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Volume 32

Issue 2

June 2015

80 TL

ISSN 1300-7777

TURKISH JOURNAL OF HEMATOLOGY • VOL.: 32

Review Article Recommendations for Risk Categorization and Prophylaxis of Invasive Fungal Diseases in Hematological Malignancies: A Critical Review of Evidence and Expert Opinion (TEO-4) Can Boğa, et al.; Adana, Aydın, İzmir, Antalya, Bursa, Trabzon, Ankara, Turkey

Research Articles A Novel Natural Product, KL-21, Inhibits Proliferation and Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Aysun Adan Gökbulut, et al.; İzmir, Kayseri, Turkey

A Possible Role for WNT5A Hypermethylation in Pediatric Acute Lymphoblastic Leukemia Özden Hatırnaz Ng, et al.; İstanbul, Antalya, Turkey

The Hematological and Molecular Spectrum of α-Thalassemias in Turkey: The Hacettepe Experience Şule Ünal, et al.; Ankara, Turkey

ISSUE: 2

Cohort Study: Central Venous Catheter-Related Complications in Children with Hematologic Diseases at a Single Center Ayhan Pektaş, et al.; Afyonkarahisar, Ankara, Turkey

JUNE 2015

The Effect of FcγRIIIA Gene Polymorphism on the Treatment of Diffuse Large B-cell Non-Hodgkin Lymphoma: A Multicenter Prospective Observational Study Nurhilal Büyükkurt, et al.; Adana, İzmir, Manisa, Turkey

Presence of Essential Hypertension or Diabetes Mellitus Is a Predictor of Intracranial Bleeding in Elderly Patients: A Study of 108 Patients with Isolated Thrombocytopenia from a Single Reference Center Rajan Kapoor, et al.; New Delhi, India

Cover Picture: Ebru Koca Güzeldere Waterfall, Düzce

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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 Eylül 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

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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 Ofset Matbaacılık Reklam Promosyon ve Tan. Hiz. San. Dış. Tic. Ltd. Şti. Tel.: +90 212 493 26 26 Topkapı, Maltepe Litros yolu, No: 24, Zeytinburnu, İstanbul, Turkey

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

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

Printing Date / Basım Tarihi 10.05.2015

Cover Picture Ebru Koca was born in 1974, Turkey. She is currently working at Başkent University Faculty of Medicine, Department of Hematology, Ankara, Turkey. Güzeldere (Beautiful Stream) Waterfall is located about 30 km away from the Düzce city center and is one of the biggest and most attractive waterfalls in Turkey. More than 30 kinds of plants and 15 kinds of wild animals can be seen. It’s under the protection of the Ministry of Forestry and Water Affairs of Turkey.

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

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AIMS AND SCOPE The Turkish Journal of Hematology is published quarterly (March, June, September, and December) by the Turkish Society of Hematology. It is an independent, non-profit peer-reviewed international English-language periodical encompassing subjects relevant to hematology. The Editorial Board of The Turkish Journal of Hematology adheres to the principles of the World Association of Medical Editors (WAME), International Council of Medical Journal Editors (ICMJE), Committee on Publication Ethics (COPE), Consolidated Standards of Reporting Trials (CONSORT) and Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). The aim of The Turkish Journal 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



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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. If you use a table or figure (or some data in a table or figure) from another source, cite the source directly in the figure or table legend. The journal is printed on acid-free paper.

Editorial Policy Following receipt of each manuscript, a checklist is completed by the Editorial Assistant. The Editorial Assistant checks that each manuscript contains all required components and adheres to the author guidelines, after which time it will be forwarded to the Editor in Chief. Following the Editor in Chief’s evaluation, each manuscript is forwarded to the Associate Editor, who in turn assigns reviewers. Generally, all manuscripts will be reviewed by at least three reviewers selected by the Associate Editor, based on their relevant expertise. Associate editor could be assigned as a reviewer along with the reviewers. After the reviewing process, all manuscripts are evaluated in the Editorial Board Meeting. Turkish Journal of Hematology’s editor and Editorial Board members are active researchers. It is possible that they would desire to submit their manuscript to the Turkish Journal of Hematology. This may be creating a conflict of interest. These manuscripts will not be evaluated by the submitting editor(s). The review process will be managed and decisions made by editor-in-chief who will act independently. In some situation, this process will be overseen by an outside independent expert in reviewing submissions from editors.


TURKISH JOURNAL OF HEMATOLOGY INSTRUCTIONS TO AUTHORS The Turkish Journal of Hematology accepts invited review articles, research articles, brief reports, 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.

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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, O’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.

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4. Book Chapter Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin: Genetics, Pathophysiology, Clinical Management. New York, Cambridge University Press, 2000. 5.Abstract Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract). 6.Letter to the Editor Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further definition of 20q deletion in myeloid leukemia using fluorescence in situ hybridization. Blood 1994;84:2821-2823. 7. Supplement Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr Transplant. 2005;9(Suppl 7):81-86

Brief Reports Abstract length: Not to exceed 150 words. Article length: Not to exceed 1200 words. Introduction: State the purpose and summarize the rationale for the study. Materials and Methods: Clearly describe the selection of the observational or experimental participants. Identify the methods and procedures in sufficient detail. Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for their use and an evaluation of their limitations. Identify all drugs and chemicals used, including generic names, doses, and routes of administration. Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify the reported findings/results. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used. Results: Present the findings/results in a logical sequence in the text, tables, and figures. Do not repeat all the findings/results in the tables and figures in the text; emphasize and/or summarize only those that are most important. Discussion: Highlight the new and important findings/results of the study and the conclusions they lead to. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by your data.

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

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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.

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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.

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CONTENTS 100

Review Article

118

Research Articles

Recommendations for Risk Categorization and Prophylaxis of Invasive Fungal Diseases in Hematological Malignancies: A Critical Review of Evidence and Expert Opinion (TEO-4) Can Boğa, Zahit Bolaman, Seçkin Çağırgan, İhsan Karadoğan, Mehmet Ali Özcan, Fahir Özkalemkaş, Rabin Saba, Mehmet Sönmez, Esin Şenol, Hamdi Akan, Murat Akova

A Novel Natural Product, KL-21, Inhibits Proliferation and Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Aysun Adan Gökbulut, Mustafa Yaşar, Yusuf Baran

127

A Possible Role for WNT5A Hypermethylation in Pediatric Acute Lymphoblastic Leukemia Özden Hatırnaz Ng, Sinem Fırtına, İsmail Can, Zeynep Karakaş, Leyla Ağaoğlu, Ömer Doğru, Tiraje Celkan, Arzu Akçay, Yıldız Yıldırmak, Çetin Timur, Uğur Özbek, Müge Sayitoğlu

136

The Hematological and Molecular Spectrum of α-Thalassemias in Turkey: The Hacettepe Experience Şule Ünal, Fatma Gümrük

144

Cohort Study: Central Venous Catheter-Related Complications in Children with Hematologic Diseases at a Single Center Ayhan Pektaş, Ateş Kara, Aytemiz Gurgey

152

The Effect of FcγRIIIA Gene Polymorphism on the Treatment of Diffuse Large B-cell Non-Hodgkin Lymphoma: A Multicenter Prospective Observational Study Nurhilal Büyükkurt, Mehmet Ali Özcan, Ülkü Ergene, Bahriye Payzın, Sunay Tunalı, Fatih Demirkan, Hayri Özsan, Özden Pişkin, Bülent Ündar

158

Presence of Essential Hypertension or Diabetes Mellitus Is a Predictor of Intracranial Bleeding in Elderly Patients: A Study of 108 Patients with Isolated Thrombocytopenia from a Single Reference Center Rajan Kapoor, Hara Prasad Pati, Manoranjan Mahapatra, Anuradha Monga

Brief Report

163

Pharmacobiological Approach for the Clinical Development of Ruxolitinib in Myeloproliferative Neoplasms Eylem Eliaçık, Ayşe Işık, Salih Aksu, Ayşegül Üner, Yahya Büyükaşık, Nilgün Sayınalp, Hakan Göker, Osman İ. Özcebe, İbrahim C. Haznedaroğlu

Case Reports

168

Possible Role of Interleukin-31/33 Axis in Imatinib Mesylate-Associated Skin Toxicity Caterina Musolino, Alessandro Allegra, Carmen Mannucci, Sabina Russo, Andrea Alonci, Valerio Maisano, Gioacchino Calapai, Sebastiano Gangemi

172

Severe Clinical Course in a Patient with Congenital Amegakaryocytic Thrombocytopenia Due to a Missense Mutation of the c-MPL Gene İkbal Ok Bozkaya, Neşe Yaralı, Pamir Işık, Rukiye Ünsal Saç, Betül Tavil, Bahattin Tunç

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175

Management of Two Juvenile Myelomonocytic Leukemia Patients According to Clinical and Genetic Features Özlem Tüfekçi, Hale Ören, Fatma Demir Yenigürbüz, Salih Gözmen, Tuba Hilkay Karapınar, Gülersu İrken

180

Ruxolitinib Treatment in a Patient with Primary Myelofibrosis Resistant to Conventional Therapies and Splenectomy: A Case Report Meltem Aylı, Muhit Özcan, Güldane Cengiz Seval

184

Letters to the Editor

186

Platelet Levels of High- and Mega-Dose Methylprednisolone Treatment in Acute Immune Thrombocytopenia Ali Ayçiçek

187

Gaucher Disease and Gaucher Cells Sevgi Gözdaşoğlu

189

Terbinafine and Neutropenia İrfan Yavaşoğlu

189

Multiple Myeloma and Alkaline Phosphatase İrfan Yavaşoğlu, Gürhan Kadıköylü, Zahit Bolaman

191

Images in Hematology

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Promyelocytic Blastic Crisis in Chronic Myeloid Leukemia During Imatinib Treatment Federico Angriman, Maria Nelly Gutierrez Acevedo, Maria Sol Rossi, Alberto Daniel Gimenez Conca, Victoria Otero, Jorge Alberto Arbelbide, Hernán Michelángelo

Thiopurine S-Methyltransferase and Methylenetetrahydrofolate Reductase Polymorphisms in Leukemia Serhan Küpeli

Disseminated Histoplasmosis in an Immunocompetent Host Presenting as Pancytopenia with Bilateral Adrenal Masses Smeeta Gajendra, Bhawna Jha, Tushar Sahni, Shalini Goel, Vimarsh Raina, Ritesh Sachdev

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Advisory Board of This Issue (June 2015) Ahmet Muzaffer Demir, Turkey Ali Bay, Turkey Alphan Küpesiz, Turkey Ana Boban, Croatia Ayşe Işık, Turkey Ayşegül Ünüvar, Turkey Bedia Ağaçhan, Turkey Betül Çatalgöl, Turkey Bülent Eser, Turkey Burhan Ferhanoğlu, Turkey Deniz Yılmaz Karapınar, Turkey Emel Özyürek, Turkey

Emmanuel Roilides, Greece Engin Ulukaya, Turkey Güçhan Alanoğlu, Turkey Güray Saydam, Turkey Halis Akalın, Turkey Handan Çipil, Turkey Hasan Tezer, Turkey Hatice Ilgın Ruhi, Turkey İbrahim Haznedaroğlu, Turkey Lebriz Yüksel Soycan, Turkey Livio Pagano, Italy Massimo Breccia, Italy

Müge Gündoğdu, Turkey Nalan Yazıcı, Turkey Nazan Sarper, Turkey Nejat Akar, Turkey Selami Koçak Toprak, Turkey Sema Karakuş, Turkey Semra Paydaş, Turkey Serap Aksoylar, Turkey Şinasi Özsoylu, Turkey Velu Nair, India


Review Article

DOI: 10.4274/tjh.2014.0277

Recommendations for Risk Categorization and Prophylaxis of Invasive Fungal Diseases in Hematological Malignancies: A Critical Review of Evidence and Expert Opinion (TEO-4) Hematolojik Malignitelerdeki İnvazif Fungal Enfeksiyon Riskinin Belirlenmesi ve Profilaksi: Kanıtlara Eleştirel Bakış ve Türk Uzman Görüşleri (TUG-4) Can Boğa1, Zahit Bolaman2, Seçkin Çağırgan3, İhsan Karadoğan4, Mehmet Ali Özcan5, Fahir Özkalemkaş6, Rabin Saba7, Mehmet Sönmez8, Esin Şenol9, Hamdi Akan10, Murat Akova11 1Başkent

University Faculty of Medicine Hospital, Department of Hematology, Adana, Turkey Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey 3Medical Park İzmir Hospital, Clinic of Hematology and Bone Marrow Transplantation Center, İzmir, Turkey 4Medstar Antalya Hospital, Clinic of Hematology and Stem Cell Transplantation, Antalya, Turkey 5Dokuz Eylül University Faculty of Medicine, Department of Hematology, İzmir, Turkey 6Uludağ University Faculty of Medicine, Department of Hematology, Bursa, Turkey 7Medstar Antalya Hospital, Clinic of Infectious Diseases, Antalya, Turkey 8Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey 9Gazi University Faculty of Medicine, Department of Infectious Diseases, Ankara, Turkey 10Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey 11Hacettepe University Faculty of Medicine, Department of Infectious Diseases, Ankara, Turkey 2Adnan

Abstract: This is the last of a series of articles on invasive fungal infections prepared by opinion leaders in Turkey. The aim of these articles is to guide clinicians in managing invasive fungal diseases in hematological malignancies and stem cell transplantation based on the available best evidence in this field. The previous articles summarized the diagnosis and treatment of invasive fungal disease and this article aims to explain the risk categorization and guide the antifungal prophylaxis in invasive fungal disease. Key Words: Hematological malignancy, Invasive fungal infections, Prophylaxis, Risk

Özet: Bu makale Türkiye’de invazif fungal enfeksiyon ile uğraşan uzmanlar tarafından hazırlanan bir seri yazının sonuncusudur. Bu makaleler hematolojik malignitelerde ve kök hücre nakli hastalarında invazif fungal hastalıkların yönetimini eldeki kanıtların ışığında en iyi hale getirmeyi amaçlamaktadır. İlk yazılar tanı ve tedaviyi özetlerken, bu makale invazif fungal hastalıkta risk kategorizasyonu ve profilaksiyi ele almaktadır. Anahtar Sözcükler: Hematolojik malignite, İnvazif fungal enfeksiyon, Profilaksi, Risk Address for Correspondence: Hamdi Akan, M.D., Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone: +90 532 424 26 40 E-mail: hamdiakan@gmail.com Received/Geliş tarihi : July 05, 2014 Accepted/Kabul tarihi : October 17, 2014

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Introduction Dr. Murat Akova Invasive fungal diseases (IFDs) continue to be an important cause of morbidity and mortality during the course of disease in patients with hematological malignancies and/or stem cell transplantation [1]. The lack of a cheap, easily applied diagnostic test with high sensitivity and specificity, as well as the serious mortality caused by the delay in diagnosis, has brought prevention of fungal infections to the forefront. While recently developed effective new antifungal medications with a wide spectrum of activity make prophylactic application attractive in high-risk patient groups, the wide use of serological and molecular biological diagnostic tools, like high-resolution computerized tomography examinations, serum galactomannan and beta-D-glucan tests, and PCR applications provides effective treatment options for selected patients in the early period [2]. The most important concerns about prophylaxis are additional costs, unwanted side effects due to antifungal use, and development of resistance due to the use of unnecessary antifungal antibiotics in many patients in order to prevent fungal infection-related mortality in one patient. Therefore, before routine antifungal prophylaxis (AFP) is given to high-risk patient groups, the abovementioned risk-benefit relationship should be necessarily reviewed. If an example is to be given, in order to decrease infection incidence by 50%, the number of patients who should receive prophylaxis is 100 in an environment where fungal infection prevalence is 2%, and this figure drops to 44 in the event that the prevalence is 4.5% [3]. Another point that should be kept in mind before application of prophylaxis is that although a decrease in fungal infection incidence and fungal-related mortality has been provided by AFP in numerous clinical studies performed to date, only 2 studies have been able to show a decrease in the general mortality of high-risk patients [4]. All these findings prove that selective action should be taken in high-risk patients regarding AFP. Determination of Risk and Targets of Prophylaxis Dr. Fahir Ă–zkalemkaĹ&#x; Patients with hematological malignancies and allogeneic stem cell transplantation are the primary risk groups for IFD [5,6]. It has been known for a long time that early treatment initiation in patients with IFD is one of the major determinants of successful treatment and decreased mortality [7,8,9]. Despite all the recent advances in diagnostic testing, the absence of rapid and reliable diagnostic tests in IFD diagnosis enhances the importance of risk determination. When the prevalence and mortality rates are taken into consideration, well-defined high-risk patients constitute the group that will benefit from the prophylactic use of an effective antifungal

Turk J Hematol 2015;32:100-117

agent. Therefore, determination of the correct risk level forms the basis of AFP. Furthermore, besides an increase in mortality, IFDs lead to delays in scheduled treatments (chemotherapy, stem cell transplantation), longer hospital stays, and increased treatment costs [10,11,12,13]. Invasive aspergillosis and invasive candidiasis are the most important entities of IFD. A third important group is invasive mucormycosis infection, with a relatively low prevalence but a high mortality rate [5,6]. Apart from common risk factors such as neutropenia and long-term myelosuppression, risk profile varies depending on the causative agent. For example, while the presence of numerous spores in inspired air and factors facilitating the passage of these spores through respiratory mucous membranes and reduced phagocytosis in the tissues play the major role in aspergillosis infections, diminished general phagocytic activity, dense colonization in the gastrointestinal tract mucosa, and mucosal damage due to chemo-/radiotherapy come to the forefront in invasive candidiasis. For mucormycosis, in addition to defective phagocytosis, other factors such as prior steroid use and the presence of metabolic acidosis should also be taken into consideration [1,5,14,15]. Hematological malignancies are widely heterogeneous in terms of risk. Among patients with hematological malignancies, acute leukemia patients, particularly those with acute myeloid leukemia (AML) receiving remission induction chemotherapy, are at significantly higher risk [6,13]. Highrisk myelodysplastic syndrome (MDS) is treated similarly to AML, as both have similar biological behavior; hence, MDS patients receiving remission induction chemotherapy are accepted to have the same risk profile as AML patients [16]. Likewise, hematopoietic stem cell transplantation patients are also heterogeneous. Risk is especially lower in patients with autologous transplantation than in those with allogeneic transplantation. Among allogeneic stem cell recipients, risk is significantly increased in those with graft-versus-host disease (GVHD) and long-term steroid use [17]. Another point that should not be forgotten is that risk profile may change over time due to the use of new treatment agents. For instance, the use of new monoclonal antibodies may alter the risk profile of chronic lymphocytic leukemia patients who are considered to be at low risk [18]. Similarly, use of new protocols in both the preparative regimen and GVHD prophylaxis in allogeneic stem cell transplantation, different stem cell sources (bone marrow, peripheral blood, umbilical cord blood), donor type (unrelated donors, relatives with perfect or partial match), stem cell manipulation (T-cell reduction), and superimposed infections (cytomegalovirus, respiratory syncytial virus infections) may lead to remarkable changes in the risk profile [10,17,19,20,21,22,23,24,25]. 101


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Apart from the comorbid conditions, the most important parameter in IFD development is neutropenia [26]. Both the depth and duration of neutropenia are important; more recently, an index taking these 2 parameters into account was reported to be important in predicting invasive mold infections [27]. In allogeneic transplants, in addition to neutropenia, development of long-term lymphopenia particularly enhances the risk of invasive aspergillosis [17]. Although specific polymorphisms (toll-like receptor 4, plasminogen alleles, dectin-1, TNF-1A) in some genes affecting natural immunity have been reported in recent years to make significant changes in invasive aspergillosis risk, these parameters are far from practical for use in the determination of treatment approach [28,29,30,31,32,33]. A recent study demonstrated that genetic deficiency of pentraxin 3 (PTX3) affects the antifungal capacity of neutrophils and may contribute to the risk of invasive aspergillosis in patients treated with hematopoietic stem cell transplantation [34]. Finally, in daily practice, particularly in Turkey, it is wise to underline the importance of environmental factors such as ‘air quality’ and personal factors like ‘colonization’ and ‘prevention’ in risk determination. Causative molds in the air may be at different concentrations in different geographic regions and may show seasonal variations in the same region [1,20,35]. In this respect, positive-pressure HEPA filters can be of critical importance in reducing the risk in risky regions and periods. Colonization during hospitalization is considered among the risk factors [36]. Other environmental control measures, hand-washing being the leading one, may reduce the risk [37]. Timing of Prophylaxis Dr. Esin Şenol There is no standard approach or recommendation regarding the optimal timing of prophylaxis in the guidelines and prophylaxis protocols. It is understood from prophylaxis studies that prophylaxis initiation times are different: at the time of hospitalization, or at the beginning or at the end of chemotherapy. In 2 important studies on primary prophylaxis using posaconazole, AML or MDS patients receiving chemotherapy had prophylaxis initiated together with chemotherapy in those not using anthracycline or 24 h after anthracycline in those using anthracycline. It was planned to be continued until resolution of neutropenia (ANC>500/ mm3), fungal infection development, or for 12 weeks [mean of 23 days (1-110 days, 29±21)]. In patients with allogeneic stem cell transplantation and GVHD, it was planned to be given at the time of development of acute GVHD of 2-4 degree or chronic disseminated GVHD at a dose of 1 mg/kg/day for acute GVHD and 0.8 mg/kg every other day for chronic GVHD, or at the beginning of 2 or more immunosuppressive agents

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without steroids, and scheduled to be continued for 112 days [38,39]. The reason for starting prophylaxis along with chemotherapy is that the oral antifungals used for prophylaxis reach their plasma saturation levels in the neutropenic period when fungal infection risk is the highest. This period is 5 days if voriconazole is used orally and 7-10 days if posaconazole is used [2,40,41]. However, there is concern that the azole antifungals mostly used for prophylaxis may interact with drugs used in chemotherapy regimens. Fluconazole prophylaxis (not involving molds) is given to allogeneic stem cell transplantation recipients before engraftment, in the beginning or immediately after the preparative regimen. There is a strong recommendation for giving prophylaxis for at least 3-6 months after engraftment; however, this period can be prolonged if treatment-related immunosuppression is caused by drugs such as corticosteroids [42]. The single remarkable end-point for the timing of prophylaxis termination for autologous stem cell transplantation recipients and for patients receiving AML/MDS chemotherapy is the resolution of neutropenia. Additionally, prophylaxis is discontinued in the event of conditions like drug intolerance, development of a new fungal infection, and drug-related side effects. However, there are still uncertainties in the timing of termination as well as the timing of initiation of prophylaxis, especially in allogeneic stem cell transplantation recipients [43]. Agents Used in Prophylaxis Dr. Mehmet Sönmez Polyene, azole, and echinocandin-class antifungal agents, including fluconazole, itraconazole, posaconazole, voriconazole, micafungin, anidulafungin, caspofungin, amphotericin B deoxycholate, and liposomal amphotericin B, have been used for AFP [38,39,44,45,46,47,48,49,50,51,52]. Toxicity, drug interactions, costs, effects of the used antifungal agent on fungal diagnostic tests, and risk of developing resistant fungal infections should be taken into account in patients receiving antifungal prophylaxis. While a metaanalysis of the studies comparing the efficacy of antifungal prophylaxis in patients receiving AML/MDS induction treatment or in those undergoing allogeneic hematopoietic stem cell transplantation showed that antifungal prophylaxis decreased IFD prevalence and IFD-related mortality, similar effects were not observed in patients undergoing autologous stem cell transplantation. The incidence of IFD, and especially the incidence of Aspergillus infections, was found to be lower in patients receiving prophylaxis for molds. However, side effectrelated discontinuation of the drug was found to be higher compared to the fluconazole-treated group. It was noted that that overall mortality was not changed [11,51,53]. Currently,


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BoÄ&#x;a C, et al: Risk and Prophylaxis in IFD

echinocandins, apart from micafungin and anidulafungin, and polyene-group antifungals are not considered to be preferable prophylactic agents in spite of their wide antifungal activity spectrum because of intravenous use, side effect profiles, costs, and absence of sufficient data on prophylactic use. Therefore, azole-group drugs are generally preferred in antifungal prophylaxis. Although itraconazole, included in this group of drugs, is an effective agent, high discontinuation rates due to gastric intolerance, drug interactions, and variable bioavailability restrict its use. Fluconazole, with the lowest rate of side effects and drug interactions and an activity spectrum limited to Candida species, is recommended in patients undergoing allogeneic hematopoietic stem cell transplantation during neutropenia, but necessitates mold testing during prophylaxis. The demonstration that posaconazole is more effective in preventing IFD development in comparison to fluconazole led to the preferential use of posaconazole prophylaxis in AML/MDS patients receiving induction treatment and in those who developed GVHD after allogeneic hematopoietic stem cell transplantation. However, the necessity of taking posaconazole on a full stomach along with food rich in fats, drug interactions, and the necessity of drug level monitoring are among the factors that restrict its usage. Likewise, while voriconazole, an azole-group drug that should be used in therapeutic levels, is recommended to be used in patients undergoing allogeneic stem cell transplantation, it requires careful monitoring of hepatotoxicity, neurotoxicity, and drug interactions [2,54,55,56,57,58,59,60]. Furthermore, voriconazole is recommended to be used for secondary prophylaxis in patients with prior Aspergillus infection who require retreatment or allogeneic hematopoietic stem cell transplantation. On the other hand, in hospitals with a mold incidence of <5% and with HEPA filtration, monitoring of patients with diagnostic tests without antifungal prophylaxis for molds may also be a suitable approach [60,61,62]. Monitoring of Prophylaxis Dr. Rabin Saba After a decision is made about AFP and the drug that will be used, the efficacy and side effects of the chosen drug should be monitored. Primarily, the interaction of the drug with food and other drugs should be evaluated. The bioavailability of voriconazole increases when taken on an empty stomach, itraconazole capsule with food, and posaconazole with fatty food. The bioavailability of proton pump inhibitors or H2 receptor blockers decreases when used together with posaconazole or itraconazole [2,63]. Considering drug interactions, special care should be taken when using triazoles, which can be both the substrate and the inhibitor of cytochrome P (CYP) 450 isoenzymes. Each drug should be considered individually. For instance, posaconazole

is metabolized by glucuronidation, not by the CYP system; however, it is a weak inhibitor of CYP 3A4. For this reason, if taken together with drugs inducing CYP enzymes, the serum concentration of triazoles other than posaconazole decreases. If taken together with triazoles, the serum concentration of drugs metabolized by CYP enzymes increases. It is contraindicated to use sirolimus with voriconazole and posaconazole [64]. When interactions with chemotherapeutic agents are considered, the best known example is the interaction between itraconazole and vincristine [65]. The increased neurotoxicity (by crossing the blood brain barrier) and the organ toxicity of vincristine is noteworthy. Antifungal drugs should also be monitored in terms of side effects [66]. While triazoles are particularly monitored regarding tolerability and hepatotoxicity, amphotericin B should be monitored in terms of infusion-related side effects, nephrotoxicity, and hypokalemia. The point that should not be forgotten is that prophylaxis should be used in conditions where the protective effects of prophylaxis are superior to the expected side effects. Discontinuation of the drug and/or switching to another drug should be considered in the case of side effects. Drug Level Monitoring While therapeutic drug monitoring generally gains importance for mold-active triazoles (itraconazole, voriconazole, and posaconazole), it is not recommended for echinocandin and polyene-group antifungals [2,63]. Measurement of serum concentrations is especially recommended in pharmacokinetically unstable patients (children, neonates, critical patients, those with organ dysfunction, etc.), in the suspicion of incompatibility, in the presence of drug interactions, when switching from the intravenous form of the drug to the oral form, and in patients with absorption problems such as diarrhea or GVHD. Itraconazole The bioavailability of itraconazole is variable and shows changes depending on the formulation. Bioavailability of the oral capsules increases with food and gastric acidity. The oral solution, which has better bioavailability, is much better absorbed when taken on an empty stomach and is not affected by gastric acidity. As the rates of breakthrough infections and mortality were found to be significantly higher at lower drug levels, it is required to maintain the serum concentrations at >8 mg/L (measuring both itraconazole and hydroxyitraconazole levels) as measured by bioassay method, at <0.5-1 mg/L by high-performance liquid chromatography and mass spectrometry, and at <17 mg/L by bioassay to minimize gastrointestinal, neurological, and hepatic toxicity [67,68]. As the drug concentration will achieve a steady state within 2 weeks, the measurements should begin after 7 days [2,63,64,65,66,67,68].

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Voriconazole As voriconazole shows nonlinear pharmacokinetics, changes in the dosage are not similarly reflected. Although the efficacy of prophylaxis monitoring has not been clearly demonstrated, serum concentrations are to be maintained at 1-5 mg/L with regard to toxicity [63,69,70]. It is recommended that serum concentrations should be measured within and after 5 days of use. Posaconazole Due to its long half-life (34 h), and because the drug concentration achieves a steady state within 7 days, the first measurement is recommended to be performed after 1 week of use [63]. The plateau concentration is recommended to be >0.7 mg/L for efficient prophylaxis [63,71,72]. It was shown that the alveolar intracellular posaconazole level was 40-50 times greater than outside the cell and this might explain the efficacy of posaconazole prophylaxis in patients with low serum posaconazole levels [73]. As the alveolar tissue concentration is important for posaconazole, it has been stated that alveolar concentration rather than serum concentration will be required to be measured in the future [63]. In the follow-up of patients receiving mold prophylaxis for fungal infections, special care should be given to diagnostic test interpretation. Notably, the sensitivity of the galactomannan test is decreased in patients receiving posaconazole and voriconazole prophylaxis [74]. A study evaluating the place of Aspergillus polymerase chain reaction and galactomannan antigen in bronchoalveolar lavage fluid in diagnosis showed that anti-mold prophylaxis decreases sensitivity [75]. On the other hand, there are studies reporting that itraconazole prophylaxis had no effect on the molecular method used [76]. Therefore, sensitivity of molecular tests should also be interpreted with caution [77,78]. Prophylaxis Failure Dr. Seçkin Çağırgan AFP failure may be defined as the development of proven or probable IFD during prophylaxis, the requirement of empirical antifungal treatment, and the necessity of discontinuing the prophylaxis drug due to side effects or patient-related reasons [39]. AFP failure due to development of an IFD may be related to the activity spectrum of the prophylactic agent, development of infection with resistant fungal pathogens, and failure to provide effective blood levels of the drug. Although fluconazole prophylaxis significantly reduces Candida infections in patients with acute myelocytic leukemia and in those undergoing allogeneic stem cell transplantation, it is accompanied with an increase in the rates of invasive aspergillosis and other mold infections, as fluconazole is not

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active against molds [39,42,44,79]. Moreover, it has been shown that fluconazole prophylaxis increases colonization and development of infection with resistant non-albicans Candida species [80,81]. The risk of aspergillosis decreases if a broadspectrum azole (itraconazole, voriconazole, or posaconazole) or an echinocandin effective against Aspergillus species is used; however, the probability of infections with other molds, especially Mucorales species, remains the same, as itraconazole, voriconazole, and echinocandins are not effective against Mucorales [82]. Failure in providing adequate serum levels is most frequently seen when using oral itraconazole and posaconazole and this may lead to prophylaxis failure and development of an IFD [2]. Particularly, patients with mucositis, nausea and vomiting, insufficient enteral intake, and diarrhea are at risk. Necessity of termination of prophylaxis or switching to another drug may be associated with patient intolerance or drug toxicity. Gastrointestinal intolerance and hepatotoxicity are the most common toxicity-related causes of AFP termination [2]. Prophylaxis using mold active agents, posaconazole being the leading one, has been demonstrated to significantly decrease the sensitivity of galactomannan testing [83,84]. Therefore, a preemptive AFP treatment approach based on galactomannan antigen monitoring will not be safe in these patients; hence, an empirical treatment approach is recommended. A detailed diagnostic study should be started in patients with prophylaxis failure if symptoms and clinical findings indicative of an IFD are present. If possible, the pathogen should be detected (microscopic examination in suitable samples, culture, histopathological examination; bronchoscopy, galactomannan testing in bronchoalveolar lavage fluid in the presence of a pulmonary lesion, etc.) [2]. Which antifungal agent should be selected in AFP failure characterized by IFD development or the requirement of initiating empirical fungal treatment? As a general rule, a change in the antifungal agent class should be considered if an IFD is suspected [2]. The majority of cases with prophylaxis failure are associated with the development of pulmonary infiltrates. During oral mold-active azole prophylaxis, switching to liposomal amphotericin B should be considered if aspergillosis is suspected (galactomannan positivity) and effective serum levels of itraconazole or posaconazole can be achieved or if drug level monitoring is not available [2,82]. If low serum levels of itraconazole or posaconazole have been shown, intravenous voriconazole can be used [2]. If galactomannan antigen testing is negative or is not available and a mold-active azole or echinocandin effective against Aspergillus strains has been used in prophylaxis, the risk of Aspergillus infection decreases; however, the probability


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of infections with other filamentous fungi, particularly Mucorales, remains, as itraconazole, voriconazole, and echinocandins are not effective against Mucorales strains. Posaconazole is active against some Mucorales strains; however, effective serum concentrations usually cannot be achieved. In these conditions, liposomal amphotericin B, with the widest spectrum of activity (Candida, Aspergillus species, Cryptococcus, Fusarium, Mucorales, and endemic fungi), is the antifungal drug that should be chosen. Azoles should not be used empirically in the case of prior azole prophylaxis [58]. Secondary Prophylaxis Dr. Mehmet Ali Özcan During cytotoxic treatment, there is a substantial risk of recurrent invasive fungal infection in patients who “survived” the first invasive fungal infection. This rate is between 16% and 33% in the published series and IFD-related mortality reaches up to 88% [85,86,87,88,89]. Application of antifungal drugs for the management of this risk is called “secondary prophylaxis”. The most important problem in this area is that there is still no prospective randomized study on this subject.

Therefore, evaluations are based on available experiences with different applications of antifungal agents. Amphotericin formulations, fluconazole, itraconazole, caspofungin, voriconazole, and posaconazole can be used in secondary prophylaxis depending on the use and success of these agents in primary treatment [90] (Table 1). While secondary prophylaxis seems to be effective according to the information obtained from case series and the few prospective secondary prophylaxis studies published to date, sufficient evidence to make a recommendation on the use of “which agent”, “what dose”, and “for how long” has not yet been provided. In manuscripts evaluating secondary prophylaxis, probable risk factors, mainly neutropenia duration, state of underlying disease, presence of GVHD, and steroid use, are found to be important. The European Conference on Infections in Leukemia guidelines recommend secondary prophylaxis with an evidence level of AII, and, instead of recommending a certain agent, they recommend that secondary prophylaxis should be based on the causative agent of the prior invasive fungal infection and treatment success [58].

Table 1. Studies on secondary prophylaxis for invasive fungal diseases (IFDs).

Source

Study

The Numbers and Characteristics of the Patients

Drug

Treatment Duration

New Breakthrough IFD

Sevilla et al. [91]

Case series

7 acute leukemia

Itraconazole + liposomal amphotericin

4-22 months

0/7

Nosari et al. [92]

Case series

24 patients; 9 patients allo, 15 patients acute leukemia

Itraconazole; itraconazole + liposomal amphotericin

Not indicated

3/24

Krüger et al. [93]

Case series

43 allo

Liposomal amphotericin B

2-54 days

0/43

Cornely et al. [85]

Prospective survey

124 patients, 14.5% surgical resections

Miscellaneous; itraconazole, voriconazole, liposomal amphotericin

Not indicated

26/124

Zhang et al. [94]

Retrospective

49 patients; 25 ALL, 10 AML

Miscellaneous; voriconazole, itraconazole, caspofungin, liposomal amphotericin

Not indicated

9/49

Cordonnier et al. [95]

Prospective open

45 patients

Voriconazole, 400 mg/ kg/day

5-180 days

2/45

IFD: Invasive fungal disease.

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In the decision-making period of secondary prophylaxis, the selected patients with sequel lesions are to be evaluated in terms of receiving chemotherapy or surgical resection before transplantation. As this group of patients is small in number in case series, it would be suitable to evaluate this subgroup separately in clinical studies.

Boğa C, et al: Risk and Prophylaxis in IFD

Environmental Protection Dr. Can Boğa The basic principle in AFP is to use drugs with proven efficacy and high evidence levels. This section of the article mainly discusses subjects associated with environmental factors.

Table 2. Recommendations of the CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC) [100].

Recommendations for Increasing Air Quality Evidence Level* Use of heating, pressure, and ventilation filters Environmental monitoring Use of portable HEPA filters Providing back-ups for technical problems such as power source Obtaining tissue biopsies and culture samples for diagnosis, in case of Aspergillus or other respiratory nosocomial infection Taking necessary preventive precautions during construction (barriers, stoppers, portable HEPA filters, etc.) Shortening the exposure time to fungal spores in transplant patients Maintenance of preventive measures when the patients leave their rooms for interventional procedures Maintaining positive pressure with respect to corridors Patient must be placed in a protective environment room with an anteroom

Category 1B Category 1B Category 2 Category 1C Category 1B Category 1B Category 1B Category 1B Category 1B Category 1C

Recommendations Related to Water Systems Prevention of damage to underground water pipes and contamination of water by soil Elimination of contaminated reservoirs Disinfection of sinks and taps Use of barrier precautions (gloves) in order to prevent the spread of infection after hand-washing Maintaining the temperature of hot water at >51 °C and cold water at <20 °C Periodically increasing the temperature of water to at least 66 °C Chlorination of water Disinfection of water tanks

Category 1B Category 1B Category 2 Category 1A Category 1C Category 2 Category 1C Category 2

Other Environmental Precautions Scrubbing the non-critical surfaces with detergents Regular scrubbing of ceilings, floors, and walls Not performing alcohol disinfestation on large surfaces Use of registered ready-to-use disinfectants Deep vacuum cleaning of the carpets and wool fabrics Avoiding the use of carpets in high-traffic zones Using carpeting in dry places (in order to avoid fungal growth), not keeping carpets in wet places for over 72 h Restriction of flowers Staff should be instructed to wear gloves when handling plants and flowers; negative pressure should be provided in the areas with dirty laundry Minimizing contact with saliva, urine, and feces of animals Hand-washing after contact with an animal Implementation of waste control procedures

Category 2 Category 2 Category 2 Category C Category 2 Category 2 Category 1B Category 2 Category 2 Category 1C Category 2 Category 2

*: Good Manufacturing Practice recommendations are taken into account. Category 1A: Strongly recommended for implementation and strongly supported by well-designed experimental, clinical, or epidemiologic studies. Category 1B: Strongly recommended for implementation and supported by certain experimental, clinical, or epidemiologic studies and a strong theoretic rationale. Category 1C: Required by state or federal regulation or representing an established association standard. Category 2: Suggested for implementation and supported by suggestive clinical or epidemiologic studies, or a theoretic rationale.

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Nosocomial fungal infections are mainly transmitted by air, and less frequently by the oral route. It is well known that hospital construction and repair activities may increase the fungal spore concentrations in the air and that they are associated with the frequency of IFDs [96,97]. Organizations That Recommend the Use of HEPA Filters in Critical Areas Areas such as bone marrow transplantation units, in which immunosuppressed patients that require protection from infectious agents are monitored, and the isolation areas in which infected patients are monitored are defined as critical areas. The US Centers for Disease Control and Prevention (CDC) and the Spanish Society of Infectious Diseases and Clinical Microbiology recommend that these areas should be separated from other areas; heating, water systems, and ventilation conditions should be specially organized; and HEPA filters changing the room air 12 times in an hour should be used (Table 2) [98]. In What Conditions Are HEPA Filters Effective? The minimum acceptable limits that can lead to the development of IFDs are debatable. It is required that HEPA filters should remove the respirable particles from the environment at least at the Good Manufacturing Practices Class D level (Table 3). It was demonstrated in a Spanish study that if the limit for the room air is 0.5 CFU/m3, or in other words if the presence of 1 fungus colony in 2 m3 of air is allowed, it can lead to infections in high-risk patients [97]. Evaluations of the Outcomes of HEPA Filter Use The results of published studies on HEPA filters are summarized as follows [98,99,100,101,102,103,104,105]: a. HEPA filters are effective to reduce the fungal load in the room air during and after construction. b. HEPA filters were found to provide a more effective protection against invasive aspergillosis than amphotericin B during and after construction. c. Acceleration of the laminar flow increases the efficacy of HEPA filters. Fungal concentration in the air is correlated with a decrease in IFD incidence. Table 3. Clean room classification arranged according to the number of particles considering the particle size [100].

Class

Particle Diameter (>0.5 µm)

Particle Diameter (>5 µm)

A

3520

20

B

35,200

29

C

352,000

2900

D

3,520,000

29,000

d. Moreover, HEPA filters were shown to improve the general quality of life after transplantation. The Efficacy of Portable HEPA Filters The CDC recommends the use of portable HEPA filters with rates of 300-800 cubic feet per minute to improve the removal process of respirable particles (Category 2). HEPA filters may be placed at different locations in and out of the room during and after construction until the surfaces are completely cleaned (Category 2). Microbiological analysis of the air samples during and immediately after construction is not recommended [98,99,100]. Problematic Issues Issues like patient-related comorbid conditions, the degree of immunosuppression, AFP, and microbiological quality of the water make it difficult to make scientific inferences. It has been reported that the gravity air-setting plate method is an applicable method in aerobiological monitoring of fungal spores. Petri plates involving Sabouraud agar media are placed in different areas of the rooms for 1 h with their lids open and each test is repeated 3 times. The samples are kept at 37 °C for 7 days. Quality Control The necessary protective precautions during transfer of the patients, primarily the hand-washing of the health care staff, and the necessary arrangements in terms of hygiene, waste control, and biosafety increase the efficacy of environmental control. Antifungal Vaccines Dr. Zahit Bolaman Aggressive chemotherapy or the use of agents leading to lymphocyte dysfunction such as rituximab and Campath and GVHD-related immunosuppression negatively affect the previous immunity achieved by vaccines in hematological malignant diseases [106]. As the risk for pneumococcal infection increases, patients are recommended to receive pneumococcal conjugate (PCV13) vaccine or pneumococcal polysaccharide vaccine (PPV23) before or during chemotherapy [107]. For inactivated influenza, hepatitis A, hepatitis B, meningococcus, conjugate haemophilus influenza, diphtheria-tetanuspertussis, human papilloma, and poliovirus vaccinations, the country’s vaccination program is taken into account. It is not recommended that acute leukemia patients under induction, consolidation, or maintenance treatment or those receiving rituximab or alemtuzumab be vaccinated with any vaccine other than pneumococcal vaccine. Live vaccines including measles-mumps-scarlet fever, shingles, chicken pox, and polio are contraindicated before or during chemotherapy [108]. The immune system is reorganized after allogeneic stem cell transplantation. Vaccination with PCV13 or PPV23, inactivated

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Table 4. Immunological properties of fungal vaccines.

Disease

Vaccine

Mechanism

Aspergillosis

HKY (heat-killed S. cerevisiae cells)* Laminarin-CRM197 conjugate* AspF antigens Cell-wall glucanase Crf1*

Incomplete information: Possibly related to the antifungal effect of protective CD4Th1 antibodies

Candida

Laminarin Fba (fructose-biphosphate-aldolase)* β-Mannan-peptide or protein conjugates Candida albicans cell-surface protein HyR1* Recombinant Als3* proteins* Recombinant Sap2* proteins Laminarin-CRM197 conjugate Mdh-1p

Direct antifungal effect of CD4Th1 antibodies Direct effect of anti-fba antibodies Opsonic, antibody-mediated Anti-Hyr1p neutralizing antibodies Antibodies developed due to Th17-Th1 activity Antibodies neutralizing Sap activity Direct anti-Candida effect of anti-β-glucan antibody Anti-Mdh-1p antibody

Cryptococcus

Laminarin-CRM197 conjugate GXM conjugate peptide mimotopes

Antibodies related to capsule structure and function Opsonic anti-capsular antibodies *: Universal.

influenza, hepatitis A and B, conjugate haemophilus influenza B, diphtheria-tetanus-pertussis, conjugate meningococcus, or inactivated polio is recommended to be performed at 3-12 months after transplantation. General rules apply for the human papilloma virus vaccine. Live measles-mumps-scarlet fever vaccine is applied if the patient is seronegative, does not have GVHD, and is not receiving immunosuppressive treatment. Attenuated live influenza, live measles, mumps and measles-varicella, BCG, live shingles, varicella, and shingles vaccines are contraindicated (Table 4, Figure 1). Data are insufficient for typhoid fever and cholera vaccines [60,108,109,110]. The aggressive therapies used in hematological malignancies cause tissue destruction and immunosuppression, sometimes resulting in death due to fungal infections. Although some patients benefit from antifungal prophylaxis, the results are not very satisfying and optimal antifungal treatment strategies can only rescue 50% of patients. This is also associated with high economic cost. As a result, novel approaches are needed. Antifungal vaccines are developed for this purpose and show their effect by stimulating humoral or cellular immunity and by dendritic cells. For the full-blown effect of an antifungal vaccine, it is important to develop them against common fungal antigens (universal vaccines) [111]. The targeted antifungal determinants and their mechanisms are shown in Table 4. Successful results have been achieved in animal

108

B

6

7

8

12

18 months

Figure 1. Vaccination program after allogeneic stem cell transplantation (modified from the Report from the International Consensus Conference on Clinical Practice in Chronic Graft-versus-host disease) [110]. DTaP: Diphtheria-tetanus-attenuated pertussis vaccine *Influenza vaccine is repeated each year.

studies on fungal cell determinants, mainly laminarin, cell surface antigen, or dendritic cell-mediated vaccination [112]. Experimental animal studies are on-going with vaccines developed against Aspergillus, Candida, Cryptococcus, and pneumocystis infections, and 3 studies are being carried out in humans on recombinant NDV-3. Although lack of standardization, reduced immunogenicity, and difficulties in the vaccination of immunosuppressive individuals reduce the development speed of fungal vaccines, initial studies are


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BoÄ&#x;a C, et al: Risk and Prophylaxis in IFD

Table 5. Pros and cons of antifungal prophylaxis. Advantages of antifungal prophylaxis

Problems with antifungal prophylaxis

The prevalence of IFD caused by Candida strains has been decreased with routine AFP.

There has been an increase in the prevalence of Aspergillus and non-albicans Candida strains with routine AFP. In particular, mucormycosis frequency has been shown to increase with broadspectrum azole prophylaxis [115,116].

The patients who will benefit from AFP can depend on the right classification of risk groups.

Risk factors and potentials for developing diseases are widely variable. Patients should be assessed individually for multiple factors; there are differences in the risk factors and classifications over time.

There are many old and new drugs with proven efficacy that The usage, doses, efficacies, and side effect profiles of these drugs can be used in AFP. are quite different from each other. In these patients, the frequently used drugs, mainly chemotherapy and immunosuppressive drugs, interact with many other drugs. Food may change the absorption rates of the drugs and there may be individual differences. AFP has been shown to be beneficial in some high-risk patients with hematological malignancies or in those undergoing stem cell transplantation.

Low IFD incidence in certain patient groups, increased risk of toxicity and resistance, and increased treatment cost due to unnecessary AFP use prevents its routine use in all patients. Patientbased risk-benefit evaluation should be performed.

Different drugs should be selected in patients according to the increased risk of developing yeast or mold infections.

The number of randomized controlled trials (RCTs) in which headto-head comparisons of several drugs are made is limited. Therefore, recommendations with high levels of evidence cannot be made in various guidelines and different recommendations are available in different guidelines. Treatment failure rates with mold-active AFP in real-life studies are higher in comparison to those in registration studies [116,117,118,119].

Meta-analysis performed in 2007 [11]: 64 RCTs, AFP (fluconazole, itraconazole, posaconazole) versus placebo Decreases in fungal-related mortality (RR: 0.66) and documented IFDs (RR: 0.69) were seen in acute leukemia patients. All-cause mortality (RR: 0.62), fungal-related mortality (RR: 0.52), and documented IFDs (RR: 0.33) decreased in allogeneic transplant patients.

Meta-analysis performed in 2007 [11]: 64 RCTs, AFP (fluconazole, itraconazole, posaconazole) versus placebo No difference could be detected in all-cause mortality (RR: 0.88) in acute leukemia patients.

Meta-analysis performed in 2012 [53]: 20 RCTs, mold-active versus fluconazole Fungal-related mortality (RR: 0.67), documented IFDs (RR: 0.71), and invasive aspergillosis risk (RR: 0.53) in both acute leukemia and allogeneic transplant patients were decreased.

Meta-analysis performed in 2012 [53]: 20 RCTs, mold-active versus fluconazole No difference could be detected in all-cause mortality in acute leukemia and allogeneic transplant patients. Side effects leading to drug discontinuation were more common in patients using moldactive drugs (RR: 1.95).

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Table 5. Continuous Advantages of Antifungal prophylaxis

Problems with antifungal prophylaxis

Meta-analysis performed in 2014 [120]: 20 RCTs, stem cell transplantations Risk of documented fungal disease (OR: 0.24), risk of systemic candidiasis (OR: 0.11), and overall need for empirical antifungal treatment (OR: 0.60) was decreased in patients receiving fluconazole in comparison to those receiving a placebo. Risk of invasive aspergillosis was lower in patients receiving itraconazole than in those receiving fluconazole (OR: 0.40). Risk of mold infections was found to be lower in those receiving micafungin than those receiving fluconazole (OR: 0.35). Voriconazole was found to be superior to fluconazole and itraconazole, and posaconazole was found to be superior to amphotericin B.

Meta-analysis performed in 2014 [120]: 20 RCTs, stem cell transplantations IFD risk under AFP was 5.1%. Most of the studies were performed using fluconazole. Comparative studies with new azoles are very limited (a single RCT).

GITMO guidelines published in 2014 [121]: Allogeneic stem cell transplantation patients are divided into 3 risk groups (high, standard, low) in 3 different periods (early, late, very late). Mold-active drugs are recommended in the high-risk group, and AFP is recommended in the standard-risk group.

Real-life data published in recent years [117]: Posaconazole comes into prominence in AML and MDS patients and in transplant patients with GVHD; although the level of evidence is low as the studies were retrospective and comparisons were made with historic controls, these data are valuable as they reflect real life.

Fluconazole: Oral + 4, cheap, good tolerability, fewer drug interactions.

Fluconazole: Anti-Candida spectrum is narrower than the candins, there is a risk of breakthrough infections (C. krusei, C. glabrata), not effective against molds. Echinocandin group: Can only be used by 4 route, expensive. Itraconazole: Can only be used by oral route, low tolerability, variable bioavailability, high drug interactions. Voriconazole: May lead to temporary visual impairment and hepatic toxicity, may interact with chemotherapy drugs. Posaconazole: Oral bioavailability is variable, may interact with chemotherapy drugs and proton pump inhibitors. Liposomal amphotericin B: Studies involving small series.

Echinocandin group: Effective against most Candida species, safety profile is high and drug interaction is low. Itraconazole: Effective against Aspergillus spp. Voriconazole: Oral + 4, wide spectrum of activity, superior to fluconazole and itraconazole in AFP. Posaconazole: Oral + 4, wide spectrum of activity. Liposomal amphotericin B: A single high dose once a week, effective and safe in AML patients and children. Drug level monitoring: Used to increase treatment success and decrease side effects, is recommended for mold-active drugs (itraconazole, voriconazole, and posaconazole).

Drug level monitoring: Not in routine use in guidelines yet.

Timing of AFP: AFP drugs that carry a risk of interaction with chemotherapy drugs are recommended to be discontinued 1 week before chemotherapy and initiated after termination of chemotherapy. If not receiving AFP is a risk for the patient, echinocandin-group drugs or liposomal amphotericin B may be given in this period.

Timing of AFP: There is no clear standard on this issue; it may be necessary to continue it until neutrophil counts exceed 500/mm3 in leukemia patients and for at least 3 months in allogeneic transplants. If the patient is still at risk (immunosuppressive treatment, etc.), the period should be prolonged.

Interaction with diagnostic antigen tests: Drugs do not affect standardized PCR-based tests.

Interaction with diagnostic antigen tests: Mold-active drugs (posaconazole, voriconazole) decrease the sensitivity of galactomannan test.

Secondary AFP: The drug that is effective in the treatment of the first attack should be preferred.

Secondary AFP: Risk of IFD extensively increases in the other chemotherapy periods of patients that had a previous IFD.

IFD: Invasive fungal disease, AFP: antifungal prophylaxis, GVHD: Graft-versus-host disease, AML: acute myeloid leukemia, MDS: myelodysplastic syndrome.

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Turk J Hematol 2015;32:100-117

promising in decreasing deaths related to fungal infections by fungal vaccinations in the future [112,113,114]. Interpretation and Problematic Areas Dr. İhsan Karadoğan Although prophylaxis in IFDs has become more evidencebased in recent years, there are still several gray areas and unresolved issues. A summary of these issues is presented in Table 5. 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.

7. Pizzo PA, Robichaud KJ, Gill FA, Witebsky FG. Empiric antibiotic and antifungal therapy for cancer patients with prolonged fever and granulocytopenia. Am J Med 1982;72:101-111.

References

10. Hachem R, Sumoza D, Hanna H, Girgawy E, Munsell M, Raad I. Clinical and radiologic predictors of invasive pulmonary aspergillosis in cancer patients: should the European Organization for Research and Treatment of Cancer/Mycosis Study Group (EORTC/MSG) criteria be revised? Cancer 2006;106:1581-1586.

1. Pagano L, Akova M, Dimopoulos G, Herbrecht R, Drgona L, Blijlevens N. Risk assessment and prognostic factors for mould-related diseases in immunocompromised patients. J Antimicrob Chemother 2011;66(Suppl 1):i5-14. 2. Akan H, Antia VP, Kouba M, Sinko J, Tanase AD, Vrhovac R, Herbrecht R. Preventing invasive fungal disease in patients with haematological malignancies and the recipients of haematopoietic stem cell transplantation: practical aspects. J Antimicrob Chemother 2013;68(Suppl 3):iii5-16. 3. Donnelly JP. Where are we with antifungal prophylaxis in AML and allogeneic HSCT? In: ESCMID Online Lecture Library. Available online at http://www.escmid.org/escmid_ library/online_lecture_library/material/?mid=2429. 4. Drgona L, Colita A, Klimko N, Rahav G, Ozcan MA, Donnelly JP. Triggers for driving treatment of at-risk patients with invasive fungal disease. J Antimicrob Chemother 2013;68(Suppl 3):iii17-24. 5. Pagano L, Caira M, Candoni A, Offidani M, Fianchi L, Martino B, Pastore D, Picardi M, Bonini A, Chierichini A, Fanci R, Caramatti C, Invernizzi R, Mattei D, Mitra ME, Melillo L, Aversa F, Van Lint MT, Falcucci P, Valentini CG, Girmenia C, Nosari A. The epidemiology of fungal infections in patients with hematologic malignancies: the SEIFEM-2004 study. Haematologica 2006;91:1068-1075. 6. Kontoyiannis DP, Marr KA, Park BJ, Alexander BD, Anaissie EJ, Walsh TJ, Ito J, Andes DR, Baddley JW, Brown JM, Brumble LM, Freifeld AG, Hadley S, Herwaldt LA, Kauffman CA, Knapp K, Lyon GM, Morrison VA, Papanicolaou G, Patterson TF, Perl TM, Schuster MG, Walker R, Wannemuehler KA, Wingard JR, Chiller TM, Pappas PG. Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, 2001-2006: overview of the Transplant-Associated Infection Surveillance Network (TRANSNET) Database. Clin Infect Dis 2010;50:1091-1100.

8. [No authors listed.] Empiric antifungal therapy in febrile granulocytopenic patients. EORTC International Antimicrobial Therapy Cooperative Group. Am J Med 1989;86(6 Pt 1):668672. 9. Morrell M, Fraser VJ, Kollef MH. Delaying the empiric treatment of Candida bloodstream infection until positive blood culture results are obtained: a potential risk factor for hospital mortality. Antimicrob Agents Chemother 2005;49:3640-3645.

11. Robenshtok E, Gafter-Gvili A, Goldberg E, Weinberger M, Yeshurun M, Leibovici L, Paul M. Antifungal prophylaxis in cancer patients after chemotherapy or hematopoietic stemcell transplantation: systematic review and meta-analysis. J Clin Oncol 2007;25:5471-5489. 12. Worth LJ, Slavin MA. Bloodstream infections in haematology: risks and new challenges for prevention. Blood Rev 2009;23:113-122. 13. Zhang P, Jiang EL, Yang DL, Yan ZS, Huang Y, Wei JL, Wang M, Ma QL, Liu QG, Zou DH, He Y, Qiu LG, Feng SZ, Han MZ. Risk factors and prognosis of invasive fungal infections in allogeneic stem cell transplantation recipients: a singleinstitution experience. Transpl Infect Dis 2010;12:316-321. 14. Martino R, Subira M. Invasive fungal infections in hematology: new trends. Ann Hematol 2002;81:233-243. 15. Rickerts V, Bohme A, Just-Nubling G. Risk factor for invasive zygomycosis in patients with hematologic malignancies. Mycoses 2002;45(Suppl 1):27-30. 16. Buckley SA, Othus M, Vainstein V, Abkowitz JL, Estey EH, Walter RB. Prediction of adverse events during intensive induction chemotherapy for acute myeloid leukemia or high-grade myelodysplastic syndromes. Am J Hematol 2014;89:423-428. 17. Wingard JR, Hsu J, Hiemenz JW. Hematopoietic stem cell transplantation: an overview of infection risks and epidemiology. Hematol Oncol Clin North Am 2011;25:101-116. 18. Montagna MT, De Giglio O, Napoli C, Lovero G, Caggiano G, Delia M, Pastore D, Santoro N, Specchia G. Invasive fungal infections in patients with hematologic malignancies (Aurora Project): lights and shadows during 18-months surveillance. Int J Mol Sci 2012;13:774-787.

111


Turk J Hematol 2015;32:100-117

19. Martino R, Subira M, Rovira M, Solano C, Vazquez L, Sanz GF, Urbano-Ispizua A, Brunet S, De la Camara R; alloPBSCT Infectious/Non-infectious Complications Subcommittees of the Grupo Español de Trasplante Hematopoyético (GETH). Invasive fungal infections after allogeneic peripheral blood stem cell transplantation: incidence and risk factors in 395 patients. Br J Haematol 2002;116:475-482. 20. Garcia-Vidal C, Upton A, Kirby KA, Marr KA. Epidemiology of invasive mold infections in allogeneic stem cell transplant recipients: biological risk factors for infection according to time after transplantation. Clin Infect Dis 2008;47:10411050. 21. Marr KA, Carter RA, Boeckh M, Martin P, Corey L. Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors. Blood 2002;100:4358-4366. 22. Omer AK, Ziakas PD, Anagnostou T, Coughlin E, Kourkoumpetis T, McAfee SL, Dey BR, Attar E, Chen YB, Spitzer TR, Mylonakis E, Ballen KK. Risk factors for invasive fungal disease after allogeneic hematopoietic stem cell transplantation: a single center experience. Biol Blood Marrow Transplant 2013;19:1190-1196. 23. Sun Y, Xu L, Liu D, Zhang X, Han W, Wang Y, Chen H, Chen Y, Wang F, Wang J, Ji Y, Tang F, Liu K, Huang XJ. Incidence of invasive fungal disease after unmanipulated haploidentical stem cell transplantation was significantly higher than that after HLA-matched sibling transplantation. Clin Microbiol Infect 201319:1029-1034. 24. Sun YQ, Xu LP, Liu DH, Zhang XH, Chen YH, Chen H, Ji Y, Wang Y, Han W, Wang JZ, Wang FR, Liu KY, Huang XJ. The incidence and risk factors of invasive fungal infection after haploidentical haematopoietic stem cell transplantation without in vitro T-cell depletion. Clin Microbiol Infect 2012;18:997-1003. 25. Li L, Wang J, Zhang W, Yang J, Chen L, Lv S. Risk factors for invasive mold infections following allogeneic hematopoietic stem cell transplantation: a single center study of 190 recipients. Scand J Infect Dis 2012;44:100-107. 26. Gerson SL, Talbot GH, Hurwitz S, Strom BL, Lusk EJ, Cassileth PA. Prolonged granulocytopenia: the major risk factor for invasive pulmonary aspergillosis in patients with acute leukemia. Ann Intern Med 1984;100:345-351. 27. Portugal RD, Garnica M, Nucci M. Index to predict invasive mold infection in high-risk neutropenic patients based on the area over the neutrophil curve. J Clin Oncol 2009;27:38493854. 28. Camargo JF, Husain S. Immune correlates of protection in human invasive aspergillosis. Clin Infect Dis 2014;59:569577. 29. Bochud PY, Chien JW, Marr KA, Leisenring WM, Upton A, Janer M, Rodrigues SD, Li S, Hansen JA, Zhao LP, Aderem

112

Boğa C, et al: Risk and Prophylaxis in IFD

A, Boeckh M. Toll-like receptor 4 polymorphisms and aspergillosis in stem-cell transplantation. N Engl J Med 2008;359:1766-1777. 30. Zaas AK, Liao G, Chien JW, Weinberg C, Shore D, Giles SS, Marr KA, Usuka J, Burch LH, Perera L, Perfect JR, Peltz G, Schwartz DA. Plasminogen alleles influence susceptibility to invasive aspergillosis. PLoS Genet 2008;4:e1000101. 31. Chai LY, de Boer MG, van der Velden WJ, Plantinga TS, van Spriel AB, Jacobs C, Halkes CJ, Vonk AG, Blijlevens NM, van Dissel JT, Donnelly PJ, Kullberg BJ, Maertens J, Netea MG. The Y238X stop codon polymorphism in the human beta-glucan receptor dectin-1 and susceptibility to invasive aspergillosis. J Infect Dis 2011;203:736-743. 32. Cunha C, Rodrigues F, Zelante T, Aversa F, Romani L, Carvalho A. Genetic susceptibility to aspergillosis in allogeneic stemcell transplantation. Med Mycol 2011;49(Suppl 1):S137143. 33. Loeffler J, Ok M, Morton OC, Mezger M, Einsele H. Genetic polymorphisms in the cytokine and chemokine system: their possible importance in allogeneic stem cell transplantation. Curr Top Microbiol Immunol 2010;341:83-96. 34. Cunha C, Aversa F, Lacerda JF, Busca A, Kurzai O, Grube M, Loffler J, Maertens JA, Bell AS, Inforzato A, Barbati E, Almeida B, Santos e Sousa P, Barbui A, Potenza L, Caira M, Rodrigues F, Salvatori G, Pagano L, Luppi M, Mantovani A, Velardi A, Romani L, Carvalho A. Genetic PTX3 deficiency and aspergillosis in stem-cell transplantation. N Engl J Med 2014;370:421-432. 35. Panackal AA, Li H, Kontoyiannis DP, Mori M, Perego CA, Boeckh M, Marr KA. Geoclimatic influences on invasive aspergillosis after hematopoietic stem cell transplantation. Clin Infect Dis 2010;50:1588-1597. 36. Blum G, Eschertzhuber S, Auberger J, Ulmer H, Geltner C, Gastl G, Nachbaur D, Lass-Florl C. Airborne fungus exposure prior to hospitalisation as risk factor for mould infections in immunocompromised patients. Mycoses 2012;55:237-243. 37. Munoz P, Burillo A, Bouza E. Environmental surveillance and other control measures in the prevention of nosocomial fungal infections. Clin Microbiol Infect 2001;7(Suppl 2):38-45. 38. Ullmann AJ, Lipton JH, Vesole DH, Chandrasekar P, Langston A, Tarantolo SR, Greinix H, Morais de Azevedo W, Reddy V, Boparai N, Pedicone L, Patino H, Durrant S. Posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease. N Engl J Med 2007;356:335-347. 39. Cornely OA, Maertens J, Winston DJ, Perfect J, Ullmann AJ, Walsh TJ, Helfgott D, Holowiecki J, Stockelberg D, Goh YT, Petrini M, Hardalo C, Suresh R, Angulo-Gonzalez D. Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia. N Engl J Med 2007;356:348-359. 40. Theuretzbacher U, Ihle F, Derendorf H. Pharmacokinetic/ pharmacodynamic profile of voriconazole. Clin Pharmacokinet 2006;45:649-663.


Boğa C, et al: Risk and Prophylaxis in IFD

41. Krishna G, Martinho M, Chandrasekar P, Ullmann AJ, Patino H. Pharmacokinetics of oral posaconazole in allogeneic hematopoietic stem cell transplant recipients with graftversus-host disease. Pharmacotherapy 2007;27:1627-1636. 42. Marr KA, Seidel K, Slavin MA, Bowden RA, Schoch HG, Flowers ME, Corey L, Boeckh M. Prolonged fluconazole prophylaxis is associated with persistent protection against candidiasis-related death in allogeneic marrow transplant recipients: long-term follow-up of a randomized, placebocontrolled trial. Blood 2000;96:2055-2061. 43. Slavin MA, Heath CH, Thursky KA, Morrissey CO, Szer J, Ling LM, Milliken ST, Grigg AP. Antifungal prophylaxis in adult stem cell transplantation and haematological malignancy. Intern Med J 2008;38:468-476. 44. Slavin MA, Osborne B, Adams R, Levenstein MJ, Schoch HG, Feldman AR, Meyers JD, Bowden RA. Efficacy and safety of fluconazole prophylaxis for fungal infections after marrow transplantation--a prospective, randomized, double-blind study. J Infect Dis 1995;171:1545-1552. 45. Goodman JL, Winston DJ, Greenfield RA, Chandrasekar PH, Fox B, Kaizer H, Shadduck RK, Shea TC, Stiff P, Friedman DJ, Powderly WG, Silber JL, Horowitz H, Lichtin A, Wolff SN, Mangan KF, Silver SM, Weisdorf D, Ho WG, Gilbert G, Buell D. A controlled trial of fluconazole to prevent fungal infections in patients undergoing bone marrow transplantation. N Engl J Med 1992;326:845-851. 46. Marr KA, Crippa F, Leisenring W, Hoyle M, Boeckh M, Balajee SA, Nichols WG, Musher B, Corey L. Itraconazole versus fluconazole for prevention of fungal infections in patients receiving allogeneic stem cell transplants. Blood 2004;103:1527-1533. 47. Wingard JR, Carter SL, Walsh TJ, Kurtzberg J, Small TN, Baden LR, Gersten ID, Mendizabal AM, Leather HL, Confer DL, Maziarz RT, Stadtmauer EA, Bolanos-Meade J, Brown J, Dipersio JF, Boeckh M, Marr KA; Blood and Marrow Transplant Clinical Trials Network. Randomized, doubleblind trial of fluconazole versus voriconazole for prevention of invasive fungal infection after allogeneic hematopoietic cell transplantation. Blood 2010;116:5111-5118. 48. Nucci M, Biasoli I, Akiti T, Silveira F, Solza C, Barreiros G, Spector N, Derossi A, Pulcheri W. A double-blind, randomized, placebo-controlled trial of itraconazole capsules as antifungal prophylaxis for neutropenic patients. Clin Infect Dis 2000;30:300-305. 49. Mattiuzzi GN, Cortes J, Alvarado G, Verstovsek S, Koller C, Pierce S, Blamble D, Faderl S, Xiao L, Hernandez M, Kantarjian H. Efficacy and safety of intravenous voriconazole and intravenous itraconazole for antifungal prophylaxis in patients with acute myelogenous leukemia or highrisk myelodysplastic syndrome. Support Care Cancer 2011;19:19-26.

Turk J Hematol 2015;32:100-117

50. Penack O, Schwartz S, Martus P, Reinwald M, Schmidt-Hieber M, Thiel E, Blau IW. Low-dose liposomal amphotericin B in the prevention of invasive fungal infections in patients with prolonged neutropenia: results from a randomized, singlecenter trial. Ann Oncol 2006;17:1306-1312. 51. Perfect JR, Klotman ME, Gilbert CC, Crawford DD, Rosner GL, Wright KA, Peters WP. Prophylactic intravenous amphotericin B in neutropenic autologous bone marrow transplant recipients. J Infect Dis 1992;165:891-897. 52. van Burik JA, Ratanatharathorn V, Stepan DE, Miller CB, Lipton JH, Vesole DH, Bunin N, Wall DA, Hiemenz JW, Satoi Y, Lee JM, Walsh TJ; National Institute of Allergy and Infectious Diseases Mycoses Study Group. Micafungin versus fluconazole for prophylaxis against invasive fungal infections during neutropenia in patients undergoing hematopoietic stem cell transplantation. Clin Infect Dis 2004;39:14071416. 53. Ethier MC, Science M, Beyene J, Briel M, Lehrnbecher T, Sung L. Mould-active compared with fluconazole prophylaxis to prevent invasive fungal diseases in cancer patients receiving chemotherapy or haematopoietic stem-cell transplantation: a systematic review and meta-analysis of randomised controlled trials. Br J Cancer 2012;106:1626-1637. 54. Vazquez L, Carreras E, Serrano D, Jarque I, Mensa J, Barberan J. Antifungal prophylaxis in the haematological patient: a practical approach. Rev Esp Quimioter 2012;25:299-304. 55. Rogers TR, Slavin MA, Donnelly JP. Antifungal prophylaxis during treatment for haematological malignancies: are we there yet? Br J Haematol 2011;153:681-697. 56. Cornely OA, Aversa F, Cook P, Jones B, Michallet M, Shea T, Vallejo C. Evaluating the role of prophylaxis in the management of invasive fungal infections in patients with hematologic malignancy. Eur J Haematol 2011;87:289-301. 57. Nucci M. Use of antifungal drugs in hematology. Rev Bras Hematol Hemoter 2012;34:383-391. 58. Maertens J, Marchetti O, Herbrecht R, Cornely OA, Flückiger U, Frêre P, Gachot B, Heinz WJ, Lass-Flörl C, Ribaud P, Thiebaut A, Cordonnier C; Third European Conference on Infections in Leukemia. European guidelines for antifungal management in leukemia and hematopoietic stem cell transplant recipients: summary of the ECIL 3--2009 update. Bone Marrow Transplant 2011;46:709-718. 59. Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Segal BH, Steinbach WJ, Stevens DA, van Burik JA, Wingard JR, Patterson TF; Infectious Diseases Society of America. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 2008;46:327-360.

113


Turk J Hematol 2015;32:100-117

60. Tomblyn M, Chiller T, Einsele H, Gress R, Sepkowitz K, Storek J, Wingard JR, Young JA, Boeckh MJ; Center for International Blood and Marrow Research; National Marrow Donor program; European Blood and Marrow Transplant Group; American Society of Blood and Marrow Transplantation; Canadian Blood and Marrow Transplant Group; Infectious Diseases Society of America; Society for Healthcare Epidemiology of America; Association of Medical Microbiology and Infectious Disease Canada; Centers for Disease Control and Prevention. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol Blood Marrow Transplant 2009;15:1143-1238. 61. Agrawal S, Hope W, Sinko J, Kibbler C. Optimizing management of invasive mould diseases. J Antimicrob Chemother 2011;66(Suppl 1):i45-53. 62. Maertens J, Groll AH, Cordonnier C, de la Camara R, Roilides E, Marchetti O. Treatment and timing in invasive mould disease. J Antimicrob Chemother 2011;66(Suppl 1):i37-43. 63. Ashbee HR, Barnes RA, Johnson EM, Richardson MD, Gorton R, Hope WW. Therapeutic drug monitoring (TDM) of antifungal agents: guidelines from the British Society for Medical Mycology. J Antimicrob Chemother 2014;69:11621176. 64. Cronin S, Chandrasekar PH. Safety of triazole antifungal drugs in patients with cancer. J Antimicrob Chemother 2010;65:410-416. 65. Moriyama B, Henning SA, Leung J, Falade-Nwulia O, Jarosinski P, Penzak SR, Walsh TJ. Adverse interactions between antifungal azoles and vincristine: review and analysis of cases. Mycoses 2012;55:290-297. 66. Girois SB, Chapuis F, Decullier E, Revol BG. Adverse effects of antifungal therapies in invasive fungal infections: review and meta-analysis. Eur J Clin Microbiol Infect Dis 2006;25:138149. 67. Glasmacher A, Prentice A, Gorschluter M, Engelhart S, Hahn C, Djulbegovic B, Schmidt-Wolf IG. Itraconazole prevents invasive fungal infections in neutropenic patients treated for hematologic malignancies: evidence from a meta-analysis of 3,597 patients. J Clin Oncol 2003;21:4615-4626. 68. Lestner JM, Roberts SA, Moore CB, Howard SJ, Denning DW, Hope WW. Toxicodynamics of itraconazole: implications for therapeutic drug monitoring. Clin Infect Dis 2009;49:928930. 69. Park WB, Kim NH, Kim KH, Lee SH, Nam WS, Yoon SH, Song KH, Choe PG, Kim NJ, Jang IJ, Oh MD, Yu KS. The effect of therapeutic drug monitoring on safety and efficacy of voriconazole in invasive fungal infections: a randomized controlled trial. Clin Infect Dis 2012;55:1080-1087. 70. Matsumoto K, Ikawa K, Abematsu K, Fukunaga N, Nishida K, Fukamizu T, Shimodozono Y, Morikawa N, Takeda Y, 114

BoÄ&#x;a C, et al: Risk and Prophylaxis in IFD

Yamada K. Correlation between voriconazole trough plasma concentration and hepatotoxicity in patients with different CYP2C19 genotypes. Int J Antimicrob Agents 2009;34:9194. 71. Eiden C, Meniane JC, Peyriere H, Eymard-Duvernay S, Le Falher G, Ceballos P, Fegueux N, Cociglio M, Reynes J, Hillaire-Buys D. Therapeutic drug monitoring of posaconazole in hematology adults under posaconazole prophylaxis: influence of food intake. Eur J Clin Microbiol Infect Dis 2012;31:161-167. 72. Ananda-Rajah MR, Grigg A, Slavin MA. Making sense of posaconazole therapeutic drug monitoring: a practical approach. Curr Opin Infect Dis 2012;25:605-611. 73. Campoli P, Al Abdallah Q, Robitaille R, Solis NV, Fielhaber JA, Kristof AS, Laverdiere M, Filler SG, Sheppard DC. Concentration of antifungal agents within host cell membranes: a new paradigm governing the efficacy of prophylaxis. Antimicrob Agents Chemother 2011;55:57325739. 74. Marchetti O, Lamoth F, Mikulska M, Viscoli C, Verweij P, Bretagne S; European Conference on Infections in Leukemia (ECIL) Laboratory Working Groups. ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients. Bone Marrow Transplant 2012;47:846-854. 75. Reinwald M, Spiess B, Heinz WJ, Vehreschild JJ, Lass-FlĂśrl C, Kiehl M, Schultheis B, Krause SW, Wolf HH, Bertz H, Maschmeyer G, Hofmann WK, Buchheidt D. Diagnosing pulmonary aspergillosis in patients with hematological malignancies: a multicenter prospective evaluation of an Aspergillus PCR assay and a galactomannan ELISA in bronchoalveolar lavage samples. Eur J Haematol 2012;89:120127. 76. Meije Y, Aguado JM, Cuenca-Estrella M. Silent and prolonged Aspergillus DNAemia in oncohematological patients receiving antifungal prophylaxis: a new phenomenon with clinical implications. Bone Marrow Transplant 2011;46:1016-1017. 77. Morrissey CO, Chen SC, Sorrell TC, Milliken S, Bardy PG, Bradstock KF, Szer J, Halliday CL, Gilroy NM, Moore J, Schwarer AP, Guy S, Bajel A, Tramontana AR, Spelman T, Slavin MA; Australasian Leukaemia Lymphoma Group and the Australia and New Zealand Mycology Interest Group. Galactomannan and PCR versus culture and histology for directing use of antifungal treatment for invasive aspergillosis in high-risk haematology patients: a randomised controlled trial. Lancet Infect Dis 2013;13:519-528. 78. Blennow O, Remberger M, Klingspor L, Omazic B, Fransson K, Ljungman P, Mattsson J, Ringden O. Randomized PCRbased therapy and risk factors for invasive fungal infection


Boğa C, et al: Risk and Prophylaxis in IFD

following reduced-intensity conditioning and hematopoietic SCT. Bone Marrow Transplant 2010;45:1710-1718.

Turk J Hematol 2015;32:100-117

80. Wingard JR, Merz WG, Rinaldi MG, Johnson TR, Karp JE, Saral R. Increase in Candida krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with fluconazole. N Engl J Med 1991;325:1274-1277.

Invasive aspergillosis before allogeneic hematopoietic stem cell transplantation: 10-year experience at a single transplant center. Biol Blood Marrow Transplant 2004;10:494-503. 88. Avivi I, Oren I, Haddad N, Rowe JM, Dann EJ. Stem cell transplantation post invasive fungal infection is a feasible task. Am J Hematol 2004;75:6-11. 89. Bjerke JW, Meyers JD, Bowden RA. Hepatosplenic candidiasis-a contraindication to marrow transplantation? Blood 1994;84:2811-2814. 90. Sipsas NV, Kontoyiannis DP. Clinical issues regarding relapsing aspergillosis and the efficacy of secondary antifungal prophylaxis in patients with hematological malignancies. Clin Infect Dis 2006;42:1584-1591.

81. Abbas J, Bodey GP, Hanna HA, Mardani M, Girgawy E, Abi-Said D, Whimbey E, Hachem R, Raad I. Candida krusei fungemia. An escalating serious infection in immunocompromised patients. Arch Intern Med 2000;160:2659-2664. 82. Barberan J, Mensa J, Llamas JC, Ramos IJ, Ruiz JC, Marín JR, Tello PB, Massana MB, Vidal JB, Vinas JM, Huelva FJ, Pons EC, Mediavilla JD, Morfa ML, Barrigón FE, Avellan PF, Lopez SG, García CG, Maraver DH, Guía AL, Jimenez JL, Chacon EM, Rubio MO, Oteyza JP, Ramírez GR, Contreras RR, Barbero AR, Tarrats MR, Felix DR, Godoy PS, Salinas AS, Alonso MA, Torroba Jde L, Ferreiras DV, Lopez LV, García JM, Perea JR, Moreno RC, Cancer RC, Abete JF, Rodrıguez JG, Gomez JG, Pedrosa EG, Baranda JM, García FJ, Camps IR, Lleti MS, Cisneros Jde L; Spanish Society of Chemotherapy. Recommendations for the treatment of invasive fungal infection caused by filamentous fungi in the hematological patient. Rev Esp Quimioter 2011;24:263-270. 83. Pagano L, Caira M, Cuenca-Estrella M. The management of febrile neutropenia in the posaconazole era: a new challenge? Haematologica 2012;97:963-965. 84. Marr KA, Laverdiere M, Gugel A, Leisenring W. Antifungal therapy decreases sensitivity of the Aspergillus galactomannan enzyme immunoassay. Clin Infect Dis 2005;40:1762-1769. 85. Cornely OA, Böhme A, Reichert D, Reuter S, Maschmeyer G, Maertens J, Buchheidt D, Paluszewska M, Arenz D, Bethe U, Effelsberg J, Lövenich H, Sieniawski M, Haas A, Einsele H, Eimermacher H, Martino R, Silling G, Hahn M, Wacker S, Ullmann AJ, Karthaus M; Multinational Case Registry of the Infectious Diseases Working Party of the German Society for Hematology and Oncology. Risk factors for breakthrough invasive fungal infection during secondary prophylaxis. J Antimicrob Chemother 2008;61:939-946. 86. Offner F, Cordonnier C, Ljungman P, Prentice HG, Engelhard D, De Bacquer D, Meunier F, De Pauw B. Impact of previous aspergillosis on the outcome of bone marrow transplantation. Clin Infect Dis 1998;26:1098-1103. 87. Fukuda T, Boeckh M, Guthrie KA, Mattson DK, Owens S, Wald A, Sandmaier BM, Corey L, Storb RF, Marr KA.

91. Sevilla J, Hernandez-Maraver D, Aguado MJ, Ojeda E, Morado M, Hernandez-Navarro F. Autologous peripheral blood stem cell transplant in patients previously diagnosed with invasive aspergillosis. Ann Hematol 2001;80:456-459. 92. Nosari A, Oreste P, Cairoli R, Montillo M, Carrafiello G, Astolfi A, Muti G, Marbello L, Tedeschi A, Magliano E, Morra E. Invasive aspergillosis in haematological malignancies: clinical findings and management for intensive chemotherapy completion. Am J Hematol 2001;68:231-236. 93. Kruger WH, Russmann B, de Wit M, Kroger N, Renges H, Sobottka I, Zander AR. Haemopoietic cell transplantation of patients with a history of deep or invasive fungal infection during prophylaxis with liposomal amphotericin B. Acta Haematol 2005;113:104-108. 94. Zhang P, Song A, Wang Z, Feng S, Qiu L, Han M. Hematopoietic SCT in patients with a history of invasive fungal infection. Bone Marrow Transplant 2009;43:533-537. 95. Cordonnier C, Rovira M, Maertens J, Olavarria E, Faucher C, Bilger K, Pigneux A, Cornely OA, Ullmann AJ, Bofarull RM, de la Camara R, Weisser M, Liakopoulou E, Abecasis M, Heussel CP, Pineau M, Ljungman P, Einsele H; Voriconazole for Secondary Prophylaxis of Invasive Fungal Infections in Patients With Allogeneic Stem Cell Transplants (VOSIFI) study group; Infectious Diseases Working Party, European Group for Blood and Marrow Transplantation. Voriconazole for secondary prophylaxis of invasive fungal infections in allogeneic stem cell transplant recipients: results of the VOSIFI study. Haematologica 2010;95:1762-1768. 96. Alberti C, Bouakline A, Ribaud P, Lacroix C, Rousselot P, Leblanc T, Derouin F; Aspergillus Study Group. Relationship between environmental fungal contamination and the incidence of invasive aspergillosis in haematology patients. J Hosp Infect 2001;48:198-206. 97. Benet T, Nicolle MC, Thiebaut A, Piens MA, Nicolini FE, Thomas X, Picot S, Michallet M, Vanhems P. Reduction of invasive aspergillosis incidence among immunocompromised patients after control of environmental exposure. Clin Infect Dis 2007;45:682-686.

79. Rotstein C, Bow EJ, Laverdiere M, Ioannou S, Carr D, Moghaddam N. Randomized placebo-controlled trial of fluconazole prophylaxis for neutropenic cancer patients: benefit based on purpose and intensity of cytotoxic therapy. The Canadian Fluconazole Prophylaxis Study Group. Clin Infect Dis 1999;28:331-340.

115


Turk J Hematol 2015;32:100-117

Boğa C, et al: Risk and Prophylaxis in IFD

98. Center for International Blood and Marrow Transplant Research (CIBMTR); National Marrow Donor Program (NMDP); European Blood and Marrow Transplant Group (EBMT); American Society of Blood and Marrow Transplantation (ASBMT); Canadian Blood and Marrow Transplant Group (CBMTG); Infectious Disease Society of America (IDSA); Society for Healthcare Epidemiology of America (SHEA); Association of Medical Microbiology and Infectious Diseases Canada (AMMI); Centers for Disease Control and Prevention (CDC). Guidelines for preventing infectious complications among hematopoietic cell transplant recipients: a global perspective. Bone Marrow Transplant 2009;44:453-558.

106. Small TN, Cowan MJ. Immunization of hematopoietic stem cell transplant recipients against vaccine-preventable diseases. Expert Rev Clin Immunol 2011;7:193-203.

99. Menegueti MG, Ferreira LR, Silva MF, Silva AS, BellissimoRodrigues F. Assessment of microbiological air quality in hemato-oncology units and its relationship with the occurrence of invasive fungal infections: an integrative review. Rev Soc Bras Med Trop 2013;46:391-396.

109. Eliakim-Raz N, Vinograd I, Zalmanovici Trestioreanu A, Leibovici L, Paul M. Influenza vaccines in immunosuppressed adults with cancer. Cochrane Database Syst Rev 2013;10:CD008983.

107. Kumar D, Chen MH, Welsh B, Siegal D, Cobos I, Messner HA, Lipton J, Humar A. A randomized, double-blind trial of pneumococcal vaccination in adult allogeneic stem cell transplant donors and recipients. Clin Infect Dis 2007;45:1576-1582. 108. Rubin LG, Levin MJ, Ljungman P, Davies EG, Avery R, Tomblyn M, Bousvaros A, Dhanireddy S, Sung L, Keyserling H, Kang I. 2013 IDSA clinical practice guideline for vaccination of the immunocompromised host. Clin Infect Dis 2014;58:e44-100.

100. Sehulster L, Chinn RY; CDC; HICPAC. Guidelines for environmental infection control in health-care facilities. Recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). MMWR Recomm Rep 2003;52:1-42.

110. Hilgendorf I, Freund M, Jilg W, Einsele H, Gea-Banacloche J, Greinix H, Halter J, Lawitschka A, Wolff D, Meisel R. Vaccination of allogeneic haematopoietic stem cell transplant recipients: report from the international consensus conference on clinical practice in chronic GVHD. Vaccine 2011;29:2825-2833.

101. Brun CP, Miron D, Silla LM, Pasqualotto AC. Fungal spore concentrations in two haematopoietic stem cell transplantation (HSCT) units containing distinct air control systems. Epidemiol Infect 2013;141:875-879.

111. Hamad M. Universal fungal vaccines: could there be light at the end of the tunnel? Hum Vaccin Immunother 2012;8:1758-1763.

102. Abdul Salam ZH, Karlin RB, Ling ML, Yang KS. The impact of portable high-efficiency particulate air filters on the incidence of invasive aspergillosis in a large acute tertiarycare hospital. Am J Infect Control 2010;38:e1-7. 103. Passweg JR, Rowlings PA, Atkinson KA, Barrett AJ, Gale RP, Gratwohl A, Jacobsen N, Klein JP, Ljungman P, Russell JA, Schaefer UW, Sobocinski KA, Vossen JM, Zhang MJ, Horowitz MM. Influence of protective isolation on outcome of allogeneic bone marrow transplantation for leukemia. Bone Marrow Transplant 1998;21:1231-1238. 104. Kruger WH, Zollner B, Kaulfers PM, Zander AR. Effective protection of allogeneic stem cell recipients against aspergillosis by HEPA air filtration during a period of construction--a prospective survey. J Hematother Stem Cell Res 2003;12:301-307. 105. National Disease Surveillance Centre. National Guidelines for the Prevention of Nosocomial Invasive Aspergillosis During Construction/Renovation Activities. Dublin, Ireland, National Disease Surveillance Center, 2002. Available online at https:// www.hpsc.ie/A-Z/MicrobiologyAntimicrobialResistance/ InfectionControlandHAI/Guidelines/File,896,en.pdf.

116

112. Dan JM, Levitz SM. Prospects for development of vaccines against fungal diseases. Drug Resist Updat 2006;9:105-110. 113. Cassone A, Casadevall A. Recent progress in vaccines against fungal diseases. Curr Opin Microbiol 2012;15:427-433. 114. Cassone A. Fungal vaccines: real progress from real challenges. Lancet Infect Dis 2008;8:114-124. 115. Rüping MJ, Heinz WJ, Kindo AJ, Rickerts V, Lass-Flörl C, Beisel C, Herbrecht R, Roth Y, Silling G, Ullmann AJ, Borchert K, Egerer G, Maertens J, Maschmeyer G, Simon A, Wattad M, Fischer G, Vehreschild JJ, Cornely OA. Forty-one recent cases of invasive zygomycosis from a global clinical registry. J Antimicrob Chemother 2010;65:296-302. 116. Auberger J, Lass-Florl C, Aigner M, Clausen J, Gastl G, Nachbaur D. Invasive fungal breakthrough infections, fungal colonization and emergence of resistant strains in high-risk patients receiving antifungal prophylaxis with posaconazole: real-life data from a single-centre institutional retrospective observational study. J Antimicrob Chemother 2012;67:2268-2273. 117. Pagano L, Caira M. The role of primary antifungal prophylaxis in patients with haematological malignancies. Clin Microbiol Infect 2014;20(Suppl 6):19-26.


BoÄ&#x;a C, et al: Risk and Prophylaxis in IFD

118. Winston DJ, Bartoni K, Territo MC, Schiller GJ. Efficacy, safety, and breakthrough infections associated with standard long-term posaconazole antifungal prophylaxis in allogeneic stem cell transplantation recipients. Biol Blood Marrow Transplant 2011;17:507-515. 119. Hoenigl M, Raggam RB, Salzer HJ, Valentin T, Valentin A, Zollner-Schwetz I, Strohmeier AT, Seeber K, WĂślfler A, Sill H, Krause R. Posaconazole plasma concentrations and invasive mould infections in patients with haematological malignancies. Int J Antimicrob Agents 2012;39:510-513.

Turk J Hematol 2015;32:100-117

121. Girmenia C, Barosi G, Piciocchi A, Arcese W, Aversa F, Bacigalupo A, Bandini G, Bosi A, Busca A, Castagnola E, Caselli D, Cesaro S, Ciceri F, Locasciulli A, Locatelli F, Mikulska M, Pagano L, Prete A, Raiola AM, Rambaldi A. Primary prophylaxis of invasive fungal diseases in allogeneic stem cell transplantation: revised recommendations from a consensus process by Gruppo Italiano Trapianto Midollo Osseo (GITMO). Biol Blood Marrow Transplant 2014:20;1080-1088.

120. Ziakas PD, Kourbeti IS, Mylonakis E. Systemic antifungal prophylaxis after hematopoietic stem cell transplantation: a meta-analysis. Clin Ther 2014;36:292-306.e1.

117


Research Article

DOI: 10.4274/tjh.2013.0381

A Novel Natural Product, KL-21, Inhibits Proliferation and Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Yeni Doğal Bir Ürün Olan KL-21 Kronik Lenfositik Lösemi Hücrelerinin Çoğalmasını İnhibe Etmekte ve Apoptozu İndüklemektedir Aysun Adan Gökbulut1, Mustafa Yaşar2, Yusuf Baran1,3 1İzmir

Institute of Technology Faculty of Science, Department of Molecular Biology and Genetics, İzmir, Turkey Natural Products, İzmir, Turkey 3Abdullah Gül University Faculty of Life and Natural Sciences, Kayseri, Turkey 2Naturin

Abstract: Objective: The aims of this study were to examine the cytotoxic and apoptotic effects of KL-21, a novel plant product (produced by Naturin Natural Products, İzmir, Turkey), on 232B4 chronic lymphocytic leukemia (CLL) cells and to determine the cytotoxic effects on healthy BEAS-2B human bronchial epithelial cells.

Materials and Methods: The cytotoxic effect of KL-21 was determined by MTT cell proliferation assay. Changes in caspase-3 enzyme activity were measured using the caspase-3 colorimetric assay. Changes in mitochondrial membrane potential were determined using the JC-1 dye-based method. Annexin V-FITC/PI double staining was performed to measure the apoptotic cell population. Effects of KL-21 on cell cycle profiles of CLL cells were investigated by flow cytometry.

Results: We detected time- and concentration-dependent increases in the cytotoxic effect of KL-21 on 232B4 CLL cells. However, we also showed that, especially at higher concentrations, KL-21 was less cytotoxic towards BEAS-2B healthy cells than towards CLL cells. Annexin-V/PI double staining results showed that the apoptotic cell population increased in 232B4 cells. Increasing concentrations of KL-21 increased caspase-3 enzyme activity and induced loss of mitochondrial membrane potential. KL-21 administration resulted in small increases in the percentage of the cells in the G0/G1 phase while it decreased the S phase cell population up to 1 mg/mL. At the highest concentration, most of the cells accumulated in the G0/G1 phase.

Conclusion: KL-21 has a growth-inhibitory effect on 232B4 CLL cells. KL-21 causes apoptosis and cell cycle arrest at G0/G1. Key Words: Apoptosis, Cell cycle arrest, Chronic lymphocytic leukemia, KL-21

Address for Correspondence: Yusuf BARAN, M.D., İzmir Institute of Technology Faculty of Science, Department of Molecular Biology and Genetics, İzmir, Turkey Phone: +90 232 750 73 15 E-mail: ybaran@gmail.com Received/Geliş tarihi : November 11, 2013 Accepted/Kabul tarihi : April 11, 2014

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Özet: Amaç: Bu çalışmanın amacı yeni bitkisel bir ürün olan KL-21’in [Naturin doğal ürünler şirketi (İzmir, Türkiye) tarafından üretilen] 232B4 kronik lenfostik lösemi (KLL) hücreleri üzerindeki sitotoksik ve apoptotik etkilerinin araştırılmasıdır. Ayrıca, KL-21’in BEAS-2B sağlıklı insan bronşial epitelyum hücreleri üzerindeki sitotoksik etkisine de bakılmıştır. Gereç ve Yöntemler: KL-21’in sitotoksik etkisine MTT hücre çoğalma testiyle bakılmıştır. Kaspaz-3 enzim aktivitesindeki ve mitokondri membran potansiyelindeki değişimlere sırasıyla kaspaz-3 kolorimetrik testi ve JC-1 boyasına dayalı bir yöntem kullanılarak bakılmıştır. Apoptotik hücre popülasyonunu belirlemek amacıyla Anneksin 5-FITC/PI ikili boyama yöntemi kullanılmıştır. KL-21’in KLL hücrelerinin hücre siklusu üzerindeki etkilerine akım sitometresi ile bakılmıştır.

Bulgular: KL-21’in KLL hücrelerinin çoğalması üzerine etkisi zamana ve doza bağımlı olarak artmıştır. Bununlar beraber, KL21’in özellikle yüksek konsantrasyonlarda KLL hücreleri ile karşılaştırıldığında BEAS-2B hücreleri üzerinde daha az sitotoksik etki gösterdiği saptanmıştır. Anneksin-5/PI ikili boyaması 232B4 hücrelerinde apoptotik hücre popülasyonunun arttığını göstermiştir. KL-21’in artan konsantrayonları kaspaz-3 enzim aktivitesini arttırmış ve mitokondri membran potansiyelindeki kayıpları indüklemiştir. KL-21 1 mg/ml konsantrasyonuna kadar G0/G1 fazındaki hücrelerin yüzdesinde küçük artışlara ve S fazındaki hücre popülasyonunda ise azalmalara neden olmaktadır. En yüksek konsantrasyonda ise hücrelerin büyük bir çoğunluğu G0/G1 fazında birikmiştir.

Sonuç: Elde edilen sonuçlar, KL-21’in 232B4 KLL hücreleri üzerinde büyümeyi inhibe edici bir etkisi olduğunu göstermiştir. Ayrıca, K-21 apoptozu indüklemekte ve G0/G1 fazında hücre siklusunun tutulumuna neden olmaktadır.

Anahtar Sözcükler: Apoptoz, Hücre siklusu tutulması, Kronik lenfositik lösemi, KL-21

Introduction Chronic lymphocytic leukemia (CLL) is a monoclonal disorder characterized by an increase in the number of functionally deficient mature CD5+ B lymphocytes in the blood, bone marrow, lymph nodes, and spleen [1,2]. CLL is most commonly observed in Western countries and affects mainly older individuals [3,4]. The main purposes of CLL treatment are to reduce cancer progression and induce apoptosis while providing quality of life for patients. Treatment approaches for CLL include chemotherapy, radiotherapy, immunotherapy, and bone marrow transplantation (BMT) with high-dose chemotherapy [4,5]. Among these treatment options, purine analogs such as fludarabine and cladribine, alkylating agents like chlorambucil, and alkylating agent/ anthracycline combinations are frequently used [6]. Rituximab, a chimeric anti-CD20 monoclonal antibody, and alemtuzumab, a monoclonal antibody against the CD52 antigen, have received attention in the treatment of CLL due to their increased specificity as compared to chemotherapy [7,8]. BMT has been reported to decrease mortality when it is applied early in the course of the disease [9]. However, BMT, especially allogeneic BMT, is not considered as an appropriate regimen for CLL patients since most CLL patients are older than 55 years [10,11]. Although all the methods used in the treatment of CLL are effective, none of the standard therapies are capable of completely eliminating CLL. On the other hand, these methods have distinct side effects in the

treatment of CLL [5]. Moreover, CLL remains an incurable disease with conventional therapies due to development of relapse or refraction [12]. For these reasons, it is necessary to improve the treatments for relapsed or refractory CLL or to develop new therapeutic agents that are less toxic and more effective for the complete elimination of CLL cells. Use of plant-derived substances such as resveratrol and quercetin has been indicated in several studies for CLL treatment, either alone or in combination with other agents [13,14]. KL-21 is a novel agent of plant origin produced by Naturin Natural Products (İzmir, Turkey) for the treatment of CLL. Ethical committee approved this study. In the present study, we aimed to investigate the possible cytotoxic and apoptotic effects of KL-21 on 232B4 CLL cells. Materials and Methods Cell Lines and Culture Conditions and Chemicals Human 232B4 CLL cells were kindly provided by Prof. Dr. Ander Rosen from Linköping University, Sweden. Healthy human BEAS-2B epithelial cells were obtained from Assist. Prof. Dr. Ali Çağır from İzmir Institute of Technology and were used as a positive control. The cells were grown and maintained in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in 5% CO2. A 10 mg/mL stock solution of KL-21 was prepared in DMSO and stored at -20 °C. The final concentration of DMSO did not exceed more than 0.1% in culture. KL-21 is a novel plant extract containing extracts from 21 plant species in different amounts. KL-21 is

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a mixture of Achillea millefolium (8.47%), Juniperus communis (7.62%), Thymus vulgaris (6.78%), Peganum harmala (6.78%), Curcuma longa (6.78%), Silybum marianum (6.78%), Urtica dioica (6.77%), Equisetum arvense (5.93%), Lavandula stoechas (5.93%), Zingiber officinale (5.08%), Fumaria officinalis (4.24%), Taraxacum officinale (4.24%), Rosmarinus officinalis (4.23%), Nigella sativa (4.23%), Cichorium endivia (3.39%), Viscum album (2.54%), Solidago virgaurea (2.54%), Hypericum perforatum (2.54%), Acorus calamus (1.69%), Valeriana officinalis (1.69%), and Melissa officinalis (1.69%). Measurement of Cell Growth by MTT Assay Antiproliferative effects of KL-21 were determined by the MTT cell proliferation assay as described. The IC50 value (the drug concentration that inhibits cell growth by 50%) of KL-21 was calculated according to cell proliferation plots [15]. Annexin-V/PI Double Staining We determined the translocation of phosphatidylserine from the inner membrane to the outer cell membrane in order to examine the apoptotic effects of KL-21 on CLL cells. Initially, 1x106 cells were treated with increasing concentrations of KL21 (0.001 to 1 mg/mL) for 48 h. After incubation, the cells were washed twice with cold phosphate buffered saline (PBS) and then homogenized with 1 mL of 1X binding buffer, and then 100 µL of this solution was added into glass tubes. Next, 5 µL of FITC annexin V and 5 µL of propidium iodide (PI) were added to the cell solutions. These samples were vortexed gently and then incubated for 15 min at room temperature in the dark. Afterwards, 400 µL of 1X binding buffer was added to each tube, and samples were analyzed by flow cytometry (BD FACSCanto Flow Cytometer, Belgium) within 1 h. Analysis of the Changes in Mitochondrial Membrane Potential We examined the loss of mitochondrial membrane potential (MMP) in response to KL-21 in 232B4 cells with the JC-1 Mitochondrial Membrane Potential Detection Kit (Cayman Chemicals, USA). Briefly, the cells (1x106 cells/2 mL), induced to undergo apoptosis, were collected by centrifugation at 180 x g for 10 min. Supernatants were removed and pellets were homogenized with 300 µL of medium, and then 30 µL of JC-1 dye was added to the cells and the cells were incubated at 37 °C in 5% CO2 for 30 min. They were centrifuged at 400 x g for 5 min, supernatants were removed, and 200 µL of assay buffer was added to the pellets and vortexed. This step was then repeated. Afterwards, all pellets were homogenized with 320 µL of assay buffer and 100 µL from each sample was added to a 96-well plate as triplicates. In healthy cells, the aggregate red form has absorption/emission maxima of 560/595 nm, whereas in apoptotic cells, the monomeric green form has absorption/emission maxima of 485/535 nm. The plate was read in these wavelengths with a fluorescence ELISA

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reader (Thermo Varioskan Spectrum, Finland). The ratio of fluorescent intensity of JC-1 monomers to fluorescent intensity of JC-1 aggregates was calculated for each concentration as well as the untreated control sample. Relative changes in cytoplasmic/mitochondrial JC-1 were then determined [16]. Analysis of Caspase-3 Activity Changes in caspase-3 activity of the cells were examined with a caspase-3 colorimetric assay kit (BioVision Research Products, USA). In short, the cells (1x106 cells/2 mL/well), induced to undergo apoptosis by KL-21, were collected by centrifugation at 180 x g for 10 min. The cells were lysed by adding 50 µL of chilled Cell Lysis Buffer and incubated on ice for 10 min before centrifugation at 10,000 x g for 1 min. Supernatants were transferred to new Eppendorf tubes, and the reaction mixture was prepared in 96-well plates by adding 50 µL of 2X reaction buffer (containing 10 mM DTT), 50 µL of sample, and 5 µL of DEVD-pNA substrate and was incubated for 2 h at 37 °C in a CO2 incubator. At the end of this period, the plate was read under 405 nm wavelengths with an ELISA reader. The absorbance values were normalized to protein concentrations determined by the Bradford assay. Cell Cycle Analysis This technique is based on the determination of amounts of dsDNA by using PI and flow cytometry. Briefly, 1x106 cells/2 mL were treated with increasing concentrations of KL21 for 48 h. After the incubation period, cells were collected by centrifugation at 260 x g for 10 min. Supernatants were removed and pellets were homogenized with 1 mL of cold PBS, and then the samples were put on ice. Afterwards, while the cells were slightly vortexed, 4 mL of cold ethanol was added to these cells, and then the mixture was put on ice. Cells fixed by this method were incubated overnight at -20 °C for analysis. The next day, the cells were centrifuged at 260 x g for 10 min, and supernatants were completely removed from the pellets. Pellets were homogenized with 1 mL of cold PBS and were centrifuged again at 260 x g for 10 min. Afterwards, cell pellets were homogenized with 1 mL of PBS containing 0.1% Triton X-100; 100 µL of RNase A (200 µg/mL) was then added to these cells and the mixture was incubated at 37 °C for 30 min. After this incubation period, 100 µL of PI (1 mg/ mL) was added to the cells. These cells were incubated at room temperature for 15 min and were then analyzed by flow cytometry. Statistical Analysis Statistical significance was determined via GraphPad Prism 6.0 software using 1-way analysis of variance (ANOVA) for MTT analyses and 2-way ANOVA for annexin V, MMP, caspase-3 activity and cell cycle analyses. P<0.05 was considered to be significant. MTT, annexin V, MMP, caspase-3, and cell cycle analysis results were shown as the means of 3 independent experiments (N).


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Results Cytotoxic Effects of KL-21 on 232B4 CLL and BEAS-2B Epithelial Cells There were decreases in the viability/proliferation of 232B4 CLL cells in a dose- and time-dependent manner (Figure 1A), while KL-21 had very little or no effect on the viability of BEAS-2B healthy cells at up to 0.1 mg/mL (Figure 1B). Although KL-21 was cytotoxic toward healthy control cells at higher concentrations (0.5 and 1 mg/mL), more than 20% of cells were alive at indicated time points. IC50 values of KL-21 for 232B4 cells at 24, 48, and 72 h were calculated from cell proliferation plots and were found to be 0.2, 0.1, and 0.08 mg/mL, respectively. On the other hand, IC50 values of KL-21 for BEAS-2B cells were found to remain at about 0.4 mg/mL at each time point. These findings indicated that CLL cells are more susceptible than BEAS-2B cells to the growth inhibitory effects of KL-21. KL-21 Induced Apoptosis in a Dose-Dependent Manner in 232B4 Cells The percentage of the apoptotic cell population (late apoptotic plus early apoptotic) of 232B4 cells was determined by flow cytometry. There were 1.16-, 1.23-, 1.28-, 1.77-, and 3.23-fold increases in percentage of apoptotic cells treated

with 0.001, 0.01, 0.05, 0.1, and 1 mg/mL KL-21, respectively, when compared to untreated controls, as shown in Figures 2A and 2B. The data showed that KL-21 triggers apoptosis in a dose-dependent manner. KL-21 Induced Loss of Mitochondrial Membrane Potential in 232B4 Cells There were 1.31-, 1.45-, 3.6-, 4.12-, and 14.4-fold increases in loss of MMP in 232B4 cells treated with 0.001, 0.01, 0.05, 0.1, and 1 mg/mL KL-21, respectively, as compared to untreated control cells (Figure 3). KL-21 Increased Caspase-3 Activity in 232B4 CLL Cells There were 1.02-, 1.03-, 1.23-, 1.52-, and 2.12-fold increases in caspase-3 enzyme activity in response to 0.001, 0.01, 0.05, 0.1, and 1 mg/mL KL-21, respectively, as compared to untreated controls (Figure 4). KL-21 Induced G0/G1 Arrest in 232B4 Cells In order to determine the possible mechanism of antiproliferative activity of KL-21, cell cycle progression of

A

A

B

BB

Figure 1. Cytotoxic effects of KL-21 on human 232B4 CLL cells (A) and BEAS-2B healthy cells (B) with statistical analysis. The IC50 value of KL-21 was calculated from cell proliferation plots. The results are the means of 3 independent experiments. The error bars represent the standard deviations. Statistical significance was determined using 1-way analysis of variance and p<0.05 was considered to be significant.

Figure 2. Evaluation of apoptosis in 232B4 cells induced by KL-21. The percentage of cells undergoing early and late apoptosis in a dose-dependent manner as compared to the control and flow cytometry analysis via annexin V-FITC/PI staining is shown (A and B). Cells in the lower right quadrant are annexin-positive/PI-negative early apoptotic cells. The cells in the upper right quadrant are annexin-positive/PIpositive late apoptotic cells. The percentage of cells annexin V-positive, PI-positive, or double positive for both annexin V and PI is indicated. The results are the means of 3 independent experiments.

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232B4 cells was examined by flow cytometry in the presence of DNase-free RNase and PI dye. As summarized in Figures 5A and 5B, treatment of 232B4 cells with KL-21 resulted in small increases in the percentage of cells in the G0/G1 phase at 0.001 to 0.1 mg/mL, but there was a significant increase in response to 1 mg/mL KL-21. Discussion CLL is an adult leukemia characterized by accumulation of malignant B cells in several parts of the body. Despite the presence of many therapeutic regimens, CLL is still an incurable disorder [2]. CLL can be effectively treated with various agents; however, these strategies have their own side effects and some patients have limited therapeutic options [5,17]. Therefore, there is a need to discover novel drugs or agents for CLL treatment.

We conducted this study to examine the cytotoxic and apoptotic effects of KL-21, a novel plant-derived product, on 232B4 CLL cells. KL-21 decreased proliferation of 232B4 CLL cells in a dose- and time-dependent manner. Moreover, we found that KL-21 did not have an antiproliferative effect on BEAS-2B cells at concentrations between 0.001 and 0.1 mg/ mL. Although KL-21 was cytotoxic towards healthy cells, CLL cells were more susceptible to the cytotoxic effects of 0.5 and 1 mg/mL KL-21. On the other hand, annexin-V/PI double staining showed dose-dependent increases in the apoptotic cell population in response to KL-21 when compared to untreated controls. To elucidate the molecular mechanism of KL-21induced apoptosis in CLL cells, we first checked the effect of KL-21 on MMP. The results revealed that KL-21 caused loss of MMP in a dose-dependent manner. In the literature, it is very well established that alterations in the structure and function of mitochondria play an important role in caspase-dependent apoptosis. Caspase-3, the executioner caspase, functions in the last step of caspase-mediated apoptosis. Therefore, we next checked the activation of caspase-3 enzyme in KL21-treated 232B4 cells. Our data demonstrated that KL-21 increased caspase-3 activity in a dose-dependent manner when compared to untreated controls. Taken together, we can conclude that KL-21 induces apoptosis in 232B4 cells through the loss of MMP and caspase-3 activation. We also investigated A

Figure 3. Percent changes in cytoplasmic/mitochondrial JC-1 in CLL cells treated with increasing concentrations of KL21. The results are the means of 3 independent experiments. The error bars represent the standard deviations. Statistical significance was determined using 2-way analysis of variance and p<0.05 was considered to be significant. B

Figure 4. Changes in caspase-3 enzyme activity in response to KL-21. The results are the means of 3 independent experiments. The error bars represent the standard deviations. Statistical significance was determined using 2-way analysis of variance and p<0.05 was considered to be significant.

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Figure 5. Effect of KL-21 on cell cycle progression. The percentage of cells in cell cycle phases are shown in the graph (A) and in quadrants (B). The results represent 3 independent experiments. Statistical significance was determined using 2-way analysis of variance and p<0.05 was considered to be significant.


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Table 1. Botanical data of plant species included in KL-21. Plant Name

Traditional and/or Medicinal Use

Active Constituents of the Plant

References

Achillea millefolium

Breast and liver cancer, hardness of uterus, warts Flavonoids, sesquiterpenoids

[21]

Urtica dioica

Diabetes, rheumatism, eczema, anemia, hair loss, Steroids, terpenoids, flavonol prostatic hyperplasia glycosides

[22]

Equisetum arvense

Bleeding from the intestine or hemorrhoids, growth-inhibitory effects on melanoma

Alkaloids, flavonoids, phenolics, tannin

[23,24]

Thymus vulgaris

Respiratory and gastrointestinal diseases

Mono- and diterpenoids, flavonoids, biphenyl compounds

[25]

Viscum album

Used as complementary medicine for solid tumors

Mistletoe lectins, viscotoxins, flavonoids, triterpene acids

[26,27]

Acorus calamus

Epilepsy, chronic diarrhea, dysentery, abdominal tumors

Sesquiterpenes and their alcohols, flavonoids, choline

[28]

Solidago virgaurea

Urinary tract, nephrolithiasis, prostate cancer

Saponins, α-tocopherol quinone, trans-phytol

[29,30]

Silybum marianum

Diabetes mellitus, hepatic disorders

Silibinin, silymarin

[31,32]

Curcuma longa

Cough, diabetic wounds, hepatic disorders, rheumatism, sinusitis, anticarcinogen

Curcumin [polyphenol], an essential oil

[33]

Rosmarinus officinalis Renal colic, dysmenorrhea, respiratory disorders  Rosmarinic acid, caffeic acid, rosmanol, different diterpenes

[34]

Lavandula stoechas

Colic and chest affections, nervous headache, biliousness, wound healing

Essential oils

[35]

Fumaria officinalis

Hepatobiliary diseases

Phenolics, alkaloids

[36,37]

Taraxacum officinale

Breast, uterine, lung tumors; hepatitis, digestive diseases

Luteolin, luteolin-7-glucoside

[38,39]

Cichorium endivia

Liver diseases

Kaempferol, kaempferol3-O-β-D-glucoside, sesquiterpenes

[40]

Zingiber officinale

Arthritis, migraine, diabetes, nausea

Gingerols, sesquiterpenes

[41]

Peganum harmala

Skin, subcutaneous tumors

Alkaloids [harmine, harmaline, harman]

[42,43]

Juniperus communis

Dyspepsia, bladder and kidney diseases

Terpinen-4-ol, sesquiterpenes

[44]

Nigella sativa

Asthma, diarrhea, dyslipidemia

Thymoquinone, alkaloids, saponin

[45]

Hypericum perforatum

Skin wounds, eczema, burns, depression

Hypericins, hyperforins, flavonoids

[46]

Valeriana officinalis

Insomnia

Valepotriates, monoterpenes, sesquiterpenes

[47]

Melissa officinalis

Used as a mild sedative, spasmolytic, antibacterial agent

Eugenol, tannin, terpenes

[48]

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the cytostatic property of KL-21 on CLL cells and observed that the treatment of CLL cells with KL-21 resulted in G0/ G1 phase arrest of cell cycle progression, especially at high concentrations. Cells may either undergo repair or enter the apoptotic pathway following the G1 phase of the cell cycle to eliminate mutated or neoplastic cells. In the present study, we observed that CLL cells undergo significant apoptosis in response to KL-21 treatment. Therefore, it can be concluded that, in addition to the mitochondrial pathway of apoptosis, G1 phase arrest may be another mechanism of apoptosis in these cells, especially at high concentrations of KL-21. By our group and some other groups it was reported that many plant-derived products have been shown to induce apoptosis in CLL cells. Honokiol, a plant product, triggers apoptosis in CLL cells via activation of caspase-3, -8, and -9, and apoptosis was further evaluated by annexin-V/PI double staining [18]. Gokbulut et al. showed that resveratrol and quercetin might block CLL growth by inducing apoptosis and cell cycle arrest [19]. The growth inhibition and induction of apoptosis in CLL cells treated with flavopiridol, which is isolated from the Indian plant Dysoxylum binectariferum, has been related to the downregulation of Bcl-2 [20]. KL-21 is a novel plant extract that is the herbal combination of 21 plant species at different percentages as described above. These plant species have been widely used in traditional medicine all over the world for the treatment of different diseases and some types of cancer. Most of them possess antiinflammatory, antiviral, antibacterial, antioxidant, and anticarcinogenic properties. Detailed information about the ingredients of KL-21 and their use in medicine is given in Table 1. As summarized in Table 1, terpenoids, flavonoids, and their derivatives are the main effective components of KL-21, similar to several plant-derived medicinal extracts. The main mechanisms of flavonoids include induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis [49]. Terpenoids possess anticarcinogenic activities and their mechanisms of action include inhibition of NF-κB signaling, induction of apoptosis, and cell cycle arrest [50,51]. Other ingredients are generally plant-specific and their effects have been explained in several studies. For instance, the antitumor activity of hypericin from Hypericum perforatum has been shown to be related to the release of cytochrome c, activation of caspase-3, and partial inhibition of protein kinase C [52,53]. In conclusion, we demonstrated that KL-21 has growthinhibitory effects on 232B4 CLL cells. KL-21 causes apoptosis and cell cycle arrest at G0/G1. While the complete mechanism of apoptosis needs to be elucidated, we have shown that KL-21 induced apoptosis by the mitochondrial/caspase-3-dependent pathway and the inhibition of cell cycle progression through the G0/G1 phase. Therefore, given its active plant constituents, the results of this study suggest that KL-21 could be a novel promising natural agent for the treatment of CLL. 124

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Acknowledgments We thank Prof. Dr. Anne Frary for critically reviewing the manuscript and the staff of the Biotechnology and Bioengineering Center of İzmir Institute of Technology for their help and technical support. Conflict of Interest Statement Dr. Yusuf Baran was on the advisory board of Naturin Natural Products from 15.09.2011 to 15.01.2012. He designed and conducted the experiments for the examination of effects of natural products on cancer and healthy cells. References 1. Chiorazzi N, Ferrarini M. B cell chronic lymphocytic leukemia: lessons learned from studies of the B cell antigen receptor. Annu Rev Immunol 2003;21:841-894. 2. Caligaris-Cappio F, Ghia P. Novel insights in chronic lymphocytic leukemia: are we getting closer to understanding the pathogenesis of the disease? J Clin Oncol 2008;26:44974503. 3. Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med 1995;333:1052-1057. 4. Veliz M, Pinilla-Ibarz J. Role of ofatumumab in treatment of chronic lymphocytic leukemia. J Blood Med 2011;2:71-77. 5. Byrd JC, Stilgenbauer S, Flinn IW. Chronic lymphocytic leukemia. Hematology 2004;2004:163-183. 6. Flinn IW, Grever MR. Chronic lymphocytic leukemia. Cancer Treat Rev 1996;96:1-13. 7. Hainsworth JD, Litchy S, Barton JH, Houston GA, Hermann RC, Bradof JE, Greco FA; Minnie Pearl Cancer Research Network. Single-agent rituximab as first-line and maintenance treatment for patients with chronic lymphocytic leukemia or small lymphocytic lymphoma: a phase II trial of the Minnie Pearl Cancer Research Network. J Clin Oncol 2003;21:17461751. 8. O’Brien SM, Kantarjian HM, Thomas DA, Cortes J, Giles FJ, Wierda WG, Koller CA, Ferrajoli A, Browning M, Lerner S, Albitar M, Keating MJ. Alemtuzumab as treatment for residual disease after chemotherapy in patients with chronic lymphocytic leukemia. Cancer 2003;98:2657-2663. 9. Dreger P, Montserrat E. Autologous and allogeneic stem cell transplantation for chronic lymphocytic leukemia. Leukemia 2002;16:985-992. 10. Esteve J, Villamor N, Colomer D, Cervantes F, Campo E, Carreras E, Montserrat E. Stem cell transplantation for chronic lymphocytic leukemia: different outcome after autologous and allogeneic transplantation and correlation with minimal residual disease status. Leukemia 2001;5:445-451.


Gökbulut AA, et al: KL-21 for Treatment of CLL

11. Berdeja JG, Jones RJ, Zahurak ML, Piantadosi S, Abrams RA, Borowitz MJ, Vogelsang GB, Noga SJ, Ambinder RF, Flinn IW. Allogeneic bone marrow transplantation in patients with sensitive low-grade lymphoma or mantle cell lymphoma. Biol Blood Marrow Transplant 2001;7:561-567. 12. Veliz M, Pinilla-Ibarz J. Treatment of relapsed or refractory chronic lymphocytic leukemia. Cancer Control 2012;19:3753. 13. Podhorecka M, Halicka D, Klimek P, Kowal M, Chocholska S, Dmoszynska A. Resveratrol increases rate of apoptosis caused by purine analogues in malignant lymphocytes of chronic lymphocytic leukemia. Ann Hematol 2011;90:173-183. 14. Russo M, Spagnuolo C, Volpe S, Mupo A, Tedesco I, Russo GL. Quercetin induced apoptosis in association with death receptors and fludarabine in cells isolated from chronic lymphocytic leukaemia patients. Br J Cancer 2010;103:642648. 15. Baran Y, Bielawski J, Gunduz U, Ogretmen B. Targeting glucosylceramide synthase sensitizes imatinib-resistant chronic myeloid leukemia cells via endogenous ceramide accumulation. J Cancer Res Clin Oncol 2011;137:1535-1544. 16. Avcı ÇB, Gündüz C, Baran Y, Şahin F, Yılmaz S, Dogan ZO, Saydam G. Caffeic acid phenethyl ester triggers apoptosis through induction of loss of mitochondrial membrane potential in CCRF-CEM cells. J Cancer Res Clin Oncol 2011;137:41-47. 17. Brown JR. The treatment of relapsed refractory chronic lymphocytic leukemia. Hematology Am Soc Hematol Educ Program 2011;2011:110-118. 18. Battle TE, Arbiser J, Frank DA. The natural product honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Blood 2005;106:690697. 19. Gokbulut AA, Apohan E, Baran Y. Resveratrol and quercetininduced apoptosis of human 232B4 chronic lymphocytic leukemia cells by activation of caspase-3 and cell cycle arrest. Hematology 2013;18:144-150. 20. König A, Schwartz GK, Mohammad RM, Al-Katib R, Gabrilove JL. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 1997;90:4307-4312. 21. Csupor-Löffler B, Hajdú Z, Zupkó I, Réthy B, Falkay G, Forgo P, Hohmann J. Antiproliferative effect of flavonoids and sesquiterpenoids from Achillea millefolium s.l. on cultured human tumour cell lines. Phytother Res 2009;23:672-676. 22. Akbay P, Basaran AA , Undeger U, Basaran N. In vitro immunomodulatory activity of flavonoid glycosides from Urtica dioica L. Phytother Res 2003;17:34-37. 23. Yoshinobu Y. Antitumor activity of crude protein extracted from Equisetum arvense LINN’E. Journal of Analytical BioScience 1992;22:421-424.

Turk J Hematol 2015;32:118-126

24. Asgarpanah J, Roohi E. Phytochemistry and pharmacological properties of Equisetum arvense L. J Med Plants Res 2012;6:3689-3693. 25. Jimenez-Arellanes A, Martinez R, García R, Leon-Diaz R, Luna-Herrera J, Molina-Salinas G, Said-Fernández S. Thymus vulgaris as a potencial source of antituberculous compounds. Pharmacologyonline 2006;3:569-574. 26. Kienle G. Influence of Viscum album L (European mistletoe) extracts on quality of life in cancer patients: a systematic review of controlled clinical studies. Integr Cancer Ther 2010;9:142-157. 27. Sabova L, Pilátová M, Szilagyi K, Sabo R, Mojzis J. Cytotoxic effect of mistletoe (Viscum album L.) extract on Jurkat cells and its interaction with doxorubicin. Phytother Res 2010;24:365368. 28. Singh R, Sharma PK, Malviya R. Pharmacological properties and ayurvedic value of Indian buch plant (Acorus calamus): a short review. Advances in Biological Research 2011;5:145-154. 29. Demir H, Açık L, Bali EB, Koç LY, Kaynak G. Antioxidant and antimicrobial activities of Solidago virgaurea extracts. Afr J Biotechnol 2009;8:274-279. 30. Gross SC, Goodarzi G, Watabe M, Bandyopadhyay S, Pai SK, Watabe K. Antineoplastic activity of Solidago virgaurea on prostatic tumor cells in an SCID mouse model. Nutr Cancer 2002;43:76-81. 31. Davis-Searles PR, Nakanishi Y, Kim NC, Graf TN, Oberlies NH, Wani MC, Wall ME, Agarwal R, Kroll DJ. Milk thistle and prostate cancer: differential effects of pure flavonolignans from Silybum marianum on antiproliferative end points in human prostate carcinoma cells. Cancer Res 2005;65:44484457. 32. Huseini HF, Larijani B, Heshmat R, Fakhrzadeh H, Radjabipour B, Toliat T, Raza M. The efficacy of Silybum marianum (L.) Gaertn. (silymarin) in the treatment of type II diabetes: a randomized, double-blind, placebo-controlled, clinical trial. Phytother Res 2006;20:1036-1039. 33. Chattopadhyay I, Biswas K, Bandyopadhyay U, Banerjee RK. Turmeric and curcumin: biological actions and medicinal applications. Curr Sci India 2004;87:44-53. 34. Al-Sereitia MR, Abu-Amerb KM, Sena P. Pharmacology of rosemary (Rosmarinus officinalis Linn.) and its therapeutic potentials. Indian J Exp Biol 1999;37:124-131. 35. Gören AC, Topçu G, Bilsel G, Bilsel M, Aydoğmus Z, Pezzuto JM. The chemical constituents and biological activity of essential oil of Lavandula stoechas ssp. stoechas. Z Naturforsch 2002;57c:797-800. 36. Neves JM, Matos C, Moutinho C, Queiroz E, Gomes LR. Ethnopharmacological notes about ancient uses of medicinal plants in Tras-os-Montes (northern of Portugal). J Ethnopharmacol 2009;124:270-283.

125


Turk J Hematol 2015;32:118-126

37. Manske RHF. The alkaloids of fumariaceous plants: xviii. Fumaria officinalis L. Can J Res 1938;16:438-444. 38. Sigstedt SC, Hooten CJ, Callewaert MC, Jenkins AR, Romero AE, Pullin MJ, Kornienko A, Lowrey TK, Slambrouck SV, Steelant WF. Evaluation of aqueous extracts of Taraxacum officinale on growth and invasion of breast and prostate cancer cells. Int J Oncol 2008;32:1085-1090. 39. Hu C, Kitts DD. Antioxidant, prooxidant, and cytotoxic activities of solvent-fractionated dandelion (Taraxacum officinale) flower extracts in vitro. J. Agric Food Chem 2003;51:301-310. 40. Chen CJ, Deng AJ, Liu C, Shi R, Qin HL, Wang AP. Hepatoprotective activity of Cichorium endivia L. extract and its chemical constituents. Molecules 2011;16:9049-9066.

GĂśkbulut AA, et al: KL-21 for Treatment of CLL

46. Silva BA, Ferreres F, Malva JO, Dias ACP. Phytochemical and antioxidant characterization of Hypericum perforatum alcoholic extracts. Food Chem 2005;90:157-167. 47. Lefebvre T, Foster BC, Drouin CE, Krantis A, Livesey JF, Jordan SA. In vitro activity of commercial valerian root extracts against human cytochrome P450 3A4. J Pharm Pharmaceut Sci 2004;72:265-273. 48. Akhondzadeh S, Noroozian M, Mohammadi M, Ohadinia S, Jamshidi A, Khani M. Melissa officinalis extract in the treatment of patients with mild to moderate Alzheimerâ&#x20AC;&#x2122;s disease: a double blind, randomised, placebo controlled trial. J Neurol Neurosurg Psychiatry 2003;74:863-866. 49. Ren W, Qiao Z, Wang H, Zhu L, Zhang L. Flavonoids: promising anticancer agents. Med Res Rev 2003;23:519-534.

41. Al-Amin ZM, Thomson M, Al-Qattan KK, Peltonen-Shalaby R, Ali M. Anti-diabetic and hypolipidaemic properties of ginger (Zingiber officinale) in streptozotocin-induced diabetic rats. Br J Nutr 2006;96:660-666.

50. Salminen A, Lehtonen M, Suuronen T, Kaarniranta K, Huuskonen J. Terpenoids: natural inhibitors of NF-B signaling with anti-inflammatory and anticancer potential. Cell Mol Life Sci 2008;65:2979-2999.

42. Mirzaei M. Treatment of natural tropical theileriosis with the extract of the plant Peganum harmala. Korean J Parasitol 2007;45:267-271.

51. Rabi T, Bishayee A. Terpenoids and breast cancer chemoprevention. Breast Cancer Res Treat 2009;115:223239.

43. Lamchouri F, Settaf A, Cherrah Y, Zemzami M, Lyoussi B, Zaid A, Atif N, Hassar M. Antitumour principles from Peganum harmala seeds. Therapie 1999;54:753-758.

52. Vandenbogaerde AL, de Witte PA. Hypericin as a natural photosensitizer with cytotoxic and antitumor effects. Phytother Res 1996;10:S150-S152.

44. Pepeljnjak S, Kosalec I, Kalodera Z, Blazevic N. Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae). Acta Pharm 2005;55:417-422.

53. Roscetti G, Franzese O, Comandini A, Bonmassar E. Cytotoxic activity of Hypericum perforatum L. on K562 erythroleukemic cells: differential effects between methanolic extract and hypericin. Phytother Res 2004;18:66-72.

45. Ali BH, Blunden G. Pharmacological and toxicological properties of Nigella sativa. Phytother Res 2003;17:299-305.

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DOI: 10.4274/tjh.2013.0296

Research Article

A Possible Role for WNT5A Hypermethylation in Pediatric Acute Lymphoblastic Leukemia WNT5A Hipermetilasyonun Çocukluk Çağı Akut Lenfoblastik Lösemideki Olası Rolü Özden Hatırnaz Ng1, Sinem Fırtına1, İsmail Can1, Zeynep Karakaş2, Leyla Ağaoğlu2, Ömer Doğru3, Tiraje Celkan4, Arzu Akçay5, Yıldız Yıldırmak6, Çetin Timur7, Uğur Özbek1, Müge Sayitoğlu1 1İstanbul

University Faculty of Medicine, Institute of Experimental Medicine Research (DETAE), Department of Genetics, İstanbul, Turkey University İstanbul Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey 3Akdeniz University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Antalya, Turkey 4İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey 5Kanuni Sultan Süleyman Research and Training Hospital, Clinic of Pediatric Hematology and Oncology, İstanbul, Turkey 6Şişli Etfal Research and Training Hospital, Clinic of Pediatric Hematology and Oncology, İstanbul, Turkey 7Medeniyet University Faculty of Medicine, Göztepe Research and Training Hospital, Clinic of Pediatric Hematology and Oncology, İstanbul, Turkey 2İstanbul

Abstract: Objective: WNT5A is one of the most studied noncanonical WNT ligands and is shown to be deregulated in different tumor types. Our aim was to clarify whether hypermethylation might be the cause of low WNT5A mRNA levels and whether we could restore this downregulation by reversing the event.

Materials and Methods: The expression of WNT5A mRNA was studied in a large acute lymphoblastic leukemia (ALL) patient group (n=86) by quantitative real-time PCR. The methylation status was detected by methylation-specific PCR (MSPCR) and bisulphate sequencing. In order to determine whether methylation has a direct effect on WNT5A expression, disease-representative cell lines were treated by 5’-aza-20-deoxycytidine. Results: Here we designed a validation experiment of the WNT5A gene, which was previously examined and found to be differentially expressed by microarray study in 31 T-cell ALL patients. The expression levels were confirmed by quantitative real-time PCR and the expression levels were significantly lower in T-cell ALL patients than in control thymic subsets (p=0.007). MSPCR revealed that 86% of the patients were hypermethylated in the WNT5A promoter region. Jurkat and RPMI cell lines were treated with 5’-aza-20-deoxycytidine and WNT5A mRNA expression was restored after treatment.

Conclusion: According to our results, WNT5A hypermethylation does occur in ALL patients and it has a direct effect on mRNA expression. Our findings show that epigenetic changes of WNT signaling can play a role in ALL pathogenesis and reversing methylation might be useful as a possible treatment of leukemia. Key Words: WNT5A, Methylation, Downregulation, Gene expression, ALL

Address for Correspondence: Müge Sayİtoğlu, M.D., İstanbul University Faculty of Medicine, Institute of Experimental Medicine Research (DETAE), Department of Genetics, İstanbul, Turkey Phone: +90 212 414 20 00-33312 E-mail: mugeay@istanbul.edu.tr Received/Geliş tarihi : September 02, 2013 Accepted/Kabul tarihi : May 27, 2014

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Özet: Amaç: WNT5A, kanonik olmayan WNT ligandlarının en çok çalışılanıdır ve farklı tümör tiplerinde fonksiyon bozukluğu gösterdiği bilinmektedir. Amacımız tespit edilen düşük WNT5A mRNA miktarının altında yatan sebebin hiper metilasyon olup olmadığını açıklığa kavuşturmak ve bu düşüşü hiper metilasyonu tersine çevirerek düzeltip düzeltemeyeceğimizi belirlemekti.

Gereç ve Yöntemler: WNT5A mRNA anlatımı, geniş bir ALL hasta gurubunda eş zamanlı kantitatif PZR ile çalışıldı (n=86). Metilasyon durumu metilasyona özgü PZR (MSPZR) ve bisülfit dizileme yöntemleri ile belirlendi. Metilasyonun WNT5A anlatımına doğrudan etkisi olup olmadığı ise hastalık özelliklerini gösteren hücre serilerine 5’-aza-20-deoxycytidine muamelesi ile gösterildi. Bulgular: Bu çalışmada daha önceki çalışmamızda mikro dizi analizi ile belirlenen düşük WNT5A anlatımının doğrulanması için mRNA anlatımı eş zamanlı kantitatif PZR yöntemi ile belirlendi ve T-ALL hastalarında kontrol timositlere göre istatistiki olarak anlamlı bir düşüş gözlendi (p=0,007). MSPZR ise hastaların %86’sında WNT5A geni promotör bölgesinin hiper metile olduğunu gösterdi. Jurkat ve RPMI hücre serileri 5’AZA ile muamele edildi ve WNT5A mRNA anlatımının yeniden arttığı belirlendi. Sonuç: Çalışmamızın sonuçlarına göre ALL hastalarında WNT5A hiper metilasyonu gözlenmektedir ve mRNA anlatımına doğrudan etkisi bulunmaktadır. Sonuçlarımız WNT sinyal ileti yolundaki epigenetik değişikliklerin ALL patogenezinde rol oynadığını göstermektedir ve metilasyonun tersine çevrilmesi lösemiler için olası bir tedavi yöntemi olarak kullanılabilir.

Anahtar Sözcükler: WNT5A, Metilasyon, Azalarak düzenlenme, Gen ekspresyonu, ALL Introduction WNT signaling is a highly conserved pathway that has critical roles in differentiation, proliferation, migration, and hematopoiesis; it is widely studied in normal and malignant development [1]. The WNT pathway is divided into 2 main paths: the canonical, which is dependent on β-catenin, and the noncanonical, which is independent of β-catenin. The molecular mechanism of the canonical WNT pathway has been studied comprehensively, whereas the exact mechanism of the noncanonical WNT pathway remains unclear. Noncanonical pathways are activated by the binding of specific Wnt ligands like Wnt4, Wnt5a, and Wnt11 to Frizzled and Disheveled as in the canonical counterpart with different downstream members. Among all the noncanonical factors, WNT5A is most widely studied, since it was shown to have roles in both canonical and noncanonical WNT pathways. WNT5A was found to be overexpressed in many solid tumors, such as in breast, prostate, colon, and lung cancers [2,3,4,5]. On the contrary, it has been shown that WNT5A is essential for proper development but that hemizygous WNT5A mice could develop normally, except for the fact that some WNT5A hemizygous mice developed B-cell leukemia [6]. Liang et al. showed that WNT5A inhibits the proliferation of B-cells and plays a tumor-suppressing role in hematologic malignancies [7]. The role of canonical WNT signaling was also shown in normal T-cell development [8], but it is not well described in malignant development of T-cell leukemia. In our previous study, we showed that canonical WNT signaling is highly deregulated in T-acute lymphoblastic leukemia (ALL) patients. To evaluate the potential role of WNT signaling in T-cell leukemogenesis, we performed

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expression analysis of key components of the WNT pathway. More than 85% of the childhood T-ALL patients showed upregulated β-catenin expression at the protein level as compared to normal human thymocytes. The impact of this upregulation was reflected in high expression of known target genes (AXIN2, c-MYC, TCF1, and LEF). When the β-CATENIN gene was silenced by small interfering RNA, the cancer cells showed higher rates of apoptosis. These results demonstrate that abnormal WNT signaling activation occurs in a significant fraction of human T-ALL cases independently of known T-ALL risk factors. We concluded that deregulated WNT signaling is a novel oncogenic event in childhood T-ALL [9]. Here we show that WNT5A is downregulated in acute lymphoblastic leukemia patients, which is caused by epigenetic silencing. This finding confirms our previous results about abnormal canonical activation of WNT in T-ALL and the controversial actions of canonical and noncanonical WNT pathways. Materials and Methods Patients and Controls Eighty-six childhood ALL patients (52 males and 34 females) were included in this study. The mean age was 8.11 years (min: 16 days, max: 17 years). Bone marrow samples were obtained from patients at the time of diagnosis. All patients were treated according to the BFM protocol. The mean WBC count was 65x109/L (min: 1x109/L, max: 600x109/L) and cases were grouped as <50x109/L [n=45, mean: (129±110) x109/L] or >50x109/L [n=27, mean: (15±11)x109/L]. As controls, CD19-positive B-cells were obtained from the healthy bone marrow of transplantation donors (n=6) and


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normal peripheral blood (n=10), and healthy thymocytes were obtained from healthy thymus tissue of patients scheduled for cardiac surgery. The control samples were sorted with a FACS Aria II cell sorter (BD, USA). Mononuclear cells were isolated by density-gradient centrifugation over Ficoll-Hypaque and contained at least 90% B cells as determined by fluorescent CD19 staining for B-cell controls and CD3 for T-cell controls. This study was approved by the local ethics committee of the Medical Faculty of İstanbul University (reference number and date: 2008/305 and 20.02.2008) and informed consent was obtained from all patients and healthy controls or their families. RNA Isolation and cDNA Synthesis Bone marrow samples were stored at -80 °C after homogenization in RTL buffer (QIAGEN, 79216). Total RNA was isolated with the QIAGEN RNeasy Protect Mini Kit (QIAGEN, 74104). RNA samples were treated with DNase (1 U/µg, Sigma, AMPD1-1KT) for possible DNA contaminations during isolation. One microgram of total RNA was used for cDNA synthesis by random hexamers and MMLV reverse transcriptase (MBI Fermentas, EPO351) according to the recommendations of the manufacturer. Analysis of Gene Expression by Real-Time Quantitative RT-PCR Quantitative PCR (QRT-PCR) was carried out on the Light Cycler Instrument 1.5 (Roche Diagnostics, Germany) with SYBR Premix Ex Taq (TAKARA, RR420A). The PCR conditions were prepared as per the instructions of the manufacturer and all samples were studied in duplicate. The specificity of amplification of the products was confirmed by melting curve analyses and agarose gel electrophoresis. The PCR program was as follows: initial denaturation at 95 °C for 7 min; amplification segment of 5 s at 95 °C, 10 s at 59 °C, and 10 s at 72 °C for 45 cycles; and melting curve segment of 15 s at 60 °C for 1 cycle. WNT5A relative expression levels were normalized to 3 reference genes (β-actin, CypA, and ABL). DNA Isolation, Bisulphate Treatment, MethylationSpecific PCR, and Bisulphate Sequencing Following the DNA isolation, bisulphate treatment was performed as described previously [10]. Bisulphate-treated DNA was purified with the Gene Clean III Kit (Qbiogene, 74104) according to the manufacturer’s recommendations. After bisulphate treatment, the samples were amplified for the proximal region of the WNT5A promoter, which has been shown to regulate WNT5A transcription of the gene, by methylation-specific PCR (MSPCR). The MSPCR primers were designed by the MethPrimer database [11]. The PCR yields were analyzed on 4% agarose gel with ethidium bromide under UV light. As the MSPCR positive control, a healthy peripheral blood sample was methylated in vitro by the SssI methylase enzyme (New England Biolabs, M0226S).

To confirm the MSPCR findings, samples that were treated with bisulphate were also sequenced directly by the Sanger sequencing method, with a primer set that was designed outside of the CpG island area. Sequence results were analyzed with the CLC Main Workbench 6.0 program as described by the manufacturer. WNT5A Gene Expression and 5-Azacytidine Treatments in Acute Leukemia Cell Lines We analyzed WNT5A methylation and expression status among the acute leukemia cell lines (Fleb14-4, Molt4, Jurkat, T-ALL1, and RPMI 8402). Methylation was confirmed by bisulphate sequencing. The WNT5A methylated cell lines Jurkat and RPMI 8402 were cultured for 4 days with 5’-aza-20deoxycytidine (Aza; Sigma-Aldrich, A3656) at 5 mM and 10 mM concentrations respectively in 24-well plates (5x105 cells/ well) supplemented with 1 mL of RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) at 37 °C in a humid atmosphere containing 5% CO2. At the 96th hour, the cells were collected for DNA and RNA isolations as previously described for MSPCR and QRT-PCR studies. Statistical Analysis Relative expression levels were calculated by the delta Ct method. Differences between the relative expression levels of cases and controls were tested with the Mann-Whitney test. The clinical characteristics according to the methylation status were analyzed by chi-square test, Fisher’s exact test, or multivariate analysis where appropriate and p≤0.05 was considered statistically significant. The Kaplan-Meier method was used to estimate survival rates. The median follow-up was 66 months (min: 1 month, max: 158 months). Overall survival was defined by the interval from the date of diagnosis to the date of death or last follow-up. Event-free survival was defined as the time from diagnosis to treatment failure, relapse, death, or last followup. Differences were compared with the 2-sided log-rank test. Multivariate survival analysis was estimated according to the Cox regression model. All statistical analyses were done with SPSS 19.0 for Windows (IBM SPSS Data Editor Inc., USA) and GraphPad Prism V (GraphPad Software Inc., USA). Results Downregulated Expression Lymphoblastic Leukemia Cases

of

WNT5A

in

Acute

The raw data of our previous expression array study [microarray data are available at http://www.ncbi.nlm.nih. gov/geo/ (accession no. GSE46170)] were reanalyzed and evaluated for the noncanonical WNT pathway members by defining a gene list obtained from public databases and web portals (NCBI Entrez Gene, WNT Homepage, http://www. stanford.edu/~rnusse/wntwindow.html). The data set was

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Figure 1. The heat map diagram of WNT5A probes in T-ALL patients and controls. Gene and samples were clustered using Euclidean distance and complete linkage method for the probe sets in 31 T-ALL patients and thymocyte subsets as controls [CD4 single positive, CD8 single positive, CD4+CD8+ double positive, Thymus (total thymus tissue), DP3- (CD4+, CD8+ double positive CD3 negative, immature single positive and CD3-, CD4-, CD8-)]. Three probe sets for WNT5A are illustrated in the heat map analysis. Green color shows downregulation and red color shows upregulation of the targeted gene. A

Hatırnaz Ng Ö, et al: WNT5A in ALL

Figure 3. MSPCR results in acute lymphoblastic leukemia patients. The samples were run on 4% agarose gel with ethidium bromide under UV light. PCR products were 178 bp long. Initial M: pUC Mix Marker 8, P: patient, M: methylated PCR, U: unmethylated PCR (patients 1, 2, 3, and 4 are methylated; patient 7 is slightly methylated; patients 5 and 6 are unmethylated), IVM: in vitro methylated control sample, Neg: negative control.

B

Figure 2. Relative WNT5A mRNA expression in B- and T-cell acute lymphoblastic leukemia patients. A) B-ALL patients’ samples were compared with healthy bone marrow samples and T-cell acute lymphoblastic leukemia patients’ were compared with healthy thymocytes (p=0.007). Each sample was studied in duplicate and threshold cycle numbers are relative to the geometric mean of 3 reference genes (ABL, β-actin, and CypA). B) Comparison of WNT5A mRNA levels between B-ALL and T-ALL samples (p<0.0001). Each sample was studied in duplicate and threshold cycle numbers are relative to the geometric mean of 3 reference genes (ABL, β-actin, and CypA). filtered to exclude genes showing minimal variation across the set of arrays from the analysis. Probe sets whose expression differed by at least 2-fold from the median in at least 20% of the arrays were retained. The heat map results showed that the WNT5A is downregulated in T-ALL patients when compared to control thymic subsets (Figure 1). To validate the downregulation of WNT5A detected in expression array analysis, quantitative mRNA expression of WNT5A was assessed by QRT-PCR for ALL patients’ samples. 130

Figure 4. Aza-20-deoxycytidine (Aza) treatment experiments in cell lines. Cell lines Jurkat and RPMI 8402 were cultured for 4 days with Aza (Sigma-Aldrich, A3656) at 5 mM and 10 mM concentrations respectively in 24-well plates (5x105 cells/well) supplemented with 1 mL of RPMI 1640 medium with 10% FBS and 1% antibiotics (penicillin and streptomycin) at 37 °C in a humid atmosphere containing 5% CO2. A) MSPCR results after 0 mM, 5 mM, and 10 mM 5’-Aza treatment of RPMI 8402 cell line. B) MSPCR results after 0 mM, 5 mM, and 10 mM 5’Aza treatment of Jurkat cell line. C) Relative WNT5A mRNA expression level after 5’-Aza treatment of RPMI 8402 cell line (5 mM, p=0.0043; 10 mM, p=0.0002). D) Relative WNT5A mRNA expression level after 5’-Aza treatment of Jurkat cell line (5 mM, p=0.004). The expression of WNT5A was found to be downregulated both in B-ALL and T-ALL patients when compared to specific control cell populations (Figure 2A), but only in T-ALL was the difference statistically significant (p=0.007). Among the


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phenotypic groups, WNT5A mRNA levels were lower in T-ALL patients than B-ALL patients and the difference was statistically significant (Figure 2B, p<0.0001). WNT5A Promoter is Heavily Methylated in Acute Lymphoblastic Leukemia Patients According to MS-PCR results, in total 84% of the ALL patients were methylated for the WNT5A promoter region (Figure 3). Among the analyzed cell lines, Fleb14-4, Molt4, Jurkat, and T-ALL1 were determined as methylated for the WNT5A promoter region and the results were also confirmed by bisulphate sequencing, both in patients and cell lines (Supplemental Figure 1). To determine the effect of this methylated region on WNT5A transcription, we treated cell lines that were also methylated for the WNT5A promoter with Aza. In the RPMI 8402 cell line 10 mM (Figures 4A and 4C, p=0.0002) and in the Jurkat cell line 5 mM (Figures 4B and 4D, p=0.004) concentrations of Aza were able to demethylate the WNT5A promoter region and the WNT5A mRNA levels were increased after treatment, which shows that the studied region regulates WNT5A transcription.

Figure 5. Kaplan-Meier estimate of probability of A) overall and B) event-free survival analysis.

Clinical Implications of WNT5A Methylation The relations between WNT5A promoter methylation and clinical findings like age (p=0.50), sex (p=0.8), white blood cell count at the time of diagnosis (p=0.55), and immunophenotype (p=0.72) were analyzed and no statistical significance was detected; the results are summarized in Table 1. The Kaplan-Meier estimate of probability both for overall (p=0.31) and event-free (p=0.36) survival according to WNT5A methylation status showed no significant difference (Figures 5A and 5B, respectively). Multivariate regression analysis revealed no significant association between clinical features and WNT5A promoter methylation. Discussion WNT5A is one of the most studied noncanonical WNT ligands and it has shown to be expressed both in normal B and T cells [7,12]. WNT5A is found overexpressed in different solid tumors, such as in lung cancer, prostate cancer, metastatic carcinomas, and squamous head and neck carcinomas [13,14]. WNT5A was also described as a potential tumor suppressor gene, able to prevent and reverse tumor genesis [7,15]. However, like in endometrial carcinomas, WNT5A mRNA expression was downregulated in some malignant tumors [16,17] and types of leukemia [18,19,20]. One of the

Supplemental Figure 1. CpG island methylation status by bisulphate sequencing in ALL-derived cell lines. A) MSPCR results of cell lines, IVM: in vitro methylated control; B) bisulphate sequencing of Jurkat cell line, red bars show CpG islands; C) bisulphate sequencing of Fleb14-4 cell line, red bars show CpG islands. 131


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Table 1. Clinical characteristics of pediatric acute lymphoblastic leukemia patients according to WNT5A methylation status.

Methylated

Unmethylated

n=73 (%)

n=13 (%)

Sex

0.80

Male

45 (61.6)

7 (53.8)

Female

28 (38.4)

6 (46.2)

Age (years)

0.50

<1

4 (5.5)

0 (0)

1-9

45 (61.6)

7 (53.8)

≥10

24 (32.9)

6 (46.2)

WBC count (x109/L)

0.55

<10

15 (20.5)

3 (23.0)

10-<50

25 (34.3)

4 (30.8)

≥50-<100

19 (26.0)

4 (30.8)

≥100

14 (19.2)

2 (15.4)

Median

44.5

17.4

Range

1.06-72.2

1.0-286

Platelets (x109/L)

0.04

<50

20 (27.4)

5 (38.5)

50-<100

12 (16.4)

0 (0)

≥100

16 (22.0)

0 (0)

Unknown

25 (34.2)

8 (61.5)

Hb (mmol/L)

1.00

<5

5 (6.8)

0 (0)

>5

38 (52.1)

3 (23.0)

Unknown

30 (41.1)

10 (77.0)

Immunophenotype

0.72

Common B

39 (53.4)

5 (38.4)

Pre-B

8 (11.0)

4 (30.8)

Pro-B

4 (5.5)

0 (0)

T-ALL

22 (30.1)

4 (30.8)

132

p value


Turk J Hematol 2015;32:127-135

Hatırnaz Ng Ö, et al: WNT5A in ALL

Table 1. Continuous

Methylated

Unmethylated

Risk group

p value 0.01

SRG

12 (16.4)

0 (0)

MRG

30 (41.1)

11 (84.6)

HRG

31 (42.4)

2 (15.4)

>5% blasts

6 (9.5)

1 (7.6)

<5% blasts

37

6

Relapse

10 (13.7)

4 (30.7)

t(12;21)

4 (5.5)

2 (15.3)

t(4;11)

3 (4.1)

0 (0)

t(9;22)

2 (2.7)

0 (0)

Day 33 BM 1.00

Translocation

WBC: White blood cell, Hb: hemoglobin, T-ALL: T-cell acute lymphoblastic leukemia, BM: bone marrow, SRG: standard risk group, MRG: medium risk group, HRG: high risk group.

most well-known mechanisms for downregulation of a gene is epigenetic modification, such as promoter hypermethylation. WNT5A is silenced in most colorectal cancer cell lines and nasal NK/T-cell lymphoma due to promoter methylation [21]. Multistep deregulation of the WNT pathway was previously shown in childhood ALL by others and by our group [7,9,18]. In this study, we detected downregulation of WNT5A gene expression in T-ALL samples by microarray and confirmed decreased mRNA levels by real-time PCR both in B-ALL and T-ALL patients. The downregulation was more dramatic in T-ALL than B-ALL patients that had approximately 4-fold higher WNT5A mRNA levels. We then showed that downregulation of WNT5A expression is caused by promoter hypermethylation, which was previously described by Gomez et al., although their methylation rates were not as high as in our study [20]. This difference may be due to the primer regions that were selected. In this study, the primers were designed from the WNT5A proximal promoter, which resides within 631 bp upstream of the major transcription start site that was shown to have the strongest promoter activity [22]. The direct effect of the methylated region on transcription was confirmed by 5’-aza treatment, and when the methylation was undone, WNT5A expression was restored. Deng et al. also showed downregulation of WNT5A mRNA expression caused

by promoter methylation, and it was restored in complete remission samples [23]. Some demethylating agents are also used in chemotherapy protocols and this may be the reason for loss of methylation in remission patients. The lack of remission samples limits our study as we could not detect the methylation status of remission samples, but we can speculate that the loss of WNT5A may cause leukemogenesis and methylation restoration might help complete remission in patients with leukemia. Hypermethylation of WNT5A has been reported previously both in T-ALL and B-ALL, but without any significant differences between the lineages. WNT5A was shown to be essential for normal T-cell development and the thymocytes of WNT5A knockout mice could rescue them from apoptosis [18], whereas the same effect could not be detected for B cells [7]. That result shows a role for noncanonical WNT signaling in cell death and T-cell homeostasis. Our results are in concordance with this finding, showing that the noncanonical pathway is downregulated specifically in T-ALL but not in B-ALL. This downregulation may cause the thymocytes to escape apoptosis and take part in leukemia development in our T-ALL cohort. Topol et al. showed that even low levels of WNT5A were able to reduce canonical WNT signaling via degradation of β-catenin, independent of GSK-3β activity [24].

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In conclusion, we have demonstrated that WNT5A, an important member of the noncanonical WNT pathway, was silenced by hypermethylation in ALL. We have also shown that this methylation has a direct effect on gene expression, which may reveal that WNT5A can be used as a specific marker to reverse leukemic development. Acknowledgments This work was supported by the Research Fund of İstanbul University (Project No. 355/03062005) and the Scientific and Technological Research Council of Turkey (TÜBİTAK, Project Nos. 106S112 and 109S395). 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. Nusse R. WNT targets: repression and activation. Trends Genet 1999;15:1-3. 2. Dejmek J, Dejmek A, Safholm A, Sjölander A, Andersson T. Wnt-5a protein expression in primary dukes B colon cancers identifies a subgroup of patients with good prognosis. Cancer Res 2005;65:9142-9146. 3. Huang CL, Liu D, Nakano J, Ishikawa S, Kontani K, Yokomise H, Ueno M. Wnt5a expression is associated with the tumor proliferation and the stromal vascular endothelial growth factor--an expression in non-small-cell lung cancer. J Clin Oncol 2005;23:8765-8773. 4. Saitoh T, Mine T, Katoh M. Frequent up-regulation of WNT5A mRNA in primary gastric cancer. Int J Mol Med 2002;9:515519. 5. Saldanha G, Ghura V, Potter L, Fletcher A. Nuclear betacatenin in basal cell carcinoma correlates with increased proliferation. Br J Dermatol 2004;151:157-164. 6. Yamaguchi TP, Bradley A, McMahon AP, Jones S. A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development 1999;126:1211-1223. 7. Liang H, Chen Q, Coles AH, Anderson SJ, Pihan G, Bradley A, Gerstein R, Jurecic R, Jones SN. Wnt5a inhibits B cell proliferation and functions as a tumor suppressor in hematopoietic tissue. Cancer Cell 2003;4:349-360. 8. Luis TC, Ichii M, Brugman MH, Kincade P, Staal FJ. Wnt signaling strength regulates normal hematopoiesis and its deregulation is involved in leukemia development. Leukemia 2012;26:414-421.

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9. Ng OH, Erbilgin Y, Firtina S, Celkan T, Karakas Z, Aydogan G, Turkkan, Yildirmak Y, Timur C, Zengin E, van Dongen JJ, Staal FJ, Ozbek U, Sayitoglu M. Deregulated WNT signaling in childhood T-cell acute lymphoblastic leukemia.Blood Cancer J 2014;4:e192. doi: 10.1038/bcj.2014.12. 10. Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 1992;89:1827-1831. 11. Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002;18:1427-1431. 12. Mulroy T, McMahon JA, Burakoff SJ, McMahon AP, Sen J. Wnt-1 and Wnt-4 regulate thymic cellularity. Eur J Immunol 2002;32:967-971. 13. Iozzo RV, Eichstetter I, Danielson KG. Aberrant expression of the growth factor Wnt-5A in human malignancy. Cancer Res 1995;55:3495-3499. 14. Rhee CS, Sen M, Lu D, Wu C, Leoni L, Rubin J, Corr M, Carson DA. Wnt and frizzled receptors as potential targets for immunotherapy in head and neck squamous cell carcinomas. Oncogene 2002;21:6598-6605. 15. Olson DJ, Gibo DM, Saggers G, Debinski W, Kumar R. Reversion of uroepithelial cell tumorigenesis by the ectopic expression of human wnt-5a. Cell Growth Differ 1997;8:417-423. 16. Bui TD, Tortora G, Ciardiello F, Harris AL. Expression of Wnt5a is downregulated by extracellular matrix and mutated c-Ha-ras in the human mammary epithelial cell line MCF10A. Biochem Biophys Res Commun 1997;239:911-917. 17. Kremenevskaja N, von Wasielewski R, Rao AS, Schofl C, Andersson T, Brabant G. Wnt-5a has tumor suppressor activity in thyroid carcinoma. Oncogene 2005;24:2144-2154. 18. Liang H, Coles AH, Zhu Z, Zayas J, Jurecic R, Kang J, Jones SN. Noncanonical Wnt signaling promotes apoptosis in thymocyte development. J Exp Med 2007;204:3077-3084. 19. Martin V, Valencia A, Agirre X, Cervera J, San Jose-Eneriz E, Vilas-Zornoza A, Rodriguez-Otero P, Sanz MA, Herrera C, Torres A, Prosper F, Roman-Gomez J. Epigenetic regulation of the non-canonical Wnt pathway in acute myeloid leukemia. Cancer Sci 2010;101:425-432. 20. Roman-Gomez J, Jimenez-Velasco A, Cordeu L, Vilas-Zornoza A, San Jose-Eneriz E, Garate L, Castillejo JA, Martin V, Prosper F, Heiniger A, Torres A, Agirre X. WNT5A, a putative tumour suppressor of lymphoid malignancies, is inactivated by aberrant methylation in acute lymphoblastic leukaemia. Eur J Cancer 2007;43:2736-2746.


Hatırnaz Ng Ö, et al: WNT5A in ALL

Turk J Hematol 2015;32:127-135

21. Ying J, Li H, Chen YW, Srivastava G, Gao Z, Tao Q. WNT5A is epigenetically silenced in hematologic malignancies and inhibits leukemia cell growth as a tumor suppressor. Blood 2007;110:4130-4132.

23. Deng G, Li ZQ, Zhao C, Yuan Y, Niu CC, Pan J, Si WK. WNT5A expression is regulated by the status of its promoter methylation in leukaemia and can inhibit leukemic cell malignant proliferation. Oncol Rep 2011;25:367-376.

22. Danielson KG, Pillarisetti J, Cohen IR, Sholehvar B, Huebner K, Ng LJ, Nicholls JM, Cheah KS, Iozzo RV. Characterization of the complete genomic structure of the human WNT-5A gene, functional analysis of its promoter, chromosomal mapping, and expression in early human embryogenesis. J Biol Chem 1995;270:31225-31234.

24. Topol L, Jiang X, Choi H, Garrett-Beal L, Carolan PJ, Yang Y. Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation. J Cell Biol 2003;162:899-908.

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Research Article

DOI: 10.4274/tjh.2014.0200

The Hematological and Molecular Spectrum of α-Thalassemias in Turkey: The Hacettepe Experience Türkiye’de Alfa Talasemilerin Hematolojik ve Moleküler Spektrumu: Hacettepe Deneyimi Şule Ünal, Fatma Gümrük Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey

Abstract: Objective: The spectrum of α-thalassemias correlates well with the number of affected α-globin genes. Additionally, combinations of the several non-deletional types of mutations with a large trans deletion comprising the 2 α-globin genes have an impact on the clinical severity. The objective of this study was to analyze the hematological and molecular data of 35 patients with Hb H disease from a single center in order to identify the genotypes of Hb H disease and genotype-phenotype correlations.

Materials and Methods: Herein, we report the hematological and mutational spectrum of patients with Hb H disease (n=35). Additionally, genotypes of α-gene mutations of 78 individuals, who were referred to our institution for α-gene screening, were analyzed.

Results: Supporting the previous data from Turkey, -α3.7 was the most common mutation among patients with Hb H disease (62.8%) and in the other 78 subjects (39.7%). Of the patients with Hb H disease, the most common genotypes were -α3.7/--20.5, -α3.7/--26.5, and -α3.7/--17.5 in 10 (28.6%), 6 (17.1%), and 6 (17.1%) patients, respectively. Another small deletion, -4.2 alpha, and several non-deletional types of α-gene mutations, namely α (-5nt): IVS-I donor site (GAG.GTG.AGG->GAG.G-----); α (PA-2): AATAAA>AATGGA, and α (cd59): GGC->GAC, were found to be associated with Hb H disease when present at trans loci of one of the large deletions given above. The combinations consisting of 1 non-deletional and 1 of the large deletional types of mutations (αTα/--) at trans loci were found to result in a more severe phenotype compared to the genotypes composed of 1 small trans deletion of a large deletion (-α/--). The combination of α (Cd59) and -- in trans was associated with severe phenotype and the disease was associated with an increase in Hb Bart’s level with null Hb H. In spite of the presence of 2 intact α-globin genes, homozygosity for PA-2 mutation resulted in severe Hb H disease.

Conclusion: This study indicated that Hb H disease is not rare in Turkey and its genotype is quite heterogeneous. Key Words: Molecular, Mutation, α-Thalassemia, Turkey

Address for Correspondence: Şule ÜNAL, M.D., Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey Phone: +90 532 526 37 49 E-mail: suleunal@hacettepe.edu.tr Received/Geliş tarihi : May 20, 2014 Accepted/Kabul tarihi : June 17, 2014

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Ünal Ş, et al: Molecular Spectrum of α-Thalassemias in Turkey

Turk J Hematol 2015;32:136-143

Özet: Amaç: Alfa (α) talasemilerin farklı klinik spektrumundan etkilenen α-globin gen sayısı sorumludur. Ayrıca delesyonel olmayan mutasyonların, iki α-globin geninin birden etkilendiği büyük delesyonel mutasyonlarla kombinasyon oluşturmasının da hastalığın klinik şiddetinde etkisi bulunmaktadır. Gereç ve Yöntemler: Burada Hb H hastalarımızın (n=35) hematolojik ve mutasyonel spektrumunu sunmaktayız. Buna ek olarak, merkezimize α-globin geninde mutasyon varlığı taraması için merkezimize gönderilen ve α-globin geni mutasyonu taşıyan 78 bireyin bulguları analiz edilmiştir. Bulgular: Çalışmamızda daha önce bildirilenleri destekler şekilde Hb H hastası grubunda (%62,8) ve 78 bireyde (%39,7) en sık mutasyon -α3,7 olarak bulunmuştur. Hemoglobin H hastalarımızda en sık genotipler -α3.7/--20.5; -α3,7/--26,5 ve -α3,7/--17,5 olarak sırasıyla 10 (%28,6), 6 (%17,1) ve 6 (%17,1) sıklıklarda bulunmuştur. Diğer bir küçük delesyon olan -4.2 (Asya tipi), delesyonel olmayan α-globin mutasyonları α (-5nt): IVS-I donor site (GAG.GTG.AGG->GAG.G-----); α (PA-2): AATAAA>AATGGA ve α (cd59): GGC->GAC; translarında büyük delesyonel bir mutasyon bulunduğunda Hb H hastalığına neden olduğu görülmüştür. Delesyonel olmayan mutasyonla büyük delesyonel tipte mutasyonların kombinasyonlarının (α Tα/--), sadece delesyonel mutasyonların kombinasyonları sonucu gelişen Hb H hastalarına göre kliniklerinin daha şiddetli olduğu gözlenmiştir (-α/--). α(Cd59) ve -- trans birlikteliğinde, (α (Cd59)/--), daha ağır bir fenotip izlenmiştir ve bu durumda Hb H bulunmayıp, hastada Hb Bart’s yüksek olarak ölçülmüştür. Homozigot PA-2 mutasyonu olan hastalar (α PA-2/α PA-2) ağır fenotipte Hb H hastaları olarak gözlenmiştir.

Sonuç: Çalışmamız Hb H hastalığının ülkemizde nadir olmadığına ve genotipinin heterojen olduğuna işaret etmektedir. Anahtar Sözcükler: Moleküler, Mutasyon, Alfa talasemiler, Türkiye

Introduction α-Thalassemia results from a genetic defect in α-globin chain synthesis, often as a consequence of deletional mutations and less frequently due to non-deletional types of mutations [1,2]. α-Thalassemias may occur worldwide; however, they are seen more commonly among populations in South East Asia, the Mediterranean region, and the Middle East [1]. The α-globin gene is located on the short arm of chromosome 16 (16p13.3) and normally there are 4 α-globin gene copies in an individual, with 2 in each allele [3]. The phenotype of α-thalassemias is directly related to the number of α-globin genes affected. α+-Thalassemias designate the status of deletion in one of the paired α-globin genes (-α/αα), whereas in α0-thalassemias both of the paired α-globin genes are deleted (--/αα). Heterozygous α+-thalassemia usually causes a silent carrier state. On the other hand, heterozygous α0-thalassemia (--/α) and homozygous α+-thalassemia (-α/α) result in hematological findings similar to α-thalassemia trait, except for the Hb A2 value, which is at the normal level or below the normal level in α-thalassemia. The co-existence of both α+-thalassemia and α0-thalassemia (-α/--) results in hemoglobin H (Hb H) disease [1]. There are also nondeletional types of mutations (αTα) resulting in Hb H disease, when a large deletional type of mutation (--) co-exists in trans (αTα/--) [4,5].

The most common deletional mutations causing α+thalassemia are -α3.7 and -α4.2, whereas the common deletional mutations causing α0-thalassemias are of 20.5-kb deletion, approximately 17.5-kb deletion (-MED-I), greater than 26.5-kb deletion (-MED-II), and approximately 18-kb deletion (-SEA) [1,4,6]. MED-II has previously been reported in a few Turkish families and from other Mediterranean populations [4]. In this study, the hematological and molecular data of 35 patients with Hb H disease from a single center were analyzed and reported in order to identify the genotypes of Hb H disease and genotype-phenotype correlations, and also to create awareness that Hb H disease is not a rare entity in Turkey. Materials and Methods Of the 788 patients who were diagnosed with thalassemia between 1981 and 2014 at our institution, 138 (17.5%) were diagnosed with Hb H disease (Table 1). Unfortunately, from those 138 patients only a total of 35 had genotype data available; those 35 were included in the current study. Splenomegaly was detected at diagnosis, during physical examination, or by ultrasonography in 40% of the patients with Hb H disease. The transfusion histories of patients with Hb H were recorded from patients’ files. Of the patients with Hb H disease, 18% received erythrocyte transfusion at least once, and 82% had no transfusion history at diagnosis and received no transfusion during follow-up. The number of transfusions ranged between

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1 and 24. One patient was on a chronic transfusion program, whereas the other patients were transfused occasionally. Ethical committee approved this study. Excluding the patients with Hb H disease, of the individuals screened for α-thalassemia mutations, 78 were found to carry an α-thalassemia mutation. The indications of α-thalassemia mutational screening among those 78 individuals were either having hypochromic microcytic erythrocytes, with normal iron status and Hb A2 below 3.5%, or being the available parent of a patient with Hb H disease. Results of hematological studies and red cell indices were analyzed. For discussion purposes, values prior to splenectomy or erythrocyte transfusion were taken into consideration. Hemoglobin A2, Hb F, and Hb H values were measured with the previously described methods [7] or high-performance liquid chromatography with the Bio-Rad Variant II system. Supravital stains for Hb H inclusions were examined in all cases [8]. Prior to 2008, α-thalassemia mutations were identified with previously described methods [7,8,9,10,11,12,13]. After 2008, mutation analyses for the α-globin gene were evaluated with the α-Globin Strip-Assay (ViennaLab, Austria), based on the reverse-hybridization technique used for detection of the 21 most common α-thalassemia mutations in the Mediterranean region. Of the 35 patients with Hb H disease, 25 have been reported previously [7]. The obtained data were evaluated with SPSS 21 (IBM Corp., Armonk, NY, USA). Normality test was performed to determine if the data were distributed in a normal fashion. For comparison between groups of more than 2, one-way ANOVA test was used. Statistical significance was determined as p values <0.05. Results Of the 35 patients with Hb H disease, the age range was 1.5-50 years at diagnosis (mean: 15.9±12.9 years). The mean values of red blood cell indices at diagnosis are summarized in Table 2a. A total of 10 different genotypes were detected in 35 patients with Hb H disease (Tables 2b and 2c.). Of the 35 patients with Hb H disease, 22 (62.8%) and 18 (51.4%) were found to have -α3.7 or --20.5 alleles, respectively (Table 3). The most common genotype was -α3.7/--20.5 in 10 (28.6%) of the patients, followed by -α3.7/--26.5 in 6 (17.1%) and -α3.7/--17.5 in 6 (17.1%). The most common 3 genotypes were distributed among 22 of the 35 patients, representing 62.8% of all genotypes found in patients with Hb H disease. The numbers of Hb H patients having other genotypes were too small to make any statistical analysis; therefore, comparison of the hematological data was made only among the patients with the 3 most common above-mentioned genotypes.

138

Statistical analyses of the mean values of red cell indices showed no significant difference among these 3 common genotypes. Hemoglobin F level was found significantly higher in -α3.7/--17.5 patients (p=0.041), whereas Hb H levels were significantly lower among patients with this genotype compared to the -α3.7/--20.5 and -α3.7/--26.5 genotypes (p=0.036). Hemoglobin A2 levels were similar among these 3 genotypes. Of the patients with Hb H disease, 26 (74.3%) were found to have deletional types of mutations, whereas 9 (25.7%) were found to have non-deletional types of mutations. Comparison of the hematological data of the Hb H patients showed that the group of patients with a genotype consisting of non-deletional types of mutations with a large trans deletion (ααT/--) had statistically lower hemoglobin values (p=0.007) compared to those who had deletional types of mutations with a large trans deletion (-α/--) (Table 4). On the other hand, the mean of Hb H levels was significantly higher in the former patients (18.1± 8.3 vs. 7.4±4.7; p=0) than the latter (Table 4). In the examination of the 78 individuals with α-thalassemia mutations other than Hb H disease, the most common genotype was -α3.7/αα in 31 patients (39.7%) (Table 5). The most common non-deletional genotype was α(PA-1)/αα in 5 of the individuals (6.4%). Of the 78 subjects, 34 (43.5%) and 21 (26.9%) were found to have -α3.7 or --20.5 alleles, respectively (Table 5). Discussion The incidence of deletional α-thalassemia (-α/αα) among newborns screened by globin gene mapping from samples obtained from cord blood at birth has been reported to be 3.6% in Turkey [14]. In other reports, the chromatographic analyses of cord blood samples of newborns in Turkey suggested that -α/αα or (αTα) thalassemia incidence was between 2.9% and 4.1% [15,16]. In a recent report from Antakya-Hatay, a city in the southern part of Turkey, 300 individuals with moderate anemia, microcytosis, and normal iron levels were tested for α-thalassemia by the aid of α-globin strip assay; of these, 97 were found to have at least 1 mutation in 4 of the α-globin genes [17]. Of these patients, the most common mutation was -α3.7 (57.3%) [17]. Similarly, Öner et al. and Çürük reported -α3.7 as the most common α-thalassemia gene Table 1. The distribution of β- and α-thalassemias between 1981 and 2014 in the Hacettepe University Division of Pediatric Hematology.

Disease

n (%)

b-thalassemia major/intermedia

650 (82.5)

Hb H

138 (17.5)

Total

788 (100)


15.9±12.9

1.5-50

Mean ± SD

Range

n=35

6.7-13.7

9.3±1.6

Age at diagnosis (years) Hb (g/dL)

48-98

63.1±9.7

MCV (fL)

15.3-20.9

17.7±1.8

4.7±0.8

RBC (x1012/L)

28.2-35.8 2.8-6.4

30.9±2.3

MCHC MCH (pg) (g/dL)

p

-α3.7/--17.5

-α3.7/--26.5

-α3.7/--20.5

Genotype

1.5-30

Range

8-28

Range

2-43

Range >0.05

13.6±15.3

Mean ± SD

n=6

18±6.5

Mean ± SD

n=6

14.8±9.6

Mean ± SD

n=10

>0.05

9-9.9

9.5±0.3

7.5-11.6

9.9±1.5

8.1-12.3

9.8±1.6

Age at diagnosis Hb (g/ (years) dL)

>0.05

48-63

56.3±5.3

52-72

61.5±7.3

51.4-77

64.6±9.6

MCV (fL)

>0.05

19.2

19.2

15.8-19.2

17.5±1.7

15.3-17.5

16.6±1.2

MCH (pg)

>0.05

35.8

35.8

29.1-32.1

30.5±1.5

28.2-30.3

29.5±1.1

>0.05

4.2-5.8

4.9±0.5

3.9-6.4

5.4±1.1

4-5.3

4.9±0.4

MCHC RBC (g/dL) (x1012/L)

9.5-34.9

>0.05

11.5

11.5

13-32.8

21.8±11.7

21.2-25.7

23.9±2.4

>0.05

1.2-1.9

1.5±0.3

0.9-1.2

1±0.2

0.9-2

1.4±0.4

Hb A2 (%)

0.5-2

0.041

0.6-4.3

2.4±1.3

0-1.1

0.7±0.4

0.5-2.2

0.9±0.6

Hb F (%)

0-4.3

1.3±0.9

Hb A2 Hb F (%) (%)

22.5±8.5 1.2±0.4

RDW

RDW

Table 2b. The age and hematological data of patients with Hb H disease with the 3 most common genotypes.

Total

Patients with Hb H

Table 2a. The age and hematological data of patients with Hb H disease with molecular diagnosis.

0.036

1.4-6

3.3±1.7

1.5-15.4

8±4.8

2.9-17

9.9±5.2

Hb H (%)

1.4-34

10.3±7.5

Hb H (%)

Ünal Ş, et al: Molecular Spectrum of α-Thalassemias in Turkey Turk J Hematol 2015;32:136-143

139


140 10-15

Range

6-48

Range

2-2.5

Range

27-50

Range

NA

NA 

NA

NA

NA

NA

30.4

30.4

NA

4.8

2.8

3.2-4.1

3.6±0.6

3.7-4.6

4.2±0.6

4.2-4.3

4.3±0.1

3.4

4.4±1.4

4-5.6

4.6±0.8

NA

NA

NA

NA

19

19

NA

NA

64

NA

NA

NA

NA

20.9

20.9

NA

Value

8.2

98

68-68

68±0

64-67

65.5±2.1

51-55

53±2.8

61-68.6

64.8±5.4

66-71

68.3±2.5

RDW

12

13,700

6.7-6.9

6.8±0.2

8.2-10

9.1±1.3

7.5-8.1

7.8±0.4

8.1-11.4

9.7±2.3

7.9-9

8.4±0.6

RBC (x1012/L)

n=1

Value

8

6-10

Range

n=1

8±2.8

Mean ± SD

n=2

38.5±16.3

Mean ± SD

n=2

2.3±0.4

Mean ± SD

n=2

27±29.6

Mean ± SD

n=2

13±2.6

Mean ± SD

n=3

MCH MCHC (pg) (g/dL)

0.5

0.8

0.7-0.9

0.8±0.1

1.1-1.8

1.5±0.5

0.6-0.7

0.7±0.1

0.6-1.3

0.9±0.5

0.6-0.9

0.8±0.2

Hb A2 (%)

2.9

NA

1-1.5

1.2±0.4

0.5-1.8

1.2±0.9

0.7-1.5

1.1±0.6

0.6-1

0.8±0.3

1-2.5

1.5±0.9

Hb F (%)

28

10.5****

8.4-19.7

14.1±8

8-13.4

10.7±3.8

12-15.5

13.8±2.5

4.4-8.6

6.5±3

15.5-34

23.2±9.6

Hb H (%)

*: α (-5nt): IVS-I donor site (GAG.GTG.AGG->GAG.G-----); **: α (PA-2): AATAAA>AATGGA; ***: α (cd59): GGC->GAC; ****: This value indicates Hb Bart’s but not Hb H for this particular patient.

α (-5nt*)/--17.5

/--20.5

α (cd59 ***)

α (PA-2**)/α (PA-2)

-α4.2/--17.5

α (PA-2**)/--20.5

-α4.2/--20.5

α (-5nt*)/--20.5

Genotype

Age at diagnosis MCV (years) Hb (g/dL) (fL)

Table 2c. The age and hematological data of patients with Hb H disease associated with rare mutations.

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Ünal Ş, et al: Molecular Spectrum of α-Thalassemias in Turkey

that was associated with Hb H disease in 25 and 32 patients, respectively [7,18]. Our study is compatible with the above stated previously published data pointing out that -α3.7 has been the most common genotype among patients with Hb H disease (62.8%).

--MED-I mutation (--17.5) has been reported as the second most common type of allele by Guvenc et al. with 15.11% frequency among the population of Adana, a city in the southern part of Turkey [21]. This is probably related to the homogeneity of the population studied in that publication.

In our study, among Hb H patients, the second most common allele was --20.5 (51.4%). This finding is in accordance with the other reports from Turkey [7,18,19]. A hydrops fetalis case due to α-thalassemia associated with homozygosity of --20.5 was also previously reported from Turkey [20].

The --MED-II deletion has been known as a genotype more common among Turkish populations [4], and it was found as the third most common allele in our study.

In the current study, the -MED-II deletion (--26.5) was found as the third most common allele among patients with Hb H disease (25.7%), which was followed by --MED-I deletion (--17.5) at 17%. Contrary to our observation, the Table 3. Distribution of mutations in 35 patients with Hb H (70 chromosomes).

Genotype

Number of chromosomes affected

-α3.7

22

-α4.2

4

α (PA-2)

6

α (-5nt)

4

α (cd59)

1

--20.5

18

--17.5

9

--26.5

6

Total

70

All of these studies suggest that the molecular pathology of Hb H disease is heterogeneous and, according to our study, the most common genotypes associated with Hb H in 35 patients who were referred to us from all over Turkey are as follows: -α3.7/--20.5 (28.6%), -α3.7/--26.5 (17.1%), and -α3.7/-17.5 (17.1%) (Table 2b). In the current study, 25.7% of the patients with Hb H disease who had a combination of large deletional and non-deletional (ααT/--) mutations were found to have statistically significantly lower Hb and higher Hb H levels compared to those of patients having combinations of large and small deletional (-α/--) types of mutations (Table 4). This finding was compatible with the previously published data [1,2,3]. This study revealed the presence of 3 different non-deletional types of mutations, namely the (-5nt), PA2, and C59 mutations. It seemed that the most common nondeletional type of combination involved in Hb H was (-5nt/--), which was found in 3 patients (8.6%) in the current study. Contrary to this, α (PA-2)/--MED-II was the most frequent non-deletional combination in a regional study by Çürük [18]. It was interesting that in spite of the presence of 2 intact α-globin genes, homozygosity for PA-2 mutation (α PA-2/α PA-2) resulted in severe Hb H disease in 2 patients (Table 2c); this was discussed elsewhere [7].

Table 4. The comparison between hematological parameters of patients with Hb H disease with deletional and non-deletional types of mutations.

Genotype

Hb (g/dL)

RBC (x1012/L) MCV (fL)

Hb A2 (%)

Hb F (%) Hb H (%)

Combination of deletional mutations* (n=26)

9.7±1.3

4.9±0.7

61.9±7.8

1.3±0.4

1.2±0.9

7.4±4.7

Combination of deletional and non-deletional mutations** (n=9)

8.4±2

4.1±0.8

67.6±13.1

0.7±0.1

1.5±0.7

18.1±8.3

p

0.007

0.026

>0.05

0

>0.05

0.001

*Of these 26 patients, 8 were below 10 years of age. **Of these 9 patients, 4 were below 10 years of age.

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Table 5. The distribution of deletional and non-deletional types of α-thalassemia mutations in 78 individuals.

Genotype

n (%)

-α3.7/α

31 (39.7)

--20.5/αα

21 (26.9)

--26.5/αα

8 (10.3)

α (PA-1)/αα

5 (6.4)

α (Cd59 G>A)/αα

4 (5.1)

α (IVS 1-5 nt)/αα

3 (3.8)

-α3.7/-α3.7

2 (2.6)

α (PA-2)/αα

1 (1.3)

α (Cd14 G>A)/αα

1 (1.3)

α (Cd14 G>A)/-α3.7

1 (1.3)

–α17.5/αα

1 (1.3)

Total

78 (100)

In this study, we did not find any of the previously described α-gene mutations from Turkey, such as -THAI, --FIL, init.cd, Cd 19, Hb Icaria, Hb Pakse, or Hb Koya Dora [14,16,17,18,19,21]. In a previous study from our center, the rate of unidentifiable mutations among individuals with α-thalassemia mutations was reported to be 2.72% [22]. In this study, all of the mutations among patients with Hb H disease were known mutations. In the previous study from our center, among individuals with α-thalassemia major, the most common 3 mutations were distributed among 69.39% of the patients [22]. In this study, it was shown that the most common 3 genotypes associated with Hb H accounted for almost 63% of the study group. In the previous reports by Altay and by Akar and Altay, related to National Hemoglobinopathy Registry data, Hb H was reported to be 3.6% (n=103) of all hemoglobinopathies in Turkey [22,23]. In our cohort study from a single center, it was shown that Hb H disease was diagnosed in 17.5% of the total 650 thalassemic patients (Table 1). The latest figure for α-thalassemia major in Turkey was reported to be 57% of 5500 patients with hemoglobinopathies [24,25]. Therefore, according to the data of our center as stated above, the total number of Hb H patients in Turkey should be around 550. The discrepancy in the rates of Hb H between 2002 data and the current study may derive from the higher awareness of the disorder in some centers in recent years, more accurate diagnoses, and/or developments in the diagnostic tools of Hb H disease and/or an increase in referral rates of anemic patients from peripheral to tertiary centers like ours. Therefore, if the figure of the current study reflects a more accurate value of

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the number of Hb H cases, we may expect to diagnose more patients in the near future. In conclusion, as our center is a referral center in the mid-Anatolia region with a patient profile from all over the Turkey, the results of our study may represent the Hb H disease rates among the overall Turkish population. Some of the data of this study were in agreement with previous reports [7,8,9,16,17,18,19,20], and our current study also indicated that the molecular spectrum of α-thalassemias is quite heterogeneous in Turkey, as all together 9 deletional and non-deletional mutations and 10 combinations of them were found to be associated with Hb H disease. In previous reports, the mutational spectra were reported to be less heterogeneous among smaller populations, such as among Cypriots and Iraqi Turks [26,27]. Although in this study the molecular pathology of Hb H disease has been addressed, the frequencies of rare genotypes associated with α-thalassemia requires more patients and further population studies, since most of the individuals screened for that purpose in the current study were parents of the patients with Hb H disease, a limiting factor in prediction of the population frequencies of several genotypes. This study also showed that Hb H disease is not uncommon in Turkey; therefore, this disease should be kept in mind in discussion of microcytic anemias and all efforts should be made for correct diagnosis of α-thalassemias. Detection of new cases will be helpful in determining the allele frequencies of different α-thalassemia mutations. Acknowledgment We would like to express our gratitude to Prof. Çiğdem Altay for her critical review of the manuscript. Dr. Emine Pürçüklü was involved in data collection and we are thankful for her dedicated efforts. 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. Vichinsky EP. Clinical manifestations of α-thalassemia. Cold Spring Harb Perspect Med 2013;3:1-10. 2. Weatherall DJ. Hemoglobinopathies worldwide: present and future. Curr Mol Med 2008;8:592-599. 3. Higgs DR, Vickers MA, Wilkie AO, Pretorius IM, Jarman AP, Weatherall DJ. A review of the molecular genetics of the human α-globin gene cluster. Blood 1989;73:1081-1104. 4. Huisman THJ, Carver MFH, Baysal E. A Syllabus of Thalassemia Mutations. Augusta, GA, USA, Sickle Cell Anemia Foundation, 1997. Available online at http://globin. bx.psu.edu/html/huisman/thals/contents.html, last access date May 2014.


Ünal Ş, et al: Molecular Spectrum of α-Thalassemias in Turkey

5. Weatherall DJ, Clegg JB (eds). The Thalassaemia Syndromes. 4th ed. Malden, MA, USA, Blackwell Science, 2001. 6. Kutlar F, Gonzalez-Redondo JM, Kutlar A, Gurgey A, Altay C, Efremov GD, Kleman K, Huisman TH. The levels of zeta, gamma, and delta chains in patients with Hb H disease. Hum Genet 1989;82:179-186. 7. Öner C, Gurgey A, Öner R, Balkan H, Gümrük F, Baysal E, Altay Ç. The molecular basis of Hb H disease in Turkey. Hemoglobin 1997;21:41-51. 8. Kulaç İ, Ünal Ş, Gümrük F. Brilliant cresyl blue staining for screening hemoglobin H disease: reticulocyte smear. Turk J Hematol 2009;26:45. 9. Oner C, Oner R, Gurgey A, Altay C. A new Turkish type of beta-thalassaemia major with homozygosity for two nonconsecutive 7.6 kb deletions of the psi beta and beta genes and an intact delta gene. Br J Haematol 1995;89:306-312. 10. Baysal E, Huisman TH. Detection of common deletional alpha-thalassemia-2 determinants by PCR. Am J Hematol 1994;46:208-213. 11. Bowden DK, Vickers MA, Higgs DR. A PCR-based strategy to detect the common severe determinants of α thalassaemia. Br J Haematol 1992;81:104-108. 12. Smetanina NS, Oner C, Baysal E, Oner R, Bozkurt G, Altay C, Gurgey A, Adekile AD, Gu LH, Huisman TH. The relative levels of alpha 2-, alpha 1-, and zeta-mRNA in HB H patients with different deletional and nondeletional alpha-thalassemia determinants. Biochim Biophys Acta 1996;1316:176-182. 13. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 1977;74:5463-5467. 14. Fei YJ, Kutlar F, Harris HF 2nd, Wilson MM, Milana A, Sciacca P, Schiliro G, Masala B, Manca L, Altay C, Gurgey A, Ma de Pablos J, Villegas A, Huisman THJ. A search for anomalies in the zeta, alpha, beta, and gamma globin gene arrangements in normal black, Italian, Turkish, and Spanish newborns. Hemoglobin 1989;13:45-65. 15. Özsoylu Ş, Malik SA. Incidence of alpha-thalassemia in Turkey. Turk J Pediatr 1982;24:235-244.

Turk J Hematol 2015;32:136-143

16. Kılınç Y, Kümi M, Gurgey A, Altay Ç. Adana bölgesinde doğan bebeklerde kordon kanı çalışması ile alfa talasemi, G6PD enzim eksikliği ve HbS sıklığının araştırılması. Doğa Tıp ve Ecz D 1986;10:162 (in Turkish). 17. Celik MM, Gunesacar R, Oktay G, Duran GG, Kaya H. Spectrum of α-thalassemia mutations including first observation of - -FIL deletion in Hatay Province, Turkey. Blood Cells Mol Dis 2013;51:27-30. 18. Çürük MA. Hb H (b4) disease in Çukurova, southern Turkey. Hemoglobin 2007;31:265-271. 19. Karakaş Z, Koç B, Temurhan S, Çilsaat G, Gencay G, Karaman S. Seçilmiş vakalarda alfa talasemi: daha az maliyetle tarama mümkün mü? In: XXXIX. Ulusal Hematoloji Kongresi ve VIII. Balkan Day of Hematology, 2013, Abstract 0597 (in Turkish). 20. Gurgey A, Altay C, Beksaç MS, Bhattacharya R, Kutlar F, Huisman TH. Hydrops fetalis due to homozygosity for α-thalassemia-1, -(α)-20.5 kb: the first observation in a Turkish family. Acta Haematol 1989;81:169-171. 21. Guvenc B, Yildiz SM, Tekinturhan F, Dincer S, Akyuzluer I, Okten S, Erkman H. Molecular characterization of alphathalassemia in Adana, Turkey: a single center study. Acta Haematol 2010;123:197-200. 22. Altay Ç. The frequency and distribution pattern of α-thalassemia mutations in Turkey. Turk J Hematol 2002;19:309-315. 23. Akar N, Altay Ç. The results of national hemoglobinopathy registry. In: XXV. National Congress of Hematology, 12-15 November 1997, İstanbul, Turkey. 24. Sağlık Bakanlığı AÇSAP. Hemoglobinopati Kontrol Programı. Türkiye Klinikleri Hematoloji-Onkoloji 2010;3:5-82 (in Turkish). 25. Canatan D. Türkiye’de hemoglobinopatilerin epidemiyolojisi. Hematolog 2014;4:11-22 (in Turkish). 26. Baysal E, Kleanthous M, Bozkurt G, Kyrri A, Kalogirou E, Angastiniotis M, Ioannou P, Huisman TH. α-Thalassemia in the population of Cyprus. Br J Haematol 1995;89:496-499. 27. Esmael A, Öztürk A, Akar N. The incidence of alphathalassemia in Iraqi Turks. Turk J Hematol 2011;28:235-236.

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Research Article

DOI: 10.4274/tjh.2013.0403

Cohort Study: Central Venous Catheter-Related Complications in Children with Hematologic Diseases at a Single Center Kohort Çalışması: Tek Bir Merkezde Hematolojik Hastalık Nedeniyle Tedavi Edilen Çocuklarda Santral Venöz Kateter Kullanımıyla İlişkili Olarak Ortaya Çıkan Komplikasyonlar Ayhan Pektaş1, Ateş Kara2, Aytemiz Gurgey3 1Afyon

Kocatepe University Faculty of Medicine Hospital, Department of Pediatrics, Afyonkarahisar, Turkey University Faculty of Medicine, İhsan Doğramacı Children’s Hospital, Clinic of Pediatric Infectious Diseases, Ankara, Turkey 3Hacettepe University Faculty of Medicine, İhsan Doğramacı Children’s Hospital, Clinic of Pediatric Hematology, Ankara, Turkey 2Hacettepe

Abstract: Objective: This study aims to document and analyze the central venous catheter (CVC)-related complications in children with hematological diseases who were treated within a single institution.

Materials and Methods: A retrospective investigation was conducted in 106 pediatric patients in whom 203 CVCs were inserted. A total of 175 catheter-related complications occurred in 5 years.

Results: The rates of clinical catheter infections, local catheter infections, venous thromboembolism, bleeding, and mechanical complications were 2.6, 1.1, 0.2, 0.2, and 0.2 per 1000 catheter days. Methicillin-resistant Staphylococcus epidermidis was the predominant infectious organism in blood and catheter cultures. The children with leukemia had a significantly higher frequency of clinical catheter infections (p=0.046). The children who underwent bone marrow transplantation had a significantly lower frequency of clinical catheter infections (p=0.043) and higher frequency of local catheter infections (p=0.003). The children with implanted catheters had a significantly lower frequency of clinical catheter infections (p=0.048). The children with thrombocytopenia had significantly fewer local catheter infections and significantly more clinical catheter infections and catheter-related bleeding (respectively p=0.001, p=0.042, and p=0.024).

Conclusion: Leukemia, bone marrow transplantation, and thrombocytopenia are risk factors for CVC-associated complications. The relatively higher number of interventions performed via permanent catheters may be responsible for the significantly increased incidence of systemic infections and mechanical injury. Key Words: Blood coagulation, Hematologic manifestation, Infection, Pediatric leukemia

Address for Correspondence: Ayhan PEKTAŞ, M.D., Afyon Kocatepe University Faculty of Medicine Hospital, Department of Pediatrics, Afyonkarahisar, Turkey Phone: +90 (272) 246 33 33 E-mail: drayhanpektas@hotmail.com Received/Geliş tarihi : December 02, 2013 Accepted/Kabul tarihi : April 14, 2014

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Özet: Amaç: Bu çalışma, tek bir merkezde hematolojik hastalık nedeniyle tedavi edilen çocuklarda santral venöz kateter (SVK) kullanımıyla ilişkili olarak ortaya çıkan komplikasyonları belirlemeyi ve incelemeyi amaçlamaktadır.

Gereç ve Yöntemler: Beş yıl boyunca, 203 SVK uygulaması yapılan 106 çocuk hastada ortaya çıkan 175 katetere bağlı komplikasyon geriye dönük olarak değerlendirilmiştir.

Bulgular: Klinik kateter enfeksiyonu, lokal kateter enfeksiyonu, venöz tromboembolizm, kanama ve mekanik komplikasyon oranları, sırasıyla, 1000 kateter gününde 2,6, 1,1, 0,2, 0,2 ve 0,2 olarak hesaplanmıştır. Metisiline dirençli Staphylococcus epidermidis, kan ve kateter kültürlerinden en çok izole edilen mikroorganizmadır. Lösemili çocuklarda klinik kateter enfeksiyonlarının sıklığı anlamlı olarak yüksektir (p=0,046). Kemik iliği transplantasyonu yapılan çocuklarda klinik kateter enfeksiyonu sıklığı anlamlı olarak düşük (p=0,043) bulunurken lokal kateter enfeksiyonu sıklığı anlamlı olarak yüksektir (p=0,003). İmplante kateteri olan çocuklarda klinik kateter enfeksiyonlarının sıklığı anlamlı olarak düşüktür (p=0,048). Trombositopenisi olan çocuklarda lokal kateter enfeksiyonları anlamlı olarak daha az görülürken klinik kateter enfeksiyonları ve katetere bağlı kanama anlamlı olarak daha sık ortaya çıkmıştır (sırasıyla p=0,001, p=0,042 ve p=0,024).

Sonuç: Lösemi, kemik iliği transplantasyonu ve trombositopeni, SVK ile ilişkili olarak çocuklarda ortaya çıkan komplikasyonlar için en önemli risk etkenleridir. Kalıcı kateterler aracılığıyla gerçekleştirilen müdahale sayısındaki artış, sistemik enfeksiyon ve mekanik hasar riskindeki anlamlı yükselişten sorumlu olabilir.

Anahtar Sözcükler: Koagülasyon, Hematolojik bulgu, Enfeksiyon, Pediatrik lösemi

Introduction Central venous catheters (CVCs) are clinical tools of the utmost importance that facilitate the administration of chemotherapy, antibiotics, blood products, fluids, and parenteral nutrition and the collection of blood samples in children with hematological diseases [1]. Tunneled, cuffed, silastic CVCs were first introduced by Broviac and colleagues and were improved subsequently by Hickman and colleagues. Afterwards, totally implantable vascular access devices (ports) were developed. These devices require less frequent care and provide more freedom and comfort for the patients [2,3]. Despite all preventive measures, systemic and local infections remain a challenge for the utilization of CVCs, including ports and external lines with the expanding use of ports [4]. Venous thromboembolism (VTE) is another serious catheter-related complication that usually appears in critically ill children [5,6]. Access-related bleeding and mechanical complications such as blockage, leakage, dislodgement, and malposition are also encountered in children with catheters [7]. The present study aims to document and analyze the CVC-related complications in pediatric hematology patients who were treated within a single institution over a period of 5 years. Since CVCs have indispensable importance in clinical practice, possible precautions for the prevention of relevant complications are also discussed.

Materials and Methods Selection of Patients The inclusion criteria were the diagnosis of hematological diseases and the administration of CVCs in pediatric patients aged less than 18 years at the study center between June 2003 and December 2007. Therefore, 136 children were initially chosen for the study. Three children with a documented infection or VTE in any location within 6 weeks, 3 children for whom CVCs were implanted at the site of a previously confirmed infection or VTE, 2 children with concurrent anticoagulant treatment, 2 children with sensitivity to antibiotics and/or anticoagulants, 3 children with bacterial endocarditis, 2 children with severe thrombocytopenia (<20x109/L), and 5 children with severe hypertension, renal dysfunction, or hepatic disease were excluded [8]. Thus, 106 children for whom 203 CVCs were inserted were finally enrolled into the study. A total of 175 catheter-related complications were analyzed retrospectively. Ethical committee approved this study. Devices and Their Use All CVC implantations were performed under general anesthesia and sterile conditions by experienced pediatric surgeons or interventional radiologists. The CVCs were inserted via the internal jugular vein using an infraclavicular approach, namely the Seldinger technique. No perioperative antimicrobial prophylaxis was administered and no serious perioperative complications were observed except one case of hemothorax. The position of the catheter tip was verified with

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an on-table chest X-ray after the insertion was completed. Either titanium-based polysulfone ports with a single lumen or multilumen tunneled catheters were used. When port failure occurred, multilumen tunneled catheters were implanted until a new port could be relocated. The catheter exit site dressings were changed each Monday, Wednesday, and Friday of the week or when the dressing became contaminated or wet. All dressing changes were done with an aseptic technique using a mask and sterile gloves. The site was cleaned with 10% povidone-iodine and covered with a sterile dressing. The catheter was flushed every other day with a standardized procedure using 3 mL of heparin/normal saline solution (100 U/mL), which was prepared daily. The catheter cap was prepared by 10% povidone-iodine application before each needle entry and was changed weekly or as needed. The ports were accessed at least once every 4 weeks using sterile techniques and during periods of prolonged, continuous use, while the needles were changed every 7 days and tubing was renewed every 3 days. Catheter care was performed by trained doctors. Definitions Clinical catheter infection is defined as fever of ≥38.5 °C without any obvious cause of fever and rigors associated with flushing of catheters without microbiologic documentation. Proven catheter infection refers to 2 catheter cultures positive for coagulase-negative Staphylococcus but negative peripheral blood culture or a positive culture for any other microorganism. Catheter-associated bacteremia denotes positive peripheral and central blood cultures. Bacteremia unrelated to catheter means a negative catheter culture but a positive peripheral culture. Catheter-related local infection refers to inflammation (redness, edema, warmth, tenderness, discharge) around the site or along the catheter tunnel [9,10]. Mechanical complications are any malposition or extravasation, while hematological complications include bleeding, hematoma, or thrombosis at the site of insertion. The diagnosis of VTE was based on the detection of thrombi and/or flow absence in noncompressible veins by Doppler ultrasonography [11]. Absolute indications for CVC removal were mechanical complications, successful completion of chemotherapy, tunnel or pocket infections, persistence of fever and positive blood cultures obtained later than 48 h after the initiation of appropriate antimicrobial therapy, septic emboli, and persistent obstruction or thrombosis of a large vein refractory to thrombolysis. Data Collection The medical records of the eligible patients were reviewed by the first author to obtain data on age, sex, primary diagnosis, duration of catheterization, indication of catheterization,

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catheter type, catheter-related complications, and clinical outcomes of these complications. There were no emergency catheter insertions. Statistical Analysis Collected data were analyzed with SPSS 11.5 (SPSS Inc., Chicago, IL, USA). Data distribution was tested by the SmirnovKolmogorov test. Data were expressed as mean±standard deviation or percent where appropriate. Parametric variables of 2 groups were compared by independent-samples t-test, whereas those of 3 groups were compared by the one-way ANOVA. Nonparametric variables of 2 groups were analyzed by Mann-Whitney U test and those of 3 groups were evaluated by the Kruskal-Wallis test and Pearson chi-square test. If the one-way ANOVA or other tests resulted in statistical significance, a post-hoc test was applied. P<0.05 was accepted to be statistically significant. Results The reviewed children had a mean age of 6.4 years (minimum-maximum: 0.2-17 years). Seventy-three children (68.9%) were boys and 33 children (31.3%) were girls. Acute lymphoblastic leukemia (50.9%, n=54) and acute myeloid leukemia (14.2%, n=15) were the most common hematological diseases, followed by aplastic anemia (6.6%, n=7), myelodysplastic syndrome (4.7%, n=5), hemophagocytic syndrome (4.7%, n=5), Fanconi aplastic anemia (5.7%, n=6), hemolytic uremic syndrome (4.7%, n=5), thalassemia major (3.8%, n=4), chronic myeloid leukemia (2.8%, n=3), and juvenile myelomonocytic leukemia (1.9%, n=2). The CVCs were either tunneled (55.2%, n=112) or implanted (44.8%, n=91). These vascular access devices were applied to administer chemotherapy (59.1%, n=120), bone marrow transplantation (27.1%, n=55), drug infusion (6.9%, n=14), total parenteral nutrition (5.4%, n=11), and plasmapheresis (1.5%, n=3). The total and mean durations of catheterization were 40,162 and 378.9 days (minimum-maximum: 1-1460 days), respectively. The rates of clinical catheter infections, local catheter infections, VTE, bleeding, and mechanical complications were 2.6, 1.1, 0.2, 0.2, and 0.2 per 1000 catheter days. Methicillin-resistant Staphylococcus epidermidis was the predominant infectious organism, isolated in 48 of 150 catheter cultures (32.0%) and 19 of 150 blood cultures (12.7%). Catheter cultures also yielded Candida albicans (6.7%, n=10), Staphylococcus saprophyticus (6.7%, n=10), and methicillin-resistant Staphylococcus aureus (6.0%, n=9). Moreover, Escherichia coli (4.7%, n=7), methicillin-sensitive Staphylococcus aureus (2.7%, n=4), and Staphylococcus epidermidis (2.7%, n=4) were detected in blood cultures. About 29.1% of the CVCs (59 of 203) were removed due to


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catheter-related complications. The most frequent causes for catheter removal were infections (42.4%) and mechanical injury (57.6%). No deaths occurred due to catheter-related complications. When compared to 37 patients with other hematological diseases, 69 patients with leukemia had significantly longer Table 1. Catheter complications in patients with leukemia and patients with other hematological diseases.

Leukemia (n=69)

Other Diseases (n=37)

p

Mean catheter duration (days)

375.1±340.1

92.0±140.0

0.001*

Clinical catheter infection

45 (65.3%)

18 (48.6%)

0.046*

Local catheter infection

10 (14.5%)

8 (21.7%)

0.075

Bleeding

4 (5.8%)

4 (10.8%)

0.001*

Venous thromboembolism

5 (7.2%)

3 (8.1%)

0.857

Mechanical complications

5 (7.2%)

4 (10.8%)

0.559

Days until complication development

108.8±196.8

43.5±102.5

0.044*

Catheter removal

12 (17.4%)

10 (27.0%)

0.246

*p<0.05 was accepted to be statistically significant.

duration of catheterization (p=0.001) and higher frequency of clinical catheter infections (p=0.046) (Table 1). When compared to the remaining patients, 28 children who underwent bone marrow transplantation had significantly shorter duration of catheterization (p=0.001), lower frequency of clinical catheter infections (p=0.043), and higher frequency of local catheter infections (p=0.003) (Table 2). When compared to 58 children with tunneled catheters, 48 children with implanted catheters had significantly longer duration of catheterization (p=0.001) and lower frequency of clinical catheter infections (p=0.048) (Table 3). When compared to 34 patients with normal platelet counts, 72 patients with thrombocytopenia had significantly lower local catheter infections (p=0.001) and significantly higher frequency of clinical catheter infections and catheter-related bleeding (p=0.042 and p=0.024) (Table 4). Discussion Despite the widespread utilization of broad-spectrum antibiotics, infections remain a significant cause of morbidity related to catheters. European studies documented that the majority of patients with bloodstream infections had catheters and defined catheter insertion as an independent risk factor for sepsis [12]. A study by Ertem et al. reported that the rate of catheter-related sepsis was 4.9 per 1000 catheter days in children with right atrial catheters [13]. In a similar study, the rate of catheter-related infection was 2.5 per 1000 catheter days in children with totally implantable CVCs [14]. A recent study indicated the rate of catheter-associated bloodstream infections as 7.4 per 1000 catheter days in children [15]. The present study indicates clinical and local infections as the most frequently encountered catheter-related complications. The rates of clinical catheter infection and

Table 2. Catheter complications in patients undergoing bone marrow transplantation and other patients.

Bone Marrow Transplantation (n=28)

Others (n=78)

p

Mean catheter duration (days)

69.6±42.7

322.5±328.9

0.001*

Clinical catheter infection

12 (42.9%)

51 (65.3%)

0.043*

Local catheter infection

10 (35.7%)

6 (7.7%)

0.003*

Venous thromboembolism

1 (3.6%)

7 (9.0%)

0.001*

Bleeding

1 (3.6%)

7 (9.0%)

0.001*

Mechanical complications

2 (7.2%)

7 (9.0%)

0.077

Days until complication development

33.2±30.2

100.8±186.1

0.001*

Catheter removal

5 (17.9%)

17 (21.8%)

0.220

*p<0.05 was accepted to be statistically significant.

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local catheter infection were respectively 2.6 and 1.1 per 1000 catheter days, corresponding to an overall rate of 3.7 episodes per 1000 catheter days. Clinical catheter infections are found to be associated with leukemia, utilization of tunneled catheters, and thrombocytopenia, whereas local catheter infections are related to bone marrow transplantation. The relatively lower rate of catheter-related infections in this study may be attributed to the adoption of highly sterile minimaltouch techniques for catheter insertion, care, and removal. Coagulase-negative staphylococci are responsible for the majority of catheter-related bloodstream infections, whereas gram-negative bacteria, enterococci, and Candida species account for the remainder of these infections [12,16]. Ertem et al. also designated coagulase-negative staphylococci and Candida species as the most common organisms, respectively accounting for 25.0% and 13.1% of catheter-related infections

in children with right atrial catheters [13]. Another study reported that gram-positive bacteria (predominantly coagulase-negative staphylococci) were present in 55% of all infections associated with totally implantable CVCs, but no fungi were detected in blood or catheter cultures [14]. Celebi et al. documented that coagulase-negative Staphylococcus was isolated in 41% of catheter-related bloodstream infections [15]. In accordance with the literature, Staphylococcus epidermidis was the predominant infectious organism in blood and catheter cultures reviewed in this study. However, Staphylococcus epidermidis was specified in only 32% of blood cultures and 13% of catheter cultures. The relatively lower incidence of staphylococcal infection may be caused by the differences in the hematological diagnoses and catheter types. Catheter-related VTE is a serious complication that usually affects critically ill children in whom catheters are applied via

Table 3. Catheter complications in patients with tunneled and implanted catheters.

Tunneled (n=58)

Implanted (n=48)

p

Mean catheter duration (days)

57.5±38.1

505.3±307.5

0.001*

Clinical catheter infection

35 (60.3%)

27 (56.3%)

0.048*

Local catheter infection

9 (15.6%)

9 (18.8%)

0.153

Venous thromboembolism

4 (6.9%)

4 (8.3%)

0.337

Bleeding

5 (8.6%)

4 (8.3%)

0.666

Mechanical complications

5 (8.6%)

4 (8.3%)

0.666

Days until complication development

27.5±28.4

152.9±224.3

0.001*

Catheter removal

12 (20.7%)

9 (18.8%)

0.416

*p<0.05 was accepted to be statistically significant.

Table 4. Catheter complications in patients with low and normal platelet counts.

Platelet Count <150x103/mm3 (n=72)

Platelet Count ≥150x103/mm3 (n=34)

p

Mean catheter duration (days)

262.4±298.0

326.9±366.3

0.355

Clinical catheter infection

50 (69.4%)

13 (38.2%)

0.042*

Local catheter infection

2 (2.8%)

16 (47.1%)

0.001*

Venous thromboembolism

6 (8.3%)

2 (5.9%)

0.571

Bleeding

8 (11.2%)

0 (0.0%)

0.024*

Mechanical complications

6 (8.3%)

3 (8.8%)

0.897

Days until complication development

78.2±155.1

131.3±228.2

0.183

Catheter removal

15 (20.8%)

7 (20.6%)

0.954

*p<0.05 was accepted to be statistically significant.

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Table 5. Literature review of infectious complications of implanted port catheters in children.

Author [Reference]

Number Underlying of Disease Subjects

Total Cath- Mean eter Days Catheter Days

Related Related Local Bloodstream Infections Infections

Shulman, 1987

31

Cancer

7198

232

4 (12.9%)

1 (3.2%)

Becton, 1988

66

Cancer

16,101

227

23 (34.8%)

Unspecified

Ross, 1988

49

Cancer

10,478

350

17 (34.7%)

Unspecified

Schmidt, 1989

41

Cancer

11,138

272

1 (2.4%)

2 (4.8%)

Mirro, 1990

93*

Cancer

Unspecified

419

10 (10.8%)

Unspecified

Severien, 1991

39

Cancer

9611

384

4 (10.3%)

Unspecified

Wiener, 1992

290*

Cancer

189,495

635

Unspecified

Unspecified

DeBacker, 1993

46

Cancer

15,024

290

2 (4.3%)

0

Abdul-Rauf, 1995

25

Sickle cell anemia 17,444

442**

15 (60%)

0

Blanchette, 1996

23

Hemophilia

15,795

687

11 (47.8%)

0

Hollyoak, 1997

73

Cancer

15,251

257

17 (23.3%)

0

Tobiansky, 1997

63

Cancer

13,293

211**

15 (23.8%)

4 (6.3%)

Al-Bassam, 1998

17

Metabolic diseases

7278

428

6 (35.3%)

0

Deerojanawong, 1998

44

Cystic fibrosis

53,057

700**

5 (11.4%)

13 (29.5%)†

Ljung, 1998

53

Hemophilia

49,290

930

9 (17.0%)

0

Miller, 1998

41

Hemophilia

44,070

930

6 (14.6%)

0

Santagostino, 1998

15

Hemophilia

5426

413**

1 (6.7%)

1 (6.7%)

Munro, 1999

134

Cancer

69,342

399**

11 (8.2%)

1 (0.7%)

Aitken, 2000

65

Cystic fibrosis

68,220

784

9 (13.8%)

0

Wildhaber et al., 2000 91 [3]

Cancer

62,488

595

2 (2.2%)

4 (4.4%)

Babu, 2002

41

Hematological diseases

Unspecified

750

2 (4.9%)

0

Hengartner et al., 2004 [23]

155

Cancer

134,773

738**

12 (7.7%)

3 (1.9%)

Loh et al., 2007 [22]

159

Cancer

75,000

407**

33 (20.8%)

5 (3.1%)

This study

106

Hematological diseases

40,162

379

104 (98.1%)

44 (41.5%)

*: Number of implanted catheters. **: Median. †: No catheter was withdrawn.

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the left subclavian vein and used for a prolonged period of time [17,18,19,20,21]. The rate of catheter-related VTE was 0.2 episodes per 1000 days in this study. Meticulous catheter care combined with the use of thrombolytic agents and relatively shorter duration of catheterization may be the underlying reasons for this relatively lower rate. Due to the low rate of catheter-related VTE, no risk factors could be identified for this complication. Previously published studies have addressed thrombocytopenia as a risk factor for catheter-related bleeding. This complication can be prevented by visualizing the catheter route and applying tamponade at the insertion site [19,22]. The rate of catheter-associated bleeding was 0.2 episodes per 1000 days in this study. Although this is a relatively low number, thrombocytopenia also emerges as a risk factor for catheter-related bleeding. A thorough review of the literature demonstrates that up to 67% of inserted catheters are removed for various reasons. When deaths and elective removals are excluded, the most frequent causes of catheter removal are infection and mechanical complications, including dislodgement, leakage, occlusion, and malposition [3,11]. Approximately 88% of right atrial catheters were removed and the most common reasons for removal were infection (42.4%) and dislodgement (32.2%) in a study by Ertem et al. [13]. Another study reported that 29% of all totally implantable catheters were removed due to infections and mechanical complications [14]. Celebi et al. showed that nearly 20% of catheters were removed and the most frequent reasons for removal were infections (44.4%) and mechanical complications (55.6%) [15]. As for the present study, 29% of the CVCs were removed due to catheter-related complications. The most frequent causes for catheter removal were infections (42.4%) and mechanical injury (57.6%). These figures are in accordance with the literature. Leukemia has been identified as a major risk factor for catheter-associated infections [17,23]. Although several studies were unable to detect such a relationship [14,22], this study also describes leukemia as an underlying factor for catheter-related infections. Leukemia precipitates qualitative and quantitative blood cell abnormalities. Furthermore, chemotherapy administered for leukemia can cause immunosuppression and thus induce infections. Bone marrow transplantation was determined as an independent risk factor for catheter-related bloodstream infections [24]. In contrast, this study shows that bloodstream infections were significantly less frequent and local infections were significantly more frequent in children undergoing bone marrow transplantation. Such a discrepancy may be caused by the utilization of implanted catheters in children undergoing bone marrow transplantation.

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Implanted catheters have been designed to lessen the need for special care and to facilitate body movements. The children with implanted catheters were less likely to develop infections and VTE, whereas mechanical complications increased in children who had implanted catheters for the treatment of hematological diseases [24,25]. Table 5 summarizes the findings of previous clinical studies that evaluated the infectious complications in children with implanted catheters [22,23,24]. Variations within study samples and designs are the major confounding factors for the interpretation of the results yielded by the present study and previously published studies. The adoption of different criteria and definitions by different health centers might have contributed to this discrepancy, as well. This study evaluates all catheter-related complications globally and aims to help pediatricians perceive the potential hazards of CVC application in children as a whole. However, its power is limited by its retrospective design and relatively small cohort size. Leukemia, bone marrow transplantation, and thrombocytopenia are risk factors for CVC-associated complications. In order to avoid catheter-related complications, previously established guidelines for catheter insertion, care, and removal should be followed carefully. Further research is warranted to clarify the risk factors for catheter-related complications. 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. Crnich CJ, Maki DG. The promise of novel technology for the prevention of intravascular device-related bloodstream infection. II. Long-term devices. Clin Infect Dis 2002;34:13621368. 2. Bollard CM, Teague LR, Berry EW, Ockelford PA. The use of central venous catheters (portacaths) in children with haemophilia. Haemophilia 2000;6:66-70. 3. Wildhaber B, Kistler W, Caflisch U. Experience with the Port-A-Cath system in children. Schweiz Med Wochenschr 2000;130:732-738. 4. Oâ&#x20AC;&#x2122;Grady NP, Alexander M, Burns LA, Dellinger EP, Garland J, Heard SO, Lipsett PA, Masur H, Mermel LA, Pearson ML, Raad II, Randolph AG, Rupp ME, Saint S; Healthcare Infection Control Practices Advisory Committee (HICPAC). Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis 2011;52:e162-193. 5. Male C, Chait P, Andrew M, Hanna K, Julian J, Mitchell L; PARKAA investigators. Central venous line-related thrombosis in children: association with central venous line location and insertion technique. Blood 2003;101:4271-4278.


Pektaş A, et al: Catheter Complications in Children

6. Merrer J, De Jonghe B, Golliot F, Lefrant JY, Raffy B, Barre E, Rigaud JP, Casciani D, Misset B, Bosquet C, Outin H, BrunBuisson C, Nitenberg G; French Catheter Study Group in Intensive Care. Complications of femoral and subclavian venous catheterization in critically ill patients: a randomized controlled trial. JAMA 2001;286:700-707. 7. Walshe LJ, Malak SF, Eagan J, Sepkowitz KA. Complication rates among cancer patients with peripherally inserted central catheters. J Clin Oncol 2002;20:3276-3281. 8. Ozier Y, Mignon A, Rosencher N. Indications for labile blood products and the physiology of transfusion in surgery. Transfus Clin Biol 2005;12:221-225. 9. Miller DL, O’Grady NP; Society of Interventional Radiology. Guidelines for the prevention of intravascular catheter-related infections: recommendations relevant to interventional radiology for venous catheter placement and maintenance. J Vasc Interv Radiol 2012;23:997-1007. 10. Raad II, Hanna HA. Intravascular catheter-related infections: new horizons and recent advances. Arch Intern Med 2002;162:871-878. 11. Male C, Chait P, Ginsberg JS, Hanna K, Andrew M, Halton J, Anderson R, McCusker P, Wu J, Abshire T, Cherrick I, Mahoney D, Mitchell L. Comparison of venography and ultrasound for the diagnosis of asymptomatic deep vein thrombosis in the upper body in children: results of the PARKAA study. Prophylactic Antithrombin Replacement in Kids with ALL treated with Asparaginase. Thromb Haemost 2002;87:593-599. 12. Bouza E, Burillo A, Muñoz P. Catheter-related infections: diagnosis and intravascular treatment. Clin Microbiol Infect 2002;8:265-274. 13. Ertem M, Yavuz G, Aysev D, Unal E, Gozdasoglu S, Tacyildiz N, Cavdar A, Cin S. Right atrial catheter-related complications in pediatric oncology patients: the situation in a developing country. Pediatr Hematol Oncol 1999;16:299-309. 14. Sarper N, Zengin E, Corapcioglu F, Tugay M. Totally implantable central venous access devices in children with hemato-oncologic malignancies: evaluation of complications and comparison of incidence of febrile episodes with similar patients without central venous access devices. Pediatr Hematol Oncol 2006;23:459-470.

Turk J Hematol 2015;32:144-151

16. Odds FC, Hanson MF, Davidson DA, Jacobsen MD, Wright P, Whyte JA, Gow NA, Jones BL. One year prospective survey of Candida bloodstream infections in Scotland. J Med Microbiol 2007;56:1066-1075. 17. Fratino G, Molinari AC, Parodi S, Longo S, Saracco P, Castagnola E, Haupt R. Central venous catheter-related complications in children with oncological/hematological diseases: an observational study of 418 devices. Ann Oncol 2005;16:648-654. 18. Yazıcı N, Akyüz C, Yalçın B, Varan A, Kutluk T, Büyükpamukçu M. Infectious complications and conservative treatment of totally implantable venous access devices in children with cancer. Turk J Pediatr 2013;55:164-171. 19. Journeycake JM, Manco-Johnson MJ. Thrombosis during infancy and childhood: what we know and what we do not know. Hematol Oncol Clin North Am 2004;18:1315-1338. 20. Male C, Julian JA, Massicotte P, Gent M, Mitchell L; PROTEKT Study Group. Significant association with location of central venous line placement and risk of venous thrombosis in children. Thromb Haemost 2005;94:516-521. 21. Revel-Vilk S, Chan A, Bauman M, Massicotte P. Prothrombotic conditions in an unselected cohort of children with venous thromboembolic disease. J Thromb Haemost 2003;1:915921. 22. Loh AHP, Chan-Hon C. Port-A-Cath insertions in acute leukemia and childhood malignancies. Asian J Surg 2007;30:193-199. 23. Hengartner H, Berger C, Nadal D, Niggli FK, Grotzer MA. Port-A-Cath infections in children with cancer. Eur J Cancer 2004;40:2542-2558. 24. Adler A, Yaniv I, Solter E, Freud E, Samra Z, Stein J, Fisher S, Levy I. Catheter-associated bloodstream infections in pediatric hematology-oncology patients: factors associated with catheter removal and recurrence. J Pediatr Hematol Oncol 2006;28:23-28. 25. Perdikaris P, Petsios K, Vasilatou-Kosmidis H, Matziou V. Complications of Hickman-Broviac catheters in children with malignancies. Pediatr Hematol Oncol 2008;25:375-384.

15. Celebi S, Sezgin ME, Cakir D, Baytan B, Demirkaya M, Sevinir B, Bozdemir SE, Gunes MA, Hacimustafaoglu M. Catheterassociated bloodstream infections in pediatric hematologyoncology patients. Pediatr Hematol Oncol 2013;30:187-194.

151


Research Article

DOI: 10.4274/tjh.2013.0367

The Effect of FcγRIIIA Gene Polymorphism on the Treatment of Diffuse Large B-cell Non-Hodgkin Lymphoma: A Multicenter Prospective Observational Study FcγRIIIA Gen Polimorfizminin Diffüz Büyük B Hücreli NonHodgkin Lenfoma Tedavisine Etkisi: Çok Merkezli Prospektif Gözlemsel Çalışma Nurhilal Büyükkurt1, Mehmet Ali Özcan2, Ülkü Ergene3, Bahriye Payzın4, Sunay Tunalı2, Fatih Demirkan2, Hayri Özsan2, Özden Pişkin2, Bülent Ündar2 1Başkent

University Faculty of Medicine, Adana Education and Research Centre, Clinic of Hematology, Adana, Turkey Eylül University Faculty of Medicine, Department of Hematology, İzmir, Turkey 3Celal Bayar University Faculty of Medicine, Department of Hematology, Manisa, Turkey 4Atatürk Training and Research Hospital, Clinic of Hematology, İzmir, Turkey 2Dokuz

Abstract: Objective: The curative treatment approach for diffuse large B-cell lymphoma (DLBCL) is controversial even in the rituximab

(R) era. The aim of this study was to examine the FcγRIIIA gene polymorphism distribution of DLBCL patients who had been treated with R-CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy. Furthermore, we investigated the impact of FcγRIIIA gene polymorphism on the overall response rate (ORR) and overall survival (OS). Materials and Methods: Patients from 3 centers in the Aegean region of Turkey who had newly diagnosed CD20-positive DLBCL were enrolled in the study. The single nucleotide polymorphisms of the FcγRIIIA gene were analyzed by real timePCR. The response to treatment was determined in the middle and at the end of the protocol. During 2 years of follow-up, the patients were clinically and radiologically evaluated for disease status every 3 months. Results: Thirty-six patients were included in the study and the distributions of F/F, V/F, and V/V types of alleles of FcγRIIIA were 25%, 50%, and 25%, respectively. Twenty-seven patients were considered as evaluable according to ORR and OS. The patients’ ORR was 87.5%, 100%, and 50% in the F/F, V/F, and V/V allele groups, respectively. We did not establish any statistically significant differences among the 3 alleles groups in respect to ORR (p=0.93). The OS within 2 years in the F/F, V/F, and V/V allele groups was 62.5%, 100%, and 100%, respectively. The OS in the F/F allele group was found to be lower than in the other 2 allele groups (p=0.01). Conclusion: The distribution of gene polymorphisms in our study group was similar to those of previous studies. While ORR was similar between the groups, our results highlight a lower OS in F/F patients compared to other allele groups of FcγRIIIA. Key Words: FcγRIIIA, Diffuse large B-cell lymphoma, Rituximab Address for Correspondence: Nurhilal Büyükkurt, M.D., Başkent University Faculty of Medicine, Adana Education and Research Centre, Clinic of Hematology, Adana, Turkey Phone: +90 322 327 27 27 E-mail: nurhilalt@yahoo.com Received/Geliş tarihi : November 02, 2013 Accepted/Kabul tarihi : February 26, 2014

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Özet: Amaç: Diffüz büyük B hücreli non-Hodgkin lenfomada (DLBCL) kür sağlayıcı tedavi yaklaşımı rituximab çağında olmamıza rağmen tartışmalı bir konudur. Bu çalışmanın amacı R-CHOP (siklofosfamid, doksorubisin, vinkristin ve prednizon) rejimi alan DLBCL hastalarında FcγRIIIA gen polimorfizminin dağılımını incelemekti. Ayrıca FcγRIIIA gen polimorfizminin tüm yanıt oranları (ORR) ve tüm yaşam (OS) üzerine olan etkisini araştırmaktı.

Gereç ve Yöntemler: Türkiye’nin Ege Bölgesi’ndeki üç merkezden yeni tanı almış CD-20 pozitif DLBCL hastaları çalışmaya dahil edildi. FcγRIIIA’daki tek gen polimorfizmi gerçek zamanlı-PCR ile incelendi. Tedaviye yanıt, planlanmış olan protokolün ortasında ve sonunda değerlendirildi. İki yıllık takip süresince her üç ayda bir hastalığın hem klinik, hem de radyolojik durumu ele alındı.

Bulgular: Çalışmaya dahil edilen 36 hastada, FcγRIIIA’nın F/F, V/F ve V/V alellerinin dağılımı sırasıyla %25, %50 ve %25’ti. ORR ve OS verilerine göre 27 hasta değerlendirilebilir olarak kabul edildi. Hastaların ORR değerleri F/F, V/F ve V/V alel gruplarına göre sırasıyla %87,5; %100 ve %50 olarak hesaplandı. Hastaların ORR değerleri açısından üç alel grubu arasında istatistiksel olarak anlamlı fark saptanmadı (p=0,93). F/F, V/F ve V/V gruplarında iki yıllık OS %62,5, %100 ve %100 bulundu. F/F alel grubunun OS’si diğer iki alel grubundakinden daha düşük bulunmuştur (p=0,01).

Sonuç: Gen polimorfizmi dağılımı sonuçlarımız önceki çalışmalarda bulunanlarla benzerdir. Gruplar arasında ORR değerleri arasında fark yokken, sonuçlarımız F/F hastalarının FcγRIIIA’nın diğer allel gruplarına göre daha kısa bir OS değerine sahip olduğunu göstermektedir. Anahtar Sözcükler: FcγRIIIA, Diffüz büyük B hücreli lenfoma, Rituksimab Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin lymphoma (NHL). It constitutes 25%-30% of NHLs [1,2]. Cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) combined with rituximab (R) is the standard treatment protocol for DLBCL. Before the introduction of rituximab, which is a human-mouse chimerical anti-CD20 monoclonal antibody, CHOP was used alone. However, it was demonstrated that the addition of rituximab to the treatment protocol improves the complete remission rate and the 5-year event-free survival rate [3]. On the other hand, several studies have shown that R-CHOP has some limitations due to tumor pathobiology. The biological pathway of rituximab in the treatment of lymphomas is still controversial. According to the results of in vivo and in vitro studies, researchers have focused on 2 mechanisms: it increases the efficacy by inducing antibodydependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC) [4,5]. Natural killer (NK) cells, macrophages, and neutrophils play major roles in ADCC. When they recognize the constant region of the Fcγ receptors (FcγR) on the surface of immunoglobulin (Ig), they activate in order to initiate the ADCC cascade [6,7]. Macrophages, NK cells, and some dendritic cells express FcγRIIIA [8]. There may be valine (V) or phenylalanine (F) at the 158th position on the FcγRIIIA gene. An in vitro study showed that these gene polymorphisms (V/V, F/F, and V/F) change the binding affinities to immunoglobulin G (IgG). V/V alleles have the

strongest affinity to IgG, whereas the F/F alleles have the lowest [9]. In the current study, we evaluated the distribution of FcγRIIIA gene single nucleotide polymorphism (SNP) in Turkish patients with DLBCL. The response rate to R-CHOP and overall survival (OS), regarding gene polymorphism, were also investigated. Materials and Methods Patients’ Characteristics and Treatment Protocol Thirty-six newly diagnosed DLBCL patients were included. The subjects were recruited from 3 hospitals in the Aegean region of Turkey over the course of 30 months. Immunohistochemistry staining was performed for all and the presence or absence of CD20 was examined. This study was approved by the Dokuz Eylül University Faculty of Medicine Hospital Ethics Committee and all patients signed an informed consent form. The International Prognostic Index (IPI) score was calculated for the prediction of prognosis at the beginning of therapy. The chemotherapy regimen was administered in 3-week intervals. On the first day, rituximab 375 mg/m2 was given by intravenous infusion over 4-6 h. On the second day, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, and vincristine 1.4 mg/m2 (upper limit of 2 mg) were given intravenously, while prednisone 100 mg/m2 was administered orally on the second day and was then continued for 5 days. While stage 1 or 2 disease was generally treated with 4 cycles

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of R-CHOP followed by involved field radiotherapy (RT), the advanced stages of disease were treated with 6 to 8 cycles of chemotherapy followed by RT if there were bulky tumors. In order to assess the response to treatment, patients were evaluated after the second or fourth cycles of R-CHOP and also after all the planned cycles were completed. Monitoring and reevaluations of patients were performed every 3 months. All evaluations were performed according to the criteria of the International Lymphoma Workshop [10]. Patients fulfilling these follow-up criteria were classified as “evaluable”. FcγRIIIA Gene Polymorphism Four milliliters of peripheral blood was collected into a tube containing EDTA. The genomic DNA was extracted and stored at -80 °C. The FcγRIIIA gene V158F polymorphism was determined by melting curve analysis after fluorescent realtime polymerase chain reaction (RT-PCR) on a Light Cycler (Roche Diagnostics, Basel, Switzerland). The RT-PCR was performed with the FcγRIIIA V158F Toolset for Light Cycler containing specific primers and fluorescent oligonucleotide probes and the Fast Start DNA Master Hybridization Probe Kit (Genes-4U, Neftenbach, Switzerland) according to the manufacturer’s instructions. The RT-PCR protocol consisted of an initial denaturation step at 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, 56 °C for 10 s, and 72 °C for 10 s. The melting protocol consisted of a waiting process at 95 °C for 30 s and at 70 °C for 1 min, and a continuous fluorescence reading from 70 to 99 °C with a rising rate of 0.1 °C per second. Statistical Analysis All available data were analyzed with SPSS 15. We performed descriptive analysis for patients’ age, sex, disease stage, extranodal involvement, bone marrow infiltration, and IPI score according to the FcγRIIIA gene allele groups. The variations of the clinical characteristics and treatment outcomes of the patients among the gene allele groups were compared with Kruskal-Wallis and Mann-Whitney U tests. The survival estimates were calculated by Kaplan-Meier technique and the differences of OS in gene allele groups were analyzed by log-rank test. All the analysis results were interpreted as statistically significant if the p-value was smaller than 0.05. Results There were 16 males (44.4%) and 20 females (55.6%). The median age was 61 (24-82) years at the time of diagnosis. The majority of patients (72.2%) had advanced stage disease (stages 3 and 4). Extranodal involvement was detected in 44.4% of the patients and B symptoms were present in 38.9%. High-intermediate and high IPI scores were observed in 36.1% (n=13) and 27.8% (n=10) of cases, respectively. One-fourth of patients had bulky lesions, and bone marrow infiltration was seen in 13.9%. The data concerning the patients’ characteristics in regard to FcγRIIIA gene alleles is exhibited in Table 1.

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The V/F allele was the most frequent type of FcγRIIIA gene (50%). The incidences of the remaining alleles were equal (25%). We did not find any statistically significant differences among gene allele groups in terms of age, sex, B symptoms, extranodal involvement, bone marrow infiltration, stage, or IPI score (p=0.94, p=0.72, p=0.5, p=0.17, p=0.89, p=0.46, p=0.22, respectively). The overall response rate (ORR) to R-CHOP was examined in 27 patients. The rest could not be reached due to various reasons such as dying before the termination of protocol or moving to another city. According to our results, there was no statistically significant difference in the response rate among the 3 allele groups (p=0.93). We performed survival analysis at the end of the 30th month. Eight patients in the F/F allele group were considered as evaluable. Three of them died within the first Table 1. Patients’ characteristics and treatment outcomes with R-CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen according to FcγRIIIA gene alleles.

F/F

V/F

V/V

Patients, no. (%)

9 (25)

18 (50)

9 (25)

Sex, no. men/women

4/5

7/11

5/4

Disease, no.

Stage 3, 4

7

13

6

Extranodal

6

8

2

Bulky

2

5

2

IPI score, no. Low Low-intermediate

0 2

5 2

2 2

High-intermediate

3

7

3

High

4

4

2

Response

Evaluable

8

15

4

CR

5

14

2

PR

2

1

0

RD

0

0

2

PD

1

0

0

Survival

Dead

3

0

0

Alive

5

15

4

CR: Complete remission, PR: partial remission, RD: refractory disease, PD: progressive disease.


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Büyükkurt N, et al: FcγRIIIA Gene Polymorphism in DLBCL

year of therapy. One of them had progressive disease after R-CHOP and received salvage therapy. Fifteen patients in the V/F allele group were evaluable and all were alive. Four of 9 patients were evaluable in the V/V allele group and none of them died during the study interval. The OS rate within 2 years was 62.5% for the F/F, 100% for the V/F, and 100% for the V/V allele group. We found statistically significant differences among the V/V-V/F and F/F allele groups for OS (p=0.01), as presented in Figure 1. Discussion Until the early 2000s, the standard therapy for DLBCL was the CHOP combination [11]. Since then, rituximab has been added, which was the first monoclonal antibody therapy approved by the Food and Drug Administration [12]. While

Figure 1. Overall survival curve of patients in each genotype.

significant improvement was achieved with rituximab, its mechanism of action is not clearly understood. In vitro and in vivo studies highlighted the importance of ADCC, CDC, and possibly the activation of the intracellular apoptosis signal pathway [4,5]. Recent studies showed that patients with follicular lymphoma, Waldenström’s macroglobulinemia, and chronic lymphocytic leukemia responded to rituximab treatment at different rates due to FcγRIIIA gene SNPs [13,14,15,16]. In 2006, Kim et al. stated that the estimated benefit of R-CHOP therapy in patients having DLBCL with the V/V allele is higher compared to patients with other allele types [8]. Ansell et al. demonstrated that rituximab improves the response rate not only when combined with chemotherapeutic agents, but also when combined with cytokines such as interleukin-12. These cytokines play a role in cellular cytotoxicity [17]. This report supported the idea that rituximab employs its anti-lymphoma effect by inducing major mediator cells of the ADCC pathway such as macrophages, NK cells, and dendritic cells. In this process, rituximab stimulates the effector cells of ADCC, which express FcγR after binding to CD20-positive B-cell lymphoma cells. FcγRIIIA is one of several types of Fcγ receptors. FcγRIIIA gene polymorphisms due to a point mutation at the 158th amino acid position may influence the responses of rituximab in patients with DLBCL. In their in vitro study, Hatjiharissi et al. indicated that individuals with FcγRIIIA V/V and V/F alleles showed higher rates of ADCC activity, because the NK cell surface has increased expression of FcγRIIIA, which leads to a remarkable affinity to rituximab [9]. Four previously published reports showed the clinical meaning of FcγRIIIA SNPs in DLBCL patients. In the first

Table 2. Summary of recent papers investigating the clinical importance of FCγRIIIA single nucleotide polymorphism (SNPs) in diffuse large B-cell lymphoma (DLBCL) patients.

Genotype Distribution

ORR (CR+PR)

OS

VV

VF

FF

VV

VF

Kim et al. [8], n=113

53 (47%)

54 (48%)

6 (5%)

50 (98%)

48 (90%) 3 (50%)

0.048

82±5* for 2 years

Váróczy et al. [11], n=51

29 (56.8%)

12 (23.5%)

10 (19.6%) 25 (86%)

10 (83%) 8 (80%)

NS

90* for 2 years

Zhang et al. [18], n=34

11 (32%)

18 (53%)

5 (15%)

15 (83%) 3 (60%)

0.04

For 3 years

Mitroviç et al. [19], n=58

16 (28%)

32 (55%)

10 (17%)

9 (82%)

12 (75%)

FF

p

27 (84%) 9 (90%)

0.36

VV

VF FF

58

69

40

75* for 2 years

ORR: Overall response rate, CR: complete remission, PR: partial remission, OS: overall survival, NS: not significant, *: total OS value of all types of FCγRIIIA.

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study, Kim et al. found a rapid response to R-CHOP in patients with the V/V allele in DLBCL compared to others allele groups [8]. The second was published by Zhang et al. They reported that V/V and V/F types of FcγRIIIA were evidently more responsive to initial R-CHOP therapy, as well as associated with longer survival [18]. The last 2 publications were made by Váróczy et al. and Mitroviç et al. [11,19]. A summary of these recent papers is depicted in Table 2. The results of the studies stated above are partially compatible with ours. They found no significance between the V/V and F/F alleles in term of event-free survival, OS, and ORR in R-CHOP treatment. As demonstrated in the last 2 papers, we did not find statistically significant differences in ORR, although OS was found significantly lower in F/F homozygous patients compared to the other 2 allele groups. According to this literature, there are 2 opposite observations on the same subject. We suppose that the reason for such contradicting results may be the different actions of the mechanism of rituximab other than the ADCC pathway, as well as different tumor pathobiologies. Our data have some limitations due to small sample size. To our knowledge, there are no other data available about this issue for Turkish patients. We suggest that these findings be assessed as preliminary data for Turkey. Our results were interpreted without drawing a precise conclusion. Acknowledgment This work was supported by a grant from the İzmir Blood Disease and Cancer Research Society. 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. Vural F, Akad Soyer N, Özen P, Dönmez A, Ocakçı S, Saydam G, Çağırgan S, Tombuloğlu M. Non-Hodgkin’s lymphoma with bone involvement: a single center experience with 18 patients. Turk J Hematol 2010;27:29-33. 2. Nitsu N. Current treatment strategy of diffuse large B-cell lymphomas. Int J Hematol 2010;92:231-237. 3. Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, Morel P, Van Den Neste E, Salles G, Gaulard P, Reyes F, Lederlin P, Gisselbrecht C. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002;346:235-242. 4. Cartron G, Watier H, Golay J, Solal-Celigny P. From the bench to bedside: ways to improve rituximab efficacy. Blood 2004;104:2635-2642.

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5. Atay D, Öztürk G, Anak S, Devecioğlu Ö, Ünüvar A, Karakaş Z, Ağaoğlu L. Rituximab therapy for refractory autoimmune thrombocytopenia in patients with systemic lupus erythematosus. Turk J Hematol 2012;29:92-93. 6. Golay J, Zafforoni L, Vaccari T, Lazzari M, Borleri GM, Bernasconi S, Tedesco F, Rambaldi A, Introna M. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 2000;95:3900-3908. 7. Flieger D, Renoth S, Beier I, Sauerbruch T, Schmidt-Wolf I. Mechanism of cytotoxicity induced by chimeric mouse human monoclonal antibody IDEC-C2B8 in CD20-expressing lymphoma cell lines. Cell Immunol 2000;204:55-63. 8. Kim DH, Jung HD, Kim JG, Lee J, Yang DH, Park YH, Do YR, Shin HJ, Kim MK, Hyun MS, Sohn SK. FCGR3A gene polymorphisms may correlate with response to frontline R-CHOP therapy for diffuse large B-cell lymphoma. Blood 2006;108:2720-2725. 9. Hatjiharissi E, Xu L, Santos DD, Hunter ZR, Ciccarelli BT, Verselis S, Modica M, Cao Y, Manning RJ, Leleu X, Dimmock EA, Kortsaris A, Mitsiades C, Anderson KC, Fox EA, Treon SP. Increased natural killer cell expression of CD16, augmented binding and ADCC activity to rituximab among individuals expressing the FcγIIIA-158 V/V and V/F polymorphism. Blood 2007;110:2561-2564. 10. Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-López A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. Report of an international workshop to standardize response criteria for non-Hodgkin’s lymphomas. J Clin Oncol 1993;17:1244-1253. 11. Váróczy L, Zilahi E, Gyetvai A, Kajtár B, Gergely L, Sipka S, Illés A. Fc-gamma-receptor IIIa polymorphism and gene expression profile do not predict the prognosis in diffuse large B-cell lymphoma treated with R-CHOP protocol. Pathol Oncol Res 2012;18:43-48. 12. Riaz W, Hernandez-Ilizaliturri FJ, Czuczman MS. Strategies to enhance rituximab anti-tumor activity in the treatment of CD20-positive B-cell neoplasms. Immunol Res 2010;46:192205. 13. Cartron G, Dacheux L, Salles G, Solal-Celigny P, Bardos P, Colombat P, Watier H. Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcγIIIAa gene. Blood 2002;99:754-758. 14. Treon SP, Hansen M, Branagan AR, Verselis S, Emmanouilides C, Kimby E, Frankel SR, Touroutoglou N, Turnbull B, Anderson KC, Maloney DG, Fox EA. Polymorphisms in FcγIIIA (CD16) receptor expression are associated with clinical response to rituximab in Waldenström’s macroglobulinemia. J Clin Oncol 2005;23:474-481.


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Turk J Hematol 2015;32:152-157

15. Farag SS, Flinn IW, Modali R, Lehman TA, Young D, Byrd JC. FcγIIIA and FcγIIIA polymorphisms do not predict response to rituximab in B-cell chronic lymphocytic leukaemia. Blood 2004;103:1472-1474.

18. Zhang W, Wang X, Li J, Duan MH, Zhou DB. Fcγ receptor IIIA polymorphisms and efficacy of rituximab therapy on Chinese diffuse large B-cell lymphoma. Chin Med J (Engl) 2010;123:198-202.

16. Carlotti E, Palumbo GA, Oldani E, Tibullo D, Salmoiraghi S, Rossi A, Golay J, Pulsoni A, Foà R, Rambaldi A. FcγIIIa and FcγIIIA polymorphisms do not predict clinical outcome of follicular non-Hodgkin’s lymphoma patients treated with sequential CHOP and rituximab. Hematologica 2007;92:11271130.

19. Mitroviç Z, Aurer I, Radman I, Ajdukoviç R, Sertiç J, Labar B. FcγIIIA and FcγIIIA polymorphisms are not associated with response to rituximab and CHOP in patients with diffuse large B-cell lymphoma. Haematologica 2007;92:998-999.

17. Ansell SM, Witzig TE, Kurtin PJ, Sloan JA, Jelinek DF, Howell KG, Markovic SN, Habermann TM, Klee GG, Atherton PJ, Erlichman C. Phase 1 study of interleukin-12 in combination with rituximab in patients with B-cell non-Hodgkin lymphoma. Blood 2002;99:64-74.

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Research Article

DOI: 10.4274/tjh.2013.0161

Presence of Essential Hypertension or Diabetes Mellitus Is a Predictor of Intracranial Bleeding in Elderly Patients: A Study of 108 Patients with Isolated Thrombocytopenia from a Single Reference Center Esansiyel Hipertansiyon ya da Diabetes Mellitus Varlığı İleri Yaş Hastalarda İntrakraniyal Kanamanın Bir Göstergesidir: Tek Referans Merkezinden Toplam 108 İzole Trombositopeni Hastasına Yönelik Bir Çalışma Rajan Kapoor1, Hara Prasad Pati1, Manoranjan Mahapatra1, Anuradha Monga2 1All

India Institute of Medical Sciences
(AIIMS), Department of Hematology, New Delhi, India India Healthcare Services (TPA), New Delhi, India

2MD

Abstract: Objective: Thrombocytopenia poses a significant problem in the elderly. Not only are there varied causes, but it is also associated with significant morbidity and mortality. We carried out a study to learn the causes of isolated thrombocytopenia in elderly patients and to correlate the severity of thrombocytopenia and bleeding manifestations with various etiologic factors and comorbidities. Materials and Methods: A total of 108 patients above 50 years of age presenting with isolated thrombocytopenia (platelet counts of <100x109/L with normal hemoglobin and total leukocyte counts) were enrolled in the study. Detailed history and clinical examinations were carried out for each patient. Complete blood counts were analyzed by automated cell counter. Peripheral smears were examined in all cases. HbsAg, anti-HCV, and anti-HIV testing by enzyme-linked immunosorbent assay was done in all patients. Wherever clinically indicated, bone marrow aspiration biopsy and cytogenetic studies were done.

Results: Out of 108 patients, 102 (94.4%) presented with bleeding tendencies. Twenty-nine (26.8%) presented with serious (World Health Organization grade 3/4) bleedings. Major findings were immune thrombocytopenic purpura in 79 (73.1%), myelodysplastic syndrome in 7 (6.5%), drug-induced thrombocytopenia in 7 (6.5%), and connective tissue disorder in 4 (3.7%) cases. Ten patients presented with intracranial bleedings. Upon logistic regression analysis, comorbidities in the form of essential hypertension and diabetes mellitus were significantly associated with occurrence of intracranial bleeding. There was no correlation of serious bleedings with platelet counts.

Address for Correspondence: Rajan Kapoor, M.D., All India Institute of Medical Sciences
 (AIIMS), Department of Hematology, New Delhi, India E-mail: rjnkapoor@yahoo.com Received/Geliş tarihi : May 08, 2013 Accepted/Kabul tarihi : December 27, 2013

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Kapoor R, et al: Isolated Thrombocytopenia in Elderly

Conclusion: Isolated thrombocytopenia in the elderly is associated with significant morbidity. Diligent clinical and laboratory evaluation is required to elucidate the cause of thrombocytopenia in the elderly. Comorbidities in this population are associated with serious bleedings and not low platelet counts as is commonly thought. Key Words: Thrombocytopenia, Elderly, Immune thrombocytopenic purpura, Intracranial bleeding Özet: Amaç: Trombositopeni ileri yaşlarda ciddi sorunlar doğurur. Bunda sadece çeşitli sebeplerin olduğunu söylemek yeterli

olmayacaktır zira önemli hastalıklarla ve ölümle de çokça ilişkilendirilir. İleri yaştaki hastalarda görülen izole trombositopeninin sebeplerini öğrenmek ve trombositopeninin şiddetiyle çeşitli etiyolojik faktörler ve beraberinde görülen ek hastalıklardaki kanamalar arasında var olan ilişkiyi göstermek amacıyla bir çalışma yürüttük. Gereç ve Yöntemler: İzole trombositopeni (normal hemoglobin ve toplam lökosit sayısı mevcut, trombosit sayısı <100x109/L) görülen 50 yaşın üstündeki toplam 108 hasta bu çalışmaya dahil edildi. Her hasta için ayrı ayrı geçmiş sorgulaması ve klinik incelemeler yapıldı. Tam kan sayımı otomatik hücre sayıcısında analiz edildi. Her olguda çevresel kan yayması incelendi. Her hastada Eliza yöntemi ile HbsAg, anti HCV ve anti HIV testleri yapıldı. Klinik olarak belirtilen her noktada kemik iliği aspirasyon biyopsisi ve sitogenetik analizler yapıldı. Bulgular: Yüz sekiz hastadan 102’sinde (%94,4) kanama eğilimi görüldü. Yirmi dokuz hastada (%26,8) ciddi kanamalar (Dünya Sağlık Örgütü 3./4. derece) yaşandı. Elde edilen ana bulgulara göre 79 olguda (%73,1) immün trombositopenik purpura, 7’sinde (%6,5) miyelodisplastik sendrom, 7’sinde (%6,5) ilaca bağlı oluşan trombositopeni ve 4’ünde de (%3,7) bağ dokusu hastalığı görüldü. Hastaların 10’unda intrakranial kanamalar yaşandı. Lojistik regresyon analizine göre esansiyel hipertansiyon ve diabetes mellitus gibi eşlik eden hastalıkların, intrakranial kanamalarla önemli ölçüde ilişkilendirildiği gösterilmiştir. Trombosit değerleriyle ciddi kanamalar arasında herhangi bir ilişki bulunamamıştır. Sonuç: İleri yaşlardaki hastalarda görülen izole trombositopeni ciddi morbidite nedenidir. Yaşlı hastalarda görülen trombositopeninin sebebini açıklamak için özenli klinik ve laboratuvar değerlendirmelerin yapılması gerekmektedir. Bu popülasyonda görülen diğer hastalıklar ciddi kanamalarla ilişkilidir, ancak genel kanının aksine düşük trombosit değerleriyle kanama arasında herhangi bir ilişkisi yoktur.

Anahtar Sözcükler: Trombositopeni, Yaşlı, İmmün trombositopenik purpura, İntrakranial kanama

Introduction Thrombocytopenia poses an especially significant problem in the elderly, where it can not only lead to life-threatening bleedings, but may also be a presenting feature of underlying hematologic malignancies, which are more common in this age group [1]. Thrombocytopenia in the elderly is associated with significant morbidity and mortality, leading to frequent hospitalizations [2]. The most common type of isolated thrombocytopenia, immune thrombocytopenic purpura (ITP), is not uncommon in the elderly. In a study of 245 adult patients, the highest age-specific incidence was found in patients above 60 years of age [3]. Compared to patients of less than 40 years of age, those older than 60 have been reported to have a higher incidence of major hemorrhagic complications of ITP and a higher ITP-related mortality [1,4]. In view of potential comorbidities such as hypertension, gastrointestinal disorders, and exposure to antiplatelet medications, older individuals have a greater risk of bleeding than children [1]. In addition to ITP, myelodysplastic syndrome (MDS) may present as isolated thrombocytopenia in the elderly [5].

However, no large-scale study of isolated thrombocytopenia in the elderly and its effect on morbidity and mortality has been reported. To analyze the clinical profile, etiology, and correlation of severity of bleeding with various etiologic factors and comorbidities in patients of more than 50 years of age with isolated thrombocytopenia, we carried out a study at a tertiary care referral center. Materials and Methods Patients and Methods All patients above the age of 50 years presenting with isolated thrombocytopenia to the outpatient department or admitted to the hospital with isolated thrombocytopenia were enrolled in the study. This study was an observational, both prospective and retrospective, single institution-based study. All studied patients had applied to the All India Institute of Medical Sciences for treatment. The study was given clearance by the ethics committee of the All India Institute of Medical Sciences, New Delhi. For the purpose of the study, isolated thrombocytopenia was defined as a platelet count of less than 100x109/L with normal total leucocyte count and hemoglobin

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concentration. Low platelet counts as counted by automated cell counter were confirmed by peripheral smear (PS) examination and any pseudothrombocytopenia was ruled out. Patients with low platelet counts (<100x109/L) by automated cell counter but normal counts by PS were excluded from the study. A detailed history and examination was recorded for all enrolled patients. Particular attention was paid to the presence and severity of bleeding symptoms, site of bleeding, and duration of illness prior to presentation. Bleeding severity was graded according to the World Health Organization (WHO) bleeding scale [6]. Detailed history regarding drug intake, including alternative medicines, was recorded. History regarding comorbidities that are prevalent in this age group, mainly hypertension, diabetes mellitus, and coronary artery disease, was also recorded. Physical examination findings of each patient were noted. The presence of bleeding symptoms and their severity was assessed. A detailed systemic evaluation, which included abdominal, respiratory, cardiac, and neurologic examination, was also carried out. Complete blood counts were analyzed by automated cell counter (Sysmex). HbsAg, anti-HCV, and anti-HIV testing by enzyme-linked immunosorbent assay was done in all patients. Thyroid function tests were done in all patients. Bone marrow aspiration and biopsy were done in the following cases: a) Abnormal findings on clinical evaluation, especially presence of lymphadenopathy and/or organomegaly. b) Abnormal findings on PS in any of the cell lines. c) Strong clinical suspicion of MDS or ITP. Bone marrow cytogenetics was also studied in cases of suspected MDS. In the presence of clinical pointers toward connective tissue disorder, especially in females, special tests like ANA, dsDNA, lupus anticoagulant, and anticardiolipin antibodies were also done. Diagnosis Immune Thrombocytopenic Purpura For the diagnosis of ITP, absence of any obvious initiating and/or underlying cause of thrombocytopenia was mandatory. Diagnosis of ITP was based on recently published diagnostic guidelines [7]. All categories of ITP (acute, persistent, and chronic) were included in the study. Bone marrow aspirate and biopsy were done in all cases. Fulfilment of the following criteria were essential for the diagnosis: 1- Normal PS examination, except isolated thrombocytopenia. Large platelets were supportive of but not mandatory for diagnosis. 2- Normal or increased megakaryocyte number on visual assessment by a hematopathologist on bone marrow biopsy. 3- Absence of any megakaryocyte dysplasia, which is indicative of myelodysplasia. 4- Absence of clinically apparent associated conditions or causes of thrombocytopenia.

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Kapoor R, et al: Isolated Thrombocytopenia in Elderly

Myelodysplastic Syndrome MDS was diagnosed on the basis of the WHO criteria [8], which require the presence of dysplasia in hematopoietic cell series or the presence of cytogenetic abnormalities typical of MDS. Drug-Induced Thrombocytopenia Drugs were implicated as a cause of isolated thrombocytopenia if there was definite prior history of ingestion of a drug known to cause thrombocytopenia along with resolution of thrombocytopenia upon discontinuation of the drug. Statistical Analysis Statistical software R 2.11.1 was used (http://www.Rproject.org). Logistic regression analysis was carried out to study correlations of binary outcomes with different variables. Results A total of 108 patients above 50 years of age were enrolled in the study. The median age was 55 years. The oldest patient was 81 years old. Fifty-six patients were female and 52 were male. Mean duration of illness prior to presentation was 140.2 days, with a range from 12 days to 224 days. According to dietary habits, 57 patients (52.7%) were vegans while the rest consumed a mixed diet. Age-wise distribution is illustrated in Table 1. One hundred and two patients had bleeding as a presenting feature. Six patients were incidentally found to have thrombocytopenia during evaluation of unrelated illness. Mucocutaneous bleedings in the form of petechiae and ecchymoses were the only symptom in 38.8% (42/108) of patients. Ear, nose, and throat (ENT) bleedings in the form of gum bleedings or epistaxis were the second most common presentation with 13.8% (15/108) of patients presenting with some form of ENT bleeding. Among the severe bleedings, gastrointestinal bleeding was the presenting feature in 13.8% (15/108) of patients. Ten patients (9.2%) presented with intracranial (IC) bleedings. Out of these, 7 were subdural bleedings, while 3 patients had intraparenchymal bleedings. The underlying diagnosis was ITP in 9 of these patients, while 1 patient had drug-induced thrombocytopenia. On grading the severity of bleeding according to the WHO bleeding scale, 63 patients (58.3%) had grade 1, while serious bleedings of grades 3 and 4 were seen in 17 (15.7%) and 12 (11.1%) patients, respectively. Essential hypertension was the most common comorbidity in the study population with 41 (38%) patients having a history of hypertension. Type 2 diabetes mellitus was present in 27 (25%) patients. Primary hypothyroidism was present in 17.5% (19/108) of patients. Median platelet count was 22x109/L, with a range from 2x109/L to 80x109/L. Breakdown of the final diagnoses of these 108 patients is given in Table 2.


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Occurrence of intracranial (IC) bleeding in patients of isolated thrombocytopenia and its correlation with other variables were analysed (Table 3). Baseline platelet count had no significant effect on occurrence of IC bleedings. Presence of essential hypertension and diabetes mellitus had a significant association with occurrence of IC bleedings. Patients with arterial hypertension were 8.33 times more likely to have IC bleeding (95% CI: 1.64-50). Similarly, diabetics were 3.7 times more likely to have IC bleeding (95% CI: 1.04-14.3). Increased age (p=0.892), male sex (OR: 2.04, 95% CI: 0.557.69), and presence of ITP (OR: 3.91, 95% CI: 0.48-32.1) were not associated with occurrence of IC bleedings. On analysis of severe (WHO grade 3/4) bleeding, essential hypertension was associated with 3.09 times higher risk (95% CI: 1.25-7.66). Patients with ITP had 4.17 times more association with severe bleeding (95% CI: 1.15-14.28). There was no significant difference in baseline platelet count among different diagnostic categories. Discussion A total of 108 patients above the age of 50 years presenting with isolated thrombocytopenia were analyzed in this study. We decided to take 50 years as the cut-off for the definition of â&#x20AC;&#x153;elderlyâ&#x20AC;? as this age better represents our elderly population. Although there are many definitions of old age, there is no Table 1. Age-wise distribution of patients with isolated thrombocytopenia.

Age Group in Years

Number

Percentage

50-59

69

63.8

60-69

22

20.3

70-79

14

12.9

>80

3

3

general agreement on the age at which a person becomes old. In a recent study of prevalence of MDS in an elderly population in Africa, the WHO took as a cut-off 50 years [9]. The most common underlying diagnosis in this age group was ITP, which was expected as the highest age-specific incidence of ITP has been reported in patients above 60 years of age [3]. Although most patients with ITP have overall good outcomes, excessive morbidity and mortality can be attributed to both bleeding and the immunosuppressive treatments to which these patients are subjected [5]. In our series, 29 patients (26.8%) presented with severe (WHO grade 3/4) bleedings. Ten patients (9.2%) had IC bleeding. Out of these, 9 had ITP, whereas in 1 patient, thrombocytopenia was induced by drugs. In a study of 47 elderly patients with ITP, the incidence of brain hemorrhage was 3/47 (6.3%) [6], which is less than the rate of 9/79 (11.3%) found in our study. Although none of the patients died due to thrombocytopenic bleedings, the incidence of IC bleedings Table 2. Diagnosis in 108 patients above 50 years of age presenting with isolated thrombocytopenia.

Diagnosis

Number Percentage

ITP

79

73.1

MDS

7

6.4

Drug-induced immune thrombocytopenia

7

6.4

Systemic lupus erythematosus

3

2.7

Hypersplenism

3

2.7

HCV-related

2

1.8

Others*

7

6.4

*: Others include non-Hodgkin lymphoma, HIV, chronic hepatitis B, postrenal transplant CMV infection, Budd-Chiari syndrome, sarcoidosis, and acute glomerulonephritis in 1 patient each. ITP: immune thrombocytopenic purpura, MDS: myelodysplastic syndrome.

Table 3. Correlation between occurrence of intracranial (IC) bleeding and other variables by logistic regression analysis; n=10.

Variable

Effect

95% CI

Statistical Significance

Baseline platelet count*

p=0.286

Not significant

Increasing age*

p=0.892

Not significant

Male vs. female

OR: 2.04

0.55-7.69

Not significant

Essential hypertension (present vs. absent)

OR: 8.33

1.64-50

Significant

Type 2 diabetes mellitus (present vs. absent)

OR: 3.7

1.04-14.3

Significant

*: Continuous variables; OR: odds ratio.

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Kapoor R, et al: Isolated Thrombocytopenia in Elderly

is much higher than that reported in younger patients. The most common comorbidities were primary hypertension and type II diabetes mellitus, which were seen in 37.9% and 25% cases, respectively. Though comorbidities in ITP have been reported in the past, none of them were specifically studied in the elderly [10]. The importance of this lies in the fact that occurrence of IC bleeding is significantly associated with the presence of comorbidities. Patients with arterial hypertension were 8.33 times more likely to have IC bleeding (95% CI: 1.64-50). Patients with diabetes mellitus were 3.7 times more likely to have IC bleeding (95% CI: 1.04-14.3). Similarly, essential hypertension was associated with 3.09 times greater risk of having severe grade III/IV bleedings (95% CI: 1.257.66). Patients with ITP had 4.17 times greater risk of severe bleedings (95% CI: 1.15-14.28) in comparison to patients with thrombocytopenia due to other etiologies. Baseline platelet count was not predictive of serious bleedings in this elderly population. To our knowledge, the detrimental effect of comorbidities specifically in the elderly population has not been reported. A study of 178 elderly patients with ITP did not report any serious bleeding complications attributable to low platelet counts [11]. In the largest reported retrospective study of ITP in the elderly, risk of severe bleeding was associated with both platelet count (p<0.001; OR: 0.973) and age (p=0.025; OR: 1.039) [12]. On the contrary, according to the results of our study, the presence of comorbidities was a predictor of serious bleedings in elderly patients with ITP. Conclusions Isolated thrombocytopenia is a common clinical problem in the elderly and is associated with significant morbidity and mortality. In patients with ITP, the presence of essential hypertension and diabetes mellitus, which are common in this age group, is associated with occurrence of IC bleeding, which leads to significant morbidity in this otherwise benign disease. Due to the retrospective nature of this study, we could not analyze any correlation between glycemic or blood pressure control and the presence of IC bleedings in this population. Another weakness of this study was that we could not assess B12 and folic acid status in our patients, which are known to be affected by dietary habits, as half of our patients were vegans. Optimal management of comorbidities in this age group is of paramount importance if we are to prevent serious bleedings in the elderly thrombocytopenic population. 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.

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References 1. Cortelazzo S, Finazzi G, Buelli M, Molteni A, Viero P, Barbui T. High risk of severe bleeding in aged patients with chronic idiopathic thrombocytopenic purpura. Blood 1991;77:31-33. 2. Danese MD, Lindquist K, Gleeson M, Deuson R, Mikhael J. Cost and mortality associated with hospitalizations in patients with immune thrombocytopenic purpura. Am J Hematol 2009;84:631-635. 3. Neylon AJ, Saunders PW, Howard MR, Proctor SJ, Taylor PR. Clinically significant newly presenting autoimmune thrombocytopenic purpura in adults: a prospective study of a population-based cohort of 245 patients. Br J Haematol 2003;122:966-974. 4. Linares M, Cervero A, Colomina P, Pastor E, Lopez A, Pérez A, Perella M, Carbonell F. Chronic idiopathic thrombocytopenic purpura in the elderly. Acta Haematol 1995;93:80-82. 5. Kuroda J, Kimura S, Kobayashi Y, Wada K, Uoshima N, Yoshikawa T. Unusual myelodysplastic syndrome with the initial presentation mimicking idiopathic thrombocytopenic purpura. Acta Haematol 2002;108:139-143. 6. Webert K, Cook RJ, Sigouin CS, Rebulla P, Heddle NM. The risk of bleeding in thrombocytopenic patients with acute myeloid leukemia. Haematologica 2006;91:1530-1537. 7. Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold DM, Bussel JB, Cines DB, Chong BH, Cooper N, Godeau B, Lechner K, Mazzucconi MG, McMillan R, Sanz MA, Imbach P, Blanchette V, Kühne T, Ruggeri M, George JN. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 2009;113:2386-2393. 8. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France, IARC, 2008. 9. World Health Organization. Definition of an Older or Elderly Person: Proposed Working Definition of an Older Person in Africa for the MDS Project. Geneva, Switzerland: WHO, ©2014. Available at http://www.who.int/healthinfo/survey/ ageingdefnolder/en/. 10. Feudjo Tepie MA, Roux GL, Beach KJ, Bennett D, Robinson NJ. Comorbidities of idiopathic thrombocytopenic purpura: a population based study. Adv Hematol 2009;2009:963506. 11. Bizzoni L, Mazzucconi MG, Gentile M, Santoro C, Bernasconi S, Chiarotti F, Foà R, Mandelli F. Idiopathic thrombocytopenic purpura (ITP) in the elderly: clinical course in 178 patients. Eur J Haematol 2006:76:210-216. 12. Zhou H, Fu R, Wang H, Zhou F, Li H, Zhou Z, Zhang L, Yang R. Immune thrombocytopenia in the elderly: clinical course in 525 patients from a single center in China. Ann Hematol 2013;92:79-87.


DOI: 10.4274/tjh.2013.0265

Brief Report

Pharmacobiological Approach for the Clinical Development of Ruxolitinib in Myeloproliferative Neoplasms Miyeloproliferatif Neoplazilerde Ruxolitinib İlacının Klinik Geliştirilmesine Farmakobiyolojik Yaklaşım Eylem Eliaçık1, Ayşe Işık1, Salih Aksu1, Ayşegül Üner2, Yahya Büyükaşık1, Nilgün Sayınalp1, Hakan Göker1, Osman İ. Özcebe1, İbrahim C. Haznedaroğlu1 1Hacettepe 2Hacettepe

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

Abstract: Ruxolitinib, a JAK1 and JAK2 inhibitor drug, has recently been approved for the treatment of patients with high- or intermediaterisk myelofibrosis with symptomatic splenomegaly. Ruxolitinib is the first clinically useful targeted therapy in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). The aim of this paper is to indicate pharmacobiological aspects of ruxolitinib within the potential context of MPNs. Pharmacobiological assessments, in addition to knowledge of the risk profile for ruxolitinib in MPNs, are required. We propose hypotheses based on our experience in a splenectomized MPN patient with hyperproliferative bone marrow and moderate fibrosis receiving ruxolitinib. We believe that a true clinical development approach for this drug should include pharmacobiological assessments for ruxolitinib in addition to the disease risk profile of MPNs.

Key Words: Myeloproliferative neoplasms, Ruxolitinib, Myelofibrosis Özet: Ruxolitinib, JAK1 ve JAK2 inhibitörü olarak işlev gören bir ilaçtır. Semptomatik splenomegalisi olan orta- veya yüksek-risk myelofibrozis hastalarında kullanımı uluslararası onam almıştır. Bu bağlamda ruxolitinib, Philadelphia kromozomu negatif myeloproliferatif neoplaziler (MPN) için klinik yararı gösterilen ilk hedefe yönelik ajan konumundadır. Bu yazının amacı, ruxolitinibin MPN’nin klinik tablolarındaki potansiyel kullanım alanları konusunda farmakobiyolojik yönleri tartışmaktır. Ruxolitinib onamları başlıca hastalık risk faktörleri üzerinden yapılmaktadır. Ancak klinik kullanımda hastalığın ve ilacın farmakobiyolojik yönlerini de dikkate alma gerekliliği vardır. Bu hipotezimizi tartışırken splenektomize bir MPN hastamızda, hiperproliferatif bir kemik iliği ve orta derecede fibrozis mevcutken uyguladığımız ruxolitinib tedavisinden elde ettiğimiz deneyimlere dayandık. İlacın gelecekte klinik geliştirilmesi gerçekleştirilirken MPN risk profili yanı sıra farmakobiyolojik değerlendirmelerin de yapılması gerektiği düşüncesindeyiz.

Anahtar Sözcükler: Myeloproliferatif neoplaziler, Ruxolitinib, Miyelofibroz

Address for Correspondence: Eylem ELİAÇIK, M.D., Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey E-mail: eylemeli@gmail.com Received/Geliş tarihi : July 30, 2013 Accepted/Kabul tarihi : December 30, 2013

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Eliaçık E, et al: Ruxolitinib in Myeloproliferative Neoplasms

Introduction Ruxolitinib, a JAK1 and JAK2 inhibitor drug, has recently been approved for the treatment of patients with high- or intermediate-risk myelofibrosis (MF) with symptomatic splenomegaly [1]. This approval in MF depends upon 2 different phase 3 randomized clinical trials (RCTs), namely COMFORT-I and COMFORT-II. COMFORT-I compared ruxolitinib with a placebo in 309 patients with MF, whereas COMFORT-II compared the drug with the best-available therapy (mostly hydroxyurea) in 219 MF patients. Both of the RCTs attained the primary endpoint of >35% reduction in spleen size, as measured by imaging techniques, at 24 or 48 weeks after ruxolitinib treatment initiations [2,3]. Clinical development of ruxolitinib is currently focused on the Philadelphia-negative myeloproliferative neoplastic disorders (Ph -MPNs) [4]. Ruxolitinib is a “JAK-STAT signaling pathway inhibitor” targeted drug with predictable pharmacobiological actions. The main function of the JAK-STAT signaling pathway is cellular proliferation in health and disease. Ruxolitinib should thus be considered as an “anti-proliferative” medicine [4,5,6,7]. Ruxolitinib has the potential to inhibit neoplastic cellular proliferation of MPNs and can cause cytopenias due to its “anti-proliferative” effects in any hematopoietic lineage. The current view of ruxolitinib in MPNs is dependent upon mainly the disease risk profile of the given MPN entity. However, this risk-only approach is not sufficient and can cause the mechanistic wrong decision that ruxolitinib is unnecessary in low-risk MPN. Likewise, ruxolitinib may be considered as ineffective, useless, harmful, or dangerous in (very) high-risk advanced/terminal MPN due to cytopenias of the drug itself. Ruxolitinib could precipitate anemia, leukopenia, and thrombocytopenia in an already pancytopenic patient with MPN. However, there are some initial clues that ruxolitinib can reverse bone marrow fibrosis in MPN if the patient population (such as cases of hyperproliferative bone marrow with splenomegaly and peripheral cytosis) is carefully selected and long-term exposure to the drug (such as 48 months) is possible [8].

revealed systemic hypertension for 35 years and the diagnosis of polycythemia vera (PV) 20 years earlier. In 1994, the patient underwent total gastrectomy and splenectomy in order to cure gastric cancer. The JAK2V617F mutation was also detected in due course. The patient was treated by phlebotomy only until 2003, and then hydroxyurea plus phlebotomy until 2008 to control the disease. At that time, the patient had acute respiratory failure due to hyperviscosity (Plt count over 4 million per mm3 and white blood cell (WBC) count of about 50,000 per mm3), deep vein thrombosis, gastrointestinal bleeding, nasal bleeding, and hydroxyurea-induced skin lesions. After emergency treatment with leukapheresis and Ara-C infusions, an effort was made to control the patient’s thrombocytosis with aa combination of hydroxyurea plus anagrelide. In the following years, the patient had several severe attacks due to hyperleukocytosis (WBC count reaching 120,000 per mm3) and extreme thrombocytosis (Plt count reaching 2 million per mm3) with hyperproliferative bone marrow (Figure 1) requiring intermediate doses of Ara-C infusions for 3-5 days. In June 2012, PEG-IFN treatment (180 µg/week) was initiated to control the PV. In October 2012, ruxolitinib (10 mg b.i.d.) was added to the treatment schedule and the dose of PEG-IFN was set as 90 µg in this dual combination. Complete blood counts were stable and the ongoing hemostatic systemic complications due to cytosis were successfully controlled with this combination treatment (Figure 2). Our patient represents a model of the ideal MPN population in which ruxolitinib should be administered, with hyperproliferative bone marrow with or without fibrosis and peripheral cytosis and organomegaly. Informed consent was obtained.

The aim of this paper is to indicate pharmacobiological aspects of ruxolitinib within the potential context of MPNs. Pharmacobiological assessments, in addition to clarification of the risk profile [9] for ruxolitinib in MPNs, are required. Current clinical challenges for ruxolitinib in MPNs are summarized in Table 1. Pharmacobiological assessments and risk profiles for ruxolitinib in MPNs are depicted in Table 2. Case Report, Methods, and Results: A Typical Myeloproliferative Neoplasms Case to Support the Hypothesis A 64-year-old female patient with elevated blood counts was evaluated in our hematology unit. Medical history 164

Figure 1. Hyperproliferative bone marrow of the patient diagnosed with JAK2V617F-positive polycythemia vera. Hypercellular bone marrow with grade 1 fibrosis and trilineage hyperplasia (100x).


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Eliaçık E, et al: Ruxolitinib in Myeloproliferative Neoplasms

Discussion Patients who have hyperproliferative bone marrow in any lineage (enhanced granulopoiesis, erythropoiesis, thrombopoiesis) plus/minus fibrosis with or without peripheral cytosis (hyperleukocytosis, polycythemia, thrombocytosis), and splenomegaly plus peripheral cytosis (hyperleukocytosis, polycythemia, thrombocytosis); all of the ruxolitinib arm of the RESPONSE trial patients; all ruxolitinib-

Figure 2. Peripheral white blood cell (upper panel) and Plt (lower panel) counts of the patient diagnosed with JAK2V617F-positive polycythemia vera. Control of the neoplastic cellular proliferation was obtained via a PEGintron + ruxolitinib combination.

receiving prefibrotic primary myelofibrosis (PMF, WHO 2008) patients; all ruxolitinib-receiving patients splenectomized for any reason; and any MPN patient to whom ruxolitinib was already administered within other trials (COMFORT-I, COMFORT-II, and others) on compassionate use could be registered and independently evaluated in the context of a ‘Ruxolitinib Pan-MPN Trial’. In this specific MPN patient population (Ruxolitinib Pan-MPN Registry), some critical clinical/laboratory evaluations including effects of ruxolitinib on white blood cell counts (control versus leukopenia) and leukostasis/infections, on hematocrit levels (control versus anemia) and hyperviscosity, on platelet levels (control versus thrombocytopenia) and leukostasis/thrombosis/ hemorrhage, on hepatic enlargement and complications of portal hypertension (particularly after splenectomy), and on hyperproliferative bone marrow neoplastic cellular proliferation and fibrosis would be assessed. MPN disease risk categories of this specific MPN patient population should be detected, as well as the established clinically important ruxolitinib effects (reduction in spleen size and MPN symptoms). A Ruxolitinib Pan-MPN Phase II study should be performed if the Step 1 Ruxolitinib Pan-MPN Registry reveals that ruxolitinib can control hyperproliferative bone marrow (enhanced granulopoiesis, erythropoiesis, thrombopoiesis), fibrosis and/or peripheral cytosis (hyperleukocytosis, polycythemia, thrombocytosis), cytosis-related acute/sub-acute complications (hyperleukocytosis, polycythemia, thrombocytosis), or hepatic enlargement and complications of portal hypertension (particularly after splenectomy), or can decrease spleen size in PV and normalize bone marrow architecture in MPNs in the long

Table 1. Current clinical challenges for ruxolitinib in myeloproliferative neoplasms (MPNs).

(Very) Low-Risk MPN

(Very) High-Risk MPN

Survival is long and may not even differ from that of the healthy population in some low-risk MPNs like ET and PV.

Profound deep (pan) cytopenias already extant in high-risk MPN could become worse after the addition of ruxolitinibinduced cytopenias.

Competitors of ruxolitinib are observation only, low-dose aspirin, phlebotomy, hydroxyurea, and anagrelide. All of them are cheaper and safer in most cases.

Inability to administer ruxolitinib at optimal dosage and duration due to cytopenias in high-risk MPN. Interruption and discontinuation of the drug (ruxolitinib cessation syndrome).

There are no data that long-term ruxolitinib may affect disease course via the prevention of bone marrow fibrosis in low-risk MPN.

Ruxolitinib may not improve already very shortened survival, morbidity, and comorbidities due to advanced/terminal MPNs. Ruxolitinib may not improve irreversible severe organ damage due to advanced/terminal MPN (hepatic failure, portal hypertension bone marrow failure, huge spleen, etc.).

MPN: myeloproliferative neoplasms, PV: polycythemia vera.

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Table 2. The need for pharmacobiological assessments in addition to the risk profile for ruxolitinib in myeloproliferative neoplasms (MPNs).

Current concerns regarding ruxolitinib

Ideal MPN subpopulation for rux- Expected outcome in the given olitinib administration population based on pharmacobiology

Ideal MPN subpopulation for ruxolitinib administration.

Increased bone marrow erythropoiesis (and/or peripheral erythrocytosis).

Suppression of neoplastic erythropoiesis (with Hb control).

Causing drug-induced leukopenia in an already leukopenic MPN patient.

Increased bone marrow granulopoiesis (and/or peripheral leukocytosis).

Suppression of neoplastic granulopoiesis (with WBC control).

Causing drug-induced thrombocytopenia in an already thrombocytopenic MPN patient.

Increased bone marrow thrombopoiesis (and/or peripheral thrombocytosis).

Suppression of neoplastic megakaryocyto-thrombopoiesis (with Plt control).

Ruxolitinib-withdrawal syndrome due to discontinuation.

Expected outcome in the given population Expected outcome in the given based on pharmacobiology. population based on pharmacobiology.

Decreasing spleen size without clinical improvement.

Expected outcome in the given population Controlling the neoplastic cellular based on pharmacobiology. growth preventing the acute cytosis complications/symptoms and reducing spleen size.

Failure to control hepatic extramedullary hematopoiesis.

Already splenectomized MPN patient for any reason and peripheral cytosis.

Controlling the complications of portal hypertension with the inhibition of hepatic extramedullary hematopoiesis.

Failure to modify MPN bone marrow architecture.

Controlling the complications of portal hypertension with the inhibition of hepatic extramedullary hematopoiesis.

Reversal and/or prevention of progression in BM fibrosis.

MPN: myeloproliferative neoplasms, WBC: white blood cell.

term in a study population whose main inclusion criteria are any MPN patient with hyperproliferative bone marrow in any lineage AND bone marrow fibrosis AND splenomegaly AND peripheral cytosis in at least one lineage (hyperleukocytosis, polycythemia, thrombocytosis), and all of the MPN patients with PMF (WHO 2008). These proposals will be tested as hypotheses on efficacy. Hypotheses on safety, including that the degree of ruxolitinibinduced anemia, leukopenia, thrombocytopenia, and related complications is lower in hyperproliferative MPN and that the tolerability and adherence of ruxolitinib with proper dosage and duration is enhanced in patients with hyperproliferative MPNs, will also be tested.

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The development of any drug from bench side to clinic is very difficult and expensive. Therefore, proper scientific strategy is absolutely necessary during the design of clinical studies. Ruxolitinib is the first clinically useful targeted therapy in Ph -MPNs. We think that a true clinical development approach for this drug should include pharmacobiological assessments for ruxolitinib in addition to the disease risk profile of MPNs. 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.


Elia癟覺k E, et al: Ruxolitinib in Myeloproliferative Neoplasms

References 1. Cervantes F, Martinez-Trillos A. Myelofibrosis: an update on current pharmacotherapy and future directions. Expert Opin Pharmacother 2013;14:873-884. 2. Harrison C, Kiladjian JJ, Al-Ali HK, Gisslinger H, Waltzman R, Stalbovskaya V, McQuitty M, Hunter DS, Levy R, Knoops L, Cervantes F, Vannucchi AM, Barbui T, Barosi G. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med 2012;366:787-798. 3. Harrison CN, Mesa RA, Kiladjian JJ, Al-Ali HK, Gisslinger H, Knoops L, Squier M, Sirulnik A, Mendelson E, Zhou X, CopleyMerriman C, Hunter DS, Levy RS, Cervantes F, Passamonti F, Barbui T, Barosi G, Vannucchi AM. Health-related quality of life and symptoms in patients with myelofibrosis treated with ruxolitinib versus best available therapy. Br J Haematol 2013;162:229-239. 4. Haznedaroglu IC. Ruxolitinib for myelofibrosis. N Engl J Med 2012;366:2032.

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5. Pardanani A, Vannucchi AM, Passamonti F, Cervantes F, Barbui T, Tefferi A. JAK inhibitor therapy for myelofibrosis: critical assessment of value and limitations. Leukemia 2011;25:218-225. 6. Tefferi A, Litzow MR, Pardanani A. Long-term outcome of treatment with ruxolitinib in myelofibrosis. N Engl J Med 2011;365:1455-1457. 7. Tefferi A, Pardanani A. Serious adverse events during ruxolitinib treatment discontinuation in patients with myelofibrosis. Mayo Clin Proc 2011;86:1188-1191. 8. Kvasnicka HM, Thiele J, Bueso-Ramos CE, Hou K, Cortes JE, Kantarjian HM, Verstovsek S. Exploratory analysis of the effect of ruxolitinib on bone marrow morphology in patients with myelofibrosis. J Clin Oncol (ASCO Meeting Abstracts) 2013;31:7030. 9. Haznedaroglu IC. The therapeutic goals of essential thrombocythemia under the clouds of over-treatment and undertreatment. Expert Opin Pharmacother 2013;14:1431-1436.

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Case Report

DOI: 10.4274/tjh.2014.0021

Possible Role of Interleukin-31/33 Axis in Imatinib MesylateAssociated Skin Toxicity İmatinib Mesilat ile İlişkili Deri Toksisitesinde İnterlökin-31/33 Aksının Olası Rolü Caterina Musolino1, Alessandro Allegra1, Carmen Mannucci2, Sabina Russo1, Andrea Alonci1, Valerio Maisano1, Gioacchino Calapai2, Sebastiano Gangemi3,4 1University

of Messina Faculty of Medicine, Department of General Surgery and Oncology, Division of Hematology, Messina, Italy Ospedaliera Universitaria Policlinico “G. Martino”, Department of Clinical and Experimental Medicine, Operative Unit of Clinical Pharmacology, Messina, Italy 3University of Messina Faculty of Medicine, Policlinic “G. Martino”, Department of Clinical and Experimental Medicine, Division of Allergy and Clinical Immunology, Messina, Italy 4IFC CNR, Messina Unit, Institute of Clinical Physiology, Messina, Italy 2Azienda

Abstract: Imatinib mesylate is a small-molecule tyrosine kinase inhibitor (TKi) designed to target c-ABL and BCR-ABL, approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Adverse cutaneous reactions induced by imatinib are frequent, generally moderate, and dose-dependent. The aim of this work was to investigate the possible contribution of interleukin (IL)-33 and IL-31, cytokines involved in disorders associated with itching, in the pathogenesis of pruritus in a patient undergoing imatinib mesylate treatment. His IL-31 and IL-33 serum levels were significantly higher than in the control group (respectively 96.6 pg/mL vs. 7.623±7.681 pg/mL and 27.566 pg/mL vs. 6.170±7.060 pg/mL). In light of these findings, imatinib mesylate-related symptoms of dermatologic toxicities might be related to the release of IL-31 and IL-33. In particular, it is supposable that TKi usage could cause keratinocyte injury, the release of IL-33, and the consequent interaction with its receptor on mast cells that induces the secretion of several factors capable of causing skin manifestations, including IL-31, a known pruritus-inducing cytokine. This report, to the best of our knowledge, is the first work describing the possible involvement of the IL-31/IL-33 axis in the pathogenesis of skin side effects related to imatinib mesylate treatment.

Key Words: Interleukin-31 (IL-31), Interleukin-33 (IL-33), Tyrosine kinase inhibitors, Imatinib mesylate, Chronic myeloid leukemia, Pruritus

Özet: İmatinib mesilat c-ABL ve BCR-ABL’yi hedeflemek için tasarlanan küçük- molekül tirozin kinaz inhibitörü (TKİ) olup kronik miyeloid lösemi ve gastrointestinal stromal tümör tedavisi için onaylanmıştır. İmatinib ile indüklenen advers kutanöz reaksiyonlar nadir, genellikle ılımlı ve doz bağımlıdır. Bu çalışmanın amacı, kaşıntı ile ilişkili bozukluklar ile ilgili sitokinler olan interlökin (IL)-33 ve IL-31’in imatinib mesilat tedavisi alan bir hastada kaşıntı patogenezine olası

Address for Correspondence: Alessandro ALLEGRA, M.D., University of Messina Faculty of Medicine, Department of General Surgery and Oncology, Division of Hematology, Messina, Italy Phone: 0039 090 221 23 64 E-mail: aallegra@unime.it Received/Geliş tarihi : January 15, 2014 Accepted/Kabul tarihi : March 13, 2014

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katkısını araştırmak idi. Hastanın serum IL-31 ve IL-33 düzeyleri kontrol grubundan anlamlı olarak yüksek idi (sırasıyla; 96,6 pg/mL vs. 7,623±7,681 pg/mL ve 27,566 pg/mL vs. 6,170±7,060 pg/mL). Bu bulgular ışığında, imatinib mesilatile ilişkili dermatolojik toksisiteler IL-31 ve IL-33 salınımı ile ilişkili olabilir. Özellikle, TKİ kullanımının keratinosit hasarı, IL-33 salınımı ve mast hücre yüzeyindeki reseptörü ile karşılıklı etkileşimi sonucu, deri bulgularına yol açma yeteneği olan, kaşıntıyı-indükleyen bir sitokin olarak bilinen IL-31’de içeren çeşitli faktörlerin sekresyonuna sebep olabileceği varsayılabilir. Bu rapor, bildiğimizce, imatinin mesilat tedavisi ile ilişkili deri yan etkilerinin patogenezinde interlökin-31/33 aksının olası rolünü tanımlayan ilk çalışmadır.

Anahtar Sözcükler: İnterlökin-31 (IL-31), İnterlökin-33 (IL-33), Tirozin kinaz inhibitörleri, İmatinib mesilat, Kronik miyeloid lösemi, Kaşıntı

Introduction Imatinib mesylate is a tyrosine kinase inhibitor (TKi) approved for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors [1]. Several case reports noted the occurrence of dose-limiting skin disorders during imatinib administration [2,3,4,5,6]. Interleukin (IL)33 is a recently recognized cytokine that appears to drive T helper type 2 (Th2) responses [7,8,9]. IL-33 has been linked to important diseases, including asthma, rheumatoid arthritis, ulcerative colitis, and metabolic, neurologic, and cardiovascular diseases. IL-31 is a member of the IL-6 family of cytokines, mainly expressed in pruritic disorders [10]. IL-31 is a Th2 cytokine that is mainly produced by the CD45RO+ cutaneous lymphocyte antigen-positive T lymphocytes. It is involved in both innate and adaptive immunity in tissues that are in close contact with the external environment, i.e. the skin [11]. Recently IL-31 has been demonstrated to be produced by human mast cells [11]; in addition, monocytes, macrophages, and monocyte-derived dendritic cells produce IL-31. Moreover, epidermal keratinocytes and dermal fibroblasts show enhanced IL-31 mRNA expression upon H2O2 stimulation [10]. Enhanced expression of IL-31 is associated with a number of diseases, including pruritic diseases such as atopic dermatitis, allergic contact dermatitis, prurigo nodularis, and chronic urticaria [12]. In a previous work we reported a significant increase of IL31 and IL-33 serum levels in a patient with a bronchoalveolar carcinoma, who had shown previous skin rash, xerosis, and pruritus during treatment with different EGFR-TK inhibitors [13]. The aim of this work was to investigate the possible contribution of IL-31 and IL-33, cytokines involved in disorders associated with itching, in the pathogenesis of pruritus in a patient undergoing imatinib mesylate treatment. Case Presentation A 73-year-old man, while being evaluated for splenomegaly, showed leukocytosis upon peripheral blood examination with low hemoglobin and normal platelet count. His past medical history included hypertension, stroke, and dyslipidemia. He had no history of drug allergy.

His provisional diagnosis was CML, which was subsequently confirmed by the presence of Philadelphia (Ph) chromosome [Ph+ t (9;22) (q34;q11)] in 100% of the cells in metaphase. He was started on cytoreduction with hydroxyurea. Subsequently, he started to take imatinib mesylate at 400 mg once daily. While on therapy, he developed pruritus. Physical examination revealed erythema of the skin associated with mild exfoliation, which affected mainly the upper and lower limbs. There was no history of application of or contact with any irritant substances. Systemic antihistamines were administered. Moreover, the patient was treated with a short course of corticosteroids along with topical clobetasol propionate. Imatinib mesylate was discontinued for 2 weeks, and the patient showed some improvement. Imatinib was restarted at 100 mg once daily and was gradually built up to 300 mg once daily with reappearance of the pruritus. On further follow-up, he had achieved complete hematologic response at 6 months, but failed to achieve a cytogenetic response or a major molecular response at 12 months. His pruritus has become constant and involves his entire body. He is unable to sleep unless medicated with sedatives. The patient is still only in complete hematological response and our intent is to shift to a second-line TKi. We evaluated IL-31 and IL-33 serum levels in this patient and in 18 sex- and age-matched healthy controls. The study was conducted according to good clinical and laboratory practice rules and the principles of the Declaration of Helsinki, and it was approved by the local ethics committee. After obtaining written informed consent, blood samples were collected to determine IL-31 and IL-33 serum levels. We used a standard sandwich ELISA kit (USCN Life Science, Houston, TX, USA). The lower limit of detection was determined as suggested by the manufacturer, as follows: (mean negative control optical density) + 2 x (StDev of negative control optical density). The absorbance was measured at 450 nm. The patient’s IL-31 and IL-33 serum levels were markedly higher than those in the control group (respectively 96.6 pg/ mL vs. 7.623±7.681 pg/mL and 27.566 pg/mL vs. 6.170±7.060 pg/mL). 169


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Discussion and Review of the Literature Imatinib mesylate is a small-molecule TKi designed to target c-ABL and BCR-ABL, but it is also able to target KIT and the platelet-derived growth factor (PDGF) receptor. Adverse cutaneous reactions induced by imatinib are frequent, generally moderate, and dose-dependent [14,15], although all grades of cutaneous reactions have been reported, ranging from exfoliative dermatitis to vesicular rash and Stevens-Johnson syndrome [16,17,18,19]. Concerning the pathogenesis of skin reactions occurring during imatinib administration, a direct effect of the tyrosine kinase inhibition on the PDGF receptor, expressed on dermal mast cells and blood vessels, was suggested [20]. The inhibition of this receptor might cause an augmentation of dermal interstitial fluid pressure with subsequent phenomena of skin edema and erythema. However, the histological evidence for an augmented number of dermal mast cells, which express a functional c-kit receptor, in cases of severe skin toxicity from imatinib mesylate seems to exclude a direct effect of the drug on mast cells themselves [21,22]. As a result, it has also been proposed that imatinib mesylate might operate as a dose-dependent inducer of chemoattractant substances able to induce pruritus [21], such as IL-33 and IL-31. IL-33 has recently been attributed to the epithelial ‘alarmin’ defense system. IL-33 is liberated by the epithelial cells in several tissues and organs, including keratinocytes, immune cells, and endothelial cells [9,23]. It has been proven that IL-33 is recognized by T1/ST2 receptors on the surface of mast cells; this results in the secretion of proinflammatory factors, including IL-6, TNF-α, and leukotrienes. Subsequently, these signals can cause changes, including vasodilatation, increased permeability of the microvasculature, and recruitment of inflammatory cells [23]. The link between pruritus and IL-31 has also been confirmed by a study showing that transgenic mice models over-expressing IL-31 developed severe pruritus and an increase in mast cells [24]. Moreover, it is probable that IL-31 may generate pruritus through the induction of a yet unknown keratinocyte-derived mediator, which subsequently activates unmyelinated C fibers in the skin [25]. It is presumable that the skin manifestations and itch caused by imatinib mesylate treatment could be related to the release of IL-31 and IL-33. It is supposable that TKi usage can cause keratinocyte injury with the release of IL33, which in turn interacts with its receptor on mast cells, leading to the secretion of several factors capable of causing skin manifestations, including IL-31 [26,27]. Finally, our finding of very high serum levels of IL-33 and IL-31 in a CML patient undergoing imatinib mesylate treatment compared to healthy controls has a more relevant

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significance in light of previous works, where we found a significant decrease of IL-33 plasma levels in patients affected by myeloproliferative disorders such as polycythemia vera and essential thrombocythemia and in subjects with other hematologic diseases [28,29,30]. For this reason, although we have no information about basal IL-31 and -33 levels in our CML patient before imatinib treatment, we think that the increase of the values of cytokines after imatinib treatment with respect to the controls is significant. In conclusion, although our report, with the description of a unique case, does not permit us to draw sure conclusions on the possible association between itch and TKi usage, further studies conducted using different TKis such as nilotinib and dasatinib will be useful to better define the role of these cytokines in these patients. 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. Savage DG, Antman KH. Imatinib mesylate – a new oral targeted therapy. N Engl J Med 2002;346:683-693. 2. Brouard MC, Prins C, Mach-Pascual S, Saurat JH. Acute generalized exanthematous pustulosis associated with STI571 in a patient with chronic myeloid leukaemia. Dermatology 2001;203:57-59. 3. Brouard M, Saurat JH. Cutaneous reactions to STI571. N Engl J Med 2001;345:618-619. 4. Drummond A, Micallef-Eynaud P, Douglas WS, Hay I, Holyoake TL, Drummond MW. A spectrum of skin reactions caused by the tyrosine kinase inhibitor imatinib mesylate (STI571, Glivec®). Br J Haematol 2003;120:907-915. 5. Breccia M, Carmosino I, Russo E, Morano SG, Latagliata R, Alimena G. Early and tardive skin adverse events in chronic myeloid leukaemia patients treated with imatinib. Eur J Haematol 2005;74:121-123. 6. Valeyrie L, Bastuji-Garin S, Revuz J, Bachot N, Wechsler J, Berthaud P, Tulliez M, Giraudier S. Adverse cutaneous reactions to imatinib (STI571) in Philadelphia chromosomepositive leukemias: a prospective study of 54 patients. J Am Acad Dermatol 2003;48:201-206. 7. Pushparaj PN, Tay HK, H’ng SC, Pitman N, Xu D, McKenzie A, Liew FY, Melendez AJ. The cytokine interleukin-33 mediates anaphylactic shock. Proc Natl Acad Sci U S A 2009;106:97739778. 8. Joshi AD, Oak SR, Hartigan AJ, Finn WG, Kunkel SL, Duffy KE, Das A, Hogaboam CM. Interleukin-33 contributes to both M1 and M2 chemokine marker expression in human macrophages. BMC Immunol 2010;11:52-60. 9. Cevikbas F, Steinhoff M. IL-33: a novel danger signal system in atopic dermatitis. J Invest Dermatol 2012;132:1326-1329.


Musolino C, et al: IL-31/-33 in Imatinib Skin Toxicity

10. Cornelissen C, Brans R, Czaja K, Skazik C, Marquardt Y, Zwadlo-Klarwasser G, Kim A, Bickers DR, Lüscher-Firzlaff J, Lüscher B, Baron JM. Ultraviolet B radiation and reactive oxygen species modulate interleukin-31 expression in T lymphocytes, monocytes and dendritic cells. Br J Dermatol 2011;165:966-975. 11. Cornelissen C, Lüscher-Firzlaff J, Baron JM, Lüscher B. Signaling by IL-31 and functional consequences. Eur J Cell Biol 2012;91:552-566. 12. Sonkoly E, Muller A, Lauerma AI, Pivarcsi A, Soto H, Kemeny L, Alenius H, Dieu-Nosjean MC, Meller S, Rieker J, Steinhoff M, Hoffmann TK, Ruzicka T, Zlotnik A, Homey B. IL-31: a new link between T cells and pruritus in atopic skin inflammation. J Allergy Clin Immunol 2006;117:411-417. 13. Gangemi S, Franchina T, Minciullo PL, Profita M, Zanghì M, David A, Adamo V. IL-33/IL-31 axis: a new pathological mechanism for epidermal growth factor receptor tyrosine kinase inhibitors-associated skin toxicity. J Cell Biochem 2013;114:2673-2676. 14. Scott LC, White JD, Reid R, Cowie F. Management of skin toxicity related to the use of imatinib mesylate (STI571, Glivec trade mark) for advanced stage gastrointestinal stromal tumours. Sarcoma 2005;9:157-160. 15. Le Nouail P, Viseux V, Chaby G, Billet A, Denoeux JP, Lok C. Drug reaction with eosinophilia and systemic symptoms (DRESS) following imatinib therapy. Ann Dermatol Venereol 2006;133:686-688. 16. van Oosterom AT, Judson IR, Verweij J, Stroobants S, Dumez H, Donato di Paola E, Sciot R, Van Glabbeke M, Dimitrijevic S, Nielsen OS. Update of phase I study of imatinib (STI5721) in advanced soft tissue sarcoma and gastrointestinal stromal tumors: a report of the EORTC Soft Tissue and Bone Sarcoma Group. Eur J Cancer 2002;38:83-87. 17. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, Heinrich MC, Tuveson DA, Singer S, Janicek M, Fletcher JA, Silverman SG, Silberman SL, Capdeville R, Kiese B, Peng B, Dimitrijevic S, Druker BJ, Corless C, Fletcher CD, Joensuu H. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002;347:472-480. 18. Verweij J, van Oosterom A, Blay JY, Judson I, Rodenhuis S, van der Graaf W, Radford J, Le Cesne A, Hogendoorn PC, di Paola ED, Brown M, Nielsen OS. Imatinib mesylate (STI-571 Glivec®, Gleevec™) is an active agent for gastrointestinal stromal tumours, but does not yield responses in other soft tissue sarcomas that are unselected for a molecular target: results from an EORTC Soft Tissue and Bone Sarcoma Group phase II study. Eur J Cancer 2003;39:2006-2011. 19. Verweij J, Casali PG, Zalcberg J, Le Cesne A, Reichardt P, Blay JY, Issels R, van Oosterom A, Hogendoorn PC, Van Glabbeke M, Bertulli R, Judson I. Progression-free survival in gastrointestinal stromal tumors with high-dose imatinib: randomised trial. Lancet 2004;364:1127-1134.

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20. Pietras K, Östman A, Sjöquist M, Buchdunger E, Reed RK, Heldin CH, Rubin K. Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors. Cancer Res 2001;61:29292934. 21. Ugurel S, Hildenbrand R, Dippel E, Hochhaus A, Schadendorf D. Dose-dependent severe cutaneous reactions to imatinib. Br J Cancer 2003;88:1157-1159. 22. Ma Y, Zeng S, Metcalfe DD, Akin C, Dimitrijevic S, Butterfield JH, McMahon G, Longley BJ. The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations. Blood 2002;99:1741-1744. 23. Enoksson M, Lyberg K, Möller-Westerberg C, Fallon PG, Nilsson G, Lunderius-Andersson C. Mast cells as sensors of cell injury through IL-33 recognition. J Immunol 2011;186:2523-2528. 24. Dillon SR, Sprecher C, Hammond A, Bilsborough J, RosenfeldFranklin M, Presnell SR, Haugen HS, Maurer M, Harder B, Johnston J, Bort S, Mudri S, Kuijper JL, Bukowski T, Shea P, Dong DL, Dasovich M, Grant FJ, Lockwood L, Levin SD, LeCiel C, Waggie K, Day H, Topouzis S, Kramer J, Kuestner R, Chen Z, Foster D, Parrish-Novak J, Gross JA. Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice. Nat Immunol 2004;5:752-760. 25. Steinhoff M, Bienenstock J, Schmelz M, Maurer M, Wie E, Biro T. Neurophysiological, neuroimmunological, and neuroendocrine basis of pruritus. J Invest Dermatol 2006;126:1705-1718. 26. Mukhopadhyay A, Do DV, Ong CT, Khoo YT, Masilamani J, Chan SY, Vincent AS, Wong PK, Lim CP, Cao X, Lim IJ, Phan TT. The role of stem cell factor and c-KIT in keloid pathogenesis: do tyrosine kinase inhibitors have a potential therapeutic role? Br J Dermatol 2011;164:372-386. 27. Belleudi F, Cardinali G, Kovacs D, Picardo M, Torrisi MR. KGF promotes paracrine activation of the SCF/c-KIT axis from human keratinocytes to melanoma cells. Transl Oncol 2010;3:80-90. 28. Gangemi S, Allegra A, Profita M, Saitta S, Gerace D, Bonanno A, Alonci A, Petrungaro A, Russo R, Musolino C. Decreased plasma levels of IL-33 could contribute to the altered function of Th2 lymphocytes in patients with polycythemia vera and essential thrombocythemia. Cancer Invest 2013;31:212-213. 29. Musolino C, Allegra A, Profita M, Alonci A, Saitta S, Russo S, Bonanno A, Innao V, Gangemi S. Reduced IL-33 plasma levels in multiple myeloma patients are associated with more advanced stage of disease. Br J Hematol 2013;160:709-710. 30. Musolino C, Allegra A, Profita M, Alonci A, Saitta S, Petrungaro A, Bonanno A, Gerace D, Calabrò L, Gangemi S. Reduction in IL-33 plasma levels might be involved in T-cell dysregulation in chronic lymphocytic leukemia. Acta Haematol 2013;131:165-166.

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Case Report

DOI: 10.4274/tjh.2013.0191

Severe Clinical Course in a Patient with Congenital Amegakaryocytic Thrombocytopenia Due to a Missense Mutation of the c-MPL Gene c-MPL Genindeki Yanlış Anlamlı Mutasyona Bağlı Konjenital Amegakaryositik Trombositopenili Bir Hastada Ağır Klinik Seyir İkbal Ok Bozkaya, Neşe Yaralı, Pamir Işık, Rukiye Ünsal Saç, Betül Tavil, Bahattin Tunç Ankara Children’s Hematology Oncology Hospital, Clinic of Pediatric Hematology, Ankara, Turkey

Abstract: Congenital amegakaryocytic thrombocytopenia (CAMT) generally begins at birth with severe thrombocytopenia and progresses to pancytopenia. It is caused by mutations in the thrombopoietin receptor gene, the myeloproliferative leukemia virus oncogene (c-MPL). The association between CAMT and c-MPL mutation type has been reported in the literature. Patients with CAMT have been categorized according to their clinical symptoms caused by different mutations. Missense mutations of c-MPL have been classified as type II and these patients have delayed onset of bone marrow failure compared to type I patients. Here we present a girl with severe clinical course of CAMT II having a missense mutation in exon 4 of the c-MPL gene who was admitted to our hospital with intracranial hemorrhage during the newborn period.

Key Words: Congenital amegakaryocytic thrombocytopenia, Thrombopoietin, c-MPL, Homozygous missense mutation, c-MPL Tryp154Arg, Amino acid change Özet: Konjenital amegakaryositik trombositopeni (KAMT) genellikle, doğumda ağır trombositopeni ile başlar ve pansitopeniye ilerler. Hastalığın nedeni trombopoetin reseptör geni olan myeloproliferatif lösemi virüs onkogenindeki (c-MPL) mutasyon olup, literatürde KAMT kliniği ile c-MPL mutasyon tipleri arasında ilişki bildirilmiş, hastalar farklı mutasyonların neden olduğu klinik belirtilere göre sınıflandırılmışlardır. c-MPL’nin yanlış anlamlı mutasyonları tip 2 olarak sınıflanmıştır. Bu hastalar tip 1 hastalar ile karşılaştırıldığında kemik iliği yetmezliğinin daha geç başladığı bildirilmiştir. Burada, yenidoğan döneminde intrakranial kanama ile hastanemize başvuran c-MPL geninde ekzon 4’de yanlış anlamlı mutasyonu olan KAMTII tanılı ağır klinik seyirli bir kız sunulmuştur.

Anahtar Sözcükler: Konjenital amegakaryositik trombositopeni, Trombopoetin, Homozigot yanlış anlamlı mutasyon, c-MPL Tryp154Arg, Amino asit değişikliği

Address for Correspondence: İkbal OK BOZKAYA, M.D., Ankara Children’s Hematology Oncology Hospital, Clinic of Pediatric Hematology, Ankara, Turkey Phone: +90 312 396 99 70 E-mail: ikbalok@yahoo.com Received/Geliş tarihi : May 31, 2013 Accepted/Kabul tarihi : December 16, 2013

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Ok Bozkaya İ, et al: Severe Course in CAMT Due to Missense Mutation

Introduction Congenital amegakaryocytic thrombocytopenia (CAMT), one of the autosomal recessive hereditary bone marrow deficiency syndromes, generally begins at birth with severe thrombocytopenia and progresses to pancytopenia [1]. Other syndromes presenting with severe thrombocytopenia at birth are thrombocytopenia with the absence of the radius bone, amegakaryocytic thrombocytopenia with radioulnar synostosis, and Paris-Trousseau syndrome. The molecular pathophysiology of CAMT was explained after the discovery of thrombopoietin (TPO) and its receptor, namely the myeloproliferative leukemia virus oncogene (c-MPL) [2]. The c-MPL gene consists of 12 exons located in the 1p34 locus. In the literature, 41 mutations in the c-MPL gene were defined [1,3,4]. Most CAMT patients have homozygous or compound heterozygous mutations in the c-MPL gene, which lead to absent or impaired reactivity to TPO [5]. Ballmaier et al. published a series of CAMT cases in 2001 and suggested 2 groups of genotype-phenotype features. CAMT I is a severe course of the disease with early development of pancytopenia due to a complete loss of function of the TPO receptor. However, CAMT II patients may show a transient increase of platelet counts during the first year of life with missense mutations of the c-MPL gene [2]. Clinical highlights of CAMT are severe thrombocytopenia secondary to ineffective thrombopoiesis and bone marrow deficiency due to a failure of early hematopoietic progenitors. This indicates the critical role of TPO in both megakaryocytopoiesis and maintenance of stem cells [4]. Thrombocytopenia or associated symptoms appear in 70% of cases at birth and 90% of cases in the first year of life [1]. We hereby present a girl with a severe clinical course of CAMT II having a missense mutation in exon 4 of the c-MPL gene who was admitted to our hospital with intracranial hemorrhage (ICH) during the newborn period. Informed consent was obtained.

Turk J Hematol 2015;32:172-174

of 6 fL. Her mother’s platelet count was normal. Bone marrow aspiration disclosed absence of megakaryocytes. She was diagnosed with CAMT. Analysis of the patient’s TPO revealed a very high level (564 pg/mL; normal range: 120±76 pg/mL); however, her parents’ TPO levels were below 32 pg/mL. Molecular analysis disclosed a homozygous missense mutation in exon 4, which causes a change in arginine instead of tryptophan at the 154th amino acid position. The same heterozygote mutation was detected in her mother, father, and 2 siblings. However, she was lost to follow-up for 2 years. Two years later, she was admitted to our intensive care unit with gastrointestinal bleeding. At that time, laboratory analysis revealed Hb of 47 g/L, WBC count of 8.2x109/L, absolute neutrophil count of 3.7x109/L, and PLT count of 5x109/L. Her bone marrow aspiration smears revealed a decline in bone marrow cellularity and erythroid and myeloid cells, in addition to a decreased number of megakaryocytes (Figure 1). Bone marrow biopsy showed 25% cellularity and a few megakaryocytes were confirmed by CD61 staining. During the follow-up period, an intraventricular shunt was placed in order to treat increased intracranial pressure due to ICH in the prenatal period. During her follow-up for the last 2 years, she has had pancytopenia. The search for a bone marrow donor was unsuccessful among both family and unrelated donors. Discussion and Review of the Literature CAMT is a rare disorder characterized by the lack of megakaryocytic progenitors in the bone marrow. Patients with CAMT are categorized according to their clinical symptoms caused by different mutations [6]. The first group, CAMT I, has total loss of TPO receptors due to homozygous nonsense mutations, deletions, and frame shift mutations, and its clinical course is more severe than that of the other

Case Presentation A 2-day-old girl was admitted to our hospital with petechiae and purpura. Her past medical history revealed that she had ICH at the 28th week of gestation as detected by fetal ultrasonography. No fetal intervention was applied. She was born by cesarean section at the 38th week of gestation with a birth weight of 2750 g. Her parents were first cousins. On physical examination, no congenital abnormalities were revealed. Cranial USG revealed ICH and head computed tomography showed a severe parenchymal hemorrhage on her first day of life. Initial laboratory studies revealed hemoglobin (Hb) of 134 g/L, white blood cell (WBC) count of 10.3x109/L, platelet (PLT) count of 6x109/L, and mean platelet volume

Figure 1. Progression of bone marrow failure in a child with CAMT. A, B, C- Bone marrow aspiration performed at 2 years of age showed no megakaryocytes in a cellular particle with erythroid and myeloid precursors without dysplasia (100x, 1000x, 1000x, respectively). D, E, F- Bone marrow aspirate showed very hypocellular results with few lymphocytes at 2.5 years of age (100x, 100x, 1000x, respectively). 173


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group. Thrombocytopenia is more serious and persistent and pancytopenia begins in the first year of life. In the second group, CAMT II, there are partially functioning TPO receptors due to homozygous or compound heterozygous missense mutations and the clinical features are mild. Transient increases may be seen in platelet counts during the course of the disease. In this group, pancytopenia is not as frequent as in patients with CAMT I [2,7,8,9]. Ballmaier and Germeshausen reported 41 different mutations detected in 58 thrombocytopenic patients with CAMT [1]. More than 60% of these mutations were located in exons 2 or 3. Missense mutations in exon 4 were obtained in 8 patients, whereas a nonsense mutation was found in only 1 patient. Our patient was found to bear a homozygous missense mutation in exon 4. Cases with missense mutations in exon 4 are categorized as CAMT II and the clinical course is expected to be milder than that of patients with CAMT I. However, our patient had a relatively severe clinical course, presenting with ICH in utero and platelet counts that did not increase during follow-up. Pancytopenia developed at the early age of 2 years. Measurement of plasma TPO levels is very important in the evaluation of CAMT, but the TPO level could not be measured routinely. TPO is produced in the liver and removed from circulation by receptor-mediated uptake and degradation [10,11]. Most TPO receptors in megakaryocytes and thrombocytes are expressed by the c-MPL gene [4]. Thrombopoiesis is remarkably decreased in patients with CAMT because of the absence of functional c-MPL expression. As a result, the plasma TPO level is frequently â&#x2030;Ľ10 times higher in children with CAMT than in controls [2]. However, TPO level is normal or slightly increased in patients with immune thrombocytopenia in which platelet destruction is the main cause of thrombocytopenia [12]. CAMT patients usually show 10- to 50-fold elevated TPO levels compared to normal donors. In our patient, the TPO level was higher than normal, but not as high as is observed in patients with CAMT I. However, her platelet count was persistently below 20x109/L and she developed pancytopenia in the very early period, which is not expected in this group of patients. The genotype-phenotype discrepancy despite relatively low levels of TPO in our patient could be caused by factors related to the host or additional genetic alterations that could not be observed during routine analysis. In conclusion, CAMT is a rare cause of thrombocytopenia in childhood. Children suspected of CAMT should be analyzed for mutations in c-MPL, confirmative for the diagnosis of CAMT. The role of the clinical phenotypes of CAMT I and CAMT II is not yet clear, especially regarding the development of bone marrow failure and the influence of other regulatory genes and epigenetic factors on the phenotype of CAMT. Acknowledgment We thank Dr. M. Germeshausen, Hannover Medical School, Germany, for analysis of the TPO levels and genetic mutations in our patient and her family members. 174

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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. Ballmaier M, Germeshausen M. Congenital amegakaryocytic thrombocytopenia: clinical presentation, diagnosis, and treatment. Semin Thromb Hemost 2011;37:673-681. 2. Ballmaier M, Germeshausen M, Schulze H, Cherkaoui K, Lang S, Gaudig A, Krukemeier S, Eilers M, Strauss G, Welte K. c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia. Blood 2001;97:139-146. 3. Savoia A, Dufour C, Locatelli F, Noris P, Ambaglio C, Rosti V, Zecca M, Ferrari S, di Bari F, Corcione A, Di Stazio M, Seri M, Balduini CL. Congenital amegakaryocytic thrombocytopenia: clinical and biological consequences of five novel mutations. Haematologica 2007;92:1186-1193. 4. Geddis AE. Congenital amegakaryocytic thrombocytopenia. Pediatr Blood Cancer 2011;57:199-203. 5. Ballmaier M, Germeshausen M. Advances in the understanding of congenital amegakaryocytic thrombocytopenia. Br J Haematol 2009;146:3-16. 6. Geddis AE. Inherited thrombocytopenias: an approach to diagnosis and management. Int J Lab Hematol 2013;35:14-25. 7. Germeshausen M, Ballmaier M, Welte K. MPL mutations in 23 patients suffering from congenital amegakaryocytic thrombocytopenia: the type of mutation predicts the course of the disease. Hum Mutat 2006;27:296. 8. King S, Germeshausen M, Strauss G, Welte K, Ballmaier M. Congenital amegakaryocytic thrombocytopenia: a retrospective clinical analysis of 20 patients. Br J Haematol 2005;131:636-644. 9. Stoddart MT, Connor P, Germeshausen M, Ballmaier M, Steward CG. Congenital amegakaryocytic thrombocytopenia (CAMT) presenting as severe pancytopenia in the first month of life. Pediatr Blood Cancer 2013;60:E94-E96. 10. Nagata Y, Shozaki Y, Nagahisa H, Nagasawa T, Abe T, Todokoro K. Serum thrombopoietin level is not regulated by transcription but by the total counts of both megakaryocytes and platelets during thrombocytopenia and thrombocytosis. Thromb Haemost 1997;77:808-814. 11. Kuter DJ, Rosenberg RD. The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit. Blood 1995;85:2720-2730. 12. Mukai HY, Kojima H, Todokoro K, Tahara T, Kato T, Hasegawa Y, Kobayashi T, Ninomiya H, Nagasawa T, Abe T. Serum thrombopoietin (TPO) levels in patients with amegakaryocytic thrombocytopenia are much higher than those with immune thrombocytopenic purpura. Thromb Haemost 1996;76:675-678.


Case Report

DOI: 10.4274/tjh.2014.0034

Management of Two Juvenile Myelomonocytic Leukemia Patients According to Clinical and Genetic Features İki Juvenil Myelomonositik Lösemili Hastanın Klinik ve Genetik Bulgulara Göre Tedavisi Özlem Tüfekçi, Hale Ören, Fatma Demir Yenigürbüz, Salih Gözmen, Tuba Hilkay Karapınar, Gülersu İrken Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey

Abstract: Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder of childhood. Major progress has been achieved in diagnosis and the understanding of the pathogenesis of JMML by identifying the genetic pathologies that occur in patients. Mutations of RAS, NF1, PTPN11, and CBL are found in approximately 80% of JMML patients. Distinct clinical features have been reported to be associated with specific gene mutations. The advent of genomic studies and recent identification of novel genetic mutations in JMML are important not only in diagnosis but also in the management and prognosis of the disease. Herein, we present 2 patients with JMML harboring different mutations, NRAS and c-CBL, respectively, with distinct clinical features and different therapeutic approaches.

Key Words: c-CBL mutation, Childhood, Juvenile myelomonocytic leukemia, NRAS mutation Özet: Juvenil myelomonositik lösemi (JMML) çocukluk çağında nadir görülen klonal myeloproliferatif bir hastalıktır. Hastalarda genetik patolojiler saptandıkça JMML’nin tanı ve patogenezini anlamada önemli ilerlemeler kaydedilmiştir. Bu hastaların yaklaşık %80’inde RAS, NF1, PTPN11 ve CBL gen mutasyonları bulunmuştur. Belirli klinik bulgular ile spesifik gen mutasyonları arasında ilişki olduğu bildirilmektedir. JMML’de genomik çalışmalardaki gelişmeler ve son yıllarda tanımlanmış yeni genetik mutasyonların saptanması sadece hastalığın tanısı için değil, tedavi ve prognozunda da önem taşımaktadır. Burada NRAS ve c-CBL mutasyonları olan iki JMML’li hasta, belirli klinik bulguları ve farklı tedavi yaklaşımları ile sunulmaktadır.

Anahtar Sözcükler: c-CBL mutasyonu, Çocukluk çağı, Juvenil myelomonositik lösemi, NRAS mutasyonu

Address for Correspondence: Hale ÖREN, M.D., Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey Phone: +90 232 412 61 41 E-mail: hale.oren@deu.edu.tr Received/Geliş tarihi : January 24, 2014 Accepted/Kabul tarihi : May 13, 2014

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TĂźfekçi Ă&#x2013;, et al: Juvenile Myelomonocytic Leukemia

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder that accounts for 2%-3% of all pediatric leukemias [1,2,3,4,5]. Clinically, patients generally present with pallor, fever, lymphadenopathy, and splenomegaly. Leukocytosis with monocytosis, circulating myeloid/ erythroid precursors, varying degrees of myelodysplasia, and thrombocytopenia are common findings found in peripheral blood [1,2,6,7]. Hypersensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor is characteristic of JMML [8]. Major progress has been achieved in diagnosis and the understanding of the pathogenesis of JMML by identifying the genetic pathologies that occur in patients. Mutations of RAS, NF1, PTPN11, and CBL, the genes involved in the RAS-MAPK pathway, are found in approximately 80% of these patients [9]. The advent of genomic studies and recent identification of novel genetic mutations in JMML are important not only in diagnosis but also in phenotypic presentation, prognosis, and clinical management of the disease [10,11,12,13,14,15,16]. In the past, the only known curative therapy for JMML was hematopoietic stem cell transplantation (HSCT), but recently it was reported that some patients have clinical improvement and long-term survival without any treatment [2,6,7,9,10,12,14,15,16]. Herein, we present 2 patients with JMML harboring different mutations, NRAS and c-CBL, respectively, with distinct clinical features and different clinical courses. Informed consent was obtained. Case Presentation Patient 1 A 9-month-old male was admitted with complaints of fever, bloody diarrhea, and recurrent lower respiratory tract infections of 3 months in duration. His physical examination was normal. The complete blood count analysis was as follows: hemoglobin of 9.4 g/dL, white blood cells of 39.7x109/L (with neutrophil predominance), and platelets of 185x109/L. Immunological and serological testing excluded immunodeficiency and viral infections. Bone marrow aspiration showed myeloid lineage predominance with slight dysmyelopoiesis. Cytogenetic analysis of the bone marrow sample revealed a normal karyotype and a negative result for the BCR/ABL fusion gene. During follow-up, he began to suffer from diarrhea, febrile episodes, and painful vasculitic skin lesions with no infectious origin. Mild thrombocytopenia, monocytosis, and myeloid precursors on peripheral blood smear appeared 6 months after his first admission. Analysis of hemoglobin electrophoresis revealed increased hemoglobin F (23%). Diagnosis of JMML was made according to the current WHO diagnostic criteria [17]. The genetic work-

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up from the peripheral blood sample revealed somatic heterozygous mutation in NRAS exon 2 p61 Q>P and a donor search for HSCT was started immediately after diagnosis. A cytoreductive treatment with 6-mercaptopurine and lowdose cytosine arabinoside was started. Prednisolone at a dose of 2 mg/kg per day was started with possible accompanying autoimmune, autoinflammatory characteristics of the disease. After the 15th day of steroid treatment, his fever, cutaneous lesions, and diarrhea attacks subsided. During follow-up, 6-mercaptopurine, low-dose cytosine arabinoside, and steroids were administered from time to time depending on his clinical features. As soon as a fully matched unrelated donor was found, allogeneic HSCT was performed. He has been in remission for 7 months. Patient 2 A 17-month-old female presented with fever, failure to thrive, and recurrent respiratory tract infections of a few months in duration. She was 71 cm tall (<3rd percentile), weighed 7.5 kg (<3rd percentile), and had an occipitofrontal head circumference of 43 cm (<3rd percentile). Physical examination revealed broad forehead, mild hypertelorism, short upturned nose, prominent philtrum, mild retrognathism, pallor, petechiae, hepatomegaly (3 cm below the costal margin), and splenomegaly (8 cm below the costal margin). The complete blood count analysis showed leukocytosis (53.7x109/L), monocytosis (11.1x109/L), anemia (Hb: 8.9 g/ dL), and thrombocytopenia (platelets: 46x109/L). Peripheral blood smear showed a leukoerythroblastic picture, dysplastic monocytes, and monocytosis (23%). Bone marrow examination revealed cellular smears with myeloid hyperplasia, features of dysmyelopoiesis, and presence of 3% blasts. Cytogenetic studies of the patient revealed a normal karyotype with absence of the BCR/ABL fusion gene, monosomy 7, or any other chromosomal abnormality. Hemoglobin F level was 3%. JMML was diagnosed according to the WHO criteria [17]. Genetic analysis of the peripheral blood samples revealed heterozygous germline mutation in c-CBL exon 8 p371 Y>H. The same genetic mutation was detected in DNA isolated from the hair follicles, indicating the germline origin of the mutation. Her diagnosis was consistent with the CBL syndrome. The family members were negative for c-CBL mutation. Cytoreductive therapy with 6-mercaptopurine (50 mg/m2 per day, given orally) was started and has continued to date. The hepatosplenomegaly decreased in size and leukocytosis regressed. The disease has been stable for 2 years. Discussion and Review of the Literature JMML is clinically a heterogeneous disease [6]. Although most patients experience an aggressive clinical course and die if not treated with HSCT, there are some patients with better clinical course and spontaneous improvement


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Tüfekçi Ö, et al: Juvenile Myelomonocytic Leukemia

[1,3,6,7,12,16]. Classically, young age at diagnosis (<2 years), platelet counts above 33x109/L, and hemoglobin F of less than 15% at diagnosis have been identified as favorable prognostic factors [3,18]. In addition to these known prognostic factors, increased molecular knowledge of the molecular pathogenesis of JMML has made it possible to outline clinical characteristics and prognosis of the disease for some mutation types [10,11,12,13,14,15,16]. Patient 1 was found to have NRAS mutation. RAS proteins are small GTP-binding signaling molecules that control cell proliferation, survival, and differentiation [19]. The activation of RAS is an essential step in the proliferation of cells for most hematopoietic growth factors [20]. Somatic activating point mutations of NRAS or KRAS genes are found in 20%-30% of patients with JMML [6]. Flotho et al. [21] reviewed the clinical and molecular data of 216 cases collected by the EWOG-MDS group and recommended prompt HSCT for every patient with JMML, except children with Noonan syndrome. There are some studies that reported lower relapse rates and better eventfree survival with RAS mutations [22,23]. RAS mutations have been reported to be associated with autoimmune phenomena [11,24,25]. Oliveira et al. [24] reported that NRAS has an immune regulatory function. The clinical syndrome of autoimmune phenomena, lymphocyte accumulation, and somatic mutations in NRAS, previously designated as ALPS type IV, is now reclassified as a new nosologic entity termed RALD, for RAS-associated autoimmune leukoproliferative disease [25,26]. Mutations causing alteration in the structure of CBL protein have recently been associated with various myeloid malignancies, including JMML [13,27,28]. Patient 2 was found to have marked growth retardation, some phenotypic features, and germline c-CBL mutation. Mutation in c-CBL exon 8 p371 Y>H is the most commonly encountered mutation detected in CBL syndrome [13,14]. Niemeyer et al. [13] reported that the common p371 Y>H mutation causing alteration in CBL protein induces cytokine-independent growth and constitutive phosphorylation only in hematopoietic cells. There are limited reports in the literature about CBL syndrome, but it is important to note that most of these studies have documented the spontaneous resolution rate of JMML in this group of patients [14,15,16]. On the other hand, these patients with CBL syndrome have also been reported to develop serious vasculopathies later in life as evidenced by optic atrophy, hypertension, cardiomyopathy, or arteritis [14]. As our patient harbored germline c-CBL mutation and had a chance of spontaneous resolution, HSCT was not planned as an initial treatment. In conclusion, JMML is phenotypically and genotypically a heterogeneous disease. Despite the aggressive clinical course observed in most patients, some patients may have a mild

clinical course. Recent developments in identifying molecular lesions have revealed the importance of genotype-phenotype correlation in this disease, which is critical for tailoring the management. Every patient with a possible diagnosis of JMML should be screened for underlying molecular lesions. Acknowledgment We are grateful to Prof. Dr. Charlotte M. Niemeyer, Dr. Mutlu Kartal-Kaess, and Dr. Annamaria Cseh for performing genetic analysis in the Division of Pediatric Hematology and Oncology and Department of Pediatrics and Adolescent Medicine, University Clinic of Freiburg, Germany, and for their contributions in the diagnosis and management of the patients. 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. Niemeyer CM, Kratz CP. Pediatric myelodysplastic syndromes and juvenile myelomonocytic leukemia: molecular classification and treatment options. Br J Hematol 2008;140:610-624. 2. Locatelli F, Nöllke P, Zecca M, Korthof E, Lanino E, Peters C, Pession A, Kabisch H, Uderzo C, Bonfim CS, Bader P, Dilloo D, Stary J, Fischer A, Revesz T, Führer M, Hasle H, Trebo M, van den Heuvel-Eibrink MM, Fenu S, Strahm B, Giorgiani G, Bonora MR, Duffner U, Niemeyer CM; European Working Group on Childhood MDS; European Blood and Marrow Transplantation Group. Hematopoietic stem cell transplantation (HSCT) in children with juvenile myelomonocytic leukemia (JMML): results of the EWOGMDS/EBMT trial. Blood 2005;105:410-419. 3. Niemeyer CM, Arico M, Basso G, Biondi A, Cantu Rajnoldi A, Creutzig U, Haas O, Harbott J, Hasle H, Kerndrup G, Locatelli F, Mann G, Stollmann-Gibbels B, van’t Veer-Korthof ET, van Wering E, Zimmermann M. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. European Working Group on Myelodysplastic Syndromes in Childhood (EWOG-MDS). Blood 1997;89:3534-3543. 4. Luna-Fineman S, Shannon KM, Atwater SK, Davis J, Masterson M, Ortega J, Sanders J, Steinherz P, Weinberg V, Lange BJ. Myelodysplastic and myeloproliferative disorders of childhood: a study of 167 patients. Blood 1999;93:459-466. 5. Sasaki H, Manabe A, Kojima S, Tsuchida M, Hayashi Y, Ikuta K, Okamura I, Koike K, Ohara A, Ishii E, Komada Y, Hibi S, Nakahata T; MDS Committee of the Japanese Society of Pediatric Hematology, Japan. Myelodysplastic syndrome in childhood: a retrospective study of 189 patients in Japan. Leukemia 2001;15:1713-1720.

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6. Yoshimi A, Kojima S, Hirano N. Juvenile myelomonocytic leukemia: epidemiology, etiopathogenesis, diagnosis, and management considerations. Paediatr Drugs 2010;12:11-21. 7. Loh ML. Recent advances in the pathogenesis and treatment of juvenile myelomonocytic leukaemia. Br J Haematol 2011;152:677-687. 8. Emanuel PD, Bates LJ, Castleberry RP, Gualtieri RJ, Zuckerman KS. Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. Blood 1991;77:925929. 9. Loh ML, Mullighan CG. Advances in the genetics of high-risk childhood B-progenitor acute lymphoblastic leukemia and juvenile myelomonocytic leukemia: implications for therapy. Clin Cancer Res 2012;18:2754-2767. 10. Yoshida N, Doisaki S, Kojima S. Current management of juvenile myelomonocytic leukemia and the impact of RAS mutations. Paediatr Drugs 2012;14:157-163. 11. Takagi M, Shinoda K, Piao J, Mitsuiki N, Takagi M, Matsuda K, Muramatsu H, Doisaki S, Nagasawa M, Morio T, Kasahara Y, Koike K, Kojima S, Takao A, Mizutani S. Autoimmune lymphoproliferative syndrome-like disease with somatic KRAS mutation. Blood 2011;117:2887-2890. 12. Matsuda K, Shimada A, Yoshida N, Ogawa A, Watanabe A, Yajima S, Iizuka S, Koike K, Yanai F, Kawasaki K, Yanagimachi M, Kikuchi A, Ohtsuka Y, Hidaka E, Yamauchi K, Tanaka M, Yanagisawa R, Nakazawa Y, Shiohara M, Manabe A, Kojima S, Koike K. Spontaneous improvement of hematologic abnormalities in patients having juvenile myelomonocytic leukemia with specific RAS mutations. Blood 2007;109:54775480. 13. Loh ML, Sakai DS, Flotho C, Kang M, Fliegauf M, Archambeault S, Mullighan CG, Chen L, Bergstraesser E, Bueso-Ramos CE, Emanuel PD, Hasle H, Issa JP, van den Heuvel-Eibrink MM, Locatelli F, Stary J, Trebo M, Wlodarski M, Zecca M, Shannon KM, Niemeyer CM. Mutations in CBL occur frequently in juvenile myelomonocytic leukemia. Blood 2009;114:18591863. 14. Niemeyer CM, Kang MW, Shin DH, Furlan I, Erlacher M, Bunin NJ, Bunda S, Finklestein JZ, Sakamoto KM, Gorr TA, Mehta P, Schmid I, Kropshofer G, Corbacioglu S, Lang PJ, Klein C, Schlegel PG, Heinzmann A, Schneider M, Starý J, van den Heuvel-Eibrink MM, Hasle H, Locatelli F, Sakai D, Archambeault S, Chen L, Russell RC, Sybingco SS, Ohh M, Braun BS, Flotho C, Loh ML. Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia. Nat Genet 2010;42:794-800.

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15. Pérez B, Mechinaud F, Galambrun C, Ben Romdhane N, Isidor B, Philip N, Derain-Court J, Cassinat B, Lachenaud J, Kaltenbach S, Salmon A, Désirée C, Pereira S, Menot ML, Royer N, Fenneteau O, Baruchel A, Chomienne C, Verloes A, Cavé H. Germline mutations of the CBL gene define a new genetic syndrome with predisposition to juvenile myelomonocytic leukaemia. J Med Genet 2010;47:686-691. 16. Matsuda K, Taira C, Sakashita K, Saito S, Tanaka-Yanagisawa M, Yanagisawa R, Nakazawa Y, Shiohara M, Fukushima K, Oda M, Honda T, Nakahata T, Koike K. Long-term survival after nonintensive chemotherapy in some juvenile myelomonocytic leukemia patients with CBL mutations, and the possible presence of healthy persons with the mutations. Blood 2010;115:5429-5431. 17. Chan RJ, Cooper T, Kratz CP, Weiss B, Loh ML. Juvenile myelomonocytic leukemia: a report from the 2nd International JMML Symposium. Leuk Res 2009;33:355-362. 18. Passmore SJ, Chessells JM, Kempski H, Hann IM, Brownbill PA, Stiller CA. Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia in the UK: a populationbased study of incidence and survival. Br J Haematol 2003;121:758-767. 19. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003;3:459-465. 20. Satoh T, Nakafuku M, Miyajima A, Kaziro Y. Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/ macrophage colony-stimulating factor, but not from interleukin 4. Proc Natl Acad Sci U S A 1991;88:3314-3318. 21. Flotho C, Kratz CP, Bergsträsser E, Hasle H, Stary J, Trebo M, van den Heuvel-Eibrink MM, Wojcik D, Zecca M, Locatelli F, Niemeyer CM; European Working Group of Myelodysplastic Syndromes in Childhood. Genotype-phenotype correlation in cases of juvenile myelomonocytic leukemia with clonal RAS mutations. Blood 2008;111:966-967. 22. Yoshida N, Yagasaki H, Xu Y, Matsuda K, Yoshimi A, Takahashi Y, Hama A, Nishio N, Muramatsu H, Watanabe N, Matsumoto K, Kato K, Ueyama J, Inada H, Goto H, Yabe M, Kudo K, Mimaya J, Kikuchi A, Manabe A, Koike K, Kojima S. Correlation of clinical features with the mutational status of GM-CSF signaling pathway-related genes in juvenile myelomonocytic leukemia. Pediatr Res 2009;65:334-340. 23. Bresolin S, Zecca M, Flotho C, Trentin L, Zangrando A, Sainati L, Stary J, de Moerloose B, Hasle H, Niemeyer CM, Te Kronnie G, Locatelli F, Basso G. Gene expression-based classification as an independent predictor of clinical outcome in juvenile myelomonocytic leukemia. J Clin Oncol 2010;28:1919-1927.


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24. Oliveira JB, Bidere N, Niemela JE, Zheng L, Sakai K, Nix CP, Danner RL, Barb J, Munson PJ, Puck JM, Dale J, Straus SE, Fleisher TA, Lenardo MJ. NRAS mutation causes a human autoimmune lymphoproliferative syndrome. Proc Natl Acad Sci U S A 2007;104:8953-8958. 25. Niemela JE, Lu L, Fleisher TA, Davis J, Caminha I, Natter M, Beer LA, Dowdell KC, Pittaluga S, Raffeld M, Rao VK, Oliveira JB. Somatic KRAS mutations associated with a human nonmalignant syndrome of autoimmunity and abnormal leukocyte homeostasis. Blood 2011;117:2883-2886.

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27. Schmidt MH, Dikic I. The Cbl interactome and its functions. Nat Rev Mol Cell Biol 2005;6:907-918. 28. Caligiuri MA, Briesewitz R, Yu J, Fleisher TA, Jaffe ES, Lenardo MJ, Rieux-Laucat F, Siegel RM, Su HC, Teachey DT, Rao VK. Novel c-CBL and CBL-b ubiquitin ligase mutations in human acute myeloid leukemia. Blood 2007;110:1022-1024.

26. Oliveira JB, Bleesing JJ, Dianzani U, Fleisher TA, Jaffe ES, Lenardo MJ, Rieux-Laucat F, Siegel RM, Su HC, Teachey DT, Rao VK. Revised diagnostic criteria and classification for the autoimmune lymphoproliferative syndrome (ALPS): report from the 2009 NIH International Workshop. Blood 2010;116:e35-40.

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Case Report

DOI: 10.4274/tjh.2013.0338

Ruxolitinib Treatment in a Patient with Primary Myelofibrosis Resistant to Conventional Therapies and Splenectomy: A Case Report Konvansiyonel Tedavilere ve Splenektomiye Refrakter Primer Myelofibrozisli Olguda Ruxolitinib Deneyimi Meltem Aylı1, Muhit Özcan2, Güldane Cengiz Seval1 1Ufuk

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

2Ankara

Abstract: A 67-year-old male patient who was diagnosed with primary myelofibrosis 4 years ago did not respond to conventional therapies. The splenomegaly progressively increased, which caused spleen infarctions and led to the decision to perform a splenectomy procedure. After splenectomy, the patient started treatment with ruxolitinib. In the first month of ruxolitinib treatment, the patient became transfusion-free and all constitutional symptoms disappeared. However, in the sixth month of ruxolitinib treatment, the disease transformed to acute myeloblastic leukemia, and the patient died 1 month later. This is the first case report that shows the effects of ruxolitinib in a splenectomized patient.

Key Words: Primary myelofibrosis, Ruxolitinib, Splenectomy Özet: Dört yıl önce primer myelofibrozis tanısı konulan 67 yaşındaki erkek hastaya uygulanan konvansiyonel tedavi yöntemleri ile sonuç alınamadı. Dalak boyutları ileri derecede arttı, hastanın tekrar ayda 4-6 ünite transfüzyon gereksinimi olmaya başladı. Bu dönemde dev boyutlara ulaşan dalakta infarktüs gelişti ve hastaya splenektomi yaptırıldı. Splenektomi sonrası hastaya ruxolitinib başlandı. Ruxolitinib tedavisinin 1. ayından itibaren hasta transfüzyon bağımsız hale geldi, tüm konstitüsyonel semptomlar ortadan kalktı. Ancak ruxolitinib tedavisinin 6. ayında hasta akut myeloblastik lösemiye (AML) transfore oldu. Ve AML tedavisinin 1. ayında hasta kaybedildi. Bu olgu splenektomi yapılmış bir hastada ruxolitinib etkisini gösteren ilk olgudur.

Anahtar Sözcükler: Primer myelofibrozis, Ruxolitinib, Splenektomi

Address for Correspondence: Güldane CENGİZ SEVAL, M.D., Ufuk University Faculty of Medicine, Department of Hematology, Ankara, Turkey E-mail: guldanecengiz@gmail.com Received/Geliş tarihi : October 03, 2013 Accepted/Kabul tarihi : May 16, 2014

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Aylı M, et al: Ruxolitinib in Refractory Primary Myelofibrosis

Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by stem cell-derived clonal myeloproliferation, hypersensitivity to cytokines, reactive bone marrow fibrosis, and extramedullary hematopoiesis. Clinical manifestations are splenomegaly, severe anemia and cytopenias, constitutional symptoms (e.g., hypercatabolic state, fatigue, night sweats, fever), cachexia, bone pain, osteosclerosis, splenic infarct, pruritus, thrombosis, bleeding, leukemic progression, and shortened survival [1]. The pathogenesis of the disease is currently not understood. PMF is a clonal disorder of the hematopoietic stem cells in which the fibrosis is a reactive process involving the interaction of multiple cytokines, such as platelet-derived growth factor (PDGF), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF). Recent studies have shown mutations that directly or indirectly lead to the deregulated activation of Janus-activated kinase 2 (JAK2). About half of patients with myelofibrosis carry a gain-of-function mutation in the Janus kinase 2 gene (JAK2 V617F) that contributes to the pathophysiology of the disease [2,3]. Conventional medications are largely palliative and rarely provide durable benefits, whereas stem cell transplantation is restricted to a small percentage of patients. These limitations underscore the need to develop more effective disease-targeted therapeutic approaches in patients with myelofibrosis. Appreciation for the activation of JAK2 and the importance of the pathogenesis of myelofibrosis has led to novel therapeutic agents targeting JAKs [4]. Ruxolitinib is an orally available and potent selective inhibitor of JAK1 and JAK2, and it is the most advanced JAK1/JAK2 inhibitor in development for the treatment of myeloproliferative neoplasms. Previous studies showed regression in splenomegaly during ruxolitinib treatment, but there has been no evidence that ruxolitinib has the same effect in splenectomized patients or what the consequences of it are in this patient population. In this case report, we present the results of ruxolitinib treatment in a JAK2 mutation-negative primary myelofibrosis patient who also had a mandatory splenectomy operation. Informed consent was obtained. Case Presentation A 67-year-old male patient presented to us 4 years ago with a 1-month history of fatigue, night sweats, and abdominal distention. Splenomegaly was observed on physical examination; his spleen was 12 cm below the costal margin. There was no lymphadenopathy. Laboratory findings were as follows: white blood cell (WBC) count was 12,600/

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mm3, hemoglobin level was 9.0 g/dL with MCV of 86 fL, hematocrit was 26%, erythrocyte count was 3.09x1012/L, platelet count was 450x109/L, and lactate dehydrogenase was 845 IU/L. Peripheral blood smear showed normocytic anemia, tear drop-shaped red blood cells (RBCs) (dacryocytes), and leukoerythroblastosis (nucleated RBCs and granulocyte precursors). The bone marrow aspirate was a dry tap. Bone marrow biopsy revealed an increased number of megakaryocytes and a moderate increase of reticulin fibers. The biopsy results were reported as myelofibrosis. Assays for JAK2 V617F and the Philadelphia chromosome were negative. Chromosomal analysis showed no abnormalities. We investigated the secondary myelofibrosis events, but all of them were negative. These findings showed that the patient had primary myelofibrosis. The prognostic score of the patient was calculated as intermediate-2 according to the International Prognostic Scoring System. Treatment of myelofibrosis-related anemia was started with androgen (danazol, 600 mg/day). After treatment with danazol for 3 months, it became clear that there was no increase in hemoglobin levels and so danazol treatment was stopped immediately. Treatment of myelofibrosisrelated anemia was then started with hydroxyurea but myelosuppression began, and so hydroxyurea treatment was also stopped. In place of hydroxyurea, treatment of myelofibrosis-related anemia was started with interferonalpha at 3 million IU subcutaneously 3 times/week, but the patient could not tolerate it. In the meantime, he became transfusion-dependent again and needed, on average, 4-6 units of erythrocyte suspension per month. Afterwards, treatment with lenalidomide (25 mg/day each 21 days of 28 days) was started. After this treatment his constitutional symptoms regressed and hemoglobin levels increased, but the splenomegaly never regressed. The patient was followed under lenalidomide treatment for about 18 months. During this period of time, he did not require any transfusions. However, in the 19th month, hemoglobin levels decreased to 6 g/dL and his spleen became enlarged. He gained weight, had night sweats, and became transfusion-dependent again after 4 months. Lenalidomide treatment was stopped and then we applied for compassionate use of ruxolitinib. During the application procedure, the patient’s spleen size increased progressively; because of trouble with spleen infarctions, a splenectomy procedure was performed. Following the first month after splenectomy, ruxolitinib at 10 mg/day was given to the patient, who had a poor prognosis and still needed erythrocyte suspensions. The dosage was carefully raised to 20 mg/day. At the beginning of ruxolitinib treatment, the constitutional symptoms regressed; the patient put on some weight and at the end of the first month he became transfusion-free. However, he did not allow us to perform a

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control bone marrow biopsy. Therefore, we could not assess the drug’s effect on fibrosis. In the second month of treatment, the weight of the patient returned to its basal level and all of his laboratory values were in the normal reference range. He started working again and had an overseas vacation that summer. He was followed asymptomatically for 6 months, but at the end of the sixth month his disease transformed to acute myeloblastic leukemia. He was rehospitalized and 7+3 chemotherapy was started. The patient died 13 days after rehospitalization because of invasive aspergillosis. Discussion and Review of the Literature PMF is a rare disease. Patients with PMF usually present with anemia and extramedullary hematopoiesis marked by hepatosplenomegaly that is often accompanied by severe constitutional symptoms. Most patients become RBC transfusion-dependent; progressive splenomegaly is often associated with drug-refractory splenic pain and abdominal discomfort, early satiety, cachexia fatigue, night sweats, and low-grade fever, and some patients develop portal hypertension. Bone marrow examination is essential for the diagnosis of PMF and typically shows granulocytic and megakaryocytic hyperplasia accompanied by variable degrees of bone marrow fibrosis and osteosclerosis [5]. Survival in PMF is affected by the presence of risk factors. In particular, 5 factors have been identified as being independently predictive of poor survival: hemoglobin below 10 g/dL, constitutional symptoms, circulating blast rates of 1% or greater, leukocyte counts of >30x109/L or <4x109/L, and presence of cytogenetic abnormalities. In the absence of these adverse features, median survival exceeds 10 years; in the presence of 1 adverse feature, median survival is shortened to 5 to 10 years; and in the presence of 2 or more adverse features, median survival is less than 5 years [6]. The pathogenesis of PMF is currently not understood. PMF is a clonal disorder of hematopoietic stem cells and fibrosis is a reactive process involving the interaction of multiple cytokines, such as PDGF, TGF-β1, bFGF, and VEGF [3,4,5], produced by CD34+ cells, megakaryocytes, and monocytes. According to the recommendations of the European LeukemiaNet, the main goals of therapy in PMF are prolongation of survival and, if possible, cure, which is currently only achieved by stem cell transplantation. If prolongation of survival or cure is not possible, symptomorientated palliation and quality of life are the main goals [7]. Until recently, most treatments provided only palliative care, with no single treatment addressing all of the complications and symptoms of the disorder. Although allogeneic stem cell transplant offers the potential for cure, it is associated with a high mortality rate, even using a protocol of reduced intensity, and thus it is only appropriate for a limited group of patients

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(e.g., younger, otherwise healthy patients with high-risk myelofibrosis). Splenomegaly is a common sign of PMF that is associated with bothersome symptoms that have a significant negative impact on patients’ quality of life. Until recently, none of the therapies used to treat myelofibrosis were particularly effective in reducing splenomegaly. Discovery of the JAK2 V617F mutation prompted the development of clinical trials using JAK2 inhibitors; overall, these agents have resulted in meaningful symptomatic improvements and reductions of splenomegaly that were otherwise not achievable with conventional therapy. The suggested mechanism of action of ruxolitinib is attenuation of cytokine signaling via the inhibition of JAK1 and JAK2, resulting in antiproliferative and proapoptotic effects. In the phase III COMFORT-I trial, ruxolitinib demonstrated durable reductions in splenomegaly in patients with myelofibrosis. The proportion of patients that achieved spleen volume reduction of ≥35% from baseline in 24 weeks was 41.9% with ruxolitinib versus 0.7% with the placebo (p<0.0001). In the phase III COMFORT-II trial, reductions in spleen volume of ≥35% were observed in 32% of patients treated with ruxolitinib at week 24 and in 28.5% at week 48 (both p<0.0001). Low toxicity, alleviation of constitutional symptoms, weight gain, and improvement of performance status, exercise capacity, and general physical condition were observed with ruxolitinib treatment [8,9]. In November 2011, the US Food and Drug Administration approved the use of the JAK1- and JAK2selective inhibitor ruxolitinib for the treatment of patients with intermediate- or high-risk myelofibrosis [10]. Favorable results of ruxolitinib treatment in patients who suffer from PMF are well known, but there is no evidence that shows beneficial effects of the same agent in PMF patients who also had a splenectomy [11]. We observed favorable results with ruxolitinib treatment for 6 months in a PMF-diagnosed patient who had a mandatory splenectomy operation. During this 6-month period, all constitutional symptoms regressed. The patient did not require any erythrocyte suspension transfusions and his clinical status improved. Additionally, no side effects were observed. However, ruxolitinib failed to prevent the disease from transforming into acute myeloblastic leukemia. Ruxolitinib did not demonstrate disease-modifying potential and is not considered a curative therapeutic option. This case report supports the palliative role of ruxolitinib, which significantly reduced constitutional symptoms in a splenectomized patient. Ruxolitinib improved the patient’s quality of life in a very short time and provided a comfortable life until the return to acute leukemia. This is suggestive of the inability of ruxolitinib to influence the natural history of the disease. The JAK2 V617F mutation may be a key factor, but not necessarily a primary factor, driving the tumorigenesis of PMF. Other molecular events may precede the acquisition


Ayl覺 M, et al: Ruxolitinib in Refractory Primary Myelofibrosis

of the JAK2 V617F mutation in PMF patients. Novel selective JAK2 inhibitors and other novel agents are the active focus for further clinical investigations. 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. Tefferi A. Myelofibrosis with myeloid metaplasia. N Engl J Med 2000;342:1255-1265. 2. Levine RL, Pardanani A, Tefferi A, Gilliland DG. Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer 2007;7:673-683. 3. Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352:1779-1790. 4. Vaddi K, Sarlis NJ, Gupta V. Ruxolitinib, an oral JAK1 and JAK2 inhibitor, in myelofibrosis. Expert Opin Pharmacother 2012;13:2397-2407. 5. Abdel-Wahab OI, Levine RL. Primary myelofibrosis: update on definition, pathogenesis, and treatment. Annu Rev Med 2009;60:233-245. 6. Cervantes F, Dupriez B, Pereira A, Passamonti F, Reilly JT, Morra E, Vannucchi AM, Mesa RA, Demory JL, Barosi G, Rumi E, Tefferi A. New prognostic scoring system for primary myelofibrosis based on a study of the International Working Group for Myelofibrosis Research and Treatment. Blood 2009;113:2895-2901.

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7. Barbui T, Barosi G, Birgegard G, Cervantes F, Finazzi G, Griesshammer M, Harrison C, Hasselbalch HC, Hehlmann R, Hoffman R, Kiladjian JJ, Kr繹ger N, Mesa R, McMullin MF, Pardanani A, Passamonti F, Vannucchi AM, Reiter A, Silver RT, Verstovsek S, Tefferi A; European LeukemiaNet. Philadelphianegative classical myeloproliferative neoplasms: critical concepts and management recommendations from European LeukemiaNet. J Clin Oncol 2011;29:761-770. 8. Verstovsek S, Mesa RA, Gotlib J, Levy RS, Gupta V, DiPersio JF, Catalano JV, Deininger M, Miller C, Silver RT, Talpaz M, Winton EF, Harvey JH, Arcasoy MO, Hexner E, Lyons RM, Paquette R, Raza A, Vadi K, Viitanen SE, Koumenis IL, Sun W, Sandor W, Kantarjian HM. A double-blind, placebocontrolled trial of ruxolitinib for myelofibrosis. N Engl J Med 2012;366:799-807. 9. Harrison C, Kiladjian JJ, Al-Ali HK, Gisslinger H, Waltzman R, Stalbovskaya V, McQuitty M, Hunter DS, Levy R, Knoops L, Cervantes F, Vannucchi AM, Barbui T, Barosi G. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N Engl J Med 2012;366:787-798. 10. Tefferi A. Primary myelofibrosis: 2013 update on diagnosis, risk-stratification, and management. Am J Hematol 2013;88:437-445. 11. Randhawa J, Ostojic A, Vrhovac R, Atallah E, Verstovsek S. Splenomegaly in myelofibrosis-new options for therapy and the therapeutic potential of Janus kinase 2 inhibitors. J Hematol Oncol 2012;5:43.

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Letters to the Editor Thiopurine S-Methyltransferase and Methylenetetrahydrofolate Reductase Polymorphisms in Leukemia Lösemide Tiyopürin S-Metiltransferaz ve Metilentetrahidrofolat Redüktaz Polimorfizmleri To the Editor, I read the paper by Belen et al. published in a recent issue of this journal with great interest [1]. They reported a 15-year-old girl with T-ALL who developed severe pancytopenia during consolidation and maintenance therapy despite dose reduction of 6-mercaptopurine (MP). They found thiopurine S-methyltransferase (TPMT) *3A/*3C polymorphisms upon TPMT genotyping. Prednisolone therapy produced a remarkable but transient bone marrow recovery in this patient. The authors should be congratulated in that they followed the recent evidence-based clinical guidance recommendations related to safe and effective thiopurine administration. The authors began treatment with standard doses and down-titrated the MP according to the results of blood counts, and they omitted some parts of Protocol Ib and the second part of the reinduction therapy. Although in a recent systematic review 30%-50% of the normal dose for thiopurines in the case of heterozygous TPMT status was recommended [2], I agree with the authors about low-dose MP administration (2.5%-10% of normal dose) when necessary, because of the possibility of the presence of undetected deficiencies in other enzymes involved in the metabolic pathway [3]. Moreover, by omitting 6-thioguanine (6TG) treatment in reinduction therapy, the authors prevented the probable additional toxicity arising from another thiopurine drug since patients with heterozygous TPMT status have higher cytosolic 6TG nucleotide levels, the cytotoxic metabolites of both MP and 6TG [4]. On the other hand, after careful reading, some concerns and questions arose regarding the paper. First of all, it is not possible to understand that the patient had an accompanying methylenetetrahydrofolate reductase (MTHFR) polymorphism from either the title or the English abstract. Only after reading the Turkish abstract and the text I did realize that the patient had concomitant TPMT *3A/*3C and MTHFR C677T and A1298C polymorphisms, which were reported to be associated with increased myelotoxicity in children with ALL [5]. Although the authors stated that they found t(11;14) in karyotyping and that PCR tests for t(4;11) and t(9;21) were negative, they did not mention t(9;22), an important prognostic abnormality in patients with ALL. Additionally, the information about the extension of the disease at the time of diagnosis, such as involvement 184

of the central nervous system (CNS) or kidneys and renal impairment due to increased tumor burden, is lacking in the case presentation. The latter two, if present, might be responsible for the protracted bone marrow suppression by increasing toxic effects of methotrexate. Furthermore, it is not clear to which risk category the patient was assigned in the treatment protocol in the text or from the figure (proper reading of the figure is impossible). I would want to learn more details regarding the clinical condition of the patient, such as whether she was given CNS radiotherapy or not, her renal function test results and especially those before methotrexate administration, and whether she completed the entire chemotherapy protocol or not. Lastly, I am curious about the follow-up results of this patient, since TPMT heterozygosity was reported to be associated with better event-free survival than in TPMT wild-type patients and thiopurine-induced cytopenias did not negatively affect the treatment outcome [6]. Conflict of Interest Statement The author of this paper has no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. Key Words: Thiopurine S-methyltransferase, Methylenetetrahydrofolate reductase, Gene polymorphisms, Leukemia, Childhood Anahtar Sözcükler: Tiyopürin S-metiltransferaz, Metilentetrahidrofolat redüktaz, Gen polimorfizmleri, Lösemi, Çocukluk çağı Serhan Küpeli Çukurova University Faculty of Medicine, Department of Pediatric Oncology and Pediatric Bone Marrow Transplantation Unit, Adana, Turkey

References 1. Belen BF, Gürsel T, Akyürek N, Albayrak M, Kaya Z, Koçak Ü. Severe myelotoxicity associated with thiopurine S-methyltransferase*3A/*3C polymorphisms in a patient with pediatric leukemia and the effect of steroid therapy. Turk J Hematol 2014;31:276-285. 2. Burnett HF, Tanoshima R, Chandranipapongse W, Madadi P, Ito S, Ungar WJ. Testing for thiopurine methyltransferase status for safe and effective thiopurine administration: a systematic review of clinical guidance documents. Pharmacogenomics J 2014;14:493-502. 3. Stocco G, Cheok MH, Crews KR, Dervieux T, French D, Pei D, Yang W, Cheng C, Pui CH, Relling MV, Evans WE. Genetic polymorphism of inosine triphosphate pyrophosphatase is a determinant of mercaptopurine metabolism and toxicity during treatment for acute lymphoblastic leukemia. Clin Pharmacol Ther 2009;85:164-172.


Letter to the Editor

4. Levinsen M, Rosthøj S, Nygaard U, Heldrup J, Harila-Saari A, Jonsson OG, Bechensteen AG, Abrahamsson J, Lausen B, Frandsen TL, Weinshilboum RM, Schmiegelow K. Myelotoxicity after high-dose methotrexate in childhood acute leukemia is influenced by 6-mercaptopurine dosing but not by intermediate thiopurine methyltransferase activity. Cancer Chemother Pharmacol 2015;75:59-66. 5. Karas-Kuzelicki N, Jazbec J, Milek M, Mlinaric-Rascan I. Heterozygosity at the TPMT gene locus, augmented by mutated MTHFR gene, predisposes to 6-MP related toxicities in childhood ALL patients. Leukemia 2009;23:971-974. 6. Lennard L, Cartwright CS, Wade R, Vora A. Thiopurine dose intensity and treatment outcome in childhood lymphoblastic leukaemia: the influence of thiopurine methyltransferase pharmacogenetics. Br J Haematol 2015;169:228-240. Address for Correspondence: Serhan KÜPELİ, M.D., Çukurova University Faculty of Medicine, Department of Pediatric Oncology and Pediatric Bone Marrow Transplantation Unit, Adana, Turkey Phone: +90 322 338 74 44 E-mail: serhankupeli@cu.edu.tr Received/Geliş tarihi: January 01, 2015 Accepted/Kabul tarihi: January 15, 2015 DOI: 10.4274/tjh.2015.0001

Reply: We would like to thank for comments on our article, entitled “Severe myelotoxicity associated with thiopurine S-methyltransferase *3A/*3C polymorphisms in a patient with pediatric leukemia and the effect of steroid therapy”, published in Turkish Journal of Hematology 2014, volume 31, issue 4, pages 276-285. Severe bone marrow suppression in our case occurred during consolidation chemotherapy which contained oral 6-mercaptopurine (6-MP), intravenous cyclophosphamide and low dose cytosine arabinoside and continued until the end of the maintenance treatment despite markedly reduced 6MP dose. It can not be attributed to renal impairment, central nervous system leukemia or methotrexate (MTX) toxicity because none of these were present during or before pancytopenia was noted. The patient is compound heterozygous for TPMT*3A and *3C polymorphisms, which is known to cause low enzyme activity and increase of intracellular thioguanine nucleotides, and lead to severe bone marrow suppression. Although Karas-Kuzelicki et al. reported that co-inheritance of MTHFR C677T with non-functional TPMT variants augments myelotoxicity during maintenance therapy with oral MTX and 6-MP in children with ALL [1], other investigators did not find significant association between these polymorphic variants

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and excessive myelotoxicity [2]. In line with these data, we did not observe severe leukopenia or hepatoxicity during the four courses of intensification therapy with high dose (5 g/m2) MTX, which was given in conjunction with adjusted dose 6-MP. This may be due to the presence of MTHFR A1298C polymorphism, which is associated with lower toxicity risk in patients with ALL. It has been suggested that linkage disequilibrium between C677T and A1298C may explain decreased toxicity during high dose MTX administration [3]. Possible effects of MTHFR C677T and A1298C polymorphisms on the stability of the TPMT enzyme were addressed in our article. The patient was assigned to the medium risk group based on ALL-BFM-95 risk stratification criteria [4]. Renal function tests and glomerular filtration rate were within normal limits during all stages of chemotherapy. In regard to the cranial radiotherapy, 12 Gy prophylactic cranial irradiation was given before maintenance therapy without causing excessive myelosuppression. The patient is in complete remission for 3 years after completion of the maintenance chemotherapy. Adding “MTHFR C677T and A1298C polymorphisms” to the title of our article was not possible due to the the word count restriction of Turkish Journal of Hematology. Please kindly be informed that ‘t(9;21)’ expression in our article in Turkish Journal of Hematology 2014, volume 31, issue 4, pages 276-285 is incorrect. The correct expression should be t(9;22). We apologize for this typing error. Burcu Belen and Türkiz Gürsel

References 1. Karas-Kuzelicki N, Jazbec J, Milek M, Mlinaric-Rascan I. Heterozygosity at the TPMT gene locus, augmented by mutated MTHFR gene, predisposes to 6-MP related toxicities in childhood ALL patients. Leukemia 2009;23:971-974. 2. Wall AM, Rubnitz JE. Pharmacogenomic effects on therapy for acute lymphoblastic leukemia in children. Pharmacogenomics J 2003;3:128-135. 3. Ongaro A, De Mattei M, Della Porta MG, Rigolin G, Ambrosio C, Di Raimondo F, Pellati A, Masieri FF, Caruso A, Catozzi L, Gemmati D. Gene polymorphisms in folate metabolizing enzymes in adult acute lymphoblastic leukemia: effects on methotrexate-related toxicity and survival. Haematologica 2009;94:1391-1398. 4. Kocak U, Gursel T, Kaya Z, Aral YZ, Albayrak M, Keskin EY, Belen B, Isik M, Oner N. ALL-BFM 95 treatment in Turkish children with acute lymphoblastic leukemia--experience of a single center. Pediatr Hematol Oncol 2012;29:130-140.

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Platelet Levels of High- and Mega-Dose Methylprednisolone Treatment in Acute Immune Thrombocytopenia Akut İmmün Trombositopenide Yüksek ve Megadoz Metilprednizolon Tedavisi ile Trombosit Seviyeleri To the Editor, High-dose methylprednisolone (HDMP) therapy was originally used by Ozsoylu et al. in the treatment of immune thrombocytopenic purpura (ITP), and it is also important in the history of Turkish hematology [1,2]. A 16-year-old male (weight: 107.5 kg, height: 167 cm, BMI: 38.5 kg/m2) presented with the complaints of oral submucosal bleeding and purpura. Complete blood count analysis revealed a platelet count of 1900/mm3, and his peripheral blood smear and bone marrow aspiration slides were consistent with ITP. Informed consent was obtained for HDMP and MDMP treatment. High-dose methylprednisolone was initiated at a dose of 1000 mg/day for 3 days followed by 750 mg/day for 4 days, with each dose administered orally early in the morning. Platelet count reached 28,000/mm3 and 31,000/mm3 on the 3rd and 7th days of treatment, respectively. Mucosal and cutaneous bleeding ceased. Methylprednisolone treatment was continued at a dose of 1 mg/kg/day for 7 days and tapered over a week. Platelet count was 22,000/mm3 on the last day, but bleeding reoccurred 10 days later and platelet count was 3900/mm3. Mega-dose methylprednisolone (MDMP) was then given at a dose of 30 mg/kg (3250 mg/day) for 3 days, and then 20 mg/kg (2125 mg/day) for 4 days orally in a single dose in the early morning. Platelet count reached 355,000/mm3, 225,000/mm3, and 381,000/mm3 after 3, 35, and 100 days, respectively (Figure 1). No corticosteroid side effects were observed in this patient [3]. There was no thrombocytopenic attack in the last 12 months.

HDMP treatment has been used as a therapeutic choice in childhood acute ITP in Turkey for a long time [2,4,5]. The term ‘HDMP’ was used for this kind of methylprednisolone administration initially, but it was later changed to MDMP since ‘HDMP’ was also used for 4-10 mg/kg doses in the literature [6]. It is reported that MDMP treatment differs from conventional corticosteroid (2 mg/kg in divided doses) and pulse methylprednisolone (1000 mg methylprednisolone infusion in 4 h) administration, not only by dose (30-100 mg/kg initially for 3 days, tapered gradually) but also the time of administration [7,8]. The doses (30 mg/kg for 3 days and 20 mg/kg for 4 days) of methylprednisolone for acute ITP treatment were also originally stated by Özsoylu and Ertürk [6]. Acute ITP is usually a benign, self-limited condition that occurs in young children, typically those younger than 10 years. In the majority of these patients, the thrombocytopenia resolves within weeks or a few months of the original manifestation. Glucocorticoid therapy in symptomatic childhood ITP patients has been suggested as an appropriate first-line therapy [2]. Therefore, I think that the results obtained with every high-dose corticosteroid cannot be compared. Based on previous experiences, I wish to conclude that MDMP is a cost-effective treatment for acute ITP, if treatment is required [7]. I would specifically like to emphasize MDMP treatment for these patients, which seems to be the most effective approach in the treatment of this disorder regardless of patient weight. In addition to some reports on intravenous immunoglobulin [9,10,11], MDMP is cheap, safe, and easily applicable in all conditions. Conflict of Interest Statement The author of this paper has no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. Key Words: Immune thrombocytopenic purpura, Methylprednisolone, Glucocorticoids, Child, Adolescent Anahtar Sözcükler: İmmün trombositopenik purpura, Metil prednizolon, Glükokortikoidler, Çocuk, Adölesan Ali Ayçiçek Kanuni Sultan Süleyman Research and Education Hospital, Clinic of Pediatric Hematology/Oncology, İstanbul, Turkey

References 1. Ozsoylu S. Bolus methylprednisolone therapy in chronic idiopathic thrombocytopenic purpura in children. Acta Haematol 1984;72:359-360. 2. Ozsoylu S, Irken G, Karabent A. High-dose intravenous methylprednisolone for acute childhood thrombocytopenic purpura. Eur J Haematol 1989;42:431-435. Figure 1. Platelet levels during high- and mega-dose methylprednisolone treatment (platelet count x 103/mm3).

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3. Özsoylu Ş. Megadose methylprednisolone treatment. Turk J Hematol 2006;23:120-121.

(MDMP)


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4. Özsoylu S, Allahverdi H, Laleli Y, Pirnar A. Platelet survival in childhood idiopathic thrombocytopenic purpura in remission. J Pediatr 1976;89:388-390. 5. Özsoylu Ş, Karabent A, Irken G, Tuncer M. Antiplatelet antibody in childhood idiopathic thrombocytopenic purpura. Am J Hematol 1991;36:82-85. 6. Özsoylu Ş, Ertürk G. Oral megadose methylprednisolone for acute childhood idiopathic thrombocytopenic purpura. Blood 1991;77:1856-1857. 7. Özsoylu Ş. Megadose methylprednisolone for childhood idiopathic thrombocytopenic purpura (ITP). Turk J Med Sci 2005;35:347-356. 8. Engin E, Kılınç Ö, Ozsoylu Ş. Sağlık personelinin serum fizyolojik ile üst solunum yolları enfeksiyonlarından korunması. Yeni Tıp Dergisi 1997;14:211-212. 9. Simpson KN, Coughlin CM, Eron J, Bussel JB. Idiopathic thrombocytopenia purpura: treatment patterns and an analysis of cost associated with intravenous immunoglobulin and anti-D therapy. Semin Hematol 1998;35(Suppl 1):5864.

Figure 1. Electron micrograph of a Gaucher cell in the liver. In the cytoplasm, large Gaucher bodies containing tubular elements (25,000x, Gc: Gaucher bodies, Ç: nucleus, G: Golgi apparatus).

10. Blackhouse G, Xie F, Levine MA, Campbell K, Assasi N, Gaebel K, O’Reilly D, Tarride J, Goeree R. Canadian costutility analysis of intravenous immunoglobulin for acute childhood idiopathic thrombocytopenic purpura. J Popul Ther Clin Pharmacol 2012;19:e166-178. 11. O’Brien SH, Ritchey AK, Smith KJ. A cost-utility analysis of treatment for acute childhood idiopathic thrombocytopenic purpura (ITP). Pediatr Blood Cancer 2007;48:173-180. Address for Correspondence: Ali AYÇİÇEK, M.D., Kanuni Sultan Süleyman Research and Education Hospital, Clinic of Pediatric Hematology/ Oncology, İstanbul, Turkey E-mail: ayciceka@hotmail.com Received/Geliş tarihi: November 07, 2014 Accepted/Kabul tarihi: December 02, 2014 DOI: 10.4274/tjh.2014.0436

Gaucher Disease and Gaucher Cells Gaucher Hastalığı ve Gaucher Hücreleri To the Editor, I read the paper entitled “Gaucher cells or pseudoGaucher cells: that’s the question” written by Gören Şahin et al. in a recent issue of this journal. The authors mentioned the main findings of Gaucher cells and pseudo-Gaucher cells in their article without calling attention to the lysosomal enzyme β-glucocerebrosidase levels [1]. Gaucher disease is inherited as an autosomal recessive disorder resulting from mutations at the glucocerebrosidase locus on chromosome 1q21. In this disorder, glucosylceramide (glucocerebroside) is stored in the reticuloendothelial system due to a deficiency of the lysosomal enzyme β-glucocerebrosidase [2]. The storage and deposition of

Figure 2. The cytoplasm of a Gaucher cell showed cytoplasmic bodies containing elongated tubular structures (72,000x, Tu: tubules).

glucocerebroside within these cells, prominently macrophages, results in the appearance of Gaucher cells, which are very large cells with a diameter of 20-80 µm, round or polyhedral. Gaucher cells have small, usually eccentrically placed nuclei and cytoplasm with characteristic wrinkles or striations. Electron microscopy reveals that the cytoplasm contains spindle or rod-shaped membrane-bound inclusion bodies of 0.6-4 µm in diameter consisting of numerous small tubules of 13-75 nm in diameter. Electron microscopy allows the identification of all stages of formation of the inclusions [3,4]. Five patients were diagnosed with Gaucher disease by the presence of many Gaucher cells in the bone marrow associated with organomegaly and cytopenias between 1964 and 1970 in our department at Ankara University. Liver biopsy was performed in one of these patients and electron microscopic study was done. Ultrastructural analysis revealed many Gaucher bodies filled with tubules 187


Letters to the Editor

Turk J Hematol 2015;32:184-190

Terbinafine and Neutropenia Terbinafin ve Nötropeni To the Editor,

Address for Correspondence: İrfan YAVAŞOĞLU, M.D., Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey Phone: +90-256-2120020 E-mail: dryavas@hotmail.com Received/Geliş tarihi: December 18, 2014 Accepted/Kabul tarihi: December 23, 2014 DOI: 10.4274/tjh.2014.0486

The article entitled “Aplastic Anemia Associated with Oral Terbinafine: A Case Report and Review of the Literature”, written by Kantarcıoğlu et al. and published in a recent issue of your journal, was quite interesting [1]. Here we would like to emphasize some relevant points. In the assessment of 425 cases by van der Klauw et al., the most common causes of drug-related agranulocytosis or neutropenia were, in order, dipyrone, mianserin, sulfasalazine, trimethoprim-sulfamethoxazole, penicillins, cimetidine, thiouracil groups, and phenylbutazone [2]. Terbinafine was not included in this list. Neutropenia associated with terbinafine is more common in women, which may be due to more fungal infections encountered and more drug usage for this purpose. To know the MCV and vitamin B12 levels in the presented patients would be useful. In our case, a patient presented with neutropenia due to terbinafine. A 64-year-old man with a history of onychomycosis presented with neutropenia after starting terbinafine at 250 mg/day [3]. In conclusion, routine hematological monitoring is not indicated, but patients should be informed of this potentially life-threatening adverse reaction. Conflict of Interest Statement The author of this paper has no conflict of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included in this manuscript. Key Words: Terbinafine, Drug, Neutropenia Anahtar Sözcükler: Terbinafin, İlaç, Nötropeni İrfan Yavaşoğlu Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey

References 1. Kantarcıoğlu B, Türköz HK, Yılmaz G, Tanrıkulu FP, Atagündüz IK, Adıgüzel C, Tuğlular TF. Aplastic anemia associated with oral terbinafine: a case report and review of the literature. Turk J Hematol 2014;31:411-416. 2. van der Klauw MM, Wilson JH, Stricker BHC. Drug-associated agranulocytosis: 20 years of reporting in The Netherlands (1974-1994). Am J Hematol 1998;57:206-211. 3. Yavaşoğlu İ, Arslan E. Neutropenia due to terbinafine. Osmangazi Tıp Dergisi 2008;30:59-62.

Reply: We are very pleased by the interesting comment and participation by Yavaşoğlu. As we already mentioned in the article oral terbinafine is a highly effective agent, which is generally well tolerated. Two large-scale postmarketing surveillance studies showed that the incidence of serious side effects was <1% [1,2]. We want to clarify that the main purpose of our report was to share the experience of handling such a difficult case with colleagues who are practicing medicine, since the definite causality of aplastic anemia with oral terbinafine cannot be established [3]. The well prepared case presented by Yavaşoğlu and Arslan is another example in this regard [4]. In addition MCV and serum vitamin B12 levels were missing in most of the cases reported in the literature, but they were both in normal ranges in our case. Bülent Kantarcıoğlu

References 1. Hall M, Monka C, Krupp P, O’Sullivan D. Safety of oral terbinafine: results of a postmarketing surveillance study in 25,884 patients. Arch Dermatol 1997;133:1213-1219. 2. O’Sullivan DP, Needham CA, Bangs A, Atkin K, Kendall FD. Postmarketing surveillance of oral terbinafine in the UK: report of a large cohort study. Br J Clin Pharmacol 1996;42:559-565. 3. Kantarcıoğlu B, Türköz HK, Yılmaz G, Tanrıkulu FP, Atagündüz IK, Adıgüzel C, Tuğlular TF. Aplastic anemia associated with oral terbinafine: a case report and review of the literature. Turk J Hematol 2014;31:411-416. 4. Yavaşoğlu İ, Arslan E. Neutropenia due to terbinafine. Osmangazi Tıp Dergisi 2008;30:59-62.

Multiple Myeloma and Alkaline Phosphatase Multipl Myelom ve Alkalen Fosfataz To the Editor, The article entitled “Bone-Specific Alkaline Phosphatase Levels among Patients with Multiple Myeloma Receiving Various Therapy Options”, written by Çetin et al. [1] and published in a recent issue of your journal, was quite interesting. Here we would like to emphasize some relevant points. 189


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This study was not homogeneous. It is important that the initial value of total alkaline phosphatase (ALP) be measured. In addition, in that study, had serum intact parathyroid hormone level, vitamin D level, or bone turnover markers been available, they would have provided further biochemical correlates of skeletal metabolism. Bone-specific ALP may increase in Paget’s disease, osteosarcoma, bone metastases of prostatic cancer (high/very high ALP values), other bone metastases, fractured bones, multiple myeloma (only when associated with fractures), osteomalacia, rickets, vitamin D deficiency (moderate rise), malignant tumors (ALP originating from tumors), renal disease (secondary hyperparathyroidism), and primary hypothyroidism [2,3]. Vitamin D deficiency is extremely common in multiple myeloma, with 40% of patients having vitamin D levels in the deficient range of levels less than 36 nmol/L, and it represents a surrogate for clinical multiple myeloma disease status [4]. Vitamin D deficiency can cause osteomalacia and those patients can develop generalized musculoskeletal pain, proximal muscle weakness, and increased risk of falls. This may not be clinically detectable but is nonetheless present and often unrecognized [5]. Conflict of Interest Statement The authors of this paper have no conflict of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included in this manuscript. Key Words: Alkaline phosphatase, Myeloma, Vitamin D deficiency Anahtar Sözcükler: Alkalen fosfataz, Myelom, D vitamini eksikliği İrfan Yavaşoğlu, Gürhan Kadıköylü, Zahit Bolaman Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey

References 1. Çetin G, Eşkazan AE, Ar MC, Aydın ŞÖ, Ferhanoğlu B, Soysal T, Başlar Z, Aydın Y. Bone-specific alkaline phosphatase levels among patients with multiple myeloma receiving

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various therapy options. Turk J Hematol 2014;31:374-380. 2. Li-Fern H, Rajasoorya C. The elevated serum alkaline phosphatase--the chase that led to two endocrinopathies and one possible unifying diagnosis. Eur J Endocrinol 1999;140:143-147. 3. Gennari L, Di Stefano M, Merlotti D, Giordano N, Martini G, Tamone C, Zatteri R, De Lucchi R, Baldi C, Vattimo A, Capoccia S, Burroni L, Geraci S, De Paola V, Calabrò A, Avanzati A, Isaia G, Nuti R. Prevalence of Paget’s disease of bone in Italy. J Bone Miner Res 2005;20:1845-1850. 4. Badros A, Goloubeva O, Terpos E, Milliron T, Baer MR, Streeten E. Prevalence and significance of vitamin D deficiency in multiple myeloma patients. Br J Haematol 2008;142:492-494. 5. Diamond T, Golombick T, Manoharan A. Vitamin D status may affect the skeletal complications of multiple myeloma. Am J Hematol 2010;85:302-303. Address for Correspondence: İrfan YAVAŞOĞLU, M.D., Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey Phone: +90-256-2120020 E-mail: dryavas@hotmail.com Received/Geliş tarihi: December 23, 2014 Accepted/Kabul tarihi: December 26, 2014 DOI: 10.4274/tjh.2014.0495

Reply: At the beginning of the study we had planned to use some other parameters with a standard multiple myeloma workup. We consulted with biostatisticians. As we compared a wide variety of patient groups in this analysis, according to the biostatisticians adding new parameters to our data would complicate the study. Therefore, we had to exclude some parameters. We thank the reviewers for their valuable comments. We agree to plan further studies with new therapy options in small patient groups adding serum intact parathyroid hormone level, vitamin D level, and bone turnover. Güven Çetin


Images in Hematology

DOI: 10.4274/tjh.2014.0084

Histopathological Image in Hematology Disseminated Histoplasmosis in an Immunocompetent Host Presenting as Pancytopenia with Bilateral Adrenal Masses Bağışıklığı Normal Bir Konakta Çift Taraflı Adrenal Kitleler ile Beliren Yaygın Histoplazmoz

Figure 1. A) Bone marrow aspirate showing numerous Histoplasma capsulatum inside the osteoclastic giant cell and macrophage (inset) (Giemsa, 100x), B) bone marrow biopsy showing Histoplasma (H&E, 100x), C) PAS staining showed yeast-like cells with bright eosinophilic structures, D) GMS staining showed clusters of fungal yeasts.

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Turk J Hematol 2015;32:191-192

A 44-year-old male presented with fever, progressive weight loss, and anorexia for 6 months. The laboratory results showed deranged renal function tests. Serum adrenocorticotropic hormone was high at 252 pg/mL (normal limit: <46 pg/mL), suggestive of primary adrenal insufficiency. Serum free light chains were elevated, kappa at 87.97 mg/L (reference range: 3.30-19.40 mg/L) and lambda at 91.77 mg/L (reference range: 5.71-26.30). Ultrasonography of the abdomen showed hepatosplenomegaly with space-occupying lesions in bilateral suprarenal regions, while endoscopy ultrasound-guided fine-needle aspiration showed necrotizing granulomatous inflammation. Work-up for tuberculosis and human immunodeficiency virus was negative. The hematological parameters showed pancytopenia. The bone marrow aspiration revealed round to oval organisms with crescent-shaped eccentric nuclei both extracellularly and intracellularly, inside the macrophages and osteoclastic giant cells (Figure 1A). Bone marrow biopsy showed the presence of intracellular and extracellular oval capsulated globose organisms spread throughout the marrow spaces (Figure 1B). Periodic acid-Schiff (PAS) staining showed these organisms as bright eosinophilic structures with clear halos around them (Figure 1C). Gomori methenamine silver (GMS) staining showed clusters of fungal yeasts, morphologically compatible with Histoplasma capsulatum (Figure 1D). The patient was started with intravenous amphotericin B followed by oral itraconazole. His condition improved with recovery of counts and improvement of renal function; he is currently doing well. Informed consent was obtained. Histoplasmosis is a fungal infectious disease caused by inhalation of spores of Histoplasma capsulatum. It may present as a self-limiting form or progressive disseminated disease. Disseminated histoplasmosis may affect almost all systems, including the reticuloendothelial system, lungs, gastrointestinal tract, renal tract, central nervous system, visual system, bone marrow, and adrenal glands [1]. Histoplasmosis presenting as a bilateral adrenal mass in an immunocompetent patient is rare. A high index of suspicion is required for the diagnosis of disseminated histoplasmosis in a patient with unexplained fever, as it may mimic other chronic illnesses

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Gajendra S, et al: Histoplasmosis as Pancytopenia&Adrenal Masses

or a neoplasm. The differential diagnoses that should be considered are tuberculosis, sarcoidosis, adrenal hemorrhage, metastatic carcinoma, and lymphoma. Despite extensive imaging, positron emission tomography scanning, and fineneedle aspiration biopsy, a definite diagnosis may not be reached [2]. Bone marrow examination is a useful diagnostic test to establish a diagnosis of disseminated histoplasmosis. In our case, a middle-aged immunocompetent patient presented with nonspecific symptoms and bilateral adrenal mass with insufficiency, the diagnosis of which was only possible with bone marrow examination. 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: Histoplasma, Pancytopenia, Adrenal masses Anahtar SĂśzcĂźkler: Histoplazma, Pansitopeni, Adrenal kitleler Smeeta Gajendra, Bhawna Jha, Tushar Sahni, Shalini Goel, Vimarsh Raina, Ritesh Sachdev Medanta The Medicity Hospital, Clinic of Pathology and Laboratory Medicine, Haryana, India E-mail: sachdev05@gmail.com Phone: 09811836806 Received/GeliĹ&#x; tarihi

: February 23, 2014

Accepted/Kabul tarihi

: April 07, 2014 References

1. Rana C, Krishnani N, Kumari N. Bilateral adrenal histoplasmosis in immunocompetent patients. Diagn Cytopathol 2011;39:294-296. 2. Narang V, Sinha T, Sandhu AS, Karan SC, Srivastava A, Sethi GS, Talwar R, Prusty P, Kotwal N, Adlakha N, Aggarwal A. Clinically inapparent bilateral adrenal masses due to histoplasmosis. Eur Urol 2009;55:518-521.


Images in Hematology

DOI: 10.4274/tjh.2014.0211

Clinical Image in Hematology Promyelocytic Blastic Crisis in Chronic Myeloid Leukemia During Imatinib Treatment İmatinib Tedavisi Sırasında Kronik Miyeloid Lösemide Gelişen Promiyelositik Blastik Kriz

Figure 1. Leukemic cell morphology of bone marrow aspiration specimen (May-Grünwald-Giemsa).

An 82-year-old woman was admitted to our hospital presenting with febrile neutropenia. She had been diagnosed with chronic myeloid leukemia 2 years ago and had been on imatinib treatment since [1]. A month before admission she presented with malaise, anemia, and mild leukopenia; a bone marrow aspirate and biopsy performed 20 days before admission showed no alterations. Imatinib dosing was adjusted but mild cytopenia persisted. The patient presented with acute abdominal pain, fever, and shaking chills to the emergency department. On physical examination the patient was awake and appeared uncomfortable. She had pain in the left lower abdominal quadrant. Petechiae were evident on the lower limbs. Complete blood count revealed anemia, severe neutropenia, and thrombocytopenia (Table 1). Overt disseminated intravascular coagulation was present. Abdominal computed tomography showed acute

diverticulitis. The patient was started on broad-spectrum antibiotics. Informed consent was obtained. A peripheral blood smear revealed more than 30% circulating promyelocytic blasts. A bone marrow aspirate and biopsy showed hypercellular marrow with myeloid hyperplasia and more than 90% myeloblasts (Figure 1). The blasts displayed hypergranular cytoplasm with bundles of Auer rods. PML/RAR-α was positive according to realtime PCR of the bone marrow. A diagnosis of promyelocytic blastic crisis was made [2,3,4,5]. 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.

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Angriman F, et al: Promyelocytic Blastic Crisis

Turk J Hematol 2015;32:193-194

Variable

On admission Day 12

Hematocrit (%)

18.4

25.6

Federico Angriman1, Maria Nelly Gutierrez Acevedo2, Maria Sol Rossi2, Alberto Daniel Gimenez Conca3, Victoria Otero3, Jorge Alberto Arbelbide3, Hernán Michelángelo2

White blood cell count (/ mL)

560

1130

1Buenos

Neutrophils (%) Lymphocytes (%) Monocytes (%)

10 85 5

0 0 30

Platelets (/mL)

13.100

4900

Blasts present in peripheral blood (%)

0

70

Bacterial blood cultures

Negative

Table 1. Results of laboratory testing.

E. coli, S. viridans

Serum creatinine (mg/dL) 1.05

0.58

LDH (units)

167

492

Prothrombin (%)

40

49

Activated PTT (s)

42

32

Fibrinogen (mg/dL)

102

141

D dimer (ng/mL)

>5000

>5000

BCR/ABL RT PCR

0.006

-

PTT: Partial thromboplastin time, LDH: lactate dehydrogenase, RT PCR: reversetranscription polymerase chain reaction.

Key Words: Acute promyelocytic leukemia, All-trans retinoic acid, BCR/ABL, PML/RAR-α, Imatinib Anahtar Sözcükler: Akut promiyelositik lösemi, All-trans retinoik asit, BCR/ABL, PML/RAR-α, İmatinib

Aires University School of Medicine, Hospital Italiano de Buenos Aires, Department of Internal Medicine, Buenos Aires, Argentina 2Hospital Italiano de Buenos Aires, Department of Internal Medicine, Buenos Aires, Argentina 3Hospital Italiano de Buenos Aires, Department of Internal Medicine, Division of Hematology, Buenos Aires, Argentina E-mail: federico.angriman@hospitalitaliano.org.ar Phone: +54-11-49590200 Ext: 4419 Received/Geliş tarihi

: July 02, 2014

Accepted/Kabul tarihi

: May 27, 2014 References

1. Faderl S, Talpaz M, Estrov Z, O’Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukemia. N Engl J Med 1999;341:164-172. 2. Calabretta B, Perrotti D. The biology of CML blast crisis. Blood 2004;103:4010-4022. 3. Shichishima T, Abe R, Satoh T, Kawaguchi M, Uchida T, Kariyone S. Promyelocytic crisis of chronic myelocytic leukemia: case report and review of the literature. Nihon Ketsueki Gakkai Zasshi 1987;50:1190-1195. 4. Abe R, Shichishima T, Kawaguchi M, Uchida T, Kariyone S. Promyelocytic crisis of chronic myelogenous leukemia without t(15;17). Cancer Genet Cytogenet 1986;21:175-179. 5. Gozzetti A, Bocchia M, Calabrese S, Pirrotta MT, Crupi R, Raspadori D, Lauria F. Promyelocytic blast crisis of chronic myelogenous leukemia during imatinib treatment. Acta Haematol 2007;117:236-237.

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Turkish Journal of Hematology Volume: 32 - Issue: 2  

Turkish Journal of Hematology Volume: 32 - Issue: 2  

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