Tjh 2016 1 by LookUs Scientific

Issue 1

March 2016

80 TL

ISSN 1300-7777

Volume 33

Review Article The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome Doruk Erkan, Jane E. Salmon; New York, United States

Research Articles Deregulated Levels of the NF-κB1, NF-κB2, and Rel Genes in Ukrainian Patients with Leukemia and Lymphoma in the Post-Chernobyl Period Hakan Savlı, et al.; Kocaeli, Turkey, Kyiv, Ukraine

Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression Tze Yan Lee, et al.; Serdang, Malaysia

Freezing of Apheresis Platelet Concentrates in 6% Dimethyl Sulfoxide: The First Preliminary Study in Turkey Soner Yılmaz, et al.; Ankara, Turkey

Comparison of the Efficiencies of Buffers Containing Ankaferd and Chitosan on Hemostasis in an Experimental Rat Model with Femoral Artery Bleeding Serkan Abacıoğlu, et al.; Osmaniye, Ankara, Zonguldak, Turkey

Cover Picture: Işıl Erdoğan Özünal Ağva, İstanbul

Editor-in-Chief

International Review Board

Reyhan Küçükkaya

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 Aytemiz Gürgey

Language Editor Leslie Demir

Senior Advisory Board Yücel Tangün Osman İlhan Muhit Özcan Teoman Soysal

Editorial Office İpek Durusu Bengü Timoçin

İstanbul Bilim University, İstanbul, Turkey

Associate Editors Ayşegül Ünüvar İstanbul University, İstanbul, Turkey

Cengiz Beyan Gülhane Military Medical Academy, Ankara, Turkey

Hale Ören Dokuz Eylül University, İzmir, Turkey

İbrahim C. Haznedaroğlu Hacettepe University, Ankara, Turkey

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

Selami Koçak Toprak Ankara University, Ankara, Turkey

Semra Paydaş Çukurova University, Adana, 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

Elif Ünal İnce Ankara University, Ankara, Turkey

İnci Alacacıoğlu Dokuz Eylül University, İzmir, Turkey

Müge Sayitoğlu İstanbul University, İstanbul, Turkey

Nil Güler Ondokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay Osmangazi University, Eskişehir, Turkey

Şule Ünal Hacettepe University, Ankara, Turkey

Veysel Sabri Hançer İstanbul Bilim University, İstanbul, Turkey

Zühre Kaya Gazi University, Ankara, Turkey

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Publishing Services

Statistic Editor Hülya Ellidokuz

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. Reyhan Küçükkaya E-mail : rkucukkaya@hotmail.com

All inquiries should be addressed to TURKISH JOURNAL OF HEMATOLOGY Address Phone Fax E-mail

: İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550 Çankaya, Ankara / Turkey : +90 312 490 98 97 : +90 312 490 98 68  : info@tjh.com.tr

ISSN: 1300-7777

Turkish Society of Hematology

Publishing Manager Sorumlu Yazı İşleri Müdürü

Ahmet Muzaffer Demir, President

Güner Hayri Özsan

Güner Hayri Özsan, General Secretary T. Tiraje Celkan, Vice President

Management Address Yayın İdare Adresi

M. Cem Ar, Research Secretary E. Naci Tiftik, Treasurer

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

Meltem Yüksel, Member İlknur Kozanoğlu, Member

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: Özgün Ofset Ticaret Ltd. Şti. Yeşilce Mah. Aytekin Sk. No: 21 34418 4. Levent / İSTANBUL

http://mc.manuscriptcentral.com/tjh

Web page www.tjh.com.tr

Printing Date / Basım Tarihi 15.02.2016

Owner on behalf of the Turkish Society of Hematology Türk Hematoloji Derneği adına yayın sahibi Ahmet Muzaffer Demir 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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Cover Picture Işıl Erdoğan Özünal was born in 1982, Turkey. She is currently working at İstanbul University Cerrahpaşa Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey.

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 Englishlanguage 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 of 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, 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. As of December 2015, The Turkish Journal of Hematology does not accept case reports. Important new findings or data about interesting hematological cases may be submitted as a brief report. 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-of-charge 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

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Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh  Web page: www.tjh.com.tr  E-mail: info@tjh.com.tr   Permissions  Requests for permission to reproduce published material should be sent to the editorial office. Editor: Professor Dr. Reyhan Diz Küçükkaya Adress: İlkbahar 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, 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 double 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). 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.

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. 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

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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. Other types of manuscripts, such as 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. Study Limitations: 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. Conclusion: The conclusion of the study should be highlighted.

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

Examples of References: 1. List all authors. Deeg HJ, Oâ&#x20AC;&#x2122;Donnel M, Tolar J. Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure immunosuppressive therapy. Blood 2006;108:1485-1491. 2.Organization as author Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in posthepatitis marrow aplasia. Lancet 1977;2:742-744. 3.Book Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger, 1961. 4. Book Chapter Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of

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Hemoglobin: Genetics, Pathophysiology, Clinical Management. New York, Cambridge University Press, 2000. 5.Abstract Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract). 6.Letter to the Editor Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further definition of 20q deletion in myeloid leukemia using fluorescence in situ hybridization. Blood 1994;84:2821-2823. 7. Supplement Alter BP. Fanconiâ&#x20AC;&#x2122;s anemia, transplantation, and cancer. Pediatr Transplant. 2005;9(Suppl 7):81-86

Brief Reports Abstract length: Not to exceed 150 words. Article length: Not to exceed 1200 words. Introduction: State the purpose and summarize the rationale for the study. Materials and Methods: Clearly describe the selection of the observational or experimental participants. Identify the methods and procedures in sufficient detail. Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for their use and an evaluation of their limitations. Identify all drugs and chemicals used, including generic names, doses, and routes of administration. Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify the reported findings/results. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used. Results: Present the findings/results in a logical sequence in the text, tables, and figures. Do not repeat all the findings/results in the tables and figures in the text; emphasize and/or summarize only those that are most important. Discussion: Highlight the new and important findings/results of the study and the conclusions they lead to. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by your data.

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 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.

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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. 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.

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unc.edu/~rowlett/units/scales/clinical_data.html. For more details, see http://www.amamanualofstyle.com/oso/public/jama/si_conversion_ table.html. Example for CBC.

Hematology component

SI units

RBC

6.7-11 x 1012/L

WBC

5.5-19.5 x109/L

Hemoglobin

116-168 g/L

PCV

0.31-0.46 L/L

MCV

39-53 fL

MCHC

300-360 g/L

MCH

19.5-25 pg

Platelets

300-700 x 109/L

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

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

Online Manuscript Submission Process The Turkish Journal of Hematology uses submission software powered by ScholarOne Manuscripts. The website for submissions to The Turkish Journal of Hematology is http://mc.manuscriptcentral.com/tjh. This system is quick and convenient, both for authors and reviewers.

Setting up an account New users to the submission site will need to register and enter their account details before they can submit a manuscript. Log in, or click the “Create Account” button if you are a first-time user. To create a new account: After clicking the “Create Account” button, enter your name and e-mail address, and then click the “Next” button. Your e-mail address is very important. Enter your institution and address information, as appropriate, and then click the “Next” Button. Enter a user ID and password of your choice, select your area of expertise, and then click the “Finish” button. If you have an account, but have forgotten your log-in details, go to “Password Help” on the journal’s online submission system and enter

your e-mail address. The system will send you an automatic user ID and a new temporary password. Full instructions and support are available on the site, and a user ID and password can be obtained during your first visit. Full support for authors is provided. Each page has a “Get Help Now” icon that connects directly to the online support system. Contact the journal administrator with any questions about submitting your manuscript to the journal (info@tjh.com.tr). For ScholarOne Manuscripts customer support, click on the “Get Help Now” link on the top right hand corner of every page on the site.

The Electronic Submission Process Log in to your author center. Once you have logged in, click the “Submit a Manuscript” link in the menu bar. Enter the appropriate data and answer the questions. You may copy and paste directly from your manuscript. Click the “Next” button on each screen to save your work and advance to the next screen.

Upload Files Click on the “Browse” button and locate the file on your computer. Select the appropriate designation for each file in the drop-down menu next to the “Browse” button. When you have selected all the files you want to upload, click the “Upload Files” button. Review your submission before sending to the journal. Click the “Submit” button when you are finished reviewing. You can use ScholarOne Manuscripts at any time to check the status of your submission. The journal’s editorial office will inform you by e-mail once a decision has been made. After your manuscript has been submitted, a checklist will then be completed by the Editorial Assistant. The Editorial Assistant will check that the manuscript contains all required components and adheres to the author guidelines. Once the Editorial Assistant is satisfied with the manuscript it will be forwarded to the Senior Editor, who will assign an editor and reviewers.

The Review Process Each manuscript submitted to The Turkish Journal of Hematology is subject to an initial review by the editorial office in order to determine if it is aligned with the journal’s aims and scope, and complies with essential requirements. Manuscripts sent for peer review will be assigned to one of the journal’s associate editors that has expertise relevant to the manuscript’s content. All accepted manuscripts are sent to a statistical and English language editor before publishing. Once papers have been reviewed, the reviewers’ comments are sent to the Editor, who will then make a preliminary decision on the paper. At this stage, based on the feedback from reviewers, manuscripts can be accepted, rejected, or revisions can be recommended. Following initial peer-review, articles judged worthy of further consideration often require revision. Revised manuscripts generally must be received within 3 months of the date of the initial decision. Extensions must be requested from the Associate Editor at least 2 weeks before the 3-month revision deadline expires; The Turkish Journal of Hematology will

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reject manuscripts that are not received within the 3-month revision deadline. Manuscripts with extensive revision recommendations will be sent for further review (usually by the same reviewers) upon their re-submission. When a manuscript is finally accepted for publication, the Technical Editor undertakes a final edit and a marked-up copy will be e-mailed to the corresponding author for review and to make any final adjustments.

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CONTENTS 1

Review Article

Research Articles

The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome Doruk Erkan, Jane E. Salmon

Deregulated Levels of the NF-κB1, NF-κB2, and Rel Genes in Ukrainian Patients with Leukemia and Lymphoma in the Post-Chernobyl Period Hakan Savlı, Ramis Ufuk Akkoyunlu, Naci Çine, Daniil F. Gluzman, Michael P. Zavelevich, Lilia M. Sklyarenko, Stella V. Koval, Deniz Sünnetçi

Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression Tze Yan Lee, Logeswaran Muniandy, Lai Kuan Teh, Maha Abdullah, Elizabeth George, Jameela Sathar, Mei I Lai

Freezing of Apheresis Platelet Concentrates in 6% Dimethyl Sulfoxide: The First Preliminary Study in Turkey Soner Yılmaz, Rıza Aytaç Çetinkaya, İbrahim Eker, Aytekin Ünlü, Metin Uyanık, Serkan Tapan, Ahmet Pekoğlu, Aysel Pekel, Birgül Erkmen, Uğur Muşabak, Sebahattin Yılmaz, İsmail Yaşar Avcı, Ferit Avcu, Emin Kürekçi, Can Polat Eyigün

Is It Possible to Predict Pulmonary Complications and Mortality in Hematopoietic Stem Cell Transplantation Recipients from Pre-Transplantation Exhaled Nitric Oxide Levels? Nurdan Köktürk, Fatma Yıldırım, Müge Aydoğdu, Şahika Zeynep Akı, Zeynep Arzu Yeğin, Zübeyde Nur Özkurt, Elif Suyanı, İpek Kıvılcım Oğuzülgen, Gülsan Türköz Sucak

A Quasi-Experimental Study Analyzing the Effectiveness of Portable High-Efficiency Particulate Absorption Filters in Preventing Infections in Hematology Patients during Construction Mehmet Özen, Gülden Yılmaz, Belgin Coşkun, Pervin Topçuoğlu, Bengi Öztürk, Mehmet Gündüz, Erden Atilla, Önder Arslan, Muhit Özcan, Taner Demirer, Osman İlhan, Nahide Konuk, İsmail Balık, Günhan Gürman, Hamdi Akan

Comparison of the Efficiencies of Buffers Containing Ankaferd and Chitosan on Hemostasis in an Experimental Rat Model with Femoral Artery Bleeding Serkan Abacıoğlu, Kemal Aydın, Fatih Büyükçam, Ural Kaya, Bahattin Işık, Muhammed Evvah Karakılıç

Brief Reports

Antifungal Treatment in Stem Cell Transplantation Centers in Turkey Hamdi Akan, Erden Atilla

Hemoglobin H Disease in Turkey: Experience from Eight Centers Selma Ünal, Gönül Oktay, Can Acıpayam, Gül İlhan, Edip Gali, Tiraje Celkan, Ali Bay, Barış Malbora, Nejat Akar, Yeşim Oymak, Tayfur Toptaş

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The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia Damla Eyüpoğlu, Süreyya Bozkurt, İbrahim Haznedaroğlu, Yahya Büyükaşık, Deniz Güven

Case Report

Secondary Neoplasms in Children with Hodgkin’s Lymphoma Receiving C-MOPP and Radiotherapy: Presentation of Four Cases Sevgi Gözdaşoğlu, Ali Pamir, Emel Ünal, İsmail Haluk Gökçora, Ömer Uluoğlu, Koray Ceyhan, Haluk Deda, Erdoğan Işıkman, Gülsan Yavuz, Nurdan Taçyıldız, Ayhan Çavdar

Letters to the Editor

First Observation of Hemoglobin G-Waimanalo and Hemoglobin Fontainebleau Cases in the Turkish Population Duran Canatan, Türker Bilgen, Vildan Çiftçi, Gülsüm Yazıcı, Serpil Delibaş, İbrahim Keser

Serum Lipids in Turkish Patients with β-Thalassemia Major and β-Thalassemia Minor Yasemin Işık Balcı, Şule Ünal, Fatma Gümrük

Extranodal Natural Killer/T-Cell Lymphoma: A Rare Nasal-Type Case Esra Sarıbacak Can, Harika Okutan, Murat Albayrak, Ünsal Han

T-Cell Lymphoma Presenting with Auricular and Parotid Gland Involvement Birgül Öneç, Alper Koç, Elif Nisa Ünlü, İlhan Ünlü, Hüseyin Yaman, Durdu Mehmet Köş

Immune Thrombocytopenia Resolved by Eltrombopag in a Carrier of Glucose-6-Phosphate Dehydrogenase Deficiency Laura Scaramucci, Pasquale Niscola, Massimiliano Palombi, Andrea Tendas, Marco Giovannini, Paolo de Fabritiis

Wernicke’s Encephalopathy in an Acute Myeloid Leukemia Patient: A Case Study Muhammet Maden, Gülsüm Pamuk, Yahya Çelik, Ercüment Ünlü

Kaleidoscopic Views in the Bone Marrow: Oxalate Crystals in a Patient Presenting with Bicytopenia Yelda Dere, Simge Erbil, Murat Sezak, Başak Doğanavşargil, Mümtaz Yılmaz, Nazan Özsan, Mine Hekimgil

An Unexpected Innocent Complication Associated with Azacitidine Treatment of Myelodysplastic Syndrome: Erythema Annulare Centrifugum Esra Turan Erkek, Sevgi Kalayoğlu Beşışık

Thrombotic Microangiopathic Hemolytic Anemia without Evidence of Hemolytic Uremic Syndrome Şinasi Özsoylu

Images in Hematology

Radiologic Image of a Child with Leukemia Who Developed Sepsis and Fulminant Thrombosis during Induction Therapy Eda Ataseven, Ömer Özden, Şebnem Yılmaz Bengoa, Handan Güleryüz, Murat Duman, Hale Ören

Hereditary Elliptocytosis with Pyropoikilocytosis Turan Bayhan, Şule Ünal, Fatma Gümrük

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REVIEW DOI: 10.4274/tjh.2015.0197 Turk J Hematol 2016;33:1-7

The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome Trombotik Anjiyopati ve Antifosfolipid Sendromunda Kompleman İnhibisyonunun Rolü Doruk Erkan, Jane E. Salmon Hospital for Special Surgery, Weill Cornell Medicine, New York, United States

Abstract

Öz

Antiphospholipid syndrome (APS) is characterized by thrombosis (arterial, venous, small vessel) and/or pregnancy morbidity occurring in patients with persistently positive antiphospholipid antibodies (aPL). Catastrophic APS is the most severe form of the disease, characterized by multiple organ thromboses occurring in a short period and commonly associated with thrombotic microangiopathy (TMA). Similar to patients with complement regulatory gene mutations developing TMA, increased complement activation on endothelial cells plays a role in hypercoagulability in aPL-positive patients. In mouse models of APS, activation of the complement is required and interaction of complement (C) 5a with its receptor C5aR leads to aPL-induced inflammation, placental insufficiency, and thrombosis. Anti-C5 antibody and C5aR antagonist peptides prevent aPL-mediated pregnancy loss and thrombosis in these experimental models. Clinical studies of anti-C5 monoclonal antibody in aPL-positive patients are limited to a small number of case reports. Ongoing and future clinical studies of complement inhibitors will help determine the role of complement inhibition in the management of aPL-positive patients.

Antifosfolipid sendromu (APS), ısrarcı antifosfolipid antikor (aPL) pozitifliği olan hastalarda görülen tromboz (arteriyel, venöz, küçük damar) ve/veya gebelik ile ilişkili morbidite ile karakterizedir. Hastalığın en şiddetli formu olan katastrofik APS, kısa süre içerisinde gelişen çoklu organ trombozları ile karakterizedir ve sıklıkla trombotik mikroanjiyopati (TMA) ile ilişkilidir. TMA geliştiren kompleman düzenleyici gen mutasyonları bulunan hastalarla benzer olarak, aPLpozitif hastalardaki hiperkoagülopatide, endotel hücrelerinde artmış kompleman aktivasyonunun rolü vardır. APS’nin fare modellerinde, kompleman aktivasyonunun olması zorunludur ve kompleman (C) 5a ile reseptörü C5aR’nin etkileşmesi aPL-ile uyarılmış yangı, plasenta yetmezliği ve tromboza neden olur. Anti-C5 antikoru ve C5aR antagonisti olan peptidler bu deneysel modellerde, aPL-aracılı gebelik kaybı ve trombozu önler. aPL-pozitif hastalarda anti-C5 monoklonal antikorun klinik kullanımı az sayıdaki olgu sunumları ile sınırlıdır. Halen devam etmekte olan ve gelecekte yapılacak klinik çalışmalar, aPL-pozitif hastaların yönetiminde kompleman inhibitörlerinin rolünü belirlemede yardımcı olacaktır.

Keywords: Antiphospholipid syndrome, Complement inhibition, Eculizumab, Thrombotic angiopathy

Anahtar Sözcükler: Antifosfolipid sendromu, Komplaman inhibisyonu, Eculizumab, Trombotik anjiyopati

Introduction Antiphospholipid syndrome (APS) is characterized by thrombosis (arterial, venous, small vessel) and/or pregnancy morbidity occurring in patients with persistently positive antiphospholipid antibodies (aPL) [1]. The current treatment in APS focuses on final thrombosis rather than the initial aPL-induced prothrombotic and proinflammatory phenotypes. In parallel to our increased understanding of the mediators and mechanisms of the aPL-induced clinical events, the blockade of early pathogenic effects of aPL on target cells (monocytes, endothelial cells, or platelets) has been increasingly investigated. The proposed mechanism of aPL-mediated thrombosis is the binding of aPL to endothelial cells [via β2-glycoprotein-I Address for Correspondence/Yazışma Adresi: Doruk ERKAN, M.D., Hospital for Special Surgery, Weill Cornell Medicine, New York, United States Phone : +90 212 774 22 91 E-mail : erkand@hss.edu

(β2GPI)] inducing a procoagulant state through different mechanisms including the expression of adhesion molecules and tissue factor (a physiologic initiator of coagulation and thrombin formation), and complement activation. In addition, products of complement activation, complement 3 (C3), C5a, and membrane attack complex (MAC), are potent mediators of platelet and endothelial cell activation; thus, the complement system is likely a critical step in the pathogenesis of APS [2]. Eculizumab, a humanized monoclonal antibody directed against C5, is approved for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) [3,4]. Given several recent case reports describing positive outcomes of severely ill aPL-positive patients treated with eculizumab, Received/Geliş tarihi: May 13, 2015 Accepted/Kabul tarihi: September 28, 2015

Erkan D and Salmon JE: The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome

the purpose of this review is to discuss the importance of the complement system in the pathogenesis of APS, and the potential role of complement inhibition to prevent organ damage in aPL-positive patients.

Complement System The complement system, composed of 30 proteins, protects the host against infections and initiates inflammation to kill microbes, remove dying cells, and dispose of immune complexes. The system is activated in a rapid fashion to opsonize or lyse a bacterium, while simultaneously triggering the release of proinflammatory and chemotactic peptides. The complement cascade can be triggered through 3 pathways: 1) the classical pathway, initiated by multivalent binding of the Fc fragments of antibody binding to the C1 complex; 2) the lectin pathway, binding specific sugars on a microbe to mannose binding lectin-associated proteases; and 3) the alternative pathway, spontaneous low-grade cleavage of C3 in plasma (Figure 1) [5,6]. These 3 pathways converge to generate C3 convertases, which cleave C3 into C3a and C3b. C3a is an anaphylatoxin that recruits and activates leukocyte effectors; C3b tags pathogens and immune complexes for opsonization. C3b leads to the

Turk J Hematol 2016;33:1-7

assembly of C5 convertase and subsequent cleavage of C5 into C5a and C5b. C5a is a potent chemotactic molecule that recruits and stimulates leukocytes and endothelial cells, triggering release of cytokines/chemokines and the expression of adhesion molecules. Binding of C5b to cell surface assembles C5b-9 MAC, which inserts itself into membranes, damages cells, and activates proinflammatory pathways [5,6]. Furthermore, complement activation products contribute to thrombosis by augmenting the inflammatory responses of leukocytes and the endothelium, which in turn potentiate coagulation [6]. Factor B, factor D, and properdin contribute to the generation of C3b directly through the alternative pathway or through the amplification loop where C3b is formed. The production of C3b, triggered from engagement of the classical or lectin pathways, is augmented through the alternative pathway amplification [7]. Because of its potency, complement activation is regulated at each step. The major regulators of the alternate pathway amplification loop are plasma proteins complement factor H (CFH) and complement factor I (CFI), and a membrane cofactor protein (MCP) (CD46) [6].

Thrombotic Microangiopathies Nocturnal Hemoglobinuria

and

Paroxysmal

Definitions Thrombotic microangiopathy (TMA) is defined as thrombosis in arterioles and capillaries, which is commonly associated with thrombocytopenia, microangiopathic hemolytic anemia, and/ or kidney failure [8]. Diseases associated with TMA are either hereditary or acquired; selected TMA syndromes are described in Table 1 [9]. Antiphospholipid antibody-positive patients, especially those with catastrophic APS [10], can develop TMA with or without medium-to-large vessel thrombosis (further discussed below). Table 1. Hereditary and acquired thrombotic microangiopathies (adapted from George and Nester [9]).

Figure 1. Human complement system. Three pathways are activated by immune complexes and apoptotic cells (classical); by microbes and stressors (lectin); and spontaneously (alternative). The effect of complement: clearance of apoptotic cells, opsonization of pathogens and immune complexes for phagocytosis, release of anaphylatoxins and lysis (shown in italics), and activation of effector cells that express receptors for C5a and/or C3a (neutrophils, monocytes, and platelets) are shown on the left. Complement inhibitors are indicated in red. Soluble inhibitors are factor I (FI), C4b-binding protein (C4BP), factor H (FH), and FH-like protein (FHL-1). Membrane-bound inhibitors include MCP (CD46), DAF (CD55), and CD59. Reprinted from Placenta 2010;31:561-567, Lynch AM, Salmon JE, Dysregulated complement activation as a common pathway of injury in preeclampsia and other pregnancy complications. Copyright (2015) with permission from Elsevier. 2

Hereditary: Thrombotic thrombocytopenic purpura (TTP) (ADAMTS13 deficiency-mediated TMA) Complement-mediated TMA Acquired (Primary): Thrombotic thrombocytopenic purpura (TTP) (ADAMTS13 deficiency-mediated TMA) Complement-mediated TMA Shiga-toxin-mediated TMA (hemolytic uremic syndrome) Acquired (Secondary): Infection Cancer Preeclampsia HELLP (hemolysis, elevated liver enzymes, and low platelets) Syndrome Autoimmune Disorders (e.g., Lupus, Systemic Sclerosis, Antiphospholipid Syndrome) Hematopoietic Stem Cell or Organ Transplantation

Turk J Hematol 2016;33:1-7

Erkan D and Salmon JE: The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome

Thrombotic thrombocytopenic purpura (TTP), which is hereditary (ADAMTS13 mutations) or acquired (antibodies against ADAMTS13), can present with a wide spectrum of manifestations including microangiopathic hemolytic anemia, thrombocytopenia, neurologic manifestations, gastrointestinal symptoms, purpura, and/or renal disease [9]. Hereditary [regulatory (CFH, CFI, or CD46) or effector (complement factor B or C3) gene mutations] or primary acquired (antibodies against factor H) complement-mediated TMA is due to the uncontrolled activation of the alternative pathway of the complement resulting in acute kidney injury and hypertension. These patients were previously classified as having “aHUS”, clinically defined as thrombocytopenia and microangiopathic hemolysis (with ADAMTS13 activity of >5% and a negative stool test for Shiga-toxin-producing infection) and one of the following: neurological symptoms, renal impairment, or gastrointestinal symptoms [9]. Hemolytic uremic syndrome (HUS) is a TMA featuring the triad of hemolytic anemia, thrombocytopenia, and acute renal impairment, mainly caused by Shiga-toxin-producing Escherichia coli. It is often preceded by bloody diarrhea (although one-third of the patients do not have bloody diarrhea), accounts for 90% of HUS cases in childhood, and does not relapse, and renal function recovers completely in >90% of cases. Traditionally, aHUS has been distinguished from HUS by the absence of diarrhea secondary to an Escherichia coli infection and a more severe clinical course [6]; however, rarely aHUS patients can present with gastrointestinal symptoms. Several other conditions can be associated with secondary TMA (Table 1). Disseminated intravascular coagulation is associated with intravascular activation and consumption of the different coagulation system components depending on the underlying cause. Preeclampsia is the onset of hypertension and proteinuria after 20 weeks of gestation; disease manifestations range from mild blood pressure elevations to severe hypertension, the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), and eclampsia (seizures). PNH is a disease of hematopoietic stem cells resulting in intravascular hemolysis, hemoglobinuria, and thromboembolism due to the deficiency of two proteins (CD55-decay accelerating factor; CD59-membrane inhibitor of reactive lysis) that inhibit the activation and cytolytic functions of the complement system [11]. Complement System in Thrombotic Microangiopathies and Paroxysmal Nocturnal Hemoglobinuria Mouse studies demonstrate that the absence of complement regulatory proteins is associated with TMA and pregnancy loss [12]. As discussed above, mutations in complement regulatory proteins result in aHUS and PNH, both associated

with microvascular endothelial cell activation, cell injury, and thrombosis [13]. Furthermore, a relationship exists between activation of the complement system and development of an imbalance in angiogenic factors. Mouse models show that C5a induces release of antiangiogenic factors [14] and complement deletion prevents placental insufficiency in mouse models of preeclampsia [15]. Excess production of an antiangiogenic factor by the placenta and inflammatory cells leads to impaired placental development and placental dysfunction [5,14,16]. In normal pregnancies, excessive complement activation is prevented by complement regulatory proteins that are highly expressed on trophoblast membranes (CD55 and CD59) and circulating complement regulatory proteins (CFH, CFI, and C4 binding protein). In preeclampsia, complement activation products (C4d and C5b-9) are detected on trophoblasts, and in vivo hypoxia enhances MAC deposition on villous trophoblasts [17]. Complement Inhibition in Thrombotic Microangiopathies and Paroxysmal Nocturnal Hemoglobinuria Eculizumab is a recombinant humanized monoclonal antibody that binds to the terminal complement protein C5, inhibits its cleavage into C5a and C5b, and prevents the generation of MAC. In PNH patients, eculizumab reduces the frequency of hemolysis, hemoglobinuria, transfusion, and thrombosis [18]. In aHUS patients, eculizumab inhibits complement-mediated TMA (resolving thrombocytopenia and TMA) and improves renal transplantation outcomes by allowing plasma exchangedependent patients to stop this treatment [4]. Eculizumab has been also used off-label in TTP patients refractory to plasmapheresis [19].

Antiphospholipid Syndrome Definitions Antiphospholipid antibodie are a heterogeneous family of autoantibodies directed against phospholipid-binding plasma proteins, most commonly β2GPI. The predominant hypothesis regarding the origin of aPL states that an incidental exposure to environmental agents with β2GPI-like peptides induces aPL in susceptible individuals (molecular mimicry) [20]. A positive aPL test is not always “clinically significant”; transient aPL positivity is not uncommon, especially during infections [21]. Documentation of aPL persistence is therefore important. High titers of anticardiolipin antibodies (aCL) and aβ2GPI as well as IgG/M isotypes are more concerning than lower titers and IgA isotypes. Whereas a positive lupus anticoagulant (LA) test is a better predictor of thrombosis than aCL and aβ2GPI, falsepositive and false-negative LA tests can occur for patients on anticoagulation [22]. Documentation of a positive LA test requires 4 criteria according to International Society of Thrombosis and 3

Erkan D and Salmon JE: The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome

Haemostasis guidelines [23]: 1) demonstration of a prolonged phospholipid-dependent coagulation screening test, such as activated partial thromboplastin time or dilute Russell viper venom time; 2) failure to correct the prolonged screening test by mixing the patient’s plasma with normal platelet-poor plasma, demonstrating the presence of an inhibitor; 3) shortening or correction of the prolonged screening test by the addition of excess phospholipid, demonstrating phospholipid dependency; and 4) exclusion of other inhibitors. Our definition of a “clinically significant” aPL profile is: 1) LA test positivity in accordance with the above guidelines [23]; 2) aCL IgG/M of ≥40 U [1]; and/or 3) aβ2GPI IgG/M of ≥40 U tested twice at least 12 weeks apart. Clinical judgment is required to determine thrombosis risk when aPL results are equivocal, e.g., LA test not measurable because the patient is anticoagulated, aCL or aβ2GPI IgG/M titers are between 20 and 39 U, and/or aCL or aβ2GPI IgA is the only positive aPL test. In patients with clinically significant aPL profiles, clinical manifestations may be varied: 1) asymptomatic aPL positivity (no history of thrombosis or pregnancy morbidity); 2) non-criteria manifestations of aPL, e.g., livedo reticularis, thrombocytopenia, hemolytic anemia, cardiac valve disease, aPLassociated nephropathy, or cognitive dysfunction; 3) pregnancy morbidity (recurrent embryonic or fetal loss, preeclampsia, placental insufficiency, and fetal growth restriction); 4) venous, arterial, or small vessel thrombosis (stroke is the most common presentation of arterial thrombosis; deep vein thrombosis is the most common venous manifestation); and 5) catastrophic APS (multiple organ thrombosis developing in a short period), which is usually associated with TMA [1,10]. Mechanisms of Antiphospholipid Antibody-Mediated Injury Antiphospholipid antibodies induce thrombosis and placental injury through multiple mechanisms. The process begins with activation or apoptosis of platelets, endothelial cells, or trophoblasts, during which phosphatidylserine (a negatively charged phospholipid) migrates from the inner to the normally electrically neutral outer cell membrane. β2GPI, which potentially exists in the circulation in a circular form [24], then binds to PS independently of aβ2GPI (via β2GPI surface receptors such as apoER2′, annexin A2, or a toll-like receptor). After this binding, the circular β2GPI opens up to expose domain I and aPL binds to β2GPI, directly stimulating cells through surface receptors. Antiphospholipid antibodies can also stimulate cells indirectly by activating the classic complement pathway. The generation of C5a induces expression of adhesion molecules and tissue factor and activation of monocytes, polymorphonuclear cells, and platelets, and it triggers the release of proinflammatory mediators (e.g., tumor necrosis factor, vascular endothelial growth factor receptor-1) and initiation of the proadhesive and prothrombotic state. Thus, multiple pathways are engaged 4

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by aPL binding to the surface. Crosslinking apoER2′ receptors antagonize endothelial nitric oxide synthase, blocking its phosphorylation and leading to increased leukocyte adhesion and thrombosis. Both nuclear factor κB and p38 mitogenactivated protein kinase play a role in the intracellular signaling cascade. Antiphospholipid antibodies also downregulate the expression of trophoblast signal transducer and activator of transcription 5 (STAT5), reducing the endometrial stromal cell production of prolactin (PRL) and insulin growth factor binding protein-1 (IGFBP-1) [25]. Patients with aPL may have thrombocytopenia, and its mechanisms include: 1) promoting platelet activation and aggregation; 2) antiplatelet antibodies directed against platelet membrane glycoproteins such as aGPIIb/IIIa [26]; and 3) platelet destruction as seen in TMA, including catastrophic APS. Thrombotic microangiopathy, which is common in catastrophic APS and/or transplant rejection, is the most characteristic lesion of APS nephropathy; the pathologic changes may be similar to other TMAs, e.g., HUS, TTP, and preeclampsia. In addition, aPLnephropathy patients can develop chronic cortical ischemia/ infarction (arteriosclerosis, arteriolosclerosis, arterial fibrous intimal hyperplasia, glomerular ischemia, interstitial fibrosis, tubular thyroidization, tubular atrophy, and/or organized thrombi with/without recanalization) [1,27,28]. Recently, it has been shown that in APS patients these vascular renal lesions are associated with the activation of the mammalian target of rapamycin pathway [29]. Given the tissue damage prominent in renal TMA, it is likely that inflammatory damage by recruited leukocytes and vascular cell activation are amplified by complement activation products generated as a consequence of the alternative pathway. Complement System in Antiphospholipid Antibody-Mediated Injury Passive transfer of human aPL results in endothelial cell activation and pregnancy loss in animal models [30,31]. Endothelial cell activation correlates with a prothrombotic phenotype in vitro and enhances thrombus formation in vivo [30,32]. Complement activation, specifically C5, is a necessary intermediary event in both thrombosis and pregnancy complications associated with aPL in rodent models [33]. Complement activation initiates and amplifies the cellular features characteristic of APS: endothelial cell activation, monocyte tissue factor expression, and platelet aggregation. Generation of C5a contributes to vascular inflammation [34,35]. Complement 5a interacts with its receptor, C5aR, to promote recruitment and activation of neutrophils (PMNs) and monocytes, and activation of EC [29]. C5a-C5aR ligation also: 1) upregulates neutrophil-derived TF expression, thought to be one mechanism of aPL-mediated coagulation and disseminated

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Erkan D and Salmon JE: The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome

thrombosis [36]; 2) leads to trophoblast injury and angiogenic factor imbalance in aPL-induced fetal injury [37]; and 3) produces lesions such as those seen in TMA in mouse models. Mice deficient in complement components C3, C5, C6, or C5a receptors are resistant to aPL-induced enhanced thrombophilia and endothelial cell activation [38]. Treatment with anti-C5 monoclonal antibody or C5aR antagonist peptides attenuates thrombosis in mouse models of APS [31]. In mouse models of surgically induced thrombus formation, complement activation plays an important role in the increased thrombosis and adhesion of leukocytes to endothelial cells caused by treatment with aPL. Heparin has anticomplement effects, as well as acting as an anticoagulant, which may explain some of its efficacy in APS [39]. In addition, mouse models of aPL-induced pregnancy loss and growth restriction show that C4, factor B, C3, C5, and C5aR are required for placental injury [30]. Complement deposition is present in human placenta from patients with APS [40]. Patients with aPL, with or without clinical manifestation of APS, show elevated circulating levels of Bb and C3a fragments [41]; the fact that the complement contributes to placental injury is suggested by the evidence for C4d on trophoblasts in patients with APS [42]. In a recent study, APS patients were found to have elevated C3a levels in plasma, but there was no correlation with the development of thrombosis [43]. Complement Inhibition in Antiphospholipid Antibody-Positive Patients Clinical studies of anti-C5 monoclonal antibody (eculizumab) in aPL-positive patients are limited to a small number of case reports. The first report was published in 2010 by Lonze et al. describing improvement of TMA after kidney transplant in an eculizumabtreated patient with a history of catastrophic aPL syndrome [44]. Of note, the patient also received systemic anticoagulation and standard immunosuppression. Velik-Salchner et al. questioned the effectiveness of eculizumab in this patient by drawing attention to the ability of heparin to inhibit complement in APS mouse models and the possibility of TMA in aPL-positive patients that does not involve complement activation [45]. In 2011, Hadaya et al. reported an aPL-positive systemic lupus erythematosus patient who underwent a living-related kidney transplantation, which was complicated by recurrent thrombotic microangiopathy [46]. Despite the standard posttransplantation regimen and daily plasma exchange, renal function did not improve. The patient received 5 weekly infusions of eculizumab, and the renal function normalized after 6 months. Darnige et al. studied aPL titers in 20 PNH patients receiving eculizumab [47]. Only 3 patients had preinfusion low-titer aCL or aÎ˛2GPI

(negative lupus anticoagulant); there was no significant change in the titers after 11 weeks of treatment. In 2012, Shapira et al. reported a catastrophic APS patient resistant to anticoagulation, immunosuppression, plasmapheresis, and rituximab; eculizumab successfully blocked complement activity, aborted progressive thrombosis, and reversed thrombocytopenia [48]. In 2013, Canaud et al. reported 3 aPL-nephropathy patients treated with eculizumab following TMA after kidney transplantation due to aPL-nephropathy recurrence [49]. Based on pre- and posttransplantation biopsies, the investigators showed that: 1) eculizumab results in remission in plasmapheresis-resistant thrombotic angiopathy related to aPL-nephropathy recurrence; 2) persistent C5b-9 deposition is commonly found in allografts developing posttransplant thrombotic microangiopathy; and 3) chronic vascular changes related to aPL may not be related to complement activation. In the same year, another case report [50] and personal communications during the 14th International Congress on aPL described catastrophic APS patients who failed to respond to eculizumab [51]. In 2014, several case reports or series described the outcomes of eculizumab-treated aPL-positive patients: 1) Bakhtar et al. described a lupus and APS patient who developed biopsy-proven TMA, thrombocytopenia, and hemolysis 3 years after livingrelated kidney transplantation; after 7 months of eculizumab, there was no evidence of TMA on biopsy and both hemoglobin and platelets were normal [52]; 2) Lonze et al. reported 3 APS patients (2 with catastrophic APS, and including the follow-up information of the first eculizumab-receiving patient reported in 2010) who were treated with anticoagulation and eculizumab prior to and following live donor renal transplantation (2 also received plasmapheresis); after a follow-up ranging from 4 months to 4 years, all patients had functioning renal allografts [53]; 3) Strakhan et al. reported another catastrophic APS patient (multiple strokes, non-ST elevation myocardial infarction, endstage renal disease due to TMA, intraretinal hemorrhage, and thrombocytopenia) who had no response to corticosteroids and plasma exchange (no heparin during the acute period); the patientâ&#x20AC;&#x2122;s condition stabilized after eculizumab [54]; 4) Zapantis et al. reported 3 APS patients with recurrent thrombosis and thrombocytopenia unresponsive to conventional therapy with significant improvement of thrombocytopenia after eculizumab administration (personal communication) [55]. Given the above reports, complement inhibition may have a role as an adjuvant or main therapy for APS patients refractory to anticoagulation; however, publication bias is a concern as well as the lack of systematic clinical studies. Thus, more mechanistic and clinical studies are needed before eculizumab can be recommended [50]. Clinicians should keep in mind that the infection risk of eculizumab is mainly with encapsulated organisms, specifically 5

Erkan D and Salmon JE: The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome

meningococcal. Patients must be immunized against Neisseria meningitidis before treatment with eculizumab. Ongoing Observational and Interventional Complement-Related Clinical Studies in Antiphospholipid Antibody-Positive Patients Potential novel approaches to target terminal complement activation include C5aR antagonists (antibodies or peptides) and soluble and targeted complement regulatory proteins. The PROMISE Study (Predictors of pRegnancy Outcome: bioMarkers In APS and Systemic lupus Erythematosus) (clinicaltrials.gov#: NCT00198068), a prospective, multicenter observational study, aims to translate findings in mice to humans and evaluate the role of complement in lupus and aPL-associated pregnancy complications. The study, which has recruited over 700 patients as of December 2014, is ongoing and will test the hypotheses that classical, alternative, and terminal complement pathway activation and dysregulation of angiogenic factors will be detected in the circulation of patients destined for pregnancy complications. The PROMISE Study has the potential to identify new biomarkers for adverse pregnancy outcomes that in addition to being good predictors of these outcomes are also part of the mechanistic process of these pregnancy complications. In the future, it may be possible to identify those patients in whom complement inhibitors are likely to prevent or modify the inflammatory-related sequelae associated with adverse pregnancy outcomes. One open-label interventional phase II prevention trial (clinicaltrials.gov#: NCT01029587) is investigating whether blocking the complement cascade with eculizumab in 10 patients with a prior history of catastrophic APS who are undergoing kidney transplant will lead to increased transplant success. Three patients included in the protocol have already been reported [44,51] and the estimated completion date was August 2015. Another open-label multicenter international phase IIa treatment trial (clinicaltrials.gov#: NCT02128269) is evaluating the safety and tolerability of an intravenous C5a inhibitor in persistently aPL-positive patients with at least one of the following non-criteria manifestations of APS: aPL-nephropathy, skin ulcers, and/or thrombocytopenia.

Conclusion Animal and human studies have confirmed the relevance of complement inhibition in many inflammatory and microthrombotic diseases including APS. Thus, complement inhibition may have a role for APS patients refractory to anticoagulation; however, more clinical data are needed. Future mechanistic and clinical studies of eculizumab and other complement inhibitors will be necessary to individualize treatment. We hope that the results from the ongoing studies will be available for further discussion at the 15th International Congress on aPL (İstanbul, Turkey) (www.apsistanbul2016.org). 6

Turk J Hematol 2016;33:1-7

Authorship Contributions Concept: Doruk Erkan, Jane E. Salmon, Design: Doruk Erkan, Jane E. Salmon, Data Collection or Processing: Doruk Erkan, Jane E. Salmon, Analysis or Interpretation: Doruk Erkan, Jane E. Salmon, Literature Search: Doruk Erkan, Jane E. Salmon, Writing: Doruk Erkan, Jane E. Salmon. Conflict of Interest: Doruk Erkan and Jane Salmon: Alexion (Clinical Trial Investigator, Advisory Board)

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16. Lynch AM, Murphy JR, Byers T, Gibbs RS, Neville MC, Giclas PC, Salmon JE, Holers VM. Alternative complement pathway activation fragment Bb in early pregnancy as a predictor of preeclampsia. Am J Obstet Gynecol 2008;198:385.1-9. 17. Rampersad R, Barton A, Sadovsky Y, Nelson DM. The C5b-9 membrane attack complex of complement activation localizes to villous trophoblast injury in vivo and modulates human trophoblast function in vitro. Placenta 2008;29:855-861. 18. Hillmen P, Hall C, Marsh JC, Elebute M, Bombara MP, Petro BE, Cullen MJ, Richards SJ, Rollins SA, Mojcik CF, Rother RP. Effect of eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med 2004;350:552-559. 19. Tsai E, Chapin J, Laurence JC, Tsai HM. Use of eculizumab in the treatment of a case of refractory, ADAMTS13-deficient thrombotic thrombocytopenic purpura: additional data and clinical follow-up. Br J Haematol 2013;162:558-559. 20. Shoenfeld Y. Etiology and pathogenetic mechanisms of the antiphospholipid syndrome unraveled. Trends Immunol 2003;24:2-4. 21. Avcin T, Toplak N. Antiphospholipid antibodies in response to infection. Curr Rheumatol Rep 2007;9:212-218. 22. Galli M, Luciani D, Bertolini G, Barbui T. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature. Blood 2003;101:1827-1832. 23. Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, De Groot PG; Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost 2009;7:1737-1740. 24. Giannakopoulos B, Krilis SA. The pathogenesis of the antiphospholipid syndrome. N Engl J Med 2013;368:1033-1044. 25. Erkan D, Salmon J, Lockshin MD. Antiphospholipid syndrome. In: Firestein GS, Budd RC, Gabriel SE, McInnes IB, Odell JR (eds). Kelley’s Textbook of Rheumatology, 9th ed. Philadelphia, Elsevier Saunders, 2013. 26. Alpert D, Mandl LA, Erkan D, Yin W, Peersche EI, Salmon JE. Anti-heparin platelet factor 4 antibodies in systemic lupus erythematosus are associated with IgM antiphospholipid antibodies and the antiphospholipid syndrome. Ann Rheum Dis 2008;67:395-401. 27. D’Cruz D. Renal manifestations of the antiphospholipid syndrome. Curr Rheumatol Rep 2009;11:52-60. 28. Gigante A, Gasperini ML, Cianci R, Barbano B, Giannakakis K, Di Donato D, Fuiano G, Amoroso A. Antiphospholipid antibodies and renal involvement. Am J Nephrol 2009;30:405-412. 29. Canaud G, Bienaimé F, Tabarin F, Bataillon G, Seilhean D, Noël LH, Dragon-Durey MA, Snanoudj R, Friedlander G, Halbwachs-Mecarelli L, Legendre C, Terzi F. Inhibition of the mTORC pathway in the antiphospholipid syndrome. N Engl J Med 2014;371:303-312. 30. Girardi G, Berman J, Redecha P, Spruce L, Thurman JM, Kraus D, Hollmann TJ, Casali P, Caroll MC, Wetsel RA, Lambris JD, Holers VM, Salmon JE. Complement C5a receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J Clin Invest 2003;112:1644-1654. 31. Pierangeli SS, Colden-Stanfield M, Liu X, Barker JH, Anderson GL, Harris EN. Antiphospholipid antibodies from antiphospholipid syndrome patients activate endothelial cells in vitro and in vivo. Circulation 1999;99:1997-2002. 32. Simantov R, LaSala JM, Lo SK, Gharavi AE, Sammaritano LR, Salmon JE, Silverstein RL. Activation of cultured vascular endothelial cells by antiphospholipid antibodies. J Clin Invest 1995;96:2211-2219. 33. Bulla R, Bossi F, Fischetti F, De Seta F, Tedesco F. The complement system at the fetomaternal interface. Chem Immunol Allergy 2005;89:149-157. 34. Giannakopoulos B, Passam F, Rahgozar S, Krilis SA. Current concepts on the pathogenesis of the antiphospholipid syndrome. Blood 2007;109:422-430. 35. Peerschke EI, Yin W, Ghebrehiwet B. Complement activation on platelets: implications for vascular inflammation and thrombosis. Mol Immunol 2010;47:2170-2175.

36. Ritis K, Doumas M, Mastellos D, Micheli A, Giaglis S, Magotti P, Rafail S, Kartalis G, Sideras P, Lambris JD. A novel C5a receptor-tissue factor crosstalk in neutrophils links innate immunity to coagulation pathways. J Immunol 2006;177:4794-4802. 37. Redecha P, Tilley R, Tencati M, Salmon JE, Kirchhofer D, Mackman N, Girardi G. Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury. Blood 2007;110:2423-2431. 38. Pierangeli SS, Girardi G, Vega-Ostertag M, Liu X, Espinola RG, Salmon J. Requirement of activation of complement C3 and C5 for antiphospholipid antibody-mediated thrombophilia. Arthritis Rheum 2005;52:2120-2124. 39. Licht C, Fremeaux-Bacchi V. Hereditary and acquired complement dysregulation in membranoproliferative glomerulonephritis. Thromb Haemost 2009:101:271-278. 40. Shamonki JM, Salmon JE, Hyjek E, Baergen RN. Excessive complement activation is associated with placental injury in patients with antiphospholipid antibodies. Am J Obstet Gynecol 2007;196:167.1-5. 41. Oku K, Amengual O, Atsumi T. Pathophysiology of thrombosis and pregnancy morbidity in the antiphospholipid syndrome. Eur J Clin Invest 2012;42:1126-1135. 42. Viall CA, Chamley LW. Histopathology in the placentae of women with antiphospholipid antibodies: a systematic review of the literature. Autoimmun Rev 2014;14:446-471. 43. Devreese K, Hoylaerts MF. Is there an association between complement activation and antiphospholipid antibody related thrombosis? Thromb Haemost 2010;104:1279-1281. 44. Lonze BE, Singer AL, Montgomery RA. Eculizumab and renal transplantation in a patient with CAPS. N Engl J Med 2010; 362:1744-1745. 45. Velik-Salchner C, Lederer W, Wiedermann F. Eculizumab and renal transplantation in a patient with catastrophic antiphospholipid syndrome: effect of heparin on complement activation. Lupus 2011;20:772. 46. Hadaya K, Ferrari-Lacraz S, Fumeaux D, Boehlen F, Toso C, Moll S, Martin PY, Villard J. Eculizumab in acute recurrence of thrombotic microangiopathy after renal transplantation. Am J Transplant 2011;11:2523-2527. 47. Darnige L, Peffault de Latour R, Zemori L, Socié G, Fischer AM, Helley D; French Society of Haematology. Antiphospholipid antibodies in patients with paroxysmal nocturnal haemoglobinuria receiving eculizumab. Br J Haematol 2011;153:789791. 48. Shapira I, Andrade D, Allen SL, Salmon JE. Brief report: induction of sustained remission in recurrent catastrophic antiphospholipid syndrome via inhibition of terminal complement with eculizumab. Arthritis Rheum 2012;64:2719-2723. 49. Canaud G, Kamar N, Anglicheau D, Esposito L, Rabant M, Noël LH, GuilbeauFrugier C, Sberro-Soussan R, Del Bello A, Martinez F, Zuber J, Rostaing L, Legendre C. Eculizumab improves posttransplant thrombotic microangiopathy due to antiphospholipid syndrome recurrence but fails to prevent chronic vascular changes. Am J Transplant 2013;13:2179-2185. 50. Mushin SA, Khianey R, Erkan D. Discordant aPTT and anti-FXa values in a catastrophic antiphospholipid syndrome patient receiving intravenous unfractionated heparin. In: 14th International Congress on Antiphospholipid Antibodies Abstract Book, 2013;1:14-76 (abstract). 51. Erkan D, Aguiar CL, Andrade D, Cohen H, Cuadrado MJ, Danowski A, Levy RA, Ortel TL, Rahman A, Salmon JE, Tektonidou MG, Willis R1, Lockshin MD. 14th International Congress on Antiphospholipid Antibodies: Task force report on antiphospholipid syndrome treatment trends. Autoimmun Rev 2014;13:685-696. 52. Bakhtar O, Thajudeen B, Braunhut BL, Yost SE, Bracamonte ER, Sussman AN, Kaplan B. A case of thrombotic microangiopathy associated with antiphospholipid antibody syndrome successfully treated with eculizumab. Transplantation 2014;98:17-18. 53. Lonze BE, Zachary AA, Magro CM, Desai NM, Orandi BJ, Dagher NN, Singer AL, Carter-Monroe N, Nazarian SM, Segev DL, Streiff MB, Montgomery RA. Eculizumab prevents recurrent antiphospholipid antibody syndrome and enables successful renal transplantation. Am J Transplant 2014;14:459-465. 54. Strakhan M, Hurtado-Sbordoni M, Galeas N, Bakirhan K, Alexis K, Elrafei T. 36-yearold female with catastrophic antiphospholipid syndrome treated with eculizumab: a case report and review of literature. Case Rep Hematol 2014;2014:704371. 55. Zapantis E, Furie R, Horowitz D. Eculizumab in antiphospholipid antibody syndrome. Arthritis Rheum 2014;66(Suppl):6(abstract).

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0190 Turk J Hematol 2016;33:8-14

Deregulated Levels of the NF-κB1, NF-κB2, and Rel Genes in Ukrainian Patients with Leukemia and Lymphoma in the Post-Chernobyl Period Çernobil Sonrası Ukraynalı Lösemi ve Lenfoma Hastalarında Değişken NF-κB1, NF-κB2 ve Rel Gen Düzeyleri Hakan Savlı1, Ramis Ufuk Akkoyunlu1, Naci Çine1, Daniil F. Gluzman2, Michael P. Zavelevich2, Lilia M. Sklyarenko2, Stella V. Koval2, Deniz Sünnetçi1 1Kocaeli University Faculty of Medicine, Department of Medical Genetics, Kocaeli, Turkey 2National Academy of Sciences of Ukraine, R.E. Kavetsky Institute of Experimental Pathology, Oncology, and Radiobiology, Kyiv, Ukraine

Abstract

Öz

Objective: Nuclear factor kappa B (NF-κB) is an important transcription factor in cancer and NF-κB activation has been seen in angiogenesis, tumor progression, and metastasis. Relationships between specific NF-κB gene networks, leukemogenesis, and radiation exposure are still unknown. Our aim was to study the expression levels of the NF-κB1, NF-κB2, and Rel genes in hematological malignancies in the post-Chernobyl period.

Amaç: Nükleer faktör kappa B (NF-κB), kanserde önemli bir transkripsiyon faktörü olup aktivasyonu anjiyogenez, tümör gelişimi ve metastazın birçok basamağında görülmektedir. Spesifik NF-κB gen ağları, lökomogenez ve radyasyon maruziyeti arasındaki ilişki halen belirsizdir. Çalışmamızda Çernobil sonrası hematolojik kanserlerde NF-κB1, NF-κB2 ve Rel genlerinin ekspresyon düzeylerini incelemeyi amaçladık.

Materials and Methods: We analyzed gene expression levels of NFκB1, NF-κB2, and Rel in 49 B-cell chronic lymphocytic leukemia, 8 B-cell non-Hodgkin’s lymphoma, 3 acute myeloid leukemia, 3 chronic myeloid leukemia, 2 hairy cell leukemia, 2 myelodysplastic syndrome, and 2 T-cell large granular lymphocytic leukemia patients using realtime polymerase chain reaction.

Gereç ve Yöntemler: Gerçek zamanlı polimeraz zincir reaksiyonu ile 49 B-hücreli kronik lenfositik lösemi, 8 B-hücreli non-Hodgkin lenfoma, 3 akut myeloid lösemi, 3 kronik myeloid lösemi, 2 tüylü hücre lösemi, 2 miyelodisplastik sendrom ve 2 T-hücreli büyük granüler lenfositik lösemi hastasında NF-κB1, NF-κB2 ve Rel gen ekspresyon düzeylerini analiz ettik.

Results: Expression levels of NF-κB1, NF-κB2, and Rel genes were found to be deregulated.

Bulgular: NF-κB1, NF-κB2 ve Rel genlerine ait ekspresyon düzeyleri değişmiş olarak saptandı.

Conclusion: These results could be accepted as specific gene traces to radiation-induced leukemia or as potential candidates for new diagnostic biomarker studies. Larger experiments and non-exposed control malignant cell populations are needed to clarify these suggestions.

Sonuç: Bu sonuçlar, radyasyonla indüklenmiş lösemilerdeki spesifik gen izleri veya yeni tanısal biyobelirteç çalışmalarına muhtemel aday önerileri olarak da kabul edilebilir. Bu düşünceleri açıklığa kavuşturmak için daha geniş deneyler ve radyasyon maruziyeti olmayan kontrol malign hücre popülasyonlarına ihtiyaç vardır.

Keywords: Chronic lymphocytic leukemia, Non-Hodgkin’s lymphoma, B-cell neoplasms, Cancer, Thrombosis, T-cell neoplasms, B-cell neoplasms, Acute leukemia, Myelodysplastic syndromes, Chronic leukemia

Anahtar Sözcükler: Kronik lenfositik lösemi, Non-Hodgkin lenfoma, B-hücreli neoplazmalar, Kanser, Tromboz, T-hücreli neoplaziler, B-hücreli neoplaziler, Akut lösemi, Myelodisplastik sendromlar, Kronik lösemi

Address for Correspondence/Yazışma Adresi: Deniz SÜNNETÇİ, PhD., Kocaeli University Faculty of Medicine, Department of Medical Genetics, Kocaeli, Turkey E-mail : sun_deniz@hotmail.com

Received/Geliş tarihi: May 14, 2014 Accepted/Kabul tarihi: October 13, 2014

Turk J Hematol 2016;33:8-14

Savlı H, et al: Deregulated Levels of NF-κB1, NF-κB2, and Rel Genes in Leukemia and Lymphoma

Introduction The derivations of signatures using proteomics and genomics are increasingly integrated in the design of prognostic and predictive markers in oncology. Some of these markers are also well-known targets for therapeutic approaches, such as bortezomib, a nuclear factor kappa B (NF-κB) inhibitor possessing clinical activity in mantle cell lymphoma patients [1]. NF-κB is an important transcription factor in immunity, cell proliferation, cell survival, and cancer [2,3,4,5]. NF-κB activation has been demonstrated in angiogenesis, tumor progression, and metastasis [6,7]. Relationships between gene networks, leukemogenesis, and radiation exposure are still unknown. Our aim was to study expression levels of the NF-κB1 gene family in Ukrainian B-cell chronic lymphocytic leukemia (B-CLL), B-cell non-Hodgkin’s lymphoma (NHL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), and T-cell large granular lymphocytic leukemia (T-cell LGLL) patients in the post-Chernobyl period.

Materials and Methods Samplesof theperipheral bloodand bonemarrow of49 B-CLL,8 B-cell NHL,3 AML,3 CML,2 HCL,2 MDS,and 2T-cell LGLL patients were obtainedfrom the R.E. Kavetsky Institute of Experimental Pathology, Oncology, and Radiobiology of the National Academy of Sciences of Ukraine in 2008 and 2009. The mean age ofthe B-CLL groupwas 58.7 years and the median was 60 years (minimum 36 yrs, maximum 87 yrs). In theB-cell NHL group, the mean age was 57.3 years and the median was 60 years (minimum 43 yrs, maximum 69 yrs). Patients were analyzed morphologically and immunocytochemically according to the new World Health Organization classification, asshown in Table 1 along with demographical data. Thecontrol group comprised the peripheral blood samples of 8 healthy donors from Ukraine. Themean age of the secontrol subjects was 45.9 years and the median was 42.5 years (minimum 27 yrs, maximum 78 yrs). All B-CLL cases under study were of the typical B-CLL immunophenotype without adverse prognostic markers such as CD38+. Total ribonucleic acid (RNA) was isolated from leukocytes using the QIAamp RNA Blood Mini Kit (QIAGEN, Valencia, CA, USA) and treated with DNase I according to the manufacturer’s instructions. Quantity and purity were checked using a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Complementary deoxyribonucleic acid (cDNA) was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Fermentas Inc., Hanover, MD, USA) from 100 ng/µL total RNA as starting material. Gene expression levels were determined by quantitative reverse transcription-polymerase chain reaction as described previously [8,9]. Standard curves were obtained using serial dilutions of the beta-globulin gene (DNA Control

Kit, Roche, Penzberg, Germany). Gene-specific primers (Table 2) were obtained from Integrated DNA Technologies (Coralville, IA, USA). Obtained gene expression values were normalized using a housekeeping gene of beta-2 microglobulin. Gene expression ratios were compared in patient and control groups using the Relative Expression Software Tool (REST). Statistical Analysis Statistical analysis was performed using independent sample t-tests to analyze the statistical significance of our results by comparing controls with B-CLL, B-cell NHL, AML, MDS, CML, HCL, and T-cell LGLL patients. The p-values are shown in Table 3.

Results The NF-κB1, NF-κB2, and Rel genes were found to be upregulated in 49 B-CLL, 8 B-cell NHL, 3 AML, and 2 HCL patients in the post-Chernobyl period (Table 3). NF-κB1 was decreased 1.301fold in B-CLL, 1.473-fold in B-cell NHL, 1.534-fold in AML, and 1.862-fold in HCL cases. NF-κB2 was upregulated 1.720-fold in B-CLL, 8.545-fold in B-cell NHL, 16.257-fold in AML, and 1.676fold in HCL cases. We found Rel expression upregulated 2.736fold in B-CLL, 4.039-fold in B-cell NHL, 65.526-fold in AML, and 6.912-fold in HCL cases. In the MDS group, NF-κB2 was found to be significantly upregulated (50.563-fold). Rel was 2.272-fold upregulated whereas NF-κB1 was 1.100-fold downregulated in the same group. In the CML group, NF-κB2 was 2.110-fold upregulated while NF-κB1 and Rel were downregulated 1.056-fold and 1.239-fold, respectively. We found downregulation of the NF-κB1, NF-κB2, and Rel genes in T-cell LGLL cases at 4.557-fold, 3.771-fold, and 2.632-fold, respectively.

Discussion We had already found deregulated levels of NF-κB in our genomic experiments on prostate cancer [10], papillary thyroid cancer [11], and leukemia [12,13] in our previous studies. Recently, our proteomic results confirmed the upregulation of NF-κB in microarray screening in a breast cancer population [14]. This is our first observation of NF-κB deregulations in hematopoietic malignancies. Transcription of proteins that promote cell survival, stimulate growth, induce angiogenesis, and reduce susceptibility to apoptosis are upregulated by NF-κB. The NF-κB signaling pathway was found activated in MDS, AML, acute lymphoblastic leukemia (ALL), CML, CLL, multiple myeloma, and lymphoma cases before. These 3 genes were defined as deregulated before 9

Savlı H, et al: Deregulated Levels of NF-κB1, NF-κB2, and Rel Genes in Leukemia and Lymphoma

Turk J Hematol 2016;33:8-14

Table 1. Patient data, clinical features, and individual gene expression ratios. Leukemia Type

Patient ID

Sex, Age

WBC

Clinical Data

NF-κB1 Gene Expression Fold Change

NF-κB2 Gene Expression Fold Change

Rel Gene Expression Fold Change

B-CLL

U4B

F/73

Le-132x109/L Ly-85%

Stage 3 Lymph nodes +, lien +, hepar +

1.528/U

2.676/U

3.444/U

B-CLL

U 11 B

F/71

Le-19.2x109/L Ly-57%

Stage 2 Lymph nodes +

1.397/U

1.366/D

5.220/U

B-CLL

U 20 B

F/46

Le-23.2x109/L Ly-64%

Stage 2 Lien +

1.818/U

15.562/U

6.653/U

B-CLL

U 23 B

M/55

Le-15.0x109/L Ly-65%

Stage 2 Lymph nodes +, lien +, 1.233/U hepar +

1.214/D

3.350/U

B-CLL

U 27 B

M/49

Le-43.3x109/L Ly-91%

Stage 2 Lymph nodes +

2.953/U

3.434/U

8.598/U

B-CLL

U 28 B

M/62

Le-105x109/L Ly-72%

Stage 1-2

1.916/D

17.630/D

1.773/D

B-CLL

U 29 B

F/72

B-CLL

34.824/U

n/a

2.740/U

B-CLL

U 30 B

M/69

B-CLL

22.816/U

n/a

1.489/U

B-CLL

U 31 B

M/36

B-CLL

2.661/U

2.497/D

1.120/U

B-CLL

U 32 B

F/59

B-CLL

1.134/U

7.362/D

1.598/D

B-CLL

U 33 B

B-CLL

1.402/D

1.693/U

2.880/D

B-CLL

U 34 B

M/67

B-CLL

3.463/U

1.240/D

2.020/U

B-CLL

U 35 B

F/66

B-CLL

12.658/U

2.908/D

2.288/U

B-CLL

U 36 B

F/87

B-CLL

1.656/D

2.990/D

1.450/D

B-CLL

U 37 B

F/63

B-CLL

2.238/U

1.310/U

2.272/U

B-CLL

U 43 B

F/50

B-CLL

1.216/U

1.257/U

1.924/U

B-CLL

U 44 B

M/74

B-CLL

1.022/U

1.079/D

5.007/U

B-CLL

U 45 B

F/57

B-CLL

2.141/D

1.569/D

1.820/U

B-CLL

U 49 B

M/57

B-CLL

18.405/U

n/a

7.180/U

B-CLL

U 51 B

M/62

B-CLL

16.772/D

n/a

2.556/U

B-CLL

U 52 B

M/64

B-CLL

1.103/U

n/a

4.211/U

B-CLL

U 54 B

F/52

B-CLL

1.327/D

3.117/D

4.737/U

B-CLL

U1P

M/70

B-CLL

2.518/U

2.657/U

2.452/U

B-CLL

U 12 P

M/57

B-CLL

3.245/D

1.729/U

1.252/U

B-CLL

U 24 P

F/53

B-CLL

2.102/U

5.657/D

1.060/U

B-CLL

U 33 P

F/36

B-CLL

1.282/D

1.301/U

4.482/U

B-CLL

U 34 P

M/85

B-CLL

1.027/D

2.549/U

2.504/U

B-CLL

U 40 P

M/62

B-CLL

1.309/D

2.868/D

1.722/U

B-CLL

U 42 P

M/56

B-CLL

1.393/D

8.574/D

1.607/U

B-CLL

U 47 P

F/71

B-CLL

1.482/D

8.938/U

6.426/U

B-CLL

U 51 P

F/70

B-CLL

1.116/D

4.959/U

8.779/U

Savlı H, et al: Deregulated Levels of NF-κB1, NF-κB2, and Rel Genes in Leukemia and Lymphoma

Turk J Hematol 2016;33:8-14

Table 1. Continuation. B-CLL

U 69 P

M/41

Preliminary diagnosis: B-CLL NHL of spleen could not be excluded

1.155/D

5.776/U

1.584/U

B-CLL

U 76 P

M/70

B-CLL

1.282/D

3.074/D

6.746/U

B-CLL

U 77 P

F/69

B-CLL

3.254/U

1.301/U

11.035/U

B-CLL

U 80 P

M/51

B-CLL

1.462/D

7.945/U

6.122/U

B-CLL

U 81 P

M/70

B-CLL

2.025/D

18.252/U

2.521/U

B-CLL

U 82 P

M/60

B-CLL

1.889/D

3.945/U

4.608/U

B-CLL

U 83 P

F/48

B-CLL

1.051/U

n/a

1.686/U

B-CLL

U 85 P

F/57

B-CLL

1.668/D

14.420/U

5.184/U

B-CLL

U 87 P

B-CLL

2.691/D

9.781/U

2.320/U

B-CLL

U 88 P

M/51

B-CLL

1.436/U

3.811/D

7.537/U

B-CLL

U 208 P

F/62

B-CLL

1.259/U

20.821/U

1.128/U

B-CLL

U 211 P

F/55

B-CLL

1.213/D

17.630/U

1.399/U

B-CLL

U7B

F/78

Stage 3 Lymph nodes +, lien +, hepar + B-CLL/PLL (NHL)

1.571/U

2.848/U

14.065/U

B-CLL

U 15 B

F/67

Stage 1-2 Lymph nodes + B-CLL/PLL (NHL)

1.524/D

2.014/D

1.978/U

B-CLL

U 41 B

M/53

Chronic lymphoproliferative disease, Waldenström macroglobulinemia

1.507/U

n/a

1.428/U

B-CLL

U 55 B

F/59

Chronic lymphoproliferative disease

1.116/D

2.567/D

2.797/U

B-CLL

U 57 B

M/78

B-CLL

1.147/D

1.064/D

3.396/U

B-CLL

U 58 B

F/57

B-CLL

2.415/U

1.454/U

5.293/U

B-cell NHL

U8B

F/43

Le-20.5x109/L Ly-76%

Lien +, hepar +

1.336/D

1.310/U

3.565/U

B-cell NHL

U 14 B

M/53

Le-15.1x109/L Ly-49%

Lymph nodes +, lien +

1.196/D

1.094/D

1.082/U

B-cell NHL

U 19 B

M/69

Le-10x109/L Ly-68%

Lymph nodes +, hepar + Large B-cell lymphoma

1.015/U

n/a

B-cell NHL

U 48 B

M/45

B-cell NHL

4.061/U

n/a

2.006/U

B-cell NHL

U 13 P

M/60

Diffuse large B-cell lymphoma plasmablastic variant

2.717/U

n/a

Le-149.9x109/L Ly-71%

B-cell NHL

U 209 P

M/62

B-cell NHL

1.207/U

37.531/U

1.552/U

B-cell NHL

U 15 P

F/60

Lymphoma of marginal zone of spleen, leukemic transition

3.660/U

n/a

7.230/U

Savlı H, et al: Deregulated Levels of NF-κB1, NF-κB2, and Rel Genes in Leukemia and Lymphoma

Turk J Hematol 2016;33:8-14

Table 1. Continuation. B-cell NHL

U 56 B

M/66

Splenic lymphoma

1.402/D

4.925/D

1.951/U

AML M5

12.484/U

1341.843/U

29,004.719/U

AML M4

n/a

4.000/U

n/a

AML M3

3.686/U

1.248/D

2.8967/U

AML

U5P

F/51

AML

U 17 B

M/68

AML

U 46 B

M/35

CML

U9B

M/20

Lymph nodes +, lien + Acceleration phase

CML

1.935/U

10.703/U

2.402/U

CML

U 26 B

F/59

Lien + Le-47.8x109/L Blast cells-19% Acceleration phase

CML

1.611/D

1.670/U

3.195/D

CML

U 47 B

M/20

CML

1.412/D

1.905/D

1.430/D

HCL

U 24 B

M/49

HCL

1.229/D

1.803/D

1.595/U

HCL

U 50 B

F/36

HCL

4.263/U

n/a

29.940/U

MDS

U3P

M/74

MDS RAEB

1.282/D

15.348/U

n/a

MDS

U 207 P

M/57

MDS RA

n/a

166.572/U

2.646/U

T-cell LGLL

U7P

M/70

T-cell LGLL

11.778/D

2.204/D

2.323/D

T-cell LGLL

U 42 B

F/78

1.763/D

n/a

2.981/D

Le-33.7x109/L Lien +, hepar + Blast cells-82%

Le-3.0x109/L Ly-70% Thrombocytopenia

NF-κB: Nuclear factor kappa B, B-CLL: B-cell chronic lymphocytic leukemia, AML: acute myeloid leukemia, MDS: myelodysplastic syndrome, CML: chronic myeloid leukemia, HCL: hairy cell leukemia, T-cell LGLL: T-cell large granular lymphocytic leukemia, NHL: non-Hodgkin’s lymphoma, WBC: white blood cell, D: downregulated, U: upregulated, F: female, M: male, n/a: not applicable.

Table 2. Primer sequences of the studied genes. Genes

Primer Sequences

Beta-2 microglobulin

(F) 5’ TGA CTT TGT CAC AGC CCA AGA TA 3’ (R) 5’ AAT CCA AAT GCG GCA TCT TC 3’

NF-κB1

(F) 5’ AGC ACG AAT GAC AGA GGC GTG TA 3’ (R) 5’ TTC TGC TTG CAA ATA GGC AAG GT 3’

NF-κB2

(F) 5’ AGA CGA GTG TGG TGA GCT TTCT 3’ (R) 5’ AGT CAG GCA TAT GCA ACA 3’

Rel

(F) 5’ TGC CGA TGA CAT AGT CGG AAT 3’ (R) 5’ GGA CAT CTG ATG GAG CTG TCT 3’

NF-κB: Nuclear factor kappa B.

in hematological malignancies [15]. Here we have observed deregulated levels in radiation-induced leukemia populations. Results supported that radiation-exposed and non-exposed hematological malignancies use the same gene pathways and are shaped around the NF-κB gene network. It was shown that losses in the 13q chromosomal region are also associated with B-CLL and these losses deregulate the NF-κB pathway [16]. Deregulation of the NF-κB pathway by gains at chromosomal loci including the NF-κB1, NF-κB2, and Rel genes was reported previously. A gain at the (2)(p16.1p14) 12

region including the Rel gene, an oncogene, was reported in 17p-deleted CLL with poor prognosis [17]. Rearrangements such as translocations and deletions occurred in 10q24 affecting the NF-κB2 gene, a protooncogene. These rearrangements are known to lead to deletion of 3’ sequences of the NF-κB2 gene and cause production of carboxy-truncated constitutively nuclear proteins that may have a role in the tumorigenesis of B-CLL and B-cell NHL at high levels [18]. Unlike its relative NF-κB2, NF-κB1 has few rearrangements reported in leukemias and lymphomas. There is evidence in the literature that NF-κB2 is involved in oncogenesis in T-cell ALL as a result of LYL1 translocation [19]. Further studies are needed to assess the NF-κB1 rearrangements leading to B-CLL and B-cell NHLs. These observations give us new clues about relationships between NF-κB deregulation in leukemias and chromosomal regions. We are planning to continue our further studies by array comparative genomic hybridization analysis to focus on fine mapping of 13q and 2p in particular. Over the last decade, the problem of association between B-CLL and ionizing radiation has become a matter of considerable scientific interest [20]. Nevertheless, the experimental studies on the relationship between ionizing radiation and CLL are limited. Lyng et al. indicated that activation of the NF-κB pathway

Savlı H, et al: Deregulated Levels of NF-κB1, NF-κB2, and Rel Genes in Leukemia and Lymphoma

Turk J Hematol 2016;33:8-14

Table 3. Gene expression levels in groups of studied patients. Number of Patients

NF-κB1 Gene Expression Change

NF-κB2 Gene Expression Change

REL Gene Expression Change

B-CLL

1.301 U p=0.569

1.720 U p=0.335

2.736 U p=0.018

B-cell NHL

1.473 U p=0.039

8.545 U p=0.391

4.039 U p=0.018

AML

1.534 U p=0.001

16.257 U p=0.698

65.526 U p=0.786

CML

1.056 D p=0.758

2.110 U p=0.99

1.239 D p=0.127

HCL

1.862 U p=0.272

1.676 U

6.912 U p=0.519

MDS

1.100 D

50.563 U p=0.72

2.272 U

T-cell LGLL

4.557 D p=0.236

3.771 D

2.632 D p=0.623

may suppress the apoptotic response in U698 cells, a malignant B-lymphocyte cell line, to ionizing radiation [21]. Activation of the NF-κB pathway by ionizing radiation induces antiapoptotic genes and inhibits apoptosis by upregulation of NF-κB genes. This was linked to proliferation and increased survival of B-CLL [22]. B-CLL and HCL cells are known to be refractory to signals activating normal B cells. B-CLL and HCL cells are stimulated by tumor necrosis factor (TNF-α) [23]. TNF-α is involved in many human tumors and associated with poor prognosis. TNF-α is produced by B-CLL and HCL cells [24] and contributes to the escape of HCL cells from apoptosis through NF-κB activation [25]. Radiation exposure results in high levels of NF-κB gene expression. We found upregulated levels of NF-κB1, NF-κB2, and Rel genes in our patients. Our results were in concordance with the previous findings above. NF-κB expressions were found significantly higher than in the controls in both AML and ALL by Kapelko-Słowik et al. before [26]. They also found lower expression levels of NF-κB in AML patients who reached complete remission compared with patients with primary resistance to chemotherapy who did not reach complete remission. These data indicated that high expression levels of NF-κB might be involved in the pathogenesis of AML and ALL [26]. There are few studies on radiation-induced leukemia populations, such as in the Chernobyl area. However, we cannot assess all etiological sources for our subjects. Levels of exposure among subjects during the Chernobyl accident remain unclear. Thus, we cannot conclude that the NF-κB pathway is the main cause of AML and ALL pathogenesis in radiationinduced forms of the disease.

Rel has the potential to transform cells in culture and is expressed in high levels in both B-cell NHL [27] and large granular lymphocytic leukemia [28]. Interestingly, we found decreased levels of Rel, NF-κB1, and NF-κB2 in our T-cell LGLL group. In our study we found NF-κB2 significantly higher in MDS cases. There is evidence in the literature that the degree of NF-κB activity is correlated with the risk of progression to AML. NF-κB activation is known to be a hallmark of high-risk MDS [27]. We obtained increased levels of NF-κB2 in CML cases. Exposure to ionizing radiation causes CML [29]. CML is characterized by t(9;22), which leads to Bcr/Abl fusion oncoprotein expression. This protein activates the NF-κB pathway. The NF-κB pathway, in turn, leads to expression of antiapoptotic proteins such as Bcl-XL and lets Bcr/Abl+ cells grow [27]. Here we have defined a positive correlation between upregulated levels of NF-κB genes in hematological malignancies related to radiation exposure. However, the limited number of patients and controls was an obstacle. Therefore, these experiments are presented here as results of a preliminary study. Similar studies should be extended to experiments in time- and dose-dependent manners in cell lines or primary cultures. We think that our results are a good starting point for drawing a network around the NF-κB genes to investigate the life cycles of hematological malignancies. 13

Savlı H, et al: Deregulated Levels of NF-κB1, NF-κB2, and Rel Genes in Leukemia and Lymphoma

Conclusion Moreover, it would be tempting to suggest that this gene region may be used as a trace of early radiation exposure leading to leukemia. Either way, the NF-κB pathway certainly deserves more attention since its overexpression is almost a rule in many solid tumors and hematopoietic malignancies. Ethics Ethics Committee Approval: Bioethics Committee of the R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of the National Academy of Sciences of Ukraine (Approval number: 5/2008), Informed Consent: It was taken. Authorship Contributions Concept: Hakan Savlı, Daniil F. Gluzman, Design: Hakan Savlı, Daniil F. Gluzman, Michael P. Zavelevich, Data Collection or Processing: Lilia M. Sklyarenko, Stella V. Koval, Analysis or Interpretation: Deniz Sünnetçi, Naci Çine, Ramis Ufuk Akkoyunlu, Lilia M. Sklyarenko, Literature Search: Deniz Sünnetçi, Ramis Ufuk Akkoyunlu, Michael P. Zavelevich, Writing: Hakan Savlı, Ramis Ufuk Akkoyunlu. 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. Financial Disclosure: The authors declared that this study has received a financial support by TÜBİTAK-NASU joint project (M/32-2008).

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RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0197 Turk J Hematol 2016;33:15-20

Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression BACH1 ve Hemoglobin E/Beta-Talasemi Globin Sunumu Korelasyonu Tze Yan Lee1, Logeswaran Muniandy1, Lai Kuan Teh1, Maha Abdullah1, Elizabeth George1,2 Jameela Sathar3, Mei I Lai1,2 1Universiti Putra Malaysia Faculty of Medicine and Health Sciences, Department of Pathology, Serdang, Malaysia 2Universiti Putra Malaysia Faculty of Medicine and Health Sciences, Genetic and Regenerative Medicine Research Centre, Serdang, Malaysia 3Ampang Hospital, Clinic of Hematology, Selangor, Malaysia

Abstract

Öz

Objective: The diverse clinical phenotype of hemoglobin E (HbE)/βthalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes.

Amaç: Hemoglobin E (HbE)/β-talaseminin çeşitli klinik fenotipleri

Materials and Methods: A total of 47 HbE/β-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data.

kompanse etmedeki rolünü inceledik.

Results: The BACH1 expression among the β-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, β-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy.

veriler ile korele edildi.

Conclusion: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/β-thalassemia. Keywords: BACH1, Gene expression, Hemoglobin E/β-thalassemia, Oxidative stress, Red blood cell parameters

klinisyenlerin hasta yönetimi esnasında zihinlerini karıştırmakla kalmamış, α- ve β-globin genotiplerinde bariz benzerlikler varken fenotiplerde farklılıklar bulunduğundan bilim insanlarının hassas eritrosit çevrenin muhafaza edilmesinde yer alan karmaşık mekanizmaları incelemelerine de ön ayak olmuştur. BTB ve CNC homoloji 1 (BACH1) proteininin eritroid hücrelerin son farklılaşması sırasında α- and β-globin gen transkripsiyonlarını ayarladığı bilinmektedir. HbE/β-talasemi hastalığındaki mutasyonlar ile her ne kadar ince ayar amaçlı ise de BACH1’in globin zincir dengesizliğini Gereç ve Yöntemler: Toplam 47 HbE/β-talasemi örneği gerçek zamanlı kantitatif polimeraz zincir reaksiyonu ile incelendi ve yaş, cinsiyet, eritrosit değişkenleri, globin gen sunumları ve bazı klinik Bulgular: β-talasemi intermedia hastalarındaki BACH1 sunumu 2-log’a kadar farklılık göstermekteydi ve yaş; α-, β- ve γ-globin gen sunum düzeyleri; ve hem oksijenaz 1 proteini ile pozitif korelasyonu vardı. Ayrıca BACH1’in retikülosit düzeyi ile negatif korelasyonu vardı ve splenektomi ile anlamlı korelasyonu bulunmaktaydı. Sonuç: Bu çalışma hem HbE/β-talasemide bulunan oksidatif stresi hem de globin zincir dengesizliğini azaltmak için BACH1 sunumunun kompansasyon mekanizması olarak artabileceğini göstermiştir. Anahtar Sözcükler: BACH1, Gen sunumu, Hemoglobin E/β-talasemi, Oksidatif stres, Eritrosit değişkenleri

Address for Correspondence/Yazışma Adresi: Mei I LaI, PhD., Universiti Putra Malaysia Faculty of Medicine and Health Sciences, Department of Pathology, Serdang, Malaysia Phone : +603-89472494 E-mail : lmi@upm.edu.my

Received/Geliş tarihi: May 18, 2014 Accepted/Kabul tarihi: August 12, 2014

Lee TY, et al: BACH1 in Hemoglobin E/Beta-Thalassemia

Introduction Hemoglobin E (HbE) is a highly common hemoglobin variant in Asia. The substitution of G>A at codon 26 of the β-globin gene not only causes a reduced and structurally abnormal HbE production but also activates a cryptic splice site, leading to abnormal messenger ribonucleic acid (mRNA) processing. The heterozygous state of HbE is asymptomatic with minimal morphological red cell changes, and homozygous HbE at most displays mild anemia. However, when this variant is co-inherited with β-thalassemia, the clinical spectrum ranges from mild anemia to severe thalassemia intermedia [1,2]. This complex phenotype is affected not only by free α-globin chain precipitations causing membrane damage and increased reactive oxygen species, but also by various secondary and tertiary modifiers including variable compensatory mechanisms available in the red cell environment. This is evident particularly in families with seemingly similar α/β genotypes but discordant phenotypes [3]. BACH1 is a heme-binding factor that regulates multiple gene expressions. By forming a multivalent deoxyribonucleic acid (DNA)-binding complex in the enhancer regions of targeted genes, BACH1 represses transcription of its target genes. However, this function is inhibited in the presence of heme [4,5]. BACH1 plays an important role in the expression of heme-responsive genes like α- and β-globin, which are the key proteins in HbE/β-thalassemia, as well as in the regulation of heme oxygenase 1 (HO-1), a stress-responsive protein [5,6,7]. As BACH1 is able to suppress the expression of α- and β-globin genes under physiological conditions, the expression of BACH1 in HbE/β-thalassemia is of interest. A study by De Franceschi et al. showed a reduced intracellular heme content in late β-thalassemic precursors, which leads to an increased expression of the BACH1 gene [8]. Perhaps the increased BACH1 expression may play a compensatory role to reduce α/β-globin chain imbalance, thereby decreasing the oxidative stress produced by the imbalance. Thus, this study investigates the correlation of BACH1 to HbE/β-thalassemia parameters, where any effects of BACH1 would most likely be manifested.

Materials and Methods Study Population HbE/β-thalassemia patients from the Ampang Hospital Thalassemia Clinic were recruited for this study. The HbE/βthalassemia patients in our cohort were either transfusionindependent or previously had transfusion record of not less than 3 months prior to recruitment for this study. Ethical approval was obtained from the Medical Research and Ethics Committee, Ministry of Health Malaysia (NMRR-10-1177-6947), and the 16

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Medical Research Ethics Committee, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM/FPSK/PADS/ T7-MJKEtikaPer/F01). All subjects had given their informed consents prior to blood collection and anonymity of all data was made possible by numerical identification throughout the study. Laboratory Studies Full Blood Count, High-Performance Liquid Chromatography, and Plasma Ferritin Study Collection of blood samples were done in BD Vacutainer spraydried K2EDTA tubes and BD Vacutainer freeze-dried lithium heparin tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and full blood count indices were analyzed using a Sysmex 5000i Automated Hematology Analyzer (Sysmex, Kobe, Japan) according to the manufacturer’s protocol. The screening of HbE/β-thalassemia was performed using the VARIANT II β-Thalassemia Short Program on the VARIANT II Hemoglobin Testing System (Bio-Rad, Hercules, CA, USA). Plasma ferritin analysis was performed using the Tina-Quant ferritin assay (Cobas, Roche Diagnostics GmbH, Mannheim, Germany) on the Hitachi 902 Automatic Chemistry Analyzer (Hitachi, Ibaraki, Japan) according to the manufacturer’s protocol. Genomic Studies Genomic DNA was extracted using the QIAamp Blood Midi Kit (QIAGEN GmbH, Hilden, Germany). Amplification refractory mutation system polymerase chain reaction (PCR) using primer sequences and PCR protocol modified from Old was performed to identify and confirm HbE and β-thalassemia mutations for each sample [9]. The β-thalassemia mutations characterized were codon 19 (A>G), IVS I-5 (G>C), IVS I-1 (G>T), codon 41/42 (-TCTT), and IVS II-654 (C>T). Genomic sequencing was done on uncharacterized samples. Co-inheritance of α-thalassemia was determined by --SEA,/-α3.7, and /-α4.2 mutation screening to minimize the occurrence of confounding factors for this study. Xmn1 polymorphisms were determined as previously described by Wong et al. [10]. Primers covering the exons of the BACH1 gene were used to sequence the gene. Primers and PCR conditions for the BACH1 gene are available upon request. Expression Analysis Ribonucleic acid (RNA) extraction from peripheral reticulocytes was performed as described previously by Lai et al. [11]. Expression levels of BACH1, HO-1, and α-, β-, and γ-globin genes were quantified using real-time quantitative reverse transcriptionPCR (Applied Biosystems, Warrington, UK). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (TaqMan Gene Expression Assay, Applied Biosystems) acts as the endogenous control in this expression study.

Lee TY, et al: BACH1 in Hemoglobin E/Beta-Thalassemia

Turk J Hematol 2016;33:15-20

Table 1. Correlation of BACH1 expression with demographic and hematological data and heme oxygenase 1 and globin gene expressions. Parameter

Mean ± 2 Standard Deviations

Range (min-max)

p-value (R2 value)

Age, years

34.36±19.02

21-57

0.006 (0.155)*

Sex (M/F)

15/32

0.891

Ethnicity (M^/C^)

45/2

Transfusion (Y^/N^)

46/1

Splenomegaly (n/s/sp)

23/19/5

0.0085*

Iron overload (Y^/N^)

39/8

0.622

Iron chelation (Y^/N^)

33/14

0.174

RBC, x106/mL

3.85±1.72

2.32-5.86

0.086 (0.064)

Hb, g/dL

7.40±2.56

4.8-10.8

0.157 (0.044)

Hct, %

23.66±7.70

15.8-36.1

0.438 (0.013)

MCV, fL

63.09±20.52

46.4-90.0

0.125 (0.052)

MCH, pg

19.61±5.96

14.5-30.4

0.491 (0.011)

MCHC, g/dL

31.12±4.12

26.0-34.6

0.051 (0.082)

RDW-SD, #

62.1±24.82

36.2-84.3

0.112 (0.055)

RDW-CV %

28.61±11.68

16.9-52.9

0.856 (0.001)

Retic #, x106/µL

3.93±23 (1)

0-53.72

0.005 (0.16)*

Retic %

28.94±152.46 (1)

0-376.8

0.01 (0.138)*

NRBC #, x103/µL

6.85±44.24 (1)

0.02-112.27

0.681 (0.004)

NRBC %

34.03±244.72 (1)

0.31-758.6

0.435 (0.014)

Plasma ferritin, ng/mL

2134.37±4141.14 (3)

153.5-8138.4

0.098 (0.06)

Hb F, %

28.79±29.36

3.8-62.2

0.569 (0.007)

Hb A2, %

11.07±34

1.3-77.8

0.721 (0.003)

Beta genotype†

0.6531

Xmn1 (+-/++)

31/13

0.139 (0.031)

Log BACH1 expression

0.418±1.932

-2.92 to 2.00

Log HO-1 expression

1.319±2.083

-2.22 to 3.95

0.001 (0.329)*

Log beta expression

-1.128±2.991

-7.04 to 1.64

0.001 (0.253)*

Log alpha expression

-1.487±3.264

-7.54 to 0.02

0.002 (0.192)*

Log gamma expression

0.1634±2.142

-2.92 to 2.56

0.001 (0.330)*

Min: Minimum, Max: maximum, M: male, F: female, M^: Malay, C^: Chinese, Y^: intermittent transfusion, N^: transfusion-independent, n: normal, s: splenomegaly, sp: splenectomized, SD: standard deviation, RBC: red blood cell, Hb: hemoglobin, Hct: hematocrit, MCV: mean cell volume, MCH: mean cell hemoglobin, MCHC: mean cell hemoglobin concentration, RDW: red cell distribution width, RDW-CV: red cell distribution width coefficient of variation, NRBC: nucleated red blood cell, Retic: reticulocytes, †HbE/IVSI-5, HbE/IVSI-1, HbE/Cd41-42, HbE/ IVSII-654, HO-1: heme oxygenase 1, (): number of samples with unknown related data, NA: not analyzed. *: All values are considered significant when p≤0.05.

Statistical Analysis

Results

IBM SPSS 20 was used to analyze the correlation of BACH1 expression to age, sex, red blood cell indices, HbF, HbA2 level, Xmn1 genotype, β-thalassemia genotypes, HO-1 expression, and α-, β-, and γ-globin genes expression. BACH1 was also correlated to clinical data such as transfusion history, splenomegaly, iron overloading, and iron chelation therapy. The parameters were analyzed using analysis of variance, Student’s t-test, Pearson’s correlation coefficient analysis, and simple linear regression analysis.

Correlation of BACH1 Gene Expression with Age, Sex, and Red Blood Cell Parameters A total of 47 unrelated HbE/beta-thalassemia samples were collected (14 males and 33 females; 45 Malays and 2 Chinese; 21-57 years old). These individuals had thalassemia intermedia with a mean hemoglobin level of 7.40±2.56 g/dL. Co-inheritance of α-thalassemia or iron-deficiency anemia was not found in our sample cohort. BACH1 expression in our samples varied up to 100.43 with a mean of 1.601±42.775 fold change and 17

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Turk J Hematol 2016;33:15-20

was positively skewed. To fit the normal distribution curve for statistical analyses, the BACH1 expression results were logtransformed with approximately 2 log-fold variation. Simple linear regression analysis showed positive association of log BACH1 expression with age (p=0.006; R2=0.155). Log BACH1 expression was not correlated to sex or most red blood cells parameters, except reticulocyte count (p=0.005; R2=0.16) and percentage (%) (p=0.01; R2=0.138). Detailed results can be found in Table 1.

polymorphism was excluded from the analysis as the numbers were too small (n=3). The efficiency of βE-globin mRNA splicing could be the main factor of β-globin expression variations, rather than β genotypes, to affect the BACH1 expression level [12]. Five samples were randomly selected for re-sequencing of the BACH1 gene. However, no polymorphisms could be detected in these samples. Polymorphisms that affect the BACH1 gene expression could be located upstream in the enhancer regions that were not sequenced.

Correlation of BACH1 Gene Expression to Hemoglobin +E/β-Thalassemia Genotypes, BACH1 Genotypes, and Xmn1 Polymorphisms

Correlation of BACH1 Gene Expression with Globin Gene Expressions and Heme Oxygenase 1 Expression

The β-thalassemia mutations present in these HbE/βthalassemia individuals were 20 IVS I-5 (G>C), 18 IVS I-1 (G>T), 7 CD41/42 (-TCTT), and 2 IVS II-654. BACH1 expression was not correlated to any β-thalassemia genotypes (p=0.6531) or Xmn1 polymorphisms (p=0.139; R2=0.031) (Table 1). The Xmn1 -/-

All positively skewed expression data were log-transformed to fit the normal distribution. Log BACH1 expression was positively correlated to log α-globin (p=0.002; R2=0.192), log β-globin (p=0.001; R2=0.253), log γ-globin (p=0.001; R2=0.330), and log HO-1 (p=0.001; R2=0.329) gene expressions (Figure 1). Correlation of BACH1 Gene Expression to Clinical Data BACH1 was not affected by transfusion history (p=0.6298), iron overloading (p=0.6216), or iron chelation therapy (p=0.1743). However, BACH1 correlation was significant in splenectomized individuals (p=0.0085) (Figure 2).

Discussion The heme-BACH1 transcription activation pathway provides a mechanism for the cellular environment to maintain a balanced homeostasis during erythroid differentiation when the production and assembly of hemoglobin actively comes together. While BACH1 functions to maintain a dormant expression of the α- and β-globin genes, it could quickly change with the increase of heme [13]. However, in HbE/β-

Figure 1. Correlations of BACH1 expression with age; reticulocyte count; α-, β-, and γ-globin gene expression; and heme oxygenase 1 gene expression. A) Correlation of BACH1 with age (p=0.006), B) correlation of BACH1 with reticulocyte number (µL) (p=0.01), C) correlation of BACH1 with α-globin expression (p=0.002), D) correlation of BACH1 with β-globin expression (p=0.001), E) correlation of BACH1 with γ-globin expression (p=0.001), F) correlation of BACH1 expression with heme oxygenase 1 expression (p=0.001). 18

Figure 2. BACH1 expression correlation to spleen sizes and splenectomized hemoglobin E/β-thalassemia individuals (p=0.0085).

Lee TY, et al: BACH1 in Hemoglobin E/Beta-Thalassemia

Turk J Hematol 2016;33:15-20

thalassemia, the expression of the β-globin gene is reduced. We studied the BACH1 gene expression in this disorder to elucidate the function of BACH1 when the α/β-globin chain ratio balance is compromised. HbE/β-thalassemia red cells are known to have increased levels of oxidative stress, which is caused by the excess free α-globin chains available and the increased labile iron pool [14,15]. Cell damage or death caused by increased available reactive oxygen species could be related to the natural cellular aging process or iron overload in HbE/β-thalassemia as excess iron absorption from the intestinal tract accumulates slowly over time [15,16,17,18]. A study by Dohi et al. showed that BACH1 is involved in the repression of premature cellular senescence induced by oxidative stress [19]. Therefore, the rise of BACH1 expression as age increases is perhaps to delay premature cellular aging due to excessive oxidative stress. As reticulocyte numbers reflect the bone marrow erythropoietic activity, the degree of ineffective erythropoiesis due to harmful precipitation of excess free α-globin chains in β-thalassemia can be phenotypically typed [20,21,22]. The negative correlation of BACH1 and reticulocyte count could reflect the role of BACH1 in repressing the excess α-globin gene to reduce the rate of ineffective erythropoiesis [6]. HbE/β-thalassemia does not only have lower βE-globin chain synthesis but also the presence of aberrantly spliced βE-globin mRNA. Higher levels of aberrant compared to correctly spliced βE-globin mRNA have been linked to increased severity [12,23]. BACH1 could function to decrease the production of βE-globin to reduce the burden of aberrantly spliced βE-globin mRNA. The non-association of beta genotypes to BACH1 expression could be due to the masking of the genotype expression by HbE. In terms of the lack of β-globin gene expression in HbE/βthalassemia, compensatory increase of γ-globin chains to form HbF with excess α-globin chains explains the indirect correlation to BACH1 expression. To date, there has not been any study done to examine the effect of BACH1 on the γ-globin gene. However, if BACH1 does have a repressive role on γ-globin gene expression, then BACH1 is maintaining cellular homeostasis by regulating the expression of the γ-globin chain. Wickramasinghe and Lee showed that large production of γ-globin chains does not necessarily protect against extensive precipitations of α-globin monomers [24]. BACH1 is also a well-known repressor of HO-1. De Franceschi et al. showed that heme and HO-1 levels were both reduced in β-thalassemia precursors compared to controls [8]. BACH1 could be repressing HO-1 in β-thalassemia to prevent the cytotoxic effect of excess free heme due to the lack of normal hemoglobin formation and also to prevent the excessive accumulation of

heme degradation products as they possess the potential to be cytotoxic beyond a certain threshold [25]. However, this mechanism could be overwhelmed in splenectomized patients. Splenectomy is performed when accelerated transfusions are required to maintain adequate hemoglobin levels in the patient [26]. A study on thalassemia intermedia patients showed severe iron decompartmentalization in the red blood cells of splenectomized patients compared to non-splenectomized patients with significantly higher levels of membrane-bound free iron, non-heme iron, and heme compounds [27]. Heme not only suppresses the function of BACH1 but has also been found to promote BACH1 degradation [28].

Conclusion BACH1 plays a role in maintaining the microcellular homeostasis in HbE/β-thalassemia by repressing the excess α-globin chains and aberrantly spliced βE-globin mRNA as well as preventing cytotoxic effects of excess free heme, although the amount expressed may not be sufficient to alleviate the severity of the HbE/β-thalassemia phenotype and it is abolished in the presence of excessive heme. Further investigations to confirm the pathways involved are necessary, perhaps by using a mouse model. Acknowledgments The authors wish to thank the Director General of Health, Malaysia, for permission to publish this paper. Funding of this work is supported by the Fundamental Research Grant Scheme, Ministry of Higher Education, Malaysia (04-04-10-837FR), and a Research University Grant Scheme, Universiti Putra Malaysia (04-02-11-1378RU), awarded to MIL. TYL is a recipient of a MyBrain scholarship from the Ministry of Higher Education (MOHE), Malaysia. Ethics Ethics Committee Approval: NMRR-10-1177-6947 and UPM/ FPSK/PADS/T7-MJKEtikaPer/F01, Informed Consent: It was taken. Authorship Contributions Concept: Mei I Lai, Design: Mei I Lai, Data Collection or Processing: Tze Yan Lee, Logeswaran Muniandy, Jameela Sathar, Analysis or Interpretation: Mei I Lai, Elizabeth George, Maha Abdullah, Lai Kuan Teh, Tze Yan Lee, Logeswaran Muniandy, Literature Search: Tze Yan Lee, Writing: Mei I Lai, Tze Yan Lee. 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. 19

Lee TY, et al: BACH1 in Hemoglobin E/Beta-Thalassemia

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14. Amer J, Goldfarb A, Fibach E. Flow cytometric analysis of the oxidative status of normal and thalassemic red blood cells. Cytometry A 2004;60:7380. 15. Prus E, Fibach E. The labile iron pool in human erythroid cells. Br J Haematol 2008;142:301-307. 16. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature 2000;408:239-247. 17. Ginzburg Y, Rivella S. β-Thalassemia: a model for elucidating the dynamic regulation of ineffective erythropoiesis and iron metabolism. Blood 2011;118:4321-4330. 18. Olivieri NF. The beta-thalassemias. N Engl J Med 1999;341:99-109. 19. Dohi Y, Ikura T, Hoshikawa Y, Katoh Y, Ota K, Nakanome A, Muto A, Omura S, Ohta T, Ito A, Yoshida M, Noda T, Igarashi K. Bach1 inhibits oxidative stressinduced cellular senescence by impeding p53 function on chromatin. Nat Struct Mol Biol 2008;15:1246-1254. 20. Cortellazzi LC, Teixeira SM, Borba R, Gervásio S, Cintra CS, Grotto HZW. Reticulocyte parameters in hemoglobinopathies and iron deficiency anemia. Rev Bras Hematol Hemoter 2003;25:97-102. 21. Lamchiagdhase P, Pattanapanyasat K, Muangsup W. Reticulocyte counting in thalassemia using different automated technologies. Laboratory Hematology 2000;6:73-78. 22. Nathan DG, Gunn RB. Thalassemia: the consequences of unbalanced hemoglobin synthesis. Am J Med 1966;41:815-830.

8. De Franceschi L, Bertoldi M, De Falco L, Santos Franco S, Ronzoni L, Turrini F, Colancecco A, Camaschella C, Cappellini MD, Iolascon A. Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in β-thalassemic erythropoiesis. Haematologica 2011;96:1595-1604.

23. Tubsuwan A, Munkongdee T, Jearawiriyapaisarn N, Boonchoy C, Winichagoon P, Fucharoen S, Svasti S. Molecular analysis of globin gene expression in different thalassaemia disorders: individual variation of β(E) pre-mRNA splicing determine disease severity. Br J Haematol 2011;154: 635-643.

9. Old JM. Hematological applications: hemoglobinopathies. Methods Mol Med 2004;92:203-219.

24. Wickramasinghe SN, Lee MJ. Observations on the relationship between gamma-globin chain content and globin chain precipitation in thalassaemic erythroblasts and on the composition of erythroblastic inclusions in HbE/ beta-thalassaemia. Eur J Haematol 1997;59:305-309.

10. Wong YC, George E, Tan KL, Yap SF, Chan LL, Tan JA. Molecular characterisation and frequency of Gg Xmn I polymorphism in Chinese and Malay beta-thalassaemia patients in Malaysia. Malays J Pathol 2006;28:1721. 11. Lai MI, Jiang J, Silver N, Best S, Menzel S, Mijovic A, Colella S, Ragoussis J, Garner C, Weiss MJ, Thein SL. Alpha-hemoglobin stabilising protein is a quantitative trait gene that modifies the phenotype of beta-thalassaemia. Br J Haematol 2006;133:675-682. 12. Watanapokasin Y, Winichagoon P, Fuchareon S, Wilairat P. Relative quantitation of mRNA in beta-thalassemia/Hb E using real-time polymerase chain reaction. Hemoglobin 2000;24:105-116. 13. Igarashi K, Sun J. The heme-BACH1 pathway in the regulation of oxidative stress response and erythroid differentiation. Antioxid Redox Signal 2006;8:107-118.

25. Shibahara S, Nakayama M, Kitamuro T, Udono-Fujimori R, Takahashi K. Repression of heme oxygenase-1 expression as a defense strategy in humans. Exp Biol Med (Maywood) 2003;228:472-473. 26. Smith CH, Erlandson ME, Stern G, Schulman I. The role of splenectomy in the management of thalassemia. Blood 1960;15:197-211. 27. Tavazzi D, Duca L, Graziadei G, Comino A, Fiorelli G, Cappellini MD. Membrane-bound iron contributes to oxidative damage of betathalassaemia intermedia erythrocytes. Br J Haematol 2001;112:48-50. 28. Hada H, Shiraki T, Watanabe-Matsui M, Igarashi K. Hemopexin-dependent heme uptake via endocytosis regulates the Bach1 transcription repressor and heme oxygenase gene activation. Biochim Biophys Acta 2014;1840:23512360.

RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0142 Turk J Hematol 2016;33:21-27

The Feasibility of Magnetic Resonance Imaging for Quantification of Liver, Pancreas, Spleen, Vertebral Bone Marrow, and Renal Cortex R2* and Proton Density Fat Fraction in Transfusion-Related Iron Overload Manyetik Rezonans Görüntülemenin Transfüzyon İlişkili Demir Birikimi Bulunan Hastalarda Karaciğer, Pankreas, Dalak, Vertebral Kemik İliği ve Böbrek Korteksi R2* ve Proton Dansite Yağ Oranı Ölçümünde Uygulanabilirliği İlkay S. İdilman1,2, Fatma Gümrük3, Mithat Haliloğlu1, Muşturay Karçaaltıncaba1,2 1Hacettepe University Faculty of Medicine, Department of Radiology, Ankara, Turkey 2Hacettepe University Faculty of Medicine, Liver Imaging Team, Ankara, Turkey 3Hacettepe University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey

Abstract

Öz

Objective: We aimed to evaluate the feasibility of quantification of liver, pancreas, spleen, vertebral bone marrow, and renal cortex R2* and magnetic resonance imaging-proton density fat fraction (MRIPDFF) and to evaluate the correlations among them in patients with transfusion-related iron overload. Materials and Methods: A total of 9 patients (5 boys, 4 girls) who were referred to our clinic with suspicion of hepatic iron overload were included in this study. All patients underwent T1-independent volumetric multi-echo gradient-echo imaging with T2* correction and spectral fat modeling. MRI examinations were performed on a 1.5 T MRI system. Results: All patients had hepatic iron overload. Severe hepatic iron overload was recorded in 5/9 patients (56%), and when we evaluated the PDFF maps of these patients, we observed an extensive patchy artifact in the liver in 4 of 5 patients (R2* greater than 671 Hz). When we performed MRI-PDFF measurements despite these artifacts, we observed artifactual high MRI-PDFF values. There was a close correlation between average pancreas R2* and average pancreas MRIPDFF (p=0.003, r=0.860). There was a significant correlation between liver R2* and average pancreas R2* (p=0.021, r=0.747), liver R2* and renal cortex R2* (p=0.020, r=0.750), and average pancreas R2* and renal cortex R2* (p=0.003, r=0.858). There was a significant negative correlation between vertebral bone marrow R2* and age (p=0.018, r=0.759). Conclusion: High iron content of the liver, especially with a T2* value shorter than the first echo time can spoil the efficacy of PDFF calculation. Fat deposition in the pancreas is accompanied by pancreatic iron overload. There is a significant correlation between hepatic siderosis and pancreatic siderosis. Renal cortical and pancreatic siderosis are correlated, too. Keywords: Iron overload, Liver, Pancreas, R2*, Magnetic resonance imaging-proton density fat fraction

Amaç: Bu çalışmada, transfüzyon ilişkili demir birikimi bulunan hastalarda karaciğer, pankreas, dalak, vertebral kemik iliği ve böbrek korteksi R2* ve manyetik rezonans görüntüleme-proton dansite yağ oranı (MRG-PDYO) ölçümünün uygulanabilirliği ve bunlar arasındaki ilişkiyi değerlendirmeyi amaçladık. Gereç ve Yöntemler: Çalışmaya, karaciğer demir birikimi şüphesi ile kliniğimize gönderilmiş olan toplam 9 hasta (5 erkek, 4 kız) dahil edildi. Tüm hastalara T2* düzeltmeli ve spektral yağ modellemeli T1 bağımsız volumetrik multi-eko gradient-eko görüntüleme yapıldı. MRG incelemeleri 1.5 T MRG sistemi ile yapıldı. Bulgular: Tüm hastalarda karaciğerde demir birikimi mevcuttu. Dokuz hastanın beşinde (%56) ağır demir birikimi saptandı ve bu hastaların PDYO haritaları incelendiğinde 4 hastada (R2* değeri 671 Hz’den fazla olanlarda) karaciğerde yaygın yamasal artefaktlar olduğu saptandı. Bu artefaktlara rağmen MRG-PDYO ölçümü yapıldığında normalden çok yüksek MRG-PDYO değerleri belirlendi. Ortalama pankreas R2* ve ortalama pankreas MRG-PDYO arasında (p=0,003, r=0,860), karaciğer R2* and ortalama pankreas R2* arasında (p=0,021, r=0,747), karaciğer R2* ve renal korteks R2* arasında (p=0,020, r=0,750) ve ortalama pankreas R2* ve renal korteks R2* arasında (p=0,003, r=0,858) anlamlı korelasyon saptandı. Vertebral kemik iliği R2* ve yaş arasında anlamlı negatif korelasyon saptandı (p=0,018, r=-0,759). Sonuç: Karaciğerdeki yüksek demir birikimi özellikle de T2* değeri ilk eko zamanından daha kısa olduğunda PDYO hesaplamasını bozabilmektedir. Pankreastaki yağ birikimi pankreatik demir birikimine eşlik edebilmektedir. Karaciğer ve pankreas siderozisi birbiri ile ilişki göstermektedir. Böbrek korteksi ve pankreas siderozisi arasında da ilişki mevcuttur. Anahtar Sözcükler: Demir birikimi, Karaciğer, Pankreas, R2*, Manyetik rezonans görüntüleme-proton dansite yağ oranı

Address for Correspondence/Yazışma Adresi: Muşturay Karçaaltıncaba, M.D., Hacettepe University Faculty of Medicine, Department of Radiology, Ankara, Turkey E-mail : musturayk@yahoo.com

Received/Geliş tarihi: April 01, 2015 Accepted/Kabul tarihi: June 15, 2015

İdilman İS, et al: Magnetic Resonance IDEAL-IQ in Transfusion-Related Iron Overload

Introduction

Materials and Methods

Iron is an essential nutrient for all human cells [1,2]. Under normal circumstances, intake and excretion of iron is balanced within a daily range of 1-2 mg [3]. There is no effective way of excretion of iron from the body; therefore, if the total amount of income exceeds outcome, such as in cases of increased intestinal absorption, long-term transfusion therapies, or excess parenteral iron treatment, total body iron increases. Transfusion-related iron overload is one of the leading causes of iron overload. It primarily depends on repetitive transfusions that bring a burden of excess iron, especially after 40 to 50 transfusions that saturate the capacity of reticuloendothelial system [4]. After saturation, iron accumulates in parenchymal organs like the liver, pancreas, myocardium, and endocrine glands, which leads to tissue damage and fibrosis [3].

Patients

Iron is mainly stored in the liver and the iron content of the liver is an indirect marker of total body iron [5]. Hence, quantification of hepatic iron content is used for guiding and monitoring iron chelation therapies in transfusion-related iron overload [1]. Current methods for quantification of hepatic iron consist of liver biopsy and imaging. Percutaneous liver biopsy is the current reference method for quantification of hepatic iron content. However, it is an invasive procedure that can bring serious complications, and acquired small sample sizes in liver biopsy are insufficient to represent the whole organ. In addition, iron overload can be organ-specific and estimation of iron accumulation in different organs such as the pancreas, spleen, vertebral bone marrow, and kidneys requires noninvasive techniques. Magnetic resonance imaging (MRI) methods including signal intensity ratio and relaxometry are promising for quantification of hepatic iron overload compared with liver biopsy [6,7,8]. However, coexistence of fat influences the measurement of hepatic iron content because of the spectral complexity of fat [9]. Vice versa, hepatic iron overload influences the measurement of hepatic fat content in chemical shift techniques due to its T2* shortening effect [10]. A recent technique, MR IDEAL-IQ (Iterative Decomposition of water and fat with Echo Asymmetry and Least square estimation), which was defined primarily for quantification of the fat fraction of tissue, quantifies R2* by correcting the fat-dependent confounding factors [11]. However, the former technique’s accuracy was found to be lower, especially in patients with T2* values below 1 ms [12]. Additionally, the feasibility of this technique was not evaluated in the estimation of pancreas, spleen, vertebral bone marrow, and renal cortex iron overload. In the present study, we aimed to evaluate the feasibility of MR IDEAL-IQ in the quantification of liver, pancreas, spleen, vertebral bone marrow, and renal cortex R2* and MRI-proton density fat fraction (MRI-PDFF) and to evaluate the correlations among them in patients with transfusion-related iron overload. 22

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This was a retrospective cross-sectional study. A total of 9 patients (5 boys, 4 girls) who were referred to our clinic with suspicion of hepatic iron overload and examined with MR IDEAL-IQ between August 2010 and November 2010 were included in the study. All of the patients had a history of repetitive transfusions, 8 of them with a diagnosis of beta thalassemia major and 1 of them with non-Hodgkin lymphoma (NHL). Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Magnetic Resonance Imaging Examination MR images were acquired with a 1.5 T HDxt MRI system (GE Healthcare, Milwaukee, WI, USA). The subjects were examined in the supine position. An 8-channel phased array body coil was used for acquisition. A 3-plane gradient echo localizer sequence was performed at the beginning of the examination. The MRI protocol included the IDEAL-IQ sequence. This is a 3D volumetric imaging sequence used to create T2* and triglyceride fat fraction maps from a single breath-hold acquisition. The technique was used to estimate R2* (1/T2*) and PDFF (watertriglyceride fat separation) in the liver simultaneously in a single acquisition. Afterwards, a correction was applied to the resulting PDFF maps to correct for T2* effects. Six gradient echoes were applied to reconstruct water and triglyceride fat images, relative triglyceride fat fractions, and R2* maps. The IDEAL-IQ sequence uses a novel “complex field map” to incorporate the T2* and field inhomogeneity effects into the overall multi-echo acquisition signal model. It was shown by Yu et al. [11] that, by acquiring a 6-echo image and estimating a complex field map using an iterative least square estimation algorithm, it is possible to achieve fat-water separation and T2* estimation in a single breath-hold acquisition. The parameters of this sequence were TR: 12.9 ms, FOV: 3540 cm, matrix: 224x160, 125 kHz bandwidth, flip angle: 5 °, and slice thickness: 5 mm. A single 3D slab with 44 to 56 slices was acquired. We acquired data sets with 6 different echoes ranging from 1.6 ms to 9.8 ms. A 2D self-calibrated parallel imaging technique called auto calibrating reconstruction of Cartesian sampling was used with an acceleration factor of 2. The images were processed using the software provided by the manufacturer to create water, fat, in-phase, opposed-phase, R2*, and fat fraction maps. Image Analysis By using a work station (AW 4.4, GE Healthcare), a radiologist placed an elliptic region of interest (ROI) of approximately 4 cm2 in Couinaud segments 5-6 on the PDFF maps and the

İdilman İS, et al: Magnetic Resonance IDEAL-IQ in Transfusion-Related Iron Overload

Turk J Hematol 2016;33:21-27

R2* maps, avoiding blood vessels, bile ducts, and artifacts. An elliptic ROI of approximately 1 cm2 for the pancreatic head, body, and tail on the PDFF maps and the R2* maps was placed for pancreatic measurements and the arithmetic mean was calculated. The same procedure was performed for the spleen with a ROI of 2 cm2, for the L1 or L2 vertebral corpus with a ROI of 2 cm2, and for the renal cortex with a ROI of 1 cm2. The patients with a T2* value under 18 ms were included in the hepatic iron overload group [13]. The patients with a T2* value under 2 ms were included in the severe hepatic iron overload group. The patients with pancreatic R2* values between 30 and 100 Hz were included in the mild pancreatic siderosis group, those between 100 and 400 Hz were included in the moderate pancreatic siderosis group, and those >400 Hz were included in the severe pancreatic siderosis group [14].

in the study population. The results are summarized in Table 1. All of the patients had hepatic iron overload. Severe hepatic iron overload was recorded in 5/9 patients (56%). When we evaluated the PDFF maps of severe hepatic iron overload patients, we observed an extensive patchy artifact in the liver in the majority of them (4/5) (Figures 1 and 2). When we performed MRI-PDFF measurements even with these artifacts, we observed unexpectedly high MRI-PDFF values. The liver R2* values of these patients were higher (range: 671.1-773.9 Hz) when compared with the patient with severe hepatic iron overload without artifacts (528.70 Hz). Mean average pancreas R2* value was 236.8 Hz (47.5-496.9 Hz) and mean average pancreas MRI-PDFF value was 14.7%

The degree of association between continuous and/or ordinal variables was calculated by Pearson correlation coefficient (r) and p<0.05 was considered significant.

Results Eight patients with beta thalassemia major and 1 patient with NHL were included in this study (male/female: 5/4). The mean age of the patients was 17.4 years (range: 13-22 years). Thalassemia patients had a diagnosis of beta thalassemia major since infancy and were treated with repetitive red blood cell transfusion and iron chelation therapy. The patient with NHL had an 8-year disease history with two bone marrow transplantations and multiple red blood cell transfusion therapies. Three of the patients had a history of myocardial iron overload, and two of them had hypogonadotropic hypogonadism. The mean BMI of the patients was 19.1 kg/m2 (range: 17.6-20.7 kg/m2). The mean ferritin level was 5933.1 ng/mL (range: 600-29950 ng/mL), mean liver R2* value was 424.8 Hz (range: 60.9-773.9 Hz), and mean liver MRI-PDFF was 22.4% (range: 1.5%-63.9%)

Figure 1. The magnetic resonance imaging-proton density fat fraction (top row) and R2* (bottom row) maps of the patients with severe hepatic iron overload. Except for Patient 1, all patients with severe hepatic iron overload demonstrated a patchy artifact in proton density fat fraction maps.

Figure 2. The magnetic resonance imaging-proton density fat fraction (top row) and R2* (bottom row) maps of the patients with milder hepatic iron overload. There is no artifact in patients with milder hepatic iron overload.

Table 1. The characteristics and magnetic resonance imaging findings of patients with transfusion-related iron overload. Subject

Sex

Age (Years)

Primary Diagnosis

Serum Ferritin Level (ng/mL)

SHIO

Liver R2* (Hz)

Liver T2* (ms) Liver MRI-PDFF (%)

NHL

29950

528.7

1.89

1.5

β-TM

600

68.7

14.56

2.1

β-TM

1232

60.9

16.42

2.2

β-TM

8177

722.6

1.38

32.6

β-TM

4709

671.1

1.49

30.5

β-TM

3106

105.4

9.49

2.7

β-TM

1381

155.9

6.41

3.6

β-TM

1931

773.9

1.29

63.9

β-TM

2312

736.4

1.36

62.6

MRI-PDFF: Magnetic resonance imaging-proton density fat fraction, NHL: non-Hodgkin lymphoma, β-TM: beta thalassemia major, SHIO: severe hepatic iron overload, M: male, F: female.

Ä°dilman Ä°S, et al: Magnetic Resonance IDEAL-IQ in Transfusion-Related Iron Overload

Turk J Hematol 2016;33:21-27

(1.2%-42.2%). All patients had pancreatic siderosis; 2 of them had mild siderosis, 5 of them had moderate siderosis, and 2 of them had severe siderosis (Table 2). There was a close correlation between average pancreas R2* and average pancreas MRI-PDFF (p=0.003, r=0.860) (Figure 3). We could not perform splenic measurements in 3 patients as they were splenectomized. Mean spleen R2* value was 142.1 Hz (29.9-224.2 Hz) and mean spleen MRI-PDFF was 2.1% (1.3%-3.1%). Mean vertebral bone marrow R2* value was 289 Hz (151.4-548.8 Hz) and mean vertebral bone marrow MRI-PDFF was 13.3% (0.1%-60%). Mean average renal cortex R2* value was 21.9 Hz (11.2-42.2 Hz) and mean average renal cortex MRI-PDFF was 0.8% (0%-1.5%).

Figure 3. Scatterplot shows the correlation between pancreatic R2* and magnetic resonance imaging-proton density fat fraction (p=0.003, r=0.860).

Figure 4. Scatterplot shows the correlation between liver R2* and average pancreas R2* (p=0.021, r=0,747).

When we evaluated the correlations among patient age, serum ferritin level, and MRI findings, we observed a significant correlation between liver R2* and average pancreas R2* (p=0.021, r=0.747) (Figure 4). There was also a significant correlation among liver R2* and renal cortex R2* (p=0.020, r=0.750) (Figure 5) and

Figure 5. Scatterplot shows the correlation between liver R2* and renal cortex R2* (p=0.020, r=0.750).

Table 2. Pancreas, spleen, vertebral bone marrow, and renal cortical R2* and magnetic resonance imaging-proton density fat fraction of the patients. Subject

Average Pancreas R2* (Hz)

Average Pancreas MRI-PDFF (%)

Average Spleen R2* (Hz)

Average Spleen MRIPDFF (%)

Vertebral Bone Marrow R2* (Hz)

Vertebral Bone Marrow MRI-PDFF (%)

Renal Cortex R2* (Hz)

Renal Cortex MRI-PDFF (%)

154.8

1.9

215.7

1.6

206.7

19.7

0.7

177

12.7

N/A

151.4

11.4

11.8

1.6

76.4

1.6

29.9

1.3

238.6

1.7

11.2

0.7

350.6

42.2

224.4

2.4

308

2.6

24.8

1.1

178.5

7.4

184

2.2

467.3

1.9

19.5

1.4

216.9

164.5

3.1

548.8

18.4

0.4

47.5

1.2

N/A

196.3

0.1

432.9

33.8

1.7

252.6

0.8

29.7

0.6

496.9

31.5

N/A

231

1.2

42.2

1.1

MRI-PDFF: Magnetic resonance imaging-proton density fat fraction, N/A: not applicable.

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Ä°dilman Ä°S, et al: Magnetic Resonance IDEAL-IQ in Transfusion-Related Iron Overload

average pancreas R2* and renal cortex R2* (p=0.003, r=0.858) (Figure 6). There was a significant negative correlation between vertebral bone marrow R2* and age (p=0.018, r=-0.759) (Figure 7). No other significant correlation was observed between patient age, serum ferritin level, and liver, pancreas, spleen, vertebral bone marrow, and renal cortex R2* and MRI-PDFF values.

Discussion Noninvasive assessment of hepatic iron content in transfusionrelated iron overload, which predominantly involves the pediatric population, is an important issue that was studied before by many investigators [6,7,8]. However, within the obesity epidemic, fat accumulation can coexist in the liver in such patients. The presence of both fat and iron in the liver has a confounding effect on the quantification of each one with MRI techniques [15]. In the present study we evaluated the

Figure 6. Scatterplot shows the correlation between average pancreas R2* and renal cortex R2* (p=0.003, r=0.858).

Figure 7. Scatterplot shows the correlation between vertebral bone marrow R2* and age (p=0.018, r=-0.759).

feasibility of a recently described method, IDEAL-IQ, for liver, pancreas, spleen, vertebral bone marrow, and renal cortex iron and fat quantification in patients with transfusion-related iron overload. Previously, Liau et al. evaluated the effect of changes in R2* caused by an intravenous infusion of super paramagnetic iron oxide contrast agent in quantification of liver fat fraction with IDEAL-IQ and observed that the IDEAL-IQ method of fat quantification is robust to changes in R2* [16]. However, the highest liver R2* value observed in this study was 212 Hz, which was distinctly lower than our patients with severe hepatic iron overload. Another study by Vasanawala et al. evaluated the clinical feasibility of weighted least squares T2* IDEAL, which is a similar technique to the one that we used, in transfusionrelated iron overload and concluded that this technique is feasible in clinical applications [12]. In that study, investigators also observed significant hepatic steatosis in the liver in three patients that had milder hepatic iron overload compared with our study. In our study, we observed artifacts and unexpectedly high MRI-PDFF values in PDFF maps of patients with a T2* value approximately under 1.6 ms with the MR-IDEAL technique. As discussed by Vasanawala et al. [12], estimation of a T2* value under the first echo time used in the technique is challenging. However, they did not mention that there is a problem in PDFF maps in such patients. In previous studies that evaluated the utility of this technique in quantification of hepatic steatosis in adults [17] and individuals predominantly consisting of pediatric patients [18] with biopsy-proven nonalcoholic fatty liver disease, the authors did not observe such high MRI-PDFF values, even in patients with severe steatosis, as we observed in the present study. Additionally, the patients in our study had no risk factors for hepatic steatosis or any hepatic steatosis, supporting additional imaging findings and confirming the inability of this technique in quantification of fat fraction in severe hepatic iron overload. All of the patients in our study had pancreatic siderosis with variable degrees. Diabetes mellitus is one of the most common endocrine problems in thalassemia patients and pathogenesis depends on beta-cell dysfunction according to increased iron deposition [19]. It has been shown that early application of chelation therapy is protective for diabetes [20]. Pancreatic iron overload causes cell death and fatty transformation in pancreatic tissue [21]. An MRI technique that is feasible for both fat fraction quantification and R2* values would be valuable in assessing the pancreas in patients with transfusion-related iron overload. In our study, we could demonstrate both pancreatic siderosis and steatosis with MRI-PDFF. In addition, we observed a significant correlation between pancreatic siderosis and pancreatic steatosis, which was presumed but not demonstrated in previous studies. 25

İdilman İS, et al: Magnetic Resonance IDEAL-IQ in Transfusion-Related Iron Overload

Papakonstantinou et al. previously investigated the correlations between hepatic, splenic, pancreatic, vertebral bone marrow, and myocardial siderosis and did not find a correlation between pancreatic siderosis and hepatic and splenic siderosis [22]. Another study by Argyropoulou et al. did not find a correlation of T2 among the liver, bone marrow, pancreas, and pituitary gland [23]. However, in our study, we observed significant correlation between hepatic siderosis and pancreatic siderosis, hepatic siderosis, and renal cortical siderosis and pancreatic siderosis and renal cortical siderosis. We could not find a correlation between liver R2* and either spleen or bone marrow R2*. On the contrary, we observed a significant negative correlation between vertebral bone marrow R2* and age, which was not mentioned in previous studies. One of the limitations of our study is the small sample size. However, there were patients with severe hepatic iron overload, which demonstrates the inability of the technique in fat quantification in short T2* values. Another limitation is the absence of histology assessment for accurate quantification of iron and fat content of the liver and other tissues. Furthermore, it is not feasible to evaluate iron and fat content of all organs with biopsy. On the other hand, we could easily demonstrate R2* values of different tissues just with an ROI placement with this technique, which is valuable in transfusion-related iron overload. In addition, we could demonstrate relationships between different tissues’ R2* values and the relationships between R2* values and fat fractions in tissues with a R2* value below approximately 671 Hz.

Conclusion High iron content of the liver, especially with a T2* value shorter than the first echo time can spoil the efficacy of PDFF calculation. Fat deposition in the pancreas is accompanied by pancreatic iron overload. There is a significant correlation between hepatic siderosis and pancreatic siderosis. Renal cortical and pancreatic siderosis are correlated, too. Acknowledgments Muşturay Karçaaltıncaba was supported by the Turkish Academy of Sciences (TÜBA) in the framework of the Young Scientist Award Program (EA-TÜBA-GEBİP/2011). Ethics Ethics Committee Approval: Hacettepe University Ethics Committee, Informed Consent: It was taken. Authorship Contributions Concept: Muşturay Karçaaltıncaba, Design: Muşturay Karçaaltıncaba, Data Collection or Processing: İlkay S. İdilman, Fatma Gümrük, Mithat Haliloğlu, Muşturay Karçaaltıncaba, 26

Turk J Hematol 2016;33:21-27

Analysis or Interpretation: İlkay S. İdilman, Literature Search: İlkay S. İdillman, Muşturay Karçaaltıncaba, Writing: İlkay S. İdilman, Muşturay Karçaaltıncaba. 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. Brittenham GM, Cohen AR, McLaren CE, Martin MB, Griffith PM, Nienhuis AW, Young NS, Allen CJ, Farrell DE, Harris JW. Hepatic iron stores and plasma ferritin concentration in patients with sickle cell anemia and thalassemia major. Am J Hematol 1993;42:81-85. 2. Alustia JM, Castiella A, De Juan MD, Emparanza JI, Artetxe J, Uranga M. Iron overload in the liver diagnostic and quantification. Eur J Radiol 2007;61:499-506. 3. Andrews NC. Disorders of iron metabolism. N Engl J Med 1999;341:19861995. 4. Siegelman ES, Mitchell DG, Semelka RC. Abdominal iron deposition: metabolism, MR findings, and clinical importance. Radiology 1996;199:13-22. 5. Angelucci E, Brittenham GM, McLaren CE, Ripalti M, Baronciani D, Giardini C, Galimberti M, Polchi P, Lucarelli G. Hepatic iron concentration and total body iron stores in thalassemia major. N Eng J Med 2000;343:327-331. 6. Gandon Y, Olivie D, Guyader D, Aubé C, Oberti F, Sebille V, Deugnier Y. Noninvasive assessment of hepatic iron stores by MRI. Lancet 2004;363:357362. 7. St Pierre TG, Clark PR, Chua-Anusorn W, Fleming AJ, Jeffrey GP, Olynyk JK, Pootrakul P, Robins E, Lindeman R. Noninvasive measurement and imaging of liver iron concentrations using proton magnetic resonance. Blood 2005;105:855-861. 8. Wood JC, Enriquez C, Ghugre N, Tyzka JM, Carson S, Nelson MD, Coates TD. MRI R2 and R2* mapping accurately estimates hepatic iron concentration in transfusion-dependent thalassemia and sickle cell disease patients. Blood 2005;106:1460-1465. 9. Sirlin CB, Reeder SB. Magnetic resonance imaging quantification of liver iron. Magn Reson Imaging Clin N Am 2010;18:359-381. 10. Westphalen AC, Qayyum A, Yeh BM, Merriman RB, Lee JA, Lamba A, Lu Y, Coakley FV. Liver fat: effect of hepatic iron deposition on evaluation with opposed-phase MR imaging. Radiology 2007;242:450-455. 11. Yu H, McKenzie CA, Shimakawa A, Vu AT, Brau AC, Beatty PJ, Pineda AR, Brittain JH, Reeder SB. Multiecho reconstruction for simultaneous water-fat decomposition and T2* estimation. J Magn Reson Imaging 2007;26:11531161. 12. Vasanawala SS, Yu H, Shimakawa A, Jeng M, Brittain JH. Estimation of liver T2 in transfusion-related iron overload in patients with weighted least squares T2 IDEAL. Magn Reson Med 2012;67:183-190. 13. Westwood M, Anderson LJ, Firmin DN, Gatehouse PD, Charrier CC, Wonke B, Pennell DJ. A single breath-hold multiecho T2* cardiovascular magnetic resonance technique for diagnosis of myocardial iron overload. J Magn Reson Imaging 2003;18:33-39. 14. Noetzli LJ, Papudesi J, Coates TD, Wood JC. Pancreatic iron loading predicts cardiac iron loading in thalassemia major. Blood 2009;114:4021-4026. 15. Sharma P, Altbach M, Galons JP, Kalb B, Martin DR. Measurement of liver fat fraction and iron with MRI and MR spectroscopy techniques. Diagn Interv Radiol 2014;20:17-26.

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İdilman İS, et al: Magnetic Resonance IDEAL-IQ in Transfusion-Related Iron Overload

16. Liau J, Shiehmorteza M, Girard OM, Sirlin CB, Bydder M. Evaluation of MRI fat fraction in the liver and spine pre and post SPIO infusion. Magn Reson Imag 2013;31:1012-1016. 17. Idilman IS, Aniktar H, Idilman R, Kabacam G, Savas B, Elhan A, Celik A, Bahar K, Karcaaltincaba M. Hepatic steatosis: quantification by proton density fat fraction with MR imaging versus liver biopsy. Radiology 2013;267:767-775. 18. Tang A, Tan J, Sun M, Hamilton G, Bydder M, Wolfson T, Gamst AC, Middleton M, Brunt EM, Loomba R, Lavine JE, Schwimmer JB, Sirlin CB. Nonalcoholic fatty liver disease: MR imaging of liver proton density fat fraction to assess hepatic steatosis. Radiology 2013;267:422-431. 19. Tiosano D, Hochberg Z. Endocrine complications of thalassemia. J Endocrinol Invest 2001;24:716-723. 20. Brittenham GM, Griffith PM, Nienhuis AW, McLaren CE, Young NS, Tucker EE, Allen CJ, Farrell DE, Harris JW. Efficacy of deferoxamine in preventing

complications of iron overload in patients with thalassemia major. N Engl J Med 1994;331:567-573. 21. Midiri M, Lo Casto A, Sparacia G, D’Angelo P, Malizia R, Finazzo M, Montalto G, Solbiati L, Lagalla R, De Maria M. MR imaging of pancreatic changes in patients with transfusion dependent beta-thalassemia major. AJR Am J Roentgenol 1999;173:187-192. 22. Papakonstantinou O, Alexopoulou E, Economopoulos N, Benekos O, Kattamis A, Kostaridou S, Ladis V, Efstathopoulos E, Gouliamos A, Kelekis NL. Assessment of iron distribution between liver, spleen, pancreas, bone marrow and myocardium by means of R2 relaxometry with MRI in patients β-thalassemia major. J Magn Reson Imag 2009;29:853-859. 23. Argyropoulou MI, Kiortsis DN, Astrakas L, Metafratzi Z, Chalissos N, Efremidis SC. Liver, bone marrow, pancreas and pituitary gland iron overload in young and adult thalassemic patients: a T2 relaxometry study. Eur Radiol 2007;17:3025-3030.

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0181 Turk J Hematol 2016;33:28-33

Freezing of Apheresis Platelet Concentrates in 6% Dimethyl Sulfoxide: The First Preliminary Study in Turkey Aferez Trombosit Konsantrelerinin Yüzde 6’lık Dimetil Sülfoksitte Dondurulması: İlk Türkiye Çalışmasının Başlatılması Soner Yılmaz1, Rıza Aytaç Çetinkaya1, İbrahim Eker2, Aytekin Ünlü3, Metin Uyanık4, Serkan Tapan4, Ahmet Pekoğlu1, Aysel Pekel5, Birgül Erkmen6, Uğur Muşabak5, Sebahattin Yılmaz1, İsmail Yaşar Avcı7, Ferit Avcu6, Emin Kürekçi2, Can Polat Eyigün7 1Gülhane Military Medical Academy, Blood Training Center and Blood Bank, Ankara, Turkey 2Gülhane Military Medical Academy, Division of Pediatric Hematology, Ankara, Turkey 3Gülhane Military Medical Academy, Department of General Surgery, Ankara, Turkey 4Gülhane Military Medical Academy, Department of Medical Biochemistry, Ankara, Turkey 5Gülhane Military Medical Academy, Division of Immunology and Allergy, Ankara, Turkey 6Gülhane Military Medical Academy, Division of Hematology, Ankara, Turkey 7Gülhane Military Medical Academy, Department of Infectious Disease and Clinical Microbiology, Ankara, Turkey

Abstract

Öz

Objective: Transfusion of platelet suspensions is an essential part of patient care for certain clinical indications. In this pioneering study in Turkey, we aimed to assess the in vitro hemostatic functions of platelets after cryopreservation. Materials and Methods: Seven units of platelet concentrates were obtained by apheresis. Each apheresis platelet concentrate (APC) was divided into 2 equal volumes and frozen with 6% dimethyl sulfoxide (DMSO). The 14 frozen units of APCs were kept at -80 °C for 1 day. APCs were thawed at 37 °C and diluted either with autologous plasma or 0.9% NaCl. The volume and residual numbers of leukocytes and platelets were tested in both before-freezing and post-thawing periods. Aggregation and thrombin generation tests were used to analyze the in vitro hemostatic functions of platelets. Flow-cytometric analysis was used to assess the presence of frozen treated platelets and their viability. Results: The residual number of leukocytes in both dilution groups was <1x106. The mean platelet recovery rate in the plasma-diluted group (88.1±9.5%) was higher than that in the 0.9% NaCl-diluted group (63±10%). These results were compatible with the European Directorate for the Quality of Medicines quality criteria. Expectedly, there was no aggregation response to platelet aggregation test. The mean thrombin generation potential of postthaw APCs was higher in the plasma-diluted group (2411 nmol/L per minute) when compared to both the 0.9% NaCl-diluted group (1913 nmol/L per minute) and the before-freezing period (1681 nmol/L per minute). The flowcytometric analysis results for the viability of APCs after cryopreservation were 94.9% and 96.6% in the plasma and 0.9% NaCl groups, respectively. Conclusion: Cryopreservation of platelets with 6% DMSO and storage at -80 °C increases their shelf life from 7 days to 2 years. Besides the increase in hemostatic functions of platelets, the cryopreservation process also does not affect their viability rates. Keywords: Frozen platelets, Flow-cytometric analysis, In vivo thrombin generation test

Amaç: Trombosit süspansiyonlarının transfüzyonu, belirli klinik endikasyonlarda hastaların tedavisinin önemli bir parçasıdır. Bu çalışma ile Türkiye’de ilk kez olmak üzere trombositlerin in vitro hemostatik fonksiyonlarının kriyopreservasyon işleminden sonra değerlendirilmesi amaçlanmıştır. Gereç ve Yöntemler: Çalışmamızda 7 ünite trombosit süspansiyonu aferez yöntemiyle elde edildi. Her aferez trombosit konsantresi (ATK) iki eşit hacime ayrıldıktan sonra %6 dimetil sülfoksit (DMSO) kullanılarak donduruldu. Dondurulmuş 14 ünite ATK -80 °C’de bir gün süre ile bekletildi. ATK’lar 37 °C’de çözdürüldükten sonra otolog plazma veya %0,9 NaCl kullanılarak dilüe edildi. ATK’ların dondurma öncesi ve çözülme işlemi sonrası; hacim, rezidüel lökosit ve trombosit sayıları incelendi. Trombositlerin in vitro hemostatik fonksiyonların incelenmesinde agregasyon ve trombin jenerasyon testleri kullanıldı. Dondurma işlemine maruz kalan ATK’lardaki trombositlerin varlığı ve bu hücrelerin canlılığını değerlendirmek için akım sitometri yöntemi kullanıldı. Bulgular: Her iki dilüsyon grubunda yer alan ATK’ların residüel lökosit sayısı 1x106’nın altındaydı. Plazma ile dilüe edilen grubun ortalama trombosit geri kazanım oranı %0,9 NaCl ile dilüe edilen gruptan daha yüksekti (%88,1±9,5’e karşılık %63±10). Bu sonuçlar Avrupa İlaç Kalite ve Sağlık Hizmetleri Direktörlüğü’nün kalite kriterlerine uygundu. Trombosit agregasyon testine beklenildiği üzere yanıt alınamadı. Dondurulup çözülerek otolog plazma ile dilüe edilen ATK’ların ortalama trombin oluşturma potansiyeli (2411 nmol/L×dakika), %0,9 NaCl ile dilüe edilenlere (1913 nmol/L×dakika) ve dondurma işlemi öncesine göre (1681 nmol/L×dakika) daha yüksek saptandı. Kriyopreservasyon işlemi sonrası plazma ve %0,9 NaCl ile dilüe edilen ATK’ların akım sitometri yöntemi ile canlılığı sırasıyla %94,9 ve %96,6 olarak bulundu. Sonuç: Trombositlerin DMSO ile kriyopreserve edilerek -80 °C saklanmaları, raf ömürlerini 7 günden 2 yıla çıkarmaktadır. Kriyopreservasyon işlemi trombositlerin in vitro hemostatik fonksiyonlarını arttırmanın yanında canlılık oranlarını da etkilememektedir. Anahtar Sözcükler: Dondurulmuş trombositler, Akım-sitometri testi, İn vivo thrombin jenerasyon testi

Address for Correspondence/Yazışma Adresi: Soner YILMAZ, M.D., Gülhane Military Medical Academy, Blood Training Center and Blood Bank, Ankara, Turkey Phone : +90 312 304 49 02 E-mail : soyilmaz@gata.edu.tr

Received/Geliş tarihi: May 07, 2014 Accepted/Kabul tarihi: October 14, 2014

Turk J Hematol 2016;33:28-33

Introduction Currently in blood banking applications, platelet concentrates (PCs) prepared through apheresis or from buffy coat should be used within 5-7 days after preparation. In order to overcome the short shelf life-related problems, studies on frozen PCs have continued since the 1970s [1]. Although the literature data on the use of cryopreserved platelets showed that in vivo cryopreserved platelet suspensions have hemostatic activities superior to those of fresh apheresis suspensions, they showed delayed responses to in vitro platelet aggregation tests. Initially, this delay was attributed to the loss of aggregation capability of platelets during the cryopreservation process. However, recent studies proved that the in vitro failure of aggregation response to agonists occurred in response to the transformation of platelets into a procoagulant phenotype by the activationdegranulation process [2,3,4]. In the last decade, the demand for frozen platelets that have a long shelf life has increased for the treatment of military casualties in the Iraq and Afghanistan campaigns. As for Turkey, freezing and storing PCs as a part of contingency plans and prevention of this valuable blood product’s disposal due to short shelf life has an importance beyond emphasis. The most commonly used cryopreservation protocol is the addition of dimethyl sulfoxide (DMSO) to PCs at a final concentration of 4%-6%, followed by removal of DMSO involving supernatant before the freezing process and finally freezing of the hyperconcentrated low volume of PCs. After the thawing process, PCs can be diluted by adding 0.9% NaCl, autologous plasma, or platelet additive solutions. This protocol can attain a gain of platelets between 70% and 80% [5]. In this study, we aimed to assess the in vitro hemostatic activity of cryopreserved platelets using different dilution methods (0.9% NaCl and autologous plasma).

Materials and Methods In August 2013, Gülhane Military Medical Academy Ethics Committee Approval was received for the assessment of in vitro hemostatic activity of cryopreserved apheresis platelet concentrates (APCs). Written informed consent was obtained from all participants. PCs were obtained using the apheresis method (Trima, Caridian BCT, Inc., Lakewood, CO, USA) from donors that met the National Blood and Blood Products criteria for the donation of APCs. APCs at a total of 200 mL were collected in acid-citrate-dextrose (ACD, NIH, Formula A, Baxter Healthcare Corp., Deerfield, IL, USA) at a ratio of 1 volume of ACD to 10 volumes of blood. APCs from all 7 donors were divided into 2 packs of 100 mL in volume each. One of each of the 100-mL packs was included in either the plasma-diluted group or the 0.9% NaCl-diluted group. Each

Yılmaz S, et al: Cryopreservation of Platelet Concentrates

group comprised 7 APCs. Before the freezing process, APCs were preserved in an automatic shaker on a horizontal plane at 2024 °C for 1 day. Each of the APCs’ volume and weight were calculated at all stages of the procedure. Apheresis Platelet Concentrate Freezing Process A 41-mL sample of plasma collected by apheresis from each donor and 0.9% NaCl were mixed with 9 mL of 27% DMSO in an empty blood bag located on a rigid ice pack for the plasmadiluted group and 0.9% NaCl-diluted groups, respectively. The resultant 50-mL mixture and 100 mL of APC were collected in a 750-mL ethyl vinyl acetate freezing bag (CryoMACS® Freezing Bag 750, Miltenyi Biotec, Teterow,Germany) through a sterile hose combining device. The final DMSO concentration in the freezing bag was 6% and the bag was centrifuged at 22 °C and 1250x g for 10 min (Thermo Fisher Scientific RC12BP, Asheville, NC, USA). A platelet pellet of 20-25 mL was obtained after removal of the supernatant and the bag was put into a cardboard freezing box and stored at -80 °C. Thawing of Frozen Apheresis Platelet Concentrates The 1-day-old frozen APCs were thawed through immersion in 37 °C water within 10 min. Either 20 mL of autologous plasma or 0.9% NaCl was added to the APCs depending on the dilution group and they were kept at room temperature for 30 min.

In Vitro Measurements All analyses were repeated in the fresh state and after diluting the APCs in the post-thaw state. Residual Leukocyte and Platelet Counts The frozen APCs were analyzed for the determination of platelet and residual leukocyte counts with a whole-blood analyzer device (ABX Pentra XL80, HORIBA ABX SAS, Montpellier, France). Platelet Aggregation Test Platelet aggregation tests were performed with a Chrono-log platelet aggregometer by the same laboratory technician and thrombocyte agonist (ADP, epinephrine, collagen, and ristocetin) responses were assessed for both dilution groups. Thrombin Generation Test Thrombin generation test (TGT) was performed with a calibrated automated thrombogram device (Thrombinoscope BV, Maastricht, the Netherlands) [6]. In this test, thrombin generation occurs in the co-presence of phospholipid and tissue factor present in the platelet supernatant and/or added reagents. The platelet-rich plasma reagent (Thrombinoscope BV) used in our test involves 1 pmol/L tissue factor. However, this reagent does not involve phospholipid and is used for assessing the presence of phospholipid in the medium. A sample of 80 µL was collected from both dilution groups. Each sample 29

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Turk J Hematol 2016;33:28-33

number and percentage. All statistical analyses were processed using SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA).

was transferred to 3 different microtitrated plates (Immulon 2 HB, Thermo Electron Corporation, Milford, MA, USA) that involved 20 µL of platelet-rich plasma reactant and 20 µL of thrombin calibrator. After the incubation of the mixture at 37 °C for 15 min, a 20-µL sample was collected and added to 20 µL of Fluo-buffer solution, and the reaction was monitored with a fluorometer. Using the Thrombinoscope program, the thrombogram curve, endogenous thrombin potential, and peak height were measured. The endogenous thrombin potential, which indicates the total amount of endogenous thrombin generated, was recorded as nmol/L per minute. The peak height, which indicates the highest measured value of thrombin, was shown as nmol/L.

Results The mean volume of APCs after dilution with autologous plasma or 0.9% NaCl was 45±3 mL. The mean platelet counts of the plasma and 0.9% NaCl groups were (123.6±13.7)x1011 (range: (106.9-143.5)x1011) and (84.6±7.6)x1011 (range: (77.8100.8)x1011), respectively (Table 1). The freeze-thaw percentage recovery was calculated according to the standard operating procedure of the Naval Blood Research Laboratory [7]. The residual leukocyte counts of all APCs were <1x106. In the 0.9% NaCl-diluted group, one sample was excluded from the study due to damage to the plastic bag. The platelet and residual leukocyte counts and the rate of platelet recovery of APCs are shown in Table 1.

Flow Cytometry Analysis Platelet samples were transferred to tubes containing K3 EDTA. CD41a FITC (BD Biosciences, San Jose, CA, USA) and 7-aminoactinomycin D (7-AAD) were used to determine viable platelets. The incubated cells were analyzed using the FACSDiva software for FACSCanto II model flow cytometry (BD Biosciences).

In all 13 fresh APC samples, platelet aggregation tests with ADP, epinephrine, and collagen were normal. There was no aggregation response to a variety of dilution ratios in any of the frozen treated platelet samples.

Statistical Analysis

TGT revealed that post-thaw APCs diluted with autologous plasma (Figure 1A) had higher endogenous thrombin potentials when compared to fresh-state samples (Figure 1B) and post-

Quantitative results were presented as mean ± standard deviation and minimum-maximum. Categorical results were presented as

Table 1. Residual leukocyte and platelet counts, and platelet recovery rates of fresh apheresis platelet concentrates and apheresis platelet concentrates after thawing. Plasma-Diluted Group

Platelet Count (x109/L)

Residual Leukocytes (x109/L) Fresh

Platelet Recovery (%)

Post-thaw

Sample 1

0.10

115.7

113.6

Sample 2

0.10

129

128.2

Sample 3

0.30

139.8

106.9

Sample 4

0.20

139.5

117.8

Sample 5

0.20

148.3

139.1

Sample 6

0.10

149

116.1

Sample 7

0.10

156.7

143.5

Mean ± SD

0.15±0.07

139.7±13.7

123.6±13.7

88.1±9.5

0.9% NaCl-Diluted Group

Sample 1

0.10

115.7

100.8

Sample 2

0.10

129

79.6

Sample 3

0.30

139.8

84.4

Sample 4

0.20

139.5

85.3

Sample 5

0.20

139.1

77.8

Sample 6

0.10

149

Sample 7*

Mean ± SD

0.16±0.07

135.3±10.5

84.6±7.6

63±10

SD: Standard deviation. *This sample was excluded from the study due to a damaged plastic bag.

Yılmaz S, et al: Cryopreservation of Platelet Concentrates

Turk J Hematol 2016;33:28-33

thaw APC samples diluted with 0.9% NaCl (Figure 1C) (2411 vs. 1681 and 1913 nmol/L per minute). The peak height values were also higher in post-thaw APC samples diluted with autologous plasma (609 vs. 350 and 338 nmol/L, respectively). As a result of flow-cytometric analysis, 99.2% of fresh APCs were stained with the CD41A thrombocyte indicator, while 97.9% were determined as viable when tested with 7-AAD nucleic acid dye. The viability rates of the post-thaw APC samples diluted with plasma and 0.9% NaCl are shown in Table 2.

Discussion In 1956, Klein et al. reported the use of previously frozen platelets in an actively bleeding thrombocytopenic patient, and since then numerous studies have been reported on both the in vitro and in vivo efficacies of cryopreserved platelets [8]. Since Schiffer et al.’s 1976 study on the use of autologous platelets for the treatment of patients with leukemia, relevant studies until the 1990s showed that the platelets were damaged to a significant extent by the freezing process, which decreased their efficacy when compared to fresh platelets [1]. These results were supported by other in vitro studies that assessed the platelets’ primary hemostatic functions [1,9,10]. However,

Figure 1A. Thrombin generation test results of plasma diluted group (sample). Endogenous thrombin potential and peak height values of apheresis platelet concentrates were 2411 nmol/L per minute and 609 nmol/L, respectively.

Khuri et al.’s 1999 report caused a shift in this paradigm; they showed that the in vivo hemostatic functions of cryopreserved APCs were superior to those of fresh preserved platelets [3]. Almost simultaneously, Bernard et al. reported the procoagulant changes in frozen treated platelet membrane surfaces [4]. Recently, it was also reported that fresh PCs with almost expired shelf lives contained platelet-derived microparticles with 50 to 100 times more potent procoagulant activity than activated platelets and they had a significant impact on the activation and continuation of the coagulation cascade [11,12]. In 2014, Johnson et al. demonstrated increased phosphatidylserine expression on cryopreserved platelet membranes and also showed that these cryopreserved APCs contained

Figure 1B. Thrombin generation test results of apheresis platelet concentrates before freezing (sample). Endogenous thrombin potential and peak height values of apheresis platelet concentrates were 1681 nmol/L per minute and 350 nmol/L, respectively.

Figure 1C. Thrombin generation test results of 0.9% NaCl-diluted group (sample). Endogenous thrombin potential and peak height values of apheresis platelet concentrates were 1913 nmol/L per minute and 338 nmol/L, respectively.

Table 2. Viability rates of fresh and post-thaw apheresis platelet concentrates. Fresh Platelets

Plasma-Diluted Group

0.9% NaCl-Diluted Group

Total Platelets (%)

Viability (%)

Sample 1

98.7

94.3

87.3

97.1

Sample 2

98.5

99.9

91.7

98.4

Sample 3

99.3

96.6

95.4

94.7

Sample 4

99.5

99.9

93.1

95.4

Sample 5

99.5

94.8

98.8

95.6

Sample 6

99.6

99.9

99.7

98.9

Sample 7*

99.8

99.9

98.5

SD: Standard deviation. *This sample was excluded from the study due to a damaged plastic bag.

Yılmaz S, et al: Cryopreservation of Platelet Concentrates

phosphatidylserine microparticles that might contribute to the increased hemostatic activity. They also presented the first in vitro phosphatidylserine-dependent coagulation and thrombin generation potentials of cryopreserved APCs by using the TGT [13]. The use of autologous plasma was the most common method for resuspending PCs after thawing until 2006 when Valeri et al. claimed that 0.9% NaCl could be used instead of autologous plasma [14]. The design of this study includes 2 different dilution groups (0.9% NaCl and autologous plasma), aimed to better delineate the dilution method that meets the quality control criteria. According to the Guide to the Preparation, Use and Quality Assurance of Blood Components: European Directorate for the Quality of Medicines & Healthcare of the Council of Europe (EDQM), frozen APCs have 3 quality control criteria (platelet recovery, residual leukocyte count, and volume) [15]. The platelet recovery rate should be higher than 40%. In our study, the mean platelet recovery rate in the plasma-diluted group (88.1±9.5%) was higher than in the 0.9% NaCl-diluted group (63±10%). However, the platelet recovery rate of both dilution groups met the EDQM quality criteria. Compared to other studies, the results of the 0.9% NaCl-diluted group were lower than in Valeri et al.’s study (74±11%), but those of the plasma-diluted group were better than in Lelkens et al.’s (77±15%) [16,17]. One potential weakness in the current study is the lower number of samples assessed. The EDQM’s criteria require that the volume of post-thaw APCs be ≥50 mL. In our study, the mean volume of APCs after thawing and dilution was 45±3 mL, which was due to the division of 200 mL of APCs into 2 packs of equal volume prior to freezing. Another quality standard requires the presence of <1x106 residual leukocytes in post-thaw APCs, which was met by all the samples in both dilution groups [15]. The maximum shelf life of PCs stored at 22 °C is 5-7 days. Below this temperature, toxic effects begin to appear in the cells. Moreover, ice crystal formation occurs at low-temperature storage (<0 °C). This formation can puncture the platelet membrane, leading to cell death. In this study, we aimed to show the effect of low temperature on the viability of platelets by flow-cytometric analysis. The mean viability rates of postthaw APC samples diluted with plasma and 0.9% NaCl were determined as 94.9% and 96.6%, respectively (Table 2). These results revealed that toxic effects of temperature could be prevented using DMSO as a cryoprotective agent. The TGT is an assay that measures the overall tendency of thrombin formation after initiation of coagulation [18]. The use of autologous plasma for the dilution of frozen thawed platelets could affect the TGT test results. Frozen APCs diluted 32

Turk J Hematol 2016;33:28-33

with autologous plasma (Figure 1A) had correspondingly higher thrombin generation potentials, as in Johnson et al.’s study [13], when compared to both fresh platelets (Figure 1B) and the APC group diluted with 0.9% NaCl (Figure 1C). These findings suggest that cryopreservation increases the platelet hemostatic activities independently from the plasma content. Due to the presence of coagulation factors, the plasma content may also provide an additional hemostatic stimulus when compared to the 0.9% NaCl-diluted group. Platelet aggregation responses were negative as expected. In Valeri et al.’s study, frozen treated platelets had a significant decrease in aggregation response irrespective of the dilution or resuspension method when compared to fresh platelets (p<0.001) [2]. On the other hand, Hornsey et al. reported that frozen treated platelets demonstrated no aggregation response [19]. However, DMSO-treated platelets were effective on kidney bleeding time in a study that investigated the correlation of in vivo and in vitro functions of fresh and stored human platelets [20]. DMSO-treated frozen platelets have been used successfully since the 1970s [1,17,21]. Khuri et al. compared the clinical effects and hemostatic efficiency of frozen and liquid-preserved platelets in patients undergoing cardiopulmonary bypass in 1999 [3]. They reported that cryopreserved platelet transfusions were superior to liquid-preserved platelets in reducing blood loss and blood transfusion requirements after cardiopulmonary bypass. They concluded that these results were probably related to the improved in vivo hemostatic activity of cryopreserved platelets [3]. In 2001, Özsan et al. studied the cryopreservation of platelets by using a cryopreserving agent and showed that sialic acid was not an alternative compound for cryopreservation [22]. In 2003, Kurt Yüksel et al. presented a case report that demonstrated that the autologous transfusion of cryopreserved platelets could be a reasonable approach in bleeding alloimmunized patients [23]. In 2001, the Netherlands Military Blood Bank implemented the use of frozen platelets in Bosnia and abandoned the walking blood bank concept. Within a 6-month period, 2 thrombocytopenic casualties with exsanguinating hemorrhage were treated with frozen platelets. After those reports, frozen platelets and frozen blood bank facilities became an essential part of military hospital standard equipment deployed by the military of the Netherlands [17]. In 2008, the Australian Defence Force embedded a surgical and intensive care team into the Netherlands-led forward health facility in Afghanistan. Twentytwo units of frozen platelets were used by these teams for 17 casualties undergoing surgery. Except in one patient, there was no clinical evidence of coagulopathy in patients treated with frozen platelets [24]. Between 2006 and 2012, 6246 cryopreserved blood products were transfused in Afghanistan; 2175 of them were erythrocyte concentrations, 3001 were fresh frozen plasma, and 1070 were frozen platelets. No transfusion

Turk J Hematol 2016;33:28-33

reactions were reported related to the use of these cryopreserved blood components [25]. The cryopreservation of platelets increases their shelf life from 7 days to 2 years when they are stored at -80 °C with the cryoprotective agent DMSO. Difficulties in the preparation of APCs and the cost of sets could be prevented with the use of cryopreserved blood products. Moreover, the availability of autologous cryopreserved platelets for patients likely to develop refractoriness to platelets or allogenic ABO- and human leukocyte antigen-compatible cryopreserved platelets is crucial for the treatment of these patients. The strategic location of Turkey mandates the urgent collaboration of the Turkish Armed Forces, the Red Crescent, and other governmental medical organizations in establishing both frozen platelet and erythrocyte stocks. Ethics Ethics Committee Approval: Gülhane Military Medical Academy Ethics Committee (Approval number: 06-05-14/37), Informed Consent: It was taken. Authorship Contributions Concept: Soner Yılmaz, İbrahim Eker, Design: Soner Yılmaz, İbrahim Eker, Data Collection or Processing: Aytekin Ünlü, Metin Uyanık, Ahmet Pekoğlu, Aysel Pekel, Birgül Erkmen, Analysis or Interpretation: Rıza Aytaç Çetinkaya, Literature Search: Serkan Tapan, Uğur Muşabak, Sebahattin Yılmaz, İsmail Yaşar Avcı, Ferit Avcu, Emin Kürekçi, Can Polat Eyigün, Writing: Aytekin Ünlü, Rıza Aytaç 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.

References 1. Schiffer CA, Aisner J, Wiernik PH. Clinical experience with transfusion of cryopreserved platelets. Br J Haematol 1976;34:377-385. 2. Valeri CR, Macgregor H, Ragno G. Correlation between in vitro aggregation and thromboxane A2 production in fresh, liquid-preserved, and cryopreserved human platelets: effect of agonists, pH, and plasma and saline resuspension. Transfusion 2005;45:596-603. 3. Khuri SF, Healey N, MacGregor H, Barnard MR, Szymanski IO, Birjiniuk V, Michelson AD, Gagnon DR, Valeri CR. Comparison of the effects of transfusions of cryopreserved and liquid preserved platelets on hemostasis and blood loss after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1999;117:172-183. 4. Barnard MR, MacGregor H, Ragno G, Pivacek LE, Khuri SF, Michelson AD, Valeri CR. Fresh, liquid preserved, and cryopreserved platelets: adhesive surface receptors and membrane procoagulant activity. Transfusion 1999;39:880-888. 5. Johnson LN, Winter KM, Reid S, Hartkopf-Theis T, Marks DC. Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution. Cryobiology 2011;62:100-106.

Yılmaz S, et al: Cryopreservation of Platelet Concentrates

6. Dargaud Y, Wolberg AS, Luddington R, Regnault V, Spronk H, Baglin T, Lecompte T, Ten Cate H, Negrier C. Evaluation of a standardized protocol for thrombin generation measurement using the calibrated automated thrombogram: an international multicenter study. Thromb Res 2012;130:929-934. 7. Naval Blood Research Laboratory. Standard Operating Procedure: Glycerolization and Deglycerolization of Red Blood Cells in a Closed System Using the Haemonetics ACP215. Plymouth, MA, USA, NBRL. Available at http://www.nbrl.org/SOP/ACP215/ACP215All.html. 8. Klein E, Toch R, Farber S, Freeman G, Fiorentino R. Hemostasis in thrombocytopenic bleeding following infusion of stored, frozen platelets. Blood 1956;11:693-699. 9. Lazarus HM, Kaniecki-Green EA, Warm SE, Aikawa M, Herzig RH. Therapeutic effectiveness of frozen platelet concentrates for transfusion. Blood 1981;57:243-249. 10. Towell BL, Levine SP, Knight WA 3rd, Anderson JL.A comparison of frozen and fresh platelet concentrates in the support of thrombocytopenic patients. Transfusion 1986;26:525-530. 11. Keuren JF, Magdeleyns EJ, Govers-Riemslag JW, Lindhout T, Curvers J. Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation. Br J Haematol 2006;134:307-313. 12. Sinauridze EI, Kireev DA, Popenko NY, Pichugin AV, Panteleev MA, Krymskaya OV, Ataullakhanov FI. Platelet microparticle membranes have 50- to 100fold higher specific procoagulant activity than activated platelets. Thromb Haemost 2007;97:425-434. 13. Johnson L, Coorey CP, Marks DC. The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserine-expressing platelets and platelet microparticles. Transfusion 2014;54:1917-1926. 14. Valeri CR, Ragno G, Khuri S. Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at -80 °C without postthaw processing. Transfusion 2005;45:1890-1898. 15. Ayhan Y, Karakoç E, Pelit NB. Guide to the Preparation, Use and Quality Assurance of Blood Components. Strasbourg, France, Council of Europe Publishing, 2008. 16. Valeri CR, Srey R, Lane JP, Ragno G. Effect of WBC reduction and storage temperature on PLTs frozen with 6 percent DMSO for as long as 3 years. Transfusion 2003;43:1162-1167. 17. Lelkens CC, Koning JG, de Kort B, Floot IB, Noorman F. Experiences with frozen blood products in the Netherlands military. Transfus Apher Sci 2006;34:289-298. 18. Castoldi E, Rosing J. Thrombin generation tests. Thromb Res 2011;127 (Suppl 3):21-25. 19. Hornsey VS, McMillan L, Morrison A, Drummond O, Macgregor IR, Prowse CV. Freezing of buffy coat-derived, leukoreduced platelet concentrates in 6 percent dimethyl sulfoxide. Transfusion 2008;48:2508-2514. 20. Rothwell SW, Maglasang P, Reid TJ, Gorogias M, Krishnamurti C. Correlation of in vivo and in vitro functions of fresh and stored human platelets. Transfusion 2000;40:988-993. 21. Schiffer CA, Aisner J, Dutcher JP, Daly PA, Wiernik PH. A clinical program of platelet cryopreservation. Prog Clin Biol Res 1982;88:165-180. 22. Özsan GH, Pişkin Ö, Demirkan F, Ateş H, Özcan MA, Ündar B. Effect of sialic acid on platelet cryopreservation. Turk J Hematol 2001;18:251-257. 23. Kurt Yüksel M, Arat M, Arslan Ö, Beksaç M, İlhan O. Autologous platelet collection and storage to support thrombocytopenia in a leukemia patient with platelet alloimmunization undergoing chemotherapy. Turk J Hematol 2003;20:233-236. 24. Neuhaus SJ, Wishaw K, Lelkens C. Australian experience with frozen blood products on military operations. Med J Aust 2010;192:203-205. 25. Holley A, Marks DC, Johnson L, Reade MC, Badloe JF, Noorman F. Frozen blood products: clinically effective and potentially ideal for remote Australia. Anaesth Intensive Care 2013;41:10-19.

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0159 Turk J Hematol 2016;33:34-40

Is It Possible to Predict Pulmonary Complications and Mortality in Hematopoietic Stem Cell Transplantation Recipients from Pre-Transplantation Exhaled Nitric Oxide Levels? Hematopoetik Kök Hücre Nakli Uygulanan Hastalarda Nakil Öncesi Ekshale Nitrik Oksit Düzeyi ile Pulmoner Komplikasyonları ve Mortaliteyi Öngörmek Mümkün müdür? Nurdan Köktürk1, Fatma Yıldırım1, Müge Aydoğdu1, Şahika Zeynep Akı2, Zeynep Arzu Yeğin2, Zübeyde Nur Özkurt2, Elif Suyanı2, İpek Kıvılcım Oğuzülgen1, Gülsan Türköz Sucak2 1Gazi University Faculty of Medicine, Department of Pulmonary Medicine, Ankara, Turkey 2Gazi University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract

Öz

Objective: Chemo/radiotherapy-induced free oxygen radicals and reactive oxygen derivatives contribute to the development of early and late transplantation-related pulmonary and extra-pulmonary complications in hematopoietic stem cell transplantation (HSCT) recipients. It has been proposed that an increase in fractional exhaled nitric oxide (FeNO) level indicates oxidative stress and inflammation in the airways. The aim of this prospective study is to evaluate the pretransplantation FeNO levels in HSCT patients and to search for its role in predicting post-transplantation pulmonary complications and mortality. Materials and Methods: HSCT patients were included in the study prospectively between October 2009 and July 2011. Pre-transplantation FeNO levels were measured with a NIOX MINO® device prior to conditioning regimens. All patients were monitored prospectively for post-transplantation pulmonary complications with medical history, physical examination, chest X-ray, and pulmonary function tests. Results: A total of 56 patients (33 autologous, 23 allogeneic) with mean age of 45±13 years were included in the study, among whom 40 (71%) were male. Pre-transplantation FeNO level of the whole study group was found to be 24±13 (mean ± standard deviation) parts per billion (ppb). The FeNO level in allogeneic HSCT recipients was 19±6 ppb while it was 27±15 ppb in autologous HSCT recipients (p=0.042). No significant correlation was found between the pre-transplantation chemotherapy and radiotherapy protocols and baseline FeNO levels (p>0.05). Posttransplantation pulmonary toxicity was identified in 12 (21%) patients and no significant relationship was found between baseline FeNO levels and pulmonary toxicity. The survival rate of the whole study group for 1 year after transplantation was 70%. No significant relationship was identified between baseline FeNO values and survival (FeNO 19±7 ppb in patients who died and 26±15 ppb in the survivors; p=0.114). Conclusion: Pre-transplantation FeNO measurement does not seem to have a role in predicting post-transplantation pulmonary complications and mortality. Keywords: Hematopoietic stem cell transplantation, Exhaled nitric oxide, Pulmonary complications, Mortality

Amaç: Hematopoetik kök hücre nakli (HKHN) hastalarında, nakil öncesi kemoterapi/radyoterapi etkisi ile oluşan serbest oksijen radikalleri ve reaktif oksijen türevleri pek çok nakil ilişkili erken ve geç pulmoner ve nonpulmoner komplikasyonun oluşmasında rol oynamaktadır. Ekshale nitrik oksit (NO) düzeyindeki artışın hava yollarındaki oksidatif stresi ve enflamasyonu yansıttığı ileri sürülmektedir. Bu prospektif çalışmada amacımız HKHN hastalarında nakil öncesi bakılan ekshale NO düzeylerinin nakil sonrası gelişen pulmoner komplikasyonlar ve sağkalım üzerine etkisinin araştırılması idi. Gereç ve Yöntemler: Ekim 2009-Temmuz 2011 tarihleri arasında HKHN uygulanan hastalar prospektif olarak çalışmaya alındılar. Nakil öncesi ekshalasyon havasında NO ölçümleri NIOX MINO® cihazı ile yapıldı. Bütün hastalar transplantasyon sonrası prospektif olarak anemnez, fizik muayene, akciğer filmi ve solunum fonksiyon testleri ile pulmoner komplikasyon açısından takip edildi. Bulgular: Çalışmaya yaş ortalamaları 45±13 yıl olan 40’ı (71%) erkek toplam 56 hasta (33 otolog, 23 allojeneik) dahil edildi. Nakil öncesi ekshale NO düzeyi tüm grupta 24±13 ppb (ortalama ± standart sapma) (median: 22; minimum-maksimum: 5-75) bulundu. Allojeneik HKHN uygulanan hastalarda ekshale NO düzeyi 19±6 ppm; otolog nakil uygulananlarda 27±15 ppm olarak bulundu (p=0,042). Nakil öncesi uygulanan kemoterapi ve radyoterapi rejimleri ile bazal NO düzeyleri arasında anlamlı korelasyon bulunmadı (p>0,05). Nakil sonrası pulmoner toksisite 12 (%21) hastada saptandı. Bazal NO değeri ile pulmoner toksisite arasında anlamlı ilişki saptanmadı. Tüm çalışma grubunda sağkalım oranı %70 olarak bulundu. Nakil öncesi ekshale NO düzeyinin sağ kalım üzerinde etkisinin olmadığı görüldü (eksitus grubunda 19±7 ppm, taburcu grubunda 26±15 ppm, p=0,114). Sonuç: HKHN hastalarında nakil öncesi ekshale NO ölçümü nakil sonrası pulmoner komplikasyon gelişimini ve mortaliteyi öngörmede kullanılabilir gözükmemektedir. Anahtar Sözcükler: Hematopoetik kök hücre nakli, Ekshale nitrik oksit, Pulmoner komplikasyonlar, Mortalite

Address for Correspondence/Yazışma Adresi: Fatma YILDIRIM, M.D., Gazi University Faculty of Medicine, Department of Pulmonary Medicine, Ankara, Turkey Phone : +90 312 202 61 19 E-mail : fatma_bodur2000@yahoo.com

Received/Geliş tarihi: April 15, 2014 Accepted/Kabul tarihi: December 16, 2014

Turk J Hematol 2016;33:34-40

Köktürk N, et al: Exhaled NO in Predicting Post-Transplantation Pulmonary Complications and Mortality in HSCT Patients

Introduction Hematopoietic stem cell transplantation (HSCT) is an important treatment option for several malignant and non-malignant hematological diseases. However, pulmonary complications such as idiopathic pulmonary syndromes, bronchiolitis obliterans organizing pneumonia (BOOP), and infections and graft-versus-host disease (GVHD) developing after bone marrow transplantation have a negative impact on outcome. Chemo/ radiotherapy-induced oxidative stress occurring prior to HSCT is claimed to contribute to development of many early and late transplantation-related pulmonary complications [1,2,3,4,5]. A marker of bronchial inflammation might guide in predicting HSCT-related pulmonary pathology. Nitric oxide (NO) is an endogenous regulator molecule that is synthesized in the body from L-arginine by the enzyme NO synthase. NO in the airways is measured after reaction with ozone by chemiluminescence method. Fractional exhaled NO (FeNO) has been shown to increase as a non-invasive marker of inflammation especially in bronchiectasis, bronchial asthma, tuberculosis, acute exacerbation of chronic obstructive pulmonary disease (COPD), and many other systemic and autoimmune diseases such as systemic lupus erythematosus, systemic sclerosis, and cirrhosis of the liver. Its level increases in parallel with the increase in the level of inflammation and tends to decline in a short period of time after anti-inflammatory therapies [6,7,8]. Our group demonstrated a significant relationship between pre-transplantation diffusion capacity of the lungs for carbon monoxide adjusted for hemoglobin (DLCO adj) levels and development of post-transplantation sinusoidal obstruction syndrome after transplantation in a previous study [9]. We hypothesized whether exhaled FeNO, a simple and non-invasive measurement that has been shown to be increased in many inflammatory conditions, might show pre-transplantation inflammation and endothelial injury and could possibly predict post-transplantation pulmonary complications and mortality.

Materials and Methods Study Subjects After receiving the approval of our institutional review board, 56 patients were enrolled in the study prospectively between October 2009 and July 2011. Inclusion criteria were age above 18 years, being a candidate for allogeneic or autologous stem cell transplantation, and signing an informed consent form to participate in the study and for the use of their medical records. Patients who were younger than 18 years of age; who smoked through the last 6 months; who had asthma, COPD, bronchial hyperreactivity, or upper or lower respiratory tract infection in the last 4 weeks; who were diagnosed to have had activation

of the underlying disease in the last 4 weeks; and who used L-arginine, phosphodiesterase inhibitors, or nitrate were excluded from the study. Graft-Versus-Host Disease Cyclosporine and methotrexate was the standard prophylaxis regimen for GVHD. The assessment and grading of acute and chronic GVHD was primarily based on clinical findings and pulmonary function test (PFT) results. Overall grade of acute GVHD and severity of organ involvement was assessed on a 0-to-4 scale according to the original Seattle criteria. Acute GVHD was considered present if a grade of at least 2 was assigned. Pulmonary toxicities were also graded on a 0-to-4 scale according to these criteria. Chronic GVHD was defined as GVHD occurring 100 days or more after HSCT. GVHD was treated with 1-2 mg/kg/day of prednisolone. Antimicrobial Prophylaxis Antimicrobial prophylaxis, which was performed with acyclovir and fluconazole, was given from the beginning of the conditioning regimen until day +180 post-transplantation for autologous and until discontinuation of the immunosuppressive therapy for allogenic HSCT recipients. All patients received trimethoprim-sulfamethoxazole orally as prophylaxis against Pneumocystis jirovecii beginning from the conditioning regimen until 1 day before stem cell infusion and from neutrophil engraftment to 6 months post-transplantation for autologous and until the discontinuation of the immunosuppressive therapy for allogenic HSCT. Fractional Exhaled Nitric Oxide Measurements FeNO levels were measured with a NIOX MINO® device prior to HSCT. Each subject inhaled NO-free air to the total lung capacity (TLC) and expired as long as possible at 4 different flow rates (50, 100, 150, and 200 mL/s) against a resistance of 10 cm H2O/L/s as previously described. The respiratory tract contains both forms of nitric oxide synthetase enzymes, inducible and endothelial. The FeNO measurement represents the total NO of the respiratory tract [10]. Pulmonary Follow-Up PFTs were routinely performed for detection of underlying ventilatory abnormalities and for assessment of baseline lung function. The following parameters were measured: forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and TLC. In order to determine non-infectious pulmonary complications, serial PFTs of patients were also performed at 3, 6, 9, and 12 months after HSCT. DLCO adj, DLCO per unit alveolar volume (DLCO VA), and DLCO adjusted for alveolar volume (DLCO VA/adj) were also measured. 35

Köktürk N, et al: Exhaled NO in Predicting Post-Transplantation Pulmonary Complications and Mortality in HSCT Patients

Pulmonary Toxicity Definitions Infectious pulmonary complications were defined as pulmonary infections with clinical signs of fever, dyspnea, and crackles, proven by radiologic infiltrates on chest X-ray and in microbiological samples. Microbiological samples were obtained from the data of microbiological analyses, including direct microscopy or culture of sputum or bronchoalveolar lavage fluid. Non-infectious pulmonary complications were defined as the new onset of an obstructive pulmonary defect clinically manifested by dyspnea on exertion, cough, or wheezing. Evidence of obstructive defect is revealed in PFTs. Non-infectious pulmonary complication was accepted as bronchiolitis obliterans syndrome (BOS), because BOS is a clinical term defined by pulmonary function changes rather than histology. Patients were classified as having BOS if they showed FVC % predicted of >80% and FEV1/FVC of <70% [11,12]. Statistical Analysis Statistics were calculated using SPSS 15.0 for Windows. Continuous variables are presented as mean±SD and categorical variables as percentages. Patients with and without pulmonary complications were compared using the chi-square test for categorical variables and the t-test and Mann-Whitney U test for continuous variables. Pearson and Spearman correlation tests were used to determine the relationship between basal FeNO levels and pulmonary complications. Values of p<0.05 were considered statistically significant. The allogeneic and autologous HSCT groups were compared according to their basal FeNO levels as high and low FeNO groups, but there was no cut-off point. Therefore, we divided the groups according to their median basal FeNO group. Pearson and Spearman correlation tests were used to determine the relationships between groups. Kaplan-Meier survival analysis was performed for comparing the progression-free survival (PFS) and overall survival among allogenic and autologous patients with low and high basal FeNO levels. A difference was considered statistically significant when p<0.05 by log-rank.

Results Patient Characteristics Fifty-six patients were included in the study; 33 received autologous and 23 received allogeneic stem cells. Forty (71%) patients were male, and the mean age of the patients was 45±13 years. The median age of the autologous HSCT group was higher than that of the allogenic HSCT group (53 vs. 34, p=0.001). The basal FeNO level of the autologous HSCT group 36

Turk J Hematol 2016;33:34-40

was higher than that of the allogenic HSCT group (26.8±15.4 vs. 18.9±6.2 ppb, p=0.042) (Tables 1 and 2). In the allogeneic and autologous HSCT groups there were no significant relations with regard to age, sex, diagnosis, exitus rate, progression rate, and total and infectious pulmonary complications for low and high basal FeNO levels (p>0.05) (Table 3). Basal PFT values of the groups with pulmonary complications and without pulmonary complications were similar (Table 4). Infectious Pulmonary Toxicities In the allogeneic HSCT group, 3 of the infections were bacterial, 2 were fungal, and 1 was viral. In the autologous HSCT group, the infectious agent was bacterial in one patient and Pneumocystis jirovecii in the other patient. All of the pulmonary infections were with febrile neutropenia and pneumonia. The incidence of invasive pulmonary aspergillosis was 3.7%. Non-Infectious Pulmonary Toxicities In the allogeneic HSCT group, BOS was diagnosed in 2 (9%) patients, and in the autologous HSCT group BOS was detected in 2 (6%) patients. In the autologous HSCT group, one patient had acute GVHD. This patient was a woman and had 2 children. This is a rare complication and it was thought to be the result of maternal antigen activation [13,14]. Survival There were no deaths in the allogeneic and autologous groups within the first 100 days after HSCT. The survival rate of the whole study group for 1 year after transplantation was 70%. The mean value of the pre-transplantation FeNO was 19±7 ppb in the exitus group, while it was 26±15 ppb in the survivors in the total study group. There was no significant relationship between baseline FeNO and survival (p=0.114). The median follow-up of 33 patients with autologous HSCT was 424 days (154-766 days). Survival rate of these patients at the end of the follow-up period was 78.8%. Six patients succumbed to their underlying disease after autologous HSCT, whereas 1 patient died due to transplant-related causes. None of patients died due to only pulmonary complications. Pre-transplantation basal FeNO levels did not have an impact on survival (p>0.05). After autologous HSCT, 4 of the 33 patients (12.1%) developed grade 3-4 pulmonary toxicity. Basal FeNO levels had no impact on pulmonary toxicity (p>0.05). The median follow-up of the 23 allogeneic HSCT recipients was 203 days (10-774 days). The survival rate of these patients at the

Turk J Hematol 2016;33:34-40

Köktürk N, et al: Exhaled NO in Predicting Post-Transplantation Pulmonary Complications and Mortality in HSCT Patients

end of the follow-up period was 56.5%. While 6 patients died due to transplant-related causes, 4 patients died of diseaserelated causes. Pre-transplantation FeNO level was 19±6 ppb in allogeneic HSCT recipients. Pre-transplantation basal FeNO levels had no impact on survival, as well (p>0.05). Seven of the 23 patients (30.4%) developed grade 3-4 pulmonary toxicity after allogeneic HSCT. One patient had grade 1-2 toxicities and

this patient was excluded from analysis. No relationship was found between the basal FeNO levels and the development of pulmonary toxicity (p>0.05). Mean PFS was 246.3±210.1 days in the allogeneic HSCT group; for autologous HSCT patients, mean PFS was 366.7±199.1. There were no significant associations between PFS and basal FeNO levels of low and high basal FeNO group patients in either the

Table 1. Baseline clinical characteristics of patients undergoing hematopoietic stem cell transplantation. Characteristics

Autologous HSCT, n=33, n (%)

Allogeneic HSCT, n=23, n (%)

Total, n=56, n (%)

Sex Male Age, years Median (range)

25 (75.7) 53 (27-64)

15 (65.2) 34 (19-62)

40 (71.0) 45 (19-64)

0.287 0.001

Average CD34 (x106/kg)

4.04±0.92

4.34±0.81

0.214

Diagnosis Multiple myeloma Hodgkin disease Non-Hodgkin lymphoma Acute myeloid leukemia Acute lymphoblastic leukemia SAA/MDS/PNH* Myeloproliferative disease

21 (63.6) 4 (12.1) 7 (21.2) 1 (3.1) -

2 (8.7) 1 (4.3) 8 (34.8) 4 (17.4) 5 (21.7) 3 (13.0)

23 (41.1) 4 (7.2) 8 (14.3) 9 (16.1) 4 (7.2) 5 (8.9) 3 (5.4)

Preparation regimes BEAM Melphalan Busulfan-cyclophosphamide TBI-cyclophosphamide Non-myeloablative regimes Others

9 (27.3) 20 (60.6) 4 (12.1)

8 (34.8) 4 (17.4) 11 (47.8) -

9 (16.1) 20 (35.7) 8 (14.3) 4 (7.2) 11 (19.6) 4 (7.2)

*SAA: Severe aplastic anemia, MDS: myelodysplastic syndrome, PNH: paroxysmal nocturnal hemoglobinuria, HSCT: hematopoietic stem cell transplantation, TBI: total body irradiation.

Table 2. Fractional exhaled nitric oxide levels, pulmonary function test measurements, and pulmonary complications. Characteristics

Autologous HSCT, n=33

Allogenic HSCT, n=23

Basal FeNO, ppb (mean ± SD)

26.8±15.4

18.9±6.2

0.042

Basal FEV1, % (mean ± SD)

94.3±12.1

96.1±15.2

0.613

Basal TLC, L (mean ± SD)

7.9±11.4

5.7±0.9

0.383

DLCO/VA adj, %

86.7±16.9

84.1±10.9

0.535

Exitus, n (%)

7 (21.0)

10 (44.0)

0.069

Progression, n (%)

10 (30.0)

4 (17.0)

0.218

Total pulmonary toxicity, n (%)

6 (18.2)

6 (26.2)

0.054

Infectious pulmonary complications

2 (6.1)

3 (13.1)

0.044

Non-infectious pulmonary complications

4 (12.1)

3 (13.1)

0.058

Acute GVHD, n (%)

1 (3.0)

7 (30.0)

FeNO: Fractional exhaled nitric oxide, FEV1: forced expiratory volume in the first second, TLC: total lung capacity, DLCO/VA adj: DLCO adjusted for alveolar volume, GVHD: graftversus-host disease, HSCT: Hematopoietic stem cell transplantation, SD: standard deviation.

Köktürk N, et al: Exhaled NO in Predicting Post-Transplantation Pulmonary Complications and Mortality in HSCT Patients

allogeneic (p=0.460) or the autologous (p=0.52) HSCT group when examined with Kaplan-Meier analysis and log-rank test.

Discussion We hypothesized that pre-transplantation FeNO could be a surrogate biomarker demonstrating pre-existing pulmonary inflammation and/or pre-transplantation injury caused by the oxidative stress due to previous chemotherapies, radiation, and infections. We therefore measured pre-transplantation FeNO levels and investigated whether they had an impact on posttransplantation pulmonary toxicity and mortality. However, our results failed to demonstrate an impact of FeNO levels on transplantation outcomes. There was neither a correlation between baseline FeNO levels and pre-transplantation induction chemotherapy and radiotherapy protocols nor posttransplantation pulmonary complications and mortality.

Turk J Hematol 2016;33:34-40

Pulmonary complications are major causes of morbidity and mortality after HSCT. Unfortunately, there is currently no established marker that is non-invasive and can predict the pulmonary complications and guide preventive strategies or risk-stratified transplant techniques. A significant correlation has been observed between the levels of serum nitrite/nitrate and host-versus-graft and graft-versus host reactions in rats [15] and humans [4,5] in previous studies. Furthermore, measurement of NO in exhaled air has been proposed for the assessment of individual “pulmonary” risk status amongst various other factors in adults. Haddad et al. [16] studied a mouse model of idiopathic pneumonia syndrome following bone marrow transplantation. They showed that alveolar macrophages, after being stimulated by allogeneic T cells of graft origin, express higher NO synthase levels and thus produce more NO. Cyclophosphamide in

Table 3. Comparison of allogeneic and autologous hematopoietic stem cell transplantation groups according to basal fractional exhaled nitric oxide. Characteristics

Allogeneic HSCT (n=23)

Autologous HSCT (n=33)

Low Basal FeNO, n=12

High Basal FeNO, n=11

Low Basal FeNO, n=17

High Basal FeNO, n=16

Age, years (mean ± SD)

34.6±14.3

36.0±12.4

0.367

50.1±10.6

52.3±6.7

0.871

Sex, male, n (%)

7 (58.0)

8 (72.7)

0.389

14 (82.4)

11 (68.6)

0.438

Diagnosis, n (%)

0.487

0.125

Multiple myeloma

1 (8.3)

1 (9.1)

8 (47.1)

13 (81.3)

Hodgkin disease

2 (11.8)

2 (12.5)

Non-Hodgkin lymphoma

1 (9.1)

6 (35.3)

1 (6.3)

Acute myeloid leukemia

3 (25.0)

5 (45.5)

1 (5.9)

Acute lymphoblastic leukemia

4 (33.3)

SAA/MDS/PNH*

2 (16.7)

3 (27.3)

Myeloproliferative disease

2 (16.7)

1 (9.1)

Exitus, n (%)

5 (41.7)

5 (45.5)

0.593

6 (35.3)

1 (6.3)

0.085

Progression, n (%)

4 (33.3)

0 (0.0)

0.056

5 (29.4)

5 (31.3)

0.603

Total pulmonary toxicity, n (%)

0.389

3 (17.7)

1 (6.3)

0.485

Infectious pulmonary complications, n (%)

0.203

1 (5.9)

1 (6.3)

0.601

Acute GVHD*, n (%)

3 (25.0)

4 (36.4)

0.444

1 (5.9)

0.515

*SAA: Severe aplastic anemia, MDS: myelodysplastic syndrome, PNH: paroxysmal nocturnal hemoglobinuria, GVHD: graft-versus-host disease, HSCT: hematopoietic stem cell transplantation, SD: standard deviation.

Table 4. Characteristics of patients with and without pulmonary toxicities. Characteristics

Pulmonary Toxicities (+), n=12

Pulmonary Toxicities (-), n=44

Basal FeNO, ppb (mean ± SD)

20±10

25±14

0.252

Basal FEV1, %

96±13

95±14

0.837

Basal TLC, L

6±1

7±10

0.551

DLCO/VA adj, %

86±16

86±15

0.980

FeNO: Fractional exhaled nitric oxide, FEV1: forced expiratory volume in the first second, TLC: total lung capacity, DLCO/VA adj: DLCO adjusted for alveolar volume, SD: standard deviation.

Turk J Hematol 2016;33:34-40

Köktürk N, et al: Exhaled NO in Predicting Post-Transplantation Pulmonary Complications and Mortality in HSCT Patients

their model stimulated superoxide production by alveolar macrophages. They concluded that the resultant higher NO and superoxide levels might have led to production of peroxynitrites and nitrotyrosines, which mediated lung damage.

until day 100 of HSCT had higher FeNO levels at day 0 than children without early respiratory pathology, suggesting the role of conditioning chemotherapy rather than the induction regimens prior to transplantation.

Several investigators assessed the usefulness of FeNO for the early detection of chronic GVHD in HSCT recipients. Kanamori et al. [17] published a case series of adults with BOOP after HSCT with FeNO levels above 36 ppb, suggesting that elevated FeNO may be indicative of pulmonary complications after HSCT. Increased FeNO production in their cases also suggests that bone marrow transplantation-related BOOP might be a manifestation of chronic GVHD. They proposed that the FeNO measurement was useful in monitoring inflammatory complications after HSCT.

In previous studies, age was noted as a predictor of FeNO in children [21,22]. However, the influence of age is controversial in healthy adults [23,24,25]. In our study, the autologous HSCT group was older than the allogenic HSCT group, and the basal FeNO level of the autologous HSCT group was higher than that of allogenic HSCT group. We attributed this difference to advanced age.

Two previous reports studied the concentration of exhaled NO in patients after lung transplantation [8,18]. Neurohr et al. studied 166 consecutive lung transplantation recipients [18]. Those patients received no induction therapy and were maintained with standard care on triple immunosuppression with corticosteroids, tacrolimus, and mycophenolate mofetil. A total of 611 FeNO measurements were classified depending on BOS stage at the time of assessment and course during a minimum follow-up of 3 months: stable non-BOS, unstable non-BOS, stable BOS, and unstable BOS. FeNO was significantly increased prior to the unstable course in comparison to the stable counterparts (non-BOS: 28.9±1.2 ppb, 16.4±0.8 ppb, and BOS: 32.5±1.3 ppb, 15.3±0.8 ppb, respectively). Their report demonstrated that elevated levels of FeNO constituted an increased risk for future BOS and preceded further deterioration in transplant recipients. Another report studying FeNO in patients after autologous HSCT is that of Qureshi et al. [19]. In their study, FeNO was significantly increased following autologous peripheral HSCT and correlated with reduction in DLCO. Mean FeNO increased from 12.54±1.32 ppb before HSCT to 21.26±1.94 ppb at 6 weeks and 25.28±3.31 ppb at 24 weeks. The exhaled FeNO was determined before and after the conditioning regimen and showed a significant and progressive increase after the conditioning, suggesting chemotherapy-related pulmonary toxicity. FeNO was also measured prior to the conditioning regimen in the current study in order to determine whether it could be used as a biomarker that defines pre-transplantation pulmonary risk status. However, FeNO unfortunately failed to demonstrate such a predictive value. Fazekas et al. [20] evaluated the correlation of FeNO and pulmonary complications in 30 pediatric HSCT patients. They measured FeNO 10 days before HSCT and at day 0, day 28, and day 60 of HSCT. Similar to our results pretransplantation, FeNO levels were not different in patients with and without post-transplantation pulmonary complications. However, children with any kind of pulmonary complications

The limitation of our study is the lack of consecutive FeNO measurements within the long-term follow-up. By serial measurement of FeNO, its potential as a non-invasive marker for continuous risk stratification of HSCT patients for determining pulmonary complications might be better identified.

Conclusion We conclude that pre-transplantation FeNO levels do not seem to be of value as a marker of post-transplantation pulmonary complications and mortality pre-transplantation. Further studies are required to designate a pre-transplantation surrogate marker of post-transplantation pulmonary toxicity. Ethics Ethics Committee Approval: Gazi University Ethics Committee (Approval number: 43/10.01.2009), Informed Consent: It was taken. Authorship Contributions Concept: Nurdan Köktürk, İpek Kıvılcım Oğuzülgen, Gülsan Türköz Sucak, Design: Nurdan Köktürk, İpek Kıvılcım Oğuzülgen, Gülsan Türköz Sucak, Data Collection or Processing: Fatma Yıldırım, Müge Aydoğdu, Şahika Zeynep Akı, Zeynep Arzu Yeğin, Zübeyde Nur Özkurt, Elif Suyanı, Analysis or Interpretation: Fatma Yıldırım, Müge Aydoğdu, Şahika Zeynep Akı, Zeynep Arzu Yeğin, Zübeyde Nur Özkurt, Elif Suyanı, İpek Kıvılcım Oğuzülgen, Gülsan Türköz Sucak, Literature Search: Fatma Yıldırım, Müge Aydoğdu, Şahika Zeynep Akı, Zeynep Arzu Yeğin, Zübeyde Nur Özkurt, Elif Suyanı, Writing: Nurdan Köktürk, Fatma Yıldırım, Müge 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. Financial Disclosure: The authors declared that this study has received financial support from Glaxosmithkline. 39

Köktürk N, et al: Exhaled NO in Predicting Post-Transplantation Pulmonary Complications and Mortality in HSCT Patients

References 1. Khurshid I, Anderson LC. Non-infectious pulmonary complications after bone marrow transplantation. Postgrad Med J 2002;78:257-262. 2. Sakaida E, Nakaseko C, Harima A, Yokota A, Cho R, Saito Y, Nishimura M. Late-onset noninfectious pulmonary complications after allogeneic stem cell transplantation are significantly associated with chronic graftversus-host disease and with the graft-versus-leukemia effect. Blood 2003;102:4236-4242. 3. Soubani AO, Miller KB, Hassoun PM. Pulmonary complications of bone marrow transplantation. Chest 1996;109:1066-1077. 4. Vora A, Monaghan J, Nuttall P, Crowther D. Cytokine mediated nitric oxide release: a common cytotoxic pathway in host-versus-graft and graftversus-host reactions? Bone Marrow Transplant 1997;20:385-389. 5. Weiss G, Schwaighofer H, Herold M, Nachbaur D, Wachter H, Niederwieser D, Werner ER. Nitric oxide formation as predictive parameter for acute graftversus host-disease after human allogeneic bone marrow transplantation. Transplantation 1995;60:1239-1244. 6. Kharitonov SA, Barnes PJ. Exhaled biomarkers. Chest 2006;130:1541-1546. 7. Smith AD, Cowan JO, Brassett KP, Herbison GP, Taylor DR. Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med 2005;352:2163-2173. 8. Antus B, Csiszer E, Czebe K, Horvath I. Pulmonary infections increase exhaled nitric oxide in lung transplant recipients: a longitudinal study. Clin Transplant 2005;19:377-382. 9. Aydoğdu M, Özyilmaz E, Köktürk N, Yeğin ZA, Özkurt ZN, Aki ZŞ, Sucak G. Is there any relationship between pulmonary function tests and post-transplant complications of allogeneic hematopoetic stem cell transplantation? Minerva Med 2012;103:189-198. 10. American Thoracic Society Workshop. ATS Workshop Proceedings: Exhaled nitric oxide and nitric oxide oxidative metabolism in exhaled breath condensate: Executive summary. Am J Respir Crit Care Med 2006;173:811813. 11. Clark JG, Crawford SW, Madtes DK, Sullivan KM. Obstructive lung disease after allogeneic marrow transplantation. Clinical presentation and course. Ann Intern Med 1989;111:368-376. 12. Colby T, Myers J. Clinical and histologic spectrum of bronchiolitis obliterans, including bronchiolitis obliterans with organizing pneumonia. Semin Respir Med 1992;13:119-133. 13. Wolin MJ, Rigor RL. Acute graft-versus-host disease in a recipient of a twin blood cell transplant. Bone Marrow Transplant 1996;17:125-126.

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14. Fidler C, Klumpp T, Mangan K, Martin M, Sharma M, Emmons R, Lu M, Kropf P. Spontaneous graft versus host disease occurring in a patient with multiple myeloma after autologous stem cell transplant. Am J Hematol 2012;87:219-221. 15. Langrehr JM, Murase N, Markus PM, Cai X, Neuhaus P, Schraut W, Simmons RL, Hoffman RA. Nitric oxide production in host-versus-graft and graftversus-host reactions in the rat. J Clin Invest 1992;90:679-683. 16. Haddad IY, Panoskaltsis-Mortari A, Ingbar DH, Yang S, Milla CE, Blazar BR. High levels of peroxynitrite are generated in the lungs of irradiated mice given cyclophosphamide and allogeneic T cells: a potential mechanism of injury after marrow transplantation. Am J Respir Cell Mol Biol 1999;20:1125-1135. 17. Kanamori H, Fujisawa S, Tsuburai T, Yamaji S, Tomita N, Fujimaki K, Miyashita A, Suzuki S, Ishigatsubo Y. Increased exhaled nitric oxide in bronchiolitis obliterans organizing pneumonia after allogeneic bone marrow transplantation. Transplantation 2002;74:1356-1358. 18. Neurohr C, Huppmann P, Leuschner S, von Wulffen W, Meis T, Leuchte H, Ihle F, Zimmermann G, Baezner C, Hatz R, Winter H, Frey L, Ueberfuhr P, Bittmann I, Behr J, Munich Lung Transplant Group. Usefulness of exhaled nitric oxide to guide risk stratification for bronchiolitis obliterans syndrome after lung transplantation. Am J Transplant 2011;11:129-137. 19. Qureshi MA, Girgis RE, Dandapantula HK, Abrams J, Soubani AO. Increased exhaled nitric oxide following autologous peripheral hematopoietic stemcell transplantation: a potential marker of idiopathic pneumonia syndrome. Chest 2004;125:281-287. 20. Fazekas T, Eickhoff P, Lawitschka A, Knotek B, Pötschger U, Peters C. Exhaled nitric oxide and pulmonary complications after paediatric stem cell transplantation. Eur J Pediatr 2012;171:1095-1101. 21. Kovesi T, Kulka R, Dales R. Exhaled nitric oxide concentration is affected by age, height, and race in healthy 9- to 12-year-old children. Chest 2008;133:169-175. 22. Buchvald F, Baraldi E, Carraro S, Gaston B, De Jongste J, Pijnenburg MW, Silkoff PE, Bisgaard H. Measurements of exhaled nitric oxide in healthy subjects age 4 to 17 years. J Allergy Clin Immunol 2005;115:1130-1136. 23. Travers J, Marsh S, Aldington S, Williams M, Shirtcliffe P, Pritchard A, Weatherall M, Beasley R. Reference ranges for exhaled nitric oxide derived from a random community survey of adults. Am J Respir Crit Care Med 2007;176:238-242. 24. Olivieri M, Talamini G, Corradi M, Perbellini L, Mutti A, Tantucci C, Malerba M. Reference values for exhaled nitric oxide (reveno) study. Respir Res 2006;7:94. 25. Liu HC, Hsu JY, Cheng YW, Chou MC. Exhaled nitric oxide in a Taiwanese population: age and lung function as predicting factors. J Formos Med Assoc 2009;108:772-777.

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0010 Turk J Hematol 2016;33:41-47

A Quasi-Experimental Study Analyzing the Effectiveness of Portable High-Efficiency Particulate Absorption Filters in Preventing Infections in Hematology Patients during Construction İnşaat Esnasında Hematoloji Hastalarında Gelişen Enfeksiyonların Önlenmesinde Portabl Yüksek Etkinlikte Partikül Emici Filtrelerin Etkinliğinin Değerlendirildiği Bir Öncesi Sonrası Çalışması Mehmet Özen1, Gülden Yılmaz2, Belgin Coşkun2, Pervin Topçuoğlu1, Bengi Öztürk3, Mehmet Gündüz1, Erden Atilla1, Önder Arslan1, Muhit Özcan1, Taner Demirer1, Osman İlhan1, Nahide Konuk1, İsmail Balık2, Günhan Gürman1, Hamdi Akan1 1Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey 2Ankara University Faculty of Medicine, Department of Infectious Diseases, Ankara, Turkey 3Ankara University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey

Abstract

Öz

Objective: The increased risk of infection for patients caused by construction and renovation near hematology inpatient clinics is a major concern. The use of high-efficiency particulate absorption (HEPA) filters can reduce the risk of infection. However, there is no standard protocol indicating the use of HEPA filters for patients with hematological malignancies, except for those who have undergone allogeneic hematopoietic stem cell transplantation. This quasi-experimental study was designed to measure the efficacy of HEPA filters in preventing infections during construction. Materials and Methods: Portable HEPA filters were placed in the rooms of patients undergoing treatment for hematological malignancies because of large-scale construction taking place near the hematology clinic. The rates of infection during the 6 months before and after the installation of the portable HEPA filters were compared. A total of 413 patients were treated during this 1-year period. Results: There were no significant differences in the antifungal prophylaxis and treatment regimens between the groups. The rates of infections, clinically documented infections, and invasive fungal infections decreased in all of the patients following the installation of the HEPA filters. When analyzed separately, the rates of invasive fungal infections were similar before and after the installation of HEPA filters in patients who had no neutropenia or long neutropenia duration. HEPA filters were significantly protective against infection when installed in the rooms of patients with acute lymphocytic leukemia, patients who were undergoing consolidation treatment, and patients who were neutropenic for 1-14 days. Conclusion: Despite the advent of construction and the summer season, during which environmental Aspergillus contamination is more prevalent, no patient or patient subgroup experienced an increase in fungal infections following the installation of HEPA filters. The protective effect of HEPA filters against infection was more pronounced in patients with acute lymphocytic leukemia, patients undergoing consolidation therapy, and patients with moderate neutropenia. Keywords: HEPA filter, Infection, Invasive fungal infection

Amaç: Hematoloji yatan hasta kliniklerinin yakınındaki inşaat ve tamiratların neden olduğu, hastalardaki artmış enfeksiyon riski büyük bir endişe kaynağıdır. Yüksek etkinlikte partikül emici (YEPE) filtrelerin kullanımı enfeksiyon riskini azaltabilir. Bununla birlikte allojenik hematopoetik kök hücre nakli yapılanlar dışında, YEPE filtrelerin hematolojik malignite hastalarında kullanımı konusunda standart bir öneri yoktur. Bu öncesi sonrası çalışma inşaat esnasında enfeksiyonların engellenmesinde YEPE filtrelerin etkinliğini ölçmek için kurgulandı. Gereç ve Yöntemler: Hematoloji kliniğinin yanında meydana gelen geniş çaplı bir inşaat nedeniyle hematolojik tümörlerin tedavisinin yapıldığı hasta odalarına portabl YEPE filtreler yerleştirildi. Portabl YEPE filtrelerin yerleştirilmesinden önceki ve sonraki 6 aydaki enfeksiyon oranları karşılaştırıldı. Bu 1 yıllık dönemde toplam 413 hasta tedavi edildi. Bulgular: Gruplar arasında antifungal profilaksi ve tedavi rejimleri açısından farklılık yoktu. YEPE filtrelerin yerleştirilmesini takiben tüm hastalarda enfeksiyon, klinik gösterilmiş enfeksiyon ve invaziv fungal enfeksiyon oranları azaldı. Ayrı ayrı analiz edildiğinde ise YEPE filtre yerleştirilmesi sonrası ve öncesi invaziv fungal enfeksiyon oranları nötropenisi olmayan veya uzun nötropeni süresi olan hastalarda benzerdi. Akut lenfoblastik lösemi hastalarının, konsolidasyon tedavisi alan hastaların ve 1 ile 14 gün arasında nötropenik kalan hastaların odalarına yerleştirilen YEPE filtreler enfeksiyonlara karşı belirgin bir şekilde koruyucuydu. Sonuç: Çevresel Aspergillus kontaminasyonunun daha sık olduğu yaz sezonuna ve ortaya çıkan inşaata rağmen, YEPE filtrelerin yerleştirilmesi sonrası dönemde ne hastalarda ne de hasta alt gruplarında fungal enfeksiyonlarda artış gözlenmedi. YEPE filtrelerin enfeksiyonlara karşı koruyucu etkisi akut lenfoblastik lösemi hastalarında, konsolidasyon tedavisi alan hastalarda ve orta derecede nötropenisi olanlarda daha belirgindi. Anahtar Sözcükler: YEPE filtre, Enfeksiyon, İnvaziv fungal enfeksiyon

Address for Correspondence/Yazışma Adresi: Mehmet ÖZEN, M.D., Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone : +90 312 595 70 99 E-mail : kanbilimci@gmail.com

Received/Geliş tarihi: January 10, 2014 Accepted/Kabul tarihi: October 17, 2014

Özen M, et al: High-Efficiency Particulate Absorption and Invasive Fungal Infections

Introduction Infectious diseases are the most common cause of mortality and morbidity in hematology inpatient clinics. The use of high-efficiency particulate absorption (HEPA) filters in bone marrow transplantation units reduces the rates of infection and transplant-related mortality in allogeneic hematopoietic stem cell transplantation (AlloHSCT) recipients [1]. The use of HEPA systems is recommended because of the high infection rates in these units [2]. Although the rates of infection are high in all neutropenic patients [3], there are no recommendations regarding the use of HEPA filters to prevent infections in nonAlloHSCT hematologic patients. Construction near hospitals is an important contributing factor in the development of invasive fungal infections (IFIs) in patients due to environmental fungal contamination, and HEPA filters are effective in preventing IFIs [4,5,6]. The use of HEPA filters can also prevent bacterial infections [7,8,9]. To our knowledge, ours is the first study to compare the ability of HEPA filters to prevent infections in various patient groups.

Materials and Methods Demolition and construction occurring near a 6-story hospital located 10 m from the hematology ward at our university provided us with the opportunity to conduct a non-randomized retrospective quasi-experimental study to evaluate the ability of HEPA filters to prevent infections in patients being treated for hematologic malignancies during the construction. All of the patients in the hematopoietic stem cell transplantation unit were excluded from the study because that unit already had HEPA filters installed. Portable H14-type HEPA filters (99.9995% effective; Uvion Air Aseptizör, Teknomar, Turkey) were installed in all the patients’ rooms on 5 May 2011. We compared the infection rates in the 6-month periods before and after the installation of the HEPA filters to evaluate whether the filters prevented infections. A total of 413 patients were treated in our hematology ward during this 1-year period. All patients were admitted to private rooms, and preventative measures against infection were taken with all patients. The 210 patients treated between 5 November 2010 and 4 May 2011 served as the control group and the 203 patients treated between 5 May 2011 and 26 October 2011 served as the intervention group. The patients in the control group were housed in rooms without HEPA filters, and the patients in the intervention group were housed in rooms with HEPA filters. We excluded patients from the study if they acquired IFIs in other wards prior to being admitted to our inpatient hematology department. We also randomly measured the level of airborne particulates in patients’ rooms to evaluate HEPA filter efficiency. The levels of particulates in the patients’ rooms were within acceptable limits. 42

Turk J Hematol 2016;33:41-47

Data were assembled from patients’ files, digital records, and records of infection from the control team. Definitions of Infections Infections were classified as microbiologically documented infections, clinically documented infections, and fevers of unknown origin (FUOs). Microbiologically documented infections were defined microbiologically in cultures either as bloodstream infections or infected foci [10,11]. Clinically documented infections in patients were defined by the presence of clinical signs of infections in the absence of positive cultures for pathogenic microorganisms [10,11]. FUOs were defined as isolated fevers with no clinical or microbiological signs of infection [10,11]. IFIs were defined according to EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria [12]. Although candidemia results were given, Candida-associated yeast infections were not considered as IFIs in this study because HEPA filters are not effective in preventing yeast infections [13]. Therefore, in our study, all cases of IFIs were mold-related. Although severe neutropenia is classically defined as neutropenia persisting for more than 7 or 10 days, many experts extend this to 14 days for IFIs [2,14]. Thus, we defined severe neutropenia as neutropenia that lasted for more than 14 days for IFIs. Statistical Analysis Numeric variables are given as medians or mean and range. The non-parametric Mann-Whitney U test was used to compare nominal variables. The categorized variables were compared using the chi-square or Fisher exact test. Data were analyzed using SPSS 16.0 for Windows and p-values of less than 0.05 were considered to be significant.

Results The control and intervention groups were similar in sex distribution, underlying hematological disease, history of fungal infections, presence of central catheter, granulocyte colonystimulating factor usage, minimum albumin levels, and severity of neutropenia (Table 1). However, patients in the intervention group tended to have a higher mean age (p=0.053). Mean hospitalization durations were longer in the control group than in the intervention group at 20 days and 15 days, respectively (p<0.05) (Table 2). The intervention group had lower incidences of IFIs, clinically documented infections, clinically documented pneumonia, and overall infections than the control group (Table 2). The rates of FUOs, all pneumonias, bacterial

Özen M, et al: High-Efficiency Particulate Absorption and Invasive Fungal Infections

Turk J Hematol 2016;33:41-47

pneumonias, fungal infections, probable IFIs, possible IFIs, microbiologically documented infections, gram-positive and gram-negative bacterial infections, candidemia, and infectionrelated mortality were similar between the groups (Table 2). The most common bacterial infections were Streptococcus in 8 patients, Escherichia coli in 6 patients, Pseudomonas in 4 patients, Staphylococcus in 4 patients, Klebsiella in 3 patients, and Salmonella, Pneumococcus, and Acinetobacter baumannii in 1 patient each in the control group, and Streptococcus in 4 patients, E. coli in 11 patents, Pseudomonas in 2 patients, Klebsiella in 4 patients, Pneumococcus in 2 patients, and Enterococcus and Staphylococcus in 1 patient each in the intervention group. HEPA filters seemed to be effective in preventing IFIs in all neutropenic patients during construction. Careful evaluation of the data revealed that HEPA filters were more effective in preventing infections in particular subgroups of hematology patients during construction. When the subgroups were

analyzed separately, the IFI-preventive effect of HEPA filters was most marked in acute lymphoid leukemia patients, especially during consolidation treatment and moderate neutropenia (114 days) (Table 3). HEPA filters did not appear to reduce the rates of IFIs in non-neutropenic patients or in patients with >14 days of neutropenia, patients undergoing induction treatment, or patients with either acute myeloid leukemia or non-acute leukemia (multiple myeloma, solid tumors, lymphoma, etc.) (Table 3). We also evaluated the patients’ hospital bills per group. The total cost of the HEPA filters, including costs of installment and service over the 6-month intervention period, was 50,975 Turkish lira (TL; equivalent to 29,809 US$ or 21,328 €) [15]. We found that all costs as given in dollars and euros per patient were decreased after HEPA filter installation, but costs as expressed in TL were not significantly different between these groups (Table 4).

Table 1. Patient characteristics.

Control Group n=210 (%)

Intervention Group n=203 (%)

p-value

Median age, years (range)

47 (18-87)

53 (20-84)

0.053

Sex, M/F (n)

137/73

118/85

0.1

Diagnosis AML ALL Bone marrow failure (SAA/PNH/MDS) Lymphoma (NHL/HL) Plasma cell disease CMPD/CML Other (solid/benign)

60 (28.6) 31 (14.8) 15 (7.1) 53 (25.2) 31 (14.8) 6 (2.9) 14 (6.7)

53 (26.1) 29 (14.3) 20 (9.9) 51 (25.1) 35 (17.2) 6 (3.0) 9 (4.4)

0.6 0.9 0.3 1 0.5 1 0.3

Treatment Induction Consolidation Other chemotherapy No chemotherapy

36 (17.1) 40 (19.0) 80 (38.1) 54 (25.7)

27 (13.3) 46 (22.7) 77 (37.9) 53 (26.1)

0.3 0.4 1.0 0.9

Catheter

38 (18.1)

36 (17.7)

0.9

Neutropenia, present >15 days neutropenia 1-14 days neutropenia

116 (54.7) 69 (59.5) 47 (40.5)

96 (45.3) 60 (62.5) 36 (37.5)

0.1 0.5 0.2

Median neutropenia duration, days (range)

11.5 (1-72)

12 (1-50)

0.1

G-CSF usage

58 (27.6)

51 (25.1)

0.6

Previous fungal infection

12 (5.7)

10 (4.9)

0.7

Antibacterial prophylaxis

5 (2.3)

4 (2.0)

1.0

Antifungal prophylaxis

18 (8.5)

12 (5.9)

0.3

Minimum albumin level

2.85±0.617

2.84±0.623

1.0

M: Male, F: female, AML: acute myeloid leukemia, ALL: acute lymphoid leukemia, SAA: severe aplastic anemia, PNH: paroxysmal nocturnal hemoglobinuria, MDS: myelodysplastic syndrome, NHL: non-Hodgkin lymphoma, HL: Hodgkin lymphoma, CMPD: chronic myeloproliferative disease, CML: chronic myeloid leukemia, CLL: chronic lymphocytic leukemia, G-CSF: granulocyte colony-stimulating factor.

Ă&#x2013;zen M, et al: High-Efficiency Particulate Absorption and Invasive Fungal Infections

Turk J Hematol 2016;33:41-47

Table 2. The effect of high-efficiency particulate absorption filters on infection rates. Control Group n (%)

Intervention Group n (%)

p-value

Hospitalization days, median (range)

20 (2-130)

15 (1-130)

0.02*

Infections

121 (57.6)

94 (46.3)

0.02*

FUOs

40 (19)

45 (22.2)

0.4

Clinically documented infections

49 (23.3)

19 (9.4)

<0.001*

Microbiologically documented infections (bacteria and fungi)

32 (15.2)

30 (14.8)

0.9

Pneumonia

39 (18.6)

30 (14.8)

0.3

Clinically documented pneumonia

27 (12.9)

13 (6.4)

0.03*

Bacterial pneumonia

21 (10)

21 (10.3)

0.9

All fungal infections

27 (12.9)

18 (8.9)

0.2

IFIs

20 (9.5)

9 (4.4)

0.04*

Probable IFIs

7 (3.3)

3 (1.5)

0.3

Possible IFIs

13 (6.2)

6 (3)

0.1

Bacterial infections (according to culture)

28 (13.3)

25 (12.3)

0.8

Gram-positive infections

18 (8.6)

15 (7.4)

0.7

Gram-negative infections

12 (5.7)

10 (4.9)

0.7

Candidemia

7 (3.3)

9 (4.4)

0.6

Infection-related mortality

17 (8.1)

10 (4.9)

0.2

FUO: Fever of unknown origin, IFI: invasive fungal infection. *: Statistically significant.

Table 3. The effect of high-efficiency particulate absorption filters on invasive fungal infections. Control Group n (%)

Intervention Group n (%)

p-value

IFIs in all neutropenic patients

20 (17.2)

7 (7.3)

0.03*

IFIs in non-neutropenic patients

0 (0)

2 (1.9)

0.5

IFIs in neutropenic patients, >14 days

13 (27.7)

7 (19.4)

0.4

IFIs in neutropenic patients, 1-14 days

7 (10.1)

0 (0)

0.02*

IFIs in AML

9 (15)

6 (11.3)

0.6

IFIs in ALL

9 (29)

1 (3.4)

0.01*

IFIs in AML induction

7 (25.9)

5 (23.8)

0.9

IFIs in AML consolidation

2 (7.7)

0 (0)

0.5

IFIs in ALL induction

2 (22.2)

1 (16.7)

1.0

IFIs in ALL consolidation

6 (42.9)

0 (0)

0.002*

IFIs in all induction

9 (25)

6 (22.2)

0.8

IFIs in all consolidation

8 (20)

0 (0)

0.001*

IFIs in non-AML non-ALL

2 (1.7)

1.0

AML: Acute myeloid leukemia, ALL: acute lymphoid leukemia, IFIs: invasive fungal infections. *: Statistically significant.

Turk Turk J Hematol 2016;33:41-47

Özen M, et al: High-Efficiency Particulate Absorption and Invasive Fungal Infections

Table 4. Financial analysis of the patients. Control Group

Intervention Group

p-value

Hospital bill per patient, TL, mean ± SD

9688±13,454

9345±15,372

0.11

All costs including HEPA per patient, TL, mean ± SD

9688±13,454

9596±15,540

0.22

All costs including HEPA per patient, $, mean ± SD

6590±9152

5611±9087

0.01*

All costs including HEPA per patient, €, mean ± SD

4548±6316

4014±6502

0.02*

TL: Turkish lira, SD: standard deviation, $: US dollar, €: Euro, HEPA: high-efficiency particulate absorption. *: Statistically significant.

Discussion Hospital construction is a significant source of serious hospitalacquired infections due to aspergillosis, with nosocomial aspergillosis outbreaks occurring primarily among neutropenic patients [16]. The period before the construction, when the HEPA filters had not yet been installed, was winter and spring, while the installed HEPA filters were used in summer and autumn. The use of HEPA filters was associated with a lack of increase in IFI rates despite both the construction and the summer months [17]. We conclude that HEPA filter installation in hematology wards is a safe option to prevent IFIs during construction. The use of HEPA filters most likely prevented the rates of infectionrelated mortality from increasing in patients treated during construction. Clinically documented infections originate from either IFIs or bacterial infections. The pulmonary system is the origin of most infections, but other systems may also be involved [18,19]. The most common bacterial agents observed in our study were similar to those reported to be most common in the literature [20]. HEPA filters prevented both IFIs and bacterial infections [21]. In our study, the rates of clinically documented pneumonia were also reduced, which may explain why the use of HEPA filters decreased the rates of clinically documented infections. The literature contains few reports about the effects of HEPA filters on patients with hematological malignancies and either clinically documented infections or clinically documented pneumonia; to our knowledge, our study is the first to report this clinical finding. During construction, the IFI rates did not increase in the subsets of patients who were at higher risk of IFIs, including those with acute myeloid leukemia, those undergoing remission-induction therapy, and/or those with neutropenia that lasted >14 days. This outcome was most likely due to the ability of HEPA filters to prevent IFIs. However, HEPA filters appeared to be most effective in preventing infections in patients with moderate duration of neutropenia, patients with acute lymphoid leukemia, and patients undergoing consolidation therapy. These groups are reported in the literature to have lower rates of IFIs [22]. This might result from a balance between the protective effects of the HEPA filters and the deleterious effects of neutropenia

duration on developing IFIs. To our knowledge, this finding has not yet been reported in the literature. In multi-center studies, the effect of HEPA filters in preventing infections may be a confounding variable, and HEPA filter effects should be taken into account. HEPA filters can reduce the exposure to Aspergillus from unfiltered air and contaminated dust by reducing the number of Aspergillus organisms in the air [23]. Aspergillus has been cultured from numerous hospital sources including horizontal surfaces, food, water supplies, and ventilation systems [24]. HEPA filters may not completely prevent IFI in high-risk patients [16]. As a result, antifungal prophylaxis should be considered as another preventive option in high-risk patient groups [6,25,26]. The effect of season on IFI is controversial. It has been reported that aspergillosis infections are most commonly seen in the summer [7]. However, one study found no seasonal effect on the rate of IFIs [27]. In our study, we were not able to evaluate seasonal effects on the incidence of IFIs because of the study design. However, Bénet et al. reported that the incidence of IFIs in hematological patients during the summer months in the absence of HEPA filters was 13.2% (9/68) [28]. We observed that the IFI incidence during the winter months in the absence of HEPA filters was 9.5% (20/210). Our study population and that of Bénet et al. [28] were similar. Thus, we compared the findings of our study with those of Bénet et al. [28] to evaluate seasonal effect on the rate of IFIs. There was no significant difference between the summer and winter IFI rates in these studies (p=0.4). In other words, the protective effects of HEPA filters against infections were independent of season. The duration of hospitalization was longer before the installation of HEPA filters than after installation. Lower incidences of infection in the intervention group during construction may have led to shorter hospital stays. Adal et al. reported that HEPA filters may be cost-effective [29]. We did not evaluate the cost-effectiveness of HEPA filters in our patients. However, we found that HEPA filter installation lowered all costs per patient in euro and dollar currencies, although costs as expressed in TL were not significantly different between these groups, probably due to the changes in exchange rates. Thus, we propose that HEPA filters may be a cost-effective option for 45

Özen M, et al: High-Efficiency Particulate Absorption and Invasive Fungal Infections

preventing infections in hematology patients, especially when construction is taking place nearby. Our study had several limitations, including its retrospective nature, a small sample size, the fact that it was conducted at a single center, and the lack of cost-benefit analysis. In addition, our confirmed IFIs rates were low, because they were not evaluated by pathology. Some studies found hypoalbuminemia to be a risk factor for Aspergillus infections [30,31]. Therefore, we evaluated minimum albumin levels in patients treated in HEPA and non-HEPA rooms. However, we did not observe any differences in albumin levels between these 2 patient groups.

Conclusion In conclusion, after the implementation of infection control measures during construction, we found that keeping immunocompromised patients in single-bed rooms with air filtration through a HEPA system could significantly reduce IFIs in low-risk patient groups. However, additional protective measurements such as antifungal prophylaxis are required to reduce the rate of infection in high-risk patient groups. Footnote The preliminary data included in this study were previously presented at the 2013 American Society of Clinical Oncology (ASCO) congress: Gurman G, Ozen M, Yilmaz G, Coskun B, Topcuoglu P, Ozturk B, Ozcan M, Arslan O, Ilhan O, Beksac M, Ismail B, Akan H. Hepa systems in hematology clinic to ameliorate the increased fungal infection risk owing to environmental changing. J Clin Oncol 2013;31 (Suppl; abstr e18009). Ethics Ethics Committee Approval: Retrospective study, Informed Consent: It was taken. Authorship Contributions Concept: Günhan Gürman, Hamdi Akan, Design: Mehmet Özen, Gülden Yılmaz, Belgin Coşkun, Data Collection or Processing: Mehmet Özen, Gülden Yılmaz, Belgin Coşkun, Pervin Topçuoğlu, Bengi Öztürk, Mehmet Gündüz, Erden Atilla, Analysis or Interpretation: Mehmet Özen, Literature Search: Önder Arslan, Muhit Özcan, Taner Demirer, Osman İlhan, Nahide Konuk, İsmail Balık, Writing: Mehmet Özen, Gülden Yılmaz, Belgin Coşkun, Hamdi Akan. 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. 46

Turk J Hematol 2016;33:41-47

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Jiménez JL, Chacón EM, Rubio MO, Oteyza JP, Ramírez GR, Contreras RR, Barbero AR, Tarrats MR, Félix DR, Godoy PS, Salinas AS, Alonso MA, Torroba Jde L, Ferreiras DV, López LV, García JM, Perea JR, Moreno RC, Cáncer RC, Abete JF, Rodríguez JG, Gómez JG, Pedrosa EG, Baranda JM, García FJ, Camps IR, Lleti MS, Cisneros Jde L; Spanish Society of Chemotherapy. Recommendations for the treatment of invasive fungal infection caused by filamentous fungi in the hematological patient. Rev Esp Quimioter 2011;24:263-270. 15. http://paracevirici.com/doviz-arsiv/merkez-bankasi/tcmb-gecmis-tarihlidoviz-kurlari-cevirici.php (accessed 29 September 2014). 16. Haiduven D. Nosocomial aspergillosis and building construction. Med Mycol 2009;47:210-216. 17. Menegueti MG, Ferreira LR, Silva MF, Silva AS, Bellissimo-Rodrigues F. Assessment of microbiological air quality in hemato-oncology units and its relationship with the occurrence of invasive fungal infections: an integrative review. Rev Soc Bras Med Trop 2013;46:391-396. 18. Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F, Denning DW, Donnelly JP, Edwards JE, Erjavec Z, Fiere D, Lortholary O, Maertens J, Meis JF, Patterson TF, Ritter J, Selleslag D, Shah PM, Stevens DA, Walsh TJ; Invasive Fungal Infections Cooperative Group of the European Organization for Research and Treatment of Cancer; Mycoses Study Group of the National Institute of Allergy and Infectious Diseases. Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin Infect Dis 2002;34:7-14. 19. Young RC, Bennett JE, Vogel CL, Carbone PP, DeVita VT. Aspergillosis. The spectrum of the disease in 98 patients. Medicine (Baltimore) 1970;49:147-173. 20. Digiorgio MJ, Fatica C, Oden M, Bolwell B, Sekeres M, Kalaycio M, Akins P, Shane C, Bako J, Gordon SM, Fraser TG. Development of a modified surveillance definition of central line-associated bloodstream infections for patients with hematologic malignancies. Infect Control Hosp Epidemiol 2012;33:865-868. 21. Furuhashi M. Efficiency of bacterial filtration in various commercial air filters for hospital air conditioning. Bull Tokyo Med Dent Univ 1978;25:147-155. 22. Camps IR. Risk factors for invasive fungal infections in haematopoietic stem cell transplantation. Int J Antimicrob Agents 2008;32(Suppl 2):119-123. 23. Sherertz RJ, Belani A, Kramer BS, Elfenbein GJ, Weiner RS, Sullivan ML, Thomas RG, Samsa GP. Impact of air filtration on nosocomial Aspergillus

infections. Unique risk of bone marrow transplant recipients. Am J Med 1987;83:709-718. 24. Anaissie EJ, Stratton SL, Dignani MC, Lee CK, Summerbell RC, Rex JH, Monson TP, Walsh TJ. Pathogenic molds (including Aspergillus species) in hospital water distribution systems: a 3-year prospective study and clinical implications for patients with hematologic malignancies. Blood 2003;101:2542-2546. 25. Vehreschild JJ, Böhme A, Buchheidt D, Arenz D, Harnischmacher U, Heussel CP, Ullmann AJ, Mousset S, Hummel M, Frommolt P, Wassmer G, Drzisga I, Cornely OA. A double-blind trial on prophylactic voriconazole (VRC) or placebo during induction chemotherapy for acute myelogenous leukaemia (AML). J Infect 2007;55:445-449. 26. Racil Z, Toskova M, Kocmanova I, Buresova L, Kouba M, Drgona L, Masarova L, Guman T, Tothova E, Gabzdilova J, Forsterova K, Haber J, Ziakova B, Bojtarova E, Rolencova M, Timilsina S, Cetkovsky P, Mayer J. Micafungin as empirical antifungal therapy in hematological patients: a retrospective, multicenter study in the Czech and Slovak Republics. Leuk Lymphoma 2013;54:1042-1047. 27. Hospenthal DR, Kwon-Chung KJ, Bennett JE. Concentrations of airborne Aspergillus compared to the incidence of invasive aspergillosis: lack of correlation. Med Mycol 1998;36:165-168. 28. Bénet T, Nicolle MC, Thiebaut A, Piens MA, Nicolini FE, Thomas X, Picot S, Michallet M, Vanhems P. Reduction of invasive aspergillosis incidence among immunocompromised patients after control of environmental exposure. Clin Infect Dis 2007;45:682-686. 29. Adal KA, Anglim AM, Palumbo CL, Titus MG, Coyner BJ, Farr BM. The use of high-efficiency particulate air-filter respirators to protect hospital workers from tuberculosis. A cost-effectiveness analysis. N Engl J Med 1994;331:169-173. 30. Baddley JW, Andes DR, Marr KA, Kontoyiannis DP, Alexander BD, Kauffman CA, Oster RA, Anaissie EJ, Walsh TJ, Schuster MG, Wingard JR, Patterson TF, Ito JI, Williams OD, Chiller T, Pappas PG. Factors associated with mortality in transplant patients with invasive aspergillosis. Clin Infect Dis 2010;50:1559-1567. 31. Perfect JR, Cox GM, Lee JY, Kauffman CA, de Repentigny L, Chapman SW, Morrison VA, Pappas P, Hiemenz JW, Stevens DA; Mycoses Study Group. The impact of culture isolation of Aspergillus species: a hospital-based survey of aspergillosis. Clin Infect Dis 200;33:1824-1833.

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0029 Turk J Hematol 2016;33:48-52

Comparison of the Efficiencies of Buffers Containing Ankaferd and Chitosan on Hemostasis in an Experimental Rat Model with Femoral Artery Bleeding Femoral Arter Kanamasında Ankaferd ve Chitosan İçeren Tamponların Hemostaz Üzerine Etkilerinin Deneysel Fare Modelinde Karşılaştırılması Serkan Abacıoğlu1, Kemal Aydın2, Fatih Büyükcam2, Ural Kaya3, Bahattin Işık4, Muhammed Evvah Karakılıç5 1Osmaniye State Hospital, Clinic of Emergency, Osmaniye, Turkey 2Dışkapı Yıldırım Beyazıt Training and Research Hospital, Clinic of Emergency, Ankara, Turkey 3Bülent Ecevit University Faculty of Medicine, Department of Emergency, Zonguldak, Turkey 4Keçiören Training and Research Hospital, Clinic of Emergency, Ankara, Turkey 5Ankara Numune Training and Research Hospital, Clinic of Emergency, Ankara, Turkey

Abstract

Öz

Objective: In the first assessment of trauma patients with major vascular injuries, we need effective and rapid-acting homeostatic materials. In this study we compare the efficiencies of Ankaferd Blood Stopper® and a chitosan linear polymer (Celox®) in an experimental rat model with femoral artery bleeding.

Amaç: Büyük damar yaralanması olan travma hastalarının ilk müdahalesinde etkin ve hızlı etkili kanama durdurucu malzemelere ihtiyaç duyulmaktadır. Biz bu çalışmamızda, femoral arter kanamalı deneysel fare modelinde Ankaferd Blood Stopper® ve chitosan lineer polymerin (Celox®) etkinliklerini karşılaştırdık.

Materials and Methods: Thirty male Wistar albino rats weighing 200250 g were divided into 3 groups: control, Ankaferd, and chitosan. The femoral artery and vein were visualized and bleeding was started by an incision. The bleeding time was recorded and categorized as ‘bleeding stopped at the second minute’, ‘bleeding stopped at the fourth minute’, and ‘unsuccessful’ if bleeding continued after the fourth minute.

Gereç ve Yöntem: Ağırlıkları 200-250 gram olan 30 erkek Wistar albino fare, kontrol, Ankaferd ve chitosan olmak üzere üç gruba ayrıldı. Femoral arter ve ven görünür hale getirildi ve bir kesi ile kanama başlatıldı. Kanama zamanı kaydedildi ve ‘kanaması ilk iki dakikada duranlar’, ‘kanaması ilk dört dakikada duranlar’ şeklinde ve eğer dördüncü dakikadan sonra kanama halen devam ediyorsa ‘başarısız’ olarak kaydedildi.

Results: In the control group, 60% of the bleeding did not stop. In the first 4 min in the Ankaferd group, the bleeding stopped in all rats; only in 1 of the rats in the chitosan group did the bleeding not stop. In stopping the bleeding in the first 4 min, Ankaferd was similar to chitosan but better than the control group; the chitosan group was similar to the control, but the p-value was close to significance.

Bulgular: Kontrol grubunda, kanamaların %60’ı durmadı. İlk dört dakikada Ankaferd grubunda tüm farelerde kanama durdu; chitosan grubunda sadece bir farede kanama durmadı. İlk dört dakikada kanamayı durdurmada Ankaferd, chitosan ile benzerdi fakat kontrol grubundan daha iyiydi; ayrıca chitosan kontrol grubuna benzerdi fakat p değeri anlamlılığa yakındı.

Conclusion: For major arterial bleeding, the main treatment is surgical bleeding control, but outside of the hospital we can use buffers containing Ankaferd and chitosan on the bleeding region. The results of this study should be supported with larger studies. Furthermore, in our study, healthy rats were used. New studies are needed to evaluate the results of hypovolemic and hypotensive cases with major artery bleeding.

Sonuç: Büyük arteryel kanamaların kontrolü için asıl tedavi cerrahi kanama kontrolüdür, fakat hastane dışı alanlarda Ankaferd ve chitosan içeren tamponlar kanama bölgesinde kullanılabilir. Araştırmamızın sonuçları daha geniş çalışmalarla desteklenmelidir. Ayrıca araştırmamızda sağlıklı fareler kullanılmıştır. Büyük arter kanamalı hipotansif ve hipovolemik olgulardaki sonuçları değerlendirmek için yeni çalışmalara ihtiyaç vardır.

Keywords: Bleeding, Ankaferd, Chitosan, Hemostasis

Anahtar Sözcükler: Kanama, Ankaferd, Chitosan, Hemostaz

Address for Correspondence/Yazışma Adresi: Serkan ABACIOĞLU, M.D., Osmaniye State Hospital, Clinic of Emergency, Osmaniye, Turkey E-mail : serkanabacioglu@gmail.com

Received/Geliş tarihi: January 22, 2014 Accepted/Kabul tarihi: September 22, 2014

Turk J Hematol 2016;33:48-52

Introduction Injury is the most frequent cause of death before the age of 45 years [1]. Major vascular injury is one of the major causes of death after trauma [2]. In the first assessment of trauma patients, in the circulation step, direct pressure should be applied to the sites of brisk external bleeding [2]. In this process, we need effective and rapid-acting materials to stop the bleeding. Some of the procedures that can be used locally are direct pressure on bleeding, fibrin glues, microporous polysaccharide hemosphere (TraumaDEX®), poly-N-acetylglucosamine (Chitin®), microporous hydrogel forming polyacrylamide (BioHemostat®), chitosan linear polymer (Celox®), and oxidized cellulose (Bloodcare®) [3,4,5,6]. In this study, we compare the efficiencies of Ankaferd Blood Stopper® (ABS) and a chitosan linear polymer (Celox®) in an experimental rat model with femoral artery bleeding. ABS is composed of folkloric herbal extracts that have been traditionally used in Anatolia as hemostatic agents (5), including Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. ABS, which contributes to the conventional methods to control bleeding, has been launched as a novel topical hemostatic agent for the management of visible hemorrhages [6,7,8,9,10]. ABS works by creating a protein network. It induces a very rapid (<1 s) formation of a cellular protein network, particularly including red blood cells and activated leukocytes within the whole blood sample, as well as within plasma and serum samples. It also induces the very rapid (<1 s) formation of vital erythroid aggregations as red blood cells clustered together to aggregate rapidly, thereby inducing a protein network formation. High-resolution scanning electron microscopy images accompanied by morphological analysis following the topical application of ABS revealed very rapid (<1 s) protein network formation within concurrent vital erythroid aggregation covering the classical coagulation cascade [11]. The overall hemostatic effects of ABS depend on the protein agglutination and polymerization modulating the erythroid aggregation and vascular endothelium. ABS also affects the distinct steps of cellular proliferation [12]. As an important advantage, ABS is also effective in patients with deficient primary and/or secondary hemostasis [13,14,15,16]. In addition to its anti-hemorrhagic properties, ABS has been shown to act as a topical biological response modifier [16]. All of these abilities not only make ABS an effective hemostatic agent, but they also confer anti-infective, anti-neoplastic, and healing modulator properties [17]. ABS has been used in a wide range of applications, from dental bleedings to gastrointestinal bleedings [18]. Chitosan (Celox®) is a non-toxic biological polysaccharide polymer of deacetylated chitin (poly [(1,4)-N-acetyl-D-glucose-

Abacıoğlu S, et al: Comparison of Ankaferd and Chitosan on Hemostasis

2-amine)]) (19). It was approved by the United State Food and Drug Administration in June 2006 with ‘Generally Recognized as Safe’ status. The positive loaded NH3+ groups interact with negative loaded platelets and red blood cells, binding them with an ionic bond [20]. This causes the aggregation of platelets in the formation of thrombus. In vitro studies have shown its positive effects in wound healing on activation of polymorphonuclear neutrophils, macrophages, and fibroblasts [21,22,23]. Chitosan has antimicrobial activity against fungi and gram-positive and gram-negative bacteria that accelerates wound healing [22,24]. Celox® is a topical compound of chitosan that is used to stop bleeding of surface injuries [25].

Materials and Methods The study was carried out with approval from the local experimental animals ethics committee (Ankara Numune Education and Research Hospital, 31.01.2011, protocol number: 2011/5). This study was performed in the Ankara Numune Education and Research Hospital animal laboratory and 30 male Wistar albino rats weighing 200-250 g were used. Rats were all fed with the same amount of feed and were fasted for 12 h before the study. Rats were divided into 3 groups as follows: in the control group (n=10), direct compression was applied to the bleeding without medication; in the Ankaferd group (n=10), direct compression was applied with ABS; and in the chitosan group (n=10), direct compression was applied with Celox®. Before the experiment, xylazine hydrochloride and ketamine were used for anesthesia. At that time, arterial blood pressure monitorization was done with a KMA®250 monitor (Petaş, İstanbul, Turkey). The right inguinal regions of the rats were wiped with Batticon and shaved, and the skin and subcutaneous tissues were cut into to reveal the femoral vein and artery. Bleeding was started with a total incision of the femoral artery and vein. Another person collected the accumulated blood with a sponge by pressing for 10 s. The sponge was removed and immediately the homeostatic material was applied (Celox® or ABS), and a constant 50 g of weight was put on this material. At this time, the timer was started. After the first minute, the bleeding was checked. If the bleeding had stopped, it was recorded as ‘bleeding stopped at the first minute’; if not, compression was continued with the same amount of material up to 2 min. After 2 min, the bleeding was checked. If the bleeding had stopped, it was recorded as ‘bleeding stopped at the second minute’; if not, compression was applied again with the same amount of material for 2 min. After these additional 2 min, the bleeding was checked. If the bleeding had stopped, it was recorded as ‘bleeding stopped at the fourth minute’. If the bleeding was still continuing, it was recorded as ‘unsuccessful’. 49

Abacıoğlu S, et al: Comparison of Ankaferd and Chitosan on Hemostasis

Turk J Hematol 2016;33:48-52

Before the rats were sacrificed under anesthesia with 100 mg/kg sodium thiopental (Pental Sodyum®, İ.E. Ulagay, İstanbul, Turkey), 3-mL blood samples were taken from the abdominal aorta in order to measure the levels of hemoglobin (Hb), hematocrit (Hct), coagulation parameters (activated partial thromboplastin time [APTT], prothrombin time [PT], and international normalized ratio [INR]), potassium (K), and calcium (Ca). Statistical Analysis Statistical analysis was performed with SPSS 18.0 for Windows. Continuous variables were expressed as mean ± standard deviation and categorical parameters were given as numbers and percentages. For comparing continuous variables among more than 2 groups, the Kruskal-Wallis test was used. For comparison of categorical variables, Fisher’s exact test was used. All calculations were 2-tailed and p<0.05 was accepted as significant.

Results Two rats were excluded from the study because their mean arterial pressures fell below 50 mmHg. Two new rats were added to the study in their place. At the end of the study, the rats that survived were sacrificed by 100 mg/kg intravenous sodium thiopental (Pental Sodyum®, İ.E. Ulagay). Mean plasma K, Ca, Hb, Hct, and platelet levels; APTT, PT, and INR values; and weights of the groups are expressed in Table 1. These parameters were similar in all groups (p>0.05). In the control group, 60% of the bleeding did not stop. In the Ankaferd group, the bleeding stopped in the first 4 min in all

rats; only in 1 rat of the chitosan group did the bleeding not stop (Table 2). The bleeding did not stop in any rats in the first minute. Among rats in which the bleeding stopped in the first 2 min, results with ABS were similar to those with chitosan (p=1.000) and to the control group (p=0.087), but the p-values were not statistically significant; chitosan results were also similar to those of the control group (p=0.211). In stopping the bleeding in the first 4 min, ABS was similar to chitosan (p=1.000) and better than the control group (p=0.011); chitosan was similar to the control group (p=0.057), but the p-value was close to significance.

Discussion Various procedures, such as direct compression, tourniquets, and clamps, are used to stop bleeding, but these methods do not always end in success. Homeostatic materials are now produced to deal with severe bleeding due to trauma. In this study, we compared 2 known homeostatic materials and direct compression without medication. There are limited studies that have compared ABS and Celox®. Aktop et al. evaluated Table 2. Homeostasis durations of the groups.

Bleeding Stopped at Second Minute

Bleeding Stopped at Fourth Minute

Unsuccessful

Control

4 (40%)

6 (60%)

Ankaferd

4 (40%)

6 (60%)

Chitosan

3 (30%)

6 (60%)

1 (10%)

Table 1. Weight and blood test results of the groups.

Normal Ranges for Rats

Control

Ankaferd

Chitosan

Weight (g)

200

206.80±9.02

208.50±5.74

206.90±7.47

0.853

Hemoglobin (mg/dL)

11-18

13.53±0.20

13.54±0.16

13.54±0.25

0.992

Hematocrit (%)

36-48

49.75±0.20

49.61±0.32

49.61±0.41

0.523

Platelets (103/µL)

500-1300

863.5±7.65

860.70±8.65

857.90±8.91

0.346

Blood urea nitrogen (mg/dL)

15-21

23.10±2.42

23.30±1.83

23.30±1.64

0.860

Creatinine (mg/dL)

0.2-0.8

0.33±0.57

0.32±0.05

0.31±0.04

0.752

Na (mmol/L)

143-156

136.60±2.01

135.90±1.52

0.576

K (mmol/L)

5.4-7

4.71±0.79

4.59±0.69

4.66±0.84

0.990

PT (s)

24.5-30.9

9.51±0.19

9.53±0.13

9.52±0.16

0.963

INR

0.82±0.02

0.817±0.02

0.81±0.02

0.619

APTT (s)

13.0-19.2

15.45±0.19

15.39±0.22

15.37±0.23

0.688

AST (U/L)

45.7-80.8

45.30±1.64

46.70±2.00

45.30±2.63

0.890

ALT (U/L)

17.5-30.2

45.40±2.12

45.70±2.00

45.60±2.32

0.605

Ca (mg/dL)

3.2-8.5

9.31±0.34

9.15±0.36

9.14±0.33

0.473

APTT: Activated partial thromboplastin time, PT: prothrombin time, INR: international normalized ratio, K: potassium, Ca: calcium, Na: sodium AST: aspartate aminotransferase, ALT: alanine aminotransferase.

Turk J Hematol 2016;33:48-52

hemostatic parameters and the early stages of healing potential with Celox® and ABS on soft tissue in warfarin-treated rats [26]. As in our study, they found no differences in hemostasis time, but they did find increased tissue factor values in the Celox®treated group. Huri et al. also found no significant difference between Celox® and ABS hemostasis time [27]. However, tissue healing has been shown to be improved with ABS. Homeostatic agents are mainly used to stop venous and small arterial bleedings, but we used them for significant arterial bleedings and showed their efficiency even in major bleeding. Ersoy et al. showed that microporous polysaccharide hemosphere shortens hemostasis time [3]. Hanks et al. compared the homeostatic efficiencies of fibrin glue and oxidized cellulose among patients who had undergone multiple surgical operations; they reported a shorter homeostatic time in the fibrin glue group, where the bleeding time was 1.6 min as opposed to 3.3 min with oxidized cellulose [28]. In our study, in the ABS group, the bleeding stopped in 40% of the rats in the first 2 min and in the remaining rats in the first 4 min. In this rat model with femoral bleeding, ABS was better than direct compression to stop the bleeding in the first 4 min (p=0.011). In 60% of the control group, the bleeding did not stop in the first 4 min, but in the ABS group, the bleeding stopped in the first 4 min in all rats. Topical homeostatic agents have additional advantages. There are some studies reporting that they reduce secondary complications in some interventions; acidic forms have antibacterial and anticandidal effects and they accelerate wound healing [28,29].

Abacıoğlu S, et al: Comparison of Ankaferd and Chitosan on Hemostasis

Bahattin Işık, Analysis or Interpretation: Fatih Büyükçam, Literature Search: Ural Kaya, Muhammed Evvah Karakılıç, Writing: Serkan Abacıoğlu, Ural Kaya, Fatih Büyükçam, Muhammed Evvah Karakılıç. 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. Committee on Injury Prevention and Control. Reducing the Burden of Injury: Advancing Prevention and Treatment. Washington, DC, USA, National Academy Press, 1999. 2. Brunett PH, Cameron PA. Trauma in adults. In: Tintinalli JE, Stapczynski JS, Ma OJ, Cline DM, Cydulka RK, Meckler GD, (eds). Tintinalli’s Emergency Medicine: A Comprehensive Study Guide. 17th ed. New York, NY, USA, McGraw Hill, 2010. 3. Ersoy G, Kaynak MF, Yilmaz O, Rodoplu U, Maltepe F, Gokmen N. Hemostatic effects of microporous polysaccharide hemosphere in a rat model with severe femoral artery bleeding. Adv Ther 2007;24:485-492. 4. Ward KR, Tiba MH, Holbert WH, Blocher CR, Draucker GT, Proffitt EK, Bowlin GL, Ivatury RR, Diegelmann RF. Comparison of a new hemostatic agent to current combat hemostatic agents in a swine model of lethal extremity arterial hemorrhage. J Trauma 2007;63:276-283. 5. Beyazit Y, Kurt M, Kekilli M, Goker H, Haznedaroglu IC. Evaluation of hemostatic effects of Ankaferd as an alternative medicine. Altern Med Rev 2010;15:329-336. 6. Uçar Albayrak C, Caliskan U, Haznedaroglu IC, Goker H. Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper. J Int Med Res 2008;36:1447-1448. 7. Sonmez M, Baltacioğlu E, Sarac O, Erkut N. The use of Ankaferd blood stopper in a patient with Glanzmann’s thrombasthenia with gingival bleeding. Blood Coagul Fibrinolysis 2010;21:382-383.

Conclusion

8. Aydin S. Haemostatic actions of the folkloric medicinal plant extract Ankaferd Blood Stopper. J Int Med Res 2009;37:279.

In conclusion, in the case of major bleeding, the main treatment is surgical bleeding control, but outside of the hospital, we have to use bleeding control procedures. Here we showed that ABS and chitosan are better than direct pressure on the bleeding region.

9. Teker AM, Korkut AY, Gedikli O, Kahya V. Prospective, controlled clinical trial of Ankaferd Blood Stopper in children undergoing tonsillectomy. Int J Pediatr Otorhinolaryngol 2009;73:1742-1745.

Study Limitations The results of this study should be supported with larger studies. Additionally, in our study, healthy rats were used. New studies are needed to evaluate the results of already hypovolemic and hypotensive subject groups in major artery bleeding. Ethics Ethics Committee Approval: Ankara Numune Education and Research Hospital, 31.01.2011, protocol number: 2011/5, Informed Consent: N/N. Authorship Contributions Concept: Serkan Abacıoğlu, Kemal Aydın, Design: Serkan Abacıoğlu, Data Collection or Processing: Fatih Büyükçam,

10. Meric Teker A, Korkut AY, Kahya V, Gedikli O. Prospective, randomized, controlled clinical trial of Ankaferd Blood Stopper in patients with acute anterior epistaxis. Eur Arch Otorhinolaryngol 2010;267:1377-1381. 11. Haznedaroglu BZ, Haznedaroglu IC, Walker SL, Bilgili H, Goker H, Kosar A, Aktas A, Captug O, Kurt M, Ozdemir O, Kirazli S, Firat HC. Ultrastructural and morphological analyses of the in vitro and in vivo hemostatic effects of Ankaferd Blood Stopper. Clin Appl Thromb Hemost 2010;16:446-453. 12. Huri E, Haznedaroglu IC, Akgul T, Astarci M, Ustun H, Germiyanoulu C. Biphasic effects of ankaferd blood stopper on renal tubular apoptosis in the rat partial nephrectomy model representing distinct levels of hemorrhage. Saudi Med J 2010;31:864-868. 13. Baykul T, Alanoglu EG, Kocer G. Use of Ankaferd Blood Stopper as a hemostatic agent: a clinical experience. J Contemp Dent Pract 2010;11:E088-E094. 14. Ercetin S, Haznedaroglu IC, Kurt M, Onal IK, Aktas A, Kurt OK, Goker H, Ozdemir O, Kirazli S, Firat HC. Safety and efficacy of Ankaferd Blood Stopper in dental surgery. UHOD 2010;20:1-5. 15. Ergenoglu MU, Yerebakan H, Kucukaksu DS. A new practical alternative for the control of sternal bleeding during cardiac surgery: Ankaferd Blood Stopper. Heart Surg Forum 2010;13:379-380. 16. Haznedaroglu IC. Molecular basis of the pleiotropic effects of Ankaferd Blood Stopper. IUBMB Life 2009;61:290.

Abacıoğlu S, et al: Comparison of Ankaferd and Chitosan on Hemostasis

17. Akalin C, Kuru S, Barlas AM, Kismet K, Kaptanoglu B, Demir A, Astarci HM, Ustun H, Ertas E. Beneficial effects of Ankaferd Blood Stopper on dermal wound healing: an experimental study. Int Wound J 2014;11:64-68. 18. Haznedaroglu BZ, Beyazit Y, Walker SL, Haznedaroglu IC. Pleiotropic cellular, hemostatic, and biological actions of Ankaferd hemostat. Crit Rev Oncol Hematol 2012;83:21-34. 19. Fischer TH, Connolly R, Thatte HS, Schwaitzberg SS. Comparison of structural and hemostatic properties of the poly-N-acetyl glucosamine Syvek Patch with products containing chitosan. Micrsosc Res Tech 2004;63:168-174. 20. Kim IY, Seo SJ, Moon HS, Yoo MK, Park IY, Kim BC, Cho CS. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv 2008;26:1-21. 21. Dai T, Tegos GP, Burkatovskaya M, Castano AP, Hamblin MR. Chitosan acetate bandage as a topical antimicrobial dressing for infected burns. Antimicrob Agents Chemother 2009;53:393-400. 22. Dai T, Tanaka M, Huang YY, Hamblin MR. Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. Expert Rev Anti Infect Ther 2011;9:857-879.

Turk J Hematol 2016;33:48-52

23. Muzzarelli RA. Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone. Carbohydrate Polymers 2009;76:167-182. 24. Muzzarelli RA. Chitins and chitosans as immunoadjuvants and nonallergenic drug carriers. Mar Drugs 2010;8:292-312. 25. Kozen BG, Kircher SJ, Henao J, Godinez FS, Johnson AS. An alternative hemostatic dressing: comparison of CELOX, HemCon, and QuikClot. Acad Emerg Med 2008;15:74-81. 26. Aktop S, Emekli-Alturfan E, Ozer C, Gonul O, Garip H, Yarat A, Goker K. Effects of Ankaferd Blood Stopper and Celox on the tissue factor activities of warfarin-treated rats. Clin Appl Thromb Hemost 2014;20:16-21. 27. Huri E, Akgül KT, Yücel MÖ, Astarcı H, Üstün H, Germiyanoğlu RC. The second step in vitro trial of Ankaferd® Bloodstopper®: comparison with other hemostatic agents. Turk J Med Sci 2011;41:7-15. 28. Hanks JB, Kjaergard HK, Hollingsbee DA. A comparison of the haemostatic effect of Vivostat patient-derived fibrin sealant with oxidised cellulose (Surgicel) in multiple surgical procedures. Eur Surg Res 2003;35:439-444. 29. Tomizawa Y. Clinical benefits and risk analysis of topical hemostats: a review. J Artif Organs 2005;8:137-142.

Brief REPORT DOI: 10.4274/tjh.2014.0012 Turk J Hematol 2016;33:53-55

Antifungal Treatment in Stem Cell Transplantation Centers in Turkey Türkiye’deki Kemik İliği Transplantasyonu Merkezlerinde Antifungal Tedavi Hamdi Akan, Erden Atilla Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract

Öz

Despite the development of various guidelines, the approach to antifungal treatment in stem cell transplantation centers differs according to country or even between centers. This led to the development of another survey that aims to understand the antifungal treatment policies of Turkish stem cell transplantation centers. Although there has been an increasing trend towards the use of diagnostic-based treatments in Turkey in the last few years, empirical treatment is still the main approach. The practices of the stem cell transplantation centers reflect the general trends and controversies in this area, while there is a considerable use of antifungal combination therapy.

Çeşitli kılavuzlara rağmen, antifungal hastalıkların tedavisine yaklaşım kök hücre nakil merkezlerinde ülkeden ülkeye, hatta aynı ülke içerisinde farklı merkezlerde farklılık göstermektedir. Bu farklılıkları belirlemek amacı ile ilk defa 2010 yılında Türkiye’deki kök hücre nakli merkezlerinde profilaksi yaklaşımlarını anlamak üzere bir anket düzenlemiştik. Bu anket, Türkiye’deki merkezlerde tedavi yaklaşımlarını anlamamıza yol açacak yeni bir çalışma yapmamızı sağladı. Genel olarak tanı-güdümlü yaklaşım giderek artma eğilimi gösterse de, ampirik yaklaşım hala ilk seçenektir. Kök hücre nakli merkezlerindeki yaklaşımlar genel eğilimlere ve tartışmalar uygun gözükse de, kombine antifungal kullanımının yaygın olduğu görülmektedir.

Keywords: Antifungal treatment, Diagnosis, Stem cell transplantation

Anahtar Sözcükler: transplantasyonu

Introduction

Results

Despite the development of various guidelines [1,2,3], approaches to antifungal treatment in stem cell transplantation (SCT) centers differ according to country and even between centers. This inspired the development of another survey aimed at understanding the antifungal treatment policies of Turkish SCT centers.

Center Characteristics

Materials and Methods Out of 28 EBMT-registered SCT centers, 26 responded to the survey (Figure 1). The questionnaire consisted of separate sections defined to understand the basic treatment approach in each center as empirical or diagnostic-driven, the use of diagnostic tools to start or end a treatment, strategies in empirical or diagnostic-driven treatment, and the use of antifungal combinations.

Antifungal

tedavi,

Teşhis,

Kemik

iliği

While 19 (73.1%) of these centers are adult SCT centers, 7 (26.9%) are pediatric SCT centers. While all centers (26) are performing allogeneic transplants, 24 centers are also performing autologous transplants. Among the 26 allogeneic centers, 24 are performing non-myeloablative, 7 non-related, and 6 cord blood cell transplants. Treatment Approach Four centers (16%) reported that they were only using empirical antifungal treatment, while 56% of the centers reported that they initially employ empirical treatment but that further treatment decisions are based on diagnostic tools such as high resolution computed tomography (HRCT) of the lungs and

Address for Correspondence/Yazışma Adresi: Hamdi AKAN, M.D., Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone : +90 312 595 73 42 E-mail : hamdiakan@gmail.com

Received/Geliş tarihi: January 10, 2014 Accepted/Kabul tarihi: March 31, 2014

Akan H and Atilla E: Antifungals in Stem Cell Transplantation

Turk J Hematol 2016;33:53-55

galactomannan (GM) (Figure 2). Twenty percent of the centers reported that they always use a diagnostic-driven approach and 8% of the centers stated that they use an empirical approach in selected cases.

When using antifungal combination therapy, 57% of the centers add voriconazole to initial amphotericin-B treatment, while 38% add caspofungin to initial amphotericin-B and 5% use voriconazole with caspofungin (Figure 4).

Salvage Therapy

End of Treatment

In non-responding patients, 70% of the centers stop the initial antifungal treatment and switch to another class of antifungal. Twenty-five percent of the centers reported that they add another antifungal to the initial treatment. Drug Selection In empirical approaches, the first drug is amphotericin-B (conventional in 6/21 centers, liposomal in 6/21 centers) in the allogeneic setting (Figure 3). This is followed by voriconazole (4/21) and caspofungin (2/21). This trend is similar in the autologous setting, but voriconazole is less commonly used in autologous transplants. Voriconazole is the main choice in proven cases in allogeneic (23/25) and in autologous (21/23) transplants. In centers treating their patients (diagnostic-driven treatment), the voriconazole (15/20), followed by the allogeneic setting. This trend is settings.

In diagnostic-driven approaches, treatment mainly stops at 90 days (23.8%), after radiological improvement (19%), or after resolution of neutropenia (14.3%). Most of the centers continue oral antifungals, especially in patients with partial radiological resolution and GVHD.

based on HRCT and GM main drug of choice is amphotericin-B (5/20), in similar in other transplant

Figure 1. Distribution of stem cell transplantation centers responding to the query.

Figure 2. Approach to the treatment of invasive fungal disease in stem cell transplantation centers. 54

It was found that 33.3% of the centers continue the antifungal treatment until the end of neutropenia in empirical treatment. Other centers reported that they use both resolution of neutropenia and other evidence such as the clinical condition, diagnostic tools, presence of graft-versus-host disease (GVHD), and pre-transplant fungal status to decide to stop the treatment.

Figure 3. The initial antifungal used for empirical treatment in stem cell transplantation centers.

Figure 4. The antifungal combinations used in centers giving salvage treatment (25% of all centers) (Am-B: amphotericin-B, CAS: caspofungin, Vori: voriconazole).

Turk J Hematol 2016;33:53-55

Patient Selection Diagnostic-driven treatment is mainly used in allogeneic settings (19/26 in allogeneic transplants, 18/24 in nonmyeloablative transplants), with a rate of 62.5% (15/24) in autologous settings.

Candida Treatment Echinocandin is the first drug of choice in established Candida infections at 17/25 centers in allogeneic and 17/23 centers in autologous transplants, followed by amphotericin-B (5/25) in allogeneic and fluconazole in autologous settings. Further Treatment In patients not responding to initial antifungal treatment, 70% of the centers stop the initial antifungal and start a new one, and 25% of the centers choose to use a combination antifungal treatment. Diagnostic Tools HRCT is routine in 23/26 centers, GM is routine in 4/26 centers, and beta-glucan and molecular diagnosis are routine in 4/26 centers. General Approach When asked about their view on empirical or diagnostic-driven approaches in patients with prolonged fever and neutropenia, 46.2% responded in favor of empirical treatment and 11.5% in favor of a diagnostic-driven approach, while 42.3% responded that the choice should be made per patient and most of them choose to use empirical treatment in high-risk patients (Figure 2).

Discussion and Conclusion Although there has been an increasing trend towards the use of diagnostic-based treatments in Turkey in the last few

Akan H and Atilla E: Antifungals in Stem Cell Transplantation

years, empirical treatment is still the main approach. HRCT is the major determinant of diagnostic-driven treatment, and while amphotericin-B is the main drug in empirical treatment, voriconazole is the main choice in diagnostic-driven treatment and proven cases. Despite the guidelines, a large number of centers are using antifungal drug combinations. Keeping in mind that there is still controversy about the selection of empirical therapy versus preemptive (diagnostic-driven) therapy, the differences between the centers in this aspect is understandable. The frequent use of antifungal combinations is interesting, but especially in centers with inadequate diagnostic tools, this approach is to be expected, especially when physicians are faced with a fungal infection that may end up having dreadful consequences. Authorship Contributions Concept: Hamdi Akan, Design: Hamdi Akan, Data Collection or Processing: Hamdi Akan, Erden Atilla, Analysis or Interpretation: Hamdi Akan, Erden Atilla, Literature Search: Hamdi Akan, Erden Atilla, Writing: Hamdi Akan, Erden Atilla. 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. Akan H. Antifungal prophylaxis in stem cell transplantation centers in Turkey. Turk J Hematol 2011;28:271-275. 2. European Society for Blood and Marrow Transplantation. ECIL 2011 Update for Antifungal Therapy. Available at http://www.ebmt.org/contents/ resources/library/ecil/pages/ecil.aspx, accessed 1 September 2013. 3. Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Mullen CA, Raad II, Rolston KV, Young JA, Wingard JR, Infectious Diseases Society of America. Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of America. Clin Infect Dis 2011;52:56-93.

Brief REPORT DOI: 10.4274/tjh.2014.0461 Turk J Hematol 2016;33:56-59

Hemoglobin H Disease in Turkey: Experience from Eight Centers Türkiye’de Hemoglobin H Hastalığı: Sekiz Merkezin Deneyimi Selma Ünal1, Gönül Oktay2, Can Acıpayam3, Gül İlhan3, Edip Gali2, Tiraje Celkan4, Ali Bay5, Barış Malbora6, Nejat Akar7, Yeşim Oymak8, Tayfur Toptaş9 1Mersin University Faculty of Medicine, Department of Pediatric Hematology, Mersin, Turkey 2Antakya Hemoglobinopathy Diagnosis, Control, and Education Center, Antakya, Turkey 3Antakya Education and Research Hospital, Antakya, Turkey 4İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology, İstanbul, Turkey 5Gaziantep University Faculty of Medicine, Department of Pediatric Hematology, Gaziantep, Turkey 6Dr. Sami Ulus Education and Research Hospital, Ankara, Turkey 7TOBB University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey 8Behçet Uz Education and Research Hospital, İzmir, Turkey 9Van Education and Research Hospital, Van, Turkey

Abstract

Öz

The purpose of this study was to research the problem of hemoglobin

Bu çalışmada, ülkemizdeki hemoglobin H (HbH) hastalığı sorununu irdelemek, bölgelere göre dağılımını tespit etmek ve sonuç olarak HbH hastalığının ülkemiz için önemini vurgulamak amaçlandı. Sekiz hemoglobinopati merkezinden toplam 273 hasta çalışmaya dahil edildi. Merkezler arasında en fazla bildirimi 232 hasta ile Antakya Hemoglobinopati Merkezi sağladı. Kalan 7 merkezden toplam 41 HbH hastası bildirildi. Eş zamanlı olarak PubMed’den Türkiye’den yayınlanan ya da Türk HbH hastalarının dahil edildiği makaleler araştırıldı ve sonuçta 198 HbH hastasını içeren toplam 16 yayına ulaşıldı. Bu çalışma, ülkemizde HbH hastalığı sorununu araştıran bir ön çalışmadır. Çalışmamıza Antakya’dan oldukça fazla hasta bildiriminin olması HbH hastalığı yönünden dikkatimizi bu bölgeye yöneltmemize neden olacaktır. Sağlık Bakanlığı önderliğinde oluşturulacak kayıt sistemleri ile tüm HbH’li hastalar kayıt altına alınmalı ve böylece HbH hastalığının ülkemiz için önemi vurgulanmalıdır.

H (HbH) disease, to reveal the distribution patterns among different health centers, and to emphasize the importance of this disease for Turkey. A total of 273 patients were included from 8 hemoglobinopathy centers. The Antakya Hemoglobinopathy Center reported 232 patients and the remaining 7 centers reported 41 patients. PubMed was also searched for published articles related to Turkish patients with HbH disease, and we found 16 articles involving a total of 198 HbH patients. Most of the patients were reported from Antakya; thus, special attention should be paid to this region. This is a preliminary study to investigate the extent of the problem of HbH disease and it emphasizes the need for hematology associations or the Ministry of Health to record all cases of HbH disease in Turkey. Keywords: Thalassemia, Hemoglobinopathy, Hemoglobin H disease

Introduction Hemoglobin H (HbH) disease occurs due to defects in 3 of the 4 alpha genes found in healthy people. HbH (--/-α) is compatible with life and usually has a similar presentation to that of thalassemia intermedia [1]. However, clinical signs vary among patients, and while some patients may need intermittent or frequent transfusions, others do not. Address for Correspondence/Yazışma Adresi: Selma ÜNAL, M.D., Mersin University Faculty of Medicine, Department of Pediatric Hematology, Mersin, Turkey E-mail : unalselma@hotmail.com

Anahtar Sözcükler: Talasemi, Hemoglobinopati, Hemoglobin H hastalığı

HbH disease is suspected in cases of unresponsiveness to iron replacement therapy and findings of microcytic anemia in complete blood counts and peripheral smears. In the early decades of the disease, most patients do not need erythrocyte transfusions. Diagnosis is established when 5%-30% HbH is detected on Hb electrophoresis. Patients with HbH disease also have 20%-40% Hb Barts in the evaluation of cord blood [2]. Brilliant cresyl staining can be used as a screening procedure where the molecular diagnosis of HbH disease is not possible [3]. Received/Geliş tarihi: December 01, 2014 Accepted/Kabul tarihi: May 12, 2015

Turk J Hematol 2016;33:56-59

Ünal S, et al. Hemoglobin H Disease in Turkey: Experience from Eight Centers

The prevalence of alpha thalassemia is 0.24% worldwide and 13,000 babies with HbH are born annually [4]. The overall incidence rate of alpha thalassemia for Turkey is reported to be 0.25%-4.1% [5,6,7]. However, according to different singlecenter studies from the south of Turkey, the frequency of alpha thalassemia ranges between 2.5% and 7.5% [4,5,6,7,8,9,10,11,12].

red blood cell count was 5.16 (x1012/L) (range: 4.07-5.8), mean MCV was 54.7 fL (range: 48-76.4), mean MCH value was 17.9 pg (range: 15-23.9), mean MCHC value was 32.2 g/dL (range: 28.434), mean RDW value was 26.7 (range: 14.6-28), mean HbA2 was 2% (range: 1.2%-2.8%), mean HbF was 2.9% (range: 0.2%3.3%), and mean HbH was 4.7% (range: 1.8%-17.9%).

As the incidence of alpha thalassemia is high, the prevalence of HbH disease may be assumed to be higher. However, due to inadequate reporting of patients with HbH disease, the exact spread and occurrence rates of HbH disease cannot be determined. Thus, the patients who were reported from eight different centers and those reported from previously published studies related to Turkish cases of HbH disease were included in the present study.

The AHDCEC reported 232 patients with HbH disease who were recorded in a data file reporting cases dating back to as far as 1993. In the evaluation of the patients from the AHDCEC, it was observed that the diagnosis of HbH disease was based on clinical findings, hemoglobin electrophoresis, and HbH detection, but alpha gene mutation analysis was not performed for any of these patients. The evaluation of data from 41 patients from the centers other than the AHDCEC revealed that only one patient reached adulthood with an age of 58. Premarital screening was performed in only one family and one other family had a history of hydrops fetalis. The centers contributing to the study and the numbers of patients from these centers are shown in Table 1.

Materials and Methods Hemoglobinopathy centers in Turkey were informed about and invited to participate in this study. Eight centers accepted the invitation and 273 patients were included in the study. Three of these centers were university hospitals and the others were state hospitals. The Antakya Hemoglobinopathy Diagnosis, Control, and Education Center (AHDCEC) reported 232 patients. The remaining 41 patients were reported from the other 7 centers. All of the data collected from these centers were evaluated retrospectively. Additionally, PubMed was searched for English publications related to Turkish patients with HbH disease. As publications in Turkish were difficult to locate and access, those sources were not included.

The PubMed search for publications on Turkish patients with HbH disease yielded 16 articles with a total of 198 patients with HbH disease (Table 2) [10,13,14,15,16,17,18,19,20,21,22,23,24, 25,26,27].

Discussion

Results

Although reports about patients with beta thalassemia and sickle cell anemia are available, insufficient data exist about patients with HbH disease, which can be accompanied by various complications and moderate or severe anemia that may require transfusions.

Of the 273 patients included in the study, 125 were female and 148 were male. Their ages ranged between 9 month and 78 years (Table 1). Mean Hb level was 8.7 g/dL (range: 7.2-10.9), mean

A total of 273 patients from 8 centers were included in this study and 232 of these patients were from Antakya, which is located in the Mediterranean region and had high malaria incidence rates,

Table 1. Centers involved in the study and number of hemoglobin H patients. Center

Reported Number of Patients (n=273)

Age

Sex

Antakya Hemoglobinopathy Diagnosis, Control, and Education Center

232

2-78 years

112 Female/ 120 Male

Other centers: - İstanbul University Cerrahpaşa Faculty of Medicine, Pediatric Hematology Department (n=13) - Gaziantep University Faculty of Medicine, Pediatric Hematology Department (n=9) - Sami Ulus Education and Research Hospital (n=8) - TOBB University Faculty of Medicine, Pediatric Hematology Department (n=6) - Mersin University Faculty of Medicine, Pediatric Hematology Department (n=3) - Behçet Uz Education and Research Hospital (n=1) - Van Education and Research Hospital (n=1)

9 months to 60 years

13 Female/ 28 Male

Total number of hemoglobin H patients

273

Ünal S, et al. Hemoglobin H Disease in Turkey: Experience from Eight Centers

Turk J Hematol 2016;33:56-59

Table 2. Articles associated with hemoglobin H disease in Turkey. Reported Center

Number of Patients

References

Hacettepe University Faculty of Medicine

138

[14,15,16,18,19,22,23,26]

İstanbul University Faculty of Medicine

[13,17,20,27]

Çukurova University Faculty of Medicine

[20,21,25]

Ege University Faculty of Medicine

[24]

Mustafa Kemal University Faculty of Medicine

[10]

Total number of patients

198

probably due to Lake Amik, until recent years. It is known that alpha thalassemia is common where malaria is endemic. It has been observed that another reason why the majority of patients with HbH disease are reported from this area is that the doctors dealing with thalassemia have been working in the region for many years, and thalassemia patients have been recorded since 1993 at the AHDCEC. The low reported number of patients from cities within the same geographic region, such as Mersin and other centers of population, may be due to the recent start of recording patients with thalassemia and HbH disease. We detected 198 patients with HbH disease when we searched articles from PubMed related to Turkish patients with HbH disease [10,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. When the results of our study were added to that number, we found 471 HbH cases to date from Turkey, and this number is fairly high (Table 2). Since the origin of the patients was not recorded in other published studies, no other region was pointed out like Antakya where HbH disease was very frequent. As the data used in this study were not obtained from every center in Turkey, this does not accurately reflect the real number of patients and data on HbH disease in Turkey. However, as many of the HbH patients were reported from Antakya, the situation of the disease in this particular region deserves attention first. In addition, it is necessary to design studies using the data from all centers in Turkey to determine the exact number of patients with HbH disease. Another important point is that cases of hydrops fetalis due to alpha thalassemia are rarely reported in Turkey [28]. All cases should be reported and families with HbH should be evaluated for their history of nonimmunological hydrops fetalis. Prenatal diagnosis should be offered to families who have history of hydrops fetalis, in utero death, or abortion.

Conclusion There are no exact data related to the prevalence of HbH disease in Turkey. However, the 471 cases that are reported based on the data from the literature and the eight centers included in this study are noteworthy. Thalassemia is very common in Turkey, and there are several centers to follow the disease. Recording of HbH cases by these centers will illustrate the urgency of the thalassemia problem in Turkey. 58

In this study, it was found that Antakya is the region where HbH disease is most frequently encountered in Turkey. More studies are required to understand the facts about alpha thalassemiaHbH disease in Turkey. Authorship Contributions Concept: Selma Ünal, Design: Selma Ünal, Data Collection or Processing: Selma Ünal, Gönül Oktay, Can Acıpayam, Gül İlhan, Edip Gali, Tiraje Celkan, Ali Bay, Barış Malbora, Nejat Akar, Yeşim Oymak, Tayfur Toptaş, Analysis or Interpretation: Selma Ünal, Literature Search: Selma Ünal, Writing: Selma 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.

References 1. Chui DH, Fucharoen S, Chan V. Hemoglobin H disease: not necessarily a benign disorder. Blood 2003;103:791-800. 2. Steinberg MH, Forget BG, Higgs DR, Weatherall DJ. Disorders of Hemoglobin. 2nd ed. Cambridge, Cambridge University, 2009. 3. Kulaç İ, Ünal Ş, Gümrük F. Brilliant cresyl blue staining for screening hemoglobin H disease: reticulocyte smear. Turk J Hematol 2009;26:45. 4. Canatan D, Oğuz N, Güvendik İ, Yıldırım S. The incidence of alphathalassemia in Antalya-Turkey. Turk J Haematol 2002;19:433-434. 5. Ozsoylu S, Malik SA. Incidence of alpha thalassemia in Turkey. Turk J Pediatr 1982;24:235-244. 6. Fei YJ, Kutlar F, Harris HF, Wilson MM, Milana A, Sciacca P, Schiliro G, Masala B, Manca L, Altay C. A search for anomalies in the zeta, alpha, beta, and gamma globin gene arrangements in normal black, Italian, Turkish, and Spanish newborns. Hemoglobin 1989;13:45-65. 7. Arcasoy A. Türkiye’de Thalassemia Taşıyıcı Sıklığı. Ankara, Turkey: Ankara Thalassemia Derneği, 1991 (in Turkish). 8. Kılınç Y. Hemoglobinopathies in Turkey. Turk J Hematol 2006;23:214-216. 9. Polat G, Yuregir GT, Aksoy K. Detection of deletional alpha thalassemia in Cukurova. Ann Med Sci 1998;7:14-17. 10. Celik MM, Gunesacar R, Oktay G, Duran GG, Kaya H. Spectrum of α-thalassemia mutations including first observation of (FIL) deletion in Hatay Province, Turkey. Blood Cells Mol Dis 2013;51:27-30. 11. Aldemir Ö, İzmirli M. Hataydaki α-talasemi genotipleri ve α-talasemi genotip frekansı. Abant Med J 2014;3:233-236 (in Turkish). 12. Kılınç Y, Kümi M, Gürgey A, Altay Ç. Adana bölgesinde doğan bebeklerde kordon kanı çalısması ile alfa talasemi, G6PD enzim eksikliği ve HbS sıklığının araştırılması. Doğa Tıp ve Ecz Der 1986;10:162 (in Turkish). 13. Aksoy M, Erdem S. Haemoglobin H disease study of an Eti-Turk family. Acta Genet Stat Med 1968;18:12-22.

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14. Ozsoylu S, Isik K. Haemoglobin H disease in a Turkish family. Scand J Haematol 1973;10:54-58.

the -(α)20.5 KBα-thal-1 deletion in two Turkish patients. Am J Hematol 1993;44:270-275.

15. Orkin SH, Old J, Lazarus H, Altay C, Gurgey A, Weatherall DJ, Nathan DG. The molecular basis of alpha-thalassemias: frequent occurrence of dysfunctional alpha loci among non-Asians with Hb H disease. Cell 1979;17:33-42.

22. Oner C, Gürgey A, Oner R, Balkan H, Gümrük F, Baysal E, Altay C. The molecular basis of Hb H disease in Turkey. Hemoglobin 1997;21:41-51.

16. Altay C, Gurgey A, Tuncbilek E. Hematological evaluation of patients with various combinations of alpha-thalassemia. Am J Hematol 1980;9:261-267. 17. Aksoy M, Kutlar A, Kutlar F, Harano T, Chen SS, Huisman TH. Hemoglobin H disease in two Turkish females and one Iranian newborn. Hemoglobin 1985;9:373-384. 18. Kutlar F, Gonzalez-Redondo JM, Kutlar A, Gurgey A, Altay C, Efremov GD, Kleman K, Huisman TH. The levels of zeta, gamma, and delta chains in patients with Hb H disease. Hum Genet 1989;82:179-186. 19. Fei YJ, Oner R, Bozkurt G, Gu LH, Altay C, Gurgey A, Fattoum S, Baysal E, Huisman TH. Hb H disease caused by a homozygosity for the AATAAA->AATAAG mutation in the polyadenylation site of the alpha 2-globin gene: hematological observations. Acta Haematol 1992;88:82-85. 20. Yüregir GT, Aksoy K, Çürük MA, Dikmen N, Fei YJ, Baysal E, Huisman TH. Hb H disease in a Turkish family resulting from the interaction of a deletional α-thalassemia-1 and a newly discovered poly A mutation. Br J Haematol 1992;80:527-532. 21. Cürük MA, Dimovski AJ, Baysal E, Gu LH, Kutlar F, Molchanova TP, Webber BB, Altay C, Gürgey A, Huisman TH. Hb Adana or α2(59)(E8)Gly-->Aspβ2, a severely unstable α1-globin variant, observed in combination with

23. Oner C, Oner R, Birben E, Balkan H, Gümrük F, Gürgey A, Altay C. HB H disease with homozygosity for red cell G6PD deficiency in a Turkish female. Hemoglobin 1998;22;157-160. 24. Durmaz AA, Akin H, Ekmekci AY, Onay H, Durmaz B, Cogulu O, Aydinok Y, Ozkinay F. A severe α thalassemia case compound heterozygous for Hb Adana in α1 gene and 20.5 kb double gene deletion. J Pediatr Hematol Oncol 2009;31:592-594. 25. Cürük MA. Hb H (β4) disease in Cukurova, southern Turkey. Hemoglobin 2007;31:265-271. 26. Ünal Ş, Gümrük F. The hematological and molecular spectrum of alpha thalassemias in Turkey: the Hacettepe experience. Turk J Hematol 2015;32:136-143. 27. Karakas Z, Koc B, Temurhan S, Elgun T, Karaman S, Asker G, Gencay G, Timur Ç, Yıldırmak ZY, Celkan T, Devecioglu Ö, Aydın F. Evaluation of alpha thalassemia mutations in cases with hypochromic microcytic anemia: Istanbul perspective. Turk J Hematol 2015. 28. Gurgey A, Altay C, Beksaç MS, Bhattacharya R, Kutlar F, Huisman TH. Hydrops fetalis due to homozygosity for alpha-thalassemia-1, -(alpha)-20.5 kb: the first observation in a Turkish family. Acta Haematol 1989;81:169171.

Brief REPORT DOI: 10.4274/tjh.2015.0237 Turk J Hematol 2016;33:60-65

The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia Kronik Myeloid Lösemide Varyant Philadelphia Translokasyonlarının Klinik Açıdan Etkisi Damla Eyüpoğlu1, Süreyya Bozkurt2, İbrahim Haznedaroğlu3, Yahya Büyükaşık3, Deniz Güven1 1Hacettepe University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey 2Hacettepe University Faculty of Medicine Cancer Institute, Basic Oncology, Ankara, Turkey 3Hacettepe University Faculty of Medicine, Division of Hematology, Ankara, Turkey

Abstract

Öz

Chronic myeloid leukemia (CML) is genetically characterized by the presence of the reciprocal translocation t(9;22) with the formation of Philadelphia (Ph) chromosome. Sometimes, the Ph translocation is generated by variant rearrangements. The prognostic impact of the variant translocations is still controversial. Among the 180 patients with Ph-positive CML who were treated in Hacettepe University Faculty of Medicine Division of Hematology, variant translocations were detected, and retrospectively clinical and prognostic features were described. Also we performed a comprehensive literature review on the prognosis of such variant cases before and after tyrosine kinase inhibitor era. Five patients (2.7%) had variant Ph chromosomes, involved in the rearrangements were chromosomes 2 (2 cases), 11, 14 and 15. Patients were treated with imatinib or dasatinib. All patients reached a stable major molecular response suggesting a prognosis not worse than standard translocation individuals. Our present data were compatible with the data of previous studies indicating no difference in the prognosis between standard and variant translocations in tyrosine kinase inhibitors era of CML.

Kronik myeloid lösemi (KML) Philadelphia (Ph) kromozomu olarak anılan t(9;22) resiprokal translokasyonuyla karakterize bir hastalıktır. Bazen Ph kromozomu varyant rearranjmanlarla ortaya çıkabilir. Varyant translokasyonların prognostik etkisi halen tartışmalıdır. Hacettepe Üniversitesi Tıp Fakültesi Hematoloji Kliniği’nde tedavi edilen 180 KML hastasından tanı anında varyant translokasyon taşıyanlar tespit edildi, klinik ve prognostik özellikleri retrospektif olarak incelendi. Ayrıca varyant olgularda tirozin kinaz inhibitörleri dönemi öncesi ve sonrasındaki prognoz üzerine geniş bir literatür taraması yapıldı. Hastalardan 5’i (%2,7) tanı anında 2. (2 olgu), 11., 14. veya 15. kromozom rearranjmanlarını içeren varyant Ph taşımaktaydı. Hastalar imatinib veya dasatinib ile tedavi edildi. Hastaların tümünde stabil bir majör moleküler yanıt elde edilmesi standart translokasyona göre daha kötü bir prognoza sahip olmadığını telkin etmektedir. Mevcut verilerimiz, daha önce yapılmış, tirozin kinaz inhibitörleri döneminde standart ve varyant translokasyonlar arasında prognoz açısından farklılık belirtmeyen çalışmalarla uyumluluk göstermektedir.

Keywords: Chronic myeloid leukemia, Variant Philadelphia, Tyrosine kinase inhibitors, Prognosis

Anahtar Sözcükler: Kronik myeloid lösemi, Varyant Philadelphia, Tirozin kinaz inhibitörleri, Prognoz

Introduction Chronic myeloid leukemia (CML) is a proliferative disorder of hematopoietic pluripotent stem cells [1]. It presents with an estimated incidence of 1/100,000 cases per year, which accounts for 15%-20% of all leukemia cases [2]. CML is genetically characterized by the presence of the reciprocal translocation t(9;22) with the formation of the Philadelphia (Ph) chromosome [3]. The BCR-ABL fusion gene encodes a constitutively active protein tyrosine kinase and it is responsible for the leukemia

phenotype through the constitutive activation of multiple signaling pathways [4]. The Ph chromosome is detected in around 90% of CML patients, among whom 5%-10% may have variant types [5]. Variant Ph chromosomes can present a simple form (involving 22q11 and one additional breakpoint) or a complex form (involving 22q11, 9q34, and at least one additional breakpoint) [6]. The aim of this study is to assess the frequency and prognosis of CML with variant Ph chromosomes. We also performed a

Address for Correspondence/Yazışma Adresi: Damla EYÜPOĞLU, M.D., Hacettepe University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey Phone : +90 507 595 24 79 E-mail : damlakarakaplan@hotmail.com

Received/Geliş tarihi: July 14, 2015 Accepted/Kabul tarihi: September 07, 2015

Turk J Hematol 2016;33:60-65

Eyüpoğlu D, et al: The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia

comprehensive literature review to understand the prognosis of such cases before and after the tyrosine kinase inhibitor (TKI) era.

Materials and Methods Study Population Between 2008 and 2014, 180 patients were diagnosed with CML at our institution. The diagnosis of CML was established on the basis of bone marrow examination and supported by cytogenetic and molecular studies. Clinical, cytogenetic, and molecular responses to TKIs were rated according to the European Leukemia Net (ELN) 2013 guidelines [7]. Cytogenetic Studies Conventional cytogenetic analysis was performed on unstimulated bone marrow specimens after 24 h of culture. Briefly, the cells were cultured and processed by conventional methods. After trypsin-Giemsa banding (GTG-banding), 20 metaphases were analyzed and karyotypes were interpreted according to the 2013 International System for Human Cytogenetic Nomenclature [8].

Results Among the 180 patients with Ph-positive CML, 5 had variant Ph chromosomes. Rearrangements involving chromosomes 2 (2 cases), 11, 14, and 15 were detected. Four patients were female, the median age was 60 (range: 49-68) years, and the median white blood cell count was 64x103/µL (24-177x103/ µL). In regard to cytogenetic characteristics, all of the variant Ph translocations were reciprocal three-way translocations that presented at diagnosis (Figure 1). One patient’s followup data (case 2) were not available. The other four patients’ median follow-up time was 38.5 months (8-65 months), and TKIs (imatinib, and dasatinib in the case of imatinib failure) were used as therapeutic agents. The main clinical parameters and cytogenetic responses are outlined in Table 1. For evaluating the literature data on the impact of the variant translocations on the prognosis and clinical features, we performed an English literature review. For this review, the PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and Web of Science (Web of Knowledge [v5.12], Thomson Reuters, http:// apps.webofknowledge.com/) databases were used. “CML AND variant philadelphia” and “CML AND variant translocation” were used as keywords. We analyzed the studies in which at least 4 cases were included and TKIs had been used as therapeutics. The literature review was conducted in May 2015. The main criteria of these studies are outlined in Table 2.

Discussion In 2%-10% of cases, the Ph translocation is generated by variant rearrangements, involving 9q34, 22q11, and one or several other genomic regions [3]. In our study, 2.7% of our patients exhibited variant Ph chromosomes, which corresponds to the lower margin of the reported range [2,5,9]. Rearrangements involving chromosomes 2 (2 cases), 11, 14, and 15 were detected in our patients. The profile of the variant translocations was similar with those of previous reports [2,10,11]. In our study, 2 out of 3 patients who had been followed for >12 months attained complete cytogenetic response (CCyR) at 12 months. All of the 4 patients for whom follow-up data were available reached major molecular response (MMR) and they were still in MMR at the last follow-up. These data do not suggest worse prognosis compared to our standard Ph patients, which has been reported before [12]. The prognostic impact of the variant translocations was reported in many studies. However, some authors have stated that the involvement of additional oncogenes could be associated with poorer prognosis [10,13,14], while the majority of related studies have confirmed no difference in the prognosis between standard and variant translocations [2,5,9,15,16,17]. The ELN recommendations do not provide any specific advice for patients with variant translocations [7]. Johansson et al. [3] mentioned that the prognostic impact of variant translocations and secondary abnormalities was heterogeneous and most likely related to several parameters, such as time of appearance, specific abnormalities, and treatment modalities. In the first such comprehensive study, El-Zimaity et al. [9] investigated the characteristics and outcomes of 44 patients with variant translocations among 721 CML patients treated

Figure 1. The karyotype of case 3; 46,XX t(9;11;22)(q34;p15;q11.2). 61

F/60

M/60

F/55

F/68

F/49

Hb (g/ dl)

12.5 11.2

12.6

13.4 12.8

Karyotype

46,XX, t(2;9;22) (q37;q34q11.2)

46,XY, t(2;9;22) (q11;q34;q11.2)

46,XX, der(9) t(9;15;22) (q34;?q;q11.2)/ der(15)

46,XX, t(14;9;22) (q31;q34;q11.2)

46,XX, t(9;11;22) (q34;p15;q11.2) 72.2

209.4

177.8

WBC (103/dL)

221

412

363

484

1548

PLT (103/ dL)

Phase at diagnosis

Low

Inter.

Low

Sokal score

Low

Inter.

Euro score

n/a

IM duration,

CCyR

n/a

PCyR

Cytogenetic response at 1st year

MMR

n/a

Molecular response at 1st year

None

n/a

Loss of PCyR at 24 mo

Cause of discontinuation

MMR /IM

n/a

MMR / Dasatinib

treatment

Current response/

Alive/8

Alive/28

Alive/50

n/a

Alive/65

Survival, mo

c) Responses at 6th month

b) Dasatinib dose: 100 mg/daily

a) IM dose: 400 mg/daily

Hb: Hemoglobin, WBC: white blood cell count, PLT: platelet count, F: female, M: male, CP: chronic phase, Inter: intermediate, HU: hydroxyurea, IM: imatinib mesylate, mo: months, n/a: not available, PCyR: partial cytogenetic response, MMR: major molecular response, CCyR: complete cytogenetic response.

Sex/Age

Case

Table 1. Main clinical, hematological, and cytogenetic characteristics of the patients.

Eyüpoğlu D, et al: The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia Turk J Hematol 2016;33:60-65

27/(5.9)

7/(8.8)

44/(6.1)

5/(6)

69/(6)

8/(8.6)

30/(5.4)

6/(7.2)

10/(6.5)

9/(3.2)

Study

Johansson et al. [3], 2001

Richebourg et al. [21], 2008

Costa et al. [22], 2006

El-Zimaity et al. [9], 2004

Valencia et al. [15], 2009

Fabarius et al. [16], 2011

Koshiyama et al. [2], 2013

Marzocchi et al. [5], 2011

Hsiao et al. [17], 2011

Stagno et al. [10], 2010

Gorusu et al. [13], 2007

Lee et al. [14], 2012

6/3 (67/33)

1/9 (10/90)

11/19 (37/63)

3/5 (37.5/62.5)

31/38 (45/55)

3/2 (60/40)

20/24 (45.5/54.5)

17/24 (41/59)

9/18 (33/67)

Sex, Female/ Male, n (%)

IM Cytotoxic agents, IM IM, NIL

No significant difference/Similara,b Compared with cytotoxic therapy, IM showeda higher response/Similara,b Higher amount of BCR-ABL/ND

1, 11, 17 1

51 (32-70)

8 and 17

3, 4, 9, 12, 14, 15

b) Euro risk score

a) Sokal risk score

Overall survival and failure-free survival were inferior/Worsea

HU, IFN, IM

No significant difference/Similara

X, 3, 4, 5, 6, 7, 11, 14, 17, 19

Increased activity of genomic rearrangement/ND

IM, IFN, cytarabine

No significant difference/Similarb

1, 3, 2, 12, 5, 17

17, 1, 11, 12, 15

HU, IFN, IM

Therapies

No significant difference/ND

Higher frequency of accelerated phase/Similar

11, 7, 19, 10, 3, 12 3, 5, 7, 8, 10

ND/ND

Heterogeneous features/ND

Differences in Clinical and Laboratory Presentation/Risk Scores

1 and 11

3, der(9), 15, 12

ins (22;9), 3, 14

The Most Frequent Ph Chromosome Variant Types in the Study

57 (29-74)

52 (33-84)

38 (13-75)

54 (16-88)

59 (50-71)

48 (27-75)

38 (27-51)

54 (12-78)

Median Age, Years (range)

ND: Not described, HU: hydroxyurea, IM: imatinib, IFN: interferon therapy, NIL: nilotinib, Ph: Philadelphia.

Variant Ph, n/(%)

Table 2. Summary of the studies describing the role of variant Philadelphia in chronic myeloid leukemia patients.

Poor

Similar

Heterogeneous

Prognostic Features

Turk J Hematol 2016;33:60-65 Eyüpoğlu D, et al: The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia

Eyüpoğlu D, et al: The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia

with imatinib. The only significant difference in clinical characteristics was a higher frequency of accelerated phase in those with variant translocations (56% vs. 38%). In a large retrospective study, Fabarius et al. [16] mentioned that there was no significant difference in the median time of CCyR (0.95 and 1.01 years), the median time to MMR (1.58 and 1.40 years), the 5-year progression-free survival (81% and 90%), and the 5-year overall survival (87% and 92%). In the study of Marzocchi et al. [5], no significant differences in complete hematological response (93% and 98%), CCyR at 12 months (70% and 78%), or MMR at 12 months (57% and 59%) were observed between both groups in terms of the initial therapy with imatinib mesylate. Hsiao et al. [17] compared the clinical features of CML patients with standard and variant translocations. Apart from the other studies, they not only included TKIs as therapeutic agents but also investigated clinical outcomes of the cytotoxic protocols. It was stated that there was no significant difference in sex, age, complete blood counts, disease status, and survival between variant and classical Ph groups. On the other hand, several studies pointed out the poor prognosis of variant translocations. Lee et al. [14] stated that variant Ph at diagnosis was associated with lower event-free survival (EFS) (p=0.02) and failure-free survival (p=0.008). Stagno et al. [10] identified that the median amount of BCRABL at diagnosis was significantly higher in the variant Ph group. After 18 months of imatinib (8 patients) or nilotinib (2 patients) treatment, 8 patients achieved suboptimal response or failed, while 7 patients had a cytogenetic or a molecular suboptimal response. As a result, the authors stated that complex variant translocations are associated with genomic instability and a more aggressive form of CML. Gorusu et al. [13] confirmed that deletions of the ABL1 or BCL locus were more prevalent in variant translocation CML cases and indicated statistically worse therapeutic responses (p<0.04) and outcomes. The impact of the variant translocations on Sokal and Euro scores was also found to differ in several studies. Some studies mentioned that there was no significant difference between patients with variant and standard Ph translocations regarding Sokal and Euro scores [2,5,9,16]. On the other hand, it was reported that intermediate Sokal risk score is more frequent in patients with variant translocations and that patients with intermediate Sokal risk had lower EFS (p=0.047) in another study [14].

Turk J Hematol 2016;33:60-65

typical t(9;22) translocation [3,18], controversies were found in respect to the prognostic meaning of variant Ph chromosomes [19,20]. Although our patient number was relatively limited, our data were coherent with the studies mentioning no difference in the prognosis between standard and variant translocations in the TKI era. Authorship Contributions Concept: Yahya Büyükaşık, Design: Yahya Büyükaşık, İbrahim C. Haznedaroğlu, Data Collection or Processing: Damla Eyüpoğlu, Yahya Büyükaşık, Analysis or Interpretation: Süreyya Bozkurt, Damla Eyüpoğlu, Literature Search: Süreyya Bozkurt, Deniz Güven, Writing: Damla Eyüpoğlu. 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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Conclusion

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9. El-Zimaity MM, Kantarjian H, Talpaz M, O’Brien S, Giles F, Garcia-Manero G, Verstovsek S, Thomas D, Ferrajoli A, Hayes K, Nebiyou Bekele B, Zhou X, Rios MB, Glassman AB, Cortes JE. Results of imatinib mesylate therapy in chronic myelogenous leukaemia with variant Philadelphia chromosome. Br J Haematol 2004;125:187-195.

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Eyüpoğlu D, et al: The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia

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11. Lin HY, Rocher LL, McQuillan MA, Schmaltz S, Palella TD, Fox IH. Cyclosporineinduced hyperuricemia and gout. N Engl J Med 1989;321:287-292.

17. Hsiao HH, Liu YC, Tsai HJ, Hsu JF, Yang WC, Chang CS, Lin SF. Additional chromosome abnormalities in chronic myeloid leukemia. Kaohsiung J Med Sci 2011;27:49-54.

12. Uz B, Buyukasik Y, Atay H, Kelkitli E, Turgut M, Bektas O, Eliacik E, Isik A, Aksu S, Goker H, Sayinalp N, Ozcebe OI, Haznedaroglu IC. EUTOS CML prognostic scoring system predicts ELN-based ‘event-free survival’ better than Euro/Hasford and Sokal systems in CML patients receiving front-line imatinib mesylate. Hematology 2013;18:247-252. 13. Gorusu M, Benn P, Li Z, Fang M. On the genesis and prognosis of variant translocations in chronic myeloid leukemia. Cancer Genet Cytogenet 2007;173:97-106. 14. Lee SE, Choi SY, Bang JH, Kim SH, Jang EJ, Byeun JY, Park JE, Jeon HR, Oh YJ, Kim M, Kim DW. The long-term clinical implications of clonal chromosomal abnormalities in newly diagnosed chronic phase chronic myeloid leukemia patients treated with imatinib mesylate. Cancer Genet 2012;205:563-571. 15. Valencia A, Cervera J, Such E, Barragán E, Bolufer P, Fuster O, Collado R, Martínez J, Sanz MA. Complex variant t (9; 22) chromosome translocations in five cases of chronic myeloid leukemia. Adv Hematol 2009;2009:187125. 16. Fabarius A, Leitner A, Hochhaus A, Müller MC, Hanfstein B, Haferlach C, Göhring G, Schlegelberger B, Jotterand M, Reiter A, Jung-Munkwitz S, Proetel U, Schwaab J, Hofmann WK, Schubert J, Einsele H, Ho AD, Falge C, Kanz L, Neubauer A, Kneba M, Stegelmann F, Pfreundschuh M, Waller CF, Spiekermann K, Baerlocher GM, Lauseker M, Pfirrmann M, Hasford J, Saussele S, Hehlmann R; Schweizerische Arbeitsgemeinschaft für Klinische

18. Al-Achkar W, Wafa A, Liehr T. A new t(9;11;20;22)(q34;p11.2;q11.21;q11) in a Philadelphia-positive chronic myeloid leukemia case. Oncol Lett 2013;5:605-608. 19. Zhao Y, Wu G, Wu K, Wu K, Liu L, Cao W, Yu X, Luo Y, Shi J, Tan Y, Huang H. Simultaneous occurrence of variant Philadelphia translocations and ABL mutations in two patients with chronic myeloid leukemia. Leuk Res 2009;33:e85-e87. 20. Chauffaille Mde L, Bandeira AC, da Silva ASG. Diversity of breakpoints of variant Philadelphia chromosomes in chronic myeloid leukemia in Brazilian patients. Rev Bras Hematol Hemoter 2015;37:17-20. 21. Richebourg S, Eclache V, Perot C, Portnoi MF, Van den Akker J, Terré C, Maareck O, Soenen V, Viguié F, Laï JL, Andrieux J, Corm S, Roche-Lestienne C; Fi-LMC Group. Mechanisms of genesis of variant translocation in chronic myeloid leukemia are not correlated with ABL1 or BCR deletion status or response to imatinib therapy. Cancer Genet Cytogenet 2008;182:95-102. 22. Costa D, Carrió A, Madrigal I, Arias A, Valera A, Colomer D, Aguilar JL, Teixido M, Camós M, Cervantes F, Campo E. Studies of complex Ph translocations in cases with chronic myelogenous leukemia and one with acute lymphoblastic leukemia. Cancer Genet Cytogenet 2006;166:89-93.

CASE REPORT DOI: 10.4274/tjh.2015.0027 Turk J Hematol 2016;33:66-70

Secondary Neoplasms in Children with Hodgkin’s Lymphoma Receiving C-MOPP and Radiotherapy: Presentation of Four Cases C-MOPP ve Radyoterapi Alan Hodgkin Lenfomalı Çocuklarda İkincil Neoplaziler: Dört Olgu Sunumu Sevgi Gözdaşoğlu1, Ali Pamir1, Emel Ünal1, İsmail Haluk Gökçora2, Ömer Uluoğlu3, Koray Ceyhan4, Haluk Deda5, Erdoğan Işıkman6, Gülsan Yavuz1, Nurdan Taçyıldız1, Ayhan Çavdar1 1Ankara University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Ankara, Turkey 2Ankara University Faculty of Medicine, Department of Pediatric Surgery, Ankara, Turkey 3Gazi University Faculty of Medicine, Department of Pathology, Ankara, Turkey 4Ankara University Faculty of Medicine, Department of Pathology, Ankara, Turkey 5TOBB University of Economics and Technology Hospital, Clinic of Neurosurgery, Ankara, Turkey 6Ankara University Faculty of Medicine, Department of Radiotherapy, Ankara, Turkey

Abstract

Öz

Patients who survive Hodgkin lymphoma (HL) are at increased risk of secondary neoplasms (SNs). A wide variety of SNs have been reported, including leukemias, non-Hodgkin’s lymphomas, and solid tumors, specifically breast and thyroid cancers. Herein we report subsequent neoplasms in four patients with HL receiving chemoradiotherapy. It is interesting that three SNs, fibrosarcoma, thyroid carcinoma, and retrobulbar meningioma, were observed in the radiation area in one of our patients. A hypopharyngeal epithelioid malignant peripheral nerve sheath tumor as an unusual secondary malignant neoplasm developed in another patient, while a benign thyroid nodule and invasive ductal breast carcinoma were observed at different times in the female patient. Follicular adenoma of the thyroid gland developed in one of our patients.

Yaşayan Hodgkin lenfomalı olgularda ikincil neoplazilerin gelişme riski yüksektir. Lösemiler, Hodgkin-dışı malign lenfomalar ve solid tümörler özellikle meme ve tiroid kanserlerini içeren çok çeşitli ikincil malign neoplaziler raporlanmıştır. Burada kemoradyoterapi alan Hodgkin lenfomalı dört olguda gelişen ikincil neoplaziler sunulmuştur. İlginç olarak hastalarımızdan birinde ışın alanında üç ikincil neoplazm; fibrosarkom, tiroid karsinom ve retrobulber meningiom, diğer bir hastamızda sıradışı ikincil malign neoplazm olarak hipofaringeal epitelyal malign periferik sinir kılıfı tümörü gelişirken kız hastada farklı zamanlarda benign tiroid nodülü ve invaziv duktal meme kanseri saptandı. Bir hastamızda ise, tiroidde folliküler adenoma gelişti.

Keywords: Secondary neoplasms, Chemoradiotherapy, Hodgkin’s lymphoma

Anahtar Sözcükler: İkincil neoplaziler, Kemoradyoterapi, Hodgkin lenfoması

Introduction Developments in chemoradiotherapy have enabled most patients with Hodgkin’s lymphoma (HL) to be cured. However, the long-term effects of the treatment include an increased risk of secondary neoplasms (SNs). SNs are defined as histologically distinct neoplasms developing at least 2 months after the completion of treatment for the primary malignancy [1]. SNs may be benign or malignant in characteristics. The occurrence of SNs following HL has now been recognized as a major problem. Among long-term survivors who received C-MOPP (cyclophosphamide, vincristine, procarbazine, and prednisone) plus radiotherapy (RT), SNs developed only in 4 out of 28 patients. In 3 respective cases, Address for Correspondence/Yazışma Adresi: Sevgi GÖZDAŞOĞLU, M.D., Kemer Sk. No: 21/78 Büyükesat, Ankara, Turkey E-mail : sgozdasoglu@hotmail.com

follicular adenoma of the thyroid gland, invasive ductal breast carcinoma, and hypopharyngeal epithelioid malignant peripheral nerve sheath tumor (EMPNST) occurred individually. In another patient, 3 neoplasms, fibrosarcoma, papillary thyroid cancer, and retrobulbar meningioma, were observed subsequently. Herein we report these cases.

Case Presentations Case 1 A 9-year-old male patient was diagnosed with clinical stage I HL with the mixed cellularity (MC) histopathological subtype and Received/Geliş tarihi: January 14, 2015 Accepted/Kabul tarihi: July 02, 2015

Turk J Hematol 2016;33:66-70

had received local RT of 40 Gy to the neck region. He relapsed 3.5 years later with clinical stage IV disease and was given 6 cycles of C-MOPP and 4 cycles of maintenance C-MOPP, for a total of 10 cycles. A fibrosarcoma developed in the radiation area 8 years following the initial treatment (Figure 1), then followed by papillary thyroid carcinoma diagnosed at 16 years and a rightsided retrobulbar meningioma at 30 years later consecutively. The patient was treated according to his neoplasms and he is alive at the present time. Case 2 A 15-year-old male patient was diagnosed with clinical stage I HL with lymphocytic predominance (LP) and received 3 cycles of C-MOPP and 40 Gy RT to the neck region. A thyroid nodule developed 27 years after the initial treatment. Surgical excision was performed and the histopathological diagnosis was follicular adenoma of the thyroid gland. The patient is presently alive with no symptoms. Case 3 A 10-year-old female patient was diagnosed with clinical stage II HL of the nodular sclerosis (NS) type and received nitrogen mustard, vinblastine, and local RT (40 Gy) to the neck region. She developed a mediastinal relapse 4 years after the initial treatment and was given cyclophosphamide (CTX), vincristine (VCR), and adriamycin (ADM) (four cycles) and C-MOPP (two cycles), and mediastinal RT at a dose of 37 Gy. She further developed a benign thyroid nodule 14 years later, which was excised. An invasive ductal carcinoma appeared in her left breast 30 years after the initial chemoradiotherapy. After the diagnosis of the breast carcinoma, the patient did not return and was lost to follow-up. Case 4 A 13-year-old male patient was diagnosed with clinical stage III HL with MC histopathology and he received 40 Gy RT to the neck region and C-MOPP plus maintenance C-MOPP (a total of 10 cycles). A swan-like neck developed 12 years after the treatment (Figure 2). An EMPNST (Figure 3) developed 30 years after the initial treatment. Although the patient received four cycles of iphosphamide and ADM combination chemotherapy, he died with progression of his malignant disease 6 months after diagnosis.

Gözdaşoğlu S, et al: Secondary Neoplasms in Children with Lymphoma

lymphomas, and solid tumors, specifically breast and thyroid cancers. Breast cancer was the most common solid tumor with an estimated actuarial incidence of 35% in women by 40 years of age [4]. In one article, these SNs occurred from 3 months to 21 years after the diagnosis of HL, with leukemias having a median latent period of 5.5 years and solid tumors 9.5 years from diagnosis [5]. Age at treatment has a major effect on the risk of SNs after therapy for HL. A cohort group of 5519 patients with HL treated during 1963-1993 was evaluated and followed for SNs, and it was found that 322 SNs occurred. Relative risks of solid cancers and of leukemia increased significantly with younger age at first treatment [6]. On the other hand, in addition to chemoradiotherapy, genetic predisposing factors such as Li-Fraumeni syndrome, neurofibromatosis, and genetic retinoblastoma further enhance the potential for developing SNs. Genetic susceptibility may play an aggravating role [7]. Thirty-nine children with previously untreated HL were treated with MOPP and RT between 1970 and 1984 in our department. The median age was 10 years (range: 3 to 15 years); 29 were males and 10 were females. The majority of the patients were at stage III-IV with a predominance of the MC histopathological subtype. Twenty-four patients received C-MOPP combination chemotherapy (10-12 cycles), whereas 14 patients were given sandwich therapy of 3 C-MOPP plus EF RT 40 Gy plus 3 C-MOPP, and one case was treated with CTX, VCR, and ADM (4 cycles) and C-MOPP (2 cycles) plus 38 Gy mediastinal RT. Eleven of the 39 patients could not be followed, while 28 patients had a complete evaluation with a median follow-up period of 234 months. Among long-term survivors, SNs occurred only in 4 out of 28 patients: follicular adenoma of the thyroid gland, invasive ductal breast carcinoma, and hypopharyngeal EMPNST, and, in one case, three neoplasms, fibrosarcoma, papillary thyroid cancer, and retrobulbar meningioma, were observed

Discussion The incidence of SNs has been extensively investigated in patients treated for HL. The observed number of SNs was 3.8 times that expected among patients treated with MOPP only, 3.2 times that expected among those treated with extended field or total nodal irradiation only, and 23.0 times that expected among those treated with MOPP and extended field or total nodal irradiation [2]. Approximately 25% of the mortality after treatment for HL is believed to be due to SNs [3]. A wide variety of SNs have been reported, including leukemias, non-Hodgkin’s

Figure 1. Histopathologic section of fibrosarcoma showing fascicles of spindle-shaped cells (H&E stain, 20x). 67

Gözdaşoğlu S, et al: Secondary Neoplasms in Children with Lymphoma

Turk J Hematol 2016;33:66-70

subsequently. All of these SNs developed in the radiation areas. The patients’ details are given in Table 1. Radiation-related solid SNs account for 80% of all SNs and demonstrate a strong relationship with RT. The risk of these solid tumors increases with the total dose of radiation, exposure at a younger age, and longer follow-up after RT [1]. Radiation-induced fibrosarcoma occurred at random intervals from 3 to 38 years after irradiation, usually after high dosages [8]. Fibrosarcoma developed 8 years after the initial treatment in the radiation area in Case 1. Fibrosarcoma was observed as the first SN in our experience with HL [9].

Figure 2. Appearance of swan-like neck.

In our patients, thyroid abnormalities occurred in 50% of the patients who received 40 Gy to the neck region. All patients without irradiation to the neck region showed normal thyroid function [10]. Radiation therapy at a young age is the major risk factor for the development of secondary thyroid cancers. The risk has been reported to be 18-fold that of the general population [1]. In Case 1, papillary thyroid cancer developed 16 years after 40 Gy radiation to the neck. Total thyroidectomy was performed and hormonal replacement treatment was given. In addition to these two malignancies, a right-sided retrobulbar meningioma developed 30 years after the initial RT. It is interesting that these 3 SNs, fibrosarcoma, papillary thyroid cancer, and meningioma, were observed consecutively in the radiation areas of the patient and all 3 tumors were completely resected. Benign thyroid lesions including follicular adenoma were also reported [11]. Follicular adenoma developed 27 years after the initial treatment in Case 2. Best et al. identified two variants at chromosome 6q21 associated with decreased basal PRDM1 expression and impaired induction of PRDM1 by radiation exposure [12]. PRDM1 encodes a zinc finger transcriptional repressor involved in a variety of cellular processes including proliferation, differentiation, and apoptosis. Loss of heterozygosity at chromosome 6q was found to be significantly more common in breast cancers following RT for HL than in sporadic breast cancers (42% vs. 10%) [12].

Figure 3. Epithelioid malignant peripheral nerve sheath tumor cells with May Grunwald-Giemsa and diaminobenzidine stainings. a) Binucleated tumor cells and cytoplasmic perinuclear small vacuoles (May Grunwald-Giemsa stain, 200x). b) Diffuse nuclear and cytoplasmic S-100 protein positivity (Diaminobenzidine stain, 40x). 68

The risk of breast cancer is high among women treated with RT for childhood HL. Excess risk has been reported in female survivors treated with high-dose, extended-volume radiation at age 30 years or younger. In patients treated with chest RT before 16 years of age, the cumulative incidence approaches 20% by age 45 years. Radiation-associated breast cancer has been reported to have more adverse clinicopathological features [1]. Our patient (case 3) was treated with 40 Gy to the neck region at the first admission and 38 Gy to the mediastinum for relapse in addition to chemotherapy.

Gözdaşoğlu S, et al: Secondary Neoplasms in Children with Lymphoma

Turk J Hematol 2016;33:66-70

Table 1. Characteristics of the patients with secondary neoplasias. No

Age/Sex

9/Male

Histopathological Type MC

Clinical Stage I

Treatment

Second Neoplasm

Latency Period (Years)

Local RT 40 Gy 3.5 years after relapse

MOPP (10 cycles)

8 16 30

Fibrosarcoma Papillary thyroid carcinoma Retrobulbar meningioma

Follicular adenoma of thyroid gland

15/Male

3 MOPP+Local RT 40 Gy

10/Female NS

Nitrogen mustard+VB+Local RT 40 Gy 4 years after RT, mediastinal relapse

13/Male

CTX+VCR+ADM (4 cycles)+MOPP (2 cycles)+Mediastinal RT 38 Gy

14 30

Benign thyroid nodule Invasive ductal breast carcinoma

III

Local RT 40 Gy+MOPP (10 cycles)

Hypopharyngeal epithelioid malignant peripheral nerve sheath tumor

Case 4 was treated with 40 Gy RT to the neck region and a total of 10 cycles of C-MOPP. A swan-like neck developed in the patient 12 years after the initial treatment and a hypopharyngeal EMPNST developed in the irradiated area 30 years after RT [13]. The epithelioid variant is an unusual form of malignant peripheral nerve sheath tumors with poor prognosis and represents approximately 5% of malignant peripheral nerve sheath tumors [14]. Adamson et al. reported two patients with malignant peripheral nerve sheath tumors of the spine after RT for HL, and despite prompt surgical resection in the patients, the tumors exhibited aggressive behavior [15]. EMPNST is uncommon but an important fatal complication of RT. There is increasing evidence that RT doses used in the past were higher than necessary. Children receiving RT have an increased risk of developing serious complications such as pulmonary or cardiac toxicity and other cancers later in life. Hence, the new concept is the use of involved side RT and involved node RT. The goal of this new concept is to reduce both treatment volume and treatment dose while maintaining efficacy and minimizing acute and late sequelae [16,17].

Conclusion All children who have received these treatments remain at risk and continued surveillance is warranted. Treatment alterations have potentially decreased the future appearance of SNs. Monitoring for the detection of late effects in adult survivors of childhood cancer necessitates good collaboration between pediatric and adult oncology units.

Ethics Informed Consent: It was taken. Authorship Contributions Surgical and Medical Practices: İsmail Haluk Gökçora, Haluk Deda, Ali Pamir, Emel Ünal, Ayhan Çavdar, Concept: Sevgi Gözdaşoğlu, Design: Ali Pamir, Sevgi Gözdaşoğlu, Data Collection or Processing: Ali Pamir, Emel Ünal, Sevgi Gözdaşoğlu, Analysis or Interpretation: Sevgi Gözdaşoğlu, Literature Search: Sevgi Gözdaşoğlu, Writing: Sevgi Gözdaşoğlu, İsmail Haluk Gökçora. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interest, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. National Cancer Institute. Late Effects of Treatment for Childhood Cancer (PDQ®). Second Cancers. Rockville, MD, USA, National Cancer Institute, 2012. Available online at http://www.cancer.gov/types/childhood-cancers/ late-effects-pdq#section/_36. 2. Green DM. Long-Term Complications of Therapy for Cancer in Childhood and Adolescence. Baltimore, MD, USA, The Johns Hopkins University Press, 1989. 3. Wolden SL, Lamborn KR, Cleary SF, Tate DJ, Donaldson SS. Second cancers following pediatric Hodgkin’s disease. J Clin Oncol 1998;16:536-544. 4. Bhatia S, Robison LL, Oberlin O, Greenberg M, Bunin G, Fossati-Bellani F, Meadows AT. Breast cancer and other second neoplasms after childhood Hodgkin’s disease. N Engl J Med 1996;334:745-751. 5. Meadows AT, Baum E, Fossati-Bellani F, Green D, Jenkin RD, Marsden B, Nesbit M, Newton W, Oberlin O, Sallan SG, Siegel S, Strong LC, Voute PA. Second malignant neoplasms in children: an update from the Late Effects Study Group. J Clin Oncol 1985;3:532-538. 6. Swerdlow AJ, Barber JA, Hudson GV, Cunningham D, Gupta RK, Hancock BW, Horwich A, Lister TA, Linch DC. Risk of second malignancy after Hodgkin’s disease in a collaborative British cohort: the relation to age at treatment. J Clin Oncol 2000;18:498-509.

Gözdaşoğlu S, et al: Secondary Neoplasms in Children with Lymphoma

7. Boice JD Jr. Cancer following irradiation in childhood and adolescence. Med Pediatr Oncol Suppl 1996;1:29-34. 8. O’Neil MB Jr, Cocke W, Mason D, Hurley EJ. Radiation induced soft-tissue fibrosarcoma: surgical therapy and salvage. Ann Thorac Surg 1982;33:624-628. 9. Gözdasoglu S, Cavdar AO, Babacan E. Late effects (including 2nd malignancy) in Turkish children with Hodgkin’s disease. In: SIOP XVIII Annual Meeting. Belgrade, Yugoslavia, Abstract Book, 1986:183-185. 10. Gözdasoglu S, Cavdar AO, Babacan E, Mengübas K, Yavuz G, Unal E, Pamir A, Ocal G, Gökcora I. Late effects of chemoradiotherapy in pediatric Hodgkin’s disease. J Chemother 1995;7:463-466. 11. Acharya S, Sarafoglou K, LaQuaglia M, Lindsley S, Gerald W, Wollner N, Tan C, Sklar C. Thyroid neoplasms after therapeutic radiation for malignancies during childhood or adolescence. Cancer 2003;97:2997-2403. 12. Best T, Li D, Skol AD, Kirchhoff T, Jackson SA, Yasui Y, Bhatia S, Strong LC, Domchek SM, Nathanson KL, Olopade OI, Huang RS, Mack TM, Conti DV,

Turk J Hematol 2016;33:66-70

Offit K, Cozen W, Robison LL, Onel K. Variants at 6q21 implicate PRDM1 in the etiology of therapy-induced second malignancies after Hodgkin lymphoma. Nat Med 2011;17:941-943. 13. Gözdaşoğlu S, Ceyhan K, Üstüner E, Akyar S, Doğan M, İçli F. Hypopharyngeal epithelioid malignant schwannoma following treatment of Hodgkin’s lymphoma. Int J Case Report Med 2015;2015:275309. 14. Reis Filho JS, Pope LZ, Balderrama CM, Fillus-Neto J, Schmitt FC. Epithelioid malignant peripheral nerve sheath tumour: case report and review of the previously published cases. Cytopathology 2002;13:54-63. 15. Adamson DC, Cummings TJ, Friedman AH. Malignant peripheral nerve sheath tumor of the spine after radiation therapy for Hodgkin’s lymphoma. Clin Neuropathol 2004;23:245-255. 16. Yeoh KW, Mikhaeel NG. Role of radiotherapy in modern treatment of Hodgkin’s lymphoma. Adv Hematol 2011;2011:258797. 17. Specht L, Yahalom J, Illidge T, Berthelsen AK, Constine LS, Eich HT, Girinsky

LETTERS TO EDITOR Turk J Hematol 2016;33:71-83

First Observation of Hemoglobin G-Waimanalo and Hemoglobin Fontainebleau Cases in the Turkish Population Türk Toplumunda Gözlenen ilk Hemoglobin G-Waimanalo ve Hemoglobin Fontainebleau Olguları Duran Canatan1,2, Türker Bilgen3, Vildan Çiftçi1, Gülsüm Yazıcı1, Serpil Delibaş2, İbrahim Keser4 1Antalya Genetic Diagnostic Center, Antalya, Turkey 2Hemoglobinopathy Diagnostic Center of Mediterranean Blood Diseases Foundation, Antalya, Turkey 3Namık Kemal University Research and Application Center for Scientific and Technological Investigations, Tekirdağ, Turkey 4Akdeniz University Faculty of Medicine, Department of Biology and Genetics, Antalya, Turkey

To the Editor, Deletional alpha thalassemia mutations can be detected by various methods such as reverse dot blot, gap-polymerase chain reaction (gap-PCR), and multiplex ligation-dependent probe amplification. Point mutations leading to abnormal hemoglobins (Hb) are also observed in common populations. When a point mutation is suspected, resequencing of the alpha genes has become a routine procedure [1]. Hb G-Waimanalo is a silent mutation characterized by a substitution to aspartic acid from asparagine at codon 64 [A64(A2), Asp>Asn] [2]. Hb Fontainebleau is a slightly unstable mutation characterized by a substitution to alanine from proline at codon 21 [A21(A2), Ala>Pro] [3]. To date, they have not been reported from Turkey [4]. Here we present two cases with abnormal Hbs. Case 1: NB, a 33-year-old female, was admitted to the hemoglobinopathy diagnostic center for premarital thalassemia testing. Her complete blood count (CBC) was found to be normal with 24.2% abnormal bands in high-performance liquid chromatography (HPLC) (Table 1). A blood sample was studied further at the genetic diagnostic center. Following DNA extraction with a commercial kit (Roche, Mannheim, Germany) and amplification of the whole beta globin gene by standard protocols of PCR and DNA sequencing (Applied Biosystems, USA), mutation was not found in the beta globin gene. Sequence analyses of alpha genes A1 and A2 were performed and an abnormal Hb in the HBA2: c.193G>A change was detected. This change in the HbA2 gene was at codon 64 GAC>AAC (Asp>Asn), known as Hb G-Waimanalo.

Case 2: ND, a 37-year-old, female, was also admitted to the hemoglobinopathy diagnostic center for premarital testing. She had normocytic anemia in CBC and abnormal bands detected at 16.2% in HPLC. This band result was found to be lower because it may have been fragmented with results of slightly unstable mutation (Table 1). Her blood was studied by the same method at the genetic diagnostic center. Mutation was not found in the beta globin gene. The HbA2 and HbA2 genes were then selectively amplified by standard protocols of PCR. DNA sequencing revealed a G to C change at nucleotide position 21 in the HbA2 gene. This mutation at codon 21 GCT>CCT (Ala>Pro) in the HbA2 gene is known as Hb Fontainebleau. Hb G-Waimanalo is an abnormal Hb and asymptomatic. It was reported in association with alpha and/or beta thalassemia [5,6]. There were no hematological findings in our case; the beta gene was found to be normal. Hb G-Waimanalo was identified in five cases in a study in China [7]. Hb Fontainebleau was described as a silent mutation for the first time in a family of Italian origin [8]. Two cases with mild microcytosis were reported in New Zealand [3]. Our patient had normocytic anemia based on CBC and lower abnormal bands by reason of a slightly unstable mutation in HPLC. Beta gene analysis was normal. So far, a total of 22 cases including 1 homozygous case without clinical findings and 11 heterozygous cases have been reported from premarital screening in the United Arab Emirates [9]. In conclusion, abnormal bands, especially in HPLC, should be investigated with sequence analysis to corroborate alpha and/or beta globin gene mutations.

Table 1. The results of complete blood count and high-performance liquid chromatography in the presented cases. Case

Hb (g/dL)

Hct (%)

RBC (1012/L)

MCV (fL)

MCH (pg)

HbA1 (%)

HbA2 (%)

HbF (%)

Abnormal bands (%)

Case 1

13.3

39.4

4.31

89.1

34.6

73.5

1.6

0.3

24.6

Case 2

11.1

37.8

4.66

83.1

24.4

72.0

1.2

0.6

16.2

Hb: Hemoglobin, Hct: hematocrit, RBC: red blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin.

LETTERS TO EDITOR

Keywords: Abnormal hemoglobins, Hemoglobin G-Waimanalo, Hemoglobin Fontainebleau Anahtar Sözcükler: Anormal hemoglobinler, Hemoglobin G-Waimanalo, Hemoglobin Fontainebleau Authorship Contributions Concept: Duran Canatan, Design: Duran Canatan, Data Collection or Processing: Serpil Delibaş, Gülsüm Yazıcı, Vildan Çiftçi, Analysis or Interpretation: Türker Bilgen, İbrahim Keser, Gülsüm Yazıcı, Vildan Çiftçi, Literature Search: Duran Canatan, Türker Bilgen, 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.

Turk J Hematol 2016;33:71-83

References 1. Harteveld CL, Higgs DR. Alpha-thalassaemia. Orphanet J Rare Dis 2010;5:13. 2. Blackwell RQ, Jim RT, Tan TG, Weng MI, Liu CS, Wang CL. Hemoglobin G Waimanalo: alpha-64 Asp leads to Asn. Biochim Biophys Acta 1973;322:27-33. 3. Brennan SO, Chan T, Ryken S, Ruskova A. A second case of Hb Fontainebleau [alpha21(B2)Ala-->Pro] in an individual with microcytosis. Hemoglobin 2009;33:258-261. 4. Akar N. An updated review of abnormal hemoglobins in the Turkish population. Turk J Hematol 2014;31:97-98. 5. Lin M, Wu JR, Yang LY, Chen H, Wang PP, Wang Q, Zheng L. Hb G-Waimanalo: occurrence in combination with alpha-thalassemia-1 Southeast Asian deletion. Blood Cells Mol Dis 2009;42:36-37. 6. Tan TG, Jim RT, Blackwell RQ. Hemoglobin G Waimanalo beta thalassemia. Hawaii Med J 1978;37:235-239. 7. Lin M, Wang Q, Zheng L, Huang Y, Lin F, Lin CP, Yang LY. Prevalence and molecular characterization of abnormal hemoglobin in eastern Guangdong of southern China. Clin Genet 2012;81:165-171. 8. Wajcman H, Blouquit Y, Gombaud-Saintonge G, Riou J, Galacteros F.Hb Fontainebleau [alpha 21(B2)Ala----pro], a new silent mutant hemoglobin. Hemoglobin 1989;13:421-428. 9. Turner A, Sasse J, Varadi A. Hb Fontainebleau (HBA2: c.64G > C) in the United Arab Emirates. Hemoglobin 2014;38:216-220.

Address for Correspondence/Yazışma Adresi: Duran CANATAN, M.D., Antalya Genetic Diagnostic Center, Antalya, Turkey E-mail : durancanatan@gmail.com

Received/Geliş tarihi: August 18, 2015 Accepted/Kabul tarihi: September 17, 2015 DOI: 10.4274/tjh.2015.0299

Serum Lipids in Turkish Patients with β-Thalassemia Major and β-Thalassemia Minor Türk β-Talasemi Majör ve β-Talasemi Minör Hastalarının Serum Lipidleri Yasemin Işık Balcı1, Şule Ünal2, Fatma Gümrük3 1Pamukkale University Faculty of Medicine, Department of Pediatric Hematology, Denizli, Turkey 2Hacettepe University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey 3Hacettepe University Faculty of Medicine, Department of Radiology, Ankara, Turkey

To the Editor, It is well-known that β-thalassemia is associated with changes in plasma lipids and lipoproteins [1,2,3]. To our knowledge, no data are available on lipid profiles in Turkish β-thalassemia major (TM) and β-thalassemia trait (TT) patients together. The aim of this study was to evaluate lipid profiles in two groups of patients with β-TM and β-TT and to compare them with healthy controls. The study included a total of 311 subjects. Group 1 included 131 β-TM patients (mean age: 16.3±7.58 years). Group 2 included 68 β-TT patients (mean age: 7.25±4.43 years). Group 3 consisted of 112 age- and sex-matched healthy controls (mean age: 9±4.7 years). Serum ferritin level was 2487±1103 (range: 661-5745) ng/mL in Group 1. In comparing the correlation between ferritin and lipid parameters, while a significantly negative relationship was detected between ferritin and highdensity lipoprotein cholesterol (HDL-C) (p=0.000, r=-0.602), a 72

significantly positive relationship was detected between ferritin and triglyceride (TG) levels (p=0.02) in TM patients. Serum lipid profiles of the 3 groups are shown in Table 1. Previous studies have shown total serum cholesterol, HDL-C, lower low-density lipoprotein cholesterol (LDL-C), and higher TG in β-TM patients compared to healthy controls [1,2,3]. In our study, we found lower serum total cholesterol, lower HDL-C, LDL-C, and higher TG in β-TM patients compared to healthy controls. The pathophysiology of hypocholesterolemia in thalassemia remains obscure, although several mechanisms have been proposed; plasma dilution due to anemia, increased cholesterol requirement associated with erythroid hyperplasia, macrophage system activation with cytokine release, and increased cholesterol uptake by the reticuloendothelial system [4,5]. Previous studies reported different variations in lipid profiles of β-TT patients [6,7]. In our study, we demonstrated

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Turk J Hematol 2016;33:71-83

Table 1. Lipid profiles and their significance in patients with β-thalassemia major, patients with β-thalassemia trait, and controls. Group 1

Group 2

Group 3

p-values

β-TM (n=131)

β-TT (n=68)

Control (n=112)

Groups 1-2

Groups 1-3

Groups 2-3

T-Chol

118.5±30.6

145.6±27.6

154.3±31.7

0.00

LDL-C

59.1±27.6

82.5±24.9

89.6±26.1

0.00

HDL-C

34.4±11.2

45.7±12.2

45.5±11.1

0.00

121.8±50.8

82.9±34.6

97.8±52.4

0.00

T-Chol: Total cholesterol, LDL-C: low-density lipoprotein cholesterol, HDL-C: high-density lipoprotein cholesterol, TG: triglyceride, β-TM: β-thalassemia major, β-TT: β-thalassemia trait, NS: non-significant.

similar lipid profiles in β-TT patients and healthy controls. Based on statistical insignificance, we considered that the effects of lipid profile on the development of atherosclerotic vessel disease were similar in both β-TT patients and the healthy control group. Serum iron and iron stores, expressed as elevated ferritin levels, have been implicated in coronary artery disease. Iron overload depletes the antioxidant and HDL-C levels. Lower HDL-C level is an important risk factor for development of coronary heart diseases [8]. We found significant relationships of serum ferritin levels with TG and HDL-C in β-TM patients. These results indicate that β-TM patients who need life-long red blood cell transfusions should receive chelation therapy not only for iron overload-induced congestive heart failure but also in order to prevent cardiovascular diseases resulting from lipid profile alterations. In conclusion, lipid profiles of β-TM patients differed from those of β-TT patients and healthy controls. The present study demonstrates that lower levels of HDL-C in β-TM should be a reason for concern for better evaluation of the cardiovascular risk factors in β-TM. In order to reduce the effects of lipid metabolism on cardiovascular disorders, an effective chelating therapy is essential in TM patients. Keywords: Thalassemia major, Thalassemia minor, Serum lipids Anahtar Sözcükler: Talasemi majör, Talasemi minör, Serum lipidleri

Authorship Contributions Concept: Yasemin Işık Balcı, Design: Yasemin Işık Balcı, Data Collection or Processing: Yasemin Işık Balcı, Şule Ünal, Fatma Gümrük, Analysis or Interpretation: Yasemin Işık Balcı, Şule Ünal, Literature Search: Yasemin Işık Balcı, Şule Ünal, Fatma Gümrük, Writing: Yasemin Işık Balcı. 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. Maioli M, Vigna GB, Tonolo G, Brizzi P, Ciccarese M, Donega P, Maioli M, Fellin R. Plasma lipoprotein composition, apolipoprotein (a) concentration and isoforms in β-thalassemia. Atherosclerosis 1997:131;127-133. 2. Mansi KM, Aburjai TA. Lipid profile in Jordanian children with β-thalassemia major. Int J Hematol 2008;18:93-98. 3. Nasr MR, Abdelmaskoud AM, Abd El-Aal KS, Mabrouk NA, Ismael WM. Plasma lipid profile and lipid peroxidation in beta-thalassemic children. J Clin Lipidol 2008;2:405-409. 4. Papanastasiou DA, Siorokou T, Haliotis FA. β-Thalassaemia and factors affecting the metabolism of lipids and lipoproteins. Haematologia (Budap) 1996;27:143-153. 5. Hashemieh M, Javadzadeh M, Sihirkavand A, Sheibani K. Lipid profile in minor thalassemic patients: a historical cohort study. Bangladesh Med Res Counc Bull 2011;37:24-27. 6. Namazi MR. Minor thalassemia as a protective factor against cerebrovascular accidents. Med Hypotheses 2002;59:361-362. 7. Maioli M, Pettinato S, Cherchi GM, Giraudi D, Pacifico A, Pupita G, Tidore MG. Plasma lipids in β-thalassemia minor. Atherosclerosis 1989;75:245-248. 8. Brizzi P, Isaja T, D’Agata A, Malaguarnera L, Malaguarnera M, Musumeci S. Oxidized LDL antibodies (OLAB) in patients with β-thalassemia major. J Atheroscler Thromb 2002;9:139-144.

Address for Correspondence/Yazışma Adresi: 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: April 26, 2015 Accepted/Kabul tarihi: August 17, 2015 DOI: 10.4274/tjh.2015.0168

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Turk J Hematol 2016;33:71-83

Extranodal Natural Killer/T-Cell Lymphoma: A Rare Nasal-Type Case Ekstranodal Natural Killer/T-Hücreli Lenfoma: Nadir Bir Nasal Tip Olgusu Esra Sarıbacak Can1, Harika Okutan1, Murat Albayrak1, Ünsal Han2 1Dışkapı Yıldırım Beyazıt Research and Training Hospital, Clinic of Hematology, Ankara, Turkey 2Dışkapı Yıldırım Beyazıt Research and Training Hospital, Clinic of Pathology, Ankara, Turkey

To the Editor, Nasal type extranodal natural killer (NK) NK-cell/T-cell lymphoma (NKTCL) is a rare extranodal lymphoma of NK-cell or T-cell origin that most commonly affects immunocompetent middle-aged men of Asian or Native American descent [1]. The pathogenesis is not understood completely, but it is related in part to infection of the tumor cells with Epstein-Barr virus (EBV) [2]. Around 6-7% of all non-Hodgkin’s lymphoma (NHL) in Southeast Asia accounts for NKTCL. However, the incidence of NHL is lower in the United States at 1.5% [3,4]. Disease within the nasal cavity has a better prognosis. Radiation therapy alone can be curative. Over 60% of patients with stage 1 disease remain in long-term remission following treatment with radiation therapy with or without chemotherapy [5]. Nasal disease may be cured with radiotherapy at a rate of 85%. However, the relapse rate is high at 25%. Therefore, it is highly crucial for this aggressive disease to be diagnosed and treated at an early stage [6,7]. In our case, a 29-year-old female complained of nasal obstruction and had a necrotizing mass in the upper middle concha at the right nasal cavity; biopsy was planned. CD56 was strongly positive (Figure 1), CD8 was positive in a few scattered cells, and CD4 was positive in the majority of infiltrating T cells in the sample of necrotic tissue pieces. TIA-1 and perforin were commonly positive. Granzyme was commonly strongly cytoplasmic-positive. Epstein-Barr encoding region (EBER) in situ hybridization analysis was done with a probe cocktail containing EBV. Early RNA transcript showed that NKTCL compatibility existed with commonly strongly nuclear-positivity in EBER infiltrating cells. Positron emission tomography-computed tomography (PET-CT) revealed pathologically heterogeneous soft tissue mucosal thickening, pushing the nasal septum slightly to the left and hypertrophy of the right ethmoid cells in the upper middle concha level of the nasal cavity, with increased metabolic uptake (SUVmax: 3.18). The patient was diagnosed with stage 1E based on PET-CT evaluation and received a total

of 38 Gy external radiotherapy at 200 cGy daily. No involvement was detected month after radiotherapy and complete response was considered to have occurred after 1 year. NKTCL of the palate and sinuses has been reported in many cases. However, the incidence of NKTCL is much lower in the United States. Nasal obstruction, bleeding, pain, or local swelling are usually observed and ulcerative, destructive lesions within extranodal sites can be produced. Often it is associated with EBV. Immunophenotypically, the tumor cells express CD2, CD3, and CD56. The cells can lack CD56 and express CD8+ T-cell antigens in some cases [7]. The course of NKTCL, nasal type, is aggressive where a 5-year overall survival ranges from 25% to 50% [8]. Symptoms of nasal type NKTCL can include nasal discharge, nasal obstruction and other nonspecific sinonasal symptoms. However, sore throat and dysphagia, also known as symptoms of nasal type NKTCL, are frequently missed and treated as viral and bacterial pharyngitis, which leads to late diagnosis. Therefore, morbidity and mortality are increased. NK/T-cell lymphoma, nasal type, is rarely observed in Turkey and early diagnosis of the disease is of vital importance.

Figure 1. Strong staining of diffuse cytoplasmic natural killer cells with CD56 (CD56, IHC, 200x).

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Turk J Hematol 2016;33:71-83

Keywords: Extranodal natural killer/T-cell lymphoma, Non-

References

Hodgkin lymphoma

1. Roschewski M, Wilson WH. EBV-associated lymphomas in adults. Best Pract Res Clin Haematol 2012;25:75-89.

Anahtar

Sözcükler:

Ekstranodal

natural

killer/T-hücreli

lenfoma, Hodgkin dışı lenfoma Authorship Contributions Concept: Esra Sarıbacak Can, Design: Esra Sarıbacak Can, Data Collection or Processing: Esra Sarıbacak Can, Analysis or Interpretation: Esra Sarıbacak Can, Harika Okutan, Literature Search: Esra Sarıbacak Can, Murat Albayrak, Writing: Esra Sarıbacak Can, Ünsal Han. 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.

2. Niedobitek G. Epstein-Barr virus infection in the pathogenesis of nasopharyngeal carcinoma. Mol Pathol 2000;53:248-254. 3. Al-Hakeem DA, Fedele S, Carlos R, Porter S. Extranodal NK/T-cell lymphoma, nasal type. Oral Oncol 2007;43:4-14. 4. Jaccard A, Hermine O. Extranodal natural killer/T-cell lymphoma: advances in the management. Curr Opin Oncol 2011;23:429-435. 5. Liu QF, Wang WH, Wang SL, Liu YP, Huang WT, Lu N, Zhou LQ, Ouyang H, Jin J, Li YX. Immunophenotypic and clinical differences between the nasal and extranasal subtypes of upper aerodigestive tract natural killer/T-cell lymphoma. Int J Radiat Oncol Biol Phys 2014;88:806-813. 6. Liang R. Diagnosis and management of primary nasal lymphoma of T-cell or NK-cell origin. Clin Lymphoma 2000;1:33-38. 7. Hasserjian RP, Harris NL. NK-cell lymphomas and leukemias: a spectrum of tumors with variable manifestations and immunophenotype. Am J Clin Pathol 2007;127:860-868. 8. Kwong YL. Natural killer-cell malignancies: diagnosis and treatment. Leukemia 2005;19:2186-2194.

Address for Correspondence/Yazışma Adresi: Esra SARIBACAK CAN, M.D., Dışkapı Yıldırım Beyazıt Research and Training Hospital, Clinic of Hematology, Ankara, Turkey E-mail : esracanercan@hotmail.com

Received/Geliş tarihi: April 27, 2015 Accepted/Kabul tarihi: September 02, 2015 DOI: 10.4274/tjh.2015.0169

T-Cell Lymphoma Presenting with Auricular and Parotid Gland Involvement Aurikula ve Parotis Bezi Tutulumu ile Seyreden T-Hücreli Lenfoma Birgül Öneç1, Alper Koç2, Elif Nisa Ünlü3, İlhan Ünlü4, Hüseyin Yaman4, Durdu Mehmet Köş2 1Düzce University Faculty of Medicine, Department of Hematology, Düzce, Turkey 2Düzce University Faculty of Medicine, Department of Internal Medicine, Düzce, Turkey 3Düzce University Faculty of Medicine, Department of Radiology, Düzce, Turkey 4Düzce University Faculty of Medicine, Department of Otorhinolaryngology, Düzce, Turkey

To the Editor, The external auditory canal is an unusual presenting site for lymphomas, with only a few case reports in the literature [1,2]. Malignant lymphomas arising from the salivary glands are also uncommon, accounting for approximately 5% of extranodal lymphomas, and the majority of them are of B-cell lineage. Primary salivary gland T-cell lymphomas are extremely rare [3,4,5]. A 63-year-old man was admitted with swelling of the left side of his face and left auricle. Considered as an infection, it was empirically treated with systemic and topical antibiotics. Examination by an otorhinolaryngologist revealed a suppurative

lesion that consisted of ulcerated areas and granulation tissue in the external auditory canal and preauricular region in addition to edema (Figure 1A). A lobulated mass lesion of 47x39 mm arising from the left parotid and extending to the left auditory canal was detected in computed tomography (CT) with accompanying lymph nodes in the left subauricular and cervical localization. Fine-needle biopsy was nondiagnostic and incisional biopsy found only severely active chronic inflammation. Finally, deep excisional biopsy revealed CD3-, CD5-, and CD30-positive and S100-negative lymphoid cells diffusely infiltrating the dermis. The case was considered as stage 2 peripheral T-cell lymphoma not otherwise specified peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS) and CHOP (cyclophosphamide,

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Turk J Hematol 2016;33:71-83

for proper assignment of lineage of salivary gland lymphomas [5]. Reactive lymphoid infiltrate located in the periphery of the lymphoma may contribute to the delay of diagnosis and larger biopsy samples are needed. Keywords: Parotid gland, T-Cell lymphoma, Auricula Anahtar Sözcükler: Parotis bezi, T-Hücreli lenfoma, Aurikula Figure 1. A) A suppurative lesion that consisted of ulcerated areas and granulation tissue was observed in the external auditory canal and preauricular region. B) Significant regression of lesion after 4 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) treatment. doxorubicin, vincristine, prednisone) protocol was started. Despite clinical improvement in the lesions (Figure 1B), CT revealed progression after the fourth cycle. The second-line treatment is ongoing with DHAP (dexamethasone, high-dose ara-C, cisplatin) at the 8th month of follow-up. The auricula, external auditory canal, and parotid glands are unusual locations for T-cell lymphoma. Presentations of lymphomas are indistinguishable from other swellings of the auricle or parotid gland and therefore a high index of suspicion should be maintained in patients who present with presumptive cutaneous infections that do not respond to antibiotic therapy in these locations. A suppurative auricular lesion suggests an infectious disease rather than a lymphoma, but primary cutaneous lymphomas and cutaneous manifestations of lymphomas must be kept in mind. Early excisional biopsy may prevent excessive waste of time with unnecessary antibiotherapies. Parotid lymphomas are most likely to be B-cell non-Hodgkin lymphoma, but non-B-cell lymphomas have a more aggressive course in all salivary gland lymphomas. Both B-cell and T-cell lymphomas share many morphological similarities; therefore, immunohistochemical analysis is required Address for Correspondence/Yazışma Adresi: Alper KOÇ, M.D., Düzce University Faculty of Medicine, Department of Internal Medicine, Düzce, Turkey E-mail : alperkoc44@hotmail.com

Authorship Contributions Concept: Birgül Öneç, Design: Birgül Öneç, Alper Koç, Data Collection or Processing: Birgül Öneç, Alper Koç, Analysis or Interpretation: Birgül Öneç, Durdu Mehmet Köş, Elif Nisa Ünlü, Literature Search: Birgül Öneç, Hüseyin Yaman, İlhan Ünlü, Writing: Birgül Öneç, Alper 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.

References 1. Marçal N, Campelos S, Dias L, Gonçalves M, Pereira G, Godinho T. Primary cutaneous CD30-positive anaplastic large-cell lymphoma of the external auditory canal. Ear Nose Throat J 2012;91:10-12. 2. González Delgado A, Argudo Marco F, Sánchez Martínez N, Sprekelsen Gassó C. T cell non-Hodgkin’s lymphoma of the external auditory canal. Acta Otorrinolaringol Esp 2008;59:200-201. 3. Yanagawa N, Osakabe M, Furuse H, Maeda K, Tamura G. Peripheral T-cell lymphoma arising from an intraglandular lymph node in the parotid gland: a case report and literature review. Pathol Int 2012;62:60-64. 4. Metikurke SH, Krishnappa R, Ramachar SM, Arora I. Primary malignant lymphoma of the parotid gland. J Cancer Res Ther 2012;8:641-643. 5. Chan JK, Tsang WY, Hui PK, Ng CS, Sin VC, Khan SM, Siu LL. T- and T/ natural killer-cell lymphomas of the salivary gland: a clinicopathologic, immunohistochemical and molecular study of six cases. Hum Pathol 1997;28:238-245. Received/Geliş tarihi: May 25, 2015 Accepted/Kabul tarihi: August 17, 2015 DOI: 10.4274/tjh.2015.0217

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Turk J Hematol 2016;33:71-83

Immune Thrombocytopenia Resolved by Eltrombopag in a Carrier of Glucose-6-Phosphate Dehydrogenase Deficiency Glukoz-6-Fosfat Dehidrogenaz Eksikliği Taşıyıcısında Eltrombopag Yanıtlı İmmün Trombositopeni Laura Scaramucci, Pasquale Niscola, Massimiliano Palombi, Andrea Tendas, Marco Giovannini, Paolo de Fabritiis Sant’Eugenio Hospital, Clinic of Hematology, Rome, Italy

To the Editor, Eltrombopag, a thrombopoietin mimetic peptide, may provide excellent clinical efficacy in steroid-refractory patients with immune thrombocytopenic purpura (ITP) [1,2]. Eltrombopag is generally well tolerated. However, its use in the particular setting of glucose-6-phosphate dehydrogenase (G6PD) and history of acute hemolytic anemia (AHA) has not been reported so far. A 51-year-old female was diagnosed as having ITP in September 2014. She was not taking any medication and her past history was negative, apart from having been diagnosed a carrier (heterozygous) of G6PD deficiency (Mediterranean variant) after a familial screening by molecular and biochemical methods. She presented with only slightly reduced (about 50%) enzyme level, belonging to World Health Organization-defined class 3 [3,4]. In the following years, the patient experienced some episodes of AHA, which were managed at outside institutions; in particular, a severe episode of AHA, probably triggered by urinary infection and antibiotics [5], had complicated her second and last delivery. The hemolytic episodes were selflimiting and resolved without sequelae. No other causes of hemolysis were documented. When the case came to our attention, a diagnosis of ITP was made; hemolytic parameters were normal, although the G6PD enzyme concentration was not measured. Oral prednisone (1 mg/kg) was given with only a transient benefit. The patient was then a candidate for elective splenectomy. However, given her extremely low platelet count, she was started in October 2014 on eltrombopag at 50 mg/day as a bridge to splenectomy. Given that, to the best of our knowledge, the use of this drug has never been reported in the particular setting of G6PD deficiency, the patient was constantly monitored. A prompt platelet increase (178x109/L) was observed 1 week after the start of treatment. After she achieved the target platelet count, the dose of eltrombopag was tapered to the lowest effective dose. The patient’s response was stabile while she remained on a dose of eltrombopag between 25 and 50 mg/day without any adverse events; in particular, no variations of hemolytic parameters were observed. As of today, after 6 months of continuous eltrombopag administration, the patient has constantly maintained the target platelet counts and she is awaiting elective splenectomy. According to our

knowledge, we report for the first time the evidence regarding the safe use of this thrombomimetic agonist, which provided an excellent treatment outcome without any adverse effects, in a steroid-refractory adult ITP patient at risk of drug-induced AHA as a G6PD-deficient heterozygous carrier. G6PD deficiency is an X-linked, hereditary genetic defect [2,3,4] for which heterozygous women, who are usually asymptomatic, have a mosaicism of normal and G6PD-deficient erythrocytes. Given the possible decreased amount of G6PD enzyme, although exceptional and only under particularly stressing conditions, such as urinary tract infections and/or the use of certain antibiotics such as nitrofurantoin, AHA may occur [5]. Although our report, being limited to a single patient, is purely anecdotal, considering the high prevalence of G6PD deficiency and the increasing use of thrombomimetic drugs, further collection of such cases would be very useful to determine the complication risks associated in this setting with the use of thrombopoietin agonists. Keywords: Idiopathic thrombocytopenic purpura, Glucose-6phosphate dehydrogenase deficiency, Thrombopoietin mimetic peptide, TMP mimetic peptide Anahtar Sözcükler: İdiyopatik immün trombositopeni, Glukoz6-fosfat dehidrogenaz eksikliği, Trombopoetin uyarıcı peptit, TPO uyarıcı peptit Authorship Contributions Concept: Laura Scaramucci, Pasquale Niscola, Massimiliano Palombi, Andrea Tendas, Marco Giovannini, Paolo de Fabritiis, Design: Laura Scaramucci, Pasquale Niscola, Massimiliano Palombi, Andrea Tendas, Marco Giovannini, Paolo de Fabritiis, Data Collection or Processing: Laura Scaramucci, Marco Giovannini, Analysis or Interpretation: Laura Scaramucci, Pasquale Niscola, Marco Giovannini, Literature Search: Pasquale Niscola, Massimiliano Palombi, Andrea Tendas, Writing: Laura Scaramucci, Pasquale Niscola, Final Approval: Paolo de Fabritiis. 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. 77

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References 1. Tripathi AK, Shukla A, Mishra S, Yadav YS, Yadav DK. Eltrombopag therapy in newly diagnosed steroid non-responsive ITP patients. Int J Hematol 2014;99:413-417. 2. Luzzatto L, Seneca E. G6PD deficiency: a classic example of pharmacogenetics with on-going clinical implications. Br J Haematol 2014;164:469-480.

Turk J Hematol 2016;33:71-83

3. No authors listed. Glucose-6-phosphate dehydrogenase deficiency. WHO Working Group. Bull World Health Organ 1989;67:601-611. 4. Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet 2008;371:64-74. 5. van de Mheen L, Smits SM, Terpstra WE, Leyte A, Bekedam DJ, van den Akker ES. Haemolytic anaemia after nitrofurantoin treatment in a pregnant woman with G6PD deficiency. BMJ Case Rep 2014:2014.

Address for Correspondence/Yazışma Adresi: Pasquale NISCOLA, M.D., Sant’Eugenio Hospital, Clinic of Hematology, Rome, Italy Phone: +39 065 100 31 41 E-mail : pniscola@gmail.com

Received/Geliş tarihi: May 01, 2015 Accepted/Kabul tarihi: August 17, 2015 DOI: 10.4274/tjh.2015.0181

Wernicke’s Encephalopathy in an Acute Myeloid Leukemia Patient: A Case Study Bir Akut Myeloid Lösemi Hastasında Wernicke Ensefalopatisi: Bir Olgu Sunumu Muhammet Maden1, Gülsüm Pamuk1, Yahya Çelik2, Ercüment Ünlü3 1Trakya University Faculty of Medicine, Department of Hematology, Edirne, Turkey 2Trakya University Faculty of Medicine, Department of Neurology, Edirne, Turkey 3Trakya University Faculty of Medicine, Department of Radiodiagnostics, Edirne, Turkey

To the Editor, Wernicke’s encephalopathy (WE) is a life-threatening disease with acute onset, resulting from thiamine deficiency. Causes are alcohol intake, malnutrition, gastric bypass surgery, human immunodeficiency virus infection, total parenteral nutrition (TPN), chronic dialysis, and cancer [1]. WE may cause neurological symptoms such as nystagmus, ophthalmoplegia, ataxia, confusion, convulsions, delirium, coma, and acute psychoses [2]. An 18-year-old female patient with refractory acute myeloid leukemia (AML) was given FLAG-IDA (fludarabine, cytosine arabinoside, idarubicin) chemotherapy protocol. As she developed typhlitis, oral intake was stopped; broad-spectrum antibiotics and TPN without any vitamin supplementation were started. She developed a fixed look to a point, chin and upper extremity spasms, and urinary incontinence on the 38th day of chemotherapy. Neurological examination showed nonlateralization, bilateral light reflexes were +/+, verbal stimuli were negative, and the reflex response to painful stimuli was positive. Laboratory results showed hemoglobin of 7.2 g/dL, leukocytes of 3380/mm3, neutrophils of 2890/mm3, platelets of 48,000/mm3, and normal blood biochemistry. The level of serum thiamine could not be measured, because the laboratory did not have the capabilities to measure it. Brain diffusion MRI showed increased signal intensity in the medial thalami (Figure 1). The patient was diagnosed with WE. She was given 1500 mg/day 78

thiamine i.v. for 3 days and 250 mg/day thiamine i.v. for another 5 days. On the fourth day of thiamine infusion, her general condition began to improve and she started giving one-word responses to verbal stimuli. Her convulsions disappeared and she started to form short sentences and walk without assistance; she was discharged on the 30th day of thiamine replacement therapy. Currently, 2 years have passed since the WE and the patient is in complete hematologic remission.

Figure 1. Coronal T2-weighted image shows symmetrical, weak, and limited increased signal intensity in the medial thalami and in the third ventricle-facing surface.

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Turk J Hematol 2016;33:71-83

In cancer patients, WE may develop because of certain chemotherapeutic agents (especially doxifluridine, ifosfamide, and 5-fluorouracil) [3], malnutrition, and thiamine depletion due to fast-growing tumor cells or gastrointestinal bypass surgery [2]. In the literature, there are a few cases of WE in AML. The diagnosis of WE in cancer patients is difficult because there are many causes of similar symptoms, such as confusion-causing hypoxia, infections, electrolyte imbalance, opioid medications, chemotherapy, brain and meningeal metastases, and delirium [4]. To make a definite diagnosis of WE, it should be clinically suspected. WE may be verified with the measurement of thiamine concentration in blood or erythrocyte transketolase activity; however, these tests are not widely available [3]. Magnetic resonance imaging (MRI) has 93% specificity and 53% sensitivity to verify the diagnosis [2]. Bilateral dorsomedial thalamus, tectal plaque, and periaqueductal gray matter signal abnormalities are observed as classical in MRI [5]. WE should be treated empirically with 500 mg of thiamine 3 times per day for 2-3 days. If there is no response, supplementation may be discontinued after 2-3 days. In case an effective response is observed, 250 mg of thiamine should be continued daily until clinical improvement [2]. Thiamine supplementation should definitely be added to TPN or the diets of hematologic malignancy patients with poor oral nutrition. When symptoms such as ataxia, confusion, or ophthalmoplegia appear in these patients, brain MRI should be immediately performed and thiamine infusion should be initiated immediately in the case of strong suspicion. Keywords: Thiamine, Wernicke’s encephalopathy, Acute myeloid leukemia

Anahtar Sözcükler: Tiamin, Wernicke ensefalopatisi, Akut myeloid lösemi Authorship Contributions Concept: Muhammet Maden, Gülsüm Pamuk, Design: Muhammet Maden, Gülsüm Pamuk, Data Collection or Processing: Muhammet Maden, Ercüment Ünlü, Yahya Çelik, Analysis or Interpretation: Muhammet Maden, Gülsüm Pamuk, Ercüment Ünlü, Yahya Çelik, Literature Search: Muhammet Maden, Gülsüm Pamuk, Writing: Muhammet Maden. 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. Papila B, Yildiz O, Tural D, Delil S, Hasiloglu ZI, Ayan F, Papila C. Wernicke’s encephalopathy in colon cancer. Case Rep Oncol 2010;3:362-367. 2. Sechi G, Serra A. Wernicke’s encephalopathy: new clinical settings and recent advances in diagnosis and management. Lancet Neurol 2007;6:442455. 3. Basu TK, Dickerson JW. The thiamin status of early cancer patients with particular reference to those with breast and bronchial carcinomas. Oncology 1976;33:250-252. 4. 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;76:10-18. 5. Zuccoli G, Siddiqui N, Bailey A, Bartoletti SC. Neuroimaging findings in pediatric Wernicke encephalopathy: a review. Neuroradiology 2010;52:523-529.

Address for Correspondence/Yazışma Adresi: Muhammet MADEN, M.D., Trakya University Faculty of Medicine, Department of Hematology, Edirne, Turkey E-mail : drmaden@yandex.com

Received/Geliş tarihi: June 24, 2015 Accepted/Kabul tarihi: September 07, 2015 DOI: 10.4274/tjh.2015.0249

Kaleidoscopic Views in the Bone Marrow: Oxalate Crystals in a Patient Presenting with Bicytopenia Kemik İliğinde Oksalat Kristalleri Birikimine Bağlı Gelişen Bisitopeni Yelda Dere1, Simge Erbil2, Murat Sezak2, Başak Doğanavşargil2, Mümtaz Yılmaz3, Nazan Özsan2, Mine Hekimgil2 1Sıtkı Koçman University Faculty of Medicine, Department of Pathology, Muğla, Turkey 2Ege University Faculty of Medicine, Department of Pathology, İzmir, Turkey 3Ege University Faculty of Medicine, Department of Nephrology, İzmir, Turkey

To the Editor, Our patient is a 24-year-old female who was admitted to the nephrology clinic of our hospital with fatigue, weakness, and swelling of the feet. From her medical history, we learned that she had two operations for nephrolithiasis at the ages of 9 and

12, and she underwent renal transplantation in 2013, but she was still on hemodialysis. Laboratory tests showed bicytopenia (anemia and leukopenia) with hemoglobin of 8.2 g/dL and white blood cell count of 3800/mm3, and she underwent a bone marrow (BM) biopsy. Microscopically, an almost complete suppression of 79

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Turk J Hematol 2016;33:71-83

hematopoietic cells with the replacement of BM cells by foreignbody reactive fibrous tissue and numerous birefringent crystalline materials were detected (Figures 1A and 1B). The crystals formed rosettes with needle-like radial extensions surrounded by foreign body-type giant cells. Under polarized light, the crystals formed multicolored rosettes (Figure 1C). In BM aspirates envelope-like crystals were found in the background of a few myeloid cells with normal morphology (Figure 1D). Based on histopathological examination integrated with clinical results, a diagnosis of hypocellular BM associated with crystal deposition concordant with oxalate crystals was made. Because of the absence of genetic tests performed to date, the patient was referred to the genetics department, and after genetic studies the diagnosis of primary hyperoxaluria was confirmed. Pancytopenia associated with BM infiltration of different deposits is a rare condition mostly associated with amyloidosis or the accumulation of iron. One of the rarest deposits in the BM is oxalate crystals due to hyperoxaluria [1,2,3]. Primary hyperoxaluria, a genetic disorder due to mutation in the alanine glyoxylate aminotransferase gene, located on chromosome 2q37.3 and resulting in the conversion of glyoxylate to oxalate, is characterized by increased production of oxalic acid because of the specific liver enzyme deficiency and generally presents with renal stones, renal or liver failure, and oxalosis [4]. Calcium oxalate may even be deposited into various tissues such as those of the retina, peripheral nerves, arterial media, and heart [4,5]. The medical history of nephrolithiasis at early ages, characteristic appearance of birefringent crystals forming rosettes in the BM, and the envelope-like forms in the BM aspirates seen in our case supported the diagnosis of primary hyperoxaluria, which is best confirmed by genetic studies and treated with liver transplantation because of the location of the abnormal enzymes in the hepatocytes. Keywords: Oxalosis, Hyperoxaluria, Bone marrow Anahtar Sözcükler: Oksalozis, Hiperoksalüri, Kemik iliği Authorship Contributions Concept: Yelda Dere, Mine Hekimgil, Design: Simge Erbil, Yelda Dere, Data Collection or Processing: Mümtaz Yılmaz,

Figure 1. A, B: Characteristic appearance of oxalate crystals in the bone marrow, H&E, 100x. C: Colorful rosette-like crystal under polarized light, H&E, 200x. D: Colorful envelope-like crystal in the bone marrow aspirate, Giemsa stain, 100x. Başak Doğanavşargil, Murat Sezak, Nazan Özsan, Analysis or Interpretation: Mine Hekimgil, Yelda Dere, Simge Erbil, Literature Search: Başak Doğanavşargil, Murat Sezak, Simge Erbil, Writing: Yelda Dere, Mine Hekimgil, Simge Erbil. 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. Walter MJ, Dang CV. Pancytopenia secondary to oxalosis in a 23-year-old woman. Blood 1998;91:4394. 2. Halil O, Farringdon K. Oxalosis: an unusual cause of leucoerythroblastic anaemia. Br J Haematol 2003;122:2. 3. Taşlı F, Özkök G, Ok ES, Soyer N, Mollamehmetoğlu H, Vardar E. Massive bone marrow involvement in an end stage renal failure case with erythropoietinresistant anemia and primary hyperoxaluria. Ren Fail 2013;35:1167-1169. 4. Hoppe B, Beck BB, Milliner DS. The primary hyperoxalurias. Kidney Int 2009;75:1264-1271. 5. Doganavsargil B, Akil I, Sen S, Mir S, Basdemir G. Autopsy findings of a case with oxalosis. Pediatr Dev Pathol 2009;12:229-232.

Address for Correspondence/Yazışma Adresi: Yelda DERE, M.D., Sıtkı Koçman University Faculty of Medicine, Department of Pathology, Muğla, Turkey Phone : +90 505 465 31 98 E-mail : yeldamorgul@gmail.com

Received/Geliş tarihi: July 01, 2015 Accepted/Kabul tarihi: September 07, 2015 DOI: 10.4274/tjh.2015.0256

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Turk J Hematol 2016;33:71-83

An Unexpected Innocent Complication Associated with Azacitidine Treatment of Myelodysplastic Syndrome: Erythema Annulare Centrifugum Miyelodisplastik Sendrom Tedavisi Seyrinde Azasitidin İlişkili Beklenmedik Masum Komplikasyon: Eritem Annulae Santrifuj Esra Turan Erkek, Sevgi Kalayoğlu Beşışık İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

To the Editor, Skin lesions accompanying hematological malignancies can be formed due to either direct tumor infiltration of the skin or indirect effects. Indirectly developing lesions may be a component of paraneoplastic syndrome. Erythema annulare centrifugum (EAC) is considered to be a hypersensitivity reaction developed against various antigens associated with infections, drugs, and endocrine diseases. EAC, rarely seen in neoplastic diseases, has been reported in lymphoma, leukemia, histiocytosis, and prostate cancer. Here we report EAC in a patient using a hypomethylating agent, azacitidine. A 69-year-old female patient was admitted to our polyclinic with weakness and ecchymosis in her legs existing for 3 months. She was considered as having refractory anemia with excess blasts-2 according to myelodysplastic syndrome (MDS) classification [1]. Because there was only hyperdiploidy in conventional cytogenetic examination, she was classified in group intermediate-2 of the International Prognostic Scoring System. She had a history of radical mastectomy and adjuvant chemoradiotherapy for breast cancer 3 years ago. She said that variously sized round and oval erythematous, itching, painless lesions had formed in the abdominal region on the 4th day of azacitidine usage (75 mg/m2/day, 7 days, s.c.) (Figure 1 and 2). There were no concomitant complaints or physical examination findings except fatigue. After azacitidine was stopped, a skin biopsy was taken. In the biopsy, mild perivascular inflammatory infiltration accompanying vascular ectasia in the papillary dermis was detected. The possibility of paraneoplastic syndrome was excluded due to the disappearance of all lesions by 1 week after cessation of treatment. During the second course of azacitidine, the lesions reoccurred on the second day. Subsequently to the second course, the patient died of sepsis, which developed after pneumonia. EAC was first defined by Darier in 1916, and it was classified into categories of superficial and deep forms by Ackerman in 1978 [2]. In the deep form, the lesions are hard and are

usually seen together with desquamation without itching. The superficial form is characterized by itchy lesions with uncertain borders and desquamation. EAC formation is associated with trauma, ectoparasites, tuberculin test, PUVA therapy (photochemotherapy), viral infections, and diabetes. There are publications reporting that EAC may be associated with Hodgkin’s lymphoma rarely and lung, colon, cervix, prostate, stomach, and ovarian cancers even more rarely [3,4]. Lesions are often observed on the trunk, proximal portions of the limbs, and the buttocks. Today EAC is defined as a characteristic hypersensitivity reaction that can be triggered by many different antigens and disappears within 1-2 weeks. Skin lesions, whose most common forms are Sweet syndrome and myeloid sarcoma, are rarely observed in MDS [5]. Azacitidine, a

Figure 1. Erythema annulare centrifugum rashes formed during treatment.

Figure 2. Skin lesions’ regression after treatment interruption. 81

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nucleoside analogue, is one of the low-density treatment options in MDS. Azacitidine usage may cause cutaneous reactions such as urticaria, skin dryness, nodules, localized hematoma at the injection area, rash, granuloma, swelling, pigmentation changes, and induration. This case was presented because no AEC development during azacitidine use in MDS had been reported previously.

Turk J Hematol 2016;33:71-83

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. Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood 2002;100:2292-2302.

Keywords: Erythema annulare centrifugum, Azacitidine, Myelodysplastic syndrome

2. Weyers W, Diaz-Cascajo C, Weyers I. Erythema annulare centrifugum: results of a clinicopathologic study of 73 patients. Am J Dermatopathol 2003;25:451-462.

Anahtar Sözcükler: Eritem annuler santrifuj, Azasitidin, Miyelodisplastik sendrom

3. Ural AU, Ozcan A, Avcu F, Kaptan K, Taştan B, Beyan C, Yalçin A. Erythema annulare centrifugum as the presenting sign of CD 30 positive anaplastic large cell lymphoma-association with disease activity. Haematologia (Budap) 2001;31:81-84.

Authorship Contributions Concept: Sevgi Kalayoğlu Beşışık, Design: Sevgi Kalayoğlu Beşışık, Data Collection or Processing: Esra Turan Erkek, Sevgi Kalayoğlu Beşışık, Analysis or Interpretation: Esra Turan Erkek, Sevgi Kalayoğlu Beşışık, Literature Search: Esra Turan Erkek, Sevgi Kalayoğlu Beşışık, Writing: Esra Turan Erkek.

4. Carlesimo M, Fidanza L, Mari E, Pranteda G, CacchiC, Veggia B, Cox MC, Camplone G. Erythema annularecentrifugum associated with mantle B-cell non-Hodgkin’s lymphoma. Acta Derm Venereol 2009;89:319-320. 5. Soppi E, Nousiainen T, Seppa A, Lahtinen R. Acute febrile neutrophilic dermatosis (Sweet’s syndrome) in association with myelodysplastic syndromes: a report of three cases and a review of the literature. Br J Haematol 1989;73:43-47.

Address for Correspondence/Yazışma Adresi: Esra TURAN ERKEK, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey E-mail : dresraturan@gmail.com

Received/Geliş tarihi: July 10, 2015 Accepted/Kabul tarihi: September 07, 2015 DOI: 10.4274/tjh.2015.0268

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Turk J Hematol 2016;33:71-83

Thrombotic Microangiopathic Hemolytic Anemia without Evidence of Hemolytic Uremic Syndrome Hemolitik Üremik Sendrom Bulguları Olmayan Trombotik Mikroanjiyopatik Hemolitik Anemi Şinasi Özsoylu Retired Professor of Pediatrics, Hematology and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society

To the Editor, In a recent issue of this journal Dr. Oymak and her colleagues presented a clinically and genetically well-studied 5-year-old boy who was seen with severe microangiopathic hemolytic anemia without laboratory findings of renal involvement despite complement factor H gene mutations [1]. Because of Yeneral’s extensive review [2] on atypical hemolytic uremic syndrome (aHUS) published recently in the Turkish Journal of Hematology, I brought it to readers’ attention that more recently some authors do not use ‘aHUS’, which was historically used to distinguish heterogeneous uncharacterized syndromes from Shiga toxin-related HUS, since the term lacks both specificity and suggested causes [3]. Though in our patient with thrombotic thrombocytopenic purpura renal involvement was documented at the beginning but not in the last two recurrences, neither serum nor urinary findings indicated kidney involvement [4]. Although the discussions of Dr. Oymak et al. are well taken, the term ‘microangiopathic hemolytic anemia’ is covering the syndrome to a large extent as suggested by George and Nester [5].

Keywords: Microangiopathy, Kidney functions, Hemolytic anemia Anahtar Sözcükler: Mikroanjiopati, Böbrek fonksiyonları, Hemolitik anemi Conflict of Interest: The author of this paper has no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

References 1. Oymak Y, Karapınar TH, Ay Y, Özcan E, Müminoğlu N, Aydın Köker S, Töret E, Berdeli A, Serdaroğlu E, Vergin C. Thrombotic microangiopathy with complement factor H gene mutations unassociated with atypical hemolytic uremic syndrome. Turk J Hematol 2015;32:275-276. 2. Yenerel MN. Atypical hemolytic uremic syndrome: differential diagnosis from TTP/HUS and management. Turk J Hematol 2014;31:216-225. 3. Özsoylu Ş. About microangiopathic hemolytic anemia. Turk J Hematol 2015;32:92. 4. Özsoylu Ş. A case of acquired thrombotic thrombocytopenic purpura: three recurrences in 25 years. Turk J Hematol 2015;32:279-280. 5. George JN, Nester CM. Syndromes of thrombotic microangiopathy. N Engl J Med 2014;371:654-666.

Address for Correspondence/Yazışma Adresi: Şinasi ÖZSOYLU, M.D., Retired Professor of Pediatrics, Hematology and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society E-mail : sinasiozsoylu@hotmail.com

Received/Geliş tarihi: August 19, 2015 Accepted/Kabul tarihi: August 24, 2015 DOI: 10.4274/tjh.2015.0301

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0046 Turk J Hematol 2016;33:84-85

Radiologic Image of a Child with Leukemia Who Developed Sepsis and Fulminant Thrombosis during Induction Therapy İndüksiyon Tedavisi Sırasında Sepsis ve Fulminan Tromboz Gelişen Lösemili Bir Çocuğun Radyolojik Görüntüsü Eda Ataseven1, Ömer Özden2, Şebnem Yılmaz Bengoa1, Handan Güleryüz3, Murat Duman4, Hale Ören1 1Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey 2Dokuz Eylül University Faculty of Medicine, Department of Pediatric Intensive Care, İzmir, Turkey 3Dokuz Eylül University Faculty of Medicine, Department of Pediatric Radiology, İzmir, Turkey 4Dokuz Eylül University Faculty of Medicine, Department of Pediatric Emergency, İzmir, Turkey

Figure 1. Thorax and abdominal computed tomography of the patient demonstrating bilateral areas of consolidation in the lung parenchyma and multiple infarcts in the left lower lobe of the lungs, in the liver, in the spleen, in the left upper lobe of the left and middle zone of the right kidney, and in some parts of the intestines (arrows).

Address for Correspondence/Yazışma Adresi: Hale ÖREN, M.D., Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey Phone : +90 232 412 61 41 E-mail : hale.oren@deu.edu.tr

Received/Geliş tarihi: January 23, 2015 Accepted/Kabul tarihi: February 02, 2015

Turk J Hematol 2016;33:84-85

Ataseven E, et al: Sepsis and Thrombosis in a Child with Acute Lymphoblastic Leukemia

In a 5-year-old girl with acute lymphoblastic leukemia (ALL), febrile neutropenia occurred in the induction phase of chemotherapy. She was not using a central venous catheter. Despite empiric antibiotics, she developed tachypnea, bilateral rales, and disseminated intravascular coagulation (DIC). Viral, bacterial, and fungal investigations were unremarkable. Thorax and abdominal computed tomography showed bilateral consolidation areas in the lungs and multiple infarcts in the left lower lobe of the lungs, the liver, the spleen, the kidneys, and the intestines (Figure 1). Heparin infusion was started. No inherited prothrombotic defect could be shown; antiphospholipid antibodies were negative. She died of pulmonary failure. Sepsis secondary to an unknown pathogen is the most common cause of mortality and the overall risk of symptomatic thrombosis is 5.2% in ALL [1,2,3]. Despite a high incidence of central nervous system and upper venous system events, widespread thromboembolism seems to be rare [3,4]. Our patient had multiple acquired risk factors, such as leukemia, concurrent administration of Escherichia coli asparaginase and prednisone, infection, and DIC. After administration of anticoagulant therapy, patients usually show improvement, but in our patient we could not reduce the occlusive events. This case is a good reminder for hematologists that the onset of neutropenic fever may be very aggressive and thrombotic events may occur rapidly and may be fulminant in children with ALL.

Keywords: Acute leukemia, Sepsis, Thrombosis Anahtar Sözcükler: Akut lösemi, Sepsis, Tromboz Authorship Contributions Concept: Eda Ataseven, Hale Ören, Design: Hale Ören, Data Collection or Processing: Eda Ataseven, Handan Güleryüz, Murat Duman, Ömer Özden, Analysis or Interpretation: Hale Ören, Şebnem Yılmaz Bengoa, Literature Search: Eda Ataseven, Hale Ören, Writing: Eda Ataseven. 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. Kulkarni KP, Marwaha RK. Mortality pattern in childhood acute lymphoblastic leukemia with sepsis as a major barrier. J Pediatr Hematol Oncol 2012;34:264-265. 2. Lund B, Åsberg A, Heyman M, Kanerva J, Harila-Saari A, Hasle H, Söderhäll S, Jónsson ÓG, Lydersen S, Schmiegelow K; Nordic Society of Paediatric Haematology and Oncology. Risk factors for treatment related mortality in childhood acute lymphoblastic leukaemia. Pediatr Blood Cancer 2011;56:551-559. 3. Caruso V, Iacoviello L, Di Castelnuovo A, Storti S, Mariani G, de Gaetano G, Donati MB. Thrombotic complications in childhood acute lymphoblastic leukemia: a meta-analysis of 17 prospective studies comprising 1752 pediatric patients. Blood 2006;108:2216-2222. 4. Nowak-Göttl U, Kenet G, Mitchell LG. Thrombosis in childhood acute lymphoblastic leukaemia: epidemiology, aetiology, diagnosis, prevention and treatment. Best Pract Res Clin Haematol 2009;22:103-114.

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0054 Turk J Hematol 2016;33:86-87

Hereditary Elliptocytosis with Pyropoikilocytosis Piropoikilositozlu Herediter Eliptositoz Turan Bayhan, Şule Ünal, Fatma Gümrük Hacettepe University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey

Figure 1. The peripheral blood smear of the patient: prominent elliptocytic erythrocytes, in addition to microcytic erythrocytes (arrows) and fragmented erythrocytes (circles).

Address for Correspondence/Yazışma Adresi: Turan BAYHAN, M.D., Hacettepe University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey E-mail : turanbayhan@yahoo.com

Received/Geliş tarihi: January 26, 2015 Accepted/Kabul tarihi: March 09, 2015

Turk J Hematol 2016;33:86-87

A 17-day-old boy was admitted because of jaundice and anemia. He was born weighing 2900 g subsequent to a term gestation as the fourth child of first-degree cousin parents. The previous history revealed the administration of phototherapy for 4 days starting from the first day of life. Complete blood count revealed hemoglobin (Hb) of 6.9 g/dL, hematocrit of 19.8%, mean corpuscular volume (MCV) of 87.5 fL, red cell distribution width (RDW) of 37%, white blood cell count of 11.4x109/L, and platelet count of 263x109/L. Corrected reticulocyte count was 5.3%. Peripheral blood smear revealed polychromasia and pyropoikilocytosis. Direct antibody test was negative. Erythrocyte glucose-6-phosphate dehydrogenase, pyruvate kinase, and pyrimidine 5’ nucleotidase levels were normal. An erythrocyte transfusion was administered with a diagnosis of non-immune hemolytic anemia and the patient was discharged at the 26th day of life with initiation of folic acid. During his outpatient followup, he required erythrocyte transfusions 2 more times and the last transfusion was performed when he was 3 months old. At a visit 3 months after the last transfusion, his blood count was as follows: Hb of 9.5 g/dL, hematocrit of 28.2%, MCV of 68.2 fL, and RDW of 30.5%. Erythrocyte osmotic fragility was found to be normal and Hb electrophoresis revealed Hb F of 6.6% and Hb A2 of 1.7%. Upon physical examination he had mild jaundice and no splenomegaly. The parents’ blood counts were within normal ranges. Peripheral blood smear revealed prominent elliptocytes and occasional microcytic and fragmented erythrocytes with poikilocytosis (Figure 1). The clinical findings and laboratory results were diagnostic for the hereditary pyropoikilocytosis (HPP) type of hereditary elliptocytosis (HE), but in vitro fragmentation testing was not performed. HE is a common hemolytic red cell membrane disease with variant clinical presentations [1]. Common mutations that cause

Bayhan T, et al: Hereditary Elliptocytosis with Pyropoikilocytosis

HE are found in the α-spectrin, β-spectrin, and protein 4.1 genes [2]. The majority of patients with HE are asymptomatic, but HPP is a severe form of HE that presents with hemolytic anemia and jaundice during the infantile period. Erythrocyte morphology in HPP resembles that of blood smears in thermal burns with poikilocytes, red blood cell fragments, microspherocytes, and elliptocytes [3]. Low MCV (25 to 75 fL) due to fragmented red blood cells is characteristic and osmotic fragility is commonly normal [1,3]. Keywords: Anemia, Elliptocytosis, Pyropoikilocytosis Anahtar Sözcükler: Anemi, Eliptositoz, Piropoikilositoz Authorship Contributions Concept: Turan Bayhan, Design: Turan Bayhan, Şule Ünal, Data Collection or Processing: Şule Ünal, Analysis or Interpretation: Fatma Gümrük, Literature Search: Şule Ünal, Fatma Gümrük, Writing: Turan Bayhan. 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. Da Costa L, Galimand J, Fenneteau O, Mohandas N. Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders. Blood Rev 2013;27:167-178. 2. Gallagher PG. Hereditary elliptocytosis: spectrin and protein 4.1R. Semin Hematol 2004;41:142-164. 3. King MJ, Zanella A. Hereditary red cell membrane disorders and laboratory diagnostic testing. Int J Lab Hematol 2013;35:237-243.

Advisory Board of This Issue (Mart 2016) Ahmet Emre Eşkazan, Turkey Ahmet Kürekçi, Turkey Ali Bay, Turkey Ali Ghasemi, Iran Amir Steinberg, USA Aytemiz Gürgey, Turkey Balint Nagy, Hungary Canan Vergin, Turkey Cengiz Beyan, Turkey Christopher Dandoy, USA Emin Kaya, Turkey Erol Atalay, Turkey Fevzi Altuntaş, Turkey Francesca Malvestiti, Italy Füsun Özdemirkıran, Turkey Gamal Abdul Hamid, Yemen Gülden Yılmaz, Turkey

Gülsün Karasu, Turkey İbrahim Haznedaroğlu, Turkey İdil Yenicesu, Turkey İlknur Kozanoğlu, Turkey İnci Alacacıoğlu, Turkey Keith R. McCrae, USA Luke Curtis, USA Mehmet Doğu, Turkey Mehmet Gündüz, Turkey Mehmet Kantar, Turkey Mesut Ayer, Turkey Muhit Özcan, Turkey Murat Akova, Turkey Murat İnanç, Turkey Mustafa Pehlivan, Turkey Mustafa Sünbül, Turkey Müge Sayitoğlu, Turkey

Namık Özbek, Turkey Nazan Sarper, Turkey Nazlı Başak, Turkey Nil Güler, Turkey Rejin Kebudi, Turkey Selami Koçak Toprak, Turkey Selma Ünal, Turkey Şule Ünal, Turkey Sümeyye Ekmekçi, Turkey Tahir Ali, Pakistan Tezer Kutluk, Turkey Türkan Patıroğlu, Turkey Vahid Afshar-Kharghan, USA Yusuf Baran, Turkey Zeynep Karakaş, Turkey