tjh-2011-1 by LookUs Scientific

Issue 1

March 2011

40 TL

ISSN 1300-7777

Volume 28

TURKISH JOURNAL OF HEMATOLOGY • VOL.: 28 ISSUE: 1 MARCH 2011

Review Article Protein profiles in leukemia

Gülşan Şanlı-Mohamed et al.; İzmir, Turkey, Utah, USA

Research Articles Apoptosis, proliferation in large B-cell lymphoma Nilay Şen Türk, et al.; Denizli, İzmir, Turkey

sEPCR and FVIII levels in infants

Filiz Şimşek Orhon et al.; Ankara, İstanbul, Turkey, New Jersey, USA

Differences in cord blood

Nilgün Akdeniz, et al.; İstanbul, Turkey

Oxidative stress and iron deficiency anemia Mehmet Aslan et al.; Van, Sanliurfa, Turkey

Genotyping of HCV in β-TM

Suleimman Ahmad Al-Sweedan et al.; Irbid, Jordan

Hyperuricemia and tumor lysis syndrome Betül Sevinir et al.; Bursa, Turkey

Cover Picture: Abdullah Hacıhanefioğlu

Editor-in-Chief

International Review Board

Aytemiz Gürgey

Nejat Akar (Turkey) Görgün Akpek (USA) Serhan Alkan (USA) Çiğdem Altay (Turkey) Koen van Besien (USA) Ayhan Çavdar (Turkey) M.Sıraç Dilber (Sweden) Ahmet Doğan (USA) Peter Dreger (Germany) Thierry Facon (France) 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)

Associate Editors

Mutlu Arat Muzaffer Demir Reyhan Diz Küçükkaya Mehmet Ertem Hale Ören Mehmet Ali Özcan Ayşegül Ünüvar Celalettin Üstün Neşe Yaralı Akif Selim Yavuz Past Editors

Erich Frank Orhan Ulutin Hamdi Akan Senior Advisory Board

Orhan Ulutin Yücel Tangün Osman İlhan Language Editors

Corinne Can Scott Evans Statistic Editor

Mutlu Hayran

Contact Information

All other inquiries should be adressed to

Editorial Correspondence should be addressed to Dr. Aytemiz Gürgey Editor-in-Chief Address: Hacettepe University Faculty of Medicine Pediatric Hematology Department 06100 Sıhhiye, Ankara/Turkey Phone: +90 312 305 41 17 Fax: +90 312 305 41 17 E-mail: agurgey@hacettepe.edu.tr

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

Turkish Society of Hematology

Editorial Secretary

İpek Durusu Bengü Timoçin Cover Picture: Abdullah Hacıhanefioğlu was born in 1962, İstanbul, Turkey. He is currently working at Kocaeli University, Department of Hematology, İzmit, Turkey.

Abdullah Kutlar (USA) Luca Malcovati (Italy) Robert Marcus (United Kingdom) Jean Pierre Marie (France) Ghulam Mufti (UK) Gerassimos A. Pangalis (Greece) Santiago Pavlovsky (Argentina) Antonio Piga (Italy) Ananda Prasad (USA) Jacob M. Rowe (Israel) Jens-Ulrich Rüffer (Germany) Norbert Schmit (Germany) Orhan Sezer (Germany) Anna Sureda (Spain) Ayalew Tefferi (USA) Nüket Tüzüner (Turkey) Catherine Verfaillie (USA) Srdan Verstovsek (USA) Claudio Viscoli (Italy)

Muhit Özcan, President Mutlu Arat, General Secretary Hale Ören, Vice President Muzaffer Demir, Research Secretary Teoman Soysal, Treasurer Fahir Özkalemkaş, Member Mehmet Sönmez, Member Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile Turkish Journal of Hematology’nin Türk Hematoloji Derneği İkdisadi İşletmesi tarafından yayınlanmasına karar vermiştir.

Web page www.tjh.com.tr

Sahibi

Türk Hematoloji Derneği adına Muhit Özcan

Sorumlu Yazı İşleri Müdürü Aytemiz Gürgey

Yayın ve Yönetim Yeri Türk Hematoloji Derneği Türk Ocağı Cad. 17/6 Cağaloğlu-Eminönü-İstanbul Üç ayda bir yayınlanan yerel bilimsel dergidir.

Publisher: AVES Yayıncılık Address: Kızılelma cad. 5/3 34096 Fındıkzade-İstanbul-Turkey Phone: +90 212 589 00 53 Fax: +90 212 589 00 94 E-mail: info@avesyayincilik.com Place of printing: ADA Ofset Matbaacılık Tic. Ltd. Şti. - 0212 567 12 42 Date of printing: February 2011 Broadcast as: Local periodical

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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. Editorial Board of Turkish Journal of Hematology works under the principles of The World Association of Medical Editors (WAME), the International Council of Medical Journal Editors (ICMJE), and Committee on Publication Ethics (COPE). The aim of the Turkish Journal Hematology is to publish original research papers of highest scientific and clinical value on hematology. Additionally, educational material, reviews on basic developments, editorial short notes, case reports, original views and letters from specialists on hematology, and hematology medicine covering their experience and comments as well as social subjects are published. General Practitioners interested in hematology, and internal medicine specialists, are also our target audience, and we will arrange the Turkish Journal of Hematology according to their needs. The Turkish Journal of Hematology is indexed in - Science Citation Index Expanded - EMBASE - Scopus - CINAHL - Index Copernicus - Gale/Cengage Learning - EBSCO - DOAJ - ProQuest - Tübitak/Ulakbim Türk Tıp Dizini Subscription Information The Turkish Journal of Hematology is sent free of charge to hematologists and academicians in our country as well as to other specialists interested in hematology. All published volumes in full text can be reached free of charge through the web site www.tjh.com.tr Adress: Ilkbahar mah. Turan Güneş Bulvarı 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: info@tjh.com.tr Permissions Requests for permission to reproduce published material should be sent to the editorial office. Editor: Prof.Dr. Aytemiz Gürgey Adress: Ilkbahar mah. Turan Güneş Bulvarı 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: 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 300 words with subheadings; objective, material and methods, results, conclusion. 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. Editorial Board of Turkish Journal of Hematology works under the principles of The World Association of Medical Editors (WAME), the International Council of Medical Journal Editors (ICMJE), and Committee on Publication Ethics (COPE). 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 300 words. Review Articles Review articles should not exceed 4,000 words in length, must include an abstract of 300 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.

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Images in Hematology 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.

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.

Preparation of Manuscript

Ethics

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 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.

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.

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

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 300 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, Materials and 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

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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;336:684-7. 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;2:742-4. 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: 780-94. 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;84:2821-3. 7. CD-ROM Anderson SC, Poulsen KB, Andersüon’s electronic atlaso hematology [CD-ROM]. Philadelphia: Lippincott Williams&Wilkins: 2002.

8. Journal article on the internet Abood S. Quality improvement intititative in nursing homes: The ANA acts in an advisory role. Am J Nurs [serial on the Internet.] 2002 n [cited 2002 Aug 12]:102(6)[about 3 p.] Available from: http:/www.nursing world.org/ AJN/2002/June/Wawatch.htm. 9. Homepage/Web site Cancer-Pain.org [homepage on the Internet]. New York: Association of Cancer Online Resources, Inc.: c2000-01 [updated 2002 May 16: cited 2002 Jul 9]. Available from: hhtp:// www.cancer-pain.org/. 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.

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

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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 Supported images .bmp .jpg .jpeg .gif .tif

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. 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

The importance of protein profiling in the diagnosis and treatment of hematologic malignancies Gülşan Şanlı-Mohamed, Taylan Turan, Hüseyin Atakan Ekiz, Yusuf Baran, İzmir, Turkey, Utah, USA

Research Articles 15

Determination of apoptosis, proliferation status and O6-methylguanine DNA methyltransferase methylation profiles in different immunophenotypic profiles of diffuse large B-cell lymphoma Nilay Şen Türk, Nazan Özsan, Vildan Caner, Nedim Karagenç, Füsun Düzcan, Ender Düzcan, Mine Hekimgil, Denizli, İzmir, Turkey

The relation between soluble endothelial protein C receptor and factor VIII levels and FVIII/sEPCR index in healthy infants Filiz Şimşek Orhon, Yonca Eğin, Betül Ulukol, Sevgi Başkan, Nejat Akar, Ankara, İstanbul, Turkey, New Jersey, USA

Differences in lymphocyte subpopulation count and function in cord, maternal and adult blood

Seroprevalence and genotyping of hepatitis C virus in multiple transfused Jordanian patients with β-thalassemia major

Evaluation of oxidative status in iron deficiency anemia through total antioxidant capacity measured using an automated method

Nilgün Akdeniz, Esin Aktaş, Gaye Erten, Sema Bilgiç, Günnur Deniz, İstanbul, Turkey

Suleimman Ahmad Al-Sweedan, Said Jaradat, Khitam Amer, Wail Hayajneh, Hazem Haddad, Irbid, Jordan

Mehmet Aslan, Mehmet Horoz, Hakim Çelik, Van, Turkey, Şanlıurfa, Turkey

Hyperuricemia and tumor lysis syndrome in children with non-Hodgkin’s lymphoma and acute lymphoblastic leukemia Betül Sevinir, Metin Demirkaya, Birol Baytan, Adalet Meral Güneş, Bursa, Turkey

Case Reports 60

PET CT imaging in extramedullary hematopoiesis and lung cancer surprise in a case with thalassemia intermedia

Treatment of intrathecal methotrexate overdose with folinic acid rescue and lumbar cerebrospinal fluid exchange:A report of two cases

Brucellosis presenting with pancytopenia due to hemophagocytic syndrome

Semra Paydaş, Özoğul Sargın, Gülfiliz Gönlüşen, Adana, Turkey

Elif Kazancı, Hüseyin Gülen, Ayşe Erbay, Canan Vergin, İzmir, Manisa, Turkey Ela Erdem, Yıldız Yıldırmak, Nurşen Günaydın, İstanbul, Turkey

Letters to the Editor 72

A child with primary gastric lymphoma and cavernous sinus involvement

Feasibility of four discriminant functions for identifying hemoglobin E disorders: Experience in 114 Thai pregnant subjects

The association between calcium dobesilate and pancytopenia in type 2 diabetes: A case report

Aeromonas sobria bacteriemia in an acute lymphoblastic leukemia case in remission

ARA-C associated pulmonary toxicity

Mehmet Akif Özdemir, Yasemin Altuner Torun, Türkan Patıroğlu, Edip Torun, Ahmet Candan Durak, Kayseri, Turkey Viroj Wiwanitkit, Nara Paritpokee, Jamsai Suwansaksri, Hainan, China, Bangkok, Thailand Aylin Cesur, Meltem Aylı, Mustafa Cesur, Sibel Ertek, Ankara, Turkey

Selami Koçak Toprak, Gül İlhan, Elçin Erdoğan, Sema Karakuş, Ankara, Turkey

Zeynep Arzu Yegin, Gülsan Türköz Sucak, Gonca Erbaş, Münci Yağcı, Ankara, Turkey

Images in Hematology 84

Niemann-Pick disease

Serap Karaman, Tiraje Celkan, İstanbul, Turkey

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Review

The importance of protein profiling in the diagnosis and treatment of hematologic malignancies Hematolojik malignitelerin tanı ve tedavisinde protein profillemenin önemi Gülşah Şanlı-Mohamed1, Taylan Turan1, Hüseyin Atakan Ekiz2, Yusuf Baran3, 1Department

of Chemistry, Science Faculty, İzmir Institute of Technology, Urla, İzmir, Turkey of Oncological Sciences, University of Utah, Salt Lake City, Utah, USA 3Department of Molecular Biology and Genetics, Science Faculty, İzmir Institute of Technology, Urla, İzmir, Turkey 2Department

Abstract Proteins are important targets in cancer research because malignancy is associated with defects in cell protein machinery. Protein profiling is an emerging independent subspecialty of proteomics that is rapidly expanding and providing unprecedented insight into biological events. Quantitative assessment of protein levels in hematologic malignancies seeks a comprehensive understanding of leukemiaassociated protein patterns for use in aiding diagnosis, follow-up treatment, and the prediction of clinical outcomes. Many recently developed high-throughput proteomic methods can be applied to protein profiling. Herein the importance of protein profiling, its exploitation in leukemia research, and its clinical usefulness in the treatment and diagnosis of various cancer types, and techniques for determining changes in protein profiling are reviewed. (Turk J Hematol 2011; 28: 1-14) Key words: Hematologic malignancies, leukemia, proteomics, protein profiling Received: November 24, 2010

Accepted: January 19, 2011

Özet Malignitelerde proteinlerin hücresel mekanizmalarında meydana gelen bozukluklardan dolayı, proteinler kanser araştırmaları için önemli hedeflerdir. Proteomiksin alt uzmanlık dalı olarak ortaya çıkan ve bağımsız bir alan olan protein profilleme biyolojik olaylara farklı bir bakış açısı sağlamak amacıyla hızla gelişmektedir. Hematolojik malignitelerdeki protein düzeylerinin kantitatif olarak değerlendirilmesi, teşhise yardımcı olması, tedavinin izlenmesi ve klinik sonuçların tahmininde mükemmel bir yaklaşım olması nedeni ile lösemi ile ilgili protein modellerinin kapsamlı bir şekilde incelenmesini amaçlamaktadır. Son dönemlerde geliştirilen yüksek verimli yöntemler protein profillemede kullanılabilir. Bu makalede, protein profillemenin önemi, lösemi araştırmalarındaki rolü, çeşitli kanser tiplerinin tanısı ve tedavisi için klinik kullanımları ve protein profilindeki değişikliklerin belirlenmesinde kullanılan teknikler değerlendirilmiştir. (Turk J Hematol 2011; 28: 1-14) Anahtar kelimeler: Hematolojik malignitiler, lösemi, proteomiks, protein profilleme Geliş tarihi: 24 Kasım 2010

Kabul tarihi: 19 Ocak 2011

Address for Correspondence: Assoc. Prof. Yusuf Baran, Department of Molecular Biology and Genetics, Science Faculty, İzmir Institute of Technology, Urla, İzmir, Turkey Phone: +90 232 750 75 15 E-mail: yusufbaran@iyte.edu.tr, iytecancer@gmail.com doi:10.5152/tjh.2011.01

Ĺ&#x17E;anlÄą-Mohamed et al. Protein profiles in leukemia

Introduction Proteins are abundant in all organisms and play key roles in most biological events as catalysts, transporters, and messengers. Thus, it is crucial to note that all research related to proteins increase our understanding of their levels, interactions, functions, modifications, regulations, and localization in cells [1,2]. Proteomics is a rapidly expanding discipline that aims to gain a comprehensive understanding of proteins. The term proteomics, which is a combination of protein and genomics, is used to define the large-scale analysis of a complete set of proteins - the chief components of cells that are responsible for the most significant metabolic pathways in cells or tissues [2-7]. The goal underlying proteomics is not only to identify all proteins in a cell, but also to identify the correlation between the genetic sequence and three-dimensional (3D) protein structure [2]. In other words, work in proteomics encompasses the investigation of proteinprotein interactions, the connection between the structure of proteins and their function, cellular processes and networks, and to improve protein separation and protein profiling techniques. Protein profiling, an emerging independent subspecialty of proteomics, is poised to provide unprecedented insight into biological events. Quantitative evaluation of protein levels can be accomplished with protein profiling, which shows us unique expression patterns (diseased vs. healthy, treated vs. untreated, experimental vs. control) at the protein level when proteins from one cell type are compared with those of another cell type. The value of protein profiling is increasing daily and there are several reasons why it is of great importance, especially as a potential tool for the early diagnosis of leukemias and other diseases. One such reason is that it provides a much better understanding of an organism, as it is not always possible or sufficient for scientists to clarify some metabolic pathways, including mechanisms of diseases, exclusively by studying the genome [2]. Additionally, there are some difficulties associated with accurately indentifying genes solely by dealing with genomic data [8]. To overcome this problem, data provided from genomic studies should be supported with data obtained from the study of proteins. Proteomics is often considered as the stage following genomics in the study of biological systems. Compared to genomics, proteomics is much

Turk J Hematol 2011; 28: 1-14

more complicated, as the proteome differs from cell to cell, and under different conditions. This is because distinct genes are expressed in distinct cell types, and to identify even a basic group of proteins produced in a cell, one needs to have a comprehensive understanding of protein-related actions [2]. Until recently, such research was carried out using mRNA analysis via different methods, including microarray technology [9,10] and serial analysis of gene expression (SAGE) [11]. On the other hand, recent studies demonstrate that mRNA analysis cannot be correlated directly with protein levels [12-17], as mRNA is not always translated into proteins [7]. Moreover, the quantity of protein formed for a given quantity of mRNA depends on both the gene that it is transcribed from and the current physiological state of the cell. As such, the level of transcription of a gene provides only a rough estimation of its extent of translation into a protein. Additionally, mRNA produced may go under rapid degradation that causes a reduction in translation, resulting in the production of less protein. In addition, some bodily fluids, such as serum and urine, have no source of mRNA under normal circumstances; therefore, proteomic technologies have emerged as an important addition to genomic studies [2]. Proteomics verifies the presence of a protein and provides a direct measure of the quantity present. An additional important reason that protein profiling is crucial is its power to analyze protein modifications. Although a particular cell may have a distinguishable set of proteins at various times or under various conditions, any particular protein may go through a wide range of alterations known as post-translational modifications, which will have critical effects on its function. Phosphorylation is an example of posttranslational modification. Structural proteins can undergo phosphorylation during cell signaling and result in the protein becoming a target for binding to or interacting with a distinct group of proteins that recognize the phosphorylated domain [18]. Ubiquitination is another post-translational type of modification. Ubiquitin is a small protein that can be affixed to certain protein substrates by means of enzymes known as E3 ubiquitin ligases [19]. Identifying which proteins are polyubiquitinated can be helpful in understanding how protein pathways are regulated. In addition to phosphorylation and ubiquitination, proteins can undergo additional modifications via methylation, acetylation, glycosyl-

Turk J Hematol 2011; 28: 1-14

ation, oxidation, sulfation, hydroxylation, nitrosylation, amidation, etc. These modifications can be assessed only at the protein level and modifications of many proteins expressed by a cell can be determined at the same time using such proteomic methods as phosphoproteomics and glycoproteomics [2]. In addition to modifications, there is no doubt that protein localization and interactions are of vital importance to their function. Mislocalization of a protein or any problem in signal transduction can turn normal cells into abnormal cells, which is a well-known paradigm in carcinogenesis. Protein profiling using proteomic methods can also be used to characterize these regulatory mechanisms. Another point that emphasizes the importance of protein profiling is that many proteins form complexes with other proteins and/or with nucleic acids, and exert their function in the presence of these molecules. In summary, protein profiling provides a much better understanding of an organism, in terms of structure and function. Use of protein profiling in the study of multiple proteins, protein forms, and protein families - almost always by comparing 2 different states (diseased vs. healthy) - is expected to expand our understanding of molecular mechanisms. Elucidation of protein-protein interactions and signaling pathways, identification of biomarkers useful for drug development, serum profiling to identify patient populations that respond to various treatments, and eventually medical diagnostics in the near future can be implemented for hematological malignancies via proteomics. Exploitation of protein profiling in leukemia research Because leukemic cells lose regulation of growth controlling mechanisms, in most cases signaling pathways involving numerous proteins are altered, as mentioned before. When this is taken into account, not surprisingly, expression patterns of growth-inducing and growth-suppressing genes change with malignant transformation [20,21]. Therefore, monitoring these changes is of great importance for understanding carcinogenesis, identifying diagnostic markers, and developing new therapeutics for leukemia. The most widely used methods for this involve examination of differential gene expression in leukemic cells by assessing the mRNA levels in the given cells. Several successful studies reported the feasibil-

Ĺ&#x17E;anlÄą-Mohamed et al. Protein profiles in leukemia

ity of this approach [22-25]. On the other hand, this methodology comes from a reductionist point of view, as it neglects the dynamic nature of protein translation and further modifications that take place beyond transcription. As such, an approach involving direct proteomic methods might be a better choice for obtaining more accurate insight into what is happening at the cellular level in leukemic cells. Although the initial changes that occur during the development of hematologic malignancies appear as little sparks igniting a larger fire, the majority of leukemias manifest with profound alterations in protein profiles. In some types of leukemia, growthsuppressive genes undergo mutations and nullify protein synthesis completely, whereas in other types aberrant proteins arise due to chromosomal rearrangement, which is not usually seen in healthy cells. Those alterations are readily detectable with current proteomic methods. What follows are examples of proteins whose cellular presence changes most significantly with malignant transformation, and from this perspective the feasibility of protein profiling for the diagnosis and therapy of hematological malignancies becomes evident. P53 has been perhaps the most famous protein for decades because of its essential role in cell cycle regulation, apoptosis and senescence. It is a transcription factor that responds to stressful conditions by inducing cell cycle arrest and apoptosis. Not surprisingly, it was reported that p53 is deleted or mutated in the majority of cancers. Experiments involving p53 knockout mice have shown that the occurrence of tumors increases with the loss of p53 function [26,27]. In some cases inactivation of p53 involves small changes such as point mutations [2830]. On the other hand, numerous other leukemias manifest with more global changes in the p53 protein structure. The protein can also be tagged for proteolytic degradation by interacting with another protein, MDM2 [31], or without the need of a degradation protein can be inactivated by exporting back to cytoplasm, inhibiting its DNA-binding functions [32]. Major structural alterations aside, p53 can be inactivated by binding to other proteins [32] and by chemical modifications, such as phosphorylation and acetylation [33,34], and we know that these alterations are traceable using proteomic methods. Furthermore, signaling networks of p53 and downstream targets can also be examined proteomically

Ĺ&#x17E;anlÄą-Mohamed et al. Protein profiles in leukemia

to gain more comprehensive insight into carcinogenesis. While p53 is a protein commonly altered in numerous cancers, there are some proteins that appear to be signature molecules for particular cancer types. Such proteins are important for diagnostic purposes and might be used in cancer therapeutics as highly selective targets. Chronic myeloid leukemia (CML) is a wonderful example. CML is characterized by accumulation of immature blasts of myeloid origin in the bloodstream and bone marrow. The main driving force of leukemogenesis in CML is reciprocal translocation between chromosomes 9 and 22, which produces a fusion protein (BCR-ABL) having constitutive tyrosine kinase activity, and, in turn, induces cell growth [35]. BCR-ABL is one of the most prominent proteins in leukemic cells and its overexpression is linked to chemotherapeutic drug resistance [36]. With the increased knowledge about this fusion protein, highly specific drugs were developed and considerable cytogenetic responses were delineated [37-39]. In the light of these advancements in leukemia therapy, it is obvious that identification of proteins responsible for malignant transformation is particularly important for the development of efficient drugs. The current literature has provided detailed insight into the pathogenesis of CML, but there are numerous other cancer types whose pathophysiology remains to be elucidated. At this point, the need for the utilization of proteomic methods rather than genomic methodologies is clear, as it provides a deeper understanding of the features of leukemic cells. The literature sheds light on the specifics of different types of leukemias, as the mechanism of CML has been described. For instance, acute lymphoblastic leukemia (ALL) cells were reported to express unique tyrosine kinases that originate from Abl kinase, which are thought to be important for malignancy [40]. In some ALL patients, chromosomal translocations create ABL1/NUP214 or P2RY8/ CRLF2 fusion proteins with oncogenic activity [41,42]. T-cell ALL cells undergo site-specific genetic alterations in the TAL1 gene due to problematic activity of the enzyme recombinase, and as a result, a truncated protein with oncogenic activity is produced [43]. Translocations involving this region were also shown to be important for ALL leukemogenesis [44-46]. Similarly, KRAS2 protein with a

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genetic insertion was shown to play a role in malignant transformation by activating the RAS-activated signaling pathway in acute myeloid leukemia (AML) cells [47]. Additionally, some cases of AML were caused by alterations in CEBPA and NPM proteins via mutations [48,49]. Proteins with role in the development of hematological malignancies are not confined to those already mentioned. There are many others identified as responsible for malignant transformation, and without any doubt there are many other proteins whose mysteries remain unsolved. Moreover, understanding the complex signaling networks amongst the proteins is no less important than identifying the proteins themselves; therefore, it is clear that proteomic techniques and protein profiling are especially valuable for obtaining a deeper understanding of malignant transformation, improved diagnostics, and more accurate prognostic predictions, and for the development of effective therapeutic options. Genomics has provided a considerable body of useful information on the alterations in cancer cells by identifying the genes responsible for tumor suppression and growth, but as long as the complex interactions of proteins and the dynamic nature of protein synthesis are overlooked, genomics will be unable to establish a complete understanding of malignancy. Because the information stored in genes is reflected in the phenotype by proteins, assessment of proteins in the cell at a given time would provide accurate and detailed insight into the specifics of the cell type being investigated. Despite advancements in leukemia therapy, we remain baffled by the complexity of cancer cells and their adaptation to current therapies; hence, we are still unable to provide an effective solution. Nonetheless, as more knowledge about the specifics of leukemic cells is obtained, such as oncogenic proteins, more effective therapies will be developed. The development of tyrosine kinase inhibitors is a wonderful example. Nowadays, survival time in CML patients is greatly prolonged due to the availability of these inhibitors [50,51]. Hopefully, as the utilization of proteomic techniques in leukemia research increases, promising new targets and treatment opportunities will emerge in a near future. The major important proteins in different types of leukemias are summarized in Table 1.

Şanlı-Mohamed et al. Protein profiles in leukemia

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Protein profiling: clinical implications Investigating protein profiles and understanding the dynamic alterations of cellular proteins are of great importance for diagnostic and therapeutic purposes in clinical settings. This is because most diseases, cancers in particular, are reflected as driven by protein alterations in cells. As such, proteomic methods enhance our understanding of the characteristics of various cancers via discovering new biomarkers, making it possible to discriminate healthy and malignant cells more accurately, development of novel therapeutics that target altered protein signaling pathways, and assessment of what changes occur at the protein level in cells in response to treatment with drugs, leading to the evaluation of therapeutic efficacy. In fact, several studies reported that protein signatures were potent discriminators of diseased and healthy cells. Such an approach used with serum proteins discriminated CML cells from healthy cells. Differences in protein levels were identified using matrix-assisted laser desorption/ionization time of flight (MALDITOF) analysis and spectrum comparisons showed that there were 31 differentially expressed proteins involved in cell growth, survival, and programmed cell death [100]. Barnidge et al. analyzed CLL patient samples to characterize CLL B-cell protein expression at the quantitative and qualitative level using two-dimensional liquid-chromatography cou-

pled to MALDI tandem mass spectrometry (2D-LCMS/MS) [101]. The power of proteomic techniques is not only limited to distinguishing the diseased state from the healthy state, but it also helps to resolve tumor subtypes. The latter is especially important for the prediction the prognostic features of cancer and for determining the therapeutic strategy to be adopted. It was previously reported that pathologic changes in hematological malignancies are correlated with the protein profiles of cells; 247 different protein spots were identified between the HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia), and Raji (B-cell Burkitt’s lymphoma) cells [102]. In another study, differentially expressed serum proteins were identified in non-Hodgkin’s lymphoma (NHL) patients before treatment, lymphnoditis patients, and healthy adults using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) [103]. Significant differences were observed in different phases of NHL. The study showed that levels of 3 main proteins could be used for the early and differential diagnosis of NHL [103]. Differences in protein levels can be used as a diagnostic marker to characterize tumors. In addition to its contribution to our knowledge of tumor subtypes, proteomics combined with

Table 1. Major important proteins in hematological malignancies Leukemias

Lymphomas

Myelomas

Type of Hematological Malignancy

Major Important Proteins

Acute lymphoblastic leukemia (ALL)

P53 (30, 52, 53), TEL/AML1 (54), E2A/PBX (PBX1) (55), BCR/ABL (56), MLL/AF4 (57), IGH/MYC (58), TCR/RBTN2 (59), TdT (60,61), ABL1/NUP214 (41), P2RY8/CRLF2 (42) and Tal1 (43) proteins

Acute myelogenous leukemia (AML)

P53 (62-66), PML/RARα (67), CEBPA (48), KRAS2 (47), BAALC (68), ERG (69), NPM1 (49), RUNX1/RUNX1T1 (70-72) and CBFB/MYH11 (73-75) proteins

Chronic lymphocytic leukemia (CLL)

P53 (76-78), CD5 (79), CD23 (80,81), CD38 (82) and ZAP70 (83) proteins

Chronic myelogenous leukemia (CML)

P53 (84), BCR/ABL (35) and MDM2 (31) proteins

Hodgkin's lymphomas

CD2, CD3, CD4, CD5, CD7 and CD8 (85), CD15, CD20 and CD30 (86,87) proteins

Non-Hodgkin's lymphomas

P53 (88, 89), CD3, CD4, CD5, CD10 (90), c-myc, BCL2 (91), API2/MLT (92), MALT1 (93) and Cyclin D1 (94), proteins

Multiple Myeloma

N-ras, K-ras (95), P53 (95,96), c-myc (97,98), and Bcl-2 (99), proteins

Ĺ&#x17E;anlÄą-Mohamed et al. Protein profiles in leukemia

other gene expression analyses can be used to characterize the tumor microenvironment, which has profound effects on tumor progression [104,105]. Identifying the characteristics of cancer subtypes leads to a better understanding of chemotherapeutic resistance as well. Use of proteomic methods to identify alterations at the protein level that confer resistance to cancer cells and nullify current chemotherapeutic options is therefore of great interest for use in the development of novel interventions with increased efficacy against resistance. Twodimensional differential gel electrophoresis (2-DE) coupled with mass spectrometry analysis between imatinib-sensitive and resistant KCL22 CML cells showed that there were 27 over-expressed and 24 under-expressed proteins involved in proliferation and apoptosis. Such data may open new ways of determining novel targets for the treatment of drugresistant leukemias [106]. However when mRNA expression levels in these proteins were examined, similar patterns were not observed, indicating that post-translational control is also very important for different protein profiles. This observation highlights the importance of proteomics, in terms of illuminating what accounts for the difference between sensitivity and resistance to chemotherapy in cells. As it helps differentiate resistant subtypes, proteomics techniques can be used for predicting chemotherapeutic susceptibility [107]. The protein content of cancer cells is also subject to changes in response to treatment with drugs and radiation. Therefore, protein profiling can be utilized to compare treated cells and untreated counterparts to better understand the therapeutic mechanisms of action and possible points of intervention for obtaining better responses to current therapies. Protein profiles are not only important for delineating drug mechanisms, but also for monitoring minimal residual disease (MRD) in cancer patients treated with various forms of anti-cancer agents. It was reported that a panel of antibodies could be used in microarrays to trace disease-specific proteins in CLL patient samples that were previously treated with rituximab [108]. A broader analysis of several different CLL-specific antigens would be more accurate, in terms of identifying MRD, than previously adopted assessment of CD20 expression, which cannot be measured accurately during the course of rituximab treatment. In addition to the current advanced techniques for the assessment of cellular proteins, this area is still

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open to development. For instance, 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling coupled with 2-DE and mass spectrometry was shown to yield more reliable results than the conventional methods of analyzing protein levels [109]. Techniques for determining changes in protein profiles Proteins are important targets for drug discovery and are therefore utilized in cancer research because there are defects in the protein machinery of cells undergoing malignant transformation. Identification of protein profiles is clinically promising in the development of potential new drugs to eradicate various malignancies, including, but not limited to, those of hematologic origin. Changes in protein profiles provide a wide variety of critical data regarding various cancers. By examining these alterations proteins that have a profound impact on the progression of diseases can be identified, making the development of individually tailored drugs possible [1]. Because of its importance, recent technologic advances have opened a new era for analyzing changes in protein profiles. To date, many of the high-throughput protein identification and characterization methods developed for proteomics have been applied to protein profiling. Among them, the most widely and efficiently used ones are mass spectrometry, sometimes in combination with different chromatographic methods [110-115], protein microarray [115-118], and high-performance liquid chromatography laser-induced fluorescence (HPLC-LIF) [119, 120]. Mass spectrometry Mass spectrometry is a technique used for the analysis of complex protein samples in order to detect a given set of proteins. Its principle depends primarily on separating ionized molecules according to their mass to charge ratios [2]. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), liquid-chromatography coupled to MALDI tandem mass spectrometry (LCMS/MS), and surface-enhanced laser desorption/ ionization mass spectrometry (SELDI MS) are of great importance to applying mass spectrometry to clinical biomedicine. For the application of mass spectrometry the first step (sample preparation) is the resolution of proteins in complex mixtures obtained from whole organisms, cell lines, tissues, or bodily fluids. The most widely used method for resolution and visualization of proteins for mass

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spectrometry is 2-DE. Chromatographic approaches could also be adopted for better separation of proteins of interest. Protein electrophoresis has remained the most effective way to resolve a complex mixture of proteins since 1970s. For the case of protein profiling, 2-DE is more applicable because complex protein mixtures such as crude cell lysates can be resolved better, not only according to molecular mass but also to isoelectric properties of proteins [121]. Two distinct characteristics of proteins facilitate resolution of protein mixtures by their net charge in the first dimension and by their molecular mass in the second dimension. In protein profiling, protein expressions of 2 given samples (diseased vs. healthy for instance) can be compared, both qualitatively and quantitatively. Differences in protein quantities are indicated by the appearance or disappearance of spots in 2-DE gel and quantitative information about protein levels can be provided by the assessment of the spot intensity in the gel. Resolution and visualization of up to ten thousand proteins in a single sample via 2-DE is a powerful approach for the analysis of samples from a variety of sources, including cell lines and body fluids. Although there are some drawbacks to this methodology, such as poor reproducibility, labor intensiveness, a slow and tedious procedure that can’t be easily automated, and dependence of the results on the expertise of the analyst, it is still an essential component of proteomics for protein profiling. Protein microarray Protein microarray is a technique used in most biomedical applications for determining the presence and quantity of proteins in a biological system. It has great potential to increase the throughput of proteomic research. Although analytical microarrays, functional microarrays, and reverse phase microarrays are 3 types of protein microarrays currently used for studying the biochemical activity of proteins, analytical microarrays are typically used to profile a complex mixture of proteins, in terms of measuring binding affinities, specificities, and protein expression levels of the proteins in the mixture [122]. It is a very powerful technique that can be used to monitor differential expression of proteins for clinical diagnosis, prediction of prognosis, and identification of targets for therapy. In general, first a library of antibodies, aptamers, or affibodies is arrayed on a glass microscope slide, and then the prepared array is probed with the protein solution. To date, protein profiling

Şanlı-Mohamed et al. Protein profiles in leukemia

using analytical microarray has been implemented successfully for the assessment of responses to environmental stress and differences between healthy and diseased tissues [123]. When positive and negative controls are used carefully there aren’t many drawbacks to this methodology, yet microarray studies are limited due to their cost. High-performance liquid chromatography laserinduced fluorescence High-performance liquid chromatography laserinduced fluorescence (HPLC-LIF) is another technique used for the detection of differentially expressed proteins by simultaneously recording spectra and chromatograms of physiological samples in a short time [124]. Though to the best of our knowledge this technique has not been utilized in hematologic malignancies, it is useful in the early diagnosis of certain types of cancer, including cervical cancer and oral cancer [119,120,124-126]. The above-mentioned technique is composed of the combination of laser-induced fluorescence, an ultra-sensitive optical method, and high-performance liquid chromatography. The principle of HPLC-LIF is based on recording the chromatographic peaks and corresponding fluorescence spectra at the same time. This makes it possible to differentiate diseased and healthy, treated and untreated, and/or experimental and control sample protein profiles, even in femto/subfemtomole quantities [126]. Even though use of the technique is restricted, as compared to the others, there is no doubt that it is a very powerful and sensitive method [125]. Drawbacks of protein profiling Although analysis of important proteins in biological systems is important, there are several drawbacks of protein profiling due to techniques like mass spectrometry in combination with separation tools such as 2-DE [114,127,128]. First of all, these techniques are labor intensive and time consuming for the analysis of proteins. Improved robotics may increase the frequency with which these techniques are utilized, as well as their efficiency. Secondly, 2-DE lacks the sensitivity to detect low quantities of proteins and therefore requires a significant quantity of biological material [129,130]. Additionally, most of the time high-quantity proteins can mask low-quantity proteins that may be important biomarkers in hematologic malignancies. To overcome this type of drawback immunodepletion and multidimensional chromatography may be a reasonable solution [131].

Şanlı-Mohamed et al. Protein profiles in leukemia

Through sample purification, low-quantity proteins may be lost due to interactions with other highquantity proteins; therefore, all steps of purification must be analyzed. Sample collection, processing, and, ultimately, sample measurement are also very crucial for the utilization of protein profiling in many clinical settings. Consistency, use of strict protocols, running replicates for each sample, and increasing

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the sample count can be a general solution to eliminating such drawbacks. Other obstacles to the use of techniques in protein profiling can be ascribed to poor resolution of spots after 2-DE, non-identification of mass spectrometry peaks, and the limitations of databanks for unknown proteins. Protein microarray [132,133] and HPLC-LIF, alternative techniques for protein profiling, have fewer drawbacks when

Table 2. Advantages and disadvantages of protein profiling techniques Different Techniques

Advantages

Disadvantages

2D Gel Electrophoresis

• High resolution

• Slow

• Quantitative

• Lacks automation

• Qualitative

• Poor reproducibility

• Sample variety (cell lines and body fluids)

• Labor intensive

• Many protein analyses in a single run

• Tedious procedure • Requires a significant quantity of sample • Lacks sensitivity for low-quantity proteins

SELDI-TOF MS

• Fast

• Limited to low-MW proteins

• Good for diagnostic patterns of disease

• Preferably used in serum samples

• Limited resolution • Accuracy of quantification is restricted MALDI-TOF MS

•Fast

• Limited databank information

• Buffer/salt tolerance

• Limited resolution of peaks

• Off-line

• Low dynamic range of detection

• Mass accuracy • Mixture of samples okay • Small and large polypeptide analysis Protein Microarrays

• Highly informative

• Difficult to achieve general binding conditions

• Qualitative

• Time consuming

• Quantitative

• Costly procedure

• Powerful technique • Target specific • Successful in improving sensitivity and specificity LC-MS/MS

• Straightforward protein identification

• Slow, but can be automated

• High sensitivity

• Slow, but can be automated it is used two

• Good resolution

• Analysis of a limited number of samples

times • Characterizes post-translational modifications HPLC-LIF

• Powerful • Time efficient • Sensitive • Fast • High detection capacity • Good for use in early diagnosis

• Low reproducibility

Şanlı-Mohamed et al. Protein profiles in leukemia

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compared with the aforementioned methods, but they are preferred less because they require welltrained experts. The advantages and disadvantages of the techniques used for protein profiling are summarized in Table 2. Conclusion and future perspectives Discovery of leukemia-associated protein patterns is an excellent tool for aiding diagnosis, follow-up treatment, and predicting clinical outcomes. In the future, the determination of individual protein profiles in hematological malignancies and other types of cancers, together with the investigation of genomic profiles is expected to contribute to the development of tailor-made treatments. Protein profiling strategies that identify the pattern of changes in protein levels are therefore not only promising for hematological diseases, but also for other types of cancers, and may lead to the development of novel tools and interventions of unprecedented diagnostic and/or prognostic value. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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Acknowledgment This study was supported by the Turkish Academy of Sciences, Outstanding Young Investigator Programme.

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Research Article

Determination of apoptosis, proliferation status and O6-methylguanine DNA methyltransferase methylation profiles in different immunophenotypic profiles of diffuse large B-cell lymphoma Diffüz büyük B-hücreli lenfomanın farklı immünofenotipik profillerinde apoptozis, proliferasyon durumu ve O6-metilguanin DNA metiltransferaz metilasyon profillerinin tespiti Nilay Şen Türk1, Nazan Özsan2, Vildan Caner3, Nedim Karagenç3, Füsun Düzcan4, Ender Düzcan1, Mine Hekimgil2 1Department

of Pathology, Faculty of Medicine, Pamukkale University, Denizli, Turkey of Pathology, Faculty of Medicine, Ege University, İzmir, Turkey 3Department of Medical Biology, Faculty of Medicine, Pamukkale University, Denizli, Turkey 4Department of Medical Genetics, Faculty of Medicine, Pamukkale University, Denizli, Turkey 2Department

Abstract Objective: Our aim was to investigate the expression of apoptosis-associated proteins (bcl-2, bcl-xl, bax, bak, bid), apoptotic index (AI) and proliferation index (PI) in germinal center B-cell-like immunophenotypic profile (GCB) and non-GCB of diffuse large B-cell lymphoma (DLBCL). Materials and Methods: The methylation status of the promoter region of O6-methylguanine-DNA yerine O6-methylguanine-DNA methyltransferase (MGMT) gene and its relation with immunophenotypic differentiation of DLBCLs were also investigated. 101 cases were classified as GCB (29 cases) or non-GCB (72 cases). Apoptosis-associated proteins and PI were determined by IHC, and TUNEL method was used to determine AI. MGMT methylation analysis was performed by real-time PCR. Results: The PI was significantly higher in GCB compared with non-GCB (p=0.011). Percentage of cells stained with bcl-6 was positively correlated with the percentage of cells expressing bcl-2 (p=0.023), AI (p=0.006) and PI (p<0.001), while a significant negative correlation was observed with the percentage of cells expressing bax (p=0.027). The percentage of cells stained with MUM1 showed a significantly positive correlation with the percentage of cells expressing bcl-xl (p=0.003), bid (p=0.002), AI (p<0.001), and PI (p=0.001). MGMT methylation analysis was performed in 95 samples, and methylated profile was found in 31 cases (32.6%). GCB was found in 6 cases (22.2%) and non-GCB was determined in 25 cases (36.8%) out of 31 with MGMT methylated samples. There was no significant association between MGMT methylation status and immunophenotypic profiles (p=0.173).

Address for Correspondence: Asst. Prof. Nilay Şen Türk, Department of Pathology, Faculty of Medicine, Pamukkale University, Denizli, Turkey Phone: +90 258 361 39 16 E-mail: sennilay@hotmail.com doi:10.5152/tjh.2010.37

Türk et al. Apoptosis, proliferation in large B-cell lymphoma

Turk J Hematol 2011; 28: 15-26

Conclusion: These results suggest that bcl-6 protein expression may be responsible for the high PI in GCB. Additionally, we found that apoptosis-associated proteins were not significantly associated with immunophenotypic profiles. (Turk J Hematol 2011; 28: 15-26) Key words: Lymphoma, B-cell, apoptosis, proliferation, methylation Received: March 1, 2010

Accepted: June 29, 2010

Özet Amaç: Diffüz büyük B-hücreli lenfoma (DBBHL)’nın germinal merkez B-hücresi benzeri (GCB) ve non-GCB profillerinde apoptozis-ilişkili proteinlerin (bcl-2, bcl-xl, bax, bak, bid) ekspresyonunu, apoptotic indeksi (AI) ve proliferasyon indeksi (PI)’ni araştırmaktır. Ayrıca, O6-methylguanine-DNA methyltransferase (MGMT) geninin promoter bölgesinin metilasyon durumunu ve onun DBBHL’nın immünofenotipik diferansiyasyonuyla ilişkisini araştırmaktır. Yöntem ve Gereçler: 101 olgu GCB (29 olgu) ve non-GCB (72 olgu) olarak sınıflandırıldı. Apoptozisilişkili proteinler ve PI immünohistokimyasal olarak saptandı ve TUNEL yöntemi AI’i belirlemek için kullanıldı. MGMT metilasyon analizi, real-time PCR ile gerçekleştirildi. Bulgular: PI, non-GCB ile karşılaştırıldığında GCB’de anlamlı şekilde yüksek saptandı (p=0.011). Bcl-6 ile p: PI, non-GCB ile karşılaştırıldığında GCB’de anlamlı şekilde yüksek saptandı (p=0.011). Bcl-6 ile pozitif boyanan hücrelerin yüzdesi bcl-2 (p=0.023), AI (p=0.006), ve PI (p<0.001) eksprese eden hücrelerin yüzdesi ile pozitif şekilde korele iken, bax eksprese eden hücrelerin yüzdesi ile negatif korelasyon gözlendi (p=0.027). MUM1 ile boyanan hücrelerin yüzdesi bcl-xl (p=0.003), bid (p=0.002), AI (p<0.001) ve PI (p=0.001) eksprese eden hücrelerin yüzdesi ile anlamlı şekilde pozitif korelasyon gösterdi. MGMT metilasyon analizi 95 örneğe uygulandı ve metilasyon profili 31 olguda (32.6%) saptandı. 31 MGMT metile örnekten 6 olgunun (22.2%) GCB ve 25 olgunun (36.8%) nonGCB olduğu belirlendi. MGMT metilasyon durumu ve immünofenotipik profiler arasında anlamlı ilişki saptanmadı (p=0.173). Sonuç: Bu bulgular, bcl-6 protein ekspresyonunun GCB’de yüksek PI’inden sorumlu olabileceğini öne sürmektedir. Ek olarak, apoptozis-ilişkili proteinlerin immünofenotipik profilerle anlamlı ilişki göstermediğini saptadık. (Turk J Hematol 2011; 28: 15-26) Anahtar kelimeler: Büyük B-hücreli lenfomalarda apoptosis ve proliferasyon Geliş tarihi: 1 Mart 2010

Kabul tarihi: 29 Haziran 2010

Introduction Diffuse large B-cell lymphoma (DLBCL) constitutes the largest group of aggressive lymphomas in adults and 30-40% of adult non-Hodgkin lymphomas (NHL) in western countries [1,2]. DLBCLs show diversity in clinical presentation, morphology and genetic and molecular properties, which suggests that these tumors represent a heterogeneous group of neoplasms rather than a single clinicopathologic entity [2,3]. Therefore, patients who are diagnosed as DLBCL could show remarkably different history, clinical behavior and outcome [1]. Different mechanisms such as deregulation of cell cycle and apoptotic pathways are involved in the pathogenesis of DLBCL. As for the molecular pathogenesis of DLBCL, distinct chromosomal translocations and aberrant somatic hypermutations as well as numerical chromosomal anomalies such as duplications and deletions, which are also common to other malignancies, have been reported [4-7].

DLBCL originates from germinal center (GC) and post-GC B-cells that normally have encountered with antigen [8,9]. Different methods are applied to determine B-cell differentiation antigens in DLBCL. In spite of the distinct advantages of cDNA and oligonucleotide microarray techniques, immunohistochemistry is a commonly used method to determine the GC B-cell-like (GCB) and non-GCB-like profiles because it is cheap and easily applied [10-12]. Hans et al. [11] reported that the classification of DLBCL into GCB and non-GCB profiles based on CD10/bcl6/ MUM1 immunophenotypic differentiation is prognostically relevant to the cDNA classification in 71% of GCB and in 88% of non-GCB. It is well known that the expression of bcl-6 and CD10 are associated with increased apoptosis and proliferation in lymphoid malignancies [13-18]. Bai et al. [10] reported that increased expression of apoptotic index (AI) in DLBCL with GCB profile is associated with high expression of the pro-apoptotic proteins (bax, bak, bid) and low expression of the antiapoptotic protein (bcl-xl). However, data related to apoptosis and pro-

Turk J Hematol 2011; 28: 15-26

liferation status in CD10/bcl-6/MUM1 immunophenotypic differentiation profiles is considerably limited. Although the immunohistochemical expression of the apoptosis-associated bcl-2 family proteins, bcl-2, bax, bak, and mcl1, was reported in DLBCLs [19-28], the expression levels of bcl-xl, bad, bid proteins and their relations with the status of apoptosis and proliferation were not extensively analyzed in these lymphomas [25]. Altered DNA methylation profile has been comprehensively studied in the pathogenesis of cancer. Compared with normal cells, cancer cells frequently demonstrate genome-wide hypomethylation, hypermethylation of tumor suppressor gene, and loss of genomic imprinting [29]. In human cancers, the gene encoding the DNA-repair enzyme O6methylguanine-DNA methyltransferase (MGMT) is not commonly mutated or deleted. Loss of MGMT expression is mainly due to epigenetic changes, specifically methylation of the promoter region [1,30]. MGMT protects cells from the toxicity of environmental and therapeutic alkylating agents, which frequently target the O6 position of guanine. Inactivation of the MGMT gene via hypermethylation of its promoter region increases sensitivity of cells to the genotoxic effect of alkylating agents both in vitro and in vivo [1,3]. Recent research has focused on the relationship between MGMTmethylation status and immunophenotypic differentiation in DLBCLs [30,31]. The aim of this study was to investigate the expression profiles of apoptosis-associated proteins (bcl-2, bcl-xl, bax, bak, bid), apoptotic index (AI), and proliferation index (PI) in GCB and non-GCB immunophenotypic profiles of DLBCL. In addition, the methylation status of the promoter region of the MGMT gene and its relation with immunophenotypic differentiation of DLBCLs were investigated.

Materials and Methods Materials A total of 101 cases of de novo DLBCLs, diagnosed according to the World Health Organization (WHO) classification [2], were obtained from the files of the Department of Pathology, Faculty of Medicine, Ege University. Clinicopathological parameters for all patients were obtained from the pathology records. Ethical committee approval was obtained for this study.

Türk et al. Apoptosis, proliferation in large B-cell lymphoma

Immunohistochemistry For immunohistochemical staining, sections of 5-μm-thickness were cut from formalin-fixed, paraffin-embedded tissue blocks and placed on electrostatic-charged, poly-L-lysine-coated slides (X-traTM, Surgipath Medical Industries, Richmond, IL, USA). Sections were dehydrated at 60ºC for a minimum of 2 hours (h). All immunostaining procedures including deparaffinization and antigen retrieval processes were performed on BenchMark XT® automated stainer (Venatana Medical Systems, USA). After counterstaining of the slides with hematoxylin in automated stainer, dehydration, incubation in xylene and mounting processes were performed manually, and immunostaining procedure was completed. Bcl-6 (dilution: 1/20, clone: P1F6, Dako SA, Glostrup, Denmark), CD10 (dilution: 1/25, clone: 56C6, Spring Bioscience, Pleasanton, CA, USA), IRF4/MUM1 (dilution: 1/25, clone: MUM1p, Dako SA, Glostrup, Denmark), bcl-2 (dilution: 1/40, clone: bcl2/100/DS, Novocastra, Newcastle upon Tyne, UK), bcl-xl (dilution: 1/20, clone: 2H12, Zymed, South San Francisco, CA, USA), bax (dilution: 1/200, code: A3533, Dako SA, Glostrup, Denmark), bak (dilution: 1/100, code: A3538, Dako SA, Glostrup, Denmark), bid (dilution: 1/100, clone: NB110-40718, Novus, Littleton, CO, USA), and Ki-67 (dilution: 1/150, clone: MIB-1, Dako SA, Glostrup, Denmark) were used as primary antibodies. Reactive lymph nodes and normal thymic tissue samples were used as positive controls. Negative controls were treated with the same immunohistochemical method by omitting the primary antibody. At least 10 fields selected on the basis that they contained immunopositive cells were counted by using the 40x objective lens on the light microscope. The number of immunopositive cells was divided by the total number of the counted cells, and the expression was defined as the percentage of positive cells. CD10, bcl-6, and MUM1 proteins were considered positive when at least 25% of neoplastic cells were immunopositive according to the previously defined criteria [12]. The CD10/bcl-6/MUM1 immunophenotypes and their designation to GCB and non-GCB profiles were determined according to the classification by Hans et al. [11]. The expressions of bcl-2, bcl-xl, bax, bak, and bid proteins were considered positive when at least 10% of neoplastic cells were immunopositive [10]. PI with

Türk et al. Apoptosis, proliferation in large B-cell lymphoma

Ki-67 was determined as the percentage of positive cells within the total number of the counted cells. Tunel Method The terminal deoxynucleotidyl-transferase (TdT)mediated in situ labeling technique (TUNEL; in situ Cell Death Detection Kit, POD, Roche) was performed on the 5-μm-thick sections of formalinfixed, paraffin-embedded tissue for demonstration of DNA fragmentation. Briefly, after deparaffinization and dehydration, slides were rinsed in phosphate-buffered saline (PBS) (pH 7.4). The peroxidase activity was blocked by incubation for 15 minutes (min) in 3% hydrogen peroxide in PBS at room temperature. Tissue sections were digested by incubation for 30 min with proteinase K (20 μg/ml) at 37ºC, and then were rinsed in PBS. TUNEL reaction mixture was prepared according to the manufacturer’s recommendations, and a mixture of 50 μL per slide was added. Slides were incubated for 1 h at 37ºC in dark. One positive control and two negative controls were included in each set of experiments. Reactive lymph nodes were used as positive controls. Negative controls were treated similarly by omitting the TdT reaction step. Slides, once again, were rinsed in PBS. To examine the slides in light microscope, 50 μl Converter-peroxidase (POD) was added per slide and slides were incubated for 30 min at 37ºC in humid and dark conditions. Slides were rinsed in PBS, and then were incubated with 3,3’-diaminobenzidine tetrahydrochloride (DAB Substrate, Roche) for 10 min at room temperature. Slides were counterstained with Harris’ hematoxylin and mounted. The evaluation of the results was performed as Bai et al. [32] had described previously. Morphologically intact TUNEL-positive cells were considered as positive and referred to as apoptotic cells. The number of apoptotic cells was recorded by using the 40X objective in at least 10 randomly selected fields. The AI was expressed as a percentage of the number of apoptotic cells within the total number of counted cells. O6- Methylguanine-DNA Methyltransferase Methylation Analysis Genomic DNAs were extracted from four or five 5-μm-thick sections of formalin-fixed, paraffinembedded tissue, using a commercial kit (QIAamp DNA Mini Kit, Qiagen, Valencia, CA). Briefly, after

Turk J Hematol 2011; 28: 15-26

deparaffinization with xylene, tissue samples were digested with proteinase K treatment. DNAs that emerged from cells were collected in the column through ethanol treatment. Cellular remnants and chemical agents were removed by washing buffer, and genomic DNA was eluted in DNAase-free buffer and stored at -20ºC. Commercial kit (EZ Methylation Gold-Kit, Zymo Research, Orange, CA) was used for bisulfite modification of isolated DNA. Briefly, DNA concentration was measured and arranged to make 500 ng/μL, and 130 μL conversion reagent was added to 20 μL DNA sample. Samples were incubated for 10 min at 98ºC and then for 2.5 h at 64ºC. Samples were transferred to column with 600 μL M-binding buffer. After homogenization, samples were centrifuged. Samples were rinsed with 100 μL M-wash buffer, and were then incubated in 200 μL M-desulfonation buffer for 20 min at room temperature. After incubation, samples were rinsed two times, and then 10 μL M-elution buffer was added. Sodium bisulfite-treated genomic DNA was stored at -20ºC. Primer and probe sequences of the promoter region of MGMT gene were used, which target the localization of 1067 -1149 bp amplicon, as described by Esteller et al. (33) (GenBank Accession Number: X61657). Final reaction volume for analysis of both methylated and unmethylated profiles was performed in 20 μL volume: 2 μl from each primer (final concentration: 0.5 μM), 2 μl TaqMan probe (final concentration: 0.2 μM), 4 μl LightCycler TaqMan Master mixture, 5 μl DNA sample, and 5 μl polymerase chain reaction (PCR)-grade water. The cycling conditions for methylation-specific PCR were: 10 min at 95ºC for Taq activation, followed by 45 cycles of 95ºC for 10 seconds (sec), 60ºC for 20 sec, and 72ºC for 1 sec for amplification. PCR products were run on a 3% agarose gel containing ethidium bromide. Statistical Analysis χ2 test, Mann-Whitney test and analysis of variance were applied for statistical analysis. MannWhitney test was used to analyze the relationship of positivity of apoptosis-related proteins, PI (Ki-67) and AI with immunophenotypic differentiation profiles (CD10/bcl6/MUM1). Spearman correlation coefficient test was used to analyze any significant relationship between PI, AI and percentage of posi-

Türk et al. Apoptosis, proliferation in large B-cell lymphoma

Turk J Hematol 2011; 28: 15-26

tive cells with apoptosis-related proteins, CD10, bcl6, and MUM1, whether evaluated as positive or negative using cut-off values. The results were considered as statistically significant when p<0.05. All the statistics were calculated using the SPSS 11.0 program (SPSS 11.0 Inc., Chicago, IL, USA) for Windows.

Results Patients [53 males (52.5%), 48 females (47.5%)] were aged between 19 and 84 (mean 55.53±14.12). Localization was nodal in 42 (41.6%) cases andextranodal in 58 (57.4%) cases, while one case was unknown. Immunohistochemical expression of bcl-6, CD10, MUM1, bcl-2, bcl-xl, bax, bak, and bid proteins was found in 51/101 (50.5%), 20/101 (19.8%), 49/101 (48.5%), 32/101 (31.7%), 8/101 (7.9%), 67/101 (66.3%), 82/101 (81.2%), and 67/99 (66.3%) cases, respectively (Figure 1 A-E). The mean PI was 46.15% (±32.82) as assessed by Ki-67 staining. The mean AI was 1.94% (±2.68) as determined by the TUNEL method (Figure 1 F). Two major immunophenotypic profiles were distinguished according to the pattern of differentiation described by Hans et al. [11]: (a) GCB immunophenotypic profile: 29 cases (CD10+: 20 cases, CD10- / bcl-6+ /MUM1-: 9 cases) and (b) non-GCB immunophenotypic profile: 72 cases (CD10- /bcl-6-: 47 cases, CD10- /bcl-6+ /MUM1+: 25 cases) (Table 1). Mann-Whitney test was used to analyze the association of two immunophenotypic differentiation profiles in relation to the AI, the expression levels of apoptosis-related proteins and PI (Ki-67) (Table 2). Compared to the non-GCB profile, the GCB profile was significantly associated with a higher PI (p=0.011). However, no other significant correlations were determined between the two major differentiation immunophenotypic profiles regarding AI and the expression levels of apoptosis-related proteins bcl-2, bcl-xl, bax, bak, and bid (p>0.3). The percentage of cells stained by CD10, bcl-6 and MUM1 were analyzed in relation to apoptosisrelated proteins, AI and PI by Spearman correlation coefficient test, regardless of previous evaluation with cut-off points to consider the results as positive or negative (Table 3). The expression of bcl6 was positively correlated with the expression of bcl2

(r=0.226, p=0.023), the AI (r=0.272, p=0.006) and the PI (r=0.515, p<0.001), but a significantly negative correlation was observed with the expression of bax (r=-0.221, p=0.027). The expression of MUM1 showed significant positive correlation with the expression of bcl-xl (r=0.295, p=0.003), bid (r=0.313, p=0.002), AI (r=0.341, p<0.001), and PI Table 1. CD10/bcl-6/MUM1 immunophenotypic differentiation profiles (Total n=101) Immunophenotypic differentiation profiles

n (%)

Germinal center B-cell-like profile CD10+

bcl-6bcl-6+

CD10-

bcl-6+

MUM1-

1(1%)

MUM1+

2 (2%)

MUM1-

13 (12.8%)

MUM1+

4 (4%)

MUM1-

9 (8.9%)

TOTAL

29 (28.7%)

Non-germinal center B-cell-like profile CD10-

bcl-6-

CD10-

bcl-6+

MUM1+

18 (17.8%)

MUM1-

29 (28.7%)

MUM1+

25 (24.8%)

TOTAL

72 (71.3%)

Table 2. The immunophenotypic differentiation profiles in relation to the apoptotic index (AI), the expression of apoptosis related proteins, and the proliferation index (PI) (Mann-Whitney test) Percentage of Immunophenotypic positive differentiation expression profile AI bcl-2 bcl-xl bax bak bid PI (Ki-67)

Mean rank

p values 0.941

1.93

GCB profile

51.33

1.91

non-GCB profile

50.87

18.52

GCB profile

53.52

15.69

non-GCB profile

49.99

1.86

GCB profile

46.95

6.16

non-GCB profile

52.63

43.07

GCB profile

45.81

50.87

non-GCB profile

53.09

58.34

GCB profile

51.36

60.80

non-GCB profile

50.85

42.00

GCB profile

45.34

51.81

non-GCB profile

51.93

59.17

GCB profile

62.66

41.69

non-GCB profile

46.31

0.561 0.341 0.257 0.937 0.297 0.011*

GCB profile: Germinal center B-cell-like profile, Non-GCB profile: Non-germinal center B-cell-like profile *indicates the statistically significant correlations (p<0.05)

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Turk J Hematol 2011; 28: 15-26

Figure 1. Immunohistochemical expression of antiapoptotic proteins (a) bcl2 and (b) bcl-xl and apoptotic proteins (c) bax, (d) bak and (e) bid in neoplastic cells of diffuse large B-cell lymphomas (a-d, x400; e, x200). (f) Staining of apoptotic cells by the TUNEL method (x400)

(r=0.330, p=0.001). Similarly, the Spearman correlation coefficient test was used to analyze the relations between apoptosis-related proteins, AI and PI. The expression of bcl-xl protein showed significant posi-

tive correlation with the expression of bak (r=0.198, p=0.047) and bid (r=0.198, p=0.049) proteins. The expression of bax protein showed significant positive correlation with the expression of bak (r=0.229,

Türk et al. Apoptosis, proliferation in large B-cell lymphoma

Turk J Hematol 2011; 28: 15-26

Table 3. Correlations between CD10, bcl-6 and MUM1 proteins and the apoptosis related proteins, the apoptotic index (AI), and the proliferation index (PI) (Spearman’s correlation test) bcl-2 CD10 bcl-6 MUM1

bcl-xl

bax

bak

bid

r=0.092

r=0.050

r=0.055

r=-0.024

r=-0.001

r=0.130

r=0.148

p=0.360

p=0.619

p=0.585

p=0.811

p=0.991

p=0.195

p=0.139

r=0.226

r=0.176

r=-0.221

r=0.123

r=0.114

r=0.272

r=0.515

p=0.023*

p=0.078

p=0.027*

p=0.220

p=0.260

p=0.006*

p<0.001*

r=0.122

r=0.295

r=0.009

r=0.152

r=0.313

r=0.341

r=0.330

p=0.225

p=0.003*

p=0.932

p=0.129

p=0.002*

p<0.001*

p=0.001*

r, Spearman’s correlation coefficient. The positive or negative sign of the Spearman’s correlation coefficient r expresses significant (p<0.05) or nonsignificant (p=0.05 or p>0.05) positive or negative correlations between two continuous variables *indicates the statistically significant correlations (p<0.05)

a Amplification Curves Case no: 1 Case no: 2 Negative template control

2.5 2.3

Fluodescence (530)

2.1 1.9 1.7 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1 2

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Cycles

b Amplification Curves Case no: 1 Case no: 2 Negative template control

Fluodescence (530)

0.39 0.34 0.29 0.24 0.19 0.14 0.09 0.04

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Cycles Figure 2. (a) Amplification curve after real-time PCR with methyleted specific primer and probe set. (b) Amplification curve after real-time PCR with unmethyleted specific primer and probe set

p=0.022) and bid (r=0.223, p=0.027) proteins. The expression of bak protein also showed significant positive correlation with the expression of bid (r=0.214, p=0.033) protein. The AI showed significant positive correlation with the PI (r=0.349, p<0.001).

MGMT promoter methylation analysis was performed in 95 patients with DLBCL (Figure 2). MGMT methylated profile was found in 31/95 (32.6%) samples while MGMT unmethylated profile was determined in 64/95 (67.4%) samples. The GCB profile was determined in 6 cases (22.2%) and non-GCB profile in 25 cases (36.8%) out of the 31 with MGMT methylated profile (Table 4). There was no significant association between MGMT promoter methylation status and the two immunophenotypic differentiation profiles (p=0.173) (Table 5). There was also no significant association between MGMT promoter methylation status and the expression levels of apoptosis-related proteins, the AI and the PI (p>0.06).

Discussion In the present study, we found that in DLBCLs, the GCB profile was significantly associated with a higher PI compared to the non-GCB profile. In addition, the percentage of cells reacting with the GC B-cell-related blc-6 protein showed significant positive correlation with PI. The correlation between bcl-6 and proliferation is not conclusive in the published data. A review of the literature indicates that bcl-6 could have a role both as stimulator or inhibitor of cell cycle progression and proliferation [16,3237]. Some in vitro studies showed that bcl-6 expression was associated with delaying cell cycle progression and decreased proliferation [15,35]. Albagli et al. [16] demonstrated that bcl-6 mediates growth suppression associated with impaired S phase progression in human U2OS osteosarcoma cells. Hosokawa et al. [35] established Ba/F3 pro-B cells carrying a human bcl-6 transgene, and revealed that

TĂźrk et al. Apoptosis, proliferation in large B-cell lymphoma

Table 4. The immunophenotypic differentiation profiles in relation to the MGMT methylation status MGMT methylation status Methylated

Total number of

Unmethylated

cases

GCB

6 (22.2%)

21 (77.8%)

non-GCB

25 (36.8%)

43 (63.2%)

Total

31 (32.6%)

64 (67.4%)

MGMT, O6- methylguanine-DNA methyltransferase GCB profile: Germinal center B-cell-like profile, Non-GCB profile: Non-germinal center B-cell-like profile

Table 5. Association between MGMT methylation status and expression of CD10/bcl-6/MUM1 proteins (Ď&#x2021;2 test) Parameter

MGMT p methylated (%) values*

CD10 expression bcl-6 expression

5 (26.3%)

26 (34.2%)

14 (29.2%)

17 (36.2%)

12 (26.7%)

19 (38.0%)

CD10/bcl-

CD10+/bcl-6-/MUM1-

6/MUM1

CD10+/bcl-6+/MUM1-

5 (38.5%)

MUM1 expression

coexpression

CD10+/bcl-6-/MUM1+

CD10+/bcl-6+/MUM1+

CD10-/bcl-6-/MUM1+

4 (23.5%)

CD10-bcl-6-/MUM1-

13 (46.4%)

CD10-/bcl-6+/MUM1-

1 (12.5%)

CD10-/bcl-6+/MUM1+

8 (34.8%)

0.514 0.469 0.242 0.729

MGMT, O6- methylguanine-DNA methyltransferase *indicates the statistically significant correlations (p<0.05)

induced bcl-6 protein downregulates the expression of cyclin A2 and inhibits cell proliferation, though Shaffer et al. [34] demonstrated that bcl-6 may induce cell cycle progression and maintain proliferation by blocking the expression of the cyclindependent kinase inhibitor p27 and by repressing blimp-1 expression, which decreases c-myc expression. Furthermore, Allman et al. [37] revealed that bcl-6 protein expression was 34-fold higher in rapidly proliferating GC B-cells than in the resting B-cells. Bai et al. [18] suggested that the resistance to antiproliferative signals through the P19 (ARF) -p53 pathway and downregulation of the expression of cyclin-dependent kinase inhibitor p27 are at least partly responsible for the association between bcl-6 and increased proliferation in DLBCL. Xu et al. [38] investigated the role of bcl-6 gene rearrangement

Turk J Hematol 2011; 28: 15-26

and bcl-6 expression in subgroups of DLBCLs and showed that DLBCLs with bcl-6 gene rearrangement had higher proliferative activity than those without bcl-6 gene rearrangement. On the basis of the previously mentioned results, results of this study suggest that bcl-6 protein expression could be responsible for high PI in the GCB profile of DLBCL. Additionally, in this study, bcl-6 expression showed significant positive correlation with the AI and anti-apoptotic protein bcl-2 expression and negative correlation with pro-apoptotic protein bax expression. In association with the proliferation of bcl-6, the data in the literature indicates that bcl-6 may have a role as stimulator or inhibitor of apoptosis [15,16,39-41]. Some studies showed that bcl-6 may protect cells from apoptosis [39,41]. Kojima et al. [39] demonstrated that bcl-6 may have a stabilizing role to protect spermatocytes from heat shockinduced apoptosis in bcl-6-deficient mice. Baron et al. [41] showed that the human programmed cell death-2 (PDCD2) gene is a target of bcl-6 repression in Epstein-Barr virus-negative Burkitt lymphoma cell line expressing high levels of bcl-6. Furthermore, they immunohistochemically demonstrated the inverse relationship between bcl-6 and PDCD2 expression in human tonsils [41]. Consequently, they proposed that bcl-6 may downregulate apoptosis by means of its repressive effects on PDCD2. However, some other studies suggested that high expression of bcl-6 may induce apoptosis [1416,40]. Albagli et al. [16] used the human osteosarcoma cell line U2OS transfected with bcl-6 and demonstrated that bcl-6 mediates dose-dependent growth suppression, which is associated with impaired S phase progression and trigger of apoptosis. Yamochi et al. [14] showed that viability of CV-1 and HeLa cells infected with a recombinant adenovirus expressing bcl-6 was markedly reduced due to apoptosis. Furthermore, induction of apoptosis by bcl-6 overexpression was preceded by downregulation of apoptosis repressors bcl-2 and bcl-xl, which suggests that bcl-6 might also regulate the expression of these apoptosis repressors [14]. Bai et al. [18] found that high expression of bcl-6 shows significant correlation with negative bcl-2 expression. They suggested that the association between increased bcl-6 expression and increased apoptosis in DLBCL might be due, at least to some extent, to the downregulation of bcl-2, which is induced by

Turk J Hematol 2011; 28: 15-26

bcl-6 overexpression [18]. In contrast to previous studies, in this study, we determined that bcl-6 expression shows a significantly positive correlation with bcl-2 expression. Moreover, we found that bcl-6 expression shows significant negative correlation with pro-apoptotic protein bax expression. It is well known that apoptosis-related proteins bcl-2 and bclxl show anti-apoptotic effect, whereas bax, bak and bid show pro-apoptotic function. These proteins, as members of the bcl-2 family, exert their specific effects by dimerizing with themselves or with each other [25]. If the balance favors the presence of free bcl-2, apoptosis is inhibited, whereas when bax predominates, apoptosis is initiated [25]. Thus, the ratio of the anti-apoptotic to the pro-apoptotic proteins determines whether a given cell will respond to or ignore an apoptotic stimulus [25]. On the basis of these results, we suggest that pro-apoptotic function of bcl-6 partly occurs independent of an anti-apoptotic effect of bcl-2 and pro-apoptotic effect of bax. In this study, no significant correlations were determined between two major immunophenotypic differentiation profiles regarding AI and the expression levels of apoptosis-related proteins bcl-2, bcl-xl, bax, bak, and bid. However the percentage of cells positive for MUM1, which is major marker of nonGCB immunophenotypic differentiation profile, showed a significantly positive correlation with the positivity percentage of anti-apoptotic protein bcl-xl, pro-apoptotic protein bid, AI, and PI. Bai et al. [10] determined that the expression of MUM1 showed significant negative correlation with the expression of bax and bid and significant positive correlation with the expression of bcl-xl. They proposed that this result may provide an explanation for the significant positive correlation between MUM1 and bcl-xl expression in their study, because the MUM1 (IRF4) gene is also a nuclear factor-Kappa B target. Nuclear factorKappa B, depending on the stimulus and the cellular context, can activate pro-apoptotic (e.g. CD95, CD95L, TRAIL receptors), anti-apoptotic (c-FLIP, bcl2, bcl-xl, c-IAP1, c-IAP2) and cell cycle (cyclin D1, cyclin D2, c-myc) genes [32]. This status may explain how MUM1 expression shows significant positive correlation with pro-apoptotic protein bid, AI, PI, as was also shown in the results of Bai et al. [10]. In this study, we determined that DLBCLs frequently express bcl-2 family member apoptosisrelated proteins as bcl-2 in 31.7%, bcl-xl in 7.9%, bax

TĂźrk et al. Apoptosis, proliferation in large B-cell lymphoma

in 66.3%, bak in 81.2%, and bid in 66.3%. Bairey et al. [25] examined the role of bcl-2 family proteins in aggressive NHLs and determined that of the entire group of 44 samples, 25 (57%) showed bcl-2 staining, 11 (25%) showed bcl-xl staining, 17 (39%) showed bax staining, and 16 (36%) showed bak staining. Among these proteins, bcl-2, the most exclusively investigated, is a potent suppressor of apoptosis and is determined in unexpectedly high levels probably in all cancers of humans [50]. Simonian et al. [43] suggested that bcl-2 and bcl-xl can downregulate or upregulate apoptosis. Sclaifer et al. [44] showed that all cases with a positive expression of bax expressed either bcl-2 or mcl-1 anti-apoptotic proteins, suggesting that the presence of bax in the tumor cells must be associated with apoptosis-inhibiting proteins, allowing malignant cell survival. Kiberu et al. [22] investigated the correlation between apoptosis, proliferation and bcl-2 expression in NHLs, and they suggested that expression of bcl-2 is not necessarily related to low levels of apoptosis, as some bcl-2positive high-grade tumors also had high levels of apoptosis. Nevertheless, they found that the majority of lymphomas expressing bcl-2 had average levels of apoptosis [22]. These results suggested that bcl-2-independent apoptosis is an important factor influencing cell death in many NHLs [22]. Overall results of previous studies [10,19,22-27] and our results indicate that the expressions of bcl-2 family proteins are variable and heterogeneous in DLBCL. The dual effect of bcl-2 family proteins may show individual variation in the pathogenesis and prognosis of DLBCLs. In this study, we found that the AI showed significant positive correlation with the PI. This result is correlated with previously reported results [10,22,45]. However, we were unable to show any relation between apoptosis-related proteins and PI. In this study, we determined that MGMT promoter methylation was detected in 32.6% of cases. MGMT methylation was reported at a frequency from 36% to 52% in Western populations in previous studies [1,3,30,46,47]. Higher rates (75.9%) were found in Middle Eastern populations [38]. This status may be explained by the differences in etiologic factors such as viral infections and exposure to environmental factors as well as differences in the genetic susceptibility. Furthermore, substantial ethnic differences existed with respect to molecular

Türk et al. Apoptosis, proliferation in large B-cell lymphoma

features of malignant tumors [31,48-50]. No significant association between MGMT promoter methylation status and the two immunophenotypic differentiation profiles was observed in this study. Al-Kuraya et al. [31] investigated the interrelationship between MGMT methylation status and immunohistochemical coexpression of CD10/bcl-6, and they did not show a significant association. Furthermore, Al-Kuraya et al. determined that their frequency of GCB profile DLBCL (13%) was somewhat lower than described in previous studies. They also found a much higher rate of MGMT methylation in their patients (75.9%) as compared with previous studies [1,3,30,31,46,47]. In our study, no significant association was found between MGMT promoter methylation status and the expression levels of apoptosis-related proteins, the AI and the PI. There is no similar previous study searching the relation of MGMT promoter methylation and expression levels of apoptosis-related proteins, the AI and the PI. Further studies could be done to elucidate the role of MGMT promote methylation in DLBCLs. In summary, these results suggest that bcl-6 protein expression may be responsible for the high PI in the GCB profile of DLBCLs. Additionally, we found that expression status of apoptosis-related bcl-2 family proteins (bcl-2, bcl-xl, bax, bak, bid) was not significantly associated with the immunophenotypic differentiation profiles. We also determined that there was no significant association between MGMT promoter methylation status and the immunophenotypic differentiation profiles of DLBCLs. Acknowledgements This research project was supported by the Turkish Society of Hematology (2007/348). Additionally, results of this study were presented at the 22nd European Congress of Pathology (4-9 September 2009, Florence, Italy) and the 19th National Congress of Pathology (7-11 October 2009, Girne, Cyprus). These presentations were supported by the Scientific Research Project Unit of Pamukkale University. The authors thank Dr. Mehmet Zencir for the statistical analysis. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, rela-

Turk J Hematol 2011; 28: 15-26

tionships, and/or affiliations relevant to the subject matter or materials included.

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Research Article

The relation between soluble endothelial protein C receptor and factor VIII levels and FVIII/sEPCR index in healthy infants Sağlıklı süt çocuklarında çözünür endotelyal protein C reseptörü ve faktor VIII düzeyleri arasında ilişki ve FVIII/sEPCR indeksi Filiz Şimşek Orhon1, Yonca Eğin2, Betül Ulukol1, Sevgi Başkan1, Nejat Akar2 11Department

of Pediatrics, Division of Social Pediatrics, School of Medicine, Ankara University, Ankara, Turkey

2Department of Pediatrics,Division of Pediatric Molecular Genetics, School of Medicine, Ankara University, Ankara, Turkey

Abstract Objective: Both soluble endothelial protein C receptor (sEPCR) and factor VIII (FVIII) seem to be potential mediators in thrombotic and inflammatory states. The aim of the present study was to determine the relation between plasma sEPCR and FVIII levels in a group of healthy Turkish infants. Materials and Methods: The study population consisted of 50 healthy infants aged 6 months (Group 1, n=23) and 12 months (Group 2, n=27) having no acute or chronic infection and/or disease. sEPCR levels and FVIII levels were measured by ELISA and one stage factor assay method, respectively. Results: The sEPCR levels of the infants aged 6 months were found higher than those of the infants aged 12 months (p<0.001). There was a correlation between sEPCR and FVIII levels of the infants in Group 1 (6-month-old infants) (r=0.678, p<0.001). FVIII/sEPCR index was 0.73±0.3 and 1.0±0.5 in Group 1 and Group 2, respectively (p=0.027). A correlation between infant age and FVIII/sEPCR index was found (r=0.312, p=0.027). Conclusion: The FVIII/sEPCR index in healthy infants reflects the physiological condition of this population. The finding showing a positive relationship between sEPCR and FVIII levels suggests a possible interaction between these mediators in healthy infants aged six months. (Turk J Hematol 2011; 28: 27-32) Key words: Soluble endothelial protein C receptor (sEPCR), factor VIII, healthy infants, thrombosis Received: March 01, 2010

Accepted: June 29, 2010

Özet Amaç: Çözünür endotelyal protein C reseptörü (sEPCR) ve faktör VIII (FVIII) trombotik ve inflamatuvar durumlarda potansiyel arabileşenler olarak görülmektedir. Bu çalışmanın amacı; bir grup sağlıklı süt çocuğunda plazma sEPCR ve FVIII düzeyleri arasındaki ilişkiyi tanımlamaktır. Yöntem ve Gereçler: Çalışma grubunu herhangi bir akut ya da kronik hastalığı ve/veya enfeksiyonu olmayan, sağlıklı 6 aylık (Grup 1, n=23) ve 12 aylık (Grup 2, n=27) çocuklar oluşturmaktadır. sEPCR düzeyleri ve FVIII düzeyleri sırasıyla; ELISA ve one stage factor metodu ile çalışılmıştır. Bulgular: Altı aylık çocukların sEPCR düzeyleri oniki aylık olanlardan daha yüksek bulunmuştur (p<0.001). Grup 1’i oluşturan 6 aylık çocuklarda sEPCR ve FVIII düzeyleri arasında bir korelasyon vardır (r=0.678, Address for Correspondence: Assoc. Prof. Filiz Şimşek Orhon, Department of Pediatrics, Division of Social Pediatrics, School of Medicine, Ankara University, 06100 Ankara, Turkey Phone: +90 312 595 72 02 E-mail: simsekfiliz@hotmail.com doi:10.5152/tjh.2011.02

Orhon et al. sEPCR and FVIII levels in infants

Turk J Hematol 2011; 28: 27-32

p<0.001). FVIII/sEPCR indeksi Grup 1’de 0.73±0.3 ve Grup 2’de 1.0±0.5 olarak bulunmuştur (p=0.027). Çocuğun yaşı ve FVIII/sEPCR indeksi arasında bir korelasyon saptanmıştır (r=0.312, p=0.027). Sonuç: Çalışmada sağlıklı çocuklarda kullanılan FVIII/sEPCR indeksi bu populasyonun fizyolojik durumunu yansıtmaktadır. Altı aylık çocuklarda sEPCR ile FVIII arasındaki pozitif ilişki bu yaş grubundaki çocuklarda bu mediatörler arasında muhtemel bir etkileşim olduğunu gösterebilir. (Turk J Hematol 2011; 28: 27-32) Anahtar kelimeler: sEPCR, Factor VIII, sağlıklı süt çocukları, tromboz Geliş tarihi: 1 Mart 2010

Kabul tarihi: 29 Haziran 2010

Introduction The protein C anticoagulant pathway is critical to both regulation of the blood coagulation process and control of the innate inflammatory response and some of its associated downstream pathologies [1,2]. The endothelial protein C receptor (EPCR) plays an important role in this pathway [3]. EPCR is preferentially expressed by endothelial cells of large blood vessels and increases the rate of protein C activation by the thrombin/thrombomodulin complex [3,4]. Increased levels of a soluble form of EPCR (sEPCR) in plasma lead to dysfunction of the EPCR-mediated coagulation [5]. sEPCR levels increase in conditions associated with considerable thrombin production such as vasculitis, sepsis and systemic lupus erythematosus [6,7]. Factor VIII (FVIII) is a plasma glycoprotein in the coagulation cascade, and it is the cofactor of factor IXa in the activation of factor X. FVIIIa can be inactivated by activated protein C [1,2]. Previous studies have shown that the coagulant activity of FVIII is increased as an acute phase reaction in thrombosis [8,9]. Elevated plasma levels of FVIII are associated with an increased risk of venous and arterial thrombosis [10,11]. Possible mechanisms thought to be associated with elevated FVIII are the enhancement of thrombin formation or the induction of acquired activated protein C resistance, but the molecular mechanisms that underlie elevated FVIII are still not clear. Although in vivo physiological mechanisms and their importance are still unknown, both EPCR and FVIIII seem to be potential mediators in thrombotic and inflammatory states because of their roles in the protein C anticoagulant pathway. Thus, the determination of the association between sEPCR and FVIII may provide new knowledge about the pathogenesis of thrombotic and inflammatory conditions. The aim of this study, therefore, was to determine the relation between plasma sEPCR and FVIII levels in a group of healthy Turkish infants.

Materials and Methods This study was conducted at the Department of Pediatrics, Divisions of Pediatric Molecular Genetics and Social Pediatrics. Ethics approval was obtained from the Ethics Committee of the School of Medicine. The study population consisted of healthy infants aged 6 and 12 months who were admitted for wellchild visits. They had no acute or chronic infection and/or disease. The written informed consent to participate was obtained from the parents of all subjects. Peripheral blood samples were collected from the subjects into tubes containing 1 ml 0.109 M trisodium citrate. Plasma was obtained by centrifugation at 2500xg for 10 minutes (min) at room temperature. Plasma specimens obtained were maintained until the date of measurement at 20±5˚C for 4 hours (h), at 2-8˚C for 24 h, and at -20˚C for 1 month. Soluble EPCR levels were determined in plasma by using sEPCR Asserachrom enzymelinked immunosorbent assay (ELISA) kits from Diagnostica Stago (Asnières, France), according to the manufacturer’s instructions. Factor VIII levels were measured concomitantly with one stage factor assay method and FVIII-absent plasma (Sigma Diagnostica Inc, St. Louis, MO). Statistical analysis Statistical analysis was performed using the SPSS 11.5. Descriptive analysis summarizing the characteristics and the levels of sEPCR and FVIII is presented. Since the plasma sEPCR and FVIII measurements were not normally distributed, nonparametric tests were conducted to compare these parameters. The Mann-Whitney U test was used to compare sEPCR and FVIII levels between the groups. Nonparametric correlations between sEPCR and FVIII levels were evaluated using the Spearman correlation test. FVIII/sEPCR index was calculated in the groups. The Student t test was used to compare FVIII/sEPCR index between the groups, and the Pearson correlation test was used to evaluate the correlation between this index and age.

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Turk J Hematol 2011; 28: 27-32

Results Totally, 50 healthy infants were studied. These infants were divided into two groups according to age. Group 1 consisted of 23 infants aged 6 months (9 boys and 14 girls), and Group 2 consisted of 27 infants aged 12 months (13 boys and 14 girls). Table 1 shows the sEPCR and FVIII levels of the infants. There was a significant difference between Group 1 and Group 2 with respect to sEPCR levels (p<0.001), with the sEPCR levels of infants aged 6 months found to be significantly higher than those of the infants aged 12 months. There was no difference between the groups with respect to the FVIII levels. Concerning all infants and groups, there was no statistical difference between boys and girls in sEPCR or FVIII levels. In the correlation analysis, there was a correlation between sEPCR and FVIII levels of the infants

in Group 1 (aged 6 months) (r=0.678, p<0.001) (Figure 1). However, no correlation was present between the sEPCR and FVIII levels in Group 2 (aged 12 months) (r=-0.251, p=0.206). With respect to all infants, the mean FVIII/sEPCR index was found as 0.88 (median: 0.85, SE: 0.06, min: 0.3, max: 3.1). There was a statistically significant difference in the FVIII/sEPCR index between the groups (p=0.027; 0.73±0.3 and 1.0±0.5 for Group 1 and Group 2, respectively). In the correlation analysis, there was a correlation between the infant age and the FVIII/sEPCR index (r=0.312, p=0.027).

Discussion This is the first study in the literature to examine the relationship between plasma sEPCR and FVIII levels in healthy infants as an indicator of the physiological condition. This study also highlights the

Table 1. sEPCR and FVIII concentrations according to groups All infants (n=50)

Group 1 (n=23)

Group 2 (n=27)

Mean ± SE

145.4±10.7

175.1±17.9

120.0±10.8

Median

112.0

130.0

102.0

Min-Max

67.0-346.0

95.0-346.0

67.0-280.0

95% CI1

123.8-166.9

137.9-212.3

sEPCR (ng/ml)

97.8-142.3

P* FVIII (U/dl)

<0.001 Mean ± SE

109.6±6.2

116.1±11.8

104.1±5.4

Median

100.5

103.9

96.6

Min-Max

42.0-284.0

72.0-209.0

95% CI1

97.2-122.0

91.6-140.6

92.9-115.2

>0.05

195% CI: Confidence intervals, * Mann-Whitney U test

300

220 200

250

180

150 100 50 0 0

200

160

FVII (% U/dl)

200

140 120 100

60 0

100 Groups

80 50 100 150 200 250 300 350 400 sEPCR (ng/ml) Group 1 (r=0.678, p<0.001)

150

0 50

100 150 200 250 300 sEPCR (ng/ml) Group 2 (r=-0.251, p=0.206)

Figure 1. The correlations between sEPCR levels and FVIII levels according to groups

0 50 100 150 200 250 300 350 400 sEPCR (ng/ml)

Group 1 Group 2

Orhon et al. sEPCR and FVIII levels in infants

relation between infant age and the FVIII/sEPCR index. Protein C activation is regulated by numerous mediators in the coagulation process, and activated protein C has a variety of anti-inflammatory activities [1]. Increased levels of sEPCR in plasma lead to dysfunction of the EPCR-mediated coagulation [5]. sEPCR levels vary among healthy subjects, and the bimodal distribution has been reported several times [12-14]. A recent study showed a negative relationship between sEPCR levels and an individual’s age; that is, sEPCR levels of healthy children were found to be higher than those of healthy adults (14). The levels of sEPCR and FVIII reflect the physiological condition of these mediators in this healthy population; however, the physiological importance and influence of sEPCR levels in vivo and the relation to FVIII are unknown. Increased plasma sEPCR levels were found associated with an increased risk of venous thrombosis and thrombin generation [12,15]. Further, it was reported that increased FVIII levels are an important risk factor for venous and arterial thrombosis [11,16]. Yürürer et al. [17] showed a negative relationship between sEPCR and FVIII levels in a group of pediatric stroke patients. They suggested that this association makes each of them regulate the action of the other, and may play a role in the stroke pathophysiology. Under the physiological circumstances, we found that the sEPCR levels of infants aged 6 months were significantly higher than of infants aged 12 months. We also found a positive relationship between sEPCR and FVIII levels in the 6-monthold healthy infants, but not in the 12-month-old infants. These findings, which indicate the physiological state in healthy infants, may reflect the interactions among the mediators in the coagulation system over the first months of life. Thrombin is a multifunctional serine protease generated at the site of vascular injury and has a key role in blood coagulation. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), members of the G protein-coupled receptors. Their expression is low in contractile vascular smooth muscle cells (SMC), but becomes markedly upregulated upon injury [18]. We suggest that the overexpression of PARs or the presence of unknown mechanisms during the early stage of life may play a role in the tendency towards thrombotic condi-

Turk J Hematol 2011; 28: 27-32

tions. We may speculate that the positive relation between sEPCR and FVIII levels in 6-month-old infants may be associated with the enhancement of thrombin formation and probably with the overexpression of PARs. Further studies are needed to evaluate the associations among the mediators in the coagulation system in a variety of healthy groups, including newborns, infants, children, and adults, under physiological circumstances. As the first in the literature, we proposed the ratio of FVIII/sEPCR as an index of the physiological status of the coagulation system. We found a correlation between infant age and the FVIII/sEPCR index in this healthy group. In our study population, this index reflects the physiological conditions regarding the coagulation pathway process under normal conditions. Although the in vivo importance of this index is unknown, determining the difference in this index between physiological and pathological conditions may confirm the potential interactions between the mediators in the coagulation process. We consider the possibility of using this index in further studies in pathologic states in which the pathogenesis involves thrombotic and inflammatory processes. In this study, we aimed primarily to determine the actual physiological condition in healthy infants. Because of the potential tendency to thrombotic states over the first months of life and the possible risk of complexity in our findings, the younger infants were not included in the study. On the other hand, the interactions among the mediators in the coagulation system over the first month of life need to be determined, in order to clarify the detailed mechanisms under the tendency of thrombosis. Therefore, further studies with the aim of assessing the relations among the mediators, such as EPCR, FVIII, other factors, PARs, or other receptors, may be conducted in healthy infants in the first month of life. Various genetic and environmental conditions have been discussed as risk factors for thrombotic events in adult and pediatric patients [19]. Functional polymorphisms in the EPCR gene may increase/decrease the risk of thrombosis, especially in carriers of prothrombotic mutations [20]. Familial clustering of high factor VIII levels in patients with venous and arterial thromboembolism and retinal artery occlusion were reported in

Orhon et al. sEPCR and FVIII levels in infants

Turk J Hematol 2011; 28: 27-32

previous studies [21-23]. These findings have pointed to genetic influences on these mediator levels. It is conceivable that the genetic polymorphisms of the FVIII and/or EPCR genes are linked with high FVIII and/or sEPCR levels in patients with thrombosis. The evaluation of these polymorphisms may be an important clinical indicator for determination of the thrombosis risk in a healthy population. In a recent study, Ay et al. [24] found no evidence of an association between observed single nucleotide polymorphisms in exons of the FVIII gene and high thrombosis levels. The findings in the literature seem to be contradictory about the polymorphisms in the genes of the mediators regarding the protein C system; therefore, further detailed studies are needed to assess these polymorphisms to determine their roles in the pathogenesis of thrombosis. In conclusion, this is the first study in the literature to examine the relationship between plasma sEPCR and FVIII levels in healthy infants and also to determine the ratio of FVIII/sEPCR as an index. Both sEPCR and FVIIII seem to be potential mediators in thrombotic and inflammatory states. The levels of sEPCR and FVIII in our study group reflect the physiological levels in this healthy population. We found a positive relationship between sEPCR and FVIII levels in 6-month-old healthy infants, but not in 12-month-old infants. This may reflect the interactions among the mediators in the coagulation system over the first months of life under physiological circumstances. We also found a correlation between infant age and the FVIII/sEPCR index in this healthy group. Although the in vivo importance of this index is unknown, we consider the possibility of using this index in further studies in pathologic states in which the pathogenesis involves thrombotic and inflammatory processes. On the other hand, future studies are needed to evaluate the physiological associations among the mediators, such as sEPCR and FVIII, in the coagulation system in a variety of healthy groups, including newborns, infants, children, and adults.

Acknowledgement: This study was supported by the Ankara University Research Fund.

Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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Esmon CT. Inflammation and the activated protein C anticoagulant pathway. Semin Thromb Hemost 2006;32:49-60. [CrossRef] Griffin JH, Fernandez JA, Gale AJ, Mosnier LO. Activated protein C. J Thromb Haemost 2007;5:73-80. [CrossRef] Fukodome K, Esmon CT. Identification, cloning and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994;269:26486-91. Regan LM, Stearns-Kurosawa DJ, Kurosawa S, Mollica J, Fukodome K, Esmon CT. The endothelial protein C receptor. J Biol Chem 1996;271:17499-503. Esmon CT. The protein C pathway. Chest 2003;124: 265-325. [CrossRef] Kurosawa S, Stearns-Kurosawa DJ, Carson CW, D’Angelo A, Della Vale P, Esmon CT. Plasma levels of endothelial cell protein C receptor are elevated in patients with sepsis and systemic lupus erythematosus: lack of correlation with thrombomodulin suggests involvement of different pathological processes. Blood 1998;91:725-7. Boomsma MM, Stearns-Kurosawa DJ, Stegeman CA, Raschi E, Meroni PL, Kurosawa S, Tervaert JW. Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener’s granulomatosis. Clin Exp Immunol 2002;128:187-94. [CrossRef] O’Donnell J, Mumford AD, Manning RA, Laffan M. Elevation of FVIII: C in venous thromboembolism is persistent and independent of the acute phase response. Thromb Haemost 2000;83:10-3. Kamphuisen PW, Eikenboom JC, Bertina RM. Elevated FVIII levels and the risk of thrombosis. Arterioscler Thromb Vasc Biol 2001;21:731-8. Rosendaal FR. High levels of factor VIII and venous thrombosis. Thromb Haemost 2000;83:1-2. Kurekci AE, Gokce H, Akar N. Factor VIII levels in children with thrombosis. Pediatr Int 2003;45:159-62. [CrossRef] Stearns-Kurosawa DJ, Swindle K, D’Angelo A, Della Vale P, Fattorini A, Caron N, Grimaux M, Woodhams B, Kurosawa S. Plasma levels of endothelial protein C receptor respond to anticoagulant treatment. Blood 2002;99:526-30. [CrossRef] Stearns-Kurosawa DJ, Burgin C, Parker D, Comp P, Kurosawa S. Bimodal distribution of soluble endothelial protein C receptor levels in healthy subjects. J Thromb Haemost 2003;4:855-6. [CrossRef] Orhon FS, Ergun H, Egin Y, Ulukol B, Baskan S, Akar N. Soluble endothelial protein C receptor levels in healthy population. J Thromb Thrombolysis 2010;29:46-51. [CrossRef] Saposnik B, Reny JL, Gaussem P, Emmerich J, Aiach M, Gandrilel S. A haplotype of the EPCR gene is associat-

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ed with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis. Blood 2004;103:1311-8. [CrossRef] Bugnicourt JM, Roussel B, Tramier B, Lamy C, Godefroy O. Cerebral venous thrombosis and plasma concentrations of factor VIII and von Willebrand factor: a case control study. J Neurol Neurosurg Psychiatry 2007;78:699-701. [CrossRef] Yururer D, Teber S, Deda G, Egin Y, Akar N. The relation between cytokines, soluble endothelial protein C receptor, and factor VIII levels in Turkish pediatric stroke patients. Clin Appl Thromb Hemost 2009;15:545-51. [CrossRef] Martorell L, Martinez-Gonzalez J, Rodriguez C, Gentile M, Calvayrac O, Badimon L. Thrombin and proteaseactivated receptors (PARs) in atherothrombosis. Thromb Haemost 2008;99:305-15. [CrossRef] Esmon CT. Basic mechanisms and pathogenesis of venous thrombosis. Blood Rev 2009;23:225-9. [CrossRef]

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Medina P, Navarro S, Estellés A, España F. Polymorphisms in the endothelial protein C receptor gene and thrombophilia. Thromb Haemost 2007;98:564-9. Schambeck CM, Hinney K, Haubitz I, Mansouri Taleghani B, Aahler D, Keller F. Familial clustering of high factor VIII levels in patients with venous thromboembolism. Arterioscler Thromb Vasc Biol 2001;21:289-92. Bank I, Libourel EJ, Middeldorp S, Hamulyak K, Van Pampus ECM, Koopman MMW, Prins MH, Van Der Meer J, Büller HR. Elevated levels of FVIII:C within families are associated with an increased risk for venous and arterial thrombosis. J Thromb Haemost 2004;3:79-84. [CrossRef] Akar N, Gökçe H. High levels of FVIII and FIX in a pediatric patient with retinal artery occlusion. Pediatr Hematol Oncol 2002;19:593-5. [CrossRef] Ay M, Dolek B, Erdem G, Devecioglu O, Gozukirmizi N. Is there any correlation between the elevated plasma levels and gene variations of factor VIII in Turkish thrombosis patients? Clin Appl Thromb Hemost 2011;17:46-50. [CrossRef]

Research Article

Differences in lymphocyte subpopulation count and function in cord, maternal and adult blood Kordon kanı, maternal ve erişkin kanda sayısal ve fonksiyonel lenfosit alt grup farklılıkları Nilgün Akdeniz, Esin Aktaş, Gaye Erten, Sema Bilgiç, Günnur Deniz Department of Immunology, Institute of Experimental Medicine, İstanbul University, İstanbul, Turkey

Abstract Objective: Phenotypical characterization and functional activity of lymphocytes and natural killer (NK) cells in cord blood (CB) were investigated, and maternal peripheral blood (MPB) values were compared to those of adult peripheral blood (APB) (control). Materials and Methods: To determine cytotoxic activity target cells (K562) were labeled with carboxyfluorescein diacetate (CFDA) or fluorescein isothiocyanate (FITC), and propidium iodide (PI) was used to label dead cells. Cell surface expression in CB, APB, and MPB cells were analyzed using flow cytometry. Results: CB and MPB mononuclear cells had similar CD45, CD34, CD4, and surface molecule for T helper cell expression, but had low-level expression of total T-lymphocyte surface molecules CD3 and CD8. CD19 and HLA-DR expression was higher in CB than in MPB. The same high-level of expression for CD19 and HLA-DR was observed in APB, as compared to MPB. All other cell surface expressions were similar in APB and MPB samples. NK (CD16+ and CD56+) cells in CB was similar to that in MPB and APB, and the level of inhibitory KIR receptors in NK cells was higher in venous CB than in MPB and APB. The only difference between MPB and APB was that the CD158a level was higher in MPB. No difference was observed in NK cells in CB and MPB, in terms of cytotoxicity. Conclusion: The present results show that there was numerical and proportional variability of lymphocytes and their subgroups in CB and APB, but no cytological difference. (Turk J Hematol 2011; 28: 33-41) Key words: NK activity, cord blood lymphocytes, flow cytometry Received: September 10, 2009

Accepted: April 30, 2010

Özet Amaç: Kordon kanındaki lenfosit ve NK hücrelerinin fonksiyonel aktivitesi ve fenotipik karakterizasyonu araştırılmış, anne kan değerleri (MPB) kontrol olarak erişkin periferik kan (APB) değerleri ile karşılaştırılmıştır. Address for Correspondence: Prof. Günnur Deniz, Department of Immunology, Institute of Experimental Medicine, İstanbul University, Vakıf Gureba Cad. Şehremini 34393 İstanbul, Turkey Phone: +90 212 414 20 97 E-mail: gdeniz@istanbul.edu.tr doi:10.5152/tjh.2011.03

Akdeniz et al. Differences in cord blood

Turk J Hematol 2011; 28: 33-41

Yöntem ve Gereçler: Sitotoksik aktivite için hedef hücreler (K562) karboksifloresandiasetat (CFDA) veya floresanizotiyosiyanat (FITC) ile işaretlenmiş, ölü hücreler ise propidiumiyodid (PI) ile saptanmıştır. Kordon, anne ve erişkin periferik kan hücre yüzey ifadeleri flow sitometri ile analiz edilmiştir. Bulgular: Kordon kanı CD45, CD34 ve CD4 ifadesi açısından maternal periferik kana benzerlik gösterirken, CD3 ve CD8 ifadesi düşük, CD19 ve HLA-DR ifadeleri ise kordon kanında maternal kana göre yüksek saptanmıştır. Maternal kana göre CD19 ve HLA-DR açısından benzer yüksek ifade erişkin kanda da gözlenmiş, diğer tüm yüzey ifadeleri erişkin ve maternal örneklerde benzer olarak saptanmıştır. Kordon kanı NK (CD16+CD56+) hücreleri maternal ve erişkin kana benzerlik gösterirken, NK hücre yüzeyinde aktivatör CD161 ve inhibitör anti-Hu-KIR ifadesi hem maternal hem de erişkin kana göre, CD158a ifadesi ise sadece erişkin kana göre yüksek saptanmıştır. Erişkin ve maternal kan karşılaştırıldığında sadece CD158a ifadesi maternal kanda yüksek olarak bulunmuştur. Kordon, erişkin ve maternal kan örneklerinde NK sitotoksisitesi açısından farklılık gözlenmemiştir. Sonuç: Bulgularımız lenfosit ve altgrupları açısından kordon kanı ile yetişkin immün sistem arasında sayı ve fonksiyonel anlamda farklılık olduğunu göstermekle birlikte, sitotoksik aktivite açısından bir farklılık olmadığını ortaya koymaktadır.(Turk J Hematol 2011; 28: 33-41) Anahtar kelimeler: NK aktivitesi, kordon kanı lenfositleri, flow sitometri Geliş tarihi: 10 Eylül 2009

Kabul tarihi: 30 Nisan 2010

Introduction Allogeneic cord blood transplantation (CBT), especially from unrelated donors, has become an extensively used treatment for patients with both malignant and nonmalignant disorders [1]. As compared to bone marrow transplantation (BMT), the advantages of CBT include ease and safety of hematopoietic collection, low risk of viral contamination, prompt availability when an unrelated donor is used, and reductions in the incidence and severity of graft-versus-host disease (GVHD) [2,3]. Several immunologic properties and peculiarities of cord blood lymphocytes (CBL) may be responsible for the reduction of GVHD following CBT [4]. In fact, CBLs are naive and characterized by a low number of interleukin-2 (IL-2), interferon (IFN)-γ, and tumor necrosis factor (TNF)-α producing cells, and have been shown to produce lower quantities of proinflammatory cytokines, such as IFN-γ and TNF-α, and to display no or markedly reduced responsiveness to allogeneic stimuli in a secondary mixed lymphocyte reaction (MLR) [5,6]. One of the reasons for the lower incidence of GVHD may be the reduced cytotoxic potential of cord blood (CB)-derived natural killer (NK) and cytotoxic T cells, as well as reduced levels of T helper 1 (Th1) cytokines, which are known to take part in the GVHD mechanistic cascade [7-10]. On the other hand, cytotoxic T and NK cells are the key effector cells that mediate the graft-versus-leukemia (GVL)

effect, which are used clinically in adoptive cellmediated immunotherapy to control minimal residual disease and for re-induction of remission in chronic myelogenous leukemia patients that relapse following allogeneic stem cell transplantation (alloSCT) [11-13]. Most clinical experimental data indicate that the greater the GVL affect after alloSCT, the higher the risk of developing GVHD [14,15]. Reducing the risk of GVHD after alloSCT, either by T cell depletion or by immunosuppression, is known to lead to an increase in leukemic relapse, which may indicate a decrease in the GVL effect [16,17]. As such, one of the main concerns in CBT was that the reduced incidence of GVHD observed following CBT would lead to a decrease in the GVL effect and a subsequent increase in the relapse rate. Nevertheless, it has been shown that CB-derived NK and lymphokine-activated killer cells are able to lyse non-cultured fresh leukemia blasts, readily respond to IL-2 and IL-12, and mediate relatively high levels of apoptosis-mediated cytotoxicity against target cell lines [18,19]. Furthermore, CB is rich in unique NK cell subsets that may possess greater potential proliferative capacity than adult peripheral blood (APB) NK cells [20]. NK cells in CB appear to be similar to those in APB, and these cells may actually have greater proliferative capacity when exposed to alloantigens or exogenous cytokines [8,18,21]. This suggests that CB may have substantial GVL potential and that CB-

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Turk J Hematol 2011; 28: 33-41

derived NK cells may be used effectively, if properly amplified, for adoptive cell-mediated immunotherapy and amplification of the GVL effect. The function of NK cells is important for the clearance of tumor cells, the removal of immunoglobulin-bound antigens, and the control of viral infections [22]. It was reported that NK function is decreased in some patients, including those with primary immunodeficiencies, those with late-stage human immunodeficiency virus (HIV) infection, and pregnant women [23,24]. NK cell function is tightly regulated by a fine balance of inhibitory and activatory signals that are delivered by a diverse array of cell surface receptors. Killer cell Ig-like receptor (KIR) binds to HLA class I molecules on the surface of target cells, and it confers inhibitory signals to NK cells [25,26]. Upon its ligation by HLA class I molecules, KIR can deliver inhibitory signals via the immune-receptor tyrosine-based inhibitory motif. As such, NK cells can recognize cells that do not express HLA class I molecules as cytotoxic target cells, and KIR plays a role in NK cellsâ&#x20AC;&#x2122; cytotoxic target discrimination [27,28]. Among the inhibitory receptors, some are specific for different groups of MHC class I alleles, while others are orphan receptors. In contrast, various activating receptors are involved in the triggering of NK cell-mediated natural cytotoxicity [25]. The present study analyzed activatory KIR: CD161 (NKR-P1A), and inhibitory KIRs: CD158a (NKAT1, KIR2DL1) and anti-human KIR (NKB1). As more is learned about NK cells and their function, and more simplified and precise means to quantify their numbers and functions become available, analysis of lymphocytes from healthy human controls and patients may become a more routine practical approach in clinical trials. Moreover, low-level NK cell activity may be useful for predicting patient outcome [23]; therefore, a suitable clinical assay for NK cell activity is necessary. The role of the functional and phenotypic characteristics of CB lymphocytes and NK cell activity against K562 in CB were investigated and compared to that in maternal peripheral blood (MPB) and APB (control). Target cells were labeled with fluorescein isothiocyanate (FITC) or carboxyfluorescein diacetate (CFDA) before contact with effector cells. The

red fluorescent dye propidium iodide (PI) was applied for the identification of dead cells.

Materials and Methods Study population CB samples (n=10) were obtained immediately postpartum from full-term, normally delivered healthy babies via cannulation of the umbilical vein and collected into heparinized tubes. Then, mononuclear cell fraction of CBL was obtained via FicollHypaque (Sigma Chemical Co., St Louis, MO, USA) density-gradient centrifugation. Mononuclear cell fractions of heparinized MPB (derived from healthy maternal donors [n=10]) and APB (derived from adult healthy donors [n=10]) were also separated by the same procedure described above. The viability of separated CB, MPB, and APB lymphocytes was measured using Trypan blue. The study protocol was approved by the Istanbul University Ethics Committee. Flow cytometric analysis Assay of cell surface markers Mononuclear cells (2Ă&#x2014;105 cells mL-1) were stained with anti-CD45-FITC/anti-CD14-PE, antiCD3-FITC, anti-CD19-PE, anti-CD4-FITC, anti-CD8PE, anti-CD16/56-PE, anti-HLA-DR-PE, anti-CD158aFITC, anti-CD161-FITC, anti-human KIR (NKB1)FITC, and anti-CD34-FITC (all obtained from Becton Dickinson, San Jose, USA). Stained cells were fixed in 2% paraformaldehyde. The controls were FITCand PE-conjugated mouse IgG1 and IgG2A (Becton Dickinson, San Jose, USA). Flow cytometric analysis was performed using FACSCalibur (Becton Dickinson, San Jose, USA). Cytotoxic activity Target cell The cell line K562, an NK-sensitive tumoral human erythroleukemia cell line, was used as target cells. Cells were grown in RPMI-1640 (GibcoBRL, UK) supplemented with 10% fetal bovine serum (FBS, Dutscher, France) and cultured for 24 h before cytometric analysis.

Akdeniz et al. Differences in cord blood

Cell labeling Two fluorescent dyes were initially tested for labeling target cells: FITC (Sigma Chemical Co., St Louis, MO, USA) at 50 μg mL-1 in PBS and CFDA (Sigma Chemical Co., St Louis, MO, USA) at 30 μg m L-1 in PBS. For FITC and CFDA labeling, target cells were adjusted to 105 cells m L-1 in PBS, incubated for 30 min at 37°C, and then rinsed 2 times with PBS. Cell viability was assessed using PI (Sigma Chemical Co., St Louis, MO, USA), which permeates only through the membrane of dead cells and emits a red fluorescence (10 μL mL-1 is the optimal concentration determined after a calibration assay). Effector cells CB, MPB, and APB mononuclear cells were obtained from heparinized blood via density gradient centrifugation over Ficoll (Sigma Chemical Co., St Louis, MO, USA), and were used as the source of NK effector cells. These effector cells were washed twice with RPMI-1640 medium (Sigma Chemical Co., St Louis, MO, USA) and resuspended to a final concentration of 5×106 cells mL-1 for NK assay. Cytotoxicity Effector and target cells were placed in 12×75mm round bottom polystyrene tubes (Falcon, NJ, USA) to yield effector:target ratios of 50:1, 25:1, and 12.5:1. Control tubes that included only target cells were assayed to identify spontaneous cell death. For maximum lysis (100% of death) target cells were incubated with 0.1% Triton X-100 (Sigma Chemical Co., St Louis, MO, USA). To enhance cell contact the mixture was centrifuged at 800 rpm for 1-2 min, and then incubated as a cell pellet in complete medium for 4 h at 37 °C under 5% CO2. After incubation, 10 μL mL-1 of PI was added to each tube for detection of dead cells, and cooled for 5-10 min on ice before acquisition. The samples were gently mixed and analyzed using flow cytometry (FACSCalibur, Becton Dickinson, San Jose, USA). Forward and side scatter parameters were adjusted to accommodate the inclusion of both target and effector cells within the acquisition gate. No cells were excluded from the analysis, and 10,000 cells were counted. Data were analyzed using BD FACSCalibur with CellQuest software (BD Bioscience, San Jose, USA).

Turk J Hematol 2011; 28: 33-41

Statistical analysis Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed by Student's t test using SPSS 11.5 version

Results Expression of cell surface molecules To characterize the subpopulations of cells undergoing expansion cells were stained with T and NK cell-associated surface markers, and flow cytometric analysis was performed. Table 1 summarizes the percentage of lymphocyte subsets observed in CB, MPB, and APB with a viability >95%. In MPB and CB mononuclear cells total leukocyte marker CD45, and stem cell marker CD34 and CD4 (marker for helper T cells) expression were similar (p= 0.068, p=0.075, p= 0.059, respectively); however, CD3, total T cell marker, and CD8 (a marker for cytotoxic T cells) were significantly lower in CB (p= 0.046, p= 0.012, respectively, Figure 1A). Expression of the B-lymphocyte marker CD19 and activation marker HLA-DR were significantly higher in CB than in MPB (p= 0.0089, p= 0.0014, respectively, Figure 1A). Expression of CD19 and HLA-DR were also significantly higher in APB than in MPB; however, there wasn’t a significant difference in the expression of CD3 and CD8 between APB and MPB (p= 0.019, p= 0.0085, respectively). CD16+ and CD56+ NK cell expression was similar in CB, MPB, and APB samples; however, activatory KIR CD161, and CD158a, and Hu-KIR cell surface molecules, which are inhibitory KIR receptors in NK cells, were more highly expressed in venous CB than in MPB (p= 0.0021, p= 0.0068, p= 0.036, respectively, Figure 1B). Compared to MPB, only CD158a expression was significantly lower in APB and there wasn’t a difference in the expression of CD161 and anti-Hu-KIR (p= 0.0046). Flow cytometric assay for NK cytotoxicity The cytotoxic potential of CB, MPB, and APB was analyzed via direct cytotoxic activity in NK-sensitive K562 cells. A flow cytometry-based assay, which correlates well with the standard chromium-release assay, was used [29]. Cells from CB, MPB, and APB were used as effector cells against the labeled target cells (K562) with 2 differ-

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Turk J Hematol 2011; 28: 33-41

Table 1 A. Lymphocyte subsets in CB and MPB Surface molecules

Table 1 B. Lymphocyte subsets in APB and MPB.

MPB

CD45+

97.2±1.5

98.2±0.5

p= 0.068

CD3+

63.8±3.6

73.2±5.2

CD4+

47.0±9.0

CD8+

41.4±5.4

23.8±5.2

36.8±4.7

Surface molecules

APB

MPB

CD45+

98.7±0.6

98.2±0.5

p= 0.073

p= 0.046

CD3+

76.0±0.8

73.2±5.2

p= 0.056

p= 0.059

CD4+

41.0±6.5

41.4±5.4

p= 0.078

p= 0.012

CD8+

36.0±2.0

36.8±4.7

p= 0.076

CD19+

17.6±5.4

9.6±0.8

p= 0.0089

CD19+

14.0±4.3

9.6±0.8

p= 0.019

HLA-DR+

19.2±6.4

8.2±4.0

p= 0.0014

HLA-DR+

14.7±4.8

8.2±4.0

p=0.0085

CD34+

1.6±1.1

1.0±0.4

p= 0.075

CD34+

1.1±0.4

1.0±0.4

p= 0.095

p= 0.094

CD16+

16.0±6.5

18.0±6.6

p= 0.088

p= 0.0021

CD161+

14.3±3.0

15.6±3.0

p= 0.0965

2.7±0.6

4.4±1.1

p= 0.0046

3.3±1.2

2.8±1.3

p= 0.098

CD16+

56+

20.0±6.6

CD161+

18.0±6.6

30.2±7.0

15.6±3.0

56+

CD158a+

8.2±2.5

4.4±1.1

p= 0.0068

CD158a+

Hu-KIR

5.6±0.4

2.8±1.3

p= 0.036

Hu-KIR

Lymphocyte subsets in CB, MPB, and APB. Phenotypic analysis of lymphocytes

isolated from CB, MPB, and APB. The percentage of positive cells was deter-

mined using FACSCalibur. The results are given as mean %±SD

mined using FACSCalibur. The results are given as mean % ± SD

100 80

% expression

20 CD3

CD4

CD8

CD19

CD34

CD45

HLA-DR

CB MPB APB

%lysis

120

* CD16/59

CD158a

12.5:1

25:1

50:1

Figure 2. Cytotoxic activity in CB, MPB, and APB

* CD161

anti-hu KIR

Figure 1. (A-B) Expression of lymphocyte surface molecules, and NK and KIR receptors in CB and MPB.

ent dyes, namely CFDA and FITC. Dead cells were identified base on PI incorporation. Effector and target cells were mixed at ratios of 12.5:1, 25:1, and 50:1, and co-incubated for 4 h at 37°C under a 5%-CO2 atmosphere; the results are shown in Table 2. Cytotoxic activity of CB, MPB, and APB increased in an effector:target ratio-dependent manner, and cytotoxicity increased as the number

of effector cells increased; however, there wasn’t a significant difference between CB, MPB, and APB cells, in terms of cytotoxic activity.

Discussion

CB T cells have been studied since it was demonstrated that recipients of related and unrelated umbilical CBT experience less acute and chronic GVHD than recipients of BMT [30]. The major theory regarding the reduced immunological response of CB lymphocytes is that CB T and NK cells are naive and exhibit decreased cytokine production and are therefore not primed for activation [31]. In the present study immunophenotypic characterization of CB mononuclear cells and cytotoxic activity of NK cells in CB were investigated. The

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Turk J Hematol 2011; 28: 33-41

Table 2. The cytotoxic effect of effector cells on FITC-labeled and CFDA-labeled K562 target cells 12.5:1 FITC CFDA

25:1 FITC

CFDA

FITC

50:1+ CFDA

APB 52.0±3.2

54.7±6.5

42.0±1.5

42.0±4.0 29.0±3.5 32.0±5.0

MPB 54.8±1.5

56.0±3.7

40.0±3.8

42.0±6.7 30.4±3.4 29.6±6.0

52.0±6.6

39.2±2.0

38.9±5.2 27.6±5.1 27.0±5.2

54.2±3.0

The cytotoxic effect of effector cells on FITC-labeled and CFDA-labeled K562 target cells. NK cytotoxic activity in CB and MPB at different effector: target ratios (12.5:1, 25:1, and 50:1) is shown as mean %±SD

phenotypic subsets of CB lymphocytes differed significantly from those of MPB lymphocytes; In comparison to MPB cells, higher expression of B-lymphocyte surface molecules, CD19, and the activation molecule HLA-DR, and lower expression of CD3 and CD8 were observed in CB cells. Similar to CB, lower expression of CD19 and HLA-DR was also noted in APB than in MPB; however, CD3 and CD8 did not differ significantly. The observed decrease in the ratio of total T-lymphocytes was proportional to the decrease in the ratio of cytotoxic T-lymphocytes, and low CD3 expression may be considered indicative of immature T-lymphocytes during the intermediate phase. There wasn’t a statistically significant difference in the expression of CD4 as a surface molecule for T helper cells between CB and MPB. In contrast to the present results, it has been reported that CB contains a higher absolute number of T, NK, and B cells than MPB. The same higher expression of NK cells was observed even in CB obtained during caesarean sections than in CB obtained during vaginal deliveries, indicating variability in NK cell numbers according to the type of delivery [32]. In CB a relative excess of naive cells among T cells and in the CD4+ and CD8+ subsets individually were reported. Similarly, the B-lymphocyte compartment had a smaller fraction of memory IgE receptor (CD23)positive cells, along with a higher percentage of the so-called B1 ontogenetically more primitive CD19+/ CD5+ B cells [33,34]. A smaller fraction of CB T cells exhibited markers of peripheral activation, such as HLA-DR, or the CCR5 subset performing tissue surveillance in the skin was absent, which may also explain the lower incidence of skin GVHD after CBT [35]. In the present study CB the NK (CD16+/CD56+) cell ratio was similar to the MPB NK cell ratio, as was previously reported for NK cells in APB and CB

[36]. The nature of CB NK cells remains controversial; despite the similar frequency of NK cells in CB and APB, CB NK cell dysfunction, as compared to APB and MPB was reported. In contrast, NK cells differentiated from CB stem cells exhibited comparable cytotoxicity as those from bone marrow or APB stem cells [37]. Although NK cell counts in CB were higher, cytotoxic activity was reported to be lower in some studies, and CB T-lymphocytes were immature and their cytotoxic activity was insufficient. Other possible mechanistic explanations for the reduction in killing by CB NK cells include relatively higher expression of inhibitory receptor complexes, including CD94/NKG2A and/or KIR [38]. This KIR is expressed in 10%-30% of CB NK cells and in the present study compared to MPB the observed higher expression of CD161, CD158a, and antihuman-KIR in CB supports the protective role of CB in GVHD. Some studies suggest that expression of NK cells triggering receptors, including CD94, KIR (CD158a/h and CD158b/j), NKp46, and NKG2D, does not differ between CB and APB NK cells, while others reported that a higher percentage of CB NK cells express the inhibitory receptor complex of CD94/ NKG2A and CD158b/j [38,39]. Considering the role of T-lymphocytes and NK cells in the pathophysiology of GVHD, the reported lower incidence of GVHD after CBT could be due to insufficient T-lymphocyte and NK cell function [40-42]. The sources for bone marrow transplantation are bone marrow, CB, and APB. Cells necessary for short- and long-term reconstitution after transplantation are thought to exist among the mononuclear cell population expressing CD34. CD34 stem cells can be isolated from CB, bone marrow, and APB and all three sources are deemed to have sufficient capacity for reconstitution of the hematopoietic system [43]. In the present study the CD34 cell ratio in CB was similar to that in MPB. Although most studies reported similar results, CD34 stem cells obtained from these sources had qualitative and quantitative differences [44,45]. Many studies reported that CB NK cells have lower-level cytotoxic function against traditional cell lines, such as K562 and Daudi, than MPB NK cells; however, low-level CB NK cell cytotoxic function may be increased by various cytokines, such as IFN-γ, IL-2, IL-7, IL-12, and IL-15 individually and in

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Turk J Hematol 2011; 28: 33-41

combination. In CBT the existence or absence of several cytokines is considered to lower the incidence of GVHD [46,47]. In the present study NK activity was measured using flow cytometry and FITC- or CFDA-labeled K562 target cells. According to the present results, no difference in cytotoxicity was observed between MPB and CB samples, in contrast to studies that reported suppression of NK cell cytotoxicity in postpartum women [48]. The same suppression pattern was observed in NK cells from premature infants, as compared to those from full-term infants [49-51], indicating that not only the number, but also the function differs according to the type of delivery. The standard method for determining NK activity is the chromium-release assay; however, this assay is not a preferred method for use in clinical laboratories for a variety of reasons, e.g. it requires the use of radioactive chromium, which is very expensive and requires specialized handling and disposal. For these reasons a new flow cytometry-based method was developed for measuring NK cell activity. Flow cytometric assays avoid the problems associated with the use of radioactive methods, and are rapid and easier to standardize. The present results support the notion that flow cytometry could be a suitable alternative for measuring NK activity [23,52]. In conclusion, the results of the present study suggest that there is numerical and proportional variability between lymphocytes and their subgroups in CB and APB, although no significant difference was observed between these 2 samples, according to cytotoxic activity. The clinical relevance of these subsets for GVHD and the development of protective immunity in allogeneic CBT is substantial, and we are conducting ongoing prospective research. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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Research Article

Evaluation of oxidative status in iron deficiency anemia through total antioxidant capacity measured using an automated method Demir eksikliği anemisinde otomatik bir yöntem kullanılarak total antioksidan kapasite ile oksidatif durumun değerlendirilmesi Mehmet Aslan1, Mehmet Horoz2, Hakim Çelik3 1Department

of Internal Medicine Clinic, Özel Erciş Çapa Medicine Center, Van, Turkey of Internal Medicine, Faculty of Medicine, Harran University, Şanlıurfa, Turkey 3Department of Clinical Biochemistry, Faculty of Medicine, Harran University, Şanlıurfa, Turkey 2Department

Abstract Objective: Oxidative stress, an increase in oxidants and/or a decrease in antioxidant capacity, is one of the potential biochemical mechanisms involved in the pathogenesis of iron deficiency anemia. The objective of this study was to evaluate the oxidative status and to determine whether there is any relationship between oxidative status and the severity of anemia in patients with iron deficiency anemia using an automated method. Materials and Methods: Twenty-six subjects with iron deficiency anemia and 20 healthy controls were enrolled in the present study. Serum total antioxidant capacity, serum total peroxide level and oxidative stress index were determined in all study subjects. Results: Serum total antioxidant capacity was significantly lower in patients with iron deficiency anemia than controls (p<0.05), while serum total peroxide level and oxidative stress index were significantly higher (both p<0.05). There was a significant correlation between hemoglobin level and serum total peroxide level, oxidative stress index and total antioxidant capacity (r=-0.504, p<0.05; r=-0.503, p<0.05; r=0.417, p<0.05, respectively). Conclusion: Increased oxidative stress may play a role in the pathogenesis of iron deficiency anemia. Supplementation of antioxidant vitamins in conjunction with iron replacement therapy may offer better responses and provide early resolution of symptoms related to iron deficiency anemia. The automated assay is a reliable and easily applied method for measurement of serum total antioxidant capacity in iron deficiency anemia. (Turk J Hematol 2011; 28: 42-6) Key words: Iron deficiency anemia, total peroxide, total antioxidant capacity, oxidative stress Received: June 03, 2010

Accepted: August 19, 2010

Özet Amaç: Oksidanlarda artış veya antioksidanlarda azalma olan oksidatif stres demir eksikliği anemisinin patogenezinde olası biyokimyasal mekanizmalardan biridir. Bu çalışmamızın amacı otomatik bir yöntem Address for Correspondence: M.D. Mehmet Aslan, Department of Internal Medicine Clinic, Özel Erciş Çapa Medicine Center, Van, Turkey Phone: +90 414 314 02 16 E-mail: m.aslan301@mynet.com doi:10.5152/tjh.2011.04

Aslan et al. Oxidative stress and iron deficiency anemia

Turk J Hematol 2011; 28: 42-6

kullanılarak demir eksikliği anemili hastalarda oksidatif durumu ve aneminin ciddiyeti ile oksidatif durum arasında herhangi bir ilişkinin olup olmadığını değerlendirmektir. Yöntem ve Gereçler: Çalışmaya 26 demir eksikliği anemili hasta ve 20 sağlıklı kişi alındı. Tüm çalışma olgularında serum total antioksidan kapasite, total peroksit seviyesi ve oksidatif stres indeksi ölçüldü. Bulgular: Demir eksikliği anemili hastalarda serum total antioksidan kapasitesi kontrol grubuna göre anlamlı derecede düşük bulunurken (p<0.05), serum total peroksit seviyesi ve oksidatif stres indeksi ise anlamlı derecede yüksek bulundu (her ikisi için p<0.05). Hemoglobin seviyeleri ile serum total peroksit seviyesi, oksidatif stres indeksi ve total antioksidan kapasite arasında anlamlı bir korelasyon vardı (sırasıyla; r= -0.504, p<0.05; r= -0.503, p<0.05; r= 0.417, p<0.05). Sonuç: Artmış oksidatif stres demir eksikliği anemisinin patogenezinde bir rol aynayabilir. Demir eksikliği anemili hastalarda demir replasman tedavisi ile birlikte antioksidan vitaminlerin verilmesi daha iyi yanıtlar sunabilir ve demir eksikliği anemisi ile ilgili semptomların erken düzelmesini sağlar. Bu otomatik test demir eksikliği anemisinde serum total antioksidan kapasitenin ölçümü için güvenilir ve kolay uygulanabilen bir yöntemdir. (Turk J Hematol 2011; 28: xx-xx) Anahtar kelimeler: Demir eksikliği anemisi, total peroksit, total antioksidan kapasite, oksidatif stres Geliş tarihi: 03 Haziran 2010

Kabul tarihi: 19 Ağustos 2010

Introduction Anemia is a pathologic condition in which there is a decrease in red blood cell mass or a decrease in the amount of hemoglobin. Iron deficiency is the most common nutritional problem worldwide, causing iron deficiency anemia (IDA) in approximately 500 to 600 million people [1]. Iron deficiency also affects the production of other Fe2+ containing proteins such as cytochromes, myoglobin, catalase (CAT) and peroxidase [2]. Thus, IDA is associated with prematurity and low birth weight during pregnancy, defects in cognitive and psychomotor development during childhood, and impaired work capacity in adulthood [3-5]. Reactive oxygen species (ROS) are oxygen-containing molecules that are produced during normal metabolism [6]. The organism has enzymatic and non-enzymatic antioxidant systems that neutralize the harmful effects of endogenous ROS products. Under certain conditions, the oxidative or antioxidative balance shifts towards the oxidative status as a result of an increase in ROS and/or impairment in the antioxidant mechanism. Thus, oxidative stress develops [7]. Increased oxidative stress in IDA subjects was reported in several studies, in which the oxidative status was evaluated using measurement of oxidants, individual antioxidants, or both [8-14]. In these studies, it has been reported that oxidants were increased and antioxidants were decreased, and as a result, the oxidative/anti-oxidative balance shifted toward the oxidative side in patients with IDA. Thus, increased oxidative stress may contribute to the pathogenesis of patients with IDA. To our best knowledge, oxidative status in IDA subjects

and its relation to the severity of anemia were not previously investigated using a measurement of total antioxidant capacity (TAC) along with oxidants and calculation of the oxidative stress index (OSI), indicators of oxidative stress that reflect the redox balance between oxidation and antioxidation. Thus, in the present study, we aimed to evaluate the oxidative status and to determine whether there is any relationship between oxidative status and the severity of anemia in IDA patients using measurements of TAC and total peroxide (TP) level and calculation of OSI.

Materials and Methods A total of 26 female subjects with IDA and 20 healthy female controls were included in the present study. The study protocol was carried out in accordance with the Helsinki Declaration as revised in 1989 and approved by the local research committee for ethics. All subjects were informed about the study protocol and written consents were obtained from all participants. Iron deficiency was defined as serum ferritin concentration <15 μg/L, indicating depleted iron stores; hemoglobin (Hb)<10 g/dl and a mean corpuscular volume (MCV) <80 fl and/or a mean corpuscular Hb concentration (MCHC) <32 g/dl. In order to exclude the presence of thalassemia trait, subjects with MCV <80 fl underwent Hb electrophoresis, and examination of the blood smear for Hb-H inclusion bodies was performed. Routine blood biochemistry, tumor marker determination, stool examination for parasites, gynecologic examination, pelvic ultrasonography, and gas-

Aslan et al. Oxidative stress and iron deficiency anemia

trointestinal endoscopic examination were performed in all subjects with IDA. Subjects with IDA were included if their biochemical, radiological and endoscopic examinations were normal. Most IDA subjects had regular menstrual bleeding; only three of them were considered to have dysfunctional menstrual bleeding. In all study subjects, IDA was considered to be secondary to the inadequate intake of an iron-containing diet despite the monthly iron loss through menstrual bleeding. Exclusion criteria Exclusion criteria included usage of supplemental vitamins and existence of diabetes mellitus, coronary artery disease, rheumatoid arthritis, malignancy, myoma uteri, systemic or local infection, hypertension, acute-chronic liver diseases, renal dysfunction, anemias other than IDA, and gastrointestinal parasitic diseases. Blood sample collection Blood samples were drawn after an overnight fast. Serum was separated by centrifugation at 1500 g for 10 minutes (min) and stored at -80°C until use. Measurement of serum total antioxidant potential Serum TAC was determined using a novel automated measurement method, developed by Erel [15]. In this method, hydroxyl radical - the most potent biological radical - is produced. In the assay, ferrous ion solution, which is present in Reagent 1, is mixed by hydrogen peroxide, which is present in Reagent 2. The sequentially produced radicals such as brown-colored dianisidinyl radical cation, produced by the hydroxyl radical, are also potent radicals. This method measures the antioxidative effect of the sample against the potent free radical reactions, which are initiated by the produced hydroxyl radical. The assay has excellent precision values lower than 3%. The results are expressed as mmol Trolox Equivalent/L. Measurement of total serum peroxide concentration Serum TP concentrations were determined by an enzymatic assay as described previously [16]. The FOX2 test system is based on oxidation of ferrous ion to ferric ion by various types of peroxides

Turk J Hematol 2011; 28: 42-6

contained within the serum samples, to produce a colored ferric-xylenol orange complex, the absorbance of which can be measured. Briefly, the FOX2 reagent was prepared by dissolving ammonium ferrous sulphate (9.8 mg) in 250 mM H2SO4 (10 ml), to give a final concentration of 250 μM ferrous ion in acid. This solution was then added to 90 ml of HPLC-grade methanol containing 79.2 mg butylated hydroxytoluene (BHT). Finally, 7.6 mg xylenol orange was added while stirring to make the final working reagent (250 μM ammonium ferrous sulphate, 100 μM xylenol orange, 25mM H2SO4 and 4 mM BHT in 90% v/v methanol in a final volume of 100 ml). The blank reagent contained all the components of the solution except ferrous sulphate. Aliquots (200 μl) of serum were mixed with 1800 ml FOX2 reagent. After incubation at room temperature for 30 min, the vials were centrifuged at 12000 g for 10 min. Absorbance of the supernatant was then determined at 560 nm. TP content of serum samples was determined as a function of the absorbance difference between sample and blank tubes using a solution of H2O2 as standard. The coefficient of variation for individual serum samples was less than 5%. Oxidative stress index The percent ratio of the TP to the TAC gave the OSI, an indicator of the degree of oxidative stress [17]. Other parameters Routine biochemical analyses were made using auto-analyzer (Aeroset®, Abbott, Germany). Complete blood count was performed using Celdyne 3700 Haematology Analyser. Serum ferritin was measured using an immunometric assay (chemiluminescence) (Aeroset®, Abbott). Statistical analysis All data were expressed as mean±standard deviation (SD). The comparisons of parameters were performed using Student’s t test, and correlation analyses were performed using Pearson’s correlation test. A p value <0.05 was accepted as significant.

Results Demographic and clinical data of the subjects are shown in Table 1. There were no significant differences between IDA subjects and controls with

Aslan et al. Oxidative stress and iron deficiency anemia

Turk J Hematol 2011; 28: 42-6

respect to age, gender and body mass index (BMI) (all p>0.05). TAC, TP level and OSI in IDA subjects and controls are shown in Table 2. TP and OSI were significantly higher in IDA subjects than controls (both p<0.05), while TAC was significantly lower (p<0.05). A significant positive correlation was observed between TAC and Hb level (r=0.417, p<0.05). On the contrary, TP level and OSI were negatively correlated with Hb level (r=-0.504, p<0.05 and r=-0.503, p<0.05, respectively).

Discussion Normal cell functions and integrity of cell structures may be broken via considerable reactivity of ROS. The organism has enzymatic, such as superoxide dismutase (SOD), CAT, glutathione peroxidase (GSH-Px), and non-enzymatic (e.g. vitamin C, vitamin E) antioxidant mechanisms that work as scavengers against ROS [7]. In the present study, we observed that the patients with IDA are exposed to increased oxidative stress, and the potency of the oxidative stress is significantly related to the severity of anemia. The increase in oxidative stress that was observed in IDA subjects in the present study resulted from both increase in oxidants and decrease in TAC. Table 1. Demographic and clinical parameters in controls and IDA patients Parameters

IDA (n=26)

Controls (n=20)

Age (years)

41±5

40±6

BMI (kg/m2)

22.4±1.8

23.2±2.1

Hb (g/dl)

7.56±1.09

15.40±1.60

p<0.001

Hct (%)

25.7±2.78

40.00±3.26

p<0.001

MCV (fl)

63.71±7.40

85.70±2.90

p<0.001

Ferritin (μg/L)

5.71±3.48

76.35±33.28

p<0.001

BMI: Body mass index; Hb: Hemoglobin; Hct: Hematocrit; MCV: Mean corpuscular volume; IDA: Iron deficiency anemia.Values are mean ± SD; ns: non significant

Table 2. Oxidative and antioxidative parameters in controls and IDA patients Parameters

IDA (n=26)

Controls (n=20)

TAC (m mol Trolox Eq./L)

1.66±0.12

1.76±0.10

p<0.05

TP (μmol H2O2 Equiv./L)

9.26±0.02

6.86±0.12

p<0.05

OSI (Arbitrary Unit)

5.57±0.16

3.89±1.20

p<0.05

Values are mean ± SD, TAC: Total antioxidant capacity; TP: Total peroxide; OSI: Oxidative stress index; IDA: Iron deficiency anemia

Oxidant levels and antioxidant enzyme activities such as SOD, GSH-Px and CAT have been evaluated in several studies [8-14]. Although conflicting results have been reported [8,10], it is generally accepted that oxidative stress is increased through increase in oxidant levels and/or decrease in antioxidant enzyme capacities in IDA. Generally, decreased GSH-Px activity along with normal or increased SOD and CAT activity are the main findings in IDA [10,11]. However, in some studies, normal GSH-Px activity along with decreased SOD and CAT activity or decrease in all of these activities was also reported [12,13]. The effects of various antioxidants in serum are additive, and the cooperation of antioxidants in human serum protects the organism against attacks by free radicals [18]. Thus, measurement of individual antioxidants may not accurately reflect the true antioxidant status of the organism. In this regard, measurement of TAC should be essential in evaluating the true antioxidant status [12,15,19,20]. The most widely used methods for oxidative status measurement are colorimetric, or involve either fluorescence or chemiluminescence [21-23]. The fluorescence and chemiluminescence methods require sophisticated techniques, and these improved systems are not present in many routine clinical biochemistry laboratories. Even when these technologies are available, their routine usage is limited [20]. In the present study, we used an automated method, which has several major advantages in comparison with the other currently available methods, to measure TAC in our study population. It is simple and cheap, and can easily be a fully automated method. It is also reliable and sensitive, and does not interact with commonly occurring serum components such as bilirubin, serum lipids and anticoagulants. Accurate measurements of TAC can be obtained in as little as 10 minutes, making this assay imminently suitable for the clinical biochemistry laboratory [18]. Although our results were in concordance with the studies mentioned above with respect to increase in oxidative stress, none of the previous studies evaluated oxidative stress using TAC measurement. To our knowledge, this is the first study in which antioxidant capacity of IDA subjects was determined using measurement of TAC along with measurement of TP level and calculation of OSI.

Aslan et al. Oxidative stress and iron deficiency anemia

In light of these findings, we concluded that oxidative stress may have a critical role in the pathogenesis of IDA and is associated with the disease severity. Supplementation of antioxidant vitamins in conjunction with iron replacement therapy may offer better responses and provide early resolution of symptoms related to IDA. In addition, the automated assay is a reliable and easily applied method for serum TAC measurement in subjects with IDA. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

DeMaeyer E, Adiels Tegman M. The prevalence of anaemia in the world. World Health Stat Q 1985;38:302-16. Rockey DC, Cello JP. Evaluation of the gastrointestinal tract in patients with iron deficiency anemia. N Engl J Med 1993;329:1691-5. [CrossRef] Lieberman E, Ryan KJ, Monson RR, Schoenbaum SC. Association of maternal hematocrit with premature labor. Am J Obstet Gynecol 1988;159:107-14. Oski FA, Honig AS, Helu B, Howanitz P. Effect of iron therapy on behavior performance in nonanemic, irondeficient infants. Pediatrics 1983;71:877-80. Basta SS, Soekirman MS, Karyadi D, Scrimshaw NS. Iron deficiency anemia and the productivity of adult males in Indonesia. Am J Clin Nutr 1979;32:916-25. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979;59:527-605. Halliwell B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 1994;344:721-4. [CrossRef] Ramachandran M, Iyer GY. Erythrocyte membrane lipid peroxidation in iron deficiency anemia. Experientia 1984;40:173-4. [CrossRef] Kumerova A, Lece A, Skesters A, Silova A, Petuhovs V. Anaemia and antioxidant defence of the red blood cells. Mater Med Pol 1998;30:12-5. Jansson LT, Perkkio MV, Willis WT, Refino CJ, Dallman PR. Red cell superoxide dismutase is increased in iron deficiency anemia. Acta Haematol 1985;74:218-21. [CrossRef] Macdougall LG. Red cell metabolism in iron deficiency anemia. The relationship between glutathione peroxi-

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12.

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14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

dase, catalase, serum vitamin E, and susceptibility of iron-deficient red cells to oxidative hemolysis. J Pediatr 1972;80:775-82. [CrossRef] Kurtoglu E, Ugur A, Baltaci AK, Undar L. Effect of iron supplementation on oxidative stress and antioxidant status in iron deficiency anemia. Biol Trace Elem Res 2003;96:117-23. [CrossRef] Isler M, Delibas N, Guclu M, Gultekin F, Sutcu R, Bahceci M, Kosar A. Superoxide dismutase and glutathione peroxidase in erythrocytes of patients with iron deficiency anemia: effects of different treatment modalities. Croat Med J 2002;43:16-9. Tekin D, Yavuzer S, Tekin M, Akar N, Cin S. Possible effects of antioxidant status on increased platelet aggregation in childhood iron-deficiency anemia. Pediatr Int 2001;43:74-7. [CrossRef] Erel O. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin Biochem 2004;37:112-9. [CrossRef] Yeni E, Gulum M, Selek S, Erel O, Unal D, Verit A, Savas M. Comparison of oxidative/antioxidative status of penile corpus cavernosum blood and peripheral venous blood. Int J Impot Res 2005;17:19-22. [CrossRef] Harma M, Harma M, Erel O. Increased oxidative stress in patients with hydatidiform mole. Swiss Med Wkly 2003;133:563-6. Wayner DD, Burton GW, Ingold KU, Barclay LR, Locke SJ. The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma. Biochim Biophys Acta 1987;924:408-19. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci (Lond) 1993;84:407-12. Erel O. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 2004;37:277-85. [CrossRef] Schlesier K, Harwat M, Bohm V, Bitsch R. Assessment of antioxidant activity by using different in vitro methods. Free Radic Res 2002;36:177-87. [CrossRef] Janaszewska A, Bartosz G. Assay of total antioxidant capacity: comparison of four methods as applied to human blood plasma. Scand J Clin Lab Invest 2002;62:231-6. [CrossRef] Prior RL, Cao G. In vivo total antioxidant capacity: comparison of different analytical methods. Free Radic Biol Med 1999;27:1173-81. [CrossRef]

Research Article

Seroprevalence and genotyping of hepatitis C virus in multiple transfused Jordanian patients with β-thalassemia major Multipl transfüzyon uygulanan β-talasemi majörlü Ürdünlü hastalarda hepatit C virüsünün seroprevalansı ve genotiplemesi Suleimman Ahmad Al-Sweedan1, Said Jaradat2, Khitam Amer2, Wail Hayajneh1, Hazem Haddad2 1Department 2Department

of Pediatrics, Jordan University of Science and Technology, Irbid, Jordan of Genetics, Jordan University of Science and Technology, Irbid, Jordan

Abstract Objective: The main objectives of this study were to investigate the prevalence of hepatitis C virus (HCV) among patients with β-thalassemia major and to determine the most prevalent genotype for this virus. Materials and Methods: One hundred twenty-two β-thalassemia major patients who were previously diagnosed at the molecular level were included. All plasma samples were tested for the presence of antibodies by ELISA. Real-time polymerase chain reaction (PCR) was used in the quantitation the HCV RNA viral loads, and consequently, patients with high virus titer were genotyped by the linear array. Results: Forty of the patients were anti-HCV positive. The prevalence of anti-HCV was significantly higher in patients who received blood transfusion before 1993 (83.7%) than in those who received it after 1993 (16.3%) (p=0.000). β-thalassemia major patients with HCV infection had significantly higher rates of elevated aspartate aminotransferase (54.4% vs 40.5%, p=0.045) and alanine aminotransferase (72.47% vs 37.47%, p=0.00) and of splenectomy (54.8% vs 45.2%, p=0.004) than β-thalassemia major patients without HCV. Conclusion: HCV genotype 4 is the commonest genotype in multi-transfused patients with β-thalassemia major in Jordan. (Turk J Hematol 2011; 28: 47-51) Key words: Virology, transfusion, thalassemia Received: August 7, 2009

Accepted: February 22, 2010

Özet Amaç: Bu çalışma kapsamında, β-talasemi majörlü hastalarda HCV prevalansının araştırılması ve bu virüsün söz konusu hastalar arasında en yaygın genotipinin belirlenmesi amaçlanmaktadır. Yöntem ve Gereçler: Önceden moleküler düzeyde tanı konan 122 β-talasemi majörlü hasta çalışmaya alınmıştır. Tüm plazma numuneleri antikor varlığına yönelik olarak ELISA ile test edilmiştir. HCV RNA Address for Correspondence: MD, MS, FAAP. Suleimman Al-Sweedan,Pediatric Hematology/Oncology/BMTKing Abdalla University HospitalFaculty of MedicineJordan University of Science & Technology, Irbid, Jordan Phone: +0799051255 E-mail: sweedan@just.edu.jo doi:10.5152/tjh.2011.05

Al-Sweedan et al. Genotyping of HCV in β-TM

Turk J Hematol 2011; 28: 47-51

viral yükler ve dolayısıyla doğrusal dizilim ile genotiplenen yüksek virüs titreli hastalar gerçek zamanlı PCR kullanarak belirlenmiştir. Bulgular: Hastaların 40’ı anti-HCV pozitif idi. Anti-HCV prevalansı, 1993’ten önce kan transfüzyonu uygulanan hastalarda (%83.7), 1993’ten sonra uygulananlara (%16.3) kıyasla anlamlı derecede daha yüksek idi (p=0.00). HCV enfeksiyonlu β-talasemi majörlü hastaların Aspartat aminotransferaz (%54.4; %40.5, p= 0.045) ve Alanin aminotransferaz (%72.47; %37.47, p= 0.00) düzeyleri HCV enfeksiyonsuz β-talasemi majörlü hastalarınkine kıyasla anlamlı derecede daha yüksek olup, bu gruptaki splenektomize hastaların sayısı anlamlı derecede daha yüksek idi (%54.8; %45.2, p= 0.004). Sonuç: Ürdün’de, multipl transfüzyon uygulanmış β-TM’li hastalarda genotip 4, en yaygın genotiptir. (Turk J Hematol 2011; 28: 47-51)

Anahtar kelimeler: Viroloji, transfüzyon, talasemi Geliş tarihi: 07 Ağustos 2009

Kabul tarihi: 22 Şubat 2010

Introduction Beta-thalassemia is an autosomal recessive disorder and occurs because of the absence or reduced synthesis of the beta globin chains [1]. Patients with beta-thalassemia major (β-TM) suffer from numerous complications such as massive hepatosplenomegaly, dental problems, leg ulcers, and high risk for acquiring blood-transmitted infections such as hepatitis B virus (HBV), human immunodeficiency virus (HIV) and particularly hepatitis C virus (HCV) [2,3]. According to estimates by the World Health Organization, 170 million people are infected with HCV worldwide [4]. HCV genome encodes a single poly protein of 3000 amino acids; it is cleaved post translationally to yield at least 10 structural and nonstructural proteins [5]. Sequence comparisons of the virus led to the identification of at least six major genotypes [6], and these differ in nucleotide sequence by more than 30% over the complete virus genome. Additionally, there are more than 50 subtypes, which also differ in nucleotides sequence by more than 20% [7]. Genotype 4 is found most commonly in the Middle East [8]. Genotyping and sub-typing for HCV are not only required for therapy initiation and monitoring, but they also assist in vaccine development [9,10]. Studies have shown that HCV genotypes 1 and 4 are more resistant to treatment with pegylated interferon and ribavirin than genotypes 2 and 3 [11]. A study conducted in Jordan among patients on regular hemodialysis showed that the prevalence of HCV infection was correlated with the history of blood transfusion before the introduction of antiHCV screening in Jordanian blood banks in 1993 [12]. HCV genotype 1a was found to be the pre-

dominant sub-type among blood donors and Jordanian hemodialysis patients as well as other Middle Eastern countries including Lebanon, Turkey, Cyprus, and Syria [13,14]. HCV genotype 4 is the most prevalent in Saudi Arabia, Egypt, Yemen, and Bahrain [15]. Patients with hemoglobinopathies have been excluded from large studies of therapy for HCV, particularly studies that include ribavirin, because of the associated blood hemolysis [16]. Both ribavirin and interferon drugs were tested on thalassemic patients with HCV infection and were shown to be effective [17]. The main objectives of this study were (i) to investigate the prevalence of HCV among Jordanian patients with β-TM, and (ii) to determine the most prevalent genotype for this virus.

Materials and Methods A hospital-based study was carried out on 122 patients pre-diagnosed with β-TM based on hemoglobin electrophoresis and molecular DNA analysis between April -December 2008. Identification of β-TM for over 150 patients, including the patients of this study, was achieved by polymerase chain reaction (PCR) amplification of beta-globin genes and direct DNA sequencing of amplified genomic DNA. All patients receive regular blood transfusions every 2-4 weeks in Princess Rahmah Governmental Hospital, Irbid, Jordan. All study subjects provided written informed consent or consent was obtained from their guardian in the case of minors, and the study was approved by the Institutional Review Board of Jordan University of Science and Technology. Blood samples were drawn from patients before the scheduled transfusion, and

Al-Sweedan et al. Genotyping of HCV in β-TM

Turk J Hematol 2011; 28: 47-51

plasma was separated from whole blood and stored at -80°C until analysis. All plasma samples were tested using the GENEDIA ELISA 3.0 kit (Green Cross Medical Science Corp, Korea) for the presence of antibodies directed against four HCV genomic regions: Core, NS3, NS4, and NS5. Total RNA was extracted from ELISA-positive samples by the Ribo-Virus spin column extraction kit (Saccace, Italy) according to the manufacturer’s specifications. Quantification of the viral load (HCVRNA) in serum was done by amplification method using the HCV Real-TM Quant MX kit (Sacace, Italy) according to the manufacturer’s specifications. The emission of a reporter dye probe specific for HCV, which is proportional to the amount of HCV RNA in the starting sample, was measured using MX4000TM (Stratagene, USA). Samples with high viral loads were genotyped using the linear array HCV genotyping test kit, version 2.0 (Roche Molecular System) according to manufacturer’s specifications. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, version 16.0) software. Differences between β-TM patients with and without HCV infection were analyzed using the t-test and chi-square test where applicable. Statistical significance was considered at a p value <0.05.

Results A total of 122 β-TM patients (67 males, 55 females) were studied, and their ages ranged from 2-40 years old (mean age: 14.5 years). A total of 12 different mutations were detected, with the IVS1-110 (G>A) mutation found to be the more frequent among Jordanians (manuscript in preparation). Forty patients were anti-HCV positive. The prevalence of anti-HCV was significantly higher in patients who received blood transfusion before 1993 (83.7%) than in those who received it after 1993 (16.3%) (p=0.00) (Table 1). β-TM patients with HCV infection had a significantly higher rate of elevated aspartate aminotransferase (AST) (54.4% vs 40.5%, p=0.045), alanine aminotransferase (ALT) (72.47% vs 37.47%, p=0.00) and of splenectomy (54.8% vs 45.2%, p=0.004) than β-TM patients without HCV (Tables 1, 2). Factors with no statistical significance between the two groups were gender, hemoglobin level, ferritin level, drug intake, and alkaline phosphatase level (Tables 1, 2).

Twenty of 40 samples with ELISA-positive test had high viral loads using the real time PCR assay. HCV genotype 4 was found in 12 patients with high viral titer. Two patients refused to participate in completing the study and genotyping array failed for the other six samples (3 of the failed samples were from patients under prolonged treatment with interferon and ribavirin).

Discussion Several studies have shown that patients with β-TM receiving chronic blood transfusions have a higher prevalence of chronic HCV infection, particularly if transfused before HCV serological testing became available [18]. Patients with hemoglobinopathies, mainly with thalassemia, have traditionally been excluded from large studies of therapy of HCV [19], particularly studies that include ribavirin because of the associated hemolysis leading to an increase in transfusion requirement, iron accumulation and the risk of iron-related toxicities [20]. A study done among 142 HCV-infected Lebanese thalassemic patients identified HCV genotype 4 as the predominant genotype among thalassemic patients, which is in agreement with our results, and this confirms the predominance of HCV genotype 4 in our country and perhaps in the Middle East [21]. Another study involving 104 thalassemic patients in Thailand showed that prevalence of HCV infection among Thai thalassemic patients was 20.2% [2], while a higher prevalence of HCV infection was observed in our study, with a frequency of 32.8%. Wanachiwanawin et al. [2] showed that patients with anti-HCV antibodies had significantly higher levels of serum AST and ALT than patients without anti-HCV antibodies (p=0.021 and 0.017, respectively), in agreement with our results, which revealed that HCV-infected patients had significantly higher levels of ALT and AST than patients without anti-HCV (p=0.045 and 0.000, respectively). A study done among 283 Jordanian hemodialysis patients showed that 98 (34.6%) patients were antiHCV-positive by ELISA, two HCV genotypes (1 and 4) were identified and HCV genotype la was predominant, whereas in our results, only genotype 4 was identified, and in six samples the genotype arrays failed (3 of the samples were from patients under extended treatment with interferon and ribavirin).

Al-Sweedan et al. Genotyping of HCV in β-TM

Turk J Hematol 2011; 28: 47-51

This may be explained by the presence of a new variant or a mixed infection that can not be detected using this genotyping kit [22]. In a study done in Iran in 2006 among 732 patients with β-TM and141 (19.3%) patients who were anti-HCV positive showed that older age (p=0.001), longer transfusion duration (p=0.000) and higher serum ferritin level (p=0.002) were significantly associated with a higher prevalence of HCV [23]. These results are in agreement with our results except for the ferritin level, since we determined no statistical significance for the association between ferritin level and HCV infection. However, our study showed that splenectomized β-TM patients have a higher prevalence of HCV infection. Splenectomized patients are probably heavily transfused with blood before splenectomy due to hypersplenism. In conclusion, HCV genotype 4 is the commonest genotype in multi-transfused patients with β-TM in Jordan. Considering the possibilities of HCV mixed genotype among patients with thalassemia, accuracy and precision should be an important concern in the detection of genotypes, which might explain the genotyping failure in six patients with high viral load. Table 1. Age discrimination and laboratory findings of patients HCV-infected

HCV-not infected

p value

19.08±6.03

12.1±7.5

<0.001

ALT (U/L)

72.47

37.47

<0.001

AST (U/L)

54.4

40.50

0.045

ALP (U/L)

209.12

213.04

0.90

Ferritin (mg/dl)

2131.95

2013.68

0.69

8.76

8.79

0.86

Age (years) (Mean±SD)

Hb (g/dl)

References 1. 2.

4. 5. 6.

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase; Hb: Hemoglobin

Table 2. Risk factors for HCV infection other than blood transfusion in β-TM patients Characteristic

HCV-infected patients (%)

HCV-not infected (%)

p value

Surgery

44.7

55.3

0.062

Deferoxamine intake

38.4

61.6

0.58

Splenectomy

54.8

45.2

0.004

Family history of β-TM

37.5

62.5

0.76

Gender Male

33.3

66.7

0.83

34.6

65.4

Female

10.

11.

12.

Sadeghi-Bojd S, Hashemi M, Karimi M. Renal tubular function in patients with β-thalassaemia major in Zahedan, southeast Iran. Singapore Med J 2008;49:410-2. Wanachiwanawin W, Luengrojanakul P, Sirangkapracha P, Leowattana W, Fucharoen S. Prevalence and clinical significance of hepatitis C virus infection in Thai patients with thalassemia. Int J Hematol 2003;78:374-8. [CrossRef] Forget BG. Thalassemia syndromes. In: Hoffman R, Benz E J, Shathl S J, Furie B, Cohen HJ, Silberstein LE, Mcglare P, editors. Hematology Basic Principles and Practice. 3rd ed. Philadelphia: Churchill Livingstone, 2000:485-510. Chevaliez S, Pawlotsky JM. Hepatitis C virus: virology, diagnosis and management of antiviral therapy. World J Gastroenterol 2007;13:2461-6. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001;345:41-51. [CrossRef] Simmonds P, Holmes EC, Cha TA, Chan SW, McOmish F, Irvine B, Beall E, Yap PL, Kolberg J, Urdea MS. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol 1993;74:2391-9. [CrossRef] Simmonds P, Alberti A, Alter HJ, Bonino F, Bradley DW, Brechot C, Brouwer JT, Chan S-W, Chayama K. A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatol 1994;19:1321-4. [CrossRef] Dixit V, Quan S, Martin P, Larson D, Brezina M, DiNello R, Sra K, Lau JY, Chien D, Kolberg J, et al. Evaluation of a novel serotyping system for hepatitis C virus: strong correlation with standard genotyping methodologies. J Clin Microbiol 1995;33:2978-83. Stuyver L, Vanarnhem W, Wyseur A, Hernandez F, Delaporte E, Maertens G. Classification of hepatitis C viruses based on phylogenetic analysis of the envelope 1 and non-structural 5B regions and identification of five additional subtypes. Proc Natl Acad Sci USA 1994;91:10134-8. [CrossRef] Maertens G, Stuyver L. Typing of hepatitis C virus isolates. In: Rizzetto M, Purcell RH, Gerin JL, Verme G, editors. Viral Hepatitis and Liver Disease. Turin: Minerva Medica, 1997:181-6. Mahaney K, Tedeschi V, Maertens G, Bisceglie A, Vergalla J, Hoofnagle J, Sallie R. Genotypic analysis of hepatitis C virus in American patients. Hepatology 1994;20:1405-14. [CrossRef] Salwa B. Hepatitis C virus infection in Jordanian haemodialysis units: serological diagnosis and genotyping. J Med Microbiol 2002;51:700-4.

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Dusheiko G, Schmilovitz H, Brown D. Hepatitis C virus genotypes: an investigation of type-specific differences in geographic origin and disease. Hepatol 1994;19:13-8. [CrossRef] Abdulkarim AS, Zein NN, Germer JJ. Hepatitis C virus genotypes and hepatitis G virus in hemodialysis patients from Syria: identification of two novel hepatitis C virus subtypes. Am J Trop Med Hyg 1998;59:571-6. Davidson F, Simmonds P, Ferguson JC. Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 59 non-coding region. J Gen Virol 1995;76:1197-204. [CrossRef] Li CK, Chan PK, Ling SC, Ha SY. Interferon and ribavirin as frontline treatment for chronic hepatitis C infection in thalassaemia major. Br J Haematol 2002;117:755-8. [CrossRef] Marco V, Iacono L, Capra M, Grutta S, Ciaccio C, Gerardi C, Maggio A, Renda D, Almasio P, Pisa R, Craxi A. Alpha interferon treatment of chronic hepatitis C in beta-thalassaemia. Gut 1993;34 (2 Suppl):S142-3. Cunningham MJ, Macklin EA, Neufeld EJ, Cohen AR. Complications of b-thalassemia major in North America. Blood 2004;104:34-9. [CrossRef]

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Strader DB. Understudied populations with hepatitis C. Hepatology 2002;36:226-36. [CrossRef] Distante S, Bjoro K, Hellum KB, Myrvang B, Berg JP, Skaug K. Raised serum ferritin predicts nonresponse to interferon and ribavirin treatment in patients with chronic hepatitis C infection. Liver 2002;22:269-75. [CrossRef] Sharara AI, Ramia S, Ramlawi F, Fares JE, Klayme S, Naman R. Genotyping of hepatitis C virus (HCV) among positive Lebanese patients: comparison of data with that from other Middle Eastern countries. Epidemiol Infect 2007;135:427-32. [CrossRef] Bdour S. Hepatitis C virus infection in Jordanian haemodialysis units: serological diagnosis and genotyping. J Med Microbiol 2002;51:700-4. Mirmomen S, Alavian SM, Hajarizadeh B, Kafaee J, Yektaparast B, Zahedi MJ, Zand V, Azami AA, Hosseini MM, Faridi AR, Davari K, Hajibeigi B. Epidemiology of hepatitis B, hepatitis C, and human immunodeficiency virus infections in patients with beta-thalassemia in Iran: a multicenter study. Arch Iran Med 2006;9:319-23.

Research Article

Hyperuricemia and tumor lysis syndrome in children with non-Hodgkin’s lymphoma and acute lymphoblastic leukemia Non-Hodgkin lenfoma ve akut lenfoblastik lösemili çocuklarda hiperürisemi ve tümör lizis sendromu Betül Sevinir1, Metin Demirkaya1, Birol Baytan2, Adalet Meral Güneş2 1Department 2Department

of Pediatric Oncology, Medical Faculty, Uludağ University, Bursa, Turkey of Pediatric Hematology, Medical Faculty, Uludağ University, Bursa, Turkey

Abstract Objective: This study aimed to examine the incidence, clinical characteristics, and outcome of hyperuricemia and tumor lysis syndrome (TLS) in children with non-Hodgkin’s lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Materials and Methods: This retrospective study included data from 327 patients (113 NHL and 214 ALL). Results: Hyperuricemia occurred in 26.5% and 12.6% of the patients with NHL and ALL, respectively. The corresponding figures for TLS were 15.9% and 0.47% (p=0.001). All hyperuricemic NHL patients had advanced disease and renal involvement was present in 53%. All hyperuricemic ALL patients had a leukocyte count >50,000 mm3 at the time of diagnosis. Among the hyperuricemic NHL and ALL patients, 96.6% and 66.6% had LDH ≥500 UI/L, respectively. Treatment consisted of hydration and allopurinol; none of the patients received urate oxidase. Among the patients that developed TLS, 26.3% had laboratory TLS, 42.1% had grade I or II TLS, and 31.6% had grade III or IV TLS. Uric acid levels returned to normal after a mean period of 3.5±2.5 and 3.05±0.8 d in NHL and ALL groups, respectively. In all, 7% of the patients with hyperuricemia required hemodialysis. None of the patients died. Conclusion: In this series the factors associated with a high-risk for TLS were renal involvement in NHL and high leucocyte count in ALL. Management with allopurinol and hydration was effective in this group of patients with high tumor burden. (Turk J Hematol 2011; 28: 52-9) Key words: Non-Hodgkin’s lymphoma, leukemia, tumor lysis syndrome, hyperuricemia, children Received: January 28, 2010

Accepted: April 30, 2010

Özet Amaç: Çalışmanın amacı NHL ve ALL’li çocuklarda hiperürisemi ve TLS sıklığını, klinik özellikleri ve sonuçlarını tanımlamaktır. Address for Correspondence: Prof. Betül Sevinir, Department of Pediatric Oncology, Medical Faculty, Uludağ University, Bursa, Turkey Phone: +90 224 295 04 21 E-mail: bsevinir@yahoo.com doi:10.5152/tjh.2011.06

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Yöntem ve Gereçler: Bu retrospektif çalışmada 113 NHL ve 214 ALL’li toplam 327 hastanın verileri değerlendirildi. Bulgular: NHL olgularının %26.5’inde, ALL olgularının %12.6’sında hiperürisemi görüldü. TLS insidansı NHL ve ALL gruplarında %15.9 ve %0.47 bulundu (p=0.001). Hiperürisemi görülen NHL olgularının tümü ileri evrede olup, %53’ünde renal tutulum vardı. Tüm hiperürisemili ALL olgularında tanıda lökosit sayımı 50.000/mm3’den yüksekti. Hiperürisemik NHL grubunun %96.6’sında, ALL grubunun %66.6’sında LDH ≥500 UI/L idi. Tedavide hidrasyon ve allopürinol uygulandı, ürat oksidaz verilen hasta olmadı. TLS gelişen olguların %26.3’ünde laboratuvar TLS, %42,1’inde grade I ve grade II TLS, %31.6’sında grade III ve IV TLS saptandı. Ürik asit düzeyleri NHL ve ALL hastalarında ortalama 3.5±2.5 ve 3.05±0.8 günde normale döndü. Hiperürisemili hastaların %7’sinde hemodiyaliz gerekti. Mortalite olmadı. Sonuç: Bu seride en yüksek TLS riski renal tutulumu olan NHL olgularında saptandı. Allopürinol ve hidrasyonun tümör yükü yüksek olan bu grupta etkili olduğu gözlendi. (Turk J Hematol 2011; 28: 52-9) Anahtar kelimeler: Non-Hodgkin lenfoma, lösemi, tümör lizis sendromu, hiperürisemi, çocuklar Geliş tarihi: 28 Ocak 2010

Kabul tarihi: 30 Nisan 2010

Introduction Tumor lysis syndrome (TLS) is a complication of cancers that are highly sensitive to cytotoxic agents, and have a high tumor burden and proliferation rate. Acute renal injury may ensue due to hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia. Uric acid is an important mediator of renal injury in TLS, and hyperuricemia is one the first signs of TLS [1,2]. Nonetheless, not all patients with hyperuricemia develop other signs of TLS. Although TLS may also occur in solid tumors, patients with nonHodgkin’s lymphoma (NHL) and acute leukemia represent the majority of cases [1-3]. In Turkey acute leukemias and lymphomas are the most frequent childhood cancers [4,5]. NHL and acute lymphoblastic leukemia (ALL) are frequently associated with a number of adverse metabolic consequences due to high cell turnover and a high response rate to cytotoxic agents. Most children in Turkey with NHL are diagnosed at an advanced stage [4,6]. As such, metabolic disturbances that require urgent treatment are frequently observed. Among these, hyperuricemia has recently emerged as an important complication associated with the use of newer therapeutic agents. Allopurinol, a xanthine oxidase inhibitor, has traditionally been used to treat hyperuricemia; it blocks the production of uric acid from xanthine and hypoxanthine without affecting the breakdown of already formed uric acid, and at the same time prevents new production. Alkalinization of urine may increase the excretion of uric acid via urine [1,2]. In recent years, recombinant urate oxidase has been introduced,

which is an enzyme that exists in many mammalians, but not humans. This enzyme allows urinary excretion of uric acid by converting it to allantoin, which is 5-10-fold more soluble in water than uric acid; however, urate oxidase causes hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) enzyme deficiency and is expensive. Hyperuricemia and acute TLS may lead to adverse consequences, including renal insufficiency or sudden death. TLS has been graded on the basis of laboratory and clinical features, with the Cairo and Bishop TLS classification systems being the most recent [7]. Additionally, guidelines for preventing morbidity and mortality have been issued, despite the lack of a consensus. In Turkey the incidence of hyperuricemia in children with NHL and ALL has not been specifically addressed and, therefore, the present study aimed to determine the frequency, grade, clinical course, and outcome of hyperuricemia in children with NHL and ALL. Moreover, the efficacy of the protocol used at our hospital for the prevention and treatment of hyperuricemia and TLS was re-assessed.

Material and Methods Patients The medical records of children with NHL and ALL that were treated at our hospital between January 1997 and December 2007 were retrospectively evaluated. Age, gender, histopathological group, disease stage, and the presence of renal involvement, and serum lactate dehydrogenase (LDH), urea, uric acid, creatinine, calcium, phosphorus, and electrolyte levels were assessed in NHL

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patients. In the patients with ALL, age, gender, leukocyte count at the time of diagnosis, risk group status, and the presence of renal involvement, and serum LDH, urea, uric acid, creatinine, calcium, phosphorus, and electrolyte levels were assessed. Spontaneous or post-chemotherapy elevation of uric acid levels and signs of acute TLS were recorded, as were the response to the routine hyperuricemia management protocol, number of days required for normalization of uric acid levels, number of patients that required hemodialysis, and mortality data. The Local Ethical Committee of Uludağ University Medical Faculty approved this retrospective study (Approval number: 2009-11/80). Chemotherapy protocol The NHL BFM-95 and ALL BFM-95 protocols were used for the treatment of NHL and ALL patients, respectively. Criteria for hyperuricemia and acute tumor lysis syndrome Serum uric acid levels above normal were considered hyperuricemia (normal range: 2.2-7.2mg/ dL). TLS was defined on the basis of Cairo and Bishop criteria [7]. Accordingly, laboratory TLS (L TLS) criteria were as follows: serum uric acid ≥8mg/

dL (476 μmol/L), serum potassium ≥6 mEqL-1 (6 mmol/L), serum phosphorus >6.5 mg/dL (2.1 mmol/L), and serum calcium <7 mg/dL (1.75 mmol/L), or a 25% increase from baseline in the first 3 parameters, or a 25% decrease from baseline in serum calcium. Fulfillment of at least 2 laboratory criteria from 3 d prior to cytotoxic treatment to 7 d after was diagnostic for LTLS. Clinical TLS (CTLS) was defined on the basis of renal, cardiac, and neurological changes (Table 1). Data for hyperuricemic patients were classified according to LTLS and CTLS criteria. Routine management protocol for hyperuricemia Routine prophylaxis and treatment were given to all the NHL and ALL patients. As such, all patients with uric acid levels at the upper limit of normal or those with a high risk of acute TLS received intravenous hydration and oral allopurinol. Hydration was given for 24 h be be fore chemotherapy and the total daily dose ranged between 2500 and 3000 mL/m-2. The urine density and pH range were set at 1010 and 6.7-7, respectively. Allopurinol - 300 and 200 mg·m-2 ·d-1 p.o. - was given in 3 divided doses to the NHL and ALL patients, respectively. None of the patients received urate oxidase.

Table 1. Cairo-Bishop TLS grading system [7] LTLS

Creatinine

Cardiac arrhythmia

Seizure

Grade 0

<1.5 × ULN

None

Grade I

1.5 × ULN

Intervention not indicated

Grade II

1.5-3 × ULN

Non-urgent medical intervention

Brief, generalized seizure(s) controlled with anticonvulsants, or infrequent focal motor seizures not interfering with daily activity

Grade III

3-6 × ULN

Symptomatic and incomplete

Seizures in which consciousness is altered, controlled medically or poorly controlled, with breakthrough generalized control, with device seizures despite medical intervention

Grade IV

>6 ≈ ULN

Life threatening (e.g. arrhythmia associated with congestive heart, failure, hypotension, syncope, shock)

Seizure of any kind that is prolonged, repetitive, or difficult to control (e.g. status epilepticus, intractable epilepsy)

Grade V

Death

ULN: Upper limit of normal; LTLS: laboratory tumor lysis syndrome

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Table 2. Patient characteristics Number of patients Median age [years] (range) Gender (n, male/female)

NHL

ALL

113

214

6.5 (0.9-17.3)

5.5 (1.16-17.5)

0.8

90/23

131/83

Stage III, IV (n, %)

110 (97.3)

High WBC* (n, %)

28 (13)

895 ± 1246

372 ± 213

0.02

64 (56)

26 (12)

Uric Acid† (mg/dL)

5.79 ± 4.13

5.81 ± 2.37

Urea† (mg/dL),

30.5 ± 35.8

26.6 ± 12.5

0.3

Creatinine† (mg/dL)

0.74 ± 0.9

0.53 ± 0.11

0.11

Renal involvement (n, %)

32 (28.2)

Hyperuricemia (n, %)

30 (26.5)

27 (12.6)

0.05

ATLS (n, %)

18 (15.9)

1 (0.47)

0.001

LDH† (UI/L) High LDH level** (n, %)

ATLS: Acute tumor lysis syndrome, *High white blood cell count (WBC) ≥50,000

mm-3,

0.2

**LDH ≥500 IU/L , †Normal laboratory ranges: UA: 2.2-7.2 mg/dL; Cr: 0.6-

1.3 mg/dL; urea: 10-50 mg/dL; LDH: 125-243 IU/L (mean ± SD)

Statistical method Age, LDH concentration, biochemical parameters, and leukocyte counts are presented as mean±standard deviation (SD). Gender, hyperuricemia, hyperleukocytosis, and the frequency of TLS are presented as percentages. The NHL and ALL groups were compared using the t-test and chisquare test to determine the significance of the differences between means, and between the subgroups with or without uricemia in each group. The findings in the NHL and ALL patients with and without hyperuricemia were statistically compared using the t-test or chi-square test. Statistical comparisons were performed using SPSS v.10.0.

Results Data obtained from a total of 327 newly diagnosed patients with NHL (n=113) and ALL (n=214) between December 1997 and January 2007 were examined. Table 2 shows the epidemiological and clinical characteristics of the patients. Mean age at the time of diagnosis was 6.5±5.2 years (range: 0.917.3 years) and 6.1±3.9 years (range: 1.16-17.5 years) in the NHL and ALL groups, respectively (P=0.8). Among the patients with NHL, 68% (n=77) had B-cell, 28% (n=31) had lymphoblastic, and 4% (n=5) had anaplastic large cell lymphoma. According to the St. Jude staging system, 2.7%

(n=3), 77% (n=87), and 20.3% (n=23) had stage II, III, and III disease, respectively. Mean LDH level was 895±1246 IU/L , with 56% of the patients having an LDH level >500 IU/L. Among the ALL patients, 65% (n=139), 16.8% (n=36), and 18.2% (n=39) were in the standard-, medium-, and high-risk categories, respectively. Mean LDH level was 372±213 IU/L, and 12% (26/214) had an LDH level >500 IU/L. In all, 28 of the children with leukemia (13%) had a leukocyte count >50,000 mm3 (range: 50,000-192,000 mm3; mean: 82,985±39,029 mm3). Mean serum urea, creatinine, and uric acid levels did not differ significantly between the NHL and ALL patients (Table 2). Serum uric acid levels were above the normal range, corresponding to hyperuricemia in 30 children (26.5%) with NHL and 27 children (12.6%) with ALL (p=0.05). TLS criteria were met by 15.9% (n=18) of the NHL patients and 1 (0.47%) ALL patient (p=0.001). The majority of hyperuricemic NHL cases (25/30, 83%) presented at initial diagnosis, of which 5 (17%) developed hyperuricemia following the first dose of chemotherapy; the corresponding figures for hyperuricemic ALL patients were 59% (16/27) and 41% (11/27), respectively. A comparison of the characteristics of the hyperuricemic and normouricemic patients is shown in Table 3. All hyperuricemic NHL patients had stage III or IV disease, and their mean urea and creatinine concentrations were higher than those in the normouricemic cases (p=0.001

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and p=0.001, respectively). In 96.6% of the hyperuricemic NHL patients LDH was >500 UI/L (mean concentration: 1286±1378 UI/L). LDH levels in the hyperuricemic cases were significantly higher than those in the normouricemic cases (p=0.001). Of the hyperuricemic NHL patients, 53% had renal involvement, as compared to 19% of the non-hyperuricemic NHL patients (p=0.001) (Table 3). Among the hyperuricemic ALL patients, 59% and 41% were in the medium- and high-risk categories, respectively. Mean LDH was 967±798 UI/L, with 66.6% of the cases (18/27) having an LDH level >500 UI/L. All hyperuricemic ALL cases had an elevated white cell count, versus 1 (0.5%) patient in the nonhyperuricemic ALL group (p=0.0001). No patient had renal involvement. LDH levels were not significantly different between the NHL and ALL patients (p=0.29), nor were creatinine or urea (p=0.15 and p=0.8, respectively). Mean serum uric acid level in the hyperuricemic NHL and ALL patients was 13.29±5.92 mg/dL and 8.82±1.4 mg/dL, respectively (p=0.0001). The maximum uric acid concentration in these 2 groups was 30.4 mg/dL and 13 mg/dL, respectively. Table 4 shows the TLS grading. None of the patient with TLS developed arrhythmia or seizure. Among the hyperuricemic NHL patients, renal involvement was observed in 77% and 25% of the cases that did and did not develop TLS, respectively (Fisher’s exact test, p=0.008). Including the 1 ALL

patient with TLS, in all 5 patients with TLS (26.3%) LTLS was diagnosed without clinical signs. Grade I, II, III, and IV CTLS was observed in 15.8%, 26.3%, 26.3%, and 5.3% of the patients, respectively. Routine treatment for hyperuricemia resulted in normalization of the uric acid level within 1-10 d. On average, normouricemia was achieved within 3.57±2.1 and 3.07±0.8 d in NHL and ALL patients, respectively (P=0.22) (Figure 1). All hyperuricemic ALL patients had their serum uric acid level normalized following the routine treatment protocol for hyperuricemia. Of the 30 NHL patients, 26 had their uric acid and other biochemical parameters normalized, while among the 4 patients that developed TLS (21%), 3 cases with grade III disease and 1 case with grade IV disease required hemodialysis due to refractory hyperphosphatemia and oligoanuria. Patients that required hemodialysis represented 7% (n=4/57) of the patient population with hyperuriceTable 4. Cairo-Bishop grading results in the TLS cases Grade (n=19)

n (%)

LTLS

5 (26.3)

Grade I

3 (15.8)

Grade II

5 (26.3)

Grade III

5 (26.3)

Grade IV

1 (5.3)

Grade V

LTLS: Laboratory tumor lysis syndrome

Table 3. Characteristics of the hyperuricemic and normouricemic patients NHL hyperuricemic (n=30)

NHL non-hyperuricemic (n=83)

ALL hyperuricemic (n=27)

ALL non-hyperuricemic (n=187)

1286±1378

753±1170

0.04

967±798

286±194

0.01

High LDH** (n, %)

29 (96.6)

35 (42.2)

0.001

18 (66.6)

11 (5.9)

0.001

Uric Acid* (mg/dL)

13.29 5.92

3.76±1.33

0.0001

8.82±1.4

5.6±4.32

0.02

Urea* (mg/dL)

54.73±62

21.54±6.24

0.001

51.71±12

20.6±2.20

0.001

Creatinine* (mg/dL)

1.22±1.78

0.56±0.15

0.001

0.72±0.0 9 0.5±0.01

0.05

Renal involvement (n, %)

16 (53)

16 (19)

0.001

High WBC† (n, %)

27 (100)

1 (0.5)

0.0001

3.07±0.8

LDH* (UI/L)

TLS (n, %) Recovery time*

18 (60) 3.57±2.1

(days) *Mean ± SD, **High LDH: ≥500 IU/L , †High WBC: ≥50,000 mm-3.

-1 (3.7)

0.22

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Uric acid level

30 25 20

ALL NHL

15 10 5 0

days

Figure 1. Comparison of serum uric acid level during treatment in NHL and ALL patients

mia. In the presented patient series no deaths were recorded during hyperuricemia or TLS.

Discussion Hyperuricemia, which is the most common pediatric metabolic emergency, is a sign of TLS that occurs in high grade NHL, B-cell ALL, and acute myelocytic leukemia patients [1,2,7]; however, it has also been reported in some solid tumors, such as neuroblastomas and germ cell tumors. It can occur spontaneously or due to tumor lysis caused by a number of therapeutic modalities, including conventional cytotoxic chemotherapy, anti-CD20 monoclonal antibodies, and radiotherapy. It can also be triggered by administration of a single dose of steroid [1,2,6,8]. The reported frequency of hyperuricemia and TLS is primarily based on single-center studies, and varies between 5% and 42%. Patients with B-cell lymphoma or Burkitt’s lymphoma were reported to experience this condition twice as frequently as patients with T-cell lymphoma [9-12]. In multi-center NHL trials of the BFM-90 and 95 protocols, 4.4% of the total study population (78 out of 1791) experienced TLS, which occurred in 8.4% of the patients in the Burkitt’s lymphoma and B-cell ALL subgroups, versus 1.9% in the T-lymphoblastic lymphoma subgroup [11]. Metabolic catabolism was reported in 34 of the 410 patients (8%) with grade III and IV NHL, and B-cell ALL that were included in the LMB-89 protocol. Severe TLS was reported in 5% of patients and 1.7% required hemodialysis. In that series high tumor burden prevailed and 14% had renal involvement [13]. In a study from Turkey clinical signs and outcomes were reported for 97 NHL

patients, including 72 cases that received LMB-89 and 25 that received a modified LMT-89 protocol. Among these patients, 89.7% had stage III or IV disease, and 34 (35%) had TLS and hyperuricemia. Severe TLS was reported in 16.5% of the patients, of which 26% required hemodialysis [14]. Among patients with B-cell NHL that received a modified NHL BFM protocol, the reported incidence of TLS was 14% [15]. In the present study’s sample 30 (26.5%) of the 113 NHL patients had hyperuricemia and 18 (15.9%) had TLS, which is consistent with previously published data, and can be explained on the basis of the similarities in the biological and clinical characteristics of the patients in Turkey. The high incidence of B-cell lymphomas and the transitional nature of Burkitt’s lymphoma between the endemic and sporadic types result in a high number of advanced cases and increases the frequency of metabolic emergencies in Turkey [16,17]. This is similar to other reports from the same geographical location. For instance, TLS was reported in 22.5% of 59 patients with NHL, approximately 50% in the form of LTLS [18]. In patients with NHL and ALL tumor burden is associated with increased severity of hyperuricemia, while TLS or the need for hemodialysis is more likely to occur with renal involvement, which has been reported in 14%-61% of patients with TLS [13,15,19]. In the present study renal involvement occurred in 19% of the NHL patients and 53% of the hyperuricemic patients; this figure was 77% in the patients that developed TLS, as compared to 25% in those that did not (p=0.008). Renal involvement not only increases the risk of acute complications, but also has adverse consequences with regard to longterm prognosis [20]. None of the ALL patients in the present study had renal involvement. More severe hyperuricemia and a high incidence of TLS in the NHL group were probably associated with bulky disease accompanied by renal involvement. Similar to risk stratification in lymphoma patients, cases with ALL and AML can be classified in terms of the risk of hyperuricemia. Hyperleukocytosis, B-cell ALL, bulky mediastinal masses, and high LDH levels are likely to be associated with increased risk of hyperuricemia in leukemic patients [1,10-13,21]. In the present study all the hyperuricemic ALL patients had high tumor burden and elevated leukocyte counts, and 66.6% had elevated LDH levels (≥500 UI/L), which highlights the importance of

Sevinir et al. Hyperuricemia and tumor lysis syndrome

tumor burden in ALL patients. To the best of our knowledge this is the first study from Turkey to compare the incidence of hyperuricemia and TLS in patients with NHL and ALL diagnosed at the same center. The present data show that there was a 2.1and 33-fold increase in hyperuricemia and TLS, respectively, in children with NHL. These findings suggest that pediatric NHL patients with advanced disease and renal involvement represent the primary risk group for TLS. Of the 19 TLS cases reported here, 26.3% (n:5) met the criteria for LTLS and none had CTLS. Among the other 14 cases, 15.8% and 26.3% were classified as grade I and grade II, respectively. The rest had grade III (26.3%) and grade IV (5.3%) CTLS. Additionally, the severity of CTLS correlated with the requirement for hemodialysis. The prognostic significance of this novel classification is yet unknown and to clarify this issue clinical outcomes should be assessed in prospective studies. Despite previous reports of mortality and high hemodialysis rates associated with allopurinol treatment in TLS, in the present study high-risk patients were treated with hydration, alkalinization, and allopurinol, and no mortality associated with TLS was recorded. Additionally, none of the patients received urate oxidase and 7% required hemodialysis. Substitution of allopurinol with urate oxidase has been reported to decrease the incidence and severity of TLS [22,23]. The cost-effectiveness of urate oxidase is a subject of continuing research worldwide [24]. Urate oxidase is administered with varying doses and durations, depending on the level of risk. The major advantages of urate oxidase are rapid fall in serum uric acid level after a single dose and no prerequisite for urinary alkalinization; however, high acquisition cost and hemolysis in patients with G6PD enzyme deficiency are the major drawbacks limiting its use [2,7,12,23]. In the present study serum uric acid levels returned to normal in a mean 3.5 d. We think that selective use of allopurinol is a rational approach in such cases when resources are limited.

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Davidson MB, Thakkar S, Hix JK, Bhandankar ND, Wong A, Schreiber MJ. Pathophysiology, clinical consequences, and treatment of tumor lysis syndrome. Am J Med 2004; 116:546-54. [CrossRef] Hochberg J, Cairo MS. Tumor lysis syndrome: Current perspective. Haematologica 2008; 93:9-13. [CrossRef] Baeksgaard L, Sorensen JB. Acute tumor lysis syndrome in solid tumors- a case report and review of the literature. Cancer Chemother Pharmacol 2003;51:187-92. Buyukpamukcu M. Non-Hodgkin’s lymphomas. In: Voute T, Kalifa C, Barret A (Eds). Cancer in children. Fourth edition, Oxford, UK: Oxford University Press; 1998;119-36. Kutluk T, Yesilipek MA. Turkish National Pediatric Cancer Registry 2002-2008 (Turkish Pediatric Oncology Group and Turkish Pediatric Hematology Society) Pediatr Blood Cancer 2009; 53:(abstract) pp25. Yaris N, Mandiracioglu A, Buyukpamukcu M. Childhood cancer in developing countries. Pediatr Hematol Oncol 2004;21:237-53. [CrossRef] Cairo M, Bishop M. Tumour lysis syndrome: new therapeutic strategies and classification. Br J Haematol 2004;127:3-11. [CrossRef] Duzova A, Cetin M, Gümrük F, Yetgin S. Acute tumour lysis syndrome following a single-dose corticosteroid in children with acute lymphoblastic leukemia. Eur J Haematol 2001;66:404-7. [CrossRef] Eldar AH, Futerman B, Abrahami G, Attias D, Barak AB, Burstein Y, Dvir R, Gabriel H, Horovitz J, Kapelushnik J, Kaplinsky H, Miskin H, Sthoeger D, Toren A, Vilk-Revel S, Weintraub M, Yaniv I, Linn S, Arush MB. Burkitt lymphoma in children. The Israeli Experience. J Pediatr Hematol Oncol 2009;31:428-36. [CrossRef] Seidemann K, Meyer U, Jansen P, Yakisan E, Rieske K, Fuhrer M, Kremens B, Schrappe M, Reiter A. Impaired renal function and tumor lysis syndrome in pediatric patients with non-Hodgkin’s lymphoma and B-ALL. Observations from the BFM-trials. Klin Padiatr 1998;210:279-84. [CrossRef] Wossmann W, Schrappe M, Meyer U, Zimmermann M, Reiter A. Incidence of tumor lysis syndrome in children with advanced stage Burkitt’s lymphoma/leukemia before and after introduction of prophylactic use of urate oxidase.Ann Hematol 2003;82:160-5. [CrossRef] Coiffier B, Altman A, Pui CH, Younes A, Cairo MS. Guidelines for the management of pediatric and adult tumor lysis syndrome: An evidence -based review. J Clin Oncol 2008;26:2767-78. [CrossRef] Patte C, Sakiroglu C, Ansoborlo S, Baruchel A, Plouvier E, Pacquement H, Babin-Boilletot A. Urate-oxidase in the prevention and treatment of metabolic complications in patients with B-cell lymphoma and leukemia, treated in the Société Française d’Oncologie Pédiatrique LMB89 protocol. Ann Oncol 2002;13:78995. [CrossRef]

Sevinir et al. Hyperuricemia and tumor lysis syndrome

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Kutluk T, Varan A, Akyüz C, Buyukpamukcu M. Clinical characteristics and treatment results of LMB/LMT regimen in children with non-Hodgkin’s lymphoma. Cancer Invest 2002;20:626-33. [CrossRef] Karadeniz C, Oguz A, Citak EC, Uluoglu O, Okur V, Demirci S, Okur A, Aksakal N. Clinical characteristics and treatment results of pediatric B-cell non-Hodgkin lymphoma patients in a single center. Pediatr Hematol Oncol 2007;24:417-30. [CrossRef] Kutluk T, Sarıalioğlu F, Göğüş S, Akyüz C, Büyükpamukçu M. Burkitt’s lymphoma in Turkish children: A retrospective analysis of 104 cases. Turk J Cancer 1993;23:81-9. Cavdar AO, Gozdasoglu S, Yavuz G, Babacan E, Unal E, Uluoglu O, Yucesan S, Magrath IT, Akar N. Burkitt’s lymphoma between African and American types in Turkish children:clinical, viral (EBV) and molecular studies. Med Pediatr Oncol 1993;21:36-42. [CrossRef] Alavi S, Arzanian MT, Abbasian MR, Ashena Z. Tumor lysis syndrome in children with non-Hodgkin’s lymphoma. Pediatr Hematol Oncol 2006;23:65-70. [CrossRef] Olgar S, Yetgin S, Cetin M, Aras T, Akhan O. Electrolyte abnormalities at diagnosis of acute lymphocytic leukemia may be a clue for renal damage in long-term period. J Pediatr Hematol Oncol 2005;27:202-6. [CrossRef] Buyukpamukcu M, Varan A, Aydın B, Kale G, Akata D, Yalcin B, Akyuz C, Kutluk T. Renal involvement of non-

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Hodgkin’s lymphoma and its prognostic effects in childhood. Nephron Clin Pract 2005;100:86-91. [CrossRef] Truong TH, Beyene J, Hitzler J, Abla O, Maloney AM, Weitzman S, Sung L. Features at presentation predict children with acute lymphoblastic leukemia at low risk for tumor lysis syndrome. Cancer 2007;110:1832-9. [CrossRef] Pui CH, Jeha S, Irwin D, Camitta B.recombinant urate oxidase (rasburicase) in the prevention and treatment of malignancy-associated hyperuricemia in pediatric and adult patients: results of a compassionate-use trial. Leukemia 2001;15:1505-9. [CrossRef] Bertrand Y, Mechinaud F, Brethon B, Mialou V, Auvrignon A, Nelken B, Notz-Carrére A. SFCE (Société Française de Lutte contre les Cancers et Leucémies de l’Enfant et de l’Adolescent) recommendations for the management of tumor lysis syndrome (TLS) with Rasburicase: An observational survey. J Pediatr Hematol Oncol 2008;30:267-71. [CrossRef] Annemans L, Moeremans K, Lamotte M, Garcia Conde J, van den Berg H, Myint H, Pieters R, Uyttebroeck A. Pan-European multicentre economic evaluation of recombinant urate oxidase (rasburicase) in prevention and treatment of hyperuricaemia and tumor lysis syndrome in haematological cancer patients. Support Care Cancer 2003;11:249-57.

Case Report

PET CT imaging in extramedullary hematopoiesis and lung cancer surprise in a case with thalassemia intermedia Talasemi intermedia olgusunda ekstramedüller hematopoezin PET CT görüntülemesi ve akciğer kanseri sürprizi Semra Paydaş1, Özoğul Sargın2, Gülfiliz Gönlüşen3 1Department

of Oncology, Faculty of Medicine, Çukurova University, Adana, Turkey of Nuclear Medicine, Faculty of Medicine, Çukurova University, Adana, Turkey 3Department of Pathology, Faculty of Medicine, Çukurova University, Adana, Turkey 2Department

Abstract Extramedullary hematopoiesis (EMH) is the production of hematopoietic precursors outside the bone marrow cavity, and it causes mass effects according to its localization. Magnetic resonance imaging (MRI) and/or computed tomography (CT) scans are used most commonly to detect EMH foci. We report herein a case with thalassemia intermedia causing paravertebral mass associated with EMH detected by CT scan. We further evaluated the case with positron emission tomography (PET) CT, and lung cancer, which was not revealed in the CT scan, was detected coincidentally. (Turk J Hematol 2011; 28: 60-2)

Key words: Extramedullary hematopoiesis, PET CT, SUV max Received: June 7, 2009

Accepted: July 3, 2009

Özet Ekstramedüller hematopoez (EMH), hematopoetik öncü hücrelerin kemik iliği dışında üretimidir ve lokalizasyonuna göre kitle etkilerine neden olur. EMH odaklarını saptamada en sık MRI ve/veya CT kullanılır. Biz burada talasemi intermedia’lı bir olguda paravertebral kitleye yol açan ve CT ile saptanan bir EMH olgusu sunduk. PET CT ile daha ileri incelemede, CT ile saptanamamış olan, akciğer kanseri saptandı. (Turk J Hematol 2011; 28: 60-2) Anahtar kelimeler: Ekstramedüller hematopoez, PET CT, SUV max Geliş tarihi: 7 Haziran 2009

Kabul tarihi: 3 Temmuz 2009

Address for Correspondence: Prof. Semra Paydaş, Department of Oncology, Faculty of Medicine, Çukurova University, Adana, Turkey Phone: +90 322 338 60 60-3142 E-mail: sepay@cu.edu.tr doi:10.5152/tjh.2011.07

Turk J Hematol 2011; 28: 60-2

PaydaĹ&#x; et al. Extramedullary hematopoiesis and PET/CT

Case A 52-year-old male admitted to the hospital for further evaluation of a paravertebral mass. He had been investigated for dyspnea in another center. Thoracic computed tomography (CT) showed paravertebral mass, and fine needle aspiration (FNA) from this mass had been reported as extramedullary hematopoiesis (EMH). The patient had a history of cigarette smoking (80 pack-years). Splenomegaly had been detected at the age of 20 years; he had no transfusion history. Physical exam showed pallor, icterus, and spleen palpable 7 cm below the left costal margin. Abnormal laboratory findings were as follows: Hb: 8.7 g/dl, Hct: 30.2%, WBC: 8.5x109 /L, platelet: 359x 109 /L, MCV: 62 fl, MCHC: 28, RDW: 29.3, ferritin: 1698, erythrocyte sedimentation rate (ESR): 5 mm/h, alkaline phosphatase (ALP): 526 IU, uric acid (UA): 8.2 mg/dl, total/direct bilirubin: 1.48/0.19 mg/dl, aspartate/alanine aminotransferase (AST/ALT): 162/275 IU/L, gamma glutamyl transpeptidase (GGT): 307 IU, lactate dehydrogenase (LDH): 649 U, HbA1: 53.20%, HbA2: 8.30%, HbF: 38.20%, and HbS: 0%. Bone marrow biopsy showed hypercellularity with erythroid hyperplasia. Written informed consent was obtained from the patient. Clinical outcome: The patient was evaluated as thalassemia intermedia and paravertebral mass due to the EMH was detected by CT scan. He was further explored for dyspnea and fatigue developing in recent months. Fluorodeoxyglucose positron emission tomography (FDG PET)/CT was performed to evaluate the extramedullary mass and to determine whether there was another focus of the extramedullary mass causing dyspnea. PET/CT showed a paravertebral mass measuring 13 cm in diameter with regular borders at the posterior-inferior mediastinum, and standardized uptake value (SUV) max of this mass was 2.5. This mass was thought to be benign in origin and was found to be compatible with EMH diagnosed by FNA. However, surprisingly, there was a small subpleural mass (SUV max 13) localized at the latero-basal segment of the lower lobe of the right lung (Figure 1). Due to the very high SUV max value, this mass was evaluated as malignant. The patientâ&#x20AC;&#x2122;s history of smoking also suggested an epithelial neoplasia. FNA biopsy was made

Figure 1. PET CT imaging of extramedullary mass (low SUV max) and coincidental lung cancer (high SUV max)

from this mass, which was reported as non-small cell lung cancer. Surgery was performed for this malignant mass, and T2N0M0 non-small cell lung cancer was detected hitopathologically.

Discussion Extramedullary hematopoiesis (EMH) occurs in various disorders and is most commonly seen in sickle cell anemia, thalassemia, hereditary spherocytosis, polycythemia rubra vera, and especially in myelofibrosis. This is a compensatory mechanism for bone marrow dysfunction. The most frequent cause of EMH is thalassemia intermedia, as seen in our case. EMH usually involves the liver, spleen and lymph nodes, and it may cause paravertebral/intrathoracic, intraabdominal or pelvic masses. These foci are generally asymptomatic but may lead to symptomatic tumor-like masses [1,2]. The most commonly used diagnostic methods for EMH are magnetic resonance imaging (MRI) and CT scans [3-5]. The underlying condition generally suggests this entity, and FNA or surgical biopsy taken from the mass supports the diagnosis of EMH. If there is no known underlying condition, careful examination of a peripheral blood smear and then the nec-

PaydaĹ&#x; et al. Extramedullary hematopoiesis and PET/CT

essary diagnostic tests including hemoglobin electrophoresis and bone marrow biopsy confirm the diagnosis. There is limited data about the detection of EMH using PET CT; thus far, only a few cases have been reported in patients with EMH [6]. We thus do not know the properties of the PET CT imaging of EMH. SUV max value is generally low and the tissue appears normal. Underlying hematopoietic disorder may suggest EMH, and a sampling of this tissue confirms the diagnosis. In our case, the CT scan showed a paravertebral mass, and FNA suggested EMH. However, dyspnea and fatigue development in the recent past and the history of heavy smoking required further evaluation. PET CT revealed a paravertebral mass with benign tumor properties. However, there was a surprise on PET, which revealed a small mass with very high SUV max value, suggesting a malignant tumor. Biopsy confirmed the non-small cell lung cancer.

Turk J Hematol 2011; 28: 60-2

References 1.

Conclusion EMH was detected as a benign-appearing mass on PET CT in a case with thalassemia intermedia. SUV max was very low in EMH, which suggested a benign tumor. PET CT also showed a small malignant mass in our case, which could not be detected by CT scan.

Meo A, Cassinerio E, Castelli R, Bignamini D, Perego L, Cappellini MD. Effect of hydroxyurea on extramedullary haematopoiesis in thalassaemia intermedia: case reports and literature review. Int J Lab Hematol 2008;30:425-31. [CrossRef] Galati MC, Raiola G, De Sanctis V, Arcuri V, Arcuri PP, Brachi S, Borsari G. Extramedullary haematopoiesis in beta thalassaemia--unusual presentations: case reports. Pediatr Endocrinol Rev 2008;6:140-3. Debard A, Demasles S, Camdessanche JP, Duband S, Mohammedi R, Antoine JC. Dural localization of extramedullary hematopoiesis. Report of a case. J Neurol 2009;256:837-8. [CrossRef] Chunduri S, Gaitonde S, Ciurea SO, Hoffman R, Rondelli D. Pulonary extramedullary hematopoiesis in patients with myelofibrosis undergoing allogeneic stem cell transplantation. Haematologica 2008;93:1593-5. [CrossRef] Monce B, Hafedh J. Management of spinal cord compression caused by extramedullary hematopoiesis in beta-thalassemia. Inter Med 2008;47:1125-8. [CrossRef] Masley C, Jacene HA, Holz A, Grand DJ, Wahl RL. Extramedullary hematopoiesis on F-18 FDG PET/CT in a case with metastatic colon carcinoma. Clin Nucl Med 2007;32:878-80. [CrossRef]

Case Report

Treatment of intrathecal methotrexate overdose with folinic acid rescue and lumbar cerebrospinal fluid exchange: A report of two cases Yüksek doz intratekal metotreksat’ın folinik asit ve beyin omurilik sıvısı değişimi ile tedavisi: İki olgu sunumu Elif Kazancı1, Hüseyin Gülen2, Ayşe Erbay3, Canan Vergin4 1Clinics of Pediatric Hematology - Oncology, Dr. Behçet Uz Children’s Hospital, İzmir, Turkey 2Department of Pediatric Hematology, Faculty of Pediatric, Celal Bayar University, Manisa, Turkey 3Clinics of Pediatric Hematology - Oncology, Dr. Behçet Uz Children’s Hospital, İzmir, Turkey 4Clinics of Pediatric Hematology - Oncology, Dr. Behçet Uz Children’s Hospital, İzmir, Turkey

Abstract We report two male cases (4- and 5-years-old) of intrathecal methotrexate overdose. The two boys with acute lymphoblastic leukemia were to receive intrathecal injection of methotrexate. Instead of the prescribed 12 mg, they both received a dose of 120 mg. The initial cerebrospinal fluid samples showed methotrexate concentration of 2.24x10-2M in case 1 and 1.32x10-2M in case 2. The cases were successfully treated with cerebrospinal fluid (CSF) exchange and intravenous folinic acid rescue. The favorable outcome in our cases suggests that CSF exchange is safe and that folinic acid rescue may be adequate to prevent sequelae in patients subjected to intrathecal MTX overdoses up to 120 mg. We propose CSF exchange and intravenous folinic acid as the mainstay of treatment. In addition to the staff ’s failure to check the drug label carefully, the marked resemblance of the two dose preparations of MTX may have been contributory. (Turk J Hematol 2011; 28: 63-7) Key words: Methotrexate, intrathecal, overdose, treatment Received: December 31, 2009

Accepted: June 22, 2010

Özet İntratekal (IT) metotreksat uygulaması, merkezi sinir sistemi lösemisinin önlenmesi ve tedavisinde yaygın olarak kullanılmaktadır. Metotreksatın yüksek dozda veya uzun süreli uygulamaları ciddi nörotoksisiteye yol açabilir. Burada, hatalı olarak yüksek dozda intratekal metotreksat verilen iki olgu sunulmaktadır. Akut lenfoblastik lösemi tanısıyla izlenen 4 ve 5 yaşlarındaki iki erkek olguya, reçete edilen 12 mg yerine yanlışlıkla 120 mg dozunda intratekal metotreksat uygulandı. Uygulama sonrası başlangıç beyin omurilik sıvısı metotreksat düzeyleri sırasıyla 2.24x10-2M ve 1.32x10-2M idi. Olgular beyin omurilik sıvısı değişimi ve intravenöz folinik asit desteği ile başarılı bir şekilde tedavi edildiler. Address for Correspondence: M.D. Elif Kazancı, Clinics of Pediatric Hematology - Oncology, Dr. Behçet Uz Children’s Hospital, İzmir, Turkey Phone: +381 21 484 3963 E-mail: asavic@uns.ac.rs doi:10.5152/tjh.2011.08

Kazancı et al. Intrathecal methotrexate overdose and treatment

Turk J Hematol 2011; 28: 63-7

Takiplerinde klinik gidişin iyi olması, 120 mg’a kadar yüksek doz intratekal metotreksat uygulanmış hastalarda, beyin omurilik sıvısı değişimi ve folinik asit kurtarma tedavisinin sekelleri önleme açısından yeterli olabileceğini göstermektedir. Bu hatalı uygulamaya asistanların ilaç dozlarını kontrol etmedeki hatalarının ve farklı dozlardaki metotreksat preparatlarının benzer kutularda üretilmesinin yol açtığı düşünülmüştür. (Turk J Hematol 2011; 28: 63-7) Anahtar kelimeler: Metotreksat, intratekal, yüksek doz, tedavi Geliş tarihi: 31 Aralık 2009

Kabul tarihi: 22 Haziran 2010

Introduction Intrathecal (IT) methotrexate (MTX) therapy is used widely for the prevention and treatment of central nervous system (CNS) leukemia [1,2]. IT injection of MTX is becoming more popular, and may be the only form of CNS-directed therapy in selected patients [2]. Exposure to MTX in high concentrations or for sustained periods is clearly neurotoxic, and various dosage recommendations have been suggested to avoid neurotoxicity [3,4]. Clinical studies indicate that adults should receive a maximal IT dose of 12.5 mg of MTX to avoid acute clinical neurotoxicity. Rescue treatments of IT MTX overdose may include cerebrospinal fluid (CSF) exchange, ventricular washout, folinic acid rescue, and the investigational agent carboxypeptidase G2, an enzyme that rapidly hydrolyzes MTX into inactive metabolites [5-9]. There are only a few reports of patients with IT MTX overdose in the literature, and the management has not been uniform. In this paper, we report two cases of IT MTX overdose. Similar reports from the literature and their management are reviewed.

Case Reports Case 1 A four-year-old boy, diagnosed with pre-B cell acute lymphoblastic leukemia (ALL) without CNS involvement, started treatment according to the TRALL-BFM 2000 protocol for standard-risk ALL. The incident occurred when he received the fourth course of a 36-hour (h) continuous infusion of highdose MTX (HD-MTX, 1 g/m2). One hour later, a dose of 120 mg MTX instead of the prescribed 12 mg was administered IT. Fifteen minutes later, the patient complained of intense pain in his legs. Sixty minutes after the IT MTX, the patient developed headache and generalized hypertonia, and then IT MTX overdose was suspected. The other physical find-

ings of the patient were normal except for anxiety and generalized hypertonia. Systemic MTX infusion was stopped immediately and exchange of lumbar CSF was started 90 minutes (min) after the IT MTX. He was returned to the operating theater and a repeat lumbar puncture was performed under general anesthesia. Twenty milliliters of dark yellow CSF was removed by gravity over a period of 20 min. No complication was observed during the procedure. CSF MTX concentrations were measured by means of an enzyme-inhibition technique [10]. The initial CSF sample had MTX concentration of 2.24 x 10-2 M. 20 ml saline was administered IT instead of CSF 1.5 h after the IT HD-MTX. Twenty milliliters of normal saline was then introduced, started approximately 1.5 h after the IT dose. Subsequently, 210 ml of CSF were removed in 5-ml portions and replaced by a corresponding volume of pre-warmed normal saline. Thus, a total volume of 230 ml was exchanged, equivalent to 200 ml/m2, over a period of 2 h and 15 min [7]. Two hours after the IT MTX, folinic acid was started intravenously (IV) at a dose of 100 mg single, and then doses of 10 mg every 6 h for 24 h [5,13,15,16]. Dexamethasone was also used at a dose of 0.15 mg/kg IV every 6 h for 2 days. CSF examination, liver and kidney functions and electrolytes were found in normal limits. At 1 and 6 months, electroencephalogram (EEG) and a brain computed tomography (CT) scan were normal. The patient remained in normal neurological status thereafter. No signs of neurotoxicity were observed during the following four years. Case 2 The second patient was a five-year-old boy with T cell ALL, without CNS involvement, in whom treatment was started according to the TRALL-BFM 2000 protocol for medium-risk ALL. During the maintenance phase (Protocol II 45th day therapy), an IT dose of 120 mg of MTX instead of the intended 12 mg was accidentally given by the pediatrics staff.

Turk J Hematol 2011; 28: 63-7

A vial with a 10-fold higher concentration of MTX had erroneously been used for the preparation of the injection solution. Ninety minutes after the IT, the patient developed headache. The mistake was discovered 2 h later, and then 200 ml of CSF were exchanged with normal saline in 5-ml portions over a 3-h period via lumbar puncture. The initial CSF sample had a MTX concentration of 1.32 x 10-2M. Two hours after the IT MTX, folinic acid was started IV at a dose of 100 mg single, and then doses of 10 mg every 6 h for 24 h. Dexamethasone was also used at a dose of 0.15 mg/kg IV every 6 h for 2 days. No neurological signs were observed before or after the CSF exchange procedure. The child remained asymptomatic in complete remission, and EEG and CT of the brain performed six months after the incident were normal. Written informed consent was obtained from both of the patients’ family.

Discussion Methotrexate (MTX) has been used extensively in the treatment of various malignancies, including lymphoblastic leukemia and lymphoma. HD-MTX with leucovorin rescue is used to treat osteosarcoma (8-12 g/m2) and ALL (≤8 g/m2). The toxic effects of MTX are myelosuppression, mucositis, nephrotoxicity, hepatotoxicity, and neurotoxicity with acute or chronic encephalopathy [11-17]. Acute encephalopathy generally develops within 5-14 days after IT MTX or HD-MTX and may include headache, nausea, emesis, lethargy, altered mental status, blurred vision, aphasia, hemiparesis, and seizure. Chronic encephalopathy develops slowly, may progress, and can permanently impair neurologic function. Transient acute encephalopathy has been clinically observed in 3%-15% of cancer patients after HD- MTX [12,15,16]. Most patients can resume MTX therapy without permanent neurological sequelae, although 10% - 56% may experience recurrence on rechallenge [12,15,16]. The pathophysiology of MTX-induced acute encephalopathy is largely unknown but does not appear to be related to MTX pharmacokinetics [12]. The usual dose for IT administration is 12-15 mg. Management of IT MTX overdose is not uniform and, in fact, cases described in the literature have been treated with different approaches [18-24]. The highest reported IT dose given to patients who survived was 650 mg

Kazancı et al. Intrathecal methotrexate overdose and treatment

[5]. The neurotoxicity of MTX is both route- and dose-dependent. There is little neurotoxicity when MTX is given orally or intravenously, as the drug does not reach significant concentration in the CSF. However, IV HD MTX has been associated with CNS toxicity, and systemic toxicity may be seen following IT administration. Common findings included behavioral abnormalities, focal sensorial and motor signs and abnormal reflexes [15]. Manifestations of IT MTX overdoses are dose-dependent. It is associated with little or mild toxicity at less than 100 mg. However, doses in excess of 100 mg, and particularly in excess of 500 mg, may cause significant morbidity and mortality [5,19]. To our knowledge, 13 cases of IT MTX overdose have been reported in the literature (Table 1). Twelve of the 13 reported incidents involved children between the ages of 2-12 years. Eight patients suffered from ALL and the other four had non-Hodgkin’s lymphoma. Nine cases received 20-125 mg of IT MTX. In one of them, folinic acid rescue was administered IT and resulted in a fatal event. Nine cases were treated with folinic acid, dexamethasone and CSF exchange. No neurologic long-term sequelae were observed in any of these nine patients [20-24]. Three cases received 600-650 mg of IT MTX and underwent ventriculolumbar perfusion or CSF exchange, which are considered effective if performed promptly [5,6,18]. In ventriculolumbar perfusion, CSF is exchanged for warmed, isotonic saline solution [6,18]. From these reports, it is obvious that severe toxicity is expected when an IT MTX overdose of more than 600 mg occurs. Carboxypeptidase G1 and G2 are investigational agents that rapidly hydrolyze MTX into inactive metabolites [9]. It has been successfully used IT for treatment of IT MTX overdose [18]. However, these agents are not commercially available at present. Incorrect prescription and failure to check the drug label carefully prior to the injection are apparently the most immediate errors [25]. In our reported cases, the marked resemblance of the two strengths of parenteral MTX preparations (i.e. 50 mg/5 ml and 500 mg/5 ml, respectively) may also have been contributory. In conclusion, our patients were treated successfully with IV high doses of folinic acid and lumbar CSF exchange. Cases receiving overdoses of IT MTX should first be treated with CSF exchange and IV

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Turk J Hematol 2011; 28: 63-7

Table 1. Reported cases of intrathecal methotrexate overdose Reference

Age (year)

IT MTX dose (mg)

Ettinger (5) *

650

Spiegel et al. (6)

625

Rescue treatment

Folinic acid dose

CSF exchange

50 mg x2 +12 mg q6 h x 2 wk

IV folinic acid + dexamethasone Ventriculolumbar perfusion

200 mg/m2 q3 h x 8 + 150 mg q3 h x 32

IV folinic acid + thymidine O’Marcaigh et al. (18)

600

Ventriculolumbar perfusion

1000 mg/m2+100 mg/m2 q3 h x 12

IV folinic acid + dexamethasone Carboxypeptidase-G2 IT Riva et al. (23)

300

IV folinic acid + dexamethasone

120 mg/m2 levo product q3 h x 8 + q6 h x 4

Lee et al. (24)

125

IV folinic acid + dexamethasone

15 mg q3 h x8 + 30 mg q6 h x12

Lee et al. (24)

125

IV folinic acid + dexamethasone

15 mg q3 h x 8 + 30 mg q6 h x 12

Jakobson et al. (7)

120

CSF exchange

90 mg + 15 mg q3 h x16

IV folinic acid Jakobson et al. (7)

100

CSF exchange

Not specified

IV folinic acid + dexamethasone Ettinger et al. (22)

IV folinic acid + dexamethasone

85 mg + 6 mg q6 h x 4

Addiego et al. (20)

CSF exchange

Not specified

IV folinic acid + dexamethasone Addiego et al. (20)

CSF exchange

12 mg q12 h x 6

IV folinic acid + dexamethasone Lampkin et al. (21)

IM folinic acid

12 mg + 6 mg q4 h x 9

Jardine et al. (19) *

IT folinic acid + IV folinic acid

100 mg q4 h x24

*: Fatal outcome; IT: Intrathecal; IV: Intravenous; MTX: Methotrexate

folinic acid rescue. More important, doctors and nurses who treat malignant diseases must be very careful in all stages of disease care, such as in the prescribing, ordering and administering of medicines and the following of adverse or unexpected effects. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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Bleyer WA, Poplack DG. Prophylaxis and treatment of leukemia in the central nervous system and other sanctuaries [Review]. Semin Oncol 1985;12:131-48. Poplack DG. Acute lymphoblastic leukemia. In: Pizzo PA, Poplack DG, editors. Principles and Practice of Pediatric Oncology. Philadelphia: Lippincott, 1993:431-81.

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Bleyer WA, Drake JC, Chabner BA. Neurotoxicity and elevated cerebrospinal-fluid methotrexate concentration in meningeal leukemia. N Engl J Med 1973;289:770-3. [CrossRef] Addiego JE Jr, Ridgway D, Bleyer WA. The acute management of intrathecal methotrexate overdose: pharmacologic rationale and guidelines. J Pediatr 1981;98:825-8. [CrossRef] Ettinger LJ. Pharmacokinetics and biochemical effects of a fatal intrathecal methotrexate overdose. Cancer 1982;50:444-50. [CrossRef] Spiegel RJ, Cooper PR, Blum RH, Speyer JL, McBride D, Mangiardi J. Treatment of massive intrathecal methotrexate overdose by ventriculolumbar perfusion. N Engl J Med 1984;311:386-8. [CrossRef] Jakobson AM, Kreuger A, Mortimer O, Henningsson S, Seidel H, Moe PJ. Cerebrospinal fluid exchange after intrathecal methotrexate overdose. A report of two cases. Acta Paediatr 1992;81:359-61. [CrossRef] Widemann BC, Balis FM, Murphy RF, Sorensen JM, Montello MJ, O’Brien M, Adamson PC[p3]. Carboxypeptidase-G2, thymidine and leucovorin rescue in cancer patients with methotrexate-induced renal dysfunction. J Clin Oncol 1997;15:2125-34.

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Adamson PC, Balis FM, McCully CL, Godwin KS, Bacher JD, Walsh TJ, Poplack DG[p4]. Rescue of experimental intrathecal methotrexate overdose with carboxypeptidase-G2. J Clin Oncol 1991;9:670-4. Falk LC, Clark DR, Kalman SM, Long TF. Enzymatic assay for methotrexate in serum and cerebrospinal fluid. Clin Chem 1976;22:785-8. Relling MV, Fairclough D, Ayers D, Crom WR, Rodman JH, Pui CH, Evans WE. Patient characteristics associated with high-risk methotrexate concentrations and toxicity. J Clin Oncol 1994;12:1667-72. Rubnitz JE, Relling MV, Harrison PL, Sandlund JT, Ribeiro RC, Rivera GK, Thompson SJ, Evans WE, Pui CH. Transient encephalopathy following high-dose methotrexate treatment in childhood acute lymphoblastic leukemia. Leukemia 1998;12:1176-81. [CrossRef] Mahoney DH Jr, Shuster JJ, Nitschke R, Lauer SJ, Steuber CP, Winick N, Camitta B. Acute neurotoxicity in children with B-precursor acute lymphoid leukemia: an association with intermediate-dose intravenous methotrexate and intrathecal triple therapy-a Pediatric Oncology Group study. J Clin Oncol 1998;16:1712-22. Atra A, Pinkerton CR, Bouffet E, Norton A, Hobson R, Imeson JD, Gerrard M; United Kingdom Children Cancer Study Group/Non-Hodgkin Lymphoma Group. Acute neurotoxicity in children with advanced stage B-non-Hodgkin’s lymphoma and B-acute lymphoblastic leukaemia treated with the United Kingdom children cancer study group 9002/9003 protocols. Eur J Cancer 2004;40:1346-50. [CrossRef] Walker RW, Allen JC, Rosen G, Caparros B. Transient cerebral dysfunction secondary to high-dose methotrexate. J Clin Oncol 1986;4:1845-50.

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Jaffe N, Takaue Y, Anzai T, Robertson R. Transient neurologic disturbances induced by high-dose methotrexate treatment. Cancer 1985;56:1356-60. [CrossRef] Packer RJ, Grossman RI, Belasco JB. High dose systemic methotrexate-associated acute neurologic dysfunction. Med Pediatr Oncol 1983;11:159-61. [CrossRef] O’Marcaigh AS, Johnson CM, Smithson WA, Patterson MC, Widemann BC, Adamson PC, McManus MJ. Successful treatment of intrathecal methotrexate overdose by using ventriculolumbar perfusion and intrathecal instillation of carboxypeptidase G2. Mayo Clin Proc 1996;71:161-5. [CrossRef] Jardine LF, Ingram LC, Bleyer WA. Intrathecal leucovorin after intrathecal methotrexate overdose. J Pediatr Hematol Oncol 1996;18:302-4. [CrossRef] Addiego E, Ridgway D, Bleyer WA. The acute management of intrathecal methotrexate overdose: pharmacologic rationale and guidelines. J Pediatr 1981;98:825-8. [CrossRef] Lampkin BC, Higgins GR, Hammond D. Absence of neurotoxicity following massive intrathecal administration of methotrexate: case report. Cancer 1967;20:1780-1. [CrossRef] Ettinger LJ, Freeman AI, Creaven PJ. Intrathecal methotrexate overdose without neurotoxicity: case report and literature review. Cancer 1978;41:1270-3. [CrossRef] Riva L, Conter V, Rizzari C, Jankovic M, Sala A, Milani M. Successful treatment of intrathecal methotrexate overdose with folinic acid rescue: a case report. Acta Paediatr 1999;88:780-2. [CrossRef] Lee ACW, Wong KW, Fong KW. Intrathecal methotrexate overdose. Acta Paediatr 1997;86:434-7. [CrossRef] Poplack DG. Massive intrathecal overdose: “Check the label twice!” N Engl J Med 1984;31:400-2. [CrossRef]

Case Report

Brucellosis presenting with pancytopenia due to hemophagocytic syndrome Hemofagositik sendroma bağlı pansitopeni ile başvuran bruselloz olgusu Ela Erdem, Yıldız Yıldırmak, Nurşen Günaydın Department of Pediatrics, Şişli Etfal Education and Research Hospital, İstanbul, Turkey

Abstract Reactive hemophagocytic syndrome is clinically characterized by fever, hepatosplenomegaly, pancytopenia, and coagulopathy, and is histologically characterized by excessive proliferation and activation of histiocytes or macrophages. It can occur with systemic infections, immunodeficiency, or underlying malignancy. Brucellosis is one of the rare causes of hemophagocytosis. Herein we report an 11-year-old male with pancytopenia due to hemophagocytosis during the course of brucellosis that responded favorably to therapy. Although rare, hemophagocytosis should be considered as a possible cause of pancytopenia in patients with brucellosis, especially in regions where brucellosis is frequently encountered. (Turk J Hematol 2011; 28: 68-71) Key words: Brucellosis, pancytopenia, hemophagocytosis Received: June 05, 2009

Accepted: September 18, 2009

Özet Reaktif hemofagositik sendrom klinik olarak ateş, karaciğer ve dalak boyutlarında artış, pansitopeni ve pıhtılaşma bozuklukları; histolojik olarak da histiyosit ya da makrofajların artmış proliferasyon ve aktivasyonları ile karakterizedir. Sistemik hastalıklar, immun yetmezlikler ve altta yatan malignitelerle birlikte olabilir. Brusella, hemofagositozun nadir nedenlerinden biridir. Brusellanın sık görüldüğü ülkelerde pansitopenisi olan hastalarda hemofagositozu hatırlatmak amacı ile, brusella seyri sırasında hemofagositoza bağlı pansitopenisi olan, uygun tedavi sonrası iyileşen 11 yaşında bir erkek hasta sunulmaktadır. (Turk J Hematol 2011; 28: 68-71) Anahtar kelimeler: Brusella, pansitopeni, hemofagositoz Geliş tarihi: 5 Haziran 2009

Kabul tarihi: 18 Eylül 2009

Address for Correspondence: Dr. Ela Erdem, Department of Pediatrics, Şişli Etfal Education and Research Hospital, İstanbul, Turkey Phone: +90 216 553 73 05 E-mail: elaerdem@yahoo.com doi:10.5152/tjh.2011.09

Turk J Hematol 2011; 28: 68-71

Introduction Hemophagocytic lymphohistiocytosis (HLH) is an unusual syndrome characterized by acute onset of fever, hepatosplenomegaly, lymphadenopathy, and jaundice, along with the pathological findings of hemophagocytosis (phagocytosis of erythrocytes, leukocytes, platelets, and their precursors by macrophages) in bone marrow and other tissues. Although de novo HLH can also be seen, it often occurs in the setting of another disorder - usually in association with malignant, infections, or autoimmune diseases that are prominently linked with Epstein-Barr virus (EBV) infection [1]. Non-viral infections and bacterial infections, including tuberculosis, are other major causes [2]. Fungal and parasitic causes of HLH have also been reported [3,4]. Brucellosis is one of the rare causes of hemophagocytosis. Herein we present a case of pancytopenia due to hemophagocytosis during the course of brucellosis.

Case Report An 11-year-old male presented to our hospital with symptoms of fever, loss of appetite, and headache, which began 5 days earlier. The patient’s parents were not consanguineous. His height was 138 cm (10th-25th percentile) and weight was 26 kg (3rd-10th percentile). His general health status was moderately good and neurological findings were normal. In physical examination the liver and spleen were 8 cm and 10 cm, respectively, and palpable below the costal margin of the midclavicular line, with multiple bilateral inguinal lymphadenopathies. He was diagnosed with pancytopenia based on the following laboratory findings: hemoglobin: 7.3 g dL; mean corpuscular volume: 77 fL; white blood cell count: 2600 mm3 (granulocyte: 900 mm3); platelet count: 85,000 mm3; SGOT: 65 U L1; SGPT: 16 U L; LDH: 1733 U L1; cholesterol: 155 mg/ dL; triglycerides: 345 mg/dL; HDL: 13 mg/dL; VLDL: 73 mg/dL; LDL: 69 mg/dL; total bilirubin: 0.5 mg/dL; total protein: 5.3 g dL; albumin: 2.5 g dL; ferritin: 3167 ng/mL; prothrombin time: 14.1 sec; prothrombin activity: 60%; partial thromboplastin time: 29.3 sec; fibrinogen: 155 mg/dL. Burr cells and cells resembling sickle cells were observed in peripheral smear.

Erdem et al. Brucellosis-related hemophagocytic syndrome

In order to exclude malignancy as a cause of pancytopenia, bone marrow aspiration was performed, which showed the presence of hemophagocytes (Figure 1). Based on the clinical, laboratory (pancytopenia, hypertriglyceridemia, hypofibrinogenemia, and elevated ferritin level), and bone marrow aspiration findings, the patient was diagnosed with secondary hemophagocytic syndrome. Serological markers were examined for EBV, cytomegalovirus, hepatitis B, parvovirus, Salmonella, and Brucella. Wright agglutination test results were positive, with a titer of 1/2560. Immediately following the bone marrow biopsy and culture to confirm the diagnosis, a course of antibiotic treatment was initiated (oral doxycycline 200 mg/day for 6 weeks and intravenous gentamycin 5 mg/kg/day for 2 weeks). Blood and bone marrow cultures remained sterile. Examination of the biopsy specimens showed that the bone marrow was normocellular and contained macrophages that phagocytized the lymphoid and erythroid elements. After 1 week of antibiotic treatment the patient was afebrile. At the end of the third week of doxycycline treatment the patient’s white blood cell count was 2480 mm3 (granulocyte: 1680 mm3), hemoglobin was 9.7 g dL, and platelet count was 175,000 mm3. Six months after treatment began the patient’s white blood cell count was 6640 mm3, neutrophils was 3600 mm3, hemoglobin was 13.8 g dL, and platelet count was 187,000 mm3. The patient remained symptom-free with progressive decrease in the titers of Wright agglutination test.

Figure 1. Bone marrow smear demonstrating hemophagocytosis by a large histiocyte (Giemsa stain, 1000×)

Erdem et al. Brucellosis-related hemophagocytic syndrome

Written informed consent was obtained from the patient's family.

Discussion HLH was first described by Scott in 1939 [5] and has since been associated with a variety of viral, bacterial, fungal, and parasitic infections, as well as collagen-vascular diseases and malignancies - particularly T-cell lymphomas. This diversity has led to the suggestion that HLH secondary to an underlying medical illness should be regarded as reactive hemophagocytic syndrome. Both sporadic and familial cases of HLH are often precipitated by acute infections, and HLH may mimic infectious illnesses such as overwhelming bacterial sepsis, which emphasizes the importance of the association between HLH and infection. As such, a better understanding of the pathophysiology of HLH may clarify the interaction between the immune system and infectious agents. Brucellosis is among the rare causes of secondary HLH, in which hematological alterations are common and pancytopenia is observed in 6% of patients [6]. Examination of bone marrow aspiration specimens shows normo-, hyper-, or hypocellularity [7]. In addition to pancytopenia, severe disorders including hemophagocytic syndrome - have also been described in association with brucellosis. Although the mechanism of hemophagocytosis induction in cases of brucellosis is not well understood, immune system derangement, with defective T-cell functioning, T-cell and monocyte hyperactivation, hypercytokinemia, and selective deficiency in cellular cytotoxicity has been reported. [8] Macrophages can be activated when they come into contact with foreign substances such as bacteria. This activation, in turn, can cause hemophagocytosis when macrophages come into contact with red blood cells, white blood cells, and platelets, leading to the clinical symptoms in the same nonspecific way as when they come into contact with foreign organisms. Although the precise mechanism remains unclear, 1 currently accepted theory suggests the role of perforin and natural killer cells in HLH subtypes [9]. Patients with perforin deficiency may have impaired defenses against intracellular pathogens, which is also critically important to the mechanism of primary HLH. Decreased natu-

Turk J Hematol 2011; 28: 68-71

ral killer cell activity results in increased T-cell activation, immediately followed by cytokine production, which leads to an inflammatory reaction, and extensive damage and associated symptoms [10]. Hemophagocytic syndrome is less common in children than in adults. A search of the literature showed that there are only 3 published reports of hemophagocytosis secondary to brucellosis in children [11-13]. Hemophagocytic syndrome in children should be differentiated from familial HLH, which is characterized by early onset, a higher prevalence of parental consanguinity, and an association with immune deficiencies, such as ChediakHigashi syndrome, Griscelli syndrome, and X-linked lymphoproliferative syndrome. Both conditions can be triggered by infections or other stimuli; however, most patients with secondary HLH have no underlying immune deficiency. Brucellosis associated with pancytopenia and evidence of reactive hemophagocytosis were first reported by Zuazu et al. in 1979 [14]. The presented case had fever, hepatosplenomegaly, and pancytopenia. Pancytopenia associated with brucellosis is attributed to hypersplenism, hemophagocytosis, and granulomatous lesions of the bone marrow, which is usually hypercellular [13,15,16]. In the present case we think that hemophagocytosis was responsible for pancytopenia. As isolation of the organism is difficult, serological tests are used for the routine diagnosis of brucellosis in most cases and the agglutination test is used as the principal test. When brucellosis is suspected in patients with negative cultures, rising agglutinin titers over 1:160 are considered diagnostic. [17] With the availability of effective therapy the mortality rate associated with brucellosis has declined. In some cases, intravenous immunoglobulin is used for treatment, which results in remission in adults and older children, particularly those with an underlying immune dysfunction; however, the role of this treatment in cases of hemophagocytosis is unclear [18]. The presented case recovered after antimicrobial treatment, without the need for intravenous immunoglobulin. Brucellosis must be considered in the differential diagnosis of malignant or benign diseases associated with hemophagocytosis, especially in geographic areas where brucella infection is common. The pathogenesis of pancytopenia in brucellosis is not clear, but appears to be multi-factorial. Although

Erdem et al. Brucellosis-related hemophagocytic syndrome

Turk J Hematol 2011; 28: 68-71

rare, hemophagocytosis should be considered a possible cause of pancytopenia in patients with brucellosis. Conflict of interest statement The authors declare that there are no conflicts of interest. None of the authors have a financial or proprietary interest in the case report preparation. All of the authors have been duly credited.

10. 11.

References 1. 2.

4. 5. 6. 7. 8.

Janka G, Stadt U: Familial and acquired hemophagocytic lymphocytic lymphohistiocytosis. Hematology 2005;1:82-8. [CrossRef] Lee SW, Wang CY, Lee BJ, Kuo CY, Kuo CL. Hemophagocytic Syndrome in Miliary Tuberculosis Presenting with Noncaseating Granulomas in Bone Marrow and Liver. JFMA 2008;107:495-9. Numata K, Tsutsumi H, Wakai S, Tachi N, Chiba S. A child case of hemophagocytic syndrome associated with cryptococcal meningitis. J Infect 1998;37:118-9. [CrossRef] Kocak N, Eren M, Yuce A, Gumruk F. Hemophagocytic syndrome associated with visceral leishmaniasis. Indian Pediatr 2004;41:605-7. Scott R, Robb-Smith A. Histiocytic medullary reticulosis. Lancet 1939;2:194-8. Ajlouni YM, Shaker K: Hematological manifestations of human brucellosis. QMJ 1998;2:41-3. Yildirmak Y, Palanduz A, Telhan L, Arapoglu M, Kayaalp N. Bone marrow hypoplasia during brucella infection. J Pediatr Hematol Oncol 2003;25:63-4. [CrossRef] Henter JI, Arico M, Elinder G, Imashuku S, Janka G.

12. 13.

14. 15. 16.

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Familial hemophagocytic lymphohistiocytosis. Primary hemophagocytic lymphohistiocytosis. Hematol Oncol Clin North Am 1998;12:417-33. [CrossRef] Risma KA, Frayer RW, Filipovich AH, Sumegi J. Aberrant maturation of mutant perforin underlies the clinical diversity of hemophagocytic lymphohistiocytosis. J Clin Invest 2006;116:182-92. [CrossRef] Arico M, Danesino C, Pende D, Moretta L. Pathogenesis of haemophagocytic lymphohistiocytosis. Br J Haematol 2001;114:761-69. [CrossRef] Karakukcu M, Patiroglu T, Ozdemir MA, Gunes T, Gumus H, Karakukcu C. Pancytopenia, a rare hematologic manifestation of brucellosis in children. J Pediatr Hematol Oncol 2004;26:803-6. Ullrich CH, Fader R, Fahner JB, Barbour SD. Brucellosis presenting as prolonged fever and hemophagocytosis. Am J Dis Child 1993;147:1037-8. Al-Eissa YA, Assuhaimi SA, Al-Fawaz IM, Higgy KE, Al-Nasser MN, Al-Mobaireek KF. Pancytopenia in children with brucellosis: clinical manifestations and bone marrow findings. Acta Haematol 1993;89:132-6. [CrossRef] Zuazu JP, Duran JW, Julia AF. Hemophagocytosis in acute brucellosis. N Engl J Med 1979;301:1185-6. [CrossRef] Akdeniz H, Irmak H, Seckinli T, Buzgan T, Demirรถz AP. Hematological manifestations in brucellosis cases in Turkey. Acta Med Okayama 1998;52:63-5. Martin-Moreno S, Soto-Guzman O, Bernaldo-de-Quiros J, Reverte-Cejudo D, Bascones-Casas C. Pancytopenia due to hemophagocytosis in patients with brucellosis: a report of four cases. J Infect Dis 1983;147:445-9. Moyer NP, Holcomb LA, Hauslert WJ. Brucella. In: Balows A, ed. Manual of Clinical Microbiology. Washington: American Society for Microbiology, 1991:457-61. Gill D, Spencer A, Cobcroft R. High dose gamma-globulin therapy in the reactive hemophagocytic syndrome. Br J Haematol 1994;88:204-6. [CrossRef]

Letter to the Editor

A child with primary gastric lymphoma and cavernous sinus involvement Primer mide lenfoması olan bir çocukta kavernöz sinüs tutulumuu Mehmet Akif Özdemir1, Yasemin Altuner Torun1, Türkan Patıroğlu1, Edip Torun2, Ahmet Candan Durak3 1Department

of Pediatric Hematology, Faculty of Medicine, Erciyes University, Kayseri, Turkey of Gastroenterology, Faculty of Medicine, Erciyes University, Kayseri, Turkey 3Department of Radiology, Faculty of Medicine, Erciyes University, Kayseri, Turkey 2Department

To the Editor, Primary gastric lymphomas are exceptionally rare gastrointestinal cancers in children [1,2]. Central nervous system involvement at the time of diagnosis is not uncommon, but there are no published reports of primary gastric lymphoma involving cavernous sinus (CS) during treatment [3,4]. We report a case of primary gastric lymphoma in a previously healthy 13-year-old boy that developed CS involvement after the second cycle of chemotherapy. He presented with a 1-month history of epigastric pain, and several episodes of hematemesis and melena. He lost 3 kg during the previous month. At presentation he looked ill, with pale conjunctiva. Physical examination showed a palpable mass in the left upper abdominal quadrant. Abdominal tomography showed diffuse thickening of the gastric rugae involving the entire fundus, a 33-mm diameter mass over the splenic vein in the corpus, and pancreatic invasion. Upper gastrointestinal endoscopy was performed and a round ulcer 3 cm

in diameter was noted in the body of the stomach. The mass was diagnosed as diffuse large B-cell NHL (CD20 positive) associated with Helicobacter pylori gastritis, based on examination of the biopsy specimen. Neoplastic cells were strongly positive for CD20 and negative for cytokeratin, desmin, CD79a, S100, and CD99. The Ki67 proliferative index was 99%. EBV serology was negative. Written informed consent was obtained from the patient. The patient was treated for the H. pylori infection using a proton pump inhibitor combined with clarithromycin and amoxicillin. Computed tomography of the chest, cranial MRI, and bone scintigraphy were normal. Bone marrow aspiration showed that there wasn’t any malignant cell infiltration. He received 2 cycles of a chemotherapeutic regimen that included rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. The initial cytologic status of CSF was normal; therefore, he did not initially receive intrathecal treatment. After the second chemotherapy cycle he presented with a 2-week history of headache and left

Address for Correspondence: M.D. Yasemin Altuner Torun, Department of Pediatric Hematology, Faculty of Medicine, Erciyes University, Kayseri, Turkey Phone: +90 352 235 77 32 E-mail: yaseminaltuner@yahoo.com doi:10.5152/tjh.2011.10

Turk J Hematol 2011; 28: 72-4

Ă&#x2013;zdemir et al. Cavernous sinus involvement in gastric lymphoma

eyeball pain. Visual acuity was 20/20 in both eyes. There was 2 mm of proptosis and complete ptosis in the left eye. Left extraocular movement was markedly limited in all directions of gaze (Figure 1a-d). MRI of the brain showed a homogeneously enhancing mass in the left CS extending into the orbital apex (Figure 2). CS syndrome was considered, based on clinical manifestations and physical findings. Lumbar puncture was performed, and cytologic examination of CSF showed large lymphoma cells with CD20 (+). Treatment was initiated with high-dose iv and intrathecal chemotherapy. Progressive ptosis of the left eye, swelling of the left orbital region with diplopia, and anesthesia developed after 3 cycles of chemotherapy. Radiation therapy was planned, but was not performed, as the patient unfortunately died 2 months after initial presentation due to progressive disease. Primary lymphoma of the stomach is extremely rare in childhood, and is frequently designated as mucosa-associated lymphoid tissue (MALT) lymphoma in adults [5]. There is no consensus concerning the best management strategy. Surgery, H. pylori eradication, chemotherapy, radiotherapy, and combined methods have all been used for treatment. Despite the rarity of primary gastric lymphoma in childhood, it should be approached aggressively [5,6]. CS involvement with neuro-ophthalmological symptoms is even more rare; only a few case reports exist in the literature [7]. The etiology of CS syndrome is neoplasm, aneurysm, thrombosis,

Figure 1. Left eye ptosis (a) and limitation in adduction, abduction and upward gaze of left eye (b-d)

Figure 2. MRI scans were revealing cavernous sinus left side enlargement and tumor extension

Özdemir et al. Cavernous Sinus Involvement in Gastric Lymphoma

carotid-cavernous fistula, pituitary apoplexy, granulomatous inflammation, and infection [4,8]. CS syndrome is suspected when retro-orbital pain is accompanied by involvement of 1 or more cranial nerves to the ocular muscles. Lymphoma may also appear as diffuse enlargement and enhancement of the CS that is similar to the appearance of metastasis. If imaging findings are not conclusive, biopsy may be necessary. A search of the literature showed that there are 13 reported cases of systemic malignant lymphoma with CS involvement, 3 of which are children [8]. To the best of our knowledge this is the first case of primary gastric lymphoma presenting with CS involvement, without an intracerebral mass or nodular lesion. Acknowledgements This study was performed in Erciyes University. Our case approved by the ethical committee of the institution for publishing. Conflict of interest statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Turk J Hematol 2011; 28: 72-4

References 1. 2.

5. 6.

Ferreri AJ, Montalbán C. Primary diffuse large B-cell lymphoma of the stomach. Crit Rev Oncol Hematol 2007;63:65-71. [CrossRef] Chieng JH, Garrett J, Ding SL, Sullivan M. Clinical presentation and endoscopic features of primary gastric Burkitt lymphoma in childhood, presenting as a protein-losing enteropathy: a case report. J Med Case Reports 2009;3:7256. [CrossRef] Choi HK, Cheon JE, Kim IO, Youn BJ, Jung AY, Shin SM, Kim WS, Yeon KM. Central skull base lymphoma in children: MR and CT features. Pediatr Radiol 2008;38:863-7. [CrossRef] Demirkaya M, Sevinir B, Ozdemir O, Nazlioğlu HO, Okan M. Lymphoma of the cavernous sinus mimicking Tolosa-Hunt syndrome in a child. Pediatr Neurol 2010;42:351-4. [CrossRef] Choe BK, Kim JY, Hwang JB, Kim HS, Jung HR, Kang YN. A case of primary gastric lymphoma in a child. J Pediatr Hematol Oncol 2006;28:296-9. [CrossRef] Moschovi M, Menegas D, Stefanaki K, Constantinidou CV, Tzortzatou-Stathopoulou F. Primary gastric Burkitt lymphoma in childhood: associated with Helicobacter pylori? Med Pediatr Oncol 2003;41:444-7. [CrossRef] Huisman TA, Tschirch F, Schneider JF, Niggli F, MartinFiori E, Willi UV. Burkitt’s lymphoma with bilateral cavernous sinus and mediastinal involvement in a child. Pediatr Radiol 2003;33:719-21. [CrossRef] Tsai WC, Ho CL. Non-Hodgkin’s lymphoma with leptomeningeal recurrence presents as a cavernous sinus syndrome. Ann Hematol 2008;87:491-2. [CrossRef]

Letter to the Editor

Feasibility of four discriminant functions for identifying hemoglobin E disorders: Experience in 114 Thai pregnant subjects Hemoglobin E bozukluklarını tanımlama için dört diskriminant fonksiyonun fizibilitesi: 114 Taylandlı hamile üzerinde deneyim Viroj Wiwanitkit1, Nara Paritpokee2, Jamsai Suwansaksri3 1Department

of Tropical Medicine, Hainan Medical College, Hainan, China Health Science and Private Academic Consultant, Bangkok, Thailand 3Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand 2Allied

To the Editor, Hemoglobin disorders, especially hemoglobin E disorder, are a major problem in Thailand. Screening of pregnant subjects is among the new public health strategies for control of hemoglobinopathy in Thailand [1]. A number of screening methods for screening in pregnant subjects, such as osmotic fragility test and dichlorophenol indophenol (DCIP) test, have been evaluated [2]. Based on the modern electronic cell counter, quick differential screenings using discriminant functions (DFs) have been widely developed. However, there are only a few papers on this topic in pregnant subjects. According to our previous study using England and Frazer’s calculation method in screening for Hb disorders in Thai pregnant subjects, good diagnostic properties were determined [3]. Recently, Ittarat et al. [4] proposed the possibility of using some modified discriminant functions (DFs) as alternative tools for screening for such disorders among the general population. However, these DFs were originally primarily applied to the dif-

ferentiation of iron deficiency anemia from betathalassemia. Only a few studies on DF properties in screening for other abnormal hemoglobins have been reported. In this study, the four most widely used DFs were evaluated for their abilities to identify HbE-containing blood samples among 114 Thai pregnant subjects. The functions evaluated were: a) F1=0.01xMCH X(MCV)2, b) F2=RDWxMCHx (MCV)2/ Hbx100, c) F3=MCV/RBC, and d) F4= MCH/RBC. The correlation between DFs and HbE was evaluated according to the previous published method of Ittarat et al. [4]. DFs demonstrating a significant difference in distinguishing Hb disorders were selected for further evaluation of diagnostic properties (sensitivity, specificity, and false positive and false negative values). Only F4 showed statistically significant differences in distinguishing between the EE group and the other groups (p<0.05) (Table 1). The sensitivity, specificity, and false positive and false negative values of using F4 in identification of the EE group were 100%, 95.2%, 4.8% and 0%, respectively. In conclusion, the four tested DFs are not good screening tools

Address for Correspondence: Prof. Viroj Wiwanitkit, Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Bangkok, Thailand Phone: 662-2564136 E-mail: wviroj@yahoo.com doi:10.5152/tjh.2011.11

Wiwanitkit et al. Identifying hemoglobin E disorders

Turk J Hematol 2011; 28: 75-6

Table 1. Mean and standard deviations of some hematologic parameters and discriminant functions of blood samples from pregnant subjects with different hemoglobin types A2A (n = 77)

EA (n = 27)

EE (n =10)

1689.3+699.8

1898.1+642.5

18.3+4.3

6.4+1.3

1587.7+542.8

1758.1+842.3

17.4+6.4

5.4+0.2

1325.5+498.6

F4*

1548.1+616.5

16.9+5.2

3.0+0.6*

thankful to all medical personnel who assisted us with this study. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References

*A significant difference was determined between EE and the other two groups (P<0.05) (ANOVA test)

for distinguishing the normal (A2A) from abnormal (disease: EE and carrier: EA) subjects. However, F4 might be used as a screening tool for disease (EE). Due to the fact that these DFs do not possess good diagnostic properties in screening for abnormal subjects and require automated analyzer, implying high screening costs, they do not appear to be appropriate screening tools for antenatal care in Thailand. Acknowledgement The authors would like to thank all pregnant subjects who participated in this study. We are also

3. 4.

Fucharoen S, Winichagoon P. Thalassemia in South East Asia: problems and strategy for prevention and control. Southeast Asian J Trop Med Public Health 1992;23:647-55. Wiwanitkit V, Suwansaksri J, Paritpokee N. Combined one-tube osmotic fragility (OF) test and dichlorophenol-indophenol (DCIP) test screening for hemoglobin disorders, an experience in 213 Thai pregnant women. Clin Lab 2002;48:525-8. Ittarat W, Ongcharoenjai S, Rayatong O, Pirat N. Correlation between some discrimination functions and hemoglobin E. J Med Assoc Thai 2000;83:259-65. Suwansaksri J, Wiwanitkit V, Paritpokee N. Screening for hemoglobin disorders in Thai pregnant women by England and Frazerâ&#x20AC;&#x2122;s calculation method. Arch Gynecol Obstet 2004;270:211-3. [CrossRef]

Letter to the Editor

The association between calcium dobesilate and pancytopenia in type 2 diabetes: A case report Tip 2 diyabet hastasında kalsiyum dobesilat ile pansitopeni ilişkisi: Bir olgu sunumu Aylin Cesur1, Meltem Aylı2, Mustafa Cesur3, Sibel Ertek4 1Department

of Biochemistry, Medical Faculty, Gazi University, Ankara, Turkey of Hematology, Medical Faculty, Ufuk University, Ankara, Turkey 3Department of Endocrinology and Metabolic Diseases, Guven Hospital, Ankara, Turkey 4Department of Endocrinology and Metabolic Diseases, Medical Faculty, Ufuk University, Ankara, Turkey 2Department

To the Editor, Diabetic patients with polypharmacy are prone to unexpected drug side effects. Chronic venous insufficiency (CVI) is frequent in the type 2 diabetic population due to advanced age and obesity, and is associated with increased podiatric risk [1,2]. Calcium dobesilate (CaD) is a widely prescribed veno-tonic drug for CVI, diabetic retinopathy, and the symptoms of hemorrhoid attacks [3]. To date, all published cases of CaD-induced agranulocytosis are >60 years of age [4,5-10]. Ibanez et al. recently reported that CaD was strongly associated with the risk of agranulocytosis [10]. Herein we present a case of CaD-induced pancytopenia in an 80-yearold male diabetic patient. We had written informed consent from the patient. The patient presented to our hospital with a fever of 39°C. He had been type 2 diabetic for 25 years and was treated with repaglinide 2mg b.i.d. and insulin glargine 20U o.p.d. He was also hypertensive and dyslipidemic, and was using atenolol 50 mg/day,

lisinopril 20 mg/day, amlodipine 10 mg/day, hydrochlorothiazide 50 mg/day, atorvastatin 10 mg/ day, and acetylsalicylic acid 100 mg/day for last 8 years. Six weeks before he presented to hospital he was diagnosed with CVI and CaD 500 mg b.i.d. was started following a cardiovascular surgery consultation. After 6 weeks of the treatment leukopenia, thrombocytopenia, and anemia were observed. His hematoxylin-eosin-stained blood smear confirmed markedly decreased platelet and leucocyte counts, accompanied by normochromic normocytic red cells (Table). CaD treatment was withdrawn because of the potential side effects. On physical examination the patient did not have organomegaly or palpable lymph nodes. His iron, ferritin, vitamin B12, and folate levels, and reticulocyte count were within normal limits, and ANA (anti nuclear antibody) and anti-double stranded DNA test results were negative. The patient was hospitalized and was prescribed intravenous ceftriaxone 2 g/day and clarithromycin 400 mg/day, following the diagnosis

Address for Correspondence: M.D. Sibel Ertek, Department of Endocrinology and Metabolic Diseases, Medical Faculty, Ufuk University, Ankara, Turkey Phone: +90 312 204 42 30 E-mail: sibelertek@yahoo.it doi:10.5152/tjh.2011.12

Cesur et al. Pancytopenia with calcium dobesilate

Turk J Hematol 2011; 28: 77-8

of pneumonia. The patient was discharged after 1 week of hospitalization with improved hemogram results (Table 1). Three months after discharge he was again admitted to our hospital with a fever of 38.6°C. It was learned that he again had started taking CaD. Pancytopenia was diagnosed, CaD was withdrawn, and the same intravenous antibiotic therapy was started again. Bone marrow biopsy and aspiration were performed from the iliac crest. Wright staining of the bone marrow aspiration smear microscopically showed normal erythroid and myeloid cell lines. The patient was discharged with a normal blood count after 1 week. In conclusion, we want to emphasize the potential for serious side effects that affect the immune system in polypharmacy diabetic patients, such as granulocytopenia, and the importance of pharmacovigilance in patients with chronic diseases and the risk of comorbidity.

References 1.

2. 3.

4. 5.

Table 1. The patient’s complete blood count during the follow-up period, with and without medication Before 6 Weeks After 1 Week After 3 months later, CaD CaD Hospitalization again with CaD use (×109 L-1)

6.2

2.4

5.93

4.0

Neutrophils (%)

Lymphocytes (%)

Basophiles (%)

Eosinophils (%)

13.1

10.3

11.4

10.8

WBC

Monocytes (%)

Hb (g dL-1) Platelet (×109 L-1)

219

122

235

101

Platelet (×109 L-1)

219

122

235

101

CaD: Calcium dobesilate; WBC: white blood cells; Hb: hemoglobin

6. 7. 8.

9. 10.

Lionis C, Erevnidou K, Antonakis N, Argyriadou S, Vlachonikolis I, Katsamouris A; CVI Research Group. Chronic venous insufficiency . A common health problem in general practice. Int Angiol 2002;21:86-92. Fowkes FGR, Ewans CJ, Lee AJ. Prevalance and risk factors of chronic venous insufficiency. Angiology 2001;52:S5-15. [CrossRef] Allain H, Ramelet AA, Polard E, Bentue-Ferrer D. Safety of calcium dobesilate in chronic venous disease, diabetic retinopathy and haemorrhoids. Drug Saf. 2004;27:649-60. [CrossRef] Zapater P, Horga JF, Garcia A. Risk of drug-induced agranulocytosis: the case of calcium dobesilate. Eur J Clin Pharmacol. 2003;58:767-72. Ibanez L, Ballarin E, Vidal X, Laporte JR. Agranulocytosis associated with calcium dobesilate clinical course and risk estimation with the case-control and the casepopulation approaches. Eur J Clin Pharmacol. 2000;56:763-7. Kulessa W, Becker EW, Berg PA. Recurrent agranulocytosis after taking calcium dobesilate. Dtsch Med Wochenschr. 1992;117:372-4. [CrossRef] Garcia Benayas E, Garcia Diaz B, Perez G. Calcium dobesilate-induced agranulocytosis. Pharm World Sci. 1997;19:251-2. [CrossRef] Azaceta G, Sáenz-Cusi A, Olave T, Palomera L. Agranulocitosis inducida por dobesilato cálcico: a propósito de un nuevo caso. An Med Interna 2000;17: 337-8. Duggal L, Thukral R, Kumar S. Calcium dobesilateinduced agranulocytosis. J Assoc Physicians India. 2005;53:320-1. Ibanez L, Vidal X, Ballarin E, Laporte JR. Populationbased drug-induced agranulocytosis. Arch Intern Med. 2005;165:869-74. [CrossRef]

Letter to the Editor

Aeromonas sobria bacteriemia in an acute lymphoblastic leukemia case in remission Remisyondaki akut lenfoblastik lösemi olgusunda Aeromonas sobria bakteriyemisi Selami Koçak Toprak, Gül İlhan, Elçin Erdoğan, Sema Karakuş Department of Hematology, School of Medicine, Başkent University, Ankara, Turkey

To the Editor, Herein we present an acute lymphoblastic leukemia (ALL) case that developed Aeromonas sobria bacteriemia while undergoing consolidation treatment. In malignant hematologic diseases Aeromonastype microorganisms are rarely encountered agents of opportunistic bacterial infection during the neutropenic period following chemotherapy [1]. A 17-yearold male patient that presented to our outpatient clinic with malaise and abdominal pain was diagnosed as ALL and the BFM95 chemotherapy protocol was commenced. A chemotherapy combination given for consolidation resulted in severe back and abdominal pain on the 21st d. The pain subsided without acute surgical intervention and mixed-type neuropathy -particularly drug-related -was considered and therefore vincristine (Vincristine Amphar Flacon 1 mg; Atabay) treatment was not continued. The patient had fever on the 30th d of the consolidation protocol, and iv imipenem (Tienam-IV Flacon 500 mg; MSD) 4×500 mg d-1 was added to the treatment after the required cultures were obtained. The

following day his fever persisted and hypoxemia developed along with deterioration of his general health status; therefore, iv vancomycin (Edicin Flacon 0.5 g; Sandoz) 2×1 g d-1 and iv ciprofloxacin (ciprofloxacin infusion, 400 mg, Biofarma) 2×400 mg d-1 iv were started. Nonetheless, the patient’s renal function deteriorated along with an associated decrease in blood pressure, and, therefore, vancomycin (Edicin Flacon 0.5 g, Sandoz) was withdrawn and iv linezolid (Zyvoxid İnfusion Solution mg mL-1, Pfizer) 2×600 mg d-1 was started. As the patient’s general health status showed no improvement, he was referred to the intensive care unit. The patient’s fever remained unresponsive and the patient died on the 32nd day of the consolidation treatment due to sepsis. The results of blood cultures obtained during febrile neutropenia showed that Aeromonas sobria was sensitive to the antibiotics that the patient was already taking. Written informed consent was obtained from the patient’s family. Although in neutropenic and immunocompromised patients Aeromonas-type bacteria generally lead to uncomplicated bacteriemia with an unknown

Address for Correspondence: M.D. Selami Koçak Toprak, Department of Hematology, School of Medicine, Başkent University, Ankara, Turkey Phone: +90 532 656 02 06 E-mail: sktoprak@yahoo.com doi:10.5152/tjh.2011.13

Toprak et al. Aeromonas sobria bacteriemia in acute leukemia

focus of infection, various skin and soft tissue infections have also been observed [1]. Among these are localized cellulitis, ecthyma gangrenosum, and clostridium-like gangrenous cellulitis. Martino et al. reported that at their department in Spain Aeromonas bacteriemia was seen in only 4 patients with hematologic malignancies [1]. In healthy humans, it has been observed that local inoculation enables to skin and soft tissue infections due to Aeromonas, while in immunocompromised cases evidence of such access cannot always be found. Nevertheless, almost 2 decades ago Sherlock et al. reported that in neutropenic cancer patients intestinal colonization of Aeromonas-type bacteria increase just as other gramnegative bacteria, and that this could be why bacteriemia is observed in such patients [2]. Indeed, it is currently thought that Aeromonas is translocated to the blood from its primary intestinal localization via the bile ducts and intestine [3]. Aeromonas is a rarely encountered infectious agent that is taken into the body via contaminated food and drink. We think the presented case that

Turk J Hematol 2011; 28: 79-80

died due to Aeromonas-related sepsis is noteworthy because of the unusual agent involved and its unusual pathway. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1.

Martino R, Santamaria A, Pericas R, Sureda A, Brunet S. Acute rhabdomyolysis and myonecrosis complicating Aeromonas bacteremia n neutropenic patients with hematologic malignancies: report of two cases. Haematologica 1997;82:692-4. Sherlock CH, Burdge DR, Smith JA. Does Aeromonas hydrophila preferentially colonize the bowels of patients with hematologic malignancies? Diagn Microbiol Infect Dis 1987;7:63-8. [CrossRef] Thomsen RN, Kristiansen MM. Three cases of bacteraemia caused by Aeromonas veronii biovar sobria. Scand J Infect Dis 2001;33:718-9. [CrossRef]

Letter to the Editor

ARA-C associated pulmonary toxicity ARA-C ilişkili akciğer toksisitesi Zeynep Arzu Yegin1, Gülsan Türköz Sucak1, Gonca Erbaş2, Münci Yağcı1 1Department 2Department

of Hematology, Faculty of Medicine, Gazi University, Ankara, Turkey of Radiology, Faculty of Medicine, Gazi University, Ankara, Turkey

To the Editor, Cytosine arabinoside (ARA-C) associated pulmonary toxicity is a well-described life-threatening complication of anti-leukemia therapy. We previously reported a 17-year-old acute leukemia patient that experienced non-cardiogenic pulmonary edema (NCPE) that was most probably due to highdose ARA-C. Initial respiratory symptoms and fever appeared on the fourth day of ARA-C infusion. High resolution computed tomography (HRCT) showed bilateral pleural effusion with diffuse ground-glass opacity. The clinical course was rigorous, leading to acute respiratory distress syndrome (ARDS) that favorably responded to prompt steroid administration [1]. Chagnon et al. reported 6 acute myeloid leukemia patients treated with intermediate- to high-dose ARA-C that developed a new pattern of pulmonary toxicity described as hypersensitivity pneumonitis [2]. Drug-induced pulmonary damage is an entity encompassing a broad spectrum of pulmonary syndromes with mild to severe symptomatology, including pneumonitis/fibrosis, hypersensitivity pneumonitis, NCPE, and ARDS [3]. ARA-C-induced pulmonary injury is usually overlooked among the turbu-

lent circumstances of febrile leukemia patients. ARA-C was suggested to be a causative agent of distinct forms of pulmonary toxicity in several reports [1,3-8]. The primary cause of ARA-C toxicity is considered to be a clinical consequence of cytokine network activation, which results in alveolar damage and increased vascular permeability, leading to capillary leakage syndrome [5,6,9]. The primary characteristics of ARA-C associated pulmonary toxicity are summarized in the Table 1. Chagnon et al. based their diagnosis of hypersensitivity pneumonitis primarily on radiological findings; however, radiological findings in a variety of clinical conditions that share a similar radiological appearance and also mimic the general perspective may be difficult to differentiate. Above all, considering the confusing diagnostic possibilities, including opportunistic infections, congestive heart failure, and transfusion-related acute lung injury, and several toxic agents, exact diagnosis of hypersensitivity pneumonitis should rely primarily on characteristic HRCT features and lymphocytosis in bronchoalveolar lavage, so as to minimize the likelihood of misdiagnosis. The diagnosis of hypersensitivity pneumonitis seems to be uncertain; only 2 patients that met the minimum requisite diagnostic

Address for Correspondence: M.D. Zeynep Arzu Yegin, Gazi University Faculty of Medicine, Department of Hematology 06500 Ankara, Türkiye Phone: + 90 312 202 63 17 E-mail: zyegin@gazi.edu.tr doi:10.5152/tjh.2011.14

Yegin et al. ARA-C associated pulmonary toxicity

Turk J Hematol 2011; 28: 81-3

Table 1. The primary characteristics of ARA-C associated pulmonary toxicity (3,4,6,8,9) Median age

39 years

Incidence

12%-20%

ARA-C dose

Intermediate/high • 1-1.5 g m-2/continuous infusion • >3 g m-2 as 2-h iv infusion per 12 h

Day of onset

1-2 weeks after chemotherapy • Usually during the initial course • Increased risk with multiple doses

Pathophysiology

Increased alveolar capillary permeability

Diagnosis

Exercise of exclusion • Heart dysfunction • Infections • Metabolic abnormalities • Cancer-related causes

Common clinical symptoms

Early onset of fever Dyspnea Hypoxemia Tachypnea Cough

Radiological findings

X-ray: Confluent alveolar consolidation HRCT: Alveolar or interstitial opacification in lower lobes surrounded by ground glass areas and/or pleural effusions

Treatment

Steroids: Response rate is 65%-80% Supportive care

ARA-C: cytosine arabinoside; h: hour; HRCT: high resolution computed tomography; iv: intravenous

criteria have been reported. Moreover, early onset of clinically evident pulmonary syndrome described in the paper might contradict the diagnosis of hypersensitivity pneumonitis, as it typically develops as a result of chronic antigenic exposure. Although the clinical condition of the patients was self-limited and they recovered without specific treatment, a longer follow-up period may be necessary for evaluation of the progress of respiratory functions when hypersensitivity pneumonitis is the definitive diagnosis [2,10]. Cytokine storm-induced pulmonary damage, which occasionally leads to a more specific immunological response, appears to be the primary causative mechanism of ARA-C associated pulmonary toxicity, irrespective of clinical presentation. Thus, in addition to early recognition of pulmonary toxicity and immediate withdrawal of chemotherapy, steroids and supportive care are reported to be com-

mon therapeutic approaches. It might be misleading to refer to this toxicity as hypersensitivity pneumonitis, as there is currently no supportive evidence. To summarize, we suggest that the wide spectrum of clinical presentation associated with ARA-C might have a common underlying mechanism, which triggers the cytokine network. ARA-C associated pulmonary toxicity might be a more comprehensive term to describe this clinical picture, which has several predisposing factors. We think the main point is to consider ARA-C in the differential diagnosis of mild to severe pulmonary symptoms in leukemia patients and to treat it appropriately, rather than describing the different clinical presentations with distinct clinical terms. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, rela-

Yegin et al. ARA-C associated pulmonary toxicity

Turk J Hematol 2011; 28: 81-3

tionships and/or affiliations relevant to the subject matter or materials included.

References 1.

3. 4.

Yegin ZA, AkÄą S, Sucak G, YagcÄą M. Cytarabine induced non cardiogenic pulmonary edema in a case of acute lymphoblastic leukemia. Int J Lab Hem 2007;29: 482-3. [CrossRef] Chagnon K, Boissel N, Raffoux E, Dombret H, Tazi A, Bergeron A. A new pattern of cytosine arabinoside induced lung toxicity. Br J Haematol 2009;147: 760-74. [CrossRef] Briasoulis E, Pavlidis N. Non cardiogenic pulmonary edema: An unusual and serious complication of anticancer therapy. Oncologist 2001;6:153-61. [CrossRef] Andersson BS, Cogan BM, Keating MJ, Estey EH, McCredie KB, Freireich EJ. Subacute pulmonary failure complicating therapy with high dose ARA-C in acute leukemia. Cancer 1985;56:2181-4. [CrossRef] Andersson BS, Luna MA, Yee C, Hui KK, Keating MJ, McCredie KB. Fatal pulmonary failure complicating

7. 8.

10.

high dose cytosine arabinoside therapy in acute leukemia. Cancer 1990;65:1079-84. [CrossRef] Forghieri F, Luppi M, Morselli M, Potenza L, Volzone F, Riva G, Imovilli A, Rivolti E, Torelli G. Cytarabine related lung infiltrates on high resolution computerized tomography: a possible complication with benign outcome in leukemic patients. Haematologica 2007;92:85-90. [CrossRef] Larouche G, Denault A, Prenovault J. Corticosteroids and serious cytarabine-induced pulmonary edema. Pharmacotherapy 2000;20:1396-9. Salvucci M, Zanchini R, Molinari AL, Zuffa E, Poletti V, Poletti G, Zaccaria A. Lung toxicity following fludarabine, cytosine arabinoside and mitoxantrone (FLAN) treatment for acute leukemia. Haematologica 2000;85:769-70. Chiche D, Pico JL, Bernaudin JF, Chouaib S, Wollman E, Arnoux A, Denizot Y, Nitenberg G. Pulmonary edema and shock after high dose aracytine-C for lymphoma; possible role of TNF-alpha and PAF. Eur Cytokine Network 1993;4:147-51. Hirshmann JV, Pipavath SNJ, Godwin JD. Hypersensitivity pneumonitis: A historical, clinical and radiologic review. Radiographics 2009;29:1921-38. [CrossRef]

Images in Hematology

Niemann-Pick disease Niemann-Pick hastalığı Serap Karaman, Tiraje Celkan Department of Pediatric Hematology and Oncology, Cerrahpaşa Medical Faculty, İstanbul University, İstanbul, Turkey

The peripheral blood smear is an easy, inexpensive, and very useful diagnostic method. Although some think that it has lost its importance following the development of new automated complete blood counts, the peripheral blood smear should remain a primary diagnostic tool for clinicians, especially pediatricians. Peripheral blood smears aid clinicians in choosing the appropriate laboratory tests for determining definitive diagnoses [1,2].

Case A 6-month-old female presented to our clinic with rectal bleeding. On physical examination hepatosplenomegaly was observed. PT and aPTT were prolonged. Her peripheral smear showed lymphocyte vacuolization (Figure 1). Together with hepatosplenomegaly, prolonged PT and aPTT, and elevated ALT and AST, storage diseases were considered in the differential diagnosis. Bone marrow aspiration showed lipid-laden macrophages (Figure 2). The diagnosis of Niemann-Pick disease was confirmed via measurement of lysosomal sphingomyelinase (0.22 nmol·h·mg-1 ptn [normal range: 0.862.8]). We have taken informed consent form from the patient.

Figure 1. Lymphocyte vacuolization in the patient with NiemannPick disease

Discussion Lymphocyte vacuolization was noted in our hepatosplenomegaly patient’s peripheral blood

Figure 2. Bone marrow aspiration specimen from the patient with Niemann-Pick disease

Address for Correspondence: M.D. Serap Karaman, Department of Pediatric Hematology and Oncology, Cerrahpaşa Medical Faculty, İstanbul University, İstanbul, Turkey Phone: +90 212 624 46 07 E-mail: drkaramans@yahoo.com doi:10.5152/tjh.2011.15

Karaman et al. Niemann-Pick disease

Turk J Hematol 2011; 28: 84-5

smear and, therefore, storage diseases were considered in the differential diagnosis. As storage cells were observed in the bone marrow aspiration specimen and lysosomal sphingomyelinase activity was low, the patient was diagnosed with NiemannPick disease. Conflict of interest statement None of the authors of this paper has a conflict of interest, including specific financial interests, rela-

tionships, and/or affiliations relevant to the subject matter or materials included.

References 1. 2.

Lewis SM, Bain BJ, Bates I. Practical Haematology. 9 th ed. London:Churcill-Livingstone 2001;19-78. Ezekowitz BA. Hematologic Manifestations of Systemic disease. In: Orkin SH, Nathan DG, Ginsburg D, et al (eds). Hematology and Infancy and Childhood. 7 th ed. Philadelphia:WB Saunders, 2009;pp1679-739.

Advisory Board of This Issue (March 2011) Ahmet Türker Çetin, Turkey Akif Yeşilipek, Turkey Ali Turhan, France Ali Uğur Ural, Turkey Ali Ünal, Turkey Aytemiz Gürgey, Turkey Betül Tavil, Turkey Bilgehan Yalçın, Turkey Burhan Turgut, Turkey Can Boğa, Turkey Celalettin Üstün, USA Cengiz Beyan, Turkey

Dilber Talia İleri, Turkey Duran Canatan, Turkey Ebru Koca, Turkey Erol Erduran, Turkey Feride İffet Şahin, Turkey Hakan Özdoğu, Turkey Hilmi Apak, Turkey İsmet Aydoğdu, Turkey Mualla Çetin, Turkey Mutlu Hayran, Turkey Muzaffer Demir, Turkey Nurdan Taçyıldız, Turkey

Nükhet Tüzüner, Turkey Önder Arslan, Turkey Sema Anak, Turkey Sema Karakuş, Turkey Şule Ünal, Turkey Tiraje Celkan, Turkey Türkan Patıroğlu, Turkey Ufuk Gündüz, Turkey Ülker Koçak, Turkey Vefki Gürhan Kadıköylü, Turkey Yusuf Baran, Turkey

Announcements 4-6 March 2011 Update on Hematology - Post-Graduate Hematology Education Antalya, Turkey 25-27 March 2011 Diagnosis and Treatment Workshop on Benign Hematology Antalya, Turkey 3-6 April 2011 EBMT-European Group for Blood and Marrow Transplantation Paris, France 11-14 May 2011 3rd International Congress of Leukemia Lymphoma Myeloma Ä°stanbul, Turkey 18-21 May 2011 17th Annual ISCT Meeting Rotterdam, Netherlands 18-21 May 2011 11th International Symposium on Myelodysplastic Syndromes Edinburgh, Scotland, United Kingdom

3-7 June 2011 2011 ASCO Annual Meeting Chicago, USA 9-12 June 2011 16th Congress of the European Hematology Association London, United Kingdom 24-26 June 2011 Turkish School of Hematology 11 - Practical Hemostasis Thrombosis Laboratory Course 2 Sivas, Turkey 9-11 September 2011 Turkish School of Hematology 12 - Stem Cell Course Ankara, Turkey 19-22 October 2011 37. Turkish National Hematology Congress Ankara, Turkey 10-13 December 2011 53rd ASH Annual Meeting and Exposition San Diego, USA 23-25 December 2011 Practical Basic Hematology Laboratory Course 1