Turkish Journal of Hematology Volume: 32 - Issue: 3 by LookUs Scientific

Volume 32

Issue 3

September 2015

80 TL

ISSN 1300-7777

Review Article Hematopoietic Stem Cell Transplantation in Adult Sickle Cell Disease: Problems and Solutions TURKISH JOURNAL OF HEMATOLOGY • VOL.: 32

Hakan Özdoğu, et al.; Adana, Turkey

Research Articles Bortezomib and Arsenic Trioxide Activity on a Myelodysplastic Cell Line (P39): A Gene Expression Study Hakan Savlı, et al.; Kocaeli, Turkey, Pisa, Catania, Italy, Budapest, Hungary

Detection and Significance of CD4+CD25+CD127dim Regulatory T Cells in Individuals with Severe Aplastic Anemia Weiwei Qi, et al.; Tianjin, China

Venous Thromboembolism after Allogeneic Pediatric Hematopoietic Stem Cell Transplantation: A Single-Center Study Fatih Azık, et al.; Ankara, Turkey

ISSUE: 3

Infections in Hospitalised Patients with Multiple Myeloma: Main Characteristics and Risk Factors Toni Valković, et al.; Rijeka, Croatia, Mostar, Bosnia and Herzegovina

SEPTEMBER 2015

Secondary Infections in Febrile Neutropenia in Hematological Malignancies: More Than Another Febrile Neutropenic Episode Aslıhan Demirel, et al.; İstanbul, Turkey

The Impact of Chemotherapy on Hepatitis B Antibody Titer in Patients with Hematological Malignancies Münci Yağcı, et al.; Ankara, Turkey

Cover Picture: Teoman Soysal Halfeti

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 Ajans Reklam Matbaacılık San. ve Tic.Ltd.Şti. Sanayi Mah. Sultan Selim Cad. Aybike Sk.No:22/-3, Kağıthane, İstanbul, Türkiye Tel: +90 212 264 38 77

http://mc.manuscriptcentral.com/tjh

Web page www.tjh.com.tr

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

Printing Date / Basım Tarihi 25.07.2015

Cover Picture Teoman Soysal was born in 1958, Turkey. He is currently working at İstanbul University Cerrahpaşa Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey. Located along the Fırat River in the southeastern Turkish district of Şanlıurfa, Halfeti was partially flooded in 1999 to build the Birecik dam. Like an underwater museum, the flooded Halfeti can be clearly seen under the crystal water.

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

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Until the area was flooded in 1999, the people lived from fishing in the Fırat River and farming, especially growing peanuts and the area’s famous black roses. Then the waters came and “new” Halfeti was built.

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 Medline - PubMed Central - Science Citation Index Expanded - EMBASE - Scopus - CINAHL - Gale/Cengage Learning - EBSCO - DOAJ - ProQuest - Index Copernicus - Tübitak/Ulakbim Turkish Medical Database - Turk Medline

Impact Factor: 0.360 Subscription Information

The Turkish Journal of Hematology is sent free-of-charge to members of Turkish Society of Hematology and libraries in Turkey and abroad. Hematologists, other medical specialists that are interested in hematology, and academicians could subscribe for only 40 $ per printed issue. All published volumes are available in full text free-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, Oâ&#x20AC;&#x2122;Donnel M, Tolar J. Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure immunosuppressive therapy. Blood 2006;108:1485-1491. 2.Organization as author Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in posthepatitis marrow aplasia. Lancet 1977;2:742-744. 3.Book Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger, 1961.

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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. Fanconiâ&#x20AC;&#x2122;s anemia, transplantation, and cancer. Pediatr Transplant. 2005;9(Suppl 7):81-86

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

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

Case reports should be structured as follows:

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

Invited Review Articles Abstract length: Not to exceed 300 words. Article length: Not to exceed 4000 words. Review articles should not include more than 100 references. Reviews should include a conclusion, in which a new hypothesis or study about the subject may be posited. Do not publish methods for literature search or level of evidence. Authors who will prepare review articles should already have published research articles on therel evant subject. The study’s new and important findings should be highlighted and interpreted in the Conclusion section. There should be a maximum of two authors for review articles.

Images in Hematology Article length: Not exceed 200 words. Authors can submit for consideration an illustration and photos that is interesting, instructive, and visually attractive, along with a few lines of explanatory text and references. Images in Hematology can include no more than 200 words of text, 5 references, and 3 figure or table. No abstract, discussion or conclusion are required but please include a brief title.

Letters to the Editor Article length: Not to exceed 500 words. Letters can include no more than 500 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title.

Tables Supply each table on a separate file. Number tables according to the order in which they appear in the text, and supply a brief caption for each. Give each column a short or abbreviated heading. Write explanatory statistical measures of variation, such as standard deviation or standard error of mean. Be sure that each table is cited in the text.

Figures Figures should be professionally drawn and/or photographed. Authors should number figures according to the order in which they appear in the text. Figures include graphs, charts, photographs, and illustrations. Each figure should be accompanied by a legend that does not exceed 50 words. Use abbreviations only if they have been introduced in the text. Authors are also required to provide the level

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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

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

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CONTENTS 195

Review Article

206

Research Articles

Hematopoietic Stem Cell Transplantation in Adult Sickle Cell Disease: Problems and Solutions Hakan Özdoğu, Can Boğa

Bortezomib and Arsenic Trioxide Activity on a Myelodysplastic Cell Line (P39): A Gene Expression Study Hakan Savlı, Sara Galimberti, Deniz Sünnetçi, Martina Canesastraro, Giuseppe Palumbo, Balint Nagy, Francesco Di Raimondo, Mario Petrini

213

Can Positron Emission Tomography and Computed Tomography Be a Substitute for Bone Marrow Biopsy in Detection of Bone Marrow Involvement in Patients with Hodgkin’s or Non-Hodgkin’s Lymphoma? Güven Çetin, M. Ali Çıkrıkçıoğlu, Tuba Özkan, Cumali Karatoprak, M. Cem Ar, Emre Eşkazan, Mesut Ayer, Abdullah Cerit, Kübra Gözübenli, Betül Börkü Uysal, Simge Erdem, Nurhan Ergül, Gamze Tatar, T. Fikret Çermik

220

Detection and Significance of CD4+CD25+CD127dim Regulatory T Cells in Individuals with Severe Aplastic Anemia Weiwei Qi, Yue Ren, Rong Fu, Huaquan Wang, Chunyan Liu, Zonghong Shao

228

Venous Thromboembolism after Allogeneic Pediatric Hematopoietic Stem Cell Transplantation: A Single-Center Study Fatih Azık, Dilek Gürlek Gökçebay, Betül Tavil, Pamir Işık, Bahattin Tunç, Duygu Uçkan

234

Infections in Hospitalised Patients with Multiple Myeloma: Main Characteristics and Risk Factors Toni Valkovič, Vedrana Gačič, Jelena Ivandič, Božo Petrov, Renata Dobrila-Dintinjana, Elizabeta Dadič-Hero, Antica Načinovič-Duletič

243

Secondary Infections in Febrile Neutropenia in Hematological Malignancies: More Than Another Febrile Neutropenic Episode Aslıhan Demirel, Fehmi Tabak, M. Cem Ar, Bilgül Mete, Şeniz Öngören, Mücahit Yemişen, Reşat Özaras, Emre Eşkazan, Zafer Başlar, Ali Mert, Teoman Soysal, Burhan Ferhanoğlu, Yıldız Aydın, Recep Öztürk

251

The Impact of Chemotherapy on Hepatitis B Antibody Titer in Patients with Hematological Malignancies Münci Yağcı, Elif Suyanı, Merih Kızıl Çakar

Case Reports

257

Occurrence of Carcinoma of the Pancreas Following Nilotinib Therapy for Chronic Myeloid Leukemia: Report of a Case with Review of the Literature Yasunobu Sekiguchi, Asami Shimada, Moe Matsuzawa, Hidenori Imai, Mutsumi Wakabayashi, Keiji Sugimoto, Noriko Nakamura, Tomohiro Sawada, Junichi Arita, Norio Komatsu, Masaaki Noguchi

263

Hypertension and Life-Threatening Bleeding in Children with Relapsed Acute Myeloblastic Leukemia Treated with FLT3 Inhibitors Deniz Yılmaz Karapınar, Nihal Karadaş, Zühal Önder Siviş, Can Balkan, Kaan Kavaklı, Yeşim Aydınok

267

Portal Vein Thrombosis of a Newborn with Corrected Total Anomalous Pulmonary Venous Return Ufuk Çakır, Dilek Kahvecioğlu, Serdar Alan, Ömer Erdeve, Begüm Atasay, Tayfun Uçar, Saadet Arsan, Hasan Çakmaklı, Mehmet Ertem, Semra Atalay

A-IX

271

Leuconostoc sp. Meningitis in a Patient Treated with Rituximab for Mantle Cell Lymphoma Hrvoje Holik, Božena Coha, Marijan Šiško, Maja Tomić-Paradžik

275

Letters to the Editor

Thrombotic Microangiopathy with Complement Factor H Gene Mutations Unassociated with Atypical Hemolytic Uremic Syndrome Yeşim Oymak, Tuba Hilkay Karapınar, Yılmaz Ay, Esin Özcan, Neryal Müminoğlu, Sultan Aydın Köker, Ersin Töret, Afig Berdeli, Erkin Serdaroğlu, Canan Vergin

276

Rapidly Growing Thyroid Mass: An Unusual Case of Acute Lymphoblastic Leukemia Füsun Özdemirkıran, Gonca Oruk, Bahriye Payzin, Zeynep Gümüş, Melike Koruyucu,  Türkan Atasever, Betül Küçükzeybek, Özgür Esen

278

Auer Rods in Chronic Myelomonocytic Leukemia Can Change the Diagnosis Smeeta Gajendra, Ranjit Kumar Sahoo

279

A Case of Acquired Thrombotic Thrombocytopenic Purpura: Three Recurrences in 25 Years Şinasi Özsoylu

281

Images in Hematology

283

Congenital Dyserythropoietic Anemia Type 2 Due to Compound Heterozygote Mutation in SEC23B Gene Fatih Demircioğlu, Mustafa Erkoçoğlu, Mustafa Dilek, Mervan Bekdaş, Sevil Göksügür, Semra Büyükkorkmaz, Seher Açar

Inclusion Body in a Leukocyte in Crimean-Congo Hemorrhagic Fever Zahit Bolaman, İrfan Yavaşoğlu, Gürhan Kadıköylü

A-X

Review Article

DOI: 10.4274/tjh.2014.0311

Hematopoietic Stem Cell Transplantation in Adult Sickle Cell Disease: Problems and Solutions Erişkin Orak Hücre Hastalığında Hematopoietik Kök Hücre Nakli: Problemler ve Çözüm Önerileri Hakan Özdoğu, Can Boğa Başkent University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Adana, Turkey

Abstract: Sickle cell disease-related organ injuries cannot be prevented despite hydroxyurea use, infection prophylaxis, and supportive therapies. As a consequence, disease-related mortality reaches 14% in adolescents and young adults. Hematopoietic stem cell transplantation is a unique curative therapeutic approach for sickle cell disease. Myeloablative allogeneic hematopoietic stem cell transplantation is curative for children with sickle cell disease. Current data indicate that long-term disease-free survival is about 90% and overall survival about 95% after transplantation. However, it is toxic in adults due to organ injuries. In addition, this curative treatment approach has several limitations, such as difficulties to find donors, transplant-related mortality, graft loss, graft-versus-host disease (GVHD), and infertility. Engraftment effectivity and toxicity for transplantations performed with nonmyeloablative reduced-intensity regimens in adults are being investigated in phase 1/2 trials at many centers. Preliminary data indicate that GVHD could be prevented with transplantations performed using reduced-intensity regimens. It is necessary to develop novel regimens to prevent graft loss and reduce the risk of GVHD. Key Words: Sickle cell disease, Hematopoietic stem cell transplantation, Graft-versus-host disease, Graft rejection, Conditioning

Özet: Günümüzde hidroksiüre kullanımı, enfeksiyon proflaksisi ve destek tedavileriyle ilgili gelişmelere rağmen orak hücre hastalığına bağlı organ hasarları engellenememekte, adölesan ve genç yetişkinlerde hastalık ilişkili mortalite %14’lere kadar ulaşmaktadır. Hematopoietik kök hücre transplantasyonu, orak hücre hastalığı tedavisinde tek küratif tedavi yaklaşımıdır. Myeloablatif allojenik hematopietik kök hücre transplantasyonu orak hücre hastalığı olan çocuklar için küratiftir. Güncel veriler çocuklarda transplant sonrası uzun dönem hastalıksız yaşam oranının %90 ve toplam yaşam oranının %95 civarında olduğunu göstermektedir. Erişkinde hazırlama rejimleri organ hasarları nedeniyle fazla toksiktir. Ayrıca bu küratif tedavi yaklaşımının donör bulma güçlüğü, transplant ilişkili mortalite, graft kaybı, graft-versus-host hastalığı (GVHH), infertilite gibi nedenlerle sınırlamaları vardır. Güncel olarak erişkinde toksisitesi azaltılmış hazırlama rejimleri (myeloablatif olmayan indirgenmiş yoğunluklu) ile yapılan transplantların faz 1/2 çalışmalar ile graft yerleşmesi üzerine etkinliği ve toksisitesi birçok merkez tarafından araştırılmaktadır. Ön veriler, toksisitesi azaltılmış rejimler ile yapılan nakillerde başarılı olarak GVHH’nin engellenebildiğini göstermiştir. Bununla birlikte yüksek graft kaybı riski devam etmektedir. Bu yüzden orak hücre hastalarında uygulanabilen, GVHH riski azaltılmış ve graft kaybının engellenebildiği yeni rejimlerin geliştirilmesi gereklidir. Anahtar Sözcükler: Orak hücre hastalığı, Hematopoietik kök hücre nakli, Graft-versus-host hastalığı, Graft kaybı, Hazırlama rejimi Address for Correspondence: Hakan ÖZDOĞU, M.D., Başkent University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Adana, Turkey Phone: +90 322 327 27 27-2024 E-mail: hakanozdogu@hotmail.com Received/Geliş tarihi : August 01, 2014 Accepted/Kabul tarihi : December 26, 2014

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Introduction Disease Overview Sickle cell disease (SCD) is a homozygous hemoglobin S disease characterized by chronic anemia and tissue injury. Annually, approximately 300,000 babies are born with SCD worldwide. One in every 600 African-Americans in the United States is affected by the disease [1]. Central to the pathogenesis of SCD is a mutation leading to abnormal polymerization of hemoglobin and formation of the characteristic sickle shape as a response to deoxygenation [2,3]. Microvascular ischemia and endothelial activation are responsible for tissue injury. To date, drug therapy for SCD is limited to fetal hemoglobinstimulating medicines and nonnarcotic and narcotic agents. Use of antisickling agents is not standard, while experience with gene therapy is insufficient. Allogeneic stem cell transplantation is a curative intervention, but it carries a high risk of morbidity and mortality. The development of transplant techniques seems to give better results. In this review, we aim to highlight questions related to transplantation procedures and to discuss our proposals. Current Reasons for Transplantation Current reasons for accepting the risk of transplantation are summarized below. First, the clinical manifestations and complications are dramatic in SCD patients. Second, despite their high costs, no treatment protocol is for protecting from complications. Third, transplantation techniques and patient care have evolved over time. Manifestations and Complications of Sickle Cell Disease SCD is characterized by hemolytic anemia, painful vaso-occlusive crisis, stroke, avascular necrosis, pulmonary hypertension, susceptibility to infections, renal failure, and thrombosis. Consequently, life expectancy is decreased. Neurologic complications develop in 27% of children with SCD and acute chest syndrome in 25% [4,5]. These tissue injuries significantly influence quality of life in most patients. Frontline Treatment Options and Disease Course The current, treatment modalities for SCD include fluid replacement, pain control using opioids and analgesics during vaso-occlusive crises, oral hydroxyurea, transfusion, and chelating therapy. Hydroxyurea is effective for reducing the frequency of painful crises and recommended for adults with recurrent episodes of acute chest syndrome [6,7]. Maintaining sickle hemoglobin levels of less than 30% by transfusion may prevent or control adverse events associated with SCD [8,9]. However, these complications may develop despite hydroxyurea use and regular blood transfusions [4,5]. Painful ischemic episodes lead to irreversible sequelae by affecting bones, joints, muscles, and the heart, intestines, kidneys, and eyes. Mean survival is 40 years in developed countries despite 196

advanced supportive care [10]. At our institute, the mean age of patients who died within the last 10 years was 30 years (unpublished data). Cost The disease also has high treatment costs. A study using data from the Florida Medicaid program showed a healthcare cost per patient per month of $1389, with significant increases in SCD-related costs from $892 in the 0-9-years cohort to $2562 in the 50-64-years cohort [11]. The cost of chronic transfusions and chelating therapy alone is $40,000 per patient per year in the United States [12,13]. Allogeneic Stem Cell Transplantation Hematopoietic stem cell transplantation (HSCT) is a unique curative therapeutic approach in SCD [14,15,16]. HSCT may improve central nervous system (CNS), pulmonary system, and pain symptoms by stabilizing organ functions [17,18]. Overall survival was reported to be 90%, disease-free survival 82%-100%, graft rejection 8%-18%, and transplant-related mortality (TRM) 4%-14% in transplantations performed mainly with myeloablative conditioning regimens with fully HLA-matched donors in various countries [15,19,20,21,22]. However, this curative treatment approach has some limitations, such as difficulties to find donors, TRM, graft loss, graft-versus-host disease (GVHD), and infertility. Indications for Transplantation Problem: Although HSCT has a curative potential in this nonmalignant disease, it is difficult to determine in which patients the risk of the procedure is acceptable. Current HSCT indications are summarized in Table 1. Extreme variation in the clinical phenotypes of SCD patients makes it difficult to predict the course of the transplantation procedure. However, certain clinical and laboratory factors may predict transplant-associated problems. These factors are renal insufficiency, age >16 years, hepatic function abnormalities, and high inflammation [5,17,22,23,24]. In addition, alloantibodies against erythrocytes and HLA antigens and excess iron load develop due to transfusions in the vast majority of patients [25]. Transcranial Doppler ultrasound was shown to be effective for determining the severity of the disease and stroke risk in children. Starting blood transfusion therapy early reduces the risk [26]. Unfortunately, no reliable method is available to predict which patients would develop stroke. On the other hand, hydroxyurea reduces the frequency and severity of vaso-occlusive crises and acute chest syndrome crises. However, organ injuries continue to occur despite hydroxyurea and current supportive therapies, and mean survival of patients is about 40 years [4,27]. The presence of organ injuries is associated with increased mortality and morbidity.

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Table 1. Indications for hematopoietic stem cell transplantation.

Indications for HSCT (patients ≤16 years)

Indications for HSCT (patients ≥16 years)

Stroke or transient CNS event

Stroke

Silent stroke with cognitive impairment

MRI imaging changes of brain parenchymal damage

MRI angiogram evidence of cerebrovascular disease

ACS requiring exchange transfusion or hospitalization

Recurrent vaso-occlusive pain crises (≥2 per year for the last 2 years), requiring hospitalizations

ACS requiring recurrent hospitalizations

Osteonecrosis of multiple joints

Stage I or II sickle lung disease

ACS requiring recurrent hospitalizations

Pulmonary hypertension

Elevated TCD

Sickle hepatopathy (including iron overload)

Red cell alloimmunization (≥2 antibodies)

Sickle-related renal insufficiency (CR ≥1.5 times the upper limit of normal or biopsy-proven )

Elevated TRV of ≥2.6 m/s

Sickle retinopathy

Red cell alloimmunization (≥2 antibodies)

Recurrent priapism

HSCT: Hematopoietic stem cell transplantation, ACS: acute chest syndrome, MRI: magnetic resonance imaging, TCD: transcranial doppler, TRV: tricuspid regurgitant velocity.

Table 2. Transplantations for sickle cell disease by donor type and overall survival according to European Society for Blood and Marrow Transplantation-Eurocord and Center for International Blood and Marrow Transplant Research.

EBMT-Eurocord CIBMTR

Transplantations for SCD

Total

611

627

Type of donor

HLA-identical

487

430

Cord blood, related and unrelated

Haploidentical donor

Other unrelated donor

SCD: Sickle cell disease, CIBMTR: Center for International Blood and Marrow Transplant Research, EBMT: European Society for Blood and Marrow Transplantation.

Proposed solution: Adult SCD patients have generally been excluded from myeloablative bone marrow transplantation

trials because of anticipated excess morbidity and mortality resulting from accumulated disease-related end-organ damage. Rational thought requires applying a curative treatment (i.e. HSCT) in the early period before organ injuries that significantly reduce the life span develop. Donor Problem: The likelihood of finding an HLA-identical sibling donor is low for SCD patients. In the United States, the estimated number of homozygous SCD patients is around 70,000-100,000 and the overall number of patients who have indications for transplantation is around 5000-7000. A total of 1200 transplantations for SCD were reported in 1986-2013 according to Center for International Blood and Marrow Transplant Research (CIBMTR) and European Society for Blood and Marrow Transplantation (EBMT) records. These rates may be stated to be much lower than needed and expected (Table 2). In a study by Hsieh et al., 24 out of 112 adult patients (21%) had suitable donors for transplantation. However, the procedure could only be performed in 10 patients (8.9%) [28]. The vast majority of healthy HLA-identical sibling donors are hemoglobin S carriers. Although there is evidence that peripheral stem cell mobilization with G-CSF is safe in

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hemoglobin S carrier donors, it should be remembered that G-CSF administration could lead to severe and sometimes fatal sickle cell crisis in carrier donors [29,30,31]. Multiorgan failure was reported in 2 from 11 cases and in 1 case it was fatal. Four people who developed complications required hospitalization. Multiorgan failure and sickle cell crisis were reported to be independent of leukocyte count and G-CSF dose (2.5-10 Îźg/kg/day), but they seem to be associated with hemoglobin S concentration [29,30,31]. Erythrocyte exchange or erythrocyte transfusion may be required to reduce the level to <30% prior to mobilization. However, there are no data for plerixafor mobilization in hemoglobin S carrier donors. Theoretically, plerixafor might be safer than G-CSF for mobilization as it does not trigger hyperleukocytosis and bone marrow hyperplasia [32]. Severe pain may begin 1 h after apheresis without mobilization due to transfusion of the erythrocytes, which wait hypoxically in the pulley of the apheresis device. Bone marrow harvesting is safe under local or general anesthesia with careful perioperative management. However, the difficulty in collecting an adequate amount of stem cells for an adult should be remembered. On the other hand, no technical difficulties for cryopreservation were reported in sickle cell carriers who could easily tolerate the procedure [28,29,30,31,33]. Proposed solution: Due to the abovementioned problems, donors who are not carriers should preferably be selected in the interest of donor safety. The hemoglobin S level should be decreased to <30% if the donor is a carrier. Bone marrow should be harvested, if possible. Plerixafor should be preferred for mobilization of carrier donors and the rinse-back procedure should be avoided in apheresis procedures. Overall, less than 25% of SCD patients have a healthy HLAidentical relative donor. HLA-identical unrelated donors can be found for only a small proportion of patients [34]. A trial of unrelated cord blood transplantation (CBT) was terminated early due to high risks of rejection and GVHD [35]. On the other hand, almost all patients have a haploidentical relative donor. Bolanos-Meade et al. showed that no patients who underwent mismatched transplantation using cyclophosphamide developed GVHD or severe immunodeficiency complications [36]. However, the risk of graft rejection is high in these patients as they are immunocompetent and have proliferative bone marrow. Conditioning regimens are designed to give a low GVHD risk. This problem may be overcome by further developing conditioning regimens (see conditioning regimens below). Stem Cell Source Problem: The ideal stem cell source is not known. Bone marrow: Bone marrow should be the preferred as the stem cell source due to the low risk of chronic GVHD

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(cGVHD). However, HLA-matched donors can be found for only a small proportion of patients. G-CSF-mobilized marrow has been used in the haploidentical (family donor) setting. The use of G-CSF-primed bone marrow grafts to reduce graft loss has been reported [36]. The difficulty in collecting a sufficient amount of stem cells in adults should be considered. In addition, there is evidence that donors of African origin have lower blood counts and reduced numbers of marrow progenitor cells [37]. Haploidentical bone marrow transplantation with posttransplant cyclophosphamide has been applied in patients with hematological malignancies for several years. The Johns Hopkins Group reported that this approach is a feasible and effective treatment with acceptable toxicity [38]. Evidence on the potential use of bone marrow from haploidentical related donors has increased in patients with SCD. BolanosMeade et al. reported promising results with haploidentical nonmyeloablative bone marrow transplantation. They reported durable engraftment and acceptable toxicity with no morbidity and mortality in their series [36]. Peripheral stem cells: They can easily be collected without general anesthesia in the outpatient setting. Their application is less traumatic and has advantages such as less need for transfusion, faster engraftment, less need for platelet suspension, and shorter hospital stays. Although peripheral stem cell products with a 10-fold higher T-cell count increase the risk of cGVHD, their engraftment-facilitating effect should be considered. Cord blood: This stem cell source has some limitations, such as difficulty to find an HLA-identical donor, GVHD, and graft rejection, despite data showing successful treatment of patients by CBT. A study from the Eurocord Cooperative Group, analyzing the outcome in 44 patients who had SCD or thalassemia major and were treated by CBT with a sibling donor, reported no fatal transplantation-related complications, suggesting that CBT with a related donor is a safe treatment for hemoglobin disorders [38]. However, CBT with matched unrelated donors will likely not be applicable to sufficient numbers of SCD patients because of a lack of suitable donors in national and worldwide registries. CBT with mismatched unrelated donors, although more feasible for pediatric patients based on the availability of 4/6 HLA-matched cord blood units, appears to be associated with a greater risk of graft rejection and GVHD on the basis of the limited data available [39]. Drawbacks such as increased rates of graft rejection, the fixed cell dose, delayed immune reconstitution, and TRM have deterred unrelated cord transplantation efforts [39]. Furthermore, CBT for adults will be limited by the necessary total nucleated cell count per kilogram of body weight for engraftment to occur and the difficulty in achieving this goal in adult patients. Double umbilical cord blood allogeneic

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transplantation with reduced-intensity conditioning is increasingly used in adults lacking a suitable related or unrelated donor [40]. However, the studies of expanded and double cord blood are inadequate. Proposed solution: Current data indicate that peripheral stem cells mobilized with G-CSF could be a suitable stem cell source for patients with SCD [32]. If the donor is a carrier, decreasing hemoglobin S level below 30% of total hemoglobin concentration might be recommended for a safer mobilization process and apheresis procedure. Hematopoietic and lymphoid reconstruction is rapidly achieved through replacement of T cells by donor peripheral stem cells. Bone marrow from haploidentical donors might be an alternative to matched related or unrelated donors, but this needs more research [40]. Mesenchymal stem cell co-infusion may be considered to prevent GVHD and/or cGVHD and graft loss [41,42,43].

Conditioning Regimens Problem: Engraftment efficacy and toxicity for transplantation performed with nonmyeloablative reduced-intensity conditioning regimens are currently being investigated in phase I/II trials at many centers. Preliminary data indicate that GVHD could be prevented by nonmyeloablative reduced-intensity regimens. However, the risk of graft loss remains high. Allogeneic bone marrow transplantation performed with an HLA-matched donor using a myeloablative-conditioning regimen can cure symptomatic children with SCD. The cure rate was reported to be 85%, the rate of TRM 7%, and the rate of graft rejection 8% in studies including approximately 200 patients [16,38,44]. The results were improved by adding ATG to the conditioning regimen, which gave a cure rate of 95% (Table 3) [45]. Adult SCD patients have to be excluded from most studies using ablative regimens due to end-organ injury-related morbidity and mortality in most patients, despite positive results in childhood.

Table 3. Myeloablative hematopoietic stem cell transplantation with matched related donors.

Reference Transplantation Number of Regimen Patients (age range, years)

Alive Acute without GVHD SCD, n (%)

Chronic GVHD

Death (cause)

Comments

Vermylen et al. [15], 1998

Bu 16 mg/kg, Cy 200 mg/kg ± TLI or ATG, CSA

50 (0.3-11)

45 (90)

10 (Gr 1); 7 limited, 2 (acute 9 (Gr 2); 3 extensive GVHD with 1 (Gr CMV and 3-4) Aspergillus; sudden death)

At 5 years after HSCT, 6 had mixed chimerism (3: >90%, 3: 50%70%)

Walters et al. [14,20], 1996, 2001

Bu 14 mg/kg, Cy 200 mg/kg, ATG or alemtuzumab, MTX, CSA

59 (3-16)

50 (85

11 (acute or chronic GVHD)

3, all GVHD

At 3.5 years after HSCT, 13 had mixed chimerism (8 with 90%-99%, 5 with 11%-74%)

Brachet et al. [19], 2004

Bu 14-16 mg/kg, Cy 200 mg/kg ± ATG, MTX, CSA

24 (2-14)

19 (79)

3 (Gr 2); 3 (Gr 3)

7 limited

1 (unknown)

At 8.7 years after HSCT, 1 had mixed chimerism

Bernaudin et al. [16], 2007

Bu 16 mg/kg, Cy 200 mg/kg ± ATG, CSA ± MTX

87 (2-22)

80 (92)

10 (Gr 2); 9 limited, 6 (sepsis, 5 (Gr 3); 2 extensive CNS 2 (Gr 4) hemorrhage, and GVHD in 4)

At 6 years after HSCT, 25%-40% had mixed chimerism (5%-95%)

ATG: Antithymocyte globulin, Bu: busulfan, CSA: cyclosporine A, Cy: cyclophosphamide, Gr: grade, MTX: methotrexate, SCD: sickle cell disease, GVHD: graft-versus-host disease.

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Özdoğu H, et al: Hematopoietic Stem Cell Transplantation in Adult Sickle Cell Disease

In the past, nonmyeloablative bone marrow transplantation strategies developed to cure SCD patients with organ dysfunction gave disappointing results [46,47]. Transplantations done with nonmyeloablative regimens were not as successful as myeloablative transplantations. The main reasons for this included the recipient’s not being fully

immunocompetent, having proliferative bone marrow, and lack of a GVHD effect. Recently, inconsistently with previous studies, several small patient series have shown promising results [28,36,48,49]. Therefore, HSCT following a reducedintensity conditioning nonmyeloablative regimen emerged as a potential treatment option (Table 4).

Table 4. Hematopoietic stem cell transplantation from matched related donors with nonmyeloablative conditioning.

Reference

Transplant Regimen

Number of Alive Acute ChronDeath Comments Patients  (age without GVHD ic  GVHD (cause) range, years) SCD

BolanosMeade et al. [36], 2012

TBI 200 cGy, 17 (15-46) Flu 150 mg/kg, Cy 14.5 mg/ kg, ATG 4.5 mg/kg, post TX Cy 100 mg/kg, tacrolimus or sirolimus

1 limited (skin)

14 haploidentical related donors, in 6 patients graft rejected; 3 matched sibling donors, no grafts rejected

Hsieh et al. [28], 2009

TBI 300 cGy, alemtuzumab 1 mg/kg, sirolimus

10 (16-45)

At 3 years after HSCT, mixed chimerism in all

Hsieh et al. [49], 2014

TBI 300 cGy, alemtuzumab 1 mg/kg, sirolimus

30 (16-65)

1 (GVHD)

At median 3.4 years 15 patients discontinued immunosuppression medication

Krishnamurti et al. [48], 2008

BU 6.4 mg/kg IV or 8 mg/kg PO, FLU 175 mg/kg, ATG, 500 cGy of TLI, CSA, MMF

7 (6-16)

1 (Gr 2, skin)

1 limited (skin)

Mixed chimerism in 4

Jacobsohn et al. [46], 2004

BU 6.4 mg/kg IV, FLU 180 mg/m2, ATG, CSA, MMF

1 (22)

1 (Gr 1)

1 extensive

1 (GVHD)

Iannone et al. [47], 2003

TBI 200 cGy, FLU 150 mg/ m2, CSA or tacrolimus, MMF

6 (3-20)

1 (Gr 2)

Transient engraftment in 5 patients for  97441 days

ATG: Antithymocyte globulin, cGy: centigray, CSA: cyclosporine A, Cy: cyclophosphamide, FLU: fludarabine, Gr: grade, IV: intravenous, MMF: mycophenolate mofetil, MTX: methotrexate, PO: per os, SCD: sickle cell disease, TBI: total body irradiation, TLI: total lymphoid irradiation, GVHD: graft-versus-host disease, HSCT: hematopoietic stem cell transplantation.

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Ă&#x2013;zdoÄ&#x;u H, et al: Hematopoietic Stem Cell Transplantation in Adult Sickle Cell Disease

In general, small proportions of engrafted donor red cells seem to be sufficient for clinical control of the disease in SCD patients [50]. Hsieh et al. reported that they achieved permanent engraftment in 26/30 patients and that no patients developed acute GVHD (aGVHD) or cGVHD with a nonmyeloablativeconditioning regimen using alemtuzumab (Campath) and low-dose total-body irradiation (TBI). The mean donor T-cell level was 48% and the myeloid chimerism level was 86%. Fifteen engrafted patients discontinued immunosuppression medication with continued stable donor chimerism and no GVHD [49]. In this study, sirolimus (rapamycin) was selected as the immunosuppressive agent instead of cyclosporine. Differently from calcineurin inhibitors, sirolimus does not block T-cell activation, but it inhibits T-cell proliferation. The activated but nonproliferated T cells become anergic, resulting in T-cell tolerance. Sirolimus accelerates the differentiation of regulatory and helper T cells and this plays a key role in immune tolerance development. Additionally, the renal toxicity of sirolimus is less than that of cyclosporine. Luznik et al. showed that high-dose cyclophosphamide given in the early posttransplantation period could lead to immune tolerance by killing proliferative alloreactive T cells and preserving nonreactive T cells. High-dose cyclophosphamide is highly toxic to lymphocytes, but hematopoietic stem cells are not affected as they are rich in the aldehyde dehydrogenase enzyme that metabolizes the drug. Posttransplantation highdose cyclophosphamide deletes alloreactive T cells without affecting nonreactive T cells or hematopoietic stem cells, thereby reducing the risk of GVHD and enabling immune reconstitution [38]. Bolanos-Meade et al. achieved bone marrow transplantation in adult SCD patients with 14 haploidentical relative donors and 3 matched sibling donors using a nonmyeloablative-conditioning regimen containing cyclophosphamide, ATG, fludarabine, TBI (200 cGy), and posttransplantation high-dose cyclophosphamide [36]. Proposed solution: Novel regimens to reduce GVHD risk and toxicity and prevent graft loss in SCD patients need to be developed. Such regimens may include the following: ATG-Fresenius: The drug used in preliminary studies is referred to as ATG-thymoglobulin (rabbit) in the literature and its half-life is about 12 h. ATG-Fresenius (rabbit) has a longer half-life (4-10 days) and would provide a significant advantage for preventing graft loss and GVHD. Busulfex (busulfan): Added to conditioning regimens at microablative doses considering organ injuries. It would open spaces in the bone marrow and help to prevent graft loss. Treosulfan: Used safely and effectively instead of busulfan in nonmyeloablative regimens in thalassemia patients due to its lower toxicity [51]. However, there is no experience of its use in SCD.

Fludarabine: Used in nonmyeloablative-conditioning regimens in pediatric and adult SCD patients with tissue injuries [52]. Sirolimus: An immunosuppressive agent with an immunotolerogenic effect [53]. It is superior to cyclosporine for providing engraftment without GVHD and reducing posterior reversible encephalopathy syndrome incidence [28,49]. Posttransplantation cyclophosphamide: Posttransplantation high-dose cyclophosphamide, which is successfully used in haploidentical transplantations, should be included in the conditioning regimen. This approach was first described by Luznik et al. Interestingly, while cyclophosphamide given in the early posttransplantation period kills proliferative alloreactive T cells, it preserves resting nonreactive T cells. Thus, immune tolerance occurs, GVHD is prevented, T-cell reconstitution is achieved in a short time, and the infection risk decreases [36]. As positive results were obtained in transplantations with HLA-unmatched donors in previous studies, it would also enable haploidentical transplantations. The donor pool would be larger and patients without fully matched donors would have a chance of being cured. Pretransplantation immune suppression: Administration of 2 cycles of dexamethasone (25 mg/m2/day for 5 days) to suppress T-cell functions, facilitate engraftment, decrease the GVHD risk, and facilitate the control of disease-related inflammation [54]. Peri- and posttransplantation low-dose steroid administration: Is included in haploidentical protocols and would decrease potential immunologic events (GVHD, graft loss, etc.) by suppressing inflammatory cytokines (TNF-Îą, IL2, and IL-6) in SCD, which is an inflammatory disease [55]. An adult patient who underwent peripheral stem cell transplantation with a conditioning regimen designed in accordance with the principles recommended above showed no transplant-related adverse events during 20 months following transplantation [56]. Another SCD patient who underwent peripheral stem cell transplantation reached day 10 months following transplantation and is being followed with no complications (unpublished data). Late Complications Problem: Development of late complications. Patients with SCD are susceptible to common transplantrelated late complications such as infertility, primary gonadal failure, primary hypothyroidism, insulin-dependent diabetes mellitus, osteoporosis, and cGVHD. Fertility is affected by multifactorial causes related to the disease and the procedure [57]. On the other hand, the course of SCDrelated complications like stroke, pulmonary hypertension,

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Özdoğu H, et al: Hematopoietic Stem Cell Transplantation in Adult Sickle Cell Disease

acute chest, nephropathy, and acute vascular necrosis is an unresolved issue for adult patients. The general prediction is that these complications can improve after transplantation. Due to the absence of available data in adult patients, we accessed pediatric data showing long-term hematological improvements after HSCT with sustained engraftment. These data have supported this expectation in most studies [58,59,60]. However, if myeloablative regimens are used, data show that SCD-related organ injuries deteriorate after transplantation over time. This was attributed to the toxic effect of drugs [58,59,60]. Proposed solution: Few published studies have evaluated infertility in patients who received a nonmyeloablative regimen. Therefore, semen cryopreservation or ovum/ zygote cryopreservation should be recommended before transplantation. Current studies indicate that other transplantrelated late complications, particularly aGVHD and cGVHD, are within acceptable limits in adults and can be managed by appropriate treatment with L-thyroxin, calcitriol, estrogen, zoledronic acid, etc. [50,58,59]. The use of nonmyeloablative reduced-intensity conditioning is mandatory for adult patients. In addition, it may require individualization of the conditioning regimens as discussed above. Further studies are required in adults for determining whether reduced-intensity conditioning regimens may offer better outcomes after HSCT. Conclusion Although results of HLA-matched sibling HSCT are encouraging, particularly in children, there are many barriers to this curative treatment option. What risk of mortality and GVHD will families accept in the early period of this benign disease? In one study, only 37% of relatives of patients accepted a 15% mortality risk; only 13% accepted a 15% mortality risk and a 15% GVHD risk [60]. Only a small proportion of patients may be candidates for transplantation due to the difficulty to find a matched sibling donor, absence of financial or psychosocial support, the family’s not accepting HSCT or the physician’s not choosing HCST as a treatment option. In one study, only 44 of 315 patients were found to have an HLA-matched sibling donor [61,62]. In addition, the likelihood of finding an unrelated fully matched donor is very low [34]. An unrelated CBT trial was terminated early due to high risks of rejection and GVHD [35]. Studies to enlarge the donor pool are required. The study of posttransplantation high-dose cyclophosphamide by Bolanos-Meade et al. is encouraging in this regard. Severe GVHD and immunodeficiency, which are seen in mismatched transplantation, were not seen in any of their patients [36].

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Research Article

DOI: 10.4274/tjh.2014.0058

Bortezomib and Arsenic Trioxide Activity on a Myelodysplastic Cell Line (P39): A Gene Expression Study Bir Miyelodisplastik Hücre Hattı (P39) Üzerine Bortezomib ve Arsenik Trioksid’in Etkileri: Bir Gen Anlatımı Çalışması Hakan Savlı1, Sara Galimberti2, Deniz Sünnetçi1, Martina Canestraro2, Giuseppe Palumbo3, Balint Nagy4, Francesco Di Raimondo3, Mario Petrini2 1Kocaeli

University Faculty of Medicine, Department of Medical Genetics, Kocaeli, Turkey University Faculty of Medicine, Department of Clinical and Experimental Medicine, Division of Hematology, Pisa, Italy 3Catania University Faculty of Medicine, Department of Clinical and Molecular Bio-Medicine, Division of Hematology, Catania, Italy 4Semmelweis University Faculty of Medicine, Department of Obstetrics and Gynecology, Budapest, Hungary 2Pisa

Abstract: Objective: We aimed to understand the molecular pathways affected by bortezomib and arsenic trioxide treatment on

myelomonocytoid cell line P39. Materials and Methods: Oligonucleotide microarray platforms were used for gene expression and pathway analysis. Confirmation studies were performed using quantitative real time PCR. Results: Bortezomib treatment has shown upregulated DIABLO and NF-κBIB (a NF-κB inhibitor) and downregulated NF-κB1, NF-κB2, and BIRC1 gene expressions. Combination treatment of the two compounds showed gene expression deregulations in concordance by the results of single bortezomib treatment. Especially, P53 was a pathway more significantly modified and a gene network centralized around the beta estradiol gene. Beta estradiol, BRCA2, and FOXA1 genes were remarkable deregulations in our findings. Conclusion: Results support the suggestions about possible use of proteasome inhibitors in the treatment of high-risk myelodysplastic syndrome (MDS). NF-κB was observed as an important modulator in leukemic transformation of MDS. Key Words: Bortezomib, Arsenic trioxide, NF-κB, MDS, Gene expression

Özet: Amaç: Bu çalışmada, bir myelomonositoid hücre hattı olan P39 üzerinde bortezomib ve arsenik trioksidin etkilediği moleküler

altyolları anlamayı amaçladık. Gereç ve Yöntemler: Gen anlatım ve altyol analizleri için oligonükleotid mikroarray platformları kullanıldı. Konfirmasyon deneyleri kantitatif gerçek zamanlı PZR ile gerçekleştirildi. Bulgular: Bortezomib işlenmesi, DIABLO ve bir NF-κB inhibitorü olan NF-κBIB’nin anlatım artışını ve NF-κB1, NF-κB2, BIRC1 genlerinin anlatım azalışını gösterdi. İki bileşiğin birlikte işlenmesi ise aynı genlerin anlatım düzensizliğini göstererek bortezomib’in tek başına işlenmesinin sonuçlarıyla uyum içindeydi. Özellikle, P53 hücre altyolu daha anlamlı bir değişikliğe uğradı ve beta estradiol geni çevresinde bir gen ağı biçimlendi. Beta estradiol, BRCA2 ve FOXA1 genlerinin düzen değişiklikleri bulgularımız içinde en dikkat çekicileriydi. Sonuç: Sonuçlar proteazom inhibitörlerinin yüksek riskli myelodisplastik sendromda (MDS) olası kullanımı düşüncesini desteklemektedir. NF-κB, MDS’nin lösemik transformasyonunda önemli bir düzenleyici olarak gözlenmiştir. Anahtar Sözcükler: Bortezomib, Arsenik trioksit, NF-κB, MDS, Gen anlatımı Address for Correspondence: Deniz SÜNNETÇİ, PhD, Kocaeli University Faculty of Medicine, Department of Medical Genetics, Kocaeli, Turkey E-mail: sun_deniz@hotmail.com Received/Geliş tarihi : February 06, 2014 Accepted/Kabul tarihi : April 18, 2014

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Introduction NF-κB is defined as an important transcription factor in immunity, cell survival, and cancer [1,2,3]. NF-κB gene activation was observed in many steps such as tumor progression and metastasis [4,5]. Relationships between NF-κB and leukemia have recently been identified through new mutations on chronic lymphocytic leukemia and specific NF-κB pathway activation of multiple myeloma [6,7]. NF-κB / Rel-blocking approaches have been proposed as antineoplastic strategies. Furthermore, the identification of specific kinases within the NF-κB activation pathway offers a selective target to address tailored therapies. Recent data provided a rationale for therapeutic approaches, which combined different NF-κB inhibitors in chronic myeloid leukemia patients [8]. NF-kB is also a nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor and it has been reported to be constitutively activated in the myelomonocytoid cell line P39 [9]. Some MDS subtypes have a high risk of developing into acute myeloid leukemia [10]. Another gene whose expression levels have been reported to play a relevant prognostic role in MDS is WT1. Changes in the expression of the WT1 gene are associated with certain types of lung, prostate, breast, and ovarian cancer. Abnormal expression of the WT1 gene also occurs in leukemia. It is unclear what role the WT1 protein plays in the development or progression of cancer [11]. We decided to assess if a compound combination (bortezomib and arsenic trioxide) able to inactivate NF-kB would be also able to down-regulate the WT1 expression. Finally, we performed microarray and real-time quantitative PCR assays to understand the gene expression pathways affected by this treatment. Materials and Methods P39 cell line (DSMZ, Zellkulturen, Braunschweig, Germany) was grown within 48 hours in RPMI 1640 medium (Gibco-LT, CA, USA) under the treatment of different concentrations of bortezomib and arsenic trioxide (ATO) as previously described in our studies [12]. Cell viability was determined by trypan blue exclusion assay, and proliferative responses were assayed by a colorimetric test based on methyl thiazoletetrazolium bromide reduction [13]. After drug exposure, signs of apoptosis were evaluated by light microscopy and the Annexin V/propidiumcytofluorimetric analysis. Reactive oxygen species (ROS) production was evaluated by dihydrorhodamine 123 (DHR) (Molecular Probes, Eugene, OR, USA) and flow-cytometry assay [12]. Samples were also evaluated by the Human Apoptosis Panel (TaqMan®, Applera, Norwalk, USA). All the experiments were repeated at least three times. Reported values represent the means ± SD. The significance of differences between

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experimental conditions was determined using the 2-tailed Student’s t test. The level of significance was p<0.05. All above cell studies were performed in our laboratories, located at Pisa University. Microarray studies and real time PCR confirmations were performed at Kocaeli University. Microarray analysis was performed using the Whole Human Genome Oligo Microarray (Agilent Technologies), encompassing more than 44,000 human DNA probes. The full list of cDNAs is available online (www.agilent.com). Protocols for sample preparation and hybridization of the mononuclear cells were adaptations of those in the Agilent Technical Manual. In short, first strand cDNA was transcribed from 300 ng of total RNA using T7Oligo (dT) Promoter Primer. Samples were transcribed in vitro and Cy-3-labeled by using a Quick-AMP labeling kit (Agilent Technologies). Following a further clean-up round (Qiagen), cRNA was fragmented into pieces ranging from 35 to 200 bases in size. Fragmented cRNA samples (1.65 ug) were hybridized onto chips by means of 17 h of incubation at 65 °C with constant rotation, followed by a two-step microarray wash of 1 min in two washing buffers (Agilent Technologies). Hybridized microarrays were scanned in a Agilent Technologies Scanner (model G2505B) and numerical results were extracted with Feature Extraction version 9.5.1.1 using 014850_D_F_20060807 grid, GE1-v5_95_Feb07 protocol and GE1_QCM_Feb07 QC metric set. The microarray data were analyzed using Gene Spring software version 6.1 (Agilent Technologies, Santa Clara, CA, USA). The fold changes were analyzed by filtering the dataset using p-value <0.01 and a signal-to-noise ratio >2 for use in t-test statistical analysis. Additional filtering (minimum 2-fold change) was applied to extract the most of these genes, which were analyzed using Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA, USA). Those genes with known gene symbols (HUGO) and their corresponding expression values were uploaded into the software. Each gene symbol was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. Networks of these genes were algorithmically generated based on their connectivity and assigned a score. The score is a numerical value used to rank networks according to how relevant they are to the genes in the input dataset but may not be an indication of the quality or significance of the network. The score takes into account the number of focus genes in the network and the size of the network to approximate how relevant this network is to the original list of focus genes. The network identified is then presented as a graph indicating the molecular relationships between genes/gene products. Genes are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). The intensity of the node color indicated the degree of up- or down-regulation. The node shapes are disclosed in corresponding figure legends. Canonical pathway analysis identified the pathways from

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Savlı H, et al: Bortezomib Upregulates NF-kB Pathway in MDS

the IPA library of canonical pathways, which were most significant to the input data set. The significance of the association between the data set and the canonical pathway was determined based on two parameters: (1) A ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway and (2) a p-value calculated using Fischer’s exact test determining the probability that the association between the genes in the data set and the canonical pathway is due to chance alone. Real time PCR confirmations were performed as described before [12,14].

the IPA analysis of our data, when P39 was co-incubated with bortezomib/ATO. Moreover, we observed SHC1, MLL, ITGAV, BCRA2, HMOX1, ICAM1, JUN, PMAP1 and beta estradiol genes were down-regulated whereas BRCA2, FOXA1, LY96 and AKR1C1 genes were up-regulated. Levels and inter relationships were defined in Figure 7.

Results Bortezomib inactivated NF-kB and exerted an antiproliferative (Figure 1) and pro-apoptotic effect (Figure 2) by blocking cell cycle in the G2 phase (Figure 3). It increased the release of reactive oxygen species (Figure 4) and downregulated the WT1 expression (Figure 5). In the untreated P39, 84 of the 93 genes involved in the apoptosis pathway and representation in the Taqman Low-Density Arrays were expressed. After treatment, bortezomib had up-regulated DIABLO and NFkBIB (NF-kB inhibitor), and down-regulated the NF-kB1, NF-kB2, and BIRC1, an anti-apoptotic gene (Figure 5). Seven gene pathways (P53, PPAR, IL6, IL2, hypoxia, Huntington’s disease, TLR) were found most significantly de-regulated in our microarray analysis. Bortezomib and ATO combination resulted in synergistic anti-proliferative and pro-apoptotic effects (Figure 6). PPAR, P53, IL6, IL2, hypoxia, Huntington’s disease, TLR and cell cycle were the pathways more significantly modified in the

Figure 1. Bortezomib exerted a significant anti-proliferative effect in a dose and time-dependent manner. 208

Figure 2. A significant increase of cell apoptotic rate was observed after treatment with the association of bortezomib (p<0.05, Fisher’s exact test). Columns, means of separate experiments; bars, standard deviation. Bortezomib exerted a significant pro-apoptotic effect (Annexin-V assay) after 48 hours incubation period. First column (CTRL) indicates the control sample without bortezomib.

Figure 3. Flow cytometry analysis of accumulation of cells with DNA content. The apoptotic cell fraction increase required the simultaneous presence of bortezomib (∗p<0.05, Fisher’s exact test). Columns, means of three separate experiments; bars, SD. Bortezomib induces a cell cycle block in G2 phase. First columns indicate the (CTRL) control samples without bortezomib.

Savlı H, et al: Bortezomib Upregulates NF-kB Pathway in MDS

Discussion We have previously detected the over expressed levels of NFκB gene in our microarray screening studies on prostate cancer, papillary thyroid cancer and leukemia [12,14,15,16,17]. This study is our first observation about the relationship between dysregulated NF-kB expression levels and MDS. P53 is a well-known cell cycle regulator and two studies have shown the relationship between NF-kB gene expression

Figure 4. ROS production after bortezomib treatment. Exposure of cells with and without bortezomib after which they were labeled with dihydrorhodamine 123 and analyzed by flow cytometry, to determine the percentage of cells displaying an increase in reactive oxygen species production (∗p<0.05 with respect to control; #p<0.05 with respect to bortezomib alone; Fisher’s exact test). Columns, means of at least three separate experiments; bars, SD. (b) Role of ROS in ATO/bortezomibmediated lethality in HL60 cells. Bortezomib induces a significant ROS production after 48 hours incubation period. First column (CTRL) indicates the control sample without bortezomib.

Figure 5. WT1 gene expression was observed as most downregulated, and BIRC1 gene expression was observed as most up-regulated. NF-kB gene was down-regulated while its inhibitor NF-kB1B gene was up-regulated. Results obtained after 48 hours incubation period.

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and lymphoblastic leukemia pathogenesis before [18,19]. Bastian et al. observed an activation of NF-κB after bortezomib treatment and the induction of apoptosis-related NF-κB target genes such as TNFαRs after concomitant treatment, indicating a possible involvement of NF-κB as a proapoptotic mediator. These findings are not in concordance with ours about the bortezomib induction in a myelodysplastic cell line (P39). It is possible that bortezomib activity has different trigger effects to NF-κB deregulation on different types of leukemic cells. Among the down-regulated genes, JUN, HSP70 and HSP90 would be relevant. Indeed, high levels of HSPs have been reported to negatively condition the overall survival of patients with high-risk MDS [20]. Other down-regulated genes were CREBBP, PMAIP1, SPP1, and some adhesion molecules, such as ICAM1. Even reduction of integrins could be a relevant effect exerted by the proteasome inhibitor due

Figure 6. Bortezomib and arsenic trioxide exert synergistic anti-proliferative and pro-apoptotic effects.

Figure 7. Significant up and down regulated gene network identified around genes after bortesomib+arsenic trioxide treatment for 48 hours to P39 cell line. 209

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to higher serum levels of ICAM1 that have been observed to be associated with high-risk MDS, negatively conditioning the survival [21]. Recently, Phase 1 studies indicated that bortezomib is a well-tolerated treatment option in acute myeloid leukemia [22]. Further studies should focus on the role of NF-kB and other pathways as key regulators to therapeutic effect of bortezomib in acute myeloid leukemia. Authors indicated that intracellular NAD* level represents a major determinant in the ability of bortezomib to induce apoptosis [23]. It is still unknown as to how many more pathways and gene networks exist on these apoptotic effects. Combinations of our two compounds showed gene expression deregulations which were in concordance with the bortezomib treatment alone. Especially, P53 was a pathway more significantly modified and same seven gene pathways were most significantly de-regulated. JUN and ICAM1 genes were found down-regulated again. Beta estradiol, BRCA2 and FOXA1 genes were remarkable in our findings. ATO and bortezomib combination resulted in a gene network centralized around the beta estradiol gene. It has been previously reported that when estrogen binds its receptor it becomes a potent mitogen in the pathogenesis of mammary carcinoma and ATO specifically inhibits expression and signaling pathway of these receptor [24]. Here we can see the ATO’s downregulation effect over this gene. Besides, protein product of beta-estradiol leads to granulocytic differentiation when itself used as a compound on human CD34+ cells [25]. We believe that the gene network around beta estradiol is important to understand relationship about differentiation treatment by ATO on AML pathogenesis. BRCA2 has been defined as a part of important pathway to prevent a subgroup of human leukemias and lymphomas that may involve non-random, characteristic gene rearrangements [26]. Upregulated levels of this pathway by the treatment of our compounds was an expected result. However, upregulated levels were extremely high as 29-fold. We found a relationship between BRCA2 and beta estradiol genes over FNG gene downregulation. FNG (Fringe) is a regulator of communication between lineage compartments during hematopoietic development [27]. We found the down regulated levels of beta estradiol were in a signal communication by BRCA2 upregulation. It means that bortezomib and ATO exposure is effective on different anti-cancer mechanisms as ‘DNA repair’ or ‘cell differentiation’, in P39 cell line. Importance of FOXA1 gene was defined before in cell transcription in different mutations in acute myeloid leukemia and also offered as a promising prognostic marker in breast

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cancer [28,29]. Upregulated level of this gene was remarkable deregulation in our experiment. It is possible to discuss similarities and relations between NF-kB and these three genes mentioned above. NF-kB is an important transciption factor as FOXA1 and its critical role is well defined in multiple myeloma [30]. Homolog recombinations in DNA repair induction by NF-κB required the target BRCA2 [31]. Estrogen modulates NFeq signaling at the promoters of inflammatory genes via estrogen receptor-B [32]. We have used compounds effective to inactivate NFkB and these affection looks deeper when we add ATO to bortezomib. Taken together, NF-kB was observed as an important modulator in leukemic transformation of MDS. These results suggest the possible use of proteasome inhibitors in treatment of high-risk MDS. In vivo trials will be useful to confirm this hypothesis. Moreover, our study was a preliminary observation of a cell line exposed to drug combinations and different leukemic cells should be studied in this manner as many dimensions of the studies would be discussed separately. This would create insight to the arising gene pathways after bortezomib and ATO exposure in leukemia, and their specific traces on networks defined on miRNA investigation and proteomic levels. 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. Nagase H, Visse R, Murphy, G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res 2006;69:562-573. 2. Bendrik C, Robertson J, Gauldie J, Dabrosin C. Gene transfer of matrix metalloproteinase-9 induces tumor regression of breast cancer in vivo. Cancer Res 2008;68:3405-3412. 3. Kato K, Hara A, Kuno T, Kitaori N, Huilan Z, Mori H, Toida M, Shibata T. Matrix metalloproteinases 2 and 9 in oral squamous cell carcinomas: manifestation and localization of their activity. J Cancer Res Clinl Oncol 2005;131:340-346. 4. Groblewska M, Siewko M, Mroczko B, Szmitkowski M. The role of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the development of esophageal cancer. Folia Histochem Cytobiol 2012;50:12-19. 5. Plummer SM, Holloway KA, Manson MM, Munks RJ, Kaptein A, Farrow S, Howells L. Inhibition of cyclo-oxygenase 2 expression in colon cells by the chemopreventive agent curcumin involves inhibition of NF-kappaB activation via the NIK/IKK signalling complex. Oncogene 1999;18:6013-6022.

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DOI: 10.4274/tjh.2013.0336

Research Article

Can Positron Emission Tomography and Computed Tomography Be a Substitute for Bone Marrow Biopsy in Detection of Bone Marrow Involvement in Patients with Hodgkin’s or Non-Hodgkin’s Lymphoma? Positron Emisyon Tomografi ve Bilgisayarlı Tomografi Hodgkin ya da Non-Hodgkin Lenfomalı Hastalarda Kemik İliği Tutulumunu Belirlemede Kemik İliği Biyopsisinin Yerini Alabilir mi? Güven Çetin1, M. Ali Çıkrıkçıoğlu2, Tuba Özkan2, Cumali Karatoprak2, M. Cem Ar3, Ahmet Emre Eşkazan3, Mesut Ayer4, Abdullah Cerit5, Kübra Gözübenli5, Betül Börkü Uysal5, Simge Erdem5, Nurhan Ergül6, Gamze Tatar6, T. Fikret Çermik6 1Bezmialem

Vakıf University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey Vakıf University Faculty of Medicine, Department of General Internal Medicine, İstanbul, Turkey 3İstanbul University Cerrahpaşa Faculty of Medicine, Department of General Internal Medicine, Division of Hematology, İstanbul, Turkey 4İstanbul Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey 5İstanbul Training and Research Hospital, Clinic of Internal Medicine, İstanbul, Turkey 6İstanbul Training and Research Hospital, Clinic of Nuclear Medicine, İstanbul, Turkey 2Bezmialem

Abstract: Objective: Positron emission tomography and computed tomography (PET/CT) has become an important part of staging and treatment evaluation algorithms of lymphoma. We aimed to compare the results of PET/CT with bone marrow biopsy (BMB) with respect to bone marrow involvement (BMI) in patients with Hodgkin’s lymphoma (HL) and aggressive non-Hodgkin’s lymphoma (aNHL).

Materials and Methods: The medical files of a total of 297 patients diagnosed with HL or aNHL and followed at the hematology clinics of 3 major hospitals in İstanbul between 2008 and 2012 were screened retrospectively and 161 patients with classical HL and aNHL were included in the study. The patients were referred for PET/CT and BMB at the initial staging. BMB was performed as the reference standard for the evaluation of BMI.

Results: There were 61 (38%) HL and 100 (62%) aNHL patients. Concordant results were revealed between PET/CT and BMB in 126 patients (78%) (52 HL, 74 aNHL), 20 with positive PET/CT and BMB results and 106 with negative PET/CT and BMB results. There were discordant results in 35 patients (9 HL, 26 aNHL), 16 of them with positive BMB and negative PET/CT results and 19 of them with negative BMB and positive PET/CT results.

Address for Correspondence: Cumali KARATOPRAK, M.D., Bezmialem Vakıf University Faculty of Medicine, Department of General Internal Medicine, İstanbul, Turkey Phone: +90 212 453 17 00-6506 E-mail: ckaratoprak@hotmail.com Received/Geliş tarihi : October 02, 2013 Accepted/Kabul tarihi : March 14, 2014

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Conclusion: We observed that PET/CT is effective to detect BMI, despite it alone not being sufficient to evaluate BMI in HL and aNHL. Bone marrow trephine biopsy and PET/CT should be considered as mutually complementary methods for detection of BMI in patients with lymphoma. In suspected focal involvement, combining biopsy and PET/CT might improve staging results.

Key Words: Positron emission tomography, Computed tomography, Bone marrow biopsy, Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma

Özet: Amaç: Positron emisyon tomografi ve bilgisayarlı tomografi (PET/BT) lenfomanın tedavi ve evreleme algoritmasının önemli bir parçası oldu. Biz çalışmamız da agresif seyirli non Hodgkin lenfoma (aNHL) ve Hodgkin lenfomalı (HL) hastalarda kemik iliği tutulumunu (KİT) göstermede kemik iliği biyopsisi (KİB) ile PET/BT sonuçlarını karşılaştırmayı amaçladık.

Gereç ve Yöntemler: Çalışmaya 2008-2012 yılları arasında İstanbul’daki 3 büyük hastanede HL ve aNHL tanısı konulan 297 hasta belirlendi. Bu hastaların dosyası retrospektif olarak tarandı ve çalışma kriterlerine uygun 161 hasta çalışmaya alındı. Hastaların başlangıç evrelemesi için PET/BT ve KİB yapıldı. KİB, KİT değerlendirmede standart referans olarak kabul edildi.

Bulgular: Hastaların 61’inde (%38) HL ve 100’ünde (%62) aNHL vardı. Değerlendirilen 126 hastada (%78) (52 HL, 74 aNHL) PET/BT ve KİB arasında uyumlu sonuçlar olduğu görüldü. Bu hastalardan 20’sinde KİT bakımından PET/CT ve KİB sonuçları pozitif ve 106 hastanın PET/CT ve KİB negatif bulundu. Diğer taraftan 35 hastada (9 HL, 26 aNHL) uyumsuzluk vardı. Bu hastaların da 16’sında KİB pozitif iken PET/BT sonuçları negatif bulundu. Geri kalan 19 hastada KİB negatif ve PET/BT sonuçları pozitif saptandı.

Sonuç: Biz aNHL ve HL hastalarını değerlendirmede tek başına yeterli olmamasına rağmen, PET/BT’nin kemik iliği infiltrasyonunu belirlemede etkili olduğunu gözlemledik. Lenfomalı hastalarda kemik iliği infiltrasyonunu belirlemede; KİB ve PET/BT birbirinin tamamlayıcısı olarak düşünülmelidir. Fokal tutulumdan şüphelenildiğinde, KİB ve PET/BT’nin birlikte yapılması hastalığın evrelemesinde daha doğru sonuçlar verebilir.

Anahtar Sözcükler: Positron emisyon tomografi, Bilgisayarlı tomografi, Kemik iliği biyopsisi, Hodgkin lenfoma, Non Hodgkin lenfoma

Introduction Hodgkin’s lymphoma (HL) and aggressive nonHodgkin’s lymphoma (aNHL) are malignant diseases of the lymphoreticular system. The modality and the duration of treatment in lymphomas are associated with the stage of the disease. In this regard, bone marrow involvement (BMI) plays a crucial role in staging [1,2]. The method considered as the gold standard today in detection of BMI is the trephine bone marrow biopsy (BMB) [3,4]. This method is more useful for diffuse patterns of bone marrow infiltration without intervening areas of normal marrow. However, the invasive nature of this procedure makes its use difficult in patients with poor general condition or tendency to bleed. Moreover, in cases of focal bone marrow infiltration with intervening areas of preserved marrow, lymphoma cells might not be detected at the point of biopsy, leading to false negative results. In this context, a diagnostic tool that eliminates the requirement of an invasive procedure and is able to visualize focal infiltration would facilitate staging. Previous studies have indicated that positron emission tomography and computed tomography (PET/CT) is 214

a convenient method for bone marrow assessment in patients with lymphoma [1,5,6]. In this study, we aimed to compare the results of PET/CT with BMB regarding BMI in patients with HL and aNHL. Materials and Methods The medical files of a total of 297 patients diagnosed with HL or aNHL and followed at the hematology clinics of 3 major hospitals in İstanbul between 2008 and 2012 were screened retrospectively and 161 patients with classical HL and aNHL were included in the study. Patients with indolent aNHL, chronic lymphocytic leukemia/small lymphocytic lymphoma, or nodular lymphocyte-predominant HL and those with secondary malignancies were excluded from the study. Written permission was obtained from the medical directors of the relevant hospitals for retrospective file screening. The study was approved by the Ethics Committee of the Bezmialem Vakıf University Medical Faculty. All BMB samples were obtained from the dorsal iliac crest (unilateral), with a length of 10-15 mm. They were fixed in Hollande’s solution and sent to the pathology department for

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histological evaluation. The BMB samples were reviewed by a hematopathologist and BMB was considered the reference standard to detect BMI. PET/CT imaging was performed using a Siemens Biograph LSO HI-REZ integrated PET/CT camera (Biograph 6, Siemens Medical Solutions, Chicago, IL, USA). PET/CT scans were obtained 60-80 min after the administration of 5.4 MBq/kg 18F-fluorodeoxyglucose (FDG). The patients fasted for at least 6 h and serum glucose levels were below 120 mg/dL in all patients. All PET/CT images were visually assessed for BMI by 2 experienced nuclear medicine physicians without the results of the BMBs at the Department of Nuclear Medicine of the İstanbul Training and Research Hospital. The uptake of FDG in the bone marrow was visually classified into 3 categories: (1) diffusely intense FDG uptake in the bone marrow, which represents diffuse involvement; (2) lesions with focal intense FDG uptake, which were suggestive of focal involvement of bone marrow; (3) no focal or diffuse increased FDG uptake in bone marrow, which represents normal bone marrow functions. Anemia, infection, or other pathological situations that could cause false positivity on PET/CT imaging were excluded by a physical examination, complete blood cell counts, biochemistry tests including acute phase reactant tests, serology tests, pulmonary function tests, echocardiography, and CT of the chest, abdomen, and pelvis. Positive PET/CT results were defined as a malignancy by nuclear medicine physicians. Statistical Analysis Numerical values are given as means and standard deviations; nominal values were compared by using kappa statistics. Sensitivity, specificity, negative and positive predictive values, and overall accuracy values of PET/CT were calculated as compared with the gold standard. Twotailed p-values of less than 0.05 were considered statistically significant. Results There were 61 (37.8%) and 100 (62.2%) patients with HL and aNHL, respectively. Table 1 summarizes patient characteristics, including age, sex, and stages of disease. BMI was detected in 36 patients (22.3%) by BMB and 39 patients (24.2%) by PET/CT. If BMB and PET/CT results supported each other, they were considered as concordant results. Regarding the type of lymphoma, this study showed a concordance between PET/CT and BMB in 126 patients (78%) (74 aNHL, 52 HL), of which 20 were positive (15 aNHL, 5 HL) and 106 were negative (59 aNHL, 47 HL). If BMB and PET/CT results conflicted with each other, they were considered as discordant results. Discordant results were observed in 35 cases (22%): 19 of them with positive

PET/CT and negative BMB results (12 aNHL, 7 HL) and 16 with negative PET/CT and positive BMB results (14 aNHL, 2 HL). Comparison of BMB and PET/CT results in the detection of BMI are summarized in Table 2 (Figure 1). In patients with HL, a statistically significant accordance was seen between BMB and PET/CT with respect to BMI (kappa value 0.446; p<0.001). The nonrandom concordance rate between the 2 methods was 44.6%. When we considered BMB as the gold standard, the sensitivity and specificity of PET/CT in HL cases were 71.4% and 87%, respectively. Positive and negative predictive values were 41.7% and 95%, respectively. Overall accuracy was 85.2%. The accordance between BMB and PET with respect to BMI in aNHL was also found to be significant (kappa value 0.355; p<0.001). The nonrandom concordance ratio between the 2 methods was 35.5%. When we utilized BMB as the gold standard, the sensitivity and specificity of PET/CT in aNHL were 51.7% and 83.0%, respectively. Positive and negative predictive values were 55.5% and 80.8%, respectively. Overall accuracy was 74%. We found a statistically significant compliance between results of BMI and PET/CT with regard to BMI in all patients (kappa value 0.392, p<0.001). A nonrandom concordance of 39.2% between the 2 methods was observed. According to BMB as the gold standard, the sensitivity and specificity of PET/CT were 55.6% and 84.8%, respectively. According to BMB, positive predictive values, negative predictive values, and general accuracy of PET/CT were 51.3%, 86.9%, and 78.3%, respectively. Table 1. Patient characteristics.

aNHL

Male

Female

16-75

18-85

III

Total (n)

100

Sex

Age, years Range Stage*

*Ann Arbor Staging System. HL: Hodgkin’s lymphoma, aNHL: aggressive non-Hodgkin’s lymphoma.

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Table 2. Cross-table for patients with bone marrow involvement (positron emission tomography and computed tomography vs. trephine biopsy).*

Patient group

Bone marrow trephine biopsy Positive Negative

PET/CT

All patients

aNHL

Positive

Negative

106

Positive

Negative

Positive

Negative

*Digits indicate patient numbers. PET/CT: Positron emission tomography and computed tomography, HL: Hodgkin’s lymphoma, aNHL: aggressive nonHodgkin’s lymphoma.

Discussion Disease staging is required to determine treatment strategies in patients with HL and aNHL. Presence or absence of BMI plays a determinative role in staging [1,4]. The gold standard in detecting BMI is the trephine BMB. However, since it is an invasive procedure and may give false negative results in cases of focal involvement, the effectiveness of noninvasive methods that are able to detect focal involvements, such as PET/CT, has also been considered [7,8]. PET/CT imaging has become an important part of lymphoma staging and treatment algorithms [7,8,9]. Moreover, the presence of persistent FDG uptake during or after chemotherapy has a high sensitivity and specificity for predicting disease recurrence [9]. Staging is a crucial step in the initial diagnostic work-up of lymphoma with the aim of determining the extent of the disease and detecting subclinical disease. Early studies revealed strong FDG involvement in the initial staging of both HL and aNHL [10,11,12]. Bangerter et al. found occult disease in 5 patients (11%) in addition to PET/CT positivity detected in 38/44 (86%) of patients with proven disease [11]. PET/CT was able to detect 128 abnormal regions, of which 11 could not be detected previously by conventional methods. PET/CT has changed the management of the disease by 14% [9]. In a prospective study by Young et al., biopsies were taken from the sites of involvement detected by PET/CT to confirm the infiltration, and it was demonstrated that PET/CT changed the disease stage in 59% of the 45 patients included in the study [13].

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d Figure 1. Positron emission tomography and computed tomography images. a) Aggressive non-Hodgkin’s lymphoma patient bone marrow involvement-positive in bone marrow biopsy and bone marrow involvement-negative in positron emission tomography and computed tomography imaging, b) Aggressive non-Hodgkin’s lymphoma patient bone marrow involvement-negative in bone marrow biopsy and bone marrow involvement-positive in positron emission tomography and computed tomography imaging, c) Hodgkin’s lymphoma patient bone marrow involvement-positive in bone marrow biopsy and positron emission tomography and computed tomography imaging together, d) Hodgkin’s lymphoma patient bone marrow involvement-negative in bone marrow biopsy and positron emission tomography and computed tomography imaging together.

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In our study we evaluated PET/CT as a substitute for BMB. According to our data, PET/CT detected 20 positive cases that were detected with BMB. Additionally, 19 (11.8%) cases that were considered false-positive according to BMB were evaluated for staging by PET/CT. At this point we accepted modified stages at the beginning of the chemotherapy. By the time biopsy results were revealed, we had started chemotherapy. We correlated these results with focal involvement. During chemotherapy we could not retake the biopsy due to it not already being in the routine practice. Nineteen discordant cases were treated with chemotherapy because bone marrow infiltration could not be excluded. The results of 16 discordant cases showed false negativity of PET/CT imaging. One of the issues in the staging of aNHL is detection of BMI [14]. Many studies have shown that PET/CT is superior to conventional imaging in evaluation of bone marrow infiltration in conjunction with histological verification [15,16]. In a study that included 50 patients (12 with HL and 38 with aNHL), an overall accuracy of 93% was attained [17]. In another study that compared 78 patients (39 with HL and 39 with aNHL), although BMI was found in 13% of the patients by PET/CT, false negativity of 5% was found. When PET/CT and BMB were compared, overall accuracy was 95% for PET/CT and 89% for BMB [8]. In a study performed by Muslimani et al., 97 patients with aNHL were studied. Wholebody PET/CT and unilateral iliac BMB were performed in the patients for initial staging, and as a result, sensitivity and specificity of PET/CT in detection of bone marrow infiltration in the patients were found to be 79% and 91%, respectively [18]. According to the study conducted by Purz et al. in pediatric patients with HL, while BMB was positive in 7 of 175 patients, BMI was detected by PET/CT in 45 patients. Moreover, a typical multifocal pattern was found in 32 of these 45 patients and involvements detected by PET/CT were reported to disappear after chemotherapy [19]. However, in a study performed by Jerusalem et al., which included 42 patients, accuracy of detection by PET/CT was 39% in indolent NHL cases [20]. This appears to be a result of low FDG uptake in some subtypes of lymphoma and of the difficulty in discriminating the infiltration pattern from physiological bone marrow uptake [21]. In a prospective study of 52 patients that compared BMB with PET/CT and CT, PET/CT was able to visualize bone marrow infiltration more accurately than CT (p<0.05) and at a comparable level to bone marrow trephine biopsy. PET/CT led to treatment changes in 8% of patients [22]. In a review performed by Haioun et al., results obtained by using PET/CT alone were in line with conventional imaging and BMB in only 80% of the cases. Using PET/CT alone yielded superior results to both methods in 8% of the cases and inferior results to both of them in 12 of the cases [23]. In a metaanalysis of 587 patients that evaluated

PET/CT results in detection of bone marrow involvement in HL and aNHL, this method was designated as a good, but not excellent, tool [24]. PET/CT is the best noninvasive imaging technique in the evaluation of the response to treatment [25]. Early studies have shown that persistence of FDG uptake after treatment was associated with high recurrence rates [26,27]. Similar results have been reported by others [28,29,30]. However, it should be considered that increased FDG uptake may also be related to active infection and inflammation. Before any treatment decision is made, PET/CT images should be in line with the clinical findings, other imaging techniques, and/ or biopsy [31]. Negative PET/CT imaging cannot rule out minimal residual disease that may lead to clinical relapse [32]. Currently, PET/CT is widely used for the evaluation of response after the completion of treatment. To a lesser extent, it is used for staging before treatment. PET/CT cannot be a substitute for CT or BMB in staging before treatment, but it provides complementary information to them. It is much better than CT in conclusive evaluation of response to treatment, because this method can discriminate living tumor tissue from fibrosis and necrosis. The predictive potential of PET/CT imaging in the follow-up of patients with lymphoma in the absence of clinical, biochemical, or radiographic evidence of disease is unknown. Large prospective studies are needed to assess the role of PET/CT imaging with regard to patient follow-up and costs [33]. In conclusion, bone marrow trephine biopsy and PET/CT should be considered as mutually complementary methods for detection of BMI in patients with lymphoma. In cases of suspected focal involvement, combining biopsy and PET/CT might improve staging results. The current literature suggests that PET/CT may be more sensitive than BMB in cases with focal involvements. Our results partly confirm the literature data. 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. Moulin-Romsee G, Hindié E, Cuenca X, Brice P, Decaudin D, Bénamor M, Brière J, Anitei M, Filmont JE, Sibon D, de Kerviler E, Moretti JL. 18F-FDG PET/CT bone/bone marrow findings in Hodgkin’s lymphoma may circumvent the use of bone marrow trephine biopsy at diagnosis staging. Eur J Nucl Med Mol Imaging 2010;37:1095-1105. 2. Devesa SS, Fears T. Non-Hodgkin’s lymphoma time trends: United States and international data. Cancer Res 1992;52(Suppl 19):5432s-5440s. 217

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Research Article

DOI: 10.4274/tjh.2013.0410

Detection and Significance of CD4+CD25+CD127dim Regulatory T Cells in Individuals with Severe Aplastic Anemia CD4+CD25+CD127dim Düzenleyici T Hücrelerinin Ağır Aplastik Anemili Bireylerde Saptanması ve Önemi Weiwei Qi, Yue Ren, Rong Fu, Huaquan Wang, Chunyan Liu, Zonghong Shao General Hospital of Tianjin Medical University, Department of Hematology, Tianjin, China

Abstract: Objective: To investigate the relationship between CD4+CD25+CD127dim regulatory T cells (Tregs) and immune imbalance in acquired severe aplastic anemia (SAA).

Materials and Methods: The quantity of CD4+CD25+CD127dim Tregs in 44 SAA patients and 23 normal controls was measured by flow cytometry. Correlations between Tregs and T cell subsets, dendritic cell (DC) subsets, granulocyte counts, and percentage of reticulocytes (RET%) were analyzed.

Results: The percentage of CD4+CD25+CD127dim Tregs in peripheral blood lymphocytes (PBLs) of untreated patients was lower than in recovery patients and normal controls (0.83±0.44% vs. 2.91±1.24% and 2.18±0.55%, respectively, p<0.05). The percentage of CD4+CD25+CD127dim Tregs in CD4+ T lymphocytes of recovery patients was higher than that of untreated patients and normal controls (9.39±3.51% vs. 7.61±5.3% and 6.83±1.4%, respectively, p<0.05). The percentage of CD4+ T lymphocytes in PBLs of untreated patients was lower than in recovery patients and normal controls (13.55±7.37% vs. 31.82±8.43% and 32.12±5.88%, respectively, p<0.05). T cell subset (CD4+/CD8+ ratio) was 0.41±0.24 in untreated patients, which was lower than in recovery patients (1.2±0.4) and normal controls (1.11±0.23) (p<0.05). DC subset (myeloid DC/plasmacytoid DC ratio, DC1/ DC2 ratio) was 3.08±0.72 in untreated patients, which was higher than in recovery patients (1.61±0.49) and normal controls (1.39±0.36) (p<0.05). The percentage of CD4+CD25+CD127dim Tregs in PBLs was positively associated with T cell subset (r=0.955, p<0.01) and negatively associated with DC subset (r=-0.765, p<0.01). There were significant positive correlations between CD4+CD25+CD127dim Tregs/PBL and granulocyte counts and RET% (r=0.739 and r=0.749, respectively, p<0.01).

Conclusion: The decrease of CD4+CD25+CD127dim Tregs in SAA patients may cause excessive functioning of T lymphocytes and thus lead to hematopoiesis failure in SAA.

Key Words: Severe aplastic anemia, Regulatory T cell, Bone marrow failure

Address for Correspondence: Weiwei QI, M.D., General Hospital of Tianjin Medical University, Department of Hematology, Tianjin, China E-mail: 4817066@qq.com Received/Geliş tarihi : December 09, 2013 Accepted/Kabul tarihi : May 07, 2014

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Özet: Amaç: Edinilmiş ağır aplastik anemilerde (AAA) rastlanan CD4+CD25+CD127dim düzenleyici T hücreleri (Tregs) ve bağışıklık

bozukluğu arasındaki ilişkiyi araştırmayı amaçladık. Gereç ve Yöntemler: Kırk dört AAA hastasındaki ve 23 normal kontroldeki CD4+CD25+CD127dim Tregs miktarıları akış sitometrisi kullanarak ölçüldü. Tregs ve T hücrelerinin alt tipleri, dentrik hücrelerin alt tipleri (DH), granülosit sayımları ve retikülosit yüzdeleri (%RET) aralarındaki ilişkiler analiz edildi. Bulgular: Tedavi edilmemiş hastaların çevresel kan lenfositlerindeki (ÇKL) CD4+CD25+CD127dim Tregs’in yüzdesi iyileşme sürecindeki hastalarla ve normal kontrollerle kıyaslandığında daha düşüktü (sırasıyla %0,83±0,44’e karşı %2,91±1,24 ve %2,18±0,55, p<0,05). İyileşme sürecindeki hastaların CD4+ T lenfositlerindeki CD4+CD25+CD127dim Tregs’in yüzdesi tedavi edilmemiş hastalarla ve normal kontrollerle kıyaslandığında daha yüksekti (sırasıyla %9,39±3,51’e karşı %7,61±5,3 ve %6,83±1,4, p<0,05). Tedavi edilmemiş hastaların CD4+ T lenfositlerin yüzdesi iyileşme sürecindeki hastalarla ve normal kontrollerle kıyaslandığında daha düşüktü (sırasıyla %13,55±7,37’ye karşı %31,82±8,43 ve 32,12±5,88, p<0,05). Tedavi edilmemiş hastalardaki T hücresi alt tipi (CD4+/CD8+ oran) 0,41±0,24 seviyesindeydi; bu oran iyileşme sürecindeki hastalarla (1,2±0,4) ve normal kontrollerle (1,11±0,23) kıyaslandığında daha düşük bir seviyeydi (p<0,05). DH alt dizini (miyeloid DH/plazmasitoid DH oranı, DH1/DH2 oranı) ise tedavi edilmemiş hastalarda 3,08±0,72 seviyesindeydi; bu oran iyileşme sürecindeki hastalarla (1,61±0,49) ve normal kontrollerle kıyaslandığında (1,39±0,36) daha yüksekti (p<0,05). ÇKL’deki CD4+CD25+CD127dim Tregs’in yüzdesi pozitif olarak T hücresi alt kümesiyle (r=0,955, p<0,01) ve negatif olarak da DH alt kümesiyle (r=-0,765, p<0,01) korele etmekteydi. CD4+CD25+CD127dim Tregs/ÇKL ve granülosit değerleri ve %RET (sırasıyla r=0,739, 0,749, p<0,01, p<0,01) arasında önemli pozitif korelasyon saptandı.

Sonuç: AAA hastalarındaki CD4+CD25+CD127dim Tregs’in azalması T lenfositlerde aşırı fonksiyona sebebiyet verebilir ve bu durum AAA’da hematopoetik yetmezliğine yol açabilir.

Anahtar Sözcükler: Ağır aplastik anemi, Düzenleyici T hücre, Kemik iliği yetmezliği Introduction Severe aplastic anemia (SAA) is a rare disease characterized by severe pancytopenia and bone marrow failure. Recent studies confirmed that aplastic anemia (AA) is an immunemediated disease characteristic of hematopoietic cells destroyed by activated T lymphocytes [1,2,3]. Activated suppressor T lymphocytes are markedly increased in SAA patients and suppress normal hematopoiesis in vitro. In addition, suppressive cytokines produced by activated T lymphocytes also play a vital role in the pathogenesis of bone marrow failure. High levels of suppressive cytokines indicate an excessive Th1 type of T cell response. Recent studies have shown that myeloid dendritic cells (DCs), which also expressed high levels of costimulatory molecules (CD86), were increased in AA and were associated with disease severity. This could explain why naive T cells are prone to differentiating into Th1 cells [4]. Furthermore, CD4+CD25+FOXP3+ regulatory T cells (Tregs) were decreased at presentation in almost all patients with AA, and a defect in Tregs might explain the increased autoreactive T cells and the development of an AA phenotype [5]. Tregs are a suppressive subset of T cells, which are essential for maintaining appropriate immune self-tolerance and balance. The main function of Tregs is their powerful capacity to suppress T effector or memory cell activation as

well as to regulate self-reactive T cells [6]. Clinical studies have demonstrated a decreased suppressive ability and quantity of Tregs in patients with autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis [7,8]. Previous studies from our group and others suggested that CD4+CD25+ Tregs were decreased in untreated SAA patients compared with recovery patients and normal controls [9,10]. However, CD25 is not a unique Treg marker because it is also present on activated T cells. Recently, studies proposed that low expression of CD127 coexpressed with Foxp3 and CD25, particularly in humans, could be used to distinguish Tregs from activated CD4+ T cells [11]. In this study, we focused on detecting the quantity of Tregs in SAA, which is considered an autoimmune disease, and we analyzed the relationship between Tregs and the severity of disease as well as T cell subset and DC subset. Materials and Methods Study Subjects A total of 44 patients with SAA were enrolled in this study, including 25 untreated cases and 19 cases in remission after immunosuppressive therapy. All were inpatients of the Hematology Department of Tianjin Medical University General Hospital, China, from January 2013 to October 2013. The diagnosis of SAA was based on International AA Study Group criteria [2]. Patients were excluded if they had 221

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congenital AA or other autoimmune diseases. Paroxysmal nocturnal hemoglobinuria (PNH) clone and bone marrow cytogenetic studies were performed for all patients. The patients’ features are shown in Tables 1, 2, and 3. Twenty-three healthy controls with a median age of 33 years (range: 2150) were also enrolled in this study, including 6 males and 17 females. The study was approved by the Ethics Committee of Tianjin Medical University, China. Informed written consent was obtained from all patients or their parents in accordance with the Declaration of Helsinki. Monoclonal Antibodies CD4-PE, CD25-FITC, CD127-APC, FITC-lineage-cocktail (CD3, CD14, CD16, CD19, CD20, CD56), HLA-DR-PerCP, CD123-PE, CD11c-APC, CD3-PE-Cy5, CD4-FITC, CD8-PE, mouse isotype controls, and lysis solution were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Measurement of Quantities of Tregs from Peripheral Blood CD4+CD25+CD127dim Tregs were identified by singleplatform 3-color flow cytometric analysis. Fresh blood samples of 200 µL processed by ethylenediaminetetraacetic acid were separated into 2 TruCount tubes (Becton Dickinson) and stained with mouse IgG1-PE, mouse IgG1-FITC, and mouse IgG1-APC as a negative control, or with CD4-PE, CD25-FITC, and CD127-APC. After incubation in the dark for 20 min at 4 °C, cells were incubated with 1 mL of lysis solution (Becton Dickinson) for 10 min at room temperature and washed 3 times with phosphate buffered saline. Each specimen was acquired on a FACS-Calibur flow cytometer and analyzed using CellQuest 3.1 software (Becton Dickinson). Measurement of T Cell Subsets and Dendritic Cell Subsets in Peripheral Blood CD3+CD4+ and CD3+CD8+ T lymphocytes were stained with CD3-PE-Cy5, CD4-FITC, and CD8-PE. Myeloid DC (DC1) was identified as lineage(-)HLA-DR(+)CD11c(+), while plasmacytoid DC (DC2) was identified as lineage(-) HLA-DR(+)CD123(+). DC1 and DC2 were stained with FITClineage-cocktail, HLA-DR-PerCP, CD123-PE, and CD11cAPC. Further staining followed the protocol described above. Statistical Analysis Statistical analysis was performed by parametric t-test. Correlation studies were performed by Spearman analysis. A value of p<0.05 was considered statistically significant. Results Decreased Numbers of Tregs in Untreated Severe Aplastic Anemia Compared with Healthy Controls The percentage of CD4+CD25+CD127dim Tregs in peripheral

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blood lymphocytes (PBLs) was 0.83±0.44% in untreated SAA patients, 2.91±1.24% in recovery patients, and 2.18±0.55% in normal controls. The percentage of CD4+CD25+CD127dim Tregs in PBLs in untreated SAA patients was lower than in recovery patients and normal controls (p<0.05), and there was no statistical difference between recovery patients and normal controls (p>0.05). The percentage of CD4+CD25+CD127dim in CD4+ T cells in recovery patients (9.39±3.51%) was higher than in untreated patients (7.61±5.3%) and normal controls (6.83±1.4%) (p<0.05), and there was no statistical difference between recovery patients and normal controls (p>0.05). The percentage of CD4+ T cells in PBLs from untreated patients (13.55±7.37%) was lower than in recovery patients (31.82±8.43%) and normal controls (32.12±5.88%) (p<0.05), while there was no statistical difference between recovery patients and normal controls (p>0.05) (Figure 1). T Cell Subsets and Dendritic Cell Subsets Were Altered in Severe Aplastic Anemia Patients T cell subset (CD4+/CD8+ ratio) was 0.41±0.24 in untreated SAA patients, which was significantly lower than in recovery patients (1.2±0.4) and normal controls (1.11±0.23) (p<0.05). There was no difference between recovery patients and normal controls (p>0.05). DC subset (myeloid DC/ plasmacytoid DC ratio, DC1/DC2 ratio) was 3.08±0.72 in untreated SAA patients, which was significantly higher than in recovery patients (1.61±0.49) and normal controls (1.39±0.36) (p<0.05). There was no difference between recovery patients and normal controls (p>0.05) (Figure 2). Table 1. Characteristics of untreated severe aplastic anemia patients.

Parameter

Number Mean±SD (range)

Untreated SAA patients

Male

Female

Age

25.60±14.60 (8-57)

Granulocytes (x109/L)

0.25±0.14 (0.02-0.47)

Hemoglobin (g/L)

74.12±13.96 (46-94)

Platelets (x109/L)

10.12±4.87 (2-19)

RET%

0.38±0.36 (0.07-1.45)

Abnormal karyotype Absent

PNH clone

Absent

Therapy

(ATG+CsA)

SAA: Severe aplastic anemia, PNH: paroxysmal nocturnal hemoglobinuria.

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Table 2. Characteristics of untreated severe aplastic anemia patients.

Case

Age/Sex

Granulocytes (x109/L)

Hemoglobin (g/L)

Platelets (x109/L)

RET%

Abnormal Chromosome

Therapy

49/M

0.12

Absent

ATG+CsA

50/M

0.16

0.25

Absent

ATG+CsA

28/F

0.47

1.11

Absent

ATG+CsA

20/M

0.39

0.6

Absent

ATG+CsA

18/M

0.38

0.3

Absent

ATG+CsA

57/M

0.35

1.11

Absent

ATG+CsA

55/M

0.24

0.62

Absent

ATG+CsA

28/M

0.14

0.22

Absent

ATG+CsA

17/M

0.05

0.19

Absent

ATG+CsA

19/M

0.38

0.23

Absent

ATG+CsA

8/F

0.17

0.12

Absent

ATG+CsA

10/F

0.46

0.43

Absent

ATG+CsA

24/M

0.07

0.31

Absent

ATG+CsA

15/M

0.04

0.15

Absent

ATG+CsA

51/F

0.28

0.09

Absent

ATG+CsA

26/M

0.31

0.26

Absent

ATG+CsA

24/M

0.43

1.45

Absent

ATG+CsA

18/M

0.02

0.09

Absent

ATG+CsA

15/F

0.39

0.5

Absent

ATG+CsA

12/F

0.37

0.13

Absent

ATG+CsA

17/F

0.26

0.07

Absent

ATG+CsA

23/F

0.26

0.08

Absent

ATG+CsA

17/F

0.11

0.18

Absent

ATG+CsA

19/M

0.13

0.51

Absent

ATG+CsA

20/M

0.25

0.46

Absent

ATG+CsA

F: Female, M: male.

Correlations between CD4+CD25+CD127dim Tregs and T Cell Subsets, Dendritic Cell Subsets, Granulocytes, and RET% in Severe Aplastic Anemia Patients The percentage of CD4+CD25+CD127dim Tregs in PBLs (CD4+CD25+CD127dim Tregs/PBL) was significantly and positively associated with T cell subset (r=0.955, p<0.01). However, CD4+CD25+CD127dim Tregs/PBL was negatively associated with DC subset (r=-0.765, p<0.01). There were positive correlations between CD4+CD25+CD127dim Tregs/ PBL and granulocyte counts and RET% (r=0.739 and r=0.749, respectively, p<0.01). There was no significant correlation between CD4+CD25+CD127dim Tregs/PBL and hemoglobin and platelets (Figure 3).

Discussion Tregs are considered to play a key role in the mechanisms of peripheral immune tolerance. Tregs use various mechanisms such as cell-to-cell contact and secretion of soluble factors to suppress immune responses. There are several specific mechanisms included, such as the release of inhibitory cytokines and immunosuppressive adenosine; inhibition of IL-2 secretion; perforin- or granzyme-dependent cytolysis of effector cells; and down-regulation of antigen present cell (APC) function via costimulation with CTLA-4 [12]. There are 2 types of Tregs: natural Tregs (nTregs), which develop in the thymus, and induced Tregs (iTregs), which differentiate

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Table 3. Characteristics of recovery severe aplastic anemia patients.

Case

Age/ Sex

Granulocytes (x109/L)

Hemoglobin Platelets (g/L) (x109/L)

RET%

Abnormal Chromosome

Therapy

20/M

1.91

0.78

Absent

ATG+CsA

9/M

2.26

0.92

Absent

ATG+CsA

7/F

4.21

1.53

Absent

ATG+CsA

8/F

8.73

101

1.29

Absent

ATG+CsA

27/M

6.12

2.46

Absent

ATG+CsA

22/M

5.24

109

2.17

Absent

ATG+CsA

26/M

3.75

102

2.08

Absent

ATG+CsA

32/M

3.19

109

1.68

Absent

ATG+CsA

16/F

3.27

2.58

Absent

ATG+CsA

11/F

5.15

1.75

Absent

ATG+CsA

14/F

1.49

3.86

Absent

ATG+CsA

39/M

1.36

1.97

Absent

ATG+CsA

31/M

1.92

3.29

Absent

ATG+CsA

18/M

6.81

1.06

Absent

ATG+CsA

19/F

5.86

0.97

Absent

ATG+CsA

45/M

2.58

102

1.69

Absent

ATG+CsA

41/M

4.67

0.89

Absent

ATG+CsA

60/M

6.13

0.95

Absent

ATG+CsA

39/M

5.61

107

1.39

Absent

ATG+CsA

F: Female, M: male.

from naive CD4+ T cells in the periphery. Foxp3 is the most specific marker of nTregs and is critical for the development and maintenance of the suppressive function of Tregs. However, Foxp3 is expressed in the nucleus of Tregs, and therefore it cannot be used to identify nTregs during isolating and purification. It was recently shown that CD127, a specific Treg surface marker, is inversely correlated with Foxp3 expression and the suppressive capacity of nTregs [13]. In the current study, we used the characteristics of constitutively high expression of CD25 and diminished expression of CD127 to discriminate Tregs from conventional CD4+ T cells. Cellular immunity is abnormal in SAA patients. Numerous studies have shown that both the quantity and the function of myeloid DCs are increased, that Th1 cells are excessively activated along with excessive secretion of Th1-type cytokines, that the ratio of CD4+/CD8+ T lymphocytes is decreased, and that cytotoxic T cells are over activated [1,4]. Currently, the consensus is that acquired AA is an autoimmune disease. AA

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pathogenesis is thought to involve damage to hematopoietic stem/progenitor cells by autoreactive T lymphocytes activated by Th1 cells. An important question is why these autoreactive T cells are not suppressed and removed by selftolerance. Furthermore, what is the influence of Tregs on the complex pathogenesis of AA? Our results demonstrated that the percentage of CD4+CD25+CD127dim Tregs in the peripheral blood was decreased in SAA patients. Furthermore, the percentage of CD4+CD25+CD127dim Tregs increased and was restored to normal levels in the patients after intensive immunosuppressive therapy. Additionally, in SAA patients with a more serious condition, we noted that their granulocyte counts and RET% were markedly decreased, DC1 and DC1/DC2 ratios were increased, CD4+/CD8+ ratios were decreased, and Tregs were decreased. After statistical analysis, we concluded that the percentage of CD4+CD25+CD127dim Tregs in PBLs was negatively associated with DC subset and positively associated with T cell subset, granulocyte counts, and RET%. These results suggested that Tregs were

Qi W, et al: Significance of Regulatory T Cells in SAA

involved in the pathogenesis of SAA and inversely associated with the severity of disease. We postulate that decreased numbers of Tregs fail to suppress the function of DCs and

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activated T cells, which impair and damage hematopoietic cells in the bone marrow. Our results also indicated that the antithymocyte globulin (ATG) plus cyclosporine therapeutic regimen induced the proliferation of Tregs. This was consistent with a previous study using in vitro cultures of normal human peripheral blood mononuclear cells treated with low-dose rabbit ATG that markedly expanded functional Tregs [14]. These findings indicated that Tregs might be protective against SAA. Furthermore, CD4+CD25+CD127dim Tregs could be used as a biomarker to determine the curative effect of treatments and evaluate the prognosis of SAA. During SAA patient recovery, the quantity of CD4+CD25+CD127dim Tregs increased gradually, suggesting a better prognosis. It is essential that the function of Tregs be studied and analysis of Tregs at different stages of SAA be conducted in the future. Recent studies have explored new methods of expanding or inducing de novo generation of Tregs, which could provide a new approach for the treatment of autoimmune diseases.

Figure 1. The quantities of Tregs and CD4+ T cells in untreated severe aplastic anemia patients (SAA), recovery patients (R-SAA), and normal controls (Health). A) The percentage of CD4+CD25+CD127dim Tregs in peripheral blood lymphocytes. B) The percentage of CD4+CD25+CD127dim Tregs in CD4+ T cells. C) The percentage of CD4+ T cells in peripheral blood lymphocytes.

Figure 2. The T cell subsets and dendritic cell subsets in untreated severe aplastic anemia patients (SAA), recovery patients (R-SAA), and normal controls (Health). A) The T cell subsets (CD4+/CD8+ ratios) in the 3 groups. B) The dendritic cell subsets (DC1/DC2 ratios) in the 3 groups. 225

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Figure 3. The correlations between CD4+CD25+CD127dim Tregs and T cell subsets, dendritic cell subsets, granulocytes, and RET% in severe aplastic anemia patients. A) The correlation between the percentage of CD4+CD25+CD127dim Tregs in peripheral blood lymphocytes (PBLs) (CD4+CD25+CD127dim/PBL) and T cells subsets (CD4+/CD8+ ratios). B) The correlation between CD4+CD25+CD127dim/PBL and dendritic cell subsets (DC1/DC2 ratios). C) The correlation between CD4+CD25+CD127dim/PBL and granulocyte count. D) The correlation between CD4+CD25+CD127dim/PBL and RET%. Cao et al. showed that Tregs from patients with autoimmune diseases could be purified, activated, and expanded similarly to those from healthy donors. Additionally, ex vivo expanded Tregs showed enhanced suppressive functions compared with Tregs freshly purified from the same patients [15]. Studies using murine models confirmed that Treg adoptive transfer is feasible and efficient for treating autoimmune disease [16]. Taken together, these studies suggest that new therapeutic strategies based on Tregs for the treatment of SAA might be beneficial. Acknowledgments This project was partly supported by the Natural Science Foundation of China (No. 81170472, No. 81370607), a Major Project of the Natural Science Foundation of Tianjin (No. 12JCZDJC21500), a Major Project of the Tianjin Cancer Research Programs (No. 12ZCDZSY17900, No. 12ZCDZSY18000), a Key Project of Tianjin Health Industry Research (No. 11KG135), and a Special Project of Health Industry Scientific Research (No. 201202017).

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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. Young NS, Scheinberg P, Calado RT. Aplastic anemia. Curr Opin Hematol 2008;15:162-168. 2. Marsh JC, Ball SE, Cavenagh J, Darbyshire P, Dokal I, GordonSmith EC, Keidan J, Laurie A, Martin A, Mercieca J, Killick SB, Stewart R, Yin JA; British Committee for Standards in Haematology. Guidelines for the diagnosis and management of aplastic anaemia. Br J Haematol 2009;147:43-70. 3. Young NS, Bacigalupo A, Marsh JC. Aplastic anemia: pathophysiology and treatment. Biol Blood Marrow Transplant 2010;16(Suppl 1):119-125. 4. Zonghong S, Meifeng T, Huaquan W, Limin X, Jun W, Rong F, Hong L, Yuhong W. Circulating myeloid dendritic cells are increased in individuals with severe aplastic anemia. Int J Hematol 2011;93:156-162.

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5. Solomou EE, Rezvani K, Mielke S, Malide D, Keyvanfar K, Visconte V, Kajigaya S, Barrett AJ, Young NS. Deficient CD4+ CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia. Blood 2007;110:1603-1606. 6. Costantino CM, Baecher-Allan CM, Hafler DA. Human regulatory T cells and autoimmunity. Eur J Immunol 2008;38:921-924. 7. La Cava A. T-regulatory cells in systemic lupus erythematosus. Lupus 2008;17:421-425. 8. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 2004;199:971979. 9. Tu M, Shao Z, Liu H, He G, Fu R, Shi J, Bai J, Cao Y, Wang H, Xing L, Cui Z, Sun J, Jia H, Chen H, Xue Y, Yang C. Quantitative changes of peripheral type 3 T helper cell and CD4+CD25+ regulatory T cells and serum TGF-Î˛1 levels in the patients with severe aplastic anemia. Chin J Hematol 2006;27:753-756.

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12. Goodman WA, Cooper KD, McCormick TS. Regulation generation: the suppressive functions of human regulatory T cells. Crit Rev Immunol 2012;32:65-79. 13. Ciebiada M, Kasztalska K, Gorska-Ciebiada M, Barylski M, Gorski P. Expression of IL-7 receptor in human peripheral regulatory T cells. Arch Med Sci 2013;9:555-560. 14. Feng X, Kajigaya S, Solomou EE, Keyvanfar K, Xu X, Raghavachari N, Munson PJ, Herndon TM, Chen J, Young NS. Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro. Blood 2008;111:3675-3683. 15. Cao T, Wenzel SE, Faubion WA, Harriman G, Li L. Enhanced suppressive function of regulatory T cells from patients with immune-mediated diseases following successful ex vivo expansion. Clin Immunol 2010;136:329-337. 16. Sun M, Yang P, Du L, Zhou H, Ren X, Kijlstra A. Contribution of CD4+CD25+ T cells to the regression phase of experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci 2010;51:383-389.

10. Shi J, Ge M, Lu S, Li X, Shao Y, Huang J, Huang Z, Zhang J, Nie N, Zheng Y. Intrinsic impairment of CD4+CD25+ regulatory T cells in acquired aplastic anemia. Blood 2012;120:16241632. 11. Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras TR, Fazekas de St Groth B, Clayberger C, Soper DM, Ziegler SF, Bluestone JA. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 2006;203:1701-1711.

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Research Article

DOI: 10.4274/tjh.2013.0066

Venous Thromboembolism after Allogeneic Pediatric Hematopoietic Stem Cell Transplantation: A Single-Center Study Allojenik Pediatrik Hematopoietik Kök Hücre Nakli Sonrasında Venöz Tromboembolizm: Bir Merkezin Deneyimi Fatih Azık, Dilek Gürlek Gökçebay, Betül Tavil, Pamir Işık, Bahattin Tunç, Duygu Uçkan Ankara Children’s Hematology Oncology Hospital, Clinic of Pediatric Hematology Oncology, Ankara, Turkey

Abstract: Objective: Venous thromboembolism (VTE) in children who undergo hematopoietic stem cell transplantation (HSCT) has high morbidity. The aim of this study is to assess the incidence of VTE in allogeneic pediatric HSCT recipients and the contribution of pretransplant prothrombotic risk factors to thrombosis.

Materials and Methods: We retrospectively evaluated 92 patients between April 2010 and November 2012 undergoing allogeneic HSCT who had completed 100 days post-HSCT. Before HSCT, coagulation profiles; acquired and inherited prothrombotic risk factors including FV G1691A (factor V Leiden), prothrombin G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, and MTHFR A1298C mutations; and serum homocysteine and lipoprotein (a), plasma antithrombin III, protein C, and protein S levels were obtained from all patients.

Results: In the screening of thrombophilia, 8 patients (9%) were heterozygous for factor V Leiden, 5 (6%) were homozygous for MTHFR 677TT, 12 (14%) were homozygous for MTHFR 1298CC, and 2 (2%) were heterozygous for prothrombin G20210A mutation. We observed VTE in 5 patients (5.4%); a prothrombotic risk factor was found in 3 out of these 5 patients, while 4 out of 5 patients had central venous catheters. It was determined there was no significant relationship between VTE and inherited prothrombotic risk factors.

Conclusion: VTE after HSCT seems to be a low-frequency event that may be due to low-dose, low-molecular-weight heparin prophylaxis, and the role of inherited prothrombotic risk factors cannot be entirely excluded without a prospective study.

Key Words: Thrombosis, Pediatric stem cell transplantation, Prothrombotic risk factors Özet: Amaç: Hematopoetik kök hücre nakli (HSCT) yapılan hastalarda venöz tromboembolizm (VTE) yüksek morbiditeye sahiptir. Bu çalışmanın amacı pediatrik allojenik HSCT alıcılarındaki VTE insidansını belirlemek ve pretransplant protrombotik risk faktörlerinin trombozdaki rolünü saptamaktır.

Address for Correspondence: Dilek GÜRLEK GÖKÇEBAY, M.D., Ankara Children’s Hematology Oncology Hospital, Clinic of Pediatric Hematology Oncology, Ankara, Turkey Phone: +90 312 596 96 00 E-mail: dilek_gurlek@yahoo.com Received/Geliş tarihi : February 23, 2013 Accepted/Kabul tarihi : November 11, 2013

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Azık F, et al: Thrombosis in Pediatric HSCT

Gereç ve Yöntemler: Nisan 2010-Kasım 2012 tarihleri arasında allojenik HSCT yapılan ve HSCT sonrası 100 gününü

tamamlayan 92 hasta retrospektif olarak değerlendirildi. Kök hücre nakli öncesinde tüm hastalardan koagülasyon profili, kazanılmış ve konjenital protrombotik risk faktörlerinden faktör V Leiden, protrombin G20210A, metilentetrahidrofolat redüktaz (MTHFR) C677T, MTHFR A1298C mutasyonları ile serum homosistein ve lipoprotein (a), plazma antitrombin III, protein C ve protein S düzeyleri alındı. Bulgular: Trombofili taramasında; toplam sekiz hasta (%9) faktör V Leiden heterozigot, beş hasta (%6) MTHFR 677TT homozigot, 12 hasta (%14) MTHFR 1298CC homozigot ve iki (%2) hasta prothrombin G20210A heterozigot mutasyonuna sahipti. Venöz tromboembolizm beş hastada (%5,4) izlendi; herhangi bir protrombotik risk faktörü beş hastadan üçünde tespit edilirken, dört hastanın santral venöz kateteri vardı. Venöz tromboembolizm ile kalıtsal protrombotik risk faktörleri arasında anlamlı ilişki saptanmadı. Sonuç: Hematopoetik kök hücre nakli sonrasında VTE; muhtemelen düşük dozda düşük moleküler ağırlıklı heparin kullanımına bağlı olarak az sıklıkta görülebilen bir komplikasyon olup, prospektif bir çalışma yapılmaksızın kalıtsal protrombotik risk faktörlerinin trombozdaki rolünün tam olarak dışlanması mümkün değildir.

Anahtar Sözcükler: Tromboz, Pediatrik kök hücre nakli, Protrombotik risk faktörleri Introduction Thrombotic events are common complications in children receiving hematopoietic stem cell transplantation (HSCT). It is suggested that endothelial damage and activation of coagulation induced by pretransplant conditioning regimens may contribute to the development of thrombotic events [1]. Previous studies evaluating coagulation profiles of patients undergoing HSCT revealed altered hemostatic parameters and increased thrombin generation during HSCT. Additionally, activation of coagulation, acquired anticoagulant deficiencies, and decreased fibrinolysis were noted in HSCT patients with microangiopathic disease [2,3]. In a pediatric study, a persistent state of coagulation was considered to cause the increase in plasma levels of thrombin-antithrombin complexes and decreased levels of plasma antithrombin III (ATIII) and protein C in the early HSCT period [4]. In addition, children receiving HSCT require long-term central venous catheters (CVCs) for administering medications, blood products, and parenteral nutrition and for daily blood sampling [5]. CVCs and inherited prothrombotic risk factors may also contribute to thrombotic complications in HSCT recipients. In the English-language literature, there are contradictory studies regarding the effect of inherited prothrombotic risk factors on thromboembolism in children undergoing HSCT [6]. In this study we evaluated the incidence of venous thromboembolism (VTE) in allogeneic pediatric HSCT recipients and the contribution of pretransplant prothrombotic risk factors to thrombosis. Materials and Methods One hundred consecutive patients underwent HSCT from April 2010 to November 2012, and 92 of these transplants were allogeneic; these cases were enrolled in the study. All patients completed 100 days post-HSCT and data were collected retrospectively. All transplants were from related

donors. Patients were admitted for various hematologic/ oncologic diseases or immune deficiency disorders. All of the patients had CVCs and they were flushed, and the dressing was changed twice weekly. Catheters remained in place until hematopoietic recovery in all patients unless the catheter was proven to be infected, had mechanical complications, or was occluded with thrombus. Before insertion of CVCs, blood samples were obtained from all patients for coagulation parameters (prothrombin time, activated partial thromboplastin time, fibrinogen, d-dimer) and inherited thrombophilic factors including factor V G1691A (factor V Leiden), prothrombin G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, and MTHFR A1298C mutations and serum homocysteine, lipoprotein (a), plasma ATIII, protein C, and protein S levels. ATIII, protein C, and protein S levels were referenced for agespecific values [7]. Cut-off values were 30 mg/dL for serum lipoprotein (a) and 12 µmol/L for serum homocysteine [8]. Patients with thrombosis were checked for family history of thrombosis. None of the patients had past history of thrombosis prior to HSCT. Written informed consent was obtained from all patients’ parents. All patients stayed in HEPA-filtered isolation rooms and received standard regimens for the prevention of infectious complications (acyclovir for herpes simplex and varicella zoster, co-trimoxazole for Pneumocystis jirovecii, metronidazole for anaerobic infections, ciprofloxacin for bacterial infections, and fluconazole for Candida prophylaxis). All cellular blood products were leukocyte-depleted and irradiated prior to transfusion. While most of the HSCT recipients received cyclosporine and methotrexate for prevention of graft-versushost disease (GVHD), a few of them with Fanconi aplastic anemia received cyclosporine and steroids. All patients received low-molecular-weight heparin (LMWH) enoxaparin at a dose of 0.8 mg/kg/day by subcutaneous injection for prevention of

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Azık F, et al: Thrombosis in Pediatric HSCT

hepatic veno-occlusive disease (VOD) for 28 days. If the patient was bleeding or developed refractory thrombocytopenia, the LMWH dose was withheld. When VOD was diagnosed according to the Seattle criteria [6], defibrotide therapy was started at a dose of 25 mg/kg/day and LMWH was stopped. Myeloid engraftment was defined as the first of 3 consecutive days when the neutrophil count was >0.5x109/L and platelet engraftment as the first of 7 consecutive days with an unsupported platelet count of >20x109/L. Echocardiography was performed in all patients before the conditioning regimen and 1 month after HSCT or before discharge. When a patient had clinically suspected thrombosis, Doppler ultrasonography (US), echocardiography, and, if required, magnetic resonance (MR) imaging and angiography were performed. Statistical Analysis Analysis of data was primarily descriptive, using standard deviations, ranges, and mean and median values. Categorical variables were analyzed using the chi-square test and continuous variables were analyzed using Student’s t test. All analyses were performed using SPSS 18 for Windows (SPSS Inc., Chicago, IL, USA). When p≤0.05, it was considered statistically significant. Results A total of 92 patients (57% males and 43% females) received allogeneic HSCT; median age was 8.5 years (range: 0.5-16 years). Forty-one (44.6%) patients had malignant, 39 (42.4%) had nonmalignant, and 12 (13%) had immunodeficiency disorders. The patients and transplant characteristics are summarized in Table 1. Hickman double lumen catheters were inserted in 88 (95%) patients and 20 (22%) of them had had port catheters previously. Hickman catheters could not be inserted in 4 (5%) of the patients because of repeated CVC insertions or radiation therapy scars; therefore they had nontunneled jugular or femoral catheters during HSCT. Twenty-two of the central lines were removed during the study period as 16 of them became infected, 4 had mechanical complications (3 ruptures, 1 malposition), and 2 were blocked. Overall, 5.4% (5 out of 92) of the patients developed VTE. One patient had cerebral sinovenous thrombosis, 1 had portal vein thrombosis, and 3 had CVC-related thrombosis (2 at the right atrium and 1 at the femoral vein). Prothrombotic risk factors were found in 3 of these 5 patients, while 4 out of 5 patients had CVCs. One of the patients who had thrombosis was heterozygous for the MTHFR C677T polymorphism and had increased serum lipoprotein (a), 1 was heterozygous for the MTHFR A1298C polymorphism, and 1 had hyperhomocysteinemia without any mutation. In 2 other patients who had thrombosis, prothrombotic risk factors could not be detected. There was no statistically significant difference between HSCT recipients who had or did not have thrombosis with regard to prothrombotic risk factors (p>0.05).

230

Table 1. Subject demographics and the characteristics of the stem cell transplants.

(%)

Number of patients

(100)

Age (median years, range)

8.5

(0.5-16)

Female

(43)

Male

(57)

(44.6)

ALL

(17.4)

AML

(22.8)

CML

(1.1)

MDS

(2.2)

Lymphoma

(1.1)

(42.4)

Aplastic anemia

(14.1)

Thalassemia

(23.9)

Congenital erythropoietic porphyria

(1.1)

Adrenoleukodystrophy

(2.2)

Infantile malignant osteopetrosis

(1.1)

(13)

Hyperimmunoglobulin E syndrome

(3.2)

Chediak–Higashi syndrome

(2.2)

Wiskott–Aldrich syndrome

(1.1)

Infantile Crohn’s disease

(1.1)

Severe combined immunodeficiency disease

(3.2)

Familial hemophagocytic lymphohistiocytosis

(2.2)

10/10

(88)

9/10

(7)

Haploidentical

(5)

Bone marrow

(85)

Peripheral blood

(11)

Bone marrow-peripheral blood

(3)

Cord blood-peripheral blood

(1)

Myeloablative

(80)

Nonmyeloablative

Sex

Diagnosis Malignancy

Nonmalignant disorders

Immunodeficiency disorders

HLA matching

Stem cell source

Conditioning regimen 18

(20)

Stem cells infused CD34+ (106/kg, range)

3.4

(0.6-12.7)

Neutrophil engraftment (median days, range)

(9-22)

Platelet engraftment (median days, range)

(10-73)

Follow-up time (median days, range)

435

(101-945)

ALL: Acute lymphoblastic leukemia, AML: acute myeloid leukemia, CML: chronic myeloid leukemia, MDS: myelodysplastic syndrome.

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Azık F, et al: Thrombosis in Pediatric HSCT

There was no previous history of VTE in first-degree relatives in any of the patients who developed VTE undergoing HSCT. None of the patients had homozygous factor V Leiden or homozygous prothrombin G20210A mutation, or deficiency of protein C, protein S, or ATIII. In total, 8 (9%) of the patients were heterozygous for the factor V Leiden mutation and none of them had thrombosis. Two (2%) patients were heterozygous for the prothrombin G20210A mutation, 5 patients (6%) were homozygous for MTHFR 677TT, 12 (14%) were homozygous for MTHFR 1298CC, and no thrombotic event occurred. Prothrombotic risk factors of the HSCT recipients are summarized in Table 2. Three of 5 patients with VTE diagnosis had immunodeficiency disorders whereas the other 2 patients had acute lymphoblastic leukemia. All of the CVC-related thrombosis occurred in a subgroup of 15 patients (27%) who developed acute GVHD of any grade following HSCT. There was no association found between patients with or without thrombosis with regard to malignant or nonmalignant diseases, stem cell sources, or myeloablative or nonmyeloablative preparative regimens. Sixteen of the 92 patients (17%) had VOD; 2 of them had mild VOD according to the Seattle criteria and received supportive treatment, and 14 of them had moderate VOD and received defibrotide therapy [6]. The median time to VTE following HSCT was 58 days (range: 25-66 days). All patients were treated with LMWH at a dose of 1 mg/kg subcutaneously twice a day. No severe bleeding complications occurred during anticoagulant therapy. The patient with cerebral sinovenous thrombosis was admitted to the hospital complaining of headache and nausea at day +66 after HSCT. She was receiving steroid therapy for acute GVHD. Papilledema was noticed on her physical examination. Although her cranial computed tomography and MR imaging results were normal, lumbar puncture

showed increased intracranial pressure (>300 mmH2O). MR venography revealed marked paucity of flow in the left transverse and sigmoid sinuses. She was treated with LMWH for 6 months, and then no obstruction was shown on MR venography and she received a prophylactic dose of acetylsalicylic acid for 6 months. The 2 patients who had right atrial thrombosis were asymptomatic; their CVCs were obstructed and thrombi were seen on echocardiography. They were treated with LMWH until CVC removal and echocardiography showed no thrombus in the right atrium. Swelling of the right leg was noticed after removal of the catheter in the patient with femoral vein thrombosis, and Doppler US showed obstruction in the femoral vein. He was treated with LMWH for 6 months and thrombosis dissolved in the second month, as shown on Doppler US. The patient with portal vein thrombosis had a second HSCT because of graft failure. Despite no inherited prothrombotic risk factor, he had pancreatitis and moderate VOD, and so he received defibrotide therapy and enoxaparin was stopped. Because he had hyperbilirubinemia and hepatomegaly, Doppler US was performed and an obstruction was seen in the right branch of the portal vein. He was treated with LMWH for 1 month and thrombosis dissolved within 2 weeks, as shown on Doppler US. Thereafter, he died from grade IV GVHD with gastrointestinal tract and skin involvement. Characteristics of patients with thrombosis are summarized in Table 3. Discussion Prior studies have suggested that thrombosis occurs in 4.4%-9% of pediatric HSCT patients, and the impacts of inherited prothrombotic risk factors remain controversial [4,9,10]. In the present study, 5 out of the 92 patients (5.4%) had VTE; prothrombotic risk factors was found in 3 patients with thrombosis, while 4 had CVCs. CVCs have improved

Table 2. Characteristics of hematopoietic stem cell transplantation recipients with and without prothrombotic risk factors.

Prothrombotic Risk Factor

Number of Patients with Thrombosis (%)

p Number of Patients without Thrombosis (%)

Heterozygous factor V Leiden mutation

0/5 (0)

8/87 (9)

>0.05

Homozygous MTHFR 677TT mutation

0/5 (0)

5/87 (6)

>0.05

Homozygous MTHFR 1298CC mutation

0/5 (0)

12/87 (14)

>0.05

Heterozygous prothrombin G20210A mutation

0/5 (0)

2/87 (2)

>0.05

Hyperhomocysteinemia

1/5 (20)

15/87 (17)

>0.05

Elevated serum lipoprotein (a)

1/5 (20)

19/87 (22)

>0.05

Protein C (%, mean ± SD)

68±10

76±21

>0.05

Protein S (%, mean ± SD)

76±12

87±30

>0.05

Antithrombin III (%, mean ± SD)

102±11

104±18

>0.05

231

232

portal vein whole body

ALL: Acute lymphoblastic leukemia, CHS: Chediak-Higashi syndrome, CVC: central venous catheter, HIES: hyperimmunoglobulin E syndrome, L/P: lumbar puncture, MR: magnetic resonance, LMWH: low molecular weight heparin, Lp (a): Iipoprotein (a), US: ultrasonography, WAS: Wiskott-Aldrich syndrome, F: female, M: male.

Resolved month

LMWH for 1 CVC Doppler US

peripheral edema swelling of

branch of the

Right main Hyperbilurubinemia, Icterus, +60 ALL M, 15.2

the end of CVC

Right atrium, No

obstruction

CVC +58 ALL M, 15

+35 CHS M, 2.9

vein leg right leg

removed

5 weeks, catheter

LMWH for 2 Echocardiography

CVC

months hyperhomocysteinemia

Resolved

Resolved LMWH for 6 Right femoral Edema of the right Swelling of the

Doppler US

CVC,

removed elevated serum Lp (a)

LMWH for 2

weeks, catheter the end of CVC obstruction

No +25 WAS M, 8.6

CVC

sinus

Right atrium,

Echocardiography

MTHFR C677T,

CVC, heterozygous for

months

aspirin for 6

Resolved

Resolved 12 LMWH for 6

months, then MTHFR A1298C

Heterozygous for MR venography

and sigmoid pressure with L/P vomiting

Left transverse Elevated intracranial Headache, HIES F, 5.3

+66

Presentation Clinical Signs Primary Day of Diagnosis Thrombosis

Site of Diagnostic Thrombosis Tool

Az覺k F, et al: Thrombosis in Pediatric HSCT

Sex, Age (years)

Table 3. Clinical and laboratory findings of patients with thrombosis.

Prothrombotic Risk Factor

Treatment

Follow-up Outcome Time (months)

Turk J Hematol 2015;32:228-233

the quality of care in children with hematological/ oncological diseases or undergoing HSCT, and thrombosis is one of their most frequent secondary complications [11]. The incidence of catheter-related thrombosis in children with underlying hematological or malignant disorders ranges from 4% to 50% [12,13,14]. Catheter-related thrombosis occurred in 4 out of 92 patients (4.3%) receiving HSCT in our study. Kn繹fler et al. evaluated inherited thrombophilia and CVC-related thrombosis with malignancy; an inherited prothrombotic risk factor was determined in 23% (17 out of 77) of the subjects and in 64% (7 out of 11) of the subjects with thrombosis. They concluded that patients with combined inherited prothrombotic risk factors were particularly at risk for thrombotic events [15]. Revel-Vilk et al. evaluated 262 patients with malignancy and there was no association found between thrombophilia and CVC-related thrombosis, but a positive family history of thrombosis was related to thrombotic episodes [16]. Similarly, in the present study, we found no difference between HSCT recipients who had or did not have VTE with regard to prothrombotic risk factors. On the contrary, Abdelkefi et al. studied 171 HSCT recipients and CVC-related thrombosis was found in 7.6% of subjects; those who had at least 1 of the inherited prothrombotic markers had a 3.3-fold increased risk of thrombosis. In addition, they found that absence of heparin prophylaxis was also associated with CVC-related thrombosis [17]. Though there is some evidence that a prophylactic continuous infusion of heparin can prevent thrombosis in patients with CVC, Lagro et al. showed that there was no effect of using prophylactic nadroparin (a LMWH) on the incidence of CVC-related thrombosis in HSCT recipients [18,19]. We used enoxaparin at a dose of 0.8 mg/kg/day for prevention from VOD for 28 days, which may have offered some protection against the development of VTE. We observed acute GVHD in 3 of 4 patients with VTE. Vascular and endothelial injury that occurs as a consequence of GVHD may contribute to inflammation, which increases the risk of VTE in allogeneic transplant recipients. Additionally, the use of immunosuppressive treatment for acute GVHD has not been fully investigated in regard to the risk of developing VTE [20]. In the present study, VTE was observed in 5.4% of pediatric HSCT recipients, which is in accordance with previous reports. We found no relationship between thrombosis and prothrombotic risk factors. We concluded that VTE seems to be a low-frequency

Azık F, et al: Thrombosis in Pediatric HSCT

event, which may be due to low-dose LMWH prophylaxis, and the role of inherited prothrombotic risk factors cannot be entirely excluded without a prospective study. Application of low-dose LMWH has been useful for preventing VTE after HSCT. Since all patients in our study had CVCs, the role of CVCs on thrombosis cannot be determined and acquired hemostatic alterations after HSCT are still speculative in VTE. In order to decrease CVC-related complications after HSCT, CVCs should be removed as soon as possible after the expected work is done. 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. Han Y, Zhu L, Sun A, Lu X, Hu L, Zhou L, Ren Y, Hu X, Wu X, Wang Z, Ruan C, Wu D. Alterations of hemostatic parameters in the early development of allogeneic hematopoietic stem cell transplantation-related complications. Ann Hematol 2011;90:1201-1208. 2. Vannucchi AM, Rafanelli D, Longo G, Bosi A, Guidi S, Saccardi R, Filimberti E, Cinotti S, Grossi A, Morfini M, Rossi-Ferrini P. Early haemostatic alterations following bone marrow transplantation: a prospective study. Haematologica 1994;79:519-525. 3. Harper PL, Jarvis J, Jennings I, Luddington R, Marcus RE. Changes in the natural anticoagulants following bone marrow transplantation. Bone Marrow Transplant 1990;5:39-42. 4. Brandão LR, Kletzel M, Boulad F, Kurtzberg J, Maloney K, Fligman I, Sison CP, Dimichele D. A prospective longitudinal multicenter study of coagulation in pediatric patients undergoing allogeneic stem cell transplantation. Pediatr Blood Cancer 2008;50:12401246. 5. Hickman RO, Buckner DC, Clift RA, Sanders JE, Stewart P, Thomas ED. A modified right atrial catheter for access to the venous system in marrow transplant recipients. Surg Gynecol Obstet 1979;148:871-875. 6. McDonald GB, Hinds MS, Fisher LD, Schoch HG, Wolford JL, Banaji M, Hardin BJ, Shulman HM, Clift RA. Veno-occlusive disease of liver and multiorgan failure after bone marrow transplantation: a cohort study of 355 patients. Ann Intern Med 1993;118:255-267. 7. Andrew M, Vegh P, Johnston M, Bowker J, Ofosu F, Mitchell L. Maturation of the hemostatic system during childhood. Blood 1992;80:1998-2005. 8. Ganji V, Kafai MR. Population references for plasma total homocysteine concentrations for US children and adolescents in the post-folic acid fortification era. J Nutr 2005;135:2253-2256. 9. Wiznitzer M, Packer RJ, August CS, Burkey ED. Neurological complications of bone marrow transplantation in childhood. Ann Neurol 1984;16:569-576.

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10. Uderzo C, Marraro G, Riva A, Bonanomi E, Vaj PL, Marchi PF, Locasciulli A, Masera G. Pulmonary thromboembolism in leukemic children undergoing bone marrow transplantation. Bone Marrow Transplant 1993;11:201-203. 11. Massicotte MP, Dix D, Monagle P, Adams M, Andrew M. Central venous catheter related thrombosis in children: analysis of the Canadian Registry of Venous Thromboembolic Complications. J Pediatr 1998;133:770-776. 12. Haire WD, Lieberman RP, Edney J, Vaughan WP, Kessinger A, Armitage JO, Goldsmith JC. Hickman catheter induced thoracic vein thrombosis. Frequency and long-term sequelae in patients receiving high-dose chemotherapy and marrow transplantation. Cancer 1990;66:900-908. 13. Conlan MG, Haire WD, Lieberman RP, Lund G, Kessinger A, Armitage JO. Catheter-related thrombosis in patients with refractory lymphoma undergoing autologous stem cell transplantation. Bone Marrow Transplant 1991;7:235-240. 14. Boraks P, Scale J, Price J, Bass G, Ethell M, Keeling D, Mahendra P, Baglin T, Marcus R. Prevention of central venous catheterassociated thrombosis using minidose warfarin in patients with haematological malignancies. Br J Haematol 1998;101:483-486. 15. Knöfler R, Siegert E, Lauterbach I, Taut-Sack H, Siegert G, Gehrisch S, Müller D, Rupprecht E, Kabus M. Clinical importance of prothrombotic risk factors in pediatric patients with malignancy--impact of central venous lines. Eur J Pediatr 1999;158:147-150. 16. Revel-Vilk S, Yacobovich J, Tamary H, Goldstein G, Nemet S, Weintraub M, Paltiel O, Kenet G. Risk factors for central venous catheter thrombotic complications in children and adolescents with cancer. Cancer 2010;16:4197-4205. 17. Abdelkefi A, Ben Romdhane N, Kriaa A, Chelli M, Torjman L, Ladeb S, Ben Othman T, Lakhal A, Guermazi S, Ben Hassen A, Ladeb F, Ben Abdeladhim A. Prevalence of inherited prothrombotic abnormalities and central venous catheter-related thrombosis in haematopoietic stem cell transplants recipients. Bone Marrow Transplant 2005;36:885-889. 18. Hentschel R, Wiescholek U, von Lengerke J, Harms E, Jorch G. Coagulation associated complications of indwelling arterial and central venous catheters during heparin prophylaxis: a prospective study. Eur J Pediatr 1999;158(Suppl 3):126-129. 19. Lagro SW, Verdonck LF, Borel Rinkes IH, Dekker AW. No effect of nadroparin prophylaxis in the prevention of central venous catheter (CVC)-associated thrombosis in bone marrow transplant recipients. Bone Marrow Transplant 2000;26:11031106. 20. Biedermann BC, Sahner S, Gregor M, Tsakiris DA, Jeanneret C, Pober JS, Gratwohl A. Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease. Lancet 2002;359:2078-2083.

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Research Article

DOI: 10.4274/tjh.2013.0173

Infections in Hospitalised Patients with Multiple Myeloma: Main Characteristics and Risk Factors Hastanede Tedavi Gören Multipl Miyelom Hastalarında Görülen Enfeksiyonlar: Temel Özellikler ve Risk Faktörleri Toni Valković1, Vedrana Gačić2, Jelena Ivandić3, Božo Petrov4, Renata Dobrila-Dintinjana5, Elizabeta Dadić-Hero6, Antica Načinović-Duletić1 1University

Hospital Centre Rijeka, Clinic of Haematology, Rijeka, Croatia Hospital Mostar, Clinic of Haematology, Mostar, Bosnia and Herzegovina 3University Hospital Centre Rijeka, Clinic of Gynaecology and Obstetrics, Rijeka, Croatia 4University Hospital Mostar, Clinic of Psychiatry, Mostar, Bosnia and Herzegovina 5University Hospital Centre Rijeka, Clinic of Oncology and Radiotherapy, Rijeka, Croatia 6Community Primary Health Centre, Primorsko-Goranska County, Rijeka, Croatia 2University

Abstract: Objective: Multiple myeloma is a common haematological malignancy and immune dysfunction is the hallmark of the disease.

It leads to an increased infection risk, which is still a major cause of mortality. The infection spectrum and characteristics have evolved with the introduction of novel agents. An understanding of risk factors that increase susceptibility to infections is critical in fighting them. This retrospective investigation aimed to establish the incidence and main characteristics of infections in non-transplanted hospitalised myeloma patients in our department over a 3-year period, as well as factors associated with infections. Materials and Methods: A total of 240 hospitalised patients with multiple myeloma (120 males and 120 females; average age: 69 years, range: 41-89 years) who were diagnosed or treated in our department from January 2008 to December 2010 were included in this study and their data were retrospectively analysed. Results: Infections were identified in 17.9% of hospitalised patients. The most common pathogen found was Pseudomonas aeruginosa. The frequency of gram-positive and gram-negative pathogens was similar. In 37.2% of cases, the agent was not isolated. The most common sites of infections were the urinary system and the blood (septicemia). The frequency of infection increased with duration of disease and the rate of reinfection was 41.9%. The patients treated with bortezomib had the highest infection occurrence. Fatal outcome occurred in 9.3% of cases. Conclusion: The factors associated with infections in this investigation were female sex, 3B clinical stage of disease, increased serum creatinine and ferritin levels, neutropenia, poor general condition, and presence of catheters. Myeloma patients with one or more of these mentioned risk factors should be monitored with particular care in order to decrease the incidence and severity of infective complications. Key Words: Multiple myeloma, Infections, Risk factors, Therapy

Address for Correspondence: Jelena IVANDIĆ, M.D., University Hospital Centre Rijeka, Clinic of Gynaecology and Obstetrics, Rijeka, Croatia Phone: +0038551421426 E-mail: jelenaivandic@yahoo.com Received/Geliş tarihi : May 16, 2013 Accepted/Kabul tarihi : February 21, 2014

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Özet: Amaç: Multipl miyelom, sık görülen bir hematolojik malignite olup, bağışıklık sistemi disfonksiyonu bu hastalığın en belirgin

özelliğidir. Bu hastalığın sebep olduğu ileri düzeyde enfeksiyon riski hala başlıca ölüm sebebidir. Enfeksiyonun spektrumu ve özellikleri, yeni geliştirilen ajanlarla birlikte değişiklik göstermektedir. Enfeksiyonlara hassasiyeti arttıran risk faktörlerinin anlaşılması, bu enfeksiyonlara karşı savaşmak için son derece önemlidir. Bu retrospektif çalışma neticesinde 3 yıldır departmanımızda yatırılarak tedavi gören ve transplantasyon yapılmamış miyelom hastalarımızda görülen enfeksiyonların temel özelliklerinin saptanması amaçlanmıştır. Gereç ve Yöntemler: Ocak 2008 ile Aralık 2010 tarihleri arasında departmanımızda tanısı konmuş ve tedavi görmüş toplam 240 multipl miyelom hastası (120 erkek ve 120 kadın; ortalama yaş: 69, yaş aralığı: 41-89) bu çalışmaya dahil edilmiştir ve bu hastalardan elde edilen veriler retrospektif olarak incelenmiştir. Bulgular: Hastanemizde yatan hastaların %17,9’unda enfeksiyona rastlandı. En sık görülen patojenin Pseudomonas aeruginosa olduğu saptandı. Gram pozitif ve gram negatif patojenlerin sıklığı aynıydı. Olguların %37,2’sinde ajan izole edilemedi. Enfeksiyon kaynağının sıklıkla üriner sistem ve kan olduğu görüldü. Hastalık süresince enfeksiyon oranının arttığı ve reenfeksiyon sıklığının % 41,9 olduğu tespit edildi. En sık enfeksiyon bortezomib ile tedavi edilen hastalarda görüldü. Olguların %9,3’ü ölümle sonuçlandı. Sonuç: Bu çalışmada enfeksiyonlarla ilişkilendirilen faktörler kadın cinsiyet, ileri evre (III 3B) hastalık, artmış serum kreatinin ve ferritin düzeyi, nötropeni, genel durumun bozukluğu ve kateterlerin varlığıydı. Bahsi geçen bu faktörlerden bir ya da ikisine sahip miyelom hastalarda görülebilecek enfektif komplikasyon oranını ve şiddetini düşürmek amacıyla hastaların özenli ve dikkatli bir bakımla izlenmesi gerekmektedir. Anahtar Sözcükler: Multipl miyelom, Enfeksiyonlar, Risk faktörleri, Tedavi Introduction Multiple myeloma (MM) is a common haematological malignancy, and immune dysfunction is one of the hallmarks of the disease. The disease-related immunological defects, as well as therapy-related immunosuppression, lead to the increased risk of infective complications, which are a major cause of mortality. Malignant plasma cells produce monoclonal paraprotein, which is non-functional and crowds out the polyclonal immunoglobulins. Hypogammaglobulinaemia, which is related to suppression of CD19+ B lymphocytes, exists in a great majority of patients with the active disease [1]. Besides hypogammaglobulinaemia, numerous immunological abnormalities have been observed, such as several defects of T lymphocytes, dendritic cells, natural killer cells, and the complement cascade [2,3,4,5,6,7]. Quantitative and qualitative disorders of neutrophils are related to myelosuppressive therapy or bone marrow infiltration [8,9]. Furthermore, several other risk factors increase susceptibility to infections in MM, such as renal failure, smoking, using of various catheters, vertebral collapse, iron overload, or use of opiate drugs [10,11]. The spectrum and characteristics of infective complications in myeloma patients have evolved with the introduction of autologous and allogeneic hematopoietic stem cell transplant (HSCT) or different novel drugs, which together cause an increasing susceptibility to fungal and viral infections [12,13,14,15].

The present study has analysed the frequency and characteristics of infective complications, as well as factors related to risk for infections, in the myeloma patients treated at our institution over a 3-year period. Materials and Methods Retrospectively, using hospital medical documentation, 240 cases of hospitalised patients with MM (120 males and 120 females; average age of 69, range of 41-89 years) who were diagnosed or treated in our department from January 2008 to December 2010 were processed. Because the majority of patients were hospitalised more than once, the total number of our cases was larger than the number of patients included in this study (37 males and 35 females, respectively). Only patients who were not treated at the time of this study with high-dosage therapy and HSCT were included. The diagnosis was established according to International Myeloma Working Group criteria [16]. The great majority of patients in this study had IgG, IgA, or a light chain myeloma; however, 1 patient with IgD and 1 patient with nonsecretory myeloma were also included. Our patients were treated fairly uniformly. VAD (vincristine, doxorubicin, and dexamethasone) and MP (oral melphalan and prednisone) regimens were mostly used as the induction therapy (according to the patient’s age and eligibility for high-dose therapy), with thalidomide-based protocols as second-line therapy and bortezomib-based protocols as third- or next-line therapy. Monotherapy with dexamethasone was used as a front-line therapy, as well as for postinduction protocol in some patients.

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The study was approved by the Medical School of Rijeka Ethics Committee. The criteria for infection used in our study were increased body temperature above the normal range (37 °C) or isolation of a microbial agent in patients who also had concomitant clinical symptoms and/or humoral signs of infections (leukocytosis, neutrophilia, marked “left shift”, or increased C reactive protein in comparison with a baseline value). The frequency and the basic characteristics of the identified infections, such as type of isolated microbial agents, site of infection, time of occurrence and outcome of infection, rate of re-infections, and type of antitumor therapy at the time of infection, were also determined. The factors that could influence the pathogenesis and incidence of infections, including sex, WHO/ECOG performance status [17], DurieSalmon stage of disease [18], International Staging System (ISS) score [19], serum creatinine level, immuneparesis defined qualitatively (decreased serum concentration of any polyclonal immunoglobulin class), neutropenia (defined as blood neutrophil count of ≤2x109/L), serum ferritin level, and presence of any catheters, were compared and statistically analysed in cases with and without infections [17,18,19]. All these parameters according to the occurrence of infections are listed in Table 1. Statistical Analysis Data were expressed as means ± standard deviations, with the sample size (n). Nominal and ordinal variables were analysed with the chi-square test. Normality of distribution of continuous variables was tested by the Kolmogorov-Smirnov test. For continuous variables with distribution significantly different from the norm, we used the median and interquartile range, and the differences between the groups for these variables were tested with the Kruskal-Wallis test and MannWhitney U test. In the symmetric distribution of continuous variables, to display the mean values and dispersion measures, we used the arithmetic mean and standard deviation, and their comparison was done with Student’s t-test. SPSS for Windows (version 17.0, SPSS Inc., Chicago, IL, USA) and Apache OpenOffice (version 3.3.0, The Apache Software Foundation, Wilmington, DE, USA) were used for statistical analyses. The values were considered statistically significant at p<0.05 (2-tailed). Results Infections were identified in 43 out of 240 (17.9%) hospitalised patients. In 27 (62.8%) cases of infection, the microbial agent was identified, while in 16 (37.2%) cases, no isolation was possible. The most common pathogen found was Pseudomonas aeruginosa. The frequency of gram-positive and gram-negative bacteria was almost the same. In 10 out of

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27 cases, more than 1 microorganism was isolated (in 9 cases 2 and in 1 case 3 agents were isolated at the same time). In all cases, the identified microorganisms were bacteria, without any proven viral or fungal isolates. The frequencies of isolated agents are shown in Table 2. The most common sites of infections were the urinary system in 16 cases (37.2%) and the blood in 7 cases (16.3%). In 4 cases more than 1 site of infection was found (urinary system and blood, as well as upper respiratory tract and blood, both in 2 cases). In 8/43 cases (18.6%), the site of infection was not identified. The determined sites of infection are summarised in Table 3. The average age in the group of patients with infections was 69 years and in those without infections was 65 years, which did not reach statistical significance (p=0.094). The average disease duration in the cases without infection was 17.7 months and in the cases with infection was 33.6 months, which was statistically significant (p=0.004). In 18 of 43 cases, infections occurred in patients who had previously suffered from infective complication, which means that the re-infection rate was 41.9%. Fatal outcome related to infection occurred in 4/43 cases (9.3%). According to the type of therapy, the highest frequency of infections occurred in the group treated with bortezomib (14/28) and the lowest with VAD protocol (5/76), as can be seen in Table 4. Our results established that the factors associated with more frequent infections were female sex (p=0.001), poor general condition (p<0.001), IIIB 3B (advanced) DurieSalmon stage of disease (p=0.007), increased serum creatinine (p=0.036), neutropenia (p=0.009), high serum ferritin level (p=0.001), and the presence of catheters, especially urinary (p<0.001). There was no difference in infection occurrence related to the patient’s age (p=0.072), ISS score (p=0.532), or presence of immuneparesis (p=0.278). Discussion To date, there have been scant data regarding the incidence and characteristics of infections in hospitalised myeloma patients, especially those who were not treated with highdose therapy and HSCT. According to our results, infections occurred in 17.9% cases of myeloma patients hospitalised in our ward during a 3-year period. The most frequently isolated bacteria were Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Enterococcus faecalis, and the numbers of gram-positive and gram-negative infections were almost equal, which is in concordance with the existing data [10,20]. These agents caused mainly urinary tract infections and bacteraemia. Contrary to recent reports that showed an increased incidence of fungal infections [12,13,14,15], none of these pathogens or fungal surrogate markers were detected in

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Table 1. Characteristics of tested parameters.

Tested Parameters

Number of Cases

Group without Infections

Group with Infections

Sex

Male Female

120 120

111 86

9 34

ECOG performance status

0 1 2 3 4

16 88 70 45 20

16 85 60 30 5

0 3 10 15 15

Durie-Salmon stage of disease

1A 2A 2B 3A 3B

6 51 1 106 59

5 47 0 89 40

1 4 1 17 19

International Staging System score

1 2 3

54 57 98

44 50 81

10 7 17

Serum creatinine level

≤100 mmol/L (f); ≤120 mmol/L (m) (normal value) >100≤175 mmol/L (f); >120≤175 mmol/L (m) >175 mmol/l (f, m)

133 52 49

117 41 35

16 11 14

Immuneparesis

Yes No No

184 46 160

148 40 138

36 6 22

Neutropenia

1.01-2x109/L 0.51-1x109/L <0.5x109/L

55 14 3

44 8 1

11 6 2

≤120 µg/L (f); ≤300 µg/L (m)

>120≤240 µg/L (f); > 300≤600 µg/L (m)

>240 µg/L (f); >600 µg/L (m)

Types of therapy

Without therapy VAD MP Thalidomide-based Dexamethasone Bortezomib-based Others

39 76 13 20 55 28 5

32 71 11 17 48 14 1

7 5 2 3 7 14 4

Presence of any catheters

Without catheters UC CVC UC, CVC, ITC CVC, ITC UC, CVC

190 32 2 2 1 8

179 22 2 0 1 0

11 10 0 2 0 8

Serum ferritin level

f: Female, m: male; VAD: vincristine, doxorubicin, dexamethasone, MP: oral melphalan and prednisone, UC: urinary catheter, CVC: central venous catheter, ITC: intrathoracic catheter.

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cases when fungal infection was part of a differential diagnosis [12,13,14,15]. This conflicting finding can be explained by the fact that our patients were not treated with high-dose therapy at the time of this study. Because of that, prolonged and severe neutropenia and mucositis, which are well-known risk factors for invasive fungal infections, occurred rarely in our group. Traditionally, the frequency of infections is the highest during the first few months of induction therapy and in the later course of advanced disease [9,20,21,22,23,24,25,26]. On the contrary, patients who respond to therapy and those in the “plateau” phase are at low risk [24]. In our sample, the Table 2. The frequencies of isolated agents.

Isolated Agent

Number of Cases

Pseudomonas aeruginosa

Escherichia coli

Enterococcus faecalis

Staphylococcus epidermidis

Proteus mirabilis

Streptococcus sp.

MRSA

Staphylococcus aureus

Streptococcus pneumoniae

Acinetobacter

Enterobacter

Clostridium difficile

Table 3. The determined sites of infection.

Sites of Infection

Number of Cases

Urinary tract

16 (37.2%)

Fever of unknown origin

8 (18.6)

Blood (sepsis)

7 (16.3%)

Lower respiratory tract

3 (6.9%)

Gastrointestinal tract

2 (4.6%)

Skin

2 (4.6%)

Sepsis and urine

2 (4.6%)

Sepsis and upper respiratory tract

2 (4.6%)

Upper respiratory tract

1 (2.3%)

Total

43 (100%)

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average disease duration in the cases without infections was significantly shorter than in the cases with infections, which implies that infections tend to occur in the later stage of relapsed or refractory disease. This result supports the idea that uncontrolled and long-lasting myeloma increases cumulative immunosuppression and represents an important infection risk factor. Moreover, these patients more often suffer from renal failure, neutropenia, bone fractures, neuropathy, and other established infection promoters. Our results suggest that these subsets of patients are also candidates for antimicrobial prophylaxis, just as the patients during the first months of induction therapy, which has been proposed by some other researchers [27,28]. The data regarding the mortality rate in myeloma patients who contract infective complications are quite limited. Considering the fact that a significant number of our patients suffered from progressive disease, poor performance status, and high incidence of bacteraemia, the mortality rate of 9.3% seems acceptable. Anderson et al. reported a significant proportion of infections after the VAD regimen, which was probably related to dexamethasone-induced T-cell immunodeficiency [29]. Thalidomide is not heavily myelotoxic and the infection risk is not too high when it is used. However, the incidences of infections and pneumonia are higher in MPT than in MP groups of patients in some (but not all) clinical studies, which can be related to the immunomodulatory functions and indirect effects on deep vein thrombosis and peripheral neuropathy [30,31,32,33,34,35]. Bortezomib is also not significantly myelotoxic, but it mediates potent immunosuppressive effects on T cells, strongly connected with herpes zoster infection and other infections [36,37,38,39,40,41]. The infection rate in patients treated with bortezomib is variable, but Mateos et al. reported that 75% of patients treated with melphalan, prednisone, and bortezomib had at least 1 episode of infection during treatment [42]. According to our results, patients Table 4. Frequency of infections based on type of antitumor therapy.

Types of Antitumor Therapy

Frequency of Infections

VAD

6.6% (5/76)

15.4% (2/13)

DEXA

12.7% (7/55)

Thalidomide-based therapy

15% (3/20)

Bortezomib-based therapy

50% (14/28)

VAD: Vincristine, doxorubicin, dexamethasone, MP: oral melphalan and predni sone, DEXA: dexamethasone.

ValkoviÄ&#x2021; T, et al: Infections in Patients with Multiple Myeloma

treated with the VAD regimen had the lowest infection incidence (6.6%), but, as already mentioned, VAD was used as the first-line therapy. The frequency of infections was higher with dexamethasone-monotherapy (12.7%) and thalidomide-based protocols (15%), both commonly used as second-line therapies. The highest rate of infections was in the â&#x20AC;&#x153;bortezomib-based protocolsâ&#x20AC;? group (50%). As explained above, bortezomib-based protocols were used in this study as salvage treatment for refractory/progressive disease in patients with severe cumulative malfunction of the immune system, which is thought to be the main reason for the highest infection rate in patients treated with bortezomib. On the other hand, no herpes zoster infection was noticed in our group at the time of study, probably because of the prophylactic use of acyclovir. Among our cases, almost 80% of all infective complications were restricted to the female sex, which is quite an interesting result. On the other hand, some authors found a higher infection rate among men [24,43]. Hargreaves et al. suggested that cigarette smoking, which was more typical for men, was one of the possible explanations for this [24]. The reasons for more frequent infections in women, especially possible differences in the infection sites, and urinary catheter application among women and men should be analysed in a future study on a representative number of patients. In our patients, the highest infection rate was observed in those with poor performance status, i.e. WHO/ECOG Groups 3 and 4. This finding was expected because this group of patients very frequently had other infection risk factors such as advanced age, progressive/uncontrolled disease, renal dysfunction, or different catheters. No difference in infection rate was found between different ISS groups of patients, but advanced Durie-Salmon Stage IIIB 3B was significantly associated with more frequent infective complications. Contrary to some other studies [43,44], this result supports the idea that large tumour burden, especially when associated with renal failure, is an important infection risk factor in myeloma patients [43,44]. Like in other groups, a positive correlation between renal dysfunction and infections was found and these patients had a significantly higher rate of infective complications [10,43,45,46,47,48,49], which should be included in the estimation of infection risk [10,43,45,46,47,48,49]. In contradiction to other studies [10,43,45,46,47,48], the prognostic importance of serum polyclonal immunoglobulin deficiency as a risk factor for infection was not confirmed in our sample [10,43,45,46,47,48]. However, the present study evaluated immuneparesis only qualitatively, not quantitatively, and the number of patients without immuneparesis was low, which could have influenced and limited our results.

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Our data confirmed the well-established fact that the degree of neutropenia in haematological diseases is related to the risk of infections [50,51]. The majority of our cases had no neutropenia. However, patients with neutrophil counts of less than 1x109/L had a higher infection rate. A previous randomised trial showed some benefits for severely neutropenic myeloma patients after HSCT treated with granulocyte colonystimulating factor (G-CSF) [52]. Although the prophylactic use of G-CSF in neutropenic myeloma patients is supported on an everyday basis, the question of whether prophylactic use or addition of G-CSF to broad-spectrum antibiotics in nontransplanted myeloma patients really improved their outcome is still open and requires new clinical trials. This study has shown that patients with high serum ferritin, and especially those with levels twice the norm (caused by an underlying disease, their treatment, blood transfusions, or the infection itself), had a greater infection incidence, which some authors have already suggested, particularly in patients treated with high-dose therapy [11,53]. This suggests that iron overload, and especially free iron, can cause immunosuppression, probably due to the damaging of polymorphonuclear and macrophage function, T cell development, and facilitation of the growth of microorganisms [54]. Indwelling central venous and urinary catheters are important devices in haematological disease management but catheter-bearing patients have impaired mucosal integrity and are at significant risk of infection complications [55,56,57]. In our group, patients with different catheters, especially urinary, had an increased infection rate in comparison to those without catheters. This result highlights the importance of our decisions and estimations of possible risk and benefit regarding the implantation of different catheters. The rate of re-infections among our patients was rather high, which implies that a previous infection represents another infection risk factor. Over the last decades we have witnessed remarkable progress in the treatment of MM, which resulted in the prolongation of survival rates. However, these accomplishments have not been achieved only because of transplant procedures and potent novel drugs, but also because of better supportive therapy including anti-infective drugs. According to our results, female sex, advanced clinical stage and WHO/ECOG performance status, neutropenia, increased levels of serum creatinine and ferritin, presence of catheters (especially urinary), and previous infections represent infection risk factors in patients with MM. Each myeloma patient, especially those with one or more of the mentioned risk factors, should be carefully and individually studied in order to decrease the incidence and severity of infective complications using different approaches.

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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. Rawstron AC, Davies FE, Owen RG, English A, Pratt G, Child JA, Jack AS, Morgan GJ. B-lymphocyte suppression in multiple myeloma is a reversible phenomenon specific to normal B-cell progenitors and plasma cell precursors. Br J Haematol 1998;100:176-183. 2. Mills KH, Cawley JC. Abnormal monoclonal antibodydefined helper/suppressor T-cell subpopulations in multiple myeloma: relationship to treatment and clinical stage. Br J Haematol 1983;53:271-275. 3. Ogawara H, Handa H, Yamazaki T, Toda T, Yoshida K, Nishimoto N, Al-ma’Quol WH, Kaneko Y, Matsushima T, Tsukamoto N, Nojima Y, Matsmuto M, Sawamura M, Murakami H. High Th1/Th2 ratio in patients with multiple myeloma. Leuk Res 2005;29:135-140. 4. Brimnes MK, Svane IM, Johnsen HE. Impaired functionality and phenotypic profile of dendritic cells from patients with multiple myeloma. Clin Exp Immunol 2006;144:76-84. 5. Brown R, Murray A, Pope B, Sze DM, Gibson J, Ho PJ, Hart D, Joshua D. Either interleukin-12 or interferon-gamma can correct the dendritic cell defect induced by transforming growth factor beta in patients with myeloma. Br J Haematol 2004;125:743-748. 6. Jaharian M, Watzl C, Issa Y, Altevogt P, Momburg F. Blockade of natural killer cell-mediated lysis by NCAM140 expressed on tumor cells. Int J Cancer 2007;120:2625-2634. 7. Kraut EH, Sagone AL Jr. Alternative pathway of complement in multiple myeloma. Am J Hematol 1981;11:335-345. 8. Rajkumar SV, Jacobus S, Callander N, Fonesca R, Vesole D, Williams ME, Abonour R, Siegel DS, Katz M, Greipp PR. A randomized trial of lenalidomide plus high-dose dexamethasone (RD) versus lenalidomide plus low-dose dexamethasone (Rd) in newly diagnosed multiple myeloma. Blood 2007;110:30. 9. Kolbe K, Domkin D, Derigs HG, Bhakdi S, Huber C, Aulitzky WE. Infectious complications during neutropenia subsequent to peripheral blood stem cell transplantation. Bone Marrow Transplant 1997;19:143-147. 10. Nucci M, Anaissie E. Infections in patients with multiple myeloma. Semin Hematol 2009;46:277-288. 11. Miceli MH, Dong L, Grazziutti ML, Fassas A, Thertulien R, Van Rhee F, Barlogie B, Anaissie EJ. Iron overload is a major risk factor for severe infection after autologous stem cell transplantation: a study of 367 myeloma patients. Bone Marrow Transplant 2006;37:857-864.

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32. Palumbo A, Bringhen S, Caravita T, Merla E, Capparella V, Callea V, Cangialosi C, Grasso M, Rossini F, Galli M, Catalano L, Zamagni E, Petrucci MT, De Stefano V, Ceccarelli M, Ambrosini MT, Avonto I, Falco P, Ciccone G, Liberati AM, Musto P, Boccadoro M. Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial. Lancet 2006;367.825-831. 33. Shustik C, Belch A, Robinson S, Rubin SH, Dolan SP, Kovacs MJ, Grewal KS, Walde D, Barr R, Wilson J, Gill K, Vickars L,Rudinskas L, Sicheri DA, Wilson K, Djurfeldt M, Shepherd LE, Ding K, Meyer RM. A randomised comparison of melphalan with prednisone or dexamethasone as induction therapy and dexamethasone or observation as maintenance therapy in multiple myeloma: NCIC CTG MY.7. Br J Haematol 2007;136:203-211. 34. Facon T, Mary JY, Hulin C, Benboubker L, Attal M, Pegourie B, Renaud M, Harousseau JL, Guillerm G, Chaleteix C, Dib M, Voillat L, Maisonneuve H, Troncy J, Dorvaux V, Monconduit M, Martin C, Casassus P, Jaubert J, Jardel H, Doyen C, Kolb B, Anglaret B, Grosbois B, Yakoub-Agha I, Mathiot C, Avet-Loiseau H; Intergroupe Francophone du Myélome. Melphalan and prednisone plus thalidomide versus melphalan and prednisone alone or reduced-intensity autologous stem cell transplantation in elderly patients with multiple myeloma (IFM 99-06): a randomised trial. Lancet 2007;370:1209-1218. 35. Barlogie B, Tricot G, Anaissie E, Shaughnessy J, Rasmussen E, van Rhee F, Fassas A, Zangari M, Hollmig K, Pineda-Roman M, Lee C, Talamo G, Thertulien R, Kiwan E, Krishna S, Fox M, Crowley J. Thalidomide and hematopoietic-cell transplantation for multiple myeloma. N Engl J Med 2006;354:1021-1030. 36. Uy GL, Peles S, Fisher NM, Tomasson MH, DiPersio JF, Vij R. Bortezomib prior to autologous transplant in multiple myeloma: effects in mobilization, engraftment, and markers of immune function. Biol Blood Marrow Transplant 2006;12(Suppl 1):116. 37. Blanco B, Pérez-Simón JA, Sánchez-Abarca LI, CarvajalVergara X, Mateos J, Vidriales B, López-Holgado N, Maiso P, Alberca M, Villarón E, Schenkein D, Pandiella A, San Miguel J. Bortezomib induces selective depletion of alloreactive T lymphocytes and decreases the production of Th1 cytokines. Blood 2006;107:3575-3583. 38. Nencioni A, Schwarzenberg K, Brauer KM, Schmidt SM, Ballestrero A, Grunebach F, Brossart P. Proteasome inhibitor bortezomib modulates TLR4-induced dendritic cell activation. Blood 2006;108:551-558. 39. Nencioni A, Garuti A, Schwarzenberg K, Cirmena G, Dal BG, Rocco I, Barbieri E, Brossart P, Patrone F, Ballestrero A. Proteasome inhibitor-induced apoptosis in human monocytederived dendritic cells. Eur J Immunol 2006;36:681-689.

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DOI: 10.4274/tjh.2013.0422

Research Article

Secondary Infections in Febrile Neutropenia in Hematological Malignancies: More Than Another Febrile Neutropenic Episode Hematolojik Maligniteli Febril Nötropeni’de İkincil Enfeksiyonlar: Yeni Bir Febril Nötropenik Ataktan Daha Fazlası Aslıhan Demirel1, Fehmi Tabak1, M. Cem Ar2, Bilgül Mete1, Şeniz Öngören2, Mücahit Yemişen1, Reşat Özaras1, Emre Eşkazan2, Zafer Başlar2, Ali Mert1, Teoman Soysal2, Burhan Ferhanoğlu2, Yıldız Aydın2, Recep Öztürk1 1İstanbul 2İstanbul

University Cerrahpaşa Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey University Cerrahpaşa Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

Abstract: Objective: Febrile neutropenic episodes (FNEs) are among the major causes of mortality in patients with hematological malignancies. Secondary infections develop either during the empirical antibiotic therapy or 1 week after cessation of therapy for a FNE. The aim of this study was to investigate the risk factors associated with secondary infections in febrile neutropenic patients.

Materials and Methods: We retrospectively analyzed 750 FNEs in 473 patients between January 2000 and December 2006. Results: Secondary infections were diagnosed in 152 (20%) of 750 FNEs. The median time to develop secondary infection was 10 days (range: 2-34 days). The duration of neutropenia over 10 days significantly increased the risk of secondary infections (p<0.001). The proportion of patients with microbiologically documented infections was found to be higher in primary infections (271/750, 36%) compared to secondary infections (43/152, 28%) (p=0.038). Age; sex; underlying disease; antibacterial, antifungal, or antiviral prophylaxis; blood transfusion or bone marrow transplantation; central venous catheter; and severity of neutropenia did not differ significantly between primary and secondary infections (p>0.05). While fever of unknown origin (p=0.005) and catheter-related bacteremia (p<0.001) were less frequently observed in secondary infections, the frequency of microbiologically (p=0.003) and clinically (p<0.001) documented infections, fungal pneumonias (p<0.001), infections related to gram-positive bacteria (p=0.04) and fungi (p<0.001), and 30-day mortality rate (p<0.001) were significantly higher in cases of secondary infections (p<0.001).

Conclusion: Secondary infections should be regarded as life-threatening complications of febrile neutropenia. Secondary infections represent a more severe and mortal complication and cannot be regarded just as another FNE.

Key Words: Secondary infection, Febrile neutropenia, Hematological malignancy, Mortality, Leukemia, Lymphoma

Address for Correspondence: Mücahit YEMİŞEN, M.D., İstanbul University Cerrahpaşa Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey E-mail: yemisenmucahit@hotmail.com Received/Geliş tarihi : December 17, 2013 Accepted/Kabul tarihi : June 17, 2014

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Özet: Amaç: Febril nötropenik atak (FNA) hematolojik hastalığı olan hastalarda ölümlerin başlıca nedenleri arasında yer almaktadır. İkincil enfeksiyonlar FNA’nın ampirik antibiyotik tedavi ya da bu tedavinin kesilmesinden bir hafta sonrasına kadar gelişebilir. Bu çalışmada amacımız, febril nötropenik hastalarda ikincil enfeksiyonları ile ilişkili risk faktörlerini araştırmaktır.

Gereç ve Yöntemler: Kliniğimizde Ocak 2000 ile Aralık 2006 tarihleri arasında 73 hastada 750 FNA geriye dönük olarak incelenmiştir.

Bulgular: Enfeksiyonlar 750 FNA’nın 152’sinde (%20) tespit edilmiştir. Sekonder enfeksiyon gelişmesi için geçen medyan süre 10 gün (1-34 gün) idi. Nötropeni süresinin 10 günün üzerinde olması ikincil enfeksiyon riskini anlamlı derecede arttırmaktadır (p<0,001). Mikrobiyolojik dökümante enfeksiyon oranı, primer enfeksiyonu (271/750, %36) olan hastalarda sekonder enfeksiyonlu hastalara (43/152, %28) göre daha yüksek olduğu bulunmuştur (p=0,038). Yaş, cinsiyet, altta yatan hastalık, antibakteriyel, antifungal veya antiviral profilaksi, kan transfüzyonu veya kemik iliği nakli, santral venöz kateter ya da nötropeni şiddeti açısından primer ve sekonder enfeksiyonu olan hastalar arasında anlamlı farklılık yoktu (p>0,05). Katetere bağlı bakteremi (p<0,001) ve nedeni bilinmeyen ateş (p=0,005) ikincil enfeksiyonlu hastalarda daha az sıklıkta gözlenirken, mikrobiyolojik (p=0,003) ve klinik olarak dökümante enfeksiyonlar (p<0,001), fungal pnömoniler (p<0,001), gram-pozitif bakteriler ile ilgili enfeksiyonlar (p=0,04) ve mantar enfeksiyonları (p<0,001) ve 30-günlük mortalite oranı (p<0,001) ikincil enfeksiyonlarda (p<0,001) anlamlı olarak yüksek bulunmuştur.

Sonuç: İkincil enfeksiyonlar febril nötropenik hastada hayatı tehdit eden komplikasyonlar olarak kabul edilmelidir. Enfeksiyonlar daha ağır ve ölümcül şekilde ortaya çıkabilir ve yeni bir febril nötropenik atak olarak kabul edilmemelidir.

Anahtar Sözcükler: Sekonder enfeksiyon, Febril nötropeni, Hematolojik malinite, Mortalite, Lösemi, Lenfoma Introduction Infections are one of the most important causes of mortality in cancer patients [1]. More than 80% of patients receiving chemotherapy experience at least 1 febrile episode during neutropenia and 5%-10% of these patients are lost despite broad-spectrum antibiotherapy [2]. These infections should be treated effectively and rapidly. Previous studies showed that infections were the cause of death for 50%-80% of acute leukemia patients and for 50% of patients with lymphoma and solid tumors [3]. Infections that were not observed at the beginning and develop either during empirical antibiotic therapy or within 1 week after cessation of therapy in neutropenic patients are called ‘secondary infections’ or ‘superinfections’. A few studies suggested that secondary infections significantly increased mortality and that various factors may predict the development of secondary infections [4]. In our study, we aimed to investigate the frequency of secondary infections in febrile neutropenic patients, the risk factors for the development of secondary infections, and the differences between the febrile neutropenic patients developing and not developing secondary infections. Materials and Methods The analysis was conducted on a pooled database of febrile neutropenia in patients with hematological malignancies followed in a 1500-bed tertiary care center by a joint study group of the Hematology and Infectious Diseases Departments in the Cerrahpaşa Medical Faculty of İstanbul University. 244

A total of 750 febrile neutropenic episodes (FNEs) in 473 patients hospitalized between January 2000 and December 2006 were included in this retrospective study. ‘Fever’ was defined as a single oral temperature of ≥38.5 °C (axillary temperature of ≥38 °C) and absence of any other factors that may lead to fever or oral temperature of ≥38 °C (axillary temperature of ≥37.5 °C) for at least 2 successive measurements performed at 4-h intervals during a 24-h followup period. ‘Neutropenia’ was defined as absolute neutrophil count of ≤500/mm3 or 500-1000/mm3 but expected to fall below 500/mm3 within 24-48 h. Only patients who responded to the initial therapy were included for analysis to clearly analyze the outcomes of primary and secondary FNE attacks. A secondary infection was defined as any episode of fever and/or infection not present at the initial evaluation that developed either during empirical therapy or within 1 week after discontinuation of therapy. The recurrence of fever in 48 h after its resolution during empirical antibiotic therapy was also considered as secondary infection [4]. FNEs were classified as microbiologically documented, clinically documented, or fever of unknown origin (FUO) if the cultures remained sterile and no diagnosis was identified on day 3. Fever related to any noninfectious cause (drug fever, activation of the underlying disease, transfusion-related) was considered ‘fever due to noninfectious causes’. Secondary infections were also classified as microbiologically documented, clinically documented, or

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FUO. Minor fungal infections such as oral candidiasis were not assessed. The first 30 days of patients’ treatment were taken into consideration for analysis. The demographic data and underlying risk factors including severity and duration of neutropenia, core temperature, and C-reactive protein values were recorded for every patient on days 0, 3, 7, 14, and 30. Primary and secondary infections if detected, isolated pathogens, and the antibiotics used were assessed. Causes of any mortality after primary or secondary infections were investigated. Secondary fungal infections were defined as infections with fungal agents starting during or 1 week after the cessation of an empirical antibiotic treatment [4]. Breakthrough infection was defined as the occurrence of an invasive fungal infection while the patient was receiving an effective antifungal drug as prophylaxis or therapy [5]. The chi-square test was used to compare statistically categorical variables and the Mann-Whitney U or t-test was used to compare continuous variables. A p-value of less than 0.05 was considered as statistically significant. Results A total of 750 primary infections were assessed. The median age of the patients was 39 years (range: 15-83 years), and there were 201 female (42.5%) and 272 male (57.5%) patients. The demographic characteristics of the patients are demonstrated in Table 1.

Since all patients with bone marrow transplantation (BMT) received routine prophylaxis, we compared nontransplanted patients with or without antibacterial/antifungal prophylaxis in terms of secondary infections developments. We found that antibacterial and antifungal prophylaxis did not increase the risk of developing secondary infections (p=0.076) (Table 2). We could not demonstrate any significant differences between nontransplanted and transplanted patients in term of secondary infections. In 152 episodes that led to the development of secondary infections, the neutrophil counts during the secondary infection were <100/mm3 for 87 (57.2%) episodes and ≥100/ mm3 for 65 (42.8%) episodes (p=0.062) (Table 2). The incidence of secondary infections varied significantly with respect to duration of neutropenia; duration of neutropenia was ≤10 days and >10 days in 7.16% and 34.1% of patients, respectively (p<0.001) (Figure 1). The respiratory system was considered to be the major site of infections in 86 primary and 53 secondary infections. Among these infections, the incidence of fungal pneumonia was found to be higher in secondary compared to primary infections (p<0.001). Table 1. Demographic characteristics of the cases with primary febrile neutropenic episodes.

Features

Number (%)

Number of patients

473

Number of episodes

750

Prior prophylactic antibiotic use was recorded in 24% (185) of all FNEs. More than 1 prophylactic agent was used in 28 of these episodes. Central venous catheters (CVCs) were present in 169 (22.5%) of the patients at the time of episode.

Female/male

201/272 (42.5/57.5)

Median age

39 (range: 15-83)

Secondary infections were determined in 152 (20%) of 750 episodes. Secondary infections developed after 2 primary episodes in 13 patients and after 3 primary episodes in 1 patient. The median age of the patients developing secondary infections was 39 years (15-79 years).

ALL

89 (18.8)

AML

153 (32.3)

CML

20 (4.2)

CLL

11 (2.3)

The mean duration of neutropenia was 12 days (1-74 days). The absolute neutrophil count was <100/mm3 and ≥100/mm3 in 472 (62.1%) and 277 (36.4%) of the episodes, respectively.

HCL

4 (0.8)

Non-Hodgkin’s lymphoma

74 (15.6)

The median time to development of secondary infection was 10 days (range: 2-34 days).

Hodgkin’s lymphoma

32 (6.8)

Burkitt’s lymphoma

12 (2.5)

Myelodysplastic syndrome

13 (2.7)

Aplastic anemia

15 (3.2)

Multiple myeloma

41 (8.7)

Others

9 (1.9)

Secondary infections developed 48 h after the resolution of fever in 131 (86%) of the episodes while they developed within 1 week after the discontinuation of antibiotherapy in 21 (13%) episodes. Factors related to secondary infection are outlined in Table 2. A comparison of diagnoses in primary and secondary infections is given in Table 3.

Underlying diseases

ALL: Acute lymphoblastic leukemia, AML: acute myeloblastic leukemia, CML: chronic myeloid leukemia, CLL: chronic lymphoid leukemia, HCL: hairy cell leukemia.

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Table 2. Factors related to secondary infection developed in out of 750 primary febrile neutropenic episodes.

Variable

Number of Patients

Secondary Infection Rate (%)

<50

324

27.7

>50

149

28.1

Female

201

26.86

Male

272

28.62

Autologous

34.9

Allogeneic

19.04

None

408

27.69

Catheter

Central venous catheter

169

23.66

0.4

Absolute neutrophil count (/mm3)

<100

471

18.4

≥100

277

0.062

Duration of neutropenia

<10 days

377

7.16

>10 days

366

34.15

Antibacterial prophylaxis

Yes

16.12

581

20.30

Antifungal prophylaxis

Yes

634

20.34

Antifungal+antibacterial±antiviral prophylaxis

Yes

7.6

648

20.21

Fever (°C)

<39

522

19.92

>39

225

21.3

ALL

22.7

AML

153

35.9

CLL

27.2

CML

NHL

22.9

12.5

Age

Sex

BVMT

Underlying disease

ALL: Acute lymphoblastic leukemia, AML: acute myeloblastic leukemia, CML: chronic myeloid leukemia, CLL: chronic lymphoid leukemia, NHL: non-Hodgkin’s lymphoma, HL: Hodgkin’s lymphoma, BMT: bone marrow transplantation.

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p 0.5

0.6

0.4

<0.001 0.21 0.076 0.008

0.3

0.09

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Table 3. Comparison of diagnoses in the primary and secondary infections.

Table 4. Distribution of etiological agents in the primary and secondary infections.

Diagnosis

Primary Infection (%)

Secondary Infection (%)

Primary Infection (n)

Secondary Infection (n)

Microbiological

0.2

Clinical

<0.001

FUO

0.005

Noninfectious fever

0.37

Total

747

152

Gram-negative E. coli Enterobacter spp. Klebsiella spp. Proteus spp. Non-typhi salmonella Morganella morganii Acinetobacter spp. Pseudomonas aeruginosa Pseudomonas putida Agrobacterium radiobacter H. influenza S. maltophilia Gram-positive MRCNS MRSA MSSA MSCNS S. viridans S. pneumoniae S. agalactiae Enterococcus spp. Gram (+) rod (C. striatum) Mycobacterium goodii Fungi Aspergillus spp. Fusarium Candida albicans Candida spp. Candida krusei Candida parapsilosis Mucormycosis P. jirovecii Viruses CMV HSV Total

127 66 5 16 2 1 1 11 19 1 0 1 4 129 64 9 13 21 6 2 1 10 3 0 4 3 1 0 0 0 0 0 0 1 0 1 261

16 6 0 3 0 0 0 0 3 0 1 0 3 14 6 1 0 0 2 0 0 4 0 1 11 1 0 3 3 1 1 1 1 2 2 0 43

FUO: Fever of unknown origin.

Neither having a CVC nor the underlying disease was associated with increased risk for secondary infections at p=0.4 and p=0.09, respectively. Catheter-related bacteremia was more prevalent in primary (102 episodes) than in secondary (11 episodes) infections (p<0.001). The etiologic agents detected in the primary and secondary infections are shown in Table 4. Gram-positive bacteria and fungi had significantly higher incidence rates in secondary compared to primary infections (p=0.04 and p<0.001). We did not detect any fungal breakthrough infection. More than 1 pathogen was identified in 20 episodes during primary infection and in 5 episodes during secondary infection. Extended spectrum beta-lactamase-producing E. coli was identified in 3 of the primary episodes and 2 of the secondary infection episodes. Vancomycin-resistant Enterococcus sp. was isolated in 1 of the primary episodes. Although the proportion of patients with microbiologically diagnosed infections was higher in primary infections (26%) than in secondary infections (19%), the difference was not significant (p=0.2). The mortality rate of patients who developed secondary infections (22.7%) was significantly higher compared to patients who did not develop secondary infections (13.5%) (p<0.001). Discussion Various studies identify acute leukemia, lymphoma, and multiple myeloma as underlying diseases at rates of 53%-62%, 22%-27%, and 3%-14%, respectively, in febrile neutropenic patients [6,7,8]. In our study, similarly to other studies, acute leukemia, chronic leukemia, lymphoma, and multiple myeloma were determined in 51.1%, 7.3%, 24.9%, and 8.7% of 472 patients, respectively. In recent studies, while gram-positive bacteria were identified at the rate of 44%-67%, gram-negative bacteria

MRCNS: Methicillin-resistant coagulase-negative staphylococci, MRSA: methicillin-resistant Staphylococcus aureus, MSSA: methicillin-sensitive Staphylococcus aureus, MSCNS: methicillin-sensitive coagulase-negative staphylococci.

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were determined at the rate of 55%-85% [8,9,10]. In our study, gram-positive and gram-negative bacteria isolated from various foci were almost evenly distributed at 49% and 48%, respectively. The use of intravascular catheters, antimicrobials such as ciprofloxacin or trimethoprim-sulfamethoxazole for prophylaxis, and the development of serious mucositis related with new chemotherapeutic agents are considered to underlie the increase in gram-positive bacteria [11,12]. The mortality rates were reported as 3%-8% in febrile neutropenia [7,8,13]. In our study, the mortality rate was 13.5% for the patients that had not developed any secondary infections. Mortality was linked to the uncontrolled, nonresponsive underlying disease in 33.9% of all deaths. Viscoli et al. estimated the mortality rate as 8%, and infection was identified as the cause in 30% of such events [8]. Since our tertiary care center is one of the biggest hospitals in the region, it is a referral center for nonresponsive and relatively late-stage patients. Intense chemotherapy programs applied to such patients may account for high mortality rates. The incidence of secondary infections was 20% in our study, similar to what is reported in the literature, which varies between 12% and 24% [14,15]. It is interesting that all of these patients had acute leukemia or had undergone bone marrow transplantation. Nucci et al. examined 46 (14%) secondary infection episodes of 333 FNEs [16]. The latest study about secondary infections was carried out by the International Antimicrobial Therapy Group, a subgroup of the European Organization for Research and Treatment of Cancer (IATG/EORTC) [4]. In this study, secondary infections were reported in 129 (15%) of 836 patients responding to empirical treatment among 1720 patients with their first FNEs [4]. Serra et al. described the duration and severity of neutropenia, while Feld et al. described long-term antibacterial therapy and lack of response to empirical treatment as significant risk factors for the development of secondary infections [14,15]. In the study performed by Akova et al., adult age, acute leukemia in the first induction phase, presence of intravascular catheter, absolute neutrophil count of <100/mm3 on day 4, and the identification of any other cause of the secondary infection apart from the clinically documented infection significantly increased the risk of developing secondary infections [4]. In our study, we found that duration of neutropenia of over 10 days significantly increased the risk for secondary infection development. A significant relationship was not shown between secondary infection development and age, sex, underlying disease, severity of neutropenia, use of catheter, or administration of prophylaxis. The incidence and the severity of infections were related with the severity and duration of neutropenia. In most 248

Demirel A, et al: Secondary Infections in Febrile Neutropenia

studies, a significant relationship was determined between the duration and severity of neutropenia and the development of secondary infections [4,14,15,16]. Pizzo et al. reported a strong correlation between the duration of neutropenia and superinfection (secondary infection). While superinfection was not observed in patients with neutropenia lasting less than 7 days, it was seen in 47% of patients with neutropenia of longer duration [17]. The frequency of secondary infections in our study was 7.16% and 34.1% in patients with neutropenia lasting â&#x2030;¤10 days and >10 days, respectively. We isolated fungal pathogens more frequently in cases of secondary infections compared to primary infections (p<0.001). Furthermore, the fungal infections documented clinically and radiologically rather than microbiologically were identified with a significantly higher rate in secondary infections compared to primary ones (p<0.001). This may be explained by the fact that prolonged neutropenia is the most important risk factor for fungal infections [18]. However, when secondary infections were examined discretely, fungal pathogens, gram-negative bacteria, and gram-positive bacteria were observed at rates of 11%, 37%, and 32%, respectively. Fungal agents accounted for 67% of the secondary infections in the study by Nucci et al., 48% in the study by Akova et al., and 24.7% in the study by Serra et al. [4,14,16]. The severity of the clinical picture of the patients included in our study and the difficulty of obtaining samples for culture due to the lack of adequate platelet counts in most of these patients may account for this difference. Since patients undergoing BMT receive antibiotic prophylaxis on a routine basis, we excluded these patients when assessing the impact of prophylaxis on the development of secondary infections. No significant relationship was determined between prophylaxis and development of secondary infections. Similarly, Serra et al. did not find any relationship between prophylaxis and development of secondary infections [14]. In the study by Akova et al., an increased rate of gram-positive infections was associated with the administration of antibacterial prophylaxis [4]. On the other hand, Nucci et al. reported that omitting quinolone prophylaxis resulted in a higher risk of secondary infections [16]. The relatively lower frequency of gram-positive bacteria may be explained by the use of quinolone prophylaxis in our study. In the study carried out by Akova et al., it was reported that the median time to develop secondary infections was approximately 10 days (range: 1-28 days) [4]. Similarly, the median time was 10 days (range: 2-34 days) in our study. Secondary infections developed under antibiotherapy in 131 of the episodes, whereas 22 episodes developed within 1 week after the discontinuation of antibiotherapy.

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In a study by Serra et al., microbiologically documented infections accounted for 39.8% of primary episodes and 90.1% of secondary infections [14]. In our study, the rate of microbiologically documented infections in primary infections was significantly higher compared to secondary infections (p=0.003). Collection of cultures before the administration of antibiotherapy could have facilitated the identification of the agents in primary infections. The rate of clinically documented infections was significantly higher among secondary infections than primary infections (p<0.001). This may be explained by the higher rate of fungal infections. The inability to perform interventions for most patients due to severe thrombocytopenia further complicates microbiological documentation. The presence of CVCs predisposes to a risk for development of bacteremia and fungemia [19]. The catheterrelated bacteremia rate was high in primary episodes in our study and methicillin-resistant coagulase-negative staphylococci (MRCNS) were identified as the most common cause. Considering that the most common agents identified in the catheter-related bacteremia cases were MRCNS, S. aureus, aerobic gram-negative bacilli, and C. albicans, our finding was consistent with the literature [20]. The frequency of catheterrelated bacteremia in primary infections was higher than in secondary infections. In some of the earlier studies on secondary infections, it was reported that the use of intravenous catheters increased the risk of developing secondary infections [4,16]. However, in our study, no significant relationship was determined, similarly to some other studies [14,15]. There are several important prognostic factors increasing mortality in febrile neutropenic patients [1]. In our study, fungal infections accounted for 33% of the cases with secondary infections and a mortal course. In the study carried out by Nucci et al., fungal infections were responsible for 55% of the mortality [16]. Consistent with that, this study also shows that the development of secondary infections in these patients is an important factor increasing mortality (p<0.001). To conclude, secondary infections were considered as common and life-threatening complications of febrile neutropenia. Duration of neutropenia over 10 days significantly increased the secondary infection risk. Fungal infections were more frequently seen in secondary infections compared to primary infections. This was considered to be related to the long duration of neutropenia and long-term use of broad-spectrum antibiotics. Mortality was also significantly increased in patients with secondary infections. Thus, secondary infections should be considered as a complicated, more mortal form of FNE with a differing clinical and etiological spectrum.

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. González-Barca E, Fernández-Sevilla A, Carratalá J, Salar A, Peris J, Grañena A, Gudiol F. Prognostic factors influencing mortality in cancer patients with neutropenia and bacteremia. Eur J Clin Microbiol Infect Dis 1999;18:539-544. 2. Glasmacher A, von Lilienfeld-Toal M, Schulte S, Hahn C, Schmidt-Wolf IG, Prentice A. An evidence-based evaluation of important aspects of empirical antibiotic therapy in febrile neutropenic patients. Clin Microbiol Infect 2005;11(Suppl 5):17-23. 3. Viscoli C. The evolution of the empirical management of fever and neutropenia in cancer patients. J Antimicrob Chemother 1998;41(Suppl D):65-80. 4. Akova M, Paesmans M, Calandra T, Viscoli C; International Antimicrobial Therapy Group of the European Organization for Research and Treatment of Cancer. A European Organization for Research and Treatment of Cancer-International Antimicrobial Therapy Group Study of secondary infections in febrile, neutropenic patients with cancer. Clin Infect Dis 2005;40:239-245. 5. Nailor MD, Chandrasekar PH. Treatment of breakthrough fungal infections: Is there one best drug strategy? Current Fungal Infection Reports 2009;3:229-235. 6. Cherif H, Björkholm M, Engervall P, Johansson P, Ljungman P, Hast R, Kalin M. A prospective, randomized study comparing cefepime and imipenem-cilastatin in the empirical treatment of febrile neutropenia in patients treated for haematological malignancies. Scand J Infect Dis 2004;36:593-600. 7. Rossini F, Terruzzi E, Verga L, Larocca A, Marinoni S, Miccolis I, Giltri G, Isella M, Parma M, Pogliani EM. A randomized clinical trial of ceftriaxone and amikacin versus piperacillin tazobactam and amikacin in febrile patients with hematological neoplasia and severe neutropenia. Support Care Cancer 2005;13:387-392. 8. Viscoli C, Cometta A, Kern WV, Bock R, Paesmans M, Crokaert F, Glauser MP, Calandra T; International Antimicrobial Therapy Group of the European Organization for Research and Treatment of Cancer. Piperacillin-tazobactam monotherapy in high-risk febrile and neutropenic cancer patients. Clin Microbiol Infect 2006;12:212-216. 9. Cordonnier C, Buzyn A, Leverger G, Herbrecht R, Hunault M, Leclercq R, Bastuji-Garin S; Club de Réflexion sur les Infections en Onco-Hématologie. Epidemiology and risk factors for gram-positive coccal infections in neutropenia: toward a more targeted antibiotic strategy. Clin Infect Dis 2003;36:149-158.

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10. Winston DJ, Lazarus HM, Beveridge RA, Hathorn JW, Gucalp R, Ramphal R, Chow AW, Ho WG, Horn R, Feld R, Louie TJ, Territo MC, Blumer JL, Tack KJ. Randomized, double-blind, multicenter trial comparing clinafloxacin with imipenem as empirical monotherapy for febrile granulocytopenic patients. Clin Infect Dis 2001;32:381-390.

15. Feld R, Goodman PJ, Higgins B. Prognostic factors for the development of superinfections in febrile neutropenic cancer patients (abstract 1695). In: Program and Abstracts of the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington, DC, American Society for Microbiology, 1992.

11. Ramphal R. Changes in the etiology of bacteremia in febrile neutropenic patients and the susceptibilities of the currently isolated pathogens. Clin Infect Dis 2004;39(Suppl 1):25-31.

16. Nucci M, Spector N, Bueno AP, Solza C, Perecmanis T, Bacha PC, Pulcheri W. Risk factors and attributable mortality associated with superinfections in neutropenic patients with cancer. Clin Infect Dis 1997;24:575-579.

12. Zinner SH. Changing epidemiology of infections in patients with neutropenia and cancer: emphasis on gram-positive and resistant bacteria. Clin Infect Dis 1999;29:490-494. 13. Bow EJ, Rotstein C, Noskin GA, Laverdiere M, Schwarer AP, Segal BH, Seymour JF, Szer J, Sanche S. A randomized, openlabel, multicenter comparative study of the efficacy and safety of piperacillin-tazobactam and cefepime for the empirical treatment of febrile neutropenic episodes in patients with hematologic malignancies. Clin Infect Dis 2006;43:447-459. 14. Serra P, Santini C, Venditti M, Mandelli F, Martino P. Superinfections during antimicrobial treatment with betalactam-aminoglycoside combinations in neutropenic patients with hematologic malignancies. Infection 1985;13(Suppl 1):115-122.

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17. Pizzo PA, Ladisch S, Ribichaud K. Treatment of gram-positive septicemia in cancer patients. Cancer 1980;45:206-207. 18. Bodey GP. Fungal infections in cancer patients. Ann N Y Acad Sci 1998;544:431-442. 19. Raad II, Bodey GP. Infectious complications of indwelling vascular catheters. Clin Infect Dis 1992;15:197-208. 20. Maki DG, Mermel LA. Infections due to infusion therapy. In: Bennett JV, Brachman PS, (eds). Hospital Infections. 4th ed. Philadelphia, Lippincott-Raven, 1998.

DOI: 10.4274/tjh.2013.0342

Research Article

The Impact of Chemotherapy on Hepatitis B Antibody Titer in Patients with Hematological Malignancies Hematolojik Maligniteli Hastalarda Kemoterapinin Hepatit B Antikor Titrelerine Etkisi Münci Yağcı, Elif Suyanı, Merih Kızıl Çakar Gazi University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract: Objective: To investigate the influence of chemotherapy (CT) on HBsAb titer in patients receiving CT due to hematological malignancy.

Materials and Methods: The data of 75 patients who received CT with the diagnosis of various hematological malignancies and who had serum HBsAb levels measured prior to and after the cessation of CT were evaluated retrospectively.

Results: The median age of the patients was 52 years (range: 16-78) with 49 (65%) males and 26 (35%) females. Median HBsAb titer decreased significantly after CT compared to the pre-CT median HBsAb titer [68 (range: 0-1000) vs. 100 (range: 6.2-1000)] (p=0.001). In subgroup analysis, median HBsAb titer decreased significantly after CT in acute leukemia patients [110 (range: 6.2-1000) vs. 67.8 (range: 0-1000)] (p=0.003) and in patients receiving intensive CT [97.2 (range: 6.2-1000) vs. 71 (range: 0-1000)] (p=0.036). The decrease in median HBsAb titer was significant in male patients (p<0.001). HBsAb became negative after CT in 9 patients who were HBcAb-negative and had lower pre-CT HBsAb levels.

Conclusion: HBsAb decreased after CT, especially in acute leukemia and male patients, and in patients receiving intensive CT.

Key Words: Hepatitis B, Resolved infection, Hepatitis B surface antibody, Hematological malignancy, Chemotherapy Özet: Amaç: Bu çalışmada, hematolojik malignite nedeniyle kemoterapi alan hastalarda kemoterapinin Anti HBs antikor titresi üzerine olan etkisinin incelenmesi amaçlanmıştır.

Gereç ve Yöntemler: Çeşitli hematolojik malignite tanısı ile kemoterapi alan ve hem kemoterapi öncesi hem de kemoterapi tamamlandıktan sonraki Anti HBs antikor titresi mevcut olan toplan 75 hasta retrospektif olarak incelendi.

Bulgular: Hastaların ortanca yaşı 52 (16-78) olup 49’u (%65) erkek ve 26’sı (%35) kadın idi. Kemoterapi sonrası ortanca Anti HBs antikor titresinde kemoterapi öncesine göre anlamlı düşme saptandı [68 (range: 0-1000) vs. 100 (range: 6,2-1000)] (p=0,001). Alt grup analizi yapıldığında, kemoterapi sonrası ortanca Anti HBs antikor titresindeki azalmanın özellikle akut lösemi hastalarında [110 (range: 6,2-1000) vs. 67.8 (range: 0-1000)] (p=0,003) ve yoğun kemoterapi alan hastalarda [97,2

Address for Correspondence: Münci YAĞCI, M.D., Gazi University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone: +90 312 202 63 17 E-mail: ayagci@gazi.edu.tr Received/Geliş tarihi : October 07, 2013 Accepted/Kabul tarihi : July 01, 2014

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Yağcı M, et al: HBsAb Titer During Chemotherapy

(range: 6,2-1000) vs. 71 (range: 0-1000)] (p=0,036) olduğu gözlendi. Ayrıca ortanca Anti HBs antikor titresindeki azalma erkek cinsiyette daha belirgin idi (p<0,001). Kemoterapi öncesi Anti Hbs antikor titresi düşük olan ve yine Anti HBc antikoru negatif olan dokuz hastada kemoterapi sonrası Anti HBs antikoru negatifleşti.

Sonuç: Anti HBs antikor titresinde kemoterapi sonrasında azalma görülmüştür ve bu azalma akut lösemi hastalarında, erkek hastalarda ve yoğun kemoterapi alan hastalarda daha belirgin olarak saptanmıştır.

Anahtar Sözcükler: Hepatit B, Geçirilmiş enfeksiyon, Hepatit B yüzey antikoru, Hematolojik malignite, Kemoterapi Introduction Hepatitis B virus (HBV) infection, existing in one-third of the world’s population, is a major health problem [1,2]. With the widespread use of various cytotoxic chemotherapies, it has arisen as a considerable clinical problem in patients receiving chemotherapy (CT) treatment. CT-induced HBV reactivation in patients with hematological malignancies leads to interruption of the treatment or even to the death of the patient due to hepatic failure [1,3,4]. The frequency of CT-induced HBV reactivation in hepatitis B surface antigen (HBsAg)-carrier patients with hematological malignancies is approximately 50% [2,4,5]. However, cases of resolved HBV infection, defined as HBsAg-seronegative, hepatitis B surface antibody (HBsAb)-positive, and/or hepatitis B core antibody (HBcAb)-positive, also carry the risk of HBV reactivation during or after the cessation of CT [1,2,3,4]. The frequency of HBV reactivation in patients with resolved infection has been investigated mostly in the context of allogeneic hematopoietic stem cell transplantation (HSCT) and varies between 11.6% and 50% [6,7,8]. In patients with resolved infection, there is usually a gradual decline of the HBsAb titer followed by the appearance of HBV DNA and HBsAg later on, leading to HBV reactivation [9,10]. From this point of view, we aimed to investigate the influence of CT on HBsAb titer in patients receiving CT due to hematological malignancy. Materials and Methods The data of 949 patients who received CT with the diagnosis of various hematological malignancies at the Gazi University Faculty of Medicine, Department of Hematology, between January 1995 and January 2012 were reviewed retrospectively. Hepatitis B serology was studied as part of the routine screening program prior to CT in our clinic. Patient files were evaluated for age, diagnosis, type and number of CT cycles, and serum HBsAg, HBsAb, HBcAb, hepatitis B e antigen (HBeAg), and hepatitis B e antibody (HBeAb). The HBsAb titer was measured by enzyme-linked immunosorbent assay (ELISA) and the cut-off point was 10 IU/L. The patients who were HBsAg-negative and had serum HBsAb levels prior to and after the cessation of CT were included in the analysis. The study was approved by the local ethics committee.

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Statistical Analysis Statistical evaluation was done using SPSS 15. Data are presented as numbers and percentages or medians and ranges, as appropriate. The chi-square test was used for evaluating categorical values and the Wilcoxon U test was used for continuous values. All p-values are 2-sided with statistical significance at the 0.05 alpha level. Results Patient characteristics are presented in Table 1. Seventyfive patients receiving CT with the diagnoses of non-Hodgkin lymphoma (n=18), Hodgkin lymphoma (n=9), chronic lymphocytic leukemia (n=6), multiple myeloma (n=11), and acute myeloid leukemia (n=31) were included in the analysis. The types of CT applied to the patients were very heterogeneous and were grouped as follows: alkylating agents and/or anthracycline-based CT (n=21), nucleoside analogbased CT (n=6), demethylating agent-based CT (n=3), immunomodulatory agent and/or proteasome inhibitor-based CT (n=7), rituximab-based CT (n=14), and acute leukemia/ high-dose CT (n=24). None of the patients had received autologous stem cell transplantation. The median age of the patients was 52 years (range: 16-78), with 49 (65%) males and 26 (35%) females. The median time from last dose of CT to first record of post-CT HBsAb titer was 52 days (range: 4-1334). The median time between pre- and post-CT HBsAb titers was 232 days (range: 42-15.619). Hepatitis B serology of the patients was as follows: all patients were HBsAg-negative, 31 (46%) of the patients were HBcAb-positive (data available for 68/75 patients), all patients were HBeAg-negative (data available for 62/75 patients), and 17 (29%) patients were HBeAb-positive (data available for 16/75 patients). Median HBsAb titer decreased significantly after CT compared to the pre-CT median HBsAb titer [100 (range: 6.2-1000) vs. 68 (range: 0-1000)] (p=0.001). In subgroup analysis, while the decrease in median HBsAb titer in the lymphoproliferative disease group had only a trend for significance [98.7 (range: 8.1-810) vs. 82.5 (range: 0-810)] (p=0.072), in acute leukemia patients the decrease in median HBsAb titer after CT was significant [110 (range: 6.2-1000) vs. 67.8 (range: 0-1000)] (p=0.003) (Table 2). The decrease in HBsAb titer was also significant

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in patients receiving intensive CT, which was applied mostly for acute leukemia patients (n=21) [97.2 (range: 6.2-1000) vs. 71 (range: 0-1000)] (p=0.036). HBsAb titer results before and after CT in other CT groups were as follows: alkylating agent and/or anthracycline-based CT, 142 (range: 20.4577.6) vs. 75.4 (range: 0-805) (p=0.068); nucleoside analogbased CT, 60.7 (range: 12.8-810) vs. 49 (range: 10.9810) (p=0.225); demethylating agent-based CT, 51.6 (range: 11.7-505.5) vs. 45.9 (range: 43-368.5) (p=0.593); immunomodulatory agent and/or proteasome inhibitor-based CT, 42 (range: 8.1-644.7) vs. 45.7 (range: 0-779) (p=0.593); rituximab-based CT, 152 (range: 11-810) vs. 103 (range: 0-553) (p=0.064) (Table 2). In univariate analysis, the decrease in HBsAb titer was not affected by age, type of treatment, HBcAb positivity, HBeAb

positivity, or time from last dose of CT to time of post-CT HBsAb titer in acute leukemia group (p>0.05). When the HBsAb titer decrease was investigated according to sex, median HBsAb titer decreased significantly for males (p<0.001). After subgroup analysis according to the disease diagnosis (lymphoproliferative disease vs. acute leukemia), the decrease was significant in males in both disease groups (p=0.04 and p=0.001). Subgroup analysis according to treatment type was also done; the decrease was significant in male patients receiving high-dose CT (p=0.009) and close to significant in male patients receiving rituximab-based CT (p=0.051). HBsAb became negative after CT in 9 patients. Nevertheless, we do not have information about HBV DNA status at that time.

Table 1. Patient characteristics.

n=75

Age, median (minimum-maximum)

52 (16-78)

Sex, M/F

49/26

Diseases, n (%)

Acute leukemia

31 (41%)

NHL

18 (24%)

9 (12%)

CLL

6 (8%)

11 (15%)

Median number of CT lines, n (minimum-maximum)

4 (1-17)

Median time from last course of CT to first record of post-CT HBsAb titer, days (minimum-maximum)

52 (4-1334)

Median time between pre- and post-CT HBsAb titers, days (minimum-maximum)

232 (42-15,619)

Types of CT, n (%)

Alkylating agents and/or anthracycline-based

21 (28%)

Nucleoside analog-based

6 (8%)

Demethylating agent-based

3 (4%)

Immunomodulatory agents and/or proteasome inhibitor-based

7 (9%)

Rituximab-based

14 (19%)

High-dose CT

24 (32%)

HBV serology

Positive HBcAb

31 (46%)

Positive HBeAb

17 (29%)

CT: Chemotherapy, CLL: chronic lymphocytic leukemia, F: female, HBsAb: hepatitis B surface antibody, HBcAb: hepatitis B core antibody, HBeAb: hepatitis B e antibody, HL: Hodgkin lymphoma, NHL: non-Hodgkin lymphoma, M: male, MM: multiple myeloma.

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Table 2. Median hepatitis B antibody levels prior to and after chemotherapy according to disease and chemotherapy types.

Disease/CT Type

Median HBsAb before CT IU/dL (minimum-maximum)

Median HBsAb after CT, IU/dL (minimum-maximum)

Acute leukemia

110 (6.2-1000)

67.8 (0-1000)

0.003

Lymphoproliferative disease

98.7 (8.1-810)

82 (0-810)

0.072

Intensive CT

97.2 (6.2-1000)

71 (0-1000)

0.036

Alkylating agents and/or anthracycline-based CT 142 (20.4-577.6)

75.4 (0-805)

0.068

Nucleoside analog-based CT

60.7 (12.8-810)

49 (10.9-810)

0.225

Demethylating agent-based CT

51.6 (11.7-505.5)

45.9 (43-368.5)

0.593

Immunomodulatory agent and/or proteasome inhibitor-based CT

42 (8.1-644.7)

45.7 (0-779)

0.593

Rituximab-based CT

152 (11-810)

103 (0-553)

0.064

CT: Chemotherapy; HBsAb: hepatitis B antibody.

However, liver function tests were normal at that time (median aspartate transaminase and alanine transaminase levels were 26 and 33 IU/L, respectively). Those patients whose HBsAb became negative after CT were not different from the patients who were HBsAb-positive in terms of sex, disease diagnosis, type of treatment, median time from last dose of CT to first record of post-CT HBsAb titer, and median time between preand post-CT HBsAb titers (p>0.05). However, the patients who became HBsAb-negative were HBcAb-negative (with 7/9 available data; p=0.013) and had lower median pre-CT HBsAb titer [22.4 (range: 11-81.6) vs. 142 (range: 6.2-1000)] (p=0.001). Discussion HBV reactivation, which is frequently encountered in patients with a diagnosis of hematological malignancy, has been well studied in the era of HSCT and CT. Seropositivity, high viral load, male sex, young age, intensive CT, corticosteroids, anthracyclines, cyclophosphamide vincristine, fludarabine, and monoclonal antibodies have been defined as risk factors for HBV reactivation [1,2,3,4,7,11]. On the other hand, effects of CT on the course of HBsAb titer have not been investigated in detail in patients receiving CT due to hematological malignancies including lymphoma, chronic leukemia, acute leukemia, multiple myeloma, and myelodysplastic syndrome. Francisci et al. studied the effect of CT on HBsAb titer in 43 patients and found that HBsAb titer decreased after CT, but in none of the patients did the HBsAb titer decrease below 10 IU/L. However, that study did not denote the type of CT, nor did it cover the subgroup analysis of hematological malignancies [12]. We retrospectively analyzed the course of HBsAb titer in our patients receiving CT with the diagnosis

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of various hematological malignancies. Similar to the findings for HBV reactivation, male patients and those receiving intensive CT were found to be more vulnerable to the decrease in HBsAb titer after exposure to CT. We also found that HBsAb decreased significantly in patients with the diagnosis of acute leukemia. Although HBV reactivation frequency reaches 50% in HBsAg carriers receiving cytotoxic CT, patients with resolved infection are also at risk of reactivation [1,2,4,6,10]. HBV reactivation has been reported in lymphoma and acute lymphoblastic leukemia patients who had resolved infections [13,14,15,16,17,18,19]. Even high HBsAb titers might not prevent HBV reactivation in these patients, especially with the use of either high-dose CT or rituximab [17,18,19]. In the present study, HBsAb decreased in all CT groups, including rituximab-based CT; however, the decrease was most significant in the intensive CT group. In our study it was observed that HBsAb became negative in 9 patients; however, their HBV DNA status at that time is not known. On the other hand, those patients were HBcAbnegative, implying that they were immunized previously, and they also had lower pre-CT HBsAb titers. The antigenic stimulation caused by the vaccine is less powerful compared to exposure to the virus itself, which makes those patients more prone to acute hepatitis B infection than HBV reactivation. The decrease in HBsAb titer was more prominent in males in this study. Male sex is a well-known risk factor for HBV reactivation [1,2,3,4,7]. Women have higher numbers of B cells and produce higher levels of antibodies than men, which might be an explanation for this difference [20,21]. The underlying mechanism in the decrease of HBsAb titer after CT is probably destruction of antibodies producing

Yağcı M, et al: HBsAb Titer During Chemotherapy

B lymphocytes by CT. After exposure to HBV, CD4+ T cells trigger activation of both cytotoxic T cells and B cells [22]. Although cytotoxic T cells are the major contributors of HBV clearance by destroying infected hepatocytes, antiviral antibody production also has a substantial role in the control of infection [22,23]. As a result, HBsAg disappears and HBsAb appears, which is defined as resolved infection [1,24]. Despite this resolution, it is now well known that HBV DNA continues to exist in the liver, peripheral mononuclear cells, and even bodily secretions in minute amounts in subjects who were exposed to HBV [1,4,24,25]. In patients with resolved infection, there is usually a gradual decline of the HBsAb titer followed by appearance of HBV DNA and HBsAg later on, leading to HBV reactivation during CT or after the cessation of CT [9,10]. Serum alanine transferase levels usually rise after the elevation of HBV DNA, which may lead to the reactivation being missed [4]. The limitations of this study are its retrospective nature, leading to the lack of information about HBV DNA status of the patients, and the fact that the types of CT and diseases were heterogeneous. However, to our knowledge, despite its limitations, this study is the first investigating the effect of CT on the course of HBsAb titer in detail. In conclusion, resolved HBV infection with HBsAb positivity is not a guarantee against HBV reactivation in patients receiving CT, and HBsAb decreases after CT, especially in acute leukemia and male patients and in patients receiving intensive CT. Therefore, patients who are at greater risk for the disappearance of HBsAb need close monitorization for the decrease and ultimate disappearance of HBsAb, appearance of HBV DNA, and HBV reactivation. 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. Yeo W, Johnson PJ. Diagnosis, prevention and management of hepatitis B virus reactivation during anticancer therapy. Hepatology 2006;43:209-220. 2. Liang R. How I treat and monitor viral hepatitis B infection in patients receiving intensive immunosuppressive therapies or undergoing hematopoietic stem cell transplantation. Blood 2009;113:3147-3153. 3. Firpi RJ, Nelson DR. Viral hepatitis: manifestations and management strategy. Hematology Am Soc Hematol Educ Program 2006:375-380. 4. Lalazar G, Rund D, Shouval D. Screening, prevention and treatment of viral hepatitis B reactivation in patients with haematological malignancies. Br J Haematol 2007;136:699712.

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5. Yağci M, Ozkurt ZN, Yeğin ZA, Aki Z, Sucak GT, Haznedar R. Hepatitus B virus reactivation in HBV-DNA negative and positive patients with hematological malignancies. Hematology 2010;15:240-244. 6. Ramos CA, Saliba RM, de Pádua Silva L, Khorshid O, Shpall EJ, Giralt S, Patah PA, Hosing CM, Popat UR, Rondon G, Nieto Y, Champlin RE, de Lima M. Resolved hepatitis B virus infection is not associated with worse outcome after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2010;16:686-694. 7. Hammond SP, Borchelt AM, Ukomadu C, Ho VT, Baden LR, Marty FM. Hepatitis B virus reactivation following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2009;15:1049-1059. 8. Knöll A, Boehm S, Hahn J, Holler E, Jilg W. Reactivation of resolved hepatitis B virus infection after allogeneic haematopoietic stem cell transplantation. Bone Marrow Transplant 2004;33:925-929. 9. Knöll A, Boehm S, Hahn J, Holler E, Jilg W. Long-term surveillance of haematopoietic stem cell recipients with resolved hepatitis B: high risk of viral reactivation even in a recipient with a vaccinated donor. J Viral Hepat 2007;14:478483. 10. Firpi RJ, Nelson DR. Management of viral hepatitis in hematologic malignancies. Blood Rev 2008;22:117-126. 11. Yağci M, Sucak GT, Haznedar R. Fludarabine and risk of hepatitis B virus reactivation in chronic lymphocytic leukemia. Am J Hematol 2000;64:233-234. 12. Francisci D, Falcinelli F, Schiaroli E, Capponi M, Belfiori B, Flenghi L, Baldelli F. Management of hepatitis B virus reactivation in patients with hematological malignancies treated with chemotherapy. Infection 2010;38:58-61. 13. Hui CK, Cheung WW, Zhang HY, Au WY, Yueng YH, Leung AY, Leung N, Luk JM, Lie AK, Kwong YL, Liang R, Lau GK. Kinetics and risk of de novo hepatitis B infection in HBsAgnegative patients undergoing cytotoxic chemotherapy. Gastroenterology 2006;131:59-68. 14. Koo YX, Tay M, Teh YE, Teng D, Tan DS, Tan IB, Tai DW, Quek R, Tao M, Lim ST. Risk of hepatitis B virus (HBV) reactivation in hepatitis B surface antigen negative/hepatitis B core antibody positive patients receiving rituximab-containing combination chemotherapy without routine antiviral prophylaxis. Ann Hematol 2011;90:1219-1223. 15. Sugauchi F, Tanaka Y, Kusumoto S, Matsuura K, Sugiyama M, Kurbanov F, Ueda R, Mizokami M. Virological and clinical characteristics on reactivation of occult hepatitis B in patients with hematological malignancy. J Med Virol 2011;83:412418.

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16. Awerkiew S, Däumer M, Reiser M, Wend UC, Pfister H, Kaiser R, Willems WR, Gerlich WH. Reactivation of an occult hepatitis B virus escape mutant in an anti-HBs positive, antiHBc negative lymphoma patient. J Clin Virol 2007;38:83-86.

21. Abdullah M, Chai PS, Chong MY, Tohit ER, Ramasamy R, Pei CP, Vidyadaran S. Gender effect on in vitro lymphocyte subset levels of healthy individuals. Cell Immunol 2012;272:214219.

17. Sera T, Hiasa Y, Michitaka K, Konishi I, Matsuura K, Tokumoto Y, Matsuura B, Kajiwara T, Masumoto T, Horiike N, Onji M. Anti-HBs-positive liver failure due to hepatitis B virus reactivation induced by rituximab. Intern Med 2006;45:721724.

22. Bertoletti A, Gehring AJ. The immune response during hepatitis B virus infection. J Gen Virol 2006;87:1439-1449.

18. Sarrecchia C, Cappelli A, Aiello P. HBV reactivation with fatal fulminating hepatitis during rituximab treatment in a subject negative for HBsAg and positive for HBsAb and HBcAb. J Infect Chemother 2005;11:189-191. 19. Ozaras R, Ar C, Ongoren S, Mete B, Tabak F, Mert A, Ozturk R. Acute hepatitis B despite a previous high titer of anti-HBs. Hepatol Int 2010;4:530-532. 20. Oertelt-Prigione S. The influence of sex and gender on the immune response. Autoimmun Rev 2012;11:479-485.

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23. Chisari FV. Cytotoxic T cells and viral hepatitis. J Clin Invest 1997;99:1472-1477. 24. Yim HJ, Lok AS. Natural history of chronic hepatitis B virus infection: what we knew in 1981 and what we know in 2005. Hepatology 2006;43(2 Suppl 1):173-181. 25. Mason AL, Xu L, Guo L, Kuhns M, Perrillo RP. Molecular basis for persistent hepatitis B virus infection in the liver after clearance of serum hepatitis B surface antigen. Hepatology 1998;27:1736-1742.

Case Report

DOI: 10.4274/tjh.2013.0322

Occurrence of Carcinoma of the Pancreas Following Nilotinib Therapy for Chronic Myeloid Leukemia: Report of a Case with Review of the Literature Kronik Miyeloid Lösemi için Kullanılan Nilotinib Tedavisi Sonrasında Gelişen Pankreas Kanseri: Bir Olgu Sunumu ve Literatür Derlemesi Yasunobu Sekiguchi1, Asami Shimada1, Moe Matsuzawa1, Hidenori Imai1, Mutsumi Wakabayashi1, Keiji Sugimoto1, Noriko Nakamura2, Tomohiro Sawada2, Junichi Arita3, Norio Komatsu4, Masaaki Noguchi1 1Juntendo

University Faculty of Medicine, Urayasu Hospital, Clinic of Hematology, Urayasu, Japan University Faculty of Medicine, Urayasu Hospital, Clinic of Clinical Laboratory, Urayasu, Japan 3The Cancer Institute of the Japanese Foundation for Cancer Research, Gastroenterology Center, Tokyo, Japan 4Juntendo University Faculty of Medicine Hospital, Department of Hematology, Tokyo, Japan 2Juntendo

Abstract: The patient, a 79-year-old Japanese man, was diagnosed with the chronic phase of chronic myeloid leukemia and begun on nilotinib therapy in April 2011. The therapeutic response was major molecular response in August. About 19 months after the start of nilotinib therapy at 400 mg/day (November 2012), an adenocarcinoma (24x20 mm) confined to the head of the pancreas developed. In February 2013, a pancreaticoduodenectomy was performed. The therapy regimen was switched to dasatinib at 100 mg/day, beginning in April. The response was still major molecular response with no recurrence of pancreatic carcinoma in July 2013. There have been 29 reported cases of secondary neoplasms associated with nilotinib therapy. These secondary neoplasms were characterized by relatively frequent occurrence of papilloma (6 cases), gastric cancer (3 cases), fibroma (3 cases), and thyroid neoplasms (2 cases). The present case, however, is the first to be reported as carcinoma of the pancreas. This report describes the case. Key Words: Chronic myeloid leukemia, Nilotinib, Secondary malignancy, Carcinoma of the pancreas

Özet: Yetmiş dokuz yaşında Japon erkek hastaya, Nisan 2011’de kronik evre kronik miyeloid lösemi tanısı konulup, nilotinib tedavisi başlandı. Ağustos’ta majör moleküler yanıt sağlandı. Nilotinib 400 mg/gün dozunda kullanılırken ve tedavi başlangıcından yaklaşık 19 ay geçmişken (Kasım 2012’de), pankreas başına sınırlı (24x20 mm) adenokanser gelişti. Şubat 2013’te pankreatikoduodenektomi yapıldı. Nisan ayından başlayarak tedavi rejimi olarak dasatinib 100 mg/gün’e geçildi. Temmuz 2013’te yanıt halen majör moleküler yanıttı ve pankreas kanserine ait nüks saptanmadı. Literatürde nilotinib tedavisi ile ilişkili olan 29 ikincil neoplazi tespit edildi. Bu ikincil neoplaziler arasında görece daha sık gözlemlenenler papilloma (6 olgu), mide kanseri (3 olgu), fibroma (3 olgu) ve tiroid neoplazileridir (2 olgu). Olgumuz, literatürde nilotinib altında iken gelişen ilk pankreas kanseri olgusudur. Bu yazıda olgu tartışması ve nilotinibe bağlı gelişen ikincil kanserlere ait literatür taraması sunulmaktadır. Anahtar Sözcükler: Kronik miyeloid lösemi, Nilotinib, İkincil malinite, Pankreas kanseri Address for Correspondence: Yasunobu SEKIGUCHI, M.D., Juntendo University Faculty of Medicine, Urayasu Hospital, Clinic of Hematology, Urayasu, Japan Phone: 047-353-3111 E-mail: yasu_sek@juntendo-urayasu.jp Received/Geliş tarihi : September 20, 2013 Accepted/Kabul tarihi : June 17, 2014

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Introduction Regarding secondary neoplasms (SNs) during imatinib therapy, a previous report described that there was no increase in incidence, while another report stated that an approximately 1.5-fold increase, though not statistically significant, was noted for patients as compared to incidence for a general population [1,2]. It has also been pointed out in a few reports that the incidence of certain types of malignant tumors has increased. An increased incidence has been documented for carcinoma of the prostate; melanoma, carcinoma of the kidney, endocrinoma, and chronic lymphoid leukemia; cancer of the prostate and breast cancer; and non-Hodgkin lymphoma and cancers of the gastrointestinal tract, lungs, and skin [3,4,5,6]. Further, SNs in patients with chronic myeloid leukemia (CML) are complicated and have been described to be probably ascribed to multiple factors [7,8]. Our extensive search of the literature revealed the time to onset of SN after start of nilotinib therapy to be unclear but that after start of imatinib therapy to be about 39 months [4]. There have been no more than a total of 29 cases of SNs associated with nilotinib therapy reported, including the present case [4,6,9,10,11,12]. As second-generation tyrosine kinase inhibitors, including nilotinib, are more potent in immunosuppressive activity than imatinib, their potential risk of SN has been pointed out [4,13]. Long-term follow-up of accumulated cases treated with nilotinib, as is the case with imatinib, is considered necessary. Pancreatitis occurring during nilotinib therapy was noted with an incidence of 1.9%, being higher as compared to imatinib and dasatinib, and it tended to be more frequent among Japanese patients [10,14,15,16]. Since chronic pancreatitis represents a risk factor for cancer of the pancreas, nilotinib may possibly constitute a risk factor for pancreatic carcinoma [17]. The present case is the first to be reported as pancreatic carcinoma that developed following nilotinib therapy. Case Presentation Patient: A Japanese man aged 79, with a chief complaint of increased leukocyte and platelet counts. Past history: He had experienced hepatic angioma and lumbar disk herniation, had an operation for acute appendicitis, was operated on for chronic sinusitis, and had been known to be hypertensive with benign prostatic hypertrophy. Family history, life history, and medication history: Unremarkable. Present illness: The patient had been receiving doxazosin mesylate and tamsulosin hydrochloride since 2003 for hypertension and

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benign prostatic hypertrophy at a nearby medical clinic. He was referred to us for medical workup in April 2011 because of increased leukocyte count (12,900/µL) and platelet count (69.8x104/µL). Laboratory findings: See Table 1. The amylase (AMY), carbohydrate antigen 19-9 (CA19-9), s-pancreas-1 antigen (SPAN-1), and pancreatic cancer-associated antigen (DUPAN-2) levels were all within their respective reference ranges. Bone marrow examination revealed a nucleated cell count of 36.0x104/µL, a megakaryocyte count of 915/µL, 1.4% blasts, and chromosomal aberrations 46,XY, t(9;22) (q34; q11.2). The patient was thus diagnosed with the chronic phase of CML and was begun on nilotinib at 400 mg/day in April 2011 (Figure 1). The dose was reduced to 400 mg/day because the patient was elderly. In August 2011, i.e. 4 months later, the peripheral blood fused gene level became Amp-CML <5 copies/assay, signaling a major molecular response (MMR) [18]. In November 2012, the patient began suffering from postprandial epigastric pain, and the following parameters were noted to be elevated: AMY: 326 IU/L, CA19-9: 38.2 U/mL, SPAN-1: 33 U/mL, and DUPAN-2: >1600 U/mL. A computed tomography (CT) scan disclosed a mass measuring 24x20 mm in the head of the pancreas but there was neither vascular abnormality nor any lymphadenopathy (Figure 2). A diagnosis of adenocarcinoma was made from needle biopsy findings of the mass in the head of the pancreas. Nilotinib was discontinued late in January 2013, and the patient had a pancreaticoduodenectomy elsewhere in the middle of February. The postoperative course was uneventful. Informed consent was obtained. In March, treatment with nilotinib at 400 mg/day was resumed, at which time the response was still MMR. However, despondency appeared from immediately after reinstitution of nilotinib, which was therefore then switched to dasatinib at 100 mg/day in April. The listless feeling disappeared and the patient progressed favorably with continued oral dasatinib. The response is still MMR and there was no recurrence of cancer of the pancreas as of July 2013. Discussion and Review of the Literature Twenty-nine cases of SN have been reported in the literature to our knowledge so far as nilotinib is concerned: 9 cases in global phase III multicenter trials, 2 cases in a Japanese phase II study, 4 cases in an overseas phase II trial, 4 cases reported by Verma et al. (3 patients receiving nilotinib switched from imatinib, and 1 patient on nilotinib as a first-line regimen), 7 cases in postmarketing clinical use surveys in Japan, and the case documented herein (Table 2) [4,9,10,11,12]. The time to onset

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Table 1. Laboratory findings upon initial examination in this department.

Table 1. Continuation M:E ratio

4.74 ↑

Peripheral blood

Blasts

1.4% ↑

WBC

12.9x109/L ↑

Myeloid series

Myelo

1.5% ↑

Promyelocytes

11.4% ↑

Band

2.0%

Myelocytes

14.0%

Seg

77.0% ↑

Metamyelocytes

9.2%

9.5% ↓

Bandform

28.8% ↑

4.0%

Segmented

7.0% ↑

6.0% ↑

Eosino. immature

7.2%

RBC

5.63x1012/L ↑

Eosino. mature

2.4%

15.1 g/dL

Baso. immature

1.2% ↑

49.3% ↑

Lymphocytes

1.4% ↓

MCV

87.6%

Plasma cells

0.2%

MCH

26.8% ↓

Megakaryocytes

0.4% ↑

Plt

698.0x109/L

Ret

1.1%

↑

Biochemistry

Erythroid series Proerythroblasts

0.6%

Basophilic

1.4%

T.P.

7.6 g/dL

Polychromatic

13.2%

Alb

4.3 g/dL

Mitotic figures

0.2%

AST

16 IU/L 15 IU/L

Whole blood major bcr-abl mRNA (TMA)-L

>626 copies/assay ↑

ALT LDH

240 IU/L ↑

ALP

141 IU/L

Bone marrow chromosomes (G-band)

g-GTP

46 IU/L

46,XY, t(9;22) (q34;q11.2)

19 cells/20 cells ↑

T-Bil

0.4 mg/dL

46,XY

1 cell/20 cells

AMY

76 IU/L

BUN

12 mg/dL

0.73 mg/dL

CRP

<0.3 mg/dL

Ferritin

12.8 ng/mL ↑

Tumor marker CA 19-9

24.2 U/mL

SPAN-1

26 U/mL

DUPAN-2

130 U/mL

Bone marrow examination Nucleated cell count

36.0x104/µL ↑

Megakaryocyte count

915/µL ↑

WBC: White blood cells, Myelo: myelocytes, Seg: segmentation, Ly: lymphocytes, Eo: eosinocytes, Ba: basocytes, RBC: red blood cells, Hb: hemoglobin, Ht: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, Plt: platelets, Ret: reticulocytes, T.P.: total protein, Alb: albumin, AST: aspartate aminotransferase, ALT: alanine aminotransferase, LDH: lactate dehydrogenase, ALP: alkaline phosphatase, g-GTP: g-guanosine triphosphate, T-Bil: total bilirubin, BUN: blood urea nitrogen, Cr: creatinine, CRP: C-reactive protein, M:E ratio: myeloid:erythroid ratio, mRNA: messenger ribonucleic acid, TMA: transcription mediated amplification.

of SN after initiation of nilotinib therapy was unclear. Of the SNs reported, papilloma in 6 cases, gastric cancer in 3 cases, fibroma in 3 cases, and thyroid neoplasms in 2 cases were relatively common, while the present case is the first to be reported as carcinoma of the pancreas. Nilotinib was given as an up-front treatment in some instances or as a second-line treatment in others. Therefore, the impact of these differences, if any, on the occurrence of secondary neoplasms remains uncertain.

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Table 2. Reported secondary neoplasms associated with nilotinib therapy.

Reference

Neoplasm

Global phase III

Papilloma of the skin: 3

multicenter trials

Fibroma molle: 2

It was also reported that an increase in the incidence of the following neoplastic changes was noted in a rat 2-year carcinogenicity study of imatinib: adenoma/adenocarcinoma of the kidney, urinary tract papilloma, adenocarcinoma of the small intestine, adenoma of the parathyroid, adrenal benign and malignant medullary tumors, papilloma/squamous cell carcinoma of the forestomach, papilloma of the clitoral gland, squamous cell carcinoma of the preputial gland, and papilloma of the preputial gland [15]. In a 2-year carcinogenicity study of dasatinib in rats, the incidence of papilloma and squamous cell carcinoma of the uterus and adenoma and adenocarcinoma of the prostate was observed to be increased at dose levels equal to or even lower than a clinical exposure level [16]. Thus, the potential risk of secondary malignancies cannot be ruled out for nilotinib, although there are no animal experimental data available at present. Verification by animal experiment would be needed. Inasmuch as nilotinib and dasatinib have greater immunosuppressive potency than imatinib, there has been a report pointing out the risk of SNs; therefore, long-term follow-up of accumulated cases treated with nilotinib, as is the case with imatinib, is considered necessary [4,13].

Reference Number

Cholesteatoma: 1

Gastric cancer: 1 Papilloma of the oral cavity: 1 Thyroid neoplasm: 1 Japanese phase II

Phagocytic histiocytosis: 1

study

Seborrheic keratosis: 1

Overseas phase II

Papilloma of the skin: 2

study

Lipoma: 1

Thyroid neoplasm: 1 Duman et al.

Lung cancer: 1 Fibroma of the skin: 1

Verma et al.

Malignant tumor: 4

Postmarketing

Gastric cancer: 2

clinical use surveys

Cancer of the bladder: 1

in Japan

Esophageal cancer: 1

6 4

Pancreatitis occurring during nilotinib therapy was reported in 1.9% of patients, and this incidence rate is higher as compared to less than 1% for imatinib, while it is unknown for dasatinib [14,15,16]. It is assumed that nilotinib acts upon an unknown intracellular pathway involved in calcium release from pancreatic acini, thereby regulating pancreatic exocrine

Ureteral cancer: 1 Neoplastic progression: 1 Present case

Cancer of the pancreas: 1

Table 3. Reports of pancreatitis, pancreatic enzyme elevation, and secondary malignancies associated with nilotinib therapy.

Global Phase III Multicenter Trials

Overseas Phase II Study

Japanese Phase Postmarketing II Study Clinical Use Surveys in Japan

Nilotinib prescribed for:

Newly diagnosed chronic phase of CML

Imatinib-refractory CML

Imatinib-refractory ImatinibCML refractory or newly diagnosed CML

Nationality

Non-Japanese

Japanese

Total no. of pts., n

556

458

n.a.

Pancreatitis, n (%)

6 (1.1%)

5 (1.1%)

2 (n.a.)

Acute pancreatitis, n (%)

4 (0.7%)

1 (2.9%)

6 (n.a.)

Lipase elevation, n (%)

46 (8.3%)

59 (12.9%)

8 (23.5%)

131 (n.a.)

Amylase elevation, n (%)

32 (5.8%)

22 (4.8%)

1 (2.9%)

28 (n.a.)

Secondary malignancies, n (%)

9 (1.6%)

4 (0.9%)

2 (5.9%)

7 (n.a.)

Reference number

CML: Chronic myeloid leukemia, n.a.: not available.

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secretion, and inhibits acinar opening/exocrine secretion by facilitating fatty acid deposition in pancreatic acinar cells, yet the underlying details remain to be clarified [19]. Thus, there is the possibility that nilotinib may be a risk factor for pancreatitis. Increased plasma lipase level was noted in 23.5% of patients treated in a Japanese phase II study, this incidence being particularly high as compared to 8.3% in global phase III multicenter trials and 12.9% in an overseas phase II trial (Table 3) [9,10,11]. There may be an interracial difference in this respect. Because chronic pancreatitis represents a risk factor for cancer of the pancreas, the possibility that nilotinib eventually may constitute a risk factor for pancreatic carcinoma and that Japanese race may also be a risk factor cannot be negated [17]. The patient reported herein had no history of pancreatitis. The present case is the first to be documented as carcinoma of the pancreas that developed following nilotinib therapy. Long-term outcome data analysis based on careful follow-up observation including periodic pancreatic enzyme checkups and by-race classification of study population is considered to be needed. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. References Figure 1. Clinical course: A diagnosis of chronic phase of chronic myeloid leukemia was made and the patient was begun on nilotinib at 400 mg/day. He obtained major molecular response. He began suffering from postprandial epigastric pain and a computed tomography scan revealed a tumor mass in the head of the pancreas. The mass was diagnosed as an adenocarcinoma. Nilotinib was discontinued and a pancreaticoduodenectomy was performed. Nilotinib at 400 mg/day was reinstituted, but this was switched to dasatinib at 100 mg/day when despondency appeared. The listless feeling then disappeared and the response is still major molecular response with no indication of pancreatic cancer recurrence.

Figure 2. Computed tomography scans of the abdomen: (a) A 24x20-mm tumor mass was noted in the head of the pancreas (red arrow). (b) The main pancreatic duct was dilated, but there was neither vascular abnormality nor lymph node swelling (red arrow).

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13. Weisberg E, Manley PW, Breitenstein W, Brüggen J, CowanJacob SW, Ray A, Huntly B, Fabbro D, Fendrich G, HallMeyers E, Kung AL, Mestan J, Daley GQ, Callahan L, Catley L, Cavazza C, Azam M, Neuberg D, Wright RD, Gilliland DG, Griffin JD. Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 2005;7:129-141. 14. Novartis Pharmaceuticals Japan [Internet]. Prescribing Information. Tokyo, Tasigna [updated April 2013; cited 1 July 2013]. Available from http://product.novartis.co.jp/tas/pi/pi_ tas_201304.pdf (in Japanese). 15. Novartis Pharmaceuticals Japan [Internet]. Prescribing Information. Tokyo, Glivec [updated October 2012; cited 1 July 2013]. Available from http://product.novartis.co.jp/gli/pi/ GLI1210.pdf (in Japanese). 16. Pharmaceutical and Medical Devices Agency [Internet]. Sprycel; Prescribing Information. Tokyo, Bristol-Myers Squibb [updated June 2013; cited 1 July 2013]. Available from http://www.info.pmda.go.jp/go/pack/4291020F1027_1_08/ (in Japanese). 17. Lowenfels AB, Maisonneuve P, Cavallini G, Ammann RW, Lankisch PG, Andersen JR, Dimagno EP, Andrén-Sandberg A, Domellöf L. Pancreatitis and the risk of pancreatic cancer. International Pancreatitis Study Group. N Engl J Med 1993;328:1433-1437. 18. Yagasaki F, Niwa T, Abe A, Ishikawa M, Kato C, Ogura K, Sasaki H, Kyo T, Jinnai I, Bessyo M, Miyamura K. Correlation of quantification of major bcr-abl mRNA between TMA (transcription mediated amplification) method and real-time quantitative PCR. Rinsho Ketsueki 2009;50:481-487. 19. Rosti G, Castagnetti F, Gugliotta G, Palandri F, Baccarani M. Physician’s guide to the clinical management of adverse events on nilotinib therapy for the treatment of CML. Cancer Treat Rev 2012;38:241-248.

Case Report

DOI: 10.4274/tjh.2014.0250

Hypertension and Life-Threatening Bleeding in Children with Relapsed Acute Myeloblastic Leukemia Treated with FLT3 Inhibitors FLT3 İnhibitörleri ile Tedavi Edilen Nüks Akut Miyeloblastik Lösemili Çocuklarda Hipertansiyon ve Hayatı Tehdit Eden Kanama Deniz Yılmaz Karapınar, Nihal Karadaş, Zühal Önder Siviş, Can Balkan, Kaan Kavaklı, Yeşim Aydınok Ege University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey

Abstract: Experiences with new multikinase inhibitors are limited, especially in children. In this report we summarize our experience with 2 patients with relapsed acute myeloblastic leukemia (AML), one with FMS-like tyrosine kinase3-internal tandem duplication mutation and the other with a single base mutation (D835Y). Both patients received sorafenib, one for 19 days and the other for 42 days, with clofarabine-including chemotherapy. One additionally received sunitinib for a total of 20 days. Both patients developed severe pancytopenia, hypertension, life-threatening bleedings from the gastrointestinal system, and, finally, intrapulmonary hemorrhage. Although both reached severe aplasia of the bone marrow without blastic infiltration, death occurred with neutropenic sepsis.

Key Words: Acute myeloblastic leukemia, FLT3, Sorafenib, Sunitinib, Children, D835Y mutation Özet: Multikinaz inhibitörleri ile deneyim, özellikle çocuklarda çok sınırlıdır. Burada, biri FMS-like tirozin kinaz 3-ITD ve diğeri tek baz mutasyonu (D835Y) taşıyan nüks akut miyeloblastik lösemi (AML) tanılı iki hastadan edindiğimiz deneyimi sunmak istedik. Her iki hasta sorafenib (biri 19, diğeri 42 gün olmak üzere) tedavisini klofarabin içeren yoğun kemoterapi protokolü ile birlikte aldı. D835Y mutasyonu bulunan hasta daha sonra 20 gün sunitinib ile tedaviye devam etti. Her iki hastada da ağır pansitopeni, hipertansiyon, yaşamı tehdit eden gastrointestinal sistem kanaması ve pulmoner kanama gelişti. Her iki hastanın kemik iliğinde ağır bir aplazi sağlanmış olsa da blastik infiltrasyon görülmeden nötropenik sepsisle hastalar kaybedildi.

Anahtar Sözcükler: Akut miyeloblastik lösemi, FLT3, Sorafenib, Sunitinib, Çocuk, D835Y mutasyonu

Address for Correspondence: Deniz YILMAZ KARAPINAR, M.D., Ege University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey Phone: +90 232 390 11 13 E-mail: dyilmazk@yahoo.com Received/Geliş tarihi : June 19, 2014 Accepted/Kabul tarihi : August 14, 2014

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Introduction The FMS-like tyrosine kinase-3 (FLT3) gene is mutated in approximately 30% of acute myeloblastic leukemia (AML) cases in adults and 10%-15% in children, especially those with normal karyotypes [1,2]. The most common mutation is internal tandem duplication (ITD). Single base mutations may also be seen, most commonly resulting in a substitution of aspartic acid with tyrosine or less commonly a histidine at residue 835 in the tyrosine kinase domain (D835). These mutations result in constitutive activation of the FLT3 receptor, hence downstream of some pathways [1,3,4]. They cause enhanced proliferation and reduced apoptosis of the myeloblasts, which contributes to leukemogenesis. Generally they are associated with leukocytosis, normal cytogenetics, lower remission and higher relapse rate, and worse survival [1,2,3,5,6]. Tyrosine kinase inhibitors (TKIs) work as competitive inhibitors of ATP, binding to its pocket in the kinase domain, and are used for targeted therapy to control tumor growth and angiogenesis [3,5,6]. Experiences with TKIs in children are restricted, showing some benefits with acceptable toxicities [7,8,9,10]. Here we report 2 pediatric relapsed AML patients treated with TKIs. Case Presentation Patient 1 was a 2-year-old girl diagnosed with AML after presenting with hyperleukocytosis and normal cytogenetics. She received chemotherapy according to the AML-BFM 2004 protocol and reached remission after the first course of induction therapy. No donor was available for stem cell transplantation and she developed relapse 5 months after remission was achieved. Combined chemotherapy including idarubicin (12 mg/m2/day for 3 days), fludarabine (30 mg/ m2/day for 4 days), and cytarabine (2 g/m2/day for 4 days) was administered. Bone marrow aspiration (BMA) on day 28 showed 90% blastic cell infiltration. In retrospective analyses of bone marrow samples, FLT3-D835Y was found to be positive at initial diagnosis. Although it disappeared after the first course of induction chemotherapy, it was found to be positive again at relapse and 28 days after IDA-FLA treatment. After getting permission from the Turkish Republic Ministry of Health and from the patient’s family, sorafenib (200 mg/ m2/day) was started as a salvage therapy. To prevent handfoot-skin toxicities, protective measures were taken, such as prophylactic extensive moisturizing by local emollients to the whole body, and the patient was advised to wear soft, thin, cotton gloves and socks on her hands and feet. Five days later combined chemotherapy including clofarabine (40 mg/m2/ day for 5 days) and cytarabine (1 g/m2/day for 5 days) was added to the sorafenib. One week later she had complaints of

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stomachache and developed hypertension. Although she had very severe pancytopenia, BMA at 28 days of chemotherapy did not show any decrease in the percentage of blastic cells. Therefore, at 32 days, sorafenib was switched to sunitinib (15 mg/m2/day). Three days after sunitinib treatment was started the hypertension increased and abdominal discomfort became prominent; several X-ray and ultrasonographic evaluations were performed and no obvious cause was identified. Electrolyte imbalance was seen (hypocalcaemia and hypokalemia; both required high amounts of intravenous replacement). She had gastrointestinal system (GIS) hemorrhage with normal prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen levels at day 7 of sunitinib treatment. It was managed with intensive support and the dose of sunitinib was lowered to 7.5 mg/m2/day. However, symptoms did not resolve and severe pancytopenia persisted with a white blood cell count below 100/mm3 and transfusion dependency. Severe hypertension was under control only after triple antihypertensive agents were added to the treatment. Very severe aplasia without blastic infiltration was seen in BMA at day 18 of sunitinib treatment. She again developed a severe upper GIS hemorrhage on day 19. Therefore, sunitinib was stopped at the 20th day. In spite of aggressive and prompt support with fresh frozen plasma and platelet and erythrocyte transfusions, she had a massive pulmonary hemorrhage, requiring intubation and mechanical ventilation. She expired with neutropenic sepsis 2 days after sunitinib was stopped. Informed consent was obtained. Patient 2 was a 12-year-old boy diagnosed with AML after presenting with hyperleukocytosis and normal cytogenetics. He received chemotherapy according to the AML-BFM 2004 protocol and achieved remission after the second course of induction therapy. He developed relapsed AML 4 months after first remission was achieved. IDA-FLA was administered. His BMA at day 28 showed 75% blastic cell infiltration. In retrospective analyses of bone marrow samples, FLT3-ITD was found to be positive at initial diagnosis. Although it disappeared after the second course of induction chemotherapy, it was found to be positive again at relapse. No hematological or molecular remission was seen after IDA-FLA treatment. On day 15, BMA showed severe aplasia. After the same formal permission procedure was followed and prophylactic measures were taken to prevent severe skin toxicities, sorafenib was started at a dose of 2x200 mg/m2/day on day 21 as salvage treatment. However, on day 28, repeated BMA showed 40% blastic cells. On day 29, 8 days after sorafenib was started, he developed hypertension and required antihypertensive therapy. Second-line relapse treatment with clofarabine (40 mg/m2/day for 5 days) and cytarabine (1 g/m2/day for 5 days) was started. He complained about severe muscle and bone

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pain and stomachache. Very severe pancytopenia occurred. BMA 15 days after second-line treatment was negative for FLT3-ITD mutation; he had then been receiving sorafenib for 42 days. After a total of 45 days of treatment with sorafenib he developed severe hypertension, abnormal renal function (urea: 176 mg/dL, creatinine: 5.9 mg/dL), severe metabolic acidosis, and intraocular and severe mouth bleeding. He was still in deep pancytopenia (WBC: 0.016x109/L, Hb: 49 g/L, Hct: 13% PLT: 17.7x109/L). Fibrinogen levels and PT and APTT were in the normal ranges. Sorafenib was ceased and intensive support with aggressive blood product supplementation was given. Because renal functions progressively worsened, renal replacement therapy with hemodialysis was started. He developed pulmonary hemorrhage; intensive support including platelet suspension, fresh frozen plasma, and recombinant factor VIIa was given. He developed neutropenic sepsis and died 12 days after cessation of sorafenib. Discussion and Review of the Literature FLT3-mutated AML patients respond poorly to conventional chemotherapy and have worse prognosis. Current studies put them into high-risk groups and they are candidates for hematopoietic stem cell transplantation. Sunitinib and sorafenib are multikinase inhibitors and were approved for treatment of renal cell and hepatocellular carcinoma. Some small case series and trials with sunitinib and especially with sorafenib alone or in combination with chemotherapy for treatment of refractory or relapsed AML for salvage therapy have been published [4,7,8,9,10,11,12,13,14]. Some of them showed that hematological remission may be achieved with acceptable toxicity profiles [4,9,10]. In this report we summarize our experience in 2 patients with relapsed AML, one with FLT3-ITD mutation and the other with D835Y. Both patients received idarubicin, fludarabine, and cytarabine combination therapy after hematological relapse occurred. Hematological remission could not be achieved. They were given TKIs with combined chemotherapy and reached severe aplasia with clearance of blastic cells. It was shown that sorafenib inhibits FLT3-ITD more potently than FLT3-D835Y, while sunitinib is equally effective against both mutant forms of FLT3 [13]. The patient with D835Y mutation thus received sunitinib since no response was seen after sorafenib. The most common toxicities of TKIs are reported as reversible skin rash, hand-foot-skin reaction, diarrhea, abdominal pain, mild myelosuppression, and electrolyte abnormalities [5,7,9,10]. Children with leukemia who received sorafenib in combination with other agents experienced grade 2 or 3 hand-foot-skin reactions very frequently, at rates of up to 100% [5,7,9,10]. In adults, one-third of patients required dose

reductions due to skin toxicity or diarrhea. Hypertension was infrequently reported in children, despite being very common in adults [7,9,10,11,12]. In these presented cases, most probably the prophylactic measures prevented skin toxicities, but the patients had very severe hypertension, which possibly contributed to the life-threatening bleedings. The effects of TKIs are related to active N-oxide metabolites [9]. These metabolites might also be responsible for the toxic effects. A much higher rate of conversion of TKIs to active metabolites was shown in children than in adults by pharmacokinetic studies [9,10]. Thus, higher exposure to the metabolites may contribute to increased toxicity among children. In our patients, persistent very severe pancytopenia did not allow us to assess remission status. Both of the patients were transfusion-dependent and intensively supported. Both patients died with neutropenic sepsis after major pulmonary hemorrhage. The complications observed in our patients suggest that efficacy of TKIs may be limited by their toxicities. In particular, myelosuppression, severe hypertension, and bleeding problems had very significant effects on the continuation of therapy and resulted in death. Experiences with new multikinase inhibitors such as sorafenib or sunitinib are limited, especially in childhood AML. The requirement for prospective randomized studies to determine the safety and efficacy of these drugs is clear. 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. Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, Sonoda Y, Fujimoto T, Misawa S. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 1996;10:1911-1918. 2. Creutzig U, van den Heuvel-Eibrink MM, Gibson B, Dworzak MN, Adachi S, de Bont E, Harbott J, Hasle H, Johnston D, Kinoshita A, Lehrnbecher T, Leverger G, Mejstrikova E, Meshinchi S, Pession A, Raimondi SC, Sung L, Stary J, Zwaan CM, Kaspers GJ, Reinhardt D; AML Committee of the International BFM Study Group. Diagnosis and management of acute myeloid leukemia in children and adolescents: recommendations from an international expert panel. Blood 2012;120:3187-3205. 3. Hatzimichael E, Georgiou G, Benetatos L, Briasoulis E. Gene mutations and molecularly targeted therapies in acute myeloid leukemia. Am J Blood Res 2013;3:29-51. 265

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4. Fiedler W, Serve H, Döhner H, Schwittay M, Ottmann OG, O’Farrell AM, Bello CL, Allred R, Manning WC, Cherrington JM, Louie SG, Hong W, Brega NM, Massimini G, Scigalla P, Berdel WE, Hossfeld DK. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 2005;105:986-993. 5. Moore AS, Kearns PR, Knapper S, Pearson AD, Zwaan CM. Novel therapies for children with acute myeloid leukemia. Leukemia 2013;27:1451-1460. 6. Fathi AT, Chen YB. Treatment of FLT3-ITD acute myeloid leukemia. Am J Blood Res 2011;1:175-189. 7. Watt TC, Cooper T. Sorafenib as treatment for relapsed or refractory pediatric acute myelogenous leukemia. Pediatr Blood Cancer 2012;59:756-757. 8. Baker SD, Zimmerman EI, Wang YD, Orwick S, Zatechka DS, Buaboonnam J, Neale GA, Olsen SR, Enemark EJ, Shurtleff S, Rubnitz JE, Mullighan CG, Inaba H. Emergence of polyclonal FLT3 tyrosine kinase domain mutations during sequential therapy with sorafenib and sunitinib in FLT3-ITD-positive acute myeloid leukemia. Clin Cancer Res 2013;19:57585768. 9. Inaba H, Rubnitz JE, Coustan-Smith E, Li L, Furmanski BD, Mascara GP, Heym KM, Christensen R, Onciu M, Shurtleff SA, Pounds SB, Pui CH, Ribeiro RC, Campana D, Baker SD. Phase I pharmacokinetic and pharmacodynamic study of the multikinase inhibitor sorafenib in combination with clofarabine and cytarabine in pediatric relapsed/refractory leukemia. J Clin Oncol 2011;29:3293-3300.

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10. Widemann BC, Kim A, Fox E, Baruchel S, Adamson PC, Ingle AM, Glade Bender J, Burke M, Weigel B, Stempak D, Balis FM, Blaney SM. A phase I trial and pharmacokinetic study of sorafenib in children with refractory solid tumors or leukemias: a Children’s Oncology Group Phase I consortium report. Clin Cancer 2012;18:6011-6022. 11. Metzelder SK, Schroeder T, Finck A, Scholl S, Fey M, Götze K, Linn YC, Kröger M, Reiter A, Salih HR, Heinicke T, Stuhlmann R, Müller L, Giagounidis A, Meyer RG, Brugger W, Vöhringer M, Dreger P, Mori M, Basara N, Schäfer-Eckart K, Schultheis B, Baldus C, Neubauer A, Burchert A. High activity of sorafenib in FLT3-ITD-positive acute myeloid leukemia synergizes with allo-immune effects to induce sustained responses. Leukemia 2012;26:2353-2359. 12. Sharma M, Ravandi F, Bayraktar UD, Chiattone A, Bashir Q, Giralt S, Chen J, Qazilbash M, Kebriaei P, Konopleva M, Andreeff M, Cortes J, McCue D, Kantarjian H, Champlin RE, de Lima M. Treatment of FLT3-ITD-positive acute myeloid leukemia relapsing after allogeneic stem cell transplantation with sorafenib. Biol Blood Marrow Transplant 2011;11:1874-1877. 13. Kancha RK, Grundler R, Peschel C, Duyster J. Sensitivity toward sorafenib and sunitinib varies between different activating and drug-resistant FLT3-ITD mutations. Exp Hematol 2007;35:1522-1526. 14. Man CH, Fung TK, Ho C, Han HH, Chow HC, Ma AC, Choi WW, Lok S, Cheung AM, Eaves C, Kwong YL, Leung AY. Sorafenib treatment of FLT3-ITD+ acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation. Blood 2012;119:5133-5143.

Case Report

DOI: 10.4274/tjh.2013.0428

Portal Vein Thrombosis of a Newborn with Corrected Total Anomalous Pulmonary Venous Return Düzeltilmiş Total Pulmoner Venöz Dönüş Anomalisi Olan Bir Yenidoğanda Portal Ven Trombozu Ufuk Çakır1, Dilek Kahvecioğlu1, Serdar Alan1, Ömer Erdeve2, Begüm Atasay2, Tayfun Uçar3, Saadet Arsan2, Hasan Çakmaklı1, Mehmet Ertem4, Semra Atalay3 1Ankara

University Faculty of Medicine, Department of Pediatrics, Ankara, Turkey University Faculty of Medicine, Department of Pediatrics, Division of Neonatology, Ankara, Turkey 3Ankara University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Cardiology, Ankara, Turkey 4Ankara University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey 2Ankara

Abstract: Total anomalous pulmonary venous return (TAPVR) is a rare and frequently isolated defect identified in 1% to 3% of all congenital heart diseases. To the best of our knowledge, portal vein thrombosis (PVT) associated with TAPVR has not been reported in the literature. We report a successfully managed PVT in a newborn with infracardiac-type TAPVR and review the literature. Anticoagulation therapies were used during the neonatal period to prevent thrombus progression. PVT should be kept in mind in TAPVR patients who have open heart repair with total correction. The treatment in each neonate should be individualized with consideration of the risk/benefit ratio.

Key Words: Total anomalous pulmonary venous return, Portal vein thrombosis, Anticoagulation therapy, Lowmolecular-weight heparin

Özet: Total anormal pulmoner venöz dönüş (TAPVD) anomalisi nadir ve çoğunlukla izole bir durumdur, bütün konjenital kalp hastalıklarının %1-3’ünü oluşturur. Bildiğimiz kadarıyla, TAPVD anomalisi ile ilişkili portal ven trombozu (PVT) literatürde daha önce yayınlanmamıştır. Burada başarı ile tedavi edilen infrakardiak tipte TAPVD anomalisine eşlik eden PVT’li bir yenidoğan olgusu literatür bilgileri ışığında sunulmaktadır. Tromboz progresyonunu engellemek için yenidoğan dönemi boyunca antikoagülan tedaviler uygulanmıştır. PVT, TAPVD anomalisi olan ve tam düzeltici açık kalp onarımı yapılan hastalarda akılda tutulmalıdır. Her bir yenidoğan için tedavi, risk ve yarar göz önünde tutularak bireyselleştirilmelidir.

Anahtar Sözcükler: Total pulmoner venöz dönüş anomalisi, Portal ven trombozu, Antikoagülan tedavi, Düşük moleküler ağırlıklı heparin

Address for Correspondence: Ufuk ÇAKIR, M.D., Ankara University Faculty of Medicine, Department of Pediatrics, Ankara, Turkey E-mail: drufukcakir@hotmail.com Received/Geliş tarihi : December 22, 2013 Accepted/Kabul tarihi : March 13, 2014

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Çakır U, et al: Total Anomalous Pulmonary Venous Return

Introduction Total anomalous pulmonary venous return (TAPVR) is an uncommon congenital cardiac anomaly with abnormal drainage of pulmonary venous blood into the systemic venous system. It may present as an isolated lesion or in combination with other cardiac defects. The clinical symptoms and signs are variable and depend on the associated cardiac anomaly. The prognosis of these patients is poor without surgical treatment. Prenatal diagnosis can offer more comprehensive perinatal management, reduce morbidity, and improve survival; however, TAPVR is rarely detected prenatally due to its low incidence and the difficulty of demonstrating the details of pulmonary venous anatomy with fetal echocardiography [1]. Postoperative systemic and pulmonary hypertension, pulmonary vein thrombosis, prolonged mechanical ventilation, heart failure, and mortality are reported complications after surgical correction of TAPVR in newborns [2,3,4]. To the best of our knowledge, portal vein thrombosis (PVT) associated with TAPVR has not been reported in the literature. We here report a successfully managed PVT in a newborn with infracardiac-type TAPVR and discuss the individualized management of this complication. Case Presentation A 38-week male term infant of 2810 g was born to a 36-year-old mother by cesarean section. He was brought to the pediatric emergency room with respiratory distress, cyanosis, and apnea on the 14th day of life and was admitted to the neonatal intensive care unit (NICU). Physical examination on admission was unrevealing, except for tachypnea and cyanosis. His hemoglobin and platelet count on admission were 12.1 g/ dL and 258x109/L, respectively. Normal chest X-ray and no response to hyperoxia testing pointed to a cardiac anomaly. Echocardiography revealed a dilated right atrium and ventricle and a small left atrium with infracardiac-type TAPVR (type 3), which was confirmed by computed tomography angiography. Informed consent was obtained. The patient underwent a total surgical correction of cardiac surgery with cardiopulmonary bypass on the 19th postnatal day. Cardiac pumping for 120 min and hypothermia for 56 min were performed during the operation. The patient received dopamine, dobutamine, and milrinone infusions with furosemide for 2 days during the postoperative period, and he was extubated on the postoperative 3rd day. On the postoperative 6th day, his physical examination revealed abdominal distension and dilatation of superficial abdominal veins. Diagnostic portal Doppler ultrasonography (USG) demonstrated a 20x14 mm thrombus in the main portal vein with 4 mm of flow at the narrowest side.

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Follow-up of the extension of thrombosis closely by Doppler USG under intravenous unfractionated heparin treatment was decided, with targeting of 50-80 s for activated partial thromboplastin time. The treatment decision was based on the location of the thrombus (main portal vein) and the probable risk of portal hypertension in the future. The choice of unfractionated heparin was made due to the recent postoperative period and the advantage of closer monitoring, in addition to easier management of complications such as bleeding. No extension or enlargement of the thrombus or any bleeding complications were observed in 2 consecutive days and the treatment was changed to subcutaneous lowmolecular-weight heparin (LMWH) at a dose of 1.7 mg/ kg twice a day [5]. Antifactor Xa level during the treatment ranged between 0.5 and 0.9 units/mL. Clinical symptoms such as abdominal distension and dilatation of superficial veins regressed in the following weeks and the patient was discharged on the postoperative 28th day. Protein C, protein S, and antithrombin III values were 59.4%, 94.9%, and 90.4%, respectively, during screening for thrombophilia. The patient was also evaluated for hereditary thrombophilic mutations and was found to be heterozygous for methylenetetrahydrofolate reductase mutation. His homocysteine level was 17.03 µmol/L and 14 µmol/L (normal range: 5-14 µmol/L) on the 28th and 44th days, respectively. Factor V Leiden and prothrombin 20210Aa mutations were not detected. LMWH was discontinued after the demonstration of complete resolution of the PVT by USG at the 8th week of treatment. For followup, he was evaluated at 6 months of age with no signs of PVT or portal hypertension. Discussion and Review of the Literature TAPVR is a rare and frequently isolated defect identified in 1% to 3% of all congenital heart diseases. In TAPVR systemic and pulmonary veins return to the right heart circulation, creating an obligate right-to-left atrial shunt to provide left ventricular preload [4]. Open heart repair with total correction, such as in our patient, is necessary in most cases. One of the most important complications of open heart repair with total correction is thrombosis, especially in pulmonary veins. Our patient developed PVT, which has not been reported previously. Neonatal PVT has been described as a rare event, but it is becoming more commonly recognized. Estimates range from 1 in 100,000 live births to 1 to 36 per 1000 NICU admissions [6]. The majority of cases remain unrecognized in the neonatal period and manifest later in childhood [5]. PVT is the major cause of extrahepatic portal hypertension and gastrointestinal bleeding in children, and the portal hypertension is most commonly the result of an organized thrombus in the portal vein [2].

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The etiology of PVT is different in neonates from that in children and adults. In adults, it is most frequently secondary to cirrhosis, whereas, in older children, PVT is related to liver transplantation, intraabdominal sepsis, splenectomy, sickle cell anemia, and antiphospholipid antibodies [5,7]. In addition, PVT secondary to pylephlebitis due to urinary tract infection was presented in an adult case [8]. PVT in neonates commonly occurs secondary to the placement of an umbilical vein catheterization (UVC) with or without infection. In a large retrospective review of 133 infants identified with PVT, an umbilical catheter was inserted in 97 (73%) and in half of them the catheter was misplaced at an intrahepatic location where venous flow rate was low. Our patient did not have a UVC, infection, or meaningful hereditary thrombophilia. We thought that congenital vascular malformation, hypothermia, and cardiac pump could be possible causes of the thrombosis in the presented case. Poor outcome was reported in 36 infants with intrahepatic lobe atrophy (30 cases) or portal hypertension (6 cases) [6]. In our case, early spontaneous resolution occurred. Scarce presenting clinical signs could be the reason why PVT is a relatively rare diagnosis in the NICU setting [6,9,10,11]. We think that the incidence of PVT in postoperative cardiac patients is not exactly known and we suggest that these patients should be followed for this possible complication. Although the clinical and laboratory signs are not prominent in neonates with PVT, dilation of abdominal superficial vessels in addition to abdominal distension may be an alarming physical finding. Thrombocytopenia can be seen at the time of diagnosis in PVT cases, but it is not specific for PVT. Consumption of platelets secondary to sepsis and disseminated intravascular coagulation may explain the low platelet count in the acute stage. In the late chronic stage, a low platelet count may be most likely secondary to hypersplenism with portal hypertension [5]. In a series by Morag et al., thrombocytopenia was reported in 26/133 infants (19.5%); in 13 of these infants, it was secondary to other conditions, including necrotizing enterocolitis or sepsis [6]. There can be mild liver dysfunction in children with PVT. The extent to which similar abnormalities occur in neonates is less clear. In a series of neonatal PVT cases, 9 out of 133 (7%) neonates had abnormal liver enzymes as an indication for ultrasound, which subsequently identified the presence of PVT [6]. Our patient did not demonstrate thrombocytopenia or elevated liver enzymes at the time of diagnosis or during his hospital stay. Prognosis of PVT is associated with ultrasound grading and grade 3 PVT may lead to lobe atrophy and portal hypertension. Although there are insufficient data to make strong recommendations regarding anticoagulation therapy

for neonatal PVT, the options include conventional anticoagulation therapy, short-term anticoagulation therapy, or close monitoring of the thrombus with objective tests and the use of anticoagulation therapy if thrombus extension occurs [5]. The treatment in each neonate should be individualized with consideration of the risk/benefit ratio [5]. We preferred anticoagulation therapy with unfractionated heparin and followed the patient closely for a possible extension during the first 2 days. After the second USG, which did not demonstrate any extension, we changed the treatment to LMWH for the ease of monitoring. Thrombolytic therapy is usually reserved for major vessel occlusion such as inferior vena cava, renal veins, or right atrium with organ compromise, and it is contraindicated in patients who underwent general surgery within the last 10 days [5]. In conclusion, PVT should be kept in mind in TAPVR cases with open heart repair with total correction. The treatment in each neonate should be individualized with consideration of the risk/benefit ratio. Patients should be followed closely and thrombolytic therapy should be reserved for major vessel occlusion with organ compromise. Anticoagulation therapy with unfractionated heparin and LMWH may be used during the neonatal period to prevent future complications. Those patients with PVT should be followed for the risk of hypertension. 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. Chen YY, Hsu CY. Prenatal diagnosis and antenatal history of total anomalous pulmonary venous return. Taiwan J Obstet Gynecol 2006;45:283-285. 2. Kohli V, Sachdev MS, Joshi RK, Joshi R. Post-op systemic hypertensive crises in an infant with total anomalous pulmonary venous connection. Indian J Pediatr 2009;76:755756. 3. Jang SI, Song JY, Kim SJ, Choi EY, Shim WS, Lee C, Lim HG, Lee CH. The recent surgical result of total anomalous pulmonary venous return. Korean Circ J 2010;40:31-35. 4. Frommelt PC, Sheridan DC, Deatsman S, Yan K, Simpson P, Frommelt MA, Litwin SB, Tweddell JS. Unobstructive total anomalous pulmonary venous return: impact of early elective repair on the need for prolonged mechanical ventilatory support. Pediatr Cardiol 2010;31:1191-1197. 5. Williams S, Chan AK. Neonatal portal vein thrombosis: diagnosis and management. Semin Fetal Neonatal Med 2011;16:329-339.

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Çakır U, et al: Total Anomalous Pulmonary Venous Return

6. Morag I, Epelman M, Daneman A, Moineddin R, Parvez B, Shechter T, Hellmann J. Portal vein thrombosis in the neonate: risk factors, course, and outcome. J Pediatr 2006;148:735739.

9. Alvarez F, Bernard O, Brunelle F, Hadchouel P, Odièvre M, Alagille D. Portal obstruction in children. I. Clinical investigation and hemorrhage risk. J Pediatr 1983;103:696702.

7. Erkan O, Bozdayi AM, Disibeyaz S, Oguz D, Ozcan M, Bahar K, Karayalcin S, Ozden A, Bozkaya H, Yurdaydin C, Uzunalimoglu O. Thrombophilic gene mutations in cirrhotic patients with portal vein thrombosis. Eur J Gastroenterol Hepatol 2005;3:339-343.

10. Kim JH, Lee YS, Kim SH, Lee SK, Lim MK, Kim HS. Does umbilical vein catheterization lead to portal venous thrombosis? Prospective US evaluation in 100 neonates. Radiology 2001;219:645-650.

8. Uz E, Özlem A, Şahın Balçık Ö, Kanbay M, Işık A, Uz B, Kaya A, Koşar A. Portal vein thrombosis secondary to Klebsiella oxytoca bacteriemia. Turk J Hematol 2010;27:213-215.

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11. Yadav S, Dutta AK, Sarin SK. Do umbilical vein catheterization and sepsis lead to portal vein thrombosis? A prospective, clinical, and sonographic evaluation. J Pediatr Gastroenterol Nutr 1993;17:392-396.

Case Report

DOI: 10.4274/tjh.2013.0149

Leuconostoc sp. Meningitis in a Patient Treated with Rituximab for Mantle Cell Lymphoma Mantle Hücreli Lenfoma Tanısı ile Rituksimab Kullanan Bir Hastada Leuconostoc Menenjiti Hrvoje Holik1, Božena Coha1, Marijan Šiško2, Maja Tomić-Paradžik3

Hospital Dr. Josip Benčević, Clinic of Internal Medicine, Slavonski Brod, Croatia Hospital Dr. Josip Benčević, Clinic of Infectious Diseases, Slavonski Brod, Croatia   3Public Health Institute of the Brod-Posavina County, Department of Microbiology, Slavonski Brod, Croatia   1General 2General

Abstract: We present a 64-year-old man who was treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemoimmunotherapy for mantle cell lymphoma and developed purulent meningitis, probably caused by Leuconostoc sp. The patient had severe hypogammaglobulinemia, which is a possible complication of rituximab therapy. To our knowledge and after reviewing the available medical literature, this is the first described case of purulent meningitis caused by Leuconostoc sp. in a patient with mantle cell lymphoma that appeared after treatment with the R-CHOP protocol. The diagnosis of purulent meningitis was based on clinical, laboratory and cytological cerebrospinal fluid findings, in addition to blood culture results in which we isolated Leuconostoc sp. The patient was treated with meropenem with full recovery.

Key Words: Rituximab, Leuconostoc, Purulent meninigitis, Mantle cell lymphoma, R-CHOP Özet: Burada mantle hücreli lenfoma tanısı ile R-CHOP (rituksimab, siklofosfamid, doksorubisin, vinkristin, prednizolon) kemoimmünoterapisi ile tedavi edilen ve muhtemelen Leuconostoc cinsi etkene bağlı pürülan menenjit gelişen 64 yaşında bir erkek hastayı sunuyoruz. Hastanın rituksimab tedavisinin olası komplikasyonlarından biri olan ağır hipogammaglobulinemisi bulunmakta olup, bildiğimiz kadarı ile ve mevcut tıbbi literatürün taranması sonrasında, olgumuz R-CHOP protokolü ile tedavi sonrası Leuconostoc cinsi etkene bağlı pürülan menejit gelişen mantle hücreli lenfoma tanılı ilk hastadır. Pürülan menenjit tanısı klinik bulgular, laboratuvar ve beyin-omurilik sıvısının sitolojik bulguları ve Leuconostoc izole ettiğimiz kan kültürünü temel alıyordu. Hasta meropenem tedavisi ile tamamen iyileşti.

Anahtar Sözcükler: Rituksimab, Leuconostoc, Pürülan menenjit, Mantle hücreli lenfoma, R-CHOP

Address for Correspondence: Hrvoje HOLIK, M.D., General Hospital Dr. Josip Benčević, Clinic of Internal Medicine, Slavonski Brod, Croatia Phone: 0038535201688 E-mail: hholik@gmail.com Received/Geliş tarihi : April 28, 2013 Accepted/Kabul tarihi : November 18, 2013

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Introduction Rituximab is a chimeric monoclonal antibody that binds to the CD20 cell surface marker on B lymphocytes. It was approved in the United States in 1997 by the Food and Drug Administration and now is used in 3 major fields: hematological oncology, prevention of transplant rejection, and some autoimmune diseases [1]. After 15 years of global use, due to expansion of its indications and duration of therapy, we begin to see side effects of rituximab therapy that were not previously known [1]. The genus Leuconostoc comprises microorganisms previously classified as Lancefield group N streptococci. They are facultative anaerobic, catalase-negative, grampositive, vancomycin-resistant organisms of cocci form [2]. In nature they are often found on the surfaces of plants, vegetables, and dairy products. They are important bacteria in todayâ&#x20AC;&#x2122;s industry, especially in food fermentation [3]. Leuconostoc sp. mainly cause opportunistic infections in immunocompromised patients [3]. Case Presentation A 64-year-old man was admitted to our hospital in March 2011 with generalized lymphadenopathy and hepatosplenomegaly. Based on the clinical and histopathological findings of a resected neck lymph node and epipharyngeal tumor, we diagnosed mantle cell lymphoma stage IV B. The patient was treated with the R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone) protocol and received 6 cycles of chemoimmunotherapy in 3-week intervals with very good response. He achieved complete remission. Serum protein electrophoresis performed at diagnosis on 25 March 2011 revealed the following: albumin: 35.58 g/L, alpha 1: 2.94 g/L, alpha 2: 6.3 g/L, beta: 7.74 g/L, gamma: 7.44 g/L. Forty days after the last application of chemoimmunotherapy, on 23 September 2011, he was admitted to the department of infectious diseases of our hospital. At admission he was in a poor condition with severe headache, fever of up to 38.8 Â°C, and double vision. Upon clinical examination we found that he was conscious and dehydrated, with neck stiffness. Based on these clinical findings, cerebrospinal fluid findings (cells: 3408/3x106/L, erythrocytes: 796x106/L, glucose: 0.9 mmol/L, Cl: 99 mmol/L, protein: 1.04 g/L, 0.87 Sayk neutrophil granulocytes, lymphocytes: 0.10, monocyte-macrophageal cells: 0.03), and the blood culture findings in which Leuconostoc sp. was isolated, he was diagnosed to have purulent meningitis. Parameningeal focus such as sinusitis, mastoiditis, otitis media, brain abscess, or spinal epidural abscess was ruled out. The patient lived in the countryside with constant contact with dairy products and other 272

potential Leuconostoc habitats, so he had the opportunity to be infected with Leuconostoc. In the blood sample results at admission there were 10.1x109/L leukocytes with 83% neutrophils, the C-reactive protein level was 131 mg/L, and the lactate dehydrogenase level was 117 U/L. Electrophoresis of serum proteins showed severe hypogammaglobulinemia (30/09/2011: albumin: 34.95 g/L, alpha 1: 1.15 g/L, alpha 2: 2.1 g/L, beta: 8.4 g/L, gamma: 3.4 g/L). Sensitivity of the isolated pathogen as tested by the Kirby-Bauer method of disk diffusion and the minimum inhibitory concentration (MIC) value for each antibiotic was determined by the E-test method. The pathogen was sensitive to all betalactam antibiotics (penicillin MIC of 0.064 mg/L), imipenem (MIC of 0.50 mg/L), macrolides, lincosamides (clindamycin MIC of 0.125 mg/L), and quinolones but was resistant to vancomycin (MIC >256 mg/L). The patient was initially treated with ceftriaxone (2x2 g intravenous), then with ampicillin (6x2 g intravenous) and gentamicin (1x240 mg intravenous), and finally with meropenem (3x2 g intravenous), according to the blood culture findings. With that therapy, his general condition improved with normalization of laboratory parameters of inflammation and he no longer had fever. Control electrophoresis of serum proteins performed before discharge showed an increase in gamma-globulin levels (serum protein electrophoresis, 14/10/2011: albumin: 46.53 g/L, alpha 1: 0.96 g/L, alpha 2: 3.84 g/L, beta: 7.04 g/L, gamma: 5.63 g/L). At the time when patient was treated our biochemistry laboratory could not performed fractions of gamma globulins, so we just have total value. The patient was discharged in good condition with full recovery. Discussion and Review of the Literature To our knowledge and after reviewing the available medical literature, we conclude that this is the first described case of purulent meningitis that was probably caused by Leuconostoc sp. in a patient with mantle cell lymphoma. Meningitis appeared after treatment with the R-CHOP protocol. For certain diagnosis of purulent meningitis caused by Leuconostoc, we lack cerebrospinal fluid culture with isolated Leuconostoc. Antibiotics having been administered before the lumbar puncture could be the reason why the cerebrospinal fluid culture was negative. The factors in favor for infection with Leuconostoc were isolation of Leuconostoc from blood culture, the patient being immunocompromised, and good and quick response to the prescribed antibiotic therapy with full recovery. In the literature, most descriptions of infections with Leuconostoc are in neonatal and other pediatric patients [4,5,6,7,8,9]. Infections with Leuconostoc in adults are rare. Leuconostoc can cause endocarditis, urinary tract infections, meningitis, intraabdominal infections, bacteremia, and septicemia.

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Central venous catheters and total parenteral nutrition are risk factors for infections with Leuconostoc, while newer findings showed that previous antibiotic treatment with vancomycin is not a risk factor for Leuconostoc infections [3,7,10]. In most cases, Leuconostoc showed good sensitivity to carbapenems (imipenem, meropenem), gentamicin, tobramycin, chloramphenicol, and the more recent daptomycin [3]. However, there is also a report of Leuconostoc resistance to carbapenem antibiotics [11]. In the literature, bacterial or viral infections associated with the use of rituximab were described in patients who were receiving maintenance therapy [1,12,13]. In randomized trials in which rituximab was not administered as maintenance therapy, there was no increase in the incidence of infections in patients who were treated with the addition of rituximab to chemotherapy as compared to chemotherapy alone [1,14]. The most common infections described in the literature are reactivation of hepatitis B virus, progressive multifocal leukoencephalopathy caused with JC polyomavirus, enterovirus encephalitis, parvovirus B19 infection, Pneumocystis jirovecii pneumonia, babesiosis, infections with West Nile virus, and cytomegalovirus [1,15]. Some of the bacterial infections associated with the use of rituximab described in the literature are Staphylococcus warneri meningitis with Strongyloides stercoralis superinfection in a patient with mantle cell lymphoma and tracheobronchitis caused by Pasteurella multocida in a patient with chronic lymphocytic leukemia [16,17]. Both patients were treated with rituximab as maintenance therapy. In patients with non-Hodgkinâ&#x20AC;&#x2122;s lymphoma who were treated with rituximab as a maintenance therapy and who had developed different degrees of hypogammaglobulinemia, staphylococci and Escherichia coli were reported as causes of bacterial infections [13]. In another study, 20% of patients treated with rituximab and chemotherapy developed infections without neutropenia. In all cases in which electrophoresis of serum proteins was performed, different degrees of hypogammaglobulinemia were observed. These patients were successfully treated with intravenous gamma globulins [18]. Here we have presented a patient who developed severe hypogammaglobulinemia after only 6 cycles of rituximab, for whom infection occurred 40 days after the last cycle. Besides application of rituximab, the potential risk factors were underlying lymphoproliferative disease and immunosuppressive therapy usage. Although rituximab is a safe drug for use, further research is necessary about its use in certain groups of patients who might develop an opportunistic infection. Controlling the value of gamma globulins during rituximab therapy might reduce the number of infections by temporary interruption of

rituximab or by giving prophylactic intravenous gamma globulins in cases of severe hypogammaglobulinemia. Expansion of indications for rituximab use and increasing duration of treatment with rituximab will certainly bring about more opportunistic infections, such as those caused by Leuconostoc. 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. Gea-Banacloche JC. Rituximab-associated infections. Semin Hematol 2010;47:187-198. 2. Lehman DC, Mahon CR, Suvarna K. Streptococcus, Enterococcus and other catalase-negative gram-positive cocci. In: Mahon CR, Lehman DC, Manuselis G, (eds). Textbook of Diagnostic Microbiology, 3rd ed. St. Louis, Saunders Elsevier, 2007. 3. Ruoff KL. Leuconostoc, Pediococcus, Stomatococcus and miscellaneous gram-positive cocci that grow aerobically. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH (eds). Manual of Clinical Microbiology, 7th ed. Washington DC, American Society for Microbiology, 1999. 4. Janow G, Lambert B, Scheiner M, Rosen O, Goldman DL, Soghier L. Leuconostoc septicemia in a preterm neonate on vancomycin therapy: case report and literature review. Am J Perinatol 2009;26:89-91. 5. Yossuck P, Miller-Canfield P, Moffett K, Graeber J. Leuconostoc spp. sepsis in an extremely low birth weight infant: a case report and review of the literature. W V Med J 2009;105:24-27. 6. Handwerger S, Horowitz H, Coburn K, Kolokathis A, Wormser GP. Infection due to Leuconostoc species: six cases and review. Rev Infect Dis 1990;12:602-610. 7. Florescu D, Hill L, Sudan D, Iwen PC. Leuconostoc bacteremia in pediatric patients with short bowel syndrome: case series and review. Pediatr Infect Dis J 2008;27:10131019. 8. Friedland IR, Snipelisky M, Khoosal M. Meningitis in a neonate caused by Leuconostoc sp. J Clin Microbiol 1990;28:2125-2126. 9. Coovadia YM, Solwa Z, van den Ende J. Meningitis caused by vancomycin-resistant Leuconostoc sp. J Clin Microbiol 1987;25:1784-1785. 10. Starr JA. Leuconostoc species-associated endocarditis. Pharmacotherapy 2007;27:766-770. 11. Deye G, Lewis J, Patterson J, Jorgensen J. A case of Leuconostoc ventriculitis with resistance to carbapenem antibiotics. Clin Infect Dis 2003;37:869-870. 273

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12. Aksoy S, Dizdar O, Hayran M, Harputluoฤ lu H. Infectious complications of rituximab in patients with lymphoma during maintenance therapy: a systematic review and metaanalysis. Leuk Lymphoma 2009;50:357-365. 13. Azad A, Campbell P. High rates of infection associated with the use of maintenance rituximab monotherapy in nonHodgkin lymphoma. Intern Med J 2009;39:778-779. 14. Rafailidis PI, Kakisi OK, Vardakas K, Falagas ME. Infectious complications of monoclonal antibodies used in cancer therapy: a systematic review of the evidence from randomized controlled trials. Cancer 2007;109:2182-2189. 15. Ganjoo KN, Raman R, Sobel RA, Pinto HA. Opportunistic enteroviral meningoencephalitis: an unusual treatable complication of rituximab therapy. Leuk Lymphoma 2009;50:673-675.

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16. Incani RN, Hernรกndez M, Cortez J, Gonzรกlez ME, Salazar YD. Staphylococcus warneri meningitis in a patient with Strongyloides stercoralis hyperinfection and lymphoma: first report of a case. Rev Inst Med Trop Sao Paulo 2010;52:169170. 17. Deming D, Silverman E. Pasteurella multocida tracheobronchitis in a patient with CLL on rituximab. Am J Hematol 2010;85:144-145. 18. Cabanillas F, Liboy I, Pavia O, Rivera E. High incidence of non-neutropenic infections induced by rituximab plus fludarabine and associated with hypogammaglobulinemia: a frequently unrecognized and easily treatable complication. Ann Oncol 2006;17:1424-1427.

Letters to the Editor Thrombotic Microangiopathy with Complement Factor H Gene Mutations Unassociated with Atypical Hemolytic Uremic Syndrome Atipik Hemolitik Üremik Sendrom ile İlişkili Olmayan Kompleman Faktör H Gen Mutasyonları Gösteren Trombotik Mikroanjiopati To the Editor, Atypical hemolytic uremic syndrome (aHUS) is a rare multigenic disorder characterized by thrombotic microangiopathy (TMA). Among the genes that are associated with aHUS, mutations in the complement factor H (CFH) gene are the most common genetic cause of the disease. Several specific gene mutations have been identified in patients with aHUS [1,2,3]. A 5-year-old boy was admitted to our hospital with fatigue. Physical examination revealed pallor and hepatomegaly. His blood pressure was within the normal range. A blood smear showed hemolysis with 10% schistocytes and polychromasia. He had anemia with 5.5 g/ dL hemoglobin level and 14.6% reticulocyte level. He had also thrombocytopenia (48,000/mm3) and elevated lactate dehydrogenase (LDH) as 1066 U/L. A direct Coombs test was negative. The other blood parameters of the patient were as follows: haptoglobin 5 mg/dL (reference range: 41-165 mg/dL), C3 122 mg/dL (reference range: 79-152 mg/dL), creatinine level 0.5 mg/dL, blood urea nitrogen 12 mg/dL, and indirect bilirubin 7 mg/dL. Urine analysis was normal. On the second day of admission, the patient’s thrombocyte count dropped to 38,000/mm3, his LDH level remained elevated, and schistocytes were still present on his peripheral blood smear. As for the blood smear and other TMA symptoms, first plasmapheresis was started, which lasted 20 weeks. Then treatment was continued with 600 mg of eculizumab weekly for the first three weeks and followed by once every 2 weeks (total 11 doses). Although the patient had TMA symptoms he didn’t have renal insufficiency. Also ADAMTS-13 activity was 48% (reference range: 40%-130%) and ADAMTS-13 antibody was negative.

p.Ala473Ala mutation due to a p.1419G>A change in exon 10 of the CFH gene (Figure 1). CFH gene mutations are the most commonly observed genetic changes in patients with aHUS and they are responsible in 20%-30% of the patients [1,2,3]. Eighty-seven CFH gene mutations associated with aHUS have been described to date [4,5]. The CFH gene mutation that we identified in our case has been associated with membranoproliferative glomerulonephritis (MPGN) and age-related macular degeneration (AMD) [6,7,8]. The location of the p.His402Tyr mutation in the functional domain of the protein suggests that it might have a pathogenic effect in patients with aHUS [5].

Four months after stopping eculizumab, the patient’s levels of hemoglobin, thrombocytes, reticulocytes, haptoglobin and LDH were 11 g/dL, 150,000/mm3, 0.87%, 46.4 mg/dL (reference range: 41-165 mg/dL) and 385 U/L, respectively. Informed consent was obtained for genetic testing and publishing the patient’s data from his parents. DNA sequencing analysis of the patient revealed a homozygous p.His402Tyr mutation due to a p.1204 C>T change in exon 9, a homozygous p.Ala307Ala mutation due to a p921A>C change in exon 7 and a heterozygous

Figure 1. Sequencing data of complement factor H gene mutations of patient. A) Homozygous p.His402Tyr mutation. B) Homozygous p.Ala307Ala mutation. C) Heterozygous p.Ala473Ala mutation.

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In conclusion CFH gene analysis was performed to confirm whether the patient had aHUS or not. However, we have found CFH gene mutations that are not specific for aHUS. Epigenetic factors might have triggered the patient’s phenotype. Also p.His402Tyr mutation may cause TMA with a milder clinic feature than that of other aHUS specific CFH gene mutations. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affliations relevant to the subject matter or materials included. Key Words: Thrombotic microangiopathy, Eculizumab, aHUS, CFH gene Anahtar Sözcükler: Trombotik mikroanjiopati, Ekulizumab, aHUS, CFH geni Yeşim Oymak1, Tuba Hilkay Karapınar1, Yılmaz Ay1, Esin Özcan1, Neryal Müminoğlu1, Sultan Aydın Köker1, Ersin Töret1, Afig Berdeli2, Erkin Serdaroğlu3, Canan Vergin1 1Dr. Behçet Uz Children’s Hospital, Clinic of Hematology, İzmir, Turkey 2Ege University Faculty of Medicine, Department of Genetics, İzmir, Turkey 3Dr. Behçet Uz Children’s Hospital, Clinic of Nephrology, İzmir, Turkey

References 1. Caprioli J, Noris M, Brioschi S, Pianetti G, Castelletti F, Bettinaglio P, Mele C, Bresin E, Cassis L, Gamba S, Porrati F,Bucchioni S, Monteferrante G, Fang CJ, Liszewski MK, Kavanagh D, Atkinson JP, Remuzzi G; International Registry of Recurrent and Familial HUS/TTP. Genetics of HUS: the impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome. Blood 2006;108:1267-1279. 2. Caprioli J, Bettinaglio P, Zipfel PF, Amadei B, Daina E, Gamba S, Skerka C, Marziliano N, Remuzzi G, Noris M; Italian Registry of Familial and Recurrent HUS/TTP. The molecular basis of familial hemolytic uremic syndrome: mutation analysis of factor H gene reveals a hot spot in short consensus repeat 20. J Am Soc Nephrol 2001;12:297-307. 3. Noris M, Caprioli J, Bresin E, Mossali C, Pianetti G, Gamba S, Daina E, Fenili C, Castelletti F, Sorosina A, Piras R, Donadelli R, Maranta R, van der Meer I, Conway EM, Zipfel PF, Goodship TH, Remuzzi G. Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype. Clin J Am Soc Nephrol 2010; 5:1844-1859. 4. Rodriguez E, Rallapalli PM, Osborne AJ, Perkins SJ. New functional and structural insights from updated mutational databases for complement factor H, factor I, membrane cofactor protein and C3. Biosci Rep 2014;34:e00146. 5. Saunders RE, Abarrategui-Garrido C, Frémeaux-Bacchi V, Goicoechea de Jorge E, Goodship TH, López Trascasa M, 276

Noris M, Ponce Castro IM, Remuzzi G, Rodríguez de Córdoba S, Sánchez-Corral P, Skerka C, Zipfel PF, Perkins SJ. The interactive Factor H-atypical hemolytic uremic syndrome mutation database and website: update and integration of membrane cofactor protein and Factor I mutations with structural models. Hum Mutat 2007;28:222-234. 6. Abrera-Abeleda MA, Nishimura C, Smith JL, Sethi S, McRae JL, Murphy BF, Silvestri G, Skerka C, Józsi M, Zipfel PF, Hageman GS, Smith RJ. Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease). J Med Genet 2006;43:582-589. 7. Hageman GS, Anderson DH, Johnson LV, Hancox LS, Taiber AJ, Hardisty LI, Hageman JL, Stockman HA, Borchardt JD, Gehrs KM, Smith RJ, Silvestri G, Russell SR, Klaver CC, Barbazetto I, Chang S, Yannuzzi LA, Barile GR, Merriam JC, Smith RT, Olsh AK, Bergeron J, Zernant J, Merriam JE, Gold B, Dean M, Allikmets R. A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci USA 2005;102:7227-7232. 8. Haines JL, Hauser MA, Schmidt S, Scott WK, Olson LM, Gallins P, Spencer KL, Kwan SY, Noureddine M, Gilbert JR, Schnetz-Boutaud N, Agarwal A, Postel EA, Pericak-Vance MA. Complement factor H variant increases the risk of agerelated macular degeneration. Science 2005;308:419-421. Address for Correspondence: Yeşim OYMAK, M.D., Dr. Behçet Uz Children’s Hospital, Clinic of Hematology, İzmir, Turkey Phone: +90 532 355 42 28 E-mail: yesimoymak@hotmail.com Received/Geliş tarihi: February 17, 2015 Accepted/Kabul tarihi: March 18, 2015 DOI: 10.4274/tjh.2015.0084

Rapidly Growing Thyroid Mass: An Unusual Case of Acute Lymphoblastic Leukemia Hızlı Büyüyen Tiroid Kitlesi: Sıradışı Bir Akut Lenfoblastik Lösemi Olgusu To the Editor, Extramedullary presentations of acute lymphoblastic leukemia (ALL) in the central nervous system (CNS), lymph nodes, gonads, spleen, and liver can also be observed. Thyroid infiltration of ALL is very rare. A 53-year-old woman was admitted to the endocrinology outpatient clinic with a lump in the throat, which increased in size over a week. Her medical and family history were nonspecific. Except sensitive and painful thyroid, physical examination findings were normal. Complete blood count values were as follows:

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Hg 131 g/L, WBC 10,200x109/L, ANC 5490x109/L and platelets 188x109/L. Sedimentation and C-reactive protein (CRP) were higher than normal range. Thyroid hormone levels were within normal range. Antithyroid peroxidase was negative. Thyroid ultrasonography showed moderate enlargement of bilateral thyroid lobes, the parenchyma was hypoechoic and nonhomogeneous. Fine needle aspirates from the thyroid revealed a few lymphocyte and few polymorphonuclear leukocyte infiltration on a necrotic floor with acute inflammation. The neck MRI showed enlargement of the right lobe and a 5x4, 5x3.5 cm, T1W hypointense, T2W hyperintense nodular lesion in the right lobe that was contrasted homogeneously (Figure 1). She was diagnosed with subacute thyroiditis and treated with 1 mg/kg/day methylprednisolone. Three weeks later, she was admitted to hospital with fever, weakness, and enlarging painful thyroid lobes. Physical examination showed pallor, enlarged and painful thyroid lobes, and mild splenomegaly. Complete blood count revealed Hb of 88 g/L, WBC of 8170x109/L, ANC of 2100x109/L and a platelet count of 22x109/L, LDH: 3119. Peripheral blood smear test revealed increased blasts (52% of cells). Bone marrow biopsy showed 80% cellularity with a diffuse, uniform infiltration of lymphoblastic cells with prominent nucleoli. Immunohistochemical staining was positive for TdT, HLA-DR, CD19, CD20, CD22, CD10 and CD38. Chromosome analysis showed 46XX. The breakpoint cluster region-abelson gene (BCR-ABL) fusion was found to be negative. The patient received induction chemotherapy with Berlin-Frankfurt-Munich (BFM) protocol following the diagnosis of precursor-B cell-ALL. In a week pain and enlargement of the thyroid partially regressed, but pain on palpation persisted. On day eight, the second FNA was performed. Pathological results were consistent with leukemic infiltration in a background of very scant colloid. After remission induction therapy, bone marrow aspiration and biopsy showed a continuous rise in diffuse blast cells. During the salvage therapy the patient

died due to progresive disease. Subacute thyroiditis is a spontaneously remitting inflammatory condition of the thyroid gland. The thyroid gland is typically enlarged two or three times the normal size and is tender to palpation. When we evaluated the patient retrospectively, we thought that the complaints due to increased thyroid volume were related to leukemic infiltration of the thyroid. Her initially normal complete blood count evolved into pancytopenia. In the literature, there are ALL patients presenting with extramedullary infiltration signs and complete blood count within normal ranges [1,2]. To the best of our knowledge this is the fourth case of B ALL with extramedullary thyroid infiltration in the literature [3,4,5]. As highlighted by the present report, performing fine-needle aspiration cytology should always be considered in the clinical context of a rapidly growing thyroid mass under treatment, and without resolving symptoms. 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: B-cell neoplasms, Acute lymphoblastic leukemias, Acute leukemia Anahtar Sözcükler: B hücre neoplazileri, Akut lenfoblastik lösemi, Akut lösemi Füsun Özdemirkıran1, Gonca Oruk2, Bahriye Payzin1, Zeynep Gümüş3, Melike Koruyucu4,  Türkan Atasever1, Betül Küçükzeybek5, Özgür Esen6 1Katip Çelebi University Faculty of Medicine, Atatürk Training and Research

Hospital, Clinic of Hematology, İzmir, Turkey 2Katip Çelebi University Faculty of Medicine, Atatürk Training and Research

Hospital, Clinic of Endocrinology, İzmir, Turkey 3Katip Çelebi University Faculty of Medicine, Atatürk Training and Research

Hospital, Clinic of Internal Medicine, İzmir, Turkey 4Katip Çelebi University Faculty of Medicine, Atatürk Training and Research

Hospital, Clinic of Radiology, İzmir, Turkey 5Katip Çelebi University Faculty of Medicine, Atatürk Training and Research

Hospital, Clinic of Pathology, İzmir, Turkey 6Bozyaka Training and Research Hospital, Clinic of Radiology, İzmir, Turkey

References 1. Asdahl PH, Warner LF, Bendix K, Hasle H. Acute renal failure and normal blood count: A rare presentation of T-cell acute lymphoblastic leukemia. Leuk Res Rep 2013;3:14-16.

Figure 1. a, b, c: Neck MRI: Enlargement of the right lobe of the thyroid, 5x4.5x3.5 cm sized T1W hypointense, T2W hyperintense nodular lesion occupying right lobe with homogeneous contrast uptake.

2. Marwaha RK, Kulkarni KP, Bansal D, Trehan A. Acute lymphoblastic leukemia masquerading as juvenile rheumatoid arthritis: diagnostic pitfall and association with survival. Ann Hematol 2010;89:249-254. 3. Foresti V, Parisio E, Scolari N, Ungaro A, Villa A, Confalonieri F. Primary hypothyroidism due to leukemic infiltration of the thyroid gland. J Endocrinol Invest 1988;11:43-45. 277

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4. Sen R, Gupta S, Batra A, Gill M, Gupta V, Marwah N. Acute lymphoblastic leukaemia (ALL) with infiltration of the thyroid: a cytological diagnosis. Endocr Pathol 2012;23:268-269. 5. Valizadeh M, Moghimi M, Feizi A, Radmand F, Piri Z. Thyroid nodule in an eighteen-year-old man as the first presentation of acute lymphoblastic leukemia. Int J Endocrinol Metab 2014;12:e17364. Address for Correspondence: Füsun ÖZDEMİRKIRAN, M.D., Katip Çelebi University Faculty of Medicine, Atatürk Training and Research Hospital, Clinic of Hematology, İzmir, Turkey E-mail: fusun75@gmail.com Received/Geliş tarihi: March 27, 2015 Accepted/Kabul tarihi: April 13, 2015 DOI: 10.4274/tjh.2015.0136

Auer Rods in Chronic Myelomonocytic Leukemia Can Change the Diagnosis Kronik Miyelomonositik Lösemide Auer Çubukları Tanıyı Değiştirebilir To the Editor, Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with overlapping morphological features of myelodysplastic and myeloproliferative disease and a potential risk of transformation to acute myeloid leukemia. Presence of Auer rods in CMML is a rare finding and the presence of an occasional Auer rod gives the diagnosis of CMML-2 in spite of the presence of <5% blasts in peripheral blood/bone marrow [1,2]. A 39-year-old female, diagnosed outside our facility with Crohn’s disease, presented with severe anemia with weakness and fatigue for 1 month. The patient had been treated previously with prednisolone at 1 mg/kg/day. Diarrhea was resolved after 2 weeks of therapy. Steroid dose was reduced and stopped after 4 months. There were no bowel symptoms and the colonoscopy done at our institution was normal. Hemoglobin was 65 g/L, total leukocyte count was 16.4x109/L, and platelet count was 192x109/L. Peripheral blood smear showed 4% blasts and promonocytes, 3% myelocytes and metamyelocytes, 26% monocytes (including abnormal forms) (Figures 1A and 1B), and 2 nucleated red blood cells/100 white blood cells. Bone marrow aspirate was hypercellular with dyspoiesis in all 3 lineages with increased monocytic cells (Figures 1C and 1D). Erythroid series showed predominantly megaloblastoid erythropoiesis with nuclear budding, multinuclearity, and cytoplasmic vacuolation. Granulocytic series showed myeloid hyperplasia with 15%-20% monocytes, 2% basophils, and 4% blasts, with an occasional blast showing an Auer rod (Figure 1C, arrows). Micromegakaryocytes and megakaryocytes with abnormal lobation and multinucleation were seen. Bone marrow biopsy was hypercellular (100%) with grade 1 reticulin fibrosis. Megakaryocytes were increased in number and showed hypolobation and multinucleation. Conventional 278

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cytogenetics showed a normal female karyotype. There was no Philadelphia chromosome or BCR/ABL fusion gene. Overall features were compatible with a diagnosis of CMML type 2. She was started on 3+7 induction chemotherapy using daunorubicin and ara-C. Bone marrow aspirate done on day 28 confirmed morphological complete remission. She underwent HLA-identical allogeneic hematopoietic stem cell transplantation from her elder brother and currently (2 years posttransplant) continues to be disease-free. Auer rods are a hallmark of acute myeloid leukemia but are occasionally seen in myelodysplastic syndrome (refractory anemia with excess blasts type 2) or CMML cases, and rarely in patients with fewer than 5% blasts [3,4]. According to the World Health Organization 2008 diagnostic criteria, the presence of Auer rods fulfills the criteria for CMML-2 irrespective of the blast count [5]. Thus, in CMML, a thorough search for Auer rods should be done for a correct diagnosis as the treatment given for CMML-2 is different from that for CMML-1 and the risk of transformation to acute leukemia is greater. We also want to emphasize that the presence of Auer rods with fewer than 5% blasts is a rare phenomenon that seems to be clinically, morphologically, and cytogenetically heterogeneous, and it could be a valuable finding for early treatment options in patients with CMML-2 if there is a HLA-identical donor. 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: Auer rod, Chronic myelomonocytic leukemia, Hematopoietic stem cell transplantation Anahtar Sözcükler: Auer çubukları, Kronik miyelomonositik lösemi, Hematopoetik kök hücre transplantasyonu

Figure 1. A, B) Peripheral blood smear showing presence of abnormal monocytes along with myelocytes and blast (B- inset). C, D) Bone marrow aspirate showing dyspoiesis in all three lineages (C) with an occasional blast showing an Auer rod (C, arrows) with increased monocytic cells (D) (Jenner and Giemsa stain, 1000x).

Letters to the Editor

Turk J Hematol 2015;32:275-280

Smeeta Gajendra1, Ranjit Kumar Sahoo2 1Medanta-The Medicity, Department of Pathology and Laboratory Medicine,

Haryana, India 2All India Institute of Medical Sciences, Department of Medical Oncology, New Delhi, India

References 1. Patnaik MM, Parikh SA, Hanson CA, Tefferi A. Chronic myelomonocytic leukaemia: a concise clinical and pathophysiological review. Br J Haematol 2014;165:273-286. 2. Xu Z, Shier L. Auer rods in a patient with chronic myelomonocytic leukemia type 2. Blood 2014;124:3828. 3. Willis MS, McKenna RW, Peterson LC, Coad JE, Kroft SH. Low blast count myeloid disorders with Auer rods: a clinicopathologic analysis of 9 cases. Am J Clin Pathol 2005;124:191-198. 4. Germing U, Strupp C, Kuendgen A, Aivado M, Giagounidis A, Hildebrandt B, Aul C, Haas R, Gattermann N. Refractory anaemia with excess of blasts (RAEB): analysis of reclassification according to the WHO proposals. Br J Haematol 2006;132:162-167. 5. Orazi A, Bennett JM, Germing U, Brunning RD, Bain BJ, Thiele J. Chronic myelomonocytic leukemia. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW, (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France, IARC Press, 2008. Address for Correspondence: Smeeta GAJENDRA, M.D., Medanta-the Medicity, Department of Pathology and Laboratory Medicine, Haryana, India Phone: +90 09013590875 E-mail: drsmeeta@gmail.com Received/Geliş tarihi: April 7, 2015 Accepted/Kabul tarihi: April 16, 2015 DOI: 10.4274/tjh.2015.0148

A Case of Acquired Thrombotic Thrombocytopenic Purpura: Three Recurrences in 25 Years Kazanılmış Trombotik Trombositopenik Purpura Olgusu: 25 Yıllık Takipte Üç Rekürens To the Editor, A 60-year-old woman with fever, lassitude, neurological symptoms, hematuria, subconjunctival bleeding, several ecchymoses, reticulocytosis, microangiopathic hemolytic anemia (with burr cells, schistocytes, and spherocytes), and thrombocytopenia was first seen in the neurology clinic in 1985. Because of her normal cerebrospinal fluid and unremarkable MRI findings with some nonspecific EEG findings, thrombotic thrombocytopenic purpura (TTP) was diagnosed based on her clinical and microangiopathic hemolytic anemia findings, as reported previously. Her

Coombs’ test and kidney and liver function results were almost normal and her moribund condition was treated with megadose methylprednisolone (MDMP) [1,2,3]. Her platelet count became normal on the fourth day of treatment; she did not require any blood transfusions and all clinical and laboratory findings were improved. Thereafter, she was seen once a year. Informed consent was obtained. Fifteen years later, at the age of 75, her symptoms recurred with angiopathic findings without known infection, drug use, mild hematuria, or urinary system infection (E. coli above 100,000/mL). She was diagnosed with recurrence of TTP and was treated with antibiotics and oral MDMP (30 mg/kg for 3 days and then 20 mg/kg for 4 days; subsequently at doses of 10, 5, 2, and 1 mg/kg each for 1 week in duration), given once around 6 a.m. Her symptoms improved in terms of anemia and microangiopathic laboratory findings, including the thrombocytopenia, within a week without admission to the hospital. Her liver and kidney functions (urea: 30 mg/ dL, creatinine: less than 1.1 mg/dL) stayed normal. In 2007, 22 years after the first attack and at the age of 82, she had another episode of microangiopathic hemolytic anemia and thrombocytopenia without a known cause. She was being treated again with oral MDMP as of the year 2000. She responded in a 1-week period and stayed symptom-free for 3 years. Later it was learned that she had died at home at the age of 85 due to heart failure. Thrombotic microangiopathic syndromes, though rare, have diverse pathophysiological pathways that can lead to microangiopathic anemia, a procoagulant state with or without damage to the kidneys and other organs, and could be acquired or hereditary [4,5]. Hereditary conditions related to hereditary ADAMTS 13 deficiency, complement H factor (CFH), complement 3-3b, complement C factor 1 (CF 1 or CD 45), cobalamin C disease, and some of the coagulation disorders such as thrombomodulin, plasminogen, and protein kinase C deficiencies could not be considered in this patient because of her age (first attack at about 60 years of age). Among the acquired disorders, Shiga toxin-mediated hemolytic and uremic syndrome (HUS), HELLP syndrome, drug immune-mediated conditions, and pregnancy-related microangiopathic syndromes could easily be rejected because of her age and no known drug history. Although ADAMTS 13 deficiency related to antibodies was not studied (it was not known in 1985 when the patient was first seen), and permission for its determination could not be obtained for the patient’s second and third flare-ups, it was the most likely diagnosis for our patient at the time to our knowledge. MDMP was given intravenously during the first attack while she was in the hospital. On the second and third recurrences, it was administered orally at home. 279

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This patient has been brought to our attention because of her prompt responses to MDMP in each recurrence and the absence of obvious organ damage, especially in terms of brain, liver, and kidney functions, until her death due to congestive heart failure at home. MDMP has been used for several immune-related disorders [6]. Since acquired TTP is most likely related to antibodies against ADAMTS 13, the response to MDMP in this disorder could easily be understood and should be studied in the future in more detail. If other patients with acquired TTP with proven ADAMTS 13 deficiency respond to MDMP treatment without organ damage and early improvement of anemia and thrombocytopenia, this treatment could be the first choice for those patients. 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: Thrombotic thrombocytopenic purpura Anahtar Sözcükler: Trombotik trombositopenik purpura Şinasi Özsoylu Retired Professor of Pediatrics, Hematology and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society

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References 1. Ozsoylu S. High-dose intravenous methylprednisolone for thrombotic thrombocytopenic purpura. Acta Haematol 1990;84:110. 2. Ozsoylu Ş, Bilgin K. Megadose methylprednisolone for the treatment of thrombocytopenic purpura. Turk J Med Sci 1994;22:129-130. 3. Ozsoylu S. Corticosteroids in the treatment of thrombotic thrombocytopenic purpura. Acta Paediatr 2003;92:1229. 4. George JN, Nester CM. Syndromes of thrombotic microangiopathy. N Engl J Med 2014;371:1847-1848. 5. Gümrük F, Özsoylu Ş. Plazma infüzyonu ile düzeltilebilen trombositopeni. Yeni Tıp Dergisi 1992;9:57-58. 6. Ozsoylu S. Mega-dose methylprednisolone in hematologic and non-hematologic disorders. Turk J Hematol 2010;27:130-131. Address for Correspondence: Şinasi ÖZSOYLU, M.D., Retired Professor of Pediatrics, Hematology and Hepatology, Honorary Fellow of American Academy of Pediatrics, Honorary Member of American Pediatric Society Phone: +90-256-2120020 E-mail: sinasiozsoylu@hotmail.com Received/Geliş tarihi: September 10, 2014 Accepted/Kabul tarihi: December 03, 2014 DOI: 10.4274/tjh.2014.0361

Images in Hematology

DOI: 10.4274/tjh.2014.0284

Histopathological Image in Hematology Inclusion Body in a Leukocyte in Crimean-Congo Hemorrhagic Fever Kırım-Kongo Kanamalı Ateşinde Lökosit İçinde İnklüzyon Cismi

Figure 1. In the cytoplasm of a lymphomonocytic cell, a blue-violet inclusion body was seen.

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A 49-year-old male patient was admitted to the emergency room with fever for 5 days and nasal bleeding. A tick bite was learned to have occurred 10 days before. On physical examination, the patient was confused, with a fever of 39 °C and widespread petechiae. In his laboratory examination, hemoglobin was 9 g/dL, WBC count was 16,000/mm3, and platelet count was 8000/mm3. In Giemsa-stained peripheral blood smear, rare schistocytes and platelets, 80% neutrophils, 18% lymphocytes, and 2% monocytes were observed. In the cytoplasm of a lymphomonocytic cell, a blue-violet inclusion body was seen (Figure 1). Informed consent was obtained. The patient was prediagnosed with Crimean-Congo hemorrhagic fever (CCHF) and accordingly administered fresh frozen plasma and platelet therapy. The patient was lost after massive gastrointestinal bleeding as a result of multiple organ failure. A positive PCR result was obtained for CCHF. Inclusion bodies in leukocytes can be seen in congenital diseases, hematological malignancies, infections (especially viral), burns, and pregnancy and after cytotoxic therapy. To the best of our knowledge, in the diagnosis of CCHF, inclusion

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Bolaman Z, et al: Leukocytes and Crimean-Congo Hemorrhagic Fever

bodies in leukocytes have not been reported previously in the literature. We suggest that they be remembered as a potential clue in diagnosis during peripheral blood smear 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: Crimean-Congo hemorrhagic fever, Leukocyte Anahtar Sözcükler: Kırım-Kongo kanamalı ateşi, Lökositr Zahit Bolaman, İrfan Yavaşoğlu, Gürhan Kadıköylü Adnan Menderes University Faculty of Medicine, Division of Hematology, Aydın, Turkey E-mail: dryavas@hotmail.com Phone: +90 256 612 18 25 Received/Geliş tarihi

: July 10, 2014

Accepted/Kabul tarihi : September 1, 2014

Images in Hematology

DOI: 10.4274/tjh.2014.0478

Quiz in Hematology

A 14-year-old female patient was admitted to our hospital because of paleness and icterus. She had been investigated at different centers for chronic anemia, icterus, and splenomegaly since age 6. She had no history of blood transfusion, drug usage, or fever. There was no consanguinity between parents, and our patientâ&#x20AC;&#x2122;s sister had similar clinical findings. On physical examination she was pale and icteric and she had markedly enlarged liver and spleen, 3 cm and 8 cm below the costal margins, respectively. Respiratory, cardiovascular, musculoskeletal, and other examinations were normal. Informed consent was obtained. The hemogram revealed Hb: 8.0 g/dL, RBC: 2.59x1012/L, Hct: 24.5%, MCV: 94.6 fL, RDW: 17.1, WBC: 7.65x109/L,

and platelets: 383x109/L. Reticulocyte count was 3.4% and peripheral blood smear revealed anisocytosis and poikilocytosis. The liver and kidney function test results were within normal limits. Serum total and indirect bilirubin and LDH levels were 5.22 mg/dL, 0.46 mg/dL, and 545 IU/L respectively. Direct Coombs test was negative. Hemoglobin electrophoresis, pyruvate kinase and G6PD, vitamin B12, and folic acid levels were normal. She was immune to HBV infection and her serological tests were negative for HCV and HIV. Bone marrow examination under light microscopy showed erythroid hyperplasia, with binucleated or multinucleated and megaloblastic normoblasts (Figures 1A and 1B):

Figure 1. A and B: Marked dyserythropoiesis, nuclear bridging, binucleation, and multinucleation of the erythroid precursors.

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Demircioğlu F, et al: Diagnosis: Congenital Dyserythropoietic Anemia Type 2

Turk J Hematol 2015;32:283-284

Diagnosis: Congenital Dyserythropoietic Anemia Type 2 Due to Compound Heterozygote Mutation in SEC23B Gene Bileşik Heterozigot SEC23B Gen Mutasyonu Olan Konjenital Diseritropetik Anemi Tip 2 Olgusu A clinical diagnosis of congenital dyserythropoietic anemia (CDA) type 2 was established after exclusion of other causes associated with dyserythropoiesis and ineffective erythropoiesis, including thalassemia syndromes, vitamin B12 and folate deficiencies, myelodysplastic syndrome, and sideroblastic anemia. Molecular analyses for CDA type 2 revealed compound heterozygote mutations in the SEC23B gene (c.325G >A, p.Glu109Lys in exon 4 and c.938 G >A, p.Arg313His in exon 8). CDA is a heterogeneous group of rare hereditary disorders of ineffective erythropoiesis and dyserythropoiesis characterized by morphologically abnormal erythroid precursors in the bone marrow. Non-erythroid hematopoietic cell morphology is normal [1]. Type 2 (CDA 2), the most common type, is autosomal recessive and typically shows marked dyserythropoiesis characterized by binucleated and multinucleated normoblasts with erythroid hyperplasia [2,3,4]. Most patients with CDA 2 show variable degrees of anemia, usually have jaundice, and may have hepatosplenomegaly. Our patient was diagnosed as having CDA type 2 by the presence of anemia, icterus, hepatosplenomegaly, binucleated and trinucleated erythroid precursors in the bone marrow, and positive SEC23B mutation. 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: Anemia, Congenital dyserythropoietic anemia type 2, SEC23B gene Anahtar Sözcükler: Anemi, Konjenital diseritropetik anemi tip 2, SEC23B geni Fatih Demircioğlu1, Mustafa Erkoçoğlu2, Mustafa Dilek2, Mervan Bekdaş2, Sevil Göksügür2, Semra Büyükkorkmaz2, Seher Açar2 1Abant 2Abant

İzzet Baysal University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Bolu, Turkey İzzet Baysal University Faculty of Medicine, Department of Pediatrics, Bolu, Turkey

Received/Geliş tarihi

: December 12, 2014

Accepted/Kabul tarihi : December 17, 2014 References 1. Kamiya T, Manabe A. Congenital dyserythropoietic anemia. Int J Hematol 2010;92:432-438. 2. Unal S, Russo R, Gumruk F, Kuskonmaz B, Cetin M, Sayli T, Tavil B, Langella C, Iolascon A, Uckan Cetinkaya D. Successful hematopoietic stem cell transplantation in a patient with

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congenital dyserythropoietic anemia type II. Pediatr Transplant 2014;18:130-133. 3. Iolascon A, Heimpel H, Wahlin A, Tamary H. Congenital dyserythropoietic anemias: molecular insights and diagnostic approach. Blood 2013;122:2162-2166. 4. Alıcı IO, Aytaç S. Images in hematology. Turk J Hematol 2005;22:155.

Advisory Board of This Issue (September 2015) A. Emre Eşkazan, Turkey Ahmet Koç, Turkey Ahmet Muzaffer Demir, Turkey Akif Yeşilipek, Turkey Ana Planinc Peraica, Croatia Ayşegül Ünüvar, Turkey Aytemiz Gürgey, Turkey Ayten Nalbant, Turkey Betül Tavil, Turkey Elena Solomou, Greece Eliane Gluckman, France Filiz Çay Şenler, Turkey Hamdi Akan, Turkey

Hayat Kumbasar, Turkey Hung Chang, Taiwan İbrahim C. Haznedaroğlu, Turkey İlker Devrim, Turkey Juan Carlos Gea-Banacloche, USA Julie Kanter, USA Meral Beksaç, Turkey Mirko Samija, Croatia Mualla Çetin, Turkey Mustafa Pehlivan, Turkey Nejat Akar, Turkey Olga Meltem Akay, Turkey Ota Fuchs, Czech Republic

Rakesh Goyal, USA Rıdvan Ali, Turkey Sercan Aksoy, Turkey Sevgi Kalayoğlu Beşışık, Turkey Shalini Shenoy, USA Şinasi Özsoylu, Turkey Şule Ünal, Turkey Tunç Fışgın, Turkey Ülker Koçak, Turkey Yusuf Baran, Turkey Zeynep Gözde Özkan, Turkey Zühre Kaya, Turkey

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