidence has decreased substantially with the introduction of vaccination [5]. Mumps infection during the course of childhood ALL has been reported to have a mild course and this was related to the intrinsic low cytopathological effect of the virus, contrasting with the severe course of measles and Varicella zoster virus infections in immunocompromised patients [3]. On the other hand, up to our knowledge, this is the first report of mumps infection coincidentally diagnosed at presentation of childhood ALL. Although, mumps infection is common in childhood, the persistent swelling of parotids by the 10th day of onset of symptoms is quite uncommon and surged a possibility of a malignant infiltration of the gland, which was confirmed by the bone marrow examination. Another interesting finding of the patient was the associated dactylitis, which is quite uncommon for a patient with mumps infection. Dactylitis can occur in seronegative arthropathies, such as psoriatic arthritis and ankylosing spondylitis, in sicklecell disease as result of a vasoocclusive crisis with bone infarcts, and in infectious conditions including tuberculosis and leprosy [6]. The most common presenting feature in patients with musculoskeletal complaints who were diagnosed to have an underlying malignancy was monoarthritis, involving the larger joints such as the elbows, knees or ankles. Juvenile idiopathic arthritis was the most frequent provisional diagnosis [7]. A malignancy should always be excluded in children with musculoskeletal symptoms, especially when the clinical pattern is not characteristic of a specific rheumatic disease. A simultaneous presence of high LDH even with normal blood cell counts, should lead to additional investigations, as in the present case. The mumps vaccine induces antibody in 96.9% of seronegative recipients and has 95% protective effect in immunocompotent children [5]. It was previously reported that the seropositivity rates for measles and mumps-specific immunoglobulin G were 90%, while the seropositivity rate for rubella was 85% in 39 previously vaccinated children before administration of chemotherapy for acute leukemia. After treatment,

rates of seropositivity for mumps antibody was reported to be as low as 18% in previously vaccinated children [8]. The immunocompromised state of the leukemic patient at diagnosis and during treatment might prone these previously vaccinated children to infection. In conclusion, acute lymphoblastic leukemia should be suspected during the atypical course of any disease during childhood. Besides, mumps infection at presentation of ALL has a mild course, like the infection that emerge during the period of leukemia treatment. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. 2. 3. 4.

5. 6. 7.

McQuone SJ. Acute viral and bacterial infections of the salivary glands. Otolaryngol Clin North Am 1999;32:793-811. Morgan WS, Castleman B. A clinicopathologic study of Mikulicz's disease. Am J Pathol 1953;29:471-503. de Boer AW, de Vaan GA. Mild course of mumps in patients with acute lymphoblastic leukaemia. Eur J Pediatr. 1989; 148: 618-9. Gurgey A, Yetgin S, Cetin M, Gümrük F, Tuncer AM, Tunçbilek E, Hiçsönmez G. Acute lymphoblastic leukemia in infants. Turk J Pediatr 2004; 46:115-9. Plotkin SA. Mumps vaccine. In: Vaccines. Plotkin SA, Orenstein WA (eds). Philadelphia, Saunders, 2004:441-70. Rothschild BM, Pingitore C, Eaton M. Dactylitis. Implications for clinical practice. Semin Arthritis Rheum 1998;28:41-7. Simonini G, Calabri GB, Falcini F. Incidence of occult cancer in children presenting with musculoskeletal symptoms: a 10-year survey in a pediatric rheumatology unit.Semin Arthritis 2000;29:348-59. Feldman S, Andrew M, Norris M, McIntyre B, Iyer R. Decline in rates of seropositivity for measles, mumps, and rubella antibodies among previously immunized children treated for acute leukemia.Clin Infect Dis 1998;27:388-90.

Case Report

120

First observation of Hb Tunis [beta124(H2) Pro>Ser] in Turkey Türkiye’de gözlenen ilk Hb Tunis [beta124(H2)Pro>Ser] olgusu Aylin Köseler1, Hasan Koyuncu2, Onur Öztürk1, Anzel BahadÕr1, Sanem Demirtepe1, Ayfer Atalay1, Erol Ömer Atalay1 1Department 2Turkish

of Biophysics, Pamukkale University Faculty of Medicine, Denizli, Turkey Ministry of Health Hemoglobinopathy Center, Denizli, Turkey

Abstract Hb Tunis [beta124(H2)Pro>Ser] was reported from Tunisia in 1988. This hemoglobin variant was detected by isoelectric focusing moving just ahead of Hb A. It cannot be identified by standard hemoglobin electrophoresis due to its similar mobility to Hb A. It has normal stability and oxygen affinity and does not produce any clinical symptoms. Here, we report a heterozygous Hb Tunis [beta124(H2)Pro>Ser] case discovered for the first time in Turkey in a premarital screening program. This hemoglobin variant can be identified with high performance liquid chromatography analysis confirmed with DNA sequencing. We emphasize in our study the importance of an interdisciplinary collaborative study at the provincial basis for the success of the hemoglobinopathy control program. (Turk J Hematol 2010; 27: 120-2) Key words: Hemoglobinopathy, abnormal hemoglobin, Hb Tunis Received: September 12, 2008

Accepted: April 8, 2009

Özet Hb Tunis [beta124(H2)Pro>Ser] ilk kez 1988 yÕlÕnda Tunus’da bildirilmiýtir. Bu hemoglobin varyantÕ alkali ve asit ortamda yapÕlan standart elektroforez yöntemleri ile ayrÕlamamaktadÕr. ûzoelektrik odaklama elektroforezinde (IEF-isoelectric focusing) ise Hb A’dan biraz daha hÕzlÕ bir davranÕý ortaya koymaktadÕr. Hb Tunis normal stabilite ve normal oksijen afinitesine sahip olup herhangi bir klinik belirti vermemektedir. Premarital tarama çalÕýmasÕnda saptanan heterozigot Hb Tunis olgumuz, Türkiye ilk kez bildirilmektedir. Bu hemoglobin varyantÕ HPLC yöntemi ile ayrÕlabilmiý ve DNA dizi analizi ile doúrulanmÕýtÕr. Bu çalÕýmamÕzda ayrÕca, hemoglobinopati kontrol programlarÕnÕn baýarÕlÕ biçimde uygulanabilmesi için yöresel düzeyde disiplinler arasÕ iýbirliúinin önemini vurgulamaktayÕz. (Turk J Hematol 2010; 27: 120-2) Anahtar kelimeler: Hemoglobinopati, anormal hemoglobin, Hb Tunis Geliý tarihi: 12 Eylül 2008

Kabul tarihi: 8 Nisan 2009

Address for Correspondence: Prof. Erol Ömer Atalay, Pamukkale Üniversitesi TÕp Fakültesi Biyofizik Anabilim DalÕ (Morfoloji) 20200 Denizli, Turkey Phone: +90 258 211 90 27 Office: +90 258 296 24 87 E-mail: eatalay@pau.edu.tr doi:10.5152/tjh.2010.11

Köseler et al. Hb Tunis case in Turkey

Turk J Hematol 2010; 27: 120-2

Introduction

Case Report

mAU

Hd Tunis

HdA0

Many hemoglobin variants other than Hb S have been reported from Turkey [1,2]. In the Denizli province of Turkey, the most frequent hemoglobin variants are Hb D-Los Angeles and Hb G-Coushatta [3]. In addition to the reviewed hemoglobin variants in Turkey, Hb D-Ouled Rabah, Hb Yaizu and Hb Beograd have also been reported from the Denizli province [4-6]. Hb Tunis [beta124(H2)Pro>Ser] was reported from Tunisia by Mrad et al. in 1988 [7]. This hemoglobin variant was detected by isoelectric focusing (IEF) moving just ahead of Hb A. It has normal stability and oxygen affinity and does not cause any clinical symptoms. It is also not detectable by conventional hemoglobin electrophoresis. The Hemoglobinopathy Control Program has been applied in our province since 2005 in collaboration with the Turkish Ministry of Health Denizli Hemoglobinopathy Center and Pamukkale University Medical Faculty, leading to prenatal diagnosis in the cases of pregnancies at risk. We report herein the first observation of Hb Tunis [beta124(H2)Pro>Ser] hemoglobin variant in Turkey from the Denizli province determined during the premarital screening program.

25 20

HdA1c

HdA2

15 10 5 0 0

121

We report in this study identification of the heterozygous Hb Tunis [beta124(H2)Pro>Ser] in a 21-year-old male living in the Denizli province, located in the Aegean region of Turkey. Written informed consent was obtained for the laboratory tests and DNA analysis from the proband, and the sample was deposited at the DNA Bank of the Department of Biophysics as an anonymous sample. Hemoglobin electrophoresis at alkaline and acid pH, DE-52 column chromatography, and non-radioactive fluorescence-based DNA sequencing as previously published were performed [8]. For the sequencing of the beta globin gene exon 3, the forward primer PAM600 (5’-CAA TGT ATC ATG CCT CTT TGC ACC-3’) and reverse primer PAM603 (5’-CAC TGA CCT CCC ACA TTC CC-3’) were used. High performance liquid chromatography (HPLC) was obtained with BioRad Variant II system, USA. Blood cell counts were done with Beckman Coulter AcT10 instrument, USA. The hemoglobin variant could not be identified by alkaline or acid pH in agarose based electrophoresis and behaved like Hb A. Results of analysis of the red blood cell parameters were as follows: Hb 15.6 g/dl, RBC 5.50 106/mm3, Hct 47.7%, MCV 87.0 fL, MCH 28.3 pg, MCHC 32.7 g/dl, and RDW 12.6%. In DE-52 microcolumn chromatography, Hb A2 was eluted (3.1%) but Hb X could not be eluted with Hb S buffer (0.2 M glycine / 0.014 M NaCl / 0.01 % KCN). Changing buffer content in the NaCl concentration does contribute to the elution of Hb X. The Hb variant produces a double peak at the Hb A window slightly slower than Hb A in HPLC analysis, as shown in Figure 1. According to the HPLC results, the Hb ratios for Hb A0, Hb X, Hb A2 and Hb A1c were 45.71%, 48.84%, 3.09% and 2.36%, respectively. DNA sequencing revealed the hemoglobin variant as a mutation at beta globin gene exon 3 codon 124 (CCA>TCA, Pro>Ser), as shown in Figure 2.

min

Figure 1. HPLC result of heterozygous Hb Tunis case

Codon 124

70 C A C C Y C A C C A G T G CA

Figure 2. DNA sequencing of hemoglobin AX (beta 124, CCA>TCA, Y=C/T)

Discussion In this study, Hb Tunis was identified by DNA sequence analysis, which confirmed the C-to-T mutation at the beta globin gene codon 124. Hb Tunis was reported once in the world populations by Mrad et al. [7] from Tunisia. To the best of our knowledge, our case is the second observation in the world population and is the first reported case in Turkey. This mutation causes the replacement of proline residue into serine. Proline has no ionizable groups, and all conformations like cisand trans- are almost isoenergetic. When proline is in the protein structure, it cannot donate a hydrogen bond for stabilizing the protein structure. On the other hand, serine is a neutral polar molecule that has a tendency to remain on the surface of the proteins. The replacement proline into serine at beta globin codon 124 does not change its standard electrophoretic characteristics at alkaline and acid medium. Only IEF electrophoresis can detect Hb Tunis [beta124(H2)Pro>Ser] as a slightly faster moving band compared to Hb A [7] due to the slight difference in the isoelectric points of the proline and serine residues. The presence of serine also changes the chromatographic behavior of the hemoglobin molecule slightly. This

122

Köseler et al. Hb Tunis case in Turkey

change cannot be observed in DE-52 microcolumn chromatography, but it can be detected by the HPLC system, which has resolution capacity. In the HPLC system, we observed a slightly slower peak at the Hb A window. This behavior is due to the weak interaction of the serine residue with the HPLC column matrix. In the HPLC system, the parameters should be standardized very carefully. If the experimental HPLC analysis parameters are not controlled and standardized, such hemoglobin variants can be bypassed easily. Beta globin gene codon 124 resides in the external region of the hemoglobin molecule and is located in the _1`1 contact region of the hemoglobin tetramer. During transition of the hemoglobin molecule from oxy-form to deoxy-form, the relative displacement of the `-chain to the _-chain is greater at the _1`2-contact than at the _1`1-contact [9]. Therefore, it is expected that the effect of the mutation on the structure and function the hemoglobin molecule is absent or very small and thus does not manifest in clinical symptoms. This heterozygous Hb Tunis case is an apparently healthy male. The place of the codon 124 with its characteristic features confirms this issue. Since homozygous Hb Tunis and other combinations with alpha and beta thalassemia are not yet reported, the clinical significance of this mutation remains unclear in such conditions. Concerning the historical background, North African populations were affected by different Mediterranean populations due to the population movements. The Turkish influence was particularly notable in Tunisia and Algeria during the 14th-19th centuries [10]. Although molecular genetic data like beta globin gene cluster haplotypes is absent, Hb Tunis could have been introduced in the Denizli province gene pool due to these historical population movements. In premarital screening programs, the nature of any hemoglobin variant detected by HPLC that is of potential clinical relevance should be confirmed by alternative techniques [11]. Premarital screening leading to prenatal diagnosis of the hemoglobinopathies has been applied in our province since 2004. Identification of the hemoglobin variants poses problems in the premarital control program in Denizli province due to the heterogeneous structure of the province. According to the results of our registered cases, Hb D-Los Angeles and Hb G-Coushatta were observed frequently [3]. Hb Beograd, Hb Yaizu and Hb D-Ouled Rabah have also been reported from our province [4-6]. We would also like to emphasize the importance of an interdisciplinary collaborative study at the provincial basis to facilitate the success of the hemoglobinopathy control program. In conclusion, we report Hb Tunis [beta124(H2)Pro>Ser] for the first time in Turkey from the Denizli province. We emphasize that the exact identification of many hemoglobin variants can be easily bypassed with the application of limited laboratory techniques like standard hemoglobin electrophoresis and even with HPLC methods. The standardization of the protocols and

Turk J Hematol 2010; 27: 120-2

algorithmic approach used in hemoglobinopathy control programs is another important issue to be addressed.

Acknowledgements The authors are grateful to the staff of the Turkish Ministry of Health Denizli Hemoglobinopathy Center and MSc student of the Biophysics Department, ÇaúÕl Coýkun, for their valuable contributions. This work was supported by the Pamukkale University Research Fund Projects (Nos. 2002TPF017, 2005SBE001, 2005SBE002, 2006SBE004 and 2006TPF001). Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. 2. 3.

10.

11.

Altay Ç. Abnormal hemoglobins in Turkey. Turk J Hematol 2002;19:63-74. Akar E, Akar N. A review of abnormal hemoglobins in Turkey. Turk J Hematol 2007;24:143-5. Atalay EÖ, Koyuncu H, Turgut B, Atalay A, YÕldÕz S, Bahadir A, Köseler A. High incidence of Hb D-Los Angeles [`121(GH4) Glu>Gln] in Denizli province, Aegean region of Turkey. Hemoglobin 2005;29:307-10. Köseler A, BahadÕr A, Koyuncu H, Atalay A, Atalay EÖ. First observation of Hb D-Ouled Rabah [beta19(B1)Asn>Lys] in the Turkish population. Turk J Hematol 2008;25:51-3. Atalay EÖ, Atalay A, Koyuncu H, Öztürk O, Köseler A, Özkan A, Demirtepe S. Rare hemoglobin variant Hb Yaizu observed in Turkey. Med Princ Pract 2008;17:321-4. Atalay A, Koyuncu H, Köseler A, Özkan A, Atalay EÖ. Hb Beograd [`121(GH4)Glu>Val, GAA>GTA] in the Turkish population. Hemoglobin 2007;31:491-3. Mrad A, Blouquit Y, Lacombe C, Blibech R, Arous N, Bardakdjian J, Kastally R, Rosa J, Galacteros F. Hb Tunis [alpha 2 beta 2 124(H2)Pro>Ser], a new beta chain variant identified by HPLC. Hemoglobin 1988;12:23-30. Köseler A, Atalay A, Koyuncu H, Turgut B, Bahadir A, Atalay EÖ. Molecular identification of a rare hemoglobin variant, Hb J-Iran [beta77(EF1)His>Asp], in Denizli province of Turkey. Turk J Hematol 2006;23:164-6. Fujita S. Oxygen equilibrium characteristics of abnormal hemoglobins: Hirose (_2`237Ser), L-Ferrara (_247Gly`2), Broussais (_290Asn`2) and Dhofar (_2`258Arg). J Clin Invest 1972;51:2520-9. Chami B, Blouquit Y, Bardakdjian-Michau J, Riou J, Wajcman H, Rosa J, Galacteros F. Hemoglobin variants in North Africa. Hemoglobin 1994;18:39-51. British Committee for Standards in Hematology. Guideline: The laboratory diagnosis of hemoglobinopathies. Br J Hematol 1998;101:783-92.

Letter to the Editor

123

Ribosomal protein S19 - 631 insertion is an Africanoriginated mutation Afrika kökenli toplumlarda ribozomal protein S19- 631 insersiyonu

Özge CumaoúullarÕ1, Ayýenur Öztürk1, Nejat Akar1, Solaf Elsayed2, Ezzat Elsobky2, Bakhouche Houcher3 1Pediatric

Molecular Genetic Department, Ankara University, Ankara, Turkey Hospital, Ain Shams University, Cairo, Egypt 3Department of Biology, University of Sétif, Faculty of Sciences, Sétif, Algeria 2Pediatric

To the Editor, Ribosomes, the organelles that catalyze protein synthesis, consist of a small 40S subunit and a large 60S subunit. RPS19 gene encodes a ribosomal protein (RP) that is a component of the 40S subunit. The protein belongs to the S19E family of RPs. It is located in the cytoplasm. Mutations in this gene cause DiamondBlackfan anemia (DBA), a constitutional erythroblastopenia characterized by absent or decreased erythroid precursors in 25% of the patients. This suggests a possible extra-ribosomal function for this gene in erythropoietic differentiation and proliferation, in addition to its ribosomal function [1,2]. The RPS19 gene is located on chromosome 19q13.2 and has six exons and spans 11 kb. The first exon is untranslated, and the start codon (AUG) is located at the beginning of exon 2 [1]. RPS19 has three annotated pseudogenes. The RPS19 gene has 196 sequence variants, of which 65 had no known pathogenicity. Recent studies have provided evidence for an association between common polymorphic markers in the RPS19 exon 1 gene -631 locus insertion (ins) GCCA, AGCC and African origin [3]. At the same location, there are two common polymorphisms, -631 ins GCCA, AGCC refsnp:34020014 [4]. As previously reported, these polymorphisms do not have any effect on phenotype.

The common polymorphism -631 ins GCCA was found in African-Americans with an allele frequency of 0.09 [3]. We aimed to study the frequency of this polymorphism in North African countries and also in Turkish Cypriots. In this study, 280 Egyptians, 105 Algerians, 92 Turkish Cypriots and 6 Hemoglobin (Hb) OArab cases were included. RPS19 gene exon 1 was amplified with “F5’TTA CTA CTC CCA CTT CCG GCC AGG GAA CAG 3’, R5’TCA GGC ACG CGC GCT CTG AGG CTT CGG CGT C3’ ” primers followed by digestion with the restriction enzymes HpyF10VI (MwoI, Fermentas, USA). HpyF10VI recognizes 5’-G C N N N N N^N N G C-3’. 3% agarose gel electrophoresis was used to show the fragments, which are 295bp, 158bp and 73bp for normal sample and 173bp, 158bp, 126bp, and 73bp for homozygous sample. In this study, we aimed to analyze the -631 ins GCCA mutation in three different Mediterranean populations, of which two were North African countries. Table 1 shows the genotype distribution in the three countries. Previously, the RPS19 gene -631 ins was reported as an African marker in African-Americans in the United States population [3]. In order to test this hypothesis, we analyzed individuals from two different North African countries. Although rare, we found this polymorphism in Algerians and Egyptians. Our finding supported the hypothesis.

Address for Correspondence: Özge CumaoúullarÕ, MD, Ankara Üniversitesi TÕp Fakültesi, Pediatrik Moleküler Genetik Bilim DalÕ Dikimevi 06100 Ankara, Turkey Phone: +90 312 595 63 48 E-mail: ozge_cuma@yahoo.com doi:10.5152/tjh.2010.12

124

CumaoúullarÕ et al. African-originated mutation RPS19 -631 ins

Turk J Hematol 2010; 27: 123-4

Table 1. RPS gene -631 GCCA insertion genotype and allele frequency in Egyptians, Algerians and Turkish Cypriots Genotype Frequency Country of origin

evant to the subject matter or materials included in this manuscript.

Allele Frequency

N/N (%)

4bpINS (%)

INS

Egypt

280

98.21 (275)

1.78 (5)

0.8

Algeria

105

96.2 (101)

3.8 (4)

1.9

North Cyprus

100 (92)

RPS gene -631 GCCA insertion genotype and allele frequency in Egyptians, Algerians and Turkish Cypriots

Further, we previously noted the Hb OArab mutation in the Turkish Cypriot population, hypothesizing that there could be an admixture of African descent [5]. We thus included samples from North Cyprus; however, we were unable to find this polymorphism in Turkish Cypriots. Hb OArab was first detected in an Arab living in Israel, then in Egypt, Aden (Yemen), Bulgaria, Romania, Hungary, among American-Africans, and in Turkey. It is believed to have originated in the Sudan and to have spread from there to west Africa and to many countries once occupied by or in close contact with the Ottoman Empire [6,7]. For this reason, we screened six Hb OArab samples but none had this polymorphism. This may be due to the few samples analyzed. In conclusion, we can say that this polymorphism is an indicator of African origin. However, it may also have spread to other Mediterranean countries, which will be the subject of another research.

References 1.

Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations rel-

Draptchinskaia N, Gustavsson P, Andersson B, Pettersson M, Willig T-N, Dianzani I, Ball S, Tchernia G, Klar J, Matsson H, Tentler D, Mohandas N, Carlsson B, Dahl N. The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia. Nature Genet 1999;21:169-75. Cmejla R, Blafkova J, Stopka T, Zavadil J, Pospisilova D, Mihal V, Petrtylova K, Jelinek J. Ribosomal protein S19 gene mutations in patients with Diamond-Blackfan anemia and identification of ribosomal protein S19 pseudogenes. Blood Cells Mol Dis 2000;26:124-32. Huang Q, Robledo S, Wilson DB, Bessler M, Mason PJ. A four base pair insertion in exon 1 of the RPS19 gene is a common polymorphism in African-Americans. Br J Haematol. 2006;135:745-6. Ramenghi U, Campagnoli MF, Garelli E, Carando A, Brusco A, Bagnara GP, Strippoli P, Izzi GC, Brandalise S, Riccardi R, Dianzani I. Diamond-Blackfan anemia: report of seven further mutations in the RPS19 gene and evidence of mutation heterogeneity in the Italian population. Blood Cells Mol Dis 2000;26:417-22. Cin S, Akar N, Arcasoy A, Çavdar A, Dedeoúlu S. Haemoglobin O Arab (B121 Glu-Lys) in Turkish Cypriot population. J Med Genet 1984;21:158. Prozorova-Zamani V, Özsoylu ü, Aksoy M, Headlee MG, Lam H, Wilson JB, Altay C, Huisman TH. Hb E and Hb E like in individuals from Turkey. Hemoglobin 1981;5:743-8. Vella F, Beale D, Lehmann H. Hemoglobin O Arab in Sudanese. Nature 1966;209:308.

Letter to the Editor

125

Lower FXII activity and thrombosis: a comment Düýük FXII aktivitesi ve tromboz: bir yorum Viroj Wiwanitkit Wiwanitkit House, Bangkhae, Bangkok Thailand

To the Editor, I read the recent publication by Rasighaemi et al. with great interest. Rasighaemi et al. noted that “Lower FXII activity is not a risk factor; rather, it simply represents a risk marker for thrombosis [1].” I have some concern regarding this work. First, there is the question of whether or not this is a good case-control study. The authors stated that a matched control group was used; however, the large discrepancy between the number of cases and controls makes it difficult to believe that good matching was actually performed. Second, a polymorphism is a population–scale phenomenon; hence, it is questionable whether the rather low number of subjects in this work can be statistically acceptable to answer the research question. Third, there is no evidence to conclude that lower FXII activity represents a risk marker for thrombosis. To conclude this, the complete set of diagnostic test evaluations (sensitivity, specificity, accuracy and predictive value) must be performed.

References 1.

Rasighaemi P, Kazemi A, Ala F, Jazebi M, Razmkhah F. Association of FXII 5’UTR 46C>T polymorphism with FXII activity and risk of thrombotic disease. Turk J Hematol 2010;27:15-9.

for other important factors that may influence a study’s results and judgements, such as gender and age; furthermore, none of them had cirrhosis, nephritic syndrome or cancer. The issue of the number of cases and controls was also consulted upon with a statistical specialist, and based on our population and study, we selected the number of cases and controls that would give us reliable results to judge. Nevertheless, we accept that in a polymorphism study, a higher number of samples would result in more reliable and accurate findings. Nearly all of the recent studies have agreed upon the great importance of the C46T genotype on FXII level. For example, in our study, not only controls but also patients had lower FXII activity in the TT genotype compared with the CT and CC genotypes. Given that there was no association between TT genotype and thrombosis risk, and the importance of the C46T polymorphism on FXII activity, we speculated that the reduced FXII activity in patients with venous thromboembolism is caused by consumption of FXII in both coagulation and fibrinolysis upon thrombosis formation, or by other agents produced in thrombotic patients that can affect FXII activity. Thus, one of the possible explanations for this finding could be consideration of FXII as a risk marker, which is a consequence of thrombosis. Sincerely, Ahmed Kazemi Iran University of Medical Sciences, Hematology Department, P.O Box 14155-6183, Tehran, Iran

Author Reply Although in case-control studies, it is better to have the same number of controls and cases, or more controls than cases, it is not an obligation. If we review the literature, there have been some studies in which control numbers were less than case numbers, such as in the study of Altieri et al. [1], which included 122 patients and 45 controls. In the present study, the controls and cases were not matched for numbers, but they were matched

References 1.

Altieri P, Devoto E, Spallarossa P, Rossettin P, Garibaldi S, Bertero G, Balbi M, Barsotti A, Brunelli C, Ghigliotti G. Acute coronary syndromes do not promote prolonged in vivo FXII dependent prothrombotic activity. Thromb Res 2005;115:65-72.

Address for Correspondence: Prof. Viroj Wiwanitkit, Wiwanitkit House, Bangkhae, Bangkok Thailand Phone: +662-4132436 E-mail: wviroj@yahoo.com doi:10.5152/tjh.2010.13

Letter to the Editor

126

IgA lambda oligoclonal gammopathy in multiple myeloma Oligoklonal gammopati ile prezante olan bir Ig A multipl miyelom olgusu ûbrahim Tek1, Dilsa MÕzrak2, Güngör Utkan1, Selami Koçak Toprak3, Hüseyin Tutkak4, Abdullah Büyükçelik1, Bülent YalçÕn1, Hakan Akbulut1, Fikri ûçli1 1Department

of Medical Oncology, Ankara University School of Medicine, Ankara, Turkey of Internal Medicine, Ankara University School of Medicine, Ankara, Turkey 3Department of Hematology, Ankara University School of Medicine, Ankara, Turkey 4Department of Clinical Immunology and Rheumatology, Ankara University School of Medicine, Ankara, Turkey 2Department

To the Editor, Multiple myeloma is a disorder in which malignant plasma cells, generally derived from one clone, accumulate in the bone marrow [1]. Electrophoresis of plasma proteins is used as a diagnostic test for this disorder [2,3]. In rare conditions, electrophoresis produces more than one narrow peak simultaneously, suggesting the existence of more than one clonal antibody and establishing a biclonal, triclonal or oligoclonal gammopathy [3]. A 56-year-old male was referred to our hospital in September 2005 with low-back pain. The physical examination was normal, except for muscle weakness of the bilateral lower extremities. The laboratory examination findings were as follows: hemoglobin 11.8 g/dl, white cell count 3.8x109 /L, erythrocyte sedimentation rate 102 mm/h and globulin 2.7 g/dl. Serum IgG was 3.98 g/dl (7-16), IgA 9.7 g/dl (0.7-4), IgM 0.43 g/dl (0.4-2.3), lambda light chain 3.94 g/L (0.9-2.1), and kappa light chain 1.25 g/L (1.7-3.78). Serum protein electrophoresis showed four clonal bands on the gamma region. Capillary immunofixation of the serum revealed the presence of IgA lambda oligoclonal gammopathy (Figure 1). The liver and kidney functions were normal. X-ray films of the bones revealed lytic areas in the skull, pelvis and the ribs. Bone marrow aspiration showed hypercellularity with approximately 30% infiltration of low differentiated plasma cells. Informed consent was obtained from the patient. A homogeneous spike-like peak in a focal region of the gamma-globulin zone indicates a monoclonal gammopathy.

Both neoplastic and non-neoplastic disorders are associated with monoclonal paraproteinemia [4]. Biclonal gammopathy is associated with similar disorders [5]. The distribution between various clinical entities, the clinical features of the patients as well as the prognosis seem to be similar to those of monoclonal gammopathies [6]. In contrast, polyclonal gammopathies may be caused by any reactive or inflammatory process [7]. They are characterized by a broad diffuse band with one or more heavy chains and kappa and lambda light chains. Protein electrophoresis is extremely valuable for recognizing cases of SPE

IgG

IgA

IgM

Kappa

Lambda

Figure 1. Capillary immunofixation of the serum revealed the presence of IgA lambda oligoclonal gammopathy (arrow)

Address for Correspondence: Assoc. Prof. Ibrahim Tek, Department of Medical Oncology, Medicana International Ankara Hospital Mobile: +90 533 638 47 88 E-mail: itekmdr@yahoo.com doi:10.5152/tjh.2010.14

Turk J Hematol 2010; 27: 126-7

monoclonal gammopathies and for following quantitative changes in spikes. M-proteins appear as tall, narrow, sharply defined peaks that reflect their structural homogeneity. They are usually located in the a or a-` region. IgA spikes are usually broader, as seen in our case, because the molecule tends to form polymers of different sizes (4). The prevalence of mono and biclonal gammopathies in the general population ranges between 1-3%, but only 1% of these are biclonal [3]. In conclusion, our case is the first report of multiple myeloma that is derived from four clones. Immunoelectrophoresis showed that all four clones included the same heavy and light chain. Conflict of interest No author of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

Tek et al. IgA lambda oligoclonal gammopathy in myeloma

127

References 1. Sirohi B, Powles R. Multiple myeloma. Lancet 2004;363:875-87. 2. Longo DL, Anderson KC. Plasma cell disorders. In: Kasper, DL, Fauci AS, Longo DL, et al., editors. Harrison’s Principles of Internal Medicine. New York: McGraw-Hill, 2005: 656-62. 3. Cohen Y, Ben-Bassat I. Biclonal gammopathies: clinical and theoretical aspects. Harefuah 1993;124:393-5, 456. 4. Berenson JR, Casciato DA. Plasma cell dyscrasias and Waldenström’s macroglobulinemia. In: Manual of Clinical Oncology. 5th ed. 2004: 458-79. 5. Alanoglu G, Ozet G, Davla K, Kuzu I, Erekul S, ûlhan O, Beksaç M. Extramedullary presentation of biclonal IgGk and IgAk multiple myeloma. Turk J Haematol 2002;19:421-5. 6. Kyle RA, Robinson RA, Katzmann JA. The clinical aspects of biclonal gammopathies. Am J Med 1981;71:999-1008. 7. O’Connell TX, Horita TJ, Kasravi B. Understanding and interpreting serum protein electrophoresis. Am Fam Physician 2005;71:105-12.

Letter to the Editor

128

Blood transfusion services in Iraq; an unfortunate field Irak’ta kan transfüzyon hizmetleri: talihsiz bir alan Abbas Hashim Abdulsalam Al-yarmouk Teaching Hospital, Laboratory Department, Hematology Unit, Baghdad, Iraq

To the Editor,

Iraq is unfortunately still delayed in the development of blood transfusion services. My tenure for over 18 months as head of the blood transfusion center in one of the major teaching hospitals in Baghdad, Iraq, Al-yarmouk Teaching Hospital, led me to this unpleasant conclusion. There is an absence of almost any type of scientific interest, e.g., no Iraqi blood banking or hematology journal and no actual scientific meetings to review and improve the work in this field. There is also a lack of proper training and continuing medical education programs for the staff. Other deficiencies in the blood transfusion services are summarized hereunder: 1. Absence of obligatory, clear and acceptable minimal standards that should be available before starting a new blood transfusion center, such as regarding building size and size and number of rooms. The blood transfusion center in Al-Yarmouk Teaching Hospital is composed of only a small part of the building housing the teaching laboratories, with a few small rooms that do not meet any written national or international standards [1]. 2. Unavailability of normal ranges for Iraqi hematological indices, as normal hemoglobin concentration or PCV %. As a result, there is continuing unfruitful debate about the lower accepted values for blood donation. 3. Absence of medical record profiling and a registration process, with absence of computerization and networking services.

Improper interviewing of blood donors due to lack of privacy, insufficient number of examining doctors and medical assistants, and inadequate history taking and physical examination. There is also insufficient attention given to the regular and onneed voluntary blood transfusion programs with self-acceptance and self-deferral issues. Chronic inadequate and irregular supply of empty blood bags, instruments and disposables. The blood transfusion center in Al-Yarmouk teaching hospital was left without blood bags for a few months in 2008 [2]. Incomplete choice of combination of tests on blood donation that should be used in each center, with absence of HCV NAT on pooled plasma, ELISA anti-CMV Ab, ELISA anti-HTLV-I Ab, and optional test for malaria. Not unexpectedly, no antibody screening is done on blood donations. The choice of cross-matching procedure has not been reviewed in the last three decades, in order to delete unneeded and confusing steps or to add essential improved ones. For example, the immediate spin (IS) step is left for a 30-minute incubation period, leading to a high rate of false positives (detecting clinically insignificant cold antibodies, causing confusion), and as a result, many needed blood transfusions are cut short because tests are not carried out only at 37°C, but also at room temperature with a long incubation period. While not supported by any scientific reference, the staff are required to do so as mentioned in the standard procedures for Iraqi blood banking. Furthermore, apart from major cross-match, there

Address for Correspondence: Abbas Hashim Abdulsalam, MD, Iraq-Baghdad-Al-yarmouk Teaching Hospital 964 Baghdad - Iraq Phone: 00 964 7904 188690 E-mail: dr.abbas77@yahoo.com doi:10.5152/tjh.2010.15

Abdulsalam A. H. Blood transfusion services in Iraq

Turk J Hematol 2010; 27: 128-9

is absence of abbreviated cross-matching, antibody screening and antibody identification tests. There is also a lack of clear emergency guidelines on abbreviated cross-match and how to shorten time of delivery, as there is no clear policy for emergency blood transfusion, and the decision is left to the local personnel who are almost always non-qualified to make such decisions. 8. In all cases, the only blood group typing available is ABO and Rh D. 9. Insufficient recipient blood sample storage time (less than 24 hours at most, and usually much shorter). 10. Apart from the conventional polyspecific DAT and IAT, absence of monospecific DAT and all antibody identification, characterization and specification tests in warm and cold immune hemolytic anemia. Antibody screening and identification panels are not yet available for routine use. 11. Chronic inadequacy in preparing and transfusing leukodepleted blood components, with a continuing erroneous

129

focus on transfusing whole blood even in cases when there is a clear indication for only blood components, as in thalassemia major patients. In conclusion, the Iraqi health authority needs to undertake a thorough and complete scientific revision with respect to all aspects of blood transfusion services with an accepted budget arrangement in order to reach the unequivocal aim of providing safe and effective blood transfusion services.

References 1.

Blood transfusion in a global context: Dave Roberts, Jean-Pierre Allain, Alan Kitchen, Stephen Field and Imelda Bates in Practical Transfusion Medicine, 3rd edition. Eds Murphy MF and Pamphilon D. Wiley-Blackwell, 2009: 251-61. Abbas Hashim Abdulsalam and Sahar Hisham Abdulrazzaq: Underestimating the expected need for blood transfusion in elective procedures; a consistent feature of blood ordering system in Iraq. British Journal of Haematology. {Article in press}.

Letter to the Editor

130

Mega-dose methylprednisolone in hematologic and non-hematological disorders

Hematolojik ve hematolojik olmayan hastalÕklarda yüksek doz metilprednizolon üinasi Özsoylu

To the Editor, The comprehensive, basic and stimulating review article entitled “A novel approach to treatment in childhood acute myeloblastic leukemia and myelodysplastic syndrome with high-dose methylprednisolone as a differentiation and apoptosis–inducing agent of myeloid leukemia cells” by Prof. Hiçsönmez in the recent issue of the Journal gives me an opportunity to clarify this mode of treatment [1]. Although high-dose corticosteroid administration (up to 1000 mg) was used prior to our recommended method of methylprednisolone (MP) treatment, the drug was infused any time of the day intravenously over a 4-hour period, which is generally called “pulse MP treatment” although this term was not used before 1960. The term “high-dose methylprednisolone” (HDMP) was used for our method of MP administration initially [2-6], following the dose recommendation by Bacigalupo et al. [7], but we changed the term to mega-dose methylprednisolone (MDMP), since HDMP was also used in the literature for 4-10 mg/kg doses. I would also like to emphasize that MDMP treatment differs from conventional corticosteroid (2 mg/kg in divided doses) HDMP and pulse and bolus MP (1000 mg infused by Bacigalupo et al. in 4 hours) administration, not only by dose (which is increased up to 100 mg/ kg, if required for 3 days initially and then tapered gradually) but also the time of administration [8]. Each MDMP dose (in 10-15 iv or at once orally, covered by honey) is given around 6 am (originally stated as before 9 am) when the corticosteroid level is

highest physiologically in the body, which seems to be important for adrenocorticotropic hormone (ACTH) and corticosteroid homeostasis (highest dose of 4950 mg was given to a patient with acquired aplastic anemia). We gave a short course of MP (30 mg/kg for 3 days followed by 20 mg/kg for 4 days) to patients with acute idiopathic thrombocytopenic purpura, in which the disease prognosis was good; the duration of treatment usually extended to months or even years, according to the severity of the disease. It seems that longer usage of MDMP treatment is better for the prevention of recurrences and relapses [9]. Thus far, more than 500 patients with different hematological (acquired aplastic anemia, DiamondBlackfan anemia, idiopathic myelofibrosis, idiopathic thrombocytopenic purpura, severe pure red cell hypoplasia, acquired thrombotic thrombocytopenic purpura, severe Coombs (+) hemolytic anemia, hypereosinophilic syndrome, paroxysmal nocturnal hemoglobinuria, Evans syndrome, idiopathic pulmonary hemosiderosis, Kasabach-Merritt syndrome, different acute leukemias, thalassemia intermedia) and non-hematological (steroid-resistant nephrotic syndrome, systemic lupus erythematosus, dermatomyositis, Kawasaki disease, mixed collagen disease, polyarteritis nodosa, rheumatic fever, rheumatoid arthritis, diaphyseal dysplasia, lymphocytic infiltration of the lung, alopecia universalis, sarcoidosis, osteopetrosis, hemangiomatosis) diseases have been treated with MDMP [10-13]. Corticosteroid side effects (hypertension, hyperglycemia, growth retardation, cushingoid appearance, etc.) were practically non-existent as reported by others [14],

Address for Correspondence: üinasi Özsoylu, MD, Beysukent, Altunýehir Sitesi No.30, Ankara, Turkey Phone: +90 312 203 55 55 E-mail: sinasiozsoylu@hotmail.com doi:10.5152/tjh.2010.16

Özsoylu ü. Mega-dose methylprednisolone

Turk J Hematol 2010; 27: 130-1

with the exception of abdominal discomfort, which occurred in half of the patients with oral MDMP treatment [15]. The above points are important and highlight that the results of every high-dose corticosteroid can not necessarily be compared. Lastly, I believe saline nose drops should be recommended to all MDMP users for the prevention of upper respiratory tract infections, as we previously suggested [16-17] so that interruptions in treatment can be prevented. In short, I believe the term “MDMP treatment” better differentiates this kind of corticosteroid administration from the other applications, a point which we have reiterated on several occasions.

References

6. 7.

10.

11. 12.

1. 2. 3. 4. 5.

Özsoylu ü, Ruacan ü. High dose bolus methylprednisolone treatment for primary myelofibrosis. Eur J Pediatr 1983;140:810. Özsoylu ü. High-dose intravenous corticosteroid for a patient with Diamond Blackfan syndrome refractory to classical prednisone treatment. Acta Haematol 1984;71:207-10. Özsoylu ü, Coýkun T, Minassazi S. High dose intravenous glucocorticoid in the treatment of childhood acquired aplastic anemia. Scand J Hematol 1984;33:309-16. Özsoylu ü, Coýkun T, Minassazi S. High dose intravenous glucocorticoid in the treatment of childhood acquired aplastic anemia. Scand J Hematol 1984;33:309-16. Özsoylu ü. High dose intravenous methylprednisolone treatment for patients with Diamond-Blackfan syndrome resistant or refractory to conventional treatment. Am J Pediatr Hematol Oncol 1988;10:217-23.

13. 14.

15. 16. 17.

131

Özsoylu ü. High dose intravenous methylprednisolone (HIVMP) in hematologic disorders. Hematol Rev 1990;4:197-207. Bacigalupo A, Giordano D, Van Lint MT, Vimercati R, Marmont AM. Bolus methylprednisolone in severe aplastic anemia. N Engl J Med 1979;30:501-2. Özsoylu ü. Megadose methylprednisolone (MDMP) for childhood idiopathic thrombocytopenic purpura (ITP). Turk J Med Sci 2005;35:347-56. Özsoylu ü, Ertürk G. Oral megadose methylprednisolone for acute childhood idiopathic thrombocytopenic purpura. Blood 1991;77:1856-7. Özsoylu ü. Megadose metilprednizolon tedavisi; resyonal kortikosteroid kullanÕmÕ. In: Karaaslan Y, KadayÕfçÕ A, editors. 2004;70-4 (in Turkish). Özsoylu ü. Megadose methylprednisolone (MDMP) treatment. Turk J Pediatr 2004;46:292. Özsoylu ü. Megadose methylprednisolone (MDMP) treatment. Turk J Hematol 2006;23:115-8. Özsoylu ü. Treatment of primary myelofibrosis. Turk J Hematol 2009;26:211-2. Bernini JC, Carrillo JM, Buchanan GR. High-dose intravenous methylprednisolone therapy for patient with Diamond-Blackfan anemia refractory to conventional doses of prednisone. J Pediatr 1995;127:654-9. Özsoylu ü. Akut ITP’de methylprednisolone tedavisi. Çocuk SaúlÕúÕ ve HastalÕklarÕ Dergisi 2006;49:78-80. Özsoylu ü. Nose drops and the common cold. Eur J Pediatr 1985;144:294. Engin E, KÕlÕnç O, Özsoylu ü. Prevention of upper respiratory infections in health personnel by using saline. Yeni TÕp Dergisi 1997;14:211-2 (in Turkish).

Letter to the Editor

132

Oseltamivir and G6PD deficiency Oseltamivir ve G6PD eksikliúi üinasi Özsoylu

To the Editor, In the recent issue of the Journal, Dr. Akar suggested that oseltamivir might be used safely in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency [1]. Since the substances that decrease reduced glutathione and membrane stability (by oxidation) are responsible for hemolysis in G6PD deficiency, I am interested to learn the reasoning for the oseltamivir reaction if it caused hemolysis?

metabolite oseltamivir carboxylate is produced. Other metabolites-oseltamivir phosphate, D3-oseltamivir phosphate, and D3-oseltamivir carboxylate - have since been detected [2,3]. There is at present no information on the possible effects of these compounds on the G6PD pathway. As a result, G6PD-deficient individuals have been excluded during the pharmacokinetic studies of oseltamivir [2]. As we stated previously, further studies are needed on this subject. Sincerely, Nejat Akar MD, Prof., MESA Hospital, Ankara, Turkey

References 1.

Akar N. A note on oseltamivir treatment in a boy with G6PD deficiency. Turk J Hematol 2010;27:48.

Author Reply

References 1.

We thank Prof. Dr. Özsoylu for his question concerning our letter that was written due to the non-existent information on oseltamivir usage in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals [1]. Dr. Özsoylu wondered about the possible mechanism of oseltamivir that may be responsible for the hemolysis in G6PD-deficient individuals. Following oral administration of oseltamivir, after de-esterification of the compound, primarily the active

Akar N. A note on oseltamivir treatment in a boy with G6PD deficiency. Turk J Hematol 2010;27:48. Holodniy M, Penzak SR, Straight TM, Davey RT, Lee KK, Goetz MB, Raisch DW, Cunningham F, Lin ET, Olivo N, Deyton LR. Pharmacokinetics and tolerability of oseltamivir combined with probenecid. Antimicrob Agents Chemother 2008;52:3013-21. Doucette KE, Aoki FY. Oseltamivir: a clinical and pharmacological perspective. Expert Opin Pharmacother 2001;2:1671-83.

Address for Correspondence: üinasi Özsoylu, MD, Beysukent, Altunýehir Sitesi No.30, Ankara, Turkey Phone: +90 312 203 5555 E-mail: sinasiozsoylu@hotmail.com doi:10.5152/tjh.2010.17

Letter to the Editor

133

FLT3 - ITD positive acute lymphocytic leukemia, does it impact on disease´s course? FLT3 – ITD pozitif akut lenfositik lösemi hastalÕúÕnÕn gidiýatÕnÕ etkiliyebilir mi? Sebastian Kobold1, Nerbil KÕlÕç1,2, John Scharlau3, Carsten Bokemeyer1, Walter Fiedler1 1Department of Oncology/Hematology/Bone Marrow Transplantation with the Section Pneumology, University Medical Center Hamburg Eppendorf, Hamburg, Germany 2University Hospital Basel, Medical Oncology/Gynaecology, Basel, Switzerland 3Clinic For Internal Medicine 2, University Hospital Bonn, Bonn, Germany

To the Editor, Fms- like tyrosine kinase 3 (FLT3) - internal tandem duplication (ITD) has been identified in up to 25% of all acute myeloid leukemia where it correlates with a very poor prognosis [1]. Since FLT3 is frequently expressed by acute lymphocytic leukemia (ALL) blasts, activating FLT3 mutations may as well occur in this disease [2]. According to the literature, only 14 of 1634 ALL-patients were tested positive for FLT3-ITD (Table 1) [2-8]. Unfortunately, survival data of those patients is lacking and conclusions on the impact of FLT3-ITD in ALL patients are inconsistent. However - in analogy to AMLit has been suggested that FLT3-ITD dramatically worsens patient´s survival [8].

We report of a 42-year-old woman with a FLT3-ITD PCR positive common B-ALL. No established negative prognostic factors were found; therefore the patient was considered at standard risk. The patient achieved a complete remission after the first course of conventional induction chemotherapy. Importantly, assessment of a patient specific molecular minimal residual disease (MRD) by PCR was repeatedly performed and scored negative. Since the first control at day 28, none of the patient specific markers could be detected. 8 months later, the patient is still in complete remission. Accordingly, FLT3-ITD might not necessarily be associated with a worse outcome. Interestingly, for all FLT3-ITD positive ALL patients where an adverse outcome had been described, other

Table 1. Overview on studies reporting FLT3-ITD- positive ALL patients and their outcome # patients screened

Study population

# FLT3-ITD +

Risk factors

Outcome

Reference

adults

high LDH, high blast count

73.7% vs 84%* complete remissions

[2]

132

children

relapsed ALL

N.S.

[3]

children

biphenotypic ALL

44 and 72 months

[9]

174

children

none

alive at time of report

[5]

143

children

N.S.

[6]

N.S.

biphenotypic ALL, high blast count

poor

[7]

449

adults

CD117 positive

N.S.

[8]

*: FLT3+ vs FLT3- N.S.: not specified

Address for Correspondence: Nerbil KÕlÕç, MD, Petersgraben 4 4057 Basel, Switzerland Phone: +41-61 3286099 Office: +41 612652525 E-mail: tatlican8@hotmail.com doi:10.5152/tjh.2010.18

134

Kobold et al. FLT3 - ITD positive ALL

negative prognostic factors such as bi-phenotypic leukemia + [7, 9], c-Kit expressing T-ALL [8] or relapsed ALL [3] have been reported. Several authors even reported no deterioration of overall survival for FL3-ITD positive standard risk ALL patients (Table 1). In addition, it has been proposed that the prognosis might be correlated with high levels of FLT3 on leukemic blasts rather than with FLT3-ITD in ALL patients [2]. In the present case of a patient with common B-ALL, FLT3ITD did not seem to impact the patient’s prognosis. This assumption is based on the persistently negative MRD assessment more than 8 months after treatment’s start. A negative MRD on week 10 most strongly correlates with a prolonged remission and cure [10]. In fact, the MRD negative group has a projected 75% cure rate [10]. In summary, ALL with FLT3/ITD might not be associated with a poor prognosis, although the very low incidence of this molecular alteration in ALL prevents a definite conclusion. Larger prospective series are necessary to finally clarify the prognostic significance of FLT3 mutations in ALL.

References 1. Kottaridis PD, Gale RE, Linch DC. Prognostic implications of the presence of FLT3 mutations in patients with acute myeloid leukemia. Leuk Lymphoma 2003;44:905-13. 2. Peng HL, Zhang GS, Gong FJ, Shen JK, Zhang Y, Xu YX, Zheng WL, Dai CW, Pei MF, Yang JJ. Fms-like tyrosine kinase (FLT) 3 and FLT3 internal tandem duplication in different types of adult leukemia: analysis of 147 patients. Croat Med J 2008;49:650-69. 3. Wellmann S, Moderegger E, Zelmer A, Bettkober M, von Stackelberg A, Henze G, Seeger K. FLT3 mutations in childhood acute lymphoblastic leukemia at first relapse. Leukemia 2005;19:467-8.

Turk J Hematol 2010; 27: 133-4

4. Xu F, Taki T, Eguchi M, Kamada N, Ishii E, Endo M, Hayashi Y. Endo and Y. Hayashi. Tandem duplication of the FLT3 gene is infrequent in infant acute leukemia. Japan Infant Leukemia Study Group. Leukemia 2000;14:945-7. 5. Nakao M, Janssen JW, Erz D, Seriu T, Bartram CR. Tandem duplication of the FLT3 gene in acute lymphoblastic leukemia: a marker for the monitoring of minimal residual disease. Leukemia 2000;14:522-4. 6. Andersson A, Paulsson K, Lilljebjörn H, Lassen C, Strömbeck B, Heldrup J, Behrendtz M, Johansson B, Fioretos T. Johansson and T. Fioretos. FLT3 mutations in a 10 year consecutive series of 177 childhood acute leukemias and their impact on global gene expression patterns. Genes Chromosomes Cancer 2008;47:64-70. 7. Xu B, Li L, Tang JH, Zhou SY. Zhou. Detection of FLT3 gene and FLT3/ITD mutation by polymerase chain reaction-single-strand conformation polymorphism in patients with acute lymphoblastic leukemia. Di Yi Jun Yi Da Xue Xue Bao 2005;25:1207-10. 8. Paietta E, Ferrando AA, Neuberg D, Bennett JM, Racevskis J, Lazarus H, Dewald G, Rowe JM, Wiernik PH, Tallman MS, Look AT. Look. Activating FLT3 mutations in CD117/KIT(+) T-cell acute lymphoblastic leukemias. Blood 2004;104:558-60. 9. Xu F, Taki T, Yang HW, Hanada R, Hongo T, Ohnishi H, Kobayashi M, Bessho F, Yanagisawa M, Hayashi Y.Hayashi. Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children. Br J Haematol 1999;105:155-62. 10. Sutton R, Venn NC, Tolisano J, Bahar AY, Giles JE, Ashton LJ, Teague L, Rigutto G, Waters K, Marshall GM, Haber M, Norris MD; Australian and New Zealand Children's Oncology Group. Clinical significance of minimal residual disease at day 15 and at the end of therapy in childhood acute lymphoblastic leukaemia. Br J Haematol 2009.

Images in Hematology

135

Pleural fluid plasmacytosis in a patient with plasma cell leukemia Plazma hücreli lösemi hastasÕnda plevral sÕvÕ plazmasitozu Raihan Sajid, Bushra Moiz, Nausheen Kamran, Salman Naseem Adil Department of Pathology and Microbiology, Aga Khan University, Karachi, Pakistan

Plasma cell Leukemia (PCL) is a rare disorder characterized by malignant proliferation of plasma cells in peripheral blood and bone marrow. There are two forms of PCL: the primary form occurring in patients without preceding multiple myeloma or monoclonal gammopathy of undetermined significance, and the secondary form arising as a leukemic transformation of multiple myeloma [1]. A 55 years old lady presented in hematology clinic with complaints of fever, cough and bone pains since last 8 months. General physical examination was unremarkable except for pallor. Initial investigations showed hemoglobin: 9.6 gm/dl, hematocrit: 27.6%, MCV: 88 fl, MCH: 27 pg, white blood cells: 11.5 x109/L and platelets: 356x109/L. Peripheral film showed normochromic normocytic anemia with marked rouleux formation. Diffuse infiltration with atypical and pleomorhic plasma cells was observed on subsequent bone marrow examination. Serum immunofixation showed IgG kappa monoclonal gammopathy. Serum IgG levels were 56.6g/L and skeletal survey demonstrated multiple lytic lesions in the skull. Creatinine level at diagnosis was 114 mol/L and calcium was 2.5 mmol/L. Based on her clinico-pathological manifestations, a diagnosis of multiple myeloma (stage IIIA according to Durie and Salmon classification) was made and the patient was started on thalidomide and dexamethasone. However, unable to tolerate the treatment due to fatigue and somnolence, she presented again with shortness of breath and productive cough in one month time. This time, her chest X-ray revealed left sided pleural effusion along with right sided hydropneumothorax. Hilar vascular congestion and a posterolateral erosive lesion in fifth right rib were also evident. (Figure 1). Subsequent pleural tap drained off 1200

ml of fluid from each side. Pleural fluid analysis an exudative picture with numerous red cells with leukocyte count of 1900 mm3 and pleural fluid cytology exhibited malignant plasma cells. (Figure 2). This was not secondary to a traumatic tap as most of the cells were plasma cells and the differential in the peripheral blood was quite different as described below. Immuno-histochemistry demonstrated CD 138 positive plasma cells (Figure 3). Complete blood counts showed Hb 7.7 g/dl, WBC 10.1 x103 / L, Neutrophils 14%, Lymphocytes 56%,Plasma cells 22%;Monocytes:8% and Eosinophils 0% and platelets 55x106/L. Peripheral film examination showed 22% plasma cells consistent with transformation to plasma cell leukemia. Serum albumin was 2.3 g/dl at the time of pleural tap and no evidence of renal failure was present at the time of progression of disease. Patient was treated with one cycle of thalidomide (reduced dosage at 100 mg/day) and dexamethasone as she refused for more aggressive chemotherapy and was discharged on this treatment but was lost to follow up. PCL constitutes 2% to 4% of all cases of plasma cell disorders. The WHO criterion for diagnosis of PCL is that plasma cells constitute more than 20% of cells in the peripheral blood with an absolute plasma cell count of more than 2,000 mm3 [2]. The primary form which arise de novo constitutes 60% of all cases while secondary type represents leukemic transformation of myeloma. Overall response to treatment is poor with median survival of less than one year. Pleural fluid plasmacytosis in association with plasma cell leukaemia has been reported infrequently in literature [3, 4]. Review of local literature also revealed few cases of plasma cell leukaemia [5,6].

Address for Correspondence: Asst. Prof. Raihan Sajid, Stadium Road 74800 Karachi, Pakistan Phone: 0092214861306 E-mail: raihan.sajid@aku.edu doi:10.5152/tjh.2010.19

136

Sajid et al. Pleural fluid plasmacytosis

Figure 1. CD 138 on pleural fluid

Turk J Hematol 2010; 27: 135-6

Figure 3. Pleural fluid plasmacytosis

Our patient was initially diagnosed with multiple myeloma; however she transformed to plasma cell leukaemia and also developed pleural fluid plasmacytosis. Pleural fluid plamacytosis seems to be an expression of this aggressive disease with poor outcome in most case reports and case series despite treatment.

References: 1. 2.

3. 4.

5. 6. Figure 2. Chest X ray

Jimenez-Zepeda VH, Dominguez VJ. Plasma cell leukemia: a rare condition. Annals of hematology 2006;85:263-277. Kyle RA, Maldonado JE, Bayrd ED. Plasma cell leukemia. Report on 17 cases. Archives of internal medicine1974; 133:813-8. Alexandrakis MG, Passam FH, Kyriakou DS, Bouros D. Pleural effusions in hematologic malignancies. Chest 2004; 125:1546-55. Suresh Attili BU, Devi Lakshm, P. P. Bapsy, K. C. Lakshm, K. Govind, D. Lokana, Saini Kamal, G. Anupam. Malignant myelomatous pleural effusion-Is onset of effusion a new prognostic factor? Turk J Hemato. 2007; 4:181-5. Prabhat D, Bijur SJ, Pathare AV. Plasma cell leukaemia--a report of two cases. Journal of postgraduate medicine. 1998; 44:47-9. Raj RS, Najeeb S, Aruna R, Pavithran K, Thomas M. Primary plasma cell leukemia occuring in the young. Indian journal of cancer 2003; 40:116-7.

Advisory Board of This Issue (June 2010) A.Nazlı Başak Ahmet Öztürk Ahmet Türker Çetin Ajlan Tükün Akif Selim Yavuz Ali Uğur Ural Ali Ünal Ayşegül Ünüvar Ayşen Timurağaoğlu Aytemiz Gürgey Burhan Turgut Bülent Eser Cengiz Beyan Çiğdem Altay David Deremer Dieter Hoelzer Dilber Talia İleri Duran Canatan Duygu Uçkan

Elif Ünal Erol Erduran Evren Özdemir Fatma Gümrük Feride Duru Ferit Avcu Fevzi Altuntaş Gina Zini Görgün Akpek Güray Saydam Güçhan Alanoğlu Gülsüm Emel Pamuk Günay Balta Hakan Göker Hale Ören Işınsu Kuzu Kaan Kavaklı Mehmet Ertem Mualla Çetin Mustafa Nuri Yenerel

Mutlu Arat Mutlu Hayran Nejat Akar Nerbil Kılıç Önder Arslan Sema Karakuş Semra Paydaş Şule Mine Bakanay Şule Ünal Tansu Sipahi Tiraje Çelkan Tunç Fışgın Uğur Özbek Ülker Koçak Vefki Gürhan Kadıköylü Yahya Büyükaşık Yener Koç Yeşim Aydınok Yurdanur Kılınç

Announcements 4-8 June 2010 2010 ASCO Annual Meeting Chicago, USA

21-23 October 2010 Lymphoma & Myeloma 2010: An International Congress on Hematologic Malignancies New York, NY, United States

10-13 June 2010 15th Congress of the EHA Barcelona, Spain

3-6 November 2010 36. Turkish National Hematology Congress Antalya, Turkey

10-14 June 2010 World Congress of the World Federation of Hemophilia Buenos Aires, Argentina

4-7 December 2010 52. ASH Annual Meeting and Exposition Orlanda, Florida, USA

25-27 June 2010 ESH-EHA Type II Tutorial on Myeloid Malignancies Istanbul, Turkey

11-14 May 2011 3. International Congress on Leukemia Lymphoma Myeloma, รปstanbul, Turkey

10-13 October 2010 The 33rd World Congress of the International Society of Hematology (ISH 2010) Jerusalem, Israel