tjh-2019-1 by LookUs Scientific

Issue 1

March 2019

E-ISSN: 1308-5263

Volume 36

Review Late Effects of Therapy in Childhood Acute Lymphoblastic Leukemia Survivors Hande Kızılocak and Fatih Okcu; İstanbul, Turkey, Houston, TX, USA

Research Articles Investigation of the Relationship Between Fok1 and Col1A1 Gene Polymorphisms and Development of TreatmentRelated Bone Complications in Children with Acute Lymphoblastic Leukemia Melek Erdem et al.; İzmir, Turkey

Evaluation of Cardiac Parameters in Bone Marrow Transplant Patients: Effect of Pulmonary Artery Pressure on Survival Ali Caner Özdöver et al.; Gaziantep, Turkey

A Frequent Mutation in the FTL Gene Causing Hyperferritinemia Cataract Syndrome in Turkish Population Is c.160A>G Burhan Balta et al.; Kayseri, Turkey

A Novel ATP6V0A2 Mutation Causing Recessive Cutis Laxa with Unusual Manifestations of Bleeding Diathesis and Defective Wound Healing İlker Karacan et al.; İstanbul, Turkey

Different Presentations of Patients with Transcobalamin II Deficiency: A Single-Center Experience from Turkey Selma Ünal et al.; Mersin, Turkey

Cover Picture: Dietmar Enko, Gernot Kriegshäuser Light Chain Myeloma with Highly Atypical Plasma Cells and Extensive Auer Rod-Like Inclusions

International Review Board

Editor-in-Chief Reyhan Küçükkaya

İstanbul, Turkey rkucukkaya@hotmail.com

Associate Editors

A. Emre Eşkazan

İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul, Turkey

Ayşegül Ünüvar

İstanbul, Turkey aysegulu@hotmail.com

Cengiz Beyan

Ufuk University, Ankara, Turkey cengizbeyan@hotmail.com

Hale Ören

Dokuz Eylül University, İzmir, Turkey hale.oren@deu.edu.tr

İbrahim C. Haznedaroğlu

Hacettepe University, Ankara, Turkey haznedar@yahoo.com

M. Cem Ar

İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul, Turkey mcemar68@yahoo.com

Selami Koçak Toprak

Ankara University, Ankara, Turkey sktoprak@yahoo.com

Semra Paydaş

Çukurova University, Adana, Turkey sepay@cu.edu.tr

Şule Ünal

Hacettepe University, Ankara, Turkey

Assistant Editors Ali İrfan Emre Tekgündüz

Dr. A. Yurtaslan Ankara Oncology Training and Research Hospital, Ankara, Turkey

Claudio Cerchione

University of Naples Federico II Napoli, Campania, Italy

Elif Ünal İnce

Ankara University, Ankara, Turkey

İnci Alacacıoğlu

Dokuz Eylül University, İzmir, Turkey

Müge Sayitoğlu

İstanbul University, İstanbul, Turkey

Nil Güler

Ondokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay

Koç University, İstanbul, Turkey

Veysel Sabri Hançer

İstinye University, İstanbul, Turkey

Zühre Kaya

Gazi University, Ankara, Turkey

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

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

Past Editors Erich Frank Orhan Ulutin Hamdi Akan Aytemiz Gürgey

Language Editor Leslie Demir

Senior Advisory Board Yücel Tangün Osman İlhan Muhit Özcan Teoman Soysal Ahmet Muzaffer Demir

Editorial Office İpek Durusu Bengü Timoçin

Publishing Services

Statistic Editor Hülya Ellidokuz

GALENOS PUBLISHER Molla Gürani Mah. Kaçamak Sk. No: 21/1, Fındıkzade, İstanbul, Turkey Phone: +90 212 621 99 25 • Fax: +90 212 621 99 27 • www. galenos.com.tr

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

All Inquiries Should be Addressed to TURKISH JOURNAL OF HEMATOLOGY Address Phone Fax E-mail

: Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613. Sok.) No: 8 06550 Çankaya, Ankara / Turkey : +90 312 490 98 97 : +90 312 490 98 68  : info@tjh.com.tr

E-ISSN: 1308-5263

Publishing Manager

Publishing House

Muhlis Cem Ar

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul, Turkey Tel: +90 212 621 99 25

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

Fax: +90 212 621 99 27 E-mail: info@galenos.com.tr Publisher Certificate Number: 14521

Online Manuscript Submission

Publication Date

http://mc.manuscriptcentral.com/tjh

05.02.2019

Web page

Cover Picture

www.tjh.com.tr

Dietmar Enko, Gernot Kriegshäuser

Owner on Behalf of the Turkish Society of Hematology

Light Chain Myeloma with Highly Atypical Plasma Cells and Extensive Auer Rod-Like Inclusions

Güner Hayri Özsan

Bone marrow aspirate smear of a 73-year-old patient with κ-type light chain myeloma (A and B). The arrow marks a plasmoblastic cell with massive Auer rod-like inclusions.

International scientific journal published quarterly. The Turkish Journal of Hematology is published by the commercial enterprise of the Turkish Society of Hematology with Decision Number 6 issued by the Society on 7 October 2008.

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AIMS AND SCOPE The Turkish Journal of Hematology is published quarterly (March, June, September, and December) by the Turkish Society of Hematology. It is an independent, non-profit peer-reviewed international English-language periodical encompassing subjects relevant to hematology. The Editorial Board of The Turkish Journal of Hematology adheres to the principles of the World Association of Medical Editors (WAME), International Council of Medical Journal Editors (ICMJE), Committee on Publication Ethics (COPE), Consolidated Standards of Reporting Trials (CONSORT) and Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). The aim of The Turkish Journal of Hematology is to publish original hematological research of the highest scientific quality and clinical relevance. Additionally, educational material, reviews on basic developments, editorial short notes, images in hematology, and letters from hematology specialists and clinicians covering their experience and comments on hematology and related medical fields as well as social subjects are published. As of December 2015, The Turkish Journal of Hematology does not accept case reports. Important new findings or data about interesting hematological cases may be submitted as a brief report. General practitioners interested in hematology and internal medicine specialists are among our target audience, and The Turkish Journal of Hematology aims to publish according to their needs. The Turkish Journal of Hematology is indexed, as follows: - PubMed Medline - PubMed Central - Science Citation Index Expanded - EMBASE - Scopus - CINAHL - Gale/Cengage Learning - EBSCO - DOAJ - ProQuest - Index Copernicus - Tübitak/Ulakbim Turkish Medical Database - Turk Medline Impact Factor: 0.650 Open Access Policy Turkish Journal of Hematology is an Open Access journal. This journal provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. Open Access Policy is based on the rules of the Budapest Open Access Initiative (BOAI) http://www.budapestopenaccessinitiative.org/. Subscription Information  The Turkish Journal of Hematology is published electronically only as of 2019. Therefore, subscriptions are not necessary. All published volumes are available in full text free-of-charge online at www.tjh.com.tr.

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Address: İlkbahar Mah., Turan Güneş Bulvarı, 613 Sok., No: 8, Çankaya, Ankara, Turkey Telephone: +90 312 490 98 97  Fax: +90 312 490 98 68 Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh  Web page: www.tjh.com.tr  E-mail: info@tjh.com.tr   Permissions  Requests for permission to reproduce published material should be sent to the editorial office. Editor: Professor Dr. Reyhan Küçükkaya Adress: Turan Güneş Bulv. İlkbahar Mah. Fahrettin Paşa Sokağı (Eski 613. Sokak) No: 8, 06550 Çankaya, Ankara, Turkey Telephone: +90 312 490 98 97  Fax: +90 312 490 98 68  Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh  Web page: www.tjh.com.tr  E-mail: info@tjh.com.tr Publisher Galenos Yayınevi Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul, Turkey Telephone : +90 212 621 99 25 Fax : +90 212 621 99 27 info@galenos.com.tr Instructions for Authors Instructions for authors are published in the journal and at www.tjh.com.tr Material Disclaimer Authors are responsible for the manuscripts they publish in The Turkish Journal of Hematology. The editor, editorial board, and publisher do not accept any responsibility for published manuscripts. If you use a table or figure (or some data in a table or figure) from another source, cite the source directly in the figure or table legend. Editorial Policy Following receipt of each manuscript, a checklist is completed by the Editorial Assistant. The Editorial Assistant checks that each manuscript contains all required components and adheres to the author guidelines, after which time it will be forwarded to the Editor in Chief. Following the Editor in Chief’s evaluation, each manuscript is forwarded to the Associate Editor, who in turn assigns reviewers. Generally, all manuscripts will be reviewed by at least three reviewers selected by the Associate Editor, based on their relevant expertise. Associate editor could be assigned as a reviewer along with the reviewers. After the reviewing process, all manuscripts are evaluated in the Editorial Board Meeting. Turkish Journal of Hematology’s editor and Editorial Board members are active researchers. It is possible that they would desire to submit their manuscript to the Turkish Journal of Hematology. This may be creating a conflict of interest. These manuscripts will not be evaluated by the submitting editor(s). The review process will be managed and decisions made by editor-in-chief who will act independently. In some situation, this process will be overseen by an outside independent expert in reviewing submissions from editors.

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

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

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

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

Perspectives in Hematology “Perspectives” are articles discussing significant topics relevant to hematology. They are more personal than a Review Article. Authors wishing to submit a Perspective in Hematology article should contact the Editor in Chief prior to submission in order to screen the proposed topic for relevance and priority. Articles submitted for “Perspectives in Hematology” must advance the hot subjects of experimental and/or clinical hematology beyond the articles previously published or in press in TJH. Perspective papers should meet the restrictive criteria of TJH regarding unique scientific and/or educational value, which will impact and enhance clinical hematology practice or the diagnostic understanding of blood diseases. Priority will be assigned to such manuscripts based upon the prominence, significance, and timeliness of the content. The submitting author must already be an expert with a recognized significant published scientific experience in the specific field related to the “Perspectives” article.

slides. Explain the internal scale and identify the staining method used. Figures should be submitted as separate files, not in the text file. Highresolution image files are not preferred for initial submission as the file sizes may be too large. The total file size of the PDF for peer review should not exceed 5 MB. Authorship Each author should have participated sufficiently in the work to assume public responsibility for the content. Any portion of a manuscript that is critical to its main conclusions must be the responsibility of at least one author. Contributor’s Statement

References: Should not include more than 50 references

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

Images in Hematology

Acknowledgments

Article length: Not to exceed 200 words.

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.

Abstract length: Not to exceed 150 words. Article length: Not to exceed 1000 words.

Authors can submit for consideration illustrations or photos that are interesting, instructive, and visually attractive, along with a few lines of explanatory text and references. Images in Hematology can include no more than 200 words of text, 5 references, and 3 figures or tables. No abstract, discussion, or conclusion is required, but please include a brief title. Letters to the Editor Article length: Not to exceed 500 words. Letters can include no more than 500 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title. The total number is usually limited to a maximum of five authors for a letter to the editor. Tables Supply each table in a separate file. Number tables according to the order in which they appear in the text, and supply a brief caption for each. Give each column a short or abbreviated heading. Write explanatory statistical measures of variation, such as standard deviation or standard error of mean. Be sure that each table is cited in the text. Figures Figures should be professionally drawn and/or photographed. Authors should number figures according to the order in which they appear in the text. Figures include graphs, charts, photographs, and illustrations. Each figure should be accompanied by a legend that does not exceed 50 words. Use abbreviations only if they have been introduced in the text. Authors are also required to provide the level of magnification for histological

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Authors are expected to disclose on the title page any commercial or other associations that might pose a conflict of interest in connection with the submitted manuscript. All funding sources that supported the work and the institutional and/or corporate affiliations of the authors should be acknowledged on the title page. Ethics When reporting experiments conducted with humans indicate that the procedures were in accordance with ethical standards set forth by the committee that oversees human subject research. Approval of research protocols by the relevant ethics committee, in accordance with international agreements (Helsinki Declaration of 1975, revised 2013 available at https://www.wma.net/policies-post/wma-declarationof-helsinki-ethical-principles-for-medical-research-involving-humansubjects/), is required for all experimental, clinical, and drug studies. Patient names, initials, and hospital identification numbers should not be used. Manuscripts reporting the results of experimental investigations conducted with humans must state that the study protocol received institutional review board approval and that the participants provided informed consent.

Non-compliance with scientific accuracy is not in accord with scientific ethics. Plagiarism: To re-publish, in whole or in part, the contents of another author’s publication as one’s own without providing a reference. Fabrication: To publish data and findings/results that do not exist. Duplication: Use of data from another publication, which includes republishing a manuscript in different languages. Salami slicing: To create more than one publication by dividing the results of a study unnecessarily. We disapprove of such unethical practices as plagiarism, fabrication, duplication, and salami slicing, as well as efforts to influence the review process with such practices as gifting authorship, inappropriate acknowledgments, and references. Additionally, authors must respect participants‘ right to privacy. On the other hand, short abstracts published in congress books that do not exceed 400 words and present data of preliminary research, and those that are presented in an electronic environment, are not considered as previously published work. Authors in such a situation must declare this status on the first page of the manuscript and in the cover letter. (The COPE flowchart is available at http://publicationethics.org.) We use iThenticate to screen all submissions for plagiarism before publication. Conditions of Publication All authors are required to affirm the following statements before their manuscript is considered: 1. The manuscript is being submitted only to The Turkish Journal of Hematology; 2. The manuscript will not be submitted elsewhere while under consideration by The Turkish Journal of Hematology; 3. The manuscript has not been published elsewhere, and should it be published in The Turkish Journal of Hematology it will not be published elsewhere without the permission of the editors (these restrictions do not apply to abstracts or to press reports for presentations at scientific meetings); 4. All authors are responsible for the manuscript’s content; 5. All authors participated in the study concept and design, analysis and interpretation of the data, and drafting or revising of the manuscript and have approved the manuscript as submitted. In addition, all authors are required to disclose any professional affiliation, financial agreement, or other involvement with any company whose product figures prominently in the submitted manuscript. Authors of accepted manuscripts will receive electronic page proofs and are responsible for proofreading and checking the entire article within two days. Failure to return the proof in two days will delay publication. If the authors cannot be reached by email or telephone within two weeks, the manuscript will be rejected and will not be published in the journal.

Copyright At the time of submission all authors will receive instructions for submitting an online copyright form. No manuscript will be considered for review until all authors have completed their copyright form. Please note, it is our practice not to accept copyright forms via fax, e-mail, or postal service unless there is a problem with the online author accounts that cannot be resolved. Every effort should be made to use the online

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

Units of Measurement Measurements should be reported using the metric system, according to the International System of Units (SI). Consult the SI Unit Conversion Guide, New England Journal of Medicine Books, 1992. An extensive list of conversion factors can be found at https://www. nist.gov/sites/default/files/documents/pml/wmd/metric/SP1038.pdf. For more details, see http://www.amamanualofstyle.com/oso/public/jama/ si_conversion_table.html.

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

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

Setting Up an Account New users to the submission site will need to register and enter their account details before they can submit a manuscript. Log in, or click the “Create Account” button if you are a first-time user. To create a new account: After clicking the “Create Account” button, enter your name and e-mail address, and then click the “Next” button. Your e-mail address is very important. Enter your institution and address information, as appropriate, and then click the “Next” Button. Enter a user ID and password of your choice, select your area of expertise, and then click the “Finish” button. If you have an account, but have forgotten your log-in details, go to “Password Help” on the journal’s online submission system and enter your e-mail address. The system will send you an automatic user ID and a new temporary password. Full instructions and support are available on the site, and a user ID and password can be obtained during your first visit. Full support for

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

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

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CONTENTS Review 1 Late Effects of Therapy in Childhood Acute Lymphoblastic Leukemia Survivors

Hande Kızılocak, Fatih Okcu; İstanbul, Turkey, Houston, TX, USA

Research Articles

Investigation of the Relationship Between Fok1 and Col1A1 Gene Polymorphisms and Development of Treatment-Related Bone Complications in Children with Acute Lymphoblastic Leukemia Melek Erdem, Özlem Tüfekçi, Sefa Kızıldağ, Şebnem Yılmaz, Deniz Kızmazoğlu, Berna Eroğlu Filibeli, Hale Ören; İzmir, Turkey

Evaluation of Cardiac Parameters in Bone Marrow Transplant Patients: Effect of Pulmonary Artery Pressure on Survival Ali Caner Özdöver, İlknur Gündeş, Melya Pelin Kırık, Handan Haydaroğlu Şahin, Murat Sucu, Mustafa Pehlivan; Gaziantep, Turkey

A Frequent Mutation in the FTL Gene Causing Hyperferritinemia Cataract Syndrome in Turkish Population Is c.-160A>G Burhan Balta, Murat Erdoğan, Aslıhan Kiraz, Serdal Korkmaz, Alperen Ağadayı; Kayseri, Turkey

A Novel ATP6V0A2 Mutation Causing Recessive Cutis Laxa with Unusual Manifestations of Bleeding Diathesis and Defective Wound Healing İlker Karacan, Reyhan Diz Küçükkaya, Fatma Nur Karakuş, Seyhun Solakoğlu, Aslıhan Tolun, Veysel Sabri Hançer, Eda Tahir Turanlı; İstanbul, Turkey

Different Presentations of Patients with Transcobalamin II Deficiency: A Single-Center Experience from Turkey Selma Ünal, Feryal Karahan, Tuğba Arıkoğlu, Asuman Akar, Semanur Kuyucu; Mersin, Turkey

Brief Report

Thalidomide for the Management of Bleeding Episodes in Patients with Hereditary Hemorrhagic Telangiectasia: Effects on Epistaxis Severity Score and Quality of Life Mehmet Baysal, Elif G. Ümit, Hakkı Onur Kırkızlar, Ali Caner Özdöver, Ahmet Muzaffer Demir; Edirne, Turkey

Images in Hematology

Peutz-Jeghers Syndrome: A Very Rare Cause of Iron Deficiency Anemia Fatma Demir Yenigürbüz, Ugur Deveci, Ebru Tuncez; Şanlıurfa, Turkey

Letters to the Editor

Bendamustine and Rituximab Treatment, Chronic Lymphocytic Leukemia, Direct Antiglobulin Test, and False Negatives Won Sriwijitalai, Viroj Wiwanitkit; Bangkok, Thailand, Pune, India

A Case of Blastic Plasmacytoid Dendritic Cell Neoplasm with Unusual Presentation Sneha Dhariwal, Monica Gupta; Dilshad Garden, Delhi, India

EBV-Related Diffuse Large B-Cell Lymphoma in a Patient with Angioimmunoblastic T-Cell Lymphoma Cem Şimşek, Başak Bostankolu, Ece Özoğul, Arzu Sağlam Ayhan, Ayşegül Üner, Yahya Büyükaşık; Ankara, Turkey

A Case of SF3B1-Positive Myelodysplastic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis Alejandro Lazo-Langner, Bekim Sadikovic; London, Ontario, Canada

Kasabach-Merritt Syndrome in an Adult: A Comment Sevgi Gözdaşoğlu; Ankara, Turkey

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Atypical Radiologic Image Characterized by Cavitary Lung Lesions in a Case of Hodgkin Lymphoma Mahmut Büyükşimşek, Semra Paydaş, Derya Gumurdulu, Cem Mirili, Ali Oğul, Abdullah Evren Yetişir, Mert Tohumcuoğlu; Adana, Turkey

Light Chain Myeloma with Highly Atypical Plasma Cells and Extensive Auer Rod-Like Inclusions Dietmar Enko, Gernot Kriegshäuser; Steyr, Graz, Austria

Aggressive Clinicopathological Course of Myeloma with t(3;16)(q21;q22) Cytogenetic Abnormality Süreyya Bozkurt, Müfide Okay, İbrahim Haznedaroğlu; İstanbul, Ankara, Turkey

The Impact of DNMT3A/FLT3-ITD/NPM1 on Patients with Acute Myeloid Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation Long Su; Changchun, China

Wilms Tumor-1 (WT1) rs16754 Polymorphism and Clinical Outcome in Acute Myeloid Leukemia Mani Ramzi, Mohamad Moghadam, Nader Cohan; Shiraz, Iran

Wilms Tumor-1 (WT1) rs16754 Polymorphism Pathum Sookaromdee, Viroj Wiwanitkit; Bangkok, Thailand, Pune, India

A Novel Mutation in a Child with Atypical Wiskott-Aldrich Syndrome Complicated by Cytomegalovirus Infection Zühre Kaya, Cansu Muluk, Şule Haskoloğlu, Lale Ş. Tufan; Ankara, Turkey

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REVIEW DOI: 10.4274/tjh.galenos.2018.2018.0150 Turk J Hematol 2019;36:1-11

Late Effects of Therapy in Childhood Acute Lymphoblastic Leukemia Survivors Çocukluk Çağı Akut Lenfoblastik Lösemi Tedavisi Sonrası Geç Yan Etkiler Hande Kızılocak1,

Fatih Okcu2

1Istanbul University-Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey 2Texas Children’s Hematology and Oncology Centers, Baylor College of Medicine, Department of Pediatrics, Division of Hematology and

Oncology, Houston, TX, USA

Abstract

Öz

Over the last 50 years, the survival rates in children with acute lymphoblastic leukemia (ALL) have increased remarkably. The optimal use of antileukemic agents in cooperative group protocols, central nervous system-directed treatment, improvements in supportive care, and recognition of biological, clinical, and treatment response characteristics that predict patients with a higher or a lower risk of treatment failure have improved 5-year event-free survival rates, reaching more than 85%, and 5-year overall survival rates, reaching more than 90%. Consequently, it has become increasingly important to characterize the occurrence of long-term late effects. ALL treatments have been associated with increased risks for adverse outcomes such as late mortality, secondary malignancies, and neurological, cardiac, endocrine, and social/psychological disorders. In recent decades, cooperative groups in Europe and in the United States have provided essential information about the long-term effects of ALL therapy, giving recommendations for screening as well as facilitating new approaches for reducing late-term morbidity and mortality. Current frontline protocols continue to examine ways to lower the intensity and amount of therapy to reduce late effects, whereas survivorship studies attempt to predict such adverse effects precisely and develop targeted prevention and treatment strategies.

Son 50 yıldaki gelişmeler ile akut lenfoblastik lösemili (ALL) çocuklardaki tedavi sonrası sağkalım oranında belirgin derecede artış saptandı. Antilösemik ilaçların akılcı kullanımı, santral sinir sistemini hedefleyen lokal tedaviler ve destek tedavisindeki gelişmeler ile 5 yıllık olaysız sağkalım %85’e ve 5 yıllık genel sağkalım %90’a ulaştı. Uzamış sağkalım süreleri, tedavinin uzun dönem yan etkilerine olan farkındalığı da arttırdı. Geç dönem mortalite, ikincil maligniteler, nörolojik, kardiyak, endokrin ve psikososyal bozukluklar ALL tedavisi sonrası sık görülen yan etkilerdir. Son yıllarda Avrupa ve Amerika Birleşik Devletleri’ndeki birçok grup ALL tedavisi sonrası uzun dönem yan etkiler konusunda çalışmalar yaptı, takip için ve uzun dönem mortalite ve morbiditeyi azaltmak için yeni yaklaşımlar önerdiler. Günümüzde kullanımda olan protokoller uzun dönem yan etkileri önlemek için tedavinin yoğunluğunu azaltmaya çalışırken, sağkalımla iligili çalışmalarda da yan etkilerin öngörülmesi ve gerekli önlem ve tedavilerin sağlanması konusu irdeleniyor. Anahtar Sözcükler: Akut lenfoblastik lösemi, Kanser tedavisi sonrası sağkalım, Geç yan etkiler

Keywords: Acute lymphoblastic leukemia, Cancer survivorship, Late effects

Introduction In the last 50 years remarkable success in treating childhood acute lymphoblastic leukemia (ALL) has been achieved through modifications of chemotherapy and radiotherapy within cooperative group trials and improved supportive care [1]. As a result, it has become obvious that survivors of childhood ALL have, in the long term, increased risks of life-threatening severe

late effects related to therapy (summarized in Table 1). In recent decades, cooperative groups in Europe and in the United States have prioritized development of treatment regimens aimed at reducing the risk for late effects without adversely impacting the cure rates. While traditional and molecular epidemiology studies are pursued to describe the growing spectrum of late effects seen in such survivors, development of interventions to prevent and treat late effects associated with significant

Address for Correspondence/Yazışma Adresi: Hande KIZILOCAK, M.D., Istanbul University-Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey Phone : +90 323-361-5798 E-mail : handekizilocak2@yahoo.com ORCID-ID: orcid.org/0000-0003-0323-2571

Received/Geliş tarihi: May 02, 2018 Accepted/Kabul tarihi: July 04, 2018

Kızılocak H and Okcu F: Late Effects in Childhood Acute Lymphoblastic Leukemia

mortality and morbidity has emerged as an important field of research. In this manuscript we present a comprehensive review of the epidemiology and burden of common late effects observed among childhood ALL survivors including secondary malignancies and neurological, cardiac, endocrine, and social/ psychological disorders.

Evolution of Acute Lymphoblastic Leukemia Therapy The optimal use of antileukemic agents in cooperative group protocols, central nervous system (CNS)-directed treatment, improvements in supportive care, and recognition of biological, clinical, and treatment response characteristics able to predict patients at higher or lower risks of treatment failures have increased 5-year event-free survival (EFS) rates to above 85% and 5-year overall survival (OS) rates to above 90% [2], while few children survived 50 years ago [3]. After the initial single agent (aminopterin) and two-drug combinations (mercaptopurine and methotrexate) produced breakthrough “temporary remissions” in the 1940s and 1950s [4,5] Pinkel and colleagues developed a multiphase ALL treatment protocol in 1962 [6]. This included remission induction, CNS-directed therapy [including both cranial irradiation and intrathecal (IT) methotrexate], intensification (consolidation), and continuation treatment using a combination

Turk J Hematol 2019;36:1-11

of 6-mercaptopurine and methotrexate. Such success inspired the development of similar clinical trials around the world, including two particularly important studies in the 1970s [7,8]. First the Berlin-Frankfurt-Munster group presented its “Protocol II” treatment, specifying a reinduction phase (i.e. the repetition of the initial remission induction therapy, which is now referred to as delayed intensification). Secondly, the Dana-Farber Cancer Center added weekly asparaginase at a high dose into its multiagent protocol. Simultaneously, in the 1970s, the use of prophylactic craniospinal radiation was the next major step in the evolution of treatment of ALL [9]. While only 2% of children with ALL have overt leukemia in the spinal fluid at diagnosis, approximately half will experience a CNS relapse if given systemic therapy alone. Craniospinal radiation led to several detrimental late effects including cognitive impairment, growth arrest, and panhypopituitarism in most of the patients [10,11]. To reduce these adverse outcomes first spine radiation was eliminated, followed by reductions in the cranial radiation dose from 24 to 18 Gy and then eventually to 12 Gy. Thus, in 1980s, cranial radiotherapy (CRT) became a standard component of successful multimodality therapy for treating and preventing CNS leukemia [12]. However, many children were still left with neurocognitive impairment that manifested as impaired processing speed, global intellectual function, and executive

Table 1. Summary of late effects in childhood acute lymphoblastic leukemia. Late effects

Associated variables

Frequency

Screening methods

References

Late mortality

Radiation, cardiopulmonary diseases, SN, recurrences

Death from any cause, 13%

Lifelong periodic ROS*/ physical exam

[15,16,17,18,19]

Secondary Malignancy

Radiation, chemotherapy (alkylating agents, anthracyclines, and etoposide)

1%-6%

Lifelong periodic ROS/ physical exam

[20,21,22,23,24,25,26,27,28,29]

Neurological/ Neurocognitive disorders

Cranial or craniospinal radiation, methotrexate (IT/IV), IT ARA-C, 20%-40% vincristine, steroids

Lifelong periodic ROS/ physical exam; neurological examination, imaging as needed

[30,31,32,33,34,35,36,37,38,39,40, 41,42,43,44,45,46,47,48,49,50]

Lifelong periodic ROS/ physical exam focused on cardiac system, periodic echocardiography, blood pressure monitoring

[52,53,54,55,56,57,58,59,60,61, 62,63,64]

Clinical dietician evaluation, healthy diet, and regular physical exercise

[66,67,68,69,70,71,72,73,74,75,76, 77,78,79,80,81,82,83,84,85,86, 87,88,89]

Increased physical activity and calcium intake

[90,91,92,93,94,95,96,97,98,99, 100,101,102,103]

Consultancy and education to parents and survivors

[15,23,104,105,106]

Cardiotoxicity

Anthracycline therapy, overweight

Anthracycline <500 mg/m2, CHF 10%, >600 mg/m2: CHF 36%

Endocrine disorders (growth hormone deficiency, precocious puberty, obesity)

Cranial radiation, younger age at diagnosis, female sex

Obesity, 12%-28%

Bone disorders (osteoporosis, osteonecrosis)

High-dose methotrexate, mercaptopurine, glucocorticoids, Symptomatic cranial radiation, low calcium osteonecrosis, intake, decreased physical 1%-38% activity, obesity

Social/Psychological Young age at diagnosis, disorders female sex ROS: Review of systems, SN: secondary neoplasm, IT: intrathecal.

Not measured well

Turk J Hematol 2019;36:1-11

function. Ultimately a number of randomized studies showed that IT chemoprophylaxis with methotrexate or “triple” therapy with cytarabine, hydrocortisone, and methotrexate could replace CRT with no impact on outcome in the long term for most patients [13]. Only those patients with overt CNS disease at the time of diagnosis and some particular ALL subtypes [T-cell ALL with hyperleukocytosis or existence of overt CNS leukemia (i.e. CNS3 status)] with high risk for CNS failure are today treated with CRT. Hematopoietic stem cell transplantation (HSCT) has been applied with curative intent for cases of relapsed or high-risk ALL beginning in the late 1970s. HSCT recipients are exposed to chemotherapy and/or radiation before HSCT (for management of primary cancer), at HSCT, and after HSCT (for graft-versushost disease and/or relapse of primary cancer), leading to early or late morbidity. Severe or life-threatening morbidity was reported in 40% of transplant cases, associated with premature mortality [14]. Ongoing recent investigational targeted therapy approaches include treatment with monoclonal antibodies such as blinatumomab and chimeric antigen receptor (CAR) T-cells. This area of research may, in the end, direct the development of therapies with high efficacy and lower risks of injury to normal tissues. At present, though, the long-term outcomes in children treated with these novel agents are unknown due to shorter follow-up times. As summarized above, the evolution of childhood ALL therapy has been a long and exciting process. While it represents a major success of cancer history with steady increases in survival over decades, a long list of associated late effects has been realized and will be described herein.

Late Mortality For childhood ALL patients 5-year OS estimates, including patients salvaged after relapse, are today >90% [3]. The Childhood Cancer Survivor Study (CCSS) cohort is a multicenter North American study examining the outcomes in childhood cancer patients who received their diagnoses between 1970 and 1999 and who survived for at least 5 years. Several reports from this cohort indicated that at 25 years from diagnosis, childhood ALL survivors have higher risks of early mortality beyond the 5-year mark [15,16,17] with a 13% cumulative incidence of death from any cause. The majority of deaths were due to leukemia recurrence (66%), yet compared to the sibling controls these patients were found to have a risk of dying from a subsequent cancer that was 15 times higher, a risk of dying from cardiac-related events that was 7 times higher, and a risk of dying from other medical conditions that was 2.6 times higher [15,16]. Mortality due to recurrence in ALL survivors was seen to decrease markedly as the follow-up was extended. Thus, at ≥10

Kızılocak H and Okcu F: Late Effects in Childhood Acute Lymphoblastic Leukemia

years from the initial diagnosis, those ALL patients still surviving are seen to have low risks of recurrence and with rare exception are cured. There is a difference in OS between survivors treated with CRT (OS: 87.3%) and survivors who were not (OS: 96.1%) [18]. In a recent study that evaluated temporal trends in late mortality in a population of 34,033 patients in the CCSS cohort, reduction of mortality due to late effects related to treatment was reported in patients treated in the 1990s compared to earlier decades [19]. Reduced treatment intensity and increased use and accuracy of screening methods were associated with reduction in late mortality in patients with ALL.

Secondary Malignancies Secondary neoplasms (SNs) are among the most critical late effects of therapy for acute leukemia. Association of irradiation with the increased risk of SNs has been reported [20]. The chemotherapy agents that are most often associated with SN development are anthracyclines, etoposide, and alkylating agents [21]. ALL survivors are at increased risk for myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), breast cancer, melanoma, CNS tumors, and non-Hodgkin’s lymphomas, as well as parotid and thyroid gland carcinomas [22,23]. The cumulative incidence of SNs for childhood ALL survivors has an overall range of 1%-11%, dependent on the treatment regimen used and the follow-up duration [22,24]. SNs are most commonly seen in skin (43%) and in the CNS (31%) [18]. Most secondary skin cancers are basal cell carcinomas, while 70% of the reported neoplasms of the CNS are meningiomas [15,18]. A retrospective study including 2169 childhood ALL patients who received treatment in 1962-1998 (median duration of followup was 18.7 years), the cumulative incidence of SNs was 4% overall at 15 years and was increased to 11% at 30 years [24]. Many of these reported late-developing SNs were “benign” neoplasms (basal cell carcinoma, n=14, 11.4%; and meningioma, n=16, 13%); upon exclusion of those diagnoses, cumulative incidence of SNs at 30 years was found to be lowered to 6%. The most common malignant SNs were 46 myeloid malignancies (AML, MDS), 3 lymphomas, 16 carcinomas excluding basal cell carcinoma, 6 sarcomas, and 22 brain tumors. Essig et al. [25] described the spectrum of late effects in 556 ALL survivors (median follow-up: 18.4 years) from the CCSS cohort who were 1-9.9 years old at diagnosis and who received a therapy similar to contemporary ALL protocols for patients with standard risk. Only six (1%) of them developed a subsequent malignancy (two melanomas, one astrocytoma, one large cell lymphoma, one leukemia, and one hepatocellular carcinoma). Radiation, either cranial or craniospinal, increases the risk of the development of secondary solid tumors. Nathan et al. [26] reported that such risk for SNs due to radiation is increased over time; they further found that neoplasms usually appeared at a 3

KÄązÄąlocak H and Okcu F: Late Effects in Childhood Acute Lymphoblastic Leukemia

minimum of 10 to 15 years following treatment. In a cohort containing 8831 pediatric patients who had been diagnosed with ALL and treated with the Childrenâ&#x20AC;&#x2122;s Cancer Group therapeutic protocols in 1983-1995, children receiving cranial radiation at 18 Gy were found to have lower risks of SNs compared to children receiving radiation at doses of 24 Gy [relative risk (RR): 1.5, with 95% confidence interval (CI) of 0.9-2.6; and RR: 3.9, with 95% CI of 1.4-11.2, respectively] [22]. The relative risk of secondary brain tumors following cranial radiation at a dose of 12 Gy, that being the dose that is used most often in the current protocols, has not yet been evaluated. As mentioned above, secondary AML can develop as well in ALL survivors, and it is seen at notably higher rates in patients who had been treated with more frequent or with higher doses of alkylating agents and epipodophyllotoxins [27]. As a result, the current regimens comprise comparatively lower cumulative doses of both cyclophosphamide and other alkylators. A recent study proposed the risk of secondary leukemia to be higher in those patients treated with higher initial doses of mercaptopurine in the maintenance phase (doses of 75 mg/m2/day compared to 50 mg/m2/day) [28]. It is still controversial whether patients having heterozygous or homozygous deficiencies of thiopurine methyltransferase face higher risks for SNs, but it is believed the risk may be partially dependent on the dose intensity of the mercaptopurine and/or its duration [29].

Neurological Outcomes Cranial Radiotherapy Survivors of childhood ALL face risks of neurocognitive late effects, which can include attention problems, impaired visualmotor function, decreased processing speed, and impaired working memory, which may affect their education achievement and quality of life (QOL) [30,31]. CNS-directed therapies, including CRT and/or IT chemotherapy, are established risk factors for impaired cognitive function, especially for younger patients [18]. In the initial 5-10 years following CRT, childhood ALL survivors have higher risks for neurocognitive skill deficits [2,32], and as they get older global brain injuries due to early CRT might decrease the cognitive reserve, putting these individuals at risk of memory impairments or early-onset dementia [3,33]. A study from the CCSS with 556 long-term ALL survivors who were CRT-naive and had been treated in the 1980s and 1990s reported that the survivors had lower overall functional statuses in spite of the fact that their neurocognitive deficits and mental health statuses were not different from those among their matched sibling donors [34]. Recent studies examining individuals treated with contemporary chemotherapy-only protocols uniformly reveal decreased performance in attention and processing speed in 20%-40% of survivors by the end of therapy [35,36,37]. 4

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Methotrexate Methotrexate (MTX) is the main agent associated with neurocognitive dysfunction in ALL survivors. A majority of published studies showed survivors receiving intravenous MTX at high does (>1 g/m2) to experience neurocognitive problems at higher rates than the ones who had been given MTX in low doses [38,39,40]. In another study, cumulative dosing of intravenous MTX heightened the risk for impairments in processing speed by increments of 3% for each 1 g/m2/dose [40]. IT MTX, and even in the absence of CRT, might be connected to changes in white matter, calcifications, cortical atrophy, and leukoencephalopathy, as well as seizures [41]. It is possible that IT cytosine arabinoside might increase the neurotoxicity of IT MTX [42]. Survivors of childhood ALL also face higher risks of fatigue and sleep disturbance [43,44], which are related to increases in neurocognitive impairments [45]. This might not only be related to side effects of drugs; it might also be due to longer hospitalization times. Folate pathway polymorphisms in the MTHFR 1298A>C and MS 2756A>G genes were reported in association with worse attention and processing speed in ALL survivors treated with contemporary protocols, suggesting a role for genetic predisposition [46,47]. Corticosteroids Corticosteroids remain a critical part of contemporary therapy for ALL and are most often administered as either prednisone or dexamethasone. While research among asthma patients suggested corticosteroids to be contributing to difficulties in cognitive functioning [48], earlier studies also showed that the particular steroid regimen chosen for treatment of ALL did not have a differential impact on neurocognitive functions in the long term [49]. The choice of corticosteroid, however, might have an effect on QOL in the long term. Toxicities that are both long-term and acute have been recorded with the usage of prednisone and dexamethasone, while findings suggest better CNS penetration of dexamethasone, which might possibly differentially influence QOL in the process of treatment and in the follow-up period. Vincristine Vincristine, another critical tool in childhood ALL treatment, has been associated with dose-dependent peripheral neuropathy. In a study with 101 survivors, 16 (15.8%) of them were found to have a combination of electrophysiological and clinical neuropathy [50]. Those who were assigned to the intermediateor high-risk arms of the chemotherapy protocol, which had cumulative vincristine doses of 36 mg/m2 or above, were found to have a ninefold increased risk for developing peripheral neuropathy in comparison to patients who had been treated according to the standard-risk arms and who received doses of 33 mg/m2 or lower. Peripheral neuropathy was related to

Turk J Hematol 2019;36:1-11

lower QOL as well, but it was not found to be associated with worsened fine or gross motor functioning. Quality of Life (QOL) QOL is a construct of multiple dimensions measuring well-being in a subjective manner. In a study that evaluated the contribution of neurocognitive dysfunction to QOL [51], 25% of the included survivors of ALL were found to be under the threshold for poor psychosocial QOL. Furthermore, 14% of them had poor physical QOL. The individuals with poor psychosocial QOL were much more likely to be males and were three times more likely to possess verbal deficits in their cognitive abilities and decreased skills for visual-motor integration. As in the general population, the family’s socioeconomic status plays a role in determining the QOL in childhood ALL patients. In particular, an association was found between low household income and poorer physical QOL, and also poorer emotional and social QOL. This implies that survivors belonging to socioeconomically disadvantaged households are especially susceptible to experience decreased QOL following the end of treatment. It is important that more attention be directed towards the determination of socioeconomic risk factors in order to assist at-risk patients and their families in overcoming barriers and accessing the resources they need to ensure the optimal psychosocial and physical functioning of their children.

Cardiotoxicity Childhood ALL survivors face risks of the development of late cardiotoxic effects of their treatments, especially after anthracycline therapy, including congestive heart failure (CHF), abnormalities of the heart valve, heart attacks, and heart epithelium inflammation [52,53]. Oxygen free radicals are produced by anthracyclines; they cause damage to cardiac myocytes, resulting in lost myofibrillar content and vacuolar degeneration, which in turn leads to fibrosis and myocardial necrosis. With the passage of time, thinning of the left ventricular wall occurs, which causes an increase in wall stress together with a decrease in myocardial contractility. Progressive cardiomyopathy might arise early on, during the first year of treatment, or it may occur later, with the diagnosis being given years after the end of the treatment [54,55]. Disease risk is dependent on the dose, CHF rates being ≤10% in those patients treated with cumulative doses of anthracyclines below 500 mg/m2 and as 36% for doses of >600 mg/m2 [56,57]. Furthermore, the risk of CHF related to the therapy is found to be altered by clinical factors such as age at time of treatment (<5 years of age), being female, preexisting heart disease, and simultaneous mediastinal irradiation [57,58,59,60]. In the CCSS cohort, cumulative 30-year incidences for CHF [4.1% (95% CI of 3.2%-5.0%)], myocardial infarction [1.3% (95% CI of 1.0%1.7%)], valvular abnormalities [4.0% (95% CI of 3.1%-4.9%)],

Kızılocak H and Okcu F: Late Effects in Childhood Acute Lymphoblastic Leukemia

and pericardial disease [3.0% (95% CI of 2.1%-3.9%)] were increased in 10,367 survivors of childhood cancer who were then young adults in comparison to their siblings who served as control subjects [61]. As the anthracycline cardiotoxicity risk is dependent on the dose, practitioners have chosen to reduce the cumulative doses administered to patients with low risk in the last decade in order to decrease the occurrence of late cardiac dysfunction [52]. Where cumulative doses of >300 mg/m2 were applied, the risk for clinical heart failure at 20 years was estimated to be almost 10%, but where doses of <300 mg/m2 were applied, the predicted risk fell to 0.5% [62]. Worsening of cardiac function was also reported in a longitudinal follow-up following doses of >300 mg/m2. In a cross-sectionally designed study reporting on 138 survivors of childhood ALL who were then in adulthood and who had a median follow-up time of 23.4 years following diagnosis, LV functions were seen to be impaired in the patients (12%) who had received low cumulative doses of anthracycline (40-120 mg/m2) in their treatment [52], implying that there does not seem to be a certain safe dose when using anthracyclines [63,64]. Therefore, it is recommended to continue monitoring for any late cardiac effects in childhood survivors of ALL who experienced anthracycline exposure in order to prevent any advancement from asymptomatic cardiac dysfunction to clinical cases of CHF. Lastly, some evidence suggests that hypertension may be another cardiac late effect among ALL survivors. In a study that included 68 survivors who received treatment for childhood ALL between 1973 and 2000 and who were evaluated for the possibility of increased body mass index (BMI) and increased risk of high blood pressure, systolic and diastolic blood pressures were increased significantly compared to the general population (systolic blood pressure: mean z-score 0.736, p<0.001; diastolic blood pressure: mean z-score 0.409, p=<0.001; BMI: mean z-score 0.483, p<0.001). Female survivors, and especially those treated with CRT, had a greater risk of being overweight and obese, increasing the risk of cardiovascular morbidity later in life [65].

Endocrine and Metabolic Outcomes Hormone Deficiencies Deficiency of growth hormone (GH) is the most commonly seen endocrinopathy following radiation therapy in survivors of ALL due to direct injury to the hypothalamus [66,67]. It is dose-dependent and mostly seen at a dose of 24 Gy; however, it can also be seen at doses as low as 18 Gy, or at a single dose of 10 Gy applied during total body irradiation (TBI) [67,68]. Craniospinal radiation not only damages the hypothalamus but also directly affects skeletal growth, resulting in growth retardation [69,70]. Along with higher doses of CRT, the risk factors for GH deficiency also comprise younger age at the time 5

Kızılocak H and Okcu F: Late Effects in Childhood Acute Lymphoblastic Leukemia

of diagnosis and being female [67,68,69]. Additional central endocrinopathies, including central adrenal insufficiency, gonadotropin insufficiency, hyperprolactinemia, or central (or secondary) hypothyroidism, have also been associated with doses of 40 Gy [71], that dose being administered rarely in treating childhood ALL. The occurrence of primary hypothyroidism can also be seen following craniospinal or cranial radiation and TBI as a result of the thyroid gland’s direct exposure to radiation, even when doses of 10 Gy are applied, which is relatively low [72]. Another late effect of CRT when administered at doses of 18-24 Gy is precocious puberty; it is seen more commonly in females [73,74,75]. In two important cohort studies, the CCSS and the National Cancer Institute Children’s Cancer Group Leukemia Follow-up Study, a normal age of menarche was reported by 92% of female ALL survivors, in comparison with 97% and 96% of the studied controls, respectively [76,77]. Chemotherapy agents are not associated with endocrine late effects associated with hypothalamic pituitary axis injury [67]. In another CCSS report, compared to sibling controls, young adult ALL survivors were three times more likely to be diagnosed with a chronic endocrine disorder. The risk among survivors who did not relapse and who were not irradiated was comparable to that of the sibling control group [15]. Obesity There are conflicting reports on whether obesity is a true late effect in ALL survivors [78,79,80,81]. Most indicate that both adult and adolescent survivors present with BMIs higher than those seen in healthy controls, while others report no significant differences. Less attention has been paid to the loss of skeletal muscle mass, which leads to sarcopenia in cancer patients and is associated with the use of high-dose glucocorticosteroid therapy. In a cross-sectionally designed study of body composition in 75 long-term ALL survivors, sarcopenic obesity was present in 32 subjects (43%) [82]. Statistically significant and clinically important differences in overall health-related QOL between subjects with and without sarcopenic obesity were stated. A recent meta-analysis showed obesity to be prevalent in survivors still in childhood or those in early adolescence in comparison with reference populations [81]. Higher BMI was found among survivors to a significant extent, independently of sex, type of treatment, or duration from therapy completion to time of assessment. In another meta-analysis evaluating BMIs of adolescent and adult survivors of childhood ALL, patients were more likely by 12%-28% to be categorized as overweight or obese in comparison with the BMIs of the general population (p<0.001) [83]. Survivors of childhood cancer are seldom given recommendations about proper nutrition in the course of and subsequent to treatment. Such individuals need further resources for weight management, which could include advice regarding physical activity and support from dieticians, during 6

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the initial stages of their treatment (and particularly while steroids are being administered) and after the treatment has concluded. In a report evaluating chronic fatigue, the Fatigue Questionnaire was administered to 290 survivors of childhood lymphomas (n=139) and ALL (n=151) [84]. Among these longterm survivors of ALL, a subgroup was identified with persistent chronic fatigue. These individuals were found to have worse depressive symptoms, pain, and anxiety and they also engaged in physical activity less frequently, all of which can contribute to obesity. Infertility Male survivors of childhood ALL in the long-term have an increased risk of experiencing infertility, gonadal dysfunction, and decreased semen quality due to the gonadotoxicity of some treatments, which include testicular irradiation and alkylating agents [85,86,87,88,89]. The relationship is dose-dependent for both patients treated with gonadal radiation at higher doses and with higher cumulative doses of alkylating agents, leading to higher risks [85]. CRT doses of 24 Gy or less do not lead to azoospermia in males [90]. In females, the CCSS reported that the RR for survivors of becoming pregnant at any time was 0.81 (95% CI: 0.730.90; p<0.001) in comparison with their female siblings. In multivariable models including survivors only, those treated with a hypothalamic/pituitary radiation doses of >30 Gy (RR, 0.61; 95% CI of 0.44-0.83) or ovarian/uterine radiation doses above 5 Gy were less apt to ever be pregnant (RR: 0.56 for 5 to 10 Gy, 95% CI of 0.37-0.85; RR: 0.18 for >10 Gy, 95% CI of 0.13-0.26) [91]. Among female survivors of cancer, the loss of primordial follicles at an accelerated rate due to damage to the gonads can cause premature ovarian failure, which can lead to gonadal failure, lowered ovarian reserve accompanied by lower antimüllerian hormone and higher gonadotrophin levels, and permanent infertility [92,93], making follow-up crucial. Additionally, the CCSS reported the occurrence of acute ovarian failure (AOF) in survivors at a rate of 6.3% [94]. The ovaries being exposed to radiation at high doses (particularly doses above 10 Gy), procarbazine, and alkylating agents were found to be important risk factors at older ages for AOF. In a retrospectively designed cohort study evaluating fertility (defined as ever being pregnant) by self-reporting in a population of 182 females and 170 sibling controls, the controls being female siblings of the survivors, deficits in fertility were recorded for survivors who had been treated with any does of CRT, and particularly those treated with it around menarche [95]. In a recent cohort (1987-2006) of French women who were survivors of childhood ALL, there was a negative correlation between fertility and alkylating agent use (p=0.01) and TBI (p=0.013) in univariate and multivariable analyses [96]. Fertility was also found with cumulative cyclophosphamide equivalent

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dose (p=0.001), fertility being seen to decrease for doses of ≥1 g/m2. Bone Toxicities Changes in bone metabolism are considered as important adverse late effects, with pain, fractures, decrease of bone mineral density (BMD), and chronic impairment of bone function [97,98]. Treatment with high-dose MTX, mercaptopurine, glucocorticoids for 2-3 years, and CRT with low calcium intake, decreased physical activity, and obesity are some of the factors that lead to low BMD [99,100]. Furthermore, bone metabolism being impaired and bone mass being decreased in ALL patients who are newly diagnosed both imply that the disease directly affects BMD with leukemic infiltration of the bone marrow [101]. Survivors can recover lost bone mass during the post-treatment period, yet some will not reach their maximum BMD acquisition potential, presenting significant bone mass deficiency [102]. The first 2 years after the cessation of ALL therapy are the most critical for bone loss, with recovery taking place after that time [103]. In a single-institution study with 845 childhood ALL survivors (median age was 31 years), 5.7% displayed osteoporosis (BMD Z-score ≤-2) [104]. No correlation was found between cumulative doses of glucocorticoids and MTX and low BMD, but more exposure to prednisone was associated with decreased BMD in the female participants of the study. The strongest risk factor for a persistently low BMD (Z-score ≤-1) during young adulthood was high-dose (≥24 Gy) cranial or craniospinal radiation, associated with an approximate 2-fold increased risk compared to no cranial radiation exposure. In another single-center study with 101 patients [105], patients with Z-score values between -1.1 and -1.9 who were below 20 years of age had a higher cumulative risk of fractures (2%) and osteonecrosis (ON) (2%). The authors observed that lean mass was positively correlated with whole body and lumbar spine BMD values in patients under 20 years of age, confirming its importance for healthy development of bone mass. The incidence of symptomatic ON shows variation overall, with percentages ranging from 1% to 38% in the literature [106]. The association between ON and corticosteroids is described with two mechanisms. The first mechanism can be represented by direct vascular damage, which is characterized by processes of inflammation, and the second mechanism is gaseous microembolization following hepatotoxicity due to corticosteroids [107]. Vascular damage might justify the increased rate of ON of the femoral head and load of the humeral head [108]. The increased rate of necrosis at the femur head might be associated with the terminal type of vascularization at this level, as the collateral circulation does not compensate for alterations of the supply of blood. The same has been described

Kızılocak H and Okcu F: Late Effects in Childhood Acute Lymphoblastic Leukemia

for the humeral head, as it has vascularization and morphology that are similar to those of the femur. A retrospective analysis of 328 patients with ALL revealed only four (1.2%) cases of ON [109]. Between the diagnosis of ALL and ON, the median time was 12.5 months (range: 12-36 months). The femoral head was involved in all cases, in one case being associated with the scapula-humeral joint. ON is more often associated with dexamethasone in patients older than 10 years compared to prednisone [110].

Social/Psychological Outcomes Childhood ALL survivors have decreased educational attainment and also are less often married, employed, and parenting children than their siblings [15,111]. Cranial irradiation is the strongest predictor of these long-term effects [15,23]. Being young at the time of diagnosis and being female have been identified as the risk factors for worse future socioeconomic status [112]. In a registry-based study from Sweden involving 213 long-term childhood ALL survivors, female survivors were reported to possess higher risks of obtaining lower education levels than their male counterparts and population controls [113]. Younger age at the time of diagnosis was in association with lowered probability of marriage and children, along with lowered level of education with lower income after adjusting for the parents’ level of education. As was expected, therapy involving cranial irradiation was the main culprit for these associations, while steroids and IV/IT MTX were also statistically significant risk factors.

Conclusion More than 90% of childhood ALL survivors survive beyond 5 years after diagnosis, while many experience a long list of late effects from therapy, associated with late mortality, worse social and academic achievement, and worse QOL. Therapeutic exposures such as HSCT, CRT, and some chemotherapy agents put a significant number of ALL survivors at risk for multiple late effects including neurological and neurocognitive dysfunction, metabolic and endocrine abnormalities, bone toxicities, SNs, cardiac damage, and social/psychological adverse effects. Current trials are focused not only on maintaining high cure rates but also on further development of ALL treatments that minimize long-term adverse effects. The Children’s Oncology Group issued evidence-based guidelines available to any oncologist (http:/www.survivorshipguidelines.org/) for detection of late effects that may lead to remediation and improvement of overall health and QOL and prevention of late mortality. Thus, life-long ideal medical care in pediatric ALL survivors should include education on late effects and advice on adherence to periodic follow-up at a pediatric oncology center with an experienced survivorship team. 7

Kızılocak H and Okcu F: Late Effects in Childhood Acute Lymphoblastic Leukemia

Acknowledgements We thank Dr. Phillip Steuber from Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX, for critically reviewing the section on the historical evolution of ALL therapy. Ethics

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12. Follin C, Erfurth EM. Long-term effect of cranial radiotherapy on pituitaryhypothalamus area in childhood acute lymphoblastic leukemia survivors. Curr Treat Options Oncol 2016;17:50. 13. Richards S, Pui CH, Gayon P; Childhood Acute Lymphoblastic Leukemia Collaborative Group (CALLCG). Systematic review and meta-analysis of randomized trials of central nervous system directed therapy for childhood acute lymphoblastic leukemia. Pediatr Blood Cancer 2013;60:185-195.

Authorship Contributions

14. Bhatia S, Armenian SH, Landier W. How I monitor long-term and late effects after blood or marrow transplantation. Blood 2017;130:1302-1314.

Concept: H.K., F.O.; Design: H.K., F.O.; Data Collection or Processing: H.K., F.O.; Analysis or Interpretation: H.K., F.O.; Literature Search: H.K., F.O.; Writing: H.K., F.O.

15. Mody R, Li S, Dover DC, Sallan S, Leisenring W, Oeffinger KC, Yasui Y, Robison LL, Neglia JP. Twenty-five-year follow-up among survivors of childhood acute lymphoblastic leukemia: a report from the Childhood Cancer Survivor Study. Blood 2008;111:5515-5523.

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

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80. Warner EL, Fluchel M, Wright J, Sweeney C, Boucher KM, Fraser A, Smith KR, Stroup AM, Kinney AY, Kirchhoff AC. A population-based study of childhood cancer survivors’ body mass index. J Cancer Epidemiol 2014;2014:531958. 81. Zhang FF, Kelly MJ, Saltzman E, Must A, Roberts SB, Parsons SK. Obesity in pediatric ALL survivors: a meta-analysis. Pediatrics 2014;133:704-715. 82. Marriott CJC, Beaumont LF, Farncombe TH, Cranston AN, Athale UH, Yakemchuk VN, Webber CE, Barr RD. Body composition in long-term survivors of acute lymphoblastic leukemia diagnosed in childhood and adolescence: a focus on sarcopenic obesity. Cancer 2018;124:12251231. 83. Nam GE, Kaul S, Wu YP, Nelson RE, Wright J, Fluchel MN, Hacking CC, Kirchhoff AC. A meta-analysis of body mass index of adolescent and adult survivors of pediatric acute lymphoblastic leukemia. J Cancer Surviv 2015;9:412-421. 84. Zeller B, Loge JH, Kanellopoulos A, Hamre H, Wyller VB, Ruud E. Chronic fatigue in long-term survivors of childhood lymphomas and leukemia: persistence and associated clinical factors. J Pediatr Hematol Oncol 2014;36:438-444. 85. Green DM, Kawashima T, Stovall M, Leisenring W, Sklar CA, Mertens AC, Donaldson SS, Byrne J, Robison LL. Fertility of male survivors of childhood cancer: a report from the Childhood Cancer Survivor Study. J Clin Oncol 2010;28:332-339. 86. Jahnukainen K, Heikkinen R, Henriksson M, Cooper TG, Puukko-Viertomies LR, Makitie O. Semen quality and fertility in adult long-term survivors of childhood acute lymphoblastic leukemia. Fertil Steril 2011;96:837-842. 87. Greaves P, Sarker S, Chowdhury K, Johnson R, Matthews J, Matthews R, Smith M, Korszun A, Gribben JG, Lister TA. Fertility and sexual function in long-term survivors of haematological malignancy: using patient-reported outcome measures to assess a neglected area of need in the late effects clinic. Br J Haematol 2014;164:526-535. 88. van Casteren NJ, van der Linden GH, Hakvoort-Cammel FG, Hahlen K, Dohle GR, van den Heuvel-Eibrink MM. Effect of childhood cancer treatment on fertility markers in adult male long-term survivors. Pediatr Blood Cancer 2009;52:108-112. 89. Haavisto A, Henriksson M, Heikkinen R, Puukko-Viertomies LR, Jahnukainen K. Sexual function in male long-term survivors of childhood acute lymphoblastic leukemia. Cancer 2016;122:2268-2276. 90. Green DM, Zhu L, Wang M, Chemaitilly W, Srivastava D, Kutteh WH, Ke RW, Sklar CA, Pui CH, Kun LE, Ribeiro RC, Robison LL, Hudson MM. Effect of cranial irradiation on sperm concentration of adult survivors of childhood acute lymphoblastic leukemia: a report from the St. Jude Lifetime Cohort Study. Hum Reprod 2017;32:1192-1201. 91. Green DM, Kawashima T, Stovall M, Leisenring W, Sklar CA, Mertens AC, Donaldson SS, Byrne J, Robison LL. Fertility of female survivors of childhood cancer: a report from the childhood cancer survivor study. J Clin Oncol 2009;27:2677-2685. 92. Gurgan T, Salman C, Demirol A. Pregnancy and assisted reproduction techniques in men and women after cancer treatment. Placenta 2008;29:152-159. 93. El-Shalakany AH, Ali MS, Abdelmaksoud AA, Abd El-Ghany S, Hasan EA. Ovarian function in female survivors of childhood malignancies. Pediatr Hematol Oncol 2013;30:328-335. 94. Green DM, Sklar CA, Boice JD Jr, Mulvihill JJ, Whitton JA, Stovall M, Yasui Y. Ovarian failure and reproductive outcomes after childhood cancer treatment: results from the Childhood Cancer Survivor Study. J Clin Oncol 2009;27:2374-2381.

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95. Byrne J, Fears TR, Mills JL, Zeltzer LK, Sklar C, Nicholson HS, Haupt R, Reaman GH, Meadows AT, Roçbison LL. Fertility in women treated with cranial radiotherapy for childhood acute lymphoblastic leukemia. Pediatr Blood Cancer 2004;42:589-597. 96. Freycon F, Trombert-Paviot B, Casagranda L, Berlier P, Bertrand Y, Plantaz D, Stephan JL, Berger C; Childhood Cancer Registry of the Rhône-Alpes Region Group. Age at birth of first child and fecundity of women survivors of childhood acute lymphoblastic leukemia (1987-2007): a study of the Childhood Cancer Registry of the Rhône-Alpes Region in France (ARCERRA). Pediatr Hematol Oncol 2015;32:273-283. 97. Haddy TB, Mosher RB, Reaman GH. Osteoporosis in survivors of acute lymphoblastic leukemia. Oncologist 2001;6:278-285. 98. Davies JH, Evans BA, Jenney ME, Gregory JW. Skeletal morbidity in childhood acute lymphoblastic leukaemia. Clin Endocrinol (Oxf) 2005;63:1-9. 99. Kohler JA, Moon RJ, Sands R, Doherty LJ, Taylor PA, Cooper C, Dennison EM, Davies JH. Selective reduction in trabecular volumetric bone mineral density during treatment for childhood acute lymphoblastic leukemia. Bone 2012;51:765-770. 100. Watsky MA, Carbone LD, An Q, Cheng C, Lovorn EA, Hudson MM, Pui CH, Kaste SC. Bone turnover in long-term survivors of childhood acute lymphoblastic leukemia. Pediatr Blood Cancer 2014;61:1451-1456. 101. Halton JM, Atkinson SA, Fraher L, Webber C, Gill GJ, Dawson S, Barr RD. Altered mineral metabolism and bone mass in children during treatment for acute lymphoblastic leukemia. J Bone Mineral Res 1996;11:1774-1783.

Kızılocak H and Okcu F: Late Effects in Childhood Acute Lymphoblastic Leukemia

lymphoblastic leukemia: results from the St. Jude Lifetime Cohort Study. Pediatr Blood Cancer 2014;61:1270-1276. 105. Molinari PCC, Lederman HM, Lee MLM, Caran EMM. Assessment of the late effects on bones and on body composition of children and adolescents treated for acute lymphocytic leukemia according to Brazilian protocols. Rev Paul Pediatr 2017;35:78-85. 106. Aricò M, Boccalatte MF, Silvestri D, Barisone E, Messina C, Chiesa R, Santoro N, Tamaro P, Lippi A, Gallisai D, Basso G, De Rossi G; Associazione Italiana di Ematologia ed Oncologia Pediatrica. Osteonecrosis: an emerging complication of intensive chemotherapy for childhood acute lymphoblastic leukaemia. Haematologica 2003;88:747-753. 107. Felson DT, Anderson JJ. A cross-study evaluation of association between steroid dose and bolus steroids and avascular necrosis of bone. Lancet 1987;1:902-906. 108. Sala A, Mattano LA Jr, Barr RD. Osteonecrosis in children and adolescents with cancer: an adverse effect of systemic therapy. Eur J Cancer 2007;43:683-689. 109. Riccio I, Pota E, Marcarelli M, Affinita MC, Di Pinto D, Indolfi C, Del Regno N, Esposito M. Osteonecrosis as a complication in pediatric patients with acute lymphoblastic leukemia. Pediatr Med Chir 2016;38:118. 110. Mattano LA, Sather HN, Trigg ME, Nachman JB. Osteonecrosis as a complication of treating acute lymphoblastic leukemia in children: a report from the Children’s Cancer Group. J Clin Oncol 2000;18:3262-3272.

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104. Gurney JG, Kaste SC, Liu W, Srivastava DK, Chemaitilly W, Ness KK, Lanctot JQ, Ojha RP, Nottage KA, Wilson CL, Li Z, Robison LL, Hudson MM. Bone mineral density among long-term survivors of childhood acute

113. Holmqvist AS, Wiebe T, Hjorth L, Lindgren A, Øra I, Moëll C. Young age at diagnosis is a risk factor for negative late socio-economic effects after acute lymphoblastic leukemia in childhood. Pediatr Blood Cancer 2010;55:698-707.

RESEARCH ARTICLE DOI: 10.4274/tjh.galenos.2018.2018.0221 Turk J Hematol 2019;36:12-18

Investigation of the Relationship Between Fok1 and Col1A1 Gene Polymorphisms and Development of Treatment-Related Bone Complications in Children with Acute Lymphoblastic Leukemia Akut Lenfoblastik Lösemili Çocuklarda Fok1 ve Col1A1 Gen Polimorfizmleri ile Tedaviye Bağlı Kemik Komplikasyonlarının Gelişimi Arasındaki İlişkinin Araştırılması Melek Erdem1, Hale Ören1

Özlem Tüfekçi1,

Sefa Kızıldağ2,

Şebnem Yılmaz1,

Deniz Kızmazoğlu1,

Berna Eroğlu Filibeli3,

1Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey 2Dokuz Eylül University Faculty of Medicine, Department of Medical Biology, İzmir, Turkey 3Dokuz Eylül University Facullty of Medicine, Department of Pediatrics, İzmir, Turkey

Abstract

Öz

Objective: In acute lymphoblastic leukemia (ALL), various clinical risk factors and genetic predispositions contribute to the development of bone complications during and after chemotherapy. In this study, we aimed to investigate whether vitamin D receptor (VDR) Fok1 and collagen protein Col1A1 Sp1-binding site gene polymorphisms, which are important in bone mineral and matrix formation, have effects on the development of bone abnormalities in childhood ALL survivors.

Amaç: Akut lenfoblastik lösemide (ALL), çeşitli klinik risk faktörleri ve genetik yatkınlıklar kemoterapi sırasında ve sonrasında kemik komplikasyonlarının gelişmesine katkıda bulunur. Bu çalışmada, kemik mineral ve matriks oluşumunda önemli olan D vitamini reseptörü (VDR) Fok1 ve kollajen proteini Col1A1 Sp1 bağlayıcı bölge gen polimorfizmlerinin, ALL’den sağ kalan çocuklarda kemik anormalliklerinin gelişimine etkilerinin olup olmadığını araştırmayı amaçladık.

Materials and Methods: Fifty children with ALL who were treated with the ALL Berlin-Frankfurt-Muenster-95 protocol between 1998 and 2008 and were followed for at least 7 years were enrolled. The control group consisted of 96 healthy children. VDR Fok1 and Col1A1 Sp1-binding site gene polymorphisms were analyzed by polymerase chain reaction and restriction fragment length polymorphism. Bone mineral density (BMD) and markers of bone metabolism were all noted. All patients who presented with pain in the joints were examined for bone pathologies while on chemotherapy or during long-term follow-up.

Gereç ve Yöntemler: 1998-2008 yılları arasında ALL tanısı alıp, ALL Berlin-Frankfurt-Muenster-95 protokolü ile tedavi edilen ve en az 7 yıl takip edilen 50 çocuk çalışmaya dahil edildi. Kontrol grubu 96 sağlıklı çocuktan oluşuyordu. VDR Fok1 ve Col1A1 Sp1 bağlayıcı bölge gen polimorfizmleri polimeraz zincir reaksiyonu ve restriksiyon fragman uzunluğu polimorfizmi ile analiz edildi. Kemik mineral yoğunluğu (KMY) ve kemik metabolizması belirteçleri kaydedildi. Eklemlerde ağrı ile başvuran tüm hastalar kemoterapi veya uzun süreli takip sırasında kemik patolojileri açısından incelendi.

Results: Low BMD (16%), osteoporosis (12%), and osteonecrosis (8%) were present in a total of 18 patients (36%). The frequency of osteonecrosis and total bone abnormalities was significantly higher in children aged ≥10 years (p=0.001). The risk of low BMD and osteonecrosis was higher in those with vitamin D deficiency. Only the Col1A1 Sp1-binding site gene polymorphism showed a significant association in ALL patients with osteonecrosis.

Bulgular: Toplam 18 hastada (%36) düşük KMY (%16), osteoporoz (%12) ve osteonekroz (%8) mevcuttu. Osteonekroz sıklığı ve toplam kemik anormallikleri 10 yaş ve üstü çocuklarda anlamlı olarak yüksek bulundu (p=0,001). D vitamini eksikliği olanlarda düşük BMD ve osteonekroz riski daha yüksekti. Gen polimorfizmleri ile ilişki incelendiğinde; sadece Col1A1 Sp1 bağlayıcı bölge gen polimorfizmi ile osteonekroz arasında anlamlı bir ilişki gösterilmiştir.

Conclusion: The development of therapy-induced bone mineral loss and osteonecrosis in children with ALL is frequent and the risk is especially higher in children aged ≥10 years and with vitamin D deficiency. The association between Col1A1 Sp1-binding site gene polymorphisms and osteonecrosis has to be assessed in a larger group of ALL survivors.

Sonuç: Sonuç olarak, ALL’den sağ kalan çocuklarda tedaviye bağlı kemik mineral kaybı ve osteonekroz gelişimi sıktır ve bu risk özellikle 10 yaş ve üstü D vitamini eksikliği olan çocuklarda daha yüksektir. Col1A1 Sp1 bağlanma bölgesi gen polimorfizmleri ve osteonekroz arasındaki ilişkiyi araştırmak için ALL’den sağ kalan bireyler üzerinde yapılacak daha geniş çaplı çalışmalara ihtiyaç vardır.

Keywords: Acute lymphoblastic leukemia, Bone mineral density, Genetic polymorphism, Osteonecrosis, Osteoporosis

Anahtar Sözcükler: Akut lenfoblastik lösemi, Kemik mineral dansitesi, Genetik polimorfizm, Osteonekrozis, Osteoporozis

Address for Correspondence/Yazışma Adresi: Özlem TÜFEKÇİ, M.D., Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey Phone : +90 505 525 21 44 E-mail : ozlemtufekci@hotmail.com ORCID-ID: orcid.org/0000-0002-0721-1025

Received/Geliş tarihi: July 04, 2018 Accepted/Kabul tarihi: September 24, 2018

Turk J Hematol 2019;36:12-18

Erdem M, et al: Fok1 and Col1A1 Gene Polymorphisms in Children with Acute Lymphoblastic Leukemia

Introduction

Materials and Methods

Cure rates for childhood acute lymphoblastic leukemia (ALL) have approached 90% with therapeutic advances over the last several decades and the number of survivors has dramatically increased over the last decades [1,2]. Many treatment-related long-term complications including impaired physical growth, neurocognitive dysfunction, cardiac abnormalities, secondary neoplasms, low bone mineral density (BMD), osteoporosis, and osteonecrosis have been reported [3,4,5]. Bone infiltration of leukemic cells, corticosteroids, methotrexate (MTX) and asparaginase exposure, poor nutrition, low vitamin D, poor muscle mass, and genetic predisposition contribute to the development or worsening of bone pathologies during or after therapy [3,4]. Multiple clinical risk factors including female sex, administration of dexamethasone, and age have also been identified to have roles in the development of osteoporosis and osteonecrosis [5,6,7,8,9,10].

Study Design and Patients

Corticosteroids, which play a critical role in ALL therapy, directly affect bone and negatively impact the skeleton by altering the hormonal axis, intestinal calcium absorption, and renal excretion of calcium. Multiple candidate gene studies have indicated several polymorphisms in genes putatively related to the development of osteonecrosis, such as SERPINE 1, vitamin D receptor (VDR), and CYP3A4 [11,12]. Vitamin D plays a major role in calcium, phosphorus, and bone metabolism and thus is an important variable in the assessment of bone health [13]. Vitamin D is an important factor that mediates its action in the body through VDR, which helps in calcium uptake or bone formation like calcium binding proteins and osteocalcin [14]. The VDR Fok1 locus polymorphism is considered to be a potential regulator of bone and calcium metabolism. Some studies have suggested significant association of Fok1 locus polymorphism with low BMD in girls, whereas others showed no such association [15,16,17,18]. The main component of bone mineral is calcium, and for bone matrix, it is collagen. Osteoporosis is mainly due to the loss of calcium and collagen degradation [19]. The Col1A1 gene encodes the alpha-1 protein chain of type I collagen, the major protein of bone [20]. Some research has focused on the Col1A1 Sp1-binding site polymorphism and Col1A1 upstream regulatory region single nucleotide polymorphisms, mainly because they can regulate the expression of the Col1A1 gene. These polymorphisms have been significantly associated with low BMD, osteoporosis, and increased fracture risk [19,20,21,22,23]. In this study, we aimed to investigate whether VDR Fok1 and collagen protein Col1A1 Sp1-binding site gene polymorphisms, which are important in bone mineral and matrix formation, have effects on the development of bone abnormalities in childhood ALL survivors.

Fifty children with ALL who were diagnosed and treated with the ALL Berlin-Frankfurt-Muenster (BFM) 95 protocol [24] between 1998 and 2008 and were followed for at least 7 years after cessation of therapy were enrolled in this study. The control group consisted of 96 healthy children of similar age and sex. The children in the control group had no malignant tumors, chronic diseases, or musculoskeletal system symptoms and also had no evidence of vitamin D deficiency or hypocalcemia. In the ALL-BFM 95 protocol, patients were stratified according to age, initial white blood cell count, day 8 response to prednisone, immunophenotype, and molecular rearrangements such as t(9;22) and t(4;11) into standard-risk (SRG), medium-risk (MRG), and high-risk (HRG) groups. SRG and MRG therapy consisted of an 8-drug induction including prednisone, consolidation with a four-times higher dose of MTX, and an 8-drug reintensification including dexamethasone. HRG patients were treated with a 5-drug induction including prednisone, followed by six intensive multiagent blocks; reintensification was similar to that for the SRG and MRG patients. Maintenance therapy consisted of daily 6-MP and weekly MTX and was continued until 2 years after initial diagnosis. Patients were evaluated for age, sex, risk group, relapse, hemopoietic stem cell transplantation (HSCT), bone metabolism markers (serum calcium, phosphorus, alkaline phosphatase, parathyroid hormone, and 25-hydroxyvitamin D levels), bone changes (clinical and radiographic findings), and Fok1 and Col1A1 Sp1-binding site gene polymorphisms. Vitamin D levels below 20 ng/mL were considered as deficiency. In this study, low BMD, osteoporosis, and osteonecrosis were investigated as bone complications. BMD and markers of bone metabolism were all screened routinely before initiation of maintenance treatment and were studied whenever clinically needed. All patients who presented with pain in the joints were examined for bone pathologies while on chemotherapy or during long-term follow-up. All of the data were noted and collected from hospital records. Diagnosis of osteonecrosis had been made based on symptoms, clinical exam findings, and radiographic studies, including plain radiographs and magnetic resonance imaging (MRI). Diagnosis of low BMD and osteoporosis was made based on clinical findings, dual-energy X-ray absorptiometry (DEXA), and radiographic studies [25]. The BMDs were measured in g/cm2 and converted to Z-scores, which represent deviation from age-matched and sex-matched normative BMDs. Mean values and standard deviations (SDs) were calculated for each patient. We deﬁned osteoporosis as a BMD ≤2 SD below the mean (Z ≤ -2) and low BMD as a BMD that is abnormal but not >2 SD below the mean (-2> Z ≤0) [36]. The term “osteopenia” was not used since “low BMD” is the preferred term for pediatric DEXA reports [26]. In our hospital 13

Erdem M, et al: Fok1 and Col1A1 Gene Polymorphisms in Children with Acute Lymphoblastic Leukemia

Turk J Hematol 2019;36:12-18

DEXA results were assessed by measuring mineral density in the L1-L4 vertebra corpus; therefore, the Z-scores in the definition of osteoporosis and low BMD were obtained by this regional analysis. The proximal femur or hip region was included according to clinical and radiologic findings.

chi-square analysis. In chi-square analysis, Fisher’s exact test, Pearson’s chi-square test, the continuity correction test, and the Fisher-Freeman-Halton test were applied according to expected values and group numbers. Statistical significance was accepted at p<0.05.

Informed consent was obtained from the parents of all patients. This study was approved by the Ethics Committee of the Dokuz Eylül University Faculty of Medicine and the work described was carried out in accordance with the Declaration of Helsinki for experiments involving humans.

Results

Genotyping Peripheral blood samples from all individuals were collected into sterile tubes containing 0.1 M EDTA and stored at -20 °C. Genomic DNA was extracted using the NucleoSpin Blood Extraction Kit (Macherey-Nagel, Duren, Germany). The genotypes were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The polymorphic locus was amplified using 5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′ as the forward and 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′ as the reverse primer. PCR (Thermocycler; MJ Research PTC-200) was performed with 35 cycles by the following steps: denaturation at 94 °C for 30 s, annealing at 61 °C for 30 s, and extension at 72 °C for 1 min. After amplification the 256-bp PCR product was digested with FastDigest Fok1 restriction endonuclease for 5 min. Digested products were analyzed on 2% agarose gel stained with ethidium bromide. The sizes of the bands were estimated using a 100-bp ladder. The genotyping was done on the basis of the presence or absence of the Fok1 site as follows: FF=265 bp; Ff=265 bp, 169 bp, and 96 bp; and ff=169 bp and 96 bp lengths, respectively. The absence of a restriction site is represented by F while the presence of a restriction site is represented by f.

The general characteristics of the patients are shown in Table 1. The median age at diagnosis was 61.5 months (min. 11, max. 204 months). Five (10%) patients had to be treated with HSCT. The median age of the patients at the time of first DEXA was 96 months (min. 35, max. 196 months). The median and mean DEXA Z-scores were 0.145 and 0.63 g/cm2, respectively. Low BMD, osteoporosis, and osteonecrosis were present in 18 patients (36%) (Table 2). Lumbosacral vertebras, femur heads, knees, and sacroiliac joints were the most affected areas on MRI. Osteonecrosis was present in one patient at the bilateral sacroiliac joints, one patient at the L4-L5 area, and two patients at the head of the left and right femur, respectively. The sex distribution among patients with bone features was not statistically significant. Rates of osteonecrosis and total bone changes were significantly higher in patients aged ≥10 years (p=0.001, p=0.029, respectively) (Table 2). When the bone metabolism markers were examined, hypocalcemia was observed in 7 patients, hypophosphatemia Table 1. Demographic and clinical data of patients. n

Male

Female

Pre-B-cell ALL

T-cell ALL

0-10

≥10

SRG

Sex

The guanine (G) to thymidine (T) gene polymorphism in the Sp1binding site in the first intron of the Col1A1 gene was determined by a PCR-based method. The primers (MBI Fermentas, Lithuania) used for PCR to amplify Col1A1 gene fragments were as follows; forward primer 5′-TAACTTCTGGACTA TTTGCGGACTTTTTGG-3′ and reverse primer 5′-GTCCAGCCCTCATCCTGGCC-3′ for the Sp1 restriction site DNA. Genotypes for Col1A1 Sp1 polymorphisms were classified as G/G homozygotes (SS), G/T heterozygotes (Ss), and T/T homozygotes (ss).

Immunophenotypes

MRG

Statistical Analysis

HRG

Yes

Complete remission

Exitus

In statistical analysis, SPSS 23.0 and Number Cruncher Statistical System 2007 were used. Categorical variables were defined with frequency and percentage; continuous variables were given as mean deviation in parametric conditions and median (min., max.) in nonparametric conditions. In the analytical review of the data, the effects of Fok1 and Col1A1 gene polymorphisms and other characteristics of the patient on low BMD, osteoporosis, osteonecrosis, and categorical variables were investigated by 14

Age (years)

Risk groups

Relapse

Results

SRG: Standard-risk group, MRG: medium-risk group, HRG: high-risk group, ALL: acute lymphoblastic leukemia.

Erdem M, et al: Fok1 and Col1A1 Gene Polymorphisms in Children with Acute Lymphoblastic Leukemia

Turk J Hematol 2019;36:12-18

was observed in 14 patients, and alkaline phosphatase elevation was observed in 12 patients. No significant difference was found in terms of risk of developing low BMD, osteoporosis, osteonecrosis, and total bone changes according to these markers. However, the risk of low BMD, osteonecrosis, and total bone changes was higher in those with vitamin D deficiency and this was statistically significant (p=0.003, p=0.004, p=0.001, respectively) (Table 3). There was no relationship between any of these bone changes and parameters that could change the duration of treatment, such as ALL risk groups, relapse status, and HSCT. Only one patient had osteonecrosis in our HSCT group. Distribution of Fok1 and Col1A1 Sp1-binding site gene polymorphisms in both groups is shown in Table 4. Fok1 polymorphism shows a significant difference between patients and the control group. In terms of the mentioned bone changes, Col1A1 gene and Fok1 Sp1-binding site gene polymorphisms did not show a significant correlation between BMD values and Z-scores. The distribution of Fok1 and Col1A1 Sp1-binding site gene polymorphisms according to low BMD, osteoporosis, and osteonecrosis is shown in Table 5. Only gene polymorphism in the Sp1-binding site of Col1A1 showed a significant association in patients with osteonecrosis (p=0.045).

On follow-up, 2 of the 18 patients needed surgical operation; others received calcium and vitamin D supplements with or without bisphosphonate replacement therapy for better quality of life. All of the patients with low BMD and osteoporosis exhibited an increase in BMD values and Z-scores in the longterm follow-up period.

Discussion In children who had completed therapy for ALL, the prevalence of BMD abnormalities was reported to be as high as 93% and the incidence for asymptomatic osteonecrosis was found to vary between 15% and 38% among survivors [27,28,29]. Recently, Vitanza et al. [27] demonstrated that 46.6% of children exhibited osteoporosis in at least one anatomic site at some time during the first 6 years after chemotherapy. In our study, low BMD (32%), osteoporosis (24%), and osteonecrosis (16%) were present in 18 out of 50 patients (36%) who were followed for more than 7 years for these features. Lumbosacral vertebras, femur heads, knees, and sacroiliac joints were the most affected areas on MRI, as identified in the literature [28,29,30]. Prior studies reported multiple clinical risk factors for the development of osteonecrosis, including female sex, older age, administration of 3 weeks of continuous rather than alternate week dexamethasone during delayed intensiﬁcation,

Table 2. Distribution of bone abnormalities according to age and sex. Low bone mineral density Osteoporosis Osteonecrosis Total

Male, n (%)*

Female, n (%)

Yes

7 (28.0)

1 (4.00)

18 (72.0)

24 (96.0)

Yes

1 (4.00)

5 (20.0)

24 (96.0)

20 (80.0)

Yes

1 (4.00)

3 (12.0)

24 (96.0)

22 (88.0)

Yes

9 (36.0)

16 (64.0)

p** 0.051 1.891 0.601 1.002

<10 years, n (%)*

≥10 years, n (%)*

5 (12.8)

3 (27.0)

34 (87.2)

8 (72.8)

4 (10.0)

2 (18.0)

35 (90.0)

9 (72.0)

0 (0.00)

4 (36,3)

39 (100.0)

7 (63.7)

9 (23.0)

9 (82.0)

30 (77.0)

2 (18.0)

p** 0.353 0.601 0.0011 0.0293

*Column percentage. **,1Fisher’s exact test, 2Pearson’s chi-square test, 3continuity correction test.

Table 3. Correlation between vitamin D deficiency and bone changes.

Table 4. Distribution of Fok1 polymorphism and Col1A1 polymorphism in patients and control group.

Gene polymorphisms

Low bone mineral density Osteoporosis Osteonecrosis Total

Vitamin D deficiency Yes, n (%)*

No, n (%)*

Yes

4 (66.7)

4 (9.1)

2 (33.3)

40 (90.9)

Yes

1 (16.7)

5 (11.4)

5 (83.3)

39 (88.6)

Yes

3 (50.0)

1 (2.3)

3 (50.0)

43 (97.7)

Yes

8 (100)

10 (22.7)

0 (0.00)

34 (77.3)

*Column percentage, **Fisher’s exact test.

p** 0.003 0.556

Patients, n (%)*

Controls, n (%)*

4 (8)

18 (18.8)

23 (46)

59 (61.5)

23 (46)

19 (19.8)

31 (62)

53 (55.2)

13 (26)

27 (28.1)

6 (12)

16 (16.7)

p**

Fok1 genotypes 0.003

Col1A1 genotypes 0.004 0.001

0.667

*Line percentage, **Pearson’s chi-square test.

Erdem M, et al: Fok1 and Col1A1 Gene Polymorphisms in Children with Acute Lymphoblastic Leukemia

Table 5. Fok1 and Col1A1 genotypes in children with bone abnormalities. Fok1 gene polymorphisms Low bone mineral density Osteoporosis Osteonecrosis Total

ff, n (%)*

Ff, n (%)*

FF, n (%)*

Yes

0 (0.0)

6 (26.0)

2 (8.60)

4 (100)

17 (74.0)

21 (91.4)

Yes

1 (25.0)

1 (4.30)

4 (17.4)

3 (75.0)

22 (95.7)

19 (82.6)

Yes

1 (25.0)

2 (8.60)

1 (4.30)

3 (75.0)

21 (91.4)

22 (95.7)

Yes

2 (50.0)

8 (34.8)

6 (26.1)

2 (50.0)

15 (65.2)

17 (73.9)

p** 0.271 0.243 0.368 0.597

Turk J Hematol 2019;36:12-18

Col1A1 gene polymorphisms GG, n (%)*

GT, n (%)*

TT, n (%)* p**

5 (16.1)

3 (23.0)

0 (0.0)

26 (83.9)

10 (77.0)

6 (100)

3 (9.70)

1 (7.70)

2 (33.3)

28 (90.3)

12 (92.3)

4 (66.7)

1 (3.20)

1 (7.70)

2 (33.3)

30 (96.8)

12 (92.3)

4 (66.7)

9 (29.0)

4 (30.7)

3 (50.0)

22 (71.0)

9 (69.3)

3 (50.0)

0.537 0.274 0.045 0.537

*Column percentage, **Fisher-Freeman-Halton.

and intensive therapy [9,10,31,32,33]. We found no relationship between these bone changes and specific risk factors such as sex distribution, duration of treatment, ALL risk groups, and relapse status. As reported in the literature, the risk of osteonecrosis and other bone complications was significantly higher in patients aged ≥10 years in our study. Age remains the strongest and most consistently identiﬁed factor, with patients 10 to 20 years old at greatest risk [5,6,7,8,9,10,27,33]. In a retrospective report on the ALL-BFM-95 trial, the osteonecrosis incidence was reported to be 8.9% in patients aged ≥10 years and even higher in those ≥15 years (16.7%) [9]. The results also did not show female sex as a significant risk factor for developing osteonecrosis and higher incidences were found to be accompanied by higher total steroid doses. For the first time, Krull et al. [30] recently demonstrated that asymptomatic osteonecrosis develops independently of radiological leukemic infiltration of bone in adolescents with ALL. Different studies have reported a list of genes effective on osteoporosis, such as VDR, Col1A1, estrogen receptor alpha, interleukin-6, and LDL receptor-related protein 5 [16,17]. The relationships between Col1A1 Sp1 polymorphism and BMD were investigated among various populations. Previous studies have shown associations between Col1A1 Sp1 polymorphisms and low BMD, osteoporosis, and increased fracture risk [20,21,22,23,34,35], while some have not reached statistical significance [36,37]. In healthy prepubertal children, there have only been a small number of studies examining possible effects of Col1A1 gene polymorphisms on BMD [38,39,40]. In our study, only gene polymorphism in the Sp1-binding site of Col1A1 showed a significant association in ALL patients with osteonecrosis, but not for other bone abnormalities. Since the main component of bone matrix is collagen, this may be an important finding that has to be assessed in a larger group of ALL survivors. VDR Fok1 locus polymorphism is considered to be a potential regulator of bone and calcium metabolism. Studies on VDR gene 16

polymorphisms of the Fok1 locus and its associations with bone mass in children have shown varied results [17,18,41,42,43]. Of note, Fok1 locus polymorphism was found to be significantly different between patients and the control group in our study. This may have resulted from the relatively limited number of subjects in both groups. We found no significant association between Fok1 genotypes and low BMD, osteoporosis, or osteonecrosis in children with ALL. In our study, the risk of low BMD, osteonecrosis, and total bone changes was higher in patients with vitamin D deficiency. Potential risk factors for decreased vitamin D in survivors of childhood cancer include poor diet, more time spent indoors, less physical activity, and administration of chemotherapy, steroids, and radiation therapy [44]. The prevalence of decreased vitamin D in children with cancer is high (29%-35%) but quite similar to what has been demonstrated in the general population [44,45]. A randomized double-blind study showed that nutritional counseling and vitamin D and calcium supplementation for 2 years offered no benefit for improving BMD among adolescent and young adult survivors of ALL [46]. Recently, a similar result was reported from a Turkish study group [47]. Depending on these findings, alternative and more aggressive strategies are needed to prevent these patients from experiencing bone complications.

Conclusion The development of therapy-induced bone mineral loss and osteonecrosis in children with ALL is frequent and the risk is higher especially in children aged ≥10 years and with vitamin D deficiency. The association between Col1A1 Sp1-binding site gene polymorphisms and osteonecrosis has to be assessed in a larger group of ALL survivors. Studies investigating the possible underlying genetic susceptibilities to certain complications are important not only for better management of complications but also for development of new individual patient-specific treatment modalities.

Turk J Hematol 2019;36:12-18

Erdem M, et al: Fok1 and Col1A1 Gene Polymorphisms in Children with Acute Lymphoblastic Leukemia

Ethics

leukemia (ALL)-experiences from trial ALL-BFM 95. Pediatr Blood Cancer 2005;44:220-225.

Ethics Committee Approval: This study was approved by the Ethics Committee of the Dokuz Eylül University Faculty of Medicine.

10. Patel B, Richards SM, Rowe JM, Goldstone AH, Fielding AK. High incidence of avascular necrosis in adolescents with acute lymphoblastic leukaemia: a UKALL XII analysis. Leukemia 2008;22:308-312.

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

11. French D, Hamilton LH, Mattano LA Jr, Sather HN, Devidas M, Nachman JB, Relling MV; Children’s Oncology Group. A PAI-1 (SERPINE1) polymorphism predicts osteonecrosis in children with acute lymphoblastic leukemia: a report from the Children’s Oncology Group. Blood 2008;111:4496-4499.

Authorship Contributions Surgical and Medical Practices: M.E., Ö.T., Ş.Y., D.K., H.Ö.; Concept: M.E., Ö.T., H.Ö.; Design: M.E., Ö.T., S.K., H.Ö.; Data Collection or Processing: M.E., Ş.Y., D.K., B.E.F.; Analysis or Interpretation: M.E., Ö.T., S.K., Ş.Y., H.Ö.; Literature Search: M.E., Ö.T., S.K., H.Ö.; Writing: M.E., Ö.T., S.K., H.Ö. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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34. Falcón-Ramírez E, Casas-Avila L, Miranda A, Diez P, Castro C, Rubio J, Gómez R, Valdés-Flores M. MSp1 polymorphism in collagen I α1 gene is associated with osteoporosis in lumbar spine of Mexican women. Mol Biol Rep 2011;38:2987-2992. 35. MacDonald HM, McGuigan FA, New SA, Campbell MK, Golden MH, Ralston SH, Reid DM. COL1A1 Sp1 polymorphism predicts perimenopausal and early postmenopausal spinal bone loss. J Bone Miner Res 2001;16:16341641. 36. Ashford RU, Luchetti M, McCloskey EV, Gray RL, Pande KC, Dey A, Kayan K, Ralston SH, Kanis JA. Studies of bone density, quantitative ultrasound, and

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RESEARCH ARTICLE DOI: 10.4274/tjh.galenos.2018.2018.0015 Turk J Hematol 2019;36:19-24

Evaluation of Cardiac Parameters in Bone Marrow Transplant Patients: Effect of Pulmonary Artery Pressure on Survival Kemik İliği Nakli Yapılan Hastalarda Kardiyak Parametrelerin Değerlendirilmesi: Pulmoner Arter Basıncının Sağkalıma Etkisi Ali Caner Özdöver1,

İlknur Gündeş1,

Melya Pelin Kırık1,

Handan Haydaroğlu Şahin2,

Murat Sucu3,

Mustafa Pehlivan4

1Gaziantep University Faculty of Medicine, Department of Internal Medicine, Gaziantep, Turkey 2Gaziantep University Faculty of Medicine, Department of Hematology, Gaziantep, Turkey 3Gaziantep University Faculty of Medicine, Department of Cardiology, Gaziantep, Turkey 4Gaziantep University Faculty of Medicine, Department of Hematology, Bone Marrow Transplant Unit, Gaziantep, Turkey

Abstract

Öz

Objective: Hematopoietic stem cell transplantation (HSCT) is a choice of treatment for malignant and non-malignant diseases. After HSCT, some complications may develop in patients. Cardiac complications are particularly important. The aim of this study was to investigate whether systolic pulmonary artery pressure (PAP) is a marker for overall survival (OS) in HSCT patients.

Amaç: Hematopoietik kök hücre transplantasyonu (HKHT) malign ve non-malign hastalıklarda kullanılan bir tedavi seçeneğidir. Nakil sonrasında bir çok komplikasyon gelişmektedir. Kardiyak komplikasyonlar önemli bir yer tutmaktadır. Bu çalışmanın amacı, sistolik pulmoner arter basıncının (PAB) nakil yapılan hastaların sağkalımına olan etkisini araştırmaktır.

Materials and Methods: In our study, 428 HSCT patients were evaluated. Ejection fraction (EF) and PAP values were investigated during symptom-oriented echocardiography in the pre-HSCT and post-HSCT periods.

Gereç ve Yöntemler: Çalışmamızda 428 HKHT yapılan hasta değerlendirildi. Ejeksiyon fraksiyonu (EF) ve PAB değerleri, semptom oryante ekokardiyografi ile post ve pretransplantasyon dönemde değerlendirildi.

Results: Pre-HSCT EF values were similar between the groups. In patients with autologous HSCT (auto-HSCT) (PAP >25 mmHg), it was found that the 5-year mortality rate was 48.6%, while in the other group (PAP <25 mmHg) the 5-year mortality was 25.5%. There was a significant association between 5-year mortality rate and PAP level (p<0.046) in the auto-HSCT group. OS was 38% in the pre-autoHSCT period with PAP values of >25 mmHg, while OS was 61% in the pre-auto-HSCT period with PAP values of <25 mmHg (p<0.001). We determined that there was a statistically significant difference between OS and PAP levels in patients with auto-HSCT. Five-year mortality rate and OS were not significantly different in patients undergoing allogeneic HSCT (allo-HSCT) (p>0.05).

Bulgular: Karşılaştırılan gruplar arasında pre-HKHT döneminde EF değerleri açısından farklılık saptanmadı. Beş yıllık mortalite otolog HKHT yapılan hastalarda PAB değeri 25 mmHg üzeri olanlarda %48,6, diğer grupta %25,5 olarak gözlendi ve istatistiksel olarak p<0,046 olarak hesaplandı. Total sağkalıma bakıldığında otolog HKHT yapılan hastalarda; nakil öncesi PAB değeri 25 mmHg üzeri olanlarda %38 iken; nakil öncesi PAB değeri 25 mmHg altında olan grupta sağkalım %61 olarak saptandı ve bu durum istatistiksel olarak anlamlı hesaplandı (p<0,001). Aynı verileri değerlendirerek total sağkalım ve PAB değeri arasında otology HKHT yapılan hastalarda anlamlı ilişki oldugunu, allojenik HKHT yapılanlar arasında istatistiksel olarak anlamlı ilişki olmadığını tespit etmiş bulunmaktayız.

Conclusion: Our results suggest that pre-HSCT PAP value is an important risk factor for mortality and OS in patients undergoing auto-HSCT.

Sonuç: Çalışmamızın sonuçlarına göre HKHT öncesi ve sonrası ölçülen PAB değeri hastalarda total sağkalım ve mortalite üzerine önemli etkisi olan bir faktör olarak karşımıza çıkmaktadır.

Keywords: Hematopoietic stem cell transplantation, Pulmonary artery pressure, Overall survival

Anahtar Sözcükler: Hematopoietik kök hücre transplantasyonu, Pulmoner arter basıncı, Total sağkalım

Address for Correspondence/Yazışma Adresi: İlknur GÜNDEŞ, M.D., Gaziantep University Faculty of Medicine, Department of Internal Medicine, Gaziantep, Turkey Phone : +90 530 010 59 77 E-mail : iknur_88_@hotmail.com ORCID-ID: orcid.org/0000-0003-3940-6705

Received/Geliş tarihi: January 08, 2018 Accepted/Kabul tarihi: October 24, 2018

Özdöver AC, et al: Effect of Pulmonary Artery Pressure on Survival

Turk J Hematol 2019;36:19-24

Introduction

Materials and Methods

Hematopoietic stem cell transplantation (HSCT) is used in the treatment of life-threatening malignant and non-malignant diseases. Allogeneic HSCT (allo-HSCT) can provide an advantage for overall survival (OS) in 15%-20% of patients with acute leukemia after induction therapy and this rate may increase to 35% when HSCT is applied during the first relapse and second remission [1]. Autologous HSCT (auto-HSCT) is a good choice of treatment for multiple myeloma; it can be applied in first- and second-line treatment and can also be used in patients with lymphoma as an effective treatment [1].

Research Strategy

Short- and long-term complications can develop after HSCT. These include nausea, vomiting, pneumonia, thyroiditis, and cardiovascular side effects [2]. Cardiac complications such as pericarditis, arrhythmia, pulmonary edema, heart failure, and sudden cardiac death developing within the first 100 days of HSCT are considered as acute cardiotoxicity. Studies have shown that post-transplant acute cardiac complications have 1.2% mortality and morbidity ranging from 5% to 43% [3]. Heart failure (HF) is the most serious of cardiac complications. HF is defined as a 10% decrease in the ejection fraction (EF) or EF of less than 50% before HSCT [4]. Clinical findings such as orthopnea, paroxysmal nocturnal dyspnea, exercise intolerance, night cough, wheezing, palpitations, and syncope may develop in HF. To date, there is no medical treatment that can regenerate scar tissue in the routine management of HF, thus increasing mortality and morbidity [5]. It is therefore important to detect HF early. In our study, all of the patients were evaluated for HF with echocardiography. Pulmonary hypertension (PH) is defined as pulmonary artery pressure (PAP) of more than 25 mmHg at rest. Right heart catheterization is used as the gold standard in diagnosis, but this method is not suitable for daily practical use [6]. In the past 30 years, prodigious technological improvements in echocardiography have increased its sensitivity for quantifying PAP and it is now used as a safe and available alternative to invasive catheterization [7].

In this study, 428 patients who underwent HSCT in the Bone Marrow Transplantation Unit of the Department of Hematology, Faculty of Medicine, at Gaziantep University in Turkey were investigated. The median age was 29 (range: 15-63) years in allo-HSCT patients and 54 (range: 17-76) years in the autoHSCT group. Males comprised 250 (58.4%) of the patients and 178 (41.6%) were female. In this retrospective analysis, the patients who underwent HSCT once between 2009 and 2016 were evaluated using the official Medulla system and by scanning files from the archives. Ethics committee approval was obtained from the Gaziantep University Medical Faculty Ethics Committee with decision number 4.4.2016/103. This study was conducted in accordance with the World Medical Association’s 2000 Declaration of Helsinki. Methods EF and PAP values were evaluated during symptom-oriented echocardiography (Vivid 9, GE, Norway) examinations performed in the pre-HSCT and post-HSCT periods. Continuous wave Doppler imaging of the tricuspid regurgitation (TR) trace was used to measure the difference in pressures between the right ventricle and right atrium. The simplified Bernoulli equation (p=4[TRmax]2) was used to calculate this pressure difference using peak TR velocity. This method correlates well with systolic PAP for right heart catheterization [9]. The cut-off EF value was 50% and that of PAP was 25 mmHg according to the 2015 European Society of Cardiology/ European Respiratory Society Guidelines for the diagnosis and treatment of PAP [10]. Statistical Analysis The analysis of data was performed using SPSS 18.0 for Windows (SPSS Inc., Chicago, IL, USA). The statistical significance of the differences between the patient groups was estimated by logistic regression analysis. The adjusted odds ratios (ORs) were calculated with a logistic regression model that checked for sex and age and are reported with 95% confidence intervals (CIs).

To date, elevation of PAP has not been reported among the cardiac complications in the European Group for Blood and Marrow Transplantation (EBMT) guidelines. In the literature, the focus is generally on PH and mortality in the post-transplant period. A study by Dandoy et al. [8] revealed that symptoms of newly developed tachypnea, hypoxia, and respiratory failure occurred following transplant. It was reported that the mortality rate in 40 cases presented in the literature was 55% and the cause of mortality was PH and its complications in 86% of those cases.

Differences in the patients group were compared using the chisquare test and the Fisher exact test when required. For statistical comparison of groups, the median test was used. OS was defined as the time period between the time of transplantation to death due to any reason. OS evaluations were performed using the Kaplan-Meier method. A p-value of less than 0.05 was accepted as significant.

The aim of this study was to investigate whether PAP is a marker of OS for HSCT patients.

Allo-HSCT was performed for 154 (36%) and auto-HSCT was performed for 274 (64%) of the 428 patients who were

Results

Ă&#x2013;zdĂśver AC, et al: Effect of Pulmonary Artery Pressure on Survival

Turk J Hematol 2019;36:19-24

enrolled in this study. General characteristics of the patients are summarized in Table 1. The patients who had undergone auto-HSCT were mostly diagnosed with multiple myeloma 175 (40.9%), followed by 87 (22.4%) with non-Hodgkin lymphoma. A total of 92 (21.5%) patients (80 allo-HSCT and 12 auto-HSCT cases) were diagnosed with acute myeloid leukemia, being the third most common disease group. The final group of patients consisted of 27 (6.3%) patients with aplastic anemia (Table 1). When the follow-up period was taken into account, it lasted between 3 and 84 months (median: 24 months) in the allo-HSCT group and between 3 and 88 months (median: 30 months) in the auto-HSCT group. In patients who underwent allo-HSCT, pre-transplant EF values were between 43% and 67% (median: 60%), while EF values in patients with auto-HSCT ranged between 35% and 70% (median: 60%). In the post-transplant period, 105 patients were evaluated using echocardiography, and 3 (2%) patients in the allo-HSCT group and 8 patients (2.9%) in the auto-HSCT group had lower EF values before transplantation. As a result of this study, it was found that EF values increased by more than 10% in post-transplant evaluations in 2 out of 45 patients (4.4%) in the allo-HSCT group and 2 out of 60 patients (3.3%) in the auto-HSCT group. In stem cell treatment, multipotent bone marrow-derived mesenchymal stem cells appear to be the most promising candidates. Bone marrow-derived mesenchymal stem cells increase proliferation and attenuate scar tissue [11]. The EF increases detected in our study were thought to be due to regeneration. Table 1. General characteristics of the patients undergoing hematopoietic stem cell transplantation. Allogeneic HSCT

Autologous HSCT

Number of patients

154

274

Age at the time of diagnosis*

29 (15-63)

54 (17-76)

Male/female

PAP was higher than 25 mmHg in 35 patients (12.8%) in the auto-HSCT group and in 22 (14.3%) in the allo-HSCT group. In the pre-transplant period, 105 patients were evaluated using percentage of forced expiratory volume in one second (FEV1 %), with a median of 83% (80%-95%) in the allo-HSCT group and 89% (82%-97%) in the auto-HSCT group. Tricuspid and mitral regurgitation ratings are shown in Table 2. Table 3 shows the association of PAP elevation with survival and effect of engraftment in auto-HSCT patients. In auto-HSCT patients, 100day mortality and 12-month mortality were not statistically significantly different between the groups with high PAP and normal PAP values (p>0.05). Five-year mortality rates were 48.6% (17 patients) and 10.9% (26 patients) in patients with a PAP of >25 mmHg and normal PAP values, respectively. This difference was statistically significant (p<0.05). Furthermore, 5-year survival was 38% (median: 33.6 months) in 35 patients Table 2. Pulmonary and cardiac characteristics of the patients undergoing hematopoietic stem cell transplantation. Allogeneic HSCT

Autologous HSCT

Number of patients

154

274

EF*

60 (43-67)

60 (35-70)

EF <50%

3 (2%)

8 (2.9%)

EF >10% increase

2/45 (4.4%)

2/60 (3.3%)

FEV1 %*

83 (80-95)

89 (82-97)

Tricuspid regurgitation >grade 1

2 (1.3%)

10 (3.6%)

Mitral regurgitation >grade 1

1 (0.7%)

3 (1.1%)

PAP mmHg >25

22 (14.3%)

35 (12.8%)

*Median. EF: Ejection fraction, PAP: pulmonary artery pressure, FEV1 %: percentage of forced expiratory volume in one second, HSCT: hematopoietic stem cell transplantation.

Table 3. Association between pulmonary artery pressure and mortality and survival in patients who underwent autologous hematopoietic stem cell transplantation. PAP >25 mmHg

PAP <25 mmHg

p-value

Number of patients

35/274 (12.8%)

239/274

Age at the time of diagnosis

63 (39-74)

53 (17-76)

Male/female

17/18

140/99

Neutrophil engraftment* (days)

11 (8-28)

11 (10-24)

0.326#

93/61

157/117

AML

ALL

NHL

175

Neutrophil engraftment* (days)

15 (10-21)

11 (9-20)

Platelet engraftment* (days)

13 (9-61)

12 (9-61)

Platelet engraftment* (days)

13 (9-61)

12 (8-61)

0.696#

100-day mortality %

11 (7.1%)

12 (4.4%)

100-Day mortality %

3 (8.6%)

9 (3.8%)

0.347@

12-month mortality %

32 (20.8%)

33 (12%)

12-Month mortality %

7 (20%)

26 (10.9%)

0.316@

Overall 5-year survival %

Overall 5-year mortality % 17 (48.6%)

61 (25.5%)

0.046@

Follow-up duration* (months)

24 (3-84)

30 (3-88)

Diagnosis

*Median. NHL: Non-Hodgkin lymphoma, AML: acute myeloid leukemia, ALL: acute lymphoid leukemia, AA: aplastic anemia, MM: multiple myeloma, HSCT: hematopoietic stem cell transplantation.

Overall 5-year survival %

38 61 (33.6 months)*

0.001&

&Log

rank test; @OR (95% CI) was adjusted by age and sex; *median; #median test. PAP: Pulmonary artery pressure.

Ă&#x2013;zdĂśver AC, et al: Effect of Pulmonary Artery Pressure on Survival

with a PAP of >25 mmHg and 61% in the other group (Figure 1). This difference was statistically significant (p<0.05). When allo-HSCT transplant patients were evaluated, there was no statistically significant difference between the two groups in terms of overall survival rates (p>0.05) (Table 4).

Discussion There are many complications after HSCT. Cardiac complications rarely cause mortality and morbidity. Early cardiovascular complications may occur due to pretransplant medical history, primary diagnosis, age, associated comorbidities, mobility regimen, and transplantation type [12]. Late cardiovascular complications may develop due to

Figure 1. Five-year overall survival (38% vs. 61%) for the whole population according to pulmonary artery pressure in autologous hematopoietic stem cell transplantation group. Table 4. Association between pulmonary artery pressure and mortality and survival in patients who underwent allogeneic hematopoietic stem cell transplantation. PAP >25 mmHg

PAP <25 mmHg

Number of patients

22/154 (14.3%)

132/154

Age at the time of diagnosis

28 (15-58) 30 (15-63)

Male/female

10/12

Neutrophil engraftment* (days)

15 (10-28) 15 (10-24)

0.706#

Platelet engraftment* (days)

13 (11-61)

13 (9-61)

0.990#

100-Day mortality %

4 (18.2%)

8 (6%)

0.075@

12-Month mortality %

6 (27.3%)

26 (19.7%) 0.440@

Overall 5-year mortality %

8 (36.3%)

42 (31.8%) 0.673@

Overall 5-year survival %

&Log

p-value

83/49

0.743&

rank test; @OR (95% CI) was adjusted by age and sex; *median; #median test. PAP: Pulmonary artery pressure.

Turk J Hematol 2019;36:19-24

cardiotoxic chemotherapy exposure, radiotherapy, and age at the time of transplantation. In particular, post-transplant cardiovascular risk increases threefold in women, and in the auto-HSCT group, the risk of cardiac dysfunction increases fourfold [12]. Therefore, pre-transplant cardiac monitoring is routinely performed. A patient who is planned to undergo HSCT has adequate cardiac reserve for transplantation when the EF is above 35%-40% [13]. In our study, only 11 patients were found to have undergone transplantation with EF below 50%. Murdych and Weisdorf [14] studied a total of 2821 patients who underwent HSCT. Cardiac complications were detected in 26 (0.9%) of these patients and 11 patients died due to HF. Decrease in EF can usually occur after treatment with cardiotoxic chemotherapeutic agents. In our study, we did not have death due to heart failure. Anthracycline-related HF risk is associated with a cumulative 500-550 mg/m2 dose in 4% and a cumulative 600 mg/m2 dose in 36%. For HSCTtreated patients, a cumulative dose of 250 mg is required for the threshold HF development limit [15]. We did not encounter anthracycline at doses exceeding the upper limits available in our study. Post-transplant beta-blockers can be used to reduce the cardiotoxic effects of anthracycline drugs [16]. The EF value increased after transplantation in 2 patients in each group. The association of this elevation with transplantation is regarded as insignificant for two reasons. The first is that no information was found on whether the patients received HF treatment, and, if so, how regular that treatment was. The second reason is that when the possibility of cardiac regeneration due to HSCT is emphasized, the probability of stem cells passing through the pulmonary artery and capillaries without any damage after transplantation is low [17]. A study by Dandoy et al. [8] revealed that symptoms of newly developed tachypnea, hypoxia, and respiratory failure occurred on the median 70th day. It was reported that the mortality rate in 40 cases presented in the literature was 55% and the cause of mortality was PH and its complications in 86% of these cases. A literature review reported that patients developed PH between 1 month and 51 years after transplantation (median: 12.6 years). In our study, no significant increase in mortality was found in the 100-day and 12-month follow-ups in both groups of allo-HSCT and auto-HSCT patients who had PAP values of >25 mmHg; however, overall 5-year survival was poor for autoHSCT patients (p<0.001). All pre-transplant patients should be evaluated with respiratory function tests. The FEV1 % value should be above 80% of the expected value, while carbon monoxide diffusion capacity (DLCO) should be greater than 50% of the expected value

Turk J Hematol 2019;36:19-24

[18,19]. In our study, the FEV1 % value was a median of 83% (80%-95%) for patients with allo-HSCT and 89% (82%-97%) for patients with auto-HSCT. The EBMT has established a risk classification for patients who have undergone allo-HSCT. The classification includes the age of the patient, the stage of disease, the type of donor, the time interval between diagnosis and transplantation, and a sex difference between donor and host [20]. In our study, we investigated the age and sex of patients and found that PAP values increase the mortality rate as an independent risk factor when age and sex are taken into the confidence interval in transplantation patients.

Conclusion The PAP value, which has not yet been investigated in acute and chronic cardiac complications, has emerged here as an important risk factor that can affect overall survival. Ethics Ethics Committee Approval: Ethics committee approval was obtained from the Gaziantep University Medical Faculty Ethics Committee with decision number 4.4.2016/103. This study was conducted in accordance with the World Medical Association’s 2000 Declaration of Helsinki. Informed Consent: Retrospective study. Authorship Contributions Concept: A.C.Ö., İ.G., M.P.; Design: A.C.Ö., İ.G., M.P., M.S.; Data Collection or Processing: A.C.Ö., İ.G., M.P., M.S., M.P.K.; Analysis or Interpretation: A.C.Ö., M.P.; Literature Search: A.C.Ö.; İ.G., M.P., M.S., M.P.K., H.H.Ş.; Writing: A.C.Ö., İ.G., M.P., M.S. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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Özdöver AC, et al: Effect of Pulmonary Artery Pressure on Survival

and Chronic Heart Failure 2012 of the European Society of Cardiology, Bax JJ, Baumgartner H, Ceconi C, Dean V, Deaton C, Fagard R, FunckBrentano C, Hasdai D, Hoes A, Kirchhof P, Knuuti J, Kolh P, McDonagh T, Moulin C, Popescu BA, Reiner Z, Sechtem U, Sirnes PA, Tendera M, Torbicki A, Vahanian A, Windecker S, McDonagh T, Sechtem U, Bonet LA, Avraamides P, Ben Lamin HA, Brignole M, Coca A, Cowburn P, Dargie H, Elliott P, Flachskampf FA, Guida GF, Hardman S, Iung B, Merkely B, Mueller C, Nanas JN, Nielsen OW, Orn S, Parissis JT, Ponikowski P; ESC Committee for Practice Guidelines. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. Eur J Heart Fail 2012;14:803869. 5. Kim J, Shapiro L, Flynn A. The clinical application of mesenchymal stem cells and cardiac stem cells as a therapy for cardiovascular disease. Pharmacol Ther 2015;151:8-15. 6. Pesto S, Begic Z, Prevljak S, Pecar E, Kukavica N, Begic E. Pulmonary hypertension - new trends of diagnostic and therapy. Med Arch 2016;70:303-307. 7. Parasuraman S, Walker S, Loudon BL, Gollop ND, Wilson AM, Lowery C, Frenneaux MP. Assessment of pulmonary artery pressure by echocardiography-A comprehensive review. Int J Cardiol Heart Vasc 2016;12:45-51. 8. Dandoy CE, Hirsch R, Chima R, Davies SM, Jodele S. Pulmonary hypertension after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013;19:1546-1556. 9. Currie PJ, Seward JB, Chan KL, Fyfe DA, Hagler DJ, Mair DD, Reeder GS, Nishimura RA, Tajik AJ. Continuous wave Doppler determination of right ventricular pressure: a simultaneous Doppler-catheterization study in 127 patients. J Am Coll Cardiol 1985;6:750-756. 10. Galiè N, Humbert M, Vachiery JL, Gibbs S, Lang I, Torbicki A, Simonneau G, Peacock A, Vonk Noordegraaf A, Beghetti M, Ghofrani A, Gomez Sanchez MA, Hansmann G, Klepetko W, Lancellotti P, Matucci M, McDonagh T, Pierard LA, Trindade PT, Zompatori M, Hoeper M. 2015 ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT). Eur Respir J 2015;46:903-975. 11. Kinkaid HY, Huang XP, Li RK, Weisel RD. What’s new in cardiac cell therapy? Allogeneic bone marrow stromal cells as “universal donor cells”. J Card Surg 2010;25:359-366. 12. Armenian SH, Sun CL, Shannon T, Mills G, Francisco L, Venkataraman K, Wong FL, Forman SJ, Bhatia S. Incidence and predictors of congestive heart failure after autologous hematopoietic cell transplantation. Blood 2011;118:6023-6029. 13. Coghlan JG, Handler CE, Kottaridis PD. Cardiac assessment of patients for haematopoietic stem cell transplantation. Best Pract Res Clin Haematol 2007;20:247-263. 14. Murdych T, Weisdorf DJ. Serious cardiac complications during bone marrow transplantation at the University of Minnesota, 1977-1997. Bone Marrow Transplant 2001;28:283-287. 15. Armenian SH, Sun CL, Vase T, Ness KK, Blum E, Francisco L, Venkataraman K, Samoa R, Wong FL, Forman SJ, Bhatia S. Cardiovascular risk factors in hematopoietic cell transplantation survivors: role in development of subsequent cardiovascular disease. Blood 2012;120:4505-4512. 16. Roziakova L, Mistrik M, Batorova A, Kruzliak P, Bojtarova E, Dubrava J, Gergel J, Mladosievicova B. Can we predict clinical cardiotoxicity with cardiac biomarkers in patients after haematopoietic stem cell transplantation? Cardiovasc Toxicol 2015;15:210-216.

ร zdรถver AC, et al: Effect of Pulmonary Artery Pressure on Survival

17. Holinski S, Heinze G, Knebel F, Borges AC, Baumann G, Rudolph B, Konertz W. Cardiac effects of experimental intravenous bone marrow cell transplantation after myocardial infarction. Ann Thorac Cardiovasc Surg 2012;18:452-457. 18. Folz RJ. Allogeneic stem cell transplant, lung disease and airflow obstruction. Am J Respir Crit Care Med 2003;168:146-147.

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19. Ho VT, Weller E, Lee SJ, Alyea EP, Antin JH, Soiffer RJ. Prognostic factors for early severe pulmonary complications after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2001;7:223-229. 20. Wang HT, Chang YJ, Xu LP, Liu DH, Wang Y, Liu KY, Huang XJ. EBMT risk score can predict the outcome of leukaemia after unmanipulated haploidentical blood and marrow transplantation. Bone Marrow Transplant 2014;49:927933.

RESEARCH ARTICLE DOI: 10.4274/tjh.galenos.2018.2018.0194 Turk J Hematol 2019;36:25-28

A Frequent Mutation in the FTL Gene Causing Hyperferritinemia Cataract Syndrome in Turkish Population Is c.-160A>G Türk Toplumunda Hiperferritinemi Katarakt Sendromuna Neden Olan FTL Genindeki Yaygın Mutasyon c.-160A>G’dir Burhan Balta1,

Murat Erdoğan1,

Aslıhan Kiraz1,

Serdal Korkmaz2,

Alperen Ağadayı3

1Kayseri Training and Research Hospital, Clinic of Medical Genetics, Kayseri, Turkey 2Kayseri Training and Research Hospital, Clinic of Hematology, Kayseri, Turkey 3Kayseri Training and Research Hospital, Clinic of Ophthalmology, Kayseri, Turkey

Abstract

Öz

Objective: Hyperferritinemia cataract syndrome (HFCS) is an autosomal dominantly inherited disease characterized by increased serum ferritin levels and bilateral cataract formation in the early period of life. Heterozygote mutations in the 5’ untranslated region of the L-ferritin gene (FTL) have been reported to cause this disease. In this study, our purpose was to research the FTL gene mutations that cause HFCS in Central Anatolia and the clinical effects of these mutations.

Amaç: Hiperferritinemi katarakt sendromu (HFKS) serum ferritin seviyelerinde artış, hayatın erken döneminde bilateral katarakt oluşumu ile karakterize otozomal dominant geçişli genetik bir hastalıktır. L-ferritin geninin (FTL) 5’ kodlanmayan bölgesindeki heterozigot mutasyonların bu hastalığa neden olduğu bildirilmiştir. Biz bu çalışmada Orta Anadolu’da HFKS’ye neden olan FTL gen mutasyonlarını ve klinik etkilerini araştırmayı amaçladık.

Materials and Methods: Seventeen patients from 6 families with high ferritin levels in performed serum measurements, those who were found to have cataracts in eye examinations, and families with vertical inheritance, since the disease is autosomal dominant, were included in the study. Exons, exon-intron boundaries, and 5’ and 3’ untranslated regions of FTL (NM_000146) were sequenced using the Sanger sequencing method. Results: The female/male ratio of the patients was 7/10. All of the patients were found to have c.-160A>G heterozygous mutation in the FTL gene. Conclusion: In the Turkish population, the prevalence of HFCS is about 1/100,000 and the commonly observed mutation is c.-160A>G mutation.

Gereç ve Yöntemler: Serum ölçümünde ferritin yüksekliği olan, göz muayenesinde kataraktı çıkan, hastalık otozomal dominant olduğu için dikey kalıtım gösteren 6 aileden 17 hasta çalışmaya dahil edildi. FTL geninin (NM_000146) ekzon, ekzon-intron bağlantı noktaları ve 5’ ve 3’ kodlamayan bölgeleri sanger dizileme yöntemi kullanılarak dizilendi. Bulgular: Hastalardaki kadın/erkek oranı 7/10 idi. Tüm hastalarda FTL geninde c.-160A>G heterozigot mutasyonu tespit edilmiştir. Sonuç: Türk popülasyonunda HFKS prevelansı yaklaşık 1/100.000’dir ve yaygın görülen mutasyon c.-160A>G’dir. Anahtar Sözcükler: Hiperferritinemi katarakt sendromu, FTL, Ferritin, Katarakt, Hiperferritinemi

Keywords: Hyperferritinemia cataract syndrome, FTL, Ferritin, Cataract, Hyperferritinemia

Introduction Cataract is defined as the opacification of the lens of the eye resulting in a decrease in vision. About 30% of congenital cataracts are due to monogenic reasons and they show autosomal dominant inheritance. To date, about 25 related

genes have been defined [1]. Hyperferritinemia cataract syndrome (HFCS) (OMIM 600886) is an autosomal dominantly inherited disease characterized by increased serum ferritin levels and bilateral cataract formation in the early period of life. It was first defined by Girelli et al. [2] and Bonneau et al. [3] and

Address for Correspondence/Yazışma Adresi: Burhan BALTA, M.D., Kayseri Training and Research Hospital, Clinic of Medical Genetics, Kayseri, Turkey Phone : +90 555 685 80 98 E-mail : burhan0629@gmail.com ORCID-ID: orcid.org/0000-0003-2672-2493

Received/Geliş tarihi: June 05, 2018 Accepted/Kabul tarihi: November 06, 2018

Balta B, et al: Frequent Mutation in the FTL Gene Causing Hyperferritinemia Cataract Syndrome

independently of each other in 1995. Heterozygote mutations in the 5’ untranslated region of the L-ferritin gene (FTL) have been reported to cause this disease [4]. To date, 37 mutations have been defined in the FTL gene [5]. There are no literature data about its prevalence in the Turkish population. However, it is estimated to be about 1:200,000 in Australia [6]. Although one case was reported from Turkey in the literature [7], there are no published research studies regarding which mutations are frequent or the clinical effects of these mutations. In this study, our purpose was to research the FTL gene mutations that cause HFCS in Central Anatolia and the clinical effects of these mutations.

Materials and Methods Study Group Seventeen patients from 6 families who were referred to the Kayseri Training and Research Hospital’s Medical Genetics Department from the Hematology and Ophthalmology Department with a prediagnosis of HFCS between June 2014 and December 2017 were included in the study. The study was carried out in the Kayseri Training and Research Hospital’s Medical Genetics Department. The study was approved by the Local Ethics Research Committee of Erciyes University with protocol number 2018/186 and conducted in accordance with the Declaration of Helsinki and good clinical practice guidelines. All subjects or their legal guardians provided written informed consent prior to participation in the study. Families with high ferritin levels in serum measurements, those who were found to have cataracts in eye examinations, and families with vertical inheritance, as the disease is autosomal dominant, were included in the study. Families with other diseases that cause high ferritin levels, such as hemochromatosis, were excluded from the study.

Turk J Hematol 2019;36:25-28

forward and the reverse primers used in the initial PCR and analyzed on an ABI 3500 Genetic Analyzer (Applied Biosystems, Hitachi, Japan). Bioinformatic analysis was conducted using the SeqScape v2.6 program.

Results Seventeen patients from 6 families were evaluated. The female/ male ratio of the patients was 7/10. The youngest patient was 1 year old, while the oldest patient was 72 years old and the average age was 33.3±19.5. Patients F1P3, F3P7, F3P8, F3P9, F5P12, and F5P16 had not undergone cataract operation yet. The patients’ cataract operation ages also differed. Patient F2P5 had been operated on at the age of 9 and patient F6P17 had been operated on at the age of 58. While the levels of ferritin were increased, the levels also differed between patients. Table 1 shows the patients’ cataract states, whether they were operated on and at what age they were operated on, information about ferritin levels, and additional findings. Ferritin levels of siblings who were under risk were checked. In addition, the patients’ total blood count and biochemical parameters are listed in Table 2. In the Sanger sequencing analysis conducted, all of the patients were found to have c.-160A>G heterozygous mutation in the FTL gene (Figure 1). This mutation is a genetic change reported in the literature previously [8]. Table 1. Patients’ cataract states, whether they were operated on and at what age they were operated on, information about ferritin levels, and additional findings. Patient Sex Age Age at cataract Ferritin FTL surgery

level (µg/L) mutation

F1P1

1354

c.-160A>G

F1P2

773

c.-160A>G

F1P3

1010

c.-160A>G

Sanger Sequencing Analysis

F2P4

1607

c.-160A>G

Genomic DNA was extracted from peripheral blood samples using a DNA isolation kit according to the manufacturer’s instructions (Zinexts Life Science Corp., Taiwan). Exons, exonintron boundaries, and 5’ and 3’ untranslated regions of FTL (NM_000146) were sequenced using appropriate primers by using the Sanger sequence method. PCR conditions were as follows: initial denaturation at 94 °C for 5 min; 35 cycles at 94 °C for 30 s and 58 °C for 45 s; 72 °C for 1 min; and a final extension at 72 °C for 5 min. The PCR products were observed with 2% agarose gel electrophoresis. PCR products with enzyme transition were purified using the Exo-SAP kit (Exo-SAP PCR purification kit, UAB Corporation, USA). The cycle sequence was amplified using BigDye Terminator (Thermo Fisher Scientific, USA), and extension products were purified using Sephadex. The product was sequenced in both strands initiating from the

F2P5

1632

c.-160A>G

F2P6

>2000

c.-160A>G

F3P7

1123

c.-160A>G

F3P8

1340

c.-160A>G

F3P9

1592

c.-160A>G

F4P10

1420

c.-160A>G

F5P11

1152

c.-160A>G

F5P12

1106

c.-160A>G

F5P13

544

c.-160A>G

F5P14

1388

c.-160A>G

F6P15

1177

c.-160A>G

F6P16

759

c.-160A>G

F6P17

1993

c.-160A>G

no.

M: Male, F: female.

Turk J Hematol 2019;36:25-28

Balta B, et al: Frequent Mutation in the FTL Gene Causing Hyperferritinemia Cataract Syndrome

Discussion Ferritin is an iron-binding protein-storing iron for vital cellular activities and it is the primary intracellular iron-storing protein in the body. The protein’s iron-free form is called apoferritin, while the iron-containing form is called holoferritin. Each apoferritin consists of 24 subunits containing an H-subunit and L-subunit. HFCS is an autosomal dominant inherited rare genetic disease. It is characterized by early-onset cataract formation due to L-ferritin accumulation in the lens. Mutations in the 5’

Figure 1. All patients had heterozygous c.-160A>G mutation in the FTL gene. Table 2. The patients’ total blood counts and biochemical parameters. n Mean ± SD WBC, x103/µL RBC,

x106/µL

7.3±1.1 5.2±0.54

Hb, g/dL

14.5±2.4

Hct,%

38.1±12.2

MCV, fL

82.3±8.7

Plt,

x103/µL

261±68.8

Creatinine, mg/dL

1.0±0.21

BUN, mg/dL

13.8±2.8

AST, U/L

24.7±7.4

ALT, U/L

23.9±13.8

Total protein, g/dL

7.3±0.52

Albumin, g/dL

4.4±0.25

Ferritin, ng/mL

1292±404

Iron, µg/dL

62.1±39.6

Iron-binding capacity, µg/dL

384±60.4

WBC: White blood cells, RBC: red blood cells, Hb: hemoglobin, Hct: hematocrit, MCV: mean corpuscular volume, Plt: platelets, BUN: blood urea nitrogen, AST: aspartate aminotransferase, ALT: alanine aminotransferase, SD: standard deviation.

untranslated region of the FTL gene located in 19q13.1 cause this disease [9,10]. The binding of iron-responsive elements (IREs) with iron-regulated cytoplasmic protein (IRP) forms a complex enabling the inhibition of L-ferritin levels in the 5’ untranslated region of the FTL gene. Mutations in the FTL gene disrupt the IREs binding with IRPs, shifting the iron-mediated downregulations of FTL translations. So far, 37 mutations have been defined in the FTL gene [5]. Frequent mutations are c.-168G>C/T/A, c.-161C>G/T/A, and c.-160A>G [9]. Small deletions have also been defined [11]. In our study, we found c.-160A>G mutation in 17 patients from 6 families assessed from the provinces of Kayseri and Nevşehir in Turkey. All of our patients had the same mutation. The families were not relatives. This mutation has been reported previously [2,8]. Tuysuz et al. [7] from Turkey also reported +32G>T change in 3 individuals from a family. In addition to having early-onset cataracts, these three patients had ferritin levels between 659 and 2000 ng/mL. In HFCS, ferritin accumulates in all cells in the body. However, it causes cataracts by becoming toxic through forming L-ferritin crystal deposit formations only in the lens. The cataract is generally early-onset, bilateral, and progressive. HFCS penetration may not be complete and patients may have only hyperferritinemia. It has been claimed that the intensity of penetrance and cataract is associated with the location of the mutation [10,12]. In our study, cataracts had not developed in patients F3P7, F3P9, and F5P12, who were relatively young. However, all of the patients had hyperferritinemia. In addition, although the same mutation was found in all our patients, the differences in cataract development age and changes in ferritin levels bring to mind that factors besides the defined mutation can contribute to the disease. One of the striking findings in this study was that the youngest patient was 1 year old and the oldest one was 72 years old. Patient F5P14, who was 72 years old, underwent several tests, including a liver biopsy. However, the patient was diagnosed with FTL gene analysis in our department. Early diagnosis can save time for planning the patient’s cataract surgery and it can prevent unnecessary tests. HFCS prevalence has been shown to be around 1/200,000 in Australia [6]. All of our patients were from the provinces of Kayseri and Nevşehir in Turkey. The total population of these two provinces is about 1,700,000. We identified 17 patients from 6 families. This shows that the minimum prevalence is about 1/100,000. However, since ferritin levels were not measured routinely in cataract cases and since this study depended on hospital data, the prevalence of this disease could probably be higher. 27

Balta B, et al: Frequent Mutation in the FTL Gene Causing Hyperferritinemia Cataract Syndrome

Study Limitations The study has some limitations. The number of patients in the study is limited. Further studies with more patients are needed to explain why they have different ferritin levels and the age of onset of cataract.

Conclusion In the presence of hyperferritinemia, patients should be assessed by a hematologist and be referred to an ophthalmologist in terms of cataracts. It should be remembered that this disease is autosomal dominantly inherited and family screening should be performed. This study is the first comprehensive research article on the analysis of HFCS and FTL gene molecular analysis from Turkey. In the Turkish population, the prevalence of HFCS is about 1/100,000 and the commonly observed mutation is c.-160A>G mutation. Ethics Ethics Committee Approval: The study was approved by the Local Ethics Research Committee of Erciyes University with protocol number 2018/186 and conducted in accordance with the Declaration of Helsinki and good clinical practice guidelines. Informed Consent: It was received. Authorship Contributions Concept: B.B.; Design: B.B.; Data Collection: B.B., M.E., A.K.; Genetic Analysis: B.B., M.E., A.K.; Hematologic Examination: S.K.; Ophthalmologic Examination: A.A.; Literature Search: A.K.; Writing: B.B., M.E., A.K. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Hansen L, Mikkelsen A, Nürnberg P, Nürnberg G, Anjum I, Eiberg H, Rosenberg T. Comprehensive mutational screening in a cohort of Danish

Turk J Hematol 2019;36:25-28

families with hereditary congenital cataract. Invest Ophthalmol Vis Sci 2009;50:3291-3303. 2. Girelli D, Corrocher R, Bisceglia L, Olivieri O, De Franceschi L, Zelante L, Gasparini P. Molecular basis for the recently described hereditary hyperferritinemia-cataract syndrome: a mutation in the iron-responsive element of ferritin L-subunit gene (the “Verona mutation”). Blood 1995;86:4050-4053. 3. Bonneau D, Winter-Fuseau I, Loiseau MN, Amati P, Berthier M, Oriot D, Beaumont C. Bilateral cataract and high serum ferritin: a new dominant genetic disorder? J Med Genet 1995;32:778-779. 4. Beaumont C, Leneuve P, Devaux I, Scoazec JY, Berthier M, Loiseau MN, Grandchamp B, Bonneau D. Mutation in the iron responsive element of the L ferritin mRNA in a family with dominant hyperferritinaemia and cataract. Nat Genet 1995;11:444-446. 5. Luscieti S, Tolle G, Aranda J, Campos CB, Risse F, Moran E, Muckenthaler MU, Sanchez M. Novel mutations in the ferritin-L iron-responsive element that only mildly impair IRP binding cause hereditary hyperferritinaemia cataract syndrome. Orphanet J Rare Dis 2013;8:30. 6. Craig JE, Clark JB, McLeod JL, Kirkland MA, Grant G, Elder JE, Toohey MG, Kowal L, Savoia HF, Chen C, Roberts S, Wirth MG, Mackey DA. Hereditary hyperferritinemia-cataract syndrome: prevalence, lens morphology, spectrum of mutations, and clinical presentations. Arch Ophthalmol 2003;121:1753-1761. 7. Tuysuz G, Ozdemir N, Sonmez E, Kannengiesser C, Celkan T. A new family with hereditary hyperferritinemia cataract syndrome. Genet Couns 2013;24:393-397. 8. Cicilano M, Zecchina G, Roetto A, Bosio S, Infelise V, Stefani S, Mazza U, Camaschella C. Recurrent mutations in the iron regulatory element of L-ferritin in hereditary hyperferritinemia-cataract syndrome. Haematologica 1999;84:489-492. 9. Alvarez-Coca-Gonzalez J, Moreno-Carralero MI, Martinez-Perez J, Mendez M, Garcia-Ros M, Moran-Jimenez MJ. The hereditary hyperferritinemiacataract syndrome: a family study. Eur J Pediatr 2010;169:1553-1555. 10. Bertola F, Veneri D, Bosio S, Battaglia P, Disperati A, Schiavon R. Hyperferritinaemia without iron overload: pathogenic and therapeutic implications. Curr Drug Targets Immune Endocr Metabol Disord 2004;4:93105. 11. Garber I, Pudek M. A novel deletion in the iron-response element of the L-ferritin gene, causing hyperferritinaemia cataract syndrome. Ann Clin Biochem 2014;51:710-713. 12. Cazzola M, Bergamaschi G, Tonon L, Arbustini E, Grasso M, Vercesi E, Barosi G, Bianchi PE, Cairo G, Arosio P. Hereditary hyperferritinemia-cataract syndrome: relationship between phenotypes and specific mutations in the iron-responsive element of ferritin light-chain mRNA. Blood 1997;90:814821.

RESEARCH ARTICLE DOI: 10.4274/tjh.galenos.2018.2018.0325 Turk J Hematol 2019;36:29-36

A Novel ATP6V0A2 Mutation Causing Recessive Cutis Laxa with Unusual Manifestations of Bleeding Diathesis and Defective Wound Healing Alışılmadık Kanama Eğilimi ve Yetersiz Yara İyileşmesi Bulgularıyla Görülen Çekinik Kutis Laksaya Sebep Olan Yeni Bir ATP6V0A2 Mutasyonu İlker Karacan1,2, Reyhan Diz Küçükkaya3, Eda Tahir Turanlı1,7

Fatma Nur Karakuş4,

Seyhun Solakoğlu4,

Aslıhan Tolun5, Veysel Sabri Hançer6,

1İstanbul Technical University, Graduate School of Science, Engineering and Technology, Department of Molecular Biology-Genetics and

Biotechnology, İstanbul, Turkey 2İstanbul Medeniyet University, Department of Molecular Biology and Genetics, İstanbul, Turkey 3İstanbul Bilim University, Faculty of Medicine, Department of Hematology, İstanbul, Turkey 4İstanbul University, İstanbul Faculty of Medicine, Department of Histology and Embryology, İstanbul, Turkey 5Boğaziçi University, Department of Molecular Biology and Genetics, İstanbul, Turkey 6İstanbul Bilim University, Department of Molecular Biology and Genetics, İstanbul, Turkey 7İstanbul Technical University, Department of Molecular Biology and Genetics, İstanbul, Turkey

Abstract

Öz

Objective: Autosomal recessive cutis laxa type IIA (ARCL2A) is a rare congenital disorder characterized by loose and elastic skin, growth and developmental delay, and skeletal anomalies. It is caused by biallelic mutations in ATP6V0A2. Those mutations lead to increased pH in secretory vesicles and thereby to impaired glycosyltransferase activity and organelle trafficking. We aimed to identify the genetic and molecular cause of the unexpected hematological findings in a Turkish family.

Amaç: Otozomal çekinik kutis laksa tip IIA (ARCL2A) nadir görülen ve doğuştan bir hastalık olup, gevşek ve elastik deri, büyüme ve gelişme geriliği, iskelet anomalileri ile karakterizedir. ATP6V0A2 genindeki biallelik mutasyonlar hastalığa sebep olmaktadır. Bu mutasyonlar salgı veziküllerinde pH artışına yol açtığından, glikoziltransferaz aktivitesi ve organel trafiği bozulur. Bu çalışmada beklenmedik hematolojik bulguları da olan Türk ailede hastalığın genetik ve moleküler sebeplerinin bulunması amaçlanmıştır.

Materials and Methods: We performed clinical, genetic, and histological analyses of a consanguineous family afflicted with wrinkled and loose skin, microcephaly, intellectual disability, cleft lip and palate, downslanting palpebral fissures, ectopia lentis, bleeding diathesis, and defective wound healing.

Gereç ve Yöntemler: Akraba ebeveynin iki çocuğunda buruşuk ve gevşek deri, mikrosefali, zihinsel yetersizlik, yarık dudak ve damak, aşağı eğimli palpebral fissür, mercek dislokasyonu, kanama eğilimi ve yetersiz yara iyileşmesi bulguları görüldü ve klinik, genetik ve histolojik analizler gerçekleştirildi.

Results: Linkage analysis using SNP genotype data yielded a maximal multipoint logarithm of odds score of 2.59 at 12q24.21-24.32. Exome sequence analysis for the proband led to the identification of novel homozygous frameshift c.2085_2088del (p.(Ser695Argfs*12)) in ATP6V0A2, within the linked region, in the two affected siblings.

Bulgular: SNP genotip verisi kullanılarak yapılan bağlantı analizi sonucunda 12q24.21-24.32 kromozomal bölgesinde en yüksek çok noktalı LOD skor olan 2,59 elde edilmiştir. Bu bölgede indeks hastanın ekzom verilerinde, her iki hastada da homozigot durumda olan ve daha önce bildirilmemiş ATP6V0A2 geninde çerçeve kaymasına yol açan c.2085_2088del (p.(Ser695Argfs*12)) varyantı tespit edildi.

Conclusion: Our patients do not have gross structural brain defects besides microcephaly, strabismus, myopia, and growth or developmental delay. Large platelets were observed in the patients and unusual electron-dense intracytoplasmic inclusions in fibroblasts and epidermal basal cells were observed in both affected and unaffected

Sonuç: Hastalarda mikrosefali dışında yapısal beyin bozukluğu, şaşılık, miyop, büyüme veya gelişme geriliği yoktur. Yalnızca hastalarda büyük hacimli trombositler, hem hasta hem de sağlıklı bireylerde ise fibroblast ve epidermal bazal hücrelerde alışılmadık elektron-yoğun

Address for Correspondence/Yazışma Adresi: Eda TAHİR TURANLI, PhD., Dr. Orhan Öcalgiray Molecular Biology-Biotechnology and Genetics Research Centre, İstanbul Technical University, Ayazağa Campus, 34469 Maslak, İstanbul, Turkey Phone : +90 212 285 72 59 E-mail : turanlie@itu.edu.tr ORCID-ID: orcid.org/0000-0002-0789-0398

Received/Geliş tarihi: September 19, 2018 Accepted/Kabul tarihi: November 26, 2018

Karacan İ, et al: Cutis Laxa with Hematological Findings

Turk J Hematol 2019;36:29-36

Abstract

Öz

family members. The patients do not have any genetic defect in the VWF gene but von Willebrand factor activity to antigen ratios were low. Clinical findings of bleeding diathesis and defective wound healing have not been reported in ARCL2A and hence our findings expand the phenotypic spectrum of the disease.

intrasitoplazmik inklüzyonlar görülmüştür. Hastalarda VWF gen bozukluğu görülmemiş olup, von Willebrand faktörü aktivite/antijen oranı düşüktür. Kanama eğilimi ve yetersiz yara iyileşmesi daha önce ARCL2A hastalığında rapor edilmemiş olup, bulgularımız hastalığın fenotipik spektrumunu genişletmektedir.

Keywords: ATP6V0A2, Cutis laxa, Wound healing, Bleeding diathesis, Whole exome sequencing

Anahtar Sözcükler: ATP6V0A2, Kutis laksa, Yara iyileşmesi, Kanama diyatezi, Tüm ekzom dizi analizi

Introduction

Materials and Methods

Cutis laxa (CL) is a group of connective tissue abnormalities characterized by sagging, inelastic, and wrinkled skin [1]. These skin anomalies result from structural defects of elastic fibers. Congenital CL can be inherited as either an X-linked recessive, autosomal dominant or autosomal recessive trait. Autosomal recessive CL is the most severe type of the disease and displays the highest clinical and genetic heterogeneity. To date, causative mutations in at least eight genes have been identified for autosomal recessive CL: ALDH18A1, ATP6V0A2, ATP6V1A, ATP6V1E1, FBLN4, FBLN5, LTBP4, and PYCR1 (MIM 219100).

Patients

Autosomal recessive cutis laxa type IIA (ARCL2A; MIM 219200) is the most common type of CL and is characterized by variable clinical features including skin abnormalities, growth and developmental delay, and skeletal, neuromuscular, and central nervous system involvement [2]. ARCL2A also shares clinical presentations with wrinkly skin syndrome (WSS; MIM 278250), which appears to be a milder form of ARCL2A. Biallelic ATP6V0A2 mutations are responsible for both ARCL2A and WSS [3].

Single nucleotide polymorphism (SNP) genotyping was performed for the mother and all six siblings using Illumina OmniExpress-24 BeadChip targeting >700,000 SNP markers. Multipoint linkage analysis was performed using the GeneHunter program within the software package EasyLinkage [6,7]. Logarithm of odds (LOD) scores were calculated assuming an autosomal recessive model, full penetrance, and a disease allele frequency of 0.001. Regions of >2 Mb and with shared homozygosity in patients were considered. A DNA sample of patient V:6 was subjected to exome sequence analysis to identify the putative causative mutation. Sequencing data were analyzed according to GATK Best Practices

Loss-of-function mutations in ATP6V0A2 eventually lead to premature intracellular aggregation of tropoelastin, which in turn prevents mature elastin deposition in the extracellular matrix. Hucthagowder et al. [4] showed that abnormal elastin biosynthesis is the key pathophysiological mechanism affecting skin and connective tissue but does not underlie the neurological and developmental findings in ARCL2A. Impaired secretion of other proteins that have functions in nervous and skeletal tissues may give rise to additional clinical manifestations [4]. In ARCL2A, other mutations affecting assembly and functioning of the complex cause phenotypes overlapping with ARCL2A [5]. We present two siblings with some manifestations of ARCL2A plus unusual findings of bleeding diathesis, defective wound healing, and bilateral ectopia lentis. We identified a causative mutation and performed microscopic studies to investigate the cellular pathology. 30

The consanguineous parents (Figure 1) have six children, and the daughter and a son (V:3 and V:6, respectively) have some manifestations of ARCL2A. The blood group of both patients is A Rh-positive. Another son (V:1) has intellectual disability (ID) only. A son had died of cardiac anomaly at the age of two months, and a miscarriage at 20 weeks was also reported.

Methods Genetic Analysis

Figure 1. Pedigree of the family. Individuals included in the genetic study are indicated with asterisks. Patients diagnosed with cutis laxa are depicted in black. P: Proband.

Karacan İ, et al: Cutis Laxa with Hematological Findings

Turk J Hematol 2019;36:29-36

recommendations using human reference genome hg19 [8]. Variants in the linked region that were possibly harmful to the protein were considered. Furthermore, variants in the 39 known inherited platelet disease genes (Table 1) were evaluated for any possible contribution to bleeding diathesis. Table 1. Inherited platelet disease-related genes.

Light Microscopy Skin samples were obtained from the anterior arm by minor surgery and fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). After a wash in buffer solution, samples were dehydrated through a graded ethanol series and then embedded in paraffin blocks. Sections of 4 µm in thickness were obtained using a microtome and collected on slides, which were subsequently stained with dyes hematoxylin and eosin, Masson trichrome, and periodic acid-Schiff (PAS).

Inherited platelet disease

Associated gene

ADP receptor defect

P2RY12

ADP receptor defect

P2RX1

Amegakaryocytic thrombocytopenia with radioulnar synostosis

HOXA11

Arthrogryposis, renal dysfunction and cholestasis syndrome

VPS33B

Autosomal-dominant macrothrombocytopenia

TUBB1

Autosomal-dominant thrombocytopenia

ACTN1

Autosomal-dominant thrombocytopenia

CYCS

Bernard-Soulier syndrome

GP5

Bernard-Soulier syndrome

GP9

Chediak-Higashi syndrome

LYST

Congenital amegakaryocytic thrombocytopenia

MPL

Familial platelet disorder/acute myeloid leukemia

RUNX1

Glanzmann thrombasthenia

ITGA2B

Glanzmann thrombasthenia

ITGB3

GPS

NBEAL2

Hermansky-Pudlak syndrome

AP3B1

Hermansky-Pudlak syndrome

BLOC1S3

Hermansky-Pudlak syndrome

BLOC1S6

Hermansky-Pudlak syndrome

DTNBP1

Hermansky-Pudlak syndrome

HPS1

Hermansky-Pudlak syndrome

HPS3

Patient V:6

Hermansky-Pudlak syndrome

HPS4

Hermansky-Pudlak syndrome

HPS5

Hermansky-Pudlak syndrome

HPS6

Macrothrombocytopenia with dyserythropoiesis

GFI1B

Mediterranean macrothrombocytopenia

GP1BA

MYH9 disorders

MYH9

Paris-Trousseau/Jacobsen syndrome

FLI1

Platelet-type pseudo-VWD

GP1BB

Quebec platelet disorder

PLAU

Scott syndrome

ANO6

Scott syndrome

ABCA1

TAR

RBM8A

THC2

ANKRD26

THC2

MASTL

Thromboxane A2 receptor defect

TBXA2R

von Willebrand disease

VWF

Wiskott-Aldrich syndrome

WAS

X-linked thrombocytopenia with dyserythropoiesis

GATA1

The proband is a 21-year-old male who was referred to our hematology clinic for evaluation of bleeding diathesis prior to a left mastoidectomy operation due to chronic suppurative mastoiditis. He had wrinkled skin, hyperpigmentation, microcephaly, dysmorphic facial features, cleft lip and palate, and ectopia lentis. Bleeding diathesis, delayed wound healing, and easy bruising was noticeable in early childhood. He had been operated on for cleft lip and palate at age 6 months and for undescended testis and inguinal hernia at age 10 years. He had a history of chronic suppurative otitis media attacks that eventually caused sensorineural hearing loss. After a mild trauma to the left tibial region at age 20 years, a deep wound developed and progressed to acute compartment syndrome. He was hospitalized, and a fasciotomy was performed. During this period, excessive bleeding requiring blood transfusion attracted attention. Complete blood count showed white blood cells of 4.87x103/µL (N: 4-10x103/µL), hemoglobin of 10.9 g/dL (N: 12-16 g/dL) with mean corpuscular volume of 72 fL (N: 8094 fL), and platelet count of 205x103/µL (N: 150-400x103/µL)

Transmission Electron Microscopy Blood samples were centrifuged at 300 x g for 15 min to collect platelets in the buffy coat. Then both these buffy coat pellets and skin samples sized about 1 mm3 were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). After washing in buffer solution, samples were postfixed in 1% OsO4 in 0.1 M sodium cacodylate buffer (pH 7.2), dehydrated through a graded ethanol series, and embedded in Epon-812 resin. Ultrathin sections were cut with an EM UC6 ultramicrotome (Leica Microsystems), and sections were collected on Formvar-coated copper grids, stained with uranyl acetate, and lead-stained to enhance contrast. Grids were viewed with a JEOL JEM 1010B transmission electron microscope operating at 80 kV, and images were obtained with a Megaview II digital camera and AnalySIS software (Soft Imaging System GmBH, Germany).

Results Clinical Findings

Karacan Ä°, et al: Cutis Laxa with Hematological Findings

with mean platelet volume of 13.6 fL (N: 9-11 fL). Hypochromic and microcytic red blood cells and large platelets were seen on peripheral blood smear. Ferritin level was low (14 ng/mL, N: 20-150), and hemoglobin electrophoresis was normal. Iron deficiency anemia was treated with oral therapy. Prothrombin time, activated partial thromboplastin time, D-dimer, fibrin degradation products, and fibrinogen activity were found to be normal. Skin bleeding time (Ivy method) was 16 min (N: 4-9 min), and PFA-100 revealed prolonged closure times; both collagen/EPI (N: 85-157 s) and collagen/ADP (N: 65-125 s) results were >300 s. Light transmission aggregometry studies showed slightly decreased aggregation with ADP, collagen, and epinephrine. Aggregation with ristocetin was normal. Flow cytometric analysis of the peripheral blood platelets revealed normal expression with CD41 (for glycoprotein IIb), CD61 (for glycoprotein IIIa), CD42a (for glycoprotein IX), and CD42b (for glycoprotein Ib) cell surface markers. von Willebrand factor (vWF) activity to antigen ratio was low (0.35, N: >0.7), and factor VIII activity was 74% (N: 50%-150%). Patient V:3 The elder patient is 35 years old. She had wrinkled skin, hyperpigmentation, microcephaly, dysmorphic facial features, cleft lip and palate, ectopia lentis, hearing loss due to chronic suppurative otitis media, bleeding diathesis (menometrorrhagia requiring oral and parenteral iron treatment), easy bruising, and defective wound healing. She had been operated on for cleft lip and palate, and has had eye operations for ectopia lentis and bilateral corneal transplantations for corneal clouding. Papillomatous lesions were noticed on her tongue. Complete blood count revealed hypochromic and microcytic anemia consistent with iron deficiency, normal white blood cell count, and differential and normal platelet count with slightly elevated mean platelet volume. Basic coagulation tests (prothrombin time, activated partial thromboplastin time, D-dimer, fibrin degradation products, and fibrinogen activity) were normal. Skin bleeding time (Ivy method) was 15 min (N: 4-9 min). Light transmission aggregometry studies showed normal aggregation with ADP, collagen, ristocetin, and epinephrine. vWF activity to antigen ratio was low (0.3, N: >0.7), and factor VIII activity was 70% (N: 50%-150%).

Turk J Hematol 2019;36:29-36

of >2 Mb, with a LOD score of 2.59. The identified disease locus was 13.7 Mb at 12q24.21-24.32. The region harbored 198 genes, of which 112 were protein coding, including ATP6V0A2. Filtering exome variants in the region resulted in four rare variants (minor allele frequency of <0.01) located in either exons or splice sites (Table 2). A four-base pair frameshift deletion (NM_012463.3, c.2085_2088del, p.(Ser695Argfs*12)) in ATP6V0A2 exon 17 was the strongest candidate since the gene is known to be responsible for ARCL2A. The variant is deduced to create a premature stop codon leading to a truncated protein of 694 native and 12 non-native amino acids instead of the wild-type 856. The variant is classified as pathogenic according to the American College of Medical Genetics and Genomics guidelines [9]. Sanger sequencing revealed that the variant segregated with the disease in the family (Figure 3). No pathogenic variant in the known inherited platelet disease genes including VWF was detected in Patient V:6. The two patients and a healthy sibling (V:5) shared a heterozygous genotype around the VWF gene region. Thus, we ruled out involvement of any VWF defect in the bleeding diathesis and defective wound healing findings in the family. Microscopy Findings We studied platelets and dermal tissues under a transmission electron microscope as well as a light microscope to investigate the underlying cause of bleeding diathesis. The most prominent finding was the presence of PAS-positive intracytoplasmic granules in a number of cells in dermal and epidermal tissues of both

Neurological examination and cranial magnetic resonance imaging (MRI) of both patients were normal, excluding structural defects besides microcephaly (Figure 2). We concluded that the disease was atypical cutis laxa. Genetic Findings Initial Sanger sequencing of platelet surface glycoprotein genes GP1BA, GP1BB, GP9, ITGA2B, and ITGB3 did not reveal any mutations in the patients. We thus performed multipoint linkage analysis to localize the disease gene. We obtained a single region 32

Figure 2. Magnetic resonance imaging of patient V:6 in axial (A and B), sagittal (C), and coronal (D) planes.

Karacan İ, et al: Cutis Laxa with Hematological Findings

Turk J Hematol 2019;36:29-36

patients (Figure 4). Under the electron microscope, the content of those granules was assessed to be similar to polyglucosan bodies, and some of them resembled those seen in Lafora disease, i.e. Lafora bodies (Figure 5). They were variable in size and contained electron-dense and lipid-like inclusions in fibroblasts, exocrine glandular epithelial cells, and epidermal basal cells.

Discussion ARCL2A is a clinically highly variable group of connective tissue disorders characterized by inelastic skin due to lack of mature elastin fibers in the extracellular matrix. Highly heterogeneous clinical findings with respect to organ involvement and severity

In both patients a decreased quantity of elastic fibers in dermis and subcutis layers was observed under the light microscope. Electron microscopic evaluation of platelets for bleeding diathesis did not show any prominent abnormality except for the presence of some large platelets (approximately 5 µm in diameter; N: 2-4 µm) in both patients. In addition, very rare lamellar inclusions were found in platelets of patient V:3 (Figure 6).

Figure 3. Chromatograms showing novel 4-bp deletion c.2085_2088del (p.(Ser695Argfs*12)) in ATP6V0A2. Sequencing was performed using the reverse primer. Deletion site is marked with a dashed line.

Figure 4. Light microscopic pictures of dermis and eccrine sweat glands with intracellular periodic acid-Schiff positive inclusions in patient V:3.

Table 2. Homozygous rare/novel variants affecting amino acid sequence at the identified disease locus. Position

Gene

Change

Highest frequency in databases

SIFT

PolyPhen

MutationTaster

chr12:117,402,598

FBXW8

c.774G>T p.Met258Ile

Novel

Tolerated

Benign

Polymorphism

chr12:121,880,461

KDM2B

c.2783A>T p.Glu928Val

0.0022 (ExAC SAS)

Deleterious

Possibly damaging

Disease causing

chr12:122,018,734

KDM2B

c.83C>G p.Thr28Arg

Novel

Tolerated

Benign

Polymorphism

chr12:124,236,859124,236,862

ATP6V0A2

c.2085_2088del p.Ser695Argfs*12

Novel

Disease causing

NA: Not applicable to this type of variants.

Karacan Ä°, et al: Cutis Laxa with Hematological Findings

have been reported [10,11]. Loss-of-function mutations in various subunits of the V-ATPase complex cause similar diseases resulting from impaired glycosyltransferase activity and organelle trafficking [5]. We identified a novel frameshift deletion in ATP6V0A2 that is deduced to result in the truncation of the protein that would cause loss-of-function for the V-ATPase complex. CL patients with biallelic ATP6V0A2 mutations generally suffer from additional findings such as growth and developmental delay as well as neurological and skeletal anomalies. Our patients do not have growth or developmental delay; their heights are 165 and 175 cm (V:3 and V:6, respectively) and secondary sexual features are fully developed. Eye features in ARCL2A are downslanting palpebral fissures, strabismus, and myopia (MIM 219200). Of those, our patients had only downslanting palpebral fissures but there was additionally ectopia lentis in both and corneal clouding in V:6, features that have not been reported as associated with the disease. Neurologic examination and cranial

Figure 5. Electron micrograph of the granules resembling polyglucosan content similar to Lafora bodies in dermal fibroblasts of patient V:3. 34

Turk J Hematol 2019;36:29-36

MRI were normal for both patients, excluding any structural defect besides microcephaly. The only neurological sign in our patients is ID. As another sibling also has ID, we investigated whether the trait could be due to a common mutation in the three siblings. We performed linkage analysis assuming all three as affected to map the putative trait locus but did not find a candidate locus. We concluded that this common trait possibly does not have the same etiology in the three siblings. Any contribution of the KDM2B variants (Table 2) in the development of ID in patients V:3 and V:6, a characteristic finding of ARCL2A, remains elusive. ID, facial dysmorphism, and microcephaly have been reported in ARCL2A [2], but not bleeding diathesis or defective wound healing. Those latter two phenotypes could be a unique manifestation of the novel ATP6V0A2 mutation in the family or due to a mutation in another gene, but we did not detect a candidate mutation that could possibly underlie the trait. Since platelet functions and levels of coagulation

Figure 6. Electron micrographs showing large platelets and rare lamellar inclusions in platelets from patient V:3.

Karacan İ, et al: Cutis Laxa with Hematological Findings

Turk J Hematol 2019;36:29-36

factors were normal in both patients, we investigated skin biopsy samples and platelets from peripheral blood by electron microscopy. Ultrastructural studies did not reveal any abnormal features in the fine structure of the platelets except for larger platelet sizes. Patient V:6 did not carry any pathogenic variant of known inherited platelet disease genes; therefore, the cause of the larger size of the platelets remains elusive. In skin tissues decreased elastin fibers were observed, which is hypothesized to underlie the wrinkled skin. Furthermore, we noticed intracytoplasmic inclusions mainly in dermal fibroblasts, exocrine glandular epithelial cells, and epidermal basal cells, but these were present also in the unaffected members of the family. The content of those inclusions remains unknown. Bleeding diathesis, easy bruising, and defective wound healing in our patients could be the consequence of the abnormal elasticity of the extracellular matrix as in the case of EhlersDanlos syndrome, in which affected skin elasticity leads to bleeding tendency. Although there was a slight decrease in platelet aggregation in patient V:6, the expression of platelet surface glycoproteins was normal. In addition, vWF activity to antigen ratios were low in our patients. A low vWF activity to antigen ratio is a typical sign of vWF disease type 2A (MIM 613554), which is caused by VWF gene defects. Since we excluded a possible contribution of VWF defects, we hypothesized that possible abnormalities in posttranslational modifications of vWF could have contributed to bleeding diathesis and defective wound healing, similar to the situation in vWF disease type 2A. vWF is subject to extensive posttranslational modifications such as sulfation, glycosylation, multimerization, and proteolytic cleavage to create functional vWF, which is required for proper platelet adhesion [12,13,14]. Wagner et al. [15] concluded that some multimerization of vWF occurs in the Golgi apparatus, and a proton pump defect can lead to incomplete multimerization as in von Willebrand disease type 2A. Alternatively, bleeding diathesis in our patients could be related to possible impaired glycosylation of platelet surface glycoproteins, as O-glycosylation of platelet surface glycoproteins is important for their functions [16]. Whether glycosylation of platelet surface glycoproteins is impaired in ARCL2A and whether these two traits (bleeding diathesis and defective wound healing) are part of ARCL2A remains to be elucidated in future studies, as it is likely that not all reported ARCL2A cases have been subjected to a thorough hematological investigation.

Conclusion We have identified a novel mutation in ATP6V0A2 causing autosomal recessive CL. Our patients presented with multisystemic CL phenotypes but strabismus, myopia, and growth or developmental delay were not observed. Brain malformations besides microcephaly were not present and additional clinical

findings of bleeding diathesis, defective wound healing, ectopia lentis, and corneal clouding have not been reported previously; hence, our findings expand the clinical and allelic spectrum of the disease. We suggest that ARCL2A patients could benefit from detailed hematological examinations, especially prior to surgical operations. Ethics Ethics Committee Approval: İstanbul Technical University, Human Research Ethical Committee, approval number: MBG.22/2014, approval date: 28 August 2014. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed Consent: Informed consent was obtained from all individual participants included in the study. Authorship Contributions Surgical and Medical Practices: R.D.K.; Concept: E.T.T.; R.D.K., A.T.; Design: E.T.T., İ.K., A.T.; Data Collection or Processing: İ.K., R.D.K., F.N.K., V.S.H.; Analysis or Interpretation: E.T.T., İ.K., R.D.K., S.S., A.T.; Literature Search: İ.K., R.D.K.; Writing: İ.K., E.T.T., R.D.K., S.S., A.T. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. Financial Disclosure: This study was supported by the Scientific and Technological Research Council of Turkey (grant number 114Z829).

References 1. Mohamed M, Kouwenberg D, Gardeitchik T, Kornak U, Wevers RA, Morava E. Metabolic cutis laxa syndromes. J Inherit Metab Dis 2011;34:907-916. 2. Morava E, Lefeber DJ, Urban Z, de Meirleir L, Meinecke P, Gillessen Kaesbach G, Sykut-Cegielska J, Adamowicz M, Salafsky I, Ranells J, Lemyre E, van Reeuwijk J, Brunner HG, Wevers RA. Defining the phenotype in an autosomal recessive cutis laxa syndrome with a combined congenital defect of glycosylation. Eur J Hum Genet 2008;16:28-35. 3. Kornak U, Reynders E, Dimopoulou A, van Reeuwijk J, Fischer B, Rajab A, Budde B, Nürnberg P, Foulquier F; ARCL Debré-type Study Group, Lefeber D, Urban Z, Gruenewald S, Annaert W, Brunner HG, van Bokhoven H, Wevers R, Morava E, Matthijs G, Van Maldergem L, Mundlos S. Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2. Nat Genet 2008;40:32-34. 4. Hucthagowder V, Morava E, Kornak U, Lefeber DJ, Fischer B, Dimopoulou A, Aldinger A, Choi J, Davis EC, Abuelo DN, Adamowicz M, Al-Aama J, BaselVanagaite L, Fernandez B, Greally MT, Gillessen-Kaesbach G, Kayserili H, Lemyre E, Tekin M, Türkmen S, Tuysuz B, Yüksel-Konuk B, Mundlos S, Van Maldergem L, Wevers RA, Urban Z. Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival. Hum Mol Genet 2009;18:2149-2165.

Karacan Ä°, et al: Cutis Laxa with Hematological Findings

5. Van Damme T, Gardeitchik T, Mohamed M, Guerrero-Castillo S, Freisinger P, Guillemyn B, Kariminejad A, Dalloyaux D, van Kraaij S, Lefeber DJ, Syx D, Steyaert W, De Rycke R, Hoischen A, Kamsteeg EJ, Wong SY, van Scherpenzeel M, Jamali P, Brandt U, Nijtmans L, Korenke GC, Chung BHY, Mak CCY, Hausser I, Kornak U, Fischer-Zirnsak B, Strom TM, Meitinger T, Alanay Y, Utine GE, Leung PKC, Ghaderi-Sohi S, Coucke P, Symoens S, De Paepe A, Thiel C, Haack TB, Malfait F, Morava E, Callewaert B, Wevers RA. Mutations in ATP6V1E1 or ATP6V1A cause autosomal-recessive cutis laxa. Am J Hum Genet 2017;100:216-227. 6. Hoffmann K, Lindner TH. easyLINKAGE-Plus--automated linkage analyses using large-scale SNP data. Bioinformatics 2005;21:3565-3567. 7. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet 1996;58:1347-1363. 8. Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, LevyMoonshine A, Jordan T, Shakir K, Roazen D, Thibault J, Banks E, Garimella KV, Altshuler D, Gabriel S, DePristo MA. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics 2013;43:1-33. 9. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and

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Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405-424. 10. Morava E, Guillard M, Lefeber DJ, Wevers RA. Autosomal recessive cutis laxa syndrome revisited. Eur J Hum Genet 2009;17:1099-1110. 11. Fischer B, Dimopoulou A, Egerer J, Gardeitchik T, Kidd A, Jost D, Kayserili H, Alanay Y, Tantcheva-Poor I, Mangold E, Daumer-Haas C, Phadke S, Peirano RI, Heusel J, Desphande C, Gupta N, Nanda A, Felix E, Berry-Kravis E, Kabra M, Wevers RA, van Maldergem L, Mundlos S, Morava E, Kornak U. Further characterization of ATP6V0A2-related autosomal recessive cutis laxa. Hum Genet 2012;131:1761-1773. 12. Wagner DD. Cell biology of von Willebrand factor. Annu Rev Cell Biol 1990;6:217-246. 13. Vischer UM, Wagner DD. von Willebrand factor proteolytic processing and multimerization precede the formation of Weibel-Palade bodies. Blood 1994;83:3536-3544. 14. Solecka BA, Weise C, Laffan MA, Kannicht C. Site-specific analysis of von Willebrand factor O-glycosylation. J Thromb Haemost 2016;14:733-746. 15. Wagner DD, Mayadas T, Marder VJ. Initial glycosylation and acidic pH in the Golgi apparatus are required for multimerization of von Willebrand factor. J Cell Biol 1986;102:1320-1324. 16. Wang Y, Jobe SM, Ding X, Choo H, Archer DR, Mi R, Ju T, Cummings RD. Platelet biogenesis and functions require correct protein O-glycosylation. Proc Natl Acad Sci U S A 2012;109:16143-16148.

RESEARCH ARTICLE DOI: 10.4274/tjh.galenos.2018.2018.0230 Turk J Hematol 2019;36:37-42

Different Presentations of Patients with Transcobalamin II Deficiency: A Single-Center Experience from Turkey Transkobalamin II Eksikliği Olan Hastaların Farklı Sunumları: Türkiye’den Tek Merkez Deneyimi Selma Ünal1,

Feryal Karahan1,

Tuğba Arıkoğlu2,

Asuman Akar3,

Semanur Kuyucu2

1Mersin University Faculty of Medicine, Department of Pediatric Hematology, Mersin, Turkey 2Mersin University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Mersin, Turkey 3Mersin University Faculty of Medicine, Department of Pediatric Infectious Diseases, Mersin, Turkey

Abstract

Öz

Objective: Transcobalamin II deficiency is a rare autosomal recessive disease characterized by decreased cobalamin availability, which in turn causes accumulation of homocysteine and methylmalonic acid. The presenting clinical features are failure to thrive, diarrhea, megaloblastic anemia, pancytopenia, neurologic abnormalities, and also recurrent infections due to immune abnormalities in early infancy.

Amaç: Transkobalamin II eksikliği homosistein ve metilmalonik asit birikimine neden olan kobalamin yararlanımının azaldığı nadir bir otozomal resesif hastalıktır. Erken dönemde gelişme geriliği, diyare, megaloblastik anemi, pansitopeni, nörolojik anormallikler ve ayrıca immün bozukluklara bağlı tekrarlayan enfeksiyonlarla karekterizedir.

Materials and Methods: Here, we report the clinical and laboratory features of six children with transcobalamin II deficiency who were all molecularly confirmed. Results: The patients were admitted between 1 and 7 months of age with anemia or pancytopenia. Unexpectedly, one patient had a serum vitamin B12 level lower than the normal range and another one had nonsignificantly elevated serum homocysteine levels. Four patients had lymphopenia, four had neutropenia and three also had hypogammaglobulinemia. Suggesting the consideration of transcobalamin II deficiency in the differential diagnosis of immune deficiency. Hemophagocytic lymphohistiocytosis was also detected in one patient. Furthermore, two patients had vacuolization in the myeloid lineage in bone marrow aspiration, which may be an additional finding of transcobalamin II deficiency. The hematological abnormalities in all patients resolved after parenteral cobalamin treatment. In followup, two patients showed neurological impairments such as impaired speech and walking. Among our six patients who were all molecularly confirmed, two had the mutation that was reported in transcobalamin II-deficient patients of Turkish ancestry. Also, a novel TCN2 gene mutation was detected in one of the remaining patients. Conclusion: Transcobalamin II deficiency should be considered in the differential diagnosis of infants with immunological abnormalities as well as cytopenia and neurological dysfunction. Early recognition of this rare condition and initiation of adequate treatment is critical for control of the disease and better prognosis.

Gereç ve Yöntemler: Bu çalışmada, transkobalamin II eksikliği tanısı moleküler olarak konfirme edilen altı hastanın klinik ve laboratuvar özellikleri sunulmuştur. Bulgular: Hastalar 1-7 ay arasında anemi veya pansitopeni ile başvurdu. Beklenmedik şekilde, bir hastanın serum vitamin B12 düzeyi normalden düşüktü ve diğer bir hastanın serum homosistein düzeyi belirgin düzeyde yüksek değildi. Dört hastada lenfopeni, dört hastada nötropeni ve üç hastada ayrıca hipogamaglobulinemi olup bu bulgular immün yetersizliğin ayırıcı tanısında transkobalamin II eksikliğinin düşünülmesi gerektiğini göstermektedir. Bir hastada hemofagositik lenfohistiyositoz saptandı. Ayrıca, iki hastanın kemik iliği aspirasyonunda myeloid seride vakuolizasyon olup transkobalamin II eksikliğinin ek bir bulgusu olabilir. Parenteral kobalamin tedavisinden sonra tüm hastaların hematolojik anormallikleri düzeldi. Takipte iki hastada konuşmada ve yürümede bozulma şeklinde nörolojik anormallikler saptandı. Tümü moleküler olarak doğrulanmış altı olgumuzdan ikisinde, transkobalamin II eksikliği olan Türk ırkına ait hastalarda bildirilen mutasyon mevcuttu. Ayrıca, kalan olgulardan birinde yeni bir TCN2 gen mutasyonu tespit edildi. Sonuç: Transkobalamin II eksikliği sitopeni ve nörolojik bozuklukların olduğu gibi immünolojik anormalliklerin de olduğu infantlarda ayırıcı tanıda düşünülmelidir. Bu nadir hastalığın erken tanınması ve uygun tedavinin başlanması hastalığın kontrolü ve daha iyi bir prognoz açısından kritiktir. Anahtar Sözcükler: Transkobalamin II, Vakuolizasyon, İmmün yetersizlik, Nörolojik bozukluk, Hemofagositik lenfohistiyositoz

Keywords: Transcobalamin II, Vacuolization, Immune deficiency, Neurological impairment, Hemophagocytic lymphohistiocytosis ©Copyright 2019 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Tuğba ARIKOĞLU, M.D., Mersin University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Mersin, Turkey Phone : +90 324 241 00 00/2024 E-mail : arikoglutugba@yahoo.com ORCID-ID: orcid.org/0000-0003-3340-571X

Received/Geliş tarihi: July 03, 2018 Accepted/Kabul tarihi: September 05, 2018

Ünal S, et al: Presentations of Transcobalamin II Deficiency

Introduction Transcobalamin II (TC) deficiency is a rare autosomal recessive disease in early infancy caused by mutations in the TCN2 gene [1]. TC is a transport protein for vitamin B12 and facilitates its cellular uptake by receptor-mediated endocytosis [1]. A deficiency of TC results in a lack of vitamin B12 entry into cells, leading to intracellular cobalamin depletion [2]. The presenting clinical features are failure to thrive, diarrhea, pancytopenia, neurologic abnormalities, and infections due to immunodeficiency [3,4,5,6]. The diagnosis is suspected based on the presence of clinical and laboratory features [5]. Patients with TC deficiency have elevated homocysteine and methylmalonic acid levels [2,7]. Treatment with parenteral vitamin B12 is highly effective on clinical and biological signs. It is important to establish the diagnosis of TC deficiency as early as possible because with a significant delay in diagnosis, and therefore treatment, the neurological abnormalities can become irreversible [8]. In the present study, we report the clinical and laboratory features of six children diagnosed with TC deficiency in our hospital.

Materials and Methods We retrospectively reviewed the medical records of six children with a diagnosis of TC deficiency in our hospital. Complete blood count, serum vitamin B12, folic acid, homocysteine and immunoglobulin levels, percentage of lymphocyte subsets, bone marrow aspiration, and molecular analysis results were obtained from medical charts. Treatment regimens given to the patients were recorded. The clinical and laboratory findings of our patients are shown in Table 1.

Results Case 1 A 2-month-old male baby was admitted with complaints of fever, cough, diarrhea, and respiratory distress. The parents were first-degree cousins. The constellation of clinical features such as prolonged fever and splenomegaly and laboratory findings (cytopenia in peripheral blood, elevated ferritin, triglyceride and liver enzymes, and hemophagocytosis in the bone marrow) suggested the diagnosis of hemophagocytic lymphohistiocytosis (HLH). Cytomegalovirus (CMV) PCR was found to be positive and he was given ganciclovir therapy. Intravenous immunoglobulin was added to the therapy due to the presence of hypogammaglobulinemia. Percentages of lymphocyte subsets were in the normal ranges. A second bone marrow aspiration demonstrated megaloblastic changes in the erythroid series. The patient’s serum vitamin B12 level was normal; however, the serum homocysteine level (23 µmol/L) was 38

Turk J Hematol 2019;36:37-42

significantly higher than normal. A genetic deficiency of TC was suspected and a homozygous TCN2 gene mutation was detected in molecular analysis. This 5304-bp deletion began 1516 bp into intron 7 and ended 1231 bp into intron 8. The deletion included all of exon 8 and caused a frameshift to produce a premature stop four codons into the new reading frame. The patient was treated with intramuscular vitamin B12, which was followed by improvement in both clinical and laboratory findings. This case was published as a case report in the literature [9]. Case 2 A 6-month-old girl presented with complaints of failure to thrive, vomiting, and diarrhea. A diagnosis of sepsis or metabolic disease was suspected and antibiotic therapy was started empirically. She had anemia, neutropenia, and thrombocytopenia. Serum vitamin B12 level was found to be normal; however, serum homocysteine was 53 µmol/L. Megaloblastic changes and vacuolization were prominent in the myeloid lineage in the bone marrow aspiration. Immunological evaluation revealed hypogammaglobulinemia. Percentages of lymphocyte subsets were in the normal range. A genetic deficiency of TC was suspected. The molecular analysis revealed c.1106+1516_1222+1231del mutation. This mutation is not listed in the Human Gene Mutation Database (Cardiff). It is a 5304-bp deletion that begins 1516 bp into intron 7 and ends 1231 bp into intron 8. The deletion includes all of exon 8 and causes a frameshift to produce a premature stop four codons into the new reading frame. The patient was treated with intramuscular vitamin B12 and oral folic acid, which was followed by improvement in hematological response, but a speech deficit was detected at 2 years of age in follow-up. Case 3 A 7-month-old boy presented with complaints of poor feeding, diarrhea, and petechiae. He was the child of first-degree cousins. He had pancytopenia. Serum vitamin B12 level was found to be normal and serum homocysteine level was high at the borderline (16 µmol/L). Bone marrow was hypocellular and megaloblastic changes were prominent in the myeloid lineage. A genetic deficiency of TC was suspected. The molecular analysis revealed c.1106+1516_1222+1231del mutation. This mutation is not listed in the Human Gene Mutation Database (Cardiff). It is a 5304-bp deletion that begins 1516 bp into intron 7 and ends 1231 bp into intron 8. The deletion includes all of exon 8 and causes a frameshift to produce a premature stop four codons into the new reading frame. The patient was treated with intramuscular vitamin B12, which was followed by improvement in hematological response, but a delay in walking was detected at 2 years of age in follow-up. Case 4 A 5-month-old girl was admitted with failure to thrive, poor feeding, vomiting, and diarrhea. She was the child of first-

Turk J Hematol 2019;36:37-42

degree cousins and had a history of sibling death. Laboratory evaluation showed pancytopenia, which required transfusions, and lymphopenia and hypogammaglobulinemia. Percentages of lymphocyte subsets were in the normal range. Serum vitamin B12 level was low (136 pg/mL) and serum homocysteine level could not be measured. CMV PCR was found to be positive. Severe combined immunodeficiency was suspected. Intravenous immunoglobulin, ganciclovir treatment, and antibacterial and antifungal prophylaxis were given. However, bone marrow aspiration showed prominent vacuolization in the myeloid lineage, which suggested Pearson syndrome, and prominent megaloblastic changes in the myeloid lineage. However, molecular analysis did not support the diagnosis of Pearson syndrome. A genetic deficiency of TC was suspected. The patient was treated with intramuscular vitamin B12 and oral folic acid with clinical and hematological improvement. After her family discontinued vitamin B12 therapy, she showed relapse with severe pancytopenia. Vitamin B12 treatment was restarted. The molecular analysis revealed a homozygous TCN2 gene mutation. Case 5 A 1-month-old male baby presented with irritability, fever, and poor feeding. He had a cleft palate and lip. He was the child of first-degree cousins. A diagnosis of sepsis was suspected and antibiotic therapy was started empirically. Complete blood count revealed macrocytic anemia, which required transfusions in follow-up. Serum vitamin B12 and folic acid levels were found to be normal. Bone marrow aspiration showed megaloblastic changes in the myeloid lineage. Serum homocysteine level was 45 Âľmol/L. A genetic deficiency of TC was suspected. Homozygous deletion of the TCN2 gene was detected in exon 8. The patient was treated with intramuscular vitamin B12, which was followed by clinical and hematological response. Case 6 A 2-month-old male baby presented with complaints of irritability, fever, oral aphthous ulcers, and diarrhea. Patent ductus arteriosus was found in echocardiography. Laboratory evaluation revealed pancytopenia. Serum vitamin B12 and folic acid levels were found to be normal. Serum homocysteine level could not be measured. Bone marrow aspiration was remarkable for megaloblastic changes in erythroid and myeloid cell precursors. A genetic deficiency of TC was suspected. The molecular analysis revealed a homozygous TCN2 gene mutation: c.106C>T (p.Q36*) (p.Gln36*). The patient was treated with intramuscular vitamin B12 and oral folic acid. He has been asymptomatic in follow-up.

Discussion Here, we report six patients with the diagnosis of TC deficiency in our institution. The present study extends our understanding

Ă&#x153;nal S, et al: Presentations of Transcobalamin II Deficiency

of this rare disease and provides additional data for the clinical and laboratory manifestations. The presenting clinical features are macrocytic anemia, pancytopenia, failure to thrive, gastrointestinal symptoms, and neurologic dysfunction [4,5,10]. In addition to the aforementioned clinical features, recurrent infections can be seen in TC deficiency due to immunological abnormalities. Specific immune abnormalities include hypogammaglobulinemia, specific antibody deficiency, neutropenia, and low T and/or B cell counts [3,5,11]. Consistent with these data, four patients had lymphopenia, four had neutropenia, and three had hypogammaglobulinemia in the present study, suggesting the consideration of TC deficiency in the differential diagnosis of immune deficiency. HLH is a syndrome characterized by uncontrolled immune response with hyperinflammation [12,13]. Acquired HLH can develop as a result of infections, malignancy, and autoimmunity. In addition, HLH is associated rarely with congenital metabolic disorders [13,14]. Case 1 was reported by Unal et al. [9] in the literature as the first case of TC deficiency presenting with HLH. Defects in cobalamin metabolism were suggested to lead to HLH due to defects in DNA synthesis, secondary NK cell functions, and immune regulation [15]. Hypogammaglobulinemia, lymphopenia, and neutrophil dysfunction have been reported in TC deficiency [3,5,11]. Therefore, it can be considered that immune changes caused by a lack of TC in our patient led to the development of secondary HLH. Potential causative mechanisms of HLH induced by defects of cobalamin metabolism merit further investigation. Despite early-onset cobalamin treatment and close monitoring of hematological parameters, patients with TC deficiency have been reported to show neurological deterioration including intellectual disability, attention deficits, tremor, myoclonus, ataxia, delay in language, and motor skills. Epilepsy responsive to antiepileptic medication was also reported [4,5]. The most common neurological complication in the literature was speech deficit [5]. Consistent with the literature, Case 2 had speech deficit and Case 3 had impairment in walking despite intramuscular cobalamin treatment. The starting age and mode of cobalamin treatment influence neurological outcome [4,5]. Patients who are treated early have had a better outcome than those inadequately treated. Nevertheless, the natural course of the disease over time might also result in late-onset neurological symptoms [4,5,8]. We initiated treatment with 1 mg of intramuscular cyanocobalamin daily in the first week and the therapy was continued with 1 mg of cyanocobalamin every second day in the second week. In follow-up, weekly injections of 1 mg of cyanocobalamin were administered based on the clinical and hematological response. In association with cyanocobalamin therapy, folic acid supplementation was also given. 39

Ünal S, et al: Presentations of Transcobalamin II Deficiency

Turk J Hematol 2019;36:37-42

Table 1. Clinical manifestations and laboratory findings of the patients. Case 1

Case 2

Age (months) at presentation

Sex

Male

Female

Consanguinity

First-degree cousins

Absent

Symptoms at presentation

Fever, cough, diarrhea, respiratory distress

Vomiting, diarrhea, failure to thrive

Additional findings

CMV PCR: (+) Hemophagocytic syndrome

Impaired in speech

Hb (g/dL)

10.4

9.6

Hct (%)

29.2

27.1

RBC

(x1012/L)

3.2

3.1

WBC (x109/L)

3.67

5.19

MCV (fL)

1.54

1.08

(x109/L)

1.86 (3.5-13.1)

3.35 (3.8-9.90)

IgG (mg/dL) IgA (mg/dL) IgM (mg/dL) IgE (IU/mL)

(at 2 months) 212 (376-685) 26 (9-30) 20 (36-77) <17

(at 6 months) 255 (304-1231) 84 (7-123) 20 (32-283) 5

CD3 CD4 CD8 CD19 CD16+56

80% (60%-85%) 66% (41%-68%) 29% (9%-23%) 13% (4%-26%) 4% (3%-23)

81% (50%-77%) 66% (33%-58%) 13% (13%-26%) 14% (13%-35%) 4% (2%-13%)

Vitamin B12 (normal: 197-866 pg/mL)

568

1281

Homocysteine (normal: 5-15 µmol/L)

Bone marrow examination

Hypocellular, increase in myeloid lineage

Vacuolization and megaloblastic changes in myeloid lineage

TCN2 mutation

c.940+283_286delTGGA;c.940+303_ 1106+764del2152insCTGG

c.1106+1516_1222 +1231del

Treatment regimen

Ganciclovir and IVIG at first admission Cyanocobalamin i.m. Oral folic acid

Platelets

(x109/L)

ANC (x109/L) ALC

Cyanocobalamin i.m. Oral folic acid

ANC: Absolute neutrophil count, ALC: absolute lymphocyte count, CMV: cytomegalovirus, Hb: hemoglobin, Htc: hematocrit, IVIG: intravenous immunoglobulin; i.m.: intramuscular, MCV: mean corpuscular volume, NA: not available, RBC: red blood cells, WBC: white blood cells.

To date, serum vitamin B12 levels have been reported to be normal in cases of TC deficiency [4,10,16]. This is due to the fact that the majority of vitamin B12 in circulation is bound to haptocorrin rather than transcobalamin. As a result, patients with TC deficiency do not show a low level of circulating vitamin B12 [1,2]. However, Schiff et al. [8] reported TC-deficient patients with low vitamin B12 levels. In the present study, Case 4 had the unusual feature of a low serum cobalamin level, similar to the cases reported in Schiff et al.’s [8] study. Homocysteine and methylmalonic acid levels are the metabolic markers of vitamin B12 deficiency [7]. The diagnosis of TC deficiency is confirmed by the measurement of these metabolic markers, although they 40

can rarely be within normal limits [10]. Consistent with these data, the homocysteine levels of Case 3 were in the upper limit of normal. Therefore, our findings indicate that a normal or low serum vitamin B12 level or a slightly elevated homocysteine level does not exclude TC deficiency. Thus, molecular analysis should be done in order to establish a firm diagnosis in subjects with suggested TC deficiency but with normal plasma homocysteine levels. Two previous studies reported vacuolization in the bone marrow aspiration of TC-deficient patients [10,17]. Similar to those previous studies, vacuolization in the myeloid lineage was seen

Ă&#x153;nal S, et al: Presentations of Transcobalamin II Deficiency

Turk J Hematol 2019;36:37-42

Table 1. Continued. Case 3

Case 4

Case 5

Case 6

Male

Female

Male

First-degree cousins

First-degree cousins; two siblings exitus

First-degree cousins

Absent

Poor feeding, diarrhea, petechiae

Vomiting, diarrhea, failure to thrive

Fever, irritability, poor feeding

Irritability, oral aphthous ulcers, fever, diarrhea

Impaired walking

CMV PCR: (+)

Cleft palate and lip

Patent ductus arteriosus

6.9

6.2

8.3

8.0

21.2

23.9

2.4

1.9

2.4

2.7

3.47

1.00

11.52

2.44

103

391

0.50

0.08

2.68

2.40

2.82 (3.8-9.90)

0.91 (3.7-9.6)

6.9 (3.5-13.10)

1.88 (3.50-13.10)

(at 5 months) 183 (294-1165) 42 (13-72) 52 (33-154) <2

(at 4 months) 925 (294-1165) 27 (13-72) 37 (33-154) 7

(at 5 months) 510 (294-1165) 71 (13-72) 45 (33-154) 5

80% (48%-75%) 52% (33%-58%) 25% (11%-25%) 16% (14%-39%) 1% (2%-14%)

479

136

229

360

Hypocellular, megaloblastic changes in myeloid lineage

Vacuolization, megaloblastic changes in myeloid lineage

Megaloblastic changes in myeloid lineage

Megaloblastic changes in bone marrow precursors

c.1106+1516_1222 +1231del

Homozygous deletion of TCN2 gene was detected in exon 8

c.106C>T (p.Q36*) (p.Gln36*) (Homozygous)

Ganciclovir and IVIG at first admission Cyanocobalamin i.m. Oral folic acid

Cyanocobalamin i.m. Oral folic acid

in the bone marrow aspirations of Cases 2 and 4 in the current study and Pearson syndrome was suspected. Vacuolization is an important finding in Pearson syndrome, which may be related to defects in mitochondrial DNA synthesis [18]. However, it may also be an additional finding in TC deficiency, resulting from a defect of cobalamin metabolism. Trakadis et al. [5] reported a series of 30 patients with TC deficiency. Molecular testing was performed for 20 families and 17 different mutations were identified. Sixteen families had homozygous and four had compound heterozygous mutations. Different intragenic deletions were identified in 15

of the 20 families. Four mutations resulted in exon skipping [19]: two involved exon 7 and one involved exon 4, while the fourth was a large deletion involving exons 1 to 7. Trakadis et al. [5] reported that a founder mutation may be present in patients of Turkish ancestry, as all presumably unrelated probands of Turkish ancestry shared the same homozygous mutation (c.1106+1516_1222+1231del). This common mutation among the Turkish population was also detected in our Cases 2 and 3, who were living in the same town. In this mutation, the deletion includes all of exon 8 and causes a frameshift to produce a premature stop four codons into the new reading frame. The other mutation in Case 6 (c.106C>T 41

Ünal S, et al: Presentations of Transcobalamin II Deficiency

(p.Q36*) (p.Gln36*) produces a premature stop four codons into the new reading frame.

Conclusion TC deficiency often presents early in life with multisystem involvement. Therefore, it should be considered in differential diagnosis of infants with cytopenia and neurological dysfunction as well as immunological abnormalities. Early recognition of this rare disease and initiation of adequate treatment is critical for disease control and better prognosis. Ethics Ethics Committee Approval: Retrospective study. Informed Consent: Retrospective study. Authorship Contributions Concept: S.Ü., F.K., T.A., A.A., S.K.; Design: S.Ü., F.K., T.A., A.A., S.K.; Data Collection or Processing: F.K., A.A., S.Ü., Analysis or Interpretation: S.Ü., T.A.; Literature Search: T.A.; Writing: S.Ü., S.K. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Turk J Hematol 2019;36:37-42

5. Trakadis YJ, Alfares A, Bodamer OA, Buyukavci M, Christodoulou J, Connor P, Glamuzina E, Gonzalez-Fernandez F, Bibi H, Echenne B, Manoli I, Mitchell J, Nordwall M, Prasad C, Scaglia F, Schiff M, Schrewe B, Touati G, Tchan MC, Varet B, Venditti CP, Zafeiriou D, Rupar CA, Rosenblatt DS, Watkins D, Braverman N. Update on transcobalamin deficiency: clinical presentation, treatment and outcome. J Inherit Metab Dis 2014;37:461-473. 6. Hitzig WH, Dohmann U, Pluss HJ, Vischer D. Hereditary transcobalamin II deficiency: clinical findings in a new family. J Pediatrics 1974;85:622-628. 7. Barshop BA, Wolff J, Nyhan WL, Yu A, Prodanos C, Jones G, Sweetman L, Leslie J, Holm J, Green R, Jacobsen DW, Cooper BA, Rosenblatt D. Transcobalamin II deficiency presenting with methylmalonic aciduria and homocystinuria and abnormal absorption of cobalamin. Am J Med Genet 1990;35:222-228. 8. Schiff M, Ogier de Baulny H, Bard G, Barlogis V, Hamel C, Moat SJ, Odent S, Shortland G, Touati G, Giraudier S. Should transcobalamin deficiency be treated aggressively? J Inherit Metab Dis 2010;33:223-229. 9. Unal S, Tezol O, Oztas Y. A novel mutation of the transcobalamin II gene in an infant presenting with hemophagocytic lymphohistiocytosis. Int J Hematol 2014;99:659-662. 10. Ratschmann R, Minkov M, Kis A, Hung C, Rupar T, Mühl A, Fowler B, Nexo E, Bodamer OA. Transcobalamin II deficiency at birth. Mol Genet Metab 2009;98:285-288. 11. Hitzig WH, Kenny AB. The role of vitamin B12 and its transport globulins in the production of antibodies. Clin Exp Immunol 1975;20:105-111. 12. Weitzman S. Approach to hemophagocytic syndromes. Hematology Am Soc Hematol Educ Program 2011;2011:178-183. 13. Jordan MB, Allen CE, Weitzman S, Filipovich AH, McClain KL. How I treat hemophagocytic lymphohistiocytosis. Blood 2011;118:4041-4052. 14. Bode SF, Lehmberg K, Maul-Pavicic A, Vraetz T, Janka G, Stadt UZ, Ehl S. Recent advances in the diagnosis and treatment of hemophagocytic lymphohistiocytosis. Arthritis Res Ther 2012;14:213.

References

15. Wu S, Gonzalez-Gomez I, Coates T, Yano S. Cobalamin C disease presenting with hemophagocytic lymphohistiocytosis. Pediatr Hematol Oncol 2005;22:717-721.

1. Watkins D, Rosenblatt DS. Inborn errors of cobalamin absorption and metabolism. Am J Med Genet C Semin Med Genet 2011;157:33-44.

16. Kaikov Y, Wadsworth LD, Hall CA, Rogers PC. Transcobalamin II deficiency: case report and review of the literature. Eur J Pediatr 1991;150:841-843.

2. Rosenblatt DS, Fenton WA. Inherited disorders of folate and cobalamin transport and metabolism. In: Budet AL, Valle D, Sly W (eds). The Metabolic and Molecular Bases of Inherited Disease. New York, McGrawHill, 2001.

17. Ünal Ş, Rupar T, Yetgin S, Yaralı N, Dursun A, Gürsel T, Çetin M. Transcobalamin II deficiency in four cases with novel mutations. Turk J Hematol 2015;32:317-322.

3. Prasad C, Rosenblatt DS, Corley K, Cairney AE, Rupar CA. Transcobalamin (TC) deficiency--potential cause of bone marrow failure in childhood. J Inherit Metab Dis 2008;31(Suppl 2):287-292.

18. Pearson HA, Lobel JS, Kocoshis SA, Naiman JL, Windmiller J, Lammi AT, Hoffman R, Marsh JC. A new syndrome of refractory sideroblastic anemia with vacuolization of marrow precursors and exocrine pancreatic dysfunction. J Pediatr 1979;95:976-984.

4. Yildirim ZK, Nexo E, Rupar T, Büyükavci M. Seven patients with transcobalamin deficiency diagnosed between 2010 and 2014: A singlecenter experience. J Pediatr Hematol Oncol 2017;39:38-41.

19. Namour F, Helfer AC, Quadros EV, Alberto JM, Bibi HM, Orning L, Rosenblatt DS, Jean-Louis G. Transcobalamin deficiency due to activation of an intra exonic cryptic splice set. Br J Haematol 2003;123:915-920.

BRIEF REPORT DOI: 10.4274/tjh.galenos.2018.2018.0190 Turk J Hematol 2019;36:43-47

Thalidomide for the Management of Bleeding Episodes in Patients with Hereditary Hemorrhagic Telangiectasia: Effects on Epistaxis Severity Score and Quality of Life Herediter Hemorajik Telenjiektazi Hastalarında Epistaksis Ataklarının Yönetimi: Talidomid Tedavisinin Epistaksis Ciddiyet Skoru ve Yaşam Kalitesi Üzerine Olan Etkileri Mehmet Baysal,

Elif G. Ümit,

Hakkı Onur Kırkızlar,

Ali Caner Özdöver,

Ahmet Muzaffer Demir

Trakya University Faculty of Medicine, Department of Hematology, Edirne, Turkey

Abstract

Öz

Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominantly inherited disorder characterized by bleeding episodes. These episodes tend to happen spontaneously and reduce the quality of life. Patients are often unresponsive to local measures. With the pathophysiological role of angiogenesis in HHT, antiangiogenic drugs including thalidomide are used to control bleeding episodes. In our study, we evaluated 6 patients with HHT, calculating their Epistaxis Severity Score (ESS) and performing a quality of life assessment with the 36-Item Short Form Health Survey Questionnaire (SF-36), and we studied the alterations of these evaluations with thalidomide treatment. Three patients were male and three were female. Mean age was 60.50 years. No side effects were observed during the treatment period. Improvements of certain SF-36 dimensions including physical functioning, physical component summary, and mental component summary and of the ESS were observed after treatment. Thalidomide may be effective to control bleeding episodes with a reasonable tolerance profile in patients with HHT.

Herediter hemorajik telenjiektazi (HHT), kanama atakları ile karakterize, nadir görülen, otozomal dominant kalıtılan bir hastalıktır. Bu ataklar spontan olarak geçekleşmekte ve hastların yaşam kalitesini düşürmektedir. Hastalar genellikle lokal cerrahi müdahalelere yanıtsızdır. HHT’deki anjiyogenezin patofizyolojik rolü ile kanama ataklarını kontrol etmek için talidomid içeren antianjiyojenik ilaçlar kullanılması düşünlmüştür. Çalışmamızda 6 HTT’li hasta değerlendirilmiştir. Hastaların talidomid tedavisi öncesinde ve sonrasında Epistaksis Ciddiyet Skoru (ESS) ve yaşam kalitesini değerlendirmek amacıyla 36 maddelik Kısa Sağlık Durumu Anketi (SF-36) değerlendirildi. Hastalarımızın 3’ü erkek olup, 3’ü kadındı. Ortalama yaşları 60,50 idi. Tedavi süresince ciddi bir yan etki izlenmedi. Tedaviden sonra ESS ve SF-36’nın fiziksel fonksiyon, fiziki bileşen özeti, ruhsal bileşen özeti gibi alt gruplarında önemli düzeyde iyileşmeler izlendi. Talidomide HHT hastalarında kanama atakların kontrol etmede makül bir yan etki profiliyle etkili olabilir.

Keywords: Hereditary hemorrhagic telangiectasia, Thalidomide, Epistaxis, Quality of life

Introduction Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant disorder characterized by telangiectasia and arteriovenous malformations of the skin, mucosal tissues, and internal organs including the gastrointestinal tract, liver, and lungs. Its prevalence is estimated to be between 1/5000 and 1/10,000 worldwide. Recurrent epistaxis due to nasal telangiectasia is the most common finding [1,2]. There are several mutations linked with the disease and the most frequent

Anahtar Sözcükler: Herediter hemorajik telenjiektazi, Thalidomide, Epistaksis, Yaşam kalitesi

mutations are reported as the ENG gene encoding endoglin and the ACVRL1 gene encoding activin A receptor type II-like kinase 1 [3]. Anemia is a very common symptom in HHT patients, not only due to bleeding from telangiectases located in the nasal mucosa but also to telangiectases located in the gastrointestinal tract, especially active in older ages. The diagnostic criteria for HHT were defined in 2000 and updated in 2011, including epistaxis, telangiectases, vascular malformations, and family history. The presence of 3 of these criteria is suggested to be sufficient for diagnosis [4]. Since epistaxis is the most common

Address for Correspondence/Yazışma Adresi: Mehmet BAYSAL, M.D., Trakya University Faculty of Medicine, Department of Hematology, Edirne, Turkey Phone : +90 284 235 76 41/2687 E-mail : drmehmetbaysal@gmail.com ORCID-ID: orcid.org/0000-0001-7681-4623

Received/Geliş tarihi: June 02, 2018 Accepted/Kabul tarihi: June 08, 2018

Baysal M, et al: Effects on Epistaxis Severity Score and Quality of Life

manifestation, patients are frequently admitted and evaluated by otolaryngologists. Invasive measures and interventions including cauterization are ineffective and the diagnosis of HHT, which is quite rare, comes into consideration. Before the identification of pathogenetic mechanisms of bleeding from telangiectases, local or systemically effective antifibrinolytics (tranexamic acid, epsilon aminocaproic acid), estrogen or progesterone preparations such as ethinylestradiol or norethisterone, and selective estrogen receptor modulators such as mestranol, norethynodrel, tamoxifen, and raloxifene have been used to control recurrent epistaxis and gastrointestinal bleeding episodes [5]. Thalidomide was first designed as a sedative and then used for nausea in pregnant women; it has been responsible for about 10,000 infants being born with phocomelia. This disastrous experience led to the further understanding of the mechanism of action of thalidomide. The effects of thalidomide on the innate and adaptive immune system as well as tumor development and angiogenesis have made thalidomide a model drug for the development of anticancer treatments. Besides the role of antiangiogenic medications in tumor genesis and cancer treatment, thalidomide and other novel endothelial and vascular growth factor inhibitors including bevacizumab are suggested to be beneficial in patients with HHT since the pathogenesis of HHT primarily relies on excessive formation of new vessels [6,7,8,9,10,11,12,13]. This has been supported with limited evidence, however, due to the rare incidence of HHT itself. In our study, we aimed to evaluate our thalidomide experience based on a bleeding score developed in a multicenter study with a large cohort of HHT patients [14] as well as their improvement in quality of life as assessed with a valid and internationally accepted scale, the 36-Item Short Form Health Survey Questionnaire (SF-36) [15].

Materials and Methods The data of six patients diagnosed with HHT according to the Curaçao HHT diagnostic criteria were recorded from their files in a retrospective manner. All patients were admitted and treated by otolaryngologists with simple epistaxis first, with cauterization and local measures like tampons and compression, and as they were observed to be refractory to such interventions they were referred to our department with consideration of a possible HHT diagnosis. All patients were refractory to local cauterization and local or systemically effective antifibrinolytics. All patients were anemic due to chronic bleeding episodes. Heavy bleedings due to nasal telangiectases were assessed with the Epistaxis Severity Score (ESS) [15], while the requirement of transfusions was based on the 2016 American Association of Blood Bank clinical guidelines on red blood cell transfusion [16]. Treatment of bleeding episodes with thalidomide was started at 50 mg daily and dose escalation to 100 mg/daily was scheduled individually according to response. Patients were evaluated every 2 months 44

Turk J Hematol 2019;36:43-47

for response assessment. Therefore, all patients started with 50 mg/daily and Patient 1, Patient 2, Patient 3, and Patient 4 were escalated to 100 mg/daily after 2 months. Patient 5 and Patient 6 did not need to escalate to 100 mg and continued with 50 mg/daily. Treatment duration was based on controlling epistaxis episodes, reducing the ESS, and improving anemia. Quality of life of the patients was assessed with the SF-36, the most commonly used such survey, which consists of two main dimensions: a physical component summary, including physical function, physical role, pain, and general health status, and a mental component summary, including social functioning, emotional role, and mental health. The scale is a selfassessment scale and is filled in by the patient in a very short time [15]. This study was approved by the Trakya University Medical Faculty’s Ethics Committee of Non-Invasive Clinical Research (2017-334). Written informed consent was obtained from all patients. Statistical calculations were performed using SPSS 22 for PC (IBM Corp., USA). The Wilcoxon signed ranks test was used for nonparametric data for two related samples.

Results Three patients were male and three were female. Mean age was 60.50±10.07 years (range: 44-74). The mean ESS score before treatment was 7.40±2.02 (range: 4.31-9.66) and after treatment it was 3.10±1.79 (range: 0.92-4.94). This finding was statistically significant (p=0.028). The change of hemoglobin level (g/dL) was also statistically significant before and after treatment (p=0.027) (Table 1). One patient reported grade one dizziness and one patient reported nausea, which resolved spontaneously without a need for drug withdrawal. Patients were frequently asked about and examined for common side effects of thalidomide such as neuropathy and constipation. No patient reported these side effects, and no patient had neutropenia or hematologic toxicity. Each patient tolerated the drug well. All SF-36 item scores were found to be increased after the treatment, which may be regarded as improvement of quality of life with thalidomide treatment. Improvements of certain SF-36 dimensions including physical functioning, physical component summary, and mental component summary were statistically significant (p=0.042, 0.048, and 0.046 respectively). Detailed assessment of each patient is summarized in Table 2 and Table 3. The change in ESS before and at the end of treatment is demonstrated in Figure 1. Table 1. Improvement in Epistaxis Severity Score and hemoglobin levels before and after treatment.

Before treatment

After treatment

p-value

ESS

7.40

3.10

0.028

Hemoglobin level, g/dL

8.81

11.51

0.027

ESS: Epistaxis Severity Score.

Baysal M, et al: Effects on Epistaxis Severity Score and Quality of Life

Turk J Hematol 2019;36:43-47

Discussion

consequence of these chronic bleeding episodes is iron deficiency and iron-deficiency anemia. Severe recurrent bleeding episodes may even lead to transfusion requirements. As the international guidelines support a restrictive transfusion approach, in our study as well as our daily practice we follow this approach and limit our transfusion practice as transfusion is not indicated until the hemoglobin level is 7-8 g/dL if the patient is hemodynamically stable and asymptomatic. We instead support the patients with iron supplements. In patients with recurrent severe bleeding episodes, parenteral agents such as iron carboxymaltose may be reasonable to also protect the gastrointestinal tract and avoid the irritation of oral iron as well as to limit transfusion. To control bleeding episodes once and for all, the pathogenesis of HHT has been researched.

As a rare disease, the prevalence of HHT is much higher than that of inherited bleeding disorders hemophilia A and B with its autosomal dominant nature. As the most common symptoms are associated with bleeding, patients are frequently evaluated by emergency departments or surgical and general practice departments and are regarded as cases of simple nose bleeds or gastrointestinal hemorrhage although an autosomal dominantly inherited disorder is present with a prominent family history that is ignored and unappreciated. The development of telangiectasia is related to aging, and as life expectancy is not affected and is even reported to be increased in recent studies, the quality of life of these patients should be a main goal of treatment [17,18]. Besides the irritative nature of mucosal bleeding, the Table 2. Characteristics of patients and response to thalidomide. Name

Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

Patient 6

Age and sex

64, F

44, M

74, F

62, F

56, M

61, M

Age at diagnosis

Heavy menstrual bleeding for female patients

Yes

N/A

Yes

N/A

Obstetric problems

1 abortus

N/A

None

1 abortus

N/A

Epistaxis

Yes

Telangiectasia

Yes

AV malformations

Hepatic

None

Hepatic

None

Family history

Yes

None

Yes

ESS before treatment

9.07

9.66

7.31

8.22

4.31

5.86

Hemoglobin level (g/dL) before treatment

8.7 g/dL

9.2 g/dL

6.7 g/dL

5.9 g/dL

11.6 g/dL

10.8 g/dL

Transfusion of erythrocytes before treatment

8/month

4/month

6/month

8/month

2/month

Dose

100 mg

50 mg

Treatment duration

1 year, ongoing

6 months, ongoing

3 months, ongoing

ESS after treatment

3.69

3.06

4.94

0.92

1.05

Hemoglobin level (g/dL) after treatment

12.3 g/dL

11.8 g/dL

9.1 g/dL

8.8 g/dL

14.0 g/dL

13.1 g/dL

Mean transfusion of erythrocytes after treatment

2/month

None

3/month

None

Curaçao diagnostic criteria

M: Male, F: female, ESS: Epistaxis Severity Score, AV: arteriovenous.

Table 3. Quality of life assessment and improvement after thalidomide. SF-36 items

SF-36 scores before treatment

SF-36 scores after treatment

p-value

Physical functioning

55.83

60.00

0.042

Physical role

45.55

45.58

0.894

Bodily pain

44.55

50.41

0.103

General health

58.33

60.00

0.576

Vitality

45.00

55.00

0.272

Social functioning

54.16

70.83

0.254

Emotional role

33.33

38.85

0.363

Mental health

52.66

66.33

0.158

Physical component summary

50.74

54.47

0.048

Mental component summary

46.57

57.75

0.046

SF-36: Short Form Health Survey Questionnaire.

Baysal M, et al: Effects on Epistaxis Severity Score and Quality of Life

Turk J Hematol 2019;36:43-47

A.M.D.; Literature Search: M.B., A.C.Ö.; Writing: M.B., E.G.Ü., H.O.K. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Kroon S, Snijder RJ, Faughan ME, Mager HJ. Systematic screening in hereditary hemorrhagic telangiectasia: a review. Curr Opin Pulm Med 2018;24:260-268.

Figure 1. Epistaxis Severity Score of the patients before and at the end of treatment. Excessive angiogenesis has been the underlying mechanism of telangiectases and vascular malformations and the inhibition of angiogenesis was sought. This idea of anti-angiogenesis has brought back one of the most unfortunate medical hazards in history: thalidomide for morning sickness in pregnancy, causing phocomelia and other malformations. Several case series and phase two clinical trials with thalidomide reported long-term improvement in epistaxis and also hematological parameters [6,7,8,9,10]. In our study, though with a limited number of patients, we observed beneficial effects of thalidomide to control bleeding episodes, reduce the need to support patients with transfusions, and therefore increase the life quality of our patients. In a recent study, a systematic analysis of 4 studies involving 43 patients with thalidomide reported beneficial effects of thalidomide to control bleeding episodes, though with a comment on the lack of an optimal treatment modality in HHT [6].

Conclusion As an underappreciated inherited disease, HHT should be within the top possibilities in the differential diagnosis of patients with recurrent/refractory and debilitating mucosal bleeding. Family history should not be overlooked as it is one solid item of the diagnostic criteria. Besides short-lived supportive measures, patients should be referred to experienced centers for therapeutic modalities. Ethics Ethics Committee Approval: Trakya University Faculty of Medicine Ethics Committee 2017-334. Informed Consent: Written informed consent was obtained from all patients. Authorship Contributions Concept: M.B., E.G.Ü.; Design: M.B., E.G.Ü.; Data Collection or Processing: M.B., A.C.Ö.; Analysis or Interpretation: H.O.K., 46

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Turk J Hematol 2019;36:43-47

Baysal M, et al: Effects on Epistaxis Severity Score and Quality of Life

15. Ware JE Jr, Sherbourne CD. The MOS 36-item short-form health survey (SF36). I. Conceptual framework and item selection. Med Care 1992;30:473482.

17. de Gussem EM, Edwards CP, Hosman AE, Westermann CJ, Snijder RJ, Faughnan ME, Mager JJ. Life expectancy of parents with hereditary haemorrhagic telangiectasia. Orphanet J Rare Dis 2016;11:46.

16. Carson JL, Guyatt G, Heddle NM, Grossman BJ, Cohn CS, Fung MK, Gernsheimer T, Holcomb JB, Kaplan LJ, Katz LM, Peterson N, Ramsey G, Rao SV, Roback JD, Shander A, Tobian AA. Clinical practice guidelines from the AABB: Red blood cell transfusion thresholds and storage. JAMA 2016;316:2025-2035.

18. Geerts L, Fantini-Hauwel C, BrugallĂŠ E, Boute O, FrĂŠnois F, Defrance L, Manouvrier-Hanu S, Petit F, Antoine P. The subjective experience of patients diagnosed with hereditary hemorrhagic telangiectasia: a qualitative study. J Genet Couns 2017;26:612-619.

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.galenos.2018.2018.0267 Turk J Hematol 2019;36:48-49

A Case of SF3B1-Positive Myelodysplastic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis Halka (Ring) Sideroblast ve Trombositozu Olan SF3B1-Pozitif Myelodisplastik/ Myeloproliferati Neoplazm Olgusu Alejandro Lazo-Langner1,2,

Bekim Sadikovic3

1Western University, Schulich School of Medicine, Department of Medicine, London, Ontario, Canada 2Western University, Schulich School of Medicine, Department of Epidemiology and Biostatistics, London, Ontario, Canada 3Western University, Schulich School of Medicine, Department of Pathology and Laboratory Medicine, London, Ontario, Canada

Figure 1. A) Peripheral blood smear (Wright’s stain, 40x) showing marked anisopoikilocytosis. B) Bone marrow aspirate (Perls’ stain, 100x) showing increased ring sideroblasts (arrowheads). C) Next-generation sequencing pileup plot showing sequencing results for location 2q33.1 (red arrowhead) indicating the presence of an SF3B1:c1986C>A mutation (black arrowhead).

A 77-year-old woman, previously maintained on phlebotomies that had been discontinued 3 years before for a purported diagnosis of iron overload, was assessed for normocytic normochromic anemia. Her blood count showed hemoglobin of 90 g/L (normal: 115-160), mean corpuscular volume of 93.2 fL (normal: 79-97), erythrocyte distribution width of 28.1% (normal: 12%-15%), and platelets of 422x109/L (normal: 150-400). Iron

studies showed elevated ferritin (491 µg/L; normal: 13-150), total iron of 14 µmol/L (normal: 7-26), transferrin saturation of 32% (normal: 11%-56%), and unsaturated iron binding capacity of 30 µmol/L (normal: 19.7-66.2). The vitamin B6 level was low (<10 nmol/L; normal: 20-96). HFE C282Y, H63D, and JAK2 V617F mutations were negative. The peripheral blood smear showed marked anisopoikilocytosis (Figure 1A; Wright’s stain, 40x). A

Address for Correspondence/Yazışma Adresi: Alejandro LAZO-LANGNER, M.D., Western University, Schulich School of Medicine, Department of Medicine, London, Ontario, Canada Phone : (519) 685-8500 Ext. 58833 E-mail : alejandro.lazolangner ORCID-ID: orcid.org/0000-0001-6869-8431

Received/Geliş tarihi: July 30, 2018 Accepted/Kabul tarihi: November 23, 2018

Turk J Hematol 2019;36:48-49

Lazo-Langner A and Sadikovic B: A Case of SF3B1-Positive Myelodysplastic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis

bone marrow aspirate and biopsy showed hypercellular marrow (70%-80%) with moderate dyserythropoiesis, minimal dysplastic changes in other lineages, and increased ring sideroblasts (Figure 1B; Perls’ stain, 100x), consistent with a myelodysplastic/ myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T; WHO 2016). The karyotype was normal. Next-generation sequencing studies reported the presence of an SF3B1:c1986C>A, p.(His662Gln) mutation (Figure 1C) with a variant allele frequency of 40.5%. SF3B1 mutations result in the disruption of mitochondrial iron metabolism and define a distinct subgroup of patients with myelodysplasia with a better prognosis than other subtypes.

Keywords: Myelodysplasia, Ring sideroblasts, Splicing factor 3b subunit 1 (SF3B1) Anahtar Sözcükler: Myelodisplazi, Halka sideroblast, Splicing (ucbirleştirme) faktor 3b altünitesi (SF3B1) Informed Consent: Received. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.galenos.2018.2018.0164 Turk J Hematol 2019;36:50-51

Peutz-Jeghers Syndrome: A Very Rare Cause of Iron Deficiency Anemia Peutz-Jeghers Sendromu: Demir Eksikliği Anemisinin Nadir Bir Nedeni Fatma Demir Yenigürbüz1,

Ugur Deveci2,

Ebru Tuncez3

1Şanlıurfa Training and Research Hospital, Clinic of Pediatric Hematology, Şanlıurfa, Turkey 2Şanlıurfa Training and Research Hospital, Clinic of Pediatric Gastroenterology, Şanlıurfa, Turkey 3Şanlıurfa Training and Research Hospital, Clinic of Medical Genetics, Şanlıurfa, Turkey

Figure 2. Similar mucocutaneous pigmentation of the patient’s mother.

Figure 1. Multiple small, flat brown-violet pigmentations on the patient’s buccal mucosa and lips and brown spotty pigmentations on the lip mucosa.

Figure 3. Polyps were revealed in the patient’s gastrointestinal endoscopic examination.

Address for Correspondence/Yazışma Adresi: Fatma DEMİR YENİGÜRBÜZ, M.D., Şanlıurfa Training and Research Hospital, Clinic of Pediatric Hematology, Şanlıurfa, Turkey Phone : +90 414 31 71 27 E-mail : ftmdmr@yahoo.com ORCID-ID: orcid.org/0000-0001-8692-2767

Received/Geliş tarihi: May 19, 2018 Accepted/Kabul tarihi: July 23, 2018

Turk J Hematol 2019;36:50-51

Demir Yenigürbüz F et al: Peutz-Jeghers Syndrome: A Very Rare Cause of Iron Deficiency Anemia

An 11-year-old boy was admitted to the clinic with a 1-year history of fatigue and abdominal pain. In physical examination, there were multiple small, flat brown-violet pigmentations on his buccal mucosa and lips and brown spotty pigmentations on the lip mucosa, present since his birth (Figure 1). His mother also had similar mucocutaneous pigmentation and was operated on for intestinal polyps (Figure 2). Laboratory findings were consistent with severe iron deficiency anemia and the fecal occult blood test was positive. Gastrointestinal endoscopic examination revealed two polyps of the stomach and three polyps of the jejunum that caused bleeding were removed with forceps (Figure 3). Histopathologic examination revealed hamartomatous polyps. The presence of brown pigmentations and multiple gastrointestinal polyps alerted us to a possible diagnosis of Peutz-Jeghers syndrome and serine/threonine kinase 11 (STK11, also called LKB1) mutation was found positive in both the patient and his mother. It is very important to conduct a thorough physical examination and to probe the family history in cases of iron deficiency anemia that is frequently encountered in children, especially in the presence of other complaints such as abdominal pain [1,2]. This allows early diagnosis of rare diseases such as Peutz-Jeghers syndrome, which leads to a high risk of developing cancer, and examination of family members for the associated complications by using advanced diagnostic tools [3,4,5].

Keywords: Peutz-Jeghers syndrome, Iron deficiency anemia, Bleeding Anahtar Sözcükler: Peutz-Jeghers sendromu, Demir eksikliği anemisi, Kanama Informed Consent: It was received. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Van Lier MG, Westerman AM, Wagner A, Looman CW, Wilson JH, de Rooij FW, Lemmens VE, Kuipers EJ, Mathus-Vliegen EM, van Leerdam ME. High cancer risk and increased mortality in patients with Peutz-Jeghers syndrome. Gut 2011;60:141-147. 2. Beggs AD, Latchford AR, Vasen HF, Moslein G, Alonso A, Aretz S, Bertario L, Blanco I, Bülow S, Burn J, Capella G, Colas C, Friedl W, Møller P, Hes FJ, Järvinen H, Mecklin JP, Nagengast FM, Parc Y, Phillips RK, Hyer W, Ponz de Leon M, Renkonen-Sinisalo L, Sampson JR, Stormorken A, Tejpar S, Thomas HJ, Wijnen JT, Clark SK, Hodgson SV. Peutz-Jeghers syndrome: a systematic review and recommendations for management. Gut 2010;59:975-986. 3. Achatz MI, Porter CC, Brugières L, Druker H, Frebourg T, Foulkes WD, Kratz CP, Kuiper RP, Hansford JR, Hernandez HS, Nathanson KL, Kohlmann WK, Doros L, Onel K, Schneider KW, Scollon SR, Tabori U, Tomlinson GE, Evans DGR, Plon SE. Cancer screening recommendations and clinical management of inherited gastrointestinal cancer syndromes in childhood. Clin Cancer Res 2017;23:107-114. 4. Vidal I, Podevin G, Piloquet H, Le Rhun M, Frémond B, Aubert D, Leclair MD, Héloury Y. Follow-up and surgical management of Peutz-Jeghers syndrome in children. J Pediatr Gastroenterol Nutr 2009;48:419-425. 5. Shah J, Sunkara T, Xiao P, Gaduputi V, Reddy M, Razia S. Peutz-Jeghers syndrome presenting as colonic intussusception: a rare entity. Gastroenterol Res 2018;11:150-153.

LETTERS TO THE EDITOR Turk J Hematol 2019;36:52-71

Kasabach-Merritt Syndrome in an Adult: A Comment Erişkin Bir Hastada Kasabach-Merritt Sendromu: Bir Yorum Sevgi Gözdaşoğlu Retired Professor

To the Editor, I read with interest the article by Vinod et al. [1] entitled “Kasabach-Merritt Syndrome in an Adult” in a recent issue of the Turkish Journal of Hematology. Related to this article, I would like to add that Özsoylu [2] reported megadose methylprednisolone (MDMP) treatment for different hematological conditions including hemangiomas, infantile hemangiomatosis, and Kasabach-Merritt syndrome. Özsoylu [2] recommended the daily use of MDMP at 30 mg/kg for 3 days, then 20 mg/kg for 4 days and subsequently 10, 5, 2, and 1 mg/kg with each dose administered for 1 week around 6 AM as a single dose [2,3]. Özsoylu [2] treated patients who were resistant to conventional corticosteroid treatment either orally or intravenously over the course of 10-15 min [2,3]. Conventional corticosteroid treatment and MDMP were comparatively studied for the treatment of hemangiomas and it was observed that MDMP was superior [4]. In our department, eight patients with infantile hemangiomas were treated with interferon-α2a plus oral prednisolone without observing severe complications. In this study, the median age was 4 months (range: 3-12 months); six of them were female and two were male. Three cases were not followed and three patients achieved complete regression while a partial regression was observed in two others. The beneficial results were obtained with interferon-α2a plus oral prednisolone treatment in these cases [5]. On the other hand, vascular endothelial growth factor (VEGF) is recognized as an essential regulator of blood vessel growth. The use of anti-VEGF abolishes this vascular endothelial growth-promoting activity in vitro [6]. Three patients with

periocular epithelioid hemangioma were treated with antiVEGF successfully [7]. Although recent studies have indicated the effectiveness of anti-VEGF treatment in these cases, there is not a significant number of clinical trials reported. Keywords: Megadose methylprednisone, Kasabach-Merritt Syndrome, VEGF Anahtar Sözcükler: Megadoz metilprednizon, Kasabach-Merritt Sendromu, VEGF Conflict of Interest: The author of this paper has no conflicts of interest including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Vinod KV, Johny J, Vadivelan M, Hamide A. Kasabach-Merritt syndrome in an adult. Turk J Hematol 2018;35:204-205. 2. Özsoylu Ş. Megadose methyl-prednisolone (MDMP) for hemangiomatosis. Turk J Hematol 2012;29:436-437. 3. Özsoylu Ş. About the treatment of Kasabach-Merritt syndrome. Turk J Hematol 2013;30:94. 4. Uysal KM, Olgun N, Erbay A, Sarıalioğlu F. High-dose oral methylprednisolone therapy in childhood hemangiomas. Pediatr Hematol Oncol 2001;18:335341. 5. Gözdaşoğlu S, Uysal Z, Ertem M, Suskan E, Kürekci E, İleri T, Azık F, Cin Ş. Bebeklik çağı Hemanjiomlarında interferon - α 2a tedavisi. In: IV. Ulusal Pediatrik Hematoloji Kongresi, 10-13 September Trabzon, Turkey, 2003. 6. Mahajan D, Miller C, Hirose K, McCullough A, Yerian L. Incidental reduction in the size of liver hemangioma following use of VEGF inhibitor bevacizumab. J Hepatol 2008;49:867-870. 7. Kahana A, Lee BJ, Flint A, Elner VM. Periocular epithelioid hemangioma: response to bevacizumab and vascular pathogenesis. JAMA Ophthal 2012;130:1209-1212.

Address for Correspondence/Yazışma Adresi: Sevgi GÖZDAŞOĞLU, M.D., Kemer sok.21/78 Çankaya Ankara, Turkey Phone : +90 312 447 80 46 E-mail : sgozdasoglu@hotmail.com ORCID-ID: orcid.org/0000-0001-7198-6880

Received/Geliş tarihi: October 17, 2018 Accepted/Kabul tarihi: October 22, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0356

LETTERS TO THE EDITOR

Turk J Hematol 2019;36:52-71

Reply to the Authors Dear Editor, We would like to thank Gözdaşoğlu [1] for showing interest in our report of adult Kasabach-Merritt syndrome (KMS) [2]. Being adult hematologists, we should admit that we have limited experience with treatment of KMS, which by and large affects infants and young children. We do not have personal experience with interferon-alpha and have not studied the efficacy of different corticosteroids in different doses and combination of prednisolone with interferon-alfa for treatment of KMS. In this regard, we thank Gözdaşoğlu [1] for sharing her personal experience (effectiveness of prednisolone + interferon-alfa 2a combination therapy) with treatment of infantile hemangiomas and also for highlighting on reports of usefulness of high dose methylprednisolone [3] and anti-VEGF (bevacizumab) therapy for treatment of hemangiomas and KMS. Clinical trials evaluating different therapies would be desirable in the future to clarify on optimal treatment of KMS. Best Regards, Kolar Vishwanath Vinod, Joseph Johny, Mehalingam Vadivelan, Abdoul Hamide

References 1. Gözdaşoğlu S. Kasabach-Merritt Syndrome in an Adult: A Comment. Turk J Hematol 2019;36:52. 2. Vinod KV, Johny J, Vadivelan M, Hamide A. Kasabach-Merritt Syndrome in an adult. Turk J Hematol 2018;35:204-205. 3. Uysal KM, Olgun N, Erbay A, Sarıolioğlu F. High-dose oral methylprednisolone therapy in childhood hemangiomas. Pediatr Hematol Oncol 2001;18:335-341.

Bendamustine and Rituximab Treatment, Chronic Lymphocytic Leukemia, Direct Antiglobulin Test, and False Negatives Bendamustin ve Rituksimab Tedavisi, Kronik Lenfositik Lösemi, Direkt Antiglobulin Test ve Yanlış Negatiflik Won Sriwijitalai1,

Viroj Wiwanitkit2

1TWS Medical Center, Bangkok, Thailand 2Dr. DY Patil University, Pune, India

To the Editor, We read “Early Direct Antiglobulin Test Negativity After Bendamustine and Rituximab Treatment in Chronic Lymphocytic Leukemia: Two Cases” [1]. Eren and Suyanı noted that “BR seems to be an important treatment of choice in terms of eliminating the poor prognostic factor of direct antiglobulin test (DAT) positivity and assuring safe cessation of steroid treatment due to rapid achievement of DAT negativity” [1]. The interesting observation of a negative DAT test should be discussed. There is another possibility that Eren and Suyanı did not mention. In their report, Eren and Suyanı noted that no titer was provided [1]. Whether the negative result is a false negative result should be discussed. For DAT

testing, the prozone phenomenon is observable and it is important to consider this phenomenon in the interpretation of unexpected false negative DAT tests [2,3,4]. Keywords: Bendamustine, Rituximab, antiglobulin test, False negatives

Leukemia,

Direct

Anahtar Sözcükler: Bendamustin, Rituksimab, Lösemi, Direkt antiglobulin testi, Yanlış negatiflik Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

LETTERS TO THE EDITOR

References 1. Eren R, Suyanı E. Early direct antiglobulin test negativity after bendamustine and rituximab treatment in chronic lymphocytic leukemia: two cases. Turk J Hematol 2018;35:312-313. 2. Hubinont PO. The prozone phenomenon in haemagglutination reactions; with special reference to the quantitative antiglobulin test. Br Med J 1951;2:708-711.

Turk J Hematol 2019;36:52-71

3. Salama A, Mueller-Eckhardt C. Elimination of the prozone effect in the antiglobulin reaction by a simple modification. Vox Sang 1982;42:157159. 4. Ceppellini R. Causes of error in interpretation of direct Coombs’ test; false negative reaction due to prozone and false positive reaction due to cold autoantibodies. Sangue 1951;24:158-159.

Address for Correspondence/Yazışma Adresi: Won SRIWIJITALAI, M.D., TWS Medical Center, Bangkok, Thailand E-mail : wonsriwi@gmail.com ORCID-ID: orcid.org/0000-0002-9542-2008

Received/Geliş tarihi: November 15, 2018 Accepted/Kabul tarihi: November 23, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0397

Reply to the Authors Dear Editor, In their comments, Won Sriwijitalai and Viroj Wiwanitkit pointed out that the possibility of false negative direct antiglobulin test (DAT) was not denoted in our paper in which we shared our experience about the achievement of early DAT negativity in patients receiving bendamustine and rituximab treatment for chronic lymphocytic leukemia [1,2]. Along with the prozone phenomenon that the authors mentioned, there are other causes of a false negative DAT, including poor washing technique, improper agitation of specimen during reaction strength determination, failure to add or delayed addition of antihuman globulin reagent, inactive antihuman globulin reagent, inappropriately concentrated red blood cell suspension, and delay in testing (3). However, we used an automated gel centrifugation method sytem to detect DAT which ensured quite accurate results. Also, the reported patients had normal hemoglobin, haptoglobulin, LDH, and indirect bilirubin levels during DAT negativity, suggesting that the existence of a high titer of antibody, the reason of prozone affect, was unfeasible. Additionally, sustained DAT negativity during follow-up led to the consideration that DAT negativity was accurate and false negative DAT was unlikely. Best Regards, Rafet Eren, Elif Suyanı

References 1. Eren R, Suyanı E. Early direct antiglobulin test negativity after bendamustine and rituximab treatment in chronic lymphocytic leukemia: two cases. Turk J Hematol 2018;35:312-313. 2. Sriwijitalai W, Wiwanitkit V. Bendamustine and rituximab treatment, chronic lymphocytic leukemia, direct antiglobulin test, and false negatives. Turk J Hematol 2019;36:53-54. 3. Zantek ND, Koepsell SA, Tharp DR Jr, Cohn CS. The direct antiglobulin test: a critical step in the evaluation of hemolysis. Am J Hematol 2012;87:707-709.

LETTERS TO THE EDITOR

Turk J Hematol 2019;36:52-71

A Case of Blastic Plasmacytoid Dendritic Cell Neoplasm with Unusual Presentation Sıradışı Başlangıçlı Bir Blastik Plazmasitoid Dendritik Hücreli Neoplazi Olgusu Sneha Dhariwal,

Monica Gupta

Delhi State Cancer Institute, Department of Oncopathology, Dilshad Garden, Delhi, India

To the Editor, Blastic plasmacytoid dendritic cell neoplasm (BPDCN) was categorized in the 2008 World Health Organization classification of hematological diseases under acute myeloid leukemia and related precursor neoplasms [1]; however, in the revised edition 2017, it is a separate entity [2]. BPDCN predominantly affects elderly patients with a mean age between the sixth and seventh decades with a male predilection (M:F=3:1), but it can occur even in children [3,4]. It has an aggressive behavior and rapid systemic dissemination. We herein report a case of BPDCN in an adolescent patient with unusual presentation. A 13-year-old male presented with fever and weakness for 10 days. The patient had a history of left testicular swelling for which he had undergone orchidectomy 2 months ago. Routine

hematological investigations revealed hemoglobin of 9.1 g/dL, total leukocyte count of 5x103/µL, platelet count of 80x103/ µL, and 18% blasts on differential count. Bone marrow aspirate showed 90% blasts with lymphoid morphology (Figure 1A). Flow cytometric immunophenotyping (FCM) analysis of bone marrow revealed 80% blasts with CD45dim and low side scatter. The cells were also positive for CD4, CD56, CD38, CD123, and CD7 and negative for CD19, CD20, CD22, CD3, CD8, CD1a, CD34, CD36, TdT, CD61, CD235a, CD13, CD33, CD14, CD64, CD36, CD117, cMPO, cCD79A, and cCD3 (Figures 1J-1P). Bone marrow biopsy revealed near total replacement by monomorphic cells, which on immunohistochemistry were positive for CD4, CD43, and CD7 and negative for CD3 and CD68 (Figures 1B-1F). Paraffin blocks of the testicular mass were reviewed and revealed a tumor comprising small monomorphic round cells with scanty

Figure 1. Bone marrow aspiration showing blast-like cells, Giemsa stain, 100x (A). Bone marrow biopsy showing replacement of marrow by monomorphic cells, hematoxylin and eosin stain, 40x (B). Immunohistochemistry on bone marrow biopsy with CD3 (C), CD7 (D), CD4 (E), and CD43 (F). Histology of testicular mass, hematoxylin and eosin stain, 20x (G), and hematoxylin and eosin stain, 40x (H). Immunohistochemistry of testicular mass with CD3 (I). Dot plots with CD45 vs. SSC showing blast population highlighted in red and lymphocytes in blue (J-P). These dot plots demonstrate the expression of CD7dim, CD4dim, CD56, and CD123. The blasts are negative for CD19, CD10, cCD3, cMPO, CD3, CD8, CD5, CD2, and HLA-DR. 128x96 mm (72x72 DPI). 55

LETTERS TO THE EDITOR

cytoplasm, round to focally indented nuclei with prominent nuclear membrane, and inconspicuous nucleoli. Few mitotic figures were seen. The seminiferous tubules were enclosed and were being indented by the tumor throughout the tissue section. These cells were positive immunohistochemically for CD4, CD43, CD56, and CD123 and negative for CD3, CD68, and CD8 (Figures 1G-1I). Based on histopathological findings, immunohistochemistry, and FCM analysis, the patient was diagnosed with BPDCN. BPDCN closely resembles the precursor of plasmacytoid dendritic cells by immunophenotyping, gene expression profiling, and biologic function [5]. Skin manifestations are the main clinical presentation of typical cases of BPDCN, seen in 64%-100% of adult cases, and the diagnosis is made on skin biopsy [2,6]. Extracutaneous involvement often occurs in the lymph nodes, peripheral blood, bone marrow, skeleton, gastrointestinal tract, central nervous system, lungs, mediastinum, and pancreas [7]. Our patient, a young male, presented with testicular involvement and without any cutaneous manifestation, which is extremely rare [8]. Two months later the patient presented with leukemialike symptoms with bone marrow involvement. FCM is a useful tool and the cells express CD4, CD43, CD45RA, CD56, CD123, CD303, CD304, TCL1, CLA, CD38, CD99, and CD31 [4,7]. Recently the TCF4 (E2-2) transcription factor represents a faithful diagnostic marker [9]. The immunonegativity of immature cells for CD14, CD15, CD36, CD64, and CD68 excludes the possibility of acute myeloid leukemia with monocytic differentiation. Cytogenetic studies may show genomic abnormalities or a normal karyotype [10]. From a pathologist’s perspective, the immunophenotyping profile should be kept in mind before considering a case of BPDCN as an acute undifferentiated leukemia or acute myeloid leukemia with monocytic differentiation. Keywords: Blastic plasmacytoid dendritic cell neoplasm, Testicular involvement, Immunophenotyping Anahtar Sözcükler: Blastik plazmositoid dendritik hücreli neoplazi, testiküler tutulum, Immünfenotipleme Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships,

Turk J Hematol 2019;36:52-71

and/or affiliations relevant to the subject matter or materials included.

References 1. Facchetti F, Jones DM, Petrella T. Blastic plasmacytoid dendritic cell neoplasm. 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, IARC Press, 2008. 2. Facchetti F, Petrella T, Pileri SA. Blastic plasmacytoid dendritic cell neoplasm. In: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Arber DA, Hasserjian R, Le Beau M, Orazi A, Siebert R (eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th edition. Lyon, IARC Press, 2017. 3. Bueno C, Almeida J, Lucio P , Marco J, Garcia R, de Pablos JM, Parreira A, Ramos F, Ruiz-Cabello F, Suarez-Vilela D, San Miguel JF, Orfao A. Incidence and characteristics of CD4(+)/HLA DRhi dendritic cell malignancies. Haematologica 2004;89:58-69. 4. Julia F, Dalle S, Duru G, Balme B, Vergier B, Ortonne N, Vignon-Pennamen MD, Costes-Martineau V, Lamant L, Dalac S, Delattre C, Déchelotte P, Courville P, Carlotti A, De Muret A, Fraitag S, Levy A, Mitchell A, Petrella T. Blastic plasmacytoid dendritic cell neoplasms: clinico immunohistochemical correlations in a series of 91 patients. Am J Surg Pathol 2014;38:673 680. 5. Shi Y, Wang E. Blastic plasmacytoid dendritic cell neoplasm: a clinicopathologic review. Arch Pathol Lab Med 2014;138:564-569. 6. Jegalian AG, Buxbaum NP, Facchetti F, Raffeld M, Pittaluga S, Wayne AS, Jaffe ES. Blastic plasmacytoid dendritic cell neoplasm in children: diagnostic features and clinical implications. Haematologica 2010;95:1873-1879. 7. Suzuki R, Nakamura S, Suzumiya J, Ichimura K, Ichikawa M, Ogata K, Kura Y, Aikawa K, Teshima H, Sako M, Kojima H, Nishio M, Yoshino T, Sugimori H, Kawa K, Oshimi K; NK-cell Tumor Studt Group. Blastic natural killer cell lymphoma/leukemia (CD56‑positive blastic tumor): prognostication and categorization according to anatomic sites of involvement. Cancer 2005;104:1022‑1031. 8. Deng W, Yang M, Kuang F, Liu Y, Zhang H, Cao L, Xie M, Yang L. Blastic plasmacytoid dendritic cell neoplasm in children: a review of two cases. Mol Clin Oncol 2017;7:709-715. 9. Ceribelli M, Hou ZE, Kelly PN, Huang DW, Wright G, Ganapathi K, Evbuomwan MO, Pittaluga S, Shaffer AL, Marcucci G, Forman SJ, Xiao W, Guha R, Zhang X, Ferrer M, Chaperot L, Plumas J, Jaffe ES, Thomas CJ, Reizis B, Staudt LM. A druggable TCF-4 and BRD4-dependent transcriptional network sustains malignancy in blastic plasmacytoid dendritic cell neoplasm. Cancer Cell 2016;30:764-778. 10. Leroux D, Mugneret F, Callanan M, Radford-Weiss I, Dastugue N, Feuillard J, Le Mée F, Plessis G, Talmant P, Gachard N, Uettwiller F, Pages MP, Mozziconacci MJ, Eclache V, Sibille C, Avet-Loiseau H, Lafage-Pochitaloff M. CD4+, CD56+ DC2 acute leukemia is characterized by recurrent clonal chromosomal changes affecting 6 major targets: a study of 21 cases by the Groupe Francais de Cytogenetique Hematologique. Blood 2002;99:41544159.

Address for Correspondence/Yazışma Adresi: MONICA GUPTA, M.D., Delhi State Cancer Institute, Department of Oncopathology, Dilshad Garden, Delhi, India Phone : +919 560 390 110 E-mail : drmonica123@gmail.com ORCID-ID: orcid.org/0000-0002-3992-1013

Received/Geliş tarihi: May 29, 2018 Accepted/Kabul tarihi: July 23, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0181

LETTERS TO THE EDITOR

Turk J Hematol 2019;36:52-71

EBV-Related Diffuse Large B-Cell Lymphoma in a Patient with Angioimmunoblastic T-Cell Lymphoma Anjiyoimmünoblastik T-Hücreli Lenfomaya Eşlik Eden EBV-İlişkili Diffüz Büyük B-Hücreli Lenfoma Cem Şimşek1,

Başak Bostankolu1,

Ece Özoğul2,

Arzu Sağlam Ayhan2,

Ayşegül Üner2,

Yahya Büyükaşık3

1Hacettepe University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey 2Hacettepe University Faculty of Medicine, Department of Pathology, Ankara, Turkey 3Hacettepe University Faculty of Medicine, Division of Hematology, Ankara, Turkey

To the Editor, Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma, accounting for approximately one-fifth of cases [1]. Epstein-Barr virus (EBV)positive B cells are present in the tumor tissue in most cases [2]. Twenty-five cases of EBV-associated B-cell lymphomas in AITL patients have been reported in the literature [1,3,4,5,6,7,8,9,10, 11,12,13,14,15]; herein, we report the 26th case. A 68-year-old female patient presented with B symptoms, multiple lymphadenopathies, and hepatosplenomegaly. Her laboratory studies were unremarkable except for normocytic anemia, eosinophilia, and increased lactate dehydrogenase. Her HIV serology was negative. Lymph node biopsy showed total effacement of the lymph node architecture with a polymorphic infiltrate composed of small to medium-sized lymphocytes, eosinophils, and occasional immunoblasts in a background of vascular proliferation (Figures 1A and 1B). Neoplastic cells were positive for CD3, CD4, PD1, and CD2. There was an extensive growth of follicular dendritic

meshwork extending beyond the germinal centers. Large immunoblastic cells were scattered and positive for CD20. There were only occasional scattered LMP-1-positive blasts. However, T-cell receptor clonality analysis revealed a single prominent band with Tvγ-5J17 primers observed initially. In a subsequent biopsy, a sharp band with fr22 primers was observed. The morphologic and immunophenotypic features of the lymphoid proliferation were consistent with AITL. Six cycles of a CVP regimen (cyclophosphamide, vincristine, and prednisolone) were administered in 6 months. Doxorubicin or any other anthracycline was not administered due to associated cardiac morbidities. Post-treatment evaluation imaging showed radiological remission. However, nearly 1 month later, and 7 months after the initial diagnosis, she presented with multiple pruritic erythematous plaques on her arms and back. B symptoms had also returned. A biopsy from the largest lesion on her forearm showed infiltration of the dermis and subcutaneous tissue with large pleomorphic cells. Subsequent B-cell lymphoma cells that developed in the background of angioimmunoblastic lymphoma were positive

Figure 1. A, B) Cervical lymph node biopsy showing angoimmunoblastic T-cell lymphoma. Effacement of lymph node architecture, infiltrate of small to-medium sized lymphocytes, eosinophils, and occasional immunoblasts in a background of vascular proliferation consistent with angioimmunoblastic T-cell lymphoma. 57

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Figure 2. Biopsy from cutaneous lesion: A, B) infiltration of the dermis and subcutaneous tissue with large pleomorphic cells; C) with CD20 positivity; D) with LMP-1 positivity. for CD20, LMP-1, and EBER (Figure 2). The diagnosis was EBVrelated diffuse large B-cell lymphoma (DLBCL) secondary to AITL. Control radiological examinations and/or bone marrow biopsies were not performed at that time. ICE (etoposide, iphosphamide, mesna, carboplatin) was started and 3 cycles were completed [16]. Rituximab (375 mg/m2 on day 1) was added to the protocol after the first cycle. A later control biopsy from skin lesions showed residual B-cell neoplasia. Control imaging also showed widespread lymphadenopathies in the neck, thorax, and abdomen. She was monitored closely without further cytotoxic treatment since she had poor performance status. The patient died of sepsis, with an overall survival of 14 months. Secondary B-cell lymphoma may complicate AITL and has a poor prognosis. Only 3 of the previously reported 25 patients were described to have an overall survival longer than 12 months. Clinicians should be alerted by new-onset symptoms or lesions in a lymphoma patient, and suspicious lesions should 58

be biopsied. The optimal treatment for either AITL or secondary DLBCL remains undefined. Could the patient have had two different lymphomas (i.e. simultaneous or composite lymphomas) at the first presentation? It is impossible to exclude the possibility that she had additional EBV-related DLBCL in some of the multiple lymphadenopathies, with enlarged spleen and liver at presentation. Even so, the message to be taken from the association of AITL and EBV-related DLBCL (either simultaneous/composite or sequential) is the same: AITL is frequently EBV-positive and this positivity may result in EBVpositive DLBCL. Therefore, clinicians should be aware of this possibility. Keywords: Angioimmunoblastic T-cell lymphoma, Secondary lymphoma, EBV-related lymphoma, Cutaneous lymphoma Anahtar Sözcükler: Anjiyoimmünoblastik T hücreli lenfoma, İkincil lenfoma, EBV-ilişkili lenfoma, Deri lenfomaları

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8. Attygalle AD, Kyriakou C, Dupuis J, Grogg KL, Diss TC, Wotherspoon AC, Chuang SS, Cabeçadas J, Isaacson PG, Du MQ, Gaulard P, Dogan A. Histologic evolution of angioimmunoblastic T-cell lymphoma in consecutive biopsies: clinical correlation and insights into natural history and disease progression. Am J Surg Pathol 2007;31:1077-1088.

References

9. Willenbrock K, Braeuninger A, Hansmann ML. Frequent occurrence of B-cell lymphomas in angioimmunoblastic T-cell lymphoma and proliferation of Epstein-Barr virus-infected cells in early cases. Br J Haematol 2007;138:733739.

1. Zettl A, Lee SS, Rüdiger T, Starostik P, Marino M, Kirchner T, Ott M, Müller-Hermelink HK, Ott G. Epstein-Barr virus-associated B-cell lymphoproliferative disorders in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified. Am J Clin Pathol 2002;117:368379. 2. Zhou Y, Attygalle AD, Chuang SS, Diss T, Ye H, Liu H, Hamoudi RA, Munson P, Bacon CM, Dogan A, Du MQ. Angioimmunoblastic T-cell lymphoma: histological progression associates with EBV and HHV6B viral load. Br J Haematol 2007;138:44-53. 3. Yang QX, Pei XJ, Tian XY, Li Y, Li Z. Secondary cutaneous EpsteinBarr virus-associated diffuse large B-cell lymphoma in a patient with angioimmunoblastic T-cell lymphoma: a case report and review of literature. Diagn Pathol 2012;7:7. 4. Abruzzo LV, Schmidt K, Weiss LM, Jaffe ES, Medeiros LJ, Sander CA, Raffeld M. B-Cell lymphoma after angioimmunoblastic lymphadenopathy - a case with oligoclonal gene rearrangements associated with Epstein-Barr virus. Blood 1993;82:241-246. 5. Park S, Noguera ME, Brière J, Feuillard J, Cayuela JM, Sigaux F, Brice P. Successful rituximab treatment of an EBV-related lymphoproliferative disease arising after autologous transplantation for angioimmunoblastic T-cell lymphoma. Hematol J 2002;3:317-320. 6. Xu Y, McKenna RW, Hoang MP, Collins RH, Kroft SH. Composite angioimmunoblastic T-cell lymphoma and diffuse large B-cell lymphoma - A case report and review of the literature. Am J Clin Pathol 2002;118:848854. 7. Hawley RC, Cankovic M, Zarbo RJ. Angioimmunoblastic T-cell lymphoma with supervening Epstein-Barr virus-associated large B-cell lymphoma. Arch Pathol Lab Med 2006;130:1707-1711.

10. Weisel KC, Weidmann E, Anagnostopoulos I, Kanz L, Pezzutto A, Subklewe M. Epstein-Barr virus-associated B-cell lymphoma secondary to FCD-C therapy in patients with peripheral T-cell lymphoma. Int J Hematol 2008;88:434440. 11. Skugor ND, Perić Z, Vrhovac R, Radić-Kristo D, Kardum-Skelin I, Jaksić B. Diffuse large B-cell lymphoma in patient after treatment of angioimmunoblastic T-cell lymphoma. Coll Antropol 2010;34:241-245. 12. Takahashi T, Maruyama R, Mishima S, Inoue M, Kawakami K, Onishi C, Miyake T, Tanaka J, Nabika T, Ishikura H. Small bowel perforation caused by Epstein-Barr virus-associated B cell lymphoma in a patient with angioimmunoblastic T-cell lymphoma. J Clin Exp Hematop 2010;50:59-63. 13. Huang J, Zhang PH, Gao YH, Qiu LG. Sequential development of diffuse large B-cell lymphoma in a patient with angioimmunoblastic T-cell lymphoma. Diagn Cytopathol 2012;40:346-351. 14. Smeltzer JP, Viswanatha DS, Habermann TM, Patnaik MM. Secondary Epstein-Barr virus associated lymphoproliferative disorder developing in a patient with angioimmunoblastic T cell lymphoma on vorinostat. Am J Hematol 2012;87:927-928. 15. Lee MH, Moon IJ, Lee WJ, Won CH, Chang SE, Choi JH, Lee MW. A case of cutaneous Epstein-Barr virus-associated diffuse large B-cell lymphoma in an angioimmunoblastic T-cell lymphoma. Ann Dermatol 2016;28:789-791. 16. Moskowitz CH, Bertino JR, Glassman JR, Hedrick EE, Hunte S, Coady-Lyons N, Agus DB, Goy A, Jurcic J, Noy A, O’Brien J, Portlock CS, Straus DS, Childs B, Frank R, Yahalom J, Filippa D, Louie D, Nimer SD, Zelenetz AD. Ifosfamide, carboplatin, and etoposide: a highly effective cytoreduction and peripheralblood progenitor-cell mobilization regimen for transplant-eligible patients with non-Hodgkin’s lymphoma. J Clin Oncol 1999;17:3776-3785.

Address for Correspondence/Yazışma Adresi: Cem ŞİMŞEK, M.D., Hacettepe University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey Phone : +90 534 296 46 84 E-mail : cemsimsek111@gmail.com ORCID-ID: orcid.org/0000-0002-7037-5233

Received/Geliş tarihi: January 13, 2018 Accepted/Kabul tarihi: August 01, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0023

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Turk J Hematol 2019;36:52-71

Atypical Radiologic Image Characterized by Cavitary Lung Lesions in a Case of Hodgkin Lymphoma Hodgkin Lenfomalı Olguda Kaviter Akciğer Lezyonları ile Karakterize Atipik Radyolojik Görüntü Mahmut Büyükşimşek1, Mert Tohumcuoğlu1

Semra Paydaş1,

Derya Gumurdulu2,

Cem Mirili1,

Ali Oğul1,

Abdullah Evren Yetişir1,

1Çukurova University Faculty of Medicine, Department of Oncology, Adana, Turkey 2Çukurova University Faculty of Medicine, Department of Pathology, Adana, Turkey

To the Editor, A 30-year-old woman was admitted to the hospital with a lump in her neck. She had no B symptoms (fever, night sweats, and weight loss) and a biopsy showed Hodgkin lymphoma (HL) of the classical type. Positron emission tomography/computed tomography (PET/CT) showed cervical and mediastinal lymph nodes of 1.5-3 cm in diameter and an invasive left parasternal mass of 4x2.5 cm. Three cycles of ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) were given and less than partial response (PR) was detected by PET/CT. Salvage chemotherapy and autologous stem cell transplantation (ASCT) were planned and 2 cycles of the DHAP regimen (cisplatin, dexamethasone, cytosine) were given. PR was detected by PET-CT, but she rejected the ASCT. Local radiotherapy was given for the residual tumor. After radiation there was no evidence of a tumor upon PET/CT imaging. One and a half years after the end of radiation she was admitted with cough, dyspnea, sputum, and fever. Thoracic CT showed cavitary lesions in the parenchyma of both lungs and atelectasis in the left lingula (Figure 1). The patient was counseled in the department of chest diseases; radiation pneumonia was not considered. The angiotensin-converting enzyme level for sarcoidosis was normal. Bronchoscopic examination, lavage, and biopsy were done. Fungal tests were found to be negative. The cavitary lesion was preferred for biopsy. The biopsy showed HL of the classical type (Figure 2) and CD30 was positive (Figure 3). A QuantiFERON test of the blood sample and tuberculosis polymerase chain reaction from biopsy material were negative. HL is not a leading diagnostic consideration when evaluating cavitary lung lesions. An extensive differential diagnosis includes vasculitis, infection, and malignancy [1]. Parenchymal lung involvement is not uncommon in HL; however, cavitary pulmonary lesions are quite unusual. Lung involvement in lymphoma is generally seen as nodule formation or consolidation. 60

Bronchoscopic evaluation is very important in these cases [2,3]. Disseminated cavitary lesions mimicking tuberculosis or other opportunistic infections in a case of HL is interesting and differential diagnosis is very important.

Figure 1. Cavitary pulmonary lesions and atelectasis in the left lingula.

Figure 2. Bronchoscopic biopsy: Hodgkin lymphoma-classical type (H&E, 200x).

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Anahtar Sözcükler: Hodgkin lenfoma, Kaviter akciğer lezyonları, Tüberküloz Informed Consent: It was received. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Grunzke M, Hayes K, Bourland W, Garrington T. Diffuse cavitary lung lesions. Pediatr Radiol 2010;40:215-218.

Figure 3. Bronchoscopic biopsy: Hodgkin lymphoma, CD30+.

2. Bieliauskas S, Reyes-Trocchia A, Krasan GP, Main C, Trupiano JK. Hodgkin lymphoma presenting as multiple cavitary pulmonary nodules with associated mediastinal adenopathy and neck mass. J Pediatr Hematol Oncol 2009;31:730-733.

Keywords: Hodgkin Tuberculosis

3. Yalçin B, Kutluk MT, Sanal O, Akyüz C, Anadol D, Cağlar M, Göçmen A, Büyükpamukçu M. Hodgkin’s disease and ataxia telangiectasia with pulmonary cavities. Pediatr Pulmonol 2002;33:399-403.

lymphoma,

Cavitary

lung

lesions,

Address for Correspondence/Yazışma Adresi: Mahmut BÜYÜKŞİMŞEK, M.D., Çukurova University Faculty of Medicine, Department of Oncology, Adana, Turkey Phone : +90 536 862 20 26 E-mail : mahmutbuyuksimsek@gmail.com ORCID-ID: orcid.org/0000-0001-6356-9059

Received/Geliş tarihi: March 29, 2018 Accepted/Kabul tarihi: July 23, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0115

Light Chain Myeloma with Highly Atypical Plasma Cells and Extensive Auer Rod-Like Inclusions Yüksek Atipik Plazma Hücreleri ve Yaygın Auer Cisimciği Benzeri İnklüzyonları Olan Hafif Zincir Myeloma Dietmar Enko1,2,

Gernot Kriegshäuser1,2

1General Hospital Steyr, Institute of Clinical Chemistry and Laboratory Medicine, Steyr, Austria 2Medical University Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria

To the Editor, A 73-year-old woman with a history of chronic kidney disease presented with fever (39.8 °C), dyspnea, and fatigue. Complete blood count showed moderate normocytic anemia with hemoglobin of 10.0 g/dL (normal range: 12.0-16.0), mild leukocytosis of 10.8x109/L (normal range: 4.0-9.0), and thrombocytopenia of 102x109/L (normal range: 150-400). Serum protein electrophoresis showed mild hypogammaglobulinemia of 6.7 g/L (normal range: 7.0-16.0). Serum immunofixation electrophoresis demonstrated monoclonal κ-type light chains without heavy chain correlates (IgG, IgM, IgA, IgD, IgE). Moreover, a serum-free light chain assay measured a high

κ-type light chain level of 2060.0 mg/L (normal range: 3.3-19.4) with a κ/λ ratio of 48.5 (normal range: 0.3-1.7). A bone marrow aspirate smear showed 40% plasma cells, many of which appeared as binuclear plasmablastic cells with nucleoli (“owl-eyed” plasma cells), bright cytoplasm, and bundles of numerous Auer rod-like cytoplasmic inclusions (Figures 1A and 1B). This unique morphology is remarkable. While the current literature describes Auer rod-like inclusions in single cases of different forms of myeloma [1,2,3,4,5], this is, to the best of our knowledge, the first report on the concomitant appearance with enlarged highly atypical “owl-eyed” plasma cells in a patient suffering from κ-type light chain myeloma. 61

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However, the prognostic value of this unusual plasma cell phenotype remains unclear. Keywords: Light chain myeloma, Plasma cells, Bone marrow aspirate Anahtar Sözcükler: Hafif zincir myeloma, Plazma hücreleri, Kemik iliği aspirasyonu Informed Consent: Was obtained from the patient. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Castoldi G, Piva N, Tomasi P. Multiple myeloma with Auer-rod-like inclusions. Haematologica 1999;84:859-860. 2. Metzgeroth G, Back W, Maywald O, Schatz M, Willer A, Hehlmann R, Hastka J. Auer rod-like inclusions in multiple myeloma. Ann Hematol 2003;82:5760. 3. Abdulsalam AH, Al-Yassin FM. Myeloma cells with Auer rod-like inclusions. Turk J Hematol 2012;29:206.

Figure 1. Bone marrow aspirate smear of a 73-year-old patient with κ-type light chain myeloma (A and B). The arrow marks a binuclear plasmablastic cell containing numerous Auer rod-like inclusions.

4. Noujaim JC, D’Angelo G. Auer rod-like inclusions in ĸ light chain myeloma. Blood 2013;122:2932. 5. Ho WK, Zantomio D. Auer rod-like inclusions in plasma cells in multiple myeloma. J Clin Pathol 2014;67:547-548.

Address for Correspondence/Yazışma Adresi: Gernot KRIEGSHÄUSER, M.D., General Hospital Steyr, Institute of Clinical Chemistry and Laboratory Medicine, Steyr, Austria Phone : +43 50554 66 25308 E-mail : gernot.kriegshaeuser@gespag.at ORCID-ID: orcid.org/0000-0003-2768-795X

Received/Geliş tarihi: June 05, 2018 Accepted/Kabul tarihi: July 09, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0197

Aggressive Clinicopathological Course of Myeloma with t(3;16) (q21;q22) Cytogenetic Abnormality t(3;16)(q21;q22) Anomalili Myeloma Olgusunda Agresif Klinikopatolojik Seyir Süreyya Bozkurt1,

Müfide Okay2,

İbrahim Haznedaroğlu2

1İstinye University Faculty of Medicine, Department of Medical Biology, İstanbul, Turkey 2Hacettepe University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Ankara, Turkey

To the Editor, Multiple myeloma (MM) is a heterogeneous disease and patients present with a wide variety of cytogenetic anomalies reflecting the nature of the disease [1]. The aim of this letter is to report a rare karyotypic abnormality with an aggressive clinical course of MM. 62

A 56-year-old male patient was admitted to the neurosurgery clinic with dorsal shoulder pain and inability to walk in April 2011. He underwent thoracic and lumbar spinal magnetic resonance imaging. Laminectomy was performed on the patient upon detecting masses at the levels of the first and seventh thoracic vertebrae. The patient was referred to our center when he was determined to have “lymphoma” based on the first evaluation

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of his biopsy material. The specimen was then reevaluated in our center. A high-grade hematopoietic neoplasia was detected. Immunophenotypic findings suggested neoplasia with plasma cell origin. Immunohistochemically, neoplastic cells were positive for CD38, MUM-1, and kappa and negative for lambda. The karyotype of the patient was identified as 44,X,-Y,del(1) (p13p35),+der(1),t(3;16)(q21;q22),-4,-13,-14,+mar[8]/46,XY[42] (Figure 1). Upon detection of newly developed lesions in the tenth thoracic vertebra in the control imaging obtained after the radiotherapy of the patient, treatment with vincristine, adriamycin, and dexamethasone (VAD) was initiated. The paraprotein levels in the patient’s serum decreased after four cycles of chemotherapy. However, the treatment was planned to be continued with only bortezomib (Velcade) and dexamethasone due to severe infection after chemotherapy and the development of decubitus infection during the follow-up of the patient. After three cycles of treatment, nodular lesions compatible with plasmacytomas appeared on his skin. The treatment strategy was changed since the patient was considered to have disease progression and had responded better to VAD chemotherapy. The patient was hospitalized due to pneumonia after chemotherapy and died due to severe sepsis. The t(3;16)(q21;q22) anomaly reported in this case is a quite rare cytogenetic anomaly and, according to the databases that we have investigated [2,3], it was reported only in three adults and one child to date [4]. One of these three adult cases was a male

patient with the diagnosis of myelodysplastic syndrome. In this patient, the translocation of t(3;16)(q21;q22) was found in the complex karyotype, as in our case [5]. The other two cases were female patients and they were diagnosed with acute myeloid leukemia that developed after acute myelomonocytic leukemia and primary tumor mantle cell lymphoma, respectively. None of the reported patients were diagnosed with MM. Therefore, we report the t(3;16)(q21;q22) anomaly, which is a quite rare translocation, in a patient with MM for the first time in this case. The prognostic effect of t(3;16)(q21;q22) is unknown. In the presented case, the anomaly of t(3;16)(q21;q22) coexisted with the anomalies of 1p deletion and monosomy 13, which are markers of progressive disease and short survival, or in other words, poor prognostic markers [6]. The clinical course of our case progressed in a manner that was consistent with poor prognosis. We believe that our case contributes to the literature since it is the first MM case with a very rarely encountered translocation. Keywords: Multiple myeloma, Rare translocations, Cytogenetic abnormality Anahtar Sözcükler: Multiple myelom, Nadir translokasyonlar, Sitogenetik anomali Informed Consent: Recevied. Conflict of Interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

References 1. Sawyer JR. The prognostic significance of cytogenetics and molecular profiling in multiple myeloma. Cancer Genet 2011;204:3-12. 2. Huret JL. Atlas of Genetics and Cytogenetics in Oncology and Haematology. 2017. Available at http://atlasgeneticsoncology.org/. 3. Mitelman F, Johansson B, Mertens F. Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, 2017. Available at http://cgap.nci. nih.gov/Chromosomes/Mitelman. 4. Huret JL. t(3;16)(q21;q22). Atlas Genet Cytogenet Oncol Haematol 2009;13:747. 5. Neri G, Daniel A, Hammond N. Chromosome 16q, eosinophilia, and leukemia. Cancer Genet Cytogenet 1985;14:371-372.

Figure 1. The patient’s karyotype.

6. Fonseca R, Bergsagel PL, Drach J, Shaughnessy J, Gutierrez N, Stewart AK, Morgan G, Van Ness B, Chesi M, Minvielle S, Neri A, Barlogie B, Kuehl WM, Liebisch P, Davies F, Chen-Kiang S, Durie BG, Carrasco R, Sezer O, Reiman T, Pilarski L, Avet-Loiseau H; International Myeloma Working Group. International Myeloma Working Group molecular classification of multiple myeloma: spotlight review. Leukemia 2009;23:2210-2221.

Address for Correspondence/Yazışma Adresi: Süreyya BOZKURT, M.D., İstinye University Faculty of Medicine, Department of Medical Biology, İstanbul, Turkey Phone : +90 850 283 60 00 E-mail : sureyyabozkurt8@gmail.com ORCID-ID: orcid.org/0000-0002-1765-9894

Received/Geliş tarihi: January 30, 2018 Accepted/Kabul tarihi: May 02, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0049

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Turk J Hematol 2019;36:52-71

The Impact of DNMT3A/FLT3-ITD/NPM1 on Patients with Acute Myeloid Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation Akut Myeloid Lösemili Hastalarda DNMT3A/FLT3-ITD/NPM1’in Allojeneik Hematopoetik Kök Hücre Transplantasyonu Sonrası Etkisi Long Su Jilin University First Hospital, Clinic of Hematology, Changchun, China

To the Editor, Recently, Ardestani et al. [1] published their excellent findings in this journal. They found that DNMT3A mutations alone do not affect the clinical outcomes of acute myeloid leukemia (AML) patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but when accompanied by FLT3-ITD mutations, the overall survival (OS) was significantly reduced and the relapse rate increased. NPM1 mutations had no impact on either relapse-free survival or OS, but there was a significant difference between AML patients with and without NPM1 mutations for relapse [1]. Integrative genomic analysis of de novo AML identified a subset of AML patients in which DNMT3A, FLT3, and NPM1 mutations coexisted at a higher frequency than would be expected from chance occurrence [2]. Our unpublished data also showed that a close association could be observed among DNMT3A, FLT3, and NPM1 mutations in patients with AML by factor analysis (p<0.05) based on 357 de novo AML patients analyzed by next-generation sequencing. A previous study demonstrated that younger (<60 years) patients with DNMT3A/FLT3/NPM1 mutations had significantly shorter event-free survival (p=0.047) and a tendency towards shorter OS (p=0.095) compared to those in other mutation groups [3]. The adverse impact of DNMT3A mutations is more pronounced than that of FLT3-ITD among patients with NPM1 mutations [3]. Accordingly, how did DNMT3A/FLT3-ITD/NPM1 triple mutations influence the prognoses of AML patients who underwent allo-HSCT in this study? What about the impact of DNMT3A or FLT3-ITD on NPM1-mutated AML patients? Recent studies reported that variant allele frequencies of the NPM1 and FLT3-ITD genes were closely related to long-term outcomes in patients with AML [4,5]. I wonder if there is information available on variant allele frequency in this subset of patients in order to re-analyze the impact of NPM1 and FLT3-ITD on the prognoses of patients following allo-HSCT.

Keywords: Acute myeloid leukemia, Genetic mutations, DNMT3A, FLT3-ITD, NPM1 Anahtar Sözcükler: Akut miyeloid lösemi, Genetik mutasyonlar, DNMT3A, FLT3-ITD, NPM1 Conflict of Interest: The author of this paper has no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Ardestani MT, Kazemi A, Chahardouli B, Mohammadi S, Nikbakht M, Rostami S, Jalili M, Vaezi M, Alimoghaddam K, Ghavamzadeh A. FLT3-ITD compared with DNMT3A R882 mutation is a more powerful independent inferior prognostic factor in adult acute myeloid leukemia patients after allogeneic hematopoietic stem cell transplantation: a retrospective cohort study. Turk J Hematol 2018;35:158-167. 2. Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, Potter NE, Heuser M, Thol F, Bolli N, Gundem G, Van Loo P, Martincorena I, Ganly P, Mudie L, McLaren S1, O’Meara S, Raine K, Jones DR, Teague JW, Butler AP, Greaves MF, Ganser A, Döhner K, Schlenk RF, Döhner H, Campbell PJ. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med 2016;374:2209-2221. 3. Loghavi S, Zuo Z, Ravandi F, Kantarjian HM, Bueso-Ramos C, Zhang L, Singh RR, Patel KP, Medeiros LJ, Stingo F, Routbort M, Cortes J, Luthra R, Khoury JD. Clinical features of de novo acute myeloid leukemia with concurrent DNMT3A, FLT3 and NPM1 mutations. J Hematol Oncol 2014;7:74. 4. Kim Y, Lee GD, Park J, Yoon JH, Kim HJ, Min WS, Kim M. Quantitative fragment analysis of FLT3-ITD efficiently identifying poor prognostic group with high mutant allele burden or long ITD length. Blood Cancer J 2015;5:336. 5. Patel SS, Kuo FC, Gibson CJ, Steensma DP, Soiffer RJ, Alyea EP, Chen YA, Fathi AT, Graubert TA, Brunner AM, Wadleigh M, Stone RM, DeAngelo DJ, Nardi V, Hasserjian RP, Weinberg OK. High NPM1-mutant allele burden at diagnosis predicts unfavorable outcomes in de novo AML. Blood 2018;131:2816-2825.

Address for Correspondence/Yazışma Adresi: Long SU, M.D., Department of Hematology, First Hospital of Jilin University, Changchun 130021, China Phone : +86-0431-88782157 E-mail : sulongjdyy@163.com ORCID-ID: orcid.org/0000-0002-5360-468X

Received/Geliş tarihi: August 04, 2018 Accepted/Kabul tarihi: October 22, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0274

Turk J Hematol 2019;36:52-71

LETTERS TO THE EDITOR

Reply to the Authors To the Editor, We appreciate Dr. Long Su for his interest in our study and his useful comments about our article published in Turkish Journal Hematology, entitled â&#x20AC;&#x153;The impact of DNMT3A/FLT3-ITD/NPM1 on patients with acute myeloid leukemia after allogeneic hematopoietic stem cell transplantationâ&#x20AC;? [1]. Our study demonstrated that DNMT3A R882 mutations are not related to inferior survival in AML patients after allogeneic HSCT. Regarding his comments, we analyzed impact of DNMT3A and FLT3-ITD on NPM1 mutated AML patients. Our finding indicated that considering NPM1 and DNMT3A mutations together, no significant difference in OS and DFS was revealed (Figure 1). FLT3-ITD mutation had a negative impact on both RFS and OS of patients with mutated NPM1. DFS and OS were significantly more favorable in patients with FLT3-ITD-/NPM1+ compared to patients with other mutation subgroups (Figure 2). Also, patients with DNMT3A/FLT3-ITD/NPM1 triple mutations demonstrated a significantly worse DFS (p=0.009) and OS (p=0.028) (Figure 3) compared to all other patients (Figure 3).

Figure 1. Survival curves of acute myeloid leukemia patients according to mutational status of DNMT3A-/NPM1: relapse free survival (left) and overall survival (right) (A=DNMT3A-/NPM1-, B=DNMT3A+/NPM1+, C=DNMT3A+/NPM1-, D=DNMT3A-/NPM1-).

Figure 2. Survival curves of acute myeloid leukemia patients according to mutational status of FLT3-ITD/NPM1: relapse free survival (left) and overall survival (right) (A=FLT3-ITD-/NPM1-, B=FLT3-ITD+/NPM1+, C=FLT3-ITD+/NPM1-, D=FLT3-ITD-/NPM1+). 65

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Figure 3. Survival curves of acute myeloid leukemia patients according to DNMT3A/FLT3-ITD/NPM1 triple mutations: relapse free survival (left) and overall survival (right) (A=DNMT3A/FLT3-ITD/NPM1, B=others). Unfortunately, in this study, information about the allele frequency of mutations was not available for most of patients, but in an ongoing prospective study, we are studying the effect of the allelic frequency of FLT3-ITD and NPM mutations on the clinical outcome of AML patients after HSCT. Although there is more agreement on the poor prognostic role of DNMT3A mutations in AML patients [2], few studies have been conducted on the effect of these mutations on the clinical outcome of patients after allogeneic transplantation and there are disagreements over the results of these limited studies [3,4]. These differences can be due to coexistence of variable cytogenetic or molecular genetic aberrations in leukemia cells that could be effective in the clinical outcome of the patients. In the present study, only three genetic aberration were tested and additional genetic abnormality might impact the outcome of the patients. Taken together, our findings indicated that FLT3-ITD may be a more powerful adverse prognostic biomarker than NPM1 and DNMT3A in AML patients after HSCT. Although the comments have allowed us to add useful information to our previous paper, our conclusion remains the same as in our published work. Best Regards, Bahram Chahardouli, Saeed Mohammadi, Mohsen Nikbakht, Shahrbano Rostami

References 1. Ardestani MT, Kazemi A, Chahardouli B, Mohammadi S, Nikbakht M, Rostami S, Jalili M, Vaezi M, Alimoghaddam K, Ghavamzadeh A. FLT3-ITD compared with DNMT3A R882 mutation is a more powerful independent inferior prognostic factor in adult acute myeloid leukemia patients after allogeneic hematopoietic stem cell transplantation: a retrospective cohort study. Turk J Hematol 2018;35:158-167. 2. Patkar N, Kodgule R, Kakirde C, Raval G, Bhanshe P, Joshi S, Chaudhary S, Badrinath Y, Ghoghale S, Kadechkar S, Khizer SH, Kannan S, Shetty D, Gokarn A, Punatkar S, Jain H, Bagal B, Menon H, Sengar M, Khattry N, Tembhare P, Subramanian P, Gujral S. Clinical impact of measurable residual disease monitoring by ultradeep next generation sequencing in NPM1 mutated acute myeloid leukemia. Oncotarget 2018;9:36613-36624. 3. Wang H, Chu TT, Han SY, Qi JQ, Tang YQ, Qiu HY, Fu CC, Tang XW, Ruan CG, Wu DP, Han Y. FLT3-ITD and CEBPA mutations predict prognosis in acute myelogenous leukemia irrespective of hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2018 [Epub ahead of print]. 4. Yang X, Shi J, Zhang X, Zhang G, Zhang J, Yang S, Wang J, Ke X, Fu L. Biological and clinical influences of NPM1 in acute myeloid leukemia patients with DNMT3A mutations. Cancer Manag Res 2018;10:2489-2497.

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Wilms Tumor-1 (WT1) rs16754 Polymorphism and Clinical Outcome in Acute Myeloid Leukemia Akut Myeloid Lösemide Wilms Tumor-1 (WT1) rs16754 Polimorfizmi ve Klinik Seyir Mani Ramzi,

Mohamad Moghadam,

Nader Cohan

Shiraz University of Medical Sciences, Hematology Research Center, Shiraz, Iran

To the Editor, Cytogenetic evaluation and risk stratification are important steps in the classification of acute myeloid leukemia (AML) and also in determining the best treatment modality. Although cytogenetically normal acute myeloid leukemia (CN-AML) represents a large group of adult patients with AML (about 45% of AML patients), several mutations in some genes including FLT3, NPM1, CEBPA, and WT1 are associated with risk assessment and clinical outcome [1,2]. The WT1 gene is located on chromosome 11p13 and was detected first as a tumor-suppressor gene in Wilm’s tumor; it also has a regulatory role in normal and malignant hematopoiesis [3]. WT1 gene mutations in CN-AML were reported in about 10% of patients and its role as a sole prognostic factor is controversial [4,5,6]. The most important WT1 gene mutations associated with AML are clustered in the hotspot region of exon 7 and a synonymous single nucleotide polymorphism (SNP), rs16754, is also located in this region. Some studies were done for evaluation of WT1 rs16754 polymorphism effects on clinical response in AML patients. Most studies confirmed that rs16754 (A>G) in the hotspot region of exon 7 is a predictive independent positive prognostic factor in AML patients. Two meta-analyses were published for evaluation of these results. Megías-Vericat et al. [7] performed the first metaanalysis in this regards with a total of 3618 included patients from 14 cohort studies. They found that the overall survival (OS) at 5 years was significantly higher in patients with the variant allele (G) together with higher disease-free survival (DFS) with the variant allele. Although they did not find any significant effect of this variant on complete remission (CR), they stated that the lack of an observable effect could have been a result of the small number of studies that evaluated it. Long et al. [8] also published a meta-analysis in this regard, addressing 12 publications with 3903 included patients. Although some of the publications and included patients in those two studies may have been the same, they found the same results for the significant effects of WT1 rs16754 polymorphism on OS and relapse-free survival. In a newly published study Petiti et al. [9] also showed that patients with mutated genotypes (GG and GA) have a significantly better OS and event-free survival (EFS) compared with patients with wild AA mutation (84 vs. 9 months for OS and 23 vs. 3 months for EFS). They discussed that they

used peptide nucleic acid-directed PCR clamping technology for this study. In a prospective study, we evaluated the effects of the rs16754 SNP on clinical outcome in CN-AML patients. From December 2012 to December 2016, a total of 108 untreated adult patients who were referred to Nemazee Hospital, Shiraz University of Medical Sciences, and diagnosed with CN-AML were included. The exclusion criterion was abnormality upon karyotyping. The diagnosis was based on peripheral blood and bone marrow aspiration confirmed by immunophenotyping and the treatment was based on standard induction chemotherapy, which consisted of daunorubicin at 45 mg/m2 on days 1 to 3 and cytarabine at 100-200 mg/m2 on days 1 to 7, followed by high doses of a cytarabine-based consolidation phase (cytarabine at 3 g/m2 every 12 h for 3 days, repeated for 3 to 6 cycles). Informed consent was obtained from all patients and the work was carried out in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki) for experiments in humans. The rs16754 (A>G) polymorphism in exon 7 was evaluated in all patients by direct sequencing of the amplified and purified PCR products. OS and DFS were assessed for evaluation of clinical outcome. The allele frequency of the A allele was 181 and that of the G allele was 35. The major genotype (AA) was found in 79 patients and a minor genotype in 29 patients (AG and GG in 23 (21.3%) and 6 (5.5%) patients, respectively). We found no significant difference between patients with the wild and variant allele based on CR and relapse as well as OS and DFS, which may be a result of the small number of included patients. The results are shown in Table 1. Although the exact reason for the effects of this SNP on clinical outcome in AML patients is unclear, different hypotheses exist, including increased rate of translation and protein folding in mutated genotypes, disequilibrium linkage of this SNP with another genetic variant that affects drug metabolism, and greater sensitivity of mutated genotypes to cytarabine [7,10]. In conclusion, the WT1 rs16754 polymorphism may be a reliable independent prognostic factor in AML and could be assessed for risk stratification in this group of patients, although more studies including larger study groups are needed for more evaluation and discussion. 67

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Table 1. Clinical and laboratory data according to rs16754 wild and minor alleles. Characteristic

rs16754 wild allele (AA) n=79

rs16754 minor allele (AG or GG) n=29

40±16

41±12

43/36

16/13

39.9±11.8

41.2±13.4

Platelet count (x109/L)

75.9±34.5

74.6±39.7

Hemoglobin level (g/L)

81±19

83±24

Bone marrow blast (%)

78.3±13.9

77.2±128

Complete remission (%)

51 (64.5%)

21 (72.4%)

Relapse after complete remission (%)

38 (48.1%)

14 (48.2%)

Mean overall survival (months)

15.7±3.2

16.1±1.6

Mean disease-free survival (months)

14.6±3.8

14.9±2.7

Age (years) Sex (M/F) Primary WBC count

(x109/L)

p>0.05 was noted according to overall survival and disease-free survival between the two groups. M: Male, F: female, WBC: white blood cell.

Keywords: Polymorphism, Acute myeloid leukemia, Clinical outcome Anahtar Sözcükler: Polimorfizm, Akut miyeloid lösemi, Klinik seyir Informed Consent: Received. Conﬂict of interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Bhatnagar B, Garzon R. The use of molecular genetics to refine prognosis in acute myeloid leukemia. Curr Hematol Malig Rep 2014;9:148-157. 2. Ghanem H, Tank N, Tabbara IA. Prognostic implications of genetic aberrations in acute myelogenous leukemia with normal cytogenetics. Am J Hematol 2012;87:69-77. 3. Ariyaratana S, Loeb DM. The role of the Wilms tumour gene (WT1) in normal and malignant haematopoiesis. Expert Rev Mol Med 2007;9:1-17. 4. Gaidzik VI, Schlenk RF, Moschny S, Becker A, Bullinger L, Corbacioglu A, Krauter J, Schlegelberger B, Ganser A, Döhner H, Döhner K. Prognostic impact of WT1 mutations in cytogenetically normal acute myeloid leukemia: a study of the German-Austrian AML Study Group. Blood 2009;113:45054511. 5. Rampal R, Figueroa ME. Wilms tumor 1 mutations in the pathogenesis of acute myeloid leukemia. Haematologica 2016;101:672-679. 6. Gaidzik V, Döhner K. Prognostic implications of gene mutations in acute myeloid leukemia with normal cytogenetics. Semin Oncol 2008;35:346355. 7. Megías-Vericat JE, Herrero MJ, Rojas L, Montesinos P, Bosó V, Moscardó F, Martínez-Cuadrón D, Poveda JL, Sanz MÁ, Aliño SF. A systematic review and meta-analysis of the impact of WT1 polymorphism rs16754 in the effectiveness of standard chemotherapy in patients with acute myeloid leukemia. Pharmacogenomics J 2016;16:30-40. 8. Long J, Fang S, Dai Q, Liu X, Zhu W, Wang S. The Wilms tumor-1 (WT1) rs16754 polymorphism is a prognostic factor in acute myeloid leukemia (AML): a meta-analysis. Oncotarget 2016;7:32079-32087. 9. Petiti J, Rosso V, Lo Iacono M, Calabrese C, Signorino E, Gaidano V, Berger M, Saglio G, Cilloni D. Prognostic significance of the Wilms’ tumor-1 (WT1) rs16754 polymorphism in acute myeloid leukemia. Leuk Res 2018;67:6-11. 10. Damm F, Heuser M, Morgan M, Yun H, Grosshennig A, Göhring G, Schlegelberger B, Döhner K, Ottmann O, Lübbert M, Heit W, Kanz L, Schlimok G, Raghavachar A, Fiedler W, Kirchner H, Döhner H, Heil G, Ganser A, Krauter J. Single nucleotide polymorphism in the mutational hotspot of WT1 predicts a favorable outcome in patients with cytogenetically normal acute myeloid leukemia. J Clin Oncol 2010;28:578-585.

Address for Correspondence/Yazışma Adresi: Nader COHAN, PhD., Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Phone : +987136473239 E-mail : cohannader@yahoo.com ORCID-ID: orcid.org/0000-0003-2464-8445

Received/Geliş tarihi: August 05, 2018 Accepted/Kabul tarihi: November 23, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0277

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Turk J Hematol 2019;36:52-71

Wilms Tumor-1 (WT1) rs16754 Polymorphism Wilms Tümör-1 (WT1) rs 16754 Polimorfizmi Pathum Sookaromdee1,

Viroj Wiwanitkit2

1TWS Medical Center, Bangkok, Thailand 2Dr. DY Patil University, Pune, India

To the Editor, The publication “Wilm’s tumor-1 (WT1) rs16754 Polymorphism and Clinical Outcome in Acute Myeloid Leukemia” was very interesting [1]. Ramzi et al. [1] noted that “WT1 rs16754 polymorphism may be a reliable independent prognostic factor”. In fact, the WT1 rs16754 polymorphism is proposed as an important prognostic marker for leukemia [2]. The application in the present study might be expected. However, there can also be interference effects of other polymorphisms (such as MDM2 [3] and long non-coding RNA GAS5 polymorphism [4]) that were not investigated that might modify the value of WT1 rs16754 polymorphism testing. Single polymorphism study might limit its application in clinical usage for prognosis prediction for leukemia patients. Keywords: Wilm’s Tumor, gene, polymorphism Anahtar Sözcükler: Wilms Tümör, gen, polimorfizm

References 1. Ramzi M, Moghadam M, Cohan N. Wilm’s tumor-1 (WT1) rs16754 polymorphism and clinical outcome in acute myeloid leukemia. Turk J Hematol 2019;36:67-68. 2. Petiti J, Rosso V, Lo Iacono M, Calabrese C, Signorino E, Gaidano V, Berger M, Saglio G, Cilloni D. Prognostic significance of the Wilms’ tumor-1 (WT1) rs16754 polymorphism in acute myeloid leukemia. Leuk Res 2018;67:6-11. 3. He X, Chen P, Yang K, Liu B, Zhang Y, Wang F, Guo Z, Liu X, Lou J, Chen H. Association of MDM2 polymorphism with risk and prognosis of leukemia: a meta-analysis. Acta Haematol 2015;133:365-371. 4. Yan H, Zhang DY, Li X, Yuan XQ, Yang YL, Zhu KW, Zeng H, Li XL, Cao S, Zhou HH, Zhang W, Chen XP. Long non-coding RNA GAS5 polymorphism predicts a poor prognosis of acute myeloid leukemia in Chinese patients via affecting hematopoietic reconstitution. Leuk Lymphoma 2017;58:19481957.

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

Received/Geliş tarihi: November 25, 2018 Accepted/Kabul tarihi: December 06, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0407

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Turk J Hematol 2019;36:52-71

A Novel Mutation in a Child with Atypical Wiskott-Aldrich Syndrome Complicated by Cytomegalovirus Infection Sitomegalovirüs Enfeksiyonu ile Komplike Atipik Wiskott-Aldrich Sendromlu Bir Çocukta Yeni Bir Mutasyon Zühre Kaya1,

Cansu Muluk1,

Şule Haskoloğlu2,

Lale Ş. Tufan3

1Gazi University Faculty of Medicine, Department of Pediatrics, Division Pediatric Hematology, Ankara, Turkey 2Ankara University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Ankara, Turkey 3Ankara University Faculty of Medicine, Department of Forensic Medicine Forensic Genetics Laboratory, Ankara, Turkey

To the Editor, We present the first described case of a young child with newly diagnosed Wiskott-Aldrich syndrome (WAS) caused by a novel mutation in the WAS gene, c.271C>T(p.Q91X), presenting with juvenile myelomonocytic leukemia (JMML)-like clinical features and cytomegalovirus (CMV) infection. The proband, a 4-month-old boy, was referred to our hospital for evaluation of eczema, bicytopenia, leukocytosis, and splenomegaly, all of 2 months’ duration. He also had a history of pneumonia. The boy’s parents were not related, and there was a family history of early childhood deaths. Physical examination revealed splenomegaly and widespread eczema, and informed consent was obtained from the parents for Figure 1. Laboratory assessment revealed hemoglobin of 8.4 g/dL and reticulocytes of 1.3%, white blood count of 19,600/mm3 (35% eosinophils, 15% monocytes on differential blood count), platelet count of 33,000/mm3, and mean platelet volume (MPV) of 10 fL. The direct Coombs test was positive for warm antibodies, and a peripheral blood smear revealed marked eosinophils, monocytes, and immature myeloid cells and giant platelets. Bone marrow examination showed myeloid hyperplasia with eosinophilia. Baseline immunoglobulin (Ig) levels were normal (IgG 317 mg/dL, IgA 14 mg/dL, and IgM 87 mg/dL). The patient was diagnosed with autoimmune hemolytic anemia and had additional diagnostic criteria that suggested JMML and WAS. He was started on prednisone at 1 mg/kg twice daily. Three weeks after initial presentation, he developed shortness of breath, fatigue, and palpitations. He developed a severe pulmonary infection that was successfully treated with trimethoprimsulfamethoxazole and ganciclovir. A PCR test for CMV was positive, with 9700 copies/mL. Molecular genetic analysis revealed a novel mutation in the WAS gene, c.271C>T(p.Q91X). The patient was diagnosed with WAS. He was scheduled for allogeneic stem cell transplantation from an unrelated donor. WAS is a rare and potentially fatal disorder of X-linked recessive inheritance that is characterized by recurrent sinopulmonary 70

infections, eczema, and microthrombocytopenia. We report here a young child with newly diagnosed WAS complicated by CMV, with clinical and laboratory findings similar to JMML. Yoshimi et al. reported seven infant boys with WAS who initially presented with leukocytosis, monocytosis, and myeloid and erythroid precursors in their peripheral blood as well as bone marrow dysplasia [1]. The authors noted that the patients’ MPV values were normal or high, which is incompatible with WAS. As we observed in our case, this clinical picture is indistinguishable from JMML. Affected patients may have variable clinical presentations due to disease-modifying genetic factors and different exposure to pathogens [2,3,4]. The causes of JMMLlike features in WAS patients are poorly understood. Recent reports suggest that such atypical features may be attributed to coexistence of viral infection or activation of WAS protein by

Figure 1. Physical examination revealed widespread eczema.

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Turk J Hematol 2019;36:52-71

a somatic mutation concomitant with RAS pathway mutations [1,2,3,4,5]. Based on these considerations, we believe that CMV infection was responsible for our patient developing a JMMLlike clinical picture and immune cytopenia. Although JMML mutational studies were not performed, persistent monocytosis, splenomegaly, and positive PCR results for CMV all support the diagnosis of CMV infection. Our experience suggests that physicians should be aware of the potential development of immune cytopenias and JMML-like features in children with WAS who contract CMV infection. Keywords: Wiskott-Aldrich syndrome, Juvenile myelomonocytic leukemia, Cytomegalovirus Anahtar Sözcükler: Wiskott-Aldrich myelomonositik lösemi, Sitomegalovirüs

sendromu,

Juvenil

Informed Consent: The patient’s parents provided written informed consent for the publication of the photograph. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships,

and/or affiliations relevant to the subject matter or materials included.

References 1. Yoshimi A, Kamachi Y, Imai K, Watanabe N, Nakadate H, Kanazawa T, Ozono S, Kobayashi R, Yoshida M, Kobayashi C, Hama A, Muramatsu H, Sasahara Y, Jakob M, Morio T, Ehl S, Manabe A, Niemeyer C, Kojima S. WiskottAldrich syndrome presenting with a clinical picture mimicking juvenile myelomonocytic leukemia. Pediatr Blood Cancer 2013;60:836-841. 2. Leblebisatan G, Bay A, Mitsuiki N, Ohara O, Honma K, Imai K, Nonoyama S. Wiskott-Aldrich syndrome mutation in two Turkish siblings with X-linked thrombocytopenia. Turk J Hematol 2011;28:139-141. 3. Sano H, Kobayashi R, Suzuki D, Yasuda K, Nakanishi M, Nagashima T, Yamada M, Kobayashi K. Wiskott-Aldrich syndrome with unusual clinical features similar to juvenile myelomonocytic leukemia. Int J Hematol 2012;96:279283. 4. Patil RB, Shanmukhaiah C, Jijina F, Bamborde S, Wasekar N, Toshniwal M, Mohite A, Patil V. Wiskott-Aldrich syndrome presenting with JMML-like blood picture and normal sized platelets. Case Rep Hematol 2016;2016:8230786. 5. Coppe A, Nogara L, Pizzuto MS, Cani A, Cesaro S, Masetti R, Locatelli F, Te Kronnie G, Basso G, Bortoluzzi S, Bresolin S. Somatic mutations activating Wiskott-Aldrich syndrome protein concomitant with RAS pathway mutations in juvenile myelomonocytic leukemia patients. Hum Mutat 2018;39:579-587.

Address for Correspondence/Yazışma Adresi: Zühre KAYA, M.D., Gazi University Faculty of Medicine, Department of Pediatrics, Division Pediatric Hematology, Ankara, Turkey Phone : +90 312 202 60 25 E-mail : zuhrekaya@gmail.com ORCID-ID: orcid.org/0000-0002-3798-7246

Received/Geliş tarihi: May 31, 2018 Accepted/Kabul tarihi: July 02, 2018 DOI: 10.4274/tjh.galenos.2018.2018.0187

Advisory Board of This Issue (March 2019) Ahmet Emre Eşkazan, Turkey Alphan Küpesiz, Turkey Ayşe Çırakoğlu, Turkey Ayşen Timurağaoğlu, Turkey Birol Baytan, Turkey Bruna Codispoti, Italy Bülent Eser, Turkey Can Boğa, Turkey Carmelo Bernabeu, Spain Ceren Hangül, Turkey Christoph Robier, Austria Claudio Cerchione, Italy Deniz Karapınar, Turkey Faruk Aktürk, Turkey Ghada M. Elsayed, Egypt Hüseyin Tokgöz, Turkey

İnci Alacacıoğlu, Turkey James Armitage, USA Jir Ping Boey, Australia John Bennett, USA Keith R. McCrae, USA Klara Dalva, Turkey Lionel Van Maldergem, France Mehmet Özen, Turkey Mehmet Yılmaz, Turkey Merve Pamukçuoğlu, Turkey Mine Hekimgil, Turkey Mustafa Büyükavcı, Turkey Mustafa Merter, Turkey Nejat Akar, Turkey Rosangela Invernizzi, Italy Saadet Akarsu, Turkey

Serap Karaman, Turkey Serdar Ceylaner, Turkey Şefik Güran, Turkey Şule Ünal, Turkey Tiraje Celkan, Turkey Türkan Patıroğlu, Turkey Ülker Koçak, Turkey Vanessa Innao, Italy Veysel Sabri Hançer, Turkey Wiem Ayed, Tunisia Yener Koç, Turkey Yeşim Aydınok, Turkey Yuichi Nakamura, Japan Yurdanur Kılınç, Turkey Zeynep Karakaş, Turkey