tjh-2010-1

Page 1

Issue 1

March 2010

40 TL

ISSN 1300-7777

Volume 27

TURKISH JOURNAL OF HEMATOLOGY • VOL.: 27 ISSUE: 1 MARCH 2010

High-dose methylprednisolone in AML and MDS Gönül Hiçsönmez; Ankara, Turkey

Mixed chimerism in thalassemia major Elif Ünal ûnce et al.; Ankara, Turkey

FXII 46C>T polymorphism and thrombosis risk Parisa Rasighaemi et al.; Tehran, Iran, United Kingdom.

HAP in immunocompromised patients Ebru ÇakÕr Edis et al.; Edirne, Turkey

Sonographic evaluation of spleen size Selim Serter et al.; Manisa, Turkey

Non-Hodgkin's lymphoma with bone involvement Filiz Vural et al.; ûzmir, Turkey

Cover Picture : Alp Can Life on the Edge

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Editor-in-Chief

International Review Board

Aytemiz Gürgey

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)

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)

Contact Information

All other inquiries should be adressed to

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

TURKISH JOURNAL OF HEMATOLOGY

Associate Editors

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

Erich Frank Orhan Ulutin Hamdi Akan Senior Advisory Board

Orhan Ulutin Yücel Tangün Osman ûlhan

Language Editor

Corinne Can Statistic Editor

Mutlu Hayran Editorial Secretary

ûpek Durusu Bengü Timoçin Cover Picture:

Alp Can was born in 1963, Paris, France. He is working at Ankara University School of Medicine. He is the second prize winner of the "35. Turkish National Hematology Congress Competition of Photography" in October 2009.

Turkish Society of Hematology

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.

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: G&D Ofset MatbaacÕlÕk Date of printing: March 2010

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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 - EBSCO - DOAJ - PROQUEST - TÜRK TIP DûZûNû 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.

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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

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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.

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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 1

A novel approach to treatment in childhood acute myeloblastic leukemia and myelodysplastic syndrome with highdose methylprednisolone as a differentiation- and apoptosis-inducing agent of myeloid leukemic cells Gönül Hiçsönmez, Ankara, Turkey

Research Articles 8

Mixed chimerism following hematopoietic stem cell transplantation in pediatric thalassemia major patients: a single center experience Elif Ünal ûnce, Mehmet Ertem, Talia ûleri, Klara Dalva, Pervin Topcuoúlu, Zümrüt Uysal, Ankara, Turkey

15

Association of FXII 5’UTR 46C>T polymorphism with FXII activity and risk of thrombotic disease

20

Hospital-acquired pneumonia in patients receiving immunosuppressive therapy

25

Sonographic evaluation of spleen size and prevalence of accessory spleen in a healthy male Turkish population

29

Non-Hodgkin’s lymphoma with bone involvement: a single center experience with 18 patients

Parisa Rasighaemi, Ahmad Kazemi, Fereidun Ala, Mohammad Jazebi, Farnaz Razmkhah, Tehran, Iran, United Kingdom Ebru ÇakÕr Edis, Osman Nuri Hatipoúlu, ûlker YÕlmam, Alper Eker, Özlem Tansel, Necdet Süt, Emre Tekgündüz, Muzaffer Demir, Edirne, Turkey Selim Serter, Cengiz Ceylan, Özüm Tunçyürek, üebnem Örgüç, Yüksel Pabuçcu, Manisa, Turkey

Filiz Vural, Nur Akad Soyer, PÕnar Özen, Ayhan Dönmez, Serkan OcakçÕ, Güray Saydam, Seçkin ÇaúÕrgan, Murat Tombuloúlu, ûzmir, Turkey

Case Reports 34

Early detection of pulmonary fungal infection by CT scan in pediatric ALL patients under chemotherapy or in post-transplantation period with primary complaint of chest pain üule Ünal, BarÕý Kuýkonmaz, Betül Tavil, Selin Aytaç Elmas, Duygu Uçkan Çetinkaya, Mualla Çetin, Mithat Haliloúlu, Fatma Gümrük, Ankara, Turkey

38

Aplastic anemia presenting as hemophagocytic lymphohistiocytosis

43

Unusual cytochemical reactivity for toluidine blue in granular acute lymphoblastic leukemia: a report of two rare cases

Tiraje Celkan, ûstanbul, Turkey

Rishu Agarwal, Ritu Gupta, Sameer Bakhshi, Atul Sharma, New Delhi, India

Images in Hematology 46

Primary cutaneous diffuse large B-Cell lymphoma, leg type Ankit Shrivastav, Bhaskar Mitra, Krishnendu Mukherjee, Kolkata, India

Letters to the Editor 48

A note on oseltamivir treatment in a boy with G6PD deficiency

49

Malignancy-associated hemophagocytosis in children

51

Leukocytosis, thrombocytosis and hypercalcemia as a triple paraneoplastic syndrome in a patient with squamous cell carcinoma of the renal pelvis

Nejat Akar, Ankara, Turkey

Sema Vural, Ela Erdem, Serap Karaman, Tiraje Celkan, Nedim Polat, Öner Doúan, Istanbul, Turkey

Hasan üenol Coýkun, Ayýegül KargÕ, Mustafa Özdoúan, Hakan Bozcuk, Burhan Savaý, Antalya, Turkey

53

Nitric oxide in beta-thalassemia minor: what factors contribute?

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Acute tumor lysis syndrome secondary to a single-dose methylprednisolone in acute lymphoblastic leukemia

Viroj Wiwanitkit, Bangkok Thailand

Tansu Sipahi, Faruk Öktem, Ayça Esra Kuybulu, Ankara, Isparta, Turkey

A-V

Review

1

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 leukemic cells Çocukluk yaýÕ akut myeloblastik lösemi ve myelodisplastik sendromunda myeloid lösemik hücrelerde farklÕlaýma ve apoptosisi saúlayan yüksek doz metilprednizolon ile yeni bir tedavi yaklaýÕmÕ Gönül Hiçsönmez

Department of Hematology, ûhsan DogramacÕ Children’s Hospital, Hacettepe University Faculty of Medicine, Ankara, Turkey

Abstract Differentiation-inducing therapy with all-trans retinoic acid significantly improved the outcome in children with acute promyelocytic leukemia (APL). Therefore, use of agents that induce differentiation of leukemic cells in non-APL children appears to be a highly promising therapeutic approach. Based on the experimental studies in mice, we have shown that short-course high-dose methylprednisolone (HDMP) treatment can induce terminal differentiation of leukemic cells in children with various subtypes of acute myeloblastic leukemia (AML-M1,-M2,-M3,-M4,-M7). It has also been shown to induce apoptosis of myeloid leukemic cells with or without differentiation. Administration of HDMP as a single agent resulted in a rapid clinical improvement, a marked decrease in blast cells in both peripheral blood and bone marrow and dramatic decreases in the size of extramedullary leukemic mass in children with AML and myelodysplastic syndrome (MDS). Addition of HDMP to cytotoxic chemotherapy regimens increased the remission rate and improved the outcome in these children. Future clinical trials with HDMP would contribute to further improvements in the treatment results in these children. (Turk J Hematol 2010; 27: 1-7) Key words: High-dose methylprednisolone, differentiation, apoptosis, acute myeloblastic leukemia, myelodysplastic syndrome, children Received: November 8, 2009

Accepted: November 19, 2009

Özet Akut Promyelositik Lösemili (APL) çocuklarda, lösemik hücrenin normal hücreye dönüýünü (farklÕlaýma) saúlayan all-trans retinioik asidin (ATRA) kullanÕlmasÕ ile tedavi sonuçlarÕnda önemli ilerlemeler elde edilmiýtir. Bu nedenle APL dÕýÕnda diúer akut myeloblastik lösemi (AML) subtiplerinde lösemik hücrelerin farklÕlaýmasÕnÕ saúlayan ilaçlarÕn kullanÕlmsÕ, bu hastalar için de önemli ve ümit verici bir tedavi yaklaýÕmÕ olacaktÕr. Fare myeloid lösemik hücrelerin steroid ile farklÕlaýmasÕnÕn saúlanabileceúini gösteren in vitro çalÕýmalara dayanarak, biz de, literatürde ilk defa olmak üzere, AML’li çocuklarÕn farklÕ subtiplerinde (AML M1,-M2,-M3,-M4,-M7), kÕsa süreli yüksek doz metilprednizolon (YDMP) tedavisi ile lösemik hücrelerin normal hücrelere dönebileceúini gösterdik. AyrÕca YDMP tedavisi, lösemik hücreden dönüýen veya direkt olarak myeloid lösemik hücrelerin apoptotik ölümüne neden olmaktadÕr. Diúer lösemi ilaçlarÕ kullanÕlmaksÕzÕn YDMP, hastalara verildikten kÕsa süre sonra , kilinik bulgularda düzelme, periferik kan ve kemik iliúi lösemi hücrelerinde önemli bir azalma ve kemik iliúi dÕýÕndaki lösemi hücre kitlesinin hÕzla küçülmesine neden

Address for Correspondence: Gönül Hiçsönmez, MD, Department of Hematology, ûhsan DogramacÕ Children’s Hospital Hacettepe University, Ankara 06100, Turkey Phone: +90 312-238 11 19 Fax: +90 312-324 49 90 E-mail: gsonmez@hacettepe.edu.tr

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Hiçsönmez G. High-dose methylprednisolone in AML and MDS

Turk J Hematol 2010; 27: 1-7

olmaktadÕr. YDMP’ nun sitotoksik tedavi protokollarÕna ilavesi ile hastalarÕn remisyon yüzdeleri artmÕý ve hastalÕksÕz yaýam süreleri uzamÕýtÕr. Bu nedenlerle, YDMP ile yapÕlacak kilinik çalÕýmalarÕn, AML ve myelodisplastik sendromlu çocuklarÕn tedavilerinden alÕnacak sonuçlara önemli katkÕsÕ olacaúÕ kanÕsÕndayÕz. (Turk J Hematol 2010; 27: 1-7) Anahtar kelimeler: Yüksek doz metilprednisolon, faklÕlaýma, apoptosis, akut myeloblastik lösemi, myelodisplastik sendrom, çocuklar Geliý tarihi: 8 KasÕm 2009

Kabul tarihi: 19 KasÕm 2009

Introduction Glucocorticoids (GCs) have been used successfully in the treatment of childhood acute lymphoblastic leukemia (ALL) since the late 1940’s. To enhance their effectiveness, higher doses of GCs (250 to 1000 mg daily) were used, mostly in adults, up to the early 1960’s. Massive doses of steroid induced hematologic improvement more rapidly in ALL patients, and the beneficial effect was noted in patients who became resistant to conventional-dose steroid and also in a few patients with acute myeloblastic leukemia (AML) [1-3). However, for a long period, the therapeutic benefits of GCs at higher doses were not evaluated in clinical trials. Recently, in in vitro studies, higher antileukemic activity [4), which may be associated with an increase in apoptosis [5,6), and favorable clinical results were obtained by increasing the dose of GCs in childhood ALL [7-12]. In contrast to ALL, in patients with AML, it is generally believed that GCs in conventional dose have limited or no effect. Despite the favorable results obtained in childhood ALL, progress in the treatment of children with AML with currently used regimens, which mainly depend on the eradication of blast cells by cytotoxic agents, remains unsatisfactory [13,14]. However, the possibility of treatment with agents that induce terminal differentiation of myeloid leukemic cells has long been proposed as a promising approach for improvement in the prognosis of patients with AML. Differentiation-inducing therapy with a derivative of vitamin A, alltrans retinoic acid (ATRA), resulted in significant improvement in the outcome in children with acute promyelocytic leukemia (APL, AML-M3) [15,16]. However, the results in non-APL patients obtained with ATRA are not encouraging. Based on the in vitro studies, which revealed that arsenic trioxide can induce differentiation and apoptosis of APL cell line NB4 and fresh APL cells with t (15;17) dose-dependently [17], it has also been incorporated in the treatment of adult APL patients. However, there are limited data regarding results in children with APL [18].

Other steroid derivative-induced differentiation and/ or apoptosis of human myeloid leukemic cells in vitro Very recently, Chow et al. [35] reported that 2-methoxyestradiol (2ME2), a natural metabolite of 17`-estradiol, can induce apoptosis in different types of AML cell lines (HL-60, NB4 and U937) and in primary human AML cells (M0, M1, M2) in vitro in a dose-dependent manner. Trafalis et al. [36], who evaluated the effects of two homo-aza-steroids (1 androgen and 1 estrogen derivative) on leukemic blasts obtained from patients with various subtypes of AML, indicated the capacity of differentiation. Several aminosteroid compounds have also been shown to inhibit proliferation and/or induce differentiation in HL-60 cells dose-dependently [37,38]. In addition, a cardiotonic steroid, bufalin, has been shown to induce differentiation of human myeloid leukemia-derived cell lines, HL-60, U937 and ML1, and leukemic cells from 4 of 20 AML patients in primary culture [39,40]. In all these studies, therapeutic potential of these different steroid derivatives as possible novel agents in the blood malignancies has been indicated [35-40]. Furthermore, in addition to another steroid derivative, 1_, 25-dihydroxyvitamin D3 and its analogs, which is well known to induce differentiation [41], isomeric pregnadienedione steroids (guggulsterones, naturally occurring plant sterols) and 16-dehydroxyprogesterone with increasing concentration have been shown recently to exert antileukemic effect by inducing differentiation and apoptosis in human AML cell lines (HL-60, NB4 and U937) by Samudio et al. [42]. These compounds also induced apoptosis in primary CD34-positive leukemia cells obtained from relapsed patients with AML-M0 and M2 [42]. Interestingly, it has been indicated that the pregnadienedione structure of these steroids could offer the potential for development of novel chemotherapeutics. Here, the results of our clinical studies with another pregnadienedione steroid derivative, MP, which induces differentiation and apoptosis of myeloid leukemic cells in vivo, will be reviewed.

GC-induced differentiation and/or apoptosis of human myeloid leukemic cells in vitro Since the early 1970’s, a number of experimental studies have shown that prednisolone (PRD) and dexamethasone (Dex) are the most potent agents for inducing differentiation of mouse myeloid leukemic cells into macrophages and granulocytes [19-22]. Moreover, a high concentration of Dex caused complete arrest of mouse myeloid leukemic cell proliferation and prolonged the survival in mice inoculated with sensitive M1 cells [23]. In further studies, differentiation- and/or apoptosisinducing effects of Dex or methylprednisolone (MP) on human primary AML cells [24-26] and myeloid leukemia cell lines (HL60, NB4, U937, HIMeg and t(8;21)-positive myeloid leukemia cells), in a dose-dependent manner, have been demonstrated in vitro [27-32]. Various effects of steroid on human and mouse myeloid leukemic cells were reviewed previously [33,34].

Short-course HDMP treatment-induced differentiation of myeloid leukemic cells in vivo Since 1988, we have shown the remarkable antileukemic effects of MP at high doses, in various subtypes of newly diagnosed and relapsed children with AML [43,44]. Children who had no infection were given MP sodium succinate (Prednol-L, MN Pharmaceutical, Turkey) orally, at a single daily dose of 20-30 mg/kg (not exceeding 1 g daily) together with an antacid. Based on the experimental studies on induction of differentiation of mice myeloid leukemic cells, we first demonstrated morphologic evidence of in vivo differentiation of myeloid leukemic cells to mature granulocytes in a boy with AML-M4 treated with HDMP alone in 1991 [45]. In our further studies, short-course (3 to 7 days) high-dose (HD)MP treatment was also shown to induce terminal differentiation of myeloid leukemic cells in children with APL (AML-M3) and in other sub-

Turk J Hematol 2010; 27: 1-7

Hiรงsรถnmez G. High-dose methylprednisolone in AML and MDS

3

types of AML (AML-M1,-M2,-M4,-M7) [46-49]. In addition to rapid reduction of blast cells, morphologic changes including presence of Auer rods in mature cells (Figure 1) and surface marker analysis of peripheral blood and/or bone marrow cells by flow cytometric studies confirmed the evidence of in vivo differentiation of myeloid leukemic cells. More recently, MP has been shown by Corsello et al. [32] in vitro to induce differentiation of AML cell lines with a t(8;21) in a dose-responsive manner. In this study, treatment of t(8;21)-positive Kasumi-1 cells with MP revealed loss of AML1-ETO protein expression in a proteasomeand GC receptor-dependent manner. Moreover, in three primary patient AML samples, AML1-ETO protein was reduced with in vitro MP treatment. MP could also be the effective agent for the targeted inhibition of dysregulated transcription factors in some other subtypes of AML. It will be interesting to explore whether or not HDMP treatment induces cellular differentiation in some other malignant diseases.

AML cell lines (Kasumi-1 and Skno-1 cells) with a t(8;21) [28,32], HL-60 and in human acute megakaryocytic leukemia (AmegL) cell lines in a dose-dependent manner [27,30]. A plant steroid, diosgenin, has also been shown to induce megakaryocytic differentiation and apoptosis in a human erythroleukemia cell line dose-dependently [54]. In addition to the results of in vitro studies and the rapid hematological and morphological improvement detected four days after HDMP treatment via induction of differentiation and apoptosis of leukemic cells, as observed in our patients with AmegL [26,49], it is important to note that addition of HDMP to induction therapy appears to be a very promising treatment approach in these patients who have poor prognosis. Suppression of the Bcl-2 by MP or Dex might indicate the role GCs in inducing apoptosis of myeloid leukemic cells [32,55,56]. Dex has also been shown to significantly suppress expression of c-myc and c-myb genes during Dex-induced differentiation of mouse myeloid leukemic cells [56,57].

Short-course HDMP treatment-induced apoptosis of myeloid leukemic cells in vivo Apoptosis (programmed cell death) has been demonstrated in vitro to be the common mode of cell death of differentiated human myeloid leukemic cells (HL-60) [50]. We have also shown that induction of apoptosis of myeloid leukemic cells with or without differentiation is possible with HDMP treatment in vivo and in vitro [26,49,51]. In addition to ultrastructural findings, light microscopic studies revealed the morphologic evidence of apoptosis in bone marrow cells obtained from different subtypes of AML children (AML-M3,-M4 and -M7) who received short-course HDMP (4 days) as a single agent. Incubation of low (10-6M) and high (10-3M) concentration of MP with freshly obtained leukemic cells from AML children resulted in a dose-dependent increase in differentiated cells and in apoptosis [26]. Previously, Blewitt et al. [52] demonstrated that GCs at suprapharmacologic concentration, in contrast to low-dose, showed massive lethal effect possibly by inducing apoptosis in human myeloid leukemic cells in vitro [52]. It was also reported that cytolethal effects of GCs (hydrocortisone, Dex, MP) are dose-dependent, and among the GC preparations tested, the most severe cytolethal effect was obtained in human lymphoid and myeloid leukemic cells when exposed to a suprapharmacologic dose of MP (10-3M) [53]. Dex- and/or MP-induced apoptosis has been demonstrated in

Therapeutic effects of HDMP in children with AML Effect of HDMP on blast cells During our long-term clinical studies, in a short-period (24 or 48 hours) after administration of HDMP treatment alone, dramatic clinical improvement (improved activities; resolution of bone pain and unexplained high fever) was observed in most of the AML children who had not received HDMP previously. In addition, rapid decrease in blast cells in both peripheral blood and bone marrow with a concomitant increase in the number of maturing and mature myeloid cells (metamyelocytes and polymorphonuclear leukocytes) was detected after short-course (4 to 7 days) HDMP without using any other antileukemic agents in different subtypes of AML children [43-49,58,59]. Changes in marrow blasts 4-7 days after HDMP treatment alone or two weeks after HDMP combined with cytosine arabinoside (Ara-c) are shown in Table 1. Marked decrease in marrow blasts was also detected in relapsed children [44]. In a patient with refractory AML, complete remission (CR) was also described by Sugawara et al. [60] using HDMP combined with granulocyte colony-stimulating factor (G-CSF). Although a similar effect was not obtained in our patients who previously received HDMP, it is important to note that 3-4 months after its discontinuation, some of the patients responded again to HDMP.

Figure 1. Four to seven days after HDMP treatment, maturing myeloid cells and granulocytes containing Auer rods are seen in the bone marrow smear of children with AML (M1,- M2,- M4) (Wright stain X 2000)

Effect of HDMP on extramedullary infiltration More importantly, dramatic decreases in the size of extramedullary leukemic infiltration (orbita, gingiva and soft tissue) were detected in 24 hours to 7 days after initiation of HDMP treatment [43-45,58,61]. Rapid decreases in orbital and spinal mass following the administration of HDMP treatment alone were also reported by others in children with AML-M2 and t(8;21) translocation [62,63]. Furthermore, we demonstrated complete resolution of pleural effusion and marked decrease in pericardial effusion in children with chronic myelomonocytic leukemia (CMML) four days after HDMP treatment alone, which also induced differentiation and apoptosis of malignant cells in the pleural effusion [64]. Based on these results, we can suggest that addition of short-course HDMP to initial treatment would be a very effective approach for the treatment of patients presenting with extramedullary infiltration Combined HDMP with cytotoxic chemotherapy In our previous study reported in 1992, addition of HDMP to mild antileukemia regimens (adriamycin and Ara-c) increased

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Hiรงsรถnmez G. High-dose methylprednisolone in AML and MDS

Turk J Hematol 2010; 27: 1-7

Table 1. Decrease in percent of marrow blasts after HDMP alone or HDMP combined with Ara-c in children with newly diagnosed AML and MDS No. of Patients

Duration of

Blast cells%

Treatment

treatment (day)

<5%

0-15%

References

37*

HDMP

4-7

12(32%)

20(54%)

58

26*

HDMP+ Ara-c

15

7(27%)

17(61%)

59

4**

HDMP+LD-Ara-c

15

4(100%)

-

66

8**

HDMP

4

7(88%)

8(100%)

79

*AML,**RAEB and RAEB-t, Ara-c (3mg/kg, 3 days a week), LD Ara-c (10 mg/m2) HDMP: High-dose methylprednisolone; LD Ara-c: Low-dose cytosine arabinoside; AML: Acute myeloblastic leukemia; MDS: Myelodysplastic syndrome; RAEB: Refractory anemia with excess of blasts; RAEB-t: RAEB in transformation

the CR rate to 84.6% in 26 newly diagnosed AML children with or without extramedullary infiltration when compared to the CR rate (62%) of historical controls [59.65], and reduced the number of early relapses occurring within 12 months (45% vs 78%) and 24 months (76% vs 90%), respectively. However, difference in five-year disease-free survival (DFS) rate was not significant (18% vs 10%). In our subsequent studies with two different treatment protocols reported in 2004, the CR rate was 87% and 89% in 23 and 45 children, respectively, who had no extramedullary infiltration at presentation [58]. By using intensive maintenance therapy, improved outcome was achieved and the five-year DFS rate was 44% and 35% , respectively. In this study, in a small number of children (n=7) who presented with myeloblastoma, five-year DFS rate was 71% [58]. However, this improvement was not noted in children with gingival infiltration. We believe that further studies in these heterogeneous group of AML patients who have large differences in outcome should be explored with combined HDMP and more intensive chemotherapy than used in our studies, in an effort to improve the outcome. HDMP treatment was well tolerated, HDMP-related toxicity was mild, and no life-threatening events occurred [58,59]. During induction therapy, after the addition of cytotoxic agents to HDMP, mortality rate was 4% in 112 children with AML [58]. However, unlike with cytotoxic agents, in a short period after initiation of HDMP, increase in white blood cell (WBC) count, which was controlled by the administration of cytotoxic drugs, was observed in 25% of the AML children and in children with myelodysplastic syndrome (MDS) as well [58,66]. This increase was not associated with the increase in peripheral blast cells. Rapid increase in WBC count following massive-dose steroid therapy was also noted in some patients with AML in previous studies [2,3]. Therefore, patients with WBC count >25x109/L at diagnosis were given chemotherapy concomitantly with HDMP administration. Since an early blast cell clearance by remission induction therapy is a major independent prognostic factor, the improved outcome in our patients could be related with the initial striking decrease in leukemic cells in the bone marrow and in the extramedullary site obtained with HDMP as a differentiating- and apoptosis-inducing agent. In addition, administration of shortcourse (4 days) HDMP treatment during induction therapy resulted in a rapid increase in T4+, T8+ T-cells and natural killer (NK) cells, possibly due to stimulation of marrow CD34+ cells, which may also contribute to the antileukemic effect of HDMP [67]. It has also been shown by Vitale et al. [68] that pharmacological concentration of MP can induce preferential and rapid in vitro differentiation of CD34+ hematopoietic precursors to NK cells.

Recently, dramatic selective blast cell reduction associated with apoptosis and improved outcome were reported by Suzuki et al. [69] in elderly patients (63-89 years) with AML secondary to MDS treated with MP alone (125 mg, body). Significant blast reduction with hematological and cytogenetic remission following HDMP (1000 mg/day) treatment alone was also demonstrated by Shimohakamada et al [70] in an adult AML patient who had pulmonary infection. However, the optimal dose of MP in inducing differentiation and apoptosis remains unknown. The stimulation of normal myelopoiesis with HDMP treatment would be an additional benefit in the treatment of these patients. Short-course (3-5 days) HDMP treatment shortens the chemotherapy-induced neutropenic period in children with AML and ALL during induction and maintenance therapy, possibly by stimulating the CD34+ hematopoietic progenitor cells [71-73]. Pretreatment with short-course HDMP, before highdose consolidation therapy, reduced the duration and severity of neutropenia in children with AML [74]. We suggest that the effect of short-course HDMP should be evaluated in non-leukemic malignancies in order to stimulate neutrophil recovery. Therapeutic effects of HDMP in children with MDS Although significant progress has been achieved in the treatment of children with MDS who could have received hematopoietic stem cell transplantation, treatment results with standard chemotherapy regimens have been generally unsatisfactory [75,76]. Since progressive impairment in differentiation of hematopoietic cell lineages is the main pathophysiological feature, there has been great interest in using differentiation-inducing agents in MDS. However, results with retinoic acid and vitamin D3 are not encouraging [77]. On the other hand, we obtained promising results in children with various subtypes of MDS (refractory anemia with excess blast (RAEB), RAEB in transformation, juvenile myelomonocytic leukemia (JMML) and CMML) with the addition of HDMP to cytotoxic chemotherapy [66,78,79], with the exception of patients with JMML and monosomy 7. As observed in AML children, a marked decrease in blast cells both in peripheral blood and bone marrow was also detected after HDMP treatment (Table 1). In addition, dramatic resolution of extramedullary infiltration (orbital, spinal and pleural), possibly due to its differentiation- and apoptosis-inducing effects, was also noted in these children [66,79]. With the use of combined HDMP and mild cytotoxic chemotherapy, higher CR rate (70%) was achieved in 17 of 25 children with different subtypes of MDS [79]. Improvement in the outcome in these children, especially in children with CMML, seems to be promising. Our preliminary data showed that short-course HDMP treatment might also be beneficial by remarkably increasing

Hiçsönmez G. High-dose methylprednisolone in AML and MDS

Turk J Hematol 2010; 27: 1-7

serum levels of interleukin-2 in children with CMML [80]. As obtained with anti-thymocyte globulin and cyclosporin A, modulation of immune response by HDMP might contribute to its beneficial effect in some patients with MDS. In contrast to other immunosuppressive agents, an advantage of the use of shortcourse HDMP and its favorable effects have been indicated in adult patients with refractory anemia [81-83]. Decrease in serum tumor necrosis factor-alpha after short-course HDMP therapy observed in children with AML and ALL might indicate that it is also effective in MDS, leading to hematologic improvements [84]. In addition, an inhibitory effect of steroids (PRD and Dex) has been shown in vitro on the production of leukemia-associated inhibitor from human myeloid leukemic cells, which has been suggested by Olofsson et al. [85] as being responsible for the suppression of normal hematopoiesis. The possibility of HDMPinduced suppression of inhibitory activity of MDS abnormal clones [86] may also contribute to its effect in MDS. These possibilities might explain the favorable response to short-course HDMP treatment obtained in our patient with hypoplastic MDS as well as in a case with hypoplastic AML [87,88]. Identification of responsive MDS patients to GC therapy by in vitro technique was first reported by Bagby et al. [89]. In that study, it was suggested that if therapeutically achievable concentrations of cortisol enhance colony growth of marrow cells and if the colonial cells are well differentiated (granulocyte maturation was observed in 2 patients), the patients will likely respond favorably to PRD therapy. Interestingly, they described responses when higher doses of PRD were used in patients who were considered in vitro non-responders. Beneficial effects of high-dose steroid in patients with MDS were also reported by others [90-94]. Based on these studies, HDMP combined with chemotherapy followed by autologous stem cell transplantation can be considered as an alternative therapeutic approach in children with MDS who are not suitable for bone marrow transplantation. In conclusion, our clinical studies indicate that addition of short-course HDMP as a differentiation- and apoptosis-inducing agent to cytotoxic chemotherapy regimens represents a promising approach in the treatment of children with AML and MDS. In further studies, its effective dosage, duration of administration and role in maintenance therapy should be evaluated. Although the factors involved in the mechanisms of HDMP effect in inducing differentiation, apoptosis and stimulation of myelopoiesis are not well known, it may be effective through complex mechanisms to target several antileukemic pathways. Further studies, enhancing the understanding of the metabolic and molecular basis of these effects, could provide important therapeutic benefit in children with AML and MDS. Based on the results of our clinical studies, short-course HDMP also deserves evaluation regarding its effect in the treatment of some other malignancies.

Acknowledgements

I would like to particularly thank Prof. Dr. M. Çetin and all my colleagues who made valuable contributions during these studies, as indicated in the list of cited references of the published papers. I also warmly thank all doctors, nurses and the technical personnel who also made valuable contributions during the long clinical follow-up period of these children. I would like to acknowledge Prof.Dr. E. Erdemli and Prof.Dr. M. Tekelioúlu from the Department of Histology at Ankara University for providing electron microscopic studies.

5

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.

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Hiçsönmez G. High-dose methylprednisolone in AML and MDS

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Research Article

8

Mixed chimerism following hematopoietic stem cell transplantation in pediatric thalassemia major patients: a single center experience Pediatrik talasemi majör hastalarÕnda hematopoetik kök hücre nakli sonrasÕ karÕýÕk tip kimerizm-tek merkez sonuçlarÕ Elif Ünal ûnce1, Mehmet Ertem1, Talia ûleri1, Klara Dalva2, Pervin Topçuoúlu2, Zümrüt Uysal1 1Department 2Department

of Pediatric Hematology, Ankara University Faculty of Medicine, Ankara, Turkey of Hematology, Ankara University Faculty of Medicine, Ankara, Turkey

Abstract Objective: Stable mixed chimerism (MC) may result in cure for thalassemia major patients following hematopoietic stem cell transplantation (HSCT), but rejection can occur. Twenty-eight HSCTs for thalassemia major were reviewed retrospectively to evaluate the clinical course of MC with possible risk factors and predictors of outcome, with a median follow-up of 1669 days (811-3576 days). Materials and Methods: Chimerism was detected by fluorescence in situ hybridization (FISH) or multiplex polymerase chain reaction depending on the sex match between the donor and the recipient. Results: Primary rejection, stable MC and full donor chimerism was detected in 3.6%, 17.8% and 78.6% of patients, respectively. Clinically, 4/5 patients with stable MC had thalassemia trait with donor chimerism as low as 14%. One patient was started on pRBC transfusions at 2.5 years postHSCT. Conclusion: Stable MC can result in cure for thalassemia major patients. The clinical picture remains as the best guide for intervention until a more reliable predictor is available. (Turk J Hematol 2010; 27: 8-14) Key words: Thalassemia major, mixed chimerism, pediatrics, chimerism, hematopoietic stem cell transplantation, nonmalignant Received: May 19, 2009

Accepted: October 20, 2009

Özet Amaç: Talasemi major (TM) hastalarÕnda hematopoetik kök hücre transplantasyonu (HKHT) sonrasÕnda stabil karÕýÕk-tip kimerizm ile kür mümkündür fakat rejeksiyonla sonuçlanabilir. TM tanÕsÕyla yapÕlmÕý 28 HKHT karÕýÕk-tip kimerizm seyri, risk faktörleri ve sonuçlarÕnÕ deúerlendirmek üzere retrospektif olarak gözden geçirildi. Yöntem ve Gereçler: ûzlem süresi ortanca 1669 gündü (811-3576 gün). Kimerizm alÕcÕ-verici cinsiyet uyumuna göre FISH veya multipleks PCR yöntemiyle bakÕldÕ. Bulgular: Primer rejeksiyon, stabil karÕýÕk tip kimerizm ve tam verici tip kimerizm sÕrasÕyla % 3.6, % 17.8 ve % 78.6 idi. 4/5 hastada % 14 kadar düýük verici kimerizmine raúmen talasemi taýÕyÕcÕlÕúÕ kliniúi vardÕ. Sadece 1 hastada HKHT sonrasÕ 2.5 yÕl sonra eritrosit ihtiyacÕ oldu. Address for Correspondence: Elif Ünal ûnce, MD, Department of Pediatric Hematology, Ankara University Faculty of Medicine, Dikimevi 06100, Ankara, Turkey Phone: +90 0312 595 69 06 E-mail: elifunal@msn.com

ûnce et al. Mixed chimerism in thalassemia major

Turk J Hematol 2010; 27: 8-14

9

Sonuç: Stabil karÕýÕk-tip kimerizm, talasemi major hastalarÕnda kür saúlayabilir ve bu hastalar için rejeksiyonu önceden gösteren bir belirteç bulunana kadar hastalarÕn kliniúi, herhangi bir müdahale için belirleyici olmalÕdÕr. (Turk J Hematol 2010; 27: 8-14) Anahtar kelimeler: Talasemi majör, karÕýÕk tip kimerizm, pediatri, kimerizm, hematopoetik kök hücre transplantasyonu, non-malign Geliý tarihi: 19 MayÕs 2009

Kabul tarihi: 20 Ekim 2009

Introduction Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for thalassemia major [1-4]. However, graft rejection is the undesired possible outcome following mixed chimerism (MC) [4-7]. The evolution of MC after its first detection has been studied as a possible predictor of outcome, mainly to intervene in time in the necessary cases in order to prevent rejection. The risk factors in the occurrence as well as the outcome of MC remain elusive. For the occurrence of MC, the conditioning regimen has been shown to be a significant risk factor in some studies [8,9], but not in all [10]. For the outcome, the early detection and severity of MC were found to be significant in predicting the course of MC [5]. In this retrospective study, we evaluated the course of chimerism in patients with thalassemia major who received HSCT in our institution to determine the possible risk factors and the possible clinical predictors of outcome of MC.

Materials and Methods Patient Population Twenty-eight thalassemia major patients received 29 HSCT from an HLA-identical family donor from April 1999 to November 2007 at Ankara University Medical Faculty Pediatric Hematopoietic Stem Cell Transplantation Unit. One patient was removed from the study because of early treatmentrelated mortality. The remaining 27 patients and 28 transplants were evaluated retrospectively for the chimerism studies. One patient received a second transplant from the same donor following primary rejection. The median follow-up was 1669 days (range: 811-3576 days), median age was 8 years (range: 2-17 years) and the male/female ratio was 16/11. The patient characteristics are summarized in Table 1. Risk classification prior to HSCT was made as per Pesaro classification [2]. All patients above three years of age had a liver biopsy done to evaluate for fibrosis.

day parenterally and was switched to peroral (PO) when the patient was clinically stable and could tolerate oral medications. CSA was tapered by 10% every other week beginning on day +60 and discontinued at +6 months if there was no evidence of GVHD. Dose adjustments were made to keep the Table 1. Patient characteristics n Number of Patients

27

Number of Transplants

28

Median Age in years (range)

%

8 (2-17)

Sex Male

17

60.7

Female

11

39.3

Class I

5

17.8

Class II

8

28.5

Class III

14

50

2nd

1

3.5

Thalassemia Minor

18

64.3

Normal

10

35.7

Bone Marrow

15

53.5

Peripheral Blood

10

35.7

Cord Blood

2

7.2

Cord Blood + Bone

1

3.6

11

39.3

2

7.2

Pesaro Protocol 26

1

3.5

Pesaro Protocol 26+ATG

14

50

Cyclosporine+Methotrexate

25

89.3

Cyclosporine

3

10.7

Risk Classification

transplant

Donor Status

Stem Cell Source

Marrow (same donor) Conditioning Regimen Busulphan+ Cyclophosphamide

Conditioning Regimen, GVHD Prophylaxis and Engraftment Patients received conditioning regimen based on their risk classification as shown in Table 2. All Class III patients received conditioning regimen as per Pesaro Protocol 26 and antithymocyte globulin (ATG) except one patient who had received Pesaro Protocol 26 without ATG. Thiotepa was used in the conditioning regimen of two patients who received cord blood transplantation. Cyclosporine (CSA) and methotrexate (MTX) were used as graft-versus-host disease (GVHD) prophylaxis in 89.3% of the patients. CSA was started on day -1 at 3 mg/kg/

Busulphan+ Cyclophosphamide+Thiotepa

GVHD Prophylaxis

GVHD: Graft versus host disease; ATG: Antithymocyte globulin

10

ûnce et al. Mixed chimerism in thalassemia major

Turk J Hematol 2010; 27: 8-14

Table 2. Conditioning regimens Risk Class

Conditioning Regimen

Dose

Days

Class I and Class II

Busulphan

480 mg/m2

-9 to -6

Cyclophosphamide

200 mg/kg

-5 to -2

Class III

mg/m2

-6

(Thiotepa)*

250

Hydroxyurea

25-30 mg/kg/day

-45 to -15

Azathioprine

2.5-3 mg/kg/day

-45 to -15

Fludarabine

120

mg/m2

-14 to -10

Busulphan

14 mg/kg (total)

-9 to -6

Cyclophosphamide

40 mg/kg (total)

-5 to -2

ATG

4-10** mg/kg/day

-4 to -1

(total)

*Thiotepa was used in 2 patients who received cord blood transplantation. For these patients, busulphan was given on days -10 to -7 ATG: Antithymocyte globulin **Thymoglobulin: 4.0-4.5 mg/kg/day - ATG-Fresenius: 9-10 mg/kg/day

CSA trough levels between 150-200 ng/ml. Class I and Class II patients received MTX at 8 mg/m2 on day +1 and 10 mg/m2 on days +3 and +6, and Class III patients received MTX at 10 mg/m2 on days +1, +3 and +6. GVHD prophylaxis was discontinued at six months postHSCT in the absence of GVHD. GVHD was graded according to the Glucksberg and Seattle consensus criteria [11,12]. Myeloid engraftment (neutrophil recovery) was defined as an absolute neutrophil count (ANC) 500/mm3 for two consecutive days after nadir. Platelet engraftment was defined as a platelet count of 20,000/mm3 independent of platelet transfusions for at least seven consecutive days. Supportive Care: All patients were cared for in laminar air flow units, and received prophylactic fluconazole and acyclovir beginning on day -1 up to 75 and 180 days after transplantation, respectively. Co-trimoxazole was administered until day -1 and then resumed after neutrophil engraftment. Ciprofloxacin was used for antibacterial prophylaxis. Granulocyte colonystimulating factor (G-CSF) was used at 5 g/kg/day intravenously in 26 of the transplants. In three of these, G-CSF was used for delayed engraftment, and for 23 patients, it was started on day +5 until the ANC was 2.5x109 cells/L for two consecutive days and then was discontinued. Chimerism Studies Patients: Patients were evaluated in two categories: [1] Twenty-eight transplants with at least one-year follow-up postHSCT were evaluated for the long-term follow-up of MC, and (2) Twenty transplants within this group with chimerism evaluation done both at the 1st month and the 3rd month postHSCT were evaluated for the evolution of early MC detected at the 1st month postHSCT. Chimerism studies were done on a routine basis only after 2001. For patients who were transplanted after that date, chimerism studies were scheduled to be done at the 1st, 3rd, 6th and 12th month postHSCT. Patients who were transplanted before 2001 had their chimerism analysis done only after 2001 at whichever postHSCT date they were at. Written informed consent was obtained from all transplant patients.

Method: Chimerism was evaluated by fluorescence in situ hybridization (FISH) in 13 patients who had sex mismatch donors, and 14 who had sex match were evaluated by single nucleotide polymorphism (SNP) analysis done by multiplex polymerase chain reaction (PCR). Quantitative multiplex PCRbased method was performed by the detection of the short tandem repeats (STRs) on specific genes. DNA was extracted from whole peripheral blood/bone marrow (PB/BM) samples using the Invitek kit (Berlin, Germany). Multiplex STR-PCR was performed on 2 ng of genomic DNA using the AmpFlSTR SGM Plus (PEBiosytems, CA, USA) or AmpFlSTR Identifier Kits (Applied Biosystems, CA, USA), which contain 10-15 STR loci plus amelogenin gene, respectively. The repeat regions detected by AmpFlSTR SGM Plus included D3S1358, HUMvWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, THO1, FGA, and amelogenin. The AmpFlSTR Identifier Kit additionally detects the D7S820, CSF1PO, D13S317, TPOX, and D5S818 repeats. Upon denaturation by deionized formamide, amplified PCR products were subjected to capillary electrophoresis in an ABI Prism 310 or ABI Prism 3100 Genetic Analyzer (Applied Biosystems) using the conditions recommended by the manufacturer. All the fluorescence signals generated from the amplicons were recorded and analyzed by the Gene Scan 3.1 or Gene Mapper ver 4.0 softwares (Applied Biosystems). In order to increase specificity, only the markers in which recipient specific peaks showed no residual fluorescence might be generated from stutter peaks or spectral overlap, and the markers that were not homozygote for any defined allele were considered as informative and taken into account for the calculations. Chimerism was quantified as suggested by Fernández-Avilés et al. [13] using the peak areas calculated by the software. For FISH analysis, CEPX SO/CEPYSG directlabeled fluorescent DNA probe kit was used (Abbott, Germany). Following the MNC isolation using Ficoll 1077, cells were fixated by Carnoy solution. Hybridization was performed as described by the manufacturer, and at least 500 cells were counted with Nikon E600 fluorescent microscope and the ratio of XX and XY signals was calculated.

ûnce et al. Mixed chimerism in thalassemia major

Turk J Hematol 2010; 27: 8-14

11

Figure 1. Summary of the chimerism outcome

MC: Mixed chimerism; FRC: Full recipient chimerism; FDC: Full donor chimerism

Table 3. Characteristics of patients with mixed chimerism Pt# Age (yrs)

Sex

Risk Classification

Stem Cell Source

Donor Trait Status

Conditioning Regimen

Time of Engraftment #of Cells Infused GVHD GVHD (Days) Prophylaxis Myeloid Platelet

Last Follow-up Chimerism (Months) (%)

Nucleated CD34 Cell + Cell Count Count (108/kg) (106/kg)

1

2

Male

Class I

Bone Marrow

Trait

BU+CTX

26

26

4.00

2.90

CSA+MTX

_

40

90

2

3

Male

Class II

Bone Marrow

Trait

BU+CTX

15

25

2.83

1.83

CSA+MTX

_

53

82

3

7

Female

Class II

Bone Marrow

Trait

BU+CTX

29

26

3.88

7.86

CSA+MTX

_

47

90

4

11

Female

Class III

Peripheral Blood

Trait

Pesaro Protocol 26+ATG

15

17

6.40

8.00

CSA+MTX

_

18

37

5

9

Male

Class III

Peripheral Blood Normal

Pesaro Protocol 26+ATG

12

20

15.00

8.37

CSA+MTX

_

88

31

BU: Busulphan; CTX: Cyclophosphamide; ATG: Antithymocyte globulin; GVHD: Graft versus host disease; CSA: Cyclosporine; MTX: Methotrexate

Definitions of Chimerism Status: MC was defined as the presence of >5% residual host hematopoietic cells. Rejection was defined as >90% residual host hematopoietic cells with relapse of thalassemia and packed red blood cell (pRBC) transfusion dependence [14]. Stable MC was defined as fluctuations in the percentage of recipient cells over time without complete loss of donor cells [15]. Patients were also evaluated as per Nesci et al.’s [5] classification. Patients were included in level I if the residual host cells were less than 10%, level II if the residual host cells were 10-30% and level III if the residual host cells were >30%. Statistical Analysis: Results are presented as medians with specified ranges of data sets. The relationship between clinical parameters and chimerism was determined using Mann-Whitney test for quantitative variables. Comparison of the percentages between two groups was performed using r2 test or Fisher’s exact test where appropriate. Differences with a P value less than 0.05 were considered significant. SPSS version 15.0 (Statistical Package for Social Sciences) statistical software was used for analyses.

Results Figure 2. Evolution of the early MC detected at 1st month postHSCT

MC: Mixed chimerism; FRC: Full recipient chimerism; FDC: Full donor chimerism. *Two patients who died after 1 year postHSCT had FDC

Long-term Follow-Up and Evaluation of Stable MC All 28 transplants were evaluated for the long-term followup. The median time for follow-up was 1669 days (range: 8113576 days). The overall survival was 92.9%. Twenty-two

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รปnce et al. Mixed chimerism in thalassemia major

Turk J Hematol 2010; 27: 8-14

Figure 3. Course of chimerism in patients with MC Table 4. Clinical course of patients with MC

Pt# 1 2 3 4 5

Median

PostHSCT

Last Hemoglobin

Hemoglobin

Day of Last

without

Last

Follow-up

Current

ABO

(g/dL)

pRBC

Transfusion

chimerism

postHSCT

Status I I C C C

(Range) 9.3 (5.4-11.1) 10.3 (4.8-12.2) 9.8 (7.8-11.4) 7.1 (5.2-9.0) 9.8 (7.4-10.7)

Transfusion 119 334 43 16 40

(g/dl) 9.8 10.8 9.4 5.2 9.3

(%) 40 53 47 18 88

(months) 90 82 90 37 31

Clinical Status Thalassemia Trait Thalassemia Trait Thalassemia Trait Thalassemia Intermedia Thalassemia Trait

I: ABO incompatible; C: ABO compatible

patients had full donor chimerism (FDC) (78.6%) and 5 had MC (17.8%), with one of these patients starting pRBC transfusions at 2.5 years postHSCT. Only one patient had primary rejection (3.6%) (Figure 1). Two patients died with infection 394 and 507 days following HSCT. One patient who had primary rejection had autologous recovery and received a second transplant 1.5 years after the first. He had FDC following the second transplant. Stable MC was detected in 17.8% (5/28) of patients and the patient characteristics are summarized in Table 3. The course of chimerism during the follow-up of these patients is shown in Figure 2. The median follow-up for patients with MC was 2548 days (974-2814 days). Four of the five patients with MC (Pts 1, 2, 3 and 5) had a clinical picture of thalassemia trait as confirmed by hemoglobin electrophoresis and with near normal hemoglobin values. Patients 3 and 5 had not required pRBC transfusions after day 43 and 40 postHSCT. Pt 1 and Pt 2 had major ABO incompatibility and required pRBC transfusions till day 119 and day 334 postHSCT, respectively. Pt 2 received erythropoietin mainly to reduce the pRBC requirements at 125 u/kg/day three times a week between days 250411 postHSCT. Following the erythropoietin treatment, hemoglobin values improved and transfusion requirements

decreased. Remarkably, Pt 1 has not required any pRBC transfusions despite very low donor chimerism of 14% detected at 72 months postHSCT. Pt 4, who has been followed for 3.1 years postHSCT with a clinical picture of thalassemia intermedia, was started on pRBC transfusions with a hemoglobin of 5.2 g/dl at 2.5 years postHSCT. Her median hemoglobin had been 7.1 g/dl (5.29.0) until then and did not require any transfusions. Erythropoietin was used between days 209-307 postHSCT with her first sign of thalassemia intermedia to improve the clinical picture with no change in hemoglobin values. No additional immunosuppressive agent was prescribed. Currently she is 3.1 years postHSCT and considered for a second HSCT. Table 4 summarizes the clinical course of patients with MC. Evolution of Early Chimerism Status Twenty patients who had chimerism studies done both at the 1st month and the 3rd month postHSCT were evaluated for the evolution of the chimerism during the first few months and results are summarized in Figure 3. At the 1st month, MC was detected in 7/20 patients (35%). At the 3rd month evaluation, only 1 of these patients (14.3%) continued to have MC and the

Turk J Hematol 2010; 27: 8-14

remaining 6 (85.7%) achieved FDC. One of the 20 patients who had FDC at the 1st month regressed to MC at the 3rd month evaluation. All patients who had FDC at the 3rd month continued to have FDC with a median follow-up of 1159 days (3941974 days). When the 7 patients who had MC at the 1st month were classified according to the criteria suggested by Nesci et al. [5], 4/7 were at MC level I and 3/7 were at MC level II. The only patient within this group who continued to have MC in the long-term was at MC level II with 89% donor chimerism at the time of the 1st month evaluation. In the long-term, when all 28 transplants were evaluated regardless of having chimerism studies done at the 1st month, 4 patients had MC at the 3rd month evaluation and all had stable MC in the long-term. Additionally, all 16 patients who had FDC at the 3rd month evaluation had FDC in the long-term. Evaluation of Risk Factors and Potential Predictors for MC The recipient’s age, sex, disease class, conditioning regimen, nucleated cell and CD34+ cells infused, time of myeloid engraftment, duration and regimen for GVHD prophylaxis, presence of acute (“ grade II) or chronic GVHD, and donor sex and thalassemia trait status were evaluated as the potential risk factors and predictors. None was found to be statistically significant.

Discussion Mixed chimerism following HSCT occurs both in malignant and non-malignant disorders, and cure or clinically stable disease can be achieved with stable MC in patients with thalassemia major [5,6,16,17]. Possible rejection is the main concern with the initial detection of MC. The evolution of MC after its first detection has been studied mainly to intervene in time to prevent possible rejection. In our study, chimerism evaluations at the first month following HSCT demonstrated 65% FDC. Of the 35% who had MC at the first evaluation, 85.7% recovered to FDC at the second evaluation done at the 3rd month postHSCT. At the first-year follow-up, 17.8% of the patients had stable MC. These findings correlate with the literature in terms of the early and later incidence of MC following HSCT for thalassemia major. Early MC was reported as 31% and 36.5% by Amrolia et al. and Nesci et al., respectively [5,14]. In the same study, at the longer follow-up (8-46 months postHSCT), stable MC was detected in 20% of the patients. Additionally, it has been shown that the early detection of MC does not always correlate with the chimerism outcome in the long-term [5,6]. In our study, at the second evaluation, which was done at 3 months, most patients who had MC initially recovered to FDC, and the only patient who did not continued to have MC at 3.1 years follow-up. Thirty-five percent of patients (7/20) had changed their chimerism status from the first evaluation to the second (6 patients to FDC and 1 patient to MC). Although the study group was very small, our study suggests that the later evaluation of chimerism (in our study the 3rd month postHSCT) might be more representative of the outcome than the first month evaluation for this group of patients.

Ăťnce et al. Mixed chimerism in thalassemia major

13

Other than the timing, severity of MC was studied as a predictor of long-term outcome of MC in the literature. Nesci et al. [5] graded the MC in three groups according to the degree of MC and evaluated the outcome for each group. In their study, MC level III, defined as having >30% recipient chimerism, correlated with rapid graft rejection within the first year following HSCT. In our study group, no patient was at MC level III at the first evaluation. Of the patients with stable MC, only two had an evaluation done at the first month. One was at MC level I and the other at MC level II. Our study group was too small to evaluate for the influence of the MC level on long-term stable chimerism; larger study groups may be helpful to draw final conclusions in this regard. The risk factors for MC have been studied by different groups. So far, only the conditioning regimen was found to have a significant role in the occurrence of MC for thalassemia major. In 1992, Nesci et al. [5] found significant influence of the conditioning regimen at the early MC evaluated at two months postHSCT. At later follow-ups, this significance disappeared but MC remained higher in the group of patients who received reduced doses of cyclophosphamide (120 mg/kg versus 200 mg/kg) (Pesaro Protocol 12 versus Protocol 6), and the reduced immunosuppression was discussed as a possible reason for the incomplete suppression of the host residual cells [5]. In another study, the busulphan levels were not found to affect the chimerism status statistically, but patients with MC tended to have lower levels of busulphan [10]. In our study, there was no significant difference between the two groups of patients receiving different conditioning regimens. Compared to the previous report by Nesci et al. [5] in 1992, the conditioning regimen used in our patients is more intense for Class III disease as well as for Class I and II. Our group used busulphan as per m2 for Class I and II patients as opposed to per kg dosing used in the Pesaro protocol. The per kg equivalent dose of busulphan used for Class I and II patients in this study was a median 19.8 mg/kg (range: 18.1 mg/kg-20.6 mg/kg). Considering the intensity of the regimens increasing over time to prevent rejection in thalassemia patients, the rate of MC seems to stay approximately the same. The role of the conditioning regimen remains unclear for the occurrence of MC. None of the other risk factors evaluated in this study including age, sex, disease class, conditioning regimen, use of G-CSF after stem cell infusion, nucleated cell and CD34+ cells infused, time of myeloid engraftment, GVHD prophylaxis regimen and duration, presence of GVHD, and sex and thalassemia trait status of the donor was found to be statistically significant. Further studies are necessary to clarify the role of the conditioning regimen and to identify the risk factors for the occurrence of MC. The issue of intervention for MC has been controversial. In this study, one patient had a donor chimerism of 14% with near normal hemoglobin values whereas another patient with 19% donor chimerism started requiring pRBC transfusions. When and how to intervene in MC remain the critical questions to be answered. Erythropoietin, donor lymphocyte infusion (DLI) and/or changing the dose of immunosuppression have been tried to augment the donor chimerism [8,14,18]. No change in the immunosuppression schedule was made in any of our patients with MC because of the stable

14

ûnce et al. Mixed chimerism in thalassemia major

hemoglobin values. Erythropoietin was tried in two patients with no increase or change in either donor chimerism or hemoglobin levels. DLI had been reported to be useful by some authors although the majority of the experience with DLI was following T-cell depleted HSCT [18]. The complications following DLI include GVHD and aplasia. The risks and benefits of this intervention should be considered carefully in view of the lack of strong evidence and clinical studies. The clinical status of the patient may be the only guidance so far for any intervention. But the question remains whether we can prevent rejection or thalassemia intermedia with the guidance of the chimerism studies. Although the answer to how and when to intervene is clearer for malignant disorders, further studies are necessary for thalassemia major. 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: 8-14

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Lucarelli G, Galimberti M, Polchi P, Giardini C, Politi P, Baronciani D, Algelucci E, Manenti F, Delfini C, Aureli G, Muretto P N. Marrow transplantation in patients with advanced thalassemia. N Engl J Med 1987;23:1050-5. Lucarelli G, Galimberti M, Polchi P, Angelucci E, Baronciani D, Giardini C, Politi P, Durazzi SM, Muretto P, Albertini F. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990;322:417-21. Lucarelli G, Weatherall DJ. For debate: bone marrow transplantation for severe thalassaemia [1]. The view from Pesaro (2). To be or not to be. Br J Haematol 1991;78:300-3. Lucarelli G, Galimberti M, Polchi P, Angelucci E, Baronciani D, Giardini C, Andreani M, Agostinelli F, Albertini F, Clift RA. Marrow transplantation in patients with thalassemia responsive to iron chelation therapy. N Engl J Med 1993;329:840-4. Nesci S, Manna M, Andreani M, Fattorini P, Graziosi G, Lucarelli G. Mixed chimerism in thalassemic patients after bone marrow transplantation. Bone Marrow Transplant 1992;10:143-6. Lucarelli G, Galimberti M, Giardini C, Polchi P, Angelucci E, Baronciani D, Erer B, Gaziev D. Bone marrow transplantation in thalassemia. The experience of Pesaro. Ann N Y Acad Sci 1998;850:270-5. Andreani M, Manna M, Lucarelli G, Tonucci P, Agostinelli F, Ripalti M, Rapa S, Talevi N, Galimberti M, Nesci S. Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia. Blood 1996;87:3494-9.

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Li CK, Chik KW, Tsang KS, Pong H, Shing MM, Yuen PM. Mixed chimerism after bone marrow transplantation for thalassemia major. Haematologica 2002;87:781-2. Ortega M, Escudero T, Caballín MR, Olivé T, Ortega JJ, Coll MD. Follow-up of chimerism in children with hematological diseases after allogeneic hematopoietic progenitor cell transplants. Bone Marrow Transplant 1999;24:81-7. Pawlowska AB, Blazar BR, Angelucci E, Baronciani D, Shu XO, Bostrom B. Relationship of plasma pharmacokinetics of highdose oral busulfan to the outcome of allogeneic bone marrow transplantation in children with thalassemia. Bone Marrow Transplant 1997;20:915-20. Glucksberg H, Storb R, Fefer A, Buckner CD, Neiman PE, Clift RA, Lerner KG, Thomas ED. Clinical manifestations of graftversus-host disease in human recipients of marrow from HL-Amatched sibling donors. Transplantation 1974;18:295-304. Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, Hows J, Thomas ED. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995;15:825-8. Fernández-Avilés F, Urbano-IspizuA A, Aymerich M, Colomer D, Rovira M, Martínez C, Nadal E, Talarn C, Carreras E, Montserrat E. Serial quantification of lymphoid and myeloid mixed chimerism using multiplex PCR amplification of short tandem repeatmarkers predicts graft rejection and relapse, respectively, after allogeneic transplantation of CD34+ selected cells from peripheral blood. Leukemia 2003;17:613-20. Amrolia PJ, Vulliamy T, Vassiliou G, Lawson S, Bryon J, Kaeda J, Dokal I, Johnston R, Veys P, Darbyshire P, Roberts IA. Analysis of chimaerism in thalassaemic children undergoing stem cell transplantation. Br J Haematol 2001;114:219-25. Lisini D, Zecca M, Giorgiani G, Montagna D, Cristantielli R, Labirio M, Grignani P, Previderè C, Di Cesare-Merlone A, Amendola G, Bergami E, Mastronuzzi A, Maccario R, Locatelli F. Donor/ recipient mixed chimerism does not predict graft failure in children with beta-thalassemia given an allogeneic cord blood transplant from an HLA-identical sibling. Haematologica 2008;93:1859-67. Huisman C, De Weger RA, De Vries L, Tilanus MG, Verdonck LF. Chimerism analysis within 6 months of allogeneic stem cell transplantation predicts relapse in acute myeloid leukemia. Bone Marrow Transplant 2007;39:285-91. Petz LD, Yam P, Wallace RB, Stock AD, De Lange G, Knowlton RG, Brown VA, Donis-Keller H, Hill LR, Forman SJ. Mixed hematopoietic chimerism following bone marrow transplantation for hematologic malignancies. Blood 1987;70:1331-7. Aker M, Kapelushnik J, Pugatsch T, Naparstek E, Ben-Neria S, Yehuda O, Amar A, Nagler A, Slavin S, Or R. Donor lymphocyte infusions to displace residual host hematopoietic cells after allogeneic bone marrow transplantation for beta-thalassemia major. J Pediatr Hematol Oncol 1998;20:145-8.

Research Article

15

Association of FXII 5’UTR 46C>T polymorphism with FXII activity and risk of thrombotic disease FXII 5'UTR 46C>T polimorfizmi ile FXII aktivitesi ve trombotik hastalÕk riskinin iliýkisi Parisa Rasighaemi1, Ahmad Kazemi1, Fereidun Ala2, Mohammad Jazebi3, Farnaz Razmkhah1 1Department

of Hematology, Iran University of Medical Sciences, Tehran, Iran Blood Transfusion Service, Birmingham, United Kingdom 3Centre of Pediatric Hemophilia, Tehran, Iran 2National

Abstract Objective: Thrombotic diseases are caused by genetic and environmental factors. There are a number of well-characterized genetic defects that lead to increased risk of thrombosis. Results from previous studies have indicated that FXII is involved in the pathogenesis of thrombophilic diseases. However, the results in this regard are highly controversial. One of the most important determinants of Plasma FXII level is 46CgT polymorphism in the FXII gene. In the present study, the risk of thrombophilic diseases related to this polymorphism was investigated in a case-control study. Material and Methods: One hundred and sixty subjects were studied: 120 patients diagnosed with thrombophilia (96 venous thromboembolism, 24 arterial thrombosis), and 40 age-gender-matched controls. For each subject, FXII activity level was measured by a one-step clotting assay with FXII-deficient plasma, and 46CaT polymorphism was genotyped using a restriction fragment length polymorphism (RFLP) method. Results: In this study, the previous observation that individuals with different genotypes for the 46 CaT polymorphism show significant differences in FXII activity levels was confirmed. Most importantly, FXII activity 68% was associated with an increased risk of venous thrombosis with an adjusted odds ratio (OR) of 4.7 (95% confidence interval [CI]: 1.03-21.1, p=0.04). However, it was not a risk factor for arterial thrombosis with adjusted OR of 5 (95% CI: 0.91-27.1, p=0.09). In CT and TT genotype, the adjusted ORs were 2 (95% CI: 0.9-4.4, p=0.11) and 2.3 (95% CI: 0.45-11, p=0.48), respectively, for patients with venous thrombosis compared with the controls. Similarly, the adjusted ORs in arterial thrombosis were 1.2 (95% CI: 0.4-3.6, p=0.76) for CT and 1.8 (95% CI: 0.2-14.9, p=0.59) for TT genotype. Thus, we did not find any association of the mutated T allele in the heterozygous or homozygous state with an increased risk of venous or arterial thrombosis. Conclusion: Lower FXII activity is not a risk factor; rather, it simply represents a risk marker for thrombosis. (Turk J Hematol 2010; 27: 15-9) Key words: Factor XII, factor XII polymorphism, venous thromboembolism, arterial thrombosis Received: August 8, 2009

Accepted: December 12, 2009

Özet Amaç: Trombotik hastalÕklar genetik ve çevresel faktörlerden kaynaklanmaktadÕr. Yüksek tromboz riskine yol açan çok sayÕda iyi karakterize edilmiý genetik defekt mevcuttur. Önceki çalÕýmalardan elde edilen sonuçlarda, FXII’nin trombofilik hastalÕklarÕn patojenezinde yer aldÕúÕ gösterilmiýtir. Bununla birlikte, sonuçlar bu bakÕmdan oldukça tartÕýmalÕdÕr. FXII genindeki 46CaT polimorfizmi FXII aktivite düzeylerini etkileyen faktörlerden birisidir. Mevcut çalÕýma kapsamÕnda, bu polimorfizm ile iliýkili trombofilik hastalÕklar riski bir vaka kontrol çalÕýmasÕnda araýtÕrÕlmÕýtÕr. Address for Correspondence: Ahmad Kazemi, PhD of Hematology, Iran University of Medical Sciences, Hematology Department, P.O Box 14155-6183, Tehran, Iran Phone: (+9821) 88052982 Fax: (+9821) 88054355 E-mail: iakazemi@iums.ac.ir

16

Rasighaemi et al. FXII 46C>T polymorphism and thrombosis risk

Turk J Hematol 2010; 27: 15-9

Yöntem ve Gereçler: Yüz altmÕý denek incelenmiýtir: 120 hastaya trombofili (96’sÕ tromboembolizm, 24’ü arteriyel tromboz) tanÕsÕ konmuý olup 40 hasta yaý ve cinsiyet açÕsÕndan eýleýtirilmiýtir. Her bir denek için, FXII aktivite düzeyi, FXII’den yoksun plazma ile tek adÕmlÕ pÕhtÕlaýma testi kullanarak ölçülmüý ve 46CaT polimorfizmi, (RFLP) yöntemi ile genotiplenmiýtir. Bulgular: 46 CaT polimorfizmi için farklÕ genotipleri olan bireylerin FXII aktivite düzeylerinde anlamlÕ farklÕlÕklar sergilediúine dair önceki gözlem, bu çalÕýmada doúrulanmÕýtÕr. Daha da önemlisi, %68 olan FXII aktivitesi, 4.7 ayarlanmÕý risk oranÕ (OR) ile venöz tromboza yönelik yüksek risk ile iliýkilendirilmiýtir (%95 güven aralÕúÕ [CI]: 1.03-21.1, p=0.04). Ancak bu, 5 ayarlanmÕý OR ile arteriyel tromboza yönelik bir risk faktörü deúildir (%95 CI: 0.91-27.1, p=0.09). Ct ve TT genotipte, ayarlanmÕý OR deúerleri, kontrollere kÕyasla venöz trombozlu hastalar için sÕrasÕyla 2 (%95 CI: 0.9-4.4, p=0.11) ve 2.3 (%95 CI: 0.45-11, p=0.48) idi. Benzer ýekilde, arteriyel trombozda ayarlanmÕý OR deúerleri CT genotip için 1.2 (%95 CI: 0.4 - 3.6, p=0.76) ve TT genotip için 1.8 (%95 CI: 0.2-14.9, p=0.59) idi. Böylelikle, heterozigot veya homozigot halde mutasyona uúramÕý T aleli ile venöz ya da arteriyel tromboza iliýkin yüksek risk arasÕnda herhangi bir iliýki tespit edilmemiýtir. Sonuç: Düýük FXII aktivitesi bir risk faktörü olmamakla birlikte, yalnÕzca tromboza yönelik bir risk göstergesini temsil etmektedir. (Turk J Hematol 2010; 27: 15-9) Anahtar kelimeler: Faktör XII, faktör XII polimorfizm, venöz tromboembolizm, arteriyel tromboz Geliý tarihi: 8 Aúustos 2009

Kabul tarihi: 12 AralÕk 2009

Introduction Thrombosis is a common cause of morbidity and mortality in industrialized nations. Both venous and arterial thrombosis can be life-threatening events, and both are of great public health importance [1]. Risk factors for thrombosis can be either genetic or acquired. Current research on complex diseases, such as ischemic stroke (IS), now focuses on identifying genetic variants that increase the susceptibility to thrombotic disorders [2-4]. Among the clotting factors studied, factor XII (FXII) levels exhibited one of the highest heritabilities (67%) and a significant positive genetic correlation with thrombotic disease [5], indicating that some of the polymorphisms and mutations that influence variation in this physiological risk factor also influence liability to thrombosis. Plasma coagulation FXII (Hageman factor) is an 80-KDa serine protease with an average plasma concentration of 30 g/ml. Contact of FXII with negatively charged surfaces leads to proteolytic cleavage and activation of the FXII molecule. The generated FXIIa can initiate activation of factor XI and also seems to participate in the conversion of plasminogen to plasmin [6,7]. While in vitro, FXII plays a central role in the initiation of coagulation and fibrinolysis, the physiological function of FXII is still under discussion. Factor XII gene spans a total of 12kb and is composed of 14 exons and 13 introns. It is reported to be located at 5q33qter in the human chromosome [8]. The concentration of plasma FXII has been known to vary widely between individuals and races [9]. Recently, a frequent CJT promoter polymorphism at nucleotide 46 of the FXII gene has been identified in which the T allele creates a novel methionine-initiating codon, which results in a lower translation efficiency and decrease in FXII plasma levels [10]. Here, the relation of plasma FXII activity and a common C46T polymorphism of the FXII gene with the risk of thrombotic disease was explored in a case-control study. The identification of these relations may help to elucidate the mechanism underlying the risk of common thrombosis and therefore may suggest preventive strategies to reduce thrombosisrelated morbidity and mortality.

Materials and Methods The study group consisted of 120 patients (65 males, 54.2%; 55 females, 45.8%) diagnosed with a thrombophilic disorder based on clinicians’ decision and 40 age-gendermatched control subjects. The patient group consisted of 96 venous thromboembolism (VTE) subjects (24 pulmonary embolism; 36 cerebral venous thrombosis; 36 deep vein thrombosis) and 24 subjects with arterial thrombosis [myocardial infarction [MI]). Patients were recruited from the hemophilia center in Iran. The patients were aged 21 to 64 years (41.9±10.8), with 73 (60.8%) individuals aged <45 and 47 (39.2%) aged >45 years. The patients who had no previous diagnosis of malignant disease or thrombophlebitis, with no history of diabetes mellitus or recent surgery, with normal renal and hepatic function, and who were not taking any drugs known to affect the coagulation system were included in the study protocol. Moreover, all of the patients were screened for FVL (factor V Leiden), prothrombin G20210A mutation, and protein C and S deficiency, and only patients with negative screening were included in the study. Control subjects were friends and spouses of patients who were recruited at the same time as the patients. They were included only if they had no personal history of thromboembolic disease, including venous and arterial thrombosis, cirrhosis, nephritic syndrome, or cancer. The control group was matched for age (20 to 60; mean 41.2±10.8; with 27 individuals (67.5%) aged <45 and 13 (32.5%) aged >45 years), and sex (22 males, 18 females). All individuals provided their informed consent to take part in the study, and the study was approved by the local ethics committee. For FXII activity determination, a one-step clotting assay with FXII-deficient plasma (Technoclone, Austria) and the activated partial thromboplastin time (aPTT) reagent (Technoclone, Austria) were used on a Sysmex Ca-1500 coagulometer. For genetic analysis, DNA was extracted by the phenolchloroform method from blood cells. Then, polymerase chain reaction (PCR) amplification of exon 1 of the FXII gene was performed using the following primer pair to produce a fragment of 142 bp. The forward primer was 5´-GAT AGG CAG CTG GAC CAA CG-3´; the reverse primer was 5´-TGA TAG CGA CCC CCC AGA AC-3´. The sequence surrounding the

Rasighaemi et al. FXII 46C>T polymorphism and thrombosis risk

Turk J Hematol 2010; 27: 15-9

C46T polymorphism contains a naturally occurring BsaHI restriction enzyme (Fermentas, Russia) site. The C46 wild type sequence (GACGCC) contains this BsaHI site, which is abolished in the T46 rare variant (GATGCC). In the presence of the C allele, BsaHI digestion yields fragments of 116 and 26 bp, whereas in the presence of the T allele, digestion does not occur. After overnight incubation of the PCR product with BsaHI restriction enzyme in 37°C, electrophoresis on 8% TBE-PAG (tris borate EDTA-polyacrylamide gel) for 90 min at 150V, and staining with ethidium-bromide for 10 min, bands were visualized on an UV transilluminator and photographed using a Polaroid land camera. Statistical analyses were performed using SPSS software version 14.0, and values of p<0.05 were considered significant. Odds ratio (OR) was also calculated as relative risk for thrombosis.

Results Of 40 tested healthy volunteers, 26 (65%) carried FXII 46C genotype, 12 (30%) were heterozygous, and 2 (5%) were homozygous for FXII 46T genotype. The allele frequencies of 46C and 46T were estimated as 0.8 and 0.2, respectively. The FXII activity levels, analyzed as a function of the 46 CJT polymorphism, showed statistically significant differences between the different genotypes (p=0.002). Genotype T/T showed the lowest levels of FXII activity (58±11.3) compared with the levels of the other genotypes (C/C genotype: 123±30.3 and C/T genotype: 97.3±23).

In both the patient and control groups, the CC genotype had the highest frequency, and TT the lowest. CC frequency was higher in controls (65%) than patients with VTE and MI (48% and 58.3%, respectively), but CT and TT frequencies were higher in patients with VTE (44% and 8%, respectively) and MI (33.3% and 8.3%, respectively) than controls (30% and 5%, respectively). The C/T allele frequency in patients with VTE and MI were 70/30 and 75/25 compared with 80/20 in controls. Therefore, the risk of thrombosis associated with the genotype CT and TT was studied. CC genotype was considered as a reference group, and the OR of thrombosis associated with the 46 CJT polymorphism for genotypes CT and TT was calculated in VTE patients (Table 1) and MI patients (Table 2) compared with the controls. The results suggest that neither genotype CT nor TT is a risk factor for arterial or venous thrombosis. In both the patient and control groups, CC demonstrated the highest FXII activity level and TT the lowest FXII activity levels. The mean values of plasma FXII activity levels were 112±32.2 in controls compared with 87±23 in VTE patients and 84±21 in MI patients. There were statistically significant differences in mean values of FXII activity levels between patients and controls (p=0.001). Thus, it was speculated that decreased FXII activity is a risk factor for thrombosis. Consequently, OR was calculated as a risk for thrombosis. Levels lower than the 10th percentile (lower than 68%) were used as a cut-off, and FXII activity >68% was considered as a reference group. The risk of thrombosis associated with FXII activity levels in VTE and MI patients is shown in Table 1 and Table 2, respectively. The results suggest that FXII activity 68% is an independent risk factor for venous thrombosis. However, it is not a risk factor for arterial thrombosis.

Table 1. Risk of VTE associated with CT, TT genotype and FXII activity Patients

Controls

OR

95% CI

P

CC

46 (48%)

26 (65%)

1*

----

----

CT

42 (44%)

12 (30%)

2

0.9-4.4

0.11

TT

8 (8%)

2 (5%)

2.3

0.45-11

0.48

----

----

1.03-21.1

0.04

FXII activity >68%

77 (80.2%)

38 (95%)

1#

FXII activity 68%

19 (19.8%)

2 (5%)

4.7

* Reference group: subjects with CC genotype, # Reference group: subjects with FXII activity >68, Reference range for FXII activity: 50% - 150%

Table 2. Risk of MI associated with CT, TT genotype and FXII activity Patients

Controls

(N=96)

(N=40)

CC

14 (58.3%)

CT TT

OR

95% CI

P

26 (65%)

1*

----

----

8 (33.3%)

12 (30%)

1.2

0.4-3.6

0.76

2 (8.3%)

2 (5%)

1.8

0.2-14.9

0.59

38 (95%)

1#

----

----

2 (5%)

5

0.91-27.1

0.09

FXII activity >68%

19 (79.2%)

FXII activity 68%

5 (20.8%)

* Reference group: subjects with CC genotype,

#Reference

17

group: subjects with FXII activity >68, Reference range for FXII activity: 50%-150%

18

Rasighaemi et al. FXII 46C>T polymorphism and thrombosis risk

Discussion It is well-known that plasma level of FXII is variable among different individuals and races due to genetic and environmental factors. Various factors such as estrogen, interleukin 6 [11], specific lipoprotein particles [12] and a dysfunctional endothelium [13] were shown to have a significant influence on FXII levels. This study confirms and extends the previous observation that the 46CJT polymorphism in the FXII promoter region correlates with lower FXII plasma activity. In this study, the allele frequency of 46 C/T was 0.8/0.2 in a small Iranian population (allele number=80), which is exactly the same as the allele frequency found by Kanaji [10] in Caucasians (allele number=40). Further, genotype frequency in that study corresponded to the genotype frequency in Austrians (who are representative of the Middle European population) reported by Endler et al. [14] in 100 healthy Austrian newborns and those in the United Kingdom found by Kohler et al. [15]. Conversely, the allele frequency of 46 C/T in Orientals has been shown to be 0.27/0.73 [10], which contradicts our results. This can be explained by racial and geographical differences. It has been reported that the plasma levels of FXII in Orientals (125.4±56.3) are lower than in Caucasians (150±30.3) because of the differences in allele frequency of 46C/T in the FXII gene between the two races [10]. Although in the present study the allele frequency of 46C/T was exactly the same as in Caucasians, the level of plasma FXII activity in this study (112±32.2) was lower than in Caucasians. It may be because of the low patient number in both studies. Furthermore, although the C46T polymorphism in the FXII gene is an important determiner of plasma FXII level, it is not the only effective factor. As previously mentioned, there are other factors that have a significant influence on FXII levels. Thus, further large scale studies considering the other effective factors on FXII levels may be needed to elucidate these differences. There is controversy over the clinical significance of FXII deficiency because most of subjects with complete deficiency of FXII have no clinical manifestations. Some authors considered low FXII levels to be associated with an increased risk for venous [1,16,17] as well as arterial thromboembolism [18-20], while other studies have reported increased FXII activity in people with acute coronary syndromes [21,22]. In some studies, no effect of FXII levels on thrombosis was observed [13,23,24]. Furthermore, there are conflicting results about the effect of polymorphism 46CJT in the FXII gene on thrombotic diseases. Previous genetic association studies showed that genotype TT increases significantly the risk for ischemic stroke [25], acute coronary artery disease [26] and cerebral venous thrombosis [27]. Conversely, there are also studies that either do not detect a genetic association between this genotype and cardiovascular risk [28] or alternatively report an association between the C/C genotype and thrombosis risk [29]. Although in this study, FXII activity 68% was associated with an increased risk of VTE, there was no association between FXII activity 68% and MI risk. Moreover, no association of the mutated T allele in the heterozygous or homo-

Turk J Hematol 2010; 27: 15-9

zygous state with an increased risk of VTE and MI was found. Therefore, it was speculated that reduced FXII activity is not the cause of thrombosis, but the result of it [30],for the following reason: Previous studies [10,14] and this study showed that the substitution of T allele at nucleotide 46 in the FXII gene reduces FXII activity because the 46T allele creates a novel methionine-initiating codon that reduces the translation efficiency of FXII. As a result, the FXII 46CJT polymorphism is not merely a marker, but rather a strong determinant of plasma FXII levels. Notably, despite the importance of the FXII genotype on FXII levels, the studies by Bach et al. [20], Athanasiadis et al. [28] these results study did not observe an association between the FXII 46T/T genotype and thrombosis. This would suggest that lower FXII activity is not a risk factor; rather, it simply represents a risk marker [30]. Endler et al. [31] demonstrated that with decreasing FXII activity, the hazard ratio for all-cause mortality and death as a result of ischemic heart disease gradually increased in an almost linear manner. However, mortality was not increased in individuals who had FXII activity that was lower than 10% of normal. While this appears paradoxical, the suggested reason is that the linear association between low FXII activity and mortality may simply reflect the progression of atherosclerosis and inflammation, and that individuals with very low FXII activity may have mutations that cause FXII deficiency but do not alter their overall survival [30]. Furthermore, FXII is a plasma protein that activates both coagulation and fibrinolysis. The level of plasma FXII is suggested to be reduced as a consequence of activation of both coagulation and fibrinolysis upon thrombus formation [30]. According to our results, FXII activity is decreased as a result of thrombosis in both arterial and venous thrombosis.

Acknowledgements This research was supported by Iran University of Medical Sciences. The authors would like to thank Mrs Sh.Hejazi and Sh.Ravanbod from Centre of Pediatric Hemophilia for technical support. 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.

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20. Bach J, Endler G, Winkelmann BR, Boehm BO, Maerz W, Mannhalter C, Hellstern P. Coagulation factor XII activity, activated factor XII, distribution of factor XII C46T gene polymorphism and coronary risk. J Thromb Haemost 2008;6:291-6. 21. Ishii K, Oguchi S, Murata M, Mitsuyoshi Y, Takeshita E, Ito D, Tanahashi N, Fukuuchi Y, Oosumi K, Matsumoto K, Kitajima M, Yamamoto M, Watanabe G, Ikeda Y, Watanabe K. Activated factor XII levels are dependent on factor XII 46C/T genotypes and factor XII zymogen levels, and are associated with vascular risk factors in patients and healthy subjects. Blood Coagul Fibrinolysis 2000;11:277-84. 22. 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 FXIIdependent prothrombotic activity. Thromb Res 2005;115:65-72. 23. Endler G, Mannhalter C, Sunder-Plassmann H, Lalouschek W, Kapiotis S, Exner M, Jordanova N, Meier S, Kunze F, Wagner O, Huber K. Homozygosity for the C T polymorphism at nucleotide 46 in 5-untranslated region of the factor XII gene protects from development of acute coronary syndrome. Br J Haematol 2001;115:1007-9. 24. Oguchi S, Ito D, Murata M, Yoshida T, Tanahashi N, Fukuuchi Y, Ikeda Y, Watanabe K. Genotype distribution of the 46C/T polymorphism of coagulation factor XII in the Japanese population: absence of its association with ischemic cerebrovascular disease. Thromb Haemost 2000;83:178-9. 25. Santamaria A, Mateo J, Tirado I, Oliver A, Belvis R, MartiFabregas J, Felices R, Soria JM, Souto JC, Fontcuberta J. Homozygosity of the T allele of the 46 CAT polymorphism in the F12 gene is a risk factor for ischemic stroke in the Spanish population. Stroke 2004;35:1795-9. 26. Santamaría A, Martínez-Rubio A, Mateo J, Tirado I, Soria JM, Fontcuberta J. Homozygosity of the T allele of the 46 CAT polymorphism in the F12 gene is a risk factor for acute coronary artery disease in the Spanish population. Haematologica 2004;89:878-9. 27. Reuner KH, Jenetzky E, Aleu A, Litfin F, Mellado P, Kloss M, Juttler E, Grau A.J, Rickmann H, Patscheke H, Lichy C. Factor XII C46T gene polymorphism and the risk of cerebral venous thrombosis. Neurology 2008;70:1-4. 28. Athanasiadis G, Esteban E, Vidal MG, Torres RC, Bahri R, Moral P. Polymorphism FXII 46C>T and cardiovascular risk: additional data from Spanish and Tunisian patients. BMC Res Notes 2009;2:154. 29. Kanaji T, Watanabe K, Hattori S, Urata M, Iida H, Kinoshita S, Kayamori Y, Kang D, Hamasaki N. Factor XII gene (F12) -4C/C polymorphism in combination with low protein S activity is associated with deep vein thrombosis. Thromb Haemost 2006;96:854-5. 30. Kanaji T. Lower factor XII activity is a risk marker rather than a risk factor for cardiovascular disease: a rebuttal. J Thromb Haemost 2008;6:1053-4. 31. Endler G, Marsik C, Jilma B, Schickbauer T, Quehenberger P, Mannhalter C. Evidence of a U-shaped association between factor XII activity and overall survival. J Thromb Haemost 2007;5:1143-8.

Research Article

20

Hospital-acquired pneumonia in patients receiving immunosuppressive therapy ûmmunsupresif tedavi alan hastalarda geliýen hastane kökenli pnömoni Ebru ÇakÕr Edis1, Osman Nuri Hatipoúlu1, ûlker YÕlmam1, Alper Eker2, Özlem Tansel2, Necdet Süt3, Emre Tekgündüz4, Muzaffer Demir4 1Department

of of 3Department of 4Department of 2Department

Pulmonary Medicine, Trakya University Faculty of Medicine, Edirne, Turkey Infectious Diseases And Clinical Bacteriology, Trakya University Faculty of Medicine, Edirne, Turkey Biostatistics and Medical Informatics, Trakya University Faculty of Medicine, Edirne, Turkey Hematology, Trakya University Faculty of Medicine, Edirne, Turkey

Abstract Objective: The aims of this study were to determine the clinical success rates, effect of neutropenia on treatment success rates, risk factors related to mortality, and survival in patients who developed hospital-acquired pneumonia (HAP) while receiving immunosuppressive therapy. Materials and Methods: Forty-three adult patients receiving immunosuppressive therapy who developed HAP were included in this prospective study. Transplantation patients and human immunodeficiency virus (HIV)-positive patients were not included. Antibiotic treatment was managed by a multidisciplinary team. The Kaplan Meier method was used for the survival analysis and Cox regression was used for the identification of mortality-related independent risk factors. The relationship between neutropenia and the clinical success rate was determined using the chi-square test. Results: Although anti-pseudomonal antibiotics were started empirically in 40 of the 43 patients (93%) at the beginning of the treatment, the most frequently isolated pathogens were Acinetobacter spp. and Escherichia coli. The success rate at the end of the treatment was 65.1%. The survival rates for the 3rd, 14th, 42nd, and 365th days were 97%, 86%, 58%, and 19%, respectively. Elevated levels of urea [Hazard Ratio=1.01 (95% CI: 1.00–1.02)] and blood glucose [HR=1.01 (95% CI: 1.00–1.02)] were found to be independent risk factors affecting survival. The treatment success rate was higher in patients without neutropenia (n=23) than in those with neutropenia (n=20) (p=0.05). Conclusion: The treatment success rate was low in patients who developed HAP while receiving immunosuppressive therapy. (Turk J Hematol 2010; 27: 20-4) Key words: Immunocompromised patients, hospital-acquired pneumonia, survival Received: July 1, 2009

Accepted: January 25, 2010

Özet Amaç: ÇalÕýmamÕzda immunsupresif tedavi alÕrken hastane kökenli pnömoni (HKP) geliýen hastalarda klinik baýarÕ oranlarÕnÕ, nötropeninin tedavi baýarÕsÕna olan etkilerini, mortalite ile iliýkili risk faktörlerini ve survi oranlarÕnÕ saptamayÕ amaçladÕk. Address for Correspondence: Asst. Prof. Ebru ÇakÕr Edis, Trakya Üniversitesi TÕp Fakültesi, Göúüs HastalÕklarÕ Anabilim DalÕ, Edirne, Türkiye Phone: +90 284 235 76 41-1123 E-mail: ebruckr@yahoo.com

Edis et al. HAP in immunocompromised patients

Turk J Hematol 2010; 27: 20-4

21

Yöntem ve Gereçler: ûmmunsupresif tedavi alÕrken HKP geliýen 45 eriýkin hasta prospektif olarak çalÕýmaya alÕndÕ. Transplant hastalarÕ ve human immunodeficiency virus (HIV)-pozitif olan hastalar çalÕýmaya alÕnmadÕ.Antibiyotik tedavisi multidisipliner olarak yönetildi. Survi analizlerinde Kaplan Meier, mortaliteyle iliýkili baúÕmsÕz risk faktörlerini saptamak için Cox regresyon uygulandÕ. Nötropeninin klinik baýarÕ oranlarÕ ile iliýkisi Chi Square yöntemiyle karýÕlaýtÕrÕldÕ. Bulgular: Ampirik tedavi olarak 43 hastanÕn 40’da (%93) antipseudomonal tedavi baýlanmasÕna raúmen en sÕk izole edilen etkenler Acinetobacter spp ve Escherichia coli idi. Tedavi sonu klinik baýarÕ oranÕ %65.1 idi. Sürvi oranlarÕ 3.,14., 42. ve 365. gün sÕrasÕyla % 97, 86, 58 ve 19 olarak bulundu. Üre yüksekliúi [Hazard Ratio=1.01 (%95 GA: 1.00-1.02)] ve kan ýekeri yüksekliúi [HR=1.01 (%95 GA: 1.00-1.02)] surviyi olumsuz etkileyen baúÕmsÕz risk faktörleri olarak bulundu. Nötropenik olmayan (n=23) hastalarda klinik baýarÕ oranlarÕ nötropenik (n=20) olanlara göre daha yüksek bulundu (p=0.05). Sonuç: ûmmunsupresif tedavi alan hastalarda geliýen HKP’lerde tedavi baýarÕ oranlarÕ düýüktür. (Turk J Hematol 2010; 27: 20-4) Anahtar kelimeler: ûmmunsupresif hasta, hastane kökenli pnömoni, sürvi Geliý tarihi: 1 Temmuz 2009

Kabul tarihi: 25 Ocak 2010

Introduction In spite of prophylactic measures and the use of widespectrum antibiotics, hospital-acquired pneumonia (HAP) is still an important cause of morbidity and mortality in patients receiving immunosuppressive therapy [1]. Algorithms for empirical antimicrobial treatment for suspected pathogens have been developed for some immunosuppressed patient groups. The principle of empirical treatment is based on an anti-pseudomonal approach, and in cases where there is no response and no pathogen is detected, treatment aimed at Aspergillus spp. or methicillin-resistant Staphylococcus aureus (MRSA) should be initiated. Invasive methods are only recommended in patients who do not respond to the initial antimicrobial treatment. Despite these structured empirical antimicrobial treatments using algorithms and the many invasive and non-invasive methods for pathogen isolation in these patients, mortality rates remain high. The aims of this study were to determine the clinical success rates, effect of neutropenia on treatment success rates, risk factors related to mortality, and survival in patients who developed HAP while receiving immunosuppressive therapy.

Materials and Methods Adult patients who developed HAP while receiving immunosuppressive therapy for solid organ tumors and hematological malignancies at the Trakya Universty Medical Faculty Hospital between March 2005 and February 2006 were included in this prospective study. a) Patients who were known to have no previous pneumonia history and whose chest X-rays showed new infiltration at least 48 hours after hospital admission (that could not be explained otherwise) were diagnosed as HAP if one of the following criteria was present [2]: • Fever (> 38°C) or hypothermia ( 36°C) • Clinical findings such as dyspnea, coughing, newonset purulent sputum, or a change in the characteristics of the sputum • Leukocytosis, leukopenia, and/or elevated C-reactive protein (CRP) (not associated with primary diseases and drug therapy) • Consolidation findings on the physical examination • Pathogen isolation in blood culture and/or sputum/transtracheal aspirates

b) In the presence of one or more of the following criteria, patients were diagnosed as having severe HAP [2]: • Arterial oxygen pressure (PaO2)/fraction of inspired oxygen (FiO2) <250 • Severe sepsis or signs of septic shock • Bilateral or multilobar involvement, cavitation, abscess or effusion c) Pneumonias that developed >4 days after hospitalization were classified as “late pneumonia” [2]. d) Neutropenia was defined as either a neutrophil count of <500/mm3 or a neutrophil count of <1000/mm3 that would be expected to decrease [2]. Patients with fever and neutropenia but without new infiltration in the chest X-ray were excluded from the study. Study Protocol Patients included in the study were evaluated prospectively. At the planning stage of the study, approval from the local ethics committee was obtained, and each patient (or his/her caregivers) gave informed consent prior to participation in the study. Patients who had had consultations with a pulmonary medicine or infectious disease specialist and who were suspected of having developed HAP according to the above criteria while receiving immunosuppressive therapy were included in this study. Demographic data, risk factors, and the date of pneumonia development were recorded. Chest X-rays, complete blood counts, biochemistry parameters, arterial blood gases, CRP levels, and cultures of blood, sputum, or tracheal aspirate were studied prior to treatment, and pleural fluid was examined in patients with pleural effusion. The etiologic diagnostic criteria were defined as follows: by isolation of a microorganism in blood cultures or pleural fluid or with isolation of adequate sputum/tracheal samples in a pure or predominant culture, which correlated with the predominant morphology in the gram stain. Computerized tomography scans of the thorax were performed when indicated, and if the pathogen could not be isolated, empiric anti-pseudomonal treatment was initiated in line with national and international guidelines [2,3]. All patients were re-evaluated after 3-5 days to determine the success of the empirical treatment. If the pathogen was isolated, specific antibiotic treatment was initiated. When necessary, the treatment regimen was redesigned in patients receiving empirical

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Edis et al. HAP in immunocompromised patients

treatment, even if the pathogen was isolated afterwards. Alternative empirical treatment was given to patients who showed no response to the initial empirical antibiotic treatment and in patients in whom the pathogen could not be isolated. Bronchoscopy and bronchial lavage were performed when required, and all of the patients were evaluated by a team of physicians from pulmonary and infectious disease specialties, as well as by the physicians responsible for the patient. Patients showing clinical improvement after treatment were discharged home and reassessed after six weeks. The followup period was one year and all patients were contacted by telephone at the end of the year to determine their survival status. In the case of death, the date of death was recorded. Statistics Statistical analyses were conducted using the SPSS 9.0 (SPSS Inc.; Chicago, IL, USA) statistical software, and descriptive statistics and frequency analysis were performed. The Kaplan-Meier analysis was used for the analysis of survival. Factors that may have independently affected mortality were assessed using the univariate Cox regression analysis, and after the univariate analysis, the variables with a p value of <0.1 were analyzed using a multivariate Cox regression model. The level of significance was set as p<0.05. The relationship between neutropenia and the clinical success rate was studied using the chi-square test.

Results A total of 43 patients [15 females (34.9%), 28 males (65.1%)] were included in the study. The mean age was 56.58 ± 15.31 years (range: 17-95 years). Severe pneumonia was observed in 27 patients (62.8%) and late pneumonia in 40 patients (93%); the mean number of days before the development of pneumonia was 24.8±20.2. Of the patients, 27 were receiving immunosuppressive therapy for hematological malignancy (14 patients had acute myeloid leukemia, 7 multiple myeloma, 4 non-Hodgkin’s lymphoma, 1 myelodysplastic syndrome, and 1 mycosis fungoides) and 16 for solid tumors (9 patients had lung cancer, 3 colon cancer, 2 stomach cancer, 1 breast cancer, and 1 nasopharyngeal cancer). Twenty patients were neutropenic and 23 were non-neutropenic at the time of diagnosis. All the patients were considered to be highrisk patients because they were receiving immunosuppressive therapy and had pneumonia. The most commonly observed patient-related risk factors were hypoalbuminemia (93%), smoking (46.5%) and advanced age (27.9%) (Table 1). Of the patients, 42 (97.6%) were given empirical treatment whilst the other patients underwent specific treatment, as the pathogen was isolated before the initiation of treatment. One patient had Acinetobacter spp. and was given cefepime in addition to an aminoglycoside. One non-neutropenic patient who developed HAP early ( 4 days) was suspected of having HAP due to aspiration, and was therefore started on parenteral ampicillin-sulbactam. The other patient had a suspected atypical pathogen and was given ceftriaxone plus clarithromycin.

Turk J Hematol 2010; 27: 20-4

Table 1. Patient-related risk factors Risk factors related to patients

N

%

Hypoalbuminemia (< 3.5 mg/dl)

40

93

Smoking

20

46.5

Age >65

12

27.9

Alcohol

8

18.6

COPD

4

9.3

CVD

4

9.3

Diabetes

3

7.0

CRF

2

4.7

COPD: Chronic obstructive pulmonary disease; CVD: Cerebrovascular disease; CRF: Chronic renal failure

Of the 43 patients, 40 were started on empirical anti-pseudomonal treatment (ceftazidime plus aminoglycoside: n=13; ceftazidime plus ciprofloxacin: n=2; piperacillin/tazobactam plus aminoglycoside: n=6; piperacillin/tazobactam plus ciprofloxacin: n=5; carbapenem plus aminoglycoside: n=14). The pathogen was isolated in 16 (37.2%) of the 43 patients. Three patients had 2 pathogens, and 1 patient had 3 pathogens. The most commonly isolated pathogens were Acinetobacter spp. (n=4), Escherichia coli (n=3), Streptococcus pneumoniae (n=2), Klebsiella spp. (n=2), Serratia spp. (n=2), Aspergillus fumigatus (n=2), MRSA (n=1), Streptococcus viridans (n=1), Staphylococcus epidermidis (n=1), Proteus spp. (n=1), Stenotrophomonas maltophilia (n=1), and Candida spp. (n=1). Twenty-one pathogens were isolated from 16 patients. Blood culture was positive in 4, sputum/aspirate culture was positive in 10, pleural samples were positive in 5, and bronchoalveolar lavage culture was positive in 2. In 12 patients, the pathogens were isolated before the initiation of empirical treatment. Six of these 12 patients had pathogens that were sensitive to the empirical treatment and the treatment regimen was not changed, whereas the regimen was changed in the other 6 patients. In 3 patients in whom the pathogen could not be isolated before initiation of the empirical antibiotic treatment, the pathogen was isolated at a later time. Glycopeptides (teicoplanin) were started in 6 patients and antifungal therapy was started in line with guidelines in 12 patients (amphotericin B: n=9; liposomal amphotericin B: n=2; Caspofungin: n=1) who were not responding to the initial antibiotic treatment [3]. Clinical success at the end of the treatment was achieved in 28 (65.1%) patients and clinical success at the end of the sixth week (follow success) was achieved in 24 (55.8%) patients. Four patients with clinical success at the end of treatment died due to the primary disease at the end of the followup period, and the pneumonia relapsed in 1 patient. When the 16 patients with isolated pathogens and the 27 patients with non-isolated pathogens were compared, the difference in the rates of clinical success at the end of treatment was significant (p=0.024). The difference between the clinical success rates at the end of the follow-up period was also significant (p=0.013). The success rate was higher in patients in whom the pathogen could not be isolated. When the 20 neutropenic and 23 non-neutropenic patients were compared, the difference in the rates of clinical success at

Edis et al. HAP in immunocompromised patients

Turk J Hematol 2010; 27: 20-4

the end of treatment was significant (p=0.05), whereas the difference between the clinical success rates at the end of the follow-up period was not significant. The success rate at the end of treatment was higher in patients who were not neutropenic. Eighteen (42%) of the 43 patients had died by the end of the six-week follow-up period. According to the Kaplan Meier survival analysis, the survival rates for the 3rd, 14th, 42nd, and 365th days were 97%, 86%, 58%, and 19%, respectively. There were no significant differences between living and exitus patients in terms of age, gender, the mean number of days before the development of pneumonia, severe pneumonia, hypotension, comorbid status, medical treatment, or radiology. Three patients suffered from pneumonia attacks in the study year while they were in the hospital for the second time to receive immunosuppressive therapy. Two patients developed community-acquired pneumonia, and 11% of the patients developed recurrent pneumonia during the year. The effect of risk factors on survival was studied using univariate Cox regression analysis. Urea and fasting blood glucose (FBG) were found to be p<0.1 and were re-analyzed using multivariate analysis; urea (p=0.012) and FBG (p=0.004) were found to be the independent factors affecting survival. Elevated levels of urea [hazard ratio (HR) (95% confidence interval): 1.01 (1.00-1.02)] and FBG [HR (95%CI): 1.01 (1.00-1.02)] were the independent risk factors adversely affecting survival.

Discussion Due to the widespread use of immunosuppressive therapies, hospital-acquired infections in patients receiving immunosuppressive therapy are an important problem for today’s clinicians. The most common pathogen found in our study was Acinetobacter spp., followed by E. coli. The most common pathogen responsible for HAP in our previous study (which included patients receiving immunosuppressive therapy and others as well) was also Acinetobacter spp. [4]. In another study, Pseudomonas spp. was the most common pathogen in patients receiving immunosuppressive therapy who developed HAP; therefore, they are a target for empirical treatment, especially in neutropenic patients [5]. However, Pseudomonas spp. were not isolated in our study. Fungal pathogens, in particular Aspergillus spp., were responsible for the HAP in patients with hematological malignancies, and they contributed to a high mortality rate [6]. In patients with a hematological malignancy, the rate of definitive Aspergillus spp. was 5-6% [7]. Aspergillus spp. was isolated in two patients (4%), and this rate was consistent with the literature. The treatment success rate was low and the mortality rate high in patients receiving immunosuppressive therapy who developed HAP [8]. We found the clinical success rates at the end of the treatment and at the end of the follow-up period to be 65% and 56%, respectively. In a study comparing the HAP of 20 immunocompetent patients with that of 54 immunocompromised patients, the mortality rates were found to be high in both groups (60% and 50%, respectively) [9]. There are many comparative studies on pathogen isolation in patients receiving immunosuppressive therapy [10]. However, despite the widespread use of non-invasive and/or broncho-

23

scopic methods to isolate pathogens, we could not find any studies in the literature on the relationship between pathogen isolation and clinical success rates in these patients [except the studies involving transplantation and human immunodeficiency virus (HIV)]. In our study, the success rate of the group in which the pathogen could not be isolated was higher than that of the group in which the pathogen was isolated (there were no significant differences between the groups in terms of age, gender, comorbid status, or APACHE II scores). It is difficult to explain this fact. Are the microorganisms responsible for pneumonia in patients with higher clinical success rates the ones that are difficult to isolate? Are the patientrelated factors more important than the pathogen-related factors in clinical success? We think that larger studies should be conducted to answer these questions. However, the higher treatment success rates in non-neutropenic patients than in neutropenic patients may be considered a finding that supports the importance of patient-related factors. According to the Kaplan Meier survival analysis, the survival rates for the 3rd, 14th, 42nd, and 365th days were 97%, 86%, 58%, and 19%, respectively. We could not find any other study in the literature with such a long follow-up period. The reason for the low one-year survival rates may be the progressive characteristics of the underlying disease in these patients and the toxic and immunosuppressive characteristics of the medications they were receiving. Although the one-year survival rates were low, 11% of the patients had recurrent pneumonia, which indicates the high susceptibility of these patients to serious infections. We found elevated urea and FBG levels to be the independent risk factors that negatively affected survival. These results show that it is necessary not only to choose the antimicrobial drugs in HAP treatment, but also to address the metabolic problems. Notably, the limitation of our study with respect to the small number of patients may have prevented us from finding other independent risk factors. Elevated urea and FBG levels have been reported in community-acquired pneumonia [11], but we could not find any studies in the literature on the factors affecting the survival of patients receiving immunosuppressive therapy who developed HAP. In conclusion, the treatment success rate was low in patients who developed HAP while receiving immunosuppressive therapy. 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. Agustí C, Rañó A, Sibila O, Torres A. Nosocomial pneumonia in immunosuppressed patients. Infect Dis Clin North Am 2003;17:785-800. 2. Ozdemir O, Tabak L, Akan H, Akcay S, Akova M, Aygun G, Gulay Z, SayÕner A, Sever M, Tasova Y, Yuce A, Zeytinoglu A. Guidelines for the management of pneumonia in adult immunocompromised patients. Turk Thoracic J 2002;3:27-42.

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Edis et al. HAP in immunocompromised patients

3. American Thoracic Society; Infectious Diseases Society of America. Guidelines for the management of adults with hospitalacquired, ventilator-associated and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005;171:388-416. 4. Cakir Edis E, Caglar T, Otkun M, Gurcan M, Hatipoglu ON, Tulay E. Causative agents of hospital-acquired pneumonia and their antimicrobial resistance. Turk J Infection 2006;20:107-10. 5. Imataki O, Tamai Y, Abe Y, Kusafuka K, Kawakami K. Three cases of necrotizing pneumonia by Pseudomonas aeruginosa infection in hematological malignancy, including dead and alive cases. Gan To Kagaku Ryoho 2007;34:793-7. 6. Lass-Flรถrl C, Dierich MP. Epidemiology and prevention of pulmonary aspergillosis in patients with hematological malignancies. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2004;47:379-83. 7. Walsh TJ, Finberg RW, Arndt C, Hiemenz J, Schwart C, Bodensteiner D, Pappas P, Seibel N, Greenberg RN, Dummer S, Schuster M, Holcenberg JS. Liposomal amphotericin B for empir-

Turk J Hematol 2010; 27: 20-4

ical therapy in patients with persistent fever and neutropenia. National Institute of Allergy and Infectious Diseases Mycoses Study Group. N Engl J Med 1999;340:764-71. 8. Fernรกndez Guerrero ML, Ramos JM, Marrero J, Cuenca M, Fernรกndez Roblas R, de Gรณrgolas M. Bacteremic pneumococcal infections in immunocompromised patients without AIDS: the impact of beta-lactam resistance on mortality. Int J Infect Dis 2003;7:46-52. 9. Saenghirunvattana S, Charoenpan P, Kitboonsri S, Aeursudkij B. Nosocomial pneumonias - a comparison among normal and compromised hosts in Thailand. J Med Assoc Thai 1992;75:26-9. 10. Pozzi E, Masiero P, Oliva A. Evaluation of the invasive techniques for diagnosing bacterial respiratory infections. J Chemother 1995;7:286-91. 11. Fine MJ, Auble TE, Yealy DM, Hanusa BH, Weissfeld LA, Singer DE. A prediction rule to identify low-risk patients with communityacquired pneumonia. N Engl J Med 1997;336:243-50.

Research Article

25

Sonographic evaluation of spleen size and prevalence of accessory spleen in a healthy male Turkish population SaúlÕklÕ Türk erkeklerinde dalak boyutu ve aksesuar dalak prevelansÕnÕn ultrasonografi ile deúerlendirilmesi Selim Serter, Cengiz Ceylan, Özüm Tunçyürek, üebnem Örgüç, Yüksel Pabuçcu Department of Radiology, Celal Bayar University, Manisa, Turkey

Abstract Objective: It is known that the measurement of splenic length in routine clinical practice is a very good indicator of actual splenic size. Knowledge of the normal range of spleen size in the population being examined is a prerequisite. Racial differences in splenic length could result in incorrect interpretation of splenic measurements. The purpose of this study was to establish the range of spleen length in a young male Turkish population. Material and Methods: A total of 2179 volunteers, healthy men aged 17-42 years, from the annual Army Reserve Officer Training Corps training camp at Manisa were included in the study. Sonographic measurements of spleen length were performed on 2179 military personnel. Presence of accessory spleen was also determined. In addition, the height, weight, and age of each volunteer were recorded. Using linear regression analysis, the relation of spleen length and body height, weight and body mass index (BMI) was evaluated. Additionally, the prevalence of accessory spleen detected on ultrasound was calculated. Results: The mean±SD height was 173,1±6,5 cm, mean weight 69,1±9,7 kg, and mean BMI 22,62±2,87. Mean spleen length was 10,76±1,8 cm. The length of the spleen was below 12,80 cm in 95% of the subjects. No statistically significant correlation (p<0.01) between spleen length and body height, weight and BMI was found. The prevalence of accessory spleen was determined as 2.5% on ultrasound screening. Conclusion: It was found that in healthy Turkish men, mean spleen length was 10,76±1,8 cm. This data should be taken into consideration when the diagnosis of splenomegaly is established in Turkish males. (Turk J Hematol 2010; 27: 25-8) Key words: Splenomegaly, ultrasonography, prevalence of accessory spleen Received: April 16, 2009

Accepted: December 9, 2009

Özet Amaç: Dalak boyutu hematolojik hastalÕklarda önemli bir parametredir. Rutin klinik pratikte dalak uzunluúu gerçek dalak boyutunun en iyi göstergesidir. Dalak boyutlarÕndaki bölgesel farklÕlÕklar doúru olmayan dalak ölçümü deúerlendirmelerine ve en sÕk karýÕlaýÕlan dalak patolojisi olan splenomegalinin yanlÕý yorumlanmasÕna neden olabilir. Dalak boyutunun normal aralÕúÕnÕn bilinmesi bu durumda önýarttÕr. Prospektif çalÕýmamÕzÕn amacÕ saúlÕklÕ genç eriýkin Türk erkeklerinde ortalama dalak uzunluúunun belirlenmesidir. Address for Correspondence: Asst. Prof. Selim Serter, Department of Radiology, Celal Bayar University, Manisa, Turkey Phone: +90 236 240 90 70 E-mail: serterselim@gmail.com

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Serter et al. Sonographic evaluation of spleen size

Turk J Hematol 2010; 27: 25-8

Yöntem ve Gereçler: Mart 1993 ve Aúustos 2006 yÕllarÕ arasÕnda 161 hastaya allojeneik hematopoetik kök hücre nakli uygulandÕ. Siklofosfamide baúlÕ hemorajik sistit geliýimi engelleyebilmek amacÕyla profilaktik olarak Mesna, hiperhidrasyon ve zorlu diürez uygulandÕ. Buna raúmen 161 olgunun 17’si aúÕr olmak üzere 49’unda hemorajik sistit geliýti. Tedavi yaklaýÕmÕ ve bulgular geriye dönük olarak deúerlendirildi. YaýlarÕ 17-42 arasÕnda 2179 saúlÕklÕ genç eriýkin asker (Manisa er eúitim merkezi) tarama ultrasongrafisi ile incelendi. OlgularÕn yaý, boy, kilolarÕ, dalak uzunluúu ve aksesuar dalak varlÕúÕ kaydedildi. Lineer regresyon analizi ile olgularÕn dalak boyutu ile kilo, boy ve BMI (vücut kitle indeksi)'leri iliýkisi deúerlendirildi. Bulgular: Olgularda ortalama boy 173,1 cm±(SD) 6,5 cm, ortalama aúÕrlÕk 69,0 kg±(SD) 9,7 kg, ortalama BMI 22,62± (SD) 2,8. ortalama dalak boyu 10,76 cm±(SD) 1,84 cm bulundu. Dalak uzunluúu ile boy, aúÕrlÕk ve BMI arasÕnda istatistiki anlamlÕ bir iliýki saptanmadÕ. (p < 0.01). ÇalÕýma grubumuzdaki olgularÕn % 2.5'inde ultrasonografi ile aksesuar dalak saptandÕ. Sonuç: US tarama ile saúlÕklÕ genç eriýkin Türk popülasyonu için dalak boyutu ortalama 10,76 cm bulundu. Türk toplumunda splenomegali deúerlendirilirken bu deúer göz önünde bulundurulmalÕdÕr. (Turk J Hematol 2009; 27: 25-8) Anahtar kelimeler: Splenomegali, ultrasonografi, aksesuar dalak sÕklÕúÕ Geliý tarihi: 16 Nisan 2009

Kabul tarihi: 9 AralÕk 2009

Introduction The presence of splenomegaly is very significant in the diagnosis of several groups of diseases: immunological and inflammatory diseases, reticuloendothelial proliferations, storage disease, and portal hypertension. While the diagnosis of gross splenomegaly is both clinically and ultrasonographically obvious, the diagnosis and monitoring of clinically impalpable mild splenomegaly by ultrasound (US) is not well established. Several complicated measurements have been described, but a single, simple sonographic measurement gives a clinically useful indication of true splenic size (1-3). Since previous US data have suggested that there may be racial differences in splenic size (1,4,5), these differences in splenic length could result in incorrect interpretation of splenic measurements. Therefore, standards of normal spleen sizes have been developed for different areas, and knowledge of the normal range of spleen size in the population is a prerequisite. On the other hand, the failure of fusion of splenic tissue results in the formation of accessory spleens, and detection of an accessory spleen is important for hematological conditions. The purpose of this study was to establish the range of splenic length and to determine the prevalence of accessory spleens in a population of healthy Turkish men.

Materials and Methods Between August 2002 and May 2003, 2179 volunteers from the Reserve Officer Training Corps annual training camp in Manisa, Turkey were included in the study. The study group consisted of men serving an obligatory military service, who were randomly selected from different regions of Turkey. The subjects did not have any conditions likely to be associated with splenic enlargement. None of the subjects had a hematologic, immunologic or any other pathology like malaria or schistosomiasis. A medical history and laboratory tests were obtained from all volunteers who also underwent physical examination. Informed consent was obtained from all participants. The study was approved by the institutional review board and met all guidelines of the institution. US examination

was performed using Siemens Sonoline US machine with a convex 3,5-5 MHz transducer. The subjects were placed in a supine or right posterior oblique position and scanned during suspended respiration. The spleen size was measured in the sagittal plane in the standard oblique coronal orientation to record the maximum length (in millimeters) of the spleen. During the examinations, the presence of accessory spleens was noted. All examinations were performed and interpreted by three experienced radiologists. Measurements were done with electronic calipers on the image monitor during the examination. In addition, height, weight and body mass index (BMI) of the subjects were recorded. Relationships between spleen length and body height, weight and BMI were evaluated using linear regression analysis. Additionally, the prevalence of accessory spleen detected by US was calculated.

Results A total of 2179 white male subjects were evaluated by US. The mean age±standard deviation (SD) of subjects was 22,4±3,6 years (range: 17-42). There was no racial variation in our study cohort. The study cohort consisted of subjects from all regions of Turkey; however, our subjects are homogeneous racially because there are no different racial groups in the country. The mean body height was 173,1±6,5 cm, mean body weight 69,1±9,7 kg, and mean BMI 22,62±2,87. Mean spleen length was 10,76±1,84 cm. No statistically significant correlations (p< 0.01) between spleen length and body height, weight and BMI were found. Pearson correlation coefficients between spleen length and body height, body weight and BMI were 0,76, 0,73 and 0,34, respectively. The length of the spleen was below 12,80 cm in 95% of the subjects. The distributions of spleen length are shown in the Figure 1. Forty-nine accessory spleens were identified in 2179 US scans, giving a prevalence of 2,2% for accessory spleen in this asymptomatic population.

Discussion Spleen size may give information about the diagnosis and course of gastrointestinal and hematologic diseases (1-3).

Serter et al. Sonographic evaluation of spleen size

Turk J Hematol 2010; 27: 25-8

1400 1200 1000

800 600 400 200

0

40,0

80,0 60,0

120,0 100,0

140,0

160,0 200,0 240,0 280,0 180,0 220,0 260,0

Figure 1. The distribution of spleen length (mm) in all participants

The x axis shows measurament of spleen length and the y axis shows number of subjects

Because of its diagnostic efficiency, US is suggested as the method of choice in the diagnosis and monitoring of splenomegaly. Thus determination of normal organ size can be significant. It is known that the measurement of splenic length in routine clinical practice is a very good indicator of actual splenic size. We have used only the splenic length measurement, since it has been shown in an autopsy series that sonographic measurements of splenic length correlate with actual spleen dimensions (3,6). A study by Loftus et al. (3) on 30 cadavers found a clear linear relationship between a sonographic measurement of splenic length and the actual length, volume, and weight as measured at autopsy. The Pearson’s product moment correlation coefficient for maximum sonographic length and the actual length was 0.831 (p<0.001). They suggested that a simple single sonographic measurement of length could be used for routine work, reserving the more complex volumetric measurement (splenic area or index) for problematic cases. This position is further bolstered by another study that showed good correlation between computerized tomography (CT) volume and splenic length measured on sonography (r=0.86, p<0.001) (7). The mean splenic length was 10,76 cm in our study group. The length of the spleen was below 12.80 cm in 95% of the subjects. This length differs from the western data. The measurements are higher than the literature measurement. Bezzera et al. (7) found maximum spleen length of 9,7 cm as the upper limit of normality in United States volunteers. Accordingly, Niederau et al. (8) from Germany reported that in 95% of the normal subjects, the length of the spleen was less than 8.7 cm and mean spleen length was 5,5±1.4 cm. Another article from Germany showed that in 95% of the normal subjects, the spleen length was less than 11 cm (9). Conversely, an article from Egypt demonstrated that Egyptian norms for US organometry of the spleen are different from those of other countries (4). The means, SD and 95th percentiles of all measurements were higher than those recorded in other studies. They suggested that Egyptian sonographers should use their own normograms in routine practice. An article from China recommends 12 cm as the

27

upper limit of normal in the Chinese population (5). We think that this can be attributed to the study cohort being comprised of young healthy male subjects. One of the possible reasons for the larger spleen size in our study group is the fact that a likely decrease in the size of the spleen caused by ageing (5) was not visible in our young case group. Moreover, Spielmann et al. (10) showed spleen length was greater than 12 cm in 31.7% of males (mean spleen length, 11,4±1,7 cm) and in 12.8% of the females (mean spleen length, 10,3±1,3 cm) among tall healthy athletes in the United States. Our results are less than the values reported in Spielmann’s study population. In children, there is an expected increase in splenic length with age and body weight and height (2). In adult populations, some reports demonstrate that splenic volume is not dependent on physical data, since it does not correlate with the patient’s weight, height or BMI. In our study, splenic length and body height, weight and BMI showed no statistically significant correlation. This is in agreement with previous studies based on linear measurement of the spleen in ultrasonograms (7-9). Accessory spleen is a common congenital anomaly. The failure of fusion of splenic tissue results in the formation of accessory spleens. The prevalence of accessory spleen detected by US was calculated as 2.2% in our study group. Mortele et al. (11) reported that accessory spleens are present in 16% of patients undergoing contrast-enhanced abdominal CT. Chen et al. (12) found accessory spleens in 5% (5/103) of patients on US examinations. Gigot et al. (13) demonstrated that preoperative CT, scintigraphy and laparoscopic exploration detected accessory spleens in 25%, 25%, and 75% of patients with hematological disorders, respectively. The detection of accessory spleen is very important since the ultimate goal of surgery for hematological disorders is the complete removal of both the spleen and accessory spleens in order to avoid disease recurrence. It is known that preoperative investigations using CT and scintigraphy are indispensable for ruling out an accessory spleen in those patients for whom splenectomy needs to be done in order to alleviate hematologic disorders. Our study revealed a lower prevalence of accessory spleens in comparison to the literature. Since US is less sensitive in detection of accessory spleens, the lower prevalence can be explained partially by the imaging method. Ultrasound is the method of choice in diagnosis and monitoring of splenomegaly because of its diagnostic efficiency. Standards for normal splenic size have been developed for different areas of the world. This first study designed to evaluate splenic size by US of healthy Turkish men found the average splenic length to be 10,76±1,84 cm. The low prevalence of accessory spleens suggests that US does not allow adequate detection of accessory spleens. In conclusion, this study provides normal standard splenometric data for a healthy Turkish adult male population. This data should be taken into consideration when the diagnosis of splenomegaly is established in Turkish males. 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.

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Serter et al. Sonographic evaluation of spleen size

References 1.

2.

3.

4.

5. 6.

Perlmutter GS. Ultrasound measurements of the spleen. In: Goldberg BB, Kurtz AB, editors. Atlas of Ultrasound Measurements. 1st ed. Chicago, IL: Yearbook Medical Publishers Inc, 1990: 126-38. Rosenberg HK, Markowtz RI, Kolberg H, Park C, Hubbard A, Bellah RD. Normal spleen size in infants and children: sonographic measurements. AJR Am J Roentgenol 1991;157:119-21. Loftus WK, Chow LT, Metreweli C. Sonographic measurement of splenic length: correlation with measurement at autopsy. J Clin Ultrasound 1999;27:71-4. El Sharkawy E, Faris R, Grumbach K, Edelman R, Clemens J, Rao M, Darwish M. Ultrasonographic measurements of the normal liver and spleen among Egyptians 10-50 years old. J Egypt Public Health Assoc 1997;72:257-83. Loftus WK, Metreweli C. Normal splenic size in a Chinese population. J Ultrasound Med 1997;16:345-7. Lamb PM, Lund A, Kanagasabay RR, Martin A, Webb JA, Reznek RH. Spleen size: how well do linear ultrasound measurements correlate with three-dimensional CT volume assessments? Br J Radiol 2002;75:573-7.

Turk J Hematol 2010; 27: 25-8

7.

8.

9.

10. 11. 12.

13.

Bezzera AS, D’Ippolito G, Faintuch S, Szejnefeld J, Ahmed M. Determination of splenomegaly by CT: Is there a place for a single measurement? AJR 2005;184:1510-3. Niederau C, Sonnenberg A, Muller J, Eckenbrecht JA, Scholten T, Fritsch WP. Sonographic measurement of the normal liver, spleen, pancreas and portal vein. Radiology 1983;149:537-42. Frank K, Linhart P, Kortsik C, Wohlenberg H. Sonographic determination of spleen size: normal dimensions in adults with healthy spleen. Ultraschall Med 1986;7:134-7. Spielmann AL, DeLong DM, Kliewer MA. Sonographic evaluation of spleen size in tall healthy athletes. AJR 2005;184:45-9. Mortele KJ, Mortele B, Silverman SG. CT features of accessory spleen. AJR 2004;183:1653-7. Chen MJ, Huang MJ, Chang VH, Wang TE, Wang HY, Chu CH SC Lin, SC Shih. Ultrasonography of splenic abnormalities. World J Gastroenterol 2005;11:4061-6. Gigot JF, Jamar F, Ferrant A, van Beers BE, Lengele B, Pauwels S, Pringot J, Kestens BJ, Gianello P, Detry R. Inadequate detection of accessory spleens and splenosis with laparoscopic splenectomy. A shortcoming of the laparoscopic approach in hematologic diseases. Surg Endosc 1998;12:101-6.

Research Article

29

Non-Hodgkin’s lymphoma with bone involvement: a single center experience with 18 patients Kemik tutulumlu Hodgkin dÕýÕ lenfoma: Onsekiz hasta ile tek merkez deneyimi Filiz Vural, Nur Akad Soyer, PÕnar Özen, Ayhan Dönmez, Serkan OcakçÕ, Güray Saydam, Seçkin ÇaúÕrgan, Murat Tombuloúlu Department of Adult Hematology, Ege University Faculty of Medicine, ûzmir, Turkey

Abstract Objective: Non-Hodgkin’s lymphoma (NHL) of bone is a rare entity. The most common histological subtype is diffuse large B cell lymphoma (DLBCL). The major presenting symptoms are soft tissue swelling, bone pain and pathological fracture. Treatment options are chemotherapy, radiotherapy, surgery, or a combination of these modalities. Materials and Methods: We retrospectively analyzed the 18 patients (11 females, 7 males) with NHL of bone who were diagnosed and treated between 1995-2005. The median age was 56.5 years. The median duration of symptoms was 4.5 months. The bone pain was the first symptom in all patients. Tru-cut biopsy was performed for diagnosis in most of the cases. Diagnosis in five patients (27.8%) required open biopsy. Results: DLBCL (77.8%) was the most common histological type among all patients. Other histological subtypes were anaplastic large cell lymphoma (11.1%), Burkitt-like lymphoma (5.6%) and marginal zone lymphoma (5.6%). According to Ann Arbor staging system, 44.4% of patients were Stage I, 11.1% were Stage II and 44.4% were Stage IV. Bone marrow involvement was determined in four patients (22.2%). All patients except one were treated with anthracycline-containing regimens and eight patients (44.4%) received rituximab combination with chemotherapy. Radiation therapy was performed as the first-line therapy in 9 (50%) patients. The median follow-up was 37 months (range, 2-124 months). Among the 17 patients who achieved complete remission, five (27.8%) relapsed. All patients were still alive. The five-year relapsefree survival was 73.5%. Conclusion: The treatment of bone lymphoma can be planned according to the stage and location of the disease. Although we had a relatively low number of patients, it could be concluded that whether or not radiation therapy is performed, rituximab in combination with systemic chemotherapy has been proven beneficial on survival. (Turk J Hematol 2010; 27: 29-33) Key words: Non-Hodgkin’s lymphoma, bone, lymphoma Received: November 13,2006

Accepted: October 10, 2009

Özet Amaç: Kemik lenfomalarÕ oldukça nadirdir ve en sÕk görülen histolojik alt tip diffüz büyük B-hücreli (DBBHL) non-Hodgkin lenfomadÕr (NHL). Hastalar çoúunlukla kemik ya da yumuýak dokuda ýiýlik, kemik aúrÕsÕ ve patolojik kÕrÕk yakÕnmalarÕ ile baývururlar. Tedavi genellikle cerrahi, radyoterapi, kemoterapi veya bunlarÕn kombinasyonu ýeklindedir. Yöntem ve Gereçler: Bu çalÕýmada, merkezimizde 1995 ve 2005 yÕllarÕ arasÕnda tanÕ alan 18 primer kemik NHL’lÕ (11 kadÕn, 7 erkek) hasta retrospektif olarak analiz edilmiýtir. HastalarÕn tanÕ anÕndaki medyan yaýÕ 56.5 (27-78) yÕl olup, medyan 4.5 (1-36) aylÕk semptom süresinden sonra kliniúe baývurmuýlardÕr. Tüm hastalarda aúrÕ semptomu birinci sÕrada yer almakla birlikte 3 hastada (%16.7) B-semptomlarÕ da saptanmÕýtÕr. TanÕ yöntemi olarak çoúunlukla “tru-cut” biyopsi (%66.7) uygulanÕrken, hastalarÕn %27.8’inde açÕk operasyonla tanÕ konulmuýtur. Address for Correspondence: Filiz Vural, MD, Ege University, Medical Faculty Adult Hematology 35100 ûzmir, Türkiye Phone: +90 0 232 390 32 02 Office: +90 232 390 46 10 E-mail: filiz.vural@ege.edu.tr

30

Vural et al. Non-Hodgkin's lymphoma with bone involvement

Turk J Hematol 2010; 27: 29-33

Bulgular: DBBHL % 77.8 (14 hasta) oranÕnda en sÕk görülen histolojik alt tip olup, %11.1’i anaplastik büyük hücreli lenfoma, %5.6’Õ Burkitt-benzeri, %5.6’Õ marjinal zon lenfoma olarak belirlenmiýtir. Ann Arbor klinik evreleme sistemine göre hastalarÕn %44.4’ü Evre-I, %11,1’i Evre-II, %44.4’ü Evre-IV olarak deúerlendirilmiý, kemik iliúi tutulumu %22.2 hastada saptanmÕýtÕr. Bir hasta dÕýÕnda tümüne CHOP veya benzeri antrasiklin içeren kemoterapi protokolleri uygulanmÕý, 8 hastada (%44.4) Rituximab tedaviye eklenmiýtir ve bu hastalarÕn hiçbirinde relaps izlenmemiýtir. %50 hastada radyoterapi birinci sÕra tedavisi olarak uygulanmÕýtÕr. Medyan 37 (2-124) aylÕk izlemde, tedavilerini tamamlayan ve tam remisyon saúlanan 17 hastanÕn 5’inde (%27.8) relaps geliýmiý olup halen tüm hastalar saúdÕr. 5 yÕllÕk hastalÕksÕz saú kalÕm oranÕ %73.5 olarak belirlenmiýtir. Sonuç: Kemik lenfomalarÕn tedavisi hastalÕúÕn evresi ve lokalizasyonuna göre planlanmalÕdÕr. Radyoterapi uygulansÕn veya uygulanmasÕn, özellikle rituximab ile birlikte sistemik kemoterapi yaýam süresini olumlu yönde etkiler görünmektedir. (Turk J Hematol 2010; 27: 29-33) Anahtar kelimeler: Hematolojik hastalÕklar, çocuk, invazif mantar infeksiyonu Geliý tarihi: 13 KasÕm 2006

Kabul tarihi: 10 AralÕk 2009

Introduction Non-Hodgkin’s lymphoma (NHL) of bone is a rare disease, accounting for approximately 5% of all NHLs and 3% of all primary bone malignancies [1,2]. There is a slight male predominance and most patients are diagnosed in their fourth or fifth decade [3]. Although very rare, bone involvement of NHLs has been determined in some cases, and primary bone lymphoma has also been seen. The most common symptoms in bone involvement are classified as bone pain, soft tissue swelling and pathologic fracture. Long bones are the most commonly affected sites [3]. Histologically, diffuse large B cell lymphomas (DLBCL) are the most prominent subtype [4-6]. Trucut or open biopsy of affected sites is generally required for the diagnosis of bone lymphoma. On full staging evaluation, most of the patients have early stage disease (stage IE-IIE) and the remaining few have advanced stage [3]. Treatment options have been defined as chemotherapy, radiotherapy, surgery, or a combination of these modalities. Traditionally, stage I or II diseases have been treated with radiotherapy [3]. Combinational therapy modalities including CHOP, CHOP-like regimen and rituximab added to these modalities have been found to be the most effective regimens in the last decade [3,7,8]. Herein, we report a retrospective analysis of 18 patients who were diagnosed as NHL with bone involvement; all patients were diagnosed and followed up between 1995 and 2005.

Materials and Methods Between 1995-2005, the records of 18 patients with NHL of bone who were diagnosed and treated in our center were analyzed retrospectively. All patients had a biopsy-proven diagnosis of lymphoma established by experienced hematopathologists. Patients were evaluated according to current World Health Organization classification [9]. Information including patient age, sex, presenting symptoms, sites of involvement, presence of B symptoms, type of diagnostic procedure, histological subtype, presence of bone marrow involvement, stage of the disease, treatment strategies, response rate, relapse pattern, and survival time were ascertained from the hospital records. Pretreatment staging evaluations included medical history, physical examination, laboratory investigation

of complete blood count and lactate dehydrogenase (LDH) level, computed tomography (CT) scan of the neck, thorax and abdomen, and staging bone marrow (BM) biopsy. The clinical stage of the disease was designated by using the Ann Arbor classification system. Patients with lymph node involvement outside the affected site and all patients with other extranodal involvement such as lung, liver, etc. were excluded from the study. B-symptoms were defined as recurrent fever of more than 38°C, night sweats and an unexplained weight loss of more than 10% of actual body weight within 6 months prior to diagnosis. Complete remission (CR) was defined as absence of disease signs and symptoms one month after the completion of treatment. Relapse was defined as the appearance of a new lesion in a patient in CR. Overall survival (OS) was calculated from the time of diagnosis to the time of death or last follow-up. Relapse-free survival (RFS) was measured from the time of diagnosis to the time of treatment failure, relapse/progression or death from lymphoma. Informed consent was obtained from all patients. Statistical Analysis All descriptive and survival analyses were performed using the SPSS software for Windows. Survival curves (OS and RFS) were calculated using Kaplan-Meier method.

Results The median age of the 18 patients was 56.5 years (range, 27-78 years). Male/female ratio was 7/11. The median duration of symptoms was 4.5 months (range, 1-36 months). The median follow-up was 37 months (range, 2-124 months). Bone pain was the first symptom in all patients. Long bones were involved in 6 patients (34%). Three patients (16.7%) had B symptoms at initial presentation. Elevation in serum LDH was detected in 5 (27%) patients. The most common diagnostic procedure applied during the first application was tru-cut biopsy (12 patients, 66.7%). Open biopsy had to be performed in 5 patients (27.8%) and 1 patient was diagnosed with lymph node biopsy. DLBCL (14 patients, 77.8%) was the most common histological type of lymphoma among all patients. Others were diagnosed as anaplastic large cell lymphoma (2 patients, 11.1%), Burkitt-like lymphoma (1 patient, 5.6%) and marginal

Vural et al. Non-Hodgkin's lymphoma with bone involvement

Turk J Hematol 2010; 27: 29-33

31

Table 1. Patients characteristics, treatment modalities and outcomes Patient Age Gender Involved B Diagnostic Histology Stages Radiotherapy Chemotherapy Response Relapse Current sites

symptoms procedure

status

1

37

M

Tibia

No

Tru-cut

DLBCL

I

Yes

R-CT

CR

No

CR

2

40

F

Clavicula

No

Open biopsy

DLBCL

IV

No

R-CT

CR

No

CR

3

63

F

Temporal

No

Tru-cut

DLBCL

IV

Yes

CT

CR

No

CR

4

56

F

Femur

Yes

Tru-cut

DLBCL,

IV

Yes

CT

CR

No

CR

5

49

F

Humerus

No

Tru-cut

DLBCL

IV

No

R-CT

CR

No

CR

6

58

F

Ulna

No

Tru-cut

BURKĂťTT-LIKE

IV

No

CT

CR

No

CR

7

58

M

Rib

No

Tru-cut

DLBCL

I

No

R-CT

CR

No

CR

8

67

M

Spine

Yes

LN biopsy

DLBCL

II

No

R-CT

CR

No

CR

9

52

F

Mandibula

No

Open biopsy

DLBCL

II

No

R-CT

CR

No

CR

10

78

F

Palatinum

No

Tru-cut

MARGINAL

I

Yes

No

CR

Yes

Alive with

11

55

F

Iliac

Yes

Tru-cut

DLBCL

IV

No

CT

CR

Yes

Alive with

ANAPLASTIC

ZONE

disease sites

12

37

M

Spine

No

Open biopsy

DLBCL

IV

Yes

CT

CR

Yes

Alive with disease

13

69

M

Femur

No

Tru-cut

DLBCL

I

Yes

CT

LFU

-

LFU

14

58

M

Ethmoid

No

Tru-cut

DLBCL

I

No

CT

CR

No

CR

15

63

F

Pelvis

No

Open biopsy

DLBCL

IV

No

CT

CR

Yes

CR

16

33

F

Sternum

No

Tru-cut

ALCL

I

Yes

CT

CR

Yes

CR

17

27

M

Acromion

No

Open biopsy

DLCBL

I

Yes

R-CT

CR

No

CR

18

57

F

Femur

No

Tru-cut

DLCBL

I

Yes

R-CT

CR

No

CR

M: Male, F: Female, DLBCL: Diffuse large B cell lymphoma, R: Rituximab, CT: Chemotherapy, ALCL: Anaplastic large cell lymphoma, CR: Complete response, LFU: Lost to follow-up

zone lymphoma (1 patient, 5.6%). According to the Ann Arbor staging system, 8 patients (44.4 %) were Stage I, 2 patients (11.1%) were Stage II and 8 patients (44.4%) were Stage IV. Bone marrow involvement was determined in 4 patients (22.2%). Since we did not have an established and documented clinical protocol, and since there was an extended period between the first and last patient, each patient was treated with different approaches, such as only radiotherapy, chemotherapy, or a combination of both. All patients except one were eventually treated with CHOP or CHOP-like anthracyclinecontaining regimens. Eight patients (44.4%) also received rituximab with chemotherapy in combination. Radiation therapy was the first-line therapy in 9 patients (50%). Only 1 patient was treated with radiotherapy alone. At the time of the latest follow-up, no patient had died and 1 patient was lost to follow-up. The OS was 100% and the RFS was 73.5% at five years (Figure 1). Five (27.8%) of the 17 patients who achieved CR at the end of the treatment relapsed; 3 of these relapsed patients showed resistance to salvage treatments and were alive with disease. The remaining 2 patients were treated with high-dose chemotherapy with autologous stem cell transplantation and achieved a second CR. Patients’ characteristics, treatment modalities and outcomes are shown in Table 1.

Discussion Bone lymphoma is an uncommon disease. Therefore, classification, staging, treatment, and prognosis of bone lymphoma are controversial. In our study, we evaluated 18 patients with NHL involving the bone, retrospectively. In this series, the median age of patients was 56.5 years, and this is similar to that in the literature [3,8-14]. Although a slight male preponderance has been reported in some studies [3,8], we found a female predominance as reported in other studies [10,11,14]. Previous studies have emphasized a predominance of long bone involvement [3]; however, the most recently reported study of a large series with 131 patients with primary bone lymphoma showed that long bones and flat bones were equally affected [15]. Bone pain, soft tissue swelling and pathologic fracture are the most common symptoms of the disease, and B symptoms are reported as in other lymphomas [3,10,12]. Our series also demonstrated an equal involvement of long and flat bones, as in the previous huge series. Diffuse large B cell lymphoma is the most common histological type reported in many studies [3,5,6,8-18]. We also established this data in our study. Gianelli et al. [18] reported a study of 28 cases with primary bone lymphomas and 26 cases with systemic lymphomas involving the bone. Two main histo-

Vural et al. Non-Hodgkin's lymphoma with bone involvement

32

1,0

Turk J Hematol 2010; 27: 29-33

OS

,8

Cum Survival (%)

RFS ,6

,4

,2

0,0 0

12

24

36

48

60

72

84

96

108

120

Month Figure 1. Kaplan-Meier estimate of overall survival and relapse-free survival of all patients

logical types were established: DLBCL and CD30 (+) anaplastic large cell lymphomas. A retrospective study with a large series by Ramadan et al. [15] showed that the most common diagnosis was DLBCL, determined in 79% of all patients. Traditionally, treatment choice of localized stages IE and IIE, according to the Ann Arbor classification, was radiation therapy alone [3]. Recently, combined therapy with chemotherapy was also recommended for localized stages of disease [6,19]. In our series, all patients except one were treated with anthracycline-containing regimens. Radiation therapy were applied to nine (50%) patients. At the end of the treatment, we observed a complete response in 17 patients; one patient was lost to follow-up. The median follow-up was 37 months. Relapses were observed in only five patients during follow-up. The five-year RFS was 73.5% . Charousset et al. [13] reported a retrospective analysis of 22 patients with primary bone lymphoma. Twelve patients were treated with chemotherapy alone and eight patients with combined therapy. The mean five-year Kaplan-Meier survival was 83.3% for the chemotherapy group and 82.5% for the combined therapy group, and the mean follow-up was 48 months. They found no significant OS difference between the chemotherapy and combined therapy groups. Recently, there has been a trend toward better survival with combined therapy with chemotherapy and radiotherapy. There have been studies demonstrating an improvement in survival in patients with primary bone lymphoma with the combined treatment [8,14,16,17]. Most recently, Ramadan et al. [15] retrospectively studied 131 patients with primary bone lymphoma and compared chemotherapy alone and combined modality with radiation. In that study, patients with advanced-stage disease who received chemotherapy plus radiotherapy actually had a poorer outcome compared with chemotherapy alone (10-year OSs were 25% and 56% , respectively). Their results did not support the

routine use of irradiation in the treatment of primary bone lymphoma. They also revealed that the addition of rituximab to chemotherapy improved the outcome. They showed that three-year progression-free survival for patients who received rituximab-CHOP chemotherapy was 88% compared with 52% for those who received standard CHOP chemotherapy without rituximab (p=0.025). In our series, eight patients (44.4%) also received rituximab in addition to chemotherapy; all of them achieved CR and none relapsed. There are other studies in the literature showing the effectiveness of rituximab in improving the outcome in patients with bone lymphoma, especially in patients who had resistant disease [20,21]. Recently, Beal et al. [12] published a study including 82 patients with bone lymphoma. They compared CHOP and CHOP plus rituximab, and demonstrated that rituximab was likely to be effective not only in chemoresistant patients but also in untreated patients. In conclusion, anthracycline-containing chemotherapy regimens, especially with rituximab and radiotherapy, seem to be effective in the treatment of NHL of bone. However, randomized, controlled large prospective clinical trials will determine the role of radiotherapy and combined therapy. 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.

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10. Dürr HR, Müller PE, Hiller E, Maier M, Baur A, Jansson V, Refior HJ. Malignant lymphoma of bone. Arch Orthop Trauma Surg 2002;122:10-6. 11. Lewis VO, Primus G, Anastasi J, Doherty D, Montag AG, Peabody TD, Simon MA. Oncologic outcomes of primary lymphoma of bone in adults. Clin Orthop Relat Res 2003;415:90-7. 12. Beal K, Allen L, Yahalom J. Primary bone lymphoma: treatment results and prognostic factors with long-term follow up of 82 patients. Cancer 2006;106:2652-6. 13. Charousset C, Anract P, Carlioz B, Babinet A, Tomeno B. Primary bone lymphoma. Retrospective immunohistochemical study of 22 cases. Rev Chir Orthop Reparatrice Appar Mot 2002;88:439-48. 14. Horsman JM, Thomas J, Hough R, Hancock BW. Primary bone lymphoma: a retrospective analysis. Int J Oncol 2006;28:1571-5. 15. Ramadan KM, Shenkier T, Sehn LH, Gascoyne RD, Connors JM. A clinicopathological retrospective study of 131 patients with primary bone lymphoma: a population based study of successively treated cohorts from the British Colombia Cancer agency. Ann Oncol 2007;18:129-35. 16. Deshmukh C, Bakshi A, Parikh P, Nair R, Pai V, Gupta S, Shaikh A, Muckaden M, Naresh K, Saikia T. Primary non-Hodgkin’s lym-

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phoma of the bone: a single institution experience. Med Oncol 2004;21:263-7. Stein ME, Kuten A, Gez E, Rosenblatt KE, Drumea K, BenShachar M, Zidan J, Haim N, Epelbaum R. Primary lymphoma of bone - a retrospective study. Experience at the Northern Israel Oncology Center (1979-2000). Oncology 2003;64:322-7. Gianelli U, Patriarca C, Moro A, Ponzoni M, Giardini R, Massimino M, Alfano RM, Armiraglio E, Nuciforo P, Bosari S, Coggi G, Parafioriti A. Lymphomas of the bone: a pathological and clinical study of 54 cases. Int J Surg Pathol 2002;10:257-66. Miller TP, Dahlberg S, Cassady JR, Adelstein DJ, Spier CM, Grogan TM, LeBlanc M, Carlin S, Chase E, Fisher RI. Chemotherapy alone compared with chemotherapy plus radiotherapy for localized intermediate- and high-grade non-Hodgkin’s lymphoma. N Engl J Med 1998;339:21-6. Savage DG, Staron R. Rituximab for bone lymphoma. Ann Intern Med 2001;134:1156-7. Achemlal L, Mikdame M, Nouijai A, Bezza A, El Maghraoui A. Dramatical improvement of chemoresistant bone lymphoma with rituximab. Clin Rheumatol 2006;25:394-5.

Case Report

34

Early detection of pulmonary fungal infection by CT scan in pediatric ALL patients under chemotherapy or in post-transplantation period with primary complaint of chest pain Göúüs aúrÕsÕ ile baývuran pediatrik akut lenfoblastik lösemi olgularÕnda kemoterapi sÕrasÕnda ya da kök hücre nakli sonrasÕ geliýen pulmoner fungal enfeksiyonun bilgisayarlÕ tomografi ile erken tayini üule Ünal1, BarÕý Kuýkonmaz1, Betul Tavil1, Selin Aytaç Elmas1, Duygu Uçkan Çetinkaya1, Mualla Çetin1, Mithat Haliloúlu2, Fatma Gümrük1 1Division

of Pediatric Hematology, Hacettepe University Faculty of Medicine, Ankara, Turkey of Radiology, Hacettepe University Faculty of Medicine, Ankara, Turkey

2Department

Abstract We describe herein four children with acute lymphoblastic leukemia who were diagnosed as pulmonary fungal infection after presenting with chest pain. The plain radiologic evaluations failed to reveal any positive findings, whereas computerized tomography (CT) scanning showed nodular opacification with or without cavitation. This experience suggests that chest pain may be an initial symptom of an invasive fungal infection in patients with leukemia, and CT scan of the lungs should be performed urgently for the early diagnosis and treatment, despite normal plain X-rays. (Turk J Hematol 2010; 27: 34-7) Key words: Leukemia, pediatric, chest pain, fungal infection, pneumothorax Received: May 8, 2009

Accepted: November 11, 2009

Özet Göúüs aúrÕsÕ yakÕnmasÕ ile baývuran dört pediatrik akut lenfoblastik lösemi olgusu pulmoner fungal enfeksiyon tanÕsÕ almÕýtÕr. Düz grafilerinde pozitif bulgu olmayan bu hastalarÕn, bilgisayarlÕ tomografi incelemelerinde pulmoner noduler opasifikasyonlar ve bazÕ hastalarda kavitasyonlar pulmoner enfeksiyon yönünden uyarÕcÕ olmuýtur. Bu deneyimimiz lösemili hastalarda pulmoner invasif enfeksiyonun ilk yakÕnmasÕnÕn göúüs aúrÕsÕ olabilecegine, düz grafiler normal olsa bile bu hastalarda bilgisayarlÕ tomografinin erken yapÕlmasÕ ile fungal enfeksiyonun erken tanÕ ve tedavi edilmesinin önemine dikkat cekmektedir. (Turk J Hematol 2010; 27: 34-7) Anahtar kelimeler: Lösemi, pediatrik, göúüs aúrÕsÕ, fungal enfeksiyon, pnömotoraks Geliý tarihi: 8 MayÕs 2009

Kabul tarihi: 11 KasÕm 2009

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

Turk J Hematol 2010; 27: 34-7

Ăœnal et al. Early detection of pulmonary fungal infection by CT scan in pediatric ALL patient

Figure 1. Peripherally localized nodule at the posterobasal segment of the right lung inferior lobe

Introduction Respiratory tract fungal infection is the most common site of invasive fungal infection in children with hematologic malignancies [1,2]. The clinical onset is not specific and usually includes prolonged fever and new or resistant pulmonary infiltrates that progress despite broad-spectrum antibacterial therapy [3]. Chest pain is a common finding of pulmonary infections of any etiology including viral, bacterial and fungal agents; however, it can also be seen in pulmonary thromboembolism and pneumothorax. Usually, roentgenographic findings do not provide any clues towards identification of the specific pathogen except in a few cases in which the late development of a cavitary lesion can suggest infection with Aspergillus species [4]. Herein, we report our experience in four children with acute lymphoblastic leukemia (ALL). The aim of this report is to emphasize the importance of chest pain as an initial complaint of pulmonary fungal infection and the superiority of pulmonary computerized tomography (CT) scanning over plain chest X-rays in the detection of pulmonary fungal involvement, which enables early diagnosis and treatment in these immunologically compromised patients.

Case Reports Written informed consent was obtained from all patients. Case 1 A 16-year-old male ALL patient relapsed four months after cessation of St. Jude Total XIII treatment protocol [5], and he was placed on BFM-95 early relapse protocol [6]. However, in the 8th month of this treatment, he had a second bone marrow relapse and was started on the FLAG-IDA regimen [7]. Ten days after completion of FLAG-IDA and on

35

Figure 2. Bilateral mosaic pattern and a subpleurally localized nodule in the superior lobe of the right lung compatible with fungal infection

the 17th day of neutropenia, while under fluconazole prophylaxis, he developed bilateral chest pain at inspiration and fever. The pulmonological examination revealed bilateral fine rales. The plain chest X-rays were normal and a pulmonary CT scan was obtained, which revealed bilateral parenchymal nodular lesions compatible with fungal infection, a peripherally localized nodule at the posterobasal segment of the right lung inferior lobe and minimal pleural and pericardial effusion (Figure 1). Liposomal amphotericin-B was initiated (3 mg/kg/day) in addition to concomitant antibacterial treatment, and the dose was later increased to 5 mg/kg/ day. Sputum and blood cultures were negative. By the third week of the antifungal treatment, repeat CT scan showed partial resolution in the nodules. However, the neutropenic period continued since a remission could not be achieved, and the antifungal treatment was continued. Voriconazole was added to the antifungal treatment in the second month of antifungal treatment, since no further resolution could be achieved in pulmonary nodules and serum Aspergillus galactomannan antigen was positive by enzyme-linked immunosorbent assay (ELISA). The patient died in the third month of antifungal treatment due to progression of leukemia and sepsis. Case 2 A 12-year-old male patient who was diagnosed as ALL at the age of six years and received St. Jude Total XIII protocol [5] had bone marrow relapse twice. The initial relapse was seven months after cessation of treatment. Due to family refusal of hematopoietic stem-cell transplantation (HSCT), the patient was placed on BFM REZ 95 protocol [6]. The patient relapsed again towards the end of the maintenance treatment. Following the FLAG-IDA regimen [7], while under fluconazole prophylaxis, he had a complaint of right shoulder pain during inspiration while afebrile and severely neutropenic (WBC was 200/mm3). The

36

Ünal et al. Early detection of pulmonary fungal infection by CT scan in pediatric ALL patient

pulmonological examination was unremarkable. Echocardiographic evaluation and plain chest X-rays were normal; the pulmonary CT scan revealed bilateral mosaic pattern and a subpleurally localized nodule in the superior lobe of the right lung compatible with fungal infection radiologically (Figure 2). Granulocyte colony-stimulating factor (G-CSF) and liposomal amphotericin-B (3 mg/kg) were initiated, in addition to antibacterial treatment. Serum galactomannan antigen, sputum and blood cultures were negative. By the second week of the treatment, chest pain recurred and repeat pulmonary CT showed enlargement of the nodule in the superior lobe of the right lung. The antifungal treatment was intensified by increasing liposomal amphotericin-B dosage to 5 mg/kg and addition of caspofungin. Antifungal treatment could not be stopped for two months since the neutropenic period continued after completion of the chemotherapy protocol. By the end of the second month of treatment, the subpleural nodule was stable and the lung parenchyme was normal. The antifungal treatment was discontinued until the patient recovered from neutropenia. However, he exhibited a central nervous system (CNS) and later a bone marrow relapse and died due to leukemia progression, neutropenic sepsis, pleural effusion, and congestive heart failure four months after termination of antifungal treatment. Case 3 A 13-year-old male with ALL experienced bone marrow relapse 10 months after cessation of St. Jude Total XIII high-risk protocol [5] and underwent HSCT from his HLA-matched sister at age 17; however, he developed grade III acute and then extensive chronic graft-versus-host disease (GVHD) following donor lymphocyte infusion. He was treated with prednisolone, cyclosporine A, mycophenolate mofetil, and extracorporal photopheresis. At month +19, the patient experienced extramedullary relapse in the CNS and the eye. BFM-REZ 95 protocol [6] was initiated. During treatment, he had a complaint of sharp chest pain at inspiration in the third week of induction while under fluconazole prophylaxis, and absolute neutrophil count was 9792/mm3. The pulmonological physical examination was unremarkable. Chest X-rays were normal. Pulmonary CT scan revealed parenchymal bilateral nodular opacities. In the sputum culture, Candida albicans was isolated (resistant to fluconazole). Since he developed acute renal failure in a previous neutropenic fever attack while under empiric liposomal amphotericin-B treatment, caspofungin (1 mg/kg) was chosen. By the third week of antifungal treatment, the repeated CT scan showed that more than 50% of the nodules in the previous CT had disappeared and the remaining nodules had decreased in size. Nevertheless, the patient relapsed five months after diagnosis of pulmonary fungal infection and died on the 18th day of the last relapse due to resistant leukemia progression. Case 4 Acute lymphoblastic leukemia was diagnosed in a 14-yearold male five years before. St Jude Total XIII remission-induction therapy was started [5]. The patient developed sudden rightsided chest pain and dyspnea during induction therapy on the 31st day of hospitalization, while under fluconazole prophylaxis, during the recovery phase from neutropenia (neutrophil count, 1500/mm3). The physical examination revealed decreased breath sounds on the right side. A chest radiograph showed

Turk J Hematol 2010; 27: 34-7

small right-sided pneumothorax with no evidence of pulmonary lesions. A chest tube was inserted and left until resolution of pneumothorax on the fifth day. The follow-up chest X-ray after removal of the chest tube was normal; however, CT scan was done since the etiology of pneumothorax was obscure and revealed nodular opacifications with cavitation. Aspergillus fumigatus was identified from bronchoalveolar lavage fluid culture. Therapy with 5 mg/kg liposomal amphotericin-B was initiated and continued for four weeks, and follow-up CT scan showed regression of the pulmonary lesions. The patient died during the maintenance phase due to intracranial bleeding following an accidental fall in the ninth month of diagnosis.

Discussion Invasive fungal infections are increasingly being observed in immunocompromised patients, particularly in those who are neutropenic, due to the use of aggressive chemotherapeutic regimens. Aspergillus has been described as the leading pathogen in pulmonary cases [1]. Up to 70% of reported patients with invasive pulmonary aspergillosis (IPA) have been reported to suffer from acute leukemia [8]. In invasive aspergillosis, the duration of neutropenia is an accepted risk factor, and recovery from neutropenia is generally associated with a favorable outcome. However, the rapidity of granulocyte recovery may, on rare occasions, be associated with adverse sequelae [9]. Todeschini et al. [9] showed that the risk of pulmonary complications significantly increased when the neutrophil concentration was more than 4500 L-1 on day 5 after deep neutropenia (neutrophil less than 100 L-1). The incidence of aspergillosis is about 4-9% in stem celltransplanted children [10]. Risk factors for aspergillosis in patients with hematological diseases are prolonged (>10 days) severe neutropenia (<500/ml), high-dose steroid treatment, allogeneic SCT, and strong immunosuppressive treatment especially in patients with GVHD after allogeneic HSCT [11]. In our experience, two of the cases (Cases 1 and 2) were severely neutropenic, whereas one was in the recovery period from neutropenia (Case 4) and the other had normal neutrophil count (Case 3) when they developed pulmonary fungal infection. However, the latter patient was severely immunocompromised because of chronic GVHD and was also very prone to infections because of the severe skin involvement of GVHD. In pulmonary fungal infections, the clinical findings are nonspecific, consisting of unremitting fever, cough, chest pain, and dyspnea [8]. The most frequent presenting clinical picture has been described as cough and fever of insidious course [12]. Subira et al. [2] showed that 85% of patients had fever and 80% of those experienced respiratory symptoms as presenting symptoms and none developed pneumothorax. Excluding Case 1, who had fever in addition to chest pain, the only complaint was chest/ shoulder pain during inspiration in the other cases. Interestingly, Case 4 was asymptomatic until pneumothorax developed and he remained symptom-free afterwards. Pneumothorax has been shown to be a severe complication of pulmonary mycetoma that has rarely been reported in patients with hematologic malignancies. Martino et al. [3] found that pneumothorax occurred in six (13%) of 46 episodes of pulmonary mycetoma.

Turk J Hematol 2010; 27: 34-7

Ünal et al. Early detection of pulmonary fungal infection by CT scan in pediatric ALL patient

The diagnostic yield of fungal culture and microscopy from bronchoalveolar lavage fluid or bronchial washings varies considerably in the literature, with an overall sensitivity of 32% and specificity of 99.7% [13]. In Case 3, sputum culture revealed C. albicans resistant to fluconazole. In Case 4, A. fumigatus was identified from bronchoalveolar lavage fluid culture. On the other hand, it has been reported that although the ELISA test does not appear to play a role in the early diagnosis of invasive aspergillosis, it is especially useful for monitoring patients receiving specific therapy [14]. However, detection of Aspergillus species in bronchoalveolar fluid by polymerase chain reaction (PCR) enables early diagnosis of IPA [15]. In a recent study, the radiological appearance of early IPA diagnosed with the aid of PCR testing was found to be correlated with mainly discrete small nodules with halo and focal ground-glass appearance in the thoracic CT scans [16]. Subira et al. [11] showed that chest X-rays are not useful for the early diagnosis of IPA [2]. Thoracic CT scan findings of macronodules, halo signs, and cavitary lesions with an air crescent sign are highly indicative of IPA. In the presented cases, CT scanning showed nodular opacifications with or without cavitation, which were consistent with fungal infection. A CT scan is considered to be one of the most important procedures for early diagnosis of IPA, and several authors have shown that the use of early CT may improve survival [2]. In our series, none of the patients had a diagnostic sign of fungal infection on plain chest X-rays and all had pulmonary fungal infection diagnosis through pulmonary CT scanning. Notably, none of the patients died due to fungal infection but from other causes, and the early detection of fungal infection might have contributed to the improvement in the fungal infection. CT scanning may also be helpful for exclusion of pulmonary thromboembolism in patients with chest pain. All four patients were under fluconazole prophylaxis when pulmonary fungal infection developed. Pulmonary fungal infection is a fulminant and highly fatal infection in patients with hematologic malignancies. The reported mortality rate is approximately 60% when it occurs during chemotherapy-induced neutropenia. Improvement in chance of survival may rely on the early recognition and prompt initiation of antifungal treatment. In conclusion, chest pain may be the first symptom of a pulmonary fungal infection, which may not be detected with chest X-ray. Pulmonary CT scanning may be very helpful in the early diagnosis and treatment of fungal infection in an immunocompromised patient whose plain chest X-rays are normal. 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.

3.

4. 5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

References 1. 2.

Maertens J, Meersseman W, Van Bleyenbergh P. New therapies for fungal pneumonia. Curr Opin Infect Dis 2009;22:183-90. Subira M, Martino R, Franquet T, Puzo C, Altés A, Sureda A, Brunet S, Sierra J. Invasive pulmonary aspergillosis in patients with hematologic malignancies: survival and prognostic factors. Haematologica 2002;87:528-34.

16.

37

Martino P, Girmenia C, Venditti M, Micozzi A, Gentile G, Raccah R, Martinelli E, Rendina E, Mandelli F. Spontaneous pneumothorax complicating pulmonary mycetoma in patients with acute leukemia. Rev Infect Dis 1990;12:611-7. Girmenia C, Donato V, Iori AP, Martino P. Spontaneous pneumothorax in patients with leukemia. Ann Intern Med 1993;118:707-8. Yetgin S, Olgar S, Aras T, Cetin M, Düzova A, Beylergil V, Akhan O, Oúuz O, Saraçbaýi O. Evaluation of kidney damage in patients with acute lymphoblastic leukemia in long-term follow-up: value of renal scan. Am J Hematol 2004;77:132-9. Eckert C, Biondi A, Seeger K, Cazzaniga G, Hartmann R, Beyermann B, Pogodda M, Proba J, Henze G. Prognostic value of minimal residual disease in relapsed childhood acute lymphoblastic leukaemia. Lancet 2001;358:1239-41. Fleischhack G, Hasan C, Graf N, Mann G, Bode U. IDA-FLAG (idarubicin, fludarabine, cytarabine, G-CSF), an effective remission-induction therapy for poor-prognosis AML of childhood prior to allogeneic or autologous bone marrow transplantation: experiences of a phase II trial. Br J Haematol 1998;102:647-55. Gorelik O, Cohen N, Shpirer I, Almoznino-Sarafian D, Alon I, Koopfer M, Yona R, Modai D. Fatal haemoptysis induced by invasive pulmonary aspergillosis in patients with acute leukaemia during bone marrow and clinical remission: report of two cases and review of the literature. J Infect 2000;41:277-82. Todeschini G, Murari C, Bonesi R, Pizzolo G, Verlato G, Tecchio C, Meneghini V, Franchini M, Giuffrida C, Perona G, Bellavite P. Invasive aspergillosis in neutropenic patients: rapid neutrophil recovery is a risk factor for severe pulmonary complications. Eur J Clin Invest 1999;29:453-7. Hovi L, Saarinen-Pihkala UM, Vettenranta K, Saxen H. Invasive fungal infections in pediatric bone marrow transplant recipients: single center experience of 10 years. Bone Marrow Transplant 2000;26:999-1004. Schuster F, Moelter C, Schmid I, Graubner UB, Kammer B, Belohradsky BH, Führer M. Successful antifungal combination therapy with voriconazole and caspofungin. Pediatr Blood Cancer 2005;44:682-5. Serrano-Gonzalez A, Merino-Arribas JM, Ruiz-Lopez MJ, CasadoFlores J. Invasive pulmonary aspergillosis with pneumopericardium and pneumothorax. Pediatr Radiol 1992;22:601-2. Reichenberger F, Habicht J, Kaim A, Dalquen P, Bernet F, Schläpfer R, Stulz P, Perruchoud AP, Tichelli A, Gratwohl A, Tamm M. Lung resection for invasive pulmonary aspergillosis in neutropenic patients with hematologic diseases. Am J Respir Crit Care Med 1998;158:885-90. Ulusakarya A, Chachaty E, Vantelon JM, Youssef A, Tancrède C, Pico JL, Bourhis JH, Fenaux P, Munck JN. Surveillance of Aspergillus galactomannan antigenemia for invasive aspergillosis by enzyme-linked immunosorbent assay in neutropenic patients treated for hematological malignancies. Hematol J 2000;1:111-6. Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F, Denning DW, Donnelly JP, Edwards JE, Erjavec Z, Fiere D, Lortholary O, Maertens J, Meis JF, Patterson TF, Ritter J, Selleslag D, Shah PM, Stevens DA, Walsh TJ.; Invasive Fungal Infections Cooperative Group of the European Organization for Research and Treatment of Cancer; Mycoses Study Group of the National Institute of Allergy and Infectious Diseases. Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin Infect Dis 2002;34:7-14. Brook O, Guralnik L, Hardak E, Oren I, Sprecher H, Zuckerman T, Engel A, Yigla M. Radiological findings of early invasive pulmonary aspergillosis in immune-compromised patients. Hematol Oncol 2009;27:102-6.

Case Report

38

Aplastic anemia presenting as hemophagocytic lymphohistiocytosis Hemofagositik lenfohistositoz olarak baýlayan aplastik anemi Tiraje Celkan Department of Pediatric Hematology-Oncology, ûstanbul University, Cerrahpaýa Faculty of Medicine, ûstanbul, Turkey

Abstract Two unusual cases of hemophagocytic lymphohistiocytosis (HLH) complicating aplastic anemia (AA) are described. Each patient had a history of preexisting acute hepatitis of unknown cause at the time of HLH diagnosis and infection-associated secondary HLH. They developed high fever and pancytopenia. Hemophagocytes were seen in the bone marrow. With steroid (in combination with etoposide and CyA in 1 patient), high fever disappeared and the patients’ liver function gradually recovered. As severe pancytopenia persisted, bone marrow became acellular and AA was diagnosed. Since HLH is known to be able to cause an aplastic bone marrow if untreated for a prolonged time, it is therefore in line that hepatitis-associated AA may also be associated with HLH. Aplastic anemia-associated HLH has been reported rarely, and problems in the diagnostic procedure are discussed. (Turk J Hematol 2010; 27: 38-42) Key words: Hemophagocytosis, aplastic anemia, hepatitis Received: August 14, 2008

Accepted: April 8, 2009

Özet Bu makalede hemofagositik sendrom (HLH) tanÕsÕ alÕp daha sonra aplastik anemi (AA) geliýen 2 hasta sunulmuýtur. Her 2 hastada da HLH tanÕsÕndan önce etyolojisi net olarak konulmamÕý akut hepatit öyküsü vardÕ ve bu enfeksiyon nedeni ile HLH’leri enfeksiyon iliýkili HLH olarak deúerlendirilmiýti. ûki olguda da ateý ve pansitopeni vardÕ. Hemofagositoz iki olguda da kemik iliúi aspirasyon materyalinde gösterilmiýti. Bir olguda steroid diúerinde steroide ek olarak etoposid ve siklosporin (CyA) ile yüksek ateý ve karaciúer fonksiyonlarÕnda düzelme saptanmaya baýlanmÕýtÕ. Tedaviye raúmen aúÕr sitopeninin devam etmesi üzerine kemik iliúinin tekrar deúerlendirilmesi sonucunda hastalara aplastik anemi tanÕsÕ konuldu. Hemofagositik sendrom uzun süreli tedavi edilmediúinde kemik iliúinde aplaziye neden olabilir, bu nedenle hepatite baúlÕ geliýen aplastik anemilerin geliýim sürecinde HLH’ da olabilir. (Turk J Hematol 2010; 27: 38-42) Anahtar kelimeler: Hemofagositoz, aplastik anemi, hepatit Geliý tarihi: 14 Aúustos 2008

Kabul tarihi: 8 Nisan 2009

Address for Correspondence: Assoc. Prof. Tiraje Celkan, Konaklar mah. üebboy sok. Armakent sitesi B2/8 4. Levent / 80620 ûstanbul, Türkiye Phone: +90 212 283 69 22 Office: +90 212 414 30 00 / 21956 E-mail: tirajecelkan@yahoo.com

Celkan T. HLH predicting aplastic anemia

Turk J Hematol 2010; 27: 38-42

Introduction Differential diagnosis of hemophagocytic lymphohistiocytosis (HLH) and aplastic anemia (AA) is very confusing for a clinician who must initiate life-saving therapy with immunosuppressive/immunomodulatory agents in time [1-3]. Brown et al. [4] reported that post-hepatitis AA typically occurs in young, previously healthy males with self-limited but severe liver inflammation with very high serum aminotransferase and bilirubin levels; profound pancytopenia follows several weeks later. We present herein our experience with two adolescent boys ages 11 and 12 years who both presented with hyperbilirubinemia and secondary HLH resulting in AA. Both diseases may have some similar immune-mediated conditions involving the activation of T lymphocytes. Moreover, immunosuppressive therapy with antithymocyte globulin (ATG) and cyclosporine (CyA) is very effective for AA, while intensive immunosuppressive therapy with ATG and CyA might be a useful strategy for steroid-resistant HLH [5]. Aplastic anemia is an immune-mediated disease that is associated with increased apoptosis of bone marrow stem cells. The increase in apoptosis is due to various cytokines that inhibit hematopoiesis, produced by activated T-cells. As is well known, HLH is characterized by a systemic activation of macrophages/histiocytes, which are induced to undergo phagocytosis of hematopoietic elements. This hyperinflammatory condition is associated with genetic inheritance, infection, malignancy, and immune deficiencies. The cardinal symptoms are prolonged fever, cytopenias, hepatosplenomegaly, and hemophagocytosis by activated, morphologically benign macrophages. Biochemical markers include elevated ferritin and triglycerides and low fibrinogen. Impaired function of natural killer (NK) and cytotoxic T-cells is characteristic. Two forms of HLH, primary (genetic) and secondary (acquired), have been reported. Secondary HLH has been reported in association with a variety of conditions [6]. Children with HLH have a higher probability of malignancy, suggesting a possible predisposing role. However, there are only rare reports of an association between AA and HLH. Generally, the diagnosis of HLH is difficult unless there is suspicion. HLH initially may masquerade as a normal infection since all symptoms may be common.

Case Reports Written informed consent was obtained from all patients. Case 1: A 12-year-old boy was followed with hepatitis in a local health center for 40 days. Serologic tests for Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex virus (HSV)-I, HSV-II, hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus (HAV), and parvovirus B-19 were either negative or consistent with prior exposure. As liver enzymes remained elevated (AST 1975 U/L, ALT 2950 U/L) and total bilirubin increased to 4.73 mg/dl, with international normalized ratio (INR) of 4.67, he was referred to our hospital. Persistent

39

pancytopenia was noticed, and he was consulted to the hematology ward after 25 days. The patient met the diagnostic criteria of HLH described by the Histiocyte Society [2]. Bone marrow aspiration was performed and showed an increased number of histiocytes with hemophagocytosis. As he was suspected to have infection-associated secondary HLH, steroid therapy was started (dexamethasone 10 mg/m2 in the beginning and tapered after 2 weeks, planned for a total period of 8 weeks). After steroid, the long-lasting fever disappeared in 2 days and there was an unexpected rapid resolution of hepatosplenomegaly (each 2 cm below the costal margin). Although the clinical manifestations, which were attributed to hypercytokinemia, were controlled with steroid, the pancytopenia persisted even after etoposide was added to the HLH regimen after a 4-week period. Bone marrow biopsy performed after 6 weeks of HLH treatment due to no response of cytopenia revealed AA. He was lost 25 days after ATG treatment due to aspergillus in the lung and pseudomonas bacteremia. Case 2: An 11-year-old boy was followed with persistent hepatitis in a local health center for 2 months until gastrointestinal bleeding was detected. Viral documentation was normal. Liver enzymes remained elevated (AST 2930 U/L, ALT 3460 U/L), INR increased to 6.3, and he was referred to our hospital. Pancytopenia and fever were noticed and he was consulted to the hematology ward after 15 days. The liver biopsy performed before hematology consultation revealed infection. The patient was diagnosed as secondary HLH after an increased number of histiocytes with hemophagocytosis were noticed in the bone marrow aspiration, supported by decreased fibrinogen and increased ferritin, which until the HLH diagnosis were thought to be due to numerous transfusions. He was given dexamethasone at a dose of 10 mg/m2. Fever and splenomegaly disappeared in the first week, but pancytopenia resulting in nearfatal infection persisted. Granulocyte colony-stimulating factor (G-CSF) was added to steroid treatment due to no hematological relief, and bone marrow biopsy was done. His parents refused any treatment for AA and the patient died due to uncontrolled bleeding and sepsis.

Discussion Both of the presented patients had no family history suggestive of familial HLH. The patients died before any mutational analysis or NK cell function studies could be performed. The diagnosis of secondary HLH, therefore, is based on fulfillment of a minimum of five clinical criteria [6]. Gupta et al. [7] mentioned in their commentary that despite the availability of genetic and immunological tests in the diagnosis of HLH, there is a lack of a confirmatory diagnostic test for acute situations. There are also no clear-cut definitions for HLH bone marrow infiltration. Three smears with at least 2 histiocytes demonstrating hemophagocytosis on each are suggested for HLH diagnosis, which were detected in both of our patients [7]. No fatty infiltration was detected during HLH diagnosis. Bone marrow examination showed normal maturation of the three series

40

Celkan T. HLH predicting aplastic anemia

Turk J Hematol 2010; 27: 38-42

Table 1. Findings of patients ( ND: not done) Case 1

Clinical findings Fever Hepatomegaly Splenomegaly Infection Jaundice Bleeding Laboratory findings Hemoglobin gr/dl At the beginning of complainment At the admission our hospital At the diagnosis of HLH At the diagnosis of AA WBC mm3 At the beginning of complainment At the admission our hospital At the diagnosis of HLH At the diagnosis of AA Platelet mm3 At the beginning of complainment At the admission our hospital At the diagnosis of HLH At the diagnosis of AA Reticulocyte AST Ă•u/L At the beginning of complainment At the admission our hospital At the diagnosis of HLH At the diagnosis of AA ALT Ă•u/L At the beginning of complainment At the admission our hospital At the diagnosis of HLH At the diagnosis of AA bilirubin At the beginning of complainment At the admission our hospital Triglyceride mg/dL At the diagnosis of HLH At the diagnosis of AA Fibrinogen g/dL At the admission our hospital At the diagnosis of HLH At the diagnosis of AA Bone marrow aspiration At the diagnosis of HLH At the diagnosis of AA Bone marow biopsy At the diagnosis of AA

Case 2

+

+

2 cm

1.5 cm

2 cm

2 cm

+

+

+

+

-

+

9.6

10.8

5.6

6.8

3.6

8.7( With transfusions)

3.7

7.5

12.600

17.000

5400

6700

3000

1400

2200

1100

178.000

186.000

98.000

79.000

6000

8000

9000

4000

<0.1

0

1108

2089

1975

1790

46

47

44

39

1963

2298

2950

1810

27

45

660

43

4.8

Not known

1.35

1.77

456

780

115

ND

ND

180

80

56

170

ND

hemophagocytic features

hemophagocytic features

Bone marrow failure, hypoplasia

Hypoplasia

devoid of hematopoietic elements,

Diminished hematopoietic elements, increased

fat and reticulum cells

showing largely fatty tissue and reticulum cells

Celkan T. HLH predicting aplastic anemia

Turk J Hematol 2010; 27: 38-42

without dysplastic features or any blast. The most prominent feature was an increase in hemophagocytosing histiocytes. Based on these findings, the patients were diagnosed as HLH and not AA. Then, after 15-21 days of HLH treatment, the bone marrow became severely hypoplastic. However, the hemophagocytic features that were apparent before the HLH regimen had disappeared in both patients. Both patients were diagnosed as having severe bone marrow failure after HLH that was similar to severe AA. Severe pancytopenia with fatty infiltration of the bone marrow is characteristic of AA, but high fever or liver dysfunction is not common if there is no coexisting infection. Hemophagocytic lymphohistiocytosis and AA represent distinct disease entities, but they have some pathologically similar aspects with activation of T lymphocytes. As mentioned in the literature, HLH can be diagnosed before or after AA [8-10]. In our patients, post-hepatitis AA, or HLH associated with an undiagnosed viral infection in a patient with AA, or a coexistence of HLH and AA could be considered in the differential diagnosis. However, no fatty change of the marrow was found in the beginning, and its cellularity was not severely depressed at the time of admission, while hemophagocytic histiocytes were prominent. These findings were characteristic of HLH rather than AA. The diagnosis of AA is often difficult because of the presence of local hemapoietically active spots that can lead to an erroneous assessment before fatty bone marrow. Thus, marrow examinations need to be repeated if pancytopenia persists. The histiocyte count is sometimes increased in AA just like plasma cells, but this is a relative increase and is not associated with hemophagocytosis. Ost et al. [11] reported that the cellularity of the marrow was decreased in some patients and this tissue was severely hypoplastic, even in some children who had not been treated with cytostatic drugs. They also reported that the histological pattern in HLH resembles that of chronic persistent hepatitis. In other words, there is a potential link between AA, HLH and hepatitis-associated AA [11]. Hyperbilirubinemia usually coexists with pancytopenia in HLH, while bone marrow failure secondary to acute hepatitis generally occurs after the hepatitis has resolved if the diagnosis is AA. Immunosuppressive therapy with ATG and CyA, which is a common treatment strategy for severe AA, is sometimes used successfully for HLH, especially for steroid-refractory HLH [5,12]. Impaired function of NK cells and cytotoxic T-cells is characteristic for both genetic and acquired forms of HLH. Frequent triggers are infectious agents, mostly viruses of the herpes group [13,14]. HLH has been identified to be related with genes encoding perforin (PRF1/FHL2), Munc 13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In order to show the relation of AA and HLH, Solomou et al. [15] studied perforin levels in patients with AA. They found that in AA disease with hematopoietic stem cells, which were destructed by activated T-cells and Th1 cytokines, perforin protein levels were very low or absent, and perforin granules were completely diminished [15]. NK cells are the predominant perforin-

41

containing cell type. As a result, NK cell cytotoxicity in these patients was significantly decreased [13]. In one of the HLH subgroups with normal perforin levels, syntaxin-deficient patients, it was shown very recently that their NK cells failed to degranulate when they encountered susceptible target cells [16,17]. Patients with HLH, however, cannot control the hyperinflammatory response which, if untreated, is fatal. Awareness of the clinical symptoms and of the diagnostic criteria of HLH is important to start life-saving therapy with immunosuppressive/ immunomodulatory agents in time. Therefore, we suggest that patients with hepatitis who develop pancytopenia and high liver enzymes that remain elevated for longer than expected should be examined with bone marrow aspiration and biopsy for demonstration of hemophagocytosis or for bone marrow failure resulting in AA. 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.

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8.

9.

Kumakura S, Ishikura H, Kobayashi S. Hemophagocytic syndrome, a possible association with aplastic anemia? Intern Med 2003;42:1262-4. Matsunawa M, Kawakami K, Hisatake J, Suzuki J, Nakamaki T, Hino K, Tomoyasu S. Hepatitis-associated aplastic anemia preceded by a hemophagocytic syndrome-like state. Rinsho Ketsueki 2003;44:1010-4. Omagari K, Ashida R, Oh-I H, Minamino Y, Sasaki O, Ozono Y. Successful treatment with cyclosporine in a case of hemophagocytic syndrome manifesting as severe liver dysfunction. Am J Med Sci 1997;314:403-7. Brown KE, Tisdale J, Barrett AJ, Dunbar CE, Young NS. Hepatitis-associated aplastic anemia. N Engl J Med 1997;336: 1059-64. Kaito K, Otsubo H, Takei Y, Usui N, Kobayashi M. Immunosuppressive therapy with antithymocyte globulin and cyclosporine for prolonged marrow failure after hemophagocytic syndrome. Ann Hematol 2003;82:699-701. Henter JI, Horne A, Aricรณ M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, Janka G. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007;48:124-31. Gupta A, Weitzman S, Abdelhaleem M. Commentary: the role of hemophagocytosis in bone marrow aspirates in the diagnosis of HLH. Pediatr Blood Cancer 2008;50:192-4. de la Serna FJ, Lopez JI, Garcia-Marcilla A, Ortiz-Conde MC, Mestre MT. Hemophagocytic syndrome causing complete bone marrow failure. Report of an extreme case of a reactive histiocytic disorder. Acta Haematol 1989;82:197-200. Stephan JL, Galambrun C, Pozzetto B, Grattard F, Bordigoni P. Aplastic anemia after Mycoplasma pneumoniae infection: a report of two cases. J Pediatr Hematol Oncol 1999;21:299-302.

42

Celkan T. HLH predicting aplastic anemia

10. Yashima A, Narigasawa Y, Ishida Y, Uchiyama T, Oyake T, Enomoto S, Kaneko J, Ono Y, Sugawara T, Numaoka H, Shimosegawa K, Murai K, Itoh S, Ito T, Kuriya S. Hemophagocytic syndrome due to miliary tuberculosis in the course of aplastic anemia. Rinsho Ketsueki 1998;39:392-7. 11. Ost A, Nilsson-Ardnor S, Henter JI. Autopsy findings in 27 children with haemophagocytic lymphohistiocytosis. Histopathology 1998;32:310-6. 12. Erduran E, Gedik Y, Sen Y, Yildiran A. Successful treatment of reactive hemophagocytic syndrome with cyclosporin A and intravenous immunoglobulin. Turk J Pediatr 2000;42:168-70. 13. Janka GE. Hemophagocytic syndromes. Blood Rev 2007; 21:245-53. 14. Gurgey A, Secmeer G, Tavil B, Ceyhan M, Kuskonmaz B, Cengiz B, Ozen H, Kara A, Cetin M, Gumruk F. Secondary hemophagocytic lymphohistiocytosis in Turkish children. Pediatr Infect Dis J 2005;24:1116-7.

Turk J Hematol 2010; 27: 38-42

15. Solomou EE, Gibellini F, Stewart B, Malide D, Berg M, Visconte V, Green S, Childs R, Chanock SJ, Young NS. Perforin gene mutations in patients with acquired aplastic anemia. Blood 2007;109:5234-7. 16. Bryceson YT, Rudd E, Zheng C, Edner J, Ma D, Wood SM, Bechensteen AG, Boelens JJ, Celkan T, Farah RA, Hultenby K, Winiarski J, Roche PA, Nordenskjöld M, Henter JI, Long EO, Ljunggren HG. Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients. Blood 2007;110:1906-15. 17. Horne A, Ramme KG, Rudd E, Zheng C, Wali Y, al-Lamki Z, Gürgey A, Yalman N, Nordenskjöld M, Henter JI. Characterization of PRF1, STX11 and UNC13D genotype-phenotype correlations in familial hemophagocytic lymphohistiocytosis. Br J Haematol 2008;143:75-83.

Case Report

43

Unusual cytochemical reactivity for toluidine blue in granular acute lymphoblastic leukemia: a report of two rare cases Granüler akut lenfoblastik lösemide toluidin mavisine yönelik olaúandÕýÕ sitokimyasal reaktivite: ûki nadir olgu raporu Rishu Agarwal1, Ritu Gupta1, Sameer Bakhshi2, Atul Sharma2 1Laboratory Oncology Unit and 2Department of Medical Oncology, Dr. B.R. Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India

Abstract Azurophilic granulation of blasts is a feature of acute myeloid leukemia (AML). Granular acute lymphoblastic leukemia (ALL) may mimic AML due to the presence of cytoplasmic granules in lymphoblasts, but cytochemistry and immunophenotyping are helpful in making the correct diagnosis. Toluidine blue (TB) is a metachromatic dye, which stains basophils and myeloid blasts that exhibit basophilic differentiation. Reactivity for TB has not been described in lymphoblasts. We herein report two cases of granular ALL with blasts exhibiting reactivity for TB that caused diagnostic dilemma. Immunophenotyping and cytogenetic studies were helpful in making a correct diagnosis. This report of two rare case highlight the reactivity of lymphoblasts with TB not hitherto described and the importance of a detailed diagnostic work-up in acute leukemia. (Turk J Hematol 2010; 27: 43-5) Key words: Granular ALL, basophilic leukemia, toluidine blue, azurophilic granules Received: November 26, 2008

Accepted: April 10, 2009

Özet BlastlarÕn azurofilik granülasyonu, akut miyeloid löseminin (AML) bir özelliúidir. Granüler akut lenfoblastik lösemi (ALL), sitoplazmik granüllerin lenfoblastlardaki varlÕúÕna baúlÕ olarak AML’ye benzeyebilir, ancak histokimya ve immünofenotipleme doúru tanÕnÕn konulmasÕnda yardÕmcÕdÕr. Toluidin mavisi (TB) bir metakromatik boya olup, bazofilik diferansiyasyon sergileyen bazofiller ve miyeloid blastlarÕ boyar. TB'ye yönelik reaktivite, lenfoblastlarda tanÕmlanmamÕýtÕr. Bu yayÕnda, tanÕ ikilemine neden olan TB’ye yönelik reaktivite sergileyen blastlar ile granüler ALL’ye yönelik iki vaka rapor edilmektedir. ûmmünofenotipleme ve sitogenetik çalÕýmalar, doúru tanÕnÕn konulmasÕnÕ saúlamÕýtÕr. Bu iki nadir vaka raporu ile ýimdiye dek tanÕmlanmamÕý TB’li lenfoblastlarÕn reaktivitesinin ve akut lösemide ayrÕntÕlÕ tanÕsal tetkikin öneminin altÕ çizilmektedir. (Turk J Hematol 2010; 27: 43-5) Anahtar kelimeler: Granüler ALL, bazofilik lösemi, toluidin mavisi, azurofilik granüller Geliý tarihi: 26 KasÕm 2008

Kabul tarihi: 10 Nisan 2009

Address for Correspondence: Ritu Gupta, MD, Assistant Professor of Laboratory Oncology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India Phone: 91-11-26588958 E-mail: drritugupta@gmail.com

44

Agarwal et al. Granular ALL with TB positivity

Introduction Granular acute lymphoblastic leukemia (ALL) is a morphological variant of ALL and accounts for approximately 2-7% of all cases of acute leukemia. Cytochemistry and immunophenotyping help to differentiate granular ALL from acute myeloid leukemia (AML). Toluidine blue (TB) is a basic dye that helps in identification of myeloid blasts showing differentiation towards basophils, which may be seen in acute basophilic leukemia, basophilic blast crisis of chronic myeloid leukemia (CML) and AML with basophilia. Reactivity for TB is not seen in lymphoblasts. The present report describes an unusual cytochemical staining pattern in lymphoblasts of granular ALL and highlights the importance of immunophenotyping and cytogenetic studies for correct diagnosis in such a scenario.

Case Reports Informed consent was taken from patients for administering any form of treatment. Case 1 A 14-year-old boy was admitted with cough and weight loss of two months’ duration. On physical examination, there was no lymphadenopathy, organomegaly or symptoms of hyperhistaminemia. His hemoglobin level was 11.6 g/dl, platelet count 1.86 x 109 /L and total leukocyte count (TLC) 58x109/L, which included 8% blasts, 51% myelocytes and metamyelocytes, 24% neutrophils, 16% lymphocytes, and 1% basophils. Bone marrow was cellular with 90% blasts, which had round to oval nuclei, 1-2 nucleoli and moderate amount of cytoplasm. Prominent 5-12 coarse non-refractile intracytoplasmic granules were seen in 35% of the blasts (Figure 1A). The blasts did not stain with myeloperoxidase but showed metachromatic granules when stained with TB (Figure 1B). On flow cytometric immunophenotyping, the blasts were positive for CD34, CD45, CD10, CD19, and CD22 and negative for CD3, CD5, CD7 CD13, CD33, CD14, CD42a, CD61, and glycophorin A. Cytogenetic studies did not reveal any chromosomal abnormality. A diagnosis of B-lineage granular ALL was made. The patient was treated with MCP-841 protocol of NCI (pred-

Turk J Hematol 2010; 27: 43-5

nisone, L-asparaginase, vincristine and daunorubicin), but the disease was resistant, remission was not achieved, and the patient died due to intracranial bleed. Case 2 A 50-year-old male presented with fatigue, weakness and fever of two months’ duration. The patient was pale but had no lymphadenopathy, organomegaly or symptoms of hyperhistaminemia. His hemoglobin level was 3.6 g/dl, platelet count 23x109 /L and TLC 17.8x109/L, which included 89% blasts, an occasional myelocyte, 10% lymphocytes, and 1% neutrophils. Bone marrow was cellular with near total replacement by blasts, which had round to oval nuclei, variable nucleoli and moderate amount of cytoplasm. Prominent coarse non-refractile intracytoplasmic granules were seen in 22% of the blasts. The blasts did not stain with myeloperoxidase but showed metachromatic granules when stained with TB. On flow cytometric immunophenotyping, the blasts were positive for CD34, CD45, CD10, CD19, CD22, and CD33 and negative for CD3, CD5, CD13, and CD14. Cytogenetic studies showed normal male karyotype. A diagnosis of B-lineage granular ALL was made. Treatment was initiated with MCP-841 protocol but the patient did not achieve complete remission and died during the induction phase of treatment.

Discussion Blasts with coarse azurophilic granules are seen in granular ALL and myeloid leukemia with basophilic differentiation, i.e. acute basophilic leukemia and blast crisis of CML with basophilic differentiation. Granular ALL, morphological variant of ALL, is characterized by presence of at least three clearly defined azurophilic granules (Each 0.5 microns or greater in diameter) in more than 5% blasts and are negative for myeloperoxidase [1]. On immunophenotyping, blasts of granular ALL usually have a precursor B-cell phenotype, although granular ALL with T-cell type has also been reported [1,2]. Blasts of myeloid leukemia with basophilic differentiation may not exhibit reactivity for myeloperoxidase and express myeloid markers such as CD33, CD13 and CD117 along with CD9 and CD25, which are usually associated with basophilic lineage of cells [3,4].

Figure 1. Peripheral blood smear showing blasts with coarse granules (A, Jenner & Giemsa, 1000X), which exhibit metachromasia with toluidine blue (B, 1000X)

Agarwal et al. Granular ALL with TB positivity

Turk J Hematol 2010; 27: 43-5

Toluidine blue is a metachromatic dye, which stains basophils and is helpful in identifying the basophilic differentiation in myeloid blasts. In the two cases described here, coarse azurophilic granules were present in the blasts, both in peripheral blood smear and bone marrow, which were negative for myeloperoxidase and positive for TB. On the basis of morphology and cytochemistry, the possibility of acute basophilic leukemia and basophilic blast crisis of CML was considered. The B-cell immunophenotype of blasts, absence of Philadelphia chromosome on conventional cytogenetic studies and bcr-abl transcripts on reverse transcriptase-polymerase chain reaction (RT-PCR) were consistent with a diagnosis of B-lineage ALL. In one of the cases, blasts were positive for CD33, but aberrant expression of myeloid markers as CD13 and CD33 is well described in ALL [5]. Toluidine blue reactivity has not been described in lymphoblasts and was the main cause of the diagnostic dilemma in our cases on morphological evaluation. TB reacts with acid mucopolysaccharides of basophils to give metachromatic staining, but the exact mechanism of its reactivity in lymphoblasts, as seen here, is not known. Lysosomes are thought to be the main content of granules in granular ALL and since acid mucopolysaccharides are present in the lysosomes, it is possible that the metachromatic staining observed in our cases was due to the reaction of TB with the acid mucopolysaccharides of the lysosomes [6-10]. This may be one of the possible explanations of TB reactivity in granular ALL, but further studies may be required to elucidate the exact mechanism. Prognostic value of granular ALL is controversial, with some studies demonstrating no prognostic significance and others attributing it a poor prognosis [6,8,11]. Neither of the patients described here achieved remission and both died despite the best supportive care. Due to the very small number of patients, it remains uncertain at present if patients with granular ALL with TB-positive blasts form a distinct subset with poor prognosis. To the best of our knowledge, reactivity of granular lymphoblasts to TB has not been described before. It is being reported here to create awareness about the existence of this phenomenon and the need for immunophenotyping in such cases.

45

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.

Cerezo L, Shuster JJ, Pullen DJ, Brock B, Borowitz MJ, Falletta JM, Crist WM, Head DR. Laboratory correlates and prognostic significance of granular acute lymphoblastic leukemia in children. Am J Clin Pathol 1991;95:526-31. 2. Tsoi WC, Lai HDH, Feng CS. T-acute lymphoblastic leukemia with cytoplasmic granules. Am J Hematol 1997;56:193-6. 3. Duchayne E, Demur C, Rubie H, Robert A, Dastugue N. Diagnosis of acute basophilic leukemia. Leuk Lymphoma 1999;32:269-78. 4. Gupta R, Jain P, Anand M. Acute basophilic leukemia. Am J Haematol 2004;76:134-8. 5. Brunning RD, Borowitz M, Matutes E, Head D, Flandrin G, Swerdlow SH, Bennett JM. Precursor B lymphoblastic leukemia / lymphoblastic lymphoma. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, WHO-Classification of tumours, editors. Pathology and Genetics of Tumors of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press, 2001: 112-3. 6. Rajnoldi AC, Invernizzi R, Biondi A, Banfi P, Zoia A, Fazio PD, Polli. Biological and clinical features of acute lymphoblastic leukemia with cytoplasmic granules or inclusions. Br J Haematol 1989;3:309-14. 7. Fradera J, Garcia EV, White JG. Acute lymphoblastic leukemia with unusual cytoplasmic granulation. Blood 1986;68:406-11. 8. Darbyshire PJ, Lilleyman JS. Granular acute lymphoblastic leukemia of childhood. J Clin Pathol 1987;40:251-3. 9. Hay CRM, Barnett D, James V, Woodcock BW, Brown MJ, Lawrence ACK. Granular acute lymphoblastic leukemia in adults. Eur J Haematol 1987;39:299-305. 10. Schwarzinger L, Fodinger M, Scherrer R, Woltz M, Mannhatter Ch, Speiser W. Hypergranular acute lymphoblastic leukemia. Ann Hematol 1993;67:301-3. 11. Invernizzi R, Rosanda C, Basso G, Conter V, di Cataldo A, Fenu S, Forni M, Frappampina V, Granchi D, Locatelli F, Miano C, Santostasi T, Cantu-Rajnoldi A. Granular acute lymphoblastic leukemia in children. "Aieop Cooperative Group for Cytology of Acute Leukemias". Haematologica 1992;77:30-4.

Images in Hematology

46

Primary cutaneous diffuse large B-Cell lymphoma, leg type Primer kutanöz diffüz büyük B hücreli lenfoma, bacak tipi

Ankit Shrivastav1, Bhaskar Mitra2, Krishnendu Mukherjee2 1Department 2Department

of Internal Medicine, Institute of Post Graduate Medical Education Research and Kolkata, India of Pathology, Institute of Post Graduate Medical Education Research and Kolkata, India

A 55-year-old male presented with a superficial mass over the right leg that had gradually progressed over the last month. The lesion was pruritic and painful. It was firm, indurated, shiny, erythematous, and raised on the surface (Figure 1). There was no lymphadenopathy or hepatosplenomegaly. All routine parameters including bone marrow biopsy were normal. An incision biopsy was taken from the lesion, which revealed superficial and deep perivascular, nodular and interstitial, bottom-heavy infiltrate comprised predominantly of lymphocytes, which showed a marked crush artifact (Figures 2-4). There were some preserved lymphocytes with enlarged, slightly hyperchromatic nuclei with basophilic nuclei. The epidermis and adnexal structures were spared. Immunological staining revealed a predominance of CD20-

positive B-cells, which were bcl-2-positive. A diagnosis of primary cutaneous diffuse large B-cell lymphoma, leg type was made. The patient was treated with the cyclophosphamide, adriamycin, vincristine, prednisone (CHOP) regimen. Rituximab was not added to the regimen as the patient could not afford it. He did not respond to the treatment and was lost to follow-up.

Figure 2. Skin biopsy showing dense, diffuse infiltrates of large cells, predomiFigure 1. Indurated swelling over the right leg

nantly lymphocytes, in the entire dermis and subcutaneous tissue giving a bottomheavy appearance. The epidermis and adnexal structures were spared (hematoxylin & eosin [H&E], X10)

Address for Correspondence: Ankit Shrivastav, MD, 242, Ajc Bose Road 700020 Kolkata, India Tel: +919883252055 E-mail: ankit.med@gmail.com

Turk J Hematol 2010; 27: 46-7

Shrivastav et al. Primary cutaneous diffuse large B-Cell lymphoma, leg type

47

Figure 3-4. Skin biopsy showing dense, diffuse infiltrates of lymphocytes in the entire dermis and subcutaneous tissue giving a bottom-heavy appearance. The epidermis and adnexal structures were spared (H&E, X40)

Primary cutaneous large B-cell lymphomas (leg type) (PCBLS) are diffuse large cell B-cell lymphomas with predominance or confluent sheets of centroblasts and immunoblasts. Characteristically, they present with tumorous skin lesions on the (lower) legs but uncommonly can arise at other sites as well1. Immunological staining shows monotypic surface immunoglobulins and/or cytoplasmic immunoglobulin. Neoplastic cells are CD20- and bcl-2- positive. An exact classification is often not possible. PCBLS represent approximately 5% of cutaneous lymphomas2. It has been proposed that most cases of large B-cell lymphoma of the leg represent large-cell lymphomas originating from the lymphocytes of the germinal center. Primary cutaneous diffuse large B-cell lymphoma, leg type must be differentiated from anaplastic large-cell lymphoma and non-lymphoid tumors such as cutaneous metastases. The clinicopathologic pattern, together with immunohistochemical and molecular features of PCBLS, allows the correct diagnosis in most of the cases.

Informed consent was obtained from the patient. 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. Vermeer MH, Geelen FA, van Haselen CW, van Voorst Vader PC, Geerts ML, van Vloten WA, et al. Primary cutaneous large B-cell lymphomas of the legs. A distinct type of cutaneous B-cell lymphoma with an intermediate prognosis. Arch Dermatol 1996;132:1304-8. 2. Zackheim HS, Vonderheid EC, Ramsay DL, LeBoit PE, Rothfleisch J, Kashani-Sabet M. Relative frequency of various forms of primary cutaneous lymphomas. J Am Acad Dermatol 2000;43:793-6.

Letter to the Editor

48

A note on oseltamivir treatment in a boy with G6PD deficiency G6PD eksikliúi olan bir erkek çocukta oseltamivir tedavisine iliýkin bir not Nejat Akar Pediatric Hematology Department, Mesa Hospital, Ankara, Turkey

To the Editor, During the pandemic influenza A, a five-year-old boy was admitted to the hospital with the complaints of high fever, cough and sneezing. Influenza virus analysis from the nasopharyngeal discharge was positive for influenza A virus. The patient previously had an acute hemolytic attack after eating fava beans, and glucose-6-phosphate dehydrogenase (G6PD) deficiency was later established. As he had proven influenza A virus, oseltamivir was started at a dose of 45 mg twice daily for five days. He was completely normal after treatment. No hemolytic anemia occurred during oseltamivir treatment. G6PD deficiency is an X-linked inherited disease. A child with G6PD deficiency is clinically and hematologically normal during his life, but in a situation of ‘oxidative challenge’, caused by either fava beans or by certain drugs, acute hemolytic ane-

mia occurs. Thus, several drugs should be avoided in G6PDdeficient subjects. Oseltamivir has not been tested on G6PD-deficient subjects, and G6PD-deficient individuals have been excluded from testing in ongoing studies. It is well known that G6PD deficiency is not rare in the Turkish population [1]. This case is reported in order to share experience, since there has been no report on the clinical usage of oseltamivir or any safety data regarding its usage in G6PDdeficient subjects. More data are necessary to reach a conclusion regarding the safety of oseltamivir therapy in G6PD-deficient individuals.

References 1. Altay Ç, Gümrük F. Red cell glucose-6-phosphate dehydrogenase deficiency in Turkey. Turk J Hematol 2008;25:1-7.

Address for Correspondence: Prof. Nejat Akar, Koru Mah. 606. Sok. Uyum Sitesi 18. Çayyolu, Ankara,Türkiye Phone: +90 312 241 39 80 E-mail: akar@medicine.ankara.edu.tr

Letter to the Editor

49

Malignancy-associated hemophagocytosis in children Çocuklarda malignite ile iliýkili hemofagositoz

Sema Vural1, Ela Erdem1, Serap Karaman1, Tiraje Celkan2, Nedim Polat3, Öner Doúan4 1Department of Pediatric Hematology/Oncology, üiýli Etfal Education and Research Hospital Clinic of Pediatrics, Istanbul, Turkey 2Department

of Pediatric Hematology/Oncology, Istanbul University Cerrahpaýa School of Medicine, Istanbul, Turkey of Pathology, üiýli Etfal Education and Research Hospital, Istanbul, Turkey 4Department of Pathology, Division of Hematopathology, Istanbul University School of Medicine, Istanbul, Turkey 3Department

To the Editor, Hemophagocytic syndrome (HPS) is a clinical entity characterized by prolonged fever, splenomegaly and cytopenias. Secondary HPS can be related to underlying conditions including malignancies. HPS in association with malignant diseases, especially lymphoma, is a well-known entity in adults but is rare in children [1-5]. We report three cases of malignancies associated with HPS according to the criteria of hemophagocytic lymphohistiocytosis (HLH)2004 [6]. All the clinical and laboratory findings of the patients are summarized in Table 1. Case 1 An 11-year-old patient was admitted with cervical lymphadenopathy. Bone marrow aspiration was normal, and the patient was diagnosed as anaplastic large cell lymphoma by lymph node biopsy. One week after initial low-dose chemotherapy, fever, hepatosplenomegaly, bicytopenia, and clinical findings of disseminated intravascular coagulopathy (DIC) developed. In spite of supportive treatment, clinical findings worsened. Because of myelosuppression, which was unexpected from chemotherapy, hyperbilirubinemia, hypertriglyceridemia, and hypofibrinogenemia, the patient was thought to have HPS. Bone marrow aspirations showed hemophagocytosis and chemotherapy was restarted. Clinical and laboratory findings had improved in 10 days.

Case 2 A 15-year-old patient was hospitalized with swellings over her face and neck, fever, hepatosplenomegaly, pancytopenia, and hypertriglyceridemia. She was diagnosed as subcutaneous panniculitis-like T-cell lymphoma by skin biopsy. Hemophagocytosis was found in bone marrow aspiration and inside tumor lesions. During the first week of treatment, the fever resolved, and regression of lesions and improvement in hematological findings were noted. Case 3 A 10-year-old patient admitted with fever, purpuric lesions, hepatomegaly, lymphadenopathies, and pancytopenia. Skin biopsy was compatible with Kaposi sarcoma, and inguinal lymph node biopsy was reported as Castleman disease. Bone marrow aspiration was hypocellular. In the second week of chemotherapy, there was an improvement in clinical and laboratory findings. Treatment was ceased for two weeks because the drug could not be provided. Fever, hepatomegaly and pancytopenia were observed again with addition of hyperferritinemia. Chemotherapy was restarted. Repeated bone marrow aspirations revealed dysplastic changes and hemophagocytosis. Hemophagocytosis disappeared in bone marrow in the first month of treatment. Unfortunately, her condition failed to improve and she died due to respiratory failure. In conclusion, although rare, HPS may manifest secondary to malignancies. In hemophagocytosis, at

Address for Correspondence: Sema Vural, MD, üiýli Etfal Eúitim ve AraýtÕrma Hastanesi, Çocuk 1 Kliniúi, ûstanbul, Türkiye Phone: +90 212 231 22 09 E-mail: sdvural@yahoo.com

50

Vural et al. Malignancy hemophagocytosis

Turk J Hematol 2010; 27: 49-50

Table 1. Clinical and laboratory manifestations of three patients with concomitant malignancies Clinical/Laboratory Findings Patient 1 Patient 2

Patient 3

Fever

+

+

+

Splenomegaly

+

+

-

Hepatomegaly

+

+

+

Lymphadenopathy

+

-

+

Rash

-

-

-

Neurological signs

-

-

-

Anemia

+

+

+

Thrombocytopenia

+

+

+

Neutropenia

+

+

+

Hypertriglyceridemia

+

+

-

Hypofibrinogemia

+

-

-

Hyperbilirubinemia

+

-

-

Hyperferritinemia

Not done

-

+

Hemophagocytosis in bone marrow

+

+

+

Hemophagocytosis in other parts

-

+

-

the time of diagnosis or during the treatment of malignancies, it is not easy to establish whether clinical findings are due to malignancy, chemotherapy or hemophagocytosis. Thus, if there is an unexpected or prolonged myelosuppression in oncology patients presenting with fever and DIC findings, HPS should be included in the differential diagnosis. Conflict of interest No author of this paper has a conflict of interest, includingspecific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included in this manuscript.

References 1. Janka G, Elinder G, Imashuku S, Schneider M, Henter JI. Infectionand malignancy-associated hemophagocytic syndromes: secondary hemophagocytic lymphohistiocytosis. Hematol Oncol Clin North Am. 1998; 12: 435-44.

2. Janka GE. Haemophagocytic lymphohistiocytosis. Hematology. 2005; 10: 104-7. 3. Wong KF, Chan JK. Reactive hemophagocytic syndrome-a clinicopathologic study of 40 patients in an Oriental population. Am J Med. 1992; 93: 177-80. 4. Takahashi N, Chubachi A, Kume M, Hatano Y, Komatsuda A, Kawabata Y, Yanagiya N, Ichikawa Y, Miura AB, Miura I. A clinical analysis of 52 adult patients with hemophagocytic syndrome: the prognostic significance of the underlying diseases. Int. J. Hematol. 2001; 74: 209-13. 5. Miyahara M, Sano M, Shibata K, Matsuzaki M, Ibaraki K, Shimamoto Y, Tokunaga O. B-cell lymphoma-associated hemophagocytic syndrome: clinicopathological characteristics. Ann Hematol. 2000; 79: 378-88. 6. Henter JI, Horne A, Aricò M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, Janka G. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer. 2007; 48: 124-31.

Letter to the Editor

51

Leukocytosis, thrombocytosis and hypercalcemia as a triple paraneoplastic syndrome in a patient with squamous cell carcinoma of the renal pelvis

Renal yassÕ hücreli karsinomda multiple pareneoplatik sendrom olarak lökositoz, trombositoz ve hiperkalsemi Hasan üenol Coýkun, Ayýegül KargÕ, Mustafa Özdoúan, Hakan Bozcuk, Burhan Savaý Department of Medical Oncology, Akdeniz University School of Medicine, Antalya, Turkey

To the Editor, Squamous cell carcinoma (SCC) of the renal pelvis is a rare malignancy [1]. Paraneoplastic syndromes are common; however, multiple paraneoplastic syndromes are rarely observed. We describe a case with triple paraneoplastic syndrome including hypercalcemia, leukocytosis and thrombocytosis as multiple hematological paraneoplastic syndromes in a patient with renal pelvis SCC. A 58-year-old male patient was admitted to clinic because of right flank pain, nausea, vomiting, and constipation. He underwent a right nephrectomy four months before and diagnosis of SCC of the renal pelvis was established. Invasion of the renal capsule and adipose tissue was seen with negative surgical border. On admission, he was clinically deteriorated. He had anemia (hemoglobin 9 mg/dl), leukocytosis (WBC 57.6 x 109/L) and thrombocytosis (576 x 109/L). Leukocyte differential count showed 85% neutrophil, 20% lymphocyte, 7% monocytes, and 1% eosinophil, respectively. Serum creatinine, calcium and parathyroid hormone levels were 2.6 g/dl (range: 0.5-1.2 mg/dl), 14 mg/dl (range: 8.8-10.8 mg/dl) and 6.47 pg/ml (range: 15-65 pg/ml), respectively. Abdominal ultrasound revealed hypoechogenic lesions in the left renal region, multiple metastases of the liver. Fine needle aspiration from the mass revealed SCC. Treatment to correct hypercalcemia was started with

saline hydration, furosemide and, zoledronate. Thorax computerized tomography and bone scan were negative for metastasis. Peripheral blood smear, bone marrow aspiration and biopsy showed no pathologic finding regarding any hematologic disorder, bone marrow metastasis or infectious disease. Unfortunately, we did not perform leukocyte alkaline phosphatase (LAP) stain or analysis of Philadelphia chromosome. Cisplatin and etoposide combination regimen was administered to the patient. Performance status of the patient decreased after two cycles of chemotherapy. Objective response was not observed. The patient died in four months with progressive disease. Hypercalcemia was under control with bisphosphonate but hematological findings continued. Squamous cell carcinoma of the renal pelvis is rare and accounts for less than 1% of urological malignancies [1]. It has poor prognosis with a median survival of 3.5 months. Coexisting urinary stone and carcinoma ranges from 18 to 100% [2]. Paraneoplastic syndrome has a very wide spectrum, from simple clinical events such as fever to complex clinical syndrome. Many different mechanisms may play a role in the pathogenesis of paraneoplastic syndrome, such as cytokines, hormones, and autoimmunity, etc. Hypercalcemia is more common in squamous histology than other histopathological subtypes [3]. Hypercalcemia has two different mechanisms: one is paraneoplastic syndrome and the other is involvement of bone. Paraneoplastic

Address for Correspondence: Assoc. Prof. Hasan üenol Coýkun, Department of Medical Oncology, Akdeniz University School of Medicine, Antalya, Turkey Phone: +90 242 249 67 37 E-mail: hscoskun@yahoo.com

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Coýkun et al. Multiple hematological paraneoplastic syndrome

hypercalcemia is closely associated with parathyroid hormonerelated peptide and interleukin-6 [4]. Hematologic paraneoplastic syndromes have a wide spectrum, from coagulopathy to abnormality of blood cells, granulocyte colony-stimulating factor (G-CSF) is a major component of the mechanism in leukocytosis [5]. Dukes et al. [6] reported paraneoplastic leukemoid reaction as a marker for transitional cell carcinoma recurrence. They

indicated the role of G-CSF in their case. Thrombocytosis can occur by complex multiple mechanisms or thrombopoietin which has a major hematologic growth factor effect on platelet development map play role. Combination of paraneoplastic syndromes is a seldom clinical feature. Double paraneoplastic syndromes are relatively common. Triple or more paraneoplastic syndrome is highly interesting. Paraneoplastic syndrome of leukocytosis, thrombocytosis and hypercalcemia associated with SCC of the skin was reported [7]. There is little published data in the literature about paraneoplastic syndrome occurring in renal pelvic carcinoma [3]. Hypercalcemia and other hematological paraneoplastic syndromes had been reported [8]. A triple paraneoplastic syndrome in a patient with renal squamous cell carcinoma had been reported by Er et al. previously [9]. We describe here in a new patient who had the same features including hypercalcemia, leukocytosis and thrombocytosis in renal SCC. We were not able to show any cytokines-, growth factor-or hormone-associated pathogenesis in the clinical management. We conclude that many of the factors discussed in the literature, such as parathyroid hormone-related peptide and G-CSF, may be underlying factors in the pathogenesis in our case. 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: 51-2

References 1. Kose F, Bal N, Ozyilkan O. Squamous cell carcinoma of the renal pelvis. Med Oncol 2009;26:103-4. 2. Holmäng S, Lele SM, Johansson SL. Squamous cell carcinoma of the renal pelvis and ureter: incidence, symptoms, treatment and outcome. Urol 2007;78:51-6. 3. Walls J, Bundred NJ. Squamous cell carcinoma of the renal pelvis associated with urinary diversion and humoral hypercalcaemic malignancy syndrome. J R Coll Surg Edinb 1992;37:207-8. 4. Takaoka S, Yamane Y, Nishiki M, Yamaguchi T, Sugimoto T. Primary pulmonary squamous cell carcinoma associated with elevated IL-6, leukocytosis, hypercalcemia, phagocytosis, reactive lymphadenopathy and glomerular mesangial cell proliferation via the production of PTH-rP and G-CSF. Intern Med 2008;47:275-9. 5. Sato K, Fujii Y, Kakiuchi T, Kasono K, Imamura H, Kondo Y, Mano H, Okabe T, Asano S, Takaku F. Paraneoplastic syndrome of hypercalcemia and leukocytosis caused by squamous carcinoma cells (T3M-1) producing parathyroid hormone-related protein, interleukin 1 alpha, and granulocyte colony-stimulating factor. Cancer Res 1989;49:4740-6. 6. Dukes JW, Tierney LM Jr. Paraneoplastic leukemoid reaction as marker for transitional cell carcinoma recurrence. Urology 2009;73:928.e17-9. 7. Kato N, Yasukawa K, Onozuka T, Kimura K. Paraneoplastic syndromes of leukocytosis, thrombocytosis, and hypercalcemia associated with squamous cell carcinoma. J Dermatol 1999;26:352-8. 8. Ma XT, Yu LW, Fu J. Paraneoplastic syndromes of hypercalcemia and leukocytosis associated with colonic metastases from squamous cell carcinoma of the lung. Int J Colorectal Dis 2008;23:129-30. 9. Er O, Coýkun Hü, AltÕnbas M, Akgün H, Çetin M, Eser B, Ünal A. Rapidly relapsing squamous cell carcinoma of the renal pelvis associated with paraneoplastic syndromes of leukocytosis, thrombocytosis and hypercalcemia. Urol Int 2001;67:175-7.

Letter to the Editor

53

Nitric oxide in beta-thalassemia minor: what factors contribute? Beta talasemi minörde nitrik oksit: hangisi katkÕ saúlar? Viroj Wiwanitkit Wiwanitkit House, Bangkhae, Bangkok Thailand

To the Editor, I read with great interest the recent publication by Bayraktar et al. [1] on nitric oxide (NO) in beta thalassemia minor. in which they reached the conclusion that “These findings confirm that plasma NO levels in betathalassemia minor patients are decreased at the time of diagnosis.” Bayraktar et al. also noted the possible usefulness of NO level in assessing the prognosis and follow-up evaluation. There are some concerns with respect to the findings in this study. First, NO level can be disturbed by many factors. For example, the underlying pathophysiological conditions of the studied patients should be discussed. Some co-disorders and drug usage might alter the NO level among the patients. In addition, differences in food intake pattern or in nitrate level might be another important underlying condition that affects NO level [2]. Second, the quality control in laboratory analysis and sample collection must be addressed. Third, there is a concern regarding the proposal of Bayraktar et al. regarding the correlation between hemolysis and NO level in the studied patients. Indeed, hemolysis is not common in beta thalassemia minor. It remains doubtful whether the included cases in this study were accurately classified into the beta thalassemia minor group.

References 1. Bayraktar N, Erkurt MA, Aydogdu I, Baflaran Y. The levels of nitric oxide in beta-thalassemia minor. Turk J Hematol 2008;25:187-9. 2. Milkowski A, Garg HK, Coughlin JR, Bryan NS. Nutritional epidemiology in the context of nitric oxide biology: a riskbenefit evaluation for dietary nitrite and nitrate. Nitric Oxide 2009 Sep 11 [Epub ahead of print].

Author Reply My response regarding the manuscript entitled “Nitric oxide in beta-thalassemia minor: what factors contribute?” is given below. Firstly: In our patients, there were no co-disorders or drug usage. Patients with acute or chronic infections, chronic inflammatory diseases, heart diseases, and other anemias were not included in the study. Patients with similar underlying pathophysiological conditions were selected. In all patients, blood samples were done in a fasting state in the morning. Thus, there were no differences in food intake pattern. However, nitric oxide levels can be affected by food intake pattern, co-disorders and drug usage [1]. Secondly: In all patients, blood samples were done in a fasting state and in the morning. Centrifuge was done and plasma was separated. Plasma was frozen (-70°C). Nitric oxide levels of all patients were measured together. Plasma nitrite/nitrate levels were measured with the Griess reaction using a spectrophotometer at 545 nm. Nitrite (0.1M sodium nitrite in water) was mixed with sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) first, followed immediately by addition of NED solution (0.1% N-1-napthylethylenediamine dihydrochloride in water). The absorbance was measured within 30 minutes. Thirdly: Heterozygous beta-thalassemia (thalassemia minor) is described as: no or mild anemia, microcytosis and hypochromia, mild hemolysis manifested by slight reticulocytosis and splenomegaly [2]. Hemolytic anemia develops in direct proportion to the deficiency of the beta globin chain that cannot be produced in thalassemia sufferers [3-6]. According to these references, hemolytic anemia may develop, particularly in conditions

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

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Wiwanitkit V. NO in beta-thalassemia minor

of physiological stress such as infection, pregnancy or surgery, and anemia may increase and present symptoms. Another possible explanation for hemolysis is the apparent decrease in spectrin content, including deficient or defective spectrin synthesis in thalassemia erythroid precursors or globin chaininduced membrane changes that lead to spectrin dissociation from the membrane [7]. Indeed, mild hemolysis occurs in beta thalassemia minor. In contrast, thalassemia intermedia and thalassemia major are probably associated with more severe degrees of hemolysis. It would have been preferable if the nitric oxide levels of the patient group were also compared with thalassemia major patients or Hb S patients as a positive control. Our small study population consisted of patients diagnosed with beta thalassemia minor. Further studies are needed in a large number of patients. Sincerely, Mehmet Ali Erkurt Inonu University Medical Faculty, Field of Hematology, Malatya, Turkey

Turk J Hematol 2010; 27: 53-4

References 1. Milkowski A, Garg HK, Coughlin JR, Bryan NS. Nutritional epidemiology in the context of nitric oxide biology: a risk-benefit evaluation for dietary nitrite and nitrate. Nitric Oxide 2009 Sep 11 [Epub ahead of print]. 2. MD Consult. Ferri: Ferri's Clinical Advisor 2010. 1st ed. Instant Diagnosis and Treatment. FRED F. FERRI, M.D., F.A.C.P. Copyright Š 2010 Elsevier. 3. Weatherall DJ. Disorders of the synthesis or function of haemoglobin. In: Weatherall DJ, Ledingham JGG, Warrell DA, editors. Williams Hematology. 2nd ed. Oxford: ELBS Oxford University Press, 1988: II: 19.108-19.130. 4. Weatherall DJ. The thalassemias. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U, editors. Williams Hematology. 6th ed. New York: McGraw-Hill, 2001:547-80. 5. Holzgreve W, Sevinchan E, Kohne E, Sevinchan S, Miny P, Horst J. Beta-thalassemia problems in the Turkish population in the F.R.G. Eur J Obstet Gynecol Reprod Biol 1990;34:137-47. 6. Aydinok Y, Oztop S, Nisli G, Kavakli K. Prevalence of beta-thalassaemia trait in 1124 students from Aegean region of Turkey. J Trop Pediatr 1997;43:184-5. 7. Yuan J, Bunyaratvej A, Fucharoen S, Fung C, Shinar E, Schrier SL. The instability of the membrane skeleton in thalassemic red blood cells. Blood 1995;86:3945-50.

Letter to the Editor

55

Acute tumor lysis syndrome secondary to a single-dose methylprednisolone in acute lymphoblastic leukemia Akut lenfoblastik lösemide tek doz metilprednizolona sekonder olarak geliýen akut tümör lizis sendromu Tansu Sipahi1, Faruk Öktem2, Ayça Esra Kuybulu3 1Department

of Pediatric Hematology, Ufuk University, Ankara, Turkey of Pediatrics, Pediatric Nephrology and Rheumatology Unit, Süleyman Demirel University Faculty of Medicine, Isparta, Turkey 3Department of Pediatrics, Süleyman Demirel University, Isparta, Turkey 2Department

To the Editor There are very few reports in the English literature regarding cases with acute lymphoblastic leukemia (ALL) who developed acute tumor lysis syndrome (ATLS) after a single dose of steroid at the beginning of the chemotherapy [1-6]. Herein, we present a child with T-cell ALL who developed ATLS after methylprednisolone (2 mg/kg) therapy. A seven-year-old, previously healthy girl was admitted to our Department of Pediatrics with a two-week history of anorexia and fever. Her physical examination showed diffuse petechiae and ecchymoses and diffuse lymphadenomegaly in the cervical, submandibular, axillary, and inguinal regions. She had mild hepatomegaly but no splenomegaly. Initial investigations showed: hemoglobin 5.9 g/dl, hematocrit (Htc) 17.5%, white cell count 1100/ mm3 (blasts 90%), platelets 15000/mm3, ALT: 22 IU/L, AST: 35 IU/L, total protein: 5.7 g/dl, albumin: 3.7 mg/dl, calcium (Ca): 7.5 mg/dl, phosphorus: 2.13 mg/dl, BUN: 11 mg/dl, creatinine: 0.5 mg/dl, uric acid: 2.6 mg/dl, sodium (Na): 130 mEq/L, potassium (K): 4.3 mEq/L, chloride: 103 mEq/L, and lactate dehydrogenase: 1294 IU/L. Chest X-ray showed right hilar lymphadenopathy (LAP). Bone marrow aspiration revealed 100% blasts (ALL L1-2). Ninety-two percent of the blasts were CD7 and CD5-positive and CD13, CD33, CD10, CD19, CD20, CD22, CD14 and HLA-DR-negative. The cerebrospinal

fluid was clear, and there were no blast cells. Erythrocyte suspension transfusion was planned but Coombs test was positive. She was given methyl prednisone (2 mg/kg; 40 mg total) before blood transfusion. Thirteen hours later her general condition worsened and she started vomiting. Serum biochemical analysis was studied again and demonstrated: hemoglobin: 8.5 g/L, Htc: 23%, WBC: 0.2x10e9/L, BUN: 60 mg/dl, creatinine: 2.2 mg/dl, uric acid: 9.8 mg/dl, Na: 138 mEq/L, K: 2.9 mEq/L, Ca: 6.4 mEq/L, and phosphorus: 7.2 mg/dl. ATLS was considered in this patient, and intravenous forced alkaline over hydration (3000 ml/m2/day), allopurinol (300 mg/m2), and furosemide (1 mg/kg/day) therapy was initiated. Blood biochemistry and complete blood cell count were monitored every 12 hours [7]. When the laboratory tests were normal on the third day, the chemotherapy program was started. She is still in remission on the St. Jude T XIII maintenance chemotherapy. In our patient, there was no evidence of ATLS prior to the methyl prednisone therapy. Thirteen hours later, azotemia, hyperuricemia, hyperphosphatemia, and hypocalcemia developed. When chemotherapy is started with the diagnosis of ALL, even if leukocyte count is not very high, a careful monitoring for ATLS must be done, and serum calcium, phosphorus, electrolytes, creatinine, and uric acid should be checked every 5-6 hours, especially during the first week of therapy.

Address for Correspondence: Prof. Tansu Sipahi, Angora Evleri, Kediseven Caddesi No: 92, Beysukent, Ümitköy, Ankara, Türkiye Phone: +90 312 204 42 34 E-mail: tansusipahi@hotmail.com

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Sipahi et al. Acute tumor lysis syndrome

Conflict of interest Informed consent was obtained from the patient and her family. 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.

Duzova A, Cetin M, GĂźmrĂźk F, Yetgin S. Acute tumour lysis syndrome following a single-dose corticosteroid in children with acute lymphoblastic leukaemia. Eur J Haematol 2001;66:404-7. Dhingra K, Newcom SR. Acute tumour lysis syndrome in nonHodgkin lymphoma induced by dexamethasone. Am J Hematol 1988;29:115-6.

Turk J Hematol 2010; 27: 55-6

3.

4.

5.

6.

7.

Sparano J, Ramirez M, Wiernik PH. Increasing recognition of corticosteroid-induced tumour lysis syndrome in non-Hodgkin lymphoma. Cancer 1990;65:1072-3. Tiley C, Grimvade D, Findlay M, Treleaven J, Height S, Catalano J. Tumour lysis following hydrocortisone prior to a blood product transfusion in T-cell lymphoblastic leukaemia. Leuk Lymphoma 1992;8:143-6. Loosveld OJ, Schouten HC, Gaillard CA. Acute tumour lysis syndrome in a patient with acute lymphoblastic leukaemia after a single dose of prednisone. Br J Haematol 1991;77:122-3. Rajagopal S, Lipton JH, Messner HA. Corticosteroid induced tumour lysis syndrome in acute lymphoblastic leukaemia. Am J Haematol 1992;41:66-7. Spinazze S, Schrijvers D. Metabolic emergencies. Clin Rev Oncol Hematol 2006;58:79-89.

Advisory Board of This Issue (March 2010) Ahmet Öztürk Ahmet Türker Çetin Akif Selim Yavuz Akif Yeýilipek Ali Uúur Ural Ayýegül Ünüvar Ayýen Timuraúaoúlu Aytemiz Gürgey Burhan Ferhanoúlu Burhan Turgut Bülent Eser Can Boúa Cengiz Beyan Elif Ünal Emel Özyürek Emre Tekgündüz Evren Özdemir Fevzi Altuntaý

Güray Saydam Güçhan Alanoúlu Gülersu ûrken Gülsüm Emel Pamuk Günay Balta Hakan Özdoúu Hale Ören Hilmi Apak ûdil Yenicesu Kaan KavaklÕ Levent Ündar Mualla Çetin Musa Karakükcü Mutlu Arat Muzaffer Demir NamÕk Özbek Nejat Akar Nurdan TaçyÕldÕz

Omar Iqbal Ömer Devecioúlu Özcan Çeneli Reyhan Diz Küçükkaya Savaý Kansoy Sedat Üstündaú Semra Paydaý Tiraje Çelkan Tunç FÕýgÕn Türkan PatÕroúlu Uúur Özbek Ülker Koçak Ünsal Özgen Vefki Gürhan KadÕköylü Yahya BüyükaýÕk Yurdanur KÕlÕnç Zahit Bolaman

Announcements 4-6 March 2010 6. Turkish National Bone Marrow Transplantation and Stem Cell Therapies Congress Antalya Turkey

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

21-24 March 2010 The 36th Annual Meeting of the European Group for Blood and Marrow Transplantation (EBMT)Hematology Medical Congress, Viena, Austria

17-19 September 2010 Turkish School of Hematology Consultation Hematological Samsun Turkey

6-9 May 2010 Turkish Society of Hematology Post-Graduate Hematology Education Congress Update on Hematological Oncology Cyprus

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

22-25 May 2010 The 56th Annual ISTH Conference Cairo, Egypt 4-8 June 2010 2010 ASCO Annual Meeting Chicago, ABD 10-13 June 2010 15th Congress of the EHA Barcelona, Spain 10-14 June 2010 World Congress of the World Federation of Hemophilia Buenos Aires, Argentina

21-23 October 2010 Lymphoma & Myeloma 2010: An International Congress on Hematologic Malignancies New York, NY, United States 3-6 November 2010 36. Turkish National Hematology Congress Antalya Turkey 4-7 December 2010 52. ASH Annual Meeting and Exposition Orlanda, Florida, ABD

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