Turkish Journal of Hematology Volume: 32 - Issue: 4

Volume 32 Issue 4 December 2015 80 TL
ISSN 1300-7777
Review Article
Chimeric Antigen Receptor T Cell Therapy in Hematology
Pınar Ataca, et al.; Ankara, Turkey
TURKISH JOURNAL OF HEMATOLOGY • VOL.: 32 ISSUE: 4 DECEMBER 2015
Research Articles
Possible Role of GADD45γ Methylation in Diffuse Large B-Cell Lymphoma: Does It Affect the Progression
and Tissue Involvement?
İkbal Cansu Barış, et al.; Denizli, Turkey
Effect of Tumor Necrosis Factor-Alpha on Erythropoietin- and Erythropoietin Receptor-Induced Erythroid
Progenitor Cell Proliferation in β-Thalassemia/Hemoglobin E Patients
Dalina I Tanyong, et al.; Nakhon Pathom, Thailand
The -137G/C Polymorphism in Interleukin-18 Gene Promoter Contributes to Chronic Lymphocytic
and Chronic Myelogenous Leukemia Risk in Turkish Patients
Serap Yalçın, et al.; Kırşehir, Ankara, Turkey
Transcobalamin II Deficiency in Four Cases with Novel Mutations
Şule Ünal, et al.; Ankara, Turkey; London, Canada
Eltrombopag for the Treatment of Immune Thrombocytopenia: The Aegean Region of Turkey Experience
Füsun Özdemirkıran, et al.; İzmir, Denizli, Aydın, Turkey
Management of Invasive Fungal Infections in Pediatric Acute Leukemia and the Appropriate Time
for Restarting Chemotherapy
Özlem Tüfekçi, et al.; İzmir, Turkey
First-Step Results of Children Presenting with Bleeding Symptoms or Abnormal Coagulation Tests
in an Outpatient Clinic
İsmail Yıldız, et al.; İstanbul, Turkey
Evaluation of Alpha-Thalassemia Mutations in Cases with Hypochromic Microcytic Anemia:
The İstanbul Perspective
Zeynep Karakaş, et al.; İstanbul, Turkey
Cover Picture:
Nejat Akar
Çeşmealtı’s Morning Serenity
4

Editor-in-Chief
Aytemiz Gürgey
Ankara, Turkey
Associate Editors
Ayşegül Ünüvar
İstanbul University, İstanbul, Turkey
M. Cem Ar
İstanbul University Cerrahpaşa Faculty of
Medicine, İstanbul, Turkey
Cengiz Beyan
Gülhane Military Medical Academy,
Ankara, Turkey
Hale Ören
Dokuz Eylül University, İzmir, Turkey
İbrahim C. Haznedaroğlu
Hacettepe University, Ankara, Turkey
İlknur Kozanoğlu
Başkent University, Adana, Turkey
Mehmet Ertem
Ankara University, Ankara, Turkey
A. Muzaffer Demir
Trakya University, Edirne, Turkey
Reyhan Diz Küçükkaya
İstanbul Bilim University, İstanbul, Turkey
Assistant Editors
A. Emre Eşkazan
İstanbul University Cerrahpaşa Faculty of
Medicine, İstanbul, Turkey
Ali İrfan Emre Tekgündüz
Dr. A. Yurtaslan Ankara Oncology Training
and Research Hospital, Ankara, Turkey
İnci Alacacıoğlu
Dokuz Eylül University, Ankara, Turkey
Nil Güler
On Dokuz Mayıs University, Samsun, Turkey
Olga Meltem Akay
Osmangazi University, Eskişehir, Turkey
Selami Koçak Toprak
Ankara University, Ankara, Turkey
Şule Ünal
Hacettepe University, Ankara, Turkey
Veysel Sabri Hançer
İstanbul Bilim University, İstanbul, Turkey
Zühre Kaya
Gazi University, Ankara, Turkey
International Review Board
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
Past Editors
Erich Frank
Orhan Ulutin
Hamdi Akan
Senior Advisory Board
Yücel Tangün
Osman İlhan
Muhit Özcan
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
Language Editor
Leslie Demir
Statistic Editor
Hülya Ellidokuz
Editorial Office
İpek Durusu
Bengü Timoçin
A-I
Publishing
Services
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Contact Information
Editorial Correspondence should be addressed to Dr. Aytemiz Gürgey
Editor-in-Chief
Address: 725. Sok. Görkem Sitesi
Yıldızevler No: 39/2, 06550 Çankaya, Ankara / Turkey
Phone : +90 312 438 14 60
E-mail : agurgey@hacettepe.edu.tr
All other inquiries should be adressed to
TURKISH JOURNAL OF HEMATOLOGY
Address: İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550 Çankaya, Ankara / Turkey
Phone : +90 312 490 98 97
Fax : +90 312 490 98 68
E-mail : info@tjh.com.tr
ISSN: 1300-7777
Turkish Society of Hematology
Teoman Soysal, President
A. Muzaffer Demir, General Secretary
Hale Ören, Vice President
İbrahim C. Haznedaroğlu, Research Secretary
Fahir Özkalemkaş, Treasurer
A. Zahit Bolaman, Member
Mehmet Sönmez, Member
Online Manuscript Submission
http://mc.manuscriptcentral.com/tjh
Web page
www.tjh.com.tr
Owner on behalf of the Turkish Society of Hematology
Türk Hematoloji Derneği adına yayın sahibi
Teoman Soysal
Publishing Manager
Sorumlu Yazı İşleri Müdürü
A. Muzaffer Demir
Management Address
Yayın İdare Adresi
Türk Hematoloji Derneği
İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550
Çankaya, Ankara / Turkey
Publishing House / Yayınevi
Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul,
Turkey Tel: +90 212 621 99 25 Faks: +90 212 621 99 27
E-posta: info@galenos.com.tr
Baskı: Senk Ajans Reklam Matbaacılık San. ve Tic.Ltd.Şti.
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İstanbul, Türkiye Tel: +90 212 264 38 77
Printing Date / Basım Tarihi
20.11.2015
Cover Picture
Nejat Akar was born in 1952, Turkey. He is currently working at TOBB-ETU Hospital,
Ankara, Turkey.
Üç ayda bir yayımlanan İngilizce süreli yayındır.
International scientific journal published quarterly.
Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile
Turkish Journal of Hematology’nin Türk Hematoloji Derneği İktisadi
İşletmesi tarafından yayınlanmasına karar vermiştir.
Çeşmealtı town is a small and pretty site in Urla, İzmir. It’s a quiet and calm fishing
harbor. When rising up every morning, the sun dances on the coast of the sea, which
is surrounded by little islands.
A-II

AIMS AND SCOPE
The Turkish Journal of Hematology is published quarterly
(March, June, September, and December) by the Turkish Society
of Hematology. It is an independent, non-profit peer-reviewed
international English-language periodical encompassing subjects
relevant to hematology.
The Editorial Board of The Turkish Journal of Hematology adheres
to the principles of the World Association of Medical Editors
(WAME), International Council of Medical Journal Editors (ICMJE),
Committee on Publication Ethics (COPE), Consolidated Standards
of Reporting Trials (CONSORT) and Strengthening the Reporting of
Observational Studies in Epidemiology (STROBE).
The aim of The Turkish Journal Hematology is to publish original
hematological research of the highest scientific quality and
clinical relevance. Additionally, educational material, reviews on
basic developments, editorial short notes, case reports, images in
hematology, and letters from hematology specialists and clinicians
covering their experience and comments on hematology and related
medical fields as well as social subjects are published.
General practitioners interested in hematology and internal medicine
specialists are among our target audience, and The Turkish Journal
of Hematology aims to publish according to their needs. The Turkish
Journal of Hematology is indexed, as follows:
- PubMed Medline
- PubMed Central
- Science Citation Index Expanded
- EMBASE
- Scopus
- CINAHL
- Gale/Cengage Learning
- EBSCO
- DOAJ
- ProQuest
- Index Copernicus
- Tübitak/Ulakbim Turkish Medical Database
- Turk Medline
Impact Factor: 0.360
Subscription Information
The Turkish Journal of Hematology is sent free-of-charge to members
of Turkish Society of Hematology and libraries in Turkey and
abroad. Hematologists, other medical specialists that are interested
in hematology, and academicians could subscribe for only 40 $ per
printed issue. All published volumes are available in full text free-ofcharge
online at www.tjh.com.tr.
Address: İlkbahar Mah., Turan Güneş Bulvarı, 613 Sok., No: 8,
Çankaya, Ankara, Turkey
Telephone: +90 312 490 98 97
Fax: +90 312 490 98 68
Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh
Web page: www.tjh.com.tr
E-mail: info@tjh.com.tr
Permissions
Requests for permission to reproduce published material should be
sent to the editorial office.
Editor: Professor Dr. Aytemiz Gürgey
Adress: Ilkbahar Mah, Turan Günes Bulvarı, 613 Sok., No: 8,
Çankaya, Ankara, Turkey
Telephone: +90 312 490 98 97
Fax: +90 312 490 98 68
Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh
Web page: www.tjh.com.tr
E-mail: info@tjh.com.tr
Publisher
Galenos Yayinevi
Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul
Telephone : 0212 621 99 25
Fax : 0212 621 99 27
info@galenos.com.tr
Instructions for Authors
Instructions for authors are published in the journal and at www.
tjh.com.tr
Material Disclaimer
Authors are responsible for the manuscripts they publish in The
Turkish Journal of Hematology. The editor, editorial board, and
publisher do not accept any responsibility for published manuscripts.
If you use a table or figure (or some data in a table or figure) from
another source, cite the source directly in the figure or table legend.
The journal is printed on acid-free paper.
Editorial Policy
Following receipt of each manuscript, a checklist is completed by
the Editorial Assistant. The Editorial Assistant checks that each
manuscript contains all required components and adheres to the
author guidelines, after which time it will be forwarded to the Editor
in Chief. Following the Editor in Chief’s evaluation, each manuscript
is forwarded to the Associate Editor, who in turn assigns reviewers.
Generally, all manuscripts will be reviewed by at least three reviewers
selected by the Associate Editor, based on their relevant expertise.
Associate editor could be assigned as a reviewer along with the
reviewers. After the reviewing process, all manuscripts are evaluated
in the Editorial Board Meeting.
Turkish Journal of Hematology’s editor and Editorial Board
members are active researchers. It is possible that they would desire
to submit their manuscript to the Turkish Journal of Hematology.
This may be creating a conflict of interest. These manuscripts will
not be evaluated by the submitting editor(s). The review process
will be managed and decisions made by editor-in-chief who will act
independently. In some situation, this process will be overseen by an
outside independent expert in reviewing submissions from editors.
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TURKISH JOURNAL OF HEMATOLOGY
INSTRUCTIONS TO AUTHORS
The Turkish Journal of Hematology accepts invited review articles,
research articles, brief reports, case reports, letters to the editor, and
hematological images that are relevant to the scope of hematology,
on the condition that they have not been previously published
elsewhere. Basic science manuscripts, such as randomized, cohort,
cross-sectional, and case control studies, are given preference. All
manuscripts are subject to editorial revision to ensure they conform
to the style adopted by the journal. There is a single blind kind of
reviewing system.
Manuscripts should be prepared according to ICMJE guidelines
(http://www.icmje.org/). Original manuscripts require a structured
abstract. Label each section of the structured abstract with the
appropriate subheading (Objective, Materials and Methods, Results,
and Conclusion). Case reports require short unstructured abstracts.
Letters to the editor do not require an abstract. Research or project
support should be acknowledged as a footnote on the title page.
Technical and other assistance should be provided on the title page.
Original Manuscripts
Title Page
Title: The title should provide important information regarding the
manuscript’s content. The title must specify that the study is a cohort
study, cross-sectional study, case control study, or randomized study
(i.e. Cao GY, Li KX, Jin PF, Yue XY, Yang C, Hu X. Comparative
bioavailability of ferrous succinate tablet formulations without
correction for baseline circadian changes in iron concentration in
healthy Chinese male subjects: A single-dose, randomized, 2-period
crossover study. Clin Ther. 2011; 33: 2054-2059).
The title page should include the authors’ names, degrees, and
institutional/professional affiliations, a short title, abbreviations,
keywords, financial disclosure statement, and conflict of interest
statement. If a manuscript includes authors from more than one
institution, each author’s name should be followed by a superscript
number that corresponds to their institution, which is listed separately.
Please provide contact information for the corresponding author,
including name, e-mail address, and telephone and fax numbers.
Running Head: The running head should not be more than 40
characters, including spaces, and should be located at the bottom of
the title page.
Word Count: A word count for the manuscript, excluding abstract,
acknowledgments, figure and table legends, and references, should
be provided not exceed 2500 words. The word count for an abstract
should be not exceed 300 words.
Conflict-of-Interest Statement: To prevent potential conflicts of
interest from being overlooked, this statement must be included in
each manuscript. In case there are conflicts of interest, every author
should complete the ICMJE general declaration form, which can be
obtained at: http://www.icmje.org/coi_disclose.pdf.
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 case reports, reviews, perspectives,
and editorials, will be published according to uniform requirements.
Provide 3-10 keywords below the abstract to assist indexers. Use
terms from the Index Medicus Medical Subject Headings List
(for randomized studies a CONSORT abstract should be provided
(http://www.consort-statement.org).
Introduction: The introduction should include an overview of the
relevant literature presented in summary form (one page), and what
ever remains interesting, unique, problematic, relevant, or unknown
about the topic must be specified. The introduction should conclude
with the rationale for the study, its design, and its objective(s).
Materials and Methods: Clearly describe the selection of observational
or experimental participants, such as patients, laboratory animals, and
controls, including inclusion and exclusion criteria and a description
of the source population. Identify the methods and procedures in
sufficient detail to allow other researchers to reproduce your results.
Provide references to established methods (including statistical
methods), provide references to brief modified methods, and provide
the rationale for using them and an evaluation of their limitations.
Identify all drugs and chemicals used, including generic names,
doses, and routes of administration. The section should include only
information that was available at the time the plan or protocol for
the study was devised (http://www.strobe-statement.org/fileadmin/
Strobe/uploads/checklists/STROBE_checklist_v4_combined.pdf).
A-IV

Statistics: Describe the statistical methods used in enough detail to
enable a knowledgeable reader with access to the original data to verify
the reported results. Statistically important data should be given in the
text, tables and figures. Provide details about randomization, describe
treatment complications, provide the number of observations, and
specify all computer programs used.
Results: Present your results in logical sequence in the text, tables, and
figures. Do not present all the data provided in the tables and/or figures in
the text; emphasize and/or summarize only important findings, results,
and observations in the text. For clinical studies provide the number
of samples, cases, and controls included in the study. Discrepancies
between the planned number and obtained number of participants
should be explained. Comparisons, and statistically important values
(i.e. P value and confidence interval) should be provided.
Discussion: This section should include a discussion of the data.
New and important findings/results, and the conclusions they lead
to should be emphasized. Link the conclusions with the goals of
the study, but avoid unqualified statements and conclusions not
completely supported by the data. Do not repeat the findings/results
in detail; important findings/results should be compared with those
of similar studies in the literature, along with a summarization. In
other words, similarities or differences in the obtained findings/results
with those previously reported should be discussed. Limitations
of the study should be detailed. In addition, an evaluation of the
implications of the obtained findings/results for future research
should be outlined.
References
Cite references in the text, tables, and figures with numbers in
parentheses. Number references consecutively according to the
order in which they first appear in the text. Journal titles should be
abbreviated according to the style used in Index Medicus (consult List
of Journals Indexed in Index Medicus). Include among the references
any paper accepted, but not yet published, designating the journal
and followed by, in press.
Examples of References:
1. List all authors.
Deeg HJ, O’Donnel M, Tolar J. Optimization of conditioning for
marrow transplantation from unrelated donors for patients with
aplastic anemia after failure immunosuppressive therapy. Blood
2006;108:1485-1491.
2.Organization as author
Royal Marsden Hospital Bone Marrow Transplantation Team. Failure
of syngeneic bone marrow graft without preconditioning in posthepatitis
marrow aplasia. Lancet 1977;2:742-744.
3.Book
Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea &
Febiger, 1961.
4. Book Chapter
Perutz MF. Molecular anatomy and physiology of hemoglobin. In:
Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of
Hemoglobin: Genetics, Pathophysiology, Clinical Management. New
York, Cambridge University Press, 2000.
5.Abstract
Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand
(thrombopoietin) stimulates tyrosine phosphorylation. Blood
1994;84:390a (abstract).
6.Letter to the Editor
Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further
definition of 20q deletion in myeloid leukemia using fluorescence in
situ hybridization. Blood 1994;84:2821-2823.
7. Supplement
Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr
Transplant. 2005;9(Suppl 7):81-86
Brief Reports
Abstract length: Not to exceed 150 words.
Article length: Not to exceed 1200 words.
Introduction: State the purpose and summarize the rationale for the
study.
Materials and Methods: Clearly describe the selection of the
observational or experimental participants. Identify the methods
and procedures in sufficient detail. Provide references to established
methods (including statistical methods), provide references to brief
modified methods, and provide the rationale for their use and an
evaluation of their limitations. Identify all drugs and chemicals used,
including generic names, doses, and routes of administration.
Statistics: Describe the statistical methods used in enough detail to
enable a knowledgeable reader with access to the original data to verify
the reported findings/results. Provide details about randomization,
describe treatment complications, provide the number of observations,
and specify all computer programs used.
Results: Present the findings/results in a logical sequence in the text,
tables, and figures. Do not repeat all the findings/results in the tables
and figures in the text; emphasize and/or summarize only those that
are most important.
Discussion: Highlight the new and important findings/results of the
study and the conclusions they lead to. Link the conclusions with the
goals of the study, but avoid unqualified statements and conclusions
not completely supported by your data.
Case Reports
Abstract length: Not to exceed 100 words.
Article length: Not to exceed 1200 words.
Case Reports can include maximum 1 figure and 1 table or 2 figures
or 2 tables.
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Case reports should be structured as follows:
Abstract
An unstructured abstract that summarizes the case.
Introduction: A brief introduction (recommended length: 1-2
paragraphs).
Case Presentation: This section describes the case in detail, including
the initial diagnosis and outcome.
Discussion:This section should include a brief review of the relevant
literature and how the presented case furthers our understanding to
the disease process.
Invited Review Articles
Abstract length: Not to exceed 300 words.
Article length: Not to exceed 4000 words.
Review articles should not include more than 100 references.
Reviews should include a conclusion, in which a new hypothesis or
study about the subject may be posited. Do not publish methods for
literature search or level of evidence. Authors who will prepare review
articles should already have published research articles on therel
evant subject. The study’s new and important findings should be
highlighted and interpreted in the Conclusion section. There should
be a maximum of two authors for review articles.
Images in Hematology
Article length: Not exceed 200 words.
Authors can submit for consideration an illustration and photos that
is interesting, instructive, and visually attractive, along with a few
lines of explanatory text and references. Images in Hematology can
include no more than 200 words of text, 5 references, and 3 figure or
table. No abstract, discussion or conclusion are required but please
include a brief title.
Letters to the Editor
Article length: Not to exceed 500 words.
Letters can include no more than 500 words of text, 5-10 references, and
1 figure or table. No abstract is required, but please include a brief title.
Tables
Supply each table on a separate file. Number tables according to the
order in which they appear in the text, and supply a brief caption
for each. Give each column a short or abbreviated heading. Write
explanatory statistical measures of variation, such as standard deviation
or standard error of mean. Be sure that each table is cited in the text.
Figures
Figures should be professionally drawn and/or photographed.
Authors should number figures according to the order in which they
appear in the text. Figures include graphs, charts, photographs, and
illustrations. Each figure should be accompanied by a legend that
does not exceed 50 words. Use abbreviations only if they have been
introduced in the text. Authors are also required to provide the level
of magnification for histological slides. Explain the internal scale and
identify the staining method used. Figures should be submitted as
separate files, not in the text file. High-resolution image files are not
preferred for initial submission as the file sizes may be too large. The
total file size of the PDF for peer review should not exceed 5 MB.
Authorship
Each author should have participated sufficiently in the work to assume
public responsibility for the content. Any portion of a manuscript that is
critical to its main conclusions must be the responsibility of at least 1 author.
Contributor’s Statement
All submissions should contain a contributor’s statement page. Each
manuscript should contain substantial contributions to idea and
design, acquisition of data, or analysis and interpretation of findings.
All persons designated as an author should qualify for authorship,
and all those that qualify should be listed. Each author should
have participated sufficiently in the work to take responsibility for
appropriate portions of the text.
Acknowledgments
Acknowledge support received from individuals, organizations,
grants, corporations, and any other source. For work involving a
biomedical product or potential product partially or wholly supported
by corporate funding, a note stating, “This study was financially
supported (in part) with funds provided by (company name) to
(authors’ initials)”, must be included. Grant support, if received,
needs to be stated and the specific granting institutions’ names and
grant numbers provided when applicable.
Authors are expected to disclose on the title page any commercial or
other associations that might pose a conflict of interest in connection
with the submitted manuscript. All funding sources that supported
the work and the institutional and/or corporate affiliations of the
authors should be acknowledged on the title page.
Ethics
When reporting experiments conducted with humans indicate that
the procedures were in accordance with ethical standards set forth
by the committee that oversees human experimentation. Approval of
research protocols by the relevant ethics committee, in accordance
with international agreements (Helsinki Declaration of 1975, revised
2002 available at http://www.wma.net/e/policy/b3.htm, “Guide for
the Care and use of Laboratory Animals” www.nap.edu/catalog/5140.
html/), is required for all experimental, clinical, and drug studies.
Patient names, initials, and hospital identification numbers should
not be used. Manuscripts reporting the results of experimental
investigations conducted with humans must state that the study
protocol received institutional review board approval and that the
participants provided informed consent.
Non-compliance with scientific accuracy is not in accord with scientific
ethics. Plagiarism: To re-publish-whole or in part-the contents
of another author’s publication as one’s own without providing a
reference. Fabrication: To publish data and findings/results that
do not exist. Duplication: Use of data from another publication,
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which includes re-publishing a manuscript in different languages.
Salamisation: To create more than one publication by dividing the
results of a study preternaturally.
We disapprove of such unethical practices as plagiarism, fabrication,
duplication, and salamisation, as well as efforts to influence the review
process with such practices as gifting authorship, inappropriate
acknowledgements, and references. Additionally, authors must
respect participant right to privacy.
On the other hand, short abstracts published in congress books that
do not exceed 400 words and present data of preliminary research,
and those that are presented in an electronic environment are not
accepted pre-published work. Authors in such situation must declare
this status on the first page of the manuscript and in the cover letter.
(The COPE flowchart is available at: http://publicationethics.org)
We use iThenticate to screen all submissions for plagiarism before
publication.
Turkish Journal of Hematology uses plagiarism screening service
to verify the originality of content submitted before publication.
Conditions of Publication
All authors are required to affirm the following statements before their
manuscript is considered: 1. The manuscript is being submitted only
to The Turkish Journal of Hematology; 2. The manuscript will not be
submitted elsewhere while under consideration by The Turkish Journal
of Hematology; 3. The manuscript has not been published elsewhere,
and should it be published in The Turkish Journal of Hematology it
will not be published elsewhere without the permission of the editors
(these restrictions do not apply to abstracts or to press reports for
presentations at scientific meetings); 4. All authors are responsible for
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A-VII

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A-VIII

CONTENTS
Review Article
285 Chimeric Antigen Receptor T Cell Therapy in Hematology
Pınar Ataca, Önder Arslan
Research Articles
295 Possible Role of GADD45γ Methylation in Diffuse Large B-Cell Lymphoma: Does It Affect the Progression and Tissue Involvement?
İkbal Cansu Barış, Vildan Caner, Nilay Şen Türk, İsmail Sarı, Sibel Hacıoğlu, Mehmet Hilmi Doğu, Ozan Çetin,
Emre Tepeli, Özge Can, Gülseren Bağcı, Ali Keskin
304 Effect of Tumor Necrosis Factor-Alpha on Erythropoietin- and Erythropoietin Receptor-Induced Erythroid Progenitor Cell
Proliferation in β-Thalassemia/Hemoglobin E Patients
Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard, Suthat Fucharoen
311 The -137G/C Polymorphism in Interleukin-18 Gene Promoter Contributes to Chronic Lymphocytic and
Chronic Myelogenous Leukemia Risk in Turkish Patients
Serap Yalçın, Pelin Mutlu, Türker Çetin, Meral Sarper, Gökhan Özgür, Ferit Avcu
317 Transcobalamin II Deficiency in Four Cases with Novel Mutations
Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin
323 Eltrombopag for the Treatment of Immune Thrombocytopenia: The Aegean Region of Turkey Experience
Füsun Özdemirkıran, Bahriye Payzın, H. Demet Kiper, Sibel Kabukçu, Gülsüm Akgün Çağlıyan, Selda Kahraman,
Ömür Gökmen Sevindik, Cengiz Ceylan, Gürhan Kadıköylü, Fahri Şahin, Ali Keskin, Öykü Arslan, Mehmet Ali Özcan,
Gülnur Görgün, Zahit Bolaman, Filiz Büyükkeçeci, Oktay Bilgir, İnci Alacacıoğlu, Filiz Vural, Murat Tombuloğlu,
Zafer Gökgöz, Güray Saydam
329 Management of Invasive Fungal Infections in Pediatric Acute Leukemia and the Appropriate Time for Restarting Chemotherapy
Özlem Tüfekçi, Şebnem Yılmaz Bengoa, Fatma Demir Yenigürbüz, Erdem Şimşek, Tuba Hilkay Karapınar, Gülersu İrken,
Hale Ören
338 First-Step Results of Children Presenting with Bleeding Symptoms or Abnormal Coagulation Tests in an Outpatient Clinic
İsmail Yıldız, Ayşegül Ünüvar, İbrahim Kamer, Serap Karaman, Ezgi Uysalol, Ayşe Kılıç, Fatma Oğuz, Emin Ünüvar
344 Evaluation of Alpha-Thalassemia Mutations in Cases with Hypochromic Microcytic Anemia: The İstanbul Perspective
Zeynep Karakaş, Begüm Koç, Sonay Temurhan, Tuğba Elgün, Serap Karaman, Gamze Asker, Genco Gençay, Çetin Timur,
Zeynep Yıldız Yıldırmak, Tiraje Celkan, Ömer Devecioğlu, Filiz Aydın
Brief Report
351 The Efficacy and Safety of Procedural Sedoanalgesia with Midazolam and Ketamine in Pediatric Hematology
Sema Aylan Gelen, Nazan Sarper, Uğur Demirsoy, Emine Zengin, Esma Çakmak
A-IX

Case Reports
355 A Hemophagocytic Lymphohistiocytosis Case with Newly Defined UNC13D (c.175G>C; p.Ala59Pro)
Mutation and a Rare Complication
Yasemin Işık Balcı, Funda Özgürler Akpınar, Aziz Polat, Fethullah Kenar, Bianca Tesi, Tatiana Greenwood, Nagihan Yalçın,
Ali Koçyiğit
359 The Use of Low-Dose Recombinant Tissue Plasminogen Activator to Treat a Preterm Infant with an Intrauterine Spontaneous
Arterial Thromboembolism
Yaşar Demirelli, Kadir Şerafettin Tekgündüz, İbrahim Caner, Mustafa Kara
363 Immune Thrombocytopenic Purpura During Maintenance Phase of Acute Lymphoblastic Leukemia: A Rare
Coexistence Requiring a High Degree of Suspicion, a Case Report and Review of the Literature
Turan Bayhan, Şule Ünal, Fatma Gümrük, Mualla Çetin
367 A Rare Complication Developing After Hematopoietic Stem Cell Transplantation: Wernicke’s Encephalopathy
Soner Solmaz, Çiğdem Gereklioğlu, Meliha Tan, Şenay Demir, Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu
Letters to the Editor
371 Downgraded Lymphoma: B-Chronic Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell Lymphoma - De
Novo Occurrence or Transformation
Smeeta Gajendra, Bhawna Jha, Shalini Goel, Tushar Sahni, Pranav Dorwal, Ritesh Sachdev
373 From Bone Marrow Necrosis to Gaucher Disease; A Long Way to Run
Neslihan Erdem, Ahmet Çizmecioğlu, İsmet Aydoğdu
374 First Observation of Hemoglobin Kansas [β102(G4)Asn→Thr, AAC>ACC] in the Turkish Population
İbrahim Keser, Alev Öztaş, Türker Bilgen, Duran Canatan
Images in Hematology
376 Mott Cells in the Peripheral Blood of a Patient with Dengue Fever
Aniya Antony, Marie Ambroise, Chokka Kiran, Mookkappan Sudhagar, Anita Ramdas
378 Diagnosis: Melanoderma after Hematopoietic Stem Cell Transplantation
Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya
2015 Index
2015 Subject Index
2015 Author Index
A-X

Review Article
DOI: 10.4274/tjh.2015.0049
Turk J Hematol 2015;32:285-294
Chimeric Antigen Receptor T Cell Therapy in Hematology
Hematolojik Malignitelerde Kimerik Antijen Reseptör-T Hücre
Tedavisi
Pınar Ataca, Önder Arslan
Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey
Abstract:
It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells
has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation.
Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are
two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified
T cells. On 1 July 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19
CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality.
This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing
process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.
Keywords: Chimeric antigen receptor T cell, Hematological malignancies
Öz:
İmmün sistemin kanser hücrelerini kontrol ve elimine etme özelliğine sahip olduğu gösterilmiştir. İmmün-kontrollü
eliminasyonda kanser aşıları ve hematopoietik kök hücre naklini içeren sellüler terapiler bulunmaktadır. Adoptif T hücre
transferi daha potent ve spesifiktir, hedef dışı toksisitesi azdır. Klinik çalışmalarda iki tür T hücresi test edilmektedir: T hücre
reseptör ve kimerik antijen reseptör (KAR) modifiye T hücreleri. 1 Temmuz 2014’te Amerikan Gıda ve İlaç Dairesi anti-CD19
ŞAR modifiye T hücre tedavisini “çığır açan tedaviler” sınıfına almıştır. Bu yeni tedavi yöntemini ve etkilerini araştıran birçok
çalışma yapılmıştır. Bu derleme adoptif immünoterapinin geçmişini, ŞAR modifiye T hücrelerini, üretim sürecini, klinik ve
preklinik çalışmaları özetlemektedir.
Anahtar Sözcükler: Kimerik antijen reseptör-T hücreleri, Hematolojik maligniteler
Introduction
Poor salvage chemotherapy success rates for refractory
hematological diseases have necessitated novel approaches.
Adoptive T-cell transfer has gained significant interest and
clinical usage in hematology because of the off target effects
of allogeneic stem cell transplantation and life threatening
graft versus host disease (GVHD). Therefore, research
efforts have sought to generate more specific T cells with
higher toxicity to tumors and not healthy targets. To achieve
curative potential, T cell immunotherapy combines potency,
specificity and persistence [1]. Early approaches to adoptive T
cell immunotherapy were based on the graft-versus-leukemia
(GVL) effect mediated by donor lymphocyte infusion (DLI)
hematopoietic stem cell transplantation (HSCT) and the
therapeutic infusion of ex vivo expanded tumor-infiltrating
Address for Correspondence: Pınar ATACA, M.D.,
Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey
E-mail: drpinarataca@gmail.com
Received/Geliş tarihi : January 23, 2015
Accepted/Kabul tarihi : April 20, 2015
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Turk J Hematol 2015;32:285-294
Ataca P, et al: Chimeric Antigen Receptors in Hematology
lymphocytes (TILs) in combination with lymphodepletion
for the treatment of advanced melanoma. However, DLI is
usually associated with life-threatening forms of GVHD, and
TILs require time-consuming procedures with unsuccessful
results [2,3]. To overcome these drawbacks, genetically
modified effector T cells have been developed as an alternative
approach. In hematological malignancies, engineered T cell
receptors (TCRs) and chimeric antigen receptors (CARs)
are new powerful T-cell based immune therapies that target
specific antigens. CAR T cells have been used successfully
in the treatment of solid and hematological malignancies
recently. In the following sections, the history of adoptive
immunotherapy, TCR gene therapy, CART cell production,
and preclinical and clinical studies will be discussed.
The Role of T Cells in Cancer and T Cell Receptor Gene
Therapy
In 1909, Paul Ehrlich first proposed that the immune defense
system identifies and eliminates tumor cells [4]. However,
recent studies revealed that the immune response may be
ineffective against tumor development due to immunological
tolerance and anergy [5]. Cancer immunoediting consists
of three stages: elimination, equilibrium and escape. In the
elimination stage, cancer is eliminated by intact innate and
adaptive immunity, whereas in the equilibrium stage, variant
tumor cells that develop genetic instability survive despite the
immune attacks. Uncontrolled proliferation of variant tumor
cells occurs in the escape stage [6].
In 1890, William B Coley observed that patients with
malignancies respond to the intratumoral inoculation of live
bacterial organisms or bacterial toxins that cause tumors
to express unique proteins that could trigger an immune
response [7]. Since the beginning of the 20 th century, research
has shown that most cancer cells carry overexpressed tumorassociated
or tumor-specific antigens that are not present on
healthy cells; this feature has led to the successful application
of adoptive T-cell transfer. The discovery of T-cell growth
factor, in vitro T-cell culture and the role of lymphodepletion
have led to T-cell based therapy studies [8]. The first successful
study on T-cell transfer immunotherapy using autologous
TILs was performed in advanced melanoma in 1990 [9]. Since
tumor infiltrating lymphocyte isolation was first attempted,
in vitro expansion and re-infusion have been shown to be
time-consuming and produce transient anti-tumor effects,
and genetic engineering methods have been applied to create
specific T cell-generated TCRs.
The TCR is a heterodimer that carries information for
defined tumor antigens and is formed by alpha and beta
chains associated with a CD3 complex (Figure 1) [10]. TCR
technology has advantages as a redirected T-cell therapy. Ideal
effector T cells match with selected tumor target antigens
through HLA recognition. The natural mechanism of T-cell
immunity is associated with a low risk of cytokine release
syndrome. The major difficulties that need to be overcome
are the low surface expression of TCRs, HLA dependency,
the and short persistence of transferred T-cells in vivo [11].
In thymic selection during the development of T cells, a few
mutated proteins are encoded by cancer-causing genetic
mutations (driver mutations), the large proportion of tumor
antigens are self antigens, and T cells have low affinity for
self antigens [12]. To create a higher avidity, selected TCRs
from immunized human HLA transgenic mice with relevant
epitopes are used along with insertion of targeted mutations
in the complementary-determining region 2 or 3 (CDR2 or
3) in the variable regions of the TCR alpha/beta chains. These
modified TCRs interact with the HLA/epitope complex [13].
However, TCRs can create unwanted alpha/beta heterodimers
between the new and endogenous TCR alpha/beta chains in a
process called mispairing, which results in low avidity [14].
TCR-modified T cells adapted for solid tumors have not been
successful in most studies (Table 1) [10].
Chimeric Antigen Receptors
The genetic modification of T cells with CARs represents
a breakthrough for gene engineering in hematological
malignancies. The first CAR concept originated from the
cloning of the TCR CD3 ζ-chain that was found to activate T
cells independently [18]. First-generation CARs included only
a single-chain variable fragment (scFv) that was constructed
from the variable heavy and variable light sequences of a
monoclonal antibody (mAb) specific for a tumor cell surface
molecule and the cytoplasmic CD3 ζ-chain signaling domain.
The initial studies were conducted in patients with HIV
infection with prolonged survival [19]. In the first-generation
Figure 1. T cell receptor (adapted from Wieczorek and Uharek [10]).
286

Ataca P, et al: Chimeric Antigen Receptors in Hematology
Turk J Hematol 2015;32:285-294
cancer studies, CAR T cells did not proliferate in vivo and
persistence was transient or the T cells were present at very
low frequencies [20]. Based on a second genetic modification,
CARs possess an antibody-based extracellular receptor
structure that binds to target cells along with intracellular
activating domains. Costimulatory protein receptors (e.g.,
CD28, CD137 (4-1BB), ICOS, CD134 (OX40), CD27, or
CD244) were added to the cytoplasmic tail of the CAR in the
second- and third-generation CARs [21] (Figure 2). Secondgeneration
CARs are constructed with one costimulatory
molecule while third-generation CARs contain more than
one additional costimulatory molecule. The antitumor effect
of CAR-T cells varies due to differences in the cytoplasmic
domain and the extracellular domain’s ability to recognize a
different epitope of the same antigen with different affinities
for each CAR construct [22]. Whether the addition of
secondary costimulation as in third-generation CARs obtains
more efficacy is still an unanswered question [23]. CARs have
several advantages: initiation of reliable high-potency signals,
HLA independency, no requirement for antigen processing,
and no competition for CD3. The number of target molecules
on tumor cells that bind to CARs is greater than the number of
major histocompatibility complex (MHC)/peptide complexes,
and the scFv has a higher binding affinity for antigens than
the TCRs [24]. Recently, Oren et al. compared the functional
properties of engineered T cells expressing native low-affinity
αβ-TCR chains with high-affinity TCR-like Ab-based CARs
targeting the same specificity and suggested that the upper
affinity threshold should be used to mediate effective functional
outcomes of engineered T cells [25]. The major disadvantage
of CARs is the massive cytokine release induced by binding
and the immunogenicity of the mouse-derived scFv portion of
the CAR complex, which may result in immune responses and
the clearance of CAR T cells. In addition to that, intracellular
molecules cannot be recognized [26].
Figure 2. Generations of CART cells (adapted from Porter et al.
[55]).
Table 1. T cell receptor clinical studies.
Antigen Tumor Effectiveness Toxicities Reference
MART 1 Melanoma 6 PRs in 20 patients Erythematous skin rash grade
1-2 (14/20), hearing loss
(10/20), uveitis (11/20)
gp100 Melanoma 3 PRs in 16 patients Erythematous skin rash grade
1-2 (15/16), hearing loss (5/16),
uveitis (4/16)
MAGE-A3
MAGE-A3/A9/A12
Melanoma
multiple myeloma
Melanoma,
synovial sarcoma,
esophageal cancer
Not evaluable Acute cardiac failure (2/2),
cytokine release syndrome,
death (2/2)
4 PRs, 1 CR in 9 patients Neurological toxicity (4/9),
death (2/9)
2009, [15]
2009, [15]
2013, [16]
2012, [17]
CR: Complete remission, PR: partial remission.
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Ataca P, et al: Chimeric Antigen Receptors in Hematology
Chimeric Antigen Receptor T Cell Manufacturing
Gene transfer technology has rapidly developed; however,
the clinical production of CARs for therapy is restricted to
specialized, licensed manufacturing facilities with stringent
rules (Good Manufacturing Process). In vitro culture systems
for T cell expansion are used to manufacture large quantities
of engineered T cells. The average production time to generate
large numbers of unselected CD4 and CD8 T cells required
for therapy is 10-14 days in clean rooms. First, peripheral
blood mononuclear cells are isolated from the patient using
leukapheresis, and T cells are selected by anti-CD3/anti-CD28
paramagnetic beads. Recent studies have demonstrated that
less differentiated T cells have superior engraftment and
antitumor activity [27]. In particular, CD8 T central memory
cells can be modified with tumor-specific CARs [28]. T
Table 2. Chimeric antigen receptor T cell trials in hematological malignancies [55].
scFv/Signaling Domain
Vector Dose Number
of Patients
CD20/CD3 Electroporation 1x10 8 /m 2 to 7 (indolent
3.3x10 9 /m 2 and MCL)
CD20 or CD19/CD3 Electroporation 10 8 /m 2 to 2x10 9 /m 2 4 (2 FL, 2
DLBCL)
Responses
Reference
2 CR, 1 PR, 4 SD 2008 [46]
2 CR after autologous
stem cell transplantation
2010 [52]
CD19/CD3 and CD28-CD3 Gammaretrovirus 2x10 7 /m 2 to
2x10 9 /m 2 6 NHL 2 SD 2011 [53]
CD19/CD28 and CD3 Gammaretrovirus 0.4-3.3x10 7 CAR
cells/kg
CD19/4-1BB and CD3 Lentivirus 1.46x10 5 to 1.6x10 7
CAR cells/kg
CD19/CD28 and CD3 Gammaretrovirus 0.3-3x10 7
CD20/CD28 and 4-1BB and
CD3
CAR cells/kg
Electroporation 1x10 8 to 3.3x10 9 /
m 2
CD19/CD28 and CD3 Gammaretrovirus 1.5-3x10 6 CAR
cells/kg
CD19/4-1BB and CD3 Lentivirus 1.4x10 6 and
1.2x10 7 CAR cells/
kg
Lewis Y/CD28 and CD3 Gammaretrovirus 1.4-9.2x10 6 CAR
cells/kg
8 CLL and
1 ALL
1 death, (ALL) B cell
aplasia, 1 reduction in
lymphadenopathy
3 CLL 2 CR, 1 PR, 3 B cell
aplasia
8 (3 FL,
4 CLL, 1
MZL)
3 (2 MZL,
1 FL)
6 objective remissions
(4 B cell aplasia)
No progression in 2
patients, 1 patient PR
5 ALL All 5 converted to MRD,
1 relapsed, 1 B cell
aplasia
2011 [54]
2011 [55]
2010 [56]
2012 [57]
2013 [58]
2 ALL 2 CR, both B cell aplasia 2013 [59]
4 AML 2 SD, 1 transient
cytogenetic remission
CD19/CD28 and CD3 Gammaretrovirus 1.5x10 7 to 1.2x10 8
total T cells/m 2 8 ALL 4 of 8 patients with
decreased B cell counts
CD19/CD28 and CD3 Gammaretrovirus 0.4-7.8x10 6 CAR
cells/kg
4 CLL, 2
DLBCL, 4
MCL
2013 [48]
2013 [60]
2 PD, 6 SD, 1 PR, 1 CR 2013 [61]
AML: Acute myeloid leukemia, ALL: acute lymphoblastic leukemia, CLL: chronic lymphocytic lymphoma, CR: complete remission, DLBCL: diffuse large B cell lymphoma,
FL: follicular lymphoma, MCL: mantle cell lymphoma, MRD: minimal residual disease, MZL: marginal zone lymphoma, NHL: non-Hodgkin lymphoma, PD: progressive
disease, PR: partial remission, SD: stable disease.
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Turk J Hematol 2015;32:285-294
cells are then transduced with a CAR-encoding viral vector.
Two vector systems, retroviral or lentiviral vectors, can be
used to transfer CAR-coding genes into T cells. Retroviral
vectors have permanent gene expression; however, the
transduction can be performed only on efficiently dividing
T cells. Lentiviral vectors can also integrate into nondividing
cells. The disadvantages of viral vectors are the expense and
experience required for production. Transposon systems
such as Sleeping Beauty 100X (SB100X) or PiggyBac (PB)
are new methods for genetic modification of T cells with
high gene expression; they are simple and inexpensive and
have large cargo capacity and low immunogenicity [10]. T
cells are expanded in culture by stimulating them using the
anti-CD3 clone OKT3 with cytokines like IL-2, IL-7, and
IL-15. Moreover, in vivo persistence can be achieved by the
overexpression of antiapoptotic proteins such as Bcl-2 or
Bcl-xL. An adequate number of CAR T cells, which remains
unknown, are then transferred to the patient using host
preparative lymphodepletion regimens based on drugs and
techniques to deplete Tregs, such as cyclophosphamide,
fludarabine, low-dose irradiation, gemcitabine, denileukin
diftitox, azacitidine, or decitabine [10,29,30]. CARs on T cells
bind to their antigen on the tumor, and activation is controlled
by the intracytoplasmic domains within the CAR. Tumor
killing can be mediated by the direct cytotoxicity of the
CD8 + CAR T cells with granzyme and perforin or cytokines
released by CD4 + CAR T cells that bypass the MHC. Longterm
eradication and prevention can be achieved by memory
CAR T cells from a single infusion [31].
Studies Involving Chimeric Antigen Receptor T Therapy
TThe ideal targets for CAR-modified T cells are expressed
on tumor cells but are not expressed on normal cells. CD19
and CD20 are attractive targets due to their specificity for
the B cell linage [32]. The first-generation CARs were not
sufficient to produce a durable immune response; they rapidly
underwent apoptosis after stimulation [33]. 19z CAR T cells
were expanded on CD19+CD80+IL15+ cells and eradicated
established systemic Raji tumors in 50% of SCID-beige mice
[34]. Second-generation CARs that express CD28-containing
costimulation in the CD19+CD80/CD86-ALL SCID-beige
tumor model showed superior in vivo tumor activity and T
cell function. CD22 is also under investigation and shows
potential [35]. Imai et al. showed that in vivo anti-CD19
chimeric receptors containing the 4-1BB signal transduction
domain had powerful antileukemic activity, destroying
CD19+ acute lymphoblastic leukemia (ALL) cell lines in an
in vivo microenvironment [33]. Target discovery for T cell
leukemias and myeloid leukemias is problematic because
blasts express the same antigens as normal hematopoietic
stem cells [36]. For myeloid leukemias, CARs directed against
CD123 have demonstrated efficacy in preclinical models;
however, vascular endothelial cells also express CD123,
which requires more investigation before clinical application
[37]. Kenderian et al. stated that anti-CD33-specific CAR
T cells exhibited significant effector functions in vitro and
resulted in eradication of leukemia and prolonged survival in
acute myeloid leukemia (AML) xenografts [38]. In multiple
myeloma, CAR-engineered natural killer cells that targeted
CS-1 protein displayed enhanced cytolysis in vitro [39].
The translation of this therapy to clinical settings involves
various antigens and malignancies, and most trials have
focused on B cell malignancies with B cell antigens CD19
and CD20 as the targets [40]. The first case report of CD19+
CAR T cells was published in 2011 by Porter et al. in relapsed
refractory chronic lymphoid leukemia [41]. In that study,
3x108 T cells were transduced using a lentiviral vector, and
the patient exhibited complete remission after 10 months.
The largest dose-optimization trial involved 27 chronic
lymphocytic leukemia (CLL) patients and found no difference
between two doses of CAR T cells (5x10 7 ) with
a complete response rate of 40% of patients [42]. In another
study, CAR-modified T cells were shown to persist for more
than 3 years with an initial response rate of 57% and complete
remission of 29%, which was more favorable as compared
to ibrutinib (an overall response rate of 71% but a complete
remission rate of 2.4%) [43]. In B cell ALL (B-ALL), Davila
et al. reported on 16 relapsed or refractory cases that were
treated with 19-28z-expressing CAR T cells with an overall
complete response rate of 88%, as compared to 44% with
salvage chemotherapy. CAR T cells persisted for 2-3 months,
and almost half of the patients proceeded to allogeneic stem
cell transplantation [44]. In 30 ALL patients treated with
CD19 CAR T cells, a 6-month event-free survival of 67% and
overall survival of 78% were achieved, and ongoing remission
for up to 2 years was possible without transplantation [45].
The underlying causes of the limited clinical efficacy of the
CAR T cells in patients with CLL compared to B-ALL include
the limited persistence of CAR T cells in CLL patients, the
inhibitory effect of the tumor microenvironment in CLL, the
lymph node-based nature of CLL, and the lower tumor burden
at treatment in patients with B-ALL [40].
Patients with B cell malignancy were first treated with
modified autologous CD20-specific T cells in 2008 by
investigators from the Fred Hutchinson Cancer Research
Center and the City of Hope National Medical Center.
T cells persisted for up to 9 weeks with 7 patients with
indolent or mantle cell lymphoma achieving partial response
(1 patient), stable disease (4 patients), or complete response
(2 patients) [46]. In 2014, an anti-CD19 chimeric antigen
receptor trial for chemotherapy-refractory diffuse large B cell
lymphoma and indolent B cell malignancies was published
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Ataca P, et al: Chimeric Antigen Receptors in Hematology
by Kochenderfer et al.; they demonstrated that 8 of 15
patients had complete response with 1-5x106 CAR T cells
transduced by gammaretrovirus [47]. The targets for CAR
therapy in multiple myeloma can be CD138, CD38, CD56,
and CS1. Unlike CD19, these targets are coexpressed on other
important cell types and result in unacceptable on-target, offtumor
toxicity. The first AML trial targeted the LeY antigen,
and only 1 of 4 patients had 23 months of stable disease
following therapy [48]. Contrary to preclinical studies, the
CD33 antigen as a target was not proven to be safe due to the
high level of toxicity against normal hematopoietic cells [49].
Phase I clinical trials involving CD123 targeting by mAbs and
immunotoxins have produced only minor clinical responses,
suggesting the need to develop more powerful AML strategies
[50]. Table 2 shows the CAR T cell therapies in hematological
malignancies [51].
Adverse Effects of Chimeric Antigen Receptor T Cell
Therapy
AAs with all therapies, the toxicity from CAR T cells may
be classified as on-target or off-target. The most common
toxicity is cytokine release syndrome (CRS). In most cases,
CRS is correlated with antitumor activity, and patients
exhibit a range of symptoms from high fever, hypoxia, and
hypotension to mild flu symptoms. The increased cytokines,
particularly IL-6 and TNF-α, are produced by dying B cells,
tumor cells, or macrophages [51]. Grupp et al. reported that
the IL-6 receptor-blocking monoclonal antibody tocilizumab
may ameliorate CRS in steroid-refractory circumstances
without compromising T cell efficacy [59]. CRS was reported
to occur in 6/13 patients with high complete response rates
with tocilizumab as an alternative treatment option. The
C-reactive protein level has been shown to be an indicator of
severe CRS [45]. Another off-target adverse effect is tumor
lysis syndrome, which is due to rapid and massive destruction
of tumor cells. Macrophage activation syndrome is another
life-threatening off-target effect of systemic inflammatory
symptoms and pancytopenia, although the mechanisms are
still unknown [42]. Several patients in CD19-CAR trials
experienced reversible obtundation, seizures, aphasia, and
mental status changes, possibly due to systemic cytokines
crossing the blood-brain barrier [51]. B cell aplasia is an
expected result of CD19-directed therapies and can be
managed by γ-globulin replacement therapy. Persistent B cell
aplasia results in an increased risk of infections [52].
Future Directions
AAdoptive T cell transfer has been used for the treatment
of malignant diseases and may be regarded as an anticancer
biopharmaceutical. A biopharmaceutical is defined as a
product that is originally natural or derived from biological
sources with industrial additions [62]. The main goals of T
cell engineering are tumor antigen targeting and an increase
in antitumor functions [1]. CAR T cell therapies are powerful
breakthrough therapies, but several challenges need to be
addressed. The optimal design of CARs remains an area of
investigation. To be useful in other disease types, tumorspecific
targets must be identified in solid tumors. T cell
trafficking to the tumor microenvironment is critical in the
moderate success against solid cancers [63]. To minimize
severe toxicity, standardized approaches to the management
of CRS should be applied [64]. B cell aplasia is still a problem
with long-term exposure and may have an economic impact on
health care. Once the B cell malignancy has been eradicated,
anti-CD19-CAR T cells should be ablated to maintain normal B
cell activity. A suicide system has been developed to eliminate
gene-modified T cells when they display unwanted toxicities,
such as the thymidine kinase gene of the herpes simplex virus
[65]. Relapse remains a challenge and may be prevented with
optimization of CAR design. Finally, in order for the therapy
to become routinely used, automation and robotic culture
technologies should be performed during the manufacturing
process instead of manual cell culture technologies [66].
The induction of adoptive immunotherapy using CAR T
cells has been successful in clinical trials, and the final goal
is to induce durable immunity against disease progression
without severe adverse effects. Whether this treatment option
will replace HSCT or be used as a bridge to HSCT in the near
future is still an unanswered question.
Concept: Önder Arslan, Design: Önder Arslan, Data
Collection or Processing: Pınar Ataca, Analysis or
Interpretation: Pınar Ataca, Literature Search: Pınar Ataca,
Writing: Pınar Ataca, Önder Arslan.
Conflict of Interest: The authors of this paper have no
conflicts of interest, including specific financial interests,
relationships, and/or affiliations relevant to the subject matter
or materials included.
References
1. Sadelain M. CAR T cell therapy: the CD19 paradigm. ASH
Annual Meeting, Orlando, FL, USA, 2014.
2. Miller JS, Warren EH, van den Brink MR, Ritz J, Shlomchik
WD, Murphy WJ, Barrett AJ, Kolb HJ, Giralt S, Bishop MR,
Blazar BR, Falkenburg JH. NCI First International Workshop
on the Biology, Prevention, and Treatment of Relapse After
Allogeneic Hematopoietic Stem Cell Transplantation: Report
from the Committee on the Biology Underlying Recurrence of
Malignant Disease Following Allogeneic HSCT: Graft-versus-
Tumor/Leukemia Reaction. Biol. Blood Marrow Transplant
2010;16:565-586.
290

Ataca P, et al: Chimeric Antigen Receptors in Hematology
Turk J Hematol 2015;32:285-294
3. Rosenberg SA, Dudley ME. Adoptive cell therapy for the
treatment of patients with metastatic melanoma. Curr Opin
Immunol 2009;21:233-240.
4. Ehrlich P. Über den jetzigen Stand der Karzinomforschung.
Ned Tijdschr Geneeskd 1909;5:273-290 (in German).
5. Kim R, Emi M, Tanabe K. Cancer immunoediting from immune
surveillance to immune escape. J Immunol 2007;121:1-14.
6. Dunn GP, Old LJ, Schreiber RD. The three Es of cancer
immunoediting. Ann Rev Immunol 2004;22:329-360.
7. Chang AE, Shu S. Current status of adoptive immunotherapy
of cancer. Crit Rev Oncol Hematol 1996;22:213-228.
8. Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE,
Old LJ, Schreiber RD. IFNγ and lymphocytes prevent primary
tumour development and shape tumour immunogenicity.
Nature 2001;410:1107-1111.
9. Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA,
Moen R, Karson EM, Lotze MT, Yang JC, Topalian SL, Merino
MJ, Culver K, Miller AD, Blaese RM, Anderson WF. Gene
transfer into humans — immunotherapy of patients with
advanced melanoma, using tumor-infiltrating lymphocytes
modified by retroviral gene transduction. N Eng J Med
1990;323:570-578.
10. Wieczorek A, Uharek L. Genetically modified T cells for the
treatment of malignant disease. Transfus Med Hemother
2013;40:388-402.
11. Jensen MC, Riddell SR. Design and implementation of
adoptive therapy with chimeric antigen receptor-modified T
cells. Immunol Rev 2014;257:127-144.
12. June CH. Principles of adoptive T cell cancer therapy J Clin
Inv 2007;117:1204-1212.
13. Stauss HJ. Immunotherapy with CTLs restricted by nonself
MHC. Immunol Today 1999;20:180-183.
14. Cohen CJ, Li YF, El-Gamil M, Robbins PF, Rosenberg SA,
Morgan RA. Enhanced antitumor activity of T cells engineered
to express T-cell receptors with a second disulfide bond.
Cancer Res 2007;67:3898-3903.
15. Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC,
Hughes MS, Kammula US, Royal RE, Sherry RM, Wunderlich
JR, Lee CC, Restifo NP, Schwarz SL, Cogdill AP, Bishop RJ,
Kim H, Brewer CC, Rudy SF, VanWaes C, Davis JL, Mathur A,
Ripley, RT, Nathan DA, Laurencot CM, Rosenberg SA. Gene
therapy with human and mouse T-cell receptors mediates
cancer regression and targets normal tissues expressing
cognate antigen. Blood 2009;114:535-546.
16. Linette GP, Stadtmauer EA, Maus MV, Rapoport AP, Levine BL,
Emery L, Litzky L, Bagg A, Carreno BM, Cimino PJ, Binder-
Scholl GK, Smethurst DP, Gerry AB, Pumphrey NJ, Bennett
AD, Brewer JE, Dukes J, Harper J, Tayton-Martin HK, Jakobsen
BK, Hassan NJ, Kalos M, June CH. Cardiovascular toxicity and
titin cross-reactivity of affinity- enhanced T cells in myeloma
and melanoma. Blood 2013;122:863-871.
17. Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins
PF, Zheng Z, Dudley ME, Feldman SA, Yang JC, Sherry RM,
Phan GQ, Hughes MS, Kammula US, Miller AD, Hessman
CJ, Stewart AA, Restifo NP, Quezado MM, Alimchandani M,
Rosenberg AZ, Nath A, Wang T, Bielekova B, Wuest SC, Akula
N, McMahon FJ, Wilde S, Mosetter B, Schendel DJ, Laurencot
CM, Rosenberg SA. Cancer regression and neurological
toxicity following anti-MAGE-A3 TCR gene therapy. J
Immunother 2013;36:133-151.
18. Irving BA, Weiss A. The cytoplasmic domain of the T cell
receptor zeta chain is sufficient to couple to receptor-associated
signal transduction pathways. Cell 1991;64:891-901.
19. Mitsuyasu RT, Anton PA, Deeks SG, Scadden DT, Connick
E, Downs MT, Bakker A, Roberts MR, June CH, Jalali S, Lin
AA, Pennathur-Das R, Hege KM. Prolonged survival and
tissue trafficking following adoptive transfer of CD4 z genemodified
autologous CD4+ and CD8+ T cells in HIV-infected
subjects. Blood 2000;96:785-793.
20. Jensen MC, Clarke P, Tan G, Wright C, Chung-Chang W, Clark
TN, Zhang F, Slovak ML, Wu AM, Forman SJ, Raubitschek A.
Human T lymphocyte genetic modification with naked DNA.
Mol Ther 2000;1:49-55.
21. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A,
June CH. T cells with chimeric antigen receptors have potent
antitumor effects and can establish memory in patients with
advanced leukemia. Sci Transl Med 2011;3:95ra73.
22. Chmielewski M, Hombach A, Heuser C, Adams GP, Abken
H. T cell activation by antibody-like immunoreceptors:
increase in affinity of the single-chain fragment domain above
threshold does not increase T cell activation against antigenpositive
target cells but decreases selectivity. J Immunol
2004;173:7647-7653.
23. Zhong XS, Matsushita M, Plotkin J, Riviere I, Sadelain M.
Chimeric antigen receptors combining 4-1BB and CD28
signaling domains augment PI3kinase/AKT/Bcl-XL activation
and CD8+ T cell-mediated tumor eradication. Mol Ther
2010;18:413-420.
24. Du X, Beers R, FitzGerald DJ, Pastan I. Differential cellular
internalization of anti-CD19 and -CD22 immunotoxins results
in different cytotoxic activity. Cancer Res 2008;68:6300-6305.
25. Oren R, Hod-Marco M, Haus-Cohen M, Thomas S, Blat D,
Duvshani N, Denkberg G, Elbaz Y, Benchetrit F, Eshhar Z,
Stauss H, Reiter Y. Functional comparison of engineered
T cells carrying a native TCR versus TCR-like antibodybased
chimeric antigen receptors indicates affinity/avidity
thresholds. J Immunol 2014;193:5733-5743.
291

Turk J Hematol 2015;32:285-294
Ataca P, et al: Chimeric Antigen Receptors in Hematology
26. Ramos CA, Dotti G. Chimeric antigen receptor (CAR)-
engineered lymphocytes for cancer therapy. Expert Opin Biol
Ther 2011;11:855-873.
27. Gattinoni L, Klebanoff CA, Restifo NP. Paths to stemness:
building the ultimate antitumour T cell. Nat Rev Cancer
2012;12:671-684.
28. Stemberger C, Dreher S, Tschulik C, Piossek C, Bet J,
Yamamoto TN, Schiemann M, Neuenhahn M, Martin K,
Schlapschy M, Skerra A, Schmidt T, Edinger M, Riddell SR,
Germeroth L, Busch DH. Novel serial positive enrichment
technology enables clinical multiparameter cell sorting. PLoS
ONE 2012;7: e35798.
29. Lamers CH, van Elzakker P, Langeveld SC, Sleijfer S, Gratama
JW. Process validation and clinical evaluation of a protocol
to generate gene-modified T lymphocytes for imunogene
therapy for metastatic renal cell carcinoma: GMP-controlled
transduction and expansion of. patient’s T lymphocytes using
a carboxy anhydrase IX-specific scFv transgene. Cytotherapy
2006;8:542-553.
30. Lamers CH, van Elzakker P, van Steenbergen SC, Luider BA,
Groot C, van Krimpen BA, Vulto A, Sleijfer S, Debets R, Gratama
JW. Long-term stability of T-cell activation and transduction
components critical to the processing of clinical batches of geneengineered
T cells. Cytotherapy 2013;15:620-626.
31. Sadelain M, Riviere I, Brentjens R. Targeting tumours with
genetically enhanced T lymphocytes. Nat Rev Cancer
2003;3:35-45.
32. Scheurmann RH, Racila E. CD19 antigen in leukemia and
lymphoma diagnosis and immunotherapy. Leuk Lymphoma
1995:18:385-397.
33. Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH,
Geiger TL, Campana D. Chimeric receptors with 4-1BB
signaling capacity provoke potent cytotoxicity against acute
lymphoblastic leukemia. Leukemia 2004;18:676-684.
34. Brentjens RJ, Latouche JB, Santos E, Marti F, Gong MC,
Lyddane C, King PD, Larson S, Weiss M, Rivière I, Sadelain
M. Eradication of systemic B-cell tumors by genetically
targeted human T lymphocytes co-stimulated by CD80 and
interleukin-15. Nat Med 2003;9:279-286.
35. Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan
CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ,
Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22
chimeric antigen receptors targeting B-cell precursor acute
lymphoblastic leukemia. Blood 2013;121:1165-1174.
36. Gasiorowski RE, Clark GJ, Bradstock K, Hart DN. Antibody
therapy for acute myeloid leukemia. Br J Haematol
2014;164:481-495.
37. Pizzitola I. Chimeric antigen receptors against CD33/CD123
antigens efficiently target primary acute myeloid leukemia
cells in vivo. Leukemia 2014;28:1596-1605.
38. Kenderian SS, Ruella M, Shestova O, Klichinsky M, Aikawa
V, Morrissette JJD, Scholler J, Song D, Porter DL, Carroll M,
June CH, Gill S. CD33 specific chimeric antigen receptor T
cells exhibit potent preclinical activity against human acute
myeloid leukemia. Leukemia (in press).
39. Chu J, Deng Y, Benson DM, He S, Hughes T, Zhang J, Peng
Y, Mao H, Yi L, Ghoshal K, He X, Devine SM, Zhang X,
Caligiuri MA, Hofmeister CC, Yu J. CS1-specific chimeric
antigen receptor CAR-engineered natural killer cells enhance
in vitro and in vivo antitumor activity against human multiple
myeloma. Leukemia 2014;28:917-927.
40. Davila ML, Bouhassira DC, Park JH, Curran KJ, Smith EL,
Pegram HJ, Brentjens R. Chimeric antigen receptors for the
adoptive T cell therapy of hematologic malignancies Int J
Hematol 2014;99:361-371.
41. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric
antigen receptor-modified T cells in chronic lymphoid
leukemia. N Engl J Med 2011;365:725-733.
42. Porter DL, Kalos M, Frey NV, Grupp SA, Loren AW, Jemision
C, Gilmore J, McConville H, Capobianchi J, Lledo L, Chew A,
Shen A, Wood PA, Litchman M, Zheng Z, Levine BL, June CH.
Randomized, phase II dose optimization study of chimeric
antigen receptor modified T cells directed against CD19
(CTL019) in patients with relapsed, refractory CLL. Abstract
873. ASH Annual Meeting, New Orleans, LA, USA, 2013.
43. Brown JR, Porter DL, O’Brien SM. Novel treatments for
chronic lymphocytic leukemia and moving forward. Am Soc
Clin Oncol Educ Book 2014:e317-e325.
44. Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K,
Chung SS, Stefanski J, Borquez-Ojeda O, Olszewska M, Qu
J, Wasielewska T, He Q, Fink M, Shinglot H, Youssif M,
Satter M, Wang Y, Hosey J, Quintanilla H, Halton E, Bernal
Y, Bouhassira DC, Arcila ME, Gonen M, Roboz GJ, Maslak
P, Douer D, Frattini MG, Giralt S, Sadelain M, Brentjens R.
Efficacy and toxicity management of 19-28z CAR T cell
therapy in B cell acute lymphoblastic leukemia. Sci Transl
Med 2014;6:224-225.
45. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin
NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD,
Melenhorst JJ, Rheingold SR, Shen A, Teachey DT, Levine BL,
June CH, Porter DL, Grupp SA. Chimeric antigen receptor
T cells for sustained remissions in leukemia. N Eng J Med
2014;371:1507-1517.
46. Till BG, Jensen MC, Wang J, Chen EY, Wood BL, Greisman
HA, Qian X, James SE, Raubitschek A, Forman SJ, Gopal
AK, Pagel JM, Lindgren CG, Greenberg PD, Riddell SR,
Press OW. Adoptive immunotherapy for indolent non-
Hodgkin lymphoma and mantle cell lymphoma using
genetically modified autologous CD20-specific T cells. Blood
2008;112:2261-2271.
292

Ataca P, et al: Chimeric Antigen Receptors in Hematology
Turk J Hematol 2015;32:285-294
47. Kochenderfer JN, Dudley ME, Kassim SH, Somerville RPT,
Carpenter RO, Stetler-Stevenson M, Yang JC, Phan GQ,
Hughes MS, Sherry RM, Raffeld M, Feldman S, Lu L, Li YF,
Ngo LT, Goy A, Feldman T, Spaner DE, Wang ML, Chen
CC, Kranick SM, Nath A, Nathan DN, Morton KE, Toomey
MA, Rosenberg SA. Chemotherapy-refractory diffuse large
B-cell lymphoma and indolent B-Cell malignancies can be
effectively treated with autologous T cells expressing an anti-
CD19 chimeric antigen receptor. J Clin Oncol (in press).
48. Ritchie DS, Neeson PJ, Khot A, Peinert S, Tai T, Tainton
K, Chen K, Shin M, Wall DM, Hönemann D, Gambell P,
Westerman DA, Haurat J, Westwood JA, Scott AM, Kravets L,
Dickinson M, Trapani JA, Smyth MJ, Darcy PK, Kershaw MH,
Prince HM. Persistence and efficacy of second generation CAR
T cell against the LeY antigen in acute myeloid leukemia. Mol
Ther 2013;21:2122-2129.
49. Larson RA, Sievers EL, Stadtmauer EA, Löwenberg B, Estey
EH, Dombret H, Theobald M, Voliotis D, Bennett JM, Richie
M, Leopold LH, Berger MS, Sherman ML, Loken MR, van
Dongen JJ, Bernstein ID, Appelbaum FR. Final report of the
efficacy and safety of gemtuzumab ozogamicin (Mylotarg) in
patients with CD33-positive acute myeloid leukemia in first
recurrence. Cancer 2005;104:1442-1452.
50. Tettamanti S, Biondi A, Biagi E, Bonnet D. CD123 AML
targeting by chimeric antigen receptors: a novel magic bullet
for AML therapeutics? Oncoimmunology 2014;3:e28835.
51. Maus MV, Grupp SA, Porter D, June CH. Antibody-modified T
cells: CARs take the front seat for hematologic malignancies.
Blood 2014;123:2625-2635.
52. Jensen MC, Popplewell L, Cooper LJ, DiGiusto D, Kalos M,
Ostberg JR, Forman SJ. Antitransgene rejection responses
contribute to attenuated persistence of adoptively transferred
CD20/CD19-specific chimeric antigen receptor redirected T
cells in humans. Biol Blood Marrow Transplant 2010;16:1245-
1256.
53. Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ, Carrum
G, Kamble RT, Bollard CM, Gee AP, Mei Z, Liu H, Grilley
B, Rooney CM, Heslop HE, Brenner MK, Dotti G. CD28
costimulation improves expansion and persistence of chimeric
antigen receptor-modified T cells in lymphoma patients. J
Clin Invest 2011;121:1822-1826.
54. Brentjens RJ, Rivière I, Park JH, Davila ML, Wang X, Stefanski
J, Taylor C, Yeh R, Bartido S, Borquez-Ojeda O, Olszewska M,
Bernal Y, Pegram H, Przybylowski M, Hollyman D, Usachenko
Y, Pirraglia D, Hosey J, Santos E, Halton E, Maslak P, Scheinberg
D, Jurcic J, Heaney M, Heller G, Frattini M, Sadelain M. Safety
and persistence of adoptively transferred autologous CD19-
targeted T cells in patients with relapsed or chemotherapy
refractory B-cell leukemias. Blood 2011;118:4817-4828.
55. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric
antigen receptor-modified T cells in chronic lymphoid
leukemia. N Engl J Med 2011;365:725-733.
56. Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-
Stevenson M, Feldman SA, Maric I, Raffeld M, Nathan DA,
Lanier BJ, Morgan RA, Rosenberg SA. Eradication of B-lineage
cells and regression of lymphoma in a patient treated with
autologous T cells genetically engineered to recognize CD19.
Blood 2010;116:4099-4102.
57. Till BG, Jensen MC, Wang J, Qian X, Gopal AK, Maloney DG,
Lindgren CG, Lin Y, Pagel JM, Budde LE, Raubitschek A,
Forman SJ, Greenberg PD, Riddell SR, Press OW. CD20-specific
adoptive immunotherapy for lymphoma using a chimeric
antigen receptor with both CD28 and 4-1BB domains: pilot
clinical trial results. Blood 2012;119:3940-3950.
58. Brentjens R, Davila ML, Riviere I, Park J, Wang X, Cowell LG,
Bartido S, Stefanski J, Taylor C, Olszewska M, Borquez-Ojeda
O, Qu J, Wasielewska T, He Q, Bernal Y, Rijo IV, Hedvat C,
Kobos R, Curran K, Steinherz P, Jurcic J, Rosenblat T, Maslak
P, Frattini M, Sadelain M. CD19-targeted T cells rapidly
induce molecular remissions in adults with chemotherapyrefractory
acute lymphoblastic leukemia. Sci Transl Med
2013;5:177ra138.
59. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL,
Rheingold SR, Teachey DT, Chew A, Hauck B, Wright JF,
Milone MC, Levine BL, June CH. Chimeric antigen receptormodified
T cells for acute lymphoid leukemia. N Engl J Med
2013;368:1509-1518.
60. Cruz CR, Micklethwaite KP, Savoldo B, Ramos CA, Lam S,
Ku S, Diouf O, Liu E, Barrett AJ, Ito S, Shpall EJ, Krance RA,
Kamble RT, Carrum G, Hosing CM, Gee AP, Mei Z, Grilley BJ,
Heslop HE, Rooney CM, Brenner MK, Bollard CM, Dotti G.
Infusion of donor-derived CD19-redirected virus-specific T
cells for B-cell malignancies relapsed after allogeneic stem cell
transplant: a phase 1 study. Blood 2013;122:2965-2973.
61. Kochenderfer JN, Dudley ME, Carpenter RO, Kassim SH, Rose
JJ, Telford WG, Hakim FT, Halverson DC, Fowler DH, Hardy
NM, Mato AR, Hickstein DD, Gea-Banacloche JC, Pavletic
SZ, Sportes C, Maric I, Feldman SA, Hansen BG, Wilder JS,
Blacklock-Schuver B, Jena B, Bishop MR, Gress RE, Rosenberg
SA. Donor-derived CD19-targeted T cells cause regression of
malignancy persisting after allogeneic hematopoietic stem cell
transplantation. Blood 2013;122:4129-4139.
62. Rader RA. (Re)defining biopharmaceutical. Nat Biotechnol
2008;26:743-751.
63. Slaney CY, Kershaw MH, Darcy PK. Trafficking of T cells into
tumors. Cancer Res 2014;74:7168-7174.
293

Turk J Hematol 2015;32:285-294
Ataca P, et al: Chimeric Antigen Receptors in Hematology
64. Lee DW, Gardner R, Porter DL, Louis CU, Ahmed N, Jensen
M, Grupp SA, Mackall CL. Current concepts in the diagnosis
and management of cytokine release syndrome. Blood
2014;124:188-195.
65. Ciceri F, Bonini C, Stanghellini MT, Bondanza A, Traversari C,
Salomoni M, Turchetto L, Colombi S, Bernardi M, Peccatori
J, Pescarollo A, Servida P, Magnani Z, Perna SK, Valtolina V,
Crippa F, Callegaro L, Spoldi E, Crocchiolo R, Fleischhauer K,
Ponzoni M, Vago L, Rossini S, Santoro A, Todisco E, Apperley
J, Olavarria E, Slavin S, Weissinger EM, Ganser A, Stadler M,
Yannaki E, Fassas A, Anagnostopoulos A, Bregni M, Stampino
CG, Bruzzi P, Bordignon C. Infusion of suicide-gene-engineered
donor lymphocytes after family haploidentical haemopoietic
stem-cell transplantation for leukaemia (the TK007 trial): a
non-randomised phase I-II study. Lancet Oncol 2009;10:489-
500.
66. Levine BL, June CH. Perspective: assembly line immunotherapy.
Nature 2013;498:S17.
294

Research Article
DOI: 10.4274/tjh.2014.0174
Turk J Hematol 2015;32:295-303
Possible Role of GADD45γ Methylation in Diffuse Large
B-Cell Lymphoma: Does It Affect the Progression and
Tissue Involvement?
Diffüz Büyük B-Hücreli Lenfomada GADD45γ
Metilasyonunun Olası Rolü: Lenfoma Progresyonunu ve
Doku Tutulumunu Etkiler mi?
İkbal Cansu Barış 1 , Vildan Caner 1 , Nilay Şen Türk 2 , İsmail Sarı 3 , Sibel Hacıoğlu 3 , Mehmet Hilmi Doğu 3 ,
Ozan Çetin 4 , Emre Tepeli 4 , Özge Can 1 , Gülseren Bağcı 1 , Ali Keskin 3
1Pamukkale University Faculty of Medicine, Department of Medical Biology, Denizli, Turkey
2Pamukkale University Faculty of Medicine, Department of Medical Pathology, Denizli, Turkey
3Pamukkale University Faculty of Medicine, Department of Hematology, Denizli, Turkey
4Pamukkale University Faculty of Medicine, Department of Medical Genetics, Denizli, Turkey
Abstract:
Objective: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma among adults and
is characterized by heterogeneous clinical, immunophenotypic, and genetic features. Different mechanisms deregulating cell
cycle and apoptosis play a role in the pathogenesis of DLBCL. Growth arrest DNA damage-inducible 45 (GADD45γ) is an
important gene family involved in these mechanisms. The aims of this study are to determine the frequency of GADD45γ
methylation, to evaluate the correlation between GADD45γ methylation and protein expression, and to investigate the relation
between methylation status and clinicopathologic parameters in DLBCL tissues and reactive lymphoid node tissues from
patients with reactive lymphoid hyperplasia.
Materials and Methods: Thirty-six tissue samples of DLBCL and 40 nonmalignant reactive lymphoid node tissues were
analyzed in this study. Methylation-sensitive high-resolution melting analysis was used for the determination of GADD45γ
methylation status. The GADD45γ protein expression was determined by immunohistochemistry.
Results: GADD45γ methylation was frequent (50.0%) in DLBCL. It was also significantly higher in advanced-stage tumors
compared with early-stage (p=0.041). In contrast, unmethylated GADD45γ was associated with nodal involvement as the
primary anatomical site (p=0.040).
Conclusion: The results of this study show that, in contrast to solid tumors, the frequency of GADD45γ methylation is higher
and this epigenetic alteration of GADD45γ may be associated with progression in DLBCL. In addition, nodal involvement is
more likely to be present in patients with unmethylated GADD45γ.
Keywords: GADD45γ, DNA methylation, Diffuse large B-cell lymphoma
Address for Correspondence: Vildan CANER, M.D.,
Pamukkale University Faculty of Medicine, Department of Medical Biology, Denizli, Turkey
Phone: +90 258 296 24 94 E-mail: vildancaner@yahoo.com, vcaner@pau.edu.tr
Received/Geliş tarihi : May 02, 2014
Accepted/Kabul tarihi : July 08, 2014
295

Turk J Hematol 2015;32:295-303
Barış Cİ, et al: GADD45γ Methylation in Diffuse Large B-Cell Lymphoma
Öz:
Amaç: Diffüz büyük B-hücreli lenfoma (DBBHL) yetişkin bireylerde Hodgkin-dışı lenfomaların en yaygın tipidir ve klinik,
immünofenotipik ve genetik özellikler açısından heterojen özellikler taşıması ile karakterizedir. DBBHL patogenezinde hücre
döngüsü ve apoptoz regülasyonunu bozan farklı mekanizmalar rol oynamaktadır. Growth arrest DNA damage-inducible 45
(GADD45γ), bu mekanizmalarda yer alan önemli bir gen ailesidir. Bu çalışmanın amaçları DBBHL doku örnekleri ve reaktif lenfoid
hiperplazili bireylerin reaktif lenfoid doku örneklerinde GADD45γ metilasyon sıklığını belirlemek, GADD45γ metilasyonu
ile protein ekspresyonu arasındaki ilişkiyi değerlendirmek ve DBBHL olgularında metilasyon durumunun klinikopatolojik
parametrelerle ilişkisini araştırmaktır.
Gereç ve Yöntemler: Bu çalışmada 36 adet DBBHL doku örnekleri ve 40 adet malign-olmayan reaktif lenfoid doku örnekleri
analiz edildi. GADD45γ metilasyon durumunu belirlemek için metilasyona-duyarlı yüksek çözünürlüklü erime eğrisi analizi
kullanıldı. GADD45γ protein ekspresyonu immünohistokimyasal analiz ile belirlendi.
Bulgular: DBBHL’de GADD45γ metilasyonunun sık olduğu belirlendi (%50). Aynı zamanda, erken evre ile karşılaştırıldığında
ileri evre tümörlerde GADD45γ metilasyonu istatistiksel olarak anlamlı düzeyde yüksekti (p=0,041). Ancak, GADD45γ metilasyon
yokluğunun primer anatomik yerleşim olarak nodal tutulumla ilişkili olduğu belirlendi (p=0,040).
Sonuç: Bu çalışmanın sonuçları solid tümörlerin aksine, DBBHL’de GADD45γ metilasyon sıklığının yüksek olduğunu ve
GADD45γ geninde gözlenen bu epigenetik değişimin, hastalığın progresyonu ile ilişkili olabileceğini göstermektedir. Buna ek
olarak, nodal tutulum daha çok GADD45γ metile olmayan olgularda gözlenmektedir.
Anahtar Sözcükler: GADD45γ, DNA metilasyonu, Diffüz büyük B-hücreli lenfoma
Introduction
Diffuse large B-cell lymphoma (DLBCL) is the most
common group of non-Hodgkin lymphomas (NHLs) and
represents 30% to 40% of all newly diagnosed NHLs in Western
countries. DLBCL represents a heterogeneous group of
neoplasms with diversity in clinical presentation, morphology,
and genetic and molecular properties [1]. It is well known
that genetic and epigenetic changes that create a difference
in gene expression profiles between normal and malign B
cells are responsible for the heterogeneity of DLBCL. Genetic
aberrations in DLBCL are chromosomal translocations,
aberrant somatic hypermutations, and copy number variations
including amplifications or deletions [2,3,4,5]. Other
differences come from epigenetic modifications such as DNA
methylation [6,7,8].
DNA methylation may lead to transcriptional silencing by
at least 3 different mechanisms: inhibition of binding of the
transcription factors to their specific sequences, a direct effect
on nucleosome positioning, and recruitment of other nuclear
factors that recognize the methylated CpG dinucleotide
blocks binding other factors including transcription factors
[9]. To date, a number of genes involved in the regulation
of DNA repair, cell cycle control, and apoptosis, such as
MGMT [10,11], DAPK1 [12], and GADD45γ [13], have been
determined as hypermethylated in DLBCL. A recent study
also showed that abnormal methylation patterns might be
seen depending on chromosomal regions, gene density, and
methylation status of neighboring genes in normal B-cell
populations and NHL [8].
The growth arrest DNA damage-inducible (GADD45) gene
family plays important roles in various cell functions such
as DNA repair, cell-cycle control, and cell growth [14]. The
members of the GADD45 gene family, GADD45γ, GADD45γ,
and GADD45γ, are evolutionarily conserved and expressed
in both fetal and adult tissues [15,16,17]. They act as stress
sensors that modulate cellular response against various
physical and environmental stress factors [14,17,18]. It is also
suggested that GADD45 proteins may provide a link between
DNA repair mechanisms and chromatin remodeling [19,20].
Although all 3 proteins have similar functions, these functions
are not identical since they have different activation pathways
depending on cell type and the source of the stress [17,21].
There are very limited data in the literature about the
role of GADD45γ in DLBCL pathogenesis. In this study,
we aimed to show the methylation status and expression
profiles of GADD45γ in DLBCL tissues and nonmalignant
reactive lymphoid node tissues (RLTs). We also focused on
the relationship between GADD45γ methylation status and
clinicopathologic parameters of DLBCL.
Tissue Samples
Materials and Methods
We analyzed 36 DLBCL tissue samples and 40 nonmalignant
RLTs that were diagnosed in the Department of Pathology of
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Turk J Hematol 2015;32:295-303
Pamukkale University between 2009 and 2012. Tissue samples
were collected from all patients before treatment. Based on
Hans’s algorithm, DLBCL cases were classified as germinal
center (GC) and non-GC in the Pathology Department
[22]. All of the patients with DLBCL were also classified by
Ann Arbor stage and International prognostic index (IPI)
score according to the previously described criteria [23,24].
This study was approved by the Institutional Review Board
of Pamukkale University and was in compliance with the
Declaration of Helsinki.
Two consecutive sections of formalin-fixed and paraffinembedded
(FFPE) tissues were used for DNA isolation
and immunohistochemistry (IHC). DNA was isolated
using a commercial kit according to the instructions of
the manufacturer (QIAamp DNA Mini Kit, QIAGEN, the
Netherlands) and IHC was performed using polyclonal
antibody against GADD45γ as described previously [25].
Methylation-Sensitive High-Resolution Melting Analysis
DNA samples underwent bisulfite treatment prior to
methylation-sensitive high-resolution melting (MS-HRM)
analysis by use of a commercial kit (EZ DNA Methylation-
Gold Kit, Zymo Research, USA). Forward and reverse primers
were as follows, respectively: 5’-CGTCGTGTTGAGTTTTGGT
and 5’-TAACCGCGAACTTCTTCCA [26]. The protocol for
identification of the amplicon by MS-HRM analysis is given
in Table 1. For the confirmation of melting temperature (Tm)
degrees in MS-HRM analysis, commercially available control
DNA samples were used (EpiTect Control DNA Set, QIAGEN).
All analyses were performed on a LightCycler 480 instrument
(Roche Diagnostics, Germany).
Immunohistochemistry
All immunostaining procedures including deparaffinization
and antigen retrieval processes were performed automatically
using the BenchMark XT automated stainer (Ventana Medical
Systems, USA). GADD45γ (dilution: 1/200, Bioss Laboratories,
USA) was used as the primary antibody. Larynx squamous cell
carcinoma tissue samples were used as positive controls while
negative controls were treated with the same IHC method by
omitting the primary antibody. Granular cytoplasmic staining
was assessed as positive. Immunohistochemical status of
GADD45γ was scored as 0 (less than 25% positive cells), +
(26% to 50% positive cells), ++ (51% to 75% positive cells), or
+++ (more than 75% positive cells).
Statistical Analysis
The methylation status and protein expression level of
GADD45γ between DLBCL patients and RLT controls was
compared using the chi-square test. The Fisher’ exact test was
used to compare the protein expression and methylation of
GADD45γ. The age-adjusted frequency ratios of GADD45γ
methylation were calculated using multiple logistic regression
analysis. P

Turk J Hematol 2015;32:295-303
Barış Cİ, et al: GADD45γ Methylation in Diffuse Large B-Cell Lymphoma
respectively. The most frequent sites of extranodal involvement
were as follows when the patients were classified according
to the anatomic site of tumor: lung (6 cases, 42.9%), bone
marrow (3 cases, 21.4%), liver (2 cases, 14.3%), and stomach
(2 cases, 14.3%).
GADD45γ Methylation
The Tm was 79±0.5 °C in the methylated region of the
GADD45γ gene while the unmethylated region had a Tm of
76±0.5 °C in MS-HRM analysis, which was also confirmed by
the control DNA samples. According to this finding, GADD45γ
methylation was present in 18 of the DLBCL patients (50%),
whereas 16 (40%) of the controls were methylated (Table 2).
Figure 1 shows the HRM analysis of GADD45γ methylation.
No statistically significant difference was observed between
DLBCL patients and controls in terms of GADD45γ methylation
status (p=0.381). While the mean age was 48.56±22.69
years in the group that had methylated GADD45γ, it was
46.50±25.06 in the unmethylated group (p=0.716). Age status
also did not significantly affect the methylation frequency
of the GADD45γ gene (p=0.407). However, the methylation
frequency in patients with advanced stage (stage 3 and 4)
disease was 17 times higher than in early stages (stage 1 and
2), which was statistically significant (p=0.041). In addition,
there was a difference in the methylation status of GADD45γ
between nodal and extranodal involvement (p=0.040). The
frequency of GADD45γ methylation in the group with high
clinical risk (IPI score 3-4) was 2.6 times higher than that in
the low clinical risk group (IPI score 0-2); however, this was
not statistically significant (p=0.298) (Table 2).
GADD45γ Protein Expression
GADD45γ protein expression was observed to be (0) in 1,
(+) in 18, (++) in 11, and (+++) in 6 of the DLBCL cases. In
controls, the numbers were 8, 30, and 2 for (0), (+), and (++),
respectively. None of the controls were (+++) for GADD45γ
protein expression. Since the numbers of samples in the
subgroups were small, samples were combined for ease of
statistical analysis. While (0) and (+) were regarded as low
protein expression, (++) and (+++) were accepted as high
Table 2. Associations of GADD45γ methylation and protein expression with clinicopathologic parameters in diffuse large
B-cell lymphoma patients.
Clinicopathologic
Parameters
GADD45g Promoter
Methylation
GADD45g Protein
Expression
Absent Present p-value Low High p-value
Total patients 18 (50) 18 (50) 19 (52.8) 17 (47.2)
Sex
Male 7 (50) 7 (50) 1.000 6 (42.9) 8 (57.1) 0.342
Female 11 (50) 11 (50) 13 (59.1) 9 (40.9)
Stage (Ann Arbor)
Early (I/II) 7 (87.5) 1 (12.5) 0.041 5 (62.5) 3 (37.5) 0.695
Advanced (III/IV) 11 (39.3) 17 (60.7) 14 (50) 14 (50)
Tumor location
Nodal 14 (63.6) 8 (36.4) 0.040 13 (59.1) 9 (40.9) 0.342
Extranodal 4 (28.6) 10 (71.4) 6 (42.9) 8 (57.1)
Cell origin
GC 9 (52.9) 8 (47.1) 0.738 11 (64.7) 6 (35.3) 0.175
Non-GC 9 (47.4) 10 (52.6) 8 (42.1) 11 (57.9)
IPI score
0-2 8 (61.5) 5 (38.5) 0.298 6 (46.2) 7 (53.8) 0.549
3-5 10 (43.5) 13 (56.5) 13 (56.5) 10 (43.5)
IPI: International prognostic index, GC: germinal center.
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Turk J Hematol 2015;32:295-303
protein expression (Figure 2). After this grouping, we found
a statistically significant difference between DLBCL patients
and controls (p

Turk J Hematol 2015;32:295-303
Barış Cİ, et al: GADD45γ Methylation in Diffuse Large B-Cell Lymphoma
Table 3. Association between GADD45γ methylation and its protein expression.
Methylated/High
Expression
Methylated/Low
Expression
Unmethylated/
High Expression
DLBCL patients 8 10 9 9
RLT controls 0 16 2 22
Total 8 26 11 31
DLBCL: Diffuse large B-cell lymphoma, RLT: reactive lymphoid node tissue.
Unmethylated/
Low Expression
GADD45γ in DLBCL patients and RLT controls. We detected
GADD45γ methylation in 50.0% of DLBCL patients. MS-HRM
used in this study was performed as previously described
[26]. Zhang et al. found that the HRM protocol had high
sensitivity, which allows the detection of low (1%) amounts
of DNA methylation for GADD45γ [17]. It is well known that
DNA derived from FFPE tissues is often degraded and the
degradation of DNA is highly dependent on the sample age.
In the present study, we used DNA samples extracted from
FFPE tissues ranging in age from 2 to 5 years for MS-HRM.
In a recent study, Kristensen et al. showed that DNA derived
from up to 30-year-old FFPE tissue can be successfully used
for DNA methylation analysis by MS-HRM [28]. Therefore,
we suggest that MS-HRM analysis could be used to detect the
methylation status of GADD45γ in FFPE tissue samples.
In non-small cell lung cancer, Na et al. reported that
GADD45γ methylation was detected in 31.6% of cases.
They also proposed that the silencing of GADD45γ by DNA
methylation might be contributing to the development of
lung cancer [29]. Bahar et al. detected GADD45γ methylation
in 58% of human pituitary adenoma cases [27]. In 82% of
patients whose tumors had no mRNA expression of GADD45γ,
they detected promoter methylation by both methylationspecific
PCR and sodium bisulfite sequencing. Ying et al.
reported the epigenetic inactivation of GADD45γ in primary
samples from various cancer types and tumor cell lines
[13]. In their study, they found that GADD45γ methylation
was more frequent in leukemia and lymphomas (16%-88%)
than solid tumors (11%-16%). In their series, 38% of primary
DLBCL tissues had GADD45γ promoter methylation, which
is concordant with our results. Our results and theirs may be
showing the specificity of GADD45γ methylation according
to the epithelial or mesenchymal origin of tumors. Another
interesting finding of our study was the increasing frequency
of GADD45γ methylation with tumor progression. We found
a significantly higher GADD45γ methylation frequency in
advanced stages than early stages. This may show that the loss
of function in the GADD45γ tumor suppressor gene by DNA
methylation plays a key role in the progression of DLBCL.
It is well known that NHLs arise in different anatomical
sites and they are considered as nodal and extranodal
lymphomas according to the site [30,31]. The differences in
clinical and biological characterizations between nodal and
extranodal involvement are still not clear, as reflected in the
heterogeneous nature of DLBCL in some sense, although
there are a number of studies focused on the differences
between lymphomas at different anatomical sites [32,33,34].
A recent study reported that primary extranodal involvement,
especially at gastrointestinal, pulmonary, and liver/pancreatic
sites, was associated with a worse outcome when compared
to nodal involvement [35]. In our series, the majority of the
patients had nodal involvement, while the remaining patients
had both nodal and extranodal involvement. It was interesting
that nodal involvement was observed in almost 80% of the
patients with no methylated GADD45γ, with significant
statistical difference, although there was no relation between
the tissue involvement and IPI score. This finding may suggest
that GADD45γ methylation status might be an important
factor for the primary site of the lymphoma. Further studies
are needed to identify the genetic and/or epigenetic differences
between nodal and extranodal involvement in DLBCL.
There is no consensus in the literature about the
relationship between GADD45γ methylation status and
protein expression levels. Ying et al. found no GADD45γ
expression in the cell lines with GADD45γ methylation
in their above mentioned study [13]. Bahar et al. found a
significant correlation between GADD45γ methylation and
low protein expression, although there was expression of
GADD45γ transcript in 9% of the patients with GADD45γ
methylation [27]. Furthermore, 18% of patients without
GADD45γ methylation did not have GADD45γ expression,
either. In the present study, we could not find an association
between GADD45γ methylation and protein expression in
51.3% of our cases. This finding may be explained by the
following potential mechanisms: first, the method we used
for the detection of methylation is not a quantitative method
and those cases with GADD45γ expression might have low
methylation levels that are not adequate for gene silencing.
A number of studies have reported no significant association
between protein expression and methylation status in
different genes such as MGMT, DLC1, GATA4, NDK2, and
RARRES1 [29,36]. It has also been reported that a gain of
DNA methylation is not always associated with gene silencing.
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Turk J Hematol 2015;32:295-303
Kulis et al. characterized the DNA methylomes in patients
with chronic lymphocytic leukemia and reported that there
was a significant correlation between gene expression and
DNA methylation levels in 4% of all CpGs [37]. In a study that
identified DNA methylation differences in different human
ethnic groups, it was shown that a gain of DNA methylation
was associated with gene repression and activation in 63.0%
and 37.0% of cases, respectively [38]. Second, our target in
GADD45γ was relatively small because large amplicon sizes
are generally unsuitable for HRM analysis. The GADD45γ
gene has a unique CpG island that contains not only the
promoter region but also exons [13,27]. Searching in the
whole GADD45γ gene should be more accurate to detect the
real methylation status. Third, since GADD45γ mutation was
very rarely detected in primary tumors [13], the inhibition
of expression might be due to other epigenetic mechanisms
than DNA methylation, such as small noncoding RNAs and
histone modifications. Finally, the polyclonal antibody that we
used for IHC due to unavailability of commercial monoclonal
antibody against GADD45γ protein might have cross-reacted
with other epitopes in colocalized protein targets [39].
In summary, we found that the frequency of GADD45γ
methylation in DLBCL was higher than that reported in solid
tumors. We also observed that the frequency of GADD45γ
methylation in advanced stages was significantly higher than
that in early stages. In comparison to nodal DLBCL, GADD45γ
was commonly methylated in extranodal DLBCL. These
findings indicated that the silencing of GADD45γ by DNA
methylation may play a role in the progression and the tissue
involvement of DLBCL. Further studies are needed to evaluate
the role of other members of the GADD45 family and their
partners in DLBCL.
Acknowledgments
This research project was supported by the Scientific
Research Project Unit of Pamukkale University (Project
No. 2012SBE007). The authors thank Dr. Mehmet Zencir
(Department of Public Health, Pamukkale University) for the
statistical analysis.
Ethics Committee Approval: This study was approved by
the Institutional Review Board of Pamukkale University and
was in compliance with the Declaration of Helsinki, Concept:
İkbal Cansu Barış, Vildan Caner, Design: İkbal Cansu Barış,
Vildan Caner, Data Collection or Processing: Nilay Şen Türk,
İsmail Sarı, Sibel Hacıoğlu, Mehmet Hilmi Doğu, Ozan Çetin,
Emre Tepeli, Özge Can, Ali Keskin, Analysis or Interpretation:
İkbal Cansu Barış, Vildan Caner, Nilay Şen Türk, Ozan Çetin,
Emre Tepeli, Özge Can, Gülseren Bağcı, Literature Search:
İkbal Cansu Barış, Vildan Caner, Nilay Şen Türk, İsmail Sarı,
Sibel Hacıoğlu, Writing: İkbal Cansu Barış, Vildan Caner,
Nilay Şen Türk, İsmail Sarı, Sibel Hacıoğlu.
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. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein
H, Thiele J, Vardiman JW. WHO Classification of Tumours of
Haematopoietic and Lymphoid Tissues, 4th ed. Lyon, France,
IARC, 2008.
2. Rosenwald A, Wright G, Leroy K, Yu X, Gaulard P, Gascoyne
RD, Chan WC, Zhao T, Haioun C, Greiner TC, Weisenburger
DD, Lynch JC, Vose J, Armitage JO, Smeland EB, Kvaloy S,
Holte H, Delabie J, Campo E, Montserrat E,Lopez-Guillermo
A, Ott G, Muller-Hermelink HK, Connors JM, Braziel R,
Grogan TM, Fisher RI, Miller TP, LeBlanc M, Chiorazzi M,
Zhao H, Yang L, Powell J, Wilson WH, Jaffe ES, Simon R,
Klausner RD, Staudt LM. Molecular diagnosis of primary
mediastinal B cell lymphoma identifies a clinically favorable
subgroup of diffuse large B cell lymphoma related to Hodgkin
lymphoma. J Exp Med 2003;198:851-862.
3. Savage KJ, Monti S, Kutok JL, Cattoretti G, Neuberg D, De
Leval L, Kurtin P, Dal Cin P, Ladd C, Feuerhake F, Aguiar RC,
Li S, Salles G, Berger F, Jing W, Pinkus GS, Habermann T,
Dalla-Favera R, Harris NL, Aster JC, Golub TR, Shipp MA.
The molecular signature of mediastinal large B-cell lymphoma
differs from that of other diffuse large B-cell lymphomas and
shares features with classical Hodgkin lymphoma. Blood
2003;102:3871-3879.
4. Nogai H, Dörken B, Lenz G. Pathogenesis of non-Hodgkin’s
lymphoma. J Clin Oncol 2011;29:1803-1811.
5. Schneider C, Pasqualucci L, Dalla-Favera R. Molecular
pathogenesis of diffuse large B-cell lymphoma. Semin Diagn
Pathol 2011;28:167-177.
6. Shaknovich R, Melnick A. Epigenetics and B-cell lymphoma.
Curr Opin Hematol 2011;18:293-299.
7. Taylor KH, Briley A, Wang Z, Cheng J, Shi H, Caldwell CW.
Aberrant epigenetic gene regulation in lymphoid malignancies.
Semin Hematol 2013;50:38-47.
8. De S, Shaknovich R, Riester M, Elemento O, Geng H,
Kormaksson M, Jiang Y, Woolcock B, Johnson N, Polo JM,
Cerchietti L, Gascoyne RD, Melnick A, Michor F. Aberration
in DNA methylation in B-cell lymphomas has a complex
origin and increases with disease severity. PLoS Genet
2013;9:e1003137.
9. Espada J, Esteller M. DNA methylation and the functional
organization of the nuclear compartment. Semin Cell Dev
Biol 2010;21:238-246.
301

Turk J Hematol 2015;32:295-303
Barış Cİ, et al: GADD45γ Methylation in Diffuse Large B-Cell Lymphoma
10. Hiraga J, Kinoshita T, Ohno T, Mori N, Ohashi H, Fukami
S, Noda A, Ichikawa A, Naoe T. Promoter hypermethylation
of the DNA-repair gene O6-methylguanine-DNA
methyltransferase and p53 mutation in diffuse large B-cell
lymphoma. Int J Hematol 2006;84:248-255.
11. Türk NŞ, Özsan N, Caner V, Karagenç N, Düzcan F, Düzcan E,
Hekimgil M. Determination of apoptosis, proliferation status
and O6-methylguanine DNA methyltransferase methylation
profiles in different immunophenotypic profiles of diffuse
large B-cell lymphoma. Turk J Hematol 2011;28:15-26.
12. Kristensen LS, Treppendahl MB, Asmar F, Girkov MS, Nielsen
HM, Kjeldsen TE, Ralfkiaer E, Hansen LL, Grønbæk K.
Investigation of MGMT and DAPK1 methylation patterns
in diffuse large B-cell lymphoma using allelic MSPpyrosequencing.
Sci Rep 2013;3:2789.
13. Ying J, Srivastava G, Hsieh WS, Gao Z, Murray P, Liao SK,
Ambinder R, Tao Q. The stress-responsive gene GADD45G
is a functional tumor suppressor, with its response to
environmental stresses frequently disrupted epigenetically in
multiple tumors. Clin Cancer Res 2005;11:6442-6449.
14. Tamura RE, de Vasconcellos JF, Sarkar D, Libermann TA,
Fisher PB, Zerbini LF. GADD45 proteins: central players in
tumorigenesis. Curr Mol Med 2012;12:634-651.
15. Schrag JD, Jiralerspong S, Banville M, Jaramillo ML, O’Connor-
McCourt MD. The crystal structure and dimerization interface
of GADD45gamma. Proc Natl Acad Sci U S A 2008;105:6566-
6571.
16. Kearsey JM, Coates PJ, Prescott AR, Warbrick E, Hall PA.
Gadd45 is a nuclear cell cycle regulated protein which
interacts with p21Cip1. Oncogene 1995;11:1675-1683.
17. Zhang W, Bae I, Krishnaraju K, Azam N, Fan W, Smith K,
Hoffman B, Liebermann DA. CR6: A third member in the
MyD118 and Gadd45 gene family which functions in negative
growth control. Oncogene 1999;18:4899-4907.
18. Tront JS, Hoffman B, Liebermann DA. Gadd45a suppresses
Ras-driven mammary tumorigenesis by activation of c-Jun
NH2-terminal kinase and p38 stress signaling resulting in
apoptosis and senescence. Cancer Res 2006;66:8448-8454.
19. Smith ML, Ford JM, Hollander MC, Bortnick RA, Amundson
SA, Seo YR, Deng CX, Hanawalt PC, Fornace AJ Jr. p53-
mediated DNA repair responses to UV radiation: studies of
mouse cells lacking p53, p21, and/or gadd45 genes. Mol Cell
Biol 2000;20:3705-3714.
20. Niehrs C, Schäfer A. Active DNA demethylation by Gadd45
and DNA repair. Trends Cell Biol 2012;22:220-227.
21. Shaulian E, Karin M. Stress-induced JNK activation
is independent of Gadd45 induction. J Biol Chem
1999;274:29595-29598.
22. Hans CP, Weisenburger DD, Grenier TC, Gascoyne RD,
Delabie J, Ott G, Müller-Hermelink HK, Campo E, Braziel
RM, Jaffe ES, Pan Z, Farinha P, Smith LM, Falini B, Banham
AH, Rosenwald A, Staudt LM, Connors JM, Armitage JO,
Chan WC. Confirmation of the molecular classification of
diffuse large B-cell lymphoma by immunohistochemistry
using a tissue microarray. Blood 2004;103:275-282.
23. Carbone PP, Kaplan HS, Musshoff K, Smithers DW, Tubiana
M. Report of the committee on Hodgkin’s disease staging
classification. Cancer Res 1971;31:1860-1861.
24. No authors listed. A predictive model for aggressive non-
Hodgkin’s lymphoma. The International Non-Hodgkin’s
Lymphoma Prognostic Factors Project. N Engl J Med
1993;329:987-994.
25. Zhu N, Shao Y, Xu L, Yu L, Sun L. Gadd45-α and Gadd45-γ
utilize p38 and JNK signaling pathways to induce cell
cycle G2/M arrest in Hep-G2 hepatoma cells. Mol Biol Rep
2009;36:2075-2085.
26. Zhang W, Li T, Shao Y, Zhang C, Wu Q, Yang H, Zhang J, Guan
M, Yu B, Wan J. Semi-quantitative detection of GADD45-
gamma methylation levels in gastric, colorectal and pancreatic
cancers using methylation-sensitive high-resolution melting
analysis. J Cancer Res Clin Oncol 2010;136:1267-1273.
27. Bahar A, Bicknell JE, Simpson DJ, Clayton RN, Farrell WE.
Loss of expression of the growth inhibitory gene GADD45γ,
in human pituitary adenomas, is associated with CpG island
methylation. Oncogene 2004;23:936-944.
28. Kristensen LS, Wojdacz TK, Thestrup BB, Wiuf C, Hager H,
Hansen LL. Quality assessment of DNA derived from up to
30 years old formalin fixed paraffin embedded (FFPE) tissue
for PCR-based methylation analysis using SMART-MSP and
MS-HRM. BMC Cancer 2009;9:453.
29. Na YK, Lee SM, Hong HS, Kim JB, Park JY, Kim DS.
Hypermethylation of growth arrest DNA-damage-inducible
gene 45 in non-small cell lung cancer and its relationship
with clinicopathologic features. Mol Cells 2010;30:89-92.
30. Harris NL. Mature B-cell neoplasms. In: Jaffe ES, Harris
NL, Stein H, Vardiman JW (eds). Pathology and Genetics:
Tumours of Haematopoietic and Lymphoid Tissues. World
Health Organization Classification of Tumours. Lyon, France,
IARC Press, 2001.
31. Zucca E, Cavalli F. Extranodal Iymphomas. Ann Oncol
2000;11(Suppl 3):219-222.
32. López-Guillermo A, Colomo L, Jiménez M, Bosch F, Villamor
N, Arenillas L, Muntañola A, Montoto S, Giné E, Colomer
D, Beà S, Campo E, Montserrat E. Diffuse large B-cell
lymphoma: clinical and biological characterization and
outcome according to the nodal or extranodal primary origin.
J Clin Oncol 2005;23:2797-2804.
302

Barış Cİ, et al: GADD45γ Methylation in Diffuse Large B-Cell Lymphoma
Turk J Hematol 2015;32:295-303
33. Krol AD, Le Cessie S, Snijder S, Kluin-Nelemans JC, Kluin PM,
Noorduk EM. Waldeyer’s ring lymphomas: a clinical study
from the Comprehensive Cancer Center West population
based NHL registry. Leuk Lymphoma 2001;42:1005-1013.
34. Toda H, Sato Y, Takata K, Orita Y, Asano N, Yoshino T.
Clinicopathologic analysis of localized nasal/paranasal diffuse
large B-cell lymphoma. PLoS One 2013;8:e57677.
35. Castillo JJ, Winer ES, Olszewski AJ. Sites of extranodal
involvement are prognostic in patients with diffuse large
B-cell lymphoma in the rituximab era: an analysis of the
surveillance, epidemiology and end results database. Am J
Hematol 2013;89:310-314.
36. Pike BL, Greiner TC, Wang X, Weisenburger DD, Hsu YH,
Renaud G, Wolfsberg TG, Kim M, Weisenberger DJ, Siegmund
KD, Ye W, Groshen S, Mehrian-Shai R, Delabie J, Chan WC,
Laird PW, Hacia JG. DNA methylation profiles in diffuse large
B-cell lymphoma and their relationship to gene expression
status. Leukemia 2008;22:1035-1043.
37. Kulis M, Heath S, Bibikova M, Queirós AC, Navarro A, Clot
G, Martínez-Trillos A, Castellano G, Brun-Heath I, Pinyol
M,Barberán-Soler S, Papasaikas P, Jares P, Beà S, Rico D, Ecker
S, Rubio M, Royo R, Ho V, Klotzle B, Hernández L,Conde
L, López-Guerra M, Colomer D, Villamor N, Aymerich
M, Rozman M, Bayes M, Gut M, Gelpí JL, Orozco M, Fan
JB, Quesada V, Puente XS, Pisano DG, Valencia A, López-
Guillermo A, Gut I, López-Otín C, Campo E, Martín-Subero
JI. Epigenomic analysis detects widespread gene-body DNA
hypomethylation in chronic lymphocytic leukemia. Nat
Genet 2012;44:1236-1242.
38. Heyn H, Moran S, Hernando-Herraez I, Sayols S, Gomez
A, Sandoval J, Monk D, Hata K, Marques-Bonet T, Wang L,
Esteller M. DNA methylation contributes to natural human
variation. Genome Res 2013;23:1363-1372.
39. Burry RW. Immunocytochemistry: A Practical Guide for
Biomedical Research. New York, Springer, 2010.
303

Research Article
DOI: 10.4274/tjh.2014.0079
Turk J Hematol 2015;32:304-310
Effect of Tumor Necrosis Factor-Alpha on Erythropoietinand
Erythropoietin Receptor-Induced Erythroid Progenitor
Cell Proliferation in β-Thalassemia/Hemoglobin E Patients
β-Talasemi/Hemoglobin E Hastalarında Tümör Nekrozlaştırıcı
Faktör-Alfa’nın Eritropoetin- ve Eritropoetin Reseptör- ile
Uyarılmış Eritroid Öncül Hücre Çoğalması Üzerine Etkisi
Dalina I Tanyong1, Prapaporn Panichob1, Wasinee Kheansaard1, Suthat Fucharoen2
1Mahidol University Faculty of Medical Technology, Department of Clinical Microscopy, Nakhon Pathom, Thailand
2Mahidol University Thalassemia Research Center, Institute of Molecular Biosciences, Nakhon Pathom, Thailand
Abstract:
Objective: Thalassemia is one of the genetic diseases that cause anemia and ineffective erythropoiesis. Increased levels
of several inflammatory cytokines have been reported in β-thalassemia and might contribute to ineffective erythropoiesis.
However, the mechanism by which tumor necrosis factor-alpha (TNF-α) is involved in ineffective erythropoiesis in thalassemic
patients remains unclear. The objective of this study is to investigate the effect of TNF-α on the erythropoietin (EPO) and
erythropoietin receptor (EPOR) expression involved in proliferation of β-thalassemia/hemoglobin (Hb) E erythroid progenitor
cells compared with cells from healthy subjects.
Materials and Methods: CD34-positive cells were isolated from heparinized blood by using the EasySep ® CD34 selection
kit. Cells were then cultured with suitable culture medium in various concentrations of EPO for 14 days. The effect of TNF-α
on percent cell viability was analyzed by trypan blue staining. In addition, the percentage of apoptosis and levels of EPOR
protein were measured by flow cytometry.
Results: Upon EPO treatment, a higher cell number was observed for erythroid progenitor cells from both healthy participants
and β-thalassemia/Hb E patients. However, a reduction of apoptosis was found in EPO-treated cells especially for β-thalassemia/
Hb E patients. Interestingly, TNF-α caused higher levels of cell apoptosis and lower levels of EPOR protein in thalassemic
erythroid progenitor cells.
Conclusion: TNF-α caused a reduction in the level of EPOR protein and EPO-induced erythroid progenitor cell proliferation.
It is possible that TNF-α could be involved in the mechanism of ineffective erythropoiesis in β-thalassemia/Hb E patients.
Keywords: Erythropoietin, β-Thalassemia/hemoglobin E, Apoptosis
Address for Correspondence: Dalina I TANYONG, M.D.,
Mahidol University Faculty of Medical Technology, Department of Clinical Microscopy, Nakhon Pathom, Thailand
E-mail: dalina.itc@mahidol.ac.th
Received/Geliş tarihi : February 21, 2014
Accepted/Kabul tarihi : May 08, 2014
304

Tanyong ID, et al: TNF Inhibited EPO-Induced Erythroid Proliferation
Turk J Hematol 2015;32:304-310
Öz:
Amaç: Talasemi anemi ve inefektif eritropoeze neden olan genetik hastalıklardan birisidir. Enflamatuvar sitokinlerin bir çoğunun
seviyelerinde artma b-talasemide gösterilmiş olup, bu durum inefektif eritropoeze katkıda bulunabilir. Ancak, tümör nekrozlaştırıcı
faktör-alfa’nın (TNF-α) talasemik hastalarda inefektif eritropoeze nasıl bir mekanizma ile neden olduğu bilinmemektedir. Bu
çalışmanın amacı b-talasemi/hemoglobin (Hb) E eritroid öncül hücrelerinde sağlıklı kontrollerin hücreleri ile karşılaştırıldığında
TNF-α’nın eritropoetin (EPO) ve eritropoetin reseptör (EPOR) sunumu üzerine etkisinin araştırılmasıdır.
Gereç ve Yöntemler: CD34-pozitif hücreler EasySep® CD34 seçim kiti yardımı ile heparinli kandan izole edildi. Hücreler 14
gün boyunca uygun kültür ortamında değişik EPO konsantrasyonlarında kültürde bekletildi. TNF-α’nın hücre canlılık yüzdesine
etkisi tripan mavisi boyası ile incelendi. Bunun yanında, apopitoz yüzdesi ve EPOR protein seviyeleri akış sitometrisi ile ölçüldü.
Bulgular: EPO tedavisi ile eritroid öncül hücrelerinin sayısında hem sağlıklı katılımcılarda hem de b-talasemi/Hb E hastalarında
artış olduğu görüldü. Ancak özellikle b-talasemi/Hb E hastalarında EPO ile muamele edilmiş hücrelerde apopitozda azalma
görüldü. İlginç olarak, TNF-α talasemik eritroid öncül hücrelerde hücre apopitoz oranında artmaya ve EPOR protein seviyelerinde
azalmaya neden oldu.
Sonuç: TNF-α EPOR protein düzeyi ve EPO ile uyarılmış eritroid öncül hücre çoğalmasında azalmaya neden oldu. b-talasemia/
Hb E hastalarında TNF-α inefektif eritropoez mekanizmasında yer alıyor olabilir.
Anahtar Sözcükler: Eritropoetin, β-Talasemi/hemoglobin E, Apopitoz
Introduction
Thalassemia is a genetic disease. The major
pathophysiological features include ineffective erythropoiesis
and anemia. In terms of ineffective erythropoiesis, the
mechanism includes increased intramedullary erythroid death
and arrested proliferation of erythroid progenitors, which
plays an important role in β-thalassemia [1]. β-Thalassemia/
hemoglobin (Hb) E is the commonest form in many Asian
countries. In Thailand, the World Health Organization
estimates that at least 100,000 new cases of the disease will
be seen in the next few decades. The pathophysiology is
more complex and the cause of the variability of the severity
remains unknown [2].
Erythropoietin (EPO) is a glycoprotein hormone
required for the survival, proliferation, and differentiation
of committed erythroid progenitor cells. The erythropoietin
receptor (EPOR) belongs to the cytokine receptor superfamily,
which includes receptors for other hematopoietic growth
factors such as interleukins, colony-stimulating factors,
and growth hormone. EPO binds to EPOR and causes the
signaling pathways to control survival and proliferation of
erythroid cells [3]. Survival signaling by EPOR is essential
for erythropoiesis and for its acceleration in hypoxic stress.
Several apparently redundant EPOR survival pathways were
identified in vitro, raising the possibility of their functional
specialization in vivo [4].
One of the most important pathophysiologies of
β-thalassemia is ineffective erythropoiesis. Inflammatory
cytokines such as tumor necrosis factor-alpha (TNF-a) were
reported to inhibit erythropoiesis in vivo and vitro [5]. TNF-a
induces an increase of apoptosis within the compartments of
immature erythroblasts and a decrease in mature erythroblasts.
However, the exact mechanism remains unclear.
The objectives of this study were to study the effect of
TNF-a on EPO and EPOR protein involved in proliferation
of erythroid progenitor cells in β-thalassemia/Hb E patients.
Blood Samples
Materials and Methods
Heparinized blood samples were collected from 5
healthy subjects and 5 β-thalassemia/Hb E patients. The
thalassemia patients in this study had the moderate to severe
type of the disease. They were transfusion-dependent and
splenectomized. However, patients had no transfusions or
iron chelation at least 3 weeks before the time of sampling.
Diagnosis of thalassemia was based on family history, red cell
indices, and hemoglobin typing. The procedures followed
were in accord with the ethical standards established by the
institution at which the experiments were performed or were
in accord with the Helsinki Declaration of 1975.
Hematological Parameters and Erythropoietin Level
Blood cells and red cell indices were analyzed with a Coulter
counter (model ZX6). Hemoglobin typing was performed
by automated high-performance liquid chromatography
(Bio-Rad). EPO level was measured by enzyme-linked
immunosorbent assay (ELISA).
Erythroid Progenitor Cell Culture and TNF-a Treatment
CD34-positive cells (105 cells/mL) were isolated from
peripheral blood mononuclear cells using the EasySep ®
CD34 selection kit, following the manufacturer’s instructions,
305

Turk J Hematol 2015;32:304-310
Tanyong ID, et al: TNF Inhibited EPO-Induced Erythroid Proliferation
and were cultured in Iscove’s modified Dulbecco’s medium
(GIBCO) supplemented with 15% human AB serum, 15%
fetal calf serum in the presence of 10 ng/mL recombinant
interleukin-3, 20 ng/mL stem cell factor, and various
concentrations of EPO (0, 0.2, 2, and 20 U/mL). For TNF-a
treatment, cells were incubated with 20 ng/mL of TNF-α and
incubated at 37 °C in 5% CO 2 for 14 days. CD34-positive cells
were checked by flow cytometry and erythroid progenitor cell
development was observed by Wright-Giemsa staining.
Total Cell and Viability Assay by Trypan Blue Staining
Trypan blue solution was used for cell viability assay.
To determine total cell count and cell viability, 20 µL of
cell suspension was mixed with 20 µL of 0.4% trypan blue
solution. Viable cells and number of total cells were counted
by hemocytometer.
Detection of Percent Apoptosis of Erythroid Progenitor
Cells
Apoptosis was assessed by flow cytometry according to the
manufacturer’s protocol. First, erythroid cultured cells were
washed with 1 mL of cold D-PBS. After centrifugation at 12,000
rpm for 5 min, 100 µL of room-temperature 1X Annexin V
binding buffer was added to the pellet. Next, 2 µL of Annexin
V-FITC and 5 µL of glycophorin A-PE antibody were mixed
into the cell suspension; this mixture was incubated for 15
min in the dark and then 100 µL of 1X Annexin V binding
buffer was again mixed into the cell suspension. Finally, the
cells were analyzed using a FAC Sort flow cytometer (BD
Biosciences, USA). At least 10,000 cells were counted in order
to determine the percentage of apoptosis.
Measuring Erythropoietin Receptor Protein by Flow
Cytometry
Erythroid progenitor cells were cultured for 14 days.
Cells were then incubated with anti-EPOR labeled with FITC
and the percentage of EPOR protein was measured by flow
cytometry.
Statistical Analysis
Results are expressed as mean ± SD. Statistical analysis was
performed using a nonparametric Kolmogorov-Smirnov test
test and Student’s t-test. Significance was set at p

Tanyong ID, et al: TNF Inhibited EPO-Induced Erythroid Proliferation
Turk J Hematol 2015;32:304-310
Role of Erythropoietin on Erythropoietin Receptor
Protein of Erythroid Progenitor Cells
The level of EPOR protein was measured by flow cytometry
and the results showed that the level of EPOR in erythroid
progenitor cells from β-thalassemia/Hb E patients was lower
than in those from healthy subjects. The highest EPOR protein
level was shown in EPO-treated erythroid cells from healthy
subjects at day 5 of culture (Figure 6).
TNF-α Inhibits Erythropoietin Receptor Protein of
Erythroid Progenitor Cells
After adding TNF-a to erythroid progenitor cells treated
with EPO, lower levels of EPOR protein were seen in erythroid
progenitor cells from both healthy subjects and β-thalassemia/
Hb E patients (Figure 7).
Figure 1. Serum erythropoietin levels of healthy control subjects
and beta thalassemia/hemoglobin E patients. *: p

Turk J Hematol 2015;32:304-310
Tanyong ID, et al: TNF Inhibited EPO-Induced Erythroid Proliferation
Discussion
β-Thalassemia/Hb E is a thalassemic syndrome that
results from co-inheritance of the hemoglobin E trait with
either β 0 or β + thalassemia. The severity of the disease is very
variable, ranging from minor through intermediate to major.
Many studies have tried to explain the severity based on
pathophysiological factors such as ineffective erythropoiesis.
Ineffective erythropoiesis is characterized by apoptosis of
the erythroid progenitor cells [6]. Many proteins have the
potential to affect erythroid proliferation and differentiation.
Interestingly, the level of serum EPO in β-thalassemia/Hb E
patients was higher than normal. A previous study reported
that cells become progressively more sensitive to EPO during
erythroid differentiation due to the appearance of EPOR [7].
In this study, the highest EPOR protein levels were seen at day
5 of culture in erythroid progenitor cells from healthy subjects;
the majority of cells were pronormoblasts. In addition, EPOR
protein levels in thalassemic patients were lower than in
healthy subjects. The level of EPOR might be associated with
the stage of erythroid cells. There are reports on the relation of
EPO and EPOR expression in other cells, such as endothelial
cells and head and neck squamous cell carcinoma [8]. In this
study, a reduction of percent cell apoptosis was found in EPOtreated
cells. The percent apoptosis of thalassemic patients was
higher than that of healthy subjects, which might be related to
ineffective erythropoiesis in β-thalassemia/Hb E patients.
Recent studies reported that cytokines could be involved
with ineffective erythropoiesis in β-thalassemia. A previous
study by our group showed that cytokines, including
TNF-α and interferon-γ, had the potential to induce nitric
oxide, involved with apoptosis of erythroid progenitor
cells from β-thalassemia/Hb E patients [9]. However, the
mechanism of TNF-α involved in EPO regulation remains
unclear. TNF-α is one of the proinflammatory cytokines that
reportedly inhibit generation of glycophorin A + cells [10],
and decreased differentiation of erythroid cells exacerbates
ineffective erythropoiesis in b-thalassemia [11]. In addition,
the serum level of TNF-α was statistical significantly higher
in postsplenectomized thalassemic patients than in normal
controls and nonsplenectomized patients, which indicated
that TNF-α could play a role in the pathogenesis of the disease
[12]. One previous study reported that the TNF-α levels of
Figure 5. Effect of tumor necrosis factor-alpha on percent cell
apoptosis of erythroid progenitor cells from healthy control
subjects (a) and β-thalassemia/hemoglobin E patients (b) after
treatment with 2 U and 20 U erythropoietin for 14 days as
analyzed by flow cytometry.
*: p

Tanyong ID, et al: TNF Inhibited EPO-Induced Erythroid Proliferation
Turk J Hematol 2015;32:304-310
b-thalassemia/Hb E patients were higher than normal in
only 13% of the patients [13]. However, many studies have
shown an increased TNF-α concentration in b-thalassemia
major patients [12,14]. It was suggested that the increase
in TNF-α could be caused by macrophage activation due
to iron overload and the antigenic stimulation induced by
chronic transfusion therapy. The activated macrophages were
selectively phagocytosing apoptotic erythroid precursors,
thereby contributing to ineffective erythropoiesis [15]. In this
study it was demonstrated that TNF-α caused higher levels
of apoptosis in b-thalassemia/Hb E erythroid progenitor cells
compared to cells from the control group. In addition, EPOR
protein in erythroid progenitor cells was inhibited by this
cytokine. This suggests that TNF-α caused a reduction of both
EPOR protein expression and EPO-induced cell proliferation
of thalassemic erythroid progenitor cells, which could be
involved in the mechanism of ineffective erythropoiesis in
b-thalassemia/Hb E patients.
Acknowledgments
This work was supported by the Thailand Research Fund
(Grant No. MRG5180127) and by a Mahidol University
Research Grant.
Ethics Committee Approval: COA No. MU-IRB
2009/252.2910, Informed Consent: It was taken, Concept:
Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard,
Suthat Fucharoen, Design: Dalina I Tanyong, Prapaporn
Panichob, Wasinee Kheansaard, Suthat Fucharoen, Data
Collection or Processing: Dalina I Tanyong, Prapaporn
Panichob, Wasinee Kheansaard, Suthat Fucharoen, Analysis
or Interpretation: Dalina I Tanyong, Prapaporn Panichob,
Wasinee Kheansaard, Suthat Fucharoen, Literature Search:
Dalina I Tanyong, Prapaporn Panichob, Wasinee Kheansaard,
Suthat Fucharoen, Writing: Dalina I Tanyong, Prapaporn
Panichob, Wasinee Kheansaard, Suthat Fucharoen.
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
Figure 7. Effect of tumor necrosis factor-alpha on erythropoietin
receptor protein levels of erythroid progenitor cells from healthy
control subjects (a) and β-thalassemia/hemoglobin E patients
(b) after treatment with 2 U and 20 U on day 5 of culture as
measured by flow cytometry.
*: p

Turk J Hematol 2015;32:304-310
Tanyong ID, et al: TNF Inhibited EPO-Induced Erythroid Proliferation
erythropoietin receptor expression to hypoxia and anemia
in head and neck squamous cell carcinoma. Clin Cancer Res
2005;11:7614-7620.
9. Kheansaard W, Panichob P, Fucharoen S, Tanyong DI.
Cytokine-induced apoptosis of beta-thalassemia/hemoglobin
E erythroid progenitor cells via nitric oxide-mediated process
in vitro. Acta Haematol 2011:126;224-230.
10. Xiao W, Koizumi K, Nishio M, Endo T, Osawa M, Fujimoto K,
Sato I, Sakai T, Koike T, Sawada KI. Tumor necrosis factor-α
inhibits generation of glycophorin A+ cell by CD34+ cells.
Exp Hematol 2002;30:1238-1247.
11. Libani IV, Guy EC, Melchiori L, Schiro R, Ramos P, Breda L,
Scholzen T, Chadburn A, Liu Y, Kernbach M, Baron-Lühr B,
Porotto M, de Sousa M, Rachmilewitz EA, Hood JD, Cappellini
MD, Giardina PJ, Grady RW, Gerdes J, Rivella S. Decreased
differentiation of erythroid cells exacerbates ineffective
erythropoiesis in β-thalassemia. Blood 2008;112:875-885.
12. Chuncharunee S, Archararit N, Hathirat P, Udomsubpayakul
U, Atchatakam V. Level of serum interleukin 6 and tumor
necrosis factor in postsplenectomized thalassemic patients. J
Med Assoc Thai 1997;80(Suppl 1):86-91.
13. Wanachiwanawin W, Wiener E, Siripanyaphinyo U,
Chinprasertsuk S, Mawas F, Fucharoen S, Wickramasinghe S,
Pootrakul P, Visudhiphan S. Serum levels of tumor necrosis
factor-alpha, interleukin-1, and interferon-gamma in beta(o)-
thalassemia/Hb E and their clinical significance. J Interferon
Cytokine Res 1999;19:105-111.
14. Lombardi G, Matera R, Minervini MM, Cascavilla N,
D’Arcangelo P, Carotenuto M, Di Giorgio G, Musto P. Serum
levels of cytokines and soluble antigens in polytransfused
patients with beta-thalassemia major: relationship to immune
status. Haematologica 1994;79:406-412.
15. Angelucci E, Bai H, Centis F, Bafti MS, Lucarelli G, Ma L,
Schrier S. Enhanced macrophagic attack on beta-thalassemia
major erythroid precursors. Haematologica 2002;87:578-
583.
310

Research Article
DOI: 10.4274/tjh.2014.0126
Turk J Hematol 2015;32:311-316
The -137G/C Polymorphism in Interleukin-18 Gene
Promoter Contributes to Chronic Lymphocytic and
Chronic Myelogenous Leukemia Risk in Turkish Patients
İnterlökin 18 Geninin Promotör Bölgesindeki -137G/C
Polimorfizmi Türk Popülasyonunda Kronik Lenfositik ve Kronik
Miyeloid Lösemi Riskini Arttırmaktadır
Serap Yalçın 1 , Pelin Mutlu 2 , Türker Çetin 3 , Meral Sarper 4 , Gökhan Özgür 3 , Ferit Avcu 3,4
1Ahi Evran University Faculty of Engineering and Architecture, Kırşehir, Turkey
2Middle East Technical University, Central Laboratory, Department of Molecular Biology and Biotechnology, Ankara, Turkey
3Gülhane Military Medical Academy, Department of Hematology, Ankara, Turkey
4Gülhane Military Medical Academy, Cancer and Stem Cell Research Center, Ankara, Turkey
Abstract:
Objective: Interleukin-18 (IL-18) is a cytokine that belongs to the IL-1 superfamily and is secreted by various immune and
nonimmune cells. Evidence has shown that IL-18 has both anticancer and procancer effects. The aim of this study was to
evaluate the relationship between IL-18 gene polymorphisms and susceptibility to chronic lymphocytic leukemias (CLL) and
chronic myelogenous leukemias (CML) in Turkish patients.
Materials and Methods: The frequencies of polymorphisms (rs61667799(G/T), rs5744227(C/G), rs5744228(A/G), and
rs187238(G/C)) were studied in 20 CLL patients, 30 CML patients, and 30 healthy individuals. The genotyping was performed
by polymerase chain reaction and DNA sequencing analysis.
Results: Significant associations were detected between the IL-18 rs187238(G/C) polymorphism and chronic leukemia. A
higher prevalence of the C allele was found in CML cases with respect to controls. The GC heterozygous and CC homozygous
genotypes were associated with risk of CML when compared with controls. However, prevalence of the C allele was not
significantly high in CLL cases with respect to controls. There was only a significant difference between the homozygous CC
genotype of CLL patients and the control group; thus, it can be concluded that the CC genotype may be associated with the
risk of CLL. Based on our data, there were no significant associations between the IL-18 rs61667799(G/T), rs5744227(C/G),
or rs5744228(A/G) polymorphisms and CLL or CML.
Conclusions: IL-18 gene promoter rs187238(G/C) polymorphism is associated with chronic leukemia in the Turkish
population. However, due to the limited number of studied patients, these are preliminary results that show the association
between -137G/C polymorphism and patients (CLL and CML). Further large-scale studies combined with haplotype and
expression analysis are required to validate the current findings.
Keywords: IL-18, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Single nucleotide polymorphisms
Address for Correspondence: Serap YALÇIN, PhD.,
Ahi Evran University Faculty of Engineering and Architecture, Kırşehir, Turkey
Phone: +90 386 280 38 08 E-mail: syalcin@ahievran.edu.tr
Received/Geliş tarihi : March 23, 2014
Accepted/Kabul tarihi : August 12, 2014
311

Turk J Hematol 2015;32:311-316
Yalçın S, et al: IL-18 Polymorphisms in CML and CLL Patients
Öz:
Amaç: İnterlökin-18 (İL-18), İL-1 süper ailesine ait bir sitokin olup, bağışıklık sistemine ait olan ve olmayan çeşitli hücrelerden
salınmaktadır. Yapılan çalışmalar, İL-18’in hem anti-kanser hem de kansere öncülük eden etkilere sahip olduğunu göstermiştir. Bu
çalışmanın amacı, kronik lenfositik lösemili (KLL) ve kronik miyeloid lösemili (KML) Türk hastalarda İL-18 gen polimorfizmleri
ilişkisini değerlendirmektir.
Gereç ve Yöntemler: İL-18 polimorfizleri (rs61667799(G/T), rs5744227(C/G), rs5744228(A/G) ve rs187238(G/C)), 20
KLL ve 30 KML hasta ve 30 sağlıklı bireyde araştırılmıştır. Genotipleme, polimeraz zincir reaksiyonu ve DNA dizi analizi ile
gerçekleştirilmiştir.
Bulgular: İL-18 geninde, rs187238(G/C) polimorfizmi ile kronik lösemi arasında anlamlı bir ilişki belirlenmiştir. KML
hastalarında kontrol grubuna göre, C allelinin daha yüksek olduğu bulunmuştur. Kontroller ile karşılaştırıldığında, GC heterozigot
ve CC homozigot genotipleri KML hastalarında risk oluşturmaktadır. Ancak, C alleli sıklığı kontrollere göre KLL olgularında
istatistiksel olarak anlamlı değildir. KLL hastaları ve kontrol grubunun homozigot CC genotipi arasında anlamlı farklılık vardır
ve bunun sonucu olarak CC genotipi, KLL hastaları için risk taşımaktadır denilebilir. Verilerimize dayanarak, KLL ve KML
hastalarında, İL-18 geninde rs61667799(G/T), rs5744227(C/G) ve rs5744228(A/G) polimorfizmleri arasında anlamlı bir ilişki
yoktur.
Sonuç: İL-18 geninin promotor bölgesindeki rs187238(G/C) polimorfizmi Türk popülasyonunda kronik lösemi ile ilişkilidir.
Ancak, yapılan bu çalışma, hasta sayısının sınırlı olması nedeniyle, -137G/C polimorfizmi ve hastalar (KLL ve KML) arasındaki
ilişkiyi gösteren bir ön çalışma niteliğindedir. Mevcut bulguları doğrulamak için, haplotip ve gen ifade düzeyi analizleri ile
birleştirilmiş daha geniş çaplı çalışmalara ihtiyaç vardır.
Anahtar Sözcükler: İL-18, Kronik lenfositik lösemi, Kronik miyeloid lösemi, Tek nükleotid polimorfizmi
Introduction
Interleukin-18 (IL-18) is a member of the IL-1 cytokine
family [1]. It is secreted by various immune and nonimmune
cells including T and B lymphocytes, activated monocytes,
macrophages, Kupffer cells, natural killer cells, and
Langerhans cells [2,3,4]. Evidence has shown that IL-18 has
both anticancer and procancer effects [5]. IL-18 can stimulate
natural killer cells and T cells promoting primarily Th1
responses, resulting in the elimination of tumor cells [6,7,8,9].
On the other hand, it has been reported that IL-18 is able to
induce angiogenesis, migration, proliferation, and immune
escape of tumor cells [10]. In models of hepatic melanoma
metastasis the IL-18 blockade reduces the adherence of
malignant cells by preventing IL-18 upregulation of vascular
endothelial adhesion-1 molecule expression [11]. Higher
expression levels of IL-18 are detected in different cancer
types, such as gastric and breast cancer [12,13]. These results
suggest that there is an association between the IL-18 gene
and cancer risk, but this still remains controversial.
The IL-18 gene is located on chromosome 11q22.2-q22.23.
A number of single nucleotide polymorphisms (SNPs) have
been identified and investigated [14]. The IL-18 gene promoter
-137G/C (rs187238) polymorphism is one of the most common
SNPs, relative to the transcriptional start site, which may alter
the expression of IL-18. This polymorphism can change the
binding site of histone 4 transcription factor-1 nuclear factor
and can have an impact on IL-18 gene activity [15].
Chronic myelogenous leukemia (CML) is a clonal bone
marrow stem cell disorder characterized by the unregulated
growth of mature granulocytes in the bone marrow and their
accumulation in the blood [16]. The formation of the BCR-ABL
fusion protein, which activates tyrosine kinase, plays a central
role in the pathogenesis of CML [17]. Chronic lymphocytic
leukemia (CLL) is the most common type of leukemia. CLL
affects B cell lymphocytes that originate in the bone marrow,
develop in the lymph nodes, and normally fight infection by
producing antibodies [18].
It has been reported that malignant proliferation of leukemic
cells is supported by a cytokine network surrounding these cells,
produced partially by the cells themselves [19]. Elevated levels
of IL-18 were observed in some leukemia patients, especially
those with acute lymphoblastic leukemia and CML [20]. On
the other hand, IL-18 receptor expression was reported mostly
from CD19 + B cells and some CD8 + T cells [21].
The aim of this study is to evaluate the frequency of IL-
18 gene promoter polymorphisms in Turkish CLL and CML
patient groups and compare them with a control group in
order to verify a correlation between the allelic variations and
the risk of CML and CLL.
Subjects
Materials and Methods
Twenty unrelated CLL patients and 30 unrelated CML
patients diagnosed clinically at the Gülhane Military Medical
312

Yalçın S, et al: IL-18 Polymorphisms in CML and CLL Patients
Turk J Hematol 2015;32:311-316
Academy Department of Hematology and a control group of
30 unrelated healthy volunteers were randomly selected from
different geographic regions of Turkey. The study protocol was
approved by the local ethics committee of Gülhane Military
Medical Academy and was conducted in accordance with the
guidelines of the Declaration of Helsinki.
Genotyping
The SNP in the promoter region of the IL-18 gene (SNP
*g/c......rs187238; *g/t..... rs61667799; *c/g.......rs5744227;
*a/g....rs5744228) was sequenced was determined by
sequencing method. Genomic DNA was isolated from the
peripheral blood by standard phenol-chloroform procedure.
The genotyping of polymorphisms was performed by
polymerase chain reaction and DNA sequencing analysis.
A 446-bp fragment was amplified using specific primers
(forward: 5’-CCAATAGGACTGATTATTCCGCA-3’ and
reverse: 5’-AGGAGGGCAAAATGCACTGG-3’). Amplification
was carried out on a Bio-RAD PCR system in 50 µL of reaction
mixture containing 10 mM dNTPs, 25 mM magnesium
chloride, 5 pmol each of forward and reverse primers, 2.5 U of
Taq DNA polymerase, 10X PCR buffer, and 50 ng of genomic
DNA. The PCR cycling conditions consisted of an initial
denaturation step at 95 °C for 5 min, followed by 30 cycles of
94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min, with a
final extension step at 72 °C for 5 min. PCR products of 446 bp
were then separated by 2% agarose gel electrophoresis at 120
V, stained by ethidium bromide (0.5 µg/mL), and visualized
under a UV transilluminator. Single-pass sequencing was
performed on each template using the forward primer. Cycle
sequencing was carried out using the BigDye Terminator v.3.1
Cycle Sequencing Kit (Applied Biosystems, USA) according
to the manufacturer’s instructions. The fluorescencelabeled
fragments were purified by sodium acetate-ethanol
precipitation method. Samples were then resuspended in
distilled water and subjected to electrophoresis in an ABI
PRISM 3100 Genetic Analyzer (Applied Biosystems).
Statistical Analysis
SPSS 16.0 (SPSS Inc., USA) was used for the statistical
analysis. Allele and genotype frequencies of alleles and
genotypes were obtained by direct count. Statistical
significance was defined as p

Turk J Hematol 2015;32:311-316
Yalçın S, et al: IL-18 Polymorphisms in CML and CLL Patients
Table 1. Genotype and allele frequencies of the -137G/C polymorphism in the control and chronic myelogenous leukemia groups.
Genotype Control (n=30) CML (n=30) p-value OR (95% CI)
GG 23 (77%) 15 (50%) 0.037 0.3043 (0.1005-0.9218)
GC 7 (23%) 11 (37%) 1.9023 (0.6171-5.8636)
CC 0 (0%) 4 (13%) 10.3585 (0.5326-201.4622)
Allele
G 53 (88%) 41 (68%) 0.032 _
C 7 (12%) 19 (32%)
CML: Chronic myelogenous leukemia.
Table 2. Genotype and allele frequencies of the -137G/C polymorphism in the control and chronic lymphocytic leukemia groups.
Genotype Control (n=30) CLL (n=20) p-value OR (95% CI)
GG 23 (77%) 14 (70%)
0.7101 (0.1981-2.5462)
GC 7 (23%) 2 (10%) 0.027
0.3651 (0.0675-1.9750)
CC 0 (0%) 4 (20%) 16.6364 (0.8428-328.3734)
Allele
G 53 (88%) 30 (75%)
C 7 (12%) 10 (25%)
0.599 _
CLL: Chronic lymphocytic leukemia.
GG genotype, 10% were heterozygous (GC), and 20% were
homozygous for the CC genotype. The G allele frequency was
found as 75% whereas C allele frequency was 25%. Among
the CML patients, 50% were found to be homozygous for the
GG genotype, 37% were heterozygous (GC), and 13% were
homozygous for the CC genotype. The G allele frequency was
found as 68% whereas C allele frequency was 32%.
A higher prevalence of the C allele was found in CML
patients with respect to the controls (p0.05). There was only a
significant difference between the homozygous CC genotype
of CLL patients and the control group (p

Yalçın S, et al: IL-18 Polymorphisms in CML and CLL Patients
Turk J Hematol 2015;32:311-316
several studies that concluded that there was no significant
association between cancer and the -137G/C polymorphism
[5,33,34]. Moreover, in one study, Monroy et al. found a
significantly reduced cancer risk with the GC/CC genotype in
Hodgkin disease [35].
These discrepant conclusions might be explained by ethnic
differences since the studies that reported increased cancer
risk were almost all carried out in Asians. On the contrary, a
trend of reduced cancer risk was found in Caucasians [27].
To our knowledge, there are not many reports describing
a comprehensive relation between -137G/C polymorphism
and susceptibility to CML and CLL. In the present study,
potential influence of the -137G/C polymorphism on both
CLL and CML susceptibility was considered in a Turkish
population. Our results showed a significantly increased
risk in heterozygous (GC) and homozygous (CC) genotypes
for CML. On the other hand, only the homozygous (CC)
genotype is associated with the risk of CLL when compared
with the controls. The results of this study may be important
since there are not many reports showing the association of
the -137G/C polymorphism with the risk of CML and CLL.
However, there are several studies that showed dysregulated
expression of IL-18 and/or IL-18 receptor in chronic B-cell
lymphoproliferative disorders [36,37]. The dysregulated
expression of IL-18 may be due to IL-18 gene promoter
polymorphisms such as -137G/C. In addition, for this study,
several limitations should be considered. First, the CML and
CLL patient numbers were small. Second, haplotype analysis
linking other IL-18 polymorphisms to IL-18 expression level
may be necessary.
In conclusion, we demonstrate that IL-18 gene promoter
-137G/C polymorphism is associated with CLL and CML in
a Turkish population. However, due to the limited number
of studied patients, these are only preliminary results that
show the association between the -137G/C polymorphism
and CLL and CML. Further large-scale studies combined with
haplotype and expression analysis are required to validate the
current findings.
Ethics Committee Approval: Ethics No: 1491-43-12/1648-
4451(GATA) Date: 06/June/2012, Concept: Serap Yalçın,
Pelin Mutlu, Ferit Avcu, Design: Serap Yalçın, Pelin Mutlu,
Ferit Avcu, Data Collection or Processing: Türker Çetin,
Meral Sarper, Gökhan Özgür, Analysis or Interpretation:
Serap Yalçın, Pelin Mutlu, Literature Search: Serap Yalçın,
Pelin Mutlu, Ferit Avcu, Writing: Serap Yalçın, Pelin Mutlu,
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. Dinarello CA. Interleukin-18. Methods 1999;19:121-132.
2. Baxevanis CN, Gritzapis AD, Papamichail M. In vivo antitumor
activity of NKT cells activated by the combination of IL-12
and IL-18. J Immunol 2003;171:2953-2959.
3. Lebel-Binay S, Berger A, Zinzindohoué F, Cugnenc P, Thiounn
N, Fridman WH, Pagès F. Interleukin-18: biological properties
and clinical implications. Eur Cytokine Netw 2000;11:15-26.
4. Tschoeke SK, Oberholzer A, Moldawer LL. Interleukin-18:
a novel prognostic cytokine in bacteria-induced sepsis. Crit
Care Med 2006;34:1225-1233.
5. Liu JM, Liu JN, Wei MT, He YZ, Zhou Y, Song XB, Ying BW,
Huang J. Effect of IL-18 gene promoter polymorphisms on
prostate cancer occurrence and prognosis in Han Chinese
population. Genet Mol Res 2013;12:820-829.
6. Günel N, Coşkun U, Sancak B, Günel U, Hasdemir O, Bozkurt
S. Clinical importance of serum interleukin-18 and nitric oxide
activities in breast carcinoma patients. Cancer 2002;95:663-667.
7. Dinarello CA. IL-18: A TH1-inducing, proinflammatory
cytokine and new member of the IL-1 family. J Allergy Clin
Immunol 1999;103:11-24.
8. Gillies SD, Young D, Lo KM, Roberts S. Biological activity
and in vivo clearance of antitumor antibody/cytokine fusion
proteins. Bioconjug Chem 1993;4:230-235.
9. Okamura H, Tsutsi H, Komatsu T, Yutsudo M, Hakura A,
Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K, Akita
K, Namba M, Tanabe F, Konishi K, Fukuda S, Kurimoto
M. Cloning of a new cytokine that induces IFN-gamma
production by T cells. Nature 1995;378:88-91.
10. Park S, Cheon S, Cho D. The dual effects of interleukin-18 in
tumor progression. Cell Mol Immunol 2007;4:329-335.
11. Dinarello CA. Interleukin-18, a proinflammatory cytokine.
Eur Cytokine Netw 2000;11:483-486.
12. Ye ZB, Ma T, Li H, Jin XL, Xu HM. Expression and significance
of intratumoral interleukin-12 and interleukin-18 in human
gastric carcinoma. World J Gastroenterol 2007;13:1747-
1751.
13. Eisse SA, Zaki SA, El-Maghraby SM, Kadry DY. Importance
of serum IL-18 and RANTES as markers for breast carcinoma
progression. J Egypt Natl Canc Inst 2005;17:51-55.
14. Tsuboi K, Miyazaki T, Nakajima M, Fukai Y, Masuda N, Manda
R, Fukuchi M, Kato H, Kuwano H. Serum interleukin-12
and interleukin-18 levels as a tumor marker in patients with
esophageal carcinoma. Cancer Lett 2004;205:207-214.
15. Gedraitis V, He B, Huang WX, Hillert J. Cloning and mutation
analysis of the human IL-18 promoter: a possible role of
polymorphisms in expression regulation. J Neuroimmunol
2001;112:146-152.
315

Turk J Hematol 2015;32:311-316
Yalçın S, et al: IL-18 Polymorphisms in CML and CLL Patients
16. Besa EC, Buehler B, Markman M, Sacher RA. Chronic
myelogenous leukemia. In: Krishnan K (ed). Medscape
Reference. WebMD. Retrieved 3 January 2014; available at
http://emedicine.medscape.com/article/199425-overview.
17. Yin CC, Abruzzo LV, Qui X, Apostolidou E, Cortes JE, Medeiros
LJ, Lu G. Del(15q) is a recurrent minor-route cytogenetic
abnormality in the clonal evolution of chronic myelogenous
leukemia. Cancer Genet Cytogenet 2009;192:18-23.
18. Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink
HK, Vardiman J, Lister TA, Bloomfield CD. World Health
Organization classification of neoplastic diseases of the
hematopoietic and lymphoid tissues: report of the Clinical
Advisory Committee meeting, Airlie House, Virginia,
November 1997. J Clin Oncol 1999;17:3835-3849.
19. Zhang B, Ma XT, Zheng GG, Li G, Rao Q, Wu KF. Expression
of IL-18 and its receptor in human leukemia cells. Leuk Res
2003;27:813-822.
20. Taniguchi M, Nagaoka K, Ushio S, Nukada Y, Okura T,
Mori T, Yamauchi H, Ohta T, Ikegami H, Kurimoto M.
Establishment of the cells useful for murine interleukin-18
bioassay by introducing murine interleukin-18 receptor
cDNA into human myelomonocytic KG-1 cells. J Immunol
Meth 1998;271:97-102.
21. Kunikata T, Torigoe K, Ushio S, Okura T, Ushio C, Yamauchi
H, Ikeda M, Ikegami H, Kurimoto M. Constitutive and
induced IL-18 receptor expression by various peripheral
blood cell subsets as determined by anti-hIL-18R monoclonal
antibody. Cell Immunol 1998;189:135-143.
22. Kalina U, Ballas K, Koyama N, Kauschat D, Miething C,
Arnemann J, Martin H, Hoelzer D, Ottmann OG. Genomic
organization and regulation of the human interleukin-18
gene. Scand J Immunol 2000;52:525-530.
23. Park H, Byun D, Kim TS, Kim YI, Kang JS, Hahm ES, Kim
SH, Lee WJ, Song HK, Yoon DY, Kang CJ, Lee C, Houh D,
Kim H, Cho B, Kim Y, Yang YH, Min KH, Cho DH. Enhanced
IL-18 expression in common skin tumors. Immunol Lett
2001;79:215-219.
24. Merendino RA, Gangemi S, Ruello A, Bene A, Losi E, Lonbardo
G, Purello-Dambrosio F. Serum levels of interleukin-18 and
sICAM-1 in patients affected by breast cancer: preliminary
considerations. Int J Biol Markers 2001;16:126-129.
25. Bushley AW, Ferrell R, McDuffie K, Terada KY, Carney ME,
Thompson PJ, Wilkens LR, Tung KH, Ness RB, Goodman MT.
Polymorphisms of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-
10, and IL-18 and the risk of ovarian cancer. Gynecol Oncol
2004;95:672-679.
26. Pratesi C, Bortolin MT, Bidoli E, Tedeschi R, Vaccher E,
Dolcetti R, Guidoboni M, Franchin G, Barzan L, Zanussi S,
Caruso C, De Paoli P. Interleukin-10 and interleukin-18
promoter polymorphisms in an Italian cohort of patients with
undifferentiated carcinoma of nasopharyngeal type. Cancer
Immunol Immunother 2006;55:23-30.
27. Yang X, Qui MT, Hu JW, Jiang F, Li M, Wang J, Zhang Q,
Yin R, Xu L. Association of interleukin-18 gene promoter
-607C>A and -137G>C polymorphisms with cancer risk: a
meta-analysis of 26 studies. PLOS ONE 2013;8:e73671.
28. Sobti RC, Shekari M, Tamandani DM, Malekzadeh K, Suri V.
Association of interleukin-18 gene promoter polymorphism
on the risk of cervix carcinogenesis in north Indian population.
Oncol Res 2008;17:159-166.
29. Liu Y, Lin N, Huang L, Xu Q, Pang G. Genetic polymorphisms
of the interleukin-18 gene and risk of prostate cancer. DNA
Cell Biol 2007;26:613-618.
30. Jaiswal PK, Singh V, Srivastava P, Mittal RD. Association
of IL-12, IL-18 variants and serum IL-18 with bladder
cancer susceptibility in North Indian population. Gene
2013;519:128-134.
31. Pan HF, Leng RX, Ye DQ. Lack of association of interleukin-18
gene promoter -607 A/C polymorphism with susceptibility to
autoimmune diseases: a meta-analysis. Lupus 2011;20:945-
951.
32. Guo JY, Qin AQ, Li RK, Yang CM, Huang FD, Huang ZY,
Guo HJ. Association of the IL-18 gene polymorphism with
susceptibility to colorectal cancer. Zhonghua Wei Chang Wai
Ke Za Zhi 2012;15:400-403.
33. Sáenz-López P, Carretero R, Vazquez F, Martin J, Sánchez E,
Tallada M, Garrido F, Cózar JM, Ruiz-Cabello F. Impact of
interleukin-18 polymorphisms -607 and -137 on clinical
characteristics of renal cell carcinoma patients. Hum Immunol
2010;71:309-313.
34. Haghshenas MR, Hosseini SV, Mahmoudi M, Saberi-Firozi M,
Farjadian S, Ghaderi A. IL-18 serum level and IL-18 promoter
gene polymorphism in Iranian patients with gastrointestinal
cancers. J Gastroenterol Hepatol 2009;24:1119-1122.
35. Monroy CM, Cortes AC, Lopez MS, D’Amelio AM Jr, Etzel
CJ, Younes A, Strom SS, El-Zein RA. Hodgkin disease risk:
role of genetic polymorphisms and gene-gene interactions in
inflammation pathway genes. Mol Carcinog 2011;50:36-46.
36. Singer MK, Assem M, Abdel Ghaffar AB, Morcos NY. Cytokine
profiling as a prognostic markers in chronic myeloid leukemia
patients. Egypt J Immunol 2011;18:37-46.
37. Airoldi I, Raffeghello L, Cocco C, Guglielmino R, Roncella S,
Fedeli F, Gambini C, Pistoia V. Heterogeneous expression of
interleukin-18 and its receptor in B-cell lymphoproliferative
disorders deriving from naive, germinal center, and memory
B lymphocytes. Clin Cancer Res 2004;10:144-154.
316

Research Article
DOI: 10.4274/tjh.2014.0154
Turk J Hematol 2015;32:317-322
Transcobalamin II Deficiency in Four Cases with Novel
Mutations
Yeni Mutasyonu Olan Dört Transkobalamin II Eksikliği Olgusu
Şule Ünal 1 , Tony Rupar 2 , Sevgi Yetgin 1 , Neşe Yaralı 3 , Ali Dursun 4 , Türkiz Gürsel 5 , Mualla Çetin 1
1Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey
2Victoria Hospital, London Health Sciences Centre, Biochemical Genetics Laboratory, London, Canada
3Ankara Children’s Hematology and Oncology Hospital, Clinic of Pediatric Hematology, Ankara, Turkey
4Hacettepe University Faculty of Medicine, Division of Metabolism and Nutrition, Ankara, Turkey
5Gazi University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey
Abstract:
Objective: Transcobalamin II deficiency is one of the rare causes of inherited vitamin B12 disorders in which the patients have
characteristically normal or high vitamin B12 levels related to the transport defect of vitamin B12 into the cell, ending up with
intracellular cobalamin depletion and high homocysteine and methylmalonic acid levels.
Materials and Methods: Herein, we describe the findings at presentation of four patients who were diagnosed to have
transcobalamin II deficiency with novel mutations.
Results: These patients with transcobalamin II deficiency were found to have novel mutations, of whom 2 had the same large
deletion (homozygous c.1106+1516-1222+1231del).
Conclusion: Transcobalamin II deficiency should be considered in differential diagnosis of any infant with pancytopenia,
failure to thrive, diarrhea, and vomiting.
Keywords: Vitamin B12, Transcobalamin II, Novel mutation, Novel deletion, Vacuolization
Öz:
Amaç: Transkobalamin II eksikliği nadir bir kalıtsal B12 vitamini bozukluğudur. Defektin B12 vitamininin transportu ile
ilgili olması nedeniyle hastalar normal ya da yüksek B12 vitamini düzeylerine eşlik eden yüksek homosistein ve metilmalonik
asit düzeylerine sahiptir.
Gereç ve Yöntemler: Bu çalışmada transkobalamin II eksikliği tanısı alan dört hasta sunulmuştur. Bu hastalarda daha önce
bildirilmemiş yeni mutasyonlar saptanmıştır.
Bulgular: Hastaların ikisinde aynı büyük delesyon olduğu görülmüştür (homozigot c.1106+1516-1222+1231del).
Sonuç: Pansitopeni, büyüme geriliği, ishal ya da kusması olan tüm bebeklerde transcobalamin II eksikliği ayırıcı tanıda
düşünülmelidir.
Anahtar Sözcükler: B12 vitamini, Transkobalamin II, Yeni mutasyon, Yeni delesyon, Vaküolizasyon
Address for Correspondence: Şule ÜNAL, M.D.,
Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey
Phone: +90 312 305 11 70 E-mail: suleunal@hacettepe.edu.tr
Received/Geliş tarihi : April 13, 2014
Accepted/Kabul tarihi : August 18, 2014
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Ünal Ş, et al: TCII Deficiency with Novel Mutations
Introduction
Among the pancytopenia etiologies during infancy, the
acquired vitamin B12 deficiency in exclusively breast-fed
infants of strictly vegan mothers and inherited vitamin B12
deficiency related to transcobalamin II deficiency should be
considered, since the treatment of both conditions is easy and
possibly life-saving [1,2]. About 30% of plasma cobalamin is
bound to transcobalamin II while the remaining part is bound
to haptocorrin, but only the part of circulating cobalamin
attached to transcobalamin II is the biologically active form
and transcobalamin II mediates the entry of cobalamin
into a variety of cell types other than hepatocytes [3,4,5].
Transcobalamin II deficiency is a rare autosomal recessive
disorder causing intracellular cobalamin depletion, which in
turn causes megaloblastic bone marrow failure, accumulation
of homocysteine and methylmalonic acid with clinical
findings of failure to thrive, diarrhea, vomiting, pancytopenia,
megaloblastic anemia, and neurological findings [2].
Homozygous or compound heterozygous mutations in
the transcobalamin II gene on chromosome 22q12.2 that
contains 9 coding exons are known to cause transcobalamin
II deficiency, including deletions, nonsense mutations, and a
mutation resulting in activation of a cryptic intronic splice site
[6,7,8,9,10,11,12].
Herein, we describe the clinical findings at presentation
and outcome of 4 patients with genetically confirmed novel
transcobalamin II gene mutations, of whom 3 had large
deletions of 1 kb and 1 had a homozygous Q36X mutation.
Materials and Methods
The clinical and laboratory findings of the patients at
presentation are summarized in Table 1. The patients were
further investigated for molecular diagnosis.
Case 1
Results
A 2-month-old girl from the southeastern part of Turkey
presented with failure to thrive (birth weight unknown;
2-month-old weight in 10 th percentile, length in 25 th
percentile, head circumference in 3 rd to 10 th percentiles),
irritability, and diarrhea for the last 20 days and was found to
have pallor, petechial rash, and no head control upon physical
examination. She was the 6 th child of first-degree cousins from
the 8 th gestation, and family history revealed that a sister of
hers had died at 1 year of age with diarrhea and vomiting and a
brother had died at 3.5 months with bleeding. Liver and renal
function tests were unrevealing. Urinalysis revealed absence of
proteinuria. Bone marrow aspiration indicated megaloblastic
changes in the erythroid and myeloid lineages and vacuolization
in the myeloid lineage. Serum vitamin B12 level was found
to be 351 pg/mL (normal range: 200-860); however, serum
homocysteine was 40 µmol/L (normal: 5.5-17) and urinary
methylmalonic acid level was twice the normal value. She was
given erythrocyte and platelet transfusions on the first day of
admission and intramuscular hydroxocobalamin was initiated
at 1000 µg/day with a possible diagnosis of transcobalamin
II deficiency. The hemogram findings on the day of vitamin
B12 treatment initiation were as follows; RBC: 2.6x10 12 /L, Hb:
7.4 g/dL, Hct: 21.3%, MCV: 80 fL, WBC: 3.8x10 9 /L, platelets:
61x10 9 /L, absolute neutrophil count (ANC): 0.3x10 9 /L, and
absolute lymphocyte count (ALC): 3.4x10 9 /L. By the 6 th
day of admission the diarrhea subsided and on the 10 th day
of admission the hemogram results improved to Hb: 8.9 g/
dL, Hct: 24.4%, MCV: 78.5 Fl, WBC: 33.2x10 9 /L, platelets:
125x10 9 /L, and ANC: 22.3x10 9 /L. Leukocytosis developed
in the absence of an infection after the initiation of vitamin
B12 treatment and subsided to the normal range in 2 weeks.
Hydroxocobalamin dosage was continued intramuscularly
on alternating days for the 2 nd week and weekly after the 3 rd
week. Folic acid at 1 mg orally was added to the treatment.
Molecular analyses revealed c.1106+1516-1222+1231del in a
homozygous state, which was a deletion of 5304 bp beginning
1516 bp into intron 7 and ending 1231 bp into intron 8,
causing deletion of all of exon 8 and a frameshift to produce a
premature stop 4 codons into the new reading frame. During
the follow-up, the family was learned to have attempted to
cease the treatment by their own intention and the patient
had similar attacks of pancytopenia and diarrhea. Both attacks
resolved after reinitiation of hydroxocobalamin. The patient
was also detected to have β-thalassemia trait (HbA2 6%)
during outpatient visits due to MCV values as low as 65.2 fL
after initiation of vitamin B12 in the absence of iron deficiency.
She is currently alive and asymptomatic at 4 years of age.
Case 2
A 28-day-old boy from the 1 st gestation of a couple of firstdegree
cousins presented with failure to thrive, poor feeding,
and vomiting. He was from the Central Anatolia region of
Turkey. Hemogram results revealed pancytopenia. Antibiotic
treatment was started empirically. He received transfusions
several times, and the bone marrow examination was
remarkable for megaloblastic changes and vacuolization in
bone marrow precursors. Serum vitamin B12 was 623 pg/mL
(normal: 200-860). Cyanocobalamin (1000 µg) was initiated
intramuscularly with a possible diagnosis of transcobalamin II
deficiency. Signs and symptoms declined after cyanocobalamin
initiation. Folic acid was added to the vitamin B12 treatment.
The control for bone marrow aspiration after vitamin B12
initiation revealed the disappearance of megaloblastic changes
and vacuolization in the myeloid lineage. The molecular
analyses was ordered and revealed c.1107-347_1222+981del in
364. This complex mutation appears to be a 1444-bp deletion
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Ünal Ş, et al: TCII Deficiency with Novel Mutations
Turk J Hematol 2015;32:317-322
Table 1. Clinical and laboratory findings of patients at presentation.
Case 1 Case 2 Case 3 Case 4
Age, sex 2 months, Female 28 days, Male 2 months, Female 3 months, Male
Symptoms at presentation
Failure to thrive,
irritability, diarrhea
Failure to thrive,
vomiting, poor feeding
Diarrhea, vomiting,
fever
Hb (g/dL) 4.8 9.5 4.3 6.5
Hct (%) 13.3 26.6 12.8 18
RBC (x10 12 /L) 1.5 2.44 NA NA
WBC (x10 9 /L) 3.6 2.1 4.7 3.2
MCV (fL) 88 109 93.3 NA
Platelets (x10 9 /L) 8 9 11 30
ANC (x10 9 /L) 0.79 0.08 0.95 NA
ALC (x10 9 /L) 2.5 1.67 4.0 NA
Vitamin B12 (normal:
200-860 pg/mL)
Homocysteine
(normal: 5.5-17 µmol/L)
351 623 Normal 677
40 NA NA 46
Spot urinary MMA analysis Twice normal NA High NA
Bone marrow examination
Genetic analyses
Treatment regimen
currently stable with
Megaloblastic changes
in myeloid and
erythroid lineages,
vacuolization in
myeloid lineage
Homozygous
c.1106+1516-
1222+1231del
Hydroxocobalamin,
1000 µg, im, weekly
Folic acid, oral, 1 mg
Megaloblastic changes
and vacuolization in
bone marrow precursors
c.1107-
347_1222+981delin 364;
this complex mutation
appears to be a 1444-bp
deletion that includes
exon 8 and a 364-bp
insertion
Cyanocobalamin,
1000 µg, im, weekly
Folic acid, oral, 1 mg
Megaloblastic
changes in bone
marrow precursors
Homozygous
c.106C>T. (Q36X)
Cyanocobalamin,
1000 µg, im,
weekly
Folic acid, orally,
1 mg
Failure to thrive,
poor feeding
Megaloblastic
changes in
myeloid lineage
Homozygous
c.1106+1516-
1222+1231del
Cyanocobalamin,
1000 µg, im,
weekly
ANC: Absolute neutrophil count, ALC: absolute lymphocyte count, NA: not available, MMA: methylmalonic acid, im: intramuscular.
that includes exon 8. There was also a 364-bp insertion. He is
currently alive at 6.5 years of age under weekly intramuscular
cyanocobalamin.
Case 3
A 2-month-old girl, from the 1 st gestation of a couple of
first-degree cousins, presented with diarrhea, vomiting, and
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Ünal Ş, et al: TCII Deficiency with Novel Mutations
fever for 1 week. Body weight and length were below the 3 rd
percentile for age. Hemogram results revealed pancytopenia.
Bone marrow examination revealed megaloblastic changes.
Sweat test by pilocarpine iontophoresis was ordered for the
diarrhea and results were positive, with a sweat chloride of 87
mEq/L. Molecular testing for cystic fibrosis for the common
21 mutations in Turkey was negative. Serum vitamin B12
was within normal laboratory limits, whereas urinary
methylmalonic acid level was 16.45 mmol/mol creatinine
(normal: 0-10). Molecular study revealed homozygous
c.106C>T p.Q36X. A C-to-T substitution at nucleotide 106
resulted in a premature stop codon. She was started on
intramuscular cyanocobalamin at 1000 µg/day on the 13 th day
of admission; by the 16 th day of admission, she was discharged
after resolution of symptoms with hemogram findings of
Hb: 10.1 g/dL, WBC: 20x10 9 /L, MCV: 89 fl and platelets:
850x10 9 /L, with continuation of treatment twice weekly.
Oral folic acid was also initiated. The sweat test was repeated
during that period and was normal. She is currently alive at 5
years of age and asymptomatic under weekly cyanocobalamin
treatment.
Case 4
A 3-month-old boy of Turkish origin from Cyprus
presented with failure to thrive and poor feeding. Blood
and bone marrow examination revealed pancytopenia,
hypersegmentation, and megaloblastic changes in the myeloid
lineage. Serum homocysteine and vitamin B12 levels were 46
µmol/L (normal: 5.5-17) and 677 pg/mL (normal: 200-860),
respectively. Cyanocobalamin was initiated intramuscularly
and the pancytopenia resolved. Molecular analyses revealed
c.1106+1516-1222+1231del in a homozygous state. The
mutation was the same as that found in Case 1.
Discussion
Transcobalamin II deficiency is a severe disorder with
intracellular cobalamin depletion [2]. Transcobalamin II
deficiency usually presents with hematological features that
overlap with vitamin B12 deficiency including pancytopenia
and megaloblastic anemia with high serum homocysteine
and methylmalonic acid levels; however, serum vitamin B12
levels are typically normal [13,14,15]. The early initiation
of treatment is very important, since pancytopenia and
gastrointestinal symptoms including vomiting and diarrhea
reverse very soon after treatment, and delay in diagnosis
and treatment may cause morbidities and mortalities
related to pancytopenia including bleeding and infection
in addition to severe and possibly permanent neurological
and retinal impairment [14]. Treatment is suggested as
hydroxocobalamin or cyanocobalamin either orally and twice
weekly or systemically and weekly with high doses of 1000
µg in order to achieve serum cobalamin levels of 1000-10.000
pg/mL, so that cobalamin can be transferred into the cell in
the absence of transcobalamin in such high serum levels [15].
Folic acid may be added to the treatment [15].
In cases 1 and 2, the bone marrow findings of vacuolization
in the myeloid lineage is interesting. Vacuolization is an
important finding in another metabolic disease, namely
Pearson syndrome, that may present with pancytopenia,
megaloblastic anemia during infancy with lactic acidosis, and
exocrine pancreas dysfunction related to a mitochondrial
defect [16]. In the literature, Ratschmann et al. provided the
bone marrow figures of their index patient of 6 weeks old
with transcobalamin II deficiency and described the changes
in the myeloid lineage as dysgranulopoiesis [12]. In those
findings, vacuolization was prominent, similar to our patients
(cases 1 and 2). Vacuolization may be an additional finding
of transcobalamin II deficient patients that may be related to
defect in the mitochondrial DNA synthesis, as well, resulting
from cobalamin deficiency.
Case 1 of the current report had an initial MCV value of 88
fL; after vitamin B12 treatment, the patient had MCV measured
as low as 67.2 fL and was further tested with hemoglobin
electrophoresis. She was found to have β-thalassemia trait.
This indicates that initial MCV values may not be macrocytic in
the presence of β-thalassemia trait; if the clinical presentation
is very suggestive of transcobalamin II deficiency, the normal
MCV values may not preclude the diagnosis. Additionally,
since case 2 was presented at the neonatal stage, MCV was
already macrocytic. These findings may indicate that a normal
MCV for age may not exclude macrocytic anemia etiologies.
Another finding is that in cases 1 and 3, after the initiation
of vitamin B12, hematological improvement occurred with
rapid and dramatic leukocytosis in case 1 and leukocytosis
and thrombocytosis in case 3. In both cases the high counts
normalized in follow-up, but our patients indicate that
initiation of therapy may cause a rapid increase of blood
counts in transcobalamin II deficient patients.
Additionally, case 3 had a transiently high sweat chloride
level that normalized after vitamin B12 treatment. Among the
etiologies that may cause a false-positive sweat chloride test,
transcobalamin II deficiency has not been reported [17,18].
Transcobalamin II deficiency may be one of the causes of falsepositive
sweat tests that has not been previously reported and
this hypothesis may require further support from additional
studies.
In cases 1, 2, and 4, patients were found to have large
deletions, and in case 3 a point mutation was detected,
all of which are reported here as novel findings. Tanner
et al. previously reported the same mutation among their
juvenile cobalamin deficiency patients with GIF mutations
together with Yassin et al. [19,20]. Both of those patients
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Ünal Ş, et al: TCII Deficiency with Novel Mutations
Turk J Hematol 2015;32:317-322
with GIF mutations were of African ancestry, and Tanner
et al. claimed that the mutation might be common in some
African populations through a founder effect [19]. The same
hypothesis may also be true for our patients (cases 1 and 4)
who have the same novel mutation, indicating a common
mutation among the Turkish population.
In conclusion, vitamin B12 deficiency has deleterious
long-term consequences and, differing from nutritional
deficiencies of vitamin B12, patients with transcobalamin II
deficiency are especially responsive to high doses of vitamin
B12 [21]. Transcobalamin II deficiency should be considered
in differential diagnosis of any infant with pancytopenia,
failure to thrive, diarrhea, and vomiting. In patients with
pancytopenia, transcobalamin II deficiency should be
considered in differential diagnosis, especially in countries
with high rates of consanguineous marriages, like Turkey.
Early initiation of high-dose vitamin B12 treatment is very
crucial not only for being potentially life-saving, but also in
order to prevent long-term neurological morbidities.
Acknowledgment
We would like to acknowledge Roger Dewar and Jennifer
Kerkhof for their contributions from the Biochemical Genetics
Laboratory, London Health Sciences Centre, Victoria Hospital,
London, Ontario, Canada.
Ethics Committee Approval: Not applicable, Informed
Consent: Informed consent was obtained from the parents,
Concept: Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali
Dursun, Türkiz Gürsel, Mualla Çetin, Design: Şule Ünal, Tony
Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel,
Mualla Çetin, Data Collection or Processing: Şule Ünal, Tony
Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel,
Mualla Çetin, Analysis or Interpretation: Şule Ünal, Tony
Rupar, Sevgi Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel,
Mualla Çetin, Literature Search: Şule Ünal, Tony Rupar, Sevgi
Yetgin, Neşe Yaralı, Ali Dursun, Türkiz Gürsel, Mualla Çetin,
Writing: Şule Ünal, Tony Rupar, Sevgi Yetgin, Neşe Yaralı, Ali
Dursun, Türkiz Gürsel, Mualla Çetin.
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. Kanra G, Cetin M, Unal S, Haliloglu G, Akça T, Akalan N, Kara
A. Answer to hypotonia: a simple hemogram. J Child Neurol
2005;20:930-931.
2. 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.
3. 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, McGraw Hill, 2001.
4. Oberley MJ, Yang DT. Laboratory testing for cobalamin
deficiency in megaloblastic anemia. Am J Hematol
2013;88:522-526.
5. Meyers PA, Carmel R. Hereditary transcobalamin II
deficiency with subnormal serum cobalamin levels. Pediatrics
1984;74:866-871.
6. Arwert F, Porck HJ, Frater-Schröder M, Brahe C, Geurts
van Kessel A, Westerveld A, Meera Khan P, Zang K, Frants
RR, Kortbeek HE, Erikkson AW. Assignment of human
transcobalamin II (TC2) to chromosome 22 using somatic
cell hybrids and monosomic meningioma cells. Hum Genet
1986;74:378-381.
7. Regec A, Quadros EV, Platica O, Rothenberg SP. The cloning
and characterization of the human transcobalamin II gene.
Blood 1995;85:2711-2719.
8. Li N, Seetharam S, Seetharam B. Genomic structure of
human transcobalamin II: comparison to human intrinsic
factor and transcobalamin I. Biochem Biophys Res Commun
1995;208:756-764.
9. Li N, Rosenblatt DS, Kamen BA, Seetharam S, Seetharam B.
Identification of two mutant alleles of transcobalamin II in an
affected family. Hum Mol Genet 1994;3:1835-1840.
10. Li N, Rosenblatt DS, Seetharam B. Nonsense mutations in
human transcobalamin II deficiency. Biochem Biophys Res
Commun 1994;204:1111-1118.
11. 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
site. Br J Haematol 2003;123:915-920.
12. 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.
13. Hakami N, Neiman PE, Canellos GP, Lazerson J. Neonatal
megaloblastic anemia due to inherited transcobalamin II
deficiency in two siblings. N Engl J Med 1971;285:1163-
1170.
14. Hall CA. The neurologic aspects of transcobalamin II
deficiency. Br J Haematol 1992;80:117-120.
321

Turk J Hematol 2015;32:317-322
Ünal Ş, et al: TCII Deficiency with Novel Mutations
15. Watkins D, Whitehead VM, Rosenblatt D. Megaloblastic
anemia. In: Nathan DG, Orkin SH (eds). Nathan and Oski’s
Hematology of Infancy and Childhood. Philadelphia, WB
Saunders, 2009.
16. Tumino M, Meli C, Farruggia P, La Spina M, Faraci M, Castana
C, Di Raimondo V, Alfano M, Pittalà A, Lo Nigro L, Russo
G, Di Cataldo A. Clinical manifestations and management
of four children with Pearson syndrome. Am J Med Genet A
2011;155:3063-3066.
17. Mishra A, Greaves R, Massie J. The relevance of sweat testing
for the diagnosis of cystic fibrosis in the genomic era. Clin
Biochem Rev 2005;26:135-153.
18. Boat TF, Acton JD. Cystic fibrosis. In: Kliegman RM, Behrman
RE, Jenson HB, Stanton BF (eds). Nelson Textbook of
Pediatrics. Philadelphia, WB Saunders, 2007.
19. Tanner SM, Li Z, Perko JD, Oner C, Cetin M, Altay C,
Yurtsever Z, David KL, Faivre L, Ismail EA, Gräsbeck R, de la
Chapelle A. Hereditary juvenile cobalamin deficiency caused
by mutations in the intrinsic factor gene. Proc Natl Acad Sci
USA 2005;102:4130-4133.
20. Yassin F, Rothenberg SP, Rao S, Gordon MM, Alpers DH,
Quadros EV. Identification of a 4-base deletion in the gene
in inherited intrinsic factor deficiency. Blood 2004;103:1515-
1517.
21. Evim MS, Erdöl Ş, Özdemir Ö, Baytan B, Güneş AM. Longterm
outcome in children with nutritional vitamin B12
deficiency. Turk J Hematol 2011;28:286-293.
322

Research Article
DOI: 10.4274/tjh.2014.0152
Turk J Hematol 2015;32:323-328
Eltrombopag for the Treatment of Immune
Thrombocytopenia: The Aegean Region of Turkey
Experience
İmmün Trombositopeni Tedavisinde Eltrombopag: Türkiye Ege
Bölgesi Deneyimi
Füsun Özdemirkıran1, Bahriye Payzın 1 , H. Demet Kiper 2 , Sibel Kabukçu 3 , Gülsüm Akgün Çağlıyan 4 ,
Selda Kahraman 5 , Ömür Gökmen Sevindik 6 , Cengiz Ceylan 7 , Gürhan Kadıköylü 8 , Fahri Şahin 2 , Ali Keskin 3 ,
Öykü Arslan 4 , Mehmet Ali Özcan 6 , Gülnur Görgün 7 , Zahit Bolaman 8 , Filiz Büyükkeçeci 2 , Oktay Bilgir 4 ,
İnci Alacacıoğlu 6 , Filiz Vural 2 , Murat Tombuloğlu 2 , Zafer Gökgöz 2 , Güray Saydam2
1Katip Çelebi University Faculty of Medicine, Atatürk Research and Education Hospital, Clinic of Hematology, İzmir, Turkey
2Ege University Faculty of Medicine, Department of Hematology, İzmir, Turkey
3Pamukkale University Faculty of Medicine, Department of Hematology, Denizli, Turkey
4Bozyaka Research and Education Hospital, Clinic of Hematology, İzmir, Turkey
5Aydın State Hospital, Clinic of Hematology, Aydın, Turkey
6Dokuz Eylül University Faculty of Medicine, Department of Hematology, İzmir, Turkey
7Tepecik Research and Education Hospital, Clinic of Hematology, İzmir, Turkey
8Adnan Menderes University Faculty of Medicine, Department of Hematology, Aydın, Turkey
Abstract:
Objective: Immune thrombocytopenia (ITP) is an immune-mediated disease characterized by transient or persistent decrease
of the platelet count to less than 100x10 9 /L. Although it is included in a benign disease group, bleeding complications may
be mortal. With a better understanding of the pathophysiology of the disease, thrombopoietin receptor agonists, which came
into use in recent years, seem to be an effective option in the treatment of resistant cases. This study aimed to retrospectively
assess the efficacy, long-term safety, and tolerability of eltrombopag in Turkish patients with chronic ITP in the Aegean region
of Turkey.
Materials and Methods: Retrospective data of 40 patients with refractory ITP who were treated with eltrombopag in the
Aegean region were examined and evaluated.
Results: The total rate of response was 87%, and the median duration of response defined as the number of the platelets
being over 50x10 9 /L was 19.5 (interquartile range: 5-60) days. In one patient, venous sinus thrombosis was observed with no
other additional risk factors due to or related to thrombosis. Another patient with complete response and irregular follow-up
for 12 months was lost due to sudden death as the result of probable acute myocardial infarction.
Conclusion: Although the responses to eltrombopag were satisfactory, patients need to be monitored closely for overshooting
platelet counts as well as thromboembolic events.
Keywords: Immune thrombocytopenia, Thrombopoietin receptor agonist, Bleeding, Eltrombopag
Address for Correspondence: Füsun ÖZDEMİRKIRAN, M.D.,
Katip Çelebi University Faculty of Medicine, Atatürk Research and Education Hospital, Clinic of Hematology, İzmir, Turkey
E-mail: fusun75@gmail.com
Received/Geliş tarihi : April 12, 2014
Accepted/Kabul tarihi : August 14, 2014
323

Turk J Hematol 2015;32:323-328
Özdemirkıran F, et al: Eltrombopag for the Treatment of Immune Thrombocytopenia
Öz:
Amaç: İmmün trombositopeni (İTP), trombositlerin immün aracılı yıkım ile kalıcı veya geçici olarak 100x10 9 /L altında olduğu
bir hastalıktır. Selim hematolojik hastalıklar içinde yer almasına rağmen kanama komplikasyonları ölümcül olabilir. Hastalığın
patofizyolojisi daha iyi anlaşılması ile son yıllarda kullanıma giren trombopoetin reseptör agonistleri, dirençli hastaların
tedavisinde etkili bir seçenek olarak görünmektedir.
Gereç ve Yöntemler: Bu çalışmada Ege Bölgesi’nde refrakter İTP tanısı ile eltrombopag ile tedavi edilen 8 farklı merkezden 40
hastanın retrospektif verileri incelenmiş ve değerlendirilmiştir.
Bulgular: Çalışmada toplam yanıt oranı %87 idi ve trombositlerin 50x10 9 /L’nin üzerine çıktığı medyan süre 19,5 (5-60) gün
saptandı. Tromboz için başka hiçbir ek risk faktörü bulunmayan bir hastada venöz sinüs trombozu gözlendi. On iki aydır tam yanıtlı
izlenen ve düzensiz takibe gelen bir diğer hasta olası akut miyokard infarktüsü sonucu gelişen ani ölüm nedeni ile kaybedildi.
Sonuç: Her ne kadar eltrombopag yanıtları tatmin edici olsa da, hızlı ilerleyen trombositemiye bağlı gelişecek tromboembolik
olaylar açısından, yakın takip ve monitorizasyon gereklidir.
Anahtar Sözcükler: İmmün trombositopeni, Trombopoetin reseptor agonisti, Kanama, Eltrombopag
Introduction
Immune thrombocytopenia (ITP) is an acquired
autoimmune disease in which antiplatelet antibodies accelerate
the destruction of platelets in the reticuloendothelial system
and is characterized by impaired platelet production, resulting
in low platelet counts [1]. Among adults, about 50 new cases
of ITP per million are diagnosed per year [2]. In adults, the
course of the disease is commonly chronic. The primary goal
of treatment is to prevent bleeding by increasing the platelet
count to a stable level while managing the few treatment-related
toxic effects. Current guidelines suggest that treatment should
only be considered in symptomatic patients with platelet
counts of less than 30x10 9 /L. Treatment is rarely indicated
for patients with platelets of >50x10 9 /L in the absence of
bleeding or predisposing comorbid conditions [1,3]. The firstline
treatment for ITP is glucocorticosteroids. For patients
who are actively bleeding or who have a contraindication
to glucocorticosteroids, intravenous immunoglobulin or
anti-D globulin can be used [4]. These drugs increase
platelet counts primarily by reducing the extent of platelet
destruction by several different mechanisms. In the case of
glucocorticosteroid treatment failure, splenectomy is the main
second-line therapy and induces a 70%-80% response rate [5].
Until recently, in patients who were refractory to or relapsing
after splenectomy or when splenectomy was contraindicated,
a variety of immunosuppressive or cytotoxic drugs (such as
vincristine, cyclophosphamide, azathioprine, cyclosporine
A, and rituximab) were common as the third-line therapy.
However, almost 30% of adults with ITP fail to respond to
these therapies and eventually develop a chronic refractory
disease [2,6,7]. All of these treatments mainly reduce
destruction of antibody-coated platelets; however, treatment
is not always effective and can be restricted by adverse effects.
ITP is often considered as benign disorder, but health-related
quality of life is poor. Most of the treatment strategies, such as
glucocorticosteroids and immunosuppressive drugs, adversely
affect quality of life. In recent years, a better understanding of
the pathophysiology of ITP has demonstrated the impaired
thrombopoiesis and has led to the development of new
therapeutic approaches. A new approach to the treatment of
ITP is based on platelet production rather than destruction of
platelets. Eltrombopag is an oral, nonpeptide, thrombopoietin
receptor (TPO-R) agonist, approved in several countries for
the treatment of chronic ITP. Eltrombopag increases platelet
production by interacting with the transmembrane domain of
the TPO-R and inducing proliferation and differentiation of
bone marrow progenitor cells in the megakaryocyte lineage
[8,9]. It can be prescribed in Turkey since November 2011.
In this study we aimed to retrospectively assess the efficacy,
long-term safety, and tolerability of eltrombopag in Turkish
patients with chronic ITP in the Aegean region of Turkey.
Materials and Methods
This study was designed as a retrospective study. A total of
40 patients who received eltrombopag treatment for refractory
chronic ITP at 8 different centers in the Aegean region of
Turkey were included.
ITP diagnosis was verified according to the International
Consensus Report on the Investigation and Management of
Primary ITP [1]. Primary ITP requires only the finding of
isolated thrombocytopenia (100x10 9 /L) with no obvious
associated medical condition [1]. Patients were aged 18
years and older and had primary ITP of more than 6 months’
duration, had baseline platelet counts of lower than 30,000/
μL, and had relapsed after one or more previous treatments for
their disorder. The form prepared for the study was sent to all
centers. Date of the first diagnosis of the patients, demographic
data, time to splenectomy, previous treatments and response
to treatments, side effects, posttreatment follow-up period,
and other such records were retrospectively evaluated. The
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Özdemirkıran F, et al: Eltrombopag for the Treatment of Immune Thrombocytopenia
Turk J Hematol 2015;32:323-328
most recent patient data were recorded in December 2013.
Bleeding was assessed with the World Health Organization
bleeding scale (grade 0: no bleeding, grade 1: petechiae,
grade 2: mild blood loss, grade 3: gross blood loss, grade 4:
debilitating blood loss) [10]. Response rates were defined
as follows: complete response when the platelet count was
100x10 9 /L, partial response when the platelet count ranged
between 30 and 100x10 9 /L with at least a 2-fold increase in
the initial platelet count, and no response when the platelet
count was 30x10 9 /L [3].
Statistical Analysis
Statistical analysis was performed using SPSS 18.0 (SPSS
Inc., Chicago, IL, USA). The Kolmogorov-Smirnov test was
used to evaluate the distribution of data. Data with normal
distribution were reported as mean ± standard deviation (SD),
while data with nonnormal distribution and nonparametric
data were reported as medians (interquartile ranges, 25%-
75%). To evaluate effect of baseline platelet counts on treatment
by eltrombopag, the Mann-Whitney U test was used. For
comparison of categorical variables, Pearson’s chi-square test
was used, or in the case of small frequencies, Fisher’s exact
test was used. Statistical significance was defined as p

Turk J Hematol 2015;32:323-328
Özdemirkıran F, et al: Eltrombopag for the Treatment of Immune Thrombocytopenia
patients having had splenectomy and those who had not were
compared by chi-square test. No difference was determined
between the patients with and without splenectomy in their
response to eltrombopag treatment (p=0.370).
In the 21 cases in which bone marrow biopsy was done
prior to treatment, bone marrow reticulin was evaluated in 2
cases as 2, in 3 cases as 1, and in 16 cases as 0. During treatment,
none of the patients showed any clinical or laboratory findings
suggesting increased bone marrow reticulin and bone marrow
biopsy was not repeated. Adverse effects due to treatment are
summarized in Table 3. Of the cases with response to treatment,
drug-related nausea developed in 2 cases and headache in 4
cases. However, drug use was continued and these adverse
effects vanished in a few weeks after the beginning of the
treatment. Platelet count was below 50x10 9 /L on the 7 th day
of treatment in a case in which erythromelalgia developed,
whereas on the 13 th day it reached 580x10 9 /L. However,
it receded back to the baseline value about 2 weeks after
termination of drug use. The patient was suggested to start the
drug again with a lower dose but refused the treatment. One
male patient at the age of 35, having venous sinus thrombosis
and showing no other additional risk factors from the point of
view of thrombosis, was receiving 50 mg eltrombopag and had
a platelet value on the 15 th day of 680x10 9 /L. Treatment was
terminated. Platelet counts receded back to below 10x10 9 /L
in 15 days; due to recurrent epistaxis and intraoral bleedings,
Table 1. Baseline characteristics of the patients.
Sex (Female/Male) 30/10
Age (years) 46.82±16.35
Baseline platelet count x10 9 /L 11.5±8.3
Final platelet count x10 9 /L 204.8±18.5
Period from diagnosis to
splenectomy (months)
Number of previous
treatments
Results are given as mean ± standard deviation.
21±36
3 (interquartile range: 3-4)
treatment was resumed with dose regulation and no thrombotic
attack was observed.
In another patient, treatment was stopped due to an
increase in transaminases. Transaminase levels were all
in normal ranges prior to eltrombopag therapy. Alanine
transaminase (ALT) and aspartate transaminase (AST) levels
of this patient had gradually increased while she was on
eltrombopag. After the ALT level had reached up to 3 times
the upper normal level, eltrombopag was stopped with a
presumptive diagnosis of toxic hepatitis possibly related
to eltrombopag. In order to clarify the etiology of elevated
transaminases and to be certain about whether this coincidence
was a side effect of eltrombopag or was another
concomitant disease, hepatitis serology and autoimmune tests
were applied. Serology results were all negative considering
hepatitis A, hepatitis B, and hepatitis C. Antimitochondrial
antibody was positive with a titer of 1/1000. Liver biopsy was
applied for further clarification of ongoing transaminitis and
it revealed autoimmune hepatitis. The elevated transaminases
were therefore not considered as a side effect of the drug,
rather being considered as an independent concomitant
autoimmune disorder. After proper treatment of autoimmune
hepatitis with steroids and azathioprine, transaminase levels
decreased to normal ranges. At the same time, platelets counts
were at a steady level between 50,000 and 70,000/µL with the
aforementioned immunosuppressive therapy and eltrombopag
was not reinitiated.
One of the patients with complete response who was
followed irregularly for 12 months was lost due to sudden
death as a result of probable acute myocardial infarction. In
the laboratory tests performed on the same day, the patient’s
platelets were measured as 120x10 9 /L. Two different patients
who both had complete response at the beginning of treatment
but whose platelet counts decreased below 10x10 9 /L in the 8 th
month and 1 st year of treatment were found to be taking iron
supplements and calcium supplements, respectively. These
patients were warned about drug and diet interactions, and
their platelet counts increased again to above 100x10 9 /L in
further follow-up.
Table 2. Outcomes of the treatment.
Total rate of response n=34 (87%)
Complete response n=24 (60%)
Partial response n=11 (27%)
No response n=5 (13%)
Number of days with of platelet counts above 50,000 (median) 19.5 (interquartile range: 5-60)
Duration of eltrombopag treatment (months) 13.78±7.51
Posttreatment mean platelet count (n=35) 204,771
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Özdemirkıran F, et al: Eltrombopag for the Treatment of Immune Thrombocytopenia
Turk J Hematol 2015;32:323-328
Table 3. Adverse effects and toxicity of treatment.
Venous sinus thrombosis
Headache
Nausea
Erythromelalgia
Acute coronary syndrome, sudden death
Target platelet counts after therapy should be between
50 and 100x10 9 /L, not normalization. In 2 patients, despite
platelet counts of 30 to 35x10 9 /L, treatment continued with
partial response since bleeding symptoms were controlled.
One patient for whom 4 different treatment options had
been previously applied with no response, and who was
progressing with intraoral bleedings recurring frequently,
received a splenectomy in the 2nd month of eltrombopag
treatment while platelet counts were over 100x10 9 /L. The
patient then started follow-up with complete response without
treatment. Treatment doses in responding patients are given in
Figure 3. In the patients with response to treatment, average
follow-up period was evaluated as 13.78±7.51 months.
Discussion
1 case
4 cases
2 cases
1 case
1 case
In this retrospective study, we have evaluated the longterm
safety, efficacy, and tolerability of eltrombopag use on
Turkish patients with chronic ITP. In this study in which the
data of 40 patients were evaluated retrospectively, the total rate
of response is 87%, where various different treatment options
such as steroids, anti-D globulin, splenectomy, intravenous
immunoglobulin, azathioprine, cyclophosphamide, danazol,
vincristine, and rituximab were applied with no response
prior to eltrombopag treatment. This rate was similar to the
rate of 80% obtained in the study of Katsutani et al., where
3 years of eltrombopag data from 19 patients were evaluated,
and to the rate of 69.6% obtained in the study of Tomiyama
et al. including 23 patients with a placebo control [11,12].
In the present study, there was no difference between the
response rates of patients with and without splenectomy,
in accordance with the literature [13,14]. Although the
responses were satisfactory, patients need to be monitored
closely regarding rapidly progressing thrombocythemia as
well as thromboembolic events.
Treatment was generally well tolerated and continued,
except for a patient who developed erythromelalgia in the
1st month of therapy and another patient who developed
autoimmune hepatitis in the 6 th month. While eltrombopag
is known to have the ability of increasing transaminases
[15], treatment was terminated in the patient who developed
autoimmune hepatitis. However, during follow-up, no decrease
in transaminases occurred despite discontinuing the drug.
This situation was thus regarded as a concomitant disease.
The common side effects of eltrombopag treatment,
headache and nausea, did not cause any termination in the
treatment and disappeared spontaneously over time. In
the literature, the incidence of thromboembolic events was
reported as 2%-4% during treatment with TPO-R agonists;
however, the rate of only 1 patient out of 40 having sinus vein
thrombosis was consistent with the literature [16]. We could
not obtain detailed information about the patient who was lost
to acute myocardial infarction in the 12th month of treatment
while being monitored with full response.
TPO-R agonists may increase the risk of developing or
progressing reticulin fiber deposition in the bone marrow
[17]. For patients on eltrombopag, peripheral blood smears
should be examined for morphological abnormalities such as
teardrop cells, nucleated red blood cells, leukoerythroblastic
pictures, dysplastic changes, or cytopenia [18]. If such
abnormalities develop or deteriorate, a bone marrow biopsy
should be performed. A loss of response or failure to maintain
a platelet response with eltrombopag treatment within the
recommended dosing range should also prompt a search for
causative factors such as myelofibrosis [18]. In our study, no
patients displayed suggestive clinical or laboratory findings
of significant increases in bone marrow reticulin during
treatment and bone marrow biopsy was not repeated. The
average follow-up period in the patients who responded to
treatment of 13.78±7.51 months was satisfactory; on the other
hand, close monitoring is recommended for thrombocythemia
and thromboembolic events. In particular, patients who begin
treatment should be informed in this regard in detail, and this
therapy is not recommended for cases that cannot be followed
closely. A decrease in eltrombopag dosage is recommended
when platelet counts exceed 200x10 9 /L and should be
completely stopped if platelet count is over 400x10 9 /L. After
discontinuation due to thrombocythemia or any other adverse
effects, patients should be monitored to detect any transient
decrease in platelet counts and to decide about further
treatment indication and dose. In the case of response loss
during follow-up in patients with an initial response, dietdrug
interactions must be questioned in detail.
Acknowledgments
We are grateful to all of the following centers for their
contributions by sharing their data and all the staff of these
centers for their contributions in preparation of the data: Katip
Çelebi University Atatürk Research and Education Hospital,
Department of Hematology, İzmir; Ege University Medical
Faculty, Department of Hematology, İzmir; Pamukkale
University Medical Faculty, Department of Hematology,
Denizli; Bozyaka Research and Education Hospital,
Department of Hematology, İzmir; Aydın State Hospital,
Aydın; Dokuz Eylül University Medical Faculty, Department of
327

Turk J Hematol 2015;32:323-328
Özdemirkıran F, et al: Eltrombopag for the Treatment of Immune Thrombocytopenia
Hematology, İzmir; Tepecik Research and Education Hospital,
Department of Hematology, İzmir; Adnan Menderes University
Medical Faculty, Department of Hematology, Aydın. We are
also grateful to Dr. Mehmet Çalan, who worked diligently in
preparation of the display of statistical data and the graphics.
Ethics Committee Approval: In our center, ethics
committee approval is not required for retrospective studies,
Concept: Füsun Özdemirkıran, Design: Bahriye Payzın,
Data Collection or Processing: H. Demet Kiper, Sibel
Kabukçu, Gülsüm Akgün Çağlıyan, Selda Kahraman, Ömür
Gökmen Sevindik, Cengiz Ceylan, Gürhan Kadıköylü, Fahri
Şahin, Ali Keskin, Öykü Arslan, Mehmet Ali Özcan, Gülnur
Görgün, Zahit Bolaman, Filiz Büyükkeçeci, Oktay Bilgir, İnci
Alacacıoğlu, Filiz Vural, Murat Tombuloğlu, Zafer Gökgöz,
Analysis or Interpretation: Güray Saydam, Literature Search:
Füsun Özdemirkıran, Writing: Füsun Özdemirkıran.
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. Provan D, Stasi R, Newland AC, Blanchette VS, Bolton-Maggs
P, Bussel JB, Chong BH, Cines DB, Gernsheimer TB, Godeau
B, Grainger J, Greer I, Hunt BJ, Imbach PA, Lyons G, McMillan
R, Rodeghiero F, Sanz MA, Tarantino M, Watson S, Young J,
Kuter DJ. International consensus report on the investigation
and management of primary immune thrombocytopenia.
Blood 2010;115:168-186.
2. Cines DB, Blanchette VS. Immune thrombocytopenic purpura.
N Engl J Med 2002;346:995-1008.
3. Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan
D, Arnold DM, Bussel JB, Cines DB, Chong BH, Cooper N,
Godeau B, Lechner K, Mazzucconi MG, McMillan R, Sanz
MA, Imbach P, Blanchette V, Kühne T, Ruggeri M, George
JN. Standardization of terminology, definitions and outcome
criteria in immune thrombocytopenic purpura of adults and
children: report from an international working group. Blood
2009;113:2386-2393.
4. Neunert C, Lim W, Crowther M, Cohen A, Solberg L Jr,
Crowther MA; American Society of Hematology. The American
Society of Hematology 2011 evidence-based practice guideline
for immune thrombocytopenia. Blood 2011;117:4190-4207.
5. Kumar S, Diehn FE, Gertz MA, Tefferi A. Splenectomy for
immune thrombocytopenic purpura: long term results
and treatment of postsplenectomy relapse. Ann Hematol
2002;81:312-319.
6. McMillan R. Classical management of refractory adult
immune (idiopathic) thrombocytopenic purpura. Blood Rev
2002;16:51-55.
7. Yang R, Han ZC. Pathogenesis and management of chronic
idiopathic thrombocytopenic purpura: an update. Int J
Hematol 2000;71:18-24.
8. Garnock-Jones KP, Keam SJ. Eltrombopag. Drugs 2009;69:567-
576.
9. Glaxo Smith Kline. Promacta (Eltrombopag) Prescribing
Information. Research Triangle Park, NC, USA, Glaxo Smith
Kline, 2012. Available at http://www.gsksource.com/gskprm/
en/US/adirect/gskprm?cmd=Product DetailPage&product_
id=1353688574915. Accessed 28 February 2013.
10. Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting
results of cancer treatment. Cancer 1981;47:207-214.
11. Katsutani S, Tomiyama Y, Kimura A, Miyakawa Y, Okamoto S,
Okoshi Y, Ninomiya H, Kosugi H, Ishii K, Ikeda Y, Hattori T,
Katsura K, Kanakura Y. Oral eltrombopag for up to three years
is safe and well-tolerated in Japanese patients with previously
treated chronic immune thrombocytopenia: an open-label,
extension study. Int J Hematol 2013;98:323-330.
12. Tomiyama Y, Miyakawa Y, Okamoto S, Katsutani S, Kimura
A, Okoshi Y, Ninomiya H, Kosugi H, Nomura S, Ozaki K,
Ikeda Y, Hattori T, Katsura K, Kanakura Y. A lower starting
dose of eltrombopag is efficacious in Japanese patients with
previously treated chronic immune thrombocytopenia. J
Thromb Haemost 2012;10:799-806.
13. Cheng G, Saleh MN, Marcher C, Vasey S, Mayer B, Aivado M,
Arning M, Stone NL, Bussel JB. Eltrombopag for management
of chronic immune thrombocytopenia (RAISE): a 6-month,
randomised, phase 3 study. Lancet 2011;377:393-402.
14. Saleh MN, Bussel JB, Cheng G, Meyer O, Bailey CK,
Arning M, Brainsky A; EXTEND Study Group. Safety and
efficacy of eltrombopag for treatment of chronic immune
thrombocytopenia: results of the long-term, open-label
EXTEND study. Blood 2013;121:537-545.
15. Zelcer S, Bussel JB. Thrombosis in patients with immune
thrombocytopenic purpura (ITP): a case series. J Thromb
Haemost 2003;1:1169 (abstract).
16. Ghanima W, Lee SY, Barsam S, Miller A, Sandset PM, Bussel JB.
Venous thromboembolism and coagulation activity in patients
with immune thrombocytopenia treated with thrombopoietin
receptor agonists. Br J Haematol 2012;158:811-814.
17. Douglas V, Tallman M, Cripe L, Peterson LC. Thrombopoietin
administered during induction chemotherapy to patients
with acute myeloid leukemia induces transient morphologic
changes that may resemble chronic myeloproliferative
disorders. Am J Clin Pathol 2002;117:844-850.
18. Cheng G. Eltrombopag, a thrombopoietin- receptor agonist
in the treatment of adult chronic immune thrombocytopenia:
a review of the efficacy and safety profile. Ther Adv Hematol
2012;3:155-164.
328

Research Article
DOI: 10.4274/tjh.2014.0035
Turk J Hematol 2015;32:329-337
Management of Invasive Fungal Infections in Pediatric
Acute Leukemia and the Appropriate Time for Restarting
Chemotherapy
Çocukluk Çağı Akut Lösemisinde İnvaziv Fungal Enfeksiyonların
Tedavisi ve Kemoterapiye Başlamanın Uygun Zamanı
Özlem Tüfekçi 1 , Şebnem Yılmaz Bengoa 1 , Fatma Demir Yenigürbüz 1 , Erdem Şimşek 2 ,
Tuba Hilkay Karapınar 1 , Gülersu İrken 1 , Hale Ören 1
1Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey
2Dokuz Eylül University Faculty of Medicine, Department of Pediatrics, İzmir, Turkey
Abstract:
Objective: Rapid and effective treatment of invasive fungal infection (IFI) in patients with leukemia is important for survival.
In this study, we aimed to describe variations regarding clinical features, treatment modalities, time of restarting chemotherapy,
and outcome in children with IFI and acute leukemia (AL).
Materials and Methods: The charts of all pediatric AL patients in our clinic between the years of 2001 and 2013 were
retrospectively reviewed. All patients received prophylactic fluconazole during the chemotherapy period.
Results: IFI was identified in 25 (14%) of 174 AL patients. Most of them were in the consolidation phase of chemotherapy
and the patients had severe neutropenia. The median time between leukemia diagnosis and definition of IFI was 122 days.
Twenty-four patients had pulmonary IFI. The most frequent finding on computed tomography was typical parenchymal
nodules. The episodes were defined as proven in 4 (16%) patients, probable in 7 (28%) patients, and possible in 14 (56%)
patients. The median time for discontinuation of chemotherapy was 27 days. IFI was treated successfully in all patients with
voriconazole, amphotericin B, caspofungin, or posaconazole alone or in combination. Chemotherapy was restarted in 50% of
the patients safely within 4 weeks and none of those patients experienced reactivation of IFI. All of them were given secondary
prophylaxis. The median time for antifungal treatment and for secondary prophylaxis was 26 and 90 days, respectively. None
of the patients died due to IFI.
Conclusion: Our data show that rapid and effective antifungal therapy with rational treatment modalities may decrease the
incidence of death and that restarting chemotherapy within several weeks may be safe in children with AL and IFI.
Keywords: Acute leukemia, Chemotherapy, Children, Fungal infection
Address for Correspondence: Hale ÖREN, M.D.,
Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey
Phone: +90 532 666 90 50 E-mail: hale.oren@deu.edu.tr
Received/Geliş tarihi : January 24, 2014
Accepted/Kabul tarihi : April 28, 2014
329

Turk J Hematol 2015;32:329-337
Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia
Öz:
Amaç: Lösemili hastalarda invaziv fungal enfeksiyonların (İFE) çabuk ve etkin tedavisi sağkalım için önemlidir. Bu çalışmada
akut lösemi (AL) ve İFE olan çocuklarda klinik bulgular, tedavi şekilleri, tekrar kemoterapiye başlama zamanı ve tedavi sonucu
gibi değişkenleri değerlendirmeyi amaçladık.
Gereç ve Yöntemler: Kliniğimizde 2001-2013 yılları arasında izlenmiş tüm AL’lı çocukların hastane kayıtları retrospektif olarak
tarandı. Tüm hastalara kemoterapi süresince proflaktik flukonazol tedavisi verildi.
Bulgular: İFE, 174 AL hastasından 25’inde (%14) saptandı. Çoğu konsolidasyon tedavisi sırasında gelişmişti ve hastalar ağır
nötropenikti. Lösemi tanısı ve İFE gelişme arasındaki ortanca süre 122 gündü. Hastaların 24’ünde pulmoner İFE vardı. Bilgisayarlı
tomografi tetkikinde en sık izlenen bulgu parenkimal nodüllerdi. İFE epizodları 4 (%16) olguda kanıtlanmış, 7 (%28) olguda
olası, 14 (%56) olguda muhtemel olarak değerlendirildi. Kemoterapiye ara verme süresi ortanca 27 gündü. İFE voriconazole,
amphotericin B, caspofungin, posaconazole tekli veya kombine tedavileri ile başarıyla tedavi edildi. Olguların %50’sinde
kemoterapiye 4 haftadan önce başlandı ve hiçbirinde İFE reaktivasyonu saptanmadı. Tümüne ikincil proflaksi verildi. Antifungal
tedavi ve sekonder proflaksi ortanca süresi sırayla 26 ve 90 gündü. Hastalardan hiçbiri İFE ile kaybedilmedi.
Sonuç: Verilerimiz AL ve İFE olan çocuklarda erken ve etkin rasyonel antifungal tedavi ile ölüm oranının azaltılabileceğini ve
birkaç hafta içinde kemoterapiye güvenle başlanabileceğini göstermektedir.
Anahtar Sözcükler: Akut lösemi, Çocukluk çağı, Fungal enfeksiyon, Kemoterapi
Introduction
The breakdown of host defense mechanisms in
immunocompromised patients leads to increased risk of lifethreatening
infections, including invasive fungal infections
(IFIs) [1,2,3,4,5,6]. Studies of pediatric populations with
hemato-oncological diseases show an incidence rate of IFI
ranging from 4.9% to 29% [7,8,9,10]. Besides causing increased
mortality and morbidity, IFIs cause a substantial delay in
treatment of acute leukemia (AL), which in turn could result
in failure of this potentially curative treatment. The optimal
time for restarting chemotherapy in these patients is not clear.
In this retrospective study, our purpose was to describe the
incidence, risk factors, clinical features, treatment modalities,
and outcome of IFIs in children with AL. We also aimed to
investigate the appropriate (optimal) time for restarting
chemotherapy in this group of patients.
Patients and Institution
Materials and Methods
This retrospective study included all acute lymphoblastic
leukemia (ALL) and acute myeloid leukemia (AML) patients,
aged 0-18 years, who developed IFI at our clinic between
January 2001 and January 2013. The patients were identified
by reviewing the medical charts of all AL patients. Children
with ALL received the BFM-95 or BFM-2000 protocol and
those with AML received the BFM-98 or BFM-2004 protocol.
Children were hospitalized in single rooms without highefficiency
air filtration systems.
The medical, microbiological, and imaging records of the
patients who met the inclusion criteria were reviewed for the
following variables:
Demographic and clinical data: Age and sex, leukemia
type, remission status, and risk group of underlying disease
at the time of diagnosis; the day and phase of treatment at
which IFI developed (remission, induction, consolidation,
maintenance); corticosteroid use 14 days prior to IFI onset;
presence of central venous catheter; presence of mucositis;
duration of neutropenia prior to IFI; use and type of primary
antifungal prophylaxis; type of symptoms and signs of the IFI.
Laboratory data: Complete blood count; fungus detection
tests including serum galactomannan (GM) antigen, direct
stains, cultures, sinus aspirate, and samples from other sites.
Radiological data: X-ray, computed tomography, and
ultrasound.
Treatment and outcome of IFI: Empiric therapy and
definitive therapy with one or a combination of antifungal
drugs; use of surgery; duration of treatment; the time from the
onset of fungal infection to the restarting of chemotherapy;
the use and type of secondary antifungal prophylaxis; the
development of reactivation of fungal infection; mortality.
According to our institutional policy, all patients with ALL
and AML receive prophylactic fluconazole (4-6 mg/kg/day)
during all phases of chemotherapy. Severe neutropenia was
defined as absolute granulocyte count of

Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia
Turk J Hematol 2015;32:329-337
The primary empiric antifungal treatment in our clinic
was either with caspofungin or liposomal amphotericin B,
depending on availability in the pharmacy of the hospital. The
antifungal agent was sometimes switched during the course of
the illness if the patient was intolerant or in culture-positive
cases, according to the drug susceptibilities of the specific
pathogen isolated.
In all cases, IFI was defined according to the guidelines of
the EORTC/MSG [11]. Proven IFI was diagnosed by a positive
fungal culture from a normally sterile site. Probable IFI was
diagnosed on the basis of a combination of host factors, clinical
and radiological features, and mycological evidence, such as
positive fungal culture, positive GM assay, or microscopy of
bronchoalveolar lavage fluid or sinus aspirate. Possible IFI was
diagnosed when the clinical and imaging findings and host
factors were consistent with IFI but there was no mycological
support.
Statistical Analysis
Statistical analyses were performed with SPSS 15.
Descriptive statistics were calculated and reported as absolute
frequencies or percentages for qualitative data and as medians
and ranges for quantitative data.
Results
A total of 174 patients were diagnosed with and treated
for AL (144 had ALL and 30 had AML) in our clinic. IFI was
diagnosed in 25 (14%) of 174 AL patients, in 12% of all ALL
cases, and in 27% of all AML cases. The characteristics of the
25 patients diagnosed with IFI are shown in Table 1. Of the 25
patients, 17 (68%) had ALL and 8 (32%) had AML. Five of the
8 AML patients (62%) and 7 of the 17 ALL patients (41%) were
allocated into the high-risk group at the time of diagnosis of
AL. The median age was 12 years (range: 0.7-17.5 years). Nine
(36%) of the patients were in the induction phase, 14 (56%)
of the patients were in the consolidation phase, and 2 (8%) of
the patients were in the maintenance phase of chemotherapy;
overall, 18 (72%) patients were in remission at the time of
diagnosis of IFI.
The median time between the leukemia diagnosis and
the definition of IFI was 122 days (range: 15-305 days).
Absolute neutrophil count was

Turk J Hematol 2015;32:329-337
Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia
21-57 days). All patients were given secondary prophylaxis
with oral voriconazole, itraconazole, or posaconazole. The
median time for secondary prophylaxis was 90 days (range:
39-429 days). Reactivation of IFI occurred in 4 patients
as pulmonary IFI; all of them were cured completely after
treatment.
The median time for discontinuation of chemotherapy was
27 days (range: 0-57 days). Chemotherapy was not restarted
in 3 patients due to refractory/progressive primary disease.
Out of 22 patients for whom chemotherapy was restarted,
the duration of cessation of chemotherapy was

Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia
Turk J Hematol 2015;32:329-337
Table 4. Disease characteristics, duration of chemotherapy discontinuation, and causes of death for the 9 patients who died.
Patients
Primary
Disease/Risk Group
Duration for
Discontinuation of
Chemotherapy
Cause of Death
1 AML/HRG NA † Refractory/progressive primary disease
2 ALL/HRG NA † Refractory/progressive primary disease
3 ALL/MRG NA † Refractory/progressive primary disease
4 ALL/HRG ≤28 days EBV related-lymphoproliferative disease
5 ALL/HRG ≤28 days Post-transplantation lymphoproliferative disease ‡ ‡
6 ALL/MRG >28 days Allogeneic bone marrow transplantation toxicity ‡ ‡
7 ALL/MRG >28 days Refractory/progressive primary disease
8 ALL/HRG >28 days Refractory/progressive primary disease
9 ALL/HRG >28 days Refractory/progressive primary disease
ALL: Acute lymphoblastic leukemia, AML: acute myeloid leukemia, HRG: high-risk group, MRG: medium-risk group, NA: not applicable.
† : Chemotherapy was not restarted in 3 patients due to refractory/progressive primary disease.
‡: Allogeneic bone marrow transplantation was performed due to wwwleukemia relapse.
years in our study. Dvorak et al. and Kobayashi et al. found
that age above 10 years on admission is a risk factor for IFI
and it has been suggested that this finding may reflect the
importance of host colonization by environmental fungi as an
important step in the development of invasive disease, with
younger patients having had less exposure time to fungal
spores in the environment [15,16,17].
The majority of our patients (88%) were severely
neutropenic at the time of diagnosis of IFI and the overall
median duration of neutropenia was longer than 10 days. The
incidence of IFI in children with leukemia was previously
found to be closely related to the type of leukemia, with AML
having a higher rate than ALL as in our study [3,7,18,19,20].
The intensive treatment and the relatively longer duration of
neutropenia in AML patients are responsible for the increased
risk of infections in this group of patients.
More than half of our patients (56%) were in the
consolidation phase at the time of diagnosis of IFI. Similarly,
Hale et al. also reported that half of IFIs were diagnosed 100-
365 days after the initial diagnosis in AL patients [12]. We
use BFM protocols and the consolidation phases of ALL and
AML in BFM protocols correspond to HD-MTX and HD-ARA
C blocks where there is increased risk of mucositis, a known
risk factor for fungal infections [21,22].
In our study, GM was positive in 2 consecutive samples of 9
patients. Adult studies and recent pediatric studies have revealed
the favorable specificity of the assay [23,24,25,26,27,28,29].
Another important diagnostic approach in identifying IFI is
the HRCT of the chest. Chest X-rays have little value in the
early stage of disease [30,31,32]. The most common sign in
our patients was typical parenchymal nodules on HRCT; halo
signs and air-crescent findings were less frequently seen. It is
important to emphasize that pulmonary lesions characteristic
for adults, such as air-crescent signs and cavitary lesions, are
rarely seen in children [33,34]. A recent retrospective analysis
of 139 pediatric invasive aspergillosis cases reported that the
most frequent diagnostic radiologic finding was nodules at a
rate of 34.6% [35].
The vast majority of IFIs in our study were due to
Aspergillus spp. and the respiratory tract was the most common
site for invasive aspergillosis. On the other hand, the absence
of Candida albicans infections was remarkable in our study,
which may be attributable to the strict use of fluconazole.
One of our patients had Candida kefyr bloodstream infection,
which is the fluconazole-resistant nonalbicans type of Candida
and may be seen in patients with neutropenia. Recent reports
have shown that infections caused by resistant Candida spp.
and molds such as Aspergillus, Fusarium, and Scedosporium
have been subsequently increased by the widespread use of
fluconazole prophylaxis [10,12,36,37]. An additional risk
factor for development of invasive aspergillosis in our study
might be the absence of effective air filtration systems in
patient rooms, as well as ongoing hospital renovation for
the last 5 years. There are many reports in the literature
suggesting an association between invasive aspergillosis and
contaminated ventilation systems, hospital construction, or
renovation [14,38,39].
333

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Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia
Empirical antifungal therapy and investigation for
IFIs should be considered for patients with persistent or
recurrent fever after 4-7 days of antibiotics [40,41]. IFI was
treated successfully in all our patients with voriconazole,
amphotericin B, caspofungin, or posaconazole alone or in
combination [42,43,44,45,46]. One of our patients with
orbitocerebral mucormycosis and aspergillosis initially did
not respond to liposomal amphotericin B, but did recover
completely after posaconazole was added to the treatment.
Combination therapy, although not recommended by
international guidelines, is often used as rescue treatment in
patients who are switched to second- or third-line antifungal
therapy [45,47]. Regarding secondary antifungal prophylaxis,
it is recommended to continue treatment with an agent and
dose effective against the isolate of the primary infection until
the end of immunosuppression [48].
An important consequence of IFI is that the relatively longer
duration of time for treatment of this severe infection causes a
significant delay in the primary treatment of AL. The optimal
time for restarting chemotherapy in these patients is not clear,
which poses a great dilemma for the physician [45]. One of
our aims in this study was to investigate the safe, appropriate
timing for restarting chemotherapy in these patients. The
median time for discontinuation of chemotherapy was 27
days in our study; chemotherapy was restarted in 50% of
the patients safely before 4 weeks and none of those patients
experienced reactivation of IFI. Similarly Nosari et al., in their
retrospective review of hematological malignancies, identified
61 adult cases of IFI and detected a median time of 27 days for
discontinuation of chemotherapy (range: 17-45 days) [49].
The decision for timing chemotherapy is generally made on an
individual basis depending on the extent of the fungal disease
and the status of the primary disease.
The mortality rate of IFI shows wide variations among
the studies reported in the literature. While earlier studies
reported IFI-related mortality rates of up to 85%, recent
studies have reported lower rates [8,39,50,51,52]. Kaya et al.
reported the rate of IFI-attributable death as 5% (1 patient)
in 21 children with AL [10]. Another previously mentioned
study from Turkey found the total mortality of IFI to be
30% in 23 patients with AL and aplastic anemia [14]. In this
study, death occurred in 36% of patients, but none of the
deaths were attributable to the IFIs themselves. This finding
may be due to increased awareness of the possibility of IFIs,
the widespread use of HRCT as an early diagnostic method,
early empirical treatment for febrile neutropenic patients, and
greater effectiveness of newer antifungal agents.
In conclusion, our study demonstrated that rapid and
effective antifungal therapy with rational treatment modalities
may decrease the incidence of death in children with AL and
IFI. Depending on the clinical status of the patient, restarting
chemotherapy within several weeks may be safe and reactivation
of IFI may be prevented with secondary prophylaxis.
Ethics Committee Approval: It is a retrospective study,
Informed Consent: It is a retrospective study, Concept: Hale
Ören, Design: Hale Ören, Özlem Tüfekçi, Gülersu İrken,
Şebnem Yılmaz Bengoa, Data Collection or Processing: Özlem
Tüfekçi, Tuba Hilkay Karapınar, Erdem Şimşek, Analysis or
Interpretation: Hale Ören, Özlem Tüfekçi, Şebnem Yılmaz
Bengoa, Literature Search: Hale Ören, Özlem Tüfekçi,
Writing: Hale Ören, Özlem Tüfekçi.
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. Bow EJ. Infection risk and cancer chemotherapy: the impact
of the chemotherapeutic regimen in patients with lymphoma
and solid tissue malignancies. J Antimicrob Chemother
1998;(Suppl 41):1-5.
2. Zaoutis TE, Heydon K, Chu JH, Walsh TJ, Steinbach WJ.
Epidemiology, outcomes, and costs of invasive aspergillosis
in immunocompromised children in the United States, 2000.
Pediatrics 2006;117:711-716.
3. Mor M, Gilad G, Kornreich L, Fisher S, Yaniv I, Levy I. Invasive
fungal infections in pediatric oncology. Pediatr Blood Cancer
2011;56:1092-1097.
4. Kurosawa M, Yonezumi M, Hashino S, Tanaka J, Nishio M,
Kaneda M, Ota S, Koda K, Suzuki N, Yoshida M, Hirayama Y,
Takimoto R, Torimoto Y, Mori A, Takahashi T, Iizuka S, Ishida
T, Kobayashi R, Oda T, Sakai H, Yamamoto S, Takahashi F,
Fukuhara T. Epidemiology and treatment outcome of invasive
fungal infections in patients with hematological malignancies.
Int J Hematol 2012;96:748-757.
5. Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert
F, Denning DW, Donnelly JP, Edwards JE, Erjavec Z, Fiere
D, Lortholary O, Maertens J, Meis JF, Patterson TF, Ritter J,
Selleslag D, Shah PM, Stevens DA, Walsh TJ; Invasive Fungal
Infections Cooperative Group of the European Organization
for Research and Treatment of Cancer; Mycoses Study
Group of the National Institute of Allergy and Infectious
Diseases. Defining opportunistic invasive fungal infections in
immunocompromised patients with cancer and hematopoietic
stem cell transplants: an international consensus. Clin Infect
Dis 2002;34:7-14.
334

Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia
Turk J Hematol 2015;32:329-337
6. Chamilos G, Luna M, Lewis RE, Bodey GP, Chemaly R,
Tarrand JJ, Safdar A, Raad II, Kontoyiannis DP. Invasive fungal
infections in patients with hematologic malignancies in a
tertiary care cancer center: an autopsy study over a 15-year
period (1989-2003). Haematologica 2006;91:986-989.
7. Rosen GP, Nielsen K, Glenn S, Abelson J, Deville J, Moore
TB. Invasive fungal infections in pediatric oncology patients:
11-year experience at a single institution. J Pediatr Hematol
Oncol 2005;27:135-140.
8. Wiley JM, Smith N, Leventhal BG, Graham ML, Graham ML,
Strauss LC, Hurwitz CA, Modlin J, Mellits D, Baumgardner R,
Corden BJ. Invasive fungal disease in pediatric acute leukemia
patients with fever and neutropenia during induction
chemotherapy: a multivariate analysis of risk factors. J Clin
Oncol 1990;8:280-286.
9. Gözdaşoğlu S, Ertem M, Büyükkeçeci Z, Yavuzdemir S,
Bengisun S, Ozenci H, Taçyildiz N, Unal E, Yavuz G, Deda G,
Aysev D. Fungal colonization and infection in children with
acute leukemia and lymphoma during induction therapy.
Med Pediatr Oncol 1999;32:344-348.
10. Kaya Z, Gürsel T, Koçak U, Aral YZ, Kalkancı A, Albayrak
M. Invasive fungal infections in pediatric leukemia patients
receiving fluconazole prophylaxis. Pediatr Blood Cancer
2009;52:470-475.
11. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards
JE, Calandra T, Pappas PG, Maertens J, Lortholary O,
Kauffman CA, Denning DW, Patterson TF, Maschmeyer G,
Bille J, Dismukes WE, Herbrecht R, Hope WW, Kibbler CC,
Kullberg BJ, Marr KA, Muñoz P, Odds FC, Perfect JR, Restrepo
A, Ruhnke M, Segal BH, Sobel JD, Sorrell TC, Viscoli C,
Wingard JR, Zaoutis T, Bennett JE; European Organization
for Research and Treatment of Cancer/Invasive Fungal
Infections Cooperative Group; National Institute of Allergy
and Infectious Diseases Mycoses Study Group (EORTC/MSG)
Consensus Group. Revised definitions of invasive fungal
disease from the European Organization for Research and
Treatment of Cancer/Invasive Fungal Infections Cooperative
Group and the National Institute of Allergy and Infectious
Diseases Mycoses Study Group (EORTC/MSG) Consensus
Group. Clin Infect Dis 2008;46:1813-1821.
12. Hale KA, Shaw PJ, Dalla-Pozza L, MacIntyre CR, Isaacs D, Sorrell
TC. Epidemiology of paediatric invasive fungal infections and
a case-control study of risk factors in acute leukaemia or post
stem cell transplant. Br J Haematol 2010;149:263-272.
13. Cesaro S, Pagano L, Caira M, Carraro F, Luciani M, Russo D,
Colombini A, Morello W, Viale P, Rossi G, Tridello G, Pegoraro
A, Nosari A, Aversa F; Hema-e-chart Group. A prospective,
multicentre survey on antifungal therapy in neutropenic
paediatric haematology patients. Mycoses 2013;56:21-25.
14. Baytan B, Güneş AM, Çelebi S, Günay Ü. Invasive fungal
diseases in children with hematologic disorders. Turk J
Hematol 2009;26:190-196.
15. Dvorak CC, Steinbach WJ, Brown JM, Agarwal R. Risks and
outcomes of invasive fungal infections in pediatric patients
undergoing allogeneic hematopoietic cell transplantation.
Bone Marrow Transplant 2005;36:621-629.
16. Kobayashi R, Kaneda M, Sato T, Ichikawa M, Suzuki D, Ariga
T. The clinical feature of invasive fungal infection in pediatric
patients with hematologic and malignant diseases: a 10-year
analysis at a single institution at Japan. J Pediatr Hematol
Oncol 2008;30:886-890.
17. Dini G, Castagnola E, Comoli P, van Tol MJ, Vossen JM.
Infections after stem cell transplantation in children: state
of the art and recommendations. Bone Marrow Transplant
2001;28(Suppl 1):18-21.
18. Castagnola E, Cesaro S, Giacchino M, Livadiotti S, Tucci F,
Zanazzo G, Caselli D, Caviglia I, Parodi S, Rondelli R, Cornelli
PE, Mura R, Santoro N, Russo G, De Santis R, Buffardi S,
Viscoli C, Haupt R, Rossi MR. Fungal infections in children
with cancer: a prospective, multicenter surveillance study.
Pediatr Infect Dis J 2006;25:634-639.
19. Castagnola E, Rossi MR, Cesaro S, Livadiotti S, Giacchino M,
Zanazzo G, Fioredda F, Beretta C, Ciocchello F, Carli M, Putti
MC, Pansini V, Berger M, Licciardello M, Farina S, Caviglia
I, Haupt R. Incidence of bacteremias and invasive mycoses
in children with acute non-lymphoblastic leukemia: results
from a multi-center Italian study. Pediatr Blood Cancer
2010;55:1103-1107.
20. Castagnola E, Caviglia I, Pistorio A, Fioredda F, Micalizzi
C, Viscoli C, Haupt R. Bloodstream infections and invasive
mycoses in children undergoing acute leukaemia treatment: a
13-year experience at a single Italian institution. Eur J Cancer
2005;41:1439-1445.
21. Cornely OA, Böhme A, Reichert D, Reuter S, Maschmeyer
G, Maertens J, Buchheidt D, Paluszewska M, Arenz D, Bethe
U, Effelsberg J, Lövenich H, Sieniawski M, Haas A, Einsele
H, Eimermacher H, Martino R, Silling G, Hahn M, Wacker
S, Ullmann AJ, Karthaus M; Multinational Case Registry of
the Infectious Diseases Working Party of the German Society
for Hematology and Oncology. Risk factors for breakthrough
invasive fungal infection during secondary prophylaxis. J
Antimicrob Chemother 2008;61:939-946.
22. Bow EJ, Kilpatrick MG, Scott BA, Clinch JJ, Cheang MS.
Acute myeloid leukemia in Manitoba. The consequences
of standard “7 + 3” remission-induction therapy followed
by high dose cytarabine postremission consolidation for
myelosuppression, infectious morbidity, and outcome.
Cancer 1994;74:52-60.
335

Turk J Hematol 2015;32:329-337
Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia
23. Dornbusch HJ, Groll A, Walsh TJ. Diagnosis of invasive fungal
infections in immunocompromised children. Clin Microbiol
Infect 2010;16:1328-1334.
24. Busca A, Locatelli F, Barbui A, Limerutti G, Serra R, Libertucci
D, Falda M. Usefulness of sequential Aspergillus galactomannan
antigen detection combined with early radiologic evaluation
for diagnosis of invasive pulmonary aspergillosis in patients
undergoing allogeneic stem cell transplantation. Transplant
Proc 2006;38:1610-1613.
25. Pfeiffer CD, Fine JP, Safdar N. Diagnosis of invasive aspergillosis
using a galactomannan assay: a meta-analysis. Clin Infect Dis
2006;42:1417-1427.
26. El-Mahallawy HA, Shaker HH, Ali Helmy H, Mostafa T,
Razak Abo-Sedah A. Evaluation of pan-fungal PCR assay
and Aspergillus antigen detection in the diagnosis of invasive
fungal infections in high risk paediatric cancer patients. Med
Mycol 2006;44:733-739.
27. Armenian SH, Nash KA, Kapoor N, Franklin JL, Gaynon PS,
Ross LA, Hoffman JA. Prospective monitoring for invasive
aspergillosis using galactomannan and polymerase chain
reaction in high risk pediatric patients. J Pediatr Hematol
Oncol 2009;31:920-926.
28. Castagnola E, Furfaro E, Caviglia I, Licciardello M, Faraci
M, Fioredda F, Tomà P, Bandettini R, Machetti M, Viscoli C.
Performance of the galactomannan antigen detection test in
the diagnosis of invasive aspergillosis in children with cancer
or undergoing haemopoietic stem cell transplantation. Clin
Microbiol Infect 2010;16:1197-1203.
29. Steinbach WJ, Addison RM, McLaughlin L, Gerrald Q,
Martin PL, Driscoll T, Bentsen C, Perfect JR, Alexander BD.
Prospective Aspergillus galactomannan antigen testing in
pediatric hematopoietic stem cell transplant recipients. Pediatr
Infect Dis J 2007;26:558-564.
30. Caillot D, Casasnovas O, Bernard A, Couaillier JF, Durand C,
Cuisenier B, Solary E, Piard F, Petrella T, Bonnin A, Couillault
G, Dumas M, Guy H. Improved management of invasive
pulmonary aspergillosis in neutropenic patients using early
thoracic computed tomographic scan and surgery. J Clin
Oncol 1997;15:139-147.
31. Blum U, Windfuhr M, Buitrago-Tellez C, Sigmund G, Herbst
EW, Langer M. Invasive pulmonary aspergillosis. MRI, CT,
and plain radiographic findings and their contribution for
early diagnosis. Chest 1994;106:1156-1161.
32. Crassard N, Hadden H, Piens MA, Pondarré C, Hadden R,
Galambrun C, Pracros JP, Souillet G, Basset T, Berthier JC,
Philippe N, Bertrand Y. Invasive aspergillosis in a paediatric
haematology department: a 15-year review. Mycoses
2008;51:109-116.
33. Thomas KE, Owens CM, Veys PA, Novelli V, Costoli V. The
radiological spectrum of invasive aspergillosis in children: a
10-year review. Pediatr Radiol 2003;33:453-460.
34. Taccone A, Occhi M, Garaventa A, Manfredini L, Viscoli
C. CT of invasive pulmonary aspergillosis in children with
cancer. Pediatr Radiol 1993;23:177-180.
35. Burgos A, Zaoutis TE, Dvorak CC, Hoffman JA, Knapp
KM, Nania JJ, Prasad P, Steinbach WJ. Pediatric invasive
aspergillosis: a multicenter retrospective analysis of 139
contemporary cases. Pediatrics 2008;121:1286-1294.
36. Marr KA, Carter RA, Crippa F, Wald A, Corey L. Epidemiology
and outcome of mould infections in hematopoietic stem cell
transplant recipients. Clin Infect Dis 2002;34:909-917.
37. Brown JM. Fungal infections in bone marrow transplant
patients. Curr Opin Infect Dis 2004;17:347-352.
38. Marr KA, Patterson T, Denning D. Aspergillosis. Pathogenesis,
clinical manifestations, and therapy. Infect Dis Clin North Am
2002;16:875-894.
39. Groll AH, Kurz M, Schneider W, Witt V, Schmidt H, Schneider
M, Schwabe D. Five-year-survey of invasive aspergillosis in a
paediatric cancer centre. Epidemiology, management and
long-term survival. Mycoses 1999;42:431-442.
40. Blyth CC, Hale K, Palasanthiran P, O’Brien T, Bennett MH.
Antifungal therapy in infants and children with proven,
probable or suspected invasive fungal infections. Cochrane
Database Syst Rev 2010;2:CD006343.
41. Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Mullen
CA, Raad II, Rolston KV, Young JA, Wingard JR, Infectious
Diseases Society of America. Clinical practice guideline for
the use of antimicrobial agents in neutropenic patients with
cancer: 2010 update by the infectious diseases society of
America. Clin Infect Dis 2011;52:56-93.
42. Maertens JA, Madero L, Reilly AF, Lehrnbecher T, Groll
AH, Jafri HS, Green M, Nania JJ, Bourque MR, Wise BA,
Strohmaier KM, Taylor AF, Kartsonis NA, Chow JW, Arndt
CA, DePauw BE, Walsh TJ; Caspofungin Pediatric Study
Group. A randomized, double-blind, multicenter study of
caspofungin versus liposomal amphotericin B for empiric
antifungal therapy in pediatric patients with persistent fever
and neutropenia. Pediatr Infect Dis J 2010;29:415-420.
43. Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis
DP, Marr KA, Morrison VA, Segal BH, Steinbach WJ, Stevens
DA, van Burik JA, Wingard JR, Patterson TF; Infectious
Diseases Society of America. Treatment of aspergillosis:
clinical practice guidelines of the Infectious Diseases Society
of America. Clin Infect Dis 2008;46:327-360.
44. Queiroz-Telles F, Berezin E, Freire A, van der Vyver A,
Chotpitayasunondh T, Konja J, Diekmann-Berndt H, Koblinger
S, Groll AH, Arrieta A; Micafungin Invasive Candidiasis
336

Tüfekçi Ö, et al: Fungal Infection in Childhood Leukemia
Turk J Hematol 2015;32:329-337
Study Group. Micafungin versus liposomal amphotericin
B for pediatric patients with invasive candidiasis: substudy
of a randomized double-blind trial. Pediatr Infect Dis J
2008;27:820-826.
45. Katragkou A, Roilides E. Best practice in treating infants and
children with proven, probable or suspected invasive fungal
infections. Curr Opin Infect Dis 2011;24:225-229.
46. Maertens J, Groll AH, Cordonnier C, de la Cámara R, Roilides
E, Marchetti O. Treatment and timing in invasive mould
disease. J Antimicrob Chemother 2011;66(Suppl 1):37-43.
47. Lass-Flörl C. Invasive fungal infections in pediatric patients: a
review focusing on antifungal therapy. Expert Rev Anti Infect
Ther 2010;8:127-135.
48. Tragiannidis A, Dokos C, Lehrnbecher T, Groll AH.
Antifungal chemoprophylaxis in children and adolescents
with haematological malignancies and following allogeneic
haematopoietic stem cell transplantation: review of the
literature and options for clinical practice. Drugs 2012;72:685-
704.
49. Nosari A, Oreste P, Cairoli R, Montillo M, Carrafiello G,
Astolfi A, Muti G, Marbello L, Tedeschi A, Magliano E, Morra
E. Invasive aspergillosis in haematological malignancies:
clinical findings and management for intensive chemotherapy
completion. Am J Hematol 2001;68:231-236.
50. Abbasi S, Shenep JL, Hughes WT, Flynn PM. Aspergillosis in
children with cancer: a 34-year experience. Clin Infect Dis
1999;29:1210-1219.
51. Riley LC, Hann IM, Wheatley K, Stevens RF. Treatment-related
deaths during induction and first remission of acute myeloid
leukaemia in children treated on the Tenth Medical Research
Council acute myeloid leukaemia trial (MRC AML10). The
MCR Childhood Leukaemia Working Party. Br J Haematol
1999;106:436-444.
52. Grigull L, Beier R, Schrauder A, Kirschner P, Loening L, Jack T,
Welte K, Sykora KW, Schrappe M. Invasive fungal infections
are responsible for one-fifth of the infectious deaths in
children with ALL. Mycoses 2003;46:441-446.
337

Research Article
DOI: 10.4274/tjh.2013.0370
Turk J Hematol 2015;32:338-343
First-Step Results of Children Presenting with Bleeding
Symptoms or Abnormal Coagulation Tests in an
Outpatient Clinic
Polikliniğe Kanama Belirtileri veya Anormal Koagülasyon
Testleri Nedeniyle Başvuran Olgularda Birinci Basamak
Değerlendirme Sonuçları
İsmail Yıldız 1 , Ayşegül Ünüvar 2 , İbrahim Kamer 3 , Serap Karaman 2 , Ezgi Uysalol 2 , Ayşe Kılıç 1 , Fatma Oğuz 4 ,
Emin Ünüvar 1
1İstanbul University İstanbul Faculty of Medicine, Department of Pediatrics, Division of Ambulatory Pediatrics, İstanbul, Turkey
2İstanbul University İstanbul Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology and Oncology, İstanbul, Turkey
3İstanbul University İstanbul Faculty of Medicine, Department of Pediatrics, İstanbul, Turkey
4İstanbul University Institute of Child Health, Division of Ambulatory Pediatrics, İstanbul, Turkey
Abstract:
Objective: Mild bleeding symptoms are commonly seen in the general population. The aim of this study was to determine
the final clinical and laboratory features of children referred for a first evaluation with a suspected bleeding disorder in the
pediatric outpatient clinic of İstanbul University.
Materials and Methods: The medical records of 26,737 outpatients who were admitted to the Division of Ambulatory
Pediatrics between 31 October 2011 and 31 October 2012 were evaluated retrospectively. Ninety-nine patients were initially
diagnosed as having probable bleeding disorders and were followed up. The symptoms of bleeding in addition to coagulation
tests were analyzed.
Results: Of the 99 patients, 52 (52.5%) were male and 47 were female, and the mean age of the entire study group was 9.1±4.1
years (minimum-maximum: 2-18 years). Major bleeding symptoms were epistaxis in 36 patients (36.4%), easy bruising in 32
(32.3%), and menorrhagia in 6 (6.1%). After initial tests ordered by the pediatrician, 36 of 99 patients (36.4%) were diagnosed
as having bleeding disorders that included von Willebrand disease in 12 (12.1%), hemophilia A or B in 9 (9.1%), and other
rare factor deficiencies in 9 (9.1%). Six patients (6.1%) were found to have combined deficiencies. Seven of 36 patients had a
family history of bleeding.
Conclusion: Among the patients referred for bleeding disorders, 36.4% were diagnosed with a bleeding disorder with the
help of primary screening tests ordered in the outpatient clinic.
Keywords: Children, Blood coagulation, Hemophilia, Inherited coagulopathies, Epistaxis, Menorrhagia
Address for Correspondence: İsmail YILDIZ, M.D.,
İstanbul University İstanbul Faculty of Medicine, Department of Pediatrics, Division of Ambulatory Pediatrics, İstanbul, Turkey
Phone: +90 212 414 20 00 E-mail: drismail810@yahoo.com
Received/Geliş tarihi : November 03, 2013
Accepted/Kabul tarihi : May 29, 2014
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Yıldız İ, et al: Evaluation of Children with Bleeding Symptoms
Turk J Hematol 2015;32:338-343
Öz:
Amaç: Hafif kanama bozukluğu belirtileri toplumda sık görülmektedir. Bu çalışmanın amacı İstanbul Üniversitesi İstanbul Tıp
Fakültesi Genel Pediatri Polikliniği’ne kanama bozukluğu şüphesi ile sevk edilen hastaların klinik ve laboratuvar özelliklerini
belirlemektir.
Gereç ve Yöntemler: 31 Ekim 2011 ile 31 Ekim 2012 tarihleri arasında kanama bozukluğu şüphesiyle yönlendirilen 99
hastanın tıbbi kayıtları incelenmiştir. Başvuru semptomları ile pıhtılaşma testlerinin sonuçları değerlendirilmiştir.
Bulgular: Olguların 47’si kız çocuğu olup ve yaş ortalaması 9,1±4,1 yıl (2-18 yıl) idi. Kanama semptomları 36 hastada (%36,4)
burun kanaması, 32 (%32,3) hastada kolay morarma ve 6 hastada (%6,1) menoraji idi. Birinci basamak testleri sonrasında, 99
hastanın 36’sında (%36,4) primer kanama bozukluğu saptandı. Bunlardan 12’sinde (%12,1) von Willebrand hastalığı, 9’unda
(%9,1) hemofili A veya B, 9’unda (%9,1) diğer nadir faktör eksiklikleri ve 6 hastada (%6,1) kombine faktör eksiklikleri saptandı.
Otuz altı hastanın 7’sinde ailede kanama öyküsü vardı.
Sonuç: Kanama bozukluğu şüphesi ile sevk edilen hastaların %36,4’ünde birinci basamak koagulasyon testleri ışığında kanama
bozukluklarından biri saptandı.
Anahtar Sözcükler: Çocuk, Koagülasyon, Hemofili, Kalıtsal koagülopatiler, Epistaksis, Menoraji
Introduction
When there is damage to the vascular wall, cessation of
bleeding without interrupting the blood flow and maintenance
of vascular integrity are ensured by hemostatic mechanisms.
Hemostasis is a multifunctional physiologic mechanism
involving the vascular wall, subendothelial tissues, platelets,
coagulation factors in plasma, and fibrinolytic factors, where
coagulants, anticoagulants, and fibrinolytic activities operate
in balance [1,2,3].
Hemostatic disorders manifesting with bleeding may
be caused by several factors including vascular issues, low
platelet counts, platelet function disorders, and disorders of
coagulation or fibrinolysis, which is due to either too much or
too fast dissolving of blood clots [1,3].
A careful history and physical examination of a patient
with bleeding symptoms leads to a correct diagnosis in 80%-
90% of patients. Adequate laboratory tests are performed
subsequently to confirm diagnosis [4,5,6].
In cases of bleeding disorders, the primary screening
tests include complete blood count, peripheral blood smear,
bleeding time test (if possible) using a platelet function
analyzer (PFA-100), prothrombin time (PT), activated partial
thromboplastin time (aPTT), thrombin time (TT), and
fibrinogen levels [5,6]. Advanced tests are carried out later
based on the pathological results from the primary screening
tests. Regardless of whether primary screening test results are
found to be normal, there may still be an underlying bleeding
disorder. In these cases, factor 13 deficiency, von Willebrand
disease (vWD) type 1, mild-type hemophilia A or B, mild factor
11 deficiency and mild deficiencies of other factors, alpha-2
anti-plasmin deficiency, plasminogen activator inhibitor-1
deficiency, collagen tissue diseases, vitamin C deficiency, and
various vascular bleeding disorders should be considered
[4,6].
Mild bleeding symptoms such as epistaxis, easy bruising,
gingival bleeding, and prolonged menstrual bleeding are
commonly seen in the general population and reported in
up to 25%-45% of healthy people [7]. Although patients who
present with these symptoms may have underlying bleeding
disorders, initial tests for bleeding etiology may yield normal
results [8,9].
The purpose of this study was to evaluate patients who
were referred to the Division of Ambulatory Pediatrics with
suspected bleeding disorders.
Materials and Methods
A total of 26,737 outpatients were admitted to the İstanbul
Faculty of Medicine’s Department of Pediatrics from 31 October
2011 to 31 October 2012. After exclusion of all patients with
immune thrombocytopenia, 115 patients with suspected
bleeding disorders were evaluated retrospectively. Thirteen of
these patients were not included because of known bleeding
disorders or they were lost during follow-up. Three patients
were excluded from the study after they were diagnosed as
having secondary thrombocytopenia caused by viral infections
or platelet function disorder. Thrombocytopenia and platelet
function disorders were not included in the evaluation.
This study was thus conducted with 99 patients (Figure 1).
All the admission symptoms, history, physical examination
findings, laboratory test results, and initial and definitive
diagnoses are based on the database from the hospital’s
automation system and the patients’ charts.
We recorded the patients’ sex, age, symptoms, site of
bleeding, duration of hemorrhage, existence of any bleeding
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Turk J Hematol 2015;32:338-343
Yıldız İ, et al: Evaluation of Children with Bleeding Symptoms
problems in the newborn period, and previous personal or
family history of bleeding disorders.
The first primary tests performed on patients suspected
of having a bleeding disorder were complete blood count,
bleeding time or PFA-100, PT, aPTT, TT, and fibrinogen
levels. A peripheral blood smear was performed in all
patients to evaluate platelet count and size, thus excluding
pseudothrombocytopenia. Bleeding time was measured either
in vitro with the PFA-100 (n=61) or in vivo by Duke’s method
(n=5). Advanced laboratory investigations were performed
on all patients whose initial tests revealed any pathological
findings. VWF antigen, ristocetin cofactor activity (Ricof),
and factor level (II, V, VII, VIII, IX, X, XI, XII, XIII) tests were
performed in the Pediatric Hematology Hemostasis Laboratory.
The patients with abnormal test results were referred to
the Pediatric Hematology and Oncology Unit for advanced
evaluation and follow-up. Tests with abnormal results were
repeated again at the next visit. The patients’ folders that
were created in the Pediatric Hematology and Oncology Unit
were evaluated for the definite diagnosis. Initial and definitive
diagnoses of these patients were recorded.
Results
A total of 26,737 outpatients were admitted to our unit
during the 1-year period of study. Ninety-nine (0.37%) patients
were initially diagnosed with probable bleeding disorders and
were followed up. Fifty-two (52.5%) patients were male and
47 (47.5%) were female, and their mean age was 9.1±4.1 years
(minimum-maximum: 2-18 years).
The most frequent symptoms were epistaxis in 36 of the
patients (36.4%), easy bruising in 32 (32.3%), prolonged
and/or massive menstrual bleeding in 6 (6.1%), and gingival
bleeding in 2 (2%) (Table 1). Duration of the symptoms
ranged from 2 days to 6 years.
According to the laboratory test results, 63 of the patients
(63.6%) had no bleeding disorders, whereas 36 (36.4%)
were diagnosed with bleeding disorders (Figure 1). The final
diagnosis included vWD type 1 in 8 (8.1%); vWD type 2
in 4 (4%); mild hemophilia A in 4 (4%); vWD type 1 and
FXI deficiency in 3 (3%); FV deficiency in 3 (3%); moderate
hemophilia A in 2 (2%); hemophilia A carrier in 2 (2%); FVII
deficiency in 2 (2%); FXI deficiency in 2 (2%); FX deficiency
in 1 (1%); FXII deficiency in 1 (1%); combined FII, VII, IX,
X, and FXII deficiency in 1 (1%); combined FV and FVIII
deficiency in 1 (1%); combined FVII and FX deficiency in 1
(1%); and hemophilia B carrier in 1 (1%) (Table 1).
Seven (19.4%) of 36 patients who were diagnosed with
bleeding disorders had a family bleeding history. Family
histories of the patients for coagulation disorders are presented
in Table 2.
Twenty-eight percent of the patients with epistaxis (10 of
36 patients; 3 cases of mild hemophilia A, 2 of vWD type 1,
1 of vWD type 2, 1 of vWD type 1+FXI deficiency, 1 of FVII
deficiency, 1 of FV deficiency, and 1 of combined FII, VII,
IX, X, and XII deficiency), 28.1% of the patients with easy
bruising (9 of 32 patients; 3 cases of vWD type 1, 1 of vWD
type 2, 1 of a hemophilia A carrier, 1 of FV deficiency, 1 of FXI
deficiency, 1 of FVII+X deficiency, and 1 of FXII deficiency),
Table 1. Characteristics of patients initially diagnosed
with bleeding disorders (n=99).
Characteristics n (%)
Sex
Female
Male
Symptoms
Epistaxis
Easy bruising
Menorrhagia
Gingival bleeding
Prolonged bleeding
Blood in stool
Hematoma under tongue
Other causes of outpatient clinic admissions
Abnormal PT/aPTT*
Examination bleeding diathesis**
Preoperative evaluation
Diagnosis
vWD type 1
vWD type 2
Mild hemophilia A
vWD type 1+factor XI deficiency
Factor V deficiency
Moderate hemophilia A
Hemophilia A carrier
Factor VII deficiency
Factor XI deficiency
Factor X deficiency
Factor XII deficiency
Factor II, VII, IX, X, and XII deficiencies
Factor V and VIII deficiency
Factor VII and X deficiency
Hemophilia B carrier
47 (47.5)
52 (52.5)
36 (36.4)
32 (32.3)
6 (6.1)
2 (2)
1 (1)
1 (1)
1 (1)
11 (11.1)
7 (7.1)
2 (2)
8 (8.1)
4 (4)
4 (4)
3 (3)
3 (3)
2 (2)
2 (2)
2 (2)
2 (2)
1 (1)
1 (1)
1 (1)
1 (1)
1 (1)
1 (1)
PT: Prothrombin time, aPTT: activated partial thromboplastin time,
vWD: von Willebrand disease.
*: Patients who presented with any symptoms but were assigned only to
abnormal prothrombin time, activated partial thromboplastin time.
**: Patients had a family history of bleeding disorders and were without
symptoms.
340

Yıldız İ, et al: Evaluation of Children with Bleeding Symptoms
Turk J Hematol 2015;32:338-343
Between 31 st of October, 2011, and 31 st of October, 2012, the number of patients admitted to the Department of
Pediatrics= 26,737
The number of cases initially diagnosed with bleeding disorders = 115 (115/26737=0.43%)
Excluded cases=16
• previously diagnosed bleeding disorders (n=2)
• lost to follow-up (n=11)
• incorrect diagnosis (n=3)
Number of cases initially diagnosed as bleeding disorders and included in our study= 99 (99/115=86.1%)
Number of cases definitively diagnosed as bleeding disorders= 36 (36/99=36.4%)
Number of cases received followed-up by the Department of Pediatric Hematology= 20 (20/36=55.5%)
Figure 1. Study flow-chart.
Table 2. Family history of the patients for coagulation disorders.
Number of Patient Diagnosis of Patient Family History of Coagulation Disorders
1 Factor XI deficiency Older sister, factor VIII and XI deficiencies
2 Factor V and VIII deficiencies Sibling, factor V and VIII deficiencies
3 Moderate-type hemophilia A Father and older brother, hemophilia A
4 vWD type 1 Sibling, vWD
5 vWD type 1 Mother, vWD
6 vWD type 2 Mother, vWD
7 vWD type 2 Uncle, hemophilia A
vWD: Von Willebrand disease.
and 33.3% of the patients with menorrhagia (2 of 6 patients;
1 a hemophilia B carrier and 1 with FX deficiency) were
diagnosed with a bleeding disorder after the first evaluation
due to clinic and laboratory results.
Unfortunately, only 20 of 36 patients with bleeding
disorders could be evaluated in the Division of Pediatric
Hematology and Oncology. When these 20 patients were
evaluated again, 16 of them (16/20, 80%) were confirmed
to have the same diagnosis that the general pediatrician had
established, whereas 4 of them had different diagnoses. One
patient with factor V and VIII deficiencies at the Division of
Ambulatory Pediatrics was diagnosed with factor V deficiency
at the Division of Pediatric Hematology and Oncology. Another
patient with probable vWD type 1 and factor XI deficiency
was diagnosed with vWD type 1. Furthermore, a patient with
probable mild-type hemophilia A and a probable hemophilia
A carrier were not diagnosed with a bleeding disorder after
the evaluation in the Division of Pediatric Hematology and
Oncology (Table 3).
Discussion
The first step for a patient with a suspected bleeding
disorder is to get a detailed medical history, such as initial
time of bleeding; history of any traumas; patient’s operation
history; circumcision history for male patients; any known
liver, kidney, or malabsorption-related disorders; and the
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Turk J Hematol 2015;32:338-343
Yıldız İ, et al: Evaluation of Children with Bleeding Symptoms
Table 3. Laboratory test results of the patients diagnosed with different bleeding disorders.
Number of
Patient
At Division of Ambulatory
Pediatrics
1 Factors V and VIII
deficiencies
At Division of Pediatric
Hematology and Oncology
Diagnosis Levels of factors Diagnosis Levels of factors
2 vWD type 1 and factor
XI deficiency
Factor V (%): 7
Factor VIII (%): 34
vWF antigen (%): 30
Factor XI (%): 28
Factor V deficiency Factor V (%): 16
Factor VIII (%): 110
vWD type 1 vWF antigen (%): 20
Factor XI (%): 80
3 Hemophilia A carrier Factor VIII (%): 46 Normal Factor VIII (%): 102
4 Mild-type hemophilia A Factor VIII (%): 24 Normal Factor VIII (%): 102
vWD: Von Willebrand disease, vWF: von Willebrand factor.
history of bleeding disorders in other family members. In
physical examination, location and type of the bleeding
and any accompanying signs should be investigated. First
diagnosis can be made for most of the patients with careful
medical history and physical examination, and final diagnosis
can be made with laboratory tests [1,2,3].
The most common congenital coagulation disorders
in childhood are vWD, hemophilia A and B, and factor XI
deficiency [1,10]. Nevertheless, rare factor deficiencies may
also be seen in childhood [11]. In our study, the patients who
had bleeding disorders were mostly diagnosed with vWD, mildtype
hemophilia A, and factor XI deficiency. In this study, vWD
was the most frequently diagnosed disease, in accordance with
the literature. The relatively high rate of rare factor deficiencies
may be explained by consanguinity of parents. In addition, our
university’s hospital is a tertiary hospital.
The most commonly seen symptoms in patients with
bleeding disorders are easy bruising, recurrent epistaxis,
prolonged bleeding after circumcision or teeth extraction,
menorrhagia, and hemarthrosis [1,4,8,12]. However, these
symptoms may vary by age. For example, in the neonatal
period, umbilical bleeding, cephalic hematoma, and
hematoma and ecchymosis at injection sites can be seen; in
infants, mucosal bleeding, easy bruising, and hemarthrosis
when the child starts to walk; and in older children, bleeding
after surgical procedures [1,7,10]. Nose, skin, and oral
mucosal bleedings are easily recognized by parents, whereas
gastrointestinal and genitourinary bleeding may not be easily
noticed. Therefore, anamnesis is very important. In our study,
the most commonly seen symptoms were epistaxis, easy
bruising, menorrhagia, and gingival bleeding. Epistaxis is a
symptom commonly seen in the general population. Epistaxis
may be a symptom of a bleeding disorder, but it may also be due
to trauma, nose-picking, sinusitis, rhinitis, nasal polyps, and
high blood pressure. In patients manifesting with recurrent
epistaxis, the rate of detecting a bleeding disorder is 5.5%-33%
[13,14]. In our study, of all of patients who were diagnosed
with a bleeding disorder, 28% of them had epistaxis. These
findings are consistent with the literature data [14].
The rate of bleeding disorders in adult patients with
menorrhagia is 15%, but in adolescent patients the rate goes up
to 10%-40% [15]. In our study, 2 patients out of 6 (33.3%) with
menorrhagia had a bleeding disorder. Therefore, in adolescents
with menorrhagia, existence of an underlying bleeding disorder
should be investigated. The final diagnosis was different in 4
cases. This shows that a second laboratory evaluation should
be done for all patients with bleeding symptoms [16].
In conclusion, rational approaches to children who present
with bleeding symptoms require detailed history taking and
careful physical examination, followed by adequate laboratory
tests to confirm the initial diagnosis. In this study, about 40%
of the children presenting with bleeding symptoms were
diagnosed with a bleeding disorder in our outpatient clinic
after the first evaluation. Additionally, an underlying bleeding
disorder should be considered in a child with menorrhagia.
Ethics Committee Approval: The study was conducted
with approval of all the faculty members in our department,
Informed Consent: The study was conducted with assessment
based on the database from hospital’s automation system and
the patient’s charts retrospectively, Concept: İsmail Yıldız,
Ayşegül Ünüvar, Design: Emin Ünüvar, Fatma Oğuz, Ayşe
Kılıç, Data Collection or Processing: İbrahim Kamer, Serap
Karaman, Ezgi Uysalol, Analysis or Interpretation: İsmail
Yıldız, Ayşegül Ünüvar, Literature Search: İsmail Yıldız,
Writing: İsmail Yıldız, Ayşegül Ünüvar.
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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Yıldız İ, et al: Evaluation of Children with Bleeding Symptoms
Turk J Hematol 2015;32:338-343
References
1. Lanzkowsky P. Disorders of platelets-hemostatic disorders.
In: Lanzkowsky P (ed). Manual of Pediatric Hematology and
Oncology. 5th ed. Amsterdam, Elsevier Academic Press, 2011.
2. Coller BS, Schneiderman PI. Clinical evaluation of
hemorrhagic disorders. In: Hoffmann R, Benz JE, Shattil JS
(eds). Hematology: Basic Principles and Practice. 4th ed.
Philadelphia, Churchill Livingstone, 2005.
3. van Herrewegen F, Meijers JC, Peters M, van Ommen CH.
Clinical practice: the bleeding child. Part II: disorders
of secondary hemostasis and fibrinolysis. Eur J Pediatr
2012;171:207-214.
4. Khair K, Liesner R. Bruising and bleeding in infants and
children-a practical approach. Br J Haematol 2006;133:221-
231.
5. Revel-Vilk S. Clinical and laboratory assessment of the bleeding
pediatric patient. Semin Thromb Hemost 2011;37:756-762.
6. Ünüvar A. Kanamalı çocukta laboratuvar testleri ve
değerlendirilmesi. Güncel Pediatri Dergisi 2007;5:42-45.
7. Sadler JE. New concept in von Willebrand disease. Annu Rev
Med 2005;56:173-191.
8. Rydz N, James PD. Why is my patient bleeding or bruising?
Hematol Oncol Clin North Am 2012;26:321-344.
9. Sarnaik A, Kamat D, Kannikeswaran N. Diagnosis and
management of bleeding disorder in a child. Clin Pediatr
(Phila) 2010;49:422-431.
10. Avcı Z, Özbek N. Diagnosis and treatment of hemorrhagic
diathesis. Turkish J Pediatr 2011;1:81-89.
11. Taşkesen M, Okur N, Okur N, Katar S, Menteş SE, Söker M.
Evaluation of fourteen patients with rare coagulation factor
deficiencies in childhood. J Child 2008;8:183-186.
12. Celkan T, Yılmaz İ, Demirel A, Çam H, Karaman S, Doğru Ö,
Apak H, Özkan A, Taştan Y, Yıldız İ. Bleeding disorders in
pediatric emergency department. Turk Arch Ped 2006;41:146-
150.
13. Gifford TO, Orlandi RR. Epistaxis. Otolaryngol Clin North
Am 2008;41:525-536.
14. Sandoval C, Dong S, Visintainer P, Ozkaynak MF, Jayabose S.
Clinical and laboratory features of 178 children with recurrent
epistaxis. J Pediatr Hematol Oncol 2002;24:47-49.
15. Rodriguez V, Alme C, Killian JM, Weaver AL, Khan SP, Simmons
PS. Bleeding disorders in adolescents with menorrhagia: an
institutional experience. Haemophilia 2013;19:101-102.
16. Bidlingmaier C, Treutwein J, Olivieri M, Kurnik K. Repeated
coagulation testing in children. Does it improve the diagnostic
value? Hamostaseologie 2011;31(Suppl 1):51-56.
343

Research Article
DOI: 10.4274/tjh.2014.0204
Turk J Hematol 2015;32:344-350
Evaluation of Alpha-Thalassemia Mutations in Cases
with Hypochromic Microcytic Anemia: The İstanbul
Perspective
Hipokromik Mikrositer Anemili Olgularda Alfa Talasemi
Mutasyonlarının Değerlendirmesi: İstanbul Perspektifi
Zeynep Karakaş 1 , Begüm Koç 1 , Sonay Temurhan 2 , Tuğba Elgün 2 , Serap Karaman 1 , Gamze Asker 3 ,
Genco Gençay 3 , Çetin Timur 4 , Zeynep Yıldız Yıldırmak 5 , Tiraje Celkan 6 , Ömer Devecioğlu 1 , Filiz Aydın 2
1İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey
2İstanbul University İstanbul Faculty of Medicine, Department of Medical Biology, İstanbul, Turkey
3İstanbul University İstanbul Faculty of Medicine, Department of Pediatrics, İstanbul, Turkey
4Göztepe Education and Research Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey
5Şişli Etfal Education and Research Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey
6İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey
Abstract:
Objective: Alpha thalassemia syndromes are caused by mutations on one or more of the four α-globin genes. Mutations could
be either more commonly deletional or non-deletional. As some deletions (3.7 and 4.2) cause α + -thalassemia, some cause
(-20.5, MED, THAI, FIL) α 0 -thalassemia. The aim of this study was to determine alpha thalassemia mutations in patients with
unsolved hypochromic microcytic anemia and to evaluate types of mutations.
Material and Methods: Two hundred six patients with hypochromic microcytic anemia were evaluated for alpha
thalassemia. A venous blood sample of 2 mL was drawn from each patient for DNA isolation. The samples were investigated
for α-thalassemia mutations by using the Vienna Lab α-Globlin StripAssay TM commercial kit.
Results: Fourteen different mutations were determined in 95 (46.1%) patients. The most common mutation was the 3.7
single gene deletion and was found in 37 patients (n=37/95, 39%). Others common mutations were the 20.5 kb double gene
deletion (n=20 patients, 21%), MED double gene deletion (n=17 patients, 17.9%), α2 IVS1 (n=10 patients, 10.5%), α2 cd142
Hb Koya Dora (n=6 patients, 6.3%), α2 polyA1 (Saudi type) (n=6 patients, 6.3%), 4.2 single gene deletion (n=4 patients, 4.2%),
α1 cd14 (n=2 patients, 2.1%), and -FIL mutation (n=2 patients 2.1%), respectively. Hb Adana, Hb Icaria, α2 init cd and α2
polyA2 (Turkish type) were found in 1% of the patients (n=1). Seven patients (7.4%) had α-thalassemia triplication. In our
study, three mutations (Hb Icaria, α1 cd14, α2 init.cd) were determined firstly in Turkey. Seven mutations (-SEA, -THAI, Hb
Constant Spring, α2 cd19, α2 cd59, α2 cd125, Hb Paksé) were not determined in this study.
Conclusion: Alpha thalassemia should be considered in the differential diagnosis of hypochromic microcytic anemia
especially in cases without iron deficiency and b-thalassemia carrier state. Genetic testing should be performed for the
suspicious cases. We also recommend that a national database with all mutations in Turkey should be created to screen the
alpha thalassemia cost-effectively.
Keywords: Anemia, Alpha thalassemia, Hb Adana, Hb Icaria, Hb Koya Dora, Mutation, Thalassemia
Address for Correspondence: Begüm KOÇ, M.D.,
İstanbul University İstanbul Faculty of Medicine, Department of Pediatric Hematology-Oncology, İstanbul, Turkey
Phone: +90 505 906 27 91 E-mail: begumsirins@hotmail.com
Received/Geliş tarihi : May 25, 2014
Accepted/Kabul tarihi : October 13, 2014
344

Karakaş Z, et al: Alpha-Thalassemia Mutations: The İstanbul Perspective
Turk J Hematol 2015;32:344-350
Öz:
Amaç: Alfa talasemi sendromları, bir ya da daha fazla a-globin genindeki mutasyonlardan kaynaklanır. Mutasyonlar genelikle
delesyonel olmakla birlikte non-delesyonel de olabilir. Bazı delesyonlar (3.7 ve 4.2) α + -talasemiye neden olurken bazıları da (-20.5,
MED, THAI, FIL) α 0 -talasemiye yol açar. Bu çalışma ile İstanbul ilinde, diğer nedenlerle açıklanamayan hipokrom mikrositer
anemili olgularda alfa talasemi mutasyonlarını belirlemeyi ve mutasyon tiplerini değerlendirmeyi amaçladık.
Gereç ve Yöntemler: Bu çalışmada 206 hasta alfa talasemi için değerlendirmeye alındı. Her hastadan DNA izolasyonu için
2 ml venöz kan örneği alındı. Strip analiz kiti (ViennaLab Diagnostics GmbH, Austria) kullanılarak alfa talasemi mutasyonları
araştırıldı.
Bulgular: Doksan beş hastada (%46,1) 14 farklı mutasyon tespit edildi. En sık saptanan mutasyon 3.7 tek gen delesyonu idi
(n=37 hasta, %39). Diğer mutasyonlar sıklık sırasına göre; 20,5 kb çift gen delesyonu (n=20, %21), MED çift gen delesyonu (n=17,
%17,9), α2 IVS1 (n=10, %10,5), α2 poly-A1 (Suudi tip) (n=6, %6,3), Hb Koya Dora (n=6, %6,3), 4.2 tek gen delesyonu (n=4,
%4,2), FIL mutasyonu (n=2, %2,1) ve α1 cd 14 (n=2, %2,1) idi. Hb Adana (n=1), Hb Ikaria (n=1), α2 init cd (n=1) ve α2 poly-A2
(Türk tipi) (n=1) hastaların %1’inde saptandı. Yedi hasta alfa talasemi gen triplikasyonu (%7,4) taşıyordu. Çalışmamızda üç
mutasyon (Hb Icaria, α1 cd14, α2 init.cd) Türkiye’de ilk kez tespit edildi. Yedi mutasyon ise (-SEA, -THAI, Hb Constant Spring,
α2 cd19, α2 cd59, α2 cd125, Hb Paksé) hastalarımızda hiç saptanmadı.
Sonuç: Alfa talasemi, hipokrom mikrositer anemilerin ayırıcı tanısında özellikle de demir eksikliği ve beta-talasemi taşıyıcılığının
saptanmadığı durumlarda akla getirilmelidir. Şüpheli olgularda genetik açıdan mutasyon taraması yapılmalıdır. Alfa talasemi
taramasını daha uygun maliyetle yapabilmek için Türkiye’de saptanan tüm alfa talasemi mutasyonlarının toplandığı ulusal bir
veritabanı oluşturulmasını önermekteyiz.
Anahtar Sözcükler: Alfa talasemi, Anemi, Hb Adana, Hb Icaria, Hb Koya Dora, Mutasyon, Talasemi
Introduction
Alpha thalassemia syndromes are inherited autosomal
recessively and caused by defects on one or more of the
4 α-globin genes (αα/αα), leading to reduced or absent
production of the alpha-globin polypeptide chains [1,2]. The
α-globin gene mutations could be either the more common
deletion (partial (α + ) deletions or total (α 0 ) deletions) or
non-deletional types. There are reported to be more than
40 deletion mutations in various studies [2,3,4]. The most
common alpha-thalassemia mutations in the world are the
3.7 single-gene deletions. While α + -thalassemia is caused by
single-gene deletions (such as 3.7 and 4.2), α 0 -thalassemia
is caused by double-gene deletions (such as -20.5, SEA,
MED, THAI, and FIL). Three-gene deletions (α + with α 0 -
thalassemia) or a combination of two-gene deletions with
a non-deletion mutation cause HbH disease. If there are
deletion mutations on 4 α-genes, Hb Bart’s hydrops fetalis
develops [5,6]. These large deletions have particularly severe
phenotypes. On the other hand, non-deletion mutations result
in structurally abnormal and instable hemoglobin variants
such as Hb Constant Spring, which is the most common, and
Hb α TSaudi α, polyA α2, Hb Koya Dora, and Hb Quong Sze
[4,7]. Non-deletion mutations may reduce α-globin chain
synthesis more severely than most of the deletion mutations
[1]. More than 70 non-deletion mutations have been reported
[8].
The clinical course of alpha thalassemia is correlated
with the number of affected α-globin genes. There are 4
clinical definitions of α-thalassemia syndromes: 1) silent
carrier, defined as heterozygous α + -thalassemia (-α/αα)
with mostly normal hemoglobin or mild hypochromic
anemia; 2) α-thalassemia trait, defined as heterozygous α 0 -
thalassemia (--/αα) or homozygous α + -thalassemia (-α/-α)
with mild anemia; 3) HbH disease, defined as compound
heterozygous α + /α 0 -thalassemia with 3 inactive α-genes (--/-
α) with moderate hemolytic anemia; and 4) Hb Bart’s, defined
as homozygous α 0 -thalassemia (--/--) with hydrops fetalis.
Silent carriers and those with α-thalassemia trait are generally
clinically asymptomatic and do not need any treatment.
Patients with HbH disease usually have moderate anemia
with hepatosplenomegaly; some of them need periodic blood
transfusion and folic acid supplementation. Hb Bart’s causes
hydrops fetalis prenatally and is fatal if not treated with
intrauterine blood transfusions [2,3,8].
The aim of this study was to determine alpha-thalassemia
mutations in patients with unsolved hypochromic microcytic
anemia and to evaluate the types of mutations.
Materials and Methods
Two hundred six individuals either having hypochromic
microcytic anemia or being parents and/or siblings of a patient
with HbH disease were referred to our institution for screening
345

Turk J Hematol 2015;32:344-350
Karakaş Z, et al: Alpha-Thalassemia Mutations: The İstanbul Perspective
of alpha thalassemia mutations in İstanbul. A venous blood
sample of 2 mL was drawn from each patient into the EDTA
tubes for DNA isolation. In vitro amplification was made with
polymerase chain reaction (PCR) multiplex method using
Biotin marked primers belonging to alpha globin encoding
gene zones. Products of the amplification process were
investigated for mutations of the alpha globulin genes using
the Vienna Lab α-Globlin Strip Assay TM commercial kit
including 21 alpha thalassemia mutations. These mutations
are shown in Table 1.
Results
Ninety-five patients (46.1%) with alpha thalassemia
mutations were identified. The patients were aged from
1 to 46 years; 52 of those were male and 43 were female.
Deletion mutations were detected in 69.3% of the patients
whereas non-deletion mutations in 30.6%. The most common
mutation was the 3.7 single gene deletion and was found in
37 patients (39%). Others common mutations were the 20.5
kb double gene deletion (n=20 patients, 21%), MED double
gene deletion (n=17 patients, 17.9%), α2 IVS1 (n=10 patients,
10.5%), α2 cd142 Hb Koya Dora (n=6 patients, 6.3%), α2
polyA1 (Saudi type) (n=6 patients, 6.3%), 4.2 single gene
deletion (n=4 patients, 4.2%), α1 cd14 (n=2 patients, 2.1%),
and -FIL mutation (n=2 patients 2.1%), respectively. Hb Adana,
Hb Icaria, α2 init cd and α2 polyA2 (Turkish type) were
found in 1% of the patients (n=1). Seven patients (7.4%) had
α-thalassemia triplication (Table 2). Some deletions (-SEA,
-THAI) and some mutations (α2 cd19, α2 cd59, α2 cd125,
Hb Paksé and Hb Constant Spring) were not determined in
this study (Table 1).
Fourteen distinct alpha thalassemia mutations were
detected in 95 patients. In the total of 190 alleles, the most
common mutation was the 3.7 single gene deletion (n=37
alleles, 19.5%). The allele frequencies of the other mutations
were: 20.5 kb double gene deletion (n=20 alleles, 10.5%), MED
double gene deletion (n=17 alleles, 8.9%), α2 IVS1 (n=10
alleles, 5.2%), α2 polyA1 (Saudi type) (n=7 alleles, 3.7%),
alpha triplication (n=7 alleles, 3.7%), Hb Koya Dora (n=6
alleles, 3.1%), 4.2 single gene deletion(n=4 alleles, 2.1%), α1
cd14 (n=2 alleles, 1%), and -FIL mutation (n=2 alleles, 1%),
respectively (Table 3). The allele frequencies of Hb Adana, Hb
Icaria, α2 init cd and α2 polyA2 (Turkish type) were found to
be 0.5% (Table 3). Three mutations (Hb Icaria, α1 cd14, α2
init.cd) were detected for the first time in Turkey.
The genetic results of the patients showed that 28 patients
(29.4%) were silent carriers, 45 patients (47.3%) had alpha
thalassemia trait, and 15 patients (15.8 %) had Hemoglobin H
disease (Table 2). Seven patients with alpha triplication were
not grouped phenotypically.
Discussion
According to reports from the World Health Organization,
at least 20% of the world’s population is alpha-thalassemia
carrier [9]. The geographic distribution of α-thalassemia
mutations is especially concentrated in the Mediterranean and
Middle Eastern regions, where up to 40% of people are carriers
[4,10]. Turkey also has a high alpha-thalassemia frequency
because of its geographic position.
Table 1. Positions of the 21 alpha-gene mutations in the
strip assay kit.
No Position Gene Mutation/Deletion
1 -α 3.7 Single-gene deletion
2 -α 4.2 Single-gene deletion
3 (α) 20.5 Double-gene deletion
4 --MED Double-gene deletion
5 α2 IVS1 5-bp deletion
6 ααα anti- 3.7 Gene triplication
7 α2 cd 142 A>C (Hb Koya Dora)
8 α2 polyA-1 AATAAA>AATAAG (Saudi type)
9 --FIL Double-gene deletion
10 α1 cd 14 G>A
11 α1 cd 59 G>A (Hb Adana)
12 α2 polyA-2 AATAAA>AATGAA (Turkish
type)
13 α2 cd 142 T>A (Hb Icaria)
14 α2 init.cd ATG>ACG
15* --THAI Double-gene deletion
16* --SEA Double-gene deletion
17* α2 cd 125 T>C (Hb Quong Sze)
18* α2 cd 142 T>C (Hb Constant Spring)
19* α2 cd 19 -G
20* α2 cd 142 A>T(Hb Paksé)
21* α2 cd 59 G>A
*: Not detected in this study.
346

Karakaş Z, et al: Alpha-Thalassemia Mutations: The İstanbul Perspective
Turk J Hematol 2015;32:344-350
The first publication on alpha-thalassemia from Turkey
was that of Özsoylu and Malik, who studied alpha-thalassemia
by column chromatography in 1982 [11]. The first screening
study of alpha-thalassemia by sensitive DNA method (gene
mapping) was published in 1989 [12]. The frequency of
alpha-thalassemia was detected at 3.6%, while the frequency
of alpha-gene triplication was also 3.6%. Arcasoy reported
that the frequency of alpha-thalassemia in Turkey was
0.25% [13]. Kılınç et al. studied the cord blood of newborns
and reported the frequency of alpha-thalassemia carriers to
Table 2. Genotypes and phenotypes of the patients with alpha-thalassemia.
Genotype Mutation Type Phenotype n (%)
α -3.7 α/α α Deletional Silent carrier 19
(αα) -MED /αα Deletional α-thal trait 14
α- 3.7 α/ α -3.7 α Deletional α-thal trait 7
(αα)- 20.5 /αα Deletional α-thal trait 13
(αα)- 20.5 /α-3.7 α Deletional HbH 5
(αα)- MED /α-4.2 α Deletional HbH 2
α- 4.2 α/α Deletional Silent carrier 1
(αα)- FIL /α-3.7 α Deletional HbH 2
Total 63 (66.3%)
α IVS1 α/αα Non-deletional Silent carrier 3
α HbKD* α/αα Non-deletional Silent carrier 4
α HbIC** α/αα Non-deletional Silent carrier 1
α PA1 α/αα Non-deletional α-thal trait 4
α IVS1 α/α IVS1 α Non-deletional α-thal trait 2
α PA1 α/α PA1 α Non-deletional HbH 1
αα cd14 /αα Non-deletional α-thal trait 1
αα cd14 /α HbKD *α Non-deletional HbH 1
α init.cd*** α/α PA1 α Non-deletional α-thal trait 1
Total 18 (19%)
α -3.7 α/α IVS1 α Deletional/non-deletional α-thal trait 2
(αα) -20.5 /α IVS1 α Deletional/non-deletional HbH 2
(αα) -MED /α PA2 α Deletional/non-deletional HbH 1
α -4.2 α/α IVS1 α Non-deletional α-thal trait 1
α -3.7 α/αα cd59 Deletional/non-deletional HbH 1
Total 7 (7.3%)
α anti-3.7 α/αα Triplication 5
α anti-3.7 α/α HbKD *α Triplication/non-deletional 1
α anti-3.7 α/α -3.7 α Triplication/deletional 1
Total 7 (7.3%)
Total 95 (100%)
*HbKD: Hb Koya Dora, **HbIC: Hb Icaria, n: patient number.
347

Turk J Hematol 2015;32:344-350
Karakaş Z, et al: Alpha-Thalassemia Mutations: The İstanbul Perspective
Table 3. The allele frequencies of the alpha thalassemia mutations in different studies from Turkey.
Alpha
Thalassemia
Mutations
İstanbul
present
study,
2014,
n=95, %
Aegean
Onay et al.,
2013,
n=229,%
[24]
Hatay
Celik et
al., 2013,
n=97,%
[19]
Isparta
Sütçü et
al., 2011,
n=9,%
[18]
Adana
Guvenc et
al., 2010,
n=225,%
[17]
Çukurova
Çürük,
2007,
n=32,%
[20]
Turkey
Oner et
al., 1997,
n=25,%
[21]
Cyprus
Baysal et
al., 1995,
n=78, %
[25]
Study
population
Patients
with HMA
Patients
with HMA
Patients
with
HMA
Material Method Strip assay Strip assay Strip
assay
- Premarital
couples/
Patients
with HMA
Strip
assay
Strip assay
Patients with
HbH
Gene
mapping by
Q probe
Patients
with HbH
-α 3.7 23.1 52.2 43.8 5.5 40.6 29.6 28 30
(α) 20.5 9.4 14.2 0.5 11.1 3.3 18.8 22 4
--MED 8.9 11 5.6 27.7 9.5 14.1 20 40
α2 IVS1
(α2 -5nt )
DNA
6.3 4 6.7 5.5 0 4.7 8 10
ααα anti 3.7 3.7 3.2 1.5 0 1.1 0 0 0
α2 cd142
(Hb Koya Dora)
3.1 1.8 0 0 0 0
α2 polyA-1 3.7 9 0.5 0 0.7 4.7 0 12
-α 4.2 2.1 0.5 0 0.6 1.6 12 12
α1 cd 14 1 0 0 0 0
--FIL 1 3.2 0.5 0 0 0 0 0
α2 polyA-2 0.5 1.4 2.5 0 2 7.8 10 4
α2 cd 142
(Hb Icaria)
0.5 0 0 0 0
α2 init.cd 0.5 0 0 0 0 0 0
Patients
with HbH
DNA
α1 cd 59
(Hb Adana)
0.5 0 0 0 6.2
n: Patients with alpha thalassemia mutations, *HMA: Hypochromic microcytic anemia.
be 2.9% in the Adana region in 1986, while Canatan et al.
reported the frequency of alpha-thalassemia as 2.5%-6.5%
in the Antalya region [14,15,16]. Guvenc et al. reported the
incidence of α-thalassemia as 7.5% in selected patients in the
Adana region [17]. The diagnosis of alpha-thalassemia is also
important in patients with unsolved hypochromic microcytic
anemia. We found a rate of alpha-thalassemia as high as 46.1%
among selected patients with hypochromic microcytic anemia
in İstanbul.
The types of alpha-thalassemia mutations are variable
depending on geographic region. Although 21 mutations were
screened, we found 14 different alpha-thalassemia mutations
in patients who lived in İstanbul. The most common mutations
were the 3.7 single-gene deletion, 20.5 double-gene deletion,
MED double-gene deletion, α2 IVS-1, 3.7 gene triplication,
Hb Koya Dora, α2 polyA1, 4.2 single-gene deletion, α1 cd 14,
and FIL mutation, respectively. These mutations were seen
in 95% of our patients, similar to reports from other parts of
Turkey (Table 3).
348

Karakaş Z, et al: Alpha-Thalassemia Mutations: The İstanbul Perspective
Turk J Hematol 2015;32:344-350
According to the study by Guvenc et al., the rate of 3.7-kb
deletion was extremely high (53.3%) in selected patients from
Adana [17]. They also performed the largest study in Turkey
with 3000 premarital couples and anemic patients, excluding
those with iron deficiency, and they detected alpha-thalassemia
mutations in 225 patients. They demonstrated 11 different
genotypes; the 3.7 single-gene deletion and MED double-gene
deletion were the most prevalent genotypes in their study.
Sütçü et al. reported that the most common alphathalassemia
mutations were the MED double-gene deletion,
20.5-kb double gene deletion, 3.7 single-gene deletion, and
α2IVS 1-5 nt, respectively, in the Isparta area in the south of
Turkey [18]. Although they tested few patients and detected
mutations in only 9 patients, their most common mutations
were similar to those of other studies in Turkey.
Celik et al. demonstrated 9 distinct mutations and the
frequencies of the mutations in Hatay [19]. They tested 330
individuals and detected mutations in 97 patients. Their
inclusion criteria for the study were similar to ours. They
reported that 3.7 single-gene deletions were the most common
mutation at 43.81%. In addition, they reported FIL doublegene
deletion for the first time in 2012 in Turkey. We also
detected FIL mutation in 2 patients in İstanbul. In their study,
deletion mutations were detected in 81.8% of the patients and
non-deletion mutations in 18.2%, whereas deletion mutations
were found in 69.3% and non-deletion mutations in 30.6% in
our study. These findings are similar to those of other studies
addressing alpha-thalassemia in the world.
HbH is the most common condition that arises from
the deletions of 3 α-globin genes (--/-α) and rarely by the
combination of deletion and non-deletion mutations affecting
the α-globin genes. There are also published studies from
Turkey about HbH disease [20,21,22,23]. Çürük reported
mutations in 32 patients with HbH disease [20]. In that study,
20 patients with HbH had 3 alpha-gene deletions, while the
remaining 12 cases were caused by the combination of alphagene
deletion and point mutation. In our study, 15 patients
were evaluated as having HbH disease; 9 of them had 3 α-globin
gene deletions, 2 of them had non-deletion mutations, and the
other cases were caused by the combination of deletion and
non-deletion mutations, as shown in Table 2.
We found a very heterogeneous distribution of alphathalassemia
mutations. This heterogeneity could be because
İstanbul is the city in Turkey receiving the most migrants. We
present the results of our study and other studies from Turkey
in Table 3 [17,18,19,20,21,24,25]. In our study, we found 3
mutations that not been reported previously in Turkey. One-third
of the mutations from the strip assay kit were not determined,
similar to other studies from different parts of Turkey.
In our study, patients were identified as being silent carriers,
having alpha-thalassemia traits, or having HbH disease on the
basis of genetic mutations. For example, patients with single-gene
deletion were defined as silent carriers. We determined clinical
definitions for the patients according to their genetic mutations.
Most silent carriers have normal hemoglobin levels in the general
population. Our results showed that the silent carriers had
mild hypochromic microcytic anemia because our study group
included only patients with hypochromic microcytic anemia.
Finally, silent carriers of alpha-thalassemia could be mostly
normal or mildly anemic, as shown in the literature [2,3,8].
Screening for the 7 most common mutations, present
in >95% of patients, is recommended in Canada [26]. The
strip assay method for alpha-thalassemia genetic testing can
be used effectively due to homogeneity of mutations in the
public. It is also cost-effective for the most commonly seen
mutations in patients with otherwise unexplained, longstanding,
hypochromic microcytic anemia.
In conclusion, we found a high rate (46.1%) of alphathalassemia
mutations among patients with long-standing
hypochromic microcytic anemia. Alpha-thalassemia should
be considered in the differential diagnosis of hypochromic
microcytic anemia, especially in cases without iron deficiency
and α-thalassemia carrier states. Genetic testing should
be performed for these suspicious cases. Furthermore, we
recommend that a national database including all mutations
in Turkey should be created to screen alpha-thalassemia
mutations cost-effectively.
Conflict of Interest Statement
The author of this paper have no conflicts of interest,
including specific financial interests, relationships, and/or
affiliations relevant to the subject matter or materials included.
Informed Consent: It was taken, Concept: Zeynep
Karakaş, Begüm Koç, Design: Zeynep Karakaş, Begüm Koç,
Data Collection or Processing: Zeynep Karakaş, Begüm Koç,
Serap Karaman, Gamze Asker, Genco Gençay, Çetin Timur,
Zeynep Yıldız Yıldırmak, Tiraje Celkan, Ömer Devecioğlu,
Analysis or Interpretation: Filiz Aydın, Sonay Temurhan,
Tuğba Elgün, Literature Search: Zeynep Karakaş, Begüm Koç,
Writing: Zeynep Karakaş, Begüm Koç.
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.
349

Turk J Hematol 2015;32:344-350
Karakaş Z, et al: Alpha-Thalassemia Mutations: The İstanbul Perspective
References
1. Harteveld CL, Higgs DR. α-Thalassaemia. Orphanet J Rare
Dis 2010;5:13.
2. Singer ST. Variable clinical phenotypes of alpha-thalassemia
syndromes. Scientific World Journal 2009;9:615-625.
3. Kohne E. Hemoglobinopathies: clinical manifestations,
diagnosis, and treatment. Dtsch Arztebl Int 2011;108:532-
540.
4. Vichinsky EP. Alpha thalassemia major--new mutations,
intrauterine management, and outcomes. Hematology Am
Soc Hematol Educ Program 2009:35-41.
5. Chui DH, Waye JS. Hydrops fetalis caused by alphathalassemia:
an emerging health care problem. Blood
1998;91:2213-2222.
6. Vichinsky EP. Clinical manifestations of α-thalassemia. Cold
Spring Harb Perspect Med 2013;3:011742.
7. Galanello R, Cao A. Gene test review. Alpha-thalassemia.
Genet Med 2011;13:83-88.
8. Rachmilewitz EA, Giardina PJ. How I treat thalassemia. Blood
2011;118:3479-3488.
9. Modell B, Darlison M. Global epidemiology of haemoglobin
disorders and derived service indicators. Bull World Health
Organ 2008;86:480-487.
10. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: an
increasing global health problem. Bull World Health Organ
2001;79:704-712.
11. Özsoylu Ş, Malik SA. Incidence of alpha thalassemia in
Turkey. Turk J Pediatr 1982;24:235-244.
12. Fei YJ, Kutlar F, Harris HF, Wilson MM, Milana A, Sciacca P,
Schiliro G, Masala B, Manca L, Altay C, Gurgey A, Huisman
TJH. A search for anomalies in the zeta, alpha, beta, and
gamma globin gene arrangements in normal black, Italian,
Turkish, and Spanish newborns. Hemoglobin 1989;13:45-
65.
13. Arcasoy A. Türkiye’de Thalassemia Taşıyıcı Sıklığı. Ankara,
Turkey, Ankara Thalassemia Association, 1991 (in Turkish).
14. Kılınç Y, Kumi M, Gürgey A, Altay C. Adana Bölgesi’nde doğan
bebeklerde kordon kanı çalışması ile β-talassemi, glukoz-
6-fosfat dehidrogenaz enzim eksikligi ve Hb S sıklığının
araştırılması. DOĞA 1986;10:162-167 (in Turkish).
15. Canatan D. Türkiye’de hemoglobinopatilerin epidemiyolojisi.
HematoLog 2014;4-1:11-23 (in Turkish).
16. Canatan D, Oğuz N, Guvendik İ, Yıldırım S. The incidence
of alpha-thalassemia in Antalya, Turkey. Turk J Haematol
2002;19:433-434.
17. Guvenc B, Yildiz SM, Tekinturhan F, Dincer S, Akyuzluer I,
Okten S, Erkman H. Molecular characterization of alphathalassemia
in Adana, Turkey: a single center study. Acta
Haematol 2010;124:197-200.
18. Sütçü R, Aylak F, Koçak H, Sipahi T, Vural H, Delibaş N. The
investigation of distribution of hereditary alpha-thalassemia
mutations in Isparta reservoir. Eur J Basic Med Sci 2011;1:28-
32.
19. Celik MM, Gunesacar R, Oktay G, Duran GG, Kaya H.
Spectrum of α-thalassemia mutations including first
observation of --FIL deletion in Hatay Province, Turkey. Blood
Cells Mol Dis 2013;51:27-30.
20. Çürük MA. Hb H (α4) disease in Çukurova, southern Turkey.
Hemoglobin 2007;31:265-271.
21. Oner C, Gürgey A, Oner R, Balkan H, Gümrük F, Baysal
E, Altay C. The molecular basis of Hb H disease in Turkey.
Hemoglobin 1997;21:41-51.
22. Yüreğir GT, Aksoy K, Çürük MA, Dikmen N, Fei YJ, Baysal E,
Huisman TH. Hb H disease in a Turkish family resulting from
the interaction of a deletional alpha-thalassemia-1 and newly
discovered poly A mutation. Br J Haematol 2008;80:527-532.
23. Çürük MA, Kilinç Y, Evrüke C, Özgünen FT, Aksoy K,
Yüreğir GT. Prenatal diagnosis of Hb H disease caused by
alpha homozygosity for the α2 poly A(AATAAA-AATAAG)
mutation. Hemoglobin 2001;25:255-258.
24. Onay H, Aykut A, Karaca E, et al. Ege bölgesinde alfa
talasemi mutasyonlarının dağılımının araştırılması. İçinde: 1.
Hematolojik Genetik Sempozyumu Bildiri Özet Kitabı, İzmir,
Türkiye, 2013, p. 95 (in Turkish).
25. Baysal E, Kleanthous M, Bozkurt G, Kyrri A, Kalogirou E,
Angastiniotis M, Ioannou P, Huisman TH. Alpha-thalassaemia
in the population of Cyprus. Br J Haematol 1995;89:496-499.
26. Waye JS, Eng B. Diagnostic testing for a-globin gene disorders
in a heterogeneous North American population. Int J Lab
Hematol 2013;35:306-313.
350

Brief Report
DOI: 10.4274/tjh.2014.0149
Turk J Hematol 2015;32:351-354
The Efficacy and Safety of Procedural Sedoanalgesia with
Midazolam and Ketamine in Pediatric Hematology
Çocuk Hematolojide Midazolam ve Ketaminle Uygulanan İşlemsel
Sedoanaljezinin Etkinliği ve Güvenilirliği
Sema Aylan Gelen, Nazan Sarper, Uğur Demirsoy, Emine Zengin, Esma Çakmak
Kocaeli University Faculty of Medicine Hospital, Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey
Abstract:
Objective: The aim of this study is to investigate the efficacy and safety of sedoanalgesia performed outside the operating
room by pediatricians trained in advanced airway management and life support.
Materials and Methods: Midazolam and ketamine were administered consecutively by intravenous route under
cardiorespiratory monitoring for painful procedures of pediatric hematology.
Results: A total of 115 patients had 237 sedoanalgesia sessions. Sedation time was 24.02±23.37 s and sedation success was
92.5% (Ramsay scores of ≥5). Patient satisfaction was high. The recovery time was 28.81±14.4 min. Although statistically
significant (p

Turk J Hematol 2015;32:351-354
Gelen SA, et al: Procedural Sedoanalgesia in Hematology
Introduction
Lumbar puncture, bone marrow aspiration/biopsy, and
intrathecal therapy are painful procedures. In patients with
leukemia, traumatic lumbar puncture due to poor patient
stabilization is a diagnostic dilemma and may cause seeding of
the blasts into the cerebrospinal fluid from circulation [1,2].
The burden of the procedure under inadequate sedoanalgesia
can lead to refusal of the diagnostic procedure or treatment
[3].
In this study, the aim was to evaluate the efficacy and safety
of procedural sedoanalgesia performed by pediatricians and
hematologists trained in advanced airway management and
life support.
Materials and Methods
This prospective study was planned by pediatric
hematologists. The ethics committee of the center approved
the study and written informed consent was obtained.
Physicians were trained in advanced life support and had a
proficient command of the characteristics and pharmacology
of the sedatives/analgesics. One of the physicians performed
the invasive procedure, and the other administered the drugs,
assisted in patient monitoring, and recorded the vital signs and
sedation and recovery times. During the lumbar punctures
and intrathecal therapy a nurse assisted in proper positioning
of the patient.
Sedation time (the period to induce sedation after the
administration of both drugs) and recovery time (the period
until the patient was awake with age-appropriate behavior
and age-appropriate oriented responses to verbal and motor
stimuli after the procedure was accomplished) were recorded.
Sedation was initiated with midazolam (0.1 mg/kg/dose by
slow intravenous infusion, maximum 10 mg) and continued
with ketamine (1 mg/kg/dose by slow intravenous bolus,
maximum 100 mg). Level of sedation was assessed according
to the modified Ramsay scale (Table 1). When the score
was 5 or 6, which was considered as satisfactory sedation,
the procedure was initiated. A score of below 5 was rated as
unsatisfactory sedation. Patients were followed by the study
nurse for 4 h for any adverse events. Severe adverse events
were defined as cardiovascular collapse, airway and respiratory
events including hypoxemia requiring resuscitation, and
allergic reactions.
Statistical Analysis
Statistical analysis was performed using SPSS 2.0 (SPSS
Inc., Chicago, IL, USA). For evaluation of demographic
characteristics descriptive statistics were used, and for
intergroup comparison of the parameters that had normal
distribution the paired samples t-test was used.
Results
Between May 2012 and May 2013, a total of 237 invasive
procedures (bone marrow biopsy/aspiration, intrathecal
chemotherapy) were performed in 115 children (9.4±4.5
years, range: 10 months to 19.5 years) with sedoanalgesia.
Median sedation time was 24.02±23.37 s (range: 1-300
s). Median recovery time was 28.81±14.45 min (range: 5-90
min). In 87% (n=207) of the sessions no additional dose
was administered. Due to prolongation of the procedures
or unsatisfactory sedation 1 additional dose of midazolam,
1 additional dose of ketamine, and 2 additional doses of
ketamine were administered in 2 (0.8%), 29 (12.2%), and 1
(0.8%) of the sedoanalgesia sessions, respectively. Out of 32
additional doses, 17 (53%) were administered due to multiple
painful procedures in the same session.
Oxygen saturation was over 90% in all the patients during
sedation and at recovery. There was no apnea, respiratory
depression, or need for assisted ventilation/intubation. None
of the patients required flumazenil administration. No severe
adverse events were observed. Vital signs are shown in Table 2.
A significant increase in systolic and diastolic blood pressure,
heart rate, and respiratory rate was observed during sedation
and when the procedure was completed compared to baseline
values (p

Gelen SA, et al: Procedural Sedoanalgesia in Hematology
Turk J Hematol 2015;32:351-354
Table 2. Cardiovascular parameters, respiratory rate, and hypersalivation in invasive procedures under midazolam/
ketamine sedoanalgesia.
Before Sedation During Sedation Procedure Completed When Awake
BPsys (mmHg) 118.4±14.5 124.4±16.3 121.9±16.9 113.7±14.8
BPdia (mmHg) 69.1±12.7 77.1±13.2 73.9±13 67.2±11.8
Heart rate (bpm) 115.6±21.7 124.9±21.1 123.3±20.3 113.8±20.5
Respiratory rate
(bpm)
21.7±6.5 22.9±6.8 23.7±7 21.5±6.3
Hypersalivation 1 (0.4%) 40 (16.9%) 58 (24.5%) 12 (5.1%)
BPsys/BPdia: Systolic/diastolic blood pressure, bpm: beats per minute. Values are given as means.
adverse event rate was 14.8% (n=35). Sedation was successful
in 92.5% (n=219) of the procedures. All the procedures
were completed successfully and all the outpatients could
be discharged on the same day. Patient satisfaction was high;
when painful procedures were repeated all the patients and/or
caregivers preferred the same sedoanalgesia.
Discussion
In developing countries during painful procedures many
centers perform no sedoanalgesia due to limited numbers
of anesthesiologists, busy operation rooms, and inadequate
training in sedoanalgesia, advanced airway management,
and life support [4]. In many studies, it has been shown
that sedation and analgesia during painful procedures were
administered with equally good results by pediatricians who
had received advanced life support training [5,6,7,8].
When midazolam and ketamine are used alone, respiratory
depression with midazolam and dysphoric reactions
(irritability, depression, etc.) with ketamine may occur. When
midazolam is used with ketamine, faster analgesia, amnesia,
and fewer side effects occur [9,10,11]. Oxygen desaturation
may increase with addition of high-dose midazolam [12].
Therefore, additional doses of ketamine are preferred. In
some previous studies with midazolam and ketamine the
incidence of oxygen desaturation was between 4.8% and
12%, whereas Ozdemir et al. reported no oxygen desaturation
[7,13,14,15,16]. In the present study none of the patients’
oxygen saturation dropped below 90%. Compared to propofol,
the combination of ketamine and midazolam was associated
with less hypoxemia [13,14].
In recent reports, similar to our findings, a significant
increase in cardiovascular parameters was seen due to
ketamine’s sympathomimetic action via inhibition of
catecholamine reuptake, but these parameters returned to
baseline values at recovery and no treatment was required
[2,14,15,16,17].
In other published studies the sedation time was similar to
that of the present study [2,14,16]. Parker et al. showed that
more than 70% of the patients woke up in 30 min, similar to
our result [9]. Short sedation and recovery time seems a good
feature of the drug combination.
Overall adverse event rate in our study was comparable
to those of some other studies [18,19,20]. Ketamine may
cause airway obstruction, laryngospasm, and aspiration by
increasing tracheal and bronchial secretions. Agents such
as atropine and glycopyrrolate can be used to reduce the
increased secretions [15,21,22]. The hypersalivation rate was
higher compared to those in the literature; this may be due to
no atropine or glycopyrrolate administration. Prone position
during and after the procedure prevented obstruction of the
airway and aspiration was not required.
Venipuncture is another painful procedure for outpatient
children, but oral, nasal, and rectal administrations of
midazolam can provide slower sedation and this may cause a
delay in the procedure. The intramuscular route is also painful
and may require additional injections. With the intravenous
route, sedoanalgesia can be achieved faster and, if necessary,
additional doses and drugs for cardiopulmonary resuscitation
can be administered easily.
Conclusion
With adherence to the published guidelines, sedoanalgesia
with intravenous midazolam and ketamine performed by two
physicians, trained in airway management and life support,
in an optimally equipped setting outside the operating room
is safe and efficient. Sedoanalgesia reduces the physical and
psychological trauma of the invasive procedures for the
patients, parents, and physicians and increases the success of
the procedures.
353

Turk J Hematol 2015;32:351-354
Gelen SA, et al: Procedural Sedoanalgesia in Hematology
Concept: Nazan Sarper, Design: Nazan Sarper, Data
Collection or Processing: Sema Aylan Gelen, Uğur Demirsoy,
Emine Zengin Esma Çakmak, Analysis or Interpretation:
Sema Aylan Gelen, Literature Search: Sema Aylan Gelen,
Nazan Sarper, Writing: Sema Aylan Gelen, Nazan Sarper.
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. Maurizi P, Russo I, Rizzo D, Chiaretti A, Coccia P, Attina G,
Ruggiero A, Riccardi R. Safe lumbar puncture under analgosedation
in children with acute lymphoblastic leukemia. Int J
Clin Oncol 2014;19:173-177.
2. Meyer S, Aliani S, Graf N, Reinhard H, Gottschling S. Sedation
with midazolam and ketamine for invasive procedures in
children with malignancies and hematological disorders: a
prospective study with reference to the sympathomimetic
properties of ketamine. Pediatr Hematol Oncol 2003;20:291-
301.
3. Sitaresmi MN, Mostert S, Schook RM, Sutaryo, Veerman AJ.
Treatment refusal and abandonment in childhood acute
lymphoblastic leukemia in Indonesia: an analysis of causes
and consequences. Psychooncology 2010;19:361-367.
4. Iannalfi A, Bernini G, Caprilli S, Lippi A, Tucci F, Messeri
A. Painful procedures in children with cancer: comparison
of moderate sedation and general anesthesia for lumbar
puncture and bone marrow aspiration. Pediatr Blood Cancer
2005;45:933-938.
5. American Society of Anesthesiologists Task Force on Sedation
and Analgesia by Non-Anesthesiologists. Practice guidelines
for sedation and analgesia by non-anesthesiologists.
Anesthesiology 2002;96:1004-1017.
6. Pitetti RD, Singh S, Pierce MC. Safe and efficacious use of
procedural sedation and analgesia by nonanesthesiologists in
a pediatric emergency department. Arch Pediatr Adolesc Med
2003;157:1090-1096.
7. Borker A, Ambulkar I, Gopal R, Advani SH. Safe and
efficacious use of procedural sedation and analgesia by nonanesthesiologists
in a pediatric hematology-oncology unit.
Indian Pediatr 2006;43:309-314.
8. Monroe KK, Beach M, Reindel R, Badwan L, Couloures KG,
Hertzog JH, Cravero JP. Analysis of procedural sedation
provided by pediatricians. Pediatr Int 2013;55:17-23.
9. Parker RI, Mahan RA, Guigliano D, Parker MM. Efficacy and
safety of intravenous midazolam and ketamine as sedation for
therapeutic and diagnostic procedures in children. Pediatrics
1997;99:427-431.
10. Pellier I, Mongrial JP, Le Moine P, Rod B, Rialland X, Granry
JC. Use of intravenous ketamine-midazolam association for
pain procedures in children with cancer: a prospective study.
Paediatr Anaesth 1999;9:61-68.
11. Marx CM, Stein J, Tyler MK, Nieder ML, Shurin SB, Blumer
JL. Ketamine-midazolam versus meperidine-midazolam for
painful procedures in pediatric oncology patients. J Clin
Oncol 1997;1:94-102.
12. Cheuk DK, Wong WH, Ma E, Lee TL, Ha SY, Lau YL, Chan
GC. Use of midazolam and ketamine as sedation for children
undergoing minor operative procedures. Support Care Cancer
2005;13:1001-1009.
13. Godoy ML, Pino AP, Córdova LG, Carrasco OJA, Castillo
MA. Sedation and analgesia in children undergoing invasive
procedures. Arch Argent Pediatr 2013;111:22-28.
14. Gottschling S, Meyer S, Krenn T, Reinhard H, Lothschuetz D,
Nunold H, Graf N. Propofol versus midazolam/ketamine for
procedural sedation in pediatric oncology. J Pediatr Hematol
Oncol 2005;27:471-476.
15. Karapinar B, Yilmaz D, Demirağ K, Kantar M. Sedation with
intravenous ketamine and midazolam for painful procedures
in children. Pediatr Int 2006;48:146-151.
16. Ozdemir D, Kayserili E, Arslanoglu S, Gulez P, Vergin C.
Ketamine and midazolam for invasive procedures in children
with malignancy: a comparison of routes of intravenous, oral
and rectal administration. J Trop Pediatr 2004;50:224-228.
17. Roback MG, Wathen JE, Bajaj L, Bothner JP. Adverse events
associated with procedural sedation and analgesia in pediatric
emergency department: a comparison of common parenteral
drugs. Acad Emerg Med 2005;12:508-513.
18. Ozkan A, Okur M, Kaya M, Kaya E, Kucuk A, Erbas M,
Kutlucan L, Sahan L. Sedoanalgesia in pediatric daily surgery.
Int J Clin Exp Med 2013;6:576-582.
19. Migdady MI, Hayajneh WA, Abdelhadi R, Gilger MA. Ketamine
and midazolam sedation for pediatric gastrointestinal
endoscopy in the Arab world. World J Gastroenterol
2011;17:3630-3635.
20. Wood M. The use of intravenous midazolam and ketamine in
pediatric dental sedation. SAAD Dig 2013;29:18-30.
21. Wathen JE, Roback MG, Mackenzie T, Bothner JP. Does
midazolam alter the clinical effects of intravenous ketamine
sedation in children? A double-blind, randomized, controlled,
emergency department trial. Ann Emerg Med 2000;36:579-588.
22. Ramaiah R, Bhananker S. Pediatric procedural sedation
and analgesia outside the operating room: anticipating,
avoiding and managing complications. Expert Rev Neurother
2011;11:755-763.
354

Case Report
DOI: 10.4274/tjh.2014.0416
Turk J Hematol 2015;32:355-358
A Hemophagocytic Lymphohistiocytosis Case with Newly
Defined UNC13D (c.175G>C; p.Ala59Pro) Mutation and a
Rare Complication
Yeni Tanımlanan UNC13D (c.175G>C; p.Ala59Pro) Mutasyonlu
Hemofagositik Lenfohistiositozlu Bir Hasta ve Nadir Komplikasyon
Yasemin Işık Balcı 1 , Funda Özgürler Akpınar 2 , Aziz Polat 1 , Fethullah Kenar 3 , Bianca Tesi 4 , Tatiana Greenwood 4 ,
Nagihan Yalçın 5 , Ali Koçyiğit 6
1Pamukkale University Faculty of Medicine, Department of Pediatric Hematology, Denizli, Turkey
2Pamukkale University Faculty of Medicine, Department of Pediatrics, Denizli, Turkey
3Pamukkale University Faculty of Medicine, Department of Otorhinolaryngology, Denizli, Turkey
4Karolinska University Hospital Huddinge, Stockholm, Sweden
5Pamukkale University Faculty of Medicine, Department of Pathology, Denizli, Turkey
6Pamukkale University Faculty of Medicine, Department of Radiology, Denizli, Turkey
Abstract:
Hemophagocytic lymphohistiocytosis (HLH) represents a severe hyperinflammatory condition with cardinal symptoms
of prolonged fever, cytopenias, hepatosplenomegaly, and hemophagocytosis by activated, morphologically benign
macrophages with impaired function of natural killer cells and cytotoxic T lymphocytes. A 2-month-old girl, who was
admitted with fever, was diagnosed with HLH and her genetic examination revealed a newly defined mutation in the
UNC13D (c.175G>C; p.Ala59Pro) gene. She was treated with dexamethasone, etoposide, and intrathecal methotrexate.
During the second week of treatment, after three doses of etoposide, it was noticed that there was a necrotic plaque
lesion on the soft palate. Pathologic examination of debrided material in PAS and Grocott staining revealed lots
of septated hyphae, which was consistent with aspergillosis infection. Etoposide was stopped and amphotericin B
treatment was given for six weeks. HLH 2004 protocol was completed to eight weeks with cyclosporine A orally. There
was no patient with invasive aspergillosis infection as severe as causing palate and nasal septum perforation during
HLH therapy. In immuncompromised patients, fungal infections may cause nasal septum perforation and treatment
could be achieved by antifungal therapy and debridement of necrotic tissue.
Keywords: Hemophagocytic lymphohistiocytosis, Invasive aspergillosis infection, UNC13D (c.175G>C; p.Ala59Pro)
Öz:
Hemofagositik lenfohistiositoz (HLH) uzamış ateş, sitopeni, hepatosplenomegali semptomları ile seyreden, active
olmuş, morfolojik olarak benign makrofaj ve doğal öldürücü hücreler ile sitotosik T lenfosit fonksiyon bozukluğu
sonucu gelişen hiperenflamatuvar bir durumdur. İki aylık düşmeyen ateş yakınması ile başvuran hasta HLH tanısı aldı
ve hastanın genetik incelemesinde UNC13D (c.175G>C; p.Ala59Pro) geninde yeni tanımlanan bir mutasyon saptandı.
Hastaya deksamatazon, etopozit ve intratekal metotreksat tedavileri başlandı. Tedavinin 2. haftasında, üç doz etopozit
aldıktan sonra, yumuşak damakta plak lezyonu fark edildi ve bu nekrotik lezyon debride edildi. Debridman
Address for Correspondence: Yasemin IŞIK BALCI, M.D.,
Pamukkale University Faculty of Medicine, Department of Pediatric Hematology, Denizli, Turkey
Phone: +90 532 547 71 79 E-mail: dryibalci@gmail.com
Received/Geliş tarihi : October 21, 2014
Accepted/Kabul tarihi : January 15, 2015
355

Turk J Hematol 2015;32:355-358
Işık Balcı Y, et al: Invasive Aspergillosis in HLH with Novel UNCD13 Mutation
materyalinin patolojik incelemesinin PAS, Grocott boyamasında aspergilloz enfeksiyonu ile uyumlu olarak çok sayıda
septalı hif görüldü. Etopozid tedavisi sonlandırılarak altı hafta boyunca amphotericin B tedavisi verildi. HLH 2004
tedavi protokolü oral siklosporin ile sekiz haftaya tamamlandı. HLH tedavisi sırasında yumuşak damak perforasyonuna
neden olacak kadar ağır aspergilloz enfeksiyonu geçiren bir olgu bildirilmemiştir. İmmünyetmezlikli hastada mantar
enfeksiyonları nazal septum perforasyonuna neden olabilmektedir ve tedavi nekrotik dokunun debridmanı ve antifungal
tedavi ile sağlanabilmektedir.
Anahtar Sözcükler: Hemofagositik lenfohistiositoz, İnvaziv aspergilloz enfeksiyonu, UNC13D (c.175G>C;
p.Ala59Pro)
Introduction
Hemophagocytic lymphohistiocytosis (HLH) is a severe
life-threatening disease precipitated by secretion of cytokines
from morphologically benign macrophages, which ends
with uncontrolled hyperinflammation with prolonged fever,
cytopenias, hepatosplenomegaly, and hemophagocytosis.
Elevation of triglycerides, ferritin, lactate dehydrogenase,
and transaminase levels and decreases in fibrinogen levels are
characteristic findings [1]. Impaired cytotoxic function of T
cells and natural killer cells is a known cause of familial forms
of HLH. The HLH-2004 protocol with immunomodulatory
and cytotoxic drugs is used for treatment of patients with HLH
[2]. Importantly, invasive infections have been reported in up
to 56% of children with HLH on chemotherapy, with invasive
fungal infections causing 50% of deaths among such cases [3].
Here we report an unusual aspergillosis infection with
palate and nasal septum perforation following chemotherapy
in a patient with familial HLH with a novel mutation in
UNC13D. Notably, the fungal infection in our patient was
treated successfully with antifungal therapy and surgical
debridement.
Case Presentation
A 2-month-old girl, born subsequent to a term gestation
with unrelated parents and an unremarkable previous history,
was referred to our clinic with unremitting fever since 1 month
despite repeated intravenous administrations of antibiotics.
There was no history of sibling death in her family. On
admission, vital signs were normal except body temperature
of 38.7 °C. The patient was pale with petechial rashes on the
lower extremities. She displayed hepatomegaly (6 cm below
costal margin) and splenomegaly (3 cm below costal margin).
Informed consent was obtained.
The patient’s laboratory findings were as follows;
hemoglobin: 63 g/L, mean corpuscular volume (MCV): 88.9
fL, total leukocyte count: 2.84x10 9 /L, thrombocyte count:
10x10 9 /L, alanine aminotransferase (ALT): 50 IU/L, aspartate
aminotransferase (AST): 62 IU/L, total bilirubin: 0.58 mg/
dL, direct bilirubin: 0.7 mg/dL, gamma glutamyl transferase:
328 U/L, albumin: 2.9 g/dL, ferritin: 2000 ng/mL, triglyceride:
617 mg/dL, LDL cholesterol: 11 mg/dL, HDL cholesterol: 7
mg/dL, lactate dehydrogenase: 379 U/L, uric acid: 2.3 mg/dL,
fibrinogen: 107 mg/dL. Her renal function tests and electrolytes
were normal. Her peripheral smear revealed 4% neutrophils,
90% lymphocytes, and 6% monocytes. Absolute neutrophil
count was 0.113x10 9 /L. No hemolysis or blasts were visible in
her peripheral blood smear. Her transaminase levels increased
on the third day of administration (AST: 280 IU/L, ALT: 265
IU/L). Serological studies for infection with Epstein-Barr
virus, parvovirus B19, cytomegalovirus, Toxoplasma gondii,
rubella, Leishmania, and hepatitis were all negative. Natural
killer cell activity and soluble IL-2 level could not be analyzed.
Numerous histiocytes showing hemophagocytosis were
observed in the bone marrow aspiration smears.
Conclusively, the patient fulfilled a required 5 out of 6
examined diagnostic criteria for the diagnosis of HLH [1].
Accordingly, the patient was treated with the HLH-2004
protocol with dexamethasone, etoposide, and cyclosporine
A. Mutation analyses, identifying a novel homozygous variant
in UNC13D (c.175G>C; p.Ala59Pro), confirmed a diagnosis
of familial HLH. The variant was not found in the healthy
population (1000 Genomes database), and it was predicted as
possibly damaging by PolyPhen-2 but as tolerated by sorting
tolerant from intolerant (SIFT). The father was a heterozygous
carrier of the mutation, while the mother could not be tested.
During the second week of treatment, after 3 doses of
etoposide at 150 mg/m 2 /dose, a grossly necrotic soft tissue
lesion was noticed on the soft palate and was successfully
excised (Figure 1). During the operation an oronasal fistula
was revealed, as well as a perforation of the caudal side of
the nasal septum along with an abscess formation in the left
vestibular floor. Although there was no microbial growth
in the necrotic material, microscopic examination of the
debrided material in PAS and Grocott staining showed
abundant septatedhyphae, consistent with aspergillosis
infection (Figure 2). Etoposide was stopped and amphotericin
B treatment was given for 6 weeks at a dosage of 3.5 mg/kg/
day. The HLH-2004 protocol was followed for 8 weeks with
cyclosporine A and dexamethasone orally.
356

Işık Balcı Y, et al: Invasive Aspergillosis in HLH with Novel UNCD13 Mutation
Turk J Hematol 2015;32:355-358
Discussion and Review of the Literature
Figure 1. Partial perforation in septal cartilage.
Figure 2. Septated hyphae with 45° angle branching in
aspergillosis (Grocott, 100 x ).
At the most recent follow-up, after 4 months, the patient
still presented with a 2-cm hepatosplenomegaly, while her
soft palate had successfully epithelialized. However, there is a
permanent deformity of her nose. Her laboratory findings were
as follows; hemoglobin: 103 g/L, MCV: 77.7 fL, total leukocyte
count: 15,280x10 9 /L, thrombocytes: 230,000x10 9 /L, ALT:
32 IU/L, AST: 12 IU/L, ferritin: 916 ng/mL, triglyceride: 421
mg/dL, LDL cholesterol: 78 mg/dL, HDL cholesterol: 20 mg/
dL, lactate dehydrogenase: 226 U/L, uric acid: 1.7 mg/dL,
fibrinogen: 233 mg/dL. Until bone marrow transplantation
she was treated with oral cyclosporine A (at the HLH-2004
protocol dosage), trimethoprim sulfamethoxazole, and
fluconazole.
Herein we describe the disease course of a patient carrying
a novel homozygous UNC13D mutation. Familial HLH cases
typically have an earlier presentation, with infectious agents
including herpes viruses such as the Epstein-Barr virus
precipitating disease. However, in our case, we did not detect an
infectious etiological agent. For treatment, chemoimmunotherapy
(etoposide, dexamethasone, cyclosporine A, and, for selected
patients, intrathecal methotrexate or corticosteroids) is
recommended, but for severe disease or familial cases hemopoietic
stem cell transplantation is life saving [4].
Opportunistic infections are a common complication of
immunosuppression caused by cytotoxic treatment of the disease
and by the disease itself. As our case illustrates, HLH patients
have a potential risk of developing invasive fungal infections that
can be severe. Aspergillus species have emerged as an important
cause of life-threatening infections in immunocompromised
patients. Highlighting the severity of invasive fungal infections,
6/12 (50%) fatal cases in a study cohort of 18 children with
primary HLH were reported to be caused by invasive fungal
infections, of which 2 cases were diagnosed with invasive
Aspergillus infection first at autopsy [5].
Aspergillus can differentiate into hyphal forms that
produce toxins damaging epithelial tissue, leading to invasion
of connective and vascular tissue by the fungi, which
subsequently can result in thrombosis and ultimately necrosis
of hard and soft tissues with perforation. Systemic antifungal
therapy and surgical resection or debridement is important
for the management of invasive sinonasalaspergillosis.
Amphotericin B, voriconazole, and caspofungincan be
considered for antifungal therapy [5]. Our case was treated
successfully with surgical debridement and 6 weeks of
amphotericin B treatment.
In the English literature, the case of a 15-year-old boy
who developed fungal infection with nasal septal perforation
after bone marrow transplantation for acute myeloid leukemia
was reported. He was also treated successfully with surgical
debridement and amphotericin B [6].
Our report represents an interesting case of familial
HLH caused by a novel homozygous UNC13D mutation and
affected by invasive sinonasal aspergillosis.
The UNC13D gene encodes for the Munc13-4 protein, a
critical effector of the exocytosis of cytotoxic granules priming
cytotoxic granule fusion. Munc13-4 deficiency impairs the
delivery of the effector proteins, perforin and granzymes, into the
target cells, resulting in defective cellular cytotoxicity and a
clinical picture that appears very similar to that of FHL-2 [7].
UNC13D mutations are present in almost 30%-40% of familial
HLH cases [8].
357

Turk J Hematol 2015;32:355-358
Işık Balcı Y, et al: Invasive Aspergillosis in HLH with Novel UNCD13 Mutation
To the best of our knowledge, no c.175G>C; p.Ala59Pro
mutation in UNC13D has been presented before in the
literature. This novel mutation may be responsible for our
patient’s severe clinical condition. However, to compare the
mutation type and clinical course, there is a need for clinical
studies.
We want to emphasize the importance of awareness of
the occurrence of potentially life-threatening invasive fungal
infections in patients with HLH. Furthermore, this highlights
the efficacy of surgical debridement and amphotericin B for
successful treatment of fungal infections with focal lesions.
Informed Consent: Informed consent was obtained,
Concept: Aziz Polat, Design: Yasemin Işık Balcı, Data
Collection or Processing: Funda Özgürler Akpınar, Analysis
or Interpretation: Bianca Tesi, Tatiana Greenwood, Fethullah
Kenar, Literature Search: Funda Özgürler Akpınar, Writing:
Funda Özgürler Akpınar, Bianca Tesi.
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
4. Horne A, Janka G, Maarten Egeler R, Gadner H, Imashuku
S, Ladisch S, Locatelli F, Montgomery SM, Webb D,
Winiarski J, Filipovich AH, Henter JI; Histiocyte Society.
Haematopoietic stem cell transplantation in haemophagocytic
lymphohistiocytosis. Br J Haematol 2005;129:622-630.
5. Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis
DP, Marr KA, Morrison VA, Segal BH, Steinbach WJ, Stevens
DA, van Burik JA, Wingard JR, Patterson TF; Infectious
Diseases Society of America. Treatment of aspergillosis:
clinical practice guidelines of the Infectious Diseases Society
of America. Clin Infect Dis 2008;46:327-360.
6. Shannon MT, Sclaroff A, Colm SJ. Invasive aspergillosis of the
maxilla in an immunocompromised patient. Oral Surg Oral
Med Oral Pathol 1990;70:425-427.
7. Cetica V, Pende D, Griffiths GM, Aricò M. Molecular basis of
familial hemophagocytic lymphohistiocytosis. Haematologica
2010;95:538-541.
8. Sieni E, Cetica V, Hackmann Y, Coniglio ML, Da Ros
M, Ciambotti B, Pende D, Griffiths G, Aricò M. Familial
hemophagocytic lymphohistiocytosis: when rare diseases
shed light on immune system functioning. Front Immunol
2014;5:167.
1. Janka GE. Hemophagocytic syndromes. Blood Rev
2007;21:245-253.
2. Henter JI, Horne A, Arico M, Horne A, Aricó M, Egeler RM,
Filipovich AH, Imashuku S, Ladisch S, McClain K, Webb D,
Winiarski J, Janka G. HLH-2004: Diagnostic and therapeutic
guidelines for hemophagocytic lymphohistiocytosis. Pediatr
Blood Cancer 2007;48:124-131.
3. Sung L, Weitzman SS, Petric M, King SM. The role of infections
in primary hemophagocytic lymphohistiocytosis: a case series
and review of the literature. Clin Infect Dis 2001;33:1644-
1648.
358

Case Report
DOI: 10.4274/tjh.2015.0016
Turk J Hematol 2015;32:359-362
The Use of Low-Dose Recombinant Tissue Plasminogen
Activator to Treat a Preterm Infant with an Intrauterine
Spontaneous Arterial Thromboembolism
Intrauterin Spontan Arteriyel Trombozlu Bir Preterm Bebeğin
Düşük Doz Rekombinan Doku Plazminojen Aktivatörü ile Tedavisi
Yaşar Demirelli, Kadir Şerafettin Tekgündüz, İbrahim Caner, Mustafa Kara
Atatürk University Faculty of Medicine, Division of Neonatology, Erzurum, Turkey
Abstract:
Neonatal thromboembolic events are rare, and only a few cases of intrauterine spontaneous arterial thromboembolisms
have been reported in the literature. Thrombolytic therapy with recombinant tissue plasminogen activator is usually
the preferred treatment because it has a short half-life, fewer systemic side effects, and a strong, specific affinity for
fibrin. Protocols vary from center to center, but there is still no consensus regarding the proper dosage or treatment
duration. Herein, we present the case of an intrauterine spontaneous arterial thromboembolism in a preterm infant
that completely resolved after being treated with low-dose recombinant tissue plasminogen activator (0.02 mg/kg/h).
Keywords: Preterm, Thromboembolism, Tissue, Plasminogen, Intrauterine arterial thromboembolism, Low dose
recombinant tPA therapy
Öz:
Yenidoğanda tromboembolik olaylar sık değildir. Literatürde spontan intrauterin arteriyel tromboz olgusu oldukça az
bildirilmiştir. Tromboemboli tedavisinde yarılanma ömrü kısa olduğu için rekombinan doku plazminojen aktivatörü
çoğunlukla tercih edilmektedir. Tedavi protokolü merkezden merkeze değişiklik göstermesine karşın, doz ve süre
konusunda bir uzlaşı yoktur. Biz burada kullanılabilecek en düşük dozlardan biriyle (0,02 mg/kg/doz) tamamen
iyileşme sağladığımız intrauterin spontan arteriyel trombozlu bir olguyu sunmak istedik.
Anahtar Sözcükler: Preterm, Tromboemboli, Doku, Plazminojen, İntrauterin arteriyel tromboembolizm, Düşük
doz rekombinant tPA tedavisi
Address for Correspondence: Kadir Şerafettin TEKGÜNDÜZ, M.D.,
Atatürk University Faculty of Medicine, Division of Neonatology, Erzurum, Turkey
Phone: +90 442 344 69 90 E-mail: k.tekgunduz@yahoo.com.tr
Received/Geliş tarihi : January 07, 2015
Accepted/Kabul tarihi : May 04, 2015
359

Turk J Hematol 2015;32:359-362
Demirelli Y, et al: Thromboembolism in a Preterm Infant
Introduction
Spontaneous arterial thromboembolisms are a serious
cause of mortality and morbidity in the neonatal period, and
the congenital, acquired, and inherited prothrombotic states
of this condition along with maternal characteristics have
been identified as significant risk factors. The hypofibrinolytic
situation in neonates, especially in premature infants, includes
hemodynamic changes during the transition from the fetal
period to the neonatal period which may predispose infants to
these types of thromboembolisms [1]. The goal of treatment is
to prevent life-threatening situations that might occur because
of the embolism and the recurrence of thrombosis while also
minimizing the risk of bleeding. Generally, recombinant tissue
plasminogen activator (rtPA) is the first choice of treatment
because it is nonantigenic, has a short half-life, produces
rapidly reversible hypocoagulability, and possesses a strong,
specific affinity for fibrin, but there is no consensus regarding
the proper dosage or treatment duration [2]. In this study,
we present the case of a premature baby with an intrauterine
spontaneous arterial thromboembolism which developed at
the level of the left brachial artery and was successfully treated
with low-dose rtPA for a short period of time.
Case Presentation
A male baby (1,530 g) was born at the 32 nd gestational week
via an emergency caesarean section because of anhydroamnios
and vaginal bleeding. The mother had not had prenatal care.
The mother was 24 years old, and neither the mother nor the
infant had any complications during the surgical procedure.
The Apgar scores at one and five minutes were 6 and 9
respectively, and the cord blood pH was normal. In addition,
there was no history of maternal diabetes nor preeclampsia.
However, the baby did receive a single dose of surfactant due
to the presence of mild respiratory distress syndrome. The
patient’s left forearm from the elbow to the fingertips had a
pale and cyanotic appearance at birth that persisted afterwards
(Figure 1), but the baby’s vital signs were normal. No brachial
arterial flow was detected from the level of the elbow nor was
there any distal radioulnar flow on Doppler ultrasonography
(USG). Furthermore, no cardiac pathology was detected on
an echocardiographic examination. The blood count was
normal, there was no polycythemia or thrombocytopenia, and
the coagulometer readings and fibrinogen values were also
within normal ranges. After obtaining the informed consent of
the infant’s parents, low-dose (0.02 mg/kg/h) rtPA (alteplase)
was administered along with low-molecular-weight heparin
(LMWH) (Clexane ® 4000 IU/0.4 mL; 100 IU/kg/dose twice
daily) in the first hour after birth. The transfontanelle USG
was normal before and after the rtPA infusion, and distal pulses
were detected by Doppler USG at the fourth hour of infusion
(Figure 2). Hence, the rtPA was discontinued although the
LMWH continued to be administered for six additional weeks.
At follow-up, no complications or thrombosis had developed,
and the screening for inherited thrombophilias [factor V
Leiden mutation, homozygous protein C and S deficiency,
prothrombin G20210A mutation, methyltetrahydrofolate
reductase (MTHFR) gene mutation, antithrombin III, factor
12, anticardiolipin antibodies, homocysteine, and lipoprotein
(a)] was normal.
Discussion and Review of the Literature
The incidence of clinically apparent neonatal thrombosis
in recent reports has varied from 5.1 per 100,000 births to 2.4
per 1,000 admissions [3,4]. Most cases of thromboembolism
during the neonatal period are due to vascular interventions;
Figure 1. View of the patient’s left forearm from the elbow to the
fingertips at birth. Note the pale, cyanotic appearance.
Figure 2. The patient experienced a complete recovery four
hours after the recombinant tissue plasminogen activator
infusion.
360

Demirelli Y, et al: Thromboembolism in a Preterm Infant
Turk J Hematol 2015;32:359-362
however, there have been very few reported cases of
intrauterine spontaneous arterial thromboembolism in the
literature [5]. Various risk factors, such as being an infant of
a diabetic mother, having polycythemia, dehydration, sepsis,
asphyxia, or oligohydroamnios, and being of the male gender
can contribute to this condition, but the pathophysiology has
not yet been fully clarified [1,6]. In the case of our patient, we
observed cyanosis in the left forearm at birth.
Saracco et al. reported that prepartum risk factors, including
emergency caesarean sections, are significantly associated
with arterial ischemic stroke in neonates, and Rashish et
al. identified decreased fetal movements, oligohydramnios,
preeclampsia, and maternal diabetes as maternal risk factors
[1,6]. In addition, they also found that when oligohydramnios
is present, decreased fetal movement may cause venous
stasis and thrombus formation. We believe that the caesarean
delivery and anhydroamnios were the primary risk factors
in our patient, but we could not determine whether the
thromboembolism occured within the uterus or during the
birth process. However, the apparent lack of necrosis in the
forearm at birth suggests that the thromboembolism occurred
just prior to delivery.
Rashish et al. hypothesized that spontaneous arterial
thromboembolisms originate in utero and develop secondary
to placental-fetal umblical pathology [1]. Furthermore, they
reported that the most common site of thromboembolisms
were, in order of frequency, the umblical artery, the aorta, and
the extremities. In our patient, the left forearm was the site of
the thromboembolism. However, no thrombophilic state was
detected in our patient, which might have been because of
intrauterine pathology.
In newborn infants, appropriate, adequate, timely
intervention of thromboembolisms is very important in order
to reduce morbidity and mortality. In the literature, there are
several studies that have reported the use of streptokinase,
urokinase, and rtPA for thrombolytic therapy, and in recent
years the popularity of rtPA has been on the rise because of
its short half-life, nonantigenic qualities, and locally specific
action on plasminogen-bound fibrin [7,8]. In addition, it
also has fewer systemic side effects than other agents used in
thrombolytic therapy [9,10]. However, this type of therapy
is associated with significant bleeding complications such as
intracranial hemorrhage, and Monagle et al. determined that
the most frequent problem was bleeding at the sites of invasive
procedures that required treatment with blood products
[11]. Furthermore, they also found a connection between
prolonged thrombolytic infusion and increased bleeding.
In our case the short duration of rtPA treatment, which was
limited to four hours, may have played a role in the prevention
of complications. However, we did inform the patient’s
parents about the possible complications before initiating the
treatment.
Many case series have been reported on the use of low
and high dose rtPa both with and without a bolus as well
as in conjuction with other anticoagulant agents, and there
is general agreement that rtPa is safe and effective. There is
still no consensus concerning the correct dosage and duration
of treatment [3,5,12]. Olgun et al. started rtPa 13 of their 22
patients (range 5 days-17 years old) with extremity or cardiac
thrombosis on low-dose treatment (0.01-0.03 mg/kg/h), and
in six of these the dosage was increased over the course of
the treatment [12]. Their findings showed that seven patients
experienced complete recovery within 4 to 36 hours and that
no significant complications were seen except for bleeding
at the vascular puncture site in two patients. However, five
patients with fibrinogen deficiency in the high-dose group
reported epistaxis and melena. In our patient, we used lowdose
rtPA (0.02 mg/kg/h) and observed a complete recovery
within four hours after the infusion without any complications.
We administered rtPA (alteplase) along with LMWH in the
first hour after birth, and this treatment has previously been
reported to be safe and effective, especially for preventing
second thrombi [13].
In conclusion, low-dose rtPA proved to be successful for
the treatment of arterial thromboembolism in our patient.
However, a randomized prospective study is needed to
determine the precise dosage and duration of this treatment
in premature newborns.
Informed Consent: Informed consent was obtained from
the parents of the patient, Concept: Yaşar Demirelli, Kadir
Şerafettin Tekgündüz, İbrahim Caner, Mustafa Kara, Design:
Yaşar Demirelli, Kadir Şerafettin Tekgündüz, Data Collection
or Processing: Yaşar Demirelli, Kadir Şerafettin Tekgündüz,
İbrahim Caner, Analysis or Interpretation: Yaşar Demirelli,
Kadir Şerafettin Tekgündüz, İbrahim Caner, Mustafa Kara,
Literature Search: Yaşar Demirelli, İbrahim Caner, Writing:
Yaşar Demirelli, Kadir Şerafettin Tekgündüz.
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. Rashish G, Paes BA, Nagel K, Chan AK, Thomas S; Thrombosis
and Hemostasis in Newborns (THiN) Group. Spontaneous
neonatal arterial thromboembolism: infants at risk, diagnosis,
treatment, and outcomes. Blood Coagul Fibrinolysis
2013;24:787-797.
361

Turk J Hematol 2015;32:359-362
Demirelli Y, et al: Thromboembolism in a Preterm Infant
2. Holden RW. Plasminogen activators: pharmacology and
therapy. Radiology 1990;174:993-1001.
3. Nowak-Göttl U, Kries von R, Göbel U. Neonatal symptomatic
thromboembolism in Germany: two year survey. Arch Dis
Child Fetal Neonatal Ed 1997;76:163-167.
4. Schmidt B, Andrew M. Neonatal thrombosis: report of a
prospective Canadian and international registry. Pediatrics
1995;96:939-943.
5. Aslam M, Guglietti D, Hansen AR. Neonatal arterial thrombosis
at birth: case report and literature review. Am J Perinatol 2008;
25:347-352.
6. Saracco P, Parodi E, Fabris C, Cecinati V, Molinari AC,
Giordano P. Management and investigation of neonatal
thromboembolic events: genetic and acquired risk factors.
Thromb Res 2009;123:805-809.
7. Michelson AD, Bovill E, Andrew M. Antithrombotic therapy
in children. Chest 1995;108:506-522.
8. Nowak-Göttl U, Auberger K, Halimeh S, Junker R, Klinge J,
Kreuz WD, Ries M, Schlegel N. Thrombolysis in newborns
and infants. Thromb Haemost 1999;(Suppl 1)82:112-116.
9. Cinà CS, Goh RH, Chan J, Kenny B, Evans G, Rawlinson J, Gill
G. Intrarterial catheter directed thrombolysis: urokinase versus
tissue plasminogen activator. Ann Vasc Surg 1999;13:571-
575.
10. Hartmann J, Hussein A, Trowitzsch E, Becker J, Hennecke KH.
Treatment of neonatal thrombus formation with recombinant
tissue plasminogen activator: six years experience and review
of the literature. Arch Dis Child Fetal Neonatal Ed 2001;85:18-
22.
11. Monagle P, Chalmers E, Chan A, DeVeber G, Kirkham F,
Massicotte P, Michelson AD; American College of Chest
Physicians. Antithrombotic therapy in neonates and children:
American College of Chest Physicians Evidence-Based
Clinical Practice Guidelines (8th Edition). Chest 2008;133(6
Suppl):887-968.
12. Olgun H, Buyukavci M, Ceviz N, Sahin IO, Yildirim ZK,
Colak A, Tekgunduz KS, Caner I. Clinical experience with
recombinant tissue plasminogen activator in the management
of intracardiac and arterial thrombosis in children. Blood
Coagul Fibrinolysis 2014;24:726-730.
13. Erdinç K, Sarıcı SÜ, Dabak O, Gürsel O, Güler A, Kürekçi
AE, Canpolat FE. A neonatal thrombosis patient treated
successfully with recombinant tissue plasminogen activator.
Turk J Hematol 2013;30:325-327.
362

Case Report
DOI: 10.4274/tjh.2014.0138
Turk J Hematol 2015;32:363-366
Immune Thrombocytopenic Purpura During Maintenance
Phase of Acute Lymphoblastic Leukemia: A Rare
Coexistence Requiring a High Degree of Suspicion, a Case
Report and Review of the Literature
Akut Lenfoblastik Lösemi İdame Tedavisi Sırasında Gelişen İmmün
Trombositopenik Purpura: Fazla Şüphe Gerektiren Nadir Bir
Birliktelik, Bir Olgu Sunumu ve Literatür Derlemesi
Turan Bayhan, Şule Ünal, Fatma Gümrük, Mualla Çetin
Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey
Abstract:
Thrombocytopenia may develop in patients with acute lymphoblastic leukemia (ALL) due to myelosuppression of
chemotherapy or relapse. Here we report a pediatric patient with ALL whose platelet counts decreased at the 102 nd
week of maintenance treatment. Thrombocytopenia was refractory to platelet infusions and bone marrow aspiration
revealed remission status for ALL along with increased megakaryocytes. The cessation of chemotherapy for 2 weeks
caused no increase in thrombocyte counts. The viral serology was unrevealing. A diagnosis of immune thrombocytopenic
purpura (ITP) was established. After administration of intravenous immunoglobulin, the thrombocytopenia resolved.
When thrombocytopenia occurs in patients with ALL in remission, ITP should be kept in mind after exclusion of the
more common etiologies.
Keywords: Acute lymphoblastic leukemia, Children, Immune thrombocytopenic purpura
Öz:
Akut lenfoblastik lösemi (ALL) tanılı hastalarda trombositopeni, kemoterapiye ikincil kemik iliği baskılanması veya
hastalığın relapsı sonucu gelişebilir. Olgumuz ALL idame tedavisinin 102. haftasında gelişen trombositopeni nedeniyle
incelendiği sırada immün trombositopenik purpura (İTP) tanısı almıştır. Trombositopeninin trombosit infüzyonuna
rağmen dirençli olması üzerine yapılan kemik iliği aspirasyonunda löseminin remisyonda olduğu ve megakaryositlerin
artmış olduğu görüldü. Kemoterapiye iki hafta ara verilmesine rağmen trombosit sayısında artma olmadı. Viral seroloji
sonuçları aktif enfeksiyon ile uyumlu değildi. Hastaya İTP tanısı konuldu. İntravenöz immünoglobulin tedavisi ile
trombositopeni düzeldi. Remisyondaki ALL hastalarında trombositopeni geliştiğinde, daha sık görülen nedenler
dışlandıktan sonra İTP de akılda bulundurulmalıdır.
Anahtar Sözcükler: Akut lenfoblastik lösemi, Çocuk, İmmün trombositopenik purpura
Address for Correspondence: Turan BAYHAN, M.D.,
Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey
Phone: +90 312 305 11 72 E-mail: turanbayhan@yahoo.com
Received/Geliş tarihi : March 30, 2014
Accepted/Kabul tarihi : May 13, 2014
363

Turk J Hematol 2015;32:363-366
Bayhan T, et al: Immune Thrombocytopenic Purpura in Acute Lymphoblastic Leukemia
Introduction
Immune thrombocytopenic purpura (ITP) is an
acquired autoimmune disorder characterized by isolated
thrombocytopenia due to increased platelet destruction
and impaired platelet production [1]. Autoimmunity in ITP
develops because of a failure in the regulatory checkpoints
of the immune system, resulting in a loss of self-tolerance to
platelet glycoproteins. The events that trigger this pathway
are largely unknown [2]. Association of ITP with hematologic
malignancies such as Hodgkin and non-Hodgkin lymphoma or
chronic lymphocytic lymphoma is a well-known phenomenon.
ITP has also been reported to accompany acute lymphoblastic
leukemia (ALL), albeit extremely rarely [3]. Herein we report
a patient with ALL who developed ITP during maintenance
therapy for ALL.
Case Presentation
A 3-year-old girl was admitted with fever, bone and joint
pain, and malaise. Complete blood count showed a hemoglobin
level of 7.4 g/dL, platelet count of 97x10 9 /L, and white blood
cell count of 3.8x10 9 /L with 34% blasts on the peripheral
blood smear. Bone marrow aspiration revealed CALLA (+)
pre-B cell ALL. A modified St. Jude Total XV protocol was
initiated with institutional modifications in the induction
phase concerning the dose of steroids, and remission was
achieved [4]. Maintenance treatment was planned according
to the patient’s low risk status [4]. Nothing was remarkable
up to the 102nd week of maintenance. After the 68 th week
of treatment, maintenance included weekly parenteral
methotrexate (40 mg/m 2 ) and daily oral 6-mercaptopurine
(75 mg/m 2 /day) with pulses of dexamethasone and vincristine
every 4 weeks until the 100 th week, after which only
6-mercaptopurine and methotrexate were given. At that time,
routine blood count showed hemoglobin of 12.8 g/dL, white
blood cell count of 5.4x10 9 /L, and platelet count of 43x10 9 /L.
Physical examination revealed no hepatosplenomegaly. She
was free of bleeding symptoms despite ecchymoses of the
lower extremities. Treatment was ceased for 2 weeks and, at
the end of 2 weeks of follow-up, thrombocytopenia persisted.
Since the platelet count had decreased to 16x10 9 /L, irradiated
and filtered platelet transfusion was administered, but the next
day the platelet count was found to still be as low as 21x10 9 /L.
Viral tests for parvovirus B19 polymerase chain reaction (PCR),
Epstein-Barr virus PCR, and cytomegalovirus PCR were all
negative. Antinuclear, antidouble-stranded DNA antibodies
and direct Coombs test were negative. Vitamin B12 and folate
levels were within normal ranges. In order to exclude the
possibility of associated hemophagocytic lymphohistiocytosis,
testing of plasma fibrinogen, serum triglyceride, and ferritin
levels was ordered and all were found to be within the normal
range. Bone marrow aspiration was performed in order to
exclude relapse of ALL. The bone marrow examination
revealed a cellular bone marrow in remission for ALL with
erythroid hyperactivity and increased megakaryocytes (up
to 9-10/field at 10 x magnification). A diagnosis of acute ITP
was established and intravenous immunoglobulin (IVIG)
therapy was given (1 g/kg/day, for 1 day). Three days after
IVIG treatment, platelet count was found to have increased to
272x10 9 /L. During follow-up, thrombocytopenia showed no
recurrence, despite continuation of the maintenance treatment
without any modification. Informed consent was obtained.
Discussion and Review of the Literature
Thrombocytopenia seen in patients with ALL is generally
secondary to chemotherapy or relapse of primary disease. Both
of these conditions manifest with reduced platelet production
[1]. Impaired megakaryocytopoiesis may also be seen in ITP,
but commonly accelerated destruction of platelets results in
increased megakaryocytes in bone marrow as a distinctive
finding of ITP [1,5]. In our patient, we did not check for
antiplatelet antibodies; however, bone marrow findings, as
well as the response of thrombocytopenia to IVIG treatment,
were strongly suggestive for the diagnosis of ITP.
Classically, the pathophysiology of ITP is attributed to
opsonization of platelets by immunoglobulin G antibodies
and then phagocytosis and destruction by macrophages in
the reticuloendothelial system within the spleen [5]. T cellmediated
immunity is also important in ITP pathogenesis [2].
Regulatory T cells (T reg cells) marked by CD4 + CD25 + Foxp3 +
have essential roles in self-tolerance by suppression of
humoral and cellular immunity response [6]. T reg cells have
been blamed for a role in ITP. Reduction in number and/or
function of circulating T reg cells in ITP patients has been
shown in several reports [1,5]. Increased numbers of CD4 +
Th17 cells and higher levels of T cell-related cytokines are
other T cell abnormalities detected in ITP [5].
In the English-language literature, 9 pediatric patients
who developed ITP subsequent to a diagnosis of ALL were
reported in 7 reports; 6 of them were on chemotherapy and
3 patients’ ITP developed after cessation of chemotherapy
(Table 1) [3,7,8,9,10,11,12]. It seems paradoxical to diagnose
ITP in patients with ALL who are under extensive immune
suppression with chemotherapeutics for the primary
disease. Because of the intensive chemotherapy used in
ALL, autoimmune diseases have rarely been reported among
patients with ALL who are under treatment [13]. Of the
reported cases, ITP was detected during the maintenance
period in 4 of the patients, in 1 patient after reinduction,
in 1 patient after induction therapy, and in 3 patients after
cessation of chemotherapy [3,7,8,9,10,11,12]. In the majority
of these reports, ITP was diagnosed during treatment with
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Bayhan T, et al: Immune Thrombocytopenic Purpura in Acute Lymphoblastic Leukemia
Turk J Hematol 2015;32:363-366
Table 1. Reported pediatric cases with immune thrombocytopenic purpura subsequent to a diagnosis of acute lymphoblastic leukemia.
Report Age at ITP
Diagnosis
Sex Chemotherapy
Protocol
Leukemia Treatment
Status at ITP Diagnosis
Rao et al., 1979 [12] 8 years Female Unspecified Maintenance with
cyclophosphamide
Interventions
for ITP
Response to
Treatment
Prednisone Responsive
Campbell et al., 1993 [7] 18 years Female Unspecified After completion of
induction (induction with
vincristine, daunorubicin,
prednisolone, L-asparaginase)
IVIG, danazol,
prednisolone
Responsive
Yenicesu et al., 2000 [3] 9 years Male St. Jude Total XIII Maintenance IVIG Responsive
Kurekci et al., 2006 [10] 5 years Male BFM-95 Maintenance IVIG,
prednisolone,
dexamethasone +
vincristine
Responsive to
dexamethasone
and vincristine
treatment
Price et al., 2006 [11] 10.2 years Female BFM-based highrisk
protocol
4 years after the end of
chemotherapy
IVIG Responsive
Price et al., 2006 [11] 16 years Male BFM-based highrisk
protocol
12 years after the end of
chemotherapy
IVIG, prednisone,
splenectomy
Responsive to
splenectomy
Price et al., 2006 [11] 13.3 years Female BFM-based highrisk
protocol
0.2 years after the end of
chemotherapy
None Asymptomatic
with mild
thrombocytopenia
Horino et al., 2009 [9] 7 years Female Protocol of Japan
Association
of Childhood
Leukemia Study
After completion of
reinduction therapy
IVIG, prednisone,
vincristine,
rituximab,
anti-D Ig,
splenectomy
Nonresponsive
Dua et al., 2012 [8] 16.6 years Female BFM-95 Maintenance Prednisone Responsive
ITP: Immune thrombocytopenic purpura, BFM: Berlin-Frankfurt-Munster, IVIG: intravenous immunoglobulin, Ig: immunoglobulin.
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Turk J Hematol 2015;32:363-366
Bayhan T, et al: Immune Thrombocytopenic Purpura in Acute Lymphoblastic Leukemia
6-mercaptopurine, similar to our case [3,8,9,10]. In 2 of
these reports, 6-mercaptopurine treatment was continued
without recurrence of ITP; in 1 case, due to resistant
thrombocytopenia, maintenance therapy was administered
with the support of IVIG; and in 1 report, continuation of
6-mercaptopurine after development of ITP was not stated
clearly [3,8,9,10]. 6-Mercaptopurine is a purine nucleoside
analogue that disturbs DNA synthesis and induces apoptosis
[14]. Purine nucleoside analogues cause profound depletion
of T cells [15]. Consequently, CD4 + CD25 + Foxp3 + cell counts
also decrease, and this will result in immune dysregulation.
This cascade has been thought of as a mechanism of ITP seen
in ALL [9,10]. In the literature, 2 patients were reported to
have developed ITP after treatment with cyclophosphamide
[9,12]. Cyclophosphamide also has suppressive effects on T reg
cells, similar to purine analogues, and this may support the
association of T reg cells with ITP in patients with ALL [9].
In conclusion, newly developed persistent
thrombocytopenia in patients with ALL may indicate ITP. After
exclusion of other common causes including recurrence of
the primary disease, chemotherapy-related myelosuppression,
folate deficiency, or viral etiologies, the coexistence of ITP
should be kept in mind as a rare etiology for unexplained
thrombocytopenia in order to initiate appropriate treatment
as early as possible.
Informed Consent: Informed consent was obtained,
Concept: Mualla Çetin, Design: Turan Bayhan, Şule Ünal,
Data Collection or Processing: Fatma Gümrük, Mualla Çetin,
Analysis or Interpretation: Şule Ünal, Literature Search:
Turan Bayhan, Fatma Gümrük, Mualla Çetin, Writing: Turan
Bayhan, Şule Ünal.
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. Kashiwagi H, Tomiyama Y. Pathophysiology and management
of primary immune thrombocytopenia. Int J Hematol
2013;98:24-33.
2. Arnold DM, Patriquin C, Toltl LJ, Nazi I, Smith J, Kelton J.
Diseases of platelet number: immune thrombocytopenia,
neonatal alloimmune thrombocytopenia, and posttransfusion
purpura. In: Hoffman R, Benz EJ, Silberstein LE, Heslop HE,
Weitz JI, Anastasi J (eds). Hematology: Basic Principles and
Practice. Philadelphia, Elsevier-Saunders, 2013.
3. Yenicesu I, Sanli C, Gürgey A. Idiopathic thrombocytopenic
purpura in acute lymphocytic leukemia. Pediatr Hematol
Oncol 2000;17:719-720.
4. Rubnitz JE, Campbell P, Zhou Y, Sandlund JT, Jeha S, Ribeiro
RC, Inaba H, Bhojwani D, Relling MV, Howard SC, Campana
D, Pui CH. Prognostic impact of absolute lymphocyte
counts at the end of remission induction in childhood acute
lymphoblastic leukemia. Cancer 2013;119:2061-2066.
5. McKenzie CG, Guo L, Freedman J, Semple JW. Cellular
immune dysfunction in immune thrombocytopenia (ITP). Br
J Haematol 2013;163:10-23.
6. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+
regulatory T cells in immunological tolerance to self and nonself.
Nat Immunol 2005;6:345-352.
7. Campbell JK, Mitchell CA. Immune thrombocytopenia
in association with acute lymphoblastic leukaemia and a
haemophagocytic syndrome. Eur J Haematol 1993;51:259-
261.
8. Dua V, Sharma JB. Immune thrombocytopenic purpura with
acute lymphoblastic leukemia an unusual association. Indian
Pediatr 2012;49:994.
9. Horino S, Rikiishi T, Niizuma H, Abe H, Watanabe Y,
Onuma M, Hoshi Y, Sasahara Y, Yoshinari M, Kazama T,
Hayashi Y, Kumaki S, Tsuchiya S. Refractory chronic immune
thrombocytopenic purpura in a child with acute lymphoblastic
leukemia. Int J Hematol 2009;90:483-485.
10. Kurekci AE, Atay AA, Demirkaya E, Sarici SU, Ozcan O.
Immune thrombocytopenic purpura in a child with acute
lymphoblastic leukemia and mumps. J Pediatr Hematol Oncol
2006;28:170-172.
11. Price V, Barnes C, Canning P, Blanchette V, Greenberg M.
Immune thrombocytopenia following successful treatment of
cancer in children. Pediatr Blood Cancer 2006;46:372-376.
12. Rao S, Pang EJ. Idiopathic thrombocytopenic purpura in acute
lymphoblastic leukemia. J Pediatr 1979;94:408-409.
13. Teachey DT, Felix CA. Development of cold agglutinin
autoimmune hemolytic anemia during treatment for pediatric
acute lymphoblastic leukemia. J Pediatr Hematol Oncol
2005;27:397-399.
14. Bar F, Sina C, Fellermann K. Thiopurines in inflammatory
bowel disease revisited. World J Gastroenterol 2013;19:1699-
1706.
15. Robak T, Korycka A, Lech-Maranda E, Robak P. Current status
of older and new purine nucleoside analogues in the treatment
of lymphoproliferative diseases. Molecules 2009;14:1183-
1226.
366

Case Report
DOI: 10.4274/tjh.2014.0412
Turk J Hematol 2015;32:367-370
A Rare Complication Developing After Hematopoietic Stem
Cell Transplantation: Wernicke’s Encephalopathy
Hematopoetik Kök Hücre Nakli Sonrası Gelişen Nadir Bir
Komplikasyon: Wernicke Ensefalopatisi
Soner Solmaz 1 , Çiğdem Gereklioğlu 2 , Meliha Tan 3 , Şenay Demir 4 , Mahmut Yeral 1 , Aslı Korur 2 , Can Boğa 1 ,
Hakan Özdoğu 1
1Adana Hospital of Başkent University, Department of Hematology, Adana, Turkey
2Adana Hospital of Başkent University, Department of Family Medicine, Adana, Turkey
3Adana Hospital of Başkent University, Department of Neurology, Adana, Turkey
4Adana Hospital of Başkent University, Department of Radiology, Adana, Turkey
Abstract:
Thiamine is a water-soluble vitamin. Thiamine deficiency can present as a central nervous system disorder known
as Wernicke’s encephalopathy, which classically manifests as confusion, ataxia, and ophthalmoplegia. Wernicke’s
encephalopathy has rarely been reported following hematopoietic stem cell transplantation. Herein, we report
Wernicke’s encephalopathy in a patient with acute myeloid leukemia who had been receiving prolonged total parenteral
nutrition after haploidentical allogeneic hematopoietic stem cell transplantation. To the best of our knowledge, this is
the first case reported from Turkey in the literature.
Keywords: Thiamine, Wernicke’s encephalopathy, Hematopoietic stem cell transplantation, Total parenteral nutrition
Öz:
Tiamin suda çözünen bir vitamindir. Tiamin eksikliği Wernicke ensefalopatisi olarak bilinen, klasik olarak konfüzyon,
ataksi ve oftalmopleji ile kendini gösteren bir merkezi sinir sistemi hastalığı olarak karşımıza çıkabilir. Hematopoetik
kök hücre nakli sonrasında gelişen Wernicke ensefalopatisi nadiren bildirilmiştir. Bu nedenle haploidentik allojenik
kök hücre naklinden sonra uzun süre total parenteral beslenme alan akut myeloid lösemili bir hastada gelişen Wernicke
ensefalopatisini sunmak istedik. Bildiğimiz kadarıyla literatürde Türkiye’den bildirilen ilk olgudur.
Anahtar Sözcükler: Tiamin, Wernicke ensefalopatisi, Hematopoetik kök hücre nakli, Total parenteral beslenme
Address for Correspondence: Soner SOLMAZ, M.D.,
Adana Hospital of Başkent University, Department of Hematology, Adana, Turkey
Phone: +90 322 327 27 27 E-mail: drssolmaz@gmail.com
Received/Geliş tarihi : October 17, 2014
Accepted/Kabul tarihi : November 25, 2014
367

Turk J Hematol 2015;32:367-370
Solmaz S, et al: Wernicke’s Encephalopathy After Hematopoietic Stem Cell Transplantation
Introduction
Thiamine is a water-soluble vitamin also known as
vitamin B1 [1]. Thiamine deficiency can present as a central
nervous system (CNS) disorder known as Wernicke’s
encephalopathy (WE), which classically manifests as
confusion, ataxia, and ophthalmoplegia [1,2]. The disease
is most frequently associated with chronic alcoholism, yet it
can also occur in relation to other forms of malnutrition or
malabsorption such as prolonged total parenteral nutrition
(TPN), total gastrectomy, gastrojejunostomy, severe anorexia,
or hyperemesis gravidarum [3]. Hematopoietic stem cell
transplantation (HSCT) does not seem to have a strong link
with WE [4]. To the best of our knowledge, this is the first
such case reported from Turkey in the literature and wanted
to report this case due to its rarity.
Case Presentation
A 19-year-old male patient diagnosed with acute myeloid
leukemia was admitted to our hospital for HSCT. After
remission had been achieved, he underwent haploidentical
HSCT from a sibling donor with a busulfan-fludarabine
conditioning regimen. During the conditioning period, the
patient was administered TPN, which is routinely used in
haploidentical HSCT; however, he developed grade 2-3 nausea
and vomiting and could not tolerate TPN. His oral intake was
also insufficient, so he received saline solution and glucosecontaining
intravenous solutions. He gradually recovered
from neutropenia on day 13 after HSCT without any adverse
events.
He was hospitalized due to diarrhea and vomiting 3 weeks
after the transplantation. On follow-up, toxic megacolon
and cytomegalovirus positivity were detected, so ganciclovir
treatment was started and oral intake was restricted until
recovery of intestinal symptoms. Efforts were made to feed
the patient by TPN with the aim of meeting his caloric needs
although he could not initially tolerate it. He was examined
for acute graft-versus-host disease (GVHD); he underwent
colonoscopy and pathologic samples were obtained, but
this examination did not reveal histological findings of
GVHD. Three weeks after his hospitalization, he developed
confusion, hallucination, strabismus, and nystagmus. A
neurology consultation was therefore done. In his neurologic
examination, he was oriented to place and person, but not to
time. He had horizontal nystagmus and lateral gaze paralysis
in the right eye, his motor power was 4/5, deep tendon reflexes
were hypoactive, Babinski reflex was negative bilaterally, he
could not cooperate with cerebellar tests, and he could not
stand up. Magnetic resonance imaging (MRI) of the brain
showed increased signal on T2-weighted and fluid-attenuated
inversion recovery (FLAIR) sequences around the aqueductus
sylvii and at the medial parts of both thalami (Figures 1a and
1b). A prediagnosis of WE was made based on the patient’s
history of inadequate oral intake and TPN use, CNS symptoms,
and specific radiologic findings. A blood sample was obtained
for testing serum thiamine level to confirm the diagnosis
before initiating therapy. Thereafter, 125 mg of thiamine
was intravenously administered daily, resulting in a rapid
improvement of the CNS symptoms within 48 h of treatment,
and parenteral treatment continued for 2 weeks. Serum
thiamine level was reported as 7.5 µg/L (normal range: 25-75
µg/L), verifying our diagnosis. During follow-up, his neurologic
findings and oral intake gradually improved, and so medical
therapy was switched to peroral treatment and maintained
with 250 mg of daily peroral thiamine. MRI revealed that the
previous increased signal around the aqueductus sylvii and at
the medial parts of both thalami on T2-weighted and FLAIR
sequences had significantly diminished (Figures 2a and 2b).
Informed consent was obtained.
Discussion and Review of the Literature
Neurological complications are fairly common in patients
undergoing HSCT and are present in 30%-39% of cases [5].
These complications may be of infectious, cerebrovascular,
toxic, immune-mediated, or metabolic origin [5]. Additionally,
several drugs routinely used during HSCT are associated with
Figure 1a. Axial fluid-attenuated inversion recovery magnetic
resonance imaging images of the brain demonstrating the
increased signal around the aqueductus sylvii.
368

Solmaz S, et al: Wernicke’s Encephalopathy After Hematopoietic Stem Cell Transplantation
Turk J Hematol 2015;32:367-370
neurological abnormalities, including cyclosporine A [5] and
tacrolimus [6]. Used alone or in combination with other agents,
methylprednisolone and ganciclovir may be responsible
for neurological findings, including disorientation, altered
mental status, visual disturbance, and coma [5]. We think
that we saved some time in making a differential diagnosis
by examining serum tacrolimus level to exclude drug toxicity
and cerebrovascular causes.
WE is characterized by the triad of altered mental status,
ataxia, and ophthalmoplegia, but only 16% of patients present
with the full classic triad of symptoms [5]. Mental status
changes are the most frequent findings in these patients
(82%), followed by ocular findings (29%) and ataxia (23%)
[5]. Ocular signs, including ophthalmoplegia, horizontal
and vertical nystagmus, and conjugate gaze palsies, are the
hallmark of WE [3]. Although almost all WE patients show
some degree of improvement after initiation of thiamine
replacement, only about 20% recover completely [4].
Furthermore, mortality increases dramatically when treatment
is delayed [4]. According to the guidelines of the European
Federation of Neurological Societies, total thiamine in blood
samples should be measured immediately before thiamine
administration to confirm suspected or manifest WE and MRI
should be used to support diagnosis [7]. Fortunately, we could
make a timely diagnosis based on clinical and radiological
findings and supported by decreased thiamine level thereafter,
and thus we could prevent mortality.
Figure 2a. Control magnetic resonance imaging 2 weeks after
the onset of the symptoms; fluid-attenuated inversion recovery
image showing the diminution of increased signal around the
aqueductus sylvii.
Figure 1b. Axial fluid-attenuated inversion recovery magnetic
resonance imaging images of the brain demonstrating the
increased signal at the medial parts of both thalami.
Figure 2b. Control magnetic resonance imaging 2 weeks after
the onset of the symptoms; fluid-attenuated inversion recovery
image showing the diminution of increased signal at the medial
parts of both thalami.
369

Turk J Hematol 2015;32:367-370
Solmaz S, et al: Wernicke’s Encephalopathy After Hematopoietic Stem Cell Transplantation
Patients receiving long-term TPN and glucose-containing
intravenous solutions require larger amounts of thiamine
to metabolize their carbohydrate intake, which can rapidly
deplete thiamine stores [3]. Studies show that a state of
depletion could develop within 18-20 days in patients
receiving a strict thiamine-free diet [5]. Almost all published
reports, except for one, concluded that prolonged TPN was the
primary risk factor for HSCT-associated WE [4]. Our patient
had received TPN for approximately 4-5 weeks in total. TPN
includes multivitamin and mineral supplementation in our
routine treatment protocol. However, we could not administer
it in this patient due to temporary lack of the concerned drugs
in the pharmacy of the hospital. The only other suggested
cause was the use of busulfan in the conditioning regimen
[4]. Similarly to data in the literature, our patient received
busulfan in the conditioning regimen and thiamine-free TPN,
and symptoms of WE emerged from day +45.
Many authors have recommended the use of a thiamine
supplement for prophylaxis against WE [4]. However, an
earlier publication from a Brazilian group reported 8 patients
who died after developing WE despite receiving prophylactic
thiamine (50 mg/day) [4]. Further studies are required to
decide on an effective prophylactic dose of thiamine and to
determine whether thiamine prophylaxis is effective in the
prevention of WE in HSCT patients [4]. This case taught us the
vital importance of vitamin supplementation in patients who
need long-term TPN. Based on these findings, we reviewed our
institutional policy about vitamin supplementation in TPN
and began adding water-soluble vitamins into TPN solutions
individually if combination preparations were not available in
the pharmacy of the hospital.
There are not routine recommendations for initial CNS
evaluation and management of the rarely occurring WE [4].
However, WE is a neurological emergency [8]. Therefore,
WE should be considered in HSCT patients, because cancer
patients are at high risk of this acute encephalopathy due
to chronic malnutrition, chemotherapy-induced nausea and
vomiting, and consumption of thiamine by rapidly growing
tumors [8].
In conclusion, differential diagnosis should consider WE
for patients who undergo HSCT and develop neurological
symptoms. Early treatment prevents high morbidity and
mortality. Therefore, thiamine supplements should be
administered to patients at high risk for WE.
Informed Consent: Informed consent was obtained,
Concept: Soner Solmaz, Can Boğa, Hakan Özdoğu,
Design: Soner Solmaz, Çiğdem Gereklioğlu, Can Boğa, Hakan
Özdoğu, Data Collection or Processing: Soner Solmaz, Çiğdem
Gereklioğlu, Meliha Tan, Şenay Demir, Mahmut Yeral, Aslı
Korur, Can Boğa, Hakan Özdoğu, Analysis or Interpretation:
Soner Solmaz, Çiğdem Gereklioğlu, Meliha Tan, Şenay
Demir, Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu,
Literature Search: Soner Solmaz, Meliha Tan, Şenay Demir,
Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu, Writing:
Soner Solmaz, Çiğdem Gereklioğlu, Meliha Tan, Şenay Demir,
Mahmut Yeral, Aslı Korur, Can Boğa, Hakan Özdoğu.
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. Trueg A, Borho T, Srivastava S, Kiel P. Thiamine deficiency
following umbilical cord blood transplant. Nutr Clin Pract
2013;28:223-225.
2. Han JW, Lim S, Shin HS, Park HJ, Jung WJ, Kwon SY, Lyu CJ.
Two cases of Wernicke’s encephalopathy in young age patients
receiving allogeneic hematopoietic stem cell transplantation.
Yonsei Med J 2012;53:1049-1053.
3. Baek JH, Sohn SK, Kim DH, Kim JG, Lee HW, Park SP, Lee
KB. Wernicke’s encephalopathy after allogeneic stem cell
transplantation. Bone Marrow Transplant 2005;35:829-830.
4. Choi YJ, Park SJ, Kim JS, Kang EJ, Choi CW, Kim BS. Wernicke’s
encephalopathy following allogeneic hematopoietic stem cell
transplantation. Korean J Hematol 2010;45:279-281.
5. Bleggi-Torres LF, de Medeiros BC, Ogasawara VS, Loddo G,
Zanis Neto J, Pasquini R, de Medeiros CR. Iatrogenic Wernicke’s
encephalopathy in allogeneic bone marrow transplantation: a
study of eight cases. Bone Marrow Transplant 1997;20:391-
395.
6. Sklar EM. Post-transplant neurotoxicity: what role do
calcineurin inhibitors actually play? AJNR Am J Neuroradiol
2006;27:1602-1603.
7. Galvin R, Bråthen G, Ivashynka A, Hillbom M, Tanasescu
R, Leone MA; EFNS. EFNS guidelines for diagnosis, therapy
and prevention of Wernicke encephalopathy. Eur J Neurol
2010;17:1408-1418.
8. Kuo SH, Debnam JM, Fuller GN, de Groot J. Wernicke’s
encephalopathy: an underrecognized and reversible cause of
confusional state in cancer patients. Oncology 2009;76:10-
18.
370

Letters to the Editor
Downgraded Lymphoma: B-Chronic
Lymphocytic Leukemia in a Known Case
of Diffuse Large B-Cell Lymphoma - De
Novo Occurrence or Transformation
Geriletilmiş Lenfoma: Diffüz Büyük B Hücreli
Lenfoma Olduğu Bilinen Bir Olguda B-Kronik
Lenfositik Lösemi- De Novo Oluşum veya
Dönüşüm
To the Editor,
Low-grade indolent lymphomas can be transformed into
high-grade aggressive lymphomas [1,2,3,4]. Very few cases
of transformation of high/intermediate-grade lymphoma to
low-grade lymphoma have been reported in the literature
[5,6]. This may arise through transformation of the original
clone or may represent a new neoplasm resulting from
additional genetic mutations that alter the growth rate,
growth pattern, and sensitivity to treatment.
A 57-year-old male diagnosed with diffuse large B-cell
lymphoma (DLBCL) (non-germinal center B-cell type) in
2002 completed 6 cycles of CHOP followed by radiotherapy.
In 2006, 18 F- fluorodeoxyglucose (FDG) positron emission
tomography/computed tomography (PET/CT) showed
no active disease. In 2007 there was recurrence in the left
obturator and external iliac nodes. Lymph node biopsy done
outside our facility showed CD20+ B-cell lymphoma. The
patient was advised to undergo intensive chemotherapy,
but was lost to follow-up. In 2010, the patient came to our
hospital with bilateral firm non-tender inguinal and right
axillary lymphadenopathy without any organomegaly.
18F PET/CT revealed heterogeneous uptake in the left
paraaortic, retrocaval, precaval, and bilateral internal
iliac nodes. A previous diagnostic lymph node biopsy was
reviewed, showing diffuse infiltration of large atypical
cells, positive for CD20, CD30, MUM1, and Bcl2 with a
Ki67 index of 80% and negative for CD3, CD5, and CD10,
which was consistent with DLBCL (Figure 1A). Biopsy
of the paraaortic mass revealed sheets of small lymphoid
cells, which were positive for CD20, CD5, and CD23 and
negative for CD3 and cyclin D1 with a low Ki67 index,
suggestive of small-cell lymphoma (Figure 1B). 18 F PET-
CT was repeated after 1 year, showing multiple FDG-avid
cervical, supraclavicular, mediastinal, axillary, abdominal,
and pelvic lymphadenopathies (Figure 1C). After 10
months, hemoglobin was 90 g/L, total leukocyte count was
21.1x10 9 /L, and platelet count was 40x10 9 /L. Peripheral
blood smear showed 84% abnormal lymphoid cells, which
were immunopositive for CD19, CD5, CD23, CD22 (dim),
CD200, and CD20 with lambda light chain restriction
and negative for CD10, FMC7, CD38, IgM, and CD103,
confirming the diagnosis of chronic lymphocytic leukemia
(CLL) (Figure 1D). The patient was started on a fludarabine,
cyclophosphamide, and rituximab (FCR) regimen. After 6
cycles of FCR, he was in complete remission and was started
on rituximab maintenance therapy.
Figure 1. A) Lymph node biopsy showing diffuse infiltration
of large atypical cells with prominent nucleoli and vesicular
chromatin, which were positive for CD20, CD30, and MUM1
with a Ki67 index of 80%. B) Biopsy from paraaortic mass
showing small-sized neoplastic cells with scant cytoplasm,
hyperchromatic nuclei, and clumped chromatin arranged in
sheets, which were positive for CD20 and CD5 and negative
for cyclin D1 with a low Ki67 index. C) 18 F-FDG PET-CT
showing multiple cervical, supraclavicular, mediastinal,
axillary, abdominal, and pelvic lymphadenopathies with gross
splenomegaly. D) Immunophenotyping of peripheral blood
smear showing 84% abnormal lymphoid cells, which were
positive for CD19, CD5, CD23, CD22 (dim), and CD200 with
lambda light chain restriction and negative for FMC7.
371

Turk J Hematol 2015;32:371-375
Letter to the Editor
The phenomenon of high- or intermediate-grade non-
Hodgkin lymphoma recurring as a low-grade lymphoma is an
uncommon form of transformation known as “downgraded”
lymphoma. This downgrading may be due to: 1) recurrence
of a low-grade lymphoma that was present as a minor
component of the initial lymphoma or in a site not biopsied,
or 2) development of a second lymphoma resulting from
chemotherapy and/or an intrinsic propensity for lymphoma
development in the patient [5,6]. Relapse in DLBCL mainly
occurs in the first 2 to 3 years, while late relapses after 5 years are
rare, occurring in 3.6% of cases. Patients with DLBCL relapse
usually have the same histology. However, relapse as indolent
lymphoma following initial DLBCL may occur in about 17%
of cases, predominantly as follicular lymphoma or rarely as
nodal marginal zone lymphoma or as extranodal mucosaassociated
lymphoid tissue lymphoma [7]. Histopathological
examination including extensive immunohistochemistry
should be done, not only when transformation is clinically
suspected but also at each recurrence because the disease
can recur as indolent lymphoma and an accurate histologic
diagnosis will contribute to a better understanding of the
pathogenesis of transformation and the start of prompt
therapy to improve the survival of the patients.
Concept: Smeeta Gajendra, Bhawna Jha, Shalini Goel,
Tushar Sahni, Pranav Dorwal, Ritesh Sachdev, Design: Smeeta
Gajendra, Bhawna Jha, Shalini Goel, Tushar Sahni, Pranav
Dorwal, Ritesh Sachdev, Data Collection or Processing:
Smeeta Gajendra, Bhawna Jha, Shalini Goel, Pranav Dorwal,
Ritesh Sachdev, Analysis or Interpretation: Smeeta Gajendra,
Bhawna Jha, Tushar Sahni, Ritesh Sachdev, Literature Search:
Smeeta Gajendra, Ritesh Sachdev, Writing: Smeeta Gajendra.
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.
Keywords: Diffuse large B-cell lymphoma, Chronic
lymphocytic leukemia, Downgraded lymphoma
Anahtar Sözcükler: Diffüz büyük B hücreli lenfoma,
Kronik lenfositik lösemi, Geriletilmiş lenfoma
Smeeta Gajendra, Bhawna Jha, Shalini Goel, Tushar Sahni, Pranav
Dorwal, Ritesh Sachdev
Medanta-The Medicity, Department of Pathology and Laboratory Medicine,
Gurgaon, India
References
1. Tsimberidou AM, Keating MJ. Richter syndrome: biology,
incidence, and therapeutic strategies. Cancer 2005;103:216-
228.
2. Rossi D, Cerri M, Capello D, Deambrogi C, Rossi FM, Zucchetto
A, De Paoli L, Cresta S, Rasi S, Spina V, Franceschetti S, Lunghi
M, Vendramin C, Bomben R, Ramponi A, Monga G, Conconi
A, Magnani C, Gattei V, Gaidano G. Biological and clinical risk
factors of chronic lymphocytic leukaemia transformation to
Richter syndrome. Br J Haematol 2008;142:202-215.
3. Montoto S, Fitzgibbon J. Transformation of indolent B-cell
lymphomas. J Clin Oncol 2011;29:1827-1834.
4. Lin P, Mansoor A, Bueso-Ramos C, Hao S, Lai R, Medeiros
LJ. Diffuse large B-cell lymphoma occurring in patients
with lymphoplasmacytic lymphoma/Waldenström
macroglobulinemia. Clinicopathologic features of 12 cases.
Am J Clin Pathol 2003;120:246-253.
5. Kerrigan DP, Foucar K, Dressler L. High-grade non-Hodgkin
lymphoma relapsing as low-grade follicular lymphoma: socalled
downgraded lymphoma. Am J Hematol 1989;30:36-
41.
6. Ogata Y, Setoguchi M, Tahara T, Takahashi M. Downgraded
non-Hodgkin’s lymphoma in the neck occurring as a
secondary malignancy. ORL J Otorhinolaryngol Relat Spec
1998;60:295-300.
7. Larouche JF, Berger F, Chassagne-Clément C, Ffrench M,
Callet-Bauchu E, Sebban C, Ghesquières H, Broussais-
Guillaumot F, Salles G, Coiffier B. Lymphoma recurrence 5
years or later following diffuse large B-cell lymphoma: clinical
characteristics and outcome. J Clin Oncol 2010;28:2094-
2100.
Address for Correspondence: Smeeta GAJENDRA, M.D.,
Medanta-The Medicity, Department of Pathology and Laboratory Medicine, Gurgaon, India
Phone: 0901 359 08 75
E-mail: drsmeeta@gmail.com
Received/Geliş tarihi : April 23, 2015
Accepted/Kabul tarihi : June 15, 2015
DOI: 10.4274/tjh.2015.0164
372

Letter to the Editor
Turk J Hematol 2015;32:371-375
From Bone Marrow Necrosis to Gaucher
Disease; A Long Way to Run
Kemik İliği Nekrozundan Gaucher Hastalığı
Tanısına Uzun Yol
To the Editor,
Bone marrow necrosis (BMN) is a disease characterized
with fever and bone pain and caused by many different
malignancies, benign diseases and drugs. We reported a case
of BMN due to diclofenac in 2006 [1]. And now we present
the same patient with a corrected diagnosis, seven years after
the first presentation.
A 26-year-old male presented with fever, bone pain,
splenomegaly, anemia, leucopenia and was diagnosed with
BMN due to diclofenac consumption. Nine months after his
initial admission, his laboratory and physical examination
were normal. Seven year after diagnosis, he was admitted to
hospital due to bone pain. He had splenomegaly, leukocyte
level was 6.22x10 9 /L, hemoglobin level was 13.7 g/dL and
thrombocyte level was 152x10 9 /L. Because of history of
BMN and reccurring splenomegaly, bone marrow aspiration
and biopsy were performed. He was diagnosed with Gaucher
disease in bone marrow biopsy and diagnosis was also
confirmed by pathology. He had low glucosylceramide level
(0.53 µkat/kg protein, normal range 2.4-3.8 µkat/kg protein)
and high chitotriosidase level (2793 µkat/kg protein, normal
range

Turk J Hematol 2015;32:371-375
Letter to the Editor
Neslihan Erdem 1 , Ahmet Çizmecioğlu 2 , İsmet Aydoğdu 3
1 Celal Bayar University Faculty of Medicine, Department of Internal Medicine,
Manisa, Turkey
2 Karaman State Hospital, Clinic of Internal Medicine, Karaman, Turkey
3 Celal Bayar University Faculty of Medicine, Department of Hematology, Manisa,
Turkey
References
1. Aydogdu I, Erkurt MA, Ozhan O, Kaya E, Kuku I, Yitmen
E, Aydin NE. Reversible bone marrow necrosis in a patient
due to overdosage of diclofenac sodium. Am J Hematol
2006;81:298.
2. Bhasin TS, Sharma S, Chandey M, Bhatia PK, Mannan R. A
case of bone marrow necrosis of an idiopathic aetiology: the
report of a rare entity with review of the literature. J Clin
Diagn Res 2012;7:525-528.
3. Rosenbaum H. Hemorrhagic aspects of Gaucher disease.
Rambam Maimonides Med J 2014;5:e0039.
Address for Correspondence: Neslihan ERDEM, M.D.,
Celal Bayar University Faculty of Medicine, Department of Internal Medicine, Manisa, Turkey
Phone: +90 555 729 88 22
E-mail: neslihnerdem@gmail.com
Received/Geliş tarihi: March 15, 2015
Accepted/Kabul tarihi: May 04, 2015
cardiac echocardiography results were within normal levels.
High-performance liquid chromatography results were as
follows; HbA1: 63.6%, HbA2: 32.8%, HbF: 0.2%. Agarose
gel electrophoresis was performed to distinguish HbA2, but
a band was identified at the level of 39.7% in HbF, G zone,
and between HbA1 and HbA2. A blood sample was transferred
to our genetic diagnostic center. Following DNA extraction
with a commercial kit (Roche, Germany) and amplification
of the whole beta globin gene by standard PCR protocols,
DNA sequencing (Applied Biosystems, USA) revealed an A
to C substitution at nucleotide position 308 (Figure 1). This
change was identified as HBB: c.308 A>C, known as Hb Kansas
in the HbVar database [5].
Hb Kansas is one of four known hemoglobins with neutral
substitutions, along with Hb Köln, Porto Alegre, and Genova
[1].
The oxygen equilibrium of Hb Kansas has two unusual
characteristics: low affinity for oxygen and low heme-heme
interaction. The low oxygen affinity of Hb Kansas should be
considered in the differential diagnosis of peripheral cyanosis,
especially in the neonatal period and in cyanotic disease and
polycythemia in the elderly.
DOI: 10.4274/tjh.2015.0123
First Observation of Hemoglobin Kansas
[β102(G4)Asn→Thr, AAC>ACC] in the
Turkish Population
Türk Toplumunda İlk Hemoglobin Kansas
[β102(G4)Asn→Thr, AAC>ACC] Gözlemi
To the Editor,
Hemoglobin (Hb) Kansas [β102 (G4) Asn→Tyr,
AAC>ACC] is an unstable abnormal hemoglobin with low
oxygen affinity and increased dissociation. Hb Kansas has
rarely been reported in the literature to date; the first case
was defined in the state of Kansas of the United States [1].
The second reported case was a newborn baby with cyanosis
from Sarajevo and the third was an elderly patient with
polycythemia from Japan [2,3]. There has been no previous
report from Turkey [4]. We herein report the first case of Hb
Kansas from Turkey, an introduction of clinical significance.
Case: A 28-year-old male patient with cyanosis of the
lips and fingertips was admitted to a hospital in the city of
Malatya. He had peripheral cyanosis of the hands and feet
on physical examination. Blood gas analysis showed low
oxygen levels. Complete blood count, blood chemistry, and
Figure 1. Hemoglobin Kansas in DNA sequencing.
374

Letter to the Editor
Turk J Hematol 2015;32:371-375
Concept: Duran Canatan, Design: Duran Canatan, Data
Collection or Processing: İbrahim Keser, Alev Öztaş, Analysis
or Interpretation: İbrahim Keser, Türker Bilgen, Literature
Search: Duran Canatan, Writing: Duran Canatan.
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.
Keywords: Abnormal hemoglobins, Hb Kansas, Turkish
population
Anahtar Sözcükler: Anormal hemoglobinler, Hb Kansas,
Türk toplumu
İbrahim Keser 1 , Alev Öztaş 2 , Türker Bilgen 3 , Duran Canatan 4
1 Akdeniz University Faculty of Medicine, Department of Biology and Genetics,
Antalya, Turkey
2 Melid Park Private Hospital, Malatya, Turkey
3 Research and Application Center for Scientific and Technological Investigations
(NABİLTEM) of Namık Kemal University, Tekirdağ, Turkey
4 Antalya Diagnostic Center of Genetic Diseases, Antalya, Turkey
References
1. Bonaventura J, Riggs A. Hemoglobin Kansas, a human
hemoglobin with a neutral amino acid substitution and an
abnormal oxygen equilibrium. J Biol Chem 1968;243:980-
991.
2. Zimmermann-Baer U, Capalo R, Dutly F, Saller E, Troxler
H, Kohler M, Frischknecht H. Neonatal cyanosis due to a
new (G)γ-globin variant causing low oxygen affinity: Hb
F-Sarajevo [(G)γ102(G4)Asn→Thr, AAC>ACC]. Hemoglobin
2012;36:109-113.
3. Morita K, Fukuzawa J, Onodera S, Kawamura Y, Sasaki
N, Fujisawa K, Ohba Y, Miyaji T, Hayashi Y, Yamazaki N.
Hemoglobin Kansas found in a patient with polycythemia.
Ann Hematol 1992;65:229-231.
4. Akar N. An updated review of abnormal hemoglobins in the
Turkish population. Turk J Hematol 2014;31:97-98.
5. Giardine B, van Baal S, Kaimakis P, Riemer C, Miller W, Samara
M, Kollia P, Anagnou NP, Chui DH, Wajcman H, Hardison
RC, Patrinos GP. HbVar database of human hemoglobin
variants and thalassemia mutations: 2007 update. Hum Mutat
2007;28:206.
Address for Correspondence: Duran CANATAN, M.D.,
Antalya Diagnostic Center of Genetic Diseases, Antalya, Turkey
E-mail: durancanatan@gmail.com
Received/Geliş tarihi: April 29, 2015
Accepted/Kabul tarihi: May 12, 2015
DOI: 10.4274/tjh.2015.0177
375

Images in Hematology
DOI: 10.4274/tjh.2015.0082
Mott Cells in the Peripheral Blood of a Patient with Dengue Fever
Dang Hummalı Bir Hastanın Periferik Kanındaki Mott Hücreleri
Turk J Hematol 2015;32:376-377
Image in Hematology
Figure 1. The top image shows a Mott cell. The bottom left image shows a similar Mott cell packed with spherical cytoplasmic
inclusions. The bottom right image shows a plasmacytoid lymphocyte with deep basophilic cytoplasm (Leishman stain;
magnification 1000 x ).
376

Turk J Hematol 2015;32:376-377
Antony A, et al: Mott Cells in the Peripheral Blood of a Patient with Dengue Fever
A 48-year-old female presented with intermittent highgrade
fever, chills, and severe myalgia for 4 days. There was
no lymphadenopathy or hepatosplenomegaly. Investigations
revealed hemoglobin concentration of 142 g/L; leucocyte
count of 3.5x10 9 /L with 54% neutrophils, 40% lymphocytes,
1% eosinophils, and 5% monocytes; and thrombocytopenia
(platelet count of 55x10 9 /L). Peripheral smear revealed
numerous plasmacytoid lymphocytes and occasional cells
with eccentrically placed nucleipacked with multiple
prominent cytoplasmic vacuoles, morphologically consistent
with Mott cells (Figure 1). Meanwhile, Dengue NS1 antigen
assay turned out to be positive. The patient was managed
conservatively and discharged after 4 days with a platelet
count of 150x10 9 /L. Peripheral smear revealed only occasional
reactive lymphocytes and the Mott cells had disappeared.
Three weeks after discharge, platelet and leucocyte counts had
improved further.
Nonmalignant reactive peripheral blood plasmacytosis
can occur in tumors, autoimmune conditions, and infections
[1]. Polyclonal peripheral blood plasmacytosis also occurs
in Dengue virus infections and is prominent during the first
week of the disease [2]. However, the transient occurrence of
Mott cells in the peripheral blood of Dengue fever patients has
not been reported previously. Our patient was not suffering
from lymphoma or multiple myeloma, which are potential
causes of peripherally circulating Mott cells.
Concept: Aniya Antony, Marie Ambroise, Anita Ramdas
Design: Aniya Antony, Marie Ambroise, Mookkappan
Sudhagar, Data Collection or Processing: Aniya Antony, Marie
Ambroise, Mookkappan Sudhagar, Anita Ramdas, Analysis
or Interpretation: Aniya Antony, Marie Ambroise, Chokka
Kiran, Anita Ramdas, Literature Search: Aniya Antony, Marie
Ambroise, Chokka Kiran, Writing: Aniya Antony, Marie
Ambroise, Chokka Kiran, Mookkappan Sudhagar.
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.
Keywords: Infection, Platelets, Lymphocytes, Viral
infection
Anahtar Sözcükler: Enfeksiyon, Platelet, Lenfosit, Viral
enfeksiyon
References
1. Jego G, Robillard N, Puthier D, Amiot M, Accard F, Pineau
D, Bataille R, Pellat-Deceunynck C. Reactive plasmacytoses
are expansions of plasmablasts retaining the capacity to
differentiate into plasma cells. Blood 1999;94:701-712.
2. Thai KT, Wismeijer JA, Zumpolle C, de Jong MD, Kersten MJ,
de Vries PJ. High incidence of peripheral blood plasmacytosis
in patients with dengue virus infection. Clin Microbiol Infect
2011;17:1823-1828.
Aniya Antony 1 , Marie Ambroise 1 , Chokka Kiran 1 ,
Mookkappan Sudhagar 2 , Anita Ramdas 1
1Pondicherry Institute of Medical Sciences, Clinic of
Pathology, Puducherry, India
2Pondicherry Institute of Medical Sciences, Clinic of General
Medicine, Puducherry, India
E-mail: aniya.antony@gmail.com
Received/Geliş tarihi : February 14, 2015
Accepted/Kabul tarihi : March 23, 2015
377

Images in Hematology
DOI: 10.4274/tjh.2014.0475
Turk J Hematol 2015;32:378-379
Quiz in Hematology
Figure 1. Patchy melanoderma lesions mimicking ecchymoses in the legs, trunk.
Figure 2. A) Skin. A slight perivascular infiltration of mononuclear inflammatory cells and dermal melanophages (arrows) are
seen (HEx200). B) An increment in melanin pigment in basal keratinocytes (arrow heads) and dermal melanophages (white
arrows) are highlighted by Fontana-Masson stain (x200).
378

Turk J Hematol 2015;32:378-379
Ünal Ş, et al: Diagnosis: Melanoderma After Hematopoietic Stem Cell Transplantation
Diagnosis: Melanoderma after Hematopoietic Stem Cell Transplantation
Hematopoetik Kök Hücre Nakli Sonrası Gelişen Melanoderma
An 8-month-oldboy diagnosed with T-B+NK- SCID underwent peripheral blood hematopoietic stem cell transplantation
(HSCT) from MSD without conditioning. However, he developed pancytopenia and became transfusion dependent by posttransplant
2 nd month. Bone marrow aspiration/biopsy revealed an aplastic marrow with 98% donor chimerism. With a diagnosis
of T-cell engraftment of the donor but no engraftment of the other lineages, a 2 nd HSCT with conditioning (BU/FLU/ATG) was
performed at post-transplant +23 rd month from the same donor. Due to hyperferritinemia pre-transplant desferoxamine was
given. On post-transplant day +2, he developed hyperpigmented patches (Figure 1). Platelet count was 22x10 9 /L and aPTT
and PT were normal. Platelet transfusion was given; however the lesions did not subside with the expected color change of
ecchymoses. Skin biopsy from medial thigh was obtained (Figure 2).
Generalized hyperpigmentation, after conditioning, is a common finding after HSCT [1]. However, in our patient the lesions
were patchy. There are few reports of melanoderma [1,2] after HSCT and in one, melanoderma was reported as a finding of
chronic GvHD [2]. Based on the absence of clinical signs of GvHD and lack of typical histological evidence, the melanoderma
in our patient was attributed to drugs used in conditioning. The transfusional iron loading may cause a generalized darkening
of the skin; however in our patient the lesions were patchy and developed just after completion of the conditioning regimen and
subsequent stem cell infusion. The patient did not develop acute or chronic GvHD signs throughout the follow-up. The lesions’
color faded after engraftment gradually, although did not disappear totally. Currently, the patient is alive at post-HSCT 6 th month.
Informed Consent: Informed consent has been obtained from the parents of the patient, Concept: Şule Ünal, İlhan Tezcan,
Şafak Güçer, Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Design: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem
Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Data Collection or Processing: Şule Ünal, İlhan Tezcan, Şafak Güçer,
Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Analysis or Interpretation: Şule Ünal, İlhan Tezcan, Şafak Güçer,
Meryem Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Literature Search: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem
Seda Boyraz, Deniz Çağdaş, Duygu Uçkan Çetinkaya, Writing: Şule Ünal, İlhan Tezcan, Şafak Güçer, Meryem Seda Boyraz, Deniz
Çağdaş, Duygu Uçkan Çetinkaya.
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.
Keywords: Hematopoietic stem cell transplantation, Melanoderma, Skin findings, SCID
Anahtar Sözcükler: Hematopoetik kök hücre nakli, Melanoderma, Deri bulguları, SCID
Şule Ünal 1 , İlhan Tezcan 2 , Şafak Güçer 3 , Meryem Seda Boyraz 4 , Deniz Çağdaş 2 , Duygu Uçkan Çetinkaya 1
1Hacettepe University Faculty of Medicine, Division of Pediatric Hematology, Ankara, Turkey
2Hacettepe University Faculty of Medicine, Division of Immunology, Ankara, Turkey
3Hacettepe University Faculty of Medicine, Division of Pediatric Pathology, Ankara, Turkey
4Hacettepe University Faculty of Medicine, Department of Pediatrics, Ankara, Turkey
Phone: +90 312 305 11 70
E-mail: suleunal@hacettepe.edu.tr
Received/Geliş tarihi : December 12, 2014
Accepted/Kabul tarihi : January 19, 2015
References
1. Aractingi S, Janin A, Devergie A, Bourges M, Socie G,
Gluckman E. Histochemical and ultrastructural study of
diffuse melanoderma after bone marrow transplantation. Br J
Dermatol 1996;134:325-331.
2. Martin-Gorgojo A, Martín JM, Gavrilova M, Monteagudo C,
Jordá-Cuevas E. Chronic graft-versus-host disease presenting
with coexisting diffuse melanoderma and hypopigmented
patches: A peculiar presentation. Eur J Dermatol. 2013;23:553-
555.
379

32 nd Volume Index / 32. Cilt Dizini
SUBJECT INDEX - KONU DİZİNİ 2015
Abnormal Hemoglobins
Chediak-Higashi / Chediak-Higashi, 90
Hemoglobin Lansing / Hemoglobin Lansing, 90
Hemoglobin Jabalpur / Hemoglobin Jabalpur, 90
Erythropoietin / Eritropoetin, 304
β-Thalassemia/hemoglobin E / β-Talasemi/hemoglobin E, 304
Apoptosis / Apopitoz, 304
Abnormal hemoglobins / Anormal hemoglobinler, 375
Hb Kansas / Hb Kansas, 375
Turkish population / Türk toplumu, 375
Acute Leukemia
Lymphoid enhancer-binding factor-1 /
Lenfoid enhansır-bağlayıcı faktör-1, 15
Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 15,277,363
Prognosis / Prognoz, 15
Wnt / Wnt, 15
Acute megakaryoblastic leukemia / Akut megakaryoblastik lösemi, 64
t(1;22) / t(1;22), 64
Acute myeloid leukemia / Akut miyeloid lösemi, 64,77
Trisomy 6 / Trizomi 6, 77
Cytogenetics / Sitogenetik, 77
Acute promyelocytic leukemia / Akut promyelositik lösemi, 97
Rhinocerebral mucormycosis / Rinoserebral mukormikozis, 97
WNT5A / WNT5A, 127
Methylation / Metilasyon, 127
Downregulation / Azalarak düzenlenme, 127
Gene expression / Gen ekspresyonu, 127
ALL / ALL, 127
Acute myeloblastic leukemia / Akut miyeloblastik lösemi, 263
FLT3 / FLT3, 263
Sorafenib / Sorafenib, 263
Sunitinib / Sunitinib, 263
Children / Çocuk, 263,329,363
D835Y mutation / D835Y mutasyonu, 263
B-cell neoplasms / B hücre neoplazileri, 277
Acute leukemia / Akut lösemi, 277,329
Chemotherapy / Kemoterapi, 329
Fungal infection / Fungal enfeksiyon, 329
Immune thrombocytopenic purpura / İmmün trombositopenik purpura, 363
Anemia
Cold-reactive antibody / Soğuk otoimmün, 86
Hemolytic anemia / Hemolitik anemi, 86,92
Breast carcinoma / Meme kanseri, 86
Microangiopathy / Mikroanjiyopati, 92
Severe aplastic anemia / Ağır aplastik anemi, 220
Regulatory T cell / Düzenleyici T hücre, 220
Bone marrow failure / Kemik iliği yetmezliği, 220
Thrombotic microangiopathy / Trombotik mikroanjiopati, 276
Eculizumab / Ekulizumab, 276
aHUS / aHUS, 276
CFH gene / CFH geni, 276
Anemia / Anemi, 284
Congenital dyserythropoietic anemia type 2 /
Konjenital diseritropetik anemi tip 2, 284
SEC23B gene / SEC23B geni, 284
Vitamin B12 / B12 vitamini, 317
Transcobalamin II / Transkobalamin II, 317
Novel mutation / Yeni mutasyon, 317
Novel deletion / Yeni delesyon, 317
Vacuolization / Vaküolizasyon, 317
Bleeding Disorders
Children / Çocuk, 338
Blood coagulation / Koagülasyon, 338
Hemophilia / Hemofili, 338
Inherited coagulopathies / Kalıtsal koagülopatiler, 338
Epistaxis / Epistaksis, 338
Menorrhagia / Menoraji, 338
Cancer
Cold-reactive antibody / Soğuk otoimmün, 86
Autoimmune hemolytic anemia / Hemolitik anemi, 86
Breast carcinoma / Meme kanseri, 86
Chronic myeloid leukemia / Kronik miyeloid lösemi, 257
Nilotinib / Nilotinib, 257
Secondary malignancy / İkincil malinite, 257
Carcinoma of the pancreas / Pankreas kanseri, 257
Chronic Leukemia
Monoclonal B lymphocytosis / Monoklonal B lenfositoz, 29
Prevalence / Prevalans, 29
Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 29,118,311
First-degree relatives / Birinci derece akraba, 29
Cytogenetics / Sitogenetik, 83
Marrow / Kemik iliği, 83
Neoplasia / Neoplazi, 83
Chronic myeloid leukemia / Kronik miyeloid lösemi, 83,257,311
Monosomy / Monozomi, 83
Apoptosis / Apoptoz, 118
Cell cycle arrest / Hücre siklusu tutulması, 118
KL-21 / KL-21, 118
Acute promyelocytic leukemia / Akut promiyelositik lösemi, 194
All-trans retinoic acid / All-trans retinoik asit, 194
BCR/ABL / BCR/ABL, 194
PML/RAR-α / PML/RAR-α, 194
Imatinib / İmatinib, 194
Nilotinib / Nilotinib, 257
Secondary malignancy / İkincil malinite, 257
Carcinoma of the pancreas / Pankreas kanseri, 257
IL-18, Polymorphism / İL-18, Polimorfizm, 311
Single nucleotide polymorphisms / Tek nükleotid polimorfizmi, 311
Coagulation
Children / Çocuk, 338
Blood coagulation / Koagülasyon, 338
Hemophilia / Hemofili, 338
Inherited coagulopathies / Kalıtsal koagülopatiler, 338
Epistaxis / Epistaksis, 338
Menorrhagia / Menoraji, 338
Granulocytic Sarcoma
Myeloid sarcoma / Miyeloid sarkom, 35
Granulocytic sarcoma / Granülositik sarkom, 35
Monoblastic sarcoma / Monoblastik sarkom, 35

32 nd Volume Index / 32. Cilt Dizini
SUBJECT INDEX - KONU DİZİNİ 2015
Hematological Malignancies
Hematological malignancy / Hematolojik malignite, 100,251
Invasive fungal infections / İnvazif fungal enfeksiyon, 100
Prophylaxis / Profilaksi, 100
Risk / Risk, 100
Secondary infection / Sekonder enfeksiyon, 243
Febrile neutropenia / Febril nötropeni, 243
Hematological malignancy / Hematolojik malinite, 243
Mortality / Mortalite, 243
Leukemia / Lösemi, 243
Lymphoma / Lenfoma, 243
Hepatitis B / Hepatit B, 251
Resolved infection / Geçirilmiş enfeksiyon, 251
Hepatitis B surface antibody / Hepatit B yüzey antikoru, 251
Chemotherapy / Kemoterapi, 251
Hemophagocytic Lymphohistiocytosis
Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiositoz, 355
Invasive aspergillosis infection / İnvaziv aspergilloz enfeksiyonu, 355
UNC13D (c.175G>C; p.Ala59Pro) / UNC13D (c.175G>C; p.Ala59Pro), 355
Immunohematology
Chimeric antigen receptor T cell / Şimerik antijen reseptör T hücreleri, 285
Hematological malignancies / Hematolojik maligniteler, 285
Iron Disorder
Iron deficiency / Demir eksikliği, 1
TMPRSS6 / TMPRSS6, 1
Matriptase-2 / Matriptaz-2, 1
Hepcidin / Hepsidin, 1
Infection Disorders
Invasive pulmonary aspergillosis / İnvasiv pulmoner aspergillozis, 73
Recombinant factor VIIa / Recombinant factor VIIa, 73
Coil embolization / Embolizasyon, 73
Children / Çocuk, 73,329
Acute leukemia / Akut lösemi, 73,329
Acute promyelocytic leukemia / Akut promyelositik lösemi, 96
Rhinocerebral mucormycosis / Rinoserebral mukormikozis, 96
Hematological malignancy / Hematolojik malignite, 100,243
Invasive fungal infections / İnvazif fungal enfeksiyon, 100
Prophylaxis / Profilaksi, 100
Risk / Risk, 100
Blood coagulation / Koagülasyon, 144
Hematologic manifestation / Hematolojik bulgu, 144
Infection / Enfeksiyon, 144,234,376
Pediatric leukemia / Pediatrik lösemi, 144
Histoplasma / Histoplazma, 191
Pancytopenia / Pansitopeni, 191
Adrenal masses / Adrenal kitleler, 191
Multiple myeloma / Multipl miyelom, 234
Risk factors / Risk faktörleri, 234
Therapy / Tedavi, 234
Secondary infection / Sekonder enfeksiyon, 243
Febrile neutropenia / Febril nötropeni, 243
Mortality / Mortalite, 243
Leukemia / Lösemi, 243
Lymphoma / Lenfoma, 243
Rituximab / Rituksimab, 271
Leuconostoc / Leuconostoc, 271
Purulent meninigitis / Pürülan menenjit, 271
Mantle cell lymphoma / Mantle hücreli lenfoma, 271
R-CHOP / R-CHOP, 271
Crimean-Congo hemorrhagic fever / Kırım-Kongo kanamalı ateşi, 281
Leukocyte / Lökositr, 281
Chemotherapy / Kemoterapi, 329
Fungal infection / Fungal enfeksiyon, 329
Platelets / Platelet, 376
Lymphocytes / Lenfosit, 376
Viral Infection / Viral enfeksiyon, 376
Leukocyte
Crimean-Congo hemorrhagic fever / Kırım-Kongo kanamalı ateşi, 281
Leukocyte / Lökositr, 281
Lymphoma
s-IL6 / s-IL6, 21
s-VEGF / s-VEGF, 21
Lymphoma / Lenfoma, 21
Overall survival / Genel sağkalım, 21
Fcγ RIIIA / Fcγ RIIIA, 152
Diffuse large B-cell lymphoma / Diffüz büyük B hücreli lenfoma, 152,295,371
Rituximab / Rituksimab, 152
Positron emission tomography / Positron emisyon tomografi, 213
Computed tomography / Bilgisayarlı tomografi, 213
Bone marrow biopsy / Kemik iliği biyopsisi, 213
Hodgkin’s lymphoma / Hodgkin lenfoma, 213
Non-Hodgkin’s lymphoma / Non Hodgkin lenfoma, 213
Rituximab / Rituksimab, 271
Leuconostoc / Leuconostoc, 271
Purulent meninigitis / Pürülan menenjit, 271
Mantle cell lymphoma / Mantle hücreli lenfoma, 271
R-CHOP / R-CHOP, 271
GADD45γ / GADD45γ, 295
DNA methylation / DNA metilasyonu, 295
Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 371
Downgraded lymphoma / Geriletilmiş lenfoma, 371
Molecular Hematology
Blood platelets / Trombositler, 58
Growth arrest-specific protein 6 / Growth arrest-specific protein 6, 58
Hemostasis / Hemostaz, 58
Apoptosis / Apoptoz, 118,304
Cell cycle arrest / Hücre siklusu tutulması, 118
Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 118,311
KL-21 / KL-21, 118
WNT5A / WNT5A, 127
Methylation / Metilasyon, 127
Downregulation / Azalarak düzenlenme, 127
Gene expression / Gen ekspresyonu, 127
ALL / ALL, 127
Fcγ RIIIA / Fcγ RIIIA, 152
Diffuse large B-cell lymphoma / Diffüz büyük B hücreli lenfoma, 152,295
Rituximab / Rituksimab, 152
Anemia / Anemi, 284
Congenital dyserythropoietic anemia type 2 /
Konjenital diseritropetik anemi tip 2, 284

32 nd Volume Index / 32. Cilt Dizini
SUBJECT INDEX - KONU DİZİNİ 2015
SEC23B gene / SEC23B geni, 284
IL-18 / İL-18, 311
Polymorphism / Polimorfizm, 311
Chronic myelogenous leukemia / Kronik miyeloid lösemi 311
Single nucleotide / Tek nükleotid 311
Polymorphisms / Polimorfizmi 311
Erythropoietin / Eritropoetin, 304
β-Thalassemia/hemoglobin E / β-Talasemi/hemoglobin E, 304
GADD45γ / GADD45γ, 295
DNA methylation / DNA metilasyonu, 295
Multiple Myeloma
Terbinafine / Terbinafin, 189
Drug / İlaç, 189
Neutropenia / Nötropeni, 189
Multiple myeloma / Multipl miyelom, 234
Infections / Enfeksiyonlar, 234
Risk factors / Risk faktörleri, 234
Therapy / Tedavi, 234
Myelomonocytic Leukemia
c-CBL mutation / c-CBL mutasyonu, 175
Childhood / Çocukluk çağı, 175
Juvenile myelomonocytic leukemia / Juvenil myelomonositik lösemi, 175
NRAS mutation / NRAS mutasyonu, 175
Auer rod / Auer çubukları, 278
Chronic myelomonocytic leukemia / Kronik miyelomonositik lösemi, 278
Myelofibrosis
Primary myelofibrosis / Primer myelofibrozis, 180
Ruxolitinib / Ruxolitinib, 180
Splenectomy / Splenektomi, 180
Myelodysplastic Syndromes
Psoriasis / Psöriazis, 87
Hypogammaglobulinemia / Hipogamaglobulinemi, 87
Monosomy 7 / Monozomi 7, 87
MDS / MDS, 87, 206
Bortezomib / Bortezomib, 206
Arsenic trioxide / Arsenik trioksit, 206
NF-κ B / NF-κ B, 206
Gene expression / Gen anlatımı 206
Myeloproliferative Disorders
Myeloproliferative neoplasms / Myeloproliferatif neoplaziler, 163
Ruxolitinib / Ruxolitinib, 163
Myelofibrosis / Miyelofibroz, 163
Neutropenia
Alkaline phosphatase / Alkalen fosfataz, 189
Myeloma / Myelom, 189
Vitamin D deficiency / D vitamin eksikliği, 189
Plasmacytoma
Blastic plasmacytoid dendritic cell neoplasm /
Blastik plazmasitoid dendritik hücre neoplazisi, 98
Cutaneous involvement / Hızlı ilerleme, 98
Sickle Cell
Sickle cell disease / Orak hücre hastalığı, 195
Hematopoietic stem cell transplantation /
Hematopoietik kök hücre nakli, 195
Graft-versus-host disease / Graft-versus-host hastalığı, 195
Graft rejection / Graft kaybı, 195
Conditioning / Hazırlama rejimi, 195
Stem Cell Transplantation
Tunneled central venous catheter / Tunelli santral venöz kateter, 51
Hematopoietic stem cell transplantation /
Hematopoetik kök hücre nakli, 51,195,367,379
Thrombosis / Tromboz, 51
Infection / Enfeksiyon, 51
Sickle cell disease / Orak hücre hastalığı, 195
Graft-versus-host isease / Graft-versus-host hastalığı, 195
Graft rejection / Graft kaybı, 195
Conditioning / Hazırlama rejimi, 195
Thrombosis / Tromboz, 228
Pediatric stem cell transplantation / Pediatrik kök hücre nakli, 288
Prothrombotic risk factors / Protrombotik risk faktörleri, 288
Melanoderma / Melanoderma, 379
Skin findings / Deri bulguları, 379
SCID / SCID, 379
Thiamine / Tiamin, 367
Wernicke’s encephalopathy / Wernicke ensefalopatisi, 367
Total parenteral nutrition / Total parenteral beslenme, 367
Thalassemia
Molecular / Moleküler, 136
Mutation / Mutasyon, 136,344
Alpha thalassemia / Alfa talasemiler, 136,344
Turkey / Türkiye, 136
Anemia / Anemi, 344
Hb Adana / Hb Adana, 344
Hb Icaria / Hb Icaria, 344
Hb Koya Dora / Hb Koya Dora, 344
Thalassemia / Talasemi, 344
Erythropoietin / Eritropoetin, 304
β-Thalassemia/hemoglobin E / β-Talasemi/hemoglobin E, 304
Apoptosis / Apopitoz, 304
Thrombosis
Venous thrombosis / Venöz tromboz, 80
Pregnancy / Hamilelik, 80
Factor V Leiden / Faktör V Leiden, 80
Streptococcal infection / Streptokok enfeksiyonu, 80
Thrombosis / Tromboz, 228
Pediatric stem cell transplantation / Pediatrik kök hücre nakli, 228
Prothrombotic risk factors / Protrombotik risk faktörleri, 228
Total anomalous pulmonary venous return /
Total pulmoner venöz dönüş anomalisi, 267
Portal vein thrombosis / Portal ven trombozu, 267
Anticoagulation therapy / Antikoagülan tedavi, 267
Low-molecular-weight-heparin / Düşük moleküler ağırlıklı heparin, 267
Preterm / Preterm, 359
Thromboembolism / Tromboemboli, 359
Tissue / Doku, 359
Plasminogen / Plazminojen, 359

32 nd Volume Index / 32. Cilt Dizini
SUBJECT INDEX - KONU DİZİNİ 2015
Intrauterine arterial thromboembolism /
İntrauterin arteriyel tromboembolizm, 359
Low dose recombinant tPA therapy /
Düşük doz rekombinant tPA tedavisi, 359
Thrombotic Thrombocytopenic Purpura
Thrombotic thrombocytopenic purpura /
Trombotik trombositopenik purpura, 279
Transcobalamin
Vitamin B12 / B12 vitamini, 317
Transcobalamin II / Transkobalamin II, 317
Novel mutation / Yeni mutasyon, 317
Novel deletion / Yeni delesyon, 317
Vacuolization / Vaküolizasyon, 317
Thrombocytopenia
Thrombocytopenia / Trombositopeni, 158
Elderly / Yaşlı, 158
Immune thrombocytopenic purpura /
İmmün trombositopenik purpura, 158,186,363
Intracranial bleeding / İntrakranial kanama 158
Congenital amegakaryocytic thrombocytopenia /
Konjenital amegakaryositik trombositopeni, 172
Thrombopoietin / Trombopoetin, 172
c-MPL / c-MPL, 172
Homozygous missense mutation / Homozigot yanlış anlamlı mutasyon, 172
c-MPL Tryp154Arg / c-MPL Tryp154Arg, 172
Amino acid change / Amino asit değişikliği, 172
Methylprednisolone / Metil prednizolon, 186
Glucocorticoids / Glükokortikoidler, 186
Child / Çocuk, 186,363
Adolescent / Adölesan, 186
Immune thrombocytopenia / İmmün trombositopeni, 323
Thrombopoietin receptor agonist / Trombopoetin reseptor agonisti, 323
Bleeding / Kanama, 323
Eltrombopag / Eltrombopag, 323
Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 363
Other
Mastocytosis / Mastositoz, 43,89
Bone mineral density / Kemik yoğunluk ölçümü, 43
Pyridinoline / Pridinolin, 43
Bone turnover / Kemik turnover, 43
Osteopenia / Osteopeni, 43
Dasatinib / Dasatinib, 68
Chylothorax / Şilotoraks, 68
Chronic myeloid leukemia / Kronik miyeloid lösemi, 68,168
Zinc deficiency / Çinko noksanlığı, 89
Oral lesions / Ağız yaraları, 93
S. aureus / S. aureus, 93
Hematology / Hematoloji, 93
Interleukin-31 (IL-31) / İnterlökin-31 (IL-31), 168
Tyrosine kinase inhibitors / Tirozin kinaz inhibitörleri, 168
Imatinib mesylate / İmatinib mesilat, 168
Pruritus / Kaşıntı 168
Thiopurine S-methyltransferase / Tiyopürin S-metiltransferaz, 184
Methylenetetrahydrofolate reductase / Metilentetrahidrofolat redüktaz, 184
Gene polymorphisms / Gen polimorfizmleri, 184
Leukemia / Lösemi, 184
Childhood / Çocukluk çağı, 184
Gaucher cells / Gaucher hücreleri, 187
Electron microscopy / Elektron mikroskopi, 187
Necrosis / Nekroz, 373
Gaucher / Gaucher, 373
Bone marrow / Kemik iliği, 373
Sedoanalgesia / Sedoanaljezi, 351
Ketamine / Ketamin, 351
Midazolam / Midazolam, 351
Invasive procedure / İnvazif işlem, 351

32 nd Volume Index / 32. Cilt Dizini
AUTHOR INDEX - YAZAR DİZİNİ 2015
A. G. Haddad..................................................80
A. H. Nassar....................................................80
A. H. Radwan.................................................80
Abdullah Cerit............................................. 213
Afig Berdeli.................................................. 276
Afra Yıldırım...................................................97
Ahmet Çizmecioğlu..................................... 374
Ahmet Emre Eşkazan........................... 213,243
Ahmet Muzaffer Demir..................................58
Akif Selim Yavuz............................................43
Alberto Daniel Gimenez Conca.................. 194
Alessandro Allegra...................................... 168
Alev Öztaş.................................................... 375
Ali Ayçiçek....................................................186
Ali Dursun................................................... 317
Ali Fettah........................................................73
Ali Keskin............................................ 295, 323
Ali Koçyiğit.................................................. 355
Ali Mert....................................................... 243
Ali T. Taher.....................................................80
Anand Chellappan..........................................85
Andrea Alonci............................................. 168
Anita Ramdas.............................................. 377
Aniya Antony.............................................. 377
Ansaf B. Yousef...............................................15
Antica Nacinovic-Duletic............................ 234
Anuradha Monga......................................... 158
Arzu Akçay.................................................. 127
Arzu Yazal Erdem...........................................87
Asami Shimada............................................ 257
Aslı Korur.................................................... 367
Aslıhan Demirel........................................... 243
Ateş Kara..................................................... 144
Ayhan Deviren................................................82
Ayhan Pektaş............................................... 144
Aysun Adan Gökbulut................................. 118
Ayşe Çırakoğlu...............................................82
Ayşe Işık....................................................... 163
Ayşe Kılıç..................................................... 338
Ayşegül Üner............................................... 163
Ayşegül Ünüvar........................................... 338
Aytemiz Gurgey........................................... 144
Aziz Polat..................................................... 355
Bahattin Tunç............................. 73,87,172,228
Bahriye Payzın............................... 152,277,323
Balint Nagy.................................................. 206
Begüm Atasay.............................................. 267
Begüm Koç.................................................. 344
Betül Börkü Uysal........................................ 213
Betül Küçükzeybek..................................... 277
Betül Tavil............................................ 172,228
Bhawna Jha........................................... 192,372
Bianca Tesi................................................... 355
Bilgül Mete.................................................. 243
Božena Coha................................................ 271
Božo Petrov.................................................. 234
Burak Erer......................................................43
Burcu Belen................................................. 185
Burhan Ferhanoğlu..................................... 243
Burhanettin Küçük.........................................58
Bülent Eser.....................................................97
Bülent Kantarcıoğlu.................................... 189
Bülent Ündar............................................... 152
Can Balkan.................................................. 263
Can Baykal......................................................43
Can Boğa.................................. 51,100,195,367
Canan Vergin............................................... 276
Carmen Mannucci....................................... 168
Caterina Musolino....................................... 168
Cem Muhlis Ar................................ 82,213,243
Cemil Ekinci...................................................35
Cengiz Bal.......................................................21
Cengiz Ceylan............................................. 323
Chanaveerappa Bammigatti............................85
Cheng-Hsu Wang...........................................68
Chien-Hong Lai..............................................68
Chokka Kiran.............................................. 377
Chunyan Liu............................................... 220
Cumali Karatoprak...................................... 213
Çiğdem Gereklioğlu.................................... 367
Çetin Timur.......................................... 127,344
Dalina I. Tanyong........................................ 304
Deniz Çağdaş............................................... 379
Deniz Sünnetçi............................................ 206
Deniz Yılmaz Karapınar.............................. 263
Dilek Gürlek Gökçebay.......................... 73,228
Dilek Kahvecioğlu....................................... 267
Dilhan Kuru...................................................82
Dilşad Sindel...................................................43
Duran Canatan............................................ 375
Duygu Kankaya..............................................35
Duygu Uçkan Çetinkaya...................... 228,379
Ebru Yılmaz Keskin..........................................1
Elif Suyanı.............................................. 29,251
Elizabeta Dadic-Hero.................................. 234
Emin Ünüvar............................................... 338
Emine Zengin.............................................. 351
Emre Tepeli................................................. 295
Erdem Şimşek.............................................. 329
Eren Gündüz..................................................21

32 nd Volume Index / 32. Cilt Dizini
AUTHOR INDEX - YAZAR DİZİNİ 2015
Erkin Serdaroğlu......................................... 276
Ersin Töret.................................................. 276
Esin Özcan.................................................. 276
Esin Şenol.................................................... 100
Esma Çakmak............................................. 351
Eylem Eliaçık.............................................. 163
Ezgi Uysalol................................................. 338
Fahir Özkalemkaş....................................... 100
Fahir Öztürk...................................................97
Fahri Şahin.................................................. 323
Fatih Azık.................................................... 228
Fatih Demircioğlu....................................... 284
Fatih Demirkan........................................... 152
Fatma Demir Yenigürbüz..................... 175,329
Fatma Gümrük................................ 64,136,363
Fatma Karaca Kara.........................................87
Fatma Oğuz................................................. 338
Federico Angriman...................................... 194
Fehmi Tabak................................................ 243
Ferit Avcu.................................................... 311
Fethullah Kenar........................................... 355
Fikriye Uras....................................................58
Filiz Aydın................................................... 344
Filiz Büyükkeçeci........................................ 323
Filiz Vural.................................................... 323
Francesco Di Raimondo.............................. 206
Funda Özgürler Akpınar............................. 355
Füsun Özdemirkıran............................ 277,323
Gamze Asker............................................... 344
Gamze Tatar................................................ 213
Genco Gençay............................................. 344
Gioacchino Calapai..................................... 168
Giuseppe Palumbo...................................... 206
Gonca Oruk................................................. 277
Gökhan Özgür............................................. 311
Guohua Yu.....................................................99
Güldane Cengiz Seval................................. 180
Gülersu İrken....................................... 175,329
Gülnur Görgün........................................... 323
Gülşah Akyol..................................................97
Gülşah Kaygusuz............................................35
Gülseren Bağcı............................................. 295
Gülsüm Akgün Çağlıyan............................. 323
Güray Saydam............................................. 323
Gürhan Kadıköylü......................... 190,282,323
Güven Çetin......................................... 190,213
H. Demet Kiper........................................... 323
H. El Farran....................................................80
Hakan Göker............................................... 163
Hakan Özdoğu................................ 51,195,367
Hakan Savlı.................................................. 206
Hale Ören............................................. 175,329
Hamdi Akan................................................ 100
Hara Prasad Pati.......................................... 158
Hasan Çakmaklı.......................................... 267
Hava Üsküdar Teke........................................21
Hayri Özsan................................................. 152
Hernán Michelángelo.................................. 194
Hidenori Imai.............................................. 257
Hikmet Eda Alışkan.......................................51
Hrvoje Holik................................................ 271
Huaquan Wang............................................ 220
Işınsu Kuzu....................................................35
İbrahim C. Haznedaroğlu............................ 163
İbrahim Caner............................................. 359
İbrahim İleri...................................................97
İbrahim Kamer............................................ 338
İbrahim Keser.............................................. 375
İdil Yenicesu.....................................................1
İhsan Karadoğan......................................... 100
İkbal Cansu Barış........................................ 295
İkbal Ok Bozkaya........................................ 172
İlhan Altan......................................................64
İlhan Tezcan................................................ 379
İnci Alacacıoğlu........................................... 323
İrfan Yavaşoğlu.................... 93,94,189,190,282
İsmail Can................................................... 127
İsmail Kırbaş...................................................73
İsmail Sarı.................................................... 295
İsmail Yıldız................................................. 338
İsmet Aydoğdu............................................ 374
Jelena Ivandic.............................................. 234
Jen-Seng Huang..............................................68
Jorge Alberto Arbelbide............................... 194
Junichi Arita................................................ 257
Kaan Kavaklı............................................... 263
Kadir Şerafettin Tekgündüz........................ 359
Keiji Sugimoto............................................. 257
Kun-Yun Yeh..................................................68
Kübra Gözübenli......................................... 213
Levent Oğuzkurt............................................51
Leyla Ağaoğlu.............................................. 127
M. Ali Çıkrıkçıoğlu..................................... 213
Mahmut Yeral......................................... 51,367
Maja Tomic-Paradžik................................... 271
Manoranjan Mahapatra.......................... 77,158
Maria Nelly Gutierrez Acevedo................... 194
Maria Sol Rossi............................................ 194
Marie Ambroise........................................... 377
Marijan Šiško............................................... 271

32 nd Volume Index / 32. Cilt Dizini
AUTHOR INDEX - YAZAR DİZİNİ 2015
Mario Petrini............................................... 206
Martina Canestraro..................................... 206
Masaaki Noguchi......................................... 257
Mehmet Ali Özcan........................ 100,152,323
Mehmet Ertem............................................ 267
Mehmet Hilmi Doğu................................... 295
Mehmet Sönmez.......................................... 100
Meliha Tan................................................... 367
Melike Koruyucu......................................... 277
Meltem Aylı................................................. 180
Meltem Özgüner............................................87
Meral Sarper................................................ 311
Merih Kızıl Çakar........................................ 251
Mervan Bekdaş............................................ 284
Meryem Seda Boyraz................................... 379
Mesut Ayer.................................................. 213
Moe Matsuzawa........................................... 257
Monika Gupta................................................77
Mookkappan Sudhagar............................... 377
Mualla Çetin.................................... 64,317,363
Muhit Özcan................................................ 180
Murat Akova................................................ 100
Murat Tombuloğlu...................................... 323
Mustafa Çetin.................................................97
Mustafa Dilek.............................................. 284
Mustafa Erkoçoğlu...................................... 284
Mustafa Kara............................................... 359
Mustafa Yaşar.............................................. 118
Mutlu Yüksek.................................................87
Mutsumi Wakabayashi................................ 257
Muzaffer Keklik..............................................97
Mücahit Yemişen......................................... 243
Müge Gökçe...................................................64
Müge Sayitoğlu............................................ 127
Münci Yağcı............................................ 29,251
Nagihan Yalçın............................................ 355
Namık Yaşar Özbek.................................. 73,87
Nazan Sarper............................................... 351
Nazmiye Yüksek.............................................87
Neryal Müminoğlu...................................... 276
Nesimi Büyükbabani......................................43
Neslihan Erdem........................................... 374
Neşe Yaralı....................................... 87,172,317
Nevruz Kurşunoğlu........................................29
Nihal Karadaş.............................................. 263
Nilay Şen Türk............................................ 295
Nilgün Sayınalp........................................... 163
Nilüfer Alpay Kanıtez.....................................43
Nita Radhakrishnan.......................................77
Noriko Nakamura....................................... 257
Norio Komatsu............................................ 257
Nurhan Ergül.............................................. 213
Nurhilal Büyükkurt..................................... 152
Oktay Bilgir................................................. 323
Olga Meltem Akay..........................................21
Osman İ. Özcebe......................................... 163
Ozan Çetin.................................................. 295
Ömer Devecioğlu......................................... 344
Ömer Doğru................................................ 127
Ömer Erdeve............................................... 267
Ömür Gökmen Sevindik............................. 323
Önder Arslan............................................... 285
Öner Doğan....................................................43
Öykü Arslan................................................ 323
Özden Hatırnaz Ng..................................... 127
Özden Pişkin............................................... 152
Özge Can..................................................... 295
Özgür Esen.................................................. 277
Özlem Arman Bilir.........................................87
Özlem Bingöl Özakpınar................................58
Özlem Tüfekçi...................................... 175,329
Pamir Işık............................................. 172,228
Pei-Hung Chang.............................................68
Pelin Mutlu.................................................. 311
Pervin Topçuoğlu...........................................35
Pınar Ataca.................................................. 285
Pranav Dorwal............................................. 372
Prapaporn Panichob.................................... 304
R. Hourani......................................................80
Rabab M. Aly..................................................15
Rabin Saba................................................... 100
Rajan Kapoor............................................... 158
Ranjit Kumar Sahoo.................................... 279
Recep Öztürk............................................... 243
Refik Tanakol.................................................43
Renata Dobrila-Dintinjana.......................... 234
Renu Saxena...................................................77
Reşat Özaras................................................ 243
Ritesh Sachdev..................................... 192,372
Rong Fu....................................................... 220
Rukiye Ünsal Saç......................................... 172
Saadet Arsan................................................ 267
Sabina Russo................................................ 168
Salih Aksu.................................................... 163
Salih Gözmen.............................................. 175
Sara Galimberti............................................ 206
Sebastiano Gangemi.................................... 168
Seçkin Çağırgan........................................... 100
Seher Açar................................................... 284
Selda Kahraman.......................................... 323

32 nd Volume Index / 32. Cilt Dizini
AUTHOR INDEX - YAZAR DİZİNİ 2015
Selin Aytaç......................................................64
Sema Aylan Gelen........................................ 351
Semra Atalay................................................ 267
Semra Büyükkorkmaz................................. 284
Serap Karaman..................................... 338,344
Serap Yalçın................................................. 311
Serdar Alan.................................................. 267
Serhan Küpeli.............................................. 184
Sevgi Gözdaşoğlu........................................ 188
Sevgi Yetgin................................................. 317
Sevil Göksügür............................................ 284
Shalini Goel.......................................... 192,372
Sibel Hacıoğlu............................................. 295
Sibel Kabukçu............................................. 323
Simge Erdem............................................... 213
Sinem Fırtına............................................... 127
Smeeta Gajendra........................... 192,279,372
Sonay Temurhan......................................... 344
Soner Solmaz............................................... 367
Sultan Aydın Köker..................................... 276
Sunay Tunalı............................................... 152
Suthat Fucharoen........................................ 304
Swaminathan Palamalai..................................85
Şenay Demir................................................ 367
Seniha Hacıhanefioğlu....................................82
Şeniz Öngören Aydın............................. 82,243
Şebnem Yılmaz Bengoa............................... 329
Şinasi Özsoylu.............................. 89,90,92,280
Şule Ünal........................... 64,136,317,363,379
Şükriye Yılmaz................................................82
T. Fikret Çermik.......................................... 213
Taner Demirci.................................................29
Tatiana Greenwood..................................... 355
Tayfun Uçar................................................. 267
Teoman Soysal........................................ 82,243
Tingguo Zhang...............................................99
Tiraje Celkan........................................ 127,344
Tomohiro Sawada........................................ 257
Toni Valkovic............................................... 234
Tony Rupar.................................................. 317
Tsung-Han Wu...............................................68
Tuba Hilkay Karapınar.................. 175,276,329
Tuba Özkan................................................. 213
Tuğba Elgün................................................ 344
Turan Bayhan.............................................. 363
Tushar Sahni........................................ 192,372
Türkan Atasever.......................................... 277
Türker Bilgen.............................................. 375
Türker Çetin................................................ 311
Türkiz Gürsel....................................... 185,317
Ufuk Çakır................................................... 267
Uğur Demirsoy............................................ 351
Uğur Özbek................................................. 127
Ülkü Ergene................................................ 152
Valerio Maisano........................................... 168
Vedrana Gacic.............................................. 234
Victoria Otero.............................................. 194
Vildan Caner............................................... 295
Vimarsh Raina............................................. 192
Wasinee Kheansaard................................... 304
Weiwei Qi.................................................... 220
Xin Huang......................................................99
Yahya Büyükaşık......................................... 163
Yasemin Işık Balcı........................................ 355
Yasunobu Sekiguchi.................................... 257
Yaşar Demirelli............................................ 359
Yelda Tarkan Argüden....................................82
Yen-Min Huang..............................................68
Yeşim Aydınok............................................. 263
Yeşim Oymak............................................... 276
Yıldız Aydın................................................. 243
Yılmaz Ay..................................................... 276
Yii-Jenq Lan....................................................68
Yue Ren....................................................... 220
Yueh-Shih Chang...........................................68
Yuqing Huo....................................................99
Yusuf Baran................................................. 118
Yusuf Ziya Demiroğlu....................................51
Zafer Başlar.................................................. 243
Zafer Gökgöz............................................... 323
Zafer Gülbaş...................................................21
Zahit Bolaman........................ 100,190,282,323
Zeynep Arzu Yeğin.........................................29
Zeynep Gümüş............................................ 277
Zeynep Karakaş.................................... 127,344
Zeynep Yıldız Yıldırmak....................... 127,344
Zeynep Yılmaz................................................29
Zifen Gao........................................................99
Zonghong Shao........................................... 220
Zübeyde Nur Özkurt......................................29
Zühal Önder Siviş........................................ 263

Advisory Board of This Issue (December 2015)
Ahmet Koç, Turkey
Akif Yeşilipek, Turkey
Ali Bay, Turkey
Ali İrfan Emre Tekgündüz, Turkey
Ali Ünal, Turkey
Alphan Küpesiz, Turkey
Attila Szvetko, Australia
Ayşegül Ünüvar, Turkey
BülentKarapınar, Turkey
Burhan Ferhanoğlu, Turkey
Can Balkan, Turkey
Canan Albayrak, Turkey
Christpher Dandoy, USA
Clare Y. Slaney, Australia
Dilber Talia İleri, Turkey
Elena Cassinerio, Italy
Elif Ünal İnce, Turkey
Gregory Kaufman, USA
Gülsüm Emel Pamuk, Turkey
Hande Çağlayan, Turkey
Kaan Kavaklı, Turkey
Luis Villela, Mexico
Marco L. Davila, USA
Nejat Akar, Turkey
Reyhan Diz Küçükkaya, Turkey
Sascha Meyer, Germany
Şebnem Yılmaz, Turkey
Semra Paydaş, Turkey
Şule Ünal, Turkey
Tiraje Celkan, Turkey
TürkanPatıroğlu, Turkey
Ülker Koçak, Turkey
Ulrike Reiss, USA
Vincenzo De Sanctis, Italy
Xunlei Kang, USA
Yurdanur Kılınç, Turkey
Yusuf Baran, Turkey
Zeynep Karakaş, Turkey

MAIN TOPICS
• Origin of Antiphospholipid Antibodies (aPL)
• Genetics of Antiphospholipid Syndrome (APS)
• Mechanism(s) of aPL-mediated Thrombosis & Pregnancy Morbidity
• Target Cells & Receptors that Interact with aPL
• Definition, Epidemiology & Natural History of APS
• Impact of APS in General Population with
Thrombosis & Pregnancy Morbidity
• Association Between APS & Other Systemic
Autoimmune Diseases, e.g., Lupus
• Thrombotic Angiopathies including Microangiopathic & Catastrophic APS
• Clinical & Prognostic Significance of
“Criteria” & “Non-criteria” aPL Tests
• Risk Stratification & Disease Measurement Criteria in APS
• Current Treatment Strategies & Treatment Trends in APS
• Role of Immunosuppressive Agents in APS
• Impact of Pediatric APS in Children with Thrombosis
• Strengths & Limitations of the Current APS Classification Criteria
• Recent Thrombosis Treatment Strategies in General Population
• Clinical Trial Design & Implementation
• Thrombotic & Obstetric APS for Patients
Abstract Submission and Registration Starts September 9, 2015
Local Executive
Committee
Ahmet Muzaffer Demir
Bahar Artım Esen
Ihsan Ertenli
Vedat Hamuryudan
Murat Inanc
Sedat Kiraz
Reyhan Kucukkaya
Seza Ozen
International Executive
Committee
Mary-Carmen Amigo, Mexico
Danieli Andrade, Brazil
Tatsuya Atsumi, Japan
Maria Laura Bertolaccini, UK
Ware Branch, USA
Robin Brey, USA
Ricard Cervera, Spain
Hannah Cohen, UK
Maria Cuadrado, UK
Phillip de Groot, Netherlands
Ronald Derksen, Netherlands
Doruk Erkan, USA
Paul Fortin, Canada
Nigel Harris, Jamaica
Graham Hughes, UK
Munther Khamashta, UK
Takao Koike, Japan
Steven Krilis, Australia
Steven Levine, USA
Roger Levy, Brazil
Michael Lockshin, USA
Samuel Machin, UK
Pier Luigi Meroni, Italy
Vittorio Pengo, Italy
Michelle Petri, USA
Jacob Rand, USA
Joyce Rauch, Canada
Robert Roubey, USA
Guillermo Ruiz-Irastorza, Spain
Jane Salmon, USA
Lisa Sammaritano, USA
Yehuda Shoenfeld, Israel
Maria Tektonidou, Greece
Angela Tincani, Italy
Denis Wahl, France
Zholi Zhang, China
Doruk Erkan, MD, MPH, Congress Chairman, Hospital for Special Surgery, Weill Cornell Medical College, New York, NY, USA
One-day Pre-congress
BOSPHORUS LUPUS LECTURES
Presented by World Renowned Physician-Scientists
Direct Flights Between
131 Cities & Istanbul
Endorsed by the Turkish Society of Rheumatology – Sponsored by GlaxoSmithKline
Program Coordinators: Doruk Erkan, MD, MPH & Murat İnanç, MD