Volume: 33 Issue: 2 by LookUs Scientific

Issue 2

June 2016

80 TL

ISSN 1300-7777

Volume 33

Review Article The Role of Angiogenesis in Haemophilic Arthropathy: Where Do We Stand and Where Are We Going? Alexandra Agapidou, et al.; Thessaloniki, Greece

Research Articles Impact of JAK2V617F Mutational Status on Phenotypic Features in Essential Thrombocythemia and Primary Myelofibrosis İpek Yönal, et al.; İstanbul, Turkey

D-index: A New Scoring System in Febrile Neutropenic Patients for Predicting Invasive Fungal Infections   Gülden Yılmaz, et al.; Ankara, Turkey

Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya Türker Bilgen, et al.; Antalya, Tekirdağ, Turkey

The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin Hadil A. Al Otair, et al.; Riyadh, Saudi Arabia

Comparison of Myelodysplastic Syndrome Prognostic Scoring Systems Özlen Bektaş, et al.; Ankara, Turkey

Platelet Dysfunction in Patients with Chronic Myeloid Leukemia: Does Imatinib Mesylate Improve It? Olga Meltem Akay, et al.; Eskişehir, Turkey

The Prognostic Significance of Soluble Urokinase Plasminogen Activator Receptor in Acute Myeloid Leukemia Nergiz Erkut, et al.; Trabzon, Turkey

Investigation of Rho-Kinase Expressions and Polymorphisms in Mantle Cell Lymphoma Patients Didar Yanardağ Açık, et al.; Gaziantep, Turkey

Prospective Audit of Blood Donor Selection Process in a Tertiary Care Hospital of a Developing Country Naila Raza; Karachi, Pakistan

Cover Picture: Dilek Argon Fog Covering the Bosphorus, İstanbul

Editor-in-Chief

International Review Board

Reyhan Küçükkaya

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

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

Past Editors Erich Frank Orhan Ulutin Hamdi Akan Aytemiz Gürgey

Language Editor Leslie Demir

Senior Advisory Board Yücel Tangün Osman İlhan Muhit Özcan Teoman Soysal

Editorial Office İpek Durusu Bengü Timoçin

İstanbul Bilim University, İstanbul, Turkey

Associate Editors Ayşegül Ünüvar İstanbul University, İstanbul, Turkey

Cengiz Beyan Gülhane Military Medical Academy, Ankara, Turkey

Hale Ören Dokuz Eylül University, İzmir, Turkey

İbrahim C. Haznedaroğlu Hacettepe University, Ankara, Turkey

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

Selami Koçak Toprak Ankara University, Ankara, Turkey

Semra Paydaş Çukurova University, Adana, Turkey

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

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

Elif Ünal İnce Ankara University, Ankara, Turkey

İnci Alacacıoğlu Dokuz Eylül University, İzmir, Turkey

Müge Sayitoğlu İstanbul University, İstanbul, Turkey

Nil Güler Ondokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay Koç University, İstanbul, Turkey

Şule Ünal Hacettepe University, Ankara, Turkey

Veysel Sabri Hançer İstanbul Bilim University, İstanbul, Turkey

Zühre Kaya Gazi University, Ankara, Turkey

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Publishing Services

Statistic Editor Hülya Ellidokuz

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

Contact Information Editorial Correspondence should be addressed to Dr. Reyhan Küçükkaya E-mail : rkucukkaya@hotmail.com

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

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

ISSN: 1300-7777

Ahmet Muzaffer Demir, President

Publishing Manager Sorumlu Yazı İşleri Müdürü

Güner Hayri Özsan, General Secretary

Güner Hayri Özsan

Turkish Society of Hematology

T. Tiraje Celkan, Vice President

Management Address Yayın İdare Adresi

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

Türk Hematoloji Derneği

Meltem Yüksel, Member İlknur Kozanoğlu, Member

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

Online Manuscript Submission

Publishing House / Yayınevi

http://mc.manuscriptcentral.com/tjh

Web page

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

www.tjh.com.tr

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

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

Printing Date / Basım Tarihi 15.05.2016

Cover Picture

Ahmet Muzaffer Demir

Dilek Argon is currently working at Academic Hospital, Division of Hematology, İstanbul, Turkey.

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

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AIMS AND SCOPE The Turkish Journal of Hematology is published quarterly (March, June, September, and December) by the Turkish Society of Hematology. It is an independent, non-profit peer-reviewed international English-language periodical encompassing subjects relevant to hematology. The Editorial Board of The Turkish Journal of Hematology adheres to the principles of the World Association of Medical Editors (WAME), International Council of Medical Journal Editors (ICMJE), Committee on Publication Ethics (COPE), Consolidated Standards of Reporting Trials (CONSORT) and Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). The aim of The Turkish Journal of Hematology is to publish original hematological research of the highest scientific quality and clinical relevance. Additionally, educational material, reviews on basic developments, editorial short notes, images in hematology, and letters from hematology specialists and clinicians covering their experience and comments on hematology and related medical fields as well as social subjects are published. As of December 2015, The Turkish Journal of Hematology does not accept case reports. Important new findings or data about interesting hematological cases may be submitted as a brief report. General practitioners interested in hematology and internal medicine specialists are among our target audience, and The Turkish Journal of Hematology aims to publish according to their needs. The Turkish Journal of Hematology is indexed, as follows: - PubMed Medline - PubMed Central - Science Citation Index Expanded - EMBASE - Scopus - CINAHL - Gale/Cengage Learning - EBSCO - DOAJ - ProQuest - Index Copernicus - Tübitak/Ulakbim Turkish Medical Database - Turk Medline Impact Factor: 0.360 Subscription Information  The Turkish Journal of Hematology is sent free-of-charge to members of Turkish Society of Hematology and libraries in Turkey and abroad. Hematologists, other medical specialists that are interested in hematology, and academicians could subscribe for only 40 $ per printed issue. All published volumes are available in full text free-of-charge online at www. tjh.com.tr.  Address: İlkbahar Mah., Turan Güneş Bulvarı, 613 Sok., No: 8, Çankaya, Ankara, Turkey Telephone: +90 312 490 98 97  Fax: +90 312 490 98 68

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Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh  Web page: www.tjh.com.tr  E-mail: info@tjh.com.tr   Permissions  Requests for permission to reproduce published material should be sent to the editorial office. Editor: Professor Dr. Reyhan Diz Küçükkaya Adress: İlkbahar Mah, Turan Günes Bulvarı, 613 Sok., No: 8, Çankaya, Ankara, Turkey  Telephone: +90 312 490 98 97  Fax: +90 312 490 98 68  Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh  Web page: www.tjh.com.tr  E-mail: info@tjh.com.tr Publisher Galenos Yayınevi Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul, Turkey Telephone : +90 212 621 99 25 Fax : +90 212 621 99 27 info@galenos.com.tr Instructions for Authors Instructions for authors are published in the journal and at www.tjh.com.tr Material Disclaimer Authors are responsible for the manuscripts they publish in The Turkish Journal of Hematology. The editor, editorial board, and publisher do not accept any responsibility for published manuscripts. If you use a table or figure (or some data in a table or figure) from another source, cite the source directly in the figure or table legend. The journal is printed on acid-free paper. Editorial Policy Following receipt of each manuscript, a checklist is completed by the Editorial Assistant. The Editorial Assistant checks that each manuscript contains all required components and adheres to the author guidelines, after which time it will be forwarded to the Editor in Chief. Following the Editor in Chief’s evaluation, each manuscript is forwarded to the Associate Editor, who in turn assigns reviewers. Generally, all manuscripts will be reviewed by at least three reviewers selected by the Associate Editor, based on their relevant expertise. Associate editor could be assigned as a reviewer along with the reviewers. After the reviewing process, all manuscripts are evaluated in the Editorial Board Meeting. Turkish Journal of Hematology’s editor and Editorial Board members are active researchers. It is possible that they would desire to submit their manuscript to the Turkish Journal of Hematology. This may be creating a conflict of interest. These manuscripts will not be evaluated by the submitting editor(s). The review process will be managed and decisions made by editor-in-chief who will act independently. In some situation, this process will be overseen by an outside independent expert in reviewing submissions from editors.

TURKISH JOURNAL OF HEMATOLOGY INSTRUCTIONS TO AUTHORS The Turkish Journal of Hematology accepts invited review articles, research articles, brief reports, letters to the editor, and hematological images that are relevant to the scope of hematology, on the condition that they have not been previously published elsewhere. Basic science manuscripts, such as randomized, cohort, cross-sectional, and case control studies, are given preference. All manuscripts are subject to editorial revision to ensure they conform to the style adopted by the journal. There is a double blind kind of reviewing system. Manuscripts should be prepared according to ICMJE guidelines (http:// www.icmje.org/). Original manuscripts require a structured abstract. Label each section of the structured abstract with the appropriate subheading (Objective, Materials and Methods, Results, and Conclusion). Letters to the editor do not require an abstract. Research or project support should be acknowledged as a footnote on the title page. Technical and other assistance should be provided on the title page. Original Manuscripts Title Page Title: The title should provide important information regarding the manuscript’s content. The title must specify that the study is a cohort study, cross-sectional study, case control study, or randomized study (i.e. Cao GY, Li KX, Jin PF, Yue XY, Yang C, Hu X. Comparative bioavailability of ferrous succinate tablet formulations without correction for baseline circadian changes in iron concentration in healthy Chinese male subjects: A single-dose, randomized, 2-period crossover study. Clin Ther. 2011; 33: 2054-2059). The title page should include the authors’ names, degrees, and institutional/ professional affiliations, a short title, abbreviations, keywords, financial disclosure statement, and conflict of interest statement. If a manuscript includes authors from more than one institution, each author’s name should be followed by a superscript number that corresponds to their institution, which is listed separately. Please provide contact information for the corresponding author, including name, e-mail address, and telephone and fax numbers. Running Head: The running head should not be more than 40 characters, including spaces, and should be located at the bottom of the title page. Word Count: A word count for the manuscript, excluding abstract, acknowledgments, figure and table legends, and references, should be provided not exceed 2500 words. The word count for an abstract should be not exceed 300 words. Conflict-of-Interest Statement: To prevent potential conflicts of interest from being overlooked, this statement must be included in each manuscript. In case there are conflicts of interest, every author should complete the ICMJE general declaration form, which can be obtained at: http://www.icmje.org/coi_disclose.pdf.

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Abstract and Keywords: The second page should include an abstract that does not exceed 300 words. For manuscripts sent by authors in Turkey, a title and abstract in Turkish are also required. As most readers read the abstract first, it is critically important. Moreover, as various electronic databases integrate only abstracts into their index, important findings should be presented in the abstract. Objective: The abstract should state the objective (the purpose of the study and hypothesis) and summarize the rationale for the study. Materials and Methods: Important methods should be written respectively. Results: Important findings and results should be provided here. Conclusion: The study’s new and important findings should be highlighted and interpreted. Other types of manuscripts, such as reviews, perspectives, and editorials, will be published according to uniform requirements. Provide 3-10 keywords below the abstract to assist indexers. Use terms from the Index Medicus Medical Subject Headings List (for randomized studies a CONSORT abstract should be provided (http:// www.consort-statement.org). Introduction: The introduction should include an overview of the relevant literature presented in summary form (one page), and what ever remains interesting, unique, problematic, relevant, or unknown about the topic must be specified. The introduction should conclude with the rationale for the study, its design, and its objective(s). Materials and Methods: Clearly describe the selection of observational or experimental participants, such as patients, laboratory animals, and controls, including inclusion and exclusion criteria and a description of the source population. Identify the methods and procedures in sufficient detail to allow other researchers to reproduce your results. Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for using them and an evaluation of their limitations. Identify all drugs and chemicals used, including generic names, doses, and routes of administration. The section should include only information that was available at the time the plan or protocol for the study was devised (http://www.strobe-statement.org/ fileadmin/Strobe/uploads/checklists/STROBE_checklist_v4_combined. pdf). Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify the reported results. Statistically important data should be given in the text, tables and figures. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used.

Results: Present your results in logical sequence in the text, tables, and figures. Do not present all the data provided in the tables and/or figures in the text; emphasize and/or summarize only important findings, results, and observations in the text. For clinical studies provide the number of samples, cases, and controls included in the study. Discrepancies between the planned number and obtained number of participants should be explained. Comparisons, and statistically important values (i.e. P value and confidence interval) should be provided. Discussion: This section should include a discussion of the data. New and important findings/results, and the conclusions they lead to should be emphasized. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by the data. Do not repeat the findings/results in detail; important findings/ results should be compared with those of similar studies in the literature, along with a summarization. In other words, similarities or differences in the obtained findings/results with those previously reported should be discussed. Study Limitations: Limitations of the study should be detailed. In addition, an evaluation of the implications of the obtained findings/ results for future research should be outlined. Conclusion: The conclusion of the study should be highlighted. References Cite references in the text, tables, and figures with numbers in parentheses. Number references consecutively according to the order in which they first appear in the text. Journal titles should be abbreviated according to the style used in Index Medicus (consult List of Journals Indexed in Index Medicus). Include among the references any paper accepted, but not yet published, designating the journal and followed by, in press. Examples of References: 1. List all authors. Deeg HJ, Oâ&#x20AC;&#x2122;Donnel M, Tolar J. Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure immunosuppressive therapy. Blood 2006;108:1485-1491. 2. Organization as author Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in post-hepatitis marrow aplasia. Lancet 1977;2:742-744. 3. Book Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger, 1961. 4. Book Chapter Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin:

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Genetics, Pathophysiology, Clinical Management. New York, Cambridge University Press, 2000. 5. Abstract Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract). 6. Letter to the Editor Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further definition of 20q deletion in myeloid leukemia using fluorescence in situ hybridization. Blood 1994;84:2821-2823. 7. Supplement Alter BP. Fanconiâ&#x20AC;&#x2122;s anemia, transplantation, and cancer. Pediatr Transplant. 2005;9(Suppl 7):81-86 Brief Reports Abstract length: Not to exceed 150 words. Article length: Not to exceed 1200 words. Introduction: State the purpose and summarize the rationale for the study. Materials and Methods: Clearly describe the selection of the observational or experimental participants. Identify the methods and procedures in sufficient detail. Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for their use and an evaluation of their limitations. Identify all drugs and chemicals used, including generic names, doses, and routes of administration. Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify the reported findings/results. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used. Results: Present the findings/results in a logical sequence in the text, tables, and figures. Do not repeat all the findings/results in the tables and figures in the text; emphasize and/or summarize only those that are most important. Discussion: Highlight the new and important findings/results of the study and the conclusions they lead to. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by your data. Invited Review Articles Abstract length: Not to exceed 300 words. Article length: Not to exceed 4000 words. Review articles should not include more than 100 references. Reviews should include a conclusion, in which a new hypothesis or study about the subject may be posited. Do not publish methods for literature search or level of evidence. Authors who will prepare review articles should already

have published research articles on therel evant subject. The study’s new and important findings should be highlighted and interpreted in the Conclusion section. There should be a maximum of two authors for review articles. Images in Hematology Article length: Not exceed 200 words. Authors can submit for consideration an illustration and photos that is interesting, instructive, and visually attractive, along with a few lines of explanatory text and references. Images in Hematology can include no more than 200 words of text, 5 references, and 3 figure or table. No abstract, discussion or conclusion are required but please include a brief title. Letters to the Editor Article length: Not to exceed 500 words. Letters can include no more than 500 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title. Tables Supply each table on a separate file. Number tables according to the order in which they appear in the text, and supply a brief caption for each. Give each column a short or abbreviated heading. Write explanatory statistical measures of variation, such as standard deviation or standard error of mean. Be sure that each table is cited in the text. Figures Figures should be professionally drawn and/or photographed. Authors should number figures according to the order in which they appear in the text. Figures include graphs, charts, photographs, and illustrations. Each figure should be accompanied by a legend that does not exceed 50 words. Use abbreviations only if they have been introduced in the text. Authors are also required to provide the level of magnification for histological slides. Explain the internal scale and identify the staining method used. Figures should be submitted as separate files, not in the text file. Highresolution image files are not preferred for initial submission as the file sizes may be too large. The total file size of the PDF for peer review should not exceed 5 MB. Authorship Each author should have participated sufficiently in the work to assume public responsibility for the content. Any portion of a manuscript that is critical to its main conclusions must be the responsibility of at least 1 author. Contributor’s Statement All submissions should contain a contributor’s statement page. Each manuscript should contain substantial contributions to idea and design, acquisition of data, or analysis and interpretation of findings. All persons designated as an author should qualify for authorship, and all those that qualify should be listed. Each author should have participated sufficiently in the work to take responsibility for appropriate portions of the text.

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Acknowledgments Acknowledge support received from individuals, organizations, grants, corporations, and any other source. For work involving a biomedical product or potential product partially or wholly supported by corporate funding, a note stating, “This study was financially supported (in part) with funds provided by (company name) to (authors’ initials)”, must be included. Grant support, if received, needs to be stated and the specific granting institutions’ names and grant numbers provided when applicable. Authors are expected to disclose on the title page any commercial or other associations that might pose a conflict of interest in connection with the submitted manuscript. All funding sources that supported the work and the institutional and/or corporate affiliations of the authors should be acknowledged on the title page. Ethics When reporting experiments conducted with humans indicate that the procedures were in accordance with ethical standards set forth by the committee that oversees human experimentation. Approval of research protocols by the relevant ethics committee, in accordance with international agreements (Helsinki Declaration of 1975, revised 2002 available at http://www.wma.net/e/policy/b3.htm, “Guide for the Care and use of Laboratory Animals” www.nap.edu/catalog/5140.html/), is required for all experimental, clinical, and drug studies. Patient names, initials, and hospital identification numbers should not be used. Manuscripts reporting the results of experimental investigations conducted with humans must state that the study protocol received institutional review board approval and that the participants provided informed consent. Non-compliance with scientific accuracy is not in accord with scientific ethics. Plagiarism: To re-publish-whole or in part-the contents of another author’s publication as one’s own without providing a reference. Fabrication: To publish data and findings/results that do not exist. Duplication: Use of data from another publication, which includes re-publishing a manuscript in different languages. Salamisation: To create more than one publication by dividing the results of a study preternaturally. We disapprove of such unethical practices as plagiarism, fabrication, duplication, and salamisation, as well as efforts to influence the review process with such practices as gifting authorship, inappropriate acknowledgements, and references. Additionally, authors must respect participant right to privacy. On the other hand, short abstracts published in congress books that do not exceed 400 words and present data of preliminary research, and those that are presented in an electronic environment are not accepted prepublished work. Authors in such situation must declare this status on the first page of the manuscript and in the cover letter. (The COPE flowchart is available at: http://publicationethics.org) We use iThenticate to screen all submissions for plagiarism before publication.

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

Hematology component

SI units

Conditions of Publication

RBC

6.7-11 x 1012/L

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

WBC

5.5-19.5 x109/L

Hemoglobin

116-168 g/L

PCV

0.31-0.46 L/L

MCV

39-53 fL

MCHC

300-360 g/L

MCH

19.5-25 pg

Platelets

300-700 x 109/L

Authors of accepted manuscripts will receive electronic page proofs and are responsible for proofreading and checking the entire article within two days. Failure to return the proof in two days will delay publication. If the authors cannot be reached by email or telephone within two weeks, the manuscript will be rejected and will not be published in the journal.

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

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

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

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

Setting up an account

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.

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.

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

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

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

Units of Measurement

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

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

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

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revision recommendations will be sent for further review (usually by the same reviewers) upon their re-submission. When a manuscript is finally accepted for publication, the Technical Editor undertakes a final edit and a marked-up copy will be e-mailed to the corresponding author for review and to make any final adjustments.

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CONTENTS 88

Review Article

Research Articles

The Role of Angiogenesis in Haemophilic Arthropathy: Where Do We Stand and Where Are We Going? Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou

Impact of JAK2V617F Mutational Status on Phenotypic Features in Essential Thrombocythemia and Primary Myelofibrosis İpek Yönal, Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz, Meliha Nalçacı, Akif Selim Yavuz, Fatma Deniz Sargın

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D-index: A New Scoring System in Febrile Neutropenic Patients for Predicting Invasive Fungal Infections   Gülden Yılmaz, Belgin Coşkun, Atilla Elhan, Alpay Azap, Hamdi Akan

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Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/ Deletion (δβ)0 Mutation in Antalya Türker Bilgen, Özden Altıok Clark, Zeynep Öztürk, M. Akif Yeşilipek, İbrahim Keser

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The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Syed M. Khurshid, Abdulaziz H. Alzeer, Abdul Kareem Al Momen, Mashael Al Shaikh, Farja Al Gahtani, Zohair A. Al Aseri, Hossam AH Abdelrazik

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Comparison of Myelodysplastic Syndrome Prognostic Scoring Systems Özlen Bektaş, Ayşegül Üner, Eylem Eliaçık, Burak Uz, Ayşe Işık, Sezgin Etgül, Süreyya Bozkurt, İbrahim Celalettin Haznedaroğlu, Hakan Göker, Nilgün Sayınalp, Salih Aksu, Haluk Demiroğlu, Osman İlhami Özcebe, Yahya Büyükaşık

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131

135

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Platelet Dysfunction in Patients with Chronic Myeloid Leukemia: Does Imatinib Mesylate Improve It? Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş Immature Reticulocyte Fraction and Absolute Neutrophil Count as Predictor of Hemopoietic Recovery in Patients with Acute Lymphoblastic Leukemia on Remission Induction Chemotherapy Shan E. Rauf, Saleem Ahmed Khan, Nadir Ali, Nabeel Khan Afridi, Maria Haroon, Ammara Arslan The Prognostic Significance of Soluble Urokinase Plasminogen Activator Receptor in Acute Myeloid Leukemia Nergiz Erkut, Ahmet Menteşe, Hasan Mücahit Özbaş, Nilay Ermantaş, Ayşegül Sümer, Asım Örem, Mehmet Sönmez Investigation of Rho-Kinase Expressions and Polymorphisms in Mantle Cell Lymphoma Patients Didar Yanardağ Açık, Mehmet Yılmaz, İbrahim Sarı, Serdar Öztuzcu, Zeynel A. Sayıner, Salih Subari, Abdullah T. Demiryürek

Prospective Audit of Blood Donor Selection Process in a Tertiary Care Hospital of a Developing Country Naila Raza

Brief Reports

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Regulatory T Cells in Patients with Idiopathic Thrombocytopenic Purpura Alev Akyol Erikçi, Bülent Karagöz, Oğuz Bilgi

157

Serum Zinc Levels in Iron Deficient Women: A Case-Control Study Onur Özhan, Neslihan Erdem, İsmet Aydoğdu, Ali Erkurt, İrfan Kuku

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Case Report

Diffuse Large B-Cell Lymphoma Presenting with Bilateral Renal Masses and Hematuria: A Case Report Şiyar Erdoğmuş, Serkan Aktürk, Zeynep Kendi Çelebi, Saba Kiremitçi, Gülşah Kaygusuz, Namık Kemal Altınbaş, Evren Üstüner, Kenan Keven

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Letters to the Editor A Comparison of Healthy Infants and Adults with Respect to Indirect Microparticle Activity and the Parameters of the Thrombin Generation Test Filiz Şimşek Orhon, Nejat Akar, Yonca Eğin, Betül Ulukol, Sevgi Başkan Comment: In Response to “Downgraded Lymphoma: B-Chronic Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell Lymphoma - De Novo Occurrence or Transformation” Burak Uz, Kadir Acar Tumor Necrosis Factor and Splenectomy İrfan Yavaşoğlu

Images in Hematology

Auer Rod in a Neutrophil in a Nonmalignant Condition Harish Chandra, Smita Chandra, Vibha Gupta, Divyaa Mahajan

Precursor B-Cell Lymphoblastic Lymphoma Presenting as a Spinal Mass at Initial Diagnosis Oğuzhan Erol, Çiğdem Tokyol, Feyzullah Akyüz, Nuran Ahu Baysal, Mehmet Sezgin Pepeler

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Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Should Be Kept in Mind in Children with Febrile Neutropenia, Oral Cavity Lesions, and Skin Rash Eda Ataseven, Şebnem Yılmaz Bengoa, Hale Ören

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REVIEW DOI: 10.4274/tjh.2016.0031 Turk J Hematol 2016;33:88-93

The Role of Angiogenesis in Haemophilic Arthropathy: Where Do We Stand and Where Are We Going? Hemofilik Artropatide Anjiyogenezin Rolü: Neredeyiz ve Nereye Gidiyoruz? Alexandra Agapidou1, Thomas Stavrakis1, Efthymia Vlachaki2, Panagiotis Anagnostis1, Sophia Vakalopoulou1 1Aristotle University, Hippokration Hospital, Second Propaedeutic Department of Internal Medicine, Thessaloniki, Greece 2Aristotle University, Hippokration Hospital, Second Department of Internal Medicine, Thessaloniki, Greece

Abstract

Öz

Haemophilia is an inherited bleeding disorder that can lead to degenerative joint arthropathy due to recurrent bleeding episodes affecting the musculoskeletal system of the patient. The cause of bleeding can be either traumatic or spontaneous. The pathogenesis of haemophilic arthropathy is unclear as many factors like iron, inflammatory cytokines, and angiogenic factors contribute to this process. Blood into joints can deteriorate the bone to such an extent that the patient experiences pain, reduction of the range of movement, and deformity of the joint, conditions that could have a great impact on quality of life. Over the years, management of haemophilic arthropathy has changed. Nowadays, early diagnosis with high resolution imaging like magnetic resonance imaging along with application of prophylaxis regimens can reduce the extent of damage to the joints. However, not all haemophilia patients have access to these interventions as cost may be prohibitive for some of them. The need for new, easy, and costeffective strategies with the ability to identify early changes could be beneficial and could make a difference in the management of haemophilic arthropathy. Understanding the mechanism of processes like angiogenesis in the mechanism of developing arthropathy could be innovative for these patients and could help in the detection of new early diagnostic and therapeutic markers.

Hemofili, hastanın kas ve iskelet sistemini etkileyen, tekrarlayan kanama atakları ile dejeneratif eklem artropatisine neden olan, kalıtsal bir kanama bozukluğudur. Kanama travma sonrasında ya da kendiliğinden olabilir. Hemofilik artropatinin patogenezi kesin bilinmemekle birlikte, demir, yangı sitokinleri ve anjiyogenik faktörlerin sürece katkıları vardır. Eklem içine kanama kemiği bir düzeye kadar bozabilir ve hasta, yaşam kalitesi üzerine büyük bir etkisi olan ağrı, hareket kısıtlılığı ve eklem deformitesi gibi durumları yaşar. Yıllar içerisinde hemofilik artropatinin yönetiminde değişiklikler olmuştur. Günümüzde manyetik rezonans görüntüleme gibi yüksek çözünürlüklü görüntüleme yöntemleri ile erken tanı ve profilaksi rejimlerinin kullanılması eklemlerdeki harabiyetin derecesini azaltmaktadır. Ancak bunların ücretleri bazı hastalar açısından sınırlayıcı olabileceğinden, tüm hastalar bu müdahalelere ulaşamaz. Erken dönemdeki değişiklikleri tespit edebilen yeni, kolay ve maliyet etkin stratejiler yararlı olabilir ve hemofilik artropatinin yönetiminde bir değişiklik yapabilir. Artropati gelişim mekanizmalarından anjiyogenez gibi süreçlerin mekanizmasının anlaşılması bu hastalar için bir yenilik olabilir ve yeni erken tanısal ve terapötik belirteçlerin bulunmasına yardımcı olabilir.

Keywords: Angiogenesis, Haemophilic endothelial growth factor, Haemophilia

arthropathy,

Vascular

Introduction Haemophilia A and B are X-linked inherited disorders respectively caused by the deficiency of coagulation factor VIII or IX [1]. Lack of those clotting factors (CFs) leads to an increased tendency to bleed at various intensities, according to the percentage of the missing CF. The system that is mainly affected by these recurrent bleeding episodes is the musculoskeletal system. Repeated joint bleeds can cause progressive destruction of the cartilage, resulting in a decreased range of motion due to pain and Address for Correspondence/Yazışma Adresi: Alexandra AGAPIDOU, M.D., Aristotle University, Hippokration Hospital, Second Propaedeutic Department of Internal Medicine, Thessaloniki, Greece E-mail : alekagapidou@yahoo.gr

Anahtar Sözcükler: Anjiyogenez, Hemofilik artropati, Vasküler endoteliyal büyüme faktörü, Hemofili

stiffness. This condition is known as haemophilic arthropathy or haemophilic joint disease (HJD) and it has a progressively negative impact on patients’ quality of life. Haemophilia is found to be associated with decreased bone mass in both adults and children [2]. Haemarthrosis, formed after repeated joint bleeds, could be prevented by providing prophylaxis to these patients by means of administering the missing CF from an early age and in a standard regimen. However, this requires good venous access and skills from the patient’s point of view along with highly specialised and properly organised structures from the Received/Geliş tarihi: January 19, 2016 Accepted/Kabul tarihi: April 15, 2016

Turk J Hematol 2016;33:88-93

health provider’s side. If a patient cannot receive prophylactic treatment, there are various other ways of chronic joint pain relief, like applying interventions such as synovectomy and arthroplasty. These are invasive surgical procedures that may be frustrating for the patient. It is important to realise that the mechanism underlying progressive haemophilic arthropathy is multifactorial and still remains unclear. Availability of an easy, quick, and low-cost test with high specificity for diagnosing HJD would be beneficial for both patients and health providers. Use of the Fracture Risk Assessment Tool for assessing fracture risk, regular bone mineral density assessment, and supplemented calcium, vitamin D, and, in specific cases, bisphosphonate intake, as well as long-term prophylactic factor replacement therapy, were suggested as means of prevention of bone loss [2]. Furthermore, various inflammatory and angiogenetic processes have been implicated in early joint bleeding and in the pathogenesis of HJD. Achieving a deeper knowledge of HJD could potentially lead to earlier diagnosis and treatment in patients with haemophilia.

Haemophilic Arthropathy Structure of the Synovial Joint The synovial joint belongs to the group of joints that have to bear a great amount of movement. In such joints, the bony surfaces are covered with articular cartilage and are connected by ligaments. The joint may be divided by an articular disc or meniscus, which is continuous in the periphery with the fibrous capsule while its free surfaces are covered by the synovial membrane. Synovial joints facilitate movement by bringing articulating bones into contact (Figure 1). The components of a synovial joint are the synovial cavity, which is the space between the bones filled with synovial fluid, and the articular capsule, which surrounds the joint and unites the articulating bones. The articular capsule also consists of two layers: the outer fibrous membrane, which may contain ligament, and the inner synovial membrane that secretes the lubricating synovial fluid. The bones of the synovial joint are covered by a layer of cartilage that functions to absorb tension and reduce friction during movement [3]. The articular capsule is highly innervated but is lacking blood vessels. The surrounding blood network provides the necessary nutritional supply [4]. HJD is the end result of a number of changes occurring in every component of the joint after repeated bleeding episodes. Bleeding into the synovial joint exposes synovial cells to blood, which is toxic for the joint. Morris et al. proposed that iron plays a substantial role in the development of haemophilic synovitis [5]. Studies by Wen et al. showed that iron is also involved in myelocytomatosis viral oncogene (MYC) and mouse double minute-2 (MDM2) homolog expression, which

Agapidou A, et al: The Role of Angiogenesis in Haemophilic Arthropathy

causes proliferation of the synovium and active inflammation [6]. Roosendaal and Lafeber observed that iron increases the expression of proinflammatory cytokines like interleukin-6 (IL-6), interleukin 1-β (IL-1β), and tumour necrosis factor-α (TNF-α) in synovial cells [7]. Histologically, it was shown that synovial inflammation incorporates three characteristics: 1) hypertrophy of the villi, 2) increased number of inflammatory cells, and 3) increased vascularisation [7,8,9]. Synovial Angiogenesis Angiogenesis is a normal process during wound healing and embryogenesis. It is also considered part of the pathophysiologic mechanisms implicated in diseases like rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, and carcinogenesis. It is regulated by various inducers and inhibitors. During inflammation, the inducers/promoters prevail over inhibitors (Figure 2). Angiogenesis takes place mainly in the bone marrow and in vascular stem cells. Angiogenetic factors like vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and fibroblast growth factor (FGF) participate in endothelial cell proliferation. In 2008, it was shown that mice with factor IX deficiency experienced delayed wound healing and increased wound angiogenesis along with subcutaneous haematoma formation days after induction of a wound. It was suggested that tissue damage induces coagulation and inflammation response [10]. For angiogenesis to be triggered, a series of events have to take place. Mediators from the synovium activate endothelial cells, which release proteolytic enzymes that act on the endothelial basement membrane and the perivascular extracellular matrix.

Figure 1. Synovial joint. 89

Agapidou A, et al: The Role of Angiogenesis in Haemophilic Arthropathy

The endothelial cells participate in the formation of primary sprouts. The lumen of the sprouts facilitate the formation of “capillary loops” followed by the synthesis of new basement membrane and new capillaries [11]. RA is among the inflammatory disorders in which increased angiogenesis is observed as well. By shedding light on the understanding of the relationship between angiogenesis and inflammatory arthritis, the role of new vessel formation in haemophilic arthropathy could be more easily comprehended. Angiogenesis is induced by several conditions like hypoxia and injury where proangiogenic molecules are secreted by tissues. Endothelial cell proliferation and migration are followed by capillary tube formation, deposition of basement membrane, and migration of smooth muscle cells. Anastomoses are created and the flow of blood is established [12]. In RA, inflammatory cells like macrophages, lymphocytes, mast cells, and fibroblasts, along with their soluble products including pro-inflammatory cytokines, TNF-α, IL-1, and IL-8, are all promoters of angiogenesis. One of the major endothelial growth factors found in the synovium of patients with RA is VEGF. In RA synovium, IL-1 and TNF-α facilitate the fibroblast expression of VEGF [13,14]. VEGF induces the endothelial cell decay-accelerating factor, which acts protectively for the cells against activated complement components and may regulate endothelial proliferation and angiogenesis [15]. Direct measurements confirm that the intra-articular environment is hypoxic in inflammatory arthritis. Hypoxia is often a feature of inflammation and is a strong inducer of VEGF. Tissue hypoxia in the rheumatoid joint results in increased VEGF messenger ribonucleic acid (mRNA) stability [16] and enhanced

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VEGF gene transcription through the binding of hypoxiainducible transcription factors such as hypoxia-induced factor-1 (HIF) and HIF-2 that are overexpressed in the synovial lining and stromal cells of RA patients relative to synovial tissues from individuals without arthritis [17]. Zimmermann in 1923 introduced the term ‘pericyte’ to describe a periendothelial support cell wrapped around the length of micro-vessels [18]. While it is known that pericytes are present in the microcirculation, their functional roles and importance in microvascular physiology has not been fully investigated. Recently pericytes have become a research area of growing interest as potential targets for pro- or antiangiogenic therapies. Vascular pericytes elongate around endothelial cells and their function is to assist in the regulation of vessel stabilisation and in endothelial cell proliferation. During angiogenesis, signals from the pericyte to the endothelial cell and vice versa are critical for the formation of the capillary sprout. Studies found that pericytes may have a leading role in newly formed capillaries. This implicates their role in endothelial cell guidance. During angiogenesis, pericytes are involved in recruitment and direct interaction with endothelial cells [19]. Moreover, they participate in the development of newly formed endothelial cells [20]. Pericyte development is usually controlled by plateletderived growth factor-B (PDGF), secreted by endothelial cells [21]. It was found that pericytes are directly involved in the process of angiogenesis as increasing pericyte coverage via Ang-2 inhibition can potentially represent an antiangiogenic tumour therapy [22,23]. Advancing the understanding of pericytes and the ability to develop pericyte-related therapies is a challenging and very promising process. Angiogenesis and Haemophilia Angiogenesis is a natural process considered as a physiologic response to inﬂammation, hypoxia, and malignancy. It may be mediated by various factors including growth factors, proinflammatory cytokines, chemokines, extracellular matrix

Figure 2. Angiogenesis in normal state and inflammation state. 90

Figure 3. Angiogenesis and hypoxia in rheumatoid arthritis. RA: Rheumatoid arthritis.

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molecules, matrix-degrading proteolytic enzymes, cellular adhesion molecules, and others [24]. One of the most important growth factors in terms of angiogenesis is VEGF. HIF acts by upregulating VEGF [17,25]. Furthermore, prostaglandin and nitric oxide act via VEGF in neovascularisation. There is an interaction between VEGF and Ang-1 that acts in favour of the newly formed vessels as a form of protection and stabilisation. On the contrary, Ang-2 antagonises Ang-1 and has a negative impact on neovascularisation (Figure 3). VEGF and PDGF are involved in the development of inflammatory joint disease, potentially by favouring cytokinerelated cartilage destruction and not synthetic cell responses associated with growth factor activity. Expression of VEGF is increased in individuals with inflammatory joint disease compared with normal controls. Among individuals with polyarthritis, concentrations of VEGF and its receptor, Ang-1, have been found to be related to inflammatory markers and bone destruction. Based on that, VEGF could play a role in haemophilic arthropathy [26]. There are a variety of factors that work in an inhibitory manner regarding formation of new vessels. Among them are cytokines like interferon-α (IFN-α), IFN-γ, IL-4, IL-12, and leukaemia inhibitory factor (LIF). Protease inhibitors like tissue inhibitor of metalloproteinase (TIMPS), plasminogen activator inhibitors (PAIs), and thrombospondin-1 inhibit capillary and new vessel formation [11,27]. Acharya et al. [28] observed that there is involvement of angiogenesis in the development of haemophilic synovitis. Sera from haemophilic subjects with joint arthropathy induced an angiogenic response in endothelial cells that was ceased by blocking VEGF while peripheral blood mononuclear cells from these subjects stimulated synovial cell proliferation, which was blocked by a humanised anti-VEGF antibody (bevacizumab). Human synovial cells, when incubated with haemophilic sera, could elicit upregulation of hypoxia-inducible factor-1A (HIF1A) mRNA, indicating that hypoxia plays an important role in the neoangiogenesis process. The onset of endothelial proliferation is based on the equilibrium between positive and negative regulators [29]. Once there is a stimulus from VEGF-A, endothelial cells became activated, proliferate, and form new vessels. However, these newly formed vessels are extremely sensitive and prone to bleeding. In order to protect them, PDGF stimulates pericytes, which migrate to the place of the angiogenesis and embrace the vessels with dendritic processes, forming a stabilising coat. The presence of pericytes is considered a sign of maturity. In another study, Zetterberg et al. [30] observed that VEGF is increased in synovial tissue from haemophilic patients. In this study, synovial tissue was obtained when HJD was

Agapidou A, et al: The Role of Angiogenesis in Haemophilic Arthropathy

already established. The increased VEGF in synovial cells from these patients showed that HJD is characterised by active angiogenesis. Vessels from HJD and control synovial tissue were found to be covered by pericytes, indicating that most of the vessels were mature. Even though the number of patients with different stages of HJD in this study was too low to understand how angiogenesis develops over time in HJD, there were indications that end-stage HJD is characterised by a chronic proinﬂammatory, proangiogenic drive, while the vessel formation is relatively slow, permitting vessels to mature and develop pericyte “protection”. Tattersall et al. [31] observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property called “angiotrophism”. Polarising macrophages in a proinflammatory manner could increase their angiotrophic stimulation of vessel sprouting. This increase was found to be dependent on macrophage Notch signalling. JAG1 expression and Notch signalling are essential for the growth of both endothelial cells and pericytes. In a recent study by Yi et al. [32], it was demonstrated that annexin a2 (Axna2) could promote the progression of RA. Axna2 plays an important role in pannus formation in RA. Cytological analysis showed that the Axna2/Axna2 receptor (Axna2/Axna2R) axis promoted new vessel formation by activation of the Hedgehog (HH) signalling pathway and increased the Patched (Ptc) and Smoothened (Smo) expression in order to upregulate the expression of the downstream metalloproteinases (MMPs), VEGF, and Ang-2. These results suggest that the effect of Axna2 might provide a new potential measure for treatment of RA and potentially HJD.

New Potential Therapies: A Step to the Future Cancer and inflammation research trials have been targeting angiogenic mediator and inhibitor pathways regarding the development of new therapeutic agents. There have been attempts to target VEGF by using synthetic VEGF and VEGFR inhibitors, anti-VEGF antibodies, and inhibitors of VEGF and VEGFR signalling, primarily in colorectal, lung, renal, and liver cancers. Bevacizumab, a human monoclonal antibody to VEGF, has been used in the treatment of various types of cancer [11]. Vatalanib, a VEGFR protein kinase inhibitor, inhibited knee arthritis in rabbits [33]. YC-1, a superoxide-sensitive stimulator of soluble guanylyl cyclase originally developed to treat hypertension and thrombosis, is also a HIF-1 inhibitor [34]. Microtubule destabilisers, such as 2-ME, as well as paclitaxel, an anticancer agent, also diminish HIF-1α expression and activity [35]. Infliximab treatment in combination with methotrexate reduced synovial VEGF expression and vascularity [36,37]. Anti91

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

TNF therapy in arthritic patients reduced Ang-1 but stimulated Ang-2 expression [38]. Recently, the anti-IL-6 receptor antibody tocilizumab decreased serum levels of VEGF [39]. Thalidomide, currently used in multiple myeloma treatment but also tried in lupus and RA, is a potent TNF-α antagonist and angiogenesis inhibitor [27,40]. Thalidomide could suppress both synovitis and angiogenesis [27], suggesting that its antiangiogenic effects may be, in part, VEGF-independent. Fumagillin is a natural product of Aspergillus fumigatus. TNP-470 and PPI2458 are synthetic derivatives of fumagillin that inhibit methionine aminopeptidase-2, an enzyme involved in angiogenesis [41].

References

It would be extremely interesting to learn if some of these treatment options could be applied to haemophilia patients in the future and if they could have an impact on the development, progression, and treatment of synovitis and haemophilic joint arthropathy.

4. Boundless Anatomy and Physiology. Nerve and Blood Supply. Available online at https://www.boundless.com/physiology/textbooks/boundlessanatomy-and-physiology-textbook/joints-8/synovial-joints-92/nerve-andblood-supply-517-16/.

Conclusion Further studies will have to clarify the mechanisms and circumstances that may be responsible for modulating the contribution of angiogenesis to HJD. It is very intriguing to consider the possibility that angiogenetic factors may play a crucial role in the pathogenesis of arthropathy seen in patients suffering from haemophilia. Finally, the possibility that there may be potential markers enabling identification of the onset as well as the progression of haemophilic synovitis deserves further investigation. Acknowledgements Many thanks to Dr. Emma Fosbury, BA, MBBS, MRCP, FRCPath, Clinical Research Fellow at the Katharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, London, UK, for her kind editing of the English language of the manuscript. Authorship Contributions Surgical and Medical Practices: Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou; Concept: Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou; Design: Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou; Data Collection or Processing: Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou; Analysis or Interpretation: Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou; Literature Search: Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou; Writing: Alexandra Agapidou, Thomas Stavrakis, Efthymia Vlachaki, Panagiotis Anagnostis, Sophia Vakalopoulou. 92

1. Mannucci PM. Haemophilia and related bleeding disorders: a story of dismay and success. Hematology Am Soc Hematol Educ Program 2002;1-9. 2. Anagnostis P, Karras SN, Goulis DG. Bone disease in patients with haemophilia A and B -- where are we now? Haemophilia 2015;21:1-3. 3. Boundless Anatomy and Physiology. Structure of Synovial Joints. Available online at https://www.boundless.com/physiology/textbooks/boundlessanatomy-and-physiology-textbook/joints-8/synovial-joints-92/structureof-synovial-joints-516-5247/.

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20. Ponce AM, Price RJ. Angiogenic stimulus determines the positioning of pericytes within capillary sprouts in vivo. Microvasc Res 2003;65:45-48.

32. Yi J, Zhu Y, Jia Y, Jiang H, Zheng X, Liu D, Gao S, Sun M, Hu B, Jiao B, Wang L, Wang K. The annexin a2 promotes development in arthritis through neovascularization by amplification Hedgehog pathway. PLoS One 2016;11:e015036.

21. Hellström M, Kalén M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-β in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 1999;126:3047-3055.

33. Grosios K, Wood J, Esser R, Raychaudhuri A, Dawson J. Angiogenesis inhibition by the novel VEGF receptor tyrosine kinase inhibitor, PTK787/ ZK222584, causes significant anti-arthritic effects in models of rheumatoid arthritis. Inflamm Res 2004;53:133-142.

22. Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 2003;111:1287-1295.

34. Yeo EJ, Chun YS, Cho YS, Kim J, Lee JC, Kim MS, Park JW. YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1. J Natl Cancer Inst 2003;95:516-525.

23. Gerald D, Chintharlapalli S, Augustin HG, Benjamin LE. Angiopoietin-2: an attractive target for improved antiangiogenic tumor therapy. Cancer Res 2013;73:1649-1657.

35. Mabjeesh NJ, Escuin D, LaVallee TM, Pribluda VS, Swartz GM, Johnson MS, Willard MT, Zhong H, Simons JW, Giannakakou P.2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell 2003;3:363-375.

19. Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res 2003;314:15-23.

24. Szekanecz Z, Koch AE. Chemokines and angiogenesis. Curr Opin Rheumatol 2001;13:202-208. 25. Taylor PC, Sivakumar B. Hypoxia and angiogenesis in rheumatoid arthritis. Curr Opin Rheumatol 2005;17:293-298. 26. Valentino LA. Blood-induced joint disease: the pathophysiology of hemophilic arthropathy. J Thromb Haemost 2010;8;1895-1902. 27. Lainer-Carr D, Brahn E. Angiogenesis inhibition as a therapeutic approach for inflammatory synovitis. Nat Clin Pract Rheumatol 2007;3:434-442. 28. Acharya SS, Kaplan RN, Macdonald D, Fabiyi OT, DiMichele D, Lyden D. Neoangiogenesis contributes to the development of hemophilic synovitis. Blood 2011;24:2484-2493. 29. Nadar SK, Karalis I, Al Yemeni E, Blann AD, Lip GY. Plasma markers of angiogenesis in pregnancy induced hypertension. Thromb Haemost 2005;94:1071-1076. 30. Zetterberg E, Palmblad J, Wallensten R, Morfini M, Melchiorre D, Holmström M. Angiogenesis is increased in advanced haemophilic joint disease and characterized by normal pericyte coverage. Eur J Haematol 2014;92:256262. 31. Tattersall IW, Du J, Cong Z, Cho BS, Klein AM, Dieck CL, Chaudhri RA, Cuervo H, Herts JH, Kitajewski J. In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment. Angiogenesis 2016;19:201-215.

36. Veale DJ, Fearon U. Inhibition of angiogenic pathways in rheumatoid arthritis: potential for therapeutic targeting. Best Pract Res Clin Rheumatol 2006;20:941-947. 37. Goedkoop AY, Kraan MC, Picavet DI, de Rie MA, Teunissen MB, Bos JD, Tak PP. Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective single-centre study. Arthritis Res Ther 2004;6:326-334. 38. Markham T, Mullan R, Golden-Mason L, Rogers S, Bresnihan B, Fitzgerald O, Fearon U, Veale DJ. Resolution of endothelial activation and downregulation of Tie2 receptor in psoriatic skin after infliximab therapy. J Am Acad Dermatol 2006;54:1003-1012. 39. Nakahara H, Song J, Sugimoto M, Hagihara K, Kishimoto T, Yoshizaki K, Nishimoto N. Anti-interleukin-6 receptor antibody therapy reduces vascular endothelial growth factor production in rheumatoid arthritis. Arthritis Rheum 2003;48:1521-1529. 40. D’Amato RJ, Loughnan MS, Flynn E, Folkman J. Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci U S A 1994;91:4082-4085. 41. Ingber D, Fujita T, Kishimoto S, Sudo K, Kanamaru T, Brem H, Folkman J. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature 1990;348:555-557.

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0136 Turk J Hematol 2016;33:94-101

Impact of JAK2V617F Mutational Status on Phenotypic Features in Essential Thrombocythemia and Primary Myelofibrosis Esansiyel Trombositemi ve Primer Miyelofibroziste JAK2V617F Mutasyonunun Fenotipik Etkileri İpek Yönal, Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz, Meliha Nalçacı, Akif Selim Yavuz, Fatma Deniz Sargın İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

Abstract

Öz

Objective: The JAK2V617F mutation is present in the majority of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The impact of this mutation on disease phenotype in ET and PMF is still a matter of discussion. This study aims to determine whether there are differences in clinical presentation and disease outcome between ET and PMF patients with and without the JAK2V617F mutation.

Amaç: Esansiyel trombositemi (ET) ve primer miyelofibrozis (PMF) tanılı hastaların büyük çoğunluğunda JAK2V617F mutasyonu bulunmaktadır. ET ve PMF’de bu mutasyonun hastalık fenotipi üzerine etkisi halen tartışılmaktadır. Bu çalışmada, JAK2V617F mutasyonunu taşıyan ve taşımayan ET ve PMF hastalarının başvuru sırasındaki klinik parametreler ve hastalık seyri açısından karşılaştırılması amaçlanmıştır.

Materials and Methods: In this single-center study, a total of 184 consecutive Philadelphia-negative chronic myeloproliferative neoplasms, 107 cases of ET and 77 cases of PMF, were genotyped for JAK2V617F mutation using the JAK2 Ipsogen MutaScreen assay, which involves allele-specific polymerase chain reaction.

Gereç ve Yöntemler: Tek merkezli olan bu çalışmada, 107 ET ve 77 PMF olmak üzere toplam 184 Philadelphia-negatif kronik miyeloproliferatif neoplazili hastada bir allel spesifik polimeraz zincir reaksiyonu olan JAK2 Ipsogen MutaScreen kullanılarak JAK2V617F mutasyonu taranmıştır.

Results: ET patients positive for JAK2V617F mutation had higher hemoglobin (Hb) and hematocrit (Hct) levels, lower platelet counts, and more prevalent splenomegaly at diagnosis compared to patients negative for the JAK2V617F mutation, but rates of major thrombotic events, arterial thrombosis, and venous thrombosis were comparable between the groups. At presentation, PMF patients with JAK2V617F mutation had significantly higher Hb and Hct levels and leukocyte counts than patients without the mutation. Similar to the findings of ET patients, thromboembolic rates were similar in PMF patients with and without theJAK2V617F mutation. For ET and PMF patients, no difference was observed in rates of death with respect to JAK2V617F mutational status. Moreover, leukemic transformation rate was not different in our PMF patients with and without JAK2V617F mutation.

Bulgular: JAK2V617F mutasyonunu taşıyan ET hastalarında, mutasyon bulunmayanlara göre tanı sırasındaki hemoglobin (Hb) ve hematokrit (Hct) düzeyleri anlamlı olarak daha yüksek, trombosit sayısı daha düşük ve splenomegali oranları daha yüksek bulunmuştur. Fakat her iki grup arasında majör trombotik olay, arteriyel tromboz ve venöz tromboz açısından fark saptanmamıştır. JAK2V617F mutasyonu bulunan PMF hastalarında ise mutasyon taşımayan gruba göre başvuru anındaki Hb, Hct ve lökosit değerleri anlamlı olarak daha yüksek saptanmıştır. PMF hastalarında, ET hastalarında olduğu gibi tromboembolik olayların JAK2V617F mutasyonundan bağımsız olduğu görülmüştür. ET ve PMF hastalarında JAK2V617F mutasyonu varlığında ölüm oranında farklılık gözlenmemiştir. Bunun yanında JAK2V617F mutasyonunu taşıyan ve taşımayan PMF hastaları arasında lösemik dönüşüm oranı açısından anlamlı bir fark bulunmamıştır.

Conclusion: We conclude that JAK2V617F-mutated ET patients express a polycythemia vera-like phenotype and JAK2V617F mutation in PMF patients is associated with a more pronounced myeloproliferative phenotype.

Sonuç: Bu çalışmanın sonucunda JAK2V617F mutasyonunu taşıyan ET hastalarında polisitemia vera benzeri fenotipin ortaya çıktığı ve bu mutasyonun varlığında PMF hastalarının daha belirgin bir miyeloproliferatif fenotiple ilişkili olduğu söylenebilir.

Keywords: JAK2V617F mutation, Essential thrombocythemia, Primary myelofibrosis

Anahtar Sözcükler: JAK2V617F mutasyonu, Esansiyel trombositemi, Primer miyelofibrozis

Address for Correspondence/Yazışma Adresi: İpek YÖNAL, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey E-mail: ipekyon@istanbul.edu.tr

Received/Geliş tarihi: March 30, 2014 Accepted/Kabul tarihi: July 14, 2014

Yönal İ, et al: Impact of JAK2V617F Mutational Status

Turk J Hematol 2016;33:94-101

Introduction

Statistical Analysis

Philadelphia-negative chronic myeloproliferative neoplasms (Ph-negative MPNs) are a heterogeneous group including 3 major diseases: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Thrombotic events are the major cause of morbidity and mortality in ET. Other complications include hemorrhage and progression to myelofibrosis or acute myeloid leukemia [1,2]. PMF is characterized by a worse life expectancy and a progressive disease course. The disease presents with classically severe anemia, massive splenomegaly, and acute leukemia [3]. JAK2V617F mutation is present in more than 95% of PV patients and approximately 50%-60% of ET and PMF patients [4]. Several studies investigated the clinical relevance of JAK2V617F mutation in ET and PMF patients [5,6,7,8,9,10]. In ET, overall survival (OS) or leukemia-free survival was found not to be affected by the presence of JAK2V617F mutation, while the influence of JAK2V617F on thrombosis or fibrotic transformation remained less clear [5,7,11,12]. Conflicting results have been reported regarding the impact on OS, leukemic transformation rate, and need for chemotherapy or splenectomy in the presence of JAK2V617F mutation [8,9,10,13]. We previously evaluated the clinical and laboratory correlates in 184 patients with Ph-negative MPNs according to the allele burden of JAK2V617F mutation (unpublished data). Herein, we investigate the usefulness of JAK2V617F mutational status for explaining phenotypic variability using the same group of patients, which includes a relatively large series of Ph-negative MPN patients.

Data were processed using SPSS 16 (SPSS Inc., Chicago, IL, USA). Continuous variables were summarized as mean [standard deviation (SD)]. Chi-square statistics were used to compare categorical variables among the different patient groups categorized according to the JAK2V617F mutational status. Analysis of continuous variables among the groups was performed using the Mann-Whitney U test. A p-value of less than 0.050 was considered to indicate statistical significance; all tests were 2-tailed.

Materials and Methods A total of 184 consecutive Ph-negative MPN patients, 107 with ET and 77 with PMF, admitted to the Division of Hematology of the İstanbul University İstanbul Medical Faculty from 1995 to 2013 were included in the study. ET and PMF patients were diagnosed based on WHO criteria [14]. Informed consent was obtained from all participants according to the local ethics committee guidelines. Complete clinical history, blood count, lactate dehydrogenase (LDH) level, and thrombotic or hemorrhagic complications were recorded. Spleen longitudinal diameters of ≥130 mm to 160 mm and of ≥160 mm on ultrasound were considered as mild and massive splenomegaly, respectively. A scale of 0-3 was used to grade reticulin fibrosis on bone marrow trephine biopsies [15]. The Dynamic International Prognostic Scoring System (DIPSS) plus was used for risk stratification in PMF [16]. Unfavorable karyotypes in PMF were defined as complex karyotype or sole or 2 abnormalities that included +8, -7/7q-, i(17q), inv(3), -5/5q-, 12p-, or 11q23 rearrangement [17]. Patients were genotyped for the JAK2V617F mutation by JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille, France), which is a TaqMan allelic discrimination assay that contains fluorescent probes specific for wild-type (617V) and mutant (617F) alleles [18].

Results A total of 184 patients (107 with ET and 77 with PMF) were included. Bone marrow fibrosis was detected in 90.7% (97 in 107) of ET and 100% of PMF patients. In ET patients, the grade of bone marrow fibrosis was scaled as follows: grade 0, 9.3%; grade 1, 62.7%; grade 2, 25.2%; and grade 3, 2.8%. All PMF patients had bone marrow fibrosis (grade 2 in 20.8% and grade 3 in 79.2%). JAK2V617F mutation was identified in 64 of 107 ET (59.8%) and 58 of 77 PMF (75.3%) patients (p=0.028). Clinical and laboratory correlates of ET patients according to JAK2V617F mutational status are summarized in Tables 1 and 2. JAK2V617F-positive and -negative ET patients showed no significant differences with respect to sex and age at diagnosis. ET patients with JAK2V617F mutation presented with higher hemoglobin (Hb) and hematocrit (Hct) levels and lower platelet count at diagnosis compared to patients without mutation (p=0.001, p=0.001, and p=0.043, respectively). The leukocyte count and LDH levels were similar for the 2 groups. The 2 groups showed no significant difference with respect to mean spleen size. However, JAK2V617F-positive ET patients presented with more prevalent splenomegaly at diagnosis compared to patients without the mutation (p=0.044). ET patients with JAK2V617F mutation showed a higher, albeit not statistically significant, rate of bleeding events compared to the JAK2V617F-negative group (15.6% and 7%, respectively; p=0.298). ET patients with and without JAK2V617F mutation showed no significant difference with respect to the degree of bone marrow fibrosis, prevalence of hydroxyurea use, and rate of splenectomy. In addition, no significant differences were observed in the use of other medical treatments in any of the categories (p>0.050). Duration of follow-up in patients with and without JAK2V617F mutation was 69.7 months (SD: 63.7) and 70.1 months (SD: 56.9), respectively (p=0.675). During follow-up, 3 of 64 (4.7%) JAK2V617F-positive ET and 2 of 43 (4.7%) JAK2V617F-negative ET patients succumbed to their disease (p=1.000). 95

YĂśnal Ä°, et al: Impact of JAK2V617F Mutational Status

Turk J Hematol 2016;33:94-101

Clinical and laboratory parameters of PMF patients classified according to genotype are outlined in Tables 3 and 4. The rate of female patients was higher in the JAK2V617Fnegative group compared to the JAK2V617F-positive group (84.2% and 46.6%, respectively; p=0.009). PMF patients with and without JAK2V617F mutation showed no significant differences with respect to age at diagnosis. At initial diagnosis, PMF patients with the JAK2V617F mutation presented with significantly higher Hb and Hct levels and leukocyte counts compared to those without the mutation (p=0.005, p=0.034, and p=0.046, respectively). Platelet count and LDH level did not differ between the 2 groups. The mean spleen size showed no significant difference among any of the categories, although PMF patients with JAK2V617F mutation showed a trend towards higher prevalence of massive splenomegaly at diagnosis compared to patients without mutation (p=0.193 and p=0.090, respectively). JAK2V617F-positive PMF patients showed a trend towards a higher prevalence of bleeding events compared to JAK2V617Fnegative PMF patients (24.1% and 5.3%, respectively; p=0.090). There was no significant difference in the prevalence of total thrombotic events, arterial thrombosis, and venous thrombosis between JAK2V617F-positive and -negative PMF patients. The degree of reticulin fibrosis, prevalence of hydroxyurea use, rate of allogeneic hematopoietic stem cell transplantation (AHSCT), and history of splenectomy did not differ in any of the categories. In addition, the 2 groups showed no significant differences in the use of other medical treatments (p>0.050).

No significant difference was observed in the distribution of karyotype categories and DIPSS-Plus risk stratification between JAK2V617F-positive and -negative PMF patients. Duration of follow-up in PMF patients with and without JAK2V617F mutation was 42 months (SD: 46.9) and 56.6 months (SD: 48.7), respectively (p=0.165). At the end of the data collection period, 11 of 58 (19%) PMF patients with JAK2V617F mutation succumbed to their disease, while the rate of death in patients without JAK2V617F mutation was 15.8% (p=1.000). During follow-up, rate of leukemic transformation was similar between the 2 categories.

Discussion In our relatively large series of patients with Ph-negative MPNs, including 107 ET patients with a mean follow-up duration of more than 5 years and 77 PMF patients with a mean follow-up duration of more than 3 years, we documented that JAK2V617F mutation correlates with disease phenotype in adult Turkish patients with ET and PMF. Our results suggest that JAK2V617F positivity in ET induces a phenotype resembling PV. Confirming previous observations, we found that ET patients with JAK2V617F mutation presented with higher Hb and Hct levels and lower platelet counts compared to unmutated patients [5,6,7,19,20,21,22]. Contrary to some previous reports yet consistent with the findings of Kittur et al. [5] and Pich et al. [22], our ET patients with JAK2V617F mutation showed no difference in leukocyte count at diagnosis as opposed to patients without the mutation [6,21]. Furthermore, in contrast to some previous reports but consistent

Table 1. Clinical and laboratory features between JAK2V617F-mutated and -unmutated patients among 107 patients with essential thrombocythemia. ET JAK2V617F-mutated, mean [SD] JAK2V617F-unmutated, mean [SD] p-value Number of patients

Age at diagnosis

49.7 [14.9]

51.7 [15.7]

0.565

Females (%)

38 (59.4%)

20 (46.5%)

0.266

Leukocytes at diagnosis (mm3)

10.196 [4.138]

9.593 [3.434]

0.483

Hb at diagnosis (g/dL)

13.6 [1.8]

12.4 [1.9]

0.001

40.7 [5.37]

36.8 [5.21]

0.001

Hct at diagnosis (%) Platelet count at diagnosis

(mm3)

LDH at diagnosis (U/L)

874.782 [320.867]

1055.116 [495.928]

0.043

453.2 [150]

462.1 [159.7]

0.927

Spleen size at diagnosis (mm)

141.7 [37.26]

132.07 [23.86]

0.126

Bone marrow fibrosis, n (%)

64 (100%)

43 (100%)

0.522

7 (10.9%)

3 (7%)

42 (65.6%)

25 (58.1%)

14 (21.9%)

13 (30.2%)

1 (1.6%)

2 (4.7%)

Follow-up duration (months)

69.7 [63.7]

70.1 [56.9]

0.675

ET: Essential thrombocythemia, Hb: hemoglobin, Hct: hematocrit, LDH: lactate dehydrogenase, SD: standard deviation.

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with the study of Vannucchi et al. [11], we observed a higher prevalence of splenomegaly in ET patients with JAK2V617F mutation than in patients without the mutation [5,6,7,20,21]. Data on ET regarding the impact of JAK2V617F mutational status on thrombotic events are conflicting. In the study by Campbell et al., JAK2V617F mutation in ET was associated with an increased frequency of venous thromboembolism, but not with arterial thrombosis [6]. In the study by Kittur et al., the presence of JAK2V617F mutation was found to be significantly associated with increased incidence of venous thrombosis during follow-up, but not with major thrombosis, arterial thrombosis, and venous thrombosis at diagnosis [5]. In contrast, Antonioli et al. reported that there was no correlation between thrombotic events and JAK2V617F mutation in ET patients [20]. In another study, there was no difference between ET patients with JAK2V617F mutation or wild-type alleles with respect to the frequency of major thrombotic events and major arterial and venous thrombosis, either at diagnosis or during follow-up [21]. Similar to the aforementioned study in ET patients, the presence of JAK2V617F mutation made no significant difference in the frequency of vascular complications at presentation [7]. In the current study, we observed no significant difference in the frequency of major thrombotic events, arterial thrombosis, and venous thrombosis between JAK2V617F-positive and -negative ET patients. In the study by Pich et al., ET patients with JAK2V617F mutation were younger than those without mutation [22]. Conversely, in several studies, the presence of JAK2V617F mutation was significantly associated with older age at diagnosis [5,7,11,21,23,24,25]. Some studies revealed no difference in age between JAK2V617F-positive and -negative ET patients [20,26]. In our study group, we found no significant difference in age among ET patients with and without JAK2V617F mutation. Moreover, in the current study, we did not determine an association between JAK2V617F mutation and sex, consistent with previous reports [5,7,11,20,21,23,24,25,26]. Alvarez-LarrĂĄn et al. reported that the presence of JAK2V617F mutation in ET patients was associated with increased LDH levels [25]. On the contrary, in another study, JAK2V617F mutation in ET did not correlate with LDH level [21]. Our ET patients with JAK2V617F mutation did not show differences in LDH level as compared to wild-type patients. To our knowledge, there is limited information about the association between JAK2V617F mutation and histological changes in bone marrow biopsy of ET patients. In a series of 103 ET patients, Pich et al. reported no significant impact of JAK2V617F mutation on bone marrow fibrosis [22]. In the current study, the presence of JAK2V617F mutation in ET did not correlate with the degree of reticulin fibrosis. Several studies investigated the association between JAK2V617F mutation in ET and major hemorrhages [7,11,20,21,25]. Confirming the findings of the aforementioned studies, our ET patients with mutant and wild-type alleles showed no differences in the rate of bleeding complications [7,11,20,21,25]. Some previous studies reported

YĂśnal Ä°, et al: Impact of JAK2V617F Mutational Status

that cytoreductive therapy requirement did not differ between ET patients with and without JAK2V617F mutation [7,21,23,24]. This finding is in line with our data showing that the prevalence of hydroxyurea use and other medical treatments was similar between JAK2V617F-mutated and -unmutated ET patients [7,21,23,24]. In ET patients, OS was shown not to be influenced by the presence of JAK2V617F mutation [5,7]. Confirming this observation, the death rate did not differ in our ET patients with and without JAK2V617F mutation. In our series of 77 PMF patients, we found a significant association between JAK2V617F mutation and the expression of a more pronounced myeloproliferative phenotype. In PMF patients, JAK2V617F mutational status contributed to laboratory abnormalities, including higher Hb level and leukocyte count, but its association with platelet count is inconsistent [19]. Our PMF patients with JAK2V617F mutation had higher Hb and Htc levels and leukocyte counts at diagnosis than those without the mutation. In contrast, in our PMF patients, platelet count at initial diagnosis did not differ with respect to the JAK2V617F mutation. Barosi et al. demonstrated the association between JAK2V617F mutational status and development of marked splenomegaly [9]. On the other hand, in this population, several other groups did not show any correlation between the presence of JAK2V617F mutation and spleen size [8,10]. In the study by Guglielmelli et al., JAK2V617F mutated and wildtype patients did not differ from each other as regards the presence of palpable splenomegaly greater than 15 cm from the left costal margin [27]. In our study, the mean spleen size did not significantly differ between JAK2V617F-positive and -negative PMF patients, although PMF patients with JAK2V617F mutation showed a trend towards higher prevalence of massive splenomegaly at diagnosis compared to patients without mutation. In PMF patients, the relationship of JAK2V617F mutation and thrombosis is controversial. In the study by Barosi et al., there was no significant difference in the rate of major thrombotic events between JAK2V617F-mutated and -unmutated PMF patients [9]. In a series of 199 PMF patients, Tefferi et al. showed no significant difference in the prevalence of thrombosis between JAK2V617F-positive and -negative PMF patients, whereas in another series of 117 PMF patients, Tefferi et al. reported the association of the presence of JAK2V617F mutation with history of thrombosis [8,13]. In the current study, the prevalence of total thrombotic events, arterial thrombosis, and venous thrombosis did not significantly differ among PMF patients with and without JAK2V617F mutation. Several studies have shown that ET patients with mutant alleles and wild-type alleles showed no significant difference with respect to age and sex [8,10,27]. In the current study, the presence of JAK2V617F mutation in PMF patients was not associated with age. However, in our study, the rate of females was higher among JAK2V617Fnegative PMF patients than JAK2V617F-positive PMF. We did 97

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Turk J Hematol 2016;33:94-101

Table 2. Clinical and laboratory features between JAK2V617F-mutated and -unmutated patients among 107 patients with essential thrombocythemia (continued). ET JAK2V617F-mutated, n (%) JAK2V617F-unmutated, n (%) p-value Number of patients

Splenomegaly group

64 (100%)

43 (100%)

0.044

No splenomegaly

34 (53.1%)

33 (76.8%)

Mild splenomegaly

17 (26.6%)

5 (11.6%)

Massive splenomegaly

13 (20.3%)

5 (11.6%)

Bleeding

10 (15.6%)

3 (7%)

0.298

Hydroxyurea

57 (89.1%)

35 (81.4%)

0.273

History of splenectomy

1 (1.6%)

1 (2.3%)

1.000

Thrombosis

26 (40.6%)

15 (34.9%)

0.692

Thrombosis group

64 (100%)

43 (100%)

0.219

No thrombosis

38 (59.4%)

28 (65.1%)

Arterial

11 (17.2%)

10 (23.3%)

Venous

14 (21.9%)

4 (9.3%)

Arterial and venous

1 (1.5%)

1 (2.3%)

Death

3 (4.7%)

2 (4.7%)

1.000

ET: Essential thrombocythemia.

Table 3. Clinical and laboratory features between JAK2V617F-positive and -negative patients among 77 primary myelofibrosis patients. PMF JAK2V617F-mutated, mean [SD] JAK2V617F-unmutated, mean [SD] p-value Number of patients

Age at diagnosis

58.1 [13.7]

52.8 [16]

0.120

Females (%)

27 (46.6%)

16 (84.2%)

0.009

16.134 [14.633]

9.726 [7.875]

0.046

11.03 [2.2]

9.4 [1.3]

0.005

32.9 [7.39]

29.4 [4.81]

0.034

Leukocytes at diagnosis

(mm3)

Hb at diagnosis (g/dL) Hct at diagnosis (%) (mm3)

423.691 [353.469]

464.526 [396.324]

0.832

LDH at diagnosis (U/L)

843 [405]

782 [364]

0.836

Spleen size at diagnosis (mm)

202.19 [44.2]

183.7 [37.3]

0.193

Bone marrow fibrosis, n (%)

58 (100%)

19 (100%)

0.330

14 (24.1%)

2 (10.5%)

44 (75.9%)

17 (89.5%)

Follow-up duration (months)

42 [46.9]

56.6 [48.7]

0.165

Platelet count at diagnosis

PMF: Primary myelofibrosis, Hb: hemoglobin, Hct: hematocrit, LDH: lactate dehydrogenase, SD: standard deviation.

not find a significant difference in LDH level between PMF patients with and without JAK2V617F mutation, in accordance with some previous reports [8,10,27]. In a study involving 117 patients with PMF, the presence of JAK2V617F mutation did not correlate with degree of reticulin fibrosis [8]. Consistent with the study by Tefferi et al., the degree of reticulin fibrosis did not differ between our PMF patients when stratified by JAK2V617F mutational status [8]. There is limited information regarding the relevance of JAK2V617F on bleeding complications in PMF patients. Tefferi et al. did not determine a statistically significant correlation between JAK2V617F mutation and bleeding history 98

[8]. However, we observed a trend towards higher prevalence of bleeding events in JAK2V617F-positive PMF patients compared to JAK2V617F-negative PMF patients (24.1% and 5.3%, respectively). In the study by Barosi et al., JAK2V617F mutational status was associated with an increased requirement for splenectomy and greater need of cytoreductive therapy in PMF patients [9]. However, in the study by Tefferi et al. involving 199 patients with PMF, no significant correlation was found between the presence of JAK2V617F mutation and need for cytoreductive therapy or splenectomy [13]. Confirming the finding of Tefferi et al., in our study, the presence of JAK2V617F mutation in

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Turk J Hematol 2016;33:94-101

Table 4. Clinical and laboratory features between JAK2V617F-positive and -negative patients among 77 primary myelofibrosis patients (continued). PMF

JAK2V617F-mutated, n (%)

JAK2V617F-unmutated, n (%)

p-value

Number of patients

Splenomegaly group

58 (100%)

19 (100%)

0.090

No splenomegaly

1 (5.3%)

Mild splenomegaly

11 (19%)

6 (31.6%)

Massive splenomegaly

47 (81%)

12 (63.2%)

Bleeding

14 (24.1%)

1 (5.3%)

0.090

Hydroxyurea

54 (93.1%)

18 (94.7%)

1.000

History of splenectomy

3 (5.2%)

1 (5.3%)

1.000

AHSCT

2 (3.4%)

1 (5.3%)

1.000

Karyotype

58 (100%)

19 (100%)

0.274

Normal

49 (84.5%)

18 (94.7%)

Favorable

7 (12.1%)

Unfavorable

2 (3.4%)

1 (5.3%)

DIPSS-Plus

58 (100%)

19 (100%)

0.143

Low risk

11 (19%)

4 (21.1%)

Intermediate-1

22 (37.9%)

5 (26.3%)

Intermediate-2

17 (29.3%)

10 (52.6%)

High risk

8 (13.8%)

Thrombosis

8 (13.8%)

3 (15.8%)

1.000

Thrombosis group

58 (100%)

19 (100%)

No thrombosis

50 (86.2%)

16 (84.2%)

Arterial

4 (6.9%)

3 (15.8%)

Venous

3 (5.2%)

Arterial and venous

1 (1.7%)

Leukemic transformation

3 (5.2%)

1 (5.3%)

1.000

Death

11 (19%)

3 (15.8%)

1.000

PMF: Primary myelofibrosis, AHSCT: allogeneic hematopoietic stem cell transplantation, DIPSS: Dynamic International Prognostic Scoring System.

PMF had no impact on the need for cytoreductive treatment or requirement for splenectomy [13]. Several studies investigated the association of JAK2V617F mutation in PMF patients with prognostic scoring systems [8,10,13,27]. In a series of 186 PMF patients, the number of JAK2V617F-positive patients in the low risk category of the Dupriez scoring system was significantly higher compared with JAK2V617F-negative patients [27]. Campbell et al. reported that Dupriez prognostic scores tended to be lower for patients positive for JAK2V617F mutation [10]. On the contrary, several groups reported no correlation between JAK2V617F mutation and Dupriez prognostic score [8,13]. To analyze whether the JAK2V617F mutational status correlated with prognostic scoring systems, we evaluated the distribution of patients in the different risk categories of the DIPSS-Plus [16]. We found no significant difference in the DIPSS-Plus risk stratification between JAK2V617F-positive and -negative PMF patients. Several studies revealed that in PMF, the presence of JAK2V617F mutation showed no correlation with presence or

distribution of cytogenetic abnormalities [8,9,10]. Confirming the aforementioned studies, in our population, we observed no significant difference in the distribution of karyotype categories between JAK2V617F-positive and -negative groups. Divergent results were reported regarding the effect of JAK2V617F mutation on OS and leukemic transformation rate in PMF patients [8,9,10,13]. We did not observe any differences in the rates of death and leukemic transformation in PMF patients with and without JAK2V617F mutation. Collectively, according to the results of our study, JAK2V617F mutation may identify distinct disease phenotypes of ET and PMF patients. Acknowledgment We thank the Molecular Hematology Laboratory staff of the İstanbul University İstanbul Medical Faculty for their assistance with sample handling. 99

Yönal İ, et al: Impact of JAK2V617F Mutational Status

Ethics Ethics Committee Approval: The study was approved by the Local Ethics Commitee of İstanbul University İstanbul Medical Faculty (file number: 2012/1571-1245), Informed Consent: Informed consent was obtained from all patients for being included in the study. Authorship Contributions Design the Research: İpek Yönal, Meliha Nalçacı, Akif Selim Yavuz, Fatma Deniz Sargın; Concept: İpek Yönal, Meliha Nalçacı, Akif Selim Yavuz; Supply Samples: İpek Yönal; Analyze the Data: İpek Yönal; Literature Search: İpek Yönal; Draft the Article: İpek Yönal, Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz; Perform the Laboratory Work: Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz; Help in Acquisition of Data: Aynur Dağlar-Aday, Başak Akadam-Teker, Ceylan Yılmaz; Revise the Article: Meliha Nalçacı, Akif Selim Yavuz, Fatma Deniz Sargın; Writing: İpek Yönal, Aynur Dağlar-Aday, Başak AkadamTeker, Ceylan Yılmaz, Meliha Nalçacı, Akif Selim Yavuz, Fatma Deniz Sargın. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. Financial Disclosure: The study was supported by the İstanbul University Scientific Research Foundation (project number: 30427).

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RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0070 Turk J Hematol 2016;33:102-106

D-index: A New Scoring System in Febrile Neutropenic Patients for Predicting Invasive Fungal Infections   D-index: Febril Nötropenik Hastalarda İnvazif Mantar Enfeksiyonlarının Tanımlanmasında Yeni Bir Skorlama Sistemi Gülden Yılmaz1, Belgin Coşkun1, Atilla Elhan2, Alpay Azap1, Hamdi Akan3 1Ankara University Faculty of Medicine, Department of Clinical Microbiology and Infectious Diseases, Ankara, Turkey 2Ankara University Faculty of Medicine, Department of Biostatistics, Ankara, Turkey 3Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract

Öz

Objective: Neutropenia is a critical risk factor for invasive fungal infections (IFIs). We retrospectively performed this study to assess the performance of the D-index, a new test that combines both the duration and the severity of neutropenia, in predicting IFIs among patients with acute myelogenous leukemia.

Amaç: İnvaziv fungal enfeksiyonların (İFE) gelişiminde nötropeni önemli bir risk faktörüdür. Biz bu çalışmayı, akut miyeloid lösemi olup, İFE gelişen hastalarda, nötropeni süresini ve sayısını birlikte değerlendiren yeni bir test olan D-indeks’in performansını ölçmek için geriye dönük olarak yaptık.

Materials and Methods: Fifteen patients with IFIs and 28 patients who did not develop IFIs were enrolled in the study. The D-index was defined as the area over the neutrophil curve, whereas the cumulativeD-index (c-D-index) was the area over the neutrophil curve from the start of neutropenia until the first clinical manifestation of IFI.

Gereç ve Yöntemler: Çalışmaya 50 tane İFE gelişen hasta, 28 tane İFE gelişmeyen hasta alındı. D-indeks nötrofil eğrisinin üzerinde kalan alan olarak, kümülatif-D-indeks (k-D-indeks) ise, nötropeninin ilk başladığı günden İFE’nin belirtilerinin başladığı ilk güne kadar çizilen nötrofil eğrisinin üzerinde kalan alan olarak belirlendi.

Results: The D-index and the c-D-index tended to be significantly higher in patients with IFIs, with medians of 10,150 (range: 400022,000) and 5300 (range: 2300-22,200), respectively (p=0.037 and p=0.003, respectively). The receiver operating characteristic analyses showed that there was a cutoff point of 3875 for the D-index in predicting IFI; the sensitivity, specificity, and positive and negative predictive values were 100%, 67.9%, 35.4%, and 100%, respectively. There was also a cutoff point of 4225 for the c-D-index in predicting IFI; the sensitivity, specificity, and positive and negative predictive values for the c-D-index were 93.3%, 71.4%, 36.6%, and 98.4%.

Bulgular: D-indeks ve k-D-indeks İFE gelişen hastalarda yüksek olma eğilimindedir. D-indeks ve k-D-indeks için değerler ortalama 10,150 (aralık: 4000-22,000) ve 5300 (aralık: 2300-22,200) olup, sırası ile p=0,037 ve p=0,003 saptandı. Yapılan analizlerde D-indeks için eşik değerin 3875 olup, duyarlılık, özgüllük, pozitif ve negatif prediktif değerleri sırası ile %100, %67,9, %35,4 ve %100 saptandı. k-D-index için eşik değer 4225 olup, duyarlılık, özgüllük, pozitif ve negatif prediktif değeri sırası ile %93,3, %71,4, %36,6 ve %98,4 saptandı.

Conclusion: The D-index and especially the c-D-index could be useful tools with high negative predictive value to exclude as well as to predict IFIs in the management of neutropenic patients.

Sonuç: D-indeks ve özellikle k-D-indeks nötropenik hastaları günlük değerlendirmede kullanılabilecek bir testtir. Negatif prediktif değerinin yüksek olması nedeni ile İFE olan hastaları erken yakalamanın yanı sıra, İFE’yi dışlamak için de etkili bulunmuştur.

Keywords: Neutropenia, D-index, Cumulative-D-index, Hematological malignancies, Invasive fungal infections

Anahtar Sözcükler: Nötropeni, D-indeks, Kümülatif-D-indeks, Hematolojik malignite, İnvaziv fungal enfeksiyon

Introduction Invasive fungal infections (IFIs) are major life-threatening infections among immunocompromised patients with hematologic malignancies. Although there has been significant progress in the management of febrile neutropenic cancer

patients related to increasing protective measures and antifungal agents, neutropenia is still a critical risk factor for IFI. Profound (<100 neutrophils/µL) and prolonged (>10 days) neutropenia is associated with a higher risk of invasive aspergillosis [1,2,3,4,5,6,7].

Address for Correspondence/Yazışma Adresi: Gülden YILMAZ, M.D., Ankara University Faculty of Medicine, Department of Clinical Microbiology and Infectious Diseases, Ankara, Turkey Phone : +90 312 508 27 15 E-mail : drguldeny@yahoo.com.tr

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Received/Geliş tarihi: February 14, 2014 Accepted/Kabul tarihi: May 21, 2014

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Several scoring systems have been developed to categorize febrile neutropenic patients into risk groups. These systems usually take neutropenia duration into account. Recently, Portugal et al. developed indexes called the D-index and the cumulative-D-index (c-D-index), which take into account both the duration and the intensity of neutropenia to predict the IFI risk [8]. We performed this study to assess the performance of these new tests in predicting IFIs among patients with acute myelogenous leukemia (AML).

Materials and Methods Patients The Department of Adult Hematology of Ankara University’s Faculty of Medicine, a 56-bed institution, is one of the main regional centers of hematology and bone marrow transplantation in Ankara. Patients with newly diagnosed AML receiving first induction or with relapsed or refractory AML, and who developed neutropenia at this center between March 2011 and March 2012, were included in the study. Among these patients, 15 patients with IFIs and 28 patients who did not develop IFIs were enrolled. We selected controls with the same underlying disease and leukemia status. IFIs were classified as possible, probable, or proven according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/ MSG) revised criteria [9]. Only the proven and probable cases were included in the study (proven: 2, probable: 13). Clinical and epidemiological data were collected by structured survey forms during daily infectious disease consultation visits. The patients who developed IFIs were compared with controls regarding age, sex, underlying disease, comorbidities, type of chemotherapy, antibacterial and antifungal prophylaxis, mortality rate, duration of neutropenia, profound neutropenia, D-index, and c-D-index. The study was approved by the local ethics committee. D-index and Cumulative-D-index Calculation The absolute neutrophil count was recorded in patients and controls. The D-index is an index based on a graph showing the absolute neutrophil counts over the course of the episode of neutropenia (Figure 1). It is geometrically the area over the neutrophil curve. The D-index was calculated as the difference between the observed area under curve (AUC) (Ao) and the expected neutrophil area (Ae) if the patient did not develop neutropenia (D-index: Ae-Ao). Ao was calculated by the trapezoidal method, while Ae is the product of 500 and the number of days with neutropenia (Ae: 500/µL x days with neutropenia). An XLA add-in, developed by Usansky et al., was used to apply the trapezoidal method [10].

We also calculated the c-D-index, which is from the start of neutropenia until the date of first clinical manifestation of IFI in patients. The date of first clinical manifestation was defined by 3 specialists (2 from the department of infectious diseases and one from the department of hematology), and then their results were compared with each other. The clinical manifestations were cough, nasal discharge, pleuritic chest pain, hemoptysis, skin nodules, and stomachache with elevated liver enzymes. Power Analysis The D-index was considered as the primary outcome variable for this study. Group sample sizes of 25 and 15 achieved 82% power to detect a difference of 5000 between the null hypothesis that both group means were 4000 and the alternative hypothesis that the mean of group 2 was 9000 with estimated group standard deviations of 5000 and 5000 and with a significance level (alpha) of 0.05 using a 2-sided Mann-Whitney U test, assuming that the actual distribution was normal. Statistical Analysis Mean ± standard deviation, median (minimum-maximum), or percentage values are given as descriptive statistics as applicable. Dichotomous variables were compared using the chisquare or Fisher’s exact test. Test of normality was assessed by Shapiro-Wilk test. Comparison of continuous variables between fungal and control groups was analyzed by Mann-Whitney U test. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the ability of the D-index and c-Dindex to predict IFI. Positive and negative predictive values were calculated by using cutoff values obtained from ROC analysis. A multiple logistic regression was performed to identify the independent risk factors of outcome variable and the adjusted odds ratio (OR) was calculated. SPSS 15.0 for Windows was used for statistical analysis. A p-value of less than 0.05 was considered significant.

Results A total of 15 patients with IFIs and 28 controls were enrolled during the 1-year study. The clinical and epidemiological data of the patients are shown in Table 1. Those that developed IFIs were older than the controls. The lung was the most common site of fungal infection (86.7%). There were no significant differences between patients and controls regarding sex, status of underlying disease, chemotherapies, and comorbidities. All patients were given fluconazole prophylaxis. The duration and the severity of neutropenia were significantly higher in IFI patients. Consequently, the D-index and the c-D-index tended to be significantly higher in patients with IFIs, with a median of 10,150 (range: 4000-22,000) and 5300 (range: 2300-22,200), respectively. 103

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Table 1. Patient demographics and clinical characteristics.

Cases (n=15)

Controls (n=28)

Age, years (mean ± SD)

52.5±15.1

42.5±14.6

0.042

Sex (n, %) Male Female

7 8

46.7 53.3

15 13

53.6 46.4

Status of underlying disease (n, %) Induction Relapse Refractory

8 5 2

53.3 33.3 13.3

23 3 2

82.1 10.7 7.1

Bone marrow transplantation (n, %)

26.7

21.4

0.719

Chemotherapy regimen (n, %) Daun+Ara-C EMA Cyclosporine

12 2 1

80 13.3 6.7

24 2 2

85.7 7.1 7.1

0.801

Comorbidity (n, %)

6.7

10.7

1.000

0.666 0.122

Duration of neutropenia (<500/µL), median (minimum-maximum)

13 (6-53)

8.5 (5-22)

0.004

Duration of profound neutropenia (<100/µL), median (minimum-maximum)

9 (3-22)

4.5 (0-16)

0.005

D-index, median (minimum-maximum)

10,150 (4000-22,000)

3200 (1350-9200)

0.037

Cumulative-D-index, median (minimum-maximum)

5300 (2300-22,200)

3200 (1350-9200)

0.003

500 400 300 200 100 0

ROC Curve

Neutrophil curve 1.0

D-index AUC 1

9 10 5 6 7 8 Duration of Neutropenia (days)

Figure 1. The D-index is an index based on a graph showing absolute neutrophil counts over the course of the episode of neutropenia [8]. The ROC analyses showed that both the D-index and the c-Dindex could be used to predict IFIs [AUC ± standard error (SE): 0.914±0.042, p<0.001 and AUC ± SE: 0.779±0.074, p=0.003, respectively; Figures 2 and 3]. There was a cutoff point of 3875 for the D-index in predicting IFIs; the sensitivity, specificity, and positive and negative predictive values were 100%, 67.9%, 35.4%, and 100%, respectively. There was also a cutoff point of 4225 for the c-D-index in predicting IFI; the sensitivity, specificity, and positive and negative predictive values for the c-D-index were 93.3%, 71.4%, 36.6%, and 98.4%.

Discussion Fungal infections are responsible for most of the deaths from infections in febrile neutropenic patients with hematological malignancies, with mortality rates of 50%-80%. Although the initiation of appropriate antifungal therapy is crucial and associated with improved outcomes, the management of 104

0.8

12 Sensitivity

No. of Neutrophils, mm3

SD: Standard deviation

0.6

0.4

0.2

0.0

0.2

0.4

0.6

0.8

1- Specificity

Figure 2. Receiver operating characteristic analyses for the D-index. antifungal treatment in this heterogeneous population is a matter of great research [3,6,11,12,13,14]. Empirical antifungal therapy, which is the administration of systemic antifungals for persistent and recurrent fever 96 h after broad-spectrum antibacterial treatment, has been the standard of care for many years. Since the fever-based approach increased antifungal usage, preemptive or diagnostic-driven antifungal therapy, which is usually guided by clinical or radiological signs and serum biomarkers (galactomannan, 1,3-beta-D-glucan,

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respectively. However, the D-index and c-D-index still tended to be significantly higher for the IFI group than the controls when age and sex were adjusted [OR: 1.54, 95% confidence interval (CI): 1.28-2.19].

ROC Curve

1.0

Sensitivity

0.8 0.6 0.4 0.2 0.0

0.0

0.2

0.4

0.6

0.8

1.0

1- Specificity Diagonal segments are produced by ties

Figure 3. Receiver operating characteristic analyses for the cumulative-D-index. polymerase chain reaction), was defined [15]. Studies comparing these 2 approaches reported that the rate of antifungal usage was reduced and no increase in mortality was observed with diagnostic-driven antifungal therapy. However, the success of this strategy depends on the availability and the performance of the tests predicting IFI [15,16,17,18]. IFIs are difficult to predict and diagnose. Host factors are important for predicting IFIs as well, as they are the determinants of the outcome [6]. Neutropenia, one of the host factors, is still a significant risk factor, and resolution of neutropenia has a key role in complete recovery from an IFI [6,19]. The duration and also the severity of neutropenia are critical, but there was no practical tool that combined both the duration and the severity of neutropenia in its evaluation approach. Recently, Portugal et al. developed the D-index and c-D-index, simple indexes to calculate, which combine both the duration and the intensity of neutropenia [8]. They reported that these indexes were superior to the duration of neutropenia for predicting IFI. Shortly afterwards, Kimura et al. also showed that early pulmonary infections in hematopoietic stem cell transplantation recipients tended to occur in patients with higher D-index and c-D-index scores [20]. In accordance with these results, higher D-index and c-D-index scores were associated with IFIs in our study. We presume that the c-Dindex score in particular, available earlier than the D-index score, has the ability to discriminate among patients with the same duration but different severities of neutropenia according to IFI development. Previous studies documented increased risks for fungal infections in older patients [11,21,22]. In this study, univariate analysis showed that the median age was higher for patients with IFIs than the controls, at 52.5±15.1 and 42.5±14.6 years,

Although 20% of the stem cell transplantation centers in Turkey use diagnostic-driven approaches, empirical treatment is still the main approach [23]. This means that a significant proportion of patients are receiving antifungal therapy unnecessarily and we need helpful tools to assess the risk of IFI besides chest computed tomography scan and the use of serum biomarkers. The galactomannan test is the only available serum biomarker at our center. It is performed twice weekly but the results are reported with a 1-week delay. Hence, the c-D-index could be integrated with other parameters to promote diagnostic-driven therapy in such centers. The negative predictive values of the D-index and c-D-index for IFI prevalence of 15% was 100% (95% CI: 89.8-102.0) and 98.4% (95% CI: 87.2-101.6) using the cutoff values of 3875 and 4225, respectively. The high negative predictive values suggest that this new tool should work to exclude invasive fungal infections. Serum biomarkers such as galactomannan and betaglucan for fungal infections have some false positives. Thus, when interpreting the results in these situations, a c-D-index of less than 4225 supports the false positivity of other biomarkers and suggests that antifungal therapy could be delayed. This study has some limitations. The first is the small number of patients. Since April 2012, AML patients with induction therapy have started to receive antifungal prophylaxis (posaconazole) regularly at our center and the impact of this prophylaxis could not be assessed in this study. The second limitation is the underlying disease, due to the fact that only patients with AML were evaluated. Thus, we could not investigate the applicability of this new test with other malignancies.

Conclusion In conclusion, this study confirms that the D-index, and in particular the c-D-index, could be useful tools to exclude as well as to predict IFIs. These cheap and simple tests stand out with high negative predictive values in daily management of neutropenic patients. Ethics Ethics Committee Approval: The study was approved by the local ethics committee, Informed Consent: N/A. Authorship Contributions Medical Practices: Belgin Coşkun; Concept: Gülden Yılmaz, Design: Alpay Azap, Hamdi Akan; Data Collection or Processing: Belgin Coşkun, Gülden Yılmaz, Atilla Elhan; Analysis or 105

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Interpretation: Gülden Yılmaz, Belgin Coşkun, Atilla Elhan, Alpay Azap, Hamdi Akan; Literature Search: Gülden Yılmaz, Belgin Coşkun, Atilla Elhan, Alpay Azap, Hamdi Akan; Writing: Gülden Yılmaz, Belgin Coşkun, Atilla Elhan, Alpay Azap, Hamdi Akan. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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10. Usansky JI, Desai A, Tang-Liu D. PK Functions for Microsoft Excel. Irvine, CA, USA, Department of Pharmacokinetics and Drug Metabolism of Allergan Inc. (available at www. boomer.org/pkin/xcel/pkf/pkf.doc). 11. Marr KA, Carter RA, Boeckh M, Martin P, Corey L. Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors. Blood 2002;100:4358-4366. 12. Li L, Wang J, Zhang W, Yang J, Chen L, Lv S. Risk factors for invasive mold infections following allogeneic hematopoietic stem cell transplantation: a single center study of 190 recipients. Scand J Infect Dis 2012;44:100-107. 13. Marr KA, Carter RA, Crippa F, Wald A, Corey L. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin Infect Dis 2002;34:909-917. 14. Upton A, Kirby KA, Carpenter P, Boeckh M, Marr KA. Invasive aspergillosis following hematopoietic cell transplantation: outcomes and prognostic factors associated with mortality. Clin Infect Dis 2007;44:531-540. 15. Maertens J, Theunissen K, Verhoef G, Verschakelen J, Logrou K, Verbeken E, Wilmer A, Verhaegen J, Boogaerts M, Van Eldere J. Galactomannan and computed tomography based preemptive antifungal therapy in neutropenic patients at high risk for invasive fungal infection: a prospective feasibility study. Clin Infect Dis 2005;41:1242-1250. 16. Cordonnier C, Pautas C, Maury S, Vekhoff A, Farhat H, Suarez F, Dhédin N, Isnard F, Ades L, Kuhnowski F, Foulet F, Kuentz M, Maison P, Bretagne S, Schwarzinger M. Empirical versus preemptive antifungal therapy for high risk, febrile, neutropenic patients: a randomized, controlled trial. Clin Infect Dis 2009;48:1042-1051. 17. Hebart H, Klingspor L, Klingebiel T, Loeffler J, Tollemar J, Ljungman P, Wandt H, Schaefer-Eckart K, Dornbusch HJ, Meisner C, Engel C, Stenger N, Mayer T, Ringden O, Einsele H. A prospective randomized controlled trial comparing PCR based and empirical treatment with liposomal amphotericin B in patients after allo-SCT. Bone Marrow Transplant 2009;43:553-561. 18. Maschmeyer G, Heinz WJ, Hertenstein B, Horst HA, Requadt C, Wagner T, Cornely OA, Löffler J, Ruhnke M; IDEA study investigators. Immediate versus deferred empirical antifungal (IDEA) therapy in high-risk patients with febrile neutropenia: a randomized, double-blind, placebo-controlled, multicenter study. Eur J Clin Microbiol Infect Dis 2013;32:679-689. 19. Cesaro S, Pagano L, Caira M, Carraro F, Luciani M, Russo D, Colombini A, Morello W, Viale P, Rossi G, Tridello G, Pegoraro A, Nosari A, Aversa F; Hemae-chart Group. A prospective, multicenter survey on antifungal therapy in neutropenic pediatric haematology patients. Mycoses 2013;56:21-25. 20. Kimura S, Oshima K, Sato K, Sato M, Terasako K, Hideki N, Kikuchi M, Okuda S, Yamazaki R, Tanaka Y, Tanihara A, Nishida J, Kanda Y. Retrospective evaluation of the area over the neutrophil curve index to predict early infection in hematopoietic stem cell transplantation recipients. Biol Blood Marrow Transplant 2010;16:1355-1361. 21. Wald A, Leisenring W, van Burik J, Bowden RA. Epidemiology of Aspergillus infections in a large cohort of patients undergoing bone marrow transplantation. J Infect Dis 1997;175:1459-1466. 22. Baddley J, Stroud T, Salzman D, Pappas P. Invasive mold infections in allogeneic bone marrow transplant recipients. Clin Infect Dis 2001;32:13191324. 23. Akan H. Antifungal treatment in stem cell transplantation centers in Turkey. In: 23rd European Congress of Clinical Microbiology and Infectious Diseases, Berlin, Germany, 27-30 April 2013.

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0242 Turk J Hematol 2016;33:107-111

Gap-PCR Screening for Common Large Deletional Mutations of β-Globin Gene Cluster Revealed a Higher Prevalence of the Turkish Inversion/Deletion (δβ)0 Mutation in Antalya β-Globin Gen Kümesini İçine Alan Büyük Delesyonel Mutasyonların Gap-PCR ile Taranması Türk Tipi İnversiyon/Delesyon (δβ)0 Mutasyonunun Antalya’da Yüksek Sıklıkta Olduğunu Gösterdi Türker Bilgen1,2, Özden Altıok Clark3, Zeynep Öztürk4, M. Akif Yeşilipek4, İbrahim Keser1 1Akdeniz University Faculty of Medicine, Department of Medical Biology and Genetics, Antalya, Turkey 2Namık Kemal University Central Research Laboratory (NABİLTEM), Tekirdağ, Turkey 3Akdeniz University Faculty of Medicine, Department of Medical Genetics, Antalya, Turkey 4Akdeniz University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Antalya, Turkey

Abstract

Öz

Objective: Although the calculated carrier frequency for point mutations of the β-globin gene is around 10% for Antalya Province, nothing is known about the profile of large deletional mutations involving the β-globin gene. In this study, we aimed to screen common deletional mutations in the β-globin gene cluster in patients for whom direct DNA sequencing was not able to demonstrate the mutation(s) responsible for the disease phenotype.

Amaç: Beta-globin genindeki nokta mutasyonlarının sıklığı Antalya bölgesi için yaklaşık %10 olarak belirlenmiş olmasına rağmen, betaglobin genini içine alan büyük delesyonel tip mutasyonların profili hakkında hiçbir şey bilinmemektedir. Bu çalışmada, DNA dizi analizi yöntemiyle beta-globin geninde hastalığın oluşmasından sorumlu mutasyon(lar) tespit edilememiş talasemili olgularda beta-globin gen kümesinde yaygın görülen büyük delesyonel mutasyonları taramayı amaçladık.

Materials and Methods: Thirty-one index cases selected with a series of selection events among 60 cases without detected β-globin gene mutation from 580 thalassemia-related cases tested by direct sequencing over the last 4 years in our diagnostic center were screened for the most common 8 different large deletional mutations of the β-globin gene cluster by gap-PCR. Results: We detected 1 homozygous and 9 heterozygous novel unrelated cases for the Turkish inversion/deletion (δβ)0 mutation in our series of 31 cases. Our study showed that the Turkish inversion/ deletion (δβ)0 mutation per se accounts for 16.6% of the unidentified causative alleles and also accounts for 1.5% of all detected mutations over the last 4 years in our laboratory. Conclusion: Since molecular diagnosis of deletional mutations in the β-globin gene cluster warrants different approaches, it deserves special attention in order to provide prenatal diagnosis and prevention opportunities to the families involved. We conclude that the Turkish inversion/deletion (δβ)0, as the most prevalent deletional mutation detected so far, has to be routinely tested for in Antalya, and the gapPCR approach has valuable diagnostic potential in the patients at risk. Keywords: Deletional mutations, Turkish inversion/deletion (δβ)0 mutation, Gap-PCR, β-Globin gene cluster

Address for Correspondence/Yazışma Adresi: Türker BİLGEN, PhD., Namık Kemal University Central Research Laboratory (NABİLTEM), Tekirdağ, Turkey E-mail : tbilgen@nku.edu.tr, tbilgen@akdeniz.edu.tr

Gereç ve Yöntemler: Son dört yıl boyunca tanı merkezimizde DNA dizi analizi yöntemiyle test edilmiş, talasemiyle ilişkilendirilen 580 olgu arasından öncelikle beta-globin geni mutasyonu belirlenememiş 60 olgu seçildi. Bu 60 olgu arasından bir seri seleksiyon işlemi uygulanarak nihai olarak belirlenmiş 31 hasta, beta-globin gen kümesinde en yaygın görülen sekiz farklı büyük delesyonel tip mutasyon için gapPCR yöntemiyle tarandı. Bulgular: Otuz bir olgudan oluşan serimiz içerisinde, Türk tipi inversiyon/delesyon (δβ)0 mutasyonu açısından heterozigot olan dokuz yeni olgu ve homozigot olan bir yeni olgu belirlendi. Çalışmamız Türk tipi inversiyon/delesyon (δβ)0 mutasyonunun, laboratuvarımızda son dört yıl boyunca tespit edilmiş tüm mutasyonların %1,5’ini ve DNA dizi analizi yöntemiyle mutasyon tespit edilemeyen alellerin ise %16,6’sını oluşturduğunu gösterdi. Sonuç: Beta-globin gen kümesinde delesyonel tip mutasyonlar farklı moleküler yöntemlerle tespit edilebilir. Bu durum prenatal teşhis ve hastalığı önleme fırsatı sağlayabildiği için özel bir ilgi gerektirmektedir. Sonuç olarak, toplumumuzda şu ana kadar belirlenmiş en sık görülen delesyonel tip mutasyon olan Türk tipi inversiyon/delesyon (δβ)0 mutasyonu Antalya’da rutin olarak test edilmelidir ve gap-PCR yöntemi risk altındaki hastalar için önemli bir tanı potansiyeline sahiptir. Anahtar Sözcükler: Delesyonel mutasyonlar, Türk tipi inversiyon/ delesyon (δβ)0 mutasyonu, Gap-PCR, Beta-globin gen kümesi Received/Geliş tarihi: June 19, 2014 Accepted/Kabul tarihi: October 28, 2014

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Bilgen T, et al: Screening of β-Globin Gene Cluster Deletions

Introduction Beta-thalassemia (β-thal) is generally caused by point mutations in the β-globin gene. However, there are at least 80 different large deletional mutations in the β-globin gene cluster described in the human hemoglobin variant (HbVar) database. While only the β-globin gene is partially or completely removed in some of those deletions, the δ-globin gene or δ- and γ-globin genes are deleted in addition to the β-globin gene in some others [1,2]. It was also stated that 10% of the β-globin gene mutations are large deletions causing phenotypes associated with β-thal [3]. The phenotypes produced by deletions in the β-globin gene cluster are classified according to the gene(s) involved, such as β-thal, δβ-thal, εγδβ-thal, and hereditary persistence of fetal hemoglobin (HPFH) [4]. Despite general carrier frequency for β-globin gene mutations being reported at 2% for Turkey and at as high as 10% for Antalya Province, large deletional mutations in the β-globin gene cluster have rarely been reported so far and there is no systemic study on mutation profiles of large deletional mutations in the β-globin gene cluster in Turkey [4,5,6,7,8,9]. On the other hand, the number of studies on variety and allelic frequencies of large deletions in the β-globin gene cluster has been growing recently [2,3,10,11,12]. Previous studies revealed that HPFH-1, HPFH-2, HPFH-3, Sicilian (δβ)0thal, Chinese Gγ(Aγδβ)0-thal, Hb Lepore, Asian-Indian inversiondeletion Gγ(Aγδβ)0-thal, and Turkish inversion-deletion (δβ)0thal mutations are among the most recurrent large deletional mutations in the β-globin gene cluster [10,13]. Detection of large deletions of the β-globin gene cluster has recently become an important issue because of its significance in evaluation of unresolved thalassemia-related cases and in disease prevention. On the other hand, commonly used diagnostic tests targeting point mutations and small insertionsdeletions of the β-globin gene are not suitable for detection of large deletional mutations. Therefore, molecular detection of large deletions needs different approaches in the laboratory. Researchers have recently applied strategies like Southern blotting, FISH, quantitative polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), and gap-PCR for molecular detection of large deletional mutations of the β-globin gene cluster [10,12,14,15,16]. Among them, gap-PCR is a fast and reliable method allowing us to detect the previously characterized mutations [3,13]. In this study, we screened patients in whom we were not able to find the underlying β-globin gene mutation(s) by direct DNA sequencing for the 8 different common deletional mutations of the β-globin gene cluster by gap-PCR.

Materials and Methods Patients Among the 580 patients who were tested in our diagnostic laboratory for β-globin gene mutations by direct DNA 108

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sequencing between July 2008 and July 2012, a total of 60 unrelated patients who had either no causative β-globin gene mutation(s) by sequencing or no detectable PCR amplification for the β-globin gene were initially selected. Being homozygous for all common intragenic single-nucleotide polymorphisms detected by sequence analyses was then used as the second inclusion criterion for its potential to indicate hemizygosity. Finally, the 31 most probable candidates were screened by gapPCR for the 8 different known deletions of the β-globin gene cluster. Out of these 31 patients included in the study, 21 had a mild phenotype without any β-globin gene mutation, while the remaining 10 were moderately to seriously affected by the disease with either one or no detected causative mutations. All hematological and clinical findings were collected with the informed consent of the patients. Hematological indices were obtained with an automated cell counter (Abbott Cell DYN3700; Abbott Laboratories, Abbott Park, IL, USA). The HbA2 and HbF levels were measured by high-performance liquid chromatography (VARIANT; Bio-Rad Laboratories, Hercules, CA, USA). Sequence Analyses and Gap-PCR Screening for the 8 Known Deletional Mutations of the β-Globin Gene Following the isolation of genomic DNA with a commercial kit (AxyPrep Blood Genomic DNA Miniprep Kit; Axygen Biosciences Inc., Union City, CA, USA), the β-globin gene was amplified as 2 PCR fragments (from the -101 position to the Poly-A signal) using 30-50 ng of genomic DNA in 25-µL reaction volumes. The PCR mixture contained 12.5 µL of 2X PCR master mix and 5 pmol of each primer (GML, Wollerau, Switzerland). The sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The deletional mutations were chosen by taking into account ethnic background and according to the published frequencies [10]. Gap-PCR protocols and the primers for the deletional mutations HPFH-1, HPFH-2, HPFH-3, Sicilian (δβ)0-thal, Chinese Gγ(Aγδβ)0-thal, Hb Lepore, Asian-Indian inversion-deletion Gγ(Aγδβ)0-thal, and Turkish inversion-deletion (δβ)0-thal were used as previously described elsewhere [13].

Results Among the 8 different known deletions of the β-globin gene cluster mentioned above, only the Turkish inversion-deletion (δβ)0 mutation was detected in 10 patients in our series. We found that 9 were heterozygous and 1 was homozygous for the Turkish inversion-deletion (δβ)0 mutation. The hematological indices and molecular findings of 7 heterozygous patients and 1 homozygous patient are summarized in Table 1. Hematological indices were not available for 2 heterozygous patients, males

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Bilgen T, et al: Screening of β-Globin Gene Cluster Deletions

as a technical limitation of gap-PCR; in addition, its usage is limited to known deletional mutations. Gap-PCR analyses of the parents of the patient would help to clarify such a situation; however, we were not able to perform this analysis in this family.

Figure 1. Representative samples of Turkish-type inversion/ deletion (δβ)0 mutation detected by gap-PCR. For reaction A testing the upstream breakage of the mutation, the upper band (742 bp) corresponds to normal results and the lower band (432 bp) to the mutation. Case 4 and Case 5 are heterozygous as both have normal and mutation-related polymerase chain reaction fragments. For reaction B testing the downstream breakage of the mutation, the upper band (700 bp) corresponds to normal results and the lower band (489 bp) to the mutation. Cases 4, 5, 6, and 7 show both normal and mutation-related polymerase chain reaction fragments, confirming that they are heterozygous for the mutation. NS: Normal sample, N: normal, M: mutation, 252x91 mm (72x72 dpi). of 24 and 16 years old. Sequence analyses of mutation-related gap-PCR bands of 10 patients showed that there was no variation in sequence or at breakpoints of the Turkish inversiondeletion (δβ)0 mutation. Sequence analyses determined that the exact breakpoints positions were 5.255,764 and 5.244,281 for upstream deletion (11,484 bp) and 5.236,654 and 5.235,062 (1592 bp) for downstream deletion according to NCBI reference sequence NC_000011.9, chromosome 11 GRCh37.p13 primary assembly.

Discussion While nearly 25 different β-globin gene mutations have been reported for Antalya Province as well as for Turkey so far, the large deletional-type mutations of the β-globin gene cluster have not been systematically investigated [4,6,17]. It has been suggested that 10% of the causative alleles cannot be easily detected by routine methods in β-thal-associated phenotypes [18]. This proportion in our survey was similar to the literature. Large deletional mutations might be somewhat responsible for this challenge. In this regard, the Turkish inversion/deletion (δβ)0 mutation per se accounts for approximately 16.6% of the unidentified causative alleles and accounts for 1.5% of all detected mutations from the last 4 years in our laboratory. Among the 10 new unrelated cases of the Turkish inversiondeletion (δβ)0 mutation detected in this study, only one seemed to be homozygous. The gap-PCR technique is able to reliably detect the heterozygous state of this type of mutation by showing both normal and mutation-related bands on agarose gel (Figure 1). On the other hand, this technique does not exclude the possibility of the presence of another larger deletional mutation such as the second mutation in the patient found as homozygous in our study. This should be considered

It has been demonstrated that the deletions in the β-globin gene cluster may cause HPFH, which is characterized by high HbF levels reducing the disease severity [19]. While the patients with deletions including the δ- and β-globin genes tend to have mild phenotypes, patients with larger deletions involving γ-globin genes have severe clinical phenotypes because of the lack of the compensatory effect of fetal Hb [20]. Furthermore, recent studies on hemoglobin switching events have revealed that there is a binding site between the δ- and γ-globin genes for BCL11, which is a repressor of γ-globin genes. The deletion of this cis-acting element seems to be related to higher HbF levels [9]. The Turkish type of inv/del (δβ)0 thalassemia was first characterized at the molecular level by Kulozik et al. in a Turkish patient living in Germany with normal HbA2 and elevated HbF levels in 1992 [21]. It was also associated with elevated HbF and normal HbA2 levels in another later study [22]. Our study revealed that 7 out of 9 patients carrying the Turkish inv/del (δβ)0 had elevated HbF levels, while the remaining 2 had normal HbF levels. This controversial observation can be explained by other factors that may modify the hematological expression of this mutation. Such a situation was reported in δβ-thalassemia before by Öner et al. [23]. This phenomenon shows that a small proportion of the carriers of the Turkish inv/del (δβ)0 mutation may not have elevated HbF levels, which should be considered in case selection for mutation screening. Another important point is that the molecular detection of large deletional mutations in the β-globin gene cluster is extremely important for families at risk and seeking prevention. Because their detection requires special attention, this type of mutation may sometimes compromise the prenatal diagnosis in laboratories used to focusing on point mutations and small ins/del-type mutations of the β-globin gene. Despite not being useful for previously uncharacterized deletions, gapPCR is the easiest and most precise way of detecting previously characterized recurrent deletions. For these reasons, and taking into account the relatively higher incidence of the Turkish-type inv/del (δβ)0 mutation in Antalya Province, we suggest that it is worthwhile to screen for this mutation in Turkish patients when the first-line diagnostic tests such as sequencing and strip assay fail to detect the causative mutation(s). Gap-PCR is the cheapest and fastest method for the detection of large deletional mutations. Nevertheless, the approach has specific requirements for being used as a diagnostic tool, such as positive controls, and the targeted mutation has to be 109

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Table 1. Hematological findings of the patients with Turkish inversion-deletion (δβ)0 mutation. Case (δβ)-Thalassemia Heterozygotes

(δβ)-Thalassemia

Age (years)/Sex

β-Globin Gene Mutation(s)

HbA2*

HbF (%)

(g/dL)

11/M

Turk inv-del (δβ)0/N

2.6 13.5

35/M

Turk inv-del (δβ)0/N

55/F

MCV

MCH (pg/cell)

RBC

RDW

(fL)

MCHC (g/dL)

(106/µL)

(%)

11.2

66.5

32.0

21.3

5.28

19.2

2.3 11.8

13.4

73.2

31.7

23.2

5.77

23.9

Turk inv-del (δβ)0/N

2.6 7.4

11.7

68.1

27.6

18.8

5.9

21.9

34/M

Turk inv-del (δβ)0/N

2.9 9.8

14.5

65.2

31.7

20.7

7.03

22.5

13/M

Turk inv-del (δβ)0/N

2.8 7.7

11.8

68.5

30.50

20.9

5.66

17.3

14/M

Turk inv-del (δβ)0/N

2.7 1.9

11.9

62.2

31.5

19.6

6.1

16.2

27/M

Turk inv-del (δβ)0/N

2.4 0

14.1

64.4

32.2

20.7

6.8

54.8

48/M

Turk inv-del (δβ)0/N Turk inv-del (δβ)0

0 100

74.8

31.7

23.7

5.5

22.3

F: Female, M: male, Hb: hemoglobin, WBC: white blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RBC: red blood cell, RDW: Red blood cell distribution width. *Normal HbA2 levels (between 1.5% and 3.8%) according to laboratory reference values.

previously well described. Having positive controls is important for optimization and validation of gap-PCR. Without welloptimized protocols, gap-PCR should not be used as a routine diagnostic method. In addition to the possibility of false negativity, positive results should also be confirmed by family study when the parents are available. We had positive controls for the Turkish-type inv/del (δβ)0 mutation prior to this study, but not for the other types of mutations that we screened. This could be considered as a limitation of our study. The other patients in whom we could detect none of the deletions screened in our study are strong candidates for screening for either other previously described but rarer or completely novel deletional mutations. Therefore, there is need for further analyses in order to resolve these cases. MLPA and array comparative genomic hybridization methods are strong tools to investigate possible novel and rare deletional mutations. MLPA is currently the more commonly used approach for detection of large deletions affecting a particular region of the genome, but its coverage is limited to the probe set designed. We are planning a MLPA study for the patients who had no positive findings in our gapPCR screening. On the other hand, not only the patients whose mutation(s) were not identified but also even homozygous patients for one particular parental β-globin gene mutation detected by sequencing or strip assay should be investigated for deletional mutations in order to find out the exact second hit leading to thalassemia intermedia or major phenotypes. 110

Ethics Ethics Committee Approval: Retrospective study, Informed Consent: It was taken. Authorship Contributions Surgical and Medical Practices: M. Akif Yeşilipek; Concept: Türker Bilgen; Design: Türker Bilgen; Data Collection or Processing: Türker Bilgen, Özden Altıok Clark, Zeynep Öztürk, M. Akif Yeşilipek, İbrahim Keser; Analysis or Interpretation: Türker Bilgen, İbrahim Keser; Literature Search: Türker Bilgen; Writing: Türker Bilgen, İbrahim Keser. 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. Giardine B, van Baal S, Kaimakis P, Riemer C, Miller W, Samara M, Kollia P, Anagnou NP, Chui DH, Wajcman H, Hardison RC, Patrinos GP. HbVar database of human hemoglobin variants and thalassemia mutations: 2007 update. Hum Mutat 2007;28:206. 2. Phylipsen M, Prior JF, Lim E, Lingam N, Vogelaar IP, Giordano PC, Finlayson J, Harteveld CL. Thalassemia in Western Australia: 11 novel deletions characterized by multiplex ligation-dependent probe amplification. Blood Cells Mol Dis 2010;44:146-151. 3. Tritipsombut J, Phylipsen M, Viprakasit V, Chalaow N, Sanchaisuriya K, Giordano PC, Fucharoen S, Harteveld CL. A single-tube multiplex gappolymerase chain reaction for the detection of eight β-globin gene cluster deletions common in Southeast Asia. Hemoglobin 2012;36:571-580.

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4. Başak AN. The molecular pathology of β-thalassemia in Turkey: the Boğaziçi University experience. Hemoglobin 2007;31:233-241. 5. Bilgen T, Arikan Y, Canatan D, Yeşilipek A, Keser I. The association between intragenic SNP haplotypes and mutations of the beta globin gene in a Turkish population. Blood Cells Mol Dis 2011;46:226-229. 6. Keser I, Sanlioglu AD, Manguoglu E, Guzeloglu Kayisli O, Nal N, Sargin F, Yesilipek A, Simsek M, Mendilcioglu I, Canatan D, Luleci G. Molecular analysis of beta-thalassemia and sickle cell anemia in Antalya. Acta Haematol 2004;111:205-210. 7. Mendilcioglu I, Yakut S, Keser I, Simsek M, Yesilipek A, Bagci G, Luleci G. Prenatal diagnosis of β-thalassemia and other hemoglobinopathies in southwestern Turkey. Hemoglobin 2011;35:47-55. 8. Öner C, Öner R, Balkan H, Gürgey A, Yalçın A, Avcu F, Altay Ç. Molecular analysis of the Turkish form of deletion-inversion (δβ)0 thalassaemia. Br J Haematol 1997;96:229-234. 9. Öner R, Öner C, Erdem G, Balkan H, Özdağ H, Erkan M, Gümrük F, Gürgey A, Altay Ç. A novel (δβ)0-thalassemia due to a ~30-kb deletion observed in a Turkish family. Acta Haematol 1996;96:232-236. 10. Gallienne AE, Dreau HM, McCarthy J, Timbs AT, Hampson JM, Schuh A, Old JM, Henderson SJ. Multiplex ligation-dependent probe amplification identification of 17 different β-globin gene deletions (including four novel mutations) in the UK population. Hemoglobin 2009;33:406-416. 11. Cui J, Azimi M, Baysdorfer C, Vichinsky EP, Hoppe CC. Application of multiplex ligation-dependent probe amplification to screen for β-globin cluster deletions: detection of two novel deletions in a multi ethnic population. Hemoglobin 2013;37:241-256.

Bilgen T, et al: Screening of β-Globin Gene Cluster Deletions 14. Mikula M, Buller-Burckle A, Gallivan M, Sun W, Franklin CR, Strom CM. The importance of β globin deletion analysis in the evaluation of patients with β thalassemia. Int J Lab Hematol 2011;33:310-317. 15. Joly P, Lacan P, Garcia C, Couprie N, Francina A. Identification and molecular characterization of four new large deletions in the β-globin gene cluster. Blood Cells Mol Dis 2009;43:53-57. 16. Voruganti I, Eng B, Waye JS. Molecular characterization of a novel 55.1 kb Gγ(Aγδβ)0-thalassemia deletion in two Canadian families. Hemoglobin 2009;33:422-427. 17. Henderson S, Timbs A, McCarthy J, Gallienne A, Van Mourik M, Masters G, May A, Khalil MS, Schuh A, Old J. Incidence of haemoglobinopathies in various populations - the impact of immigration. Clin Biochem 2009;42:1745-1756. 18. Gu X, Zeng Y. A review of the molecular diagnosis of thalassemia. Hematology 2002;7:203-209. 19. Lee ST, Yoo EH, Kim JY, Kim JW, Ki CS. Multiplex ligation-dependent probe amplification screening of isolated increased HbF levels revealed three cases of novel rearrangements/deletions in the β-globin gene cluster. Br J Haematol 2010;148:154-160. 20. Phylipsen M, Amato A, Cappabianca MP, Traeger-Synodinos J, Kanavakis E, Basak N, Galanello R, Tuveri T, Ivaldi G, Harteveld CL, Giordano PC. Two new β-thalassemia deletions compromising prenatal diagnosis in an Italian and a Turkish couple seeking prevention. Haematologica 2009;94:1289-1292. 21. Kulozik AE, Bellan-Koch A, Kohne E, Kleihauer E. A deletion/inversion rearrangement of the beta-globin gene cluster in a Turkish family with delta beta zero-thalassemia intermedia. Blood 1992;79:2455-2459.

12. So CC, So AC, Chan AY, Tsang ST, Ma ES, Chan LC. Detection and characterisation of β-globin gene cluster deletions in Chinese using multiplex ligation-dependent probe amplification. J Clin Pathol 2009;62:1107-1111.

22. Babashah S, Jamali S, Mahdian R, Nosaeid MH, Karimipoor M, Alimohammadi R, Raeisi M, Maryami F, Masoudifar M, Zeinali S. Detection of unknown deletions in β-globin gene cluster using relative quantitative PCR methods. Eur J Haematol 2009;83:261-269.

13. Craig JE, Barnetson RA, Prior J, Raven JL, Thein SL. Rapid detection of deletions causing δβ thalassemia and hereditary persistence of fetal hemoglobin by enzymatic amplification. Blood 1994;83:1673-1682.

23. Öner C, Gurgey A, Altay C, Kutlar F, Huisman TH. Variation in the level of fetal hemoglobin in (δβ)0-thalassemia heterozygotes with different numbers of α-globin genes. Am J Hematol 1990;34:230-231.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0312 Turk J Hematol 2016;33:112-118

The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin Profilaktik Enoksaparin Alan Sepsis Hastalarında Doku Faktör Yolak İnhibitörü Düzeyleri Hadil A. Al Otair1, Abdel Galil M. Abdel Gader2, Syed M. Khurshid1, Abdulaziz H. Alzeer1, Abdul Kareem Al Momen3, Mashael Al Shaikh4, Farja Al Gahtani3, Zohair A. Al Aseri5, Hossam A.H. Abdelrazik5 1King Saud University College of Medicine, King Khalid University Hospital, Department of Critical Care, Riyadh, Saudi Arabia 2King Saud University College of Medicine, King Khalid University Hospital, Department of Physiology, Riyadh, Saudi Arabia 3King Saud University College of Medicine, King Khalid University Hospital, Department of Medicine, Riyadh, Saudi Arabia 4King Saud University College of Medicine, King Khalid University Hospital, Department of Pharmacy, Riyadh, Saudi Arabia 5King Saud University College of Medicine, King Khalid University Hospital, Department of Emergency, Riyadh, Saudi Arabia

Abstract

Öz

Objective: Sepsis syndrome is usually accompanied by activation of blood coagulation mechanisms. Earlier studies found deficiencies of the 3 main natural anticoagulants, antithrombin, protein C, and protein S. However, none of these inhibitors block tissue factor, the prime trigger of coagulation during sepsis that is controlled specifically by the tissue factor pathway inhibitor (TFPI). The aim of this study was to characterize the fluctuations in the levels of natural anticoagulants, particularly TFPI, in the course of sepsis and to find out their association with the anticoagulant action of the lowmolecular-weight heparin enoxaparin.

Amaç: Sepsis sendromuna genellikle kan pıhtılaşma sisteminin aktivasyonu eşlik eder. İlk çalışmalar ana doğal 3 antikoagülan olan antitrombin, protein C ve protein S eksikliği bulmuştur. Bununla birlikte, bu inhibitörlerin hiç biri doku faktörü bloke etmez, sepsis sırasındaki koagülasyon tetiklenişi özelllikle doku faktör yolak inhibitörü (DFYİ) ile kontrol edilir. Bu çalışmanın amacı sepsis sırasındaki doğal antikoagülan ve özellikle DFYİ düzeyi dalgalanmalarını karakterize etmek ve bunların düşük moleküler ağırlıklı heaprin enoksaparinin antikoagülan eylemi ile ilişkilerini öğrenmekti.

Materials and Methods: We studied 51 consecutive patients with sepsis. Blood samples were collected from patients at baseline (0 h) and at 4, 12, and 24 h after enoxaparin administration. The following assays were undertaken using commercial kits: activated partial thromboplastin time, prothrombin time, thrombin time, total and free TFPI, protein C and protein S, antithrombin, fibrinogen, and anti-factor Xa.

Gereç ve Yöntemler: Ardışık 51 sepsis hastası çalışmaya alındı. Taban (0 saat) ve enoksaparin verimesinden 4, 12, 24 saat sonra kan örnekleri alındı. Aşağıdaki deneyler ticari kitleri kullanılarak yapılmıştır; parsiyel tromboplastin zamanı, protrombin zamanı, trombin zamanı, toplam ve serbest DFYİ, protein C ve protein S, antitrombin, fibrinojen, ve aktif anti-faktör Xa.

Results: Before enoxaparin administration, there was significant prolongation of the prothrombin time and activated partial thromboplastin time, and this remained the case in the 3 subsequent samples. There was marked reduction in the levels of antithrombin, protein C, and total and free protein S to below control values throughout the study. In contrast, plasma levels of both total and free TFPI were markedly elevated and increased after enoxaparin therapy. Anti-factor Xa levels were within the therapeutic range throughout. There was no difference in TFPI levels between those patients who died and those who survived.

Bulgular: Enoksaparin uygulamadan önce ptorombin zamanı ve aktif parsiyel protrombin zamanında önemli uzama vardı. Bu durum sonraki 3 örneklemde de devam etti. Çalışma boyunca antitrombin, protein C, toplam ve serbest protein S seviyeleri değerlerinde kontrollere göre belirgin bir azalma oldu. Buna karşılık, hem toplam hem de serbest plazma DFYİ değerleri belirgin biçimde yükseldi ve enoksaparin tedavisinden sonra arttı. Anti faktör Xa düzeyleri terapötik aralık içindeydi. Vefat eden ve sağ kalan hastalar arasında DFYİ düzeyi açısından fark yoktu.

Conclusion: Sepsis triggered marked release of TFPI from endothelial cells. This persisted and was increased further following the administration of enoxaparin. In contrast, there was marked consumption of the natural coagulation inhibitors antithrombin, protein C, and protein S. These results go some way towards explaining why the therapeutic use of recombinant TFPI fails to correct sepsisassociated coagulopathy. Keywords: Coagulation, Sepsis, Enoxaparin Address for Correspondence/Yazışma Adresi: Hadil A. AL OTAIR, M.D., King Saud University College of Medicine, King Khalid University Hospital, Department of Critical Care, Riyadh, Saudi Arabia Phone : +96611-4692253 E-mail : hadil.alotair@live.com

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Sonuç: Sepsis, endotel hücrelerinden belirgin DFYİ salınımı ile tetiklenir. Bu, enoksaparin uygulmasını takiben kalıcı olmuş ve daha da artmıştır. Bunun aksine, doğal koagülasyon inhibitörleri antitrombin, protein C ve protein S’nin belirgin tüketimi vardı. Bu sonuçlar, tedavi amaçlı rekombinant DFYİ kullanımının sepsis ilişkili koagülopatiyi düzeltmek için neden başarısız olduğunu doğru biçimde açıklamaktadır. Anahtar Sözcükler: Koagülasyon, Sepsis, Enoksaparin Received/Geliş tarihi: August 03, 2014 Accepted/Kabul tarihi: January 15, 2015

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Introduction

Materials and Methods

Sepsis syndrome results from a host reaction to infection that triggers the systemic inflammatory response syndrome, which, on one hand, activates procoagulation mechanisms, and, on the other, shuts down fibrinolysis, leading to the formation of fibrin microthrombi in microcirculation and multiple organ failure [1,2]. In its worst form, the interaction between inflammation and the coagulation system may lead to the development of disseminated intravascular coagulation [3,4].

Study Population

Over the last 3 decades, numerous reports have emerged that describe disturbances in the measured levels of coagulation parameters in patients with sepsis [5,6,7,8,9]. Much emphasis has been focused on the deficiencies of the 3 main natural coagulation inhibitors: antithrombin (AT), activated protein C, and tissue factor pathway inhibitor (TFPI) [9,10,11,12]. This led to numerous clinical therapeutic trials of administering these inhibitors to patients with sepsis. Some success was initially obtained with the administration of activated protein C, but later on, the PROWESS-SHOCK trial showed an increased risk of bleeding with the use of activated protein C, with no mortality benefit. Similarly, trials with AT and recombinant TFPI generated disappointing results [13,14]. The resultant procoagulant state associated with sepsis has also been recognized as an important risk factor for venous thromboembolism in critically ill patients [14,15]. Therefore, deep vein thrombosis prophylaxis is considered of utmost importance and is practiced with vigilance in intensive care units (ICU) using unfractionated heparin and low-molecular-weight heparin (LMWH) [16,17]. LMWH exerts its antithrombotic effect mainly by inhibiting activated factor X (FXa) and to a lesser degree AT [18]. Nevertheless, failure of deep vein thrombosis prophylaxis in critically ill patients has been well described [17,19]. The reason for this is thought to be multifactorial and one possible proposed explanation could be related to lower anticoagulant effect (as assessed by anti-FXa activity) in these patients, despite appropriate LMWH dosage [20]. The recent availability of more precise assay techniques for the measurement of the natural anticoagulants, particularly total and free TFPI and protein S, encouraged us to monitor the fluctuations of natural anticoagulants in patients with sepsis, in a way that no study has done before, to find a possible explanation for why past trials administering natural anticoagulants to patients with sepsis failed. Therefore, the aim of this study was to assess the levels of natural anticoagulants, particularly total and free TFPI, in patients with sepsis and septic shock and to find out the association between these fluctuations and the anticoagulant action of the LMWH enoxaparin.

Fifty-one consecutive patients were studied; 29 were male and 22 female, with a mean age of 51Âą20.8 years. All were admitted to the ICU of King Khalid University Hospital, Riyadh, with sepsis or septic shock. Sepsis is defined as systemic inflammatory response syndrome due to infection [1,2]. Septic shock is defined as severe sepsis-induced hypotension that persists despite adequate fluid resuscitation [1,6]. Exclusion criteria were patients younger than 18 years old, body weight of <45 kg or >148 kg, renal insufficiency (creatinine clearance of <30 mL/min), active bleeding, platelet count of <75,000 mm3, INR of >2, activated partial thromboplastin time (APTT) of >2 times the upper normal, therapeutic anticoagulation, pregnancy, porcine hypersensitivity, and administration of unfractionated heparin or LMWH prior to enrollment in the study. Controls (n=42) were healthy individuals (28 males) selected from blood donors, academic staff, and volunteers from the general public. Their ages ranged from 21 to 62 years (mean: 47.4). They were not taking any form of medication at the time of blood sampling. The study was approved by the Institutional Review Board of the College of Medicine-King Saud University. Written informed consent was obtained from all patients or their next of kin. Data Collection A data entry form was used for the collection of patientsâ&#x20AC;&#x2122; demographic data and clinical information as well as laboratory results. Enoxaparin (Clexane R, Aventis Pharma, Frankfurt, Germany), which is a LMWH (4500 Da) isolated from porcine intestinal mucosa and used as sodium salt, was injected subcutaneously at a dose of 0.5 mg/kg in the thighs of all eligible patients after obtaining the baseline blood samples within 1 h of the diagnosis of sepsis [20,21]. Measurements of coagulation tests for APTT, prothrombin time (PT), and thrombin time (TT), as well as the levels of natural anticoagulants including total and free TFPI, protein S, protein C, and AT, were repeated 4, 12, and 24 h after the administration of enoxaparin. Blood Collection and Processing A total of 9.5 mL of blood was carefully collected into vacutainer tubes containing 0.5 mL of sodium citrate (3.8%, 0.129 mol/L; Terumo, Tokyo, Japan) at 0 h, before the administration of the first enoxaparin dose (the baseline sample), and at 4, 12, and 24 h thereafter. Blood samples were mixed gently and transferred immediately to the Coagulation Research Laboratory, Physiology Department, College of Medicine, King Saud University. 113

Al Otair AH, et al: Tissue Factor Pathway Inhibitor in Patients with Sepsis

The blood sample tubes were centrifuged at 3000 rpm (1000×g) for 15 min in a refrigerated (4-6 °C) centrifuge (Jouan Centrifuge Series, France). Platelet-poor plasma was separated using plastic pipettes and aliquots and immediately stored at -80 °C, until analysis in batches at a later date. Before assays were performed plasma specimens were thawed at 37 °C for 15 min. Laboratory Assays Coagulation screening tests included APTT, PT, and TT. PT was measured using a Stago STA Analyzer and STA Neoplastine CI 5 (freeze-dried rabbit brain thromboplastin with heparin inhibitor). For APTT, the STA PTT Automate 5 Kit was used. TT was measured using the STA thrombin kits with calcium thrombin reagent (approximately 1.5 NIH U/mL, freeze dried). The coefficient of variation (CV) varied from 5% for APTT to 2% for PT and TT. Plasma fibrinogen was measured by a turbidimetric method [22] and the CV varied between 6% and 8%. Anti-FXa was assayed by a colorimetric kit (Rotachrom HBPM/LMWH Assay, Diagnostica Stago, Asnières-sur-Seine, France). Coagulation inhibitors were assayed using an automated coagulometer (Stago STAT 4) and reagents were supplied by Diagnostica Stago, Asnières-sur-Seine, France: TFPI [Asserachrom Enzyme-Linked Immunosorbent Assay (ELISA) Kit] [23], total and free protein S (Asserachrom Protein S ELISA Kit), protein C (Asserachrom Protein C ELISA Kit), and AT (Stachrom Antithrombin Kit), with CV of 5% or less for total TFPI, free TFPI, total protein S, free protein S, and protein C and 4% for AT. STA-Liquid Anti-Xa for use with the STA Compact (Diagnostica Stago, France) was used for the quantitative determination of the potentiating effect of LMWH on antithrombin by recording the anti-FXa activity in plasma using a chromogenic substrate. Results were expressed as percent activity and according to the manufacturer’s instructions. Statistical Methods The Mann-Whitney U test was used to compare means for 2 independent groups. The chi-square test or Fisher’s test was used as appropriate to compare the percentages for 2 categorical variables. A p-value of less than 0.05 indicated statistical significance. SPSS 15 for Windows was used for the analysis and for the drawing of the bar graphs.

Results Pneumonia was the most common diagnosis (37.2%) in the study population, followed by urosepsis and abdominal sepsis (11.8% each). Thirteen patients (25.5%) developed septic shock and were started on vasopressors. Nine (69.2%) of them died during hospitalization, 10 died during the first week, and 6 patients died 2 weeks later. None died during the study period. This accounted for a mortality rate of 31.4% (Table 1). A definite infective organism was identified in 22 patients. 114

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To facilitate comparisons between subjects and to reduce the day-to-day variation in individuals, the results of each test were expressed as percentage of normal pooled plasma. On arrival to the Accident and Emergency Department and before receiving any treatment, the baseline blood tests showed prolongation of PT and APTT; the TT did not fluctuate significantly. Significant prolongation of both PT and APTT persisted in the 3 subsequent samples (4, 8, and 24 h), with the APTT prolongation getting worse in the subsequent samples (Figure 1). The plasma fibrinogen levels were significantly elevated above normal control values at baseline and in the 3 subsequent samples (local laboratory reference values: 150-400 mg/dL) (Figure 2). There was significant reduction in the levels of the natural anticoagulants AT, protein C, and total and free protein S below control values from baseline and in the 3 subsequent samples (4, 12, and 24 h) (Figure 3). Table 1. Descriptive statistics of study population. n=51 Age (years)

51±20.8

Sex Male Female

29 28

BMI (kg/m2)

29±3

APACHE II Mean ± SD Range

24±4 16-29

Diagnosis Abdominal sepsis Pneumonia Bronchiectasis Diabetic foot Urosepsis Central nervous system infections Others

6 19 4 2 6 3 11

Comorbid conditions Hematological conditions Malignancy Diabetic mellitus Hypertension Ischemic heart disease Cerebral vascular accident Chronic kidney disease Chronic liver disease Transplant Others

4 3 12 12 5 6 2 2 3 2

Vasopressor support

Death

APACHE II: Acute Physiology and Chronic Health Evaluation II, BMI: body mass index, SD: standard deviation.

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In contrast to the above 3 natural coagulation inhibitors, the plasma levels of total and free TFPI were markedly elevated above control values (local laboratory reference value: 60.7±16.9 ng/ mL) throughout the study period. The mean level of total TFPI was 73.0±39.0 ng/mL at baseline, and it remained significantly elevated at 4 h (101.9±55.5 ng/mL), 12 h (91.2±55.1 ng/mL), and 24 h (85.7±55.5 ng/mL). A similar trend was noted in the fluctuations of free TFPI, whose levels were also elevated

significantly, but much more so than total TFPI, to almost 4 times the control levels upon arrival to the Accident and Emergency Department (30.0±17.1 ng/mL) (Figure 4). When the patients who had sepsis (n=38) were compared to patients with septic shock (n=13), we noted higher PT after 4 h and higher free TFPI after 4 and 12 h of enoxaparin administration (Table 2).

Table 2. Comparison between the hemostatic variables in patients with sepsis and septic shock.

Patients with sepsis n=38

Patients with septic shock n=13

p-value

PT (s)

0h 4h 12 h 24 h 0h 4h 12 h 24 h 0h 4h 12 h 24 h 0h 4h 12 h 24 h 0h 4h 12 h 24 h

17.14±6.23 17.27±3.82 16.74±3.17 15.84±2.22 43.69±13.53 50.50±14.33 45.68±10.26 45.21±15.57 18.81±13.27 19.75±9.65 16.35±2.37 15.77±2.46 617.93±244.43 642.44±198.64 696.81±162.90 673.79±160.72 78.17±21.23 80.96±23.23 80.33±19.75 78.25±20.25

17.92±3.61 19.55±3.70 18.07±3.76 16.68±3.58 43.15±8.43 51.90±8.62 49.08±10.61 49.55±13.17 16.56±2.15 17.37±2.50 16.67±1.99 18.20±6.62 606.57±183.42 630.64±159.77 600.08±194.75 674.50±200.68 76.01±22.59 71.57±25.06 70.08±20.54 77.07±21.77

0.096 0.020* 0.166 0.988 0.834 0.380 0.209 0.340 0.854 0.895 0.508 0.221 0.907 0.736 0.144 0.814 0.734 0.321 0.072 0.937

0h 4h 12 h 24 h 0h 4h 12 h 24 h 0h 4h 12 h 24 h 0h 4h 12 h 24 h 0h 4h 12 h 24 h

62.35±28.33 64.30±28.93 67.66±24.18 64.08±22.21 61.41±21.37 61.90±34.25 60.55±20.33 64.52±23.41 44.89±17.06 39.71±15.14 44.07±18.15 43.82±19.61 77.12±37.53 94.00±47.58 85.15±49.15 79.91±43.88 29.48±16.52 36.40±15.52 29.80±16.54 27.78±15.79

62.21±23.38 58.85±26.55 61.50±23.27 68.07±25.42 57.92±15.25 53.81±19.36 53.16±18.48 61.64±24.15 43.21±15.67 41.81±21.51 40.50±19.34 47.0±19.05 86.78±35.30 117.42±53.07 97.61±44.63 146.00±214.23 38.57±16.28 50.71±16.28 43.30±19.66 34.53±21.60

0.982 0.478 0.254 0.738 0.604 0.439 0.132 0.649 0.907 0.937 0.594 0.707 0.417 0.084 0.157 0.598 0.589 0.004* 0.049* 0.478

APTT (s)

TT (s) Fibrinogen (mg/dL) AT III (%) Protein C (%) Protein S-total (%) Protein S-free (%) TFPI-total (ng/mL)

TFPI-free (ng/mL)

PT: Prothrombin time, APTT: activated partial prothrombin time, TT: thrombin time, AT: antithrombin, TFPI: tissue factor pathway inhibitor.

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The plasma level of anti-FXa at 4 h was 0.52±0.11 IU/mL, at 12 h was 0.5±0.07 IU/mL, and at 24 h was 0.59±0.11 IU/mL; all were within the prophylactic range (0.2-0.5 IU/mL) [24].

200 180 160

Percentage (%)

140 120 100 80 60 40 20 0

Baseline

PT%

4 Hours APPT%

12 Hours TT%

24 Hours

PT: Prothrombin time, APTT: activated partial prothrombin time, TT: thrombin time

Figure 1. Comparison of PT%, APTT%, and TT% at baseline and at 4, 12, and 24 h after the administration of enoxaparin. 900.0 800.0 700.0

Fibrogen mg/dL

600.0 500.0 400.0 300.0 200.0 100.0 0.0

Baseline

4 Hours

12 Hours

24 Hours

Controls

Figure 2. Comparison of fibrinogen at baseline and at 4, 12, and 24 h after the administration of enoxaparin. 120 100 80 Percentage (%)

60 40 20 0

Baseline

4 Hours

12 Hours

24 Hours

Controls

Prot C AT Prot S Total Prot 5 FREE Prot C: Protein C, AT: Anti thrombin, Prot S Total: Protein S total, Prot S FREE: Protein S free

Figure 3. Comparison of plasma levels of protein C, AT, and total and free protein S at baseline and at 4, 12, and 24 h after the administration of enoxaparin. 180 160

ng/mL

140 120 100 80 60 40 20 0

Baseline

4 Hours TFPI T

12 Hours

24 Hours

TFPI F

TPFI T: Tissue factor pathway inhibitor-Total, TPFI F: Tissue factor pathway inhibitor-Free

Figure 4. Comparison of the plasma levels of total and free TFPI at baseline and at 4, 12, and 24 h after the administration of enoxaparin. 116

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Comparing the measured hemostatic variables in survivors and nonsurvivors, there were only 3 isolated significant findings: lower levels in nonsurvivors of TT (15.9±2.48% in nonsurvivors versus 21.74±10.2% in survivors, p=0.04), AT (74.36±17.6% in nonsurvivors versus 106.5±22.59%, p=0.01), and protein C (62.51±21.19% in nonsurvivors versus 90.0±26.87%, p=0.03) in the 24-h samples.

Discussion The findings of the current study revealed marked derangement of the coagulation system in patients with sepsis and septic shock, in the form of significant prolongation of results of both the screening tests of the intrinsic (APTT) and extrinsic (PT) coagulation pathways that persisted after enoxaparin administration. There was also very significant consumption of the natural anticoagulants protein C, AT, and total and free protein S. On the other hand, we noted with much interest that the baseline levels of both total and free TFPI were elevated above healthy control values and increased further after the administration of enoxaparin. Numerous previous studies have examined the fluctuations of the circulating levels of hemostatic parameters in septic syndrome. In this respect, natural coagulation inhibitors AT, activated protein C, TFPI, and thrombomodulin received much attention and almost all studies found marked reduction in their blood levels [3,4]. Our study is in accordance with these studies and showed lower levels of AT, protein C, and total and free protein S at baseline and 4, 12, and 24 h after enoxaparin administration. AT is the main inactivator of thrombin and also inhibits the activated forms of FIX, FX, and XI. Protein C, in the presence of protein S, inhibits the activated forms of FVIII and FV. In our patients, the levels of these inhibitors remained below control values throughout the study period, which suggested their consumption in the face of the activated coagulation in these septic patients. However, none of these 3 inhibitors act on tissue factor, the prime trigger of coagulation in vivo [25] and whose expression in septic patients is markedly enhanced by proinflammatory cytokines on the surface of endothelial cells and monocytes [9,11,25,26]. The prime and specific physiological inhibitor of tissue factor is TFPI, which is a proteinase inhibitor generated mainly from the microvascular endothelium and that circulates in 2 forms: 80% bound to lipoproteins and 20% in the physiologically active free form [26,27]. TFPI also inhibits FXa directly and indirectly by blocking action of the FVIIa/TF complex [10,27].

Turk J Hematol 2016;33:112-118

In an early report, Gando et al. [25], who measured both TF and TFPI daily for 4 days, concluded that tissue factor production is not balanced by concurrent production of TFPI and that underlies the resulting activation of the coagulation system. The design of the current study is different from that of Gando et al. [25] and we have undertaken multiple measurements over the first 24 h of admission, which we think is a critical period in the management of septic patients. We also carried out more detailed measurements of both total TFPI and free TPFI [26]. We noted with much interest the remarkable elevation in the levels of both total and free TFPI above the healthy control levels on admission, indicating that the prime inhibitor of the tissue factor does in fact show a very active response to the presumed excessive sepsis-induced generation of tissue factor. The administration of prophylactic doses of enoxaparin resulted in significant inhibition of FXa at 4, 12, and 24 h and was associated with further rise in the circulating levels of both forms of TFPI. Free TFPI exhibited more remarkable elevation (4 times the control levels) than total TFPI. This was taken to indicate that sepsis, in its own right, must be a strong trigger to the release of TFPI from the vascular endothelium. This release process must have approached its maximum degree following the administration of enoxaparin, which is known to be most potent in the mobilization and release of TFPI from the vascular endothelium as compared to other LMWHs [28,29]. Interestingly, some studies have reported not only reduced levels of natural coagulation inhibitors but also impairment of their function [2,28]. If this is indeed the case, and until the mechanism of this impairment of function is delineated, no benefit should be expected of the therapeutic uses of genetically engineered recombinant natural coagulation inhibitors. In the present study, we noted a trend towards higher TFPI levels and particularly free TFPI in patients with septic shock as compared to patients with sepsis. This could represent a more exaggerated release of TFPI in these patients with more severe disease. However, the number of patients in this group was small and perhaps future studies with larger number of patients with septic shock are needed to confirm these observations. Unlike other studies, in which most of the patients were receiving vasopressors [30,31], we found that enoxaparin administered in prophylactic doses resulted in significant inhibition of FXa. This suggests the presence of additional factors that contribute to the failure of deep venous thrombosis prophylaxis in patients with sepsis. One possibility could be the lower levels of Na as described before [9,10,11,12] and confirmed by our findings. In conclusion, the main finding of the current study is the remarkable elevation in the plasma levels of both total and free TFPI in septic patients at baseline. The levels of both forms of the inhibitor remained elevated throughout the first 24 h

Al Otair AH, et al: Tissue Factor Pathway Inhibitor in Patients with Sepsis

with further elevation after enoxaparin administration. This observation would help to explain why the administration of recombinant TFPI did not affect the course and outcome of sepsis and septic shock. Acknowledgment This study was supported by the College of Medicine Research Centre and the Deanship of Scientific Research of King Saud University, Riyadh, Saudi Arabia. All authors disclosed that there is no conflict of interest and that this study was not sponsored by any drug company. We are also grateful to Mr. M.A. Hamid and Mr. Lugman El-Sid for their technical assistance, and to the nurses of the Accident and Emergency and ICUs of the Department of Critical Care, King Khalid University Hospital, Riyadh, for the collection of blood samples. Ethics Ethics Committee Approval: The study was approved by the Institutional Review Board of the College of Medicine-King Saud University. Informed Consent: A written informed consent was obtained from all patients or their next of kin. Authorship Contributions Concept: Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Abdulaziz H. Alzeer, Mashael Al Shaikh; Design: Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Abdulaziz H. Alzeer, Mashael Al Shaikh; Data Collection or Processing: Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Syed M. Khurshid, Abdulaziz H. Alzeer, Abdul Kareem Al Momen, Mashael Al Shaikh, Farja Al Gahtani, Zohair A. Al Aseri, Hossam A.H. Abdelrazik; Analysis or Interpretation: Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Syed M. Khurshid, Abdulaziz H. Alzeer, Abdul Kareem Al Momen, Mashael Al Shaikh, Farja Al Gahtani, Zohair A. Al Aseri, Hossam A.H. Abdelrazik; Literature Search: Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Syed M. Khurshid, Abdulaziz H. Alzeer; Writing: Hadil A. Al Otair, Abdel Galil M. Abdel Gader, Syed M. Khurshid, Abdulaziz H. Alzeer. 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. Okazaki Y, Matsukawa A. Pathophysiology of sepsis and recent patents on the diagnosis, treatment and prophylaxis for sepsis. Recent Pat Inflamm Allergy Drug Discov 2009;3:26-32. 2. Levi M, van der Poll T. Inflammation and coagulation. Crit Care Med 2010;38(2 Suppl):S26-34. 3. Levi M, de Jonge E, Van der Poll T. Sepsis and disseminated intravascular coagulation. J Thromb Thrombolysis 2003;16:43-47. 4. Okabayashi K, Wada H, Ohta S, Shiku H, Nobori T, Maruyama K. Hemostatic markers and the sepsis-related organ failure assessment score in patients with disseminated intravascular coagulation in an intensive care unit. Am J Hematol 2004;76:225-229.

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5. Gando S, Nanzakaki S, Sasaki S. Activation of the extrinsic coagulation pathway in patients with severe sepsis and septic shock. Crit Care Med 1998;26:2005-2009.

18. Frydman A. Low-molecular-weight heparin: an overview of their pharmacodynamics, pharmacokinetics and metabolism in humans. Haemostasis 1996;26:24-38.

6. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, Osborn TM, Nunnally ME, Townsend SR, Reinhart K, Kleinpell RM, Angus DC, Deutschman CS, Machado FR, Rubenfeld GD, Webb SA, Beale RJ, Vincent JL, Moreno R; Surviving Sepsis Campaign Guidelines Committee including the Pediatric Subgroup. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med 2013;39:165-228.

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21. Alban S, Welzel D, Hemker HC. Pharmacokinetic and pharmacodynamic characterization of a medium-molecular-weight heparin in comparison with UFH and LMWH. Semin Thromb Hemost 2002;28:369-378.

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22. Ellis BC, Stransky AA. A quick and accurate method for the determination of fibrinogen in plasma. J Lab Clin Med 1961;58:477-488.

20. Jochberger S, Mayr V, Luckner G, Fries DR, Mayr AJ, Friesenecker BE, Lorenz I, Hasibeder WR, Ulmer H, Schobersberger W, Dünser MW. Antifactor Xa activity in critically ill patients receiving antithrombotic prophylaxis with standard dosages of certoparin: a prospective, clinical study. Crit Care 2005;9:R541-548.

9. Stief TW, Ijagha O, Weiste B, Herzum I, Renz H, Max M. Analysis of hemostasis alterations in sepsis. Blood Coagul Fibrinolysis 2007;18:179-186.

23. Alban S, Gastpar R. In vitro influence of heparin on plasma total and free tissue factor pathway inhibitor (TFPI) as measured by ELISA. Pharm Pharmacol Lett 2000;10:51-54.

10. Smithies MN, Weaver CB. Role of the tissue factor pathway in the pathogenesis and management of multiple organ failure. Blood Coagul Fibrinolysis 2004;15(Suppl 1):S11-20.

24. Weitz JI. Antithrombotic drugs. In: Hoffman F, Benz EJ, Shattil SJ (eds). Hematology: Basic Principles and Practice. 5th ed. Philadelphia, Churchill Livingstone, 2009.

11. Creasey AA, Reinhart K. Tissue factor pathway inhibitor activity in severe sepsis. Crit Care Med 2001;29(7 Suppl):S126-129.

25. Gando S, Kameue T, Morimoto Y, Matsuda N, Hayakawa M Kemmotsu O. Tissue factor production not balanced by tissue factor pathway inhibitor in sepsis promotes poor prognosis. Crit Care Med 2002;30:1729-1734.

12. Fourrier F. Coagulation inhibitors in severe sepsis: state of the art. Rev Med Interne 2003;24:295-304. 13. Angus DC, Crowther MA. Unraveling severe sepsis: why did OPTIMIST fail and what’s next? JAMA 2003;290:256-258. 14. Silva E, de Figueiredo LF, Colombari F. PROWESS-SHOCK trial: a protocol overview and perspectives. Shock 2010;34(Suppl 1):48-53. 15. Cook D, Crowther M, Meade M, Rabbat C, Griffith L, Schiff D, Geerts W, Guyatt G. Deep vein thrombosis in medical-surgical critically ill patients: prevalence, incidence, and risk factors. Crit Care Med 2005;33:1565-1571. 16. Junqueira DR, Perini E, Penholati RR, Carvalho MG. Unfractionated heparin versus low molecular weight heparin for avoiding heparin-induced thrombocytopenia in postoperative patients. Cochrane Database Syst Rev 2012;9:CD007557. 17. Fraisse F, Holzapfel L, Couland JM, Simonneau G, Bedock B, Feissel M, Herbecq P, Pordes R, Poussel JF, Roux L. Nadroparin in the prevention of deep vein thrombosis in acute decompensated COPD. The Association of Non-University Affiliated Intensive Care Specialist Physicians of France. Am Rev Resp Crit Care Med 2000;161:1109-1114.

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26. Abraham E. Tissue factor inhibition and clinical trial results of tissue factor pathway inhibitor in sepsis. Crit Care Med 2000;29(9 Suppl):S31-33. 27. Broze GJ Jr, Girard TJ. Tissue factor pathway inhibitor: structure-function. Front Biosci (Landmark Ed) 2012;17:262-280. 28. Alban S, Gastpar R. Plasma levels of total and free tissue factor pathway inhibitor (TFPI) as individual pharmacological parameters of various heparins. Thromb Haemost 2001;85:824-829. 29. Naumnik B, Rydzewska-Rosołowska A, Myśliwiec M. Different effects of enoxaparin, nadroparin, and dalteparin on plasma TFPI during hemodialysis: a prospective crossover randomized study. Clin Appl Thromb Hemost 2011;17:480-486. 30. Fourrier F. Hemostasis disorders in severe infections: state of the art. Med Mal Infect 2006;36:304-313. 31. Delle Karth G, Geppert A, Joukhadar C, Graf S, Berger R, Hülsmann M, Spitzauer S, Pabinger I, Heinz G. Prophylactic anticoagulation with enoxaparin: Is the subcutaneous route appropriate in the critically ill. Crit Care Med 2003;31:1405-1409.

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0455 Turk J Hematol 2016;33:119-126

Comparison of Myelodysplastic Syndrome Prognostic Scoring Systems Miyelodisplastik Sendrom Prognostik Skorlama Sistemlerinin Kıyaslanması Özlen Bektaş1, Ayşegül Üner2, Eylem Eliaçık1, Burak Uz1, Ayşe Işık1, Sezgin Etgül1, Süreyya Bozkurt2, İbrahim Celalettin Haznedaroğlu1, Hakan Göker1, Nilgün Sayınalp1, Salih Aksu1, Haluk Demiroğlu1, Osman İlhami Özcebe1, Yahya Büyükaşık1 1Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey 2Hacettepe University Faculty of Medicine, Department of Pathology, Ankara, Turkey

Abstract

Öz

Objective: Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disease. Patients are at risk of developing cytopenias or progression to acute myeloid leukemia. Different classifications and prognostic scoring systems have been developed. The aim of this study was to compare the different prognostic scoring systems.

Amaç: Miyelodisplastik sendrom (MDS) klonal bir hematopoetik kök

Materials and Methods: One hundred and one patients who were diagnosed with primary MDS in 2003-2011 in a tertiary care university hospital’s hematology department were included in the study. Results: As the International Prognostic Scoring System (IPSS), World Health Organization Classification-Based Prognostic Scoring System (WPSS), MD Anderson Prognostic Scoring System (MPSS), and revised IPSS (IPSS-R) risk categories increased, leukemia-free survival and overall survival decreased (p<0.001). When the IPSS, WPSS, MPSS, and IPSS-R prognostic systems were compared by Cox regression analysis, the WPSS was the best in predicting leukemia-free survival (p<0.001), and the WPSS (p<0.001) and IPSS-R (p=0.037) were better in predicting overall survival. Conclusion: All 4 prognostic systems were successful in predicting overall survival and leukemia-free survival (p<0.001). The WPSS was found to be the best predictor for leukemia-free survival, while the WPSS and IPSS-R were found to be the best predictors for overall survival. Keywords: Myelodysplastic syndrome, International Prognostic Scoring System, MD Anderson Prognostic Scoring System, World Health Organization Classification-Based Prognostic Scoring System, Revised International Prognostic Scoring System

hücre hastalığıdır. Hastalarda sitopeni veya akut miyeloid lösemi gelişmesi riski söz konusudur. Farklı sınıflandırma ve prognostik skorlama sistemleri geliştirilmiştir. Bu çalışmanın amacı, farklı prognostik skorlama sistemlerinin karşılaştırılmasıdır. Gereç ve Yöntemler: Üçüncü basamak üniversite hastanesi, hematoloji bölümünde 2003-2011 yılları arasında tanı alan 101 primer MDS hastası çalışmaya dahil edildi. Bulgular: Uluslararası Prognostik Skorlama Sistemi (UPSS), Dünya Sağlık Örgütü Sınıflandırması Bazlı Prognostik Skorlama Sistemi (DPSS), MD Anderson Prognostik Skorlama Sistemi (MPSS) ve yeniden düzenlenmiş UPSS (UPSS-D) risk kategorileri arttıkça lösemisiz sağkalım ve toplam sağkalım azalıyordu (p<0,001). UPSS, DPSS, MPSS ve UPSS-R Cox regresyon analizi ile karşılaştırıldığında, DPSS’nin lösemisiz sağkalımı (p<0,001), DPSS (p<0,001) ve UPSS-D’nin (p=0.037) toplam sağkalımı daha iyi öngördüğü tespit edildi. Sonuç: Dört prognostik skorlama sistemi de toplam sağkalımı ve lösemisiz sağkalımı başarılı şekilde öngörüyordu (p<0.001). DPSS’nin lösemisiz sağkalımın, DPSS ve UPSS-D’nin toplam sağkalımın en iyi öngöreni olduğu tespit edildi. Anahtar Sözcükler: Miyelodisplastik sendrom, Uluslararası Prognostik Skorlama Sistemi, MD Anderson Prognostik Skorlama Sistemi, Dünya Sağlık Örgütü Sınıflandırması Bazlı Prognostik Skorlama Sistemi, Yeniden Düzenlenmiş Uluslararası Prognostik Skorlama Sistemi

Address for Correspondence/Yazışma Adresi: Özlen BEKTAŞ, M.D., Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone : +90 532 543 05 75 E-mail : ozlenbektas@hotmail.com

Received/Geliş tarihi: November 20, 2014 Accepted/Kabul tarihi: December 23, 2014

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Introduction

Materials and Methods

Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic stem cell disorders with heterogeneous morphological, clinical, and survival characteristics. Common features include cytopenia(s), dysplasia of one or more major myeloid series, ineffective hematopoiesis, and an increased risk of acute myeloid leukemia [1].

One hundred and one patients who were diagnosed with primary MDS during 2003-2011 and suitable for all of the prognostication systems were included in the study. We used 101 routinely managed patients regardless of whether they were on MDS-specific treatment or not. Patient information was accessed from patient chart reviews. Each patient was categorized according to the MDS-FAB and 2001 WHO classification systems according to their bone marrow aspiration and biopsy specimens. We did not use 2008 WHO classification since the WPSS was validated only for the 2001 WHO classification system. Criteria for inclusion were: age >18 years, primary MDS patients, and marrow and peripheral blood blast counts of <20%. Exclusion criteria were: CMML, secondary MDS, and marrow or peripheral blood blast counts of ≥20%.

In 1982, the first classification of MDS was developed by the French-American-British (FAB) group. This was a morphological classification based on the degree of dysplasia and blasts in the bone marrow, without biological basis [2]. The World Health Organization (WHO) rearranged the classification of MDS-FAB in 2001 and 2008. Several parameters with prognostic significance were added in the 2008 version: number of cytopenias, dysplasia in one or more series, and presence of genetic abnormalities [3]. Following diagnosis and classification of MDS, prognostic staging should be made to plan the treatment [4]. The International MDS Risk Analysis Workshop developed the International Prognostic Scoring System (IPSS), recognizing the bone marrow blast percentage, cytogenetic status, and number and degree of cytopenias as the most important prognostic markers in MDS [5]. The IPSS is the most widely used prognostic scoring system [4]. It was designed based on untreated and primary MDS patients [5]. The WHO category, cytogenetics, and transfusion requirements were identified as the most important prognostic indicators in MDS and the WHO Classification-Based Prognostic Scoring System (WPSS) was developed by Malcovati et al. [6]. The WPSS was also designed based on untreated patients and it does not include secondary MDS patients. Kantarjian et al. developed a new classification model to overcome the limitations existing in both prior prognostic systems of MDS, which includes patients’ performance status, age, number and degree of cytopenias, cytogenetics, bone marrow blast percentage, and transfusion needs [7]. The MD Anderson Prognostic Scoring System (MPSS) is a system that can be applied to primary and secondary MDS and chronic myelomonocytic leukemia (CMML). The International Working Group for Prognosis in MDS project was initiated due to limitations of the IPSS and the revised IPSS (IPSS-R) was developed. The IPSS-R considers bone marrow blast percentage, cytogenetics, and number and degree of cytopenias. This prognostic system also does not include secondary MDS and was developed based on untreated patients [8]. In this study, we aimed to compare the different prognostication systems and determine the most appropriate system for routine clinical practice. 120

To analyze the prognosis, we used 4 different prognostic systems: the IPSS, WPSS, MPSS, and IPSS-R. Leukemia transformation and death were recorded as events and the first developed event was recorded. Event-free survival was defined as the duration from the time of diagnosis until the time of developing an event or the last follow-up time, leukemia-free survival (LFS) was defined as the duration from the time of diagnosis until the time of developing leukemia (marrow or peripheral blood blast count of ≥20%) or the last follow-up time, and overall survival (OS) was defined as the duration from the time of diagnosis until death or the last follow-up time. Last follow-up date and condition were recorded as the last condition. This investigation was approved by the Local Ethics Committee of Hacettepe University. Statistical Methods Data analysis was performed using SPSS 11.5 for Windows. Continuous data were presented as mean ± standard deviation or median (range). Categorical data were presented as numbers and percentages. For the IPSS, WPSS, MPSS, and IPSS-R, the LFS, OS, and life expectancy were evaluated with Kaplan-Meier survival analysis using the log-rank test. Life expectancy; 1-, 3-, and 5-year survival rates; and 95% confidence intervals (CIs) were calculated for each variable category. The prediction capacities of the IPSS, WPSS, MPSS, and IPSS-R for LFS and OS were compared with multivariate Cox proportional hazard regression analysis. For each variable, the hazard ratios and 95% CIs were calculated. A value of p<0.05 was considered statistically significant.

Bektaş Ö, et al: Comparison of Myelodysplastic Syndrome Prognostic Scoring Systems

Turk J Hematol 2016;33:119-126

Results Patient Characteristics The present study consisted of 101 patients; 44 of them (43.6%) were male and 57 (56.4%) were female. The mean age of the patients was 64±14.77 years. Transfusion support was given to 26 (23%) patients; hypomethylating agents were used in 21.2% of patients (n=24; 23 of them were on 5-azacytidine and 1 was on decitabine), lenalidomide in 0.9% of patients (n=1), and erythropoietin in 2.7% of patients (n=3); and 4.4% (n=5) of patients had undergone allogeneic bone marrow transplantation. The follow-up period for the patients ranged between 0 and 92 months with an average of 21.2 months. Cytogenetic classification of the patients according to the IPSS was as follows: 66 (58.4%) of good risk, 17 (15%) of intermediate risk, and 18 (15.9%) of poor risk. MDS subgroup distributions according to both the MDS-FAB classification and the 2001 WHO classification are shown in Table 1. Patients were evaluated by 4 different prognostic systems. Accordingly, the risk distributions of the patients are shown in Table 2. During the follow-up period, 34.7% (n=35) of patients experienced an event. The first event was leukemic transformation in 20.8% of the patients (n=21), while death was the first event in 13.9% (n=14) of the patients. Median time to event was 15.25 months. Median leukemic progression time was 8.28 months. Total death rate was 29.7% (n=30). The estimated OS and LFS durations were 55.93±10.19 and 56.52±10.29 months, respectively. In all 101 patients the average life expectancy was 55.9 months (95% CI: 45.77-66.09), and 1-, 3-, and 5-year OS rates were found as 77.5%, 57.5%, and 57.5%, respectively. The OS and median survival times were significantly reduced as the degree of risk increased regardless of which classification system was used (p<0.001) (Table 3, Figures 1a, 1b, 1c, 1d).

In all 4 classification systems, the LFS was reduced as the degree of risk increased (p<0.001). The 1-, 3-, and 5-year leukemia survival rates in all subjects were 76%, 60.1%, and 60.1%, respectively, and the average LFS time was found to be 56.52 months (95% CI: 46.2-66.8) (Table 4, Figures 2a, 2b, 2c, 2d). When the efficacies of the IPSS, WPSS, MPSS, and IPSS-R prognostic systems in predicting LFS were compared, the WPSS showed the best performance (p<0.001, hazard ratio [HR]: 2.1, 95% CI: 1.543-2.858). The WPSS (p<0.001, HR: 2.461, 95% CI: 1.812-3.343) and IPSS-R (p=0.037, HR: 1.460, 95% CI: 1.0242.081) systems were better than the others in predicting OS. Table 1. Distribution of patients according to FrenchAmerican-British and World Health Organization 2001 classification systems. Variables

n=101

MDS FAB classification RA

48 (47.5%)

RARS

17 (16.8%)

RAEB-I

22 (21.8%)

RAEB-II

14 (13.9%)

MDS 2001 WHO classification RA

11 (10.9%)

RARS

6 (5.9%)

RCMD

34 (33.7%)

RCMD-RS

9 (8.9%)

5q (-)

5 (5%)

RAEB-I

21 (20.8%)

RAEB-II

15 (14.9%)

MDS: Myelodysplastic syndrome, WHO: World Health Organization, FAB: FrenchAmerican-British, RAEB: refractory anemia with excess blasts, RARS: refractory anemia with ring sideroblasts, RA: refractory anemia, RCMD-RS: refractory cytopenia with multilineage dysplasia and ring sideroblasts, RCMD: refractory cytopenia with multilineage dysplasia.

Table 2. Distribution of patients by risk groups.

IPSS

WPSS

MPSS

IPSS-R

Very low risk

8 (7.9%)

18 (17.8%)

Low risk

31 (30.7%)

28 (27.7%)

23 (22.8%)

Intermediate (or Int-I) risk

41 (40.6%)

25 (24.8%)

31 (30.7%)

25 (24.8%)

Intermediate-II risk

20 (19.8%)

24 (23.8%)

High risk

9 (8.9%)

26 (25.7%)

18 (17.8%)

Very high risk

11 (10.9%)

17 (16.8%)

IPSS: International Prognostic Scoring System, MPSS: MD Anderson Prognostic Scoring System, WPSS: World Health Organization Classification-Based Prognostic Scoring System, IPSS-R: Revised International Prognostic Scoring System, NA: not applicable.

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Turk J Hematol 2016;33:119-126

Figure 1. Kaplan-Meier curves show rates of overall survival (OS) for International Prognostic Scoring System (IPSS) (a), World Health Organization-Based Prognostic Scoring System (WPSS) (b), MD Anderson Prognostic Scoring System (MPSS) (c), and Revised International Prognostic Scoring System (IPSS-R) (d). a

Figure 2. Kaplan-Meier curves show rates of leukemia-free survival (LFS) for International Prognostic Scoring System (IPSS) (a), World Health Organization Classification-Based Prognostic Scoring System (WPSS) (b), MD Anderson Prognostic Scoring System (MPSS) (c), and Revised International Prognostic Scoring System (IPSS-R) (d). 122

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Turk J Hematol 2016;33:119-126

Table 3. Overall survival according to International Prognostic Scoring System, World Health Organization Classification-Based Prognostic Scoring System, MD Anderson Prognostic Scoring System, and Revised International Prognostic Scoring System. Variables

Survival Rates (%)

Mean Survival

Log-rank

p-value

1 year

3 years

5 years

IPSS

37.10

<0.001

Low

100.0

85.9

68.38 (58.68-78.08)

Intermediate-I

81.6

76.2

65.05 (52.72-77.38)

Intermediate-II

47.9

8.0

21.51 (8.66-34.36)

High

10.17 (7.33-13.01)

WPSS

60.42

<0.001

Very low

100.0

56.72 (40.81-72.63)

Low

96.3

89.4

76.39 (58.26-94.51)

Intermediate

88.9

72.15 (58.25-86.05)

High

62.2

20.05 (15.02-25.08)

Very high

7.26 (5.66-8.86)

MPSS

44.02

<0.001

Low

100.0

70.85 (63.85-77.84)

Intermediate-I

100

75.2

70.23 (56.98-83.48)

Intermediate-II

56.3

30.8

27.85 (15.46-40.24)

High

24.3

8.1

17.45 (3.87-31.04)

IPSS-R

56.56

<0.001

Very low

100.0

80.0

47.00 (38.93-55.06)

Low

94.7

69.75 (61.73-77.76)

Intermediate

73.8

69.33 (50.43-88.23)

High

51.3

20.46 (14.00-26.92)

Very high

38.2

9.5

11.33 (7.41-15.25)

General

77.5

57.5

55.93 (45.77-66.09)

72.84

0.000

Discussion The current prognostication systems have been criticized for some specific properties. They were developed in untreated cohorts and they have generally not been tested in treated cohorts except for the IPSS-R. Neukirchen et al. demonstrated the value of the IPSS-R for patients treated with induction chemotherapy and/or allogeneic stem cell transplantation in their validation study [9]. Currently there are many widely available treatment alternatives in MDS. Therefore, we thought that these systems should be tested in a modern routinely

managed MDS cohort. The IPSS and IPSS-R are mostly criticized because they were developed in untreated patient cohorts that do not reflect current patient profiles [10]. The MPSS is mainly criticized for the inclusion of secondary and therapyrelated MDS and MDS/myeloproliferative disease cases, which are now considered separate entities [11,12]. The WPSS was initially criticized for arbitrariness of transfusion dependence. However, it was revised to include stable hemoglobin thresholds instead of this arbitrary definition [13]. It is still criticized for low reproducibility of WHO classification of subentities with low blast counts. 123

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Turk J Hematol 2016;33:119-126

Table 4. Leukemia-free survival according to International Prognostic Scoring System, World Health Organization ClassificationBased Prognostic Scoring System, MD Anderson Prognostic Scoring System, and Revised International Prognostic Scoring System. Variables

Survival Rates (%)

Mean Survival

Log-rank

p-value

1 year

3 years

5 years

IPSS

37.16

0.000

Low

100.0

85.9

68.38 (58.68-78.08)

Intermediate-I

82.4

76.9

64.99 (52.53-77.46)

Intermediate-II

35.9

9.0

18.73 (4.38-33.08)

High

38.1

9.32 (5.58-13.07)

WPSS

47.87

0.000

Very low

100.0

75.0

56.72 (40.81-72.63)

Low

96.3

89.4

76.00 (57.34-94.66)

Intermediate

90.9

84.8

73.19 (60.07-86.31)

High

52.9

16.93 (11.25-22.61)

Very high

6.42 (4.41-8.43)

MPSS

39.41

0.000

Low

100.0

70.20 (64.50-64.50)

Intermediate-I

96.7

78.9

71.15 (3.10-56.69)

Intermediate-II

49.9

31.2

26.06 (4.62-12.08)

High

26.8

8.9

17.34 (2.09-30.09)

IPSS-R

54.34

0.000

Very low

100.0

80.0

47.00 (38.93-55.06)

Low

94.7

69.44 (61.17-77.71)

Intermediate

90.9

74.6

69.52 (60.60-88.45)

High

37.5

17.17 (9.47-24.86)

Very high

35.3

9.86 (5.64-14.09)

General

76.0

60.1

56.52 (46.23-66.81)

84.30

0.000

In spite of these critiques, there is no doubt that these systems are useful in routine clinical practice. But which one(s) deserve the most credit? In our study 101 MDS patients appropriate for all prognostic systems were evaluated with the IPSS, WPSS, MPSS, and IPSS-R. The median age of the patients was 64 years, which is lower than in Western populations; younger age at diagnosis was also seen in some Asian countries, as Matsuda et al. and Kuendgen et al. demonstrated [1,14]. This is the first study to compare these 124

4 prognostic scoring systems in MDS. All 4 prognostic systems were successful in predicting OS and LFS (p<0.001). When the systems were compared, the WPSS was found to be the best predictor for LFS, while the WPSS and IPSS-R were found to be the best predictors for OS. Equal efficacies of IPSS-R and WPSS in our practice implies that our hematopathologists are quite capable of separating single-lineage dysplasia from multilineage dysplasia and refractory anemia with excess blasts (RAEB)-I from RAEB-II. Unfortunately, this capability may not be available in every setting.

Turk J Hematol 2016;33:119-126

Bektaş Ö, et al: Comparison of Myelodysplastic Syndrome Prognostic Scoring Systems

There are several studies that compared prognostic scoring systems. Voso et al. compared the IPSS, WPSS, and IPSS-R in their IPSS-R validation study and found that the IPSS-R predicted OS better than the other systems [15]. Reis-Alves et al. showed that only IPSS-R score was an independent risk factor in terms of OS in their comparison of the IPSS, WPSS, and IPSS-R [16]. In our study, the WPSS and IPSS-R may have estimated OS better since the hemoglobin cut-off was accepted as lower than in the other systems in both these scoring systems (<9 g/dL in males and <8 g/dL in females for WPSS; 8-10 g/ dL [1 point] and <8 g/dL [1.5 point] for IPSS-R). This may be especially true for low-risk patients since the main predictor of mortality is marrow failure in low-risk patients and leukemic transformation in high-risk patients. When the advanced age and frailty of many MDS patients are taken into consideration, the lower hemoglobin threshold may better reflect the impact of anemia on health. In our study, the WPSS was found to be best in reflecting LFS. This may be due to the fact that it depends on the MDS-WHO classification. This classification reflects leukemia transformation risk very well [17,18]. The MPSS is a dynamic scoring system like the WPSS and predicts survival at any time during follow-up. It can be used for chronic myelomonocytic leukemia and secondary MDS if prognostic assessment is required [7]. This study has some handicaps inherent to its retrospective nature. Additionally, it would be better to include a higher number of patients in future analyses. Ethics Ethics Committee Approval: This investigation was approved by the Local Ethics Committee of Hacettepe University, Informed Consent: Not applicable (retrospective study). Authorship Contributions Pathological Processing: Ayşegül Üner; Cytogenetic Processing: Süreyya Bozkurt; Data Collection or Processing: Özlen Bektaş, Ayşegül Üner, Eylem Eliaçık, Burak Uz, Ayşe Işık, Sezgin Etgül, Süreyya Bozkurt, İbrahim Celalettin Haznedaroğlu, Hakan Göker, Nilgün Sayınalp, Salih Aksu, Haluk Demiroğlu, Osman İlhami Özcebe, Yahya Büyükaşık; Analysis or Interpretation: Özlen Bektaş, Yahya Büyükaşık; Literature Search: Özlen Bektaş, Yahya Büyükaşık; Writing: Özlen Bektaş, Yahya Büyükaşık.

References 1. Matsuda A, Germing U, Jinnai I, Araseki K, Kuendgen A, Strupp C, Iwanaga M, Miyazaki Y, Hata T, Bessho M, Gattermann N, Tomonaga M. Differences in the distribution of subtypes according to the WHO classification 2008 between Japanese and German patients with refractory anemia according to the FAB classification in myelodysplastic syndromes. Leuk Res 2010;34:974-980. 2. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982;51:189-199. 3. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, Harris NL, Le Beau MM, Hellstrom-Lindberg E, Tefferi A, Bloomfield CD. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009;114:937-951. 4. Mittelman M, Oster HS, Hoffman M, Neumann D. The lower risk MDS patient at risk of rapid progression. Leuk Res 2010;34:1551-1555. 5. Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D, Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997;89:2079-2088. 6. Malcovati L, Germing U, Kuendgen A, Della Porta MG, Pascutto C, Invernizzi R, Giagounidis A, Hildebrandt B, Bernasconi P, Knipp S, Strupp C, Lazzarino M, Aul C, Cazzola M. Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes. J Clin Oncol 2007;25:3503-3510. 7. Kantarjian H, O’Brien S, Ravandi F, Cortes J, Shan J, Bennett JM, List A, Fenaux P, Sanz G, Issa JP, Freireich EJ, Garcia-Manero G. Proposal for a new risk model in myelodysplastic syndrome that accounts for events not considered in the original International Prognostic Scoring System. Cancer 2008;113:1351-1361. 8. Greenberg PL, Tuechler H, Schanz J, Sanz G, Garcia-Manero G, Sole F, Bennett JM, Bowen D, Fenaux P, Dreyfus F, Kantarjian H, Kuendgen A, Levis A, Malcovati L, Cazzola M, Cermak J, Fonatsch C, Le Beau MM, Slovak ML, Krieger O, Luebbert M, Maciejewski J, Magalhaes SM, Miyazaki Y, Pfeilstocker M, Sekeres M, Sperr WR, Stauder R, Tauro S, Valent P, Vallespi T, van de Loosdrecht AA, Germing U, Haase D. Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012;120:2454-2465. 9. Neukirchen J, Lauseker M, Blum S, Giagounidis A, Lubbert M, Martino S, Siragusa S, Schlenk RF, Platzbecker U, Hofmann WK, Gotze K, Palumbo GA, Magrin S, Kundgen A, Aul C, Hildebrandt B, Hasford J, Kobbe G, Haas R, Germing U. Validation of the revised international prognostic scoring system (IPSS-R) in patients with myelodysplastic syndrome: a multicenter study. Leuk Res 2014;38:57-64. 10. Komrokji RS, Zhang L, Bennett JM. Myelodysplastic syndromes classification and risk stratification. Hematol Oncol Clin North Am 2010;24:443-457. 11. Cazzola M, Della Porta MG, Travaglino E, Malcovati L. Classification and prognostic evaluation of myelodysplastic syndromes. Semin Oncol 2011;38:627-634.

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12. Vardiman JW, Arber DA, Brunning RD, Larson RA. Therapy-related myeloid neoplasms. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC, 2008.

Prognostic Scoring System (IPSS) predicts survival and leukemic evolution of myelodysplastic syndromes significantly better than IPSS and WHO Prognostic Scoring System: validation by the Gruppo Romano Mielodisplasie Italian Regional Database. J Clin Oncol 2013;31:2671-2677.

13. Malcovati L, Della Porta MG, Strupp C, Ambaglio I, Kuendgen A, Nachtkamp K, Travaglino E, Invernizzi R, Pascutto C, Lazzarino M, Germing U, Cazzola M. Impact of the degree of anemia on the outcome of patients with myelodysplastic syndrome and its integration into the WHO classificationbased Prognostic Scoring System (WPSS). Haematologica 2011;96:1433-1440.

16. Reis-Alves SC, Traina F, Harada G, Campos PM, Saad ST, Metze K, LorandMetze I. Immunophenotyping in myelodysplastic syndromes can add prognostic information to well-established and new clinical scores. PLoS One 2013;8:e81048.

14. Kuendgen A, Matsuda A, Germing U. Differences in epidemiology of MDS between Western and Eastern countries: ethnic differences or environmental influence? Leuk Res 2007;31:103-104. 15. Voso MT, Fenu S, Latagliata R, Buccisano F, Piciocchi A, Aloe-Spiriti MA, Breccia M, Criscuolo M, Andriani A, Mancini S, Niscola P, Naso V, Nobile C, Piccioni AL, D’Andrea M, D’Addosio A, Leone G, Venditti A. Revised International

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17. Vardiman JW. Hematopathological concepts and controversies in the diagnosis and classification of myelodysplastic syndromes. Hematology Am Soc Hematol Educ Program 2006;199-204. 18. Cermak J, Michalova K, Brezinova J, Zemanova Z. A prognostic impact of separation of refractory cytopenia with multilineage dysplasia and 5q-syndrome from refractory anemia in primary myelodysplastic syndrome. Leuk Res 2003;27:221-229.

RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0213 Turk J Hematol 2016;33:127-130

Platelet Dysfunction in Patients with Chronic Myeloid Leukemia: Does Imatinib Mesylate Improve It? Kronik Miyelositer Lösemili Hastalarda Trombosit Disfonksiyonu: İmatinib Mesilat Düzeltir mi? Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey

Abstract

Öz

Objective: The aim of this study was to investigate the effects of imatinib mesylate on platelet aggregation and adenosine triphosphate (ATP) release in chronic myeloid leukemia patients.

Amaç: Bu çalışmanın amacı kronik miyelositer lösemili hastalarda imatinib mesilatın trombosit agregasyonu ve adenozin trifosfat (ATP) salınımı üzerine etkilerini araştırmaktır.

Materials and Methods: Platelet aggregation and ATP release induced by 5.0 mM adenosine diphosphate, 0.5 mM arachidonic acid, 1.0 mg/ mL ristocetin, and 2 µg/mL collagen were studied by whole blood platelet lumi-aggregometer in 20 newly diagnosed chronic myeloid leukemia patients before and after imatinib mesylate treatment.

Gereç ve Yöntemler: Yirmi yeni tanı almış kronik miyelositer lösemili hastada imatinib mesilat tedavisi öncesi ve sonrası 5,0 mM adenozin difosfat, 0,5 mM araşidonik asit, 1,0 mg/mL ristosetin ve 2 µg/mL kollagen ile indüklenen trombosit agregasyon ve ATP salınımı çalışılmıştır.

Results: At the time of diagnosis, 17/20 patients had abnormal platelet aggregation results; 8 (40%) had hypoactivity, 6 (30%) had hyperactivity, and 3 (15%) had mixed hypo- and hyperactivity. Repeat platelet aggregation studies were performed after a mean of 19 months (min: 5 months-max: 35 months) in all patients who received imatinib mesylate during this period. After therapy, 18/20 (90%) patients had abnormal laboratory results; 12 (60%) had hypoactive platelets, 4 (20%) had mixed hypo- and hyperactive platelets, and 2 (10%) had hyperactive platelets. Three of the 8 patients with initial hypoactivity remained hypoactive, while 2 developed a mixed picture, 2 became hyperactive, and 1 normalized. Of the 6 patients with initial hyperactivity, 4 became hypoactive and 2 developed a mixed pattern. All of the 3 patients with initial hypo- and hyperactivity became hypoactive. Finally, 2 of the 3 patients with initial normal platelets became hypoactive while 1 remained normal. There was a significant decrease in ristocetin-induced platelet aggregation after therapy (p<0.001), while platelet aggregation and secretion induced by other agonists showed no difference after treatment (p>0.05).

Bulgular: Tanı sırasında, 17/20 hasta anormal trombosit agregasyon sonuçlarına sahip idi; sekizinde (%40) hipoaktivite, altısında (%30) hiperaktivite ve üçünde (%15) miks hipo- ve hiperaktivite saptandı. Ortalama 19 ay (min: 5 ay-maks: 35 ay) imatinib mesilat kullanımı sonrası tüm hastalarda trombosit agregasyon testleri tekrarlandı. Tedavi sonrası, 18/20 (%90) hasta anormal laboratuvar sonuçlarına sahip idi; 12’si (%60) hipoaktif trombositler, dördü (%20) miks hipo- ve hiperaktif trombositler ve ikisi (%10) hiperaktif trombositlere sahip idi. Başlangıçta hipoaktivitesi olan sekiz hastanın üçü hipoaktif kalır iken ikisi miks bir görüntü geliştirdi, ikisi hiperaktif oldu ve biri normalize oldu. Başlangıçta hiperaktivitesi olan altı hastanın dördü hipoaktif oldu ve ikisi miks patern geliştirdi. Başlangıçta hipo- ve hiperaktivitesi olan üç hastanın tamamı hipoaktif oldu. Son olarak, başlangıçta normal trombositleri olan üç hastanın ikisi hipoaktif olur iken biri normal kaldı. Ristosetin ile indüklenen trombosit agregasyonunda tedavi sonrası anlamlı azalma (p<0,001) olur iken diğer agonistler ile indüklenen trombosit agregasyon ve sekresyonu tedavi sonrası farklılık göstermedi (p>0,05).

Conclusion: These findings indicate that a significant proportion of chronic myeloid leukemia patients have different patterns of platelet function abnormalities and imatinib mesylate has no effect on these abnormalities, with a significant impairment in ristocetin-induced platelet aggregation.

Sonuç: Bulgularımız kronik miyelositer lösemili hastaların önemli bir çoğunluğunun farklı paternde trombosit fonksiyon anormalliklerine sahip olduğunu ve imatinib mesilatın ristosetin ile indüklenen trombosit fonksiyonunda azalma dışında bu anormallikler üzerinde etkisi olmadığını göstermiştir.

Keywords: Platelet aggregation, Chronic myeloid leukemia, Imatinib mesylate

Anahtar Sözcükler: Trombosit agregasyonu, Kronik miyelositer lösemi, İmatinib mesilat

Address for Correspondence/Yazışma Adresi: Olga Meltem AKAY, M.D., Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey   E-mail : olga.akay@hotmail.com

Received/Geliş tarihi: May 28, 2014 Accepted/Kabul tarihi: September 30, 2014

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Akay OM, et al: Platelet Dysfunction in Chronic Myeloid Leukemia and Imatinib Mesylate

Turk J Hematol 2016;33:127-130

Introduction

Whole Blood Platelet Lumi-Aggregometry

Imatinib mesylate (Gleevec or Glivec, Novartis, Basel, Switzerland) is the first tyrosine kinase (TK) inhibitor active against BCR-ABL, c-ABL, ARG, PDGF-r, and c-KIT. TKs are important signaling enzymes for the cellular regulation of proliferation, differentiation, survival, function, and motility, and various tumors overexpress TKs, leading to uncontrolled mitogenic signals to neoplastic cells [1]. Imatinib mesylate has considerable antineoplastic activity in patients with chronic myeloid leukemia (CML) and some solid tumors [2].

Whole blood platelet lumi-aggregometry studies were performed on the citrate tubes at the time of diagnosis and repeated following imatinib therapy in all patients. Platelet aggregation (measured as the increase in impedance) and release were simultaneously measured using a whole blood lumi-aggregometer (Model 560-Ca, Chrono-log Corporation, USA) according to the manufacturer’s instructions. The agonists used and their final concentrations were, in sequence, adenosine diphosphate (ADP; Chrono Par 384, Chrono-log Corporation), 5 µM; arachidonic acid (AA; Chrono Par 390), 0.5 mM; ristocetin (Chrono Par 396), 1.0 mg/mL; and collagen (Chrono Par 385), 2 µg/mL. Platelet function testing on all samples was completed within 2 h of collection. Our laboratory reference ranges for platelet aggregation (ohm) and adenosine triphosphate (ATP) release (nmol) were 10-22 ohm and 0.3-2 nmol for ADP, 10-28 ohm and 0.6-3 nmol for AA, 10-32 ohm and 0.3-2 nmol for collagen, and 3-19 ohm for ristocetin.

Thromboembolic and bleeding complications are the leading causes of morbidity and mortality in myeloproliferative neoplasms, particularly polycythemia vera and essential thrombocythemia, although these occur least frequently in patients with CML [3]. Abnormalities of platelet function arising from the clonal proliferation of hematopoietic cells including megakaryocyte precursors are regarded as the main origin of thrombo-hemorrhagic episodes [4]. Considering the reduction of BCR-ABL+ clones in response to imatinib mesylate and the recovery of normal hematopoietic stem and progenitor cells in the bone marrow [5], we performed platelet aggregation studies in CML patients who were treated with imatinib mesylate to investigate the effect of this drug on platelet function.

Statistics

Materials and Methods

Statistical analysis was performed using IBM SPSS 20.0 (IBM Corp., Armonk, NY, USA). The Shapiro-Wilk test was used to test normality for continuous variables. Nonnormally distributed variables were compared with the Wilcoxon test for paired data and presented as medians (quartiles). P<0.05 was accepted as statistically significant.

Patients

Results

A total of 20 newly diagnosed chronic-phase CML patients who started treatment with imatinib were enrolled. The diagnosis of CML was made by the demonstration of Philadelphia chromosome positivity and compatible hematological findings in peripheral blood and bone marrow. Imatinib was used as the fırst-line treatment in all patients. It was started at a dose of 400 mg daily. Dose modifications were allowed according to toxicity and treatment efficacy, ranging from 200 to 800 mg.

The median age of the total group of patients was 43 years (range: 29-71); there were 10 females and 10 males. Median platelet counts before and after treatment were 339.50x109/L (range: 227.50-527.25) and 225.50x109/L (range: 171.00259.25), respectively.

Sample Collection Venous blood was collected from patients under light tourniquet through 19-gauge needles into vacutainers (Becton Dickinson). A 3-mL di-potassium EDTA (1.5 mg/mL) sample was collected fırst followed by two 4.5-mL 3.2% tri-sodium citrate (0.105 M) vacutainers. The collection was performed early in the morning following a light breakfast. Subjects with known bleeding or other systemic disorders such as renal, hepatic, and endocrine diseases, and those who had taken aspirin or other nonsteroidal anti-inflammatory agents within 10 days prior to blood sampling were excluded. Automated cell counts were performed on the EDTA sample tube with a Beckman Coulter Gen-S SM (USA) automated blood-counting device. 128

As described by Manoharan et al. [6], platelets were considered to be hyperactive if at least one result (aggregation or ATP release with one agonist) was above the reference range and hyporeactive if at least one result (aggregation or ATP release with one agonist) was below the reference range. Mixed hypoand hyperactive platelets were considered present when at least one result (aggregation or ATP release) was below and above the reference range, respectively. At the time of diagnosis, 17/20 patients had abnormal platelet aggregation results; 8 (40%) had platelet hypoactivity, 6 (30%) had platelet hyperactivity, and 3 (15%) had mixed hypo- and hyperactivity. After a mean of 19 months (min: 5 months-max: 35 months), repeat platelet aggregation studies were performed in all patients who received imatinib during this period. A major molecular response was achieved in 17 (85%) of the patients at the time

Turk J Hematol 2016;33:127-130

Akay OM, et al: Platelet Dysfunction in Chronic Myeloid Leukemia and Imatinib Mesylate

of retesting. After imatinib therapy, 18/20 (90%) patients had abnormal laboratory results; 13 (65%) had hypoactive platelets, 3 (15%) had mixed hypoa and hyperactive platelets, and 2 (10%) had hyperactive platelets. Three of the 8 patients with initial hypoactivity remained hypoactive, while 2 developed a mixed picture, 2 became hyperactive, and 1 normalized. Of the 6 patients with initial hyperactivity, 5 became hypoactive and 1 developed a mixed pattern. All of the 3 patients with initial hypo- and hyperactivity became hypoactive. Finally, 2 of the 3 patients with initial normal platelets became hypoactive while 1 remained normal (Figure 1). When we compared pretreatment and post treatment platelet aggregation values induced by ADP, AA, ristocetin, and collagen, we found that there was a significant decrease in ristocetininduced platelet aggregation (p<0.001) after treatment, while pre- and post treatment platelet aggregation responses to the other agonists were not significantly different (p>0.05). There was also no significant difference between pretreatment and post treatment platelet secretion values induced by ADP, AA, and collagen (Table 1). We did not notice a significant correlation between platelet count and platelet aggregation and secretion results induced by any of the agonists used. Moreover, platelet

responses showed no correlation with the length of time on imatinib and none of the studied patients experienced bleeding.

Discussion In the present study, we demonstrated that a significant proportion of CML patients (85%) have different patterns of platelet dysfunction and imatinib therapy has neither a positive nor a negative impact on these functional defects. The number of studies investigating the effect of CML therapy on platelet abnormalities is very limited. In one study including 6 CML patients, plasma levels of beta-TG and PF4 were not reduced and platelet aggregation did not improve following normalization of leukocyte and platelet counts after busulfan or hydroxyurea [7]. In contrast, Barbui et al. [8] reported a normalization of spontaneous platelet aggregation and improvement of collagen-induced aggregation but a persistent dense granule storage deficiency after busulfan therapy. Data evaluating the effects of imatinib on platelet function are also still limited. In a recent study by Quintas-Cardama et al. [9], 5 of the 15 evaluable CML patients on imatinib had normal platelet aggregation and 10 (66%) had impaired AA-induced platelet aggregation, including 2 (13%) with impaired epinephrineinduced aggregation. Our results suggest that imatinib does not have either a positive or a negative impact on platelet function, with a significant impairment in ristocetin-induced platelet aggregation. It is known that, during ristocetin-induced platelet aggregation, ristocetin binds to the platelet surface through its phenolic groups. Being positively charged, the bound ristocetin reduces the net negative charge on the platelet surface and permits a closer contact between platelets. This, in turn, permits the von Willebrand factor to bridge between platelets, resulting in agglutination. Imatinib, either by altering ristocetinâ&#x20AC;&#x2122;s phenolic Table 1. Comparison of platelet aggregation and secretion results induced by agonists before and after imatinib therapy in chronic myeloid leukemia patients (n=20). Before therapy

After therapy

Median (25%-75%)

p-value

ADP (ohm)

14.5 (8.5-21)

8.5 (4.25-16)

0.079

AA (ohm)

12 (9-19.75)

13 (3.25-18.75)

0.380

Ristocetin (ohm)

13 (9.25-22.75)

5.5 (3-8.75)

0.002

Collagen (ohm)

26 (20-28.75)

0.251

ADP (nmol)

0.65 (0.31-1.09)

0.5 (0.3-0.94)

0.481

AA (nmol)

1.4 (0.9-1.9)

0.83 (0.55-1.16) 0.370

Collagen (nmol)

0.55 (0.3-0.84)

0.67 (0.47-1.07)

Platelet aggregation

Platelet secretion

Figure 1. Platelet aggregation (ohm) and secretion (nmol) values induced by agonists before and after imatinib therapy in CML patients (n=20). Reference interval for impedance (ohm) or release (nmol) of the agonist is shown by dotted lines in each chart. ADP: Adenosine diphosphate, AA: arachidonic acid.

0.083

ADP: Adenosine diphosphate, AA: arachidonic acid

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Akay OM, et al: Platelet Dysfunction in Chronic Myeloid Leukemia and Imatinib Mesylate

groups or by occupying its binding sites on the platelet surface, may cause a decrease in ristocetin-induced platelet aggregation. Moreover, since ristocetin does bind to the platelet, the decrease in platelet counts after imatinib treatment may be another explanation for impaired ristocetin-induced platelet aggregation results. However, these hypotheses must be confirmed with further in vitro studies [10,11]. We speculated that myelosuppression under imatinib treatment depends on the fact that after reduction of clonal hematopoiesis in response to treatment, normal hematopoietic stem and progenitor cells have to recover from preexisting suppression by the malignant clone and re-expand in the bone marrow. This may be related to changes in the growth pattern of megakaryocytes and a certain improvement of platelet function and activation after therapy. However, imatinib treatment did not improve most of the patterns of platelet function abnormalities while significantly increasing hypoactivity of platelets. The existence of platelet function abnormalities even in patients who achieved a major molecular response with imatinib in our study led us to assume that normal hematopoiesis is not fully restored in a substantial portion of CML patients despite the achievement of the desired response. Imatinib mesylate is designed as a TK inhibitor active against BCR-ABL [12,13], but it also inhibits other TKs, such as PDGF-r and c-KIT [14,15]. Inhibitory effects of the drug on platelet function may be partly explained by the inhibition of platelet TKs.

Conclusion In conclusion, these findings indicate that a significant proportion of CML patients have different patterns of platelet function abnormalities, which must be further investigated. However, one important limitation of this study is that we only studied platelet function tests in CML patients without a control group including imatinib-treated non-CML patients such as patients with gastrointestinal stromal tumors or hypereosinophilic syndrome. Subsequent studies should investigate how imatinib changes the function of platelets in patients with normal hematopoiesis. Ethics Ethics Committee Approval: Eskişehir Osmangazi University Ethics Committee, Informed Consent: It was taken.

Turk J Hematol 2016;33:127-130

or Processing: Olga Meltem Akay, Zafer Gülbaş; Analysis or Interpretation: Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş; Literature Search: Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş; Writing: Olga Meltem Akay, Fezan Mutlu, Zafer Gülbaş. 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. Hensley ML, Ford JM. Imatinib treatment: specific issues related to safety, fertility, and pregnancy. Semin Hematol 2003;40:21-25. 2. Agis H, Jaeger E, Doninger B, Sillaber C, Marosi C, Drach J, Schwarzinger I, Valent P, Oehler L. In vivo effects of imatinib mesylate on human haematopoietic progenitor cells. Eur J Clin Invest 2006;36:402-408. 3. Schafer AI. Bleeding and thrombosis in myeloproliferative disorders. Blood 1984;64:1-12. 4. Wehmeier A, Schneider W. Megakaryocytes and platelets as the main cause for vascular events in chronic myeloproliferative disorders. Hamostaseologie 1996;16:151-163. 5. Appel S, Balabanov S, Brümmendorf TM, Brossart P. Effects of imatinib on normal hematopoiesis and immune activation. Stem Cells 2005;23:1082-1088. 6. Manoharan A, Gemmell R, Brighton T, Dunkley S, Lopez K, Kyle P. Thrombosis and bleeding in myeloproliferative disorders: identification of at-risk patients with whole blood platelet aggregation studies. Br J Haematol 1999;105:618-625. 7. Wehmeier A, Scharf RE, Fricke S, Schneider W. A prospective study of hemostatic parameters in relation to the clinical course of myeloproliferative disorders. Eur J Haematol 1990;45:191-197. 8. Barbui T, Bassan R, Viero P, Cortelazzo S, Dini E. Platelet function after busulfan in chronic myeloproliferative disorders. Haematologica 1983;68:469-477. 9. Quintas-Cardama A, Han X, Kantarjian H, Cortes J. Tyrosine kinase inhibitorinduced platelet dysfunction in patients with chronic myeloid leukemia. Blood 2009;114: 261-263. 10. Coller BS, Gralnick HR. Studies on the mechanism of ristocetin-induced platelet agglutination. Effects of structural modification of ristocetin and vancomycin. J Clin Invest 1977;60:302-312. 11. Kattlove HE, Gomez MH. Studies on the mechanism of ristocetin-induced platelet aggregation. Blood 1975;45:91-96. 12. Druker BJ, Lydon NB. Lessons learned from the development of an Abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000;105:3-7. 13. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of BCR-ABL positive cells. Nat Med 1996;2:561-566.

Authorship Contributions

14. Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, Lydon NB. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000;295:139-145.

Medical Practices: Olga Meltem Akay; Concept: Olga Meltem Akay; Design: Olga Meltem Akay, Zafer Gülbaş; Data Collection

15. Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. Inhibition of c-kit receptor tyrosine kinase activity by STI571, a selective tyrosine kinase inhibitor. Blood 2000;96:925-932.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0379 Turk J Hematol 2016;33:131-134

Immature Reticulocyte Fraction and Absolute Neutrophil Count as Predictor of Hemopoietic Recovery in Patients with Acute Lymphoblastic Leukemia on Remission Induction Chemotherapy Remisyon İndüksiyon Kemoterapisi Alan Akut Lenfoblastik Lösemi Hastalarında Hematopoietik Toparlanmanın Öngörülmesinde İmmatür Retikülosit Fraksiyonu ve Mutlak Nötrofil Sayısı Shan E. Rauf, Saleem Ahmed Khan, Nadir Ali, Nabeel Khan Afridi, Maria Haroon, Ammara Arslan Armed Forces Institute of Pathology, Department of Hematology, Rawalpindi, Pakistan

Abstract

Öz

Objective: Acute lymphoblastic leukemia (ALL) encompasses a group of lymphoid neoplasms that are more common in children and arise from B-and T-lineage lymphoid precursor cells. The immature reticulocyte fraction (IRF), a new routine parameter in hematology analyzers, can give an indication of hemopoietic recovery like absolute neutrophil count (ANC). The purpose of this study was to evaluate IRF in excess of 5% was considered as IRF recovery.

Amaç: Akut lenfoblastik lösemi (ALL) çocuklarda daha sık görülen ve B- ve T-lenfoid öncül hücre dizilerinden kaynaklanan bir grup lenfoid neoplaziyi kapsamaktadır. Hematolojik incelemede yeni rutin bir parametre olan immatür retikülosit fraksiyonu (İRF), mutlak nötrofil sayısı (MNS) gibi hematopoietik toparlanma hakkında fikir oluşturabilir. Bu çalışmanın amacı remisyon indüksiyon kemoterapisi almakta olan ALL hastalarında İRF ve MNS toparlanmasını değerlendirmektir. Gereç ve Yöntemler: Bu tanımlayıcı çalışmada, remisyon indüksiyon tedavisi alan 45 ALL hastasından tedavinin birinci günü başlayarak, toparlanmaya kadar günaşırı 2,5-3 mL EDTA’lı tüpe kan alınmış ve Sysmex XE-5000 ile inceleme yapılmıştır. MNS toparlaması, ardışık üç sayımda MNS değerinin >0,5x109/L olduğu günlerden ilki olarak tanımlanmıştır. İRF toparlanması İRF’nin %5’i aşması olarak belirlenmiştir.

Materials and Methods: In this descriptive study, 2.5 to 3 mL of EDTA blood of 45 ALL patients undergoing the remission induction phase of their treatment was sampled and analyzed with a Sysmex XE-5000 on day 1 and every second day thereafter until the day of recovery. ANC of >0.5x109/L on the day corresponding to the first of the three consecutive counts was considered as the day of ANC recovery. IRF recovery was an IRF in excess of 5%. Results: The mean age of the patients was 12.04±5.30 years; 25 patients (55.6%) were male and 20 patients (44.4%) were female. On day 1 of induction remission, the mean IRF value was 9.68±1.41, while the mean ANC value was 0.077±0.061. Mean recovery day for IRF was 11.84±7.44 and mean recovery day for ANC was 17.67±8.77 (twotailed p-value <0.0001 with 95% confidence interval). By day 28, out of 45 patients 36 (80%) showed ANC recovery, while 41 (91%) showed IRF recovery. The remaining patients who had not shown recovery by day 28 were further followed up and all of them showed recovery of both parameters by day 39.

Bulgular: Hastaların ortalama yaşı 12,04±5,30 yıldı; 25 hasta (%55,6) erkek ve 20 hasta (%44,4) kadındı. İndüksiyon tedavisinin birinci gününde, ortalama İRF değeri 9,68±1,41 iken, ortalama MNS değeri 0,077±0,061 idi. İRF için ortalama toparlanma süresi 11,84±7,44 gün ve MNS için ortalama toparlanma süresi ise 17,67±8,77 gündü (p değeri <0,0001, %95 güven aralığı içinde). Yirmi sekizinci günde, 45 hastanın 36’sında (%80) MNS toparlanması varken 41’inde (%91) İRF toparlanması bulunmaktaydı. Yirmi sekizinci günde toparlanması bulunmayan hastaların takibine devam edildi ve 39. günde bu hastaların tamamında her iki parametre açısından da toparlanma tespit edildi.

Conclusion: This study concluded that postinduction bone marrow hemopoietic recovery was earlier by IRF than ANC in children with ALL on chemotherapy.

Sonuç: Bu çalışma kemoterapi alan ALL’li çocuklarda indüksiyon tedavisi sonrasında kemik iliği hematopoietik toparlanmanın İRF’de MNS’ye göre daha erken olduğunu göstermiştir.

Keywords: Acute lymphoblastic leukemia, Lymphoid cell neoplasm, Hematopoiesis, Chemotherapy

Anahtar Sözcükler: Akut lenfoblastik lösemi, Lenfoid hücreli neoplazi, Hematopoiez, Kemoterapi

Address for Correspondence/Yazışma Adresi: Shan E. RAUF, M.D., Armed Forces Institute of Pathology, Department of Hematology, Rawalpindi, Pakistan Phone : 92-333-563 19 29 E-mail : shan.e.rauf673@gmail.com

Received/Geliş tarihi: September 24, 2014 Accepted/Kabul tarihi: April 20, 2015

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Rauf SE, et al: IRF and ANC as Predictors of Hemopoietic Recovery in ALL Patients

Introduction Leukemia is the most common malignancy of childhood and acute lymphoblastic leukemia (ALL) accounts for up to 75% to 80% of the leukemia cases in the world [1]. In the Pakistani population, the frequency of ALL in children and adults combined is 32% of all malignancies [2]. The first case of leukemia in an adult was reported in 1845 by John Hughes Bennett and in children by Henry Fuller in 1846 [2]. Over the last 50 years many new modalities of diagnosis and treatment of leukemia have evolved, leading to improved survival [3,4]. Chemotherapy is the initial treatment of choice in most patients of ALL and is divided into the following stages: remission induction, consolidation or intensification, and maintenance (continuation) therapy, with central nervous system prophylaxis generally provided in each stage. The aim of remission induction therapy is to induce a complete remission. The initial response to remission induction therapy is one of the most important prognostic factors in ALL [5]. The cytotoxic chemotherapeutic agents cause marrow suppression, making patients prone to anemia, bleeding, and infections. The main cause of death in two-thirds of the patients is infection, mostly fungal [6]. During the period of marrow suppression, extensive monitoring of blood counts is required to assess hemopoietic recovery. The hemopoietic recovery can be assessed by conventional parameters like absolute neutrophil count (ANC) recovery or the newer but less commonly used parameter of immature reticulocyte fraction (IRF) recovery. The ANC has >96% sensitivity to predict bone marrow recovery after chemotherapy [7]. The IRF is now being widely used in different centers as an early predictor for hemopoietic recovery in place of the more traditional parameter of ANC recovery, which appears later in the induction phase. According to a prior study, IRF shows early bone marrow recovery in 78% of cases as compared to ANC [8] and has a sensitivity of 92% [9]. Reticulocytes reflect the erythropoietic activity of the bone marrow and were traditionally assessed by manual microscopic method; however, this method is subject to high variability. Today it is measured more objectively by flow cytometry-based hematology analyzers, which measure the messenger ribonucleic acid (RNA) content and the maturity of reticulocytes. The fluorescence of reticulocytes is dependent on the RNA content of the reticulocytes. Immature reticulocytes with higher RNA content will have maximum fluorescence, while reticulocytes with lower RNA content produce minimum fluorescence. On the basis of fluorescence intensity signals, reticulocytes are classified into 3 maturation stages: lowfluorescence reticulocytes (LFRs), medium-fluorescence reticulocytes (MFRs), and high-fluorescence reticulocytes (HFRs). The IRF is the combination of HFRs and MFRs and its fraction in excess of 5% is postulated as a reliable marker for hemopoietic recovery [8]. 132

Turk J Hematol 2016;33:131-134

Flow cytometry-based hematology analyzers are now being used in most of the large diagnostic centers of Pakistan, whereas assessment of hemopoietic recovery is still based on the conventional parameter of ANC. Unfortunately, due to lack of published data in regard to the importance of the IRF during treatment of ALL patients, this parameter is not being used effectively to monitor patients’ marrow status and so far has not been used as a protocol in Pakistan. The aim of this study is to evaluate IRF as an earlier indicator of bone marrow recovery than ANC in patients with ALL on remission induction chemotherapy.

Materials and Methods This descriptive study was carried out in the Department of Hematology at the Armed Forces Institute of Pathology, Rawalpindi, over a period of 1 year from January 2013 to January 2014. Sampling was done based on a consecutive non probability sampling technique. All diagnosed ALL patients undergoing remission induction chemotherapy of both genders were included. Remission induction as per the UKALL 2003 protocol was given, comprising dexamethasone, vincristine, L-asparaginase, and intrathecal methotrexate. Relapsead patients and those undergoing reinduction were excluded. For each included patient, 2.5 to 3 mL of EDTA anticoagulated blood was sampled and analyzed on day 1 and every second day thereafter until the day of recovery. For each sample of blood, complete blood counts along with differential leukocyte count (for calculation of ANC) and reticulocyte parameters were noted after running the sample on the flow cytometrybased hematology analyzer Sysmex XE-5000. ANC of more than >0.5x109/L on the day corresponding to the first of three consecutive scores was considered as ANC recovery. IRF recovery was an IRF (MFR+HFR) in excess of 5%. As a control, 2.5 to 3 mL of EDTA anticoagulated blood of normal healthy individuals was also examined for reticulocyte parameters on the Sysmex XE-5000. All the collected data were analyzed with SPSS 19.0. The mean and standard deviation were calculated for quantitative variables like age, ANC, and IRF, and comparisons of means were carried out by paired samples t-test. For qualitative variables like gender, frequency and percentage was calculated.

Results A total of 45 patients were included in this study. The majority of the patients were 11-20 years of age. Mean age of the patients was 12.04±5.30 years, and 25 patients (55.6%) were male and 20 patients (44.4%) were female. Mean IRF value on day 1 of induction remission was 9.68±1.41, mean ANC value on day 1 of induction remission was 0.077±0.061, mean recovery day for IRF was 11.84±7.44, and mean recovery

Rauf SE, et al: IRF and ANC as Predictors of Hemopoietic Recovery in ALL Patients

Turk J Hematol 2016;33:131-134

day for ANC was 17.67±8.77 (two-tailed p-value <0.0001, 95% confidence interval). Mean values of different variables of ANC and IRF are shown in Table 1. Out of 45 patients, 40 (88.9%) patients showed earlier IRF recovery as compared to ANC. Four (8.9%) patients had the same day of recovery by both IRF and ANC while one (2.2%) had a later IRF recovery than ANC. The recovery days of each patient for both ANC and IRF are shown in Figure 1. By day 28, 41 (91%) patients showed IRF recovery, and ANC recovery was seen in 36 (80%) patients. All those patients not showing recovery by day 28 were further followed up and all of them showed recovery by day 39, as shown in Table 1 and Figure 1.

Discussion Chemotherapy for ALL has not changed much over the years, except for a few variations in different centers. The chemotherapeutic agents used in remission induction therapy usually cause severe myelosuppression in these patients. This critical period is variable in patients and requires critical care, supportive therapy, and regular monitoring. Many patients succumb to severe sepsis and bleeding in this period. Recovery of bone marrow from myelosuppression is an indicator of likely hematological remission. The ANC has traditionally been used as an early predictor of bone marrow recovery. However, with the advent of the latest flow cytometry-based hematological analyzers, the IRF is being increasingly used for this purpose.

The IRF is an accurate and reliable parameter easily obtained from automated cell counters such as the Sysmex XE-Series [9]. Several studies showed that the immature reticulocytes detected by flow cytometry are earlier indicators of bone marrow recovery than the detection of ANC in post chemotherapy patients with acute leukemia [10,11]. Although our study was limited in time and sample size, we were able to reach similar conclusions to those published by George et al., who found that immature reticulocytes indicate engraftment, and the use of immature reticulocytes might enable the cessation of antibiotics and growth factors, which could lead to earlier discharge from the hospital and cost savings [9]. The Spanish Multicentric Study Group for hemopoietic recovery also concluded that a rise in IRF indicates hemopoietic recovery [12] and IRF recovery was seen in 91.2% of ALL patients on remission induction before ANC recovery. Luczynski et al. in their study stated that IRF was the first sign of hemopoietic recovery and might be used as a parameter of bone marrow function in clinical studies [13]. Das et al. in 2006 also showed IRF as the earlier predictor of bone marrow recovery as compared to ANC in childhood malignancies [10]. The median day for IRF recovery was 21, while for ANC recovery it was 23 [12]. In a study done in Bangladesh, IRF showed 78% similar or earlier recovery in patients with ALL on remission induction chemotherapy. The mean day for IRF recovery was 16.6±4.6 while it was 23.3±5.7 for ANC recovery, indicating that IRF is an earlier predictor of bone marrow hemopoietic recovery than ANC [8]. In our study, mean recovery days were 11.84±7.44 for IRF and 17.67±8.77 for ANC, showing earlier IRF recovery by an average of 6 days, while it was shown to be 4 days earlier by Grazziutti et al. [14]. This prediction of early recovery by the simple and reproducible parameter of IRF can have significant impact on the management of patients.

Conclusion Figure 1. Recovery day of each patient by both immature reticulocyte fraction and absolute neutrophil count.

This study concluded that the IRF shows earlier hemopoietic recovery as compared to the current practice of ANC for the monitoring of ALL patients on remission induction chemotherapy.

Table 1. Mean values of different variables. Variable

Number

Minimum

Maximum

Mean ± SD

Two-tailed p-value

IRF on day 1 of remission induction

7.0

12.1

9.68±1.41

ANC on day 1 of remission induction

0.01

0.3

0.077±0.061

Day of recovery for IRF

11.84±7.44

<0.0001

Day of recovery for ANC

17.67±8.77

<0.0001

IRF: Immature reticulocyte fraction, ANC: absolute neutrophil count, SD: standard deviation.

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This early laboratory indicator of hemopoietic recovery will guide clinicians to make early and important therapeutic decisions in such patients. Nowadays, the IRF is offered by most new hematology analyzers. Moreover, this test is a simple, quick, reproducible, and reliable parameter in the automated hematology analyzers. Authorship Contribution Concept: Shan E. Rauf, Saleem Ahmed Khan; Design: Shan E. Rauf, Saleem Ahmed Khan; Data Collection and Processing: Shan E. Rauf, Nabeel Khan Afridi; Analysis and Interpretation: Shan E. Rauf, Nadir Ali; Literature Search: Shan E. Rauf, Saleem Ahmed Khan, Nadir Ali, Nabeel Khan Afridi, Maria Haroon, Ammara Arslan; Writing: Shan E. Rauf, Saleem Ahmed Khan, Nadir Ali, Nabeel Khan Afridi, Maria Haroon, Ammara Arslan. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Turk J Hematol 2016;33:131-134

5. Schrappe M, Reiter A, Ludwig WD, Harbott J, Zimmermann M, Hiddemann W, Niemeyer C, Henze G, Feldges A, Zintl F, Kornhuber B, Ritter J, Welte K, Gadner H, Riehm H. Improved outcome in childhood acute lymphoblastic leukemia despite reduced use of anthracyclines and cranial radiotherapy: results of trial ALL-BFM 90. German-Austrian-Swiss ALL-BFM Study Group. Blood 2000;95:3310-3322. 6. Asim M, Zaidi A, Ghafoor T, Qureshi Y. Death analysis of childhood acute lymphoblastic leukemia; an experience at Shoukat Khanum Memorial Cancer Hospital and Research Centre, Pakistan. J Pak Med Assoc 2011;61:666-670. 7. Hijiya N, Onciu M, Howard SC, Zhang Z, Cheng C, Sandlund JT, Khyzer EP, Behm FG, Pui CH. Utility of automated counting to determine absolute neutrophil counts and absolute phagocyte counts for pediatric cancer treatment protocols. Cancer 2004;101:2681-2686. 8. Yesmin MS, Sultana T, Roy CK, Rehman MQ, Ahmed ANN. Immature reticulocyte fraction as a predictor of bone marrow recovery in children with acute lymphoblastic leukemia on remission induction phase. Bangladesh Med Res Counc Bull 2011;37:57-60. 9. George P, Wyre RM, Bruty SJ, Sweetenham JW, Duncombe AS. Automated immature reticulocyte counts are early markers of engraftment following autologous PBSC transplantation in patients with lymphoma. J Hematother Stem Cell Res 2000;9:219-223. 10. Das R, Dip NB, Garewal G, Marwaha RK, Vohra H. Automated reticulocyte response is a good predictor of bone-marrow recovery in pediatric malignancies. Pediatr Hematol Oncol 2006;3:299-305.

References

11. Norhana JF, De Souza CA, Vigorito AC. Immature reticulocytes as an early predictor of engraftment in autologous and allogeneic bone marrow transplantation. Clin Lab Haematol 2003;25:47-54.

1. Rana ZA, Rabbani MW, Sheikh MA, Khan AA. Outcome of childhood acute lymphoblastic leukemia after induction therapy-3 year experience at a single paediatric oncology centre. J Ayub Med Coll 2009;21:150-153.

12. [No authors listed.] Flow cytometric reticulocyte quantification in the evaluation of hematopoietic recovery. Spanish Multicentric Study Group for Hematopoietic Recovery. Eur J Haematol 1994;53:293-297.

2. Yasmeen N, Ashraf S. Childhood acute lymphoblastic leukemia; epidemiological and clinicopathological features. J Pak Med Assoc 2009;59:150-153.

13. Luczynski W, Ratomski K, Wysocka J, Krawczuk-Rybuk M, Jankiewicz P. Immature reticulocyte fraction (IRF)--an universal marker of hemopoiesis in children with cancer? Adv Med Sci 2006;51:188-190.

3. Mehrzad VA, Liaghat L, Ashrafi F, Tazhibi M, Hajalikhani M, Alijanian N. The mortality and response rate after FLANG regimen in patients with refractory/relapsed acute leukemia. Adv Biomed Res 2012;1:54.

14. Grazziutti ML, Dong L, Miceli MH, Cottler-Fox M, Krishna SG, Fassas A, van Rhee F, Barlogie BM, Anaissie EJ. Recovery from neutropenia can be predicted by the immature reticulocyte fraction several days before neutrophil recovery in autologous stem cell transplant recipients. Bone Marrow Transplant 2006;37:403-409.

4. Campana D, Coustan-Smith E. Measurement of treatment response in childhood acute leukemia. Korean J Hematol 2012;47:245-254.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2014.0405 Turk J Hematol 2016;33:135-140

The Prognostic Significance of Soluble Urokinase Plasminogen Activator Receptor in Acute Myeloid Leukemia Akut Miyeloid Lösemili Hastalarda Solubl Ürokinaz Plazminojen Aktivatör Reseptörünün Prognozdaki Önemi Nergiz Erkut1,2, Ahmet Menteşe3,4, Hasan Mücahit Özbaş2, Nilay Ermantaş2, Ayşegül Sümer4, Asım Örem4, Mehmet Sönmez2 1Kanuni Training and Research Hospital, Clinic of Hematology, Trabzon, Turkey 2Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey 3Karadeniz Technical University, Vocational School of Health Sciences, Program of Medical Laboratory Techniques, Trabzon, Turkey 4Karadeniz Technical University Faculty of Medicine, Department of Medical Biochemistry, Trabzon, Turkey

Abstract

Öz

Objective: The soluble urokinase plasminogen activator receptor (suPAR) is a soluble form of the urokinase plasminogen activator receptor expressed in various immune and cancer cells. The levels of suPAR have been demonstrated to correlate with prognosis in various cancers. This study was intended to investigate serum suPAR levels and their effect on prognosis in patients with acute myeloid leukemia (AML).

Amaç: Solubl ürokinaz plazminojen aktivatör reseptörü (süPAR) çeşitli immün sistem ve kanser hücrelerinde eksprese edilen ürokinaz plazminojen aktivatör reseptörün çözünür formudur. Çeşitli kanserlerde süPAR düzeyinin prognoz ile ilişkili olduğu gösterilmiştir. Bu çalışmada akut miyeloid lösemili (AML) hastalarda süPAR düzeyi ve prognoz üzerine olan etkisinin araştırılması planlandı.

Materials and Methods: Thirty newly diagnosed patients with AML and 29 healthy individuals were enrolled. Serum suPAR levels were analyzed by enzyme-linked immunosorbent assay.

Gereç ve Yöntemler: Çalışmaya yeni tanı almış 30 AML’li hasta ve 29 sağlıklı birey dahil edildi. Serum süPAR düzeyi enzyme-linked immunosorbent assay yöntemi ile analiz edildi.

Results: Serum suPAR levels were significantly higher in patients with AML than in healthy individuals (9±5.9 ng/mL and 2.4±1.4 ng/mL, respectively; p<0.001). Positive correlation was determined between suPAR levels and white blood cell counts (p<0.01). Serum suPAR levels were lower in patients who achieved complete response than in patients not achieving complete response (5.5±2.2 ng/mL and 12±6.6 ng/mL, respectively; p<0.001). The median overall survival was longer in patients with serum suPAR levels below 6.71 ng/mL than in those with serum suPAR levels above 6.71 ng/mL (12.6±13.2 months and 1.71±0.6 months, respectively; p=0.02). Multivariate Cox regression analysis showed that suPAR had independent prognostic value (95% confidence interval: 1.029-6.259; p<0.05) in AML.

Bulgular: Serum süPAR düzeyi AML’li hastalarda sağlıklı bireylere göre önemli derecede daha yüksek tespit edildi (9±5,9 ng/mL, 2,4±1,4 ng/mL, sırasıyla, p<0,001). süPAR düzeyi ile lökosit sayısı arasında pozitif bir korelasyon izlendi (p<0,01). Serum süPAR düzeyi, tam remisyona giren hastalarda tam remisyona girmeyen hastalara göre daha düşüktü (5,5±2,2 ng/mL, 12±6,6 ng/mL, sırasıyla, p<0,001). Toplam yaşam süresi, serum süPAR düzeyi 6,71 ng/mL’nin altında olan hastalarda, 6,71 ng/mL üstünde olanlara göre daha uzundu (12,6±13,2 ay, 1,71±0,6 ay, sırasıyla, p=0,02). AML’de çok değişkenli Cox regresyon analizi süPAR düzeyinin bağımsız prognostik değere sahip olduğunu gösterdi (%95 güven aralığı: 1,029-6,259; p<0,05).

Conclusion: Serum suPAR levels can be used as a prognostic marker in AML.

Sonuç: AML’li hastalarda serum süPAR düzeyi prognostik bir belirteç olarak kullanılabilir.

Keywords: Soluble urokinase plasminogen activator receptor, Acute myeloid leukemia, Prognosis

Anahtar Sözcükler: Solubl ürokinaz plazminojen aktivatör reseptörü, Akut miyeloid lösemi, Prognoz

Address for Correspondence/Yazışma Adresi: Nergiz ERKUT, M.D., Kanuni Training and Research Hospital, Clinic of Hematology, Trabzon, Turkey Phone : +90 462 341 56 56 E-mail : drnusta@hotmail.com

Received/Geliş tarihi: October 10, 2014 Accepted/Kabul tarihi: May 04, 2015

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Erkut N, et al: Levels of Soluble Urokinase Plasminogen Activator Receptor in Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is a heterogeneous neoplastic disorder characterized by uncontrolled proliferation of hematopoietic stem cells [1]. Although 70%-80% of patients younger than 60 years of age achieve complete remission (CR), only 30%-40% obtain long-term survival. Moreover, CR is only observed in 10%-15% of elderly patients [2]. The pathogenesis of AML involves various disorders, such as mutations in transcription factors or epigenetic modifiers, aberrant signaling pathways, overexpression of the multidrug resistance gene, abnormal immune function, and abnormalities in the bone marrow microenvironment [3]. Prognostic factors include advanced age, poor performance status, high white blood cell (WBC) count, existence of prior myelodysplastic syndrome and myeloproliferative disease, previous history of cytotoxic therapy, and particularly cytogenetics and molecular genetic changes [4,5]. The urokinase plasminogen activator receptor (uPAR) is a glycoprotein consisting of 274 amino acids with a molecular weight of 55-60 kDa attached to the plasma membrane via a glycosylphosphatidylinositol anchor protein [6]. uPAR is expressed in neutrophils, lymphocytes, monocytes, macrophages, fibroblasts, and endothelial and some tumor cells [7,8,9]. The soluble urokinase plasminogen activator receptor (suPAR) is a soluble form of uPAR found in serum, plasma, urine, and other body fluids [10]. suPAR affects cancer progression through adhesion, migration, chemotaxis, proteolysis, and invasion [11]. Several studies have demonstrated that suPAR increases in some cancers and is associated with poor prognosis [12]. This study was intended to investigate serum suPAR levels and their effect on prognosis in patients with AML.

Materials and Methods Thirty newly diagnosed patients with AML and 29 healthy individuals presenting to the Deparment of Hematology, Faculty of Medicine, Karadeniz Technical University between January 2009 and July 2011 were enrolled in this study. The eligibility criterion was age between 18 and 80 years. Patients with a history of solid cancer or other hematological cancer, the presence of active infection, or active inflammatory disease were excluded. Venous blood specimens collected from both patient and control groups were placed into biochemical separator-containing tubes. Blood samples were centrifuged at 3000 rpm for 10 min and serum was stored at -80 °C for investigation of suPAR levels. All AML patients were diagnosed according to the World Health Organization classification system [13] and categorized into three groups (i.e. low risk, intermediate risk, and high risk) according to the National Comprehensive Cancer Network guidelines [14]. 136

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Patients aged ≤60 years or 61-65 years with good performance status were treated with the standard regimen [cytarabine, 24-h continuous intravenous (IV) infusion, 100 mg/m2, days 1-7; idarubicin, 30-min IV infusion, 12 mg/m2, days 1-3]. Patients with acute promyelocytic leukemia were treated with all-transretinoic acid (ATRA) plus idarubicin therapy (ATRA, orally, 45 mg/m2 per day in two divided doses until CR was achieved; idarubicin, 30-min IV infusion, 12 mg/m2, days 2, 4, 6, and 8). Elderly patients were treated with low-dose chemotherapy [low-dose cytarabine, subcutaneous (SC), 10 mg/m2, twice a day, days 1-10; or 5-azacytidine, SC, 75 mg/m2, days 1-7]. Remission status was evaluated after the completion of cancer therapy according to conventional criteria. Patients were followed for 2 years, monthly for the first year and every third month in the following year. Measurement of Soluble Urokinase Plasminogen Activator Receptor Levels The levels of serum suPAR were determined by enzyme-linked immunosorbent assay kit (ViroGates A/S, Denmark) according to the manufacturer’s protocols. The absorbance of samples was measured at 450 nm using a VERSA max tunable microplate reader (designed by Molecular Devices, USA). The results were expressed as ng/mL. The minimum detection limit of the assay was estimated to be 0.1 ng/mL. Statistical Analysis All analyses were carried out using SPSS 21.0. Descriptive statistical analysis was performed for all studied variables. Data were tested for normal distribution using the KolmogorovSmirnov test. Statistical comparisons between the patient and control groups were carried out using the Mann-Whitney test and chi-square test. The associations between serum suPAR levels and hemoglobin (Hb) or hematocrit levels and white blood cell (WBC) or platelet count were examined by Spearman correlation analysis. The area under the receiver operating characteristic (ROC) curve was used to compare the discriminative power of suPAR levels in the diagnosis of AML. Estimates of overall survival (OS) were calculated using the Kaplan-Meier method. The logrank test was used to analyze the effect on survival time of each variable. The Cox regression model was applied for multivariate analysis. Linear regression analysis was used to investigate the relationship between serum suPAR levels and sex, patient age, WBC count, French-American-British (FAB) classification, and Fms-like tyrosine receptor kinase-3 (FLT-3) mutation. A value of p<0.05 was considered statistically significant.

Results Thirty patients with AML and 29 healthy controls were included in the study. There were no statistical differences in term of age or sex between the two groups. Risk groups included 6

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patients at good risk, 19 at intermediate risk, and 5 at poor risk. At the end of the 2-year follow-up, 26 patients had died and 4 survived. Fourteen patients exhibited CR after remissioninduction chemotherapy, while CR was not achieved in the other 16. Table 1 shows the general characteristics and laboratory findings for both patients and healthy individuals. Serum suPAR levels were significantly higher in patients with AML than in healthy individuals (9±5.9 ng/mL and 2.4±1.4 ng/ mL, respectively; p<0.001) (Figure 1). Positive correlation was determined between suPAR levels and WBC count in patients with AML (p<0.01) (Figure 2), whereas there was no correlation between suPAR levels and Hb levels or platelet count. There was no significant difference in serum suPAR levels between patients aged ≤60 and >60 years (7.6±4.4 ng/mL and 12.3±8 ng/mL, respectively; p>0.05). Serum suPAR levels were lower in patients who achieved CR than in patients not achieving CR (5.5±2.2 ng/ mL and 12±6.6 ng/mL, respectively; p<0.001) (Figure 3). In AML patients, the area under the ROC curve for suPAR was 0.938 [95% confidence interval (CI): 0.843-0.984]. For the optimum diagnostic cut-off value of 2.79 ng/mL, the sensitivity and specificity were 96.67% and 79.31%, respectively (Figure 4).

The median OS of AML patients was 4.16 months (range: 0-32.9 months). In the Kaplan-Meier analysis and the Cox regression model, patients with high serum suPAR levels showed a trend toward poorer survival (p=0.02). The median OS was longer in patients with serum suPAR levels below 6.71 ng/mL than in those with serum suPAR levels above 6.71 ng/mL (12.6±13.2 months and 1.71±0.6 months, respectively; p=0.02) (Figure 5). WBC count had no significant effect on OS (p=0.9) (Figure 6). In linear regression analysis, sex, patient age, WBC count, FAB classification (i.e. M2, M3, M4, M5), and FLT-3 mutation were not associated with serum suPAR levels (p>0.05). Multivariate Cox regression analysis showed that suPAR had independent prognostic value (95% CI: 1.029-6.259; p<0.05) in AML. When the suPAR cut-off level was considered as 6.71 ng/mL, mortality risk was 2.5-fold higher in patients with levels above the cut-off limit. 30.00 25.00 20.00 15.00 10.00 5.00 0.00

30.00 25.00 20.00 15.00 10.00 5.00 0.00

Figure 1. Soluble urokinase plasminogen activator receptor concentrations in serum from acute myeloid leukemia patients and healthy controls. The dotted line indicates the mean value plus 3 standard deviations of healthy control serum (6.6 ng/mL). suPAR: Soluble urokinase plasminogen activator receptor, AML: acute myeloid leukemia, SD: standard deviation.

Figure 3. Soluble urokinase plasminogen activator receptor concentrations in serum from acute myeloid leukemia patients with no complete remission and acute myeloid leukemia patients with complete remission. The dotted line indicates the mean value plus 3 standard deviations of serum of acute myeloid leukemia patients with complete remission (12.1 ng/mL). suPAR: Soluble urokinase plasminogen activator receptor, AML: acute myeloid leukemia, SD: standard deviation, CR: complete remission.

30.00 25.00 20.00 15.00 10.00 5.00 0.00

Figure 2. Correlations between soluble urokinase plasminogen activator receptor levels and white blood cell count in acute myeloid leukemia patients. WBC: White blood cell, suPAR: soluble urokinase plasminogen activator receptor, AML: acute myeloid leukemia.

Figure 4. The receiver operating characteristic curves of acute myeloid leukemia patients according to soluble urokinase plasminogen activator receptor levels. suPAR: Soluble urokinase plasminogen activator receptor, AUC: area under the curve. 137

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Turk J Hematol 2016;33:135-140

Table 1. Characteristics of acute myeloid leukemia patients. Parameters

n=30

Median age (minimum-maximum), years

52 (24-80)

â&#x2030;¤60 years, n (%)

22 (73.3)

>60 years, n (%)

8 (26.7)

Sex, n (%) Female

17 (56.7)

Male

13 (43.3)

Risk groups, n (%)

Figure 5. Kaplan-Meier curves of acute myeloid leukemia patients according to soluble urokinase plasminogen activator receptor levels. suPAR: Soluble urokinase plasminogen activator receptor.

Good risk

6 (20)

Intermediate risk

19 (63.3)

Poor risk

5 (16.7)

Classification of AML, n (%) AML with recurrent genetic abnormalities

7 (23.3)

t(15;17)(q22;q12); PML-RARA

3 (10)

t(8;21)(q22;q22); RUNX1-RUNX1T1

2 (6.7)

t(16;16)(p13.1q22); CBFB-MYH11

1 (3.3)

Inv(3)(q21q26.2); RPN1-EVI1

1 (3.3)

AML with MDS-related changes

5 (16.7)

AML, therapy-related

1 (3.3)

AML not otherwise categorized

16 (53.2)

AML minimally differentiated

1 (3.3)

AML without maturation

1 (3.3)

AML with maturation

7 (23.3)

Acute myelomonocytic leukemia

6 (20)

Acute monocytic leukemia

1 (3.3)

Figure 6. Kaplan-Meier curves of acute myeloid leukemia patients according to white blood cell count. WBC: White blood cell.

Other

Discussion

WBC count (x109/L)

62 (3.6-218)

The urokinase-mediated plasminogen activation (uPA) system plays an important role in tissue remodeling, angiogenesis, proteolysis, migration, chemotaxis, invasion, and metastasis [15,16,17]. The uPA system consists of uPA, uPAR, plasminogen, and plasminogen activator inhibitor [18]. In vitro studies have shown that suPAR is associated with cell adhesion, migration, and proliferation [19,20]. Elevated suPAR levels have been determined in solid cancers such as ovarian [21], endometrial, cervical [22], breast [23], stomach [24], colon [25], and non-small cell lung cancer [26]. Positive associations between serum suPAR levels and soluble serum CD138, creatinine, Î˛2 microglobulin, stage of disease, and extramedullary bone marrow involvement have been reported in patients with multiple myeloma [27].

Hb (g/dL)

9.9 (5-17.5)

Platelets (x109/L)

64 (9-198)

FLT-3 mutation, n (%)

6 (20)

In acute leukemia, circulating blast cells provide an important advantage for studying proteins expressed on the tumor 138

1 (3.3)

Laboratory findings at baseline, median (minimum-maximum)

Treatment regimen, n (%) Cytarabine plus idarubicin (10 CR, 11 without CR)

21 (70)

ATRA plus idarubicin (3 CR)

3 (10)

5-azacitidine (1 CR, 4 without CR)

5 (16.7)

Low-dose cytarabine (1 without CR)

1 (3.3)

Remission-induction chemotherapy response, n (%) CR

14 (46.7)

No CR

16 (53.3)

AML: Acute myeloid leukemia, MDS: myelodysplastic syndrome, WBC: white blood cell, Hb: hemoglobin, FLT-3: Fms-like tyrosine receptor kinase, ATRA: all-trans-retinoic acid, CR: complete response.

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cell surface. Lanza et al. demonstrated that uPAR (CD87) expression increased in patients with AML and was associated with mucocutaneous infiltration, hepatosplenomegaly, lymphadenopathy, and central nervous system involvement [28]. The levels of suPAR in the plasma of mice during the growth of xenografted cell lines were significantly related to tumor volume [29]. Mustjoki et al. reported that increased suPAR levels were correlated with number of circulating tumor cells in AML and that serum suPAR levels decreased rapidly after cytotoxic treatment [30]. Aref et al. further demonstrated that serum suPAR levels were significantly higher in AML patients as compared to controls [31]. Similarly, in our study, serum suPAR levels significantly increased in patients with AML compared to healthy individuals. In addition, suPAR was observed to possess high sensitivity and speciﬁcity in patients with AML in ROC analysis. There was a positive correlation between suPAR levels and number of circulating WBCs. Therefore, we think that the production of suPAR is related to blast cells in the peripheral circulation. Lomholt et al. demonstrated that elevated suPAR levels were independent prognostic factors in patients with colorectal cancer [32]. Another study showed that high suPAR levels were associated with poor outcome in patients with breast cancer independent of tumor size, estrogen receptor status, and lymph node status [23]. On the other hand, Begum et al. reported that preoperative plasma suPAR levels were not correlated with prognosis for stage III ovarian cancer patients [33]. In our study, serum suPAR levels were significantly higher in patients who did not achieve CR than in patients achieving CR. More importantly, high suPAR levels were associated with poor prognosis in patients with AML. When the suPAR cut-off level was considered as 6.71 ng/mL, mortality risk was 2.5-fold higher in patients with levels above the cut-off limit. Sex, patient age, WBC count, FAB classification (i.e. M2, M3, M4, M5) and FLT-3 mutation were not associated with serum suPAR levels (p>0.05). Serum suPAR levels were an independent prognostic indicator for the OS of patients with AML.

Conclusion In conclusion, our study indicates that suPAR increases in patients with AML and this situation is associated with poorer survival. suPAR can thus be used as a diagnostic and prognostic biomarker in AML and may help in the developing of specific therapeutic targets. However, further studies are required to assess the clinical relevance of suPAR.

Authorship Contributions Concept: Nergiz Erkut, Mehmet Sönmez; Design: Nergiz Erkut, Mehmet Sönmez; Data Collection or Processing: Nergiz Erkut, Ahmet Menteşe, Hasan Mücahit Özbaş, Nilay Ermantaş, Ayşegül Sümer, Asım Örem, Mehmet Sönmez; Analysis or Interpretation: Nergiz Erkut, Ahmet Menteşe; Literature Search: Nergiz Erkut, Ahmet Menteşe, Hasan Mücahit Özbaş, Nilay Ermantaş, Ayşegül Sümer, Asım Örem, Mehmet Sönmez; Writing: Nergiz Erkut, Ahmet Menteşe, Hasan Mücahit Özbaş, Nilay Ermantaş, Ayşegül Sümer, Asım Örem, Mehmet Sönmez. 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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Ethics

13. Liesveld JL, Lichtman MA. Acute myelogenous leukemia. In: Kaushansky K, Lichtman MA, Beutler E, Kipps TJ, Seligsohn U, Prchal JT (eds). Williams Hematology. 8th ed. New York, McGraw-Hill, 2010.

Ethics Committee Approval: The study was approved by the Local Ethics Committee of the Karadeniz Technical University Faculty of Medicine, and was conducted in accordance with the Declaration of Helsinki. Informed consent was taken from all patients and healthy subjects.

14. O’Donnell MR, Tallman MS, Abboud CN, Altman JK, Appelbaum FR, Arber DA, Attar E, Borate U, Coutre SE, Damon LE, Lancet J, Maness LJ, Marcucci G, Martin MG, Millenson MM, Moore JO, Ravandi F, Shami PJ, Smith BD, Stone RM, Strickland SA, Wang ES, Gregory KM, Naganuma M. National Comprehensive Cancer Network. Acute myeloid leukemia. Version 2.2013. J Natl Compr Netw 2013;11:1047-1055.

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15. Pöllanen J, Stephens RW, Vaheri A. Directed plasminogen activation at the surface of normal and malignant cells. Adv Cancer Res 1991;57:273-328. 16. Andreasen PA, Egelund R, Petersen HH. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci 2000;57:25-40. 17. Castellino FJ, Ploplis VA. Structure and function of the plasminogen/plasmin system. Thromb Haemost 2005;93:647-654. 18. de Bock CE, Wang Y. Clinical significance of urokinase-type plasminogen activator receptor (uPAR) expression in cancer. Med Res Rev 2004;24:13-39. 19. Wilhelm OG, Wilhelm S, Escott GM, Lutz V, Magdolen V, Schmitt M, Rifkin DB, Wilson EL, Graeff H, Brunner G. Cellular glycosylphosphatidylinositolspecific phospholipase D regulates urokinase receptor shedding and cell surface expression. J Cell Physiol 1999;180:225-235. 20. Mizukami IF, Todd RF 3rd. A soluble form of the urokinase plasminogen activator receptor (suPAR) can bind to hematopoietic cells. J Leulocyte Biol 1998;64:203-213. 21. Wahlberg K, Høyer-Hansen G, Casslen B. Soluble receptor for urokinase plasminogen activator in both full-length and a cleaved form is present in high concentration in cystic fluid from ovarian cancer. Cancer Res 1998;58:3294-3298. 22. Riisbro R, Stephens RW, Brünner N, Christensen IJ, Nielsen HJ, Heilmann L, von Tempelhoff GF. Soluble urokinase plasminogen activator receptor in preoperatively obtained plasma from patients with gynecological cancer or benign gynecological diseases. Gynecol Oncol 2001;82:523-531. 23. Riisbro R, Christensen IJ, Piironen T, Greenall M, Larsen B, Stephens RW, Han C, Høyer-Hansen G, Smith K, Brünner N, Harris AL. Prognostic significance of soluble urokinase plasminogen activator receptor in serum and cytosol of tumor tissue from patients with primary breast cancer. Clin Cancer Res 2002;8:1132-1141. 24. Fidan E, Mentese A, Ozdemir F, Deger O, Kavgaci H, Karahan SC, Aydin F. Diagnostic and prognostic significance of CA IX and suPAR in gastric cancer. Med Oncol 2013;30:540. 25. Stephens RW, Nielsen HJ, Christensen IJ, Thorlacius-Ussing O, Sørensen S, Danø K, Brünner N. Plasma urokinase receptor levels in patients with colorectal cancer: relationship to prognosis. J Natl Cancer Inst 1999;91:869-874.

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26. Pappot H, Høyer-Hansen G, Rønne E, Hansen HH, Brünner N, Danø K, Grøndahl-Hansen J. Elevated plasma levels of urokinase plasminogen activator receptor in non-small cell lung cancer patients. Eur J Cancer 1997;33:867-872. 27. Rigolin GM, Tieghi A, Ciccone M, Bragotti LZ, Cavazzini F, Della Porta M, Castagnari B, Carroccia R, Guerra G, Cuneo A, Castoldi G. Soluble urokinasetype plasminogen activator receptor (suPAR) as an independent factor predicting worse prognosis and extra-bone marrow involvement in multiple myeloma patients. Br J Haematol 2003;120:953-959. 28. Lanza F, Castoldi GL, Castagnari B, Todd RF 3rd, Moretti S, Spisani S, Latorraca A, Focarile E, Roberti MG, Traniello S. Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukemic cells. Br J Haematol 1998;103:110-123. 29. Holst-Hansen C, Hamers MJ, Johannessen BE, Brünner N, Stephens RW. Soluble urokinase receptor released from human carcinoma cells: a plasma parameter for xenograft tumour studies. Br J Cancer 1999;81:203-211. 30. Mustjoki S, Sidenius N, Sier CFM, Blasi F, Elonen E, Alitalo R, Vaheri A. Soluble urokinase receptor levels correlate with number of circulating tumor cells in acute myeloid leukemia and decrease rapidly during chemotherapy. Cancer Research 2000;60:7126-7132. 31. Aref S, El-Sherbiny M, Mabed M, Menessy A, El-Refaei M. Urokinase plasminogen activator receptor and soluble matrix metalloproteinase-9 in acute myeloid leukemia patients: a possible relation to disease invasion. Hematology 2003;8:385-391. 32. Lomholt AF, Christensen IJ, Høyer-Hansen G, Nielsen HJ. Prognostic value of intact and cleaved forms of the urokinase plasminogen activator receptor in a retrospective study of 518 colorectal cancer patients. Acta Oncol 2010;49:805-811. 33. Begum FD, Høgdall CK, Kjaer SK, Christensen L, Blaakaer J, Bock JE, Glud E, Høyer-Hansen G, Ring-Larsen H, Høgdall EV. The prognostic value of plasma soluble urokinase plasminogen activator receptor (suPAR) levels in stage III ovarian cancer patients. Anticancer Res 2004;24:1981-1986.

RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0193 Turk J Hematol 2016;33:141-147

Investigation of Rho-Kinase Expressions and Polymorphisms in Mantle Cell Lymphoma Patients Mantle Hücreli Lenfoma Hastalarında Rho-Kinaz Ekspresyonları ve Polimorfizmlerinin Araştırılması Didar Yanardağ Açık1, Mehmet Yılmaz1, İbrahim Sarı2, Serdar Öztuzcu3, Zeynel A. Sayıner4, Salih Subari4, Abdullah T. Demiryürek5 1Gaziantep University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Gaziantep, Turkey 2Gaziantep University Faculty of Medicine, Department of Pathology, Gaziantep, Turkey 3Gaziantep University Faculty of Medicine, Department of Medical Biology, Gaziantep, Turkey 4Gaziantep University Faculty of Medicine, Department of Internal Medicine, Gaziantep, Turkey 5Gaziantep University Faculty of Medicine, Department of Medical Pharmacology, Gaziantep, Turkey

Abstract

Öz

Objective: Mantle cell lymphoma (MCL) is a rare but aggressive form of B-cell non-Hodgkin lymphoma characterized by excessive expression of cyclin D1. Intracellular signaling enzyme Rho-kinase (ROCK) can contribute to cellular migration, proliferation, and differentiation, as well as tumor development and metastasis. However, ROCK gene and protein expressions or polymorphisms have never been investigated in MCL patients. The purpose of this study was to investigate the role of ROCK gene and protein expressions in MCL patients. We also examined ROCK2 gene polymorphisms in this study.

Amaç: Mantle hücreli lenfoma (MHL) siklin D1’in aşırı ekspresyonuyla karakterize B-hücreli Hodgkin dışı lenfomanın nadir fakat agresif bir şeklidir. İntraselüler sinyal enzimi olan Rho-kinaz (ROCK), hücre migrasyonu, proliferasyonu, farklılaşması yanında tümör gelişimi ve metastazına da katkıda bulunur. Fakat MHL hastalarında ROCK gen ve protein ekspresyonları veya polimorfizmleri araştırılmamıştır. Bu çalışmanın amacı, MHL hastalarında ROCK gen ve protein ekspresyonlarının rolünü araştırmaktı. Biz bu çalışmada ROCK2 gen polimorfizmleri de araştırdık.

Materials and Methods: A total of 60 patients with MCL and 60 healthy controls were included in this retrospective study. Hematoxylin and eosin-stained lymph node tissue slides in the entire archive were reevaluated and used for immunohistochemistry, gene expression, and polymerase chain reaction studies.

Gereç ve Yöntemler: Bu retrospektif çalışmaya 60 MHL hastası ve 60 sağlıklı kontrol dahil edildi. Bütün arşivde hematoksilin ve eosin boyalı lenf düğümü kesitleri yeniden incelendi ve immünohistokimya, gen ekspresyonu ve polimeraz zincir reaksiyonu çalışmaları için kullanıldı.

Results: In immunohistochemical studies, there were significant increases in ROCK1 (p=0.0009) and ROCK2 (p<0.0001) protein expressions in MCL patients when compared with the control group. Although a marked increase in ROCK1 gene expression (p=0.0215) was noted, no significant change was observed in ROCK2 gene expression in MCL patients. Seven ROCK2 polymorphisms were studied, but the results showed no significant differences between the groups.

Bulgular: İmmünohistokimyasal çalışmada, kontrol grubuyla karşılaştırıldığında MHL hastalarında ROCK1 (p=0,0009) ve ROCK2 protein ekspresyonlarında (p<0,0001) anlamlı artış vardı. MHL hastalarında ROCK1 gen ekspresyonunda (p=0,0215) anlamlı artış bulunmasına karşın ROCK2 gen ekspresyonunda anlamlı değişiklik gözlenmedi. Yedi ROCK2 polimorfizmi çalışıldı, fakat sonuçlar gruplar arasında anlamlı farklılıklar göstermedi.

Conclusion: This is the first study to show that ROCK1 gene and ROCK protein expressions may contribute to the development of MCL.

Sonuç: Bu çalışma, ROCK1 gen ve ROCK protein ekspresyonlarının MHL gelişimine katkısı olabileceğini gösteren ilk çalışmadır.

Keywords: Lymphoma, Expression, Polymorphism, Rho-kinase

Anahtar Sözcükler: Lenfoma, Ekspresyon, Polimorfizm, Rho-kinaz

Address for Correspondence/Yazışma Adresi: Didar YANARDAĞ AÇIK, M.D., Gaziantep University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Gaziantep, Turkey Phone : +90 532 157 76 56 E-mail : didaryanardag@gmail.com

Received/Geliş tarihi: May 11, 2015 Accepted/Kabul tarihi: August 24, 2015

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Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell-type nonHodgkin lymphoma characterized by cyclin D1 overexpression and occurs more commonly in advanced ages and in males [1,2]. MCL is a rare subtype accounting for about 6% of all non-Hodgkin lymphoma cases. During the development of MCL, via t(11;14) (q13;q32) translocation, the BCL1 gene (11q13) and immunoglobulin heavy chain gene (14q32) come together, and hence BCL1 is upregulated. As a consequence of this translocation, excessive synthesis of cyclin D1 protein promotes the cell cycle progression (to S-/G2-phase) via cyclindependent kinase 4 and 6 activation in an uncontrolled manner. A minority (about 5%) of cases are cyclin D1-negative, and these cases often exhibit high expression of cyclin D2 or D3 [3]. Phenotypically, MCL is positive for the B-cell markers CD5, CD20, and CD79a. In MCL cells, CD10 and CD23 are usually negative. There is also overexpression of SOX-11 in the nuclei in most cases [4]. Overexpression of SOX-11 has been described as a diagnostic marker for MCL, with the absence of SOX-11 a characteristic of indolent MCL [5]. No single genetic lesion that can give rise to MCL has been identified. Molecular studies including single nucleotide polymorphisms (SNPs) have revealed a large number of chromosomal alterations in MCL [6,7,8,9]. Several copy number aberrations have been found to be correlated with genomic complexity in MCL cases [10]. Most patients are diagnosed at an advanced stage, and extranodal sites are often involved [11]. Even though patients with MCL often respond to therapy, the responses are usually partial and most patients eventually relapse [12]. There is currently no proven curative therapy and no standard of care has been established for initial or subsequent lines of therapy. Therefore, ideal treatment regimens for MCL are still being investigated and studies indicate that intracellular signaling pathways may be important targets in the treatment of MCL. Rho-kinase (ROCK) signaling has been implicated in various cellular functions downstream of Rho GTPases. Rho GTPases are important regulators of cancer cell proliferation, survival, invasion, and metastasis. More recently, crucial functions of Rho GTPases in the regulation of tumor stroma, including endothelial cells, immune cells, and cancer-associated fibroblasts, as well as in the formation of microvesicles, have been reported [13]. ROCK is a serine-threonine protein kinase with multiple downstream effects. Two isoforms of ROCK protein, ROCK1 and ROCK2, have been characterized. The ROCK isoforms are encoded by separate genes on human chromosomes 18q11 (ROCK1) and 2p24 (ROCK2) [14,15]. ROCK is substantially involved in a wide range of fundamental cellular functions, such as proliferation, differentiation, adhesion, contraction, 142

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metabolism, and apoptosis. ROCK signaling enhances myosinmediated contractility and drives amoeboid migration, which is associated with certain types of carcinoma, lymphomas, and leukemia [15,16,17]. Increased expression of the ROCK proteins promotes tumor cell proliferation and contributes to the metastatic behavior of some cancers [15]. Several of the ROCK substrates are prominent players in the development of cancer and its associated phenotypes. For example, the tumor suppressor phosphatase and tensin homolog (PTEN), which is frequently inactivated in melanoma, as well as c-Jun N-terminal Kinase (JNK)-interacting protein-3, an inhibitor of JNK signaling that is upregulated in melanoma, are inhibited by ROCK phosphorylation [17]. It has been shown that the sustained activation of ROCK is sufficient to induce cell cycle progression and increase cyclin D1 expression in NIH 3T3 fibroblasts [18]. Furthermore, ROCK activation also increases the expression of cyclin D1 in vascular smooth muscle cells [19]. In this study, the contribution of both ROCK isoforms in MCL was investigated. We also explored the possible role of the ROCK gene and protein expressions in MCL and tested the hypothesis that genetic variations in the ROCK2 gene may increase the risk of MCL.

Materials and Methods Patients In the present study, tissue samples of 60 patients diagnosed with MCL between 2006 and 2012, and those of 60 healthy adults who underwent lymph node biopsy for any reason but were not diagnosed with any malignant disease and were reported to have only hyperplasia by the pathology department, were investigated retrospectively. The study was approved by the local ethics committee. Clinical and laboratory information at the date of first diagnosis was recorded and overall survival was calculated as time from diagnosis to death or to the date when the patient was seen for the last time. Patients were identified from the pathological records and all cases were confirmed by histological evaluation. All demographic and clinical characteristics as well as prognostic factors of the study cases were collected from files. The prognosis of patients was based on the Mantle Cell Lymphoma International Prognostic Index (MIPI), which is calculated on the basis of four independent prognostic factors (age, performance status, serum lactate dehydrogenase level, and leukocyte count). Immunohistochemistry Formalin-fixed, paraffin wax-embedded blocks from each case were selected for immunohistochemical studies using the antibodies against ROCK1 and ROCK2. Hematoxylin and eosin-stained lymph node tissue slides were used for immunohistochemistry. Control tissue sections were made from the lymph node biopsies of the healthy subjects. Sections of

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4 µm were cut from paraffin-embedded tissue blocks onto silane-coated slides. Sections were heated to 60 °C for 20 min prior to deparaffinization with xylene solution. Sections were then stained using the Bond Polymer Refine Detection Kit (Bond #DS9800) in an automated slide processing system (Bond-Max, Leica Microsystems, Buffalo Grove, IL, USA). ROCK1 (rabbit monoclonal, EP786Y, ab45171, Abcam, Cambridge, UK) and ROCK2 (rabbit polyclonal, ab71598, Abcam, Cambridge, UK) were used for ROCK1 and ROCK2 immunostaining, respectively. The percentage of cells staining was evaluated and intensity (–, +, ++, or +++) was scored from 0 to 3 [20]. DNA Isolation and Genotyping DNA isolation was done with the paraffin blocks using the QIAamp DNA FFPE Tissue Kit (Cat. No. 56404). Obtained DNA was measured with a UV spectrophotometer (Epoch Biotek, Winooski, VT, USA) and prepared for the study. Various SNPs in the gene region coding ROCK2 were investigated. Criteria for the choice of SNPs used were: 1) relatively high minor allele frequencies in Caucasians; 2) location within the exonic and intronic sites that could potentially impact ROCK expression and function; and 3) suitability for the Fluidigm dynamic array chip designing, i.e. with no high G/C levels. Reference numbers of SNPs for the ROCK2 gene were rs2290156 in intron 30, rs965665 in intron 3, rs10178332 in intron 3, rs2230774 (Thr431Asn) in exon 10, rs2230774 (Thr431Ser) in exon 10, rs6755196 in intron 1, and rs726843 in intron 13. Polymorphisms were analyzed in genomic DNA using the 96.96 Dynamic Array on the BioMark HD system (Fluidigm, South San Francisco, CA, USA). Digital PCR Analysis software (Fluidigm, South San Francisco, CA, USA) was used to process the data after the reaction [21].

Software Inc., San Diego, CA, USA). For comparisons of the differences between mean values of two groups, the unpaired Student t-test was used. The chi-square test for independence and Fisher exact tests were used for calculation of the significance of differences in genotype and allele frequencies. The Pearson test was used to identify the correlations. The Mann-Whitney U test was used to detect significant differences between immunohistochemical scores and compare the gene expression data between groups. All statistical tests and p-values were two-sided, and p<0.05 was considered statistically significant.

Results Demographic and clinical characteristics of MCL patients and controls are outlined in Table 1. There were no statistically significant differences between patients and control groups in terms of sex and age distribution. Immunohistochemical study of the lymph node tissues revealed that ROCK1 and ROCK2 staining was more marked in the patient group (Figure 1). A widespread stronger positivity for ROCK1 and ROCK2 staining was observed in the cytoplasm of the lymph node cells from MCL patients. The ROCK distribution displayed a similar pattern between control and MCL sections. There were marked increases in ROCK1 (1.72±1.08, p=0.0009) and ROCK2 (2.58±0.62, p<0.0001) staining scores in the lymph nodes of the patient group when compared to controls (1.07±0.66 for ROCK1 and 1.28±0.69 for ROCK2; Figure 2). Correlations between the prognostic factors and ROCK in MCL patients are shown in Table 2. It was found that there were significant negative correlations between number of drug therapies and ROCK1 and ROCK2 protein expressions. However, positive correlation was found between age and ROCK1 expression. We also noted a positive correlation

Gene Expression Ribonucleic acid (RNA) was extracted from formalin-fixed, paraffin wax-embedded blocks using the High Pure RNA Isolation Kit (Cat. No. 03 270 289 001, Roche Diagnostics, Mannheim, Germany) as described by the manufacturer. The obtained RNA was prepared for the study by being measured with UV spectrophotometry. cDNA synthesis was performed with the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) according to manufacturer’s protocol. Gene expression analysis was then done using a BioMark HD device (Fluidigm, South San Francisco, CA, USA) that utilizes a fluorescent PCR method. Data were analyzed using the 2-ΔCt method according to the following formula: ΔCt=CtROCK-CtGAPDH, where Ct=threshold cycle [22]. Statistical Analysis Data were expressed as mean ± standard deviation (SD) or percentage unless otherwise indicated. Statistical analysis was performed using GraphPad InStat version 3.05 (GraphPad

Figure 1. Histopathologic images of ROCK staining. Immunohistochemical staining for lymph node tissues with ROCK1 in control (a) and in mantle cell lymphoma patients (b), and ROCK2 staining in control (c) and in mantle cell lymphoma patients (d). Original magnification 200x. 143

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between ROCK1 and ROCK2 expressions in MCL patients (Table 2). No significant differences were found between MCL patients and the control group in terms of 7 ROCK2 gene polymorphisms (Table 3). There was a marked increase in ROCK1 gene expression in the patient group when compared to controls (p=0.0215). However, no significant change was observed in ROCK2 gene expression (p=0.9194; Figure 3).

Discussion This study provides the first evidence that ROCK1 and ROCK2 protein expressions and ROCK1 gene expression were increased in MCL patients. However, no marked change in ROCK2 gene expression was observed. There were also no significant associations between ROCK2 gene polymorphisms and MCL cases. 3.0 2.5 2.0 1,5 1.0 0.5 0.0

Figure 2. Comparison of the immunohistochemical scores for lymph node ROCK1 and ROCK2 staining in healthy controls (n=60, white bars) and in patients with mantle cell lymphoma (n=60, black bars). Values are given as mean ± SEM. *p=0.0009 and p<0.0001 values were obtained for ROCK1 and ROCK2, respectively.

Figure 3. Comparison of the lymph node ROCK1 and ROCK2 gene messenger ribonucleic acid expressions in healthy controls (white bars, n=41) and in patients with mantle cell lymphoma (black bars, n=44). Values are given as mean ± SEM. *p=0.0215 and p=0.9194 values were obtained for ROCK1 and ROCK2 gene, respectively. 144

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Information regarding underlying biology and pathogenesis constantly increases, forming the basis of molecularly targeted treatment approaches in MCL [23]. Increased protein expressions of two ROCK isoforms have been found to be associated with different types of cancer [15,24]. In the present study, elevation Table 1. Demographic and clinical characteristics of the study cases. Cases with MCL (n=60) 61.9±10.9

Age (years)a Sex (n, %) Male 42 (70.0) Female 18 (30.0) Stageb (n, %) I-II 4 (6.6) III-IV 56 (93.4) Chemotherapy protocol (n, %) 26 (68.4) R-CHOP 6 (15.7) R-HIPERCVAD R-CVP 3 (7.89) R-CEOP 2 (5.26) R-FCM 1 (2.63) Average number of drug therapy (n) R-CHOP 5.5 R-HIPERCVAD 3.2 R-CVP 5.3 R-CEOP 3.5 R-FCM 1 Response to treatment (n) CR 15 PR 7 REFR 12 RLPS 20 Average survival (months) Female 22.8 Male 21.7 Tissue sample (n, %) Lymphadenopathy 30 (50.0) 13 (21.6) Bone marrow 6 (10.0) Stomach 4 (6.6) Tonsil Nasopharynx 3 (5.0) 2 (3.3) Rectum 1 (1.6) Oral mucosa 1 (1.6) Orbital mass

Controls (n=60)

p-value

58.6±10.4

0.0910

37 (61.7) 23 (38.3)

0.4413

aData are mean ± standard deviation. bStaging was carried out according to Ann Arbor staging system. CR: Complete remission, PR: partial response, REFR: refractory, RLPS: relapse, LAP: lymphadenopathy, R-CHOP: rituximab, cyclophosphamide, hydroxydaunorubicin (doxorubicin/adriamycin), oncovin (vincristine), and prednisone, R-CVP: rituximab, cyclophosphamide, vincristine, and prednisone, R-CEOP: R-CHOP with etoposide substituted for doxorubicin, R-HIPERCVAD: rituximab, cyclophosphamide, vincristine, adriamycin (doxorubicin), dexamethasone, alternating with methotrexate and cytarabine, R-FCM: rituximab, fludarabine, cyclophosphamide, methotrexate, MCL: mantle cell lymphoma.

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Table 2. Significant correlations between the prognostic factors and Rho-kinase protein expressions in mantle cell lymphoma patients. Prognostic factors

Correlation coefficient (r)

Coefficient of determination (r2)

p-value

Age ↔ ROCK1

0.260

0.067

0.044

Number of drug therapy ↔ ROCK1

-0.394

0.155

0.026

Number of drug therapy ↔ ROCK2

-0.456

0.207

0.009

ROCK1 ↔ ROCK2

0.559

0.312

<0.0001

ROCK: Rho-kinase.

Table 3. Genotype and allele distributions of ROCK2 gene polymorphisms in patients and control groups. Gene SNP

Genotype/allele

Control

MCL patients

ROCK2 rs2290156

GG/GC/CC G/C

29/25/2 83/29

28/22/4 78/30

0.9698+, 0.6719‡ 0.8703

ROCK2 rs965665

CC/CG/GG C/G

41/5/5 87/15

36/9/1 81/11

0.2586+, 0.2185‡ 0.6746

ROCK2 rs10178332

AA/AC/CCA/C

45/8/3 98/14

44/9/0 97/9

1.0000+, 0.2432‡ 0.3829

ROCK2 rs2230774 (Thr431Asn)

AA/AC/CC A/C

15/30/10 60/50

14/28/12 56/52

1.0000+, 0.7793‡ 0.7927

ROCK2 rs2230774 (Thr431Ser)

GG/GC/CC G/C

40/16/0 96/16

39/15/0 93/15

1.0000+ 1.0000

ROCK2 rs6755196

GG/GA/AA G/A

34/17/3 86/23

36/16/0 88/16

0.9461+, 0.2397‡ 0.2940

ROCK2 rs726843

TT/TC/CC T/C

16/28/12 60/52

13/26/15 52/56

0.8206+, 0.5927‡ 0.5032

*Numbers do not always add up to total numbers because of missing values in the BioMark dynamic array system. +Comparison between heterozygous genotype and homozygous wild-type genotype. ‡Comparison between homozygous variant genotype and homozygous wild-type genotype. ROCK: Rho-kinase, MCL: mantle cell lymphoma.

of the ROCK1 protein expression in MCL patients may be due to increase in the ROCK1 gene expression. However, we found an increase in ROCK2 protein, but not gene, expression in MCL patients, suggesting that other mechanisms are involved in the ROCK2 protein expression. The underlying mechanism of this observation is currently unknown, and it may require further evaluation with other techniques. Lane et al. [25] investigated the expressions of ROCK1 and ROCK2 in human breast cancer and showed that expression of ROCK1, at both messenger RNA (mRNA) and protein levels, is much higher in human breast tumor tissue compared with normal tissue. Conversely, ROCK2 levels do not seem to vary significantly between normal and tumor tissue, although a significant decrease was seen in ROCK2 mRNA levels in patients who died from breast cancer [25]. ROCK1 is also highly expressed in tumor tissues from osteosarcoma patients [26]. High expression of ROCK2 protein has been found to be associated with more aggressive behavior in hepatocellular carcinomas [27]. Elevated ROCK2 protein expression levels have also been reported in colon and bladder cancers and are associated with shorter disease-free survival in patients with bladder cancer [28,29]. Collectively, these data may indicate that ROCK is a potential therapeutic target in MCL.

It is known that reactive oxygen species (ROS) can directly act on the Rho/ROCK signaling pathway [30]. The RhoA/ ROCK pathway may also modulate ROS generation. ROCK is documented to stimulate expression of NADPH oxidase and consequent generation of ROS [31]. Continued oxidative stress can lead to chronic inflammation, which in turn could mediate cancer [32]. It has been shown that application of the specific ROCK inhibitors produces suppression of tumor formation, growth, and metastasis [33,34,35], while specific activation of ROCK signaling has been shown to lead to increased tumor cell dissemination and angiogenesis [36]. It was also reported that ROCK inhibitors inhibited the growth of cancer cells and their invasion, and increased their sensitivity to chemotherapeutics [34,37,38]. Taken together, these findings imply that ROCK inhibitors may be beneficial in targeted cancer treatment. We have observed a marked positive correlation of ROCK1 protein expression with age of the patients. However, no correlation was found between ROCK1 and ROCK2 protein expressions between overall and disease-free survival. These data may imply that ROCK has no marked effect on survival in these patients. In addition, there were significant negative correlations between 145

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ROCK1 and ROCK2 expressions and number of drug therapies in the present study. The underlying reason for this negative correlation is not known, but these findings may suggest that short duration of intensive chemotherapy may lead to increased ROCK1 and ROCK2 expressions.

References

There are only limited numbers of published studies related to ROCK polymorphisms in humans. A recent study demonstrated that ROCK2 gene polymorphisms are significantly associated with colorectal cancer [39] or metastases of breast cancer [40]. However, we have found no support for a role of the studied variants in the ROCK2 gene in risk of MCL in the present study. This may be due to the differences in pathogenesis between different types of cancer as well as the small number of cases in the present study.

3. Wlodarska I, Dierickx D, Vanhentenrijk V, Van Roosbroeck K, Pospísilová H, Minnei F, Verhoef G, Thomas J, Vandenberghe P, De Wolf-Peeters C. Translocations targeting CCND2, CCND3, and MYCN do occur in t(11;14)negative mantle cell lymphomas. Blood 2008;111:5683-5690.

Conclusion In summary, our data strongly suggest that ROCK expressions may contribute to the development of MCL. This study provides novel insights into mechanisms of lymphomagenesis. Our findings may provide an important insight into the future development or use of potential therapeutic approaches, such as ROCK inhibitors, for patients with MCL. The results of the present study may also imply that upregulation of ROCK may represent a prognostic factor in MCL, and ROCK may be a potential target for MCL diagnosis and therapy. Further studies are also required to verify these findings in a larger cohort. Ethics Ethics Committee Approval: The study was approved by the local ethics committee, Informed Consent: It was taken. Authorship Contributions Surgical and Medical Practices: Didar Yanardağ Açık, Mehmet Yılmaz, Zeynel A. Sayıner, Salih Subari; Concept: Didar Yanardağ Açık, Mehmet Yılmaz; Design: Mehmet Yılmaz, Abdullah T. Demiryürek; Data Collection or Processing: Didar Yanardağ Açık, Mehmet Yılmaz, İbrahim Sarı, Serdar Öztuzcu, Zeynel A. Sayıner, Salih Subari, Abdullah T. Demiryürek; Analysis or Interpretation: Didar Yanardağ Açık, Mehmet Yılmaz; İbrahim Sarı, Serdar Öztuzcu, Abdullah T. Demiryürek; Literature Search: Didar Yanardağ Açık, Abdullah T. Demiryürek; Writing: Didar Yanardağ Açık, Mehmet Yılmaz, Abdullah T. Demiryürek. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. Financial Disclosure: This study was funded by a project (TF.12.38) from Gaziantep University, Turkey. 146

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18. Croft DR, Olson MF. The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms. Mol Cell Biol 2006;26:46124627.

30. Jin L, Ying Z, Webb RC. Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta. Am J Physiol Heart Circ Physiol 2004;287:H1495-H1500.

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31. Higashi M, Shimokawa H, Hattori T, Hiroki J, Mukai Y, Morikawa K, Ichiki T, Takahashi S, Takeshita A. Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system. Circ Res 2003;93:767-775.

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21. Oguz E, Alasehirli B, Pehlivan Y, Onat AM, Oztuzcu S, Ozkara E, Kisacik B, Camci C, Demiryürek AT. Association between Rho-kinase (ROCK2) gene polymorphisms and Behçet’s disease. Transl Res 2012;160:428-434.

33. Imamura F, Mukai M, Ayaki M, Akedo H. Y-27632, an inhibitor of rhoassociated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. Jpn J Cancer Res 2000;91:811816.

22. Erkutlu I, Cigiloglu A, Kalender ME, Alptekin M, Demiryurek AT, Suner A, Ozkaya E, Ulasli M, Camci C. Correlation between Rho-kinase pathway gene expressions and development and progression of glioblastoma multiforme. Tumour Biol 2013;34:1139-1144.

34. Nakajima M, Katayama K, Tamechika I, Hayashi K, Amano Y, Uehata M, Goto N, Kondo T. WF-536 inhibits metastatic invasion by enhancing the host cell barrier and inhibiting tumour cell motility. Clin Exp Pharmacol Physiol 2003;30:457-463.

23. Noel MS, Friedberg JW, Barr PM. Novel agents in mantle cell lymphoma. Best Pract Res Clin Haematol 2012;25:191-200.

35. Isler D, Ozaslan M, Karagoz ID, Kilic IH, Karakok M, Taysi S, Guler I, Cakmak A, Demiryurek AT. Antitumoral effect of a selective Rho-kinase inhibitor Y-27632 against Ehrlich ascites carcinoma in mice. Pharmacol Rep 2014;66:114-120.

24. Hahmann C, Schroeter T. Rho-kinase inhibitors as therapeutics: from pan inhibition to isoform selectivity. Cell Mol Life Sci 2010;67:171-177. 25. Lane J, Martin TA, Watkins G, Mansel RE, Jiang WG. The expression and prognostic value of ROCK I and ROCK II and their role in human breast cancer. Int J Oncol 2008;33:585-593.

36. Croft DR, Sahai E, Mavria G, Li S, Tsai J, Lee WM, Marshall CJ, Olson MF. Conditional ROCK activation in vivo induces tumor cell dissemination and angiogenesis. Cancer Res 2004;64:8994-9001.

26. Liu X, Choy E, Hornicek FJ, Yang S, Yang C, Harmon D, Mankin H, Duan Z. ROCK1 as a potential therapeutic target in osteosarcoma. J Orthop Res 2011;29:1259-1266.

37. Relja B, Meder F, Wang M, Blaheta R, Henrich D, Marzi I, Lehnert M. Simvastatin modulates the adhesion and growth of hepatocellular carcinoma cells via decrease of integrin expression and ROCK. Int J Oncol 2011;38:879-885.

27. Wong CC, Wong CM, Tung EK, Man K, Ng IO. Rho-kinase 2 is frequently overexpressed in hepatocellular carcinoma and involved in tumor invasion. Hepatology 2009;49:1583-1594.

38. Yang X, Di J, Zhang Y, Zhang S, Lu J, Liu J, Shi W. The Rho-kinase inhibitor inhibits proliferation and metastasis of small cell lung cancer. Biomed Pharmacother 2012;66:221-227.

28. Kamai T, Tsujii T, Arai K, Takagi K, Asami H, Ito Y, Oshima H. Significant association of Rho/ROCK pathway with invasion and metastasis of bladder cancer. Clin Cancer Res 2003;9:2632-2641.

39. Sari I, Berberoglu B, Ozkara E, Oztuzcu S, Camci C, Demiryurek AT. Role of Rho-kinase gene polymorphisms and protein expressions in colorectal cancer development. Pathobiology 2013;80:138-145.

29. Vishnubhotla R, Sun S, Huq J, Bulic M, Ramesh A, Guzman G, Cho M, Glover SC. ROCK-II mediates colon cancer invasion via regulation of MMP-2 and MMP-13 at the site of invadopodia as revealed by multiphoton imaging. Lab Invest 2007;87:1149-1158.

40. Kalender ME, Demiryürek S, Oztuzcu S, Kizilyer A, Demiryürek AT, Sevinc A, Dikilitas M, Yildiz R, Camci C. Association between the Thr431Asn polymorphism of the ROCK2 gene and risk of developing metastases of breast cancer. Oncol Res 2010;18:583-591.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2015.0094 Turk J Hematol 2016;33:148-152

Prospective Audit of Blood Donor Selection Process in a Tertiary Care Hospital of a Developing Country Gelişmekte Olan Bir Ülkede Üçüncü Basamak Bir Hastanede Kan Bağışçı Seçim Sürecinin İleriye Dönük Denetlenmesi Naila Raza Liaquat National Hospital & Medical College, Department of Hematology, Karachi, Pakistan

Abstract

Öz

Objective: The aim of this study was to emphasize the significance of internal audits of the blood donor selection process and documentation in a resource-limited country by assessing compliance with the established protocols, and to identify weak areas in the process.

Amaç: Bu çalışmanın amacı kaynakları kısıtlı bir ülkede kan donörü seçimi sürecinin hastane içinde denetlenmesinin ve belgelenmesinin önemini vurgulamak ve sürecin zayıf noktalarının tespitini yapmaktır.

Materials and Methods: This audit reviewed the donor selection process at the blood bank of Liaquat National Hospital & Medical College, Karachi, over a 6-month period. Seven variables selected as performance indicators were graded as very good (%90-100%), good (80%-89%), satisfactory (70%-79%), or unacceptable (<70%). Blood bank staff was asked for feedback and suggestions.

Gereç ve Yöntemler: Bu denetleme ile 6 ay boyunca Liaquat National Hospital & Medical College, Karachi Kan Bankası’nda donör seçim süreci gözden geçirdi. Verimliliğini belirlemek için kullanılan 6 değişken şu şekilde derecelendirildi, çok iyi (%90-100), iyi (%80-89), yeterli (%70-79) ve kabul edilemez (<%70). Kan bankası çalışanlarına geri bildirimleri ve önerileri soruldu.

Results: Documentation of donor demographics was not within the acceptable range (documentation rates of 65.14%), donor status records were satisfactory (77.64%), and donor physical exam records were graded as good (86.34%). Five performance indicators were graded as very good (90%-100%).

Bulgular: Donör bilgilerinin belgelenmesi kabul edilebilir düzeyde değildi (%65,14), donörlerin durumunun kayıtları yeterliydi (%77,64), donörlerin fizik muayene kayıtları iyi olarak derecelendirildi (%86,34). Beş verimlilik belirteci çok iyi olarak derecelendirildi (%90-100).

Conclusion: The audit proved productive in identifying major causes of irregularities in documentation and in making valuable suggestions for their rectification.

Sonuç: Denetleme belgelendirme konusundaki düzensizliğin ana sebeplerini belirlemede ve bunların düzeltilmesinde değerli önerilerde bulunma konusunda verimli oldu.

Keywords: Medical audit, Transfusion medicine, Donor selection

Anahtar Sözcükler: Tıbbi denetleme, Transfüzyon tıbbı, Donör seçimi

Introduction Documentation and record-keeping play integral roles in transfusion medicine from every step of the vein-to-vein chain of blood donation to the dispatch of blood components. Regular medical audits are a part of quality assurance programs in transfusion medicine and a means of continuous assessment and improvement of existing systems. For conducting audits of clinical laboratories, a written set of questions in the form of a checklist is used, evaluation of which indicates whether the laboratory is performing its procedures according to its documented policies and standard operating procedures and on time. Historically, audits done in blood banks were focused

on clinical uses of blood components to ensure appropriate use, minimize wastage, and reduce the risk of transfusiontransmissible diseases. Developing countries like Pakistan depend heavily on non-remunerated blood donors as only 10% of blood donations are collected from voluntary donors [1]. Donor deferrals based on pre-donation assessment and workup acts as a deterrent for future donations, especially among first-time donors [2]. The World Health Organization (WHO) calls for a quality system to be put in place for blood donor selection criteria, staff training, and documentation [3]. A donor questionnaire is the key tool in donor selection for assessing donor health and safety and in reducing the risk of transmission

Address for Correspondence/Yazışma Adresi: Naila RAZA, M.D., Liaquat National Hospital & Medical College, Department of Hematology, Karachi, Pakistan Phone : 9221-34557897 E-mail : drnailarahman@yahoo.com

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Received/Geliş tarihi: February 24, 2015 Accepted/Kabul tarihi: July 09, 2015

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Turk J Hematol 2016;33:148-152

of infections. Timely counseling with regular reminders can help in re-recruiting short-term temporarily deferred donors back into the donor pool. Prior to these efforts, we have to ensure that donor screening records are properly maintained. There are no published data on internal audits done on donor screening processes in Pakistan. The objective of our study is to assess compliance with the established protocols for blood donor selection processes and documentation, to identify weak areas in these processes, and to recommend improvements in the system based on feedback obtained from blood bank staff.

Materials and Methods As a part of quality system improvement we planned a prospective 6-month internal audit of the donor recruitment process and documentation at the blood bank of a tertiary care hospital in Karachi, Pakistan, from January to June 2014. An audit plan was devised and checklists were prepared with the help of a toolkit developed by the Directorate General of Health Services, Dhaka WHO, July 2008 [4]. The audit involved the review of premises and the donor selection process as per checklists and scrutiny of donor records for documentation. The audit plan and checklists are shown in Figure 1 and Table 1. Donor records were grouped into group A (donors deferred before donation), group B (donors rejected after donation: seropositive cases), and group C (donors selected for donation: seronegative cases). We selected documentation of 7 parameters as performance indicators: donor demographics, donor status, general physical exam, hemoglobin estimation, informed consent, reason of deferral, and notification of seropositive cases. For each group, performance was graded as very good, good, satisfactory, or unacceptable by maintaining a high level of scoring documentation rates of 90%-100%, 80%-89%, 70%79%, and <70%, respectively. Based on the results, feedback was obtained from the blood bank staff responsible for conducting interviews of donors to determine common causes of nonconformance. A list of recommendations for appropriate changes in the current system was designed and submitted to the head of the blood transfusion services of the institute at the end of this exercise.

the checklist were met. Equipment and materials were being properly maintained as the department is ISO-9001:2000 certified. Exceptions were absence of privacy for asking questions related to sexual behavior and lack of written material for donor self-deferral. Staff members were qualified and trained. Review of donor records from January 2014 to June 2014 showed that out of 10,041 prospective blood donors, 1027 donors belonged to group A, 496 to group B, and 8518 to group C. Donor demographic records were inadequately maintained (documentation rate: 65.14%) as donor identification card numbers and area of residency were not always documented in all 3 groups. This was followed by donor status (documentation rate: 77.64%) and vital statistics (documentation rate: 86.34%) in that order. See Supplement 1 for review of Total Donor Screening Forms and Comparison of Documentation rate among 3 groups of Donor Selection Forms. Among deferred donors, the reason of deferral was mentioned in all cases but the donor notification rate was 89.51%. The main reason cited for not documenting the identification card number was not asking for it due to its nonavailability at the time of donation, and the area of residency was missed due to ignorance about the exact zonal divisions of the city. Irregularities in documentation of donorâ&#x20AC;&#x2122;s vital statistics were mainly due to bypassing the set standard operating procedures.

Discussion This audit gave insight into the existing practices of the donor selection process at our institute in particular and in developing countries in general. Random checks were conducted to evaluate the premises and processes using checklists and direct observations. Although the overall performance and documentations were good, some important issues were highlighted. Lack of privacy for conducting donor interviews was a concern identified in our study and mentioned by Kumar

The study was conducted after obtaining approval from the instituteâ&#x20AC;&#x2122;s ethics review committee. Data analysis was performed using descriptive statistics. Numerical data are shown as percentages.

Results In this study, inspection of the donor area was found to be satisfactory as almost all the prerequisites mentioned in

Figure 1. Audit plan. 149

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Turk J Hematol 2016;33:148-152

Table 1. Checklists for donor selection. 1.

Sections

Checklist for Donor Room

Available

Premises

Separate

√

Clean

√

Air conditioned

√

Airy

√

Well lit

√

Donor rest room 2.

Equipment/Material

Emergency Kit

Not Available

√

Weighing machine

√

Blood pressure and pulse monitor

√

Thermometer

√

Analyzer for hemoglobin estimation

√

Sterile alcohol swabs

√

Gloves

√

Band aid

√

Anticoagulant tubes

√

Disposible syringes

√

Waste bin

√

Blood bags

√

Blood bags sealer

√

Blood bags stripper

√

Blood bags shaker

√

Oxygen cylinder with regulator and mask

√

Inj. Adrenaline

√

Inj. Hydrocortisone

√

Inj. Pherimine maleate

√

Inj. Calcium gluconate

√

25% dextrose water

√

5% dextrose water 500 mL

√

Checklist for Donor Selection Process

Yes

Are written sops for donor selection process available?

√

Are donor selection criteria defined?

√

Is separate donor interview room available?

√

Is educational material for self-assessment available?

√

Is procedure explained to donor?

√

Are full aseptic measures taken?

√

Are instructions for postphlebotomy care and possible adverse reactions given?

√

Is refreshment provided?

√

et al. in a similar study from India [5]. Absence of proper infrastructure and space limitations are common problems faced by health centers in developing countries. Review of donor records showed some nonconformance in donor demographics including irregularities in donor identification numbers and area of residency in all 3 groups. The purpose of the former is donor traceability and that of the latter is use for epidemiological data. Use of mobile numbers for contacting donors has become the norm as it is much easier and 150

a quick method for donor notification that can safely replace identification numbers. However, its documentation rate needs to be 100%, especially in deferred donors with seropositive status (group B); in our case, this rate was 94.50%. Area of residency has also lost credibility as people lack awareness of exact zonal locations due to formation of new localities and the constant expansion of the city. Our study showed a shortcoming as per documentation rate for donor status (77%). Omission of data regarding donor status

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Turk J Hematol 2016;33:148-152

Supplement 1. Comparison of documentation rate among 3 groups of donor selection forms andÂ total donor screening forms reviewed. Audit of donor selection forms (January-June 2014). Total Donor Screening Forms Reviewed

10,041

Complete forms

8213 (81.79%)

*Incomplete forms

1828 (18.20%)

Donors deferred before donation (Group A)

1027

Complete forms

417 (40.60%)

*Incomplete forms

610 (59.39%)

Donors deferred after donation (seronegative) (Group B)

496

Complete forms

390 (78.62%)

*Incomplete forms

106 (21.37%)

Donors selected for donation (seropositive) (Group C)

8518

Complete forms

7411 (86.95%)

*Incomplete forms

1112 (13.04%)

*Computerized national identity card number and/or area of residency excluded

Comparison of documentation rate among 3 groups of donor selection forms. Performance Indicators

Group A (n=1027)

Group B (n=496)

Group C (n=8518)

Donor demographics

Incomplete Records n

Documentation Rate %

Incomplete Records n

Documentation Rate %

Cumulative %

ID card number

1027

496

00.00

8518

00.00

65.14

Documentation Rate % 00.00

Area of residency

1027

00.00

496

00.00

8518

00.00

Qualification

92.60

99.59

274

96.78

Ethnicity

95.52

99.39

148

98.26

Resident status

97.07

96.97

99.76

Contact number

98.05

98.58

100

239

76.72

106

78.62

1909

77.58

77.64

General physical examination

415

59.59

99.19

99.54

86.11

Hb estimation

100

99.77

99.92

Donor status Voluntary vs. replacement Vital statistics

Informed consent

100

Deferral reason

100

Donor notification

100

89.15

N/A

94.57

can be overcome by using different-colored forms for voluntary donors or by keeping separate registers. In this way, the focus can be directed towards voluntary donors with regular reminders for donations, thus facilitating the donor recruitment program. Documentation rate of vital signs collectively was good (86%). Documentation of the remaining 4 indicators was satisfactory. Feedback from blood bank staff was obtained to determine the most likely causes for omitted data. Failure to document identification number and area of residency was due to a silent

understanding among staff about their triviality; hence, this information was not being collected from donors. Donor status was not noted mostly due to inattention and the incongruous location of the question window in the proforma according to the staff. Documentation rate of vital statistics was selectively poor in group A (documentation rate: 59.59%). This was attributed to bypassing of normal standard operating procedures of conducting a physical exam first, followed by hemoglobin estimation, by some new staff members due to ignorance of 151

Raza N: Audit of Blood Donor Selection Process

the protocol. Notifying donors about the potential presence of transfusion-transmissible disease is a major responsibility of blood banks. In our study, the donor notification documentation rate was good (94.5%). Failure to inform donors were due to no response when called, wrong mobile numbers, and failure to document the mobile number, in that order. Thus, instead of identity card number, at least two contact phone numbers should be noted to ensure a 100% donor notification record. Conclusion and Recommendations The donor selection process is a vital link in the chain of blood collection, screening, and transfusion. A detailed audit of this program showed certain gaps in the documentation process. The following recommendations are made to minimize chances of lacunae and further improve the system: • Privacy must be provided for conducting donor interviews. • Donor identity card number and area of residency may be removed from the donor pro forma and replaced by two valid contact phone numbers. • Separate registers or color-coded forms can be used for voluntary blood donors. • Mini-audits of selected areas must be done apart from the annual external audits to improve quality. • Refresher courses for blood bank staff should be conducted regularly. • Introduction of electronic record-keeping in blood banks is vital for easy data retrieval.

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Acknowledgment The author would like to thank the staff of the blood bank of Liaquat National Hospital & Medical College for their assistance and cooperation in conducting the audit of the department. Ethics Ethics Committee Approval: Ethical Review Committee, Liaquat National Hospital, Institute for Postgraduate Medical Studies and Health Sciences, Karachi, Pakistan (App No. 0181-2014 LNH-ERC). Informed Consent: It was taken. Conflict of Interest: The author of this paper has no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. National AIDS Control Program, Ministry of Health. National Blood Policy & Strategic Framework 2008-2012 for Blood Transfusion Services in Pakistan. Islamabad, Pakistan, Government of Pakistan, 2007. Available online at http://www.nacp.gov.pk/introduction/ National_Blood_policy_&_ strategic_framework-BT.pdf. 2. Custer B, Chinn A, Hirschler NV, Busch MP, Murphy EL. The consequences of temporary deferral on future whole blood donation. Transfusion 2007;47:1514-1523. 3. World Health Organization. Blood Donor Selection Guidelines on Assessing Donor Suitability for Blood Donation. Geneva, Switzerland, WHO, 2012. Available online at http://apps.who.int/iris/ bitstream/10665/76724/1/9789241548519_eng.pdf?ua=1. 4. Musa SAJM, Hasan MA. Develop Toolkit for Monitoring and Quality Assurance of Safe Blood Transfusion. Dhaka, Bangladesh, WHO, 2008. 5. Kumar A, Sharma S, Ingole N, Gangane N. An audit of blood bank services. J Edu Health Promot 2014;3:11.

Brief REPORT DOI: 10.4274/tjh.2015.0335 Turk J Hematol 2016;33:153-155

Regulatory T Cells in Patients with Idiopathic Thrombocytopenic Purpura İdiyopatik Trombositopenik Purpura Olgularında Düzenleyici T Hücreler Alev Akyol Erikçi, Bülent Karagöz, Oğuz Bilgi Gülhane Military Medical Academy, Haydarpaşa Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey

Abstract

Öz

Objective: Immune thrombocytopenic purpura (ITP) is an immunemediated bleeding disorder in which platelets are opsonized by autoantibodies and destroyed by an Fc receptor-mediated phagocytosis by the reticuloendothelial system within the spleen. Autoimmune processes are also considered in the pathogenesis of this disorder. CD4+CD25+FoxP3+ regulatory T (Treg) cells and CD8+CD28Treg cells have roles in autoimmune diseases. We investigated these regulatory cells in ITP patients.

Amaç: İmmün trombositopenik purpura (İTP) trombositlerin otoantikorlar tarafından opsonize edildiği ve retiküloendotelyal sistem tarafından Fc reseptör aracılı fagositoz ile dalakta yıkıldığı immün kaynaklı bir kanama bozukluğudur. Bu bozukluğun patogenezinde otoimmün süreçler de sorumlu tutulmaktadır. CD4+CD25+Foxp3+ regulatuvar T (Treg) hücreleri ve CD8+CD28- Treg hücreler otoimmün hastalıklarda rol oynamaktadır. Çalışmamızda İTP’li hastalarda bu regülatuvar hücreleri araştırdık.

Materials and Methods: We included 22 ITP patients and 16 age-matched healthy subjects. CD4+CD25+FoxP3+ Treg cells and CD8+CD28- cells were investigated by three-color flow cytometry. The ratios of these cell populations to total lymphocytes were calculated. Statistical analysis was carried out with the Mann-Whitney U test.

Gereç ve Yöntemler: İTP’li 22 hasta ile yaş uyumlu 16 sağlıklı birey dahil edildi. CD4+CD25+Foxp3+ Treg hücreler ve CD8+CD28- hücreler üç renkli akım sitometri ile çalışıldı. Bu hücre popülasyonunun tüm lenfositlere oranı hesaplanmıştır. İstatiktiksel değerlendirmede MannWhitney U testi kullanılmıştır.

Results: CD4+CD25+ Treg cells were 9.69±3.70% and 12.99±5.58% in patients with ITP and controls, respectively. CD4+CD25highFoxP3+ cells were 27.72±19.74% and 27.55±23.98% in ITP patients and controls, respectively. The percentages of both of these cell types were not statistically significant when compared to the control group.

Bulgular: CD4+CD25+ Treg hücreler İTP’de ve kontrol grubunda %9,69±3,70 ve %12,99±5,58 saptandılar. CD4+CD25 yüksek FoxP3+ hücreler ise İTP’de ve kontrol grubunda %27,72±19,74 ve %27,55±23,9 olarak saptandı. Her iki hücre tipi de kontrol grubu ile karşılaştırıldığında istatiktiksel olarak anlamlı bulunmamıştır.

Conclusion: We did not find any differences in ratios of CD4+CD25+FoxP3+ Treg cells or CD8+CD28- T cells in lymphocytes between patients and healthy subjects. We conclude that these circulatory cells are not different in ITP, but further studies are needed to explore the putative roles of these regulatory cells.

Sonuç: Lenfositlerdeki CD4+CD25+Foxp3+ Treg hücreler ve CD8+CD28- T hücrelerdeki oranlarında fark bulamadık. Biz çalışmamızda İTP’de dolaşan regulatuvar hücrelerde fark bulamadık ama daha geniş kapsamlı çalışmalara ihtiyaç vardır.

Keywords: Idiopathic thrombocytopenic purpura, Regulatory T cells

Introduction Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in association with increased platelet destruction and impaired platelet production. It is mediated by IgG antiplatelet autoantibodies in which the targets are platelet membrane glycoproteins (GPs), such as GPIIb/IIIa and GPIb/IX. CD4+CD25+ regulatory T (Treg) cells and CD8+CD28- T lymphocytes have major roles in self-tolerance. To maintain the immune tolerance

Anahtar Sözcükler: İdiopatik trombositopenik purpura, Düzenleyici T hücreleri

and to prevent autoimmune disease, CD4+CD25+FoxP3+ Treg cells, CD4+ T cells with high expression of CD25, and transcription factor forkhead box P3 (FoxP3), also referred to as FoxP3 regulatory T cells, play an important role. Treg cells account for approximately 5% of circulating CD4+ T cells. Decreased numbers of Treg cells have been reported in patients with various autoimmune diseases, including ITP, rheumatoid arthritis, and systemic lupus erythematosus [1,2,3,4,5].

Address for Correspondence/Yazışma Adresi: Alev AKYOL ERİKÇİ, M.D., Gülhane Military Medical Academy, Haydarpaşa Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey Phone : +90 532 733 03 14 E-mail : aleverikci@yahoo.com

Received/Geliş tarihi: September 22, 2015 Accepted/Kabul tarihi: October 08, 2015

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Turk J Hematol 2016;33:153-155

In the case of Treg deficiency, peripheral tolerance can fail, leading to the development of autoimmunity. The purpose of this study was to evaluate Treg cells in previously untreated newly diagnosed ITP cases.

Anti-CD4/anti-FoxP3/anti-CD25 triple staining was uved for CD4+CD25+ Treg cell counts. CD4+CD25high lymphocytes were gated and then CD4+CD25highFoxP3+ cells were calculated in CD4/FoxP3 histograms.

Materials and Methods

Statistical Analysis

Flow Cytometry

Statistical analysis was performed using SPSS. The MannWhitney U test was used to investigate immunological parameters of ITP patients and for comparisons with data of healthy subjects.

Peripheral blood samples were obtained and studied while still fresh. Flow cytometry was used to count CD4+CD25+ Treg cells and CD8+CD28- suppressive cells. Flow cytometry was performed on a Becton Dickinson FACSCalibur. Data were obtained and analyzed using CellQuest software. Monoclonal Antibodies Antihuman monoclonal antibodies conjugated with fluorochromes and appropriate isotype controls were used: fluorescein isothiocyanate (FITC) conjugated anti-CD28 (BD Pharmingen Catalog No: 555728), anti-CD4 (Caltag Laboratories Catalog No: MHCD0401), phycoerythrin-cyanine 5 (PC5) conjugated anti-CD8 (eBioscience Catalog No: 15-0088), antiCD25 (BD Pharmingen Catalog No: 555433), and phycoerythrin (PE) conjugated anti-FoxP3 (eBioscience Catalog No: 12-4776). Cell Preparation and Surface Staining Human peripheral blood mononuclear cells were isolated using Histopaque (Sigma Catalog No: 1077) gradient centrifugation. Aliquots of 100 µL were transferred to polypropylene test tubes (12x75 mm; BD Bioscience Catalog No: 352052) and 20 µL of conjugated monoclonal antibodies or isotype controls was added to each tube. Flow cytometric analysis was performed by BD FACSCalibur after the appropriate staining protocol. FoxP3 Staining CD4 and CD25 surface staining was carried out. The CD4+CD25 tube was then washed with cold PBS and resuspended, 1 mL of freshly prepared fixation/permeabilization working solution was added, and the tube was incubated at 4 °C for 30-60 min in the dark and washed twice by adding 2 mL of 1X permeabilization buffer. Next, 20 µL of PE conjugated antihuman FoxP3 antibody in 1X permeabilization buffer was added and the tube was incubated at 4 °C for 30 min in the dark. Washing was repeated twice with 2 mL of 1X permeabilization buffer. After resuspension, analysis was performed by flow cytometry. Analysis CD8+CD28- cell percentages were evaluated using antiCD28/anti-CD8 double staining in lymphocyte-gated cells. CD8+CD28- cells, CD8+CD28+ cells, and the ratio of these cells were calculated. 154

Results We enrolled 22 previously untreated patients newly diagnosed with ITP (19 males, 3 females) and 16 age-matched controls (13 males, 3 females). All of the patients were admitted to our outpatient clinic. Thrombocytopenia was newly detected and they had received no previous treatment. The patients were investigated for possible causes of thrombocytopenia. Viral serology and other underlying autoimmune diseases were screened. Demographic findings are illustrated in Table 1. We performed bone marrow aspiration and biopsy in the relatively elderly patients (patients numbers 5, 9, and 17). No pathological findings such as dysplasia were detected. Findings were consistent Table 1. Patients’ characteristics Patient number

Age

Sex

Platelets (x109/L)

Male

Female

Male

Female

Male

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with ITP, including normal or increased megakaryocytes. CD4+CD25+ Treg cells and CD4+CD25highFoxP3+ cells were calculated in lymphocytes. CD4+CD25+ Treg cells were 9.69±3.70% and 12.99±5.58% in patients with ITP and controls, respectively. CD4+CD25highFoxP3+ cells were 27.72±19.74% and 27.55±23.98% in ITP patients and controls, respectively. Both of these cell counts were not statistically different between groups. We also detected no statistically significant difference in CD8+CD28- suppressor cells between ITP patients and controls (12.50±9.40% and 11.77±4.64%, respectively).

Discussion Treg cells suppress effector T cell activation, which leads to induction of immune tolerance [6]. For this reason it is assumed that failure of the regulatory T cell system may induce autoimmunity [7,8,9]. There are increasing numbers of studies demonstrating that decreased frequency of Treg cells has a role in ITP. Liu et al. reported that the percentage of Treg cells was significantly decreased in ITP patients with active disease in which no remission was achieved [10]. Sakakura et al. reported variations in Treg amounts according to platelet counts. In patients with low platelet counts there was no reduction in the percentage of Treg cells when compared to those with platelet counts over 100,000/µL [11]. In the study by Yu et al., defective circulating CD25 Treg cells were detected in patients with chronic ITP [12]. However, there are also studies that failed to detect any differences in Treg frequencies of patients with ITP compared to healthy controls [13,14]. Similar to our results, Mazzucco et al. detected no significant difference between Treg cell and platelet counts in patients with ITP and the control group [15]. In our study we investigated previously untreated newly diagnosed ITP patients. We detected no significant difference in Treg cell frequencies in ITP patients and controls. We think that further studies are needed to explore the putative roles of these regulatory cells, especially in terms of long-term follow-ups and response to treatments. Ethics Informed Consent: It was taken.

Erikçi; Analysis or Interpretation: Bülent Karagöz, Literature Search: Bülent Karagöz, Oğuz Bilgi, Alev Akyol Erikçi; Writing: Alev Akyol Erikçi, Bülent Karagöz. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. Financial Disclosure: Contribution of the Turkish Society of Hematology.

References 1. Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002;346:995-1008. 2. Semple JW, Freedman J. Increased antiplatelet T helper lymphocyte reactivity in patients with autoimmune thrombocytopenia. Blood 1991;78:2619-2625. 3. Kuwana M, Kaburaki J, Ikeda Y. Autoreactive T cells to platelet GPIIb-IIIa in immune thrombocytopenic purpura: role in production of anti-platelet autoantibody. J Clin Invest 1998;102:1393-1402. 4. Ogawara H, Handa H, Morita K, Hayakawa M, Kojima J, Amagai H, Tsumita Y, Kaneko Y, Tsukamoto N, Nojima Y, Murakami H. High Th1/Th2 ratio in patients with chronic idiopathic thrombocytopenic purpura. Eur J Haematol 2003;71:283288. 5. Semple JW, Milev Y, Cosgrave D, Mody M, Hornstein A, Blanchette V, Freedman J. Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity. Blood 1996;87:4245-4254. 6. Itoh M, Takahashi T, Sakaguchi N, Kuniyasu Y, Shimizu J, Otsuka F, Sakaguchi S. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol 1999;162:5317-5326. 7. Nugent DJ. Immune thrombocytopenic purpura of childhood. Hematology 2006;2006:97-103. 8. Cruvinel WM, Mesquita D Jr, Araujo JAP, Salmazi KC, Kallas EG, Andrade LEC. Natural regulatory T cells in rheumatic diseases. Rev Bras Reumatol 2008;48:342355. 9. Sakaguchi S, Ono M, Setoguchi R, Yagi H, Hori S, Fehervari Z, Shimizu J, Takahashi T, Nomura T. Foxp3+CD25+CD4+ natural regulatory T cells in dominant selftolerance and autoimmune disease. Immunol Rev 2006;212:8-27. 10. Liu B, Zhao H, Poon MC, Han Z, Gu D, Xu M, Jia H, Yang R, Han ZC. Abnormality of CD4+CD25+ regulatory T cells in idiopathic thrombocytopenic purpura. Eur J Haematol 2007;78:139-143. 11. Sakakura M, Wada H, Tawara I, Nobori T, Sugiyama T, Sagawa N, Shiku H. Reduced Cd4+Cd25+ T cells in patients with idiopathic thrombocytopenic purpura. Thromb Res 2007;120:187-193. 12. Yu J, Heck S, Patel V, Levan J, Yu Y, Bussel JB, Yazdanbakhsh K. Defective circulating CD25 regulatory T cells in patients with chronic immune thrombocytopenic purpura. Blood 2008;112:1325-1328. 13. Andersson PO, Stockelberg D, Jacobsson S, Wadenvik H. A transforming growth factor-β1-mediated bystander immune suppression could be associated with remission of chronic idiopathic thrombocytopenic purpura. Ann Hematol 2000;79:507-513.

Authorship Contributions

14. Nishimoto T, Kuwana M. CD4+CD25+Foxp3+ regulatory T cells in the pathophysiology of immune thrombocytopenia. Semin Hematol 2013;50(Suppl 1):S43-S49.

Concept: Alev Akyol Erikçi; Design: Alev Akyol Erikçi; Data Collection or Processing: Bülent Karagöz, Oğuz Bilgi, Alev Akyol

15. Mazzucco KL, Junior LM, Lemos NE, Wieck A, Pezzi A, Laureano AM, Amorin B, Valim V, Silla L, Daudt LE, Marostica PJ. Assessment of regulatory T cells in childhood immune thrombocytopenic purpura. ISRN Hematol 2013;2013:143687.

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Brief REPORT DOI: 10.4274/tjh.2015.0206 Turk J Hematol 2016;33:156-158

Serum Zinc Levels in Iron Deficient Women: A Case-Control Study Demir Eksikliği Anemisi Olan Kadınlarda Serum Çinko Seviyesinin Değerlendirilmesi: Olgu Kontrol Çalışması Onur Özhan1, Neslihan Erdem2, İsmet Aydoğdu3, Ali Erkurt4, İrfan Kuku4 1Çukurova Dr. Aşkım Tüfekçi State Hospital, Clinic of Endocrinology, Adana, Turkey 2Celal Bayar University Faculty of Medicine, Department of Internal Medicine, Manisa, Turkey 3Celal Bayar University Faculty of Medicine, Department of Hematology, Manisa, Turkey 4İnönü University Faculty of Medicine, Department of Hematology, Malatya, Turkey

Abstract

Öz

Since similar symptoms and findings can be seen in the deficiencies of both iron and zinc, we aimed to evaluate the serum zinc levels of women with iron deficiency anemia (IDA). This study was conducted with women with iron deficiency and a healthy control group. When serum zinc levels were compared, they were found to be lower in the IDA group, which was statistically significant. With the help of these studies, iron and zinc treatment instead of only iron replacement may be considered in cases of iron deficiency.

Demir ve çinko eksikliği benzer belirti ve bulgularla giden hastalıklar olması sebebiyle, demir eksikliği anemisi (DEA) olan kadınlarda serum çinko düzeylerinin değerlendirilmesini planladık. Bu çalışma DEA’lı kadınlar ile sağlıklı kontrol olarak alınan kadınlar üzerinde yapıldı. Serum çinko düzeyleri karşılaştırıldığında, DEA grubunda istatistiksel olarak düşük saptandı. Bu çalışmaların yardımıyla sadece demir değil, demir ve çinkonun beraber tedavisi demir eksikliği olan olgularda düşünülebilir.

Keywords: Iron, Zinc, Women, Iron deficiency, Anemia

Anahtar Sözcükler: Demir, Çinko, Kadın, Demir eksikliği, Anemi

Introduction Iron deficiency anemia (IDA) is the most common anemia around the world and a public health concern in developing countries that still suffer from malnutrition problems [1,2]. Similar findings and symptoms affecting various systems in the body may be found both in iron and zinc deficiency; moreover, coexistence of these deficiencies can exaggerate the symptoms. However, there are not enough studies about zinc levels in adult anemic subjects. Therefore, we aimed to evaluate the serum zinc levels of women with IDA and investigate whether serum zinc levels in women with iron deficiency were low or not.

Materials and Methods Thirty women between 18 and 60 years of age with iron deficiency who had presented to our outpatient clinics were enrolled as the patient group and a healthy group consisting of 30 women with the same age range served as the control group. The study was conducted in the İnönü University Faculty of Medicine, Department of Internal Medicine. Women with history of infection within 1 month, or with chronic diseases were excluded.

Diagnosis criteria for iron deficiency were hemoglobin below 12 g/dL and serum ferritin level below 20 µg/dL [3]. The normal values of serum zinc levels were between 70 and 120 µg/dL [4]. Iron and iron-binding capacity were measured by the calorimetric method with an Olympus OSR6186 (Germany), whereas serum ferritin levels were measured by nephelometric method with a 33 Dade Behring (Germany). Complete blood count analysis was performed with a Beckman Coulter LH 750 analyzer (USA). Serum zinc levels were measured by atomic absorption method with the PerkinElmer Analyst 800 (Germany). Statistics Results were given as ± standard deviation, and with 95% safety and distribution. Statistical analysis was conducted with SPSS and the independent sample t-test.

Results Thirty healthy women and 30 women with iron deficiency were included in this study. Ages of the women were between 18 and 60; mean age was 38.4±10.5 years in the IDA group and 39.8±12.5 years in the control group. There was no statistical difference between the groups (p>0.05).

Address for Correspondence/Yazışma Adresi: Neslihan ERDEM, M.D., Celal Bayar University Faculty of Medicine, Department of Internal Medicine, Manisa, Turkey Phone : +90 555 729 88 22 E-mail : neslihnerdem@gmail.com

156

Received/Geliş tarihi: May 17, 2015 Accepted/Kabul tarihi: November 18, 2015

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Özhan O, et al: Serum Zinc Levels in Iron Deficiency in Women

Mean hemoglobin level was 10.1±1.4 g/dL in the IDA group and 14.1±0.5 g/dL in the control group. There was a statistically significant difference between the groups (p<0.001). Hematologic parameters of each group are given in Table 1. Serum iron, ferritin, and transferrin saturation levels, which are the parameters helping in the diagnosis of IDA, were higher in the control group compared to the IDA group. There was a statistically significant difference between the groups (p<0.001). Iron levels and iron storage parameters are given in Table 2. When the control and IDA groups were compared, serum zinc levels were found to be decreased as serum iron levels decreased. Minimum serum zinc level was 34 µg/dL while the maximum was 84 µg/dL in the IDA group, and mean serum zinc level was 55.8±10.8. In the IDA group, serum zinc level was in the normal range (70-120 µg/dL) in only three of the patients. The remaining 27 patients’ serum zinc levels were below 70 µg/dL. On the other hand, in the control group, only one individual had a serum zinc level below 70 µg/dL, while the remaining subjects’ serum zinc levels were in the normal range. There was a statistically significant difference between the groups (p<0.0001). Serum zinc levels of the IDA and control groups are compared in Table 2.

Discussion Although trace elements are found in minimal quantities, they have important roles in homeostasis. Two of the most important trace elements are iron and zinc. IDA is still a serious problem in Turkey and around the world [1,5,6]. Considering many etiological factors like low socioeconomic status, malnutrition, high-fiber diet, pica disorder, parasitic infections, and milk

allergies, it is not a surprise to see zinc deficiency and iron deficiency at the same time [2,7,8]. Coexistence of iron and zinc deficiency has attracted the interest of researchers and there have been many studies about this subject. Furthermore, this situation had led to the following question: Are there any interactions between these two elements? One of the reasons for iron deficiency occurring with zinc deficiency, other than diet, is the increase in production of Zn-protoporphyrin and usage of zinc instead of iron in the protoporphyrin structure [9]. There are also other hypotheses that zinc deficiency can cause iron deficiency. It has been shown that, in animal studies with Table 2. Iron levels, zinc levels, and iron storage parameters of the groups. IDA group

Control group

p-value

Serum iron level (µg/dL) Distribution

21.4±11.3* 6-46**

99.7±14.9 72-134

p<0.001

Ferritin (µg/L) Distribution

8.01±1.6 2.1-8.4

96.9±19.02 60-141

p<0.001

Transferrin saturation (%) Distribution

5.6±3.04 1.6-12.2

32.5±4.2 25.5-43.2

p<0.001

Serum zinc level (µg/dL)

55.8±10.8

80.2±8.6

p<0.001

IDA: Iron deficiency anemia. *Mean values are given as ± standard deviation. **Minimum-maximum levels.

Table 1. Hematologic parameters of the iron deficiency anemia and control groups. IDA group

Control group

p-value

Hb (g/dL) Distribution

10.1±1.4* 6.5-12.0**

14.1±0.5 13.1-15.3

p<0.001

Hct (%) Distribution

31.1±3.8 21.3-36.7

42.1±1.6 39.6-45.0

p<0.001

WBCs (/mm3) Distribution

7019.6±2228.2 2200-14,400

6633.3±1305.5 4100-9000

p>0.05

Platelets (/mm3) Distribution

318,994.3±98,236.04 94,000-522,000

293,566.7±52,937.6 180,000-369,000

p>0.05

MCV (fL) Distribution

70.2±6.5 55.9-80

87.1±2.7 80.8-92.8

p<0.001

MCH (pg) Distribution

23.1±2.9 15.8-27.8

29.2±1.01 32.8-34.4

p<0.001

MCHC (g/dL) Distribution

32.3±1.4 28.3-36.0

33.4±0.4 32.8-34.4

p<0.001

IDA: Iron deficiency anemia, Hb: hemoglobin, WBC: white blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration. *Mean values are given as ± standard deviation. **Minimum-maximum levels.

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mice and rats with zinc deficiency, bone marrow erythrocyte progenitors and plasma erythropoietin levels are decreased [10,11,12]. Furthermore, there are also hypotheses that zinc deficiency can make the erythrocytes vulnerable to oxidative stress, which can cause anemia [13,14]. In another study, serum zinc levels were measured in children between 1 and 14 years of age with iron deficiency. The serum zinc levels were lower in the IDA group than the control group (p=0.017). There was a statistically significant difference between the two groups, as in our study. As a result, it has been suggested that serum zinc levels should be checked in children with iron deficiency [15]. Serum zinc levels were studied in children with iron deficiency in Ankara. When zinc deficiency was accepted as levels below 2 SDs of mean levels of the control group, among the 100 anemic patients 23 patients had zinc levels of less than 1 SD and 19 patients had zinc levels of less than 2 SDs of the mean levels. These results were compatible with our results. It was also suggested that zinc deficiency should be evaluated in patients with IDA, because of the similarity of the symptoms of these two deficiencies [7]. In Arcasoy’s study in 1985, histopathological changes causing iron and zinc deficiency in intestinal mucosa were reversed with zinc treatment and the absorption of zinc and iron were improved [2]. Because of the lack of studies regarding this subject in adult women and the high prevalence of IDA in this age group, we conducted a study with women between 18 and 60 years of age. We tried to find out whether zinc deficiency coexists with iron deficiency or not. Although the studies we have mentioned here mostly involved children, our study has shown similar results. A study conducted in Vietnam showed that underprivileged women were at increased risk of insufficient micronutrient intake due to poor diet quality [16]. The effects of supplementation in children have also been studied. It was found that supplementation with iron plus zinc improved serum zinc and plasma ferritin [17]. Similar findings and symptoms affecting various systems in the body may be found both in iron and zinc deficiencies; therefore, the levels of zinc must be carefully evaluated in cases of iron deficiency. More importantly, as iron deficiency is still an existing problem in Turkey, further studies investigating the interactions between these elements must be performed. We suggest that serum zinc levels should be evaluated in adult women with IDA, but further studies are needed to evaluate the benefit of simultaneous zinc and iron treatment instead of only iron treatment in this age group. Ethic This study is ethically approved by İnönü University’s Local Ethics Committee. 158

Turk J Hematol 2016;33:156-158

Authorship Contributions Surgical and Medical Practices: Onur Özhan, İsmet Aydoğdu; Ali Erkurt, İrfan Kuku; Concept: Onur Özhan, Neslihan Erdem, İsmet Aydoğdu; Design: Onur Özhan, Neslihan Erdem, İsmet Aydoğdu; Data Collection or Processing: Onur Özhan, İsmet Aydoğdu; Analysis or Interpretation: Onur Özhan, İsmet Aydoğdu; Literature Search: Onur Özhan, Neslihan Erdem, İsmet Aydoğdu; Writing: Onur Özhan, Neslihan Erdem, İsmet Aydoğdu. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Berçem İ, İçağasıoğlu D, Cevit Ö, Ergür AT, Berçem G, Gültekin A, Sütçü İ. The prevalence of iron deficiency and iron deficiency anemia in adolescents. Türkiye Klinikleri Pediatri Dergisi 1999;8:15-20. 2. Arcasoy A. İnsan sağlığında çinkonun önemi. TÜBİTAK Bilim ve Teknik Dergisi 1996;12:56 (in Turkish). 3. Türk Hematoloji Derneği. Ulusal Tedavi Kılavuzu 2011 Yetişkinde Demir Eksikliği. Ankara, Turkey, Türk Hematoloji Derneği, 2011 (in Turkish). 4. Tomita H. Trace Elements in Clinical Medicine: Proceedings of the Second Meeting of the International Society for Trace Element Research in Humans (ISTERH). 28 August–1 September 1989, Tokyo, Japan. 5. Gülez P, Kayserili E, Tosun A, Eryılmaz N. Demir eksikliği anemisinde eritrosit parametrelerinin karşılaştırılması. Klinik Bilimler ve Doktor 1998;4:875-877 (in Turkish). 6. Koç A, Erol Ö, Kösecik M, Vural H, Tatlı MM, Ataş A, Avcı Ş. Şanlıurfa ili 1216 yaş arası çocuklarda demir eksikliği araştırması. Klinik Bilimler ve Doktor 1997;3:871-873 (in Turkish). 7. Prasad AS. Zinc deficiency in women, infants and children. J Am Coll Nutr 1996;15:113-120. 8. Prasad AS. Biochemistry of Zinc. New York, NY, USA, Plenum, 1993. 9. Hastka J, Lassere JJ, Schwarzbeck, Hehlmann R. Central role of zinc protoporphyrin in staging iron deficiency. Clin Chem 1994;40:768-773. 10. King LE, Fraker PJ. Zinc deficiency in mice alters myelopoiesis and hematopoiesis. J Nutr 2002;132:3301-3307. 11. King LE, Frentzel JW, Mann JJ, Fraker PJ. Chronic zinc deficiency in mice disrupted T cell lymphopoiesis and erythropoiesis while B cell lymphopoiesis and myelopoiesis were maintained. J Am Coll Nutr 2005;24:494-502. 12. Konomi A, Yokoi K. Zinc deficiency decreases plasma erythropoietin concentration in rats. Biol Trace Elem Res 2005;107:289-292. 13. Powell SR. The antioxidant properties of zinc. J Nutr 2000;130: 1447-1454. 14. O’Dell BL. Role of zinc in plasma membrane function. J Nutr 2000;130:14321436. 15. Ece A, Uyanık BS, İşcan A, Ertan P, Yiğitoğlu MR. Increased serum copper and decreased serum zinc levels in children with iron deficiency anemia. Biol Trace Elem Res 1997;59:31-39. 16. Nguyen PH, Nguyen H, Gonzalez-Casanova I. Micronutrient intakes among women of reproductive age in Vietnam. PLoS One 2014;9:e89504. 17. Fallahi E, Kimiagar M, Nazari A, Hasanvand MA, Seifi M. Effect of zinc and iron supplementation on indicators of iron, zinc and vitamin A status of primary school children. Pak J Biol Sci 2007;10:1088-1092.

CASE REPORT DOI: 10.4274/tjh.2015.0238 Turk J Hematol 2016;33:159-162

Diffuse Large B-Cell Lymphoma Presenting with Bilateral Renal Masses and Hematuria: A Case Report Bilateral Renal Kitle ve Hematüri ile Prezente Olmuş Diffüz Büyük B Hücreli Lenfoma Olgusu Şiyar Erdoğmuş1, Serkan Aktürk1, Zeynep Kendi Çelebi1, Saba Kiremitçi2, Gülşah Kaygusuz2, Namık Kemal Altınbaş3, Evren Üstüner3, Kenan Keven1 1Ankara University Faculty of Medicine, Department of Nephrology, Ankara, Turkey 2Ankara University Faculty of Medicine, Department of Pathology, Ankara, Turkey 3Ankara University Faculty of Medicine, Department of Radiology, Ankara, Turkey

Abstract

Öz

Renal involvement is most often seen in conjunction with multisystemic, disseminated lymphoma either by direct extension from a retroperitoneal mass or via hematogenous spread. Primary lymphoma of the kidney is not a common entity and it is a controversial issue on account of the absence of lymphatic tissues in the normal kidney. In this case report, we describe a 19-year-old male with hematuria, acute kidney injury, and bilateral renal masses due to massive lymphomatous infiltration of the kidneys, which was diagnosed as diffuse large B-cell non-Hodgkin lymphoma by Tru-Cut biopsy.

Lenfomada renal tutulum sıklıkla multisistemik olarak, retroperitoneal kitlenin direkt komşuluğu yoluyla veya hematojen yayılım şeklinde ortaya çıkar. Böbrekte lenfatik doku yokluğu nedeniyle primer böbrek lenfoması nadir görülen ve tartışmalı bir durumdur. Bu olguda; hematüri, akut böbrek hasarı ve böbreklerin masif lenfomatöz infiltrasyonuna bağlı bilateral renal kitle ile prezente olmuş ve böbrek biyopsisi ile diffüz büyük B hücreli non-Hodgkin lenfoma tanısı almış 19 yaşında erkek hasta sunulmaktadır.

Keywords: Acute kidney injury, Hematuria, Lymphoma, Renal biopsy, Renal masses

Anahtar Sözcükler: Akut böbrek hasarı, Hematüri, Lenfoma, Renal biyopsi, Renal kitle

Introduction Primary renal lymphoma (PRL) is a very rare disease and a controversial issue because the kidneys do not normally contain lymphatic tissue [1,2,3,4,5]. In general, renal lymphoma is most often seen along with dissemination of systemic disease and clinically silent. Occasionally, patients present nonspecific signs and symptoms as well flank pain, weight loss, fever, hematuria, and palpable mass [6]. Acute renal failure due to lymphomatous infiltration of the kidney has rarely been reported [7,8,9,10,11,12,13,14,15]. In this case report, we describe a 19-year-old male who presented with painless hematuria, acute kidney injury, and bilateral renal masses.

Case Presentation A 19-year-old male patient was admitted to the Nephrology Department of Ankara University Faculty of Medicine due to painless hematuria, bilateral renal masses, and acute kidney

injury for further investigations. He was first evaluated at another center for hematuria. There was one episode of hematuria, which had subsided spontaneously. Abdominal ultrasonography had revealed bilateral diffuse renal masses and the patient was referred to our center for further examination. On admission, there was no history of fever, weight loss, night ssweats and any other health problem. The patient’s physical examination findings were unremarkable. In particular, there was no peripheral lymphadenopathy or hepatosplenomegaly. Also the kidneys were not palpable. Laboratory tests revealed white blood cell count of 7.7x109/L, hemoglobin of 11.6 g/dL, platelet count of 315x109/L, serum blood urea nitrogen of 17 mg/dL, serum creatinine of 1.5 mg/dL (normal range: 0.5-0.9), serum uric acid of 7.6 mg/dL (normal range: 2.4-5.7), serum lactate dehydrogenase of 1042 U/L (normal range: 125-220), and serum ferritin of 749 ng/mL (normal range: 11-307). Erythrocyte sedimentation rate was 52 mm/h and C-reactive protein level was 27.5 mg/L (normal: <3). Urinalysis showed density of 1010,

Address for Correspondence/Yazışma Adresi: Şiyar ERDOĞMUŞ, M.D., Ankara University Faculty of Medicine, Department of Nephrology, Ankara, Turkey Phone : +90 312 508 21 68 E-mail : si.yar21@hotmail.com

Received/Geliş tarihi: June 12, 2015 Accepted/Kabul tarihi: September 28, 2015

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pH of 5.5, protein of 15 mg/dL, and glucose negative, and urine microscopy showed 4 leukocytes and 3 erythrocytes per highpower field. Viral serology tests were negative. C3 and C4 as well as quantitative immunoglobulin levels were all within normal limits with negative antinuclear antibody and antineutrophil cytoplasmic antibody tests. Urinary ultrasonography demonstrated bilaterally enlarged kidneys without obstruction (right: 16x8 cm, left: 15.5x8 cm) and numerous solid hypoechoic nodular cortical masses in both kidneys (largest of 6.5x5.5 cm in size, numerous variably sized masses) with perirenal and paraaortic multiple enlarged lymph nodes (largest <2.5 cm in size). Contrast-enhanced computerized tomography (CT) scanning of the abdomen confirmed bilaterally enlarged kidneys, bilateral variably sized multiple hypodense renal masses, and paraaortic, paracaval multiple enlarged lymph nodes (Figure 1A). In addition, the liver was slightly enlarged with normal parenchyma while the size of the spleen and parenchyma was normal. 18F-fluorodeoxyglucose Thereafter, positron emission tomography-computed tomography (18F-FDG PET-CT) was performed for staging and its role in the differential diagnosis. It was performed to examine the entire body, revealing an abnormal accumulation in the thyroid gland, anterior mediastinum, bilateral hilar, right parasternal lymph node, left subdiaphragmatic lymph node, bilateral renal cortices, and multiple abdominal paraaortic, paracaval, and aortocaval lymph nodes (Figures 1B and 1C).

A percutaneous tru-cut biopsy of the kidney was performed and histopathological examination showed extensive infiltration of the renal parenchyma by atypical lymphoid cells (Figures 2A and 2B). Immunohistochemical studies demonstrated positive staining of the neoplastic cells for CD20, CD10, bcl-6 (Figures 2C, 2D, 2E, and 2G) and negative results for MUM1 (Figure 2F) and Bcl-2. The ki-67 proliferation index of neoplastic cells was 80% (Figure 2H). EBER in situ hybridization was negative. A diagnosis of diffuse large B-cell non-Hodgkin lymphoma (NHL) was made. To exclude lymphoma involvement of the bone marrow, the patient underwent bone marrow biopsy and there was not bone marrow infiltration.

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and skin [16,17]. The most common site of genitourinary involvement is the kidney, usually in patients with intermediate and high-grade B-cell type NHL or American Burkitt lymphoma. Additionally, extranodal involvement of lymphoma is seen in most patients at the time of diagnosis [18,19,20]. Primary renal NHL is not a common clinical entity and it is a disputed issue owing to the absence of lymphoid tissue in normal kidneys. Malbrain et al. [8] suggested the use of some criteria for the diagnosis of PRL. These include: 1) Renal failure as the initial presentation, 2) Bilateral enlargement of the kidneys without obstruction and other organ or nodal involvement, 3) Diagnosis only made by renal biopsy, 4) Absence of other causes

Figure 1. Computed tomography with intravenous contrast reveals enlargement of both kidneys with bilateral renal masses and paraaortic, paracaval lymph nodes (arrows) (A); 18F-fluorodeoxyglucose positron emission tomographycomputed tomography fusion images showed very intense diffuse fluorodeoxyglucose uptake in bilaterally enlarged kidneys (B); maximum intensity projection images of positron emission tomography-computed tomography scan demonstrated multifocal increased 18F-fluorodeoxyglucose uptake in the thyroid, mediastinum, and kidneys (C).

Subsequently, the patient was transferred to the department of hematology for treatment and chemotherapy regimen as well; cyclophosphamide, adriamycin, vincristine, prednisolone, and rituximab (CHOP+R) were started. After a cycle of chemotherapy, the patient’s renal functions returned to normal.

Discussion and Review of the Literature Extranodal spread of lymphoma often affects the gastrointestinal tract, liver, central nervous system, genitourinary tract (e.g., kidney, testis, ovary), bone, bone marrow, lungs, breast, thyroid, 160

Figure 2. Atypical large lymphoid cells infiltrating the renal interstitium (A, B) (H&E, 65x, 830x), immunohistochemical CD20 (C, D) (37x, 479x), CD10 (E) (506x), and BCL6 expression of the neoplastic cells (G) (333x). MUM1 was negative (F) (397x). Ki67 immunostaining showed high proliferation index (H) (282x).

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Erdoğmuş Ş, et al: Diffuse Large B-Cell Lymphoma Presenting with Bilateral Renal Masses and Hematuria

of renal failure, and 5) Rapid improvement of renal function after radiotherapy or systemic chemotherapy. Our patient presented with one episode of hematuria, which had subsided spontaneously, and bilateral involvement of the kidneys. His blood tests showed slight renal function impairment (serum creatinine: 1.5 mg/dL). Furthermore, there were not any causes of renal failure such as obstructive uropathy, hypercalcemia, uric acid nephropathy, volume depletion, and nephrotoxic drugs. The diagnosis of diffuse large B-cell NHL was established by Tru-Cut biopsy of the kidney. After a cycle of chemotherapy, his creatinine level returned to normal. The patient had massive infiltration of the kidneys along with thyroid gland infiltration, mediastinal involvement, and multiple enlarged lymph nodes in different sites. Consequently, our patient fulfilled four of the above criteria, and if we had used these criteria, we could not have accepted the diagnosis of PRL. A variety of benign and malignant masses can involve the kidneys in a bilateral fashion. For example, metastatic disease, lymphoproliferative disorders, adult polycystic kidney disease, and angiomyolipoma are more commonly found in a bilateral fashion compared with transitional cell carcinomas or oncocytomas [21]. Several radiologic options exist for the evaluation of renal masses, although CT scan is the most common imaging modality used for the evaluation of renal lymphoma. Usually, definitive diagnosis of renal masses is made by renal biopsy. Urban and Fishman [22] reported that the most commonly encountered pattern of involvement in patients with renal lymphoma is multiple renal masses that are mostly bilateral. Other patterns include renal invasion from contiguous retroperitoneal tumors, perirenal masses, and diffuse renal infiltration [18,22,23]. Our patient presented with bilateral renal enlargement and renal masses in ultrasonography and CT scan. The patient’s diagnosis was made by ultrasonography-guided renal biopsy. Whole-body imaging with 18F-FDG PET-CT is obligatory to assess the extent of disease by detecting unexpected extranodal sites of disease or for exclusion of disease in the presence of nonspecific extranodal CT findings [24,25]. In the present case, in addition to the CT findings, involvement of the thyroid gland and mediastinum was determined by 18F-FDG PET-CT. In conclusion, in this case, we present bilateral renal masses due to massive lymphomatous infiltration of the kidneys, which was diagnosed as diffuse large B-cell NHL by tru-cut biopsy. The presence of extrarenal involvement in the thyroid gland and mediastinal, hilar, subcarinal, and multiple abdominal lymph nodes made the diagnosis of PRL debatable. Physicians should be aware of the probability of lymphoma in the differential diagnosis of renal masses.

Ethics Informed Consent: It was taken. Authorship Contributions Concept: Kenan Keven, Şiyar Erdoğmuş; Design: Serkan Aktürk, Zeynep Kendi Çelebi, Şiyar Erdoğmuş; Data Collection or Processing: Evren Üstüner, Namık Kemal Altınbaş, Saba Kiremitçi, Gülşah Kaygusuz, Şiyar Erdoğmuş; Analysis or Interpretation: Şiyar Erdoğmuş; Literature Search: Şiyar Erdoğmuş; Writing: Şiyar Erdoğmuş. 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. Arranz Arija JA, Carrion JR, Garcia TR, Tejedor A, Perez-Manga G, Tardio J, Menarguez FJ. Primary renal lymphoma: report of 3 cases and review of the literature. Am J Nephrol 1994;14:148-153. 2. Stallone G, Infante B, Manno C, Campobasso N, Pannarale G, Schenna FP. Primary renal lymphoma does exist: case report and review of the literature. J Nephrol 2000;13:367-372. 3. Porcaro AB, D’Amico A, Novella G, Curti P, Ficarra V, Antoniolli SZ, Martignoni G, Matteo B, Malossini G. Primary lymphoma of the kidney. Report of a case and update of the literature. Arc Ital Urol Androl 2002;74:44-47. 4. Gellrich J, Hakenberg OW, Naumann R, Manseck A, Lossnitzer A, Wirth MP. Primary renal non-Hodgkin’s lymphoma - a difficult differential diagnosis. Onkologie 2002;25:273-277. 5. Hart S, Ellimoottil C, Shafer D, Mehta V, Turk TM. A case of primary renal lymphoma. Urology 2012;80:763-765. 6. Hartman DS, David CJ Jr, Goldman SM, Friedman AC, Fritzsche P. Renal lymphoma: radiologic-pathologic correlation of 21 cases. Radiology 1982;144:759-766. 7. Mills NE, Goldenberg AS, Liu D, Feiner HD, Gallo G, Gray C, Lustbader I. B-cell lymphoma presenting as infiltrative renal disease. Am J Kidney Dis 1992;19:181-184. 8. Malbrain ML, Lambrecht GL, Daelemans R, Lins RL, Hermans P, Zachee P. Acute renal failure due to bilateral lymphomatous infiltrates. Primary extranodal non-Hodgkin’s lymphoma (p-EN-NHL) of the kidneys: does it really exist? Clin Nephrol 1994;42:163-169. 9. Obrador GT, Price B, O’Meara Y, Salant DJ. Acute renal failure due to lymphomatous infiltration of the kidneys. J Am Soc Nephrol 1997;8:13481354. 10. Ozaltin F, Yalçin B, Orhan D, Sari N, Caglar M, Besbas N, Bakkaloglu A. An unusual cause of acute renal failure: renal lymphoma. Pediatr Nephrol 2004;19:912-914. 11. Diskin CJ, Stokes TJ, Dansby LM, Radcliff L, Carter TB, Graves E, Byron D, Cook WJ. Acute renal failure due to a primary renal B-cell lymphoma. Am J Kidney Dis 2007;50:885-889. 12. Lopez R. Acute renal failure due to a primary renal B-cell lymphoma. Am J Kidney Dis 2008;52:808-809. 13. Zbiti N, Alhamany Z, Houssaini TS, Rhou H, Benamar L, Ezaitouni F, Bayahia R, Ouzeddoun N. Acute renal failure due to malignant lymphoma infiltration. Nephrol Ther 2010;6:602-605. 14. Al-Salam S, Shaaban A, Alketbi M, Haq NU, Abouchacra S. Acute kidney injury secondary to renal large B-cell lymphoma: role of early renal biopsy. Int Urol Nephrol 2011;43:237-240.

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15. Domazetovski I, Jovanovic R, Kostadinova-Kunovska S, Duganovska S, Labachevski B, Nikolov I, Ivanovski N, Sikole A, Petrushevska G. Acute renal failure in a patient with diffuse large B-cell lymphoma: case report. Prilozi 2012;33:231-238. 16. Lopez-Guillermo A, Colomo L, Jimenez M, Bosch F, Villamor N, Arenillas L, Muntanola A, Montoto S, Gine E, Colomer D, Bea S, Campo E, Montserrat E. Diffuse large B-cell lymphoma: clinical and biological characterization and outcome according to the nodal or extranodal primary origin. J Clin Oncol 2005;23:2797-2804. 17. Economopoulos T, Papageorgiou S, Rontogianni D, Kaloutsi V, Fountzilas G, Tsatalas C, Pavlidis N, Pectasides D, Papageorgiou E, Dimopoulos M; Hellenic Cooperative Oncology Group. Multifocal extranodal non-Hodgkin lymphoma: a clinicopathologic study of 37 cases in Greece, a Hellenic Cooperative Oncology Group study. Oncologist 2005;10:734-738. 18. Sheth S, Ali S, Fishman E. Imaging of renal lymphoma: patterns of disease with pathologic correlation. Radiographics 2006;26:1151-1168. 19. Strauss S, Libson E, Schwartz E, Peylan-Ramu N, Lebensart PD, Bloom RA, Itzchak Y. Renal sonography in American Burkitt lymphoma. AJR Am J Roentgenol 1986;146:549-552.

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20. Richards MA, Mootoosamy I, Reznek RH, Webb JA, Lister TA. Renal involvement in patients with non-Hodgkin’s lymphoma: clinical and pathological feature in 23 cases. Hematol Oncol 1990;8:105-110. 21. Roy A, Le O, Silverman PM, Kundra V. Common and uncommon bilateral renal masses. Cancer Imaging 2012;12:205-211. 22. Urban BA, Fishman EK. Renal lymphoma: CT patterns with emphasis on helical CT. Radiographics 2000;20:197-212. 23. El-Sharkawy MS, Siddiqui N, Aleem A, Diab AA. Renal involvement in lymphoma: prevalence and various patterns of involvement on abdominal CT. Int Urol Nephrol 2007;39:929-933. 24. Even-Sapir E, Lievshitz G, Perry C, Herishanu Y, Lerman H, Metser U. Fluorine-18 fluorodeoxyglucose PET/CT patterns of extranodal involvement in patients with non-Hodgkin lymphoma and Hodgkin’s disease. Radiol Clin North Am 2007;45:697-709. 25. Dhull VS, Sharma P, Singla S, Faizi NA, Thulkar S, Bal C, Kumar R. Extensive extranodal involvement of rare sites in non Hodgkin’s lymphoma detected on 18F- FDG PET-CT: a case report. Nucl Med Mol Imaging 2013;47:125129.

LETTERS TO EDITOR Turk J Hematol 2016;33:163-166

A Comparison of Healthy Infants and Adults with Respect to Indirect Microparticle Activity and the Parameters of the Thrombin Generation Test Sağlıklı Süt Çocukları ve Erişkinlerin İndirekt Mikropartikül Aktivitesi ve Trombin Jenerasyon Parametrelerine Göre Karşılaştırılması Filiz Şimşek Orhon1, Nejat Akar2, Yonca Eğin2, Betül Ulukol1, Sevgi Başkan1 1Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Social Pediatrics, Ankara, Turkey 2Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Pediatric Molecular Genetics, Ankara, Turkey

To the Editor, Microparticles express phospholipids and support thrombin generation, which increases with age [1,2]. In a recently published study, we showed age-dependent changes in thrombin generation parameters in a healthy infant population aged 1-24 months [3]. The aim of this present study was to compare the levels of both indirect microparticle activity and thrombin generation parameters of healthy infants from our recent study to those of a healthy adult population. The adult population consisted of medical students of the Ankara University School of Medicine. Blood was collected into tubes containing 1 mL of 0.109 M trisodium citrate. For indirect microparticle activity, plasma samples were studied using the STA-PROCOAGPPL Kit (Diagnostica Stago Inc., Asnières sur Seine, France). Plasma samples were measured using thrombin generation kits, including a Thrombin Calibrator, PPP-Reagent 5 pM, and the FluCa-Kit (Diagnostica Stago). Thrombin generation curves were calculated using Thrombinoscope software (Thrombinoscope BV, Maastricht, the Netherlands). The following parameters were derived from the curves: lag time (LT, min), time to initiation of thrombin generation; endogenous thrombin potential (ETP, nmol/L/min), area under the thrombin generation curve; peak thrombin activity (peak, nmol/L); and time to peak thrombin generated (TTP, min). Statistical analysis was performed using Statistical Package for the Social Sciences 16. A total of 58 healthy adults (23 males and 35 females; mean age: 23.2±0.4 years) were admitted to the study. In our recent study, 85 healthy infants (51 males and 34 females; mean age: 12.6±8.3 months) were studied. The indirect microparticle activity in the infant group was significantly lower than that of the adult group (p<0.001). The ETP and peak levels in the infant group were significantly lower than those of adults. Furthermore, the TTP levels of the adult group were lower than those of infants (p=0.001) (Table 1).

Physiologic concentrations of coagulation proteins gradually increase after birth [4]. Karlaftis et al. showed that procoagulant phospholipid activity was increased in neonates and decreased in children aged 1-16 years [5]. We show that the levels of indirect microparticle activity are increased in healthy adults as compared to healthy infants. This may suggest that aging is correlated to an increase in the indirect microparticle activity, and also possibly to its procoagulant and proinflammatory features. Thrombin generation is influenced by different variables like age, sex, body mass index, genetic factors, and acquired conditions [6,7]. In a previous study, the ETP values of children were found to be lower than those of adults [8]. Positive correlations were found for age versus thrombin generation parameters in calibrated automated thrombography in two recent studies [9,10]. We showed that ETP and peak levels were higher in adults as compared to infants. Thus, we suggest that ETP and peak levels, the main parameters of thrombin generation, increase gradually from infancy to adulthood. As for limitations, our adult group was not adequate for representing all ages of the adult population and there was a difference between the groups Table 1. Data on indirect microparticle activity and thrombin generation parameters of the study groups. Healthy Infants (n=85)*

Healthy Adults (n=58)*

p**

Microparticle release time (s)

31.7±7.5

39.7±9.8

0.001

Lag time (min)

3.2±0.8

3.1±0.7

0.357

ETP (nmol/L/min)

1363.6±262.2

1691.5±378.1

0.001

Peak (nmol/L)

256.5±79.7

358.4±79.9

0.001

TTP (min)

6.7±1.7

5.4±0.9

0.001

*Values are presented as mean ± standard deviation, **t-test. ETP: Endogenous thrombin potential, peak: peak thrombin activity, TTP: time to peak thrombin generated.

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in terms of sex ratios. However, we may conclude that plasma from adults may be more procoagulant than that of infants. Our findings may confirm the presence of a regulation mechanism in the coagulation parameters throughout the course of life. Keywords: Infant, Adult, Microparticle, Thrombin

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References 1. Chironi GN, Boulanger CM, Simon A, Dignat-George F, Freyssinet JM, Tedgui A. Endothelial microparticles in diseases. Cell Tissue Res 2009;335:143-151. 2. Brummel-Ziedins KE, Everse SJ, Mann KG, Orfeo T. Modeling thrombin generation: plasma composition based approach. J Thromb Thrombolysis 2014;37:32-44.

Anahtar Sözcükler: Süt çocuğu, Erişkin, Mikropartikül, Trombin

3. Orhon FS, Egin Y, Ulukol B, Baskan S, Akar N. Evaluation of indirect microparticle activity and parameters of thrombin generation test in healthy infants. Thromb Res 2014;133:281-284.

Authorship Contributions

4. Kenet G, Krumpel A, Nowak-Gottl U. Bleeding issues in neonates, infants and young children. Thromb Res 2009;123(Suppl 2):S35-S37.

Study Conception and Design: Nejat Akar, Filiz Şimşek Orhon; Acquisition and Blood Collection: Filiz Şimşek Orhon, Sevgi Başkan; Laboratory Analysis: Yonca Eğin; Interpretation of Data: Nejat Akar, Filiz Şimşek Orhon; Literature Search: Filiz Şimşek Orhon, Betül Ulukol; Drafting and Writing: Filiz Şimşek Orhon.

5. Karlaftis V, Attard C, Summerhayes R, Monagle P, Ignjatovic V. The microparticle-specific procoagulant phospholipid activity changes with age. Int J Lab Hem 2014;36:e41-e43.

8. Haidl H, Cimenti C, Leschnik B, Zach D, Muntean W. Age-dependency of thrombin generation measured by means of calibrated automated thrombography (CAT). Thromb Haemost 2006;95:772-775.

Financial Disclosure: This study was supported in part by the Ankara University Research Fund.

6. Castoldi E, Rosing J. Thrombin generation tests. Thromb Res 2011;127(Suppl 3):S21-S25. 7. Butenas S, van’t Veer C, Mann KG. “Normal” thrombin generation. Blood 1999;94:2169-2178.

9. Schneider T, Siegemund T, Siegemund R, Petros S. Thrombin generation and rotational thromboelastometry in the healthy adult population. Hamostaseologie 2015;35:181-186. 10. Wu J, Zhao HR, Zhang HY, Ge YL, Qiu S, Zhao J, Song Y, Zhao JZ, Lu SS. Thrombin generation increasing with age and decreasing with use of heparin indicated by calibrated automated thrombogram conducted in Chinese. Biomed Environ Sci 2014;27:378-384.

Address for Correspondence/Yazışma Adresi: Filiz ŞİMŞEK ORHON, M.D., Ankara University Faculty of Medicine, Department of Pediatrics, Divisions of Social Pediatrics, Ankara, Turkey Phone : +90 312 595 72 02 E-mail : simsekfiliz@hotmail.com

Received/Geliş tarihi: September 29, 2015 Accepted/Kabul tarihi: December 11, 2015 DOI: 10.4274/tjh.2015.0341

Comment: In Response to “Downgraded Lymphoma: B-Chronic Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell Lymphoma - De Novo Occurrence or Transformation” Yorum: Cevap Olarak “Geriletilmiş Lenfoma: Diffüz Büyük B-Hücreli Lenfoma Olduğu Bilinen Bir Olguda B-Kronik Lenfositik Lösemi - De Novo Oluşum veya Dönüşüm” Burak Uz, Kadir Acar Gazi University Faculty of Medicine, Department of Internal Medicine, Division of Adult Hematology, Ankara, Turkey

To the Editor, We read the letter submitted by Gajendra et al. with deep interest [1]. The authors described a patient diagnosed with diffuse large B-cell lymphoma (DLBCL) non-germinal center B-cell type in 2002 who received 6 cycles of cyclophosphamide, adriamycin, vincristine, and prednisolone (CHOP) followed by 164

radiotherapy. He was well for nearly 5 years, but subsequently his disease locally relapsed. Unfortunately, a planned intensive salvage regimen could not be given because the patient was lost to follow-up. In 2010, despite not being given any treatment modality, he presented with small lymphocytic lymphoma. Finally, 22 months thereafter, he was diagnosed with Rai stage IV chronic lymphocytic leukemia and 6 cycles of fludarabine,

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cyclophosphamide, and rituximab (FCR) were administered, resulting in complete remission. As is known, indolent or low-tumor-burden lymphomas may transform into aggressive or high-tumor-burden lymphoma forms in a process called “Richter transformation”. Although rare, the reverse process may also occur with unknown mechanisms. At this point, there are two main hypotheses that can be suggested: initially, there are two existing malignant clones, and successful eradication of the rapidly proliferating clone with intensive therapy results in the survival of the less rapidly growing clone, which may eventually lead to relapsed disease even many years following the diagnosis [2]; or, less probable, a separate secondary malignant clone that is distinct from the initial clone might appear [3]. Previously, two downgraded lymphoma cases were reported [2,3] after the successful treatment of underlying diffuse non-Hodgkin lymphoma, 3 and 14 years following the initial diagnosis. This well-described patient was accepted as having late-relapsed (~5 years later) DLBCL, which transformed into a “downgraded lymphoma” without lymphoma-specific therapy. DLBCL patients generally relapse in the first 2 or 3 years following treatment. The largest series of patients with DLBCL who relapsed ≥5 years following diagnosis was reported by a French group [4]; 3.6% of their cohort had a late relapse and those patients had some distinct clinical features, including localized disease (63%), favorable International Prognostic Index score (82%), and extranodal involvement (65%) at diagnosis. At the time of relapse, 83% had DLBCL histology, while 17% had indolent histology. Additionally, having an indolent component at diagnosis (44.4%) was significantly associated with indolent histology at relapse. However, nearly all the late-relapsed patients with initial good-risk disease were treated adequately with anthracycline-based combined chemotherapy. Late-relapsed DLBCL patients have poor outcomes; therefore, they must be treated promptly with rituximab plus chemotherapy or (if possible) autologous stem cell transplantation [4]. In the French experience, all late-relapsed patients were heavily treated and the patients experienced their relapse a median of 7.4 years after diagnosis [4]. As an interesting aside, the present patient could not be administered any treatment for 3 years after the confirmation of DLBCL relapse and he presented with downgraded lymphoma. We could not understand why the patient’s relapsed high-grade lymphoma resolved without any treatment attempts. Spontaneous remission of DLBCL is exceedingly rare, with only a handful of such cases reported [5,6,7,8]. Given the unexplained clinical course of DLBCL in this

patient, a probable infectious agent or nonprescription usage of traditional medicinal plants inducing antitumor response by modulating the immune system against lymphomatous cells should be sought in his medical history. Keywords: Diffuse large B-cell lymphoma, Downgraded lymphoma Anahtar Sözcükler: Diffüz büyük B-hücreli lenfoma, Geriletilmiş lenfoma Authorship Contributions Concept: Burak Uz; Design: Burak Uz; Data Collection or Processing: Burak Uz, Kadir Acar; Analysis or Interpretation: Burak Uz, Kadir Acar; Literature Search: Burak Uz, Kadir Acar; Writing: Burak Uz. 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. Gajendra S, Jha B, Goel S, Sahni T, Dorwal P, Sachdev R. Downgraded lymphoma: B-chronic lymphocytic leukemia in a known case of diffuse large B-cell lymphoma-de novo occurrence or transformation. Turk J Hematol 2015;32:371-372. 2. Kerrigan DP, Foucar K, Dressler L. High-grade non-Hodgkin lymphoma relapsing as low-grade follicular lymphoma: so-called downgraded lymphoma. Am J Hematol 1989;30:36-41. 3. Ogata Y, Setoguchi M, Tahara T, Takahashi M. Downgraded non-Hodgkin’s lymphoma in the neck occurring as a secondary malignancy. ORL J Otorhinolaryngol Relat Spec 1998;60:295-300. 4. Larouche JF, Berger F, Chassagne-Clement C, Efrench M, Callet-Bauchu E, Sebban C, Ghesquieres H, Broussais-Guillaumot F, Salles G, Coiffier B. Lymphoma recurrence 5 years or later following diffuse large B-cell lymphoma: clinical characteristics and outcome. J Clin Oncol 2010;28:20942100. 5. Mizuno T, Ishigaki M, Nakajima K, Matsue T, Fukushima M, Minato H, Nojima N, Atsushi S, Ishigami K, Atsumi H, Ito T, Iguchi M, Usuda D, Okamura H, Urashima S, Asano M, Fukuda A, Izumi Y, Takekoshi N, Kanda T. Spontaneous remission of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly. Case Rep Oncol 2013;6:269-274. 6. Buckner TW, Dunphy C, Fedoriw YD, van Deventer HW, Foster MC, Richards KL, Park SI. Complete spontaneous remission of diffuse large B-cell lymphoma of the maxillary sinus after concurrent infections. Clin Lymphoma Myeloma Leuk 2012;12:455-458. 7. Tamás L, Sári E, Répássy G, Szabó P, Bagdi E, Krenács L, Demeter J. Spontaneous remission in localized diffuse large B-cell lymphoma. Pathol Oncol Res 2011;17:779-784. 8. Watari J, Saitoh Y, Fujiya M, Nakamura K, Inaba Y, Okamoto K, Tanabe H, Yasuda A, Miyokawa N, Kohgo Y. Spontaneous remission of primary diffuse large B-cell gastric lymphoma. J Gastroenterol 2005;40:414-420.

Address for Correspondence/Yazışma Adresi: Burak UZ, M.D., Received/Geliş tarihi: December 30, 2015 Gazi University Faculty of Medicine, Department of Internal Medicine, Division of Adult Hematology, Ankara, Turkey Accepted/Kabul tarihi: January 06, 2016 Phone : +90 312 202 55 79 E-mail : burakuz78@gmail.com; burakuz@yahoo.com DOI: 10.4274/tjh.2015.0452

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Turk J Hematol 2016;33:163-166

Tumor Necrosis Factor and Splenectomy Tümör Nekrozis Faktör ve Splenektomi İrfan Yavaşoğlu Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey

To the Editor, The article entitled “Effect of Tumor Necrosis Factor-Alpha (TNF-α) on Erythropoietin- and Erythropoietin ReceptorInduced Erythroid Progenitor Cell Proliferation in β-Thalassemia/ Hemoglobin E Patients”, written by Tanyong et al. [1] and published in a recent issue of your journal, was quite interesting. Here we would like to emphasize some relevant points. Splenectomy can increase the release of TNF-α and cell apoptosis in experimental and clinical studies in different diseases [2,3,4]. Increased serum TNF-α was reported in E/b-Thal patients, particularly after splenectomy [3,4]. In sickle cell disease presenting with functional asplenia, increased amounts of TNF-α, indicative of monocyte activation, and increased serum C-reactive protein levels were reported [5]. Banyatsuppasin et al. suggested the role of the spleen in controlling mononuclear phagocytic activity in E/b-Thal patients [6]. TNF-α play roles as an inducer and effector of monocyte activation [6]. Additionally, TNF-α returned to normal after 12, 6, and 3 months of deferiprone treatment [7]. Therefore, chelation treatment can affect apoptosis independently of splenectomy. It might be important to know the effect of chelation treatment and splenectomy on tumor necrosis factor in the study of Tanyong et al. [1] based on all these investigations stated above [2,3,4,5,6,7]. Keywords: Thalassemia, Tumor necrosis factor, Splenectomy Anahtar Sözcükler: Talasemi, Tümör nekrozis faktör, Splenektomi

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

References 1. Tanyong D, Panichob P, Kheansaard W, Fucharoen S. Effect of tumor necrosis factor-alpha on erythropoietin- and erythropoietin receptor-induced erythroid progenitor cell proliferation in β-thalassemia/hemoglobin E patients. Turk J Hematol 2015;32:304-310. 2. Hiroyoshi T, Tsuchida M, Uchiyama K, Fujikawa K, Komatsu T, Kanaoka Y, Matsuyama H. Splenectomy protects the kidneys against ischemic reperfusion injury in the rat. Transpl Immunol 2012;27:8-11. 3. Chuncharunee S, Archararit N, Hathirat P, Udomsubpayakul U, Atichartakarn V. Levels of serum interleukin-6 and tumor necrosis factor-a in postsplenectomized thalassemic patients. J Med Assoc Thai 1997;80(Suppl 1):S86-S91. 4. Wanachiwanawin W, Wiener E, Siripanyaphinyo U, Chinprasertsuk S, Mawas F, Fucharoen S, Wickramasinghe S, Pootrakul P, Visudhiphan S. Serum levels of tumor necrosis factor-α, interleukin-1, and interferon-γ in β0thalassemia/HbE and their clinical significance. J Interferon Cytokine Res 1999;19:105-111. 5. Belcher JD, Marker PH, Weber JP, Hebbel RP. Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion. Blood 2000;96:2451-2459. 6. Banyatsuppasin W, Butthep P, Atichartakarn V, Thakkinstian A, Archararit N, Pattanapanyasat K, Chuncharunee S. Activation of mononuclear phagocytes and its relationship to asplenia and phosphatidylserine exposing red blood cells in hemoglobin E/β-thalassemia patients. Am J Hematol 2011;86:89-92. 7. Del Vecchio GC, Schettini F, Piacente L, De Santis A, Giordano P, De Mattia D. Effects of deferiprone on immune status and cytokine pattern in thalassaemia major. Acta Haematol 2002;108:144-149.

Address for Correspondence/Yazışma Adresi: İrfan YAVAŞOĞLU, M.D., Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey Phone : +90 256 212 00 20 E-mail : dr_yavas@yahoo.com

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Received/Geliş tarihi: January 23, 2016 Accepted/Kabul tarihi: January 26, 2016 DOI: 10.4274/tjh.2016.0040

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0275 Turk J Hematol 2016;33:167

Auer Rod in a Neutrophil in a Nonmalignant Condition Malign Olmayan Bir Durumda Nötrofilde Görülen Auer Cisimciği Harish Chandra, Smita Chandra, Vibha Gupta, Divyaa Mahajan Himalayan Institute of Medical Sciences, Department of Pathology, Dehradun, India

the cytoplasm of a neutrophil, along with features of dysmyelopoiesis (Figure 1). Bone marrow aspiration was done, which was unremarkable and showed normoblastic maturation. To the best of our knowledge, no case has been reported in the literature with Auer rods in a nonmalignant condition. Therefore, the present case is being reported, which shows an Auer rod in a polymorph in a case of typhoid fever. Keywords: Auer rods, Neutrophil, Typhoid fever Anahtar Sözcükler: Auer cisimciği, Nötrofil, Tifo Ethics Informed Consent: It was taken. Authorship Contributions Concept: Harish Chandra, Smita Chandra; Design: Figure 1. Neutrophil shows an Auer rod at 100x (Jenner-Giemsa stain), 270x203 Harish Chandra, Smita Chandra; Data Collection or mm (72x72 dpi). Processing: Vibha Gupta, Divyaa Mahajan; Analysis or Interpretation: Harish Chandra, Smita Chandra, Auer rods are normally observed in immature myeloid precursors Vibha Gupta; Literature Search: Harish Chandra, Smita Chandra, including myeloblasts and promyelocytes in cases of acute Vibha Gupta, Divyaa Mahajan; Writing: Harish Chandra, Smita myeloid leukemia, while cases have rarely reported Auer rods in Chandra. polymorphs in acute myeloid leukemia [1,2]. Conflict of Interest: The authors of this paper have no conflicts A 19-year-old female presented with high-grade fever and of interest, including specific financial interests, relationships, abdominal pain for 1 week. Her laboratory investigations and/or affiliations relevant to the subject matter or materials revealed hemoglobin of 65 g/L, red blood cell count of included. 3.3x1012/L, mean cell volume of 96.2 fL, white blood cell count of 8.5x109/L, and platelet count of 23x109/L. She was References 1. Dawson MA, Whitehead S. Mature neutrophils with multiple Auer rods: found to be positive for Salmonella Typhi antigen O in 1:160 a rarity in normal karyotype acute myeloid leukaemia. Br J Haematol dilutions (slide agglutination by Beacon Diagnostics, India). An 2007;137:86. interesting finding was observed during her peripheral blood 2. Dmitrienko S, Vercauteren S. Auer rods in mature granulocytes of a patient examination, which showed an Auer rod-like structure within with mixed lineage leukemia. Blood 2012;119:4348.

Address for Correspondence/Yazışma Adresi: Harish CHANDRA, M.D., Himalayan Institute of Medical Sciences, Department of Pathology, Dehradun, India E-mail : drharishbudakoti31@yahoo.co.in

Received/Geliş tarihi: July 24, 2015 Accepted/Kabul tarihi: October 13, 2015

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IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2015.0294 Turk J Hematol 2016;33:168-169

Precursor B-Cell Lymphoblastic Lymphoma Presenting as a Spinal Mass at Initial Diagnosis İlk Tanı Sırasında Spinal Kitle ile Prezente olan Prekürsör B-Hücreli Lenfoblastik Lenfoma Oğuzhan Erol1, Çiğdem Tokyol1, Feyzullah Akyüz2, Nuran Ahu Baysal3, Mehmet Sezgin Pepeler4 1Afyon Kocatepe University Faculty of Medicine, Department of Pathology, Afyonkarahisar, Turkey 2Park Hospital, Clinic of Neurosurgery, Afyonkarahisar, Turkey 3Afyonkarahisar Public Hospital, Clinic of Hematology, Afyonkarahisar, Turkey 4Gazi University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Figure 1. Lymphoid cells with irregular nuclei, dispersed nuclear chromatin, prominent nucleoli, and scant cytoplasm (H&E, 400x).

Figure 2. Diffuse expression of TdT in tumor cells (200x).

An 18-year-old male presented to the emergency department of our hospital with complaints of bilateral leg numbness and weakness since about a month. Magnetic resonance imaging of the spine revealed an extramedullary extradural mass at the T9-T11 level causing marked spinal cord compression. Emergent surgery was performed. An epidural mass was seen after laminectomy and partially removed. Microscopic examination showed a diffuse infiltration of small- to medium-sized lymphoid cells with irregular nuclei, dispersed nuclear chromatin, prominent nucleoli, and scant cytoplasm in adipose tissue (Figure 1). Immunohistochemical examination demonstrated that tumor cells stained positively for TdT, CD34,

CD10, CD20, CD79a, Pax-5, CD45, and Bcl-2 (Figure 2). Ki-67 showed immunoreactivity of 80% of tumor cells. Bone marrow and blood involvements were not detected. These findings led us to the diagnosis of precursor B-cell lymphoblastic lymphoma. He was given combination chemotherapy of R-HCVAD (rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone, cytarabine, mesna, methotrexate). After the second dose of chemotherapy, complete response was achieved as assessed by positron emission tomography/computed tomography scan. The spinal cord is an extremely rare initial site of involvement for B-cell lymphoblastic lymphoma. To our knowledge, there are only 3 reported cases in the English literature (Table 1) [1,2,3].

Address for Correspondence/Yazışma Adresi: Çiğdem TOKYOL, M.D., Afyon Kocatepe University Faculty of Medicine, Department of Pathology, Afyonkarahisar, Turkey Phone : +90 272 246 33 04 E-mail : ctokyol@yahoo.com

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Received/Geliş tarihi: August 12, 2015 Accepted/Kabul tarihi: September 17, 2015

Erol O, et al: Spinal Lymphoblastic Lymphoma

Turk J Hematol 2016;33:168-169

Table 1. Cases of isolated primary B-cell lymphoblastic lymphoma of the spine. Reference

Age

Location

Sex

First Manifestations

Treatment

Khalid et al. [1]

Thoracic spine

Female

Back pain and numbness

S+CT

Esin et al. [2]

Thoracic spine

Female

Acute walking difficulty in pregnancy

S+CT

Park et al. [3]

Thoracolumbar spine

Male

Back pain

S+CT+RT

Present case

Thoracic spine

Male

Bilateral leg numbness and weakness

S+CT

S: Surgery, CT: chemotherapy, RT: radiotherapy.

Lymphoblastic lymphoma should be included in the differential diagnosis of spinal masses.

Erol; Analysis or Interpretation: Çiğdem Tokyol; Literature Search: Oğuzhan Erol; Writing: Oğuzhan Erol.

Acknowledgment

Presented at the 25th National Congress of Pathology 2015, Bursa, Turkey. Keywords: B-cell lymphoblastic lymphoma, Thoracic spine, Spinal cord compression Anahtar Sözcükler: B-hücreli lenfoblastik lenfoma, Torasik vertebra, Spinal kord basısı Authorship Contributions Surgical and Medical Practices: Feyzullah Akyüz, Nuran Ahu Baysal, Mehmet Sezgin Pepeler; Concept: Çiğdem Tokyol; Design: Çiğdem Tokyol; Data Collection or Processing: Oğuzhan

References 1. Khalid I, Rival J, Salama ME, Banghar PK, Janakiraman N. Unusual presentations of hematologic malignancies: Case 2. Precursor B-cell lymphoblastic lymphoma presenting as spinal cord compression. J Clin Oncol 2004;22:1331-1333. 2. Esin S, Tarim E, Abali H, Kardes O, Kocer EN, Alkan O. Management of precursor B-lymphoblastic lymphoma/leukaemia of thoracic spine in a pregnancy presenting with acute paraplegia. J Obstet Gynaecol 2012;32:485-486. 3. Park DA, Park SG, Kim SW. Solitary lymphoblastic lymphoma of the thoracic spine. J Korean Neurosurg Soc 2012;52:564-566.

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IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2014.0470 Turk J Hematol 2016;33:170-171

Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Should Be Kept in Mind in Children with Febrile Neutropenia, Oral Cavity Lesions, and Skin Rash Febril Nötropeni, Oral Kavitede Lezyonlar ve Deri Döküntüsü Olan Çocuklarda StevensJohnson Sendromu/Toksik Epidermal Nekrolizis Akılda Tutulmalıdır Eda Ataseven, Şebnem Yılmaz Bengoa, Hale Ören Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey

Figure 1. Erosions and crusts on the lips and hemorrhagic ulcers in the oral cavity. A red papular rash spread to the shoulders and the back.

Figure 2. Regression of the lesions on the lips, oral cavity, and back.

A 14-year-old boy was diagnosed with acute lymphoblastic leukemia. Febrile neutropenia developed during induction. Imipenem and teicoplanin were started because of severe mucositis. Viral tests and bacterial cultures were unrevealing. On follow-up, a painful papular rash had appeared and oral mucositis had become worse (Figure 1). Stevens-Johnson syndrome (SJS)/ toxic epidermal necrolysis (TEN) was suspected. Intravenous immunoglobulin (IVIG) at 1 g/kg/day and methylprednisolone at 1 mg/kg/day were started. The lesions regressed in 1 week (Figure 2). Skin biopsy was consistent with SJS/TEN. Informed consent was obtained.

may occur after taking a new medication or may rarely have an infectious origin. Our patient had no predisposing conditions other than taking chemotherapeutic drugs and antibiotics. The mortality rate is high in SJS/TEN [1,3]. Rapid withdrawal of the probable causative drug(s) is important. Use of IVIGs and corticosteroids is reported as the most commonly used therapy in childhood [1,4]. Systematic review of adult treatments for SJS and TEN did not show any benefit of these agents on mortality rates [3]. Cyclosporine, plasmapheresis, and tumor necrosis factor-alpha inhibitors have been also reported among other treatment options [1,2,3,4,5].

SJS and TEN are rare diseases characterized by fever and mucosal and cutaneous lesions [1]. It is defined as SJS when epidermal involvement affects less than 10% of the body surface area, as SJS/TEN overlap when the skin detachment ranges from 10% to 30%, and as TEN when it involves more than 30% [1,2]. It

Keywords: Acute leukemia, Stevens-Johnson syndrome, Toxic epidermal necrolysis Anahtar Sözcükler: Akut lösemi, Stevens-Johnson sendromu, Toksik epidermal nekrolizis

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

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Received/Geliş tarihi: December 08, 2014 Accepted/Kabul tarihi: January 27, 2015

Turk J Hematol 2016;33:170-171

Authorship Contributions Concept: Hale Ören, Eda Ataseven; Design: Hale Ören, Eda Ataseven; Data Collection: Şebnem Yılmaz Bengoa, Eda Ataseven, Analysis or Interpretation: Hale Ören, Eda Ataseven; Literature Search: Şebnem Yılmaz Bengoa, Eda Ataseven; Writing: Hale Ören, Eda Ataseven. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Ataseven E, et al: Stevens-Johnson/Toxic Epidermal Necrolysis

References 1. Ferrandiz-Pulido C, Garcia-Patos V. A review of causes of Stevens-Johnson syndrome and toxic epidermal necrolysis in children. Arch Dis Child 2013;98:998-1003. 2. Mockenhaupt M. The current understanding of Stevens-Johnson syndrome and toxic epidermal necrolysis. Expert Rev Clin Immunol 2011;7:803-813. 3. Roujeau J, Bastuji-Garin S. Systematic review of treatments for StevensJohnson syndrome and toxic epidermal necrolysis using the SCORTEN score as a tool for evaluating mortality. Ther Adv Drug Saf 2011;2:87-94. 4. Spies M, Sanford AP, Aili Low JF, Wolf SE, Herndon DN. Treatment of extensive toxic epidermal necrolysis in children. Pediatrics 2001;108:11621168. 5. Scott-Lang V, Tidman M, McKay D. Toxic epidermal necrolysis in a child successfully treated with infliximab. Pediatr Dermatol 2014;31:532-534.

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Advisory Board of This Issue (June 2016) Ahmet Muzaffer Demir, Turkey Ahmet Öztürk, Turkey Akif Selim Yavuz, Turkey Anne-Mette Hvas, Denmark Aurore Keutgens, Belgium Ayşegül Ünüvar, Turkey Aytemiz Gürgey, Turkey Barbara Bain, UK Bülent Eser, Turkey Bülent Kantarcıoğlu, Turkey Cem Ar, Turkey Cengiz Beyan, Turkey Davut Albayrak, Turkey Debra Hoppensteadt, USA Dunja Mihajlovic, Serbia Eman Mosad, Egypt Emre Tekgündüz, Turkey Eric Berntorp, Sweden Erol Atalay, Turkey Fahir Özkalemkaş, Turkey

Fatemah Mohammadi-Nasrabadi, Iran Fatih Demirkan, Turkey Fatma Aktepe, Turkey Fatma Hüsniye Dilek, Turkey Ferit Avcu, Turkey Geetha Narayanan, India Gluseppe Saglio, Italy Halis Akalın, Turkey Hayri Özsan, Turkey Hüseyin Gülen, Turkey Hyun Kyung Kim, Korea İbrahim Haznedaroğlu, Turkey İrfan Yavaşoğlu, Turkey İsmail Sarı, Turkey Jawed Fareed, USA Joachim Deeg, USA John Bennett, USA Jun-Sook Ha, Korea Kaan Kavaklı, Turkey Kansu Büyükafşar, Turkey

Mahmut Bayık, Turkey Manuel Olivares, Chile Muhit Özcan, Turkey Naci Tiftik, Turkey Nicola Pavan, Italy Oral Nevruz, Turkey Praveen Papareddy, Sweden Raja Ramachandran, India Şule Ünal, Turkey Theodore Tulchinsky, Israel Türkan Patıroğlu, Turkey Ulrich Germing, Germany Usama Gergis, USA Veysel Sabri Hançer, Turkey Yehia Abed, Palestine Ying Ju, China Yusuf Baran, Turkey Zahra Sepehri, Iran