Page 1

Issue 2

June 2017

80 TL

ISSN 1300-7777

Volume 34

Research Articles Poor Prognosis in Acute Myeloid Leukemia Patients with Monosomal Karyotypes Junqing Xu, et al.; Yantai, China

The Effect of Suppressed Levels of Uninvolved Immunoglobulins on the Prognosis of Symptomatic Multiple Myeloma Murat Sarı, et al.; İstanbul, Turkey

Gonadotoxic Effects of Nilotinib in Chronic Myeloid Leukemia Treatment Dose in a Mouse Model Güldane Cengiz Seval, et al.; Ankara, Turkey

Influence of Folate-Related Gene Polymorphisms on High-Dose Methotrexate-Related Toxicity and Prognosis in Turkish Children with Acute Lymphoblastic Leukemia Burcu Yazıcıoğlu, et al.; Ankara, Turkey

Association Between N363S and BclI Polymorphisms of the Glucocorticoid Receptor Gene (NR3C1) and Glucocorticoid Side Effects During Childhood Acute Lymphoblastic Leukemia Treatment Meriç Kaymak Cihan, et al.; Ankara, Turkey

Cytomegalovirus Infection and Treatment in Allogeneic Hematopoietic Stem Cell Transplantation: A Retrospective Study from a Single Institution in an Endemic Area Hsin-Chen Lin, et al.; Taichung, Taiwan

Could Neutrophil CD64 Expression Be Used as a Diagnostic Parameter of Bacteremia in Patients with Febrile Neutropenia? Nur Efe İris, et al.; İstanbul, Turkey

Cover Picture: Emre Ali Acar et al. Pathological Fracture in Odontoid Process in Multiple Myeloma

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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, Turkey rkucukkaya@hotmail.com

Associate Editors Ayşegül Ünüvar

İstanbul University, İstanbul, Turkey aysegulu@hotmail.com

Cengiz Beyan TOBB University of Economics and Technology, Ankara, Turkey cengizbeyan@hotmail.com

Hale Ören

Dokuz Eylül University, İzmir, Turkey hale.oren@deu.edu.tr

İbrahim C. Haznedaroğlu

Hacettepe University, Ankara, Turkey haznedar@yahoo.com

M. Cem Ar

İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul, Turkey mcemar68@yahoo.com

Selami Koçak Toprak

Ankara University, Ankara, Turkey sktoprak@yahoo.com

Semra Paydaş

Çukurova University, Adana, Turkey sepay@cu.edu.tr

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: 21/1, 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

Publishing Manager Sorumlu Yazı İşleri Müdürü Güner Hayri Özsan

Management Address Yayın İdare Adresi Türk Hematoloji Derneği

Publishing House / Yayınevi

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

Online Manuscript Submission

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul, Turkey Tel: +90 212 621 99 25 Faks: +90 212 621 99 27 E-posta: info@galenos.com.tr

http://mc.manuscriptcentral.com/tjh

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

Web page

Printing Date / Basım Tarihi

www.tjh.com.tr

01.05.2017

Owner on behalf of the Turkish Society of Hematology Türk Hematoloji Derneği adına yayın sahibi Ahmet Muzaffer Demir Üç ayda bir yayımlanan İngilizce süreli yayındır. International scientific journal published quarterly. Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile Turkish Journal of Hematology’nin Türk Hematoloji Derneği İktisadi İşletmesi tarafından yayınlanmasına karar vermiştir.

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Cover Picture Emre Ali Acar et al., Pathological Fracture in Odontoid Process in Multiple Myeloma Fracture line in odontoid process in 3-dimensional cervical tomography imaging.


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 Open Access Policy Turkish Journal of Hematology is an Open Access journal. This journal provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. Open Access Policy is based on the rules of the Budapest Open Access Initiative (BOAI) http://www.budapestopenaccessinitiative.org/. 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.


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

 Permissions
 Requests for permission to reproduce published material should be sent to the editorial office. Editor: Professor Dr. Reyhan 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 FOR 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 reviewing system. Review articles are solicited by the Editorin-Chief. Authors wishing to submit an unsolicited review article should contact the Editor-in-Chief prior to submission in order to screen the proposed topic for relevance and priority. The Turkish Journal of Hematology does not charge any article submission or processing charges. 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 and 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 and should not exceed 2500 words. The word count for the abstract should not exceed 300 words.

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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 www.icmje.org/downloads/coi_disclosure.pdf. Abstract and Keywords: The second page should include an abstract that does not exceed 300 words. For manuscripts sent by authors in Turkey, a title and abstract in Turkish are also required. As most readers read the abstract first, it is critically important. Moreover, as various electronic databases integrate only abstracts into their index, important findings should be presented in the abstract. Objective: The abstract should state the objective (the purpose of the study and hypothesis) and summarize the rationale for the study. Materials and Methods: Important methods should be written respectively. Results: Important findings and results should be provided here. Conclusion: The study’s new and important findings should be highlighted and interpreted. Other types of manuscripts, such as reviews, brief reports, 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 whatever 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 square brackets. 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 followed by “in press”. Examples of References: 1. List all authors Deeg HJ, O’Donnel M, Tolar J. Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure of 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.

4. Book Chapter Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin: Genetics, Pathophysiology, Clinical Management. New York, Cambridge University Press, 2000. 5. Abstract Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract). 6. Letter to the Editor Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further definition of 20q deletion in myeloid leukemia using fluorescence in situ hybridization. Blood 1994;84:2821-2823. 7. Supplement Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr Transplant 2005;9(Suppl 7):81-86. Brief Reports Abstract length: Not to exceed 150 words. Article length: Not to exceed 1200 words. Introduction: State the purpose and summarize the rationale for the study. Materials and Methods: Clearly describe the selection of the observational or experimental participants. Identify the methods and procedures in sufficient detail. Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for their use and an evaluation of their limitations. Identify all drugs and chemicals used, including generic names, doses, and routes of administration. Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify the reported findings/results. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used. Results: Present the findings/results in a logical sequence in the text, tables, and figures. Do not repeat all the findings/results in the tables and figures in the text; emphasize and/or summarize only those that are most important. Discussion: Highlight the new and important findings/results of the study and the conclusions they lead to. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by your data. Invited Review Articles Abstract length: Not to exceed 300 words.

3. Book

Article length: Not to exceed 4000 words.

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

Review articles should not include more than 100 references. Reviews should include a conclusion, in which a new hypothesis or study about the

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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 the relevant 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 to exceed 200 words. Authors can submit for consideration illustrations or photos that are 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 figures or tables. No abstract, discussion, or conclusion is 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 in 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 one author. Contributor’s Statement All submissions should contain a contributor’s statement page. Each statement should contain substantial contributions to idea and design, acquisition of data, and analysis and interpretation of findings. All

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persons designated as an author should qualify for authorship, and all those that qualify should be listed. Each author should have participated sufficiently in the work to take responsibility for appropriate portions of the text. Acknowledgments Acknowledge support received from individuals, organizations, grants, corporations, and any other source. For work involving a biomedical product or potential product partially or wholly supported by corporate funding, a note stating, “This study was financially supported (in part) with funds provided by (company name) to (authors’ initials)”, must be included. Grant support, if received, needs to be stated and the specific granting institutions’ names and grant numbers provided when applicable. Authors are expected to disclose on the title page any commercial or other associations that might pose a conflict of interest in connection with the submitted manuscript. All funding sources that supported the work and the institutional and/or corporate affiliations of the authors should be acknowledged on the title page. Ethics When reporting experiments conducted with humans indicate that the procedures were in accordance with ethical standards set forth by the committee that oversees human subject research. 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/en/30publications/10policies/b3/; “Guide for the Care and Use of Laboratory Animals” available at 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, in 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 republishing a manuscript in different languages. Salami slicing: To create more than one publication by dividing the results of a study unnecessarily. We disapprove of such unethical practices as plagiarism, fabrication, duplication, and salami slicing, as well as efforts to influence the review process with such practices as gifting authorship, inappropriate acknowledgments, and references. Additionally, authors must respect participants‘ 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 considered as previously published work. Authors in such a situation must declare this status on the first page of the manuscript and in the cover letter.

An extensive list of conversion factors can be found at https://www.unc. edu/~rowlett/units/. For more details, see http://www.amamanualofstyle. com/oso/public/jama/si_conversion_table.html.

(The COPE flowchart is available at http://publicationethics.org.)

Abbreviations and Symbols

We use iThenticate to screen all submissions for plagiarism before publication.

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.

Conditions of Publication All authors are required to affirm the following statements before their manuscript is considered: 1. The manuscript is being submitted only to The Turkish Journal of Hematology; 2. The manuscript will not be submitted elsewhere while under consideration by The Turkish Journal of Hematology; 3. The manuscript has not been published elsewhere, and should it be published in The Turkish Journal of Hematology it will not be published elsewhere without the permission of the editors (these restrictions do not apply to abstracts or to press reports for presentations at scientific meetings); 4. All authors are responsible for the manuscript’s content; 5. All authors participated in the study concept and design, analysis and interpretation of the data, and drafting or revising of the manuscript and have approved the manuscript as submitted. In addition, all authors are required to disclose any professional affiliation, financial agreement, or other involvement with any company whose product figures prominently in the submitted manuscript. Authors of accepted manuscripts will receive electronic page proofs and are responsible for proofreading and checking the entire article within two days. Failure to return the proof in two days will delay publication. If the authors cannot be reached by email or telephone within two weeks, the manuscript will be rejected and will not be published in the journal.

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

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.

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

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.

Units of Measurement

The Electronic Submission Process

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.

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.

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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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CONTENTS 126

Research Articles Poor Prognosis in Acute Myeloid Leukemia Patients with Monosomal Karyotypes Junqing Xu, Baohua Huang, Xiaoqian Liu, Yuanfeng Zhang, Yinghui Liu, Liming Chen, Yanyan Luan, Nannan Li, Xiaoxia Chu

131

The Effect of Suppressed Levels of Uninvolved Immunoglobulins on the Prognosis of Symptomatic Multiple Myeloma Murat Sarı, Selma Sarı, Meliha Nalçacı

137

Gonadotoxic Effects of Nilotinib in Chronic Myeloid Leukemia Treatment Dose in a Mouse Model Güldane Cengiz Seval, Sinan Özkavukçu, Murat Seval, Meltem Aylı

143

Influence of Folate-Related Gene Polymorphisms on High-Dose Methotrexate-Related Toxicity and Prognosis in Turkish Children with Acute Lymphoblastic Leukemia Burcu Yazıcıoğlu, Zühre Kaya, Sezen Güntekin Ergun, Ferda Perçin, Ülker Koçak, İdil Yenicesu, Türkiz Gürsel

151

Association Between N363S and BclI Polymorphisms of the Glucocorticoid Receptor Gene (NR3C1) and Glucocorticoid Side Effects During Childhood Acute Lymphoblastic Leukemia Treatment Meriç Kaymak Cihan, Halil Gürhan Karabulut, Nüket Yürür Kutlay, Hatice Ilgın Ruhi, Ajlan Tükün, Lale Olcay

159

Cytomegalovirus Infection and Treatment in Allogeneic Hematopoietic Stem Cell Transplantation: A Retrospective Study from a Single Institution in an Endemic Area Hsin-Chen Lin, Shao-Min Han, Wen-Li Hwang, Cheng-Wei Chou, Kuang-Hsi Chang, Zhi-Yuan Shi, Chieh-Lin Jerry Teng

167

Could Neutrophil CD64 Expression Be Used as a Diagnostic Parameter of Bacteremia in Patients with Febrile Neutropenia? Nur Efe İris, Taner Yıldırmak, Habip Gedik, Funda Şimşek, Demet Aydın, Naciye Demirel, Osman Yokuş

174

Brief Report

179

181

183

TET2, ASXL1, IDH1, and IDH2 Single Nucleotide Polymorphisms in Turkish Patients with Chronic Myeloproliferative Neoplasms Nur Soyer, Burçin Tezcanlı Kaymaz, Melda Cömert Özkan, Çağdaş Aktan, Ali Şahin Küçükaslan, Fahri Şahin, Buket Kosova, Güray Saydam

Images in Hematology Lack of Early Inflammation Signs of Acute Compartment Syndrome in an Immunodeficient Patient Burcu Belen, Özlem Çakıcı, Melikşah Uzakgider, Haldun Öniz, Meral Türker, Berna Atabay, Barış Malbora, Levent Karapınar Pathological Fracture in Odontoid Process in Multiple Myeloma Emre Ali Acar, Ufuk Demirci, Yüksel Pabuşçu, Hayriye Mine Miskioğlu, İsmet Aydoğdu A Rare Complication of Congenital Afibrinogenemia: Bone Cysts Ali Fettah, Dilek Gürlek Gökçebay, Vildan Çulha, Neşe Yaralı, Bahattin Tunç, Namık Özbek

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184

Letters to the Editor Disappearance of Acquired Hemophilia A after Complete Remission in a Multiple Myeloma Patient Vanessa Innao, Alessandro Allegra, Rosalba Morreale, Sabina Russo, Caterina Musolino

186

Bing-Neel Syndrome with Detectable MYD88 L265P Gene Mutation as a Late Relapse Following Autologous Hematopoietic Stem Cell Transplantation for Waldenström’s Macroglobulinemia Anna J. Kopiñska, Grzegorz Helbig, Anna Kocléga, Sławomira Kyrcz-Krzemieñ

188

Concomitant Presence of CD5-Positive Diffuse Large B-Cell Lymphoma and Monoclonal B Cells with the “CLL Immunophenotype” - Is It Richter’s Transformation? Sabina Langer, Jasmita Dass, Suchi Mittal, Shyam Aggarwal

190

Non-Leukemic Granulocytic Sarcoma Presenting with Multiple Lymphadenopathies Ayfer Gedük, Esra T. Demirsoy, Süheyla U. Bozkurt, Zafer Gülbaş, Serkan İşgören

192

A Case of Leukemia Cutis with Acute Myeloid Leukemia on Azacitidine Therapy Asude Kara, Aslı Akın Belli, Volkan Karakuş, Yelda Dere, Erdal Kurtoğlu

194

Unique Presentation of Leukemic Cutaneous CD3/TCR- Phenotype T-Cell Lymphoma with Complete Remission after Allogeneic Stem Cell Transplantation Hatice Şanlı, Bengü Nisa Akay, Seçil Saral, Aylin Okçu Heper, Pervin Topçuoğlu

196

Is the Game Over or Starting Again? The Role of the Transplant Team in Genetic Counseling for Adult Sickle Cell Disease Recipients Pelin Aytan, Çiğdem Gereklioğlu, Mahmut Yeral, Aslı Korur, Süheyl Asma, İlknur Kozanoğlu, Hakan Özdoğu, Can Boğa

197

Assessment of Quality of Life of Chronic Myeloid Leukemia Patients by Using the EORTC QLQ-C30 Mehmet Can Uğur, Yaşar Bekir Kutbay, Özge Özer Kaya, Cengiz Ceylan

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0255 Turk J Hematol 2017;34:126-130

Poor Prognosis in Acute Myeloid Leukemia Patients with Monosomal Karyotypes Monozomal Karyotipi Olan Akut Miyeloid Lösemili Hastalarda Kötü Prognoz Junqing Xu1, Baohua Huang2, Xiaoqian Liu1, Yuanfeng Zhang1, Yinghui Liu1, Liming Chen1, Yanyan Luan1, Nannan Li1, Xiaoxia Chu1 Qindao University Medical College, Affiliated Yantai Yuhuangding Hospital, Department of Hematology, Yantai, China Qindao University Medical College, Affiliated Yantai Yuhuangding Hospital, Laboratory of Hematology, Yantai, China

1 2

Abstract

Öz

Objective: This study aimed to investigate the clinical characteristics and prognostic significance of monosomal karyotypes (MKs) in patients with acute myeloid leukemia (AML).

Amaç: Bu çalışmada akut myeloid lösemi (AML) hastalarında monozomal karyotipe (MK) sahip olmanın klinik özellikleri ve prognostik önemini araştırmak hedeflenmiştir.

Materials and Methods: We retrospectively analyzed the clinical data for 498 patients with AML, of whom 233 (46.8%) had an abnormal karyotype, including 42 with MKs (8.4%) and 70 with a complex karyotype (CK) (14.1%).

Gereç ve Yöntemler: Retrospektif olarak 498 AML hastasının klinik verileri incelenmiştir. Bu hastalardan 233’ünde (%46,8) anormal karyotip saptanmış olup, bunların da 42’sinde (%8,4) MK ve 70’inde kompleks karyotip (KK) bulunmuştur.

Results: Patients with MKs were older (median age 62.5 vs. 52 years, p=0.003) and had lower median hemoglobin levels (62.5 vs. 77 g/L, p=0.009) and lower white blood cell counts (7.0×109/L vs. 11.7×109/L, p=0.008). Univariate analysis showed that patients with MKs or CKs had shorter overall survival than patients without these karyotypes (median survival time 7.3 vs. 26.3 months for MK, p<0.001, and 14.8 vs. 26.3 months for CK, p<0.001). In multivariable analysis for overall survival, MK and National Comprehensive Cancer Network prognostic group were the only significant factors.

Bulgular: Hastalardan MK’ya sahip olanlar daha yaşlı olup (ortanca 62,5 vs. 52 yıl; p=0,003), daha düşük ortanca hemoglobin düzeyine (62,5 vs. 77 g/L; p=0,009) ve daha düşük beyaz küre sayısına (7,0×109/L vs. 11,7×109/L, p=0,008) sahipti. Ünivaryant analizde MK ya da KK’ya sahip olan hastaların diğer hastalara göre toplam sağkalımı daha düşük bulundu. (MK için ortanca sağkalım süresi 7,3 vs. 26,3 ay, p<0,001 ve CK için 14,8 vs. 26,3 ay, p<0,001). Toplam sağkalım için yapılan, multivaryant analizde ise MK ve “National Comprehesnsive Cancer Network” prognostik grubu anlamlı bulunan faktörlerdi.

Conclusion: MK is an independent risk factor for poor prognosis in AML patients.

Sonuç: AML’li hastalarda MK kötü prognoza işaret eden bağımsız bir risk faktörüdür.

Keywords: Leukemia, Monosomal karyotype, Prognosis

Anahtar Sözcükler: Lösemi, Monozomal karyotip, Prognoz

Introduction Acute myeloid leukemia (AML) is a malignant clonal disease originating from myeloid hematopoietic stem/progenitor cells, with high heterogeneity in terms of clinical manifestations, histopathology, cytogenetics, molecular genetics, and immunophenotypes. Accurate evaluation of the prognosis of patients with AML is important for establishing clinical therapeutic protocols. About 50%-60% of AML patients have cytogenetic abnormalities. A complex karyotype (CK) is an independent unfavorable prognostic factor in AML patients, and recent studies

have demonstrated that patients with a monosomal karyotype (MK) have shorter survival than those with CKs [1,2,3]. However, MK has not been included in the traditional prognostic scoring systems. This study investigated the prognostic significance of MKs and CKs in a cohort of 498 patients with AML.

Materials and Methods Study Samples and Data Sources The study was approved by the Ethics Committees of Yuhuangding Hospital and was conducted according to the

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Xiaoxia CHU, M.D., Qindao University Medical College, Affiliated Yantai Yuhuangding Hospital, Department of Hematology, Yantai, China Phone : +86-535-6691999-82303 E-mail : xiaoxiachu66@126.com

126

Received/Geliş tarihi: July 05, 2016 Accepted/Kabul tarihi: October 03, 2016


Turk J Hematol 2017;34:126-130

Xu J, et al: Poor Prognosis in Acute Myeloid Leukemia Patients with Monosomal Karyotypes

guidelines of the Declaration of Helsinki. Due to recent wide application of cytogenetic analysis in our hospital, a total of 498 patients diagnosed with AML from July 2001 to July 2013 were included in this study. All patients were strictly reevaluated according to the 2008 World Health Organization criteria [4]. Standard daunomycin (DA) (DA at 45 mg/m2/day for 3 days plus cytosine arabinoside at 150 mg/m2/day for 7 days) or mitoxantrone (MA) (MA at 6-8 mg/m2/day for 3 days plus cytosine arabinoside at 150 mg/m2/day for 7 days) protocols were given to 452 patients as induction therapy. The clinical efficacy was evaluated according to the revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in AML [5]. For patients who achieved a complete response (CR), the above two regimens were continued, or regimens based on moderate-dose cytosine arabinoside at 2 g/m2/day for 3 days were used for consolidation and intensification therapies. Patients without CR received MAC (mitoxantrone at 6-8 mg/m2/day for 3 days plus cytosine arabinoside at 150 mg/m2/day for 7 days plus cyclophosphamide at 400 mg/m2/day on days 2 and 5) or FLAG (fludarabine at 30 mg/m2/day for 5 days plus cytosine arabinoside at 1-2 g/m2/day for 5 days plus recombinant granulocyte-colony stimulating factor at 300 µg/day for 5 days) as a second induction therapy. Forty-six (9.2%) patients were treated with palliative therapies, mainly including blood transfusion and hydroxycarbamide, to reduce white blood cells. All patients were followed up to 31 March 2014. The median follow-up time was 24 months (range: 2-145 months). The overall survival (OS) time was defined as the time from diagnosis to the last date of follow-up or the date of death. Cytogenetic Analysis Cytogenetic analysis was performed at first diagnosis for all patients. Bone marrow cells were collected from all patients and cultured for 24-48 h, and then used for routine preparation of slices for banding of G fragments. Karyotypes were determined according to the International System for Human Cytogenetic Nomenclature (ISHCN 2009). One abnormal clone was defined as cells with ≥20 nuclear metakinesis phases and ≥2 cells with the same chromosome increase or structural rearrangement, or ≥3 cells with the same chromosome deletion in chromosome detection. MK was defined as ≥2 distinct autosomal monosomies or one autosomal monosomy combined with an abnormal structure in one clone [1], and CK was defined as ≥3 chromosome abnormalities in one clone [6]. Statistical Analysis Clinical and laboratory parameters at diagnosis or first referral were analyzed statistically using SPSS 18.0. Numerical variables were summarized as medians and ranges. Categorical variables

were compared using chi-square statistics, and continuous variables were compared between different categories using Mann-Whitney U tests. Survival was measured from the date of diagnosis to the date of death or the last known follow-up and estimated using the Kaplan-Meier method. Survival data were compared using log-rank tests. Multivariate analysis was conducted using a Cox proportional hazards regression model. All p-values were two-tailed and statistical significance was set at p<0.05.

Results Clinical and Laboratory Characteristics of Patients with Monosomal Karyotypes Of the 498 patients with AML, 271 (54.4%) were males and 227 (45.6%) were females, with a median age of 54 years (12-89 years). Twenty-two (4.4%) patients had a history of myelodysplastic syndrome or myeloproliferative neoplasms. All patients had analyzable chromosome karyotypes. Of the 498 AML patients, 233 (46.8%) had abnormal karyotypes, including 42 with MKs (8.4% of all patients and 18.0% of those with an abnormal karyotype) and 70 with CKs (14.1% of all patients and 30.0% of those with an abnormal karyotype). In addition, of the 42 patients with MKs, 36 (85.7%) also met the criteria for CK, while 36 of the 70 patients with CKs (51.4%) also met the criteria for MK. Compared with patients without MKs, patients with MKs were older and had lower hemoglobin levels and lower white blood cell counts. Moreover, AML patients with histories of myelodysplastic syndrome or myeloproliferative neoplasms were more likely to have MKs than de novo AML patients (p=0.038). Of the 42 patients with MKs, 15 had one chromosome monosomy concomitant with structural abnormality and 27 (64.3%) had ≥2 chromosome monosomies. The most common monosomies were -7 (12 cases, 28.6%), -17 (9 cases, 21.4%), -20 (7 cases, 16.7%), and -5 (6 cases, 14.3%) (Table 1). Treatment Effect Analysis Of the 498 AML patients, 11 died of severe pre-chemotherapeutic complications, including three patients with MKs and eight patients without MKs. Thirty-five (7.1%) patients were treated with palliative therapy. Of the 452 (90.8%) AML patients treated with induction therapy, 35 (7.7%) had MKs and 417 (92.3%) were without MKs. CR was achieved in 20% of patients with MKs compared with 61.4% of patients without MKs. Patients with MKs had significantly shorter OS than those without MKs (p<0.001). There was no difference in the outcome of patients with one monosomy combined with one structural abnormality and those with 2 monosomies (p=0.226). 127


Xu J, et al: Poor Prognosis in Acute Myeloid Leukemia Patients with Monosomal Karyotypes

Turk J Hematol 2017;34:126-130

Table 1. The clinical features of patients with acute myeloid leukemia

AML, n (%)

MK-, n (%)

MK+, n (%)

p-value

No.

498

456

42

271 (54)

245 (54)

26 (62)

0.309

Median (range)

54 (12-89)

52 (12-89)

62.5 (26-87)

0.003

≤60

321 (64.5)

301 (66.0)

20 (47.6)

0.017

177 (35.5)

155 (34.0)

22 (52.4)

Median (range)

11.0 (0.19-386.5)

11.7 (0.19-386.5)

7.0 (0.32-59)

0.008

≤100

439 (88.2)

397 (87.1)

42 (100)

0.026

>100

59 (11.8)

59 (12.9)

0

Median (range)

77 (22-148)

77 (22-146)

62.5 (32-148)

0.009

≤60

119 (23.9)

99 (21.7)

20 (47.6)

0.000

>60

379 (76.1)

357 (78.3)

22 (52.4)

Median (range)

39.5 (1-627)

39 (1-627)

44.5 (4-394)

0.375

≤100

419 (84.1)

384 (84.2)

35 (83.3)

0.882

>100

79 (15.9)

72 (15.8)

7 (16.7)

LDH [U/L, median (range)]

378 (81-5986)

372 (81-5986)

474 (147-2103)

0.395

MDS/MPN history

Yes

22 (4.4)

17 (3.7)

5 (11.9)

No

476 (95.6)

439 (96.3)

37 (88.1)

CK

0.000

Yes

70 (14.1)

34 (7.5)

36 (85.7)

No

428 (85.9)

422 (92.5)

6 (14.3)

NCCN grouping

Better risk

50 (10.4)

50 (11.0)

0

Intermediate risk Poor risk

353 (70.9) 95 (19.1)

350 (76.7) 56 (12.3)

3 (7.1) 39 (92.9)

Sex Male Age, years 

>60 WBC count, x10 /L 9

Hemoglobin, g/L

Platelets, x109/L

0.038

0.000

AML: Acute myeloid leukemia, MK: monosomal karyotype: WBC: white blood cell, LDH: lactate dehydrogenase, MDS/MPN: myelodysplastic syndrome/myeloproliferative neoplasms, CK: complex karyotype, NCCN: National Comprehensive Cancer Network.

Prognostic Significance of Monosomal Karyotypes in Acute Myeloid Leukemia Patients Univariate analysis indicated that OS was poorer in patients with MKs compared with those without (median survival time 7.3 vs. 26.3 months, p<0.001) (Figure 1) and in patients with CKs compared with those without (median survival time 14.8 vs. 26.3 months, p<0.001) (Figure 2). Age (p=0.017) and National Comprehensive Cancer Network (NCCN) prognostic grouping (p<0.001) were also identified as prognostic factors in AML patients. CK, MK, and NCCN prognostic group were included in 128

a Cox regression risk model analysis, which identified MK as a prognostic factor independent of NCCN prognostic grouping in AML patients (Table 2). The results indicated that MK, rather than CK, was a risk factor for poor prognosis in AML patients.

Discussion Breems et al. [1] previously showed that AML patients aged 15-60 years with MKs had a poorer prognosis than those with CKs, with a 4-year OS rate of only 4%. In 2010, the Southwest Oncology Group found a similar 4-year OS rate of 3% for


Xu J, et al: Poor Prognosis in Acute Myeloid Leukemia Patients with Monosomal Karyotypes

Turk J Hematol 2017;34:126-130

Figure 1. Survival curves of patients with and without monosomal karyotypes. The overall survival was poorer in patients with monosomal karyotypes than those without (median survival time 7.3 vs. 26.3 months, p<0.001).

Figure 2. Survival curves of patients with and without complex karyotypes. The overall survival was poorer in patients with complex karyotypes than those without (median survival time 14.8 vs. 26.3 months, p<0.001).

MK: Monosomal karyotype.

CK: Complex karyotype.

Table 2. Analysis results using a Cox regression risk model in patients with acute myeloid leukemia. Factors

HR

95% CI

p-value

NCCN prognostic group

1.334

1.124-1.583

0.001

MK (Y/N)

2.630

1.847-3.743

0.000

CK (Y/N)

0.383

Age (≤60 years/>60 years)

0.285

AML: Acute myeloid leukemia, HR: hazard ratio, MK: monosomal karyotype, CK: complex karyotype, NCCN: National Comprehensive Cancer Network.

patients with MKs among 1344 AML patients aged 16-88 years [7]. The results of the current study are consistent with those previous reports, with a median survival time and 3-year OS rate for MK+ AML patients of 7.3 months and 6.2%, respectively. The incidence of MKs in this study was 8.4%, which was consistent with the 9%-15% reported in other studies [1,2,8,9,10,11]. AML patients with MKs in this study were older and had lower white blood cell counts and hemoglobin levels than those without MKs, consistent with the results of Kayser et al. [2] and in line with the age-related increase in cytogenetic abnormalities. However, the mechanisms responsible for MKs are still unclear, though they may be associated with deletions or mutations in potential multiple drug resistance genes and TP53 [12,13,14]. MK + AMLs were significantly associated with TP53 alterations, which appear to be one molecular basis for this purely descriptive cytogenetic subset [13].

Patients with MKs have a poor response to routine chemotherapy, a higher recurrence rate, and shorter long-term survival. However, some studies found that high-dose cytosine arabinoside-based chemotherapeutic regimens could improve long-term survival in MK + AML patients to some extent [15,16]. Fang et al. [17] also suggested that allogeneic hematopoietic stem cell transplantation could increase the 4-year diseasefree survival rate of MK + AML patients to 25%. Although this was still lower than the 56% reported for MK - AML patients, it was obviously higher than the 3-9% for patients receiving chemotherapy alone. Subsequent studies also confirmed that transplantation could improve long-term survival in MK + AML patients, but it was associated with disadvantages such as a high recurrence rate and short median time to recurrence [2,18,19].

Conclusion In conclusion, this study confirmed that MK was a poor prognostic factor in patients with AML independent of age, CK, and NCCN prognostic grouping. When combined with cytogenetics and NCCN prognostic grouping, MK status could further improve the prognostic classification accuracy in patients with AML. Ethics Ethics Committee Approval: The study was approved by the Ethics Committees of Yuhuangding Hospital. Authorship Contributions Concept: X.C., J.X.; Design: X.C., J.X. B.H.; Data Collection or Processing: J.X., B.H., X.L., Y.Z., Y.L., L.C., Y.L., N.L., X.C.; Analysis or 129


Xu J, et al: Poor Prognosis in Acute Myeloid Leukemia Patients with Monosomal Karyotypes

interpretation: J.X., X.L.; Literature Search: J.X., B.H., X.L., Y.Z., Y.L., L.C., Y.L., N.L., X.C.; Writing: J.X., B.H., X.L., Y.Z., Y.L., L.C., Y.L., N.L., X.C. Conflict of Interest: No conflict of interest was declared by the authors. Financial Disclosure: This research was supported by the Natural Science Foundation of Shandong Province (No: ZR2015HL035, ZR2015HL074), Beijing Medical Award Foundation (YJHYXKYJJ-105), Yantai Technology Development Projects (No: 2014WS024), and Youth Foundation of Yantai Yuhuangding Hospital (No: 201404, 201405).

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Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008;358:1909-1918. 9. Radmacher MD, Marcucci G, Ruppert AS, Mrózek K, Whitman SP, Vardiman JW, Paschka P, Vukosavljevic T, Baldus CD, Kolitz JE, Caligiuri MA, Larson RA, Bloomfield CD; Cancer and Leukemia Group B. Independent confirmation of a prognostic gene-expression signature in adult acute myeloid leukemia with a normal karyotype: a Cancer and Leukemia Group B study. Blood 2006;108:1677-1683. 10. Perrot A, Luquet I, Pigneux A, Mugneret F, Delaunay J, Harousseau JL, Barin C, Cahn JY, Guardiola P, Himberlin C, Recher C, Vey N, Lioure B, OjedaUribe M, Fegueux N, Berthou C, Randriamalala E, Béné MC, Ifrah N, Witz F; Groupe Ouest-Est des Leucémies Aiguës et Maladies du Sang. Dismal prognostic value of monosomal karyotype in elderly patients with acute myeloid leukemia: a GOELAMS study of 186 patients with unfavorable cytogenetic abnormalities. Blood 2011;118:679-685. 11. Yang XF, Sun AN, Yin J, Cai CS, Tian XP, Qian J, Chen SN, Wu DP. Monosomal karyotypes among 1147 Chinese patients with acute myeloid leukemia: prevalence features and prognostic impact. Asian Pacific J Cancer Prev 2012;13:5421-5426. 12. Ahn HK, Jang JH, Kim K, Kim HJ, Kim SH, Jung CW, Kim DH. Monosomal karyotype in acute myeloid leukemia predicts adverse treatment outcome and associates with high function multidrug resistance activity. Am J Hematol 2012;87:37-41. 13. Rücker FG, Schlenk RF, Bullinger L, Kayser S, Teleanu V, Kett H, Habdank M, Kugler CM, Holzmann K, Gaidzik VI, Paschka P, Held G, von Lilienfeld-Toal M, Lübbert M, Fröhling S, Zenz T, Krauter J, Schlegelberger B, Ganser A, Lichter P, Döhner K, Döhner H. TP53 alterations in acute myeloid leukemia with complex karyotype correlate with specific copy number alterations, monosomal karyotype, and dismal outcome. Blood 2012;119:2114-2121. 14. Gaillard JB, Chiesa J, Reboul D, Arnaud A, Brun S, Donadio D, Exbrayat C, Wickenhauser S, Bourquard P, Jourdan E, Lavabre-Bertrand T. Monosomal karyotype routinely defines a poor prognosis subgroup in acute myeloid leukemia and is frequently associated with TP53 deletion. Leuk Lymphoma 2012;53:336-337. 15. Löwenberg B, Pabst T, Vellenga E, van Putten W, Schouten HC, Graux C, Ferrant A, Sonneveld P, Biemond BJ, Gratwohl A, de Greef GE, Verdonck LF, Schaafsma MR, Gregor M, Theobald M, Schanz U, Maertens J, Ossenkoppele GJ; Dutch-Belgian Cooperative Trial Group for Hemato-Oncology (HOVON) and Swiss Group for Clinical Cancer Research (SAKK) Collaborative Group. Cytarabine dose for acute myeloid leukemia. N Engl J Med 2011;364:10271036. 16. Medeiros BC, Othus M, Appelbaum FR. Cytarabine dose for acute myeloid leukemia. N Engl J Med 2011;364:2167-2168. 17. Fang M, Storer B, Estey E, Othus M, Zhang L, Sandmaier BM, Appelbaum FR. Outcome of patients with acute myeloid leukemia with monosomal karyotype who undergo hematopoietic cell transplantation. Blood 2011;118:1490-1494. 18. Oran B, Dolan M, Cao Q, Brunstein C, Warlick E, Weisdorf D. Monosomal karyotype provides better prognostic prediction after allogeneic stem cell transplantation in patients with acute myelogenous. Biol Blood Marrow Transplant 2011;17:356-364. 19. Yanada M, Kurosawa S, Yamaguchi T, Yamashita T, Moriuchi Y, Ago H, Takeuchi J, Nakamae H, Taguchi J, Sakura T, Takamatsu Y, Waki F, Yokoyama H, Watanabe M, Emi N, Fukuda T. Prognosis of acute myeloid leukemia harboring monosomal karyotype in patients treated with or without allogeneic hematopoietic cell transplantation after achieving complete remission. Haematologica 2012;97:915-918.


RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0161 Turk J Hematol 2017;34:131-136

The Effect of Suppressed Levels of Uninvolved Immunoglobulins on the Prognosis of Symptomatic Multiple Myeloma Hastalıkla İlişkili Olmayan İmmünglobulin Düzeylerindeki Baskılanmanın Multipl Miyelomda Prognoz Üzerine Etkisi Murat Sarı, Selma Sarı, Meliha Nalçacı İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, İstanbul, Turkey

Abstract

Öz

Objective: The majority of multiple myeloma (MM) patients have high levels of monoclonal immunoglobulin in the serum and/or urine and suppressed levels of the uninvolved immunoglobulins. The prognostic significance of this phenomenon has not been assessed sufficiently. In this study, our aim is to evaluate the prognostic significance of uninvolved immunoglobulin suppression measured by nephelometry in patients with new symptomatic MM and the association with other features of the disease. Materials and Methods: Between August 2003 and February 2015, 137 patients who were referred for the treatment of newly diagnosed symptomatic myeloma to the Hematology Department polyclinics of the İstanbul University İstanbul Faculty of Medicine were prospectively included and had available pretreatment immunoglobulin levels measured by nephelometry. Results: Suppression of at least one uninvolved immunoglobulin was observed in 87% of patients and this situation was slightly more common in patients with immunoglobulin A myeloma but had no statistical significance (p>0.05). Uninvolved immunoglobulin suppression was also more common among patients who had bone marrow plasma cell infiltration of ≥40% and presented with anemia and hypercalcemia (p<0.05). The overall survival time was shorter in patients with positive calcium-renal-anemia-bone criteria and International Staging System stage 3 compared with others (p<0.05). Factors that were independently associated with inferior survival in the multivariate analysis included patients with estimated glomerular filtration rate of <60 mL/min, age of >65 years, lactate dehydrogenase of >300 IU/L, bone marrow plasma cells of ≥40%, and β2-microglobulin of >3.5 mg/dL (p<0.05). Conclusion: In this study, 13.1% of MM patients had preserved levels of uninvolved immunoglobulins. We observed that patients who had preserved uninvolved immunoglobulin levels had better treatment responses and better pathologic signs, but statistical significance could not be shown. Conversely, patients with suppression of even one of the uninvolved immunoglobulins had a shorter survival, but similarly, statistical significance could not be shown. Keywords: Multiple myeloma, Prognostic factors, Serum immunoglobulins, Nephelometric measurement, M-protein

Amaç: Multipl miyelom (MM) tanılı hastaların büyük çoğunluğunda serum ve/veya idrarda yüksek seviyelerde saptanan monoklonal immünglobulinler mevcut iken hastalıkla ilişkili olmayan immünglobulin düzeylerinde ise baskılanma mevcuttur. MM’de bu fenomenin prognostik önemi yeteri kadar değerlendirilmemiştir. Biz bu çalışmada, nefelometrik ölçümler ile belirlenmiş, hastalıkla ilişkili olmayan immünglobulin düzeylerindeki baskılanmanın yeni tanı semptomatik MM hastalarında prognoz ve hastalığın diğer özellikleri ile ilişkisini incelemeyi amaçladık. Gereç ve Yöntemler: Ağustos 2003 ve Şubat 2015 tarihleri arasında İstanbul Üniversitesi İstanbul Tıp Fakültesi, İç Hastalıkları Anabilim Dalı, Hematoloji Bilim Dalı Polikliniği’ne yeni semptomatik miyelom tanısı ile başvuran 137 hasta retrospektif olarak çalışmaya alındı. Bu hastaların hepsinde tedavi öncesi nefelometrik yöntemler ile bakılmış immünglobulin düzeyleri mevcuttu. Bulgular: Hastaların %87’sinde hastalıkla ilişkili olmayan immünglobulin düzeylerinden en az birinde baskılanma mevcuttu ve immünglobulin A miyelom tanılı hastalarda istatistiksel olarak anlamlı olduğu saptanamasa da bu durum daha sık görüldü. Kemik iliği plazma hücre infiltrasyonu ≥%40 bulunan, anemi ve hiperkalsemi ile başvuran hastalarda hastalıkla ilişkili olmayan immünglobulin düzeylerinde baskılanma daha sık görüldü (p<0,05). Hiperkalsemi, böbrek yetmezliği, anemi, kemik hastalığı kriterleri pozitif olan ve Uluslararası Evreleme Sistemi evre 3 hastalığı olan hastaların diğerlerine göre genel sağkalımı daha kısa idi (p<0,05). Çoklu değişkenli analizler sonucunda; tahmini glomerüler filtrasyon hızı <60 mL/dk, yaş >65, laktat dehidrogenaz >300 IU/L, kemik iliği plazma hücre infiltrasyon oranı ≥%40 ve β2mikroglobulin >3,5 mg/dL gibi faktörlerin sağkalım açısından olumsuz birer faktörler olduğu ortaya çıktı (p<0,05). Sonuç: Bu çalışmada hastaların %13,1’inde immünglobulin düzeylerinin korunmuş olduğu görüldü. İmmünglobulin düzeyi korunmuş hastaların daha iyi tedaviye yanıt oranlarına ve daha iyi patolojik bulgulara sahip olduğu görüldü ama immünglobulin düzey korunmasının hastalar açısından bağımsız olumlu bir prognostik faktör olduğu gösterilemedi. Aynı şekilde immünglobulin baskılanması olan hastaların yaşam süresi daha kısa bulundu, ancak bu da bağımsız olumsuz bir risk faktörü olarak belirlenemedi. Anahtar Sözcükler: Multipl miyelom, Prognostik faktörler, Serum immünglobulin, Nefelometrik ölçüm, M-protein

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Murat SARI, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, İstanbul, Turkey Phone : +90 543 338 93 39 E-mail : drmuratsari@gmail.com

Received/Geliş tarihi: April 29, 2016 Accepted/Kabul tarihi: October 24, 2016

131


Sarı M, et al: Uninvolved Immunoglobulin Suppression and Prognosis of Myeloma

Introduction Excessive amounts of a monoclonal immunoglobulin (Ig) or parts of Igs are produced and secreted in multiple myeloma (MM) [1]. Around 97% of patients with MM have high levels of these monoclonal proteins (M-proteins), which can be detected through protein electrophoresis in serum and/or urine. Quantification of Igs with nephelometry is extensively used and has been validated as a method of assessment of Ig levels. Current studies have shown that suppression of uninvolved Igs in monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM increases the risk of progression to symptomatic MM [2,3,4,5]. However, there are limited data and studies on the prognostic significance of uninvolved Ig suppression in patients with symptomatic MM. Lately, novel immunoassays that measure serum concentrations of the Ig heavy-chain/light-chain subsets IgG kappa, IgG lambda, IgA kappa, and IgA lambda have been developed; this method identifies suppression of uninvolved Ig of the same isotype as the tumor and shows prognostic significance in patients MM [6]. However, this method is not widely available yet. In our study, we aimed to assess the prognostic significance and association of the suppression of uninvolved Igs with other features of the disease, as measured using nephelometry.

Materials and Methods Patients and Follow-Up Between August 2003 and February 2015, 137 patients who were referred to the Hematology Department polyclinics of the İstanbul University İstanbul Faculty of Medicine for the treatment of newly diagnosed symptomatic myeloma were prospectively included and had available pretreatment (before initiation of any antimyeloma therapy and within 1 month of the diagnosis of MM) Ig levels measured by nephelometry. Biochemical tests of patients were conducted at the Central Clinical Biochemistry Laboratory of the İstanbul University İstanbul Faculty of Medicine Hospital (Roche Modular P chemistry analyzer). In this retrospective analysis, which includes a 12-year period, only cut-off values of lactate dehydrogenase (LDH) were changed due to changing of the analyzer (before reference values were 230-420 IU/L; now they are 135-250 IU/L). Some patients were treated with classic chemotherapy as a first-line therapy. Vincristine, adriamycin, and dexamethasone or melphalanprednisolone regimens were used as classical chemotherapy. The majority of the patients were treated with novel agents that were bortezomib-based regimens as a first-line therapy. Deceased patients were identified using the Republic of Turkey Ministry of Health Mortality Reporting System, outpatient file records, and the epicrisis, and final statuses of surviving patients were confirmed by phone. 132

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Suppression of Igs was defined as a reduction of an uninvolved Ig (IgM and IgA levels in the case of IgG myeloma) below the lower normal limit, which for IgG was <700 mg/dL, for IgA was <70 mg/dL, and for IgM was <40 mg/dL. Renal function was assessed using the estimated glomerular filtration rate (eGFR), which was calculated using the modified Modification of Diet in Renal Disease formula and serum creatinine levels. Hypercalcemia was defined as a corrected serum calcium level of ≥11.5 mg/dL, and Hb of <10g/dL was accepted as anemia. The International Myeloma Working Group criteria were used for assessing response to treatment [7]. For the purpose of the current analysis, complete response was defined as confirmed negative immunofixation of serum and urine. Very good partial response included patients with ≥90% reduction of the M-spike and urine M-spike of <100 mg/day, as well as patients with no paraprotein in serum or urine electrophoresis but with positive immunofixation. A partial response was defined as a ≥50% decrease in serum M-protein concentration and a ≥90% decrease in urine M-protein excretion. Statistical Analysis SPSS 17.0 was used for the statistical analysis of this study. Comparisons for categorical variables among different groups were made using the chi-square test and Fisher’s exact test when appropriate. Overall survival (OS) was measured from the date of treatment initiation until the date of death or date of last followup. Progression-free survival (PFS) was calculated from the date of initiation of therapy until the date of the first evidence of disease progression or death. Patients without evidence of progressive disease were censored at the date of last followup. Time to the realization of the event showed the treatment results (time-to-event), Kaplan-Meier curves were plotted, and comparisons among groups were made using the log-rank test. For the multivariate analysis, factors associated with time-toevent were introduced into a Cox proportional hazards model. Values of p<0.05 were considered statistically significant.

Results We evaluated 137 patients with newly diagnosed symptomatic MM. Table 1 shows the disease features of the patients with suppressed uninvolved Igs and of those with preserved uninvolved Igs. The median age of the whole study group was 62 years (range: 26-97 years). Suppression of at least one uninvolved Ig was observed in 87% of patients (only 13% of all patients had a preserved uninvolved Ig), and at least two suppressed uninvolved Igs were found in 69% of the patients. Suppression of at least one uninvolved Ig was slightly more common in patients with IgA myeloma (96% vs. 83.7% for IgG myeloma and 95.2% for light-chain myeloma, p>0.05).


Sarı M, et al: Uninvolved Immunoglobulin Suppression and Prognosis of Myeloma

Turk J Hematol 2017;34:131-136

Table 1. Characteristics of the patients in the analysis, of those with preserved immunoglobulins, those with suppression of one uninvolved immunoglobulin, and those with suppression of more than one uninvolved immunoglobulin. All patients (n=137)

Sex Male Female Age (median) Age >65 years IgG myeloma IgA myeloma IgM myeloma IgD myeloma Light-chain only Serum creatinine ≥2 mg/dL eGFR <60 mL/min Treatment type Classical chemotherapy Classical chemotherapy + tx Novel agents Novel agents + tx Hb <10 g/dL (%) Plt <130,000/µL Stage ISS-1 ISS-2 ISS-2 Osteolytic bone disease Plasmacytoma Corrected serum calcium ≥11.5 mg/dL LDH >300 IU/L BMPCs ≥40% CRAB One CRAB criterion Two CRAB criteria Three/four CRAB criteria Treatment Unresponsive ≥Partial response Complete response

Uninvolved Igs preserved

One uninvolved Ig suppressed

At least two Igs suppressed

p

78 (57%) 10 (13%) 59 (43%) 8 (14%) 62 years (range: 26-97 years) 50 5 (10%) 86 (62.8%) 14 (16%) 26 (19%) 1 (4%) 1 (0.7%) 1 3 (2.2%) 1 (33%) 21 (15.3%) 1 (4%) 29 (21%) 3 (10%) 62 (45%) 6 (10%)

18 (23%) 7 (12%)

50 (64%) 44 (74%)

>0.05 -

10 (20%) 16 (18%) 7 (27%) 0 0 2 (10%) 3 (10%) 10 (16%)

35 (70%) 56 (64%) 18 (69%) 0 2 (67%) 18 (86%) 23 (80%) 46 (74%)

>0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05

28 (20%) 5 (4%) 80 (58%) 24 (18%) 73 (53%) 16 (11%)

6 (21%) 2 (40%) 8 (10%) 2 (8%) 5 (7%) 0%

6 (21%) 1 (20%) 14 (18%) 4 (17%) 11 (15%) 2 (13%)

16 (58%) 2 (40%) 58 (72%) 18 (75%) 57 (78%) 14 (87%)

>0.05 >0.05 >0.05 >0.05 0.01 >0.05

42 (31%) 38 (28%) 57 (41%) 92 (67%) 34 (25%) 18 (13%) 16 (12%) 38 (27.7%) 126/137 (92%) 63 (46%) 44 (32%) 19 (14%)

6 (14%) 6 (16%) 6 (11%) 8 (9%) 9 (26%) 0 (0%) 4 (25%) 1 (3%)

10 (24%) 7 (18%) 8 (14%) 18 (20%) 9 (26%) 2 (11%) 1 (6%) 3 (8%)

26 (62%) 25 (66%) 43 (75%) 66 (71%) 16 (48%) 16 (89%) 11 (69%) 34 (89%)

>0.05 >0.05 >0.05 <0.05 0.02 <0.05 >0.05 <0.001

12 (19%) 3 (7%) 0 (0%)

17 (27%) 5 (11%) 2 (11%)

34 (54%) 36 (82%) 17 (89%)

0.001 0.02 0.03

16 (11%) 121 (88%) 48 (35%)

1 (6%) 17 (12%) 9 (19%)

3 (19%) 22 (18%) 11 (23%)

12 (75%) 82 (68%) 28 (58%)

>0.05 >0.05 0.05

BMPCs: Bone marrow plasma cells, ISS: International Staging System, LDH: lactate dehydrogenase, CRAB: calcium-renal-anemia-bone (calcium ≥11.5 mg/dL, serum creatinine ≥2 mg/ dL, hemoglobin <10 g/dL, osteolytic bone disease), eGFR: estimated glomerular filtration rate, Hb: hemoglobin, Plt: platelets; tx: transplantation, Ig: immunoglobulin.

Anemia (hemoglobin of <10 g/dL; p<0.05), hypercalcemia (Ca of ≥11.5 mg/dL; p<0.05), and osteolytic bone disease (p<0.05) were more frequent in patients with suppressed uninvolved Igs, as shown in Table 1. Renal dysfunction (eGFR of <60 mL/

min per 1.73 m2) was seen in 45% of patients and was more commonly seen in patients with suppressed uninvolved Igs, albeit without a significant relationship (p>0.05). Suppression of the uninvolved Igs was more frequent in patients with 133


SarÄą M, et al: Uninvolved Immunoglobulin Suppression and Prognosis of Myeloma

Thirty-five percent of all patients (n=48) had a complete response to treatment, 17.5% (n=24) had a very good partial response, 35.8% (n=49) had a partial response, and 11.7% (n=16) did not respond to treatment. Eighty-eight percent of all patients had a partial or higher response to treatment. Although groups with preserved uninvolved Igs had partial responses or better to treatment, statistical significance could not be seen (97.4% versus 88% and 87.2%, p>0.05). Twenty percent of all patients received conventional chemotherapy, 4% had conventional chemotherapy and autologous stem cell transplantation, 58% received newgeneration novel agents, and 18% had new-generation novel agents and autologous stem cell transplantation in the form of first-line therapy. Clinical recurrence was seen in 25 (20.6%) patients, 11 of whom died; the remaining 14 patients were still living (14/84 patients). The median follow-up time for living patients without relapse was 48.6 months (range: 40.2-57.1 months). Relapsed patients were more commonly from the group with at least two uninvolved Igs suppressed (19 patients). However, there was no statistically significant relationship between relapse and uninvolved Ig suppression. Although relapse and death were more commonly seen in the suppressed uninvolved Ig group, statistical significance was not seen. Death occurred in a total of 53 patients. The median OS time was 76 months (range: 44.2-106 months). Diagnosis and the first 6 months of treatment are very important for survival. The death rate and survival rate was 3% and 97% in the first 3 months, respectively. After 6 months, the death rate was 6% and the cumulative survival rate was 94%. Thirteen deaths occurred among 137 patients in the 1-year follow-up period. The mortality rate was 10% and the cumulative survival rate was 90%. The cumulative survival rate fell to 82% at 2 years, 70% at 3 years, 55% at 5 years, and 30% at 10 years. 134

Patients with bone marrow plasma cells (BMPCs) of â&#x2030;Ľ40% had a median survival time shorter than the other patients (42.6 vs. 83.2 months, p<0.05), as shown in Figure 2. The OS time was shorter in patients with positive CRAB criteria than in patients without CRAB criteria. There was a significantly shorter OS time in patients with 3 and 4 positive CRAB criteria than in the other patients (37 months, p<0.05), as shown in Figure 3. OS was significantly shorter in patients with ISS stage 3 compared with other stages (42 months vs. 83.2 and 84.9 1.0 0.8 Cum Survival

Ninety-two percent of all patients had at least one positive calcium-renal-anemia-bone (CRAB) criterion met. One, 2, 3, or 4 positive CRAB criteria were seen more commonly in patients with at least two Igs suppressed and this was statistically significant (p<0.001). In addition, CRAB criteria were seen less commonly in those with uninvolved preserved Igs; 3 or 4 positive CRAB criteria were also not seen in this group (p<0.05).

The median OS was 85.5 months for patients with preserved uninvolved Igs and was 62.6 months for patients with at least one Ig suppressed; however, no statistical significance could be detected, as shown in Figure 1. The median OS time was shorter among patients with at least two uninvolved Ig suppressed than in the other patients (55.2 months; range: 36.2-74.1 months).

0.6 0.4 0.2 0.0 0

25

100 50 75 Survival (months)

125

Figure 1. Kaplan-Meier survival estimate regarding overall survival for patients with preserved immunoglobulins and for patients with suppressed immunoglobulins. 1.0 0.8 Cum Survival

extensive bone marrow infiltration (p<0.001). Furthermore, 31% of patients had International Staging System (ISS) stage 1, 28% had ISS stage 2, and 41% of patients had ISS stage 3 disease. Although advanced-stage disease was more commonly seen in patients with suppressed uninvolved Ig (at least one was suppressed in 85.7% vs. 84.2% vs. 89.5% of patients with ISS-1, -2, and -3 disease, respectively, p>0.05), no statistically significant relationship was detected.

Turk J Hematol 2017;34:131-136

0.6

0.4 0.2

0.0 0

25

75 100 50 Survival (months)

125

Figure 2. Kaplan-Meier survival estimate regarding overall survival for patients with bone marrow plasma cells of â&#x2030;Ľ40%. BMPC: Bone marrow plasma cell.


Sarı M, et al: Uninvolved Immunoglobulin Suppression and Prognosis of Myeloma

Turk J Hematol 2017;34:131-136

months). There was a significant difference in terms of survival between Ig levels of preservation and suppression and ISS stage 1 and stage 3 disease (p<0.05). The median PFS of all patients was 41.2 months (range: 34-48.4 months). The median PFS of patients with preserved uninvolved Ig was 63.1 months (range: 46.5-79.6 months), and patients with at least one uninvolved Ig suppressed had 38.8 months of PFS time (range: 29.4-48.1 months) (p>0.05). We performed a multivariate analysis to adjust for the impact of uninvolved Ig suppression and other well-defined prognostic factors on survival. We found no significant relationship between uninvolved Ig suppression or preservation and survival. Other factors that were independently associated with inferior survival in the multivariate analysis included ISS stage 3 disease with 2.76 times greater periodic risk of death than stage 1 disease [95% confident interval (CI): 1.36 to 5.59], while patients with eGFR of <60 mL/min had 2.28 times greater periodic risk (95% CI: 1.32-3.95) and patients aged >65 years had 2.11 times greater periodic risk (95% CI: 1.23-3.63). Patients with LDH of <300 IU/L had a 2.5 times reduced periodic risk of 1.0

Cum Survival

0.8 0.6

0.4 0.2 0.0 0

25

100 50 75 Survival (months)

125

Figure 3. Kaplan-Meier survival estimate regarding overall survival for patients with calcium-renal-anemia-bone criteria. CRAB: Calcium-renal-anemia-bone.

Table 2. Multivariate analysis of factors associated with overall survival in 137 patients.  

HR

95% CI

p

ISS-1

1

-

-

ISS-3

2.76

1.36-5.59

0.005

LDH <300 IU/L

0.41

0.19-0.89

0.02

eGFR <60 mL/min

2.28

1.32-3.95

0.003

β2-microglobulin <3.5 mg/dL

0.40

0.20-0.78

0.008

Cox regression analysis. HR: Heart rate CI: confidence interval, ISS: International Staging System, LDH: lactate dehydrogenase, eGFR: estimated glomerular filtration rate.

death. Due to change of analyzer, previously obtained values of >300 IU/L with the other analyzer were considered normal in the statistical analysis, but despite this LDH of >300 IU/L still appeared to be a poor prognostic factor. Again, BMPCs below 40% were associated with a 2-fold reduction in terms of risk of death. For patients with β2-microglobulin of <3.5 mg/dL, periodic risk of death was reduced by 2.5 times, as summarized in Table 2.

Discussion In this study, we aimed to assess the prognostic significance and association of the suppression of uninvolved Igs with other features of the disease, as measured using nephelometry. Only 13% of all patients had preserved uninvolved Ig levels and the rest of the patients (87%, 119 of 137 patients) had suppression of at least one uninvolved Ig. Suppression of at least one uninvolved Ig was slightly more common in patients with IgA myeloma (96% vs. 83.7% for IgG myeloma and 95.2% for lightchain myeloma; p>0.05). Our results were similar to the report of Kyle et al. [1] with a large cohort of 1027 patients with symptomatic MM from the mayo clinic and the results of the study by Kastritis et al. [8], which consisted of 1755 patients, but we found no statistically significant relationship between the type of MM and suppression of uninvolved Ig levels. In terms of clinical features of MM, response rates to treatment, and survival rates, we saw that the preserved uninvolved Ig group had better results than the other groups; however, we detected no statistically significant difference between the groups. Pathologic features of MM like renal failure, thrombocytopenia, and eGFR of <60 mL/min were more often found in patients with suppressed uninvolved Ig, but statistical significance could not be found. Negative MM features such as anemia, stage-3 disease, osteolytic bone disease, hypercalcemia, LDH of >300 IU/L, BMPCs of ≥40%, and positive CRAB pathologies were more commonly seen in patients with suppressed uninvolved Ig (p<0.05). Positive responses to treatment (≥partial response and complete response) were more common in patients with preserved uninvolved Ig and revealed statistical significance (p<0.05). Very few studies have investigated the prognostic importance of the preservation of uninvolved Igs. Some information comes from small series that included patients treated in the era before today’s novel agents [9,10]. Our study, which comprised 137 patients from a single center, is the first in Turkey on this subject. Kastritis et al. [8] showed the positive prognostic effects of preservation of uninvolved Igs on OS and disease-free survival. Better survival rates of patients with preserved uninvolved Ig levels were also found in our study, but a statistically significant difference was not observed. The most comprehensive study [8] on this issue included patients treated between 1990 and 2012, when novel agents were not being used effectively. Many of the patients of our study (75.9%) who were treated between 2003 135


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and 2015 were treated with new-generation agents. Treating a large portion of the patients with new-generation agents could be the reason for the lack of statistically significant differences because this situation could eliminate the negative effects of uninvolved Ig suppression on survival. This result could highlight the necessity of new-generation chemotherapeutic drugs as first-line therapy for MM. Thus, unfavorable prognostic outcomes due to suppression of uninvolved Igs may be eliminated. We found that higher levels of Ig were conserved in patients with a lower proportion of BMPC infiltration. There may be a positive relationship between normal plasma cell myeloma compartment and Ig level protection. It has been recognized that an extensive “abnormal” plasma cell population is associated with a high risk of progression in symptomatic disease in patients with asymptomatic myeloma or MGUS [11]. CRAB findings were not available in 11 of our patients, but these patients’ diseases had progressed rapidly and treatment was started. There was no suppression of uninvolved Ig in 3 of these patients, whereas the remaining 8 patients had uninvolved Ig suppression. CRAB pathologies were more common in myeloma patients with suppressed uninvolved Igs. Smoldering myeloma is known to form in a heterogeneous group of patients. Although the majority of patients have slow progression, some patients who are accepted as having early myeloma or CRAB-negative myeloma show an aggressive course. There is no molecular factor distinguishing these two groups, which are clinically and biologically different. The risk of progression is said to be associated with tumor burden. The fact that 28.8% of patients with smoldering myeloma are at high risk raises the issue of starting treatment early [12]. The presence of suppressed uninvolved Igs can be a clue to the necessity of starting treatment for patients who are diagnosed as having CRABnegative myeloma.

Conclusion In conclusion, 13% of 137 patients with symptomatic MM had preserved uninvolved Ig levels. Patients with preserved uninvolved Igs had better response rates and pathologic findings but the preservation of the uninvolved Igs in patients was not an independent prognostic factor. In the same way, survival of patients with suppressed uninvolved Igs was shorter, but this could not be determined independently as a negative risk factor.

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Authorship Contributions Concept: M.S., M.N.; Design:  M.S., M.N.; Data Collection or Processing: M.S., S.S.; Analysis or Interpretation: M.S., M.N., S.S.; Literature Search: M.S; Writing: M.S., M.N. 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. Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri A, Fonseca R, Rajkumar SV, Offord JR, Larson DR, Plevak ME, Therneau TM, Greipp PR. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003;78:21-33. 2. Cesana C, Klersy C, Barbarano L, Nosari AM, Crugnola M, Pungolino E, Gargantini L, Granata S, Valentini M, Morra E. Prognostic factors for malignant transformation in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. J Clin Oncol 2002;20:1625-1634. 3. Blade J, Dimopoulos M, Rosinol L, Rajkumar SV, Kyle RA. Smoldering (asymptomatic) multiple myeloma: current diagnostic criteria, new predictors of outcome, and follow-up recommendations. J Clin Oncol 2010;28:690-697. 4. Kastritis E, Terpos E, Moulopoulos L, Spyropoulou-Vlachou M, Kanellias N, Eleftherakis-Papaiakovou E, Gkotzamanidou M, Migkou M, Gavriatopoulou M, Roussou M, Tasidou A, Dimopoulos MA. Extensive bone marrow infiltration and abnormal free light chain ratio identifies patients with asymptomatic myeloma at high risk for progression to symptomatic disease. Leukemia 2013;27:947-953. 5. Katzmann JA, Clark R, Kyle RA, Larson DR, Therneau TM, Melton LJ 3rd, Benson JT, Colby CL, Dispenzieri A, Landgren O, Kumar S, Bradwell AR, Cerhan JR, Rajkumar SV. Suppression of uninvolved immunoglobulins defined by heavy/light chain pair suppression is a risk factor for progression of MGUS. Leukemia 2013;27:208-212. 6. Bradwell A, Harding S, Fourrier N, Mathiot C, Attal M, Moreau P, Harousseau JL, Avet-Loiseau H. Prognostic utility of intact immunoglobulin Ig’κ/Ig’λ ratios in multiple myeloma patients. Leukemia 2013;27:202-207. 7. Durie BG, Harousseau JL, Miguel JS, Bladé J, Barlogie B, Anderson K, Gertz M, Dimopoulos M, Westin J, Sonneveld P, Ludwig H, Gahrton G, Beksac M, Crowley J, Belch A, Boccadaro M, Cavo M, Turesson I, Joshua D, Vesole D, Kyle R, Alexanian R, Tricot G, Attal M, Merlini G, Powles R, Richardson P, Shimizu K, Tosi P, Morgan G, Rajkumar SV; International Myeloma Working Group. International uniform response criteria for multiple myeloma. Leukemia 2006;20:1467-1473. 8. Kastritis E, Zagouri F, Symeonidis A, Roussou M, Sioni A, Pouli A, Delimpasi S, Katodritou E, Michalis E, Michael M, Hatzimichael E, Vassou A, Repousis P, Christophoridou A, Kartasis Z, Stefanoudaki E, Megalakaki C, Giannouli S, Kyrtsonis MC, Konstantopoulos K, Spyroupoulou-Vlachou M, Terpos E, Dimopoulos MA; Greek Myeloma Study Group. Preserved levels of uninvolved immunoglobulins are independently associated with favorable outcome in patients with symptomatic multiple myeloma. Leukemia 2014;28:2075-2079.

Acknowledgment

9. Alexanian R, Migliore PJ. Normal immunoglobulins in multiple myeloma: effect of melphalan chemotherapy. J Lab Clin Med 1970;75:225-233.

We are grateful to Dr. Nalçacı for her support and technical assistance.

10. Pruzanski W, Gidon MS, Roy A. Suppression of polyclonal immunoglobulins in multiple myeloma: relationship to the staging and other manifestations at diagnosis. Clin Immunol Immunopathol 1980;17:280-286.

Ethics

11. Radbruch A, Muehlinghaus G, Luger EO, Inamine A, Smith KG, Dörner T, Hiepe F. Competence and competition: the challenge of becoming a longlived plasma cell. Nat Rev Immunol 2006;6:741-750.

Ethics Committee Approval: Retrospective study; Informed Consent: N/A. 136

12. Ghobrial IM, Landgren O. How I treat smoldering multiple myeloma. Blood 2014;124:3380-3388.


RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0092 Turk J Hematol 2017;34:137-142

Gonadotoxic Effects of Nilotinib in Chronic Myeloid Leukemia Treatment Dose in a Mouse Model Kronik Miyeloid Lösemi Tedavi Dozunda Nilotinib’in Gonadotoksik Etkilerinin Fare Modelinde Gösterilmesi Güldane Cengiz Seval1, Sinan Özkavukçu2, Murat Seval3, Meltem Aylı1 Ufuk University Faculty of Medicine, Department of Hematology, Ankara, Turkey Ankara University Faculty of Medicine, Department of Obstetrics and Gynecology, Center of Assisted Reproduction, Ankara, Turkey 3 Ankara University Faculty of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey 1 2

Abstract

Öz

Objective: Tyrosine kinase inhibitors may have deleterious effects on spermatogenesis or folliculogenesis, resulting in male or female subfertility. The aim of this study is to determine the effect of nilotinib, which is used routinely to treat chronic myeloid leukemia, on spermatogenesis and folliculogenesis by using histopathological parameters.

Amaç: Tirozin kinaz inhibitörlerinin; potansiyel gonadotoksisite ve döllenme üzerindeki olası etkileri hakkında kesin veriler bulunmamaktadır. Bu çalışmanın amacı sağlıklı fare modelinde nilotinib tedavisinin fertilite üzerine etkilerini araştırmaktır.

Materials and Methods: Ten male and ten female mice were orally treated with nilotinib at 20 mg/kg body weight dissolved in drinking water daily for 2 months.

Bulgular: Folikül sayılarında nilotinib verilen dişi fare grubunda kontrol grubuna göre istatistiksel olarak anlamlı bir azalma olduğu gözlendi (268±110 vs. 170±60; p=0,03). Kontrol ve nilotinib gruplarındaki farelerden alınan her seminifer tübül örneğinde spermatid veya spermatozoa olan aktif spermatogenez gözlendi. Spermatojenik aktivite indeksi her iki grupta da benzerdi.

Results: When compared with the control group, a statistically significant decrease was demonstrated in the total follicle numbers of the female mice in the nilotinib group (268±110 vs. 170±60; p=0.03). Active spermatogenesis was observed in each tubule sample taken from the mice in the control and nilotinib groups. Spermatogenic activity was similar in the two groups. Conclusion: We have demonstrated that even though spermatogenesis is preserved, folliculogenesis is inhibited by the usage of a continuous nilotinib treatment dose in chronic myeloid leukemia.

Gereç ve Yöntemler: On erkek ve on dişi fareye 20 mg/kg/gün nilotinib içme suyuna katılarak 2 ay süre ile verildi.

Sonuç: Bizim çalışmamız ile kronik miyeloid lösemide tedavi dozunda ve uzun süreli nilotinib kullanımı ile spermatogenez korunurken folikülogenezin baskılandığı gösterilmiştir. Anahtar Sözcükler: Kronik miyeloid lösemi, Nilotinib, Doğurganlık, Fare, Gonad

Keywords: Chronic myeloid leukemia, Nilotinib, Fertility, Mouse, Gonads

Introduction The nilotinib molecule (AMN107) was first described in 2005 by Weisberg et al. [1]. It is a new imatinib-based aminopyrimidine that inhibits BCR-Abl (breakpoint cluster region-Abelson) signalization in the same way that imatinib does [2]. It inhibits not only BCR-Abl, but also other tyrosine kinases such as c-kit, platelet-derived growth factor receptor A/B (PDGFR A/B), Arg

(Abelson-related gene), and c-fms (colony-stimulating factor-1 receptor). It is known that those sets of proteins have key roles in gonadal development, implantation, and fetal development [3]. Stem cell factor (SCF)/c-kit is expressed in human ovaries during follicular development, and during inhibition with anti-c-kit antibodies and c-kit receptor antagonists, the number of atretic

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Güldane Cengiz Seval, M.D., Ufuk University Faculty of Medicine, Department of Hematology, Ankara, Turkey E-mail : guldanecengiz@gmail.com

Received/Geliş tarihi: March 07, 2016 Accepted/Kabul tarihi: July 28, 2016

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follicles increases [4]. PDGF protein is also found in the oocytes of primordial and developing follicles, and blocking PDGF decreases the amount of primary follicles. This suggests that PDGF plays a role in the process of transition from a primordial follicle to a primary follicle [4]. However, there are reported cases of healthy deliveries in the literature where one of the parents was undergoing imatinib treatment. It has been proven that c-kit and its ligand SCF have an effective role in testicular development, migration, proliferation, and survival of germ cells in studies with rodents [5]. Additionally, PDGF is also an important mediator in the maturation of Leydig cells [6]. Therefore, it is thought that inhibition of these developmental signaling pathways might have negative effects on the production of testosterone and normal spermatogenesis [7]. Although androgen levels were decreased in animal studies, pregnancy and live birth can be achieved while male chronic myeloid leukemia (CML) patients are using imatinib [8,9]. Therefore, the reproductive toxicity of nilotinib and other tyrosine kinase inhibitors (TKIs) is still debatable. On the basis of these findings, we propose that continuous nilotinib treatment may affect folliculogenesis and spermatogenesis in a healthy mouse model. To the best of our knowledge, this is the first identification of nilotinib’s effect on fertility in the mouse and the first study using quantitative measurements of folliculogenesis and spermatogenesis.

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specially produced for small experimental animals, and fresh water. This study was carried out with the permission of the local ethics committee with decision number 67 and date 18.03.2013. Experimental Groups The experimental study comprised 4 groups, each consisting of 10 randomly picked mice: Group I (n=10): female mice given nilotinib. Group II (n=10): male mice given nilotinib. Group III (n=10): control female mice. Group IV (n=10): control male mice. Each mouse received 0.4 mg of nilotinib (Tasigna™, Novartis Pharma, Basel, Switzerland) per day dissolved in drinking water for 2 months. The dose of nilotinib (20 mg/kg, oral) was based on the plasma concentration measured in clinical trials of the dose used today [10]. For this dose, the active ingredient in the 200 mg capsule was dissolved in drinking water, and after making sure that the mixture was homogeneous, 0.4 mL of the mixture was extracted and added to the drinking water of the mice in the nilotinib groups. At the same time, the mice in the control groups (n=10 females and 10 males) received only drinking water.

Materials and Methods

Histological Evaluation

A total of 40 mice (20 males, 20 females; C57bI6 type, Kobay Animal Experiment Laboratory, Ankara, Turkey) were used in our study, which were 5 weeks old, weighed 20-22 g, and had never copulated before.

At the end of 2 months, mice were sacrificed by cervical dislocation and their ovaries and testicles were processed as described below.

During the whole study, experimental subjects were kept in custom-made standard cages made for mice, which had plastic bottoms and sides and wire fencing covering the tops. A group of mice (10 each) were put in each cage. The bottoms of the cages were always kept covered with dry shavings. Shavings were replaced once every 2 days. During the experiment, the mice were kept under optimum laboratory conditions [22±1 °C, 12-h light/dark cycle (07:00/19:00)] and were fed with fabricated pellets containing 21% crude protein, which were

Ovarian tissues were fixed in 10% (w/v) neutral-buffered formalin and embedded in paraffin. Serial sections of 5 µm were then made on a microtome (Leica RM 2125RT, Wetzlar, Germany) for histological evaluation and mounted on glass slides. The slides were stained with hematoxylin and eosin (H&E) (Merck, Darmstadt, Germany) as described below and analyzed for morphological grading and follicle count. Follicles were classified as primordial, primary, or secondary (antral) follicles and primordial follicles were counted for evaluation of ovarian reserve. Follicle numbers in primordial, primary, and secondary phases were recorded

Table 1. Classification of ovarian follicles [26]. Follicular stage

Definition

Primordial

Simple squamous epithelium cells around small, undeveloped oocyte

Primary

Developing oocyte and simple cuboidal epithelium cells around distinct nucleolus, no follicular antrum

Secondary (antral)

Developing oocyte and multiple layered granulosa cells around big nucleolus, with small-sized antrum

Tertiary (graafian)

A fully developed oocyte and cumulus oophorus in a follicle with an antral field

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in every five sections where the oocyte nucleus was visible in the section, to avoid repetitive evaluations of the same follicle. Follicular atresia incidence was morphologically scored among follicle stages (Table 1). Shrunken oocyte and/or pyknotic granulosa cells were considered as signs of atresia. For testicles, tissues were fixed in Bouin’s fixative (750 mL of picric acid, 250 mL of 40% formaldehyde, 5 mL of glacial acetic acid) for 2 h at room temperature followed by paraffin embedding and serial sections of 5 µm were made on a microtome (Leica RM 2125RT) for histological evaluation. Spermatogenic scoring and tubule diameter measurements were determined using AxioCam MRc5 software (Carl Zeiss, Göttingen, Germany). The sections were stained with H&E. Briefly, sections were deparaffinized with xylol and rehydrated in decreasing concentrations of alcohol and distilled water before hematoxylin staining for 3 min. Following eosin staining for 1 min, excess eosin was washed off with increasing concentrations of alcohol. Slides were covered with a cover slip using Entellan® (Electron Microscopy Sciences, Hatfield, PA, USA). Twenty-four transversely sectioned seminiferous tubules were assessed under 400x magnification and tubule diameters were measured under 200x magnification. Statistical Analysis Data were analyzed using SPSS 16. The results were given as average ± standard deviation. The t-test was used in evaluation of the relationship between numeric variables. A value of p<0.05 was considered statistically significant.

Results Histological Evaluation of Ovarian Functions One mouse in the nilotinib group was excluded from statistical analysis because of death on the 15th day of the study. When compared with the control group, a statistically significant decrease was observed in the total follicle numbers of the female mice in the nilotinib group (268±110 vs. 170±60; p=0.03). Follicle distributions of mice in the control and nilotinib groups are shown in Figure 1. Although there were no statistically significant differences between the two groups for the numbers of primordial, secondary, and tertiary follicles, the primary follicle numbers in the nilotinib group were significantly lower than in the control group (168±56 vs. 68±25; p=0.02) (Table 2).

In the nilotinib group, ovarian structure was observed to be irregular. It was seen that the gap between the cortex and medulla was narrower than it would normally be. The follicles, which should be organized from the periphery to the medulla in terms of the level of development in their normal hierarchical order, were sparse and scattered in the nilotinib group (Figure 2). Histological Evaluation of Testicular Functions The morphological appearances of the testicles were similar in the control and nilotinib groups (Figure 3). No pathological symptoms were observed in the tubular or interstitial areas. Active spermatogenesis was observed in each tubule sample taken from the mice in the control and nilotinib groups. Spermatogenic activity was similar in both groups (Table 3; p=0.241). No statistical difference was observed between the mean diameter values of seminiferous tubules in the control and nilotinib groups (Table 3; p=0.475)

Discussion To the best of our knowledge, this is the first study that postulated the effects of nilotinib with a continuous CML treatment dose on fertility by quantitative histopathological measurement of spermatogenesis and folliculogenesis in a healthy mouse model. We evaluated the effect of nilotinib on ovarian reserve by the change of follicular number. As expected, the follicle numbers of the female mice were significantly decreased in the nilotinib group compared to the control group (268±110 vs. 170±60; p=0.03). While a statistically significant decrease occurred in the numbers of primary follicles in the nilotinib group (168±56 vs.

Figure 1. Follicle distributions of female mice in control and nilotinib groups.

Table 2. Number of follicles in nilotinib and control groups (mean ± standard deviation).  

Primordial follicles

Primary follicles

Secondary follicles

Tertiary follicles

Total follicles

Control group Nilotinib group

218±47 164±29

168±56 68±25

87±29 60±23

64±24 44±12

268±110 170±60

p

0.17

0.02*

0.32

0.33

0.03*

*: Statistical difference related to the changes between the groups, p<0.05.

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Table 3. Comparison of spermatogenesis in control and nilotinib groups (mean ± standard deviation); no statistical difference in terms of intergroup changes at p>0.05.  

n

Diameter of seminiferous tubules (µm)

Control group Nilotinib group

10 10

190.61±8.33 194.32±7.26

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of oocytes and stimulate the transition of primordial follicles to a growing state [11]. Treatment with TKIs might inhibit recruitment of primordial follicles and could lead to irregular menstrual cycles or even amenorrhea [12]. Schultheis et al. [12] reported no effects of tyrosine kinase inhibition with 150 mg/kg/day imatinib for 2 months on folliculogenesis in the ovaries of Bcr-Abl xenografted mice. However, unlike in mice, development of primary ovarian deficiency was reported in the second year of treatment in a 28-year-old female CML patient using imatinib [13]. There is no study in the literature considering the effects of second-generation TKIs on folliculogenesis. The effects on estrous cycle, pregnancy, and copulation were observed in mature female mice after administration of >60 mg/kg/day nilotinib [http://multimediacapsule.thomsonone.com/novartis/ nilotinib-tfr-asco]. On the basis of these data, nilotinib does not cause female infertility [14]. However, the clinical data confirming this claim consist of 2 cases [15,16]. In the case report that Conchon et al. [16] published in 2009, a 30-yearold woman with CML using nilotinib had two healthy births once in 2 years. In a single-center retrospective analysis of the pregnancy incidences among female CML patients using TKIs, it was reported that 1 out of 25 pregnancies was achieved under nilotinib and a healthy baby was born [15]. Several cases of successful pregnancies have been reported with TKIs; however, the usage of any TKI during pregnancy must be avoided in light of the present data.

Figure 2. Hematoxylin and eosin-stained ovaries with sections from control [A) primordial (arrow), B) primary, and C) secondary follicles] and nilotinib [D) degenerated primary, E) degenerated primary, and F) degenerated primary and secondary follicles] groups. Bars indicate 50 µm. A

B

Figure 3. Hematoxylin and eosin-stained sections of nilotinib group testicle (A) and a detailed seminiferous tubule (B). Bars indicate 50 µm. 68±25; p=0.02), a similar follicular stage was observed between the nilotinib and control groups (p>0.05). Primordial follicles are the dormant pool of female gametes from which all mature oocytes for ovulation and fertilization originate. There is evidence from organ culture and animal experiments that PDGF and KIT-ligand promote the growth 140

Our study has revealed the effects of nilotinib on spermatogenesis with histopathological parameters. Measuring the seminiferous tubule diameter is a crucial parameter in terms of showing testicular reserves. While the mean seminiferous tubule diameter in the control group was 190.6±8.3 µm, it was measured as 194.3±7.3 µm in the nilotinib group (p=0.475). The spermatogenic activity, which is calculated by grading according to the existence of germ cells, motivic phase, spermatids, and spermatozoa, was similar in both of the groups. Our findings in this mouse model indicate that nilotinib has no effect on spermatogenesis, since there was a normal succession of sperm production in the testes. PDGFR and KIT-ligand play critical roles in regulating the development and functional control of the testis. TKIs interfere with various maturation processes in animal models, including gonocyte migration, growth of the testis, formation of spermatogonial stem cells, and Leydig cell survival [7]. Nurmio et al. [7] reported that postnatal testicular development was permanently affected in rats when PDGFR and c-kit tyrosine kinases were inhibited by oral imatinib (150 mg/kg/day) during the first postnatal week. The age of the mice might be another explanation for the failure to detect an effect of nilotinib on


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the testes in our study. It was suggested that some limited phenotypes of mice with single mutations probably indicate different explanations for this redundancy of the tyrosine protein kinase pools in testis mutations [17]. Experiments with large numbers of animals including copulation models are needed. A decrease of total epididymal weights was reported in adult male mice that were given 180 mg/kg/day nilotinib, and deleterious effects on fertility leading to decrease in the number of sperm and sperm motility have been observed [http://multimediacapsule.thomsonone.com/novartis/nilotinibtfr-asco]. On the basis of these data, it is currently reported that men taking nilotinib might experience negative effects on fertility [14]. In the literature, oligozoospermia was seen with the imatinib treatment of an 11-year-old male CML patient [6]. However, there are also male CML patients in the literature who had healthy children while taking imatinib. Between the years 2003 and 2014, a total of 109 pregnancies were reported among male CML patients on imatinib and 103 of these pregnancies resulted in healthy babies [8,15,18,19,20,21,22]. The treatment of CML is based on chronic tyrosine kinase inhibition. Imatinib mesylate, which is a Bcr-Abl TKI, has been used since 2001 and has become the cornerstone of CML treatment. In 2006, second-generation TKIs (nilotinib, dasatinib) were put into clinical practice. In the recently published Stop Imatinib Study, it was reported that imatinib treatment might safely be stopped among patients who maintained major molecular response [23]. These positive consequences increased the expectations of many patients of reproductive age who wished to have a child. However, there are no certain data related to the probable effects of TKIs on potential gonadotoxicity and fertility. Among TKIs, only the effect of imatinib was demonstrated, and these data from animal models have been conflicting. Young patients with hematological malignancies should be advised about the risks of treatment affecting their reproductive potential and the chances of a successful outcome of pregnancy. Cryopreservation of sperm and ovarian tissues or embryos before treatment is a significant option for patients who want to have a child in the future. However, this should be handled cautiously since malignant cells might infiltrate the tissues that are meant to be autotransplanted in the future. It is important to note that ovarian tissue involvement may also be seen in patients with CML [24]. Even testicular involvement was reported in the blastic phase of CML cases [25]. Considering all these possibilities, we preferred to use a healthy mouse model in our study in order to avoid leukemic infiltration; thus, it was possible for us to determine only the effects of the drug on the gonads.

Conclusion In this study, the effect of nilotinib on spermatogenesis and folliculogenesis in a healthy mouse model was evaluated by using histopathological parameters. We have shown that even though spermatogenesis is preserved, folliculogenesis is inhibited by the usage of a continuous CML treatment dose of nilotinib. There is a need for further randomized and controlled studies with primate and human models to elucidate the effects of nilotinib and endocrinopathies by hormonal measurements, such as estradiol, testosterone, follicle-stimulating hormone, luteinizing hormone, and anti-Müllerian hormone. Acknowledgments Funding: We are grateful for support from the Turkish Lymphoma-Myeloma Association. Award: The abstract (Abstract ID #71346) of this article was selected to receive an American Society of Hematology Abstract Achievement Award at the 56th Annual Meeting of the American Society of Hematology. Ethics Ethics Committee Approval: Kobay DHL A.Ş. Local Ethics Committee, Approval Number: 67, Approval Date: 18.03.2013, Informed Consent: N/A. Authorship Contributions Concept: M.A., G.C.S.; Design: M.A., G.C.S.; Data Collection or Processing: G.C.S., S.Ö., M.S.; Analysis or Interpretation: G.C.S., S.Ö., M.S.; Literature Search: G.C.S., S.Ö., M.S., M.A.; Writing: G.C.S., S.Ö., M.S., M.A. Conflict of Interest: No conflict of interest was declared by the authors. Financial Disclosure: The Turkish Association supported this study.

Lymphoma-Myeloma

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0007 Turk J Hematol 2017;34:143-150

Influence of Folate-Related Gene Polymorphisms on High-Dose Methotrexate-Related Toxicity and Prognosis in Turkish Children with Acute Lymphoblastic Leukemia Folat İlişkili Gen Polimorfizmlerinin Akut Lenfoblastik Lösemili Türk Çocuklarında Yüksek Doz Metotreksat İlişkili Toksisite ve Prognoz Üzerine Etkisi Burcu Yazıcıoğlu1, Zühre Kaya1, Sezen Güntekin Ergun2, Ferda Perçin2, Ülker Koçak1, İdil Yenicesu1, Türkiz Gürsel1 Gazi University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey Gazi University Faculty of Medicine, Department of Genetics, Ankara, Turkey

1 2

Abstract

Öz

Objective: High-dose methotrexate (HD-MTX) is widely used in the consolidation phase of childhood acute lymphoblastic leukemia (ALL), but the roles that polymorphisms in folate-related genes (FRGs) play in HD-MTX toxicity and prognosis in children with ALL are not understood. The aims of this study were to investigate the frequencies of polymorphisms in the genes for thymidylate synthase (TS), methionine synthase reductase (MTRR), and methylene tetrahydrofolate reductase (MTHFR) in Turkish children with ALL and to assess associations between these polymorphisms and HD-MTXrelated toxicity and leukemia prognosis in this patient group.

Amaç: Yüksek doz metotreksat (YD-MTX) çocukluk çağı akut lenfoblastik lösemi (ALL) tedavisinin yoğunlaştırma fazında yaygın olarak kullanılmaktadır, ancak ALL’li çocuklarda YD-MTX’in toksisite ve prognozunda rol oynayan folat ilişkili genlerdeki (FİG) polimorfizmlerin rolü iyi bilinmemektedir. Bu çalışmanın amacı; ALL’li Türk çocuklarındaki timidilat sentaz (TS), metionin sentaz redüktaz (MTRR) ve metilen tetrahidrofolat redüktazı (MTHFR) kodlayan genlerdeki polimorfizm sıklığını araştırmak, bu hasta grubunda polimorfizmler ile YD-MTX’e bağlı toksisite ve lösemi prognozu arasındaki ilişkiyi değerlendirmektir.

Materials and Methods: FRG polymorphisms were assessed by realtime polymerase chain reaction. Survival status, MTX levels, and toxicity data were retrieved from 106 patients’ charts. Results: The allele frequencies for the FRG polymorphisms were as follows: TS 2R 41.0%, 3R 57.0%, and 4R 2.0%; MTRR 66A 42.4% and 66G 57.6%; MTHFR 677C 59.3% and 677T 40.7%; and MTHFR 1298A 58.1% and 1298C 41.9%. At the 48th hour of HD-MTX infusion, serum MTX was significantly higher in patients who had TS 2R/3R/4R variants as compared to those with wild-type TS (p<0.05). No significant differences were detected with respect to event-free survival or toxicity between wild-type and other FRG variants. Conclusion: The frequencies of FRG polymorphisms in Turkish children with ALL are similar to those reported in other Caucasian populations. This is the first published finding of the TS 3R/4R variant in the Turkish population. The results indicate that HD-MTX can be tolerated by leukemic children with some polymorphic variants of FRG; thus, it may prevent future risk of leukemic relapse. Keywords: Methotrexate, Toxicity, Prognosis, Folate-related gene polymorphisms

Gereç ve Yöntemler: FİG ilişkili polimorfizmler polimeraz zincir reaksiyonu ile incelenmiştir. Hastane dosyasından 106 hastanın yaşam durumu, MTX düzeyleri ve toksisite verileri kaydedilmiştir. Bulgular: FİG ilişkili polimorfizmlerin sıklığı sırasıyla TS 2R %41,0, 3R %57,0 ve 4R %2,0; MTRR 66A %42,4 ve 66G %57,6; MTHFR 677C %59,3 ve 677T %40,7; ve MTHFR 1298A %58,1 ve 1298C %41,9’dur. TS 2R/3R/4R varyant tipini taşıyan çocuklarda YD-MTX infüzyonunun 48. saatindeki serum MTX düzeyi yaban tipini taşıyanlardan daha yüksek oranda bulunmuştur (p<0,05). FİG ilişkili polimorfik varyantlar ile yabanıl tipleri arasında olaysız sağkalım ve toksisite yönünden anlamlı farklılık saptanmamıştır. Sonuç: ALL’li Türk çocuklarındaki FİG ilişkili polimorfizm sıklığı beyaz ırk popülasyonuna benzer oranda tespit edilmiştir. Türk popülasyonunda TS 3R/4R varyantı ilk kez bildirilmiştir. Sonuçlarımız FİG ilişkili bazı polimorfik varyantları taşıyan lösemili çocuklarda YD-MTX’in tolere edilebileceğine ve böylece gelecekteki relapsı önleyebileceğine işaret etmektedir. Anahtar Sözcükler: Metotreksat, Toksisite, Prognoz, Folat-ilişkili gen polimorfizmleri

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Zühre KAYA, M.D., Gazi University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey E-mail : zuhrekaya@gazi.edu.tr

Received/Geliş tarihi: January 05, 2016 Accepted/Kabul tarihi: March 25, 2016

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Introduction

Analysis of Folate-Related Genes

Methotrexate (MTX) is a folate antagonist that impairs DNA synthesis and methylation reactions in cells. The metabolism of MTX is influenced by a number of polymorphisms in folaterelated genes (FRGs) that encode the enzymes thymidylate synthase (TS), methionine synthase reductase (MTRR), and methylene tetrahydrofolate reductase (MTHFR) [1]. Currently, high-dose (HD) MTX is recommended for all patients with acute lymphoblastic leukemia (ALL) and for any patient with lymphoma or osteosarcoma [2,3,4]. However, there is no consensus on effective doses of HD-MTX in these patients. Recent research efforts have focused on determining the influence of different polymorphic enzyme variants on MTX toxicity and prognosis in children with ALL [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21,22,23,24,25,26,27,28,29,30,31,32,33]. Some have reported that several polymorphic variants of FRGs may be linked to relapse and HD-MTX-related toxicity in children with leukemia [5,6,7,8,9,10,11,12,13,14,15,16,17,21,22,23,24,25,26,27,28, 29], whereas others have found that this treatment is protective against leukemia and there is no association between FRGs and toxicity [18,19,20,30,31,32,33].

While TS and MTRR gene polymorphisms were analyzed in all 106 patients, findings for MTHFR polymorphisms were only recorded for the 43 patients with complete clinical data (i.e. significant data were missing for the remaining patients).

The frequencies of these gene polymorphisms vary widely among different races, ranging from 5% to 50% depending on the enzyme type [21,24,25,26,34,35]. To date, only two studies have examined the link between polymorphisms in MTHFR genes and leukemia in Turkish children [3,36]. The aims of this study were to determine the frequencies of TS, MTRR, and MTHFR polymorphisms in Turkish children with ALL and to evaluate possible associations with HD-MTX toxicity and survival in this patient group.

Materials and Methods The study involved 106 children with ALL who were treated with the ALL-Berlin-Frankfurt-Munster (BFM) 95 protocol at our clinic between 1998 and 2014. The study protocol was approved by Gazi University Faculty of Medicine review board. Patients were assigned to risk groups and treated according to the ALLBFM 95 protocol, as described previously [37]. All patients received four courses of 5 g/m2 MTX together with 25 mg/m2 mercaptopurine during consolidation phases. Fifteen patients treated prior to 2003 had received intermediate doses of ARA-C (ID-ARA-C) at 200 mg/m2 with HD-MTX+MP in the consolidation phase. Serum MTX levels were measured at the 24th, 36th, 42nd, and 48th hours of infusion. Effective serum MTX levels according to the BFM 95 protocol were defined as ≤150 µmol/L, ≤3 µmol/L, ≤1 µmol/L, and ≤0.4 µmol/L for the 24th, 36th, 42nd, and 48th hours, respectively. Serum MTX levels and treatment-related toxicity data were retrieved from the patients’ charts retrospectively. National Cancer Institute criteria were used to evaluate toxicity. 144

DNA Extraction DNA was isolated from a blood sample from each patient according to the NucleoSpin® blood kit protocol (MachereyNagel, Düren, Germany). The concentration and quality of DNA were analyzed by spectrophotometer (NanoDrop ND 1000, Thermo Fisher Scientific, Waltham, MA, USA). The primers used to detect the MTRR gene 66 A>G polymorphism were 5’-AAGGCCATCGCAGAAGACAT-3’ and 5’-CCATTGAACAAACACATTTCTG-3. The primers used to detect the tandem repeat sequence in the enhancer region (TSER) of the TS gene were 5’-AACTGTGCTGCTGGCTTAGAGAA-3’ and 5’-ATGTCGGACTCTCCACTGCG-3’. To identify the MTRR gene 66 A>G polymorphism, a 220bp target gene region amplified using specific primers was incubated with AflIII restriction enzyme overnight at 37 °C. The resulting product was loaded on 3% agarose gel and subjected to electrophoresis. Examination of the bands on the gel revealed a 220-bp band for the AA genotype and 203- and 17-bp bands for the GG genotype. To identify TSER polymorphism of the TS gene, the amplified products of the primers above were loaded on 3% agarose gel and subjected to electrophoresis. A 578-bp band indicated the 2R/2R genotype, a 606-bp band and a 578-bp band indicated 2R/3R, a 606-bp band indicated 3R/3R, and a 634-bp band and a 606-bp band indicated 3R/4R. Real-Time Polymerase Chain Reaction The MTHFR mutations of C677T and A1298C were amplified and detected by TaqMan probes using a real-time polymerase chain reaction (PCR) kit (SNP, Biotech, Ankara, Turkey). For the procedure, 20.5 µL of master mix and 0.3 µL of hot-start Taq DNA polymerase were added to a PCR tube, and 4.5 µL of the patient’s DNA suspension (100 µL) was then added. The following PCR program was performed: an initial denaturation step at 95 °C for 10 min, followed by 32 cycles of denaturation at 95 °C for 15 s, and annealing at 60 °C for 1 min. Allelic discrimination was facilitated by software analysis of the fluorescence data. Statistical Analysis Data were statistically analyzed using SPSS 15.0. Genotype frequencies of the TS, MTRR, and MTHFR polymorphisms in FRGs were compared with previously reported findings for these enzymes in Turkish populations [3,34,35,36]. Differences between groups were analyzed using the Mann-Whitney U


Yazıcıoğlu B, et al: Polymorphisms in Folate-Related Genes in Turkish Children

Turk J Hematol 2017;34:143-150

levels at other time points for each of the polymorphic variants of the FRGs assessed.

test. The chi-square test was used to analyze categorical data. Survival rates, including event-free survival (EFS) and overall survival (OS), were investigated using Kaplan-Meier analysis. Events were defined as relapse or death from any cause. OS was defined as time from initiation of treatment to death or the date of the last follow-up. Values of p<0.05 were considered statistically significant.

Toxicity Evaluation After a total of 424 HD-MTX treatment courses in the 106 cases, grade III/IV severe anemia developed in 8.7% of the patients, leukopenia in 23.9%, neutropenia in 34.8%, and

Results

Table 1. Demographic features of the pediatric patients with acute lymphoblastic leukemia.

The demographic features of 106 children with ALL are shown in Table 1.

Feature

No. of patients (%) n=106

Genotype and Allele Frequencies of Folate-Related Genes

Sex Male

67 (63.2%)

The results for allele frequencies of polymorphisms in FRGs were TS 2R 41.0%, 3R 57.0%, and 4R 2.0%; MTRR 66A 42.4% and 66G 57.6%; MTHFR 677C 59.3% and 677T 40.7%; and MTHFR 1298A 58.1% and 1298C 41.9% (Table 2).

Female

39 (36.8%)

Median age (range) in years

5 (1-17)

B-cell

63 (59.4%)

Serum Methotrexate Levels

T-cell

15 (14.2%)

*Others

28 (26.4%)

Leukemia subtype

Table 3 shows the serum MTX levels at different time points for the groups of patients with TS, MTRR, and MTHFR polymorphic variants. At the 48th hour of HD-MTX infusion, serum MTX was significantly higher in patients who had TS 2R/3R/4R variants as compared to those with wild-type TS (p<0.05). There were no such statistical differences at the other time points. There were also no statistically significant differences between serum MTX

Risk group Standard risk

29 (27.4%)

Medium risk

61 (57.5%)

High risk

16 (15.1%)

*Others: Myeloid antigen-positive acute lymphoblastic leukemia (T lineage or B lineage).

Table 2. Genotype and allele frequencies for folate-related gene polymorphisms in the 106 children with acute lymphoblastic leukemia.

No. of patients (%) n=106

No. of patients (%) n=106

MTRR 66 A>G

MTHFR 677 C>T

AA AG GG

22 (20.8) 46 (43.4) 38 (35.8)

CC CT TT

15 (34.8) 21 (48.8) 7 (16.4)

Allele

Allele

A

90 (42.4)

C

51 (59.3)

G

122 (57.6)

T

35 (40.7)

TS

MTHFR 1298A>C

Genotype

Genotype

2R>2R

18 (17.0)

AA

5 (34.8)

2R>3R

51 (48.1)

AC

20 (46.5)

3R>3R

35 (33.0)

CC

8 (18.7)

3R>4R

2 (1.9)

Allele

Allele

A

50 (58.1)

2R

87 (41.0)

C

36 (41.9)

3R

121 (57.0)

4R

4 (2.0)

TS: Thymidylate synthase, MTRR: methionine synthase reductase, MTHFR: methylene tetrahydrofolate reductase.

145


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Table 3. Serum levels of methotrexate at different infusion times with the patients grouped by folate-related gene polymorphisms. Serum methotrexate cut-off levels

TS n (%)

MTRR 66 n (%)

MTHFR 677 n (%)

MTHFR 1298 n (%)

2R/2R (n=18)

2R/3R/4R (n=88)

AA (n=22)

AG/GG (n=84)

CC (n=15)

CT/TT (n=21)

AA (n=15)

AC/CC (n=21)

24th hour (>150 µmol/L)

1 (5.5)

5 (5.6)

0

6 (7.1)

1 (6.6)

3 (14.2)

3 (20.0)

1 (4.7)

36th hour (>3 µmol/L)

3 (16.6)

8 (9.1)

2 (9.0)

9 (10.7)

2 (13.3)

3 (14.2)

3 (20.0)

2 (9.5)

42nd hour (>1 µmol/L)

2 (11.1)

10 (11.3)

1 (4.5)

11 (13.1)

2 (13.3)

5 (23.8)

2 (13.3)

5 (23.8)

48th hour (>0.4 µmol/L)

1 (5.5)*

27 (30.6)*

7 (31.8)

21 (25.0)

6 (40.0)

10 (47.6)

7 (46.6)

9 (42.8)

TS: Thymidylate synthase, MTRR: methionine synthase reductase, MTHFR: methylene tetrahydrofolate reductase. *: p<0.05 for the comparison of TS groups.

Table 4. Comparison of grade III/IV toxicity findings with the patients grouped by folate-related gene polymorphisms. Type of grade III/IV toxicity

TS n (%)

2R/2R (n=18)

MTRR 66 n (%) 2R/3R/4R (n=88)

AA (n=22)

AG/GG (n=84)

MTHFR 677 n (%)

MTHFR 1298 n (%)

CC (n=15)

AA (n=15)

CT/TT (n=21)

AC/CC (n=21)

Hematological toxicity Anemia

1 (5.5)

3 (3.4)

0

4 (4.7)

0

1 (4.7)

0

1 (4.7)

Leukopenia

2 (11.1)

9 (10.2)

2 (9.0)

9 (10.7)

0

0

0

0

Neutropenia

4 (22.2)

12 (13.6)

3 (13.6)

13 (15.4)

0

0

0

0

Thrombocytopenia

1 (5.5)

0

0

1 (1.1)

0

0

0

0

Total

8 (44.4)

24 (27.2)

5 (22.7)

37 (44.0)

0

1 (4.7)

0

1 (4.7)

Nonhematological toxicity Mucositis

3 (16.6)

12 (13.6)

4 (18.1)

11 (13.1)

0

1 (4.7)

0

1 (4.7)

Hepatic

0

2 (2.2)

0

2 (2.3)

0

1 (4.7)

0

1 (4.7)

Renal

3 (16.6)

13 (14.7)

5 (22.7)

13 (15.4)

1 (6.6)

3 (14.2)

2 (13.3)

2 (9.5)

Neurologic

0

1 (1.1)

0

1 (1.1)

0

1 (4.7)

0

0

Total

6 (33.3)

28 (31.8)

9 (40.9)

27 (32.1)

1 (6.6)

6 (28.5)

2 (13.3)

4 (19.0)

TS: Thymidylate synthase, MTRR: methionine synthase reductase, MTHFR: methylene tetrahydrofolate reductase.

thrombocytopenia in 2.2%. Grade III/IV severe hepatic toxicity was recorded in 4.3% of the patients following HD-MTX, and renal toxicity in 11.3%. There were no significant differences between the respective wild-type groups and other FRG variants regarding hematologic and nonhematological toxicities (p>0.05). The toxicity findings for the 106 patients are shown in Table 4. Only one child with MTX encephalopathy carried the GG variant for MTRR, TT for MTHFR 677, and 2R/3R for TS. This patient rapidly recovered with aminophylline administration after 72 h of HD-MTX treatment. No severe mucositis was observed in patients who received only HD-MTX in the ALL-BFM 95 protocol, whereas grade III-IV mucositis had been observed previously in 15 (14.1%) of 106 patients who had received HDMTX together with intermediate-dose ARA-C in the ALL-BFM 95 protocol in our clinic. 146

Survival Status Twelve (11.3%) of the 106 children died, 8 (7.6%) due to relapse or refractory disease and 4 (3.7%) due to infections during follow-up (median 58 months). No deaths occurred in patients who received HD-MTX during consolidation therapy. Of the 106 patients who achieved complete remission, 19 (17.9%) relapsed after a median of 26.2 months. The relapse rates were 16/19 (84.2%) for TS genotypes (2R/3R, 3R/3R, and 3R/4R), 17/19 (89.4%) for MTRR genotypes (AG and GG), 6/19 (31.5%) for MTHFR 677 genotypes (CT and TT), and 2/19 (10.5%) for MTHFR 1298 genotypes (AC and CC). However, there were no significant differences with respect to relapse rates, EFS, or OS between the groups with and without polymorphic variants of FRG (Figures 1-4).


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Figure 1. Kaplan-Meier estimate of event-free survival of the patients who carried thymidylate synthase polymorphisms.

Yazıcıoğlu B, et al: Polymorphisms in Folate-Related Genes in Turkish Children

Figure 3. Kaplan-Meier estimate of event-free survival of the patients who carried methylene tetrahydrofolate reductase 677 polymorphisms. MTHFR: Methylene tetrahydrofolate reductase.

Figure 2. Kaplan-Meier estimate of event-free survival of the patients who carried methionine synthase reductase polymorphisms.

Discussion MTX is a key component of consolidation and maintenance treatment for childhood ALL [2]. However, some patients cannot tolerate HD-MTX, and in these cases the treatment can cause toxicity and discontinuation of chemotherapy, which may increase relapse risk in a small number of patients [5,8,23,24,25,29]. Our study is the first to have investigated the frequencies of FRG polymorphisms and to have assessed associations between these polymorphisms and HD-MTXrelated toxicity and outcomes in Turkish children with ALL. The respective frequencies of the TS 2R/2R, 2R/3R, and 3R/3R variants in our patients were 17.0%, 48.1%, and 33.0%. While these are not different from findings in the healthy Turkish population and other Caucasian populations [34,38], higher frequencies for the TS 3R/3R variant (66% to 76%) and lower rates for other variants (2R/2R 1% to 3%; 2R/3R 22% to 29%) compared to the Turkish population were reported in a study

Figure 4. Kaplan-Meier estimate of event-free survival of the patients who carried methylene tetrahydrofolate reductase 1298 polymorphisms. MTHFR: Methylene tetrahydrofolate reductase.

of Indonesian children and in other reports from Japanese and Chinese populations [21,34]. We detected the TS 3R/4R variant in only two patients, and ours is the first published finding of this variant in the Turkish population. One of these patients died due to cardiac and hepatic toxicity after salvage chemotherapy for relapse. The other child was diagnosed with high-risk leukemia, was treated with allogeneic stem cell transplantation, and is currently in remission. The same genetic pattern was previously described in 20 leukemic children who were receiving the Children’s Cancer Group-1891 protocol; 7 (35%) of these patients relapsed and the TS 3R/4R genotype was associated with significantly greater relapse risk in that study [23]. Our limited data confirm the previous observation that some patients with the TS 3R/4R genotype who achieve limited benefit from chemotherapy alone should ultimately undergo transplantation. 147


Yazıcıoğlu B, et al: Polymorphisms in Folate-Related Genes in Turkish Children

The frequencies of the MTRR AA, AG, and GG variants in our sample were 20.8%, 43.4%, and 35.8%, respectively. Similarly, the corresponding rates reported for healthy vs. leukemic children from Slovenia were 18.2% vs. 22.1%, 52.7% vs. 52.9%, and 29.1% vs. 25.0% [38]. The only previous investigation of MTRR polymorphism frequency in the Turkish population was conducted in children with stroke [35]; however, the frequency of homozygous GG variant (4%) for this enzyme in that study was lower than we observed in ours. Interestingly, one of our patients with the MTRR GG variant developed MTX encephalopathy. In our pediatric ALL patients, the frequencies of the MTHFR 677 CC, CT, and TT variants were 48.8%, 41.8%, and 9.3%, respectively, and the frequencies of the MTHFR 1298 AA, AC, and CC variants were 51.2%, 37.2%, and 11.6%, respectively. These results are comparable to those previously reported for children with ALL in the Turkish population [3] and other Caucasian populations [39]. There are conflicting results regarding the roles of the TS, MTRR, and MTHFR gene polymorphisms in leukemia prognosis [5,8,9, 15,18,23,24,25,26,27,28,32,33,38]. Some studies have indicated that these variants play protective roles [32,33,38], whereas others have shown that they are linked to increased rates of relapse and drug resistance [5,6,7,8,9,15,18,23,24,25,26, 27,28]. Among these gene polymorphisms, the higher enzyme activity of the TS 3R/3R variant led to the diminished MTX effect and enhanced drug resistance. A study performed at the Dana Farber Institute revealed that 32 of 205 children with ALL who were followed for 12 years developed relapse and/or died, and most of these children had the TS 3R/3R variant [24]. Similarly, an investigation of 246 children with ALL at the St. Jude Medical Center demonstrated that in high-risk cases, the TS 3R/3R polymorphism was significantly associated with development of relapse [25]. Another study performed by the BFM group in Germany investigated 40 ALL patients with relapse who received BFM 86-90 chemotherapy and found no significant difference in EFS between the TS 3R/3R and TS 2R/3R variants [26]. The Dana Farber and St. Jude centers applied MTX at single dosages of 4 g/m2 and 2 g/m2, respectively, whereas the German BFM group applied a cumulative dose of MTX with 5 g/m2 administered per cycle and repeated four times with 2-week intervals [24,25,26]. Thus, it has been suggested that the higher MTX dosage applied by the German group overcame the enhanced enzymatic activity of the TS 3R/3R genotype and reached sufficient therapeutic concentrations [26]. In accord with these data, we observed no significant differences in EFS between patients with or without polymorphic variants of TS and other enzymes, and this finding supports the effect of HD-MTX in our study. Studies have yielded contradictory results regarding associations between HD-MTX-related toxicity and FRG polymorphisms in children with ALL [3,9,10,11,12,13,14,15,16,17,18,19,20, 27,28,29,30,31]. Kantar et al. [3] reported that the MTHFR 148

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A1298C polymorphism caused severe hematological toxicity in patients with higher serum MTX levels, specifically anemia (62.5% of 37 cases), thrombocytopenia (51.5%), and aspartate aminotransferase elevation (11.8%). In contrast, a metaanalysis of all 14 studies on MTX suggested that less toxic effects were seen in cases with the MTHFR A1298 C polymorphism [14]. Most of these studies indicated that the MTHFR C677T gene polymorphism causes oral mucositis, myelosuppressive effects, and liver, intestinal, and skin toxicities, and also leads to an increased relapse rate in children with ALL [6,7,8,9,10,11,12, 13,14,15,16,17,18,19,22]. Only one report from Turkey has not confirmed this association [3]. In contrast to other research, we observed a somewhat higher rate of renal toxicity in patients who carried the MTHFR 677CT/TT genotypes compared to wildtype MTHFR enzymes [3,11,12,13,14,22,23]. Nevertheless, our previous report indicated that when severe renal toxicity occurs shortly after HD-MTX administration in children with leukemia, it resolves almost completely with time [37]. In addition to these findings, severe mucositis was also reported in other studies of children with ALL who carried MTHFR 677TT and MTRR 66GG [22,30]; however, neither of these publications specified whether HD-MTX was used alone or in combination with ARA-C. We observed lower frequencies of severe mucositis only in patients who carried TS 2R/2R, MTHFR CT/TT, and MTRR AA genotypes and received HD-MTX together with ID-ARA-C; however, in our clinic we have not used ARA-C since 2003 because research has indicated that this treatment has no effect on relapse rate [40]. Of three recent studies that examined the influence of TS polymorphism on chemotherapy toxicity, one revealed no significant abnormality and the others indicated that TS 2R and 3R allele polymorphisms were significantly associated with lower frequencies of leukocytopenia, thrombocytopenia, and peripheral neuropathy [6,23,29]. In accord with these findings, none of our patients with polymorphic variants of FRGs developed significant toxicities despite the fact that those with TS 2R/3R/4R variants had higher MTX levels at the 48-h time point. The conflicting results among these studies may be related to differences in MTX dosages, toxicity grades, or drug combinations. The small sample size of our study population is the main limitation of the present findings. We observed no significant differences between wild-type and other FRG variants with respect to toxicity or relapse rate, but further investigations with larger patient numbers are needed.

Conclusion In conclusion, the frequencies of the TS, MTRR, and MTHFR polymorphisms in Turkish children with ALL are similar to those reported for other Caucasian populations. Our study is the first published finding of the TS 3R/4R variant in the Turkish population. Our results indicate that HD-MTX can be tolerated by leukemic children with some polymorphic variants of FRGs and thus it may prevent future risk of leukemic relapse.


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Acknowledgment This study was supported in part by Grant No. 01/2011-31 from Gazi University. Ethics Ethics Committee Approval: The study protocol was approved by Gazi University Faculty of Medicine review board, Informed Consent: It was received. Authorship Contributions Concept: B.Y., Z.K., S.G.E., F.P., Ü.K., İ.Y., T.G.; Design: B.Y., Z.K., S.G.E., F.P., Ü.K., İ.Y., T.G.; Data Collection or Processing: B.Y., Z.K., S.G.E., F.P., Ü.K., İ.Y., T.G.; Analysis or Interpretation: B.Y., Z.K., S.G.E., F.P., Ü.K., İ.Y., T.G.; Literature Search: B.Y., Z.K., S.G.E., F.P., Ü.K., İ.Y., T.G.; Writing: B.Y., Z.K., S.G.E., F.P., Ü.K., İ.Y., T.G. Conflict of Interest: No conflict of interest was declared by the authors. Financial Disclosure: The authors declared that this study was supported by the Medical Faculty of Gazi University (Scientific Reserach Project No: 01/2011-31).

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0253 Turk J Hematol 2017;34:151-158

Association Between N363S and BclI Polymorphisms of the Glucocorticoid Receptor Gene (NR3C1) and Glucocorticoid Side Effects During Childhood Acute Lymphoblastic Leukemia Treatment Glukokortikoid Reseptör Geninin (NR3C1) N363S ve BclI Polimorfizmleri ile Çocukluk Çağı Akut Lenfoblastik Lösemi Tedavisi Sırasında Görülen Glukokortikoid Yan Etkileri Arasındaki İlişki Meriç Kaymak Cihan1, Halil Gürhan Karabulut2, Nüket Yürür Kutlay2, Hatice Ilgın Ruhi2, Ajlan Tükün2, Lale Olcay1 Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Clinic of Pediatrics, Division of Pediatric Hematology-Oncology, Ankara, Turkey 2 Ankara University Faculty of Medicine, Department of Medical Genetics, Ankara, Turkey 1

Abstract

Öz

Objective: Glucocorticoids (GCs) are the key drugs for the treatment of pediatric acute lymphoblastic leukemia (ALL). Herein, investigation of the relationship between the N363S and BclI polymorphisms of the GC receptor gene (NR3C1) and the side effects of GCs during pediatric ALL therapy was aimed.

Amaç: Çocukluk çağı akut lenfoblastik lösemisinde (ALL), glukokortikoidler (GC) tedavinin ana yapı taşını oluşturmaktadırlar. Bu çalışmada pediatrik ALL tedavisi sırasında GC’lerin yan etkileri ile GC reseptör geninin (NR3C1) N363S ve BclI polimorfizmleri arasındaki ilişkinin araştırılması amaçlandı.

Materials and Methods: N363S and BclI polymorphisms were analyzed in 49 patients with ALL treated between 2000 and 2012. The control group consisted of 46 patients with benign disorders. The side effects of GCs noted during the induction and reinduction periods were evaluated retrospectively according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events, version 4.0.

Gereç ve Yöntemler: N363S ve BclI polimorfizmleri 2000 ile 2012 yılları arasında tedavi edilmiş 49 ALL hastasında incelendi. Kontrol grubu benign hastalıkları olan 46 çocuktan oluşturuldu. İndüksiyon ve reindüksiyon sırasında görülen GC yan etkileri Ulusal Kanser Enstitüsü’nün “Advers Etkiler için Ortak Terminoloji Kriterleri” (CTCAE version 4,0), kullanılarak dosya ve hemşire gözlemlerinden geriye dönük olarak incelendi.

Results: The BclI allele and genotype frequencies were found similar in the two groups. No N363S polymorphism was detected in either of the groups. During induction, dyspepsia was found more frequently in the CG than in the CC (wild-type) genotype (36.4% vs. 5.3%, p=0.018) and depression symptoms more frequent in patients with the G allele (CG+GG) than the CC genotype (39.3% vs. 10.5%, p=0.031). During reinduction, Cushingoid changes, dyspepsia, and depression symptoms were more frequent in patients with the G allele (CG+GG) than in patients with the CC genotype (48.1% vs. 17.6%, p=0.041; 29.6% vs. 0.0%, p=0.016; 40.7% vs. 11.8%, p=0.040, respectively).

Bulgular: BclI alel ve genotip sıklığı açısından iki grup arasında fark yoktu. N363S polimorfizmi her iki grupta da tespit edilmedi. İndüksiyon sırasında, dispeptik yakınmalar CG genotipinde CC (yaban tip) genotipine göre daha sık görüldü (%36,4-%5,3; p=0,018) ve depresyon semptomları G alelini taşıyanlarda (CG+GG), CC genotipine göre daha sık tespit edildi (%39,3-%10,5; p=0,031). Reindüksiyon sırasında, Cushingoid değişiklikler, dispeptik yakınmalar ve depresyon semptomları G aleli taşıyanlarda (CG+GG) CC genotipine göre daha fazla tespit edildi (sırasıyla %48,1-%17,6, p=0,041; %29,6-%0,0 p=0,016; %40,7-%11,8; p=0,040).

Conclusion: In our study, patients with the BclI polymorphism were found to have developed more frequent side effects. We think that the BclI polymorphism should be considered while designing individualized therapies in childhood ALL.

Sonuç: Çalışmamızda, BclI polimorfizmine sahip bireyler daha sık yan etki gösterdiler. Çocukluk çağı ALL tedavisinde bireysel tedaviler geliştirilirken BclI polimorfizminin de göz önünde bulundurulmasının gerektiğini düşünüyoruz.

Keywords: Acute lymphoblastic leukemia, Glucocorticoid receptor gene, BclI and N363S polymorphisms

Anahtar Sözcükler: Akut lenfoblastik lösemi, Glukokortikoid reseptör geni, BclI ve N363S polimorfizmleri

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Lale Olcay M.D., Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Clinic of Pediatrics, Division of Pediatric Hematology-Oncology, Ankara, Turkey Phone: +90 532 760 09 82 E-mail: laleolcay@hotmail.com.tr

Received/Geliş tarihi: July 02, 2016 Accepted/Kabul tarihi: February 07, 2017

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Kaymak Cihan M, et al: BclI and N363S Polymorphisms and Pediatric Acute Lymphoblastic Leukemia

Introduction Glucocorticoids (GCs) are key drugs for the treatment of pediatric acute lymphoblastic leukemia (ALL) [1,2]. Their antileukemic effects occur through the induction of apoptosis and/or cell cycle arrest [3]. However, they give rise to severe side effects, which also show individual variation. Therefore, in this study we focused on individual genetic differences that may lead to increased sensitivity to GCs and their side effects. GC downstream effects are mediated through the process of binding to an intracytoplasmic glucocorticoid receptor (GR). This receptor gene (NR3C1) is located on the long arm of the fifth chromosome (5q31.3). A number of GR polymorphisms have been detected in recent years [4,5]. The 1220A/G transition that results in the substitution of asparagine to serine at codon 363 (N363S polymorphism) and the substitution of cytosine (C) to guanine (G) at intron 2 (BclI polymorphism) are related to high sensitivity to GCs [6]. The C allele is the most frequently occurring and thus can be considered the wild-type allele [7]. These polymorphisms cause personal variability in the sensitivity and responses to GCs [8]. In a study by Huizenga et al. [5], the carriers of the N363S polymorphism had lower cortisol levels after low-dose dexamethasone (0.25 mg) suppression tests. Cuzzoni et al. [6] showed that the BclI polymorphism caused increased sensitivity to methylprednisolone in human lymphocytes in vitro. Lin et al. [9] found that the frequency of N363S allele carriers in subjects with coronary artery disease was particularly high. Di Blasio et al. [10] found that among obese patients, heterozygous N363S carriers had significantly higher body mass index (BMI) when compared to wild-type homozygotes. The BclI polymorphism also plays an important role in certain psychiatric diseases [11,12,13]. The mechanism of GC sensitivity is still incompletely understood but the hypothesis that the BclI polymorphism changes GR dimerization and interactions between transcription factors is suggested [14]. There is variability in the treatment responses and side effects in patients with ALL [15,16], including GC treatment. While there have been a number of investigations of the GC response and GC receptor mutations and polymorphisms [3,17,18,19,20,21], there are a limited number of studies about the side effects of GCs in relation to GC receptor polymorphisms [22,23,24,25,26]. We hypothesized that the N363S and BclI polymorphisms could be important in the variability of side effects of GCs among patients with ALL. We think that this information will be important for improving personal treatment modalities in the future.

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group (R 3.0.1. open source program), based on the literature data [20]. Accordingly, a total of 49 patients (age: 1.4-17 years; 31 males, 18 females), 14 and 35 of whom were treated with the St Jude TXIII and Berlin-Frankfurt-Münster (BFM)-TR ALL 2000 protocols (the latter is modified from BFM 95) respectively between 2000 and 2012, were included in the study. Each patient enrolled in this study received GCs during the induction and reinduction phases. The patients received steroids as prednisolone at a daily dosage of 40 mg/m2 between day 1 and day 29 during the induction and reinduction phases in the St Jude TXIII protocol [2]. The types of steroids administered during the BFM-TR ALL 2000 protocol were prednisolone at 60 mg/ m2/day for 31 days in high-risk and 36 days in standard-risk and intermediate-risk patients during induction (protocol I/ phase I) and dexamethasone at 10 mg/m2/day during protocol II/phase I (reinduction). Forty-six patients aged 1.2-17.5 years with immune thrombocytopenia, nutritional anemia, or acute infections were included in the control group. Analysis of Glucocorticoid-Induced Side Effects We collected clinical and laboratory data retrospectively from patient files and classified potential side effects of GCs according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events, version 4.0 (CTCAE v.4) [27], which were defined as follows:

Metabolic and Nutritional Disorders: Hyperglycemia (fasting glucose value >100 mg/dL), hypertriglyceridemia (blood triglyceride value >150 mg/dL), obesity (BMI >25). Endocrine Disorders: Cushingoid changes (buffalo hump obesity, striations, adiposity, hypertension, diabetes, and osteoporosis). Gastrointestinal and Hepatic Disorders: Dyspepsia (an uncomfortable and often painful feeling in the stomach, burning stomach, bloating, heartburn, nausea, and vomiting), oral mucositis, gastric hemorrhage, and elevation in alanine aminotransferase, aspartate transaminase, and blood bilirubin levels.

Study Population

Musculoskeletal and Connective Tissue Disorders: Arthralgia, arthritis, generalized muscle weakness, myalgia, avascular necrosis, osteoporosis. Hip joint magnetic resonance imaging (MRI) was performed for detection of avascular necrosis if the patient had hip pain during or at the end of the induction and reinduction phases. Bone mineral densitometry (BMD) for osteoporosis was performed at the time of diagnosis and at the end of the induction and reinduction phases. At the end of induction 32 patients and at the end of reinduction 34 patients were investigated for BMD. Z-scores (corrected for age) between -1 and -2.5 and Z-scores below -2.5 were considered “osteopenia” and “osteoporosis,” respectively.

The necessary sample size was calculated to be at least 40 individuals in each group, with 80% power and 0.05 type I error while anticipating a deviation of ±20% in the control

Psychiatric Disorders: Depression symptoms (melancholic feelings of grief or unhappiness, change in sleep patterns, pessimistic thoughts, thoughts of death).

Materials and Methods

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Kaymak Cihan M, et al: BclI and N363S Polymorphisms and Pediatric Acute Lymphoblastic Leukemia

Nervous System Disorders: Leukoencephalopathy. Eye Disorders: Blurred vision, cataract, glaucoma. Cardiac Disorders: Sinus bradycardia, sinus tachycardia. Vascular Disorders: Hypertension, thrombosis (superficial and deep vein). Infections: Number of febrile neutropenia (FEN) attacks. Genetic Analysis Peripheral blood samples were collected from all patients when they were in remission. Genomic DNA isolation was performed using the standard salting-out method. N363S and BclI genotypes were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism. PCR was performed in total volumes of 20 µL containing 0.1 µg of genomic DNA, 10 pmol of each primer (forward: 5’-CCAGTAATGTAACACTGCCCC-3’, reverse: 5’-TTCGACCAGGGGAAGTTCAGA-3’ for N363S and forward: 5’-GAGAAATTCACCCCTACCAAC-3’, reverse: 5’-AGAGCCCTATTCTTCAAACTG-3’ for BclI), 0.2 mM of each dNTP, 10 mM Tris, 50 mM KCl, 1.5 mM MgCl2, and 1 U of Taq polymerase. PCR conditions were as follows: initial denaturation at 95 °C for 4 min and then 30 cycles of 95 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. Restriction digestion was performed in total volumes of 20 µL containing 10 µL of PCR product and 2 U of restriction enzyme (Tsp5091 for N363S and BclI for BclI). Reaction mixtures were incubated overnight at 65 °C for Tsp5091 and at 55 °C for BclI. Genotypes were determined by 2% agarose gel electrophoresis of restriction digests. In the absence of the N363S polymorphism, a 357-bp PCR product gave restriction fragments of 135, 73, 70, 60, and 19 bp, whereas the polymorphic allele produced four fragments of 135, 92, 70, and 60 bp. For the BclI polymorphism, 418-bp PCR products gave two fragments of 263 and 151 bp, whereas the polymorphic allele remained uncut. Statistical Analysis Data analysis was done using SPSS 11.5 for Windows. The Kolmogorov-Smirnov test was used to investigate whether the distribution of discrete numerical variables was near normal. Descriptive statistics for discrete numerical variables were shown as a median (minimum-maximum). The number of cases and the categorical variables were shown as frequency percentage (%). For comparison of the groups, the Mann-Whitney U test was used when the number of independent groups was 2 and the Kruskal-Wallis test was used when the number was >2. We tested for Hardy-Weinberg equilibrium for allele and genotype frequencies and they were found to be in equilibrium in both the patient and control groups. Categorical variables were investigated by Pearson chisquare or Fisher exact test. For the comparison of allele and

genotype frequencies of the control and patient groups and for comparison of side effect frequencies between the genotypes we used the Pearson chi-square test and Fisher exact chi-square test. The Cochran Q test was used to investigate whether there was significant change in osteoporosis incidence during followup of patients. Values of p<0.05 were considered statistically significant.

Results We did not find any statistically significant difference between the control and patient groups as to age (8.7 vs. 7.0 years, p=0.33) and sex (male and female: 47.8% vs. 63.3% and 52.2% vs. 36.7%, p=0.130, respectively) distribution. There was no N363S polymorphism in either the patient or the control group (Table 1). The heterozygosity rates of the BclI polymorphism in the control and patient groups were 37% (n=17) and 46.9% (n=23) while the homozygosity rates were 4.3% (n=2) and 12.2% (n=6), respectively. C allele frequencies in the control and patient groups were 69.8% and 59.7% and G allele frequencies 30.2% and 40.3%, respectively. There was no statistically significant difference between the two groups as to allele and polymorphism frequency (Table 1). Induction Phase Because two patients had received the induction phase of ALL therapy at another center, we performed induction phase investigations on 47 patients. In Table 2 the frequencies of adverse events seen during induction are listed. We performed hip MRI for 9 patients and only one of them had grade I avascular necrosis. Dyspeptic symptoms were more frequent among patients with CG rather than CC genotype (36.4% vs. 5.3%, p=0.018) (Table 3). Depression symptoms were more frequent among polymorphism carriers (CG+GG genotypes) than non-carriers (39.3% vs. 10.5%, p=0.031) (Table 3). There was no significant difference between polymorphism carriers and non-carriers as to hyperglycemia, hypertriglyceridemia, Cushingoid changes, liver function test abnormality, arthralgia, myalgia, muscle weakness, sinus tachycardia, thrombosis, number of FEN attacks, osteopenia and osteoporosis, and hypertension frequency (Table 2). Reinduction Phase Because five patients had been treated at another center, we analyzed the side effects of 44 patients during the reinduction phase. In Table 2 frequencies of adverse events seen during induction are listed. Nine patients were investigated by hip MRI for avascular necrosis. Three patients had osteonecrosis and two of them underwent bilateral hip joint replacement operations. At the end of reinduction we investigated 34 patients based 153


Kaymak Cihan M, et al: BclI and N363S Polymorphisms and Pediatric Acute Lymphoblastic Leukemia

Turk J Hematol 2017;34:151-158

Table 1. Distribution of N363S and BclI polymorphisms in control and patient groups.  

Control group (n=46) Case number (%)

Patients (n=49) Case number (%)

p

Odds ratioξ (Confidence interval: 95%)

N363S

AA

46 (100.0)

49 (100.0)

-

-

BclI

-

-

-

-

CC

27 (58.7)

20 (40.8)

-

1.000

CG

17 (37.0)

23 (46.9)

0.240†

1.664 (0.710-3.900)

GG

2 (4.3)

6 (12.2)

0.137‡

3.857 (0.705-21.090)

CG+GG

19 (41.3)

29 (59.1)

0.123

1.895 (0.839-4.281)

BclI

-

-

-

-

C

44 (69.8)

43 (59.7)

-

1.169

29 (40.3)

0.220†

1.532 (0.764-3.163)

G

19 (30.2)

Pearson chi-square test, Fisher exact chi-square test,  bivariate logistic regression analysis. Allele and genotype frequencies were in Hardy-Weinberg equilibrium in both the patient and control groups.  †

ξ

on BMD for osteoporosis. Eight (23.5%) patients had no osteopenia or osteoporosis, but the corrected (for age) Z-scores of 14/34 patients (41.2%) were between -1 and -2.5. Twelve of 34 patients (35.3%) developed osteoporosis and one of these cases was grade 3 (limiting self-care) according to CTCAE v.4. Cushingoid changes (54.3% vs. 17.6%, p=0.043), depression symptoms (50.0% vs. 11.8%, p=0.011), and dyspepsia (36.4% vs. 0.0%, p=0.008) were more frequent among patients with the CG genotype than those with the CC genotype (Table 4). Cushingoid changes (48.1% vs. 17.6%, p=0.041), dyspepsia (29.6% vs. 0.0%, p=0.016), and depression symptoms (40.7% vs. 11.8%, p=0.040) were more frequent among polymorphism carriers (CG+GG genotypes) than non-carriers (Table 4). There was no significant difference between polymorphism carriers and non-carriers as to hyperglycemia, hypertriglyceridemia, liver function test abnormality, arthralgia, myalgia, muscle weakness, sinus tachycardia, and thrombosis frequency (Table 4).

Table 2. Frequencies of adverse events seen during the induction and reinduction phases. Adverse events

Induction (n=47)

Reinduction (n=44)

Hyperglycemia, n (%)

21 (44.7)

36 (81.8)

Hypertriglyceridemia, n (%)

10 (21.3)

5 (11.4)

Cushingoid changes, n (%)

10 (21.2)

16 (77.2)

Increased liver function tests, n (%)

36 (76.6)

34 (77.3)

Dyspepsia, n (%)

9 (19.2)

8 (18.2)

GIS bleeding, n (%)

3 (6.4)

0 (0.0)

Arthralgia, n (%)

9 (19.2)

7 (15.9)

Muscle weakness, n (%)

10 (21.4)

10 (22.7)

Myalgia, n (%)

12 (25.6)

13 (29.5)

Hypertension, n (%)

7 (14.9)

4 (9.1)

In total, 22 patients were investigated regularly for BMD at the time of diagnosis, at the end of induction, and at reinduction. There was no significant change between BMD at diagnosis, at the end of induction, and at the end of reinduction (Table 5).

Depression symptoms, n (%)

13 (27.6)

13 (29.5)

Obesity (BMI>25), n (%)

1 (2.1)

2 (4.6)

Discussion

Blurred vision, n (%)

2 (4.3)

1 (2.3)

Some children with leukemia experience the side effects of steroids much more so than others, even in an exaggerated manner. GC receptor polymorphisms (N363S and BclI) may cause a genetic predilection to increased sensitivity to GCs, and their early recognition may help clinicians to modify the steroid doses in future trials. In this study, in which we searched for the presence of GR polymorphisms that yield high sensitivity to GCs, we found that carriers of the BclI polymorphism were more prone to side effects of GCs like Cushingoid changes, dyspepsia, and depression symptoms for the first time in the literature as far as we know.

Glaucoma, n (%)

1 (2.1)

1 (2.3)

Sinus tachycardia, n (%)

6 (12.8)

5 (11.8)

Sinus bradycardia, n (%)

1 (2.1)

3 (6.8)

Superficial vein thrombosis, n (%)

6 (12.8)

1 (2.3)

Deep vein thrombosis, n (%)

1 (2.1)

1 (2.3)

Febrile neutropenia, n (%)

32 (68.1)

22 (50.0)

154

GIS: Gastrointestinal system, BMI: body mass index.


Kaymak Cihan M, et al: BclI and N363S Polymorphisms and Pediatric Acute Lymphoblastic Leukemia

Turk J Hematol 2017;34:151-158

We found that the N363S polymorphism was absent in our population, consistent with the results in Asian populations in which no N363S polymorphism was found [28]. Likewise, it was recently reported in a Turkish population that the frequency of N363S G allele carriers was 0.5% [29]. In contrast to our results, in Europe, the frequency of carrying at least one G allele of the N363S polymorphism was reported to range from 4% to 9% [26,28], highlighting the ethnic factors in its carriage. The C allele of the BclI polymorphism is the most frequent allele and this is considered to be the wild type [7]. The frequencies of the C and G alleles of the BclI polymorphism

are about 65% and 35%, respectively [7]. We found similar results to those in the literature in the control and patient groups with regard to C and G allele frequency (Table 1). Marino et al. [24] reported the frequencies of GC side effects during induction and reinduction in children with ALL who were treated according to the Italian BFM ALL 2000 study protocol. During the induction phase they found the frequency of depression as 69.4%, Cushingoid changes as 50.0%, hypertriglyceridemia as 8.3%, hyperglycemia as 22.2%, and hypertension as 5.6%. During the reinduction phase, they found the frequency of depression as 58.3%, Cushingoid changes as

Table 3. Side effect distributions of patient group during induction phase according to genotypes of BclI polymorphisms. Adverse events

CC (n=19) Frequency (%)

CG (n=22) Frequency (%)

GG (n=6) Frequency (%)

CG+GG (n=28) Frequency (%)

p1

p2

OR1

OR2

Hyperglycemia (n=21)

Positive Negative

9 (47.4) 10 (52.6)

10 (45.5) 12 (54.5)

2 (33.3) 4 (66.7)

12 (42.9) 16 (57.1)

0.830†

0.760†

-

0.833

Hypertrigliseridemia (n=10)

Positive Negative

5 (26.3) 14 (73.7)

3 (13.6) 19 (86.4)

2 (33.3) 4 (66.3)

5 (17.9) 23 (82.1)

0.455†

0.496‡

-

0.609

Cushingoid changes (n=10)

Positive Negative

4 (21.1) 15 (78.9)

5 (22.7) 17 (77.3)

1 (16.7) 5 (83.3)

6 (21.4) 22 (78.6)

0.949†

1.000‡

-

1.023

Increased liver function tests (n=36)

Positive Negative

16 (84.2) 3 (15.8)

15 (68.2) 7 (31.8)

5 (83.3) 1 (16.7)

20 (71.4) 8 (28.6)

0.441†

0.485‡

-

0.469

Dyspepsia (n=9)

Positive Negative

1 (5.3)a 18 (94.7)

8 (36.4)a 14 (63.6)

0 (0.0) 6 (100)

8 (28.6) 20 (71.4)

0.018†

0.064‡

10.286

7.2

Arthralgia (n=9)

Positive Negative

4 (21.1) 15 (78.9)

3 (13.6) 19 (86.4)

2 (33.3) 4 (66.7)

5 (17.9) 23 (82.1)

0.534†

1.000‡

-

0.815

Muscle weakness (n=10)

Positive Negative

3 (15.8) 16 (84.2)

6 (27.3) 16 (72.7)

1 (16.7) 5 (83.3)

7 (25.0) 21 (75.0)

0.641†

0.718‡

-

1.778

Myalgia (n=12)

Positive Negative

5 (26.3) 14 (73.7)

6 (27.3) 16 (72.7)

1 (16.7) 5 (83.3)

7 (25.0) 21 (75.0)

0.865†

1.000‡

-

0.933

Osteopenia and osteoporosis (n=21/32)

Positive Negative

7/14 (50) 7 /14(50)

12/16 (75.0) 4/16 (25.0)

2/2 (100) 0/2 (0.0)

14/18 (77.8) 4/18 (22.2)

0.203†

0.142‡

-

3.5

Sinus tachycardia (n=6)

Positive Negative

4 (21.1) 15 (78.9)

1 (4.5) 21 (95.5)

1 (16.7) 5 (83.3)

2 (7.1) 26 (92.9)

0.274†

0.204‡

-

0.288

Thrombosis (n=7)

Positive Negative

5 (26.3) 14 (73.7)

2 (9.1) 20 (90.9)

0 (0.0) 6 (100)

2 (7.1) 26 (92.9)

0.166†

0.102‡

-

0.215

Hypertension (n=7)

Positive Negative

3 (15.8) 16 (84.2)

4 (18.2) 18 (81.9)

0 (0.0) 6 (100)

4 (14.3) 24 (85.7)

0.535†

1.000‡

-

0.889

Depression symptoms (n=13)

Positive Negative

2 (10.5) 17 (89.5)

9 (40.9) 13 (59.1)

2 (33.3) 4 (66.7)

11 (39.3) 17 (60.7)

0.090†

0.031†

-

5.5

FEN (n=32)

Positive Negative

11 (57.9) 8 (42.1)

18 (81.8) 4 (18.2)

3 (50.0) 3 (50.0)

21 (75.0) 7 (25.0)

0.156†

0.217†

-

2.182

FEN: Febrile neutropenia. p1: p-value from the comparison of CC, CG, and GG genotypes. p2: p-value from the comparison of CC and CG+GG genotypes. †Pearson’s chi-square test. Fisher’s exact chi-square test. a: There is a significant difference between CC and CG genotype (p<0.05). OR1: Odds ratio for comparison of CC and CG genotypes.

OR2: Odds ratio for comparison of CC and CG+GG genotypes.

155


Kaymak Cihan M, et al: BclI and N363S Polymorphisms and Pediatric Acute Lymphoblastic Leukemia

Turk J Hematol 2017;34:151-158

Table 4. Side effect distributions of patient group during reinduction phase according to genotypes of BclI polymorphism Adverse events

CC (n=17) Frequency (%)

CG (n=22) Frequency (%)

GG (n=5) Frequency (%)

CG+GG (n=27) Frequency (%)

p1

p2

OR1

OR2

Hyperglycemia (n=8)

Positive Negative

2 (11.8) 15 (88.2)

6 (27.3) 16 (72.7)

0 (0.0) 5 (100.0)

6 (22.2) 21 (77.8)

0.246†

0.455†

-

2.143

Hypertriglyceridemia (n=5)

Positive Negative

2 (11.8) 15 (88.2)

2 (9.1) 20 (90.9)

1 (20.0) 4 (80.0)

3 (11.1) 24 (88.9)

0.784†

1.000‡

-

0.938

Cushingoid changes (n=16)

Positive Negative

3 (17.6)a,b 14 (82.4)

12 (54.5)a 10 (45.5)

1 (20.0) 4 (80.0)

13 (48.1)b 14 (51.9)

0.043†

0.041‡

5.600

4.333

Increased liver function tests (n=34)

Positive Negative

12 (70.6) 5 (29.4)

18 (81.8) 4 (18.2)

4 (80.0) 1 (20.0)

22 (81.5) 5 (18.5)

0.700†

0.473†

-

1.833

Dyspepsia (n=8)

Positive Negative

0 (0.0)a,b 17 (100.0)

8 (36.4)a 14 (63.6)

0 (0.0) 5 (100.0)

8 (29.6)b 19 (70.4)

0.008†

0.016†

-

-

Arthralgia (n=7)

Positive Negative

3 (17.6) 14 (82.4)

3 (13.6) 19 (86.4)

1 (20.0) 4 (80.0)

4 (14.8) 23 (85.2)

0.911†

1.000‡

-

0.812

Muscle weakness (n=10)

Positive Negative

3 (17.6) 14 (82.4)

6 (27.3) 16 (72.7)

1 (20.0) 4 (80.0)

7 (25.9) 20 (74.1)

0.767†

0.716‡

-

1.633

Myalgia (n=13)

Positive Negative

7 (41.2) 10 (58.8)

5 (22.7) 17 (77.3)

1 (20.0) 4 (80.0)

6 (22.2) 21 (77.8)

0.404†

0.180†

-

0.408

Osteopenia and osteoporosis (n=26/34)

Positive Negative

10/14 (71.4) 4/14 (28.6)

11/15 (73.3) 4/15 (26.7)

5/5 (100.0) 0/5 (0.0)

16/20 (80.0) 4/20 (20.0)

0.403†

0.689‡

-

1.6

Sinus tachycardia (n=5)

Positive Negative

0 (0.0) 17 (100.0)

5 (22.7) 17 (77.3)

0 (0.0) 5 (100.0)

5 (18.5) 22 (81.5)

0.060†

0.139‡

-

-

Depression symptoms (n=13)

Positive Negative

2 (11.8)a,b 15 (88.2)

11 (50.0)a 11 (50.0)

0 (0.0) 5 (100.0)

11 (40.7)b 16 (59.3)

0.011†

0.040†

7.500

5.156

FEN (n=22)

Positive Negative

8 (47.1) 9 (52.9)

12 (54.5) 10 (45.5)

2 (40.0) 3 (60.0)

14 (51.9) 13 (48.1)

0.802†

1.000‡

-

1.212

FEN: Febrile neutropenia. p1: p-value from the comparison of CC, CG, and GG genotypes. p2: p-value from the comparison of CC and CG+GG genotypes. †Pearson’s chi-square test. ‡ Fisher’s exact chi-square test. a: There is a significant difference between CC and CG genotype (p<0.05). b: There is a significant difference between CC and CG+GG genotype (p<0.05). OR1: Odds ratio for comparison of CC and CG genotypes. OR2: Odds ratio for comparison of CC and CG+GG genotypes.

Table 5. Distribution of bone mineral densitometry results of the patient group. Bone mineral densitometry

Diagnosis

Induction

Reinduction

p

case number (%)

case number (%)

case number (%)

Normal

9 (40.9)

8 (36.4)

7 (31.8)

0.576†

Z: -1 to -2.5

8 (36.4)

8 (36.4)

8 (36.4)

-

Z: Below -2.5 and a decrease in height

4 (18.2)

5 (22.7)

6 (27.3)

-

Osteoporosis limiting self-care

1 (4.5)

1 (4.5)

1 (4.5)

-

Cochran Q test.

58.3%, hyperglycemia as 22.2%, gastritis as 11.1%, hypertension

N363S and BclI polymorphisms are known to make patients

as 2.7%, and hypertriglyceridemia as 0%. We have encountered

more prone to the side effects of the GCs [28,29,30,31]. The

some adverse events less frequently (Cushingoid changes,

effects of GR gene mutations and polymorphisms on treatment

depression symptoms) or more frequently (hypertriglyceridemia,

success and drug resistance were also recently investigated and

hyperglycemia, and hypertension) than Marino et al. [24].

controversial results were obtained [3,20,21,26]. In our study

The differences between our results and those of Marino et

we did not investigate the effect of the BclI polymorphism

al. [24] may be due to ethnic and genetic differences. Both

on treatment success. Alternatively, we focused on the side

156


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Kaymak Cihan M, et al: BclI and N363S Polymorphisms and Pediatric Acute Lymphoblastic Leukemia

effects of GCs and their relation with GR gene polymorphisms. In the literature there are few studies about the side effects of GCs and GR polymorphisms. In Marino et al.’s [24] study no relationship was found between the polymorphisms (N363S and BclI) and appetite, weight gain, Cushingoid changes, depression, anxiety, emotional lability, neuromuscular weakness, myalgia, or severity of infections in pediatric ALL patients during the induction and reinduction periods. Eipel et al. [26] found that the BclI polymorphism was not associated with hepatotoxicity, glucose metabolism abnormalities, central nervous system or behavioral abnormalities, or hypertension. We found no relation between Cushingoid changes and the BclI polymorphism during the induction phase, but a more frequent Cushingoid appearance was seen in patients with CG+GG than in patients with the CC genotype during the reinduction phase (Table 3), in contrast to the results of Marino et al. [24]. In their study Eipel et al. [26] did not analyze Cushingoid appearance as a side effect. Other studies [32,33] found higher levels of subcutaneous fat tissue in BclI polymorphism carriers than in non-carriers. Since Cushingoid changes are related to changes in the distribution of subcutaneous fat tissue, we think that our findings support these findings of previous studies [32,33]. During the reinduction period we used high-dose dexamethasone, which is more potent than prednisolone [23]. The discrepancy in the relationship between BclI polymorphism and Cushingoid appearance during the induction period while the opposite was observed during the reinduction phase may be due to the potency of dexamethasone. We found that dyspepsia was more frequent in BclI polymorphism carriers than non-carriers during both phases (Tables 3 and 4). We could not find any information about the relationship between GR polymorphisms and dyspepsia or gastrointestinal disorders in the English literature. Therefore, our study seems to be the first study in the literature to report on this aspect, as well. In our study, depression symptoms were more frequent in BclI polymorphism carriers during both phases (Tables 3 and 4). GCs are known to be related to adverse psychiatric side effects during pediatric ALL treatment [33,34]. Felder-Puig et al. [23] and Eipel et al. [26] found no relation between the BclI polymorphism and adverse psychiatric side effects in pediatric ALL patients. On the other hand, there are reports showing that the BclI polymorphism was more frequent in patients with major depression [11,12]. Therefore, our results are consistent with those in the literature [11,12]. Osteopenia and osteoporosis were more frequent in the CG and GG than in the CC genotype during the two phases, although the difference was not significant (Tables 3 and 4). Similarly, te Winkel et al. [25] reported no relationship between the BclI polymorphism and BMD in 69 pediatric ALL patients. In conclusion, in our sample, the adequacy of which was confirmed by power analysis, we found that the N363S polymorphism was absent and the BclI polymorphism prevailed, and patients who were carriers of the BclI polymorphism were

more prone to side effects of GCs like Cushingoid changes, dyspepsia, and depression symptoms. As far as we know, our study is the first to determine any relation between the BclI polymorphism and symptoms of depression and dyspepsia as side effects of GCs in childhood. The absence of the N363S polymorphism in our study sample in contrast with reports from other countries also highlights the variabilities of side effects of GCs in patients from different ethnic and geographic regions. In our study, a larger number of patients with definite side effects (including those with sinus tachycardia and osteopenia/ osteoporosis) and those consuming standard types of steroids could have provided further information about the relation between side effects and the BclI polymorphism during ALL treatment, which was the main limitation of our study. However, despite these limitations, this pilot study draws attention to the role of the BclI polymorphism on the development of particularly the aforementioned side effects during ALL treatment in Turkey and emphasizes that its role should be validated in further ethnic, regional, and global studies while designing individual treatment modalities. Acknowledgments This study is based on Meriç Kaymak Cihan’s dissertation at Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital. We are grateful to Professor İnci Ergürhan İlhan and Professor Nilgün Kurucu for presenting their helpful comments as members of the dissertation jury, to our nurses for drawing blood from the patients, to Doctor Fatma Balcı for helping to collect blood from the patients, and to our patients for participating in the study and their parents for kindly giving permission for their children to participate in the study. Ethics Ethics Committee Approval: The study was approved by the ethics committee of Dr. Abdurrahman Yurtaslan Oncology Education and Training Hospital; Informed Consent: Written informed consent was obtained from all participants or their parents according to the Declaration of Helsinki. The participants attended voluntarily. Authorship Contributions Medical Practices: M.K.C., L.O.; Data Collection and Processing: M.K.C., L.O.; Genetic Analysis: H.G.K., N.Y.K., H.I.R., A.T.; Literature Research: M.K.C., H.G.K., N.Y.K., H.I.R., A.T., L.O.; Analysis or Interpretation: M.K.C., H.G.K., N.Y.K., H.I.R., A.T., L.O.; Writing: M.K.C., H.G.K., N.Y.K., H.I.R., A.T., L.O. 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. 157


Kaymak Cihan M, et al: BclI and N363S Polymorphisms and Pediatric Acute Lymphoblastic Leukemia

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17. Hillmann AG, Ramdas J, Multanen K, Norman MR, Harmon JM. Glucocorticoid receptor gene mutations in leukemic cells acquired in vitro and in vivo. Cancer Res 2000;60:2056-2062. 18. Catts VS, Farnsworth ML, Haber M, Norris MD, Lutze-Mann LH, Lock RB. High level resistance to glucocorticoids, associated with a dysfunctional glucocorticoid receptor, in childhood acute lymphoblastic leukemia cells selected for methotrexate resistance. Leukemia 2001;15:929-935. 19. Irving JA, Minto L, Bailey S, Hall AG. Loss of heterozygosity and somatic mutations of the glucocorticoid receptor gene are rarely found at relapse in pediatric acute lymphoblastic leukemia but may occur in a subpopulation early in the disease course. Cancer Res 2005;65:9712-9718. 20. Labuda M, Gahier A, Gagné V, Moghrabi A, Sinnett D, Krajinovic M. Polymorphisms in glucocorticoid receptor gene and the outcome of childhood acute lymphoblastic leukemia (ALL). Leuk Res 2010;34:492-497. 21. Namazi S, Zareifar S, Monabati A, Ansari S, Karimzadeh I. Evaluating the effect of 3 glucocorticoid receptor gene polymorphisms on risk relapse in 100 Iranian children with acute lymphoblastic leukemia: a case-control study. Clin Ther 2011;33:280-290. 22. Eipel OT, Németh K, Török D, Csordás K, Hegyi M, Ponyi A, Ferenczy A, Erdélyi DJ, Csóka M, Kovács GT. The glucocorticoid receptor gene polymorphism N363S predisposes to severe toxic side effects during pediatric acute lymphoblastic leukemia (ALL) therapy. Int J Hematol 2013;97:216-222. 23. Felder-Puig R, Scherzer C, Baumgartner M, Ortner M, Aschenbrenner C, Bieglmayer C, Voigtländer T, Panzer-Grümayer ER, Tissing WJ, Koper JW, Steinberger K, Nasel C, Gadner H, Topf R, Dworzak M. Glucocorticoids in the treatment of children with acute lymphoblastic leukemia and Hodgkin’s disease: a pilot study on the adverse psychological reactions and possible associations with neurobiological, endocrine and genetic markers. Clin Cancer Res 2007;13:7093-7100. 24. Marino S, Verzegnassi F, Tamaro P, Stocco G, Bartoli F, Decorti G, Rabusin M. Response to glucocorticoids and toxicity in childhood acute lymphoblastic leukemia: role of polymorphisms of genes involved in glucocorticoid response. Pediatr Blood Cancer 2009;53:984-991.

9. Lin RC, Wang XL, Morris BJ. Association of coronary artery disease with glucocorticoid receptor N363S variant. Hypertension 2003;41:404-407.

25. te Winkel ML, van Beek RD, de Muinck Keizer-Schrama SM, Uitterlinden AG, Hop WC, Pieters R, van den Heuvel-Eibrink MM. Pharmacogenetic risk factors for altered bone mineral density and body composition in pediatric acute lymphoblastic leukemia. Haematologica 2010;95:752-759.

10. Di Blasio AM, van Rossum EF, Maestrini S, Berselli ME, Tagliaferri M, Podestà F, Koper JW, Liuzzi A, Lamberts SW. The relation between two polymorphisms in the glucocorticoid receptor gene and body mass index, blood pressure and cholesterol in obese patients. Clin Endocrinol (Oxf) 2003;59:68-74.

26. Eipel O, Hegyi M, Csordas K, Nemeth K, Luczay A, Török D, Csoka M, Erdeyi D, Kovacs G. Some GCR polymorphisms (N363S, ER22/23K, and Bcl-1) may influence steroid-induced toxicities and survival rates in children with ALL. J Ped Hematol Oncol 2016;38:334-339.

11. van Rossum EF, Binder EB, Majer M, Koper JW, Ising M, Modell S, Salyakina D, Lamberts SW, Holsboer F. Polymorphisms of the glucocorticoid receptor gene and major depression. Biol Psychiatry 2006;59:681-688.

27. National Institutes of Health. Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0. Washington, U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute, 2010.

12. Krishnamurthy P, Romagni P, Torvik S, Gold PW, Charney DS, Detera-Wadleigh S, Cizza G; P.O.W.E.R. (Premenopausal, Osteoporosis Women, Alendronate, Depression) Study Group. Glucocorticoid receptor gene polymorphisms premenopausal women with major depression. Horm Metab Res 2008;40:194198. 13. Zobel A, Jessen F, von Widdern O, Schuhmacher A, Höfels S, Metten M, Rietschel M, Scheef L, Block W, Becker T, Schild HH, Maier W, Schwab SG. Unipolar depression and hippocampal volume: impact of DNA sequence variants of the glucocorticoid receptor gene. Am J Med Genet B Neuropsychiatr Genet 2008;147:836-843.

28. Koper JW, van Rossum EF, van der Akker EL. Glucocorticoid receptor polymorphisms and haplotypes and their expression in health and disease. Steroids 2014;92:62-73. 29. Kaya Z, Caglayan S, Akkiprik M, Aral C, Ozisik G, Ozata M, Ozer A. Impact of glucocorticoid receptor gene (NR3C1) polymorphisms in Turkish patients with metabolic syndrome. J Endocrinol Invest 2016;39:557-566. 30. Roussel R, Reis AF, Dubois-Laforgue D, Bellanné-Chantelot C, Timsit J, Velho G. The N363S polymorphism in the glucocorticoid receptor gene is associated with overweight in subjects with type 2 diabetes mellitus. Clin Endocrinol (Oxf) 2003;59:237-241.

14. Savic D, Knezevic G, Damjanovic S, Antic J, Matic G. GR gene BclI polymorphysm changes the path, but not the level, of dexamethasone-induced cortisol suppression. J Affect Disord 2014;168:1-4.

31. Manenschijn L, van den Akker EL, Lamberts SW, van Rossum EF. Clinical features associated with glucocorticoid receptor polymorphisms. Ann N Y Acad Sci 2009;1179:179-198.

15. Lauten M, Stanulla M, Zimmermann M, Welte K, Riehm H, Schrappe M. Clinical outcome of patients with childhood acute lymphoblastic leukemia and an initial leukemic blast count less than 1000 per microliter. Klin Padiatr 2001;213:169174.

32. Tremblay A, Bouchard L, Bouchard C, Després JP, Drapeau V, Pérusse L. Longterm adiposity changes are related to glucocorticoid receptor polymorphism in young females. J Clin Endocrinol Metab 2003;88:3141-3145.

16. Schrappe M, Reiter A, Zimmermann M, Harbott J, Ludwig WD, Henze G, Gadner H, Odenwald E, Riehm H. Long term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Münster. Leukemia 2000;14:2205-2222.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0225 Turk J Hematol 2017;34:159-166

Cytomegalovirus Infection and Treatment in Allogeneic Hematopoietic Stem Cell Transplantation: A Retrospective Study from a Single Institution in an Endemic Area Allojeneik Hematopoietik Kök Hücre Transplantasyonunda Sitomegalovirüs Enfeksiyonu ve Tedavisi: Endemik Bir Alanda Tek Merkezden Retrospektif Bir Çalışma Hsin-Chen Lin1, Shao-Min Han1, Wen-Li Hwang1, Cheng-Wei Chou1, Kuang-Hsi Chang2, Zhi-Yuan Shi3, Chieh-Lin Jerry Teng1,4,5 Taichung Veterans General Hospital, Department of Medicine, Division of Hematology-Medical Oncology, Taichung, Taiwan China Medical University, Department of Public Health, Taichung, Taiwan 3 Taichung Veterans General Hospital Division of Infectious Diseases, Department of Medicine, Taichung, Taiwan 4 Tunghai University, Department of Life Science, Tunghai, Taichung, Taiwan 5 Chung Shan Medical University Faculty of Medicine, Division of Hematology, Taichung, Taiwan 1 2

Abstract

Öz

Objective: Although Cytomegalovirus (CMV) infection is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the risk factors for CMV reactivation and treatment failure in CMV endemic areas have remained unclear. This study investigated the risk factors for CMV reactivation among allo-HSCT recipients in an area where CMV is highly endemic.

Amaç: Sitomegalovirüs (CMV) enfeksiyonu, allojeneik kök hücre transplantasyonu (allo-KHT) sonrası majör bir komplikasyon olmasına rağmen CMV’nin endemik olduğu alanlarda CMV reaktivasyonu ve tedavi başarısızlığı için risk faktörleri belirsizliğini korumaktadır. Bu çalışma CMV’nin büyük ölçüde endemik olduğu bir alanda allo-KHT alıcılarında CMV reaktivasyonu için risk faktörlerini araştırmıştır.

Materials and Methods: Medical records of 82 allo-HSCT recipients from a CMV endemic area were retrospectively reviewed. The patients were stratified into two groups: those with CMV reactivation (n=32) and those without CMV reactivation (n=50). We investigated possible variables associated with CMV reactivation and treatment failure.

Gereç ve Yöntemler: CMV endemik olduğu bir alandan 82 allo-KHN alıcısının tıbbi kayıtları retrospektif olarak incelendi. Hastalar iki gruba ayrıldı: CMV reaktivasyonu olan (n=32) ve CMV reaktivasyonu olmayan (n=50). CMV reaktivasyonu ve tedavi başarısızlığı ile ilişkili olası değişkenler araştırıldı.

Results: Univariate analyses showed that non-remission disease status [hazard ratio (HR): 2.15; p=0.032] and ≥grade III acute graftversus-host disease (GVHD) (HR: 3.07; p=0.002) were associated with CMV reactivation. Multivariate analysis further demonstrated that older age (HR: 1.03; p=0.029) and ≥grade III acute GVHD (HR: 2.98; p=0.012) were associated with CMV reactivation. Overall survival time seemed lower among patients with CMV reactivation than among patients without CMV reactivation, although the difference was not statistically significant (p=0.165). The absence of ≥grade III acute GVHD was associated with successful CMV treatment in the current study (odds ratio: 4.40; p=0.008).

Bulgular: Tek değişkenli analiz CMV reaktivasyonunun remisyonolmayan hastalık durumu [risk oranı (RO): 2,15; p=0,032) ve ≥grade III akut graft versus host hastalığı (GVHH) (RO: 3,07; p=0,002) ile ilişkili olduğunu gösterdi. Çok değişkenli analiz ayrıca CMV reaktivasyonunun ileri yaş (RO: 1,03; p=0,029) ve ≥grade III akut GVHH (RO: 2,98; p=0,012) ile ilişkili olduğunu gösterdi. Genel sağkalım CMV reaktivasyonu olan hastalarda CMV reaktivasyonu olmayan hastalardan daha düşük görünmekle birlikte fark istatistiksel olarak anlamlı değildi (p=0,165). Bu çalışmada, ≥grade III akut GVHH yokluğu başarılı CMV tedavisi ile ilişkili idi (olasılık oranı: 4,40; p=0,008).

Conclusion: Prophylactic anti-CMV therapy might need to be considered for allo-HSCT recipients who have ≥grade III GVHD.

Sonuç: Profilaktik anti-CMV tedavisinin ≥grade III GVHH olan alloKHT alıcılarında dikkate alınması gerekebilir.

Keywords: Allogeneic hematopoietic stem cell transplantation, Cytomegalovirus, Graft-versus-host disease, Taiwan

Anahtar Sözcükler: Allojeneik hematopoietik kök hücre transplantasyonu, Sitomegalovirüs, Graft versus host hastalığı, Tayvan

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Chieh-Lin Jerry TENG, M.D., Taichung Veterans General Hospital, Department of Medicine, Division of Hematology-Medical Oncology, Taichung, Taiwan Phone: +886-4-23592525 E-mail: drteng@vghtc.gov.tw

Received/Geliş tarihi: June 17, 2016 Accepted/Kabul tarihi: September 06, 2016

159


Lin HC, et al: Cytomegalovirus in Taiwanese Allo-Hematopoietic Stem Cell Transplantation Recipients

Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) not only improves survival times in patients with acute myeloid leukemia [1] and acute lymphoid leukemia [2], but may also be the only curative therapy for very severe aplastic anemia [3]. Nonetheless, the morbidity and mortality that are associated with allo-HSCT limit its clinical application and efficacy. In addition to graft-versus-host disease (GVHD), infection remains one of the most important complications after allo-HSCT [4]. The incidence of each infection in allo-HSCT recipients varies depending on the time since transplantation. During the neutropenic phase, the principal pathogens are bacteria and Candida species. In contrast, Cytomegalovirus (CMV) reactivation is the major infectious complication between 30 and 100 days after transplantation. Infections in the late phase are relatively heterogeneous, which is associated with the presence and severity of GVHD [5]. Among the different infectious complications that occur in patients undergoing allo-HSCT, the clinical entity of CMV infection is unique. Reactivation of CMV appears in 60% of seropositive allo-HSCT recipients. Without appropriate treatment, asymptomatic CMV reactivation eventually progresses to symptomatic CMV diseases, which can result in death, especially in immunocompromised hosts. Typically, CMV mainly affects the lungs and gastrointestinal tract [6]. However, CMV retinitis is also common, occurring in 5% of high-risk pediatric allo-HSCT recipients [7]. Although the incidence of symptomatic CMV diseases has decreased significantly because of universal prophylaxis or preemptive therapy, this life-threatening complication still develops in 30% of all allo-HSCT recipients [8]. In addition, CMV seroprevalence is quite endemic [9]. The strategies of CMV prophylaxis and treatment can be entirely different for allo-HSCT recipients in CMV endemic areas and those in nonendemic areas. Moreover, it remains unclear whether ganciclovir in combination with CMV immunoglobulin is more effective than ganciclovir alone for the treatment of CMV reactivation; further investigation is necessary.

Turk J Hematol 2017;34:159-166

institutional review board, informed consent was not required from the patients because of the retrospective study design. Medical records were evaluated for 86 consecutive ≥18-yearold patients who received allo-HSCT at our institution for various hematological diseases from February 2010 to November 2015. Patients without regular follow-up (n=2) and those who died before successful engraftment (n=2) were excluded. The remaining 82 patients were included in the analyses of this study. The median follow-up time for these 82 patients was 513 days (range: 23 to 2045 days). The clinical characteristics of all of the patients are shown in Table 1. The mean ± standard deviation age of our study cohort was 41.98±14.57 years. Acute myeloid leukemia (47.6%) was the major underlying disease that required allo-HSCT. Regarding CMV serostatus, 92.68% (76/82) of recipients were CMV-seropositive before allo-HSCT, while 85.37% (70/82) of donors were CMV-seropositive. Complete remission could not be defined in patients with aplastic anemia (n=9) and chronic myeloid leukemia (n=3). The median OS time was not reached in this study cohort. Conditioning Regimen In this study, the non-myeloablative conditioning regimen was provided irrespective of the patient’s underlying disease. It consisted of total body irradiation (TBI) (200 cGy, day -7) and the administration of fludarabine (30 mg/m2/day, from day -6 to day -2) and cyclophosphamide (10 mg/kg/day, from day -5 to day -2). As compared with the non-myeloablative regimen, the myeloablative regimens in the current study were relatively heterogeneous. A TBI-based conditioning regimen (TBI: 1200 cGy, 6 fractions, from day -6 to day -4; cyclophosphamide: 60 mg/kg/day, from day -3 to day -2) was used for patients with acute lymphoblastic leukemia. BuCy2 was routinely delivered to patients with acute myeloid leukemia, myelodysplasia syndrome, or chronic myeloid leukemia [10]. Lymphoma patients who received a myeloablative preparative regimen were conditioned using BEAM [11]. In terms of haploidentical transplantation, we followed the Johns Hopkins protocol [12]. Graft-Versus-Host Disease Prophylaxis

Materials and Methods

We used cyclosporine as the major immunosuppressant. A trough level of 150-250 ng/mL was the targeted concentration. Myfortic acid was used since day -2 at a dose of 720 mg twice daily and was generally discontinued on day 60. With the exception of the patients undergoing haploidentical transplantation, patients received short-course methotrexate at 15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11. Antithymoglobulin (ATG) was routinely given to patients without matched sibling donors at 2 mg/kg/day from day -4 to -2.

Patients

Cytomegalovirus Monitoring and Treatment

The review board of Taichung Veterans General Hospital approved this study. According to the regulations of the

Our allo-HSCT protocol did not contain CMV antiviral prophylaxis. Because Taiwan is an endemic area for CMV infection, blood

We conducted this retrospective study to address these issues, specifically by investigating the risk factors for CMV reactivation among allo-HSCT recipients in an area where CMV is highly endemic. We additionally compared the overall survival (OS) time in patients with and without CMV reactivation. Finally, factors associated with CMV treatment failure were also analyzed.

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Turk J Hematol 2017;34:159-166

Lin HC, et al: Cytomegalovirus in Taiwanese Allo-Hematopoietic Stem Cell Transplantation Recipients

donation volunteers do not routinely check their CMV serostatus. Since getting CMV-negative blood products is difficult, to avoid further CMV infection, all allo-HSCT recipients in our study only received leukodepleted and irradiated blood products if blood transfusion was needed. We used quantitative polymerase chain reaction with a COBAS AmpliPrep/COBAS TaqMan CMV system (Roche Molecular Systems, Inc., Branchburg, NJ, USA) to detect the serum CMV viral load. The cutoff of negativity was set as <3333 copies/mL after internal adjustment and validation. Serum CMV viral load was generally monitored once every 1 to 2 weeks. CMV treatment with ganciclovir at 5 mg/kg/day was initiated preemptively in patients with a serum CMV viral load of >3333 copies/mL or with symptomatic CMV infection. Further, 81.3% (26/32) of patients with CMV reactivation also received CMV immunoglobulin simultaneously at a dose of 10,000 units per day on every other day, for a total of five doses. CMV treatment was defined as having been successful if the symptoms of CMV infection disappeared completely and the serum viral load became negative. Efficacy Assessments The patients were stratified into two groups: those with CMV reactivation (n=32) and those without CMV reactivation (n=50). Clinical characteristics, causes of death, and OS were compared between these two groups of patients. We attempted to identify variables associated with CMV reactivation and the failure of CMV treatment. Only the first episode of CMV reactivation was investigated in the current study. Statistical Analysis Student t-tests and Fisher exact tests were used to compare nominal and ordinal variables between patients with and without CMV reactivation. Differences in OS were assessed using the log-rank test. Cox proportional hazards regression was used to investigate variables that were potentially associated with CMV reactivation and successful CMV treatment, as quantified in terms of hazard ratios (HRs) and accompanying 95% confidence intervals (CIs). Data are summarized as mean ± standard deviation where appropriate. P<0.05 was regarded as indicating statistical significance. All statistical analyses were conducted using SPSS 20.0 (IBM Corp., Armonk, NY, USA).

diseases (p=0.951), donor types (p=0.332), CMV serology status (p=0.176), ATG use (p=0.076), presence of ≥grade III acute GVHD (p=0.940), and disease status before allo-HSCT (p=0.101) did not differ significantly between the two groups of patients (Table 1). Risk Factors Associated with Cytomegalovirus Reactivation The average time until the first CMV reactivation was 42.1±31.0 days. Univariate analyses showed that a disease status of noncomplete remission (HR: 2.15; 95% CI: 1.07 to 4.43; p=0.032) and ≥grade III acute GVHD (HR: 3.07; 95% CI: 1.53 to 6.16; p=0.002) were associated with CMV reactivation. Multivariate analysis further demonstrated that older age (HR: 1.03; 95% CI: 1.00 to 1.06; p=0.029) and ≥grade III acute GVHD (HR: 2.98; 95% CI: 1.27 to 6.95; p=0.012) were significantly associated with increased risks of CMV reactivation (Table 2). Patients with Cytomegalovirus Reactivation Had a Trend Toward Inferior Overall Survival The median OS time for patients with CMV reactivation was 508 days. However, median OS time was not reached during the follow-up period in patients without CMV reactivation. Although a trend toward inferior OS times was observed in patients with CMV, the difference was not significantly significant (p=0.165) (Figure 1). Cause of Death Analyses Overall, 36 patients (43.9%) died in our study cohort. The mortality rates of patients with and without CMV reactivation were 53.1% (17/32) and 38.0% (19/50), respectively. For patients with CMV reactivation, CMV infection remained the major cause of death, accounting for 41.2% (7/17) of deaths. In contrast,

Results Comparisons of Clinical Characteristics between Patients with and without Cytomegalovirus Reactivation Patients without CMV reactivation were younger than patients with CMV reactivation (38.74±13.29 years vs. 47.03±15.23 years; p=0.011). Additionally, more patients in the no-CMVreactivation group received myeloablative conditioning regimens (78.0% vs. 53.1%; p=0.032). Sex (p=0.899), underlying

Figure 1. The median overall survival time for patients with Cytomegalovirus (CMV) reactivation was 508 days. However, the median survival time was not reached during the follow-up period for patients without CMV reactivation. Although a trend toward inferior overall survival was observed in patients with CMV reactivation, the difference was not statistically significant (p=0.165). 161


Lin HC, et al: Cytomegalovirus in Taiwanese Allo-Hematopoietic Stem Cell Transplantation Recipients

Turk J Hematol 2017;34:159-166

Table 1. Comparisons of clinical characteristics between patients with and without Cytomegalovirus reactivation.

Age, years

Mean ± SD

All patients

CMV reactivation

No CMV reactivation

n=82

n=32

n=50

p

41.98±14.57

47.03±15.23

38.74±13.29

0.011*

Sex (n, %)

Diagnosis (n, %)

Donor type (n, %)

CMV serology (n, %)

Conditioning regimen

TBI or chemo-based regimen (n, %)

ATG (n, %)

Disease status (n, %)

162

0.899§ Male

43

52.4%

16

50.0%

27

54.0%

-

Female

39

47.6%

16

50.0%

23

46.0%

-

-

-

-

-

-

-

-

0.951§

AML

39

47.6%

16

50.0%

23

46.0%

-

ALL

20

24.4%

8

25.0%

12

24.0%

-

Lymphoma

8

9.8%

2

6.3%

6

12.0%

-

CML

3

3.7%

1

3.1%

2

4.0%

-

Aplastic anemia

9

11.0%

4

12.5%

5

10.0%

Multiple myeloma

2

2.4%

1

3.1%

1

2.0%

-

MDS

1

1.2%

0

0.0%

1

2.0%

-

-

-

-

-

-

-

-

0.332§

Matched sibling

27

32.9%

7

21.9%

20

40.0%

-

Matched, unrelated

21

25.6%

9

28.1%

12

24.0%

-

Mismatched, unrelated

29

35.4%

13

40.6%

16

32.0%

-

Haploidentical

5

6.1%

3

9.4%

2

4.0%

-

-

-

-

-

-

-

0.176§

D+/R+

66

80.5%

23

71.9%

43

86.0%

-

D+/R-

4

4.9%

1

3.1%

3

6.0%

-

D-/R+

10

12.2%

7

21.9%

3

6.0%

-

D-/R-

2

2.4%

1

3.1%

1

2.0%

-

-

-

-

-

-

-

-

0.032§

Myeloablative

56

68.3%

17

53.1%

39

78.0%

-

Non-myeloablative

21

25.6%

11

34.4%

10

20.0%

-

Haploidentical

5

6.1%

4

12.5%

1

2.0%

-

-

-

-

-

-

-

-

0.963§

TBI-based

19

23.2%

8

25.0%

11

22.0%

-

Chemo-based

63

76.8%

24

75.0%

39

78.0%

-

-

-

-

-

-

-

-

0.076§

Yes

56

68.3%

26

81.3%

30

60.0%

-

No

26

31.7%

6

18.8%

20

40.0%

-

-

-

-

-

-

-

-

0.101§

CR

52

63.4%

20

62.5%

32

64.0%

-

Non-CR

18

22.0%

10

31.3%

8

16.0%

-

Others

12

14.6%

2

6.3%

10

20.0%

-


Lin HC, et al: Cytomegalovirus in Taiwanese Allo-Hematopoietic Stem Cell Transplantation Recipients

Turk J Hematol 2017;34:159-166

Table 1. Continued. ≥Grade 3 aGVHD (n, %)

-

-

-

-

-

-

-

0.940§

Yes

17

20.7%

6

18.7%

11

22.0%

-

No

65

79.3%

26

81.3%

39

78.0%

-

CMV: Cytomegalovirus, SD: standard deviation, AML: acute myeloid leukemia, ALL: acute lymphoid leukemia, CML: chronic myeloid leukemia, MDS: myelodysplastic syndrome, D: donor, R: recipient, TBI: total body irradiation, ATG: antithymoglobulin, CR: complete remission, aGVHD: acute graft-versus-host disease. *indicates Student t-test; §indicates Fisher exact test.

Table 2. Risk factors associated with Cytomegalovirus reactivation. Univariate

Age

HR

95% CI

1.01

0.99

Multivariate p

HR

95% CI

p

1.04

0.273

1.03

1.00

1.06

0.029

Sex

Male vs. female

1.26

0.65

2.44

0.489

1.08

0.53

2.21

0.838

Donor type

Donors other than matched sibling donors vs. matched sibling donors

2.21

0.97

5.05

0.060

1.83

0.76

4.44

0.179

CMV serology

Donor (+) vs. donor (-)

1.02

0.4

2.63

0.968

0.79

0.27

2.31

0.661

Recipient (+) vs. recipient (-)

1.36

0.33

5.64

0.677

0.62

0.13

2.97

0.55

Conditioning regimen

Myeloablative vs. non-myeloablative

2.03

0.88

4.66

0.097

2.37

0.89

6.33

0.085

ATG

Yes vs. no

0.67

0.34

1.32

0.247

0.93

0.41

2.13

0.865

Disease status

Non-CR vs. CR

2.15

1.07

4.34

0.032

1.10

0.64

1.91

0.732

≥Grade 3 aGVHD

Yes vs. no

3.07

1.53

6.16

0.002

2.98

1.27

6.95

0.012

a

Myeloablative regimen includes conventional myeloablative regimens and regimen specific for haploidentical allogeneic hematopoietic stem cell transplantation.

a

CMV: Cytomegalovirus, HR: hazard ratio, CI: confidence interval, ATG: antithymoglobulin, CR: complete remission, aGVHD: acute graft-versus-host disease.

52.6% of deaths in patients without CMV reactivation were attributable to relapse of the underlying disease. The average OS time was 142.4±92.5 days among patients who died of CMV infection and 285.6±210.7 days among patients who died of their underlying diseases (Table 3). Variables Associated Infection Treatment

with

Successful

Cytomegalovirus

Because of the limited number of patients in the study cohort, we only conducted univariate analyses, the results of which are shown in Table 4. Briefly, the absence of ≥grade III acute GVHD was the only variable that was significantly associated with successful CMV treatment in the current study (odds ratio: 4.40; 95% CI: 1.48 to 13.15; p=0.008). Notably, use of CMV immunoglobulin was not significantly associated with CMV treatment success (odds ratio: 2.57; 95% CI: 0.58 to 11.50; p=0.217).

Discussion CMV serostatus is the most important factor for CMV reactivation in patients undergoing allo-HSCT [13]. In an analysis of a Portuguese cohort that comprised 85.81% CMVseropositive recipients and 73.27% CMV-seropositive donors,

Table 3. Causes of death in patients undergoing allogeneic hematopoietic stem cell transplantation. CMV reactivation, (n=17)

No CMV reactivation, (n=19)

n (%)

n (%)

CMV infection

7 (41.2)

0 (0)

GVHD

2 (11.8)

7 (36.8)

Underlying diseases

6 (35.3)

10 (52.6)

Others

2 (11.8)

2 (10.5)

CMV: Cytomegalovirus, GVHD: graft-versus-host disease.

Sousa et al. [14] observed that 60.3% of patients developed CMV infection after allo-HSCT. In our study, 92.68% of recipients and 85.37% of donors were CMV-seropositive, but the incidence of CMV reactivation was only 39.02%. One possible explanation for this lower incidence of CMV reactivation could be the lower incidence of acute GVHD in the present study. Acute GVHD occurred in 70.8% of patients in the Portuguese cohort, yet only 20.73% (17/82) of our patients had ≥grade III acute GVHD. Moreover, among all of the variables investigated in the present study, ≥grade III acute GVHD was the only variable that was a significant risk factor for CMV reactivation in both 163


Lin HC, et al: Cytomegalovirus in Taiwanese Allo-Hematopoietic Stem Cell Transplantation Recipients

Turk J Hematol 2017;34:159-166

Table 4. Variables associated with successful Cytomegalovirus treatment by univariate analysis (n=32). Age

OR

95% CI

p

0.99

0.96

1.02

0.548

Sex

Male vs. female

0.89

0.34

2.31

0.809

Donor type

Donors other than matched sibling donors vs. matched sibling donor

4.58

0.61

34.56

0.140

Donor (+) vs. donor (-)

1.67

0.48

5.83

0.422

Recipient (+) vs. recipient (-)

22.87

0.01

56,684.9 0.432

Condition regimen

Myeloablativea vs. non-myeloablative

1.84

0.65

5.26

0.254

ATG

Yes vs. no

0.36

0.12

1.07

0.066

Disease status

Non-CR vs. CR

2.08

0.77

5.60

0.148

CMV viral load (copies/mL)

<10,000 vs. ≥10,000

1.26

0.29

5.54

0.762

Use of CMV immunoglobulins

Yes vs. no

2.57

0.58

11.50

0.217

≥Grade 3 aGVHD

No vs. yes

4.40

1.48

13.15

0.008

CMV serology

Myeloablative regimen includes conventional myeloablative regimens and regimen specific for haploidentical allogeneic hematopoietic stem cell transplantation.

a

CMV: Cytomegalovirus, OR: odds ratio, CI: confidence interval, ATG: antithymoglobulin, CR: complete remission, aGVHD: acute graft-versus-host disease.

univariate (p=0.002) and multivariate (p=0.012) analyses (Table 2). Our result is confirmed by a study of Cohen et al. [15], in which GVHD was also identified as a risk factor for first CMV reactivation in allo-HSCT recipients (HR=5.091, p=0.021). In addition to GVHD, conditioning regimens could be also associated with CMV reactivation in patients receiving allo-HSCT. However, evidence concerning the association between conditioning regimens and CMV reactivation has been inconsistent. Although Cohen et al. [15] identified the myeloablative preparative regimen as a risk factor, George et al. [13] considered the non-myeloablative regimen to be an independent predictor of CMV reactivation. Interestingly, the type of preparative regimen did not correlate with CMV reactivation in our study. Additionally, our data did not demonstrate an association between the use of ATG and CMV reactivation, either. This result, however, is not consistent with the prior findings of Wu et al. [16]. Small study cohort and short follow-up time could be among the reasons for these inconsistent findings, suggesting that further studies are required. Previous studies also investigated whether CMV reactivation results in inferior outcomes in allo-HSCT patients. Sousa et al. [14] found that CMV infection was associated with inferior median post-transplantation survival in allo-HSCT recipients. However, the data from our cohort did not show a statistically significant difference in OS between patients with and without CMV reactivation. This result is similar to the prior findings of Liu et al. [17], who found no survival disadvantage for patients with CMV infection (p=0.699) or CMV disease (p=0.093). Notably, CMV infection is not entirely a poor prognostic predictor for allo-HSCT. On the contrary, CMV reactivation is considered 164

to reduce the risk of relapse in patients with acute myeloid leukemia after allo-HSCT [18,19]. This protective effect might partially reverse the inferior outcomes of CMV reactivation in patients undergoing allo-HSCT. Additionally, the good efficacy of CMV treatment could potentially also be responsible for the non-inferiority of OS in patients with CMV infection. In our study cohort, the rate of successful CMV treatment for the first reactivation was as high as 87.50%. In terms of CMV treatment, although the rates of clearance of CMV viremia are similar with oral valganciclovir and ganciclovir in the post-allo-HSCT population [20], ganciclovir remains the drug of choice in our allo-HSCT setting. Moreover, most of the patients with CMV reactivation (81.3%) in our study cohort were treated with CMV immunoglobulin simultaneously. However, whether CMV immunoglobulin could facilitate successful CMV treatment remains controversial. A study by Ranganathan et al. [21] revealed that prophylactic CMV immunoglobulin decreased risk of CMV infection, but not CMV disease, after lung transplantation in pediatric patients. The univariate analyses of our data also did not demonstrate a significant association between the use of CMV immunoglobulin and the success of CMV treatment (odds ratio: 2.57; 95% CI: 0.58 to 11.50; p=0.217). More studies are required to identify the role and efficacy of CMV immunoglobulin in allo-HSCT. Among the 32 patients with first CMV reactivation in our study cohort, treatment failure occurred in four patients (4/32, 12.50%). This result raises another important issue: the identification of individuals who are at higher risk of treatment failure and possibly require CMV prophylaxis. CMV prophylaxis by valganciclovir appears to fail as a means of improving CMV disease-free and invasive infection-free survival in allo-HSCT


Turk J Hematol 2017;34:159-166

Lin HC, et al: Cytomegalovirus in Taiwanese Allo-Hematopoietic Stem Cell Transplantation Recipients

recipients [22]. A phase 2 study by Chemaly et al. [23] showed that letermovir prophylaxis effectively reduced the incidence of CMV infection after allo-HSCT. Its impact on OS, however, was not clear. CMV antiviral prophylaxis was not part of the routine of our allo-HSCT protocol. Notably, our results showed that patients with ≥grade III GVHD were more refractory to CMV treatment (odds ratio: 4.40; 95% CI: 1.48 to 13.15; p=0.008), suggesting that patients with ≥grade III GVHD could potentially benefit from valganciclovir prophylaxis. However, more evidence is needed before jumping to any conclusions. The major limitations of the current study are its retrospective study design and the small number of patients. In addition, the active immunosuppressants of each patient at the time at which either CMV or GVHD occurs are very heterogeneous. The current study could not precisely identify the impact of immunosuppression on CMV reactivation. Moreover, the role of CMV immunoglobulin in either preemptive or targeted treatment remains uncertain after our analyses. Studies with prospective and randomized-control designs, focusing on more particular clinical scenarios, are urgently needed for this unmet clinical need.

Conclusion Older age, non-complete remission disease status, and ≥grade III GVHD were risk factors for CMV reactivation in allo-HSCT recipients. The presence of ≥grade III acute GVHD could be associated with CMV treatment failure. Prophylactic anti-CMV therapy needs to be considered in allo-HSCT recipients with ≥grade III GVHD. Ethics Ethics Committee Approval: Institutional Review Board of the Taichung Veterans General Hospital (TCVGH-IRB) Taiwan, CE16033B; Informed Consent: Informed consent was not required due to retrospective study. Authorship Contributions Concept: H.C.L., C.L.J.T.; Design: H.C.L., C.L.J.T.; Data Collection or Processing: H.C.L., S.M.H., W.L.H., C.W.C., K.H.C., Z.Y.S., C.L.J.T.; Analysis or Interpretation: K.H.C., C.L.J.T; Literature Search: H.C.L., S.M.H., W.L.H., C.W.C., K.H.C., Z.Y.S., C.L.J.T.; Writing: C.L.J.T. 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.2016.0123 Turk J Hematol 2017;34:167-173

Could Neutrophil CD64 Expression Be Used as a Diagnostic Parameter of Bacteremia in Patients with Febrile Neutropenia? Nötrofil CD64 Ekspresyonu Febril Nötropenik Hastaların Bakteriyemisinde Tanı Kriteri Olarak Kullanılabilir mi? Nur Efe İris1, Taner Yıldırmak2, Habip Gedik2, Funda Şimşek2, Demet Aydın3, Naciye Demirel3, Osman Yokuş3 İstanbul Bilim University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey Okmeydanı Training and Research Hospital, Clinic of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey 3 Okmeydanı Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 1 2

Abstract

Öz

Objective: The aim of this study is to investigate if neutrophil CD64 expression in febrile neutropenia patients could be used as an early indicator of bacteremia. Materials and Methods: All consecutive patients older than 18 years of age who had developed febrile neutropenia episodes due to hematological malignancies were included in the study. Those patients who had significant growth in their blood cultures constituted the case group, while those who had febrile neutropenia without any growth in their cultures and who did not have any documented infections formed the control group. Blood culture bottles were incubated in the Bact ALERT 3D system (bioMerieux, France), identification and susceptibility testing were performed using an automated broth microdilution method (VITEK 2, bioMerieux), and CD64 expression analysis was performed by the flow cytometry method. C-reactive protein (CRP) was measured by turbidimetric methods (Biosystems, Spain) and erythrocyte sedimentation rate (ESR) was measured by the Wintrobe method. Results: In total, we prospectively evaluated 31 febrile episodes. The case group consisted of 17 patients while the control group included 14 patients. CD64 was found on neutrophils of the case group patients with a mean count of 8006 molecules/cell and of control group with a mean count of 2786 molecules/cell. CD64 levels of the case group were significantly higher than those of the control group (p=0.005). In the differentiation of the case group from the control group, a 2500 cut-off value for CD64 had significant [AUC=0.792 (0.619-0.965)] predictive value (p=0.001). In the prediction of patients with a 2500 cut-off value for CD64, sensitivity was 94.1%, positive predictive value was 76.2%, specificity was 64.3%, and negative predictive value was 90.0%. CRP levels and ESR values did not differ significantly between the groups (p=0.005). Conclusion: Neutrophil CD64 expression could be a good predictor as an immune parameter with high sensitivity and a negative predictive value for bacteremia in febrile neutropenic patients. Keywords: Neutrophil, CD64, Bacteremia, Febril neutropenia, Diagnostic parameter

Amaç: Çalışmanın amacı febril nötropenik hastalarda nötrofil CD64 ekspresyonunun bakteriyemi tanısında erken bir tanımlayıcı olarak kullanılıp kulanılamayacağını araştırmaktır. Gereç ve Yöntemler: Hematolojik malignite tanısıyla izlenip febril nötropeni atağı geçiren, 18 yaşından büyük olgular çalışmaya alındı. Olgulardan kan kültürlerinde anlamlı üreme saptanan grup olgu grubunu, febril nötropenik olup kültürlerinde üreme olmayan ve dökümante edilen klinik enfeksiyon bulgusu da olmayan olgular da kontrol grubunu oluşturdu. Kan kültürleri Bact ALERT 3D sisteminde inkübe edildi (bioMerieux, Fransa), identifikasyon ve antibiyotik duyarlılık testi otomatize broth mikrodilüsyon yöntemi (VİTEK 2) ile çalışıldı. CD64 ekspresyon analizi de flow sitometri yöntemi ile çalışıldı. C-reaktif protein (CRP) turbidimetrik yöntemle ölçüldü (Biosystems, İspanya), eritrosit sedimantasyon hızı da (ESH) Wintrobe yöntemi ile çalışıldı. Bulgular: Prospektif olarak 31 febril nötropenik atağı değerlendirildi. Olgu grubu 17 hastadan, kontrol grubu 14 hastadan oluşuyordu. Olgu grubundaki hastalarda nötrofiller üzerindeki CD64 molekülü sayısı ortalama 8006 molekül/hücre ve kontrol grubunda ise ortalama 2786 molekül/hücre idi. Olgu grubundaki CD64 düzeyleri kontrol grubunun değerlerinden anlamlı olarak yüksek bulundu (p=0,005). Olgu grubunun kontrol grubundan ayrımında CD64 için 2500 cut off değeri anlamlı bulundu [AUC=0,792 (0,619-0,965)] prediktif değer (p=0,001). Hastaların tahmininde CD64 için 2500 lük cut off değerinin duyarlılığı %94,1; pozitif prediktif değeri %76,2, duyarlılığı %64,3, negatif prediktif değeri %90 olarak hesaplandı. Gruplar arasında CRP düzeyleri ve ESH değerleri arasında anlamlı bir farklılık saptanmadı (p=0,005). Sonuç: Nötrofil CD64 ekspresyonu febril nötropenik hastalarda bakteriyeminin tanısında yüksek duyarlılıklı ve negatif prediktif değere sahip iyi bir parametre olabilir. Anahtar Sözcükler: Nötrofil, CD64, Bakteriyemi, Febril nötropeni, Diyagnostik parametre

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Nur EFE İRİS, M.D., İstanbul Bilim University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey Phone : +90 212 361 88 00 E-mail : nurefeiris@yahoo.com

Received/Geliş tarihi: March 24, 2016 Accepted/Kabul tarihi: June 27, 2016

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Introduction Patients with neutropenia are very prone to bacterial infections, and especially in severely neutropenic patients, it is difficult to diagnose bacterial infection due to the lack of local signs of infection. Fever may be the only sign. For patients with febrile neutropenia, bacteremia is a very severe clinical presentation because these patients can easily progress to sepsis and septic shock. Positive blood culture is the gold standard for bacteremia but there are some difficulties. Difficulty in exclusion of infection is harmful in febrile neutropenia patients with suspected sepsis, as continuation of broad-spectrum antibiotics for presumptive bacterial infection frequently leads to unnecessary treatment and the possibility of multiresistant organisms. It brings toxicity, allergic reactions, and increased cost, too [1,2,3]. Thus, we need an improved diagnostic test for detecting bacterial infection and bacteremia in febrile neutropenic patients in the early period. Acute phase reactants like C-reactive protein (CRP) have been used as indicators of bacterial infections since the 1970s. However, CRP has some limitations; for example, it is elevated in noninfectious processes. Procalcitonin (PCT) is a more reliable test for sepsis, especially as it has a very good negative predictive value for sepsis. PCT is more widely used in the diagnosis of severe sepsis and bacterial infections. It is also used in monitoring the success of antimicrobial treatment. PCT is secreted upon exposure to endotoxin. However, it might be increased in noninfectious conditions, too, like severe congestive heart failure and acute pancreatitis [2,3,4,5,6,7]. Many investigators have used various hematologic and biochemical markers and cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, soluble IL-1ra, IL-2 receptor, IL-6, IL-8, IL-10, and markers of complement-activation for this purpose. Another approach in the early diagnosis of sepsis is neutrophil CD64 expression [1,8,9,10,11,12,13,14]. Neutrophils are very important cells for antibacterial immunity. Their basic function is phagocytosis. The surface receptors of neutrophils recognize bacterial antigens and this interaction activates the neutrophils to phagocytosis. Phagocytosis is facilitated by various receptors for immunoglobulin-G (IgG) and neutrophils can express three classes of IgG receptors. Fc gamma receptor I (Fc gamma RI) is recognized by the monoclonal antibody CD64 [12]. CD64, a high-affinity immunoglobulin Fc gamma RI, is constitutively expressed on monocytes but upregulated during acute-phase reactions on polymorphonuclear neutrophils (PMNs). Measured on PMNs, CD64 expression is a sensitive biomarker for bacterial infection [2,12,15,16,17]. In febrile neutropenic patients, can we use neutrophil CD64 expression as a rapid diagnostic tool as an indicator of bacteremia? The aim of our study was to answer this question. 168

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Our goal was to determine if neutrophil CD64 expression has a significant correlation with microbiologically documented bacterial infections and if neutrophil CD64 expression can predict the results of a patient’s blood culture.

Materials and Methods Subjects This single-center prospective study was carried out at the Okmeydanı Training and Research Hospital in İstanbul, Turkey. All consecutive patients between February 2013 and July 2013, who were older than 18 years of age and who had developed febrile neutropenic episodes during chemotherapy due to hematological cancers in the hematology department, were included in the study. Febrile neutropenia was defined as an oral temperature of >38.3 °C or two consecutive readings of >38 °C for 2 h and an absolute neutrophil count of <0.5x109/L or a count expected to fall below 0.5x109/L. We collected data including sex, age, comorbidity, and suspected origin of fever, and we collected clinical and laboratory data including body temperature, blood pressure, complete blood count, liver enzymes, creatinine, prothrombin time, CRP, chest radiographs, and urine, sputum, and blood cultures. Thirty-one episodes of febrile neutropenia in patients undergoing chemotherapy were selected independently of the type of underlying hematological disease. I. Case group: This group included patients with a confirmed diagnosis of sepsis, which was defined by positive blood culture results. II. Control group: This group included patients without sepsis, with suspected infection but negative blood culture results. Single episodes of febrile neutropenia were considered; thus, the same patient may have been included more than once. Patients undergoing chemotherapy and with fever but with absolute neutrophil counts of >1.0x109/L were excluded from the study. We performed blood cultures for each patient in the first episode of the febrile neutropenic period and before starting antibiotic therapy. We placed 10 mL of blood in aerobic and anaerobic blood culture bottles and incubated them in the Bact ALERT 3D system (bioMerieux, France). Identification and susceptibility testing were performed using an automated broth microdilution method (VITEK 2, bioMerieux). We measured neutrophil CD64 expression by flow cytometry following the manufacturer’s instructions. The BD QuantiBRITE CD64/CD45 Phycoerythrin Florescence Quantitation Kit (Becton Dickinson, USA) was used. Quantitative flow cytometric analysis of the ethylenediaminetetraacetic acid specimens was performed with the FACSCalibur machine (Becton Dickinson). The cytometer


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was routinely optimized using CaliBRITE beads (Becton Dickinson). Before each analysis, QuantiBRITE polyethylene (PE) beads conjugated with four predefined levels of PE molecules were used to construct a standard linear regression curve. The amount of PE molecules bound per cell standard for the absolute number of fluorochrome antibody binding sites per cell was calculated. In principle, each QuantiBRITE grade antibody molecule is conjugated to one PE fluorochrome molecule. The instrument measures the CD64 antigen, which is one of three Fc receptors for immunoglobulins, including human Fc gamma RII (CD32) and human Fc gamma RIII (CD16) antigens, present on the surface of leukocytes. QuantiBRITE kits contain calibration beads for the fluorescent labels. CD64 was measured using the BD QuantiBRITE CD64 PE/ CD45 PerCP assay (Becton Dickinson). The assay was carried out essentially as described in the product instructions. Samples consisting of 50 µL of blood were stained in polystyrene tubes of 12x75 mm by adding 20 µL of staining reagent and incubating at room temperature (20-25 °C) for 30 min in the dark. Then 1.0 mL of FACS lysing solution was added to each tube, and the tubes were vortexed at low speed for approximately 1 or 2 s and incubated at room temperature for approximately 5 min in the dark. The level of CD64 expression, reported as antibodies bound per cell, was determined as described in the product instructions. Serum concentrations of CRP were measured by turbidimetric methods (Biosystems, Spain). Erythrocyte sedimentation rate (ESR) was measured by the Wintrobe method. Statistical Analysis Our primary aim was the comparison of two groups for their neutrophil CD64 expressions. We determined the standard effect size as 1.05 for the two groups before the study. It was decided to include 14 patients for each group as the minimum, with 80% power and 5% confidence interval. Three more patients were kept as a reserve for the case group. In the descriptive statistics of the data, mean, standard deviation, median, lowest value, highest value, and percentage were used. The distribution of the variables was measured with the Kolmogorov-Smirnov test. The Mann-Whitney U test was used for analyzing the quantitative data. Effect level and cut-off values were assessed with ROC curves. For correspondence analysis, the kappa test was used. SPSS 22.0 was used for analyses.

culture results and 14 involved negative results. Regarding the blood cultures, coagulase-negative methicillin-resistant Staphylococcus was isolated in eight episodes (all patients had clinical signs and this organism was isolated in three blood culture bottles), Escherichia coli in five (ESBL-positive E. coli in two), Pseudomonas aeruginosa in two, Klebsiella pneumoniae in one, and Kluyvera ascorbata in one. CD64 was found on neutrophils of case group patients at a mean count of 8006 and median count of 7786 molecules/cell and on the neutrophils of the control group at a mean count of 2876 and median count of 2452 molecules/cell (Figure 1). CD64 was found on neutrophils of gram-negative bacteremia patients at a mean count of 8562 molecules/cell and on neutrophils of gram-positive bacteremia patients at a mean count of 7380 molecules/cell. The mean CRP level was 82 mg/L in the positive culture group and it was 43 mg/L in the negative culture group (Figure 2). The mean ESR level was 79 mm/h in the positive culture group and 82 mm/h in the negative culture group (Figure 3). CRP and ESR levels did not differ significantly among the groups (p=0.084 and p=0.005; Table 1). The mean total leukocyte count was 0.618x103/µL and the mean neutrophil count was 0.245x103/µL in the group with positive Table 1. Characteristics of the study group. Characteristics

Number of the patients (n=19)

Age (years)

47.9 (±11.4)

Sex (male/female)

11 male/8 female

Acute myeloid leukemia

13

Acute lymphoblastic leukemia

4

Non-Hodgkin lymphoma

2

Results Thirty-one episodes of febrile neutropenia were analyzed; analyzed episodes that corresponded to patients with two or more episodes of neutropenia were considered as separate events and were included as such in the analysis. Among the 31 included events, 17 episodes involved positive blood

Figure 1. Box plot graph shows the comparison between CD64 expression (molecules/cell) in the case group (mean ± standard deviation: 8006±4243; median: 7786) and the control group (mean ± standard deviation: 2876±2149; median: 2452) (p=0.001). 169


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blood culture (case group), while mean total leukocyte count was 0.706x103/µL and mean neutrophil count was 0.124x103/µL in the group with negative blood culture (control group) (Figure 4). CD64 levels of the bacteremia group were significantly higher than those of the control group (p=0.005). In the distinction of the study group from the control group, CD64 levels had a significant [AUC=0.866 (0.737-0.994)] predictive value (p=0.001; Table 2). In the differentiation of the case group from the control group, a cut-off value of 2500 molecules/cells for CD64 had a significant [AUC=0.792 (0.619-0.965)] predictive value (p=0.001). In the prediction of patients with a 2500 cut-off value for CD64, sensitivity was 94.1%, positive predictive value was 76.2%, specificity was 64.3%, and negative predictive value was 90.0% (Figure 5). CD64 expression in cases of gram-negative bacteremia was higher than in cases of gram-positive bacteremia, but this was a statistically nonsignificant difference (p=0.564). Table 2. Sensitivity, specificity, and predictive values of CD64. 95% CI AUC

p Lower-upper

CD64

0.866

0.737-0.994

0.001

CD64 >2500

0.792

0.619-0.965

0.006

Sensitivity

94.1%

Positive predictive value

76.2%

Specificity

64.3%

Negative predictive value

90.0%

ROC Curve

If bacteremia is not quickly treated in febrile neutropenia patients, it becomes a significant risk factor for septic shock. That is why it is crucial to get positive blood culture results as early as possible. The activation of neutrophils is evidence for the presence of bacteria in the circulation. For this reason, being able to show the activation of neutrophils on the first day of the febrile neutropenia episode is of vital importance and can serve as an alarm much earlier than the culture results. Showing the expression of CD64 on the surface with flow cytometry is the earliest indicator of neutrophil activation [1,2,12,13,14,15,16,17]. Is it possible to identify febrile neutropenia in patients with bacteremia before their microbiological culture results are obtained, and is there a test that would help us predict that the cause of fever in febrile neutropenic patients is an infection? Could CD64, an indicator of neutrophil activation, be used for this purpose? In this study, which was performed with the aim of answering these questions, the results that we obtained favor our hypotheses. CD64 levels of the bacteremia group were significantly higher than those of the control group and, in the distinction of the groups, CD64 levels had a significant predictive value. In the prediction of patients with a 2500 cut-off value for CD64 (molecules/cells), sensitivity was 94.1%, positive predictive value was 76.2%, specificity was 64.3%, and negative predictive value was 90.0%.

Sedimentation

CI: Confidence interval.

Discussion

Group Figure 2. Box plot graph shows the comparison between C-reactive protein (mg/L) in the case group (mean ± standard deviation: 82±86.4; median: 48) and the control group (mean ± standard deviation: 43.4±47.5; median: 23) (p=0.084). 170

Figure 3. Box plot graph shows the comparison between erythrocyte sedimentation rate (mm/h) in the case group (mean ± standard deviation: 79.6 ±26.3; median: 88) and the control group (mean ± standard deviation: 82.7±27.5; median: 74) (p=0.827).


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Figure 4. Box plot graph shows the comparison between neutrophils and leukocytes (/µL) in the case group (mean ± standard deviation: 246±144 and median: 230; mean ± standard deviation: 795±906 and median: 650) and the control group (mean ± standard deviation: 218±167 and median: 150; mean ± standard deviation: 706±246 and median: 715) (p=0.827). 1.0

patients in our study, their benefits in the differential diagnosis of an infection would be limited [5,21,22,23,24].

0.8

In the literature, in a study that was very similar to ours, it was investigated whether CD64 expression was a predictor for positive blood cultures in febrile neutropenic children or not and furthermore CRP and ESR were compared. Median CD64 indices were found to be similar both in the negative and the positive blood culture groups. A correlation could not be identified between CD64 index and positive blood culture. Furthermore, for CRP levels, there was no statistically significant difference between the study groups with positive and negative blood cultures and the control group [25].

0.6

0.4

0.2

0.0

0.0

0.2

0.4

06

08

1.0

Figure 5. Sensitivity and specificity of CD64 levels. Sepsis causes significant morbidity and mortality among febrile neutropenic patients. Up to 35% of sepsis patients have no identified microbiological agent and therefore diagnosis and empiric therapy have to be based on other parameters [18,19,20]. The lack of specific criteria makes the search for sensitive and specific diagnostic biomarkers crucial. As measurements of markers like CRP and PCT can be obtained much earlier than culture results, they are very useful in the early diagnosis of sepsis. However, their sensitivities are low. CRP and ESR are markers that indicate inflammation, and malignancy ranks first among the conditions that result in their elevation. To this end, in patients with malignancies, like the febrile neutropenia

In a meta-analysis evaluating neutrophil CD64 expression as a marker for bacterial infection, patients from all age groups were considered and CD64 expression on neutrophils was identified as a useful cell-based diagnostic tool for the diagnosis of bacterial infections. The sensitivity of neutrophil CD64 expression was found as 79% and its specificity as 91%. Based on the evaluation made in this meta-analysis, 13 prospective studies were analyzed and among them only those studies enrolling adult patients, those in which CD64 measurements were performed with flow cytometry, and those which confirmed the presence of an infection with blood culture positivity were shown to prove the sensitivity and specificity of CD64 [26]. In a study investigating the benefit of using neutrophil CD64 expression to discriminate between exacerbation of the disease and the addition of an infection to the clinical presentation in inflammatory autoimmune diseases, neutrophil CD64 expression was reported to have a sensitivity of 94.4% and a 171


Efe İris N, et al: Is CD64 Diagnostic for Bacteremia?

specificity of 88.9% [27]. Furthermore, neutrophil CD64 density was higher in gram-negative bacterial infections despite not reaching levels of statistical significance and this was explained by the activation of neutrophils by different bacterial products through different pathways. In our study, we found that patients who had growth of gram-negative bacteria also had higher levels of CD64 expression despite the fact that this was not statistically significant. If the results of further studies with higher numbers of patients support our results, it could provide us with an extremely useful diagnostic opportunity that might guide us in the choice of antibiotherapy. According to our study, neutrophil CD64 expression is a good diagnostic marker for bacteremia in febrile neutropenic patients. However, an infection marker should be biochemically stable because it might be necessary to keep blood samples stored, but flow cytometric analysis is not available for this situation. The advantages of CRP when compared with CD64 are rapid quantitation and easy handling. The quantitative flow cytometric analysis of CD64 was applied in this study. CD64 could be developed into a routine clinical test with high reproducibility and easy handling so that it can be used easily as a biomarker of bacteremia for febrile neutropenia patients.

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Authorship Contributions Concept: N.E.İ., T.Y.; Design: N.E.İ., T.Y.; Data Collection or Processing:  N.E.İ., T.Y. H.G., F.Ş., D.A., N.D., O.Y.; Analysis or Interpretation: N.E.İ., T.Y.; Literature Search: N.E.İ., T.Y.; Writing: N.E.İ. 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. Ng PC, Li G, Chui KM, Chu WC, Li K, Wong RP, Chik KW, Wong E, Fok TF. Neutrophil CD64 is a sensitive diagnostic marker for early-onset neonatal infection. Pediatr Res 2004;56:796-803. 2. Cid J, Aguinaco R, Sanchez R, Garcia-Pardo G, Llorente A. Neutrophil CD64 expression as a marker of bacterial infection: a systematic review and metaanalysis. J Infect 2010;60:313-319. 3. Simon L, Gauvin F, Amre DK, Saint-Louis P, Lacroix J. Serum procalcitonin and C-reactive protein levels as markers of bacterial infection: a systematic review and meta-analysis. Clin Infect Dis 2004;39:206-217. 4. Davis BH. Improved diagnostic approaches to infection/sepsis detection. Expert Rev Mol Diagn 2005;5:193-207. 5. Koeze J, Hendrix MG, Van den Bergh FA, Brouwer RM, Zijlstra JG. In critically ill patients the procalcitonin level can be misleading. Crit Care 2011;15:422.

In the literature there are some studies that demonstrated that CD64, when measured as an index, has high sensitivity and specificity for infection and monitoring sepsis [28,29,30]. The CD64 index was measured using a Leuko64kit (Trillium Diagnostics, Bangor, ME, USA) and a BD FACSCalibur running QuantiCALC software (Verity Software House, Topsham, ME, USA) in these studies. Many modern hematology analyzers contain flow cytometers and are capable of doing CD4 subsetting, and incorporation of CD64 index testing into these platforms will further simplify this test [28].

6. Chiwakata CB, Manegold C, Bönicke L, Wease I, Jülch C, Dietrich M. Procalcitonin as a parameter of disease severity and risk of mortality in patients with Plasmodium falciparum malaria. J Infect Dis 2001;183:1161-1164.

The main limitation of our study is the number of patients included. We recommend performing more studies with a higher number of patients. In this way it will be possible to obtain more powerful data on using CD64 and predicting bacteremia.

11. Diacom CC, Balaceami A, Moroşan E. Sepsis biomarkers. Past, present and future. Farmacia 2015;63:811-815.

Conclusion It is obvious that more studies focused on neutrophil CD64 expression for the diagnosis of bacteremia in febrile neutropenic patients are needed. According to our results, neutrophil CD64 expression is a good diagnostic tool for the diagnosis of bacteremia in febrile neutropenia patients, and CD64 is superior to CRP and ESR.

7. Wacker C, Prkno A, Brunkorst FM, Schlattmann P. Procalcitonin as a diagnostic marker for sepsis: a systematic review and meta-analysis. Lancet Infect Dis 2013;13:426-435. 8. Schutz AN. Infectious disease biomarkers: non-antibody-based host responses. In: Tang WW, Stratton CW (eds). Advanced Techniques in Diagnostic Microbiology. Berlin, Springer, 2013. 9. Nelson GE, Mave V, Gupta A. Biomarkers for sepsis: a review with special attention to India. Biomed Res Int 2014;2014:264351. 10. Prucha M, Bellingan G, Zazula R. Sepsis biomarkers. Clin Chim Acta 2015;440:97-103.

12. Vincent JL, Beunnier M. Diagnostic and prognostic markers in sepsis. Expert Rev Anti Infect Ther 2013;11:265-275. 13. Hoffmann JJ. Neutrophil CD64: a diagnostic marker for infection and sepsis. Clin Chem Lab Med 2009;47:903-916. 14. Hoffmeyer F, Witte K, Schmidt RE. The high-affinity Fc gamma RI on PMN: regulation of expression and signal transduction. Immunology 1997;92:544-552. 15. Schiff DE, Rae J, Martin TR, Davis BH, Curnutte JT. Increased phagocyte Fc gammaRI expression and improved Fc gamma receptor-mediated phagocytosis after in vivo recombinant human interferon-gamma treatment of normal human subjects. Blood 1997;90:3187-3194.

Ethics 


16. Qureshi SS, Lewis SM, Gant VM, Treacker D, Davis BH, Brown KA. Increased distribution and expression of CD64 on blood polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS). Clin Exp Immunol 2001;125:258-265.

Ethics Committee Approval: The study protocol was approved by Ethics Committee of Okmeydanı Training and Research Hospital, Informed Consent: It was received from patients.

17. Danikas DD, Karakantza M, Theodorou GL, Sakellarapoulos GC, Gogos CA. Prognostic value of phagocytic activity of neutrophils and monocytes in sepsis. Correlation to CD64 and CD14 antigen expression. Clin Exp Immunol 2015;154:87-97.

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18. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303-1310. 19. Dellinger RP, Carlet JM, Masur H, Gerlach H, Calandra T, Cohen J, GeaBanacloche J, Keh D, Marshall JC, Parker MM, Ramsay G, Zimmerman JL, Vincent JL, Levy MM; Surviving Sepsis Campaign Management Guidelines Committee. Surviving sepsis campaign guidelines for management of severe sepsis and septic shock. Crit Care Med 2004;32:858-873. 20. Ho BC, Bellomo R, McGain F, Jones D, Naka T, Wan L, Braitberg G. The incidence and outcome of septic shock patients in the absence of earlygoal directed therapy. Crit Care 2006;10:R80. 21. Skvarc M, Stubljar D, Rogina P, Kaasch AJ. Non-culture-based methods to diagnose bloodstream infection: does it work? Eur J Microbiol Immunol (Bp) 2013;2:97-104. 22. Au-Yong A. Towards evidence-based emergency medicine: best BETs from the Manchester Royal Infirmary. BET 2: C-reactive protein in the diagnosis of bacteraemia. Emerg Med J 2012;29:423-424. 23. St. Louis P. Markers of sepsis: a review. Point of Care 2007;6:165-169. 24. Blomkalns AL. Sick or Not Sick? : Evolving Biomarkers for Severe Bacterial Infection. New Concepts And Emerging Technologies for Emergency Physicians. Cincinnati, Emergency Medicine Cardiac Research and Education Group, 2007.

Efe İris N, et al: Is CD64 Diagnostic for Bacteremia?

25. Barbosa GG, Farias MG, Ludwig HC, Stensmann I, Fernandes MV, Michalowski MB, Daudt LE. Could CD64 expression be used as a predictor of positive culture results in children with febrile neutropenia? Rev Bras Hematol Hemoter 2015;37:395-399. 26. Dilli D, Oğuz ŞS, Dilmen U, Köker MY, Kızılgün M. Predictive values of neutrophil CD64 expression compared with interleukin-6 and C-reactive protein in early diagnosis of neonatal sepsis. J Clin Lab Anal 2010;24:363370. 27. Fang DH, Fan CH, Li J, An Q, Yao H, Ji Q, Niu G. Ratios of CD64 expressed on neutrophils, monocytes, and lymphocytes may be a novel method for diagnosis of neonatal sepsis. J Infect Dev Ctries 2015;9:175-181. 28. Streimish I, Bizzarro M, Northrup V, Wang C, Renna S, Koval N, Li FY, Ehrenkranz R, Rinder HM, Bhandari V. Neutrophil CD64 as a diagnostic marker in neonatal sepsis. Pediatr Infect Dis J 2012;31:777-781. 29. Gros A, Roussel M, Sauvadet E, Gacouin A, Marque S, Chimot L, Lavoue S, Camus C, Fest T, Le Tulzo Y. The sensitivity of neutrophil CD64 expression as a biomarker of bacterial infection is low in critically ill patients. Intensive Care Med 2012;38:445-452. 30. Cid J, Garcia-Pardo G, Aquinaco R, Sanches R, Llorente A. Neutrophil CD64: diagnostic accuracy and prognostic value in patients presenting to the emergency department. Eur J Clin Microbiol Infect Dis 201;30:845-852.

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BRIEF REPORT DOI: 10.4274/tjh.2016.0401 Turk J Hematol 2017;34:174-178

TET2, ASXL1, IDH1, and IDH2 Single Nucleotide Polymorphisms in Turkish Patients with Chronic Myeloproliferative Neoplasms Kronik Miyeloproliferatif Neoplazi Tanılı Türk Hastalarda TET2, ASXL1, IDH1 ve IDH2 Tek Nükleotid Polimorfizmleri Nur Soyer1, Burçin Tezcanlı Kaymaz2, Melda Cömert Özkan1, Çağdaş Aktan2, Ali Şahin Küçükaslan2, Fahri Şahin1, Buket Kosova2, Güray Saydam1 Ege University Faculty of Medicine, Department of Hematology, İzmir, Turkey Ege University Faculty of Medicine, Department of Medical Biology, İzmir, Turkey

1 2

Abstract

Öz

We aimed to determine the genotype distribution, allele frequency, and prognostic impact of IDH1/2, TET2, and ASXL1 single nucleotide polymorphisms (SNPs) in myeloproliferative neoplasms (MPNs). TET2 (rs763480), ASXL1 (rs2208131), and IDH1 (rs11554137) variant homozygous genotype frequencies were found at rates of 1.5%, 9.2%, and 2.3%, respectively. No IDH2 SNP was identified. IDH1 and TET2 frequencies were 5% in essential thrombocythemia (ET) and 1.7% in ET and 5% in primary myelofibrosis (PMF), respectively. ASXL1 frequencies were 8.3%-10% in MPN subgroups. The TET2 mutant allele T and ASXL1 mutant allele G had the highest frequencies with 0.272 in the PMF and 0.322 in the polycythemia vera (PV) group, respectively. There was no impact of the SNPs on prognosis. IDH1 frequency in MPNs was found similar to the literature. ASXL1 frequencies were similar between ET, PV, and PMF patients. The ASXL1 and TET2 allele frequencies of the Turkish population are similar to those of the European population. The role of SNPs in MPNs might be further evaluated in larger multicenter studies.

Bu çalışmada biz ASXL1, TET2, IDH1/2 genlerindeki tek nükleotid polimorifizmlerin (SNP) alel sıklığını, genotipik dağılımını ve prognostik etkisini saptamayı amaçladık. TET2 (rs763480), ASXL1 (rs2208131) ve IDH1 (rs11554137) varyant homozigot genotip sıklığı sırasıyla %1,5, %9,2 ve %2,3 saptandı. IDH2 SNP saptanmadı. IDH1 sıklığı ET’de %5 ve TET2 sıklığı ET’de %1,7 ve PMF’te %5 idi. ASXL1 sıklığı ise MPN alt gruplarında %8,3-10’du. En yüksek TET2 mutant allel T ve ASXL mutant allel G sıklığı sırasıyla PMF’te 0,272 ve PV’de 0,322 olarak saptandı. SNP’lerin prognoz üzerine etkisi yoktu. MPN’de IDH1 sıklığı literatür ile uyumlu bulundu. ASXL1 sıklığı PV, PMF ve ET alt gruplarında benzerdi. Türklerde ASXL1 ve TET2 allel sıklığı Avrupalılar ile benzer saptandı. MPN’lerde SNP’lerin rolü, büyük ve çok merkezli çalışmalarda değerlendirilmelidir. Anahtar Sözcükler: TET2, ASXL1, IDH1, IDH2, Tek nükleoitid polimorfizm, Ph-negatif Miyeloproliferatif neoplaziler

Keywords: TET2, ASXL1, IDH1, IDH2, Single nucleotide polymorphisms, Ph-negative myeloproliferative neoplasms

Introduction Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal disorders classified as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). MPNs are dependent on hypersensitivity or anomalies in cytokine regulation [1,2]. Some genes have been reported to be involved in the pathogenesis of MPNs, such as IDH1 (isocitrate dehydrogenase), IDH2, TET2 (ten-eleven translocation 2), and ASXL1 (additional sex combs-like 1).

IDH1/2 encodes enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate [3]. The frequency of IDH1/2 mutation was 4% in MPNs, 0.8% in ET, 1.9% in PV, and 4.1% in PMF [4,5]. These mutations in PMF were reported as independent predictors of leukemic transformation [6]. ASXL1 encodes a member of the chromatin-binding proteins and is involved in epigenetic regulation of gene expression [7]. ASXL1 mutations are rare in ET and PV (<7%) but are frequent

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Nur SOYER, M.D., Ege University Faculty of Medicine, Department of Hematology, İzmir, Turkey Phone : +90 232 390 35 18 E-mail : drakadnur@yahoo.com

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Received/Geliş tarihi: October 9, 2016 Accepted/Kabul tarihi: February 20, 2017


Turk J Hematol 2017;34:174-178

Soyer N, et al: Single Nucleotide Polymorphisms in Chronic Myeloproliferative Neoplasms

in PMF (19-40%) [3,7]. Worsened survival was reported in PMF patients with mutated ASXL1 [8]. TET proteins are enzymes that can convert 5-methylcytosine to 5-hydroxymethylcytosine [9]. TET2 mutation frequency was 16% in PV, 5% in ET, and 17% in PMF. Previous research did not identify an impact of TET2 mutations on survival or leukemic transformation [10]. Although the prognostic impact of these mutations has been investigated in some studies, there is still limited information available [6,8,10]. In this study, we focused only on single nucleotide polymorphisms (SNPs) in the ASXL1, TET2, and IDH1/2 genes. We aimed to determine the genotype distribution, allele frequency, and prognostic impact of selected SNPs in MPNs.

Materials and Methods The study included 130 MPN patients. The diagnosis of MPNs was performed based on World Health Organization and International Working Group criteria [11]. The Ege University Local Ethics Committee approved the study (13-5.1/8, 15.07.2013). Patient data were collected from the patient files. SNP analysis was performed on DNA derived from peripheral blood samples that were collected into tubes containing EDTA between February 2008 and September 2009 and were stored at -80 °C until the DNA extraction. DNA extraction was performed using the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche) according to the manufacturer’s instructions. After DNA isolation, the purity and concentration of DNA was measured using a NanoDrop spectrophotometer (Thermo Scientific, USA) at 260 nm and 280 nm.

taking the interval from the date of diagnosis to death or last contact. The log-rank test was used to compare the survival data.

Results The demographic features of patients at the time of diagnosis are shown in Table 1. IDH2 (rs121913503) and IDH2 (rs267606870) SNPs were not detected in any of the patient groups; all of the cases were genotyped as wild-type homozygous (GG and CC, respectively, for the rs numbers). Two (1.5%) of all, 12 (9.2%) of all, and 3 (2.3%) of all patients were variant homozygous for TET2 (rs763480), ASXL1 (rs2208131), and IDH1 (rs11554137) SNPs, respectively (Table 2). The TET2 mutant allele T frequency was 0.218 in the PV, 0.20 in the ET, and 0.272 in the PMF group. The ASXL1 mutant allele G frequency was 0.322 in the PV, 0.308 in the ET, and 0.25 in the PMF group. The IDH1 mutant allele T frequency was 0.073 in the PV, 0.108 in the ET, and 0.045 in the PMF group. The median follow-up time was 8 years (range: 1-25). The estimated 10-year survival rate was 71% for ET, 87.3% for PV, and 71% for PMF patients (Figure 1). We did not find any significant differences between the diagnostic subgroups and the 10-year survival rates. At the time of the analysis, 5 and 10 patients had leukemic transformation and fibrotic transformation, respectively. There were no significant differences between the sexes, diagnoses, JAK2 mutation status (positive/negative), thrombosis status at diagnosis, survival rates, leukemic and fibrotic transformation, and genotyping results of ASXL1, TET2, and IDH1 (Table 3).

All SNP analysis was carried out with ready-to-use TaqMan assays from Life Technologies (USA): IDH1 [rs11554137(C/T conversion)], IDH2 [rs121913503-(G/A conversion); rs267606870-(C/G conversion)], TET2 [rs763480-(A/T conversion)], and ASXL1 [rs2208131-(A/G conversion). Realtime polymerase chain reaction (PCR) reactions were performed according to the manufacturer’s instructions using the ABI 7500 Fast Real-Time PCR instrument (Applied Biosystems, USA). The real-time PCR cycling conditions were: 95 °C for 10 min for enzyme activation, 40 cycles of 95 °C for 15 s (denaturation), and 60 °C for 1 min (annealing/extension). All assays were evaluated and genotyped using SDS software. Statistical Analysis Hardy-Weinberg equilibrium was used for each SNP. All p-values were two sided and p≤0.05 indicated statistical significance. Categorical and continuous variables were compared with chisquare statistics and the Mann-Whitney U test, respectively. Survival analysis was performed by the Kaplan-Meier method,

Figure 1. Overall survival of different myeloproliferative neoplasm subgroups. ET: Essential thrombocythemia, PV: polycythemia vera, PMF: primary myelofibrosis.

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Table 1. The demographic features of myeloproliferative neoplasm patients at the time of diagnosis. No of patients

MPN (n=130)

PV (n=48)

ET (n=60)

PMF (n=22)

Age at diagnosis, years (range)

63.5 (16-81)

63.5 (16-74)

63 (18-81)

65 (31-74)

Sex, M/F

72/58

30/18

29/31

13/9

Thrombosis at diagnosis or before diagnosis, yes/no

19/111

6/42

10/50

3/19

Bleeding at diagnosis, yes

5

2

2

1

18.7 (3.1-73)

18.7 (7.1-40.2)

8.1 (4.8-35)

33.9 (3.1-73)

White blood cell count, x10 /L (range) 9

Platelet count, x10 /L (range)

270.7 (88-2000)

270.7 (95-1380)

906.5 (325-2000)

350 (88-1436)

Hemoglobin level, g/L (range)

18 (6-23.4)

18 (11.4-23.4)

13.6 (8.8-16)

13.8 (6-16.2)

LDH levels, normal/high

70/52

26/18

40/17

4/17

Splenomegaly, yes/no

40/90

20/28

4/56

16/6

Hepatomegaly, yes/no

18/112

8/40v

2/58

8/14

JAK2V617F positive/negative

87/43

46/2

30/30

11/11

Secondary malignancies, yes

2

0

2

0

9

LDH: Lactate dehydrogenase, MPN: myeloproliferative neoplasm, ET: essential thrombocythemia, PMF: primary myelofibrosis.

Table 2. Genotype status of IDH1, IDH2, TET2, and ASXL1 single nucleotide polymorphisms in myeloproliferative neoplasm and myeloproliferative neoplasm subgroups. MPN (n=130)

PV (n=48)

ET (n=60)

PMF (n=22)

IDH2 (rs121913503) Wild-type homozygous

130

48

60

22

IDH2 (rs267606870) Wild-type homozygous

130

48

60

22

IDH1 (rs11554137) Wild-type homozygous Heterozygous Variant homozygous

111 (85.4%) 16 (12.3%) 3 (2.3%)

41 (85.4%) 7 (14.6%) 0 (0%)

50 (83.3%) 7 (11.7%) 3 (5%)

20 (90.9%) 2 (9.1%) 0 (0%)

TET2 (rs763480) Wild-type homozygous Heterozygous Variant homozygous

75 (57.7%) 53 (40.8%) 2 (1.5%)

27 (56.2%) 21 (43.8%) 0 (0%)

37 (61.7%) 22 (36.7%) 1 (1.7%)

11 (50%) 10 (45.5%) 1 (5%)

ASXL1 (rs2208131) Wild-type homozygous Heterozygous Variant homozygous

63 (48.5%) 55 (42.3%) 12 (9.2%)

21 (43.8%) 23 (47.9%) 4 (8.3%)

29 (48.3%) 25 (41.7%) 6 (10%)

13 (59.1%) 7 (31.8%) 2 (9.1%)

MPN: Myeloproliferative neoplasm, ET: essential thrombocythemia, PV: polycythemia vera, PMF: primary myelofibrosis

Discussion In this study, we aimed to determine the genotype distribution, allele frequency, and prognostic impact of selected SNPs in MPNs. IDH mutation frequency was 2.5% in MPNs, 0.8% in ET, 1.9% in PV, 4.1% in PMF, and 1% in post-ET/PV-myelofibrosis patients [5]. Another study reported 3.70% IDH1 (G105G allele), 1.85% IDH2 R140Q, and 0.92% IDH2 (G145G allele) mutation in MPNs [12]. The frequency of the IDH1 (rs11554137) SNP in our cases was similar to that in the literature. TET2 mutation frequency was 7.2%-13% in MPNs and was similar across different MPN subgroups. JAK2-positive patients (17%) had significantly higher TET2 mutation frequency [10,13]. 176

In our series, TET2 (rs763480) frequency was higher in PMF patients. ASXL1 mutation frequency was 10% in MPN patients. These mutations are rare in ET and PV (<7%) but frequent in PMF (19%-40%) [3,7]. In JAK2- and MPL-negative MPN patients, TET2 and ASXL1 mutation frequencies were 8% [14]. ASXL1 mutation frequency was 24.7% in PMF and 8.4% in ET patients [15]. ASXL1 mutation frequency was 12%-13% in PMF patients [16]. The frequency of the ASXL1 (rs2208131) SNP was 9.2% and this was similar between ET, PV, and PMF patients. We did not find any previous study that evaluated ASXL1 (rs2208131) and TET2 (rs763480) SNPs in MPNs.


Soyer N, et al: Single Nucleotide Polymorphisms in Chronic Myeloproliferative Neoplasms

Turk J Hematol 2017;34:174-178

Table 3. Genotype status of IDH1, TET2, and ASXL1 single nucleotide polymorphisms and clinical and laboratory correlations. IDH1 (rs11554137)

p

TET2 (rs763480)

p

ASXL1 (rs2208131)

p

Wild-type (n=63)

Heterozygous and variant homozygous (n=67)

NS

31/32

41/26

NS

21

NS

21

31

NS

37

23

NS

29

27

NS

NS

11

11

NS

13

9

NS

2

NS

11

8

NS

9

10

NS

75/36

12/7

NS

50/25

37/18

NS

44/19

43/24

Overall survival (10-year)

79.8%

62.2%

NS

75.3%

80.4%

NS

84.7%

70.4%

Leukemic transformation

4

1

NS

4

1

NS

3

2

NS

Fibrotic transformation

9

1

NS

6

4

NS

7

3

NS

Wild-type Heterozygous and variant (n=75) homozygous (n=55)

Wild-type (n=111)

Heterozygous and variant homozygous (n=19)

Sex (M/F)

61/50

11/8

NS

37/38

35/20

PV

41

7

NS

27

ET

50

10

NS

PMF

20

2

Thrombosis at diagnosis

17

JAK2, mutated vs. wild-type

NS NS

NS: Not significant, PV: polycythemia vera, ET: essential thrombocythemia, PMF: primary myelofibrosis.

There was no relationship between TET2, IDH1, and ASXL1 SNPs and clinical and laboratory factors in our study. An impact of TET2 mutation on survival and leukemic transformation was not shown [10]. In normal karyotype acute myeloid leukemia, the IDH1 (rs11554137) SNP was an adverse prognostic factor [17]. In PMF, there was a significantly negative impact of IDH mutations on survival [6]. ASXL1 mutations were identified in patients with PMF or post-ET/PV-myelofibrosis and associated with poor survival [8,9,15]. Whether the TET2, IDH1, and ASXL1 SNPs possibly confer any prognostic impact in MPN patients requires further evaluation. The TET2 mutant allele T and ASXL1 mutant allele G had the highest frequencies at 0.272 in the PMF and 0.322 in the PV group, respectively. The ASXL1 and TET2 mutational statuses in the Turkish population are similar to those of the European population according to HapMap CEU data reporting 0.322 G mutant allele and 0.372 T mutant allele frequencies, respectively (hapmap.ncbi.nlm.nih.gov/). One of the limitations of this study is the small sample size. Since we did not evaluate MPL and CALR mutations, we could not assess whether there was a relationship between CALR and MPL mutations and these SNPs. Since we had no control group, we could not perform a comparison for these SNPs between patients and controls.

Conclusion IDH1 frequency in MPNs was found to be similar to the rate reported in the literature. ASXL1 frequencies were similar

between ET, PV, and PMF patients. We did not find an impact of the SNPs on survival, JAK2 status, or leukemic and fibrotic transformation. These findings suggest that IDH1 is a rare SNP in MPNs. The role of SNPs in MPNs might be further evaluated in larger multicenter studies. Authorship Contributions Surgical and Medical Practices: N.S., M.C.Ö., G.S.; Concept: N.S., B.T.K., B.K., G.S.; Design: N.S., B.T.K., B.K., G.S.; Data Collection or Processing: N.S., B.T.K., M.C.Ö., Ç.A., A.Ş.K., F.Ş., B.K., G.S.; Analysis or Interpretation: N.S., B.T.K., Ç.A., A.Ş.K.; Literature Search: N.S., B.T.K.; Writing: N.S., G.S. 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. Delhommeau F, Pisani DF, James C, Casadevall N, Constantinescu S, Vainchenker W. Oncogenic mechanisms in myeloproliferative disorders. Cell Mol Life Sci 2006;63:2939-2953. 2. Campbell PJ, Green AR. The myeloproliferative disorders. N Engl J Med 2006;355:2452-2466. 3. Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 2010;24:1128-1138. 4. Pardanani A, Lasho TL, Finke CM, Mai M, McClure RF, Tefferi A. IDH1 and IDH2 mutation analysis in chronic and blast phase myeloproliferative neoplasms. Leukemia 2010;24:1146-1151. 5. Tefferi A, Lasho TL, Abdel-Wahab O, Guglielmelli P, Patel J, Caramazza D, Pieri L, Finke CM, Kilpivaara O, Wadleigh M, Mai M, McClure RF, Gilliland

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DG, Levine RL, Pardanani A, Vannucchi AM. IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase essential thrombocythemia, polycythemia vera or myelofibrosis. Leukemia 2010;24:1302-1309. 6. Tefferi A, Jimma T, Sulai NH, Lasho TL, Finke CM, Knudson RA, McClure RF, Pardanani A. IDH mutations in primary myelofibrosis predict leukemic transformation and shortened survival: clinical evidence for leukemogenic collaboration with JAK2V617F. Leukemia 2012;26:475-480. 7. Carbuccia N, Murati A, Trouplin V, Brecqueville M, Adélaïde J, Rey J, Vainchenker W, Bernard OA, Chaffanet M, Vey N, Birnbaum D, Mozziconacci MJ. Mutations of ASXL1 gene in myeloproliferative neoplasms. Leukemia 2009;23:2183-2186. 8. Brecqueville M, Rey J, Bertucci F, Coppin E, Finetti P, Carbuccia N, Cervera N, Gelsi-Boyer V, Arnoulet C, Gisserot O, Verrot D, Slama B, Vey N, Mozziconacci MJ, Birnbaum D, Murati A. Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms. Genes Chromosomes Cancer 2012;51:743-755. 9. Abdel-Wahab O, Tefferi A, Levine RL. Role of TET2 and ASXL1 mutations in the pathogenesis of myeloproliferative neoplasms. Hematol Oncol Clin North Am 2012;26:1053-1064. 10. Tefferi A, Pardanani A, Lim KH, Abdel-Wahab O, Lasho TL, Patel J, Gangat N, Finke CM, Schwager S, Mullally A, Li CY, Hanson CA, Mesa R, Bernard O, Delhommeau F, Vainchenker W, Gilliland DG, Levine RL. TET2 mutations and their clinical correlates in polycythemia vera, essential thrombocythemia and myelofibrosis. Leukemia 2009;23:905-911. 11. Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia 2008;22:14-22.

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12. Chotirat S, Thongnoppakhun W, Wanachiwanawin W, Auewarakul CU. Acquired somatic mutations of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) in preleukemic disorders. Blood Cells Mol Dis 2015;54:286-291. 13. Patriarca A, Colaizzo D, Tiscia G, Spadano R, Di Zacomo S, Spadano A, Villanova I, Margaglione M, Grandone E, Dragani A. TET2 mutations in Ph-negative myeloproliferative neoplasms: identification of three novel mutations and relationship with clinical and laboratory findings. Biomed Res Int 2013;2013:929840. 14. Martínez-Avilés L, Besses C, Álvarez-Larrán A, Torres E, Serrano S, Bellosillo B. TET2, ASXL1, IDH1, IDH2, and c-CBL genes in JAK2- and MPL-negative myeloproliferative neoplasms. Ann Hematol 2012;91:533-541. 15. Yonal-Hindilerden I, Daglar-Aday A, Akadam-Teker B, Yilmaz C, Nalcaci M, Yavuz AS, Sargin D. Prognostic significance of ASXL1, JAK2V617F mutations and JAK2V617F allele burden in Philadelphia-negative myeloproliferative neoplasms. J Blood Med 2015;6:157-175. 16. Abdel-Wahab O, Pardanani A, Patel J, Wadleigh M, Lasho T, Heguy A, Beran M, Gilliland DG, Levine RL, Tefferi A. Concomitant analysis of EZH2 and ASXL1 mutations in myelofibrosis, chronic myelomonocytic leukemia and blast-phase myeloproliferative neoplasms. Leukemia 2011;25:1200-1202. 17. Wagner K, Damm F, Göhring G, Görlich K, Heuser M, Schäfer I, Ottmann O, Lübbert M, Heit W, Kanz L, Schlimok G, Raghavachar AA, Fiedler W, Kirchner HH, Brugger W, Zucknick M, Schlegelberger B, Heil G, Ganser A, Krauter J. Impact of IDH1 R132 mutations and an IDH1 single nucleotide polymorphism in cytogenetically normal acute myeloid leukemia: SNP rs11554137 is an adverse prognostic factor. J Clin Oncol 2010;28:23562364.


IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0255 Turk J Hematol 2017;34:179-180

Lack of Early Inflammation Signs of Acute Compartment Syndrome in an Immunodeficient Patient İmmün Yetmezlikli Bir Olguda Erken Enflamasyon Bulgularının Görülmediği Akut Kompartman Sendromu Burcu Belen1, Özlem Çakıcı2, Melikşah Uzakgider3, Haldun Öniz4, Meral Türker5, Berna Atabay5, Barış Malbora5, Levent Karapınar3 Tepecik Training and Research Hospital, Clinic of Pediatric Hematology and Oncology, İzmir, Turkey Tepecik Training and Research Hospital, Clinic of Pediatrics, İzmir, Turkey 3 Tepecik Training and Research Hospital, Clinic of Orthopedics, İzmir, Turkey 4 Tepecik Training and Research Hospital, Clinic of Pediatric Oncology, İzmir, Turkey 5 Tepecik Training and Research Hospital, Clinic of Pediatric Hematology, İzmir, Turkey 1 2

Figure 1. Acute compartment syndrome of upper extremity after restoration of white blood cells.

Figure 2. Front forearm fasciotomy was performed for treatment of acute compartment syndrome.

Acute compartment syndrome (ACS) is defined as the continuous elevation of interstitial tissue pressure within an osteofascial envelope to nonphysiological levels. It can be reversible if it is recognized early; however, it may progress to permanent disability. Therefore, early recognition and treatment is critical for optimal outcomes [1]. Pain, pallor, paresthesia, paralysis, and pulselessness (the ‘five Ps’) are reliable symptoms

of ACS; however, the lack of them may be challenging in immunodeficient patients [2,3]. Here we present upper extremity ACS in an 18-year-old male patient with non-Hodgkin lymphoma. He was admitted with antecubital vein thrombosis during gram-negative sepsis without overt signs of inflammation in the affected arm while he was neutropenic. With the increase in white blood cells, first

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Burcu BELEN, M.D., Tepecik Training and Research Hospital, Clinic of Pediatric Hematology and Oncology, İzmir, Turkey Phone : +90 532 581 45 51 E-mail : fatmaburcu.belen@ikc.edu.tr

Received/Geliş tarihi: June 30, 2015 Accepted/Kabul tarihi: November 11, 2015

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inflammatory findings of cellulitis and soon after that upper extremity ACS became evident (Figure 1). The ACS was assumed to be caused by the increased pressure of the compartment following superficial thrombosis that may have led to obstruction of venous flow accompanied by cellulitis of the forearm. Front forearm fasciotomy was performed with primary fixation of the ruptured flexor digitorum profundus muscle in combination with intravenous antibiotherapy (Figure 2). Due to lack of initial inflammation signs in immunodeficient patients, ACS diagnosis is particularly difficult. Early recognition and expeditious surgical treatment are essential to obtain a good clinical outcome and prevent permanent disability. Keywords: Acute compartment syndrome, Inflammation, Immune deficiency Anahtar Sözcükler: Akut kompartman sendromu, Enflamasyon, İmmün yetmezlik

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Informed Consent: Was obtained from the patients. 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. Prasarn ML, Oulette EA. Acute compartment syndrome of the upper extremity. J Am Acad Orthop Surg 2011;19:49-58. 2. Erdös J, Dlaska C, Szatmary P, Humenberger M, Vecsei V, Hajdu S. Acute compartment syndrome in children: a case series in 24 patients and review of the literature. Int Orthop 2011;35:569-575. 3. Davidson DJ, Shaukat YM, Jenabzadeh R, Gupte CM. Spontaneous bilateral compartment syndrome in a HIV-positive patient. BMJ Case Rep 2013:bcr2013-202651.


IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2016.0020 Turk J Hematol 2017;34:181-182

Pathological Fracture in Odontoid Process in Multiple Myeloma Multipl Miyelom Tanılı Hastada Saptanan Patolojik Odontoid Kırığı Emre Ali Acar1, Ufuk Demirci1, Yüksel Pabuşçu2, Hayriye Mine Miskioğlu3, İsmet Aydoğdu3 Celal Bayar University Faculty of Medicine, Department of Internal Medicine, Manisa, Turkey Celal Bayar University Faculty of Medicine, Department of Radiology, Manisa, Turkey 3 Celal Bayar University Faculty of Medicine, Department of Hematology, Manisa, Turkey 1 2

Figure 1. Fracture line in odontoid process (white arrow), diffuse osteolytic lesions (black arrows).

Figure 2. Fracture line in odontoid process (white arrow), diffuse osteolytic lesions (black arrow).

A 72-year-old male was hospitalized with suspicion of multiple myeloma (MM) due to anemia (hemoglobin: 9.7 g/dL), renal failure (glomerular filtration rate: 8 mL/min), hypercalcemia (calcium: 11.3 mg/dL), and lytic bone lesions. He was diagnosed with MM because of increase in monoclonal lambda light chain in urine immunofixation and plasma cells (70%) in bone marrow aspiration. Fluorescence in situ hybridization and cytogenetic tests were not performed. During his chemotherapy treatment, he was operated on due to left knee septic arthritis. After the operation, he did not have any complaints for 5 weeks. After this time, he had sudden pain, restriction of movement in the neck, and temporary unconsciousness. In brain and cervical tomography there was a fracture that shifted the odontoid

process and C1 vertebra, causing minimal pressure on the medulla spinalis (Figures 1-3). MM usually presents with anemia (73%), bone lesions (80%), and renal failure (20%-40%). Bone involvement includes diffuse lytic lesions and osteopenia, mostly seen in the skull, vertebrae, and long bones. The frequency of lesions is associated with poor prognosis [1]. Vertebral involvement is seen in 60% of MM patients. Eighty percent of this involvement is seen between the T6 and L4 levels [2]. As seen in our case, fractures of C2 vertebrae are rare. Our patient is receiving lenalidomide because he had no response to 4 cycles of vincristine, cyclophosphamide, dexamethasone chemotherapy. In conclusion, fractures of C2

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Ufuk DEMİRCİ, M.D., Celal Bayar University Faculty of Medicine, Department of Internal Medicine, Manisa, Turkey Phone : +90 505 518 92 20 E-mail : ufukdemirci3232@gmail.com

Received/Geliş tarihi: January 14, 2016 Accepted/Kabul tarihi: September 19, 2016

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Turk J Hematol 2017;34:181-182

vertebrae should be considered in MM patients when restriction of neck movement and temporary unconsciousness occur. Keywords: Myeloma, Infection, Atlas, Fracture Anahtar Sözcükler: Miyelom, Enfeksiyon, Atlas, Kırık Informed Consent: It was obtained from the patient. 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. Hameed A, Brady JJ, Dowling P, Clynes M, O’Gorman P. Bone disease in multiple myeloma: pathophysiology and management. Cancer Growth Metastasis 2014;7:33-42.

Figure 3. Fracture line in odontoid process in 3-dimensional cervical tomography imaging.

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2. Tosi P. Diagnosis and treatment of bone disease in multiple myeloma: spotlight on spinal involvement. Scientifica (Cairo) 2013;2013:104546.


IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0382 Turk J Hematol 2017;34:183

A Rare Complication of Congenital Afibrinogenemia: Bone Cysts Konjenital Afibrinojenemide Nadir Bir Komplikasyon: Kemik Kisti Ali Fettah, Dilek Gürlek Gökçebay, Vildan Çulha, Neşe Yaralı, Bahattin Tunç, Namık Özbek Ankara Children’s Hematology and Oncology Research Hospital, Clinic of Pediatric Hematology, Ankara, Turkey

Figure 1. Direct radiography of the tibia. A 12-year-old male patient diagnosed with congenital afibrinogenemia presented to our center with pain, swelling, and ecchymosis in his leg after trauma. His past medical history revealed that he had been diagnosed with afibrinogenemia shortly after birth because of umbilical bleeding. Laboratory tests at admission revealed prolonged prothrombin time and activated partial thromboplastin time, and almost undetectable fibrinogen levels. A bone scan and radiograms of both legs showed multiple cystic lesions in the tibiae (Figure 1). Magnetic resonance imaging (MRI) of the legs also showed multicystic lesions with septae formation involving metaphyseal-diaphyseal junctions (Figure 2). Bone cysts, one of the rare complications of afibrinogenemia, frequently appear in the contiguity of the cortex or trabeculae in the diaphysis of long bones, particularly the femora, tibiae, and humeri, and should be considered in patients who suffer rheumatic pains of the extremities [1,2]. Intraosseous hemorrhage, usually at the entrance of the nutrient artery, causes intraosseous cysts. Large cysts, especially in weightbearing bones, may cause pathological fractures [2,3]. Wholebody MRI might be useful to evaluate the lesions. We want

Figure 2. Magnetic resonance imaging of the lower extremities. to emphasize the importance of on-demand therapy and MRI in determining bone cysts. However, the role of secondary prophylaxis needs to be evaluated. Keywords: Afibrinogenemia, Bone cysts, Child, Rare Anahtar Sözcükler: Afibrinojenemi, Kemik kistleri, Çocuk, Nadir Informed Consent: Our patient’s parent gave consent. 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. Van Meegeren ME, De Rooy WJ, Schreuder HW, Brons PP. Bone cysts in patients with afibrinogenaemia: a literature review and two new cases. Haemophilia 2014;20:244-248. 2. Lagier R, Bouvier CA, Van Strijthem N. Skeletal changes in congenital fibrinogen abnormalities. Skeletal Radiol 1980;5:233-239. 3. Zenny JC, Chevrot A, Sultan Y, Godefroy D, Horreard P, Pallardy G. Intraosseus hemorrhagic lesions in congenital afibrinogenemia. J Radiol 1981;62:263-266.

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

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

Received/Geliş tarihi: November 06, 2015 Accepted/Kabul tarihi: February 08, 2016

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LETTERS TO THE EDITOR Turk J Hematol 2017;34:184-199

Disappearance of Acquired Hemophilia A after Complete Remission in a Multiple Myeloma Patient Multipl Miyelomlu Bir Hastada Tam Remisyon Sonrası Edinsel Hemofilinin Kaybolması Vanessa Innao, Alessandro Allegra, Rosalba Morreale, Sabina Russo, Caterina Musolino Messina University Faculty of Medicine, Department of Human Pathology and the Adult Developmental, Division of Hematology, Messina, Italy

To the Editor, Acquired inhibitors of blood coagulation are endogenously produced pathologic substances that either react directly with clotting factors or inhibit their reactions. Acquired hemophilia A (AHA) is caused by polyclonal inhibitory immunoglobulins G (predominantly IgG1 and IgG4) against factor VIII (FVIII). They react with A2, A3, or C2 domains of the FVIII molecule, blocking its interactions with active factor IX, phospholipids, and von Willebrand factor. A disturbed proportion of CD4+ Th1 to Th2 cells plays a role in autoantibody production and reactivity [1,2]. FVIII inhibitors have most often been associated with autoimmune diseases, drugs, immunosuppressive therapies, malignant neoplasms, or obstetric accidents in the postpartum period [3]. Here we report a rare case of a patient with AHA and multiple myeloma, where the disappearance of the hematologic malignancy induced by chemotherapy resulted in a regression of the coagulation disorder. A 67-year-old man came under our observation for plasmacytoma. Significantly, his past medical history included both high blood pressure and hip replacement. His concomitant medication was only lacidipine. In 2009 the patient underwent cholecystectomy without bleeding. In 2014, he underwent a surgical excision of basal cell carcinoma in the pectoral region without bleeding. In 2014 the patient was diagnosed, at a different hematological center, with congenital mild hemophilia A and monoclonal gammopathy of undetermined significance IgG kappa. At that time, FVIII was 29.1%. He also underwent a desmopressin test, which displayed an increase of FVIII activity (15% at basal level) up to 90% after 60 min. Elevation of IgG (2319 mg/dL) was present, while the rest of the immunoglobulins were within normal values. IgG k-type monoclonal protein was detected in the serum and urine. In 2015, a diagnosis of plasmacytoma resulted from a biopsy of abnormal tissue performed during vertebroplasty for a 184

pathological L3 fracture. The orthopedic intervention was performed with premedication with recombinant FVIII. He came, therefore, to our attention presenting with 28% FVIII activity, prothrombin time within normal limits, and prolonged activated partial thromboplastin time (44.1 s). Serum protein electrophoretic analysis showed a monoclonal peak within γ-globulin, increased IgG, and suppression of all other components. Serum protein electrophoresis showed a monoclonal free k-light chain. Bone marrow biopsy, performed after infusion therapy of recombinant FVIII, showed plasma cells at 50%. Therefore, a diagnosis of multiple myeloma IgG kappa, stage IIA DurieSalmon, I ISS, was made. He began treatment with intravenous bortezomib and oral melphalan and prednisone, associated with monthly infusions of bisphosphonates, for 6 cycles in total. Revaluation of the disease documented stringent complete response (sCR), but we also witnessed the normalization of blood coagulation parameters, while FVIII activity was 98%. The patient was then mobilized with high-dose cyclophosphamide and autotransplanted, retaining the sCR to this day. Diagnosis of AHA can be difficult. It is often unrecognized or misdiagnosed as other acquired hemorrhagic disorders, such as disseminated intravascular coagulation, acquired inhibitors against von Willebrand factor, and acquired factor XIII deficiency [4]. The association of acquired hemophilia and multiple myeloma is extremely rare; there are only four such reports in the literature [5,6,7,8]. However, prompt diagnosis of this acquired bleeding disorder is essential for management, aimed at hemorrhage control and inhibitor suppression. In our case, the misdiagnosis led not only to a delayed diagnosis of the real coagulation disorder, but probably also to a delay even of hematologic malignancy, effectively responsible for the coagulation dysfunction. As said above, both solid tumors and hematologic malignancies, especially lymphoproliferative diseases, are able to predispose or be associated with this antibody’s growth [9].


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A small number of works investigated the genetic basis of AHA, due to immune tolerance breakdown. Pavlova et al. [10] pointed out higher frequencies of human leukocyte antigen class II alleles DRB*16 (odds ratio [OR]: 10.2) and DQB*0502 (OR: 2.5). In a different work, the CTLA-4 +49G allele was increased in these patients with an OR of 2.17 and these data were not confirmed in congenital hemophilia. In conclusion, it is clear that in our case the reestablishment of immunocompetence, complete remission of multiple myeloma, and disappearance of AHA are closely related events. In fact, previous works have demonstrated that the treatment of the underlying cancer led to the disappearance of the inhibitor in 22% of patients [11]. Finally, as multiple myeloma is a treatable but not curable disease, it will be interesting to keep closely monitoring coagulation in the patient in the case of relapse, to evaluate if it can lead to the reappearance of FVIII inhibitors. Keywords: Acquired hemophilia, Multiple myeloma, Factor VIII, Autoantibody, Coagulation disorder Anahtar Sözcükler: Edinsel hemofili, Multipl miyelom, Faktör VIII, Otoantikor, Koagülasyon bozukluğu 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. Reding MT. Immunological aspects of inhibitor development. Haemophilia 2006;(Suppl 6):30-35. 2. Sakura Y, Takeda T. Acquired hemophilia A: a frequently overlooked autoimmune hemorrhagic disorder. J Immunol Res 2014;2014:320674. 3. Franchini M, Gandini G, Di Paolantonio T, Mariani G. Acquired hemophilia A: a concise review. Am J Hematol 2005;80:55-63. 4. Sari I, Erkurt MA, Ifran A, Kaptan K, Beyan C. Multiple myeloma presenting with acquired factor VIII inhibitor. Int J Hematol 2009;90:166-169. 5. Muzaffar J, Katragadda L, Haider S, Javed A, Anaissie E, Usmani S. Rituximab and intravenous immunoglobulin (IVIG) for the management of acquired factor VIII inhibitor in multiple myeloma: case report and review of literature. Int J Hematol 2012;95:102-106. 6. Ross C, Kuriakose K, Aswath GS, Subramanian S. Management of acquired haemophilia bleed in the backdrop of multiple myeloma. Indian J Hematol Blood Transfus 2015;31:151-153. 7. Saburi M, Ohtsuka E, Itani K, Nagamatsu K, Ikebe T, Miyazaki Y, Ogata M, Saburi Y. Development of acquired hemophilia A during treatment of multiple myeloma with lenalidomide. Rinsho Ketsueki 2015;56:496-500. 8. Dunn AL. Malignancy in patients with hemophilia: a review of the literature. Haemophilia 2010;16:427-436. 9. Lesley R, Xu Y, Kalled SL, Hess DM, Schwab SR, Shu HB, Cyster JG. Reduced competitiveness of autoantigen-engaged B cells due to increased dependence on BAFF. Immunity 2004;4:441-453. 10. Pavlova A, Zeitler H, Scharrer I, Brackmann HH, Oldenburg J. HLA genotype in patients with acquired haemophilia A. Haemophilia 2010;16:107-112. 11. Sallah S, Wan JY. Inhibitors against factor VIII in patients with cancer. Analysis of 41 patients. Cancer 2001;91:1067-1074.

Address for Correspondence/Yazışma Adresi: Alessandro ALLEGRA, M.D., Messina University Faculty of Medicine, Department of Human Pathology and the Adult Developmental, Division of Hematology, Messina, Italy Phone : 0039 090 2212364 E-mail :aallegra@unime.it

Received/Geliş tarihi: April 09, 2016 Accepted/Kabul tarihi: January 09, 2017 DOI: 10.4274/tjh.2016.0146

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Bing-Neel Syndrome with Detectable MYD88 L265P Gene Mutation as a Late Relapse Following Autologous Hematopoietic Stem Cell Transplantation for Waldenström’s Macroglobulinemia Waldenström Makroglobulinemi Tanısıyla Otolog Kök Hücre Nakli Uygulanan Hastada MYD88 L265P Mutasyonu Pozitif Bing-Neel Sendromu ile Seyreden Geç Nüks Anna J. Kopińska, Grzegorz Helbig, Anna Koclęga, Sławomira Kyrcz-Krzemień Silesia University Faculty of Medicine in Katowice, Department of Hematology and Bone Marrow Transplantation, Katowice, Poland

To the Editor, Waldenström’s macroglobulinemia (WM) affects a proportion of patients diagnosed with lymphoplasmacytic lymphoma with bone marrow involvement and Immunoglobulin M (IgM) monoclonal gammopathy of any concentration [1]. The direct central nervous system (CNS) infiltration by malignant lymphoid cells is a rare complication of WM known as BingNeel syndrome (BNS) [2]. The MYD88 L265P point mutation is detected in about 90% of WM patients and may serve as a marker in distinguishing WM from other lymphomas [3]. It is noteworthy that this mutation has recently been detected in the cerebrospinal fluid (CSF) of patients with BNS [4]. A 67-year-old female with an 8-year history of WM presented with quadriplegia. At initial diagnosis her bone marrow was infiltrated by plasmacytoid lymphocytes with expression of typical WM surface markers (CD5+, CD10+, CD19+, CD20+, CD22+, CD23-, CD43+, CD79a+, CD200+, kappa+, lambda-). A monoclonal spike at 23 g/L was demonstrated in serum electrophoresis (SPE) and serum immunofixation (IFE) detected IgM kappa protein. She received cladribine with cyclophosphamide. As a result, she achieved complete response and was autotransplanted. Seven years later, the patient started complaining of disturbed gait. Neurological examination showed quadriplegia and ataxia. Magnetic resonance imaging (MRI) of the brain was not performed due to the presence of a pacemaker. Bone marrow aspirate was free of WM. CSF analysis identified lymphoplasmocytoid cells with the WM immunophenotype. A monoclonal spike of IgM kappa was demonstrated in CSF but not in SPE/IFE. The MYD88 L265P gene mutation was found in the CSF, but not in the marrow. She received intrathecal therapy with intravenous high-dose methotrexate and ifosfamide. While still on therapy, she progressed 2 months later. A complete blood count revealed extremely elevated white blood cells (356x109/L). Blood and marrow smears revealed >90% plasmacytoid lymphocytes with WM surface markers. The MYD88 L265P mutation was detected in her blood. SPE and IPE confirmed the presence of M-protein at a high level (33 g/L). She received palliative care. 186

BNS is a rare complication of WM and may have different clinical features. The diagnosis often remains challenging and includes the combination of CSF cytology and flow cytometry, MRI, and the detection of the MYD88 L265P mutation. A consensus on the diagnostic algorithm, recommended treatment, and response criteria was published recently [5]. This novel mutation may be helpful in monitoring minimal residual disease in BNS after treatment; however, this claim is based on a single report [6]. Of note is that the presence of the MYD88 mutation in the CSF is not synonymous with BNS. Its detection may result from blood contamination as small lymphocytes cross the bloodbrain barrier [7]. Moreover, other CNS lymphomas may harbor this mutation and therefore it is not specific [8]. The choice of treatment strategy for patients with BNS is still a matter of debate. The majority of patients were treated with systemic chemotherapy combined with intrathecal chemotherapy [9]. It was recently suggested that treatment with ibrutinib may be successful in patients with BNS [10]. Long-term treatment for WM may result in the development of secondary hematological malignancies [1]. However, a leukemic transformation of WM has not been reported so far. Acknowledgments: The authors thank Hanna Makula-Łasica and Grazyna Nowak from the Institute of Hematology and Transfusion Medicine of Warsaw, Poland, for helping with the analysis of the MYD mutation in CSF. Keywords: Bing-Neel syndrome, Waldenström’s macroglobulinemia, Central nervous system, MYD88 L265P mutation, Cerebrospinal fluid, Leukemia Anahtar Sözcükler: Bing-Neel sendromu, Waldenström makroglobulinemisi, Merkezi sinir sistemi, MYD88 L265P mutasyonu, Beyin-omurilik sıvısı, Lösemi 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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References 1. Gertz MA, Fonseca R, Rajkumar SV. Waldenström macroglobulinemia. Oncologist 2000;5:63-67. 2. Bing J, Neel A. Two cases of hyperglobulinemia with affection of the central nervous system on a toxi-infection basis. Acta Med Scand 1936;88:492-496. 3. Treon SP, Xu L, Yang G, Zhou Y, Liu X, Cao Y, Sheehy P, Manning RJ, Patterson CJ, Tripsas C, Arcaini L, Pinkus GS, Rodig SJ, Sohani AR, Harris NL, Laramie JM, Skifter DA, Lincoln SE, Hunter ZR. MYD88 L265P somatic mutation in Waldenström macroglobulinemia. N Engl J Med 2012;367:826-833. 4. Poulain S, Boyle EM, Roumier C, Demarquette H, Wemeau M, Geffroy S, Herbaux C, Bertrand E, Hivert B, Terriou L, Verrier A, Pollet JP, Maurage CA, Onraed B, Morschhauser F, Quesnel B, Duthilleul P, Preudhomme C, Leleu X. MYD88 L265P mutation contributes to the diagnosis of Bing-Neel syndrome. Br J Haematol 2014;167:506-513. 5. Minnema MC, Kimby E, D’Sa S, Fornecker LM, Poulain S, Snijders TJ, Kastritis E, Kremer S, Fitsiori A, Simon L, Davi F, Lunn M, Castillo JJ, Patterson CJ, Le Garff-Tavernier M, Costopoulos M, Leblond V, Kersten MJ, Dimopoulos MA, Treon SP. Guideline for the diagnosis, treatment and response criteria for Bing-Neel syndrome. Haematologica 2017;102:43-51. 6. Frustaci AM, Rusconi C, Picardi P, Veronese S, Montillo M, Cairoli R, Tedeschi A. Bing Neel syndrome in a previously untreated patient with Waldenström’s macroglobulinemia: contribution of MYD88 L265P mutation on cerebrospinal fluid. Clin Lymphoma Myeloma Leuk 2016;1:e7-9.

7. Simon L, Fitsiori A, Lemal R, Dupuis J, Carpentier B, Boudin L, Corby A, Aurran-Schleinitz T, Gastaud L, Talbot A, Leprêtre S, Mahe B, Payet C, Soussain C, Bonnet C, Vincent L, Lissandre S, Herbrecht R, Kremer S, Leblond V, Fornecker LM. Bing-Neel syndrome, a rare complication of Waldenström’s macroglobulinemia: analysis of 44 cases and review of the literature. A study on behalf of the French Innovative Leukemia Organization (FILO). Haematologica 2015;100:1587-1594. 8. Nakamura T, Tateishi K, Niwa T, Matsushita Y, Tamura K, Kinoshita M, Tanaka K, Fukushima S, Takami H, Arita H, Kubo A, Shuto T, Ohno M, Miyakita Y, Kocialkowski S, Sasayama T, Hashimoto N, Maehara T, Shibui S, Ushijima T, Kawahara N, Narita Y, Ichimura K. Recurrent mutations of CD79B and MYD88 are the hallmark of primary central nervous system lymphomas. Neuropathol Appl Neurobiol 2016;42:279-290. 9. Castillo JJ, D’Sa S, Lunn MP, Minnema MC, Tedeschi A, Lansigan F, Palomba ML, Varettoni M, Garcia-Sanz R, Nayak L, Lee EQ, Rinne ML, Norden AD, Ghobrial IM, Treon SP. Central nervous system involvement by Waldenström macroglobulinemia (Bing-Neel syndrome): a multi-institutional retrospective study. Br J Haematol 2016;172:709-715. 10. Cabannes-Hamy A, Lemal R, Goldwirt L Poulain S, Amorim S, Pérignon R, Berger J, Brice P, De Kerviler E, Bay JO, Sauvageon H, Beldjord K, Mourah S, Tournilhac O, Thieblemont C. Efficacy of ibrutinib in the treatment of BingNeel syndrome. Am J Hematol 2016;91:e17-e19.

Address for Correspondence/Yazışma Adresi: Grzegorz HELBIG, M.D., Silesia University Faculty of Medicine in Katowice, Department of Hematology and Bone Marrow Transplantation, Katowice, Poland Phone: +48322591310 E-mail: ghelbig@o2.pl

Received/Geliş tarihi: November 20, 2016 Accepted/Kabul tarihi: December 15, 2016 DOI: 10.4274/tjh.2016.0452

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Concomitant Presence of CD5-Positive Diffuse Large B-Cell Lymphoma and Monoclonal B Cells with the “CLL Immunophenotype” - Is It Richter’s Transformation? CD5-Pozitif Diffüz Büyük B-Hücreli Lenfoma ve “KLL İmmünfenotipinde” Monoklonal B Hücrelerin Birlikteliği - Richter Transformasyonu mudur? Sabina Langer, Jasmita Dass, Suchi Mittal, Shyam Aggarwal Sir Ganga Ram Hospital, Clinic of Hematology, New Delhi, India

To the Editor, The presence of diffuse large B-cell lymphoma (DLBCL) with a concomitant unsuspected population of B cells with chronic lymphoid leukemia (CLL) phenotype is very rare with no antecedent history of CLL. This may represent cases of de novo Richter’s transformation or the coexistence of two neoplasms [1]. In cases where the monoclonal B-cell population does not exceed 5x109/L, this may represent DLBCL with concomitant monoclonal B-cell lymphocytosis (MBL) of the CLL phenotype. The coexistence of MBL of the CLL phenotype creates a diagnostic conundrum, especially in an unusual case of CD5+ DLBCL as it may be a de novo CD5+ DLBCL or Richter transformation [2]. The former has an aggressive course compared to de novo DLBCLs [3,4]. CLL has been known to occur synchronously or metachronously with hairy cell leukemia [5] and DLBCL [6]. We present here a case of clinically aggressive de novo CD5+ DLBCL with an unsuspected second population of CLL-like MBL detected on flow cytometry. A 57-year-old male presented with history of fever for 3 months and the presence of mediastinal lymphadenopathy, hepatosplenomegaly, and raised serum lactate dehydrogenase levels. The complete blood count revealed hemoglobin of 9.1 g/dL, total leukocyte count of 9600/µL, and platelet count of 16,000/µL. A peripheral smear revealed a leukoerythroblastic blood picture with 30 nRBCs/100 WBCs, left shift, and 6% abnormal lymphoid cells (neutrophils: 43%, lymphocytes: 42%, monocytes: 8%, myelocytes: 3%, and metamyelocytes: 4%). The bone marrow aspirate showed 10%-15% abnormal lymphoid cells, which were 3-5 times the size of a small mature lymphocyte with a moderate amount of deep blue cytoplasm, round nuclei with irregularity of membranes in some, and coarsely clumped chromatin (Figure 1A). Immunophenotyping of the bone marrow specimen revealed two distinct populations of cells: CD19+ small-sized lymphocytes (red) and CD19+ large lymphoid cells (Figure 1B). The small cells (red) showed the CLL immunophenotype with 188

kappa light chain restriction and CD19+ large cells (blue), which also showed surface kappa restriction but were CD5+, CD10+, and CD23- (Figures 1C-1F). The monoclonal B-cell count was 653/µL and hence the small cells represent MBL with the CLL phenotype. The large B cells suggested marrow infiltration by DLBCL. The bone marrow biopsy showed abnormal large lymphoid cells and interstitial infiltrate of small lymphocytes. Immunohistochemistry revealed that large cells were positive for CD20 (Figure 2A) and CD5 (Figure 2B) and negative for CD3, CD23, and CD10. The lymphoid cells were negative for cyclin D1 (Figure 2C). The Ki-67 staining of large lymphoid cells showed a high proliferative index (~90%). The small lymphoid cells present interstitially showed positivity for CD20, CD23, and CD5 (Figures 2A, 2B, and 2D). The immunohistochemical marker p53 seen in transformed CLL [6] was negative. Therefore, we concluded that there was incidentally detected MBL in this patient who otherwise had CD5+ DLBCL. This patient received 2 doses of injection rituximab without any additional chemotherapy as he had hepatic dysfunction and poor ejection fraction. He had an aggressive disease course and succumbed within 10 days. This report describes a rare situation where MBL was detected concomitantly with a CD5+ DLBCL. Gene mutation studies are necessary to differentiate these two entities. This case also calls for a consensus on reporting such cases. Keywords: Richter’s syndrome, Flow cytometry, Chronic lymphocytic leukemia, CD5-positive, Diffuse large B-cell lymphoma Anahtar Sözcükler: Richter sendromu, Akım sitometri, Kronik lenfositik lösemi, CD5- pozitif, Diffüz büyük B-hücreli lenfoma 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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Figure 1. A) Bone marrow aspirate with 10%-15% abnormal lymphoid cells (3-5 times the size of a small mature lymphocyte with moderate amount of deep blue cytoplasm, round nuclei with irregularity of membranes in some, and coarsely clumped chromatin (Wright-Giemsa, 1000x). B) Flow cytometric immunophenotyping of the bone marrow specimen gated on CD19 vs. forward scatter. The small B-lymphoid cells are in red while the large B-lymphoid cells are blue. C) CD5 vs. CD23 plot: the small B-lymphoid cells (red) show a coexpression of CD5 and CD23 while the large lymphoid cells (blue) are positive for CD5 at a higher intensity than small lymphoid cells and are negative for CD23. D) CD10 vs. CD20 plot: the small B-lymphoid cells (red) show dim CD20 and are negative for CD10. Large lymphoid cells (blue) are dimly positive for CD20 and show CD10 coexpression. E) CD22 vs. FMC7 plot: the small B-lymphoid cells (red) show dim CD22 and are negative for FMC7. Large lymphoid cells (blue) are positive for CD22 at a higher intensity than small lymphoid cells and are dimly positive for FMC7. F) Kappa vs. lambda plot: both the small lymphoid cells and large lymphoid cells show kappa light chain restriction.

Figure 2. CD20 immunostaining highlights the large B cells and some small lymphoid cells are also positive (200x). B) Both large and small lymphoid cells show CD5 expression (200x). C) Cyclin D1 is negative in both small and large lymphoid cells (400x). D) The small lymphoid cells present interstitially are positive for CD23 (400x). 189


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References 1. Parikh SA, Rabe KG, Call TG, Zent CS, Habermann TM, Ding W, Leis JF, Schwager SM, Hanson CA, Macon WR, Kay NE, Slager SL, Shanafelt TD. Diffuse large B-cell lymphoma (Richter syndrome) in patients with chronic lymphocytic leukaemia (CLL): a cohort study of newly diagnosed patients. Br J Haematol 2013;162:774-782. 2. Kroft SH, Dawson DB, McKenna RW. Large cell lymphoma transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma. A flow cytometric analysis of seven cases. Am J Clin Pathol 2001;115:385-395. 3. Parikh SA, Kay NE, Shanafelt TD. How we treat Richter syndrome. Blood 2014;123:1647-1657.

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4. Tsimberidou AM, O’Brien S, Khouri I, Giles FJ, Kantarjian HM, Champlin R, Wen S, Do KA, Smith SC, Lerner S, Freireich EJ, Keating MJ. Clinical outcomes and prognostic factors in patients with Richter’s syndrome treated with chemotherapy or chemoimmunotherapy with or without stem-cell transplantation. J Clin Oncol 2006;24:2343-2351. 5. Verma V, Giri S, Bhatt VR, Amador-Ortiz A, Armitage JO. Synchronous or metachronous hairy cell leukemia and chronic lymphocytic leukemia: a case series and literature review. Front Oncol 6:270. 6. Wei Q, Sebastian S, Papavassiliou P, Rehder C, Wang E. Metachronous/ concomitant B-cell neoplasms with discordant light-chain or heavy-chain isotype restrictions: evidence of distinct B-cell neoplasms rather than clonal evolutions. Hum Pathol 2014;45:2063-2076.

Address for Correspondence/Yazışma Adresi: Sabina Langer, M.D, Sir Ganga Ram Hospital, Clinic of Haematology, New Delhi, India Phone : +91 981 139 5249 E-mail : sabinapost@yahoo.com

Received/Geliş tarihi: November 19, 2016 Accepted/Kabul tarihi: February 15, 2017 DOI: 10.4274/tjh.2016.0451

Non-Leukemic Granulocytic Sarcoma Presenting with Multiple Lymphadenopathies Çoklu Lenfadenopati ile Ortaya Çıkan Alösemik Granülositik Sarkom Ayfer Gedük¹, Esra T. Demirsoy¹, Süheyla U. Bozkurt², Zafer Gülbaş³, Serkan İşgören⁴ ¹Kocaeli University Faculty of Medicine, Department of Hematology, Kocaeli, Turkey ²Marmara University Faculty of Medicine, Training and Research Hospital, Department of Pathology, İstanbul, Turkey ³Anadolu Medical Center, Bone Marrow Transplantation Center, Kocaeli, Turkey ⁴Kocaeli University Faculty of Medicine, Department of Nuclear Medicine, Kocaeli, Turkey

To the Editor, Granulocytic sarcoma (GS) is a rare tumor with poor prognosis that is composed of primitive myeloid cells, localized in extramedullary sites. The incidence is 2.5%-9.1% in acute myeloid leukemia (AML) patients and it may also occur in association with a myeloproliferative neoplasm or myelodysplastic disorders. The most common locations are the skin, lymph nodes, gastrointestinal tract, bones, and soft tissues [1]. A 68-year-old man presented with the complaint of bilateral inguinal swelling. On physical examination bilateral cervical, axillary, and inguinal multiple lymphadenopathies (LAP), approximately 2-3 cm in diameter, were noted. Initial workup revealed normal liver and renal functions; negative viral serology for epstein-barr virus, cytomegalovirus, HIV, and hepatitis B/C; and mild neutropenia (1020/µL). An excisional LAP biopsy revealed disruption of the normal lymph nodal architecture by a diffuse monomorphic infiltrate comprising medium-sized mononuclear cells with a high nuclear cytoplasmic ratio and fine chromatin pattern. In immunohistochemical study, these cells were positive for CD34, CD43, CD117, and myeloperoxidase 190

and negative for Tdt, CD3, CD5, CD20, CD15, CD30, CD56, EMA, and Pax-5. The Ki-67 proliferation index was 45% and EBER was negative. Leukemic infiltration could not be detected in the bone marrow examination. Cytogenetic analysis was negative for t(9,22), t(15,17), t(8,21), and inv16 but positive for NPM1 and 11q23 rearrangement. The results confirmed the diagnosis of GS (Figures 1A-1C). A positron emission tomography/ computed tomography (PET/CT) scan showed multiple hypermetabolic lymph nodes (SUVmax: 11.8) in bilateral cervical, axillary, paracardiac, and both common iliac areas (Figure 1D). Cytosine-arabinoside plus idarubicin (3+7 regimen) was started. PET/CT was repeated after chemotherapy and revealed partial response (Figure 1E). The persistent disease was confirmed by an excisional LAP biopsy and a fludarabine, cytarabine, G-CSF and idarubicin regimen was started. Since complete metabolic response was detected in the follow-up PET/CT, he underwent a matched related donor reduced-intensity conditioning hematopoietic stem cell transplantation (HSCT) (Figure 1F). He engrafted successfully and has had no recurrent GS for 8 months since the transplant. Although it is well recognized that GS can cause localized lymphadenopathy, manifestation as bilateral multiple LAP is


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Figure 1. A) Large neoplastic cells infiltrating paracortical areas of the lymph node (hematoxylin and eosin, 400x). B) Immunohistochemistry showing CD34 immunoreactivity in the cytoplasm of the neoplastic cells (CD34 stain, 400x). C) Immunohistochemistry showing myeloperoxidase immunoreactivity in the cytoplasm of the neoplastic cells (myeloperoxidase stain, 400x). D) Initial positron emission tomography/computed tomography (PET/CT) presents multiple hypermetabolic lymph nodes. E) PET/CT after 3+7 regimen shows partial response. F) PET/CT after fludarabine, cytarabine, G-CSF, and idarubicin regimen shows complete metabolic response. rare [2]. Nandedkar et al. [3] reported a case of GS presenting with bilateral multiple LAP without evidence of leukemia, which is similar to our patient. In these two cases the interesting point is a myeloid malignancy presenting with only lymphatic spread. There is no consensus on the treatment of non-leukemic GS. Common practice suggests AML-like induction chemotherapies as first-line treatment and allogeneic HSCT for relapsed/ refractory disease. Radiotherapy and surgery are also options in selected cases [4]. In a study by Chevallier et al. [5] that assessed the outcome of 30 patients with non-leukemic GS who underwent allogeneic HSCT, 5-year overall survival and

leukemia-free survival were 48% and 36%, respectively. In another study, a significantly longer event-free survival rate was detected in patients treated with chemotherapy and allogeneic HSCT [6]. These data suggest that allogeneic HSCT is efficient as a consolidation regimen for GS. In our case, PET/CT was used for disease detection and in the monitoring of treatment response, which is an effective imaging tool for GS [7]. In conclusion, we present a case of GS with lymphatic spread like a lymphoma. Keywords: Granulocytic sarcoma, Leukemia, Lymphadenopathy Anahtar Sözcükler: Granülositik sarkom, Lösemi, Lenfadenopati 191


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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. Pileri SA, Ascani S, Cox MC, Campidelli C, Bacci F, Piccioli M, Piccaluga PP, Agostinelli C, Asioli S, Novero D, Bisceglia M, Ponzoni M, Gentile A, Rinaldi P, Franco V, Vincelli D, Pileri A Jr, Gasbarra R, Falini B, Zinzani PL, Baccarani M. Myeloid sarcoma: clinico-pathologic, phenotypic and cytogenetic analysis of 92 adult patients. Leukemia 2007;21:340-350. 2. Neiman RS, Barcos M, Berard C, Bonner H, Mann R, Rydell RE, Bennett JM. Granulocytic sarcoma: a clinicopathologic study of 61 biopsied cases. Cancer 1981;48:1426-1437.

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3. Nandedkar S, Kawatra M, Malukani K. Myeloid sarcoma de novo presenting as generalized lymphadenopathy. Clin Cancer Investig J 2013;2:86. 4. Yilmaz AF, Saydam G, Sahin F, Baran Y. Granulocytic sarcoma: a systematic review. Am J Blood Res 2013;3:265-270. 5. Chevallier P, Labopin M, Cornelissen J, Socié G, Rocha V, Mohty M; ALWP of EBMT. Allogeneic hematopoietic stem cell transplantation for isolated and leukemic myeloid sarcoma in adults: a report from the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation. Haematologica 2011;96:1391-1394. 6. Antic D, Elezovic I, Milic N, Suvajdzic N, Vidovic A, Perunicic M, Djunic I, Mitrovic M, Tomin D. Is there a “gold” standard treatment for patients with isolated myeloid sarcoma? Biomed Pharmacother 2013;67:72-77. 7. Lee EYP, Anthony MP, Leung AYH, Loong F, Khong PL. Utility of FDG PET/CT in the assessment of myeloid sarcoma. AJR Am J Roentgenol 2012;198:11751179.

Address for Correspondence/Yazışma Adresi: Ayfer GEDÜK, M.D., Kocaeli University Faculty of Medicine, Department of Hematology, Kocaeli, Turkey Phone : +90 262 303 73 83 E-mail : ayfergeduk@hotmail.com

Received/Geliş tarihi: October 31, 2016 Accepted/Kabul tarihi: February 9, 2017 DOI: 10.4274/tjh.2016.0428

A Case of Leukemia Cutis with Acute Myeloid Leukemia on Azacitidine Therapy Akut Miyeloid Lösemide Azasitidin Tedavisi Altında Lösemi Kutis Olgusu Asude Kara1, Aslı Akın Belli1, Volkan Karakuş2, Yelda Dere3, Erdal Kurtoğlu4 Department of Dermatology, Muğla Sıtkı Koçman University Training and Research Hospital, Muğla, Turkey Department of Hematology, Muğla Sıtkı Koçman University Training and Research Hospital, Muğla, Turkey 3 Department of Pathology, Muğla Sıtkı Koçman University Faculty of Medicine, Mugla, Turkey 4 Department of Hematology, Antalya Training and Research Hospital, Antalya, Turkey 1 2

To the Editor, Leukemia cutis (LC) is an extramedullary form of leukemia. The frequency and onset age of LC depends on the subtype of underlying leukemia. Clinical presentations of LC can be variable but it generally appears as nodules and plaques [1]. Herein, we report a case of LC with acute myeloid leukemia on azacitidine therapy.

not done. In the conventional cytogenetic analysis done before the treatment, 20 metaphases were detected. Six of them had “add” (46,XY,add(8)(q24)[6]) and 10 were diploids and 4 were hypodiploids. In the fluorescence in situ hybridization (FISH) analysis [5q31, t(15;17)(q22-24;q21), trisomy 8, t(9;22) (q34;q11.2), inv(16)(p13q22), del7q31, del/inv11q23, monosomy 7, t(8;21)(q21.3;q22), and del20q], there was no abnormality.

A 70-year-old male presented with spontaneous ecchymoses and weakness in the Hematology Outpatient Clinic. Physical examination was normal. Laboratory tests were as follows: hemoglobin: 4.1 g/dL, leukocyte count: 17.600/mm3, platelet count: 57.000/mm3, and lactate dehydrogenase: 605 U/L. Peripheral blood smear revealed 60% myeloblasts. Eighty percent of blastic infiltration and positive staining with myeloperoxidase (MPO) were detected in the bone marrow biopsy. The patient was diagnosed with acute myelomonocytic leukemia (AML-M4) with the morphological and immunopathological findings. Flow cytometry of the bone marrow or peripheral blood was

After four cycles of therapy with azacitidine (75 mg/m2 daily for 7 days in a month), the need for red blood cells decreased but the need for platelets remained. Furthermore, some skin lesions appeared on the trunk. On dermatological examination, multiple discrete, violaceous-erythematous papules and nodules were observed on the trunk (Figures 1a and 1b). Histopathological examination of the skin lesions showed blastic cell infiltration with large pleomorphic nuclei and narrow cytoplasm in the dermis and also positive staining with CD34, CD117, and MPO (Figures 1c and 1d). The patient was diagnosed with LC with AML-M4 relapsed in the bone marrow synchronously and the

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leukemia generally improves skin lesions. Unfortunately, skin involvement of leukemia indicates poor prognosis [3]. Azacitidine is an approved and well-tolerated drug in the treatment of elderly AML patients in particular [4]. Recently, we noticed that LC cases have been reported in patients with chronic myeloid leukemia and myelodysplastic syndrome on azacitidine therapy [5]. Similarly, azacitidine therapy at the current dosage may have been insufficient in our patient and thus the cutaneous involvement developed. We want to emphasize that patients developing LC on azacitidine therapy should be accepted as refractory to the therapy and salvage therapy should be planned. Keywords: Acute myeloid leukemia, Azacitidine, Leukemia cutis Anahtar Sözcükler: Akut miyeloid lösemi, Azasitidin, Lösemi kutis 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 Figure 1. a) Multiple discrete, erythematous papules and nodules on the trunk. b) Closer view of the erythematous papules on the abdomen. c) Blastic cells with pleomorphic nuclei and narrow cytoplasm in the dermis (hematoxylin & eosin, original magnification 400x). d) Positive staining of the blasts with myeloperoxidase (original magnification 40x). therapy regimen was changed from azacitidine to cytosinearabinoside. The same FISH panel was obtained. LC is a rare disease characterized by leukemic cell infiltration in the dermis, subcutaneous tissue, and blood vessels. The frequency of LC is about 2%-3% in patients with AML [2]. LC mimics various dermatoses. Histopathological analysis of the lesions has an important role in the diagnosis [3]. There is no special treatment for LC and the treatment of the underlying

1. Wagner G, Fenchel K, Back W, Schulz A, Sachse MM. Leukemia cutis epidemiology, clinical presentation, and differential diagnoses. J Dtsch Dermatol Ges 2012;10:27-36. 2. Seok DK, Kee SY, Ko SY, Lee JH, Kim HY, Kim IS, Seo HY. Leukemia cutis in a patient with acute monocytic leukemia diagnosed simultaneously with hepatocellular carcinoma: a case study. Oncol Lett 2013;6:1319-1322. 3. Whittaker SJ. Cutaneous lymphomas and lymphocytic infiltrates. In: Burns T, Breathnach S, Cox N, Griffiths C, (eds). Rook’s Text Book of Dermatology. Oxford, Wiley-Blackwell, 2010. 4. Sadashiv SK, Hilton C, Khan C, Rossetti JM, Benjamin HL, Fazal S, Sahovic E, Shadduck RK, Lister J. Efficacy and tolerability of treatment with azacitidine for 5 days in elderly patients with acute myeloid leukemia. Cancer Med 2014;3:1570-1578. 5. Infante MS, Muñoz C, Heras C, Foncillas MA, González I, Lucea I, Echavarria E, Aramendi T, Hernández JA. Leukemia cutis in myelodysplastic syndrome and chronic myelomonocytic leukemia patients treated with adjusted dose 5-azacitidine. Eur J Dermatol 2015;25:622-623.

Address for Correspondence/Yazışma Adresi: Asude KARA, M.D., Muğla Sıtkı Koçman University Faculty of Medicine, Training and Research Hospital, Department of Dermatology, Muğla, Turkey Phone: +90 505 251 21 42 E-mail: asudekara@yahoo.com.tr

Received/Geliş tarihi: June 15, 2016 Accepted/Kabul tarihi: February 09, 2017 DOI: 10.4274/tjh.2016.0220

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Unique Presentation of Leukemic Cutaneous CD3/TCR- Phenotype T-Cell Lymphoma with Complete Remission after Allogeneic Stem Cell Transplantation Lösemik Kutanöz T Hücreli Lenfomanın CD3/TCR- Fenotipi ile Nadir Prezentasyonu ve Allojenik Kök Hücre Transplantasyonu ile Tam Remisyon Hatice Şanlı1, Bengü Nisa Akay1, Seçil Saral1, Aylin Okçu Heper2, Pervin Topçuoğlu3 Ankara University Faculty of Medicine, Department of Dermatology and Venereology, Ankara, Turkey Ankara University Faculty of Medicine, Department of Pathology, Ankara, Turkey 3 Ankara University Faculty of Medicine, Department of Heamatology, Ankara, Turkey 1 2

To the Editor, A 49-year-old male was admitted to our department with a 3-year history of pruritus and severe xerosis. Dermatological examination revealed squamation and ichthyosis with dark lamella involving the trunk and extremities (Figure 1). Physical examination revealed bilateral lower extremity edema. Histopathological examination of the ichthyotic skin showed perivascular dermal lymphoid cell infiltration in the superficial dermis and nodular and dense lymphoid cellular infiltration in the deep dermis. Lymphoid cells were CD3+ and CD20- with a loss of CD7 expression. There were numerous (>25%) large, anaplastic cells with CD30 positivity among the lymphoid infiltrate. Clonal expansion of T cells in the lesional skin was demonstrated. Complete blood count revealed 61.5x109/L (reference range: 4.511) leukocytes with 15% Sezary cells. The axillary lymph node was consistent with N3 mycosis fungoides (MF) involvement. Thorax, abdominal, and pelvic computed tomography was normal. Bone marrow biopsy revealed involvement with positive clonality.

of cyclophosphamide (120 mg/kg) and total body irradiation (12 Gy) from an HLA-identical sibling donor. Cyclosporine A plus short-term methotrexate was given for graft-versus-host disease (GVHD) prophylaxis. Chronic sclerodermoid GVHD developed 1 year later and extracorporeal photopheresis was started for GVHD with complete response. After allo-HSCT, clonal T cells disappeared and skin lesions resolved completely. There are only 5 patients showing negative TCR/CD3 complex in the literature and these patients were reported to have SS and atypical skin lesions, mainly non-erythrodermic leukemic variants, papuloerythroderma of Ofuji, prurigo nodularis, atopic dermatitis, papular xanthomatosis, and poikiloderma atrophicans vascularelike lesions [3,4,5,6,7]. Allo-HSCT has been proven to be an effective therapy in MF/SS, demonstrating a decrease in the relapse rate and an overall increase in disease-free survival compared with conventional therapy. In a series of MF/SS transplants, Molina et al. [8] observed complete

Flow cytometric analysis of the peripheral blood revealed the CD3/ TCR complex in only 10% of the T cells. The CD4/CD8 ratio was 23 among T cells lacking CD3 expression and 1.6 among CD3+ cells. The patient met the international criteria for Sezary syndrome [(SS); stage IVB, T4N3M1B2] and was diagnosed with ichthyosiform MF with large cell transformation with atypical flow cytometric phenotype [1,2]. According to the 2014 National Comprehensive Cancer Network Clinical Practice Guidelines [2], treatment was initiated as extracorporeal photopheresis, interferon-alpha-2a 3, and psoralen-UVA as first-line treatment without any response. The patient was unresponsive to polychemotherapy with gemcitabine and cisplatin and also three cycles of pralatrexate treatment. The patient underwent allogeneic hematopoietic peripheral stem cell transplantation (allo-HSCT) with an ablative conditioning regimen 194

a

b

Figure 1. Ichthyotic plaques over anterior surface (a), and posterior surface (b) of lower extremities, edema and varicose dilatations of superficial veins are visible.


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remission of skin lesions in 100% of patients after allo-HSCT. Duarte et al. [9] reported that 1 year after allo-HSCT, 42% of their patients remained in remission. Use of total skin electron beam as a debulking agent before conditioning with non-myeloablative alloHSCT may reduce the severity of post-transplantation cutaneous GVHD [10].

3. Shimauchi T, Sugita K, Nakamura M, Tokura Y. Leukaemic cutaneous T-cell lymphoma-manifesting papuloerythroderma with CD3(-) CD4(+) phenotype. Acta Derm Venereol 2010;90:68-72.

In conclusion, our case is the first CD3-/TCR-SS patient presenting with generalized ichthyosis. All the patients with this immunophenotype are reported to have SS with intriguing skin lesions. These patients may require early initiation of more aggressive therapies. In our patient, allo-HSCT treatment resulted in cure and remission in a follow-up period of 3 years.

5. Taniguchi T, Sugaya M, Miyagaki T, Kogure A, Takekoshi T, Asano Y, Mitsui H, Sato S. Leukaemic variant of cutaneous T-cell lymphoma with CD3- CD4+ phenotype. Clin Exp Dermatol 2012;37:443-445.

Keywords: Mycosis fungoides, Ichthyosiform, Sezary syndrome, Anaplastic, CD3/TCRAnahtar Sözcükler: Mikozis fungoides, İktiyotik, Sezary sendromu, Anaplastik, CD3/TCRConflict 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. Wilcox RA. Cutaneous T-cell lymphoma: 2016 update on diagnosis, riskstratification, and management. Am J Hematol 2016;91:151-165. 2. https://www.nccn.org/professionals/physician_gls/pdf/nhl.pdf. accessed at 27/11/2016.

4. Lima M, Velho G, Alves R, Cunha M, Teixeira Mdos A, Canelhas A, Almeida J, Sachse F, Queiros ML, Santos AH, Fonseca S, Goncalves V, Massa A, Orfao A, Justica B. Atopic dermatitis-like non-erythrodermic leukemic variant of CD3(-/+dim) CD4(+) cutaneous T-cell lymphoma preceded by cutaneous papular xanthomatosis. Leuk Lymphoma 2004;45:597-603.

6. Tokura Y, Yagi H, Seo N, Takagi T, Takigawa M. Nonerythrodermic, leukemic variant of cutaneous T-cell lymphoma with indolent clinical course: Th2type tumor cells lacking T-cell receptor/CD3 expression and coinfiltrating tumoricidal CD8 T cells. J Am Acad Dermatol 2000;43:946-954. 7. Fischer MB, Hauber I, Födinger M, Wolf HM, Thon V, Donath P, Eibl MM, Knobler RM. Defective TCR surface expression associated with impaired TCR beta-chain assembly in a patient with cutaneous T-cell lymphoma. J Invest Dermatol 1995;104:537-540. 8. Molina A, Zain J, Arber DA, Angelopolou M, O’Donnell M, Murata-Collins J, Forman SJ, Nademanee A. Durable clinical, cytogenetic, and molecular remissions after allogeneic hematopoietic cell transplantation for refractory Sezary syndrome and mycosis fungoides. J Clin Oncol 2005;23:6163-6171. 9. Duarte RF, Canals C, Onida F, Gabriel IH, Arranz R, Arcese W, Ferrant A, Kobbe G, Narni F, Deliliers GL, Olavarria E, Schmitz N, Sureda A. Allogeneic hematopoietic cell transplantation for patients with mycosis fungoides and Sezary syndrome: a retrospective analysis of the Lymphoma Working Party of the European Group for Blood and Marrow Transplantation. J Clin Oncol 2010;28:4492-4499. 10. Duvic M, Donato M, Dabaja B, Richmond H, Singh L, Wei W, Acholonu S, Khouri I, Champlin R, Hosing C. Total skin electron beam and non-myeloablative allogeneic hematopoietic stem-cell transplantation in advanced mycosis fungoides and Sezary syndrome. J Clin Oncol 2010;28:2365-2372.

Address for Correspondence/Yazışma Adresi: Seçil SARAL, M.D., Ankara University Faculty of Medicine, Department of Dermatology and Venereology, Ankara, Turkey Phone : +90 505 432 46 82 E-mail : secilsaral@gmail.com

Received/Geliş tarihi: October 03, 2016 Accepted/Kabul tarihi: December 21, 2016 DOI: 10.4274/tjh.2016.0395

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Is the Game Over or Starting Again? The Role of the Transplant Team in Genetic Counseling for Adult Sickle Cell Disease Recipients Oyun Bitti mi Yoksa Yeniden mi Başlıyor? Erişkin Orak Hücre Hastalığı Olan Alıcılarda Genetik Danışmanlıkta Nakil Ekibinin Rolü Pelin Aytan1, Çiğdem Gereklioğlu2, Mahmut Yeral1, Aslı Korur1, Süheyl Asma1, İlknur Kozanoğlu1, Hakan Özdoğu1, Can Boğa1 Başkent University Training and Research Hospital, Bone Marrow and Stem Cell Transplantation Center, Clinic of Hematology, Adana, Turkey Başkent University Faculty of Medicine, Department of Family Medicine, Adana, Turkey

1 2

To the Editor, Hematopoietic stem cell transplantation (HSCT) with nonmyeloablative regimens has become more feasible and it is currently performed more frequently in adult patients with sickle cell disease (SCD) [1]. Hsieh [1] reported successful outcomes for adults with a regimen of alemtuzumab and low-dose total body irradiation (TBI) in transplants from human leukocyte antigen (HLA)-matched sibling donors. We have obtained sustained full chimerism in 13 adults with protocols including antithymocyte globulin (ATG) and busulfan without any significant complications (unpublished data). For unrelated donors, the ongoing STRIDE study uses conditioning regimens containing ATG, fludarabine, and busulfan. Bolaños-Meade et al. [2] achieved a cure with HLA-haploidentical transplants performed with ATG, fludarabine, and low-dose TBI. Given that these protocols may allow assisted or spontaneous pregnancies, fertility issues need to be examined [3] because the patient may think that he/she is completely cured after allogeneic transplantation and that he/she no longer carries the hemoglobin S (Hb S) gene. This erroneous belief may lead to the delivery of a homozygous Hb SS infant if the partner has a high risk of being a Hb S gene carrier when marriages of close relatives occur, as in some populations like Eti-Turks. SCD is the most common hereditary disease worldwide. The impaired microcirculation caused by rigid erythrocytes leads to considerable mortality and severe morbidity if not managed appropriately [4]. The only proven curative therapy is HSCT. However, this treatment carries high risk for infertility as a major complication for young patients [5]. Factors predisposing to infertility in these patients include delayed puberty, priapism, and gonadal dysfunction with increased abnormal spermatozoa, seminal vesicle and prostate gland abnormalities, decreased ejaculate volume, lower testosterone levels, and low sperm counts in males [6,7,8]. Conditioning regimens including alemtuzumab and low-dose TBI are reported not to irreparably impair spermatogenesis, although there are insufficient 196

data about non-myeloablative conditioning including ATG and busulfan [9]. When young SCD patients want to marry after full recovery with transplantation, some of them tend to hide their disease intentionally or due to a lack of awareness. A complete blood count and hemoglobin electrophoresis test as part of the screening done before marriage may give normal results. Consequently, affected germ cells may be overlooked. This may result in giving birth to affected children. The transplant team is responsible for providing sufficient information about these issues. Keywords: Adult, Sickle cell anemia, Genetic counseling, Transplantation Anahtar Sözcükler: Erişkin, Orak hücreli anemi, Genetik danışmanlık, Transplantasyon 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. Hsieh MM. A standard nonmyeloablative transplantation regimen for adults with sickle cell disease: are we there yet? Biol Blood Marrow Transplant 2016;22:397-398. 2. Bolaños-Meade J, Fuchs EJ, Luznik L, Lanzkron SM, Gamper CJ, Jones RJ, Brodsky RA. HLA-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease. Blood 2012;120:4285-4291. 3. Tichelli A, Rovó A. Fertility issues following hematopoietic stem cell transplantation. Expert Rev Hematol 2013;6:375-388. 4. Karacaoglu PK, Asma S, Korur A, Solmaz S, Buyukkurt NT, Gereklioglu C, Kasar M, Ozbalcı D, Unal S, Kaya H, Gurkan E, Yeral M, Sariturk Ç, Boga C, Ozdogu H. East Mediterranean region sickle cell disease mortality trial: retrospective multicenter cohort analysis of 735 patients. Ann Hematol 2016;95:993-1000. 5. Özdoğu H, Boğa C. Hematopoietic stem cell transplantation in adult sickle cell disease: problems and solutions. Turk J Hematol 2015;32:195-205.


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6. Oncofertility Consortium. Sickle Cell Anemia. Chicago, Northwestern University, 2014. Available online at http://oncofertility.northwestern.edu/ resources/sickle-cell-anemia. 7. Agbaraji VO, Scott RB, Leto S, Kingslow LW. Fertility studies in sickle cell disease: semen analysis in adult male patients. Int J Fertil 1988;33:347-352.

8. Abudu EK, Akanmu SA, Soriyan OO, Akinbami AA, Adediran A, Adeyemo TA, Okany CO. Serum testosterone levels of HbSS (sickle cell disease) male subjects in Lagos, Nigeria. BMC Res Notes 2011;4:298. 9. Gharwan H, Neary NM, Link M, Hsieh MM, Fitzhugh CD, Sherins RJ, Tisdale JF. Successful fertility restoration after allogeneic hematopoietic stem cell transplantation. Endocr Pract 2014;20:157-161.

Address for Correspondence/Yazışma Adresi: Pelin AYTAN, M.D., Başkent University Training and Research Hospital, Bone Marrow and Stem Cell Transplantation Center, Clinic of Hematology, Adana, Turkey Phone: +90 322 327 27 27-20-28 E-mail:drpelinaytan@gmail.com

Received/Geliş tarihi: September 02, 2016 Accepted/Kabul tarihi: December 01, 2016 DOI: 10.4274/tjh.2016.0355

Assessment of Quality of Life of Chronic Myeloid Leukemia Patients by Using the EORTC QLQ-C30 Kronik Miyeloid Lösemili Hastaların Yaşam Kalitesinin EORTC QLQ-C30 Anketi Kullanılarak Değerlendirilmesi Mehmet Can Uğur1, Yaşar Bekir Kutbay2, Özge Özer Kaya2, Cengiz Ceylan3 Tepecik Training and Research Hospital, Clinic of Internal Medicine, İzmir, Turkey Tepecik Training and Research Hospital, Genetic Diagnostic Center, İzmir, Turkey 3 Tepecik Training and Research Hospital, Clinic of Hematology, İzmir, Turkey 1 2

To the Editor, Depression is determined in 15%-25% of patients with cancer and it is accepted as a comorbid problem with poor prognosis. The quality of life of these patients is determined to be poor [1,2]. We aimed to study the quality of life of patients using new forms of imatinib, dasatinib, or nilotinib. We analyzed 56 chronic myeloid leukemia patients followed in the İzmir Tepecik Training and Research Hospital Department of Hematology. Patients were followed from 2005 to 2015. We included patients who were >18 years of age, BCR-ABL-positive based on polymerase chain reaction results, using first- or second-generation tyrosine kinase inhibitors (TKIs) in the last 6 months, and in the chronic phase of the disease. The Turkish version of the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-C30 (EORTC QLQ-C30) [3], the Turkish version of the Hospital Anxiety and Depression Scale [4], and the General Health Questionnaire [5] were administered to patients one-on-one. The study received approval from the ethics committee.

The demographic data and laboratory values are provided in Table 1. In our study, we found no statistical significance between firstand second-generation TKIs. We also compared dasatinib and nilotinib as subgroups of the second generation and we found statistical significance for dasatinib against nilotinib for general life quality, emotional and cognitive functions, and fatigue parameters. Keywords: Cytogenetic, Chronic myeloid leukemia, Molecular hematology, Life-quality, Dasatinib, Nilotinib Anahtar Sözcükler: Sitogenetik, Kronik miyeloid lösemi, Moleküler hematoloji, Yaşam kalitesi, Dasatinib, Nilotinib 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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Table 1. Demographic datas, laboratory findings, follow up, Hospital Anxiety and Depression Scale, General Health Questionnaire, Eastern Cooperative Oncology Group and Karnofsky scores, general medical, functional and symptom scales of European Organization for Research on Treatment of Cancer Questionnaires Quality of Life-C30 between 1st generation tyrosine kinase inhibitor and 2nd generation tyrosine kinase inhibitor (dasatinib and nilotinib). 1. Generation

2. Generation Dasatinib

Nilotinib

p value1

Total

p value2

56.7±1.2

53.5±1.5

57.7±1.5

0.17

53.3±1.4

0.29

Male

16

5

6

11

-

Female

19

2

3

5

-

Hemoglobin (g/dL)

11.9±1.8

11.5±1.5

11.4±2.7

0.40

11.3±1.9

0.81

Platelet (/uL)

298000±1.5

212000±6.0

342000±1.8

0.33

287000±1.5

0.71

Leukocyte (/uL)

76050±1.1

51300±9.0

101000±7.5

0.11

84080±8.0

0.30

Follow up (month)

57.2±34.6

74.8±3.0

61±2.5

0.07

65.2±28.0

0.15

ECOG

0.17±0.5

0.0±0.0

1.0±1.4

0.09

0.26±0.7

0.84

Karnofsky

95.5±8.5

93.3.±5.7

85.0±17.0

0.21

93.3±10.4

0.24

HADs-A

8±4.1

8.6±4.1

10.5±2.0

0.18

8.3±4

0.76

HADs-D

5.8±4.1

8.3±3.2

10.5±4.2

0.72

6.1±4.4

0.86

GHQ

22.3±5.3

20.6±2.0

26.5±9.1

0.08

23.2±6.1

0.83

57.2±22.1

68.1±18.1

39.6±22.6

0.016*

52.1±24.8

0.95

Age (year) Gender (n)

Scales of EORTC-QoL-C30 General medical scale

Functional Scales of EORTC-QoL-C30 Physical scale

73.8±17.0

81.4±2.2

68±2.0

0.142

73.9±21.4

0.31

Role scale

88.6±19.0

98.2±4.7

80.5±2.4

0.252

88.2±20.1

0.70

Emotional scale

77.9±23.7

95.5±7.8

68.7±20.4

0.005*

80.4±20.8

0.63

Cognitive scale

78.5±21.9

96.4±6.0

95.2±32.3

0.023*

78.9±28.7

0.37

Social scale

87.8±20.4

94.6±9.8

79.1±16.5

0.091

85.9±15.7

0.60

Symptom Scales of EORTC-QoL-C30 Fatique

31.3±21.3

19±1.3

50.3±1.9

0.005*

36.6±23.2

0.67

Nausea

13.5±19.5

5.3±9.8

9.7±1.6

0.681

7.8±13.5

0.21

Pain

20.3±23.6

10.7±1.3

33.3±2.6

0.114

24.3±24.0

0.40

Dispnea

11.4±19.0

7.1±12.1

25±33.0

0.408

17.1±26.9

0.11

Insomnia

15.7±21.9

7.1±12.1

30.5±37

0.351

20.3±30.5

0.07

Loss of appetite

13.9±17.6

14.2±13.3

22.2±23.1

0.606

18.7±19.3

0.75

Constipation

12.1±17.5

10.7±1.3

30.5±2.4

0.114

21.8±22.1

0.58

Diarrhea

10.7±22.1

3.5±9.4

16.6±2.1

0.299

10.9±18.1

0.83

44.4±3.2

0.091

31.2±33.5

0.05

Financial effect

18.5±25.9

14.2±2.8

*p<0.05, p value : Between dasatinib and nilotinib, p value : Between 1 generation and 2 generation tyrosine kinase inhibitor, 1

2

st

nd

HADs: Hospital Anxiety and Depression Scale, ECOG: Eastern Cooperative Oncology Group, EORTC-QoL: European Organization for Research on Treatment of Cancer Questionnaires Quality of Life-C30, GHQ: General Health Questionnaire.

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References 1. Wilson KG, Chochinov HM, Shirko MG, Allard P, Chary S, Gagnon PR, Macmillan K, De Luca M, O’Shea F, Kuhl D, Fainsinger RL, Clinch JJ. Depression and anxiety disorders in palliative cancer care. J Pain Symptom Manage 2007;33:118-129. 2. Lloyd-Williams M, Friedman T. Depression in palliative care patients-a prospective study. Eur J Cancer Care (Engl) 2001;10:270-274.

3. Guzelant A, Goksel T, Ozkok S, Tasbakan S, Aysan T, Bottomley A. The European Organization for Research and Treatment of Cancer QLQ-C30: an examination into the cultural validity and reliability of the Turkish version of the EORTC QLQC30. Eur J Cancer Care Engl 2004;13:135-144. 4. Aydemir O. Validity and reliability of Turkish version of Hospital Anxiety and Depression scale. Turkish Journal of Psychiatry 1997;8:280-287. 5. Kilic C. General health questionnaire: a validity and reliability study. Turkish Journal of Psychiatry 1996;7:3-9.

Address for Correspondence/Yazışma Adresi: Mehmet Can UĞUR, M.D., Tepecik Training and Research Hospital, Clinic of Internal Medicine, İzmir, Turkey Phone : +90 505 886 11 26 E-mail : med.can@hotmail.com

Received/Geliş tarihi: October 18, 2016 Accepted/Kabul tarihi: December 22, 2016 DOI: 10.4274/tjh.2016.0409

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Advisory Board of This Issue (June 2017) Ahmed Mohamed Hasanin, Egypt Ahmet Emre Eşkazan, Turkey Akif Selim Yavuz, Turkey Ali Uğur Ural, Turkey Arbil Açıkalın, Turkey Ayşen Timurağaoğlu, Turkey Canan Vergin, Turkey Carlo Gambacorti-Passerini, Italy Deniz Karapınar, Turkey Ebru Koca, Turkey Ekrem Ünal, Turkey Elif Birtaş Ateşoğlu, Turkey Emel Gürkan, Turkey Erol Erduran, Turkey Evangelos Terpos, Greece Fablo Efficace, Italy Farhad Ravandi, USA Fatih Demirkan, Turkey

Giorgina Specchia, Italy Gülsün Karasu, Turkey Gustavo Göhringer de Almedia Barbosa, Brazil Hale Ören, Turkey Halis Akalın, Turkey İsmail Sarı, Turkey Iyad Ali, Palestine John Bennett, USA Jonathan Kell, USA Jorge Castillo, USA Levent Ündar, Turkey Mahmut Yeral, Turkey Marie Ambroise, India Marina Cavazzana, France Mehmet Fatih Azık, Turkey Meltem Aylı, Turkey Meltem Kurt Yüksel, Turkey

Meral Beksaç, Turkey Monique Minnema, Netherlands Müge Sayitoğlu, Turkey Mustafa Pehlivan, Turkey Mutlu Arat, Turkey Nazan Sarper, Turkey Özden Ng Hatırnaz, Turkey Pervin Topçuoğlu, Turkey Rauf Haznedar, Turkey Reyhan Küçükkaya, Turkey Sanjeev Sharma, India Silvia Riva, Italy Siok-Keen Tey, Australia Soner Gürsoy, Turkey Türkan Patıroğlu, Turkey Vildan Özkocaman, Turkey Zühre Kaya, Turkey

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