Turkish Journal of Hematology Volume: 32 - Issue: 4

Volume 32

Issue 4

December 2015

80 TL

ISSN 1300-7777

Review Article Chimeric Antigen Receptor T Cell Therapy in Hematology Pınar Ataca, et al.; Ankara, Turkey TURKISH JOURNAL OF HEMATOLOGY • VOL.: 32

Research Articles Possible Role of GADD45γ Methylation in Diffuse Large B-Cell Lymphoma: Does It Affect the Progression and Tissue Involvement? İkbal Cansu Barış, et al.; Denizli, Turkey

Effect of Tumor Necrosis Factor-Alpha on Erythropoietin- and Erythropoietin Receptor-Induced Erythroid Progenitor Cell Proliferation in β-Thalassemia/Hemoglobin E Patients Dalina I Tanyong, et al.; Nakhon Pathom, Thailand

The -137G/C Polymorphism in Interleukin-18 Gene Promoter Contributes to Chronic Lymphocytic and Chronic Myelogenous Leukemia Risk in Turkish Patients Serap Yalçın, et al.; Kırşehir, Ankara, Turkey

Transcobalamin II Deficiency in Four Cases with Novel Mutations Şule Ünal, et al.; Ankara, Turkey; London, Canada

Eltrombopag for the Treatment of Immune Thrombocytopenia: The Aegean Region of Turkey Experience Füsun Özdemirkıran, et al.; İzmir, Denizli, Aydın, Turkey

ISSUE: 4

Management of Invasive Fungal Infections in Pediatric Acute Leukemia and the Appropriate Time for Restarting Chemotherapy Özlem Tüfekçi, et al.; İzmir, Turkey

DECEMBER 2015

First-Step Results of Children Presenting with Bleeding Symptoms or Abnormal Coagulation Tests in an Outpatient Clinic İsmail Yıldız, et al.; İstanbul, Turkey

Evaluation of Alpha-Thalassemia Mutations in Cases with Hypochromic Microcytic Anemia: The İstanbul Perspective Zeynep Karakaş, et al.; İstanbul, Turkey

Cover Picture: Nejat Akar Çeşmealtı’s Morning Serenity

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Editor-in-Chief

International Review Board

Aytemiz Gürgey

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

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

Past Editors Erich Frank Orhan Ulutin Hamdi Akan

Language Editor Leslie Demir

Ankara, Turkey

Associate Editors Ayşegül Ünüvar İstanbul University, İstanbul, Turkey

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

Cengiz Beyan Gülhane Military Medical Academy, Ankara, Turkey

Hale Ören Dokuz Eylül University, İzmir, Turkey

İbrahim C. Haznedaroğlu Hacettepe University, Ankara, Turkey

İlknur Kozanoğlu Başkent University, Adana, Turkey

Mehmet Ertem Ankara University, Ankara, Turkey

A. Muzaffer Demir Trakya University, Edirne, Turkey

Reyhan Diz Küçükkaya İstanbul Bilim University, İstanbul, Turkey

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

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

İnci Alacacıoğlu Dokuz Eylül University, Ankara, Turkey

Nil Güler On Dokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay Osmangazi University, Eskişehir, Turkey

Selami Koçak Toprak Ankara University, Ankara, Turkey

Şule Ünal Hacettepe University, Ankara, Turkey

Veysel Sabri Hançer İstanbul Bilim University, İstanbul, Turkey

Zühre Kaya

Statistic Editor Hülya Ellidokuz

Senior Advisory Board Yücel Tangün Osman İlhan Muhit Özcan

Gazi University, Ankara, Turkey

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Publishing Services

Editorial Office İpek Durusu Bengü Timoçin

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

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

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

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

Publishing Manager Sorumlu Yazı İşleri Müdürü A. Muzaffer Demir

Management Address Yayın İdare Adresi

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

Publishing House / Yayınevi

Online Manuscript Submission

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul, Turkey Tel: +90 212 621 99 25 Faks: +90 212 621 99 27 E-posta: info@galenos.com.tr Baskı: Senk Ajans Reklam Matbaacılık San. ve Tic.Ltd.Şti.

Sanayi Mah. Sultan Selim Cad. Aybike Sk.No:22/-3, Kağıthane, İstanbul, Türkiye Tel: +90 212 264 38 77

http://mc.manuscriptcentral.com/tjh

Web page www.tjh.com.tr

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

Printing Date / Basım Tarihi 20.11.2015

Cover Picture Nejat Akar was born in 1952, Turkey. He is currently working at TOBB-ETU Hospital, Ankara, Turkey.

Üç ayda bir yayımlanan İngilizce süreli yayındır.

International scientific journal published quarterly.

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

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Çeşmealtı town is a small and pretty site in Urla, İzmir. It’s a quiet and calm fishing harbor. When rising up every morning, the sun dances on the coast of the sea, which is surrounded by little islands.

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

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

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

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

Impact Factor: 0.360 Subscription Information

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

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

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

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

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

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

If you use a table or figure (or some data in a table or figure) from another source, cite the source directly in the figure or table legend.

The journal is printed on acid-free paper.

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

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

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

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

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

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

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

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

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

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

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

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

Materials and Methods: Important methods should be written respectively.

Results: Important findings and results should be provided here.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Examples of References: 1. List all authors.

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

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

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

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

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

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

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

Brief Reports Abstract length: Not to exceed 150 words.

Article length: Not to exceed 1200 words.

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

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

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

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

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

Case Reports Abstract length: Not to exceed 100 words.

Article length: Not to exceed 1200 words.

Case Reports can include maximum 1 figure and 1 table or 2 figures or 2 tables.

Case reports should be structured as follows:

Abstract An unstructured abstract that summarizes the case.

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

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

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

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

Article length: Not to exceed 4000 words.

Review articles should not include more than 100 references.

Reviews should include a conclusion, in which a new hypothesis or study about the subject may be posited. Do not publish methods for literature search or level of evidence. Authors who will prepare review articles should already have published research articles on therel evant subject. The study’s new and important findings should be highlighted and interpreted in the Conclusion section. There should be a maximum of two authors for review articles.

Images in Hematology Article length: Not exceed 200 words.

Authors can submit for consideration an illustration and photos that is interesting, instructive, and visually attractive, along with a few lines of explanatory text and references. Images in Hematology can include no more than 200 words of text, 5 references, and 3 figure or table. No abstract, discussion or conclusion are required but please include a brief title.

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

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

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

Figures Figures should be professionally drawn and/or photographed.

Authors should number figures according to the order in which they appear in the text. Figures include graphs, charts, photographs, and illustrations. Each figure should be accompanied by a legend that does not exceed 50 words. Use abbreviations only if they have been introduced in the text. Authors are also required to provide the level

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of magnification for histological slides. Explain the internal scale and identify the staining method used. Figures should be submitted as separate files, not in the text file. High-resolution image files are not preferred for initial submission as the file sizes may be too large. The total file size of the PDF for peer review should not exceed 5 MB.

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

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

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

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

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

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

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

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

which includes re-publishing a manuscript in different languages.

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

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

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

(The COPE flowchart is available at: http://publicationethics.org) We use iThenticate to screen all submissions for plagiarism before publication.

Turkish Journal of Hematology uses plagiarism screening service to verify the originality of content submitted before publication.

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

Authors of accepted manuscripts will receive electronic page proofs and are responsible for proofreading and checking the entire article within two days. Failure to return the proof in two days will delay publication.

If the authors cannot be reached by email or telephone within two weeks, the manuscript will be rejected and will not be published in the journal.

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

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

Units of Measurement Measurements should be reported using the metric system, according to the International System of Units (SI). Consult the SI Unit Conversion Guide, New England Journal of Medicine Books, 1992.

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

Hematology component

SI units

RBC

6.7-11 x 1012/L

WBC

5.5-19.5 x109/L

Hemoglobin

116-168 g/L

PCV

0.31-0.46 L/L

MCV

39-53 fL

MCHC

300-360 g/L

MCH

19.5-25 pg

Platelets

300-700 x 109/L

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

Abbreviations and Symbols Use only standard abbreviations. Avoid abbreviations in the title and abstract. The full term for an abbreviation should precede its first use in the text, unless it is a standard abbreviation. All acronyms used in the text should be expanded at first mention, followed by the abbreviation in parentheses; thereafter the acronym only should appear in the text. Acronyms may be used in the abstract if they occur 3 or more times therein, but must be reintroduced in the body of the text. Generally, abbreviations should be limited to those defined in the AMA Manual of Style, current edition. A list of each abbreviation (and the corresponding full term) used in the manuscript must be provided on the title page.

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

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

Setting up an account New users to the submission site will need to register and enter their account details before they can submit a manuscript. Log in, or click the “Create Account” button if you are a first-time user. To create a

new account: After clicking the “Create Account” button, enter your name and e-mail address, and then click the “Next” button. Your e-mail address is very important. Enter your institution and address information, as appropriate, and then click the “Next” Button. Enter a user ID and password of your choice, select your area of expertise, and then click the “Finish” button.

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

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A-VIII

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CONTENTS

285

Review Article

295

Research Articles

Chimeric Antigen Receptor T Cell Therapy in Hematology Pınar Ataca, Önder Arslan

Possible Role of GADD45γ Methylation in Diffuse Large B-Cell Lymphoma: Does It Affect the Progression and Tissue Involvement? İkbal Cansu Barış, Vildan Caner, Nilay Şen Türk, İsmail Sarı, Sibel Hacıoğlu, Mehmet Hilmi Doğu, Ozan Çetin, Emre Tepeli, Özge Can, Gülseren Bağcı, Ali Keskin

304

Effect of Tumor Necrosis Factor-Alpha on Erythropoietin- and Erythropoietin Receptor-Induced Erythroid Progenitor Cell Proliferation in β-Thalassemia/Hemoglobin E Patients Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard, Suthat Fucharoen

311

The -137G/C Polymorphism in Interleukin-18 Gene Promoter Contributes to Chronic Lymphocytic and Chronic Myelogenous Leukemia Risk in Turkish Patients Serap Yalçın, Pelin Mutlu, Türker Çetin, Meral Sarper, Gökhan Özgür, Ferit Avcu

317

Transcobalamin II Deficiency in Four Cases with Novel Mutations Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin

323

Eltrombopag for the Treatment of Immune Thrombocytopenia: The Aegean Region of Turkey Experience Füsun Özdemirkıran, Bahriye Payzın, H. Demet Kiper, Sibel Kabukçu, Gülsüm Akgün Çağlıyan, Selda Kahraman, Ömür Gökmen Sevindik, Cengiz Ceylan, Gürhan Kadıköylü, Fahri Şahin, Ali Keskin, Öykü Arslan, Mehmet Ali Özcan, Gülnur Görgün, Zahit Bolaman, Filiz Büyükkeçeci, Oktay Bilgir, İnci Alacacıoğlu, Filiz Vural, Murat Tombuloğlu, Zafer Gökgöz, Güray Saydam

329

Management of Invasive Fungal Infections in Pediatric Acute Leukemia and the Appropriate Time for Restarting Chemotherapy Özlem Tüfekçi, Şebnem Yılmaz Bengoa, Fatma Demir Yenigürbüz, Erdem Şimşek, Tuba Hilkay Karapınar, Gülersu İrken, Hale Ören

338

First-Step Results of Children Presenting with Bleeding Symptoms or Abnormal Coagulation Tests in an Outpatient Clinic İsmail Yıldız, Ayşegül Ünüvar, İbrahim Kamer, Serap Karaman, Ezgi Uysalol, Ayşe Kılıç, Fatma Oğuz, Emin Ünüvar

344

Evaluation of Alpha-Thalassemia Mutations in Cases with Hypochromic Microcytic Anemia: The İstanbul Perspective Zeynep Karakaş, Begüm Koç, Sonay Temurhan, Tuğba Elgün, Serap Karaman, Gamze Asker, Genco Gençay, Çetin Timur, Zeynep Yıldız Yıldırmak, Tiraje Celkan, Ömer Devecioğlu, Filiz Aydın

Brief Report

351

The Efficacy and Safety of Procedural Sedoanalgesia with Midazolam and Ketamine in Pediatric Hematology Sema Aylan Gelen, Nazan Sarper, Uğur Demirsoy, Emine Zengin, Esma Çakmak

A-IX

Case Reports

355

A Hemophagocytic Lymphohistiocytosis Case with Newly Defined UNC13D (c.175G>C; p.Ala59Pro) Mutation and a Rare Complication Yasemin Işık Balcı, Funda Özgürler Akpınar, Aziz Polat, Fethullah Kenar, Bianca Tesi, Tatiana Greenwood, Nagihan Yalçın, Ali Koçyiğit

359

The Use of Low-Dose Recombinant Tissue Plasminogen Activator to Treat a Preterm Infant with an Intrauterine Spontaneous Arterial Thromboembolism Yaşar Demirelli, Kadir Şerafettin Tekgündüz, İbrahim Caner, Mustafa Kara

363

Immune Thrombocytopenic Purpura During Maintenance Phase of Acute Lymphoblastic Leukemia: A Rare Coexistence Requiring a High Degree of Suspicion, a Case Report and Review of the Literature Turan Bayhan, Şule Ünal, Fatma Gümrük, Mualla Çetin

367

A Rare Complication Developing After Hematopoietic Stem Cell Transplantation: Wernicke’s Encephalopathy Soner Solmaz, Çiğdem Gereklioğlu, Meliha Tan, Şenay Demir, Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu

371

Letters to the Editor

373

From Bone Marrow Necrosis to Gaucher Disease; A Long Way to Run Neslihan Erdem, Ahmet Çizmecioğlu, İsmet Aydoğdu

374

First Observation of Hemoglobin Kansas [β102(G4)Asn→Thr, AAC>ACC] in the Turkish Population İbrahim Keser, Alev Öztaş, Türker Bilgen, Duran Canatan

Images in Hematology

376

Mott Cells in the Peripheral Blood of a Patient with Dengue Fever Aniya Antony, Marie Ambroise, Chokka Kiran, Mookkappan Sudhagar, Anita Ramdas

378

Diagnosis: Melanoderma after Hematopoietic Stem Cell Transplantation Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya

2015 Index

2015 Subject Index 2015 Author Index

Downgraded Lymphoma: B-Chronic Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell Lymphoma - De Novo Occurrence or Transformation Smeeta Gajendra, Bhawna Jha, Shalini Goel, Tushar Sahni, Pranav Dorwal, Ritesh Sachdev

A-X

DOI: 10.4274/tjh.2015.0049

Review Article

Turk J Hematol 2015;32:285-294

Chimeric Antigen Receptor T Cell Therapy in Hematology Hematolojik Malignitelerde Kimerik Antijen Reseptör-T Hücre Tedavisi Pınar Ataca, Önder Arslan Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract: It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation.

Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified T cells. On 1 July 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality.

This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.

Keywords: Chimeric antigen receptor T cell, Hematological malignancies

Öz: İmmün sistemin kanser hücrelerini kontrol ve elimine etme özelliğine sahip olduğu gösterilmiştir. İmmün-kontrollü eliminasyonda kanser aşıları ve hematopoietik kök hücre naklini içeren sellüler terapiler bulunmaktadır. Adoptif T hücre transferi daha potent ve spesifiktir, hedef dışı toksisitesi azdır. Klinik çalışmalarda iki tür T hücresi test edilmektedir: T hücre reseptör ve kimerik antijen reseptör (KAR) modifiye T hücreleri. 1 Temmuz 2014’te Amerikan Gıda ve İlaç Dairesi anti-CD19 ŞAR modifiye T hücre tedavisini “çığır açan tedaviler” sınıfına almıştır. Bu yeni tedavi yöntemini ve etkilerini araştıran birçok çalışma yapılmıştır. Bu derleme adoptif immünoterapinin geçmişini, ŞAR modifiye T hücrelerini, üretim sürecini, klinik ve preklinik çalışmaları özetlemektedir.

Anahtar Sözcükler: Kimerik antijen reseptör-T hücreleri, Hematolojik maligniteler

Introduction Poor salvage chemotherapy success rates for refractory hematological diseases have necessitated novel approaches.

Adoptive T-cell transfer has gained significant interest and clinical usage in hematology because of the off target effects of allogeneic stem cell transplantation and life threatening graft versus host disease (GVHD). Therefore, research

efforts have sought to generate more specific T cells with higher toxicity to tumors and not healthy targets. To achieve curative potential, T cell immunotherapy combines potency, specificity and persistence [1]. Early approaches to adoptive T cell immunotherapy were based on the graft-versus-leukemia (GVL) effect mediated by donor lymphocyte infusion (DLI) hematopoietic stem cell transplantation (HSCT) and the therapeutic infusion of ex vivo expanded tumor-infiltrating

Address for Correspondence: Pınar ATACA, M.D., Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey E-mail: drpinarataca@gmail.com Received/Geliş tarihi : January 23, 2015 Accepted/Kabul tarihi : April 20, 2015

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lymphocytes (TILs) in combination with lymphodepletion for the treatment of advanced melanoma. However, DLI is usually associated with life-threatening forms of GVHD, and TILs require time-consuming procedures with unsuccessful results [2,3]. To overcome these drawbacks, genetically modified effector T cells have been developed as an alternative approach. In hematological malignancies, engineered T cell receptors (TCRs) and chimeric antigen receptors (CARs) are new powerful T-cell based immune therapies that target specific antigens. CAR T cells have been used successfully in the treatment of solid and hematological malignancies recently. In the following sections, the history of adoptive immunotherapy, TCR gene therapy, CART cell production, and preclinical and clinical studies will be discussed.

The Role of T Cells in Cancer and T Cell Receptor Gene Therapy In 1909, Paul Ehrlich first proposed that the immune defense system identifies and eliminates tumor cells [4]. However, recent studies revealed that the immune response may be ineffective against tumor development due to immunological tolerance and anergy [5]. Cancer immunoediting consists of three stages: elimination, equilibrium and escape. In the elimination stage, cancer is eliminated by intact innate and adaptive immunity, whereas in the equilibrium stage, variant tumor cells that develop genetic instability survive despite the immune attacks. Uncontrolled proliferation of variant tumor cells occurs in the escape stage [6].

In 1890, William B Coley observed that patients with malignancies respond to the intratumoral inoculation of live bacterial organisms or bacterial toxins that cause tumors to express unique proteins that could trigger an immune response [7]. Since the beginning of the 20th century, research has shown that most cancer cells carry overexpressed tumorassociated or tumor-specific antigens that are not present on healthy cells; this feature has led to the successful application of adoptive T-cell transfer. The discovery of T-cell growth factor, in vitro T-cell culture and the role of lymphodepletion have led to T-cell based therapy studies [8]. The first successful study on T-cell transfer immunotherapy using autologous TILs was performed in advanced melanoma in 1990 [9]. Since tumor infiltrating lymphocyte isolation was first attempted, in vitro expansion and re-infusion have been shown to be time-consuming and produce transient anti-tumor effects, and genetic engineering methods have been applied to create specific T cell-generated TCRs. The TCR is a heterodimer that carries information for defined tumor antigens and is formed by alpha and beta chains associated with a CD3 complex (Figure 1) [10]. TCR technology has advantages as a redirected T-cell therapy. Ideal effector T cells match with selected tumor target antigens through HLA recognition. The natural mechanism of T-cell immunity is associated with a low risk of cytokine release 286

Ataca P, et al: Chimeric Antigen Receptors in Hematology

syndrome. The major difficulties that need to be overcome are the low surface expression of TCRs, HLA dependency, the and short persistence of transferred T-cells in vivo [11].

In thymic selection during the development of T cells, a few mutated proteins are encoded by cancer-causing genetic mutations (driver mutations), the large proportion of tumor antigens are self antigens, and T cells have low affinity for self antigens [12]. To create a higher avidity, selected TCRs from immunized human HLA transgenic mice with relevant epitopes are used along with insertion of targeted mutations in the complementary-determining region 2 or 3 (CDR2 or 3) in the variable regions of the TCR alpha/beta chains. These modified TCRs interact with the HLA/epitope complex [13].

However, TCRs can create unwanted alpha/beta heterodimers between the new and endogenous TCR alpha/beta chains in a process called mispairing, which results in low avidity [14].

TCR-modified T cells adapted for solid tumors have not been successful in most studies (Table 1) [10].

Chimeric Antigen Receptors The genetic modification of T cells with CARs represents a breakthrough for gene engineering in hematological malignancies. The first CAR concept originated from the cloning of the TCR CD3 Îś-chain that was found to activate T cells independently [18]. First-generation CARs included only a single-chain variable fragment (scFv) that was constructed from the variable heavy and variable light sequences of a monoclonal antibody (mAb) specific for a tumor cell surface molecule and the cytoplasmic CD3 Îś-chain signaling domain.

The initial studies were conducted in patients with HIV infection with prolonged survival [19]. In the first-generation

Figure 1. T cell receptor (adapted from Wieczorek and Uharek [10]).

Turk J Hematol 2015;32:285-294

Ataca P, et al: Chimeric Antigen Receptors in Hematology

cancer studies, CAR T cells did not proliferate in vivo and persistence was transient or the T cells were present at very low frequencies [20]. Based on a second genetic modification, CARs possess an antibody-based extracellular receptor structure that binds to target cells along with intracellular activating domains. Costimulatory protein receptors (e.g., CD28, CD137 (4-1BB), ICOS, CD134 (OX40), CD27, or CD244) were added to the cytoplasmic tail of the CAR in the second- and third-generation CARs [21] (Figure 2). Secondgeneration CARs are constructed with one costimulatory molecule while third-generation CARs contain more than one additional costimulatory molecule. The antitumor effect of CAR-T cells varies due to differences in the cytoplasmic domain and the extracellular domain’s ability to recognize a different epitope of the same antigen with different affinities for each CAR construct [22]. Whether the addition of secondary costimulation as in third-generation CARs obtains more efficacy is still an unanswered question [23]. CARs have several advantages: initiation of reliable high-potency signals, HLA independency, no requirement for antigen processing, and no competition for CD3. The number of target molecules on tumor cells that bind to CARs is greater than the number of major histocompatibility complex (MHC)/peptide complexes, and the scFv has a higher binding affinity for antigens than the TCRs [24]. Recently, Oren et al. compared the functional properties of engineered T cells expressing native low-affinity αβ-TCR chains with high-affinity TCR-like Ab-based CARs targeting the same specificity and suggested that the upper affinity threshold should be used to mediate effective functional outcomes of engineered T cells [25]. The major disadvantage of CARs is the massive cytokine release induced by binding

and the immunogenicity of the mouse-derived scFv portion of the CAR complex, which may result in immune responses and the clearance of CAR T cells. In addition to that, intracellular molecules cannot be recognized [26].

Figure 2. Generations of CART cells (adapted from Porter et al.

[55]).

Table 1. T cell receptor clinical studies.

Antigen

Tumor

Effectiveness

Toxicities

Reference

MART 1

Melanoma

6 PRs in 20 patients

Erythematous skin rash grade 1-2 (14/20), hearing loss (10/20), uveitis (11/20)

2009, [15]

gp100

Melanoma

3 PRs in 16 patients

Erythematous skin rash grade 1-2 (15/16), hearing loss (5/16), uveitis (4/16)

2009, [15]

MAGE-A3

Melanoma multiple myeloma

Not evaluable

Acute cardiac failure (2/2), cytokine release syndrome, death (2/2)

2013, [16]

MAGE-A3/A9/A12

Melanoma, synovial sarcoma, esophageal cancer

4 PRs, 1 CR in 9 patients

Neurological toxicity (4/9), death (2/9)

2012, [17]

CR: Complete remission, PR: partial remission.

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Ataca P, et al: Chimeric Antigen Receptors in Hematology

Chimeric Antigen Receptor T Cell Manufacturing Gene transfer technology has rapidly developed; however, the clinical production of CARs for therapy is restricted to specialized, licensed manufacturing facilities with stringent rules (Good Manufacturing Process). In vitro culture systems for T cell expansion are used to manufacture large quantities of engineered T cells. The average production time to generate

large numbers of unselected CD4 and CD8 T cells required for therapy is 10-14 days in clean rooms. First, peripheral blood mononuclear cells are isolated from the patient using leukapheresis, and T cells are selected by anti-CD3/anti-CD28 paramagnetic beads. Recent studies have demonstrated that less differentiated T cells have superior engraftment and antitumor activity [27]. In particular, CD8 T central memory cells can be modified with tumor-specific CARs [28]. T

Table 2. Chimeric antigen receptor T cell trials in hematological malignancies [55].

scFv/Signaling Domain

Vector

Dose

Number of Patients

Responses

Reference

CD20/CD3

Electroporation

1x108/m2 to 3.3x109/m2

7 (indolent and MCL)

2 CR, 1 PR, 4 SD

2008 [46]

CD20 or CD19/CD3

Electroporation

108/m2 to 2x109/m2 Â

4 (2 FL, 2 DLBCL)

2 CR after autologous 2010 [52] stem cell transplantation

CD19/CD3 and CD28-CD3

Gammaretrovirus

2x107/m2 to 2x109/m2

6 NHL

2 SD

2011 [53]

CD19/CD28 and CD3

Gammaretrovirus

0.4-3.3x107 CAR cells/kg Â

8 CLL and 1 ALL

1 death, (ALL) B cell aplasia, 1 reduction in lymphadenopathy

2011 [54]

CD19/4-1BB and CD3

Lentivirus

1.46x105 to 1.6x107 3 CLL CAR cells/kg

2 CR, 1 PR, 3 B cell aplasia

2011 [55]

CD19/CD28 and CD3

Gammaretrovirus

0.3-3x107 CAR cells/kg

8 (3 FL, 4 CLL, 1 MZL)

6 objective remissions (4 B cell aplasia)

2010 [56]

CD20/CD28 and 4-1BB and CD3

Electroporation

1x108 to 3.3x109/ m2

3 (2 MZL, 1 FL)

No progression in 2 patients, 1 patient PR

2012 [57]

CD19/CD28 and CD3

Gammaretrovirus

1.5-3x106 CAR cells/kg

5 ALL

All 5 converted to MRD, 1 relapsed, 1 B cell aplasia

2013 [58]

CD19/4-1BB and CD3

Lentivirus

1.4x106 and 1.2x107 CAR cells/ kg

2 ALL

2 CR, both B cell aplasia

2013 [59]

Lewis Y/CD28 and CD3

Gammaretrovirus

1.4-9.2x106 CAR cells/kg

4 AML

2 SD, 1 transient cytogenetic remission

2013 [48]

CD19/CD28 and CD3

Gammaretrovirus

1.5x107 to 1.2x108 total T cells/m2

8 ALL

4 of 8 patients with decreased B cell counts

2013 [60]

CD19/CD28 and CD3

Gammaretrovirus

0.4-7.8x106 CAR cells/kg

4 CLL, 2 DLBCL, 4 MCL

2 PD, 6 SD, 1 PR, 1 CR

2013 [61]

AML: Acute myeloid leukemia, ALL: acute lymphoblastic leukemia, CLL: chronic lymphocytic lymphoma, CR: complete remission, DLBCL: diffuse large B cell lymphoma, FL: follicular lymphoma, MCL: mantle cell lymphoma, MRD: minimal residual disease, MZL: marginal zone lymphoma, NHL: non-Hodgkin lymphoma, PD: progressive disease, PR: partial remission, SD: stable disease.

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cells are then transduced with a CAR-encoding viral vector.

Two vector systems, retroviral or lentiviral vectors, can be used to transfer CAR-coding genes into T cells. Retroviral vectors have permanent gene expression; however, the transduction can be performed only on efficiently dividing T cells. Lentiviral vectors can also integrate into nondividing cells. The disadvantages of viral vectors are the expense and experience required for production. Transposon systems such as Sleeping Beauty 100X (SB100X) or PiggyBac (PB) are new methods for genetic modification of T cells with high gene expression; they are simple and inexpensive and have large cargo capacity and low immunogenicity [10]. T cells are expanded in culture by stimulating them using the anti-CD3 clone OKT3 with cytokines like IL-2, IL-7, and IL-15. Moreover, in vivo persistence can be achieved by the overexpression of antiapoptotic proteins such as Bcl-2 or Bcl-xL. An adequate number of CAR T cells, which remains unknown, are then transferred to the patient using host preparative lymphodepletion regimens based on drugs and techniques to deplete Tregs, such as cyclophosphamide, fludarabine, low-dose irradiation, gemcitabine, denileukin diftitox, azacitidine, or decitabine [10,29,30]. CARs on T cells bind to their antigen on the tumor, and activation is controlled by the intracytoplasmic domains within the CAR. Tumor killing can be mediated by the direct cytotoxicity of the CD8+ CAR T cells with granzyme and perforin or cytokines released by CD4+ CAR T cells that bypass the MHC. Longterm eradication and prevention can be achieved by memory CAR T cells from a single infusion [31].

Studies Involving Chimeric Antigen Receptor T Therapy TThe ideal targets for CAR-modified T cells are expressed on tumor cells but are not expressed on normal cells. CD19 and CD20 are attractive targets due to their specificity for the B cell linage [32]. The first-generation CARs were not sufficient to produce a durable immune response; they rapidly underwent apoptosis after stimulation [33]. 19z CAR T cells were expanded on CD19+CD80+IL15+ cells and eradicated established systemic Raji tumors in 50% of SCID-beige mice [34]. Second-generation CARs that express CD28-containing costimulation in the CD19+CD80/CD86-ALL SCID-beige tumor model showed superior in vivo tumor activity and T cell function. CD22 is also under investigation and shows potential [35]. Imai et al. showed that in vivo anti-CD19 chimeric receptors containing the 4-1BB signal transduction domain had powerful antileukemic activity, destroying CD19+ acute lymphoblastic leukemia (ALL) cell lines in an in vivo microenvironment [33]. Target discovery for T cell leukemias and myeloid leukemias is problematic because blasts express the same antigens as normal hematopoietic stem cells [36]. For myeloid leukemias, CARs directed against

Turk J Hematol 2015;32:285-294

CD123 have demonstrated efficacy in preclinical models; however, vascular endothelial cells also express CD123, which requires more investigation before clinical application [37]. Kenderian et al. stated that anti-CD33-specific CAR T cells exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in acute myeloid leukemia (AML) xenografts [38]. In multiple myeloma, CAR-engineered natural killer cells that targeted CS-1 protein displayed enhanced cytolysis in vitro [39].

The translation of this therapy to clinical settings involves various antigens and malignancies, and most trials have focused on B cell malignancies with B cell antigens CD19 and CD20 as the targets [40]. The first case report of CD19+ CAR T cells was published in 2011 by Porter et al. in relapsed refractory chronic lymphoid leukemia [41]. In that study, 3x108 T cells were transduced using a lentiviral vector, and the patient exhibited complete remission after 10 months.

The largest dose-optimization trial involved 27 chronic lymphocytic leukemia (CLL) patients and found no difference between two doses of CAR T cells (<5x107 versus >5x107) with a complete response rate of 40% of patients [42]. In another study, CAR-modified T cells were shown to persist for more than 3 years with an initial response rate of 57% and complete remission of 29%, which was more favorable as compared to ibrutinib (an overall response rate of 71% but a complete remission rate of 2.4%) [43]. In B cell ALL (B-ALL), Davila et al. reported on 16 relapsed or refractory cases that were treated with 19-28z-expressing CAR T cells with an overall complete response rate of 88%, as compared to 44% with salvage chemotherapy. CAR T cells persisted for 2-3 months, and almost half of the patients proceeded to allogeneic stem cell transplantation [44]. In 30 ALL patients treated with CD19 CAR T cells, a 6-month event-free survival of 67% and overall survival of 78% were achieved, and ongoing remission for up to 2 years was possible without transplantation [45].

The underlying causes of the limited clinical efficacy of the CAR T cells in patients with CLL compared to B-ALL include the limited persistence of CAR T cells in CLL patients, the inhibitory effect of the tumor microenvironment in CLL, the lymph node-based nature of CLL, and the lower tumor burden at treatment in patients with B-ALL [40].

Patients with B cell malignancy were first treated with modified autologous CD20-specific T cells in 2008 by investigators from the Fred Hutchinson Cancer Research Center and the City of Hope National Medical Center.

T cells persisted for up to 9 weeks with 7 patients with indolent or mantle cell lymphoma achieving partial response (1 patient), stable disease (4 patients), or complete response (2 patients) [46]. In 2014, an anti-CD19 chimeric antigen receptor trial for chemotherapy-refractory diffuse large B cell lymphoma and indolent B cell malignancies was published

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by Kochenderfer et al.; they demonstrated that 8 of 15 patients had complete response with 1-5x106 CAR T cells transduced by gammaretrovirus [47]. The targets for CAR therapy in multiple myeloma can be CD138, CD38, CD56, and CS1. Unlike CD19, these targets are coexpressed on other important cell types and result in unacceptable on-target, offtumor toxicity. The first AML trial targeted the LeY antigen, and only 1 of 4 patients had 23 months of stable disease following therapy [48]. Contrary to preclinical studies, the CD33 antigen as a target was not proven to be safe due to the high level of toxicity against normal hematopoietic cells [49].

Phase I clinical trials involving CD123 targeting by mAbs and immunotoxins have produced only minor clinical responses, suggesting the need to develop more powerful AML strategies [50]. Table 2 shows the CAR T cell therapies in hematological malignancies [51].

Adverse Effects of Chimeric Antigen Receptor T Cell Therapy AAs with all therapies, the toxicity from CAR T cells may be classified as on-target or off-target. The most common toxicity is cytokine release syndrome (CRS). In most cases, CRS is correlated with antitumor activity, and patients exhibit a range of symptoms from high fever, hypoxia, and hypotension to mild flu symptoms. The increased cytokines, particularly IL-6 and TNF-α, are produced by dying B cells, tumor cells, or macrophages [51]. Grupp et al. reported that the IL-6 receptor-blocking monoclonal antibody tocilizumab may ameliorate CRS in steroid-refractory circumstances without compromising T cell efficacy [59]. CRS was reported to occur in 6/13 patients with high complete response rates with tocilizumab as an alternative treatment option. The C-reactive protein level has been shown to be an indicator of severe CRS [45]. Another off-target adverse effect is tumor lysis syndrome, which is due to rapid and massive destruction of tumor cells. Macrophage activation syndrome is another life-threatening off-target effect of systemic inflammatory symptoms and pancytopenia, although the mechanisms are still unknown [42]. Several patients in CD19-CAR trials experienced reversible obtundation, seizures, aphasia, and mental status changes, possibly due to systemic cytokines crossing the blood-brain barrier [51]. B cell aplasia is an expected result of CD19-directed therapies and can be managed by γ-globulin replacement therapy. Persistent B cell aplasia results in an increased risk of infections [52].

Future Directions AAdoptive T cell transfer has been used for the treatment of malignant diseases and may be regarded as an anticancer biopharmaceutical. A biopharmaceutical is defined as a product that is originally natural or derived from biological 290

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sources with industrial additions [62]. The main goals of T cell engineering are tumor antigen targeting and an increase in antitumor functions [1]. CAR T cell therapies are powerful breakthrough therapies, but several challenges need to be addressed. The optimal design of CARs remains an area of investigation. To be useful in other disease types, tumorspecific targets must be identified in solid tumors. T cell trafficking to the tumor microenvironment is critical in the moderate success against solid cancers [63]. To minimize severe toxicity, standardized approaches to the management of CRS should be applied [64]. B cell aplasia is still a problem with long-term exposure and may have an economic impact on health care. Once the B cell malignancy has been eradicated, anti-CD19-CAR T cells should be ablated to maintain normal B cell activity. A suicide system has been developed to eliminate gene-modified T cells when they display unwanted toxicities, such as the thymidine kinase gene of the herpes simplex virus [65]. Relapse remains a challenge and may be prevented with optimization of CAR design. Finally, in order for the therapy to become routinely used, automation and robotic culture technologies should be performed during the manufacturing process instead of manual cell culture technologies [66].

The induction of adoptive immunotherapy using CAR T cells has been successful in clinical trials, and the final goal is to induce durable immunity against disease progression without severe adverse effects. Whether this treatment option will replace HSCT or be used as a bridge to HSCT in the near future is still an unanswered question.

Concept: Önder Arslan, Design: Önder Arslan, Data Collection or Processing: Pınar Ataca, Analysis or Interpretation: Pınar Ataca, Literature Search: Pınar Ataca, Writing: Pınar Ataca, Önder Arslan.

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

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Research Article

DOI: 10.4274/tjh.2014.0174 Turk J Hematol 2015;32:295-303

Possible Role of GADD45γ Methylation in Diffuse Large B-Cell Lymphoma: Does It Affect the Progression and Tissue Involvement?

Diffüz Büyük B-Hücreli Lenfomada GADD45γ Metilasyonunun Olası Rolü: Lenfoma Progresyonunu ve Doku Tutulumunu Etkiler mi? İkbal Cansu Barış1, Vildan Caner1, Nilay Şen Türk2, İsmail Sarı3, Sibel Hacıoğlu3, Mehmet Hilmi Doğu3, Ozan Çetin4, Emre Tepeli4, Özge Can1, Gülseren Bağcı1, Ali Keskin3 1Pamukkale

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

Abstract: Objective: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma among adults and is characterized by heterogeneous clinical, immunophenotypic, and genetic features. Different mechanisms deregulating cell cycle and apoptosis play a role in the pathogenesis of DLBCL. Growth arrest DNA damage-inducible 45 (GADD45γ) is an important gene family involved in these mechanisms. The aims of this study are to determine the frequency of GADD45γ methylation, to evaluate the correlation between GADD45γ methylation and protein expression, and to investigate the relation between methylation status and clinicopathologic parameters in DLBCL tissues and reactive lymphoid node tissues from patients with reactive lymphoid hyperplasia.

Materials and Methods: Thirty-six tissue samples of DLBCL and 40 nonmalignant reactive lymphoid node tissues were analyzed in this study. Methylation-sensitive high-resolution melting analysis was used for the determination of GADD45γ methylation status. The GADD45γ protein expression was determined by immunohistochemistry.

Results: GADD45γ methylation was frequent (50.0%) in DLBCL. It was also significantly higher in advanced-stage tumors compared with early-stage (p=0.041). In contrast, unmethylated GADD45γ was associated with nodal involvement as the primary anatomical site (p=0.040).

Conclusion: The results of this study show that, in contrast to solid tumors, the frequency of GADD45γ methylation is higher and this epigenetic alteration of GADD45γ may be associated with progression in DLBCL. In addition, nodal involvement is more likely to be present in patients with unmethylated GADD45γ.

Keywords: GADD45γ, DNA methylation, Diffuse large B-cell lymphoma Address for Correspondence: Vildan CANER, M.D., Pamukkale University Faculty of Medicine, Department of Medical Biology, Denizli, Turkey Phone: +90 258 296 24 94 E-mail: vildancaner@yahoo.com, vcaner@pau.edu.tr Received/Geliş tarihi : May 02, 2014 Accepted/Kabul tarihi : July 08, 2014

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Barış Cİ, et al: GADD45γ Methylation in Diffuse Large B-Cell Lymphoma

Öz: Amaç: Diffüz büyük B-hücreli lenfoma (DBBHL) yetişkin bireylerde Hodgkin-dışı lenfomaların en yaygın tipidir ve klinik, immünofenotipik ve genetik özellikler açısından heterojen özellikler taşıması ile karakterizedir. DBBHL patogenezinde hücre döngüsü ve apoptoz regülasyonunu bozan farklı mekanizmalar rol oynamaktadır. Growth arrest DNA damage-inducible 45 (GADD45γ), bu mekanizmalarda yer alan önemli bir gen ailesidir. Bu çalışmanın amaçları DBBHL doku örnekleri ve reaktif lenfoid hiperplazili bireylerin reaktif lenfoid doku örneklerinde GADD45γ metilasyon sıklığını belirlemek, GADD45γ metilasyonu ile protein ekspresyonu arasındaki ilişkiyi değerlendirmek ve DBBHL olgularında metilasyon durumunun klinikopatolojik parametrelerle ilişkisini araştırmaktır.

Gereç ve Yöntemler: Bu çalışmada 36 adet DBBHL doku örnekleri ve 40 adet malign-olmayan reaktif lenfoid doku örnekleri analiz edildi. GADD45γ metilasyon durumunu belirlemek için metilasyona-duyarlı yüksek çözünürlüklü erime eğrisi analizi kullanıldı. GADD45γ protein ekspresyonu immünohistokimyasal analiz ile belirlendi.

Bulgular: DBBHL’de GADD45γ metilasyonunun sık olduğu belirlendi (%50). Aynı zamanda, erken evre ile karşılaştırıldığında ileri evre tümörlerde GADD45γ metilasyonu istatistiksel olarak anlamlı düzeyde yüksekti (p=0,041). Ancak, GADD45γ metilasyon yokluğunun primer anatomik yerleşim olarak nodal tutulumla ilişkili olduğu belirlendi (p=0,040).

Sonuç: Bu çalışmanın sonuçları solid tümörlerin aksine, DBBHL’de GADD45γ metilasyon sıklığının yüksek olduğunu ve GADD45γ geninde gözlenen bu epigenetik değişimin, hastalığın progresyonu ile ilişkili olabileceğini göstermektedir. Buna ek olarak, nodal tutulum daha çok GADD45γ metile olmayan olgularda gözlenmektedir.

Anahtar Sözcükler: GADD45γ, DNA metilasyonu, Diffüz büyük B-hücreli lenfoma Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common group of non-Hodgkin lymphomas (NHLs) and represents 30% to 40% of all newly diagnosed NHLs in Western countries. DLBCL represents a heterogeneous group of neoplasms with diversity in clinical presentation, morphology, and genetic and molecular properties [1]. It is well known that genetic and epigenetic changes that create a difference in gene expression profiles between normal and malign B cells are responsible for the heterogeneity of DLBCL. Genetic aberrations in DLBCL are chromosomal translocations, aberrant somatic hypermutations, and copy number variations including amplifications or deletions [2,3,4,5]. Other differences come from epigenetic modifications such as DNA methylation [6,7,8].

DNA methylation may lead to transcriptional silencing by at least 3 different mechanisms: inhibition of binding of the transcription factors to their specific sequences, a direct effect on nucleosome positioning, and recruitment of other nuclear factors that recognize the methylated CpG dinucleotide blocks binding other factors including transcription factors [9]. To date, a number of genes involved in the regulation of DNA repair, cell cycle control, and apoptosis, such as MGMT [10,11], DAPK1 [12], and GADD45γ [13], have been determined as hypermethylated in DLBCL. A recent study also showed that abnormal methylation patterns might be seen depending on chromosomal regions, gene density, and

296

methylation status of neighboring genes in normal B-cell populations and NHL [8].

The growth arrest DNA damage-inducible (GADD45) gene family plays important roles in various cell functions such as DNA repair, cell-cycle control, and cell growth [14]. The members of the GADD45 gene family, GADD45γ, GADD45γ, and GADD45γ, are evolutionarily conserved and expressed in both fetal and adult tissues [15,16,17]. They act as stress sensors that modulate cellular response against various physical and environmental stress factors [14,17,18]. It is also suggested that GADD45 proteins may provide a link between DNA repair mechanisms and chromatin remodeling [19,20].

Although all 3 proteins have similar functions, these functions are not identical since they have different activation pathways depending on cell type and the source of the stress [17,21].

There are very limited data in the literature about the role of GADD45γ in DLBCL pathogenesis. In this study, we aimed to show the methylation status and expression profiles of GADD45γ in DLBCL tissues and nonmalignant reactive lymphoid node tissues (RLTs). We also focused on the relationship between GADD45γ methylation status and clinicopathologic parameters of DLBCL. Materials and Methods Tissue Samples We analyzed 36 DLBCL tissue samples and 40 nonmalignant RLTs that were diagnosed in the Department of Pathology of

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Pamukkale University between 2009 and 2012. Tissue samples were collected from all patients before treatment. Based on Hans’s algorithm, DLBCL cases were classified as germinal center (GC) and non-GC in the Pathology Department [22]. All of the patients with DLBCL were also classified by Ann Arbor stage and International prognostic index (IPI) score according to the previously described criteria [23,24].

This study was approved by the Institutional Review Board of Pamukkale University and was in compliance with the Declaration of Helsinki.

Two consecutive sections of formalin-fixed and paraffinembedded (FFPE) tissues were used for DNA isolation and immunohistochemistry (IHC). DNA was isolated using a commercial kit according to the instructions of the manufacturer (QIAamp DNA Mini Kit, QIAGEN, the Netherlands) and IHC was performed using polyclonal antibody against GADD45γ as described previously [25].

Methylation-Sensitive High-Resolution Melting Analysis DNA samples underwent bisulfite treatment prior to methylation-sensitive high-resolution melting (MS-HRM) analysis by use of a commercial kit (EZ DNA MethylationGold Kit, Zymo Research, USA). Forward and reverse primers were as follows, respectively: 5’-CGTCGTGTTGAGTTTTGGT and 5’-TAACCGCGAACTTCTTCCA [26]. The protocol for identification of the amplicon by MS-HRM analysis is given in Table 1. For the confirmation of melting temperature (Tm) degrees in MS-HRM analysis, commercially available control DNA samples were used (EpiTect Control DNA Set, QIAGEN).

All analyses were performed on a LightCycler 480 instrument (Roche Diagnostics, Germany).

Immunohistochemistry All immunostaining procedures including deparaffinization and antigen retrieval processes were performed automatically using the BenchMark XT automated stainer (Ventana Medical Systems, USA). GADD45γ (dilution: 1/200, Bioss Laboratories, USA) was used as the primary antibody. Larynx squamous cell carcinoma tissue samples were used as positive controls while negative controls were treated with the same IHC method by omitting the primary antibody. Granular cytoplasmic staining was assessed as positive. Immunohistochemical status of GADD45γ was scored as 0 (less than 25% positive cells), + (26% to 50% positive cells), ++ (51% to 75% positive cells), or +++ (more than 75% positive cells).

Statistical Analysis The methylation status and protein expression level of GADD45γ between DLBCL patients and RLT controls was compared using the chi-square test. The Fisher’ exact test was used to compare the protein expression and methylation of GADD45γ. The age-adjusted frequency ratios of GADD45γ methylation were calculated using multiple logistic regression analysis. P<0.05 was considered to be statistically significant.

Results Clinicopathologic Parameters The median ages were 67.5 (range: 24-80) and 28.00 (range: 1-79) years in DLBCL patients and RLT controls,

Table 1. High-resolution melting protocol for GADD45g methylation.

Analysis Mode

Cycle

Target (°C)

Hold Time Ramp Rate (°C/s)

Acquisition Mode

Preincubation

1

95

10 min

4.4

-

Amplification

95

10 s

4.4

-

50

60

15 s

2.2

-

72

10 s

4.4

Single

High-resolution melting

95

1 min

4.4

-

40

1 min

2.2

-

65

1s

1

-

95

-

0.02

Continuous

Cooling

1

40

10 s

-

-

297

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respectively. The most frequent sites of extranodal involvement were as follows when the patients were classified according to the anatomic site of tumor: lung (6 cases, 42.9%), bone marrow (3 cases, 21.4%), liver (2 cases, 14.3%), and stomach (2 cases, 14.3%).

GADD45γ Methylation The Tm was 79±0.5 °C in the methylated region of the GADD45γ gene while the unmethylated region had a Tm of 76±0.5 °C in MS-HRM analysis, which was also confirmed by the control DNA samples. According to this finding, GADD45γ methylation was present in 18 of the DLBCL patients (50%), whereas 16 (40%) of the controls were methylated (Table 2).

Figure 1 shows the HRM analysis of GADD45γ methylation.

No statistically significant difference was observed between DLBCL patients and controls in terms of GADD45γ methylation status (p=0.381). While the mean age was 48.56±22.69 years in the group that had methylated GADD45γ, it was 46.50±25.06 in the unmethylated group (p=0.716). Age status also did not significantly affect the methylation frequency

of the GADD45γ gene (p=0.407). However, the methylation frequency in patients with advanced stage (stage 3 and 4) disease was 17 times higher than in early stages (stage 1 and 2), which was statistically significant (p=0.041). In addition, there was a difference in the methylation status of GADD45γ between nodal and extranodal involvement (p=0.040). The frequency of GADD45γ methylation in the group with high clinical risk (IPI score 3-4) was 2.6 times higher than that in the low clinical risk group (IPI score 0-2); however, this was not statistically significant (p=0.298) (Table 2).

GADD45γ Protein Expression GADD45γ protein expression was observed to be (0) in 1, (+) in 18, (++) in 11, and (+++) in 6 of the DLBCL cases. In controls, the numbers were 8, 30, and 2 for (0), (+), and (++), respectively. None of the controls were (+++) for GADD45γ protein expression. Since the numbers of samples in the subgroups were small, samples were combined for ease of statistical analysis. While (0) and (+) were regarded as low protein expression, (++) and (+++) were accepted as high

Table 2. Associations of GADD45γ methylation and protein expression with clinicopathologic parameters in diffuse large B-cell lymphoma patients.

Clinicopathologic Parameters

GADD45g Promoter Methylation

GADD45g Protein Expression

Absent

Present

p-value

Low

High

p-value

Total patients

18 (50)

18 (50)

19 (52.8)

17 (47.2)

Sex

Male

7 (50)

7 (50)

1.000

6 (42.9)

8 (57.1)

0.342

Female

11 (50)

11 (50)

13 (59.1)

9 (40.9)

Stage (Ann Arbor)

Early (I/II)

7 (87.5)

1 (12.5)

0.041

5 (62.5)

3 (37.5)

0.695

Advanced (III/IV)

11 (39.3)

17 (60.7)

14 (50)

14 (50)

Tumor location

Nodal

14 (63.6)

8 (36.4)

0.040

13 (59.1)

9 (40.9)

0.342

Extranodal

4 (28.6)

10 (71.4)

6 (42.9)

8 (57.1)

Cell origin

GC

9 (52.9)

8 (47.1)

0.738

11 (64.7)

6 (35.3)

0.175

Non-GC

9 (47.4)

10 (52.6)

8 (42.1)

11 (57.9)

0-2

8 (61.5)

5 (38.5)

0.298

6 (46.2)

7 (53.8)

0.549

3-5

10 (43.5)

13 (56.5)

13 (56.5)

10 (43.5)

IPI score

IPI: International prognostic index, GC: germinal center.

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protein expression (Figure 2). After this grouping, we found a statistically significant difference between DLBCL patients and controls (p<0.001) (Table 2).

A

High-level expression of GADD45γ was present in 37.5% of early and 50% of advanced stage DLBCL patients. There was no significant relation between the protein expression level and the stage of DLBCL (p=0.695). Similarly, we did not find a significant association between the protein expression level and other clinicopathologic parameters (Table 2).

Association of GADD45γ Methylation Status with GADD45γ Protein Expression Among 18 DLBCL patients with methylated GADD45γ, we observed the high expression and the low expression of GADD45γ in 8 (44.4%) and 10 (55.6%) patients, respectively.

The high expression of GADD45γ was determined in 9 (50.0%) patients whose tumors had no methylated GADD45γ.

Although we observed an association between GADD45γ methylation status and protein expression level in 48.7% of all patients included in this study, no significant correlation between the protein expression level and the status of methylation was observed (p=0.695) (Table 3).

B

Discussion

C

As major stress sensors of cells, GADD45 proteins might be key players of cancer development and progression. Although several studies have focused on the relationship between GADD45γ gene expression and methylation in hematologic malignancies and solid tumors, there are limited data in the literature about the involvement of GADD45γ methylation and protein expression in DLBCL development [13,26,27]. To our knowledge, this is the first study investigating the association between the methylation and the level of protein expression of D

Figure 1. The high-resolution melting curves for GADD45γ methylation in diffuse large B-cell lymphoma, patients. a. Unmethylated GADD45γ DNA had a melting peak at 76±0.5 °C, b. Methylated GADD45γ DNA had a melting peak at 79±0.5 °C, c. Negative control (PCR-grade water was used instead of template DNA).

Figure 2. Representative immunohistochemical detection of GADD45γ in diffuse large B-cell lymphoma (A-D). Diffuse large B-cell lymphoma comprising large neoplastic lymphoid cells with strong GADD45γ staining intensity (Score: +++) (A), with moderate GADD45γ staining intensity (Score: ++) (B), with weak GADD45γ staining intensity (Score: +) (C), and with no GADD45γ staining (Score: 0) (D) (original magnification 200x).

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Table 3. Association between GADD45γ methylation and its protein expression.

Methylated/High Expression

Methylated/Low Expression

Unmethylated/ High Expression

Unmethylated/ Low Expression

DLBCL patients

8

10

9

9

RLT controls

0

16

2

22

Total

8

26

11

31

DLBCL: Diffuse large B-cell lymphoma, RLT: reactive lymphoid node tissue.

GADD45γ in DLBCL patients and RLT controls. We detected GADD45γ methylation in 50.0% of DLBCL patients. MS-HRM used in this study was performed as previously described [26]. Zhang et al. found that the HRM protocol had high sensitivity, which allows the detection of low (1%) amounts of DNA methylation for GADD45γ [17]. It is well known that DNA derived from FFPE tissues is often degraded and the degradation of DNA is highly dependent on the sample age.

In the present study, we used DNA samples extracted from FFPE tissues ranging in age from 2 to 5 years for MS-HRM. In a recent study, Kristensen et al. showed that DNA derived from up to 30-year-old FFPE tissue can be successfully used for DNA methylation analysis by MS-HRM [28]. Therefore, we suggest that MS-HRM analysis could be used to detect the methylation status of GADD45γ in FFPE tissue samples.

In non-small cell lung cancer, Na et al. reported that GADD45γ methylation was detected in 31.6% of cases.

They also proposed that the silencing of GADD45γ by DNA methylation might be contributing to the development of lung cancer [29]. Bahar et al. detected GADD45γ methylation in 58% of human pituitary adenoma cases [27]. In 82% of patients whose tumors had no mRNA expression of GADD45γ, they detected promoter methylation by both methylationspecific PCR and sodium bisulfite sequencing. Ying et al. reported the epigenetic inactivation of GADD45γ in primary samples from various cancer types and tumor cell lines [13]. In their study, they found that GADD45γ methylation was more frequent in leukemia and lymphomas (16%-88%) than solid tumors (11%-16%). In their series, 38% of primary DLBCL tissues had GADD45γ promoter methylation, which is concordant with our results. Our results and theirs may be showing the specificity of GADD45γ methylation according to the epithelial or mesenchymal origin of tumors. Another interesting finding of our study was the increasing frequency of GADD45γ methylation with tumor progression. We found a significantly higher GADD45γ methylation frequency in advanced stages than early stages. This may show that the loss of function in the GADD45γ tumor suppressor gene by DNA methylation plays a key role in the progression of DLBCL. It is well known that NHLs arise in different anatomical sites and they are considered as nodal and extranodal 300

lymphomas according to the site [30,31]. The differences in clinical and biological characterizations between nodal and extranodal involvement are still not clear, as reflected in the heterogeneous nature of DLBCL in some sense, although there are a number of studies focused on the differences between lymphomas at different anatomical sites [32,33,34].

A recent study reported that primary extranodal involvement, especially at gastrointestinal, pulmonary, and liver/pancreatic sites, was associated with a worse outcome when compared to nodal involvement [35]. In our series, the majority of the patients had nodal involvement, while the remaining patients had both nodal and extranodal involvement. It was interesting that nodal involvement was observed in almost 80% of the patients with no methylated GADD45γ, with significant statistical difference, although there was no relation between the tissue involvement and IPI score. This finding may suggest that GADD45γ methylation status might be an important factor for the primary site of the lymphoma. Further studies are needed to identify the genetic and/or epigenetic differences between nodal and extranodal involvement in DLBCL. There is no consensus in the literature about the relationship between GADD45γ methylation status and protein expression levels. Ying et al. found no GADD45γ expression in the cell lines with GADD45γ methylation in their above mentioned study [13]. Bahar et al. found a significant correlation between GADD45γ methylation and low protein expression, although there was expression of GADD45γ transcript in 9% of the patients with GADD45γ methylation [27]. Furthermore, 18% of patients without GADD45γ methylation did not have GADD45γ expression, either. In the present study, we could not find an association between GADD45γ methylation and protein expression in 51.3% of our cases. This finding may be explained by the following potential mechanisms: first, the method we used for the detection of methylation is not a quantitative method and those cases with GADD45γ expression might have low methylation levels that are not adequate for gene silencing.

A number of studies have reported no significant association between protein expression and methylation status in different genes such as MGMT, DLC1, GATA4, NDK2, and RARRES1 [29,36]. It has also been reported that a gain of DNA methylation is not always associated with gene silencing.

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Kulis et al. characterized the DNA methylomes in patients with chronic lymphocytic leukemia and reported that there was a significant correlation between gene expression and DNA methylation levels in 4% of all CpGs [37]. In a study that identified DNA methylation differences in different human ethnic groups, it was shown that a gain of DNA methylation was associated with gene repression and activation in 63.0% and 37.0% of cases, respectively [38]. Second, our target in GADD45γ was relatively small because large amplicon sizes are generally unsuitable for HRM analysis. The GADD45γ gene has a unique CpG island that contains not only the promoter region but also exons [13,27]. Searching in the whole GADD45γ gene should be more accurate to detect the real methylation status. Third, since GADD45γ mutation was very rarely detected in primary tumors [13], the inhibition of expression might be due to other epigenetic mechanisms than DNA methylation, such as small noncoding RNAs and histone modifications. Finally, the polyclonal antibody that we used for IHC due to unavailability of commercial monoclonal antibody against GADD45γ protein might have cross-reacted with other epitopes in colocalized protein targets [39].

In summary, we found that the frequency of GADD45γ methylation in DLBCL was higher than that reported in solid tumors. We also observed that the frequency of GADD45γ methylation in advanced stages was significantly higher than that in early stages. In comparison to nodal DLBCL, GADD45γ was commonly methylated in extranodal DLBCL. These findings indicated that the silencing of GADD45γ by DNA methylation may play a role in the progression and the tissue involvement of DLBCL. Further studies are needed to evaluate the role of other members of the GADD45 family and their partners in DLBCL. Acknowledgments This research project was supported by the Scientific Research Project Unit of Pamukkale University (Project No. 2012SBE007). The authors thank Dr. Mehmet Zencir (Department of Public Health, Pamukkale University) for the statistical analysis.

Ethics Committee Approval: This study was approved by the Institutional Review Board of Pamukkale University and was in compliance with the Declaration of Helsinki, Concept: İkbal Cansu Barış, Vildan Caner, Design: İkbal Cansu Barış, Vildan Caner, Data Collection or Processing: Nilay Şen Türk, İsmail Sarı, Sibel Hacıoğlu, Mehmet Hilmi Doğu, Ozan Çetin, Emre Tepeli, Özge Can, Ali Keskin, Analysis or Interpretation: İkbal Cansu Barış, Vildan Caner, Nilay Şen Türk, Ozan Çetin, Emre Tepeli, Özge Can, Gülseren Bağcı, Literature Search: İkbal Cansu Barış, Vildan Caner, Nilay Şen Türk, İsmail Sarı, Sibel Hacıoğlu, Writing: İkbal Cansu Barış, Vildan Caner, Nilay Şen Türk, İsmail Sarı, Sibel Hacıoğlu.

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

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34. Toda H, Sato Y, Takata K, Orita Y, Asano N, Yoshino T. Clinicopathologic analysis of localized nasal/paranasal diffuse large B-cell lymphoma. PLoS One 2013;8:e57677.

35. Castillo JJ, Winer ES, Olszewski AJ. Sites of extranodal involvement are prognostic in patients with diffuse large B-cell lymphoma in the rituximab era: an analysis of the surveillance, epidemiology and end results database. Am J Hematol 2013;89:310-314.

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Research Article

DOI: 10.4274/tjh.2014.0079 Turk J Hematol 2015;32:304-310

Effect of Tumor Necrosis Factor-Alpha on Erythropoietinand Erythropoietin Receptor-Induced Erythroid Progenitor Cell Proliferation in β-Thalassemia/Hemoglobin E Patients β-Talasemi/Hemoglobin E Hastalarında Tümör Nekrozlaştırıcı Faktör-Alfa’nın Eritropoetin- ve Eritropoetin Reseptör- ile Uyarılmış Eritroid Öncül Hücre Çoğalması Üzerine Etkisi Dalina I Tanyong1, Prapaporn Panichob1, Wasinee Kheansaard1, Suthat Fucharoen2 1Mahidol 2Mahidol

University Faculty of Medical Technology, Department of Clinical Microscopy, Nakhon Pathom, Thailand University Thalassemia Research Center, Institute of Molecular Biosciences, Nakhon Pathom, Thailand

Abstract: Objective: Thalassemia is one of the genetic diseases that cause anemia and ineffective erythropoiesis. Increased levels of several inflammatory cytokines have been reported in β-thalassemia and might contribute to ineffective erythropoiesis.

However, the mechanism by which tumor necrosis factor-alpha (TNF-α) is involved in ineffective erythropoiesis in thalassemic patients remains unclear. The objective of this study is to investigate the effect of TNF-α on the erythropoietin (EPO) and erythropoietin receptor (EPOR) expression involved in proliferation of β-thalassemia/hemoglobin (Hb) E erythroid progenitor cells compared with cells from healthy subjects.

Materials and Methods: CD34-positive cells were isolated from heparinized blood by using the EasySep® CD34 selection kit. Cells were then cultured with suitable culture medium in various concentrations of EPO for 14 days. The effect of TNF-α on percent cell viability was analyzed by trypan blue staining. In addition, the percentage of apoptosis and levels of EPOR protein were measured by flow cytometry.

Results: Upon EPO treatment, a higher cell number was observed for erythroid progenitor cells from both healthy participants and β-thalassemia/Hb E patients. However, a reduction of apoptosis was found in EPO-treated cells especially for β-thalassemia/ Hb E patients. Interestingly, TNF-α caused higher levels of cell apoptosis and lower levels of EPOR protein in thalassemic erythroid progenitor cells.

Conclusion: TNF-α caused a reduction in the level of EPOR protein and EPO-induced erythroid progenitor cell proliferation.

It is possible that TNF-α could be involved in the mechanism of ineffective erythropoiesis in β-thalassemia/Hb E patients.

Keywords: Erythropoietin, β-Thalassemia/hemoglobin E, Apoptosis

Address for Correspondence: Dalina I TANYONG, M.D., Mahidol University Faculty of Medical Technology, Department of Clinical Microscopy, Nakhon Pathom, Thailand E-mail: dalina.itc@mahidol.ac.th Received/Geliş tarihi : February 21, 2014 Accepted/Kabul tarihi : May 08, 2014

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Öz: Amaç: Talasemi anemi ve inefektif eritropoeze neden olan genetik hastalıklardan birisidir. Enflamatuvar sitokinlerin bir çoğunun seviyelerinde artma b-talasemide gösterilmiş olup, bu durum inefektif eritropoeze katkıda bulunabilir. Ancak, tümör nekrozlaştırıcı faktör-alfa’nın (TNF-α) talasemik hastalarda inefektif eritropoeze nasıl bir mekanizma ile neden olduğu bilinmemektedir. Bu çalışmanın amacı b-talasemi/hemoglobin (Hb) E eritroid öncül hücrelerinde sağlıklı kontrollerin hücreleri ile karşılaştırıldığında TNF-α’nın eritropoetin (EPO) ve eritropoetin reseptör (EPOR) sunumu üzerine etkisinin araştırılmasıdır.

Gereç ve Yöntemler: CD34-pozitif hücreler EasySep® CD34 seçim kiti yardımı ile heparinli kandan izole edildi. Hücreler 14 gün boyunca uygun kültür ortamında değişik EPO konsantrasyonlarında kültürde bekletildi. TNF-α’nın hücre canlılık yüzdesine etkisi tripan mavisi boyası ile incelendi. Bunun yanında, apopitoz yüzdesi ve EPOR protein seviyeleri akış sitometrisi ile ölçüldü.

Bulgular: EPO tedavisi ile eritroid öncül hücrelerinin sayısında hem sağlıklı katılımcılarda hem de b-talasemi/Hb E hastalarında artış olduğu görüldü. Ancak özellikle b-talasemi/Hb E hastalarında EPO ile muamele edilmiş hücrelerde apopitozda azalma görüldü. İlginç olarak, TNF-α talasemik eritroid öncül hücrelerde hücre apopitoz oranında artmaya ve EPOR protein seviyelerinde azalmaya neden oldu.

Sonuç: TNF-α EPOR protein düzeyi ve EPO ile uyarılmış eritroid öncül hücre çoğalmasında azalmaya neden oldu. b-talasemia/ Hb E hastalarında TNF-α inefektif eritropoez mekanizmasında yer alıyor olabilir.

Anahtar Sözcükler: Eritropoetin, β-Talasemi/hemoglobin E, Apopitoz Introduction Thalassemia is a genetic disease. The major pathophysiological features include ineffective erythropoiesis and anemia. In terms of ineffective erythropoiesis, the mechanism includes increased intramedullary erythroid death and arrested proliferation of erythroid progenitors, which plays an important role in β-thalassemia [1]. β-Thalassemia/ hemoglobin (Hb) E is the commonest form in many Asian countries. In Thailand, the World Health Organization estimates that at least 100,000 new cases of the disease will be seen in the next few decades. The pathophysiology is more complex and the cause of the variability of the severity remains unknown [2].

Erythropoietin (EPO) is a glycoprotein hormone required for the survival, proliferation, and differentiation of committed erythroid progenitor cells. The erythropoietin receptor (EPOR) belongs to the cytokine receptor superfamily, which includes receptors for other hematopoietic growth factors such as interleukins, colony-stimulating factors, and growth hormone. EPO binds to EPOR and causes the signaling pathways to control survival and proliferation of erythroid cells [3]. Survival signaling by EPOR is essential for erythropoiesis and for its acceleration in hypoxic stress.

Several apparently redundant EPOR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo [4].

One of the most important pathophysiologies of β-thalassemia is ineffective erythropoiesis. Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a) were

reported to inhibit erythropoiesis in vivo and vitro [5]. TNF-a induces an increase of apoptosis within the compartments of immature erythroblasts and a decrease in mature erythroblasts.

However, the exact mechanism remains unclear.

The objectives of this study were to study the effect of TNF-a on EPO and EPOR protein involved in proliferation of erythroid progenitor cells in β-thalassemia/Hb E patients.

Materials and Methods Blood Samples Heparinized blood samples were collected from 5 healthy subjects and 5 β-thalassemia/Hb E patients. The thalassemia patients in this study had the moderate to severe type of the disease. They were transfusion-dependent and splenectomized. However, patients had no transfusions or iron chelation at least 3 weeks before the time of sampling.

Diagnosis of thalassemia was based on family history, red cell indices, and hemoglobin typing. The procedures followed were in accord with the ethical standards established by the institution at which the experiments were performed or were in accord with the Helsinki Declaration of 1975.

Hematological Parameters and Erythropoietin Level Blood cells and red cell indices were analyzed with a Coulter counter (model ZX6). Hemoglobin typing was performed by automated high-performance liquid chromatography (Bio-Rad). EPO level was measured by enzyme-linked immunosorbent assay (ELISA).

Erythroid Progenitor Cell Culture and TNF-a Treatment CD34-positive cells (105 cells/mL) were isolated from peripheral blood mononuclear cells using the EasySep® CD34 selection kit, following the manufacturer’s instructions, 305

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and were cultured in Iscove’s modified Dulbecco’s medium (GIBCO) supplemented with 15% human AB serum, 15% fetal calf serum in the presence of 10 ng/mL recombinant interleukin-3, 20 ng/mL stem cell factor, and various concentrations of EPO (0, 0.2, 2, and 20 U/mL). For TNF-a treatment, cells were incubated with 20 ng/mL of TNF-α and incubated at 37 °C in 5% CO2 for 14 days. CD34-positive cells were checked by flow cytometry and erythroid progenitor cell development was observed by Wright-Giemsa staining.

Total Cell and Viability Assay by Trypan Blue Staining Trypan blue solution was used for cell viability assay.

To determine total cell count and cell viability, 20 µL of cell suspension was mixed with 20 µL of 0.4% trypan blue solution. Viable cells and number of total cells were counted by hemocytometer.

Detection of Percent Apoptosis of Erythroid Progenitor Cells Apoptosis was assessed by flow cytometry according to the manufacturer’s protocol. First, erythroid cultured cells were washed with 1 mL of cold D-PBS. After centrifugation at 12,000 rpm for 5 min, 100 µL of room-temperature 1X Annexin V binding buffer was added to the pellet. Next, 2 µL of Annexin V-FITC and 5 µL of glycophorin A-PE antibody were mixed into the cell suspension; this mixture was incubated for 15 min in the dark and then 100 µL of 1X Annexin V binding buffer was again mixed into the cell suspension. Finally, the cells were analyzed using a FAC Sort flow cytometer (BD Biosciences, USA). At least 10,000 cells were counted in order to determine the percentage of apoptosis.

Measuring Erythropoietin Receptor Protein by Flow Cytometry Erythroid progenitor cells were cultured for 14 days.

Cells were then incubated with anti-EPOR labeled with FITC and the percentage of EPOR protein was measured by flow cytometry.

Statistical Analysis Results are expressed as mean ± SD. Statistical analysis was performed using a nonparametric Kolmogorov-Smirnov test test and Student’s t-test. Significance was set at p<0.05.

Results Hematological Data and Level of Erythropoietin from Healthy Subjects and β-Thalassemia/Hemoglobin E Patients Hematological parameters are summarized in Table 1.

Serum EPO level was measured by ELISA and is shown in Figure 1. The level of serum EPO in β-thalassemia/Hb E cases was statistically significant higher than in healthy subjects.

Role of Erythropoietin on Cell Proliferation of Erythroid Progenitor Cells The effect of EPO on cell proliferation of erythroid progenitor cells was investigated by trypan blue staining. The results suggested that EPO increased the number of erythroid progenitor cells in a dose- and time-dependent manner. In addition, thalassemic patients had higher cell numbers than healthy subjects (Figure 2).

TNF-α Inhibits Erythropoietin-Induced Erythroid Cell Proliferation The effect of TNF-α on EPO-induced erythroid progenitor cell proliferation was studied. TNF-a caused a reduction of erythroid progenitor cells in both groups; however, this effect was stronger among β-thalassemia/Hb E patients (Figure 3).

Role of Erythropoietin on Apoptosis of Erythroid Progenitor Cells Various concentrations of EPO were added to erythroid progenitor cells and percent cell apoptosis was analyzed by flow cytometry. EPO caused a reduction of percent apoptosis in erythroid progenitor cells in a dose-dependent manner. β-Thalassemia/Hb E cells had a higher percentage of apoptosis than the cells of healthy subjects (Figure 4).

TNF-a Induced Apoptosis in Erythropoietin-Treated Cells The effect of TNF-a on induction of apoptosis of erythroid progenitor cells treated with EPO showed that percent apoptosis of TNF-a-treated cells was statistically significant higher than in the control in cells treated with both 2 U/mL and 20 U/mL EPO (Figure 5).

Table 1. Hematological parameters of healthy subjects and beta-thalassemia/hemoglobin E patients.

Sample

Age RBC (years) (x106/ µL)

Hb (g/dL)

Hct (%)

MCV (fL)

MCH (pg)

MCHC (pg)

RDW (%)

Healthy controls

36.4±4.5

4.7±0.2

13.1±0.7

40.2±2.3

88.9±3.1

29.1±0.8

32.7±0.4

13.2±0.7 7.4±2.8

269±62.0

β-Thal/ Hb E patients

22±2.1

4.5±0.6

8.2±1.4

25.1±4.0

59.3±3.9

17.9±1.3

30.7±1.3

24.7±1.4 7.9±1.0

350±111.0

Hb: Hemoglobin, Thal: thalassemia.

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Role of Erythropoietin on Erythropoietin Receptor Protein of Erythroid Progenitor Cells

TNF-α Inhibits Erythropoietin Receptor Protein of Erythroid Progenitor Cells

The level of EPOR protein was measured by flow cytometry and the results showed that the level of EPOR in erythroid progenitor cells from β-thalassemia/Hb E patients was lower than in those from healthy subjects. The highest EPOR protein level was shown in EPO-treated erythroid cells from healthy subjects at day 5 of culture (Figure 6).

After adding TNF-a to erythroid progenitor cells treated with EPO, lower levels of EPOR protein were seen in erythroid progenitor cells from both healthy subjects and β-thalassemia/ Hb E patients (Figure 7).

Figure 1. Serum erythropoietin levels of healthy control subjects and beta thalassemia/hemoglobin E patients. *: p<0.05 compared with healthy subjects.

Figure 3. Effect of TNF-a on cell count of erythroid progenitor cells treated with 2 U EPO (a) and 20 U (c) of healthy and 2 U (b) and 20 U (d) of b-thalassemia/Hb E.

a

b

Figure 2. Cell count of erythroid progenitor cells from healthy control subjects a) and β-thalassemia/hemoglobin E patients b) after treatment with various concentrations of erythropoietin for 14 days.

Figure 4. Percent cell apoptosis of erythroid progenitor cells from healthy control subjects a) and β-thalassemia/hemoglobin E patients b) after treatment with various concentrations of erythropoietin for 14 days as analyzed by flow cytometry. *: p<0.05 compared with day 7.

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Discussion β-Thalassemia/Hb E is a thalassemic syndrome that results from co-inheritance of the hemoglobin E trait with either β0 or β+ thalassemia. The severity of the disease is very variable, ranging from minor through intermediate to major.

Many studies have tried to explain the severity based on pathophysiological factors such as ineffective erythropoiesis.

Ineffective erythropoiesis is characterized by apoptosis of the erythroid progenitor cells [6]. Many proteins have the potential to affect erythroid proliferation and differentiation.

Interestingly, the level of serum EPO in β-thalassemia/Hb E patients was higher than normal. A previous study reported that cells become progressively more sensitive to EPO during erythroid differentiation due to the appearance of EPOR [7].

In this study, the highest EPOR protein levels were seen at day 5 of culture in erythroid progenitor cells from healthy subjects; the majority of cells were pronormoblasts. In addition, EPOR

Figure 5. Effect of tumor necrosis factor-alpha on percent cell apoptosis of erythroid progenitor cells from healthy control subjects (a) and β-thalassemia/hemoglobin E patients (b) after treatment with 2 U and 20 U erythropoietin for 14 days as analyzed by flow cytometry. *: p<0.05 compared with the control.

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protein levels in thalassemic patients were lower than in healthy subjects. The level of EPOR might be associated with the stage of erythroid cells. There are reports on the relation of EPO and EPOR expression in other cells, such as endothelial cells and head and neck squamous cell carcinoma [8]. In this study, a reduction of percent cell apoptosis was found in EPOtreated cells. The percent apoptosis of thalassemic patients was higher than that of healthy subjects, which might be related to ineffective erythropoiesis in β-thalassemia/Hb E patients.

Recent studies reported that cytokines could be involved with ineffective erythropoiesis in β-thalassemia. A previous study by our group showed that cytokines, including TNF-α and interferon-γ, had the potential to induce nitric oxide, involved with apoptosis of erythroid progenitor cells from β-thalassemia/Hb E patients [9]. However, the mechanism of TNF-α involved in EPO regulation remains unclear. TNF-α is one of the proinflammatory cytokines that reportedly inhibit generation of glycophorin A+ cells [10], and decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in b-thalassemia [11]. In addition, the serum level of TNF-α was statistical significantly higher in postsplenectomized thalassemic patients than in normal controls and nonsplenectomized patients, which indicated that TNF-α could play a role in the pathogenesis of the disease [12]. One previous study reported that the TNF-α levels of

Figure 6. Erythropoietin receptor protein of erythroid progenitor cells from healthy control subjects and β-thalassemia/ hemoglobin E patients after treatment with 2 U (a) and 20 U (b) erythropoietin for various times as measured by flow cytometry. *: p<0.05 compared with the healthy control.

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b-thalassemia/Hb E patients were higher than normal in only 13% of the patients [13]. However, many studies have shown an increased TNF-α concentration in b-thalassemia major patients [12,14]. It was suggested that the increase in TNF-α could be caused by macrophage activation due to iron overload and the antigenic stimulation induced by chronic transfusion therapy. The activated macrophages were selectively phagocytosing apoptotic erythroid precursors, thereby contributing to ineffective erythropoiesis [15]. In this study it was demonstrated that TNF-α caused higher levels of apoptosis in b-thalassemia/Hb E erythroid progenitor cells compared to cells from the control group. In addition, EPOR protein in erythroid progenitor cells was inhibited by this cytokine. This suggests that TNF-α caused a reduction of both EPOR protein expression and EPO-induced cell proliferation of thalassemic erythroid progenitor cells, which could be involved in the mechanism of ineffective erythropoiesis in b-thalassemia/Hb E patients.

Acknowledgments This work was supported by the Thailand Research Fund (Grant No. MRG5180127) and by a Mahidol University Research Grant.

Ethics Committee Approval: COA No. MU-IRB 2009/252.2910, Informed Consent: It was taken, Concept: Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard, Suthat Fucharoen, Design: Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard, Suthat Fucharoen, Data Collection or Processing: Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard, Suthat Fucharoen, Analysis or Interpretation: Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard, Suthat Fucharoen, Literature Search: Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard, Suthat Fucharoen, Writing: Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard, Suthat Fucharoen.

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

References 1. Round D, Rachmilewitz E. Beta-thalassemia. N Engl J Med 2005;353:1135-1146.

2. Gibbons R, Higgs DR, Olivieri NF, Wood WG. The β and δβ thalassemia in association with structural hemoglobin variants. In: Weatherall DJ, Clegg JB (eds). The Thalassemia Syndromes. 4th ed. Oxford, Blackwell Science, 2001.

3. Lappin TR, Maxwell AP, Johnston PG. EPO’s alter ego: erythropoietin has multiple actions. Stem Cells 2002;20:485492.

4. Koulnis M, Porpiqlia E, Porpiqlia PA, Liu Y, Hallstrom K, Hidalgo D, Socolovsky M. Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways.

Blood 2012;119:1228-1239.

5. Ribeil JA, Arlet JB, Dussiot M, Moura IC, Courtois G, Hermine O. Ineffective erythropoiesis in β-thalassemia. Scientific World Journal 2013;2013:394295.

6. Rivella S. Ineffective erythropoiesis and thalassemia. Curr Opin Hematol 2009;16:187-194.

Figure 7. Effect of tumor necrosis factor-alpha on erythropoietin receptor protein levels of erythroid progenitor cells from healthy control subjects (a) and β-thalassemia/hemoglobin E patients (b) after treatment with 2 U and 20 U on day 5 of culture as measured by flow cytometry.

7. Trincavelli ML, Da Pozzo E, Ciampi O, Cuboni S, Daniele S, Abbracchio MP, Martini C. Regulation of erythropoietin receptor activity in endothelial cells by different erythropoietin (EPO) derivatives: an in vitro study. Int J Mol Sci 2013;14:2258-2281.

8. Winter SC, Shah KA, Campo L, Turley H, Leek R, Corbridge RJ, Cox JG, Harris AL. Relation of erythropoietin and

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9. Kheansaard W, Panichob P, Fucharoen S, Tanyong DI. Cytokine-induced apoptosis of beta-thalassemia/hemoglobin E erythroid progenitor cells via nitric oxide-mediated process in vitro. Acta Haematol 2011:126;224-230.

10. Xiao W, Koizumi K, Nishio M, Endo T, Osawa M, Fujimoto K, Sato I, Sakai T, Koike T, Sawada KI. Tumor necrosis factor-α inhibits generation of glycophorin A+ cell by CD34+ cells.

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11. Libani IV, Guy EC, Melchiori L, Schiro R, Ramos P, Breda L, Scholzen T, Chadburn A, Liu Y, Kernbach M, Baron-Lühr B, Porotto M, de Sousa M, Rachmilewitz EA, Hood JD, Cappellini MD, Giardina PJ, Grady RW, Gerdes J, Rivella S. Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in β-thalassemia. Blood 2008;112:875-885.

12. Chuncharunee S, Archararit N, Hathirat P, Udomsubpayakul U, Atchatakam V. Level of serum interleukin 6 and tumor necrosis factor in postsplenectomized thalassemic patients. J Med Assoc Thai 1997;80(Suppl 1):86-91.

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Research Article

DOI: 10.4274/tjh.2014.0126 Turk J Hematol 2015;32:311-316

The -137G/C Polymorphism in Interleukin-18 Gene Promoter Contributes to Chronic Lymphocytic and Chronic Myelogenous Leukemia Risk in Turkish Patients İnterlökin 18 Geninin Promotör Bölgesindeki -137G/C Polimorfizmi Türk Popülasyonunda Kronik Lenfositik ve Kronik Miyeloid Lösemi Riskini Arttırmaktadır Serap Yalçın1, Pelin Mutlu2, Türker Çetin3, Meral Sarper4, Gökhan Özgür3, Ferit Avcu3,4 1Ahi

Evran University Faculty of Engineering and Architecture, Kırşehir, Turkey East Technical University, Central Laboratory, Department of Molecular Biology and Biotechnology, Ankara, Turkey 3Gülhane Military Medical Academy, Department of Hematology, Ankara, Turkey 4Gülhane Military Medical Academy, Cancer and Stem Cell Research Center, Ankara, Turkey 2Middle

Abstract: Objective: Interleukin-18 (IL-18) is a cytokine that belongs to the IL-1 superfamily and is secreted by various immune and

nonimmune cells. Evidence has shown that IL-18 has both anticancer and procancer effects. The aim of this study was to evaluate the relationship between IL-18 gene polymorphisms and susceptibility to chronic lymphocytic leukemias (CLL) and chronic myelogenous leukemias (CML) in Turkish patients.

Materials and Methods: The frequencies of polymorphisms (rs61667799(G/T), rs5744227(C/G), rs5744228(A/G), and rs187238(G/C)) were studied in 20 CLL patients, 30 CML patients, and 30 healthy individuals. The genotyping was performed by polymerase chain reaction and DNA sequencing analysis.

Results: Significant associations were detected between the IL-18 rs187238(G/C) polymorphism and chronic leukemia. A higher prevalence of the C allele was found in CML cases with respect to controls. The GC heterozygous and CC homozygous genotypes were associated with risk of CML when compared with controls. However, prevalence of the C allele was not significantly high in CLL cases with respect to controls. There was only a significant difference between the homozygous CC genotype of CLL patients and the control group; thus, it can be concluded that the CC genotype may be associated with the risk of CLL. Based on our data, there were no significant associations between the IL-18 rs61667799(G/T), rs5744227(C/G), or rs5744228(A/G) polymorphisms and CLL or CML. Conclusions: IL-18 gene promoter rs187238(G/C) polymorphism is associated with chronic leukemia in the Turkish population. However, due to the limited number of studied patients, these are preliminary results that show the association between -137G/C polymorphism and patients (CLL and CML). Further large-scale studies combined with haplotype and expression analysis are required to validate the current findings.

Keywords: IL-18, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Single nucleotide polymorphisms

Address for Correspondence: Serap YALÇIN, PhD., Ahi Evran University Faculty of Engineering and Architecture, Kırşehir, Turkey Phone: +90 386 280 38 08 E-mail: syalcin@ahievran.edu.tr Received/Geliş tarihi : March 23, 2014 Accepted/Kabul tarihi : August 12, 2014

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Yalçın S, et al: IL-18 Polymorphisms in CML and CLL Patients

Turk J Hematol 2015;32:311-316

Öz: Amaç: İnterlökin-18 (İL-18), İL-1 süper ailesine ait bir sitokin olup, bağışıklık sistemine ait olan ve olmayan çeşitli hücrelerden salınmaktadır. Yapılan çalışmalar, İL-18’in hem anti-kanser hem de kansere öncülük eden etkilere sahip olduğunu göstermiştir. Bu çalışmanın amacı, kronik lenfositik lösemili (KLL) ve kronik miyeloid lösemili (KML) Türk hastalarda İL-18 gen polimorfizmleri ilişkisini değerlendirmektir.

Gereç ve Yöntemler: İL-18 polimorfizleri (rs61667799(G/T), rs5744227(C/G), rs5744228(A/G) ve rs187238(G/C)), 20 KLL ve 30 KML hasta ve 30 sağlıklı bireyde araştırılmıştır. Genotipleme, polimeraz zincir reaksiyonu ve DNA dizi analizi ile gerçekleştirilmiştir.

Bulgular: İL-18 geninde, rs187238(G/C) polimorfizmi ile kronik lösemi arasında anlamlı bir ilişki belirlenmiştir. KML hastalarında kontrol grubuna göre, C allelinin daha yüksek olduğu bulunmuştur. Kontroller ile karşılaştırıldığında, GC heterozigot ve CC homozigot genotipleri KML hastalarında risk oluşturmaktadır. Ancak, C alleli sıklığı kontrollere göre KLL olgularında istatistiksel olarak anlamlı değildir. KLL hastaları ve kontrol grubunun homozigot CC genotipi arasında anlamlı farklılık vardır ve bunun sonucu olarak CC genotipi, KLL hastaları için risk taşımaktadır denilebilir. Verilerimize dayanarak, KLL ve KML hastalarında, İL-18 geninde rs61667799(G/T), rs5744227(C/G) ve rs5744228(A/G) polimorfizmleri arasında anlamlı bir ilişki yoktur.

Sonuç: İL-18 geninin promotor bölgesindeki rs187238(G/C) polimorfizmi Türk popülasyonunda kronik lösemi ile ilişkilidir.

Ancak, yapılan bu çalışma, hasta sayısının sınırlı olması nedeniyle, -137G/C polimorfizmi ve hastalar (KLL ve KML) arasındaki ilişkiyi gösteren bir ön çalışma niteliğindedir. Mevcut bulguları doğrulamak için, haplotip ve gen ifade düzeyi analizleri ile birleştirilmiş daha geniş çaplı çalışmalara ihtiyaç vardır.

Anahtar Sözcükler: İL-18, Kronik lenfositik lösemi, Kronik miyeloid lösemi, Tek nükleotid polimorfizmi Introduction Interleukin-18 (IL-18) is a member of the IL-1 cytokine family [1]. It is secreted by various immune and nonimmune cells including T and B lymphocytes, activated monocytes, macrophages, Kupffer cells, natural killer cells, and Langerhans cells [2,3,4]. Evidence has shown that IL-18 has both anticancer and procancer effects [5]. IL-18 can stimulate natural killer cells and T cells promoting primarily Th1 responses, resulting in the elimination of tumor cells [6,7,8,9].

On the other hand, it has been reported that IL-18 is able to induce angiogenesis, migration, proliferation, and immune escape of tumor cells [10]. In models of hepatic melanoma metastasis the IL-18 blockade reduces the adherence of malignant cells by preventing IL-18 upregulation of vascular endothelial adhesion-1 molecule expression [11]. Higher expression levels of IL-18 are detected in different cancer types, such as gastric and breast cancer [12,13]. These results suggest that there is an association between the IL-18 gene and cancer risk, but this still remains controversial.

The IL-18 gene is located on chromosome 11q22.2-q22.23.

A number of single nucleotide polymorphisms (SNPs) have been identified and investigated [14]. The IL-18 gene promoter -137G/C (rs187238) polymorphism is one of the most common SNPs, relative to the transcriptional start site, which may alter the expression of IL-18. This polymorphism can change the binding site of histone 4 transcription factor-1 nuclear factor and can have an impact on IL-18 gene activity [15].

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Chronic myelogenous leukemia (CML) is a clonal bone marrow stem cell disorder characterized by the unregulated growth of mature granulocytes in the bone marrow and their accumulation in the blood [16]. The formation of the BCR-ABL fusion protein, which activates tyrosine kinase, plays a central role in the pathogenesis of CML [17]. Chronic lymphocytic leukemia (CLL) is the most common type of leukemia. CLL affects B cell lymphocytes that originate in the bone marrow, develop in the lymph nodes, and normally fight infection by producing antibodies [18].

It has been reported that malignant proliferation of leukemic cells is supported by a cytokine network surrounding these cells, produced partially by the cells themselves [19]. Elevated levels of IL-18 were observed in some leukemia patients, especially those with acute lymphoblastic leukemia and CML [20]. On the other hand, IL-18 receptor expression was reported mostly from CD19+ B cells and some CD8+ T cells [21].

The aim of this study is to evaluate the frequency of IL18 gene promoter polymorphisms in Turkish CLL and CML patient groups and compare them with a control group in order to verify a correlation between the allelic variations and the risk of CML and CLL. Materials and Methods Subjects Twenty unrelated CLL patients and 30 unrelated CML patients diagnosed clinically at the Gülhane Military Medical

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Academy Department of Hematology and a control group of 30 unrelated healthy volunteers were randomly selected from different geographic regions of Turkey. The study protocol was approved by the local ethics committee of Gülhane Military Medical Academy and was conducted in accordance with the guidelines of the Declaration of Helsinki.

Genotyping The SNP in the promoter region of the IL-18 gene (SNP *g/c......rs187238; *g/t..... rs61667799; *c/g.......rs5744227; *a/g....rs5744228) was sequenced was determined by sequencing method. Genomic DNA was isolated from the peripheral blood by standard phenol-chloroform procedure.

The genotyping of polymorphisms was performed by polymerase chain reaction and DNA sequencing analysis.

A 446-bp fragment was amplified using specific primers (forward: 5’-CCAATAGGACTGATTATTCCGCA-3’ and reverse: 5’-AGGAGGGCAAAATGCACTGG-3’). Amplification was carried out on a Bio-RAD PCR system in 50 µL of reaction mixture containing 10 mM dNTPs, 25 mM magnesium chloride, 5 pmol each of forward and reverse primers, 2.5 U of Taq DNA polymerase, 10X PCR buffer, and 50 ng of genomic DNA. The PCR cycling conditions consisted of an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min, with a final extension step at 72 °C for 5 min. PCR products of 446 bp were then separated by 2% agarose gel electrophoresis at 120 V, stained by ethidium bromide (0.5 µg/mL), and visualized under a UV transilluminator. Single-pass sequencing was

performed on each template using the forward primer. Cycle sequencing was carried out using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, USA) according to the manufacturer’s instructions. The fluorescencelabeled fragments were purified by sodium acetate-ethanol precipitation method. Samples were then resuspended in distilled water and subjected to electrophoresis in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

Statistical Analysis SPSS 16.0 (SPSS Inc., USA) was used for the statistical analysis. Allele and genotype frequencies of alleles and genotypes were obtained by direct count. Statistical significance was defined as p<0.05.

Results Genotypes of the CML and CLL patients and the controls were determined by using DNA sequencing methodology for promoter polymorphisms. Figure 1 shows the chromatograms, which contain DNA fragments representing homozygous wild-type, heterozygous, and homozygous mutant genotypes (rs187238 G/C). The genotype and allele distributions of the controls versus CML and CLL patients are given in Tables 1 and 2, respectively.

Among the control group subjects, 77% were found to be homozygous for the GG genotype and 23% were heterozygous.

There were no subjects with the CC genotype. The G allele frequency was 88% and C allele frequency was 12%. Among the CLL patients, 70% were found to be homozygous for the

Figure 1. Representative chromatograms of sequenced PCR products, 1: homozygous mutant genotype (CC), 2: homozygous wild genotype (GG), 3: heterozygous genotype (GC).

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Yal癟覺n S, et al: IL-18 Polymorphisms in CML and CLL Patients

Table 1. Genotype and allele frequencies of the -137G/C polymorphism in the control and chronic myelogenous leukemia groups.

Genotype

Control (n=30)

CML (n=30)

p-value

OR (95% CI)

GG

23 (77%)

15 (50%)

0.037

0.3043 (0.1005-0.9218)

GC

7 (23%)

11 (37%)

1.9023 (0.6171-5.8636)

CC

0 (0%)

4 (13%)

10.3585 (0.5326-201.4622)

G

53 (88%)

41 (68%)

C

7 (12%)

19 (32%)

Allele 0.032

_

CML: Chronic myelogenous leukemia.

Table 2. Genotype and allele frequencies of the -137G/C polymorphism in the control and chronic lymphocytic leukemia groups.

Genotype

Control (n=30)

CLL (n=20)

GG

23 (77%)

14 (70%)

GC

7 (23%)

2 (10%)

CC

0 (0%)

4 (20%)

G

53 (88%)

30 (75%)

C

7 (12%)

10 (25%)

p-value

OR (95% CI) 0.7101 (0.1981-2.5462)

0.027

0.3651 (0.0675-1.9750) 16.6364 (0.8428-328.3734)

Allele 0.599

_

CLL: Chronic lymphocytic leukemia.

GG genotype, 10% were heterozygous (GC), and 20% were homozygous for the CC genotype. The G allele frequency was found as 75% whereas C allele frequency was 25%. Among the CML patients, 50% were found to be homozygous for the GG genotype, 37% were heterozygous (GC), and 13% were homozygous for the CC genotype. The G allele frequency was found as 68% whereas C allele frequency was 32%.

A higher prevalence of the C allele was found in CML patients with respect to the controls (p<0.05; Table 1). The GC heterozygous and CC homozygous genotypes were associated with the risk of CML when compared with the controls (OR: 1.9023; 95% CI: 0.6171-5.8636 for GC genotype and OR: 10.3585; 95% CI: 0.5326-201.4622 for CC genotype). These results indicate that individuals that are either heterozygous (GC) or homozygous for the CC genotype are associated with the development of CML in this Turkish population. However, prevalence of the C allele was not significantly high in CLL patients with respect to controls (p>0.05). There was only a significant difference between the homozygous CC genotype of CLL patients and the control group (p<0.05; Table 2). The CC homozygous genotype is associated with the risk of CLL when compared with controls (OR: 16.6364; 95% CI: 0.8428328.3734). This indicates that homozygosity for the CC genotype was associated with the development of CLL in the

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studied patient group. There were no significant associations between the IL-18 rs61667799(G/T), rs5744227(C/G), or rs5744228(A/G) polymorphisms and CLL or CML. Discussion The -137G/C (rs187238) SNP in the promoter region of the IL-18 gene has a confirmed impact on gene activity and expression in tissues [15,22]. Some previous studies have suggested that IL-18 might act as a protumor factor in the progress of several tumors. The IL-18 protein has been shown to be overexpressed expressed in common skin tumors [1,23].

On the other hand, higher IL-18 levels both in the tumor region and in the serum were detected in metastatic gastric and breast cancer [12,13,24]. Furthermore, linkage between -137G/C and -607C/A polymorphisms of the IL-18 gene and progression of ovarian cancer and nasopharyngeal carcinoma was reported [25,26].

Based on 21 different studies, carriers of the variant C allele for -137G/C polymorphism were only reported to have a significant increased cancer risk compared with carriers of the G allele in nasopharyngeal carcinoma [27]. In the dominant CC genotype, some single studies suggested that the -137G/C polymorphism contributed to the susceptibility to certain cancer types, such as cervical [28], prostate [29], bladder [30], esophageal [31], and colorectal [32]. There are also

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Yalçın S, et al: IL-18 Polymorphisms in CML and CLL Patients

several studies that concluded that there was no significant association between cancer and the -137G/C polymorphism [5,33,34]. Moreover, in one study, Monroy et al. found a significantly reduced cancer risk with the GC/CC genotype in Hodgkin disease [35].

These discrepant conclusions might be explained by ethnic differences since the studies that reported increased cancer risk were almost all carried out in Asians. On the contrary, a trend of reduced cancer risk was found in Caucasians [27].

To our knowledge, there are not many reports describing a comprehensive relation between -137G/C polymorphism and susceptibility to CML and CLL. In the present study, potential influence of the -137G/C polymorphism on both CLL and CML susceptibility was considered in a Turkish population. Our results showed a significantly increased risk in heterozygous (GC) and homozygous (CC) genotypes for CML. On the other hand, only the homozygous (CC) genotype is associated with the risk of CLL when compared with the controls. The results of this study may be important since there are not many reports showing the association of the -137G/C polymorphism with the risk of CML and CLL. However, there are several studies that showed dysregulated expression of IL-18 and/or IL-18 receptor in chronic B-cell lymphoproliferative disorders [36,37]. The dysregulated expression of IL-18 may be due to IL-18 gene promoter polymorphisms such as -137G/C. In addition, for this study, several limitations should be considered. First, the CML and CLL patient numbers were small. Second, haplotype analysis linking other IL-18 polymorphisms to IL-18 expression level may be necessary.

In conclusion, we demonstrate that IL-18 gene promoter -137G/C polymorphism is associated with CLL and CML in a Turkish population. However, due to the limited number of studied patients, these are only preliminary results that show the association between the -137G/C polymorphism and CLL and CML. Further large-scale studies combined with haplotype and expression analysis are required to validate the current findings.

Ethics Committee Approval: Ethics No: 1491-43-12/16484451(GATA) Date: 06/June/2012, Concept: Serap Yalçın, Pelin Mutlu, Ferit Avcu, Design: Serap Yalçın, Pelin Mutlu, Ferit Avcu, Data Collection or Processing: Türker Çetin, Meral Sarper, Gökhan Özgür, Analysis or Interpretation: Serap Yalçın, Pelin Mutlu, Literature Search: Serap Yalçın, Pelin Mutlu, Ferit Avcu, Writing: Serap Yalçın, Pelin Mutlu, Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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Research Article

DOI: 10.4274/tjh.2014.0154 Turk J Hematol 2015;32:317-322

Transcobalamin II Deficiency in Four Cases with Novel Mutations Yeni Mutasyonu Olan Dört Transkobalamin II Eksikliği Olgusu Şule Ünal1, Tony Rupar2, Sevgi Yetgin1, Neşe Yaralı3, Ali Dursun4, Türkiz Gürsel5, Mualla Çetin1 1Hacettepe

University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey Hospital, London Health Sciences Centre, Biochemical Genetics Laboratory, London, Canada 3Ankara Children’s Hematology and Oncology Hospital, Clinic of Pediatric Hematology, Ankara, Turkey 4Hacettepe University Faculty of Medicine, Division of Metabolism and Nutrition, Ankara, Turkey 5Gazi University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey 2Victoria

Abstract: Objective: Transcobalamin II deficiency is one of the rare causes of inherited vitamin B12 disorders in which the patients have characteristically normal or high vitamin B12 levels related to the transport defect of vitamin B12 into the cell, ending up with intracellular cobalamin depletion and high homocysteine and methylmalonic acid levels.

Materials and Methods: Herein, we describe the findings at presentation of four patients who were diagnosed to have transcobalamin II deficiency with novel mutations.

Results: These patients with transcobalamin II deficiency were found to have novel mutations, of whom 2 had the same large deletion (homozygous c.1106+1516-1222+1231del).

Conclusion: Transcobalamin II deficiency should be considered in differential diagnosis of any infant with pancytopenia, failure to thrive, diarrhea, and vomiting.

Keywords: Vitamin B12, Transcobalamin II, Novel mutation, Novel deletion, Vacuolization

Öz: Amaç: Transkobalamin II eksikliği nadir bir kalıtsal B12 vitamini bozukluğudur. Defektin B12 vitamininin transportu ile

ilgili olması nedeniyle hastalar normal ya da yüksek B12 vitamini düzeylerine eşlik eden yüksek homosistein ve metilmalonik asit düzeylerine sahiptir.

Gereç ve Yöntemler: Bu çalışmada transkobalamin II eksikliği tanısı alan dört hasta sunulmuştur. Bu hastalarda daha önce bildirilmemiş yeni mutasyonlar saptanmıştır.

Bulgular: Hastaların ikisinde aynı büyük delesyon olduğu görülmüştür (homozigot c.1106+1516-1222+1231del).

Sonuç: Pansitopeni, büyüme geriliği, ishal ya da kusması olan tüm bebeklerde transcobalamin II eksikliği ayırıcı tanıda düşünülmelidir.

Anahtar Sözcükler: B12 vitamini, Transkobalamin II, Yeni mutasyon, Yeni delesyon, Vaküolizasyon

Address for Correspondence: Şule ÜNAL, M.D., Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey Phone: +90 312 305 11 70 E-mail: suleunal@hacettepe.edu.tr Received/Geliş tarihi : April 13, 2014 Accepted/Kabul tarihi : August 18, 2014

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Ünal Ş, et al: TCII Deficiency with Novel Mutations

Introduction Among the pancytopenia etiologies during infancy, the acquired vitamin B12 deficiency in exclusively breast-fed infants of strictly vegan mothers and inherited vitamin B12 deficiency related to transcobalamin II deficiency should be considered, since the treatment of both conditions is easy and possibly life-saving [1,2]. About 30% of plasma cobalamin is bound to transcobalamin II while the remaining part is bound to haptocorrin, but only the part of circulating cobalamin attached to transcobalamin II is the biologically active form and transcobalamin II mediates the entry of cobalamin into a variety of cell types other than hepatocytes [3,4,5].

Transcobalamin II deficiency is a rare autosomal recessive disorder causing intracellular cobalamin depletion, which in turn causes megaloblastic bone marrow failure, accumulation of homocysteine and methylmalonic acid with clinical findings of failure to thrive, diarrhea, vomiting, pancytopenia, megaloblastic anemia, and neurological findings [2].

Homozygous or compound heterozygous mutations in the transcobalamin II gene on chromosome 22q12.2 that contains 9 coding exons are known to cause transcobalamin II deficiency, including deletions, nonsense mutations, and a mutation resulting in activation of a cryptic intronic splice site [6,7,8,9,10,11,12].

Herein, we describe the clinical findings at presentation and outcome of 4 patients with genetically confirmed novel transcobalamin II gene mutations, of whom 3 had large deletions of 1 kb and 1 had a homozygous Q36X mutation.

Materials and Methods The clinical and laboratory findings of the patients at presentation are summarized in Table 1. The patients were further investigated for molecular diagnosis.

Results Case 1 A 2-month-old girl from the southeastern part of Turkey presented with failure to thrive (birth weight unknown; 2-month-old weight in 10th percentile, length in 25th percentile, head circumference in 3rd to 10th percentiles), irritability, and diarrhea for the last 20 days and was found to have pallor, petechial rash, and no head control upon physical examination. She was the 6th child of first-degree cousins from the 8th gestation, and family history revealed that a sister of hers had died at 1 year of age with diarrhea and vomiting and a brother had died at 3.5 months with bleeding. Liver and renal function tests were unrevealing. Urinalysis revealed absence of proteinuria. Bone marrow aspiration indicated megaloblastic changes in the erythroid and myeloid lineages and vacuolization in the myeloid lineage. Serum vitamin B12 level was found

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to be 351 pg/mL (normal range: 200-860); however, serum homocysteine was 40 µmol/L (normal: 5.5-17) and urinary methylmalonic acid level was twice the normal value. She was given erythrocyte and platelet transfusions on the first day of admission and intramuscular hydroxocobalamin was initiated at 1000 µg/day with a possible diagnosis of transcobalamin II deficiency. The hemogram findings on the day of vitamin B12 treatment initiation were as follows; RBC: 2.6x1012/L, Hb: 7.4 g/dL, Hct: 21.3%, MCV: 80 fL, WBC: 3.8x109/L, platelets: 61x109/L, absolute neutrophil count (ANC): 0.3x109/L, and absolute lymphocyte count (ALC): 3.4x109/L. By the 6th day of admission the diarrhea subsided and on the 10th day of admission the hemogram results improved to Hb: 8.9 g/ dL, Hct: 24.4%, MCV: 78.5 Fl, WBC: 33.2x109/L, platelets: 125x109/L, and ANC: 22.3x109/L. Leukocytosis developed in the absence of an infection after the initiation of vitamin B12 treatment and subsided to the normal range in 2 weeks.

Hydroxocobalamin dosage was continued intramuscularly on alternating days for the 2nd week and weekly after the 3rd week. Folic acid at 1 mg orally was added to the treatment.

Molecular analyses revealed c.1106+1516-1222+1231del in a homozygous state, which was a deletion of 5304 bp beginning 1516 bp into intron 7 and ending 1231 bp into intron 8, causing deletion of all of exon 8 and a frameshift to produce a premature stop 4 codons into the new reading frame. During the follow-up, the family was learned to have attempted to cease the treatment by their own intention and the patient had similar attacks of pancytopenia and diarrhea. Both attacks resolved after reinitiation of hydroxocobalamin. The patient was also detected to have β-thalassemia trait (HbA2 6%) during outpatient visits due to MCV values as low as 65.2 fL after initiation of vitamin B12 in the absence of iron deficiency.

She is currently alive and asymptomatic at 4 years of age.

Case 2 A 28-day-old boy from the 1st gestation of a couple of firstdegree cousins presented with failure to thrive, poor feeding, and vomiting. He was from the Central Anatolia region of Turkey. Hemogram results revealed pancytopenia. Antibiotic treatment was started empirically. He received transfusions several times, and the bone marrow examination was remarkable for megaloblastic changes and vacuolization in bone marrow precursors. Serum vitamin B12 was 623 pg/mL (normal: 200-860). Cyanocobalamin (1000 µg) was initiated intramuscularly with a possible diagnosis of transcobalamin II deficiency. Signs and symptoms declined after cyanocobalamin initiation. Folic acid was added to the vitamin B12 treatment.

The control for bone marrow aspiration after vitamin B12 initiation revealed the disappearance of megaloblastic changes and vacuolization in the myeloid lineage. The molecular analyses was ordered and revealed c.1107-347_1222+981del in 364. This complex mutation appears to be a 1444-bp deletion

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Table 1. Clinical and laboratory findings of patients at presentation.

Case 1

Case 2

Case 3

Case 4

Age, sex

2 months, Female

28 days, Male

2 months, Female

3 months, Male

Symptoms at presentation

Failure to thrive, irritability, diarrhea

Failure to thrive, vomiting, poor feeding

Diarrhea, vomiting, Failure to thrive, fever poor feeding

Hb (g/dL)

4.8

9.5

4.3

6.5

Hct (%)

13.3

26.6

12.8

18

RBC (x1012/L)

1.5

2.44

NA

NA

WBC (x109/L)

3.6

2.1

4.7

3.2

MCV (fL)

88

109

93.3

NA

Platelets (x109/L)

8

9

11

30

ANC (x109/L)

0.79

0.08

0.95

NA

ALC (x109/L)

2.5

1.67

4.0

NA

Vitamin B12 (normal: 200-860 pg/mL)

351

623

Normal

677

Homocysteine (normal: 5.5-17 µmol/L)

40

NA

NA

46

Spot urinary MMA analysis

Twice normal

NA

High

NA

Bone marrow examination

Megaloblastic changes in myeloid and erythroid lineages, vacuolization in myeloid lineage

Megaloblastic changes and vacuolization in bone marrow precursors

Megaloblastic changes in bone marrow precursors

Megaloblastic changes in myeloid lineage

Genetic analyses

Homozygous c.1106+15161222+1231del

c.1107Homozygous 347_1222+981delin 364; c.106C>T. (Q36X) this complex mutation appears to be a 1444-bp deletion that includes exon 8 and a 364-bp insertion

Homozygous c.1106+15161222+1231del

Treatment regimen currently stable with

Hydroxocobalamin, 1000 µg, im, weekly Folic acid, oral, 1 mg

Cyanocobalamin, 1000 µg, im, weekly Folic acid, oral, 1 mg

Cyanocobalamin, 1000 µg, im, weekly

Cyanocobalamin, 1000 µg, im, weekly Folic acid, orally, 1 mg

ANC: Absolute neutrophil count, ALC: absolute lymphocyte count, NA: not available, MMA: methylmalonic acid, im: intramuscular.

that includes exon 8. There was also a 364-bp insertion. He is currently alive at 6.5 years of age under weekly intramuscular cyanocobalamin.

Case 3 A 2-month-old girl, from the 1st gestation of a couple of first-degree cousins, presented with diarrhea, vomiting, and

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fever for 1 week. Body weight and length were below the 3rd percentile for age. Hemogram results revealed pancytopenia.

Bone marrow examination revealed megaloblastic changes.

Sweat test by pilocarpine iontophoresis was ordered for the diarrhea and results were positive, with a sweat chloride of 87 mEq/L. Molecular testing for cystic fibrosis for the common 21 mutations in Turkey was negative. Serum vitamin B12 was within normal laboratory limits, whereas urinary methylmalonic acid level was 16.45 mmol/mol creatinine (normal: 0-10). Molecular study revealed homozygous c.106C>T p.Q36X. A C-to-T substitution at nucleotide 106 resulted in a premature stop codon. She was started on intramuscular cyanocobalamin at 1000 µg/day on the 13th day of admission; by the 16th day of admission, she was discharged after resolution of symptoms with hemogram findings of Hb: 10.1 g/dL, WBC: 20x109/L, MCV: 89 fl and platelets: 850x109/L, with continuation of treatment twice weekly.

Oral folic acid was also initiated. The sweat test was repeated during that period and was normal. She is currently alive at 5 years of age and asymptomatic under weekly cyanocobalamin treatment.

Case 4 A 3-month-old boy of Turkish origin from Cyprus presented with failure to thrive and poor feeding. Blood and bone marrow examination revealed pancytopenia, hypersegmentation, and megaloblastic changes in the myeloid lineage. Serum homocysteine and vitamin B12 levels were 46 µmol/L (normal: 5.5-17) and 677 pg/mL (normal: 200-860), respectively. Cyanocobalamin was initiated intramuscularly and the pancytopenia resolved. Molecular analyses revealed c.1106+1516-1222+1231del in a homozygous state. The mutation was the same as that found in Case 1.

Discussion Transcobalamin II deficiency is a severe disorder with intracellular cobalamin depletion [2]. Transcobalamin II deficiency usually presents with hematological features that overlap with vitamin B12 deficiency including pancytopenia and megaloblastic anemia with high serum homocysteine and methylmalonic acid levels; however, serum vitamin B12 levels are typically normal [13,14,15]. The early initiation of treatment is very important, since pancytopenia and gastrointestinal symptoms including vomiting and diarrhea reverse very soon after treatment, and delay in diagnosis and treatment may cause morbidities and mortalities related to pancytopenia including bleeding and infection in addition to severe and possibly permanent neurological and retinal impairment [14]. Treatment is suggested as hydroxocobalamin or cyanocobalamin either orally and twice weekly or systemically and weekly with high doses of 1000 µg in order to achieve serum cobalamin levels of 1000-10.000 320

pg/mL, so that cobalamin can be transferred into the cell in the absence of transcobalamin in such high serum levels [15].

Folic acid may be added to the treatment [15].

In cases 1 and 2, the bone marrow findings of vacuolization in the myeloid lineage is interesting. Vacuolization is an important finding in another metabolic disease, namely Pearson syndrome, that may present with pancytopenia, megaloblastic anemia during infancy with lactic acidosis, and exocrine pancreas dysfunction related to a mitochondrial defect [16]. In the literature, Ratschmann et al. provided the bone marrow figures of their index patient of 6 weeks old with transcobalamin II deficiency and described the changes in the myeloid lineage as dysgranulopoiesis [12]. In those findings, vacuolization was prominent, similar to our patients (cases 1 and 2). Vacuolization may be an additional finding of transcobalamin II deficient patients that may be related to defect in the mitochondrial DNA synthesis, as well, resulting from cobalamin deficiency.

Case 1 of the current report had an initial MCV value of 88 fL; after vitamin B12 treatment, the patient had MCV measured as low as 67.2 fL and was further tested with hemoglobin electrophoresis. She was found to have β-thalassemia trait.

This indicates that initial MCV values may not be macrocytic in the presence of β-thalassemia trait; if the clinical presentation is very suggestive of transcobalamin II deficiency, the normal MCV values may not preclude the diagnosis. Additionally, since case 2 was presented at the neonatal stage, MCV was already macrocytic. These findings may indicate that a normal MCV for age may not exclude macrocytic anemia etiologies.

Another finding is that in cases 1 and 3, after the initiation of vitamin B12, hematological improvement occurred with rapid and dramatic leukocytosis in case 1 and leukocytosis and thrombocytosis in case 3. In both cases the high counts normalized in follow-up, but our patients indicate that initiation of therapy may cause a rapid increase of blood counts in transcobalamin II deficient patients.

Additionally, case 3 had a transiently high sweat chloride level that normalized after vitamin B12 treatment. Among the etiologies that may cause a false-positive sweat chloride test, transcobalamin II deficiency has not been reported [17,18].

Transcobalamin II deficiency may be one of the causes of falsepositive sweat tests that has not been previously reported and this hypothesis may require further support from additional studies.

In cases 1, 2, and 4, patients were found to have large deletions, and in case 3 a point mutation was detected, all of which are reported here as novel findings. Tanner et al. previously reported the same mutation among their juvenile cobalamin deficiency patients with GIF mutations together with Yassin et al. [19,20]. Both of those patients

Ünal Ş, et al: TCII Deficiency with Novel Mutations

with GIF mutations were of African ancestry, and Tanner et al. claimed that the mutation might be common in some African populations through a founder effect [19]. The same hypothesis may also be true for our patients (cases 1 and 4) who have the same novel mutation, indicating a common mutation among the Turkish population.

In conclusion, vitamin B12 deficiency has deleterious long-term consequences and, differing from nutritional deficiencies of vitamin B12, patients with transcobalamin II deficiency are especially responsive to high doses of vitamin B12 [21]. Transcobalamin II deficiency should be considered in differential diagnosis of any infant with pancytopenia, failure to thrive, diarrhea, and vomiting. In patients with pancytopenia, transcobalamin II deficiency should be considered in differential diagnosis, especially in countries with high rates of consanguineous marriages, like Turkey.

Early initiation of high-dose vitamin B12 treatment is very crucial not only for being potentially life-saving, but also in order to prevent long-term neurological morbidities.

Acknowledgment We would like to acknowledge Roger Dewar and Jennifer Kerkhof for their contributions from the Biochemical Genetics Laboratory, London Health Sciences Centre, Victoria Hospital, London, Ontario, Canada.

Ethics Committee Approval: Not applicable, Informed Consent: Informed consent was obtained from the parents, Concept: Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin, Design: Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin, Data Collection or Processing: Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin, Analysis or Interpretation: Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin, Literature Search: Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin, Writing: Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin.

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

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2. Schiff M, Ogier de Baulny H, Bard G, Barlogis V, Hamel C, Moat SJ, Odent S, Shortland G, Touati G, Giraudier S. Should transcobalamin deficiency be treated aggressively? J Inherit Metab Dis 2010;33:223-229.

3. Rosenblatt DS, Fenton WA. Inherited disorders of folate and cobalamin transport and metabolism. In: Budet AL, Valle D, Sly W (eds). The Metabolic and Molecular Bases of Inherited Disease. New York, McGraw Hill, 2001.

4. Oberley MJ, Yang DT. Laboratory testing for cobalamin deficiency in megaloblastic anemia. Am J Hematol 2013;88:522-526.

5. Meyers PA, Carmel R. Hereditary transcobalamin II deficiency with subnormal serum cobalamin levels. Pediatrics 1984;74:866-871.

6. Arwert F, Porck HJ, Frater-Schröder M, Brahe C, Geurts van Kessel A, Westerveld A, Meera Khan P, Zang K, Frants RR, Kortbeek HE, Erikkson AW. Assignment of human transcobalamin II (TC2) to chromosome 22 using somatic cell hybrids and monosomic meningioma cells. Hum Genet 1986;74:378-381.

7. Regec A, Quadros EV, Platica O, Rothenberg SP. The cloning and characterization of the human transcobalamin II gene.

Blood 1995;85:2711-2719.

8. Li N, Seetharam S, Seetharam B. Genomic structure of human transcobalamin II: comparison to human intrinsic factor and transcobalamin I. Biochem Biophys Res Commun 1995;208:756-764.

9. Li N, Rosenblatt DS, Kamen BA, Seetharam S, Seetharam B. Identification of two mutant alleles of transcobalamin II in an affected family. Hum Mol Genet 1994;3:1835-1840.

10. Li N, Rosenblatt DS, Seetharam B. Nonsense mutations in human transcobalamin II deficiency. Biochem Biophys Res Commun 1994;204:1111-1118.

11. Namour F, Helfer AC, Quadros EV, Alberto JM, Bibi HM, Orning L, Rosenblatt DS, Jean-Louis G. Transcobalamin deficiency due to activation of an intra exonic cryptic splice site. Br J Haematol 2003;123:915-920.

12. Ratschmann R, Minkov M, Kis A, Hung C, Rupar T, Mühl A, Fowler B, Nexo E, Bodamer OA. Transcobalamin II deficiency at birth. Mol Genet Metab 2009;98:285-288.

13. Hakami N, Neiman PE, Canellos GP, Lazerson J. Neonatal megaloblastic anemia due to inherited transcobalamin II deficiency in two siblings. N Engl J Med 1971;285:11631170.

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Hall CA. The neurologic aspects of transcobalamin II deficiency. Br J Haematol 1992;80:117-120.

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15. Watkins D, Whitehead VM, Rosenblatt D. Megaloblastic anemia. In: Nathan DG, Orkin SH (eds). Nathan and Oski’s Hematology of Infancy and Childhood. Philadelphia, WB Saunders, 2009.

16. Tumino M, Meli C, Farruggia P, La Spina M, Faraci M, Castana C, Di Raimondo V, Alfano M, Pittalà A, Lo Nigro L, Russo G, Di Cataldo A. Clinical manifestations and management of four children with Pearson syndrome. Am J Med Genet A 2011;155:3063-3066.

17. Mishra A, Greaves R, Massie J. The relevance of sweat testing for the diagnosis of cystic fibrosis in the genomic era. Clin Biochem Rev 2005;26:135-153.

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21. Evim MS, Erdöl Ş, Özdemir Ö, Baytan B, Güneş AM. Longterm outcome in children with nutritional vitamin B12 deficiency. Turk J Hematol 2011;28:286-293.

Research Article

DOI: 10.4274/tjh.2014.0152 Turk J Hematol 2015;32:323-328

Eltrombopag for the Treatment of Immune Thrombocytopenia: The Aegean Region of Turkey Experience İmmün Trombositopeni Tedavisinde Eltrombopag: Türkiye Ege Bölgesi Deneyimi Füsun Özdemirkıran1, Bahriye Payzın1, H. Demet Kiper2, Sibel Kabukçu3, Gülsüm Akgün Çağlıyan4, Selda Kahraman5, Ömür Gökmen Sevindik6, Cengiz Ceylan7, Gürhan Kadıköylü8, Fahri Şahin2, Ali Keskin3, Öykü Arslan4, Mehmet Ali Özcan6, Gülnur Görgün7, Zahit Bolaman8, Filiz Büyükkeçeci2, Oktay Bilgir4, İnci Alacacıoğlu6, Filiz Vural2, Murat Tombuloğlu2, Zafer Gökgöz2, Güray Saydam2 1Katip

Çelebi University Faculty of Medicine, Atatürk Research and Education Hospital, Clinic of Hematology, İzmir, Turkey University Faculty of Medicine, Department of Hematology, İzmir, Turkey 3Pamukkale University Faculty of Medicine, Department of Hematology, Denizli, Turkey 4Bozyaka Research and Education Hospital, Clinic of Hematology, İzmir, Turkey 5Aydın State Hospital, Clinic of Hematology, Aydın, Turkey 6Dokuz Eylül University Faculty of Medicine, Department of Hematology, İzmir, Turkey 7Tepecik Research and Education Hospital, Clinic of Hematology, İzmir, Turkey 8Adnan Menderes University Faculty of Medicine, Department of Hematology, Aydın, Turkey 2Ege

Abstract: Objective: Immune thrombocytopenia (ITP) is an immune-mediated disease characterized by transient or persistent decrease of the platelet count to less than 100x109/L. Although it is included in a benign disease group, bleeding complications may be mortal. With a better understanding of the pathophysiology of the disease, thrombopoietin receptor agonists, which came into use in recent years, seem to be an effective option in the treatment of resistant cases. This study aimed to retrospectively assess the efficacy, long-term safety, and tolerability of eltrombopag in Turkish patients with chronic ITP in the Aegean region of Turkey.

Materials and Methods: Retrospective data of 40 patients with refractory ITP who were treated with eltrombopag in the Aegean region were examined and evaluated.

Results: The total rate of response was 87%, and the median duration of response defined as the number of the platelets being over 50x109/L was 19.5 (interquartile range: 5-60) days. In one patient, venous sinus thrombosis was observed with no other additional risk factors due to or related to thrombosis. Another patient with complete response and irregular follow-up for 12 months was lost due to sudden death as the result of probable acute myocardial infarction.

Conclusion: Although the responses to eltrombopag were satisfactory, patients need to be monitored closely for overshooting platelet counts as well as thromboembolic events.

Keywords: Immune thrombocytopenia, Thrombopoietin receptor agonist, Bleeding, Eltrombopag Address for Correspondence: Füsun ÖZDEMİRKIRAN, M.D., Katip Çelebi University Faculty of Medicine, Atatürk Research and Education Hospital, Clinic of Hematology, İzmir, Turkey E-mail: fusun75@gmail.com Received/Geliş tarihi : April 12, 2014 Accepted/Kabul tarihi : August 14, 2014

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Öz: Amaç: İmmün trombositopeni (İTP), trombositlerin immün aracılı yıkım ile kalıcı veya geçici olarak 100x109/L altında olduğu bir hastalıktır. Selim hematolojik hastalıklar içinde yer almasına rağmen kanama komplikasyonları ölümcül olabilir. Hastalığın patofizyolojisi daha iyi anlaşılması ile son yıllarda kullanıma giren trombopoetin reseptör agonistleri, dirençli hastaların tedavisinde etkili bir seçenek olarak görünmektedir.

Gereç ve Yöntemler: Bu çalışmada Ege Bölgesi’nde refrakter İTP tanısı ile eltrombopag ile tedavi edilen 8 farklı merkezden 40 hastanın retrospektif verileri incelenmiş ve değerlendirilmiştir.

Bulgular: Çalışmada toplam yanıt oranı %87 idi ve trombositlerin 50x109/L’nin üzerine çıktığı medyan süre 19,5 (5-60) gün saptandı. Tromboz için başka hiçbir ek risk faktörü bulunmayan bir hastada venöz sinüs trombozu gözlendi. On iki aydır tam yanıtlı izlenen ve düzensiz takibe gelen bir diğer hasta olası akut miyokard infarktüsü sonucu gelişen ani ölüm nedeni ile kaybedildi.

Sonuç: Her ne kadar eltrombopag yanıtları tatmin edici olsa da, hızlı ilerleyen trombositemiye bağlı gelişecek tromboembolik olaylar açısından, yakın takip ve monitorizasyon gereklidir.

Anahtar Sözcükler: İmmün trombositopeni, Trombopoetin reseptor agonisti, Kanama, Eltrombopag Introduction Immune thrombocytopenia (ITP) is an acquired autoimmune disease in which antiplatelet antibodies accelerate the destruction of platelets in the reticuloendothelial system and is characterized by impaired platelet production, resulting in low platelet counts [1]. Among adults, about 50 new cases of ITP per million are diagnosed per year [2]. In adults, the course of the disease is commonly chronic. The primary goal of treatment is to prevent bleeding by increasing the platelet count to a stable level while managing the few treatment-related toxic effects. Current guidelines suggest that treatment should only be considered in symptomatic patients with platelet counts of less than 30x109/L. Treatment is rarely indicated for patients with platelets of >50x109/L in the absence of bleeding or predisposing comorbid conditions [1,3]. The firstline treatment for ITP is glucocorticosteroids. For patients who are actively bleeding or who have a contraindication to glucocorticosteroids, intravenous immunoglobulin or anti-D globulin can be used [4]. These drugs increase platelet counts primarily by reducing the extent of platelet destruction by several different mechanisms. In the case of glucocorticosteroid treatment failure, splenectomy is the main second-line therapy and induces a 70%-80% response rate [5].

Until recently, in patients who were refractory to or relapsing after splenectomy or when splenectomy was contraindicated, a variety of immunosuppressive or cytotoxic drugs (such as vincristine, cyclophosphamide, azathioprine, cyclosporine A, and rituximab) were common as the third-line therapy.

However, almost 30% of adults with ITP fail to respond to these therapies and eventually develop a chronic refractory disease [2,6,7]. All of these treatments mainly reduce destruction of antibody-coated platelets; however, treatment is not always effective and can be restricted by adverse effects.

ITP is often considered as benign disorder, but health-related quality of life is poor. Most of the treatment strategies, such as 324

glucocorticosteroids and immunosuppressive drugs, adversely affect quality of life. In recent years, a better understanding of the pathophysiology of ITP has demonstrated the impaired thrombopoiesis and has led to the development of new therapeutic approaches. A new approach to the treatment of ITP is based on platelet production rather than destruction of platelets. Eltrombopag is an oral, nonpeptide, thrombopoietin receptor (TPO-R) agonist, approved in several countries for the treatment of chronic ITP. Eltrombopag increases platelet production by interacting with the transmembrane domain of the TPO-R and inducing proliferation and differentiation of bone marrow progenitor cells in the megakaryocyte lineage [8,9]. It can be prescribed in Turkey since November 2011.

In this study we aimed to retrospectively assess the efficacy, long-term safety, and tolerability of eltrombopag in Turkish patients with chronic ITP in the Aegean region of Turkey.

Materials and Methods This study was designed as a retrospective study. A total of 40 patients who received eltrombopag treatment for refractory chronic ITP at 8 different centers in the Aegean region of Turkey were included.

ITP diagnosis was verified according to the International Consensus Report on the Investigation and Management of Primary ITP [1]. Primary ITP requires only the finding of isolated thrombocytopenia (100x109/L) with no obvious associated medical condition [1]. Patients were aged 18 years and older and had primary ITP of more than 6 months’ duration, had baseline platelet counts of lower than 30,000/ μL, and had relapsed after one or more previous treatments for their disorder. The form prepared for the study was sent to all centers. Date of the first diagnosis of the patients, demographic data, time to splenectomy, previous treatments and response to treatments, side effects, posttreatment follow-up period, and other such records were retrospectively evaluated. The

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most recent patient data were recorded in December 2013.

Bleeding was assessed with the World Health Organization bleeding scale (grade 0: no bleeding, grade 1: petechiae, grade 2: mild blood loss, grade 3: gross blood loss, grade 4: debilitating blood loss) [10]. Response rates were defined as follows: complete response when the platelet count was 100x109/L, partial response when the platelet count ranged between 30 and 100x109/L with at least a 2-fold increase in the initial platelet count, and no response when the platelet count was 30x109/L [3].

Statistical Analysis Statistical analysis was performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). The Kolmogorov-Smirnov test was used to evaluate the distribution of data. Data with normal distribution were reported as mean ± standard deviation (SD), while data with nonnormal distribution and nonparametric data were reported as medians (interquartile ranges, 25%75%). To evaluate effect of baseline platelet counts on treatment by eltrombopag, the Mann-Whitney U test was used. For comparison of categorical variables, Pearson’s chi-square test was used, or in the case of small frequencies, Fisher’s exact test was used. Statistical significance was defined as p<0.05.

was initiated at 50 mg/day for all patients and the dose was regulated in accordance with their response to treatment. The distribution of the last drug dose is given in Figure 3. Treatment was stopped in 5 cases since no response was obtained. Two patients were lost due to intracranial hemorrhage in the first month of the treatment. Patient characteristics are reported in Table 1.

Mean platelet count before treatment was 11.5x109/ L±8.3x109/L. The total rate of response was 87% and in the cases with response the median period in which the number of platelets reached over 50x109/L was determined as 19.5 (interquartile range: 5-60) days (Table 2).

As for response to treatment, whether or not there was any sex-related difference was studied by chi-square test; no significant difference was determined for response to treatment (p=0.629). Whether the baseline platelets had any significance in patients’ response to treatment was analyzed

by Mann-Whitney U test and it was seen that baseline platelet count was not important in response to treatment (p=0.531).

From the viewpoint of response to eltrombopag treatment, the 40

Results

35%

In total, 40 patients, 30 of them women, from 8 centers in the Aegean region of Turkey were included in the study.

All of them had received glucocorticosteroids at various doses as first-line treatment, and splenectomy was implemented in 28 cases as second-line treatment due to resistance against steroid treatment. Splenectomy was not implemented in 12 cases because of incompatibility for surgery due to comorbid disease or the patient’s disapproval. Prior to eltrombopag treatment, bleeding scale scores were evaluated for each patient and previous treatment numbers were revised and recorded (Figure 1). Median previous treatment number was found to be 3 (interquartile range: 3-4) (Figure 2). Eltrombopag

Number of patients

32.5%

30

20

20%

10

7.5% 2.5%

2.00

3.00 4.00 5.00 6.00 7.00 Number of treatments received

Figure 2. Number of treatments prior to eltrombopag.

Bleeding scale No bleeding

Grade 1

Grade 2

2.5%

Treatment dose Grade3

n=15 (47%) n=12 (37%)

n=5 (16%)

Figure 1. Bleeding data of the patients prior to eltrombopag treatment.

75 mg

50 mg

25 mg

Figure 3. Eltrombopag requirement of patients.

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patients having had splenectomy and those who had not were compared by chi-square test. No difference was determined between the patients with and without splenectomy in their response to eltrombopag treatment (p=0.370).

In the 21 cases in which bone marrow biopsy was done prior to treatment, bone marrow reticulin was evaluated in 2 cases as 2, in 3 cases as 1, and in 16 cases as 0. During treatment, none of the patients showed any clinical or laboratory findings suggesting increased bone marrow reticulin and bone marrow biopsy was not repeated. Adverse effects due to treatment are summarized in Table 3. Of the cases with response to treatment, drug-related nausea developed in 2 cases and headache in 4 cases. However, drug use was continued and these adverse effects vanished in a few weeks after the beginning of the treatment. Platelet count was below 50x109/L on the 7th day of treatment in a case in which erythromelalgia developed, whereas on the 13th day it reached 580x109/L. However, it receded back to the baseline value about 2 weeks after termination of drug use. The patient was suggested to start the drug again with a lower dose but refused the treatment. One male patient at the age of 35, having venous sinus thrombosis and showing no other additional risk factors from the point of view of thrombosis, was receiving 50 mg eltrombopag and had a platelet value on the 15th day of 680x109/L. Treatment was terminated. Platelet counts receded back to below 10x109/L in 15 days; due to recurrent epistaxis and intraoral bleedings,

Table 1. Baseline characteristics of the patients.

Sex (Female/Male) Age (years) Baseline platelet count x109/L Final platelet count x109/L Period from diagnosis to splenectomy (months) Number of previous treatments

30/10 46.82±16.35 11.5±8.3 204.8±18.5 21±36 3 (interquartile range: 3-4)

Results are given as mean ± standard deviation.

treatment was resumed with dose regulation and no thrombotic attack was observed.

In another patient, treatment was stopped due to an increase in transaminases. Transaminase levels were all in normal ranges prior to eltrombopag therapy. Alanine transaminase (ALT) and aspartate transaminase (AST) levels of this patient had gradually increased while she was on eltrombopag. After the ALT level had reached up to 3 times the upper normal level, eltrombopag was stopped with a presumptive diagnosis of toxic hepatitis possibly related to eltrombopag. In order to clarify the etiology of elevated transaminases and to be certain about whether this coincidence was a side effect of eltrombopag or was another concomitant disease, hepatitis serology and autoimmune tests were applied. Serology results were all negative considering hepatitis A, hepatitis B, and hepatitis C. Antimitochondrial antibody was positive with a titer of 1/1000. Liver biopsy was applied for further clarification of ongoing transaminitis and it revealed autoimmune hepatitis. The elevated transaminases were therefore not considered as a side effect of the drug, rather being considered as an independent concomitant autoimmune disorder. After proper treatment of autoimmune hepatitis with steroids and azathioprine, transaminase levels decreased to normal ranges. At the same time, platelets counts were at a steady level between 50,000 and 70,000/µL with the aforementioned immunosuppressive therapy and eltrombopag was not reinitiated.

One of the patients with complete response who was followed irregularly for 12 months was lost due to sudden death as a result of probable acute myocardial infarction. In the laboratory tests performed on the same day, the patient’s platelets were measured as 120x109/L. Two different patients who both had complete response at the beginning of treatment but whose platelet counts decreased below 10x109/L in the 8th month and 1st year of treatment were found to be taking iron supplements and calcium supplements, respectively. These patients were warned about drug and diet interactions, and their platelet counts increased again to above 100x109/L in further follow-up.

Table 2. Outcomes of the treatment.

Total rate of response

n=34 (87%)

Complete response

n=24 (60%)

Partial response

n=11 (27%)

No response

n=5 (13%)

Number of days with of platelet counts above 50,000 (median)

19.5 (interquartile range: 5-60)

Duration of eltrombopag treatment (months)

13.78±7.51

Posttreatment mean platelet count (n=35)

204,771

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Table 3. Adverse effects and toxicity of treatment.

Venous sinus thrombosis

1 case

Headache

4 cases

Nausea

2 cases

Erythromelalgia

1 case

Acute coronary syndrome, sudden death

1 case

Target platelet counts after therapy should be between 50 and 100x109/L, not normalization. In 2 patients, despite platelet counts of 30 to 35x109/L, treatment continued with partial response since bleeding symptoms were controlled.

One patient for whom 4 different treatment options had been previously applied with no response, and who was progressing with intraoral bleedings recurring frequently, received a splenectomy in the 2nd month of eltrombopag treatment while platelet counts were over 100x109/L. The patient then started follow-up with complete response without treatment. Treatment doses in responding patients are given in Figure 3. In the patients with response to treatment, average follow-up period was evaluated as 13.78±7.51 months.

Discussion In this retrospective study, we have evaluated the longterm safety, efficacy, and tolerability of eltrombopag use on Turkish patients with chronic ITP. In this study in which the data of 40 patients were evaluated retrospectively, the total rate of response is 87%, where various different treatment options such as steroids, anti-D globulin, splenectomy, intravenous immunoglobulin, azathioprine, cyclophosphamide, danazol, vincristine, and rituximab were applied with no response prior to eltrombopag treatment. This rate was similar to the rate of 80% obtained in the study of Katsutani et al., where 3 years of eltrombopag data from 19 patients were evaluated, and to the rate of 69.6% obtained in the study of Tomiyama et al. including 23 patients with a placebo control [11,12].

In the present study, there was no difference between the response rates of patients with and without splenectomy, in accordance with the literature [13,14]. Although the responses were satisfactory, patients need to be monitored closely regarding rapidly progressing thrombocythemia as well as thromboembolic events.

Treatment was generally well tolerated and continued, except for a patient who developed erythromelalgia in the 1st month of therapy and another patient who developed autoimmune hepatitis in the 6th month. While eltrombopag is known to have the ability of increasing transaminases [15], treatment was terminated in the patient who developed autoimmune hepatitis. However, during follow-up, no decrease in transaminases occurred despite discontinuing the drug.

This situation was thus regarded as a concomitant disease.

The common side effects of eltrombopag treatment, headache and nausea, did not cause any termination in the treatment and disappeared spontaneously over time. In the literature, the incidence of thromboembolic events was reported as 2%-4% during treatment with TPO-R agonists; however, the rate of only 1 patient out of 40 having sinus vein thrombosis was consistent with the literature [16]. We could not obtain detailed information about the patient who was lost to acute myocardial infarction in the 12th month of treatment while being monitored with full response.

TPO-R agonists may increase the risk of developing or progressing reticulin fiber deposition in the bone marrow [17]. For patients on eltrombopag, peripheral blood smears should be examined for morphological abnormalities such as teardrop cells, nucleated red blood cells, leukoerythroblastic pictures, dysplastic changes, or cytopenia [18]. If such abnormalities develop or deteriorate, a bone marrow biopsy should be performed. A loss of response or failure to maintain a platelet response with eltrombopag treatment within the recommended dosing range should also prompt a search for causative factors such as myelofibrosis [18]. In our study, no patients displayed suggestive clinical or laboratory findings of significant increases in bone marrow reticulin during treatment and bone marrow biopsy was not repeated. The average follow-up period in the patients who responded to treatment of 13.78±7.51 months was satisfactory; on the other hand, close monitoring is recommended for thrombocythemia and thromboembolic events. In particular, patients who begin treatment should be informed in this regard in detail, and this therapy is not recommended for cases that cannot be followed closely. A decrease in eltrombopag dosage is recommended when platelet counts exceed 200x109/L and should be completely stopped if platelet count is over 400x109/L. After discontinuation due to thrombocythemia or any other adverse effects, patients should be monitored to detect any transient decrease in platelet counts and to decide about further treatment indication and dose. In the case of response loss during follow-up in patients with an initial response, dietdrug interactions must be questioned in detail.

Acknowledgments We are grateful to all of the following centers for their contributions by sharing their data and all the staff of these centers for their contributions in preparation of the data: Katip Çelebi University Atatürk Research and Education Hospital, Department of Hematology, İzmir; Ege University Medical Faculty, Department of Hematology, İzmir; Pamukkale University Medical Faculty, Department of Hematology, Denizli; Bozyaka Research and Education Hospital, Department of Hematology, İzmir; Aydın State Hospital, Aydın; Dokuz Eylül University Medical Faculty, Department of

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Hematology, İzmir; Tepecik Research and Education Hospital, Department of Hematology, İzmir; Adnan Menderes University Medical Faculty, Department of Hematology, Aydın. We are also grateful to Dr. Mehmet Çalan, who worked diligently in preparation of the display of statistical data and the graphics.

Ethics Committee Approval: In our center, ethics committee approval is not required for retrospective studies, Concept: Füsun Özdemirkıran, Design: Bahriye Payzın, Data Collection or Processing: H. Demet Kiper, Sibel Kabukçu, Gülsüm Akgün Çağlıyan, Selda Kahraman, Ömür Gökmen Sevindik, Cengiz Ceylan, Gürhan Kadıköylü, Fahri Şahin, Ali Keskin, Öykü Arslan, Mehmet Ali Özcan, Gülnur Görgün, Zahit Bolaman, Filiz Büyükkeçeci, Oktay Bilgir, İnci Alacacıoğlu, Filiz Vural, Murat Tombuloğlu, Zafer Gökgöz, Analysis or Interpretation: Güray Saydam, Literature Search: Füsun Özdemirkıran, Writing: Füsun Özdemirkıran.

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

References 1. Provan D, Stasi R, Newland AC, Blanchette VS, Bolton-Maggs P, Bussel JB, Chong BH, Cines DB, Gernsheimer TB, Godeau B, Grainger J, Greer I, Hunt BJ, Imbach PA, Lyons G, McMillan R, Rodeghiero F, Sanz MA, Tarantino M, Watson S, Young J, Kuter DJ. International consensus report on the investigation and management of primary immune thrombocytopenia.

Blood 2010;115:168-186.

2. Cines DB, Blanchette VS. Immune thrombocytopenic purpura.

N Engl J Med 2002;346:995-1008.

3. Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold DM, Bussel JB, Cines DB, Chong BH, Cooper N, Godeau B, Lechner K, Mazzucconi MG, McMillan R, Sanz MA, Imbach P, Blanchette V, Kühne T, Ruggeri M, George JN. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 2009;113:2386-2393.

4. Neunert C, Lim W, Crowther M, Cohen A, Solberg L Jr, Crowther MA; American Society of Hematology. The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia. Blood 2011;117:4190-4207.

5. Kumar S, Diehn FE, Gertz MA, Tefferi A. Splenectomy for immune thrombocytopenic purpura: long term results and treatment of postsplenectomy relapse. Ann Hematol 2002;81:312-319.

6. McMillan R. Classical management of refractory adult immune (idiopathic) thrombocytopenic purpura. Blood Rev 2002;16:51-55.

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7. Yang R, Han ZC. Pathogenesis and management of chronic idiopathic thrombocytopenic purpura: an update. Int J Hematol 2000;71:18-24.

8. Garnock-Jones KP, Keam SJ. Eltrombopag. Drugs 2009;69:567576.

9. Glaxo Smith Kline. Promacta (Eltrombopag) Prescribing Information. Research Triangle Park, NC, USA, Glaxo Smith Kline, 2012. Available at http://www.gsksource.com/gskprm/ en/US/adirect/gskprm?cmd=Product DetailPage&product_ id=1353688574915. Accessed 28 February 2013.

10. Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer 1981;47:207-214.

11. Katsutani S, Tomiyama Y, Kimura A, Miyakawa Y, Okamoto S, Okoshi Y, Ninomiya H, Kosugi H, Ishii K, Ikeda Y, Hattori T, Katsura K, Kanakura Y. Oral eltrombopag for up to three years is safe and well-tolerated in Japanese patients with previously treated chronic immune thrombocytopenia: an open-label, extension study. Int J Hematol 2013;98:323-330.

12. Tomiyama Y, Miyakawa Y, Okamoto S, Katsutani S, Kimura A, Okoshi Y, Ninomiya H, Kosugi H, Nomura S, Ozaki K, Ikeda Y, Hattori T, Katsura K, Kanakura Y. A lower starting dose of eltrombopag is efficacious in Japanese patients with previously treated chronic immune thrombocytopenia. J Thromb Haemost 2012;10:799-806.

13. Cheng G, Saleh MN, Marcher C, Vasey S, Mayer B, Aivado M, Arning M, Stone NL, Bussel JB. Eltrombopag for management of chronic immune thrombocytopenia (RAISE): a 6-month, randomised, phase 3 study. Lancet 2011;377:393-402.

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Saleh MN, Bussel JB, Cheng G, Meyer O, Bailey CK, Arning M, Brainsky A; EXTEND Study Group. Safety and efficacy of eltrombopag for treatment of chronic immune thrombocytopenia: results of the long-term, open-label EXTEND study. Blood 2013;121:537-545.

15. Zelcer S, Bussel JB. Thrombosis in patients with immune thrombocytopenic purpura (ITP): a case series. J Thromb Haemost 2003;1:1169 (abstract).

16. Ghanima W, Lee SY, Barsam S, Miller A, Sandset PM, Bussel JB. Venous thromboembolism and coagulation activity in patients with immune thrombocytopenia treated with thrombopoietin receptor agonists. Br J Haematol 2012;158:811-814.

17. Douglas V, Tallman M, Cripe L, Peterson LC. Thrombopoietin administered during induction chemotherapy to patients with acute myeloid leukemia induces transient morphologic changes that may resemble chronic myeloproliferative disorders. Am J Clin Pathol 2002;117:844-850.

18. Cheng G. Eltrombopag, a thrombopoietin- receptor agonist in the treatment of adult chronic immune thrombocytopenia: a review of the efficacy and safety profile. Ther Adv Hematol 2012;3:155-164.

Research Article

DOI: 10.4274/tjh.2014.0035 Turk J Hematol 2015;32:329-337

Management of Invasive Fungal Infections in Pediatric Acute Leukemia and the Appropriate Time for Restarting Chemotherapy Çocukluk Çağı Akut Lösemisinde İnvaziv Fungal Enfeksiyonların Tedavisi ve Kemoterapiye Başlamanın Uygun Zamanı Özlem Tüfekçi1, Şebnem Yılmaz Bengoa1, Fatma Demir Yenigürbüz1, Erdem Şimşek2, Tuba Hilkay Karapınar1, Gülersu İrken1, Hale Ören1 1Dokuz 2Dokuz

Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey Eylül University Faculty of Medicine, Department of Pediatrics, İzmir, Turkey

Abstract: Objective: Rapid and effective treatment of invasive fungal infection (IFI) in patients with leukemia is important for survival.

In this study, we aimed to describe variations regarding clinical features, treatment modalities, time of restarting chemotherapy, and outcome in children with IFI and acute leukemia (AL).

Materials and Methods: The charts of all pediatric AL patients in our clinic between the years of 2001 and 2013 were retrospectively reviewed. All patients received prophylactic fluconazole during the chemotherapy period.

Results: IFI was identified in 25 (14%) of 174 AL patients. Most of them were in the consolidation phase of chemotherapy and the patients had severe neutropenia. The median time between leukemia diagnosis and definition of IFI was 122 days.

Twenty-four patients had pulmonary IFI. The most frequent finding on computed tomography was typical parenchymal nodules. The episodes were defined as proven in 4 (16%) patients, probable in 7 (28%) patients, and possible in 14 (56%) patients. The median time for discontinuation of chemotherapy was 27 days. IFI was treated successfully in all patients with voriconazole, amphotericin B, caspofungin, or posaconazole alone or in combination. Chemotherapy was restarted in 50% of the patients safely within 4 weeks and none of those patients experienced reactivation of IFI. All of them were given secondary prophylaxis. The median time for antifungal treatment and for secondary prophylaxis was 26 and 90 days, respectively. None of the patients died due to IFI.

Conclusion: Our data show that rapid and effective antifungal therapy with rational treatment modalities may decrease the incidence of death and that restarting chemotherapy within several weeks may be safe in children with AL and IFI.

Keywords: Acute leukemia, Chemotherapy, Children, Fungal infection

Address for Correspondence: Hale ÖREN, M.D., Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey Phone: +90 532 666 90 50 E-mail: hale.oren@deu.edu.tr Received/Geliş tarihi : January 24, 2014 Accepted/Kabul tarihi : April 28, 2014

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Öz: Amaç: Lösemili hastalarda invaziv fungal enfeksiyonların (İFE) çabuk ve etkin tedavisi sağkalım için önemlidir. Bu çalışmada akut lösemi (AL) ve İFE olan çocuklarda klinik bulgular, tedavi şekilleri, tekrar kemoterapiye başlama zamanı ve tedavi sonucu gibi değişkenleri değerlendirmeyi amaçladık.

Gereç ve Yöntemler: Kliniğimizde 2001-2013 yılları arasında izlenmiş tüm AL’lı çocukların hastane kayıtları retrospektif olarak tarandı. Tüm hastalara kemoterapi süresince proflaktik flukonazol tedavisi verildi.

Bulgular: İFE, 174 AL hastasından 25’inde (%14) saptandı. Çoğu konsolidasyon tedavisi sırasında gelişmişti ve hastalar ağır nötropenikti. Lösemi tanısı ve İFE gelişme arasındaki ortanca süre 122 gündü. Hastaların 24’ünde pulmoner İFE vardı. Bilgisayarlı tomografi tetkikinde en sık izlenen bulgu parenkimal nodüllerdi. İFE epizodları 4 (%16) olguda kanıtlanmış, 7 (%28) olguda olası, 14 (%56) olguda muhtemel olarak değerlendirildi. Kemoterapiye ara verme süresi ortanca 27 gündü. İFE voriconazole, amphotericin B, caspofungin, posaconazole tekli veya kombine tedavileri ile başarıyla tedavi edildi. Olguların %50’sinde kemoterapiye 4 haftadan önce başlandı ve hiçbirinde İFE reaktivasyonu saptanmadı. Tümüne ikincil proflaksi verildi. Antifungal tedavi ve sekonder proflaksi ortanca süresi sırayla 26 ve 90 gündü. Hastalardan hiçbiri İFE ile kaybedilmedi.

Sonuç: Verilerimiz AL ve İFE olan çocuklarda erken ve etkin rasyonel antifungal tedavi ile ölüm oranının azaltılabileceğini ve birkaç hafta içinde kemoterapiye güvenle başlanabileceğini göstermektedir.

Anahtar Sözcükler: Akut lösemi, Çocukluk çağı, Fungal enfeksiyon, Kemoterapi Introduction The breakdown of host defense mechanisms in immunocompromised patients leads to increased risk of lifethreatening infections, including invasive fungal infections (IFIs) [1,2,3,4,5,6]. Studies of pediatric populations with hemato-oncological diseases show an incidence rate of IFI ranging from 4.9% to 29% [7,8,9,10]. Besides causing increased mortality and morbidity, IFIs cause a substantial delay in treatment of acute leukemia (AL), which in turn could result in failure of this potentially curative treatment. The optimal time for restarting chemotherapy in these patients is not clear.

In this retrospective study, our purpose was to describe the incidence, risk factors, clinical features, treatment modalities, and outcome of IFIs in children with AL. We also aimed to investigate the appropriate (optimal) time for restarting chemotherapy in this group of patients.

Materials and Methods Patients and Institution This retrospective study included all acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients, aged 0-18 years, who developed IFI at our clinic between January 2001 and January 2013. The patients were identified by reviewing the medical charts of all AL patients. Children with ALL received the BFM-95 or BFM-2000 protocol and those with AML received the BFM-98 or BFM-2004 protocol.

Children were hospitalized in single rooms without highefficiency air filtration systems.

The medical, microbiological, and imaging records of the patients who met the inclusion criteria were reviewed for the following variables:

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Demographic and clinical data: Age and sex, leukemia type, remission status, and risk group of underlying disease at the time of diagnosis; the day and phase of treatment at which IFI developed (remission, induction, consolidation, maintenance); corticosteroid use 14 days prior to IFI onset; presence of central venous catheter; presence of mucositis; duration of neutropenia prior to IFI; use and type of primary antifungal prophylaxis; type of symptoms and signs of the IFI. Laboratory data: Complete blood count; fungus detection tests including serum galactomannan (GM) antigen, direct stains, cultures, sinus aspirate, and samples from other sites.

Radiological data: X-ray, computed tomography, and ultrasound.

Treatment and outcome of IFI: Empiric therapy and definitive therapy with one or a combination of antifungal drugs; use of surgery; duration of treatment; the time from the onset of fungal infection to the restarting of chemotherapy; the use and type of secondary antifungal prophylaxis; the development of reactivation of fungal infection; mortality.

According to our institutional policy, all patients with ALL and AML receive prophylactic fluconazole (4-6 mg/kg/day) during all phases of chemotherapy. Severe neutropenia was defined as absolute granulocyte count of <500/mm3.

Serum GM assay could not be done routinely for all patients as the assay was not always available in the hospital laboratory.

A positive result was based on 2 consecutive samples with a GM index of greater than or equal to 0.5.

Radiologic evaluation was performed after 5 days of fever, including high-resolution computed tomography (HRCT) of the chest and paranasal sinuses, in addition to cardiac echocardiogram.

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The primary empiric antifungal treatment in our clinic was either with caspofungin or liposomal amphotericin B, depending on availability in the pharmacy of the hospital. The antifungal agent was sometimes switched during the course of the illness if the patient was intolerant or in culture-positive cases, according to the drug susceptibilities of the specific pathogen isolated.

In all cases, IFI was defined according to the guidelines of the EORTC/MSG [11]. Proven IFI was diagnosed by a positive fungal culture from a normally sterile site. Probable IFI was diagnosed on the basis of a combination of host factors, clinical and radiological features, and mycological evidence, such as positive fungal culture, positive GM assay, or microscopy of bronchoalveolar lavage fluid or sinus aspirate. Possible IFI was diagnosed when the clinical and imaging findings and host factors were consistent with IFI but there was no mycological support.

Statistical Analysis Statistical analyses were performed with SPSS 15.

Descriptive statistics were calculated and reported as absolute frequencies or percentages for qualitative data and as medians and ranges for quantitative data.

Results A total of 174 patients were diagnosed with and treated for AL (144 had ALL and 30 had AML) in our clinic. IFI was diagnosed in 25 (14%) of 174 AL patients, in 12% of all ALL cases, and in 27% of all AML cases. The characteristics of the 25 patients diagnosed with IFI are shown in Table 1. Of the 25 patients, 17 (68%) had ALL and 8 (32%) had AML. Five of the 8 AML patients (62%) and 7 of the 17 ALL patients (41%) were allocated into the high-risk group at the time of diagnosis of AL. The median age was 12 years (range: 0.7-17.5 years). Nine (36%) of the patients were in the induction phase, 14 (56%) of the patients were in the consolidation phase, and 2 (8%) of the patients were in the maintenance phase of chemotherapy; overall, 18 (72%) patients were in remission at the time of diagnosis of IFI. The median time between the leukemia diagnosis and the definition of IFI was 122 days (range: 15-305 days).

Absolute neutrophil count was <500/mm3 in 86% of patients.

The median time for duration of neutropenia was 13 days (range: 0-47 days). All of the patients were febrile at the time of diagnosis. Of the 25 AL patients with IFI, 23 patients had isolated pulmonary IFI, 1 patient had isolated orbitocerebral aspergillosis infection, and 1 patient had both orbitocerebral and pulmonary Mucor and aspergillosis infection. The episodes of IFI were defined as proven in 4 (16%) patients, probable in 7 (28%) patients, and possible in 14 (56%) patients (Table 2).

Only 7 (29%) of the 24 patients with pulmonary IFI had positive pulmonary auscultatory findings. All of the patients with pulmonary IFI had positive findings on pulmonary HRCT, the most common being typical parenchymal nodules in 17 (70%) of 24 patients. Two patients had both halo signs and air-crescent findings on HRCT, while 1 patient had only halo signs. Air-crescent findings alone were present in 2 cases. The remaining 4 patients had consolidation areas on HRCT. Eleven (45%) of the 24 patients with pulmonary IFI had concomitant positive chest X-ray findings at the time of diagnosis. Of the 11 patients with positive chest X-ray findings, 9 patients had parenchymal infiltration, 2 patients had nodular infiltration, and 1 had pleural effusion. GM was screened in 21 patients and, of those, 9 (42%) had positive GM antigenemia.

IFI was treated successfully in all patients with voriconazole, amphotericin B, caspofungin, or posaconazole alone or in combination. The 2 patients with orbitocerebral IFI needed surgery. The initial treatment was voriconazole in 11 patients, liposomal amphotericin B in 7 patients, caspofungin in 4 patients, and conventional amphotericin B in 3 patients. The median time for total antifungal treatment was 26 days (range: Table 1. Characteristics of 25 children diagnosed with an invasive fungal infection.

Characteristics

Median age, years (range)

12 (0.7-17.5)

Sex Male (%) Female (%)

17 (68) 8 (32)

Baseline diagnosis (n=25) ALL (%) AML (%)

17 (68) 8 (32)

Treatment phase Induction (%) Consolidation (%) Maintenance (%) Predisposing factors (n=25) Steroid treatment (%) ANC<500 (%) Central venous catheter (%)

9 (36) 14 (56) 2 (8)

21 (84) 22 (88) 16 (64)

Median duration of neutropenia, days (range)

13 (0-47)

Presence of symptoms (n=25) Fever (%) Cough (%) Chest pain (%)

25 (100) 15 (60) 8 (32)

ALL: Acute lymphoblastic leukemia, AML: acute myeloid leukemia, ANC: absolute neutrophil count.

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21-57 days). All patients were given secondary prophylaxis with oral voriconazole, itraconazole, or posaconazole. The median time for secondary prophylaxis was 90 days (range: 39-429 days). Reactivation of IFI occurred in 4 patients as pulmonary IFI; all of them were cured completely after treatment.

improvement (no pathological signs in HRCT) was 20 days (range: 7-180 days) in the patients for whom chemotherapy was started after 28 days, while it was 17 days (range: 8-48 days) in those for whom chemotherapy was started before 28 days.

Death occurred in 9 (36%) patients (Table 4). Six of the patients who died were in the high-risk group.

The median time for discontinuation of chemotherapy was 27 days (range: 0-57 days). Chemotherapy was not restarted in 3 patients due to refractory/progressive primary disease.

Out of 22 patients for whom chemotherapy was restarted, the duration of cessation of chemotherapy was <14 days in 5 (23%) patients and 14-28 days in 6 (27%) patients (Table 3).

Overall, chemotherapy was restarted in 50% of the patients safely before 4 weeks, and none of those patients experienced reactivation of IFI. For those for whom the chemotherapy was restarted after 28 days, the median time for discontinuation of chemotherapy was 35 days (range: 30-57 days). Regarding the outcome of the primary disease, the leukemia was cured in 9 of 11 patients for whom chemotherapy was started before 28 days and in 7 of 11 patients for whom the chemotherapy was started after that time. The median time for radiological

Discussion In the present retrospective study, IFI was defined in 14% of 174 children with AL. The incidence of IFI has been reported as between 1.3% and 25% in pediatric patients with hematological malignancies [4,7,12,13]. Taking into account the patient population and the conditions of the health center, our rate is very similar to the rate of another retrospective study from Turkey, which reported the incidence rate of IFI as 13.6% [10]. Another Turkish study reported the incidence rate of proven and probable IFI as 14.3% in patients with AML, ALL, and aplastic anemia [14].

Previous studies have suggested that older age is a risk factor for IFI in children [7,15,16]. The median age was 12

Table 2. Classification, etiology, and sites of fungal infection.

Etiology

Number of Isolates

Site of Infection

Candida kefyr

1

Blood

Trichosporon spp.

1

Lung

Aspergillus spp.

2

Orbitocerebral

1

Orbitocerebral

7

Lung

14

Lung

Proven yeast/mold infection

(n=4)†

Mucor Probable mold infection

(n=7)‡

Aspergillus spp.

Possible mold infection (n=14) Aspergillus spp.

†: One patient had both orbitocerebral and pulmonary Mucor and aspergillosis infection. ‡: Galactomannan was positive in all of the cases.

Table 3. Time for discontinuation and restarting of chemotherapy in 22 patients.

Parameter

Median time for discontinuation of chemotherapy, days (range)

27 (0-57)

Time for restarting chemotherapy (n=22)†

<14 days (%)

5 (23)

14-28 days (%)

6 (27)

>28 days (%)

11 (50)

†: Chemotherapy was not restarted in 3 patients due to refractory/progressive primary disease.

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Table 4. Disease characteristics, duration of chemotherapy discontinuation, and causes of death for the 9 patients who died.

Patients

Primary Disease/Risk Group

Duration for Discontinuation of Chemotherapy

Cause of Death

1

AML/HRG

NA†

Refractory/progressive primary disease

2

ALL/HRG

NA†

Refractory/progressive primary disease

3

ALL/MRG

NA†

Refractory/progressive primary disease

4

ALL/HRG

≤28 days

EBV related-lymphoproliferative disease

5

ALL/HRG

≤28 days

Post-transplantation lymphoproliferative disease‡‡

6

ALL/MRG

>28 days

Allogeneic bone marrow transplantation toxicity‡‡

7

ALL/MRG

>28 days

Refractory/progressive primary disease

8

ALL/HRG

>28 days

Refractory/progressive primary disease

9

ALL/HRG

>28 days

Refractory/progressive primary disease

ALL: Acute lymphoblastic leukemia, AML: acute myeloid leukemia, HRG: high-risk group, MRG: medium-risk group, NA: not applicable. †: Chemotherapy was not restarted in 3 patients due to refractory/progressive primary disease. ‡: Allogeneic bone marrow transplantation was performed due to wwwleukemia relapse.

years in our study. Dvorak et al. and Kobayashi et al. found that age above 10 years on admission is a risk factor for IFI and it has been suggested that this finding may reflect the importance of host colonization by environmental fungi as an important step in the development of invasive disease, with younger patients having had less exposure time to fungal spores in the environment [15,16,17].

The majority of our patients (88%) were severely neutropenic at the time of diagnosis of IFI and the overall median duration of neutropenia was longer than 10 days. The incidence of IFI in children with leukemia was previously found to be closely related to the type of leukemia, with AML having a higher rate than ALL as in our study [3,7,18,19,20].

The intensive treatment and the relatively longer duration of neutropenia in AML patients are responsible for the increased risk of infections in this group of patients.

More than half of our patients (56%) were in the consolidation phase at the time of diagnosis of IFI. Similarly, Hale et al. also reported that half of IFIs were diagnosed 100365 days after the initial diagnosis in AL patients [12]. We use BFM protocols and the consolidation phases of ALL and AML in BFM protocols correspond to HD-MTX and HD-ARA C blocks where there is increased risk of mucositis, a known risk factor for fungal infections [21,22].

In our study, GM was positive in 2 consecutive samples of 9 patients. Adult studies and recent pediatric studies have revealed the favorable specificity of the assay [23,24,25,26,27,28,29].

Another important diagnostic approach in identifying IFI is

the HRCT of the chest. Chest X-rays have little value in the early stage of disease [30,31,32]. The most common sign in our patients was typical parenchymal nodules on HRCT; halo signs and air-crescent findings were less frequently seen. It is important to emphasize that pulmonary lesions characteristic for adults, such as air-crescent signs and cavitary lesions, are rarely seen in children [33,34]. A recent retrospective analysis of 139 pediatric invasive aspergillosis cases reported that the most frequent diagnostic radiologic finding was nodules at a rate of 34.6% [35].

The vast majority of IFIs in our study were due to Aspergillus spp. and the respiratory tract was the most common site for invasive aspergillosis. On the other hand, the absence of Candida albicans infections was remarkable in our study, which may be attributable to the strict use of fluconazole.

One of our patients had Candida kefyr bloodstream infection, which is the fluconazole-resistant nonalbicans type of Candida and may be seen in patients with neutropenia. Recent reports have shown that infections caused by resistant Candida spp. and molds such as Aspergillus, Fusarium, and Scedosporium have been subsequently increased by the widespread use of fluconazole prophylaxis [10,12,36,37]. An additional risk factor for development of invasive aspergillosis in our study might be the absence of effective air filtration systems in patient rooms, as well as ongoing hospital renovation for the last 5 years. There are many reports in the literature suggesting an association between invasive aspergillosis and contaminated ventilation systems, hospital construction, or renovation [14,38,39].

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Empirical antifungal therapy and investigation for IFIs should be considered for patients with persistent or recurrent fever after 4-7 days of antibiotics [40,41]. IFI was treated successfully in all our patients with voriconazole, amphotericin B, caspofungin, or posaconazole alone or in combination [42,43,44,45,46]. One of our patients with orbitocerebral mucormycosis and aspergillosis initially did not respond to liposomal amphotericin B, but did recover completely after posaconazole was added to the treatment.

Combination therapy, although not recommended by international guidelines, is often used as rescue treatment in patients who are switched to second- or third-line antifungal therapy [45,47]. Regarding secondary antifungal prophylaxis, it is recommended to continue treatment with an agent and dose effective against the isolate of the primary infection until the end of immunosuppression [48].

An important consequence of IFI is that the relatively longer duration of time for treatment of this severe infection causes a significant delay in the primary treatment of AL. The optimal time for restarting chemotherapy in these patients is not clear, which poses a great dilemma for the physician [45]. One of our aims in this study was to investigate the safe, appropriate timing for restarting chemotherapy in these patients. The median time for discontinuation of chemotherapy was 27 days in our study; chemotherapy was restarted in 50% of the patients safely before 4 weeks and none of those patients experienced reactivation of IFI. Similarly Nosari et al., in their retrospective review of hematological malignancies, identified 61 adult cases of IFI and detected a median time of 27 days for discontinuation of chemotherapy (range: 17-45 days) [49].

The decision for timing chemotherapy is generally made on an individual basis depending on the extent of the fungal disease and the status of the primary disease.

The mortality rate of IFI shows wide variations among the studies reported in the literature. While earlier studies reported IFI-related mortality rates of up to 85%, recent studies have reported lower rates [8,39,50,51,52]. Kaya et al. reported the rate of IFI-attributable death as 5% (1 patient) in 21 children with AL [10]. Another previously mentioned study from Turkey found the total mortality of IFI to be 30% in 23 patients with AL and aplastic anemia [14]. In this study, death occurred in 36% of patients, but none of the deaths were attributable to the IFIs themselves. This finding may be due to increased awareness of the possibility of IFIs, the widespread use of HRCT as an early diagnostic method, early empirical treatment for febrile neutropenic patients, and greater effectiveness of newer antifungal agents.

In conclusion, our study demonstrated that rapid and effective antifungal therapy with rational treatment modalities 334

Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia

may decrease the incidence of death in children with AL and IFI. Depending on the clinical status of the patient, restarting chemotherapy within several weeks may be safe and reactivation of IFI may be prevented with secondary prophylaxis.

Ethics Committee Approval: It is a retrospective study, Informed Consent: It is a retrospective study, Concept: Hale Ören, Design: Hale Ören, Özlem Tüfekçi, Gülersu İrken, Şebnem Yılmaz Bengoa, Data Collection or Processing: Özlem Tüfekçi, Tuba Hilkay Karapınar, Erdem Şimşek, Analysis or Interpretation: Hale Ören, Özlem Tüfekçi, Şebnem Yılmaz Bengoa, Literature Search: Hale Ören, Özlem Tüfekçi, Writing: Hale Ören, Özlem Tüfekçi.

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

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Research Article

DOI: 10.4274/tjh.2013.0370 Turk J Hematol 2015;32:338-343

First-Step Results of Children Presenting with Bleeding Symptoms or Abnormal Coagulation Tests in an Outpatient Clinic Polikliniğe Kanama Belirtileri veya Anormal Koagülasyon Testleri Nedeniyle Başvuran Olgularda Birinci Basamak Değerlendirme Sonuçları İsmail Yıldız1, Ayşegül Ünüvar2, İbrahim Kamer3, Serap Karaman2, Ezgi Uysalol2, Ayşe Kılıç1, Fatma Oğuz4, Emin Ünüvar1 1İstanbul

University İstanbul Faculty of Medicine, Department of Pediatrics, Division of Ambulatory Pediatrics, İstanbul, Turkey University İstanbul Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology and Oncology, İstanbul, Turkey 3İstanbul University İstanbul Faculty of Medicine, Department of Pediatrics, İstanbul, Turkey 4İstanbul University Institute of Child Health, Division of Ambulatory Pediatrics, İstanbul, Turkey 2İstanbul

Abstract: Objective: Mild bleeding symptoms are commonly seen in the general population. The aim of this study was to determine the final clinical and laboratory features of children referred for a first evaluation with a suspected bleeding disorder in the pediatric outpatient clinic of İstanbul University.

Materials and Methods: The medical records of 26,737 outpatients who were admitted to the Division of Ambulatory Pediatrics between 31 October 2011 and 31 October 2012 were evaluated retrospectively. Ninety-nine patients were initially diagnosed as having probable bleeding disorders and were followed up. The symptoms of bleeding in addition to coagulation tests were analyzed.

Results: Of the 99 patients, 52 (52.5%) were male and 47 were female, and the mean age of the entire study group was 9.1±4.1 years (minimum-maximum: 2-18 years). Major bleeding symptoms were epistaxis in 36 patients (36.4%), easy bruising in 32 (32.3%), and menorrhagia in 6 (6.1%). After initial tests ordered by the pediatrician, 36 of 99 patients (36.4%) were diagnosed as having bleeding disorders that included von Willebrand disease in 12 (12.1%), hemophilia A or B in 9 (9.1%), and other rare factor deficiencies in 9 (9.1%). Six patients (6.1%) were found to have combined deficiencies. Seven of 36 patients had a family history of bleeding.

Conclusion: Among the patients referred for bleeding disorders, 36.4% were diagnosed with a bleeding disorder with the help of primary screening tests ordered in the outpatient clinic.

Keywords: Children, Blood coagulation, Hemophilia, Inherited coagulopathies, Epistaxis, Menorrhagia

Address for Correspondence: İsmail YILDIZ, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Pediatrics, Division of Ambulatory Pediatrics, İstanbul, Turkey Phone: +90 212 414 20 00 E-mail: drismail810@yahoo.com Received/Geliş tarihi : November 03, 2013 Accepted/Kabul tarihi : May 29, 2014

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Yıldız İ, et al: Evaluation of Children with Bleeding Symptoms

Öz: Amaç: Hafif kanama bozukluğu belirtileri toplumda sık görülmektedir. Bu çalışmanın amacı İstanbul Üniversitesi İstanbul Tıp Fakültesi Genel Pediatri Polikliniği’ne kanama bozukluğu şüphesi ile sevk edilen hastaların klinik ve laboratuvar özelliklerini belirlemektir.

Gereç ve Yöntemler: 31 Ekim 2011 ile 31 Ekim 2012 tarihleri arasında kanama bozukluğu şüphesiyle yönlendirilen 99 hastanın tıbbi kayıtları incelenmiştir. Başvuru semptomları ile pıhtılaşma testlerinin sonuçları değerlendirilmiştir.

Bulgular: Olguların 47’si kız çocuğu olup ve yaş ortalaması 9,1±4,1 yıl (2-18 yıl) idi. Kanama semptomları 36 hastada (%36,4) burun kanaması, 32 (%32,3) hastada kolay morarma ve 6 hastada (%6,1) menoraji idi. Birinci basamak testleri sonrasında, 99 hastanın 36’sında (%36,4) primer kanama bozukluğu saptandı. Bunlardan 12’sinde (%12,1) von Willebrand hastalığı, 9’unda (%9,1) hemofili A veya B, 9’unda (%9,1) diğer nadir faktör eksiklikleri ve 6 hastada (%6,1) kombine faktör eksiklikleri saptandı.

Otuz altı hastanın 7’sinde ailede kanama öyküsü vardı.

Sonuç: Kanama bozukluğu şüphesi ile sevk edilen hastaların %36,4’ünde birinci basamak koagulasyon testleri ışığında kanama bozukluklarından biri saptandı.

Anahtar Sözcükler: Çocuk, Koagülasyon, Hemofili, Kalıtsal koagülopatiler, Epistaksis, Menoraji Introduction When there is damage to the vascular wall, cessation of bleeding without interrupting the blood flow and maintenance of vascular integrity are ensured by hemostatic mechanisms.

Hemostasis is a multifunctional physiologic mechanism involving the vascular wall, subendothelial tissues, platelets, coagulation factors in plasma, and fibrinolytic factors, where coagulants, anticoagulants, and fibrinolytic activities operate in balance [1,2,3].

Hemostatic disorders manifesting with bleeding may be caused by several factors including vascular issues, low platelet counts, platelet function disorders, and disorders of coagulation or fibrinolysis, which is due to either too much or too fast dissolving of blood clots [1,3].

A careful history and physical examination of a patient with bleeding symptoms leads to a correct diagnosis in 80%90% of patients. Adequate laboratory tests are performed subsequently to confirm diagnosis [4,5,6].

In cases of bleeding disorders, the primary screening tests include complete blood count, peripheral blood smear, bleeding time test (if possible) using a platelet function analyzer (PFA-100), prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen levels [5,6]. Advanced tests are carried out later based on the pathological results from the primary screening tests. Regardless of whether primary screening test results are found to be normal, there may still be an underlying bleeding disorder. In these cases, factor 13 deficiency, von Willebrand disease (vWD) type 1, mild-type hemophilia A or B, mild factor 11 deficiency and mild deficiencies of other factors, alpha-2 anti-plasmin deficiency, plasminogen activator inhibitor-1 deficiency, collagen tissue diseases, vitamin C deficiency, and

various vascular bleeding disorders should be considered [4,6].

Mild bleeding symptoms such as epistaxis, easy bruising, gingival bleeding, and prolonged menstrual bleeding are commonly seen in the general population and reported in up to 25%-45% of healthy people [7]. Although patients who present with these symptoms may have underlying bleeding disorders, initial tests for bleeding etiology may yield normal results [8,9].

The purpose of this study was to evaluate patients who were referred to the Division of Ambulatory Pediatrics with suspected bleeding disorders.

Materials and Methods A total of 26,737 outpatients were admitted to the İstanbul Faculty of Medicine’s Department of Pediatrics from 31 October 2011 to 31 October 2012. After exclusion of all patients with immune thrombocytopenia, 115 patients with suspected bleeding disorders were evaluated retrospectively. Thirteen of these patients were not included because of known bleeding disorders or they were lost during follow-up. Three patients were excluded from the study after they were diagnosed as having secondary thrombocytopenia caused by viral infections or platelet function disorder. Thrombocytopenia and platelet function disorders were not included in the evaluation.

This study was thus conducted with 99 patients (Figure 1).

All the admission symptoms, history, physical examination findings, laboratory test results, and initial and definitive diagnoses are based on the database from the hospital’s automation system and the patients’ charts.

We recorded the patients’ sex, age, symptoms, site of bleeding, duration of hemorrhage, existence of any bleeding 339

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problems in the newborn period, and previous personal or family history of bleeding disorders.

The first primary tests performed on patients suspected of having a bleeding disorder were complete blood count, bleeding time or PFA-100, PT, aPTT, TT, and fibrinogen levels. A peripheral blood smear was performed in all patients to evaluate platelet count and size, thus excluding pseudothrombocytopenia. Bleeding time was measured either in vitro with the PFA-100 (n=61) or in vivo by Duke’s method (n=5). Advanced laboratory investigations were performed on all patients whose initial tests revealed any pathological findings. VWF antigen, ristocetin cofactor activity (Ricof), and factor level (II, V, VII, VIII, IX, X, XI, XII, XIII) tests were performed in the Pediatric Hematology Hemostasis Laboratory.

The patients with abnormal test results were referred to the Pediatric Hematology and Oncology Unit for advanced evaluation and follow-up. Tests with abnormal results were repeated again at the next visit. The patients’ folders that were created in the Pediatric Hematology and Oncology Unit were evaluated for the definite diagnosis. Initial and definitive diagnoses of these patients were recorded.

Results A total of 26,737 outpatients were admitted to our unit during the 1-year period of study. Ninety-nine (0.37%) patients were initially diagnosed with probable bleeding disorders and were followed up. Fifty-two (52.5%) patients were male and 47 (47.5%) were female, and their mean age was 9.1±4.1 years (minimum-maximum: 2-18 years).

The most frequent symptoms were epistaxis in 36 of the patients (36.4%), easy bruising in 32 (32.3%), prolonged and/or massive menstrual bleeding in 6 (6.1%), and gingival bleeding in 2 (2%) (Table 1). Duration of the symptoms ranged from 2 days to 6 years.

According to the laboratory test results, 63 of the patients (63.6%) had no bleeding disorders, whereas 36 (36.4%) were diagnosed with bleeding disorders (Figure 1). The final diagnosis included vWD type 1 in 8 (8.1%); vWD type 2 in 4 (4%); mild hemophilia A in 4 (4%); vWD type 1 and FXI deficiency in 3 (3%); FV deficiency in 3 (3%); moderate hemophilia A in 2 (2%); hemophilia A carrier in 2 (2%); FVII deficiency in 2 (2%); FXI deficiency in 2 (2%); FX deficiency in 1 (1%); FXII deficiency in 1 (1%); combined FII, VII, IX, X, and FXII deficiency in 1 (1%); combined FV and FVIII deficiency in 1 (1%); combined FVII and FX deficiency in 1 (1%); and hemophilia B carrier in 1 (1%) (Table 1).

Seven (19.4%) of 36 patients who were diagnosed with bleeding disorders had a family bleeding history. Family histories of the patients for coagulation disorders are presented in Table 2.

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Twenty-eight percent of the patients with epistaxis (10 of 36 patients; 3 cases of mild hemophilia A, 2 of vWD type 1, 1 of vWD type 2, 1 of vWD type 1+FXI deficiency, 1 of FVII deficiency, 1 of FV deficiency, and 1 of combined FII, VII, IX, X, and XII deficiency), 28.1% of the patients with easy bruising (9 of 32 patients; 3 cases of vWD type 1, 1 of vWD type 2, 1 of a hemophilia A carrier, 1 of FV deficiency, 1 of FXI deficiency, 1 of FVII+X deficiency, and 1 of FXII deficiency), Table 1. Characteristics of patients initially diagnosed with bleeding disorders (n=99).

Characteristics

n (%)

Sex Female Male

47 (47.5) 52 (52.5)

Symptoms Epistaxis Easy bruising Menorrhagia Gingival bleeding Prolonged bleeding Blood in stool Hematoma under tongue Other causes of outpatient clinic admissions Abnormal PT/aPTT* Examination bleeding diathesis** Preoperative evaluation

36 (36.4) 32 (32.3) 6 (6.1) 2 (2) 1 (1) 1 (1) 1 (1)

11 (11.1) 7 (7.1) 2 (2)

Diagnosis vWD type 1 vWD type 2 Mild hemophilia A vWD type 1+factor XI deficiency Factor V deficiency Moderate hemophilia A Hemophilia A carrier Factor VII deficiency Factor XI deficiency Factor X deficiency Factor XII deficiency Factor II, VII, IX, X, and XII deficiencies Factor V and VIII deficiency Factor VII and X deficiency Hemophilia B carrier

8 (8.1) 4 (4) 4 (4) 3 (3) 3 (3) 2 (2) 2 (2) 2 (2) 2 (2) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1)

PT: Prothrombin time, aPTT: activated partial thromboplastin time, vWD: von Willebrand disease. *: Patients who presented with any symptoms but were assigned only to abnormal prothrombin time, activated partial thromboplastin time. **: Patients had a family history of bleeding disorders and were without symptoms.

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Between 31st of October, 2011, and 31st of October, 2012, the number of patients admitted to the Department of Pediatrics= 26,737 The number of cases initially diagnosed with bleeding disorders = 115 (115/26737=0.43%) Excluded cases=16 • previously diagnosed bleeding disorders (n=2) • lost to follow-up (n=11) • incorrect diagnosis (n=3) Number of cases initially diagnosed as bleeding disorders and included in our study= 99 (99/115=86.1%)

Number of cases definitively diagnosed as bleeding disorders= 36 (36/99=36.4%)

Number of cases received followed-up by the Department of Pediatric Hematology= 20 (20/36=55.5%)

Figure 1. Study flow-chart.

Table 2. Family history of the patients for coagulation disorders.

Number of Patient

Diagnosis of Patient

Family History of Coagulation Disorders

1

Factor XI deficiency

Older sister, factor VIII and XI deficiencies

2

Factor V and VIII deficiencies

Sibling, factor V and VIII deficiencies

3

Moderate-type hemophilia A

Father and older brother, hemophilia A

4

vWD type 1

Sibling, vWD

5

vWD type 1

Mother, vWD

6

vWD type 2

Mother, vWD

7

vWD type 2

Uncle, hemophilia A

vWD: Von Willebrand disease.

and 33.3% of the patients with menorrhagia (2 of 6 patients; 1 a hemophilia B carrier and 1 with FX deficiency) were diagnosed with a bleeding disorder after the first evaluation due to clinic and laboratory results.

Unfortunately, only 20 of 36 patients with bleeding disorders could be evaluated in the Division of Pediatric Hematology and Oncology. When these 20 patients were evaluated again, 16 of them (16/20, 80%) were confirmed to have the same diagnosis that the general pediatrician had established, whereas 4 of them had different diagnoses. One patient with factor V and VIII deficiencies at the Division of Ambulatory Pediatrics was diagnosed with factor V deficiency at the Division of Pediatric Hematology and Oncology. Another

patient with probable vWD type 1 and factor XI deficiency was diagnosed with vWD type 1. Furthermore, a patient with probable mild-type hemophilia A and a probable hemophilia A carrier were not diagnosed with a bleeding disorder after the evaluation in the Division of Pediatric Hematology and Oncology (Table 3).

Discussion The first step for a patient with a suspected bleeding disorder is to get a detailed medical history, such as initial time of bleeding; history of any traumas; patient’s operation history; circumcision history for male patients; any known liver, kidney, or malabsorption-related disorders; and the

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Table 3. Laboratory test results of the patients diagnosed with different bleeding disorders.

Number of Patient

At Division of Ambulatory Pediatrics

At Division of Pediatric Hematology and Oncology

Diagnosis

Levels of factors

Diagnosis

Levels of factors

1

Factors V and VIII deficiencies

Factor V (%): 7 Factor VIII (%): 34

Factor V deficiency

Factor V (%): 16 Factor VIII (%): 110

2

vWD type 1 and factor XI deficiency

vWF antigen (%): 30 Factor XI (%): 28

vWD type 1

vWF antigen (%): 20 Factor XI (%): 80

3

Hemophilia A carrier

Factor VIII (%): 46

Normal

Factor VIII (%): 102

4

Mild-type hemophilia A

Factor VIII (%): 24

Normal

Factor VIII (%): 102

vWD: Von Willebrand disease, vWF: von Willebrand factor.

history of bleeding disorders in other family members. In physical examination, location and type of the bleeding and any accompanying signs should be investigated. First diagnosis can be made for most of the patients with careful medical history and physical examination, and final diagnosis can be made with laboratory tests [1,2,3].

The most common congenital coagulation disorders in childhood are vWD, hemophilia A and B, and factor XI deficiency [1,10]. Nevertheless, rare factor deficiencies may also be seen in childhood [11]. In our study, the patients who had bleeding disorders were mostly diagnosed with vWD, mildtype hemophilia A, and factor XI deficiency. In this study, vWD was the most frequently diagnosed disease, in accordance with the literature. The relatively high rate of rare factor deficiencies may be explained by consanguinity of parents. In addition, our university’s hospital is a tertiary hospital.

The most commonly seen symptoms in patients with bleeding disorders are easy bruising, recurrent epistaxis, prolonged bleeding after circumcision or teeth extraction, menorrhagia, and hemarthrosis [1,4,8,12]. However, these symptoms may vary by age. For example, in the neonatal period, umbilical bleeding, cephalic hematoma, and hematoma and ecchymosis at injection sites can be seen; in infants, mucosal bleeding, easy bruising, and hemarthrosis when the child starts to walk; and in older children, bleeding after surgical procedures [1,7,10]. Nose, skin, and oral mucosal bleedings are easily recognized by parents, whereas gastrointestinal and genitourinary bleeding may not be easily noticed. Therefore, anamnesis is very important. In our study, the most commonly seen symptoms were epistaxis, easy bruising, menorrhagia, and gingival bleeding. Epistaxis is a symptom commonly seen in the general population. Epistaxis may be a symptom of a bleeding disorder, but it may also be due to trauma, nose-picking, sinusitis, rhinitis, nasal polyps, and high blood pressure. In patients manifesting with recurrent

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epistaxis, the rate of detecting a bleeding disorder is 5.5%-33% [13,14]. In our study, of all of patients who were diagnosed with a bleeding disorder, 28% of them had epistaxis. These findings are consistent with the literature data [14].

The rate of bleeding disorders in adult patients with menorrhagia is 15%, but in adolescent patients the rate goes up to 10%-40% [15]. In our study, 2 patients out of 6 (33.3%) with menorrhagia had a bleeding disorder. Therefore, in adolescents with menorrhagia, existence of an underlying bleeding disorder should be investigated. The final diagnosis was different in 4 cases. This shows that a second laboratory evaluation should be done for all patients with bleeding symptoms [16].

In conclusion, rational approaches to children who present with bleeding symptoms require detailed history taking and careful physical examination, followed by adequate laboratory tests to confirm the initial diagnosis. In this study, about 40% of the children presenting with bleeding symptoms were diagnosed with a bleeding disorder in our outpatient clinic after the first evaluation. Additionally, an underlying bleeding disorder should be considered in a child with menorrhagia.

Ethics Committee Approval: The study was conducted with approval of all the faculty members in our department, Informed Consent: The study was conducted with assessment based on the database from hospital’s automation system and the patient’s charts retrospectively, Concept: İsmail Yıldız, Ayşegül Ünüvar, Design: Emin Ünüvar, Fatma Oğuz, Ayşe Kılıç, Data Collection or Processing: İbrahim Kamer, Serap Karaman, Ezgi Uysalol, Analysis or Interpretation: İsmail Yıldız, Ayşegül Ünüvar, Literature Search: İsmail Yıldız, Writing: İsmail Yıldız, Ayşegül Ünüvar.

Conflict of Interest: 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.

Yıldız İ, et al: Evaluation of Children with Bleeding Symptoms

References 1. Lanzkowsky P. Disorders of platelets-hemostatic disorders.

In: Lanzkowsky P (ed). Manual of Pediatric Hematology and Oncology. 5th ed. Amsterdam, Elsevier Academic Press, 2011.

2. Coller BS, Schneiderman PI. Clinical evaluation of hemorrhagic disorders. In: Hoffmann R, Benz JE, Shattil JS (eds). Hematology: Basic Principles and Practice. 4th ed.

Philadelphia, Churchill Livingstone, 2005.

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9. Sarnaik A, Kamat D, Kannikeswaran N. Diagnosis and management of bleeding disorder in a child. Clin Pediatr (Phila) 2010;49:422-431.

10. Avcı Z, Özbek N. Diagnosis and treatment of hemorrhagic diathesis. Turkish J Pediatr 2011;1:81-89.

11. Taşkesen M, Okur N, Okur N, Katar S, Menteş SE, Söker M. Evaluation of fourteen patients with rare coagulation factor deficiencies in childhood. J Child 2008;8:183-186.

3. van Herrewegen F, Meijers JC, Peters M, van Ommen CH. Clinical practice: the bleeding child. Part II: disorders of secondary hemostasis and fibrinolysis. Eur J Pediatr 2012;171:207-214.

12. Celkan T, Yılmaz İ, Demirel A, Çam H, Karaman S, Doğru Ö, Apak H, Özkan A, Taştan Y, Yıldız İ. Bleeding disorders in pediatric emergency department. Turk Arch Ped 2006;41:146150.

4. Khair K, Liesner R. Bruising and bleeding in infants and children-a practical approach. Br J Haematol 2006;133:221231.

13. Gifford TO, Orlandi RR. Epistaxis. Otolaryngol Clin North Am 2008;41:525-536.

5. Revel-Vilk S. Clinical and laboratory assessment of the bleeding pediatric patient. Semin Thromb Hemost 2011;37:756-762.

6. Ünüvar A. Kanamalı çocukta laboratuvar testleri ve değerlendirilmesi. Güncel Pediatri Dergisi 2007;5:42-45.

7. Sadler JE. New concept in von Willebrand disease. Annu Rev Med 2005;56:173-191.

8. Rydz N, James PD. Why is my patient bleeding or bruising?

Hematol Oncol Clin North Am 2012;26:321-344.

14. Sandoval C, Dong S, Visintainer P, Ozkaynak MF, Jayabose S. Clinical and laboratory features of 178 children with recurrent epistaxis. J Pediatr Hematol Oncol 2002;24:47-49.

15. Rodriguez V, Alme C, Killian JM, Weaver AL, Khan SP, Simmons PS. Bleeding disorders in adolescents with menorrhagia: an institutional experience. Haemophilia 2013;19:101-102.

16. Bidlingmaier C, Treutwein J, Olivieri M, Kurnik K. Repeated coagulation testing in children. Does it improve the diagnostic value? Hamostaseologie 2011;31(Suppl 1):51-56.

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Research Article

DOI: 10.4274/tjh.2014.0204 Turk J Hematol 2015;32:344-350

Evaluation of Alpha-Thalassemia Mutations in Cases with Hypochromic Microcytic Anemia: The İstanbul Perspective Hipokromik Mikrositer Anemili Olgularda Alfa Talasemi Mutasyonlarının Değerlendirmesi: İstanbul Perspektifi Zeynep Karakaş1, Begüm Koç1, Sonay Temurhan2, Tuğba Elgün2, Serap Karaman1, Gamze Asker3, Genco Gençay3, Çetin Timur4, Zeynep Yıldız Yıldırmak5, Tiraje Celkan6, Ömer Devecioğlu1, Filiz Aydın2 1İstanbul

University İstanbul Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey University İstanbul Faculty of Medicine, Department of Medical Biology, İstanbul, Turkey 3İstanbul University İstanbul Faculty of Medicine, Department of Pediatrics, İstanbul, Turkey 4Göztepe Education and Research Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey 5Şişli Etfal Education and Research Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey 6İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey 2İstanbul

Abstract: Objective: Alpha thalassemia syndromes are caused by mutations on one or more of the four α-globin genes. Mutations could

be either more commonly deletional or non-deletional. As some deletions (3.7 and 4.2) cause α+-thalassemia, some cause (-20.5, MED, THAI, FIL) α0 -thalassemia. The aim of this study was to determine alpha thalassemia mutations in patients with unsolved hypochromic microcytic anemia and to evaluate types of mutations.

Material and Methods: Two hundred six patients with hypochromic microcytic anemia were evaluated for alpha thalassemia. A venous blood sample of 2 mL was drawn from each patient for DNA isolation. The samples were investigated for α-thalassemia mutations by using the Vienna Lab α-Globlin StripAssay TM commercial kit.

Results: Fourteen different mutations were determined in 95 (46.1%) patients. The most common mutation was the 3.7 single gene deletion and was found in 37 patients (n=37/95, 39%). Others common mutations were the 20.5 kb double gene deletion (n=20 patients, 21%), MED double gene deletion (n=17 patients, 17.9%), α2 IVS1 (n=10 patients, 10.5%), α2 cd142 Hb Koya Dora (n=6 patients, 6.3%), α2 polyA1 (Saudi type) (n=6 patients, 6.3%), 4.2 single gene deletion (n=4 patients, 4.2%), α1 cd14 (n=2 patients, 2.1%), and -FIL mutation (n=2 patients 2.1%), respectively. Hb Adana, Hb Icaria, α2 init cd and α2 polyA2 (Turkish type) were found in 1% of the patients (n=1). Seven patients (7.4%) had α-thalassemia triplication. In our study, three mutations (Hb Icaria, α1 cd14, α2 init.cd) were determined firstly in Turkey. Seven mutations (-SEA, -THAI, Hb Constant Spring, α2 cd19, α2 cd59, α2 cd125, Hb Paksé) were not determined in this study.

Conclusion: Alpha thalassemia should be considered in the differential diagnosis of hypochromic microcytic anemia especially in cases without iron deficiency and b-thalassemia carrier state. Genetic testing should be performed for the suspicious cases. We also recommend that a national database with all mutations in Turkey should be created to screen the alpha thalassemia cost-effectively.

Keywords: Anemia, Alpha thalassemia, Hb Adana, Hb Icaria, Hb Koya Dora, Mutation, Thalassemia Address for Correspondence: Begüm KOÇ, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey Phone: +90 505 906 27 91 E-mail: begumsirins@hotmail.com Received/Geliş tarihi : May 25, 2014 Accepted/Kabul tarihi : October 13, 2014

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Öz: Amaç: Alfa talasemi sendromları, bir ya da daha fazla a-globin genindeki mutasyonlardan kaynaklanır. Mutasyonlar genelikle delesyonel olmakla birlikte non-delesyonel de olabilir. Bazı delesyonlar (3.7 ve 4.2) α+-talasemiye neden olurken bazıları da (-20.5, MED, THAI, FIL) α0-talasemiye yol açar. Bu çalışma ile İstanbul ilinde, diğer nedenlerle açıklanamayan hipokrom mikrositer anemili olgularda alfa talasemi mutasyonlarını belirlemeyi ve mutasyon tiplerini değerlendirmeyi amaçladık.

Gereç ve Yöntemler: Bu çalışmada 206 hasta alfa talasemi için değerlendirmeye alındı. Her hastadan DNA izolasyonu için 2 ml venöz kan örneği alındı. Strip analiz kiti (ViennaLab Diagnostics GmbH, Austria) kullanılarak alfa talasemi mutasyonları araştırıldı.

Bulgular: Doksan beş hastada (%46,1) 14 farklı mutasyon tespit edildi. En sık saptanan mutasyon 3.7 tek gen delesyonu idi (n=37 hasta, %39). Diğer mutasyonlar sıklık sırasına göre; 20,5 kb çift gen delesyonu (n=20, %21), MED çift gen delesyonu (n=17, %17,9), α2 IVS1 (n=10, %10,5), α2 poly-A1 (Suudi tip) (n=6, %6,3), Hb Koya Dora (n=6, %6,3), 4.2 tek gen delesyonu (n=4, %4,2), FIL mutasyonu (n=2, %2,1) ve α1 cd 14 (n=2, %2,1) idi. Hb Adana (n=1), Hb Ikaria (n=1), α2 init cd (n=1) ve α2 poly-A2 (Türk tipi) (n=1) hastaların %1’inde saptandı. Yedi hasta alfa talasemi gen triplikasyonu (%7,4) taşıyordu. Çalışmamızda üç mutasyon (Hb Icaria, α1 cd14, α2 init.cd) Türkiye’de ilk kez tespit edildi. Yedi mutasyon ise (-SEA, -THAI, Hb Constant Spring, α2 cd19, α2 cd59, α2 cd125, Hb Paksé) hastalarımızda hiç saptanmadı.

Sonuç: Alfa talasemi, hipokrom mikrositer anemilerin ayırıcı tanısında özellikle de demir eksikliği ve beta-talasemi taşıyıcılığının saptanmadığı durumlarda akla getirilmelidir. Şüpheli olgularda genetik açıdan mutasyon taraması yapılmalıdır. Alfa talasemi taramasını daha uygun maliyetle yapabilmek için Türkiye’de saptanan tüm alfa talasemi mutasyonlarının toplandığı ulusal bir veritabanı oluşturulmasını önermekteyiz.

Anahtar Sözcükler: Alfa talasemi, Anemi, Hb Adana, Hb Icaria, Hb Koya Dora, Mutasyon, Talasemi Introduction Alpha thalassemia syndromes are inherited autosomal recessively and caused by defects on one or more of the 4 α-globin genes (αα/αα), leading to reduced or absent production of the alpha-globin polypeptide chains [1,2]. The α-globin gene mutations could be either the more common deletion (partial (α+) deletions or total (α0) deletions) or non-deletional types. There are reported to be more than 40 deletion mutations in various studies [2,3,4]. The most common alpha-thalassemia mutations in the world are the 3.7 single-gene deletions. While α+-thalassemia is caused by single-gene deletions (such as 3.7 and 4.2), α0-thalassemia is caused by double-gene deletions (such as -20.5, SEA, MED, THAI, and FIL). Three-gene deletions (α+ with α0thalassemia) or a combination of two-gene deletions with a non-deletion mutation cause HbH disease. If there are deletion mutations on 4 α-genes, Hb Bart’s hydrops fetalis develops [5,6]. These large deletions have particularly severe phenotypes. On the other hand, non-deletion mutations result in structurally abnormal and instable hemoglobin variants such as Hb Constant Spring, which is the most common, and Hb αTSaudiα, polyA α2, Hb Koya Dora, and Hb Quong Sze [4,7]. Non-deletion mutations may reduce α-globin chain synthesis more severely than most of the deletion mutations [1]. More than 70 non-deletion mutations have been reported [8].

The clinical course of alpha thalassemia is correlated with the number of affected α-globin genes. There are 4 clinical definitions of α-thalassemia syndromes: 1) silent carrier, defined as heterozygous α+-thalassemia (-α/αα) with mostly normal hemoglobin or mild hypochromic anemia; 2) α-thalassemia trait, defined as heterozygous α0thalassemia (--/αα) or homozygous α+-thalassemia (-α/-α) with mild anemia; 3) HbH disease, defined as compound heterozygous α+/α0-thalassemia with 3 inactive α-genes (--/α) with moderate hemolytic anemia; and 4) Hb Bart’s, defined as homozygous α0-thalassemia (--/--) with hydrops fetalis.

Silent carriers and those with α-thalassemia trait are generally clinically asymptomatic and do not need any treatment.

Patients with HbH disease usually have moderate anemia with hepatosplenomegaly; some of them need periodic blood transfusion and folic acid supplementation. Hb Bart’s causes hydrops fetalis prenatally and is fatal if not treated with intrauterine blood transfusions [2,3,8].

The aim of this study was to determine alpha-thalassemia mutations in patients with unsolved hypochromic microcytic anemia and to evaluate the types of mutations.

Materials and Methods Two hundred six individuals either having hypochromic microcytic anemia or being parents and/or siblings of a patient with HbH disease were referred to our institution for screening

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of alpha thalassemia mutations in İstanbul. A venous blood sample of 2 mL was drawn from each patient into the EDTA tubes for DNA isolation. In vitro amplification was made with polymerase chain reaction (PCR) multiplex method using Biotin marked primers belonging to alpha globin encoding gene zones. Products of the amplification process were investigated for mutations of the alpha globulin genes using the Vienna Lab α-Globlin Strip Assay TM commercial kit including 21 alpha thalassemia mutations. These mutations are shown in Table 1.

Results Ninety-five patients (46.1%) with alpha thalassemia mutations were identified. The patients were aged from 1 to 46 years; 52 of those were male and 43 were female.

Deletion mutations were detected in 69.3% of the patients whereas non-deletion mutations in 30.6%. The most common mutation was the 3.7 single gene deletion and was found in 37 patients (39%). Others common mutations were the 20.5 kb double gene deletion (n=20 patients, 21%), MED double gene deletion (n=17 patients, 17.9%), α2 IVS1 (n=10 patients, 10.5%), α2 cd142 Hb Koya Dora (n=6 patients, 6.3%), α2 polyA1 (Saudi type) (n=6 patients, 6.3%), 4.2 single gene deletion (n=4 patients, 4.2%), α1 cd14 (n=2 patients, 2.1%), and -FIL mutation (n=2 patients 2.1%), respectively. Hb Adana, Hb Icaria, α2 init cd and α2 polyA2 (Turkish type) were found in 1% of the patients (n=1). Seven patients (7.4%) had α-thalassemia triplication (Table 2). Some deletions (-SEA, -THAI) and some mutations (α2 cd19, α2 cd59, α2 cd125, Hb Paksé and Hb Constant Spring) were not determined in this study (Table 1).

Discussion According to reports from the World Health Organization, at least 20% of the world’s population is alpha-thalassemia carrier [9]. The geographic distribution of α-thalassemia mutations is especially concentrated in the Mediterranean and Middle Eastern regions, where up to 40% of people are carriers [4,10]. Turkey also has a high alpha-thalassemia frequency because of its geographic position.

Table 1. Positions of the 21 alpha-gene mutations in the strip assay kit.

No

Position

Gene Mutation/Deletion

1

-α3.7

Single-gene deletion

2

-α4.2

Single-gene deletion

3

(α)20.5

Double-gene deletion

4

--MED

Double-gene deletion

5

α2 IVS1

5-bp deletion

6

ααα anti-3.7

Gene triplication

7

α2 cd 142

A>C (Hb Koya Dora)

8

α2 polyA-1

AATAAA>AATAAG (Saudi type)

9

--FIL

Double-gene deletion

10

α1 cd 14

G>A

Fourteen distinct alpha thalassemia mutations were detected in 95 patients. In the total of 190 alleles, the most common mutation was the 3.7 single gene deletion (n=37 alleles, 19.5%). The allele frequencies of the other mutations were: 20.5 kb double gene deletion (n=20 alleles, 10.5%), MED double gene deletion (n=17 alleles, 8.9%), α2 IVS1 (n=10 alleles, 5.2%), α2 polyA1 (Saudi type) (n=7 alleles, 3.7%), alpha triplication (n=7 alleles, 3.7%), Hb Koya Dora (n=6 alleles, 3.1%), 4.2 single gene deletion(n=4 alleles, 2.1%), α1 cd14 (n=2 alleles, 1%), and -FIL mutation (n=2 alleles, 1%), respectively (Table 3). The allele frequencies of Hb Adana, Hb Icaria, α2 init cd and α2 polyA2 (Turkish type) were found to be 0.5% (Table 3). Three mutations (Hb Icaria, α1 cd14, α2 init.cd) were detected for the first time in Turkey.

11

α1 cd 59

G>A (Hb Adana)

12

α2 polyA-2

AATAAA>AATGAA (Turkish type)

13

α2 cd 142

T>A (Hb Icaria)

14

α2 init.cd

ATG>ACG

15*

--THAI

Double-gene deletion

16*

--SEA

Double-gene deletion

17*

α2 cd 125

T>C (Hb Quong Sze)

18*

α2 cd 142

T>C (Hb Constant Spring)

The genetic results of the patients showed that 28 patients (29.4%) were silent carriers, 45 patients (47.3%) had alpha thalassemia trait, and 15 patients (15.8 %) had Hemoglobin H disease (Table 2). Seven patients with alpha triplication were not grouped phenotypically.

19*

α2 cd 19

-G

20*

α2 cd 142

A>T(Hb Paksé)

21*

α2 cd 59

G>A

*: Not detected in this study.

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Karakaş Z, et al: Alpha-Thalassemia Mutations: The İstanbul Perspective

The first publication on alpha-thalassemia from Turkey was that of Özsoylu and Malik, who studied alpha-thalassemia by column chromatography in 1982 [11]. The first screening study of alpha-thalassemia by sensitive DNA method (gene mapping) was published in 1989 [12]. The frequency of

alpha-thalassemia was detected at 3.6%, while the frequency of alpha-gene triplication was also 3.6%. Arcasoy reported that the frequency of alpha-thalassemia in Turkey was 0.25% [13]. Kılınç et al. studied the cord blood of newborns and reported the frequency of alpha-thalassemia carriers to

Table 2. Genotypes and phenotypes of the patients with alpha-thalassemia.

Genotype

Mutation Type

Phenotype

n (%)

α-3.7 α/α α

Deletional

Silent carrier

19

(αα)-MED/αα

Deletional

α-thal trait

14

α-3.7 α/ α-3.7 α

Deletional

α-thal trait

7

(αα)-20.5/αα

Deletional

α-thal trait

13

(αα)-20.5/α-3.7 α

Deletional

HbH

5

(αα)-MED/α-4.2 α

Deletional

HbH

2

α-4.2

Deletional

Silent carrier

1

(αα)-FIL/α-3.7 α

Deletional

HbH

2

Total

63 (66.3%)

αIVS1α/αα

Non-deletional

Silent carrier

3

αHbKD* α/αα

Non-deletional

Silent carrier

4

αHbIC**α/αα

Non-deletional

Silent carrier

1

αPA1α/αα

Non-deletional

α-thal trait

4

αIVS1α/αIVS1α

Non-deletional

α-thal trait

2

αPA1α/α PA1α

Non-deletional

HbH

1

ααcd14/αα

Non-deletional

α-thal trait

1

ααcd14/αHbKD*α

Non-deletional

HbH

1

αinit.cd***α/αPA1α

Non-deletional

α-thal trait

1

Total

18 (19%)

α-3.7 α/αIVS1α

Deletional/non-deletional

α-thal trait

2

(αα)-20.5/αIVS1α

Deletional/non-deletional

HbH

2

(αα)-MED/αPA2α

Deletional/non-deletional

HbH

1

α-4.2α/αIVS1α

Non-deletional

α-thal trait

1

α-3.7

Deletional/non-deletional

HbH

1

Total

7 (7.3%)

αanti-3.7α/αα

Triplication

5

αanti-3.7α/αHbKD*α

Triplication/non-deletional

1

αanti-3.7α/α-3.7 α

Triplication/deletional

1

Total

7 (7.3%)

Total

95 (100%)

α/α

α/ααcd59

*HbKD: Hb Koya Dora, **HbIC: Hb Icaria, n: patient number.

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Table 3. The allele frequencies of the alpha thalassemia mutations in different studies from Turkey.

Alpha Thalassemia Mutations

İstanbul present study, 2014, n=95, %

Aegean Onay et al., 2013, n=229,% [24]

Hatay Celik et al., 2013, n=97,% [19]

Isparta Sütçü et al., 2011, n=9,% [18]

Adana Guvenc et al., 2010, n=225,% [17]

Çukurova Çürük, 2007, n=32,% [20]

Turkey Oner et al., 1997, n=25,% [21]

Cyprus Baysal et al., 1995, n=78, % [25]

Study population

Patients with HMA

Patients with HMA

Patients with HMA

-

Premarital couples/ Patients with HMA

Patients with Patients Patients HbH with HbH with HbH

Material Method Strip assay

Strip assay

Strip assay

Strip assay

Strip assay

Gene mapping by Q probe

DNA

DNA

-α3.7

23.1

52.2

43.8

5.5

40.6

29.6

28

30

(α)20.5

9.4

14.2

0.5

11.1

3.3

18.8

22

4

--MED

8.9

11

5.6

27.7

9.5

14.1

20

40

α2 IVS1 (α2-5nt)

6.3

4

6.7

5.5

0

4.7

8

10

ααα anti3.7

3.7

3.2

1.5

0

1.1

0

0

0

α2 cd142 (Hb Koya Dora)

3.1

1.8

0

0

0

0

α2 polyA-1

3.7

9

0.5

0

0.7

4.7

0

12

-α4.2

2.1

0.5

0

0.6

1.6

12

12

α1 cd 14

1

0

0

0

0

--FIL

1

3.2

0.5

0

0

0

0

0

α2 polyA-2

0.5

1.4

2.5

0

2

7.8

10

4

α2 cd 142 (Hb Icaria)

0.5

0

0

0

0

α2 init.cd

0.5

0

0

0

0

0

0

α1 cd 59 (Hb Adana)

0.5

0

0

0

6.2

n: Patients with alpha thalassemia mutations, *HMA: Hypochromic microcytic anemia.

be 2.9% in the Adana region in 1986, while Canatan et al. reported the frequency of alpha-thalassemia as 2.5%-6.5% in the Antalya region [14,15,16]. Guvenc et al. reported the incidence of α-thalassemia as 7.5% in selected patients in the Adana region [17]. The diagnosis of alpha-thalassemia is also important in patients with unsolved hypochromic microcytic anemia. We found a rate of alpha-thalassemia as high as 46.1% among selected patients with hypochromic microcytic anemia in İstanbul.

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The types of alpha-thalassemia mutations are variable depending on geographic region. Although 21 mutations were screened, we found 14 different alpha-thalassemia mutations in patients who lived in İstanbul. The most common mutations were the 3.7 single-gene deletion, 20.5 double-gene deletion, MED double-gene deletion, α2 IVS-1, 3.7 gene triplication, Hb Koya Dora, α2 polyA1, 4.2 single-gene deletion, α1 cd 14, and FIL mutation, respectively. These mutations were seen in 95% of our patients, similar to reports from other parts of Turkey (Table 3).

Turk J Hematol 2015;32:344-350

Karakaş Z, et al: Alpha-Thalassemia Mutations: The İstanbul Perspective

According to the study by Guvenc et al., the rate of 3.7-kb deletion was extremely high (53.3%) in selected patients from Adana [17]. They also performed the largest study in Turkey with 3000 premarital couples and anemic patients, excluding those with iron deficiency, and they detected alpha-thalassemia mutations in 225 patients. They demonstrated 11 different genotypes; the 3.7 single-gene deletion and MED double-gene deletion were the most prevalent genotypes in their study.

Sütçü et al. reported that the most common alphathalassemia mutations were the MED double-gene deletion, 20.5-kb double gene deletion, 3.7 single-gene deletion, and α2IVS 1-5 nt, respectively, in the Isparta area in the south of Turkey [18]. Although they tested few patients and detected mutations in only 9 patients, their most common mutations were similar to those of other studies in Turkey.

Celik et al. demonstrated 9 distinct mutations and the frequencies of the mutations in Hatay [19]. They tested 330 individuals and detected mutations in 97 patients. Their inclusion criteria for the study were similar to ours. They reported that 3.7 single-gene deletions were the most common mutation at 43.81%. In addition, they reported FIL doublegene deletion for the first time in 2012 in Turkey. We also detected FIL mutation in 2 patients in İstanbul. In their study, deletion mutations were detected in 81.8% of the patients and non-deletion mutations in 18.2%, whereas deletion mutations were found in 69.3% and non-deletion mutations in 30.6% in our study. These findings are similar to those of other studies addressing alpha-thalassemia in the world.

HbH is the most common condition that arises from the deletions of 3 α-globin genes (--/-α) and rarely by the combination of deletion and non-deletion mutations affecting the α-globin genes. There are also published studies from Turkey about HbH disease [20,21,22,23]. Çürük reported mutations in 32 patients with HbH disease [20]. In that study, 20 patients with HbH had 3 alpha-gene deletions, while the remaining 12 cases were caused by the combination of alphagene deletion and point mutation. In our study, 15 patients were evaluated as having HbH disease; 9 of them had 3 α-globin gene deletions, 2 of them had non-deletion mutations, and the other cases were caused by the combination of deletion and non-deletion mutations, as shown in Table 2.

We found a very heterogeneous distribution of alphathalassemia mutations. This heterogeneity could be because İstanbul is the city in Turkey receiving the most migrants. We present the results of our study and other studies from Turkey in Table 3 [17,18,19,20,21,24,25]. In our study, we found 3 mutations that not been reported previously in Turkey. One-third

of the mutations from the strip assay kit were not determined, similar to other studies from different parts of Turkey.

In our study, patients were identified as being silent carriers, having alpha-thalassemia traits, or having HbH disease on the basis of genetic mutations. For example, patients with single-gene deletion were defined as silent carriers. We determined clinical definitions for the patients according to their genetic mutations.

Most silent carriers have normal hemoglobin levels in the general population. Our results showed that the silent carriers had mild hypochromic microcytic anemia because our study group included only patients with hypochromic microcytic anemia.

Finally, silent carriers of alpha-thalassemia could be mostly normal or mildly anemic, as shown in the literature [2,3,8].

Screening for the 7 most common mutations, present in >95% of patients, is recommended in Canada [26]. The strip assay method for alpha-thalassemia genetic testing can be used effectively due to homogeneity of mutations in the public. It is also cost-effective for the most commonly seen mutations in patients with otherwise unexplained, longstanding, hypochromic microcytic anemia.

In conclusion, we found a high rate (46.1%) of alphathalassemia mutations among patients with long-standing hypochromic microcytic anemia. Alpha-thalassemia should be considered in the differential diagnosis of hypochromic microcytic anemia, especially in cases without iron deficiency and α-thalassemia carrier states. Genetic testing should be performed for these suspicious cases. Furthermore, we recommend that a national database including all mutations in Turkey should be created to screen alpha-thalassemia mutations cost-effectively.

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

Informed Consent: It was taken, Concept: Zeynep Karakaş, Begüm Koç, Design: Zeynep Karakaş, Begüm Koç, Data Collection or Processing: Zeynep Karakaş, Begüm Koç, Serap Karaman, Gamze Asker, Genco Gençay, Çetin Timur, Zeynep Yıldız Yıldırmak, Tiraje Celkan, Ömer Devecioğlu, Analysis or Interpretation: Filiz Aydın, Sonay Temurhan, Tuğba Elgün, Literature Search: Zeynep Karakaş, Begüm Koç, Writing: Zeynep Karakaş, Begüm Koç.

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

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4. Vichinsky EP. Alpha thalassemia major--new mutations, intrauterine management, and outcomes. Hematology Am Soc Hematol Educ Program 2009:35-41.

5. Chui DH, Waye JS. Hydrops fetalis caused by alphathalassemia: an emerging health care problem. Blood 1998;91:2213-2222.

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7. Galanello R, Cao A. Gene test review. Alpha-thalassemia.

Genet Med 2011;13:83-88.

8. Rachmilewitz EA, Giardina PJ. How I treat thalassemia. Blood 2011;118:3479-3488.

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10. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: an increasing global health problem. Bull World Health Organ 2001;79:704-712.

11. Özsoylu Ş, Malik SA. Incidence of alpha thalassemia in Turkey. Turk J Pediatr 1982;24:235-244.

12. Fei YJ, Kutlar F, Harris HF, Wilson MM, Milana A, Sciacca P, Schiliro G, Masala B, Manca L, Altay C, Gurgey A, Huisman TJH. A search for anomalies in the zeta, alpha, beta, and gamma globin gene arrangements in normal black, Italian, Turkish, and Spanish newborns. Hemoglobin 1989;13:4565.

13. Arcasoy A. Türkiye’de Thalassemia Taşıyıcı Sıklığı. Ankara, Turkey, Ankara Thalassemia Association, 1991 (in Turkish).

14. Kılınç Y, Kumi M, Gürgey A, Altay C. Adana Bölgesi’nde doğan bebeklerde kordon kanı çalışması ile β-talassemi, glukoz6-fosfat dehidrogenaz enzim eksikligi ve Hb S sıklığının araştırılması. DOĞA 1986;10:162-167 (in Turkish).

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17. Guvenc B, Yildiz SM, Tekinturhan F, Dincer S, Akyuzluer I, Okten S, Erkman H. Molecular characterization of alphathalassemia in Adana, Turkey: a single center study. Acta Haematol 2010;124:197-200.

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Brief Report

DOI: 10.4274/tjh.2014.0149 Turk J Hematol 2015;32:351-354

The Efficacy and Safety of Procedural Sedoanalgesia with Midazolam and Ketamine in Pediatric Hematology Çocuk Hematolojide Midazolam ve Ketaminle Uygulanan İşlemsel Sedoanaljezinin Etkinliği ve Güvenilirliği Sema Aylan Gelen, Nazan Sarper, Uğur Demirsoy, Emine Zengin, Esma Çakmak Kocaeli University Faculty of Medicine Hospital, Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey

Abstract: Objective: The aim of this study is to investigate the efficacy and safety of sedoanalgesia performed outside the operating room by pediatricians trained in advanced airway management and life support.

Materials and Methods: Midazolam and ketamine were administered consecutively by intravenous route under cardiorespiratory monitoring for painful procedures of pediatric hematology.

Results: A total of 115 patients had 237 sedoanalgesia sessions. Sedation time was 24.02±23.37 s and sedation success was 92.5% (Ramsay scores of ≥5). Patient satisfaction was high. The recovery time was 28.81±14.4 min. Although statistically significant (p<0.01) increases in systolic and diastolic blood pressure, heart rate, and respiratory rate were observed without clinical importance, they improved without any intervention. No severe adverse events were observed.

Conclusion: Sedoanalgesia with intravenous midazolam and ketamine for pediatric hematology and oncology patients’ painful minor invasive procedures performed in an optimally equipped setting outside the operating room by pediatricians trained and certificated in advanced airway management and life support is effective and safe.

Keywords: Sedoanalgesia, Ketamine, Midazolam, Invasive procedure Öz: Amaç: Çalışmamızın amacı, ileri yaşam desteği konusunda eğitimli pediatristler tarafından ameliyathane dışında gerçekleştirilen ağrılı işlemlerde uygulanan sedoanaljezinin etkinliği ve güvenilirliğinin araştırılmasıdır.

Gereç ve Yöntemler: Pediatrik hematolojinin ağrılı işlemlerinde, kalp-solunum monitorizasyonu yapılarak midazolam ve ketamin ardarda intravenöz yolla uygulanmıştır.

Bulgular: Yüz on beş hastaya 237 sedoanaljezi uygulandı. Sedasyon süresi 24,02±23,37 saniye, sedasyon başarısı %92,5 idi (Ramsay skoru ≥5). Hasta memnuniyeti yüksekti. Uyanma süresi 28,81±14,45 dakika idi. İşlemler sırasında sistolik ve diastolik tansiyon artışı, kalp hızı ve solunum sayısında artış istatistiksel olarak anlamlı (p<0,01) bulunmasına rağmen, klinik olarak anlamlı değildi ve herhangi bir girişim yapılmadan düzeldi. Ciddi yan etki gözlenmedi.

Sonuç: Pediatrik hematoloji ve onkoloji hastalarının ağrılı minör invazif işlemlerinde, intravenöz midazolam ve ketamin ile sedoanaljezi; uygun donanıma, monitorizasyona sahip bir ortamda ve ileri yaşam desteği konusunda eğitimli ve sertifikalı pediatristler tarafından gereğinde yapıldığında, ameliyathane dışında da etkin ve güvenilirdir.

Anahtar Sözcükler: Sedoanaljezi, Ketamin, Midazolam, İnvazif işlem Address for Correspondence: Sema Aylan GELEN, M.D., Kocaeli University Faculty of Medicine Hospital, Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey Phone: +90 532 596 25 54 E-mail: semaylan@hotmail.com Received/Geliş tarihi : April 10, 2014 Accepted/Kabul tarihi : October 17, 2014

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Introduction

Results

Lumbar puncture, bone marrow aspiration/biopsy, and intrathecal therapy are painful procedures. In patients with leukemia, traumatic lumbar puncture due to poor patient stabilization is a diagnostic dilemma and may cause seeding of the blasts into the cerebrospinal fluid from circulation [1,2].

The burden of the procedure under inadequate sedoanalgesia can lead to refusal of the diagnostic procedure or treatment [3].

Between May 2012 and May 2013, a total of 237 invasive procedures (bone marrow biopsy/aspiration, intrathecal chemotherapy) were performed in 115 children (9.4Âą4.5 years, range: 10 months to 19.5 years) with sedoanalgesia.

In this study, the aim was to evaluate the efficacy and safety of procedural sedoanalgesia performed by pediatricians and hematologists trained in advanced airway management and life support.

Materials and Methods This prospective study was planned by pediatric hematologists. The ethics committee of the center approved the study and written informed consent was obtained.

Physicians were trained in advanced life support and had a proficient command of the characteristics and pharmacology of the sedatives/analgesics. One of the physicians performed the invasive procedure, and the other administered the drugs, assisted in patient monitoring, and recorded the vital signs and sedation and recovery times. During the lumbar punctures and intrathecal therapy a nurse assisted in proper positioning of the patient.

Sedation time (the period to induce sedation after the administration of both drugs) and recovery time (the period until the patient was awake with age-appropriate behavior and age-appropriate oriented responses to verbal and motor stimuli after the procedure was accomplished) were recorded.

Sedation was initiated with midazolam (0.1 mg/kg/dose by slow intravenous infusion, maximum 10 mg) and continued with ketamine (1 mg/kg/dose by slow intravenous bolus, maximum 100 mg). Level of sedation was assessed according to the modified Ramsay scale (Table 1). When the score was 5 or 6, which was considered as satisfactory sedation, the procedure was initiated. A score of below 5 was rated as unsatisfactory sedation. Patients were followed by the study nurse for 4 h for any adverse events. Severe adverse events were defined as cardiovascular collapse, airway and respiratory events including hypoxemia requiring resuscitation, and allergic reactions.

Statistical Analysis Statistical analysis was performed using SPSS 2.0 (SPSS Inc., Chicago, IL, USA). For evaluation of demographic characteristics descriptive statistics were used, and for intergroup comparison of the parameters that had normal distribution the paired samples t-test was used.

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Median sedation time was 24.02Âą23.37 s (range: 1-300 s). Median recovery time was 28.81Âą14.45 min (range: 5-90 min). In 87% (n=207) of the sessions no additional dose was administered. Due to prolongation of the procedures or unsatisfactory sedation 1 additional dose of midazolam, 1 additional dose of ketamine, and 2 additional doses of ketamine were administered in 2 (0.8%), 29 (12.2%), and 1 (0.8%) of the sedoanalgesia sessions, respectively. Out of 32 additional doses, 17 (53%) were administered due to multiple painful procedures in the same session.

Oxygen saturation was over 90% in all the patients during sedation and at recovery. There was no apnea, respiratory depression, or need for assisted ventilation/intubation. None of the patients required flumazenil administration. No severe adverse events were observed. Vital signs are shown in Table 2.

A significant increase in systolic and diastolic blood pressure, heart rate, and respiratory rate was observed during sedation and when the procedure was completed compared to baseline values (p<0.01). However, these increases were not clinically significant, and after recovery, they returned to reference values (p<0.01). There was hypersalivation during sedation, when the procedure was completed, and when awake in 16.9%, 24.5%, and 5.1% of the sessions, respectively (p<0.01).

Simply wiping the secretions was enough; no aspiration was required. After the procedures in 6.8% (n=16), 5.1% (n=12), 1.7% (n=4), and 1.3% (n=3) of the sessions, hallucinations, vomiting, agitation, and pain at the procedure site were respectively recorded, but patients recovered without any therapeutic intervention. Hallucinations were seen during recovery but they were transient and self-limited. The overall Table 1. Sedation score (modified Ramsay score).

Sedation Description of Clinical Status Level 1

Fully awake, anxious

2

Calm, adequate cooperation

3

Arousable to verbal commands

4

Arousable to mild stimulation/vigorous reaction to painful stimulus

5

Slow/incomplete reaction to painful physical stimulation

6

No reaction to painful stimulation

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Table 2. Cardiovascular parameters, respiratory rate, and hypersalivation in invasive procedures under midazolam/ ketamine sedoanalgesia.

Before Sedation

During Sedation

Procedure Completed

When Awake

BPsys (mmHg)

118.4±14.5

124.4±16.3

121.9±16.9

113.7±14.8

BPdia (mmHg)

69.1±12.7

77.1±13.2

73.9±13

67.2±11.8

Heart rate (bpm)

115.6±21.7

124.9±21.1

123.3±20.3

113.8±20.5

Respiratory rate (bpm)

21.7±6.5

22.9±6.8

23.7±7

21.5±6.3

Hypersalivation

1 (0.4%)

40 (16.9%)

58 (24.5%)

12 (5.1%)

BPsys/BPdia: Systolic/diastolic blood pressure, bpm: beats per minute. Values are given as means.

adverse event rate was 14.8% (n=35). Sedation was successful in 92.5% (n=219) of the procedures. All the procedures were completed successfully and all the outpatients could be discharged on the same day. Patient satisfaction was high; when painful procedures were repeated all the patients and/or caregivers preferred the same sedoanalgesia.

Discussion In developing countries during painful procedures many centers perform no sedoanalgesia due to limited numbers of anesthesiologists, busy operation rooms, and inadequate training in sedoanalgesia, advanced airway management, and life support [4]. In many studies, it has been shown that sedation and analgesia during painful procedures were administered with equally good results by pediatricians who had received advanced life support training [5,6,7,8].

When midazolam and ketamine are used alone, respiratory depression with midazolam and dysphoric reactions (irritability, depression, etc.) with ketamine may occur. When midazolam is used with ketamine, faster analgesia, amnesia, and fewer side effects occur [9,10,11]. Oxygen desaturation may increase with addition of high-dose midazolam [12].

Therefore, additional doses of ketamine are preferred. In some previous studies with midazolam and ketamine the incidence of oxygen desaturation was between 4.8% and 12%, whereas Ozdemir et al. reported no oxygen desaturation [7,13,14,15,16]. In the present study none of the patients’ oxygen saturation dropped below 90%. Compared to propofol, the combination of ketamine and midazolam was associated with less hypoxemia [13,14].

In recent reports, similar to our findings, a significant increase in cardiovascular parameters was seen due to ketamine’s sympathomimetic action via inhibition of catecholamine reuptake, but these parameters returned to baseline values at recovery and no treatment was required [2,14,15,16,17].

In other published studies the sedation time was similar to that of the present study [2,14,16]. Parker et al. showed that more than 70% of the patients woke up in 30 min, similar to our result [9]. Short sedation and recovery time seems a good feature of the drug combination.

Overall adverse event rate in our study was comparable to those of some other studies [18,19,20]. Ketamine may cause airway obstruction, laryngospasm, and aspiration by increasing tracheal and bronchial secretions. Agents such as atropine and glycopyrrolate can be used to reduce the increased secretions [15,21,22]. The hypersalivation rate was higher compared to those in the literature; this may be due to no atropine or glycopyrrolate administration. Prone position during and after the procedure prevented obstruction of the airway and aspiration was not required.

Venipuncture is another painful procedure for outpatient children, but oral, nasal, and rectal administrations of midazolam can provide slower sedation and this may cause a delay in the procedure. The intramuscular route is also painful and may require additional injections. With the intravenous route, sedoanalgesia can be achieved faster and, if necessary, additional doses and drugs for cardiopulmonary resuscitation can be administered easily.

Conclusion With adherence to the published guidelines, sedoanalgesia with intravenous midazolam and ketamine performed by two physicians, trained in airway management and life support, in an optimally equipped setting outside the operating room is safe and efficient. Sedoanalgesia reduces the physical and psychological trauma of the invasive procedures for the patients, parents, and physicians and increases the success of the procedures.

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Gelen SA, et al: Procedural Sedoanalgesia in Hematology

Concept: Nazan Sarper, Design: Nazan Sarper, Data Collection or Processing: Sema Aylan Gelen, Uğur Demirsoy, Emine Zengin Esma Çakmak, Analysis or Interpretation: Sema Aylan Gelen, Literature Search: Sema Aylan Gelen, Nazan Sarper, Writing: Sema Aylan Gelen, Nazan Sarper.

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

References 1. Maurizi P, Russo I, Rizzo D, Chiaretti A, Coccia P, Attina G, Ruggiero A, Riccardi R. Safe lumbar puncture under analgosedation in children with acute lymphoblastic leukemia. Int J Clin Oncol 2014;19:173-177.

2. Meyer S, Aliani S, Graf N, Reinhard H, Gottschling S. Sedation with midazolam and ketamine for invasive procedures in children with malignancies and hematological disorders: a prospective study with reference to the sympathomimetic properties of ketamine. Pediatr Hematol Oncol 2003;20:291301.

3. Sitaresmi MN, Mostert S, Schook RM, Sutaryo, Veerman AJ. Treatment refusal and abandonment in childhood acute lymphoblastic leukemia in Indonesia: an analysis of causes and consequences. Psychooncology 2010;19:361-367.

4. Iannalfi A, Bernini G, Caprilli S, Lippi A, Tucci F, Messeri A. Painful procedures in children with cancer: comparison of moderate sedation and general anesthesia for lumbar puncture and bone marrow aspiration. Pediatr Blood Cancer 2005;45:933-938.

5. American Society of Anesthesiologists Task Force on Sedation and Analgesia by Non-Anesthesiologists. Practice guidelines for sedation and analgesia by non-anesthesiologists.

Anesthesiology 2002;96:1004-1017.

6. Pitetti RD, Singh S, Pierce MC. Safe and efficacious use of procedural sedation and analgesia by nonanesthesiologists in a pediatric emergency department. Arch Pediatr Adolesc Med 2003;157:1090-1096.

7. Borker A, Ambulkar I, Gopal R, Advani SH. Safe and efficacious use of procedural sedation and analgesia by nonanesthesiologists in a pediatric hematology-oncology unit.

Indian Pediatr 2006;43:309-314.

10. Pellier I, Mongrial JP, Le Moine P, Rod B, Rialland X, Granry JC. Use of intravenous ketamine-midazolam association for pain procedures in children with cancer: a prospective study.

Paediatr Anaesth 1999;9:61-68.

11. Marx CM, Stein J, Tyler MK, Nieder ML, Shurin SB, Blumer JL. Ketamine-midazolam versus meperidine-midazolam for painful procedures in pediatric oncology patients. J Clin Oncol 1997;1:94-102.

12. Cheuk DK, Wong WH, Ma E, Lee TL, Ha SY, Lau YL, Chan GC. Use of midazolam and ketamine as sedation for children undergoing minor operative procedures. Support Care Cancer 2005;13:1001-1009.

13. Godoy ML, Pino AP, Córdova LG, Carrasco OJA, Castillo MA. Sedation and analgesia in children undergoing invasive procedures. Arch Argent Pediatr 2013;111:22-28.

14. Gottschling S, Meyer S, Krenn T, Reinhard H, Lothschuetz D, Nunold H, Graf N. Propofol versus midazolam/ketamine for procedural sedation in pediatric oncology. J Pediatr Hematol Oncol 2005;27:471-476.

15. Karapinar B, Yilmaz D, Demirağ K, Kantar M. Sedation with intravenous ketamine and midazolam for painful procedures in children. Pediatr Int 2006;48:146-151.

16. Ozdemir D, Kayserili E, Arslanoglu S, Gulez P, Vergin C. Ketamine and midazolam for invasive procedures in children with malignancy: a comparison of routes of intravenous, oral and rectal administration. J Trop Pediatr 2004;50:224-228.

17. Roback MG, Wathen JE, Bajaj L, Bothner JP. Adverse events associated with procedural sedation and analgesia in pediatric emergency department: a comparison of common parenteral drugs. Acad Emerg Med 2005;12:508-513.

18. Ozkan A, Okur M, Kaya M, Kaya E, Kucuk A, Erbas M, Kutlucan L, Sahan L. Sedoanalgesia in pediatric daily surgery.

Int J Clin Exp Med 2013;6:576-582.

19. Migdady MI, Hayajneh WA, Abdelhadi R, Gilger MA. Ketamine and midazolam sedation for pediatric gastrointestinal endoscopy in the Arab world. World J Gastroenterol 2011;17:3630-3635.

20. Wood M. The use of intravenous midazolam and ketamine in pediatric dental sedation. SAAD Dig 2013;29:18-30.

8. Monroe KK, Beach M, Reindel R, Badwan L, Couloures KG, Hertzog JH, Cravero JP. Analysis of procedural sedation provided by pediatricians. Pediatr Int 2013;55:17-23.

21. Wathen JE, Roback MG, Mackenzie T, Bothner JP. Does midazolam alter the clinical effects of intravenous ketamine sedation in children? A double-blind, randomized, controlled, emergency department trial. Ann Emerg Med 2000;36:579-588.

9. Parker RI, Mahan RA, Guigliano D, Parker MM. Efficacy and safety of intravenous midazolam and ketamine as sedation for therapeutic and diagnostic procedures in children. Pediatrics 1997;99:427-431.

22.

Ramaiah R, Bhananker S. Pediatric procedural sedation and analgesia outside the operating room: anticipating, avoiding and managing complications. Expert Rev Neurother 2011;11:755-763.

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Case Report

DOI: 10.4274/tjh.2014.0416 Turk J Hematol 2015;32:355-358

A Hemophagocytic Lymphohistiocytosis Case with Newly Defined UNC13D (c.175G>C; p.Ala59Pro) Mutation and a Rare Complication Yeni Tanımlanan UNC13D (c.175G>C; p.Ala59Pro) Mutasyonlu Hemofagositik Lenfohistiositozlu Bir Hasta ve Nadir Komplikasyon Yasemin Işık Balcı1, Funda Özgürler Akpınar2, Aziz Polat1, Fethullah Kenar3, Bianca Tesi4, Tatiana Greenwood4, Nagihan Yalçın5, Ali Koçyiğit6 1Pamukkale

University Faculty of Medicine, Department of Pediatric Hematology, Denizli, Turkey University Faculty of Medicine, Department of Pediatrics, Denizli, Turkey 3Pamukkale University Faculty of Medicine, Department of Otorhinolaryngology, Denizli, Turkey 4Karolinska University Hospital Huddinge, Stockholm, Sweden 5Pamukkale University Faculty of Medicine, Department of Pathology, Denizli, Turkey 6Pamukkale University Faculty of Medicine, Department of Radiology, Denizli, Turkey 2Pamukkale

Abstract: Hemophagocytic lymphohistiocytosis (HLH) represents a severe hyperinflammatory condition with cardinal symptoms of prolonged fever, cytopenias, hepatosplenomegaly, and hemophagocytosis by activated, morphologically benign macrophages with impaired function of natural killer cells and cytotoxic T lymphocytes. A 2-month-old girl, who was admitted with fever, was diagnosed with HLH and her genetic examination revealed a newly defined mutation in the UNC13D (c.175G>C; p.Ala59Pro) gene. She was treated with dexamethasone, etoposide, and intrathecal methotrexate.

During the second week of treatment, after three doses of etoposide, it was noticed that there was a necrotic plaque lesion on the soft palate. Pathologic examination of debrided material in PAS and Grocott staining revealed lots of septated hyphae, which was consistent with aspergillosis infection. Etoposide was stopped and amphotericin B treatment was given for six weeks. HLH 2004 protocol was completed to eight weeks with cyclosporine A orally. There was no patient with invasive aspergillosis infection as severe as causing palate and nasal septum perforation during HLH therapy. In immuncompromised patients, fungal infections may cause nasal septum perforation and treatment could be achieved by antifungal therapy and debridement of necrotic tissue.

Keywords: Hemophagocytic lymphohistiocytosis, Invasive aspergillosis infection, UNC13D (c.175G>C; p.Ala59Pro)

Öz: Hemofagositik lenfohistiositoz (HLH) uzamış ateş, sitopeni, hepatosplenomegali semptomları ile seyreden, active olmuş, morfolojik olarak benign makrofaj ve doğal öldürücü hücreler ile sitotosik T lenfosit fonksiyon bozukluğu sonucu gelişen hiperenflamatuvar bir durumdur. İki aylık düşmeyen ateş yakınması ile başvuran hasta HLH tanısı aldı ve hastanın genetik incelemesinde UNC13D (c.175G>C; p.Ala59Pro) geninde yeni tanımlanan bir mutasyon saptandı.

Hastaya deksamatazon, etopozit ve intratekal metotreksat tedavileri başlandı. Tedavinin 2. haftasında, üç doz etopozit aldıktan sonra, yumuşak damakta plak lezyonu fark edildi ve bu nekrotik lezyon debride edildi. Debridman Address for Correspondence: Yasemin IŞIK BALCI, M.D., Pamukkale University Faculty of Medicine, Department of Pediatric Hematology, Denizli, Turkey Phone: +90 532 547 71 79 E-mail: dryibalci@gmail.com Received/Geliş tarihi : October 21, 2014 Accepted/Kabul tarihi : January 15, 2015

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Işık Balcı Y, et al: Invasive Aspergillosis in HLH with Novel UNCD13 Mutation

materyalinin patolojik incelemesinin PAS, Grocott boyamasında aspergilloz enfeksiyonu ile uyumlu olarak çok sayıda septalı hif görüldü. Etopozid tedavisi sonlandırılarak altı hafta boyunca amphotericin B tedavisi verildi. HLH 2004 tedavi protokolü oral siklosporin ile sekiz haftaya tamamlandı. HLH tedavisi sırasında yumuşak damak perforasyonuna neden olacak kadar ağır aspergilloz enfeksiyonu geçiren bir olgu bildirilmemiştir. İmmünyetmezlikli hastada mantar enfeksiyonları nazal septum perforasyonuna neden olabilmektedir ve tedavi nekrotik dokunun debridmanı ve antifungal tedavi ile sağlanabilmektedir.

Anahtar Sözcükler: Hemofagositik lenfohistiositoz, İnvaziv aspergilloz enfeksiyonu, UNC13D (c.175G>C; p.Ala59Pro)

Introduction Hemophagocytic lymphohistiocytosis (HLH) is a severe life-threatening disease precipitated by secretion of cytokines from morphologically benign macrophages, which ends with uncontrolled hyperinflammation with prolonged fever, cytopenias, hepatosplenomegaly, and hemophagocytosis.

Elevation of triglycerides, ferritin, lactate dehydrogenase, and transaminase levels and decreases in fibrinogen levels are characteristic findings [1]. Impaired cytotoxic function of T cells and natural killer cells is a known cause of familial forms of HLH. The HLH-2004 protocol with immunomodulatory and cytotoxic drugs is used for treatment of patients with HLH [2]. Importantly, invasive infections have been reported in up to 56% of children with HLH on chemotherapy, with invasive fungal infections causing 50% of deaths among such cases [3].

Here we report an unusual aspergillosis infection with palate and nasal septum perforation following chemotherapy in a patient with familial HLH with a novel mutation in UNC13D. Notably, the fungal infection in our patient was treated successfully with antifungal therapy and surgical debridement.

Case Presentation A 2-month-old girl, born subsequent to a term gestation with unrelated parents and an unremarkable previous history, was referred to our clinic with unremitting fever since 1 month despite repeated intravenous administrations of antibiotics.

There was no history of sibling death in her family. On admission, vital signs were normal except body temperature of 38.7 °C. The patient was pale with petechial rashes on the lower extremities. She displayed hepatomegaly (6 cm below costal margin) and splenomegaly (3 cm below costal margin).

Informed consent was obtained.

The patient’s laboratory findings were as follows; hemoglobin: 63 g/L, mean corpuscular volume (MCV): 88.9 fL, total leukocyte count: 2.84x109/L, thrombocyte count: 10x109/L, alanine aminotransferase (ALT): 50 IU/L, aspartate aminotransferase (AST): 62 IU/L, total bilirubin: 0.58 mg/ dL, direct bilirubin: 0.7 mg/dL, gamma glutamyl transferase: 328 U/L, albumin: 2.9 g/dL, ferritin: 2000 ng/mL, triglyceride:

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617 mg/dL, LDL cholesterol: 11 mg/dL, HDL cholesterol: 7 mg/dL, lactate dehydrogenase: 379 U/L, uric acid: 2.3 mg/dL, fibrinogen: 107 mg/dL. Her renal function tests and electrolytes were normal. Her peripheral smear revealed 4% neutrophils, 90% lymphocytes, and 6% monocytes. Absolute neutrophil count was 0.113x109/L. No hemolysis or blasts were visible in her peripheral blood smear. Her transaminase levels increased on the third day of administration (AST: 280 IU/L, ALT: 265 IU/L). Serological studies for infection with Epstein-Barr virus, parvovirus B19, cytomegalovirus, Toxoplasma gondii, rubella, Leishmania, and hepatitis were all negative. Natural killer cell activity and soluble IL-2 level could not be analyzed.

Numerous histiocytes showing hemophagocytosis were observed in the bone marrow aspiration smears.

Conclusively, the patient fulfilled a required 5 out of 6 examined diagnostic criteria for the diagnosis of HLH [1].

Accordingly, the patient was treated with the HLH-2004 protocol with dexamethasone, etoposide, and cyclosporine A. Mutation analyses, identifying a novel homozygous variant in UNC13D (c.175G>C; p.Ala59Pro), confirmed a diagnosis of familial HLH. The variant was not found in the healthy population (1000 Genomes database), and it was predicted as possibly damaging by PolyPhen-2 but as tolerated by sorting tolerant from intolerant (SIFT). The father was a heterozygous carrier of the mutation, while the mother could not be tested.

During the second week of treatment, after 3 doses of etoposide at 150 mg/m2/dose, a grossly necrotic soft tissue lesion was noticed on the soft palate and was successfully excised (Figure 1). During the operation an oronasal fistula was revealed, as well as a perforation of the caudal side of the nasal septum along with an abscess formation in the left vestibular floor. Although there was no microbial growth in the necrotic material, microscopic examination of the debrided material in PAS and Grocott staining showed abundant septatedhyphae, consistent with aspergillosis infection (Figure 2). Etoposide was stopped and amphotericin B treatment was given for 6 weeks at a dosage of 3.5 mg/kg/ day. The HLH-2004 protocol was followed for 8 weeks with cyclosporine A and dexamethasone orally.

Işık Balcı Y, et al: Invasive Aspergillosis in HLH with Novel UNCD13 Mutation

Turk J Hematol 2015;32:355-358

Discussion and Review of the Literature Herein we describe the disease course of a patient carrying a novel homozygous UNC13D mutation. Familial HLH cases typically have an earlier presentation, with infectious agents including herpes viruses such as the Epstein-Barr virus precipitating disease. However, in our case, we did not detect an infectious etiological agent. For treatment, chemoimmunotherapy (etoposide, dexamethasone, cyclosporine A, and, for selected patients, intrathecal methotrexate or corticosteroids) is recommended, but for severe disease or familial cases hemopoietic stem cell transplantation is life saving [4].

Figure 1. Partial perforation in septal cartilage.

Opportunistic infections are a common complication of immunosuppression caused by cytotoxic treatment of the disease and by the disease itself. As our case illustrates, HLH patients have a potential risk of developing invasive fungal infections that can be severe. Aspergillus species have emerged as an important cause of life-threatening infections in immunocompromised patients. Highlighting the severity of invasive fungal infections, 6/12 (50%) fatal cases in a study cohort of 18 children with primary HLH were reported to be caused by invasive fungal infections, of which 2 cases were diagnosed with invasive Aspergillus infection first at autopsy [5].

Aspergillus can differentiate into hyphal forms that produce toxins damaging epithelial tissue, leading to invasion of connective and vascular tissue by the fungi, which subsequently can result in thrombosis and ultimately necrosis of hard and soft tissues with perforation. Systemic antifungal therapy and surgical resection or debridement is important for the management of invasive sinonasalaspergillosis.

Amphotericin B, voriconazole, and caspofungincan be considered for antifungal therapy [5]. Our case was treated successfully with surgical debridement and 6 weeks of amphotericin B treatment.

Figure 2. Septated hyphae with 45° angle branching in aspergillosis (Grocott, 100x).

At the most recent follow-up, after 4 months, the patient still presented with a 2-cm hepatosplenomegaly, while her soft palate had successfully epithelialized. However, there is a permanent deformity of her nose. Her laboratory findings were as follows; hemoglobin: 103 g/L, MCV: 77.7 fL, total leukocyte count: 15,280x109/L, thrombocytes: 230,000x109/L, ALT: 32 IU/L, AST: 12 IU/L, ferritin: 916 ng/mL, triglyceride: 421 mg/dL, LDL cholesterol: 78 mg/dL, HDL cholesterol: 20 mg/ dL, lactate dehydrogenase: 226 U/L, uric acid: 1.7 mg/dL, fibrinogen: 233 mg/dL. Until bone marrow transplantation she was treated with oral cyclosporine A (at the HLH-2004 protocol dosage), trimethoprim sulfamethoxazole, and fluconazole.

In the English literature, the case of a 15-year-old boy who developed fungal infection with nasal septal perforation after bone marrow transplantation for acute myeloid leukemia was reported. He was also treated successfully with surgical debridement and amphotericin B [6].

Our report represents an interesting case of familial HLH caused by a novel homozygous UNC13D mutation and affected by invasive sinonasal aspergillosis.

The UNC13D gene encodes for the Munc13-4 protein, a critical effector of the exocytosis of cytotoxic granules priming cytotoxic granule fusion. Munc13-4 deficiency impairs the delivery of the effector proteins, perforin and granzymes, into the target cells, resulting in defective cellular cytotoxicity and a clinical picture that appears very similar to that of FHL-2 [7].

UNC13D mutations are present in almost 30%-40% of familial HLH cases [8].

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Işık Balcı Y, et al: Invasive Aspergillosis in HLH with Novel UNCD13 Mutation

To the best of our knowledge, no c.175G>C; p.Ala59Pro mutation in UNC13D has been presented before in the literature. This novel mutation may be responsible for our patient’s severe clinical condition. However, to compare the mutation type and clinical course, there is a need for clinical studies.

4. Horne A, Janka G, Maarten Egeler R, Gadner H, Imashuku S, Ladisch S, Locatelli F, Montgomery SM, Webb D, Winiarski J, Filipovich AH, Henter JI; Histiocyte Society.

Haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis. Br J Haematol 2005;129:622-630.

We want to emphasize the importance of awareness of the occurrence of potentially life-threatening invasive fungal infections in patients with HLH. Furthermore, this highlights the efficacy of surgical debridement and amphotericin B for successful treatment of fungal infections with focal lesions.

Informed Consent: Informed consent was obtained, Concept: Aziz Polat, Design: Yasemin Işık Balcı, Data Collection or Processing: Funda Özgürler Akpınar, Analysis or Interpretation: Bianca Tesi, Tatiana Greenwood, Fethullah Kenar, Literature Search: Funda Özgürler Akpınar, Writing: Funda Özgürler Akpınar, Bianca Tesi.

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

References 1. Janka GE. Hemophagocytic 2007;21:245-253.

syndromes.

Blood

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2. Henter JI, Horne A, Arico M, Horne A, Aricó M, Egeler RM, Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D, Winiarski J, Janka G. HLH-2004: Diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007;48:124-131.

3. Sung L, Weitzman SS, Petric M, King SM. The role of infections in primary hemophagocytic lymphohistiocytosis: a case series and review of the literature. Clin Infect Dis 2001;33:16441648.

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5. Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Segal BH, Steinbach WJ, Stevens DA, van Burik JA, Wingard JR, Patterson TF; Infectious Diseases Society of America. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 2008;46:327-360.

6. Shannon MT, Sclaroff A, Colm SJ. Invasive aspergillosis of the maxilla in an immunocompromised patient. Oral Surg Oral Med Oral Pathol 1990;70:425-427.

7. Cetica V, Pende D, Griffiths GM, Aricò M. Molecular basis of familial hemophagocytic lymphohistiocytosis. Haematologica 2010;95:538-541.

8. Sieni E, Cetica V, Hackmann Y, Coniglio ML, Da Ros M, Ciambotti B, Pende D, Griffiths G, Aricò M. Familial hemophagocytic lymphohistiocytosis: when rare diseases shed light on immune system functioning. Front Immunol 2014;5:167.

Case Report

DOI: 10.4274/tjh.2015.0016 Turk J Hematol 2015;32:359-362

The Use of Low-Dose Recombinant Tissue Plasminogen Activator to Treat a Preterm Infant with an Intrauterine Spontaneous Arterial Thromboembolism Intrauterin Spontan Arteriyel Trombozlu Bir Preterm Bebeğin Düşük Doz Rekombinan Doku Plazminojen Aktivatörü ile Tedavisi Yaşar Demirelli, Kadir Şerafettin Tekgündüz, İbrahim Caner, Mustafa Kara Atatürk University Faculty of Medicine, Division of Neonatology, Erzurum, Turkey

Abstract: Neonatal thromboembolic events are rare, and only a few cases of intrauterine spontaneous arterial thromboembolisms have been reported in the literature. Thrombolytic therapy with recombinant tissue plasminogen activator is usually the preferred treatment because it has a short half-life, fewer systemic side effects, and a strong, specific affinity for fibrin. Protocols vary from center to center, but there is still no consensus regarding the proper dosage or treatment duration. Herein, we present the case of an intrauterine spontaneous arterial thromboembolism in a preterm infant that completely resolved after being treated with low-dose recombinant tissue plasminogen activator (0.02 mg/kg/h).

Keywords: Preterm, Thromboembolism, Tissue, Plasminogen, Intrauterine arterial thromboembolism, Low dose recombinant tPA therapy

Öz: Yenidoğanda tromboembolik olaylar sık değildir. Literatürde spontan intrauterin arteriyel tromboz olgusu oldukça az bildirilmiştir. Tromboemboli tedavisinde yarılanma ömrü kısa olduğu için rekombinan doku plazminojen aktivatörü çoğunlukla tercih edilmektedir. Tedavi protokolü merkezden merkeze değişiklik göstermesine karşın, doz ve süre konusunda bir uzlaşı yoktur. Biz burada kullanılabilecek en düşük dozlardan biriyle (0,02 mg/kg/doz) tamamen iyileşme sağladığımız intrauterin spontan arteriyel trombozlu bir olguyu sunmak istedik.

Anahtar Sözcükler: Preterm, Tromboemboli, Doku, Plazminojen, İntrauterin arteriyel tromboembolizm, Düşük doz rekombinant tPA tedavisi

Address for Correspondence: Kadir Şerafettin TEKGÜNDÜZ, M.D., Atatürk University Faculty of Medicine, Division of Neonatology, Erzurum, Turkey Phone: +90 442 344 69 90 E-mail: k.tekgunduz@yahoo.com.tr Received/Geliş tarihi : January 07, 2015 Accepted/Kabul tarihi : May 04, 2015

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Demirelli Y, et al: Thromboembolism in a Preterm Infant

Introduction Spontaneous arterial thromboembolisms are a serious cause of mortality and morbidity in the neonatal period, and the congenital, acquired, and inherited prothrombotic states of this condition along with maternal characteristics have been identified as significant risk factors. The hypofibrinolytic situation in neonates, especially in premature infants, includes hemodynamic changes during the transition from the fetal period to the neonatal period which may predispose infants to these types of thromboembolisms [1]. The goal of treatment is to prevent life-threatening situations that might occur because of the embolism and the recurrence of thrombosis while also minimizing the risk of bleeding. Generally, recombinant tissue plasminogen activator (rtPA) is the first choice of treatment because it is nonantigenic, has a short half-life, produces rapidly reversible hypocoagulability, and possesses a strong, specific affinity for fibrin, but there is no consensus regarding the proper dosage or treatment duration [2]. In this study, we present the case of a premature baby with an intrauterine spontaneous arterial thromboembolism which developed at the level of the left brachial artery and was successfully treated with low-dose rtPA for a short period of time.

(Figure 2). Hence, the rtPA was discontinued although the LMWH continued to be administered for six additional weeks.

At follow-up, no complications or thrombosis had developed, and the screening for inherited thrombophilias [factor V Leiden mutation, homozygous protein C and S deficiency, prothrombin G20210A mutation, methyltetrahydrofolate reductase (MTHFR) gene mutation, antithrombin III, factor 12, anticardiolipin antibodies, homocysteine, and lipoprotein (a)] was normal.

Discussion and Review of the Literature The incidence of clinically apparent neonatal thrombosis in recent reports has varied from 5.1 per 100,000 births to 2.4 per 1,000 admissions [3,4]. Most cases of thromboembolism during the neonatal period are due to vascular interventions;

Case Presentation A male baby (1,530 g) was born at the 32nd gestational week via an emergency caesarean section because of anhydroamnios and vaginal bleeding. The mother had not had prenatal care.

The mother was 24 years old, and neither the mother nor the infant had any complications during the surgical procedure.

The Apgar scores at one and five minutes were 6 and 9 respectively, and the cord blood pH was normal. In addition, there was no history of maternal diabetes nor preeclampsia.

However, the baby did receive a single dose of surfactant due to the presence of mild respiratory distress syndrome. The patient’s left forearm from the elbow to the fingertips had a pale and cyanotic appearance at birth that persisted afterwards (Figure 1), but the baby’s vital signs were normal. No brachial arterial flow was detected from the level of the elbow nor was there any distal radioulnar flow on Doppler ultrasonography (USG). Furthermore, no cardiac pathology was detected on an echocardiographic examination. The blood count was normal, there was no polycythemia or thrombocytopenia, and the coagulometer readings and fibrinogen values were also within normal ranges. After obtaining the informed consent of the infant’s parents, low-dose (0.02 mg/kg/h) rtPA (alteplase) was administered along with low-molecular-weight heparin (LMWH) (Clexane® 4000 IU/0.4 mL; 100 IU/kg/dose twice daily) in the first hour after birth. The transfontanelle USG was normal before and after the rtPA infusion, and distal pulses were detected by Doppler USG at the fourth hour of infusion

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Figure 1. View of the patient’s left forearm from the elbow to the fingertips at birth. Note the pale, cyanotic appearance.

Figure 2. The patient experienced a complete recovery four hours after the recombinant tissue plasminogen activator infusion.

Turk J Hematol 2015;32:359-362

Demirelli Y, et al: Thromboembolism in a Preterm Infant

however, there have been very few reported cases of intrauterine spontaneous arterial thromboembolism in the literature [5]. Various risk factors, such as being an infant of a diabetic mother, having polycythemia, dehydration, sepsis, asphyxia, or oligohydroamnios, and being of the male gender can contribute to this condition, but the pathophysiology has not yet been fully clarified [1,6]. In the case of our patient, we observed cyanosis in the left forearm at birth.

Saracco et al. reported that prepartum risk factors, including emergency caesarean sections, are significantly associated with arterial ischemic stroke in neonates, and Rashish et al. identified decreased fetal movements, oligohydramnios, preeclampsia, and maternal diabetes as maternal risk factors [1,6]. In addition, they also found that when oligohydramnios is present, decreased fetal movement may cause venous stasis and thrombus formation. We believe that the caesarean delivery and anhydroamnios were the primary risk factors in our patient, but we could not determine whether the thromboembolism occured within the uterus or during the birth process. However, the apparent lack of necrosis in the forearm at birth suggests that the thromboembolism occurred just prior to delivery.

Rashish et al. hypothesized that spontaneous arterial thromboembolisms originate in utero and develop secondary to placental-fetal umblical pathology [1]. Furthermore, they reported that the most common site of thromboembolisms were, in order of frequency, the umblical artery, the aorta, and the extremities. In our patient, the left forearm was the site of the thromboembolism. However, no thrombophilic state was detected in our patient, which might have been because of intrauterine pathology.

In newborn infants, appropriate, adequate, timely intervention of thromboembolisms is very important in order to reduce morbidity and mortality. In the literature, there are several studies that have reported the use of streptokinase, urokinase, and rtPA for thrombolytic therapy, and in recent years the popularity of rtPA has been on the rise because of its short half-life, nonantigenic qualities, and locally specific action on plasminogen-bound fibrin [7,8]. In addition, it also has fewer systemic side effects than other agents used in thrombolytic therapy [9,10]. However, this type of therapy is associated with significant bleeding complications such as intracranial hemorrhage, and Monagle et al. determined that the most frequent problem was bleeding at the sites of invasive procedures that required treatment with blood products [11]. Furthermore, they also found a connection between prolonged thrombolytic infusion and increased bleeding.

In our case the short duration of rtPA treatment, which was limited to four hours, may have played a role in the prevention of complications. However, we did inform the patient’s

parents about the possible complications before initiating the treatment.

Many case series have been reported on the use of low and high dose rtPa both with and without a bolus as well as in conjuction with other anticoagulant agents, and there is general agreement that rtPa is safe and effective. There is still no consensus concerning the correct dosage and duration of treatment [3,5,12]. Olgun et al. started rtPa 13 of their 22 patients (range 5 days-17 years old) with extremity or cardiac thrombosis on low-dose treatment (0.01-0.03 mg/kg/h), and in six of these the dosage was increased over the course of the treatment [12]. Their findings showed that seven patients experienced complete recovery within 4 to 36 hours and that no significant complications were seen except for bleeding at the vascular puncture site in two patients. However, five patients with fibrinogen deficiency in the high-dose group reported epistaxis and melena. In our patient, we used lowdose rtPA (0.02 mg/kg/h) and observed a complete recovery within four hours after the infusion without any complications.

We administered rtPA (alteplase) along with LMWH in the first hour after birth, and this treatment has previously been reported to be safe and effective, especially for preventing second thrombi [13].

In conclusion, low-dose rtPA proved to be successful for the treatment of arterial thromboembolism in our patient.

However, a randomized prospective study is needed to determine the precise dosage and duration of this treatment in premature newborns.

Informed Consent: Informed consent was obtained from the parents of the patient, Concept: Yaşar Demirelli, Kadir Şerafettin Tekgündüz, İbrahim Caner, Mustafa Kara, Design: Yaşar Demirelli, Kadir Şerafettin Tekgündüz, Data Collection or Processing: Yaşar Demirelli, Kadir Şerafettin Tekgündüz, İbrahim Caner, Analysis or Interpretation: Yaşar Demirelli, Kadir Şerafettin Tekgündüz, İbrahim Caner, Mustafa Kara, Literature Search: Yaşar Demirelli, İbrahim Caner, Writing: Yaşar Demirelli, Kadir Şerafettin Tekgündüz.

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

References 1. Rashish G, Paes BA, Nagel K, Chan AK, Thomas S; Thrombosis and Hemostasis in Newborns (THiN) Group. Spontaneous neonatal arterial thromboembolism: infants at risk, diagnosis, treatment, and outcomes. Blood Coagul Fibrinolysis 2013;24:787-797.

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2. Holden RW. Plasminogen activators: pharmacology and therapy. Radiology 1990;174:993-1001.

3. Nowak-Göttl U, Kries von R, Göbel U. Neonatal symptomatic thromboembolism in Germany: two year survey. Arch Dis Child Fetal Neonatal Ed 1997;76:163-167.

4. Schmidt B, Andrew M. Neonatal thrombosis: report of a prospective Canadian and international registry. Pediatrics 1995;96:939-943.

5. Aslam M, Guglietti D, Hansen AR. Neonatal arterial thrombosis at birth: case report and literature review. Am J Perinatol 2008; 25:347-352.

6. Saracco P, Parodi E, Fabris C, Cecinati V, Molinari AC, Giordano P. Management and investigation of neonatal thromboembolic events: genetic and acquired risk factors.

Thromb Res 2009;123:805-809.

7. Michelson AD, Bovill E, Andrew M. Antithrombotic therapy in children. Chest 1995;108:506-522.

8. Nowak-Göttl U, Auberger K, Halimeh S, Junker R, Klinge J, Kreuz WD, Ries M, Schlegel N. Thrombolysis in newborns and infants. Thromb Haemost 1999;(Suppl 1)82:112-116.

9. Cinà CS, Goh RH, Chan J, Kenny B, Evans G, Rawlinson J, Gill G. Intrarterial catheter directed thrombolysis: urokinase versus tissue plasminogen activator. Ann Vasc Surg 1999;13:571575.

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Demirelli Y, et al: Thromboembolism in a Preterm Infant

10. Hartmann J, Hussein A, Trowitzsch E, Becker J, Hennecke KH. Treatment of neonatal thrombus formation with recombinant tissue plasminogen activator: six years experience and review of the literature. Arch Dis Child Fetal Neonatal Ed 2001;85:1822.

11. Monagle P, Chalmers E, Chan A, DeVeber G, Kirkham F, Massicotte P, Michelson AD; American College of Chest Physicians. Antithrombotic therapy in neonates and children: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest 2008;133(6 Suppl):887-968.

12. Olgun H, Buyukavci M, Ceviz N, Sahin IO, Yildirim ZK, Colak A, Tekgunduz KS, Caner I. Clinical experience with recombinant tissue plasminogen activator in the management of intracardiac and arterial thrombosis in children. Blood Coagul Fibrinolysis 2014;24:726-730.

13. Erdinç K, Sarıcı SÜ, Dabak O, Gürsel O, Güler A, Kürekçi AE, Canpolat FE. A neonatal thrombosis patient treated successfully with recombinant tissue plasminogen activator.

Turk J Hematol 2013;30:325-327.

Case Report

DOI: 10.4274/tjh.2014.0138 Turk J Hematol 2015;32:363-366

Immune Thrombocytopenic Purpura During Maintenance Phase of Acute Lymphoblastic Leukemia: A Rare Coexistence Requiring a High Degree of Suspicion, a Case Report and Review of the Literature Akut Lenfoblastik Lösemi İdame Tedavisi Sırasında Gelişen İmmün Trombositopenik Purpura: Fazla Şüphe Gerektiren Nadir Bir Birliktelik, Bir Olgu Sunumu ve Literatür Derlemesi Turan Bayhan, Şule Ünal, Fatma Gümrük, Mualla Çetin Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey

Abstract: Thrombocytopenia may develop in patients with acute lymphoblastic leukemia (ALL) due to myelosuppression of chemotherapy or relapse. Here we report a pediatric patient with ALL whose platelet counts decreased at the 102nd week of maintenance treatment. Thrombocytopenia was refractory to platelet infusions and bone marrow aspiration revealed remission status for ALL along with increased megakaryocytes. The cessation of chemotherapy for 2 weeks caused no increase in thrombocyte counts. The viral serology was unrevealing. A diagnosis of immune thrombocytopenic purpura (ITP) was established. After administration of intravenous immunoglobulin, the thrombocytopenia resolved.

When thrombocytopenia occurs in patients with ALL in remission, ITP should be kept in mind after exclusion of the more common etiologies.

Keywords: Acute lymphoblastic leukemia, Children, Immune thrombocytopenic purpura

Öz: Akut lenfoblastik lösemi (ALL) tanılı hastalarda trombositopeni, kemoterapiye ikincil kemik iliği baskılanması veya hastalığın relapsı sonucu gelişebilir. Olgumuz ALL idame tedavisinin 102. haftasında gelişen trombositopeni nedeniyle incelendiği sırada immün trombositopenik purpura (İTP) tanısı almıştır. Trombositopeninin trombosit infüzyonuna rağmen dirençli olması üzerine yapılan kemik iliği aspirasyonunda löseminin remisyonda olduğu ve megakaryositlerin artmış olduğu görüldü. Kemoterapiye iki hafta ara verilmesine rağmen trombosit sayısında artma olmadı. Viral seroloji sonuçları aktif enfeksiyon ile uyumlu değildi. Hastaya İTP tanısı konuldu. İntravenöz immünoglobulin tedavisi ile trombositopeni düzeldi. Remisyondaki ALL hastalarında trombositopeni geliştiğinde, daha sık görülen nedenler dışlandıktan sonra İTP de akılda bulundurulmalıdır.

Anahtar Sözcükler: Akut lenfoblastik lösemi, Çocuk, İmmün trombositopenik purpura

Address for Correspondence: Turan BAYHAN, M.D., Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey Phone: +90 312 305 11 72 E-mail: turanbayhan@yahoo.com Received/Geliş tarihi : March 30, 2014 Accepted/Kabul tarihi : May 13, 2014

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Bayhan T, et al: Immune Thrombocytopenic Purpura in Acute Lymphoblastic Leukemia

Introduction Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disorder characterized by isolated thrombocytopenia due to increased platelet destruction and impaired platelet production [1]. Autoimmunity in ITP develops because of a failure in the regulatory checkpoints of the immune system, resulting in a loss of self-tolerance to platelet glycoproteins. The events that trigger this pathway are largely unknown [2]. Association of ITP with hematologic malignancies such as Hodgkin and non-Hodgkin lymphoma or chronic lymphocytic lymphoma is a well-known phenomenon.

ITP has also been reported to accompany acute lymphoblastic leukemia (ALL), albeit extremely rarely [3]. Herein we report a patient with ALL who developed ITP during maintenance therapy for ALL. Case Presentation A 3-year-old girl was admitted with fever, bone and joint pain, and malaise. Complete blood count showed a hemoglobin level of 7.4 g/dL, platelet count of 97x109/L, and white blood cell count of 3.8x109/L with 34% blasts on the peripheral blood smear. Bone marrow aspiration revealed CALLA (+) pre-B cell ALL. A modified St. Jude Total XV protocol was initiated with institutional modifications in the induction phase concerning the dose of steroids, and remission was achieved [4]. Maintenance treatment was planned according to the patientâ&#x20AC;&#x2122;s low risk status [4]. Nothing was remarkable up to the 102nd week of maintenance. After the 68th week of treatment, maintenance included weekly parenteral methotrexate (40 mg/m2) and daily oral 6-mercaptopurine (75 mg/m2/day) with pulses of dexamethasone and vincristine every 4 weeks until the 100th week, after which only 6-mercaptopurine and methotrexate were given. At that time, routine blood count showed hemoglobin of 12.8 g/dL, white blood cell count of 5.4x109/L, and platelet count of 43x109/L. Physical examination revealed no hepatosplenomegaly. She was free of bleeding symptoms despite ecchymoses of the lower extremities. Treatment was ceased for 2 weeks and, at the end of 2 weeks of follow-up, thrombocytopenia persisted.

Since the platelet count had decreased to 16x109/L, irradiated and filtered platelet transfusion was administered, but the next day the platelet count was found to still be as low as 21x109/L. Viral tests for parvovirus B19 polymerase chain reaction (PCR), Epstein-Barr virus PCR, and cytomegalovirus PCR were all negative. Antinuclear, antidouble-stranded DNA antibodies and direct Coombs test were negative. Vitamin B12 and folate levels were within normal ranges. In order to exclude the possibility of associated hemophagocytic lymphohistiocytosis, testing of plasma fibrinogen, serum triglyceride, and ferritin levels was ordered and all were found to be within the normal

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range. Bone marrow aspiration was performed in order to exclude relapse of ALL. The bone marrow examination revealed a cellular bone marrow in remission for ALL with erythroid hyperactivity and increased megakaryocytes (up to 9-10/field at 10x magnification). A diagnosis of acute ITP was established and intravenous immunoglobulin (IVIG) therapy was given (1 g/kg/day, for 1 day). Three days after IVIG treatment, platelet count was found to have increased to 272x109/L. During follow-up, thrombocytopenia showed no recurrence, despite continuation of the maintenance treatment without any modification. Informed consent was obtained.

Discussion and Review of the Literature Thrombocytopenia seen in patients with ALL is generally secondary to chemotherapy or relapse of primary disease. Both of these conditions manifest with reduced platelet production [1]. Impaired megakaryocytopoiesis may also be seen in ITP, but commonly accelerated destruction of platelets results in increased megakaryocytes in bone marrow as a distinctive finding of ITP [1,5]. In our patient, we did not check for antiplatelet antibodies; however, bone marrow findings, as well as the response of thrombocytopenia to IVIG treatment, were strongly suggestive for the diagnosis of ITP. Classically, the pathophysiology of ITP is attributed to opsonization of platelets by immunoglobulin G antibodies and then phagocytosis and destruction by macrophages in the reticuloendothelial system within the spleen [5]. T cellmediated immunity is also important in ITP pathogenesis [2].

Regulatory T cells (Treg cells) marked by CD4+CD25+Foxp3+ have essential roles in self-tolerance by suppression of humoral and cellular immunity response [6]. Treg cells have been blamed for a role in ITP. Reduction in number and/or function of circulating Treg cells in ITP patients has been shown in several reports [1,5]. Increased numbers of CD4+ Th17 cells and higher levels of T cell-related cytokines are other T cell abnormalities detected in ITP [5].

In the English-language literature, 9 pediatric patients who developed ITP subsequent to a diagnosis of ALL were reported in 7 reports; 6 of them were on chemotherapy and 3 patientsâ&#x20AC;&#x2122; ITP developed after cessation of chemotherapy (Table 1) [3,7,8,9,10,11,12]. It seems paradoxical to diagnose ITP in patients with ALL who are under extensive immune suppression with chemotherapeutics for the primary disease. Because of the intensive chemotherapy used in ALL, autoimmune diseases have rarely been reported among patients with ALL who are under treatment [13]. Of the reported cases, ITP was detected during the maintenance period in 4 of the patients, in 1 patient after reinduction, in 1 patient after induction therapy, and in 3 patients after cessation of chemotherapy [3,7,8,9,10,11,12]. In the majority of these reports, ITP was diagnosed during treatment with

8 years 18 years

9 years 5 years

10.2 years 16 years 13.3 years

7 years

16.6 years

Rao et al., 1979 [12]

Campbell et al., 1993 [7]

Yenicesu et al., 2000 [3]

Kurekci et al., 2006 [10]

Price et al., 2006 [11]

Price et al., 2006 [11]

Price et al., 2006 [11]

Horino et al., 2009 [9]

Dua et al., 2012 [8]

Female

Female

Female

Male

Female

Male

Male

Female

Female

Sex

BFM-95

Protocol of Japan Association of Childhood Leukemia Study

BFM-based highrisk protocol

BFM-based highrisk protocol

BFM-based highrisk protocol

BFM-95

St. Jude Total XIII

Unspecified

Unspecified

Chemotherapy Protocol

Maintenance

After completion of reinduction therapy

0.2 years after the end of chemotherapy

12 years after the end of chemotherapy

4 years after the end of chemotherapy

Maintenance

Maintenance

After completion of induction (induction with vincristine, daunorubicin, prednisolone, L-asparaginase)

Maintenance with cyclophosphamide

Leukemia Treatment Status at ITP Diagnosis

ITP: Immune thrombocytopenic purpura, BFM: Berlin-Frankfurt-Munster, IVIG: intravenous immunoglobulin, Ig: immunoglobulin.

Age at ITP Diagnosis

Report

Prednisone

IVIG, prednisone, vincristine, rituximab, anti-D Ig, splenectomy

None

IVIG, prednisone, splenectomy

IVIG

IVIG, prednisolone, dexamethasone + vincristine

IVIG

IVIG, danazol, prednisolone

Prednisone

Interventions for ITP

Responsive

Nonresponsive

Asymptomatic with mild thrombocytopenia

Responsive to splenectomy

Responsive

Responsive to dexamethasone and vincristine treatment

Responsive

Responsive

Responsive

Response to Treatment

Table 1. Reported pediatric cases with immune thrombocytopenic purpura subsequent to a diagnosis of acute lymphoblastic leukemia.

Bayhan T, et al: Immune Thrombocytopenic Purpura in Acute Lymphoblastic Leukemia Turk J Hematol 2015;32:363-366

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Bayhan T, et al: Immune Thrombocytopenic Purpura in Acute Lymphoblastic Leukemia

6-mercaptopurine, similar to our case [3,8,9,10]. In 2 of these reports, 6-mercaptopurine treatment was continued without recurrence of ITP; in 1 case, due to resistant thrombocytopenia, maintenance therapy was administered with the support of IVIG; and in 1 report, continuation of 6-mercaptopurine after development of ITP was not stated clearly [3,8,9,10]. 6-Mercaptopurine is a purine nucleoside analogue that disturbs DNA synthesis and induces apoptosis [14]. Purine nucleoside analogues cause profound depletion of T cells [15]. Consequently, CD4+CD25+Foxp3+ cell counts also decrease, and this will result in immune dysregulation.

This cascade has been thought of as a mechanism of ITP seen in ALL [9,10]. In the literature, 2 patients were reported to have developed ITP after treatment with cyclophosphamide [9,12]. Cyclophosphamide also has suppressive effects on Treg cells, similar to purine analogues, and this may support the association of Treg cells with ITP in patients with ALL [9].

In conclusion, newly developed persistent thrombocytopenia in patients with ALL may indicate ITP. After exclusion of other common causes including recurrence of the primary disease, chemotherapy-related myelosuppression, folate deficiency, or viral etiologies, the coexistence of ITP should be kept in mind as a rare etiology for unexplained thrombocytopenia in order to initiate appropriate treatment as early as possible.

Informed Consent: Informed consent was obtained, Concept: Mualla Çetin, Design: Turan Bayhan, Şule Ünal, Data Collection or Processing: Fatma Gümrük, Mualla Çetin, Analysis or Interpretation: Şule Ünal, Literature Search: Turan Bayhan, Fatma Gümrük, Mualla Çetin, Writing: Turan Bayhan, Şule Ünal.

Conflict of Interest: 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.

4. Rubnitz JE, Campbell P, Zhou Y, Sandlund JT, Jeha S, Ribeiro RC, Inaba H, Bhojwani D, Relling MV, Howard SC, Campana D, Pui CH. Prognostic impact of absolute lymphocyte counts at the end of remission induction in childhood acute lymphoblastic leukemia. Cancer 2013;119:2061-2066.

5. McKenzie CG, Guo L, Freedman J, Semple JW. Cellular immune dysfunction in immune thrombocytopenia (ITP). Br J Haematol 2013;163:10-23.

6. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and nonself. Nat Immunol 2005;6:345-352.

7. Campbell JK, Mitchell CA. Immune thrombocytopenia in association with acute lymphoblastic leukaemia and a haemophagocytic syndrome. Eur J Haematol 1993;51:259261.

8. Dua V, Sharma JB. Immune thrombocytopenic purpura with acute lymphoblastic leukemia an unusual association. Indian Pediatr 2012;49:994.

9. Horino S, Rikiishi T, Niizuma H, Abe H, Watanabe Y, Onuma M, Hoshi Y, Sasahara Y, Yoshinari M, Kazama T, Hayashi Y, Kumaki S, Tsuchiya S. Refractory chronic immune thrombocytopenic purpura in a child with acute lymphoblastic leukemia. Int J Hematol 2009;90:483-485.

10. Kurekci AE, Atay AA, Demirkaya E, Sarici SU, Ozcan O. Immune thrombocytopenic purpura in a child with acute lymphoblastic leukemia and mumps. J Pediatr Hematol Oncol 2006;28:170-172.

11. Price V, Barnes C, Canning P, Blanchette V, Greenberg M. Immune thrombocytopenia following successful treatment of cancer in children. Pediatr Blood Cancer 2006;46:372-376.

12. Rao S, Pang EJ. Idiopathic thrombocytopenic purpura in acute lymphoblastic leukemia. J Pediatr 1979;94:408-409.

References

13. Teachey DT, Felix CA. Development of cold agglutinin autoimmune hemolytic anemia during treatment for pediatric acute lymphoblastic leukemia. J Pediatr Hematol Oncol 2005;27:397-399.

1. Kashiwagi H, Tomiyama Y. Pathophysiology and management of primary immune thrombocytopenia. Int J Hematol 2013;98:24-33.

14. Bar F, Sina C, Fellermann K. Thiopurines in inflammatory bowel disease revisited. World J Gastroenterol 2013;19:16991706.

2. Arnold DM, Patriquin C, Toltl LJ, Nazi I, Smith J, Kelton J. Diseases of platelet number: immune thrombocytopenia, neonatal alloimmune thrombocytopenia, and posttransfusion purpura. In: Hoffman R, Benz EJ, Silberstein LE, Heslop HE, Weitz JI, Anastasi J (eds). Hematology: Basic Principles and Practice. Philadelphia, Elsevier-Saunders, 2013.

15. Robak T, Korycka A, Lech-Maranda E, Robak P. Current status of older and new purine nucleoside analogues in the treatment of lymphoproliferative diseases. Molecules 2009;14:11831226.

3. Yenicesu I, Sanli C, Gürgey A. Idiopathic thrombocytopenic purpura in acute lymphocytic leukemia. Pediatr Hematol Oncol 2000;17:719-720.

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Case Report

DOI: 10.4274/tjh.2014.0412 Turk J Hematol 2015;32:367-370

A Rare Complication Developing After Hematopoietic Stem Cell Transplantation: Wernicke’s Encephalopathy Hematopoetik Kök Hücre Nakli Sonrası Gelişen Nadir Bir Komplikasyon: Wernicke Ensefalopatisi Soner Solmaz1, Çiğdem Gereklioğlu2, Meliha Tan3, Şenay Demir4, Mahmut Yeral1, Aslı Korur2, Can Boğa1, Hakan Özdoğu1 1Adana

Hospital of Başkent University, Department of Hematology, Adana, Turkey Hospital of Başkent University, Department of Family Medicine, Adana, Turkey 3Adana Hospital of Başkent University, Department of Neurology, Adana, Turkey 4Adana Hospital of Başkent University, Department of Radiology, Adana, Turkey 2Adana

Abstract: Thiamine is a water-soluble vitamin. Thiamine deficiency can present as a central nervous system disorder known as Wernicke’s encephalopathy, which classically manifests as confusion, ataxia, and ophthalmoplegia. Wernicke’s encephalopathy has rarely been reported following hematopoietic stem cell transplantation. Herein, we report Wernicke’s encephalopathy in a patient with acute myeloid leukemia who had been receiving prolonged total parenteral nutrition after haploidentical allogeneic hematopoietic stem cell transplantation. To the best of our knowledge, this is the first case reported from Turkey in the literature.

Keywords: Thiamine, Wernicke’s encephalopathy, Hematopoietic stem cell transplantation, Total parenteral nutrition

Öz: Tiamin suda çözünen bir vitamindir. Tiamin eksikliği Wernicke ensefalopatisi olarak bilinen, klasik olarak konfüzyon, ataksi ve oftalmopleji ile kendini gösteren bir merkezi sinir sistemi hastalığı olarak karşımıza çıkabilir. Hematopoetik kök hücre nakli sonrasında gelişen Wernicke ensefalopatisi nadiren bildirilmiştir. Bu nedenle haploidentik allojenik kök hücre naklinden sonra uzun süre total parenteral beslenme alan akut myeloid lösemili bir hastada gelişen Wernicke ensefalopatisini sunmak istedik. Bildiğimiz kadarıyla literatürde Türkiye’den bildirilen ilk olgudur.

Anahtar Sözcükler: Tiamin, Wernicke ensefalopatisi, Hematopoetik kök hücre nakli, Total parenteral beslenme

Address for Correspondence: Soner SOLMAZ, M.D., Adana Hospital of Başkent University, Department of Hematology, Adana, Turkey Phone: +90 322 327 27 27 E-mail: drssolmaz@gmail.com Received/Geliş tarihi : October 17, 2014 Accepted/Kabul tarihi : November 25, 2014

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Solmaz S, et al: Wernicke’s Encephalopathy After Hematopoietic Stem Cell Transplantation

Introduction Thiamine is a water-soluble vitamin also known as vitamin B1 [1]. Thiamine deficiency can present as a central nervous system (CNS) disorder known as Wernicke’s encephalopathy (WE), which classically manifests as confusion, ataxia, and ophthalmoplegia [1,2]. The disease is most frequently associated with chronic alcoholism, yet it can also occur in relation to other forms of malnutrition or malabsorption such as prolonged total parenteral nutrition (TPN), total gastrectomy, gastrojejunostomy, severe anorexia, or hyperemesis gravidarum [3]. Hematopoietic stem cell transplantation (HSCT) does not seem to have a strong link with WE [4]. To the best of our knowledge, this is the first such case reported from Turkey in the literature and wanted to report this case due to its rarity.

Case Presentation A 19-year-old male patient diagnosed with acute myeloid leukemia was admitted to our hospital for HSCT. After remission had been achieved, he underwent haploidentical HSCT from a sibling donor with a busulfan-fludarabine conditioning regimen. During the conditioning period, the patient was administered TPN, which is routinely used in haploidentical HSCT; however, he developed grade 2-3 nausea and vomiting and could not tolerate TPN. His oral intake was also insufficient, so he received saline solution and glucosecontaining intravenous solutions. He gradually recovered from neutropenia on day 13 after HSCT without any adverse events.

He was hospitalized due to diarrhea and vomiting 3 weeks after the transplantation. On follow-up, toxic megacolon and cytomegalovirus positivity were detected, so ganciclovir treatment was started and oral intake was restricted until recovery of intestinal symptoms. Efforts were made to feed the patient by TPN with the aim of meeting his caloric needs although he could not initially tolerate it. He was examined for acute graft-versus-host disease (GVHD); he underwent colonoscopy and pathologic samples were obtained, but this examination did not reveal histological findings of GVHD. Three weeks after his hospitalization, he developed confusion, hallucination, strabismus, and nystagmus. A neurology consultation was therefore done. In his neurologic examination, he was oriented to place and person, but not to time. He had horizontal nystagmus and lateral gaze paralysis in the right eye, his motor power was 4/5, deep tendon reflexes were hypoactive, Babinski reflex was negative bilaterally, he could not cooperate with cerebellar tests, and he could not stand up. Magnetic resonance imaging (MRI) of the brain showed increased signal on T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequences around the aqueductus

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sylvii and at the medial parts of both thalami (Figures 1a and 1b). A prediagnosis of WE was made based on the patient’s history of inadequate oral intake and TPN use, CNS symptoms, and specific radiologic findings. A blood sample was obtained for testing serum thiamine level to confirm the diagnosis before initiating therapy. Thereafter, 125 mg of thiamine was intravenously administered daily, resulting in a rapid improvement of the CNS symptoms within 48 h of treatment, and parenteral treatment continued for 2 weeks. Serum thiamine level was reported as 7.5 µg/L (normal range: 25-75 µg/L), verifying our diagnosis. During follow-up, his neurologic findings and oral intake gradually improved, and so medical therapy was switched to peroral treatment and maintained with 250 mg of daily peroral thiamine. MRI revealed that the previous increased signal around the aqueductus sylvii and at the medial parts of both thalami on T2-weighted and FLAIR sequences had significantly diminished (Figures 2a and 2b).

Informed consent was obtained.

Discussion and Review of the Literature Neurological complications are fairly common in patients undergoing HSCT and are present in 30%-39% of cases [5].

These complications may be of infectious, cerebrovascular, toxic, immune-mediated, or metabolic origin [5]. Additionally, several drugs routinely used during HSCT are associated with

Figure 1a. Axial fluid-attenuated inversion recovery magnetic resonance imaging images of the brain demonstrating the increased signal around the aqueductus sylvii.

Solmaz S, et al: Wernickeâ&#x20AC;&#x2122;s Encephalopathy After Hematopoietic Stem Cell Transplantation

Turk J Hematol 2015;32:367-370

neurological abnormalities, including cyclosporine A [5] and tacrolimus [6]. Used alone or in combination with other agents, methylprednisolone and ganciclovir may be responsible for neurological findings, including disorientation, altered mental status, visual disturbance, and coma [5]. We think that we saved some time in making a differential diagnosis by examining serum tacrolimus level to exclude drug toxicity and cerebrovascular causes.

WE is characterized by the triad of altered mental status, ataxia, and ophthalmoplegia, but only 16% of patients present with the full classic triad of symptoms [5]. Mental status changes are the most frequent findings in these patients (82%), followed by ocular findings (29%) and ataxia (23%) [5]. Ocular signs, including ophthalmoplegia, horizontal and vertical nystagmus, and conjugate gaze palsies, are the hallmark of WE [3]. Although almost all WE patients show some degree of improvement after initiation of thiamine replacement, only about 20% recover completely [4].

Furthermore, mortality increases dramatically when treatment is delayed [4]. According to the guidelines of the European Federation of Neurological Societies, total thiamine in blood samples should be measured immediately before thiamine administration to confirm suspected or manifest WE and MRI should be used to support diagnosis [7]. Fortunately, we could make a timely diagnosis based on clinical and radiological findings and supported by decreased thiamine level thereafter, and thus we could prevent mortality.

Figure 1b. Axial fluid-attenuated inversion recovery magnetic resonance imaging images of the brain demonstrating the increased signal at the medial parts of both thalami.

Figure 2a. Control magnetic resonance imaging 2 weeks after the onset of the symptoms; fluid-attenuated inversion recovery image showing the diminution of increased signal around the aqueductus sylvii. â&#x20AC;¨

Figure 2b. Control magnetic resonance imaging 2 weeks after the onset of the symptoms; fluid-attenuated inversion recovery image showing the diminution of increased signal at the medial parts of both thalami.

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Turk J Hematol 2015;32:367-370

Solmaz S, et al: Wernicke’s Encephalopathy After Hematopoietic Stem Cell Transplantation

Patients receiving long-term TPN and glucose-containing intravenous solutions require larger amounts of thiamine to metabolize their carbohydrate intake, which can rapidly deplete thiamine stores [3]. Studies show that a state of depletion could develop within 18-20 days in patients receiving a strict thiamine-free diet [5]. Almost all published reports, except for one, concluded that prolonged TPN was the primary risk factor for HSCT-associated WE [4]. Our patient had received TPN for approximately 4-5 weeks in total. TPN includes multivitamin and mineral supplementation in our routine treatment protocol. However, we could not administer it in this patient due to temporary lack of the concerned drugs in the pharmacy of the hospital. The only other suggested cause was the use of busulfan in the conditioning regimen [4]. Similarly to data in the literature, our patient received busulfan in the conditioning regimen and thiamine-free TPN, and symptoms of WE emerged from day +45.

Many authors have recommended the use of a thiamine supplement for prophylaxis against WE [4]. However, an earlier publication from a Brazilian group reported 8 patients who died after developing WE despite receiving prophylactic thiamine (50 mg/day) [4]. Further studies are required to decide on an effective prophylactic dose of thiamine and to determine whether thiamine prophylaxis is effective in the prevention of WE in HSCT patients [4]. This case taught us the vital importance of vitamin supplementation in patients who need long-term TPN. Based on these findings, we reviewed our institutional policy about vitamin supplementation in TPN and began adding water-soluble vitamins into TPN solutions individually if combination preparations were not available in the pharmacy of the hospital.

There are not routine recommendations for initial CNS evaluation and management of the rarely occurring WE [4].

However, WE is a neurological emergency [8]. Therefore, WE should be considered in HSCT patients, because cancer patients are at high risk of this acute encephalopathy due to chronic malnutrition, chemotherapy-induced nausea and vomiting, and consumption of thiamine by rapidly growing tumors [8].

In conclusion, differential diagnosis should consider WE for patients who undergo HSCT and develop neurological symptoms. Early treatment prevents high morbidity and mortality. Therefore, thiamine supplements should be administered to patients at high risk for WE. Informed Consent: Informed consent was obtained, Concept: Soner Solmaz, Can Boğa, Hakan Özdoğu,

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Design: Soner Solmaz, Çiğdem Gereklioğlu, Can Boğa, Hakan Özdoğu, Data Collection or Processing: Soner Solmaz, Çiğdem Gereklioğlu, Meliha Tan, Şenay Demir, Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu, Analysis or Interpretation: Soner Solmaz, Çiğdem Gereklioğlu, Meliha Tan, Şenay Demir, Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu, Literature Search: Soner Solmaz, Meliha Tan, Şenay Demir, Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu, Writing: Soner Solmaz, Çiğdem Gereklioğlu, Meliha Tan, Şenay Demir, Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu.

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

References 1. Trueg A, Borho T, Srivastava S, Kiel P. Thiamine deficiency following umbilical cord blood transplant. Nutr Clin Pract 2013;28:223-225.

2. Han JW, Lim S, Shin HS, Park HJ, Jung WJ, Kwon SY, Lyu CJ. Two cases of Wernicke’s encephalopathy in young age patients receiving allogeneic hematopoietic stem cell transplantation.

Yonsei Med J 2012;53:1049-1053.

3. Baek JH, Sohn SK, Kim DH, Kim JG, Lee HW, Park SP, Lee KB. Wernicke’s encephalopathy after allogeneic stem cell transplantation. Bone Marrow Transplant 2005;35:829-830.

4. Choi YJ, Park SJ, Kim JS, Kang EJ, Choi CW, Kim BS. Wernicke’s encephalopathy following allogeneic hematopoietic stem cell transplantation. Korean J Hematol 2010;45:279-281.

5. Bleggi-Torres LF, de Medeiros BC, Ogasawara VS, Loddo G, Zanis Neto J, Pasquini R, de Medeiros CR. Iatrogenic Wernicke’s encephalopathy in allogeneic bone marrow transplantation: a study of eight cases. Bone Marrow Transplant 1997;20:391395.

6. Sklar EM. Post-transplant neurotoxicity: what role do calcineurin inhibitors actually play? AJNR Am J Neuroradiol 2006;27:1602-1603.

7. Galvin R, Bråthen G, Ivashynka A, Hillbom M, Tanasescu R, Leone MA; EFNS. EFNS guidelines for diagnosis, therapy and prevention of Wernicke encephalopathy. Eur J Neurol 2010;17:1408-1418.

8. Kuo SH, Debnam JM, Fuller GN, de Groot J. Wernicke’s encephalopathy: an underrecognized and reversible cause of confusional state in cancer patients. Oncology 2009;76:1018.

Letters to the Editor Downgraded Lymphoma: B-Chronic Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell Lymphoma - De Novo Occurrence or Transformation Geriletilmiş Lenfoma: Diffüz Büyük B Hücreli Lenfoma Olduğu Bilinen Bir Olguda B-Kronik Lenfositik Lösemi- De Novo Oluşum veya Dönüşüm

confirming the diagnosis of chronic lymphocytic leukemia (CLL) (Figure 1D). The patient was started on a fludarabine, cyclophosphamide, and rituximab (FCR) regimen. After 6 cycles of FCR, he was in complete remission and was started on rituximab maintenance therapy.

To the Editor, Low-grade indolent lymphomas can be transformed into high-grade aggressive lymphomas [1,2,3,4]. Very few cases of transformation of high/intermediate-grade lymphoma to low-grade lymphoma have been reported in the literature [5,6]. This may arise through transformation of the original clone or may represent a new neoplasm resulting from additional genetic mutations that alter the growth rate, growth pattern, and sensitivity to treatment.

A 57-year-old male diagnosed with diffuse large B-cell lymphoma (DLBCL) (non-germinal center B-cell type) in 2002 completed 6 cycles of CHOP followed by radiotherapy.

In 2006, 18F- fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) showed no active disease. In 2007 there was recurrence in the left obturator and external iliac nodes. Lymph node biopsy done outside our facility showed CD20+ B-cell lymphoma. The patient was advised to undergo intensive chemotherapy, but was lost to follow-up. In 2010, the patient came to our hospital with bilateral firm non-tender inguinal and right axillary lymphadenopathy without any organomegaly.

18F PET/CT revealed heterogeneous uptake in the left paraaortic, retrocaval, precaval, and bilateral internal iliac nodes. A previous diagnostic lymph node biopsy was reviewed, showing diffuse infiltration of large atypical cells, positive for CD20, CD30, MUM1, and Bcl2 with a Ki67 index of 80% and negative for CD3, CD5, and CD10, which was consistent with DLBCL (Figure 1A). Biopsy of the paraaortic mass revealed sheets of small lymphoid cells, which were positive for CD20, CD5, and CD23 and negative for CD3 and cyclin D1 with a low Ki67 index, suggestive of small-cell lymphoma (Figure 1B). 18F PETCT was repeated after 1 year, showing multiple FDG-avid cervical, supraclavicular, mediastinal, axillary, abdominal, and pelvic lymphadenopathies (Figure 1C). After 10 months, hemoglobin was 90 g/L, total leukocyte count was 21.1x109/L, and platelet count was 40x109/L. Peripheral blood smear showed 84% abnormal lymphoid cells, which were immunopositive for CD19, CD5, CD23, CD22 (dim), CD200, and CD20 with lambda light chain restriction and negative for CD10, FMC7, CD38, IgM, and CD103,

Figure 1. A) Lymph node biopsy showing diffuse infiltration of large atypical cells with prominent nucleoli and vesicular chromatin, which were positive for CD20, CD30, and MUM1 with a Ki67 index of 80%. B) Biopsy from paraaortic mass showing small-sized neoplastic cells with scant cytoplasm, hyperchromatic nuclei, and clumped chromatin arranged in sheets, which were positive for CD20 and CD5 and negative for cyclin D1 with a low Ki67 index. C) 18F-FDG PET-CT showing multiple cervical, supraclavicular, mediastinal, axillary, abdominal, and pelvic lymphadenopathies with gross splenomegaly. D) Immunophenotyping of peripheral blood smear showing 84% abnormal lymphoid cells, which were positive for CD19, CD5, CD23, CD22 (dim), and CD200 with lambda light chain restriction and negative for FMC7.

371

Letter to the Editor

Turk J Hematol 2015;32:371-375

The phenomenon of high- or intermediate-grade nonHodgkin lymphoma recurring as a low-grade lymphoma is an uncommon form of transformation known as “downgraded” lymphoma. This downgrading may be due to: 1) recurrence of a low-grade lymphoma that was present as a minor component of the initial lymphoma or in a site not biopsied, or 2) development of a second lymphoma resulting from chemotherapy and/or an intrinsic propensity for lymphoma development in the patient [5,6]. Relapse in DLBCL mainly occurs in the first 2 to 3 years, while late relapses after 5 years are rare, occurring in 3.6% of cases. Patients with DLBCL relapse usually have the same histology. However, relapse as indolent lymphoma following initial DLBCL may occur in about 17% of cases, predominantly as follicular lymphoma or rarely as nodal marginal zone lymphoma or as extranodal mucosaassociated lymphoid tissue lymphoma [7]. Histopathological examination including extensive immunohistochemistry should be done, not only when transformation is clinically suspected but also at each recurrence because the disease can recur as indolent lymphoma and an accurate histologic diagnosis will contribute to a better understanding of the pathogenesis of transformation and the start of prompt therapy to improve the survival of the patients.

Concept: Smeeta Gajendra, Bhawna Jha, Shalini Goel, Tushar Sahni, Pranav Dorwal, Ritesh Sachdev, Design: Smeeta Gajendra, Bhawna Jha, Shalini Goel, Tushar Sahni, Pranav Dorwal, Ritesh Sachdev, Data Collection or Processing: Smeeta Gajendra, Bhawna Jha, Shalini Goel, Pranav Dorwal, Ritesh Sachdev, Analysis or Interpretation: Smeeta Gajendra, Bhawna Jha, Tushar Sahni, Ritesh Sachdev, Literature Search: Smeeta Gajendra, Ritesh Sachdev, Writing: Smeeta Gajendra.

Conflict of Interest: 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.

Keywords: Diffuse large B-cell lymphoma, Chronic lymphocytic leukemia, Downgraded lymphoma Anahtar Sözcükler: Diffüz büyük B hücreli lenfoma, Kronik lenfositik lösemi, Geriletilmiş lenfoma

Smeeta Gajendra, Bhawna Jha, Shalini Goel, Tushar Sahni, Pranav Dorwal, Ritesh Sachdev Medanta-The Medicity, Department of Pathology and Laboratory Medicine, Gurgaon, India

References 1. Tsimberidou AM, Keating MJ. Richter syndrome: biology, incidence, and therapeutic strategies. Cancer 2005;103:216228.

2. Rossi D, Cerri M, Capello D, Deambrogi C, Rossi FM, Zucchetto A, De Paoli L, Cresta S, Rasi S, Spina V, Franceschetti S, Lunghi M, Vendramin C, Bomben R, Ramponi A, Monga G, Conconi A, Magnani C, Gattei V, Gaidano G. Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome. Br J Haematol 2008;142:202-215.

3. Montoto S, Fitzgibbon J. Transformation of indolent B-cell lymphomas. J Clin Oncol 2011;29:1827-1834.

4. Lin P, Mansoor A, Bueso-Ramos C, Hao S, Lai R, Medeiros LJ. Diffuse large B-cell lymphoma occurring in patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia. Clinicopathologic features of 12 cases.

Am J Clin Pathol 2003;120:246-253.

5. Kerrigan DP, Foucar K, Dressler L. High-grade non-Hodgkin lymphoma relapsing as low-grade follicular lymphoma: socalled downgraded lymphoma. Am J Hematol 1989;30:3641.

6. Ogata Y, Setoguchi M, Tahara T, Takahashi M. Downgraded non-Hodgkin’s lymphoma in the neck occurring as a secondary malignancy. ORL J Otorhinolaryngol Relat Spec 1998;60:295-300.

7. Larouche JF, Berger F, Chassagne-Clément C, Ffrench M, Callet-Bauchu E, Sebban C, Ghesquières H, BroussaisGuillaumot F, Salles G, Coiffier B. Lymphoma recurrence 5 years or later following diffuse large B-cell lymphoma: clinical characteristics and outcome. J Clin Oncol 2010;28:20942100.

Address for Correspondence: Smeeta GAJENDRA, M.D., Medanta-The Medicity, Department of Pathology and Laboratory Medicine, Gurgaon, India Phone: 0901 359 08 75 E-mail: drsmeeta@gmail.com

Received/Geliş tarihi : April 23, 2015 Accepted/Kabul tarihi : June 15, 2015 DOI: 10.4274/tjh.2015.0164

372

Letter to the Editor

From Bone Marrow Necrosis to Gaucher Disease; A Long Way to Run Kemik İliği Nekrozundan Gaucher Hastalığı Tanısına Uzun Yol To the Editor, Bone marrow necrosis (BMN) is a disease characterized with fever and bone pain and caused by many different malignancies, benign diseases and drugs. We reported a case of BMN due to diclofenac in 2006 [1]. And now we present the same patient with a corrected diagnosis, seven years after the first presentation.

A 26-year-old male presented with fever, bone pain, splenomegaly, anemia, leucopenia and was diagnosed with BMN due to diclofenac consumption. Nine months after his initial admission, his laboratory and physical examination were normal. Seven year after diagnosis, he was admitted to hospital due to bone pain. He had splenomegaly, leukocyte level was 6.22x109/L, hemoglobin level was 13.7 g/dL and thrombocyte level was 152x109/L. Because of history of BMN and reccurring splenomegaly, bone marrow aspiration and biopsy were performed. He was diagnosed with Gaucher disease in bone marrow biopsy and diagnosis was also confirmed by pathology. He had low glucosylceramide level (0.53 µkat/kg protein, normal range 2.4-3.8 µkat/kg protein) and high chitotriosidase level (2793 µkat/kg protein, normal range <40 µkat/kg protein). His treatment was started with imiglucerase. When we retrospectively revaluated the first bone marrow aspiration which had been made 7 years before, we saw that Gaucher cells were also present (Figure 1).

Turk J Hematol 2015;32:371-375

Gaucher disease is an autosomal ressesive, familial disease which presents with hepatosplenomegaly, skin pigmentation, bone lesions, anemia, leukopenia and thrombocytopenia.

Interestingly, our patient was diagnosed with BMN and after 7 years, his diagnosis was corrected as Gaucher diesase with new bone marrow biopsy and reevaluation of the first bone marrow biopsy. BMN is characterized by necrosis of the medullary stroma and myeloid tissues in bone marrow, because of failure of microcirculation. Chemotherapy, microvascular infarction, tumor necrosis factor, and thrombosis were blamed for the pathophysiology [2]. It’s characterized with presence of amorphous esosinophilic material and ghost-like haematopoietic cells with irregular cell membranes. As seen in Figure 1, amorphous and corrupted appearance of the cells misled us. But when inspected carefully, typical Gaucher cells can be seen. Gaucher disease is characterized by accumulation of glucosylceramide in spleen, liver and bone marrow due to lysosomal glucocerebrosidase deficiency [3]. There is usually latency in diagnosis of Gaucher disease because of its rarity and not thinking of the disease in the first step but in further steps. In our patient, BMN due to diclofenac consumption was diagnosed in the first place, but after recurring splenomegaly and bone pain, he was reevaluated and diagnosed with Gaucher disease. Gaucher disease can be easily confused with other hematological diseases and can be overlooked. This leads us to the conclusion that diagnosis of a disease is a long way and if we have doubts about the diagnosis or unexpected changes are present, we should check the diagnosis and make further investigations. Gaucher disease must be kept in mind while making differential diagnosis in patients with splenomegaly, bone pain and pancytopenia.

Concept: Neslihan Erdem, Ahmet Çizmecioğlu, İsmet Aydoğdu, Design: Neslihan Erdem, Ahmet Çizmecioğlu, İsmet Aydoğdu, Data Collection or Processing: Neslihan Erdem, Ahmet Çizmecioğlu, İsmet Aydoğdu, Analysis or Interpretation: Neslihan Erdem, Ahmet Çizmecioğlu, İsmet Aydoğdu, Literature Search: Neslihan Erdem, Ahmet Çizmecioğlu, İsmet Aydoğdu, Writing: Neslihan Erdem, Ahmet Çizmecioğlu, İsmet Aydoğdu.

Conflict of Interest: 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.

Keywords: Necrosis, Gaucher, Bone marrow

Figure 1. Gaucher cells, striated and fibrillary like cytoplasm with small dense nucleus.

Anahtar Sözcükler: Nekroz, Gaucher, Kemik iliği

373

Letter to the Editor

Turk J Hematol 2015;32:371-375

Neslihan Erdem1, Ahmet Çizmecioğlu2, İsmet Aydoğdu3 1Celal Bayar University Faculty of Medicine, Department of Internal Medicine,

Manisa, Turkey 2Karaman State Hospital, Clinic of Internal Medicine, Karaman, Turkey 3Celal Bayar University Faculty of Medicine, Department of Hematology, Manisa,

Turkey

References 1. Aydogdu I, Erkurt MA, Ozhan O, Kaya E, Kuku I, Yitmen E, Aydin NE. Reversible bone marrow necrosis in a patient due to overdosage of diclofenac sodium. Am J Hematol 2006;81:298.

2. Bhasin TS, Sharma S, Chandey M, Bhatia PK, Mannan R. A case of bone marrow necrosis of an idiopathic aetiology: the report of a rare entity with review of the literature. J Clin Diagn Res 2012;7:525-528.

3. Rosenbaum H. Hemorrhagic aspects of Gaucher disease.

Rambam Maimonides Med J 2014;5:e0039.

Address for Correspondence: Neslihan ERDEM, M.D., Celal Bayar University Faculty of Medicine, Department of Internal Medicine, Manisa, Turkey Phone: +90 555 729 88 22 E-mail: neslihnerdem@gmail.com

Received/Geliş tarihi: March 15, 2015 Accepted/Kabul tarihi: May 04, 2015

cardiac echocardiography results were within normal levels.

High-performance liquid chromatography results were as follows; HbA1: 63.6%, HbA2: 32.8%, HbF: 0.2%. Agarose gel electrophoresis was performed to distinguish HbA2, but a band was identified at the level of 39.7% in HbF, G zone, and between HbA1 and HbA2. A blood sample was transferred to our genetic diagnostic center. Following DNA extraction with a commercial kit (Roche, Germany) and amplification of the whole beta globin gene by standard PCR protocols, DNA sequencing (Applied Biosystems, USA) revealed an A to C substitution at nucleotide position 308 (Figure 1). This change was identified as HBB: c.308 A>C, known as Hb Kansas in the HbVar database [5].

Hb Kansas is one of four known hemoglobins with neutral substitutions, along with Hb Köln, Porto Alegre, and Genova [1].

The oxygen equilibrium of Hb Kansas has two unusual characteristics: low affinity for oxygen and low heme-heme interaction. The low oxygen affinity of Hb Kansas should be considered in the differential diagnosis of peripheral cyanosis, especially in the neonatal period and in cyanotic disease and polycythemia in the elderly.

DOI: 10.4274/tjh.2015.0123

First Observation of Hemoglobin Kansas [β102(G4)Asn→Thr, AAC>ACC] in the Turkish Population Türk Toplumunda İlk Hemoglobin Kansas [β102(G4)Asn→Thr, AAC>ACC] Gözlemi To the Editor, Hemoglobin (Hb) Kansas [β102 (G4) Asn→Tyr, AAC>ACC] is an unstable abnormal hemoglobin with low oxygen affinity and increased dissociation. Hb Kansas has rarely been reported in the literature to date; the first case was defined in the state of Kansas of the United States [1].

The second reported case was a newborn baby with cyanosis from Sarajevo and the third was an elderly patient with polycythemia from Japan [2,3]. There has been no previous report from Turkey [4]. We herein report the first case of Hb Kansas from Turkey, an introduction of clinical significance.

Case: A 28-year-old male patient with cyanosis of the lips and fingertips was admitted to a hospital in the city of Malatya. He had peripheral cyanosis of the hands and feet on physical examination. Blood gas analysis showed low oxygen levels. Complete blood count, blood chemistry, and

374

Figure 1. Hemoglobin Kansas in DNA sequencing.

Letter to the Editor

Turk J Hematol 2015;32:371-375

Concept: Duran Canatan, Design: Duran Canatan, Data Collection or Processing: İbrahim Keser, Alev Öztaş, Analysis or Interpretation: İbrahim Keser, Türker Bilgen, Literature Search: Duran Canatan, Writing: Duran Canatan.

Conflict of Interest: 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.

Keywords: Abnormal hemoglobins, Hb Kansas, Turkish population Anahtar Sözcükler: Anormal hemoglobinler, Hb Kansas, Türk toplumu İbrahim

Keser1,

Alev

Öztaş2,

Türker

Bilgen3,

Duran

Canatan4

1Akdeniz University Faculty of Medicine, Department of Biology and Genetics,

Antalya, Turkey 2Melid Park Private Hospital, Malatya, Turkey 3Research and Application Center for Scientific and Technological Investigations

(NABİLTEM) of Namık Kemal University, Tekirdağ, Turkey 4Antalya Diagnostic Center of Genetic Diseases, Antalya, Turkey

References

2. Zimmermann-Baer U, Capalo R, Dutly F, Saller E, Troxler H, Kohler M, Frischknecht H. Neonatal cyanosis due to a new (G)γ-globin variant causing low oxygen affinity: Hb F-Sarajevo [(G)γ102(G4)Asn→Thr, AAC>ACC]. Hemoglobin 2012;36:109-113.

3. Morita K, Fukuzawa J, Onodera S, Kawamura Y, Sasaki N, Fujisawa K, Ohba Y, Miyaji T, Hayashi Y, Yamazaki N. Hemoglobin Kansas found in a patient with polycythemia.

Ann Hematol 1992;65:229-231.

4. Akar N. An updated review of abnormal hemoglobins in the Turkish population. Turk J Hematol 2014;31:97-98.

5. Giardine B, van Baal S, Kaimakis P, Riemer C, Miller W, Samara M, Kollia P, Anagnou NP, Chui DH, Wajcman H, Hardison RC, Patrinos GP. HbVar database of human hemoglobin variants and thalassemia mutations: 2007 update. Hum Mutat 2007;28:206.

Address for Correspondence: Duran CANATAN, M.D., Antalya Diagnostic Center of Genetic Diseases, Antalya, Turkey E-mail: durancanatan@gmail.com

Received/Geliş tarihi: April 29, 2015 Accepted/Kabul tarihi: May 12, 2015 DOI: 10.4274/tjh.2015.0177

1. Bonaventura J, Riggs A. Hemoglobin Kansas, a human hemoglobin with a neutral amino acid substitution and an abnormal oxygen equilibrium. J Biol Chem 1968;243:980991.

375

Images in Hematology

DOI: 10.4274/tjh.2015.0082

Turk J Hematol 2015;32:376-377

Image in Hematology Mott Cells in the Peripheral Blood of a Patient with Dengue Fever Dang Hummal覺 Bir Hastan覺n Periferik Kan覺ndaki Mott H羹creleri

Figure 1. The top image shows a Mott cell. The bottom left image shows a similar Mott cell packed with spherical cytoplasmic inclusions. The bottom right image shows a plasmacytoid lymphocyte with deep basophilic cytoplasm (Leishman stain; magnification 1000x).

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Turk J Hematol 2015;32:376-377

A 48-year-old female presented with intermittent highgrade fever, chills, and severe myalgia for 4 days. There was no lymphadenopathy or hepatosplenomegaly. Investigations revealed hemoglobin concentration of 142 g/L; leucocyte count of 3.5x109/L with 54% neutrophils, 40% lymphocytes, 1% eosinophils, and 5% monocytes; and thrombocytopenia (platelet count of 55x109/L). Peripheral smear revealed numerous plasmacytoid lymphocytes and occasional cells with eccentrically placed nucleipacked with multiple prominent cytoplasmic vacuoles, morphologically consistent with Mott cells (Figure 1). Meanwhile, Dengue NS1 antigen assay turned out to be positive. The patient was managed conservatively and discharged after 4 days with a platelet count of 150x109/L. Peripheral smear revealed only occasional reactive lymphocytes and the Mott cells had disappeared.

Three weeks after discharge, platelet and leucocyte counts had improved further.

Nonmalignant reactive peripheral blood plasmacytosis can occur in tumors, autoimmune conditions, and infections [1]. Polyclonal peripheral blood plasmacytosis also occurs in Dengue virus infections and is prominent during the first week of the disease [2]. However, the transient occurrence of Mott cells in the peripheral blood of Dengue fever patients has not been reported previously. Our patient was not suffering from lymphoma or multiple myeloma, which are potential causes of peripherally circulating Mott cells.

Concept: Aniya Antony, Marie Ambroise, Anita Ramdas Design: Aniya Antony, Marie Ambroise, Mookkappan Sudhagar, Data Collection or Processing: Aniya Antony, Marie Ambroise, Mookkappan Sudhagar, Anita Ramdas, Analysis or Interpretation: Aniya Antony, Marie Ambroise, Chokka Kiran, Anita Ramdas, Literature Search: Aniya Antony, Marie Ambroise, Chokka Kiran, Writing: Aniya Antony, Marie Ambroise, Chokka Kiran, Mookkappan Sudhagar.

Antony A, et al: Mott Cells in the Peripheral Blood of a Patient with Dengue Fever

Conflict of Interest: 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.

Keywords: infection

Infection,

Platelets,

Lymphocytes,

Viral

Anahtar Sözcükler: Enfeksiyon, Platelet, Lenfosit, Viral enfeksiyon References 1. Jego G, Robillard N, Puthier D, Amiot M, Accard F, Pineau D, Bataille R, Pellat-Deceunynck C. Reactive plasmacytoses are expansions of plasmablasts retaining the capacity to differentiate into plasma cells. Blood 1999;94:701-712.

2. Thai KT, Wismeijer JA, Zumpolle C, de Jong MD, Kersten MJ, de Vries PJ. High incidence of peripheral blood plasmacytosis in patients with dengue virus infection. Clin Microbiol Infect 2011;17:1823-1828.

Aniya Antony1, Marie Ambroise1, Chokka Kiran1, Mookkappan Sudhagar2, Anita Ramdas1 1Pondicherry Institute of Medical Sciences, Clinic of Pathology, Puducherry, India 2Pondicherry Institute of Medical Sciences, Clinic of General Medicine, Puducherry, India E-mail: aniya.antony@gmail.com Received/Geliş tarihi

: February 14, 2015

Accepted/Kabul tarihi : March 23, 2015

377

Images in Hematology

DOI: 10.4274/tjh.2014.0475

Turk J Hematol 2015;32:378-379

Quiz in Hematology

Figure 1. Patchy melanoderma lesions mimicking ecchymoses in the legs, trunk.

Figure 2. A) Skin. A slight perivascular infiltration of mononuclear inflammatory cells and dermal melanophages (arrows) are seen (HEx200). B) An increment in melanin pigment in basal keratinocytes (arrow heads) and dermal melanophages (white arrows) are highlighted by Fontana-Masson stain (x200).

378

Ünal Ş, et al: Diagnosis: Melanoderma After Hematopoietic Stem Cell Transplantation

Turk J Hematol 2015;32:378-379

Diagnosis: Melanoderma after Hematopoietic Stem Cell Transplantation Hematopoetik Kök Hücre Nakli Sonrası Gelişen Melanoderma

An 8-month-oldboy diagnosed with T-B+NK- SCID underwent peripheral blood hematopoietic stem cell transplantation (HSCT) from MSD without conditioning. However, he developed pancytopenia and became transfusion dependent by posttransplant 2nd month. Bone marrow aspiration/biopsy revealed an aplastic marrow with 98% donor chimerism. With a diagnosis of T-cell engraftment of the donor but no engraftment of the other lineages, a 2nd HSCT with conditioning (BU/FLU/ATG) was performed at post-transplant +23rd month from the same donor. Due to hyperferritinemia pre-transplant desferoxamine was given. On post-transplant day +2, he developed hyperpigmented patches (Figure 1). Platelet count was 22x109/L and aPTT and PT were normal. Platelet transfusion was given; however the lesions did not subside with the expected color change of ecchymoses. Skin biopsy from medial thigh was obtained (Figure 2).

Generalized hyperpigmentation, after conditioning, is a common finding after HSCT [1]. However, in our patient the lesions were patchy. There are few reports of melanoderma [1,2] after HSCT and in one, melanoderma was reported as a finding of chronic GvHD [2]. Based on the absence of clinical signs of GvHD and lack of typical histological evidence, the melanoderma in our patient was attributed to drugs used in conditioning. The transfusional iron loading may cause a generalized darkening of the skin; however in our patient the lesions were patchy and developed just after completion of the conditioning regimen and subsequent stem cell infusion. The patient did not develop acute or chronic GvHD signs throughout the follow-up. The lesions’ color faded after engraftment gradually, although did not disappear totally. Currently, the patient is alive at post-HSCT 6th month.

Informed Consent: Informed consent has been obtained from the parents of the patient, Concept: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Design: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Data Collection or Processing: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Analysis or Interpretation: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Literature Search: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Writing: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya.

Conflict of Interest: 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.

Keywords: Hematopoietic stem cell transplantation, Melanoderma, Skin findings, SCID Anahtar Sözcükler: Hematopoetik kök hücre nakli, Melanoderma, Deri bulguları, SCID Şule Ünal1, İlhan Tezcan2, Şafak Güçer3, Meryem Seda Boyraz4, Deniz Çağdaş2, Duygu Uçkan Çetinkaya1 1Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey 2Hacettepe University Faculty of Medicine, Division of Immunology, Ankara, Turkey 3Hacettepe University Faculty of Medicine, Division of Pediatric Pathology, Ankara, Turkey 4Hacettepe University Faculty of Medicine, Department of Pediatrics, Ankara, Turkey Phone: +90 312 305 11 70 E-mail: suleunal@hacettepe.edu.tr Received/Geliş tarihi

: December 12, 2014

Accepted/Kabul tarihi : January 19, 2015 References 1. Aractingi S, Janin A, Devergie A, Bourges M, Socie G, Gluckman E. Histochemical and ultrastructural study of diffuse melanoderma after bone marrow transplantation. Br J Dermatol 1996;134:325-331.

2. Martin-Gorgojo A, Martín JM, Gavrilova M, Monteagudo C, Jordá-Cuevas E. Chronic graft-versus-host disease presenting with coexisting diffuse melanoderma and hypopigmented patches: A peculiar presentation. Eur J Dermatol. 2013;23:553555.

379

32nd Volume Index / 32. Cilt Dizini SUBJECT INDEX - KONU DİZİNİ 2015

Abnormal Hemoglobins Chediak-Higashi / Chediak-Higashi, 90 Hemoglobin Lansing / Hemoglobin Lansing, 90 Hemoglobin Jabalpur / Hemoglobin Jabalpur, 90 Erythropoietin / Eritropoetin, 304 β-Thalassemia/hemoglobin E / β-Talasemi/hemoglobin E, 304 Apoptosis / Apopitoz, 304 Abnormal hemoglobins / Anormal hemoglobinler, 375 Hb Kansas / Hb Kansas, 375 Turkish population / Türk toplumu, 375 Acute Leukemia Lymphoid enhancer-binding factor-1 / Lenfoid enhansır-bağlayıcı faktör-1, 15 Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 15,277,363 Prognosis / Prognoz, 15 Wnt / Wnt, 15 Acute megakaryoblastic leukemia / Akut megakaryoblastik lösemi, 64 t(1;22) / t(1;22), 64 Acute myeloid leukemia / Akut miyeloid lösemi, 64,77 Trisomy 6 / Trizomi 6, 77 Cytogenetics / Sitogenetik, 77 Acute promyelocytic leukemia / Akut promyelositik lösemi, 97 Rhinocerebral mucormycosis / Rinoserebral mukormikozis, 97 WNT5A / WNT5A, 127 Methylation / Metilasyon, 127 Downregulation / Azalarak düzenlenme, 127 Gene expression / Gen ekspresyonu, 127 ALL / ALL, 127 Acute myeloblastic leukemia / Akut miyeloblastik lösemi, 263 FLT3 / FLT3, 263 Sorafenib / Sorafenib, 263 Sunitinib / Sunitinib, 263 Children / Çocuk, 263,329,363 D835Y mutation / D835Y mutasyonu, 263 B-cell neoplasms / B hücre neoplazileri, 277 Acute leukemia / Akut lösemi, 277,329 Chemotherapy / Kemoterapi, 329 Fungal infection / Fungal enfeksiyon, 329 Immune thrombocytopenic purpura / İmmün trombositopenik purpura, 363 Anemia Cold-reactive antibody / Soğuk otoimmün, 86 Hemolytic anemia / Hemolitik anemi, 86,92 Breast carcinoma / Meme kanseri, 86 Microangiopathy / Mikroanjiyopati, 92 Severe aplastic anemia / Ağır aplastik anemi, 220 Regulatory T cell / Düzenleyici T hücre, 220 Bone marrow failure / Kemik iliği yetmezliği, 220 Thrombotic microangiopathy / Trombotik mikroanjiopati, 276 Eculizumab / Ekulizumab, 276 aHUS / aHUS, 276 CFH gene / CFH geni, 276 Anemia / Anemi, 284 Congenital dyserythropoietic anemia type 2 / Konjenital diseritropetik anemi tip 2, 284 SEC23B gene / SEC23B geni, 284

Vitamin B12 / B12 vitamini, 317 Transcobalamin II / Transkobalamin II, 317 Novel mutation / Yeni mutasyon, 317 Novel deletion / Yeni delesyon, 317 Vacuolization / Vaküolizasyon, 317 Bleeding Disorders Children / Çocuk, 338 Blood coagulation / Koagülasyon, 338 Hemophilia / Hemofili, 338 Inherited coagulopathies / Kalıtsal koagülopatiler, 338 Epistaxis / Epistaksis, 338 Menorrhagia / Menoraji, 338 Cancer Cold-reactive antibody / Soğuk otoimmün, 86 Autoimmune hemolytic anemia / Hemolitik anemi, 86 Breast carcinoma / Meme kanseri, 86 Chronic myeloid leukemia / Kronik miyeloid lösemi, 257 Nilotinib / Nilotinib, 257 Secondary malignancy / İkincil malinite, 257 Carcinoma of the pancreas / Pankreas kanseri, 257 Chronic Leukemia Monoclonal B lymphocytosis / Monoklonal B lenfositoz, 29 Prevalence / Prevalans, 29 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 29,118,311 First-degree relatives / Birinci derece akraba, 29 Cytogenetics / Sitogenetik, 83 Marrow / Kemik iliği, 83 Neoplasia / Neoplazi, 83 Chronic myeloid leukemia / Kronik miyeloid lösemi, 83,257,311 Monosomy / Monozomi, 83 Apoptosis / Apoptoz, 118 Cell cycle arrest / Hücre siklusu tutulması, 118 KL-21 / KL-21, 118 Acute promyelocytic leukemia / Akut promiyelositik lösemi, 194 All-trans retinoic acid / All-trans retinoik asit, 194 BCR/ABL / BCR/ABL, 194 PML/RAR-α / PML/RAR-α, 194 Imatinib / İmatinib, 194 Nilotinib / Nilotinib, 257 Secondary malignancy / İkincil malinite, 257 Carcinoma of the pancreas / Pankreas kanseri, 257 IL-18, Polymorphism / İL-18, Polimorfizm, 311 Single nucleotide polymorphisms / Tek nükleotid polimorfizmi, 311 Coagulation Children / Çocuk, 338 Blood coagulation / Koagülasyon, 338 Hemophilia / Hemofili, 338 Inherited coagulopathies / Kalıtsal koagülopatiler, 338 Epistaxis / Epistaksis, 338 Menorrhagia / Menoraji, 338 Granulocytic Sarcoma Myeloid sarcoma / Miyeloid sarkom, 35 Granulocytic sarcoma / Granülositik sarkom, 35 Monoblastic sarcoma / Monoblastik sarkom, 35

32nd Volume Index / 32. Cilt Dizini SUBJECT INDEX - KONU DİZİNİ 2015

Hematological Malignancies Hematological malignancy / Hematolojik malignite, 100,251 Invasive fungal infections / İnvazif fungal enfeksiyon, 100 Prophylaxis / Profilaksi, 100 Risk / Risk, 100 Secondary infection / Sekonder enfeksiyon, 243 Febrile neutropenia / Febril nötropeni, 243 Hematological malignancy / Hematolojik malinite, 243 Mortality / Mortalite, 243 Leukemia / Lösemi, 243 Lymphoma / Lenfoma, 243 Hepatitis B / Hepatit B, 251 Resolved infection / Geçirilmiş enfeksiyon, 251 Hepatitis B surface antibody / Hepatit B yüzey antikoru, 251 Chemotherapy / Kemoterapi, 251 Hemophagocytic Lymphohistiocytosis Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiositoz, 355 Invasive aspergillosis infection / İnvaziv aspergilloz enfeksiyonu, 355 UNC13D (c.175G>C; p.Ala59Pro) / UNC13D (c.175G>C; p.Ala59Pro), 355 Immunohematology Chimeric antigen receptor T cell / Şimerik antijen reseptör T hücreleri, 285 Hematological malignancies / Hematolojik maligniteler, 285 Iron Disorder Iron deficiency / Demir eksikliği, 1 TMPRSS6 / TMPRSS6, 1 Matriptase-2 / Matriptaz-2, 1 Hepcidin / Hepsidin, 1 Infection Disorders Invasive pulmonary aspergillosis / İnvasiv pulmoner aspergillozis, 73 Recombinant factor VIIa / Recombinant factor VIIa, 73 Coil embolization / Embolizasyon, 73 Children / Çocuk, 73,329 Acute leukemia / Akut lösemi, 73,329 Acute promyelocytic leukemia / Akut promyelositik lösemi, 96 Rhinocerebral mucormycosis / Rinoserebral mukormikozis, 96 Hematological malignancy / Hematolojik malignite, 100,243 Invasive fungal infections / İnvazif fungal enfeksiyon, 100 Prophylaxis / Profilaksi, 100 Risk / Risk, 100 Blood coagulation / Koagülasyon, 144 Hematologic manifestation / Hematolojik bulgu, 144 Infection / Enfeksiyon, 144,234,376 Pediatric leukemia / Pediatrik lösemi, 144 Histoplasma / Histoplazma, 191 Pancytopenia / Pansitopeni, 191 Adrenal masses / Adrenal kitleler, 191 Multiple myeloma / Multipl miyelom, 234 Risk factors / Risk faktörleri, 234 Therapy / Tedavi, 234 Secondary infection / Sekonder enfeksiyon, 243 Febrile neutropenia / Febril nötropeni, 243 Mortality / Mortalite, 243 Leukemia / Lösemi, 243 Lymphoma / Lenfoma, 243 Rituximab / Rituksimab, 271

Leuconostoc / Leuconostoc, 271 Purulent meninigitis / Pürülan menenjit, 271 Mantle cell lymphoma / Mantle hücreli lenfoma, 271 R-CHOP / R-CHOP, 271 Crimean-Congo hemorrhagic fever / Kırım-Kongo kanamalı ateşi, 281 Leukocyte / Lökositr, 281 Chemotherapy / Kemoterapi, 329 Fungal infection / Fungal enfeksiyon, 329 Platelets / Platelet, 376 Lymphocytes / Lenfosit, 376 Viral Infection / Viral enfeksiyon, 376 Leukocyte Crimean-Congo hemorrhagic fever / Kırım-Kongo kanamalı ateşi, 281 Leukocyte / Lökositr, 281 Lymphoma s-IL6 / s-IL6, 21 s-VEGF / s-VEGF, 21 Lymphoma / Lenfoma, 21 Overall survival / Genel sağkalım, 21 Fcγ RIIIA / Fcγ RIIIA, 152 Diffuse large B-cell lymphoma / Diffüz büyük B hücreli lenfoma, 152,295,371 Rituximab / Rituksimab, 152 Positron emission tomography / Positron emisyon tomografi, 213 Computed tomography / Bilgisayarlı tomografi, 213 Bone marrow biopsy / Kemik iliği biyopsisi, 213 Hodgkin’s lymphoma / Hodgkin lenfoma, 213 Non-Hodgkin’s lymphoma / Non Hodgkin lenfoma, 213 Rituximab / Rituksimab, 271 Leuconostoc / Leuconostoc, 271 Purulent meninigitis / Pürülan menenjit, 271 Mantle cell lymphoma / Mantle hücreli lenfoma, 271 R-CHOP / R-CHOP, 271 GADD45γ / GADD45γ, 295 DNA methylation / DNA metilasyonu, 295 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 371 Downgraded lymphoma / Geriletilmiş lenfoma, 371 Molecular Hematology Blood platelets / Trombositler, 58 Growth arrest-specific protein 6 / Growth arrest-specific protein 6, 58 Hemostasis / Hemostaz, 58 Apoptosis / Apoptoz, 118,304 Cell cycle arrest / Hücre siklusu tutulması, 118 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 118,311 KL-21 / KL-21, 118 WNT5A / WNT5A, 127 Methylation / Metilasyon, 127 Downregulation / Azalarak düzenlenme, 127 Gene expression / Gen ekspresyonu, 127 ALL / ALL, 127 Fcγ RIIIA / Fcγ RIIIA, 152 Diffuse large B-cell lymphoma / Diffüz büyük B hücreli lenfoma, 152,295 Rituximab / Rituksimab, 152 Anemia / Anemi, 284 Congenital dyserythropoietic anemia type 2 / Konjenital diseritropetik anemi tip 2, 284

32nd Volume Index / 32. Cilt Dizini SUBJECT INDEX - KONU DİZİNİ 2015

SEC23B gene / SEC23B geni, 284 IL-18 / İL-18, 311 Polymorphism / Polimorfizm, 311 Chronic myelogenous leukemia / Kronik miyeloid lösemi 311 Single nucleotide / Tek nükleotid 311 Polymorphisms / Polimorfizmi 311 Erythropoietin / Eritropoetin, 304 β-Thalassemia/hemoglobin E / β-Talasemi/hemoglobin E, 304 GADD45γ / GADD45γ, 295 DNA methylation / DNA metilasyonu, 295 Multiple Myeloma Terbinafine / Terbinafin, 189 Drug / İlaç, 189 Neutropenia / Nötropeni, 189 Multiple myeloma / Multipl miyelom, 234 Infections / Enfeksiyonlar, 234 Risk factors / Risk faktörleri, 234 Therapy / Tedavi, 234 Myelomonocytic Leukemia c-CBL mutation / c-CBL mutasyonu, 175 Childhood / Çocukluk çağı, 175 Juvenile myelomonocytic leukemia / Juvenil myelomonositik lösemi, 175 NRAS mutation / NRAS mutasyonu, 175 Auer rod / Auer çubukları, 278 Chronic myelomonocytic leukemia / Kronik miyelomonositik lösemi, 278 Myelofibrosis Primary myelofibrosis / Primer myelofibrozis, 180 Ruxolitinib / Ruxolitinib, 180 Splenectomy / Splenektomi, 180 Myelodysplastic Syndromes Psoriasis / Psöriazis, 87 Hypogammaglobulinemia / Hipogamaglobulinemi, 87 Monosomy 7 / Monozomi 7, 87 MDS / MDS, 87, 206 Bortezomib / Bortezomib, 206 Arsenic trioxide / Arsenik trioksit, 206 NF-κ B / NF-κ B, 206 Gene expression / Gen anlatımı 206 Myeloproliferative Disorders Myeloproliferative neoplasms / Myeloproliferatif neoplaziler, 163 Ruxolitinib / Ruxolitinib, 163 Myelofibrosis / Miyelofibroz, 163 Neutropenia Alkaline phosphatase / Alkalen fosfataz, 189 Myeloma / Myelom, 189 Vitamin D deficiency / D vitamin eksikliği, 189 Plasmacytoma Blastic plasmacytoid dendritic cell neoplasm / Blastik plazmasitoid dendritik hücre neoplazisi, 98 Cutaneous involvement / Hızlı ilerleme, 98 Sickle Cell Sickle cell disease / Orak hücre hastalığı, 195 Hematopoietic stem cell transplantation /

Hematopoietik kök hücre nakli, 195 Graft-versus-host disease / Graft-versus-host hastalığı, 195 Graft rejection / Graft kaybı, 195 Conditioning / Hazırlama rejimi, 195 Stem Cell Transplantation Tunneled central venous catheter / Tunelli santral venöz kateter, 51 Hematopoietic stem cell transplantation / Hematopoetik kök hücre nakli, 51,195,367,379 Thrombosis / Tromboz, 51 Infection / Enfeksiyon, 51 Sickle cell disease / Orak hücre hastalığı, 195 Graft-versus-host isease / Graft-versus-host hastalığı, 195 Graft rejection / Graft kaybı, 195 Conditioning / Hazırlama rejimi, 195 Thrombosis / Tromboz, 228 Pediatric stem cell transplantation / Pediatrik kök hücre nakli, 288 Prothrombotic risk factors / Protrombotik risk faktörleri, 288 Melanoderma / Melanoderma, 379 Skin findings / Deri bulguları, 379 SCID / SCID, 379 Thiamine / Tiamin, 367 Wernicke’s encephalopathy / Wernicke ensefalopatisi, 367 Total parenteral nutrition / Total parenteral beslenme, 367 Thalassemia Molecular / Moleküler, 136 Mutation / Mutasyon, 136,344 Alpha thalassemia / Alfa talasemiler, 136,344 Turkey / Türkiye, 136 Anemia / Anemi, 344 Hb Adana / Hb Adana, 344 Hb Icaria / Hb Icaria, 344 Hb Koya Dora / Hb Koya Dora, 344 Thalassemia / Talasemi, 344 Erythropoietin / Eritropoetin, 304 β-Thalassemia/hemoglobin E / β-Talasemi/hemoglobin E, 304 Apoptosis / Apopitoz, 304 Thrombosis Venous thrombosis / Venöz tromboz, 80 Pregnancy / Hamilelik, 80 Factor V Leiden / Faktör V Leiden, 80 Streptococcal infection / Streptokok enfeksiyonu, 80 Thrombosis / Tromboz, 228 Pediatric stem cell transplantation / Pediatrik kök hücre nakli, 228 Prothrombotic risk factors / Protrombotik risk faktörleri, 228 Total anomalous pulmonary venous return / Total pulmoner venöz dönüş anomalisi, 267 Portal vein thrombosis / Portal ven trombozu, 267 Anticoagulation therapy / Antikoagülan tedavi, 267 Low-molecular-weight-heparin / Düşük moleküler ağırlıklı heparin, 267 Preterm / Preterm, 359 Thromboembolism / Tromboemboli, 359 Tissue / Doku, 359 Plasminogen / Plazminojen, 359

32nd Volume Index / 32. Cilt Dizini SUBJECT INDEX - KONU DİZİNİ 2015

Intrauterine arterial thromboembolism / İntrauterin arteriyel tromboembolizm, 359 Low dose recombinant tPA therapy / Düşük doz rekombinant tPA tedavisi, 359 Thrombotic Thrombocytopenic Purpura Thrombotic thrombocytopenic purpura / Trombotik trombositopenik purpura, 279 Transcobalamin Vitamin B12 / B12 vitamini, 317 Transcobalamin II / Transkobalamin II, 317 Novel mutation / Yeni mutasyon, 317 Novel deletion / Yeni delesyon, 317 Vacuolization / Vaküolizasyon, 317 Thrombocytopenia Thrombocytopenia / Trombositopeni, 158 Elderly / Yaşlı, 158 Immune thrombocytopenic purpura / İmmün trombositopenik purpura, 158,186,363 Intracranial bleeding / İntrakranial kanama 158 Congenital amegakaryocytic thrombocytopenia / Konjenital amegakaryositik trombositopeni, 172 Thrombopoietin / Trombopoetin, 172 c-MPL / c-MPL, 172 Homozygous missense mutation / Homozigot yanlış anlamlı mutasyon, 172 c-MPL Tryp154Arg / c-MPL Tryp154Arg, 172 Amino acid change / Amino asit değişikliği, 172 Methylprednisolone / Metil prednizolon, 186 Glucocorticoids / Glükokortikoidler, 186 Child / Çocuk, 186,363 Adolescent / Adölesan, 186 Immune thrombocytopenia / İmmün trombositopeni, 323 Thrombopoietin receptor agonist / Trombopoetin reseptor agonisti, 323 Bleeding / Kanama, 323

Eltrombopag / Eltrombopag, 323 Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 363 Other Mastocytosis / Mastositoz, 43,89 Bone mineral density / Kemik yoğunluk ölçümü, 43 Pyridinoline / Pridinolin, 43 Bone turnover / Kemik turnover, 43 Osteopenia / Osteopeni, 43 Dasatinib / Dasatinib, 68 Chylothorax / Şilotoraks, 68 Chronic myeloid leukemia / Kronik miyeloid lösemi, 68,168 Zinc deficiency / Çinko noksanlığı, 89 Oral lesions / Ağız yaraları, 93 S. aureus / S. aureus, 93 Hematology / Hematoloji, 93 Interleukin-31 (IL-31) / İnterlökin-31 (IL-31), 168 Tyrosine kinase inhibitors / Tirozin kinaz inhibitörleri, 168 Imatinib mesylate / İmatinib mesilat, 168 Pruritus / Kaşıntı 168 Thiopurine S-methyltransferase / Tiyopürin S-metiltransferaz, 184 Methylenetetrahydrofolate reductase / Metilentetrahidrofolat redüktaz, 184 Gene polymorphisms / Gen polimorfizmleri, 184 Leukemia / Lösemi, 184 Childhood / Çocukluk çağı, 184 Gaucher cells / Gaucher hücreleri, 187 Electron microscopy / Elektron mikroskopi, 187 Necrosis / Nekroz, 373 Gaucher / Gaucher, 373 Bone marrow / Kemik iliği, 373 Sedoanalgesia / Sedoanaljezi, 351 Ketamine / Ketamin, 351 Midazolam / Midazolam, 351 Invasive procedure / İnvazif işlem, 351

32nd Volume Index / 32. Cilt Dizini AUTHOR INDEX - YAZAR DİZİNİ 2015

A. G. Haddad..................................................80 A. H. Nassar....................................................80 A. H. Radwan.................................................80 Abdullah Cerit............................................. 213 Afig Berdeli.................................................. 276 Afra Yıldırım...................................................97 Ahmet Çizmecioğlu..................................... 374 Ahmet Emre Eşkazan........................... 213,243 Ahmet Muzaffer Demir..................................58 Akif Selim Yavuz............................................43 Alberto Daniel Gimenez Conca.................. 194 Alessandro Allegra....................................... 168 Alev Öztaş.................................................... 375 Ali Ayçiçek....................................................186 Ali Dursun................................................... 317 Ali Fettah........................................................73 Ali Keskin............................................ 295, 323 Ali Koçyiğit.................................................. 355 Ali Mert....................................................... 243 Ali T. Taher.....................................................80 Anand Chellappan..........................................85 Andrea Alonci............................................. 168 Anita Ramdas.............................................. 377 Aniya Antony.............................................. 377 Ansaf B. Yousef...............................................15 Antica Nacinovic-Duletic............................ 234 Anuradha Monga......................................... 158 Arzu Akçay.................................................. 127 Arzu Yazal Erdem...........................................87 Asami Shimada............................................ 257 Aslı Korur.................................................... 367 Aslıhan Demirel........................................... 243 Ateş Kara..................................................... 144 Ayhan Deviren................................................82 Ayhan Pektaş............................................... 144 Aysun Adan Gökbulut................................. 118 Ayşe Çırakoğlu...............................................82 Ayşe Işık....................................................... 163 Ayşe Kılıç..................................................... 338 Ayşegül Üner............................................... 163 Ayşegül Ünüvar........................................... 338 Aytemiz Gurgey........................................... 144 Aziz Polat..................................................... 355 Bahattin Tunç............................. 73,87,172,228 Bahriye Payzın............................... 152,277,323 Balint Nagy.................................................. 206 Begüm Atasay.............................................. 267 Begüm Koç.................................................. 344 Betül Börkü Uysal........................................ 213 Betül Küçükzeybek..................................... 277

Betül Tavil............................................ 172,228 Bhawna Jha........................................... 192,372 Bianca Tesi................................................... 355 Bilgül Mete.................................................. 243 Božena Coha................................................ 271 Božo Petrov.................................................. 234 Burak Erer......................................................43 Burcu Belen................................................. 185 Burhan Ferhanoğlu..................................... 243 Burhanettin Küçük.........................................58 Bülent Eser.....................................................97 Bülent Kantarcıoğlu.................................... 189 Bülent Ündar............................................... 152 Can Balkan.................................................. 263 Can Baykal......................................................43 Can Boğa.................................. 51,100,195,367 Canan Vergin............................................... 276 Carmen Mannucci....................................... 168 Caterina Musolino....................................... 168 Cem Muhlis Ar................................ 82,213,243 Cemil Ekinci...................................................35 Cengiz Bal.......................................................21 Cengiz Ceylan............................................. 323 Chanaveerappa Bammigatti............................85 Cheng-Hsu Wang...........................................68 Chien-Hong Lai..............................................68 Chokka Kiran.............................................. 377 Chunyan Liu............................................... 220 Cumali Karatoprak...................................... 213 Çiğdem Gereklioğlu.................................... 367 Çetin Timur.......................................... 127,344 Dalina I. Tanyong........................................ 304 Deniz Çağdaş............................................... 379 Deniz Sünnetçi............................................ 206 Deniz Yılmaz Karapınar.............................. 263 Dilek Gürlek Gökçebay.......................... 73,228 Dilek Kahvecioğlu....................................... 267 Dilhan Kuru...................................................82 Dilşad Sindel...................................................43 Duran Canatan............................................ 375 Duygu Kankaya..............................................35 Duygu Uçkan Çetinkaya...................... 228,379 Ebru Yılmaz Keskin..........................................1 Elif Suyanı.............................................. 29,251 Elizabeta Dadic-Hero.................................. 234 Emin Ünüvar............................................... 338 Emine Zengin.............................................. 351 Emre Tepeli................................................. 295 Erdem Şimşek.............................................. 329 Eren Gündüz..................................................21

32nd Volume Index / 32. Cilt Dizini AUTHOR INDEX - YAZAR DİZİNİ 2015

Erkin Serdaroğlu......................................... 276 Ersin Töret.................................................. 276 Esin Özcan.................................................. 276 Esin Şenol.................................................... 100 Esma Çakmak............................................. 351 Eylem Eliaçık.............................................. 163 Ezgi Uysalol................................................. 338 Fahir Özkalemkaş....................................... 100 Fahir Öztürk...................................................97 Fahri Şahin.................................................. 323 Fatih Azık.................................................... 228 Fatih Demircioğlu....................................... 284 Fatih Demirkan........................................... 152 Fatma Demir Yenigürbüz..................... 175,329 Fatma Gümrük................................ 64,136,363 Fatma Karaca Kara.........................................87 Fatma Oğuz................................................. 338 Federico Angriman...................................... 194 Fehmi Tabak................................................ 243 Ferit Avcu.................................................... 311 Fethullah Kenar........................................... 355 Fikriye Uras....................................................58 Filiz Aydın................................................... 344 Filiz Büyükkeçeci........................................ 323 Filiz Vural.................................................... 323 Francesco Di Raimondo.............................. 206 Funda Özgürler Akpınar............................. 355 Füsun Özdemirkıran............................ 277,323 Gamze Asker............................................... 344 Gamze Tatar................................................ 213 Genco Gençay............................................. 344 Gioacchino Calapai..................................... 168 Giuseppe Palumbo...................................... 206 Gonca Oruk................................................. 277 Gökhan Özgür............................................. 311 Guohua Yu.....................................................99 Güldane Cengiz Seval................................. 180 Gülersu İrken....................................... 175,329 Gülnur Görgün........................................... 323 Gülşah Akyol..................................................97 Gülşah Kaygusuz............................................35 Gülseren Bağcı............................................. 295 Gülsüm Akgün Çağlıyan............................. 323 Güray Saydam............................................. 323 Gürhan Kadıköylü......................... 190,282,323 Güven Çetin......................................... 190,213 H. Demet Kiper........................................... 323 H. El Farran....................................................80 Hakan Göker............................................... 163 Hakan Özdoğu................................ 51,195,367

Hakan Savlı.................................................. 206 Hale Ören............................................. 175,329 Hamdi Akan................................................ 100 Hara Prasad Pati.......................................... 158 Hasan Çakmaklı.......................................... 267 Hava Üsküdar Teke........................................21 Hayri Özsan................................................. 152 Hernán Michelángelo.................................. 194 Hidenori Imai.............................................. 257 Hikmet Eda Alışkan.......................................51 Hrvoje Holik................................................ 271 Huaquan Wang............................................ 220 Işınsu Kuzu....................................................35 İbrahim C. Haznedaroğlu............................ 163 İbrahim Caner............................................. 359 İbrahim İleri...................................................97 İbrahim Kamer............................................ 338 İbrahim Keser.............................................. 375 İdil Yenicesu.....................................................1 İhsan Karadoğan......................................... 100 İkbal Cansu Barış........................................ 295 İkbal Ok Bozkaya........................................ 172 İlhan Altan......................................................64 İlhan Tezcan................................................ 379 İnci Alacacıoğlu........................................... 323 İrfan Yavaşoğlu.................... 93,94,189,190,282 İsmail Can................................................... 127 İsmail Kırbaş...................................................73 İsmail Sarı.................................................... 295 İsmail Yıldız................................................. 338 İsmet Aydoğdu............................................ 374 Jelena Ivandic.............................................. 234 Jen-Seng Huang..............................................68 Jorge Alberto Arbelbide............................... 194 Junichi Arita................................................ 257 Kaan Kavaklı............................................... 263 Kadir Şerafettin Tekgündüz........................ 359 Keiji Sugimoto............................................. 257 Kun-Yun Yeh..................................................68 Kübra Gözübenli......................................... 213 Levent Oğuzkurt............................................51 Leyla Ağaoğlu.............................................. 127 M. Ali Çıkrıkçıoğlu..................................... 213 Mahmut Yeral......................................... 51,367 Maja Tomic-Paradžik................................... 271 Manoranjan Mahapatra.......................... 77,158 Maria Nelly Gutierrez Acevedo................... 194 Maria Sol Rossi............................................ 194 Marie Ambroise........................................... 377 Marijan Šiško............................................... 271

32nd Volume Index / 32. Cilt Dizini AUTHOR INDEX - YAZAR DİZİNİ 2015

Mario Petrini............................................... 206 Martina Canestraro..................................... 206 Masaaki Noguchi......................................... 257 Mehmet Ali Özcan........................ 100,152,323 Mehmet Ertem............................................ 267 Mehmet Hilmi Doğu................................... 295 Mehmet Sönmez.......................................... 100 Meliha Tan................................................... 367 Melike Koruyucu......................................... 277 Meltem Aylı................................................. 180 Meltem Özgüner............................................87 Meral Sarper................................................ 311 Merih Kızıl Çakar........................................ 251 Mervan Bekdaş............................................ 284 Meryem Seda Boyraz................................... 379 Mesut Ayer.................................................. 213 Moe Matsuzawa........................................... 257 Monika Gupta................................................77 Mookkappan Sudhagar............................... 377 Mualla Çetin.................................... 64,317,363 Muhit Özcan................................................ 180 Murat Akova................................................ 100 Murat Tombuloğlu...................................... 323 Mustafa Çetin.................................................97 Mustafa Dilek.............................................. 284 Mustafa Erkoçoğlu...................................... 284 Mustafa Kara............................................... 359 Mustafa Yaşar.............................................. 118 Mutlu Yüksek.................................................87 Mutsumi Wakabayashi................................ 257 Muzaffer Keklik..............................................97 Mücahit Yemişen......................................... 243 Müge Gökçe...................................................64 Müge Sayitoğlu............................................ 127 Münci Yağcı............................................ 29,251 Nagihan Yalçın............................................ 355 Namık Yaşar Özbek.................................. 73,87 Nazan Sarper............................................... 351 Nazmiye Yüksek.............................................87 Neryal Müminoğlu...................................... 276 Nesimi Büyükbabani......................................43 Neslihan Erdem........................................... 374 Neşe Yaralı....................................... 87,172,317 Nevruz Kurşunoğlu........................................29 Nihal Karadaş.............................................. 263 Nilay Şen Türk............................................ 295 Nilgün Sayınalp........................................... 163 Nilüfer Alpay Kanıtez.....................................43 Nita Radhakrishnan.......................................77 Noriko Nakamura....................................... 257

Norio Komatsu............................................ 257 Nurhan Ergül.............................................. 213 Nurhilal Büyükkurt..................................... 152 Oktay Bilgir................................................. 323 Olga Meltem Akay..........................................21 Osman İ. Özcebe......................................... 163 Ozan Çetin.................................................. 295 Ömer Devecioğlu......................................... 344 Ömer Doğru................................................ 127 Ömer Erdeve............................................... 267 Ömür Gökmen Sevindik............................. 323 Önder Arslan............................................... 285 Öner Doğan....................................................43 Öykü Arslan................................................ 323 Özden Hatırnaz Ng..................................... 127 Özden Pişkin............................................... 152 Özge Can..................................................... 295 Özgür Esen.................................................. 277 Özlem Arman Bilir.........................................87 Özlem Bingöl Özakpınar................................58 Özlem Tüfekçi...................................... 175,329 Pamir Işık............................................. 172,228 Pei-Hung Chang.............................................68 Pelin Mutlu.................................................. 311 Pervin Topçuoğlu...........................................35 Pınar Ataca.................................................. 285 Pranav Dorwal............................................. 372 Prapaporn Panichob.................................... 304 R. Hourani......................................................80 Rabab M. Aly..................................................15 Rabin Saba................................................... 100 Rajan Kapoor............................................... 158 Ranjit Kumar Sahoo.................................... 279 Recep Öztürk............................................... 243 Refik Tanakol.................................................43 Renata Dobrila-Dintinjana.......................... 234 Renu Saxena...................................................77 Reşat Özaras................................................ 243 Ritesh Sachdev..................................... 192,372 Rong Fu....................................................... 220 Rukiye Ünsal Saç......................................... 172 Saadet Arsan................................................ 267 Sabina Russo................................................ 168 Salih Aksu.................................................... 163 Salih Gözmen.............................................. 175 Sara Galimberti............................................ 206 Sebastiano Gangemi.................................... 168 Seçkin Çağırgan........................................... 100 Seher Açar................................................... 284 Selda Kahraman........................................... 323

32nd Volume Index / 32. Cilt Dizini AUTHOR INDEX - YAZAR DİZİNİ 2015

Selin Aytaç......................................................64 Sema Aylan Gelen........................................ 351 Semra Atalay................................................ 267 Semra Büyükkorkmaz................................. 284 Serap Karaman..................................... 338,344 Serap Yalçın................................................. 311 Serdar Alan.................................................. 267 Serhan Küpeli.............................................. 184 Sevgi Gözdaşoğlu........................................ 188 Sevgi Yetgin................................................. 317 Sevil Göksügür............................................ 284 Shalini Goel.......................................... 192,372 Sibel Hacıoğlu.............................................. 295 Sibel Kabukçu............................................. 323 Simge Erdem............................................... 213 Sinem Fırtına............................................... 127 Smeeta Gajendra............................ 192,279,372 Sonay Temurhan......................................... 344 Soner Solmaz............................................... 367 Sultan Aydın Köker..................................... 276 Sunay Tunalı............................................... 152 Suthat Fucharoen........................................ 304 Swaminathan Palamalai..................................85 Şenay Demir................................................ 367 Seniha Hacıhanefioğlu....................................82 Şeniz Öngören Aydın............................. 82,243 Şebnem Yılmaz Bengoa............................... 329 Şinasi Özsoylu.............................. 89,90,92,280 Şule Ünal........................... 64,136,317,363,379 Şükriye Yılmaz................................................82 T. Fikret Çermik.......................................... 213 Taner Demirci.................................................29 Tatiana Greenwood..................................... 355 Tayfun Uçar................................................. 267 Teoman Soysal........................................ 82,243 Tingguo Zhang...............................................99 Tiraje Celkan........................................ 127,344 Tomohiro Sawada........................................ 257 Toni Valkovic............................................... 234 Tony Rupar.................................................. 317 Tsung-Han Wu...............................................68 Tuba Hilkay Karapınar.................. 175,276,329 Tuba Özkan................................................. 213 Tuğba Elgün................................................ 344 Turan Bayhan.............................................. 363 Tushar Sahni........................................ 192,372 Türkan Atasever.......................................... 277

Türker Bilgen.............................................. 375 Türker Çetin................................................ 311 Türkiz Gürsel....................................... 185,317 Ufuk Çakır................................................... 267 Uğur Demirsoy............................................ 351 Uğur Özbek................................................. 127 Ülkü Ergene................................................ 152 Valerio Maisano........................................... 168 Vedrana Gacic.............................................. 234 Victoria Otero.............................................. 194 Vildan Caner............................................... 295 Vimarsh Raina............................................. 192 Wasinee Kheansaard................................... 304 Weiwei Qi.................................................... 220 Xin Huang......................................................99 Yahya Büyükaşık......................................... 163 Yasemin Işık Balcı........................................ 355 Yasunobu Sekiguchi.................................... 257 Yaşar Demirelli............................................ 359 Yelda Tarkan Argüden....................................82 Yen-Min Huang..............................................68 Yeşim Aydınok............................................. 263 Yeşim Oymak............................................... 276 Yıldız Aydın................................................. 243 Yılmaz Ay..................................................... 276 Yii-Jenq Lan....................................................68 Yue Ren....................................................... 220 Yueh-Shih Chang...........................................68 Yuqing Huo....................................................99 Yusuf Baran................................................. 118 Yusuf Ziya Demiroğlu....................................51 Zafer Başlar.................................................. 243 Zafer Gökgöz............................................... 323 Zafer Gülbaş...................................................21 Zahit Bolaman........................ 100,190,282,323 Zeynep Arzu Yeğin.........................................29 Zeynep Gümüş............................................ 277 Zeynep Karakaş.................................... 127,344 Zeynep Yıldız Yıldırmak....................... 127,344 Zeynep Yılmaz................................................29 Zifen Gao........................................................99 Zonghong Shao........................................... 220 Zübeyde Nur Özkurt......................................29 Zühal Önder Siviş........................................ 263

Advisory Board of This Issue (December 2015) Ahmet Koç, Turkey Akif Yeşilipek, Turkey Ali Bay, Turkey Ali İrfan Emre Tekgündüz, Turkey Ali Ünal, Turkey Alphan Küpesiz, Turkey Attila Szvetko, Australia Ayşegül Ünüvar, Turkey BülentKarapınar, Turkey Burhan Ferhanoğlu, Turkey Can Balkan, Turkey Canan Albayrak, Turkey Christpher Dandoy, USA

Clare Y. Slaney, Australia Dilber Talia İleri, Turkey Elena Cassinerio, Italy Elif Ünal İnce, Turkey Gregory Kaufman, USA Gülsüm Emel Pamuk, Turkey Hande Çağlayan, Turkey Kaan Kavaklı, Turkey Luis Villela, Mexico Marco L. Davila, USA Nejat Akar, Turkey Reyhan Diz Küçükkaya, Turkey Sascha Meyer, Germany

Şebnem Yılmaz, Turkey Semra Paydaş, Turkey Şule Ünal, Turkey Tiraje Celkan, Turkey TürkanPatıroğlu, Turkey Ülker Koçak, Turkey Ulrike Reiss, USA Vincenzo De Sanctis, Italy Xunlei Kang, USA Yurdanur Kılınç, Turkey Yusuf Baran, Turkey Zeynep Karakaş, Turkey

MAIN TOPICS • Origin of Antiphospholipid Antibodies (aPL) • Genetics of Antiphospholipid Syndrome (APS) • Mechanism(s) of aPL-mediated Thrombosis & Pregnancy Morbidity • Target Cells & Receptors that Interact with aPL • Definition, Epidemiology & Natural History of APS • Impact of APS in General Population with Thrombosis & Pregnancy Morbidity • Association Between APS & Other Systemic Autoimmune Diseases, e.g., Lupus • Thrombotic Angiopathies including Microangiopathic & Catastrophic APS

• Clinical & Prognostic Significance of “Criteria” & “Non-criteria” aPL Tests • Risk Stratification & Disease Measurement Criteria in APS • Current Treatment Strategies & Treatment Trends in APS • Role of Immunosuppressive Agents in APS • Impact of Pediatric APS in Children with Thrombosis • Strengths & Limitations of the Current APS Classification Criteria • Recent Thrombosis Treatment Strategies in General Population • Clinical Trial Design & Implementation • Thrombotic & Obstetric APS for Patients

Abstract Submission and Registration Starts

Local Executive Committee Ahmet Muzaffer Demir Bahar Artım Esen Ihsan Ertenli Vedat Hamuryudan Murat Inanc Sedat Kiraz Reyhan Kucukkaya Seza Ozen

International Executive Committee Mary-Carmen Amigo, Mexico Danieli Andrade, Brazil Tatsuya Atsumi, Japan Maria Laura Bertolaccini, UK Ware Branch, USA Robin Brey, USA Ricard Cervera, Spain Hannah Cohen, UK

Maria Cuadrado, UK Phillip de Groot, Netherlands Ronald Derksen, Netherlands Doruk Erkan, USA Paul Fortin, Canada Nigel Harris, Jamaica Graham Hughes, UK Munther Khamashta, UK Takao Koike, Japan Steven Krilis, Australia

September 9, 2015

Steven Levine, USA Roger Levy, Brazil Michael Lockshin, USA Samuel Machin, UK Pier Luigi Meroni, Italy Vittorio Pengo, Italy Michelle Petri, USA Jacob Rand, USA Joyce Rauch, Canada Robert Roubey, USA

Guillermo Ruiz-Irastorza, Spain Jane Salmon, USA Lisa Sammaritano, USA Yehuda Shoenfeld, Israel Maria Tektonidou, Greece Angela Tincani, Italy Denis Wahl, France Zholi Zhang, China

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