Volume: 33 Issue: 3 by LookUs Scientific

Issue 3

September 2016

80 TL

ISSN 1300-7777

Volume 33

Review Article Clinical Interpretation of Genomic Variations Müge Sayitoğlu; İstanbul, Turkey

Research Articles The Mutation Profile of Calreticulin in Patients with Myeloproliferative Neoplasms and Acute Leukemia Jingyi Wang, et al.; Shandong, China

Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms   Neslihan Andıç, et al.; Eskişehir, Aydın, Turkey

Cytokine Contents in Chronic Lymphocytic Leukemia: Association with ZAP70 Expression Nilgün Işıksaçan, et al.; İstanbul, Turkey

The Changing Epidemiology of Bloodstream Infections and Resistance in Hematopoietic Stem Cell Transplantation Recipients Mücahit Yemişen, et al.; İstanbul, Turkey

BK Virus-Hemorrhagic Cystitis Following Allogeneic Stem Cell Transplantation: Clinical Characteristics and Utility of Leflunomide Treatment Young Hoon Park, et al.; Incheon, Republic of Korea

A Randomized Study Comparing the Efficacy of Three Hepatitis B Vaccine Induction Regimens in Adult Patients with Hematological Malignancies Zübeyde Nur Özkurt, et al.; Ankara, Turkey

Cover Picture: Mehmet Sönmez Trabzon, Turkey

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 Koç University, İstanbul, 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

Ahmet Muzaffer Demir, President

Publishing Manager Sorumlu Yazı İşleri Müdürü

Güner Hayri Özsan, General Secretary

Güner Hayri Özsan

Board of Directors

T. Tiraje Celkan, Vice President

Management Address Yayın İdare Adresi

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

Türk Hematoloji Derneği

Meltem Yüksel, Member İlknur Kozanoğlu, Member

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

Online Manuscript Submission

Publishing House / Yayınevi

http://mc.manuscriptcentral.com/tjh

Web page

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

www.tjh.com.tr

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

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

Printing Date / Basım Tarihi 15.08.2016

Cover Picture

Ahmet Muzaffer Demir

Mehmet Sönmez is currently working at Karadeniz Technical University, School of Medicine, Department of Hematology, Trabzon, Turkey.

Üç ayda bir yayımlanan İngilizce süreli yayındır. International scientific journal published quarterly. Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile Turkish Journal of Hematology’nin Türk Hematoloji Derneği İktisadi İşletmesi tarafından yayınlanmasına karar vermiştir.

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AIMS AND SCOPE The Turkish Journal of Hematology is published quarterly (March, June, September, and December) by the Turkish Society of Hematology. It is an independent, non-profit peer-reviewed international English-language periodical encompassing subjects relevant to hematology. The Editorial Board of The Turkish Journal of Hematology adheres to the principles of the World Association of Medical Editors (WAME), International Council of Medical Journal Editors (ICMJE), Committee on Publication Ethics (COPE), Consolidated Standards of Reporting Trials (CONSORT) and Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). The aim of The Turkish Journal 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: 0.827 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 Yayınevi Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul, Turkey Telephone : +90 212 621 99 25 Fax : +90 212 621 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 should complete the ICMJE general declaration form, which can be obtained at: http://www.icmje.org/coi_disclose.pdf.

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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 post-hepatitis 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 Hemoglobin:

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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. Highresolution 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 prepublished 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.

Hematology component

SI units

Conditions of Publication

RBC

6.7-11 x 1012/L

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.

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

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.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.

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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.

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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 172

Review Article

180

Research Articles

Clinical Interpretation of Genomic Variations Müge Sayitoğlu

The Mutation Profile of Calreticulin in Patients with Myeloproliferative Neoplasms and Acute Leukemia Jingyi Wang, Jianguo Hao, Na He, Chunyan Ji, Daoxin Ma

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Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms Neslihan Andıç, Mustafa Ünübol, Eren Yağcı, Olga Meltem Akay, İrfan Yavaşoğlu, Vefki Gürhan Kadıköylü, Ali Zahit Bolaman

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Cytokine Contents in Chronic Lymphocytic Leukemia: Association with ZAP70 Expression Nilgün Işıksaçan, Suzan Çınar, Esin Aktaş Çetin, Melih Aktan, Günnur Deniz

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Finding the Optimal Conditioning Regimen for Relapsed/Refractory Lymphoma Patients Undergoing Autologous Hematopoietic Cell Transplantation: A Retrospective Comparison of BEAM and High-Dose ICE Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür Kayıkçı, Gamze Durgun, Şerife Kocubaba, Fevzi Altuntaş

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223

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The Changing Epidemiology of Bloodstream Infections and Resistance in Hematopoietic Stem Cell Transplantation Recipients Mücahit Yemişen, İlker İnanç Balkan, Ayşe Salihoğlu, Ahmet Emre Eşkazan, Bilgül Mete, M. Cem Ar, Şeniz Öngören, Zafer Başlar, Reşat Özaras, Neşe Saltoğlu, Ali Mert, Burhan Ferhanoğlu, Recep Öztürk, Fehmi Tabak, Teoman Soysal BK Virus-Hemorrhagic Cystitis Following Allogeneic Stem Cell Transplantation: Clinical Characteristics and Utility of Leflunomide Treatment Young Hoon Park, Joo Han Lim, Hyeon Gyu Yi, Moon Hee Lee, Chul Soo Kim A Randomized Study Comparing the Efficacy of Three Hepatitis B Vaccine Induction Regimens in Adult Patients with Hematological Malignancies Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı

Brief Reports

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Results of Four-Year Rectal Vancomycin-Resistant Enterococci Surveillance in a Pediatric Hematology-Oncology Ward: From Colonization to Infection Hacer Aktürk, Murat Sütçü, Ayper Somer, Serap Karaman, Manolya Acar, Ayşegül Ünüvar, Sema Anak, Zeynep Karakaş, Aslı Özdemir, Kutay Sarsar, Derya Aydın, Nuran Salman

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Radiation-Induced Tumor Lysis Syndrome in Chronic Lymphocytic Leukemia Ali Alkan, Tuğçe Kütük, Ebru Karcı, Arzu Yaşar, Ayşe Hiçsönmez, Güngör Utkan

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A Case of Superwarfarin Poisoning Due to Repetitive Occupational Dermal Rodenticide Exposure in a Worker

Zehra Narlı Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, Berna Ateşağaoğlu, Meral Beksaç

254

Letters to the Editor

Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı

255

Cerebral Sinovenous Thrombosis Mimicking Intracranial Mass

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A Rare Cause of Unexplained Refractory Iron Deficiency Anemia: Unicentric Plasma-Cell Type Castleman’s Disease

Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden, İbrahim Öner Doğan, Fatih Beşışık

259

Images in Hematology

Erdoğan Nohuz, Sharif Kullab, Albane Ledoux-Pilon, Cécile Moluçon-Chabrot, Maël Albaut, Luisa De Simone, Xavier Durando

261

Endothelial Cells, Ankaferd Hemostat, and Estradiol

Yasemin Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu

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An Unusual Congenital Anomaly in Fanconi Aplastic Anemia: Congenital Lobar Emphysema

Ali Fettah, Gökçe Pınar Reis, Soner Sertan Kara, Tekin Aksu, Afak Durur Karakaya, Mahmut Subaşı, Atilla Çayır

A Primary Bone Diffuse Large B-Cell Lymphoma with Ocular Adnexal Involvement

Derya Özyörük, Neslihan Karakurt, Arzu Yazal Erdem, Suna Emir, Bahattin Tunç, Neşe Yaralı, Namık Özbek

Vaginal Lymphoma: A Possible Cause of Genital Hemorrhage

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Advisory Board of This Issue (September 2016) Agnieszka Bojarska Junak, Poland Ahmet Emre Eşkazan, Turkey Ahmet Soysal, Turkey Akif Selim Yavuz, Turkey Alessandro Allegra, Italy Anıl Tombak, Turkey Ayşegül Ünüvar, Turkey Bülent Kantarcıoğlu, Turkey Can Balkan, Turkey Canan Baydemir, Turkey Celalettin Üstün, USA Cem Ar, Turkey Dae-Young Kim, Korea Darko Antic, Serbia Dilber Talia İleri, Turkey Dilek Sevgi, Turkey Dorothy Funk, USA

Düzgün Özatlı, Turkey Eda Tahir Turanlı, Turkey Elif Ünal, Turkey Eva Posfai, Hungary Fortunato Morabito, Italy François Vincent, France Gökhan Aygün, Turkey Güray Saydam, Turkey Hakan Özdoğu, Turkey Hakim Ouled Haddou, France Hüsnü Pullukçu, Turkey İbrahim Haznedaroğlu, Turkey İbrahim Köker, Turkey İhsan Karadoğan, Turkey Mahmut Töbü, Turkey Mehmet Ali Özcan, Turkey Mehmet Sönmez, Turkey

Nazan Sarper, Turkey Nicolaus Kröger, Germany Özgür Mehtap, Turkey Qing Ma, USA Reşat Özaras, Turkey Şule Ünal, Turkey Takahiko Nakane, Japan Takahiro Yamauchi, Japan Tunç Fışgın, Turkey Türkan Patıroğlu, Turkey Umberto Gianelli, Italy Varinder Kaur, USA Veysel Sabri Hançer, Turkey Volkan Hazar, Turkey Yener Koç, Turkey Yusufhan Yazır, Turkey Zühre Kaya, Turkey

REVIEW DOI: 10.4274/tjh.2016.0149 Turk J Hematol 2016;33:172-179

Clinical Interpretation of Genomic Variations Genomik Varyasyonların Klinik Yorumlanması Müge Sayitoğlu İstanbul University Faculty of Medicine, Institute of Experimental Medicine, Department of Genetics, İstanbul, Turkey

Abstract

Öz

Novel high-throughput sequencing technologies generate largescale genomic data and are used extensively for disease mapping of monogenic and/or complex disorders, personalized treatment, and pharmacogenomics. Next-generation sequencing is rapidly becoming routine tool for diagnosis and molecular monitoring of patients to evaluate therapeutic efficiency. The next-generation sequencing platforms generate huge amounts of genetic variation data and it remains a challenge to interpret the variations that are identified. Such data interpretation needs close collaboration among bioinformaticians, clinicians, and geneticists. There are several problems that must be addressed, such as the generation of new algorithms for mapping and annotation, harmonization of the terminology, correct use of nomenclature, reference genomes for different populations, rare disease variant databases, and clinical reports.

Yeni dizileme teknolojileri, tek genli ve/veya kompleks kalıtılan hastalıklarla ilgili genlerinin haritalanması, kişiye özel tedavi ve farmakogenomik alanları için yüksek verimde ve büyük ölçekte genomik data üreten teknolojilerdir. Yeni nesil dizileme, hastaların tanı ve tedavi yanıtlarını değerlendirecek moleküler izlem süreçlerinde kullanılmak üzere, hızlı bir şekilde rutin uygulamada yerini bulmaktadır. Yeni nesil dizileme platformları çok büyük ölçekte genetik varyasyon datası üretmektedir ve bu varyasyonların klinik olarak anlamlandırılması çok zordur. Klinik yorumlamalar, hekimler ile biyoinformatik ve genetik uzmanlarının yakın işbirliğine ihtiyaç duymaktadır. Yeni haritalama ve hizalama araçlarına duyulan ihtiyaç, terminolojinin harmonizasyonu, genetik isimlendirmenin doğru kullanımı, farklı populasyonlar için referans genom datasının bulunmaması, nadir hastalıklar için genomik veri bankalarının eksikliği ve klinik raporlama, halen aşılması gereken sorunlar arasında bulunmaktadır. Anahtar Sözcükler: Genetik varyasyon, Dizileme, Genomik data, Klinik yorum.

Keywords: Genetic variation, Sequencing, Genomic data, Clinical interpretation

Introduction Next-generation sequencing (NGS) methods provide cheap and solid genomic data and are used extensively for de novo sequencing, disease mapping, quantifying of expression levels, and population genetic studies [1,2,3,4,5]. They can also be applied to complex disorders [6], personalized treatment, and pharmacogenomics [7,8,9]. The medical genetics field translates high-throughput genetic data to clinical settings in order to improve diagnostic efficiency and treatment decision-making [10,11]. The interpretation of the clinical significance of genomic variants in a given patient or in patients’ family members is the main challenge of resequencing. In the last decade, several diseases and syndromes have been analyzed by NGS, hundreds of disease-associated genes have been found, and novel targeted therapies have been developed. The most powerful contribution of NGS [particularly whole-exome sequencing (WES) and whole-genome sequencing (WGS)] is the description of new candidate signaling pathways involved in the pathogenesis of a

clinical condition that will help with prevention, diagnosis, and therapeutic opportunities. High-throughput sequencing can be implemented within different applications including WGS, WES, ribonucleic acid sequencing (RNA-seq), or targeted sequencing [12]. Commercially available NGS platforms are generally employed with similar steps for all these approaches: generation of sequencing libraries, sequencing simultaneously in a massively parallel fashion, and data analysis [12]. Whole-exome sequencing studies have been commonly used to identify the responsible gene of a clinical phenotype. The WGS approach holds major advantages for the detection of variations not only in the exome but also in the noncoding regulatory regions for complex and/or multigenic diseases. The analysis of WGS data is complicated by the amount of information and challenges in elimination of common genetic variations. Wholeexome sequencing studies require rough bioinformatics analysis

Address for Correspondence/Yazışma Adresi: Müge SAYİTOĞLU, M.D., İstanbul University Faculty of Medicine, Institute of Experimental Medicine, Department of Genetics, İstanbul, Turkey Phone : +90 212 414 22 00-33312 E-mail : mugeay@istanbul.edu.tr

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Received/Geliş tarihi: April 13, 2016 Accepted/Kabul tarihi: August 07, 2016

Turk J Hematol 2016;33:172-179

work and experts and national reference sequences to evaluate the population-based genetic variants, along with large budgets. Alternatively, WES is relatively cost-efficient and is able to discover disease-related rare variants in coding regions and splice sites. There are several variations that have been successfully identified by WES in monogenic diseases. On the other hand, the exome represents less than 1% of the genome and such analysis will be excluding noncoding genomic regions such as regulatory regions, repetitive sequences, or noncoding RNAs. Using gene panels in NGS studies is an alternative option that restricts screening to selected genetic regions. Although it may simplify the scale of the analysis and interpretation, incidental findings still require attention. The most suitable NGS approach for routine clinical applications is ampliconbased/targeted sequencing. Most genetic disorders have allelic and locus heterogeneity, which means that one disease may arise from different genetic variations within the same gene or different genes. Due to the genetic heterogeneity, it takes longer to obtain the genetic test result, which leads to a delay in diagnosis. Amplicon-based NGS has provided a major advantage for the molecular analysis of heterogeneous genetic disorders, including hereditary cancers [13]. RNA-seq quantifies the amount of transcripts (all transcribed isoforms) and gives a chance to evaluate the whole RNA repertoire of a specific cell or tissue. The biggest limitation of RNA-seq is the “noncoding RNAs”; most of the genome is transcribed but the majority of these transcripts are not translated into proteins [14]. Accurate Use of the Terminology: Is It a Polymorphism or a Disease-Related Variation? In common use, a DNA polymorphism is a heritable variation that is present in >1% of the population and increasingly detected by next-generation resequencing. One of two or more alternate forms of a locus (alleles) may result from the changes in the nucleotide sequence [single nucleotide polymorphisms (SNPs)], deletions, insertions, or other structural rearrangements. According to the novel terminology the term ‘SNP’ is used as single nucleotide variation (SNV) [15]. A genome contains repetitive sequences differing in copy numbers (i.e. copy number variations) between individuals. Polymorphic variations may or may not have phenotypic effects and they are valuable tools for genetic mapping studies including linkage and association studies of diseases. The vast majority of these variations (more than 90%) have been found to be localized in the noncoding genomic regions and are possibly involved in regulation of gene expression. On the other hand, a mutation is defined as a DNA variant detectable in <1% of the population and generally having phenotypic consequences.

Müge Sayitoğlu. Clinical Interpretation of Genomic Variations

The alternative use of the terms of “polymorphism” and “mutation” for an event (a difference compared with a reference standard) commonly leads to misinterpretation. This problem also can affect the accuracy of clinical interpretation and the functional relationship between a phenotype/disease and a genomic sequence. It is critical to establish clear nomenclature and guidelines regarding the identified genomic variations and their reporting. The definition of a “genetic variant” is currently in use to describe differences in comparison to a reference standard. This term can include neutral, benign, functional, pathogenic, deleterious, damaging, disease-associated, or causal definitions. Uniform terminology is recommended to correct the interpretation of a variant and to share correct genomic information. The Human Genome Variation Society (HGVS; http://varnomen.hgvs.org/) established a standard gene variant nomenclature and it is recommended for use as the primary guideline for determining variants [16]. Each genome has nearly 4 million genetic variations and each exome covers nearly 13,000 SNVs. The challenge facing researchers and clinicians is to depict the biological and clinical significance of these variants and transfer this information to clinical practice. Basic resequencing data analysis includes base calling, mapping, variant calling, and annotation steps (Figure 1). Every step in the variant interpretation process has limitations and difficulties including variation type, sample source and quality, and clinical heterogeneity, among other factors.

1) Base Calling (Image Processing) Next-generation sequencing platforms are able to generate millions of reads to reduce the costs. However, despite the technological progress, NGS results are adversely affected by biochemical and signal acquisition mistakes. Next-generation sequencing platforms have different performance levels that

Figure 1. Main steps of re-sequencing data analysis. 173

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rely on complex interactions of the chemistry, the hardware, and the optical sensors that they use. For example, in the Illumina system, the images that are acquired from the instruments are prepared and analyzed to determine the base incorporated in the complementary strand. In this process, the ordering of nucleotides in a template from the noisy signals is referred to as base calling [17]. In other words, base calling converts the fluorescence signals into actual sequence data with quality scores. Base calling accuracy is measured by a Q score (Phred quality score), which is a common metric to assess the accuracy of a sequencing run. The Q score is defined as the logarithmically related base calling error probability (Q=-10 log P/log 10) [18]. For example, if Q=40 for a sequencing run, this is equal to the probability of an incorrect base call of 1 in 10,000 times, or with 99.99% base calling accuracy or a lower Q score of 10 means, there is the probability of an incorrect call in 1 of 10 bases. Low Q scores lead to false positive variant calls and need resequencing. Errors arising from NGS data are generally due to base calling and alignment applications. Moreover, low coverage sequencing (<5×) includes the high probability that only one of the two chromosomes of a diploid individual has been sampled at a specified site. Another option to improve base calling accuracy could be to increase the sequencing cycles (coverage=resequencing copies). Several base calling strategies have been developed to infer the correct base more reliably and to perform base calling faster [19].

2) Mapping (Alignment to a Reference Genome) After successful base calling, the next step is the mapping or alignment of the sequenced genomic region. The main challenge is to accurately find the true location of each read from a large quantity of reference data and then to distinguish the technical sequencing errors and disease-related genetic variation within the sample. Next-generation sequencing platforms generally produce short reads (~200-300 bp) and we need to align or map these fragments to a reference sequence to find the corresponding part of the short reads. There are some limitations to mapping: 1) Reference sequences can be very long; for example, it is ~3 billion bases for humans and it is a crucial task to find the matching short region. 2) Since the sequences are short they can align to several places that have similar DNA sequences (such as repetitive sequences) in the genome. 3) It is not possible to get a perfect alignment because of in/del variations in the genome, so there will be some mismatches or gaps during the mapping. Mappers perform global or local alignments with respect to the approach; for example, WGS and WES data need appropriate reference sequences to find all the genetic variations. RNA sequencing data can be mapped to the full reference sequence, or to a special transcriptome reference. Mapping necessitates computational time and critical computational requirements. There are several tools (e.g., BWA, SAM, GATK (http://www. broadinstitute. org/gatk/), Bowtie, or RMAP) that are available 174

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and designed for aligning DNA, RNA, or proteins [20,21,22,23,24].

3) Variant Calling Variant calling is an important procedure for resequencing deep sequencing analysis. Next-generation sequencing platforms generate huge amounts of genetic variation data; the main challenge is to discriminate a small subset of functionally important variants. When analyzing WES or WGS data, comparison with a correct reference plays a pivotal role. Determining the genetic variation of a reference genome from the target genome allows the identification of the disease-related genetic variations. Variant calling predicts the nucleotide differences versus a reference sequence (genome or transcriptome) at a given position, generally accompanied by an estimate of variant frequency and confidence intervals. Integrity of the alignment has a crucial role in variant detection; if the sequence is incorrectly aligned, it may lead to spontaneous errors in variant calling. Genomic variants, such as SNVs, insertions, deletions, and in/dels (the occurrence of an insertion and deletion at the same time) can be identified by various analysis pipelines [25]. “SNV calling” identifies single nucleotide variable sites, whereas “genotype calling” determines the genotype for each individual at each site. To reduce the uncertainty associated with SNV calling is to increase the coverage (at >20× coverage). In association studies, sequencing many individuals at a low depth, rather than sequencing fewer individuals at a high depth, could maximize mapping power. Some of the genomic regions are difficult to interpret, such as homopolymer regions (a sequence of identical bases, like AAAA or TTTTTTTT), or simple repeats (minisatellitevariable number of tandem repeats and microsatellite-short tandem repeats). Bioinformaticians use VCF files, “Variant Call Format”, to store the gene sequence variations.

4) Annotation and Prioritization of a Variation Several challenges arise for NGS-based diagnostic and research efforts in the identification of all genetic variations. Because of the increased complexity of data analysis and clinical interpretation of the data, it is best to use some universally accepted recommendations like those of the American College of Medical Genetics and Genomics (ACMG), EuroGentest, and the European Society of Human Genetics [25,26,27]. The ACMG recommends that both “mutation” and “polymorphism” can be replaced by “variant” with the following modifiers: pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), and benign (B) [26] (Figure 2). The “likely” term is used to define certainty greater than 90% of a variant either being disease-causing or benign. Pathogenic Variant If the previously identified or novel variation has substantial evidence that it causes a disease with a known or unknown mechanism, is called a “pathogenic” variant. These kinds of

Turk J Hematol 2016;33:172-179

Müge Sayitoğlu. Clinical Interpretation of Genomic Variations

American College of Medical Genetics and Genomics suggested additional criteria including very strong, strong, and moderate support for being pathogenic, and likely pathogenic and likely benign variations. Nonsense mutations, frame shifts, exonic deletions, and promoter variations (very strong) are generally assumed to cause loss of function in the genes. These kinds of variations lead to reduced or absent gene function and nonsense-mediated decay of an altered transcript. These kinds of variations are expected to affect the clinical findings.

Figure 2. Recommended terms for interpretation of clinical variants. variations are generally nonsense mutations, frame shift variations, or splice site alterations. Likely Pathogenic Variant If the previously identified or novel variation is consistent with the diagnosis, it exists in the conserved genomic region, functional studies showed impaired gene product, or the function of the gene is known to be associated with a specific phenotype, the variation is called a “likely pathogenic” variant. Uncertain Significance Variant If a variant cannot be classified as pathogenic or benign, it is called a “variant of uncertain clinical significance” (VUS). It can be a missense variation, an in-frame deletion, or an insertion. These kinds of novel variations can cause confusion during interpretation and reporting. If there is no other variant identified, VUS should be highlighted in the report. Likely Benign Variant If a variant presents at high frequency in random individuals and is not a high penetrant or a disease-causing variant, it is called a “likely benign” variant. There is no absolute frequency threshold to classify that a variant is likely benign or likely pathogenic. This depends on the disease model, clinical characteristics, etc. These can be novel or previously reported variations with possible neutral effects. Generally, likely benign variants have enough evidence that they are not the cause of the disease, and the segregation analysis of haplotypes in affected and unaffected family members can support this finding. Benign Variant If a previously reported variation is present at a higher frequency in the general population, it is called a “benign” variant. These variations are nonpathogenic and have neutral effects.

Splice site variations may cause exon skipping and shortening or inclusion of intronic material due to loss or recreation of donor/ acceptor splice sites. These kinds of variations are predicted to lead to a null effect that needs additional functional analysis (RNA or protein). A missense variation is mostly known to be pathogenic; it alters the protein function or the nucleotide change and may disrupt the splice site. It can be detected by in silico prediction tools and then concluded to be a disease-related variation. Missense variations should be evaluated with minor allele frequency (MAF) values, which refer the second most common allele that occurs in a given population. The MAF value provides information to differentiate between common and rare variants in the population. If the determined missense variation has a low MAF value (<0.5%), it might be evaluated as a diseaserelated variation. Although an index case might have the variation that is supporting the disease association, if the parents do not have it, it can be concluded as a “de novo” variation. However, in all cases, a detailed family history and verification of paternity is needed. Furthermore, due to the germline mosaicism possibility, the same disease may affect more than one sibling. If there is only one affected proband and no previous history in a family, scientists should consider sequencing the unaffected parents of the proband to identify de novo mutations. Another issue to be aware of is “compound heterozygosity”, and especially for autosomal recessive inherited disorders it should be carefully analyzed. Paternal validation is needed to understand the genetic background of different variations within the same gene, which come from the mother and the father of the index case.

4.1. Database Search: Population- and Disease-Based There are large numbers of databases in use, including both population-based and disease-based databases. Eliminating known variants that are present in public (dbSNP) and inhouse variant databases and published projects such as the 1000 Genomes Project [28], EXAC, and the Exome Sequencing 175

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Project (ESP6500) [29] is a very helpful strategy to reduce the candidate list of disease-related variations. Population-based databases (such as the 1000 Genomes Project or ESP) have been created both for large and small local populations [30,31]. They are useful to obtain the frequencies of the variations. Disease databases mainly contain the variants of a specific disease or phenotype [32]. There are some limitations to these databases. For example, there is no absolute frequency threshold for a given variant, many populations are not represented, and there is no information about the phenotype. Limited numbers of locus-specific databases also exist but those are not available for most genes, there are contradictory data between databases, and they may not be updated. For correct data interpretation, researchers should check the updates of the databases, confirm that HGVS nomenclature is being used, and read the relevant publications [16,33]. Gene- or disease-oriented biomedical information can be found from the OMIM website (Online Mendelian Inheritance in Man- http://www.omim.org), in published scientific articles (PubMed- http://www.ncbi.nlm.nih.gov/pubmed), and in mutation databases (HGMD, Human Gene Mutation Databasehttp://www.hgmd.cf.ac.uk/ac/index.php). Clinicians can interpret a variant when it is reported and track the genotype-phenotype correlation, population frequency of the variation, and clinical assertions. Clinical laboratories should increase their collaboration with clinicians to better understand the effect of the variation on the phenotype. The ClinVar

database (http://www.ncbi.nlm.nih.gov/clinvar/) archives reports of the relationships among medically important human variations and phenotypes. It has access to dbSNP and dbVar and includes information about the location of variation and phenotypic descriptions included in MedGen (http://www.ncbi. nlm.nih.gov/medgen). ClinVar is an interactive tool that can be divided into submitter, variation, phenotype, interpretation, and evidence. ClinVar represents the interpretation of a single allele, compound heterozygotes, haplotypes, and combinations of alleles in different genes [34,35]. Searching for previously published scientific and medical studies is also a valuable tool for the annotation of a detected variant. Researchers should be aware of using older versions of nomenclature in published reports. The given information about the index case, affected family members, and the size of the family should be carefully noted to avoid incorrect data.

4.2. In Silico Functional Prediction A variety of algorithms (SIFT, PolyPhen, Provean, CADD, Condel, GERP, SNAP, SNPs&Go, PhyloP, and MutationTaster) are used to determine the effect of variations and that can be done at the nucleotide, amino acid, protein, and transcript/splice variant levels (Table 1). Mainly they have been developed to estimate the deleterious effect of a variant on a protein. The most common use of these tools is to predict the impact of a missense variation on a protein and to predict the effect of the variation on splicing. The prediction depends on the location, evolutionary conservation, amino acid charge, 2D and 3D calculations of the

Table 1. Representative in silico prediction tools and web pages. In Silico Prediction Tools Missense prediction SIFT

http://sift.jcvi.org

Mutation Tester

http://www.mutationtester.org

Mutation Assessor

http://mutationassessor.org

PolyPhen2

http://genetics.bwh.harvard.edu/pph2

MutPred

http://mutpred.mutdb.org

nsSNP Analyzer

http://snpanalyzer.uthsc.edu

Condel

http://bg.upf.edu/fannsdb/help/condel.html

CADD

http://cadd.gs.washington.edu/score

Provean

http://provean.jcvi.org/index.php

Splice site prediction Gene Splicer

http://www.cbcb.umd.edu/software/GeneSplicer/gene_spl.html

Human Splicing Finder

http://umd.be/HSF/

Net Gene 2

http://www.cbs.dtu.dk/services/NetGene2

Conservation prediction PhastCons

http://compgen.bscb.cornell.edu/phast/

PhyloP

http://compgen.bscb.cornell.edu/phast/

GERP

http://mendel.stanford.edu/sidowlab/downloads/gerp/index.html

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effect on protein structure, and biochemical consequences of the amino acid substitution. Some of these tools are used for the prediction of the effects on splicing and loss or creation of the splice sites. As a limitation of in silico tools, variable and incompatible interpretation results are derived from different algorithms and the use of multiple programs is recommended because of the differing performances of the tools [36].

4.3. Clinical Interpretation and Reporting of NextGeneration Sequencing Results Interpretation and reporting of candidate genetic variations is the biggest challenge in NGS data analysis and reporting processes. Genetic testing based on WGS often results in several variations that are not directly clinically actionable. The reportable variations should be classified as pathogenic (P), likely pathogenic (LP), a variant of uncertain significance (VUS), likely benign (LB), or benign (B) as described by the ACMG. Misinterpretation of data may be due to annotation errors, analytical errors, ethnicity effects (differential MAF values), reduced reproducibility in consideration of low-level mutations, nomenclature or terminology differences, and variable databases. International guidelines and recommendations developed to standardize and regulate deep sequencing are good references for researchers and clinicians [37]. Some general recommendations are summarized below to exclude possible incidental findings and ensure correct clinical interpretation and reporting of NGS results. Data Quality (Base Calling and Mapping) - If the Q score is low (Q30 score is lower than 70%) and total coverage is lower than 80%, sequencing should be repeated. - Different algorithms generate different outputs. Since the accuracy of the annotation depends on the success of the mapping, it is best to use at least two algorithms for mapping. - Previous NGS data generated from the same laboratory (inhouse data) are valuable to evaluate and exclude variations that arise from technical effects or poor quality of amplicon design. In-house laboratory data provide simplified analysis to exclude false variants (false positivity). - Next-generation sequencing data aim to achieve a high diagnostic yield to achieve high coverage in all genomic regions covered. If genetic variation is detected by NGS with low coverage, resequencing should be repeated, and clear communication with the clinician is required if the test results cannot be used to exclude a particular clinical diagnosis.

Müge Sayitoğlu. Clinical Interpretation of Genomic Variations

NGS, Sanger sequencing, or another proper technique to exclude false positive results. Validation results should be included in the NGS report. - All variants should be annotated and reported with regard to the gene name; gene symbol; heterozygous, homozygous, or compound heterozygous condition; nucleotide changes in coding regions; and amino acid changes in proteins according to the HGVS [16]. Mutalyzer is useful software to check the nomenclature for variations (https://mutalyzer.nl/). Each report should include the reference sequence and the use of unique nomenclature is critical; “g” represents the genomic sequence, “c” represents the coding sequence, “p” represents protein and “m” represents mitochondria, and the first translational codon (ATG) is the starting point. The universal reference genome (hg18, hg19, or hg38) and the latest versions should be used to give the correct genomic coordinates and it should cover the 5’ and 3′ untranslated regions and promoter regions (http://www. ncbi.nlm.nih.gov/refseq/) [38,39]. - Reports should state the limitations of each specific NGS test regarding the detection of different kinds of mutations. - The reference genome, software, and databases (COSMIC, ClinVar, dbSNP, etc.) that are used should be specified in the report. If the variant was previously identified, the functional and clinical significance of the variation should be stated referring to the COSMIC database, the HGMD, or a scientific publication. - For diagnostic purposes, only genes with a known (i.e. published and confirmed) relationship between the aberrant genotype and the pathology should be included in the analysis. The NGS test results should be included with the disease name, its targets, the names of the genes tested, their reportable ranges, the analytical sensitivity and specificity, and, if possible, the diseases not relevant to the clinical phenotype that could be caused by mutations in the tested genes [40]. - For diagnostic purposes, all pathogenic and likely pathogenic variants have to be reported. Whether or not variants of unknown significance (VUS) are reported will depend on local practice. Researchers should be very cautious if detailed laboratory analysis has not been performed and this should be included in the report. If no variation has been defined other than a VUS, it should be highlighted in the report. In that case, clinicians are strongly suggested to discuss the result with a clinical geneticist and it is acceptable to request additional analysis (parental testing, etc.) in order to facilitate the interpretation of the result (http://www.acgs.uk.com). The latter has to be clear for laboratory scientists, as well as for the referring clinicians [40].

Reporting

Conclusions

- Next-generation sequencing results should not be transferred to clinical reports and practice without acceptable validation. It is essential to confirm the variation from a new DNA sample by

In medical use of genetic discoveries, it is quite important to improve the standards of data collection and sharing to define a systematic method for the clinical annotation and interpretation 177

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of genomic and phenotypic variations. Data-sharing platforms like the Undiagnosed Diseases Network (UDN- https://www. genome.gov) or Matchmaker Exchange Network (http://www. matchmakerexchange.org) for researchers of rare diseases and clinicians for sharing clinical phenotypes and sequencing data, which may allow for identification of other patients with the same phenotype, help us to understand the functional relevance of the variant that is obtained and reported [41,42].

variations. Amplicon sequencing is a highly targeted approach for analyzing genetic variation in specific genomic regions.

Next-generation sequencing technology is being used as a diagnostic tool because of the expanded utility and reduced costs. Targeted sequencing offers better running times, costs, datasets, and coverage compared to WES or WGS. However, there are still many concerns about the application of NGSbased diagnostics. The challenges and clinical applications of NGS results have been discussed here. These include the accumulation and storage of huge amounts of genomic data, the need for bioinformatics experts, the need for national reference genomes, reimbursement of sequencing costs, and, of course, clinical interpretation of novel and VUS results.

Variation: Now used for mutations and polymorphisms, a change in the DNA or RNA sequence compared to a reference genome.

Glossary Allele: Alternative form of a given locus. Annotation: DNA annotation or genome annotation is the identification of the locations of genes and all of the coding regions in a genome and determination of their function. Frameshift variation: Genetic variation caused by indels (insertions or deletions) of a number of nucleotides in DNA. Missense variation: A single nucleotide variation that leads to amino acid substitution and a codon change. Also called nonsynonymous substitution. Nonsense variation: A single nucleotide variation that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and usually nonfunctional protein product. Deep sequencing: Indicates that the total number of reads is many times larger than the length of the sequence under study. Depth: In DNA sequencing refers to the number of times a nucleotide is read during the sequencing process. Coverage: The average number of reads representing a given nucleotide in the reconstructed sequence. Whole-genome sequencing (WGS): A laboratory process that determines the complete DNA sequence of an organism’s genome at a single time. Whole-exome sequencing (WES): A technique for sequencing all the expressed genes in a genome (known as the exome). Amplicon (targeted) sequencing: Amplicon sequencing refers to ultradeep sequencing of PCR products for analyzing genetic 178

Pathogenic: Anything that can produce disease. DNA polymorphism: A heritable variation that is present in >1% of the population and increasingly detected by next-generation resequencing. Mutation: DNA variants detectable in <1% of the population.

SNV: Single nucleotide variation.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0220 Turk J Hematol 2016;33:180-186

The Mutation Profile of Calreticulin in Patients with Myeloproliferative Neoplasms and Acute Leukemia Miyeloproliferatif Neoplazisi ve Akut Lösemisi Olan Hastalarda Kalretikülin Mutasyon Profili Jingyi Wang1,2, Jianguo Hao3, Na He1, Chunyan Ji1, Daoxin Ma1 1Qilu Hospital of Shandong University, Department of Hematology, Shandong, China 2Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Department of Hematology, Shandong, China 3General Hospital of Shandong Stell Group Company, Department of Surgery, Shandong, China

Abstract

Öz

Objective: Calreticulin (CALR) plays important roles in cell proliferation, apoptosis, and immune responses. CALR mutations were described recently in Janus kinase 2 gene (JAK2)-negative or MPLnegative primary myelofibrosis (PMF) and essential thrombocythemia (ET) patients. CALR trails JAK2 as the second most mutated gene in myeloproliferative neoplasms (MPNs). However, little is known about CALR mutation in Chinese patients with leukemia. In the present study, a cohort of 305 Chinese patients with hematopoietic neoplasms was screened for CALR mutations, with the aim of uncovering the frequency of CALR mutations in leukemia and MPNs.

Amaç: Kalretikülin (CALR) hücre çoğalması, apoptoz ve immün yanıtlarda önemli rol oynar. CALR mutasyonları yakın zamanda Janus kinaz 2 (JAK2) veya MPL geni negatif primer miyelofibroz (PMF) ve esansiyel trombositemi (ET) hastalarında tanımlanmıştır. CALR JAK2’yi takiben miyeloproliferatif neoplazilerde (MPN) ikinci sıklıkta görülen mutant gendir. Ancak, Çinli lösemi hastalarında CALR mutasyonları hakkında bilgi sınırlıdır. Bu çalışmada, hematopoetik neoplazisi olan 305 Çinli hasta CALR mutasyonları, bu mutasyonların lösemi ve MPN hastalarındaki sıklığının ortaya çıkarılması için taranmıştır.

Materials and Methods: Polymerase chain reaction and direct sequencing were performed to analyze mutations of CALR in 305 patients with hematopoietic malignancies, including 135 acute myeloid leukemia patients, 57 acute lymphoblastic leukemia patients, and 113 MPN patients.

Gereç ve Yöntemler: Polimeraz zincir reaksiyonu ve direkt dizileme yöntemi 135 akut miyeloid lösemi, 57 akut lenfoblastik lösemi ve 113 MPN olmak üzere toplam 305 hematopoetik malinitesi olan hastada CALR mutasyonlarını analiz etmede kullanılmıştır.

Results: CALR mutations were found in 10.6% (12 of 113) of samples from patients with MPNs. CALR mutations were determined in 11.3% (6 of 53), 21.7% (5 of 23), and 9.1% (1/11) of patients with ET, PMF, and unclassifiable MPN, respectively.

Bulgular: CALR mutasyonu MPN hastalarının %10,6’sında (12/113) tespit edilmiştir. Ayrıca bu mutasyonlar ET, PMF ve sınıflandırılamayan MPN hastalarında sırasıyla %11,3 (6/53), %21,7 (5/23) ve %9,1 (1/11) olarak bulunmuştur.

Conclusion: We showed that MPN patients carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels compared to those with mutated JAK2. However, all of the leukemia patients had negative results for CALR mutations.

Sonuç: CALR mutasyonu taşıyan MPN hastaları JAK2 pozitif olanlara göre tanı anında daha yüksek trombosit sayısı ve daha düşük hemoglobin düzeylerine sahip olduklarını gösterdik. Ancak, lösemi hastalarının tamamında CALR mutasyonları negatif tespit edildi.

Keywords: Calreticulin mutation, Myeloproliferative neoplasms, Leukemia

Anahtar Sözcükler: Kalretikülin mutasyonu, Miyeloproliferatif neoplazi, Lösemi

Address for Correspondence/Yazışma Adresi: Daoxin MA, M.D., Qilu Hospital of Shandong University, Department of Hematology, Shandong, China Phone : +86 531 82169887 E-mail : daoxinma@sdu.edu.cn

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Received/Geliş tarihi: May 28, 2015 Accepted/Kabul tarihi: September 02, 2015

Turk J Hematol 2016;33:180-186

Introduction Somatic frameshift mutations in exon 9 of calreticulin (CALR) have been identified in a large proportion of JAK2- or MPL-negative myeloproliferative neoplasm (MPN) patients, including those with primary myelofibrosis (PMF) and essential thrombocythemia (ET) [1,2]. The CALR gene, located on chromosome 19p13.3, encodes a 48-kDa protein that consists of three domains: the amino terminal N-domain (residues 1-180), central proline-rich P-domain (residues 181-290), and carboxyl terminal C-domain (residues 291-400). The CALR protein is localized primarily in the endoplasmic reticulum through its C-terminal KDEL motif [3], but it is also found in the cell membrane, cytoplasm, and extracellular matrix [4,5]. Functionally, CALR is believed to participate in Ca2+ homeostasis as a calcium-binding protein, handling misfolded proteins, cell adhesion, immune response to cancer, and phagocytosis [4,6,7,8,9,10,11,12,13]. CALR-knockout mice are born dead and display impaired cardiac development, whereas postnatal overexpression also leads to cardiac defects [14,15]. Therefore, CALR regulates key cellular functions like proliferation and apoptosis. CALR also plays an important role in immune responses [16]. Mutations of CALR were found essential for the diagnosis and prognosis of MPNs in recent years. All CALR mutations seen so far in MPNs mainly involve exon 9 and are somatic insertions or deletions. Two mutation variants (type 1 and type 2) were the most frequent: type 1 (c.1179_1230del) resulted from a 52-bp deletion, more frequent in PMF, and type 2 (c.1234_1235insTTGTC) resulted from a 5-bp TTGTC insertion [1]. Andrikovics et al. demonstrated that CALR mutations are found in about one-fourth of patients with ET or PMF and are associated with distinct clinical characteristics, and another study also found that CALR mutations are associated with younger age, more severe anemia, higher white blood cell (WBC) and platelet counts, lower Dynamic International Prognostic Scoring System Plus scores, and better survival compared to subjects with JAK2 mutations [17,18]. Similar to MPNs, acute leukemia, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), is a group of disorders characterized by abnormal clonal proliferation and immune imbalance. To investigate whether CALR mutations were present in myeloid neoplasms, Andrikovics et al. detected JAK2, CALR, and MPL genes in 289 cases of ET and 99 cases of PMF, and they reported that in ET, 154 (53%) JAK2V617F mutation-positive, 96 (33%) CALR mutationpositive, 9 (3%) MPL mutation-positive, and 30 triple-negative (11%) cases were identified, while in PMF 56 (57%) JAK2V617F mutation-positive, 25 (25%) CALR mutation-positive, 7 (7%) MPL mutation-positive, and 11 (11%) triple-negative cases were identified [18]. Qiao et al. screened CALR mutations in 104 AML patients, 55 chronic myeloid leukemia (CML) patients, 7 chronic

Wang J, et al: Calreticulin in Myeloproliferative Neoplasms and Leukemia

myelomonocytic leukemia patients, and 8 myelodysplastic syndrome (MDS) patients. Although most of these patients had negative results, one AML patient was found to harbor a CALR mutation (c.1179_1230del) without JAK2V617F or MPL W515L/K mutations [19]. Unlike AML, ALL is a heterogeneous malignancy caused by the clonal proliferation of lymphocytes. However, no data about the mutation frequency of CALR in ALL patients have been reported to date. Therefore, in the present study, a cohort of 305 Chinese patients with hematopoietic neoplasms was screened for CALR mutations, with the aim of uncovering the frequency of CALR mutations in leukemia and MPNs. The results demonstrate that CALR mutation status is an important diagnostic factor in MPN patients without JAK2 mutation while it is negative in leukemia patients.

Materials and Methods Subjects and Ethics Statement Bone marrow or peripheral blood samples from 113 MPN patients were collected at Qilu Hospital of Shandong University between August 2012 and November 2014, including cases of ET (n=53), polycythemia vera (PV; n=20), PMF (n=23), MDS/MPN (n=6), and unclassifiable MPN (MPN-U; n=11). We also obtained bone marrow samples from 192 patients with other hematopoietic neoplasms including AML (n=135) and ALL (n=57). These patients were all newly diagnosed before treatment. The characteristics of the patients at the time of sampling are presented in Tables 1 and 2. The patients with AML were treated with standard induction chemotherapy (anthracycline and cytarabine). The patients with ALL were treated with standard induction chemotherapy (vincristine, daunorubicin, L asparaginase, and prednisone). Bone marrow mononuclear cells (BMMCs) or peripheral blood mononuclear cells (PBMCs) were obtained from patients using density-gradient centrifugation with the FicollHypaque technique (Ficoll, Pharmacia LKB Biotechnology Inc., Piscataway, NY, USA). The samples were then stored at -80 °C. The present study was approved by the Ethics Committee of Qilu Hospital, Shandong University (Jinan, China). Written informed consent was obtained from all participants for treatment and the cryopreservation of bone marrow and peripheral blood according to the Declaration of Helsinki. Genomic DNA Isolation, Polymerase Amplification, and Sequencing

Chain

Reaction

Genomic DNA samples from BMMCs or PBMCs of patients were extracted using the TIANGEN DNA Extraction Kit (TIANamp Genomic DNA Kit, Beijing, China). Oligonucleotide primers targeting exon 9 of CALR were used to amplify a 377-bp product: forward 5’ - CTG GCA CCA TCT TTG ACA ACT T - 3’, reverse 5’ GGC CTC TCT ACA GCT CGT C - 3’. Polymerase chain reaction 181

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(PCR) was performed in a volume of 25 µL containing 150 ng of DNA, 12.5 µL of PCR master mix, 400 nM each of forward and reverse primers, and ddH2O. Cycling parameters consisted of an initial denaturation at 94 °C for 2 min; 40 cycles of denaturation at 94 °C for 15 s, annealing at 56 °C for 30 s, and extension at 72 °C for 45 s; and a final extension at 72 °C for 1 min. PCR products were purified (QIAquick PCR Purification Kit, QIAGEN, Valencia, CA, USA) and subjected to bidirectional sequencing. Mutations were identified using Mutation Surveyor Software (Soft Genetics, LLC, State College, PA, USA). JAK2V617F mutation burden was assessed using a quantitative PCR-based allelic discrimination assay. Real-time quantitative PCR was conducted using an ABI Prism 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s instructions. The primers and probes were as follows: JAK2-PCR-Primer-F: AAG CTT TCT CAC AAG CAT TTG GTT T, JAK2-PCR-Primer-R: AGA AAG GCA TTA GAA AGC CTG TAG TT, MGB probe sequence: JAK2-Probe-WT: VIC- TCT CCA CAG ACA CAT AC; JAK2-Probe-V617F: FAM- TCC ACA GAA ACA TAC (all the primers and probes were synthesized by Invitrogen, USA). The real-time PCR contained, in a final volume of 10 µL, 1 µL of DNA, 5 µL of Universal PCR Master Mix, 0.4 µL of Primer-F and Primer-R, 0.2 µL of Probe-WT, 0.2 µL of Probe-V617F, and 2.8 µL of distilled water. PCR reaction was done at 50 °C for 2 min and 95 °C for 15 min, followed by 45 cycles of 95 °C for 30 s and 62 °C for 1 min. The fluorescence signal was collected at 62 °C while ROX Reference Dye was used as a background to normalize the fluorescent signal. The cycle threshold (Ct) value of VIC or FAM reflects the number of wild-type or mutant JAK2V617F gene DNAs, denoted as Ct VIC and Ct FAM. JAK2V617F was considered as positive when CtFAM was lower than 38. Statistical Analysis

Turk J Hematol 2016;33:180-186

samples t-test was used to compare continuous variables. Chisquare or Fisher exact tests were used for dichotomous variables. The clinical characteristics of the leukemia and MPN patients, including sex, age, WBC count, and other factors, are presented in Tables 1 and 2. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).

Results The Profile of CALR Mutations in MPN Patients Mutant CALR in MPNs is a result of frameshift mutations, caused by exon 9 deletions or insertions; the type 1 variant, a 52-bp deletion (c.1179_1230del), and type 2 variant, a 5-bp TTGTC insertion (c.1234_1235insTTGTC), constitute more than 80% of these mutations. In our study, a total of 10.6% of patients (12 of 113) with MPNs were demonstrated to harbor CALR mutations. The CALR mutation was found in 11.3% (6 of 53) of ET, 21.7% (5 of 23) of PMF, and 9.1% (1/11) of MPN-U patients, respectively (Table 3). Moreover, CALR mutations were found in 24.0% of JAK2V617F-negative ET patients (6 of 25) and 35.7% of JAK2V617F-negative PMF patients (5 of 14). No CALR mutation was found in patients with PV. For mutation types, a total of 5 distinct variants of CALR mutation, including 4 deletions and 1 insertion, were identified (Figure 1). c.1179_1230del, which resulted from a 52-bp deletion, and c.1234_1235insTTGTC, which resulted from a 5-bp insertion, were the most frequent CALR mutations. The two mutations accounted for 50% (6 of 12) and 25% (3 of 12) in all cases with mutant CALR, respectively. For ET patients, the two mutations were 50% (3 of 6) and 50% (3 of 6), respectively. For PMF patients, the two mutations were 60% (3 of 5) and 0% (0 of 5), respectively. Moreover, we also identified other kinds of deletions of CALR genetic variation: c.1239_1257del (1/12) and c.1183_1228del (1/12) were found

The Kolmogorov-Smirnov test was performed to test whether variables were normally distributed. Then the independentTable 1. Clinical characteristics of 192 patients with acute leukemia. Characteristics

AML

ALL

Patients, n Age at study entry, years Males, n (%) Bone marrow blasts at diagnosis, % WBC count, x103 cells/mm3 AML subtype, n (%) M2 M3 M4 M5

135 42.4±16.1a 71 (52.6) 74.0±0.0a 32.1±27.8a 16 (11.9) 40 (29.6) 29 (21.5) 50 (37.0)

57 37.3±16.5a 29 (50.9) 89.1±13.8a 45.1±27.1a

a: Data are presented as mean ± standard deviation. WBC: White blood cell, AML: acute

myeloid leukemia, ALL: acute lymphoblastic leukemia.

182

Figure 1. Sequencing results of CALR mutations in patients with myeloproliferative neoplasms.

Wang J, et al: Calreticulin in Myeloproliferative Neoplasms and Leukemia

Turk J Hematol 2016;33:180-186

in ET patients, and c.1183_1216del (1/12) was found in a MPN-U patient. The Profile of CALR Mutations in Leukemia Patients To investigate whether CALR mutations were present in other hematopoietic neoplasms, we screened 135 patients with AML and 57 patients with ALL. However, no CALR exon 9 mutations were found in any of these patients. One single nucleotide polymorphism (SNP) of CALR, rs143880510 (Figure 2), was found in one ALL patient. Clinical Features of Patients with CALR Mutations All of the 20 PV patients and 6 MDS/MPN patients had wildtype CALR. ET patients with mutant CALR had lower WBC counts (7.5±4.1×109/L; p<0.001), lower hemoglobin levels (137±34.2

Figure 2. CALR rs143880510 single nucleotide polymorphism in one acute lymphoblastic leukemia patient.

g/L; p=0.002), and higher platelet counts (982±24.2×109/L; p<0.001) than those with mutant JAK2V617F (14±11.0×109/L, 145±21.4 g/L, 515±31.6×109/L). Similarly, PMF patients with mutant CALR showed lower hemoglobin levels (66.5±14.1 g/L; p=0.001) than mutant JAK2 patients (150±25.7 g/L; p=0.001). ET patients with mutant CALR were significantly younger (44.0±15.1 years; p<0.001) than those with mutant JAK2 (56.2±12.9). No significant difference was identified between ET patients with mutant CALR and mutant JAK2 in terms of sex (Table 4). There was no significant difference in sex, age, WBC count, or platelet count between PMF patients with mutant CALR and mutant JAK2 (Table 4).

Discussion Since the first description of myeloproliferative diseases by Dameshek in 1951 [20], there has been a consecutive progression in the understanding of these disease conditions characterized by abnormal bone marrow hyperplasia. Apart from the characterization of the Philadelphia chromosome in CML, the discovery of JAK2V617F mutation in 2005 [21,22] is the most thrilling development in the molecular diagnosis of Phnegative MPNs. The subsequently reported somatic mutation in

Table 2. Clinical characteristics of 113 patients with myeloproliferative neoplasms. Characteristics

PMF

MDS/MPN

MPN-U

Patients, n Males, n Age at study entry, years CALR-mutant JAK2-mutant Nonmutated JAK2/CALR

53 21 52.4±42.1a 6 (11.3) 28 (52.8) 19 (47.2)

20 11 47.4±37.2a 0 (0.0) 15 (75) 5 (25)

23 15 58.4±14.2a 5 (21.7) 9 (39.1) 9 (60.9)

6 4 42.4±26.1a 0 (0.0) 1 (16.7) 5 (83.3)

11 4 36.4±26.2a 1 (8.9) 10 (90.1) 0 (0.0)

a: Data are presented as mean ± standard deviation. ET: Essential thrombocythemia, PV: polycythemia vera, PMF: primary myelofibrosis, MDS: myelodysplastic syndrome,

MPN: myeloproliferative neoplasm, MPN-U: unclassifiable-myeloproliferative neoplasm.

Table 3. CALR exon 9 mutation profile in 12 myeloproliferative neoplasm patients. Patient No.

Clinical Diagnosis

Age

Sex

CALR Exon 9 Frameshift Mutations

QL-A2

Female

c.1179_1230del

QL-A30

PMF

Male

c.1179_1230del

QL-A83

Male

c.1179_1230del

QL-A76

PMF

Male

c.1179_1230del

QL-A88

PMF

Male

c.1179_1230del

QL-A97

Female

c.1179_1230del

QL-A16

Male

c.1234_1235insTTGTC

QL-A57

Female

c.1234_1235insTTGTC

QL-A63

Female

c.1234_1235insTTGTC

QL-A73

MPN-U

Male

c.1183-1216del

QL-A100

PMF

Male

c.1183-1228del

QL-A108

PMF

Female

c.1239-1257del

ET: Essential thrombocythemia, PMF: primary myelofibrosis, MPN-U: unclassifiable-myeloproliferative neoplasm.

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JAK2 exon 12 [23], though much less prevalent in the patients, is considered as another robust molecular marker for Ph-negative MPNs, and especially for PV patients. The mutations in JAK2, MPL, and CALR are driver mutations, and they all activate the JAK2 pathway, but additional recurrent somatic mutations in several genes (TET2, ASXL1, DNMT3A, CBL, LNK, IDH1/2, IKF1, EZH2, TP53, SRSF2), encoding transcriptional and epigenetic regulators and signaling proteins, occur in MPNs. These additional mutations modulate disease progression and can also occur as primary mutations, but it is now convincingly demonstrated that MPNs can be initiated from a single JAK2V617F hematopoietic stem cell. JAK mutations have also emerged in other hematologic diseases, and the majority of the pathogenic mutations in JAK2 (also in JAK1 and JAK3) localize in or near the pseudokinase domain. To date, somatic frameshift mutations in exon 9 of CALR have been identified in a large proportion of JAK2- and MPL-negative PMF and ET patients. In a study of 617 PMF patients by Rumi et al., 399 (64.7%) carried JAK2V617F, 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF) [24]. Kim et al. investigated mutation profiles of CALR, JAK2, and MPL in 199 Korean patients with MPNs. The overall frequency of CALR mutations was 12.6%; it was most frequent in MPN-U cases (37.5%), followed by ET (17.7%) and PMF (14.8%). CALR mutations were not found in PV or acute panmyelosis with myelofibrosis. CALR and JAK2 or MPL mutations were mutually exclusive [25]. Wu et al. also found two kinds of CALR mutations, c.1179_1230del and c.1234_1235insTTGTC, in Chinese patients with MPNs, and female patients showed a predisposition to CALR mutation [26]. Li et al. studied 1088 Chinese patients with MPNs including ET (n=234) and PMF (n=50) without JAK2V617F or MPL exon 10 mutations. CALR mutation was detected in 53% of subjects with ET and 56% of subjects with PMF, and 152 CALR mutations were identified clustering into 15 types, including deletions (n=8), insertions (n=3), and complex indels (n=4) [27].

Turk J Hematol 2016;33:180-186

In our study, mutations in CALR were present in 12 of 113 patients with Ph-negative MPNs (10.6%). Mean while, an overwhelming majority (75%) of the CALR mutation pattern still lies in c.1179_1230del and c.1234_1235insTTGTC. CALR mutations were present in 12 of 50 MPN patients without JAK2 mutations (24%). Among patients with ET, those with CALR mutations, as compared with those with JAK2V617F mutations, presented with significantly higher platelet counts and lower hemoglobin levels. Whereas the frequency of CALR mutations in MPNs is quite consistent in recent studies, it is unclear whether CALR mutations occur in up to 8.3% of patients with MDS (10 of 120 MDS patients) as reported by Nangalia et al. [2], or are infrequent in MDS (none of 73) and AML (none of 254) patients as reported by Klampfl et al. [1]. This inconsistency could be due to the relatively small number of investigated patients. Therefore, in addition to MPNs, the mutation profile of CALR in other hematopoietic diseases such as leukemia and MDS has been given more attention than in the past. CALR mutations were identified in 2 of 527 MDS patients (0.38%). None of 328 patients with MDS were found to have CALR mutations. Two of 199 patients with AML following MDS had mutated CALR, and the frequency of CALR mutations is very low in MDS, supporting the use of CALR mutations as a diagnostic marker for ET and PMF patients [28]. Recently in a Chinese study, Cui et al. sequenced CALR mutations in 14 patients who met the WHO criteria for chronic neutrophilic leukemia (CNL) and found that 1 of 14 CNL patients had a CALR mutation (c.1154-1155insTTGTC) [29]. No CALR mutations were found in 62 patients with ALL [2]. However, little attention has been paid to AML and no data about the mutation frequency of CALR in Chinese ALL patients have been reported until now. Therefore, we screened 135 AML patients and 57 ALL patients. However, no CALR exon 9 mutations were found in any of these patients. Only one of the leukemia patients was found to have a CALR SNP, rs143880510. To date, detection of CALR mutations in peripheral blood has been used as a diagnostic tool in the same way that tests for JAK2 mutations have simplified and improved the accuracy of diagnosis of patients with MPNs. However, in order to develop novel therapeutic drugs, further research is needed to explore

Table 4. Clinical characteristics of essential thrombocythemia and primary myelofibrosis patients with CALR and JAK2 mutation. Characteristics Patients, n Males, n Age, years WBC count (x109/L) Hemoglobin (g/L) Platelets (x109/L) a:

PMF

CALR+

JAK2+

CALR+

JAK2+

6 2 44.0±15.1a 7.5±4.1a 137±34.2a 982±24.2a

28 11 56.2±12.9a 14±11.0a 145±21.4a 515±31.6a

0.784 0.001 0.001 0.002 0.001

5 4 59.8±3.6a 10.42±3.6a 66.5±14.1a 171±35.2a

9 5 61.9±7.4a 15.12±3.6a 150±25.7a 603±56.2a

0.58 0.626 0.612 0.001 0.091

Data are presented as mean ± standard deviation. ET: Essential thrombocythemia, PMF: primary myelofibrosis.

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Wang J, et al: Calreticulin in Myeloproliferative Neoplasms and Leukemia

the relationship between the pathogenesis of MPNs and the function of CALR.

3. Michalak M, Groenendyk J, Szabo E, Gold LI, Opas M. Calreticulin, a multiprocess calcium-buffering chaperone of the endoplasmic reticulum. Biochem J 2009;417:651-666.

Conclusion

4. Gold LI, Eggleton P, Sweetwyne MT, Van Duyn LB, Greives MR, Naylor SM, Michalak M, Murphy-Ullrich JE. Calreticulin: non-endoplasmic reticulum functions in physiology and disease. FASEB J 2010;24:665-683.

In summary, our data from this cohort of Chinese patients with MPNs confirmed that CALR mutations were novel molecular markers in JAK2V617F-negative MPNs. Patients with the c.1179_1230del and c.1234_1235insTTGTC mutations have shown distinct clinical characteristics, but further research is required to confirm this result. Acknowledgment This work was supported by grants from the National Natural Science Foundation of China (No. 81470319, No. 81170515). Ethics Ethics Committee Approval: The present study was approved by the Ethics Committee of Qilu Hospital, Shandong University (Jinan, China); Informed Consent: Written informed consent was obtained from all participants for treatment and the cryopreservation of bone marrow and peripheral blood according to the Declaration of Helsinki. Authorship Contributions Medical Practices: Jingyi Wang, Jianguo Hao, Na He; Concept: Daoxin Ma, Chunyan Ji; Design: Daoxin Ma, Chunyan Ji; Data Collection or Processing: Jingyi Wang, Jianguo Hao, Na He, Chunyan Ji, Daoxin Ma; Analysis or Interpretation: Daoxin Ma, Chunyan Ji; Literature Search: Jingyi Wang, Jianguo Hao, Na He, Chunyan Ji, Daoxin Ma; Writing: Jingyi Wang, Jianguo Hao, Na He, Chunyan Ji, Daoxin Ma. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0041 Turk J Hematol 2016;33:187-195

Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms Miyeloproliferatif Hastalığı Olan 294 Türk Hastanın Klinik Verileri Neslihan Andıç1, Mustafa Ünübol2, Eren Yağcı3, Olga Meltem Akay1, İrfan Yavaşoğlu2, Vefki Gürhan Kadıköylü2, Ali Zahit Bolaman2 1Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey 2Adnan Menderes University Faculty of Medicine, Department of Hematology, Aydın, Turkey 3Osmangazi University Faculty of Medicine, Department of Internal Medicine, Eskişehir, Turkey

Abstract

Öz

Objective: Myeloproliferative neoplasms (MPNs) share common clonal stem cells but show significant differences in their clinical courses. The aim of this retrospective study was to evaluate thrombotic and hemorrhagic complications, JAK2 status, gastrointestinal and cardiac changes, treatment modalities, and survival in MPNs in Turkish patients.

Amaç: Miyeloproliferatif hastalıklar (MPH) ortak klonal bir kök hücreden köken almalarına karşın klinik seyirleri belirgin farklılıklar göstermektedir. Bu retrospektif çalışmanın amacı MPH’lardaki trombotik ve hemorajik komplikasyonların, JAK2 mutasyon durumunun, gastrointestinal ve kardiyak değişikliklerin, tedavi şekillerinin ve yaşam sürelerinin incelenmesidir.

Materials and Methods: Medical files of 294 patients [112 essential thrombocythemia (ET), 117 polycythemia vera (PV), 46 primary myelofibrosis, and 19 unclassified MPN cases] from 2 different universities in Turkey were examined.

Gereç ve Yöntemler: Türkiye’nin iki farklı üniversite hastanesinden 294 hastanın [112 esansiyel trombositemi (ET), 117 polisitemia vera (PV), 46 primer miyelofibozis, 19 sınıflanamayan MPH] kayıtları incelenmiştir.

Results: Older age, higher leukocyte count at diagnosis, and JAK2 mutation positivity were risk factors for thrombosis. Platelet count over 1000x109/L was a risk factor for hemorrhagic episodes. Hydroxyurea treatment was not related to leukemic transformation. Median follow-up time was 50 months (quartiles: 22.2-81.75) in these patients. Patients with primary myelofibrosis had the shortest survival of 137 months when compared with 179 months for ET and 231 months for PV. Leukemic transformation, thromboembolic events, age over 60 years, and anemia were found to be the factors affecting survival.

Bulgular: İleri yaş, tanıda yüksek lökosit sayısı JAK2 mutasyon pozitifliği tromboz için risk faktörü olarak bulunmuştur. Trombosit sayımının 1000x109/L üzerinde olması kanama komplikasyonları açısından risk faktörüdür. Hidroksiüre tedavisi lösemik dönüşümle ilişkili bulunmamıştır. Bu hastalarda: Medyan takip süresi 50 ay (22,281,75 çeyrekler) idi. Primer miyelofibrozisli hastalar ET için 179 ay ve PV için 231 ay olan yaşam süreleri ile karşılaştırıldığında 137 ay ile en kısa yaşam süresine sahip hastalardır. Lösemik transformasyon, tromboembolik olaylar, 60 yaş üstü olmak ve anemi yaşam süresinin etkileyen faktörler olarak bulunmuştur.

Conclusion: Thromboembolic complications are the most important preventable risk factors for morbidity and mortality in MPNs. Drug management in MPNs is done according to hemoglobin and platelet counts. Based on the current study population our results support the idea that leukocytosis and JAK2 positivity are more important risk factors for thrombosis than hemoglobin and platelet values.

Sonuç: Tromboembolik komplikasyonlar MPH’da en önemli önlenebilir mortalite ve morbidite nedenidir. İlaç düzenlemeleri çoğunlukla hemoglobin ve trombosit sayımlarına göre yapılmaktadır. Bu çalışmamızdaki hasta popülasyonundan elde ettiğimiz veriler lökositoz ve JAK2 pozitifliğinin hemoglobin ve trombosit sayımlarından daha önemli risk faktorleri olduğu savını desteklemektedir.

Keywords: Myeloproliferative neoplasms, Survival, Thrombosis, Treatment

Anahtar Sözcükler: Miyeloproliferatif hastalıklar, Sağkalım, Tromboz, Tedavi

Address for Correspondence/Yazışma Adresi: Neslihan ANDIÇ, M.D., Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey Phone : +90 532 518 22 63 E-mail : neslihandic@yahoo.com

Received/Geliş tarihi: January 20, 2015 Accepted/Kabul tarihi: September 15, 2015

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Introduction According to the revised World Health Organization (WHO) classification, BCR-ABL-negative chronic myeloproliferative disorders are now referred to as myeloproliferative neoplasms (MPNs) [1,2]. MPNs share common clonal stem cells and phenotypic differences occur due to different molecules affecting signal transduction. The JAK2V617F mutation is an acquired point mutation causing valine-to-phenylalanine substitution at codon 617 on the JAK2 gene. JAK2V617F will be referred to as JAK2 mutation in the text. JAK2 mutations affecting the JAK-STAT signal transduction pathway are found in 90%-95% of patients with polycythemia vera (PV) [3], 50%70% of patients with essential thrombocythemia (ET), and 40%-50% of patients with primary myelofibrosis (PMF) [4]. JAK2 mutations cannot be used in distinguishing one MPN from another but are useful in excluding reactive hematocrit and platelet elevations and reactive myelofibrosis. Absence of JAK2 mutations cannot exclude the diagnosis of PV, ET, or PMF. Some clinical criteria and bone marrow findings are required for the diagnosis of JAK2-negative MPN [2]. Prognosis of MPNs is determined by thromboembolic and hemorrhagic complications and progression to myelofibrosis and acute leukemia. The cumulative rate of nonfatal thrombosis in PV is 3.8 events per 100 persons per year, and in ET the rate of fatal and nonfatal thrombotic events ranges from 2% to 4% of patient years. Primary myelofibrosis seems less susceptible for thrombotic events as the cumulative percentage is 2.23% per patient years [5]. Age and previous thrombosis are known risk factors for future thrombosis in MPNs. Leukocytosis and JAK2 mutation are shown to be additional risk factors. Extreme thrombocytosis (count over 1000 or 1500x109/L) was found to be related to hemorrhagic complications but not thrombosis [5]. Other complications like gastrointestinal ulcers and echocardiographic changes are also reported. Their importance in the course of the disease is only partially understood [6,7]. Hydroxyurea and anagrelide are the most commonly used drugs in the treatment of MPN. Hydroxyurea was shown to reduce the incidence of thrombotic events in several studies, but there is some evidence that it may increase the risk of leukemic transformation [8]. Anagrelide is effective in reducing platelet counts in ET and PV patients who are resistant or intolerant to hydroxyurea. Risk increment of leukemia has not been shown for this drug [9]. The aim of this study is to evaluate thrombotic and hemorrhagic complications, JAK2 status, gastrointestinal and cardiac changes, treatment modalities, and survival in MPN cases.

Materials and Methods The medical files of patients diagnosed with Philadelphia chromosome-negative chronic myeloproliferative disease 188

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(CMPD) and MPN between 2003 and 2013 were retrospectively examined. Two centers in Turkey entered the study: Eskişehir Osmangazi University and Adnan Menderes University. Diagnoses were made according to PV Study Group and WHO recommendations. The WHO criteria were revised in 2005 after the discovery of JAK2 mutations. In the revision of WHO criteria made in 2008, ‘CMPD’ was changed to ‘MPN’. Patients with significant fibrosis in the bone marrow but otherwise clinically diagnosed with ET by the primary clinician were placed in the category of unclassified MPN and will be referred to here as MPN(u) patients. We included MPN(u) patients in the statistical analysis done for the whole patient group, like statistics of risk factors for thrombosis. On the other hand, we did not include MPN(u) in one-to-one comparisons with the three MPN groups (ET, PV, and PMF) because we wanted to compare the patients with exact diagnoses. The study was approved by the local ethics committees of both universities. Patients above the age of 16 at the time of diagnosis were enrolled in the study. All consecutively admitted patients during the mentioned period were taken into consideration. Clinical and laboratory parameters were recorded. JAK2 status and other cytogenetic abnormalities and bone marrow findings were evaluated. Treatment modalities, thrombotic and hemorrhagic complications, and gastrointestinal and cardiac findings were noted. Proteins C and S were studied by the Siemens BCSX coagulometric method and antithrombin was studied by Siemens BNII nephelometric method. Factor V Leiden and prothrombin gene mutations were studied with a Roche 480 II LightCycler by the real-time polymerase chain reaction (PCR) method. Bone marrow samples were cultured in 24-48 h in standard 10 µg/ mL colcemid solution without mitogen in order to perform conventional bone marrow cytogenetics. Twenty metaphases were evaluated. A locus-specific LSI D20S108 (20q12) probe was used for fluorescence in situ hybridization (FISH) analysis and 200 cells (metaphase/interphase) were evaluated. JAK2V617F mutations were studied by real-time PCR method with a Roche 480 II LightCycler using the TIB Molbiol LightMix Kit. Bone marrow aspirates and biopsies were evaluated in the pathology and hematology departments of both universities. Increases in megakaryocytes and grades of reticulin fibrosis were defined according to the WHO classification of tumors [10,11]. Statistical Analysis Statistical tests were performed using IBM SPSS 20.0 for Windows and p<0.05 was considered significant. The ShapiroWilk test was performed for testing normality. The chi-square test was used to compare categorical variables and the KruskalWallis test was used for continuous variables not normally distributed, followed by Dunn’s post hoc test. Survival was

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Andıç N, et al: Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms

assessed using Kaplan-Meier analysis and the log-rank test was used for univariate comparisons. The effect of prognostic factors on survival was analyzed by Cox proportional hazards regression models.

Results A total of 294 patients’ medical files were eligible for the study; 143 (48.6%) patients were female and 151 (51.4%) were male. Median age was 60 years (quartiles: 48-79), with a minimum of 16 and maximum of 84 years. Sex and age were not statistically different between patient groups. The number of patients diagnosed with ET was 112 (38.1%), with PV was 117 (39.8%), and with PMF was 46 (15.6%). Nineteen patients who were diagnosed with and treated for ET by the primary physician were later classified as having MPN(u). These patients had no leukoerythroblastosis in peripheral blood and their median platelet count was 1146x109/L (quartiles: 844-1416). All of them had megakaryocytic proliferation in their bone marrow and had neither dysplasia nor prominent granulocytic and erythroid proliferation. Median follow-up time was 68 months (quartiles: 15-81). Six of them had grade 2-3 and 13 of them had grade 3 reticulin fibrosis in their bone marrow. We could not classify these cases as ET or PMF so we classified them as MPN(u). The longest follow-up period was 311 months (median: 43 months; quartiles: 15.7-77.2). In 103 patients there were no comorbidities, while 155 patients had hypertension and/ or diabetes mellitus, and 53 patients had diseases including chronic obstructive pulmonary disease, liver failure, renal failure, congestive heart disease, and arrhythmias including atrial fibrillation. One patient had lung cancer and another

had prostate cancer. Eighty-five patients (28%) were smoking cigarettes. The clinical and laboratory characteristics of patients are summarized in Table 1. Splenomegaly and hepatomegaly were significantly more frequent in PMF than in other MPNs. Hemoglobin was significantly higher in PV than in other groups and significantly lower in PMF than in other groups. Platelet count was higher in ET than in other groups. Lactate dehydrogenase (LDH), potassium, and uric acid values were higher in PMF compared to other MPN subtypes. Median LDH was above normal limits in all study groups. Bone marrow findings at diagnosis are summarized in Table 2. Megakaryocytes were more prominent in ET and reticulin fibrosis was more profound in PMF than in other MPNs, as expected. It was found that 58.5% (38 patients) of ET patients, 86.2% (50 patients) of PV patients, and 70.6% (12 patients) of PMF patients were positive for JAK2 mutation. Patients with PV were carrying JAK2 mutations significantly more so than patients with ET (p<0.001). There were no statistically significant differences between ET and PMF and PV and PMF regarding JAK2 mutation. Conventional cytogenetics and FISH analysis of the bone marrow revealed that 2 patients with ET had trisomy 8 and 1 patient had 5q-, while 3 patients with PV had 20q-, 1 patient had 13q-, and 1 patient had trisomy 20. One patient with ET who had trisomy 8 had developed acute myeloid leukemia. Thromboembolic complications were seen in 36% (n=108) of patients, while 41.1% of ET patients (n=46), 35% of PV patients (n=41), and 32% of PMF patients (n=15) had thrombotic events. Six other patients who had thromboembolic events were later reclassified as having MPN(u). Most of the thrombotic events were in cerebral arteries (37 out of 108). Approximately half of

Table 1. Clinical and laboratory characteristics of patients with myeloproliferative neoplasm.* Splenomegaly (% within MPNs)

Hemoglobin (g/dL)

Platelets (x109/L)

LDH (U/L)

Uric acid (mg/dL)

PV 63 (53.8%)

17.9 (16.2-18.8)

598 (354-873)

697.4 (457-697.4)

6.59 (5.5-7.3)

ET 32 (28.6%)

13.6 (12.3-14.8)

1022 (854-1394)

680 (458-697)

6.5 (5.4-6.59)

PMF 36 (78.3%)

9.3 (7.05-10.5)

239 (83-660)

p-value PV-ET

<0.001

PV-PMF

<0.001

PV-ET

<0.001

PV-ET

<0.001

PV-PMF

<0.001

PMF-ET

<0.001

PV-ET

<0.001

PV-PMF

<0.001

PMF-ET

<0.001

PV-ET

>0.05

751 (437-1360) PV-PMF

>0.05

PMF-ET

0.026

PV-ET

>0.05

PV-PMF

>0.05

PMF-ET

0.014

6.5 (6.4-7.75)

PV: Polycythemia vera, ET: essential thrombocythemia, PMF: primary myelofibrosis, LDH: lactate dehydrogenase, *all results are reported as medians (25th-75th quartiles).

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these cerebral arterial occlusions were seen in ET patients (16 out of 37). Coronary artery disease was the second most common thrombotic complication (32 out of 108). Ten patients had deep vein thrombosis (9.3%). Twelve patients (11.1%) had thrombosis in an intraabdominal vein. Ten patients had both arterial and venous thrombotic attacks and most of them were ET patients (8 out of 10). Diagnostic groups, sex, treatment modality, and bone marrow findings did not differ between patients with or without thrombosis. Older age, higher leukocyte count at diagnosis, and JAK2 mutation positivity were risk factors for thrombosis after univariate analysis. Data are shown in Table 3. Hemorrhagic complications were seen in 34.4% (n=101) of patients. Almost all (94%) patients with hemorrhagic complications had mucocutaneous or gastrointestinal tract bleeds. There was no statistical significance between MPN groups regarding the frequency and the source of bleeding. Hemoglobin values were significantly lower in patients with hemorrhagic episodes than patients without hemorrhage (p=0.012). Medians and quartiles were 13.1 g/dL (10.3-17) and 14.6 g/dL (12.7-17.1), respectively. Platelets count over 1000x109/L was a risk factor for hemorrhagic episodes; 30.1% (n=59) of patients with platelet count less than or equal to 1000x109/L had hemorrhagic events, whereas 42.9% (n=42) of patients with platelet count over 1000x109/L had hemorrhagic episodes (p=0.030). Leukocyte counts, fibrinogen levels, and

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treatment modalities were not statistically different between patients with and without hemorrhage. Electrocardiogram results were considered as normal in 78.2% (n=230) of cases. Atrial fibrillation was present in 7.4% (n=22) and signs of ischemia in 8.2% (n=24) of patients. Echocardiography was performed in 95 patients. Forty-one (43.2%) of them had cardiac valve abnormalities and 10 (10.5%) had pulmonary hypertension. Upper gastrointestinal endoscopy was performed in 80 patients. Gastritis and duodenitis were frequent findings (56 patients, 70%). Nine patients (11.3%) had ulcers. Nine patients had esophageal varices. Helicobacter pylori testing was done in 56 patients and 53.6% of them were positive. Treatment modalities are shown in Table 4. Hydroxyurea was the first choice in ET, PV, and PMF cases. Anagrelide was mainly used in ET. Patients who were receiving anagrelide treatment were significantly younger than the patients receiving hydroxyurea treatment [49.5 years (39.7-63) and 60 years (50-69), respectively, p<0.001]. Interferon alpha was used only in 6.5% of MPN patients. Acetylsalicylic acid was used in approximately 80% of PV and ET cases. Anticoagulant drugs were administered after a thromboembolic event in 34 patients (29.1% of thromboembolic events) and the frequency was not different between MPN groups (p>0.05). Thirty-six patients (12.2%) were treated with phlebotomy alone, and 159 patients (54%) received any kind of

Table 2. Bone marrow findings at diagnosis.

PMF

67 (59.3%)

85 (78.7%)

15 (32.6%)

Increased number of megakaryocytes (% within MPNs)

p-value

PV-ET

<0.001

PV-PMF

0.002

PMF-ET

<0.001

Reticular fibrosis (% within MPNs) 0

64 (54.7%)

57 (50.9%)

18 (15.4%)

36 (32.1%)

22 (18.8%)

19 (17%)

9 (19.6%)

3 Blasts >5% (Number of patients)

13 (11.1%)

37 (80.4%)

PV: Polycythemia vera, ET: essential thrombocythemia, PMF: primary myelofibrosis, MPNs: myeloproliferative neoplasms.

Table 3. Risk factors for thrombosis.

Patients with a Thrombotic Event

Patients without a Thrombotic Event

p-value

Leukocytes, median (percentiles), x109/L

13.8 (10.2-24.2)

12.2 (8.7-17)

0.001

Age, median (percentiles), years

63.5 (53-71)

57 (45-68)

0.002

JAK2 mutation (%)* Negative

7 (14.8%)

35 (33.9%)

Heterozygous mutant

36 (76.6%)

62 (60.2%)

Homozygous mutant

4 (8.6%)

6 (5.9%)

*Percentage of patients with or without JAK2 mutation.

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0.041

Andıç N, et al: Clinical Features of 294 Turkish Patients with Chronic Myeloproliferative Neoplasms

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MPN groups (p<0.001). Leukemic transformation shortened the survival significantly at 78 vs. 210 months (p<0.001). Mean overall survival of patients with any thromboembolic event was significantly shorter than that of patients without thromboembolic events. In subgroup analysis, the same effects of thromboembolic events were seen in ET patients but not in PV and PMF patients. Arterial or venous nature of the thrombi did not affect the survival time in MPNs. Hemorrhagic complications did not have any significant effect on survival. Survival time was significantly shorter when the patient’s age was 60 years or older at diagnosis (p=0.001). Patients with hemoglobin levels lower than 13 g/dL lived significantly shorter than those with hemoglobin of 13 g/dL or higher (p=0.001). Leukocyte and platelet counts had no significant effects on survival.

cytoreductive therapy along with phlebotomy. Seven patients developed acute myeloid leukemia during follow-up. Three of them had ET, 2 of them had PV, and 2 of them had MPN(u). Patients who developed leukemia were not different from others by means of sex, megakaryocyte number, or grade of reticulin fibrosis in the initial bone marrow. Among 3 patients with excess (>5%) blasts in the initial bone marrow, 1 developed leukemia and the other 2 were diagnosed with PMF. Receiving previous treatment with hydroxyurea was not found to be a risk factor for leukemic transformation. Median follow-up time of patients receiving hydroxyurea treatment was 50 months (quartiles: 22.281.75). One patient with ET previously treated with hydroxyurea showed transition to myelofibrosis. Forty-four patients died during follow-up. Among diseaserelated deaths, thromboembolic events were the main cause for ET patients and progression of the disease was the main cause for PMF patients.

We did not find any significant effect of bleeding events or JAK2 mutation status on survival. Treatment methods including acetylsalicylic acid and anticoagulant drugs were not effective on survival for the overall patient population.

Factors affecting survival in MPN are shown in Table 5. Overall survival of the PMF patients was shorter than in the other Table 4. Treatment modalities in myeloproliferative neoplasms.

Hydroxyurea (% within MPN group)

101 (86.3%)

PMF

Total Number of Patients

p-value PV-ET >0.05

91 (81.3%)

22 (47.8%)

232 (78.9%)

PV-PMF <0.001 PMF-ET <0.001

Anagrelide (% within MPN group)

PV-ET <0.001

7 (6%)

37 (33%)

2 (4.3%)

54 (18.4%)

Interferon alpha (% within MPN group)

6 (5.1%)

6 (5.4%)

4 (8.7%)

19 (6.5%)

Acetylsalicylic acid

(% within MPN group)

90 (76.9%)

91 (81.3%)

25 (54.3%)

PV-PMF >0.05 PMF-ET <0.001

>0.05 PV-ET

>0.05

PV-PMF <0.001 PMF-ET <0.001

PV: Polycythemia vera, ET: essential thrombocythemia, PMF: primary myelofibrosis, MPNs: myeloproliferative neoplasms.

Table 5. Factors affecting survival in myeloproliferative neoplasm cases.

Mean Value of Overall Survival (Months)

95% Confidence Interval

p-value

231

(179-283)

PV-ET

179

(169-197)

PV-PMF <0.001

>0.05

PMF

137

(91-182)

PMF-ET <0.001

Leukemic transformation

(34-123)

<0.001

No leukemic transformation

210

(180-241)

Thromboembolic events

187

(145-229)

0.017

No thromboembolic events

208

(173-244)

Age <60 years

221

(184-258)

0.006

Age ≥60 years

141

(118-163)

Hemoglobin <13 g/dL

159

(124-194)

0.001

Hemoglobin ≥13 g/dL

236

(197-275)

PV: Polycythemia vera, ET: essential thrombocythemia, PMF: primary myelofibrosis, MPNs: myeloproliferative neoplasms.

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Discussion This retrospective study was aimed at evaluating the characteristics of MPNs in Turkish patients. Two centers contributed to the study and 294 patients were enrolled. Median age was 60 years and the female/male ratio was 0.9. These two findings were consistent with the literature [12,13]. Although there is knowledge in the literature that ET is more common in women and PV more common in men, we did not find any difference in sex between MPN groups [3,14]. Splenomegaly and hepatomegaly were common findings in physical examination. They were seen in almost 80% of patients with PMF, more commonly than in the other MPN groups. This finding is consistent with the literature, but our frequencies in PV and ET are higher than those reported in other studies [13,14,15]. Even minimal enlargement in the spleen, like 130 mm in a male patient, was noted as splenomegaly in this study. This could be the reason for higher splenomegaly rates in ET and PV patients compared with the literature. Hemoglobin levels were higher in PV and platelet levels were higher in ET, and they were both lower in PMF, as expected (p<0.001). The increased serum LDH levels in PMF may reflect increased disease bulk as well as the proliferative activity associated with the accumulation of additional genetic lesions [16]. In 2005 Baxter et al. showed JAK2 mutation in 97% of PV, 57% of ET, and 50% of PMF cases [17]. Three other groups showed high frequency of JAK2 mutation in MPNs in the same year [18,19,20]. We found JAK2 mutation in 86.2% of the PV group, 58.5% of the ET group, and 70.6% of PMF group. The frequency of JAK2 mutation in the PMF group was higher than that reported in the literature. All these patients with PMF were JAK2 heterozygous mutant. Cytogenetic analysis of the bone marrow was performed for 61 patients. Among these patients, 8 (13%) of them had genetic abnormalities. The number of patients with genetic abnormalities was not high enough to make prognostic assumptions about these abnormalities. Thromboembolic complications were seen in 36.7% of all MPN patients in our study. In the literature, thromboembolic complications at diagnosis range from 7% to 57% and rise to 41%-91% during follow-up [13,21,22,23,24]. In our study, thromboembolic complications were more frequent in ET cases, but the difference did not reach statistical significance. Both in PV and ET arterial thrombotic events were much more frequent than venous ones. This finding was consistent with prospective studies [25,26]. In patients with ET, arterial events were mostly located in cerebral arteries. In the study of PĂłsfai et al., 18 out of 102 ET patients had stroke. They found that stroke was not related to platelet number but rather to JAK2 mutation status [27]. Both the frequency of stroke in ET and the risk factors were similar to our results. In patients with PV, cardiac events were more frequent than in patients with ET or PMF. Ten patients 192

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had both arterial and venous vascular events, and 7 of these patients had ET. Incidence of thrombotic events in PMF patients was as high as in other MPN groups. There was no significant difference between venous and arterial thrombotic events in PMF patients. Barbui et al. published a study in 2010 reporting fewer thromboembolic events in PMF compared to other MPNs [28]. In our study, we included thromboembolic events at both diagnosis and follow-up, and this might have affected the results. After univariate analysis, older age, higher leukocyte count at diagnosis, and JAK2 positivity were risk factors for thrombosis. Increasing age is shown to be a risk factor in large cohorts of patients with MPNs [25,29]. In the ECLAP study, risk increment was shown in PV patients above the age of 65, and in the IPSET-Thrombosis study age above 60 was shown to be an independent risk factor in ET patients [25,29]. In our study, the polycythemia vera199 age of patients with thrombosis was 63.5 years. Leukocytes and especially neutrophils play a major role in inflammatory response and activation of the coagulation system [30]. Barbui et al. found leukocytosis as an independent risk factor for arterial thrombosis in MPN [31]. Increased baseline leukocyte count was shown be a risk factor for thrombosis in ET patients in other large cohort studies [32,33,34]. In our study the median baseline leukocyte count was 13.8x109/L in patients with thrombosis. Leukocyte count higher than 15x109/L was shown to be a risk factor for cardiac events in PV patients [35]. We did not find any significant relationship between platelet and hemoglobin levels and thrombotic events. Cytotoxic therapy is recommended in patients who are at a high risk of thrombosis [9]. Based on our findings we think, like many other authors, that leukocytosis is a novel high risk factor for thrombosis and cytotoxic therapy should be started in patents with persistent leukocytosis, and during follow-up, leukocyte count target is as important as hemoglobin and platelet targets. JAK2 positivity was shown to increase both arterial and venous thrombosis by 2-fold in ET patients [36]. Barbui et al. showed the risk increment in PMF patients with JAK2 positivity and showed that there is an even higher incidence of thrombotic events if the mutation is together with leukocytosis [28]. In our study, 36.7% of the patients who were heterozygous mutant for JAK2 had a thrombotic event. On the other hand, only 16.7% of JAK2 mutation-negative patients had thrombosis (p=0.041). Allele burden was not different between cases with and without thrombosis, which is controversial in some studies [37]. De Grandis et al. recently showed that there is an abnormal adhesion of red blood cells to the subendothelial protein laminin via the JAK2V617F pathway in PV patients [38]. There are other studies showing that platelet and leukocyte functions and plasma hypercoagulation markers are altered by JAK2 mutation in a prethrombotic way [39,40]. Today, JAK2

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mutation monitoring during follow-up is not recommended [9]. Based on our results and knowledge of the literature, JAK2 mutation positivity could be another high-risk factor for thrombosis along with age, previous thrombosis, and leukocytosis. However, regarding the high frequency of positive results, we cannot recommend cytotoxic treatment for every JAK2 mutation-positive patient. A total of 101 (34.4%) patients had hemorrhagic episodes. Almost all episodes were mucocutaneous or gastrointestinal. This finding is consistent with the literature [13,22]. The only risk factor for hemorrhage was platelet count over 1000x109/L. Elliot and Tefferi reached the same result [41]. It is thought that during extreme thrombocytosis the reduced levels of high-molecular-weight von Willebrand (vW) factor causes acquired vW disease, which is responsible for bleeding tendency. Frequency of hemorrhagic and thrombotic episodes was virtually the same among the MPN groups. Hydroxyurea was the most commonly used agent in all MPN groups. Anagrelide was almost always used in ET cases. Patients with ET who were using anagrelide were significantly younger than the other ET patients. Our treatment choices correlated nicely with the current recommendations. Hydroxyurea is the initial choice of treatment because of its proven efficiency, especially in reducing thrombotic risk [42,43]. However, hydroxyurea is recommended to be used with caution in young patients regarding the data showing a risk increment of leukemia in long-term usage of hydroxyurea by Kiladjian et al. [8]. In the ANAHYDRET study, it was shown that anagrelide is as effective as hydroxyurea [44]. Secondary leukemia has not been reported with anagrelide treatment yet. Interferon alpha was the least commonly used agent in all MPN groups, probably because of its parenteral usage and poor tolerability. In our study, we did not find any relation between the complications of MPN and the treatment methods. Survival was longest in PV and shortest in PMF cases. Polycythemia vera has a life expectancy of 10 to 20 years according to the literature and our finding was consistent with this knowledge [45]. Although we found that patients with PMF had the shortest survival, their mean overall survival was 11.4 years, which is longer than 5.5 years as reported in the literature [46]. None of the PMF cases transformed to leukemia and this could be one of the reasons for the finding above. Life expectancy of ET patients ranges from 13 to 22.3 years according to the literature [14,47,48]. In our ET patient group, mean overall survival was 14.9 years. Although the survival of ET patients was shorter than that of PV patients, this was not statistically significant. Older age, leukocytosis, and low hemoglobin and high platelet count were found to be related to survival in ET patients in the literature [48,49]. In our study, patients older than 60 years at

diagnosis had shorter survival than those younger than 60. We also found that anemia (defined as hemoglobin of <13 g/dL) was related to shorter survival. Leukemic transformation shortened survival significantly, as expected. However, there were only 7 patients who developed leukemia. Therefore, risk factors for leukemic transformation and their effects on survival could not be examined properly. Patients with thromboembolic events had shorter overall survival. This effect of thromboembolism was most significantly seen in the ET subgroup. This finding is consistent with other reports [48,50]. ET is a disease that has a life expectancy of 20 years, as mentioned before, and thromboembolism is a preventable complication of ET, so therapy should be directed toward preventing thromboembolism in ET. In conclusion, thromboembolic complications are the most important preventable risk factors for morbidity and mortality in MPNs. Leukocytosis and JAK2 positivity are risk factors for thrombosis and may be more important than the elevated hemoglobin levels and platelet counts. Ethics Ethics Committee Approval: Study was approved by Ethics Committees of Eskişehir Osmangazi University and Aydın Adnan Menderes University; Informed Consent: Informed consent was not applicable. Authorship Contributions Medical Practices: Mustafa Ünübol, Eren Yağcı, Neslihan Andıç; Concept: Neslihan Andıç, Mustafa Ünübol, Eren Yağcı, Olga Meltem Akay; Design: Neslihan Andıç, Mustafa Ünübol, Eren Yağcı, Olga Meltem Akay; Data Collection or Processing: Neslihan Andıç, Mustafa Ünübol, Eren Yağcı, Olga Meltem Akay, İrfan Yavaşoğlu, Vefki Gürhan Kadıköylü, Ali Zahit Bolaman; Analysis or Interpretation: Neslihan Andıç, Vefki Gürkan Kadıköylü; Literature Search: Neslihan Andıç, Mustafa Ünübol, Eren Yağcı, Olga Meltem Akay, İrfan Yavaşoğlu, Vefki Gürhan Kadıköylü, Ali Zahit Bolaman; Writing: Neslihan Andıç, Mustafa Ünübol, Eren Yağcı, Olga Meltem Akay, İrfan Yavaşoğlu, Vefki Gürhan Kadıköylü, Ali Zahit Bolaman. 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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44. Gisslinger H, Gotic M, Holowiecki J, Penka M, Thiele J, Kvasnicka HM, Kralovics R, Petrides PE; ANAHYDRET Study Group. Anagrelide compared with hydroxyurea in WHO-classified essential thrombocythemia: the ANAHYDRET Study, a randomized controlled trial. Blood 2013;121:1720-1728.

36. Lussana F, Caberlon S, Pagani C, Kamphuisen PW, Büller HR, Cattaneo M. Association of V617F Jak2 mutation with the risk of thrombosis among patients with essential thrombocythaemia or idiopathic myelofibrosis: a systematic review. Thromb Res 2009;124:409-417.

45. Passamonti F, Rumi E, Pungolino E, Malabarba L, Bertazzoni P, Valentini M, Orlandi E, Arcaini L, Brusamolino E,Pascutto C, Cazzola M, Morra E, Lazzarino M. Life expectancy and prognostic factors for survival in patients with polycythemia vera and essential thrombocythemia. Am J Med 2004;117:755-761.

37. Vannucchi AM, Antonioli E, Guglielmelli P, Longo G, Pancrazzi A, Ponziani V, Bogani C, Ferrini PR, Rambaldi A, Guerini V, Bosi A, Barbui T; MPD Research Consortium. Prospective identification of high-risk polycythemia vera patients based on JAK2V617F allele burden. Leukemia 2007;21:1952-1959. 38. De Grandis M, Cambot M, Wautier MP, Cassinat B, Chomienne C, Colin Y, Wautier JL, Le Van Kim C, El Nemer W. JAK2V617F activates Lu/BCAMmediated red cell adhesion in polycythemia vera through an EpoRindependent Rap1/Akt pathway. Blood 2013;121:658-665. 39. Arellano-Rodrigo E, Alvarez-Larrán A, Reverter JC, Villamor N, Colomer D, Cervantes F. Increased platelet and leukocyte activation as contributing mechanisms for thrombosis in essential thrombocythemia and correlation with the JAK2 mutational status. Haematologica 2006;91:169-175. 40. Falanga A, Marchetti M, Vignoli A, Balducci D, Russo L, Guerini V, Barbui T. V617F JAK-2 mutation in patients with essential thrombocythemia: relation to platelet, granulocyte, and plasma hemostatic and inflammatory molecules. Exp Hematol 2007;35:702-711. 41. Elliott MA, Tefferi A. Thrombosis and haemorrhage in polycythaemiavera and essential thrombocythaemia. Br J Haematol 2005;128:275-290. 42. Berk PD, Goldberg JD, Donovan PB, Fruchtman SM, Berlin NI, Wasserman LR. Therapeutic recommendations in polycythemia vera based on Polycythemia Vera Study Group protocols. Semin Hematol 1986;23:132-143. 43. Cortelazzo S, Finazzi G, Ruggeri M, Vestri O, Galli M, Rodeghiero F, Barbui T. Hydroxyurea for patients with essential thrombocythemia and a high risk of thrombosis. N Engl J Med 1995;332:1132-1136.

46. Tefferi A. Myelofibrosis with myeloid metaplasia. N Engl J Med 2000;342:1255-1265. 47. Chim CS, Kwong YL, Lie AK, Ma SK, Chan CC, Wong LG, Kho BC, Lee HK, Sim JP, Chan CH, Chan JC, Yeung YM, Law M, Liang R. Long-term outcome of 231 patients with essential thrombocythemia: prognostic factors for thrombosis, bleeding, myelofibrosis, and leukemia. Arch Intern Med 2005;165:2651-2658. 48. Gangat N, Wolanskyj AP, McClure RF, Li CY, Schwager S, Wu W, Tefferi A. Risk stratification for survival and leukemic transformation in essential thrombocythemia: a single institutional study of 605 patients. Leukemia 2007;21:270-276. 49. Girodon F, Dutrillaux F, Broséus J, Mounier M, Goussot V, Bardonnaud P, Chrétien ML, Maynadié M. Leukocytosis is associated with poor survival but not with increased risk of thrombosis in essential thrombocythemia: a population-based study of 311 patients. Leukemia 2010;24:900-903. 50. Barbui T, Thiele J, Passamonti F, Rumi E, Boveri E, Ruggeri M, Rodeghiero F, d’Amore ES, Randi ML, Bertozzi I, Marino F, Vannucchi AM, Antonioli E, Carrai V, Gisslinger H, Buxhofer-Ausch V, Müllauer L, Carobbio A, Gianatti A, Gangat N, Hanson CA, Tefferi A. Survival and disease progression in essential thrombocythemia are significantly influenced by accurate morphologic diagnosis: an international study. J Clin Oncol 2011;29:3179-3184.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0047 Turk J Hematol 2016;33:196-201

Retrospective Study of Incidence and Prognostic Significance of Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation: Influence of Corticosteroid Therapy Allojenik Hematopoetik Kök Hücre Nakli Sonrası Eozinofilinin Sıklığı ve Prognostik Öneminin Değerlendirildiği Geriye Dönük Çalışma: Kortikosteroid Tedavisinin Etkisi Wataru Yamamoto1, Eriko Ogusa1, Kenji Matsumoto1, Atsuo Maruta1, Yoshiaki Ishigatsubo2, Heiwa Kanamori1 1Kanagawa Cancer Center, Department of Hematology, Yokohama, Japan 2Yokohama City University Graduate Faculty of Medicine, Department of Internal Medicine and Clinical Immunology, Yokohama, Japan

Abstract

Öz

Objective: The clinical significance of eosinophilia after allogeneic hematopoietic stem cell transplantation is controversial. This study aimed to retrospectively study the impact of eosinophilia on the outcome of allogeneic hematopoietic stem cell transplantation by taking into account the influence of corticosteroid therapy.

Amaç: Allojenik hematopoetik kök hücre nakli sonrası eozinofilinin önemi tartışmalıdır. Bu çalışma kortikosteroid tedavisinin etkisini hesaba katarak, eozinofilinin allojenik hematopoetik kök hücre naklinin sonuçları üzerine etkisini geriye dönük değerlendirmeyi amaçladık.

Materials and Methods: We retrospectively studied 204 patients with acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome who underwent allogeneic hematopoietic stem cell transplantation from January 2001 to December 2010.

Gereç ve Yöntemler: Ocak 2001’den Aralık 2010’a kadar akut myeloid lösemi, akut lenfoblastik lösemi ve myelodisplastik sendrom tanısıyla allojenik hematopoetik kök hücre nakli olan 204 hastayı geriye dönük değerlendirdik.

Results: The median age was 43 years (minimum-maximum: 1765 years). Myeloablative conditioning was used in 153 patients and reduced intensity conditioning was employed in 51 patients. Donor cells were from bone marrow in 132 patients, peripheral blood in 34, and cord blood in 38. Eosinophilia was detected in 71 patients and there was no significant predictor of eosinophilia by multivariate analysis. There was no relationship between occurrence of eosinophilia and the incidence or grade of acute graft-versus-host disease when the patients were stratified according to corticosteroid treatment. Although eosinophilia was a prognostic factor for 5-year overall survival by univariate analysis, it was not a significant indicator by multivariate analysis.

Bulgular: Ortanca yaş 43 (aralık: 17-65 yaş) idi. Yüz elli üç hastada miyeloablatif, 51 hastada azaltılmış yoğunluklu hazırlama rejimi uygulandı. Kök hücre kaynağı 132 hastada kemik iliği, 34 hastada periferik kan ve 38 hastada kordon kanıydı. Yetmiş bir hastada eozinofili saptandı ve çoklu değişken analizinde eozinofiliyi anlamlı olarak öngörecek bir belirteç saptanmadı. Hastalar kortikosteroid tedavisine göre gruplandığında eozinofili gelişimi ile akut graftverus-host hastalığı sıklığı ya da derecesi arasında bağlantı yoktu. Tek değişkenli analizde eozinofili 5 yıllık genel sağkalım açısından prognostik bir faktör olmasına karşın, çok değişkenli analizde anlamlı bir belirteç değildi.

Conclusion: These results suggest that the clinical significance of eosinophilia in patients receiving allogeneic hematopoietic stem cell transplantation should be assessed with consideration of systemic corticosteroid administration. Keywords: Eosinophilia, Allogeneic hematopoietic stem cell transplantation, Corticosteroid therapy, Prognostic factor, Graftversus-host disease

Address for Correspondence/Yazışma Adresi: Wataru YAMAMOTO, M.D., Kanagawa Cancer Center, Department of Hematology, Yokohama, Japan Phone : 81-45-391-5761 E-mail : watadesu@yahoo.co.jp

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Sonuç: Bu sonuçlar allojenik hematopoetik kök hücre nakli olan hastalarda eozinofilinin klinik öneminin, sistemik kortikosteroid uygulamasını dikkate alarak değerlendirilmesi gerektiğinin düşündürmektedir. Anahtar Sözcükler: Eozinofili, Allojenik hematopoetik kök hücre nakli, Kortikosteroid tedavisi, Prognostik faktör, Graft-versus-host hastalığı

Received/Geliş tarihi: January 23, 2015 Accepted/Kabul tarihi: November 18, 2015

Turk J Hematol 2016;33:196-201

Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction

Results

Proliferation of eosinophils is induced by stimulation with cytokines [1] and eosinophilia occurs in various clinical settings. Eosinophilia is often found in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a relationship between eosinophilia and the outcome and/or graft-versus-host disease (GVHD) has been reported [2,3,4,5,6,7,8,9,10]. However, the role of corticosteroid (CS) therapy should be taken into consideration with regard to evaluation of eosinophilia after allo-HSCT, because it is known that eosinophilia is influenced by such drugs [11,12]. Therefore, we retrospectively studied the impact of eosinophilia on the outcome of allo-HSCT by taking into account the influence of CS therapy.

A total of 204 patients received allo-HSCT for AML, ALL, or MDS. The median follow-up period was 5.7 years and patients’ clinical characteristics are shown in Table 1. The median age was 43 years (minimum-maximum: 17-65 years) and there were 102 patients of each sex. The underlying disease was AML in 126 patients, ALL in 63, and MDS in 15. Myeloablative conditioning was used in 153 patients and reduced intensity conditioning was employed in 51 patients. Donor cells were from bone marrow in 132 patients, peripheral blood in 34, and cord blood in 38.

Materials and Methods Patients who underwent allo-HSCT for hematologic malignancies from January 2001 to December 2010 at the Kanagawa Cancer Center were retrospectively investigated. We defined eosinophilia as a peripheral blood eosinophil count of >500 µL on more than one occasion, while systemic steroid therapy meant CS administration at more than 0.5 mg/kg/day within 100 days after allo-HSCT. Standard-risk disease was defined as acute myeloid leukemia (AML)/acute lymphoblastic leukemia (ALL) in the first or second remission and myelodysplastic syndrome (MDS) without leukemic transformation, while highrisk disease was defined as all others. Grading of acute GVHD was done according to established criteria [13]. Statistical Analysis Statistical analyses were performed with R software (version 2.11.1; R Development Core Team). Differences between groups were analyzed by the Wilcoxon rank sum test or Fisher’s exact test, as was appropriate for univariate analysis and logistic regression analysis for multivariate analysis. Overall survival (OS) was calculated from the date of transplantation to the date of death from any cause or the date of last follow-up. Non-relapse mortality was defined as death without disease relapse or resistance. Time-to-event curves were drawn according to the Kaplan-Meier method and the statistical significance of differences in survival was assessed by the log-rank test. Prognostic factors included age, sex mismatch, disease risk, conditioning regimen, GVHD prophylaxis, donor type, cytomegalovirus infection, CS therapy, and eosinophilia. Either the Cox proportional hazard model or the Fine-Gray proportional hazard model was used for analysis. Death without relapse was considered to be a competing risk for relapse, relapse was a competing risk for non-relapse mortality, and relapse and death without GVHD were competing risks for GVHD.

Eosinophilia was detected in 71 patients (34.8%). Its appearance was associated with total body irradiation (TBI), unrelated donor, and CS administration within 100 days after transplantation by univariate analysis. However, no significant predictors of eosinophilia were identified by multivariate analysis (Table 1). Among the 204 patients, 90 patients (44%) received systemic CS therapy. The reason for CS treatment was acute GVHD in 76 patients, engraftment syndrome in 4, interstitial pneumonia in 4, organizing pneumonia in 3, disease relapse in 1, diffuse alveolar hemorrhage in 1, and vasculitis in 1. The incidence of eosinophilia within 100 days after transplantation was higher in patients without CS (47/114 patients, 41.2%) than in patients with CS (24/90, 26.7%) (p=0.038). Among the 90 patients with CS, 11 were first given CS therapy after the appearance of eosinophilia. The frequency of eosinophilia was higher among patients who were not given CS therapy before eosinophilia appeared than among patients who were already receiving CS therapy (58/125 patients, 46.4% vs. 13/79, 16.5%, respectively; p<0.001). The cumulative incidence of grade II-IV and grade III-IV acute GVHD was 45.1% and 18.6%, respectively. Table 2 shows the patients stratified according to CS treatment, GVHD grade, and occurrence of eosinophilia. There was no significant relationship between the grade of acute GVHD and occurrence of eosinophilia when patients were stratified by CS administration. The OS, cumulative incidence of relapse, and non-relapse mortality rate are stratified according to eosinophilia and systemic CS therapy in Figures 1 and 2, respectively. Patients with eosinophilia had a higher 5-year OS compared to those without eosinophilia (59.8%, 95% confidence interval [CI]: 48.9-73.1 vs. 45.4%, 95% CI: 37.5-55.0; p=0.016) (Figure 1A). In contrast, patients receiving CS therapy had a lower 5-year OS compared to those without CS therapy (38.9%, 95% CI: 29.9-50.7 vs. 60.9%, 95% CI: 52.3-70.9; p<0.001) (Figure 2A). However, there was no significant difference in 5-year OS between patients with or without eosinophilia among those who received CS therapy (47.3%, 95% CI: 30.373.9 vs. 33.7%, 95% CI: 23.9-47.7, respectively; p=0.200) (Figure 3A). Similarly, there was no difference in the 5-year 197

Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation

OS between patients with and without eosinophilia among those not receiving CS therapy (66.3%, 95% CI: 53.6-82.1 vs. 57.4%, 95% CI: 46.5-70.8, respectively; p=0.139) (Figure 3A). Furthermore, the cumulative incidence of relapse showed no significant association with eosinophilia (Figures 1B, 2B, and 3B). However, non-relapse mortality was significantly higher in patients receiving CS therapy (Figures 2C and 3C). According to univariate analysis, eosinophilia was a good prognostic indicator for 5-year OS (hazard ratio: 0.6, 95% CI: 0.4-0.9; p=0.017), but it was not an independent prognostic indicator by multivariate analysis (hazard ratio: 0.8, 95% CI: 0.5-1.3; p=0.385). Finally, high-risk disease, unrelated donor, and CS therapy were adverse prognostic indicators for 5-year OS according to multivariate analysis (Table 3).

Turk J Hematol 2016;33:196-201

Discussion In the present study, we investigated the incidence and clinical implications of eosinophilia occurring within 100 days after alloHSCT, and we also analyzed the prognostic value of eosinophilia in relation to the influence of CS administration. The incidence of eosinophilia (34.8%) in our patient cohort was comparable with that in previous reports, although the definition of eosinophilia varies among studies. It is well known that a decrease of eosinophils is caused by CS administration [10,11]; hence, we assessed the clinical implications of eosinophilia in relation to systemic CS administration. The incidence of eosinophilia was significantly lower in patients receiving CS treatment compared with that for those without CS treatment in the present study, and the same result for patients with acute GVHD has already been described [2]. In our study, MDS, use of TBI, and transplantation from an unrelated donor were also associated with a lower incidence of eosinophilia, but the reasons for these associations are unknown. The relationship between eosinophilia and acute GVHD after alloHSCT remains controversial. We could not find any association between eosinophilia and acute GVHD among patients with or without CS therapy in this study. Some previous reports suggested that eosinophilia is significantly related to the onset of acute GVHD [2,9,10]. However, Aisa et al. reported that the onset of eosinophilia within 100 days after allo-HSCT is associated with a lower rate of grade II-IV acute GVHD (43% vs. 98%; p<0.001) [3]. They speculated that this association between eosinophilia and a lower incidence of severe acute GVHD may reflect the immunosuppressive effect of Th2 cytokines, which induce eosinophilia.

Figure 1. Outcome stratified according to the presence/absence of eosinophilia. A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality.

A relationship between the occurrence of eosinophilia after alloHSCT and a good prognosis [2,3,4,5,6] has been reported previously.

Figure 2. Outcomes stratified according to corticosteroid administration. A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality.

Figure 3. Outcomes stratified according to eosinophilia and corticosteroid therapy. A) Overall survival, B) cumulative incidence of relapse, C) non-relapse mortality.

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Table 1. Patient characteristics and predictors of eosinophilia within 100 days after transplantation. Eosinophilia (+) Eosinophilia (-) (n=71) (n=133) Age. median (range)

Univariate Analysis

Multivariate Analysis

HR (95% CI)

43 (17-65)

43 (18-64)

Male

1.0

Female

1.5 (0.8-2.8)

Acute myelogenous leukemia

1.0

Acute lymphoblastic leukemia

Myelodysplastic syndrome

Standard risk High risk

0.541

Sex 0.240

1.2 (0.6-2.3)

0.633

0.1 (0.0-0.9)

0.022

1.0

0.5 (0.3-1.0)

Myeloablative

104

1.0

Reduced-intensity

1.6 (0.8-3.2)

1.0

Yes

124

0.3 (0.1-0.8)

Cyclosporine+sMTX

1.0

Tacrorimus+sMTX

0.6 (0.3-1.3)

1.0

Unrelated

0.5 (0.3-1.0)

Bone marrow

1.0

Peripheral blood

Cord blood

Match Mismatch

Disease -

Disease risk at transplantation 0.068

Conditioning regimen 0.175

Total body irradiation 1.0 0.009

0.4 (0.2-1.0)

0.058

GVHD prophylaxis* 0.196

Donor type 1.0 0.039

0.7 (0.4-1.3)

0.236

1.0 (0.4-2.4)

1.000

0.5 (0.2-1.3)

0.174

1.0

0.7 (0.3-1.3)

Other

122

1.0

Recipient negative and donor positive

2.9 (0.7-14.9)

Other

108

1.0

Female to male

0.7 (0.3-1.7)

Grade 0-I

1.0

Grade II-IV

0.8 (0.4-1.4)

1.0

Yes

0.5 (0.3-1.0)

Stem cell source -

HLA disparity* 0.272

Cytomegarovirusâ&#x20AC; 0.098

Sex mismatch 0.441

Acute GVHD 0.381

0.038

0.6 (0.3-1.1)

CS administration 100 days after transplantation 1.0 0.090

*Data is uncertain in one case. â&#x20AC; Data is uncertain in eleven cases. HR: Hazard ratio, CI: confidence interval, GVHD: graft-versus-host disease, sMTX: short-term methotrexate, CS: corticosteroid.

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Only one study showed that there was no correlation between eosinophilia and the outcome of cord blood transplantation in adults [8]. In the present study, eosinophilia was associated with a better outcome by univariate analysis, but this was not confirmed by multivariate analysis. Since the incidence of eosinophilia differs among patients with or without CS treatment, we also analyzed its effect on prognosis in patients stratified according

Turk J Hematol 2016;33:196-201

to systemic CS administration, but there was no significant impact of eosinophilia on the outcome in either group. Finally, multivariate analysis showed that high-risk disease, unrelated donor, and CS therapy were adverse predictors of survival with statistical significance. However, there is a limitation in that we did not treat eosinophilia and CS administration as timedependent covariates in multivariate analyses.

Table 2. Distribution of patients with acute graft-versus-host disease. Acute GVHD

Corticosteroid (+) (n=90)

Grade

Eosinophilia (+) (n=24)

Eosinophilia (-) (n=66)

III

Corticosteriod (-) (n = 114) Eosinophilia (+) (n=47)

Eosinophilia (-) (n=67)

0.709

0.424

GVHD: Graft-versus-host disease.

Table 3. Prognostic factors for overall survival. Variables

Univariate Analysis HR (95% CI)

Multivariate Analysis

HR (95% CI)

0.600

Age (years) <50

1.0

â&#x2030;Ľ50

1.1 (0.7-1.7)

Sex mismatch Other

1.0

Female to male

1.6 (1.0-2.5)

1.0 0.068

1.4 (0.8-2.2)

0.224

Disease risk Standard risk

1.0

High risk

2.8 (1.9-4.2)

1.0 <0.001

2.7 (1.8-4.2)

<0.001

Conditioning regimen Myeloablative

1.0

Reduced-intensity

0.9 (0.5-1.4)

0.550

0.882

GVHD prophylaxis Cyclosporine+sMTX

1.0

Tacrorimus+sMTX

1.0 (0.7-1.6)

Donor type Related

1.0

Unrelated

1.8 (1.2-2.6)

1.0 0.006

1.9 (1.2-2.9)

0.003

Cytomegarovirus Other

1.0

Recipient negative and donor positive

0.7 (0.3-2.0)

0.547

0.017

1.2 (0.8-1.9)

<0.001

1.8 (1.2-2.6)

Eosinophilia Present

1.0

Absent

1.7 (1.1-2.6)

1.0 0.440

CS administration Absent

1.0

Present

2.1 (1.4-3.1)

1.0

HR: Hazard ratio, CI: confidence interval, GVHD: graft-versus-host disease, sMTX: short-term methotrexate, CS: corticosteroid.

200

0.006

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Yamamoto W, et al: Eosinophilia after Allogeneic Hematopoietic Stem Cell Transplantation

Especially after allo-HSCT, systemic CS administration is done in patients who develop various complications, such as acute GVHD or pulmonary complications. Imahashi et al. reported that eosinophilia has an independent influence on the prognosis of patients with acute GVHD receiving CS treatment, but not that of patients without CS treatment [2]. Taking our results together with these findings raises the possibility that the severity of acute GVHD and CS therapy for GVHD may strongly influence transplantation outcomes regardless of eosinophilia. Furthermore, systemic CS administration is often done for alloHSCT patients with severe complications in addition to acute GVHD, and eosinophilia may be suppressed in those patients. Our findings about non-relapse mortality in the presence or absence of CS treatment support this interpretation. In addition to the relationship between eosinophilia and acute GVHD, there have been several reports on the pathogenesis of eosinophilia in the setting of allo-HSCT. Akhtari et al. reported that eosinophilia is observed in patients with eosinophilic pulmonary syndrome after allo-HSCT [14], but there were no specific causes of eosinophilia in our cohort. In conclusion, eosinophilia after allo-HSCT was not related to the outcome of transplantation or the incidence of acute GVHD in patients with or without systemic CS therapy, although this study had some limitations because it was a retrospective investigation conducted at a single institution. Acknowledgment This study was supported by a grant from the Kanagawa Health Foundation. Ethics Ethics Committee Approval: Retrospective study; Informed Consent: It was taken. Authorship Contributions Surgical and Medical Practices: Wataru Yamamoto, Heiwa Kanamori; Concept: Wataru Yamamoto, Heiwa Kanamori; Design: Wataru Yamamoto, Heiwa Kanamori; Data Collection or Processing: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Analysis or Interpretation: Wataru Yamamoto, Heiwa Kanamori; Literature Search: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Analysis or Interpretation: Wataru Yamamoto, Heiwa Kanamori; Literature Search: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto,

Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori; Writing: Wataru Yamamoto, Eriko Ogusa, Kenji Matsumoto, Atsuo Maruta, Yoshiaki Ishigatsubo, Heiwa Kanamori. 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. Rothenberg ME. Eosinophilia. N Engl J Med 1998;338:1592-1600. 2. Imahashi N, Miyamura K, Seto A, Watanabe K, Yanagisawa M, Nishiwaki S, Shinba M, Yasuda T, Kuwatsuka Y, Terakura S, Kodera Y. Eosinophilia predicts better overall survival after acute graft-versus-host-disease. Bone Marrow Transplant 2010;45:371-377. 3. Aisa Y, Mori T, Nakazato T, Shimizu T, Yamazaki R, Ikeda Y, Okamoto S. Blood eosinophilia as a marker of favorable outcome after allogeneic stem cell transplantation. Transpl Int 2007;20:761-770. 4. Kim DH, Popradi G, Xu W, Gupta V, Kuruvilla J, Wright J, Messner HA, Lipton JH. Peripheral blood eosinophilia has a favorable prognostic impact on transplant outcomes afterallogeneic peripheral blood stem cell transplantation. Biol Blood Marrow Transplant 2009;15:471-482. 5. Sato T, Kobayashi R, Nakajima M, Iguchi A, Ariga T. Significance of eosinophilia after stem cell transplantation as a possible prognostic marker for favorable outcome. Bone Marrow Transplant 2005;36:985-991. 6. Nakane T, Nakamae H, Hirose A, Nakamae M, Koh H, Hayashi Y, Nishimoto M, Umemoto Y, Yoshimura T, Bingo M, Okamura H, Yoshida M, Ichihara H, Aimoto M, Terada Y, Nakao Y, Ohsawa M, Hino M. Eosinophilia, regardless of degree, is related to better outcomes after allogeneic hematopoietic stem cell transplantation. Intern Med 2012;51:851-858. 7. Ahmad I, LabbĂŠ AC, Chagnon M, Busque L, Cohen S, Kiss T, Lachance S, Roy DC, Sauvageau G, Roy J. Incidence and prognostic value of eosinophilia in chronic graft-versus-host disease after nonmyeloablative hematopoietic cell transplantation. Biol Blood Marrow Transplant 2011;17:1673-1678. 8. Tomonari A, Takahashi S, Ooi J, Tsukada N, Konuma T, Kato S, Kasahara S, Iseki T, Tojo A, Asano S. Blood eosinophilia after unrelated cord blood transplantation for adults. Bone Marrow Transplant 2008;42:63-65. 9. Basara N, Kiehl MG, Fauser AA. Eosinophilia indicates the evolution to acute graft-versus-host disease. Blood 2002;100:3055. 10. McNeel D, Rubio MT, Damaj G, Emile JF, Belanger C, Varet B, Brousse N, Hermine O, Buzyn A. Hypereosinophilia as a presenting sign of acute graft-versus-host disease after allogeneic bone marrow transplantation. Transplantation 2002;74:1797-1800. 11. Walsh GM. Eosinophil apoptosis: mechanisms and clinical relevance in asthmatic and allergic inflammation. Br J Haematol 2000;111:61-67. 12. Schleimer RP. Effects of glucocorticosteroids on inflammatory cells relevant to their therapeutic applications in asthma. Am Rev Respir Dis 1990;141:5969. 13. Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, Hows J, Thomas ED. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995;15:825-828. 14. Akhtari M, Langston AA, Waller EK, Gal AA. Eosinophilic pulmonary syndrome as a manifestation of GVHD following hematopoietic stem cell transplantation in three patients. Bone Marrow Transplant 2009;43:155158.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0469 Turk J Hematol 2016;33:202-208

Cytokine Contents in Chronic Lymphocytic Leukemia: Association with ZAP70 Expression Kronik Lenfositik Lösemi Sitokin İçeriği: ZAP70 Ekspresyonu ile İlişkisi Nilgün Işıksaçan1, Suzan Çınar2, Esin Aktaş Çetin2, Melih Aktan3, Günnur Deniz2 1Bakırköy Dr. Sadi Konuk Training and Research Hospital, Central Laboratory, İstanbul, Turkey 2İstanbul University Institute of Experimental Medicine, Department of Immunology, İstanbul, Turkey 3İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

Abstract

Öz

Objective: Chronic lymphocytic leukemia (CLL) is a disease that shows varying clinical progression, and expression of the protein tyrosine kinase ZAP70 has been described as a very valuable prognostic factor. Patients with ZAP70 positivity are characterized by worse clinical course and significantly shorter progression-free and overall survival. In this study, intracytoplasmic interferon gamma (IFN-γ) and interleukin-4 (IL-4) content of T, B, and CLL cells in CLL patients and their correlations with Rai staging and ZAP70 positivity were investigated.

Amaç: Kronik lenfositik lösemi (KLL) klinik olarak çok farklı seyir gösterebilen bir hastalıktır ve tirozin kinaz ZAP70’in ekspresyonu, çok değerli bir prognostik faktör olarak tanımlanmıştır. ZAP70 pozitif hastalar, kötü klinik seyir, belirgin olarak kısa hastalıksız geçen süre ve düşük sağkalım oranı ile karakterizedir. Bu çalışmada KLL hastalarının T, B ve KLL hücrelerinde intrasitoplazmik interferon gama (IFN-y) ve interlökin-4 (IL-4) içeriği, Rai evrelemesi ve ZAP70 pozitifliği arasındaki korelasyon araştırılmıştır.

Materials and Methods: CLL patients newly diagnosed or in followup at the İstanbul University İstanbul Medical Faculty Hematology Department were included in this study. These patients were classified according to Rai staging and ZAP70 expression. IL-4, IFN-γ, and ZAP70 expressions in peripheral blood T, B, and CLL cells were measured by four-color flow cytometry. Results: There was a statistically significant correlation between advanced disease and ZAP70 positivity. IL-4-secreting T cells were significantly increased; however, IFN-γ secretion was significantly decreased in CLL patients compared to healthy individuals, whereas IL-4-secreting B cells were significantly diminished in contrast to T cells. Conclusion: These findings suggest damage in the cellular immunity and that IL-4 might lead to many complications and may be important in disease progression. Keywords: ZAP70, Interleukin-4, Interferon gamma, T cells, B cells, Chronic lymphocytic leukemia

Gereç ve Yöntemler: Bu çalışmaya İstanbul Üniversitesi İstanbul Tıp Fakültesi Hematoloji Kliniği’nde tanısı yeni konan veya takip edilmekte olan KLL hastaları dahil edilmiştir. Bu hastalar Rai evreleme sistemi ve ZAP70 ekspresyonlarına göre sınıflandırılmıştır. Periferik kan T, B ve KLL hücrelerinin IL-4, IFN-g ve ZAP70 ekspresyonları 4 renkli flow sitometri ile ölçülmüştür. Bulgular: Hastalığın evresindeki ilerleme ile ZAP70 pozitifliği arasında istatistiksel olarak anlamlı bir korelasyon bulunmaktadır. KLL hastalarının sağlıklı bireylerle kıyaslandığında IL-4 salgılayan T hücreleri sayısı anlamlı olarak artmış, ancak IFN-g salgıları anlamlı olarak azalmış, IL-4 salgılayan B hücre sayısı ise T hücrelerinin aksine anlamlı olarak azalmıştır. Sonuç: Bu bulgular, hücresel immünitede hasarın olabileceğini ve IL-4’ün birçok komplikasyonlara yol açarak hastalığın ilerlemesinde önemli olabileceğini düşündürtmektedir. Anahtar Sözcükler: ZAP70, Interlökin-4, Interferon gama, T hücreleri, B hücreleri, Kronik lenfositik lösemi hücreleri

Address for Correspondence/Yazışma Adresi: Günnur DENİZ, PhD., İstanbul University Institute of Experimental Medicine, Department of Immunology, İstanbul, Turkey Phone : +90 212 414 20 00 (pbx) 33306 E-mail : gdeniz@istanbul.edu.tr

202

Received/Geliş tarihi: December 06, 2014 Accepted/Kabul tarihi: April 20, 2015

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Işıksaçan N, et al: Cytokines’ Association with ZAP70 in Chronic Lymphocytic Leukemia

Introduction Chronic lymphocytic leukemia (CLL) is a disease that shows varying clinical progression. The Rai and Binet classification systems are useful to predict treatment requirements and survival rates for CLL patients, but current classifications fail to distinguish patients who may develop aggressive disease [1,2,3,4]. The somatic mutations in the immunoglobulin heavy chain variable (IGVH) region are very valuable prognostic factors and, in CLL cases, IGVH-mutated patients have a better clinical outcome while nonmutated patients have a poorer prognosis and impaired response to chemotherapy [5,6,7]. The protein tyrosine kinase zeta-associated protein (ZAP70) is a protein tyrosine kinase in the T cell receptor signal transduction system that can be detected in CLL cells. Studies have pointed out a correlation between ZAP70 expression in CLL cells and IGVH status, and showed that ZAP70 positive patients have an aggressive course, an immediate treatment requirement, longer therapy time, and lower survival rates [8,9,10]. Expression of ZAP70 has been described as a very valuable prognostic factor [2,8]. Many researchers have recently examined the cytokine content of the T cells in CLL patients and emphasized the significance of the T cell activity in the prognosis of the disease [11,12]. Interleukin-4 (IL-4) production by T cells has been shown to significantly increase in patients with progressive disease [11,12]. In light of these data, it was suggested that in CLL, the type 1 T cell cytokine profile is converted to the type 2 T cell profile in the advanced stages of the disease [13]. The aim of this study was to evaluate the expression of ZAP70 changing during disease progression, the intracellular interferon gamma (IFN-γ) and IL-4 content of T and B lymphocytes and the CLL cell subset (CD5+CD19+) in CLL patients and healthy subjects, and ZAP70 correlation with cytokine production.

Materials and Methods Study Population Twenty-eight patients in follow-up at the İstanbul University İstanbul Medical Faculty Hematology Department were included in the study. Patients were diagnosed according to the CLL diagnosis criteria published in 1996 by a study group supported by the National Cancer Institute. Clinical data and follow-up files of all the patients included in the study were gathered. All patients received written information about the study, including ethics committee approval, before the study was initiated. Twenty-eight CLL patients (18 males, 10 females) and 10 healthy individuals (3 males, 7 females) were involved in this study (Table 1). The ZAP70 expression was measured for all CLL cases and cytokine levels were measured for 12 CLL cases and the 10

healthy volunteers. The ages of the patients were between 36 and 81 (59±11) years, and the mean values were 60±12 years for males and 56±7 years for females. Peripheral Blood Mononuclear Cell Isolation and Flow Cytometric Analyses of Lymphocyte Subsets Samples were processed using a whole-blood lysis method to analyze lymphocyte subsets. Heparinized blood samples were collected from patient and healthy donors and stained with anti-CD19-fluorescein isothiocyanate (FITC), anti-CD3allophycocyanin (APC), anti-CD5-TRI-COLOR (TC), anti-CD45FITC, anti-CD14-phycoerythrin (PE), anti-CD23-PE, anti-CD38PE, and PE, FITC, TC, or APC conjugated isotype control (IC) monoclonal antibodies (mAbs) (all from Caltag Laboratories, Austria) for 30 min at room temperature. Lysis was performed with FACS Lysing Solution (BD Biosciences, USA). After washing cells with phosphate-buffered saline (PBS), stained cells were fixed in 1% paraformaldehyde and the cells were analyzed with BD FACSCalibur with CellQuest Software (BD Biosciences). For the intracytoplasmic ZAP70 expression, the cells were labeled with anti-CD19-FITC and anti-CD5-TC mAbs (Caltag Laboratories) for 30 min at room temperature. After the lysing period, cells were washed with PBS and were fixed and permeabilized with paraformaldehyde/saponin solution (Cytofix&Cytoperm Kit, BD Biosciences), and then were stained with PE-conjugated-IgG1 or PE-labeled ZAP70 (Caltag Laboratories) mAbs for 30 min at room temperature and analyzed by flow cytometry. Each analysis was performed using at least 5000 cells gated in the region of the B cell population, and the cells were analyzed with BD FACSCalibur with CellQuest Software (BD Biosciences). ZAP70 expression was investigated in CLL patients for positivity; 20% was used as the cut-off [9]. Table 1. Demographic and clinical characteristics of patient group. Variables

CLL (n=28)

Age, years

59±11

Sex, female, n (%)

10 (35.7)

Sex, male, n (%)

18 (64.3)

Rai stage 0-1, n (%)

15 (43.9)

Rai stage 2-4, n (%)

13 (19.3)

Zap70 expression, %

23.5

CD38 expression, %

5.9

CD23 (n=10), %

≥20

Values are presented as mean ± standard deviation, median (interquartile range), or number or percentage of patients. CLL: Chronic lymphocytic leukemia. Rai staging: Stage 0: lymphocytosis, Stage 1: lymphocytosis with lymphadenopathy; Stage 2: lymphocytosis with either hepatomegaly or splenomegaly, Stage 3: lymphocytosis and anemia (hemoglobin <11 g/dL), Stage 4: lymphocytosis and thrombocytopenia (<100,000/mm3) with or without lymphadenopathy, hepatomegaly, splenomegaly, or anemia.

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CD19+ cells were analyzed for the expression of CD5, CD38, and ZAP70 (Figure 1A). Most of the CD19+ B cells expressed CD5; however, there was only one patient expressing both CD5 and CD38 (Figures 1B and 1C, respectively). Expression of CD5 showed heterogeneity among the patients. Intracytoplasmic ZAP70 together with CD5 was expressed in different levels in CD19+ B cells (Figures 1D, 1E, and 1F). Intracytoplasmic Cytokine Staining

After incubation, PBMCs were washed with PBS solution and stained with anti-CD19-PE and anti-CD3-APC mAbs for 30 min (Caltag Laboratories) for determining the cytokine secretion of T and B cells. Cells were washed with PBS and then fixed and permeabilized with paraformaldehyde/saponin solution (Cytofix&Cytoperm Kit, BD Biosciences). After washing, the cells were stained with FITC conjugated IC (IgG1), anti-IL-4FITC, and anti-IFN-γ-FITC (Caltag Laboratories) mAbs for 30 min at room temperature. After washing, cells were resuspended in 1% paraformaldehyde (Sigma Chem. Co.) and analyzed by flow cytometry. Each sample was acquired with a BD FACSCalibur (BD Biosciences) and analyzed with the instrument’s operating software, CellQuest (BD Biosciences). Statistical Analysis Statistical analysis was performed using a standard nonparametric Mann-Whitney U test using SPSS 17.0 for Windows. The results are presented as median values and p<0.05 was accepted as the statistical significance level.

Results Increased Expression of ZAP70 in Chronic Lymphocytic Leukemia Patients ZAP70 levels in CD19+CD5+ CLL patients did not show any differences according to sex (p>0.05). Seventeen patients were

SSC

Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Hypaque (Sigma Chem. Co., USA) density gradient centrifugation, and cells were washed twice in Hank’s balanced salt solution. The cells were finally adjusted to a final concentration of 1x106 cells/mL in complete RPMI-1640 medium (Sigma Chem. Co.) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), streptomycin (100 mg/ mL), gentamicin (50 mg/mL), and 50 µM 2-mercaptoethanol. Freshly purified PBMCs were washed and 1x106 cells/mL were stimulated for 18 h by a combination of phorbol ester, phorbol12-myristate-13-acetate (50 ng/mL), and Ca2+ ionophore (ionomycin, 250 ng/mL) in a 24-well round-bottom plate (all from Sigma Chem. Co.). The combination of these two stimuli was used to achieve the strongest stimulus for intracytoplasmic cytokine secretion. Monensin (Sigma Chem. Co.) was added at a final concentration of 1 µM to the cultures in the final 4 h.

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Figure 1. Gating strategy of chronic lymphocytic leukemia. Peripheral blood mononuclear cells from chronic lymphocytic leukemia patients (n=28) were stained for CD5, CD19, CD38, and ZAP70 mAbs and analyzed by flow cytometry. CD19+ cells were gated versus SSC (A). Representative dot-plot analyses for the expression of a patient negative (B) and positive (C) for CD38 with CD5+ in CD19+ cells are shown. In the CD19+ B cell population, CD5+ZAP70+ (D, E) and CD5+ZAP70- (F) plots from three different patients with chronic lymphocytic leukemia are also indicated. The numbers indicate the proportion of cells positive for indicated markers. 204

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Işıksaçan N, et al: Cytokines’ Association with ZAP70 in Chronic Lymphocytic Leukemia

positive for ZAP70; 8 of them were Rai 0-1 and 9 of them were Rai 2-4. However, 7 patients negative for ZAP70 were Rai 0-1 and 4 patients for Rai 2-4. When patients are divided into two groups according to Rai staging system, the first group includes 15 patients in stages 0 and 1, and the second group includes 13 patients in stages 2 and 4. The difference between ZAP70 expression in the first and second Rai groups was statistically significant (p<0.02) (Figure 2). CD38 Expression with ZAP70 Increase in CD38 expression correlates with increased lymphocyte proliferation and disease progression. CD38 expression is important in prognosis when combined with ZAP70. All patients were analyzed for CD38 expression, and an expression level of ≥30% was accepted as positive [14]. In CD19+ B cell populations, 26 patients were CD5+CD38-; however, only one patient was CD5+CD38+ and also ZAP70 positive (data not shown). Increased IL-4 and Decreased IFN-γ Levels in Chronic Lymphocytic Leukemia Patients In the first step, CLL patients were divided into two main groups according to their CD19, CD5, and ZAP70 expression

Figure 2. Expression of ZAP70+ cells according to Rai classification system. Chronic lymphocytic leukemia patients were divided into two groups according to the Rai staging system. Summarized data showing the percentages of ZAP70 expression in chronic lymphocytic leukemia patients and significant differences are indicated. ZAP70 expression was 23.5% (3.02-85.55) [median (min-max)] in Rai 0-1 as the early stage and 51.84% (6.8199.05) in Rai 2-4 as the late stage among 28 chronic lymphocytic leukemia patients.

(CD19+CD5+ZAP+ and CD19+CD5+ZAP-). Secondly, CD3+ T, CD19+ B, and CD19+CD5+ cells of CD19+CD5+ZAP+ and CD19+CD5+ZAP- CLL patients were evaluated for their intracellular IL-4 and IFN-γ contents. IL-4 levels of CD3+ T cells in ZAP70+ and ZAP70- patients were significantly increased in both groups compared to healthy individuals (p<0.004 and p<0.002, respectively). In contrast, IFN-γ secretion of CD3+ T cells was significantly decreased in both groups compared to healthy individuals (p<0.006 and p<0.002, respectively). However, IL-4 and IFN-γ levels did not differ in CD3+ T cells of ZAP+ and ZAP- CLL patients (Figures 3A and 3B). IL-4 levels of CD19+ B cells showed significant decreases in ZAP70- CLL patients compared to healthy subjects (p<0.004), whereas IL-4 levels between ZAP70+ and ZAP70- patients did not show any difference. IFN-γ levels of CD19+ B cells in ZAP70+ patients did not show any difference in comparison to ZAP70patients, but both patient groups had significantly lower IFN-γ levels than healthy subjects (p<0.04 and p<0.02, respectively) (Figures 3A and 3B). There was no significant difference in IL-4 and IFN-γ expression in CD19+CD5+ cells in ZAP70+ and ZAP70- patients (Figures 3A and 3B). The IL-4/IFN-γ ratios in CD3+ T, CD19+, and CD19+CD5+ B cells were analyzed and no significant difference for the expression of ZAP70 was observed (data not shown).

Figure 3. Comparison of intracellular IFN-γ and IL-4 production of CD3+ T, CD19+ B, and CD19+CD5+ cells between ZAP70+ and ZAP70+ chronic lymphocytic leukemia patients and healthy subjects. Intracellular cytokine levels were measured in stimulated peripheral blood mononuclear cells obtained from chronic lymphocytic leukemia patients (n=28) and healthy individuals (n=10). IL-4 and IFN-γ secretions were analyzed by flow cytometry. Results are shown as median (range) deviation. 205

Işıksaçan N, et al: Cytokines’ Association with ZAP70 in Chronic Lymphocytic Leukemia

Discussion Chronic lymphocytic leukemia is leukemia of small, mature B cells; it mostly affects adults over 65 years of age and it is the most common form of lymphoid malignancy. The Rai and Binet classification systems are useful to predict treatment requirements and survival for CLL patients, but the prognostic value of these classifications is limited in early stage cases. From 30% to 40% of patients in the early stage may develop aggressive disease and die in a short time period [15,16]. It has been shown that for ZAP70 positivity, when a cut-off value of 20% was used, CLL patients could be classified into two groups: those with levels of <20% had increased survival time and decreased chance of disease progression [9]. Two main techniques for measuring ZAP70 by flow cytometry involve comparing ZAP70 expression in B cells either against an isotype control or against ZAP70 expression in T cells [1,17]. In this study, B cell surface marker CD19 and CLL-specific surface marker CD5 were used and ZAP70 expressions in the doublepositive CD19+CD5+ cells were evaluated; ZAP70 expressions of T cells were easily excluded. A great deal of information can be extracted about the presence and status of CLL from the ZAP70 expression in the Rai staging system. The statistically significant difference of ZAP70 expression in stages 2 and 4 compared to early stages with significantly more ZAP70 patients in Rai stages 2 and 4 in comparison to Rai stages 0 and 1 is consistent with the literature and was evaluated as a marker of poor diagnosis. CD38 is a glycoprotein found on hematopoietic cells including B cells; it is a marker of cell activation and also functions in proliferation and activation [18]. Association between ZAP70 as an IGVH mutation status and CD38 has been investigated [19]. In contrast to high expression of ZAP70 (53%), decreased CD38 expression in CLL patients might be related to the variability during the disease. The combination of ZAP70 and CD38 expression gives complementary prognostic information, whereas this analysis allows us to identify three isolated B-CLL patient subgroups with good, intermediate, and poor prognosis to decide how to treat them, especially in early clinical stages of the disease. ZAP70 expression is stable during the course of the disease and it can be relatively easily measured in diagnostic laboratories with flow cytometers in contrast to altered CD38 expression, which is known as an independent prognostic factor in CLL [20]. It has been found that age and stage at presentation predict prognosis in survival in newly diagnosed patients with CLL [21]. Studies about age at diagnosis and ZAP70 levels in CLL patients showed no association. Similar to recent findings, there was no relation between ZAP70 and sex in our study [22,23,24]. CLL cells are nondividing monoclonal neoplastic CD5+ B cells and they are arrested at the G0 phase of the cell cycle. Failure 206

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to induce apoptosis plays a role in accumulation of the leukemic cells rather than cell proliferation. CD19+CD5+ CLL cells are greatly influenced by T cell-derived cytokines such as IL-1-alpha, IL-1-beta, IL-6, IL-7, IL-8, IL-10, IL-13, IFN-γ, and transforming growth factor (TGF)-β, which mediate interactions between B and T cells [25,26]. Apoptosis, the programmed cell death, can be activated in different cell types in response to a number of physiologically relevant stimuli. CLL CD5+ B cells are arrested in G0 and display enhanced survival in vivo, whereas they undergo spontaneous apoptosis in vitro [27]. Changes of the cytokine network in malignant cells and T cells due to various reasons may lead to expansion of the B cell clones. The formation of a new cytokine network may govern cell proliferation and apoptosis in CLL patients. Studies indicate the development of secondary neoplasias, infectious complications, and autoimmune disorders in advanced disease due to T cell defects and alterations in the cytokine content [12]. IL-4 and IFN-γ are suggested to regulate leukocyte adhesion molecule-1, whereas they take part in the interaction of tumor cells and immune effector cells, and T cell dysfunction in the advanced stage of the disease; therefore, altered cytokine profiles may affect the pathogenesis of the disease [28]. The cytokine microenvironment plays a critical role for Th1 and Th2 polarization, and B-CLL cells express several cytokines such as TGF, IL-10, and IL-4. It is suggested that in patients with B-CLL, with or without ZAP-70 overexpression, distinct cytokine patterns are triggered. It is possible that the IL-4, IL-10, and tumor necrosis factor-α cytokine profile of ZAP-70+ CD4 lymphocytes might favor B-CLL cell growth and survival and, in contrast, the IFN-γ and IL-2 profile in CD4 lymphocytes from ZAP-70- B-CLL patients might promote an antitumor cytotoxic CD8+ T lymphocyte and NK cell response (29). Our observations of high levels of IL-4 but diminished IFN-γ content of CD3+ T cells in CLL patients suggest that the Th1 cytokine profile turns into the Th2 cytokine profile in CLL patients. Several studies indicate the dominancy of Th1 in the early stage of the disease, but in the advanced stage Th1 decreases and Th2 increases. Th1/Th2 ratio is lower in the advanced stage of the disease than in early stages [12] and it has been suggested that the transformation of Th1 to Th2 is the reason for disease progression [13]. Our findings support the other recent findings, and the transformation of Th1 to Th2 might be related to ZAP70 expression. The cause of Th1 and Th2 count alteration in cancer immunity is not clear; however, the shifting of the Th1 cytokine profile to Th2 seems to be associated with ZAP70. The role of cytokines in CLL is a new topic and it should be analyzed further. High levels of ZAP70 expression and IL-4 secretion might be indicative of poor prognosis, and increased levels of IL-4 may be caused by infections and autoimmune diseases that accompany

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Işıksaçan N, et al: Cytokines’ Association with ZAP70 in Chronic Lymphocytic Leukemia

the disease. On the other hand, IL-4 can lead to the release of the growth factors that are increased in the cytoplasm of CLL cells. Acknowledgments This work was supported by the Scientific Research Projects Coordination Unit of İstanbul University, project number T697/30062005. It was presented at the 20th National Immunology Congress held in Cyprus in 2009 (best poster award, poster reference no. 97).

8. Wiestner A, Rosenwald A, Barry TS, Wright G, Davis RE, Henrickson SE, Zhao H, Ibbotson RE, Orchard JA, Davis Z, Stetler-Stevenson M, Raffeld M, Arthur DC, Marti GE, Wilson WH, Hamblin TJ, Oscier DG, Staudt LM. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 2003;101:4944-4951. 9. Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, Marcé S, López-Guillermo A, Campo E, Montserrat E. ZAP-70 expression as a surrogate for immunoglobulin-variable region mutations in chronic lymphocytic leukemia. N Eng J Med 2003;348:1764-1775.

Ethics

10. Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG, Neuberg DS, Flinn IW, Rai KR, Byrd JC, Kay NE, Greaves A, Weiss A, Kipps TJ. ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Eng J Med 2004;351:893-901.

Ethics Committee Approval: The study protocol was approved by the local ethical committee; Informed Consent: It was taken.

11. Rossman ED, Lewin N, Jeddi-Tehrani M, Osterborg A, Mellstedt H. Intracellular T cell cytokines in patients with B cell chronic lymphocytic leukaemia (B-CLL). Eur J Haematol 2002;68:299-306.

Authorship Contributions

12. Podhorecka M, Dmoszynska A, Rolinski J, Wasik E. T type 1/type 2 subsets balance in B-cell chronic lymphocytic leukemia-the three-color flow cytometry analysis. Leuk Res 2002;26:657-660.

Concept and Design: Melih Aktan, Günnur Deniz, Nilgün Işıksaçan; Data Collection or Processing: Melih Aktan, Nilgün Işıksaçan; Analysis: Suzan Çınar, Nilgün Işıksaçan, Esin Aktaş Çetin; Interpretation: Suzan Çınar, Nilgün Işıksaçan, Günnur Deniz; Literature Search: Nilgün Işıksaçan; Writing: Nilgün Işıksaçan, Suzan Çınar, Esin Aktaş Çetin, Melih Aktan, Günnur Deniz. 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: This work was supported by the Scientific Research Projects Coordination Unit of İstanbul University, project number T697/30062005.

References 1. Orchard JA, Ibbotson RE, Davis Z, Wiestner A, Rosenwald A, Thomas PW, Hamblin TJ, Staudt LM, Oscier DG. ZAP-70 expression and prognosis in chronic lymphocytic leukemia. Lancet 2004;363:105-111. 2. Dürig J, Nückel H, Cremer M, Führer A, Halfmeyer K, Fandrey J, Möröy T, Klein-Hitpass L, Dührsen U. ZAP-70 expression is a prognostic factor in chronic lymphocytic leukemia. Leukemia 2003;17:2426-2434. 3. Dighiero G. CLL biology and prognosis. Hematology Am Soc Hematol Educ Program 2005;278-284.

13. Mantovani G, Maccio A, Esu S, Lai P, Massa E, Dessi D. Levels of IL-4, IL-10 and INF-γ in the serum and in the PBMC culture supernatants from 31 patients with hematological malignancies. Br J Haematol 1998;102:118. 14. Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, Buchbinder A, Budman D, Dittmar K, Kolitz J, Lichtman SM, Schulman P, Vinciguerra VP, Rai KR, Ferrarini M, Chiorazzi N. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840-1847. 15. Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS. Clinical staging of chronic lymphocytic leukemia. Blood 1975;46:219-234. 16. Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O’Brien S, Rai KR. National Cancer Institute-Sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996;87:4990-4997. 17. Kay S, Herishanu Y, Pick M, Rogowski O, Baron S, Naparstek E, Polliack A, Deutsch VR. Quantitative flow cytometry of ZAP-70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome. Cytometry B Clin Cytom 2006;70:218-226. 18. Hamblin T. Chronic lymphocytic leukemia: one disease or two? Ann Hematol 2002;81:299-303. 19. Sagatys EM, Zhang L. Clinical and laboratory prognostic indicators in chronic lymphocytic leukemia. Cancer Control 2012;19:18-25. 20. Üre U, Ar MC, Başlar Z, Soysal T, Öngören S, Gülseven M, Aydın Y, Ülkü B, Tüzüner N, Ferhanoğlu B. The effect of ZAP-70 expression on disease progression in early-stage (Binet A) B-CLL patients. Balkan Med J 2009;26:317-321.

4. Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Eng J Med 1995;333:1052-1057.

21. Thurmes P, Call T, Slager S, Zent C, Jenkins G, Schwager S, Bowen D, Kay N, Shanafelt T. Comorbid conditions and survival in unselected, newly diagnosed patients with chronic lymphocytic leukemia. Leuk Lymphoma 2008;49:49-56.

5. Hamblin TJ, Davis Z, Gardier A, Oscier DG, Stevenson FK. Unmutated IgV(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848-1854.

22. Assem M, Abel Hamid T, Kohla S, Arsanyos S. The prognostic significance of combined expression of ZAP-70 and CD38 in chronic lymphocytic leukemia. J Egypt Natl Canc Inst 2009;21:287-297.

6. Maloum K, Davi F, Merle-Béral H, Pritsch O, Magnac C, Vullier F, Dighiero G, Troussard X, Mauro FF, Bénichou J. Expression of unmutated VH genes is a detrimental prognostic factor in chronic lymphocytic leukemia. Blood 2000;96:377-379.

23. Shanafelt TD, Rabe KG, Kay NE, Zent CS, Jelinek DF, Reinalda MS, Schwager SM, Bowen DA, Slager SL, Hanson CA, Call TG. Age at diagnosis and utility of prognostic testing in patients with chronic lymphocytic leukemia. Cancer 2010;116:4777-4787.

7. Morilla A, Gonzalez de Castro D, Del Giudice I, Osuji N, Else M, Morilla R, Brito Babapulle V, Rudenko H, Matutes E, Dearden C, Catovsky D, Morgan GJ. Combinations of ZAP-70, CD38 and IGHV mutational status as predictors of time to first treatment in CLL. Leuk Lymphoma 2008;49:2108-2115.

24. Hus I, Podhorecka M, Bojarska-Junak A, Roliński J, Schmitt M, Sieklucka M, Wasik-Szczepanek E, Dmoszyńska A. The clinical significance of ZAP-70 and clinical CD38 expression in B cell chronic lymphocytic leukemia. Ann Oncol 2006;17:683-690.

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25. Hoffbrand AV, Panayiotidis P, Reittie J, Ganeshaguru K. Autocrine and paracrine growth loops in chronic lymphocytic leukemia. Semin Hematol 1993;30:306-317.

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28. Jewell AP, Yong KL, Worman CP, Giles FJ, Goldstone AH, Lydyard PM. Cytokine induction of leucocyte adhesion molecule-1 (LAM-1) expression on chronic lymphocytic leukemia cells. Leukemia 1992;6:400-404.

26. Pistoria V. Production of cytokines by human B cells in health and disease. Immunol Today 1997;18:343-350.

29. Monserrat J, Sánchez MÁ, de Paz R, Díaz D, Mur S, Reyes E, Prieto A, de la

27. Ghamlouch H, Ouled-Haddou H, Damaj G, Royer B, Gubler B, Marolleau JP. A combination of cytokines rescues highly purified leukemic CLL B-cells from spontaneous apoptosis in vitro. PLoS One 2013;8:60370.

subset distribution and cytokine expression in CD4 T lymphocytes in ZAP-70

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RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0214 Turk J Hematol 2016;33:209-215

Finding the Optimal Conditioning Regimen for Relapsed/ Refractory Lymphoma Patients Undergoing Autologous Hematopoietic Cell Transplantation: A Retrospective Comparison of BEAM and High-Dose ICE Otolog Hematopoetik Kök Hücre Nakli Yapılan Nüks/Dirençli Lenfoma Hastalarında BEAM ve Yüksek Doz ICE Rejimlerinin Geriye Dönük Karşılaştırılması Onur Esbah1, Emre Tekgündüz2, Itır Şirinoğlu Demiriz2, Sinem Civriz Bozdağ2, Ali Kaya2, Ayşegül Tetik2, Ömür Kayıkçı2, Gamze Durgun2, Şerife Kocubaba2, Fevzi Altuntaş2 1Ankara Oncology Hospital, Clinic of Medical Oncology, Ankara, Turkey 2Ankara Oncology Hospital, Hematology and Stem Cell Transplantation Unit, Ankara, Turkey

Abstract

Öz

Objective: High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (AHCT) is a well-defined treatment modality for relapsed/refractory non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma (HL). Although there are several options in terms of conditioning regimens before AHCT, no one treatment is accepted as a standard of care. This study aimed to compare different conditioning regimens for the treatment of NHL and HL.  Materials and Methods: Medical records of 62 patients who had undergone AHCT following BEAM (BCNU, etoposide, cytarabine, and melphalan) and high-dose ICE (hICE; ifosfamide, carboplatin, and etoposide) conditioning regimens were analyzed retrospectively and compared in terms of efficacy and adverse effects.    Results: The study included a total of 29 and 33 patients diagnosed with relapsed/refractory NHL and HL, respectively. Patients received BEAM (n=37) or hICE (n=25) regimens for conditioning. One-year overall survival was 73±6% in all patients. One-year overall survival was 71±8% and 74±9% in the BEAM and hICE groups, respectively (p=0.86). The incidences of nausea/vomiting (grade ≥2) (84% vs. 44.7%; p=0.04) and mucositis (grade ≥2) (13% vs. 3%; p=0.002) were higher in the hICE group compared to the BEAM group. In addition, we witnessed significantly more hepatotoxicity of grade ≥2 (40% vs. 2.7%; p<0.005) and nephrotoxicity of grade ≥2 (48% vs. 2.7%; p<0.005) among patients who received hICE. Significantly more patients (n=4; 25%) in the hICE group experienced veno-occlusive disease (VOD) compared to the BEAM arm, where no patients developed VOD (p=0.01). Conclusion: There was no difference in terms of overall survival between the BEAM and hICE groups. We observed significantly more adverse effects among patients treated with hICE. The BEAM regimen seems to be superior to hICE in terms of toxicity profile with comparable efficacy in patients with relapsed/refractory NHL and HL. Keywords: Relapsed/refractory lymphoma, Hematopoietic stem cell transplantation, Conditioning regimen

Amaç: Otolog kök hücre nakli (OKHN) destekli yüksek doz kemoterapi relaps/refrakter non-Hodgkin lenfoma (NHL) ve Hodgkin lenfoma (HL) tedavisinde uygulanan bir yöntemdir. Hazırlama rejimleri çok çeşitli olabilse de OKHN öncesinde henüz hiçbirisi standart olarak kabul edilmemiştir. Gereç ve Yöntemler: BEAM ve yüksek doz ICE (hICE) sonrasında OKHN olan 62 hastanın tıbbi kayıtları retrospektif olarak analiz edildi ve etkinlik ile yan etki profili açısından karşılaştırıldı. Bulgular: Çalışmaya toplamda 29 relaps/refrakter NHL ve 33 HL olgusu dahil edildi. Hazırlama rejimleri BEAM (n=37) ve hICE (n=25) idi. Bir yıllık genel sağkalım (GS) %73±%6 idi. BEAM ve hICE gruplarında ise 1 yıllık GS oranı sırasıyla %71±%8 ve %74±%9 olarak bulundu (p=0,86). Bulantı/kusma (derece ≥2) insidansı (%84 vs %44,7; p=0,04) ve mukozit (derece ≥2) insidansı (%13 vs %3; p=0,002) hICE grubunda daha yüksek oranda görüldü. İlaveten, hICE alan hastalarda istatistiksel olarak derece ≥2 hepatotoksisite (%40 vs %2,7; p<0,005) ve derece ≥2 nefrotoksisite (%48 vs %2,7; p<0,005) daha fazla oranda gözlendi. hICE grubunda veno-oklüzif hastalık (VOH) sıklığı (n=4; 25%) BEAM grubu ile karşılaştırıldığında istatistiksel olarak anlamlı düzeyde daha yüksekti (p=0,01). BEAM grubunda VOH görülmedi. Sonuç: GS oranları her iki grup arasında farklı bulunmadı ancak hICE grubunda anlamlı oranda yan etki sıklığı artmıştır. Relaps/refrakter NHL ve HL hastalarında benzer etkinlik ile BEAM rejimi toksisite profili açısından hICE rejiminden üstün olarak kabul edilebilir. Anahtar Sözcükler: Nüks/dirençli lenfoma, Hematopoetik kök hücre nakli, Hazırlama rejimi

Address for Correspondence/Yazışma Adresi: Itır ŞİRİNOĞLU DEMİRİZ, M.D., Ankara Oncology Hospital, Hematology and Stem Cell Transplantation Unit, Ankara, Turkey Phone : +90 0532 296 89 98 E-mail : dritir@hotmail.com

Received/Geliş tarihi: May 28, 2014 Accepted/Kabul tarihi: January 19, 2015

209

Esbah O, et al: Comparison of Conditioning Regimens: BEAM vs. High-Dose ICE

Introduction About 50% and 20% of patients presenting with non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma (HL) will not be cured after initial combination chemotherapy, respectively [1,2]. High-dose chemotherapy combined with autologous hematopoietic stem cell transplantation (AHCT) is an accepted treatment option for relapsed/refractory chemosensitive NHL/ HL patients [3,4]. Predictive markers for post-AHCT outcome are chemosensitivity, number of chemotherapy lines before AHCT, disease status at the time of AHCT, relevant prognostic scores for histological subtypes of lymphoma, and time of relapse following first-line therapy (<12 months vs. >12 months) [5,6,7,8]. The best conditioning regimen before AHCT in patients with relapsed/refractory lymphoma is an undefined issue. Commonly used regimens in this scenario are BEAM (BCNU, etoposide, cytarabine, and melphalan) [8,9], BEAC (BCNU, etoposide, cytarabine, cyclophosphamide) [9], highdose ICE (hICE; ifosfamide, carboplatin, and etoposide) [10], CMV (cyclophosphamide, melphalan, and etoposide) [11], CBV (cyclophosphamide, BCNU, and etoposide), combination regimens including total body irradiation (TBI) [12], and rituximab or I131-tositumomab combined with BEAM [13]. Few studies were reported comparing conditioning regimens in terms of toxicity and efficacy [9,13,14,15,16]. As we are unaware of any study comparing hICE and BEAM, we retrospectively analyzed our lymphoma patients who had undergone AHCT and received either hICE or BEAM regimens as conditioning.

Materials and Methods Patient Characteristics The clinical and laboratory records of all consecutive relapsed/ refractory HL/NHL patients who were treated with AHCT between 2010 and 2012 were retrospectively analyzed. We did not use any exclusion criteria. All patients gave informed consent for all aspects of AHCT and the institutional review board approved the study. Mobilization Strategy We used a step-by-step mobilization strategy. Granulocytecolony stimulating factor (G-CSF; filgrastim or lenograstim) at a dose of 10 µg/kg/day in two divided doses is our first-line mobilization protocol. A progenitor cell yield of <2×106/kg CD34+ cells was defined as mobilization failure. G-CSF alone was used in patients who received not more than two lines of chemotherapy and did not need chemotherapy for tumor control. Patients who failed mobilization with G-CSF alone, were heavily pretreated, or needed chemotherapy for debulking received G-CSF (10 µg/kg/day; filgrastim or lenograstim) plus chemotherapy for mobilization (second-line mobilization). Patients who failed two lines of mobilization received G-CSF 210

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combined with plerixafor as third-line mobilization. The details of mobilization with G-CSF plus plerixafor can be found elsewhere [17]. Patients who still failed mobilization with the aforementioned protocols received autologous bone marrow transplant. Patients who needed second-line mobilization protocols received various chemotherapy regimens like ASHAP (doxorubicin, methylprednisolone, high-dose cytarabine, and cisplatin), R-ASHAP (rituximab-ASHAP), R-ICE (rituximab, ifosfamide, carboplatin, and etoposide), VIGEPP (vinorelbine, gemcitabine, procarbazine, and prednisone), DHAP (dexamethasone, cytarabine, and cisplatin), and cyclophosphamide (4 g/m2) [18,19,20,21,22]. High-Dose Chemotherapy Regimens and Treatment Protocol BEAM was our preferred conditioning regimen before AHCT. Currently, BCNU and intravenous melphalan are unavailable on the Turkish market. Both drugs are exported from the European Union under the supervision of the Drug and Pharmacy Agency of Turkey. We used hICE as the second choice when one or both of these aforementioned drugs were temporarily unavailable for technical reasons. The BEAM regimen included BCNU at 300 mg/ m2 on day -7, etoposide at 200 mg/m2 and cytarabine at 200 mg/m2 on days -6 to -3, and melphalan at 140 mg/m2 on day -2. Patients in the hICE regimen group received ifosfamide at 2.5 g/ m2 (total dose: 15 g/m2; IV infusion over 2 h), etoposide at 250 mg/m2 (total dose: 1.5 g/m2; IV infusion over 2 h), carboplatin at 250 mg/m2 (total dose: 1.5 g/m2; IV infusion over 4 h), and mesna at 3.5 g/m2 (total dose: 21 g/m2) in evenly divided daily doses on days -8 to -3. Patients received subcutaneous G-CSF (5 µg/kg/day) from day +1 of AHCT until neutrophil engraftment (>500/mm3). Platelet transfusions were given if platelet counts were <10,000/mm3 without risk factors for bleeding. Erythrocyte suspensions were given to patients with anemia-related symptoms or hemoglobin values below 8 g/dL. All patients received levofloxacin at 400 mg/day, fluconazole at 200 mg/day, and valacyclovir at 1000 mg/day until neutrophil engraftment. Response and Toxicity Evaluation Responses before and after AHCT were evaluated according to revised international working group criteria [23]. Chemosensitive disease was defined as achievement of at least partial remission (PR) following salvage chemotherapy. Chemoresistant disease was defined as inability to achieve PR or observation of progressive disease. Positron emission tomography scanning was not used. Toxicities were evaluated according to Common Terminology Criteria for Adverse Events v3.0 [24]. Followup examinations were carried out at day +30 after AHCT. Thereafter, surveillance examinations were done every 3 months

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for the first 2 years, every 6 months for the next 3 years, and then annually. Definition of Engraftment, Febrile Neutropenia, and VenoOcclusive Disease Neutrophil engraftment was defined as the first of 3 consecutive days on which the absolute neutrophil count exceeded 500/ mm3 without G-CSF support. Platelet engraftment was defined as the first day of 7 consecutive days on which platelet count exceeded 20,000/mm3 without platelet transfusion [25]. We used Infectious Disease Society of America [26] and Seattle [27] criteria for defining febrile neutropenia and veno-occlusive disease, respectively. Management of Febrile Neutropenia The details of our protocol can be found elsewhere [28]. Briefly, patients with febrile neutropenia who were not responding to broad-spectrum antibiotics for 72 h were evaluated for opportunistic fungal infections. Patients who had hemodynamic instability and/or two consecutive positive serum galactomannan assays (ELISA: optical density of ≥0.5) and/or thorax computerized tomography findings suggesting invasive pulmonary aspergillosis (nodules with/without halo sign, air crescent sign, and cavitation) supported by mycological cultures received antifungal treatment. Patients with mycological evidence of Aspergillus spp. were treated with voriconazole. All others received caspofungin. Calculation of Direct Treatment Costs of Conditioning Regimens Direct drug costs of BEAM and hICE conditioning regimens were calculated based on an average patient with a body surface area of 1.7 m2 as of October 2013. Statistical Analysis Descriptive statistics are presented as median and minimummaximum. Comparisons of continuous variables between the two groups were performed using the nonparametric MannWhitney U test. Proportions were compared using the chisquare test. Survival analysis was calculated with Kaplan-Meier analysis. A p-value below 0.05 was considered to be statistically significant.

Results The demographic and clinical characteristics of the study cohort are summarized in Table 1. Fifteen (40%) and 5 (20%) patients had primary refractory disease following their firstline chemotherapy in the BEAM and hICE groups, respectively (p=0.09). While 6 patients in the BEAM group received standard-dose ICE (sICE) as rescue before AHCT, no patient in the hICE arm was treated with sICE as salvage chemotherapy.

Esbah O, et al: Comparison of Conditioning Regimens: BEAM vs. High-Dose ICE

Fourteen (38%) and 6 (25%) patients of the BEAM and hICE arms had refractory disease at AHCT, respectively. Twenty-six, 28, 8 patients were mobilized with G-CSF alone, G-CSF plus chemotherapy, G-CSF plus plerixafor, respectively. There were no significant differences in terms of conditioning regimens among patient groups (p=0.5 both for HL and NHL patients). The treatment arms were also similar according to age, sex, stage, previous radiotherapy, chemotherapy history, and disease status at AHCT and mobilization protocol. On the other hand, the BEAM group had significantly worse performance status compared to the hICE arm (p=0.011) (Table 1). Mobilization success, engraftment kinetics, and side effect profiles of the conditioning regimens are given in Table 2. The BEAM and hICE treatment arms were similar in terms of infused stem cells, median days with febrile neutropenia, engraftment kinetics, and duration of hospitalization. We observed significantly more adverse effects (grade ≥2) in terms of nausea/ vomiting, mucositis, hepatotoxicity, and nephrotoxicity among patients treated with hICE conditioning compared to patients who received BEAM. Significantly more patients (n=4; 25%) in the hICE group experienced veno-occlusive disease compared to the BEAM arm, where no patients developed veno-occlusive disease (p=0.01). The treatment arms were comparable according to diarrhea rate (p=0.09). Relapse rates following BEAM and hICE conditioning regimens were 13.5% (5/37) and 32% (8/25) (p=0.07). One patient of the BEAM arm died before day 30 following AHCT as a result of sepsis. Additionally, two patients (one patient in each arm) died before day 100. The reasons for mortality were sepsis/ engraftment failure and Cytomegalovirus pneumonia in the patients of the BEAM and hICE groups, respectively. Transplantrelated mortality for the entire cohort on day 100 was 4.8% (BEAM: 5.4%; hICE: 4%; p=0.8). Following AHCT, 5 (13.5%) and 8 (32%) patients of the BEAM and hICE arms relapsed (p=0.07). Three-year disease-free survival (DFS) and overall survival (OS) rates were 52±10% and 57±6% in the whole study cohort, respectively. There was no difference in terms of 3-year DFS rates according to conditioning regimens (BEAM: 63±13%; hICE: 42±15%; p=0.187) (Figure 1). Three-year OS was 56.8±8% and 58±10% in the BEAM and hICE groups, respectively (p=0.781) (Table 2, Figure 2). Direct treatment costs of hICE and BEAM regimens were found to be 1721 and 582 euro, respectively.

Discussion Although many different conditioning regimens for relapsed/ refractory HL and NHL have been proposed, none of them can be considered as a standard of care [8,9,10,11,12]. Different types of hICE conditioning regimens were described according 211

Esbah O, et al: Comparison of Conditioning Regimens: BEAM vs. High-Dose ICE

to large ranges of cumulative dosages and administrations of drugs [10,29]. To our knowledge, there is no direct comparison in the literature of BEAM and hICE chemotherapy regimens in patients who have undergone AHCT for relapsed/refractory lymphoma. In the current study, we observed statistically significant differences in terms of toxicity favoring the BEAM regimen compared to hICE. It was not surprising that nausea and vomiting were more frequent in the hICE arm, as ifosfamide and carboplatin have high emetogenic potential. Mucositis is an important toxicity of BCNU and etoposide [30,31]. According to several studies, BCNU-related mucositis rates were higher when the BCNU dose was increased from 450 to 600 mg/m2 [30]. The BCNU dose in the BEAM conditioning arm was 300 mg/m2 in our study. The total doses of etoposide were 1500 mg/m2 and 800 mg/m2 in the hICE and BEAM arms, respectively. The higher etoposide dose may be responsible for the higher mucositis rate observed in the hICE arm. Nephrotoxicity was significantly higher in the hICE group (p<0.005). Nephrotoxicity was seen

Turk J Hematol 2016;33:209-215

only in one patient in the BEAM group. This is not an unexpected finding because carboplatin and ifosfamide are well-known nephrotoxic agents [32]. Hepatotoxicity was also more frequent in the hICE group than the BEAM group (p<0.005). Patients on hICE experienced significantly more veno-occlusive disease compared to the BEAM arm. This may have occurred as a result of the higher total dose of etoposide in hICE compared to BEAM [31]. In addition, ifosfamide may create an extra burden on the liver, resulting in high rates of hepatotoxicity [33]. As no patient in the hICE arm received sICE as a salvage before AHCT, we think that the observed toxicity cannot be attributed to previous exposure to the same drugs at standard doses. In Turkey, direct treatment costs of ICE and BEAM regimens in a patient with an average body surface area of 1.7 m2 are 1721 and 582 euro, respectively. Indirect costs of chemotherapy like hospitalization, treatment of infections, or adverse effects are not included here. The BEAM regimen was more advantageous in terms of cost.

Table 1. Demographic and clinical characteristics of patient cohorts.

BEAM (n=37)

hICE (n=25)

0.5

NHL DLBCL/FL/MCL PTCL/ALCL

19 7/6/1 4/1

10 5/3/1 1/-

0.5

Male/female

22/15

17/8

0.49

Median age, years (min-max)

38 (17-70)

40 (20-64)

0.76

Stage (I/II/III/IV)

4/11/17/5

3/4/11/7

0.35

ECOG (I/II/III/IV)

4/23/10/-

11/9/5/-

0.011

Previous radiotherapy

9 (24%)

4 (16%)

0.33

Previous lines of chemotherapy (1st/2nd/3rd/4th)

2/19/14/2

2/17/6/-

0.37

Response to first-line chemotherapy Primary refractory disease Chemosensitive disease (at least PR)

15 (40%) 22 (60%)

5 (20%) 20 (80%)

0.09

First-line salvage chemotherapy

DHAP (n=15) ASHAP (n=1) sICE (n=6) ABVD (n=4) BEACOPP (n=1) Hyper-CVAD (n=1)

DHAP (n=9) ASHAP (n=10) CVP (n=1) CHOP (n=1) R-FC (n=1) R-MINE (n=1) Hyper-CVAD (n=1) VIGEPP (n=1)

Disease status at AHCT (CR/PR/refractory)

17/6/14

16/3/6

0.490

Mobilization (G-CSF/G-CSF+chemotherapy/G-CSF+plerixafor

15/19/3

11/9/5

0.37

HL: Hodgkin’s lymphoma, NHL: non-Hodgkin’s lymphoma, DLBCL: diffuse large B-cell lymphoma, FL: follicular lymphoma, MCL: mantle cell lymphoma, ECOG: The Eastern Cooperative Oncology Group, PTCL: peripheral T cell lymphoma ALCL: anaplastic large cell lymphoma, G-CSF: granulocyte-colony stimulating factor, AHCT: autologous hematopoietic stem cell transplantation, PR: partial remission, CR: complete remission, ASHAP: doxorubicin, methylprednisolone, high-dose cytarabine, and cisplatin, R-ASHAP: rituximab-ASHAP, R-ICE: rituximab, ifosfamide, carboplatin, and etoposide, VIGEPP: vinorelbine, gemcitabine, procarbazine, and prednisone, DHAP: dexamethasone, cytarabine, and cisplatin, sICE: standarddose ICE.

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Esbah O, et al: Comparison of Conditioning Regimens: BEAM vs. High-Dose ICE

Head-to-head comparisons of different conditioning regimens in relapsed/refractory lymphoma patients before AHCT are scarce. Jo et al. observed superior OS and event-free survival at 2 years in patients on BEAM compared to BEAC regimens (62.4% vs. 32.1% and 62.4% vs. 28.6%, respectively). However, diarrhea and mucositis were more frequent in patients of the BEAM arm [9]. In their single-center analysis, Jantunen et al. reported similar efficacy of BEAM and BEAC conditioning regimens in terms of

OS and progression-free survival in patients undergoing AHCT for NHL, but BEAM was found more toxic to the gastrointestinal system [16]. In recent years the BEAM regimen was also compared with the CEB (carboplatin, etoposide, and bleomycin) regimen with better OS in favor of BEAM [14], but other studies reported conflicting results [15]. Salar et al. reported Spanish GEL/TAMO registry data including 395 consecutively autografted diffuse large B-cell lymphoma (DLBCL) patients.

Figure 1. Kaplan-Meier plots of disease-free survival following autologous hematopoietic stem cell transplantation according to conditioning regimens. Three-year disease-free survival rates were 63±13% (BEAM) vs. 42±15% (hICE) (p=0.187).

Figure 2. Kaplan-Meier plots of overall survival following autologous hematopoietic stem cell transplantation according to conditioning regimens. Three-year overall survival rates were 56±8% (BEAM) vs. 58±10% (hICE) (p=0.781).

Table 2. Mobilization yield, engraftment kinetics, efficacy, and toxicity profiles of conditioning regimens.

BEAM (n=37)

hICE (n=25)

CD34+ cell counts, median (min-max)

5.9 (2.5-16.7)

5.7 (3.5-11.29)

0.36

Febrile neutropenic days, median (min-max)

4 (1-10)

3 (1-12)

0.79

Neutrophil engraftment, days, median (min-max)

11 (8-19)

12 (9-34)

0.24

Platelet engraftment, days, median (min-max)

12 (8-16)

12 (10-25)

0.24

Hospitalization days, median (min-max)

24 (12-67)

26 (20-50)

0.53

Nausea/vomiting, n (grade ≥2)

0.04

Diarrhea, n (grade ≥2)

0.09

Mucositis, n (grade ≥2)

0.002

Nephrotoxicity, n, (grade ≥2)

<0.05

Hepatotoxicity, n, (grade ≥2)

<0.05

Veno-occlusive disease, n (any grade)

0.01

Transplant-related mortality Day 30 Day 100

1 (2.7%) 2 (5.4%)

1 (4%)

0.8

Posttransplantation relapse

5 (13.5%)

8 (32%)

0.07

Disease-free survival (3 years)

63±13%

42±15%

0.187

Overall survival (3 years)

56±8%

58±10%

0.781

213

Esbah O, et al: Comparison of Conditioning Regimens: BEAM vs. High-Dose ICE

The main message of that study was that chemotherapy-only conditioning regimens (BEAM, BEAC, or CBV) significantly improved 8-year OS compared to TBI + cyclophosphamide [15]. Recently, the rituximab-BEAM (R-BEAM) conditioning regimen was compared to the I131-tositumomab-BEAM (B-BEAM) in a phase III randomized study in relapsed, chemosensitive DLBCL patients. Two-year progression-free survival and OS rates were comparable, but B-BEAM was found to be more toxic in terms of mucositis [13]. Although the observation period of our study cohort is limited, 3-year OS rates were similar in the BEAM and hICE arms (56±8% vs. 58±10%; p=0.781). There was a trend for lower relapse rates following BEAM compared to hICE (13.5% vs. 32%; p=0.07). There were more patients with primary refractory disease (40% vs. 20%) and refractory disease at AHCT (38% vs. 24%) in the BEAM arm compared to patients receiving hICE conditioning. The aforementioned points underline the strong antitumor effect of the BEAM regimen compared to hICE. Taking the cost and safety advantages of BEAM over hICE in addition to similar short-term DFS and OS into account, it seems reasonable to suggest that BEAM seems to be a better option than hICE for conditioning in relapsed/refractory lymphoma patients undergoing AHCT. Our study has several limitations that make it difficult to draw firm conclusions, such as the limited number of patients, retrospective design, and heterogeneous lymphoma subtypes of the cohort. As we included patients with HL and various pathologic subgroups of NHL (DLBCL, follicular lymphoma, mantle cell lymphoma, peripheral T cell lymphoma, and anaplastic large cell lymphoma), generalization of our findings may not be appropriate for specific patient populations with lymphoma. We also had a very limited number of patients with each subtype of lymphoma, making disease-specific statistical evaluation of hICE and BEAM conditioning regimens impossible. Although the BEAM treatment arm included more patients with poor performance status, the toxicity profile of BEAM was lower compared to ICE. This point again emphasizes that BEAM is a safe and effective conditioning regimen even for patients with poor performance. In conclusion, the current retrospective study showed that BEAM seems to be a better option compared to hICE as a conditioning regimen in relapsed/refractory lymphoma patients before AHCT with similar efficacy but low toxicity. Although there was no difference in 3-year DFS and OS, the nausea/ vomiting, mucositis, nephrotoxicity, and hepatotoxicity rates were significantly higher in the hICE group compared to the BEAM group. Prospective studies with homogeneous patient populations and incorporating novel agents in the therapeutic armamentarium will be very informative in the search for the optimal conditioning regimen in specific lymphoma subtypes in the future. 214

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Ethics Ethics Committee Approval: Ethical Committee approval has been not taken because it is retrospective research; Informed Consent: It was taken. Authorship Contributions Medical Practices: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür Kayıkçı, Gamze Durgun, Şerife Kocubaba, Fevzi Altuntaş; Concept: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz; Design: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz; Data Collection or Processing: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür Kayıkçı, Gamze Durgun, Şerife Kocubaba, Fevzi Altuntaş; Analysis or Interpretation: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, Sinem Civriz Bozdağ; Literature Search: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür Kayıkçı, Gamze Durgun, Şerife Kocubaba, Fevzi Altuntaş; Writing: Onur Esbah, Emre Tekgündüz, Itır Şirinoğlu Demiriz, Sinem Civriz Bozdağ, Ali Kaya, Ayşegül Tetik, Ömür Kayıkçı, Gamze Durgun, Şerife Kocubaba, Fevzi Altuntaş. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0378 Turk J Hematol 2016;33:216-222

The Changing Epidemiology of Bloodstream Infections and Resistance in Hematopoietic Stem Cell Transplantation Recipients Hematopoetik Kök Hücre Nakli Alıcılarında Kan Akım Enfeksiyonu ve Direnç Epidemiyolojisindeki Değişim Mücahit Yemişen1, İlker İnanç Balkan1, Ayşe Salihoğlu2, Ahmet Emre Eşkazan2, Bilgül Mete1, M. Cem Ar2, Şeniz Öngören2, Zafer Başlar2, Reşat Özaras1, Neşe Saltoğlu1, Ali Mert1, Burhan Ferhanoğlu3, Recep Öztürk1, Fehmi Tabak1, Teoman Soysal2 1İstanbul University Cerrahpaşa Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey 2İstanbul University Cerrahpaşa Faculty of Medicine, Department of Internal Medicine, Division of Heamatology, İstanbul, Turkey 3Koç University Faculty of Medicine, American Hospital, Clinic of Internal Medicine, Division of Heamatology, İstanbul, Turkey

Abstract

Öz

Objective: Patients receiving hematopoietic stem cell transplantation (HSCT) are exposed to highly immunosuppressive conditions and bloodstream infections (BSIs) are one of the most common major complications within this period. Our aim, in this study, was to evaluate the epidemiology of BSIs in these patients retrospectively.

Amaç: Hematopoetik kök hücre transplantasyonu (HKHT) yapılan hastaların bağışıklık sistemi ciddi şekilde baskılanmıştır ve kan akımı enfeksiyonları (KAE) bu süre içinde karşılaşılan majör komplikasyonlardan biridir. Bu çalışmada amacımız, geriye dönük olarak bu hastalarda KAE’lerinin epidemiyolojisini değerlendirmektir.

Materials and Methods: The epidemiological properties of 312 patients with HSCT were retrospectively evaluated.

Gereç ve Yöntemler: HKHT yapılan 312 hastanın epidemiyolojik özellikleri retrospektif olarak değerlendirildi.

Results: A total of 312 patients, followed between 2000 and 2011, who underwent autologous (62%) and allogeneic (38%) HSCT were included in the study. The most common underlying malignancies were multiple myeloma (28%) and Hodgkin lymphoma (21.5%). A total of 142 (45%) patients developed at least 1 episode of BSI and 193 separate pathogens were isolated from the blood cultures. There was a trend of increase in the numbers of BSIs in 2005-2008 and a relative increase in the proportion of gram-positive infections in recent years (2009-2011), and central venous catheter-related BSI was found to be most common source. Coagulase-negative staphylococci (49.2%) and Acinetobacter baumannii (8.8%) were the most common pathogens. Extended-spectrum beta-lactamase-producing strains were 23% and 22% among Escherichia coli and Klebsiella spp. isolates, respectively. Quinolone resistance was detected in 10% of Enterobacteriaceae. Resistance to carbapenems was not detected in Enterobacteriaceae, while it was seen at 11.1% and 23.5% in Pseudomonas and Acinetobacter strains, respectively.

Bulgular: 2000 ve 2011 yılları arasında otolog (%62) ve allojeneik (%38) HKHT yapılan 312 hasta, çalışmaya dahil edildi. Çalışmaya dahil edilen hasta grupları en sık multipl miyelom (%28) ve Hodgkin lenfoma (%21,5) tanılı hastalar idi. Yüz kırk iki hastada (%45) en az bir kez KAE gelişmiş ve kan kültürlerinden 193 ayrı patojen elde edilmiştir. KAE’lerde 2005-2008 yılları arası bir artışın yanında, 2009-20011 yılları arasında da gram pozitiflerde göreceli bir artış da saptanmış ve en sık KAE kaynağı santral venöz kataterler olarak tespit edilmiştir. Koagülaz negatif staphylococi (%49,2) ve Acinetobacter baumannii (%8,8), kan kültürlerinden en sık elde edilen patojenlerdir. Genişlemiş spektrumlu beta laktamaz üretimi Escherichia coli ve Klebsiella spp. suşları arasında sırası ile %23 ve %22 idi. Kinolon dirençli Enterobacteriaceae oranı %10 olarak tespit edilmiştir. Pseudomonas ve Acinetobacter suşlarında karbapenem direni sırasıyla, %11,1 ve %23,5 iken, Enterobacteriaceae grubunda karbapenemlere hiç direnç saptanmamıştır.

Conclusion: A shift was detected from gram-negative bacteria to gram-positive in the etiology over the years and central lines were the most common sources of BSIs.

Sonuç: Yıllar içinde, gram negatif bakterilerden gram pozitiflere doğru bir kayma gözlenirken, en sık KAE kaynağı santral kataterler olarak saptanmıştır.

Keywords: Hematopoietic stem cell transplantation, Bloodstream infection, Epidemiology, Resistance, Central venous catheter

Anahtar Sözcükler: Hematopoetik kök hücre nakli, Kan akımı enfeksiyonu, Epidemiyoloji, Direnç, Santral venöz kateter

Address for Correspondence/Yazışma Adresi: Mücahit YEMİŞEN, M.D., İstanbul University Cerrahpaşa Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey Phone : +90 212 414 30 95 E-mail: yemisenmucahit@hotmail.com

216

Received/Geliş tarihi: September 24, 2014 Accepted/Kabul tarihi: December 15, 2014

Turk J Hematol 2016;33:216-222

Introduction Bloodstream infection (BSI) is the most common infectious problem in patients undergoing hematopoietic stem cell transplantations (HSCTs). Depending on the protocol used for transplantation and the duration of neutropenia, approximately 13%-60% of patients develop BSIs, which can result in delays in chemotherapies, extension of admission period, and increased costs of antimicrobial therapy against target organisms [1,2]. The differences in results of these studies are probably due to different study designs, study populations, conditioning regimens, and prophylactic antibiotic protocols [1]. Beside neutropenia, the other risk factors for BSI include age, underlying disease, presence of a central catheter, severe graftversus-host disease (GVHD), mucositis, and steroid use [1,3,4]. The etiology of BSIs has changed and showed different patterns in the past years. While gram-negative BSIs among neutropenic cancer patients were formerly the leading cause of bacteremia, the etiology of BSIs in this patient population has become predominantly gram-positive, and especially viridans group streptococci and coagulase-negative staphylococci, over the last 2 decades [5,6]. Besides this shift, resistance rates and patterns also started to change and more resistant microorganisms are now found as the causes of BSIs. For example, the emergence of fluoroquinolone-resistant bacteria, increase in multidrugresistant gram-negative bacteria, increase in nosocomial methicillin-resistant Staphylococcus aureus infections, and emergence of extended-spectrum beta-lactamase (ESBL) producers have all been reported in the literature in neutropenic patients [3]. Due to the diversity of the causative microorganisms of BSIs in patients with HSCT, information about etiology and antibiotic susceptibility of BSIs is important to initiate effective antibiotic treatment, a parameter that has been shown to be closely associated with survival in bacteremic patients [7]. In this study, we aimed to assess the etiology and clinical characteristics of BSIs in patients with hematological malignancies undergoing HSCT over a 12-year period. We also evaluate the risk factors, resistance patterns, and sources of BSIs in this group of patients as a secondary objective.

Materials and Methods Patients A total of 312 patients who underwent autologous and allogeneic bone marrow transplantation in the Stem Cell Transplantation Unit of the Ä°stanbul University CerrahpaĹ&#x;a Medical School from 1 January 2000 to 31 December 2011 were included in the study. Data on demographic features of the patients, underlying disease, disease status prior to HSCT, HSCT protocols, prophylaxis regimens, and emerging resistance profiles of bacteremia were

YemiĹ&#x;en M, et al: Epidemiology of Bloodstream Infections

retrospectively analyzed. The data of the patients were recorded from the initiating day of conditioning until the 100th day after transplantation. Hematologic Definitions All patients were followed in isolated single rooms equipped with high-efficiency particulate air filters and underwent central venous catheter (CVC) insertion. Conditioning was done using standard protocols such as cyclophosphamide alone or in combination with total body irradiation for allogeneic transplantation and CBV (cyclophosphamide, VP-16, BCNU) or BEAM (BCNU, VP-16, cytarabine, melphalan) for autologous stem cell transplantation. Almost all allogeneic transplantations were done from HLA-identical sibling or matched unrelated donors. Neutrophil engraftment was defined as the first of 3 consecutive days on which the absolute neutrophil count remained at or above 500/mm3 after stem cell infusion. GVHD diagnosis and staging were performed according to previously established criteria [8,9]. Microbiological Definitions We obtained at least 2 blood cultures from all febrile neutropenic patients and initiated an antipseudomonal antibiotic. Febrile neutropenia was investigated and managed according to the Infectious Disease Society of America guidelines [10,11]. BSI (mono or poly) and catheter-associated BSI were accepted according to the established criteria [12,13,14]. Antimicrobial susceptibility tests of bacteria obtained from blood cultures were evaluated by the disk diffusion method according to the current Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) criteria [15]. Intermediate sensitivity or resistance results were accepted as resistant. The screening of multidrug-resistant phenotypes including methicillin-resistant Staphylococcus aureus, ampicillin- and vancomycin-resistant enterococci, ESBL production, and carbapenemase production was conducted according to CLSI recommendations [16,17]. Multidrug resistance was defined as acquired nonsusceptibility to at least 1 agent in 3 or more antimicrobial categories; extensive drug resistance was defined as nonsusceptibility to at least 1 agent in all but 2 or fewer antimicrobial categories, and pandrug resistance was defined as nonsusceptibility to all agents in all antimicrobial categories [18]. Statistical Analysis The categorical data were compared by chi-square tests, and p<0.05 was accepted as significant. Factors predicting bacteremia and mortality were analyzed by logistic regression analyses. 217

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Turk J Hematol 2016;33:216-222

Results A total of 312 patients were included in the study. The number of female patients was 137 (44%) and the mean age was 39 years (minimum-maximum: 12-73 years). The most common underlying conditions of the patients were multiple myeloma in 87 (28%) and Hodgkin lymphoma in 67 (21.5%). The number of patients who underwent autologous and allogeneic HSCT was 194 (62%) and 118 (38%), respectively. The stem cell source was peripheral blood in 295 (94.5%) patients and bone marrow in 17 (5.5%) patients. The mean time to neutrophil engraftment was 14 days and the number of patients having detectable cytomegalovirus-DNA was 38 (12.2%). The number of patients having acute GVHD equal to or above stage 2 was 36 (11.5%). Table 1 shows the characteristics of the patients. We obtained a total of 193 microbial isolates from patients’ blood cultures; of these 193 isolates, 12 were obtained after the conditioning regimen (before infusion of cells), 140 were obtained after infusion of cells (before neutrophil engraftment), and 41 were obtained after engraftment. Table 2 shows the properties of the isolates obtained from blood cultures. Gram-positive, gram-negative, and fungal isolates obtained from the blood cultures were 112 (58%), 74 (38.3%), and 7 (3.7%), respectively. A total of 142 (45.5%) of 312 patients developed at least 1 episode of BSI. Of these 142 patients, 68 had autologous and 74 had allogeneic HSCT. In our study, 106 patients developed 1 episode of BSI, 32 patients had 2 episodes, 3 patients had 3 episodes, and 1 patient had 4 episodes. The numbers of monomicrobial and polymicrobial episodes were 168 and 14, respectively. The source of BSI was determined as CVC-associated for 151 (78.2%) isolates while no source could be determined for the remaining isolates. Of those 151 CVCassociated isolates, 69.5% were gram-positive bacteria. The most frequently isolated gram-positive bacteria were coagulase-negative staphylococci with 95 isolates, and then Streptococcus spp. with 8, S. aureus with 5, Enterococcus spp. with 2, and gram-positive rods with 2 isolates. The numbers of gram-negative isolates obtained from blood cultures were as follows: Acinetobacter baumannii, 17; Stenotrophomonas maltophilia, 14; Escherichia coli, 13; Klebsiella spp., 9; Pseudomonas aeruginosa, 9; and other gram-negative bacteria, 12 isolates. A total of 7 fungal isolates comprised 3 Candida parapsilosis, 1 Candida tropicalis, 1 Fusarium spp., and 2 Candida spp. isolates. Between 2000 and 2005, the number of gram-negative isolates was greater than the number of gram-positive isolates; after 2005, gram-positive isolates increased in frequency and became the major causative group for BSIs. Figure 1 shows the etiology of BSIs (gram-positive, gram-negative, and fungal) according to year; there was a trend of increase in the numbers of BSIs 218

Figure 1. Evolution of bloodstream infection etiology. Table 1. Characteristics of the patients. Characteristics

n (%)

Total number of patients

312

Patients with BSI

142 (45.5%)

Patients without BSI

170 (54.5%)

Median age

39 (12-73)

Sex

Male

175 (56%)

Female

137 (44%)

Underlying disease

87 (28%)

67 (21.5%)

NHL

44 (14%)

AML

42 (13.5%)

ALL

39 (12.5%)

CML

19 (6%)

Others

14 (4.5%)

Type of transplantation

Autologous

194 (62%)

Allogeneic

118 (38%)

Graft source

Peripheral blood

295 (94.5%)

Bone marrow

17 (5.5%)

Mean duration of neutrophil engraftment (days)

14 (7-36)

Comorbid conditions

13 (4.1%)

Hepatic

11 (3.5%)

Cardiac

11 (3.5%)

Pulmonary

6 (1.9%)

Solid tumor

4 (1.2%)

Rheumatic disease

3 (0.9%)

Crude mortality (in 100 days)

40 (12.8%)

BSI: Bloodstream infection, MM: multiple myeloma, HL: Hodgkin lymphoma, NHL: non-Hodgkin lymphoma, AML: acute myeloid leukemia, ALL: acute lymphoblastic leukemia, CML: chronic myeloid leukemia, DM: diabetes mellitus.

Yemişen M, et al: Epidemiology of Bloodstream Infections

Turk J Hematol 2016;33:216-222

in 2005-2008 and also a relative increase in the proportion of gram-positive BSIs in more recent years (2009-2011).

carbapenems; and no strains were resistant to aminoglycosides or ciprofloxacin. Of the 17 A. baumannii strains, 4 were resistant to carbapenems; 3 to aminoglycosides, ceftazidime, and cefepime; 2 to piperacillin/tazobactam; and 1 to ciprofloxacin. All S. maltophilia strains were susceptible to trimethoprim/sulfamethoxazole. Among all gram-negative strains, the rate of multidrug-resistant bacteria was 12.1%, and the rate of extensively drug resistant bacteria was 8.1%. We did not identify any pandrug resistance in our study. Table 3 shows the resistance patterns of the gram-negative bacteria obtained from blood cultures.

The number of ESBL-producing Enterobacteriaceae isolates was 6 (20.6%). Among the 13 E. coli isolates, 3 were ESBL-producing and 4 were resistant to ciprofloxacin, 5 to aminoglycosides, 2 to cefepime, and 3 to third-generation cephalosporin and piperacillin/tazobactam. For Klebsiella spp., 2 isolates were resistant to third-generation cephalosporins, 3 to piperacillin/ tazobactam, and 2 to cefepime, and 2 were ESBL-producing. We found no resistant isolates for aminoglycosides or ciprofloxacin. The proportion of isolates that were ESBL-producing among E. coli and Klebsiella spp. was 23% and 22%, respectively. No resistance to carbapenems was observed.

Among all gram-positive bacteria, 95 (84.8%) were coagulasenegative staphylococci, and only 3 (3.1%) strains were susceptible to methicillin. In 5 S. aureus strains, only 1 was resistant to methicillin, and the remaining were susceptible. Among the 8 Streptococcus strains isolated, 7 were viridans group streptococci and 1 was group A beta-hemolytic streptococcus.

In the P. aeruginosa group, 2 strains were resistant to ceftazidime; 1 was resistant to piperacillin/tazobactam, cefepime, and Table 2. Etiology and source of bloodstream infections. Bacteria

<Day 0

Day 0 to Eng

>Eng

Total CVC

Source Unknown

MRCNS

MSCNS

Enterococcus sp.

Streptococcus sp.

MRSA

MSSA

Other gram-positives

Escherichia coli

Klebsiella sp.

Pseudomonas aeruginosa

Acinetobacter baumannii

Stenotrophomonas maltophilia

Other gram-negatives

Candida parapsilosis

Other fungi

Total

12 (6.2%)

140 (72.5%)

41 (21.3%)

193

151 (78.2%)

42 (21.8%)

BSI: Bloodstream infection, Eng: engraftment, CVC: central venous catheter, MRCNS: methicillin-resistant coagulase-negative Staphylococcus, MSCNS: methicillin-susceptible coagulase-negative Staphylococcus, MRSA: methicillin-resistant Staphylococcus aureus, MSSA: methicillin-sensitive Staphylococcus aureus.

Table 3. Resistance pattern of gram-negative isolates. AK

CIP

CAZ

CTX/ CRO

TZP

FEP

ESBL

IPM/MEM

MDR

XDR

PDR

Total

Escherichia coli

Klebsiella sp.

Pseudomonas aeruginosa

Acinetobacter baumannii

Others

AK: Amikacin, CIP: ciprofloxacin, CAZ: ceftazidime, CTX/CRO: cefotaxime/ceftriaxone, TZP: piperacillin/tazobactam, FEP: cefepime, ESBL: extended-spectrum beta-lactamase, IPM/ MEM: imipenem/meropenem, MDR: multidrug-resistant, XDR: extensively drug resistant, PDR: pandrug resistant.

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Univariate analysis to determine risk factors for bacteremia identified HSCT type, any comorbidity, duration of engraftment longer than 10 days, and GVHD grade of 2-4 (p<0.05). In multivariate analyses, only the type of HSCT (allogeneic) was associated with bacteremia (p<0.05). The crude death rate in the 100 days after transplantation was 12.8%. Univariate analysis of risk factors for mortality revealed association with type of HSCT, presence of bacteremia, degree of GVHD, and engraftment period longer than 10 days (p<0.05). Only allogeneic HSCT was associated with mortality in multivariate analysis (p<0.05). The highest mortality rate was observed in the patients who had bacteremia due to S. maltophilia.

Discussion Bloodsteam infections remains the main challenge for patients undergoing HSCT. Cappellano et al. reported the rate of bacteremia as 27% in 315 allogeneic HSCT patients [19]. In the studies of Poutsiaka et al. and Mikulska et al., the rates of bacteremia were found to be 43.6% and 38.4%, respectively [1,20]. In our study the rate of bacteremia was 45.5% among 312 HSCT patients. Several features of the microorganisms obtained from the blood cultures of our patients changed over the study period. Between 2000 and 2005, the isolates of BSIs were predominantly gramnegative. After 2005, parallel to similar reports, this pattern switched to a gram-positive predominance, the majority of isolates being coagulase-negative staphylococci. As indicated in previous reports, this can be explained by the different conditioning regimens used for transplantation, antibiotic prophylaxis, or changing of global bacterial resistance [3]. Coagulase-negative staphylococci are usually the most frequently isolated gram-positive bacteria, while other grampositive bacteria such as Enterococcus spp. have been reported to be more frequent in other studies [19,21,22]. In our study, the number of gram-positive bacteria other than coagulasenegative staphylococci was limited, so those resistance rates were not taken into account. In the present study, A. baumannii and S. maltophilia isolates were predominant among gram-negative bacteria. Most bacteremia cases in our unit due to A. baumannii and S. maltophilia were found to be associated with insertion of central catheters. However, after 2007, only one case of bacteremia associated with these pathogens was reported. This change may be explained by the establishment of a team at our center for placement of central catheters in 2007. After the implementation of that team, the rates of bacteremia due to these two isolates sharply decreased. However, the rate of BSIs due to coagulase-negative staphylococcus is still high. The association between S. maltophilia and central catheters 220

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in HSCT patients was reported in some studies: in the study by Chaplow et al., an outbreak of S. maltophilia was found to be associated with CVCs [23]. In another study, Williamson et al. reported the source of bacteremia due to nonfermentative gram-negative bacteria to be a consequence of central catheters [24]. However, Labarca et al. attributed mucositis rather than central catheterization to be the source of S. maltophilia [25]. ESBL production among Enterobacteriaceae was 20.6% in the study group, while it was around 30% in other patients at our hospital; Mikulska et al. found it to be over 40% among HSCT patients [20]. Carbapenemase-producing Enterobacteriaceae have not been observed in any of our patients. However, recently we had a carbapenemase-producing Klebsiella (CPK) outbreak in neutropenic patients in our hematology unit, and Zuckerman et al. also reported an outbreak of CPK in HSCT patients [26]. Only 10% of the gram-negative bacteria were resistant to ciprofloxacin in the study. This rate is lower than that noted in other units of our hospital and those in other studies [3,20,27,28]. Busca et al. reported that quinolone use may induce the resistance rate against quinolone itself and even against some other antibiotics [28]. However, despite routine use of quinolone prophylaxis, increased rates of resistance have not been observed in patients undergoing prophylaxis. In previous reports, BSI in patients who had undergone HSCT was generally found to be associated with age, late stage of underlying disease, GVHD, steroid use, mucositis, and central catheters [1,3,29]. In our study, we only identified central catheters as a source of BSI. In 93.75% of gram-positive bacteremia and 54.7% of gram-negative cases, the source was found to be associated with the presence of a CVC. In a few studies, the main source of BSI in HSCT patients was CVCassociated; Yuen et al. reported the rate of CVC-associated BSI to be 38% in the postengraftment period [30]. The rate of CVCassociated BSI was found to be 23% in the study of Liu et al [3]. In our series, the leading etiology was coagulase-negative staphylococci (95/193; 49%). In all of these patients the suggested source was a CVC. In gram-negative cases, however, CVCs were considered as the source in nearly half of the cases. In multivariate analysis, bacteremia and death were found to be associated with the type of transplantation (allogeneic). We were not surprised by this result, because allogeneic transplantation is a highly immunosuppressive condition and other factors such as GVHD, immunosuppressive treatment for GVHD, long duration of neutropenia, and long time for engraftment are also associated with allogeneic transplantation. In our study, the mortality rate of patients having bacteremia due to S. maltophilia was found to be higher than with other bacteria. This may be due to the lack of activity of initial antibiotic treatment against S. maltophilia.

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Our study had some limitations. It was performed retrospectively and, due to missing data, some patients had to be excluded. The initial empiric antibiotic treatment might have influenced the resistance of the bacteria, but we could not account for the effect of empiric antibiotic treatment. The impact of the stem cell source on bacteremia in allogeneic HSCT recipients could not be analyzed since almost all patients received peripheral stem cells from fully matched donors. In conclusion, BSI in HSCT recipients is still a great problem. The global switch from a gram-negative etiology to a grampositive one was also observed in our study. In addition to other gram-negative bacteria, A. baumannii and S. maltophilia were frequent causes of bacteremia but were generally not covered by initial empirical therapy. Accordingly, we observed a higher rate of mortality due to S. maltophilia bacteremia. It is generally difficult to identify the source of bacteremia in HSCT patients. In our study, CVCs were the only source suggested and they were usually associated with unusual pathogens. However, with a dedicated CVC team and the use of a catheter-care bundle, we could reduce the rate of catheter-related BSIs. HSCT recipients are especially at risk of CVC-related BSIs, which may include difficult-to-treat pathogens. Ethics Ethics Committee Approval: Retrospective study; Informed Consent: It was taken. Authorship Contributions Concept: Mücahit Yemişen, İlker İnanç Balkan; Design: Mücahit Yemişen, Ahmet Emre Eşkazan; Data Collection or Processing: Mücahit Yemişen, İlker İnanç Balkan, Ayşe Salihoğlu, Ahmet Emre Eşkazan, Bilgül Mete, M. Cem Ar, Şeniz Öngören, Zafer Başlar, Reşat Özaras, Neşe Saltoğlu, Ali Mert, Burhan Ferhanoğlu, Recep Öztürk, Fehmi Tabak, Teoman Soysal; Analysis or Interpretation: Mücahit Yemişen, İlker İnanç Balkan, Ayşe Salihoğlu, Ahmet Emre Eşkazan, Bilgül Mete, M. Cem Ar, Şeniz Öngören, Zafer Başlar, Reşat Özaras, Neşe Saltoğlu, Ali Mert, Burhan Ferhanoğlu, Recep Öztürk, Fehmi Tabak, Teoman Soysal; Literature Search: Bilgül Mete, Ayşe Salihoğlu; Writing: Mücahit Yemişen. 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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25. Labarca JA, Leber AL, Kern VL, Territo MC, Brankovic LE, Bruckner DA, Pegues DA. Outbreak of Stenotrophomonas maltophilia bacteremia in allogeneic bone marrow transplant patients: role of severe neutropenia and mucositis. Clin Infect Dis 2000;30:195-197. 26. Zuckerman T, Benyamini N, Sprecher H, Fineman R, Finkelstein R, Rowe JM, Oren I. SCT in patients with carbapenem resistant Klebsiella pneumoniae: a single center experience with oral gentamicin for the eradication of carrier state. Bone Marrow Transplant 2011;46:1226-1230. 27. Mitchell AE, Derrington P, Turner P, Hunt LP, Oakhill A, Marks DI. Gramnegative bacteremia (GNB) after 428 unrelated donor bone marrow transplants (UD-BMT): risk factors, prophylaxis, therapy and outcome. Bone Marrow Transplant 2004;33:303-310. 28. Busca A, Cavecchia I, Locatelli F, Dâ&#x20AC;&#x2122;Ardia S, De Rosa FG, Marmont F, Ciccone G, Baldi I, Serra R, Gaido E, Falda M. Blood stream infections after allogeneic stem cell transplantation: a single-center experience with the use of levofloxacin prophylaxis. Transpl Infect Dis 2012;14:40-48. 29. Marena C, Zecca M, Carenini ML, Bruschi A, Bassi ML, Olivieri P, Azzaretti S, Locatelli F. Incidence of, and risk factors for, nosocomial infections among hematopoietic stem cell transplantation recipients, with impact on procedure-related mortality. Infect Control Hosp Epidemiol 2001;22:510-517. 30. Yuen KY, Woo PC, Hui CH, Luk WK, Chen FE, Lie AK, Liang R. Unique risk factors for bacteremia in allogeneic bone marrow transplant recipients before and after engraftment. Bone Marrow Transplant 1998;21:1137-1143.

RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0131 Turk J Hematol 2016;33:223-230

BK Virus-Hemorrhagic Cystitis Following Allogeneic Stem Cell Transplantation: Clinical Characteristics and Utility of Leflunomide Treatment Allojenik Kök Hücre Transplantasyonu Sonrası BK Virüs Hemorajik Sistiti: Klinik Özellikleri ve Leflunomid Tedavisinin Etkisi Young Hoon Park, Joo Han Lim, Hyeon Gyu Yi, Moon Hee Lee, Chul Soo Kim Inha University Faculty of Medicine and Hospital, Department of Hematology-Oncology, Incheon, Republic of Korea

Abstract

Öz

Objective: BK virus-hemorrhagic cystitis (BKV-HC) is a potential cause of morbidity and mortality in patients having undergone allogeneic stem cell transplantation (Allo-SCT). We analyzed the clinical features of BKV-HC following Allo-SCT and reported the utility of leflunomide therapy for BKV-HC. Materials and Methods: From January 2005 to June 2014, among the 69 patients that underwent Allo-SCT in our institution, the patients who experienced BKV-HC were investigated retrospectively. Results: HC was observed in 30 patients (43.5%), and among them, 18 of the cases (26.1%) were identified as BKV-HC. The median age of the patients (12 males and 6 females) was 45 years (minimummaximum: 13-63). Patients received Allo-SCT for acute myeloid leukemia (n=11), aplastic anemia (n=4), myelodysplastic syndrome (n=2), and non-Hodgkin lymphoma (n=1). The donor types were human leukocyte antigen (HLA)-matched sibling donor for six patients, HLA-matched unrelated donor for nine, and haploidentical familial donor for two. The median onset and duration of BKV-HC was on day 21 after transplantation (minimum-maximum: 7-97) and 22 days (minimum-maximum: 6-107). Eleven patients (62.1%) had grade I-II HC and seven patients (38.9%) had grade III-IV (highgrade) HC. Among the seven patients who had high-grade HC, one had complete response, one had partial response, and five had no response. Among the five nonresponders, one died of BKV-HC associated complications. The remaining four patients were treated with leflunomide, achieving complete response (n=2) and partial response (n=2). The median duration from the start of leflunomide therapy to response was 13 days (minimum-maximum: 8-17 days). All patients tolerated the leflunomide treatment well, with three patients having mild gastrointestinal symptoms, including anorexia and abdominal bloating. Conclusion: BKV-HC was commonly observed in patients with HC following Allo-SCT. In high-grade BKV-HC patients who do not respond to supportive care, leflunomide may be a feasible option without significant toxicity. Keywords: BK virus, Hemorrhagic cystitis, Allogeneic stem cell transplantation, Leflunomide

Amaç: BK-virüs hemorajik sistiti (BKV-HS) allojenik kök hücre nakli (Allo-KHN) uygulanan hastalarda morbidite ve mortalitenin önemli bir nedenidir. Bu çalışmada Allo-KHN sonrası BKV-HS olan olguların klinik özellikleri ve leflunomid tedavisinin BKV-HS’deki etkinliği araştırılmıştır.

Öz

Gereç ve Yöntemler: Kliniğimizde Ocak 2005-Haziran 2014 arası AlloKHN uygulanmış 69 hastada, BKV-HS geçirmiş olanlar retrospektif olarak değerlendirildi. Bulgular: Otuz hastada (%43,5) HS gözlendi. Bu olguların 18’inde (%26,1) BKV-HS’si saptandı. Hastaların (12’si erkek, altısı kadın) medyan yaşı 45 (13-63) idi. Hastalara akut miyeloid lösemi (n=11), aplastik anemi (n=4), miyelodisplastik sendrom (n=2) ve non-Hodgkin lenfoma (n=1) nedeni ile Allo-KHN uygulanmıştı. Altısında insan lökosit antijeni (İLA)-uygun kardeş, dokuzunda İLA-uygun akraba dışı donör ve ikisinde haplo-identik donör kullanılmıştı. Transplant sonrası BKV-HS medyan başlangıç zamanı 21 gün (7-97 gün), medyan süresi 22 gün (6-107 gün) idi. On bir olguda (%62,1) derece I-II, yedi olguda (%38,9) derece III-IV (yüksek derecede) HS saptandı. Yüksek derece HS’li yedi hastanın, birinde tam yanıt, birinde kısmi yanıt elde edilirken, beş hastada yanıt alınamadı. Yanıt alınmayan beş hastanın birisi BKV-HS ilişkili komplikasyonlardan kaybedildi. Geri kalan dört hasta leflunomid ile tedavi edildi. Bu hastaların ikisinde tam yanıt, ikisinde kısmi yanıt elde edildi. Leflunomidin başlangıcından itibaren medyan yanıt süresi 13 gündü (8-17 gün). Tüm hastalar leflunomidi iyi tolere ederken, üç hastada anoreksi ve abdominal gaz şikayetleri dahil hafif şiddetli gastrointestinal yan etkiler gözlendi. Sonuç: Allo-KHN sonrası izlemde BKV-HS yaygın olarak gözlenmiştir. Destek tedavisine yanıt vermeyen yüksek derece BKV-HS’li olgularda leflunomid, anlamlı toksisitesi olmaksızın bir seçenek olabilir. Anahtar Kelimeler: BK virüs, Hemorajik sistit, Allojenik kök hücre transplantasyonu, Leflunomid

Address for Correspondence/Yazışma Adresi: Chul Soo KIM, M.D., Received/Geliş tarihi: March 23, 2015 Inha University Faculty of Medicine and Hospital, Department of Hematology-Oncology, Incheon, Republic of Korea Accepted/Kabul tarihi: December 21, 2015 Phone : +82-32-890-2581 E-mail : cskimmd@inha.ac.kr

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Park YH, et al: BK Virus-Hemorrhagic Cystitis

Introduction Hemorrhagic cystitis (HC) is a potential cause of morbidity and mortality in patients that have undergone allogeneic stem cell transplantation (Allo-SCT) [1,2,3]. Its incidence ranges from 5% to 68% of Allo-SCT recipients, with severe-grade hematuria in 29%-44% of cases [3,4,5,6,7]. Variable etiologies for the development of HC in Allo-SCT recipients include noninfectious and infectious causes. As an infectious cause of HC, BK virusHC (BKV-HC) occurs later after transplantation, usually in the post-engraftment period [3]. The BKV, a member of the family Polyomaviridae, is typically acquired in childhood and embedded in urothelial cells of the urinary tract in the latent dormant stage [8]. BKV reactivation is commonly associated with HC in Allo-SCT settings, occurring in 10% to 25% of patients [8]. The clinical symptoms of BKV-HC vary to a great extent in Allo-SCT recipients from asymptomatic hematuria to massive hemorrhage leading to urinary obstruction and renal failure [2,9,10]. Previous studies demonstrated that BKV-HC is associated with not only increased morbidity and but also increased mortality in Allo-SCT patients [6,7,11,12], and studies have also defined potential risk factors for the development of BKV-HC [4,5,13,14,15], most of which have not been observed consistently in several reports. Leflunomide, an immunomodulatory agent with antiviral activity, has been found effective against cytomegalovirus (CMV), herpes simplex, and BKV based on in vitro data [6,16,17]. In renal allografts, leflunomide has been widely used to treat biopsy-proven BKV nephropathy [18,19], but it has not been well studied in Allo-SCT settings. Only two reports showed satisfactory results of leflunomide therapy in the treatment of BKV-HC after Allo-SCT [20,21]. In this retrospective study, we report the incidence, severity, and outcome of clinical BKV-HC in patients who underwent Allo-SCT to treat variable hematologic diseases. Furthermore, we report high-grade BKV-HC patients who achieved favorable response to leflunomide therapy.

Materials and Methods Patients A total of 69 patients underwent Allo-SCT in our institution from January 2005, when BKV polymerase chain reaction (PCR) testing became clinically available, to June 2014. Baseline demographic and transplantation data were collected, including age, sex, underlying disease, conditioning regimen, stem cell source, donor type, prophylaxis to infection and graft-versus-host disease (GVHD), time to engraftment, presence and grade of GVHD, and survival at last follow-up. Patients received either myeloablative or reducedintensity conditioning regimen according to disease status, age, or comorbidities. As prophylaxis against HC, hyperhydration 224

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(intravenous isotonic saline over 3 L/m2 per day) with forced diuresis was used to prevent toxicity of conditioning regimen for Allo-SCT. In addition, for patients receiving a preparative regimen containing cyclophosphamide (Cy), 2-mercaptoethane sulfonate (MESNA) was given prior to the Cy administration and thereafter as a continuous infusion until the last dose of Cy. Acute GVHD and chronic GVHD were diagnosed and graded according to previously published criteria [22,23]. Diagnosis of BK Virus-Hemorrhagic Cystitis HC was defined as the presence of sustained hematuria and urinary symptoms after the beginning of conditioning therapy in the absence of gynecological-related bleeding, generalized bleeding diathesis, and urinary tract infection. Severity of HC was graded according to the following criteria [7]: grade 0 (no hematuria), grade I (microscopic hematuria), grade II (macroscopic hematuria), grade III (macroscopic hematuria with presence of blood clots), and grade IV (macroscopic hematuria with clots and renal impairment due to urinary obstruction). Grades III and IV were defined as high-grade HC. The date of onset of HC was defined as the first day of symptoms or laboratory evidence appearing after transplant. Routine urinalysis was performed at least twice a week during hospitalization and thereafter at outpatient visits. For patients who had urinary symptoms or gross hematuria, BKV testing by a qualitative PCR-based method was performed with the urine specimen to identify the presence of virus. BK Virus Polymerase Chain Reaction Test Each urine sample was submitted to DNA extraction using a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturerâ&#x20AC;&#x2122;s instructions. A primer was designed for a highly conserved region of gene large T antigen to obtain a 160bp amplicon from the BKV genome using the GeneAmp 2720 Thermal Cycler (ABI, Foster City, CA, USA). PCR of urine for BKV viruria had a sensitivity of 90% and a specificity of 96.5% (cutoff value: 160 copies/mL). Urine Cytology Each urine specimen was processed for cytological evaluation by centrifugation. After removal of the supernatant, the sediment was examined for clarity. Cytospin slides were fixed in 95% alcohol, stained by the Papanicolaou method, and observed for the presence of urine decoy cells (characterized by a groundglass appearance with an enlarged nucleus, which is occupied by a homogeneous basophilic inclusion surrounded by chromatin) by a well-trained pathologist. Treatment and Criteria of Response All patients diagnosed with BKV-HC received supportive treatment, including hyperhydration with normal saline, forced

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diuresis, urine alkalinization, analgesics, and blood transfusion to maintain platelet counts at â&#x2030;Ľ50x109/L and a hemoglobin level of â&#x2030;Ľ8 g/dL. Foley catheterization and bladder irrigation were considered for patients with high-grade HC who had no response (NR) to supportive care. Leflunomide therapy was indicated as follows: 1) grade III-IV HC; 2) no change or worsening in urinary symptoms or grade of hematuria during supportive care within 2 weeks; 3) no abnormality in liver function tests; 4) absolute neutrophil count of >1.0x109/L; and 5) no abnormality on chest radiology. For the patients receiving leflunomide therapy, leflunomide at 100 mg/day orally was used as a loading dose for 5 days, followed by maintenance doses of 20 mg/day until resolution of hematuria and urinary symptoms. Clinical response was defined as follows: complete response (CR), completely improved in symptoms with absence of hematuria; partial response (PR), downgrading of severity with persistent hematuria; NR, unchanged or worsening urinary symptoms or grade of hematuria; refractoriness, NR even after about 2 weeks of supportive care. The response was evaluated after 20 days of leflunomide treatment.

extensive GVHD, two patients showed corticosteroid-refractory chronic GVHD and were treated with mycophenolate mofetil, but failed to respond.

Statistical Analysis

Treatment

The cumulative incidence of BKV-HC was estimated with the interval starting at Allo-SCT until the day of the first PCRpositive urine sample. The Mann-Whitney test and chi-square test were used for comparisons for continuous and categorical data, respectively, between the patients with low-grade BKV-HC and those with high-grade BKV-HC. Univariate and multivariate analyses of risk factors for BKV-HC occurrence were performed using the Cox proportional hazard model. SPSS 14.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses, and all were two-sided. Statistical significance was defined as p<0.05. This study was approved by the Institutional Review Board of the Inha University Hospital.

The majority of the patients received supportive treatment, including intravenous hydration and/or blood transfusion (Table 2). A urinary catheter was inserted for three patients with HC of grade III and two patients with HC of grade IV. We slightly tapered the immunosuppressant by monitoring the serum trough level in low-grade BKV-HC patients with no evidence of GVHD or grade I-II GVHD. In contrast, in the patients with high-grade BKV-HC, reduction or withdrawal of the immunosuppressant was not done as a treatment option for BKV-HC due to the risk of GVHD. After supportive care, nine patients (50.0%) achieved CR, four patients (22.2%) PR, and five patients (27.8%) NR. All patients with low-grade HC showed better responses than PR to supportive treatment. Out of the seven patients who had highgrade HC, one had CR, one PR, and five NR. Response (CR+PR) rate in patients with low-grade HC was significantly higher than in those with high-grade HC (100% vs. 28.6%, p=0.004). Among the five nonresponders, one died of BKV-HC-associated renal failure with no evidence of progression of underlying disease. The remaining four patients were treated with oral leflunomide as salvage therapy.

Results Patient Characteristics and Transplant Outcome The demographic characteristics of the HC patients are presented in Table 1. In our study, a total of 30 of 69 (43.5%) patients who underwent Allo-SCT developed HC. Of these, 18 (26.1%) were diagnosed with BKV-HC and were included in this study. Seven (38.9%) patients received antithymoglobulin for in vivo T-cell depletion. Nine patients (50%) had acute GVHD, and two patients (11.1%) had grade III-IV GVHD. These two patients received systemic glucocorticoid therapy (1-2 mg/kg prednisone) without additional immunosuppressant agents and showed a good response to steroid therapy. Twelve patients (66.7%) had chronic GVHD, and seven patients (38.9%) showed extensive chronic GVHD. Among the seven patients with

Clinical Features of BK Virus-Hemorrhagic Cystitis The clinical characteristics of BKV-HC are detailed in Table 2. Eleven patients (62.1%) had low-grade HC and seven patients (38.9%) had high-grade HC. The median onset and duration of BKV-HC was on day 21 (minimum-maximum: 7-97) after transplantation and 22 days (minimum-maximum: 6-107), respectively. There was no significant difference between the onset in patients with low-grade HC or high-grade HC (21 vs. 21 days, p=0.633). In two patients (11.1%), BKV-HC developed before neutrophil engraftment. Nine (50.0%) patients had concomitant cytomegalovirus viremia, and among them, four patients received ganciclovir treatment. Of patients with acute GVHD (n=9), seven (77.7%) developed acute GVHD after onset of BKV-HC and, conversely, two (22.3%) developed BKV-HC after the diagnosis of acute GVHD. Four (22%) patients had urinary cytologic changes compatible with BKV.

Risk Factors for BK Virus-Hemorrhagic Cystitis Occurrence In univariate analysis, HLA mismatching and acute GVHD grades II-IV were associated with a higher risk of developing BKV-HC (p=0.034 and p<0.001, respectively). In multivariate analysis, only acute GVHD grades II-IV were independent risk factors for BKV-HC (hazard ratio 3.26, 95% CI 1.42-13.5, p=0.002) in this study (Table 3). 225

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Table 1. Demographic characteristics of the hemorrhagic cystitis patients and posttransplant outcomes. Variable

BKV-HC (n=18)

Non-BKV-HC (n=12)

Age, years

45 (13-63)

42 (21-66)

Sex Male Female

12 (66.7) 6 (33.3)

7 (58.3) 5 (41.7)

Diagnosis Acute myeloid leukemia Aplastic anemia Myelodysplastic syndrome Non-Hodgkin lymphoma

11 (61.1) 4 (22.2) 2 (11.1) 1 (5.6)

6 (50) 2 (16.7) 3 (25) 1(8.3)

Type of donor Matched sibling Matched unrelated Haploidentical familial

7 (38.9) 9 (50.0) 2 (11.1)

8 (66.7) 3 (25) 1 (8.3)

Donor source Peripheral blood stem cell Bone marrow stem cell

18 (100) 0 (0)

11 (91.7) 1 (8.3)

Conditioning regimen Busulfan/fludarabine Busulfan/cyclophosphamide Fludarabine/melphalan Cyclophosphamide/fludarabine Cyclophosphamide/total body irradiation

8 (44.4) 5 (27.8) 3 (16.7) 1 (5.6) 1 (5.6)

6 (50) 5 (41.7) 0 (0) 1 (8.3) 0 (0)

T-cell depletion None In vivo

11 (61.1) 7 (38.9)

6 (50) 6 (50)

Engraftment Neutrophil engraftment (ANC >0.5x109/L) Platelet engraftment (>20x109/L)

18 (100) 14 (77.7)

11 (91.7) 9 (75)

Acute GVHD Grade II-IV

9 (50.0) 6 (33.3)

7 (58.3) 3 (25)

Chronic GVHD None Limited Extensive

6 (33.3) 5 (27.8) 7 (38.9)

4 (33.3) 5 (41.7) 3 (25)

Values are presented as median (minimum-maximum) or number (%). HC: Hemorrhagic cystitis, ANC: absolute neutrophil count, GVHD: graft-versus-host disease.

Leflunomide Therapy Detailed information about the four patients receiving leflunomide therapy is summarized in Table 4. Of the four patients, three had acute myeloid leukemia and one had highrisk myelodysplastic syndrome. All patients had acute GVHD before development of BKV-HC. After leflunomide treatment, two (50%) patients achieved CR and two (50%) achieved PR. One CR patient and one PR patient had a negative PCR for BKV, but BKV in urine still remained detectable in one patient achieving CR. The median duration from the start of leflunomide therapy to response was 13 days (minimum-maximum: 8-17). 226

The dose of leflunomide was not reduced for any patients. All patients tolerated the leflunomide treatment well, with three patients having mild gastrointestinal symptoms, including anorexia and abdominal bloating. No significant adverse effects, such as hepatotoxicity, skin reactions, diarrhea, bone marrow suppression, or pneumonia, were observed during leflunomide treatment. There was no recurrence of hematuria in the two patients achieving CR after discontinuation of leflunomide therapy. Of the patients achieving PR, one died of leukemia relapse 16.3 months after Allo-SCT. The remaining three patients are still alive without hematuria.

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Table 2. Characteristics of BK virus-hemorrhagic cystitis and treatment outcomes. Characteristics Onset, days after Allo-SCT, median (minimum-maximum)

21 (7-97)

Duration, days, median (minimum-maximum)

22 (6-107)

Platelet count at the onset of BKV-HC (x109/L), median (minimum-maximum)

64 (17-260)

Platelet unit transfusions, median (minimum-maximum)

10 (0-48)

RBC unit transfusions, median (minimum-maximum)

0 (0-10)

Serum creatinine at the onset of BKV-HC (mg/dL), median (minimum-maximum)

1.15 (0.52-2.57)

Severity, n (%) I/II III/IV

5 (27.8)/6 (33.3) 5 (27.8)/2 (11.1)

Concomitant CMV antigenemia, n (%)

9 (50)

Treatment, n (%) Intravenous hyperhydration Quinolone antibiotics (ciprofloxacin or levofloxacin) Blood transfusion (RBC or platelet) Insertion of urinary catheter Cystoscopy Continuous bladder irrigation with water Leflunomide

18 (100) 9 (50.0) 12 (66.7) 5 (27.8) 1 (5.6) 2 (11.1) 4 (22.2)

Outcome after initial therapy, n (%) Complete response Partial response No response

9 (50.0) 4 (22.2) 5 (27.8)

Survival, n (%) Alive Dead Relapse/refractory disease GVHD BKV-HC related renal failure

11 (61.1) 7 (38.9) 4 (22.2) 2 (11.1) 1 (5.6)

BKV-HC: BK virus hemorrhagic cystitis, Allo-SCT: allogeneic stem cell transplantation, RBC: red blood cell, CMV: cytomegalovirus, GVHD: graft-versus-host disease.

Table 3. Univariate and multivariate analysis for development of BK virus hemorrhagic cystitis. Characteristics

Univariate

Multivariate

HR (95% CI)

p-value

HR (95% CI)

p-value

Sex Male

1.46 (0.98-6.33)

0.097

Age at transplant

2.11 (1.22-5.38)

0.241

HLA match Mismatched donor

2.23 (1.41-3.56)

0.034

Conditioning regimen intensity Myeloablative conditioning

2.01 (1.10-3.88)

0.092

Conditioning agents Cyclophosphamide-containing regimen

1.98 (0.89-5.31)

0.366

GVHD prophylaxis T-cell depletion

0.75 (0.43-1.24)

0.082

Acute GVHD Grades II-IV

3.52 (1.68-7.83)

<0.001

3.26 (1.42-7.52)

0.002

HR: Hazard ratio, CI: confidence interval, HLA: human leukocyte antigen, GVHD, graft-versus-host disease.

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Table 4. Clinical characteristics of the patients receiving leflunomide therapy. Â

Patient 1

Patient 2

Patient 3

Patient 4

Age (years)/Sex

40/M

49/M

50/F

52/M

Diagnosis

AML

MDS

AML

Donor type

MSD

MUD

MSD

Conditioning

Bu/Flu

Bu/Cy

GVHD prophylaxis

CsA/MTX

T-cell depletion

Yes

Onset of BKV-HC (days)

Severity of HC

Grade III

Acute GVHD

Grade II (skin)

Grade II (skin, liver)

Grade I (skin)

Grade II (skin)

Chronic GVHD

Extensive

Limited

CMV antigenemia

Yes

Response after therapy

PCR positivity for BKV after therapy

Positive

Negative

Positive

Negative

Adverse effects

Nausea (mild)

Abdominal bloating

None

Duration to response (days)

Outcome

Alive

Dead (due to leukemia relapse)

Alive

M: Male, F: female, AML: acute myeloid leukemia, MDS: myelodysplastic syndrome, MSD: matched sibling donor, MUD: matched unrelated donor, Bu: busulfan, Flu: fludarabine, Cy: cyclophosphamide, CsA: cyclosporine, MTX: methotrexate, Allo-SCT: allogeneic stem cell transplantation, BKV-HC: BK virus hemorrhagic cystitis, GVHD: graft-versus-host disease, CMV: cytomegalovirus, CR: complete response, PR: partial response, PCR: polymerase chain reaction.

Discussion Our results, with an overall incidence of HC following Allo-SCT of 43.5%, are consistent with other studiesâ&#x20AC;&#x2122; findings, which reported frequencies of HC following SCT ranging from 5% to 68% [3,4,5,6,7]. BKV was identified in 60.0% of cases (18 of 30 patients) by a qualitative PCR-based assay. A study of 22 Allo-SCT patients who experienced HC showed that the most frequent virus detected was BKV, with an incidence of 54.5% of patients, followed by JC virus and CMV [24]. In a study of 102 children who underwent Allo-SCT for malignancies and nonmalignant diseases, HC occurred in 26 patients (25.5%), and among them, BKV was identified in 21 (80.8%) patients [25]. These findings demonstrated that HC is a frequent complication after Allo-SCT and BKV is mainly responsible for HC in Allo-SCT recipients. Hemorrhagic cystitis after SCT frequently caused prolongation of hospitalization and occasionally death [10,24,26]. Gilis et al. demonstrated that the median duration of hospitalization for Allo-SCT was significantly longer for patients developing BKV-HC compared with those without BKV-HC (50 vs. 40 days, p<0.001) [26]. In addition, in a series of 12 patients with BKVHC, HC-associated renal failure was the main cause of death in two (16.7%) patients, who failed to respond to any treatment including administration of intravenous cidofovir [24]. In our study, among the seven high-grade BKV-HC patients, one died of BKV-HC-related renal failure. These observations suggest 228

that prompt alternative treatment should be considered in patients with BKV-HC, and especially high-grade HC, who had refractoriness to initial supportive treatment. Currently, there is no established antiviral drug for the treatment of BKV-HC, although reduction in immunosuppression is necessary for clearance of the BKV. Salvage treatment with antiviral agents, including cidofovir, leflunomide, and fluoroquinolones, has been considered in renal transplant recipients when reduction of immunosuppression alone could not improve renal function, with no antiviral agents shown to be effective in randomized controlled studies [18,27,28,29]. In Allo-SCT settings, prior reports showed that treatment with varying doses of cidofovir, having activity against CMV, adenovirus, and polyomaviruses, was feasible [30,31,32]. However, the potential nephrotoxicity of cidofovir, resulting in direct tubular toxicity, still remains a major obstacle to its use. Leflunomide, an immunomodulatory agent, has modest antipolyomavirus activity in vitro, although the precise mechanism of action remains to be fully understood [6,17]. In renal transplant patients, leflunomide has been used to treat BKV nephropathy, resulting in a significant decline in the BK viral load with relatively stable renal function without the toxicity of leflunomide [10,18]. Thus far, there are limited clinical data on the utility of leflunomide therapy in the treatment of BKV-HC patients in Allo-SCT settings. In a series of 14 adult patients with BKV-HC undergoing Allo-SCT, leflunomide therapy showed favorable results for BKV-HC treatment with no serious adverse effects, with CR and PR being seen in 50% and 37.5%

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of patients, respectively [21]. In a pilot study with five pediatric patients with severe BKV-HC after Allo-SCT, significantly shorter duration of BKV-HC (p<0.01) and decreased BK viral loads in blood (p<0.01) and urine (p=0.03) were observed after leflunomide therapy compared with historical controls [20]. In this study, CR was achieved in 50% of the patients receiving leflunomide therapy. In addition, potential adverse effects associated with leflunomide treatment, including hepatotoxicity and bone marrow suppression, were not observed in any treated patients. Our results were similar to previous reports regarding leflunomide therapy in adult patients. These data suggest that leflunomide is effective in the treatment of posttransplant highgrade BKV-HC, especially in cases refractory to supportive care. This study has potential limitations, mostly stemming from its small sample size and retrospective design. Several studies have indicated that BK viruria was quantitatively related to the occurrence of HC after Allo-SCT and clinical response to antiviral therapy may be linked with reduction of viral loads in blood and/or urine [20,21,26,33]. However, we performed only a qualitative test in urine samples, but BKV was still detectable in one patient achieving CR after leflunomide therapy, suggesting that complete clearance of the virus in urine may not be necessary in BKV-HC patients treated with leflunomide. In summary, BKV-HC is a common complication of Allo-SCT and is associated with significant morbidity, especially in high-grade BKV-HC. In high-grade BKV-HC patients who fail to respond to supportive care, leflunomide therapy may be a feasible treatment option without significant toxicity. Prospective randomized controlled trials are warranted to evaluate the efficacy and safety of leflunomide in the treatment of BKV-HC after Allo-SCT. Ethics Ethics Committee Approval: This study was approved by the Institutional Review Board of the Inha University Hospital; Informed Consent: It was taken. Authorship Contributions Surgical and Medical Practices: Young Hoon Park, Hyeon Gyu Yi, Chul Soo Kim; Concept: Young Hoon Park, Chul Soo Kim; Design: Young Hoon Park, Chul Soo Kim; Data Collection or Processing: Young Hoon Park, Joo Han Lim, Hyeon Gyu Yi, Moon Hee Lee, Chul Soo Kim; Analysis or Interpretation: Young Hoon Park, Joo Han Lim, Hyeon Gyu Yi, Moon Hee Lee; Literature Search: Young Hoon Park, Joo Han Lim, Hyeon Gyu Yi, Moon Hee Lee, Chul Soo Kim; Writing: Young Hoon Park, Joo Han Lim, Hyeon Gyu Yi, Moon Hee Lee, Chul Soo Kim. 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. Gargiulo G, Orlando L, Alberani F, Crabu G, Di Maio A, Duranti L, Errico A, Liptrott S, Pitrone R, Santarone S, Soliman C, Trunfio A, Selleri C, Bruno B, Mammoliti S, Pane F. Haemorrhagic cystitis in haematopoietic stem cell transplantation (HSCT): a prospective observational study of incidence and management in HSCT centres within the GITMO network (Gruppo Italiano Trapianto Midollo Osseo). Ecancermedicalscience 2014;8:420. 2. Siguier M, Sellier P, Bergmann JF. BK-virus infections: a literature review. Med Mal Infect 2012;42:181-187. 3. Yang CC, Hurd DD, Case LD, Assimos DG. Hemorrhagic cystitis in bone marrow transplantation. Urology 1994;44:322-328. 4. Yaghobi R, Ramzi M, Dehghani S. The role of different risk factors in clinical presentation of hemorrhagic cystitis in hematopoietic stem cell transplant recipients. Transplant Proc 2009;41:2900-2902. 5. Sencer SF, Haake RJ, Weisdorf DJ. Hemorrhagic cystitis after bone marrow transplantation. Risk factors and complications. Transplantation 1993;56:875-879. 6. Dropulic LK, Jones RJ. Polyomavirus BK infection in blood and marrow transplant recipients. Bone Marrow Transplant 2008;41:11-18. 7. Bedi A, Miller CB, Hanson JL, Goodman S, Ambinder RF, Charache P, Arthur RR, Jones RJ. Association of BK virus with failure of prophylaxis against hemorrhagic cystitis following bone marrow transplantation. J Clin Oncol 1995;13:1103-1109. 8. dela Cruz J, Pursell K. BK virus and its role in hematopoietic stem cell transplantation: evolution of a pathogen. Curr Infect Dis Rep 2014;16:417. 9. Raval M, Gulbis A, Bollard C, Leen A, Chemaly R, Shpall E, Lahoti A, Kebriaei P. Evaluation and management of BK virus-associated nephropathy following allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2011;17:1589-1593. 10. Pahari A, Rees L. BK virus-associated renal problems--clinical implications. Pediatr Nephrol 2003;18:743-748. 11. Arthur RR, Shah KV, Baust SJ, Santos GW, Saral R. Association of BK viruria with hemorrhagic cystitis in recipients of bone marrow transplants. N Engl J Med 1986;315:230-234. 12. Razonable RR, Eid AJ. Viral infections in transplant recipients. Minerva Med 2009;100:479-501. 13. Leung AY, Mak R, Lie AK, Yuen KY, Cheng VC, Liang R, Kwong YL. Clinicopathological features and risk factors of clinically overt haemorrhagic cystitis complicating bone marrow transplantation. Bone Marrow Transplant 2002;29:509-513. 14. Mori Y, Miyamoto T, Kato K, Kamezaki K, Kuriyama T, Oku S, Takenaka K, Iwasaki H, Harada N, Shiratsuchi M, Abe Y, Nagafuji K, Teshima T, Akashi K. Different risk factors related to adenovirus- or BK virus-associated hemorrhagic cystitis following allogeneic stem cell transplantation. Biol Blood Marrow Transplant 2012;18:458-465. 15. Rorije NM, Shea MM, Satyanarayana G, Hammond SP, Ho VT, Baden LR, Antin JH, Soiffer RJ, Marty FM. BK virus disease after allogeneic stem cell transplantation: a cohort analysis. Biol Blood Marrow Transplant 2014;20:564-570. 16. Zaman RA, Ettenger RB, Cheam H, Malekzadeh MH, Tsai EW. A novel treatment regimen for BK viremia. Transplantation 2014;97:1166-1171. 17. Farasati NA, Shapiro R, Vats A, Randhawa P. Effect of leflunomide and cidofovir on replication of BK virus in an in vitro culture system. Transplantation 2005;79:116-118. 18. Leca N. Leflunomide use in renal transplantation. Curr Opin Organ Transplant 2009;14:370-374. 19. Josephson MA, Gillen D, Javaid B, Kadambi P, Meehan S, Foster P, Harland R, Thistlethwaite RJ, Garfinkel M, Atwood W, Jordan J, Sadhu M, Millis MJ, Williams J. Treatment of renal allograft polyoma BK virus infection with leflunomide. Transplantation 2006;81:704-710.

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20. Wu KH, Weng T, Wu HP, Peng CT, Sheu JN, Chao YH. Effective treatment of severe BK virus-associated hemorrhagic cystitis with leflunomide in children after hematopoietic stem cell transplantation: a pilot study. Pediatr Infect Dis J 2014;33:1193-1195. 21. Chen XC, Liu T, Li JJ, He C, Meng WT, Huang R. Efficacy and safety of leflunomide for the treatment of BK virus-associated hemorrhagic cystitis in allogeneic hematopoietic stem cell transplantation recipients. Acta Haematol 2013;130:52-56. 22. Przepiorka D, Anderlini P, Saliba R, Cleary K, Mehra R, Khouri I, Huh YO, Giralt S, Braunschweig I, van Besien K, Champlin R. Chronic graft-versushost disease after allogeneic blood stem cell transplantation. Blood 2001;98:1695-1700. 23. Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, Hows J, Thomas ED. 1994 Consensus conference on acute GVHD grading. Bone Marrow Transplant 1995;15:825-828. 24. Kim SY, Lee JW, Lee KM, Cho BS, Eom KS, Kim YJ, Lee S, Min CK, Kim HJ, Cho SG, Kim DW, Min WS, Kim CC. Viruria in adult hemorrhagic cystitis patients following allogeneic hematopoietic stem cell transplantation and implication of antiviral treatment. Korean J Hematol 2007;42:114-121. 25. Gorczynska E, Turkiewicz D, Rybka K, Toporski J, Kalwak K, Dyla A, Szczyra Z, Chybicka A. Incidence, clinical outcome, and management of virusinduced hemorrhagic cystitis in children and adolescents after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2005;11:797-804. 26. Gilis L, Morisset S, Billaud G, Ducastelle-Lepretre S, Labussiere-Wallet H, Nicolini FE, Barraco F, Detrait M, Thomas X, Tedone N, Sobh M, Chidiac C, Ferry T, Salles G, Michallet M, Ader F; Lyon BK Virus Study Group. High burden of BK virus-associated hemorrhagic cystitis in patients undergoing

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allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2014;49:664-670. 27. Vats A, Shapiro R, Singh Randhawa P, Scantlebury V, Tuzuner A, Saxena M, Moritz ML, Beattie TJ, Gonwa T, Green MD, Ellis D. Quantitative viral load monitoring and cidofovir therapy for the management of BK virus-associated nephropathy in children and adults. Transplantation 2003;75:105-112. 28. Kadambi PV, Josephson MA, Williams J, Corey L, Jerome KR, Meehan SM, Limaye AP. Treatment of refractory BK virus-associated nephropathy with cidofovir. Am J Transplant 2003;3:186-191. 29. Thamboo TP, Jeffery KJ, Friend PJ, Turner GD, Roberts IS. Urine cytology screening for polyoma virus infection following renal transplantation: the Oxford experience. J Clin Pathol 2007;60:927-930. 30. Gonzalez-Fraile MI, Canizo C, Caballero D, Hernandez R, Vazquez L, Lopez C, Izarra A, Arroyo JL, de la Loma A, Otero MJ, San Miquel JF. Cidofovir treatment of human polyomavirus-associated acute haemorrhagic cystitis. Transpl Infect Dis 200;3:44-46. 31. Held TK, Biel SS, Nitsache A, Kurth A, Chen S, Gelderblom HR, Sieqert W. Treatment of BK virus-associated hemorrhagic cystitis and simultaneous CMV reactivation with cidofovir. Bone Marrow Transplant 2000;26:347-350. 32. Hatakeyama N, Suzuki N, Kudoh T, Hori T, Mizue N, Tsutsumi H. Successful cidofovir treatment of adenovirus-associated hemorrhagic cystitis and renal dysfunction after allogenic bone marrow transplant. Pediatr Infect Dis J 2003;22:928-929. 33. Leung AY, Suen CK, Lie AK, Liang RH, Yuen KY, Kwong YL. Quantification of polyoma BK viruria in hemorrhagic cystitis complicating bone marrow transplantation. Blood 2001;98:1971-1978.

RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0079 Turk J Hematol 2016;33:231-235

A Randomized Study Comparing the Efficacy of Three Hepatitis B Vaccine Induction Regimens in Adult Patients with Hematological Malignancies Erişkin Hematolojik Maligniteli Hastalarda Üç Tip Hepatit B Aşılama Rejimini Karşılaştıran Randomize Bir Çalışma Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı Gazi University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract Objective: Non-responsiveness to hepatitis B virus (HBV) vaccines is not rare in hemato-oncological patients due to disease-associated or treatment-induced immune suppression. Although different strategies have been employed to improve the response rates, to date there is not an approved schedule for HBV immunization in patients with hematological malignancies. We designed a prospective randomized study to evaluate the efficacy of 3 different induction regimens for HBV vaccination.

Öz Amaç: Hematolojik maliniteli hastalarda hastalık veya tedavi ilişkili immün baskılanma yüzünden Hepatit B virüsü (HBV) aşısı yanıtsızlığı nadir değildir. Aşı yanıtını düzeltecek yöntemler denenmiş olsa da hematolojik maligniteli hastalarda kabul görmüş bir protokol henüz mevcut değildir.

Öz

Gereç ve Yöntemler: Üç farklı HBV aşılama rejimini karşılaştıran

Materials and Methods: In the standard-dose (SD) group, total vaccine dose delivered was 40 µg and patients were vaccinated with 20 µg at weeks 0 and 4. In the high-dose dose-intensive (HDDI) group, total vaccine dose delivered was 80 µg and patients were vaccinated with 40 µg at weeks 0 and 4. In the high-dose time-intensive (HDTI) group, total vaccine dose delivered was 80 µg and patients were vaccinated with 20 µg at weeks 0, 2, 4, and 6.

ileriye dönük ve randomize bir çalışma tasarlandı. Standart doz (SD)

Results: In a cohort of 114 patients, 38.6% responded to HBV vaccination. The response rate in the SD arm, HDDI arm, and HDTI arm was 26.2%, 29.7%, and 44.4%, respectively (p>0.05). Age was the only variable identified as having a negative impact on response.

%38,6 bulundu. Yanıt oranı SD, YDDY ve YDSU kolu için yanıt sırası ile

Conclusion: Short of achieving statistical significance, a higher response rate was observed in the HDTI arm. Therefore, this study supports a high-dose, time-intensive HBV vaccine induction regimen in patients with hematological malignancies who are not on chemotherapy.

Sonuç: İstatistiksel anlamı olmasa da YDSU kolunda HBV aşı yanıtı

Keywords: Hepatitis B, Vaccine, Hematological malignancies

Anahtar Sözcükler: Hepatit B, Aşılama, Hematolojik malignite

Introduction Hepatitis B virus (HBV) infection, the most common chronic viral infection in the world, can have serious clinical complications ranging from fulminant hepatitis to cirrhosis and hepatocellular

grubuna toplam 40 µg olmak üzere 0. ve 4. haftada 20 µg HBV aşısı yapıldı. Yüksek doz-doz yoğun (YDDY) gruba toplam 80 µg HBV aşısını 0. ve 4. haftada 40 µg uygulandı. Yüksek doz-sık uygulama (YDSU) grubuna toplam 80 µg HBV aşısı 0., 2., 4. ve 6. haftalarda 20 µg yapıldı. Bulgular: Yüz on dört hastayı içeren bu çalışmada HBV aşısına yanıt %26,2, %29,7 ve %44,7 bulundu (p>0,05). Aşı yanıtı üzerine olumsuz etkili tek değişkenin yaş olduğu saptandı. oransal olarak daha yüksek bulundu. Bu çalışmada elde edilen sonuçlar hematolojik maligniteli hastalarda yüksek doz ve daha sık uygulanan HBV aşılama rejiminin daha etkin olduğunu düşündürmektedir.

carcinoma [1]. Vaccination is most effective in preventing HBV infection and complications. The complete vaccine series induce protective antibody levels in more than 95% of infants, children, and young adults. Protection has been estimated to last at least 20 years and is possibly lifelong [2]. It is generally held that patients

Address for Correspondence/Yazışma Adresi: Zübeyde Nur ÖZKURT, M.D., Gazi University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone : +90 312 202 63 17 E-mail : zubeydenurozkurt@yahoo.com

Received/Geliş tarihi: February 10, 2015 Accepted/Kabul tarihi: September 28, 2015

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with hematological malignancies are immunosuppressed, either as a result of the underlying hematological malignancy or due to treatment with chemotherapy. Immunosuppressive diseases like hematological malignancies are a risk factor for nonresponsiveness to HBV vaccination. Despite low response rates, it is recommended that HBV-naive patients with hematological malignancies be immunized against HBV [3].

Chemotherapy regimens were categorized into 4 groups, namely alkylating agent-based, purine analog-based, chemo immunotherapy, and acute leukemia type, in an attempt to evaluate the effect of various drugs with different modes of action on vaccine response. Disease status at the time of vaccination was evaluated as untreated, in complete remission, or stable disease.

Although a rapid and effective strategy for HBV immunization of patients with hematological malignancies is highly desirable, to date there is not an approved schedule for these patients. Different strategies have been employed to improve the response rates in immunologically compromised patients, including HIVinfected adult patients, and in patients with chronic kidney disease [4,5,6]. We designed a prospective randomized study to evaluate the efficacy of 3 different induction regimens for HBV immunization in patients with hematological malignancies. In this study we aim to compare the results of the 3 different induction regimens for HBV vaccine at week 8. The basis of this deviation of omitting the consolidation dose at month 6, the current standard of care for routine HBV vaccination, was to decrease the drop-out rate due to stem cell transplantation, progression, relapse, death, frequent infections, and the effect of infection, antibiotics, and intravenous immunoglobulin on vaccination, and to allow more patients to be recruited into the study.

Before vaccination blood was drawn for complete blood count and IgG, IgA, and IgM levels. Absolute CD4, CD8, CD3+ CD56+, and CD4+ CD25+ counts were determined by flow cytometry (Becton Dickinson, FACSCalibur). Anti-HBs levels were planned to be measured at week 8. Patients who completed the vaccine schedule and had an anti-HBs serology at week 8 were considered eligible for response evaluation.

Materials and Methods

Group 2: High-dose dose-intensive (HDDI): Patients were vaccinated with 40 µg at weeks 0 and 4; total vaccine dose was 80 µg.

Patients with hematological malignancies followed by the Division of Hematology at Gazi University in Turkey between January 2008 and December 2013 were included in the study. Inclusion criteria were: a. Age >18 years b. Eastern Cooperative Oncology Group performance status ≥2, c. Negative serology for HBsAg, anti-HBc, and anti-HBs, d. Negative serology for hepatitis C and HIV, e. Chemotherapy-naive patients, f. Patients who achieved remission or stable disease after chemotherapy, g. In subjects with a history of treatment, a time interval of at least 3 months from the last dose of chemotherapy and/or radiotherapy. Exclusion criteria were: a. History of prior HBV vaccination, b. Evidence of ongoing systemic infection, c. Ongoing immunosuppressive therapy, d. History of prior stem cell transplantation, e. History of a non-hematological malignancy, except adequately treated squamous or basal cell carcinoma of the skin or cervical carcinoma in situ. 232

Study Protocol All patients participating in the study received a recombinant HBV vaccine (Genhevac B, Sanofi Pasteur) in the deltoid region, intramuscularly. Patients were randomized (1:1:1 ratio) into 1 of the 3 groups below to receive the hepatitis B vaccine. Groups were named based on the total vaccine dose delivered and vaccination frequency. Group 1: Standard dose (SD): Patients were vaccinated with 20 µg at weeks 0 and 4; total vaccine dose was 40 µg.

Group 3: High-dose time-intensive (HDTI): Patients were vaccinated with 20 µg at weeks 0, 2, 4, and 6; total vaccine dose was 80 µg. Patients who achieved anti-HBs levels of >10 IU/L were defined as vaccine responders. The primary endpoint was seroprotection rate at week 8 and the secondary endpoint was comparison of antibody levels in 3 different HBV vaccination regimens. The study protocol was approved by the Local Ethics Committee of Gazi University Faculty of Medicine and written informed consent was obtained from all subjects prior to study entry. Statistical Analysis Statistical evaluation was done with SPSS 15. Data were described as numbers and percentages or medians and minimum-maximum, as appropriate. Chi-square test was used for evaluating categorical values, and Kruskal-Wallis and oneway ANOVA tests were used for continuous values in patient groups. All p-values were 2-sided with statistical significance at 0.05 alpha levels. Logistic regression analysis was used for multivariate analysis to evaluate the factors affecting the vaccination response.

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Özkurt ZN, et al: A Step Further for Improving Hepatitis B Vaccine Response

Results

Response Evaluation at Week 8

A total of 124 patients were randomized during the study period. Ten patients were excluded from the study due to cerebrovascular event before the first dose of the vaccine (1 patient) or not fulfilling the criteria for response evaluation after vaccination (9 patients). Characteristics of the patients are summarized in Tables 1 and 2. Significant differences existed only in CD4/CD8 ratio between the 3 vaccine groups.

At the end of the study, 114 patients were eligible for response evaluation. Overall, 44 of 114 patients responded, with a response rate of 38.6% at week 8. On the other hand, 61.4% of the patients did not respond to HBV vaccination. The response rate in the SD arm, HDDI arm, and HDTI arm was 26.2%, 29.7%, and 44.4%, respectively (Figure 1). The high response rate in the HDTI arm did not translate to statistical significance, possibly due to relatively low patient numbers (p>0.05). The median antibody concentration (MAC) in the SD arm, HDDI arm, and HDTI arm was 46.6 IU/L (12.4-706), 73.95 IU/L (14.7-779), and 47.4 IU/L (11.6-779), respectively (Figure 2). The differences in MAC between groups were not statistically significant. Among the variables evaluated, only age had a negative impact on response (p<0.001). Other clinical variables such as sex, disease type, disease status, treatment status, type of chemotherapy, radiotherapy, time from diagnosis to vaccination, and time from last treatment to vaccination had no impact on antibody response. Similarly IgG, IgM, and IgA levels and absolute CD4, CD8, CD3+ CD56+, and CD4+ CD25+ numbers did not influence the response. No serious adverse effects attributable to vaccination were identified.

Table 1. Baseline demographic characteristics of the study patients.

Group 1

Group 2

Group 3

Age

54 (22-78)

57 (25-78)

54 (20-80)

0.83

Sex (Male/ Female)

20/22

25/12

17/18

0.25

Patient (n)

CLL

NHL

0.92

HCL

PCD

Treatment

0.66

Yes

Radiotherapy

0.76

Yes

DTV (months)

10.5 (0-144)

11 (0-83)

12.5 (0-89)

0.96

LTTV (months)

6 (3-82)

6 (2-64)

9 (3-89)

0.77

Values are expressed as medians and minimum-maximum where necessary. CLL: Chronic lymphocytic leukemia, NHL: non-Hodgkin lymphoma, HD: Hodgkin’s disease, AL: acute leukemia, HCL: hairy cell leukemia, PCD: plasma cell disorder, DTV: time from diagnosis to vaccination, LTTV: time from last treatment to vaccination.

Figure 1. Response rates to 3 different vaccination regimens at week 8.

Table 2. Baseline laboratory characteristics.

Group 1

Group 2

Group 3

Hb (g/dL)

12.9 (8.9-17.3)

13.55 (9.6-18)

13.8 (9.8-16.8)

0.58

WBC (µL)

8455 (3540-87,530)

6885 (2100-93,300)

7200 (3370-258,101)

0.76

IgG (mg/dL)

1060 (589-2330)

1015 (256-2110)

1110 (595-5130)

0.33

IgA (mg/dL)

147 (22.9-518)

102 (11.6-551)

178.5 (14.7-426)

0.38

IgM (mg/dL)

70.5 (17-3630)

57 (5.3-2640)

66.9 (21.8-152)

0.64

CD4 (µL)

412 (110-8993)

440.5 (89-2038)

528 (126-2006)

0.5

CD8 (µL)

702 (147-9811)

521(64-2600)

562 (197-1639)

0.56

CD4/CD8

0.73 (0.14-1.96)

0.92 (0.28-2.57)

0.93 (0.21-4.6)

0.045

123 (43-765)

135 (13-810)

100 (4-699)

0.96

33 (3-578)

31 (5-183)

42 (6-532)

0.78

CD3+

CD56+

(µL)

CD4+ CD25+ (µL)

All values are expressed as medians and minimum-maximum. WBC: White blood cell, Hb: hemoglobin.

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trials in patients with hematological malignancies is very limited and mostly confined to pediatric patients with acute leukemia [8,9,10,11]. The data supporting HBV vaccination almost completely come from general vaccination strategies and no evidence-based recommendations for the dose, frequency, and timing of HBV vaccination in adult hematological patients are available.

600.0

500.0

400.0

300.0

200.0

100.0

00.0 1

Figure 2. Median antibody concentration response to 3 different regimens at week 8.

Discussion In this study, overall response rate, defined as anti-HBs of >10 IU/L at week 8, was 38.6%. The response rate in the SD arm, HDDI arm, and HDTI arm was 26.2%, 29.7%, and 44.4%, respectively. Short of achieving statistical significance, a higher response rate was observed in the HDTI arm. The MAC in the SD arm, HDDI arm, and HDTI arm was not statistically significant. Among the variables evaluated, only age had a negative impact on response. HBV reactivation is a common complication in HBsAg-positive patients undergoing immunosuppressive anticancer therapy. The clinical consequences of HBV reactivation are observed as asymptomatic liver function disturbances, liver failure, and delay or premature cessation of chemotherapy courses with adverse prognostic consequences for the hematological disease. It is strongly recommended that all hemato-oncological patients be screened for HBV markers and immunization against hepatitis B be performed in HBV-naive patients when appropriate [3,7]. Conducting HBV vaccination trials in adult patients with hematological malignancies is troublesome. Heterogeneity of both the underlying hematological conditions and chemotherapy regimens, maintenance therapies, relapse of the disease, and salvage regimens including high-dose chemotherapy with stem cell support make the situation more complex. Therefore, recruiting a sufficient number of patients for randomized trials and multivariate analysis requires the active collaboration of centers, especially in developing countries. As a result of these difficulties, the number of HBV vaccination 234

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The risk of HBV transmission, just after the diagnosis or during the chemotherapy of patients, is high due to frequent transfusions and interventions. Immediate vaccination is highly desirable; however, disease and chemotherapy may compromise antibody response. If the vaccination is postponed until after the chemotherapy, disease and chemotherapy-related immunosuppression are lessened and probability of response increases, but active protection from HBV during the high-risk period is missed. Accelerated vaccination strategies could be useful for atrisk groups in terms of rapid seroconversion and increasing adherence. Studies conducted in high-risk healthy adults, drug users, lung transplantation candidates, and HIV-infected patients elicited similar or better anti-HBs responses and could be advantageous for the short term in this population. However, additional studies on long-term protection and effectiveness of accelerated schedules are necessary [12,13,14,15]. Non-responsiveness to HBV vaccine is not rare in hematooncological patients due to disease-associated or treatmentinduced immune suppression. There are a number of means to augment the immune response to HBV immunization in nonresponders, including adding additional doses, doubling the vaccine dose, intradermal injection of the vaccine, combination with granulocyte-macrophage colony-stimulating factor (GMCSF), and use of new, more immunogenic HBV vaccines [16]. We have identified 2 studies on improving serological response to HBV vaccine in adult patients with hematological malignancies. First, Pullukcu et al. carried out a non-randomized study in 42 HBV-naive hematology patients during chemotherapy. Patients were administered a 20 µg HBV vaccine on days 0, 14, and 28. Overall, 23.8% of the patients responded to this accelerated schedule during chemotherapy [17]. The second study was a randomized one comparing the efficacy of a single dose of 40 µg HBV vaccine with one course of 40 µg HBV vaccine after 5 µg/kg recombinant GM-CSF injection in 94 patients with lymphoproliferative disorders (LPDs). Although the seroprotection rate was higher in the GM-CSF + HBV vaccine group (25.5% vs. 17%), the difference did not reach a significant level. In multivariate analysis, age was the only predictor of achieving a seroprotective response. The authors concluded that in LPDs, the response to HBV vaccine is impaired and GM-CSF may enhance the response rate to HBV vaccine [18].

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Özkurt ZN, et al: A Step Further for Improving Hepatitis B Vaccine Response

The available data are far from allowing definite recommendations. However, some useful information about the dose, frequency, and timing of the vaccination may be collected for the design of future studies in adult patients with hematological disease. First of all, response to standard HBV vaccination is impaired and doubling the vaccine dose per injection does not increase the response rate at week 8. On the other hand, frequent antigenic stimulation seems to induce better immune response than a standard schedule. Thus, an accelerated vaccination schedule is feasible in this patient group.

References

The durability of the response, even in stable disease conditions, is another factor that needs to be taken into consideration. In our previous study, some of the patients lost their seroprotective anti-HBs responses beginning at the sixth month of the vaccination [18]. Therefore, we suggest that a single booster dose of vaccine should be given to responders at the sixth month of vaccination. Finally, response rate to HBV vaccination during chemotherapy is low even though a frequent injection scheme is used [17]. Moreover, anti-HBs response may be lost during a chemotherapy course [19]. Whether patients who lost anti-HBs response during chemotherapy need re-vaccination or a single booster dose of vaccine to reinduce antibody production is not known. The findings from this study suggest a time-intensive approach, at 20 µg biweekly of 3-4 doses of HBV vaccine, for the design of future studies of adult patients with hematological disease who are not on chemotherapy. Ethics Ethics Committee Approval: The study protocol was approved by the Local Ethics Committee of Gazi University Faculty of Medicine; Informed Consent: Written informed consent was obtained from all subjects prior to study entry. Authorship Contributions Medical Practices: Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı; Concept: Zübeyde Nur Özkurt, Münci Yağcı; Design: Münci Yağcı, Zübeyde Nur Özkurt; Data Collection or Processing: Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı; Analysis or Interpretation: Zübeyde Nur Özkurt, Elif Suyanı, Münci Yağcı; Literature Search: Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı; Writing: Zübeyde Nur Özkurt, Elif Suyanı, Rauf Haznedar, Münci Yağcı. 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.

1. Trépo C, Chan HL, Lok A. Hepatitis B virus infection. Lancet 2014;384:20532063. 2. WHO. Hepatitis B. Fact Sheet No. 204. Geneva, WHO, 2014. 3. Lalazar G, Rund D, Shouval D. Screening, prevention and treatment of viral hepatitis B reactivation in patients with haematological malignancies. Br J Haematol 2007;136:699-712. 4. Rey D, Piroth L, Wendling MJ, Miailhes P, Michel ML, Dufour C, Haour G, Sogni P, Rohel A, Ajana F, Billaud E, Molina JM, Launay O, Carrat F; ANRS HB04 B-BOOST study group. Safety and immunogenicity of double-dose versus standard-dose hepatitis B revaccination in non-responding adults with HIV-1 (ANRS HB04 B-BOOST): a multicentre, open-label, randomised controlled trial. Lancet Infect Dis 2015;15:1283-1291. 5. Tajiri K, Shimizu Y. Unsolved problems and future perspectives of hepatitis B virus vaccination. World J Gastroenterol 2015:21;7074-7083. 6. McNulty CA, Bowen JK, Williams AJ. Hepatitis B vaccination in predialysis chronic renal failure patients a comparison of two vaccination schedules. Vaccine 2005;14:4142-4147. 7. Yağci M, Ozkurt ZN, Yeğin ZA, Aki Z, Sucak GT, Haznedar R. Hepatitis B virus reactivation in HBV-DNA negative and positive patients with hematological malignancies. Hematology 2010;15:240-244. 8. Goyal S, Pai SK, Kelkar R, Advani SH. Hepatitis B vaccination in acute lymphoblastic leukemia. Leuk Res 1998;22:193-195. 9. Yetgin S, Tunç B, Koç A, Toksoy HB, Ceyhan M, Kanra G. Two booster dose hepatitis B virus vaccination in patients with leukemia. Leuk Res 2001;25:647-649. 10. Somjee S, Pai S, Parikh P, Banavali S, Kelkar R, Advani S. Passive active prophylaxis against Hepatitis B in children with acute lymphoblastic leukemia. Leuk Res 2002;26:989-992. 11. Yetgin S, Tavil B, Aytac S, Kuskonmaz B, Kanra G. Unexpected protection from infection by two booster hepatitis B virus vaccination in children with acute lymphoblastic leukemia. Leuk Res 2007;31:493-496. 12. Jin H, Tan Z, Zhang X, Wang B, Zhao Y, Liu P. Comparison of accelerated and standard hepatitis B vaccination schedules in high-risk healthy adults: a meta-analysis of randomized controlled trials. PLoS One 2015;10:e0133464. 13. Shah DP, Grimes CZ, Nguyen AT, Lai D, Hwang LY. Long-term effectiveness of accelerated hepatitis B vaccination schedule in drug users. Am J Public Health 2015;105:36-43. 14. Galar A, Engelson BA, Kubiak DW, Licona JH, Boukedes S, Goldberg HJ, Baden LR,Marty FM, Issa NC. Serologic response to hepatitis B vaccination among lung transplantation candidates. Transplantation 2014;27:676-679. 15. Mena G, Llupià A, García-Basteiro AL, Díez C, León A, García F, Bayas JM. Assessing the immunological response to hepatitis B vaccination in HIVinfected patients in clinical practice. Vaccine 2012;30:3703-3709. 16. Chen DS. Hepatitis B vaccination: the key towards elimination and eradication of hepatitis B. J Hepatol 2009;50:805-816. 17. Pullukcu H, Ertem E, Karaca Y, Yamazhan T, Sertoz RY, Altuglu I. Efficacy of accelerated hepatitis B vaccination program in patients being actively treated for hematologic malignancies. Int J Infect Dis 2008;12:166-170. 18. Yagci M, Acar K, Sucak GT, Yamaç K, Haznedar R. Hepatitis B virus vaccine in lymphoproliferative disorders: a prospective randomized study evaluating the efficacy of granulocyte-macrophage colony stimulating factor as a vaccine adjuvant. Eur J Haematol 2007;79:292-296. 19. Yağcı M, Suyanı E, Kızıl Çakar M. The impact of chemotherapy on hepatitis B antibody titer in patients with hematological malignancies. Turk J Hematol 2015;32:251-256.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0242 Turk J Hematol 2016;33:236-243

Reliability and Validity of the Turkish Version of the PedsQL 3.0 Cancer Module for 2- to 7-Year-Old and the PedsQL 4.0 Generic Core Scales for 5- to 7-Year-Old: The Hacettepe University Experience Çocuklar için Yaşam Kalitesi Ölçeği Kanser Modülü Türkçe Versiyonunun 2-7 Yaşları Arasındaki Çocuklarda ve Genel Skalası’nın 5-7 Yaşları Arasındaki Çocuklarda Geçerlik ve Güvenilirliği: Hacettepe Üniversitesi Deneyimi Vesile Yıldız Kabak1, Yavuz Yakut1, Mualla Çetin2, Tülin Düger1 1Hacettepe University Faculty of Health Sciences, Department of Physical Therapy and Rehabilitation, Ankara, Turkey 2Hacettepe University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey

Abstract

Öz

Objective: The aim of this study was to investigate the reliability and validity of the Turkish version of the Pediatric Quality of Life Inventory (PedsQL) 3.0 Cancer Module for 2- to 7-year-old and the PedsQL 4.0 Generic Core Scales for 5- to 7-year-old in childhood cancer.

Amaç: Çalışmamızın amacı, çocukluk çağı kanserlerinde Çocuklar için Yaşam Kalitesi Ölçeği (ÇİYKÖ) 3.0 Kanser Modülü Türkçe versiyonunun 2-7 yaşları arasındakiler için ve ÇİYKÖ 4.0 Genel Skalası’nın 5-7 yaşları arasındakiler için geçerliliğini ve güvenilirliğini araştırmaktır.

Materials and Methods: The PedsQL 3.0 Cancer Module and PedsQL 4.0 Generic Core Scales were administered to children with cancer and their parents at Hacettepe University. Internal consistency was determined by using Cronbach’s alpha and test-retest reliability was determined by using the intraclass correlation coefficient (ICC). Construct validity was assessed by comparing the results of the PedsQL 3.0 Cancer Module with those of the PedsQL 4.0 Generic Core Scales.

Gereç ve Yöntemler: ÇİYKÖ 3.0 Kanser Modülü ve ÇİYKÖ 4.0 Genel Skalası Hacettepe Üniversitesi’ndeki kanserli çocuklara ve ebeveynlerine uygulanmıştır. Cronbach’s alpha kullanılarak iç tutarlılık, sınıf içi korelasyon katsayısı (SKK) kullanılarak test-retest güvenirliği belirlenmiştir. Yapı geçerliliği ÇİYKÖ 3.0 Kanser Modülü ile ÇİYKÖ 4.0 Genel Skalası sonuçlarının karşılaştırılmasıyla değerlendirilmiştir.

Results: Cronbach’s alpha of the PedsQL 3.0 Cancer Module varied from 0.803 to 0.873 and that of the PedsQL 4.0 Generic Core Scales from 0.665 to 0.841. Test-retest ICC values of the PedsQL 3.0 Cancer Module varied from 0.877 to 0.949 and those of the PedsQL 4.0 Generic Core Scales from 0.681 to 0.824. The correlation of the PedsQL 3.0 Cancer Module with subscale scores of the PedsQL 4.0 Generic Core Scales showed that there were excellent to fair correlations between the two scales. The relationship between parent proxy-report and child self-report of the PedsQL 3.0 Cancer Module had very good correlation (r=0.694, p<0.001), as did the PedsQL 4.0 Generic Core Scales (r=0.540, p=0.002).

Bulgular: ÇİYKÖ 3.0 Kanser Modülü’nün Cronbach’s alpha değerleri 0,803-0,873 arasında, ÇİYKÖ 4.0 Genel Skalası’nın ise 0,665-0,841 arasında değişiklik göstermektedir. Test-retest SKK değerleri ise ÇİYKÖ 3.0 Kanser Modülü’nde 0,877-0,949 arasında, ÇİYKÖ 4.0 Genel Skalası’nda ise 0,681-0,824 arasında değişiklik göstermektedir. ÇİYKÖ Kanser Modülü ile Genel Skalası’nın alt başlık skorlarının korelasyonlarının mükemmel-orta düzeyde olduğu gösterilmiştir. ÇİYKÖ Kanser Modülü Çocuk Formu ile Ebeveyn Formu arasındaki ilişki çok iyi düzeyde korele (r=0,694, p<0,001), ÇİYKÖ Genel Skalası’nda ise iyi düzeyde koreledir (r=0,540, p=0,002).

Conclusion: This study demonstrated the reliability, validity, and feasibility of the Turkish version of the PedsQL 3.0 Cancer Module in 2- to 4-year-old and 5- to 7-year-old and the PedsQL 4.0 Generic Core Scales in 5- to 7-year-old in childhood cancer.

Sonuç: Bu çalışma ÇİYKÖ 3.0 Kanser Modülü’nün Türkçe versiyonunun 2-4 yaş ile 5-7 yaşları arasındaki ve ÇİYKÖ 4.0 Genel Skalası Türkçe versiyonunun 5-7 yaşları arasındaki kanserli çocuklarda geçerli, güvenilir ve uygulanabilir olduğunu göstermiştir.

Keywords: Pediatric Quality of Life Inventory, Validity, Reliability, Children, Cancer

Anahtar Sözcükler: Çocuklar için Yaşam Kalitesi Ölçeği, Geçerlilik, Güvenirlik, Çocuk, Kanser

Address for Correspondence/Yazışma Adresi: Vesile YILDIZ KABAK, M.D., Hacettepe University Faculty of Health Sciences, Department of Physical Therapy and Rehabilitation, Ankara, Turkey Phone : +90 312 305 25 25/180 E-mail : vesile_yldz@hotmail.com

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Received/Geliş tarihi: June 16, 2015 Accepted/Kabul tarihi: December 29, 2015

Turk J Hematol 2016;33:236-243

Introduction Quality of life (QOL) has been described as a subjective term and is defined as a person’s sense of social, emotional, and physical well-being and his/her ability to function in ordinary tasks of daily living [1,2,3,4]. Therefore, a health-related quality of life (HRQOL) instrument should include physical, mental, and social health dimensions [5,6]. It is increasingly acknowledged as an important health outcome measure in clinical trials and health service research and evaluation [7]. Disease-specific HRQOL assessment instruments have been developed to determine the impact of disease and treatment on the quality of patients’ life. However, there are a limited number of instruments designed to measure the HRQOL of pediatric patients with cancer [8,9,10]. The Pediatric Quality of Life Inventory (PedsQL), which has both generic and disease-specific modules as well as patient and parent versions, is one of the very few instruments that is widely used to assess HRQOL among children and adolescents between the ages of 2 and 18 years [3,11]. It is brief, can be applied in 5-15 min, and can be scored easily [12,13]. Evaluation is conducted by both children and parents; children aged 5 to 18 years are asked to evaluate their own HRQOL (child self-report) and the parents of children aged 2 to 18 years are asked to evaluate their child’s HRQOL (parent proxy-report) [14]. The PedsQL 4.0 Generic Core Scales were specifically designed for application in both healthy and patient populations. The PedsQL 3.0 Cancer Module was designed to measure HRQOL dimensions specific to pediatric cancers [7]. This instrument has already been validated in English [15], German [16], Chinese [11], Japanese [14], Urdu [17], and Portuguese [8]. In Turkey, the validity and reliability of the PedsQL 4.0 Generic Core Scales for 8- to 12-year-old and 13- to 18-year-old children were evaluated, as was the PedsQL 3.0 Cancer Module for 8- to 12-year-old children [18,19,20]. Uneri et al. investigated the reliability and validity of the Turkish translation of the PedsQL 4.0 Generic Core Scales for 2- to 4-year-old and 5- to 7-year-old Turkish children. They found that the validity of the parent proxy-reports for the two age groups was sufficient, whereas the validity of the child selfreport of the 5- to 7-year-old age group was low [12]. The aim of this study was to investigate the reliability and validity of the Turkish version of the PedsQL 3.0 Cancer Module for 2- to 4-year-old and 5- to 7-year olds and the reliability and validity of the Turkish version of the PedsQL 4.0 Generic Core Scales for 5- to 7-year-old in childhood cancer.

Materials and Methods Patients and Setting This study was developed in Turkey. We recruited children with cancer and their parents from Hacettepe University Hospital.

Yıldız Kabak V, et al: Turkish Version of the PedsQL at Hacettepe University

Children between the ages of 2 and 7 who were diagnosed with cancer at least 2 months earlier, who agreed to participate in the study, and who had good verbal communication were included. Children with comorbid disease, major developmental disorders, and neurologic problems were excluded from the study. The 2- to 4-year-old age group consisted of 43 children and their parents. The 5- to 7-year-old age group consisted of 31 children and their parents. Informed consent was obtained from all individual participants included in the study. Instruments PedsQL 4.0 Generic Core Scales The PedsQL 4.0 Generic Core Scales were designed by Varni et al. in 1999 as a HRQOL measurement for children and adolescents aged between 2 and 18 years [13]. It consists of 23 items: physical functioning (8 items), emotional functioning (5 items), social functioning (5 items), and school functioning (5 items). Child self-reports include 3 age groups: 5-7 years (young children), 8-12 years (children), and 13-18 years (teens). The parent proxyreport, however, includes 4 age groups: 2-4 (toddlers), 5-7, 8-12, and 13-18. The response scale is a 5-point Likert scale for all age groups, except for the 5- to 7-year-old’ version. Items are reverse-scored and linearly transformed to a 0-100 scale, so that higher scores indicate better HRQOL. The response scale of the 5- to 7-year-old version is completed with the help of an interviewer and simplified to a 3-point scale (0, 2, and 4 points). The child answers the items with the help of a visual scale (happy, neutral, and sad faces). The PedsQL 4.0 computes the scale scores as well as the Psychosocial Health Summary Scores by adding the sum of points from the Emotional, Social, and School Functioning Subscales and dividing them by the total number of items answered [21,22]. PedsQL 3.0 Cancer Module The PedsQL 3.0 Cancer Module was developed and tested for validity and reliability by Varni et al. [3,22,23]. This module consists of 27 items: pain and hurt (2 items), nausea (5 items), procedural anxiety (3 items), treatment anxiety (3 items), worry (3 items), cognitive problems (5 items), perceived physical appearance (3 items), and communication (3 items). The format, instructions, Likert response options, and scoring method are similar to those of the PedsQL 4.0 Generic Core Scales [22]. This scale comprises two parallel forms for child and for parent. Higher scores indicate better HRQOL [21]. Translation and Cross-Cultural Adaptation This study was conducted in these phases: 1. Translation, validation, and reliability of the PedsQL 3.0 Cancer Module for 2- to 4-year-old parent proxy-report and 5- to 7-year-old children’s form and parent proxy-report. 237

Yıldız Kabak V, et al: Turkish Version of the PedsQL at Hacettepe University

2. Translation, validation, and reliability of the PedsQL 4.0 Generic Core Scales for 5- to 7-year-old children’s form. In the translation process, we used the guidelines for crosscultural adaptation and we obtained permission for the Turkish version from Varni et al. (Mapi Research Trust) [4,24]. Approval of the study was obtained from the Ethics Committee of Hacettepe University (GO 14/455). The original English instruments (PedsQL 4.0 Generic Core Scale /5-7 years and PedsQL 3.0 Cancer Module/2-4 and 5-7 years) were translated independently into Turkish. Two translations from English to Turkish were done by two different and independent native Turkish translators. The Turkish translations were then compared for inconsistencies. The two translations were then retranslated, also blindly and independently, into English by two native English speakers. The Turkish version was then jointly reviewed by a bilingual team, including the four translators, three physical therapists, and a physician, to assess the necessity of cultural adaptation. The Turkish version was compared with the original English version to detect possible errors of interpretation and nuances that might have been missed. The final stage of the adaptation process was to test the prefinal version. Ten children were tested in this stage. The results eliminated the necessity for Turkish cultural adaptation. Statistical Analysis Reliability Two common forms of reliability are test-retest reliability and internal consistency. For test-retest reliability, the forms were applied in 7-day intervals. We used the intraclass correlation coefficient (ICC) to evaluate test-retest reliability. The ICC can vary from 0.00 to 1.00, where values of 0.60 to 0.80 are regarded as evidence of good reliability and those above 0.80 indicate excellent reliability [25,26,27]. The internal consistency of a scale relates to its homogeneity. The coefficient of internal consistency is mainly assessed with Cronbach’s alpha. It is suggested that the value of alpha should be above 0.80 for acceptance as high internal consistency [28]. Validity In this study, construct validity was assessed by comparing the responses to the PedsQL 3.0 Cancer Module to the results of the PedsQL 4.0 Generic Core Scales. Construct validity coefficients (r) were accepted as follows: 0.81-1.0 as excellent, 0.61-0.80 very good, 0.41-0.60 good, 0.21-0.40 fair, and 0-0.20 poor. Construct validity was measured by Pearson’s correlation coefficient [29]. All assessments were repeated 7 days later by the physical therapist. Means and standard deviations were determined to describe the demographic data of the patients. All statistical 238

Turk J Hematol 2016;33:236-243

analyses were done with IBM-SPSS 22.0 for Windows. A probability value of p<0.05 was considered to indicate a significant effect.

Results Demographic Characteristics The total sample consisted of 74 children with cancer, aged 2 to 7 years old, and their parents. While 60% of the children had leukemia, the others had different cancers (lymphoma, brain tumors, rhabdomyosarcoma, Wilms’ tumor, osteosarcoma, and Ewing’s sarcoma). The number of children in the 2- to 4-yearold group was 43 (58.10%) and in the 5- to 7-year-old group was 31 (41.89%). Characteristics of the groups are summarized in Table 1. Subscale and total scores of the 2- to 4-year-old and 5- to 7-year-old groups are summarized in Table 2. Reliability Internal Consistency Reliability The internal consistency reliability alpha coefficient (Cronbach’s coefficient alpha) of the PedsQL 3.0 Cancer Module for the 2to 4-year-old parents’ form was 0.803, and those for the 5- to 7-year-old parents’ and children’s forms were 0.867 and 0.817, respectively. Cronbach’s coefficient alpha values of the PedsQL 4.0 Generic Core Scales 5- to 7-year-old total score were 0.704 for the parents’ form and 0.712 for the children’s form. Test-Retest Reliability For the 2- to 4-year-old parents’ form, the test-retest ICC value of the PedsQL 3.0 Cancer Module total score was 0.877 [95% confidence interval (CI)=0.774-0.934]. For the 5- to 7-year-old, ICC values of the parents’ and children’s forms were 0.949 (95% CI=0.898-0.975) and 0.889 (95% CI=0.780-0.945), respectively. The ICC values of the PedsQL 4.0 Generic Core Scales total scores for parents’ and children’s forms were 0.824 (95% CI=0.5990.928) and 0.681 (95% CI=0.425-0.837), respectively. Total Subscale Correlation Good and very good correlations were found between total scores of the scales and the subscale scores, as shown in Table 3. Validity Construct Validity Examination of the correlation of the 2- to 4-year-old parent proxy-report of the PedsQL 3.0 Cancer Module with the subscale scores of the PedsQL 4.0 Generic Core Scales showed that there were excellent to fair correlations between the two scales, except for the subscales of ‘worry’ and ‘school-related problems’. Table 4 indicates the intercorrelations between the PedsQL 3.0

Yıldız Kabak V, et al: Turkish Version of the PedsQL at Hacettepe University

Turk J Hematol 2016;33:236-243

Table 1. Demographic characteristics of the samples.

2- to 4-year-old (n=43)

5- to 7-year-old (n=31)

Sex

Girl

51.2

45.2

Boy

48.8

54.8

Current treatment status

On-treatment

18.6

9.7

Off-treatment

18.6

45.2

Antibiotic treatment

62.8

32.3

Respondent parent

Mother

86.0

83.9

Father

9.3

9.7

Other

4.65

6.5

Education level of the parent

Illiterate

9.3

3.2

Primary school

20.9

19.4

Secondary school

4.7

9.7

High school

30.2

22.6

University

34.9

38.7

Mean

The age of the children (years)

3.24

0.12

6.27

0.77

The age of the parents (years)

31.81

0.93

37.0

7.95

Total duration after diagnosis (months)

11.95

1.65

18.60

18.15

Body mass index (kg/m2)

16.05

0.4

16.54

3.22

SD: Standard deviation.

Table 2. Pediatric Quality of Life Inventory 3.0 Cancer Module and 4.0 Generic Core Scale Scores.

2- to 4-year-old

5- to 7-year-old

Parent Proxy-Report

Child Self-Report

Mean

72.49

21.03

75.29

20.27

76.66

25.37

PedsQL 3.0 Cancer Module Pain and hurt Nausea

72.23

21.75

57.43

29.89

67.24

25.05

Procedural anxiety

38.19

28.52

50.25

29.95

61.36

31.25

Treatment anxiety

59.12

35.39

76.96

32.57

84.30

28.14

Worry

70.26

35.00

80.25

31.97

73.85

24.96

Cognitive problems

71.81

23.08

81.45

21.74

78.83

20.45

Perceived physical appearance

78.83

25.80

68.80

30.07

72.89

28.07

Communication

62.90

38.01

64.67

42.55

77.66

27.64

Total score

64.51

18.27

68.16

22.05

73.33

17.85

70.98

19.99

68.64

26.86

71.73

20.44

PedsQL 4.0 Generic Core Scale Physical functioning Emotional functioning

60.47

20.23

70.48

23.03

74.63

17.37

Social functioning

84.40

18.89

78.53

23.03

82.82

19.10

School-related problems

67.57

33.50

59.90

30.17

66.55

26.23

Total score

70.53

15.22

70.32

18.85

73.73

16.08

SD: Standard deviation, PedsQL: Pediatric Quality of Life Inventory.

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Turk J Hematol 2016;33:236-243

Table 3. Pearson’s correlation coefficients between total scores and subscale scores of the scales.

PedsQL 3.0 Cancer Module Subscale Scores PH

PPA

Total scores (5-7 years old) Parent proxy-report

0.533*

0.949**

0.709**

0.768**

0.709**

0.512*

0.864**

0.744**

Child self-report

0.408*

0.829**

0.664**

0.622**

0.701**

0.702**

0.516*

0.816**

Total scores (2-4 years old) Parent proxy-report

0.393*

0.621**

0.671**

0.744**

0.620**

0.588**

0.746**

0.736**

Child self-report

PedsQL 4.0 Generic Core Scale Subscale Scores

SRP

Parent proxy-report

0.678**

0.761**

0.647**

0.817**

Child self-report

0.908**

0.748**

0.743**

0.526*

Total scores (5-7 years old)

*p<0.0.5, **p<0.001. PH: Pain and hurt, N: nausea, PA: procedural anxiety, TA: treatment anxiety, W: worry, CP: cognitive problems, PPA: perceived physical appearance, C: communication. PF: Physical functioning, EF: emotional functioning, SF: social functioning, SRP: school-related problems, PedsQL: Pediatric Quality of Life Inventory.

Table 4. Pearson’s correlation coefficients between subscale scores of the 2- to 4-year-old parent proxy-report of the Pediatric Quality of Life Inventory 3.0 Cancer Module and Pediatric Quality of Life Inventory 4.0 Generic Core Scales. PedsQL 3.0 Cancer Module, 2- to 4-year-old

PedsQL 4.0 Generic Core Scales, 2- to 4-year-old Physical Functioning Emotional Functioning

Social Functioning

School-Related Problems

Total Score

Pain and hurt

0.539**

0.335*

0.365*

-0.212

0.511**

Nausea

0.239

0.411**

0.436**

-0.559

0.396**

Procedural anxiety

0.427**

0.393**

0.208

-0.689

0.461**

Treatment anxiety

0.380*

0.628**

0.496**

-0.671

0.574**

Worry

-0.039

0.086

-0.095

-0.694

-0.042

Cognitive problems

0.395*

0.277

0.441**

-0.359

0.493**

Perceived physical appearance

0.421**

0.353*

0.636**

-0.656

0.552**

Communication

0.541**

0.416**

0.352*

-0.733

0.546**

Total score

0.559**

0.567**

0.547**

-0.828*

0.685**

*p<0.0.5, **p<0.001. PedsQL: Pediatric Quality of Life Inventory.

Cancer Module and PedsQL 4.0 Generic Core Scales for the 2- to 4-year-old parent proxy-report. Examination of the correlation of the 5- to 7-year-old parent proxy-report of the PedsQL 3.0 Cancer Module with the subscale scores of the PedsQL 4.0 Generic Core Scale showed that there were very good to fair correlations between the two scales. Table 5 indicates the intercorrelations between the PedsQL 3.0 Cancer Module and the PedsQL 4.0 Generic Core Scales for the 5- to 7-year-old parent proxy-report. Table 6 indicates the intercorrelation between the PedsQL 3.0 Cancer Module and PedsQL 4.0 Generic Core Scales for 5- to 7-year-old children’s self-reports. When the relationship between parents’ and children’s forms for the 5- to 7-year-old PedsQL 3.0 Cancer Module was analyzed, a very good correlation was found (r=0.694, p<0.001). For the 5240

to 7-year-old PedsQL 4.0 Generic Core Scales, a good correlation was found between parents’ and children’s forms, which was also statistically significant (r=0.540, p=0.002).

Discussion This study demonstrated the reliability, validity, and feasibility of the Turkish version of the PedsQL 3.0 Cancer Module in 2- to 4-year-old and 5- to 7-year-old and the PedsQL 4.0 Generic Core Scales in 5- to 7-year-old with childhood cancers. The analyses support the reliability and validity of the instrument as a child self-report and parent proxy-report HRQOL measurement instrument for Turkish pediatric cancer patients. The PedsQL is brief and easy to complete. The PedsQL self-report and proxy-report internal consistency reliabilities generally exceeded the recommended minimum

Yıldız Kabak V, et al: Turkish Version of the PedsQL at Hacettepe University

Turk J Hematol 2016;33:236-243

Table 5. Pearson’s correlation coefficients between subscale scores of the 5- to 7-year-old parent proxy-report of the Pediatric Quality of Life Inventory 3.0 Cancer Module and Pediatric Quality of Life Inventory 4.0 Generic Core Scales. PedsQL 3.0 Cancer Module, 5- to 7-year-old

PedsQL 4.0 Generic Core Scale, 5- to 7-year-old

Physical Functioning Emotional Functioning

Social Functioning

School-Related Problems

Total Score

Pain and hurt

0.455*

0.179

-0.120

0.060

0.296

Nausea

0.227

0.625**

0.399*

0.583*

0.586*

Procedural anxiety

0.197

0.418*

0.357

0.578*

0.460*

Treatment anxiety

-0.136

0.739**

0.566*

0.674*

0.468*

Worry

-0.005

0.769**

0.578*

0.701*

0.583*

Cognitive problems

0.038

0.649**

0.690**

0.706**

0.503*

Perceived physical appearance

0.103

0.713**

0.536*

0.513*

0.545*

Communication

0.154

0.604**

0.312

0.627*

0.510*

Total score

0.179

0.810**

0.562*

0.795**

0.694**

*p<0.0.5, **p<0.001. PedsQL: Pediatric Quality of Life Inventory.

Table 6. Pearson’s correlation coefficients between subscale scores of the 5-to 7- year old child self-report of the Pediatric Quality of Life Inventory 3.0 Cancer Module and Pediatric Quality of Life Inventory 4.0 Generic Core Scales. PedsQL 3.0 Cancer Module, 5- to 7-year-old

PedsQL 4.0 Generic Core Scales, 5- to 7-year-old

Physical Functioning

Emotional Functioning

Social Functioning

School-Related Problems

Total Score

Pain and hurt

0.214

0.363*

-0.024

0.233

0.244

Nausea

0.542*

0.389*

0.384

0.399

0.546*

Procedural anxiety

0.278

0.349

0.120

0.226

0.316

Treatment anxiety

0.484

0.740**

0.391*

0.588*

0.605**

Worry

0.429*

0.390*

0.484*

0.519*

0.546*

Cognitive problems

0.386*

0.368*

0.342

0.655*

0.520*

Perceived physical appearance

0.274

0.241

0.369

0.097

0.350

Communication

0.395*

0.528*

0.371

0.366

0.468*

Total score

0.586*

0.623**

0.452*

0.585*

0.681**

*p<0.0.5, **p<0.001. PedsQL: Pediatric Quality of Life Inventory.

alpha coefficient standard of 0.70 for group comparisons. Across the ages, the PedsQL 4.0 Generic Core Scales Total Score for both child self-report and parent proxy-report approached or exceeded an alpha of 0.90, recommended for individual patient analysis, making the Total Scale Score suitable as a summary score for the primary analysis of HRQOL outcome in clinical trials and other group comparisons [4]. Uneri et al. investigated the reliability and validity of the Turkish version of the PedsQL 4.0 Generic Core Scales for 5- to 7-yearold Turkish children. They found that internal consistency reliability alpha coefficients (Cronbach’s coefficient alpha) of the total scale score for parent-proxy report and for the child’s self-report of the 5- to 7-year-old age group were 0.86 and 0.80. The validity of the parent-proxy report was found to be sufficient, whereas the validity of the child self-report was low. When the concordance between the child self-report and

parent-proxy reports was analyzed, statistically significant but low correlations were found between the total scores [12]. The reason for this situation, similar to our study, may be the low number of patients included. This can be considered as a limitation of our study. In our study, we found that the Cronbach’s coefficient alpha values of the PedsQL 4.0 Generic Core Scales total score varied from 0.665 to 0.841 for 5- to 7-year-old, test-retest ICC values were 0.681-0.824 (good), and the correlations between total score and subscale scores were good to excellent. Eiser and Morse stated that, in practice, values greater than 0.50 might be considered as acceptable in chronic disease of childhood QOL measurements [30]. Thus, the reliability of the PedsQL 4.0 Generic Core Scales for 5- to 7-year-old may be considered as acceptable. For the PedsQL 4.0 Generic Core Scales the correlation between parent proxy-report and child self-report of 5- to 7-year-old was 241

Yıldız Kabak V, et al: Turkish Version of the PedsQL at Hacettepe University

significant and good (r=0.540, p=0.002). This lower correlation and internal consistency of the 5- to 7-year-old age group may be related to school functioning. In Turkey, children of this age do not go to school yet, and children with chronic diseases quit school. Therefore, most of the children in this group had difficulties understanding school functioning questions and some parents mentioned that their children were too young to understand some of the questions. Only 16 parents and children answered the school functioning questions, which might be due to misunderstanding some questions (for example, forgetting things). This indicated that not only are some modifications to the school functioning questions for children aged 5-7 years necessary, but also that the parent-proxy report and child selfreport should be applied together in this age group. The PedsQL 3.0 Cancer Module Scales internal consistency reliabilities generally exceeded the recommended minimum alpha coefficient standard of 0.70 for group comparison for child self-report for ages 8-18 years and parent proxy-report for ages 2-18 years. For self-report at ages 5-7 years, only the procedural anxiety and treatment anxiety scales met the 0.70 standard and for most scales values were in the range of 0.80 to 0.90 [4]. In our study, Cronbach’s coefficient alpha of the PedsQL 3.0 Cancer Module for 2- to 7-year-old was from 0.803 to 0.73 for the parent-proxy reports and the child self-reports (high). Test-retest ICC values were 0.877 to 0.949 (excellent) and the correlations between total score and subscale scores for 5- to 7-year-old were higher than those for 2- to 4-year-old. The Turkish version of the PedsQL 3.0 Cancer Module in children aged 2-7 years can be applied for the Turkish population. The intercorrelations between the PedsQL 3.0 Cancer Module and PedsQL 4.0 Generic Core Scales parent proxy-reports were in the range of moderate to high, except for ‘worry’ and ‘schoolrelated problems’ in 2- to 4-year-old, and except for ‘pain and hurt’ and ‘physical functioning’ in 5- to 7-year-old. In Turkey, many children between 2 and 4 years old do not go to school or kindergarten. Therefore, not all parents answered schooling questions. At these ages, on the other hand, children do not know about their disease and its treatment. In 5- to 7-yearold, subscales and their questions of the cancer module do not correlate with physical situations. According to the results of 5- to 7-year-old, while parents correlated physical dysfunctions with ‘pain and hurt’, children correlated them with ‘nausea’. In this self-report, ‘procedural anxiety’ and ‘perceived physical appearance’ do not correlate with Generic Core Scale subscales, and the ‘social functioning subscale’ of the Generic Core Scale does not correlate with Cancer Module subscales. These low correlations in subscales might be due to the small number of items that compose the subscales, the low level of schooling in these ages, or the absence of physical function subscales in the cancer module. Total scale score may be suitable as a summary 242

Turk J Hematol 2016;33:236-243

score for the primary analysis of the PedsQL Inventory in clinical trials and other group comparisons. Parent-child agreement about QOL is controversial in the literature [30]. Some studies reported high agreement, whereas others reported low agreement [31,32,33]. In our study, there was an excellent correlation between the parent-proxy report and child self-report for 5- to 7-year-old with the PedsQL 3.0 Cancer Module. Although patient self-report is considered the standard for measuring perceived HRQOL, it is the parent’s perception of the child’s HRQOL that may influence healthcare utilization [34]. In clinical practice, there may be circumstances in which the child is too young to understand the questions, or too ill and unwilling to complete an instrument. Therefore, in cases in which pediatric patients are not able to provide self-reports, reliable and valid parent proxy-report instruments are needed [35]. In conclusion, our results indicated that the Turkish versions of the PedsQL 4.0 Generic Core Scales for 5- to 7-year-old and the PedsQL 3.0 Cancer Module for 2- to 7-year-old are easy to understand, reliable, and valid instruments in the Turkishspeaking population. These instruments may be utilized as outcome measures in pediatric cancer clinical trials, research, and clinical practice for HRQOL outcome assessment. However, we suggest that the parent proxy-report and child self-report should be used together for 5- to 7-year-old. Further studies should focus on testing the responsiveness and reliability of the PedsQL in patients who continue or finish treatments and in long-term follow-up measurements. Ethics Ethics Committee Approval: The approval of the Ethic Committee of the Hacettepe University was obtained about this study (GO 14/455); Informed Consent: It was taken. Authorship Contributions Concept: Tülin Düger; Design: Mualla Çetin; Data Collection or Processing: Vesile Yıldız Kabak; Analysis or Interpretation: Yavuz Yakut, Vesile Yıldız Kabak; Literature Search: Vesile Yıldız Kabak; Writing: Tülin Düger. Conflict of interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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4. Varni JW, Burwinkle TM, Katz ER, Meeske K, Dickinson P. The PedsQL in pediatric cancer: reliability and validity of the Pediatric Quality of Life Inventory Generic Core Scales, Multidimensional Fatigue Scale, and Cancer Module. Cancer 2002;94:2090-2106. 5. Pal DK. Quality of life assessment in children: a review of conceptual and methodological issues in multidimensional health status measures. J Epidemiol Community Health 1996;50:391-396.

Yıldız Kabak V, et al: Turkish Version of the PedsQL at Hacettepe University

19. Memik NÇ, Ağaoğlu B, Coşkun A, Karakay I. Çocuklar için Yaşam Kalitesi Ölçeği’nin 8-12 yaş çocuk formunun geçerlik ve güvenirliği. Turk J Child Adolesc Ment Health 2008;15:87-98. 20. Tanir MK, Kuguoglu S. Turkish validity and reliability of a pediatric quality of life cancer module for children aged 8-13 and parents. Asian Pac J Cancer Prev 2011;12:125-130.

6. Vance YH, Morse RC, Jenney ME, Eiser C. Issues in measuring quality of life in childhood cancer: measures, proxies, and parental mental health. J Child Psychol Psychiatry 2001;42:661-667.

21. Varni JW, Limbers CA, Burwinkle TM. Impaired health related quality of life in children and adolescents with chronic conditions: a comparative analysis of 10 disease clusters and 33 disease categories/severities utilizing the PedsQL 4.0 Generic Core Scales. Health Qual Life Outcomes 2007;5:43.

7. Ji Y, Chen S, Li K, Xiao N, Yang X, Zheng S, Xiado X. Measuring health-related quality of life in children living in mainland China: feasibility, reliability and validity of the Chinese Mandarin version of PedsQL 4.0 Generic Core Scales and 3.0 Cancer Module. Health QualLife Outcomes 2011;9:103.

22. Varni JW, Katz ER, Seid M, Quiggins DJ, Freidman-Bender A, Castro CM. The Pediatric Cancer Quality of Life Inventory (PCQL). I. Instrument development, descriptive statistics, and cross-informant variance. J Behav Med 1998;21:179-204.

8. Scarpelli AC, Paiva SM, Pordeus IA, Ramos-Jorge ML, Varni JW, Allison PJ. Measurement properties of the Brazilian version of the Pediatric Quality of Life Inventory (PedsQL) cancer module scale. Health Qual Life Outcomes 2008;6:7.

23. Varni JW, Katz ER, Seid M, Quiggins DJ, Freidman-Bender A, Castro CM. The pediatric cancer quality of life inventory-32 (PCQL-32): I. Reliability and validity. Cancer 1998;82:1184-1196.

9. Eiser C, Havermans T, Craft A, Kernahan J. Development of a measure to assess the perceived illness experience after treatment for cancer. Arch Dis Child 1995;72:302-307. 10. Fenny D, Furlong W, Barr RD, Torrance GW, Resenbaum P, Weitzman S. A comprehensive multiattribute system for classifying the health status of survivors of childhood cancer. J Clin Oncol 1992;10:923-928. 11. Lau JT, Yu XN, Chu Y, Shing MM, Wong EM, Leung TF, Li CK, Fok TF, Mak WW. Validation of the Chinese version of the Pediatric Quality of Life Inventory (PedsQL) Cancer Module. J Pediatr Psychol 2010;35:99-109. 12. Uneri OS, Agaoglu B, Coskun A, Memik NC. Validity and reliability of Pediatric Quality of Life Inventory for 2-to 4-year-old and 5-to 7-year-old Turkish children. Qual Life Res 2008;17:307-315. 13. Varni JW, Seid M, Rode CA. The PedsQL: the measurement model for the Pediatric quality of life inventory. Med Care 1999;37:126-139. 14. Tsuji N, Kakee N, Ishida Y, Asami K, Tabuchi K, Nakadate H, Iwai T, Maeda M, Okamura J, Kazama T, Terao Y, Ohyama W, Yuza Y, Kaneko T, Manabe A, Kobayashi K, Kamibeppu K, Matsushima E. Validation of the Japanese version of the Pediatric Quality of Life Inventory (PedsQL) Cancer Module. Health Qual Life Outcomes 2011;9:22. 15. Parsons SK, Brown AP. Evaluation of quality of life of childhood cancer survivors: a methodological conundrum. Med Ped Oncol 1998;(Suppl 1):4653. 16. Felder-Puig R, Frey E, Proksch K, Varni JW, Gadner H, Topf R. Validation of the German version of the Pediatric Quality of Life Inventory (PedsQL) in childhood cancer patients off treatment and children with epilepsy. Qual Life Res 2004;13:223-234.

24. Beaton DE, Bombardier C, Guillemin F, Ferraz MB. Guidelines for the process of cross-cultural adaptation of self-report measures. Spine 2000;25:3186-3191. 25. Shrout PE, Fleiss JL. Intraclass correlations: uses in assessing rater reliability. Psychol Bull 1979;86:420-428. 26. PortneyLG, WatkinsMP. Foundations of Clinical Research: Application to Practice. Norwalk, CT,USA, Appleton&Lange, 1993. 27. De Jong Z, van der Heijde D, McKenna SP, Whalley D. The reliability and construct validity of the RAQoL: a rheumatoid arthritis-specific quality of life instrument. Br J Rheumatol 1997;36:878-883. 28. BellamyN. Musculoskeletal Clinical Metrology. Boston, MA, USA, Kluwer Academic, 1993. 29. Feise RJ, Menke JM. Functional rating index: a new valid and reliable instrument to measure the magnitude of clinical change in spinal conditions. Spine 2001;26:78-8. 30. Eiser C, Morse R. Quality of-life measures in chronic disease of childhood. Health Technol Assess 2001;5:1-157. 31. Varni JW, Limbers CA, Burwinkle TM. Parent proxy-report of their children’s health-related quality of life: an analysis of 13,878 parents’ reliability and validity across age subgroups using the PedsQL™ 4.0 Generic Core Scales. Health Qual Life Outcomes 2007;5:2. 32. Varni JW, Limbers CA, Burwinkle TM. How young can children reliably and validly self-report their health-related quality of life?: An analysis of 8,591 children across age subgroups with the PedsQL™ 4.0 Generic Core Scales. Health Qual Life Outcomes 2007;5:1. 33. Cremeens J, Eiser C, Blades M. Factors influencing agreement between child self-report and parent proxy reports on the Pediatric Quality of Life Inventory (PedsQL) generic core scale. Health Qual Life Outcomes 2006;4:58.

17. Chaudhry Z, Siddiqui S. Health related quality of life assessment in Pakistani paediatric cancer patients using PedsQL 4.0 generic core scale and PedsQL 3.0 cancer module. Health Qual Life Outcomes 2012;10:52.

34. Varni JW, Setoguchi Y. Screening for behavioural and emotional problems in children and adolescents with congenital or acquired limb deficiencies. Am J Dis Child 1992;146:103-107.

18. Memik NÇ, Ağaoğlu B, Coşkun A, Üneri ÖŞ, Karakata I. Çocuklar için Yaşam Kalitesi Ölçeği’nin 8-13 yaş ergen formunun geçerlik ve güvenirliliği. Turk Psikiyatri Derg 2007;18:353-363.

35. Tomlinson D, Hinds PS, Bartels U, Hendershot E, Sung L. Parent report of quality of life for pediatric patients with cancer with no realistic change of cure. J Clin Oncol 2011;29:639-645.

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BRIEF REPORT DOI: 10.4274/tjh.2015.0368 Turk J Hematol 2016;33:244-247

Results of Four-Year Rectal Vancomycin-Resistant Enterococci Surveillance in a Pediatric Hematology-Oncology Ward: From Colonization to Infection Bir Pediatrik Hematoloji-Onkoloji Ünitesinde Vankomisine Dirençli Enterokok Kolonizasyon ve Enfeksiyonu: Dört Yıllık Sürveyans Sonuçları Hacer Aktürk1, Murat Sütçü1, Ayper Somer1, Serap Karaman2, Manolya Acar1, Ayşegül Ünüvar2, Sema Anak2, Zeynep Karakaş2, Aslı Özdemir3, Kutay Sarsar4, Derya Aydın4, Nuran Salman1 1İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Infectious Diseases, İstanbul, Turkey 2İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey 3İstanbul University İstanbul Faculty of Medicine, Infection Control Committee, İstanbul, Turkey 4İstanbul University İstanbul Faculty of Medicine, Department of Clinical Microbiology, İstanbul, Turkey

Abstract Objective: To investigate the clinical impact of vancomycinresistant enterococci (VRE) colonization in patients with hematologic malignancies and associated risk factors. Materials and Methods: Patients colonized and infected with VRE were identified from an institutional surveillance database between January 2010 and December 2013. A retrospective case-control study was performed to identify the risk factors associated with development of VRE infection in VRE-colonized patients. Results: Fecal VRE colonization was documented in 72 of 229 children (31.4%). Seven VRE-colonized patients developed subsequent systemic VRE infection (9.7%). Types of VRE infections included bacteremia (n=5), urinary tract infection (n=1), and meningitis (n=1). Enterococcus faecium was isolated in all VRE infections. Multivariate analysis revealed severe neutropenia and previous bacteremia with another pathogen as independent risk factors for VRE infection development in colonized patients [odds ratio (OR): 35.4, confidence interval (CI): 1.7-72.3, p=0.02 and OR: 20.6, CI: 1.3-48.6, p=0.03, respectively]. No deaths attributable to VRE occurred. Conclusion: VRE colonization has important consequences in pediatric cancer patients. Keywords: Colonization, Infection, Pediatric malignancy, Vancomycinresistant enterococci

Öz Amaç: Hematolojik malignitesi olan pediatrik hastalarda vankomisine Öz dirençli enterokok (VDE) kolonizasyonunun klinik öneminin araştırılması ve eşlik eden risk faktörlerinin değerlendirilmesi amaçlanmıştır. Gereç ve Yöntemler: Ocak 2010-Aralık 2013 tarihleri arasında yapılan VDE sürveyans kayıtlarından faydalanılarak VRE ile kolonize ve/veya enfekte olmuş hastalar belirlenmiştir. VDE ile kolonize hastalarda VRE enfeksiyonu gelişimi ile ilişkili risk faktörlerini belirlemek amacıyla retrospektif olgu-kontrol çalışması yapılmıştır. Bulgular: Çalışma süresince yatırılan 229 hastanın 72’sinde (%31,4) rektal VRE kolonizasyonu saptanmıştır. VRE kolonize hastaların yedisinde (%9,7) kolonizasyon sonrası sistemik VRE enfeksiyonu gelişmiştir. Beş hastada bakteriyemi (n=5), bir hastada idrar yolu enfeksiyonu (n=1) ve bir hastada menenjit (n=1) gözlenmiştir. Tüm enfeksiyonlarda ilgili klinik örneklerde Enterococcus faecium üretilmiştir. Multivaryant analiz sonucunda, ciddi nötropeni ve başka bir patojene bağlı bakteriyemi öyküsü VDE kolonize hastalarda VDE enfeksiyonu gelişimi için risk faktörleri olarak bulunmuştur [odds ratio (OR): 35,4; güven aralığı (GA): 1,7-72,3; p=0,02 ve OR: 20,6; GA: 1,3-48,6; p=0,03; sırasıyla]. VDE enfeksiyonuna bağlı mortalite saptanmamıştır. Sonuç: Pediatrik kanser hastalarında VDE kolonizasyonunun önemli klinik sonuçları olduğu gözlenmiştir. Anahtar Sözcükler: Kolonizasyon, Enfeksiyon, Pediatrik malignite, Vankomisine dirençli enterokok

Address for Correspondence/Yazışma Adresi: Serap KARAMAN, M.D., Received/Geliş tarihi: November 04, 2015 İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey Accepted/Kabul tarihi: February 29, 2016 Phone : +90 212 414 20 00 E-mail : drkaramans@yahoo.com

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Aktürk H, et al: Vancomycin-Resistant Enterococci Infections in Pediatric Malignancies

Introduction Children with cancer are at high risk of developing systemic infections by the microorganisms that colonize their own intestinal system [1,2]. Vancomycin-resistant enterococci (VRE) are health care-associated opportunistic pathogens. Limited data exist on the incidence of subsequent VRE infection development among VRE-colonized pediatric cancer patients and associated risk factors, which were investigated in this study.

Materials and Methods All patients admitted to the pediatric hematology/oncology ward were sampled within 48-72 h after admission and weekly thereafter as part of institutional rectal VRE surveillance. An infection control nurse assigned by the Hospital Infection Control Committee (HICC) prospectively tracked the results of rectal surveillance and all health care-associated infections occurring in the hematology/oncology ward. VRE-colonized and VRE-infected patients were identified from the HICC surveillance database retrospectively. Detailed clinical and laboratory features of these patients were collected from their medical records. The overall rate of VRE colonization and the subsequent infection occurrence throughout the study period were determined. To identify the risk factors associated with VRE infection occurrence in a colonized patient, a retrospective case-control study was performed. Patients were defined as VRE-

colonized (VRE-C) when the culture of the rectal swab yielded VRE in the absence of any clinical specimens positive for VRE [3]. Systemic VRE infection (VRE-I) was defined as isolation of VRE from a clinical specimen together with signs and symptoms of infection. Statistical analysis was performed with SPSS 21.0 for Windows. Parameters were compared between groups with the chi-square test, Fisher exact test, or Mann-Whitney U test. Variables with a p-value of ≤0.1 in univariate analysis were fitted to perform logistic regression analysis to identify independent risk factors associated with VRE infection occurrence.

Results A total of 229 children were admitted to the hematology/ oncology ward. Fecal VRE-C was documented in 72 of these patients (31.4%). Excluding eight patients who were transferred from the pediatric intensive care unit, 89% of the patients were colonized during their stay in the hematology/oncology ward. Species determination could be performed in 32 VRE-colonized patients: Enterococcus faecium was isolated in 28 patients, Enterococcus gallinarum in 2 patients, and nontypeable Enterococcus in 2 patients. VRE-I was detected in 7 patients, all of whom were previously colonized with VRE. The overall rate of VRE-I developing in patients with VRE-C was 9.7%. VRE bacteremia was detected in five patients (6.9%). Other VRE infections were urinary

Table 1. Demographic and clinical characteristics of vancomycin-resistant enterococci-colonized patients who developed systemic vancomycin-resistant enterococci infection and those who did not.

VRE-Colonized Patients Who Did Not Develop VRE Infection (n=65)

VRE-Colonized Patients Who Developed VRE Infection (n=7)

Age, months; mean ± SD

77.7±6.1

45.2±12.9

0.16

Sex, n (%) Male Female

38 (58.5) 27 (41.5)

2 (28.6) 5 (71.4)

0.13

Underlying malignancy, n (%) ALL AML Solid tumor

33 (50.8) 15 (23.1) 17 (26.2)

2 (28.6) 3 (42.9) 2 (28.6)

0.67

Duration of time between admission and determination of VRE colonization, days; mean ± SD

27.8±3.9

25.5±7.5

0.85

Severe neutropenia (<100/mm3), n (%)

11 (16.9)

5 (71.4)

<0.001

Previous bacteremia with another pathogen, n (%)

16 (24.6)

6 (85.7)

0.001

Previous PICU admission, n (%)

12 (18.5)

3 (42.9)

0.15

Invasive procedures, n (%) CVC/port Surgery TPN

62 (95.4) 34 (35.1) 26 (40)

7 (100) 6 (54.5) 6 (85.7)

0.56 0.20 0.02

VRE: Vancomycin-resistant enterococci, ALL: acute lymphoblastic leukemia, AML: acute myeloid leukemia, PICU: pediatric intensive care unit, CVC: central venous catheter, TPN: total parenteral nutrition, SD: standard deviation.

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tract infection in one patient and meningitis in one patient. Enterococcus faecium was isolated in all patients with VRE-I. The mean duration of time from identification of VRE colonization to development of a VRE infection was 32.4±8.6 days (median: 25 days, range: 10-73 days). Univariate analysis of demographic and clinical variables associated with development of VRE-I among patients with VRE-C is presented in Table 1. Duration of neutropenia was significantly longer in patients with VRE-I than in patients with VRE-C (12.8±1.4 days vs. 38.5±7.3 days; p=0.016). Similarly, total parenteral nutritional support was found to be received for a longer time by patients with VRE-I (9.37±0.8 days vs. 15.33±5.5 days; p=0.04). Antimicrobials used among patients are shown in Table 2. Four out of seven patients were receiving a glycopeptide antibiotic when a systemic VRE infection was diagnosed. Multivariate analysis revealed severe neutropenia and history of previous bacteremia with another pathogen as independent risk factors for development of VRE infection in a VRE-colonized patient with cancer (Table 3). All VRE infections were treated with linezolid. No VREattributable deaths occurred during systemic VRE infections. However, crude mortality was higher in patients who suffered from VRE infection than those who did not (2/7 (28.6%) vs. 4/65 (6.2%), respectively; p=0.04).

Discussion Overall, 31.4% of patients admitted to our hematology/oncology ward were colonized with VRE. Colonization rates in other studies ranged from 4.7% to 38% [4,5,6,7]. Systemic VRE infections develop mostly in VRE-colonized patients [8,9], although some contrary cases may exist rarely [5,10]. In this study, about 1 in 10 VRE-colonized patients developed subsequent systemic VRE

Turk J Hematol 2016;33:244-247

infection (9.7%). Studies evaluating cancer patients reported a range of 13% to 61% rate of progression of VRE colonization to VRE bacteremia [6,10,11,12,13]. Results of univariate analysis revealed some risk factors associated with VRE-I development. One of them was severe neutropenia (<100/mm3) and longer duration of neutropenia, which was consistent with the literature [4,7,12,13]. The presence and a longer duration of total parenteral nutritional support were more likely to be present in VRE-I patients. It increases the risk of infection due to the use of an invasive central line, tendency for a longer hospitalization, and disadvantageous effects on gastrointestinal flora [14,15]. Its association with VRE infection in our univariate analysis may merely indicate illness severity of the patients, which is the main predisposing factor for VRE bacteremia [10,16]. In casecontrol studies, colonization and infection with VRE have been associated with exposure to several antibiotics, especially to glycopeptides [8,17]. In the current study, VRE-I patients had longer duration of glycopeptide treatment and more frequently received glycopeptides after detection of VRE colonization, similar to literature findings [7,10,13]. After multivariate analysis, severe neutropenia and history of previous bacteremia with another pathogen remained as independent risk factors for development of VRE infection in a colonized patient. Previous bacteremia episodes were not evaluated in similar reports. It may be proposed that a previous blood stream infection history indicates the patient’s severity of illness, which in turn increases the risk of a VRE infection. Moreover, a bacteremia episode itself might have caused clinical deterioration of the patients, predisposing them to the progression of VRE colonization to a systemic infection. It was reported that active screening leads to reduced VRE colonization, infection, and reduced costs [18]. In a comparative

Table 2. Antimicrobial use in vancomycin-resistant enterococci-colonized patients who developed systemic vancomycinresistant enterococci infection and those who did not.

VRE-Colonized Patients Who Did Not Develop VRE Infection (n=65)

VRE-Colonized Patients Who Developed VRE Infection (n=7)

Number of antibiotics administered within last 3 weeks, mean ± SD

2.9±0.1

3.4±0.2

0.23

Administration of ≥3 antibiotics, n (%)

41 (67.2)

6 (85.7)

0.31

Antimicrobial treatment, n (%) Carbapenems Glycopeptides Aminoglycosides Amphotericin B Glycopeptide treatment days, mean ± SD Administration of glycopeptides after detection of VRE colonization, n (%)

38 (58.5) 43 (66.2) 49 (75.4) 11 (16.9) 8.7±0.6 30 (46.2)

6 (85.7) 7 (100) 5 (71.4) 4 (57.1) 12.8±1.2 6 (85.7)

0.16 0.06 0.81 0.03 0.02 0.04

VRE: Vancomycin-resistant enterococci, SD: standard deviation.

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Table 3. Multivariate analysis of risk factors for development of vancomycin-resistant enterococci infection in patients colonized with vancomycin-resistant enterococci. Variables Severe neutropenia

(<100/mm3)

Previous bacteremia with another pathogen

OR (95% CI)

0.02

35.4 (1.7-72.3)

0.03

20.6 (1.3-48.6)

OR: Odds ratio, CI: confidence interval.

study, the rate of VRE infection was found to be lower in a hospital with active screening [19]. It increases awareness of the staff and leads to improvement in compliance with control measures. Thus, by detecting all carriers, onward transmission of colonization may be prevented and resultant VRE infections may be reduced. Our study revealed a remarkable incidence of VRE colonization and subsequent progression to infection, indicating the ongoing clinical importance of VRE in this high-risk population.

Conclusion This study provides the four-year incidence of VRE colonization and its clinical impact on pediatric malignancy patients. Furthermore, it evaluates risk factors for progression from colonization to infection, which may help physicians to identify VRE-colonized patients at high risk of developing systemic infection. Ethics Ethics Committee Approval: The study protocol was approved by Ethics Committee of İstanbul University, İstanbul Medical Faculty; Informed Consent: Not required. Authorship Contributions Surgical and Medical Practices: Hacer Aktürk, Murat Sütçü, Ayper Somer, Serap Karaman, Manolya Acar, Ayşegül Ünüvar, Sema Anak, Zeynep Karakaş, Aslı Özdemir, Kutay Sarsar, Derya Aydın, Nuran Salman; Concept: Hacer Aktürk, Murat Sütçü; Design: Hacer Aktürk, Murat Sütçü; Data Collection or Processing: Hacer Aktürk, Murat Sütçü, Ayper Somer, Serap Karaman, Manolya Acar, Ayşegül Ünüvar, Sema Anak, Zeynep Karakaş, Aslı Özdemir, Kutay Sarsar, Derya Aydın, Nuran Salman; Analysis or Interpretation: Hacer Aktürk, Murat Sütçü, Serap Karaman, Ayper Somer, Nuran Salman; Literature Search: Hacer Aktürk, Murat Sütçü, Ayper Somer, Serap Karaman, Manolya Acar, Ayşegül Ünüvar, Sema Anak, Zeynep Karakaş, Aslı Özdemir, Kutay Sarsar, Derya Aydın, Nuran Salman; Writing: Hacer Aktürk. 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. Schimpff SC, Young VM, Greene WH, Vermeulen GD, Moody MR, Wiernik PH. Origin of infection in acute nonlymphocytic leukemia: significance of hospital acquisition of potential pathogens. Ann Intern Med 1972;77:707-714. 2. Fainstein V, Rodriguez V, Turck M, Hermann G, Rosenbaum B, Bodey GP. Patterns of oropharyngeal and fecal flora in patients with acute leukemia. J Infect Dis 1981;144:10-18. 3. Olivier CN, Blake RK, Steed LL, Salgado CD. Risk of vancomycin-resistant Enterococcus (VRE) bloodstream infection among patients colonized with VRE. Infect Control HospEpidemiol 2008;29:404-409. 4. Ford CD, Lopansri BK, Haydoura S, Snow G, Dascomb KK, Asch J, Bo Petersen F, Burke JP. Frequency, risk factors, and outcomes of vancomycin-resistant Enterococcus colonization and infection in patients with newly diagnosed acute leukemia: different patterns in patients with acute myelogenous and acute lymphoblastic leukemia. Infect Control Hosp Epidemiol 2015;36:47-53. 5. Suntharam N, Lankford MG, Trick WE, Peterson LR, Noskin GA. Risk factors for acquisition of vancomycin-resistant enterococci among hematologyoncology patients. Diagn Microbiol Infect Dis 2002;43:183-188. 6. Worth LJ, Thursky KA, Seymour JF, Slavin MA. Vancomycin-resistant Enterococcus faecium infection in patients with hematologic malignancy: patients with acute myeloid leukemia are at high-risk. Eur J Haematol 2007;79:226-233. 7. Dubberke ER, Hollands JM, Georgantopoulos P, Augustin K, DiPersio JF, Mundy LM, Khoury HJ. Vancomycin-resistant enterococcal bloodstream infections on a hematopoietic stem cell transplant unit: are the sick getting sicker? Bone Marrow Transplant 2006;38:813-819. 8. Zaas AK, Song X, Tucker P, Perl TM. Risk factors for development of vancomycin-resistant enterococcal bloodstream infection in patients with cancer who are colonized with vancomycin-resistant enterococci. Clin Infect Dis 2002;35:1139-1146. 9. Kang Y, Vicente M, Parsad S, Brielmeier B, Pisano J, Landon E, Pettit NN. Evaluation of risk factors for vancomycin-resistant Enterococcus bacteremia among previously colonized hematopoietic stem cell transplant patients. Transpl Infect Dis 2013;15:466-473. 10. Matar MJ, Safdar A, Rolston KV. Relationship of colonization with vancomycin-resistant enterococci and risk of systemic infection in patients with cancer. Clin Infect Dis 2006;42:1506-1507. 11. Matar MJ, Tarrand J, Raad I, Rolston KV. Colonization and infection with vancomycin-resistant enterococcus among patients with cancer. Am J Infect Control 2006;34:534-536. 12. Weinstock DM, Conlon M, Iovino C, Aubrey T, Gudiol C, Riedel E, Young JW, Kiehn TE, Zuccotti G. Colonization, bloodstream infection, and mortality caused by vancomycin-resistant enterococcus early after allogeneic hematopoietic stem cell transplant. Biol Blood Marrow Transplant 2007;13:615-621. 13. Husni R, Hachem R, Hanna H, Raad I. Risk factors for vancomycin-resistant Enterococcus (VRE) infection in colonized patients with cancer. Infect Control Hosp Epidemiol 2002;23:102-103. 14. Guglielmi FW, Boggio-Bertinet D, Federico A, Forte GB, Guglielmi A, Loguercio C, Mazzuoli S, Merli M, Palmo A, Panella C, Pironi L, Francavilla A. Total parenteral nutrition-related gastroenterological complications. Dig Liver Dis 2006;38:623-642. 15. Kuehnert MJ, Jernigan JA, Pullen AL, Rimland D, Jarvis WR. Association between mucositis severity and vancomycin-resistant enterococcal bloodstream infection in hospitalized cancer patients. Infect Control Hosp Epidemiol 1999;20:660-663. 16. Dever LL, China C, Eng RH, O’Donovan C, Johanson WG Jr. Vancomycinresistant Enterococcus faecium in a Veterans Affairs Medical Center: association with antibiotic usage. Am J Infect Control 1998;26:40-46. 17. Tacconelli E, Cataldo MA. Vancomycin-resistant enterococci (VRE): transmission and control. Int J Antimicrob Agents 2008;31:99-106. 18. Humphreys H. Controlling the spread of vancomycin-resistant enterococci. Is active screening worthwhile? J Hosp Infect 2014;88:191-198. 19. Price CS, Paule S, Noskin GA, Peterson LR. Active surveillance reduces the incidence of vancomycin-resistant enterococcal bacteremia. Clin Infect Dis 2003;37:921-928.

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BRIEF REPORT DOI: 10.4274/tjh.2015.0259 Turk J Hematol 2016;33:248-250

Radiation-Induced Tumor Lysis Syndrome in Chronic Lymphocytic Leukemia Kronik Lenfositik Lösemide Radyasyon İlişkili Tümör Lizis Sendromu Ali Alkan1, Tuğçe Kütük2, Ebru Karcı1, Arzu Yaşar1, Ayşe Hiçsönmez2, Güngör Utkan1 1Ankara University Faculty of Medicine, Department of Medical Oncology, Ankara, Turkey 2Ankara University Faculty of Medicine, Department of Radiation Oncology, Ankara, Turkey

Abstract Tumor lysis syndrome (TLS) is an important oncological emergency that is usually observed with hematological malignancies and rarely with solid tumors. It can be induced either by therapy or spontaneously. Radiotherapy-induced TLS has been rarely reported in the literature. Here we present a patient with a diagnosis of metastatic prostate cancer and chronic lymphocytic leukemia complicated with TLS during palliative radiotherapy. Keywords: Chronic lymphocytic leukemia, Radiation, Tumor lysis syndrome

Introduction Tumor lysis syndrome (TLS) is one of the important oncological emergencies in oncology practice, especially in lymphoproliferative malignancies. Aggressive solid tumors with shorter doubling time can be complicated with TLS. Although it is generally triggered by cytotoxic therapy, it can also be observed spontaneously in cases of bulky tumors. Radiation as a cause of TLS has been rarely reported in the literature [1,2,3]. Here we present a patient with 2 primary malignancies experiencing TLS during palliative radiotherapy.

Case Presentation A 69-year-old male patient, without any comorbidities, presented with fullness in the left axilla. The initial examination showed lymphocytosis and lymphadenopathies in the bilateral axillary and inguinal region. There was neither hepatosplenomegaly nor pathological lymph nodes in the abdominal and thoracic cavity. Lymphocytosis and basket cells were seen in the blood smear. Excisional biopsy from the left axilla showed infiltration by CD5-positive cells. Immunohistochemistry was consistent with a diagnosis of chronic lymphocytic leukemia (CLL)

Öz Tümör lizis sendromu (TLS) sıklıkla hematolojik malignitelerde görülen, nadiren solid tümörlerde karşımıza çıkabilen bir onkolojik acildir. Tedavi ilişkili olabileceği gibi spontan olarak da ortaya çıkabilir. Radyasyon ilişkili TLS literatürde nadiren bildirilmiştir. Burada, kronik lenfositik lösemi ve prostat kanseri tanısıyla izlenen hastada palyatif radyoterapi ile indüklenen TLS olgusu sunulmaktadır. Anahtar Sözcükler: Kronik lenfositik lösemi, Radyasyon, Tümör lizis sendromu

Öz

infiltration. The bone marrow biopsies supported the diagnosis with hypercellularity and diffuse interstitial infiltration of small atypical lymphoid cells. The patient had been followed with a diagnosis of CLL for 2 years. Due to initial stage 1 disease, the patient had been periodically followed without medical therapy. In a routine outpatient visit, the clinically asymptomatic patient was evaluated with lymph node excisional biopsy due to progression of the pathological lymph nodes in order to exclude Richter’s transformation. In addition, bone marrow sampling was performed. Pathology revealed small atypical lymphoid cells consistent with an ongoing lymphoproliferative disease and infiltration of an adenocarcinoma as a second primary malignancy. Further immunohistochemical staining was inconclusive for the origin of the tumor. The age of the patient, osteoblastic metastatic lesions detected in a bone scan, and prostate-specific antigen (PSA) level of 1712 ng/mL led us to order a work-up for prostate cancer. Transrectal prostate biopsy showed prostate adenocarcinoma with a Gleason score of 10. The patient was treated with goserelin (10.8 mg SC, every 12 weeks) and bicalutamide (50 mg daily). The analysis of bone scans before hormonal therapy showed metastatic

Address for Correspondence/Yazışma Adresi: Ali ALKAN, M.D., Ankara University Faculty of Medicine, Department of Medical Oncology, Ankara, Turkey Phone : +90 312 595 73 21 E-mail : alkanali@yahoo.com

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Received/Geliş tarihi: July 02, 2015 Accepted/Kabul tarihi: March 23, 2016

Alkan A, et al: Radiation-Induced Tumor Lysis Syndrome

Turk J Hematol 2016;33:248-250

lesions in the bilateral scapula, thoracic vertebral column, pelvis, bilateral humerus, and femur. For skeletal metastatic disease, zoledronic acid (4 mg intravenous, every 4 weeks) was started and palliative radiotherapy was planned for painful metastatic lesions in the thoracic vertebrae. Radiotherapy to T3-6 and the right scapula with a total of 30 Gy divided into 10 fractions was planned. Pathological lymph nodes associated with CLL, ranging between 1 and 3 cm in diameter in the bilateral inguinal areas, axilla, neck, and hilum, were noted for followup. On day 7 of radiotherapy the patient complained about mild nausea, progressive malaise, and perioral numbness. Physical examination was normal except for pathological lymph nodes with minimal regression and paleness. The largest lymph node in the right axilla had regressed from 3 to 2 cm and the other pathological nodes were stable. The hilar fullness in chest X-ray was stable. The only medications used were drugs for prostate cancer (bicalutamide and goserelin), lansoprazole for dyspepsia, and dexamethasone (8 mg), which was planned with the radiotherapy. The laboratory evaluation revealed acute renal failure complicated with TLS (Table 1). The PSA value was stable. The patient was hospitalized and aggressively hydrated with 0.9% NaCl at 500 mL/h in the first 6 h of follow-up with monitorization of hourly urine output. In the initial evaluation, due to hypocalcemia (5.2 mg/dL) with neuromuscular symptoms and prolonged QT interval of 0.50 s, the patient was treated with calcium gluconate replacement under cardiac monitorization. Laboratory results were checked at 4-h intervals. Until we could use rasburicase, allopurinol was the initial specific therapy for TLS. The dosage was titrated according to glomerular filtration rate and a 0.2 mg/kg single dose of rasburicase could be added to therapy on day 3 of hospitalization. The patient’s symptoms and renal dysfunction progressively improved (Table 1). The patient was free of any findings of infection or septicemia. The leukocytosis was linked to the dexamethasone therapy, which

Figure 1. Digitally reconstructed radiograph of the scapula field including axillary lymph nodes, supraclavicular lymph nodes, and the upper mediastinal lymph node area.

was planned during radiotherapy for anti-edema prophylaxis. After stopping the drug, leukocytosis improved. Analysis of the dose-volume histogram showed that during palliative radiotherapy to the thoracal vertebrae, mediastinal, axillary, and supraclavicular lymph nodes were also affected with maximums of 24.8 Gy (mean: 19.8), 34.2 Gy (mean: 20), and 28.1 Gy (mean: 19.1), respectively. A digitally reconstructed radiograph of the scapula field included axillary lymph nodes, supraclavicular lymph nodes, and the upper mediastinal lymph node area (Figure 1). After improvement of TLS, reanalysis of the patient for progression of CLL showed minimal regression of the pathological mediastinal and axillary lymph nodes. There was no progression in other lymph nodes or new organomegaly. With stable clinical findings of CLL and history of lymph node resection three months ago without any aggressive form of lymphoproliferative disease or Richter transformation, rebiopsy was not planned. After six months of follow-up the patient was stable for CLL and the PSA level progressively decreased.

Discussion and Review of the Literature TLS is an oncological emergency that results from massive tumor lysis due to therapy or spontaneous bursting of tumor cells. The release of intracellular electrolytes into circulation causes an electrolyte imbalance and, as a result, acute renal failure. Intracellular nucleic acid catabolism and as a result Table 1. The laboratory parameters before and after radiotherapy and after treatment. Laboratory Parameters

Before Radiotherapy

Day 6 of Radiotherapy

After Therapy

Leukocytes (x109/L)

219

867

424

Hemoglobin (g/L)

129

Platelets (x109/L)

177

298

113

BUN (mmol/L)

11.07

27.85

9.12

Creatinine (µmol/L)

102.5

624.1

159.1

Sodium (mmol/L)

140

132

143

Potassium (mmol/L)

3.7

6.1

3.7

Calcium (mmol/L)

4.4

2.1

3.2

Phosphorus (mmol/L)

1.36

2.33

1.26

Albumin (g/L)

Corrected calcium (mmol/L)

4.6

2.5

3.8

Uric acid (µmol/L)

398.5

868.4

249.8

ALT (U/L, reference: 7-40)

AST (U/L, reference: 5-40)

LDH (U/L, reference: 210-425)

320

732

443

ALT: Alanine aminotransferase, AST: aspartate aminotransferase, BUN: blood urea nitrogen, LDH: lactate dehydrogenase.

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hyperuricemia further contribute to renal dysfunction. The clinical presentation may range from nonspecific malaise and nausea to sudden cardiac death. TLS is diagnosed by both laboratory and clinical findings. Laboratory TLS is defined as the imbalance of two or more electrolytes (hypocalcemia, hyperuricemia, hyperphosphatemia, hyperkalemia), whereas clinical TLS is defined as laboratory TLS plus one clinical finding (increased creatinine, arrhythmia/sudden death, seizure) [4,5,6,7]. In the literature, radiation has been rarely reported as a cause of TLS. Most reported cases are associated with hematological pathologies and splenic radiation is the most often related scenario [8]. Total body irradiation before allogeneic stem cell transplantation has been related to TLS [9]. TLS as a complication of radiation in solid tumors has been rarely reported. Palliation of bone metastasis from malignant melanoma [10] and prostate carcinoma [3] has been discussed. The outcomes of the patients were good and there were no mortalities as a result of complications. After the diagnosis of a second primary tumor, palliative radiotherapy was indicated due to bone pain refractory to analgesia. The radiotherapy plan for the right scapula and thoracal vertebrae involved the right axilla and mediastinum incidentally, where pathological lymph nodes had been documented. The regression of axillary pathological lymph nodes and stable PSA values after radiotherapy led us to a link between TLS and the radiated pathological lymph nodes. CLL with a leukocyte value of <50,000/µL is accepted as low risk for TLS and there is no recommended precaution except for hydration [11]. Due to the absence of any therapy for CLL, there was no precaution for TLS. Similar to the cases in the literature, our patient improved progressively with supportive care. To the best of our knowledge, our experience is the first reported TLS case related to radiation in a CLL patient. While our case gives important clues about a rare complication of radiation, it also reminds us of the importance of radiation planning in a patient with lymphadenopathies. Ethics Informed Consent: It was taken.

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Authorship Contributions Concept: Ali Alkan, Tuğçe Kütük, Ebru Karcı, Arzu Yaşar, Ayşe Hiçsönmez, Güngör Utkan; Design: Ali Alkan, Tuğçe Kütük, Ebru Karcı, Arzu Yaşar, Ayşe Hiçsönmez, Güngör Utkan; Data Collection or Processing: Ali Alkan, Tuğçe Kütük, Ebru Karcı, Arzu Yaşar, Ayşe Hiçsönmez, Güngör Utkan; Analysis or Interpretation: Ali Alkan, Tuğçe Kütük, Ebru Karcı, Arzu Yaşar, Ayşe Hiçsönmez, Güngör Utkan; Literature Search: Ali Alkan, Tuğçe Kütük, Ebru Karcı, Arzu Yaşar, Ayşe Hiçsönmez, Güngör Utkan; Writing: Ali Alkan, Tuğçe Kütük, Ebru Karcı, Arzu Yaşar, Ayşe Hiçsönmez, Güngör Utkan. 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. Rostom AY, El-Hussainy G, Allam A. Tumor lysis syndrome following hemibody irradiation for metastatic breast cancer. Ann Oncol 2000;11:13491351. 2. Noh GY, Choe DH, Kim CH, Lee JC. Fatal tumor lysis syndrome during radiotherapy for non-small-cell lung cancer. J Clin Oncol 2008;26:60056006. 3. Kaplan MA, Kucukoner M, Alpagat G, Isikdogan A. Tumor lysis syndrome during radiotherapy for prostate cancer with bone and bone marrow metastases without visceral metastasis. Ann Saudi Med 2012;32:306-308. 4. Mirrakhimov AE, Ali AM, Khan M, Barbaryan A. Tumor Lysis Syndrome in Solid Tumors: An up to Date Review of the Literature. Rare Tumors 2014;6:5389. 5. Cairo MS, Bishop M. Tumour lysis syndrome: new therapeutic strategies and classification. Br J Haematol 2004;127:3-11. 6. Howard SC, Jones DP, Pui CH. The tumor lysis syndrome. N Engl J Med 2011;364:1844-1854. 7. Wilson FP, Berns JS. Tumor lysis syndrome: new challenges and recent advances. Adv Chronic Kidney Dis 2014;21:18-26. 8. Chen SW, Hwang WS, Tsao CJ, Liu HS, Huang GC. Hydroxyurea and splenic irradiation-induced tumourlysis syndrome: a case report and review of the literature. J Clin Pharm Ther 2005;30:623-625. 9. Fleming DR, Henslee-Downey PJ, Coffey C. Radiation induced acute tumor lysis syndrome in the bone marrow transplant setting. Bone Marrow Transplant 1991;8:235-236. 10. Dar L, Gendelman O, Amital H. Tumor lysis syndrome presenting in a patient with metastatic melanoma treated with radiation therapy. Isr Med Assoc J 2014;16:456-457. 11. Cairo MS, Coiffier B, Reiter A, Younes A: TLS Expert Panel. Recommendations for the evaluation of risk and prophylaxis of tumourlysis syndrome (TLS) in adults and children with malignant diseases: an expert TLS panel consensus. Br J Haematol 2010;149:578-586.

BRIEF REPORT DOI: 10.4274/tjh.2015.0433 Turk J Hematol 2016;33:251-253

A Case of Superwarfarin Poisoning Due to Repetitive Occupational Dermal Rodenticide Exposure in a Worker Tekrarlayan Mesleksel Deri Maruziyetine Bağlı Süpervarfarin Zehirlenmesi Gelişen Bir İşçi Olgusu Zehra Narlı Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, Berna Ateşağaoğlu, Meral Beksaç Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract Superwarfarin poisoning is usually due to chronic occult small-dose exposures and can easily be misdiagnosed and may lead to serious complications. The diagnosis can be confirmed by a concordant history and analyses of blood and urine specimens with the liquid chromatography with tandem mass spectrometry (LC-MS/MS) technique. Several months of continuous treatment with high doses of daily oral vitamin K, as well as other supportive measures, are warranted, especially when repeated laboratory measurements to help predict the treatment period are not available. In this paper, a case of superwarfarin poisoning due to chronic repetitive occupational dermal exposure to commercial rodenticides is presented. Keywords: Superwarfarin, Acquired coagulopathies, Vitamin K

Introduction A 40-year-old man without any prior disease was referred to our outpatient clinics with a one-month history of recurrent epistaxis, ecchymoses, and hemarthroses. Administration of fresh frozen plasma (FFP) in the emergency room corrected the markedly abnormal international normalized ratio (INR), prothrombin time (PT), and activated partial thromboplastin time (aPTT) temporarily. His past medical and family histories were unremarkable. There were no bleeding episodes following surgery or trauma. Laboratory analyses repeatedly revealed a sustained deficiency of vitamin K-dependent clotting factors with the following results: factor II, 26.9%; factor VII, 0.1%; factor IX, 13.3%; factor X, 23.5%; factor V, 82%; factor VIII, 131%; INR, 4.04; PT, 48.5 s; aPTT, 48.3 s; D-dimer, 21 ng/mL; fibrinogen, 5.01 g/L. PT and aPTT normalized in the dilution assay, excluding any acquired inhibitors. He had a hypochromic microcytic anemia concordant with chronic blood loss. The platelet count was 164x109/L. The

Öz Süpervarfarin zehirlenmesi genellikle, fark edilmeyen küçük dozlarda kronik maruziyete bağlı olarak gelişir ve kolaylıkla yanlış tanı konarak ciddi komplikasyonlara yol açabilir. Tanı, uyumlu bir hikaye varlığında kan ve idrar örneklerinin sıvı kromatografisi ve tandem kitle spektrometresi (LC-MS/MS) tekniği ile analiz edilmesi yoluyla doğrulanabilir. Özellikle de, tedavi süresine karar verilebilmesi için tekrarlanan laboratuvar ölçümlerinin yapılmasının mümkün olmadığı durumlarda, aylar boyunca yüksek dozda günlük oral K vitamini verilmesi gereklidir. Bu yazıda, ticari rodentisitlerle tekrarlayan mesleksel deri maruziyetine bağlı Özolarak süpervarfarin zehirlenmesi gelişen bir olgu sunulmaktadır. Anahtar Sözcükler: Süpervarfarin, Kazanılmış koagülopatiler, K vitamini

absence of schistocytes in the peripheral blood smear excluded disseminated intravascular coagulation. Liver function tests and hepatobiliary ultrasound were normal. Endoscopic examinations of the gastrointestinal tract revealed normal mucosa. After excluding all other possible causes, intoxication with superwarfarin remained the diagnosis of exclusion. Blood and urine specimens were analyzed at the biochemistry laboratory of the Forensic Medicine Institute in Ankara and revealed a high blood level of superwarfarin, 32 ng/dL, and a positive urine test. Liquid chromatography with tandem mass spectrometry (LC-MS/ MS), which allows for the identification, characterization, and quantification of chemical compounds in a specimen based on their molecular masses and fragmentation patterns [1], was used in analyses. However, various special methods do exist for the simultaneous analysis of multiple hydroxycoumarin rodenticides in a specimen [2]. Our laboratory could quantify the blood level of superwarfarin, but could give only a qualitative result for the urine specimens. We were not able to identify the specific superwarfarin compound in the blood samples, as this requires some special techniques and expertise.

Address for Correspondence/Yazışma Adresi: Zehra NARLI ÖZDEMİR, M.D., Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone : +90 312 595 70 99

Received/Geliş tarihi: December 16, 2015 Accepted/Kabul tarihi: March 07, 2016

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Narlı Özdemir Z, et al: A Case of Superwarfarin Poisoning Due to Repetitive Occupational Dermal Rodenticide Exposure in a Worker

Thereafter, the patient, who had been working in a car-washing facility, admitted that he was exposed to various brands of commercial rodenticides in this facility and did not take the necessary precautions (i.e. gloves and masks) while handling and scattering the rodenticidal pellets and pastes, in contrast to his coworkers, none of whom had similar symptoms. As previously reported, long-term repetitive exposure to rodenticides via direct skin contact was the probable route of systemic absorption in this case [3].

Turk J Hematol 2016;33:251-253

superwarfarin with the longest half-life, which is brodifacoum. As we could not diagnose the patient earlier, he was given vitamin K supplementation at lower doses and shorter periods than recommended. Supplemental vitamin K doses of up to 100 mg daily for 12 months have been reported [7]. Our patient was a nonobese male, weighing 72 kg and 175 cm in height. In obese patients, considering the lipid solubility of these compounds, longer periods and higher doses may be required.

Superwarfarins are lipid-soluble long-acting coumarin derivatives available since the 1970s as strong rodenticides and they cause prolonged anticoagulation both in rats and human [4]. The second-generation anticoagulant rodenticides including brodifacoum, bromadiolone, difenacoum, difethialone, and flocoumafen have longer elimination half-lives and greater accumulation and persistence due to a greater affinity to binding sites in the liver [5]. Among these compounds, brodifacoum has been reported to have the longest plasma and liver elimination half-lives, which are 91.7 days and 307.4 days, respectively [5].

Superwarfarin poisoning due to accidental, occupational, and criminal exposures has been reported and should be suspected when an acquired bleeding disorder due to persistent deficiency of vitamin K-dependent clotting factors and a lack of sustained response to treatment with conventional doses of vitamin K are present [8]. Rodent infestation and food contamination are important sources of superwarfarin poisoning in poor socioeconomic settings. Long-term vitamin K supplementation is the recommended treatment [6]. Intravenous administration of vitamin K is often necessary initially. However, the risk of anaphylactic reactions should be considered [9].

The common commercial rodenticidal formulations marketed in Turkey are in pellet and paste forms and contain 0.005% (w/w) brodifacoum, 0.005% (w/w) difenacoum, or 0.0025% (w/w) difethialone. Given the extremely dilute concentrations of these compounds in the commercial formulations, only a huge amount of a single-dose exposure may cause important side effects. In concordance, fatalities from a single-dose exposure are reported to be very rare [2]. However, regarding the long elimination half-lives and lipid solubility of these compounds, repetitive exposures to small doses of these formulations may result in chronic poisoning.

This paper aims to refresh awareness about this clinical scenario that can easily be misdiagnosed and may lead to serious complications. Chronic occult small-dose exposures might be overlooked and should be meticulously investigated. The diagnosis can be confirmed by a concordant history and analyses of blood and urine specimens with the LC-MS/MS technique in the absence of warfarin therapy. Several months of continuous treatment with high doses of daily oral vitamin K, as well as other supportive measures, are warranted, especially when repeated laboratory measurements to help predict the treatment period are not available.

Our patient had intermittent episodes of bleeding together with prolonged PT approximately every 3-4 weeks after the outpatient clinic visit during a 6-month period. In every visit he was prescribed FFP and 10 mg of parenteral phytomenadione, and he continued taking oral phytomenadione of 10 mg daily for 7-10 days. At the end of six months his coagulation tests normalized and bleeding episodes disappeared.

Acknowledgments

The half-life of superwarfarin can be estimated by repeated measurements of blood levels in order to predict the treatment period [6]. In our case, the Forensic Medicine Institute laboratory was the only available place able to perform these kinds of analyses in the area. However, as it is subject to governmental regulations, we could not get permission for repeated measurements. Due to the presence of chronic exposure to more than one product and failure to identify the specific superwarfarin molecule with laboratory tests, it was hard to predict the outcome in our case. However, considering the long wash-out period of our case, we can speculate that the patient might have had more exposure to a product containing the 252

We are grateful for the exchange of experience provided by Dr. Reyhan Diz Küçükkaya during the diagnostic stages of this case. Ethics Ethics Committee Approval: Not applicable; Informed Consent: It was taken. Authorship Contributions Medical Practices: Zehra Narlı Özdemir, Mustafa Merter, Mehmet Gündüz, Meral Beksaç; Concept: Zehra Narlı Özdemir, Uğur Şahin, Meral Beksaç; Analysis or Interpretation: Zehra Narlı Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, Berna Ateşağaoğlu, Meral Beksaç; Literature Search: Zehra Narlı Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, Berna Ateşağaoğlu, Meral Beksaç; Writing: Zehra Narlı Özdemir, Uğur Şahin, Mustafa Merter, Mehmet Gündüz, Berna Ateşağaoğlu, Meral Beksaç.

Turk J Hematol 2016;33:251-253

NarlÄą Ă&#x2013;zdemir Z, et al: A Case of Superwarfarin Poisoning Due to Repetitive Occupational Dermal Rodenticide Exposure in a Worker

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. Vogeser M, Parhofer KG. Liquid chromatography tandem-mass spectrometry (LC-MS/MS)-technique and applications in endocrinology. Exp Clin Endocrinol Diabetes 2007;115:559-570. 2. Schaff JE, Montgomery MA. An HPLC-HR-MS-MS method for identification of anticoagulant rodenticides in blood. J Anal Toxicol 2013;37:321-325. 3. Voitsekhovskii VV, Pivnik AV, Bitiutskaia LG, Protsko TT. Acquired hemorrhagic coagulopathy due to contact with the rodenticide brodifacoum in the Nutcracker bait. Ter Arkh 2012;84:66-71.

4. Altay S, Cakmak HA, Boz GC, Koca S, Velibey Y. Prolonged coagulopathy related to coumarin rodenticide in a young patient: superwarfarin poisoning. Cardiovasc J Afr 2012;23:9-11. 5. Vandenbroucke V, Bousquet-Melou A, De Backer P, Croubels S. Pharmacokinetics of eight anticoagulant rodenticides in mice after single oral administration. J Vet Pharmacol Ther 2008;31:437-445. 6. Boettcher S, Wacker A, Moerike K, Kopp HG, Jaschonek K, Grobosch T, Kanz L, Salih HR. Acquired coagulopathy caused by intoxication with the superwarfarin-type anticoagulant rodenticide flocoumafen. Eur J Haematol 2011;86:173-175. 7. Weitzel JN, Sadowski JA, Furie BC, Moroose R, Kim H, Mount ME, Murphy MJ, Furie B. Surreptitious ingestion of a long-acting vitamin K antagonist/ rodenticide, brodifacoum: clinical and metabolic studies of three cases. Blood 1990;76:2555-2559. 8. Poovalingam V, Kenoyer DG, Mahomed R, Rapiti N, Bassa F, Govender P. Superwarfarin poisoning-a report of 4 cases. S Afr Med J 2002;92:874-876. 9. Spahr JE, Maul JS, Rodgers GM. Superwarfarin poisoning: a report of two cases and review of the literature. Am J Hematol 2007;82:656-660.

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A Primary Bone Diffuse Large B-Cell Lymphoma with Ocular Adnexal Involvement Oküler Adneks Tutulumu Olan Kemiğin Primer Diffüz Büyük B Hücreli Lenfoması Rafet Eren1, Ceyda Aslan1, Cihan Gündoğan2, Osman Yokuş1, Mehmet Hilmi Doğu1, Elif Suyanı1 1İstanbul Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 2İstanbul Training and Research Hospital, Clinic of Nuclear Medicine, İstanbul, Turkey

To the Editor, Primary bone lymphomas (PBLs) [1,2] and ocular adnexal (OA) lymphomas [3,4] are rare types of extranodal lymphomas. Coexistence of these two rare entities without lymph node infiltration has not been reported previously. A 55-year-old man presented with left hip pain without a history of trauma. His medical history and physical examination did not reveal any remarkable findings. The X-ray radiographs of the pelvis and left hip showed multiple lytic lesions. Body 18fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) demonstrated multiple osteolytic bone lesions in the left zygomatic bone, vertebral column, bilateral iliac bones, left caput femoris, and trochanter major of the femur with increased 18F-FDG uptake (SUVmax: 31) (Figure 1a). Tru-Cut biopsy of the caput femoris showed atypical lymphoid cells that were pancreatin (-), s-100 (-), CD138 (+), CD30 (-), CD20 (+), CD3 (-), CD5 (-), CD10 (-), bcl6 (+), and MUM-1 (+), consistent with diffuse large B-cell lymphoma. Laboratory data were as follows: erythrocyte sedimentation rate, 37 mm/h; lactate dehydrogenase level, 405 U/L; hemoglobin, 12 g/dL; white blood cell count, 6.74x109/L; platelet count, 250x109/L; normal liver and renal function tests. Bone marrow aspirate and core biopsy were normal. A rituximab, cyclophosphamide, doxorubicine, vincristine, and prednisolone (R-CHOP) regimen was planned. However, prior to the first chemotherapy day, the patient was admitted with swelling of the eyelids, exophthalmos, proptosis, and vision loss in his left eye that developed progressively over 3 days. Magnetic resonance imaging (MRI), which was performed two months after the initial 18F-FDG PET/CT, showed an orbital mass with diameters of 36x21x38 mm eroding the superior wall of the orbita and adjacent soft tissue (Figure 1b). After the detection of orbital involvement, investigations for central nervous system (CNS) involvement were negative. Chemotherapy treatment was commenced immediately without doing a biopsy because of the patient’s vision loss. After 4 cycles of R-CHOP chemotherapy, the patient’s left hip pain, left eye swelling, exophthalmos, and proptosis resolved completely, but his vision did not improve. Control 18F-FDG PET/CT showed marked regression of bone 254

lesions with decreased 18F-FDG uptake (SUVmax: 4.6). Orbital MRI also showed that the mass had regressed to 14x12 mm in size. After obtaining this response, 2 cycles of R-CHOP, radiotherapy to the left orbita, and two cycles of high-dose methotrexate for CNS prophylaxis were planned. Considering the extensive lytic bone lesions and recent emergence of the OA tumor, the primary site of the disease must have been the bones in the presented case. Thus, the diagnosis can be categorized as PBL with OA involvement. An orbital mass was detected two months after diagnosis by means of MRI, but not by 18F-FDG PET/CT performed at diagnosis. It would be speculative to claim that such a large mass had arisen in a two month period. Taking into account that MRI is the gold standard imaging technique in evaluation of OA tumors [3], the orbital mass, which was probably small at the beginning, could not have been noticed on 18F-FDG PET/CT. This case emphasizes that a high suspicion index of OA involvement in PBL cases with any symptoms regarding the eyes and prompt assessment of the patients with MRI might prevent undesirable consequences.

Figure 1a. 18Fluorodeoxyglucose positron emission tomography/ computed tomography image of the patient at diagnosis.

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Authorship Contributions Surgical and Medical Practices: Rafet Eren, Elif Suyanı; Concept: Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı; Design: Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı; Data Collection or Processing: Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı; Analysis or Interpretation: Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı; Literature Search: Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı; Writing: Rafet Eren, Ceyda Aslan, Cihan Gündoğan, Osman Yokuş, Mehmet Hilmi Doğu, Elif Suyanı.

Figure 1b. Magnetic resonance imaging image of orbital adnexal mass.

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

Keywords: Primary bone lymphoma, Ocular adnexal lymphoma,

References

Diffuse large B-cell lymphoma

1. Messina C, Christie D, Zucca E, Gospodarowicz M, Ferreri AJ. Primary and secondary bone lymphomas. Cancer Treat Rev 2015;41:235-246.

Anahtar Sözcükler: Primer kemik lenfoma, Oküler adneks

2. Kitsoulis P, Vlychou M, Papoudou-Bai A, Karatzias G, Charchanti A, Agnantis NJ, Bai M. Primary lymphomas of bone. Anticancer Res 2006;26:325-337.

lenfoma, Diffüz büyük B hücreli lenfoma Ethics Ethics Committee Approval: Not applicable; Informed Consent: Not applicable.

3. Ponzoni M, Govi S, Licata G, Mappa S, Giordano Resti A, Politi LS, Spagnuolo L, Di Cairano E, Doglioni C, Ferreri AJ. A reappraisal of the diagnostic and therapeutic management of uncommon histologies of primary ocular adnexal lymphoma. Oncologist 2013;18:876-884. 4. Woolf DK, Ahmed M, Plowman PN. Primary lymphoma of the ocular adnexa (orbital lymphoma) and primary intraocular lymphoma. Clin Oncol (R Coll Radiol) 2012;24:339-344.

Address for Correspondence/Yazışma Adresi: Elif SUYANI, M.D., İstanbul Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey Phone : +90 212 459 63 04 E-mail : elifsuyani@hotmail.com

Received/Geliş tarihi: December 12, 2015 Accepted/Kabul tarihi: March 25, 2016 DOI: 10.4274/tjh.2015.0424

Cerebral Sinovenous Thrombosis Mimicking Intracranial Mass İntrakranial Kitleyi Taklit Eden Serebral Sinovenöz Tromboz Derya Özyörük, Neslihan Karakurt, Arzu Yazal Erdem, Suna Emir, Bahattin Tunç, Neşe Yaralı, Namık Özbek Ankara Children’s Hematology and Oncology Training and Research Hospital, Ankara, Turkey

To the Editor, Cerebral sinovenous thrombosis is rare in children [1]. Most common signs and symptoms are seizure, lethargy, and headache [1,2,3]. We herein report a case diagnosed as cerebral sinovenous thrombosis mimicking an intracranial mass and presenting with increased intracranial pressure symptoms in an adolescent girl.

A 16-year-old girl was admitted to our emergency service with complaints of headache, vomiting, and confusion. Her past medical history was unremarkable. Physical examination revealed facial paralysis and motor weakness on the left side. Magnetic resonance (MR) imaging disclosed a mass (65x42x55 mm) in the right temporal lobe shifting the midline structure from right to left (Figure 1). Because of herniation findings, surgery was performed immediately. Histopathologic investigation showed 255

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Figure 1. Cranial magnetic resonance images of the patient demonstrate a heterogeneous parenchymal lesion with increased T2 signal intensity in the right temporal lobe causing compression of the third lateral ventricle, basal ganglion, and thalamus with sylvian fissure, sulcal effacement, shifting midline structure, and significant edema. hematoma, gliosis, inflammation, and vasculitis. The laboratory examination revealed iron deficiency anemia (white blood cells: 12x109/L, Hb: 7.2 g/dL, mean corpuscular volume: 49 fL, red cell distribution width: 20%, platelets: 570x109/L, ferritin: 4.4 ng/mL). Other hematologic tests and coagulation panels were determined to be normal. Prothrombotic markers such as protein C, protein S, antithrombin III, plasminogen, heparin cofactor II, factor VIII, factor XII, lipoprotein (a), fibrinogen, homocysteine, anticardiolipin IgG, lupus anticoagulant levels, prothrombin 20210G, and factor V Leiden were normal. Tumor markers were negative. Cranial MR venography demonstrated stasis and occlusion in the middle distal part of the transverse sinus, sigmoid sinus, and internal jugular vein. The patient had no signs of nephrotic syndrome, infection, or vasculitis. She was diagnosed with transverse sinus thrombosis and anticoagulated with low-molecular-weight heparin. Iron supplementation was administered for six months. Cases of thrombosis presenting with signs of intracranial mass are rarely reported in the literature. Kim et al. [4] reported a 54-yearold male patient who was operated on for an intracranial tumor; however, the final diagnosis was thrombus in the aneurysm of the middle cerebral artery. In another report, a 20-month-old girl presented with rapidly progressing hemiparesis on the left side. Cranial MR revealed an abscess or glial tumor-like lesion in the right thalamus; however, pathological investigation of the stereotactic biopsy specimen did not show any signs of malignancy. On day 14 of admission, control cranial MR showed thrombosis in the superior sagittal sinus and transverse sinus [5]. The association of iron deficiency anemia with sinus thrombosis has been reported previously in children [6]. Although secondary thrombocytosis has been implicated in cerebral venous sinus

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thrombosis associated with iron deficiency, two cases with normal platelet count have also been described [7]. As not all cases of iron-related thrombotic events occur in patients with concomitant high platelet counts, other pathogenic mechanisms have been proposed. One of these explanations is that iron deficiency may contribute to a hypercoagulable state by affecting blood flow patterns within the vessels because of reduced deformability and increased viscosity of microcytic red blood cells. Furthermore, anemic hypoxia secondary to iron deficiency has been suggested to precipitate situations of increased metabolic stress, in particularly in vulnerable areas of the brain supplied by end arteries [8]. In our patient, iron deficiency may also have contributed to the development of thrombosis. In conclusion, cerebral sinovenous thrombosis associated with increased intracranial pressure symptoms may mimic intracranial masses in children. Keywords: Intracranial mass, Cerebral sinovenous thrombosis, Increased intracranial pressure Anahtar Sözcükler: İntrakranial kitle, Serebral sinovenöz tromboz, Artmış intrakranial basınç

References 1. Hedlund GL. Cerebral sinovenous thrombosis in pediatric practice. Pediatr Radiol 2013;43:173-188. 2. Suppiej A, Gentilomo C, Saracco P, Sartori S, Agostini M, Bagna R, Bassi B, Giordano P, Grassi M, Guzzetta A, Lasagni D, Luciani M, Molinari AC, Palmieri A, Putti MC, Ramenghi LA, Rota LL, Sperlì D, Laverda AM, Simioni P. Stroke working group of the Italian Registry of Pediatric Thrombosis. Paediatric arterial ischaemic stroke and cerebral sinovenous thrombosis. First report from the Italian Registry of Pediatric Thrombosis (R. I. T. I., Registro Italiano Trombosi Infantili). Thromb Haemost 2015;113:1270-1277. 3. Ichord RN, Benedict SL, Chan AK, Kirkham FJ, Nowak-Göttl U. International Paediatric Stroke Study Group. Paediatric cerebral sinovenous thrombosis: findings of the International Paediatric Stroke Study. Arch Dis Child 2015;100:174-179. 4. Kim YJ, Jeun SS, Park JH. Thrombosed large middle cerebral artery aneurysm mimicking an intra-axial brain tumor: case report and review of literature. Brain Tumor Res Treat 2015;3:39-43. 5. Haug V, Linder-Lucht M, Zieger B, Korinthenberg R, Mall V, Mader I. Unilateral venous thalamic infarction in a child mimicking a thalamic tumor. J Child Neurol 2009;24:105-109. 6. Sébire G, Tabarki B, Saunders DE, Leroy I, Liesner R, Saint-Martin C, Husson B, Williams AN, Wade A, Kirkham FJ. Cerebral venous sinus thrombosis in children: risk factors, presentation, diagnosis and outcome. Brain 2005;128:477-489. 7. Kinoshita Y, Taniura S, Shishido H, Nojima T, Kamitani H, Watanebe T. Cerebral venous sinus thrombosis associated with iron deficiency: two case reports. Neurol Med Chir (Tokyo) 2006;46:589-593. 8. Franchini M, Targher G, Montagnana M, Lippi G. Iron and thrombosis. Ann Hematol 2008;87:167-173.

Address for Correspondence/Yazışma Adresi: Derya ÖZYÖRÜK, M.D., Ankara Children’s Hematology and Oncology Training and Research Hospital, Ankara, Turkey Phone : +90 505 633 52 74 E-mail : dozyoruk@yahoo.com

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Received/Geliş tarihi: January 22, 2016 Accepted/Kabul tarihi: March 21, 2016 DOI: 10.4274/tjh.2016.0038

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A Rare Cause of Unexplained Refractory Iron Deficiency Anemia: Unicentric Plasma-Cell Type Castleman’s Disease Tedaviye Dirençli Demir Eksikliği Anemisinin Nadir Bir Nedeni: Unisentrik Plazma Hücreli Tip Castleman Hastalığı Sevgi Kalayoğlu Beşışık1, İpek Yönal Hindilerden1, Fehmi Hindilerden2, İbrahim Öner Doğan3, Fatih Beşışık4 ¹İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 2İstanbul Bakırköy Sadi Konuk Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 3İstanbul University İstanbul Faculty of Medicine, Department of Pathology, İstanbul, Turkey 3İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Gastroenterohepatology, İstanbul, Turkey

To the Editor, Castleman’s disease (CD) is an uncommon benign lymphoproliferative disorder characterized by enlargement of hyperplastic lymph nodes with abnormal interfollicular vascular growth [1]. It is clinically categorized as unicentric disease or multicentric disease. Histopathologic variants of CD include hyaline-vascular type, plasma-cell (PC) type, and mixed form [2]. CD presents with features ranging from asymptomatic lymphadenopathy to systemic manifestations such as serious infections, anemia, and nerve damage [3]. In CD, hepcidin secretion induced by IL-6 is the main cause of anemia [4,5]. Anemia is more common in multicentric CD and is rarely reported in unicentric CD [3]. Few cases of unicentric PC type CD associated with iron deficiency anemia (IDA) have been reported [6,7,8,9]. To our knowledge, there is only one previous reported case of adult unicentric PC type CD located in the abdomen and presenting with IDA [9]. We describe an adult with occult unicentric PC type CD of the abdomen presenting with iron-refractory anemia (IRA) and achieving dramatic response to curative resection. A 47-year-old man was referred with a 5-year history of IRA. Blood analysis showed Hb of 8 g/dL, mean corpuscular volume of 70 fL, red cell distribution width of 17%, and platelet count of 478,000/mm3. Biochemical tests were as follows: serum ferritin, 371 µg/L; transferrin saturation, 8.6%; and erythrocyte sedimentation rate (ESR), 110 mm/h (reference range: 0-20). Serum protein electrophoresis revealed polyclonal gammopathy with gamma globulin of 2.16 g/dL. The soluble transferrin receptor/log10 ferritin index of 2.3 indicated the presence of combined IDA and anemia of inflammation (AI) [10]. Underlying chronic inflammatory diseases were excluded. Upper and lower gastrointestinal endoscopic evaluations and bone marrow examination were normal. Positron emission tomographycomputed tomography showed a soft tissue mass with diffuse fluorodeoxyglucose uptake with an SUVmax of 11.39 and a

craniocaudal length of 8 cm extending from the hepatogastric ligament and with a maximal diameter of 4.4x4.3 cm in the axial plane at the portal region. He underwent exploratory laparotomy and the mass was completely excised. Histopathological examination of the mass revealed greater retention of the nodal architecture with increased secondary lymphoid follicles, vascularization of germinal centers, and expansion of mantle zones. The interfollicular region contained sheets of CD38-positive mature-appearing plasma cells and increased postcapillary venules. Plasma cells expressed polytypic immunoglobulins, light and heavy chains (Figure 1). These findings were compatible with a diagnosis of PC type CD. Four months after complete resection, laboratory tests had completely normalized (Hb 14 g/dL, mean

Figure 1. Histopathological examination of the resected specimen. Greater retention of the nodal architecture with increased secondary lymphoid follicles, and vascularization of germinal centers (a: 40x), germinal centers fed by prominent vessels; lollipop-like appearance (b: 200x). The interfollicular region contained sheets of mature-appearing plasma cells and increased postcapillary venules (c: 400x; d: 200x). 257

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corpuscular volume 83 fL, and ESR 10 mm/h). He has been free of disease for more than 26 months since the resection.

Writing: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden, İbrahim Öner Doğan, Fatih Beşışık.

Previously reported PC type CD patients with abdominal involvement and IDA aged between 11 and 29 years [6,7,9], making our patient the oldest reported patient. PC type CD with abdominal involvement may show an indolent course, often leading to great delay in diagnosis. We report an unusual case of unicentric PC type CD in which the patient suffered from systemic manifestations of anemia for five years. After surgical resection, the anemia completely resolved. CD should be included in the differential diagnosis of chronic, unexplained inflammation associated with combined IDA and AI.

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

Keywords: Iron deficiency anemia, Unicentric plasma-cell type, Castleman’s disease Anahtar Sözcükler: Demir eksikliği anemisi, Unisentrik plazma hücreli tip, Castleman hastalığı Ethics Informed Consent: It was taken. Authorship Contributions Concept: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden, İbrahim Öner Doğan, Fatih Beşışık; Design: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden; Data Collection or Processing: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden, İbrahim Öner Doğan, Fatih Beşışık; Analysis or Interpretation: İpek Yönal Hindilerden; Literature Search: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden; Draft the Article: Sevgi Kalayoğlu Beşışık, İpek Yönal Hindilerden, Fehmi Hindilerden; Revise the Article: Sevgi Kalayoğlu Beşışık, İbrahim Öner Doğan, Fatih Beşışık;

References 1. Castleman B, Iverson L, Menendez VP. Localized mediastinal lymph node hyperplasia resembling thymoma. Cancer 1956;9:822-830. 2. Keller AR, Hochholzer L, Castleman B. Hyaline-vascular and plasma-cell types of giant lymph node hyperplasia of the mediastinum and other locations. Cancer 1972;29:670-683. 3. Bjarnason I, Cotes PM, Knowles S, Reid C, Wilkins R, Peters TJ. Giant lymph node hyperplasia (Castleman’s disease) of the mesentery. Observations on the associated anemia. Gastroenterology 1984;87:216-223. 4. Yoshizaki K, Matsuda T, Nishimoto N, Kuritani T, Taeho L, Aozasa K, Nakahata T, Kawai H, Tagoh H, Komori T. Pathogenic significance of interleukin-6 (IL-6/ BSF-2) in Castleman’s disease. Blood 1989;74:1360-1367. 5. Song SN, Tomosugi N, Kawabata H, Ishikawa T, Nishikawa T, Yoshizaki K. Down-regulation of hepcidin resulting from long-term treatment with an anti-IL-6 receptor antibody (tocilizumab) improves anemia of inflammation in multicentric Castleman disease. Blood 2010;116:3627-3634. 6. Yin L, Lu XY, Xu F, Li AJ, Wu MC. Unicentric Castleman’s disease presenting with growth retardation and iron deficiency anemia. Am J Med Sci 2012;343:426428. 7. Chandrakasan S, Bakeer N, Mo JQ, Cost C, Quinn CT. Iron-refractory microcytic anemia as the presenting feature of unicentric Castleman disease in children. J Pediatr 2014;164:928-930. 8. Suh JH, Hong SH, Jeong SC, Park CB, Choi KB, Shin OR, Choi SY. Anemia resolved by thoracoscopic resection of a mediastinal mass: a case report of unicentric Castleman’s disease. J Thorac Dis 2015;7:189-193. 9. Vinzio S, Ciarloni L, Schlienger JL, Rohr S, Méchine A, Goichot B. Isolated microcytic anemia disclosing a unicentric Castleman disease: The interleukin-6/hepcidin pathway? Eur J Intern Med 2008;19:367-369. 10. Punnonen K, Irjala K, Rajamäki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. Blood 1997;89:1052-1057.

Address for Correspondence/Yazışma Adresi: İpek YÖNAL HİNDİLERDEN, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey Phone : +90 535 687 59 92 E-mail : ipekyonal@yahoo.com.tr

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Received/Geliş tarihi: February 28, 2016 Accepted/Kabul tarihi: March 28, 2016 DOI: 10.4274/tjh.2016.0083

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0112 Turk J Hematol 2016;33:259-260

Vaginal Lymphoma: A Possible Cause of Genital Hemorrhage Vajinal Lenfoma: Olası Bir Genital Kanama Nedeni Erdoğan Nohuz1, Sharif Kullab2, Albane Ledoux-Pilon3, Cécile Moluçon-Chabrot4, Maël Albaut1, Luisa De Simone1, Xavier Durando2 1General Hospital of Thiers, Clinic of Obstetrics and Gynecology, Thiers, France 2Centre Jean Perrin, Clinic of Medical Oncology, Clermont-Ferrand, France 3Estaing University Hospital, Department of Pathology, Clermont-Ferrand, France 4Estaing University Hospital, Department of Hematology, Clermont-Ferrand, France

Figure 1. A) Grenz zone (arrows): CD20-positive immunoreactivity in neoplastic cells (25x). B: Immunohistochemical analysis of paraffinembedded sections of the mass lesion showing tumor cells expressing the CD20 molecule (400x).

A 59-year-old patient complaining of vaginal bleeding and puruloid discharge was admitted to our gynecology department. Speculum examination showed a vaginal fungating necrotic ulcerated mass. There was no palpable lymphadenopathy or hepato-splenomegaly on physical examination. Transvaginal ultrasound and abdominopelvic computed tomography demonstrated a bulky vaginal mass approximately 5x4x3 cm in diameter involving the bladder and the rectovaginal septum. With the patient’s approval, a punch biopsy was performed and failed to establish the diagnosis (small and necrotic samples that were not representative of the lesion). Histopathological diagnosis was obtained after a second biopsy performed under general anesthesia. Immunohistochemistry

showed that tumor cells were positive for CD20, CD30, MUM1, and bcl-6 and were negative for bcl-2, EMA, CD10, and CD30 (Figure 1). The patient was diagnosed with primary vaginal diffuse large-B-cell non-Hodgkin lymphoma (NHL) and underwent 8 courses of rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone immunochemotherapy. Complete remission was achieved without any relapse at 18 months’ follow-up. Primary vaginal NHL represents less than 1% of genital neoplasms [1,2,3]. Early and accurate diagnosis significantly influences the prognosis [4,5]. It should be considered in differential diagnosis of patients with vaginal bleeding. A deep biopsy may be required.

Address for Correspondence/Yazışma Adresi: Erdoğan NOHUZ, M.D. General Hospital of Thiers, Clinic of Obstetrics and Gynecology, Thiers, France E-mail : enohuz@yahoo.fr

Received/Geliş tarihi: March 09, 2015 Accepted/Kabul tarihi: November 09, 2015

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Nohuz E, et al: Vaginal Lymphoma

Keywords: Non-Hodgkin’s lymphoma, Vaginal B-cell lymphoma, Postmenopausal bleeding, Vaginal discharge Anahtar Sözcükler: Non-Hodgkin lenfoma, Vajinal B-hücreli lenfoma, Postmenopozal kanama, Vajinal akıntı Ethics Informed Consent: It was taken. Authorship Contributions Surgical and Medical Practices: Erdoğan Nohuz, Cécile Moluçon-Chabrot; Concept: Erdoğan Nohuz, Maël Albaut; Design: Erdoğan Nohuz, Sharif Kullab; Data Collection or Processing: Erdoğan Nohuz, Albane Ledoux-Pilon, Luisa De Simone; Analysis or Interpretation: Erdoğan Nohuz, Xavier Durando; Literature Search: Erdoğan Nohuz, Sharif Kullab, Albane Ledoux-Pilon, Cécile Moluçon-Chabrot, Maël Albaut, Luisa De Simone, Xavier Durando; Writing: Erdoğan Nohuz.

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References 1. Kosari F, Daneshbod Y, Parwaresch R, Krams M, Wacker HH. Lymphomas of the female genital tract: a study of 186 cases and review of the literature. Am J Surg Pathol 2005;29:1512-1520. 2. Ragupathy K, Bappa L. Primary vaginal non-Hodgkin lymphoma. J Low Genit Tract Dis 2013;17:326-329. 3. Dane C, Dane B, Kalli E, Erginbaş M, Çetin A. Primary uterine lymphoma. Turk J Haematol 2004;21:39-43. 4. Nohuz E, Albaut M, Kullab S, Fattouh M, Tamburro S, Dauplat MM, Benoît C, Durando X. What is your diagnosis? J Turk Ger Gynecol Assoc 2014;15:262-263. 5. Ameri M, Memarian A, Behtash N, Karimi Zarchi M. The importance of re-examination with deep biopsies in diagnosing cervical malignancies despite multiple negative pathology reports: A case report. Int J Surg Case Rep 2015;14:48-49.

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0143 Turk J Hematol 2016;33:261-262

Endothelial Cells, Ankaferd Hemostat, and Estradiol Endotel Hücreleri, Ankaferd ve Estradiol Yasemin Ardıçoğlu1, Nejat Akar1, İbrahim Haznedaroğlu2 1TOBB-ETÜ Hospital, Ankara, Turkey 2Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Figure 1A. A. Human umbilical vein endothelial cells adhered to each other, within seconds, just after the application of Ankaferd hemostat (5 µL).

Figure 1B. Reversible vital endothelial cell adherence/aggregation in human umbilical vein endothelial cells 24 h after application of Ankaferd hemostat (5 µL).

We previously demonstrated the effects of Ankaferd hemostat (AH) on human umbilical vein endothelial cells (HUVECs) in Turkish Journal of Hematology [1]. Endothelial cells adhered to each other within seconds and critical intracellular transcription factors were activated just after the application of AH (5 µL) to the HUVECs (Figure 1A). Rapid vital endothelial cell adherence/ aggregation is reversible and could be reversed within 24 h (Figure 1B).

cells remain unknown. Since HUVECs express estrogen receptor (ER) beta [4] and rapid HUVEC cellular responses to estrogen can be mediated by estrogen binding to ER [5], we herein aimed to investigate the estradiol content of AH. Estradiol concentration is found to be very high in AH (1452.6 pg/mL), whereas progesterone level is 6.06 ng/mL. Those results suggest novel hypotheses that shall be tested in future investigations regarding the interrelationships of vascular endothelial cells, hemostasis, and estradiol inside AH.

We further determined that the cellular effects of AH on HUVECs are clinically important in pharmacobiological hemostasis [1,2,3]. Endothelial cells are involved in a range of pathophysiological processes including hemostasis, inflammation, and angiogenesis [4], all of which are directly related to the effects of AH [1,2,3]. However, the relevant receptors on the surface of HUVECs and the molecules inside the content of AH affecting the endothelial

Keywords: Endothelium, Ankaferd, Estradiol Anahtar Sözcükler: Endotel, Ankaferd, Estradiol Authorship Contributions Concept: İbrahim Haznedaroğlu, Nejat Akar; Design: Yasemin Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu; Data Collection

Address for Correspondence/Yazışma Adresi: İbrahim HAZNEDAROĞLU, M.D., Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey E-mail : haznedar@yahoo.com, ichaznedaroglu@gmail.com

Received/Geliş tarihi: April 02, 2015 Accepted/Kabul tarihi: May 12, 2015

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or Processing: Yasemin Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu; Analysis or Interpretation: Yasemin Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu; Literature Search: Yasemin Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu; Writing: Yasemin Ardıçoğlu, Nejat Akar, İbrahim Haznedaroğlu. 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. Yılmaz E, Güleç Ş, Torun D, Haznedaroğlu İC, Akar N. The effects of Ankaferd® Blood Stopper on transcription factors in HUVEC and the erythrocyte protein profile. Turk J Hematol 2011;28:276-285.

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2. Karabiyik A, Güleç S, Yilmaz E, Haznedaroglu I, Akar N. Reversible proteaseactivated receptor 1 downregulation mediated by Ankaferd Blood Stopper inducible with lipopolysaccharides inside the human umbilical vein endothelial cells. Clin Appl Thromb Hemost 2011;17:165-170. 3. Karabıyık A, Yılmaz E, Güleç S, Haznedaroğlu I, Akar N. The Dual Diverse Dynamic Reversible Effects of Ankaferd Blood Stopper on EPCR and PAI-1 Inside Vascular Endothelial Cells with and without LPS Challenge. Turk J Hematol 2012;29:361-366. 4. Toth B, Saadat G, Geller A, Scholz C, Schulze S, Friese K, Jeschke U. Human umbilical vascular endothelial cells express estrogen receptor beta (ERβ) and progesterone receptor A (PR-A), but not ERα and PR-B. Histochem Cell Biol 2008;130:399-405. 5. Russell KS, Haynes MP, Sinha D, Clerisme E, Bender JR. Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc Natl Acad Sci U S A 2000;97:59305935.

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0384 Turk J Hematol 2016;33:263-264

An Unusual Congenital Anomaly in Fanconi Aplastic Anemia: Congenital Lobar Emphysema Fanconi Aplastik Anemisinde Nadir Bir Konjenital Anomali: Konjenital Lober Amfizem Ali Fettah1, Gökçe Pınar Reis1, Soner Sertan Kara2, Tekin Aksu3, Afak Durur Karakaya4, Mahmut Subaşı5, Atilla Çayır6 1Erzurum Regional Training and Research Hospital, Clinic of Pediatric Hematology, Erzurum, Turkey 2Erzurum Regional Training and Research Hospital Clinic of Pediatric Infectious Disease, Erzurum, Turkey 3Dr. Abdurrahman Yurtaslan Oncology Training and Research Hospital, Clinic of Pediatric Hematology, Ankara, Turkey 4Erzurum Regional Training and Research Hospital, Clinic of Radiology, Erzurum, Turkey 5Erzurum Regional Training and Research Hospital, Clinic of Thoracic Surgery, Erzurum, Turkey 6Erzurum Regional Training and Research Hospital, Clinic of Endocrinology, Erzurum, Turkey

Figure 1. Posterior anterior lung radiography imaging of the patient.

Figure 2. Computerized tomography imaging of the patient.

A 7-year-old girl who presented with epistaxis was examined due to pancytopenia. Her medical history revealed that she had respiratory distress in the neonatal period. She was born to a second-degree consanguineous marriage. Physical examination revealed short stature, microcephaly, microphthalmia, and hypo/ hyperpigmented lesions on the trunk and extremities. She did not have tachypnea, but she had decreased breathing sounds in the left lung. A laboratory work-up revealed hemoglobin of 5.4 g/dL, mean corpuscular volume of 103/fL, leukocyte count of 2.7x109/L, and thrombocyte count of 11x109/L. A chromosomal breakage test with diepoxybutane was compatible with Fanconi anemia (FA). Posteroanterior chest X-ray showed hyperinflation of the left lung (Figure 1). Chest computed tomography revealed

emphysematous changes in the upper part of the left lung, compatible with congenital lobar emphysema (Figure 2). FA is a rare autosomal recessive disorder and presents with numerous organ abnormalities, progressive cytopenia, and susceptibility to several malignancies [1,2]. Although absent lung lobes and abnormal pulmonary drainage have been reported [3], congenital lobar emphysema has not been presented as an accompanying pathology with FA. It is striking that the patient had no prominent respiratory symptoms since the newborn period. Congenital lobar emphysema’s association with FA has not been reported previously and it could be in coexistence or have an association with FA.

Address for Correspondence/Yazışma Adresi: Ali FETTAH, M.D., Erzurum Regional Training and Research Hospital, Clinic of Pediatric Hematology, Erzurum, Turkey Phone : +90 505 675 05 86 E-mail : alifettah@gmail.com

Received/Geliş tarihi: November 09, 2015 Accepted/Kabul tarihi: February 08, 2016

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Fettah A, et al: Congenital Lobar Emphysema in Fanconi Anemia

Keywords: Fanconi, Anemia, Congenital lobar emphysema Anahtar Sözcükler: Fanconi, Anemi, Konjenital lober amfizem Ethics Informed Consent: It was taken. Authorship Contributions Surgical and Medical Practices: Tekin Aksu, Afak Durur Karakaya, Mahmut Subaşı, Atilla Çayır; Concept: Ali Fettah, Gökçe Pınar Reis; Design: Ali Fettah, Gökçe Pınar Reis; Data Collection or Processing: Ali Fettah, Gökçe Pınar Reis, Soner Sertan Kara, Tekin Aksu, Afak Durur Karakaya, Mahmut Subaşı, Atilla Çayır; Analysis or Interpretation: Ali Fettah, Gökçe Pınar Reis, Soner

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Sertan Kara; Literature Search: Ali Fettah, Gökçe Pınar Reis, Soner Sertan Kara; Writing: Ali Fettah, Soner Sertan Kara. 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. Unal S, Ozbek N, Kara A, Alikaşifoğlu M, Gümrük F. Five Fanconi anemia patients with unusual organ pathologies. Am J Hematol 2004;77:50-54. 2. Auerbach AD. Fanconi anemia and its diagnosis. Mutat Res 2009;668:4-10. 3. Bessler M, Mason PJ, Daniel CL, Wilson DB. Inheritedbone marrow failure syndromes. In: Nathan DG, Oski FA (eds). Hematology of Infancy and Childhood. Philadelphia, Saunders, 2015.