Tjh 2018 2

Issue 2

June 2018

80 TL

ISSN 1300-7777

Volume 35

Research Articles The Role of CD200 and CD43 Expression in Differential Diagnosis Between Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma Mesude Falay et al.; Ankara, Turkey

Association of Interleukin-2-330T/G and Interleukin-10-1082A/G Genetic Polymorphisms with B-Cell Non-Hodgkin Lymphoma in a Cohort of Egyptians Hala Aly Abdel Rahman, et al.; Cairo, Egypt

Myelodysplastic Syndrome in Pakistan: Clinicohematological Characteristics, Cytogenetic Profile, and Risk Stratification Rafia Mahmood et al.; Rawalpindi, Pakistan

Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated Peptide in Plasma Cell Dyscrasias Tamar Tadmor, et al.; Haifa, Israel

Acute Traumatic Coagulopathy: The Value of Histone in Pediatric Trauma Patients Emel Ulusoy, et al.; Ä°zmir, Turkey

Cover Picture: Shivangi Harankhedkar et al.

Pleomorphic Multinucleated Plasma Cells Simulating Megakaryocytes in an Anaplastic Variant of Myeloma

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International Review Board

Editor-in-Chief Reyhan Küçükkaya

İstanbul, Turkey rkucukkaya@hotmail.com

Associate Editors Ayşegül Ünüvar

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

Claudio Cerchione

University of Naples Federico II Napoli, Campania, Italy

Elif Ünal İnce

Ankara University, Ankara, Turkey

İnci Alacacıoğlu

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 Ahmet Muzaffer Demir

Editorial Office İpek Durusu Bengü Timoçin

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

İstinye 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

: Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613. Sok.) 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

Publishing House / Yayınevi Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul, Turkey Tel: +90 212 621 99 25 Fax: +90 212 621 99 27 E-mail: info@galenos.com.tr

Muhlis Cem Ar

Management Address Yayın İdare Adresi

Print: Özgün Ofset Ticaret Ltd. Şti.

Türk Hematoloji Derneği

Yeşilce Mah. Aytekin Sok. No: 21 34418 4. Levent, İstanbul-Turkey

Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613. Sok.) No: 8 06550 Çankaya, Ankara / Turkey

Phone: +90 212 280 0009

Online Manuscript Submission

25.05.2018

Printing Date / Basım Tarihi

http://mc.manuscriptcentral.com/tjh

Cover Picture

Web page www.tjh.com.tr

Shivangi Harankhedkar et al., Pleomorphic Multinucleated Plasma Cells Simulating Megakaryocytes in an Anaplastic Variant of Myeloma

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

Panel of photomicrographs: A) May-Grünwald Giemsa stained bone marrow aspirate smear (100x) showing pleomorphic cells, with multilobation and multinuclearity, with prominent inclusions (red arrows) and abundant basophilic cytoplasm, and absence of perinuclear hof.

Güner Hayri Özsan International scientific journal published quarterly. Üç ayda bir yayımlanan İngilizce süreli yayındır.

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

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

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

The aim of The Turkish Journal of Hematology is to publish original hematological research of the highest scientific quality and clinical relevance. Additionally, educational material, reviews on basic developments, editorial short notes, images in hematology, and letters from hematology specialists and clinicians covering their experience and comments on hematology and related medical fields as well as social subjects are published. As of December 2015, The Turkish Journal of Hematology does not accept case reports. Important new findings or data about interesting hematological cases may be submitted as a brief report.

General practitioners interested in hematology and internal medicine specialists are among our target audience, and The Turkish Journal of Hematology aims to publish according to their needs. The Turkish Journal of Hematology is indexed, as follows: - PubMed Medline - PubMed Central - Science Citation Index Expanded - EMBASE - Scopus - CINAHL - Gale/Cengage Learning - EBSCO - DOAJ - ProQuest - Index Copernicus - Tübitak/Ulakbim Turkish Medical Database - Turk Medline Impact Factor: 0.686 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 http://www.icmje.org/downloads/coi_disclosure.zip 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 (https://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.

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

4. Book Chapter Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin:

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Genetics, Pathophysiology, Clinical Management. New York, Cambridge University Press, 2000.

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

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

7. Supplement Alter BP. 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.

Article length: Not to exceed 4000 words.

Review articles should not include more than 100 references. Reviews should include a conclusion, in which a new hypothesis or study about the subject may be posited. Do not publish methods for literature search or level of evidence. Authors who will prepare review articles should already have published research articles on 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.

Perspectives in Hematology “Perspectives” are articles discussing significant topics relevant to hematology. They are more personal than a Review Article. Authors wishing to submit a Perspective in Hematology article should contact the Editor in Chief prior to submission in order to screen the proposed topic for relevance and priority. Articles submitted for “Perspectives in Hematology” must advance the hot subjects of experimental and/or clinical hematology beyond the articles previously published or in press in TJH. Perspective papers should meet the restrictive criteria of TJH regarding unique scientific and/or educational value, which will impact and enhance clinical hematology practice or the diagnostic understanding of blood diseases.

Priority will be assigned to such manuscripts based upon the prominence, significance, and timeliness of the content. The submitting author must already be an expert with a recognized significant published scientific experience in the specific field related to the “Perspectives” article.

Abstract length: Not to exceed 150 words.

Article length: Not to exceed 1000 words.

References: Should not include more than 50 references 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. High-

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resolution image files are not preferred for initial submission as the file sizes may be too large. The total file size of the PDF for peer review should not exceed 5 MB. Authorship Each author should have participated sufficiently in the work to assume public responsibility for the content. Any portion of a manuscript that is critical to its main conclusions must be the responsibility of at least 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 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 2013 available at https://www.wma.net/policies-post/wma-declarationof-helsinki-ethical-principles-for-medical-research-involving-humansubjects/), 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.

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

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

Conditions of Publication All authors are required to affirm the following statements before their manuscript is considered: 1. The manuscript is being submitted only to The Turkish Journal of Hematology; 2. The manuscript will not be submitted elsewhere while under consideration by The Turkish Journal of Hematology; 3. The manuscript has not been published elsewhere, and should it be published in The Turkish Journal of Hematology it will not be published elsewhere without the permission of the editors (these restrictions do not apply to abstracts or to press reports for presentations at scientific meetings); 4. All authors are responsible for the manuscript’s content; 5. All authors participated in the study concept and design, analysis and interpretation of the data, 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.

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All accepted manuscripts become the permanent property of The Turkish Journal of Hematology and may not be published elsewhere, in whole or in part, without written permission.

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

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

An extensive list of conversion factors can be found at https://www. nist.gov/sites/default/files/documents/pml/wmd/metric/SP1038.pdf. For more details, see http://www.amamanualofstyle.com/oso/public/jama/ si_conversion_table.html.

Abbreviations and Symbols Use only standard abbreviations. Avoid abbreviations in the title and abstract. The full term for an abbreviation should precede its first use in the text, unless it is a standard abbreviation. All acronyms used in the text should be expanded at first mention, followed by the abbreviation in parentheses; thereafter the acronym only should appear in the text.

Acronyms may be used in the abstract if they occur 3 or more times therein, but must be reintroduced in the body of the text. Generally, abbreviations should be limited to those defined in the AMA Manual of Style, current edition. A list of each abbreviation (and the corresponding full term) used in the manuscript must be provided on the title page.

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

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

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

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

Full instructions and support are available on the site, and a user ID and password can be obtained during your first visit. Full support for authors is provided. Each page has a “Get Help Now” icon that connects

directly to the online support system. Contact the journal administrator with any questions about submitting your manuscript to the journal (info@tjh.com.tr). For ScholarOne Manuscripts customer support, click on the “Get Help Now” link on the top right-hand corner of every page on the site.

The Electronic Submission Process Log in to your author center. Once you have logged in, click the “Submit a Manuscript” link in the menu bar. Enter the appropriate data and answer the questions. You may copy and paste directly from your manuscript.

Click the “Next” button on each screen to save your work and advance to the next screen.

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CONTENTS

94

Research Articles

99

Association of Interleukin-2-330T/G and Interleukin-10-1082A/G Genetic Polymorphisms with B-Cell Non-Hodgkin Lymphoma in a Cohort of Egyptians Hala Aly Abdel Rahman, Mervat Mamdooh Khorshied, Ola Mohamed Reda Khorshid, Heba Mahmoud Mourad; Cairo, Egypt

109

Myelodysplastic Syndrome in Pakistan: Clinicohematological Characteristics, Cytogenetic Profile, and Risk Stratification Rafia Mahmood, Chaudry Altaf, Parvez Ahmed, Saleem Ahmed Khan, Hamid Saeed Malik; Rawalpindi, Pakistan

116

Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated Peptide in Plasma Cell Dyscrasias Tamar Tadmor, Ilana Levy, Zahava Vadasz; Haifa, Israel

122

Acute Traumatic Coagulopathy: The Value of Histone in Pediatric Trauma Patients Emel Ulusoy, Murat Duman, Aykut Çağlar, Tuncay Küme, Anıl Er, Fatma Akgül, Hale Çitlenbik, Durgül Yılmaz, Hale Ören; İzmir, Turkey

129

Brief Report

The Role of CD200 and CD43 Expression in Differential Diagnosis Between Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma Mesude Falay, Berna Afacan Öztürk, Kürsad Güneş, Yasin Kalpakçı, Simten Dağdaş, Funda Ceran, Gülsüm Özet; Ankara, Turkey

Use of a High-Purity Factor X Concentrate in Turkish Subjects with Hereditary Factor X Deficiency: Post Hoc Cohort Subanalysis of a Phase 3 Study Ahmet F. Öner, Tiraje Celkan, Çetin Timur, Miranda Norton, Kaan Kavaklı; Van, İstanbul, İzmir, Turkey; Hertfordshire, United Kingdom

Images in Hematology

134

Flaming Plasma Cell Leukemia Reza Ranjbaran, Habibollah Golafshan; Shiraz, Iran

135

Improvement of Cutaneous Anaplastic Large Cell Lymphoma by Brentuximab Vedotin Monotherapy Takashi Onaka, Tomoya Kitagawa, Chika Kawakami, Akihito Yonezawa; Fukuoka, Japan

Letters to the Editor

137

Glomerular and Tubular Functions in Transfusion-Dependent Thalassemia Pathum Sookaromdee, Viroj Wiwanitkit; Bangkok, Thailand; Hainan, China

138

Use of Plerixafor to Mobilize a Healthy Donor Infected with Influenza A Mahmut Yeral, Pelin Aytan, Can Boğa; Adana, Turkey

A-IX

139

Influenza A Infection and Stem Cell Mobilization Sora Yasri, Viroj Wiwanitkit; Bangkok, Thailand; Hainan, China

141

Primary Mediastinal Large B-Cell Lymphoma As an Incidental Finding: Report of a Case İpek Yönal-Hindilerden, Fehmi Hindilerden, Serkan Arslan, İbrahim Öner Doğan, Meliha Nalçacı; İstanbul, Turkey

142

A Rare Late Complication of Port Catheter Implantation: Embolization of the Catheter Işık Odaman Al, Cengiz Bayram, Gizem Ersoy, Kazım Öztarhan, Alper Güzeltaş, Taner Kasar, Ezgi Uysalol, Başak Koç, Ali Ayçiçek, Nihal Özdemir; İstanbul, Turkey

144

Nuclear Projections in Neutrophils for Supporting the Diagnosis of Trisomy 13 Şebnem Kader, Mehmet Mutlu, Filiz Aktürk Acar, Yakup Aslan, Erol Erduran; Trabzon, Turkey

145

Intravascular Large B-Cell Lymphoma of the Gallbladder Bülent Çetin, Nalan Akyürek, Yavuz Metin, Feryal Karaca, İrem Bilgetekin, Ahmet Özet; Rize, Ankara, Adana, Turkey

147

Successful Treatment of Chronic Lymphocytic Leukemia Multifocal Central Nervous System Involvement with Ibrutinib Anna Christoforidou, Georgios Kapsas, Zoe Bezirgiannidou, Spyros Papamichos, Ioannis Kotsianidis; Alexandroupolis, Greece

150

Pleomorphic Multinucleated Plasma Cells Simulating Megakaryocytes in an Anaplastic Variant of Myeloma Shivangi Harankhedkar, Ruchi Gupta, Khaliqur Rahman; Uttar Pradesh, India

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Advisory Board of This Issue (June 2018) Ahmet Emre Eşkazan, Turkey Antonio Medina Almeida, Portugal Arbil Açıkalın, Turkey Argiris Symeonidis, Greece Canan Albayrak, Turkey Claudio Cerchione, Italy Donato Mannina, Italy Emanuele Angelucci, Italy Erdal Kurtoğlu, Turkey Figen Atalay, Turkey Güldane Cengiz Seval, Turkey Hüseyin Gülen, Turkey Jayadev Manikkam Umakanthan, USA

Melek Ergin, Turkey Meltem Kurt Yüksel, Turkey Mine Hekimgil, Turkey Muhit Özcan, Turkey Muhlis Cem Ar, Turkey Mustafa Pehlivan, Turkey Mustafa Yıldırım, Turkey Müge Sayitoğlu, Turkey Mükerrem Safalı, Turkey Münci Yağcı, Turkey Namık Özbek, Turkey Nükhet Tüzüner, Turkey Özgür Mehtap, Turkey

Özgür Rosti, Turkey Pervin Topçuoğlu, Turkey Prajwal Dhakal, USA Rauf Haznedar, Turkey Reyhan Küçükkaya, Turkey Robert F. Cornell, USA Tayfur Toptaş, Turkey Teoman Soysal, Turkey Ufuk Demirci, Turkey Yavuz Bilgin, Turkey Yookarin Khonglah, India Zehra Çoban, Turkey Zühre Kaya, Turkey

RESEARCH ARTICLE DOI: 10.4274/tjh.2017.0085 Turk J Hematol 2018;35:94-98

The Role of CD200 and CD43 Expression in Differential Diagnosis between Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma Kronik Lenfositik Lösemi ve Mantle Hücreli Lenfoma Ayırıcı Tanısında CD200 ve CD43 Ekspresyonunun Rolü Mesude Falay,

Berna Afacan Öztürk,

Kürşad Güneş,

Yasin Kalpakçı,

Simten Dağdaş,

Funda Ceran,

Gülsüm Özet

University Ministry of Health, Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey

Abstract

Öz

Objective: Atypical chronic lymphocytic leukemia (CLL) is most frequently confused with mantle cell lymphoma (MCL). Several markers may contribute to the diagnosis of CLL. However, there is no consensus on which markers are needed to be used in flow cytometry for the diagnosis of CLL. The aim of the present study was to investigate the role of CD43 and CD200 markers in the differential diagnosis between CLL and MCL.

Amaç: İmmünfenotip olarak atipik kronik lenfositik lösemi (KLL) ile mantle cell lenfoma (MCL) sıklıkla karışabilmektedir. KLL tanısı için birçok marker kullanılmaktadır, ancak akım sitometride KLL tanısı için tam bir konsensüs oluşmamıştır. Bu çalışmada KLL ve MCL ayırıcı tanısında CD43 ve CD200 ifadeleri araştırılmıştır.

Materials and Methods: To address this issue, 339 consecutive patients with CLL and MCL were included in the flow cytometry lymphoproliferative disease panel for evaluation of CD43 and CD200 expressions, but not in the Matutes scoring system.

Results: CD200 was expressed in 97.3% of atypical CLL cases, whereas it was dimly expressed in only 6.1% of MCL cases. CD43 expression was 95.7% in atypical CLL cases. In the MCL cases, its expression rate was 39.4%.

Conclusion: CD43 and CD200 were found to be more valuable markers than CD22, CD79b, and FMC7. CD43 and CD200 could also be considered as definitive markers in atypical CLL patients, for whom the Matutes scoring system remains ineffective.

Gereç ve Yöntemler: Matutes skorlama sisteminde olmayan CD43 ve CD200’ü akım sitometri lenfoproliferatif hastalık paneline dahil ederek 339 KLL ve MCL olgusunda incelenmiştir.

Bulgular: Atipik KLL olgularının %97,3’ünde CD200 pozitifken MCL olgularının ise sadece %6,1’inde düşük oranda ifade ediliyordu.

CD43’te atipik KLL olgularının %95,7’sinde ifade edilirken MCL olgularının %39,4’ünde donuk ifade ediliyordu.

Sonuç: CD43 ve CD200; CD22, CD79b ve FMC7’ye göre daha anlamlı bulundu. CD43 ve CD200 Matutes skorlama sistemi skorunun yetersiz kaldığı KLL olgularının tanısında tamamlayıcı marker olarak kullanılabilir.

Anahtar Sözcükler: Kronik lenfositik lösemi, Mantle cell lenfoma, İmmünfenotiplendirme, CD200, CD43

Keywords: Chronic lymphocytic leukemia, Mantle cell lymphoma, Immunophenotyping, CD200, CD43

Introduction The World Health Organization (WHO) classification of hematolymphoid system neoplasms is based on clinical, morphological, immunophenotypic, and genetic features.

Mature B-cell lymphoproliferative diseases (LPDs) account for more than 80% of hematolymphoid neoplasms [1]. Chronic

lymphocytic leukemia (CLL) is the most frequent type of LPD [1,2]. Genetics has no role in the diagnosis of CLL, although there are numerous genetic abnormalities. The presence of persistent clonal B lymphocytosis (>5x109/L lymphocytes) for more than 3 months is needed to make a diagnosis of CLL. It has characteristic morphological features, as well as immunophenotypic features in flow cytometry [1,2,3,4]. These include CD5+CD19+, CD23+,

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Mesude FALAY, M.D., University Ministry of Health, Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey Phone : +90 545 408 90 08 E-mail : mesudey@gmail.com ORCID-ID: orcid.org/0000-0001-7846-3476

94

Received/Geliş tarihi: March 01, 2017 Accepted/Kabul tarihi: July 07, 2017

Falay M, et al: CLL and MCL Immunophenotyping

Turk J Hematol 2018;35:94-98

weak surface membrane immunoglobulins (sIg), and absent or low expression of CD79b and FMC7 [3,4]. Immunophenotyping has a major role in the diagnosis of CLL. However, CLL is a quite heterogeneous disease; for this reason, it can be difficult to diagnose [3,4,5,6,7]. Accordingly, a scoring system for the diagnosis of CLL was first defined in 1994 by Matutes et al.

[8]. This scoring system consists of five parameters: CD5, CD22, CD23, FMC7, and sIg. In 1997, Moreau et al. [9] replaced CD22 by CD79b in the scoring system. According to this scoring system, a score of 4-5 indicates typical CLL and a score of 3 indicates atypical CLL, whereas a score of 0-2 excludes CLL [8,9]. Atypical CLL is most frequently confused with mantle cell lymphoma (MCL), which co-expresses CD5 and CD19 similarly to CLL [4,10,11,12,13,14,15,16]. Generally, MCL is more aggressive and requires a different therapeutic approach; therefore, differential diagnosis between these two diseases should be performed precisely. Histochemical or molecular tests [cyclin D1, SOX11, t(11;14)] can be used for differential diagnosis [4,12]. Molecular tests are not easily available, and they are time-consuming and more expensive. For this reason, reliable additional new markers have been investigated in cases in which the Matutes score is inadequate. Several markers such as CD200 and CD43 may contribute to the diagnosis of CLL. However, there is no consensus on which markers are needed to be used in flow cytometry for the diagnosis of CLL. In the present study, we aimed to investigate the role of markers that were included in our LPD panel in flow cytometry but not in the Matutes scoring system in the differential diagnosis between CLL and MCL.

Materials and Methods Patients and Samples The present study retrospectively evaluated the medical records of 339 patients diagnosed with CLL (n=306) and MCL (n=33) according to the WHO criteria [1]. For all patients, data on complete blood count and peripheral blood (PB) and/or bone marrow (BM) smear performed for morphological assessments were obtained. All atypical CLL patients were evaluated for cyclin D1 and/or t(11;14). Diagnosis of MCL was confirmed by immunohistochemical detection of cyclin D1 in BM biopsies or detection of t(11;14) by fluorescence in situ hybridization.

SOX11 expression was not evaluated.

Flow Cytometry Immunophenotyping For flow cytometric study, fresh PB/BM samples were drawn into 4-mL K3-EDTA tubes (BD Vacutainer, USA) and studied immediately. Cells in suspension (2x106 cells in 50-100 µL per tube) from the PB and BM samples were stained with monoclonal antibodies (MoAbs) directed against cell surface markers via a stain-lyse-and-then-wash direct immunofluorescence method [17]. The MoAbs used for labeling in flow cytometry were obtained

from Beckman Coulter (BC, USA). A five-color staining was applied for all samples using the following fluorochrome-conjugated antibodies. MoAbs including fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin-Texas red (ECD), phycoerythrin cyanine 5 (PC5), and allophycocyanin (APC) were used for all patients: CD45/CD5/CD10/CD19/CD23, CD19/CD103/CD22/ CD11c/CD25, CD5/CD20/sIgk/sIgλ CD45, CD19/CD3/CD79b/ CD22, and CD19/CD43/CD200/CD38. A tube containing Ig isotype controls for FITC/PE/ECD/PC5/APC was used for all patients. Data were immediately obtained at the end of sample staining using a flow cytometer (Navios, BC, USA) and Kaluza Flow Cytometry Analysis Software (BC, USA). For each sample, data from at least 10x104 events per tube were obtained. Instrument alignment was confirmed daily using an alignment control bead (Flow-Check, BC, USA). The accuracy and precision of cell counts were tested using international quality controls purchased from the United Kingdom National External Quality Assessment Scheme (UK NEQAS LI, Sheffield, UK) (z-score range of -2.0 to 2.0). Briefly, CD19+ B cells were selected (at least 2000 events according to the threshold of the isotype control) from the data file using conventional gating strategies (forward and side scatter and the pattern of CD19 expression). As recommended by the British Committee for Standards in Haematology guideline [2], a cutoff value of 30% of lymphoid cells was accepted to indicate a positive result with a given antibody using the Kaluza software.

The Matutes scoring was defined as ≥30% cell surface expression.

In all patients, the same fluorescent-labeled MoAbs were used to ensure that the Matutes scoring was accurate. Diagnosis of LPD was established according to the WHO classification based on clinical data and morphologic, immunophenotypic, and genetic criteria. The revised Matutes scoring system [9], based on the immunophenotypic analysis of five membrane markers (CD5, CD23, FMC7, sIg, CD79b), was used to classify all patients. This scoring system assigns 1 point each for expression of CD5, CD23, and sIg and for lack of expression of CD79b and FMC7. A score of ≥4 indicates typical CLL patients and a score of 3 or a lack of CD23 indicates atypical CLL patients. In all patients, cyclin D1 and/or t(11;14) was used for the differential diagnosis. Diagnosis of MCL was confirmed by cyclin D1 and/or t(11;14).

Statistical Analysis Data analysis was performed using SPSS 15 for Windows (SPSS Inc., Chicago, IL, USA). Descriptive statistics were expressed as numbers and percentages. Categorical data were analyzed by multivariate forward stepwise regression analysis, Pearson’s chisquare test, or Fisher’s exact test as appropriate. A p-value of less than 0.05 was considered statistically significant.

Results The evaluation of 339 patients (100 females, 239 males) with mean age of 68±10.4 years (range: 31-87 years) revealed 95

Falay M, et al: CLL and MCL Immunophenotyping

that median PB lymphocyte count at diagnosis was 19.8x109 lymphocytes/L (range: 0.8-274x109 lymphocytes/L). Of the patients, 306 (90.26%) had CLL and 33 (9.74%) had MCL (Table 1). According to the Matutes scoring of CLL patients, 121 (40%) patients had a score of ≥4 (of whom 105 (34.3%) had a score of 4 and 16 (5.2%) had a score of 5), 178 (58.2%) patients had a score of 3, 6 (2%) patients had a score of 2, and 1 patient (0.3%) had a score of 1 (Table 2). There was no significant difference between the typical and atypical CLL patients in terms of morphological evaluation. In all atypical CLL cases, cyclin D1 and/or t(11;14) were negative. The Matutes scores of the MCL patients with positive cyclin D1 and/or t(11;14) were 3 in 7 (21.2%) patients, 2 in 11 (33.3%) patients, and 1 in 15 (45.5%) patients. There were no MCL patients with a score of ≥4. Regarding CD22, CD79b, FMC7, and CD23 expressions in the Matutes score, CD23 expression was negative in 11 (3.5%) CLL patients (3 had typical CLL and 8 had atypical CLL), whereas it was positive in 6 (21.2%) MCL patients CD23 expression was not diagnostic for CLL but it was significantly more expressed in CLL patients (p<0.001). CD22, CD79b, and FMC7 expressions were highly positive in atypical CLL patients (96.2%, 81.6%, and 97.3%, respectively) (Table 3); however, the difference was not significant in the differential diagnosis between CLL and MCL (p=1.000, p=0.431, and p=1.000, respectively). CD79b expression was also positive in 38.8% of the CLL patients. No significant difference was found between the CLL and MCL patients regarding sIg expression intensity (p=0.385). Evaluations of CD38, CD43, and CD200 expressions were included in the LPD panel but not in the Matutes scoring system (Table 2). While CD38 expression was moderate to strong in 93.9% of the MCL patients, it was dimly expressed in 24% of both atypical and typical CLL patients (p<0.001). When CD43 expression was evaluated, 95.7% of the patients with atypical CLL and 98.3% of the patients with typical CLL had moderate to strong expression.

Among the MCL patients, CD43 expression was dim to moderate in 39.4% (p<0.001). When CD200 expression was evaluated, it was moderate to strong in 95.8% of the CLL patients (3.6% had

Turk J Hematol 2018;35:94-98

negative expression), whereas it was dimly expressed only in 2 MCL patients (6.1%) (p<0.001; Table 2). There was no significant difference in CD200 expressions between the atypical and typical CLL patients. In the differential diagnosis of MCL and atypical CLL patients, multivariate forward stepwise regression analysis revealed the most determinant marker to be CD200 (p<0.001, 95% CI; Table 4).

Table 2. Mantle cell lymphoma and chronic lymphocytic leukemia patients’ Matutes scores.

Matutes score

MCL (n) %

CLL (n) %

1

(15) 45.5

(1) 0.3

2

(11) 33.3

(6) 2.0

3

(7) 21.2

(178) 58.2

4

(-) 0

(105) 34.3

5

(-) 0

(16) 5.2

CLL: Chronic lymphocytic leukemia, MCL: mantle cell lymphoma.

Table 3. Distribution of cases by marker positivity in the differential diagnosis of mantle cell lymphoma and atypical chronic lymphocytic leukemia score of ≤3, chronic lymphocytic leukemia score of ≥4.

MCL (n=33)

Atypical CLL score of ≤3

p p

(n=185) 60%

(n=121) 40%

CD20

31 (93.9%) 184 (99.5%)

0.061

113 (93.4%) 1.000

CD22

32 (97.0%) 177 (96.2%)

1.000

90 (74.4%)

CD23

6 (21.2%)

<0.001

118 (96.7%) <0.001

CD79b

25 (75.8%) 151 (81.6%)

0.431

47 (38.8%)

<0.001

CD25

10 (30.3%) 111 (60.0%)

0.002

77 (63.6%)

0.002

CD38

31 (93.9%) 185 (24.9%)

<0.001

29 (24.0%)

<0.001

CD200

2 (6.1%)

180 (97.3%)

<0.001

117 (96.7%) <0.001

sIg

33 (100%)

74 (40%)

0.385

97 (80%)

CD43

13 (39.4%) 177 (95.7%)

<0.001

119 (98.3%) <0.001

CD11C

11(33.3%)

116 (62.7%)

0.002

87 (71.9%)

<0.001

n=339

FMC7

32 (97%)

180 (97.3%)

1.000

51 (42.1%)

<0.001

Age, years (range)

68.0±10.4 (45-89)

Sex

MCL: Mantle cell lymphoma, CLL: chronic lymphocytic leukemia, sIg: surface membrane immunoglobulins.

Female, n (%)

100 (29.4%)

Male, n (%)

239 (70.6%)

CLL, n (%)

306 (90.26%)

MCL, n (%)

33 (9.74%)

White blood cells, x103

19.8 (8-274)

Lymphocytes, x103

17.3 (6.8-240)

Hemoglobin, g/dL

12.7±2.27

Platelets, x103

202 (19-403)

Table 1. Demographic features.

Variables

CLL: Chronic lymphocytic leukemia, MCL: mantle cell lymphoma.

96

177 (97.0%)

1.000

0.410

Table 4. Multivariate analysis for mantle cell lymphoma and atypical chronic lymphocytic leukemia discrimination.

Odds ratio

95% Confidence interval Lower

Upper

Wald

p

CD200

1317.886

79.380

21879.729

25.115

<0.001

CD38

31.909

2.446

416.220

6.984

0.008

CD43

17.632

1.766

176.091

5.974

0.015

Falay M, et al: CLL and MCL Immunophenotyping

Turk J Hematol 2018;35:94-98

Discussion The diagnosis of CLL is easy in the presence of characteristic immunophenotypic features (CD5+CD19+ dual-positive, CD23+, CD22-/low, CD79b-/low, sIg low, FMC7-, and CD20 low).

However, it is difficult to make a differential diagnosis of CLL from MCL when immunophenotypic features are not typical.

In the present study, CD43 and CD200 expressions, which were included in the LPD panel but not in the Matutes scoring system, were found significant in the differential diagnosis between CLL and MCL. Immunophenotyping by flow cytometry, which is a frequently used method, is beneficial in the distinction of CLL from MCL [3,4,15]. However, there may be a problem for atypical immunophenotypes in which the Matutes score is â&#x2030;¤3. Therefore, it may be particularly difficult to distinguish some MCL cases from atypical CLL cases. CD23 positivity is the most characteristic feature of CLL [10,11]. Earlier studies have reported that CD23 negativity is a reliable marker in the distinction between CLL and MCL [15]. In the present study, while 2.1% of the typical CLL patients were CD23-negative, 21.2% of the MCL patients with positive t(11;14) were CD23-positive. However, according to our findings, CD23 alone was not efficient to make a differential diagnosis between CLL and MCL [12,13,16]. On the other hand, FMC7, which is an epitope of CD20, was expressed in 42.1% of the typical CLL patients and 97.3% of the atypical CLL patients.

Similarly, the level of CD22 expression was closely correlated with CD20. CD79b expression was also positive in 38.8% of the CLL patients, which was considered in normal ranges. The percent positivity and intensity of CD79b expression in MCL, atypical CLL, and typical CLL is still controversial. CD22 and FMC7 expressions are generally higher in MCL patients, whereas in the present study, they were higher in both the CLL and MCL patients. For this reason, the majority of the patients (58.2%) were classified as having atypical CLL when Matutes scoring was used. Earlier studies stated that FMC7, CD79b, and CD22 are not efficient in making a differential diagnosis [1,17,18,19]. Every manufacturer produces MoAbs in different clones and different stains. There is a need for validation and standardization studies on these MoAbs. At this point, the present study had a limitation because the results were not checked with the use of different MoAbs of different clones from different manufacturers.

With regard to CD38, CD43, and CD200, which were not included in the Matutes scoring system, the different results obtained in the present study between the CLL and MCL patients could be partially explained by the individual differences among the patients as well as the absence of specific techniques and procedures in the flow cytometry. In the present study, CD38 expression was higher in the MCL patients than in the CLL patients (p<0.001) but heterogeneous in the CLL patients; thus, it was difficult to standardize. In addition, the LPD may have

a fluctuating course [20,21,22,23]. All of these factors need to be taken into account while making a differential diagnosis between CLL and MCL. CD43 expression was first defined in 1999 by Harris et al. [24] for the classification of malignant lymphomas. In the present study, CD43 expression was higher in the CLL patients compared with that in the MCL patients and it was quite effective in accurate classification of the patients having Matutes scores of â&#x2030;¤3 according to the classical classification (p<0.001) [25,26,27].

In the present study, while 95.8% of the CLL patients showed moderate to strong CD200 expression (3.6% had negative expression), 6.1% of the MCL patients showed positive CD200 expression (p<0.001). There was no significant difference in CD200 expressions between the atypical and typical CLL patients. Moreover, CD200 was constantly expressed in the typical CLL patients and was an excellent marker for its differential diagnosis from MCL, as previously shown in other studies [14,15,16,17,18,19].

Study Limitation The limitation of the present study was to not evaluate CD200 and CD43 expressions in other LPD groups. If these expressions were evaluated in other LPD groups, other diseases besides CLL and MCL would have also been evaluated with regard to CD200 and CD43 expressions. However, as the number of patients with other diseases was low in the present study, they were not included.

Conclusion In conclusion, there has not been a single marker identified yet to make a definite diagnosis of CLL by flow cytometry.

Therefore, new markers for the differential diagnosis of CLL are under investigation. The results of the present study revealed that CD43 and CD200 in particular were more valuable markers than CD22, CD79b, and FMC7, which are within the scope of the Matutes scoring system. CD43 and CD200 could also be considered as definitive markers in atypical CLL patients for whom the Matutes scoring system remains ineffective. However, as with the other markers, their heterogeneous distribution and different rates of expression may still be in question. For this reason, large-scale harmonization studies are needed for patients with various diseases by defining standardized sample preparation and staining, as well as specific techniques.

Identification of a new scoring system following these studies would also be beneficial.

Ethics Ethics Committee Approval: Ankara Numune Education and Training Hospital Ethics Committee (decision number: 12.06.2015-1028).

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Informed Consent: Retrospective study.

Authorship Contributions Surgical and Medical Practices: G.Ö., B.A.Ö., K.G.; Concept: M.F.; Design: M.F.; Data Collection or Processing: M.F.; Analysis or Interpretation: M.F., Y.K., S.D, F.C; Literature Search: M.F.; Writing: M.F. 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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3. Costa ES, Pedreira CE, Barrena S, Lecrevisse Q, Flores J, Quijano S, Almeida J, del Carmen García-Macias M, Bottcher S, Van Dongen JJ, Orfao A. Automated pattern-guided principal component analysis vs expert-based immunophenotypic classification of B-cell chronic lymphoproliferative disorders: a step forward in the standardization of clinical immunophenotyping. Leukemia 2010;24:1927-1933.

4. Braylan RC. Impact of flow cytometry on the diagnosis and characterization of lymphomas, chronic lymphoproliferative disorders and plasma cell neoplasias. Cytometry A 2004;58:57-61.

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8. Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A, Que TH, Catovsky D. The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia 1994;8:1640-1645.

9. Moreau EJ, Matutes E, A’Hern RP, Morilla AM, Morilla RM, Owusu-Ankomah KA, Seon BK, Catovsky D. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin Pathol 1997;108:378-382.

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expression of CD23, CD20, and CD38 with platelet count predicts for the presence of the t(11;14) translocation of mantle cell lymphoma. Cytometry B Clin Cytom 2011;80:230-237.

11. Kroft SH. Uncovering clinically relevant phenotypic variations in malignancies: CD23 in mantle cell lymphoma. Am J Clin Pathol 2008;130:159-161.

12. Schlette E, Fu K, Medeiros LJ. CD23 expression in mantle cell lymphoma: clinicopathologic features of 18 cases. Am J Clin Pathol 2003;120:760-766.

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21. Matrai Z. CD38 as a prognostic marker in CLL. Hematology 2005;10:39-46.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2017.0106 Turk J Hematol 2018;35:99-108

Association of Interleukin-2-330T/G and Interleukin-10-1082A/G Genetic Polymorphisms with B-Cell Non-Hodgkin Lymphoma in a Cohort of Egyptians Bir Mısırlı Hasta Kohortunda İnterlökin-2-330T/G ve İnterlökin-10-1082A/G Genetik Polimorfizmlerinin B-Hücreli Hodgkin Dışı Lenfoma ile İlişkisi Hala Aly Abdel Rahman1,

Mervat Mamdooh Khorshied1,

Ola Mohamed Reda Khorshid2,

Heba Mahmoud Mourad1

Cairo University Kasr Alainy Faculty of Medicine, Department of Clinical and Chemical Pathology, Cairo, Egypt Cairo University National Cancer Institute, Department of Medical Oncology, Cairo, Egypt

1 2

Abstract

Öz

Objective: Polymorphisms in the interleukin (IL)-2 and IL-10 genes are known to be associated with susceptibility to different immunedysregulated disorders and cancers such as non-Hodgkin lymphoma (NHL). To explore the possible association between IL-2-330T/G and IL10-1082A/G single-nucleotide polymorphisms and the susceptibility to B-cell NHL (B-NHL) in Egyptians, we conducted a case-control study.

Amaç: İnterlökin (IL)-2 ve IL-10 genlerindeki polimorfizmlerin değişik immün bozukluklar ve Hodgkin dışı lenfoma (HDL) gibi kanserlere duyarlılık ile ilişkili olduğu bilinmektedir. Mısırlılardaki IL-2-330T/G ve IL-10-1082A/G tek nükleotid polimorfizmleri ile B hücreli HDL’ye (B-HDL) duyarlılık arasındaki olası ilişkinin araştırılması için bir olgukontrol çalışması yapılmıştır.

Materials and Methods: Genotyping of the studied genetic variations was done for 100 B-NHL patients as well as 100 age- and sex-matched healthy controls.

Results: The IL-2 variant allele occurred at a significantly higher rate in patients than controls and was associated with susceptibility to B-NHL [odds ratio (OR): 1.91, 95% confidence interval (CI): 1.28-2.85].

It was also associated with advanced performance status score. IL-2 polymorphism conferred an almost threefold increased risk of diffuse large B-cell lymphoma (OR: 2.64, 95% CI: 1.35-5.15) and a fourfold increased risk of indolent subtypes (OR: 4.34, 95% CI: 1.20-15.7). The distribution of IL-10-1082A/G genotypes in our patients was close to that of the controls. Co-inheritance of the variant genotypes of IL-2 and the common genotype of IL-10 conferred an almost sixfold increased risk (OR: 5.75, 95% CI: 1.39-23.72), while co-inheritance of the variant genotypes of IL-2 and IL-10 conferred fivefold increased risk of B-NHL (OR: 5.43, 95% CI: 1.44-20.45). The variant genotypes of IL-2-330T/G and IL-10-1082A/G had no effect on the disease-free survival of B-NHL patients.

Conclusion: The present study highlights the possible involvement of the IL-2-330T/G genetic polymorphism in the susceptibility to B-NHL in Egypt, especially indolent subtypes. Moreover, IL-10-1082A/G is not a molecular susceptibility marker for B-NHL in Egyptians.

Keywords: Interleukin-2-330T/G, rs2069762, Interleukin-10-1082A/G, rs1800896, B-cell non-Hodgkin lymphoma, Egypt

Gereç ve Yöntemler: Bahsedilen genetik varyasyonlar için, 100 B-HDL hastası ve yaş ile cinsiyet uyumlu 100 sağlıklı kontrole genotipleme yapıldı.

Bulgular: IL-2 varyant alleli hastalarda kontrollere göre anlamlı olarak daha yüksekti ve B-HDL duyarlılığı ile ilişkili bulundu [olasılık oranı (OO): 1,91, %95 güven aralığı (GA): 1,28-2,85). Ayrıca bunun ileri performans skoru ile de ilişkili olduğu görüldü. IL-2 polimorfizminin diffüz büyük B hücreli lenfoma için yaklaşık üç kat (OO: 2,64; %95 GA: 1,35-5,15) ve yavaş seyirli (indolan) alt tiplerde dört kat artış doğurmaktaydı (OO: 4,34, %95 GA: 1,20-15,7). IL-10-1082A/G genotiplerinin dağılımı hastalar ve kontrollerde benzerdi. IL-2 varyant genotipleri ile IL-10’un sık rastlanan genotiplerinin eş kalıtımı yaklaşık altı kat artmış risk (OO: 5,75, %95 GA: 1,39-23,72) yaratmaktayken, IL-2 ve IL-10 varyant genotiplerinin eş kalıtımı B-HDL riskinde beş kat artışa (OO: 5,43, %95 GA: 1,44-20,45) neden olmaktaydı. IL-2330T/G ve IL-10-1082A/G variant genotiplerinin B-HDL hastalarında hastalıksız sağkalım üzerine etkisi yoktu.

Sonuç: Bu çalışma Mısır’da, IL-2-330T/G genetik polimorfizmlerinin özellikle yavaş seyirli B-HDL’ye yatkınlık ile olası ilişkisini vurgulamaktadır. Ayrıca Mısırlılarda IL-10-1082A/G, B-HDL için duyarlı bir moleküler belirteç değildir.

Anahtar Sözcükler: İnterlökin-2-330T/G, rs2069762, İnterlökin-101082A/G, rs1800896, B-hücreli Hodgkin dışı lenfoma, Mısır

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Mervat MAMDOOH KHORSHIED, M.D., Cairo University Kasr Alainy Faculty of Medicine, Department of Clinical and Chemical Pathology, Cairo, Egypt Phone : +202 235 644 80 E-mail : mervatkhorshied@hotmail.com ORCID-ID: orcid.org/0000-0003-2052-3768

Received/Geliş tarihi: March 14, 2017 Accepted/Kabul tarihi: July 07, 2017

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Rahman HAA., et al: IL-2 and -10 Polymorphisms and B-NHL

Introduction Despite the fact that there are some proven non-Hodgkin lymphoma (NHL) risk factors, the etiology of NHL still warrants extensive investigations [1]. Interleukin-2 (IL-2) has multiple opposing functions in the immune system. It plays a master role in T-cell growth and activation and in natural killer cellmediated immune responses [2]. It has been reported to have antitumor effects through its contribution in the development of regulatory T cells, as well as expansion and apoptosis among activated T cells [3]. It is postulated that low production of IL-2 can suppress the antitumor response via the antibodydependent cellular cytotoxicity (ADCC) seen in NHL patients, thus increasing the susceptibility to develop NHL [4].

IL-10 has both immunosuppressive and antiangiogenic functions.

It thus has tumor-promoting as well as tumor-suppressing properties [5]. It may protect malignant cells through the inhibition of cytotoxic T lymphocyte-mediated tumor-specific cell lysis. Thus, IL-10 has an important role in carcinogenesis and it is postulated that it affects cancer risk, specifically for NHL [6]. The IL-10 promotor region may influence its expression and consequently alter susceptibility to NHL and disease outcome.

It has been hypothesized that decreased production of IL-10 may increase the risk of NHL by less effectively downregulating the production of proinflammatory cytokines [7]. Accordingly, genetic factors that downregulate IL-10 production may provide a proinflammatory medium that favors lymphomagenesis [8].

However, other studies have hypothesized that IL-10, which is a B-cell stimulatory cytokine, could promote lymphomagenesis [9].

Therefore, these conflicting findings suggest that dysregulation in IL-10 in general could be a pivotal factor in NHL development.

The aim of the current work was to study the possible role of IL2-330T/G (rs2069762) and IL-10-1082A/G (rs1800896) singlenucleotide polymorphisms (SNPs) as genetic risk factors for B-cell NHL (B-NHL) in a group of Egyptian patients.

Materials and Methods Study Population This case-control study included 100 adult Egyptian B-NHL patients recruited from the Department of Medical Oncology, National Cancer Institute (NCI), Cairo University. These comprised either de novo cases or patients attending the NCI for follow-up. There were 54 males and 46 females. Their ages ranged between 20 and 83 years with a mean age of 52.7 years.

One hundred unrelated age- and sex-matched volunteers were included in the study as a control group. The research protocol was approved by the Research Ethics Committee of the Kasr Al Ainy Faculty of Medicine, Cairo University. From all participants, informed consent was obtained in writing, and all procedures 100

Turk J Hematol 2018;35:99-108

were in accordance with the 1964 Helsinki Declaration.

Diagnosis and subtyping of B-NHL was performed according to the World Health Organization classification of 2008. Patients were subjected to thorough clinical examinations, as well as laboratory investigations and radiological work-up for proper clinical assessment. The demographic and clinical features of the B-NHL patients are presented in Table 1.

Genotyping of IL-2-330T/G (rs2069762) and IL-10-1082A/G (rs1800896) DNA extraction from peripheral blood leukocytes was done with the GeneJET Whole Blood Genomic DNA Purification Mini Kit (Fermentas Life Sciences, Canada) according to the manufacturer’s instructions. Samples were stored in the elution buffer at -20 °C until use.

Detection of the IL-2-330T/G (rs2069762) SNP was performed with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique according to Cavet et al.

[10]. The primer set used was as follows: forward, 5’-TAT TCA CAT GTT CAG TGT AGT TCT-3’; and reverse, 5’-AGA CTG ACT GAA TGG ATG TAG GTG-3’. Amplification was performed in a thermocycler (PerkinElmer 9700; PerkinElmer, USA) using the following program: 94 °C (5 min); then 30 cycles of 94 °C (1 min), 48° C (1 min), and 72 °C (1 min); and a final extension step for 8 min at 72 °C. This SNP abolishes the restriction site that can be recognized by the MaeI restriction enzyme; accordingly, the T allele was restricted into two bands of 124 and 64 bp, while the G allele remained a 188-bp band.

Genotyping of the IL-10-1082A/G (rs1800896) SNP was performed by allele-specific PCR (ARMS) technique [11]. The following primers were used: F-5’-AGCAACACTCCTCGTCGCAAC, with either B1-5’-CCTATCCCTACTTCCCCC (G allele) or B2-5’CCTATCCCTACTTCCCCT (A allele). The thermocycler program applied was 95 °C (10 min); then 30 cycles of 94 °C (30 s), 60 °C (1 min), and 72 °C (1 min); and a final extension step for 7 min at 72 °C. The AA genotype was identified by a single 153-bp band in tube B2, while the homozygous variant (GG) showed a 153-bp band in tube B1. The heterozygous variant (AG) was identified by a 153-bp band in both tubes. To validate our results, re-genotyping of 40 samples with respect to case-control status was performed. The results were interpreted blindly and found to be 100% concordant.

Treatment Regimen and Response to Therapy All patients received the standard protocol treatment for NHL at the NCI of Cairo University. Diffuse large B-cell lymphoma (DLBCL) patients were treated according to stage and bulkiness.

Non-bulky (<10 cm) stage I-II cases including extranodal presentations received 4 cycles of R-CHOP/21 days [rituximab at

Rahman HAA., et al: IL-2 and -10 Polymorphisms and B-NHL

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Table 1. Demographic and clinical data of B-cell non-Hodgkin lymphoma patients at presentation and their response to therapy.

B-NHL patients (n=100)

Item Male

Sex Female B-symptoms: Fever, night sweats, weight loss Lymphadenopathy Cervical Axillary Inguinal

Groups of lymph Abdominal nodes involved

Para-aortic Submandibular Mesenteric

54/100 46/100 25/100 81/100 61/100 42/100 38/100 33/100 24/100 20/100 9/100

Extranodal involvement <2 ≥2

Splenomegaly Hepatomegaly Clinical stage I & II III & IV

79/100 21/100 40/100 38/100 23/100 77/100

PS

375 mg/m2, cyclophosphamide at 750 mg/m2, doxorubicin at 50 mg/m2, vincristine at 2 mg total dose, and prednisone at 100 mg for 5 days, followed by involved field radiotherapy (IFRT)]. Stage III or IV patients received 6-8 cycles of R-CHOP guided by the patient’s response by positron emission tomography–computed tomography, which was done after 4 cycles. Patients with initial bulky disease received IFRT after their chemotherapy cycles.

Follicular lymphoma of stage I and II was treated with IFRT only, while stages III and IV were treated if patients met the Groupe d’Etude des Lymphomes Folliculaires criteria for initiation of treatment. Mantle cell lymphoma patients were treated with R-CHOP alternated with R-DHAP. Therapeutic responses were assessed according to Oken et al. [12].

Statistical Analysis Data management and analysis were performed using SPSS 21. Data were explored for normality using the KolmogorovSmirnov test and the Shapiro-Wilk test. Comparisons between groups for parametric numeric variables were done using the Student t-test, while for non-parametric numeric variables, comparisons were done by the Mann-Whitney U test. Chi-square or Fisher exact tests were used for comparing categorical data.

For risk estimation, the odds ratio (OR) and 95% confidence interval (CI) were calculated. The Kaplan-Meier method was used to assess disease-free survival (DFS). Differences between survival curves were evaluated for statistical significance with the log-rank test. All p-values are two-sided and p<0.05 was considered significant.

Score <2 Score ≥2

61/100 39/100

IPI risk group Low Intermediate/High

29/100 71/100

Results

44/78 34/78

The genotypic and allelic frequencies of the IL-2-330T/G and IL10-1082A/G SNPs in B-NHL patients and controls are presented in Tables 2 and 3. The genotypic distribution of the studied SNPs was in agreement with Hardy-Weinberg equilibrium (p>0.05).

IPI risk groups for DLBCL subtype (n=78) Low/Intermediate low (1, 2) Intermediate high/High (3, 4)

Histological aggressiveness Indolent Aggressive

21/100 79/100

Regimen of treatment Chemotherapy Chemotherapy & radiotherapy No treatment

78/100 17/100 5/100

Response to treatment CR Non-CR PR PD SD Unavailable

59/100 27/100 17/100 4/100 6/100 14/100

PS: Performance status, IPI: International Prognostic Index, CR: complete remission, PR: partial remission, PD: progressive disease, SD: stable disease, B-NHL: B-cell nonHodgkin lymphoma.

The IL-2-330T/G variant genotypes (TG and GG) are associated with B-NHL risk, and the risk was higher for the indolent subtypes.

Statistical comparison revealed that a performance status score of ≥2 was more common in patients harboring the variant genotypes (Supplementary Tables 1 and 2). The distribution of the variant genotypes of IL-10-1082A/G (AG and GG) did not differ between B-NHL patients and controls. Extranodal involvement of ≥2 sites was statistically more common in patients having the common genotype (Supplementary Tables 3 and 4). Combined genotype analysis showed that B-NHL risk increased almost sixfold in those having the variant genotypes of IL-2-330T/G and the common genotype of IL-10-1082A/G (AA), while co-inheritance of the variant genotypes of both SNPs was associated with fivefold increased risk of B-NHL (OR: 5.43, 95% CI: 1.44-20.45).

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Table 2. Distribution of interleukin-2-330T/G and interleukin-10-1082A/G genotypes in B-cell non-Hodgkin lymphoma patients and controls.

Genotypes

IL-2-330T/G

IL-10-1082A/G

Combined genotypes analysis, IL-2/IL-10

Controls, n (%)

B-NHL patients, n (%)

OR

95% CI

p-value

TT

42 (42%)

20 (20%)

(1) Ref.

TG

26 (26%)

38 (38%)

3.07

1.48-6.37

0.003

GG

32 (32%)

42 (42%)

2.76

1.36-5.57

0.005

TG & GG

58 (58%)

80 (80%)

2.90

1.54-5.44

0.001

T allele

0.55

0.39

G allele

0.45

0.61

1.91

1.28-2.85

<0.001

AA

28 (28%)

26 (26%)

(1) Ref.

GA

59 (59%)

51 (51%)

1.91

0.80-4.52

0.144

GG

13 (13%)

23 (23%)

0.93

0.49-1.79

0.830

GA & GG

72 (72%)

74 (74%)

1.11

0.59-2.07

0.750

A allele

0.575

0.515

G allele

0.425

0.485

0.79

0.53-1.16

0.228

TT/AA

3 (3%)

12 (12%)

(1) Ref.

TT/GA &/or GG

17 (17%)

30 (30%)

2.27

0.56-9.17

0.251

TG &/or GG / AA

23 (23%)

16 (16%)

5.75

1.39-23.72

0.016

TG &/or GG / GA &/or GG

57 (57%)

42 (42%)

5.43

1.44-20.45

0.012

OR: Odds ratio, 95% CI: 95% confidence interval, Ref.: reference, B-NHL: B-cell non-Hodgkin lymphoma, IL: interleukin.

Table 3. Distribution of interleukin-2-330T/G and interleukin-10-1082A/G genotypes in indolent and aggressive subtypes of B-cell non-Hodgkin lymphoma patients and controls.

Controls (n=100)

Genotypes

Indolent B-NHL OR (n=21) (95% CI)

IL-2-330 T/G

TT

42 (42%)

3 (14.3%)

TG & GG

58 (58%)

18 (85.7%)

IL-10-1082 A/G

AA

28 (28%)

3 (14.3%)

GA & GG

72 (72%)

18 (85.7%)

p-value

4.34 (1.2-15.71)

0.017

2.33 (0.64-8.54)

0.191

Aggressive B-NHL (n=79) 17 (21.5%) 62 (78.5%) 23 (29.1%) 56 (70.9%)

OR (95% CI)

p-value

2.64 (1.35-5.15)

0.004

0.95 (0.49-1.82)

0.870

OR: Odds ratio, 95% CI: 95% confidence interval, B-NHL: B-cell non-Hodgkin lymphoma, IL: interleukin.

Regarding the potential role of these SNPs as molecular prognostic markers, the 3-year and 5-year DFS rates were estimated. The 3-year DFS rate for the variant genotypes (GG or TG) of IL-2330T/G was 65.4% versus 69.2% for the common genotype (TT), while the 5-year DFS rate for the variant genotypes (GG or TG) was 45.3% versus 69.2% for the common genotype (TT) with no statistically significant difference (p=0.211). The 3-year DFS rate for the variant genotypes (GG or AG) of IL-10-1082A/G was 60.7% versus 79.5% for the common genotype (AA), while the 5-year DFS rate for the variant genotypes (GG or AG) was 49.1% versus 39.8% for the common genotype (AA), which was statistically insignificant (p=0.205). Other potential prognostic 102

factors, such as the patientsâ&#x20AC;&#x2122; age at diagnosis, sex, clinical stage, performance status, International Prognostic Index score, extranodal involvement, and histopathological subtypes, did not affect the DFS of our B-NHL patients (Supplementary Table 5).

Discussion The relationship between the IL-2-330T/G SNP and NHL remains ambiguous. Some studies showed that the variant (G) allele correlates with decreased IL-2 production in vivo [13]. It has been suggested that reduced IL-2 levels may downregulate the antitumor response through ADCC and thus increase the risk of NHL [4]. In the present study, 38% of B-NHL patients had the

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heterozygous genotype (TG), while 42% had the homozygous genotype (GG). These frequencies differed from those reported

by Song et al. [4], being 56.2% and 12.7% for the TG and GG genotypes in Chinese NHL patients. This might be due to ethnicity. In the study presented here, the frequency of the variant genotypes was significantly higher in patients than controls and was associated with increased risk of B-NHL among Egyptians. This is in agreement with the study of Song et al. [4] involving Chinese patients.

The IL-2-330T/G polymorphism was associated with advanced performance status score. Otherwise, there was no association between the IL-2-330T/G SNP and sex, presenting symptoms, or other clinical and laboratory features, as well as response to therapy. Song et al. [4] could not find any association between the IL-2-330T/G SNP and clinical features in the Chinese patients in their study. Based on the clinical behavior of the disease, our patients were stratified into cases of indolent and aggressive lymphomas. IL-2-330T/G polymorphic genotypes were found to confer threefold increased risk of DLBCL, and the increase in risk for indolent B-NHL was fourfold.

Being an anti-inflammatory cytokine, the main functions of IL10 are suppression of cytokine synthesis in Th1 cells as well as downregulation of cytotoxic and cell-mediated inflammatory responses [14]. It acts as an autocrine growth factor that upregulates BCL-2 expression in some cases of B-cell neoplasms [15]. High IL-10 levels were shown to be associated with poor outcomes and shorter survival in B-NHL patients [16,17].

Genetic polymorphisms in the promotor area of the IL-10 gene have been reported to influence IL-10 levels. IL-10-1082 common (A) and variant (G) alleles respectively correlate with low and high IL-10 expression levels [18]. Several studies have investigated the association of IL-10 gene polymorphisms and NHL susceptibility, reporting conflicting results. In the current study, 74% of B-NHL patients harbored this genetic variation, with 51% being heterozygous (AG) and 23% homozygous (GG). These frequencies agree with those previously reported in Australian patients, being 51% and 29% for AG and GG variant genotypes, respectively [19]. Similarly, Lan et al. [20] found the AG and GG genotypes in 52% and 23% of their female American B-NHL patients, and these frequencies were close to those of their controls. Extranodal involvement (i.e. the involvement of ≥2 extranodal sites) was more prominent in patients having the common genotype. Otherwise, there were no statistical differences between patients harboring the common or the variant genotypes. Lech-Maranda et al. [24] found that DLBCL patients harboring the variant genotypes had slightly higher complete remission (CR) rates. They stated that patients with

Rahman HAA., et al: IL-2 and -10 Polymorphisms and B-NHL

elevated cytokine levels had significantly lower CR rates.

IL-10-1082A/G variant genotypes (AG and GG) were not associated with susceptibility to either indolent or aggressive B-NHL subtypes. Similar results were reported by Talaat et al. [21], who concluded that IL-10-1082A/G polymorphic genotypes could not be considered as a genetic risk factor for DLBCL in Egyptians. Moreover, the studies of Kube et al. [22] and Berglund et al. [23] revealed that the IL-10-1082A/G SNP was not associated with susceptibility to aggressive B-NHL in German or Swedish populations, respectively. Contrary to our results, Purdue et al. [19] found that the frequency of the variant genotypes conferred increased risk of DLBCL. LechMaranda et al. [24] reported a similar frequency of the variant genotypes in France, which was statistically significant when compared to controls. They considered the IL-10-1082A/G SNP as a genetic risk factor for DLBCL in the French population. Lan et al. [20] stated that the GG homozygous variant genotype was significantly associated with an increased risk for DLBCL in female Americans. However, Cunningham et al. [25] reported that the low-producing IL-10-1082 AA genotype was significantly higher in patients with aggressive lymphoma compared to controls.

Combined genotype analysis showed that B-NHL risk was increased when IL-2-330T/G variant genotypes were coinherited with either common or variant genotypes of IL10-1082A/G. Accordingly, we assume that B-NHL risk can be attributed to the IL-2 rather than the IL-10 SNP. Regarding DFS, none of the potentially known prognostic factors affected the DFS of B-NHL patients. Furthermore, the polymorphic genotypes of either IL-2-330T/G or IL-10-1082A/G had no effect on the 3- and 5-year DFS rates of these patients.

Study Limitations The relatively small sample size of this study is a limitation of the present work. Larger sample size is recommended to validate our results regarding the role of the studied SNPs as molecular risk factors for B-NHL and to clarify their impact on therapeutic response and disease course. Furthermore, IL-2 and IL-10 levels should have been examined to conclude the association between the examined variations and NHL.

Conclusion The current study highlights the possible involvement of the IL-2-330T/G SNP in susceptibility to B-NHL. Moreover, IL-101082A/G is not a molecular susceptibility marker for B-NHL in Egyptians.

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Turk J Hematol 2018;35:99-108

and all procedures were performed in accordance with the 1964

Ethics Ethics Committee Approval: The research protocol was

Helsinki Declaration.

approved by the Research Ethics Committee of the Departments

Informed Consent: Informed written consent was obtained

of Clinical Pathology and Medical Oncology, Cairo University,

from all participants prior to enrollment in the study.

Supplementary Table 1. Comparison between B-cell non-Hodgkin lymphoma patients having wild genotype and polymorphic genotypes of interleukin-2-330T/G regarding their clinical data.

IL-2 wild genotype (n=20)

Item No (%)

IL-2 polymorphic genotypes (n=80)

p-value

No (%)

Sex Male Female

12/20 (60%) 8/20 (40%)

42/80 (52.5%) 38/80 (47.5%)

0.547

B-symptoms

6/20 (30%)

19/80 (23.8%)

0.564

Lymphadenopathy

Groups of lymph nodes involved

17/20 (85%)

64/80 (80%)

0.610

Cervical

14/20 (70%)

47/80 (58.8%)

0.356

Axillary

8/20 (40%)

34/80 (42.5%)

0.939

Inguinal

8/20 (40%)

30/80 (37.5%)

0.937

Submandibular

6/20 (30%)

14/80 (17.5%)

0.211

Abdominal

8/20 (40%)

25/80 (31.3%)

0.457

Mesenteric

4/20 (20%)

5/80 (6.3%)

0.055

Para-aortic

8/20 (40%)

16/80 (20%)

0.061

Extranodal involvement <2 â&#x2030;Ľ2

18/20 (90%) 2/20 (10%)

61/80 (76.3%) 19/80 (23.7%)

0.230

Splenomegaly

8/20 (40%)

32/80 (40%)

1.0

Hepatomegaly

6/20 (30%)

32/80 (40%)

0.453

Clinical stage I & II III & IV

8/20 (40%) 12/20 (60%)

15/80 (18.8%) 65/80 (81.2%)

0.071

PS Score <2 Score â&#x2030;Ľ2

16/20 (80%) 4/20 (20%)

45/80 (56.3%) 35/80 (43.7%)

0.05*

IPI risk group Low Intermediate/high

9/20 (45%) 11/20 (55%)

20/80 (25%) 60/80 (75%)

0.078

IPI risk groups for DLBCL subtype Low/Intermediate low (1, 2) Intermediate high/high (3, 4)

12/17 (70.6%) 5/17 (29.4%)

32/61 (52.5%) 29/61 (47.5%)

0.183

Treatment outcome CR Non-CR (PR, PD, SD) Unavailable

12/20 (60%) 5/20 (25%) 3/20 (15%)

47/80 (58.8%) 22/80 (27.5%) 11/80 (13.7%)

0.971

3/20 (15%) 17/20 (85%)

18/80 (22.5%) 62/80 (77.5%)

0.461

Pathology Indolent Aggressive

*p-value <0.05 = significant, PS: Performance status, IPI: International Prognostic Index, CR: complete remission, PR: partial remission, PD: progressive disease, SD: stable disease, IL: interleukin, DLBCL: Diffuse large B-cell lymphoma.

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Supplementary Table 2. Comparison between B-cell non-Hodgkin lymphoma patients with wild genotype and those with polymorphic genotypes of interleukin-2-330T/G regarding their hematological data.

Item

IL-2 wild genotype Range

Hemogram

Median

IL-2 polymorphic genotypes Mean ± SD

Range

Median

Mean ± SD

p-value

Hb, g/dL

9.60-16.70 12.80

12.59±1.83

5-17

11.85

11.68±2.34

0.092

TLC, x103/cm3

1.50-50

8.55

10.64±9.87

1.90-80

7.55

8.89±8.73

0.274

Plts, x103/cm3

133-491

300.50

281.25±113.37

14-675

278.50

295.40±138.87

0.829

130-3531

239

499.45±751.66

135-3664

359

521.09±626.45

0.196

LDH, IU/L

Hb: Hemoglobin, TLC: total leukocyte count, LDH: lactate dehydrogenase, Plts: platelets, IL: interleukin.

Supplementary Table 3. Comparison between B-cell non-Hodgkin lymphoma patients with wild genotype and those with polymorphic genotypes of interleukin-10-1082A/G regarding their clinical data.

Item

Sex Male Female B-symptoms Lymphadenopathy

Groups of lymph nodes involved

Cervical Axillary Inguinal Submandibular Abdominal Mesenteric Para-aortic

Extranodal involvement <2 ≥2 Splenomegaly Hepatomegaly Clinical stage I & II III & IV PS Score <2 Score ≥2 IPI risk group Low Intermediate/High IPI risk groups for DLBCL subtype Low/Intermediate low (1, 2) Intermediate high/High (3, 4) Treatment outcome CR Non-CR (PR, PD, SD) Unavailable Pathology Indolent Aggressive

IL-10 wild genotype (n=26)

IL-10 polymorphic genotypes (n=74)

No. (%)

No. (%)

11/26 (42.3%) 15/26 (57.7%)

43/74 (58.1%) 31/74 (41.9%)

0.164

7/26 (26.9%) 22/26 (84.6%) 15/26 (57.7%) 13/26 (50%) 13/26 (50%) 3/26 (11.5%) 10/26 (38.5%) 1/26 (3.9%) 4/26 (15.4%)

18/74 (24.3%) 59/74 (79.7%) 46/74 (62.2%) 29/74 (39.2%) 25/74 (33.8%) 17/74 (23%) 23/74 (31.1%) 8/74 (10.8%) 20/74 (27%)

0.792 0.585 0.688 0.337 0.143 0.210 0.491 0.439 0.232

16/26 (61.5%) 10/26 (38.5%) 10/26 (38.5%) 10/26 (38.5%)

63/74 (85.1%) 11/74 (14.9%) 30/74 (40.5%) 28/74 (37.8%)

4/26 (15.4%) 22/26 (84.6%)

19/74 (25.7%) 55/74 (74.3%)

0.283

14/26 (53.9%) 12/26 (46.1%)

47/74 (63.5%) 27/74 (36.5%)

0.385

4/26 (15.4%) 22/26 (84.6%)

25/74 (33.8%) 49/74 (66.2%)

0.075

12/23 (52.2%) 11/23 (47.8%)

32/55 (58.2%) 23/55 (41.8%)

0.626

14/26 (53.8%) 8/26 (30.8%) 4/26 (15.4%)

45/74 (60.8%) 19/74 (25.7%) 10/74 (13.5%)

0.822

3/26 (11.5%) 23/26 (88.7%)

18/74 (24.3%) 56/74 (75.7%)

0.169

p-value

0.011* 0.852 0.955

*p-value <0.05=significant.

PS: Performance status, IPI: International Prognostic Index, CR: complete remission, PR: partial remission, PD: progressive disease, SD: stable disease, DLBCL: Diffuse large B-cell, IL: interleukin.

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Supplementary Table 4. Comparison between B-cell non-Hodgkin lymphoma patients with wild genotype and those with polymorphic genotypes of interleukin-10-1082A/G regarding their hematological data.

IL-10 wild genotype

Item

IL-10 polymorphic genotypes

p-value

Range

Median

Mean ± SD

Range

Median

Mean ± SD

5-16.70

12

11.47±2.36

6.50-17

12

12±2.24

0.366

TLC, x103/cm3 3.20-80

7.30

10.18±14.46

1.50-50

8

8.91±6.04

0.368

Plts, x103/cm3 23-675

294

287.96±160.88

14-644

278.50

294.19±124.04

0.771

350

539.42±582.06

130-3664

297.50

508.79±674.97

0.346

p-value

Hb, g/dL

Hemogram

LDH, IU/L

155-2922

Hb: Hemoglobin, TLC: total leukocyte count, LDH: lactate dehydrogenase, Plts: platelets, IL: interleukin, SD: standard deviation.

Supplementary Table 5. Disease-free survival of B-cell non-Hodgkin lymphoma patients.

Factors

All Age Sex Stage IPI IPI for DLBCL B-symp.

PS Spleen Liver Extranodal Hb TLC Plts

106

<60 ≥60 Male Female I & II III & IV Intermediate/High Low Interm. high/High Low/Interm/low No Yes <2 ≥2 No Yes No Yes <2 ≥2 Abnormal Normal <11,000 ≥11,000 ≤150 >150

Number of cases

Number of relapses

3 years

5 years

Median

100 71 29 54 46 23 77 71 29 34 44 75 25 61 39 60 40 62 38 79 21 57 43 79 21 17 83

22 16 6 12 10 4 18 17 5 8 6 16 6 11 11 12 10 12 10 18 4 15 7 16 6 3 19

61.8 68.5 57.1 61.6 67.2 71.1 65.1 63.3 76.7 49.8 77.0 68.7 60.9 78.2 48.7 71.9 57.4 70.7 59.3 66.0 64.9 56.1 76.3 66.9 84.4 85.7 61.9

49.6 56.2 28.6 35.2 67.2 71.1 46.8 49.7 38.4 33.2 57.7 61.8 22.9 57.9 36.5 46.2 47.8 49.5 49.4 46.4 64.9 48.1 66.8 50.2 36.8 64.3 45.8

56.5 130.6 56.5 130.6 47.4 56.5 56.5 47.4 31.8 130.6 36.8 230.0 31.8 56.5 36.8 56.5 36.8 56.5 36.8 36.8 130.6 56.5

0.318 0.989 0.868 0.903 0.156 0.689 0.153 0.840 0.541 0.932 0.178 0.433 0.333

Rahman HAA., et al: IL-2 and -10 Polymorphisms and B-NHL

Turk J Hematol 2018;35:99-108

Supplementary Table 5. Continue.

LDH Pathology IL-2 IL-10

Elevated

61

16

64.6

60.0

130.6

Normal

39

6

77.0

38.5

47.4

Aggressive

79

15

64.6

48.4

56.5

Indolent

21

7

68.0

56.7

230.8

TT

20

2

69.2

69.2

-

TG & GG

80

20

65.4

45.3

56.5

AA

26

4

79.5

39.8

-

AG & GG

74

18

60.7

49.1

47.4

0.616 0.889 0.211 0.205

Hb: Hemoglobin, TLC: total leukocyte count, LDH: lactate dehydrogenase, Plts: platelets, IL: interleukin, IPI: International Prognostic Index.

Authorship Contributions Surgical and Medical Practices: O.M.R.K.; Concept: H.A.R., O.M.R.K.; Design: M.M.K., H.A.A.R. O.M.R.K.; Data Collection or Processing: H.M.M.; Analysis or Interpretation: M.M.K., H.A.R.; Literature Search: M.M.K., H.M.M.; Writing: M.M.K., H.M.M., H.A.R., O.M.R.K. 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.2017.0130 Turk J Hematol 2018;35:109-115

Myelodysplastic Syndrome in Pakistan: Clinicohematological Characteristics, Cytogenetic Profile, and Risk Stratification Pakistan’da Myelodisplastik Sendrom: Klinikohematolojik Özellikler, Sitogenetik Profil ve Risk Stratifikasyonu Rafia Mahmood,

Chaudry Altaf,

Parvez Ahmed,

Saleem Ahmed Khan,

Hamid Saeed Malik

Armed Forces Institute of Pathology, Department of Hematology, Rawalpindi, Pakistan

Abstract

Öz

Objective: Myelodysplastic syndrome (MDS) is a group of bone marrow diseases that not only have variable morphological presentation and heterogeneous clinical courses but also have a wide range of cytogenetic abnormalities. Clinicohematological parameters have a significant role in diagnosis and along with identification of cytogenetic abnormalities are important for prognostic scoring and risk stratification of patients to plan management and make treatment decisions. This study aimed to determine the clinicohematological characteristics, cytogenetic abnormalities, and risk stratification of newly diagnosed de novo MDS patients.

Amaç: Myelodisplastik sendrom (MDS) sadece değişken morfolojik prezentasyona ve heterojen klinik seyre değil geniş sitogenetik anormallikler yelpazesine de sahip olan bir grup kemik iliği hastalığıdır.

Klinikohematolojik parametreler tanıda önemli role sahiptir ve sitogenetik anormalliklerin tanımlanması ile birlikte prognostik skorlamada ve yönetimi planlamak ve tedavi kararlarını vermek için risk stratifikasyonunda önemlidir. Bu çalışma, yeni tanı de novo MDS hastalarında klinikohematolojik özellikler, sitogenetik anormallikler ve risk stratifikasyonunu belirlemeyi amaçlamıştır.

Materials and Methods: This cross-sectional study was conducted in the Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi, from January 2013 to January 2017. Patients were diagnosed on the basis of World Health Organization criteria for MDS, clinicohematological parameters were noted, and cytogenetic analysis was performed. Risk stratification was done using the Revised International Prognostic Scoring System.

Results: A total of 178 cases of MDS were analyzed, including 119 males (66.9%) and 59 females (33.1%). The median age was 58 years.

The most common presenting feature was anemia in 162 (91%) of the patients. MDS with multilineage dysplasia was the most common diagnosis, seen in 103 (57.9%) patients. A normal karyotype was seen in 95 (53.4%), while 83 (46.6%) showed clonal karyotypic abnormalities at diagnosis. Of these, the common abnormalities found were trisomy 8, complex karyotype, and del 5q. Risk stratification revealed low-risk disease in 73 (41%) patients.

Conclusion: Cytogenetic analysis showed the normal karyotype to be the most common while risk stratification revealed a predominance of low-risk disease at the time of presentation.

Keywords: Myelodysplastic syndrome, International Prognostic Scoring System

Cytogenetics,

Revised

Gereç ve Yöntemler: Bu kesitsel çalışma Rawalpindi Silahlı Kuvvetler Patoloji Enstitüsü Hematoloji Departmanı’nda Ocak 2013’ten Ocak 2017 tarihine kadar sürdürülmüştür. Hastalar Dünya Sağlık Örgütü MDS kriterlerine göre teşhis edildi, klinikohematolojik parametreler not edildi ve sitogenetik analiz yapıldı. Risk stratifikasyonu Revize Uluslararası Prognostik Skorlama Sistemi kullanılarak yapıldı.

Bulgular: Toplam 178 MDS olgusu, 119 erkek (%66,9) ve 59 kadın (%33,1) analiz edildi. Medyan yaş 58 idi. Başvuruda en sık görülen belirti olguların 162’sinde (%91) anemi idi. En sık tanı MDS çoklu seride displazi olup 103 (%57,9) hastada görüldü. Teşhiste normal karyotip 95 (%53,4) olguda görülürken 83 (%46,6) olgu klonal karyotipik anormallikler gösterdi. Bunlar arasında, en sık görülenler trizomi sekiz, kompleks karyotip ve del5q idi. Risk stratifikasyonu 73 (%41) hastada düşük-risk hastalık ortaya koydu.

Sonuç: Sitogenetik analiz en sık normal karyotipi gösterirken risk stratifikasyonu tanı sırasında düşük-risk hastalığın çoğunlukta olduğunu ortaya koymuştur.

Anahtar Sözcükler: Myelodisplastik sendrom, Sitogenetik, Revize Uluslararası Prognostik Skorlama Sistemi

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Rafia MAHMOOD, M.D., Armed Forces Institute of Pathology, Department of Hematology, Rawalpindi, Pakistan Phone : 923 365 182 270 E-mail : rafiamahmood@hotmail.com ORCID-ID: orcid.org/0000-0002-5394-9290

Received/Geliş tarihi: March 27, 2017 Accepted/Kabul tarihi: May 31, 2017

109

Mahmood R, et al: Myelodysplastic Syndrome in Pakistan

Introduction Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal stem cell disorders characterized by peripheral blood cytopenias, dysplasia, and ineffective hematopoiesis [1].

Patients have a variable clinical course and there is an increased risk of myeloid leukemic transformation [2]. It is a disease of the elderly; its incidence increases with age. It is slightly more common in males with a male:female ratio of 1.4:1 [3]. While a few patients may be detected incidentally when a routine blood count reveals unexpected cytopenia, most present with symptoms and signs of bone marrow failure. Notable findings include fatigue due to anemia, infections, and bleeding [4].

Morphologic dysplasia is the hallmark of the disease [5]. Dysplasia may be seen in any or all of the three lineages [6]. The World Health Organization (WHO) has classified the myelodysplastic syndromes based on the number of cytopenias, dysplasia in a single lineage or in multiple lineages, cytogenetics, the number of blast cells, and the presence or absence of ring sideroblasts [5].

In addition to morphologic heterogeneity, MDS cases show profound heterogeneity in their genetic presentation [7]. More than half the patients show clonal chromosomal abnormalities with a predominance of unbalanced abnormalities [8].

Cytogenetic analysis not only has an important role in diagnosis where certain chromosomal abnormalities are considered presumptive evidence of MDS, but also has important prognostic implications. These cytogenetic abnormalities can be detected by conventional metaphase karyotyping. However, fluorescent in situ hybridization (FISH) has been seen to have a much higher sensitivity for detection of del 5q [9]. These cytogenetic findings serve as a basis for the characterization of cytogenetic subgroups [10].

Over time, a better understanding of the biology of disease has shown cytogenetics to be an important prognostic parameter [11]. The Revised International Prognostic Scoring System (R-IPSS) refines risk group definitions, aiming for better prediction of individual prognosis. The parameters included are the degree of cytopenias, the number of blast cells, and the cytogenetic subgroup [12]. The prognostication of patients based on individualized risk assessment not only predicts disease progression but also provides an important tool in planning management and making treatment decisions [13].

Most studies regarding MDS are from Western populations.

Disease biology, clinical presentations, and cytogenetic findings are different and distinctive for population groups and can show noticeable differences in geographic prevalence around the world. The present study was designed with an aim to see the clinicohematological features, cytogenetic profile, and risk stratification of the patients of Pakistan (an Asian population) 110

Turk J Hematol 2018;35:109-115

as so far there is a lack of data on MDS in our region. This will help to determine treatment protocols and prognosis.

Materials and Methods Patients This study was a cross-sectional analysis conducted in the Department of Hematology of the Armed Forces Institute of Pathology, Rawalpindi, from January 2013 to January 2017.

All patients were Pakistanis, of Asian origin, belonging to different ethnic groups including Punjabis, Pashtuns, Sindhis, Balochis, Kashmiris, and those from Gilgit-Baltistan. Patients were between the ages of 30 and 85 years. These patients were newly diagnosed with MDS and had no previous history of any treatment. Patients who had failed culture (did not yield at least 20 metaphases) in cytogenetic analysis were excluded from the study. All subjects were thoroughly informed about the study and written informed consent was obtained.

Clinicohematological Parameters Detailed history was recorded and complete physical examination was done. Symptoms and signs were noted. Complete blood count, peripheral blood film, and bone marrow examination were done and patients were diagnosed as having MDS based on the WHO criteria.

Cytogenetics and FISH Cytogenetic analysis was performed by using the conventional G banding technique. A bone marrow specimen of 3 mL was collected in sodium heparin. Metaphase chromosomes were banded using the conventional Giemsa trypsin banding technique and karyotyped according to the International System for Human Cytogenetic Nomenclature criteria. At least twenty metaphases were analyzed with the CytoVision semiautomated image analysis and capture system.

Interphase FISH studies were performed on blood or bone marrow specimens processed by standard methods for cultured samples. The MetaSystems XL 5q31/5q33 probe (10 ÂľL) was applied to the target on the slide. A total of 500 nuclei were analyzed per probe set by using a fluorescent microscope with an orange green spectrum filter.

Risk Stratification The patients were risk-stratified according to the R-IPSS. Statistical Analysis Collected data were entered and analyzed using SPSS 20 (IBM Corp., Armonk, NY, USA). Quantitative variables, i.e. age, hemoglobin (Hb), platelet count, and absolute neutrophil count (ANC), have been presented as mean Âą standard deviation.

Mahmood R, et al: Myelodysplastic Syndrome in Pakistan

Turk J Hematol 2018;35:109-115

Qualitative variables, i.e. sex, cytogenetics, and risk category, have been presented as frequency and percentage.

Ethical Approval This study was approved by the Ethical Review Committee of the Armed Forces Institute of Pathology, Rawalpindi. Informed written consent was received from the patients.

Results A total of 178 patients were diagnosed as having de novo MDS. The median age of the patients was 58 years. Out of 178 patients, 119 (66.9%) were male, while the remaining 59 (33.1%) patients were female.

The most common presenting clinical feature was pallor, followed by symptoms of fatigue, recurrent infections, and bruising/bleeding; 118 (66%) of the patients were transfusiondependent at the time of presentation. Mean Hb was 6.4 g/dL and mean platelet count was 97x109/L, while the mean ANC was 2.1x109/L. Table 1 shows the clinicohematological parameters of our patients. We classified our patients according to the 2016 revised WHO classification: 103 (57.9%) of the patients were in the MDS-MLD (MDS with multilineage dysplasia) category while 36 (20.2%) cases were classified as MDS-SLD (MDS with single lineage dysplasia), 16 (8.9%) as MDS-EB1 (MDS with excess blasts-1), 12 (6.7%) as MDS-EB2 (MDS with excess blasts-2), 6 (3.4%) as MDS with isolated del (5q), 3 (1.7%) as MDS-RSSLD (MDS with ring sideroblasts with single lineage dysplasia), and 2 (1.1%) as MDS-RS-MLD (MDS with ring sideroblasts with multilineage dysplasia).

(4.5%) had double cytogenetic abnormalities while 19 (10.7%) had a complex karyotype. Of the cytogenetic abnormalities seen, the most commonly found was trisomy 8 in 23 (12.9%) cases, followed by del 5q in 13 (7.3%), monosomy 7 in 10 (5.6%), loss of Y in 5 (2.8%), del 11q in 5 (2.8%), del 20q in 4 (2.2%), del 7q in 3 (1.7%) and i(17q) in 1 (0.6%) patient. Other abnormalities, including translocations, hyperdiploidy, hypodiploidy, deletions, and monosomies, were seen in 8 (4.5%) of the patients. del 5q was detected in 8 patients based on conventional cytogenetics while in 5 patients it was missed by conventional cytogenetics and detected by FISH. Each parameter was assessed and scored according to the R-IPSS. Based on the score, the patients were stratified into five distinct risk groups. In the very-low-risk group, there were 17 (9.6%) patients, while there were 73 (41%) patients in the low-risk group, 48 (27.1%) patients in the intermediate-risk group, 24 (13.5%) patients in the high-risk group, and 16 (9.1%) patients in the very-high-risk group.

A normal karyotype was seen in 95 (53.4%) cases, while 83 (46.6%) patients showed clonal karyotypic abnormalities at diagnosis (Figure 1 and 2). Of these, 56 (31.4%) had single and 8 Table 1. Clinicohematological parameters of the patients.

Parameters

n=178

%

Hb <10 g/dL

174

97.8

Platelets <100x109/L

99

55.6

ANC <1.5x10 /L

69

38.8

Unicytopenia

45

25.3

Bicytopenia

69

38.8

Pancytopenia

64

35.9

Single lineage

49

27.5

Multilineage

129

72.5

PB <1%, BM <5%

150

84.8

PB 2%-4%, BM 5%-9%

16

8.9

PB 5%-19%, BM 10%-19%

12

6.2

5

2.7

9

Cytopenia

Dysplasia

Blasts

Ring sideroblasts >15%

Hb: Hemoglobin, ANC: absolute neutrophil count, PB: peripheral blood, BM: bone marrow.

Figure 1. Cytogenetics of the patients.

Figure 2. Common karyotypic abnormalities of the patients.

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Discussion Myelodysplastic syndromes show not only clinical heterogeneity and genetic diversity but also a highly variable clinical course [14]. Over the last decade a better understanding of the biology of MDS has led to the identification of genetic molecular factors that have diagnostic value as well as roles in determining the disease course and prognosis [15]. Conventional cytogenetics of all newly diagnosed MDS patients is thus of paramount significance as it is an important component in risk-stratifying the patients [16]. FISH has a much higher sensitivity and has improved the detection of genomic aberrations in MDS, especially del 5q [9].

To our knowledge, there are no comprehensive data available on the clinicohematological features, cytogenetic profiles, and risk stratification of MDS patients from our part of the country. The Armed Forces Institute of Pathology is a tertiary care institute and a referral center in the north of Pakistan. It caters to a large number of patients from all over the country from very different ethnic backgrounds. Our study aims to help clinicians in structuring treatment decisions in light of cytogenetically based risk stratification.

In our study, the median age of the patients was 58 years.

Similar findings have been reported in local studies. However, a much higher age, 71 years, was reported by Greenberg et al.

[11] in a Western population. There is a major difference in the age of presentation of our patients and the Western population.

These differences may be attributable to racial and geographic differences and differences in disease biology in different

populations. Among our patients, males were more common as compared to females (66.9% vs. 33.1%). The male-to-female ratio was 2:1. Our observation coincides with the findings of Sultan and Irfan [17], who reported a sex ratio of 1.6:1. Deeg et al. [18] also reported a male predominance.

The most common presenting clinical findings of pallor followed by fatigue observed in our study are consistent with those reported by Narayanan [19] in the Indian population. Of our patients, 66% were transfusion-dependent at the time of presentation. A similar frequency of 58% was reported in the Italian population, while Greenberg et al. [11] reported 32% of the patients to be transfusion-dependent based on data from eleven countries. Mean Hb was 6.4 g/dL. Chaubey et al. [20] demonstrated mean Hb of 6.8 g/dL, which is in accordance with our findings. In another study, Voso et al. [12] reported mean Hb of 9.9 g/dL in Italian patients. There is a striking difference in presenting Hb levels and transfusion dependency in our population as compared to the Western populations studied by Greenberg et al. [11]. This may be due to the fact that Pakistan is a developing country and patients present late as they do not have early access to tertiary care medical facilities. However, these differences in presentation, with more than two-thirds of our patients being transfusion-dependent, may affect the overall treatment plan. These patients need further stratification by evaluation of their erythropoietin levels, which will guide further management. In patients with low erythropoietin levels (less than 200 IU/L), early institution of erythropoietin therapy predicts the response. Erythropoietin therapy not only improves Hb levels but also enhances the quality of life without the risks associated with blood transfusions. Iron chelation will also be an

Table 2. Comparison of clinicohematological characteristics with national and international studies.

Parameters

Our study

Rashid et al.

[21]

Sultan and Irfan [17]

Ehsan and Chaubey Aziz et al.

[22] [20]

Narayanan [19]

Avgerinou et al.

[23]

Voso et al.

[12]

Greenberg et al.

[11]

Median age (years)

58

60

64

-

42

67

74

71

71

M:F ratio

2:1

1.4:1

1.7:1

1.6:1

-

2.3:1

2.4:1

1.1:1

1.5:1

Fatigue (%)

91

-

60

92.5

-

90

55

-

-

Bleeding/bruising (%)

13.5

-

20

42.5

-

33.3

8

-

-

Fever/infection (%)

25.3

-

33.3

55

-

31.7

15

-

-

Pallor (%)

92

-

37.7

-

-

75

-

-

-

Mean Hb (g/dL)

6.4

-

7.7

6.5

6.8

5.5

9.5

9.9

-

Mean platelet (x109/l)

97

-

82.7

59.6

84.5

-

158

152

-

Mean ANC (x10 /l)

2.1

-

3.0

-

-

-

3.94

1.9

-

Transfusion dependent %

66

-

-

-

-

-

-

58

32

BM <5%

84.8

69

-

-

-

-

-

68

65

BM 5-10%

8.9

18.3

-

-

-

-

-

23

19

BM >10%

6.2

12.7

-

-

-

-

-

9

16

381

-

-

-

-

-

-

317

-

9

Blasts

Median LDH (IU/L)

Hb: Hemoglobin, ANC: absolute neutrophil count, BM: bone marrow, LDH: lactate dehydrogenase.

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Table 3. Cytogenetic profile in comparison with national and international studies.

Parameters

Our study

Rashid et al. [21]

Chaubey et al. [20]

Narayanan [19]

Chen et al. [25]

Lee et al. [24]

Avgerinou et al. [23]

Voso et al. [12]

Haase et al. [8]

Normal karyotype

53.4

57.7

52.5

65.4

62.9

56

61.6

61

49

Abnormal karyotype

46.6

42.3

47.5

34.6

37.1

44

38.4

39

51

Complex karyotype

10.7

15.5

-

-

-

15.1

7.6

6

-

Trisomy 8

12.9

9.9

7.5

-

9.5

5.9

8.3

5

8.4

Del 5q

7.3

2.8

10

21.1

4.6

1.7

2.7

10.5

15.1

Monosomy 7

5.6

-

15

-

1.6

1.7

3

2

8

Loss of Y

2.8

2.8

-

5.8

-

2.5

5.8

-

2.8

del 11q

2.8

1.4

-

-

-

-

-

-

1.1

del 20q

2.2

1.4

-

-

5.4

-

2.2

5

3.6

del 7q

1.1

4.2

-

7.7

-

-

-

-

3.1

Table 4. Comparison of cytogenetic subgroups and risk stratification.

AFIP n=178 %

Narayanan [19] n=52 %

Greenberg et al. [11] n=7012 %

Voso et al. [12] n=380 %

Very good

5.6

5.8

4

3

Good

62.9

86.5

72

77

Intermediate

15.2

7.7

13

13

Poor

12.9

-

4

4

Very poor

3.4

-

7

3

Very low

9.6

9.6

19

38

Low

41.0

34.6

38

33

Intermediate

27.1

36.5

20

18

High

13.5

19.2

13

7

Very high

9.1

-

10

4

Cytogenetic subgroup

Risk stratification

AFIP: Armed Forces Institute of Pathology.

important consideration in patients who have received multiple transfusions.

of our patients, while Rashid et al. [21] reported a frequency of 15.5%.

In our study, the mean platelet count was 97x109/L, while a mean platelet count of 100.5x109/L was reported in Indians [20] and 152x109/L [12] in the Italian population. The mean ANC in our study population was (2.1Âą1.8)x109/L, which correlates with the median ANC of 1.9x109/L reported by Voso et al. [12]. The clinicohematological characteristics of our study population are compared with those of national and international studies in Table 2.

Table 3 shows a comparison of the cytogenetic profile with national and international data. In our study, the most common cytogenetic abnormality was trisomy 8 in 12.9% followed by del 5q in 7.3% and monosomy 7 in 5.6% of the patients.

Rashid et al. [21] reported trisomy 8 to be the most common cytogenetic abnormality with a frequency of 9.9%. However, they reported a much lower frequency of del 5q in 2.8% of the patients. This difference may be due to the difference in the cytogenetic methodology adopted, as we used FISH for detection of del 5q in addition to conventional cytogenetics, as FISH has higher sensitivity. Chaubey et al. [20] reported monosomy 7 as the most frequent cytogenetic abnormality detected in 15%, followed by del 5q in 10% and trisomy 8 in 7.5% of Indian patients. In the Italian population [12], the most common karyotypic abnormality reported is del 5q in 10.5%, while much lower frequencies of 5% for trisomy 8 and 2% for

On cytogenetic analysis, a normal karyotype was seen in 95 patients (53.4%), while 83 (46.6%) patients showed clonal karyotypic abnormalities at diagnosis. Chromosomal abnormalities were detected in 34.6% of cases by Narayanan [19], 39% by Voso et al. [12], 42.3% by Rashid et al. [21], 47.5%

by Chaubey et al. [20], and 48% by Cao et al. [9]. A complex karyotype, which carries poor overall survival, was seen in 10.7%

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Mahmood R, et al: Myelodysplastic Syndrome in Pakistan

Turk J Hematol 2018;35:109-115

monosomy 7 have been reported. Identification of patients with del 5q is particularly important as these patients are candidates for treatment with the immunomodulatory drug lenalidomide.

Early initiation of treatment with lenalidomide not only leads to transfusion independence but also induces cytogenetic remission in this subgroup of patients.

Authorship Contributions

The R-IPSS score is particularly useful in clinical decision-making and selection of appropriate treatment options while at the same time providing prognostic information and predicting outcome in response to disease-modifying therapies. Upon risk stratification by the R-IPSS, as shown in Table 4, most of our patients (41%) were in the low-risk category, followed by 27.1% of the patients in the intermediate-risk category. These findings are in accordance with the findings of Greenberg et al. [11], who reported 38% of their patients in the low-risk followed by 20% in the intermediaterisk and 19% in the very-low-risk category. However, in an Italian study, Voso et al. [12] reported 38% in the very-low-risk category, followed by 33% in the low-risk and 18% in the intermediate-risk category. Those patients in the high-risk and very-high-risk groups need stringent regular monitoring as they have poor prognosis and are potentially more likely to have disease progression and transformation into acute myeloid leukemia.

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.

Conclusion Cytogenetic analysis showed the normal karyotype to be the most common while among the cytogenetic abnormalities detected trisomy 8 was the most common. Risk stratification revealed a predominance of low-risk disease at the time of presentation. The results of our study are in accordance with other local studies with a few differences, which may be due to differences in the method of detection of chromosomal abnormalities. However, there are differences with studies in other parts of the world. These differences may be attributable to geographical and ethnic differences in disease biology and genetics. As MDS has a heterogeneous clinical course, genetic characterization of all newly diagnosed MDS patients is important not only for diagnosis but also for risk stratification so that individualized treatment can be instituted to improve survival and for predicting outcome.

Acknowledgment We are grateful for the technical support provided by Parvez Iqbal.

Ethics Ethics Committee Approval: Research Ethics and Academics Department, Armed Forces Institute of Pathology, Rawalpindi, Pakistan.

Informed Consent: Informed written consent was received from the patients.

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Surgical and Medical Practices: R.M., C.A.; Concept: R.M.; Design: R.M., H.S.M.; Data Collection or Processing: R.M.; Analysis or Interpretation: R.M.; Literature Search: R.M., C.A. P.A., S.A.K.; Writing: R.M., C.A.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2018.0022 Turk J Hematol 2018;35:116-121

Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated Peptide in Plasma Cell Dyscrasias Plazma Hücreli Diskraziye Myeloid Kökenli Baskılayıcı Hücreler ve Latent Asosiye Peptit Ekprese Eden Monositlerin Hiyerarşik Katılımı Tamar Tadmor1,2

Ilana Levy3,

Zahava Vadasz2,4

Bnai-Zion Medical Center, Clinic of Hematology, Haifa, Israel The Ruth and Bruce Rappaport Faculty of Medicine, Clinic of Hematology, Haifa, Israel 3 Bnai-Zion Medical Center, Clinic of Internal Medicine B, Haifa, Israel 4 Bnai-Zion Medical Center, Clinic of Allergy and Clinical Immunology, Haifa, Israel 1 2

Abstract

Öz

Objective: Plasma cell dyscrasias (PCDs) are disorders of plasma cells having in common the production of a monoclonal M-protein.

They include a spectrum of conditions that may represent a natural progression of the same disease from monoclonal gammopathy of unknown significance to asymptomatic and symptomatic multiple myeloma, plasma cell leukemia, and Waldenström’s macroglobulinemia. In PCDs, the immune system is actively suppressed through the secretion of suppressive factors and the recruitment of immune suppressive subpopulations. In this study, we examined the expression of two subpopulations of cells with immunosuppressive activity, monocytic myeloid-derived suppressor cells (MDSCs) and monocytes expressing latency-associated peptide (LAP), in patients with different PCDs and in healthy volunteers.

Amaç: Plazma hücreli diskrazi (PHD), monoklonal M-proteinin üretimine sahip olan plazma hücrelerinin bozukluklarıdır. Aynı hastalığın, önemi bilinmeyen monoklonal gammopatiden, asemptomatik ve semptomatik multipl myeloma, plazma hücreli lösemi ve Waldenström makroglobulinemiye doğru doğal ilerlemesini temsil edebilen bir dizi spektrum içerir. PHD’lerde, baskılayıcı faktörlerin salgılanması ve bağışıklık baskılayıcı alt popülasyonların katılımı ile bağışıklık sistemi aktif olarak baskılanır. Bu çalışmada, PHD’lerin ve sağlıklı gönüllülerin, immün baskılayıcı aktiviteye sahip iki alt popülasyonundaki; monositik myeloid kökenli baskılayıcı hücreler (MKBH) ve latent asosiye peptit (LAP) eksprese eden monositlerin, ekspresyonunu incelenmiştir.

Materials and Methods: A total of 27 consecutive patients with PCDs were included in this study. Nineteen healthy volunteers served as controls.

Gereç ve Yöntemler: Bu çalışmaya PHD’li toplam 27 hasta dahil edildi. On dokuz sağlıklı gönüllü, kontrol olarak kullanılmıştır.

Bulgular: Hastalık aktivitesi ile immünosüpresif aktivitesi olan monositler arasında hiyerarşik bir ilişki gözlenmiştir.

Results: We observed a hierarchical correlation between disease activity and the presence of monocytes with immunosuppressive activity.

Sonuç: Bu sonuçlar LAP anlatımı gösteren MKBH’lerin ve monositlerin, PHD’lerde farklı rollere sahip olduğunu ve tümör aktivitesi ve kitle biyobelirteçleri olarak kullanılabileceğini düşündürmektedir.

Conclusion: These results suggest that MDSCs and monocytes expressing LAP have diverging roles in PCDs and may perhaps serve as biomarkers of tumor activity and bulk.

Anahtar Sözcükler: Multiple myelom, Önemi bilinmeyen monoklonal gammopati, Myeloid kökenli baskılayıcı hücreler, Latent asosiye peptit

Keywords: Multiple myeloma, Monoclonal gammopathy of unknown significance, Myeloid-derived suppressor cells, Latency-associated peptide

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Tamar TADMOR, M.D., Bnai-Zion Medical Center, Clinic of Hematology, Haifa, Israel Phone : +972 48359407 E-mail : tamar.tadmor@b-zion.org.il ORCID-ID: orcid.org/0000-0002-3435-8612

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Received/Geliş tarihi: January 13, 2018 Accepted/Kabul tarihi: March 23, 2018

Turk J Hematol 2018;35:116-121

Tadmor T, et al: Hierarchical Involvement of MDS Cells and Monocytes Expressing LAP in PCD

Introduction Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells of granulocytic or monocytic origin, which accumulate in a number of disorders including solid tumors and hematological malignancies in particular [1,2]. MDSCs inhibit T-cell proliferation and cytokine secretion, favoring the recruitment of regulatory T cells (Tregs), and are part of the immune regulatory subpopulations of cells responsible for inhibition of the immune response, thereby facilitating tumor escape [1,2].

Latency-associated peptide (LAP) is the N-terminal propeptide of the transforming growth factor beta (TGF-β) precursor, which binds noncovalently to TGF-β, forming a latent TGF-β complex.

When released into the extracellular milieu, LAP forms small latent complexes with TGF-β1 [3,4,5]. TGF-β-LAP complexes are present on the surface of various immune cells and have been shown to play a role in immune regulation, promoting the conversion of naive to activated Tregs, which induce Tregassociated immunosuppression [3,4,5].

Bolzoni et al. [6] studied the function of CD14/CD16+ monocyte subpopulations sorted from the bone marrow of patients with monoclonal gammopathies at different stages of disease. In this report, monocytes isolated from patients with multiple myeloma (MM) showed activity that contributed to enhanced osteoclast activation.

MM is the second most common hematological malignancy in the United States and is invariably preceded by monoclonal gammopathy of unknown significance (MGUS). Myeloma cells are critically dependent on the tumor microenvironment for their survival, progression, and proliferation, and a number of recent studies have concentrated on targeted therapy of tumor niche pathways [7,8,9].

MM is also associated with immune dysfunction, and several reports have demonstrated increased numbers of MDSCs in the bone marrow microenvironment, which contributes to immunosuppression and tumor invasion [10,11,12,13,14,15,16].

Recently, we studied two immune subpopulations, monocytic MDSCs and LAP-expressing monocytes, in the peripheral blood of patients with different plasma cell dyscrasias (PCDs) and in healthy volunteers and compared their frequencies.

Materials and Methods A total of 27 consecutive patients with PCDs, classified according to the International Myeloma Working Group as published in 2009 and updated in 2014-2015 [14,15] and seen in the Hematology Unit of the Bnai Zion Medical Center in Haifa, Israel, between 2013 and 2015 were included in this study. For patients with plasma

cell leukemia, diagnosis was based on the percentage (≥20%) and absolute number (≥2x109/L) of plasma cells in the peripheral blood, while Waldenström’s macroglobulinemia (WM) was defined on the basis of the presence of immunoglobulin M monoclonal gammopathy and ≥10% bone marrow lymphoplasmacytic infiltration [17,18,19,20].

The cohort included 8 patients with MGUS, 14 with symptomatic MM, 2 with plasma cell leukemia, and 3 with WM. Nineteen healthy volunteers served as controls.

All samples were taken from treatment-naive patients, before starting any therapy.

Written informed consent was obtained from all patients and the study was approved by the hospital’s ethics committee.

Materials Mononuclear cells were enriched from whole blood using the Ficoll-Hypaque gradient (Lymphoprep, Oslo, Norway).

Fluorescence-activated cell sorting analysis was performed on these mononuclear cells using the following antibodies: antiCD45 PC-5 (PE-Cy5), anti-CD14 PE (phycoerythrin), and antiHLA-DR FITC (fluorescein) (BD Biosciences, San Jose, CA, USA).

For staining, 0.5-1x106 mononuclear cells were stained and incubated at room temperature for 30 min in the dark with the above antibodies according to the manufacturer’s instructions in 100 µL of PBS followed by red blood cell lysis (VersaLyse, Beckman Coulter, Inc., Marseille, France). In addition, MDSCs were characterized using antibodies to CD124 [interleukin (IL)-4Ra], which is the common receptor for interleukin-4 (IL4). CD14+/HLA-DRneg/low cells were also gated for expression of LAP using anti-LAP (clone 27232), obtained from R&D Systems (Minneapolis, MN, USA).

Data were acquired with a Beckman Coulter Cytomics FC 500 flow cytometer and analyzed with CXP Software, version 2.2.

(Beckman Coulter, Brea, CA, USA).

Statistical Analysis All values were expressed as mean ± standard error of the mean.

For flow-cytometry data, values between groups of data were tested for statistical significance.

The chi-square test was performed to determine whether data were normally distributed and a two-tailed t-test was then applied to the results. Significant p-values were those less than 0.05.

Results The patient cohort included 11 males (41%) and 16 females (59%); median age at diagnosis was 61 years (range: 45-86).

All patients were diagnosed and followed at the same medical center. Patients’ characteristics are presented in Table 1.

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Monocytic MDSC Expression

LAP Expression

The mean number of circulating monocytic MDSCs in the peripheral blood was defined by coexpression of positive CD14+ and dim expression of HLA-DR. The average expression was 5.9% (3.7%-8.1%) for the MGUS cohort, 12.5% (6.7%-27.2%) for MM patients, 18.4% (14.6%-22%) in plasma cell leukemia cases, 17.8% (16.5%-19%) in WM cases, and 5.5% (2.4%-7.9%) in healthy controls.

The mean number of circulating monocyte/LAP+ cells in the peripheral blood was defined by coexpression of positive CD14+ and LAP. The average expression was 6.5% (3.7%-9.1%) for the MGUS cohort, 15.1% (12.1%-44%) for MM patients, 19% (13.5%-23.2%) in plasma cell leukemia cases, 19.7% (16.9%23%) in WM cases, and 7.2% (5.9%-9.5%) in healthy controls.

No significant difference was observed between MGUS patients and healthy volunteers (p=0.8), but results were significant for other PCDs (p=0.018) (Figures 2a and 2b).

No significant difference was observed between MGUS patients and healthy volunteers (p=0.39), but the comparison with cases of PCD was significant (p=0.002) (Figure 1a). Next, we analyzed the monocyte subpopulation coexpressing CD124+, another marker of MDSCs. Results obtained using mean numbers for healthy controls and patients with MGUS, MM, plasma cell leukemia, and WM were 8.1% (6.1%-11%), 4.4% (1.6%-7.1%), 15.7% (2.5%-17.5%), 18.4% (14.5%-22.3%), and 19.7% (18.5%-20.9%), respectively (Figure 1b).

Results were statistically significant for all PCDs when compared to healthy controls (p=0.03).

Discussion Substantial advances in understanding the biology of PCD progression have been achieved through the study of the bone marrow microenvironment [8]. The bone marrow niche appears to play an important role in the differentiation, proliferation, migration, and survival of plasma cells. It is composed of a heterogeneous cellular compartment that includes stromal cells, osteoblasts, osteoclasts, endothelial cells, and immune cells [13].

Intercellular interaction appears to induce immune dysfunction,

Table 1. Patients’ demographic, clinical, and laboratory characteristics.

Characteristics

MM

WM

PCL

MGUS

Healthy controls

Sex Male Female

5 (36%) 9 (64%)

1 (33%) 2 (67%)

1 (50%) 1 (50%)

4 (50%) 4 (50%)

6 (32%) 13 (68%)

Age

67.3±13.4

74.3±6.7

75.5±4.9

67.9±15.5

48.1±18.8

Hemoglobin (g/dL)

10.8±1.7

11.4±3.6

11.4±3.6

12.1±2.7

13.2±1.4

Creatinine (mg/dL)

1.4±1.3

0.9±0.3

0.9±0.1

1.4±1.3

0.8±0.2

Calcium (mg/dL)

9.5±1.9

10.4±1.3

9.8±0.4

8.9±1.5

9.5±0.2

Albumin (g/dL)

3.7±0.7

3.9±0.9

4.1±0.5

3.8±0.8

4.3±0.4

Beta-2-microglobulin (mg/L)

8.1±7.3

2.9±1.1

2.3±0

3.2±1.6

Unknown

M spike IgG kappa g/dL IgG lambda g/dL IgA kappa g/dL IgA lambda g/dL IgM kappa g/dL IgM lambda g/dL FLC kappa Kappa/lambda ratio FLC lambda Kappa/lambda ratio

5 (36%) 2.9±3.3 2 (14%) 2.7±3.1 1 (7%) 0.1±0 4 (28%) 57.3±66.0 2 (14%) 0.007±0.009

2 (67%) 0.45±0.07 1 (33%) 1.0±0 -

1 (50%) 0.1±0 1 (50%) Unknown -

8 (100%) 1.1±0.98 -

-

MM: Multiple myeloma, WM: Waldenström’s macroglobulinemia, PCL: plasma cell leukemia, MGUS: monoclonal gammopathy of unknown significance.

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which is also an important feature of MGUS and MM and may promote progression from a premalignant state to malignancy [8,10,21,22,23].

Monocytes, macrophages, and mesenchymal stromal cells play a role in MM pathogenesis, where they support the survival and proliferation of neoplastic myeloma cells [25,26,27].

MDSCs are a heterogeneous population of immature myeloid cells at different stages of maturation; they play a role in cancer tolerance and function as an immunosuppressive cell subpopulation [2].

Several studies have analyzed the frequency and function of MDSCs in MM, indicating that they promote both myeloma growth and osteoclast activity and are involved in cross-talk with Treg cells, resulting in their expansion in the bone marrow microenvironment [28,29,30,31].

We hypothesize that the enhanced activity of a monocyte subpopulation with immunosuppressive activity may play a role in patients with PCDs. We were able to demonstrate that, in parallel to disease progression from MGUS to MM and plasma cell leukemia, the number of monocytic MDSCs appears to increase and they may express more IL-4R, which is critical for suppression of MDSC function through the L4Ra-STAT6 pathway and thereby indicative of greater immune-related activity [32].

The preliminary results that we report here are in keeping with those of a recent study that also demonstrated increased activity of CD14/CD16+ monocytes in different monoclonal gammopathies in a hierarchical pattern. Indeed, these CD14/ C16+ monocytes isolated from MM patients appear to contribute to bone disease and osteoclastogenesis via IL-21 overexpression [6].

Recently, a novel regulatory cell subset population has also been described: Tregs and immature dendritic cells that express human LAP (LAP+) [3,4,5,33,34,35]. To date, LAP+ expression on monocytes or monocytic MDSCs has not yet been studied extensively, but based on our lab’s preliminary results, showing high expression of LAP on the surface of CD14+ mononuclear cells isolated from patients with ankylosing spondylitis [35], we decided to examine this phenomenon in patients with PCDs. Here we indeed show that monocytes isolated from these patients have higher positive expression of LAP and that the frequency of its expression was correlated with disease progression.

Our results may have additional significance for biomarkers of disease activity and we are currently initiating a study analyzing these two subpopulations after therapy in symptomatic patients with PCDs.

Figure 1. Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls. a) Coexpression of CD14+/HLA-DR+dim. b) Coexpression of CD14+/CD124+, both representing the average of myeloid-derived suppressor cell (MDSC) percentage identified in the peripheral blood of each cohort. c) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by MDSCs in monoclonal gammopathy of unknown significance, multiple myeloma, and plasma cell leukemia patients.

MM: Multiple myeloma, MGUS: monoclonal gammopathy of unknown significance, MDSC: Myeloid-derived suppressor cell, LAP: latencyassociated peptide, WM: Waldenström’s macroglobulinemia.

Figure 2. Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls for the expression of latency-associated peptide (LAP) on monocytes. a) Coexpression of CD14+/ LAP+. Results represent the average percentage identified in the blood of each cohort. b) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by monocytes/ LAP+ cells in a healthy control and a multiple myeloma patient.

LAP: Latency-associated peptide.

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In addition, it has been reported that when effective therapy for PCD is given, as with immunoregulatory lenalidomide [36,37,38,39] and more recently treatment with daratumumab [40], immunosuppressive MDSCs, Tregs, and Bregs are reduced while the expression of CD4+ T-helper cells and CD8+ cytotoxic T cells is increased, supporting a numerical correlation between their frequency and disease activity.

Our study obviously has several limitations, including the limited size of the cohort, the fact that these immunosuppressive populations were isolated from peripheral blood and not bone marrow, and the lack of functional assays.

Conclusion In conclusion, we observed a hierarchical correlation between the subtypes of PCD categories and the recruitment of two subpopulations of monocytes, monocytic MDSCs and monocytes expressing LAP, with immunosuppressive activity. These results suggest that MDSCs and LAP play diverging roles in PCDs and may have potential roles as markers of tumor activity. Our results require further validation and we are now performing a subsequent study to validate them and analyze the effect of therapy on these two subpopulations.

Ethics Ethics Committee Approval: The study was approved by the hospital’s ethics committee.

Informed Consent: Written informed consent was obtained from all patients.

Authorship Contributions Medical Practices: T.T., I.L., Z.V.; Concept: T.T.; Design: T.T., Z.V.; Data Collection or Processing: Z.V., I.L.; Analysis or Interpretation: T.T., Z.V.; Literature Search: T.T.; Writing: T.T., I.L., Z.V. 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.2017.0444 Turk J Hematol 2018;35:122-128

Acute Traumatic Coagulopathy: The Value of Histone in Pediatric Trauma Patients Akut Travma İlişkili Koagülopati: Pediatrik Travma Hastalarında Histonun Yeri Emel Ulusoy1, Murat Duman1, Durgül Yılmaz1, Hale Ören3

Aykut Çağlar1,

Tuncay Küme2,

Anıl Er1,

Fatma Akgül1,

Hale Çitlenbik1,

Dokuz Eylül University Faculty of Medicine, Department of Pediatric Emergency Care, İzmir, Turkey Dokuz Eylül University Faculty of Medicine, Department of Biochemistry, İzmir, Turkey 3 Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey 1 2

Abstract

Öz

Objective: Acute traumatic coagulopathy occurs after trauma with impairment of hemostasis and activation of fibrinolysis. Some endogenous substances may play roles in this failure of the coagulation system. Extracellular histone is one such molecule that has recently attracted attention. This study investigated the association between plasma histone-complexed DNA (hcDNA) fragments and coagulation abnormalities in pediatric trauma patients.

Amaç: Akut travma ilişkili koagülopati; travma sonrası ortaya çıkan, hemostazda bozulma ve fibrinoliz aktivasyonudur. Koagülasyon sistemindeki bu bozuklukta bazı endojen moleküller rol oynamaktadır.

Histon bu moleküllerden bir tanesi olup son dönemlerde dikkat çekmeye başlamıştır. Bu çalışmada, pediatrik travma olgularında histon-kompleks DNA (hcDNA) fragmanları ile koagülasyon anormallikleri arasındaki ilişkinin incelenmesi amaçlanmıştır.

Materials and Methods: This prospective case-control study was conducted in pediatric patients with trauma. Fifty trauma patients and 30 healthy controls were enrolled. Demographic data, anatomic injury characteristics, coagulation parameters, computerized tomography findings, trauma, and International Society on Thrombosis and Haemostasis disseminated intravascular coagulation (ISTH DIC) scores were recorded. Blood samples for hcDNA were collected and assessed by enzyme-linked immunosorbent assay.

Gereç ve Yöntemler: Bu çalışma pediatrik travma olgularında yapılmış prospektif olgu-kontrol çalışmasıdır. Çalışmaya 50 hasta ve 30 kontrol olgusu dahil edildi. Tüm hastaların demografik verileri, travmanın özellikleri, koagülasyon parametreleri, bilgisayarlı tomografi sonuçları, travma skorları ve Dissemine İntravasküler Koagülasyon skoru (DİKS) kaydedildi. hcDNA düzeyi için kan örnekleri alınarak enzim ilintili immün test ile değerlendirildi.

Results: Thirty-two patients had multiple trauma, while 18 patients had isolated brain injury. hcDNA levels were significantly higher in trauma patients than healthy controls (0.474 AU and 0.145 AU, respectively). There was an association between plasma hcDNA levels and trauma severity. Thirteen patients had acute coagulopathy of trauma shock (ACoTS). ACoTS patients had higher plasma histone levels than those without ACoTS (0.703 AU and 0.398 AU, respectively).

Plasma hcDNA levels were positively correlated with the ISTH DIC score and length of stay in the intensive care unit and were negatively correlated with fibrinogen level.

Conclusion: This study indicated that hcDNA levels were increased in pediatric trauma patients and associated with the early phase of coagulopathy. Further studies are needed to clarify the role of hcDNA levels in mortality and disseminated intravascular coagulation.

Keywords: Acute traumatic coagulopathy, Children, Histone, Trauma

Bulgular: Hastaların 32’sinde çoklu travma, 18’inde izole kafa travması mevcuttu. hcDNA düzeyi travma olgularında sağlıklı kontrollere göre istatistiksel olarak anlamlı yüksek bulundu (0,474 AU and 0,145 AU, sırasıyla). Plazma hcDNA düzeyi ile travma ciddiyeti arasında anlamlı ilişki saptandı. On üç hastada akut travma ilişkili koagülopati saptanmış olup, bu hastaların akut travma ilişkili koagülopati olmayanlara göre daha yüksek plazma histon düzeyine sahip oldukları görüldü (0,703 AU and 0,398 AU, sırasıyla). Plazma hcDNA düzeyinin, DİKS ve yoğun bakımda kalış süresi ile pozitif; fibrinojen düzeyi ile negatif korelasyon gösterdiği bulundu.

Sonuç: Bu çalışmada, pediatrik travma olgularında hcDNA düzeyinin arttığı ve koagülopatinin erken fazıyla ilişkili olduğu gösterilmiştir. hcDNA’nın dissemine intravasküler koagülasyon ve mortalite oranını belirlemedeki yerini ortaya koymak için ileri çalışmalara ihtiyaç vardır.

Anahtar Sözcükler: Akut travma ilişkili koagülopati, Çocuk, Histon, Travma

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Hale ÖREN, M.D., Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey Phone : +90 232 412 60 01 E-mail : hale.oren@deu.edu.tr ORCID-ID: orcid.org/0000-0001-5760-8007

122

Received/Geliş tarihi: December 11, 2017 Accepted/Kabul tarihi: March 23, 2018

Turk J Hematol 2018;35:122-128

Introduction Trauma is the leading cause of visits to pediatric emergency departments [1]. The majority of pediatric trauma is minor, but it remains an important cause of morbidity and mortality in childhood [2]. While massive bleeding is less common in the pediatric trauma cohort, coagulation abnormalities have been described in 10% to 77% of patients [3]. In this regard, identification of coagulopathy and early intervention are important in severely injured trauma patients [4,5].

Acute traumatic coagulopathy (ATC) is an endogenous process that occurs after trauma with the impairment of hemostasis and activation of fibrinolysis [6]. Patients with ATC frequently meet the criteria for disseminated intravascular coagulation (DIC). Currently available data suggest that ATC reflects the early phase of DIC in trauma patients [7,8]. Three major factors that are associated with subsequent development of ATC are hemodilution, hypothermia, and acidosis, and its complex nature is exacerbated by shock and tissue injury [9,10].

Accumulating evidence supports an important role of different interactions between coagulation and inflammation in ATC. Damage-associated molecules such as histone-complexed DNA (hcDNA) fragments released after trauma play a significant role in the balance of the coagulation system [11,12]. There are five types of histones; all have alkaline structures. Histones form an organized pattern with DNA in the cell nucleus by neutralizing the acidic residues of the DNA [13]. Complex structures of DNA, histones, and cell-specific granular proteins, known as neutrophil extracellular traps (NETs), can be released into circulation after stimulation by inflammatory cytokines [14,15].

During NET osis, which is a pathogen-induced cell death causing NET release or tissue damage, nuclear and plasma membranes dissolve or rupture and nuclear materials are released into the circulation [12,16].

DIC can be subdivided into two different phenotypes: fibrinolytic (hemorrhagic) and antifibrinolytic (thrombotic).

The antifibrinolytic phenotype is associated with plasminogen activator inhibitor-1 and is seen in sepsis or the late phase of trauma, while the fibrinolytic phenotype leads to coagulopathy including primary and secondary fibrin(ogen)olysis in the early phase of trauma [7]. NETs, contributing factors to coagulopathy in the early stage of trauma, have various effects on the vascular endothelium, platelets, erythrocytes, and coagulation proteins [14,15]. Although NETs contain different components like neutrophil granule enzymes and bactericidal molecules, the main structure consists of DNA and histones [16]. hcDNA plays a role in coagulopathy by increasing thrombin formation, activating platelets, stimulating endothelial activation, inhibiting tissue factor pathway inhibitor, causing thrombocytopenia, decreasing fibrinogen, inhibiting anticoagulant protein C activation, and stimulating factor XII-mediated thrombin generation

Ulusoy E, et al: Acute Traumatic Coagulopathy and Histone

[15,17,18,19,20,21,22,23,24]. In addition, hcDNA complexes, having an integrated linkage between inflammation and coagulation, augment thrombin generation to a greater extent than histones alone [15].

To date, few studies have been done assessing the extracellular hcDNA fragment levels in trauma patients [12,25], while there are no studies investigating this in the pediatric trauma population. The aim of this study was to investigate the relationship of histone with coagulopathy in pediatric trauma patients and also to analyze coagulopathy frequency and its relationship with clinical findings.

Materials and Methods Study Population This is a prospective case-control study conducted among pediatric patients (1-16 years old) with multiple trauma or isolated brain injury in a pediatric emergency department between August 2014 and August 2015. Multiple trauma was defined as injury to more than 1 body system, or at least 2 serious injuries to 1 body system [26]. Fifty trauma patients were enrolled in the study. Patients with bleeding diathesis, liver disease, arrival to the trauma center >2 h after injury and/or >40 mL/kg intravenous fluid given before arrival to the hospital, and usage of any drugs including antiplatelet drugs or anticoagulants were all excluded.

Demographic data, patient characteristics, vital and anatomic injury characteristics were recorded.

signs,

The control group consisted of 30 children who were evaluated in the outpatient clinic of our hospital for routine well-child visits. None of the children had a history of drug usage, chronic systemic disease, or any major trauma in the last 6 months.

Scoring Systems Four scoring systems were used to assess patients upon admission: the Glasgow Coma scale (GCS) [27], the Pediatric Trauma score (PTS) [28], the Injury Severity score (ISS) [29], and the International Society on Thrombosis and Haemostasis (ISTH) DIC score [30]. GCS scores were classified as mild (1415), moderate (9-13), or severe (3-8) to describe the level of consciousness. The PTS score was determined with six parameters; the minimum score is -6 and the maximum score is +12. Trauma severity is inversely correlated with PTS score and a score of 8 or less indicates the need for trauma services.

The ISS consists of six body regions and produces values from 0 to 75. Major trauma is signified by an ISS score of greater than 15. If an injury is assigned an Abbreviated Injury Scale score of 6 (unsurvivable injury), the ISS score is automatically assigned as 75. According to the ISTH DIC scale, overt DIC was diagnosed if the total score was ≥5. The ISTH DIC score includes platelet 123

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count, fibrinogen level, prothrombin time (PT), and fibrin degradation products. Age-appropriate reference ranges within our trauma center were used to determine prolonged PT (11.214.4 s) and activated partial thromboplastin time (aPTT) (age 1-3 years: 30.6-39.9 s, age 4-7 years: 28.8-38.9 s, age 8-14 years: 28.1-39.1 s, age 14-18 years: 26.0-36.6 s) levels. The presence of acute coagulopathy of trauma shock (ACoTS) was defined as prolonged PT and/or aPTT according to the age-appropriate references ranges [31,32,33,34,35].

The threshold for defining anemia is hemoglobin at or below the 2.5th percentile for age, race, and sex [36]. Hypothermia is graded as mild (36-34 °C), moderate (34-32 °C), or severe (<32 °C) [37].

Biochemical Analysis Biochemical parameters of the study population were assessed on admission. Venous blood gas, routine biochemistry, complete blood count, PT, aPTT, fibrinogen, and D-dimer were evaluated based on normal laboratory reference ranges in the hospital.

Peripheral venous blood samples were collected in blood tubes with EDTA for hcDNA. Tubes were centrifuged at 1200 x g for 10 min and plasma samples were stored at -80 °C until analysis.

Plasma nucleosome levels were measured with the Cell Death Detection ELISAPLUS commercial kit based on the principle of sandwich enzyme immunoassay (Catalog No: 1774425, Roche Diagnostics, Mannheim, Germany). Results were reported as absorbance units (AU).

Ethical Approval The study protocol was designed in compliance with the Declaration of Helsinki. Informed consent was obtained from parents or legal guardians before enrollment in the study.

The study was begun after receiving the approval of the Ethics Committee of the Dokuz Eylül University Faculty of Medicine.

Turk J Hematol 2018;35:122-128

points for separating the ACoTS group from the healthy control group. Bonferroni correction was used for multiple comparisons.

Results Fifty trauma patients and 30 healthy controls were enrolled in the study. The study group and control children were comparable in terms of age and sex distribution (Table 1).

Falls were the most frequent cause of injury; the second most common was motor vehicle accidents (Table 2).

Eighteen (36%) patients had isolated brain injury while 32 (64%) patients had multiple trauma. Three patients had liver laceration, 3 patients had spleen laceration, and 1 patient had renal and spleen laceration. Twenty-one patients had anemia and none of the patients had thrombocytopenia. Although no patient had overt DIC, 13 patients had ACoTS. Ten patients had only prolonged PT, 1 patient had only prolonged aPTT, and 2 patients had both. The median level of PT was 13.0 s (12.314.2 s) and the median level of aPTT was 27.6 s (23.7-30.4 s) in trauma patients. There were significant differences between patients with ACoTS [PT: 15.6 s (14.9-16.9 s), aPTT: 31.8 s (27.640.0 s)] and those without ACoTS [PT: 12.6 s (12.1-13.4 s), aPTT: 25.9 s (22.3-29.1 s)] according to hemostasis parameters (p=0.000 and p=0.001, respectively). Nineteen patients (38%) were admitted to the intensive care unit (ICU). Emergency endotracheal intubation was performed for 15 patients. The overall mortality rate was 6%. Clinical characteristics of the patients are shown in Table 3.

When we evaluated patients according to the ISS, we determined that 21 patients had scores over 16 and only three patients had 75 points. Those three patients died.

Table 1. Demographic variables of patients and controls.

Patients (n=50)

Controls (n=30)

Statistical Analysis

Age (IQR)

6.5 (3.0-11.5)

6.5 (3.8-9.5) 0.913*

Statistical analysis was performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Power hoc analysis was performed to evaluate the sample size. Data are presented as medians with interquartile ranges (IQRs) and 25th-75th percentiles. Histograms were used to assess the normality of sample distributions. The Kruskal-Wallis test was used for analyzing plasma hcDNA levels among different groups. The Mann-Whitney U test was used for comparing two groups. The chi-square test was used for comparing group ratios. Correlations between parameters were computed through Pearson correlation analysis. All t-tests were two-tailed and group differences or correlations with p<0.05 were considered to be statistically significant. Receiver operating curve (ROC) analysis was used to detect the optimal cut-off

Males, n (%)

33 (66.0)

20 (66.7)

124

p-value

0.951**

All data presented as median (IQR); *Mann-Whitney U test was used; **chi-square test was used.

IQR: Interquartile range.

Table 2. Trauma mechanisms in the whole patient group.

Trauma mechanism, n (%)

Falls

20 (40)

Motor vehicle accidents

11 (22)

Pedestrians struck by a motor vehicle

9 (18)

Bicycle crashes

7 (14)

Others

3 (6)

Ulusoy E, et al: Acute Traumatic Coagulopathy and Histone

Turk J Hematol 2018;35:122-128

Table 3. Clinical and laboratory characteristics of the patients.

n=50 (%)

Multiple trauma Isolated brain injury

32 (64) 18 (36)

Bone fracture

26 (52)

Open wounds

25 (50)

Hypotension

4 (8)

Blood transfusion

2 (4)

Pathological findings on CT* Cranial Abdominal Thorax

31 (62) 7 (14) 28 (56)

Endotracheal intubation

15 (30)

Prolonged PT

12 (24)

Prolonged aPTT

3 (6)

Increased D-dimer

47 (94)

Decreased fibrinogen

2 (4)

Metabolic acidosis

30 (60)

Hypothermia

2 (4)

ICU stay

19 (38)

Mortality

3 (6)

*Pathological findings: Cranial CT: Facial-calvarial fracture, subarachnoid hemorrhage, epidural hemorrhage, subdural hemorrhage, intraparenchymal hemorrhage, contusion, pneumocephalus. Thorax CT: Pneumothorax, pulmonary contusion, fracture.

Abdominal CT: Spleen/renal/liver laceration, intraabdominal bleeding, intraabdominal fluid collection, fracture.

CT: Computerized tomography, PT: prothrombin time, aPTT: activated partial thromboplastin time, ICU: intensive care unit.

Plasma hcDNA levels were significantly higher in trauma patients [0.474 AU (0.184-0.841 AU)] than in healthy controls [0.145 AU (0.086-0.361 AU)] (p=0.008). ACoTS patients [0.703 AU (0.3010.897 AU)] had higher plasma histone levels than those without ACoTS [0.398 AU (0.130-0.802 AU)]. We found significant differences between hcDNA levels and groups according to the GCS, PTS, ISS, and D-dimer (Table 4), but we did not find any differences of hcDNA levels in terms of trauma type (p=0.338). ROC curve analyses of hcDNA were performed. ROC analysis revealed an optimal cut-off point at 0.186 AU for separating the ACoTS patients from the control group. The sensitivity and specificity were 76.0% and 66.4%, respectively. The area under the curve for hcDNA was 0.679 (p=0.008) (Figure 1).

Plasma hcDNA levels were significantly correlated with ISTH DIC score (r=0.433, p=0.002) and length of stay in the ICU (r=0.314, p=0.026) in the whole study group. There was a negative correlation between hcDNA levels and PTS score (r=0.464, p=0.001). When we investigated coagulation parameters, we found a positive correlation between hcDNA levels and D-dimer levels (r=0.597, p≤0.001) and a negative correlation with fibrinogen (r=-0.342, p=0.015).

While most of the patients with organ laceration had higher

Table 4. Histone-complexed DNA levels of patients and controls. hcDNA levels (AU) Patients Controls Patients

GCS

PTS ISS ACoTS No ACoTS D-dimer PT

APTT

Fibrinogen

(n=50)

0.474 (0.184-0.841)

(n=30)

0.145 (0.086-0.361)

Male (n=17)

0.463 (0.094-0.853)

Female (n=33)

0.485 (0.243-0.802)

14-15 (n=27)

0.246 (0.095-0.709)

9-13 (n=11)

0.785 (0.666-0.932)

3-8 (n=12)

0.527 (0.252-0.871)

9-12 (n=21)

0.246 (0.098-0.747)

0-8 (n=29)

0.688 (0.404-0.864)

16-75 (n=26)

0.695 (0.379-0.865)

0-15 (n=24)

0.243 (0.094-0.652)

(n=13)

0.703 (0.301-0.897)

(n=37)

0.398 (0.130-0.802)

>0.5 (n=47)

0.503 (0.231-0.843)

≤0.5 (n=3)

0.094 (0.038-0.101)

>14.4 s (n=12)

0.767 (0.368-0.902)

≤14.4 s (n=38)

0.359 (0.119-0.793)

>Age-appropriate references range (n=3)

0.324 (0.094-0.324)

Normal (n=47)

0.485 (0.196-0.841)

<1.8 g/L (n=2)

0.892 (0.785-0.892)

≥1.8 g/L (n=48)

0.442 (0.160-0.838)

p-value 0.008* 0.759*

0.009**

0.007* 0.004* 0.044* 0.008* 0.013*

0.854

0.097

All data are presented as median (IQR); *Mann-Whitney U test was used; **KruskalWallis test was used. hcDNA: Histone-complexed DNA; GCS: Glasgow Coma Scale; PTS: Pediatric Trauma Score; ISS: Injury Severity Score; ACoTS: acute coagulopathy of trauma shock; PT: prothrombin time; aPTT: activated partial thromboplastin time; IQR: interquartile range.

Figure 1. Receiver operating characteristic curve analyses of histone-complexed DNA. 125

Ulusoy E, et al: Acute Traumatic Coagulopathy and Histone

hcDNA levels than the median level in the trauma group, we did not find any differences in hcDNA levels according to pathological findings in computerized tomography (CT) of the brain, thorax, or abdomen (p=0.342, p=0.229, and p=0.071, respectively).

Discussion In the present study, blood levels of hcDNA fragments and ATC were assessed in pediatric trauma patients. The findings showed that extracellular histone correlates with ATC and also trauma severity.

In this study, ACoTS was determined in 13 (26%) patients. In the literature there is a wide range, varying from 10% to 71%, for the incidence of coagulopathy on admission after severe trauma in the pediatric population [3]. Routine coagulation tests such as PT, aPTT, international normalized ratio, fibrinogen, or fibrinogen degradation product have been used to determine the presence of coagulopathy, but Mann et al. [38] showed that these tests do not indicate whole coagulation system abnormalities because of reflecting only 4% of thrombin production. Different studies have been performed to clarify mechanisms of coagulopathy after trauma. In a large retrospective study it was found that large-volume resuscitation with fluid during the management of shock causes dilution of plasma proteins and coagulation factors [39], but Brohi et al. [35] also showed that coagulopathy could occur before excessive fluid resuscitation. Acidosis and hypothermia can be easily observed in patients with especially severe trauma, which cause clotting and platelet dysfunction.

Dirkmann et al. [40] showed that if both of them existed, a synergistic effect occurred on coagulopathy and mortality was increased. These factors increase the coagulopathy risks after trauma, but the exact nature of this process is still not clear and correction of acidosis and hypothermia does not always correct the associated coagulopathy. This has led researchers to continue investigating the additional underlying mechanisms. Damageassociated molecules play a significant role in the balance of the coagulation system in critically ill children [10,14]. In the current study, we demonstrated the increase of hcDNA in the early phase of coagulopathy without existing DIC in pediatric trauma patients.

This study showed that plasma hcDNA levels were higher in the trauma group than in healthy controls. This increase occurs because of nuclear proteins being released out of the cell membrane with cells dying in critically ill patients and in cases of trauma [41]. As is well known, nuclear and plasma membranes must be damaged for the release of intranuclear substances like histone or DNA to occur after mechanical trauma [42]. In this regard, extracellular hcDNA levels must be higher as trauma becomes more serious with growing tissue damage. Kutcher et al. [25] showed that critically injured adult trauma patients with high hcDNA levels had higher 126

Turk J Hematol 2018;35:122-128

ISS and lower GCS scores. In another study of adults, Johansson et al. [12] found a correlation between the circulating hcDNA levels and ISS values. In accordance with the literature, this study shows a relationship between plasma hcDNA levels and trauma severity according to GCS, PTS, and ISS scores. According to the GCS, the highest hcDNA level was seen in the moderate GCS group.

Multiple organ injuries were mainly found in the moderate GCS group. Among these trauma patients, only 2 patients had serious organ injuries other than head trauma in the severe GCS group. The amount of tissue damage was highest in the moderate GCS group and lowest in the mild GCS group, consistent with histone levels.

The main finding of our study was that plasma hcDNA levels were significantly correlated with coagulation parameters that indicate coagulopathy in the pediatric trauma population.

After tissue injury, histone moves out of the cell membrane and increases activated protein C (aPC). In turn, aPC inhibits FV, FVIII, and PAI-1, thereby creating hypocoagulation and hyperfibrinolysis [43]. The late phase of trauma can be complicated with hypercoagulability and thromboembolic events like prothrombotic states after depletion of aPC stores as reported in septic patients [44]. In addition, extracellular histone activates platelets by TLR2 and TLR4 to cause platelet aggregation [17]. In experimental models with mice, histone injection caused coagulopathy and bleeding with prolonged PT, decreased fibrinogen, and fibrin deposition [45]. In this respect, it seems that histone plays roles in both pro- and anticoagulant processes. Two human studies examined blood histone levels and coagulopathy in adult trauma patients [12,25]. The present study demonstrated a hypocoagulopathic phase at the early stage of trauma having an association between histone levels and increased PT and aPTT and decreased fibrinogen.

Another result presented here is plasma hcDNA levels being correlated with length of stay in the ICU in the whole study group. A relationship between elevated histone levels and days of mechanical ventilation was found in trauma patients by Kutcher et al. [25]. This result is not surprising considering that patients with high histone levels had high trauma severity and coagulopathy. The effects of histone on lung tissue were also shown in human and animal models. High histone levels caused 1.8-fold higher incidence of acute lung injury [25] along with pulmonary edema, hemorrhage, and microvascular thrombosis after injection of histone in animal models [46]. Nakahara et al. [45] showed that extracellular histones caused platelet aggregation, thrombotic occlusion of pulmonary capillaries, and right-sided heart failure. We could not show a relationship between histone levels and thorax, cranial, or abdominal CT findings. Because these patients had multiple trauma, we could not isolate any organ systems from the other tissues. When these patients had higher ISS values, we attributed it to the release of histone from dying cells that could not be shown

Turk J Hematol 2018;35:122-128

Ulusoy E, et al: Acute Traumatic Coagulopathy and Histone

by imaging methods. We are unable to discuss whether a high histone level is a marker for mortality due to the small size of our patient population.

4. Rourke C, Curry N, Khan S, Taylor R, Raza I, Davenport R, Stanworth S, Brohi K. Fibrinogen levels during trauma hemorrhage, response to replacement therapy, and association with patient outcomes. J Thromb Haemost 2012;10:1342-1351.

Treatment approaches have undergone more investigation since the understanding of the important role of NETs in coagulopathy. NETs may represent an attractive target for antithrombotic therapy. In the literature, prevention of histone toxicity on platelets and protection against histone-induced thrombocytopenia by heparin were demonstrated [17,18].

Nakahara et al. [45] also demonstrated that recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism. New therapeutic approaches have caused excitement, with the better understanding of NETs including hcDNA contributing to the pathophysiology of coagulopathy.

5. Epstein DS, Mitra B, Cameron PA, Filtzgerald M, Rosenfeld JV. Normalization of coagulopathy is associated with improved outcome after isolated traumatic brain injury. J Clin Neurosci 2016;29:64-69.

Conclusion In conclusion, this study indicated that hcDNA levels increase in pediatric trauma patients associated with coagulopathy. There was an association between plasma hcDNA levels and trauma severity according to GCS, PTS, and ISS scores. There was also a significant correlation between hcDNA levels and length of stay in the ICU. Further studies are needed to clarify the role of high hcDNA levels in determining the functional significance of these changes in therapy, DIC, and prediction of mortality.

Ethics Ethics Committee Approval: The study was begun after receiving the approval of the Ethics Committee of the Dokuz Eylül University Faculty of Medicine.

Informed Consent: Informed consent was obtained from parents or legal guardians before enrollment in the study.

Authorship Contributions Concept: E.U., H.Ö.; Design: E.U., F.A., H.Ç.; Data Collection or Processing: E.U., A.Ç., A.E.; Analysis or Interpretation: E.U., M.D., D.Y., A.Ç., T.K.; Literature Search: H.Ö., M.D.; Writing: E.U., H.Ö., M.D., T.K. Conflicts 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.

6. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie RC, Pittet JF. Acute coagulopathy of trauma: hypoperfusion induces systemic anticoagulation and hyperfibrinolysis. J Trauma 2008;64:1211-1217.

7. Gando S, Otomo Y. Local hemostasis, immunothrombosis, and systemic disseminated intravascular coagulation in trauma and traumatic shock. Crit Care 2015;19:72.

8. Gando S. Acute coagulopathy of trauma shock and coagulopathy of trauma: a rebuttal. You are now going down the wrong path. J Trauma 2009;67:381-383.

9. Frith D, Goslings JC, Gaarder C, Maegele M, Cohen MJ, Allard S, Johansson PI, Stanworth S, Thiemermann C, Brohi K. Definition and drivers of acute traumatic coagulopathy: clinical and experimental investigations. J Thromb Haemostasis 2010;8:1919-1925.

10. Davenport R. Pathogenesis of acute traumatic coagulopathy. Transfusion 2013;53(Suppl 1):23-27.

11. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K, Hauser CJ. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 2010;464:104-107.

12. Johansson PI, Windelov NA, Rasmussen LS, Sørensen AM, Ostrowski SR. Blood levels of histone-complexed DNA fragments are associated with coagulopathy, inflammation and endothelial damage early after trauma. J Emerg Trauma Shock 2013;6:171-175.

13. Felsenfeld G, Groudine M. Controlling the double helix. Nature 2003;421:448-453.

14. Kim JE, Lee N, Gu JY, Yoo HJ, Kim HK. Circulating levels of DNA histone complex and dsDNA are independent prognostic factors of disseminated intravascular coagulation. Thromb Res 2015;135:1064-1069.

15. Wisher JW, Becker RC. Antithrombotic therapy: new areas to understand efficacy and bleeding. Expert Opin Ther Targets 2014;18:1427-1434.

16. Gould TJ, Lysov Z, Liaw PC. Extracellular DNA and histones: double-edged swords in immunothrombosis. J Thromb Haemost 2015;13(Suppl 1):82-91.

17. Semeraro F, Ammollo CT, Morrissey JH, Dale GL, Friese P, Esmon NL, Esmon CT. Extracellular histones promote thrombin generation through plateletdependent mechanisms: involvement of platelet TLR2 and TLR4. Blood 2011;118:1952-1961.

18. Fuchs TA, Bhandari AA, Wagner DD. Histones induce rapid and profound thrombocytopenia in mice. Blood 2011;118:3708-3714.

19. Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood 2014;123:2768-2776.

20. Ammollo CT, Semeraro F, Xu J, Esmon NL, Esmon CT. Extracellular histones increase plasma thrombin generation by impairing thrombomodulindependent protein C activation. J Thromb Haemost 2011;9:1795-1803.

21. Komissarov AA, Florova G, Idell S. Effects of extracellular DNA on plasminogen activation and fibrinolysis. J Biol Chem 2011;286:4194941962.

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24. Altincicek B, Stotzel S, Wygrecka M, Preissner KT, Vilcinskas A. Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects. J Immunol 2008;181:2705-2712.

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31. Macleod JB, Lynn M, McKenney MG, Cohn SM, Murtha M. Early coagulopathy predicts mortality in trauma. J Trauma 2003;55:39-44.

32. Johansson PI, Sørensen AM, Perner A, Welling KL, Wanscher M, Larsen CF, Ostrowski SR. Disseminated intravascular coagulation or acute coagulopathy of trauma shock early after trauma? An observational study.

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33. Johansson PI, Stensballe J, Rasmussen LS, Ostrowski SR. High circulating adrenaline levels at admission predict increased mortality after trauma. J Trauma Acute Care Surg 2012;72:428-436.

34. Curry NS, Davenport RA, Hunt BJ, Stanworth SJ. Transfusion strategies for traumatic coagulopathy. Blood Rev 2012;26:223-232.

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BRIEF REPORT DOI: 10.4274/tjh.2017.0446 Turk J Hematol 2018;35:129-133

Use of a High-Purity Factor X Concentrate in Turkish Subjects with Hereditary Factor X Deficiency: Post Hoc Cohort Subanalysis of a Phase 3 Study Kalıtsal Faktör X Eksikliği Olan Türk Hastalarda Yüksek Saflıkta Faktör X Konsantresi Kullanımı: Faz 3 Çalışmasının Post Hoc Kohort Alt Analizi Ahmet F. Öner1,

Tiraje Celkan2,

Çetin Timur3,

Miranda Norton4,

Kaan Kavaklı5

Yüzüncü Yıl University Faculty of Medicine, Department of Pediatric Hematology, Van, Turkey İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey 3 İstanbul Medeniyet University, Göztepe Training and Research Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey 4 Bio Products Laboratory Ltd., Elstree, Hertfordshire, United Kingdom 5 Ege University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey 1 2

Abstract

Öz

Hereditary factor X (FX) deficiency is a rare bleeding disorder more prevalent in countries with high rates of consanguineous marriage. In a prospective, open-label, multicenter phase 3 study, 25 IU/kg plasmaderived factor X (pdFX) was administered as on-demand treatment or short-term prophylaxis for 6 months to 2 years. In Turkish subjects (n=6), 60.7% of bleeds were minor. A mean of 1.03 infusions were used to treat each bleed, and mean total dose per bleed was 25.38 IU/kg. Turkish subjects rated pdFX efficacy as excellent or good for all 84 assessable bleeds; investigators judged overall pdFX efficacy to be excellent or good for all subjects. Turkish subjects had 51 adverse events; 96% with known severity were mild/moderate, and 1 (infusionsite pain) was possibly pdFX-related. These results demonstrate that 25 IU/kg pdFX is safe and effective in this Turkish cohort (ClinicalTrials.gov identifier: NCT00930176).

Kalıtsal faktör X (FX) eksikliği, akraba evliliklerinin yüksek oranda görüldüğü ülkelerde daha sık olan nadir bir kanama bozukluğudur.

Prospektif, açık etiketli, çok merkezli bir faz 3 çalışmada 6 ay ila 2 yıl boyunca gerektiği zaman veya kısa dönemli profilaktik olarak 25 IU/ kg plazma kaynaklı FX (pdFX) uygulanmıştır. Türk hastalarda (n=6) kanamaların %60,7’si hafiftir. Her kanamayı tedavi etmek için ortalama 1,03 infüzyon gerekmiş ve kanama başına ortalama toplam doz 25,38 IU/kg olmuştur. Türk hastalar değerlendirilebilir 84 kanamanın tümü için pdFX etkililiğini mükemmel veya iyi olarak derecelendirmiştir; araştırmacılar genel pdFX etkililiğinin tüm hastalarda mükemmel veya iyi olduğu kararına varmıştır. Türk hastalarda 51 advers olay gözlenmiştir; şiddeti bilinenlerin %96’sı hafif/orta derecededir ve 1’i (infüzyon bölgesi ağrısı) muhtemelen pdFX ile ilişkili olmuştur. Bu sonuçlar 25 IU/kg pdFX kullanımının bu Türk kohortunda güvenli ve etkili olduğunu ortaya koymaktadır (ClinicalTrials.gov tanımlayıcısı: NCT00930176).

Keywords: Clinical trial, Clotting factor concentrate, Efficacy, Factor X deficiency, Orphan drug, Safety

Introduction Hereditary factor X (FX) deficiency (FXD) is a rare, autosomal recessive coagulation disorder most prevalent in countries with high rates of consanguineous marriage [1,2,3,4,5,6,7,8]. Patients with severe FXD commonly present with bleeding into joints, muscles, or mucous membranes [1,3]. Hereditary FXD is often

Anahtar Sözcükler: Klinik çalışma, Pıhtılaşma faktörü konsantresi, Etkililik, Faktör X eksikliği, Yetim ilaç, Güvenlilik

treated with fresh-frozen plasma (FFP) or prothrombin complex concentrates (PCCs) [9,10], but single-factor concentrates, when available, are recommended for treatment of rare bleeding disorders [11].

A high-purity, high-potency, plasma-derived FX concentrate (pdFX; Bio Products Laboratory Ltd., Elstree, UK) is approved in

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Miranda NORTON, PhD., Bio Products Laboratory Ltd., Elstree, Hertfordshire, United Kingdom Phone : +44 20 8957 2661 E-mail : miranda.norton@bpl.co.uk ORCID-ID: orcid.org/0000-0003-4011-9877

Received/Geliş tarihi: December 12, 2017 Accepted/Kabul tarihi: March 15, 2018

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Turk J Hematol 2018;35:129-133

the USA and the EU for on-demand treatment and bleeding episode control in subjects aged ≥12 years with hereditary FXD [12]. pdFX efficacy and safety were demonstrated in 5 subjects with hereditary FXD undergoing surgery [13] and in 16 subjects with hereditary FXD in a phase 3 trial conducted in the USA, the UK, Spain, Germany, and Turkey [14].

Bleeds were categorized as menorrhagic, covert, or overt, and pdFX efficacy for each bleed was categorized as “excellent,” “good,” “poor,” or “unassessable” [14]. An independent data review committee evaluated each bleed for assessability and severity.

PK assessments were performed at baseline and 6 months or after ≥1 bleed had been treated with pdFX as described previously [16]. Plasma FX:C levels were measured via a onestage clotting assay, and incremental recovery and half-life were calculated.

This analysis evaluated pdFX use in the Turkish cohort (a homogeneous subgroup in terms of the F10 mutation) from the phase 3 trial [14].

Materials and Methods

Safety and tolerability assessments included adverse events (AEs), infusion-site reactions, thrombogenicity markers, and viral serology. FX inhibitor development was analyzed using activated partial thromboplastin time-based inhibitor screens and the Nijmegen-Bethesda assay.

This was a post hoc analysis of 6 Turkish subjects enrolled in a prospective, open-label, multicenter, nonrandomized phase 3 study (ClinicalTrials.gov identifier, NCT00930176; EudraCT identifier, 2009 0111145-18) [14] with independent ethics committee approval for each study center, conducted in accordance with good clinical practice guidelines [15]. All subjects provided written informed consent.

Results The Turkish cohort (Table 1) had a history of severe bleeds treated using FFP or PCCs; one subject (17%) and 3 subjects (50%, including the only subject with moderate FXD) had received >150 days of exposure to FFP and PCCs, respectively.

All 6 subjects had the same homozygous missense mutation in the F10 gene (p.Gly262Asp), including 3 who were known relatives.

As reported previously [14], enrolled subjects were aged ≥12 years with moderate or severe hereditary FXD (FX activity [FX:C] <5 IU/dL) with ≥1 spontaneous/menorrhagic bleed in the previous 12 months treated with FFP, PCCs, or a factor IX/X concentrate. Subjects received on-demand pdFX at 25 IU/kg for 6 months to 2 years until ≥1 bleed had been treated; pdFX was also used as short-term preventative therapy and presurgical prophylaxis [13].

Hemostatic Efficacy Of 92 pdFX-treated bleeds (range, 12-19; Figure 1), 84 were eligible for primary efficacy analysis (Table 2). The median number of bleeds was 1.05 per subject per month overall (range, 0.8-1.2), and 1.1 bleeds per month for the subject with moderate FXD. The majority of bleeds (60.7%) were minor. Major bleeds (39.3% of all episodes) included spontaneous bleeding, injury, and menorrhagia.

Assessments pdFX efficacy, pharmacokinetics (PK), and safety were assessed for the Turkish cohort as previously described for the overall cohort [14,16]; optional F10 genotyping was also performed [17].

Subjects evaluated treatment efficacy for each bleed, and investigators evaluated treatment efficacy for each subject.

Table 1. Subjects’ demographics and clinical characteristics (Turkish cohort; n=6).

Subject number

Age

Sex

Bleeding history†

Basal FX:C (IU/dL)* Joint

Muscle

Menorrhagia

Other‡

Severe FX deficiency (plasma FX:C <1 IU/dL) 1

20

M

<1

N

Y

NA

Y

2

19

F

<1

N

N

Y

Y

3

14

F

<1

Y

Y

Y§

Y

4

17

F

<1

N

N

Y

Y

5

17

F

<1

N

N

Y

N

Y

N

NA

Y

Moderate FX deficiency (plasma FX:C ≥1 but <5 IU/dL) 6

12

M

1

*Lowest level recorded in subject’s lifetime (including during the study), †Includes all bleeds within the year prior to study entry and all significant bleeds in the subject’s lifetime, ‡Includes gastrointestinal, mucosal (not menorrhagia), pelvic, and unknown, §This subject was documented as having a history of heavy menstrual bleeding; this had previously been reported as “no” due to lack of specific bleed details within the past year or in the subject’s lifetime.

FX: Factor X, FX:C: factor X activity, N: no; Y: yes, NA: not applicable.

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Turk J Hematol 2018;35:129-133

Table 2. Characteristics of assessable* bleeding episodes (n=84) treated with plasma-derived FX and analyzed (Turkish cohort).

Number (%) of bleeds All subjects

Subject 1

Subject 2

Subject 3

Subject 4

Subject 5

Subject 6

84

18

14

11

16

11

14

Menorrhagic

48 (57.1)

0

13

11

13

11

0

Covert

26 (31.0)

9

0

0

3

0

14

Overt

10 (11.9)

9

1

0

0

0

0

Mucosal

58 (69.0)

9

14

11

13

11

0

Joint

13 (15.5)

4

0

0

2

0

7

Muscle

11 (13.1)

5

0

0

1

0

5

Kidney

2 (2.4)

0

0

0

0

0

2

Menorrhagia

48 (57.1)

0

13

11

13

11

0

Spontaneous

21 (25.0)

10

1

0

2

0

8

Injury

15 (17.9)

8

0

0

1

0

6

Major

33 (39.3)

6

0

5

7

1

14

Minor

51 (60.7)

12

14

6

9

10

0

Total bleeds Bleed type

Bleed location

Bleed cause

Bleed severity*

*As assessed by the data review committee.

Subject-rated efficacy was “excellent” or “good” for each of the 84 pdFX-treated assessable bleeds. Investigators rated pdFX efficacy (on-demand, preventative, or surgical) as “excellent” in 4 subjects (67%) and “good” for 2 subjects (33%).

FX:C PK parameters following single intravenous pdFX doses did not differ significantly between baseline and repeat PK assessment visits. Mean pdFX incremental recovery was slightly lower in the Turkish cohort than the overall cohort (1.77 vs. 2.00 IU/dL per IU/kg, respectively), while the mean terminal half-life was similar (29.7 vs. 29.4 h, respectively).

Figure 1. Summary of bleeding episodes treated with plasmaderived FX (Turkish cohort). pdFX: plasma-derived FX.

A total of 95 pdFX infusions (94 exposure days) were administered (mean total dose, 22,596 IU or 389 IU/kg) to treat a bleed (n=94) or for short-term preventative use (n=1) (Table 3). A mean of 1.03 infusions were used to treat each bleed, and mean total dose per bleed was 25.38 IU/kg. All 6 Turkish subjects completed the study and then received on-demand pdFX compassionate use for 1 year. During this time, 1 subject experienced a subdural hematoma successfully treated with pdFX, followed by weekly pdFX prophylaxis (2000 IU; ~30.8 IU/kg).

Safety and Tolerability Of 51 AEs reported by the Turkish subjects, 44 of 46 (96%) with known severity were mild or moderate. The most frequently reported AE was upper respiratory tract infection (9 events in 4 subjects, none of which were considered by the investigators to be related to pdFX). Of the 51 AEs, 1 event in 1 subject (mild infusion-site pain) was considered possibly pdFX-related; no AEs were considered probably or very likely pdFX-related, and no AEs resulted in death.

There were no inhibitors to FX, viral seroconversions, or hypersensitivity reactions to pdFX. No evidence of thrombotic events or clinical signs of thrombogenicity were observed.

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Turk J Hematol 2018;35:129-133

Table 3. Summary of plasma-derived FX infusions (Turkish cohort).

Infusions (n)

Total dose (IU)

Total dose (IU/kg)

Mean

15.8

22,596

388.94

Median (range)

16.0 (12-19)

25,457 (12,312-31,308)

401.97 (284.6-484.9)

Mean

0.95

NC

23.27

Median (range)

0.98 (0.7-1.1)

NC (NC)

22.59 (18.7-30.4)

Treatments of bleeds

Mean

15.7

22,444

386.45

Median (range)

16 (12-19)

25,001 (12,312-31,308)

394.5 (284.6-484.9)

Mean

0.94

NC

23.12

Median (range)

0.95 (0.7-1.1)

NC (NC)

22.15 (18.7-30.4)

Mean

1.0

912

14.95

Use per month

Mean

0.06

NC

0.87

Total use

Use per month

Use per month

Preventative use*

*Data refer to a single infusion; therefore, medians and ranges are not presented. The single preventative dose was given following an injury to the subject’s leg, prior to the appearance of swelling.

NC: Not calculated.

During the year of compassionate use, no product-related AEs were reported. One pdFX infusion was given to treat bleeding due to a urinary tract infection during pregnancy, with no adverse effect on the baby.

Discussion This post hoc analysis demonstrated the efficacy, PK, and safety of pdFX in Turkish subjects with moderate or severe hereditary FXD. One subject with moderate FXD (FX:C 1 IU/dL) nonetheless had severe bleeding diathesis based on his bleeding and treatment history.

The Turkish cohort required fewer infusions to treat each bleed than the overall study cohort [14] (mean, 1.03 vs. 1.21 doses) and consequently a lower total dose per bleed (mean, 25.38 vs.

31.00 IU/kg). The percentage of minor bleeds was higher in the Turkish cohort than in the overall study population (60.7% vs.

47.1%), and preventative use was much lower (mean, 0.06 vs. 1.64 infusions per month). The slightly lower mean pdFX incremental recovery among Turkish subjects versus the overall study population [16] may derive from the small sample size. Across 94 exposure days, only 1 AE in 1 subject was considered by the investigators to be possibly treatment-related.

All Turkish subjects had a homozygous F10 mutation (p.Gly262Asp) resulting in an identical amino acid substitution. A recent study of 12 Turkish patients with severe FXD identified p.Gly262Asp in 11 of 12 patients (92%), this mutation being associated with severe bleeding symptoms, suggesting the 132

potential value of mutational screening analysis in Turkey and certain areas of Iran [18]. Other regional studies have also suggested a correlation between genotype and clinical manifestations of hereditary FXD [9,19]; additional studies are needed, however, to confirm these findings.

Conclusion In conclusion, pdFX is the first highly purified FX concentrate developed for patients with hereditary FXD. The treatment success rate observed in Turkish subjects (100%) was comparable with that in the overall study population (98.4%) [14]. As hereditary FXD is a rare disorder, this post hoc analysis is limited by a small sample size. Nevertheless, these results demonstrate that 25 IU/kg pdFX was safe and effective in Turkish patients with moderate or severe hereditary FXD for on-demand treatment of bleeding episodes.

Acknowledgments Fiona Fernando, PhD, and Alexandra W. Davis (Ashfield Healthcare Communications, Middletown, CT, USA) drafted and revised the manuscript based on input from authors, and Dena McWain (Ashfield Healthcare Communications) copyedited and styled the manuscript per journal requirements. The authors would like to thank the data review committee (Drs. Jørgen Ingerslev [Aarhus University Hospital, Shejby, Denmark], Carol Kasper [University of Southern California School of Medicine, Los Angeles, CA, USA], and John Hanley [Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK]) for their role in the study.

Turk J Hematol 2018;35:129-133

Öner AF, et al: Use of pdFX in Turkish Subjects

Ethics

6. Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004;104:1243-1252.

Ethics Committee Approval: Ege University Medical Faculty Clinical Trials Ethics Committee (approval number: 10-11.1/14).

7. Fışgın T, Balkan C, Celkan T, Kılınç Y, Türker M, Timur Ç, Gürsel T, Kürekçi E, Duru F, Küpesiz A, Olcay L, Yılmaz Ş, Özgen Ü, Ünüvar A, Ören H, Kavaklı K. Rare coagulation disorders: a retrospective analysis of 156 patients in Turkey. Turk J Hematol 2012;29:48-54.

Informed Consent: All subjects provided written informed consent.

Authorship Contributions Surgical and Medical Practices: A.F.Ö., T.C., Ç.T., K.K.; Concept: M.N.; Design: M.N.; Data Collection or Processing: A.F.Ö., T.C., Ç.T., M.N., K.K.; Analysis or Interpretation: M.N., K.K.; Literature Search: M.N.; Writing: M.N. Conflict of Interest: M.N. is an employee of Bio Products Laboratory Ltd. K.K. has received investigational support from Bio Products Laboratory Ltd. Other authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Financial Disclosure: Bio Products Laboratory (Elstree, UK) provided support for this study and funding for medical writing and editorial support in the development of this manuscript. A.F.Ö.: Received educational support from Pfizer.

M.N.: Employee of Bio Products Laboratory. K.K.: Advisory board member for Bayer, Novo Nordisk, Pfizer, and Shire; received educational and investigational support from Bayer, Bio Products Laboratory, CSL Behring, Novo Nordisk, Octapharma, Pfizer, and Shire.

8. Menegatti M, Peyvandi F. Factor X deficiency. Semin Thromb Hemost 2009;35:407-415.

9. Karimi M, Vafafar A, Haghpanah S, Payandeh M, Eshghi P, Hoofar H, Afrasiabi A, Gerdabi J, Ardeshiri R, Menegatti M, Peyvandi F. Efficacy of prophylaxis and genotype-phenotype correlation in patients with severe Factor X deficiency in Iran. Haemophilia 2012;18:211-215.

10. Mumford AD, Ackroyd S, Alikhan R, Bowles L, Chowdary P, Grainger J, Mainwaring J, Mathias M, O’Connell N; BCSH Committee. Guideline for the diagnosis and management of the rare coagulation disorders: a United Kingdom Haemophilia Centre Doctors’ Organization guideline on behalf of the British Committee for Standards in Haematology. Br J Haematol 2014;167:304-326.

11. Giangrande P, Seitz R, Behr-Gross ME, Berger K, Hilger A, Klein H, Schramm W, Mannucci PM. Kreuth III: European consensus proposals for treatment of haemophilia with coagulation factor concentrates. Haemophilia 2014;20:322-325.

12. Bio Products Laboratory. Coagadex® Prescribing Information. Available online at http://www.coagadex.com/download/Coagadex_PI_10-2015.pdf.

Accessed 17 October 2017.

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14. Austin SK, Kavakli K, Norton M, Peyvandi F, Shapiro A; FX Investigators Group. Efficacy, safety, and pharmacokinetics of a new high-purity factor X concentrate in subjects with hereditary factor X deficiency. Haemophilia 2016;22:419-425.

15. Dixon JR Jr. The International Conference on Harmonization Good Clinical Practice Guideline. Qual Assur 1998;6:65-74.

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16. Austin SK, Brindley C, Kavakli K, Norton M, Shapiro A; FX Investigators Group. Pharmacokinetics of a high-purity plasma-derived factor X concentrate in subjects with moderate or severe hereditary factor X deficiency. Haemophilia 2016;22:426-432.

2. Khair K, Kumar P, Mathias M, Efford J, Liesner R. Successful use of BPL factor X concentrate in a child with severe factor X deficiency. J Haem Pract 2014;1:8-10.

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IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2016.0388 Turk J Hematol 2018;35:134

Flaming Plasma Cell Leukemia Alevsi Plazma Hücreli Lösemi Reza Ranjbaran,

Habibollah Golafshan

Shiraz University of Medical Sciences, School of Paramedical Sciences, Diagnostic Laboratory Sciences and Technology Research Center, Shiraz, Iran

A 58-year-old man presented with anemia and splenomegaly.

Peripheral blood smear indicated rouleaux formation along with 28% mononuclear cells with reddish-purple peripheral cytoplasm suspicious for plasma cells (PCs). Flow cytometric immunophenotyping of the peripheral blood revealed a large mononuclear population positive for CD38, CD138, and CD20 and negative for CD45, CD19, and CD56. Intracytoplasmic staining of kappa and lambda light-chains demonstrated lambda restriction. The serum protein electrophoresis pattern illustrated normal density in the γ-globulin region but an increase of about threefold in the β-globulin fraction.

Regarding these findings, the patient was more likely to be diagnosed with IgA monoclonal gammopathy [1]. However, a definitive diagnosis was made by immunonephelometric evaluation of serum immunoglobulins.This assay revealed 810 mg/dL IgG, 1595 mg/dL IgA, and 22 mg/dL IgM with a free kappa/lambda ratio of 0.14.

PCs have particular morphological features including ovalshaped structure and eccentric nuclei. IgA-secreting PCs have cytoplasm with a pinkish tinge associated with the presence of abundant glycoprotein and ribosomes and are totally known as flame cells (Figure 1). PC leukemia, in particular the IgA variant, is a rare and aggressive type of PC dyscrasia [2]. A well-prepared peripheral blood smear can be very helpful in diagnosing and determining the next diagnostic approach.

Keywords: Plasma cell leukemia, Flame cell, Immunoglobin A Anahtar Sözcükler: Plazma hücre lösemi, Alevsi hücre, İmmunoglobulin A

Figure 1. Flame cells.

Informed Consent: It was received.

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. Attaelmannan M, Levinson SS. Understanding and identifying monoclonal gammopathies. Clin Chem 2000; 46:1230-1238.

2. Singh T, Premalata C, Sajeevan K, Jain A, Batra U, Saini K, Satheesh C, Babu KG, Lokanatha D. IgA plasma cell leukemia. J Lab Physicians 2009;1:19-21.

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Reza RANJBARAN, M.D., Shiraz University of Medical Sciences, Faculty of Paramedical Sciences, Diagnostic Laboratory Sciences and Technology Research Center, Shiraz, Iran Phone : +98 917 074 9518 E-mail : reza_ranjbaran2009@yahoo.com ORCID-ID: orcid.org/0000-0002-8890-0999

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Received/Geliş tarihi: September 26, 2016 Accepted/Kabul tarihi: January 24, 2017

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2017.0448 Turk J Hematol 2018;35:135-136

Improvement of Cutaneous Anaplastic Large Cell Lymphoma by Brentuximab Vedotin Monotherapy Kutanöz Anaplastik Büyük Hücreli Lenfomada Brentuksimab Vedotin Monoterapisi ile Düzelme Takashi Onaka1,

Tomoya Kitagawa1,

Chika Kawakami2,

Akihito Yonezawa1

Kokura Memorial Hospital, Clinic of Hematology, Kitakyushu, Fukuoka, Japan University of Occupational and Environmental, Department of Dermatology, Fukuoka, Japan

1 2

Brentuximab vedotin (BV) is an antibody-drug conjugate composed of a CD30-directed monoclonal antibody and monomethyl auristatin E [1]. BV monotherapy showed good response rates for cases of refractory and relapsed anaplastic large cell lymphoma (ALCL), but only a few case reportsare available for cutaneous localized ALCL (cALCL). We herein report the treatment with BV of relapsed cALCL with an excellent response.

An 82-year-old female with relapsed cALCL had generalized erythema accompanied by desquamation and could not extend her fingers enough (Figure 1), with no lymph node lesions. Due to the previous treatment with radiation, steroid ointment, and systemic chemotherapy, we chose BV monotherapy for her, dosing at 1.8 mg/kg every 21 days. After the third infusion, her generalized erythemaand her finger movement were improved (Figure 2). She did not have any severe adverse effects or infusion reaction except for hematologic toxicity (leukocytopenia). She has finished 6 courses of BV infusion and maintained remission of skin lesions. There are several reports that showed the effectiveness of BV treatment for cALCL [2,3], but the optimal treatment interval and cycles, and the necessity of maintenance therapy by using BV, are unclear. Further studies are needed to evaluate BV treatment in cases of cALCL.

Figure 1. Generalized erythema accompanied by desquamation before treatment with brentuximab vedotin.

Figure 2. Improvement of skin erythema accompanied by desquamation after 4 cycles of brentuximab vedotin. ©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Takashi ONAKA, M.D., Kokura Memorial Hospital, Clinic of Hematology, Kitakyushu, Fukuoka, Japan T: 81-093-511-2000 E-mail : takashionaka3@gmail.com ORCID-ID: orcid.org/0000-0002-3149-2584

Received/Geliş tarihi: December 14, 2017 Accepted/Kabul tarihi: January 26, 2018

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Onaka T, et al: Improvement of Cutaneous ALCL by Brentuximab Vedotin Monotherapy

Turk J Hematol 2018;35:135-136

Keywords: Brentuximab vedotin, Cutaneous ALCL

References

Anahtar Sözcükler: Brentuksimab vetodin, Kutanöz ABHL

1. Katz J, Janik JE, Younes A. Brentuximab vedotin (SGN-35). Clin Cancer Res 2011;17:6428-6436.

Informed Consent: Informed consent 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.

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2. Desai A, Telang GH, Olszewski AJ. Remission of primary cutaneous anaplastic large cell lymphoma after a brief course of brentuximab vedotin. Ann Hematol 2013;92:567-568.

3. Kaffenberger BH, Kartono Winardi F, Frederickson J, Porcu P, Wong HK. Periocular cutaneous anaplastic large cell lymphoma clearance with brentuximab vedotin. J Clin Aesthet Dermatol 2013;6:29-31.

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Glomerular and Tubular Functions in Transfusion-Dependent Thalassemia Transfüzyona Bağımlı Talasemide Glomerüler ve Tübüler Fonksiyonlar Pathum Sookaromdee1,

Viroj Wiwanitkit2

TWS Primary Care Center, Bangkok, Thailand Hainan Medical University, Department of Tropical Medicine, Haikou, Hainan, China

1 2

To the Editor, Annayev et al. [1] reported their interesting observations in the publication entitled “Glomerular and Tubular Functions in Children and Adults with Transfusion-Dependent Thalassemia” (TDT). They concluded that “subclinical renal injury may be present in TDT patients” [1]. We would like to share ideas and experiences from our setting in Southeast Asia where transfusion-dependent beta-thalassemia is very common. Renal dysfunction is not uncommon in our thalassemic patients and the degree of dysfunction varies [2]. In fact, the varying degree of renal dysfunction in thalassemia patients is well known [3,4].

Patients with different variants of thalassemia have different degrees of renal dysfunction [3,4,5]. Ong-ajyooth et al. [5] noted that “The mechanism leading to the damage is not known but it might be related to increased oxidative stress secondary to tissue deposition of iron, as indicated by the raised levels of serum and urine MDA”. Improved renal function is also observed after stem cell transplantation therapy [6].

Keywords: Glomerular, Tubular, Thalassemia Anahtar Sözcükler: Glomerüler, Tübüler, Talasemi

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. Annayev A, Karakaş Z, Karaman S, Yalçıner A, Yılmaz A, Emre S. Glomerular and tubular functions in children and adults with transfusion-dependent thalassemia. Turk J Hematol 2018;35:66-70.

2. Sumboonnanonda A, Malasit P, Tanphaichitr VS, Ong-ajyooth S, Sunthornchart S, Pattanakitsakul S, Petrarat S, Assateerawatt A, Vongjirad A. Renal tubular function in beta-thalassemia. Pediatr Nephrol 1998;12:280283.

3. Nickavar A, Qmarsi A, Ansari S, Zarei E. Kidney function in patients with different variants of beta-thalassemia. Iran J Kidney Dis 2017;11:132-137.

4. Uzun E, Balcı YI, Yüksel S, Aral YZ, Aybek H, Akdağ B. Glomerular and tubular functions in children with different forms of beta thalassemia. Ren Fail 2015;37:1414-1418.

5. Ong-ajyooth L, Malasit P, Ong-ajyooth S, Fucharoen S, Pootrakul P, Vasuvattakul S, Siritanaratkul N, Nilwarangkur S. Renal function in adult beta-thalassemia/Hb E disease. Nephron 1998;78:156-161.

6. Sumboonnanonda A, Sanpakit K, Piyaphanee N. Renal tubule function in beta-thalassemia after hematopoietic stem cell transplantation. Pediatr Nephrol 2009;24:183-187.

Address for Correspondence/Yazışma Adresi: Pathum SOOKAROMDEE, M.D., TWS Primary Care Center, Bangkok, Thailand Phone : 662 448 7892 E-mail : pathumsook@gmail.com ORCID-ID: orcid.org/0000-0002-8859-5322

Received/Geliş tarihi: March 04, 2018 Accepted/Kabul tarihi: March 08, 2018 DOI: 10.4274/tjh.2018.0083

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Reply to the Authors: To the Editor, We thank Drs. Sookaromdee and Wiwanitkit for their interest and contribution to our article. There is a growing evidence of renal dysfunction in patients with thalassemia. Although the process is multifactorial (the disease itself with regular transfusion, iron accumulation in the parenchyma and toxicity of chelators), oxidative stress seems to be the main mechanism of renal damage.

Several studies have shown the beneficial effects of antioxidants (curcumin, glutamine) in prevention of chemotherapy-induced nephrotoxicity by decreasing oxidative damage. Considering the significantly increased life expectancy of thalassemia patients with long-term complications, we think the role and effects of antioxidant treatments in routine follow-up of the thalassemia patients should be investigated in prospective studies.

Best Regards Zeynep Karakaş, Serap Karaman

Use of Plerixafor to Mobilize a Healthy Donor Infected with Influenza A İnfluenza A ile Enfekte Olan Sağlıklı Bir Vericinin Plerixafor ile Mobilizasyonu Mahmut Yeral,

Pelin Aytan,

Can Boğa

Başkent University Adana Practice and Research Center, Adult Bone Marrow Transplantation Center, Adana, Turkey

To the Editor, The combined use of plerixafor and granulocyte-colony stimulating factor (G-CSF) improves mobilization in poor mobilizers. However, there are limited data available on the use of plerixafor in healthy donors [1,2]. The effects of influenza A infection on stem cell mobilization are not known.

A 46-year-old male was selected as an HLA-matched donor for a patient diagnosed with acute myeloid leukemia (AML). Donor assessment was performed in accordance with the standard operating procedure prepared for JACIE (SOP: BMT-CU-006, Donor Assessment and Safety). The donor was given 10 mg/kg/ day G-CSF. He developed a dry persistent cough, chills, fever of 39 °C, fatigue, and flu-like symptoms on day 3 of G-CSF administration. The donor was considered to have an upper respiratory tract infection, which could not be attributed to only G-CSF administration. The family members of the donor were found to have similar symptoms. Thus, blood and urine cultures were obtained and he was started on levofloxacin in addition to paracetamol; G-CSF was continued. A respiratory tract virus panel was performed on a nasal smear using a PCRbased technique. The peripheral blood leukocyte count was 22,000/µL but CD34+ cells represented just 0.07% of all cells (11/µL) on day 5 of G-CSF administration; this was considered to reflect “poor mobilization”. Therefore, 0.24 mg/kg plerixafor 138

was administered “just in time,” in addition to G-CSF, on night 5, after the donor had been given all necessary information and informed consent had been obtained. Two hours after the 11th dose of G-CSF, the leukocyte count was 45,000/µL, of which 0.33% (148/µL) were CD34+ cells. Peripheral stem cell apheresis was performed using the Donor Spectra Optia Apheresis System (Terumo BCT, Lakewood, CO, USA). A total of 15.20x108 nuclear cells/kg were collected. The product contained 3.92x106 CD34+ cells/kg, 14.91x107 CD3+ cells/kg, 17.36x107 CD19+ cells/kg, and 7.17x107 CD56+ cells/kg. G-CSF was discontinued after an adequate number of stem cells had been collected, but the fever persisted. Oseltamivir at 150 mg twice daily was then prescribed for the donor because the respiratory tract virus panel examination revealed influenza A infection. The fever became controlled 24 h after oseltamivir administration. The plerixafor procedure was considered to have permitted “sufficient mobilization” in a healthy donor who could not be mobilized with G-CSF probably because of his influenza infection.

Many factors including age, sex, body mass index, baseline leukocyte count, and comorbid conditions may compromise mobilization [3]. Although certain viral infections may cause poor mobilization, data on the influence of influenza in this context are rather limited [4]. Cytokine production or cytokine storm developing during influenza infection may be presumed to impair stem cell mobilization [5]. A combination of G-CSF and

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Turk J Hematol 2018;35:137-151

plerixafor can be used to treat mobilization failure and is usually well tolerated [6,7]. The only option upon stem cell mobilization failure with G-CSF is bone marrow harvesting. Our donor was given plerixafor “just in time”; he had an active infection and did not consent to bone marrow harvesting. While plerixafor is usually used for mobilization in lymphoma or myeloma patients, literature data are available about its use in allogeneic settings [8]. Stem cells in numbers adequate for safe transplantation were collected in a single procedure.

This report indicates that influenza A may suppress the hematopoietic system, negatively affecting stem cell mobilization. The problem may be overcome by plerixafor administration.

Keywords: Plerixafor, Influenza A, Healthy donor Anahtar Sözcükler: Plerixafor, İnfluenza A, Sağlıklı verici 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. Eyre TA, King AJ, Peniket A, Rocha V, Collins GP, Pawson R. Partial engraftment following plerixafor rescue after failed sibling donor peripheral blood stem cell harvest. Transfusion 2014;54:1231-1234.

2. Gattillo S, Marktel S, Rizzo L, Malato S, Malabarba L, Coppola M, Assanelli A, Milani R, De Freitas T, Corti C, Bellio L, Ciceri F. Plerixafor on demand in ten healthy family donors as a rescue strategy to achieve an adequate graft for stem cell transplantation. Transfusion 2015;55:1993-2000.

3. Hölig K. G-CSF in healthy allogeneic stem cell donors. Transfus Med Hemother 2013;40:225-235.

4. Rohn A, Kessler HH, Valentin T, Linkesch W, Neumeister P. Prophylactic oseltamivir treatment for prevention of donor-recipient influenza A H1N1 virus transmission does not compromise stem cell mobilization or engraftment. Bone Marrow Transplant 2011;46:312-313.

5. Teijaro JR, Walsh KB, Rice S, Rosen H, Oldstone MB. Mapping the innate signaling cascade essential for cytokine storm during influenza virus infection. Proc Natl Acad Sci U S A 2014;111:3799-3804.

6. Hauge AW, Haastrup EK, Sengeløv H, Minulescu L, Dickmeiss E, FischerNielsen A. Addition of plerixafor for CD34+ cell mobilization in six healthy stem cell donors ensured satisfactory grafts for transplantation. Transfusion 2014;54:1055-1058.

7. Flomenberg N, Devine SM, Dipersio JF, Liesveld JL, McCarty JM, Rowley SD, Vesole DH, Badel K, Calandra G. The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood 2005;106:1867-1874.

8. Namdaroglu S, Korkmaz S, Altuntas F. Management of mobilization failure in 2017. Transfus Apher Sci 2017;56:836-844.

Address for Correspondence/Yazışma Adresi: Mahmut YERAL, M.D., Başkent University Adana Practice and Research Center, Adult Bone Marrow Transplantation Center, Adana, Turkey Phone : +90 322 327 27 27-2023 E-mail : drmyeral@gmail.com ORCID-ID: orcid.org/0000-0002-9580-628X

Received/Geliş tarihi: August 14, 2017 Accepted/Kabul tarihi: January 22, 2018 DOI: 10.4274/tjh.2017.0304

Influenza A Infection and Stem Cell Mobilization İnfluenza A Enfeksiyonu ve Kök Hücre Mobilizasyonu Sora Yasri1,

Viroj Wiwanitkit2

KMT Primary Care Center, Bangkok, Thailand Hainan Medical University, Department of Tropical Medicine, Haikou, Hainan, China

1 2

To the Editor, We read the publication entitled “Use of Plerixafor to Mobilize a Healthy Donor Infected with Influenza A” and found it to be very interesting [1]. Yeral et al. [1] mentioned that “The effects of influenza A infection on stem cell mobilization are not known” and concluded that “This report indicates that influenza A may suppress the hematopoietic system, negatively affecting stem cell mobilization. The problem may be overcome by plerixafor administration” [1]. This article may provide a new observation and confirm the usefulness of plerixafor in

achieving stem cell mobilization. Nevertheless, it should be noted that this is not the first case of stem cell transplantation in which the donor has influenza A infection. Lee et al. [2] reported stem cell transplantation from a related donor infected with influenza H1N1 2009 and in that case the transplantation was completely done without noting any problem of stem cell mobilization due to the influenza virus. Regardless of using plerixafor, however, stem cell transplantation in cases in which the donor has influenza infection is a considerable challenge and it is questionable whether the procedure should be done then or not.

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Keywords: Influenza, Infection, Stem cell

References

Anahtar Sözcükler: İnfluenza, Enfeksiyon, Kök hücre

1. Yeral M, Aytan P, Boğa C. Use of plerixafor to mobilize a healthy donor infected with influenza A. Turk J Hematol 2018;35:139-140.

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

2. Lee SH, Cheuh H, Yoo KH, Kim YJ, Sung KW, Koo HH, Kim DH, Kim SJ, Kim K, Jang JH, Jung CW. Hematopoietic stem cell transplantation from a related donor infected with influenza H1N1 2009. Transpl Infect Dis 2011;13:548550.

Address for Correspondence/Yazışma Adresi: Sora YASRI, M.D., KMT Primary Care Center, Bangkok, Thailand Phone : 662 257 89 63 E-mail : sorayasri@outlook.co.th ORCID-ID: orcid.org/0000-0001-8292-6656

Received/Geliş tarihi: March 23, 2014 Accepted/Kabul tarihi: August 12, 2014 DOI: 10.4274/tjh.2018.0089

Reply to the Authors: To the Editor, We read the recent letter by Yasri and Wiwanitkit [1] regarding our manuscript with great interest. We are pleased with their contributions and comments. The literature data with regard to the effect of influenza A on hematopoietic cell mobilization is limited to only several case reports [2,3].

However it would not be incorrect to relate mobilization failure to Influenza in a donor who has no prior diseases, who is not using any kind of medication or substance, and who is considered to be healthy in clinical and laboratory evaluations before mobilization.

Mobilization failure may be associated with cytokine increase, presence of viremia and viral titers. Lee et al. [2] reported three donors who were infected with influenza. Mobilization was postponed for a short period of time in one donor due to poor mobilization risk.

Two donors could be mobilized with granulocyte-colony stimulating factor. Nevertheless one of the donors could not be regarded as good mobilized. Because despite two days of apheresis procedure, the collected CD34+ cells from the healthy donor were ≤2×10 6/kg.

With our case we aimed to point out that influenza A may affect mobilization negatively and this condition may be overcome with plerixafor. It should be known that mortality is inevitable in a patient who received myeloablative conditioning regimen without stem cells.

Best Regards Mahmut Yeral, Pelin Aytan, Can Boğa

References 1. Yasri S, Wiwanitkit V. Influenza A Infection and Stem Cell Mobilization, Turk J Hematol 2018;35:137-153.

2. Lee SH, Cheuh H, Yoo KH, Kim YJ, Sung KW, Koo HH, Kim DH, Kim SJ, Kim K, Jang JH, Jung CW. Hematopoietic stem cell transplantation from a related donor infected with influenza H1N1 2009. Transpl Infect Dis 2011;13:548-550.

3. Rohn A, Kessler HH, Valentin T, Linkesch W, Neumeister P. Prophylactic oseltamivir treatment for prevention of donor-recipient influenza A H1N1 virus transmission does not compromise stem cell mobilization or engraftment. Bone Marrow Transplant. 2011;46:312-313.

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Primary Mediastinal Large B-Cell Lymphoma As an Incidental Finding: Report of a Case Tesadüfen Tanı Konulan Primer Mediastinal Büyük B Hücreli Lenfoma Olgusu İpek Yönal-Hindilerden1,

Fehmi Hindilerden2,

Serkan Arslan3,

İbrahim Öner Doğan4,

Meliha Nalçacı1

¹İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 2 Dr. Sadi Konuk Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 3 Dr. Sadi Konuk Training and Research Hospital, Clinic of Radiology, İstanbul, Turkey 4 İstanbul University İstanbul Faculty of Medicine, Department of Pathology, İstanbul, Turkey

To the Editor, A 21-year-old female was examined for an incidentally detected left parahilar mass on chest radiograph which was taken at the time of job application (Figure 1a). Thoracic computed tomography revealed a mass of 10x9x5 cm with irregular lobulated borders in the anterior mediastinum invading the pericardium (Figure 1b). Histopathological examination of the anterior mediastinotomy material revealed large neoplastic B cells staining positive for CD20 and MUM-1, negative for CD10, and with a high Ki-67 proliferation index (80%-90%) (Figure 2). On positron-emission tomography scan, only the mediastinal mass showed increased fludeoxyglucose uptake (SUVmax: 18) (Figure 1c). Final diagnosis was stage 1A primary mediastinal large B-cell lymphoma (PMBCL). After 6 cycles of R-CHOP, PET scan showed partial anatomical and metabolic response. R-CHOP was completed to 8 cycles followed by mediastinal radiation. She has now been disease-free for 2 years.

PMBCL, accounting for 2%-4% of all non-Hodgkin lymphomas, often presents as a bulky anterior mediastinal mass and often

Figure 2. Histopathological examination of the mass. a) Diffuse neoplastic infiltration on a partially sclerotic background (hematoxylin and eosin stain, 40x). b) The clear-cell appearance of the tumor cells (hematoxylin and eosin stain, 100x). c) The appearance of round nuclei (centroblast-like) and clear cytoplasm (hematoxylin and eosin stain, 400x). d) Infiltrated cells with CD20 expression (hematoxylin and eosin stain, 400x).

Figure 1. Radiological findings of primary mediastinal B-cell lymphoma. a) Appearance of the left parahilar mass on chest plain film. b) Thorax computed tomography depicts a mass of 10x9x5 cm in the anterior mediastinum with irregular lobulated borders invading the pericardium. c) Positron-emission tomography scan shows increased fludeoxyglucose uptake in the tumor.

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invades surrounding structures such as the heart, lungs, pleura, and superior vena cava [1,2]. Patients often present with cough, dyspnea, chest pain, and superior vena cava syndrome [3].

R-CHOP plus consolidative mediastinal radiation is often an option [4]. Herein, we report a rare case of asymptomatic PMBCL with bulky mediastinal mass in which the patient achieved complete remission after R-CHOP and mediastinal radiation.

References 1. Savage KJ. Primary mediastinal large B-cell lymphoma. Oncologist 2006;11:488-495.

2. Bhatt VR, Mourya R, Shrestha R, Armitage JO. Primary mediastinal large B-cell lymphoma. Cancer Treat Rev 2015;41:476-485.

3. Abou-Elella AA, Weisenburger DD, Vose JM, Kollath JP, Lynch JC, Bast

Keywords: Mediastinal neoplasm, B-cell lymphoma, PMBCL

MA, Bierman PJ, Greiner TC, Chan WC, Armitage JO. Primary mediastinal

Anahtar Sözcükler: Mediastinal kitle, B hücreli lenfoma, PMBCL

Nebraska Lymphoma Study Group. J Clin Oncol 1999;17:784-790.

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.

large B-cell lymphoma: a clinicopathologic study of 43 patients from the

4. Giri S, Bhatt VR, Pathak R, Bociek RG, Vose JM, Armitage JO. Role of radiation therapy in primary mediastinal large B-cell lymphoma in rituximab era: a US population-based analysis. Am J Hematol 2015;90:1052-1054.

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: İpek YÖNAL-HİNDİLERDEN, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey E-mail : ipekyonal@hotmail.com ORCID-ID: orcid.org/0000-0003-3020-850X

Received/Geliş tarihi: February 08, 2016 Accepted/Kabul tarihi: March 25, 2016 DOI: 10.4274/tjh.2016.0057

A Rare Late Complication of Port Catheter Implantation: Embolization of the Catheter Nadir Görülen Bir Port Kateter Geç Komplikasyonu: Kateter Embolizasyonu Işık Odaman Al1, Cengiz Bayram1, Gizem Ersoy1, Başak Koç1, Ali Ayçiçek1, Nihal Özdemir1

Kazım Öztarhan2,

Alper Güzeltaş3,

Taner Kasar3,

Ezgi Uysalol1,

1University of Health Sciences, İstanbul Kanuni Sultan Süleyman Training and Research Hospital, Clinic of Pediatric Hematology and Oncology,

İstanbul, Turkey 2University of Health Sciences, İstanbul Kanuni Sultan Süleyman Training and Research Hospital, Clinic of Pediatric Cardiology, İstanbul, Turkey 3University of Health Sciences, İstanbul Mehmet Akif Ersoy Thoracic and Cardiovascular Surgery Training and Research Hospital, Clinic Pediatric Cardiology, İstanbul, Turkey

To the Editor, Children with cancer need long-term venous access due to the long duration of therapy. Long-term totally implantable port devices (TIPDs) are widely used in these patients for administration of chemotherapeutic agents, parenteral nutrition, fluids, and blood products [1,2]. Fracture and embolism of TIPDs are rare complications but may cause serious results and mortality, including pulmonary artery embolism, sepsis, arrhythmias, and perforation of the caval vein [3,4,5]. Herein, we present a 9-yearold male patient with pre-B acute lymphoblastic leukemia who was admitted to the outpatient pediatric hematology and oncology clinic at the 13th month of maintenance therapy due to new onset of non-flushing catheter. The patient had no other complaints.

On posterior anterior chest X-ray, the catheter was found to be disconnected from its reservoir (Figure 1). Echocardiography 142

Figure 1. Chest X-ray showing disconnection of the catheter from its reservoir.

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and thorax computed tomography angiography of the patient revealed the embolization of the catheter to the left pulmonary artery (Figure 2). The embolized catheter was removed using an interventional endovascular procedure under general anesthesia through the femoral vein by an interventional cardiologist (Figure 3). Our case report highlights a rarely encountered complication of TIPDs, which may be undiagnosed due to its rarity and lack of symptoms in some patients, leading to serious complications.

Keywords: Acute Complication Anahtar Sözcükler: Komplikasyon

lymphoblastic Akut

leukemia,

lenfoblastik

lösemi,

Catheter, Kateter,

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. Kurul S, Saip P, Aydin T. Totally implantable venous-access ports: local problems and extravasation injury. Lancet Oncol 2002;3:684-692.

2. Intagliata E, Basile F, Vecchio R. Totally implantable catheter migration and its percutaneous retrieval: case report and review of the literature. G Chir 2017;37:211-215.

3. Kassar O, Hammemi R, Ben Dhaou M, Kammoun S, Elloumi M. Spontaneous fracture and migration of a totally implanted port device to pulmonary artery in acute leukemia child. J Pediatr Hematol Oncol 2017;39:103-105.

Figure 2. Thorax computed tomography angiography of the patient showing the embolization of the catheter to the left pulmonary artery.

4. Surov A, Buerke M, John E, Kösling S, Spielmann RP, Behrmann C. Intravenous port catheter embolization: mechanisms, clinical features, and management. Angiology 2008;59:90-97.

5. Ribeiro RC, Monteiro AC, Menezes QC, Schettini ST, Vianna SM. Totally implantable catheter embolism: two related cases. Sao Paulo Med J 2008;126:347-349.

Figure 3. Removal of the catheter with an interventional endovascular procedure from pulmonary artery. ©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Cengiz BAYRAM M.D., University of Health Sciences, İstanbul Kanuni Sultan Süleyman Training and Research Hospital, Clinic of Pediatric Hematology and Oncology, İstanbul, Turkey Phone : +90 505 839 60 92 E-mail : cengizbayram2013@gmail.com ORCID-ID: orcid.org/0000-0003-2153-0628

Received/Geliş tarihi: March 30, 2017 Accepted/Kabul tarihi: April 20, 2017 DOI: 10.4274/tjh.2017.0134

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Nuclear Projections in Neutrophils for Supporting the Diagnosis of Trisomy 13 Trisomi 13 Tanısını Desteklemede Nötrofillerdeki Nükleer Çıkıntılar Şebnem Kader1,

Mehmet Mutlu1,

Filiz Aktürk Acar1,

Yakup Aslan1,

Erol Erduran2

1Karadeniz Technical University Faculty of Medicine, Division of Neonatology, Trabzon, Turkey 2Karadeniz Technical University Faculty of Medicine, Division Pediatric Hematology, Trabzon, Turkey

To the Editor, Trisomy 13 is a rare genetic disorder characterized by severe multiple congenital anomalies. Structural anomalies of neutrophils may be supportive for the diagnosis of trisomy 13.

A newborn was born by vaginal delivery after 29 weeks of pregnancy. Physical examination revealed symmetric growth restriction, low-set hypoplastic ears, aplasia cutis congenita areata on the vertex, postaxial polydactyly of the foot, bilateral microphthalmia, an umbilical cord cyst, and heart murmurs.

Echocardiography showed truncus arteriosus type I. Review of the peripheral blood smear revealed two or more small threadlike pedunculated projections attached to the surface of the nuclei in more than 60% of the neutrophils (Figure 1). The diagnosis of trisomy 13 was made by chromosomal analysis. The infant died at 2 days of life because of massive pulmonary hemorrhage.

The presence of threadlike pedunculated projections attached to the surface of the nuclei of neutrophils was described in trisomy of the D group of chromosomes (13, 14, and 15) and also in trisomy 18 [1,2]. Two or more nuclear projections detected in more than 15% of neutrophils may be highly suggestive of these trisomies [3]. We suggest that identification of characteristic structural anomalies of neutrophils on a blood smear may be used for supporting the diagnosis of these trisomies.

Figure 1. Peripheral blood smear showing threadlike pedunculated projections attached to the surface of the nuclei of neutrophils.

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. Huehns ER, Lutzner M, Hecht F. Nuclear abnormalities of the neutrophils in D1 (13-15)-trisomy syndrome. Lancet 1964;1:589-590.

Keywords: Trisomy 13, Blood smear, Neutrophilic nuclear projections

2. Kahwash BM, Nowacki NB, Kahwash SB. Aberrant (barbed-wire) nuclear projections of neutrophils in trisomy 18 (Edwards syndrome). Case Rep Hematol 2015;2015:163857.

Anahtar Sözcükler: Trisomi 13, Periferik yayma, Nötrofilik nükleer projeksiyon

3. Lakovschek IC, Streubel B, Ulm B. Natural outcome of trisomy 13, trisomy 18, and triploidy after prenatal diagnosis. Am J Med Genet A 2011;155:26262633.

©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Mehmet MUTLU, M.D., Karadeniz Technical University Faculty of Medicine, Division of Neonatology, Trabzon, Turkey Phone : +90 532 633 27 49 E-mail : drmehmetmutlu38@hotmail.com ORCID-ID: orcid.org/0000-0003-3666-3159

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Received/Geliş tarihi: June 06, 2017 Accepted/Kabul tarihi: July 26, 2017 DOI: 10.4274/tjh.2017.0227

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Turk J Hematol 2018;35:137-151

Intravascular Large B-Cell Lymphoma of the Gallbladder Safra Kesesinin İntravasküler Diffüz Büyük Hücreli Lenfoması Bülent Çetin1,

Nalan Akyürek2,

Yavuz Metin3,

Feryal Karaca4,

İrem Bilgetekin5,

Ahmet Özet6

1Recep Tayyip Erdoğan University Faculty of Medicine, Department of Internal Medicine, Division of Medical Oncology, Rize, Turkey 2Gazi University Faculty of Medicine, Department of Pathology, Ankara, Turkey 3Recep Tayyip Erdoğan University Faculty of Medicine, Department of Radiology, Rize, Turkey 4Adana Numune Training and Research Hospital, Clinic of Radiation Oncology, Adana, Turkey 5Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Clinic of Internal Medicine, Division of Medical Oncology,

Ankara, Turkey 6Gazi University Faculty of Medicine, Department of Internal Medicine, Division of Medical Oncology, Ankara, Turkey

To the Editor, Intravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal B-cell lymphoma characterized by the growth of lymphoma cells within the lumina of small vessels. Two major patterns of clinical presentation have been recognized: the first is in European countries, with brain and skin involvement, and the second in Asian countries, where patients typically present with multiorgan failure, hepatosplenomegaly, pancytopenia, and hemophagocytic syndrome [1,2,3,4,5]. Primary IVLBCL of the gallbladder is exceedingly rare.

A 60-year-old male patient was admitted to the hospital with fever, abdominal pain, and weight loss. Physical examination showed an epigastric mass of approximately 4 cm in diameter and the absence of hepatosplenomegaly and lymphadenopathy.

Laboratory tests revealed anemia (hemoglobin: 10 g/dL), with normal leukocytes and platelets. Peripheral smear showed normocytic-normochromic anemia without any abnormal cells.

Increases in liver function tests were positive laboratory findings (aspartate aminotransferase: 240 U/L, alanine aminotransferase: 240 U/L, alkaline phosphatase: 740 U/L, gamma-glutamyl transferase: 80 U/L, total bilirubin/direct bilirubin: 2.06/1.2 mg/ dL). Contrast-enhanced abdominal computerized tomography (CT) for further evaluation revealed a greater curvature-based mass of 8x5x5.5 cm in size, at the level of the distal gastric corpus, significantly narrowing the gastric lumen (Figures 1A and 1B). CT also showed hypodense areas in liver segments 5 and 8 and gallbladder stones, the largest being 1.5 cm in diameter. Dynamic liver magnetic resonance imaging (MRI) was performed to characterize the liver lesions. MRI revealed calculous cholecystitis, choledocholithiasis, and a mass lesion of 6.5x3 cm in size, thought to be based on the greater curvature at the corpus of the stomach. With no signs of distant metastasis, the patient primarily underwent both cholecystectomy and partial gastrectomy. Surgical biopsy of liver lesions revealed nonspecific inflammatory changes and no evidence of a tumor, while histologic examination confirmed a gastrointestinal

Figure 1. A, B) Axial computerized tomography image showing a greater curvature-based intraluminal gastric mass (white arrows), stones in the gallbladder (asterisk), and a vague hypodense area, which was proven to be caused by cholangitis, in segment 5 of the liver (black arrow). C) Intravascular B-cell lymphoma. The numerous dilated blood vessels were filled with large, atypical, centroblast-like lymphoid cells (hematoxylin and eosin, 400x). D) CD20-positive atypical lymphoid cells (400x). stromal tumor (GIST) of the stomach. Histological analysis of the cholecystectomy material showed cells with irregular nuclear contours and open chromatin confined to small vessels, characteristic of the IVLBCL phenotype. These cells were strongly positive for CD20 stain (Figures 1C and 1D). Since intravascular infiltrations are easily missed on hematoxylin and eosin-stained sections, bone marrow and liver biopsy slides were also stained 145

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by CD20 and no evidence of intravascular lymphoma was found. A whole-body integrated positron electron tomographyCT scan for tumor staging showed diffusely increased uptake of 18F-fludeoxyglucose in the liver (SUVmax: 7.0) and multiple lymph node lesions including the submandibular, preauricular, cervical, and jugular lymph nodes (SUVmax: 8.3). He was treated with six cycles of an R-CHOP regimen. He did not show any evidence of recurrence (normal gastroscopy and CT scan) at 36 months of follow-up.

IVLBCL usually occurs in adults in the sixth and seventh decades.

The tumor is often clinically unsuspected and can be easily overlooked on biopsy. The diagnosis is most commonly made at autopsy. The lymphoma cells are generally large with round nuclei and prominent nucleoli. The malignant cells uniformly express pan-B-cell antigens (CD20, CD79a) and variably express other antigens such as CD5 (38%) and CD10 (13%) [2]. There are no pathognomonic laboratory or radiologic abnormalities associated with IVLBCL. Abdominal CT and MRI findings of our patient with IVLBCL were nonspecific. What is the pathogenic mechanism for simultaneous presentation of gallbladder intravascular B-cell lymphoma with GIST? A unifying hypothesis supports a single underlying genetic instability that could have led to both diseases. The finding of two different neoplasms in our patient seems to be coincidental rather than related to the same pathogenic triggering. Central nervous system symptoms, skin manifestations, bone marrow involvement, and hemophagocytic syndrome are the most common clinical and laboratory abnormalities, but these were not seen in our case. Our patient presented with nonspecific symptoms and laboratory abnormalities. The ability of IVLBCL to involve any organ system further makes it very difficult to suspect this condition in a patient with a rare presentation such as ours.

Turk J Hematol 2018;35:137-151

Keywords: Intravascular large B-cell lymphoma, Gallbladder, Gastrointestinal stromal tumor Anahtar Sözcükler: İntravasküler büyük hücreli lenfoma, Safra kesesi, Gastrointestinal stromal tümör 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. Ferreri AJ, Campo E, Seymour JF, Willemze R, Ilariucci F, Ambrosetti A, Zucca E, Rossi G, López-Guillermo A, Pavlovsky MA, Geerts ML, Candoni A, Lestani M, Asioli S, Milani M, Piris MA, Pileri S, Facchetti F, Cavalli F, Ponzoni M; International Extranodal Lymphoma Study Group (IELSG). Intravascular lymphoma: clinical presentation, natural history, management and prognostic factors in a series of 38 cases, with special emphasis on the “cutaneous variant.” Br J Haematol 2004;127:173-183.

2. Ferreri AJ, Dognini GP, Campo E, Willemze R, Seymour JF, Bairey O, Martelli M, De Renz AO, Doglioni C, Montalbán C, Tedeschi A, Pavlovsky A, Morgan S, Uziel L, Ferracci M, Ascani S, Gianelli U, Patriarca C, Facchetti F, Dalla Libera A, Pertoldi B, Horváth B, Szomor A, Zucca E, Cavalli F, Ponzoni M; International Extranodal Lymphoma Study Group (IELSG). Variations in clinical presentation, frequency of hemophagocytosis and clinical behavior of intravascular lymphoma diagnosed in different geographical regions.

Haematologica 2007;92:486-492.

3. Murase T, Nakamura S. An Asian variant of intravascular lymphomatosis: an updated review of malignant histiocytosis-like B-cell lymphoma. Leuk Lymphoma 1999;33:459-473.

4. Murase T, Nakamura S, Kawauchi K, Matsuzaki H, Sakai C, Inaba T, Nasu K, Tashiro K, Suchi T, Saito H. An Asian variant of intravascular large B-cell lymphoma: clinical, pathological and cytogenetic approaches to diffuse large B-cell lymphoma associated with haemophagocytic syndrome. Br J Haematol 2000;111:826-834.

5. Shimazaki C, Inaba T, Nakagawa M. B-cell lymphoma-associated hemophagocytic syndrome. Leuk Lymphoma 2000;38:121-130.

Address for Correspondence/Yazışma Adresi: Bülent ÇETİN, M.D., Recep Tayyip Erdoğan University Faculty of Medicine, Department of Internal Medicine, Division of Medical Oncology, Rize, Turkey Phone : +90 505 884 26 94 E-mail : caretta06@hotmail.com ORCID-ID: orcid.org/0000-0001-8628-0864

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Received/Geliş tarihi: July 24 , 2017 Accepted/Kabul tarihi: January 26, 2018 DOI: 10.4274/tjh.2017.0276

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Turk J Hematol 2018;35:137-151

Successful Treatment of Chronic Lymphocytic Leukemia Multifocal Central Nervous System Involvement with Ibrutinib Kronik Lenfositik Löseminin Multifokal Santral Sinir Sistemi Tutulumunun İbrutinib ile Başarılı Tedavisi Anna Christoforidou1,

Georgios Kapsas2,

Zoe Bezirgiannidou1,

Spyros Papamichos1,

Ιoannis Kotsianidis1

1Democritus University of Thrace, Department of Hematology, Alexandroupolis, Greece 2Democritus University of Thrace, Department of Radiology, Alexandroupolis, Greece

To the Editor, Central nervous system involvement (CNSi) is rare in the course of chronic lymphocytic leukemia (CLL). The frequency ranges from 0.8% to 1% [1], and it is often underreported. Diagnosis is challenging and there is no consensus on the optimal therapy or survival. CNSi manifests as either leptomeningeal infiltration or a focal parenchymal lesion, or both [1]. We describe the case of a CLL patient who progressed with parenchymal CNS involvement and was successfully treated with ibrutinib.

A 71-year-old woman was followed without treatment at the hematology clinic for 12 years for asymptomatic CLL, Binet stage I, exhibiting slowly progressive lymphocytosis and mild hepatosplenomegaly. In March 2016 she presented with expressive aphasia, memory problems, confusion, and headache, but no B symptoms. Neurological examination confirmed the mental and speech impairment but was otherwise unremarkable.

Thoracic and abdominal computed tomography scan showed no lymphadenopathy or progression of visceromegaly. Her complete blood count was unchanged compared to the previous year with WBC lymphocytes at 14,652x109/L, Htc at 45%, and platelets at 144x109/L, with typical CLL morphology and immunophenotype (CD19 83% with CD5+/CD23+/CD20+low/CD38-/sIglow) and unmutated p53. IGH mutational analysis showed a mutated clone with IGHV3-7/IGHD1-26/IGHJ4 rearrangement. Serum chemistry was normal apart from elevated lactate dehydrogenase at 303 U/L (upper normal limit: 248 U/L). Antinuclear antibody and rheumatoid factor were negative; C-reactive protein, C3, and C4 levels were within the normal limits. Magnetic resonance imaging (MRI) showed a contrast-enhanced irregularly shaped mass of 22x17x16 mm in the left frontal lobe with intense edema and midline shift (Figure 1A). Lumbar puncture showed 5/µL nucleated cell count, 5/µL erythrocytes, 0.4 g/L protein, and no monoclonal B lymphocytes (CD5/CD19) by flow cytometry. Extensive investigations for infection with cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, herpes simplex virus, ortoxoplasma antibodies as well as PCR for Cytomegalovirus DNA were negative in both serum and cerebrospinal fluid. She was referred to a neurosurgeon but the

Figure 1. A) Initial presentation of the enhancing lesion in the left frontal lobe (thick arrow), with considerable perilesional edema. B, C, D) After one and four rituximab plus a high-dose methylprednisolone cycles there was a reduction of the enhancing lesion (thin arrow) and edema; however, new enhancing lesions appeared in the left frontal operculum and the right middle cerebellar peduncle (arrowheads). E) Brain magnetic resonance imaging 5 months after ibrutinib therapy demonstrates complete resolution of the cerebellar lesion and F) minimal enhancement in the area of the lesion in the left frontal operculum (arrow). G) Dynamic susceptibility contrast perfusion imaging. Comparison between the enhancing lesion and the normal contralateral side demonstrates an overshooting of the intensity curve of the lesion above the baseline (arrow). This phenomenon is suggestive of lymphoma [149x172 mm (72x72 DPI)]. patient was reluctant to undergo a core biopsy of the brain lesion. However, dynamic susceptibility contrast MRI perfusion imaging displayed a signal intensity curve overshooting above the baseline that was suggestive of lymphoma (Figure 1G) [2].

Considering the above findings, the patient was treated in an exploratory fashion with rituximab plus a high-dose 147

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Turk J Hematol 2018;35:137-151

methylprednisolone (RHDM) regimen (rituximab at 500 mg/m2 i.v. and methylprednisolone at 1g iv for 4 days). After 2 monthly cycles, the neurological symptoms partially regressed, but her MRI findings deteriorated with a new lesion on the left frontal lobe, although the original lesion was impressively smaller (Figures 1B and 1C). Continued RHDM resulted in a decrease of lymphocytosis to 10.9x109/L, but repeat MRI showed an atypical pattern of older lesions receding coupled with the appearance of new ones in multiple cerebral sites (Figure 1D). Since we did not have proof of whether the infiltrating neoplastic cells were identical to the original leukemic clone or a manifestation of Richter’s syndrome (RS), second-line treatment was a challenge.

The patient was switched to ibrutinibat 420 mg per day, based on the recent reports of ibrutinib’s CNS penetration and effectiveness, even in high-grade lymphomas. Three months later there was a partial improvement in the MRI findings and no new lesions. Currently on the 15th month of ibrutinib therapy, she is completely symptom-free , shows partial response of CLL and stable neuroimaging improvement, 21 months after initial CNS involvement (Figure 1E, 1F).

or another solid tumor. Strati et al. [1] reviewed 33 patients with CLL CNSi and, among them, 11 out of 12 patients with CNS RS had later developed systematic disease [1]. Our patient did not at any point develop systematic Richter’s syndrome and has an excellent clinical course during the 21 months of follow up which is suggestive of a CLL rather than RS origin of the CNSi. The treatment outcome of clinically apparent CNSi is unclear, as most studies are retrospective. The management ranges from CLL therapy alone [5] to CNS irradiation, intrathecal chemotherapy, and intensive CNS-lymphoma modalities. Intrathecal rituximab has been used in several case reports and in a small study for high-grade CNS lymphomas but never in CLL [6]. In a recent study the median overall survivalof patients with CLL or RS brain involvement was 12 and 11 months, respectively [1]. On the contrary, a cohort of 30 French patients had much better overall survivalof 65% at 5 years [7]. Ibrutinib is an oral Bruton tyrosine kinase inhibitor approved for B-CLL [8]. It is a small molecule that crosses the blood-brain barrier with promising results in CNS lymphoma, as shown in some cases of mantle cell lymphoma [9,10,11], in Waldenström macroglobulinemia patients [12,13,14], and, more importantly, in a phase I study of 20 patients with relapsed/refractory CNS lymphoma showing 75% overall response rate, including 8 complete responses, although responses were relatively short-lived [15]. Ibrutinib has a convenient outpatient oral administration scheme with minimal toxicity and is an attractive option for CNS lymphoma

Autopsy studies have found leukemic meningitis and parenchymal brain involvement in up to 20% of CLL patients, but clinical syndromes are very rarely reported [3], with the first ever case published by Solal-Celigny et al. [4]. CNSi is diagnosed by neuroimaging, cerebrospinal fluid evaluation, and core tissue biopsy that differentiate between CLL, Richter’s transformation,

Table 1. Characteristics of published cases of ibrutinib-treated chronic lymphocytic leukemia central nervous system involvement.

Patient 4 [16]

Patient 5 [7]

Patient 6 [7]

Patient 7 [17]

Patient 8 (present case)

Median Median of of 106 106 months* months*

Median of 106 months*

N/A*

N/A*

N/A*

12 years

C

B

C

A

N/A

N/A

C

A

CLL progression at CNSi diagnosis

Yes

No

Yes

No

N/A

N/A

N/A

No

CNSi presentation

Nodular enhancement of left parietal lobe with nonspecific periventricular T2 hyperintensities

Leukemic meningitis

Leukemic meningitis

Thickening of optic nerves and chiasma; FLAIR hyperintensities with nodular lesion of internal occipitotemporal region

N/A

N/A

Cervical myelopathy with expansion of the spinal cord from C2 to C7

Multifocal parenchymal masses, with biggest one at 22x1x16 mm in the left frontal lobe

Del17p

Yes

Yes

No

Yes

N/A

N/A

N/A

No

CNS response to ibrutinib

MRI normalization

CR

CR

MRI near normalization

N/A

N/A

MRI MRI near normalization normalization

Duration of response to ibrutinib (months)

9

14

8

9

N/A

N/A

18

Patient 1 [16]

Patient 2 [16]

Time since CLL diagnosis

Median of 106 months*

Binet stage at CNSi

Patient 3 [16]

*Patients 1-6 were mentioned in the French cohort study [7], but only patients 1-4 had a detailed description in a separate publication [16].

MRI: Magnetic resonance imaging, CNSi: central nervous system involvement, CLL: chronic lymphocytic leukemia.

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compared to traditional intensive chemotherapy and/or intrathecal therapy.

So far, there are seven published cases of CLL with CNSi treated with ibrutinib monotherapy (Table 1): two with nodular masses [7,16], four with leptomeningeal disease [7,16], and one with cervical myelopathy [17]. None of these patients underwent brain biopsy. All patients received the standard dose of 420 mg/day and all of them responded with sustained complete responseor partial response, with a median follow-up of 8 to 18 months. Our patient had multiple brain masses and shows an ongoing response to second line ibrutinib monotherapy for a total of 21 months as per December 2017, when the latest brain MRI was performed.

In conclusion, this case further supports the efficacy of ibrutinib in CLL with CNSi, suggesting a potential future change in the frontline management and also the outcome of this rare condition.

chemotherapy. Presse Med 1983;12:2323-2325.

5. Benjamini O, Jain P, Schlette E, Sciffman JS, Estrov Z, Keating M. Chronic lymphocytic leukemia with central nervous system involvement: a high-risk disease? Clin Lymphoma Myeloma Leuk 2013;13:338-341.

6. Rubenstein JL, Fridlyand J, Abrey L, Shen A, Karch J, Wang E, Issa S, Damon L, Prados M, McDermott M, O’Brien J, Haqq C, Shuman M. Phase I study of intraventricular administration of rituximab in patients with recurrent CNS and intraocular lymphoma. J Clin Oncol 2007;25:1350-1356.

7. Wanquet A, Birsen R, Bonnet C, Boubaya M, Choquet S, Dupuis J, Lepretre S, Re D, Fahri J, Michallet AS, Ysebaert L, Lemal R, Lamy T, Delarue R, Troussard X, Cymbalista F, Levy V, Dietrich PY, Leblond V, Aurran-Schleinitz T. Management of central nervous system involvement in chronic lymphocytic leukaemia: a retrospective cohort of 30 patients. Br J Haematol 2017;176:37-49.

8. Byrd JC, Furman RR, Coutre SE, Flinn IW, Burger JA, Blum KA, Grant B, Sharman JP, Coleman M, Wierda WG, Jones JA, Zhao W, Heerema NA, Johnson AJ, Sukbuntherng J, Chang BY, Clow F, Hedrick E, Buggy JJ, James DF, O’Brien S. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med 2013;369:32-42.

9. Tucker DL, Naylor G, Kruger A, Hamilton MS, Follows G, Rule SA. Ibrutinib is a safe and effective therapy for systemic mantle cell lymphoma with central nervous system involvement - a multi-centre case series from the United Kingdom. Br J Haematol 2017;178:327-329.

Keywords: Chronic lymphocytic leukemia, Central nervous system, CNS, Ibrutinib

10. Bernard S, Goldwirt L, Amorim S, Brice P, Briere J, de Kerviler E, Mourah S, Sauvageon H, Thieblemont C. Activity of ibrutinib in mantle cell lymphoma patients with central nervous system relapse. Blood 2015;126:1695-1698.

Anahtar Sözcükler: Kronik lenfositik lösemi, Santral sinir sistemi, SSS, İbrutinib

11. Gonzalez-Bonet LG, Garcia-Boyero R, Gaona-Morales J. Mantle cell lymphoma with central nervous system involvement simulating bilateral subdural hematomas. World Neurosurg 2017;99:808.

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. Strati P, Uhm JH, Kaufmann TJ, Nabhan C, Parikh SA, Hanson CA, Chaffee KG, Call TG, Shanafelt TD. Prevalence and characteristics of central nervous system involvement by chronic lymphocytic leukemia. Haematologica 2016;101:458-465.

12. Cabannes-Hamy A, Lemal R, Goldwirt L, Poulain S, Amorim S, Perignon 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;91:17-19.

13. 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 macroglobulinaemia (Bing-Neel syndrome): a multi-institutional retrospective study. Br J Haematol 2016;172:709-715.

14. Mason C, Savona S, Rini JN, Castillo JJ, Xu L, Hunter ZR, Treon SP, Allen SL. Ibrutinib penetrates the blood brain barrier and shows efficacy in the therapy of Bing Neel syndrome. Br J Haematol 2017;179:339-341.

2. Mangla R, Kolar B, Zhu T, Zhong J, Almast J, Ekholm S. Percentage signal recovery derived from MR dynamic susceptibility contrast imaging is useful to differentiate common enhancing malignant lesions of the brain. AJNR Am J Neuroradiol 2011;32:1004-1010.

15. Grommes C, Pastore A, Gavrilovic I, Kaley T, Nolan C, Omuro AM, Wolfe J, Pentsova E, Hatzoglou V, Mellinghoff I, DeAngelis L. Single-agent ibrutinib in recurrent/refractory central nervous system lymphoma. Blood 2016;128:783.

3. Barcos M, Lane W, Gomez GA, Han T, Freeman A, Preisler H, Henderson E. An autopsy study of 1206 acute and chronic leukemias (1958 to 1982). Cancer 1987;60:827-837.

16. Wanquet A, Birsen R, Lemal R, Hunault M, Leblond V, Aurran-Schleinitz T. Ibrutinib responsive central nervous system involvement in chronic lymphocytic leukemia. Blood 2016;127:2356-2358.

4. Solal-Celigny P, Schuller E, Courouble Y, Gislon J, Elghozi D, Boivin P. Cerebromeningeal location of chronic lymphoid leukemia. Rapid immunochemical diagnosis and complete remission by intrathecal

17. Tam CS, Kimber T, Seymour JF. Ibrutinib monotherapy as effective treatment of central nervous system involvement by chronic lymphocytic leukaemia.

Br J Haematol 2017;176:829-831.

Address for Correspondence/Yazışma Adresi: Anna CHRISTOFORIDOU, M.D., Democritus University of Thrace, Department of Hematology, Alexandroupolis, Greece Phone : +30 255 135 15 11 E-mail : annachristof@yahoo.gr ORCID-ID: orcid.org/0000-0002-3979-8318

Received/Geliş tarihi: August 20, 2017 Accepted/Kabul tarihi: January 26, 2017 DOI: 10.4274/tjh.2017.0313

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Pleomorphic Multinucleated Plasma Cells Simulating Megakaryocytes in an Anaplastic Variant of Myeloma Anaplastik Variant Myelomda Megakaryositi Taklit Eden Pleomorfik Multinükleer Plazma Hücreleri Shivangi Harankhedkar,

Ruchi Gupta,

Khaliqur Rahman

Sanjay Gandhi Post Graduate Institute of Medical Sciences, Department of Hematology, Lucknow, Uttar Pradesh, India

To the Editor, Myeloma cells are notorious for their morphological variations, which range from mature-appearing plasma cells to other poorly differentiated forms. The pleomorphic or anaplastic variants are its uncommon rare variants, which may pose a diagnostic dilemma in unprecedented cases. These anaplastic variants may mimic high-grade lymphomas, leukemia, or even metastatic carcinomas [1,2]. Anaplastic plasma cells may be seen at diagnosis or evolve during the terminal phase of the disease [3]. The correlation of this morphological variant with treatment outcome is controversial, but it is believed to be a harbinger of aggressive disease [4,5]. Herein we report the case of an unsuspected multiple myeloma, where bone marrow examination revealed the presence of bizarre plasma cells simulating megakaryocytes.

An asymptomatic 65-year-old diabetic male presented with bicytopenia. Complete blood count analysis showed hemoglobin of 7 g/dL, total leukocyte count of 6.3x109/L, and 51x109/L platelets. The peripheral smear showed the presence of occasional circulating plasma cells with minimal rouleaux formation. Bone marrow examination revealed proliferation of highly pleomorphic cells with multinucleation, simulating megakaryocytes. Cells had moderate to abundant basophilic cytoplasm, while nuclei were multilobulated, with open chromatin and prominent nucleoli, along with a few intranuclear basophilic inclusions (Figure 1A). Serum protein electrophoresis revealed monoclonal protein of 0.19 g/dL, which was confirmed to be IgA kappa on immunofixation (Figure 1B). The kappa/ lambda ratio was 427.6 and the β2 microglobulin level was 21.9 mg/L. Immunophenotypically, the cells expressed CD38, CD138, CD56, and CD200 (Figures 1C-1E). FISH analysis, performed after magnetic bead enrichment of plasma cells, showed the presence of del(13q14.3). The patient was unfortunately lost to follow-up.

Anaplastic multiple myeloma (AMM) is a rare morphological variant of multiple myeloma, the true incidence of which is largely unknown [1,2,6,7]. In the early 1990s, Allen and Coleman [3] reviewed 108 cases of anaplastic myeloma, 68 of which 150

showed the presence of extramedullary disease. Other salient characteristics of AMM, which have been observed by other authors, too, include a younger age atpresentation, cytopenias, predilection for IgA myelomas, and aggressive clinical course [4,7,8,9]. Bahmanyar et al. [10] reviewed the genetic features of 11 cases of AMM for the presence of myeloma-associated genetic abnormalities and compared them with 188 newly diagnosed non-anaplastic variants of MM. They observed significantly higher frequencies of 1q21 amplification, 17p(p53) deletion, and t(4,14). Additionally, the presence of complex karyotype, del(13q14.3), t(1;19), and near tetraploidy has also been reported [8,9,10]. The treatment outcome of this variant is considered poor as per the older literature; however, patients treated with triple-drug chemotherapeutic regimens in the modern era have shown sustained responses [1,5,9].

To conclude, awareness of these variants in myeloma is important for an accurate diagnosis. In cases where myeloma cells show extreme “de-differentiation”, a multidisciplinary

Figure 1. Panel of photomicrographs: A) May-Grünwald Giemsa stained bone marrow aspirate smear (100x) showing pleomorphic cells, with multilobation and multinuclearity, with prominent inclusions (red arrows) and abundant basophilic cytoplasm, and absence of perinuclear hof; B) serum immunofixation highlighting presence of IgA kappa monoclonal protein; C, D, E) panel of dot plots documenting these atypical plasma cells to be positive for CD38, CD138, CD200, and CD56 and negative for CD45.

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approach with the addition of immunophenotyping in the diagnostic armamentarium is recommended. With the advent of triple-drug regimens in myeloma therapy and autologous bone marrow transplantation, the outcome of this variant needs to be re-addressed inlarger studies.

Keywords: Myeloma, Anaplastic, Megakaryocytes Anahtar Sözcükler: Myeloma, Anaplastik, Megakaryosit 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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©Copyright 2018 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Ruchi GUPTA, M.D., Sanjay Gandhi Post Graduate Institute of Medical Sciences, Department of Hematology, Lucknow, Uttar Pradesh, India Phone : 800 490 4799 E-mail : ruchipgi@yahoo.co.in ORCID-ID: orcid.org/0000-0003-3427-9188

Received/Geliş tarihi: September 04, 2017 Accepted/Kabul tarihi: February 06, 2018 DOI: 10.4274/tjh.2017.0329

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