Turkish Journal of Hematology Volume: 32 - Issue: 1 by LookUs Scientific

Issue 1

March 2015

40 TL

ISSN 1300-7777

Volume 32

Review Article Iron-Refractory Iron Deficiency Anemia Ebru Yılmaz Keskin, et al.; Samsun, Ankara, Turkey

Research Articles Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients Rabab M. Aly, et al.; Mansoura, Egypt

Are the High Serum Interleukin-6 and Vascular Endothelial Growth Factor Levels Useful Prognostic Markers in Aggressive Non-Hodgkin Lymphoma Patients? Hava Üsküdar Teke, et al.; Eskişehir, Kocaeli, Turkey

Prevalence of Monoclonal B Lymphocytosis in First-Degree Relatives of Chronic Lymphocytic Leukaemia Patients in Turkey Taner Demirci, et al.; Ankara, Turkey

Myeloid Sarcomas: A Clinicopathologic Study of 20 Cases Gülşah Kaygusuz, et al.; Ankara, Turkey

Osteoporosis and Osteopathy Markers in Patients with Mastocytosis Nilüfer Alpay Kanıtez, et al.; İstanbul, Turkey

Tunnelled Central Venous Catheter-Related Problems in the Early Phase of Haematopoietic Stem Cell Transplantation and Effects on Transplant Outcome Mahmut Yeral, et al.; Adana, Ankara, Turkey

Cover Picture: Güçhan Alanoğlu Rumeli Castle, İstanbul

Editor-in-Chief

International Review Board

Aytemiz Gürgey

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  Istanbul 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

Language Editor Leslie Demir

Ankara, Turkey

Associate Editors Ayşegül Ünüvar İstanbul University, İstanbul, Turkey

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

Cengiz Beyan Gülhane Military Medical Academy, Ankara, Turkey

Hale Ören Dokuz Eylül University, İzmir, Turkey

İbrahim C. Haznedaroğlu Hacettepe University, Ankara, Turkey

İlknur Kozanoğlu Başkent University, Adana, Turkey

Mehmet Ertem Ankara University, Ankara, Turkey

A. Muzaffer Demir Trakya University, Edirne, Turkey

Reyhan Diz Küçükkaya İstanbul Bilim University, İstanbul, 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

İnci Alacacıoğlu Dokuz Eylul University, Ankara, Turkey

Nil Güler On Dokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay Osmangazi University, Eskişehir, Turkey

Selami Koçak Toprak Ankara University, Ankara, Turkey

Şule Ünal Hacettepe University, Ankara, Turkey

Veysel Sabri Hançer İstanbul Bilim University, İstanbul, Turkey

Zühre Kaya

Statistic Editor Hülya Ellidokuz

Senior Advisory Board Yücel Tangün Osman İlhan Muhit Özcan

Gazi University, Ankara, Turkey

A-I

Publishing Services

Editorial Office İpek Durusu Bengü Timoçin

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. Aytemiz Gürgey Editor-in-Chief  Address: 725. Sok. Görkem Sitesi  Yıldızevler No: 39/2, 06550 Çankaya, Ankara / Turkey Phone : +90 312 438 14 60 E-mail : agurgey@hacettepe.edu.tr

All other inquiries should be adressed to TURKISH JOURNAL OF HEMATOLOGY Address: İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550 Çankaya, Ankara / Turkey Phone : +90 312 490 98 97 Fax : +90 312 490 98 68  E-mail : info@tjh.com.tr ISSN: 1300-7777

Turkish Society of Hematology Teoman Soysal, President  A. Muzaffer Demir, General Secretary Hale Ören, Vice President  İbrahim C. Haznedaroğlu, Research Secretary Fahir Özkalemkaş, Treasurer  A. Zahit Bolaman, Member  Mehmet Sönmez, Member

Publishing Manager Sorumlu Yazı İşleri Müdürü A. Muzaffer Demir

Management Address Yayın İdare Adresi

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

Publishing House / Yayınevi

Online Manuscript Submission

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul, Turkey Tel: +90 212 621 99 25 Faks: +90 212 621 99 27 E-posta: info@galenos.com.tr Baskı: Senk Ajans Reklam Matb. San. ve Tic. Ltd. Şti. Sanayi Mahallesi, Sultan Selim Caddesi Aybike Sokak, Cihad İş Merkezi No:22/-3 Kağıthane-İstanbul-Türkiye Tel.: +90 212 264 38 77

http://mc.manuscriptcentral.com/tjh

Web page www.tjh.com.tr

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

Printing Date / Basım Tarihi 10.02.2015

Cover Picture

Güçhan Alanoğlu was born in 1959, Turkey. She is currently working in the Department of Hematology, Süleyman Demirel University Faculty of Medicine, Isparta, 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.

A-II

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 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, case reports, 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. 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 - 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: 0.340 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-ofcharge 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 Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh  Web page: www.tjh.com.tr  E-mail: info@tjh.com.tr

A-III

Permissions

Requests for permission to reproduce published material should be sent to the editorial office. Editor: Professor Dr. Aytemiz Gürgey  Adress: Ilkbahar Mah, Turan Günes Bulvarı, 613 Sok., No: 8, Çankaya, Ankara, Turkey  Telephone: +90 312 490 98 97  Fax: +90 312 490 98 68  Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh  Web page: www.tjh.com.tr  E-mail: info@tjh.com.tr Publisher Galenos Yayinevi Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul Telephone : 0212 621 99 25 Fax : 0212 621 99 27 info@galenos.com.tr

Instructions for Authors Instructions for authors are published in the journal and at www. tjh.com.tr

Material Disclaimer Authors are responsible for the manuscripts they publish in The Turkish Journal of Hematology. The editor, editorial board, and publisher do not accept any responsibility for published manuscripts. 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, case 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 single 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). Case reports require short unstructured abstracts. 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.

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. 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.

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.

A-IV

Other types of manuscripts, such as case reports, 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. 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.

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

Examples of References: 1. List all authors. Deeg HJ, Oâ&#x20AC;&#x2122;Donnel M, Tolar J. Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure immunosuppressive therapy. Blood 2006;108:1485-1491. 2.Organization as author Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in posthepatitis marrow aplasia. Lancet 1977;2:742-744. 3.Book Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger, 1961.

A-V

4. Book Chapter Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin: Genetics, Pathophysiology, Clinical Management. New York, Cambridge University Press, 2000. 5.Abstract Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract). 6.Letter to the Editor Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further definition of 20q deletion in myeloid leukemia using fluorescence in situ hybridization. Blood 1994;84:2821-2823. 7. Supplement Alter BP. 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.

Case Reports Abstract length: Not to exceed 100 words. Article length: Not to exceed 1200 words. Case Reports can include maximum 1 figure and 1 table or 2 figures or 2 tables.

Case reports should be structured as follows:

Abstract An unstructured abstract that summarizes the case. Introduction: A brief introduction (recommended length: 1-2 paragraphs). Case Presentation: This section describes the case in detail, including the initial diagnosis and outcome. Discussion:This section should include a brief review of the relevant literature and how the presented case furthers our understanding to the disease process.

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

A-VI

of magnification for histological slides. Explain the internal scale and identify the staining method used. Figures should be submitted as separate files, not in the text file. High-resolution image files are not preferred for initial submission as the file sizes may be too large. The total file size of the PDF for peer review should not exceed 5 MB.

Authorship Each author should have participated sufficiently in the work to assume public responsibility for the content. Any portion of a manuscript that is critical to its main conclusions must be the responsibility of at least 1 author.

Contributor’s Statement All submissions should contain a contributor’s statement page. Each manuscript should contain substantial contributions to idea and design, acquisition of data, or analysis and interpretation of findings. All persons designated as an author should qualify for authorship, and all those that qualify should be listed. Each author should have participated sufficiently in the work to take responsibility for appropriate portions of the text.

Acknowledgments Acknowledge support received from individuals, organizations, grants, corporations, and any other source. For work involving a biomedical product or potential product partially or wholly supported by corporate funding, a note stating, “This study was financially supported (in part) with funds provided by (company name) to (authors’ initials)”, must be included. Grant support, if received, needs to be stated and the specific granting institutions’ names and grant numbers provided when applicable. Authors are expected to disclose on the title page any commercial or other associations that might pose a conflict of interest in connection with the submitted manuscript. All funding sources that supported the work and the institutional and/or corporate affiliations of the authors should be acknowledged on the title page.

Ethics When reporting experiments conducted with humans indicate that the procedures were in accordance with ethical standards set forth by the committee that oversees human experimentation. Approval of research protocols by the relevant ethics committee, in accordance with international agreements (Helsinki Declaration of 1975, revised 2002 available at http://www.wma.net/e/policy/b3.htm, “Guide for the Care and use of Laboratory Animals” www.nap.edu/catalog/5140. html/), is required for all experimental, clinical, and drug studies. Patient names, initials, and hospital identification numbers should not be used. Manuscripts reporting the results of experimental investigations conducted with humans must state that the study protocol received institutional review board approval and that the participants provided informed consent. Non-compliance with scientific accuracy is not in accord with scientific ethics. Plagiarism: To re-publish-whole or in part-the contents of another author’s publication as one’s own without providing a reference. Fabrication: To publish data and findings/results that do not exist. Duplication: Use of data from another publication,

which includes re-publishing a manuscript in different languages. Salamisation: To create more than one publication by dividing the results of a study preternaturally. We disapprove of such unethical practices as plagiarism, fabrication, duplication, and salamisation, as well as efforts to influence the review process with such practices as gifting authorship, inappropriate acknowledgements, and references. Additionally, authors must respect participant right to privacy. On the other hand, short abstracts published in congress books that do not exceed 400 words and present data of preliminary research, and those that are presented in an electronic environment are not accepted pre-published work. Authors in such situation must declare this status on the first page of the manuscript and in the cover letter. (The COPE flowchart is available at: http://publicationethics.org) We use iThenticate to screen all submissions for plagiarism before publication. Turkish Journal of Hematology uses plagiarism screening service to verify the originality of content submitted before publication.

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

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

A-VII

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

Units of Measurement Measurements should be reported using the metric system, according to the International System of Units (SI). Consult the SI Unit Conversion Guide, New England Journal of Medicine Books, 1992. An extensive list of conversion factors can be found at http://www. unc.edu/~rowlett/units/scales/clinical_data.html. For more details, see http://www.amamanualofstyle.com/oso/public/jama/si_conversion_ table.html. Example for CBC.

Hematology component

SI units

RBC

6.7-11 x 1012/L

WBC

5.5-19.5 x109/L

Hemoglobin

116-168 g/L

PCV

0.31-0.46 L/L

MCV

39-53 fL

MCHC

300-360 g/L

MCH

19.5-25 pg

Platelets

300-700 x 109/L

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

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

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

Setting up an account New users to the submission site will need to register and enter their account details before they can submit a manuscript. Log in, or click the “Create Account” button if you are a first-time user. To create a

new account: After clicking the “Create Account” button, enter your name and e-mail address, and then click the “Next” button. Your e-mail address is very important. Enter your institution and address information, as appropriate, and then click the “Next” Button. Enter a user ID and password of your choice, select your area of expertise, and then click the “Finish” button. If you have an account, but have forgotten your log-in details, go to “Password Help” on the journal’s online submission system and enter your e-mail address. The system will send you an automatic user ID and a new temporary password. Full instructions and support are available on the site, and a user ID and password can be obtained during your first visit. Full support for authors is provided. Each page has a “Get Help Now” icon that connects directly to the online support system. Contact the journal administrator with any questions about submitting your manuscript to the journal (info@tjh.com.tr). For ScholarOne Manuscripts customer support, click on the “Get Help Now” link on the top right hand corner of every page on the site.

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

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

The Review Process Each manuscript submitted to The Turkish Journal of Hematology is subject to an initial review by the editorial office in order to determine if it is aligned with the journal’s aims and scope, and complies with essential requirements. Manuscripts sent for peer review will be assigned to one of the journal’s associate editors that has expertise relevant to the manuscript’s content. All manuscripts are single-blind peer reviewed. All accepted manuscripts are sent to a statistical and English language editor before publishing. Once papers have been reviewed, the reviewers’ comments are sent to the Editor, who will then make a preliminary decision on the paper. At this stage, based on the feedback from reviewers, manuscripts can be accepted, rejected, or

A-VIII

revisions can be recommended. Following initial peer-review, articles judged worthy of further consideration often require revision. Revised manuscripts generally must be received within 3 months of the date of the initial decision. Extensions must be requested from the Associate Editor at least 2 weeks before the 3-month revision deadline expires; The Turkish Journal of Hematology will reject manuscripts that are not received within the 3-month revision deadline. Manuscripts with extensive revision recommendations will be sent for further review (usually by the same reviewers) upon their re-submission. When a manuscript is finally accepted for publication, the Technical Editor undertakes a final edit and a marked-up copy will be e-mailed to the corresponding author for review and to make any final adjustments.

Submission of Revised Papers When revising a manuscript based on the reviewers’ and Editor’s feedback, please insert all changed text in red. Please do not use track changes, as this feature can make reading difficult. To submit revised manuscripts, please log into your author center at ScholarOne Manuscripts. Your manuscript will be stored under “Manuscripts with Decisions”. Please click on the “Create a Revision” link located to the right of the manuscript title. A revised manuscript number will be created for you; you will then need to click on the “Continue Submission” button. You will then be guided through a submission process very similar to that for new manuscripts. You will be able to amend any details you wish. At stage 6 (“File Upload”), please delete the file for your original manuscript and upload the revised version. Additionally, please upload an anonymous cover letter, preferably in table format, including a point-by-point response to the reviews’ revision recommendations. You will then need to review your paper as a PDF and click the “Submit” button. Your revised manuscript will have the same ID number as the original version, but with the addition of an R and a number at the end, for example, TJH-2011-0001 for an original and TJH-2011-0001.R1, indicating a first revision; subsequent revisions will end with R2, R3, and so on. Please do not submit a revised manuscript as a new paper, as revised manuscripts are processed differently. If you click on the “Create a Revision” button and receive a message stating that the revision option has expired, please contact the Editorial Assistant at info@tjh.com.tr to reactivate the option.

English Language Editing All manuscripts are professionally edited by an English language editor prior to publication.

Online Early The Turkish Journal of Hematology publishes abstracts of accepted manuscripts online in advance of their publication in print. Once an accepted manuscript has been edited, the authors have submitted any final corrections, and all changes have been incorporated, the manuscript will be published online. At that time the manuscript will receive a Digital Object Identifier (DOI) number. Both forms can be found at www.tjh.com.tr. Authors of accepted manuscripts will receive electronic page proofs directly from the printer, and are responsible for proofreading and checking the entire manuscript, including tables, figures, and references. Page proofs must be returned within 48 hours to avoid delays in publication.

CONTENTS 1

Review Article

Research Articles

Iron-Refractory Iron Deficiency Anemia Ebru Yılmaz Keskin, İdil Yenicesu

Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients Rabab M. Aly, Ansaf B. Yousef

Are the High Serum Interleukin-6 and Vascular Endothelial Growth Factor Levels Useful Prognostic Markers in Aggressive Non-Hodgkin Lymphoma Patients? Hava Üsküdar Teke, Eren Gündüz, Olga Meltem Akay, Cengiz Bal, Zafer Gülbaş

Prevalence of Monoclonal B Lymphocytosis in First-Degree Relatives of Chronic Lymphocytic Leukaemia Patients in Turkey Taner Demirci, Zeynep Arzu Yeğin, Nevruz Kurşunoğlu, Zeynep Yılmaz, Elif Suyanı, Zübeyde Nur Özkurt, Münci Yağcı

Myeloid Sarcomas: A Clinicopathologic Study of 20 Cases Gülşah Kaygusuz, Duygu Kankaya, Cemil Ekinci, Pervin Topçuoğlu, Işınsu Kuzu

Osteoporosis and Osteopathy Markers in Patients with Mastocytosis Nilüfer Alpay Kanıtez, Burak Erer, Öner Doğan, Nesimi Büyükbabani, Can Baykal, Dilşad Sindel, Refik Tanakol, Akif Selim Yavuz

Tunnelled Central Venous Catheter-Related Problems in the Early Phase of Haematopoietic Stem Cell Transplantation and Effects on Transplant Outcome Mahmut Yeral, Can Boğa, Levent Oğuzkurt, Hikmet Eda Alışkan, Hakan Özdoğu, Yusuf Ziya Demiroğlu

Brief Report

Growth Arrest-Specific 6 (Gas6) and TAM Receptors in Mouse Platelets Fikriye Uras, Burhanettin Küçük, Özlem Bingöl Özakpınar, Ahmet Muzaffer Demir

Case Reports

Acute Megakaryoblastic Leukemia with t(1;22) Mimicking Neuroblastoma in an Infant Müge Gökçe, Selin Aytaç, Şule Ünal, İlhan Altan, Fatma Gümrük, Mualla Çetin

Dasatinib-Related Chylothorax Yen-Min Huang, Cheng-Hsu Wang, Jen-Seng Huang, Kun-Yun Yeh, Chien-Hong Lai, Tsung-Han Wu, Pei-Hung Chang, Pei-Hung Chang, Yueh-Shih Chang, Yii-Jenq Lan

Successful Off-Label Use of Recombinant Factor VIIa and Coil Embolization in an Adolescent with Massive Hemoptysis Due to Invasive Pulmonary Aspergillosis Dilek Gürlek Gökçebay, Ali Fettah, İsmail Kırbaş, Bahattin Tunç, Namık Yaşar Özbek

Trisomy Chromosome 6 as a Sole Cytogenetic Abnormality in Acute Myeloid Leukemia Monika Gupta, Nita Radhakrishnan, Manoranjan Mahapatra, Renu Saxena

A-IX

Letters to the Editor

Recurrent Monosomies Confirmed by Interphase FISH in Three Chronic Myeloid Leukemia Cases Yelda Tarkan Argüden, Dilhan Kuru, Ayşe Çırakoğlu, Şükriye Yılmaz, Şeniz Öngören Aydın, Cem Muhlis Ar, Ayhan Deviren, Teoman Soysal, Seniha Hacıhanefioğlu

An Unusual Presentation of Metastatic Breast Carcinoma as Cold Autoimmune Hemolytic Anemia Anand Chellappan, Chanaveerappa Bammigatti, Swaminathan Palamalai

A Child with Psoriasis, Hypogammaglobulinemia, and Monosomy 7-Positive Myelodysplastic Syndrome Namık Özbek, Arzu Yazal Erdem, Özlem Arman Bilir, Fatma Karaca Kara, Mutlu Yüksek, Neşe Yaralı, Meltem Özgüner, Nazmiye Yüksek, Bahattin Tunç

Clinical Picture in Hematology Şinasi Özsoylu

About Chediak-Higashi, Hemoglobin Lansing, and Hemoglobin Jabalpur Şinasi Özsoylu

About Microangiopathic Hemolytic Anemia Şinasi Özsoylu

Oral Lesions and Hematology İrfan Yavaşoğlu

Follicular Lymphoma İrfan Yavaşoğlu

Images in Hematology

Rapid Progression of Blastic Plasmacytoid Dendritic Cell Neoplasm without Extracutaneous Manifestation Guohua Yu, Xin Huang, Yuqing Huo, Tingguo Zhang, Zifen Gao

Post-Partum Ovarian Vein Thrombosis: Combined Effect of Infection and Factor V Leiden Mutation H. El Farran, A. G. Haddad, A. H. Radwan, A. H. Nassar, R. Hourani, Ali T. Taher

Rhinocerebral Mucormycosis in a Patient with Acute Promyelocytic Leukemia Muzaffer Keklik, Afra Yıldırım, Fahir Öztürk, İbrahim İleri,  Gülşah Akyol, Mustafa Çetin, Bülent Eser

A-X

Review Article

DOI: 10.4274/tjh.2014.0288

Iron-Refractory Iron Deficiency Anemia Demir Tedavisine Dirençli Demir Eksikliği Anemisi Ebru Yılmaz Keskin1, İdil Yenicesu2 1Samsun 2Gazi

Education and Research Hospital, Clinic of Pediatric Hematology and Oncology, Samsun, Turkey University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey

Abstract: Iron is essential for life because it is indispensable for several biological reactions, such as oxygen transport, DNA synthesis, and cell proliferation. Over the past few years, our understanding of iron metabolism and its regulation has changed dramatically. New disorders of iron metabolism have emerged, and the role of iron as a cofactor in other disorders has begun to be recognized. The study of genetic conditions such as hemochromatosis and iron-refractory iron deficiency anemia (IRIDA) has provided crucial insights into the molecular mechanisms controlling iron homeostasis. In the future, these advances may be exploited to improve treatment of both genetic and acquired iron disorders. IRIDA is caused by mutations in TMPRSS6, the gene encoding matriptase-2, which downregulates hepcidin expression under conditions of iron deficiency. The typical features of this disorder are hypochromic, microcytic anemia with a very low mean corpuscular volume of erythrocytes, low transferrin saturation, no (or inadequate) response to oral iron, and only a partial response to parenteral iron. In contrast to classic iron deficiency anemia, serum ferritin levels are usually low-normal, and serum or urinary hepcidin levels are inappropriately high for the degree of anemia. Although the number of cases reported thus far in the literature does not exceed 100, this disorder is considered the most common of the “atypical” microcytic anemias. The aim of this review is to share the current knowledge on IRIDA and increase awareness in this field. Key Words: Iron deficiency, TMPRSS6, Matriptase-2, Hepcidin

Özet: Demir, oksijenin taşınması, DNA sentezi ve hücre çoğalması gibi çeşitli biyolojik reaksiyonlar için vazgeçilmez olduğundan, yaşam için zorunludur. Demir metabolizması ve bu elementin düzenlenmesiyle ilgili bilgilerimiz, son yıllarda belirgin şekilde değişmiştir. Demir metabolizması ile ilgili yeni bozukluklar tanımlanmış ve demirin başka bozuklukların kofaktörü olduğu anlaşılmaya başlamıştır. Hemokromatozis ve demir tedavisine dirençli demir eksikliği anemisi (IRIDA; “iron-refractory iron deficiency anemia”) gibi genetik durumlar üzerinde yapılan çalışmalar, vücuttaki demir dengesini kontrol eden moleküler mekanizmalar ile ilgili önemli ipuçları sunmuştur. Bu ilerlemeler, gelecekte, hem genetik hem de kazanılmış demir bozukluklarının daha etkili şekilde tedavi edilmesi amacıyla kullanılabilir. IRIDA, demir eksikliği ile giden durumlarda, hepsidin üretimini baskılayan matriptaz-2’yi kodlayan TMPRSS6 genindeki mutasyonlardan kaynaklanmaktadır. Hastalığın tipik özellikleri, hipokrom, mikrositer anemi, çok düşük ortalama eritrosit hacmi, oral demir tedavisine yanıtsızlık (veya yetersiz yanıt) ve parenteral demire kısmi yanıttır. Klasik demir eksikliği anemisinin aksine, serum ferritin değeri genellikle hafif düşük ya da normal aralıkta; serum ve idrar hepsidin değerleri ise, aneminin derecesi ile orantısız şekilde yüksek bulunur. Şimdiye kadar literatürde bildirilmiş olguların sayısı 100’ü geçmediği halde, IRIDA’nın, “atipik” mikrositik anemilerin en sık nedeni olduğu düşünülmektedir. Bu derlemenin amacı, IRIDA hakkındaki güncel bilgileri araştırıcılar ile paylaşmak ve bu alandaki farkındalıklarını arttırmaktır. Anahtar Sözcükler: Demir eksikliği, TMPRSS6, Matriptaz-2, Hepsidin

Address for Correspondence: Ebru YILMAZ KESKİN, M.D., Samsun Education and Research Hospital, Clinic of Pediatric Hematology and Oncology, Samsun, Turkey Phone: +90 505 558 36 11 E-mail: ebruyilmaz81@hotmail.com Received/Geliş tarihi : July 11, 2014 Accepted/Kabul tarihi : November 12, 2014

Turk J Hematol 2015;32:1-14

Keskin Y覺lmaz E, et al: Iron-Refractory Iron Deficiency Anemia

Introduction

Pathophysiology

Iron deficiency anemia (IDA) has been described for centuries. As a very frequent disorder, IDA constitutes a serious public health problem. It usually develops due to low intake of dietary iron; in the presence of hypochromic, microcytic anemia, it is the first underlying cause to be considered. However, some inherited conditions with variable clinical characteristics may also result in microcytic anemia by causing defective iron metabolism. Iron-refractory iron deficiency anemia (IRIDA; OMIM #206200), which was described only recently, is one such disorder [1].

Iron-refractory iron deficiency anemia develops due to mutations in TMPRSS6, which is the gene encoding matriptase-2. Matriptase-2 is a type II transmembrane serine protease expressed mainly in hepatocytes (Figure 1) [1,3,29]. It plays a role in downregulating hepcidin expression in liver cells. TMPRSS6-mutant mice were found to have microcytic anemia and elevated hepcidin levels disproportionate to the degree of anemia [30].

Iron-refractory iron deficiency anemi develops due to mutations in TMPRSS6, the gene encoding matriptase-2. Its typical mode of inheritance is autosomal recessive, and IRIDA is characterized clinically by hypochromic, microcytic anemia with an inadequate response to oral iron and an only partial response to parenteral iron [1,2]. To date, over 50 TMPRSS6 mutations in individuals with the IRIDA phenotype have been reported [1,3,4,5,6,7,8,9, 10,11,12,13,14,15,16,17,18,19,20,21]. The mostly normal growth and development of IRIDA patients and their almost normal hematologic findings in adulthood prevent the precise determination of IRIDA frequency. However, IRIDA is considered the most frequent disorder among both congenital iron metabolism disorders and atypical microcytic anemias [22]. We think that this review will increase awareness of this issue and facilitate recognition of cases of this autosomal recessive inherited disorder. History Although the genetic basis of IRIDA was elucidated only recently, the disorder was first described clinically in the early 1980s [1,23]. Buchanan et al. reported IDA in 3 siblings who did not respond to oral ferrous sulfate therapy. Additionally, there was no history of poor dietary iron intake or gastrointestinal blood loss. None of the 3 children had any clinical or laboratory findings suggestive of a generalized malabsorption disorder. Notably, intramuscular iron dextran injection resulted in an only partial hematologic response; however, serum ferritin levels increased, indicating a rise in iron stores, although serum iron levels remained low. The authors suggested that the specific iron absorption disorder may partially explain the phenotype, and an additional disorder in iron utilization may result in the partial response to intramuscular iron. The occurrence of the disorder in the 3 siblings pointed toward the hereditary nature of the disease. Cases reported subsequently supported this observation [24,25,26,27,28]. However, 27 years passed before the genetic basis of the disease was finally elucidated.

Similar to the findings in TMPRSS6-mutant mice, the hepcidin levels in serum, plasma, and urine samples of IRIDA patients were found to be either within or above the normal ranges of those in healthy adults [1,3,4]. In classic IDA, hepcidin levels decrease markedly to promote intestinal iron absorption [31,32]. Thus, normal-to-elevated hepcidin levels observed in IRIDA patients reflect the inability to appropriately regulate this protein. Hepcidin, the master regulator of iron metabolism, is a small peptide synthesized in the liver. In cases of iron loading, hepcidin expression is induced to inhibit intestinal iron uptake, whereas iron deficiency results in reduced hepcidin expression to increase the availability of iron for erythropoiesis [33,34]. Hepcidin exerts its iron regulatory effects by binding to ferroportin (a cellular iron exporter expressed on the basolateral membrane of enterocytes) on the plasma membrane of macrophages and in hepatocytes. Ferroportin is the only known cellular iron exporter. Because hepcidin binding leads to the internalization and degradation of ferroportin in lysosomes, dietary iron absorption and mobilization of iron from macrophage stores are decreased if hepcidin levels are elevated [35,36,37,38]. Therefore, the decrease in ferroportin expression through hepcidin can explain the development of iron deficiency and unresponsiveness to oral iron in IRIDA cases. Before it can be utilized in erythropoiesis, parenteral iron administered in the form of iron-carbohydrate complexes must be processed and exported by macrophages. This export step is also ferroportin-dependent. Therefore, inappropriately high hepcidin levels can additionally explain the sluggish and incomplete response to parenteral iron observed in IRIDA cases. In a cell-model study, matriptase-2 was found to regulate hepcidin expression by cleaving hemojuvelin (HJV), a protein found on the plasma membrane of hepatocytes, to promote the expression of hamp, the gene encoding hepcidin. Therefore, the final effect of matriptase-2 is attenuation of hepcidin activation (Figure 2) [39]. HJV promotes hepcidin expression through a pathway involving bone morphogenetivc proteins (BMPs). Following the interaction of BMPs with their specific receptors on the cell surface, sons of mothers against decapentaplegic (SMAD) proteins become phosphorylated owing to the serine/threonine activity of BMP receptors.

Keskin Y覺lmaz E, et al: Iron-Refractory Iron Deficiency Anemia

SMAD proteins are a class of intracellular signaling molecules. Following phosphorylation, SMAD proteins bind to SMAD4, a common mediator. The heteromeric complexes formed translocate to the nucleus and regulate the transcription of target genes, among which is hamp [40,41]. Therefore, loss of matriptase-2 activity caused by TMPRSS6 mutations results in hepcidin overexpression. In healthy fetuses and neonates, because of the rapid growth and expansion of the red cell compartment, hepcidin gene expression is drastically repressed [42,43]. However, the role of matriptase-2 in this repression was not elucidated until recently. In their very recent study, Willemetz et al. found that in TMPRSS6-/- fetuses, liver Hamp1 mRNA expression was up to 60 times higher than that in control mice, in which hepcidin expression was only barely detectable [44]. It was noteworthy that TMPRSS6-/- fetuses and newborns had lower iron content, mean corpuscular erythrocyte volume (MCV), and hemoglobin (Hb), indicating microcytic anemia secondary to iron deficiency in mutant mice. However, the red blood cell (RBC) levels were unaffected. These observations suggest that the requirement of matriptase-2 for hepcidin suppression begins in utero, and its deficiency leads to microcytic anemia. Despite the laboratory findings of iron deficiency in individuals with TMPRSS6 polymorphisms or mutations suggested to be mediated by the inability to suppress hepcidin expression appropriately, 2 population-based studies have found that the affected iron and erythrocyte parameters are at least partially independent of hepcidin levels [45,46]. Similarly, Lehmberg et al. reported that the urinary hepcidin levels of patients with the IRIDA phenotype and TMPRSS6 mutations were either below the normal range or undetectable [19]. These observations suggest that, in the presence of TMPRSS6 mutations, underlying mechanisms other than the inability to downregulate hepcidin expression may be responsible for the laboratory findings of iron deficiency. Studies to elucidate those mechanisms are underway.

Figure 1. Schematic representation of matriptase-2, encoded by the TMPRSS6 gene. N: amino-terminus, C: carboxyterminus, TM: transmembrane domain; SEA: sea urchin sperm protein, enteropeptidase agrin, CUB: complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenic protein 1 domain, L: low density lipoprotein receptor class A domain (LDLR), Serine Protease: serine protease domain, red triangle: cleavage activation site.

Turk J Hematol 2015;32:1-14

Clinical Presentation Iron-refractory iron deficiency anemia can present with various clinical and laboratory characteristics. Similarly, there is a discrepancy in the response to treatment among patients. However, individuals with IRIDA are usually diagnosed during childhood. Patients most commonly present with mild to moderate anemia, and their growth and development are normal. The key features of the disease are: 1) congenital hypochromic, microcytic anemia; 2) very low MCV (patients present with marked microcytosis and hypochromia that are disproportionate to the degree of anemia); 3) low transferrin saturation; 4) abnormal iron absorption; 5) defective iron utilization (as evidenced by sluggish and incomplete response to parenteral iron); and 6) an inheritance mode compatible with autosomal recessive transmission [1]. When laboratory findings of the IRIDA cases reported to date are reviewed, it can be noted that anemia was detected in the first blood count testing performed in most of the individuals (children); this testing usually occurred before the age of 2 years. Because the subjects are usually healthy children with normal growth and development, blood count testing was typically not ordered due to a specific indication but was rather performed as a part of routine screening. The recent findings of Willemetz et al. in TMPRSS6 knockout mice may shed light on the beginning of laboratory findings associated with IRIDA. They suggest that (mild) microcytic anemia may actually be present in utero and at birth in IRIDA cases [44]. Recently, de Falco et al. reported the findings of a Turkish female infant who had a molecular diagnosis of IRIDA (testing

Figure 2. Schematic model of hepcidin regulation by matriptase-2. BMP: bone morphogenetic protein, BMPR: BMP receptor, HJV: hemojuvelin, R-SMAD: receptor-associated Son of Mothers against Decapentaplegic proteins. In conditions of iron deficiency, matriptase-2 modulates hepcidin signaling by cleaving HJV from the hepatocyte plasma membrane, resulting in decreased hepcidin production. 3

Turk J Hematol 2015;32:1-14

Keskin Y覺lmaz E, et al: Iron-Refractory Iron Deficiency Anemia

Table 1. Literature review of iron-refractory iron deficiency anemia cases with homozygous or compound heterozygous TMPRSS6 mutations. Reference

Case Mutation 1; Number# Mutation 2

Ethnic Origin

Age Hb MCV (years) (g/L) (fL)

TSI (%)

Ferritin Hepcidin Treatment (ng/mL) (nmol/L)+

Finberg et al.,

Turkish

461*

1828*

Northern

1.1

European

25.0

4055*

23.0

3329*

Northern

European

113*

p.Lys636fs; p.Lys636fs

2008 [1]

Finberg et al.,

p.Ala605fs; p.Glu527fs

2008 [1]

Finberg et al.,

p.Gly442Arg; p.Asp521Asn

2008 [1] Finberg et al.,

p.Asp622fs; p.Asp622fs

Nigerian

p.Tyr355X; p.Glu461fs

African

1.3

English

<5%

25.1

oral/iv Fe

Sardinian

100

17.1

oral Fe?/iv Fe

234

57.8

oral Fe/iv Fe

100

129

oral Fe?/iv Fe

127

184

25.4

oral Fe?/iv Fe

139

49.1

oral Fe?/iv Fe

123

5069*

oral/iv Fe/EPO

119

388

5350*

oral/iv Fe

2008 [1] Finberg et al., 2008 [1] Guillem et al.,

American 1

2008 [4] Melis et al.,

p.Tyr393X; p.Arg599X

p.Ser288fs; p.Ser288fs

2008 [3]

Ramsay et al.,

p.Ala118Asp; p.Pro686fs

Spanish

2009 [5] Silvestri et al.,

p.Asp521As; p.Glu522Lys

French

0.8

100

158

oral/iv Fe

c.2278- 1G> C (splicing);

Indian

29.1

oral/iv Fe

Swiss

3.0

12.2

oral/iv Fe

2009 [6] Edison et al., 2009 [7] Tchou et al.,

c.2278- 1G> C (splicing) 1

2009 [8] Tchou et al.,

p.Ser304Leu; g.29139_ 30192 del 1054

p.Ser623Thr; p.Ser623Thr

Italian

9.8

oral Fe

p.Asp521Asn; p.Glu522Lys

100

347

443

iv Fe

p.Leu235Pro; p.Tyr418Cys

oral Fe

0.6

115

oral Fe

iv Fe

2009 [8] Kannengiesser et al., 2009 [9] Kannengiesser et al., 2009 [9] Kannengiesser et al., 2009 [9]

p.Glu114Lys; p.Pro765Ala

Turk J Hematol 2015;32:1-14

Keskin Y覺lmaz E, et al: Iron-Refractory Iron Deficiency Anemia

Continued

Table 1. Reference

Case Mutation 1; number# Mutation 2

Ethnic Origin

Age (years)

Hb (g/L)

MCV (fL)

TSI (%)

Ferritin (ng/mL)

Hepcidin (nmol/L)+

Treatment

Kannengiesser

p.Ala605Pro fsX8;

230

4288

iv Fe

p.Ala605Pro fsX8

iv Fe

p.Glu527fs; p.Leu674Phe

Belgian

1.8

829.8*

oral Fe

0.8

1222.4*

oral Fe

et al., 2009 [9] Beutler et al.,

2010 [12] Beutler et al.,

p.Leu166X; p.Leu166X

Dutch

6292.5*

iv Fe

p.Tyr141Cys; p.Tyr141Cys

Lebanese

3.3

200*

oral Fe

p.Tyr141Cys; p.Tyr141Cys

Indian

3.7

9.8

oral/iv Fe

p.Ile212Thr; p.Arg271Gln

Italian

3.7

5.6

oral/iv Fe

p.Ser304Leu; p.Ser304Leu

Arab

106

112

17.8

oral/iv Fe

105

9.4

8.9

oral/iv Fe

6.2

7.6

oral/iv Fe

2010 [12] Altamura et al., 2010 [11] De Falco et al., 2010 [10] De Falco et al., 2010 [10] De Falco et al., 2010 [10]

De Falco et al.,

p.Leu166fs; p.Gln229fs15

Australian

2.3

5.8

oral/iv Fe

p.Trp247fs; p.Trp247fs

Greek

4.2

oral/iv Fe

10.4

oral/iv Fe

3.1

oral/iv Fe

3.3

101

oral/iv Fe

2010 [10] De Falco et al., 2010 [10] De Falco, 2010

De Falco et al.,

p.Ser561X; p.Ser561X

Arab

p.Cys510Ser; p.Ser570fs

Algerian

105

228

oral/iv Fe

p.Cys702Phe; p.Val795Ile

Dutch

12.9

oral/iv Fe

p.Gly603Arg; p.Gly603Arg

Portuguese

45.5

oral Fe

p.Ser288fs; p.Ser288fs

Sardinian

0.4

102

oral

2010 [10] Cuijpers et al., 2010 [13] Palare et al., 2010 [14] Cau et al., 2012 [54]

Fe+ascorbic acid/iv Fe

Choi et al., 2012 1

p.Gly603Arg;

[15]

c.658+1G>T (splicing)

Guillem et al.,

2012 [16] Guillem et al.,

p.Ala605Pro fsX8;

Korean

101

3.5

oral/iv Fe

Algerian

230

1.54

oral/iv Fe

0.02

oral Fe

0.5

115

0.03

oral Fe

101

0.02

oral/iv Fe

p.Ala605Pro fsX8 2

p.Leu235Pro; p.Tyr418Cys

French

2012 [16] Guillem et al.,

p.Glu114Lys; p.Pro765Ala

French

2012 [16]

Turk J Hematol 2015;32:1-14

Keskin Y覺lmaz E, et al: Iron-Refractory Iron Deficiency Anemia

Continued

Table 1. Reference

Case Mutation 1; number# Mutation 2

Ethnic Origin

Age (years)

Hb (g/L)

MCV (fL)

TSI (%)

Ferritin Hepcidin (ng/ (nmol/L)+ mL)

Treatment

Pellegrino et al.,

Italian

oral/iv Fe

p.Gly442Arg;

French

348

51.1

oral Fe

p.Glu522Lys

Canadian

195

71.2

oral Fe

p.Lys636fs; p.

Turkish

0.5

oral/iv Fe

Turkish

0.6

0.03

oral/iv Fe/EPO

2012 [17]

p.Ser304Leu; c.1001-11_10013del (splicing)

Khuong-Quang

et al., 2013 [20] Lehmberg et al.,

2013 [19] Lehmberg et al.,

Lys636fs 1

2013 [19]

p.Lys636fs; p. Lys636fs

mg/dL creatinine

Lehmberg et al.,

2013 [19] Lehmberg et al.,

2 3

et al., 2013 [21]

German

p.Gln571Glnfs;

Turkish

p.Gln571Glnfs 2

et al., 2013 [21] Y覺lmaz-Keskin

p.Ser561Ter; p. Trp590Ter

et al., 2013 [21] Y覺lmaz-Keskin

Turkish

0.8

1.9

p. Lys636fs

2013 [19] Y覺lmaz-Keskin

p.Lys636fs;

p.Arg599X;

Turkish

p. Arg599X 1

IVS10 +1 G>A;

Turkish

0.37 mg/dL

oral Fe/iv Fe/

creatinine

EPO

1.4

undetectable

oral Fe

1.4

oral Fe

7.6

2.36

oral/iv Fe

5.8

<2%

7.40

oral/iv Fe

0.7

7.76

oral?/iv Fe

10.2

3.60

oral Fe

7.3

<2%

15.88

oral Fe

3.5

<2%

2.77

oral Fe/iv Fe/

p.Cys510>Arg

oral Fe+ascorbic acid

Notes. TSI: transferin saturation index, np: not present, iv: intravenous, EPO: erythropoietin. # : number of cases reported with the same genotype (within the same kindred) in the study. +: hepcidin is a non-standardized parameter, and results can therefore only be compared within a study using the same assay. *: hepcidin in urine (ng/mg creatinine).

was ordered because of a family history of IRIDA) at the age of 3 months before she developed an overt IRIDA phenotype [47]. The physical examination findings of the infant were normal at birth, with the birth weight being appropriate for the gestational age. The follow-up data of the same infant were later reported in another study, which stated that findings of a typical IRIDA phenotype were obvious when she was 4 months old [21]. In the evaluation of the cases regarding treatment, a temporary rise in serum ferritin was observed after (parenteral) iron therapy [3,4,8,21,48]. Once administered, intravenous (i.v.) iron in a colloidal form (iron gluconate) enters reticuloendothelial cells [49,50]. Following sequestration of iron in these cells (as evidenced by an increase in the serum ferritin level), a portion of the iron probably reaches plasma transferrin despite high hepcidin levels and can be utilized in

erythropoiesis; this results in partial correction of anemia and a slight increase in MCV. Indeed, the uptake of colloidal iron by reticuloendothelial cells results in an increase in the serum ferritin level; however, upon binding of iron to transferrin in the plasma, both the iron load in macrophages and the serum ferritin concentration gradually decrease again. In their study including 11 children with IRIDA, Akin et al. reported their findings following i.v. iron administration [51]. The Hb and serum ferritin levels of the patients increased to 9.5 g/dL and 24 ng/mL, respectively, at 6 weeks after the first therapy. Although the level of Hb was steady, ferritin levels continued to increase up to 30 ng/mL and 47 ng/mL at 6 months after the first week and 6 weeks after the second therapy, respectively. Thus, the authors suggested that i.v. iron should be administered only once in IRIDA cases because its continued administration would be of no benefit

Finberg et al., 2008 [1]

Kannengiesser et al., 2009 [9]

Beutler et al., 2010 [12]

Palare et al., 2010 [14]

Pellegrino et al., 2012 [17]

Jaspers et al., 2013 [18]

114

106

Portuguese 9

105 np

106

103

np np

1.4

Age Hb (years) (g/L)

Portuguese 5

Dutch

African American

Nigerian

Ethnic Origin

78.9

77 np

MCV (fL)

np np

TSI (%)

116

121

19.6

42 np

Ferritin (ng/mL)

TSI/hepcidin: 1.6%/nM TSI/hepcidin: 1.14%/nM

1627* 797.5*

Hepcidin (nmol/L)+

oral/iv Fe

none

oral/iv Fe

oral Fe

oral Fe none

iv Fe

Treatment

Notes: Although IRIDA typically displays autosomal recessive mode of inheritance, the few cases with IRIDA phenotype reported to harbor only a single pathogenic TMPRSS6 mutation might reflect the possible effect of environmental factors, modulating polymorphisms, a low-expressing allele, or involvement of defects in other genes . Besides, the current Sanger sequencing method misses certain defects in the exons or introns of the gene or its regulatory regions [59]. TSI: transferin saturation index, np: not present, iv: intravenous. # : number of cases reported with the same genotype (within the same kindred) in the study. +: hepcidin is a non-standardized parameter, and results can therefore only be compared within a study using the same assay. *: hepcidin in urine (ng/mg creatinine). §: non-pathogenic mutation.

p.His369Asn

c.1869- 21C>G (splicing)

p.Asp521Asn

p.Leu166X; p.Arg446Trp§

p.Arg112Leu

c.1369 + 4 A>T (splicing)

p.Arg774Cys

p.Gly713fs

Case Mutation Number#

Reference

Table 2. Literature review of iron-refractory iron deficiency anemia cases with a heterozygous TMPRSS6 mutation identified.

Keskin Yılmaz E, et al: Iron-Refractory Iron Deficiency Anemia Turk J Hematol 2015;32:1-14

Table 3. Main characteristics of rare microcytic anemias secondary to inherited disorders of iron metabolism or heme synthesis.

IRIDA (TMPRSS6)

Disorder (involved gene)

Variable

Skin symptoms

Anemia

symptoms

Neurologic

Child

Age at

presentation

AR#

Inheritance

described (n)

Aceruloplasminemia (CP) Mild

Yes

40-50 years

AR/AD

20-100

Hypotransferrinemia (TF)

20-100

Variable

5-20

DMT1 deficiency (DMT1)

Patients

Variable

Child

5-20

Sideroblastic anemia (STEAP3) Variable

No+

Child

AR/AD

STEAP3

Severe

Child

20-100

SLC25A38

Sideroblastic anemia (SLC25A38)

DMT1

Mild

anemia-mild†

Variable

Yes

Childd

>100

ALAS2

XLc

5-20

ABCB7

XLSA with ataxia (ABCB7)

Transferrin

XLSA (ALAS2)

Mild

Adult

GLRX5

Sideroblastic anemia (GLRX5)

Matriptase-2

mild

No anemia-

Yes

child

Newborn/

Mostly AD

>100

FECH

EPP (FECH)

protein

hemolytic

Severe-

Yes

Newborn

GATA1

XLCEP (GATA1)

Involved

Turk J Hematol 2015;32:1-14

Keskin Yılmaz E, et al: Iron-Refractory Iron Deficiency Anemia

Turk J Hematol 2015;32:1-14

Unresponsiveness to oral iron therapy is considered one of the hallmarks of IRIDA; however, some recent studies have reported the correction (at least partially) of hematological parameters after prolonged and/or high-dose oral iron therapy [12,16,20,21,53]. With high-dose oral iron (6-10 mg/kg/day elemental iron) for up to 17 months, acceptable Hb levels were achieved in some of these cases, although microcytosis, hypoferremia, and low transferrin saturation persisted in most of the cases. Recently, a 5-month-old Sardinian female infant with a homozygous TMPRSS6 mutation who was unresponsive to oral iron and partially responsive to i.v. iron displayed a marked increase in the Hb level (up to 121 g/L) following the use of the combination of oral iron and ascorbic acid for 3 months [54]. If confirmed in more patients, this combination treatment may offer an alternative in the treatment of patients with IRIDA and simplify their management.

a: iron loading may be secondary to erythrocyte transfusions. b: measurement after erythrocyte transfusions. c: in some families, only affected women were reported, because the defect is lethal in men. d: neurologic symptoms manifest in childhood, however, anemia may develop later in young adolescence. Modified from [59].

No Iron loading

in terms of increasing Hb levels. Additionally, in their study, Khuong-Quang et al. reported 2 siblings who presented with very high ferritin levels at admission in the absence of iron treatment [20]. These observations point to the variability of the genotype-phenotype correlations in IRIDA cases [52].

â&#x20AC; : anemia resolves by pyridoxine treatment in most XLSA patients.

No Yes Variable No Yes Possiblea Variable Yes Yes

No No No sideroblasts

Ring

Notes: DMT1: divalent metal transporter 1, XLSA: X-linked sideroblastic anemia, EPP: erythropoietic protoporphyria, XLCEP: X-linked congenital erythropoietic porphyria, AD: autosomal dominant, AR: autosomal recessive, XL: X-linked, TSI: transferin saturation index. #: AD mode of inheritance also described. *: hepcidin/TSI ratio is greater than the upper limit of the reference range in the absence of inflammation. +: gonadal dysfunction.

No Yes Yes Yes Yes Yes No

Yes

No data No data No data No data No data Normal-elevatedb Low-normal Low High* Hepcidin

No data

Variable High High Low-normal Ferritin

high

Normal High Higha Variable

No data

Normal Low-normal High

Normal Low-normal <10% TSI

Table 3.

Low-normal

100%

Normal High High High

Normal-

High

Continued

Keskin YÄąlmaz E, et al: Iron-Refractory Iron Deficiency Anemia

Because of the paucity of IRIDA cases reported to date, data concerning the clinical course and long-term followup of these individuals are limited. Nevertheless, it has been observed that the low Hb levels in early childhood increased to acceptable values in adulthood in those few cases that could be followed [3]. However, some of the laboratory findings indicating iron deficiency (low MCV, mean corpuscular Hb [MCH], serum iron, and transferrin saturation) had persisted. Notably, the ferritin levels of these patients tended to increase with age. Because iron is needed during childhood for body growth, particularly for red cell mass expansion, the less severe anemia phenotype in these individuals in adulthood was explained by the consequence of the greater availability of the limited amount of dietary iron for erythropoiesis. Genetics IRIDA was first associated with a genetic locus on the long arm of chromosome 22 (22q12.3-13.2) in the Sardinian family members reported by Melis et al. [3]. In the affected individuals, the disorder could be attributed to a mutation in the homozygous state arising in a common ancestor. Subsequently, other families with the IRIDA phenotype were evaluated and a recessive mode of inheritance arising from mutations in the same genetic locus was confirmed [1]. Included within the critical chromosome 22 region was the gene TMPRSS6, which encodes matriptase-2, a protein belonging to the type II transmembrane serine protease family. This group contains a short cytoplasmic amino terminal tail, a transmembrane region, a stem region with several structural domains, and a carboxy-terminus serine protease domain (Figure 1) [29,55].

Turk J Hematol 2015;32:1-14

IRIDA can be found in individuals from a range of ethnic backgrounds; to date, there is no evidence for a significant founder effect. All of the reported mutations are predicted to cause functional loss in the encoded protein. Most of them are missense mutations; however, frameshift, intronic, and nonsense mutations, as well as one large in-frame deletion, have also been reported (Tables 1 and 2) [1,3,4,5,6,7,8,9, 10,11,12,13,14,15,16,17,18,19,20,21]. The mutations are spread throughout the gene sequence, and they disrupt not only the serine protease catalytic activity but also other domains participating in protein-protein interactions [1,3,4,5,6,10,11,16]. In vitro studies have demonstrated that causative TMPRSS6 mutations are associated with reduced inhibitory activity on the hepcidin promoter compared with the wild-type proteins [5,6,10]. In the parents of IRIDA patients with TMPRSS6 mutations, as expected, normal erythrocyte and serum iron parameters were reported because the typical transmission mode of the disorder is autosomal recessive. However, in some family members of individuals with IRIDA, iron deficiency was reported under certain clinical conditions, an observation inconsistent with recessive transmission. As an example, Hartman et al. reported intramuscular iron use in the maternal aunt of 2 affected siblings during pregnancy and the persistence of iron deficiency even after hysterectomy. Additionally, she responded poorly to an oral iron absorption test [26]. We were also informed of anemia worsening in the mothers of our patients with IRIDA (unpublished data). These women were all heterozygous carriers of a pathogenic TMPRSS6 mutation, and at times other than during pregnancy, they had acceptable complete blood count results. Similarly, the maternal grandmother of 2 children with IRIDA was reported to have required regular intramuscular iron therapy throughout adulthood [24]. Supporting these observations, TMPRSS6haploinsufficient mice were found to be more susceptible to iron deficiency under conditions of iron restriction or an increased iron requirement, such as pregnancy [56,57]. However, it remains unclear whether certain environmental factors, a low-expressing allele, a combination of modulating polymorphisms, or defects in other genes can explain this observation. Common genetic variants such as single-nucleotide polymorphisms (SNPs) in the TMPRSS6 gene in several populations have been associated with changes in erythrocyte and iron parameters, such as the Hb level, MCV, MCH, serum iron level, and transferrin saturation [56,57,58,59,60]. TMPRSS6 SNP rs855791, which shows the strongest association with these parameters, is characterized by a missense change in the serine protease domain. This variant

Keskin Y覺lmaz E, et al: Iron-Refractory Iron Deficiency Anemia

was found to be less effective in suppressing hepcidin levels in vitro and was shown to influence serum iron parameters in healthy individuals [56]. Another recent study evaluated the predisposition to IDA in the Chinese population and found 2 TMPRSS6 polymorphisms (rs855791 and rs4820268) to be genetic risk factors for iron deficiency and IDA [61]. Interestingly, Nie et al. recently reported a 10-year-old Chinese female with a triallelic polymorphism of TMPRSS6 (homozygous for c.757 A>G and heterozygous for c.2207 T>C [rs855791]), who presented with severe hypochromic, microcytic anemia (Hb 58 g/L) at the age of 15 months [62]. The patient had laboratory findings consistent with IRIDA, including inappropriately high hepcidin levels, and was unresponsive to both oral and i.v. iron. The c.757 A>G polymorphism in the homozygous state was previously reported in a 27-year-old Japanese female with mild anemia (Hb: 108 g/L) [63]. The identification of a triallelic polymorphism resulting in an IRIDA phenotype is unique to the case reported by Nie et al. Despite these observations, current guidelines recommend IRIDA due to TMPRSS6 defect to be diagnosed with certainty only when the subject is homozygous or compound heterozygous for pathogenic TMPRSS6 mutations [59]. Differential Diagnosis In an individual with hypochromic, microcytic anemia, acquired causes of iron deficiency or congenital causes, mainly thalassemia syndromes, are the most common underlying pathology. Upon confirmation of iron deficiency, the next step is evaluation of acquired causes of iron deficiency, such as poor dietary iron intake, on-going blood loss, and chronic inflammatory conditions. In an untreated patient with iron deficiency, 2 initial laboratory patterns may suggest an IRIDA diagnosis: 1) very low MCV (range: 45-65 fL) relative to the degree of anemia (Hb range: 60-80 g/L); and 2) marked hypoferremia and low transferrin saturation (usually <5%) in the presence of a slightly low or even normal serum ferritin level. Subjects with iron deficiency will usually be treated with therapeutic doses of oral iron. A poor or absent response to therapy is most commonly associated with poor compliance to treatment, inadequate dosing, or duration of therapy. If these conditions are avoided and adequate oral iron is taken by the patient, the absence of a hematologic response should indicate first a possible defect in iron absorption. Among the causes of poor iron absorption are achlorhydria and duodenal damage, such as celiac sprue. In addition, because hepcidin production is not solely dependent on iron stores in the body but also on inflammatory stimulants [34,64], both anemia of chronic inflammation (ACI) and IRIDA are associated with hepcidin

Turk J Hematol 2015;32:1-14

Keskin Yılmaz E, et al: Iron-Refractory Iron Deficiency Anemia

elevation and result in impaired export of iron from duodenal enterocytes into the plasma. In ACI, inflammatory cytokines stimulate hepcidin expression, resulting in a decline of iron mobilization from its stores to be used in erythropoiesis [65]. Therefore, serum iron levels are found to be low in ACI, a condition that also occurs in IRIDA. However, because ACI is an acquired disorder of iron utilization, iron stores are typically normal or elevated [66], while in IRIDA, a true systemic iron deficiency state is present due to the life-long defect in intestinal iron absorption.

and sometimes invasive, examinations. This condition can generally be proven easily considering the knowledge in the literature. In spite of the paucity of IRIDA cases reported so far, this disorder may in fact be more common in countries with frequent consanguineous marriages like Turkey, in which the rate of consanguineous marriages was reported as 22% [67]. Therefore, an increased awareness of the clinical and laboratory characteristics of IRIDA is important in such populations, particularly in terms of reducing the number of unnecessary (and possibly invasive) examinations.

If oral iron challenge results indicate poor iron absorption and the onset of iron deficiency is in infancy or childhood, IRIDA is the likely diagnosis. Today, the only diagnostic test for IRIDA is genetic analysis of the TMPRSS6 gene for causative mutations. In fact, measuring serum or urinary hepcidin levels assists in distinguishing IRIDA from classic IDA. However, no approved hepcidin assay is available yet for routine clinical use. Once a hepcidin assay becomes available, the diagnosis of IRIDA may be simpler.

Conflict of Interest Statement

Hereditary iron metabolism disorders other than IRIDA may also cause hypochromic, microcytic anemia. Among these are divalent metal transporter 1 (DMT1) deficiency, congenital hypotransferrinemia, some hereditary forms of sideroblastic anemia, and aceruloplasminemia. Certain clinical and laboratory characteristics of these disorders allow their differentiation from IRIDA (Table 3) [2,59]. Conclusion The prevalence of IRIDA seems to be more frequent than predicted and more heterogeneous than previously thought, indicating that the “classical” severe homozygous form may in fact be just one of several forms of the disorder, with some cases of mild microcytic anemia actually also belonging to this disorder. A few points may help in a “classical” IRIDA diagnosis in the clinical practice of hematologists and pediatricians: if present, the familial nature of the disease; the presence of atypical iron parameters not in accordance with classical IDA (such as low-normal or normal serum ferritin levels accompanying very low transferrin saturation); and the absence of the expected hematologic response after the use of oral iron. If there is a high suspicion of IRIDA, the diagnosis can be confirmed with demonstration of the mutations in the TMPRSS6 gene, testing that is currently available only at some specialized laboratory centers. The acceptable Hb levels of IRIDA patients in adulthood and normal growth and development during their childhood make the recognition of these individuals difficult. The long time interval that passed before the establishment of an IRIDA diagnosis in the cases followed supports this situation. These patients are mostly diagnosed after very detailed,

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. Finberg KE, Heeney MM, Campagna DR, Aydinok Y, Pearson HA, Hartman KR, Mayo MM, Samuel SM, Strouse JJ, Markianos K, Andrews NC, Fleming MD. Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA). Nat Genet 2008;40:569-571. 2. Camaschella C, Poggiali E. Inherited disorders of iron metabolism. Curr Opin Pediatr 2011;23:14-20. 3. Melis MA, Cau M, Congiu R, Sole G, Barella S, Cao A, Westerman M, Cazzola M, Galanello R. A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease that suppresses hepcidin production, in familial iron deficiency anemia refractory to oral iron. Haematologica 2008;93:1473-1479. 4. Guillem F, Lawson S, Kannengiesser C, Westerman M, Beaumont C, Grandchamp B. Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency. Blood 2008;112:2089-2091. 5. Ramsay AJ, Quesada V, Sanchez M, Garabaya C, Sarda MP, Baiget M, Remacha A, Velasco G, Lopez-Otin C. Matriptase-2 mutations in iron-refractory iron deficiency anemia patients provide new insights into protease activation mechanisms. Hum Mol Genet 2009;18:3673-3683. 6. Silvestri L, Guillem F, Pagani A, Nai A, Oudin C, Silva M, Toutain F, Kannengiesser C, Beaumont C, Camaschella C, Grandchamp B. Molecular mechanisms of the defective hepcidin inhibition in TMPRSS6 mutations associated with iron-refractory iron deficiency anemia. Blood 2009;113:5605-5608. 7. Edison ES, Athiyarath R, Rajasekar T, Westerman M, Srivastava A, Chandy M. A novel splice site mutation c.2278 (-1) G>C in the TMPRSS6 gene causes deletion of the substrate binding site of the serine protease resulting in refractory iron deficiency anaemia. Br J Haematol 2009;147:766-769.

Turk J Hematol 2015;32:1-14

8. Tchou I, Diepold M, Pilotto PA, Swinkels D, Neerman-Arbez M, Beris P. Haematologic data, iron parameters and molecular findings in two new cases of iron-refractory iron deficiency anaemia. Eur J Haematol 2009;83:595-602. 9. Kannengiesser C, Guillem F, Silvestri L, Oudin C, Marfaing A, Chaiba-Berrouche L, Donadieu J, Toutain F, da Silva M, Isidor B, Marguerite G, Aguila-Martinez P, Camaschella C, Beaumont C, Grandchamp B. Allelic heterogeneity of TMPRSS6 mutations in IRIDA. Am J Hematol 2009;84:272. 10. De Falco L, Totaro F, Nai A, Pagani A, Girelli D, Silvestri L, Piscopo C, Campostrini N, Dufour C, Al Manjomi F, Minkov M, Van Vuurden DG, Feliu A, Kattamis A, Camaschella C, Iolascon A. Novel TMPRSS6 mutations associated with iron-refractory iron deficiency anemia (IRIDA). Hum Mutat 2010;31:1390-1405. 11. Altamura S, D’Alessio F, Selle B, Muckenthaler MU. A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA. Biochem J 2010;431:363-371. 12. Beutler E, Van Geet C, te Loo DM, Gelbart T, Crain K, Truksa J, Lee PL. Polymorphisms and mutations of human TMPRSS6 in iron deficiency anemia. Blood Cells Mol Dis 2010;44:16-21. 13. Cuijpers ML, Wiegerinck ET, Brouwer R, de Witte TJ, Swinkels DW. Iron deficiency anaemia due to a matriptase-2 mutation. Ned Tijdschr Geneeskd 2010;154:1038 (in Dutch with English abstract). 14. Palare M, Ferrao A, Relvas L, Bento C, Morais A. TMPRSS6 gene - Two new nonsense mutations associated with IRIDA. Haematologica 2010;95(Suppl 2):704 (abstract 1820). 15. Choi HS, Yang HR, Song SH, Seo JY, Lee KO, Kim HJ. A novel mutation Gly603Arg of TMPRSS6 in a Korean female with iron-refractory iron deficiency anemia. Pediatr Blood Cancer 2012;58:640-642. 16. Guillem F, Kannengiesser C, Oudin C, Lenoir A, Matak P, Donadieu J, Isidor B, Mechinaud F, Aguilar-Martinez P, Beaumont C, Vaulont S, Grandchamp B, Nicolas G. Inactive matriptase-2 mutants found in IRIDA patients still repress hepcidin in a transfection assay despite having lost their serine protease activity. Hum Mutat 2012;33:1388-1396. 17. Pellegrino RM, Coutinho M, D’Ascola D, Lopes AM, Palmieri A, Carnuccio F, Costa M, Zecchina G, Saglio G, Costa E, Barbot J, Porto G, Pinto JP, Roetto A. Two novel mutations in the TMPRSS6 gene associated with iron-refractory irondeficiency anaemia (IRIDA) and partial expression in the heterozygous form. Br J Haematol 2012;158:668-672. 18. Jaspers A, Caers J, Le Gac G, Ferec C, Beguin Y, Fillet G. A novel mutation in the CUB sequence of matriptase-2 (TMPRSS6) is implicated in iron-resistant iron deficiency anaemia (IRIDA). Br J Haematol 2013;160:564-565.

Keskin Yılmaz E, et al: Iron-Refractory Iron Deficiency Anemia

19. Lehmberg K, Grosse R, Muckenthaler MU, Altamura S, Nielsen P, Schmid H, Graubner U, Oyen F, Zeller W, Schneppenheim R, Janka GE. Administration of recombinant erythropoietin alone does not improve the phenotype in iron refractory iron deficiency anemia patients. Ann Hematol 2013;92:387-394. 20. Khuong-Quang DA, Schwartzentruber J, Westerman M, Lepage P, Finberg KE, Majewski J, Jabado N. Iron refractory iron deficiency anemia: presentation with hyperferritinemia and response to oral iron therapy. Pediatrics 2013;131:620-625. 21. Yılmaz-Keskin E, Sal E, de Falco L, Bruno M, Iolascon A, Koçak Ü, Yenicesu İ. Is the acronym IRIDA acceptable for slow responders to iron in the presence of TMPRSS6 mutations? Turk J Pediatr 2013;55:479-484. 22. Camaschella C. How I manage patients with atypical microcytic anaemia. Br J Haematol 2013;160:12-24. 23. Buchanan GR, Sheehan RG. Malabsorption and defective utilization of iron in three siblings. J Pediatr 1981;98:723-728. 24. Mayo MM, Samuel SM. Iron deficiency anemia due to a defect in iron metabolism: a case report. Clin Lab Sci 2001;14:135-138. 25. Brown AC, Lutton JD, Pearson HA, Nelson JC, Levere RD, Abraham NG. Heme metabolism and in vitro erythropoiesis in anemia associated with hypochromic microcytosis. Am J Hematol 1988;27:1-6. 26. Hartman KR, Barker JA. Microcytic anemia with iron malabsorption: an inherited disorder of iron metabolism. Am J Hematol 1996;51:269-275. 27. Andrews NC. Iron deficiency: lessons from anemic mice. Yale J Biol Med 1997;70:219-226. 28. Galanello R, Cau M, Melis MA, Deidda F, Cao A, Cazzola M. Studies of NRAMP2, transferrin receptor and transferrin genes as candidate genes for human hereditary microcytic anemia due to defective iron absorption and utilization. Blood 1998;92(Suppl 1):669a (abstract). 29. Velasco G, Cal S, Quesada V, Sanchez LM, Lopez-Otin C. Matriptase-2, a membrane-bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins. J Biol Chem 2002;277:37637-37646. 30. Du X, She E, Gelbart T, Truksa J, Lee P, Xia Y, Khovananth K, Mudd S, Mann N, Moresco EM, Beutler E, Beutler B. The serine protease TMPRSS6 is required to sense iron deficiency. Science 2008;320:1088-1092. 31. Kemna EH, Tjalsma H, Podust VN, Swinkels DW. Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications. Clin Chem 2007;53:620-628. 32. Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Immunoassay for human serum hepcidin. Blood 2008;112:4292-4297.

Keskin Yılmaz E, et al: Iron-Refractory Iron Deficiency Anemia

33. Nemeth E, Ganz T. Regulation of iron metabolism by hepcidin. Annu Rev Nutr 2006;26:323-342. 34. Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, Loreal O. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 2001;276:7811-7819. 35. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004;306:2090-2093. 36. Nemeth E, Preza GC, Jung CL, Kaplan J, Waring AJ, Ganz T. The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study. Blood 2006;107:328-333. 37. De Domenico I, Nemeth E, Nelson JM, Phillips JD, Ajioka RS, Kay MS, Kushner JP, Ganz T, Ward DM, Kaplan J. The hepcidin-binding site on ferroportin is evolutionarily conserved. Cell Metab 2008;8:146-156. 38. De Domenico I, Ward DM, Langelier C, Vaughn MB, Nemeth E, Sundquist WI, Ganz T, Musci G, Kaplan J. The molecular mechanism of hepcidin-mediated ferroportin down-regulation. Mol Biol Cell 2007;18:2569-2578. 39. Silvestri L, Pagani A, Nai A, De Domenico I, Kaplan J, Camaschella C. The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin. Cell Metab 2008;8:502-511. 40. Miyazono K, Maeda S, Imamura T. BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine Growth Factor Rev 2005;16:251-263. 41. Babitt JL, Huang FW, Wrighting DM, Xia Y, Sidis Y, Samad TA, Campagna JA, Chung RT, Schneyer AL, Woolf CJ, Andrews NC, Lin HY. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat Genet 2006;38:531-539. 42. Nicolas G, Bennoun M, Porteu A, Mativet S, Beaumont C, Grandchamp B, Sirito M, Sawadogo M, Kahn A, Vaulont S. Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci U S A 2002;99:4596-4601. 43. Courselaud B, Pigeon C, Inoue Y, Inoue J, Gonzalez FJ, Leroyer P, Gilot D, Boudjema K, Guguen-Guillouzo C, Brissot P, Loreal O, Ilyin G. C/EBPα regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism. Cross-talk between C/EBP pathway and iron metabolism. J Biol Chem 2002;277:41163-41170. 44. Willemetz A, Lenoir A, Deschemin JC, Lopez-Otin C, Ramsay AJ, Vaulont S, Nicolas G. Matriptase-2 is essential for hepcidin repression during fetal life and postnatal development in mice to maintain iron homeostasis. Blood 2014;124:441-444.

Turk J Hematol 2015;32:1-14

45. Traglia M, Girelli D, Biino G, Campostrini N, Corbella M, Sala C, Masciullo C, Vigano F, Buetti I, Pistis G, Cocca M, Camaschella C, Toniolo D. Association of HFE and TMPRSS6 genetic variants with iron and erythrocyte parameters is only in part dependent on serum hepcidin concentrations. J Med Genet 2011;48:629-634. 46. Galesloot TE, Geurts-Moespot AJ, den Heijer M, Sweep FC, Fleming RE, Kiemeney LA, Vermeulen SH, Swinkels DW. Associations of common variants in HFE and TMPRSS6 with iron parameters are independent of serum hepcidin in a general population: a replication study. J Med Genet 2013;50:593-598. 47. De Falco L, Bruno M, Keskin EY, Yenicesu I, Vitiello F, Iolascon A. The role of TMPRSS6 causing IRIDA during fetal and neonatal life. Am J Hematol 2013;88:80 (abstract). 48. Hartman KR, Finberg KE, Merino ME. Iron resistant iron deficiency anemia: long term follow-up of 5 patients. ASPHO Annual Meeting Abstracts, San Diego, 2009 (abstract). 49. Cazzola M, Ponchio L, de Benedetti F, Ravelli A, Rosti V, Beguin Y, Invernizzi R, Barosi G, Martini A. Defective iron supply for erythropoiesis and adequate endogenous erythropoietin production in the anemia associated with systemic-onset juvenile chronic arthritis. Blood 1996;87:4824-4830. 50. Martini A, Ravelli A, Di Fuccia G, Rosti V, Cazzola M, Barosi G. Intravenous iron therapy for severe anaemia in systemiconset juvenile chronic arthritis. Lancet 1994;344:10521054. 51. Akin M, Atay E, Oztekin O, Karadeniz C, Karakus YT, Yilmaz B, Erdogan F. Responsiveness to parenteral iron therapy in children with oral iron-refractory iron-deficiency anemia. Pediatr Hematol Oncol 2014;31:57-61. 52. De Falco L, Silvestri L, Kannengiesser C, Morán E, Oudin C, Rausa M, Bruno M, Aranda J, Argiles B, Yenicesu I, Falcon-Rodriguez M, Yilmaz-Keskin E, Kocak U, Beaumont C, Camaschella C, Iolascon A, Grandchamp B, Sanchez M. Functional and clinical impact of novel Tmprss6 variants in iron-refractory iron-deficiency anemia patients and genotypephenotype studies. Human Mutat 2014;35:1321-1329. 53. De Falco L, Bruno M, Keskin EY, Yenicesu I, Vitiello F, Iolascon A. Responsiveness to oral iron therapy of two IRIDA patients. Am J Hematol 2013;88:84-85 (abstract). 54. Cau M, Galanello R, Giagu N, Melis MA. Responsiveness to oral iron and ascorbic acid in a patient with IRIDA. Blood Cells Mol Dis 2012;48:121-123. 55. Netzel-Arnett S, Hooper JD, Szabo R, Madison EL, Quigley JP, Bugge TH, Antalis TM. Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer. Cancer Metastasis Rev 2003;22:237-258.

Turk J Hematol 2015;32:1-14

56. Nai A, Pagani A, Silvestri L, Camaschella C. Increased susceptibility to iron deficiency of Tmprss6haploinsufficient mice. Blood 2010;116:851-852. 57. Finberg KE, Whittlesey RL, Fleming MD, Andrews NC. Down-regulation of Bmp/Smad signaling by Tmprss6 is required for maintenance of systemic iron homeostasis. Blood 2010;115:3817-3826. 58. Delbini P, Vaja V, Graziadei G, Duca L, Nava I, Refaldi C, Cappellini MD. Genetic variability of TMPRSS6 and its association with iron deficiency anaemia. Br J Haematol 2010;151:281-284. 59. Donker AE, Raymakers RA, Vlasveld LT, van Barneveld T, Terink R, Dors N, Brons PP, Knoers NV, Swinkels DW. Practice guidelines for the diagnosis and management of microcytic anemias due to genetic disorders of iron metabolism or heme synthesis. Blood 2014;123:3873-3886. 60. Soranzo N, Spector TD, Mangino M, Kuhnel B, Rendon A, Teumer A, Willenborg C, Wright B, Chen L, Li M, Salo P, Voight BF, Burns P, Laskowski RA, Xue Y, Menzel S, Altshuler D, Bradley JR, Bumpstead S, Burnett MS, Devaney J, Doring A, Elosua R, Epstein SE, Erber W, Falchi M, Garner SF, Ghori MJ, Goodall AH, Gwilliam R, Hakonarson HH, Hall AS, Hammond N, Hengstenberg C, Illig T, Konig IR, Knouff CW, McPherson R, Melander O, Mooser V, Nauck M, Nieminen MS, Oâ&#x20AC;&#x2122;Donnell CJ, Peltonen L, Potter SC, Prokisch H, Rader DJ, Rice CM, Roberts R, Salomaa V, Sambrook J, Schreiber S, Schunkert H, Schwartz SM, Serbanovic-Canic J, Sinisalo J, Siscovick DS, Stark K, Surakka I, Stephens J, Thompson JR, Volker U, Volzke H, Watkins NA, Wells GA, Wichmann HE, Van Heel DA, Tyler-Smith C, Thein SL, Kathiresan S, Perola M, Reilly MP, Stewart AF, Erdmann J, Samani NJ, Meisinger C, Greinacher A, Deloukas P, Ouwehand WH, Gieger C. A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium. Nat Genet 2009;41:1182-1190.

Keskin YÄąlmaz E, et al: Iron-Refractory Iron Deficiency Anemia

61. An P, Wu Q, Wang H, Guan Y, Mu M, Liao Y, Zhou D, Song P, Wang C, Meng L, Man Q, Li L, Zhang J, Wang F. TMPRSS6, but not TF, TFR2 or BMP2 variants are associated with increased risk of iron-deficiency anemia. Hum Mol Genet 2012;21:2124-2131. 62. Nie N, Shi J, Shao Y, Li X, Ge M, Huang J, Zhang J, Huang Z, Li D, Zheng Y. A novel tri-allelic mutation of TMPRSS6 in iron-refractory iron deficiency anaemia with response to glucocorticoid. Br J Haematol 2014;166:300-303. 63. Sato T, Iyama S, Murase K, Kamihara Y, Ono K, Kikuchi S, Takada K, Miyanishi K, Sato Y, Takimoto R, Kobune M, Kato J. Novel missense mutation in the TMPRSS6 gene in a Japanese female with iron-refractory iron deficiency anemia. Int J Hematol 2011;94:101-103. 64. Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003;101:2461-2463. 65. Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pedersen BK, Ganz T. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest 2004;113:1271-1276. 66. Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med 2005;352:1011-1023. 67. Koc I. Prevalence and sociodemographic correlates of consanguineous marriages in Turkey. J Biosoc Sci 2008;40:137148.

Research Article

DOI: 10.4274/tjh.2013.0140

Prognostic Significance of Lymphoid EnhancerBinding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients Mısırlı Yetişkin Akut B Lenfoblastik Lösemi Hastalarında Görülen Lenfoid Enhansır-Bağlayıcı Faktör-1 Ekspresyonunun Prognostik Önemi Rabab M. Aly1, Ansaf B. Yousef2 1Mansoura 2Mansoura

University Faculty of Medicine, Department of Clinical Pathology, Mansoura, Egypt University Faculty of Medicine, Department of Internal Medicine, Mansoura, Egypt

Abstract: Objective: Lymphoid enhancer-binding factor-1 (LEF-1) is a key transcription factor of wingless-type (Wnt) signaling in various tumors and it is associated with a number of malignant diseases such as leukemia. We explored the expression profile of LEF-1 in acute lymphoblastic leukemia (ALL) and determined its specific prognostic significance in this disease.

Materials and Methods: We studied LEF-1 expression in 56 newly diagnosed B-acute ALL adult patients using realtime quantitative polymerase chain reaction to investigate whether LEF-1 expression was associated with clinical patient characteristics and treatment outcomes.

Results: High LEF-1 expression was associated with significantly poorer disease-free survival (p=0.03) and overall survival (p=0.005). Patients with high LEF-1 expression had a significantly higher relapse rate compared with low LEF-1 expression (p=0.01). Conclusion: We provide evidence that high LEF-1 expression is a prognostic marker in adult B-acute ALL patients. Key Words: Lymphoid enhancer-binding factor-1, Acute lymphoblastic leukemia, Prognosis, Wnt Özet: Amaç: Lenfoid enhansır-bağlayıcı faktör-1 (LEF-1), çeşitli tümörlerde wingless-type (Wnt)’yi işaret eden ana transkripsiyon faktörü olmakla birlikte lösemi gibi birçok ölümcül hastalıkla da ilişkilendilir. Biz bu çalışmada, LEF-1’in akut lenfoblastik lösemideki (ALL) ekspresyon profilini araştırdık ve LEF-1’in bu hastalık genelindeki özel prognostik önemini saptadık. Gereç ve Yöntemler: B-ALL hastalığı tanısı yeni konmuş 56 yetişkin hastada LEF-1 ekspresyonunu çalıştık ve LEF-1 ekspresyonunun klinik hasta özellikleriyle ve tedavi sonuçlarıyla ilişkili olup olmadığını araştırmak için gerçek-zamanlı kantitatif polimeraz zincir reaksiyonu uyguladık. Address for Correspondence: Rabab M. ALY, M.D., Mansoura University Faculty of Medicine, Department of Clinical Pathology, Mansoura, Egypt E-mail: rababzeadah@yahoo.com Received/Geliş tarihi : April 18, 2013 Accepted/Kabul tarihi : September 27, 2013

Aly MR, et al: Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients

Turk J Hematol 2015;32:15-20

Bulgular: Yüksek LEF-1 ekspresyonu olan hastalarda hastalıksız sağkalım (p=0,03) ve genel sağkalım (p=0,005) daha kısaydı.

Düşük LEF-1 ekspresyonlu hastalarla kıyaslandığında, yüksek LEF-1 ekspresyonunlu hastalarda anlamlı olarak daha yüksek nüks oranları gözlendi (p=0,01). Sonuç: Bu çalışma sonucunda, yüksek LEF-1 ekspresyonunun yetişkin B-ALL hastalarında prognostik belirteç olduğuna yönelik kanıt sunuyoruz.

Anahtar Sözcükler: Lenfoid enhansır-bağlayıcı faktör-1, Akut lenfoblastik lösemi, Prognoz, Wnt Introduction The outcome of adult B-precursor acute lymphoblastic leukemia (B-ALL) has considerably improved because of identification of clinical and genetic risk factors stratifying patients into different treatment groups [1]. Common risk factors in B-ALL include white blood cell (WBC) count, the immunophenotype of B-ALL, response to induction therapy, level of minimal residual disease, age, and cytogenetic as well as molecular genetic aberrations [2]. High-risk cytogenetics in B-ALL mainly comprise the Philadelphia chromosome with balanced translocation t(9; 22) (q34; q11) and the BCR-ABL fusion gene in approximately 20% to 36% of cases [3] and translocation t(4; 11) (q21; q23) with the MLL-AF4 fusion gene in approximately 6% to 9% of cases [4]. Patients lacking clinical and molecular risk factors are considered to be of standard risk. The relapse rate is still approximately 40% to 50% and relapse is not predictable with pretreatment markers in the standard-risk patient group without established risk factors [5,6]. The identification of new prognostic factors is of particular interest for this subgroup of ALL. Moreover, it may help to develop novel targeted therapies for these patients. It was previously demonstrated that ordered expression of lymphoid enhancer-binding factor-1 (LEF-1) is necessary for normal hematopoietic stem cell function in mice, and that LEF1 overexpression induces acute myeloid leukemia (AML) [7]. LEF-1 plays a crucial role in the development of B and T lymphocytes as well as neutrophilic granulocytes [8,9]. In different hematologic malignancies, including lymphomas, chronic lymphocytic leukemia, and ALL and AML [10,11,12,13,14], LEF-1 was found to be highly expressed. In vitro studies revealed a prosurvival effect of LEF-1 in an AML1-ETO-positive leukemic cell line, primary chronic lymphocytic leukemia cells, and murine T-cell lymphomas [11,12,15]. LEF-1 is a member of the lymphoid enhancer factor/Tcell factor (LEF/TCF) family (LEF-1, TCF-1, TCF-3, and TCF-4) of the HMG transcription factors [16]. LEF-1 acts as a central transcription mediator of Wingless-type (Wnt) signaling, regulating cell cycle, and growth-relevant genes like cyclin D1 and c-myc [17,18,19]. The Wnt pathway has

been implicated in leukemic transformation and was shown to promote proliferation and survival of leukemic cells in vitro [15,20,21]. Studies on LEF-1 in hematopoietic development have been mainly restricted to the lymphoid lineages, where LEF-1 has functions in T-cell development and affects proliferation and apoptosis in pro-B cells [8]. Recent reports, however, also discuss specific functions of LEF-1 independent of Wnt signaling, suggesting a more complex role of LEF-1 in the development of hematopoietic tissues [15]. The human LEF-1 gene spans at least 140 kb and contains 12 exons and 11 introns, with a large third intron (about 75 kb) that may contain an alternative exon. The LEF-1 gene encodes at least 2 isoforms. LEF-1 is a sequence-specific DNA binding protein that binds to a functionally important site in the T-cell receptor-alpha enhancer through an HMG domain and confers maximal enhancer activity [22]. Materials and Methods Patients Fifty-six Egyptian patients (43 males and 13 females) diagnosed with B-ALL at the Oncology Center of Mansoura University Hospital were enrolled in this study. The study was approved by the Institutional Review Board of the Mansoura University Hospital. All patients gave informed consent to morphological and molecular examination. Diagnosis of ALL was based on morphologic and immunophenotypic criteria. Heparinized bone marrow (BM) and peripheral blood samples were collected prior to treatment. Minimal residual disease was effectively monitored by use of BM samples. Clinical and hematological parameters were determined. All included patients received the same treatment protocol, approved by the oncology team of the oncology center. It included 6 weeks of induction, 2 weeks of consolidation, and 120 weeks of continuation therapy. Induction therapy consisted of vincristine (VCR), daunomycin, asparaginase, etoposide (VP-16), and aracytin (Ara-C) in addition to triple intrathecal (IT) therapy including 2 courses of high-dose methotrexate (HDMTX), 6-mercaptopurine (6-MP), and dexamethasone (Dex). Continuation therapy consisted of extended triple IT therapy and 15 cycles of an 8-week course of VP16+cyclophosphamide (CTX), 6-MP+MTX, MTX+Ara-C, Dex+VCR, VP-16+Ara-C, 6-MP+HDMTX, VP-16+Ara-C, and

Aly MR, et al: Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients

Dex+VCR. During continuation therapy, reinduction was given in the form of VCR, daunomycin, Dex, HDMTX, 6-MP, and triple IT therapy. Complete remission was defined as mononuclear BM containing less than 5% blast cells and showing evidence of normal maturation of other marrow elements after induction chemotherapy. Relapse was defined by the appearance of more than 5% lymphoblasts in a single BM aspirate or leukemic cell infiltration in extramedullary organs [23]. Methods RNA Extraction and cDNA Synthesis High-quality RNA was extracted using the RNeasy Mini Kit in accordance with the manufacturer’s instructions (QIAGEN, Valencia, CA, USA). The concentration, quality, and purity of RNA were measured by UV spectrophotometer at 260/280 nm. The integrity and the size distribution of total RNA were checked by electrophoresis on 1.5% agarose gel. cDNA synthesis reaction was performed with 25 µL of total RNA, 2.5 µL of reverse transcriptase, 5 µL of RT, 4 µL of dNTPs, 5 µL of random primers, and 8.5 µL of water. This mixture was then incubated at 42 °C for 1 h. The reaction was inactivated by heating at 95 °C for 5 min. Analyses of Gene Expression by Real-Time-Polymerase Chain Reaction The LEF-1 gene and reference gene GAPDH were quantified according to the real-time quantitative polymerase chain reaction (RT-PCR) method using the Applied 7700 sequence detection system (TaqMan; PerkinElmer Applied Biosystems, Foster City, CA, USA). RT-PCR was performed in a MicroAmp optical 96-well plate with 10 µL of the cDNA solution, 1.0 µM of forward primer, 1.0 µM of reverse primer, 10 µL of dH2O, 0.5 µL of probe, and 25 µL of universal master mix. The sequences of forward and reverse primers for measurement of LEF-1 expression were as follows: LEF1 probe, 5-FAM-CCAGATTCTTGGCAGAAGGTGGCATTAMRA; LEF-1 forward, 5-AATGAGAGCGAATGTCGTTGC; and LEF-1 reverse, 5-GCTGTCTTTCTTTCCGTGCTA. The sequence of the primers and probe GAPDH control were: GAPDH forward 5’-GAAGGTGAAGGTCGGAGTC-3’; GAPDH reverse 5’ GAAGATGGTGATGGGATTTC-3; and GAPDH VIC-CAAGCTTCCCGTTCTCAGCC-TAMRA.

of the primer, primer, probe,

All samples were analyzed in duplicate. The variation of the duplicate measurements was extremely small compared to the variation between different samples. For each patient, the relative mRNA expression levels of LEF-1 were calculated using the comparative cycle time (Ct) method [24]. The target PCR Ct value, which is the cycle number at which emitted

Turk J Hematol 2015;32:15-20

fluorescence exceeds 10 times the standard deviation of baseline emissions, was normalized to the GAPDH PCR Ct value by subtracting the GAPDH Ct value from the target PCR Ct value. The mRNA expression level relative to GAPDH for each target PCR was calculated using the following equation: relative mRNA expression= 2-(Ct target-Ct GAPDH). Statistical Analysis SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for all calculations. Clinical features across groups were compared using the χ2 or 2-sided Fisher exact test for categorical data and the nonparametric Mann-Whitney U test for continuous variables. Survival curves were calculated by the Kaplan-Meier method. Multivariate analyses were performed using the Cox proportional hazards model for survival, including the following variables in the full model: LEF-1 expression, age, WBC count, and immunophenotype. P<0.05 was considered statistically significant. Results We determined LEF-1 expression in 56 patients with newly diagnosed B-ALL. There were no significant correlations between LEF-1 expression levels and clinical, laboratory, or immunophenotypic characteristics (Table 1). Based on the detection of LEF-1 median expression level (1.73), patients were divided into the low-expression group (LEF-1 expression level of <1.73) and high-expression group (LEF-1 expression level of >1.73). In the Cox regression analysis, LEF-1 expression was the most significant prognostic factor for disease-free survival (DFS) (p=0.001); the other significant factors predicting DFS were age and immunophenotype (Table 2). We analyzed the influence of LEF-1 expression on the prognosis of B-ALL patients. Patients with high LEF-1 expression, as compared to patients with low LEF-1 expression, showed significantly lower remission rates (p=0.02). Patients with high LEF-1 expression were significantly associated with a higher relapse rate (p=0.01, Table 3). Table 3 also showed that, at 3 years, the estimated overall survival (OS) was 58.9% in patients with low LEF1 expression, which was higher than in patients with high expression (33.9%) (p=0.005). Adverse prognosis associated with high LEF-1 expression was observed in terms of 3-year DFS (high LEF-1, 23.4% vs. low LEF-1 expression, 51.0%; p=0.03). Discussion The prognostic markers in B-ALL have a significantly important role in the development of new molecular therapies for these patients. In the current study, we have used a

Aly MR, et al: Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients

Turk J Hematol 2015;32:15-20

quantitative real-time PCR assay to analyze the role of LEF-1 expression in patients with B-ALL. Table 1. LEF-1 expression and clinical characteristics in acute lymphoblastic leukemia (ALL) cases.

High LEF-1 Low LEF-1 p (n=22) (n=34) Age (years) Median Range

45 18-49

38 19-46

0.09

Sex (%) Male

68.1

70.5

0.73

Immunophenotype Precursor B-ALL Common ALL

15 (68.2%) 7 (31.8%)

25 (73.5%) 9 (26.5%)

0.07

WBC count (x109/L) Median

ALL: acute lymphoblastic leukemia, B-ALL: B-precursor acute lymphoblastic leukemia, WBC: white blood cell

Table 2. Multivariate analysis of LEF-1 expression for disease-free survival (DFS) in acute lymphoblastic leukemia (ALL) patients.

HR 95% CI p Age

0.9

0.7-1.2

0.04

WBCs

1.3

0.6-1.5

0.06

Immunophenotype Precursor B-ALL vs. common ALL

1.7

1.2-1.9

0.02

LEF-1 expression High vs. low

3.1

1.7-4.3

0.001

ALL: acute lymphoblastic leukemia, B-ALL: B-precursor acute lymphoblastic leukemia, WBC: white blood cell, HR: hazard ratio

In this study, we have identified high LEF-1 expression as an independent prognostic factor associated with a high risk of relapse and lower DFS in B-ALL patients. High LEF-1 expression was independently prognostic for lower OS, with a 3-year survival rate of only 33.9% for patients with high LEF-1 expression compared to 58.9% in the patient group with low LEF-1. Several studies found effects of LEF-1 expression on the degree of malignancy in neoplastic diseases. LEF-1 appears to mediate tumor growth and invasion ability in androgenindependent prostate cancer [25]. Moreover, Nguyen et al. found that LEF-1 also mediates cell invasion in breast cancer [26]. Deregulated LEF-1 expression may also be an important step in the development of neoplastic diseases. LEF-1 is involved in B and T lymphocyte development [8,27]. The causes and effects of abnormal LEF-1 expression likely depend on the cellular context and differentiation stage. The diverse functions of LEF-1 in normal and malignant hematopoiesis are reflected by recent reports that high LEF-1 expression is associated with inferior outcomes in B-ALL [28]. Metzeler et al. reported a larger increase of the expression of LEF-1 in ALL samples than in AML, reflecting the higher expression of the transcription factor in lymphoid tissue [29]. Several studies found that deregulated expression of LEF-1 could induce B-ALL [7,8]. Our study showed no significant association of clinical characteristics and high LEF-1 expression. Conversely, in patients with T-ALL, LEF-1 was characterized by distinctive clinical features, including a younger age at diagnosis and a trend toward improved OS in children treated with contemporary T-ALL combination therapy [30]. Current risk stratification is based primarily on clinical variables, immunophenotyping, detection of cytogenetic or molecular lesions, and early response to therapy [31]. Importantly, our study shows that high LEF-1 expression was also associated with poor DFS. This was in agreement with K端hnl et al., who found that high LEF-1 expression identifies B-ALL patients with inferior DFS [28]. In multivariate analysis, the association between LEF-1 expression and outcome showed that high LEF-1

Table 3. LEF-1 expression and clinical outcome.

High LEF-1 expression

Low LEF-1 expression

Complete response, CR/total, no. (%)

16/22 (72.7)

31/34 (91.1)

0.02

Relapse, Relapse/total, no. (%)

9/16 (56.2)

11/31 (35.4)

0.01

3-year Overall survival, % (n=56)

33.9

58.9

0.005

3-year Disease-free survival, % (n=47)

23.4

51.0

0.03

Aly MR, et al: Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients

expression is more significant than other prognostic factors for DFS, such as for age, leukocyte count at presentation, or immunophenotype; this indicates that high LEF-1 expression at diagnosis might be useful in identifying patients with a high risk of treatment failure. A study of T-ALL patients with inactivating LEF-1 mutations showed a trend toward a favorable OS [30]. Conversely, in patients with myelodysplastic syndrome, advanced disease and poor prognosis were associated with downregulation of LEF-1, probably reflecting the impaired maturation of myeloid progenitors associated with loss of LEF-1 function [15,32]. In leukemic cells, LEF-1 enhanced self-renewal properties and survival in vitro and was shown to confer leukemogenic potential in a mouse model [7,12,15]. Furthermore, patients with high LEF-1 expression should be considered for new molecular directed therapies, especially agents targeting the Wnt pathway [33].

Turk J Hematol 2015;32:15-20

6. Brüggemann M, Raff T, Flohr T, Gökbuget N, Nakao M, Droese J, Lüschen S, Pott C, Ritgen M, Scheuring U, Horst HA, Thiel E, Hoelzer D, Bartram CR, Kneba M; German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia. Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia. Blood 2006;107:1116-1123. 7. Petropoulos K, Arseni N, Schessl C, Stadler CR, Rawat VP, Deshpande AJ, Heilmeier B, Hiddemann W, QuintanillaMartinez L, Bohlander SK, Feuring-Buske M, Buske C. A novel role for Lef-1, a central transcription mediator of Wnt signaling, in leukemogenesis. J Exp Med 2008;205:515-522. 8. Reya T, O’Riordan M, Okamura R, Devaney E, Willert K, Nusse R, Grosschedl R. Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism. Immunity 2000;13:15-24.

Conflict of Interest Statement

9. Skokowa J, Cario G, Uenalan M, Schambach A, Germeshausen M, Battmer K, Zeidler C, Lehmann U, Eder M, Baum C, Grosschedl R, Stanulla M, Scherr M, Welte K. LEF-1 is crucial for neutrophil granulocytopoiesis and its expression is severely reduced in congenital neutropenia. Nat Med 2006;12:11911197.

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.

10. Gelebart P, Anand M, Armanious H, Peters AC, Dien Bard J, Amin HM, Lai R. Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma. Blood 2008;112:51715179.

In summary, we provide evidence that high LEF-1 expression has adverse prognostic significance and thus may provide a valuable new approach to molecular-targeted therapy in B-ALL patients.

References 1. Gökbuget N, Hoelzer D. Treatment of adult acute lymp Belirteç hoblastic leukemia. Semin Hematol 2009;46:64-75. 2. Rowe JM. Optimal management of adults with ALL. Br J Haematol 2009;144:468-483. 3. Burmeister T, Schwartz S, Bartram CR, Gökbuget N, Hoelzer D, Thiel E; GMALL study group. Patients’ age and BCR/ABL frequency in adult B-precursor ALL: a retrospective analysis from the GMALL study group. Blood 2008;112:918-919. 4. Thomas X, Boiron JM, Huguet F, Dombret H, Bradstock K, Vey N, Kovacsovics T, Delannoy A, Fegueux N, Fenaux P, Stamatoullas A, Vernant JP, Tournilhac O, Buzyn A, Reman O, Charrin C, Boucheix C, Gabert J, Lhéritier V, Fiere D. Outcome of treatment in adults with acute lymphoblastic leukemia: analysis of the LALA-94 trial. J Clin Oncol 2004;22:40754086. 5. Gökbuget N, Hoelzer D, Arnold R, Böhme A, Bartram CR, Freund M, Ganser A, Kneba M, Langer W, Lipp T, Ludwig WD, Maschmeyer G, Rieder H, Thiel E, Weiss A, Messerer D. Treatment of adult ALL according to protocols of the German Multicenter Study Group for Adult ALL (GMALL). Hematol Oncol Clin North Am 2000;14:1307-1325.

11. Spaulding C, Reschly EJ, Zagort DE, Yashiro-Ohtani Y, Beverly LJ, Capobianco A, Pear WS, Kee BL. Notch1 co-opts lymphoid enhancer factor 1 for survival of murine T-cell lymphomas. Blood 2007;110:2650-2658. 12. Gutierrez A Jr, Tschumper RC, Wu X, Shanafelt TD, EckelPassow J, Huddleston PM 3rd, Slager SL, Kay NE, Jelinek DF. LEF-1 is a prosurvival factor in chronic lymphocytic leukemia and is expressed in the preleukemic state of monoclonal B-cell lymphocytosis. Blood 2010;116:2975-2983. 13. Simon M, Grandage VL, Linch DC, Khwaja A. Constitutive activation of the Wnt/beta-catenin signalling pathway in acute myeloid leukemia. Oncogene 2005;24:2410-2420. 14. Wang W, Ji P, Steffen B, Metzger R, Schneider PM, Halfter H, Schrader M, Berdel WE, Serve H, Müller-Tidow C. Alterations of lymphoid enhancer factor-1 isoform expression in solid tumors and acute leukemias. Acta Biochim Biophys Sin (Shanghai) 2005;37:173-180. 15. Müller-Tidow C, Steffen B, Cauvet T, Tickenbrock L, Ji P, Diederichs S, Sargin B, Köhler G, Stelljes M, Puccetti E, Ruthardt M, deVos S, Hiebert SW, Koeffler HP, Berdel WE, Serve H. Translocation products in acute myeloid leukemia activate the Wnt signaling pathway in hematopoietic cells. Mol Cell Biol 2004;24:2890-2904.

Turk J Hematol 2015;32:15-20

Aly MR, et al: Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients

16. Nawshad A, Hay ED. TGFβ3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development. J Cell Biol 2003;163:1291-1301. 17. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW. Identification of c-MYC as a target of the APC pathway. Science 1998;281:1509-1512. 18. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006;127:469-480. 19. Gehrke I, Gandhirajan RK, Kreuzer KA. Targeting the WNT/ beta-catenin/TCF/LEF1 axis in solid and haematological cancers: multiplicity of therapeutic options. Eur J Cancer 2009;45:2759-2767. 20. Khan NI, Bradstock KF, Bendall LJ. Activation of Wnt/betacatenin pathway mediates growth and survival in B-cell progenitor acute lymphoblastic leukemia. Br J Haematol 2007;138:338-348. 21. Lu D, Zhao Y, Tawatao R, Cottam HB, Sen M, Leoni LM, Kipps TJ, Corr M, Carson DA. Activation of the Wnt signaling pathway in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 2004;101:3118-3123. 22. Hovanes K, Li TW, Munguia JE, Truong T, Milovanovic T, Lawrence Marsh J, Holcombe RF, Waterman ML. β-Cateninsensitive isoforms of lymphoid enhancer factor-1 are selectively expressed in colon cancer. Nat Genet 2001;28:53– 57. 23. Lanzokowsky PH. Leukemia. In: Lanzokowsky P (ed). Manual of Pediatric Hematology and Oncology. 2nd ed. New York, Churchill Livingstone, 2005. 24. Meijerink J, Mandigers C, van de Locht L, Tönnissen E, Goodsaid F, Raemaekers J. A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real time PCR. J Mol Diagn 2001;3:5561. 25. Li Y, Wang L, Zhang M, Melamed J, Liu X, Reiter R, Wei J, Peng Y, Zou X, Pellicer A, Garabedian MJ, Ferrari A, Lee P. LEF1 in androgen-independent prostate cancer: regulation of androgen receptor expression, prostate cancer growth, and invasion. Cancer Res 2009;69:3332-3338.

26. Nguyen A, Rosner A, Milovanovic T, Hope C, Planutis K, Saha B, Chaiwun B, Lin F, Imam SA, Marsh JL, Holcombe RF. Wnt pathway component LEF1 mediates tumor cell invasion and is expressed in human and murine breast cancers lacking ErbB2 (her-2/neu) overexpression. Int J Oncol 2005;27:949956. 27. Okamura RM, Sigvardsson M, Galceran J, Verbeek S, Clevers H, Grosschedl R. Redundant regulation of T cell differentiation and TCRα gene expression by the transcription factors LEF-1 and TCF-1. Immunity 1998;8:11-20. 28. Kühnl A, Gökbuget N, Kaiser M, Schlee C, Stroux A, Burmeister T, Mochmann LH, Hoelzer D, Hofmann WK, Thiel E, Baldus CD. Overexpression of LEF1 predicts unfavorable outcome in adult patients with B-precursor acute lymphoblastic leukemia. Blood 2011;118:6362-6367. 29. Metzeler KH, Heilmeier B, Edmaier KE, Rawat VP, Dufour A, Döhner K, Feuring-Buske M, Braess J, Spiekermann K, Büchner T, Sauerland MC, Döhner H, Hiddemann W, Bohlander SK, Schlenk RF, Bullinger L, Buske C. High expression of lymphoid enhancer-binding factor-1 (LEF1) is a novel favorable prognostic factor in cytogenetically normal acute myeloid leukemia. Blood 2012;120:2118-2126. 30. Gutierrez A, Sanda T, Ma W, Zhang J, Grebliunaite R, Dahlberg S, Neuberg D, Protopopov A, Winter SS, Larson RS, Borowitz MJ, Silverman LB, Chin L, Hunger SP, Jamieson C, Sallan SE, Look AT. Inactivation of LEF1 in T-cell acute lymphoblastic leukemia. Blood 2010;115:2845-2851. 31. Pui CH, Robison LL, Look AT. Acute lymphoblastic leukemia. Lancet 2008;371:1030-1043. 32. Pellagatti A, Marafioti T, Paterson JC, Malcovati L, Della Porta MG, Jädersten M, Pushkaran B, George TI, Arber DA, Killick S, Giagounidis A, Hellström-Lindberg E, Cazzola M, Wainscoat JS, Boultwood J. Marked downregulation of the granulopoiesis regulator LEF1 is associated with disease progression in the myelodysplastic syndromes. Br J Haematol 2009;146:86-90. 33. Minke KS, Staib P, Puetter A, Gehrke I, Gandhirajan RK, Schlösser A, Schmitt EK, Hallek M, Kreuzer KA. Small molecule inhibitors of WNT signaling effectively induce apoptosis in acute myeloid leukemia cells. Eur J Haematol 2009;82:165-175.

Research Article

DOI: 10.4274/tjh.2013.0325

Are the High Serum Interleukin-6 and Vascular Endothelial Growth Factor Levels Useful Prognostic Markers in Aggressive Non-Hodgkin Lymphoma Patients? Agresif Non-Hodgkin Lenfomalı Hastalarda, Serum İnterlökin-6 ve Vasküler Endoteliyal Büyüme Faktörü Düzeyleri Kullanışlı Prognostik Belirteçler midir? Hava Üsküdar Teke1, Eren Gündüz1, Olga Meltem Akay1, Cengiz Bal2, Zafer Gülbaş3 1Osmangazi

University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey University Faculty of Medicine, Department of Biostatistics, Eskişehir, Turkey 3Anadolu Health Center, Bone Marrow Transplantation Center, Kocaeli, Turkey 2Osmangazi

Abstract: Objective: Pro-inflammatory and pro-angiogenic cytokines play an important role in the pathogenesis of lymphoma, and recent studies have shown that cytokines can be used as prognostic markers. Non-Hodgkin lymphoma (NHL) patients with high levels of serum interleukin-6 (s-IL6) and serum vascular endothelial growth factor (s-VEGF) have poor prognosis and shorter survival time. We aimed to determine pre-treatment levels of s-IL6 and s-VEGF and their relation with known prognostic markers, especially International Prognostic Index (IPI) scores, and to examine their effects on overall survival in newly diagnosed, untreated aggressive NHL patients.

Materials and Methods: The study included 51 newly diagnosed NHL patients and 17 healthy controls. Blood samples were obtained to study s-IL6 and s-VEGF cytokine levels. Results: Patients with aggressive NHL diagnosis had higher s-VEGF and s-IL6 levels than the healthy population. If the s-IL6 levels of patients were above the cut-off levels, the overall survival time was shorter. There was no relation between s-VEGF and overall survival time. Conclusion: s-IL6 is an independent prognostic factor that may be included in IPI risk classification. In addition to the s-IL6 level, age, erythrocyte sedimentation rate, beta-2 microglobulin, WHO performance status, and IPI score are independent prognostic factors that are effective, especially for overall survival, in the clinical follow-up of NHL patients.

Key Words: s-IL6, s-VEGF, Lymphoma, Overall survival Özet: Amaç: Pro-inflamatuvar ve pro-angiogenik sitokinlerin, lenfomanın patogenezinde önemli rolleri olduğu kadar son yıllarda yapılan çalışmalarda sitokinlerin prognostik birer belirteç olarak da kullanılabileceği gösterilmiştir. s-IL6 ve s-VEGF düzeyleri Address for Correspondence: Hava ÜSKÜDAR TEKE, M.D., Osmangazi University Faculty of Medicine, Department of Hematology, Eskişehir, Turkey Phone: +90 222 239 29 79-3854 E-mail: havaus@yahoo.com Received/Geliş tarihi : September 24, 2013 Accepted/Kabul tarihi : December 16, 2013

Turk J Hematol 2015;32:21-28

Üsküdar Teke H, et al: s-IL6 and s-VEGF, Lymphoma

yüksek olan non-Hodgkin lenfoma (NHL) hastalarında prognoz daha kötüdür ve genel sağkalımları daha kısadır. Bu çalışmada yeni tanı almış, tedavi verilmemiş NHL hastalarında tedavi öncesi s-IL6 ve s-VEGF düzeylerinin belirlenmesi, bu sitokinlerin bilinen prognostik belirteçler ve özellikle International Prognostic Index (IPI) değerleri ile ilişkisinin saptanması ve genel sağkalım üzerine etkilerinin incelenmesi amaçlanmıştır.

Gereç ve Yöntemler: Çalışmaya 51 yeni tanı konmuş NHL hastası ve 17 sağlıklı kontrol alındı. s-IL6 ve s-VEGF sitokin düzeylerini değerlendirmek için kan örnekleri toplandı.

Bulgular: NHL ile izlenen hastalarda s-VEGF ve s-IL6 düzeyleri sağlıklı popülasyondan yüksektir. Hastaların özellikle s-IL6 düzeyleri cut-off değerin üzerinde ise genel sağkalımları kısa olmaktadır. s-VEGF ile genel sağkalım arasında ilişki saptanmamıştır. Sonuç: s-IL6, IPI risk sınıflamasına dahil edilebilir bağımsız bir prognostik faktördür. s-IL6 düzeyine ilave olarak, yaş, ESR, beta-2 mikroglobulin, WHO performans statüsü ve IPI skoru NHL hastalarının klinik takibinde özellikle genel sağkalım üzerine etkili olan bağımsız birer prognostik faktördür. Anahtar Sözcükler: s-IL6, s-VEGF, Lenfoma, Genel sağkalım Introduction Various clinical and laboratory parameters were included in the prognostic definition of non-Hodgkin lymphoma (NHL) and are used in clinical follow-up. Pro-inflammatory and pro-angiogenic cytokines play an important role in the pathogenesis of lymphoma, and recent studies have shown that cytokines can be used as prognostic markers. Interleukin (IL)-6, which is one of these cytokines, is a lymphoid growth factor and is also an important protein for the immune system, hematopoiesis, and inflammation. It is responsible for the B symptoms in lymphoma [1,2,3]. Angiogenesis, the formation of new blood vessels, is required for the growth and spread of cancer cells [4]. Angiogenesis is regulated by a variety of positive and negative angiogenic molecules. The most capable and the most important angiogenic molecule is vascular endothelial growth factor (VEGF). Lymphoma tumor cells express VEGF and have been shown to promote survival, proliferation, and metastasis via autocrine mechanisms [5]. NHL patients with high levels of serum (s)-IL6 and s-VEGF have poor prognosis and short survival [6]. In addition, the pre-treatment s-IL6 and s-VEGF levels of NHL patients are correlated with life expectancy, and both of these are independent and important prognostic indicators for all International Prognostic Index (IPI) groups [6,7]. However, there is no information available for patients from the Turkish population. In this study, we aimed to determine pre-treatment levels of s-IL6 and s-VEGF and their relation with known prognostic markers, especially IPI levels, and to examine their effects on overall survival (OS) in newly diagnosed, untreated aggressive NHL patients in a Turkish population. Materials and Methods Patients and Controls This study was performed between April 2006 and October 2007 in the Hematology Division of the Eskişehir Osmangazi

University (ESOGU) Medical Faculty’s Internal Medicine Department. The approval of the ESOGU Medical Faculty Ethics Committee and informed consent from subjects, including all necessary explanations, were obtained for this study. The study included 51 newly diagnosed NHL patients. Forty-five patients had diffuse large B-cell lymphoma, 4 had mantle cell lymphoma, and 2 had peripheral T-cell lymphoma. The patients consisted of individuals who were not being treated and had not used steroids in the last 1 month. The patients were followed from the time of diagnosis until October 2012 in terms of life expectancy. The control group consisted of 17 healthy individuals who were not taking any medication, did not have any acute or chronic disease, and had not had fever in the last 1 week. Patients receiving chemotherapy, using steroids in the last 1 month, or not agreeing to participate were excluded from the study. In the control group, people taking any medication, having any acute or chronic disease, or having fever in the last 1 week were excluded from the study. Clinical and Laboratory Evaluations In all cases, blood samples were taken in the morning after at least 8-12 h of fasting. Routine tests were performed immediately. For cytokine study, blood samples were again obtained in the morning after at least 8-12 h of fasting. We used empty tubes for serum and tubes containing EDTA for plasma samples. As soon as blood samples were put into tubes, they were transferred to icy media and forwarded to the laboratory within 5-10 min. Plasma and serum were separated by centrifuging at 3000 rpm and 4 °C for 10 min and were stored at -75 °C until use. All sera were then warmed to room temperature; s-IL6 and s-VEGF cytokine levels were studied with Panomics Company ProcartaTM Human Cytokine multiplex kits with the Luminex platform. Statistical Evaluation For statistical evaluation of the findings of this study, SPSS 15.0 for Windows was used and p<0.05 was considered

Turk J Hematol 2015;32:21-28

Üsküdar Teke H, et al: s-IL6 and s-VEGF, Lymphoma

statistically significant at a 95% confidence interval. Quantitative variables were given as mean ± standard deviation. Assumptions of normality were tested with the Shapiro-Wilk test. We used parametric tests for data with normal distribution and non-parametric tests for data with non-normal distribution. The independent samples t-test and Mann-Whitney U test were used for comparison of 2 independent groups. Spearman’s correlation coefficients were used to determine the relationships between the variables. The Kaplan-Meir method was used for survival analysis. A Cox regression model was used for multivariate analysis of the parameters that were effective on prognosis. Results

patients with H-I or H IPI scores and advanced Ann Arbor staging (p<0.001 and p=0.014, respectively; Table 1). Correlation of s-VEGF and s-IL6 with Overall Survival in Non-Hodgkin Lymphoma Patients The mean survival time of the patients based on the Kaplan-Meir method was 51.9 months (Figures 1 and 2). When the cut-off value of s-VEGF was taken as 11 pg/mL, OS was 50.8 months in the group with values below the cutoff and 50.9 months in the group with values above the cutoff (p=0.926; Figure 3). When the cut-off value of s-IL6 was taken as 1.28 pg/mL, OS was 66.2 months in the group with values below the cut-off and 38.1 months in the group with values above the cut-off (p=0.004; Figure 4). When these

Concentrations of s-VEGF and s-IL6 and Characteristics of Non-Hodgkin Lymphoma Patients When the s-VEGF median value (11 pg/mL) was taken as the cut-off value, there was no difference between the groups that were above and below the cut-off for B symptoms, age, stage, erythrocyte sedimentation rate (ESR), lactate dehydrogenase (LDH) level, mortality, beta-2 microglobulin level, or IPI score (p>0.05). In patients with higher s-VEGF levels, more profound lymphopenia (p=0.034), poor performance status (p=0.027), and higher platelet levels (p=0.035) were observed. When the s-IL6 median value (1.28 pg/mL) was taken as the cut-off value, more advanced age, presence of B symptoms (p=0.022), advanced stage (p=0.003), poor performance status (p=0.001), high IPI score (p=0.016), increased incidence of mortality (p=0.003), more profound lymphopenia (p=0.008), higher LDH levels (p=0.006), higher platelet counts (p=0.047), and lower albumin levels (p=0.014) were observed in patients with s-IL6 levels above the cut-off. When patients were grouped according to categories of over or below 60 years; WHO performance status of 0-1 or 2-4; serum LDH levels normal or above normal; Ann Arbor stage of I-II or III-IV; IPI score of low (L), low-intermediate (L-I), highintermediate (H-I), or high (H); and presence of B symptoms and were compared according to median s-VEGF and s-IL6, significantly higher values of s-IL6 were only observed in

Figure 1. Mean overall survival (OS) based on s-VEGF levels.

Cumulative survival (%)

The median age was 52 years (min: 18, max: 90) in the patient group and 48 years (min: 40, max: 63) in the control group. The median s-VEGF levels were 11 pg/mL (min: 1.28 pg/mL, max: 159.75 pg/mL) in the patient group and 1.27 pg/ mL (min: 1.00 pg/mL, max: 29.4 pg/mL) in the control group (p=0.002). The median levels of s-IL6 were 1.28 pg/mL (min: 1.10 pg/mL, max: 18.01 pg/mL) in the patient group and 1.27 pg/mL in the control group (p=0.001).

Cumulative survival (%)

s-VEGF and s-IL6 Levels in Patients and Healthy Controls

Figure 2. Mean overall survival (OS) based on s-IL6 levels.

Turk J Hematol 2015;32:21-28

patients were analyzed according to IPI score, no significant difference in survival was found between the patients with s-VEGF and s-IL6 values above or below the cut-off values in patients with IPI scores of L and L-I (p=0.952, p = 0.282, respectively; Figures 5 and 6). In patients with IPI scores of H-I and H, while there was no difference between the groups with s-VEGF levels above and below the cut-off (p=0.420), significantly lower survival was seen in the group of patients with s-IL6 levels above the cut-off (p=0.035; Figures 7 and 8). Survival was found shorter in patients aged ≥60 years (34

Figure 3. Overall survival (OS) curves of patients with lymphoma based on serum s-VEGF level (cut-off level: 11 pg/ mL; p=0.926).

Figure 4. Overall survival (OS) curves of patients with lymphoma based on serum s-IL6 level (cut-off level: 1.28 pg/ mL; p=0.004).

Üsküdar Teke H, et al: s-IL6 and s-VEGF, Lymphoma

months) and longer in patients younger than 60 years (62.5 months) (p=0.005; Figure 9). Prognostic Factors in Multivariate Analysis of NonHodgkin Lymphoma Patients Multivariate analysis performed using Cox regression analysis showed no relation between survival and s-VEGF, age, sedimentation, beta-2 microglobulin, ECOG performance, or high IPI scores (p>0.05). NHL patients with high s-IL6 levels, advanced age, elevated ESR, high beta-2 microglobulin, poor ECOG performance, and high IPI scores had a shorter

Figure 5. Overall survival (OS) curves of International Prognostic Index (IPI) risk group L + L-I patients with lymphoma based on serum s-VEGF level (cut-off level: 11 pg/ mL; p=0.952).

Figure 6. Overall survival (OS) curves of International Prognostic Index (IPI) risk group L + L-I patients with lymphoma based on serum s-IL6 level (cut-off level: 1.28 pg/ mL; p=0.282).

Üsküdar Teke H, et al: s-IL6 and s-VEGF, Lymphoma

duration of life and poorer prognosis. These parameters are independent risk factors in the follow-up of NHL patients (Table 2). Discussion s-IL6 and s-VEGF levels of NHL patients are found to be higher than those of normal healthy controls [6]. In our study, both s-IL6 and s-VEGF levels of the NHL patient group were significantly higher than those of healthy controls. Age is an important prognostic factor for both morbidity and mortality in NHL patients. In our study, overall survival time was significantly shorter in the patient group of ≥60 years of age.

Figure 7. Overall survival (OS) curves of International Prognostic Index (IPI) risk group H-I + H patients with lymphoma based on serum s-VEGF level (cut-off level: 11 pg/ mL; p=0.420).

Figure 8. Overall survival (OS) curves of International Prognostic Index (IPI) risk group H-I + H patients with lymphoma based on the serum s-IL6 level (cut-off level: 1.28 pg/mL; p=0.035).

Turk J Hematol 2015;32:21-28

IPI score is an important prognostic marker for follow-up of patients with particularly aggressive lymphoma [8]. Recent studies showed that some prognostic factors may be included in the IPI or may be used in combination. These cytokines include s-IL6 and s-VEGF [5,6,7]. Pre-treatment levels of s-IL6 and s-VEGF are correlated with the survival of NHL patients, and both cytokines are important independent predictors of prognosis for all IPI risk groups. Disease-free survival time and OS time are shorter in NHL patient groups, especially among those with s-IL6 and s-VEGF values above the cut-off [6,9]. In our study, we found significantly shorter OS in the patient group with s-IL6 levels above the cut-off. When the IPI risk groups were divided into 2 as L + L-I and H-I + H, we found significantly shorter life expectancy and poorer prognosis in the H-I + H group patients, and especially those with s-IL6 levels above the cut-off. Unlike other studies, we did not find a difference between s-VEGF levels and the OS time or IPI risk group. As a result, s-IL6 is an independent prognostic factor that may be included in IPI risk classification. In NHL patients, LDH levels, bulky mass, beta-2 microglobulin levels, performance status, presence of B symptoms, age, advanced stage of disease, and extranodal involvement are well-known and commonly used prognostic factors. There is usually a significant correlation among poor performance status, high LDH levels, large bulky mass, and presence of B symptoms in patients having high levels of s-IL6 and s-VEGF [6,10,11]. When the median s-VEGF value was taken as the cut-off (11 pg/mL), we did not find any differences in B symptoms, age, stage, ESR, LDH, mortality, or beta-2 microglobulin level between groups below and above the cut-off. We found more profound lymphopenia, poorer performance status, and higher platelet counts in the patient group with higher s-VEGF levels. When the median s-IL6

Figure 9. Overall survival (OS) time in patients with advanced age (≥60 years; p=0.005).

Turk J Hematol 2015;32:21-28

Üsküdar Teke H, et al: s-IL6 and s-VEGF, Lymphoma

Table 1. Correlation of serum levels of s-VEGF and s-IL6 with clinical prognostic factors in lymphoma.

Characteristic

Median VEGF level, pg/mL

p-value/ u-value

30 21

11.77 8.75

0.673/293

38 13

9.91 11.77

0.745/232

1.27 1.96

0.088/171

Serum LDH level Normal >Normal

19 32

6.95 12.45

0.374/258.5

1.27 1.37

0.679/283.5

Ann Arbor stage I-II III-IV

21 30

8.67 12.45

0.220/251

1.27 1.88

<0.001/135.5

IPI L + L-I H-I + H

26 25

8.36 13.14

0.322/272.5

1.27 1.96

0.014/199.5

B symptoms +

12 39

5.66 13.14

0.230/180

1.27 1.45

0.328/191.5

Age (years) ≥60 <60 WHO performance status 0-1 2-4

Median IL-6 level, pg/mL

p-value/ u-value

No. of patients

1.45 1.27

0.250/257

Mann-Whitney U test, LDH: lactate dehydrogenase, IPI: International Prognostic Index

Table 2. Multivariate analysis of serum s-IL6, age, erythrocyte sedimentation rate, beta-2 microglobulin, WHO performance status, and International Prognostic Index (IPI) score.

p-value

%95 Cl for OR (lower-upper)

s-IL6

0.478

<0.001

1.613

(1.245-2.088)

Age

0.141

<0.001

1.151

(1.069-1.240)

ESR

-0.061

<0.001

0.941

(0.910-0.973)

Beta-2 microglobulin

0.408

0.023

1.532

(1.059-2.136)

WHO performance status

-2.944

0.001

0.03

(0.000-0.054)

IPI

3.522

0.013

33.858

(2.104-544.19)

IPI: International Prognostic Index, ESR: erythrocyte sedimentation rate

value was taken as the cut-off (1.28 pg/mL), we found more advanced age, more presence of B symptoms, more advanced stage, poorer performance status, higher IPI scores, increased

mortality, more profound lymphopenia, higher LDH, higher platelet count, and lower serum albumin in the patient group with s-IL6 levels above the cut-off. When patients

Turk J Hematol 2015;32:21-28

Üsküdar Teke H, et al: s-IL6 and s-VEGF, Lymphoma

were grouped in categories of <60 years or ≥60 years, WHO performance status of 0-1 or 2-4, serum LDH levels normal or above normal, Ann Arbor stage I-II or stage III-IV, IPI score of L + L-I or H + H-I, and presence or absence of B symptoms and compared according to median s-VEGF and s-IL6 levels, we only found significantly higher s-IL6 levels in patients with H + H-I IPI scores and advanced Ann Arbor stage. We did not find any similar relationships between s-VEGF level and LDH, B symptoms, age, stage, ESR, LDH, mortality, beta-2 microglobulin level, or the value of the IPI score as were seen in the study of Xia et al. [11]. It should be kept in mind that the patient group with s-IL6 levels above the cut-off values may proceed to lymphopenia, hypoalbuminemia, advanced stage, B symptoms, poor performance status, thrombocytosis, elevated LDH, and high mortality. Disease-free survival time and OS time are short in NHL patient groups, especially those with s-IL6 and s-VEGF values above the cut-off. These 2 cytokines can be used separately or in combination as independent prognostic factors [6,12,13,14]. In our study, we found strong correlations between s-IL6, age, ESR, beta-2 microglobulin, WHO performance status, and IPI score and OS. This shows that when high s-IL6, high ESR, high beta-2 microglobulin, poor WHO performance status, and high IPI score are detected in NHL patients, their duration of life may be predicted to be shorter. However, we could not find a difference between s-VEGF and survival due to the small patient sample. Conclusion Patients with aggressive NHL diagnosis have higher s-VEGF and s-IL6 levels than healthy populations. In particular, if s-IL6 levels of patients are above the cut-off, OS is shorter. There was no relation between s-VEGF and OS. In addition to the s-IL6 level, age, ESR, beta-2 microglobulin, WHO performance status, and IPI score are independent prognostic factors that are effective, especially for OS, in the clinical follow-up of NHL patients. NHL patients with high levels of s-IL6 and s-VEGF have been reported to have poor prognosis and short survival before. However, to the best of our knowledge, there is no information available for patients from a Turkish population. We think that this is the novelty of our study. Conflict of Interest Statement 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. Voorzanger N, Toituou R, Garcia E, Delecluse HJ, Rousset F, Joab I, Favrot MC, Blay JY. Interleukin (IL)-10 and IL-6 are produced in vivo in non-Hodgkin’s lymphoma cells and act as cooperative growth factors. Cancer Res 1996;56:5499-5505. 2. Aydin F, Yilmaz M, Ozdemir F, Kavgaci H, Yavuz MN, Yavuz AA. Correlation of serum IL-2, IL-6 and IL-10 with international prognostic index in patients with aggressive non-Hodgkin’s lymphoma. Am J Clin Oncol 2002;25:570-572. 3. Gaiolla R, Domingues M, Melo L, Oliveira D. Serum levels of interleukins 6, 10, and 13 before and after treatment of classic Hodgkin lymphoma. Arch Pathol Lab Med 2011;135:483-489. 4. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669-676. 5. Rijhijarvi S, Nurmi H, Holte H, Björkholm M, Fluge O, Pedersen ML, Rydström K, Jerkeman M, Mikael E, Leppa S. High serum vascular endothelial growth factor level is an adverse prognostic factor for high-risk diffuse large B-cell lymphoma patients treated with dose-dense chemoimmunotherapy. Eur J Haematol 2012;89:395-402. 6. Niitsu N, Okamoto M, Nakamine H, Yoshino T, Tamaru J, Nakamura S, Higashihara M, Hirano M. Simultaneous elevation of the serum concentrations of vascular endothelial growth factor and interleukin-6 as independent predictors of prognosis in aggressive non-Hodgkin’s lymphoma. Eur J Haematol 2002;68:91-100. 7. Pedersen ML, Klausen WT, Davidsen HU, Johnsen EH. Early changes in serum IL-6 and VEGF levels predict clinical outcome following first-line therapy in aggressive nonHodgkin’s lymphoma. Ann Hematol 2005;84:510-516. 8. [No authors listed.] A predictive model for aggressive nonHodgkin’s lymphomas. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. N Engl J Med 1993;329:987-994. 9. Salven P, Teerenhovi L, Joensuu H. A high pre-treatment serum vascular endothelial growth factor concentration is associated with poor outcome in non-Hodgkin’s lymphoma. Blood 1997;90:3167-3172. 10. Duan Y, Li G, Hu HX. Clinical significance of serum lactate dehydrogenase, β2-microglobulin and vascular endothelial growth factor level detection in patients with nonHodgkin’s lymphoma. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2012;20:608-610. 11. Xia Y, Sun XF, Zhang CQ, Zhen ZJ, Wang ZH, Wang ZQ. Primary study of relationship between serum level of VEGF and non-Hodgkin’s lymphoma in children and adolescent patients. Ai Zheng 2004;23:1448-1450.

Turk J Hematol 2015;32:21-28

12. Potti A, Ganti AK, Kargas S, Koch M. Immunohistochemical detection of C-kit (CD-117) and vascular endothelial growth factor (VEGF) overexpression in mantle cell lymphoma. Anticancer Res 2002;22:2899-2901. 13. Fabre-Guillevin E, Tabrizi R, Coulon V, Monnereau A, Eghbali H, Soubeyran I, Soubeyran P. Aggressive non-Hodgkin’s lymphoma: concomitant evaluation of interleukin-2, soluble interleukin-2 receptor, interleukin-4, interleukin-6, interleukin-10 and correlation with outcome. Leuk Lymphoma 2006;47:603-611.

Üsküdar Teke H, et al: s-IL6 and s-VEGF, Lymphoma

14. Fayad L, Cabanillas F, Talpaz M, McLaughlin P, Kurzrock R. High serum interleukin-6 levels correlate with a shorter failure-free survival in indolent lymphoma. Leuk Lymphoma 1998;30:563-571.

Research Article

DOI: 10.4274/tjh.2013.0288

Prevalence of Monoclonal B Lymphocytosis in FirstDegree Relatives of Chronic Lymphocytic Leukemia Patients in Turkey Türkiye’de Kronik Lenfositik Lösemi Tanılı Hastaların Birinci Derece Akrabalarında Monoklonal B Lenfositoz Sıklığının Araştırılması Taner Demirci1, Zeynep Arzu Yeğin2, Nevruz Kurşunoğlu2, Zeynep Yılmaz2, Elif Suyanı2, Zübeyde Nur Özkurt2, Münci Yağcı2 1Gazi

University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey

2Gazi

University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract: Objective: Monoclonal B lymphocytosis (MBL) is considered to be a precursor state for chronic lymphocytic leukemia (CLL). This study was planned to evaluate the MBL prevalence in first-degree relatives of CLL patients in Turkey, which is considered to be an ethnic and geographic bridge between the Eastern and Western worlds. Materials and Methods: A total of 136 volunteers [median age: 40 (17-77) years; male/female: 60/76] from 61 families were included. Flow cytometry analysis by 4-colour staining was used for MBL diagnosis.

Results: MBL was demonstrated in 17 cases (12.5%). A total of 14 cases (10.3%) were classified as CLL-like MBL, while 3 (2.2%) exhibited a non-CLL-like phenotype. The prevalence of MBL was 12.72% in subjects aged less than 40 years, 12.28% in subjects between 40 and 60 years, and 40% in subjects over 60 years, without statistical significance (p>0.05). A total of 115 cases were evaluated for intermarriage, which was observed in 19 cases (16.5%). The prevalence of MBL did not differ based on intermarriage status (p>0.05). Conclusion: The current report is the first MBL prevalence study in a Eurasian population that demonstrates a similar distribution pattern of MBL in Anatolian CLL kindreds. Further efforts should be made to refine our understanding of the natural history and clinical outcomes of MBL.

Key Words: Monoclonal B lymphocytosis, Prevalence, Chronic lymphocytic leukemia, First-degree relatives Özet: Amaç: Monoklonal B lenfositozun (MBL) kronik lenfositik lösemi (KLL) için öncül bir durum olduğu düşünülmektedir. Bu çalışma doğu ve batı dünyası arasında etnik ve coğrafi köprü olduğu düşünülen Türkiye’de KLL hastalarının birinci derece akrabalarında MBL sıklığının araştırılması için tasarlanmıştır. Address for Correspondence: Taner Demİrcİ, M.D., Gazi University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey Phone: +90 312 202 63 17 E-mail: tnrdemirci@gmail.com Received/Geliş tarihi : August 25, 2013 Accepted/Kabul tarihi : December 25, 2013

Turk J Hematol 2015;32:29-34

Demirci T, et al: MBL in Anatolia: A Report from the Bridge of East&West

Gereç ve Yöntemler: Çalışmaya 61 aileden olmak üzere toplam 136 gönüllü [ortalama yaş 40 (17-77); erkek/kadın: 60/76] dahil edilmiştir. MBL tanısı içi dört farklı boyama ile akım sitometri analiz yöntemi kullanılmıştır. Bulgular: Monoklonal B lenfositoz 17 (%12,5) olguda tespit edilmiştir. Olguların 14’ü (%10,3) KLL benzeri MBL fenotipi olarak sınıflandırılırken kalan 3 olgu (%2,2) KLL dışı MBL olarak sınıflandırılmıştır. MBL prevalansı 40 yaş altı olgularda %12,72, 40-60 yaş arası olgularda %12,28, 60 yaş üstü olgularda %40 olarak gözlenmiştir ve istatistiksel olarak anlamlı değildir (p>0,05). Akraba evliliği yönünden 115 olgu değerlendirilmiş olup bunlardan 19’unda (%16,5) akraba evliliği öyküsü mevcuttu. MBL prevalansı akraba evliliği durumu ile farklılık göstermiyordu (p>0,05). Sonuç: Bu araştırma Avrasya topluluğunda yapılan ve aynı etnik özellikleri taşıdığı Anadolu kökenli KLL hastalarının akrabalarında MBL dağılım şeklini gösteren ilk MBL prevalans çalışmasıdır. MBL’nin doğal gelişimini ve klinik sonuçlarını daha iyi anlayabilmemiz için daha fazla çaba harcanmalıdır. Anahtar Sözcükler: Monoklonal B lenfositoz, Prevalans, Kronik lenfositik lösemi, Birinci derece akraba Introduction Chronic lymphocytic leukemia (CLL) is the most common haematological malignancy, which accounts for 30% of all leukemias in the Western world [1,2,3,4,5,6,7,8]. Despite knowledge of the classical risk factors of male sex, advanced age, white race, and a family history of CLL, the aetiology and pathogenesis remain largely unknown [3,4,5,6,7,9]. In previous reports, the presence of familial clustering in CLL has been widely confirmed. Approximately 12% of patients with CLL report a family history of a lymphoproliferative disease, while 6-9% have a relative who has also CLL [2,3,4,10]. Based on epidemiological studies, first-degree relatives of CLL patients have 3-8 times greater risk for the development of CLL [2]. Diagnostic criteria for CLL are the presence of monoclonal B lymphocytes with CD5, CD19, and CD23 expression, and weak or no expression of CD20, CD79b, FMC7, and surface immunoglobulin. The monoclonality should be represented in the majority of leukocytes with an absolute lymphocyte count (ALC) of >5x109/L [7,11,12,13,14]. Monoclonal B lymphocytosis (MBL) is considered to be a precursor state for CLL, similarly to the association of monoclonal gammopathy of undetermined significance (MGUS) and plasma cell myeloma. According to the current data, CLL is accepted to be preceded by a MBL state. A prediagnostic B cell clone was demonstrated in 98% of CLL patients 77 months before the CLL diagnosis [7,9,15]. MBL is defined as the presence of a clonal B cell population in the context of an absolute B cell count (ABCC) of <5x109/L, no history of autoimmune disease, and no evidence of lymphadenopathy and organomegaly on physical examination [3,7,9,12,13,14,15,16,17,18,19]. Monoclonal B lymphocytosis is subclassified into 3 immunophenotypic categories: CLL-like (CD5+23+), atypical CLL (CD5+20bright), and non-CLL (CD5). The most common MBL subtype is CLL-like MBL, while atypical and non-CLL types of MBL account for only 15%30% of all MBL cases [7,9,13,14,15,16]. MBL and CLL share a similar genetic profile. Deletion of 13q and trisomy 12 are 30

identified in MBL at similar frequencies to CLL, but the higher risk abnormalities such as deletions of 11q and 17p appear to be rare in MBL. To our knowledge, 88%-96% of MBLs have mutated immunoglobulin heavy variable group genes, with intraclonal heterogeneity similar to that of CLL [2,13,15]. The prevalence of MBL is dependent largely on the characteristics of the study population and the sensitivity of flow cytometry methods. The lack of standardisation in this context complicates determination of the true prevalence, as highly sensitive flow cytometry methods reveal higher estimates. As a result, prevalence in the general adult population is reported to be 0.1%-14%, indicating a wide range [14,17]. MBL is more common in CLL families. The prevalence of MBL among first-degree relatives of CLL patients ranges between 12% and 18% [1,10,14]. At present, detailed information on epidemiological characteristics of MBL is not available. In particular, MBL status in Asia or Africa is entirely unknown, although CLL is reported to be less common in Eastern parts of the world [2,3,4,7]. This study was projected to evaluate the MBL prevalence in first-degree relatives of CLL patients in Turkey, which is considered to be an ethnic and geographic bridge between the Eastern and Western worlds. Materials and Methods A total of 136 first-degree volunteer relatives [median age: 40 (17-77) years; male/female: 60/76] from 61 CLL families were included. We utilised the recently defined diagnostic criteria for MBL [12]: 1) detection of a disease-specific immunophenotype or an overall kappa (κ)/lambda (λ) ratio of >3:1 or <0.3:1, 2) stable monoclonal B cell population over a 3-month period, and 3) absence of lymphadenopathy, organomegaly, and autoimmune or infectious diseases, and B lymphocyte counts of <5x109/L. MBL was classified as CLLlike (CD5+23+) or non-CLL (CD5-) types. Flow cytometry analysis by 4-colour staining was performed using peripheral blood collected into EDTA. All samples were analysed on a FACSCalibur flow cytometer

Demirci T, et al: MBL in Anatolia: A Report from the Bridge of East&West

(Becton Dickinson). Monoclonal antibodies IgM fluorescein isothiocyanate (FITC), IgD phycoerythrin (PE), and IgG FITC were purchased from BD Pharmingen and all the rest from Becton Dickinson. A total of 200,000 events per tube were acquired. Sequential gating strategy was used as previously described [3,20]. A 2-step analysis method was used for MBL diagnosis. The initial panel consisted of CD5 allophycocyanin (APC) and CD19 peridin-chlorophyll protein (PerCP)/anti-κ FITC/ anti-λ PE. Cells were first evaluated by biparametric graphics based on CD5 and CD19 expressions (Figure 1A). R1 and R2 represented CD5-CD19+ and CD5+CD19+ cells, respectively. Selected cells on R1 and R2 were then analysed for κ and λ expressions. R3/R5 and R4/R6 showed κ- and λ-positive cells, respectively (Figures 1B and 1C). Monoclonality was detected by light chain restriction, which was defined as κ/λ of >3:1 or <0.3:1. Whole blood count and second panels were performed in cases of B cell clonality in the initial panel. The second panel was arranged based on the presence of lymphocytosis. If lymphocytosis was detected, the panel included CD3 FITC/CD3 control PE/CD19 PerCP; CD20 FITC/CD5 PE/CD19 PerCP; CD10 FITC/CD38 PE/CD19 PerCP; FMC7 FITC/CD22 PE/CD19 PerCP; CD11a FITC/ CD23 PE/CD19 PerCP; IgM FITC/IgD PE/CD19 PerCP; and IgG FITC/CD79b PE/CD19 PerCP. If lymphocyte count was found to be normal, the analysis was switched to CD20 FITC/ CD79b PE/CD19 PerCP; FMC7 FITC/CD23 PE/CD19 PerCP; and CD5 APC profile.

Turk J Hematol 2015;32:29-34

A total of 115 cases were evaluated for intermarriage, which was observed in 19 cases (16.5%). The prevalence of MBL did not differ based on intermarriage status (p>0.05). The geographic distribution of the 115 subjects was also evaluated. A total of 51 (44.3%) were from Central Anatolia, 2 (1.7%) from West Anatolia, 4 (3.5%) from South Anatolia, 19 (16.5%) from East Anatolia, 3 (2.6%) from South-East Anatolia, 34 (29.6%) from North Anatolia, 1 (0.9%) from North-West Anatolia, and 1 (0.9%) from Cyprus. MBL was more common in South (25%) and Central (17.6%) Anatolia. No MBL cases from North, West, and South-East Anatolia or Cyprus were found. The difference in MBL prevalence among geographic regions of Turkey was not found to be significant (p>0.05). Characteristics of the studied subjects are detailed in Table 1. As the prevalence of MBL is indicated to vary based on age distribution [14,17], a total of 117 subjects including 16 MBL cases were divided into age groups defined as <40 years, 40-60 years, and >60 years. The prevalence of MBL was 12.72% (7/55) in subjects aged less than 40 years, 12.28% (7/57) in subjects between 40 and 60 years, and 40% (2/5) in subjects over 60

The study was approved by the Ethics Committee of the Turkish Ministry of Health and informed consent was received from all participants. Statistical Analysis Statistical analysis of the data was performed with SPSS 15 (SPSS Inc., Chicago, IL, USA). Continuous variables were presented as median values, whereas categorical variables were presented as frequencies and percentages. Differences between categorical variables were evaluated with chi-square or Fisher’s exact test. Continuous variables were compared by Mann-Whitney U test for 2 independent groups or KruskalWallis test for 3 or more groups. P-values of less than 0.05 were considered to be statistically significant.

κ/λ : 2.3

κ/λ : 4.14

Results MBL was demonstrated in 17 cases (12.5%). A total of 14 cases (10.3%) were classified as CLL-like MBL, while 3 (2.2%) exhibited the non-CLL-like phenotype. Median ALC was found to be 2.2x109/L (1.5-3.9) in MBL cases and 2.25x109/L (1.5-3) in normal subjects (p>0.05). Median ABCC in MBL cases was 1.48x109/L (0.3-2.8). Prevalence of MBL was not statistically different in male and female subjects (15% vs. 10.5%, respectively) (p>0.05) (Figure 2).

Figure 1. A) Evaluation of CD5 and CD19 expressions by biparametric graphics. R1 and R2 represent CD5-CD19+ and CD5+CD19+ cells, respectively. B) R3 shows CD5-CD19+κ+ and R4 shows CD5-CD19+λ+ cells. C) R5 shows CD5+CD19+κ+ and R6 shows CD5+CD19+λ+ cells. 31

Turk J Hematol 2015;32:29-34

Demirci T, et al: MBL in Anatolia: A Report from the Bridge of East&West

Table 1. Characteristics of the studied subjects. 8/76 9/60

Figure 2. Monoclonal B lymphocytosis in male and female subjects.

7/55

7/57

Characteristics

Value

Age (years), median (range)

40 (17-77)

Age groups (years), n=117, n (%) <20 20-29 30-39 40-49 50-59 60-69 >70

2 (1.7%) 19 (16.2%) 34 (29.1%) 38 (32.5%) 19 (16.2%) 3 (2.6%) 2 (1.7%)

Sex (male/female), n

60/76

CD5+/CD19+ κ/λ, median (range)

1.66 (0.41-5.10)

CD5-/CD19+ κ/λ, median (range)

1.63 (0.69-3.88)

Frequency of MBL, n (%) CD5-/CD19+ CD5+/CD19+

17 (12.5%) 3 (2.2%) 14 (10.3%)

Absolute lymphocyte count (ACL) in 2200 (1500-3900) MBL(+) cases, /µL Absolute lymphocyte count (ACL) in 2250 (1500-3000) MBL(-) cases, /µL 2/5

Figure 3. Monoclonal B lymphocytosis among age groups. years, without statistical significance (p>0.05) (Figure 3). Discussion The present study investigated the prevalence of MBL in 136 first-degree relatives from 61 families of patients with CLL. MBL was demonstrated in 17 cases (12.5%). A total of 14 cases (10.3%) were classified as CLL-like MBL, while 3 (2,2%) displayed the non-CLL phenotype. Variations in MBL prevalence can be attributed to study design, study population, and sensitivity of flow cytometric techniques [16]. Despite the variability in prevalence estimates, it is obvious that MBL is more common among the elderly and first-degree relatives of CLL patients. The prevalence of MBL in our cohort seems to confirm the previously reported data [1,2,3,7,10,21]. As intermarriage is frequently encountered in certain regions of Anatolia, we did analyse the possible impact of intermarriage on MBL prevalence in CLL families. However, we did not find any difference in MBL prevalence between families with and without intermarriage.

Intermarriage, n=115, n (%) Yes No

19 (16.5%) 96 (83.5%)

Geographic distribution, n=115, n (%) Central Anatolia West Anatolia South Anatolia East Anatolia South-East Anatolia North Anatolia North-West Anatolia Cyprus

51 (44.3%) 2 (1.7%) 4 (3.5%) 19 (16.5%) 3 (2.6%) 34 (29.6%) 1 (0.9%) 1 (0.9%)

κ: kappa, λ: lambda, MBL: monoclonal B lymphocytosis.

The reported prevalence of CLL-like MBL has increased significantly over time, representing up to 85% of all MBL cases. However, even in studies using very sensitive flow cytometry methods, the prevalence of CD5- MBL is considered to be around 2.3%, in concordance with our results [19,21]. The lack of large clinical series for CD5- MBL might be due to less peripheral blood involvement of lymphoproliferative diseases other than CLL. The age and sex distribution of MBL is similar to that of CLL, as prevalence correlates with a male predominance

Turk J Hematol 2015;32:29-34

Demirci T, et al: MBL in Anatolia: A Report from the Bridge of East&West

and older age [15,21,22]. We also demonstrated an increase in MBL prevalence in elderly subjects, without statistical significance. This insignificance might be explained by the small sample size, as there were only 5 individuals who were >60 years in our study group. Similarly, we did not find any significant difference in MBL prevalence between males and females. The clinical subgroups of CLL-like MBL can be defined as clinical MBL and low-count MBL (defined as <0.15x109 MBL cells/L). Clinical MBL refers to the MBL cases presented with lymphocytosis. The second category is represented by the MBLs discovered while screening [18,22]. Rawstron et al. investigated 1520 subjects with a normal blood count and 2228 subjects with lymphocytosis. A total of 185 subjects with CLLlike MBL and lymphocytosis were monitored for a median of 6.7 years. Progressive lymphocytosis occurred in 51 (28%), progressive CLL developed in 28 (15%), and chemotherapy was required in 13 (7%). The ABCC was the only independent prognostic factor associated with progressive lymphocytosis. Treatment requirements developed in subjects with clinical MBL at a rate of 1.1% per year, which is similar to the rate of progression to myeloma seen in patients with MGUS [13]. Median ALC was found to be 2.2x109/L (1.5-3.9) and median ABCC was 1.48x109/L (0.3-2.8) in our MBL cases. We could not make a comment on the outcome of MBLs in this study, as follow-up data are not currently available. Rossi et al. compared 123 clinical MBL and 154 Rai 0 CLL patients according to clinical and biological profiles. The best B cell thresholds for the lowest and highest risk of progression to CLL were respectively defined as <1.2x109/L and >3.7x109/L [18]. Shanafelt et al. identified a B cell threshold of 11x109/L for the best prediction of survival in a study of 459 patients with a clonal CLL-like cell population. Similarly, Molica et al. investigated 1158 patients with newly diagnosed Binet A CLL and identified an ALC of 11.5x109/L and ABCC of 10x109/L as the best B cell thresholds for treatment requirement [15,23,24]. Generally, the use of ABCC rather than ALC for the diagnosis of CLL or MBL is recommended; however, a specific cut-off value that could be used for the discrimination of these entities has not been defined yet. Definite comment about the progression risk cannot be made, as MBL patients were not prospectively identified or followed [7,9,13]. The current report is the first MBL prevalence study in a Eurasian population that demonstrates a similar distribution pattern of MBL in Anatolian CLL kindreds. Further efforts should be made to refine our understanding of the natural history and clinical outcomes of MBL. Conflict of Interest Statement 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. Marti GE, Carter P, Abbasi F, Washington GC, Jain N, Zenger VE, Ishibe N, Goldin L, Fontaine L, Weissman N, Sgambati M, Fauget G, Bertin P, Vogt RF Jr, Slade B, Noguchi PD, StetlerStevenson MA, Caporaso N. B-cell monoclonal lymphocytosis and B-cell abnormalities in the setting of familial B-cell chronic lymphocytic leukemia. Cytometry B Clin Cytom 2003;52:1-12. 2. Brown JR. Inherited predisposition to chronic lymphocytic leukemia. Exp Rev Hematol 2008;1:51-61. 3. Matos DM, Ismael SJ, Scrideli CA, de Oliveira FM, Rego EM, Falcão RP. Monoclonal B-cell lymphocytosis in first-degree relatives of patients with sporadic (non-familial) chronic lymphocytic leukemia. Br J Haematol 2009;147:339-346. 4. Slager SL, Kay NE. Familial chronic lymphocytic leukemia: what does it mean to me? Clin Lymphoma Myeloma 2009;9(Suppl 3):194-197. 5. Goldin LR, Landgren O, Marti GE, Caporaso NE. Familial aspects of chronic lymphocytic leukemia, monoclonal B-cell lymphocytosis (MBL), and related lymphomas. European J Clin Med Oncol 2010;2:119-126. 6. Goldin LR, Slager SL, Caporaso NE. Familial chronic lymphocytic leukemia. Curr Opin Hematol 2010;17:350355. 7. Shim YK, Middleton DC, Caporaso NE, Rachel JM, Landgren O, Abbasi F, Raveche ES, Rawstron AC, Orfao A, Marti GE, Vogt RF. Prevalence of monoclonal B-cell lymphocytosis: a systematic review. Cytometry B Clin Cytom 2010;78(Suppl 1):10-18. 8. Seifert M, Sellmann L, Bloehdorn J, Wein F, Stilgenbauer S, Dürig J, Küppers R. Cellular origin and pathophysiology of chronic lymphocytic leukemia. J Exp Med 2012;209:21832198. 9. Landgren O, Albitar M, Ma W, Abbasi F, Hayes RB, Ghia P, Marti GE, Caporaso NE. B-cell clones as early markers for chronic lymphocytic leukemia. N Engl J Med 2009;360:659-667. 10. Abbasi F, Longo NS, Lipsky PE, Raveche E, Schleinitz TA, Stetler-Stevenson M, Caporaso N, Marti G. B-cell repertoire and clonal analysis in unaffected first degree relatives in familial chronic lymphocytic leukemia kindred. Br J Haematol 2007;139:820-823. 11. Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O’Brien S, Rai KR. National Cancer Institute-Sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996;87:49904997. 12. Marti GE, Rawstron AC, Ghia P, Hillmen P, Houlston RS, Kay N, Schleinitz TA, Caporaso N. International Familial CLL Consortium. Diagnostic criteria for monoclonal B-cell lymphocytosis. Br J Haematol 2005;130:325-332.

Turk J Hematol 2015;32:29-34

13. Rawstron AC, Bennett FL, O’Connor SJ, Kwok M, Fenton JA, Plummer M, de Tute R, Owen RG, Richards SJ, Jack AS, Hillmen P. Monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia. N Engl J Med 2008;359:575-583. 14. Mowery YM, Lanasa MC. Clinical aspects of monoclonal B-cell lymphocytosis. Cancer Control 2012;19:8-17. 15. Kern W, Bacher U, Haferlach C, Dicker F, Alpermann T, Schnittger S, Haferlach T. Monoclonal B-cell lymphocytosis is closely related to chronic lymphocytic leukemia and may be better classified as early-stage CLL. Br J Haematol 2012;157:86-96. 16. Rachel JM, Zucker ML, Fox CM, Plapp FV, Menitove JE, Abbasi F, Marti GE. Monoclonal B-cell lymphocytosis in blood donors. Br J Haematol 2007;139:832-836. 17. Nieto WG, Almeida J, Romero A, Teodosio C, López A, Henriques AF, Sánchez ML, Jara-Acevedo M, Rasillo A, González M, Fernández-Navarro P, Vega T, Orfao A. Primary Health Care Group of Salamanca for the Study of MBL. Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach. Blood 2009;114:33-37. 18. Rossi D, Sozzi E, Puma A, De Paoli L, Rasi S, Spina V, Gozzetti A, Tassi M, Cencini E, Raspadori D, Pinto V, Bertoni F, Gattei V, Lauria F, Gaidano G, Forconi F. The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukemia and is recapitulated by biological risk factors. Br J Haematol 2009;146:64-75.

Demirci T, et al: MBL in Anatolia: A Report from the Bridge of East&West

19. Rawstron AC. Occult B-cell lymphoproliferative disorders. Histopathology 2011;58:81-89. 20. Rawstron AC, Kennedy B, Evans PA, Davies FE, Richards SJ, Haynes AP, Russell NH, Hale G, Morgan GJ, Jack AS, Hillmen P. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy. Blood 2001;98:29-35. 21. Goldin LR, Lanasa MC, Slager SL, Cerhan JR, Vachon CM, Strom SS, Camp NJ, Spector LG, Leis JF, Morrison VA, Glenn M, Rabe KG, Achenbach SJ, Algood SD, Abbasi F, Fontaine L, Yau M, Rassenti LZ, Kay NE, Call TG, Hanson CA, Weinberg JB, Marti GE, Caporaso NE. Common occurrence of monoclonal B-cell lymphocytosis among members of highrisk CLL families. Br J Haematol 2010;151:152-158. 22. Ghia P, Caligaris-Cappio F. Monoclonal B-cell lymphocytosis: right track or red herring? Blood 2012;119:4358-4362. 23. Shanafelt TD, Kay NE, Jenkins G, Call TG, Zent CS, Jelinek DF, Morice WG, Boysen J, Zakko L, Schwager S, Slager SL, Hanson CA. B-cell count and survival: differentiating chronic lymphocytic leukemia from monoclonal B-cell lymphocytosis based on clinical outcome. Blood 2009;113:4188-4196. 24. Molica S, Mauro FR, Giannarelli D, Lauria F, Cortelezzi A, Brugiatelli M, Liso V, Cuneo A, Foà R. Differentiating chronic lymphocytic leukemia from monoclonal B-lymphocytosis according to clinical outcome: on behalf of the GIMEMA chronic lymphoproliferative diseases working group. Haematologica 2011;96:277-283.

Research Article

DOI: 10.4274/tjh.2013.0087

Myeloid Sarcomas: A Clinicopathologic Study of 20 Cases Miyeloid Sarkomlar: 20 Olguluk Klinikopatolojik Çalışma Gülşah Kaygusuz1, Duygu Kankaya1, Cemil Ekinci1, Pervin Topçuoğlu2, Işınsu Kuzu1 1Ankara 2Ankara

University Faculty of Medicine, Department of Pathology, Ankara, Turkey University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract: Objective: Myeloid sarcoma is a tumoral mass of mature or immature myeloid blasts in extramedullary anatomic locations.

It can be seen de novo or in association with acute myeloid leukemia, myeloproliferative neoplasias, or myelodysplastic syndrome. Isolated myeloid sarcoma can be seen as a relapse in cases with allogenic bone marrow transplantation. Although it may involve any tissue in the body, the most common locations are skin, soft tissues, lymph nodes, and the gastrointestinal tract. Immunohistochemically, most cases show myelomonocytic or pure monoblastic differentiation. We reviewed the clinicopathological features of 20 cases of myeloid sarcoma diagnosed in our institute in view of the literature. Materials and Methods: The cases diagnosed between 2005 and 2012 at the Ankara University Faculty of Medicine, Department of Pathology, were selected. Clinicopathological findings including the age and sex of the patients; symptoms; anatomic location; accompanying hematological disease; and the morphological, immunohistochemical, and cytogenetic features of the cases were noted. Results: Sixteen of the patients were male and 4 were female. The median age at diagnosis was 47 years. The most commonly involved locations were the lymph nodes and skin. Immunohistochemically, eleven cases were of the myelomonocytic and 7 cases were of the myeloid phenotype, whereas 2 cases showed pure monoblastic differentiation. The median follow-up period for the 18 cases with known clinical data was 33 weeks. Five patients died of the disease in an average of 36 weeks. Conclusion: Myeloid sarcoma is a rare presentation of leukemias, myeloproliferative neoplasias, or myelodysplastic syndrome, composed of immature myelomonocytic cells in extramedullary tissues. It may present with variable morphological and phenotypic features, always creating a challenge in pathological diagnosis. Key Words: Myeloid sarcoma, Granulocytic sarcoma, Monoblastic sarcoma

Özet: Amaç: Miyeloid sarkom, ekstramedüller anatomik bölgelerde matür veya immatür miyeloid blastların oluşturduğu

tümöral kitledir. De novo veya akut miyeloid lösemi, miyeloproliferatif neoplaziler ya da miyelodisplastik sendrom ile birlikte görülebilir. Tek başına miyeloid sarkoma, allojenik kemik iliği transplantasyonu yapılan olgularda nüks şeklinde görülebilir. Vücutta herhangi bir dokuda gelişebilmekle birlikte en sık tutulan bölgeler deri, yumuşak dokular, lenf nodülü ve gastrointestinal sistemdir. İmmünohistokimyasal olarak olguların birçoğu miyelomonositik ya da saf monoblastik farklılaşma gösterir. Bu makalede kurumumuzda tanı alan 20 miyeloid sarkoma olgusunun klinikopatolojik özellikleri kaynaklar ışığında gözden geçirilmiştir. Address for Correspondence: Gülşah KAYGUSUZ, M.D., Ankara University Faculty of Medicine, Department of Pathology, Ankara, Turkey Phone: +90 312 595 81 14/595 81 03 E-mail: gulsah@gmail.com Received/Geliş tarihi : March 11, 2013 Accepted/Kabul tarihi : October 21, 2013

Turk J Hematol 2015;32:35-42

Kaygusuz G, et al: Myeloid Sarcoma

Gereç ve Yöntemler: 2005-2012 yılları arasında Ankara Üniversitesi Tıp Fakültesi Patoloji departmanında tanı alan olgular seçilmiştir. Hastaların yaş, cinsiyet, semptomlar, anatomik yerleşim, eşlik eden hematolojik hastalık bilgileri ve olguların morfolojik, immünohistokimyasal ve sitogenetik özellikleri kayıt edilmiştir.

Bulgular: On altı hasta erkek, 4 hasta kadındır. Tanı anındaki ortalama yaş 47’dir. En sık tutulan bölgeler lenf nodları ve deridir. İmmünohistokimyasal olarak 11 olguda miyelomonositik ve 7 olguda miyeloid fenotip, 2 olguda pür monoblastik farklılaşma saptanmıştır. Klinik verileri bilinen 18 olgu için ortalama takip süresi 33 hafta bulunmuştur. Hastalardan 5’i hastalığı nedeniyle kaybedilmiştir.

Sonuç: Miyeloid sarkom, ekstramedüller dokularda immatür miyelomonositik hücrelerin oluşturduğu; lösemiler, miyeloproliferatif neoplaziler veya miyelodisplastik sendromların nadir bir prezentasyonudur. Daima patolojik tanı güçlüğü yaratacak şekilde değişken morfolojik ve fenotipik bulgularla ortaya çıkabilir.

Anahtar Sözcükler: Miyeloid sarkom, Granülositik sarkom, Monoblastik sarkom Introduction Myeloid sarcoma (MS) is a tumoral mass composed of mature or immature myeloid blasts in extramedullary anatomic locations. There is a predilection for males with the median age of 56 years (1-89 years) [1]. MS can be seen de novo or in association with acute myeloid leukemia (AML), myeloproliferative neoplasias (MPNs), or myelodysplastic syndrome (MDS) [2,3,4,5,6]. It has been observed most commonly in pediatric AML cases (30%) and is less frequently seen among adult AML patients (2%-5%) [7]. MS can be the first manifestation of AML, or it can arise as a recurrence of treated AML. While it can be reported from any anatomic region, the most common locations are the skin, soft tissues, lymph nodes, and gastrointestinal tract [7]. Most cases show myelomonocytic or pure monoblastic differentiation. The differential diagnosis of MS includes a large group of entities according to the clinical history, age of the patient, or information on preexisting or concomitant myeloid neoplasia. The clinicopathological features of 20 cases of MS are reviewed in this manuscript along with previously published reports. Materials and Methods The cases diagnosed as MS between 2005 and 2012 at the Ankara University Faculty of Medicine, Department of Pathology, were selected. Clinicopathological findings including the age and sex of the patients; symptoms; anatomic location; accompanying hematological disease; the morphological, immunohistochemical, and cytogenetic features of the cases; and the follow-up data are summarized in Table 1. Informed consent was obtained. Results Sixteen of the patients were male and 4 were female. The median age at diagnosis was 47 years (range: 22-82 years). In 7 cases, more than 1 anatomic region was involved. The most commonly involved locations were the lymph nodes (9

cases) and skin (3 cases), while the breast (2 cases), paratubal tissue (2 cases), and soft tissues of pectoral or lumbar (2 cases) regions were the next most commonly involved sites. The rest of the locations were as follows: thoracic vertebra (1 case), submandibular gland (1 case), minor salivary gland (1 case), pleura (1 case), anterior mediastinum (1 case), retroperitoneum (1 case), liver (1 case), small bowel (1 case), gingiva (1 case), and ovary (1 case) (Table 1). The main symptoms of the patients were related to the site of the lesions. Regional pain (6 cases), weakness (4 cases), palpable mass (4 cases), lymphadenopathy (3 cases), weight loss (2 cases), fecal incontinence (1 case), ileus (1 case), skin rash (1 case), mouth sore (1 case), vomiting (1 case), and hypermenorrhea (1 case) were the recorded symptoms and complaints. Ten patients presented with preexisting or concurrent AML (1 case primary myelofibrosis-based) and 2 with chronic myeloid leukemia (CML). Three cases were presented as de novo disease (Table 1). In 5 of the cases there were no bone marrow or clinical data revealing the association of primary myeloid neoplasia. The disease developed following allogenic bone marrow transplantation in 3 of 13 cases. Tuberculosis, myxoid liposarcoma, and colonic adenocarcinoma were simultaneously diagnosed second pathologies in 3 MS cases. In one case, blastic cells were noted in the wall of a Morgagni cyst (Figure 1A). A diffuse infiltration pattern was noted in other extranodal locations (Figures 1B and 1D). One case showed infiltration of adenocarcinoma of the colon intermingled with MS (case 15; Figure 1C). Morphologically, 6 of 9 cases were characterized by the complete effacement of the lymph node, while 1 case had paracortical infiltration (case 11) and 1 case had sinusoidal infiltration (case 16) (Figure 1E). Morphological signs of aggressive behavior of the tumors, such as angiolymphatic invasion (1 case; Figure 1F), starry sky appearance (Figure 2A; 1 case), crushing artifacts (2 cases; Figure 2B), necrosis (2 cases; Figure 2C, 2D), and marked nuclear pleomorphism (1 case; Figure 2E, 2F), were present on histopathological examination (Table 1).

Kaygusuz G, et al: Myeloid Sarcoma

Non-necrotic granulomas were observed in a case showing sinusoidal infiltration of the lymph node (Figure 2C). In almost all of the tumors, the mitotic rate was high (Figure 2E). Immunohistochemically, variable expressions of myeloid markers myeloperoxidase (MPO), CD34, CD68/PGM1, CD117, lysozyme, CD45, terminal deoxynucleotidyl transferase (TDT), CD4, CD15, CD56, CD31, CD10, and Bcl2 were observed (Figures 3A-3F). Eleven cases were of the myelomonocytic and 7 cases were of the myeloid phenotype, whereas 2 cases showed pure monoblastic differentiation (Table 1). Cytogenetic analyses were routinely performed from bone marrow aspirates. Cytogenetic data were present for 10 cases in our series. The 4 cases with positive cytogenetic abnormalities all had concurrent bone marrow involvement at diagnosis of MS (Table 1): 1 case with t(8;21) (q22;q22) (case 18); 1 case with FLT3-ITD heterozygous mutation and del(22q11) (case 12); 1 case with t(9;22) (q34;q11), inv(17) (q10), and trisomy 8 (case 10); and 2 cases with t(9;22) (q34;q11). Although 4 of them had concurrent AML with MS and 2 cases were de novo, there were no cytogenetic abnormalities detected in 6 cases. Cytogenetic data were not available in 10 cases.

Turk J Hematol 2015;32:35-42

by production of myeloperoxidase [8]. It occurs most often in males at the median age of 56 years (range: 1-89 years) [1]. Male sex predominance was also reported with a median age of 47, ranging between 22 and 82 years. Besides its most common locations in the skin, soft tissue, lymph nodes, and gastrointestinal tract, MS may involve any organ or system in the body [7]. The symptoms are substantially related with the anatomic location that is involved. The lymph nodes were the most frequent localizations involved in our series. MS may develop as de novo disease, or it may occur in combination with myeloid neoplasias (AML, MPNs, or MDS) in 1/3 of the reported cases [2,3,4,5,6]. MS can be the first manifestation of AML, or it can arise as a relapse of treated AML. It may occur as a first sign of blastic crisis in CML [9]. Pileri et al. reported that 4 of 92 MS cases had preexisting or concurrent primary myelofibrosis (PMF) in their series [3]. Neiman et al. reported MS development in 5% of PMF cases in their series [10]. In this presented series, we have seen 10 cases of AML, 1 of PMF and 2 that were CML-based. Three of our

The median follow-up period for the 18 cases with known clinical data was 33 weeks (range: 1-144 weeks). Five patients died of the disease in an average of 36 weeks (range: 1-80 weeks) (Table 1). Discussion MS, also known as granulocytic/monoblastic sarcoma or extramedullary myeloid tumor, was first known as chloroma, with its macroscopically greenish color caused

Figure 1. A, B, D) Diffuse blastic infiltration in the wall of a Morgagni cyst, in the small intestinal mucosa, and in the dermis (H&E 400x, 200x, 400x, respectively). C) Blastic cells (black arrow) intermingled with adenocarcinoma of the colon (H&E 400x). E) Case 16 showed sinusoidal infiltration of the blastic cells mixed with megakaryocytes (black arrow; H&E 400x). F) Angiolymphatic invasion was seen (black arrows; H&E 400x).

Figure 2. A, B) Starry sky appearance and crushing artifact were noted (H&E 100x). C) Granuloma with blastic cell infiltration (black arrows; H&E 100x). D, E) Necrosis (black arrows) and numerous mitoses were found (H&E 200x, 400x, respectively). F) Pleomorphic blastic cells (H&E 1000x).

Figure 3. Immunohistochemical CD34 (A), CD45 (B), MPO (C), CD117 (D), Tdt (E), and CD68 (F) expression of the neoplastic cells (400x, 400x, 400x, 400x, 200x, 400x, respectively).

Painful mass

Multiple LAPs

28/M

29/M

49/M

52/M

54/M

60/M

61/M

appetite

Weakness, loss of

mass

Weakness, pectoral

Cervical LAP

N/A

Chest pain

LN, liver

Pectoral soft tissue, LN

Retroperitoneum

anterior mediastinum

Skin,

pleura

Concurrent AML

CML

N/A

De novo

N/A

Concurrent AML

N/A

47/M

LN,

Chest pain

numerous mitoses

Paracortical infiltration,

necrosis CD45, CD68

CD34, CD68, MPO

Tdt

numerous mitoses Diffuse infiltration,

MPO, CD34, CD45,

Diffuse infiltration,

CD45

lysozyme, CD4,

CD68, MPO,

CD31, CD117, Tdt

Diffuse infiltration

CD34, CD45, MPO,

angiolymphatic invasion

lysozyme, MPO

CD68, CD15,

CD34, CD117,

lysozyme

MPO, CD68, CD56,

CD34, CD117

MPO, CD117, CD10

BCL2

MPO, CD117, CD34,

Tdt

Phenotype (IHC)

Diffuse infiltration,

Diffuse infiltration

numerous mitoses

Diffuse infiltration,

numerous mitoses

crushing artifact,

44/M

CML

Thoracal vertebra

Diffuse infiltration,

fecal incontinence

Weakness,

41/M

Diffuse infiltration, starry

mitoses sky, numerous mitoses

Concurrent AML

crushing artifact,

7 months pleomorphism, numerous

Diffuse infiltration,

Diffuse infiltration

Morphology

Preexisting AML of

N/A

De novo/ association with MN

Submandibular gland

22/M

N/A

Age/ Symptoms at Site sex diagnosis

Case

Table 1. The clinicopathological features of the cases.

(-)

trisomy 8

inv(17)(q10),

Ph (+),

N/A

Ph (+)

(-)

N/A

36/DOD

4/DOD

60/AWD

4/AWD

12/AWD

4/AWD

144/AWD

80/DOD

36/AWD

N/A

Cytogenetic Survival report (weeks)/ disease status at last follow-up

Turk J Hematol 2015;32:35-42

Kaygusuz G, et al: Myeloid Sarcoma

62/M

67/M

71/M

82/M

30/F

34/F

36/F

29/F

Hypermenorrhea

Mammary mass

vomiting

Abdominal pain,

Mammary mass

N/A

anorexia

Preexisting AML of 1 month

paratubal tissue

84 months

Preexisting AML of

8 months

Preexisting AML of

De novo

Concurrent AML

De novo

N/A

Concurrent AML

Bilateral ovaries,

Breast, skin

Paratubal tissue

Breast, gingiva

Mesenteric LN

gland

Right lumbar pain,

Skin, minor salivary

mouth wounds

Small intestine

subcutaneous region

Right lumbar

Weakness, skin eruption,

Ileus

Subcutaneous nodules

numerous mitoses

wall of Morgagni cyst,

Diffuse infiltration in the

Diffuse infiltration

numerous mitosis

Diffuse infiltration,

Diffuse infiltration

granulomas

Sinus infiltration,

adenocarcinoma

mixed with colon

Diffuse infiltration

numerous mitoses

CD34

MPO, CD45, Tdt,

MPO, lysozyme

CD117

MPO, lysozyme,

CD15, lysozyme

CD4, CD68

lysozyme

CD4, CD15, CD68,

CD117

Diffuse infiltration,

MPO, CD68, CD34,

numerous mitoses

CD68

CD34, MPO, CD117,

Diffuse infiltration,

mitoses

necrosis, numerous

Diffuse infiltration,

N/A

(-)

(+)

t(8;21) (q22;q22)

(-)

N/A

(-)

N/A

del(22q11)

FLT3-ITD (+),

1/AWD

8/AWD

84/AWD

1/AWD

N/A

60/DOD

3/AWD

1/DOD

64/AWD

M: male, F: female, LAP: lymphadenopathy, N/A: not available, LN: lymph node, MN: myeloid neoplasia, AML: acute myeloid leukemia, CML: chronic myeloid leukemia, IHC: immunohistochemistry, MPO: myeloperoxidase, AWD: alive with disease, DOD: dead of disease.

61/M

Kaygusuz G, et al: Myeloid Sarcoma Turk J Hematol 2015;32:35-42

Turk J Hematol 2015;32:35-42

patients presented with de novo disease. It has been suggested that the bone marrow involvement can be determined in approximately 10 months in all de novo MS cases [9]. Our 3 de novo patients were lost 1 to 4 weeks following their diagnoses. During their short follow-up period, bone marrow involvement could not be demonstrated. MS developed within an average of 25 months (range: 1-84 months) in cases of preexisting AML. Isolated MS can be seen as the recurrent form of the disease in cases with allogenic bone marrow transplantation. Clark et al. reported the frequency as 8%-20% in their series [11]. Three of the MS patients in our series had recurrence of the disease during their follow-up period after allogenic bone marrow transplantation. Tuberculosis, myxoid liposarcoma, and colonic adenocarcinoma were coincidentally present in 3 MS cases in the current series. Rare cases with association of MS and colon adenocarcinoma were previously reported by Rocca et al., proposing the association with p53 deletion in intestinal stem cells [12]. Myeloid sarcomas are immature cell proliferations that are morphologically composed of myeloblasts, monoblasts/ promonocytes, and rarely promyelocytes. A variable number of mature myeloid cells may be present. Necrosis, numerous mitoses, and tingible-body macrophages can be present [3]. The neoplastic cells may infiltrate lymph nodes in an intrasinusoidal or diffuse pattern. In extranodal localizations, the infiltration pattern is generally diffuse, or the tumor cells constitute cohesive groups in a sclerotic stroma, resembling metastatic carcinoma. In our series, 6 of 9 cases were characterized by the complete effacement of the normal lymph node structure, while 1 case had paracortical and 1 case had sinusoidal infiltration. All of these patterns may create difficulty at diagnosis in de novo cases. Non-necrotic granulomas were present in the case with nodal sinusoidal infiltration, creating a diagnostic difficulty. The patientsâ&#x20AC;&#x2122; clinical history did not reveal previously defined tuberculosis or any other granulomatous disease. Granulomatous reaction is rarely seen in conjunction with MS. Baksh et al. reported a case of leukemia cutis with granulomatous reaction, suggesting that is caused by the host response to damaged elastin fibers and other tissue stromal elements produced by leukemic infiltration [13]. MSs were also called granulocytic sarcoma and monoblastic sarcoma in the past, according to the morphological features [1,14]. This morphological classification was abandoned with molecular and cytogenetic classifications. A majority of cases have myelomonocytic or pure monoblastic morphology and immunophenotype. MS with erythroid and megakaryoblastic differentiation or extramedullary acute promyelocytic

Kaygusuz G, et al: Myeloid Sarcoma

leukemia rarely occurs [15,16,17,18]. Immunophenotypic characteristics of MSs are also parallel to the morphological features. Various expressions of CD68/KP1, MPO, CD117, CD99, CD68/PG-M1, and lysozyme, concordant with the myelomonocytic differentiation, can be seen. Other than these markers, CD34, TdT, CD56, CD30, glycophorin A, and CD4 are also useful for diagnosis [19]. CD43 and lysozyme are sensitive but not specific for the diagnosis. CD68 and MPO are commonly expressed, whereas CD34 is negative, especially in cases with monocytic differentiation [20,21]. Variable CD45 expression can be seen, and CD33 and CD117 can be used to demonstrate myeloid nature [22,23]. Parallel with the previous reports, most of our patients carried the myelomonocytic (11 cases) or myeloid (7 cases) phenotypes, whereas 2 showed pure monoblastic differentiation. Cytogenetic abnormalities, particularly monosomy 7, trisomy 8, MLL rearrangement, and inv(16), can be seen in about 55% of MS cases. t(8;21) (q22;q22) occurs in the pediatric age group more so than in adults [1]. In our series, cytogenetic analyses revealed 1 case with t(8;21) (q22;q22); 1 case with FLT3-ITD heterozygous mutation and del(22q11); 1 case with t(9;22) (q34;q11), inv(17) (q10), and trisomy 8; and 2 cases with t(9;22) (q34;q11). There were no cytogenetic changes in 6 cases. Conventional cytogenetics cannot be used as a routine procedure for MS, especially for de novo cases before the diagnosis. Performing cytogenetic examination for MS can be difficult if there is no bone marrow or peripheral blood involvement. It is difficult to speculate about the cytogenetic results in our series as we obtained very limited data. In 4 cases, although there was concurrent AML, conventional cytogenetics did not give any abnormalities. These cases could represent AML without any cytogenetic abnormalities. The differential diagnosis of MS can be especially difficult in small biopsies. In this category, MS should be differentiated from lymphomas, particularly diffuse large B-cell lymphoma, lymphoblastic lymphoma, blastic variants of mantle cell lymphoma, and Burkittâ&#x20AC;&#x2122;s lymphoma. The morphological diagnosis of MS can be easier if an extramedullary hematopoiesis picture is present with erythroid precursors, megakaryocytes, and eosinophilic myelocytes mixed with neoplastic cells. Immunohistochemically, the expression of B-cell markers such as CD20 and CD79a is rarely seen in MS, but it should be noted that cases with t(8;21) (q22;q22) may express PAX5, CD19, and CD79a [14,19]. Since MS may express T-cell markers (such as CD43, CD45, CD4, and CD7), immunohistochemical expressions of MPO, lysozyme, and CD68 should be analyzed for their distinction from T-cell lymphomas [24]. The cutaneous neoplastic infiltrates that are positive for CD56 and CD4 should be differentiated from blastic plasmacytoid dendritic cell neoplasms. The presence of MPO and lysozyme expression as well as negative T-cell leukemia 1 staining favors the diagnosis of MS [25].

Kaygusuz G, et al: Myeloid Sarcoma

Histiocytic sarcomas are rare tumors, which may be seen in extranodal sites and have common clinicopathological features with AML. Negative staining with CD33 and CD13 is helpful for their diagnosis. Malignant melanomas and poorly differentiated carcinomas are the other tumors that need to be differentiated from MS. Immunohistochemistry with S100, Melan-A, HMB-45, and AE1/AE3 can be helpful in providing the correct diagnosis. Since non-monocytic MSs are mostly CD99-positive, other blue round cell tumors of the pediatric age such as Ewing sarcoma/PNET or medulloblastoma may mimic MS. The myeloid and monocytic markers may help in diagnosis, but flow cytometric analysis of the specimen should be performed [20]. Studies concerning the prognosis and therapy response of MSs are limited. The therapy options are local radiotherapy, systemic chemotherapy, immunotherapy, and donor lymphocyte infusion for the allogenic transplanted cases [11]. Patients are generally treated with induction chemotherapy and allogenic/autologous bone marrow transplantation. Success in therapy in the long term is very low (10%), and the mortality associated with therapy is high (>60%) [12]. In our series, the mean follow-up period was 33 weeks (range: 1-144 weeks) for 18 of the patients with known follow-up data. Five of those died from the disease within an average of 36 weeks (range: 1-80 weeks) following their diagnosis. In conclusion, MS is a rare presentation of leukemias, myeloproliferative neoplasias, or myelodysplastic syndrome, composed of immature myelomonocytic cells in extramedullary tissues. It may present with variable morphological and phenotypic features, always creating a challenge in pathological diagnosis. In addition to its diagnostic difficulty, overcoming the disease is also crucial, as it results in poor survival. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. References 1. Pileri SA, Orazi A, Falini B. Myeloid sarcoma. 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 Tissue. 4th ed. Lyon, France, IARC, 2008. 2. Koc Y, Miller KB, Schenkein DP, Daoust P, Sprague K, Berkman E. Extramedullary tumors of myeloid blasts in adults as a pattern of relapse following allogeneic bone marrow transplantation. Cancer 1999;85:608-615.

Turk J Hematol 2015;32:35-42

3. Pileri SA, Ascani S, Cox MC, Campidelli C, Bacci F, Piccioli M, Piccaluga PP, Agostinelli C, Asioli S, Novero D, Bisceglia M, Ponzoni M, Gentile A, Rinaldi P, Franco V, Vincelli D, Pileri A Jr, Gasbarra R, Falini B, Zinzani PL, Baccarani M. Myeloid sarcoma: clinico-pathologic, phenotypic and cytogenetic analysis of 92 adult patients. Leukemia 2007;21:340-350. 4. Imamura T, Matsuo S, Yoshihara T, Chiyonobu T, Mori K, Ishida H, Nishimura Y, Kasubuchi Y, Naya M, Morimoto A, Hibi S, Imashuku S. Granulocytic sarcoma presenting with severe adenopathy (cervical lymph nodes, tonsils, and adenoids) in a child with juvenile myelomonocytic leukemia and successful treatment with allogeneic bone marrow transplantation. Int J Hematol 2004;80:186-189. 5. Elenitoba-Johnson K, Hodges GF, King TC, Wu CD, Medeiros LJ. Extramedullary myeloid cell tumors arising in the setting of chronic myelomonocytic leukemia: a report of two cases. Arch Pathol Lab Med 1996;120:62-67. 6. Hancock JC, Prchal JT, Bennett JM, Listinsky CM. Trilineage extramedullary myeloid cell tumor in myelodysplastic syndrome. Arch Pathol Lab Med 1997;121:520-523. 7. Byrd JC, Edenfield WJ, Shields DJ, Dawson NA. Extramedullary myeloid cell tumors in acute nonlymphocytic leukemia: a clinical review. J Clin Oncol 1995;13:1800-1816. 8. King A. A case of chloroma. Monthly Journal of the Medical Society 1853;17:17. 9. Morita K, Nakamine H, Inoue R, Takano M, Takeda M, Enomoto Y, Kasai T, Nonomura A, Tanaka H, Amano I, Morii T, Kimura H. Case Report: Autopsy case of primary myelofibrosis in which myeloid sarcoma was the initial manifestation of tumor progression. Pathol Int 2012;62:433437. 10. Neiman RS, Barcos M, Berard C, Bonner H, Mann R, Rydell RE, Bennett JM. Granulocytic sarcoma: a clinicopathologic study of 61 biopsied cases. Cancer 1981;48:1426-1437. 11. Clark WB, Strickland SA, Barrett AJ, Savani BN. Extramedullary relapses after allogeneic stem cell transplantation for acute myeloid leukemia and myelodysplastic syndrome. Haematologica 2010;95:860-863. 12. Rocca BJ, Ambrosio MR, Gozzetti A, Bocchia M, Leoncini L, Lazzi S. Myeloid sarcoma and adenocarcinoma of the large bowel as collision tumors: a case report. Histol Histopathol 2012;27:941-947. 13. Baksh FK, Nathan D, Richardson W, Kestenbaum T, Woodroof J. Leukemia cutis with prominent giant cell reaction. Am J Dermatopathol 1998;20:48-52. 14. Traweek ST, Arber DA, Rappaport H, Brynes RK. Extramedullary myeloid cell tumors. An immunohistochemical and morphologic study of 28 cases. Am J Surg Pathol 1993;17:1011-1019.

Turk J Hematol 2015;32:35-42

15. Hirose Y, Masaki Y, Shimoyama K, Sugai S, Nojima T. Granulocytic sarcoma of megakaryoblastic differentiation in the lymph nodes terminating as acute megakaryoblastic leukemia in a case of chronic idiopathic myelofibrosis persisting for 16 years. Eur J Haematol 2001;67:194-198. 16. Vega-Ruiz A, Faderl S, Estrov Z, Pierce S, Cortes J, Kantarjian H, Ravandi F. Incidence of extramedullary disease in patients with acute promyelocytic leukemia: a single-institution experience. Int J Hematol 2009;89:489-496. 17. Wang HY, Huang LJ, Liu Z, Garcia R, Li S, Galliani CA. Erythroblastic sarcoma presenting as bilateral ovarian masses in an infant with pure erythroid leukemia. Hum Pathol 2011;42:749-758. 18. Kyaw TZ, Maniam JA, Bee PC, Chin EF, Nadarajan VS, Shanmugam H, Kadir KA. Myeloid sarcoma: an unusual presentation of acute promyelocytic leukemia causing spinal cord compression. Turk J Hematol 2012;29:278-282. 19. Campidelli C, Agostinelli C, Stitson R, Pileri SA. Myeloid sarcoma: extramedullary manifestation of myeloid disorders. Am J Clin Pathol 2009;132:426-437. 20. Klco JM, Welch JS, Nguyen TT, Hurley MY, Kreisel FH, Hassan A, Lind AC, Frater JL. State of the art in myeloid sarcoma. Int J Lab Hem 2011;33:555-565.

Kaygusuz G, et al: Myeloid Sarcoma

21. Chang H, Brandwein J, Yi QL, Chun K, Patterson B, Brien B. Extramedullary infiltrates of AML are associated with CD56 expression, 11q23 abnormalities and inferior clinical outcome. Leuk Res 2004;28:1007-1011. 22. Alexiev BA, Wang W, Ning Y, Chumsri S, Gojo I, Rodgers WH, Stass SA, Zhao XF. Myeloid sarcomas: a histologic, immunohistochemical, and cytogenetic study. Diagn Pathol 2007;2:42. 23. Hoyer JD, Grogg KL, Hanson CA, Gamez JD, Dogan A. CD33 detection by immunohistochemistry in paraffin-embedded tissues: a new antibody shows excellent specificity and sensitivity for cells of myelomonocytic lineage. Am J Clin Pathol 2008;129:316-323. 24. Lewis RE, Cruse JM, Sanders CM, Webb RN, Suggs JL. Aberrant expression of T-cell markers in acute myeloid leukemia. Exp Mol Pathol 2007;83:462-463. 25. Petrella T, Meijer CJ, Dalac S, Willemze R, Maynadie M, Machet L, Casasnovas O, Vergier B, Teitell MA. TCL1 and CLA expression in agranular CD4/CD56 hematodermic neoplasms (blastic NK-cell lymphomas) and leukemia cutis. Am J Clin Pathol 2004;122:307-313.

Research Article

DOI: 10.4274/tjh.2013.0170

Osteoporosis and Osteopathy Markers in Patients with Mastocytosis Sistemik Mastositozda Osteoporoz ve Osteopati Belirteçleri Nilüfer Alpay Kanıtez1, Burak Erer1, Öner Doğan2, Nesimi Büyükbabani2, Can Baykal3, Dilşad Sindel4, Refik Tanakol5, Akif Selim Yavuz6 1İstanbul

University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Rheumatology, İstanbul, Turkey University İstanbul Faculty of Medicine, Division of Pathology, İstanbul, Turkey 3İstanbul University İstanbul Faculty of Medicine, Division of Dermatology, İstanbul, Turkey 4İstanbul University İstanbul Faculty of Medicine, Division of Physical Treatment and Rehabilitation, İstanbul, Turkey 5İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Endocrinology, İstanbul, Turkey 6İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 2İstanbul

Abstract: Objective: Osteoporosis, osteosclerosis, and lytic bone lesions have been observed in patients with systemic mastocytosis

(SM). We examined bone mineral density (BMD) biochemical turnover markers and serum tryptase levels in SM, which is considered a rare disease. Materials and Methods: Seventeen adult patients (5 females, 12 males; median age: 33 years, range: 20-64) with mastocytosis were included in this study. We investigated the value of quantitative ultrasound (QUS) of the calcaneus in the assessment of BMD in SM patients, as well as BMD of the lumbar spine (L1-L4), femoral neck, and distal radius using dual energy x-ray absorptiometry (DXA) and plasma tryptase levels, biochemical markers of bone turnover. Results: At lumbar spine L1-L4, the femoral neck, and the distal radius or as calcaneus stiffness, 12 of 17 patients had T-scores of less than -1 at least at 1 site, reflecting osteopenia. Three of 17 patients had T-scores showing osteoporosis (T-score <-2.5). There was no relationship between DXA and bone lesion severity. We also found a significant positive correlation between tryptase levels and disease severity, as well as between disease severity and pyridinoline (p<0.01 by Spearman’s test). Conclusion: DXA and calcaneal QUS may not be appropriate techniques to assess bone involvement in SM patients because of the effects of osteosclerosis. This study further shows that the osteoclastic marker pyridinoline is helpful in patients with severe disease activity and sclerotic bone lesions to show bone demineralization. Key Words: Mastocytosis, Bone mineral density, Pyridinoline, Bone turnover, Osteopenia

Özet: Amaç: Sistemik mastositozlu (SM) hastalarda osteoporoz, osteoskleroz ve litik kemik lezyonları görülebilir. Bu çalışmada

nadir bir hastalık olan SM’de, kemik yoğunluk ölçümü (BMD), serumda kemik ‘turnover’ belirteçleri ve triptaz düzeyi araştırılmıştır.

Gereç ve Yöntemler: Çalışmaya 5’i kadın, 12’si erkek olmak üzere median yaşları 33 (20-64) olan toplam 17 hasta dahil edilmiştir. Hastaların BMD’leri lomber vertebra (L1-L4), femur boynu ve distal radiustan dual enerji x-ray absorbitesi (DXA) Address for Correspondence: Nilüfer Alpay Kanıtez, M.D., İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Rheumatology, İstanbul, Turkey Phone: +90 212 414 20 00 E-mail: nalpay@istanbul.edu.tr Received/Geliş tarihi : May 14, 2013 Accepted/Kabul tarihi : September 19, 2013

Turk J Hematol 2015;32:43-50

Alpay Kanıtez N, et al: Osteopathy in Mastocytosis

yöntemi, kalkaneustan ise kantitatif ultrasonografi (USG) yöntemi kullanılarak ayrı ayrı hesaplanmıştır. Serum triptaz düzeyleri ile biyokimyasal kemik ‘turnover’ belirteçlerileri arasındaki ilişki araştırılmıştır.

Bulgular: Hastaların 12 tanesinde osteopeni (incelenen kemik alanlarının en az birinde T skoru <-1), 3 tanesinde ise osteoporoz (T skoru <-2,5) saptanmıştır. Kemik lezyonlarının şiddeti ile DXA sonuçları arasında korelasyon gösterilememiştir. Triptaz ve pridinolin seviyeleri ile hastalık şiddeti arasında pozitif korelasyon bulunmuştur (p<0,01).

Sonuç: Osteosklerotik kemik lezyonları sebebiyle SM’li hastalarda kemik tutulumu değerlendirmesi için DXA ve kalkaneus USG uygun teknikler değildir. Osteoklastik aktivite belirteçleri olan pridinolin, şiddetli hastalık aktivitesi ve kemik sklerozlarını değerlendirmede faydalıdır.

Anahtar Sözcükler: Mastositoz, Kemik yoğunluk ölçümü, Pridinolin, Kemik turnoverı, Osteopeni Introduction Mastocytosis is characterized by an abnormal accumulation of mast cells (MCs) in at least one organ system [1]. MCderived mediators and disruptive infiltration of MCs cause symptoms in mastocytosis. The clinical forms of mastocytosis are based on specific criteria such as bone marrow pathology, organ involvement, and measurement of serum tryptase [2,3]. Mastocytosis was defined in 7 categories: cutaneous mastocytosis (CM), indolent systemic mastocytosis (ISM) including bone marrow mastocytosis and smoldering systemic mastocytosis (SSM), systemic mastocytosis (SM) with associated clonal hematological non-MC-lineage disease, aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), mast cell sarcoma (MCS), and extracutaneous mastocytoma [2]. Skeletal manifestations such as osteoporosis, osteosclerosis, and lytic lesions have been observed in patients with SM, reflecting different numbers of infiltrating MCs and variation in the active substance they secrete [4]. In a recent study using dual energy X-ray absorptiometry (DXA), it was reported that SM patients with more severe disease had significantly higher bone mineral density (BMD) at the spine L1-L4 and femoral neck than patients with less severe disease [5]. However, DXA may be an inappropriate technique for assessing bone involvement in SM patients with osteosclerosis that has been observed, in addition to osteopenia in patients with SM. The purpose of this study was to assess BMD with a different technique in patients with SM and to investigate whether specific markers of mastocytosis and features of clinical disease correlate with BMD. Informed consent was obtained. Materials and Methods Patients Seventeen adult patients, 12 males and 5 females with mastocytosis, were included in this study. Patient evaluations included history, physical assessment, determination of plasma tryptase levels and c-kit mutation (D816V), skin biopsy, and bone marrow biopsy and aspirate. The diagnosis and classification of mastocytosis were based on WHO criteria [2]. Subgroups were defined as less severe disease (CM, ISM) 44

and more severe disease (SSM, ASM, MCL). The subjects participating in the study had no endocrine disorders, were not menopausal women, did not receive any medication affecting the bones (including steroids and estrogens), and had no history of traumatic bone fractures. Information about risk factors of osteoporosis, including physical inactivity, tobacco-smoking habits, previous fractures, age at menarche and menopause, number of pregnancies, and consumption of milk, cheese, coffee, and medications, was obtained from individual questionnaires. According to the information obtained from the questionnaire and laboratory analysis, patients who had any risk factors for osteoporosis and those with abnormal serum parathyroid hormone (PTH) levels were excluded. In patients with low vitamin D levels, vitamin D was substituted with cholecalciferol orally and biochemical tests were done after correction of the vitamin D level. Bone Mineral Measurements BMD was measured by DXA using a Hologic QDR 4500 (Hologic, Bedford, MA, USA). Scans were performed at 3 different sites for all patients: lumbar spine (L1-L4), femoral neck, and distal radius. DXA measures the areal BMD by using ionizing radiation with photon beams at 2 different energy levels. Results were expressed as g/cm2 and T-scores representing standard deviations from young, healthy control subjects. T-scores were classified according to the WHO criteria as normal (T-scores >-1), osteopenia (-1< T-scores <-2.5) or osteoporosis (T-scores <-2.5). Simultaneously, quantitative ultrasound (QUS) measurements of the calcaneus were carried out using an Achilles Express ultrasound device (Sahara sonometer, Hologic). Calcaneal QUS involves placing ultrasound transducers on either side of the calcaneus, one acting as a wave transmitter and the other acting as the receiver. These devices measure 3 main types of parameters: broadband ultrasound attenuation (BUA), speed of sound (SOS), and the stiffness index. The stiffness index combines the parameters of SOS and BUA. Results were expressed as BMD and T-scores. Laboratory Measurements Serum calcium was measured by colorimetric assay, 25-OH vitamin D by radioimmunoassay, intact PTH by chemiluminescence assay, osteocalcin by radioimmunoassay,

Turk J Hematol 2015;32:43-50

Alpay Kanıtez N, et al: Osteopathy in Mastocytosis

bone-specific alkaline phosphatase (BAP) by immunoassay, and tryptase by commercial fluoroenzyme immunoassay. Pyridinoline and deoxypyridinoline were measured in morning urine samples by high-performance liquid chromatography and values were corrected for urine excretion of creatinine. Radiographs and Body Mass Index Radiographs of the bones that better reflect osteopenia and/ or osteosclerosis, including the spine (T4-L5), pelvis, femur, and humerus, were assessed by radiologists. Concurrently observed osteolytic and osteosclerotic lesions were interpreted as more severe bone disease than only observed osteolytic lesions. For severity of bone disease, bone radiological findings were arbitrarily documented for statistical analysis as follows: 1, normal; 2, lytic lesions; 3, severe lytic lesions and sclerotic lesions. Radiological findings were compared with BMD values. Body mass index was calculated as body weight divided by height squared. Statistical Analysis Result were expressed as median (minimum-maximum). Test statistics were computed using the Mann-Whitney U test. Correlation coefficients and significance were calculated by Spearman’s test to assess the differences between groups. For all the tests, a 2-tailed p-value of <0.05 was considered statistically significant. Statistical analyses were performed using SPSS 13 on Windows NT. Results Patient’s Demographics and Characteristics The overall characteristics of the patients by diagnostic category are shown in Table 1. The age of patients ranged from 20 to 64 years of age. Five of the 17 patients were female. Four patients had CM, 9 patients had ISM, 2 patients had SSM, 1 patient had ASM, and 1 patient had MCL. Bone Mineral Density The prevalence of low T-scores is shown in Table 2. Overall, 52% of patients (9 of 17 with complete data; BMD measurements at lumbar spine L1-L4, femoral neck, and distal radius and calcaneus stiffness) had T-scores between -1 and -2.5 for at least 1 site. Seventeen percent (3 of 17) had T-scores of less than -2.5. The statistical means of BMD for different areas and biochemical parameters of bone turnover and tryptase levels are shown in Table 3. The mean T-score calculated for the lumbar spine L1-L4 was lowest (-1.8±1.1) in the ISM group; 6 of 9 patients (66%) had T-scores between -1 and -2.5 and 2 patients (22%) had T-scores of less than -2.5. The mean femoral neck T-score was found to be the lowest (-0.1±0.5) in the SSM group. Two patients had femoral neck bone density of less than -1, and those patients were diagnosed with ISM. Six patients (35%) had low T-scores at the distal radius (2 with ISM and 2 with SSM). One of them

with SSM had a T-score of less than -2.5. Eight patients (47%) had T-scores of calcaneus stiffness of less than -1. Significant differences for values of lateral spine BMD and all areas of radius BMD were found between female and male groups (p<0.05). Although not statistically significant, BMD of the spine and femur was increased in patients with more severe disease compared to patients with less severe disease. The results of calcaneal QUS were similar to results acquired by DXA. There was no relationship among history of hypotensive episodes, dyspepsia, diarrhea, and clinical severity. No significant correlation was found among clinical severity or serum tryptase level or any bone turnover marker and BMD calculated with not only DXA but also calcaneal QUS. Bone Turnover Markers The levels of serum ALP, tryptase, and urine pyridinoline were found to be significantly different among patient groups defined by disease severity as less severe and as more severe (respectively p<0.01, p<0.05, and p<0.01). Patients with less severe disease had lower pyridinoline and tryptase levels than patients with more severe disease (Figure 1). There was a significant positive correlation between disease severity and pyridinoline as well as tryptase levels for both sexes (p<0.01). The same correlation was shown between tryptase and pyridinoline (p<0.01) (Figure 2). Besides tryptase level, a positive correlation was found between pyridinoline and severity of radiological findings. There was no relationship between BMD and biochemical parameters of bone formations (BAP, osteocalcin, pyridinoline, deoxypyridinoline). Although

200 180 160 140 120 Tryptase*

100

Pyridinoline*

80 60 40 20 0 CM

ISM

SSM

ASM

MCL

Figure 1. Urine pyridinoline levels (reference interval: 20-52 pmol/µmol creatinine for men, 25-63 pmol/µmol creatinine for women) and serum tryptase levels (reference interval: <13.5 µg/L) in accordance with clinical form of mastocytosis. Median values for the CM, ISM, and SSM groups containing more than 1 patient are given. *: µg/L, **: pmol/µmol creatinine.

Turk J Hematol 2015;32:43-50

Alpay Kan覺tez N, et al: Osteopathy in Mastocytosis

a positive correlation was found between serum ALP levels and disease severity (p<0.01), there was no correlation between BAP and disease severity. It was speculated that the source of elevated serum ALP levels in patients with severe disease may be partly hepatic. Radiological Findings

Discussion

No finding of bone fracture was seen on radiographs. There were lytic and sclerotic bone lesions, especially in the lumbar spine and femoral neck. Three of 17 patients (1 with SSM, 1 with ASM, and 1 with MCL) had lytic and sclerotic lesions in at least 1 site in bone radiographs. Other patients with ASM had multiple lytic bone lesions. Four of 9 patients with ISM had lytic bone lesions in at least 1 site. There were no lesions in radiographs of the patients with CM. Besides osteolytic lesions, patients with severe disease had more osteosclerotic lesions on their radiographs than patients with less severe disease. The relationships among T-scores of the spine and femoral neck

creatinine

Figure 2. Scatter plot shows that there was a positive correlation between serum tryptase levels and urine pyridinoline levels (p<0.01).

Figure 3. Spine T-scores of all patients are shown according to radiological severity. Radiological findings are documented as 1: normal, 2: lytic lesions, and 3: severe lytic lesions and sclerotic lesions. 46

and radiological findings of those bone areas for each patient are shown in Figure 3. The patients with less severe disease had lower T-scores compared to patients with more severe disease. Considering radiological findings, the reason for this is the increased osteosclerosis in more severe disease.

The frequency of bone changes varies with the clinical form of SM. The most common radiological findings associated with SM consist of concurrent osteosclerotic and osteolytic lesions (45%) [2]. According to previous studies, trabecular and cortical bone turnover increases in the regions of MC accumulation [6]. In trabecular or cortical bone, accelerated bone remodeling is remarkable, with expansive peritrabecular fibrosis, osteoidosis, increased numbers of osteoblasts and osteoclasts, and extension of osteoclastic resorbing surfaces [7]. Excess MCs are associated with accelerated bone loss and remodeling states. Heterogeneous groups of mediators such as the granule-associated mediators (histamine, heparin, and neutral proteases) and lipid-derived mediators (lipoxygenase, cyclooxygenase, leukotrienes, and prostaglandin D2) are released from MCs [8]. Histamine, the most important mediator produced and stored, has been shown to modulate osteoclastic activity. Thereby, the MCs may lead to osteoclastic bone resorption by increasing osteoclasts [9]. Large-sized osteolyses or/and severe osteoporosis causing pathologic fractures is one of the C findings defining disease stage and severity [2,5]. Radiographic methods, including roentgenography, magnetic resonance imaging, nuclear bone scan, and bone densitometry, have been employed to assess the patterns and severity of skeletal involvement [10,11]. DXA is currently the most frequently used instrument for measuring BMD [12]. Previous studies showed that DXA is unable to detect changes in trabecular bone microstructure in relation to changes in its mechanical properties [13]. Although DXA is a useful technique to detect bone mineral density in patients with osteolytic lesions, patients with severe disease had more sclerotic changes, especially in the trabecular bone of the pelvis and the thoracolumbar spine. Those patients had an increased level of bone density in trabecular bone and low bone density in the distal radius. There was not a statistically significant correlation between BMD of any area and disease severity. DXA is probably not adequate to detect bone changes in patients with aggressive mastocytosis because osteosclerotic lesions may occur concurrently and cause higher BMD scores despite the occurrence of osteopenia [4,5]. There is increased interest in calcaneal QUS for osteoporosis screening as an additional method because it predicts fracture risk, is portable, and is relatively inexpensive [14]. Ultrasound parameter values are typically lower in osteoporotic bone

Turk J Hematol 2015;32:43-50

Alpay Kanıtez N, et al: Osteopathy in Mastocytosis

than in healthy bone [15,16]. On the other hand, there is no consensus regarding its accuracy for identifying patients with osteoporosis, and osteosclerosis is also a problem in screening for osteoporosis in patients with mastocytosis, as in our results [17]. Therefore, calcaneal QUS does not seem to be a useful method compared to DXA in estimating bone involvement in patients with mastocytosis. Direct radiography was found to be more useful for showing bone involvement in our patients. Both osteosclerotic and osteolytic lesions can be shown on radiographs, especially in severe disease. Although there were no findings of fracture in the radiographies of our patients, pathological fractures may be seen in mastocytosis patients [18,19]. Biochemical markers of bone turnover, such as pyridinoline and BAP, may increase in other diseases, such as hyperthyroidism, suggesting an increase in osteoclastic and osteoblastic activity [20]. Johansson et al. found that the level of serum ALP was significantly higher in mastocytosis patients with severe osteoporosis [4]. However, BAP is a more specific marker because serum ALP may be elevated in severe cases with hepatic involvement. We used BAP, osteocalcin, pyridinoline, and deoxypyridinoline as bone turnover markers and observed that patients with severe skeletal lesions had greater increases in levels of BAP and pyridinoline than patients with mild skeletal lesions. The relationship between BAP and

disease severity was not statistically significant, whereas there was a significant correlation between pyridinoline and disease severity (p<0.01). Kushnir-Sukhov et al. showed that elevated tryptase levels were associated with greater bone density in patients with mastocytosis [5]. We could not confirm this relationship in this study. Both our study and the study done by KushnirSukhov (21 patients) had a small number of patients because mastocytosis is a very rare disease. Tryptase is a well-established and important disease-related marker that should be determined in patients with suspected mastocytosis [21,22]. Higher tryptase values increase the likelihood of multiorgan involvement. Moreover, tryptase levels in SM are thought to correlate with the burden of neoplastic MCs [23]. In our study, there was a similar correlation between disease severity and serum tryptase and urine pyridinoline; patients with severe bone lesions had a greater elevation of pyridinoline levels. According to a previous study, tryptase may activate matrix metalloproteinases (MMPs) [24]. MMP can degrade collagens and may increase the pyridinoline cross-linked carboxyterminal telopeptide of type 1 collagen. Therefore, it may be speculated that one cause of osteolytic bone lesions may be tryptase associated with elevated pyridinoline levels; however, this hypothesis is insufficient to explain osteosclerosis in more severe disease.

Table 1. General characteristics of patients with mastocytosis.

Patients (n=17)

CM (n=4)

ISM (n=9) SSM (n=2)

ASM (n=1)

MCL (n=1)

Age, years

33 (20-64)

30 (20-49)

33 (23-51)

45.5 (40-51)

64.0

47.0

Months of disease

12 (1-120)

10.5 (1-120)

10 (1-108)

16 (8-24)

12.0

14.0

Females

5 (29%)

2 (50%)

2 (22%)

1 (50%)

None

Body mass index

25 (22-33)

25.5 (22-32)

25 (23-30)

30.5 (22-33)

23.0

26.0

Body weight

75 (58-98)

82.5 (65-98)

75 (58-96)

89 (76-98)

67.0

76.0

History of smoking*

13.1±18.7

12.5±15.0

5.6±13.0

42.5±24.7

None

37.0

History of HE

9 (52%)

2 (50%)

3 (33%)

2 (100%)

1 (100%)

History of dyspepsia

12 (70%)

1 (25%)

7 (77%)

2 (100%)

1 (100%)

History of diarrhea

6 (35%)

1 (25%)

3 (33%)

1 (50%)

None

1 (100%)

Hepatomegaly

8 (47%)

1 (25%)

3 (33%)

2 (100%)

1 (100%)

Splenomegaly

4 (23%)

None

2 (100%)

1 (100%)

Lymphadenomegaly

2 (11%)

None

1 (100%)

Urticaria pigmentosa

15 (88%)

4 (100%)

9 (100%)

1 (50%)

1 (100%)

none

Values represent median (minimum-maximum). HE: Hypotensive episodes, CM: cutaneous mastocytosis, ISM: indolent systemic mastocytosis, SSM: smoldering systemic mastocytosis, ASM: aggressive systemic mastocytosis, MCL: mast cell leukemia , *: pack-years.

Turk J Hematol 2015;32:43-50

Alpay Kanıtez N, et al: Osteopathy in Mastocytosis

Table 2. T-scores of the lumbar spine, femoral neck, distal radius, and QUS of the calcaneus for each mastocytosis category. T-scores calculated with DXA were obtained for all patients but calcaneal QUS could not performed for 1 patient because there was anatomical mismatch between the device and the patient’s heel.

Diagnostic Category

Spine L1-L4

Femoral Neck

Distal Radius

Calcaneus Stiffness

T*<-1

T<-2.5

T<-1

T<2.5

T<-1

T<-2.5

T<-1

Patients (n=17) (5 females, 12 males)

10 (58%)

2 (11%)

6 (35%)

1 (5%)

8 (47%)

Less SD (n=13) (4 females, 9 males)

9 (69%)

2 (15%)

4 (30%)

6 (46%)

CM (n=4) (2 females, 2 males)

1 (25%)

ISM (n=9) (2 females, 7 males)

8 (88%)

2 (22%)

4 (44%)

5 (55%)

More SD (n=4) (1 female, 3 males)

1 (25%)

2 (50%)

1 (25%)

2 (50%)

SSM (n=2) (1 female, 1 male)

1 (50%)

2 (100%)

1 (50%)

2 (100%)

ASM (n=1 male)

MCL (n=1 male)

-**

CM: Cutaneous mastocytosis, ISM: indolent systemic mastocytosis, SSM: smoldering systemic mastocytosis, ASM: aggressive systemic mastocytosis, MCL: mast cell leukemia, Less SD: less severe disease including CM and ISM, More SD: more severe disease including SSM, ASM, MCL. *: T-scores,**:calcaneus stiffness could not be measured in this case.

Table 3. Biochemical parameters and BMD of spine, femoral neck, total radius, and calcaneus for each mastocytosis category.

ISM

SSM

ASM MCL

All

5.25 (3-7)

87.3 (3-200)

144 (117-171)

80.0

200.0

80 (3-200)

Osteocalcin (ng/mL) 8.2 (3.7-15.2)

3.7 (1-13)

19.6 (11.2-28)

2.1

8.8

5.8 (1-28)

bALP (U/L)

32 (25-64)

42.5 (28-60)

42.3 (35-50)

47.5

185

42.5 (25-185)

PYR¶

16.6 (11-27.2)

48.4 (22.6-78.6)

86.3

114.6

161.7

48.4 (11-161.7)

DPYR

6.3 (5.2-12.6)

6.4 (5.4-13.7)

12.5

6.5

11.1

7.1 (5.2-13.7)

Spine BMD

0.972 (0.956-1.135)

0.884 (0.720-1.114)

0.946 (0.944-0.949) 1.465

1.409

0.956 (0.720-1.465)

Lateral spine BMD

0.802 (0.496-1.121)

0.725 (0.444-0.905)

0.853 (0.728-0.979) 1.052

2.634

0.728 (0.444-2.634)

Femur BMD

1.014 (0.912-1.216)

0.993 (0.662-1.271)

0.966 (0.955-0.978) 1.323

1.301

0.993 (0.662-1.323)

Total radius BMD

0.587 (0.564-0.633)

0.590 (0.430-0.735)

0.477 (0.461-0.493) 0.654

0.590

0.590 (0.430-0.735)

Calcaneus BMD

0.570 (0.435-0.742)

0.466 (0.366-0.794)

0.331 (0.297-0.366) 0.587

0.484 (0.297-0.794)

Tryptase

(µg/L)¶

Values represent median (minimum-maximum). ¶: There was a significant relationship among patient groups according to disease severity (respectively p<0.05 and p<0.01). *: Calcaneus stiffness could not be measured in this case. PYR: Pyridinoline (pmol/µmol creatinine, normal values for men: 20-52, for women: 25-63), DPYR: deoxypyridinoline (pmol/µmol creatinine, normal values for men: 4-11, for women: 6-13), BMD: bone mineral density, CM: cutaneous mastocytosis, ISM: indolent systemic mastocytosis, SSM: smoldering systemic mastocytosis, ASM: aggressive systemic mastocytosis, MCL: mast cell leukemia. Normal values of bALP: 12-42, osteocalcin: 2-22 ng/mL, tryptase: 13.5 µg/L.

Alpay Kanıtez N, et al: Osteopathy in Mastocytosis

In conclusion, this study confirmed that SM can affect bone remodeling in various ways and demonstrated the need for radiography in bone evaluation. BMD measured by DXA or calcaneal QUS may not be decreased in patients with osteopenia due to osteosclerotic bone lesions seen on plain radiography. In follow-up, increase in serial BMD measurements related to the osteosclerotic lesions in patients with SM may be used to determine the progression of bone involvement. High pyridinoline levels may indicate high bone turnover in mastocytosis and are associated with more severe bone lesions. However, it is not clear whether high pyridinoline levels are the result of elevated tryptase levels. The number of patients studied here is rather small; thus, further work is needed. Conflict of Interest Statement 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. Valent P, Akin C, Sperr WR, Mayerhofer M, Födinger M, Fritsche-Polanz R, Sotlar K, Escribano L, Arock M, Horny HP, Metcalfe DD. Mastocytosis: pathology, genetics, and current options for therapy. Leuk Lymphoma 2005;46:35-48. 2. Horny HP, Metcalfe DD, Bennett JM, Bain BJ, Akin C, Escribano L, Valent P. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds). World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissue. 4th ed. Lyon, France, IARC, 2008. 3. Valent P, Horny HP, Escribano L, Longley BJ, Li CY, Schwartz LB, Marone G, Nuñez R, Akin C, Sotlar K, Sperr WR, Wolff K, Brunning RD, Parwaresch RM, Austen KF, Lennert K, Metcalfe DD, Vardiman JW, Bennett JM. Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leuk Res 2001;25:603-625. 4. Johansson C, Roupe G, Lindstedt G, Mellström D. Bone density, bone markers and bone radiological features in mastocytosis. Age Ageing 1996;25:1-7. 5. Kushnir-Sukhov NM, Brittain E, Reynolds JC, Akin C, Metcalfe DD. Elevated tryptase levels are associated with greater bone density in a cohort of patients with mastocytosis. Int Arch Allergy Immunol 2006;139:265-270. 6. Valent P, Akin C, Sperr WR, Escribano L, Arock M, Horny HP, Bennett JM, Metcalfe DD. Aggressive systemic mastocytosis and related mast cell disorders: current treatment options and proposed response criteria. Leuk Res 2003;27:635-641. 7. Chiappetta N, Gruber B. The role of mast cells in osteoporosis. Semin Arthritis Rheum 2006;36:32-36.

Turk J Hematol 2015;32:43-50

8. Fallon MD, Whyte MP, Teitelbaum SL. Systemic mastocytosis associated with generalized osteopenia. Histopathological characterization of the skeletal lesion using undecalcified bone from two patients. Hum Pathol 1981;12:813-820. 9. Brumsen C, Papapoulos SE, Lentjes EG, Kluin PM, Hamdy NA. A potential role for the mast cell in the pathogenesis of idiopathic osteoporosis in men. Bone 2002;31:556-561. 10. Weide R, Ehlenz K, Lorenz W, Walthers E, Klausmann M, Pflüger KH. Successful treatment of osteoporosis in systemic mastocytosis with interferon alpha-2b. Ann Hematol 1996;72:41-43. 11. Lehmann T, Beyeler C, Lämmle B, Hunziker T, Vock P, Olah AJ, Dahinden C, Gerber NJ. Severe osteoporosis due to systemic mast cell disease: successful treatment with interferon alpha2B. Br J Rheumatol 1996;35:898-900. 12. Lewiecki EM, Borges JL. Bone density testing in clinical practice. Arq Bras Endocrinol Metabol 2006;50:586-595. 13. Nazarian A, Cory E, Müller R, Snyder BD. Shortcomings of DXA to assess changes in bone tissue density and microstructure induced by metabolic bone diseases in rat models. Osteoporos Int 2009;20:123-132. 14. Khaw KT, Reeve J, Luben R, Bingham S, Welch A, Wareham N, Oakes S, Day N. Prediction of total and hip fracture risk in men and women by quantitative ultrasound of the calcaneus: EPICNorfolk prospective population study. Lancet 2004;363:197202. 15. Prins SH, Jørgensen HL, Jørgensen LV, Hassager C. The role of quantitative ultrasound in the assessment of bone: a review. Clin Physiol 1998;18:3-17. 16. Danese RD, Licata AA. Ultrasound of the skeleton: review of its clinical applications and pitfalls. Curr Rheumatol Rep 2001;3:245-248. 17. Nayak S, Olkin I, Liu H, Grabe M, Gould MK, Allen IE, Owens DK, Bravata DM. Meta-analysis: accuracy of quantitative ultrasound for identifying patients with osteoporosis. Ann Intern Med 2006;144:832-841. 18. Pusl T, Kenngott S, Bartl R, Baur A, Ludolph-Hauser D, Juengst D. A case of systemic mastocytosis associated with severe osteoporosis and pathologic fractures. Eur J Intern Med 2004;15:537-539. 19. Inaoui R, Petit B, Jaccard A, Bertin P, Trèves R. Aggressive systemic mastocytosis. Joint Bone Spine 2003;70:64-66. 20. Van de Ven AC, Erdtsieck RJ. Changes of bone mineral density, quantitative ultrasound parameters and markers of bone turnover during treatment of hyperthyroidism. Neth J Med 2008;66:428-432.

Turk J Hematol 2015;32:43-50

21. Schwartz LB, Sakai K, Bradford TR, Ren S, Zweiman B, Worobec AS, Metcalfe DD. The alpha form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis. J Clin Invest 1995;96:2702-2710. 22. Akin C, Metcalfe DD. Surrogate markers of disease in mastocytosis. Int Arch Allergy Immunol 2002;127:133-136. 23. Sperr WR, Jordan JH, Fiegl M, Escribano L, Bellas C, Dirnhofer S, Semper H, Simonitsch-Klupp I, Horny HP, Valent P. Serum tryptase levels in patients with mastocytosis: correlation with mast cell burden and implication for defining the category of disease. Int Arch Allergy Immunol 2002;128:136-141.

Alpay Kan覺tez N, et al: Osteopathy in Mastocytosis

24. Iddamalgoda A, Le QT, Ito K, Tanaka K, Kojima H, Kido H. Mast cell tryptase and photoaging: possible involvement in the degradation of extra cellular matrix and basement membrane proteins. Arch Dermatol Res 2008;300:69-76.

Research Article

DOI: 10.4274/tjh.2013.0278

Tunnelled Central Venous Catheter-Related Problems in the Early Phase of Haematopoietic Stem Cell Transplantation and Effects on Transplant Outcome Hematopoetik Kök Hücre Alıcılarında Tünelli Santral Venöz Kateter Problemleri ve Erken Dönem Nakil Komplikasyonları Üzerine Etkileri Mahmut Yeral1, Can Boğa1, Levent Oğuzkurt2, Hikmet Eda Alışkan3, Hakan Özdoğu1, Yusuf Ziya Demiroğlu4 1Başkent

University Faculty of Medicine, Adana Adult Bone Marrow Transplantation Centre, Department of Hematology, Adana, Turkey University Faculty of Medicine, Department of Radiology, Ankara, Turkey 3Başkent University Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey 4Başkent University Faculty of Medicine, Department of Infectious Disease and Clinical Microbiology, Ankara, Turkey 2Başkent

Abstract: Objective: Haematopoietic stem cell recipients need central venous catheters (CVCs) for easy administration of intravenous

fluid, medications, apheresis, or dialysis procedures. However, CVCs may lead to infectious or non-infectious complications such as thrombosis. The effect of these complications on transplantation outcome is not clear. This manuscript presents the complication rates of double-lumen tunnelled CVCs and their effect on transplantation outcome. Materials and Methods: Data from 111 consecutive patients, of whom 75 received autologous and 36 received allogeneic peripheral blood stem cell transplantations, were collected retrospectively. The data were validated by the Record Inspection Group of the related JACIE-accredited transplantation centre. Results: Thrombosis developed in 2.7% of recipients (0.9 per 1000 catheter days). Catheter-related infection was identified in 14 (12.6%) patients (3.6 per 1000 catheter days). Coagulase-negative Staphylococcus was the most common causative agent. Engraftment time, rate of 100-day mortality, and development of grade II-IV graft-versus-host disease were not found to be associated with catheter-related complications. Conclusion: These results indicate that adverse events related with tunnelled CVCs are manageable and have no negative effects on transplant outcome. Key Words: Tunnelled central venous catheter, Haematopoietic stem cell transplantation, Thrombosis, Infection

Özet: Amaç: Hematopoetik kök hücre nakli alıcıları damar içi mayi, ilaç, aferez ve diyaliz işlemleri için santral venöz kateterlere (SVK) ihtiyaç duyarlar. Ancak SVK’ların enfeksiyöz ve tromboz gibi enfeksiyöz olmayan komplikasyonları vardır. Bu Address for Correspondence: Mahmut YERAL, M.D, Başkent University Faculty of Medicine, Adana Adult Bone Marrow Transplantation Centre, Department of Hematology, Adana, Turkey E-mail: drmyeral@gmail.com Received/Geliş tarihi : August 17, 2013 Accepted/Kabul tarihi : November 8, 2013

Turk J Hematol 2015;32:51-57

Yeral M, et al: Catheter Problems and Effects on Transplant

komplikasyonların nakil sürecine etkisi net değildir. Bu çalışma ile çift lümen tünelli santral kateter komplikasyonlarının sıklığı ve nakil sürecine etkisi araştırılmıştır.

Gereç ve Yöntemler: Geriye dönük olarak 75 otolog ve 39 allojeneik periferik kök hücre nakli uygulanan toplam 111 hastanın verileri toparlandı. Verilerin doğruluğu JACIE akredite merkezimizin kayıt denetleme grubu tarafından kontrol edildi. Bulgular: Olguların %2,7’sinde tromboz gelişti (1000 kateter gününde 0,9). Hastaların 14’ünde (%12,6) kateter ilişkili enfeksiyon tesbit edildi (1000 kateter gününde 3,6). Koagulaz negatif Stafilokoklar en sık etken idi. Kateter ilişkili komplikasyonlar ile engrafman zamanı, evre II-IV graft versus host hastalığı ve 100 günlük mortalite arasında bir ilişki saptanmadı. Sonuç: Sonuçlarımız nakil hastalarında tünelli SVK komplikasyonlarının yönetilebilir olduğunu ve bu komplikasyonların nakil üzerine olumsuz bir etki yaratmadığını göstermektedir.

Anahtar Sözcükler: Tunelli santral venöz kateter, Hematopoietik kök hücre transplantasyonu, Tromboz, Enfeksiyon Introduction Haematopoietic stem cell recipients need central venous catheters (CVCs) for chemotherapy, parenteral nutrition, and blood infusion. A wide range of catheter types can be used. However, the type, number of lumens, diameter, and insertion site of the catheter should be appropriate for the procedure, as these affect the frequency of CVC-related complications [1,2,3]. Tunnelled CVC is considered appropriate for neutropenic and bone marrow transplant patients. Doublelumen tunnelled CVCs have a lower risk of infection than nontunnelled catheters [4]. These are long-term catheters that allow for the simultaneous infusion of physically incompatible drugs, blood products, and stem cells. Long-term, continuous total parenteral nutrition can be provided via these catheters. There is limited literature on the complications of tunnelled CVC in haematopoietic stem cell recipients. The effects of these complications on the outcome parameters of transplant recipients remain to be identified. This study aimed to determine the frequency of tunnelled CVC complications in patients who underwent peripheral blood stem cell transplantation and to analyse the effects of complications on engraftment time, 100-day mortality, and acute graft-versus-host disease (GVHD). Materials and Methods Study Plan This was a retrospective, cross-sectional study; it included 111 patients who underwent haematopoietic peripheral blood stem cell transplantation between May 2011 and January 2013. Data were collected from a previously authorised and validated Hospital Information Management System (Nucleus, Monad, Ankara, Turkey). The data were validated by the Record Inspection Group of the related department. The primary endpoint of the study was the rate of catheter-related complications. Haemorrhage, thrombosis, and infection data obtained from the record system were

calculated according to the number of catheterisations and per 1000-catheter days. Secondary endpoints were the effects of complications on thrombocyte and neutrophil engraftment, 100-day mortality, and development of acute GVHD. The study included patients who were considered eligible for autologous and allogeneic peripheral blood haematopoietic stem cell transplantation by the clinical directors of the transplant centre. Patient eligibility was evaluated according to the Republic of Turkey Regulations on Tissue and Stem Cell Transplantation and FACT-JACIE Standards (version 5.2) [5]. Patients who had a purulent skin infection of the chest or arm during catheter application, those who had chest trauma, those who received radiotherapy in the chest region, and those of paediatric age (<16 years) were excluded from the study. The study was approved by the Başkent University Research Committee. Timing of Placement, Replacement, or Removal of Catheters Tunnelled CVCs were placed in patients just prior to starting a conditioning regime for peripheral blood haematopoietic stem cell transplantation. Naturally, these CVCs were not used for harvesting peripheral blood cells. Clinicians continually reviewed the need for central venous access in individual patients. Catheters were replaced only for clinical indications like clinical infection or purulence at the insertion site. Selection and Placement of Catheters All CVCs were inserted in the Interventional Radiology Unit by a radiologist or a specialised nurse only. Complete blood count and coagulation tests were performed before the procedure for all patients who received a double-lumen, 18-cm-long, tunnelled CVC (Cath, Medcomp, Harleysville, PA, USA). Prophylactic antibiotics at the time of insertion of the CVC, in-line filters, or anti-infective/microbial lock prophylaxis were not used.

Turk J Hematol 2015;32:51-57

Yeral M, et al: Catheter Problems and Effects on Transplant

The Interventional Radiology Unit inserted CVCs using an ultrasound image-guided percutaneous technique. The right internal jugular vein was preferred for first access; however, the left internal jugular was used in the case of a problem on the right side. The vein was punctured with an 18-G needle under ultrasonography guidance [6]. Care of the Catheters A sterile gauze dressing, secured with adhesive tape, was only to be changed by a catheter nurse until sufficient healing had taken place (following adherence of the cuff; usually within 3 weeks of insertion). During this period, the gauze was changed by the catheter nurse at least every 48 h. Flushing was used after placement prior to and after fluid infusion or injection, and prior to and after blood drawing. The patients carried responsibility for the catheter following hospital discharge. Culture Collection Procedure A 10-mL blood sample was collected from the peripheral vein and from each lumen of the catheter simultaneously into an aerobic blood culture bottle. Culture bottles were incubated in a BACTEC 9240 (Becton Dickinson Microbiology Systems, Sparks, MD, USA) automatic culture device. Direct stained preparations were made from blood culture bottles, which gave a positive result from the system. The cultures used media containing 5% sheep blood and eosin methylene blue. Cultures were evaluated after incubation at 37 °C for 24-48 h. Conventional methods and BBL crystal identification systems (Becton Dickinson Microbiology Systems) were used to identify bacteria. Antibiotic sensitivity was determined via the Kirby-Bauer disc diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute.

Identification of Complications If the same agent grew in the blood culture collected from the catheter lumen and the peripheral blood, or if the growth in the blood culture collected from the catheter lumen occurred 2 h prior to that in peripheral blood, it was considered a catheter-related blood-stream infection. If erythema, increased temperature, or sensitivity occurred within a 2-cm periphery of the insertion site of the catheter, it was considered a catheter-exit-site infection, irrespective of whether the blood culture contained bacterial growth [7]. Thrombosis as detected by Doppler ultrasonographic examination was evaluated to obtain more objective and reliable data and thus avoid misevaluation. Statistical Analysis Statistical analysis was performed using SPSS 17.0. For each continuous variable, normality was checked using Kolmogorov-Smirnov and Shapiro-Wilk tests. Comparisons between groups were applied using a one-way Student’s t-test for normally distributed data, and the Mann-Whitney test was used for data not normally distributed. Categorical variables between groups were analysed using the chi-square test. Statistical significance was accepted at p<0.05. Results Transplant patients in the study included 40 females (36%) and 71 males (64%), with a median age of 49 years (range: 16-64). Of the patients, 75 (67.6%) underwent an autologous transplant while 36 (32.4%) underwent an allogeneic transplant. Diagnoses and stem cell transplant types are presented in Table 1. All 111 catheters placed in the patients were tunnelled central catheters. Of these, 100 (90%) were placed in the right internal jugular vein and 11 (10%) in the left internal

Table 1. Distribution of patients who underwent catheter placement according to primary disease and haematopoietic stem cell transplantation type.

Primary Disease

Total, n (%)

Allogeneic PBSCT, n

Autologous PBSCT, n

Acute myeloblastic leukemia

15 (14)

Multiple myeloma

50 (45)

Non-Hodgkin’s lymphoma

23 (21)

Hodgkin’s lymphoma

9 (8)

Acute lymphoblastic leukemia

8 (7)

Other

6 (5)

Total

111 (100)

PBSCT: Peripheral blood stem cell transplantation.

Turk J Hematol 2015;32:51-57

Yeral M, et al: Catheter Problems and Effects on Transplant

jugular vein. No catheters were placed in the subclavian veins. Catheters placed in the internal jugular veins had a median size of 10 F (range: 8-14 F). Total catheter dwell time was 3322 catheter days. Median catheter dwell time per catheter was 26 (range: 15-106) days; the median was 14 (15-85) days for autologous transplants and 30 (15-106) days for allogeneic transplants (p<0.05). Of the 111 catheters used during bone marrow transplantation, 108 (92.7%) were removed when they were no longer needed. Two catheters were removed due to infection and thrombosis complications, and 1 catheter (0.9%) exited spontaneously. The incidences of tunnelled CVC-related complications in transplant recipients are shown in Table 2. Analysis according to catheterisation day showed complications in a total of 26 (23.4%) patients (7.8 complications per 1000 catheter days). Mild haemorrhage, in the form of leakage, was observed in 2 patients (1.8%). Three of 111 patients developed symptomatic thrombosis attack (2.7%), representing a rate of 0.9 per 1000 catheter days. Symptomatic thrombosis rates of the autologous and allogeneic groups were 2 (2.7%) and 1 (2.8%), respectively. Fibrin sheaths, which cause catheter dysfunction, were Table 2. The incidences of tunnelled CVC-related complications in transplant recipients.

Complications

Incidence, n (%)

Events in 1000 catheter days, n

Haemorrhage

2 (1.8)

0.6

Fibrin sheaths

7 (6.3)

2.1

Thrombosis

3 (2.7)

0.9

Infection Exit site Blood stream

14 (12.6) 2 (1.8) 12 (10.8)

4.2 0.6 3.6

Total

26 (23.4)

7.8

CVC: Central venous catheter.

identified in 7 (6.3%) patients (2.1 per 1000 catheter days). There was no significant difference between the allogeneic and autologous sub-groups in terms of the frequencies of catheter-related thrombosis and fibrin sheaths (p>0.05). Catheter-related infections were identified in 14 (12.6%) patients. Of these infections, 2 (1.8%) were considered exitsite infections. During follow-up, catheter-related bloodstream infection was detected in 12 (10.8%) patients (3.6 per 1000 catheter days), which represented 9.3% and 13.9% of autologous and allogeneic transplant recipients, respectively. One (8.4%) infection was caused by Corynebacterium jeikeium, 2 (16.6%) were caused by Escherichia coli, and the remaining 9 catheter-related blood-stream infections (75%) were caused by coagulase-negative Staphylococcus spp. The relationship between complications and catheter days was assessed. There was no significant relationship with thrombosis (p>0.05), but there was a statistically significant relationship between duration of catheter dwelling and infection (p<0.05). Outcome parameters according to type of stem cell transplantation are documented in Table 3. All patients achieved absolute neutrophil counts of â&#x2030;Ľ0.5x109/L at a median of day 12 (range: 8-17) and platelet counts of â&#x2030;Ľ20x109/L at a median of day 11 (range: 8-23). In the autologous and allogeneic sub-groups, the neutrophil engraftment times were 12 (8-17) and 12 (10-17) days, respectively; thrombocyte engraftment times were 11 (8-21) and 12 (9-23) days, respectively. We observed no significant difference between the autologous and allogeneic groups in terms of engraftment time (p>0.05). Grade II-IV acute GVHD was identified in 4 (11.1%) patients who underwent allogeneic stem cell transplants. Analyses showed that infection and thrombosis did not delay engraftment times. No evidence of acute GVHD was observed in patients who developed catheter-related blood-stream infection. The overall 100-day mortality rate was 3.6% in all stem cell recipients. None of the mortalities were related to catheter complications.

Table 3. Outcome parameters according to type of stem cell transplantation.

Type of Transplantation

Days until neu. engraftment, median (range)

Days until plt. engraftment, median (range)

100-day mortality rate, n (%)

Autologous

12 (8-17)

11 (8-21)

2 (2.6)

Allogeneic

12 (10-17)

12 (9-23)

2 (5.5)

Total

12 (8-17)

11 (8-23)

4 (3.6)

Neu: neutrophil, plt: platelet.

Yeral M, et al: Catheter Problems and Effects on Transplant

Discussion Tunnelled CVCs differ from other catheters in the sense that one part of the catheter remains in subcutaneous tissue after the venous entrance level. This tunnel theoretically provides stability and serves as a barrier against skin-related infections. If high-flow venous access is necessary for the patient, and if this vascular access is required for at least 3 weeks, a tunnelled catheter is preferred. Catheters can have some complications. Mechanical complications in the early period include conditions such as haematoma, haemorrhage, artery adjustments, and pneumothorax. Previous studies show that, when implanted by experienced personnel using imaging techniques, the incidence of complications is low [1,6,8]. In the present study, tunnelled central catheters were inserted by experienced staff of the Interventional Radiology Unit under ultrasonographic guidance. In our study, haemorrhage in the form of leakage was observed in 2 catheters as a mechanical complication. Leakage was controlled by local compression without further intervention. A review of the literature showed that infection levels in patients with a haematological malignity with tunnelled or non-tunnelled CVCs varied from 4.5% to 20.8% [9,10,11]. The frequency of infection in patients with haematological malignancy was higher than in those with solitary tumours [12,13]. In acute myeloid leukemia patients, the catheterrelated bacteraemia rate was 31.5%, and 69.6% had a mortal course after stem cell infusion [14]. In a large patient series that included mostly acute leukemia patients (690 Hickman catheters), catheter-related bacteraemia was found in 2.9 per 1000 catheter days [15]. The incidence of Hickman catheterrelated infections in cancer patients was reported to be 5.987.2 per 1000 catheter days [16]. In contrast, the incidence of catheter-related infections was 2.7% in intensive care unit patients who were placed with short-term CVCs (5.9 per 1000 catheter days) [17]. Our rate of catheter-related infections was lower than those in intensive care unit patients and haematological and non-haematological malignancy patients with temporary CVCs. Blood-stream infection rates were similar to those in previous reports of complications of tunnelled CVCs. Catheter infection levels in allogeneic transplant patients were higher than those in autologous catheter patients. This is likely caused by the use of immune suppressive agents in allogeneic transplants, catheter use time, and more frequent catheter manipulations. Nevertheless, there was no significant difference between the 2 groups. Two cases of exit-site infections were controlled via local treatment and care. A wide spectrum of microorganisms can cause catheter-related infections. However, the frequency of the agents varies according to catheter placement location and

Turk J Hematol 2015;32:51-57

type. The primary source of infection is contamination from the skin flora of both the patient and healthcare personnel. It was reported that 34.1%-50% of catheter-related infections are caused by coagulase-negative Staphylococcus, while 9.9%-20% are caused by S. aureus [17,18,19,20]. In our study, most of the catheter-related infections were caused by coagulase-negative Staphylococcus, suggesting that efficient catheter care or antimicrobial prophylaxis with levofloxacin for transplantation procedure could prevent gram-negative infections. Fibrin sheath formation around the catheter is one of the most important problems that disrupt catheter functions. This has been reported to occur in 56% of patients with short-term catheters [21,22,23]. A fibrin sheath was identified in 7 of our patients. Selective t-PA was infused into the catheter in 5 patients; mechanical intervention via a guide wire was used in the remaining 2 patients. Catheter function was re-established in all patients following the procedure. The type of malignancy, chemotherapy type, catheter type, placement location, catheter usage errors, and inappropriate care are risk factors for thrombosis. Most cases of catheterrelated thrombosis are asymptomatic. A thrombosis incidence of 17%-18% was reported in tunnelled CVCs in solid organ tumours [13,24]. The incidence of symptomatic thrombosis is 1.2%-13% in patients with a haematological malignancy and central catheter [25]. We report here much lower thrombosis rates than did previous studies on tunnelled or non-tunnelled CVCs. All 3 of our thrombosis cases developed 21 days after placement of catheters. The type of transplantation had no significant effect on the rate of complications. However, it should be noted that our series included relatively few thrombosis cases. The risk of thrombosis and infection in Hickman catheters in patients who underwent chemotherapy infusion was approximately 5 times greater than that for implanted ports [26]. Despite the lower complication rates, that type of catheter cannot be considered in haematopoietic peripheral blood stem cell transplantation, especially in allogeneic transplants. Therefore, we did not use implanted venous catheters in our patients. Catheter-related complications, mostly infections, can lead to local or systemic inflammatory conditions. All inflammatory states may contribute to the development of GVHD. In this study, catheter-related exit-site or blood-stream infections were not remarkable observations in the allogeneic transplant recipients who developed grade II-IV GVHD. At the 100 days of follow-up, 2 patients had been lost due to disease progression, 1 due to hepatic veno-occlusive disease, and 1 due to lung infection. Catheter-related complications had no effect on the mortality rate in transplant patients. Three catheters were removed or exited spontaneously before completion of the survey. One catheter in a patient who

Turk J Hematol 2015;32:51-57

Yeral M, et al: Catheter Problems and Effects on Transplant

developed thrombosis was removed because it was not recanalised despite thrombolytic treatment. One of the catheters exited spontaneously. The other catheter was colonised by Corynebacterium jeikeium, which is a hospital-acquired microorganism resistant to multiple antimicrobial agents. In conclusion, double-lumen tunnelled CVC is considered appropriate for preparation regimens, medical treatments, stem cell infusions, and parenteral support in autologous and allogeneic stem cell transplantation patients. Our data indicate that the acceptable incidence of complications in our case series did not have a negative effect on neutrophil or thrombocyte engraftment time and did not increase GVHD or mortality rates. However, further prospective multi-centre studies of catheter-related complications and early-period morbidity and mortality are warranted. Conflict of Interest Statement The authors declare that they have no conflict of interest. All authors agree to allow the journal to review data if requested. This study was approved by the Başkent University Institutional Review Board (Project No. KA13/109) and supported by the Başkent University Research Fund. References 1. McGee DC, Gould MK. Preventing complications of central venous catheterization. N Engl J Med 2003;348:1123-1133. 2. Polderman KH, Girbes AR. Central venous catheter use. Part 2: infectious complications. Intensive Care Med 2002;28:18-28. 3. Polderman KH, Girbes AJ. Central venous catheter use. Part 1: mechanical complications. Intensive Care Med 2002;28:1-17. 4. Randolph AG, Cook DJ, Gonzales CA, Brun-Buisson C. Tunneling short-term central venous catheters to prevent catheter-related infection: a meta-analysis of randomized, controlled trials. Crit Care Med 1998;26:1452-1457. 5. FACT, JACIE. Guidance to Accompany the FACT-JACIE International Standards for Cellular Therapy Product Collection, Processing, and Administration. 5th ed., Version 5.2. 2011.

9. Cortelezzi A, Moia M, Falanga A, Pogliani EM, Agnelli G, Bonizzoni E, Gussoni G, Barbui T, Mannucci PM. CATHEM Study Group. Incidence of thrombotic complications in patients with haematological malignancies with central venous catheters: a prospective multicentre study. Br J Haematol 2005;129:811-817. 10. van Rooden CJ, Schippers EF, Barge RM, Rosendaal FR, Guiot HF, van der Meer FJ, Meinders AE, Huisman MV. Infectious complications of central venous catheters increase the risk of catheter-related thrombosis in hematology patients: a prospective study. J Clin Oncol 2005;23:2655-2660. 11. Nouwen JL, Wielenga JJ, van Overhagen H, Laméris JS, Kluytmans JA, Behrendt MD, Hop WC, Verbrugh HA, de Marie S. Hickman catheter-related infections in neutropenic patients: insertion in the operating theater versus insertion in the radiology suite. J Clin Oncol 1999;17:1304. 12. Rotstein C, Brock L, Roberts RS. The incidence of first Hickman catheter-related infection and predictors of catheter removal in cancer patients. Infect Control Hosp Epidemiol 1995;16:451-458. 13. Anderson AJ, Krasnow SH, Boyer MW, Cutler DJ, Jones BD, Citron ML, Ortega LG, Cohen MH. Thrombosis: the major Hickman catheter complication in patients with solid tumor. Chest 1989;95:71-75. 14. Lukenbill J, Rybicki L, Sekeres MA, Zaman MO, Copelan A, Haddad H, Fraser T, DiGiorgio MJ, Hanna R, Duong H, Hill B, Kalaycio M, Sobecks R, Bolwell B, Copelan E. Defining incidence, risk factors, and impact on survival of central lineassociated blood stream infections following hematopoietic cell transplantation in acute myeloid leukemia and myelodysplastic syndrome. Biol Blood Marrow Transplant 2013;19:720-724. 15. Newman KA, Reed WP, Schimpff SC, Bustamante CI, Wade JC. Hickman catheters in association with intensive cancer chemotherapy. Support Care Cancer 1993;1:92-97. 16. Koolen DA, van Laarhoven HW, Wobbes T, Punt CJ. Singlecentre experience with tunnelled central venous catheters in 150 cancer patients. Neth J Med 2002;60:397-401.

6. Randolph AG, Cook DJ, Gonzales CA, Pribble CG. Ultrasound guidance for placement of central venous catheters: a metaanalysis of the literature. Crit Care Med 1996;24:2053-2058.

17. Walz JM, Memtsoudis SG, Heard SO. Prevention of central venous catheter bloodstream infections. J Intensive Care Med 2010;25:131-138.

7. Mermel LA, Allon M, Bouza E, Craven DE, Flynn P, O’Grady NP, Raad II, Rijnders BJ, Sherertz RJ, Warren DK. Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America. Clin Infect Dis 2009;49:1-45.

18. Abdelkefi A, Torjman L, Ladeb S, Othman TB, Achour W, Lakhal A, Hsairi M, Kammoun L, Hassen AB, Abdeladhim AB. Randomized trial of prevention of catheter-related bloodstream infection by continuous infusion of low-dose unfractionated heparin in patients with hematologic and oncologic disease. J Clin Oncol 2005;23:7864-7870.

8. Fares LG 2nd, Block PH, Feldman SD. Improved house staff results with subclavian cannulation. Am Surg 1986;52:108111.

19. Safdar N, Maki DG. The pathogenesis of catheter-related bloodstream infection with noncuffed short-term central venous catheters. Intensive Care Med 2004;30:62-67.

Yeral M, et al: Catheter Problems and Effects on Transplant

20. Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK. NHSN annual update: antimicrobialresistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infect Control Hosp Epidemiol 2008;29:996-1011. 21. Oguzkurt L, Tercan F, Torun D, Yildirim T, Z端mr端tdal A, Kizilkilic O. Impact of short-term hemodialysis catheters on the central veins: a catheter venographic study. Eur J Radiol 2004;52:293-299. 22. Reddy AS, Lang EV, Cutts J, Loh S, Rosen MP. Fibrin sheath removal from central venous catheters: an internal snare manoeuvre. Nephrol Dial Transplant 2007;22:1762-1765.

Turk J Hematol 2015;32:51-57

24. Anderson AJ, Krasnow SH, Boyer MW, Raucheisen ML, Grant CE, Gasper OR, Hoffmann JK, Cohen MH. Hickman catheter clots: a common occurrence despite daily heparin flushing. Cancer Treat Rep 1987;71:651-653. 25. Boersma RS, Jie KS, Verbon A, van Pampus EC, Schouten HC. Thrombotic and infectious complications of central venous catheters in patients with hematological malignancies. Ann Oncol 2008;19:433-442. 26. Ng F, Mastoroudes H, Paul E, Davies N, Tibballs J, Hochhauser D, Mayer A, Begent R, Meyer T. A comparison of Hickman line- and Port-a-Cath-associated complications in patients with solid tumours undergoing chemotherapy. Clin Oncol (R Coll Radiol) 2007;19:551-556.

23. Baskin JL, Reiss U, Wilimas JA, Metzger ML, Ribeiro RC, Pui CH, Howard SC. Thrombolytic therapy for central venous catheter occlusion. Haematologica 2012;97:641-650.

Brief Report

DOI: 10.4274/tjh.2013.0097

Growth Arrest-Specific 6 (Gas6) and TAM Receptors in Mouse Platelets Fare Trombositlerinde “Growth Arrest-Specific 6” (Gas6) ve TAM Reseptörleri Fikriye Uras1, Burhanettin Küçük1, Özlem Bingöl Özakpınar1, Ahmet Muzaffer Demir2 1Marmara 2Trakya

University Faculty of Pharmacy, Department of Biochemistry, İstanbul, Turkey University Faculty of Medicine, Department of Hematology, Edirne, Turkey

Abstract: Objective: Growth arrest-specific 6 (Gas6) is a newly discovered vitamin K-dependent protein, which is a ligand for TAM receptors [Tyro3 (Sky), Axl, and Mer] from the tyrosine kinase family. Gas6 knockout mice were resistant to venous and arterial thrombosis. There are contradictory reports on the presence of Gas6 and its receptors in mouse platelets. The objective of this study was to investigate whether Gas6 and its receptors were present in mouse platelets or not.

Materials and Methods: Specific pathogen-free BALB/c male and female mice of 8-10 weeks old and 25-30 g in weight were anesthetized under light ether anesthesia and blood samples were taken from their hearts. RNAs were isolated from isolated platelets, and then mRNAs encoding Gas6 and TAM receptors were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA, but not those of Mer, because of the absence of any commercial ELISA kit for mouse specimens. Results: RT-PCR results indicated the presence of mRNAs encoding Gas6 and Mer in mouse platelets. However, although RTPCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro3 (Sky). Receptor protein levels of Axl and Tyro3 were below the detection limits of the ELISA method. Conclusion: We found the presence of mRNAs encoding Gas6 and the receptor Mer in mouse platelets, but not Axl and Tyro3. Gas6, Axl, and Tyro3 protein levels were below the detection limits of the ELISA. The presence of mRNA is not obvious evidence of protein expression in platelets that have no nucleus or DNA. Further studies are required to clarify the presence of Gas6/TAM receptors in platelets using real-time PCR and more sensitive immunological methods, and future studies on mechanisms will indicate whether the Gas6/TAM pathway is a strategy for treatment of disorders.

Key Words: Blood platelets, Growth arrest-specific protein 6, Hemostasis Özet: Amaç: Gas6 proteini (growth arrest specific 6), K vitaminine bağımlı protein ailesinin yeni bir üyesi olup reseptör tirozin kinazlardan TAM reseptörlerinin [Tyro3 (Sky), Axl ve Mer] bir ligandıdır. Gas6 geni susturulmuş farelerin, venöz tromboembolizme daha dirençli oldukları görülmüştür. Fare trombositlerinde Gas6 proteininin var olup olmadığı konusunda birbiriyle çelişkili yayınlar bulunmaktadır. Biz bu araştırmada, fare trombositlerinde Gas6 proteini ve reseptörlerinin varlığını hem mRNA hem de protein seviyesinde araştırmayı amaçladık. Address for Correspondence: Fikriye Uras, M.D., Marmara University Faculty of Pharmacy, Department of Biochemistry, İstanbul, Turkey E-mail: furas@marmara.edu.tr Received/Geliş tarihi : March 17, 2013 Accepted/Kabul tarihi : June 24, 2013

Turk J Hematol 2015;32:58-63

Uras F, et al: Gas6 and TAM Receptors

Gereç ve Yöntemler: Spesifik patojen içermeyen 8-10 haftalık, 25-30 gr ağırlığındaki Balb-C ırkı dişi/erkek fareler hafif eter anestezisi altında bayıltıp, kalpten kan örnekleri alındı. Önce trombositlerden mRNA’lar elde edildi. Daha sonra ters transkripsiyon-polimeraz zincir reaksiyonu (RT-PCR) yöntemiyle Gas6 ve TAM reseptörlerinin herbirini kodlayan mRNA’lar araştırıldı. Trombositlerdeki Gas6 ve TAM reseptör proteinleri ELISA yöntemiyle ölçüldü. Ancak farede Mer protein seviyesini ölçen bir ELISA kiti henüz üretilmediği için Mer seviyesi ölçülemedi. Bulgular: RT-PCR sonuçları fare trombositlerinde Gas6 ve Mer’i kodlayan mRNA’ların var olduğunu gösterdi. Çeşitli sıcaklık ve döngülerde RT-PCR deneyleri yapıldığı halde, Axl ve Tyro3’e (Sky) ait mRNA varlığı saptanamadı. Gas6, Axl ve Tyro3 (Sky) protein seviyeleri ELISA yöntemlerinin en düşük ölçüm sınırlarının altındaydı. Sonuç: Biz fare trombositlerinde, Gas6 ve onun reseptörlerinden sadece Mer’i kodlayan mRNA’ların var olduğunu bulduk ama Axl ve Tyro3’ü kodlayan mRNA’ların varlığını saptayamadık. Protein olarak Gas6, Axl ve Tyro3 seviyeri, ELISA yöntemlerinin ölçüm limitlerinin altındaydı. Trombositlerde mRNA’ların var olması, protein sentezinin burada yapıldığının kesin bir kanıtı değildir. Çünkü trombositlerde DNA yoktur. Trombositlerde Gas6/TAM reseptörlerinin varlığını daha iyi aydınlatmak için Gerçek Zamanlı-PCR ve yüksek duyarlılığa sahip immunolojik yöntemlerle yapılacak ileri araştırmalara ihtiyaç vardır. Bu moleküllerin fizyolojik mekanizması aydınlatıldığında hastalıkların tedavisinde yeni bir strateji olabilir.

Anahtar Sözcükler: Trombositler, Growth arrest-specific protein 6, Hemostaz Introduction The number of newly discovered vitamin K-dependent proteins has been increasing. These include osteocalcin, matrix Gla protein, Gla-rich protein, periostin, and growth arrestspecific gene 6 (Gas6). The Gas6 protein was first described as a vitamin K-dependent protein by Manfioletti et al. in 1993 [1]. Human Gas6 was mapped to chromosome 13q34 [2]. Gas6, a 75-kDa protein, has 44% sequence homology to natural anticoagulant protein S, but has no anticoagulant activity [1,3]. Both Gas6 and protein S have affinity to TAM receptors [Tyro3 (Sky), Axl, and Mer] from the receptor tyrosine kinase family [4]. Unlike the other vitamin K-dependent proteins, the primary site of synthesis for Gas6 is not the liver. However, it is expressed in endothelial cells [1], vascular smooth muscle cells [5], bone marrow cells [6], and normal and malignant hematopoietic cells [7]. Gas6 has structural domains: a carboxy-terminal domain that is similar to the sex hormone-binding globulin, 4 epidermal growth factor-like domains, and a γ-carboxylated amino-terminal domain (Gla domain) consisting of 11 glutamic acid residues [8,9]. The Gla domain, which forms complexes with calcium ions, is unique to vitamin K-dependent proteins. In the endoplasmic reticulum, some glutamate residues are posttranslationally modified by a vitamin K-dependent reaction [10]. Without the Gla domain the clotting factors cannot bind to phospholipid receptors on the surface of platelets. This binding is essential for the activation of the coagulation pathway [11,12,13]. In the absence of carboxylation, the coagulation process becomes defective [14,15]. In a conformationally specific manner, Gla residues can coordinate themselves to bind to cell membranes [16]. This interaction was shown to mediate both Gas6 and protein S binding to apoptotic cells [10].

It has been shown that Gas6 has a role in some physiological processes including cell growth arrest, bone resorption, phagocytosis of apoptotic cells, cell survival, cell proliferation, cell migration, and cell adhesion [1,17,18,19,20,21,22]. Gas6 binds to TAM receptors with markedly different affinities [23,24]. Axl was first isolated from chronic myelogenous leukemia patients in 1991 [25]. It was detected in some organs and cell lines including hematopoietic, mesenchymal, and epithelial cells [26]. In 1994, Tyro3 (Sky) was reported as a novel receptor [27]. The genomic structure is very similar to human Tyro3 and it is expressed in embryonic cells [28]. Mer was first identified in 1994 and its mRNA is present in bone marrow and monocytes [29,30]. Gas6 knockout mice experiments showed that these mice were resistant to venous and arterial thrombosis [31]. It was also shown that TAM receptor knockout mice were resistant to thrombosis and degradation of platelet aggregation [32]. It was suggested that Gas6 increases the tendency to thrombosis by leading to platelet plaque stabilization. The role of Gas6 and its receptors on platelets is uncertain. There is disagreement as to whether synthesis of Gas6 in mouse platelets plays a role in these alterations. Some research groups have reported contradictory results for the presence of Gas6 and its receptors in mouse platelets. The presence of Gas6 and its 3 receptors was shown in both human and mouse platelets [31]. Chen et al. found the presence of Gas6 in mice platelets and only Mer from the TAM receptors [33]. Gould et al. showed the presence of each of the 3 receptors of TAM in mouse platelets [34]. It is obvious that there are contradictory findings related to the presence of Gas6 and its receptors in platelets. The objective of the present study was to clarify disagreement on the existence of Gas6 and TAM receptors in platelets.

Turk J Hematol 2015;32:58-63

Materials and Methods Mice Specific pathogen-free BALB/c male and female mice of 8-10 weeks old and 25-30 g in weight were obtained from the Experimental Research and Animal Laboratory of Marmara University (İstanbul, Turkey). All of the experimental procedures were conducted according to the guidelines of the Animal Care and Use Committee of Marmara University. Isolation of Mice Platelets Mice were anesthetized under light ether anesthesia and blood samples were collected from their hearts into tubes containing a 1:9 ratio of sodium citrate (3.2%). Platelet-rich plasma was isolated after centrifugation of whole blood at 200 x g for 10 min. The supernatant was transferred into an Eppendorf tube for centrifugation at 1000 x g for 10 min. Platelet pellets were suspended in a HEPES-Tyrode buffer (pH 7.4) containing 1 µg/mL prostacyclin and then centrifuged at 1000 x g for 10 min. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from platelets using a NucleoSpin RNA XS kit in line with the manufacturer’s instructions (Macherey-Nagel, Düren, Germany). The primers, obtained from Invitrogen, were as follows: those that generated a 400bp Gas6 PCR product, 5′-CGG CAT TCC CTT CAA GGA GAG T-3′ (Gas6 forward; 1459-1480) and 5′-CTC AAC TGC CAG GAC CAC CAA CT-3′ (Gas6 backward; 1836-1868); those that generated a 238-bp Mer PCR product [35], 5′GCA GGG ACT TAC AAA GAG CTT TCT-3′ (Mer forward; 1309-1332) and 5′-AGC CGA GGA TGA TGA ACA TAG AGT-3′ (Mer backward; 1542-1566); those that generated a 400-bp Axl PCR product [36], 5′-AGG CTC ATT GGC GTC TGT T-3′ (Axl forward; 2004-2033) and 5′-ATC GCT CTT GCT GGT GTA G-3′ (Axl backward; 2385-2403); and those that generated a 445-bp Tyro3 PCR product [37], 5′GGA AGA GAC GCA AGG AGA C-3′ (Tyro3 forward; 16001620) and 5′-ATG GGA ATG GGG AGA CGA C-3′ (Tyro3 backward; 2027-2045). For each sample, 3 µL of total RNA was used. RT-PCR was performed with the QIAGEN OneStep PCR kit (QIAGEN, Düsseldorf, Germany) according to the manufacturer’s instructions. The same application for all genes was performed with reverse transcription at 50 °C for 30 min, PCR activation at 95 °C for 15 min, and final extension at 72 °C for 10 min. The following parameters for amplification were selected: Gas6, 38 cycles, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min; Mer, 35 cycles, same as the procedure for Gas6 except for annealing at 55 °C for 30 s; Tyro3, 38 cycles, same as the procedure for Gas6 except for annealing at 52 °C for 30 s; Axl, 35 cycles, same as the procedure for Gas6 except for annealing at 55 °C

Uras F, et al: Gas6 and TAM Receptors

for 30 s. Using 2% agarose gel, PCR products were subjected to electrophoresis (100 V) and visualized with ethidium bromide (1%). These experiments were repeated 3 times with different sets of animals. Enzyme-Linked Immunosorbent Assay (ELISA) Concentrations of Gas6, Axl, and Tyro3 in mouse plasma and platelets were measured with DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s recommendations. Due to the lack of a commercial ELISA kit for Mer, we were unable to assay it. In brief, a 96-well microtiter plate was coated and incubated overnight at 4 °C with 100 µL of capture antibody. Phosphate-buffered saline Tween-20 (PBST) was used as the washing buffer, comprising phosphate-buffered saline (PBS) containing 0.1% Tween-20. All washing steps were carried out 5 times between steps. Wells were blocked with 1% bovine serum albumin (BSA) in PBST for 1.5 h at room temperature. Platelets were lysed by freezing/thawing [13] and 100 µL of calibrators or samples was added to the wells in 10-, 20-, or 40-fold dilutions. Wells were incubated for 1 h at 37 °C and the manufacturer’s instructions were then followed. The reaction was stopped after 20 min of incubation by addition of 100 µL of 2 N H2SO4. Optical density was measured at 450 nm on an ELISA reader. Statistical Analysis Descriptive statistics were reported as median (25th percentile-75th percentile) for mouse plasma Gas6 concentrations. Results Platelets isolated in blood and flow cytometric analysis demonstrated the high purity (>93%) of the platelet preparation when we used CD41 as a marker. After isolation of total RNA from the platelets, the RT-PCR studies were carried out with suitable primers belonging to Gas6 and TAM receptors. Gas6 (400 bp) and Mer receptor (238 bp) gene amplification products were detected in mouse platelets, indicating the presence of mRNAs of Gas6 and Mer (Figure 1). However, for Axl and Tyro3, no appreciable PCR products were detected, which indicated no significant mRNA of Axl or Tyro3 in platelets. When we used brain tissue as a positive control, the primers and RT-PCR worked well for Axl and Tyro3 (Figure 2). ELISA was used to measure protein levels of Gas6, Tyro3, and Axl in platelets. Mouse plasma was used for a positive control and PBS-BSA was used as a negative control. The analytical sensitivity of the assays was 0.7 ng/mL. Gas6 concentration was measured as 26 ng/mL (22.7-29.1) [median (25th percentile-75th percentile)] in the mouse plasma (n=6) and <0.7 ng/mL in platelets, which was lower than the

Turk J Hematol 2015;32:58-63

Uras F, et al: Gas6 and TAM Receptors

Discussion Gas6 knockout mice studies showed that these mice were resistant to venous and arterial thrombosis and had normal hemostasis parameter values [31]. In parallel with these results, it was shown that TAM receptor knockout mice are resistant to thrombosis and degradation of platelet aggregation. It was suggested that Gas6 increases a tendency to thrombosis by leading to platelet plaque stabilization [32]. The role of Gas6 and its receptors on platelet functions is not clear.

Figure 1. Expression of Gas6 (400 bp) and Mer (238 bp) gene amplifications in mouse platelet by reverse transcriptionpolymerase chain reaction (RT-PCR).

The changes in platelet aggregation or activation have important roles in the pathophysiology of various diseases. There is disagreement as to whether synthesis of Gas6 in mouse platelets plays a role in these alterations. Some research groups have reported contradictory results for the presence of Gas6 and its receptors in mouse platelets. Angelillo-Scherer et al. showed the presence of Gas6 and its 3 receptors in both human and mouse platelets by RT-PCR [32]. In the current study, we found the presence of mRNAs encoding Gas6 and receptor Mer in mouse platelets, but not Axl or Tyro3. Protein Gas6, Axl, and Tyro3 levels were below the detection limits of the ELISA methods. Chen et al. reported only the presence of receptor Mer in both human and mouse platelets using the western blot and RT-PCR methods [33]. They did not detect the presence of mRNAs for Axl and Tyro3. In Mer knockout mice, they observed impaired platelet functions and a decrease in the responsiveness to low-concentration agonists. Based on these findings, they considered that receptor Mer plays a role in the regulations of platelet functions. Sather et al. observed impaired platelet aggregation in Mer knockout mice [38]. Ishimoto and Nakano reported the presence of Gas6 in rat platelets by ELISA [39]. Moreover, in another study, Balogh et al. used mass spectroscopy and ELISA for measuring Gas6 in human plasma and platelets [40]. They reported that Gas6 levels were 13-23 ng/mL in human plasma by ELISA. However, in the same study they did not detect Gas6 protein in human platelets.

Figure 2. Expression of Gas6 (400 bp) and TAM receptors in mouse brain by reverse transcription-polymerase chain reaction (RT-PCR).

As a result of RT-PCR reactions, in the current study the presence of mRNAs of Gas6 and Mer was observed in platelets. It is known that platelets do not have DNA, but only RNA. Hence, the presence of mRNA is not obvious evidence of protein synthesis. Although RT-PCR reactions were performed at various temperatures and cycles, no visual band was detected for either Tyro3 or Axl. This agrees with the findings of Chen et al. [33].

detection limit and very close to the negative control value of PBS-BSA. These results showed us that the Gas6 protein level in the platelets was below the detection limit of ELISA. In the mouse platelets, the protein levels of Axl and Tyro3 were also below the detection limit (0.7 ng/mL) of the methods.

One limitation of the current study is that we were unable to measure Mer levels as protein because of the absence of any commercial ELISA kit for mouse Mer. We found that the levels of Gas6 and receptors Axl and Tyro3 in platelets were below the measurable limits of ELISA. 61

Turk J Hematol 2015;32:58-63

Uras F, et al: Gas6 and TAM Receptors

Further studies are required to clarify the presence of Gas6 and TAM receptors in platelets using real-time PCR and more sensitive immunological methods, as well as studies of their effects on physiological mechanisms. The effects of Gas6 and TAM receptors in clot stabilization are a promising field of research. Further experiments in humans are required to form conclusions as to whether Gas6 may be a reliable pharmacological target in thrombosis, hemostasis, restenosis, and atherosclerosis. Conflict of Interest Statement 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. Manfioletti G, Brancolini C, Avanzi G, Schneider C. The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol 1993;13:4976-4985. 2. Saccone S, Marcandalli P, Gostissa M, Schneider C, Della Valle G. Assignment of the human GAS6 gene to chromosome 13q34 by fluorescence in situ hybridization. Genomics 1995;30:129-131. 3. Dahlback B, Villoutreix BO. Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition. Arterioscler Thromb Vasc Biol 2005;25:1311-1320. 4. Stitt TN, Conn G, Gore M, Lai C, Bruno J, Radziejewski C, Mattsson K, Fisher J, Gies DR, Jones PF, Masiakowski P, Ryan TE, Tobkes NJ, Chen DH, DiStefano PS, Long GL, Basilico C, Goldfarb MP, Lemke G, Glass DJ, Yancopoulos GD. The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases. Cell 1995;80:661-667. 5. Nakano T, Higashino K, Kikuchi N, Kishino J, Nomura K, Fujita H, Ohara O, Arita H. Vascular smooth muscle cellderived, Gla-containing growth-potentiating factor for Ca2+mobilizing growth factors. J Biol Chem 1995;270:5702-5705. 6. Avanzi GC, Gallicchio M, Cavalloni G, Gammaitoni L, Leone F, Rosina A, Boldorini R, Monga G, Pegoraro L, Varnum B, Aglietta M. GAS6, the ligand of Axl and Rse receptors, is expressed in hematopoietic tissue but lacks mitogenic activity. Exp Hematol 1997;25:1219-1226. 7. Neubauer A, Fiebeler A, Graham DK, O’Bryan JP, Schmidt CA, Barckow P, Serke S, Siegert W, Snodgras, HR, Huhn D, Liu ET. Expression of Axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis. Blood 1994;84:19311941.

8. Villoutreix BO, Garcia de Frutos P, Lovenklev M, Linse S, Fernlund P, Dahlback B. SHBG region of the anticoagulant cofactor protein S: secondary structure prediction, circular dichroism spectroscopy, and analysis of naturally occurring mutations. Proteins 1997;29:478-491. 9. Tjwa M, Bellido-Martin L, Lin Y, Lutgens E, Plaisance S, Bono F, Delesque-Touchard N, Hervé C, Moura R, Billiau AD, Aparicio C, Levi M, Daemen M, Dewerchin M, Lupu F, Arnout J, Herbert JM, Waer M, García de Frutos P, Dahlbäck B, Carmeliet P, Hoylaerts MF, Moons L. Gas6 promotes inflammation by enhancing interactions between endothelial cells, platelets and leukocytes. Blood 2008;111:4096-4105. 10. Hafizi S, Dahlback B. Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily. FEBS J 2006;273:5231-5244. 11. Tetik S, Uras F, Eksioglu-Demiralp E, Turay Yardimci K. Lowdensity lipoprotein specifically binds glycoprotein IIb/IIIa. A flow cytometric method for ligand-receptor interaction. Clin Appl Thromb Hemost 2008;14:210-219. 12. Ozsavci D, Yardimci T, Demirel GY, Demiralp E, Uras F, Onder E. Flow cytometric assay of platelet glycoprotein receptor numbers in hypercholesterolemia. Platelets 2002;13:223229. 13. Ozsavci D, Yardimci T, Demirel GY, Uras F, Hekim N, Ulutin ON. Apo-A1 binding to platelets detected by flow cytometry. Thromb Res 2001;103:117-122. 14. Uras F, Uras AR, Yardimci T, Sardana MK. Determination of the N-terminal amino acid sequence of the purified prothrombin from a patient with liver cirrhosis. Thromb Res 2000;99:277283. 15. Koldas M, Uras F. Avidin-biotin ELISA for measurement of prothrombin in human plasma. Thromb Res 2001;102:221227. 16. Mann KG, Lawson JH. The role of the membrane in the expression of the vitamin K-dependent enzymes. Arch Pathol Lab Med 1992;116:1330-1336. 17. Nakamura YS, Hakeda Y, Takakura N, Kameda T, Hamaguchi I, Miyamoto T, Kakudo S, Nakano T, Kumegawa M, Suda T. Tyro 3 receptor tyrosine kinase and its ligand, Gas6, stimulate the function of osteoclasts. Stem Cells 1998;16:229-238. 18. Hall MO, Obin MS, Heeb MJ, Burgess BL, Abrams TA. Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epithelial cells. Exp Eye Res 2005;81:581-591. 19. Healy AM, Schwartz JJ, Zhu X, Herrick BH, Varnum B, Farber HW. Gas6 promotes Axl-mediated survival in pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 2001;280:L1273-1281.

Uras F, et al: Gas6 and TAM Receptors

20. Li R, Chen J, Hammonds G, Phillips H, Armanini M, Wood P, Bunge R, Godowski PJ, Sliwkowski MX, Mather JP. Identification of Gas6 as a growth factor for human Schwann cells. J Neurosci 1996;16:2012-2019. 21. Fridell YW, Villa J Jr, Attar EC, Liu ET. GAS6 induces Axlmediated chemotaxis of vascular smooth muscle cells. J Biol Chem 1998;273:7123-7126. 22. Tjwa M, Moons L, Lutgens E. Pleiotropic role of growth arrest-specific gene 6 in atherosclerosis. Curr Opin Lipidol 2009;20:386-392. 23. Nagata K, Ohashi K, Nakano T, Arita H, Zong C, Hanafusa H, Mizuno K. Identification of the product of growth arrestspecific gene 6 as a common ligand for Axl, Sky, and Mer receptor tyrosine kinases. J Biol Chem 1996;271:3002230027. 24. Sun WS, Fujimoto J, Tamaya T. Coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine endometrial cancers. Ann Oncol 2003;14:898-906. 25. Oâ&#x20AC;&#x2122;Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, Espinosa R, Le Beau MM, Earp HS, Liu ET. Axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 1991;11:5016-5031.

Turk J Hematol 2015;32:58-63

31. Angelillo-Scherrer A, de Frutos P, Aparicio C, Melis E, Savi P, Lupu F, Arnout J, Dewerchin M, Hoylaerts M, Herbert J, Collen D, Dahlback B, Carmeliet P. Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis. Nat Med 2001;7:215-221. 32. Angelillo-Scherrer A, Burnier L, Flores N, Savi P, DeMol M, Herbert JM, Lemke G, Goff SP, Matsushima GK, Earp HS, Vesin C, Hoylaerts MF, Plaisance S, Collen D, Conway EM, Wehrle-Haller B, Carmeliet P. Role of Gas6 receptors in platelet signaling during thrombus stabilization and implications for antithrombotic therapy. J Clin Invest 2005;115:237-246. 33. Chen C, Li Q, Darrow AL, Wang Y, Derian CK, Yang J, de Garavilla L, Andrade-Gordon P, Damiano BP. Mer receptor tyrosine kinase signaling participates in platelet function. Arterioscler Thromb Vasc Biol 2004;24:1118-1123. 34. Gould WR, Baxi SM, Schroeder R, Peng YW, Leadley RJ, Peterson JT, Perrin LA. Gas6 receptors Axl, Sky and Mer enhance platelet activation and regulate thrombotic responses. J Thromb Haemost 2005;3:733-741. 35. Chan MC, Mather JP, McCray G, Lee WM. Identification and regulation of receptor tyrosine kinases Rse and Mer and their ligand Gas6 in testicular somatic cells. J Androl 2000;21:291-302.

26. Faust M, Ebensperger C, Schulz AS, Schleithoff L, Hameister H, Bartram CR, Janssen JW. The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis. Oncogene 1992;7:1287-1293.

36. Xiong W, Chen Y, Wang H, Wang H, Wu H, Lu Q, Han D. Gas6 and the Tyro 3 receptor tyrosine kinase subfamily regulate the phagocytic function of Sertoli cells. Reproduction 2008;135:77-87.

27. Ohashi K, Mizuno K, Kuma K, Miyata T, Nakamura T. Cloning of the cDNA for a novel receptor tyrosine kinase, Sky, predominantly expressed in brain. Oncogene 1994;9:699705.

37. Katagiri M, Hakeda Y, Chikazu D, Ogasawara T, Takato T, Kumegawa M, Nakamura K, Kawaguchi H. Mechanism of stimulation of osteoclastic bone resorption through Gas6/ Tyro 3, a receptor tyrosine kinase signaling, in mouse osteoclasts. J Biol Chem 2001;276:7376-7382.

28. Crosier PS, Freeman SA, Orlic D, Bodine DM, Crosier KE. The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis. Exp Hematol 1996;24:318323. 29. Jia R, Hanafusa H. The proto-oncogene of v-eyk (v-ryk) is a novel receptor-type protein tyrosine kinase with extracellular Ig/GN-III domains. J Biol Chem 1994;269:1839-1844. 30. Graham DK, Dawson TL, Mullaney DL, Snodgrass HR, Earp HS. Cloning and mRNA expression analysis of a novel human protooncogene, c-mer. Cell Growth Differ 1994;5:647-657.

38. Sather S, Kenyon KD, Lefkowitz JB, Liang X, Varnum BC, Henson PM, Graham D. A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation. Blood 2007;109:1026-1033. 39. Ishimoto Y, Nakano T. Release of a product of growth arrestspecific gene 6 from rat platelets. FEBS Lett 2000;466:197199. 40. Balogh I, Hafizi S, Stenhoff J, Hansson K, Dahlback B. Analysis of Gas6 in human platelets and plasma. Arterioscler Thromb Vasc Biol 2005;25:1280-1286.

Case Report

DOI: 10.4274/tjh.2013.0189

Acute Megakaryoblastic Leukemia with t(1;22) Mimicking Neuroblastoma in an Infant Nöroblastomu Taklit Eden t(1;22) Pozitif Akut Megakaryoblastik Lösemili Bir Süt Çocuğu Müge Gökçe, Selin Aytaç, Şule Ünal, İlhan Altan, Fatma Gümrük, Mualla Çetin Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey

Abstract: Acute megakaryoblastic leukemia (AMKL) with t(1;22) (p13;q13) is an extremely rare subtype of acute myeloid leukemia that is almost always described in infants. t(1;22) (p13;q13)-positive AMKL with extramedullary infiltration has been previously reported only once in the literature. Herein, we report a 3-month-old infant presenting with a pelvic mass and pancytopenia suggesting neuroblastoma. Bone marrow evaluation revealed t(1;22)-positive AMKL that responded well to a regimen containing high-dose cytarabine. Key Words: Acute megakaryoblastic leukemia, t(1;22), Acute myeloid leukemia

Özet: t(1;22) (p13;q13) pozitif akut megakaryoblastik lösemi (AMKL) olgularının neredeyse hepsi sadece süt çocukluğu döneminde bildirilmiş olup, akut miyeloid löseminin son derece nadir bir alt tipidir. Bu olgu sunumunda, pelvik kitle ve pansitopeni ile başvuran ve tanı anında nöroblastomu taklit eden üç aylık bebek sunulmaktadır. Hastanın kemik iliği incelemesi ile t(1;22) pozitif AMKL tanısı konulmuş ve yüksek doz sitarabin içeren tedavi rejimine hasta iyi cevap vermiştir. Anahtar Sözcükler: Akut megakaryoblastik lösemi, t(1;22), Akut miyeloid lösemi Introduction Acute megakaryoblastic leukemia (AMKL) is a rare but heterogeneous subtype of acute myeloid leukemia (AML) with diverse morphological and cytogenetic features. Being more common in children than in adults, it constitutes 4%20% of pediatric AML cases with increasing rates during infancy [1,2,3]. AMKL predominates in children with Down syndrome and is associated with somatic GATA1 mutations [4]. Baruchel et al. first reported the nonrandom association between t(1;22) (p13;q13) and infant AMKL [5]. Just after that, the fusion gene OTT-MAL was identified in patients with t(1;22) [6]. Address for Correspondence: Şule Ünal, M.D., Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey Phone: +90 312 305 11 70 E-mail: suleunal@hacettepe.edu.tr Received/Geliş tarihi : May 30, 2013 Accepted/Kabul tarihi : July 10, 2013

Herein, we report a 3-month-old infant who presented with a pelvic mass and pancytopenia suggesting neuroblastoma, who was diagnosed with AMKL with t(1;22) based on a detailed work-up. Case Presentation A 3-month-old girl was referred to our hospital due to hepatosplenomegaly and pancytopenia, which were noticed during admission, with the complaints of irritability and intractable crying for 1 week. She was the first child of a healthy couple. The prenatal and natal histories were unremarkable.

Gรถkรงe M, et al: Acute Megakaryoblastic Leukemia with t(1;22)

She was irritable and pale. Her weight and height were 5 kg (in the 25th percentile according to age) and 59 cm (in the 50th percentile according to age), respectively. No ecchymoses or petechiae were noted. Cardiovascular and respiratory system evaluations were normal. The abdomen was distended and the liver and spleen were palpable 3 and 2 cm below the costal margins, respectively. A firm mass of a diameter of 2x3 cm was palpable in the right lower quadrant. Complete blood count revealed hemoglobin of 6.8 g dL-1, platelet count of 8x109 L-1, and leukocyte count of 12.9x109 L-1, with a differential count of 65% lymphocytes, 33% blasts, 1% metamyelocytes, and 1% neutrophils. Serum urea-creatinine and liver function tests were all normal. The lactate dehydrogenase level was 2294 IU L-1. Abdominal ultrasonography yielded multiple hypoechogenic nodules in the liver, packed lymph nodes in the portal area, and a solid mass behind the right psoas major muscle. Abdominal magnetic resonance imaging (MRI) confirmed the ultrasonographic findings (Figure 1). With the preliminary diagnosis of neuroblastoma or hepatoblastoma, serum alphafetoprotein (AFP) and urine vanillylmandelic acid (VMA) were analyzed. AFP was 64 IU L-1 (range: 0.5-5.5 IU L-1) and VMA was 36.4 mg g creatinine-1 (normal value: <27 for <12 months of age). Bone marrow aspirate showed overt hypocellularity and scarce myeloblasts with cytoplasmic blebbing without rosette formation. Bone marrow biopsy exhibited an increase in the reticulin fibers and fibrosis (Figure 2). Additionally, CD3,

Figure 1. Abdominal magnetic resonance imaging (MRI) with solid mass behind the right psoas major muscle.

Turk J Hematol 2015;32:64-67

CD20, TdT, CD1a, and AE1-AE3 were negative and CD68, CD34, and S100 were positive in a few cells, while neuronspecific enolase, chromogranin A, and PGP 9.5 were lightly positive in a few cells and synaptophysin was negative. Flow cytometric analysis demonstrated a blast gate of 51% with positivity of CD13, CD33, CD117, and CD42 but negativity of MPO. Conventional cytogenetic evaluation from bone marrow aspirate demonstrated 34-45, XX, t(1;22) (p13;q13) [14]/45,XX. Evaluating both the clinical presentation and the results of the bone marrow analysis, the diagnosis was AMKL (AML FAB M7). No blasts were detected in the cerebrospinal fluid. An AML BFM 2004 protocol regimen containing high-dose cytarabine was started [7]. Abdominal ultrasonography performed 6 weeks after the initiation of chemotherapy yielded no mass, neither in the liver nor in the neighborhood of the psoas major muscle. She had no matched sibling or unrelated donor. She recently completed therapy and is in complete remission at the second year after diagnosis. Discussion and Review of the Literature AMKL constitutes 4.1%-15.3% of pediatric AML cases in large collaborative studies [8,9,10], and prominent hepatosplenomegaly and bone marrow fibrosis are the characteristic features of this subtype [11]. AMKL in patients with Down syndrome and without Down syndrome are 2 major subgroups of AML. Down syndrome patients with somatic mutations in GATA1 have favorable prognosis, with 90% remission rates and 60%-70% event-free survival [12]. However, AMKL patients without Down syndrome have been reported to have different cytogenetics and poor prognosis. Presence of t(1;22) (p13;q13) cytogenetic anomaly is as low

Figure 2. Bone marrow biopsy with increase in reticulin fibers and fibrosis. 65

Turk J Hematol 2015;32:64-67

Gökçe M, et al: Acute Megakaryoblastic Leukemia with t(1;22)

as <1% among all AML patients and 70% of these patients present in the first year of life [13]. The overexpression of the fusion oncogene (OTT-MAL) in the presence of t(1;22) leads to NOTCH signaling deregulation with c-mpl activation [14]. On the other hand, it has been speculated that this protooncogene may also modulate chromatin organization and HOX differentiation pathways. Additional cytogenetic anomalies and/or hyperdiploid clones have been exhibited in 60% of patients [15]. Its prognosis has been thought of as poor, but recent data reported long-term survivors after intensive chemotherapy [16]. In previous reports, the frequency of extramedullary involvement and granulocytic tumors showed wide ranges of 12%-49% and 7%-18%, respectively [3,17,18]. In a previous report from our center, 40% of 127 children with AML were found to have extramedullary infiltration (EMI) at diagnosis and, in this series, FAB M2 and M4+M5 subtypes constituted 31% and 25% of all cases with EMI. None of the patients with EMI were found to have M7 morphology [3]. Presentation with granulocytic sarcoma in AMKL with t(1;22) (p13;q13) has been reported only in a 7-month-old infant until now [13]. Due to the paranasal location of the granulocytic sarcoma in that case, she was misdiagnosed with Burkitt lymphoma, but bone marrow aspiration revealed the diagnosis of AML M7. In the present case, the solid mass behind the right psoas major muscle mimicking neuroblastoma disappeared just after the first cycle of intensive acute nonlymphocytic leukemia-directed therapy and was defined as EMI or granulocytic sarcoma. To our knowledge, this is the second infant with t(1,22) (p13;q13)-positive AMKL presenting with extensive extramedullary involvement that disappeared immediately after chemotherapy. It should be kept in mind that AMKL with t(1,22) (p13;q13) might mimic solid tumors such as neuroblastoma and patients should be thoroughly investigated for bone marrow involvement. Patients who are unresponsive to other regimens and have atypical presentations of solid tumors must be evaluated for the presence of EMI of AML. Conflict of Interest Statement 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. Tallman MS, Neuberg D, Bennett JM, Francois CJ, Paietta E, Wiernik PH, Dewald G, Cassileth PA, Oken MM, Rowe JM. Acute megakaryocytic leukemia: the Eastern Cooperative Oncology Group experience. Blood 2000;96:2405-2411. 2. Martinez-Climent JA. Molecular cytogenetics of childhood hematological malignancies. Leukemia 1997;11:1999-2021.

3. Hicsonmez G, Cetin M, Tuncer AM, Yenicesu I, Aslan D, Ozyürek E, Unal S. Children with acute myeloblastic leukemia presenting with extramedullary infiltration: the effects of high-dose steroid treatment. Leuk Res 2004;28:25-34. 4. Wechsler J, Greene M, McDevitt MA, Anastasi J, Karp JE, Le Beau MM, Crispino JD. Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome. Nat Genet 2002;32:148-152. 5. Baruchel A, Daniel MT, Schaison G, Berger R. Nonrandom t(1;22) (p12-p13;q13) in acute megakaryocytic malignant proliferation. Cancer Genet Cytogenet 1991;54:239-243. 6. Mercher T, Coniat MB, Monni R, Mauchauffe M, Nguyen Khac F, Gressin L, Mugneret F, Leblanc T, Dastugue N, Berger R, Bernard OA. Involvement of a human gene related to the Drosophila spen gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia. Proc Natl Acad Sci U S A 2001;98:5776-5779. 7. Creutzig U, Zimmermann M, Bourquin JP, Dworzak MN, Fleischhack G, Graf N, Klingebiel T, Kremens B, Lehrnbecher T, von Neuhoff C, Ritter J, Sander A, Schrauder A, von Stackelberg A, Stary J, Reinhardt D. Randomized trial comparing liposomal daunorubicin with idarubicin in induction for pediatric acute myeloid leukemia: results from Study AML-BFM 2004. Blood 2013;122:37-43. 8. Zeller B, Gustafsson G, Forestier E, Abrahamsson J, Clausen N, Heldrup J, Hovi L, Jonmundsson G, Lie SO, Glomstein A, Hasle H. Nordic Society of Paediatric Haematology and Oncology (NOPHO). Acute leukemia in children with Down syndrome: a population-based Nordic study. Br J Haematol 2005;128:797-804. 9. Rao A, Hills RK, Stiller C, Gibson BE, de Graaf SS, Hann IM, O’Marcaigh A, Wheatley K, Webb DK. Treatment for myeloid leukemia of Down syndrome: population-based experience in the UK and results from the Medical Research Council AML 10 and AML 12 trials. Br J Haematol 2006;132:576-583. 10. Hama A, Yagasaki H, Takahashi Y, Nishio N, Muramatsu H, Yoshida N, Tanaka M, Hidaka H, Watanabe N, Yoshimi A, Matsumoto K, Kudo K, Kato K, Horibe K, Kojima S. Acute megakaryoblastic leukemia (AMKL) in children: a comparison of AMKL with and without Down syndrome. Br J Haematol 2008;140:552-561. 11. Lion T, Haas OA, Harbott J, Bannier E, Ritterbach J, Jankovic M, Fink FM, Stojimirovic A, Herrmann J, Riehm HJ. The translocation t(1;22) (p13;q13) is a nonrandom marker specifically associated with acute megakaryocytic leukemia in young children. Blood 1992;12:3325-3330. 12. Gamis AS. Acute myeloid leukemia and Down syndrome evolution of modern therapy--state of the art review. Pediatr Blood Cancer 2005;44:13-20.

Gökçe M, et al: Acute Megakaryoblastic Leukemia with t(1;22)

13. Torres L, Lisboa S, Vieira J, Cerveira N, Santos J, Pinheiro M, Correia C, Bizarro S, Almeida M, Teixeira MR. Acute megakaryoblastic leukemia with a four-way variant translocation originating the RBM15-MKL1 fusion gene. Pediatr Blood Cancer 2011;56:846-849. 14. Mercher T, Raffel GD, Moore SA, Cornejo MG, Baudry-Bluteau D, Cagnard N, Jesneck JL, Pikman Y, Cullen D, Williams IR, Akashi K, Shigematsu H, Bourquin JP, Giovannini M, Vainchenker W, Levine RL, Lee BH, Bernard OA, Gilliland DG. The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model. J Clin Invest 2009;119:852-864. 15. Huret JL. t(1;22)(p13;q13). Atlas of Genetics and Cytogenetics in Oncology and Haematology. 2001. Available at: http:// atlasgeneticsoncology.org/Anomalies/t0122.html, accessed 25 February 2013.

Turk J Hematol 2015;32:64-67

16. Reinhardt D, Diekamp S, Langebrake C, Ritter J, Stary J, Dworzak M, Schrauder A, Zimmermann M, Fleischhack G, Ludwig WD, Harbott J, Creutzig U. Acute megakaryoblastic leukemia in children and adolescents, excluding Down’s syndrome: improved outcome with intensified induction treatment. Leukemia 2005;19:1495-1496. 17. Jenkin RD, Al-Shabanah M, Al Nasser A, El-Solh H, Aur R, Al Sudairy R, Mustafa MM, Al Fawaz I, Gray A, da Cunha M, Ayas M, Al Mahr M, Kofide A, Mahgoub AN, Rifai S, Belgaumi A, Al Jefri A, Al Musa A, Sabbah R. Extramedullary myeloid tumors in children: the limited value of local treatment. J Pediatr Hematol Oncol 2000;22:34-40. 18. Bisschop MM, Revesz T, Bierings M, van Weerden JF, van Wering ER, Hählen K, van der Does-van den Berg A. Extramedullary infiltrates at diagnosis have no prognostic significance in children with acute myeloid leukemia. Leukemia 2001;15:46-49.

Case Report

DOI: 10.4274/tjh.2012.0196

Dasatinib-Related Chylothorax Dasatinib İlişkili Şilotoraks Yen-Min Huang1,2, Cheng-Hsu Wang1,3, Jen-Seng Huang1, Kun-Yun Yeh1, Chien-Hong Lai1, Tsung-Han Wu1, Pei-Hung Chang1, Pei-Hung Chang1, Yueh-Shih Chang1, Yii-Jenq Lan1 1Chang

Gung Memorial Hospital, Clinic of Internal Medicine, Division of Hematology-Oncology, Keelung, Taiwan Gung Memorial Hospital, Clinic of Internal Medicine, Division of Hematology, Linkou, Taiwan 3Chang Gung University Faculty of Medicine, School of Traditional Chinese Medicine, Taoyuan, Taiwan 2Chang

Abstract: Dasatinib is a potent second-generation tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia. The most common adverse event associated with dasatinib therapy is fluid retention, including pleural effusion. Dasatinib-related chylothorax has rarely been reported. The clinical manifestations, pathophysiology, management, and prognosis are not fully understood. Here we report a 40-year-old woman presenting with chylothorax following dasatinib use. We propose the hypothesis of its mechanism as well as offering a review of the relevant literature.

Key Words: Dasatinib, Chylothorax, Chronic myeloid leukemia Özet: Dasatinib kronik miyeloid lösemi tedavisinde kullanılan etkin bir ikinci kuşak tirozin kinaz inhibitörüdür. Dasatinib tedavisi ile ilişkili en sık görülen yan etki plevral effüzyonu da içeren sıvı retansiyonudur. Dasatinib ile ilişkili şilotoraks nadiren bildirilmiştir. Klinik belirtileri, patofizyolojisi, yönetimi ve prognozu tam olarak anlaşılamamıştır. Burada dasatinib kullanımı sonrasında şilotoraks gelişimi ile başvuran 40 yaşında bir kadın sunulmuştur. Dasatinib ile ilişkili şilotoraks gelişiminin mekanizmasının yanı sıra, konu ile ilgili literatür derlemesinden de bahsedilmektedir.

Anahtar Sözcükler: Dasatinib, Şilotoraks, Kronik miyeloid lösemi

Introduction Dasatinib (Sprycel®) is a highly potent small-molecule inhibitor of BCR-ABL and Src family tyrosine kinases, which is indicated for the treatment of adults with newly diagnosed chronic-phase chronic myeloid leukemia (CML), resistance or intolerance to prior CML therapy, or Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Pleural effusion was a common complication in 14%32% of all patients in clinical trials [1,2,3]. However, cases of

dasatinib-related chylothorax have rarely been reported. The pathophysiology and management of chylothorax remain unclear. It is essential to provide more information about dasatinib-related chylothorax since dasatinib is increasingly being used due to its effectiveness in the treatment of CML and Ph+ ALL. Case Presentation A 40-year-old female, a housewife, had been diagnosed with CML with b2a2 BCR-ABL fusion 8 years ago. The initial

Address for Correspondence: Yii-Jenq LAN, M.D., Chang Gung Memorial Hospital, Clinic of Internal Medicine, Division of Hematology-Oncology, Keelung, Taiwan Phone: +886-2-24329292 Ext. 2360 E-mail: liting@adm.cgmh.org.tw Received/Geliş tarihi : December 15, 2012 Accepted/Kabul tarihi : April 4, 2013

Huang YM, et al: Dasatinib-Related Chylothorax

Turk J Hematol 2015;32:68-72

presentation was peripheral leukocytosis with splenomegaly. She received 400 mg imatinib daily after the diagnosis and was titrated to 300 mg twice a day due to slow log reduction of BCR-ABL after 2 years. The log reduction achieved major molecular response half a year later. Four years after the diagnosis, the treatment was shifted to 50 mg of dasatinib twice a day because of intolerable knee pain. Her knee pain improved soon and the log reduction achieved complete molecular response (CMR) after 3 years of dasatinib treatment. Her liver and renal functions were monitored regularly and were within normal ranges.

by distinctly binding to active and inactive ABL-kinase [5]. It was first used as second-line treatment for imatinib-resistant or -intolerant patients with CML [6]. Later, dasatinib was considered as first-line therapy for patients with CML, and further clinical trials are presently continuing [1]. Common adverse effects of dasatinib include body fluid retention (all grades, up to 35%), skin rash (all grades, 10% to 20%), and diarrhea (all grades, 3% to 31%) [7]. Among body fluid retention, pleural effusion was seen in 14%-32% of all patients in clinical trials of dasatinib [1,2,3]. The pathophysiology of dasatinib-related chylothorax is still not fully understood.

Forty months after dasatinib use, the patient complained of progressive dyspnea and aggressive cough. Chest radiography revealed elevation of the right hemidiaphragm and small pleural effusion of the left (Figure 1A). Computed tomography (CT) confirmed prominent right pleural effusion and a small amount over the left side, as well as pericardial effusion. Echocardiography revealed adequate left ventricular function. Liver cirrhosis and splenomegaly were excluded by abdominal sonography. Ascites formation was excluded by the above CT and sonographic studies. Chest ultrasound-guided thoracentesis of bilateral pleural fluid yielded exudative effusion to be compared with her serum total protein concentration (6.4 g/dL) and lactic dehydrogenase (LDH) concentration (244 U/L) (Table 1) according to Light’s criteria [4]. Bilateral chylothorax was determined based on the milky yellow appearance and elevated triglyceride concentration (right: 263 mg/dL, left: 536 mg/dL) of the pleural effusion. Her serum triglyceride was 80 mg/dL, similar to her previous levels. Culture was negative for bacteria and tuberculosis. Cytology of the pleural effusion showed lymphocytes and macrophage/mesothelial cells. The patient denied history of trauma or surgery. The etiologies of chylothorax including trauma, surgery, infection, or malignancy were not likely. Under the suspicion of drug-related chylothorax, dasatinib was discontinued. Diuretics and steroids were prescribed for symptom control. Her pleural effusion improved after 9 days of treatment (Figure 1B). Dasatinib was resumed 2 weeks after discontinuation and the pleural effusion recurred soon under the treatment of diuretics and steroids (Figure 1C). Due to intractable chylothorax even after repeated thoracentesis, dasatinib was discontinued again and the patient was later switched to nilotinib. The pleural effusion resolved gradually (Figure 1D). After several months of nilotinib use, no further symptoms of pleural effusion were experienced. Informed consent was obtained.

Chylothorax is caused by chyle leakage from the thoracic duct into the pleural space, which results from disruption or obstruction of the thoracic duct. Chyle typically contains high levels of triglycerides and often has a turbid and milky appearance. Triglyceride concentrations of pleural effusion greater than 110 mg/dL (1.24 mmol/L) strongly support the diagnosis [8]. In this case, the patient’s triglyceride concentration levels were 536 mg/dL in the left pleural space and 263 mg/dL in the right. Those findings supported the diagnosis of bilateral chylothorax. The most common etiology of chylothorax is surgery or trauma [9], accounting

Discussion and Review of the Literature Dasatinib (Sprycel®) is a second-generation BCR-ABL tyrosine kinase inhibitor that targets most imatinib-resistant BCR-ABL mutations (except the T315I and F317V mutants)

Figure 1. Chest radiography: A) initial small pleural effusion; B) 9 days later after diuretics and steroid treatment; C) recurrent pleural effusion after dasatinib was resumed; D) 6 weeks after dasatinib interruption. 69

Turk J Hematol 2015;32:68-72

Huang YM, et al: Dasatinib-Related Chylothorax

Table 1. Pleural effusion studies.

Pleural Effusion Left

Right

Appearance

Turbid

Color

Yellow

Specific gravity

1.027

1.028

Protein

Positive

WBC

109/L

3.02

2.27

RBC

1012/L

12.0

7.0

Neutrophil

Lymphocyte

Monocyte

No bacteria seen

Gram stain Glucose

mg/dL

5.83

5.77

Total protein

g/dL

Lactic dehydrogenase

U/L

3.27

3.36

Triglyceride

mg/dL

6.06

2.97

for nearly half of all cases. Nontraumatic etiologies include malignancy, sarcoidosis, retrosternal goiter, amyloidosis, superior vena cava thrombosis, benign tumors, congenital duct abnormalities, and diseases of the lymph vessels such as yellow nail syndrome, lymphangioleiomyomatosis, and hemangiomatosis [10]. Hematologic malignancies including lymphoma, chronic lymphocytic leukemia, and Waldenström macroglobulinemia were reported to be associated with chylothorax [11,12,13,14,15]. Among the above hematologic malignancies, non-Hodgkin’s lymphoma is the most common. Only one case report mentioned CML and chylothorax, which was Adams-Oliver syndrome-related [16]. There is no current evidence of the association between the natural course of CML and chylothorax. In our case, the patient’s pleural effusion was regarded as concordant exudate according to an analysis of chylothorax [17]. The causes of this kind of chylothorax include lymphoma, tuberculosis, empyema, and idiopathic/ biliopleural fistula. Infection was not likely as the culture results for tuberculosis and bacteria were negative. There was no evidence of lymphoma. It is most likely that the cause of the chylothorax was dasatinib therapy.

Dasatinib-related pleural effusions are generally lymphocyte-predominant exudates [18]. These findings suggest that the etiology of pleural effusions is different from that of body fluid retention. The exact mechanism of pleural effusions remains unclear. The possible mechanisms potentially include inhibiting platelet-derived growth factor receptor beta (PDGFR-β) expressed in pericytes, which is involved in the regulation of angiogenesis [19]. The defect of PDGFR-β is associated with the formation of abnormal initial lymphatics in human lymphedema distichiasis, whereas another report suggested that platelet-derived growth factor (PDGF)-BB and its receptor, PDGFR-β, are directly lymphangiogenic [20,21]. This mechanism was reported in Gorham’s disease-related chylothorax [22]. Src kinase inhibition by dasatinib is also possibly related to changes of vascular endothelial growth factor-mediated vascular permeability and stability of the pleural epithelium [23,24]. The PDGF-signaling pathway stimulates tumor cell proliferation, angiogenesis, and pericyte recruitment to tumor blood vessels. Furthermore, ligated integrins recruit several nonreceptor tyrosine kinases, including focal adhesion kinase, integrin-linked kinase, and Src-family kinases, among others [25]. In congenital chylothorax of human fetuses, human integrin defect genes were found [26]. If the presentation of Src kinases changes, it may lead to defects of integrin and further chylothorax. The management of dasatinib-related pleural effusion including diuretics and a short course of prednisone (40 mg daily for 4 days) was suggested [18]. Once-daily dosing would reduce incidence of pleural effusions versus twice-daily dosing. There is no established standard treatment for chylothorax. In an earlier report, 138 patients were treated with dasatinib as second-line treatment after imatinib failure [27]. Fortyeight patients had pleural effusion and 1 of them developed recurrent chylous effusions, which required 12 treatments of thoracentesis. In another report, 40 patients were treated with dasatinib and 6 of them had pleural effusion [28]. One patient had right-sided chylothorax after about 1 year of dasatinib treatment and improved after drug interruption. In our case, the patient was treated with thoracentesis, prednisolone, and diuretics first, but the pleural effusions recurred soon after dasatinib was resumed. The second disappearance of chylothorax after discontinuing dasatinib again suggests the association between dasatinib and chylothorax. We therefore changed her treatment to another tyrosine kinase inhibitor, nilotinib. Now she maintains her CMR status. In conclusion, in patients under dasatinib treatment who develop chylothorax, dasatinib-related chylothorax should be considered. Dasatinib should be discontinued if the chylothorax is intractable. Further investigation to define the

Huang YM, et al: Dasatinib-Related Chylothorax

pathogenesis of dasatinib-related chylothorax is warranted since dasatinib is increasingly being used. Conflict of Interest Statement 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. Acknowledgment The authors thank all the members of the Cancer Center, Chang Gung Memorial Hospital, Keelung, for their invaluable help. References 1. Kantarjian HM, Shah NP, Cortes JE, Baccarani M, Agarwal MB, Undurraga MS, Wang J, Ipiña JJ, Kim DW, Ogura M, Pavlovsky C, Junghanss C, Milone JH, Nicolini FE, Robak T, Van Droogenbroeck J, Vellenga E, Bradley-Garelik MB, Zhu C, Hochhaus A. Dasatinib or imatinib in newly diagnosed chronic-phase chronic myeloid leukemia: 2-year followup from a randomized phase 3 trial (DASISION). Blood 2012;119:1123-1129. 2. Porkka K, Khoury HJ, Paquette RL, Matloub Y, Sinha R, Cortes JE. Dasatinib 100 mg once daily minimizes the occurrence of pleural effusion in patients with chronic myeloid leukemia in chronic phase and efficacy is unaffected in patients who develop pleural effusion. Cancer 2010;116:377-386. 3. Lilly MB, Ottmann OG, Shah NP, Larson RA, Reiffers JJ, Ehninger G, Müller MC, Charbonnier A, Bullorsky E, Dombret H, Brigid Bradley-Garelik M, Zhu C, Martinelli G. Dasatinib 140 mg once daily versus 70 mg twice daily in patients with Ph-positive acute lymphoblastic leukemia who failed imatinib: results from a phase 3 study. Am J Hematol 2010;85:164-170. 4. Light RW, Macgregor MI, Luchsinger PC, Ball WC Jr. Pleural effusions: the diagnostic separation of transudates and exudates. Ann Intern Med 1972;77:507-513. 5. Keam SJ. Dasatinib: in chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. BioDrugs 2008;22:59-69. 6. Abbott BL. Dasatinib: from treatment of imatinib-resistant or -intolerant patients with chronic myeloid leukemia to treatment of patients with newly diagnosed chronic phase chronic myeloid leukemia. Clin Ther 2012;34:272-281. 7. Bristol-Myers Squibb. Product Information: SPRYCEL(R) Oral Tablets, Dasatinib Oral Tablets. Princeton, NJ, USA, BristolMyers Squibb, 2010. 8. McGrath EE, Blades Z, Anderson PB. Chylothorax: aetiology, diagnosis and therapeutic options. Respir Med 2010;104:1-8. 9. Doerr CH, Allen MS, Nichols FC 3rd, Ryu JH. Etiology of chylothorax in 203 patients. Mayo Clin Proc 2005;80:867-870.

Turk J Hematol 2015;32:68-72

10. STI571 Prospective International Randomized Trial (SPIRIT 2). Available at http://www.spirit-cml.org/spirit-2-home.aspx; accessed 24 August 2012. 11. Doerr CH, Staats BA, Markovic SN. Chylothorax in chronic lymphocytic leukemia patient. Am J Hematol 2002;70:237240. 12. Scholz GA, Sirbu H, Semrau S, Anders K, Mackensen A, Spriewald BM. Persisting right-sided chylothorax in a patient with chronic lymphocytic leukemia: a case report. J Med Case Rep 2011;5:492. 13. Thomas LC, Maida MJ, Martinez-Outschoorn U, FilickoO’Hara J, Morris GJ. Chronic lymphocytic leukemia/small lymphocytic lymphoma with pancytopenia and chylothorax. Semin Oncol 2011;38:165-170. 14. Rizzo S, Campagnoli M. Chylothorax as a complication of Waldenström’s disease. Eur J Respir Dis 1984;65:371-372. 15. Antón Aranda E. Chylothorax complicating Waldenström macroglobulinemia. Arch Bronconeumol 2001;37:155-156 (article in Spanish). 16. Farrell SA, Warda LJ, LaFlair P, Szymonowicz W. Adams-Oliver syndrome: a case with juvenile chronic myelogenous leukemia and chylothorax. Am J Med Genet 1993;47:1175-1179. 17. Agrawal V, Doelken P, Sahn SA. Pleural fluid analysis in chylous pleural effusion. Chest 2008;133:1436-1441. 18. Brixey AG, Light RW. Pleural effusions due to dasatinib. Curr Opin Pulm Med 2010;16:351-356. 19. 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. 20. Petrova TV, Karpanen T, Norrmén C, Mellor R, Tamakoshi T, Finegold D, Ferrell R, Kerjaschki D, Mortimer P, Ylä-Herttuala S, Miura N, Alitalo K. Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat Med 2004;10:974-981. 21. Cao R, Björndahl MA, Religa P, Clasper S, Garvin S, Galter D, Meister B, Ikomi F, Tritsaris K, Dissing S, Ohhashi T, Jackson DG, Cao Y. PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell 2004;6:333345. 22. Mavrogenis AF, Zambirinis CP, Dimitriadis PA, Tsakanikas A, Papagelopoulos PJ. Gorham-Stout disease. J Surg Orthop Adv 2010;19:85-90. 23. Breccia M, Alimena G. Pleural/pericardic effusions during dasatinib treatment: incidence, management and risk factors associated to their development. Expert Opin Drug Saf 2010;9:713-721.

Turk J Hematol 2015;32:68-72

Huang YM, et al: Dasatinib-Related Chylothorax

24. Carragher NO, Westhoff MA, Fincham VJ, Schaller MD, Frame MC. A novel role for FAK as a protease-targeting adaptor protein: regulation by p42 ERK and Src. Curr Biol 2003;13:1442-1450.

27. Quintás-Cardama A, Kantarjian H, O’Brien S, Borthakur G, Bruzzi J, Munden R, Cortes J. Pleural effusion in patients with chronic myelogenous leukemia treated with dasatinib after imatinib failure. J Clin Oncol 2007;25:3908-3914.

25. Legate KR, Fassler R. Mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails. J Cell Sci 2009;122:187-198.

28. Bergeron A, Réa D, Levy V, Picard C, Meignin V, Tamburini J, Bruzzoni-Giovanelli H, Calvo F, Tazi A, Rousselot P. Lung abnormalities after dasatinib treatment for chronic myeloid leukemia: a case series. Am J Respir Crit Care Med 2007;176:814-818.

26. Ma GC, Liu CS, Chang SP, Yeh KT, Ke YY, Chen TH, Wang BB, Kuo SJ, Shih JC, Chen M. A recurrent ITGA9 missense mutation in human fetuses with severe chylothorax: possible correlation with poor response to fetal therapy. Prenat Diagn 2008;28:1057-1063.

Case Report

DOI: 10.4274/tjh.2014.0347

Successful Off-Label Use of Recombinant Factor VIIa and Coil Embolization in an Adolescent with Massive Hemoptysis Due to Invasive Pulmonary Aspergillosis İnvaziv Pulmoner Aspergillozise Bağlı Masif Hemoptizi Görülen Adölesan Bir Hastada, Endikasyon Dışı Rekombinan Faktör VIIa’nın ve Coil Emobolizasyonunun Başarılı Kullanımı Dilek Gürlek Gökçebay1, Ali Fettah1, İsmail Kırbaş2, Bahattin Tunç1, Namık Yaşar Özbek1 1Ankara 2Turgut

Children’s Hematology and Oncology Hospital, Clinic of Pediatric Hematology, Ankara, Turkey Özal University Faculty of Medicine, Department of Interventional Radiology, Ankara, Turkey

Abstract: Invasive fungal infections have turned out to be a significant cause of morbidity and mortality in pediatric patients with malignant disorders. Massive hemoptysis, a rare complication of invasive pulmonary aspergillosis, may threaten the lives of patients, usually during the resolution of neutropenia. In this report, we describe a patient with massive hemoptysis due to invasive pulmonary aspergillosis whose bleeding was controlled successfully with off-label use of recombinant factor VIIa and subsequent coil embolization of the right pulmonary artery. Key Words: Invasive pulmonary aspergillosis, Recombinant factor VIIa, Coil embolization, Children, Acute leukemia

Özet: İnvaziv fungal enfeksiyonlar pediatrik malin hastalıklarda morbidite ve mortalitenin önemli bir nedenidir. Masif hemoptizi de sıklıkla nötropenin düzelme aşamasında görülen, invaziv pulmoner aspergillozisin hayatı tehdit edici nadir bir komplikasyonudur. Bu yazıda invaziv pulmoner aspergillozise bağlı masif hemoptizi gelişen bir hastada endikasyon dışı recombinant FVIIa kullanımı ardından sağ pulmoner artere coil embolizasyon uygulanarak kanama kontrolu sağlanan bir olgu sunulmuştur. Anahtar Sözcükler: İnvasiv pulmoner aspergillozis, Recombinant factor VIIa, Embolizasyon, Çocuk, Akut lösemi Introduction The incidence of invasive fungal infections is increasing worldwide in patients with acute leukemia due to the use of more aggressive chemotherapy regimens that result in severe neutropenia [1,2]. Despite the availability of new antifungal agents, the global death rate remains quite high because of the

difficulty in establishing diagnoses [3]. Invasive pulmonary aspergillosis (IPA) is a severe mycosis characterized by acute invasion of the pulmonary vessels, leading to local parenchymal destruction, hemorrhage, thrombosis, and widespread hematogenous dissemination [4]. Massive hemoptysis is a life-threatening complication of IPA as we reported previously [5]. We herein report another adolescent

Address for Correspondence: Ali FETTAH, M.D., Ankara Children’s Hematology and Oncology Hospital, Clinic of Pediatric Hematology, Ankara, Turkey Phone: +90 505 675 05 86 E-mail: alifettah@gmail.com Received/Geliş tarihi : September 1, 2014 Accepted/Kabul tarihi : October 17, 2014

Turk J Hematol 2015;32:73-76

Gürlek Gökçebay D, et al: rFVIIa and Coil Embolization in Pulmonary Aspergillosis

patient with massive hemoptysis treated successfully with offlabel use of recombinant factor VIIa (rFVIIa) and subsequent coil embolization of the right pulmonary artery. To the best of our knowledge, this is the first report concerning transarterial coil embolization for control of massive life-threatening hemoptysis in children with IPA. Informed consent was obtained.

time, her hemoglobin level dropped from 9.5 g/dL to 6.5 g/dL. Hemostasis was achieved with rFVIIa given 3 times at doses of 90 µg/kg at 2-h intervals. In the meantime, she received consultation from the interventional radiology department; the right bronchial artery was selectively embolized with a coil immediately after the last factor VIIa infusion (Figure 2). There was no recurrence during the 3 months of follow-up.

Case Presentation

Discussion and Review of the Literature

A 16-year-old previously healthy girl was admitted to our hospital with ongoing menorrhagia for the past 2 weeks and headache and vomiting for the past 2 days. Her physical examination revealed normal findings except for signs of meningeal irritation. Complete blood count revealed hemoglobin of 8.1 g/dL, white blood cell count of 64.4x109/L, and platelet count of 70x109/L. Her peripheral blood and bone marrow aspiration smears showed FAB L2type lymphoblasts. Blastic infiltration was evident in her cerebrospinal fluid examination. Bone marrow cytogenetic analysis revealed hypodiploidy in 2 of the 17 metaphases. She was diagnosed with acute lymphoblastic leukemia (ALL) with central nervous system involvement, and ALL-IC BFM 2009 induction therapy was initiated.

Hemoptysis may be caused by chronic inflammatory lung diseases including bronchiectasis, tuberculosis, and aspergillosis [6]. Massive hemoptysis has been described as the expectoration of an amount of blood ranging from 100 to >1000 mL over 24 h; in 90% of cases of massive hemoptysis, the bleeding originates from the bronchial arterial circulation. It is a medical emergency that requires prompt evaluation and management. Conservative management of massive hemoptysis carries a mortality rate of as high as 50%-85% [7,8,9].

On follow-up, she had not achieved bone marrow remission at days 15, 33, or 52. She was diagnosed with resistant ALL and therefore received the IDA-FLAG regimen according to our institution’s decision (fludarabine at 30 mg/m2/day for 4 days; cytarabine at 2 g/m2/day for 4 days; idarubicin at 12 mg/m2/ day on days 2, 3, and 4; and granulocyte-colony stimulating factor at 5 µg/kg/day). After the IDA-FLAG regimen, she developed prolonged neutropenic fever. Meanwhile, due to infiltration of the lungs on chest radiography, she was treated with broad-spectrum antibiotics. Due to persistent fever, computed tomography (CT) of the thorax was performed, revealing 2 nodules in the right lung surrounded by a halo of ground-glass attenuation. At this point, serum galactomannan antigen was also positive, and so, considering the existence of IPA, voriconazole was added to the treatment. After resolution of the neutropenic period, her bone marrow was found to be in remission and repeated thorax CT showed persistence of IPA with 2 cavities in the right lung (Figure 1). Thorax CT angiography showed no evidence of vessel invasion at this stage. Therefore, she received the FLAG protocol under voriconazole treatment. One week after the FLAG treatment, at day 37 of the voriconazole treatment, she developed massive hemoptysis with coughing during a thrombocytopenic period (platelet count: 9x109/L). Otolaryngological examination revealed bleeding from the subepiglottic area. Despite receiving adequate platelet and cryoprecipitate support, about 300 mL of fresh bleeding was observed within 90 min. During that 74

Hemoptysis due to fungal invasion of the pulmonary

Figure 1. Thorax computed tomography with halo sign.

Figure 2. Coil embolization of the right bronchial artery.

Turk J Hematol 2015;32:73-76

Gürlek Gökçebay D, et al: rFVIIa and Coil Embolization in Pulmonary Aspergillosis

vessels is reported at rates of up to 58% during the neutropenic period in patients with IPA [10]. However, massive hemoptysis usually occurs during the phase of bone marrow recovery, as in one of our previously reported patients [5]. The pathogenesis of this phenomenon is not fully understood. Chemotherapyinduced severe neutropenia results in an immunodeficient state facilitating infection with filamentous mycosis. Following recovery of the bone marrow, the neutrophils are chemoattracted to the lung regions infected with fungi that enhance the local inflammatory response and release of proteolytic enzymes. These may play roles in the invasion of blood vessels by filamentous mycosis [4,10,11]. The initial approach to treating life-threatening massive hemoptysis is maintenance of the airway and oxygenation. After stabilizing the hemodynamic status, topical application of antifibrinolytic agents, adrenaline, or thrombin-fibrinogen solutions into the bleeding bronchi via bronchoscopy can be performed; however, the efficacy of those treatments is uncertain [12]. rFVIIa has been widely used as an off-label drug for patients with critical bleeding. It acts with tissue factor and leads to thrombin generation via the extrinsic clotting pathway. It also activates factor X on the surface of activated platelets, which is called “thrombin burst” [13]. The main concern with the administration of rFVIIa is the potential for inappropriate thrombosis. In an earlier study, thromboembolic adverse events were reported at 5.3% in patients who received rFVIIa for refractory hemorrhage. No difference was found between placebo-treated and rFVIIa-treated patients for thromboembolic events [14]. In a case report concerning the successful use of rFVIIa in the treatment of an adult patient with massive life-threatening hemoptysis due to chronic necrotizing Aspergillus infection, 2 administrations of rFVIIa at doses of 30 µg/kg were used and the hemoptysis was successfully resolved without recurrence [15]. Similarly, another report revealed the utility of rFVIIa in the critical refractory bleeding of 2 adult patients [16]. We could not find a pediatric case in the English literature similar to that of our present patient who responded to treatment with factor VIIa. Various embolic materials, such as polyvinyl alcohol particles, coils, and microspheres, have been used for selective bronchial and nonbronchial systemic arterial embolization in patients with hemoptysis [8]. The use of liquid agents, such as alcohol, is not recommended because of very fast delivery, possible tracheal or bronchial necrosis related to extremely distal occlusion of the bronchial arteries, and occlusion of collateral vessels [17,18]. On the other hand, using coils prevents repeat embolization if hemoptysis recurs. In addition, coils cause proximal occlusion, which promotes the development of new collateral circulation from

the surrounding vessels [19]. Dohen-Bécue et al. reported 4 patients with massive hemoptysis due to IPA [20]. Two of them received embolization, but the procedure was only successful in one of them and the other patient died. In our case, 3 doses of rFVIIa (90 µg/kg) were enough to control the massive bleeding. However, due to continuing leakage, we could not exclude a recurrence; therefore, selective right bronchial arterial coil embolization was performed. No complications or recurrent hemoptysis occurred. To the best of our knowledge, this is the first pediatric case in the English literature of massive hemoptysis due to IPA treated successfully with the aid of rFVIIa infusion and transarterial coil embolization. In conclusion, in emergency circumstances, we suggest off-label use of rFVIIa to control bleeding, followed by coil embolization to achieve durable hemostasis if necessary. Conflict of Interest Statement 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. Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Mullen CA, Raad II, Rolston KV, Young JA, Wingard JR. Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2011;52:56-93. 2. Azap A, Bozkurt GY, Yüksel MK, Kutlu H, Topçuoğlu P, Aypak A, Akan H. Secondary infections in cancer patients with febrile neutropenia. Turk J Hematol 2012;29:254-258. 3. de Pauw BE, Viscoli C. Managing invasive fungal infections: relying on clinical instincts or on a rational navigation system? J Antimicrob Chemother 2011;66:55-58. 4. Albelda SM, Talbot GH, Gerson SL, Miller WT, Cassileth PA. Pulmonary cavitation and massive hemoptysis in invasive pulmonary aspergillosis. Am Rev Respir Dis 1985;131:115120. 5. Avci Z, Alioglu B, Anuk D, Ozbek OY, Azap OK, Ozbek N. Double invasive fungal infection and typhlitis in children with acute lymphoblastic leukemia. Pediatr Hematol Oncol 2008;25:99-106. 6. Caillot D, Casasnovas O, Bernard A, Couaillier JF, Durand C, Cuisenier B, Solary E, Piard F, Petrella T, Bonnin A, Couillault G, Dumas M, Guy H. Improved management of invasive pulmonary aspergillosis in neutropenic patients using early thoracic computed tomographic scan and surgery. J Clin Oncol 1997;15:139-147.

Turk J Hematol 2015;32:73-76

7. Johnson JL. Manifestations of hemoptysis. Postgrad Med 2002;112:101-106. 8. Yoon W. Embolic agents used for bronchial artery embolization in massive haemoptysis. Expert Opin Pharmacother 2004;5:361-367. 9. Jean-Baptiste E. Clinical assessment and management of massive hemoptysis. Crit Care Med 2000;28:1642-1647. 10. Pagano L, Ricci P, Nosari A, Tonso A, Buelli M, Montillo M, Cudillo L, Cenacchi A, Savignana C, Melillo L. Fatal haemoptysis in pulmonary filamentous mycosis: an underevaluated cause of death in patients with acute leukemia in haematological complete remission. A retrospective study and review of the literature. Gimema Infection Program (Gruppo Italiano Malattie Ematologiche dell’Adulto). Br J Haematol 1995;89:500-505. 11. Denning DW. Invasive aspergillosis. Clin Infect Dis 1998;26:781-805. 12. Lordon JL, Gascoigne A, Corris PA. The pulmonary physician in critical care • Illustrative case 7: assessment and management of massive haemoptysis. Thorax 2003;58:814819. 13. Bain J, Lewis D, Bernard A, Hatton K, Reda H, Flynn J. Implementation of an off-label recombinant factor VIIa protocol for patients with critical bleeding at an academic medical center. J Thromb Thrombolysis 2014;38:447-452. 14. Levy JH, Fingerhut A, Brott T, Langbakke IH, Erhardtsen E, Porte RJ. Recombinant factor VIIa in patients with coagulopathy secondary to anticoagulant therapy, cirrhosis, or severe traumatic injury: review of safety profile. Transfusion 2006;46:919-933.

Gürlek Gökçebay D, et al: rFVIIa and Coil Embolization in Pulmonary Aspergillosis

15. Samarzija M, Srdic D, Redzepi G, Plestina S, Mazuranic I, Jakopovic M, Miculinic N. Recombinant factor VIIa in massive haemoptysis associated with chronic necrotising aspergillosis. Coll Antropol 2008;32:999-1002. 16. Mitrovic M, Elezovic I, Suvajdzic-Vukovic N, Antic D. Successful non-standard approaches to massive hemoptysis in invasive pulmonary aspergillosis. Srp Arh Celok Lek 2012;140:505-507. 17. Remy J, Voisin C, Ribet M. Treatment, by embolization, of severe or repeated hemoptysis associated with systemic hypervascularization. Nouv Presse Med 1973;2:2060-2068 (article in French). 18. Khalil A, Fartoukh M, Bazot M, Parrot A, Marsault C, Carette MF. Systemic arterial embolization in patients with hemoptysis: initial experience with ethylene vinyl alcohol copolymer in 15 cases. Am J Roentgenol 2010;194:104-110. 19. Chapman SA, Holmes MD, Taylor DJ. Unilateral diaphragmatic paralysis following bronchial artery embolization. Chest 2001;118:269-270. 20. Dohen-Bécue F, Salez F, Ramon P, Leblond-Tillie I, Wallaert B, Bauters A, Tonnel AB. Management of hemoptysis in invasive pulmonary aspergillosis. Rev Mal Respir 1998;15:791-796 (article in French with English abstract).

Case Report

DOI: 10.4274/tjh.2013.0107

Trisomy Chromosome 6 as a Sole Cytogenetic Abnormality in Acute Myeloid Leukemia Akut Miyeloid Lösemide Tek Bir Sitogenetik Anormallik Olarak Kromozom 6 Trizomisi Monika Gupta, Nita Radhakrishnan, Manoranjan Mahapatra, Renu Saxena All India Institute of Medical Sciences, Department of Hematology, New Delhi, India

Abstract: Identification of cytogenetic abnormalities plays an important role in the diagnosis and prognosis of leukemias. Isolated trisomy 6 is a rare abnormality, the prognostic significance of which is not well established. We report one case of acute myeloid leukemia (AML-M5 variant) with trisomy 6 as the sole cytogenetic abnormality. Previously, trisomy 6 has been reported in aplastic anemia, myelodysplastic syndrome, and AML, usually associated with hypocellular marrow. However, our patient had a very short history and hypercellular marrow infiltrated with blasts. We report this case due to the rarity of the condition. More studies are required to ascertain the role of trisomy 6 in the development of leukemia as well as in prognosis.

Key Words: Acute myeloid leukemia, Trisomy 6, Cytogenetics Özet: Sitogenetik anormalliklerin tanımlanması lösemilerin tanı ve prognozunda önemli bir rol oynar. İzole trizomi 6, prognostik önemi iyi belirlenmemiş olan nadir bir anormalliktir. Tek sitogenetik anormallik olarak trizomi 6’ya sahip bir akut miyeloid lösemi (AML-M5 varyantı) olgusunu sunuyoruz. Trizomi 6 daha önce aplastik anemi, miyelodisplastik sendrom ve genellikle hiposelüler ilik ile ilişkilendirilmiş AML’de bildirilmiştir. Ancak hastamızın çok kısa bir geçmişi ve blastlar ile infiltre hiperselüler bir iliği vardı. Bu durumun nadirliği nedeni ile bu olguyu rapor ediyoruz. Hem lösemi gelişiminde trizomi 6’nın rolünü tespit etmek, hem de prognoz için daha fazla çalışma gereklidir.

Anahtar Sözcükler: Akut miyeloid lösemi, Trizomi 6, Sitogenetik Introduction Identification of cytogenetic abnormalities plays an important role in the diagnosis and prognosis of leukemias. However, apart from recurrent cytogenetic abnormalities, the role of other rare abnormalities is not well known. Numerical aberrations as the only karyotypic anomalies, including single or multiple losses or gains, are found in approximately 15%

of all cytogenetically abnormal hematological malignancies [1]. Among them, isolated trisomy of chromosome 6 is a rare abnormality. It is difficult to determine the prognostic significance of trisomy 6 in acute myeloid leukemia (AML) because of the paucity of clinical publications [2]. Here we present a case of AML with karyotypic abnormality of trisomy 6.

Address for Correspondence: Monika Gupta, M.D., All India Institute of Medical Sciences, Department of Hematology, New Delhi, India Phone: +91- 9013770590 E-mail: monikashashwat@hotmail.com Received/Geliş tarihi : March 23, 2013 Accepted/Kabul tarihi : January 27, 2014

Turk J Hematol 2015;32:77-79

Gupta M, et al: Cytogenetic Abnormalities in Leukemias

Case Presentation A 21-year-old female reported to the Department of Hematology, All India Institute of Medical Sciences, New Delhi, India, with complaints of generalized weakness and fatigue of 2 weeks in duration. She was found to be pale and had received packed red cell transfusions during this period. On general examination she had anemia, fever, and sternal tenderness. There was no lymphadenopathy or bleeding manifestations. On systemic examination, she had hepatomegaly 6 cm below the costal margin, but the spleen was not palpable. Complete blood count revealed a hemoglobin level of 5.4 g%, total leucocyte count of 56.3x103/µL, and platelet count of 85x103/µL. Peripheral smear showed 25% blasts of monocytoid morphology. Bone marrow aspirates and touch preparation revealed hypercellular marrow filled with monoblasts and promonocytes. On cytochemistry, blasts were positive for myeloperoxidase, nonspecific esterase, and Sudan Black B and negative for periodic acid-Schiff and acid phosphatase. Bone marrow biopsy showed 100% cellularity with diffuse replacement by blasts. Immunophenotyping was performed by 6-color flow cytometry using a BD FACSCanto (Becton Dickinson, San Jose, CA, USA). Gated blast populations of cells were positive for CD13, CD33, CD34, HLA-DR, cMPO, CD64, and CD10. Bone marrow morphology, cytochemistry, and immunophenotyping were consistent with the diagnosis of AML-M5. Cytogenetic analysis was performed on a short-term unstimulated bone marrow culture with and without colcemid using standard cytogenetic techniques [3]. G-banded metaphases using trypsin and Giemsa staining were analyzed using an automated karyotyping system (MetaSystems GmbH, Altlußheim, Germany). The karyotype was reported according to the 2009 International System of Human Cytogenetic Nomenclature [4]. Analysis of 20 metaphases showed trisomy 6 in 12 metaphases while the remaining 8 metaphases were normal: 47, XX, +6 (12)/46,XX (8) (Figure 1).

Figure 1. GTG banding 47,XX, +6.

She was started on 7+3 induction with cytarabine at a dose of 100 mg/m2/day as a continuous infusion over 24 h for 7 days and daunorubicin at 60 mg/m2/day as a short infusion for 3 days. After the first induction, bone marrow on day 14 revealed persistence of blasts (34%). Hence, she was given a second induction with mitoxantrone and high-dose cytarabine (HAM). After treatment with HAM, the patient developed septicemia with cardiac dysfunction and bilateral fungal pneumonia, for which she was treated appropriately. After blood counts recovered, bone marrow testing was repeated and she was found to be in remission. She then received 2 more cycles of high-dose cytarabine as postremission therapy. She recovered uneventfully, and is presently being followed up with on a regular basis. Counselling regarding HLA-typing with her sibling and possible need for bone marrow transplantation was given, but this was deferred due to monetary constraints. Informed consent was obtained. Discussion and Review of the Literature Gains and losses of whole chromosomes are frequently found in hematological malignancies, either identified as solitary abnormalities at diagnosis or superimposed on other abnormalities in later stages of the disease [1]. Trisomy 6 as the sole karyotypic abnormality is a rare but recognized finding in hematological disorders. The mechanisms by which trisomies contribute to leukemogenesis are largely unknown, but 2 processes have been suggested. The first is a gene dosage effect as a direct result of the trisomy, with the extra copies of the gene leading to overexpression. The second is underlying cryptic gene rearrangement or mutation of genes on the additional chromosome [5]. A literature search reveals that only 14 cases of AML and 5 cases of myelodysplastic syndrome (MDS) have been reported with isolated trisomy 6. It has been seen in patients with aplastic anemia (AA), MDS, and AML, and is very rarely seen in childhood mixed-lineage leukemia, lymphoblastic transformation of chronic myeloid leukemia, and chronic myeloproliferative disorders. In most of the published cases, patients usually had hypocellular marrow with erythroid dysplasia or they proceeded from AA to AML [6,7]. It has also been reported as an associated finding along with other cytogenetic abnormalities. In contrast to this, our patient presented with a short duration of pallor and fever and was found to have hypercellular bone marrow with replacement of normal marrow elements by blasts. In our case, blasts were positive for CD34 and HLA-DR, similar to previous cases, which indicates the primitive nature of the blasts [8]. Response to treatment was also poor as our patient failed to respond to the first induction with persistence of blasts in the marrow at day 14. However, our patient went into remission with the second induction.

Gupta M, et al: Cytogenetic Abnormalities in Leukemias

Turk J Hematol 2015;32:77-79

Due to the limited number of cases reported in the literature, the role of trisomy 6 as the sole cytogenetic abnormality is not clear, but from the data available, it is likely that this clonal cytogenetic abnormality is associated with the primitive nature of blasts and poor response to treatment [1,9]. More studies are required to ascertain the role of trisomy 6 in the development of leukemia as well as in prognosis.

3. Korf BR. Cancer genetics. In: Dracopoli NC, Haines JL, Korf BR, Moir DT, Morton CC, Seidman CE, Seidman JG, Smith DR (eds). Current Protocols in Human Genetics. New York, John Wiley & Sons, 1994.

Conclusions

5. McCullough SJ, Neat MJ, Power M, Oâ&#x20AC;&#x2122;Marcaigh AO. Trisomy 6 in a child with acute megakaryoblastic leukemia (AML-M7). Cancer Genet Cytogenet 2004;154:190-192.

Isolated trisomy 6, although found in many hematological disorders, is rare as a sole abnormality in patients with AML. Although it has been reported more commonly in AA and MDS, it can also be observed in patients with de novo AML. The prognostic significance of this abnormality is not well established at present. Conflict of Interest Statement 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. Mohamed AN, Varterasian ML, Dobin SM, McConnell TS, Wolman SR, Rankin C, Willman CL, Head DR, Slovak ML. Trisomy 6 as a primary karyotypic aberration in hematologic disorders. Cancer Genet Cytogenet 1998;106:152-155.

4. Shaffer LG, Slovak M, Campbell L (eds). ISCN: An International System of Human Cytogenetic Nomenclature. Basel, S. Karger, 2009.

6. Ma ESK, Wan TSK. +6 or trisomy 6. Atlas Genet Cytogenet Oncol Haematol 2004;8:253-254. 7. Kelly MJ, Meloni-Ehrig AM, Manley PE, Altura RA. Poor outcome in a pediatric patient with acute myeloid leukemia associated with a variant t(8;21) and trisomy 6. Cancer Genet Cytogenet 2009;189:48-52. 8. Moormeier JA, Rubin CM, Le Beau MM, Vardiman JW, Larson RA, Winter JN. Trisomy 6: a recurring cytogenetic abnormality associated with marrow hypoplasia. Blood 1991;77:13971398. 9. Jonveaux P, Fenaux P, Berger R. Trisomy 6 as the sole chromosome abnormality in myeloid disorders. Cancer Genet Cytogenet 1994;74:150-152.

2. Choi J, Song J, Kim SJ, Choi JR, Kim SJ, Min YH, Park TS, Cho SY, Kim MJ. Prognostic significance of trisomy 6 in an adult acute myeloid leukemia with t(8;21). Cancer Genet Cytogenet 2010;202:141-143.

DOI: 10.4274/tjh.2014.0266

Letter to the Editor

Post-Partum Ovarian Vein Thrombosis: Combined Effect of Infection and Factor V Leiden Mutation Post-Partum Yumurtalık Ven Trombozu: Enfeksiyon ve Faktör V Leiden Mutasyonunun Birleşik Etkisi H. El Farran1, A. G. Haddad1, A. H. Radwan1, A. H. Nassar2, R. Hourani3, Ali T. Taher1 1American

University of Beirut Medical Center, Department of Internal Medicine, Beirut, Lebanon University of Beirut Medical Center, Department of Obstetrics and Gynecology, Beirut, Lebanon 3American University of Beirut Medical Center, Department of Diagnostic Radiology, Beirut, Lebanon 2American

To the Editor, Ovarian vein thrombosis (OVT) is a rare complication of pregnancy that mainly affects women in their 3rd or 4th decade. Numerous etiologies have been proposed, including ones of idiopathic origin. Early therapy with anticoagulants can be lifesaving; hence, a high index of suspicion is important in order to avoid serious complications such as pulmonary embolism (14%), sepsis, and death [1]. In this letter, an attempt at uncovering one of the etiologies to further solidify our understanding of the disease is made.

A 34-year-old female, gravida 3, para 3, presented on the fifth day after an uncomplicated normal vaginal delivery with right lower quadrant pain, fever, and chills. She had been diagnosed with Behçet’s disease 8 years ago and was maintained on colchicine and steroids for 2 years, which were stopped later. The patient denied previous episodes of deep vein thrombosis (DVT), as well as family history of hypercoagulable diseases. The course of her pregnancy was uneventful except for a positive rectovaginal culture for beta-hemolytic group B streptococci, for which she received

Figure 1. Coronal (A and C) and axial (B) contrast-enhanced multidetector computerized tomography images of the abdomen and pelvis demonstrate an enlarged hypodense right ovarian vein (white arrows, A and B) representing the thrombosed vein, extending from the right adnexa to the inferior vena cava at the level of the right renal hilum. There are severe perivascular inflammatory changes in the retroperitoneum (arrowhead, A). The right ovary is enlarged and heterogeneously enhanced (arrow, C). Address for Correspondence: Ali T. TAHER, M.D., American University of Beirut Medical Center, Department of Internal Medicine, Beirut, Lebanon Phone +961.1.350000 E-mail: ataher@aub.edu.lb Received/Geliş tarihi : June 28, 2014 Accepted/Kabul tarihi : September 10, 2014

Turk J Hematol 2015;32:80-81

Farran EH, et al: Post-Partum Ovarian Vein Thrombosis: Combined Effect of Infection and Factor V Leiden Mutation

prophylactic antibiotics before delivery. She was found to be heterozygous for factor V Leiden. Computed tomography (CT) of the abdomen with IV contrast revealed enlargement of the right adnexa with heterogeneous enhancement and surrounding fat streaking and fluid. It also showed a tubular structure arising from this adnexa extending into the inferior vena cava, where a small filling defect was noted and associated with surrounding fat streaking and fluid (Figure 1). The constellation of findings was suggestive of infection involving the right adnexa with associated thrombophlebitis of the right ovarian vein. The patient was admitted for antibiotics and was discharged on anticoagulation. Repeat CT scan showed gradual resolution of the thrombus. Informed consent was obtained. The topic of thrombosis in pregnancy has long been the subject of thorough investigation. In western Europe and the United States, maternal thromboembolism is the leading cause of pregnancy-related death [2]. Postpartum, the risk of venous thromboembolism is believed to be increased 20-fold. It is especially significant in the first week after birth [3]. Around one-third of pregnancy-related DVTs occur after delivery [4]. In addition, a link between DVT and infection has been suggested [5]. One of the proposed mechanisms is the alteration of endothelial function in blood vessels after an underlying insult [6,7]. In a recent case series, acute infection in community settings was linked to DVT. The incidence was not found to be related to any specific kind of infection and the conclusion was that acute infection could precipitate DVT [8]. In a systematic review conducted in 2006 about thrombophilia in pregnancy, patients who were heterozygous for factor V Leiden were found to have an odds ratio of 8.32 (CI: 5.44-12.7) [9]. Behçet’s disease should also be kept in mind as a contributing prothrombotic state. It is plausible that this patient’s group B streptococcal infection could have increased her risk of OVT as she had had 2 previous uneventful pregnancies, keeping in mind her increased risk of thrombosis. Further studies to solidify this notion are needed. Conflict of Interest Statement 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.

Key Words: Venous thrombosis, Pregnancy, Factor V Leiden, Streptococcal infection Anahtar Sözcükler: Venöz tromboz, Hamilelik, Faktör V Leiden, Streptokok enfeksiyonu References 1. Sharma P, Abdi S. Ovarian vein thrombosis. Clin Radiol 2012;67:893-898. 2. Chang J, Elam-Evans LD, Berg CJ, Herndon J, Flowers L, Seed KA, Syverson CJ. Pregnancy-related mortality surveillance-United States, 1991-1999. MMWR Surveill Summ 2003;52:1-8. 3. Heit JA, Kobbervig CE, James AH, Petterson TM, Bailey KR, Melton LJ 3rd. Trends in the incidence of venous thromboembolism during pregnancy or postpartum: a 30year population-based study. Ann Intern Med 2005;143:697706. 4. James AH, Tapson VF, Goldhaber SZ. Thrombosis during pregnancy and the postpartum period. Am J Obstet Gynecol 2005;193:216-219. 5. Bhagat K, Moss R, Collier J, Vallance P. Endothelial “stunning” following a brief exposure to endotoxin: a mechanism to link infection and infarction? Cardiovasc Res 1996;32:822-829. 6. Lozinguez O, Arnaud E, Belec L, Nicaud V, Alhenc-Gelas M, Fiessinger JN, Aiach M, Emmerich J. Demonstration of an association between Chlamydia pneumoniae infection and venous thromboembolic disease. Thromb Haemost 2000;83:887-891. 7. Koster T, Rosendaal FR, Lieuw-A-Len DD, Kroes AC, Emmerich JD, van Dissel JT. Chlamydia pneumoniae IgG seropositivity and risk of deep-vein thrombosis. Lancet 2000;355:1694-1695. 8. Smeeth L, Cook C, Thomas S, Hall AJ, Hubbard R, Vallance P. Risk of deep vein thrombosis and pulmonary embolism after acute infection in a community setting. Lancet 2006;367:10751079. 9. Robertson L, Wu O, Langhorne P, Twaddle S, Clark P, Lowe GD, Walker ID, Greaves M, Brenkel I, Regan L, Greer IA. Thrombosis: Risk and Economic Assessment of Thrombophilia Screening (TREATS) Study. Thrombophilia in pregnancy: a systematic review. Br J Haematol 2006;132:171-196.

DOI: 10.4274/tjh.2014.0374

Letter to the Editor

Recurrent Monosomies Confirmed by Interphase FISH in Three Chronic Myeloid Leukemia Cases Üç Kronik Miyeloid Lösemi Olgusunda İnterfaz FISH ile Doğrulanan Tekrarlayan Monozomiler Yelda Tarkan Argüden1, Dilhan Kuru1, Ayşe Çırakoğlu1, Şükriye Yılmaz1, Şeniz Öngören Aydın2, Cem Muhlis Ar2, Ayhan Deviren1, Teoman Soysal2, Seniha Hacıhanefioğlu1 1İstanbul 2İstanbul

University Cerrahpaşa Faculty of Medicine, Department of Medical Biology, İstanbul, Turkey University Cerrahpaşa Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

To the Editor, Although chronic myeloid leukemia (CML) is characterized by the Philadelphia (Ph) chromosome, which is the result of t(9;22) (q34;q11) or its variants, 10%-20% of cases have additional cytogenetic abnormalities. The most common additional abnormalities are loss of the Y chromosome, +8, +Ph, and i(17q). Since these additional chromosome abnormalities are signs of disease progression, it is important to perform cytogenetic analyses periodically in patients with CML [1]. We have published our results on clonal chromosome abnormalities other than the Ph chromosome in Ph+ and Ph- cells of CML patients who were followed in our center a few years ago [2,3]. Monosomies were the most frequently observed chromosome abnormalities in these reports. In some cases, there were recurrent monosomies in more than one sample. To evaluate the significance of these recurrent monosomies, we performed fluorescence in situ hybridization (FISH) analysis for 3 of the previously reported patients with recurrent monosomies. Informed consent was obtained before the study. For FISH testing, chromosomes 8, 10, 17, and 20 were selected, since they had been the most common monosomies in our earlier publications. All 3 patients in this study were under imatinib therapy except for patient 2, who was receiving interferon at the time of the first sampling.

Conventional cytogenetic techniques were performed to examine the marrow samples. GTL-banded metaphases were examined according to International System for Human Cytogenetic Nomenclature guidelines [4]. Twenty metaphases were studied whenever possible. Cytocell Aquarius alpha satellite probes were used according to the manufacturer’s instructions for FISH. Two hundred interphase cells were counted by 2 different researchers. Normal karyotyped blood or marrow samples were used as control cases. Results of the cytogenetic and FISH analyses are given in Table 1. In the first 3 samples of patient 1, monosomy of chromosomes 8, 10, and 20 was confirmed by FISH. In the fourth sample of this patient, we performed FISH for chromosomes 10 and 20 despite their absence in the karyotype to see whether there was a hidden monosomy that we could not show by karyotyping. FISH indeed revealed a hidden monosomy for chromosome 20, but not for chromosome 10. In the first 2 samples of patient 2, monosomy of chromosome 17 was observed in cytogenetic analysis and confirmed by FISH. However, in the third sample, -17 could be shown neither in the karyotype nor by FISH. Notably, percentages of -17 obtained by cytogenetics and FISH were compatible in each of the samples. In the first sample, -17 was detected cytogenetically in 3 out of 12 cells (25%), and it was found in 46% of the interphases by FISH. In the second sample, -17 was found in 12% and 31% of the cells in cytogenetic and FISH analysis, respectively. In the third, it was absent

Address for Correspondence: Yelda Tarkan ARGÜDEN, M.D., İstanbul University Cerrahpaşa Faculty of Medicine, Department of Medical Biology, İstanbul, Turkey Phone: +90 212 414 30 34 E-mail: yeldata@istanbul.edu.tr Received/Geliş tarihi : September 22, 2014 Accepted/Kabul tarihi : October 15, 2014

Turk J Hematol 2015;32:82-84

Argüden YT, et al: Recurrent Monosomies Confirmed by Interphase FISH in 3 Chronic Myeloid Leukemia Cases

Table 1. Cytogenetic and fluorescence in situ hybridization results.

Case no.

Sample no.

Chromosome numbers analyzed by FISH

Cytogenetic Results

Monosomy percentages by FISH Cases, %

Case 1

Case 2

Case 3

Controls, %

38~46,X,-Y [4], t(9;22) (q34;q11) [6], -10[4], -15 [4], -16 [3] [cp15]/46,XY [8]

37~47,XY, -Y [3], -3 [3], -7 [4], -8 [3], -9 [3], -10 [4], -11 [3], -14 [3], -15 [3], -18 [4], -20 [4], +mar1 [3], +mar2 [2] [cp16]/46,XY [13]

10 20

16.5 40

7 14

35~45,XY, -4 [3], -8 [4], -9 [3], -10 [5], -13 [6], -14 [3], -15 [5], -16 [4], -18 [3], -21 [3], -22 [3] [cp12]/46,XY [6]

8 10

12 14

6.4 7

46,XY, t(9;22) [3]/46,XY [13]

10* 20*

5.5 43.5

7 14

1**

39~46,XX, t(9;22) (q34;q11) [12], -17 [3], -21 [3] [cp12]

15.6

41~46,XX, -9 [3], t(9;22) (q34;q11) [25], -14 [5], -15 [4], -17 [3], -18 [4], -21 [3] [cp25]

15.6

35~46,XX, -3 [5], -7 [4], -8 [5], t(9;22) (q34;q11) [18], -10 [3], -12 [3], -13 [5], -15 [4], -19 [3], -20 [6], -21 [4], -22 [6] [cp18]

8 10 17* 20

9 9 11 15

6.4 7 15.6 14

44~38,XY, -6 [3], -8 [5], -20 [4] [cp8]/32,X, +1, +7, 8 +10, +19, +20 [cp2]/46,XY [9] 20

12 8.5

6.4 14

40~46,XY, -8 [3], -9 [4], -13 [3],-20 [3] [cp7]/46,XY [20]

13 9

6.4 14

8 20

*: Not in the karyotype. **: Under interferon therapy. FISH: Fluorescence in situ hybridization

in cytogenetic preparations as well as in FISH study. -17 may be an important candidate marker to be followed by FISH in the course of CML since it is one of the additional minor-route chromosome abnormalities in the clonal evolution of Ph+ CML [1]. Furthermore, it leads to the loss of the p53 gene localized on 17p, which is known to be involved in CML progression [1]. In patient 3, -8 was confirmed by FISH, but -20 could not be demonstrated by FISH despite its presence in the karyotype. FISH results were in line with those of cytogenetics in some samples while not in others. This, once again, highlights the importance of concurrent use of different techniques (i.e. FISH and conventional cytogenetics) in cancer samples to

increase the detection capability and improve the reliability of the results. Studies with larger numbers of patient samples and longer follow-up are required to establish the impact of certain monosomies on the disease course. Conflict of Interest Statement 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. Key Words: Cytogenetics, Marrow, Neoplasia, Chronic myeloid leukemia, Monosomy Anahtar Sözcükler: Sitogenetik, Kemik iliği, Neoplazi, Kronik miyeloid lösemi, Monozomi

Turk J Hematol 2015;32:82-84

Argüden YT, et al: Recurrent Monosomies Confirmed by Interphase FISH in 3 Chronic Myeloid Leukemia Cases

References 1. Fioretos T, Johansson B. Chronic myeloid leukemia. In: Heim S, Mitelman F (eds). Cancer Cytogenetics. 3rd ed. Hoboken, NJ, USA, Wiley-Blackwell, 2009. 2. Öngören Ş, Tarkan-Argüden Y, Ar MC, Yılmaz Ş, Üre Ü, Kuru D, Eşkazan AE, Güven GS, Çetin G, Çırakoğlu A, Başlar Z, Deviren A, Aydın Y, Hacıhanefioğlu S, Ferhanoğlu B, Tüzüner N, Ülkü B, Soysal T. Clonal chromosomal abnormalities in Philadelphia-negative cells and their clinical significance in patients with chronic myeloid leukemia: results of a single center. Türkiye Klinikleri J Med Sci 2009;29:321-330.

3. Tarkan-Arguden Y, Ar MC, Yılmaz S, Ongoren S, Kuru D, Ure U, Cırakoglu A, Eskazan AE, Guven GS, Cetin G, Purisa S, Baslar Z, Deviren A, Aydın Y, Hacıhanefioglu S, Ferhanoglu B, Tuzuner N, Ulku B, Soysal T. Cytogenetic clonal evolution in patients with chronic myeloid leukemia. Biotechnol Biotechnol Eq 2009;23:1515-1520. 4. International Standing Committee on Human Genetic Nomenclature. Neoplasia. In: Shaffer LF, Tommerup N (eds). ISCN: An International System for Human Cytogenetic Nomenclature. Basel, Switzerland, S. Karger, 2005.

Letter to the Editor

DOI: 10.4274/tjh.2014.0373

An Unusual Presentation of Metastatic Breast Carcinoma as Cold Autoimmune Hemolytic Anemia

Metastatik Meme Kanserinin Soğuk Tipte Otoimmun Hemolitik Anemi Olarak Sıradışı Başvurusu Anand Chellappan, Chanaveerappa Bammigatti, Swaminathan Palamalai Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Department of Internal Medicine, Puducherry, India

To the Editor, Cold autoimmune hemolytic anemia is a rare disease caused by cold-reactive antibodies. It has been reported in patients with lymphoproliferative disorders and autoimmune and infectious diseases, and rarely with solid tumors [1,2,3]. Herein we bring to your attention a patient with metastatic breast carcinoma who presented with cold autoimmune hemolytic anemia and showed a dramatic improvement following treatment with steroids. A 50-year-old woman presented with complaints of dyspnea, easy fatigability, and palpitations of 1 week in duration. Dyspnea had worsened rapidly and was present even at rest. She had been diagnosed with metastatic left breast carcinoma, stage T4b N1 M1, and had been treated with left mastectomy and chemotherapy. She was receiving second-line adjuvant chemotherapy with gemcitabine and capecitabine. She was also a diabetic on metformin and glibenclamide. There was no history of intake of any other drug. She had severe pallor and mild icterus at presentation. Her hemoglobin was 28 g/L and peripheral smear showed agglutinated RBCs, nucleated RBCs, and polychromatophils. Total leucocyte count was 9.2x109/L (neutrophils: 62%, lymphocytes: 30%, band forms: 3%, myelocytes: 4%, metamyelocytes: 1%) and platelet count was 165x109/L. Reticulocyte count was 7%. Direct antiglobulin test (DAT) was 3+ (graded on a scale from 0 indicating no agglutination to 4+ indicating solid agglutination). DAT was positive for C3d and negative for IgG. Liver function tests showed mild elevation of bilirubin (total bilirubin: 2.2 mg/ dL and direct bilirubin: 0.7 mg/dL on day 1; total bilirubin:

Figure 1. Bone marrow aspirate showing clusters of atypical epithelial cells consistent with metastatic adenocarcinoma deposits. 4.2 mg/dL and direct bilirubin: 0.8 mg/dL on day 2). Urine hemoglobin was positive. Serum LDH level was 360 IU/L (reference range: 60-200 IU/L). Antinuclear antibody, HIV, and hepatitis B and C serologies were negative. Serum B12, folate, and ferritin levels were normal. Serum haptoglobin and cold agglutinin titers could not be measured due to the lack of this facility in our institute. Bone marrow aspirate revealed clusters of atypical epithelial cells along with few osteoclasts, consistent with metastatic adenocarcinoma deposits (Figure 1). She was given one packed cell transfusion at presentation and was started on prednisolone at 1 mg/kg body weight. She had a dramatic clinical improvement and did not require further transfusions. Her hemoglobin had improved to 70 g/L at 2 weeks.

Address for Correspondence: Anand CHELLAPPAN, M.D., Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Department of Internal Medicine, Puducherry, India E-mail: anandthedoc@gmail.com Received/Geliş tarihi : September 22, 2014 Accepted/Kabul tarihi : November 17, 2014

Turk J Hematol 2015;32:85-86

Chellappan A, et al: An Unusual Presentation of Metastatic Breast Carcinoma as Cold Autoimmune Hemolytic Anemia

Cold agglutinin disease is a form of autoimmune hemolytic anemia characterized by the presence of circulating antibodies, usually of the IgM type. These cold antibodies have little, if any, activity at body temperature but display increasing affinity for RBCs as the temperature decreases towards 0 °C. They require complement activation and fixation for hemolysis to occur. Cold autoimmune hemolytic anemia can be primary when there is no underlying systemic disorder. Secondary cold autoimmune hemolytic anemia is usually reported in patients with leukemias and lymphomas. However, it has also been described in cancers of the lung, colon, cervix, and breast [4,5]. In most of these cases the cancer had already metastasized at presentation. The pathogenetic mechanism is poorly understood [6]. Some of the proposed hypotheses include release of tumor-associated antigens mimicking RBC antigens, production of autoantibodies by the tumor itself, and adsorption of immune complexes on the erythrocyte membrane [6]. It is important to distinguish autoimmune hemolytic anemia from other non-immune causes of anemia like bone marrow infiltration by the tumor, since the treatment and prognosis vary accordingly. Our patient had evidence of bone marrow metastasis at presentation. Autoimmune hemolysis was confirmed by a positive direct antiglobulin test. Drug-induced hemolytic anemia is another important cause of autoimmune hemolysis. The most common drugs implicated are antimicrobials (e.g., cefotetan, ceftriaxone, and piperacillin). The only way to support a diagnosis of druginduced hemolytic anemia is to see if a hematological remission occurs after withdrawal of the drug. Informed consent was obtained. Autoimmune hemolytic anemia requires treatment in most cases. There are few studies addressing the treatment of cold autoimmune hemolytic anemia. This can include pharmacotherapy with corticosteroids, immunosuppressive drugs, and rituximab, among other agents [7]. Varying results have been obtained with the use of these agents. Steroid treatment is found to be much less effective in paraneoplastic autoimmune hemolytic anemia than in idiopathic autoimmune hemolytic anemia. Steroids are able to control hemolysis in only 15% of cases of cold hemagglutinin disease [8]. Treatment of the underlying malignancy and avoidance of cold exposure form important aspects of treatment. The improvement in hemoglobin in our case could be attributed to the treatment of the underlying breast malignancy and the use of steroids.

In conclusion, autoimmune hemolytic anemia is a potentially treatable cause of anemia in a patient with breast malignancy and the clinician should be aware of this complication. Conflict of Interest Statement 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. Key Words: Cold-reactive antibody, Autoimmune hemolytic anemia, Breast carcinoma Anahtar Sözcükler: Soğuk otoimmün, Hemolitik anemi, Meme kanseri References 1. Al-Matham K, Alabed I, Zaidi SZA, Qushmaq KA. Cold agglutinin disease in fibrolamellar hepatocellular carcinoma: a rare association with a rare cancer variant. Ann Saudi Med 2011;31:197-200. 2. Okada T, Kubota K, Kita J, Kato M, Sawada T. Hepatocellular carcinoma with chronic B-type hepatitis complicated by autoimmune hemolytic anemia: a case report. World J Gastroenterol 2007;13:4401-4404. 3. Cao L, Kaiser P, Gustin D, Hoffman R, Feldman L. Cold agglutinin disease in a patient with uterine sarcoma. Am J Med Sci 2000;320:352-354. 4. Adorno G, Girelli G, Perrone MP, Arista MC, Coluzzi S, Masi M, Giudiceandrea P, Papa G. A metastatic breast carcinoma presenting as autoimmune hemolytic anemia. Tumori 1991;77:447-448. 5. Calderoni A, Altermatt HJ, Pirovino M. Autoimmune processes as paraneoplastic manifestations in familial breast carcinoma. Dtsch Med Wochenschr 1994;119:1194-1198 (in German with English abstract). 6. Spira MA, Lynch EC. Autoimmune hemolytic anemia and carcinoma: an unusual association. Am J Med 1979;67:753-758. 7. Lechner K, Jäger U. How I treat autoimmune haemolytic anemias in adults. Blood 2010;116:1831-1838. 8. Visco C, Barcellini W, Maura F, Neri A, Cortelezzi A, Rodeghiero F. Autoimmune cytopenias in chronic lymphocytic leukemia. Am J Hematol 2014;89:1055-1062.

Letter to the Editor

DOI: 10.4274/tjh.2014.0308

A Child with Psoriasis, Hypogammaglobulinemia, and Monosomy 7-Positive Myelodysplastic Syndrome Miyelodisplastik Sendrom, Psöriazis, Hipogamaglobulinemi ve Monozomi 7’si Olan Bir Çocuk Miyelodisplastik Sendrom Olgusu Namık Özbek1, Arzu Yazal Erdem1, Özlem Arman Bilir1, Fatma Karaca Kara2, Mutlu Yüksek3, Neşe Yaralı1, Meltem Özgüner4, Nazmiye Yüksek5, Bahattin Tunç1 1Ankara

Children’s Hematology and Oncology Hospital, Clinic of Pediatric Hematology, Ankara, Turkey Children’s Hematology and Oncology Hospital, Clinic of Biochemistry, Ankara, Turkey 3Bülent Ecevit University Faculty of Medicine, Department of Children’s Immunology, Ankara, Turkey 4Yıldırım Beyazıt University Faculty of Medicine, Department of Histology Embryology, Ankara, Turkey 5Bülent Ecevit University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey 2Ankara

To the Editor, A 3.5-year-old girl was admitted to our hospital with psoriasis, hypogammaglobulinemia, and pancytopenia present since 2 years of age. Before admission, due to the decreased number of B-lymphocytes and decreased immunoglobulin (Ig) levels (IgG: 196 mg/dL, IgM: 18.1, IgA: 26.8 mg/dL), she was diagnosed with autosomal recessive hypogammaglobulinemia and received intravenous Ig every month. She was referred to us after detection of 12% blasts and monosomy 7 in a bone marrow (BM) aspiration specimen. Her parents were cousins and 2 elder sisters had died of infection at 3 and 6 months of age, one of whom had had pancytopenia and hepatosplenomegaly. Physical examination revealed normal growth and diffuse psoriatic lesions. Laboratory investigations revealed hemoglobin of 8.5 g/dL, mean corpuscular volume of 92 fL, reticulocyte count of 0.4%, leukocyte count of 3.7x109/L (absolute neutrophil count of 0.4x109/L), and platelet count of 28x109/L with 2% blasts on peripheral smear. Lymphocyte subset analysis revealed 1% CD19+ cells. T-lymphocyte stimulation by phytohemagglutinin disclosed normal results. Virological, microbiological, and immunological studies; DEB test; and ferritin, vitamin B12, and folic acid levels were all unrevealing. Bone marrow aspiration revealed 15% blasts and BM biopsy revealed 10%-15% CD34+ blastic cells and trilineage

dysplasia. A clonal population with monosomy 7 was detected in 70% of metaphases studied. She was diagnosed with myelodysplastic syndrome (MDS) and refractory anemia with excess of blasts (RAEB). After 3 months, a BM study before BM transplantation (BMT) showed 23% blasts, revealing progression to acute myeloblastic leukemia (AML). After AML-BFM induction treatment, she underwent BMT from her fully matched uncle. Interestingly, her psoriatic lesions disappeared, and she is doing well 12 months after the BMT. The only report in the literature similar to ours presented 3 children with diagnoses of hypo-/agammaglobulinemia and B-lymphocytopenia, followed by refractory anemia and monosomy 7 [1]. One of them progressed to RAEB after 6 years, similar to our patient progressing to AML. Another report described secondary transient MDS in a patient with X-linked agammaglobulinemia and a clonal abnormality in the BM [del(6q), -9 and der(11)] that disappeared within 1.5 years [2]. Secondary MDS or features mimicking MDS may be seen during viral infections, in patients with genetic abnormalities including microdeletion 22q11.2, and in nonmalignant disorders like juvenile rheumatoid arthritis, polyarteritis nodosa, and idiopathic thrombocytopenic purpura [3,4,5,6,7]. Informed consent was obtained. A current hypothesis claims that in genetically predisposed persons, nonspecific stimulation of T-cells

Address for Correspondence: Arzu YAZAL ERDEM, M.D., Ankara Children’s Hematology and Oncology Hospital, Clinic of Pediatric Hematology, Ankara, Turkey Phone +90 312 596 96 00 E-mail: arzu.erdem@gmail.com Received/Geliş tarihi : July 23, 2014 Accepted/Kabul tarihi : November 7, 2014

Turk J Hematol 2015;32:87-88

Özbek N, et al: A Child with Psoriasis, Hypogammaglobulinemia, and Monosomy 7-Positive Myelodysplastic Syndrome

amplifies epidermal growth in psoriasis [8]. In the literature, the only relevant paper revealed MDS in an adult with psoriasis that occurred after etanercept treatment [9]. Although the number, distribution, and activity of T-cells were normal in our patient, the disappearance of psoriatic lesions after BMT may indicate an intrinsic T-cell defect,which is also a new finding. The promising outcome of BMT in our patient may also indicate the role of allogeneic mesenchymal stem cells in the healing of psoriasis. A recent study reported aberrant proliferative activity, increased apoptosis rate, and different gene expression profiles in bone marrow mesenchymal stem cells (BMMSCs) obtained from psoriatic patients, which lead to defective immune response [10]. The disappearance of psoriatic lesions in our patient may have been due to the immunomodulatory effect of allogeneic BMMSCs. Conflict of Interest Statement 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. Key Words: Monosomy 7, MDS

Psoriasis,

Hypogammaglobulinemia,

Anahtar Sözcükler: Psöriazis, Hipogamaglobulinemi, Monozomi 7, MDS References 1. Srivannaboon K, Conley ME, Coustan-Smith E, Wang WC. Hypogammaglobulinemia and reduced numbers of B-cells in children with myelodysplastic syndrome. J Pediatr Hematol Oncol 2001;23:122-125. 2. Narula G, Currimbhoy Z. Transient myelodysplastic syndrome in X-linked agammaglobulinemia with a novel Btk mutation. Pediatr Blood Cancer 2008;51:826-828.

3. Yetgin S, Çetin M, Yenicesu İ, Özaltın F, Uçkan D. Acute parvovirus B19 infection mimicking juvenile myelomonocytic leukemia. Eur J Hematol 2000;65:276-278. 4. Özbek N, Derbent M, Olcay L, Yılmaz Z, Tokel K. Dysplastic changes in the peripheral blood of children with microdeletion 22q11.2. Am J Hematol 2004;77:126-131. 5. Yetgin S, Ozen S, Saatci U, Bakkaloglu A, Besbas N, Kirel B. Myelodysplastic features in juvenile rheumatoid arthritis. Am J Hematol 1997;54:166-169. 6. Yetgin S, Ozen S, Yenicesu İ, Çetin M, Bakkaloğlu A. Myelodysplastic features in polyarteritis nodosa. Pediatr Hematol Oncol 2001;2:157-160. 7. Olcay L, Yetgin S, Okur H, Erekul S, Tuncer M. Dysplastic changes in idiopathic thrombocytopenic purpura and the effect of corticosteroids to increase dysplasia and cause hyperdiploid macropolycytes. Am J Hematol 2000;65:99104. 8. Bos JD, de Rie MA, Teunissen MB, Piskin G. Psoriasis: dysregulation of innate immunity. Br J Dermatol 2005;152:1098-1107. 9. Knudson RM, Tefferi A, Pittelkow MR, Davis MD. Development of myelodysplastic syndrome evolving to acute myeloid leukemia in a patient receiving etanercept for psoriasis. J Am Acad Dermatol 2011;65:673-674. 10. Hou R, Liu R, Niu X, Chang W, Yan X, Wang C, Li J, An P, Li X, Yin G, Zhang K. Biological characteristics and gene expression pattern of bone marrow mesenchymal stem cells in patients with psoriasis. Exp Dermatol 2014;23:521-523.

Letter to the Editor

DOI: 10.4274/tjh.2014.0359

Clinical Picture in Hematology Hematoloji’de Klinik Tanımlama Üzerine

Şinasi Özsoylu Retired Professor of Pediatrics, Hematology, and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society

To the Editor, I am writing this letter concerning the “Clinical Picture in Hematology” entitled “Isolated Zinc Deficiency Causing Severe Microcytosis and Sideroblastic Anemia” by Shweta et al. in a recent issue of this journal [1]. Although we have seen several cases of zinc and iron deficiency with geophagia, hepatosplenomegaly, growth retardation, and hypogonadism (Tayanç-Reimann-Prasad syndrome) [2] and zinc deficiency with acrodermatitis enteropathica and immunodeficiency, the authors’ case looked completely different than these other syndromes, with high ferritin and bone marrow ring sideroblasts (low zinc level was most likely an associated finding). Although the authors’ working diagnosis of “myelodysplastic syndrome (MDS, refractory anemia with ring sideroblasts)” seems to be the best choice, I would like to question probable pyridoxine administration in mineral supplements that contained copper and zinc “amongst” others. Among other possibilities, hepcidin, heme oxygenase heterozygosity [3], chronic liver disorders [4], and the presence of α-thalassemia (hereditary or acquired because of the proband’s origin of birth and erythrocyte morphology) should be investigated by hemoglobin electrophoresis at pH 6.4, and H inclusion bodies should be determined for a better

explanation in this very unusual case, which might shed light on iron and zinc metabolism, sideroblastic anemia, and iron homeostasis at large, I believe. Conflict of Interest Statement The author of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. Key Words: Microcytosis, Zinc deficiency Anahtar Sözcükler: Mikrositosis, Çinko noksanlığı References 1. Shweta G, Prantesh J, Shashvat S. Isolated zinc deficiency causing severe microcytosis and sideroblastic anemia. Turk J Hematol 2014;31:339-341. 2. Say B, Ozsoylu S, Berkel I. Geophagia associated with iron deficiency anemia, hepatosplenomegaly, hypogonadism and dwarfism. Clin Pediatr (Phila) 1969;8:661-668. 3. Poss KD, Tonegawa S. Heme oxygenase 1 is required for mammalian iron reutilization. Proc Natl Acad Sci USA 1997;94:10919-10924. 4. Göksu N, Özsoylu S. Hepatic and serum levels of zinc, copper and magnesium in childhood cirrhosis. J Ped Gastroenterol Nutr 1986;5:459-462.

Address for Correspondence: Şinasi ÖZSOYLU, M.D., Retired Professor of Pediatrics, Hematology, and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society E-mail: sinasiozsoylu@hotmail.com Received/Geliş tarihi : September 10, 2014 Accepted/Kabul tarihi : September 16, 2014

Letter to the Editor

DOI: 10.4274/tjh.2014.0393

About Chediak-Higashi, Hemoglobin Lansing, and Hemoglobin Jabalpur Chediak-Higashi, Hemoglobin Lansing ve Hemoglobin Jabalpur Hakkında Şinasi Özsoylu Retired Professor of Pediatrics, Hematology, and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society

To the Editor, I would like to express my concerns about some of the published letters in a recent issue of this journal. About “A Rare Cause of Recurrent Oral Lesions:

Dr. Çolak and her colleagues [4] also added another hemoglobin to the Turkish hemoglobinopathy library with their letter entitled “First Observation of Hemoglobin Jabalpur [Beta3 (NA3) Leu >Pro] in the Turkish

Chediak-Higashi Syndrome” by Karabel et al. [1], I feel

Population”. Since findings of reticulocyte count, plasma

that unbelievably low neutrophil counts (157/L ?) and/or

hemoglobin level, peripheral smear, etc. were not given, the

coinciding presence of another hereditary disorder such as

cause of mild anemia in the proband and his mother should

acatalasemia [2] should also be looked for as a cause of oral

be studied.

lesions.

Conflict of Interest Statement

It was nice seeing the addition of “Hemoglobin Lansing

The author of this paper have no conflicts of interest,

(Alpha) [HBA2 CD87 (HIS>GLU) (C>A)] in a Turkish

including specific financial interests, relationships, and/

Individual Resulting from Another Nucleotide Substitution”

or affiliations relevant to the subject matter or materials

to the hemoglobinopathy library in our country by Akar et

included.

al. [3]. More interestingly, these studies were carried out in a hematologically almost normal female (hemoglobin: 13.1 g/dL; MCV: 95 fL) for premarital counseling! The authors should give an explanation for this counseling in a female in the absence of family history and the presence of normal

Key Words: Chediak-Higashi, Hemoglobin Lansing, Hemoglobin Jabalpur Anahtar Sözcükler: Chediak-Higashi, Hemoglobin Lansing, Hemoglobin Jabalpur

hemoglobin level.

Address for Correspondence: Şinasi ÖZSOYLU, M.D., Retired Professor of Pediatrics, Hematology, and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society E-mail: sinasiozsoylu@hotmail.com Received/Geliş tarihi : October 02, 2014 Accepted/Kabul tarihi : October 13, 2014

Turk J Hematol 2015;32:90-91

ÖZsoylu Ş, About Chediak-Higashi, Hemoglobin Lansing, and Hemoglobin Jabalpur

References

1. Karabel M, Kelekçi S, Sen V, Karabel D, Aliosmanoglu C, Söker M. A rare cause of recurrent oral lesions: ChediakHigashi syndrome. Turk J Hematol 2014;31:313-314.

In Turkey, several issues including a mass screening program for the prevention of hemoglobinopathies have been taken since 2000. A hemoglobinopathy control (prevention) program was begun in 33 provinces in 2003 according to the regulations released by the Turkish Ministry of Health [1].

2. Takahara S, Hamilton HB, Neel JV, Kobara TY, Ogura Y, Nishimura ET. Hypocatalasemia: a new genetic carrier state. J Clin Invest 1960;39:610-619. 3. Akar N, Torun D, Öztürk A. Hemoglobin Lansing (alpha) [HBA2 CD87 (HIS>GLU) (C>A)] in a Turkish individual resulting from another nucleotide substitution. Turk J Hematol 2014;31:317-318. 4. Colak A, Toprak B, Yararbas K, Akyol F, Ceylan C. First observation of hemoglobin Jabalpur [beta 3 (NA3) Lac>Pro] in the Turkish population. Turk J Hematol 2014;31:319320.

Our patient was subjected to hemoglobin electrophoresis prior to marriage. When abnormal hemoglobin was detected, she was sent for genetic counseling to our center. Nejat Akar, M.D., Prof. TOBB-ETU Hospital, Clinic of Pediatrics, Ankara, Turkey Reference 1. Canatan D, Aydinok Y, Kılınç Y, Karakaş Z, Şaşmaz İ, Apak H, Sarper N. National Thalassemia Prevention Campaign: The Talotır Project. Turk J Hematol 2013;30:91-92.

Letter to the Editor

DOI: 10.4274/tjh.2014.0382

About Microangiopathic Hemolytic Anemia Mikroanjiopatik Hemolitik Anemi Üzerine Şinasi Özsoylu Retired Professor of Pediatrics, Hematology, and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society

To the Editor, I have read with interest the extensive review article by Yenerel on atypical hemolytic uremic syndrome (aHUS) in a recent issue of this journal [1]. I would like to bring to attention the point that more recently some authors do not use the term ‘aHUS’, which was historically used to distinguish heterogeneous, uncharacterized syndromes from Shiga toxin-related HUS (ST-HUS), since the term lacks both specificity and suggested causes [2]. I would also like to emphasize the term ‘hereditary’ instead of ‘congenital’ thrombotic thrombocytopenic purpura (TTP). I believe that ‘hereditary’ is more appropriate since hereditary factors are involved in these conditions. ‘Congenital’ seems to be more appropriate for conditions without gene involvement such as congenital syphilis, congenital tuberculosis, congenital rubella, etc., as I have brought to attention on several earlier occasions [3,4]. In the pathogenesis of thrombotic microangiopathic hemolytic anemia (TMHA) syndromes, endothelial injury and complement regulation (fluid phase and membrane attack) are causes of attacks that should not be omitted. Although eculizumab was emphasized, which is currently available as an anticomplementary agent, it is extremely expensive and may be limited among patients with C5 mutations [2,5].

It should be noted that glucocorticoids are standard treatment for TMHA. We have also used mega-dose methylprednisolone successfully in a patient on 3 different occasions. Rituximab and other immunosuppressive agents are appropriate when the clinical course is complicated. Conflict of Interest Statement The author of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. Key Words: Microangiopathy, Hemolytic anemia Anahtar Sözcükler: Mikroanjiyopati, Hemolitik anemi References 1. Yenerel MN. Atypical hemolytic uremic syndrome: differential diagnosis from TTP/HUS and management. Turk J Hematol 2014;31:216-225. 2. George JN, Nester CM. Syndromes of thrombotic microangiopathy. New Engl J Med 2014;371:654-666. 3. Özsoylu Ş. Acquired hemophilia. Turk J Hematol 2014; 31: 434. 4. Gümrük F, Özsoylu Ş. Plazma infüzyonu ile düzeltilebilen trombositopeni. Yeni Tıp Dergisi 1992;9:57-58 (in Turkish). 5. Noris M, Galbusera M, Gastoldi S, Macor P, Banterla F, Bresin E, Tripodo C, Bettoni S, Donadelli R, Valoti E, Tedesco F, Amore A, Coppo R, Ruggenenti P, Gotti E, Remuzzi G. Dynamics of complement activation in aHUS and how to monitor eculizumab therapy. Blood 2014;124:1715-1726.

Address for Correspondence: Şinasi ÖZSOYLU, M.D., Retired Professor of Pediatrics, Hematology, and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society E-mail: sinasiozsoylu@hotmail.com Received/Geliş tarihi : September 24, 2014 Accepted/Kabul tarihi : September 30, 2014

Letter to the Editor

DOI: 10.4274/tjh.2014.0381

Oral Lesions and Hematology Ağız Yaraları ve Hematoloji İrfan Yavaşoğlu Adnan Menderes University Faculty of Medicine, Department of Hematology, Aydın, Turkey

To the Editor, The letter entitled “A Rare Cause of Recurrent Oral Lesions: Chediak-Higashi Syndrome”, written by Karabel et al. and published in a recent issue of your journal, was quite interesting [1]. Here we would like to emphasize some relevant points. A number of systemic diseases, including hematologic disorders, do have manifestations in the orofacial region. Although non-pathognomonic, these manifestations may often represent the initial sign of the underlying hematopoietic disease. Oral ulcers are often idiopathic (recurrent aphthous stomatitis). However, oral ulcers may be findings of gluten enteropathy, inflammatory bowel disease, or Behçet’s disease. In addition, the cause may be deficiency of vitamin B12/folate/iron in 1 in 4 cases [2]. In particular, such lack of uptake may be the cause in patients who present with mental retardation. Therefore, mean corpuscular volume is important. Chediak-Higashi syndrome usually presents in infancy or early childhood; infections involving the lungs, skin, and mucous membranes are commonly encountered. Dental caries and periodontal disease are also common. The most frequent offending organism is

S. aureus. Prophylactic trimethoprim/sulfamethoxazole may be useful. Ascorbic acid (200 mg/day) can also be given [3]. Conflict of Interest Statement The author of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. Key Words: Oral Lesions, S. aureus, Hematology Anahtar Sözcükler: Ağız yaraları, S. aureus, Hematoloji References 1. Karabel M, Kelekçi S, Şen V, Karabel D, Aliosmanoğlu Ç, Söker M. A rare cause of recurrent oral lesions: ChediakHigashi syndrome. Turk J Hematol 2014;3:313-314. 2. Adeyemo TA, Adeyemo WL, Adediran A, Akinbami AJ, Akanmu AS. Orofacial manifestation of hematological disorders: hemato-oncologic and immuno-deficiency disorders. Indian J Dent Res 2011;22:688-697. 3. Dinauer M, Coates T. Disorders of phagocyte function. In: Hoofman R, Bennz E, Silberstein L, Heslop H, Weitz J, Anastasi J (eds). Hematology: Basic Principles and Practice. 6th ed. Philadelphia, PA, USA, Elsevier Saunders, 2013.

Address for Correspondence: İrfan YAVAŞOĞLU, M.D., Adnan Menderes University Faculty of Medicine, Department of Hematology, Aydın, Turkey E-mail: dryavas@hotmail.com Received/Geliş tarihi : September 24, 2014 Accepted/Kabul tarihi : September 25, 2014

Letter to the Editor

DOI: 10.4274/tjh.2014.0380

Follicular Lymphoma Foliküler Lenfoma İrfan Yavaşoğlu Adnan Menderes University Faculty of Medicine, Department of Hematology, Aydın, Turkey

To the Editor,

References

The article entitled “Renal Infiltration of Follicular Lymphoma”, written by Petkovic et al. and published in one of the recent issues of your journal, was quite interesting [1]. Here we would like to emphasize some relevant points. Extranodal disease is relatively common; any organ may be involved. The most common sites of extranodal disease include the bone marrow, skin, gastrointestinal tract, and bone [2]. Diagnosis of primary renal lymphoma based on the axillary involvement may not be accurate. There are two Follicular Lymphoma International Prognostic Index (FLIPI) scores: FLIPI scores 1 and 2. FLIPI 1 consists of age, Ann Arbor stage, hemoglobin level, serum LDH level, and number of nodal sites. FLIPI 2 consists of β2 microglobulin, bone marrow involvement, hemoglobin level, largest diameter of lymph node, and age [3,4]. Treatment may be delayed if the following conditions are not present: B symptoms or pruritus, rapid generalized disease progression, marrow involvement, life-threatening organ involvement, renal infiltration, or bone lesions [5]. The results of the Primary Rituximab and Maintenance (PRIMA) study demonstrated an advantage in progressionfree survival for rituximab maintenance therapy offered after initial chemoimmunotherapy [2]. The patient should receive maintenance rituximab treatment. Conflict of Interest Statement The author of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. Key Words: Follicular lymphoma, FLIPI, Rituximab Anahtar Rituksimab

Sözcükler:

Foliküler

lenfoma,

FLIPI,

1. Petkovic I, Krstic M, Pejcic I, Vrbic S, Stojnev S, Cvetanovic A, Balic M, Todorovic M. Renal infiltration of follicular lymphoma. Turk J Hematol 2014;3:315-316. 2. Gribben J. Clinical manifestations, staging, and treatment of follicular lymphoma. In: Hoffman R, Benz EJ Jr, Silberstein LE, Heslop H, Weitz J, Anastasi J. Hematology: Basic Principles and Practice. 6th ed. Philadelphia, PA, USA, Elsevier Saunders, 2013. 3. Solal-Céligny P, Roy P, Colombat P, White J, Armitage JO, Arranz-Saez R, Au WY, Bellei M, Brice P, Caballero D, Coiffier B, Conde-Garcia E, Doyen C, Federico M, Fisher RI, Garcia-Conde JF, Guglielmi C, Hagenbeek A, Haïoun C, LeBlanc M, Lister AT, Lopez-Guillermo A, McLaughlin P, Milpied N, Morel P, Mounier N, Proctor SJ, Rohatiner A, Smith P, Soubeyran P, Tilly H, Vitolo U, Zinzani PL, Zucca E, Montserrat E. Follicular Lymphoma International Prognostic Index. Blood 2004;104:1258-1265. 4. Federico M, Bellei M, Marcheselli L, Luminari S, LopezGuillermo A, Vitolo U, Pro B, Pileri S, Pulsoni A, Soubeyran P, Cortelazzo S, Martinelli G, Martelli M, Rigacci L, Arcaini L, Di Raimondo F, Merli F, Sabattini E, McLaughlin P, SolalCéligny P. Follicular Lymphoma International Prognostic Index 2: A new prognostic index for follicular lymphoma developed by the international follicular lymphoma prognostic factor project. J Clin Oncol 2009;27:4555-4562. 5. Ardeshna KM, Smith P, Norton A, Hancock BW, Hoskin PJ, MacLennan KA, Marcus RE, Jelliffe A, Vaughan G, Hudson, Linch DC; British National Lymphoma Investigation. Longterm effect of a watch and wait policy versus immediate systemic treatment for asymptomatic advanced-stage nonHodgkin lymphoma: a randomised controlled trial. Lancet 2003;362:516-522.

Turk J Hematol 2015;32:94-95

Yavaşoğlu İ, Follicular Lymphoma

Reply Thank you very much for your kind invitation to reply to comments on our article entitled “Renal Infiltration of Follicular Lymphoma” which has been published in your respected journal in volume 31 issue 3 in 2014. We presented a rare case of follicular lymphoma (FL) which was diagnosed after nephrectomy of the involved kidney which is well documented in histopathology/immunochistochemistry pictures. The diagnosis was set up after CT scan which showed a renal mass mimicking renal cell carcinoma (RCC) but even so we avoided to use of the term primary renal lymphoma since we were not sure if it was FL which originated from the kidney or it was the infiltration of the kidney from the surrounding perirenal tissue. However, this is not relevant since it was extranodal FL for sure, which is quite rare to be found in the renal region of the body. After a surgery, performed CT scans of the body did not show the disease spreading so we decided to use a PET/CT scan, before making a decision on eventually “watch and wait” strategy. PET/CT scan found axillary lymph nodes to be positive with a high SUV (max 7). The situation became debatable since the grade of FL was 3A and SUV was high and we assumed that FL might be transforming in a more aggressive variant such as diffuse large B-cell lymphoma (DLBCL). Both FLIPI indexes were counted for the patient but we mentioned only FLIPI1 since there was a limited space for word counts for letters to the editor, so we did not have a place to comment on every aspect of analysis we have done. Our single institution decision was to treat the patient with R-CHOP. This can be debatable and we already commented in the text about our arguments to initiate induction therapy (grade 3 A and B can be treated like DLBCL as was appointed by one of the leading authorities in the field of FL treatment-Prof Ghielmini and there are publications that support the use of combined surgery+immunochemotherapy in renal lymphoma involvement but only if one kidney is involved [1], which was the situation with our patient). We already gave our arguments pro and contra the use of anthracyclines. The patient received 8 cycles and achieved a complete remission which was PET/CT verified. If complete remission and long PFS is to be achieved, rituximab in combination with chemotherapy such as CHOP or bendamustine should be used [I, B] [2,3]. CVP combination results in inferior PFS, but no impact on OS was observed between these chemotherapy regimens [4]. Rituximab maintenance for 2 years improves PFS (75% versus 58% after 3 years, p<0.0001) [I, B] [5], whereas a shorter maintenance period results in inferior benefit [5,6]. Our patient underwent rituximab maintenance which still lasts; once again word counts were the major limiting factors to comment on every aspect of the treatment. Kind regards, Dr. Ivan Petkovic References 1. Cupisti A, Riccioni R, Carulli G, Paoletti S, Tognetti A, Meola M, Francesca F, Barsotti G, Petrini M. Bilateral primary renal lymphoma treated by surgery and chemotherapy. Nephrol Dial Transplant 2004;19:1629-1633. 2. Rummel MJ, Niederle N, Maschmeyer G, Banat GA, von Grünhagen U, Losem C, Kofahl-Krause D, Heil G, Welslau M, Balser C, Kaiser U, Weidmann E, Dürk H, Ballo H, Stauch M, Roller F, Barth J, Hoelzer D, Hinke A, Brugger W; Study group indolent Lymphomas (StiL). Bendamustine plus rituximab versus CHOP plus rituximab as first-line treatment for patients with indolent and mantlecell lymphoma: an open-label, multicentre, randomised, phase 3 non-inferiority trial. Lancet 2013;381:1203-1210. 3. Flinn IW, van der Jagt R, Kahl BS, Wood P, Hawkins TE, Macdonald D, Hertzberg M, Kwan YL, Simpson D, Craig M, Kolibaba K, Issa S, Clementi R, Hallman DM, Munteanu M, Chen L, Burke JM. Randomized trial of bendamustine rituximab or R CHOP/R-CVP in first-line treatment of indolent NHL or MCL: the BRIGHT study. Blood 2014;123:2944-2952. 4. Federico M, Luminari S, Dondi A, Tucci A, Vitolo U, Rigacci L, Di Raimondo F, Carella AM, Pulsoni A, Merli F, Arcaini L, Angrilli F, Stelitano C, Gaidano G, Dell’olio M, Marcheselli L, Franco V, Galimberti S, Sacchi S, Brugiatelli M. R-CVP versus R-CHOP versus R-FM for the initial treatment of patients with advanced-stage follicular lymphoma: results of the FOLL05 trial conducted by the Fondazione Italiana Linfomi. J Clin Oncol 2013;31:1506-1513. 5. Salles GA, Seymour JF, Feugier P, Offner F, Lopez-Guillermo A, Belada D, Xerri L, Bouabdallah R, Catalano J, Pauline B, Caballero D, Haioun C, Pedersen LM, Delmer A, Simpson D, Leppa S, Soubeyran P, Hagenbeek A, Casanovas O, Intragumtornchai T, Ferme C, Gomes de Silva M, Sebban C, Lister A, Estell JA, Milone G, Sonet A, Coiffer B, Tilly H. Updated 6 year follow-up of the PRIMA study confirms the benefit of 2-year rituximab maintenance in follicular lymphoma patients responding to frontline immunochemotherapy. Blood 2013; 122: abstr. 509. 6. Taverna CJ, Martinelli G, Hitz F, Mingrone W, Pabst T, Cevreska L, del Giglio A, Vanazzi A, Laszlo D, Raats J, Rauch D, Vorobiof DA, Lohri A, Zucca E, Biaggi Rudolf C, Rondeau S, Rusterholz C, Ghielmini M. Rituximab maintenance treatment for a maximum of 5 years in follicular lymphoma: results of the randomized phase III trial SAKK 35/03. ASH 2013; 122; abstr. 508.

Images in Hematology

DOI: 10.4274/tjh.2014.0048

Clinical Image in Hematology Rhinocerebral Mucormycosis in a Patient with Acute Promyelocytic Leukemia Rinoserebral Mukormikozis GeliĹ&#x;en Bir Akut Promyelositik LĂśsemi Olgusu

Figure 1. Yellow-black discoloration of upper palate.

Figure 2. Paranasal sinus CT scan revealed a defect of the nasal septum and skin and subcutaneous edema.

Turk J Hematol 2015;32:96-97

Keklik M, et al: Mucormycosis with Acute Promyelocytic Leukemia

A 63-year-old male presented with headache and weakness lasting for 2 months. Laboratory studies revealed the following: hemoglobin of 7.1 g/dL, white blood cells of 105x109/L, and platelets of 34x109/L. A diagnosis of acute promyelocytic leukemia was made by peripheral blood smear and bone marrow evaluations including morphological and genetic studies. The patient received one cycle of a chemotherapy regimen including cytarabine and idarubicin. During the aplastic phase, on day +6 from the end of chemotherapy, the patient developed fever, swelling on the left side of the face, infraorbital edema, and yellow-black discoloration of the upper palate (Figure 1). A paranasal sinus CT scan revealed a defect of the nasal septum and skin, and subcutaneous edema was seen at the maxillary and nasal level (Figure 2). The patient underwent surgical debridement. Histopathological assessment of the debridement specimen showed mucormycosis-associated hyphae. Culture of the nasal discharge was positive for Mucor spp. Liposomal amphotericin B was initiated at 5 mg/kg/day, but the patient died on the 30th hospital day. Informed consent was obtained. Mucormycosis is rapidly progressive and mortality for this infection is high [1,2,3,4]. Hematologic malignancies, longterm corticosteroid use, and immunosuppressive therapies are predisposing factors for mucormycosis. For management, mucormycosis should be considered early in high-risk patients, and surgical debridement together with effective antifungal therapy should be applied as soon as possible. Conflict of Interest Statement

Muzaffer Keklik1, Afra Yıldırım2, Fahir Öztürk3, İbrahim İleri3,  Gülşah Akyol1, Mustafa Çetin1, Bülent Eser1 1Erciyes

University Faculty of Medicine, Department of Hematology, Kayseri, Turkey 2Erciyes University Faculty of Medicine, Department of Radiology , Kayseri, Turkey 3Erciyes University Faculty of Medicine, Department of Internal Medicine, Kayseri, Turkey Received/Geliş tarihi

: February 2, 2014

Accepted/Kabul tarihi

: April 7, 2014 References

1. Tedeschi A, Montillo M, Cairoli R, Marenco P, Cafro A, Oreste P, Nosari A, Morra E. Prior invasive pulmonary and cerebellar mucormycosis is not a primary contraindication to perform an autologous stem cell transplantation in leukemia. Leuk Lymphoma 2002;43:657-659. 2. Petrikkos G, Skiada A, Lortholary O, Roilides E, Walsh TJ, Kontoyiannis DP. Epidemiology and clinical manifestations of mucormycosis. Clin Infect Dis 2012;54(Suppl 1):23-34. 3. Ibrahim AS, Spellberg B, Walsh TJ, Kontoyiannis DP. Pathogenesis of mucormycosis. Clin Infect Dis 2012;54(Suppl 1):16-22. 4. Kara IO, Tasova Y, Uguz A, Sahin B. Mucormycosis-associated fungal infections in patients with haematologic malignancies. Int J Clin Pract 2009;63:134-139.

promyelositik

lösemi,

Images in Hematology

DOI: 10.4274/tjh.2013.0368

Histopathological Image in Hematology Rapid Progression of Blastic Plasmacytoid Dendritic Cell Neoplasm without Extracutaneous Manifestation Deri Dışı Bulgu Olmaksızın Blastik Plazmasitoid Dendritik Hücre Neoplazisinin Hızlı İlerlemesi

Figure 1. Macroscopic, microscopic, and immunophenotypic findings of the case: (A) Multiple purple skin nodules on the back of his left shoulder; (B) the ill-defined mass had a solid and tan-white cut surface and was firm in consistency with visible infiltration of subcutaneous tissue; (C) H&E staining shows diffuse infiltration of the tumor cells in the dermis and subcutis (4x); immunophenotypic examination revealed that the tumor cells were (D) CD4-positive, (E) CD56-positive, and (F) CD123-positive.

Turk J Hematol 2015;32:98-99

Yu G, et al: BPDCN without Extracutaneous Manifestation

A 58-year-old Chinese male presented with a 3-month history of multiple purple nodules on the back of his left shoulder and on his back. The skin lesions initially appeared as maculopapules and grew progressively. Upon admission, the purplish nodules measured from 1 cm to 6 cm in diameter (Figure 1A). Blood results and image examination were normal. Tumor cells were not found in the examination of bone marrow aspiration and biopsy. With the patient’s approval, a skin biopsy was performed. On gross examination, the cut surface of the skin mass was tan-white with obscure boundaries and a firm consistency (Figure 1B). Histological examination demonstrated a dense and diffuse infiltrate of monomorphous medium-sized cells in the dermis and subcutis (Figure 1C). Immunohistochemistry showed that tumor cells were positive for CD4 (Figure 1D), CD56 (Figure 1E), CD123 (Figure 1F), and CD43 and were negative for CD3, CD5, CD20, CD30, granzyme B, and TdT. In situ hybridization testing for the Epstein-Barr virus was negative. The patient was diagnosed with blastic plasmacytoid dendritic cell neoplasm and received 4 courses of CHOP (cyclophosphamide + adriamycin + leurocristine + prednisone) chemotherapy. After the treatment, he suffered persistent high fever and pancytopenia and died 3 months later. Informed consent was obtained.

Conflict of Interest Statement 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. Key Words: Blastic plasmacytoid dendritic cell neoplasm, Cutaneous involvement Anahtar Sözcükler: Blastik plazmasitoid dendritik hücre neoplazisi, Hızlı ilerleme Guohua Yu1,2, Xin Huang3, Yuqing Huo4, Tingguo Zhang1, Zifen Gao3 1Shandong

University Faculty of Medicine, Department of Pathology, Jinan, China 2Medical College of Qingdao University, Affiliated Yantai Yuhuangding Hospital, Clinic of Pathology, Yantai, China 3Peking University Faculty of Medical Science, Health Science Center, Department of Pathology, Beijing, China 4Affiliated Hospital, Binzhou Medical College, Department of Oncology, Binzhou, China E-mail: zifengao@sohu.com Received/Geliş tarihi

: November 3, 2013

Accepted/Kabul tarihi

: March 19, 2014

Advisory Board of This Issue (March 2015) Ahmet Emre Eşkazan, Turkey Ali Ünal, Turkey Alma Cili, Albania Ayşe Çırakoğlu, Turkey Björn Dahlback, Sweden Bülent Kantarcıoğlu, Turkey Carlo Visco, Italy Cem Akın, USA Cengiz Beyan, Turkey Deniz Yılmaz Karapınar, Turkey Dina Fouad, Egypt Elif Ünal, Turkey Emel Gürkan, Turkey Emre Tekgündüz, Turkey Guimei Qu, China Gülsan Sucak Türköz, Turkey Hasan Göksoy, Turkey

Hongbo Wang, China Kaan Kavaklı, Turkey Long Su, China Luigia de Falco, Italy Maria Manara, Bologna Mehmet Akın, Turkey Mehmet Özen, Turkey Mehmet Şencan, Turkey Melda Cömert, Turkey Melih Aktan, Turkey Milena Velizarova, Bulgaria Mükerrem Safalı, Turkey Musa Karakükçü, Turkey Mustafa Pehlivan, Turkey Nejat Akar, Turkey Olga Meltem Akay, Turkey Oral Nevruz, Turkey

Özden Pişkin, Turkey Reşat Özaras, Turkey Said Abdou, Egypt Sema Anak, Turkey Semra Paydaş, Turkey Sevgi Kalayoğlu Beşışık, Turkey Shashvat Sukhal, USA Shigehisa Mori, Japan Şule Ünal, Turkey Tunç Fışgın, Turkey Veysel Sabri Hançer, Turkey Vivi Srivastava, India Yeşim Aydınok, Turkey Yusuf Baran, Turkey Zekaver Odabaşı, Turkey