Turkish Journal of Hematology Volume: 37 Issue: 1 / 2020
Turkish Journal of Hematology Volume: 37 Issue: 1 / 2020
Turkish Journal of Hematology Volume: 37 Issue: 1 / 2020
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Volume 37 Issue 1
March 2020
E-ISSN: 1308-5263
Review
Acute Graft-Versus-Host Disease: A Brief Review
Elifcan Aladağ et al.; Ankara, Samsun, Turkey
Research Articles
Therapeutic Potentials of Inhibition of Jumonji C Domain-containing Demethylases in Acute Myeloid Leukemia
Duygu Koca et al.; İzmir, Eskişehir, Sakarya, Turkey
Effects of the Proteasome Inhibitor Bortezomib in Combination with Chemotherapy for the Treatment of Mantle
Cell Lymphoma: A Meta-analysis
Shi-Jun Li et al.; Tianjin, China
Predictive Values of Early Suppression of Tumorigenicity 2 for Acute GVHD and Transplant-related Complications
after Allogeneic Stem Cell Transplantation: Prospective Observational Study
Ayako Matsumura et al.; Kanagawa, Japan
First-time Blood Donors Are Double-edged Swords for Blood Transfusion Centers: A Retrospective Study in
Southwest Iran
Hamid Reza Niazkar et al.; Gonabad, Tehran, Yasuj, Iran
Percentages of CD4+CD8+ Double-positive T Lymphocytes in the Peripheral Blood of Adults from a Blood Bank in
Bogotá, Colombia
Miguel S. Gonzalez-Mancera et al.; Bogotá, Colombia
1
Cover Picture:
Eda Ataseven, Dilek Ece,
Nazan Özsan, Mehmet Kantar,
İzmir, Turkey
Vacuolated Blasts in the Bone Marrow
of a Child with Rhabdomyosarcoma
Editor-in-Chief
Reyhan Küçükkaya
İstanbul, Turkey
rkucukkaya@hotmail.com
Associate Editors
A. Emre Eşkazan
İstanbul University-Cerrahpaşa,
İstanbul, Turkey
Ayşegül Ünüvar
İstanbul University, İstanbul, Turkey
aysegulu@hotmail.com
Cengiz Beyan
cengizbeyan@hotmail.com
Hale Ören
Dokuz Eylül University, İzmir, Turkey
hale.oren@deu.edu.tr
İbrahim C. Haznedaroğlu
Hacettepe University, Ankara, Turkey
haznedar@yahoo.com
Selami Koçak Toprak
Ankara University, Ankara, Turkey
sktoprak@yahoo.com
Semra Paydaş
Çukurova University, Adana, Turkey
sepay@cu.edu.tr
Şule Ünal
Hacettepe University, Ankara, Turkey
Assistant Editors
Ali İrfan Emre Tekgündüz
Dr. A. Yurtaslan Ankara Oncology Training
and Research Hospital, Ankara, Turkey
Claudio Cerchione
University of Naples Federico II Napoli,
Campania, Italy
Elif Ünal İnce
Ankara University, Ankara, Turkey
İnci Alacacıoğlu
Dokuz Eylül University, İzmir, Turkey
Müge Sayitoğlu
İstanbul University, İstanbul, Turkey
Nil Güler
Ondokuz Mayıs University, Samsun, Turkey
Olga Meltem Akay
Koç University, İstanbul, Turkey
Veysel Sabri Hançer
İstinye University, İstanbul, Turkey
Zühre Kaya
Gazi University, Ankara, Turkey
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
Aytemiz Gürgey
Senior Advisory Board
Yücel Tangün
Osman İlhan
Muhit Özcan
Teoman Soysal
Ahmet Muzaffer Demir
TOBB University of Economics and Technology Hospital, Ankara, Turkey
Maryland School of Medicine, Baltimore, USA
Cedars-Sinai Medical Center, USA
Ankara, Turkey
University of Chicago Medical Center, 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
University of Colorado Anschutz Medical Campus, 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
Medical College of Georgia at Augusta 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
University Medical Center Hamburg, Germany
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 Efe
A-I
Publishing
Services
GALENOS PUBLISHER
Molla Gürani Mah. Kaçamak Sk. No: 21/1, Fındıkzade, İstanbul, Turkey
Phone: +90 212 621 99 25 • Fax: +90 212 621 99 27 • www. galenos.com.tr
Contact Information
Editorial Correspondence should be addressed to Dr. Reyhan Küçükkaya
E-mail : rkucukkaya@hotmail.com
All Inquiries Should be Addressed to
TURKISH JOURNAL OF HEMATOLOGY
Address : Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613. Sok.) No: 8 06550 Çankaya, Ankara / Turkey
Phone : +90 312 490 98 97
Fax : +90 312 490 98 68
E-mail : info@tjh.com.tr
E-ISSN: 1308-5263
Publishing Manager
Muhlis Cem Ar
Management Address
Türk Hematoloji Derneği
Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613. Sok.)
No: 8 06550 Çankaya, Ankara / Turkey
Online Manuscript Submission
http://mc.manuscriptcentral.com/tjh
Web Page
www.tjh.com.tr
Owner on Behalf of the Turkish Society
of Hematology
Güner Hayri Özsan
Publishing House
Molla Gürani Mah. Kaçamak Sk. No: 21,
34093 Fındıkzade, İstanbul, Turkey
Tel: +90 212 621 99 25
Fax: +90 212 621 99 27
E-mail: info@galenos.com.tr
Publisher Certificate Number: 14521
Publication Date
17.02.2020
Cover Picture
Eda Ataseven, Dilek Ece, Nazan Özsan, Mehmet Kantar,
İzmir, Turkey
Vacuolated Blasts in the Bone Marrow of a Child with
Rhabdomyosarcoma
Bone marrow aspiration smear showed immature cells with
disseminated intranuclear/intracytoplasmic vacuolization.
International scientific journal published quarterly.
The Turkish Journal of Hematology is published by the commercial enterprise
of the Turkish Society of Hematology with Decision Number 6 issued by the
Society on 7 October 2008.
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 of Hematology is to publish original
hematological research of the highest scientific quality and clinical
relevance. Additionally, educational material, reviews on basic
developments, editorial short notes, images in hematology, and letters
from hematology specialists and clinicians covering their experience and
comments on hematology and related medical fields as well as social
subjects are published. As of December 2015, The Turkish Journal of
Hematology does not accept case reports. Important new findings or data
about interesting hematological cases may be submitted as a brief report.
General practitioners interested in hematology and internal medicine
specialists are among our target audience, and The Turkish Journal of
Hematology aims to publish according to their needs. The Turkish Journal
of Hematology is indexed, as follows:
- PubMed Medline
- PubMed Central
- Science Citation Index Expanded
- EMBASE
- Scopus
- CINAHL
- Gale/Cengage Learning
- EBSCO
- DOAJ
- ProQuest
- Index Copernicus
- Tübitak/Ulakbim Turkish Medical Database
- Turk Medline
- Hinari
- GOALI
- ARDI
- OARE
Impact Factor: 0.779
Open Access Policy
Turkish Journal of Hematology is an Open Access journal. This journal
provides immediate open access to its content on the principle that
making research freely available to the public supports a greater global
exchange of knowledge.
Open Access Policy is based on the rules of the Budapest Open Access
Initiative (BOAI) http://www.budapestopenaccessinitiative.org/.
Subscription Information
The Turkish Journal of Hematology is published electronically only as of
2019. Therefore, subscriptions are not necessary. All published volumes are
available in full text free-of-charge online at www.tjh.com.tr.
Address: Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613.
Sok.) No: 8 06550 Çankaya, Ankara / Turkey
Telephone: +90 312 490 98 97
Fax: +90 312 490 98 68
Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh
Web page: www.tjh.com.tr
E-mail: info@tjh.com.tr
Permissions
Requests for permission to reproduce published material should be sent to
the editorial office.
Editor: Professor Dr. Reyhan Küçükkaya
Adress: Turan Güneş Bulv. İlkbahar Mah. Fahreddin Paşa Sokağı (eski 613.
Sok.) No: 8 06550 Çankaya, Ankara / Turkey
Telephone: +90 312 490 98 97
Fax: +90 312 490 98 68
Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh
Web page: www.tjh.com.tr
E-mail: info@tjh.com.tr
Publisher
Galenos Yayınevi
Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul, Turkey
Telephone : +90 212 621 99 25
Fax : +90 212 621 99 27
info@galenos.com.tr
Instructions for Authors
Instructions for authors are published in the journal and at www.tjh.com.tr
Material Disclaimer
Authors are responsible for the manuscripts they publish in The Turkish
Journal of Hematology. The editor, editorial board, and publisher do not
accept any responsibility for published manuscripts.
If you use a table or figure (or some data in a table or figure) from another
source, cite the source directly in the figure or table legend.
Editorial Policy
Following receipt of each manuscript, a checklist is completed by the
Editorial Assistant. The Editorial Assistant checks that each manuscript
contains all required components and adheres to the author guidelines,
after which time it will be forwarded to the Editor in Chief. Following the
Editor in Chief’s evaluation, each manuscript is forwarded to the Associate
Editor, who in turn assigns reviewers. Generally, all manuscripts will be
reviewed by at least three reviewers selected by the Associate Editor, based
on their relevant expertise. Associate editor could be assigned as a reviewer
along with the reviewers. After the reviewing process, all manuscripts are
evaluated in the Editorial Board Meeting.
Turkish Journal of Hematology’s editor and Editorial Board members are
active researchers. It is possible that they would desire to submit their
manuscript to the Turkish Journal of Hematology. This may be creating
a conflict of interest. These manuscripts will not be evaluated by the
submitting editor(s). The review process will be managed and decisions
made by editor-in-chief who will act independently. In some situation, this
process will be overseen by an outside independent expert in reviewing
submissions from editors.
A-III
TURKISH JOURNAL OF HEMATOLOGY
INSTRUCTIONS FOR AUTHORS
The Turkish Journal of Hematology accepts invited review articles, research
articles, brief reports, letters to the editor, and hematological images that
are relevant to the scope of hematology, on the condition that they have
not been previously published elsewhere. Basic science manuscripts,
such as randomized, cohort, cross-sectional, and case-control studies,
are given preference. All manuscripts are subject to editorial revision
to ensure they conform to the style adopted by the journal. There is a
double-blind reviewing system. Review articles are solicited by the Editorin-Chief.
Authors wishing to submit an unsolicited review article should
contact the Editor-in-Chief prior to submission in order to screen the
proposed topic for relevance and priority.
The Turkish Journal of Hematology does not charge any article submission
or processing charges.
Manuscripts should be prepared according to ICMJE guidelines (http://
www.icmje.org/). Original manuscripts require a structured abstract. Label
each section of the structured abstract with the appropriate subheading
(Objective, Materials and Methods, Results, and Conclusion). Letters to
the editor do not require an abstract. Research or project support should
be acknowledged as a footnote on the title page. Technical and other
assistance should be provided on the title page.
Original Manuscripts
Title Page
Title: The title should provide important information regarding the
manuscript’s content. The title must specify that the study is a cohort
study, cross-sectional study, case-control study, or randomized study (i.e.
Cao GY, Li KX, Jin PF, Yue XY, Yang C, Hu X. Comparative bioavailability
of ferrous succinate tablet formulations without correction for baseline
circadian changes in iron concentration in healthy Chinese male
subjects: A single-dose, randomized, 2-period crossover study. Clin Ther
2011;33:2054-2059).
The title page should include the authors’ names, degrees, and institutional/
professional affiliations and a short title, abbreviations, keywords, financial
disclosure statement, and conflict of interest statement. If a manuscript
includes authors from more than one institution, each author’s name
should be followed by a superscript number that corresponds to their
institution, which is listed separately. Please provide contact information
for the corresponding author, including name, e-mail address, and
telephone and fax numbers.
Important Notice: The title page should be submitted separately.
Running Head: The running head should not be more than 40 characters,
including spaces, and should be located at the bottom of the title page.
Word Count: A word count for the manuscript, excluding abstract,
acknowledgments, figure and table legends, and references, should be
provided and should not exceed 2500 words. The word count for the
abstract should not exceed 300 words.
Conflict of Interest Statement: To prevent potential conflicts of
interest from being overlooked, this statement must be included in each
manuscript. In case there are conflicts of interest, every author should
complete the ICMJE general declaration form, which can be obtained at
http://www.icmje.org/downloads/coi_disclosure.zip
Abstract and Keywords: The second page should include an abstract
that does not exceed 300 words. For manuscripts sent by authors in
Turkey, a title and abstract in Turkish are also required. As most readers
read the abstract first, it is critically important. Moreover, as various
electronic databases integrate only abstracts into their index, important
findings should be presented in the abstract.
Objective: The abstract should state the objective (the purpose of the
study and hypothesis) and summarize the rationale for the study.
Materials and Methods: Important methods should be written
respectively.
Results: Important findings and results should be provided here.
Conclusion: The study’s new and important findings should be
highlighted and interpreted.
Other types of manuscripts, such as reviews, brief reports, and
editorials, will be published according to uniform requirements.
Provide 3-10 keywords below the abstract to assist indexers. Use
terms from the Index Medicus Medical Subject Headings List
(for randomized studies a CONSORT abstract should be provided: http://
www.consort-statement.org).
Introduction: The introduction should include an overview of the
relevant literature presented in summary form (one page), and whatever
remains interesting, unique, problematic, relevant, or unknown about
the topic must be specified. The introduction should conclude with the
rationale for the study, its design, and its objective(s).
Materials and Methods: Clearly describe the selection of observational
or experimental participants, such as patients, laboratory animals, and
controls, including inclusion and exclusion criteria and a description of the
source population. Identify the methods and procedures in sufficient detail
to allow other researchers to reproduce your results. Provide references to
established methods (including statistical methods), provide references to
brief modified methods, and provide the rationale for using them and
an evaluation of their limitations. Identify all drugs and chemicals used,
including generic names, doses, and routes of administration. The section
should include only information that was available at the time the plan
or protocol for the study was devised (https://www.strobe-statement.
org/fileadmin/Strobe/uploads/checklists/STROBE_checklist_v4_combined.
pdf).
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.
Study Limitations: Limitations of the study should be detailed. In
addition, an evaluation of the implications of the obtained findings/
results for future research should be outlined.
Conclusion: The conclusion of the study should be highlighted.
References
Cite references in the text, tables, and figures with numbers in square
brackets. Number references consecutively according to the order in
which they first appear in the text. Journal titles should be abbreviated
according to the style used in Index Medicus (consult List of Journals
Indexed in Index Medicus). Include among the references any paper
accepted, but not yet published, designating the journal followed by “in
press”.
Examples of References:
1. List all authors
Deeg HJ, O’Donnel M, Tolar J. Optimization of conditioning for marrow
transplantation from unrelated donors for patients with aplastic anemia
after failure of immunosuppressive therapy. Blood 2006;108:1485-1491.
2. Organization as author
Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of
syngeneic bone marrow graft without preconditioning in post-hepatitis
marrow aplasia. Lancet 1977;2:742-744.
3. Book
Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger,
1961.
4. Book Chapter
Perutz MF. Molecular anatomy and physiology of hemoglobin. In:
Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin:
Genetics, Pathophysiology, Clinical Management. New York, Cambridge
University Press, 2000.
5. Abstract
Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin)
stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract).
6. Letter to the Editor
Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further
definition of 20q deletion in myeloid leukemia using fluorescence in situ
hybridization. Blood 1994;84:2821-2823.
7. Supplement
Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr Transplant
2005;9(Suppl 7):81-86.
Brief Reports
Abstract length: Not to exceed 150 words.
Article length: Not to exceed 1200 words.
Introduction: State the purpose and summarize the rationale for the study.
Materials and Methods: Clearly describe the selection of the observational
or experimental participants. Identify the methods and procedures in
sufficient detail. Provide references to established methods (including
statistical methods), provide references to brief modified methods, and
provide the rationale for their use and an evaluation of their limitations.
Identify all drugs and chemicals used, including generic names, doses, and
routes of administration.
Statistics: Describe the statistical methods used in enough detail to
enable a knowledgeable reader with access to the original data to verify
the reported findings/results. Provide details about randomization,
describe treatment complications, provide the number of observations,
and specify all computer programs used.
Results: Present the findings/results in a logical sequence in the text,
tables, and figures. Do not repeat all the findings/results in the tables and
figures in the text; emphasize and/or summarize only those that are most
important.
Discussion: Highlight the new and important findings/results of the
study and the conclusions they lead to. Link the conclusions with the
goals of the study, but avoid unqualified statements and conclusions not
completely supported by your data.
Invited Review Articles
Abstract length: Not to exceed 300 words.
Article length: Not to exceed 4000 words.
Review articles should not include more than 100 references. Reviews
should include a conclusion, in which a new hypothesis or study about the
subject may be posited. Do not publish methods for literature search or level
of evidence. Authors who will prepare review articles should already have
A-V
published research articles on the relevant subject. The study’s new and
important findings should be highlighted and interpreted in the Conclusion
section. There should be a maximum of two authors for review articles.
Perspectives in Hematology
“Perspectives” are articles discussing significant topics relevant to
hematology. They are more personal than a Review Article. Authors
wishing to submit a Perspective in Hematology article should contact the
Editor in Chief prior to submission in order to screen the proposed topic for
relevance and priority. Articles submitted for “Perspectives in Hematology”
must advance the hot subjects of experimental and/or clinical hematology
beyond the articles previously published or in press in TJH. Perspective
papers should meet the restrictive criteria of TJH regarding unique
scientific and/or educational value, which will impact and enhance clinical
hematology practice or the diagnostic understanding of blood diseases.
Priority will be assigned to such manuscripts based upon the prominence,
significance, and timeliness of the content. The submitting author must
already be an expert with a recognized significant published scientific
experience in the specific field related to the “Perspectives” article.
Abstract length: Not to exceed 150 words.
Article length: Not to exceed 1000 words.
References: Should not include more than 50 references
Images in Hematology
Article length: Not to exceed 200 words.
Authors can submit for consideration illustrations or photos that are
interesting, instructive, and visually attractive, along with a few lines of
explanatory text and references. Images in Hematology can include no
more than 200 words of text, 5 references, and 3 figures or tables. No
abstract, discussion, or conclusion is required, but please include a brief
title.
Letters to the Editor
Article length: Not to exceed 500 words.
Letters can include no more than 500 words of text, 5-10 references, and
1 figure or table. No abstract is required, but please include a brief title.
The total number is usually limited to a maximum of five authors for a
letter to the editor.
Tables
Supply each table in a separate file. Number tables according to the order
in which they appear in the text, and supply a brief caption for each.
Give each column a short or abbreviated heading. Write explanatory
statistical measures of variation, such as standard deviation or standard
error of mean. Be sure that each table is cited in the text.
Figures
Figures should be professionally drawn and/or photographed. Authors
should number figures according to the order in which they appear in the
text. Figures include graphs, charts, photographs, and illustrations. Each
figure should be accompanied by a legend that does not exceed 50 words.
Use abbreviations only if they have been introduced in the text. Authors
are also required to provide the level of magnification for histological
slides. Explain the internal scale and identify the staining method used.
Figures should be submitted as separate files, not in the text file. Highresolution
image files are not preferred for initial submission as the file
sizes may be too large. The total file size of the PDF for peer review should
not exceed 5 MB.
Authorship
Each author should have participated sufficiently in the work to assume
public responsibility for the content. Any portion of a manuscript that
is critical to its main conclusions must be the responsibility of at least
one author.
Contributor’s Statement
All submissions should contain a contributor’s statement page. Each
statement should contain substantial contributions to idea and design,
acquisition of data, and analysis and interpretation of findings. All
persons designated as an author should qualify for authorship, and all
those that qualify should be listed. Each author should have participated
sufficiently in the work to take responsibility for appropriate portions of
the text.
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 subject research. Approval of
research protocols by the relevant ethics committee, in accordance
with international agreements (Helsinki Declaration of 1975, revised
2013 available at https://www.wma.net/policies-post/wma-declarationof-helsinki-ethical-principles-for-medical-research-involving-humansubjects/),
is required for all experimental, clinical, and drug studies.
Patient names, initials, and hospital identification numbers should not
be used. Manuscripts reporting the results of experimental investigations
A-VI
conducted with humans must state that the study protocol received
institutional review board approval and that the participants provided
informed consent.
Non-compliance with scientific accuracy is not in accord with scientific
ethics. Plagiarism: To re-publish, in whole or in part, the contents of
another author’s publication as one’s own without providing a reference.
Fabrication: To publish data and findings/results that do not exist.
Duplication: Use of data from another publication, which includes republishing
a manuscript in different languages. Salami slicing: To create
more than one publication by dividing the results of a study unnecessarily.
We disapprove of such unethical practices as plagiarism, fabrication,
duplication, and salami slicing, as well as efforts to influence the
review process with such practices as gifting authorship, inappropriate
acknowledgments, and references. Additionally, authors must respect
participants‘ right to privacy.
On the other hand, short abstracts published in congress books that do
not exceed 400 words and present data of preliminary research, and those
that are presented in an electronic environment, are not considered as
previously published work. Authors in such a situation must declare this
status on the first page of the manuscript and in the cover letter.
(The COPE flowchart is available at http://publicationethics.org.)
We use iThenticate to screen all submissions for plagiarism before
publication.
Conditions of Publication
All authors are required to affirm the following statements before their
manuscript is considered: 1. The manuscript is being submitted only
to The Turkish Journal of Hematology; 2. The manuscript will not be
submitted elsewhere while under consideration by The Turkish Journal
of Hematology; 3. The manuscript has not been published elsewhere,
and should it be published in The Turkish Journal of Hematology it will
not be published elsewhere without the permission of the editors (these
restrictions do not apply to abstracts or to press reports for presentations
at scientific meetings); 4. All authors are responsible for the manuscript’s
content; 5. All authors participated in the study concept and design,
analysis and interpretation of the data, and drafting or revising of the
manuscript and have approved the manuscript as submitted. In addition,
all authors are required to disclose any professional affiliation, financial
agreement, or other involvement with any company whose product
figures prominently in the submitted manuscript.
Authors of accepted manuscripts will receive electronic page proofs and
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A-VII
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A-VIII
CONTENTS
Review
1 Acute Graft-Versus-Host Disease: A Brief Review
Elifcan Aladağ, Engin Kelkitli, Hakan Göker; Ankara, Samsun, Turkey
Research Articles
5 Therapeutic Potentials of Inhibition of Jumonji C Domain-containing Demethylases in Acute Myeloid Leukemia
Duygu Koca, Nurcan Hastar, Selin Engür, Yağmur Kiraz, Gizem Tuğçe Ulu, Demet Çekdemir, Yusuf Baran; İzmir, Eskişehir, Sakarya, Turkey
13 Effects of the Proteasome Inhibitor Bortezomib in Combination with Chemotherapy for the Treatment of Mantle Cell Lymphoma:
A Meta-analysis
Shi-Jun Li, Jian Hao, Yu Mao, Yu-Ling Si; Tianjin, China
20 Predictive Values of Early Suppression of Tumorigenicity 2 for Acute GVHD and Transplant-related Complications after Allogeneic Stem Cell
Transplantation: Prospective Observational Study
Ayako Matsumura, Takuya Miyazaki, Takayoshi Tachibana, Taiki Ando, Megumi Koyama, Satoshi Koyama, Yoshimi Ishii, Hiroyuki Takahashi,
Yuki Nakajima, Ayumi Numata, Wataru Yamamoto, Kenji Motohashi, Maki Hagihara, Kenji Matsumoto, Shin Fujisawa, Hideaki Nakajima;
Kanagawa, Japan
30 First-time Blood Donors Are Double-edged Swords for Blood Transfusion Centers: A Retrospective Study in Southwest Iran
Hamid Reza Niazkar, Akbar Dorgalaleh, Fariba Rad; Gonabad, Tehran, Yasuj, Iran
36 Percentages of CD4+CD8+ Double-positive T Lymphocytes in the Peripheral Blood of Adults from a Blood Bank in Bogotá, Colombia
Miguel S. Gonzalez-Mancera, Natalia I. Bolaños, Manuel Salamanca, Guillermo A. Orjuela, Ayda N. Rodriguez, John M. Gonzalez;
Bogotá, Colombia
Perspectives in Hematology
42 WHO 2016 Definition of Chronic Myeloid Leukemia and Tyrosine Kinase Inhibitors
İbrahim C. Haznedaroğlu, Işınsu Kuzu, Osman İlhan; Ankara, Turkey
Brief Report
48 Blastic Plasmacytoid Dendritic Cell Neoplasia: A Single Center Experience
Ahu Senem Demiröz, Cuyan Demirkesen, Ayşe Salihoğlu, Nükhet Tüzüner; İstanbul, Turkey
Images in Hematology
53 Kasabach-Merritt Syndrome in an Adult
Milan Pantelic, Masa Pantelic, Petar Djuric, Katarina Markovic, Tamara Vucinic, Jovan Todor Juloski; Belgrade, Serbia
55 Platelet Satellitism
Yasemin Ardıçoğlu Akışın, Nejat Akar; Ankara, Turkey
Letters to the Editor
57 Assessment of Patients with von Willebrand Disease with ISTH/BAT and PBQ Scores
Fatma Burcu Belen Apak, Elif Gülsüm Ümit, Yağmur Zengin, Melike Sezgin Evim, Ekrem Ünal, Hasan Mücahit Özbaş, Can Acıpayam; Ankara,
Edirne, Bursa, Kayseri, Giresun, Kahramanmaraş, Turkey
A-IX
59 Children with Iron Deficiency Anemia Have a Tendency to Hypercoagulation: An Evaluation by Thromboelastography
Ceren Kılcı, Lale Olcay, Beril Özdemir, Ali Fettah, Meriç Yavuz Çolak; Ankara, Turkey
62 Successful Hemostasis with Extended Half-life Recombinant Factor VIII in Circumcision
Başak Koç, Metin İshak Öztürk, Bülent Zülfikar; İstanbul, Turkey
64 Accidental High-dose Intrathecal Treatment: Late Results of a Patient
Tiraje Celkan, Evrim Çifçi Sunamak; İstanbul, Turkey
65 CMV-specific T-Cells for Treatment of CMV Infection after Hematopoietic Stem Cell Transplantation in a Pediatric Case:
First Application in Turkey
Sevil Celilova, Ersin Toret, Başak Aksoy Adaklı, Ercüment Ovalı, Ceyhun Bozkurt; İstanbul, Turkey
67 Comparison of Different Culture Conditions for Mesenchymal Stem Cells from Human Umbilical Cord Wharton’s Jelly for Stem Cell Therapy
Yu Bao, Shumin Huang, Zhengyan Zhao; Zhejiang, China
70 Vacuolated Blasts in the Bone Marrow of a Child with Rhabdomyosarcoma
Eda Ataseven, Dilek Ece, Nazan Özsan, Mehmet Kantar; İzmir, Turkey
72 T-Cell Lymphoblastic Lymphoma Showing Aberrant Synaptophysin Expression in a Child
Nazım Emrah Koçer, Bermal Hasbay, Fazilet Kayaselçuk; Adana, Turkey
74 Hydroxyurea-induced Tooth Discoloration
Muhammed Okuyucu, Memiş Hilmi Atay; Samsun, Turkey
75 Rare Presentation of Herpes Virus Lesions in a Case of Acute Pre-B Lymphoblastic Leukemia
Eylem Kaymaz, Zeliha Güzelküçük, Melek Işık, Neşe Yaralı; Ankara, Turkey
A-X
REVIEW
DOI: 10.4274/tjh.galenos.2019.2019.0157
Turk J Hematol 2020;37:1-4
Acute Graft-Versus-Host Disease: A Brief Review
Akut Graft Versus Host Hastalığı: Kısa Bir Derleme
Elifcan Aladağ 1 , Engin Kelkitli 2 , Hakan Göker 1
1Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey
2Ondokuz Mayıs University Faculty of Medicine, Department of Hematology, Samsun, Turkey
Abstract
Graft‐versus‐host disease (GvHD) is an important complication
that can be observed after allogeneic hematopoietic stem cell
transplantation (allo-HSCT). Acute GvHD (aGvHD) is seen after allo-
HSCT and the incidence of aGvHD is around 30%-50%. aGvHD
prophylaxis is essential in patients undergoing allo-HSCT. Initial
therapy for aGvHD is steroids. Prognosis is poor in aGvHD patients
not responding to steroids. In this article, the pathobiology, clinical
findings, prophylaxis, and treatment of aGvHD will be summarized.
Keywords: Graft-versus-host disease, Acute, Chronic
Öz
Akut graft‐versus‐host hastalığı (GvHD), allojeneik hematopoetik
kök hücre nakli (alloHKHN) sonrasında görülebilen önemli bir
komplikasyondur. Akut GvHD (aGvHD) insidansı yaklaşık %30-50
oranında görülmektedir. AlloHKHN yapılan hastalarda GvHD proflaksisi
önemlidir. aGvHD gelişen hastalarda başlangıç tedavisi steroiddir.
Steroide yanıtsız aGvHD’de prognoz kötüdür. Bu yazıda aGvHD
patobiyolojisi, klinik bulguları, profilaksisi ve tedavisi özetlenecektir.
Anahtar Sözcükler: Graft‐versus‐host hastalığı, Akut, Kronik
Acute Graft-Versus-Host Disease (GvHD)
Acute graft‐versus‐host disease (aGvHD) is seen after allogeneic
hematopoietic stem cell transplantation (allo-HSCT) [1,2,3,4].
The incidence of aGvHD is around 30%-50% in HLA fully
matched allo-HSCT [1]. aGvHD is also common in haploidentical
and matched unrelated donor transplantation [1,2].
Pathobiology
In 1966, Billingham detailed the biology of GvHD development
as a three-stage process: a) the graft/donor should contain
immunologically competent cells, b) the recipient/host must
have tissue antigens not expressed in donor cells, and c) the
recipient should be unable to mount an immune response to
effectively eliminate the donor cells [3,5]. Hence, during allo-
HSCT, after conditioning the host, tissue antigens of the recipient
are expressed to the donor T-cells, which leads to donor T-cell
activation, expression, and enhanced immune response to the
host; in other words, aGvHD occurs [1,2,3,4]. The mechanism
underlying tissue damage in aGvHD is massive inflammatory
cytokine secretion. Proinflammatory cytokines [tumor necrosis
factor (TNF)-α, interleukin (IL)-1β, and IL-6] are seen, as well
as the increased expression of the receptor repertoire (pattern
recognition receptors) on antigen-presenting cells [6].
Risk Factors
The most important risk factor for aGvHD is HLA mismatch.
Other risk factors include sex disparity between donor and
recipient, the intensity of the conditioning regimen, increased
age, multiparous female donors, ineffective GvHD prophylaxis,
and the source of the graft. A study showed that aGvHD
was significantly more common with total body irradiation
involving a myeloablative regimen and peripheral stem cell
transplantation from a fully matched related donor. In that
study, the use of tacrolimus and methotrexate for GvHD
prophylaxis was associated with a significant increase in GvHD
risk compared to a cyclosporine-methotrexate combination [1].
Clinical Manifestations
GvHD can be acute or chronic based on the clinical presentation
and its occurrence after or before 100 days after allo-HSCT. aGvHD
may occur beyond this arbitrary cut-off of 100 days. The widely
accepted National Institutes of Health consensus criteria have
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Hakan Göker M.D., Hacettepe University Faculty of Medicine,
Department of Hematology, Ankara, Turkey
Phone : +90 505 822 99 16
E-mail : hgoker1@yahoo.com ORCID: orcid.org/0000-0002-1039-7756
Received/Geliş tarihi: April 14, 2019
Accepted/Kabul tarihi: September 2, 2019
1
Aladağ E, et al: Acute Graft-Versus-Host Disease: A Brief Review Turk J Hematol 2020;37:1-4
been used to classify GvHD. GvHD is divided into four subclasses:
1) Classic aGvHD: Diagnostic and distinctive features of chronic
GvHD (cGvHD) are absent. Clinical features of aGvHD and present
within 100 days of allo-HSCT or donor lymphocyte infusion
(DLI). 2) Persistent and/or recurrent late-onset aGvHD: Features
of classic aGvHD without diagnostic manifestations of cGvHD
occurring beyond 100 days after allo-HSCT or DLI. 3) Classic
cGvHD: Present at any time after HSCT. Diagnostic and
distinctive features of cGvHD are present without aGvHD. 4)
Overlap syndrome: Features of both cGvHD and aGvHD can be
seen [4,7].
Clinically significant aGvHD may be cumbersome, affecting
both morbidity and mortality [1,2,3,4]. The staging and grading
of aGvHD can be seen in Table 1 [4]. The timely diagnosis of
aGvHD is important. Hence, numerous novel biomarkers have
been recently studied for timely diagnosis. These diagnostic
and prognostic markers include systemic biomarkers
(microRNAs, suppression of tumorigenicity 2), biomarkers of
immune activation [TNF receptor 1, IL-7, B-cell activating factor
(sBAFF)], and organ-specific biomarkers [REG3α (regenerating
islet-derived 3-α], S100, TIM (T-cell immunoglobulin domain
and mucin domain), cytokeratin-18, hepatocyte growth factor,
and skin-derived anti-leukoproteinase, otherwise known as
elafin). However, there is no specific GvHD biomarker in routine
use [8].
Prevention
The most important step for the prevention of GvHD is
minimizing risk factors with donor selection and a preparative
regimen [2,3,4]. GvHD prophylaxis is essential for patients
undergoing allo-HSCT [4]. Guidelines for GvHD prophylaxis have
been proposed by the European Group for Blood and Marrow
Transplantation and European LeukemiaNet [9].
The most common form of GvHD prophylaxis has been the
combination of cyclosporine and a short course of methotrexate,
which demonstrated improved survival compared to either
drug alone. Both cyclosporine and tacrolimus decreased the
proliferation of T-lymphocytes [4]. Tacrolimus plus methotrexate
is better in decreasing the risk for aGvHD than the combination
of cyclosporine and methotrexate, particularly in unrelated
HSCT [10]. Both regimens are considered as cornerstones for
most GvHD prevention strategies for patients receiving allo-
HSCT [11,12]. The effects of the addition of corticosteroids to the
combination of cyclosporine and a short course of methotrexate
have shown conflicting results [13,14,15]. Calcineurin inhibitors
and methotrexate form the main backbone of prophylactic
treatment.
Treatment
The choice of initial therapy for aGvHD depends on the organs
involved, the severity of symptoms, and the prophylactic
regimen used. Topical steroids are the most commonly used skindirected
therapy for grade I aGvHD. Antihistamines may also be
used. Bacigalupo et al. showed that steroid treatment of grade
I GvHD prevents progression to grade II GvHD, but not to grade
III-IV GvHD [16]. Initial therapy for grade II-IV aGvHD consists of
high-dose glucocorticoid steroids. Steroid treatment is effective
in approximately half of the patients; those with more severe
aGvHD are less likely to respond. Treatment is usually started with
the equivalent of 1-2 mg/kg/day of prednisone and then tapered
after a decrease in GvHD signs or symptoms. The transplantationrelated
mortality rate is high in non-responders in the first 5
Table 1. The clinical manifestation, staging, and grading of aGvHD.
Organ Clinical manifestation Stage
Skin
Gastrointestinal tract
Liver
Erythematous, maculopapular rash
involving palms and soles; may become
confluent
Severe disease: bullae
Nausea, vomiting, abdominal cramps,
diarrhea, ileus, distension, bleeding
Hyperbilirubinemia and increased alkaline
phosphatase
Stage 1=<25% rash
Stage 2=25%-50% rash
Stage 3=Generalized erythroderma
Stage 4=Bullae
Stage 1=Diarrhea >500 mL/day
Stage 2=Diarrhea >1000 mL/day
Stage 3=Diarrhea >1500 mL/day
Stage 4=Ileus, bleeding
Stage 1=Bilirubin 2-3 mg/dL
Stage 2=Bilirubin 3.1-6 mg/dL
Stage 3=Bilirubin 6.1-15 mg/dL
Stage 4=Bilirubin >15 mg/dL
Grade Skin Gut Liver
I Stage 1-2 0 0
II Stage 1-3 1 and/or 1
III Stage 2-3 2-4 and/or 2-3
IV Stage 2-4 2-4 and/or 2-4
2
Turk J Hematol 2020;37:1-4
Aladağ E, et al: Acute Graft-Versus-Host Disease: A Brief Review
days of steroid use. Several agents have been added to steroids
in comparative studies but no evidence supports the use of these
in combination for aGvHD therapy. The best complete response
rate was obtained with mycophenolate in combination with
other agents (etanercept, etc.) with steroids [17]. Recently the
US Food and Drug Administration approved ruxolitinib, a JAK 1/2
inhibitor, and it has been used with considerable success in the
treatment of steroid-refractory aGvHD [18].
Unfortunately, there is no standard indication or timing for the
initiation of second-line therapy for aGvHD. Many agents have
been tested alone or in combination with corticosteroids with
limited sustained efficacy [4].
There are few guidelines in the literature regarding secondline
cGvHD treatment. Extracorporeal photopheresis
(ECP), mycophenolate mofetil, sirolimus, everolimus, rituximab,
and ibrutinib are available options. ECP is recommended in the
treatment of steroid-resistant aGvHD [19] and was found to
result in overall response rates of 50% to 65%.
Table 2 provides a brief summary of some of the current novel
second-line strategies for steroid-refractory aGvHD.
Conclusion
aGvHD leads to significant morbidity and mortality. Therefore, it
is crucial to prevent its development. New therapy strategies for
both prevention and treatment are needed. aGvHD is a leading
cause of late morbidity and mortality. The standard treatment is
steroid therapy and a calcineurin inhibitor may also be added.
Further treatment strategies need to be developed for the
treatment of aGvHD.
Authorship Contributions
Concept: E.A., E.K., H.G.; Design: E.A., E.K., H.G.; Data Collection
or Processing: E.A., E.K., H.G.; Analysis or Interpretation: E.A.,
E.K., H.G.; Literature Search: E.A., E.K., H.G., Writing: E.A., E.K.,
H.G.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
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Table 2. Summary of some of the current novel second-line
strategies.
Agent Comment Refs.
Mycophenolate
mofetil
Sirolimus
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Infliximab,
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A potent inhibitor of lymphocyte
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Therapy of GvHD with humanized
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immunoglobulin G1 (etanercept) and
IL-1 receptor antagonist (IL-1Ra) has
shown some promise.
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4
RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2019.2019.0083
Turk J Hematol 2020;37:5-12
Therapeutic Potentials of Inhibition of Jumonji C Domaincontaining
Demethylases in Acute Myeloid Leukemia
Akut Myeloid Lösemide Demetilaz İçeren Jumanji C Domainin İnhibisyonunun Terapötik
Potansiyeli
Duygu Koca 1 , Nurcan Hastar 1 , Selin Engür 2 , Yağmur Kiraz 1 , Gizem Tuğçe Ulu 1 , Demet Çekdemir 3 , Yusuf Baran 1
1İzmir Institute of Technology, Department of Molecular Biology and Genetics, İzmir, Turkey
2Anadolu University Faculty of Pharmacy, Department of Pharmacology, Eskişehir, Turkey
3Sakarya University Faculty of Medicine, Department of Hematology, Sakarya, Turkey
Abstract
Objective: Acute myeloid leukemia (AML) is a complex disease
affected by both genetic and epigenetic factors. Histone methylation
and demethylation are types of epigenetic modification in chromatin
remodeling and gene expression. Abnormal expression of histone
demethylases is indicated in many types of cancer including AML.
Although many commercial drugs are available to treat AML, an
absolute cure has not been discovered yet. However, inhibition
of demethylases could be a potential cure for AML. Methylstat is
a chemical agent that inhibits the Jumonji C domain-containing
demethylases.
Materials and Methods: The cytotoxic and apoptotic effects of
methylstat and doxorubicin on HL-60 cells were detected by MTT
cell viability assay, double staining of treated cells with annexin-V/
propidium iodide, and caspase-3 activity assay. Mitochondrial activity
was analyzed using JC-1 dye. The expression levels of the BCL2 and
BCL2L1 anti-apoptotic genes in HL-60 cells were determined using
real-time polymerase chain reaction (PCR). Lastly, the cytostatic effect
was determined by cell cycle analysis.
Results: In our research, cytotoxic, cytostatic, and apoptotic effects
of methylstat on human HL-60 cells were investigated. Cytotoxic
and cytostatic analyses revealed that methylstat decreased cell
proliferation in a dose-dependent cytotoxic manner and arrested HL-
60 cells in the G2/M and S phases. Methylstat also induced apoptosis
through the loss of mitochondrial membrane potential and increases
in caspase-3 enzyme activity. The expression levels of BCL2 and BCL2L1
were also decreased according to real-time PCR results. Finally, the
combination of methylstat with doxorubicin resulted in synergistic
cytotoxic effects on HL-60 cells.
Conclusion: Taken together, these results demonstrate that methylstat
may be a powerful candidate as a drug component of AML treatment
protocols.
Keywords: Acute myeloid leukemia, Methylstat, Jumonji C domain,
Histone methylation
Öz
Amaç: Akut myeloid lösemi (AML) hem genetik hem de epigenetik
faktörlerden etkilenen kompleks bir hastalıktır. Histon metilasyonu
ve demetilasyonu, kromatin yeniden yapılanması ve gen ekspresyonu
tayinindeki önemli epigenetik modifikasyonlarındandır. Histon
demetilazların anormal ekspresyonu, AML dahil birçok kanser türünde
etkilidir. AML’yi tedavi etmek için birçok ticari ilaç mevcut olmasına
rağmen, kesin tedavisi bulunmamaktadır. Bu nedenle, demetilazların
inhibisyonu AML için potansiyel bir tedavi olabilir. Methylstat,
demetilaz içeren Jumonji C domain-inhibe eden bir kimyasal maddedir.
Gereç ve Yöntemler: Methylstat ve doksorubisinin HL-60 hücreleri
üzerindeki sitotoksik ve apoptotik etkisi, sırasıyla MTT canlılık testi,
annexin-V/propidium iyodür çift boyaması ve kaspaz-3 aktivite
testi ile saptandı. Ayrıca, hücrelerdeki mitokondriyal aktivite JC-1
boyası kullanılarak analiz edildi. HL-60 hücrelerinde BCL2 ve BCL2L1
apoptotik olmayan genlerin ekspresyon seviyeleri, gerçek zamanlı
polimeraz zincir reaksiyonu (PZR) kullanılarak belirlendi. Son olarak,
sitostatik etki hücre döngüsü analizi ile belirlenmiştir.
Bulgular: Araştırmamızda, methylstatın HL-60 hücreleri üzerindeki
sitotoksik, sitostatik ve apoptotik etkileri olduğu belirlenmiştir.
Sitotoksik ve sitostatik analizlerde methylstatın, HL-60 hücrelerinde
doza bağlı hücre çoğalmasını azalttığını ve G2/M ve S fazlarındaki
sitotoksik etkisinin arttığı ortaya koydu. Methylstat ayrıca,
mitokondriyal membran potansiyelini düşürdüğü ve kaspaz-3 enzim
aktivitesindeki artışlarla apoptozu indüklemiştir. Ayrıca, gerçek
zamanlı PZR sonuçlarına göre BCL2 ve BCL2L1 genlerinin ekspresyon
seviyeleri azalmaktadır. Son olarak, methylstatın doksorubisin ile
kombinasyonu, HL-60 hücreleri üzerinde sinerjik sitotoksik etkilere yol
açmıştır.
Sonuç: Methylstatın AML için tam bir tedavi bulmak için bir ilaç
bileşeni olarak güçlü bir aday olabileceğini göstermiştir.
Anahtar Sözcükler: Akut miyeloid lösemi, Methylstat, Jumonji C
domeini, Histon metilasyonu
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Yusuf Baran, M.D., İzmir Institute of Technology, Department of
Molecular Biology and Genetics, İzmir, Turkey
Phone : +90 232 750 73 00
E-mail : ybaran@gmail.com ORCID: orcid.org/0000-0001-8354-780X
Received/Geliş tarihi: February 25, 2019
Accepted/Kabul tarihi: December 11, 2019
5
Koca D, et al: Therapeutic Potentials of Inhibition of Jumonji C Domain-containing Demethylases in Acute Myeloid Leukemia Turk J Hematol 2020;37:5-12
Introduction
Acute myeloid leukemia (AML) is a disease caused by the
rapid proliferation of neoplastic cells [1]. AML is determined
by the accumulation of blast cells and the blocking of the
differentiation of myeloid cells in bone marrow. This abnormal
proliferation leads to the disruption of bone marrow function
and the maturation of white blood cells that are necessary
for the immune system [2,3]. Treatment of AML is provided
by cytotoxic drugs and bone marrow transplantation [4].
However, the exact treatment of AML is still unclear [5]. AML
is known as a genetic disorder resulting from chromosomal
translocations. However, developing depth genome sequencing
shows that epigenetic factors and abnormalities also have a
role in the progression of AML [6]. According to cytogenetic
tests, ~50% of AML patients possess normal karyotypes
[7]. These outcomes revealed that not only chromosomal
alterations but also epigenetic abnormalities might have roles
in the progression of AML [8]. Histone-modifying enzymes play
crucial roles during the dynamic regulation of gene expression
and cell identification [9]. As histone-modifying enzymes,
histone demethylases have a potent role in the regulation of
gene expression through modulation of histone methylation.
Recently, overexpression of several histone demethylases has
been observed in many types of cancer. Different drugs are
developing to regulate DNA methylation, which is a critical
point for targeted cancer therapy and decreasing drug
resistance of cancer cells [10,11].
There are two families of histone demethylases. The LSD family
has two subfamilies known as LSD1 (also known as KDM1A) and
LSD2 (also known as KDM1B). These enzymes contain an amine
oxidase-like domain and are flavin-dependent demethylases
[12,13]. The second family of histone demethylases comprises the
catalytic Jumonji C (JmjC) domain-containing demethylases. The
enzymatic reaction mechanism of demethylases containing the
JmjC domain requires two cofactors, Fe(II) and 2-oxoglutarate.
The JmjC domain behaves differently in terms of its reaction
mechanism. In contrast to LSD demethylases, JmjC achieves
removal of trimethyl marks. The biochemical and biological
functions of the JmjC domain are interesting for cancer
treatment due to the regulation of chromatin remodeling and
epigenetic factors that provide genome stability [14,15].
In recent studies, overexpression of several JmjC domaincontaining
histone demethylases (JHDMs) was determined for
many types of cancer, including leukemia [16,17]. Therefore,
JHDMs could be a therapeutic target for AML treatment. The
compounds that inhibit JHDMs have potential as candidate
anti-cancer agents [18]. In this study, we aimed to examine
the cytotoxic, cytostatic, and apoptotic effects of methylstat, a
selective inhibitor of a large set of JHDMs, on AML cells.
Materials and Methods
Cell Culture
Human HL-60 cells were obtained from the ATCC. The required
medium was RPMI-1640, containing 10% fetal bovine serum
and 1% penicillin and streptomycin. The cells were cultured in
a CO 2
incubator with adjusted conditions of 5% CO 2
and 37 °C.
Every 3 days, the cells were passaged and fresh medium was
provided in order to properly maintain the cells.
Reagents, Drugs, and Compound
Methylstat was dissolved in dimethyl sulfoxide (DMSO) and
the final concentration of methylstat was 50 mM. Doxorubicin
hydrochloride (injectable lyophilized powder form, Teva
Pharmaceutical Industries, Pharmachemie BV) was kindly
provided by Dr. Gökmen Sevindik from Dokuz Eylül University,
İzmir, Turkey. It was prepared by dissolving powder in sterile
molecular biology water and the final concentration was
obtained as 3.4 mM. Required concentrations were calculated
and necessary dilutions were carried out with complete medium
MTT Cell Viability Assay
3-[4.5-Dimethylthiazol-2-yl]-2.5-Diphenyltetrazolium bromide
(MTT) is an auxiliary agent that gives a yellow color when
dissolved in phosphate-buffered saline (PBS). The main stock
solution was prepared in a concentration of 5 mg/mL. To sterilize
the solution, it was filtered through a 0.45-µM filter inside the
hood, and 1x10 4 HL-60 cells were inoculated in wells of 96-well
plates. Methylstat was diluted in determined concentrations
and added to wells. After 72 h, 20 µL of MTT solution was added
to each well. Thus, the ratio of MTT was adjusted to 1:10. The
96-well plates were incubated at 37 °C in an incubator with 5%
CO 2
for 3 h. At the end of incubation, plates were centrifuged
at 1800 rpm for 10 min. Supernatants were removed by gentle
tapping and 150 µL of DMSO was added as a solvent to dissolve
the formazan salts. Plates were shaken on a shaker at 150 rpm
for 5 min to totally dissolve all the crystals. The absorbance was
measured at 570 nM by using a spectrophotometer (Thermo
Multiskan Spectrum). This MTT assay was applied to HL-60 cells
with doxorubicin with the same procedure. For MTT experiments,
the following application doses were used: 50, 25, 12.5, 6.2, 3.1,
1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 µM.
Double Staining of Treated Cells with Annexin-V/Propidium
Iodide (PI)
In healthy cells, phosphatidylserines (PSs) are located in the
inner part of the membrane, while in apoptotic cells, the
asymmetry of the cell membrane is destroyed and PSs face an
extracellular matrix of cells. Annexin-V has high affinity to PSs.
Thus, apoptosis can be detected with the help of the binding
of annexin-V to PSs. On the other hand, while living cells are
impermeable to propidium iodide (PI), membranes of dead cells
6
Turk J Hematol 2020;37:5-12
Koca D, et al: Therapeutic Potentials of Inhibition of Jumonji C Domain-containing Demethylases in Acute Myeloid Leukemia
are permeable to PI. Therefore, double staining provides us an
understanding of whether the apoptosis is early or late [19]. HL-
60 cells were grown in the absence and presence of methylstat
and incubated at 37 °C and 5% CO 2
for 72 h. The cells were
then collected into 15-mL Falcon tubes and washed with PBS.
To obtain pellets, tubes were centrifuged at 800 rpm for 5 min
and the cells were washed with cold PBS two times. Obtained
pellets were resuspended with 1X annexin binding buffer and
transferred to properly labeled FACS tubes, and 5 µL of FITC and
5 µL of PI were added to each sample. Tubes were incubated
for 15 min in the dark. At the end of incubation, 400 µL of 1X
annexin binding buffer was added and the results were obtained
by using flow cytometry (FACSCanto, BD, USA).
Detection of Caspase-3 Activity
The caspase family, including cysteine-dependent, aspartatespecific
proteinases, has a role in apoptosis. Therefore, the
detection of caspase-3 activity is important in the determination
of apoptotic signals [20]. Colorimetric determination of
caspase-3 activity was determined with the help of a BioVision
assay. In this assay, DEVD-pNA (chromophore p-nitroaniline) is
the prepared substrate for the caspase-3 enzyme. When the
caspase-3 enzyme cleaves pNA from DEVD by recognizing the
DEVD sequence, pNA gives an emission in spectrophotometry.
In our experiments, 1x10 6 cells were seeded in 6-well plates
and incubated in the absence or presence of increasing
concentrations of methylstat. After 72 h of incubation at 37
°C in an incubator with 5% CO 2
, cells were collected in Falcon
tubes and centrifuged at 1000 rpm for 10 min. Supernatants
were taken into Eppendorf tubes and 100 µL of lysis buffer
was added to each sample. They were incubated for 10 min
on ice. Centrifugation was adjusted to 4 °C and the samples
were centrifuged at 10,000 x g for 1 min. Supernatants were
taken into other labeled Eppendorf tubes and 100 µL of lysis
buffer was added. Halves of the supernatants were stored at
-80 °C for the Bradford assay, and 50 µL of each sample was
transferred to a 96-well plate. Dithiothreitol (DTT) was added to
2X reaction buffer (10 mM DTT in 50 µL of reaction buffer) and
5 µL of DEVD-pNA was added to each sample, and the plates
were incubated at 37 °C with 5% CO 2
in the dark. By using a
spectrophotometer, the absorbance of samples was measured
at 405 nM. These absorbance values were used to determine
protein concentrations by standardization with Bradford assay
absorbance.
Determination of Loss of Mitochondrial Membrane Potential
Mitochondria play a crucial role during apoptosis. Cells lose their
mitochondrial membrane potential (MMP). In order to detect
this loss, a dye is utilized called 5,5′,6,6′-tetrachloro-1,1′,3,3′-
tetraethyl benzimidazol carbocyanine iodide (JC-1). It is a
cytofluorimetric, lipophilic, and cationic dye. In healthy cells, this
dye aggregates in the mitochondria and gives a red fluorescence,
whereas in apoptotic or dead cells, the dye cannot pass through
mitochondria and stays in the cytoplasm in monomer form.
This monomer form of the dye gives a green fluorescence [21].
Briefly, 1x10 6 cells were seeded in 6-well plates and increasing
concentrations of methylstat were added to the cells. After 72
h of incubation at 37 °C and 5% CO 2
, the cells were centrifuged
at 1000 rpm for 10 min. Pellets were dissolved in 300 µL of
complete medium and 30 µL of JC-1 dye was added to each
sample. They were incubated for 30 min at 37 °C. Samples were
centrifuged at 400 x g for 5 min and washed with assay buffer
twice. At the end, 300 µL of assay buffer was added and samples
were placed in a black 96-well plate in triplicate. Using a
spectrophotometer (Thermo, Varioskan Flash), the samples were
measured at 485 and 535 nm for green and at 560 and 595 nm
for red fluorescence. The ratios of green/red fluorescence were
used to analyze the apoptosis rate.
Cell Cycle Analysis
Determination of DNA contents of cells was carried out with the
help of propidium iodide (PI) dye that specifically binds to DNA.
The DNA content decreases in dead cells compared to living cells,
so the level of PI gives information about the percentage of
cells in the phases of the cell cycle [22]. For this analysis, 1x10 6
cells per 2 mL were added to 6-well plates and incubated in the
absence or presence of increasing concentrations of methylstat
at 37 °C and 5% CO 2
. Collected cells were centrifuged at 1200
rpm for 5 min. Supernatants were discarded and PBS was added.
In order to fix the cells, cold absolute ethanol (incubated at
-20 °C) was added and the cells were incubated at -20 °C deep
freeze overnight. The following day, the cells were centrifuged
at 1200 rpm for 10 min at 4 °C and washed with cold PBS.
Pellets were resuspended with 200 µL of PBS with 0.1% Triton-X
100, and then 20 µL of RNase-A enzyme (200 µg/mL in dH 2
O)
was added to each sample and they were incubated at 37 °C for
30 min. At the end of incubation, cells were stained with 20 µL
of PI dye (1 µg/mL in dH 2
O) and incubated for 10 min at room
temperature. They were analyzed using a flow cytometer.
cDNA Synthesis and Real-time PCR (RT-PCR)
In order to see the effect of methylstat on the expression
levels of anti-apoptotic BCL2 and BCL2L1, RT-PCR was carried
out. First, 1x10 6 cells/2 mL medium were seeded into 6-well
plates and incubated for 72 h at 37 °C and 5% CO 2
. The
NucleoSpin RNA kit was used to isolate RNAs according to
the manufacturer’s instructions. RNA amounts were measured
by NanoDrop (260/280 and 260/230 ratios), and 1000 ng of
RNA was used for the synthesis of cDNA. Other components
of the PCR mixture were random hexamer primer, buffer,
dNTP mix, RNase inhibitor, and reverse transcriptase. The
mixtures were incubated at 42 °C for 1 h before incubation
at 72 °C for 10 min. These synthesized cDNAs were applied
to analyze the changes in expression levels of the BCL2 and
7
Koca D, et al: Therapeutic Potentials of Inhibition of Jumonji C Domain-containing Demethylases in Acute Myeloid Leukemia
Turk J Hematol 2020;37:5-12
BCL2L1 genes. The forward primer sequence of the BCL2 gene
was 5’-GCACCTGCACACCTGGAT-3’ and the reverse primer
sequence of BCL2 was 5’-AGCCAGGAGAAATCAAACAGAG-3’,
while the forward primer sequence of the BCL2L1 gene was
5’-AGCCTTGGATCCAGGAGAA-3’ and the reverse primer was
5’-GCTGCATTGTTCCCATAGAGT-3’. Reaction mixtures were
prepared to carry out RT-PCR. The utilized Thermo Scientific
DyNAmo Flash SYBR® Green qPCR Kit mixtures contain 2X
master mix and 50X ROX reference passive dye, and 10 µL of
master mix, 5 µL of primer (diluted 1/10), and 5 µL of cDNA (5
ng/µL) were added to Eppendorf tubes for each sample. RT-PCR
conditions were adjusted according to the Thermo Scientific
DyNAmo Flash SYBR® Green qPCR Kit. Annealing and melting
curve temperatures were 60 °C and 40 °C for both the BCL2 and
BCL2L1 genes, respectively. The expression level of the GAPDH
gene was used as an internal positive control. The formula of
target gene delta C t
/reference gene delta C t
was used in the
calculations. Graphics were plotted with the control sample as
100 and other samples were calculated according to the control
sample.
control cells (which are double-negative on the lower left side
of Figure 3).
Methylstat Stimulates Loss of MMP in HL-60 Cells in a Dose-
Dependent Fashion
During apoptosis, the loss of MMP is a crucial mediator.
Consequently, the loss of MMP for HL-60 cells for the same
concentrations of methylstat was detected with the JC-1
Mitochondrial Membrane Potential Assay Kit. Considering the
results, the loss of MMP was increased 2.5-, 17-, 20-, and 27-
fold in HL-60 cells exposed to 3, 5, 10, and 20 μM methylstat,
respectively, with respect to the control group (Figure 4).
Statistical Analysis
Statistical analyses and graphs were generated using GraphPad
Prism. The statistical significance was detected using one-way
analysis of variance (ANOVA) for MTT analysis, annexin, MMP,
and caspase-3 enzyme activity and two-way ANOVA for the
expression levels of BCL2 and BCL2L1 and the MTT analysis of
the synergistic effects of methylstat and doxorubicin. A value of
p<0.05 was considered to be statistically significant and a value
of p<0.001 was considered to be highly statistically significant.
Statistics were analyzed using GraphPad Prism 6 for Windows.
Results
Cytotoxic Effects of Methylstat on HL-60 Cells
Cytotoxic effects of methylstat on HL-60 cells were indicated
by MTT cell proliferation assay. There were dose-dependent
decreases in the proliferation of HL-60 cells in response to
methylstat. The IC 50
value of methylstat for HL-60 cells was
calculated as 1.7 μM for 72 h (Figure 1).
Figure 1. Cytotoxic effects of methylstat on HL-60 acute myeloid
leukemia (AML) cells. The IC 50 value of methylstat is calculated as
1.7 µM by plotted graphs of cell proliferation. Three independent
experiments were conducted for data points. The error bars show
the standard deviations. Statistical significance was detected by
using one-way analysis of variance and p<0.05 was considered to
be significant.
Methylstat Induced Apoptosis in HL-60 Cells in a Dose-
Dependent Manner
Methylstat-induced apoptosis in HL-60 cells was demonstrated
by annexin V and PI staining. Changes in apoptotic cell
populations by incremental concentrations of methylstat were
detected by flow cytometry. There were 1.6-, 10-, 20-, and 22-
fold increases in the apoptotic cell population in response to 3,
5, 10, and 20 μM methylstat, respectively, as compared to the
untreated control (Figure 2). The contour plots with quadrant
gates also indicated that the cell population shifts toward the
late apoptotic and early apoptotic quadrants as compared to
Figure 2. Apoptotic effects of methylstat on HL-60 cells:
percentage of dose-dependent apoptotic cell population as
compared to untreated cells. The results are the means of 3
independent experiments and the error bars show the standard
deviations. Statistical significance was detected by using oneway
analysis of variance and p<0.001 was considered to be highly
significant.
8
Turk J Hematol 2020;37:5-12
Koca D, et al: Therapeutic Potentials of Inhibition of Jumonji C Domain-containing Demethylases in Acute Myeloid Leukemia
Methylstat Induces Caspase-3 Enzyme Activity in a Dose-
Dependent Manner in HL-60 Cells
In order to examine methylstat-induced apoptosis, caspase-3
enzyme activity was also determined in HL-60 cells exposed
to the same concentrations of methylstat using a caspase-3
colorimetric assay kit. There were 1.3-, 1.9-, 2.1-, and 1.9-fold
increases of caspase-3 enzyme activity with respect to the
untreated control group in HL-60 cells treated with 3, 5, 10,
and 20 μM methylstat, respectively (Figure 5).
Methylstat Induced Apoptosis by Downregulating Expression
Levels of the BCL2 and BCL2L1 Genes in HL-60 Cells
The expression levels of the anti-apoptotic genes BCL2 and
BCL2L1 for HL-60 cells treated with 5,10, and 20 μM methylstat
decreased by 78%, 86%, and 91% and by 24%, 85%, and 77%,
respectively, as compared to the control (Figure 6).
Methylstat Arrested Cell Cycle Progression in G2/M and S
Phases in HL-60 Cells
The cytostatic effects of methylstat were displayed by DNasefree
RNase and PI staining using flow cytometry. As shown in
Figure 7, methylstat-treated HL-60 cells have increased cell
populations in the S and G2/M phases as the concentration
increases, while the percentage of cells arrested at the G0/G1
phase decreases. These results indicate that methylstat arrests
the cell division cycle in the S and G2/M phases.
Synergistic Effects of Methylstat and Doxorubicin on HL-60 Cells
Figure 3. Dot plot diagrams obtained by flow-cytometric analysis
of treated HL-60 cells after double staining with annexin V-FITC
and PI. Annexin-V FITC-A and PI-A contour plots via quadrant
gates show four populations: intact cells in lower-left quadrant,
FITC-negative/PI-negative; early apoptotic cells in lower-right
quadrant, FITC-positive/PI-negative; late apoptotic or necrotic
cells in upper-right quadrant, FITC-positive/PI-positive; necrotic
cells in upper-left quadrant, FITC-negative/PI-positive.
Doxorubicin is known as a common treatment option for AML.
We examined the possible synergistic effect of the combination
of doxorubicin and methylstat on HL-60 cells. With this aim,
HL-60 cells were exposed to increasing concentrations of
doxorubicin together with IC 20
concentrations of methylstat.
There were significant synergistic effects of this combination as
compared to either agent alone (Figure 8).
Discussion
The importance of epigenetic modifications increases with
large-scale research. Histone-modifying enzymes are the subject
of special interest because they are main players in epigenetics.
Histone methylation and demethylation play important roles in
diverse pathological and biological events, including cancers.
The reversal of histone demethylations can be a potential
Figure 4. Effects of methylstat on loss of mitochondrial membrane
potential. The data are indicated as the means of at least two
independent experiments and the error bars show the standard
deviations. Statistical significance was detected by using oneway
analysis of variance and p<0.001 was considered to be highly
significant.
Figure 5. Effects of methylstat on caspase-3 enzyme activity.
The data are indicated as the means of at least two independent
experiments and the error bars show the standard deviations.
Statistical significance was detected by using one-way analysis of
variance and p<0.05 was considered to be significant.
9
Koca D, et al: Therapeutic Potentials of Inhibition of Jumonji C Domain-containing Demethylases in Acute Myeloid Leukemia
Turk J Hematol 2020;37:5-12
Figure 6. Changes in mRNA levels of anti-apoptotic BCL2 and BCL2L1 genes. Statistical significance was detected by using two-way
analysis of variance and p<0.001 was considered to be highly significant.
Figure 7. Effects of methylstat on cell cycle progression of HL-
60 cells. Three independent experiments were conducted for data
points.
treatment strategy for cancer due to the overexpression of
histone demethylases in various cancer types [23].
Over the last decade, many studies have focused on the JmjC
family of histone demethylases. A number of researchers
have indicated the association between JHDMs and several
physiological and pathological circumstances including cancer,
inflammation, development, metabolism, and neurological
disorders. The relevance of dysregulation of JHDMs in various
Figure 8. Synergistic cytotoxic effects of doxorubicin in
combination with methylstat (72 h). The results are the means of
4 independent experiments and error bars indicate the standard
deviations. Statistical significance was detected by using oneway
analysis of variance and p<0.001 was considered to be highly
significant.
types of cancer makes them a potential candidate to target. If
abnormal JmjC demethylase activity is modulated, it can lead
to normal transcriptional arrangements. Therefore, JmjC domain
inhibitors might have therapeutic potentials for the treatment
of cancer [24,25,26,27].
Here, we addressed the particular question of whether a
selectively active inhibitor of JHDMs has anti-proliferative
effects. There is no significant knowledge about the relevance
10
Turk J Hematol 2020;37:5-12
Koca D, et al: Therapeutic Potentials of Inhibition of Jumonji C Domain-containing Demethylases in Acute Myeloid Leukemia
of JHDMs for AML. In this study, we aimed to detect the
cytotoxic, cytostatic, and apoptotic effects of methylstat
on HL-60 acute pre-myelocytic leukemia cells. The cells
were treated with increasing concentrations of methylstat.
Methylstat showed cytotoxic/antiproliferative effects in a
dose-dependent manner. To determine the apoptotic effects
of methylstat, annexin FITC/PI double staining, caspase-3
enzyme activity, and the loss of MMP were studied (72 h).
With respect to these apoptotic assays, methylstat exhibited
a dose-dependent induction of apoptosis and increased the
percentage of apoptotic cells as compared to untreated
controls. The mRNA expression levels of the foremost antiapoptotic
genes, BCL2 and BCL2L1, were detected by RT-
PCR. The results supported the previous apoptotic assays and
indicated that BCL2 and BCL2L1 expressions were decreased
with incremental concentrations of methylstat. Besides the
induction of apoptosis, cytostatic effects were checked to
see how methylstat mediates cell cycle arrest in HL-60 cells.
Incubation of HL-60 cells for 72 h with methylstat led to cell
cycle arrest at the G2/M and S phases. On the other hand, to
increase the therapeutic effects and to reduce the side effects,
it might be better to combine conventional therapies with
epigenetic modifiers such as demethylase inhibitors. Therefore,
in our study, we combined methylstat with the most common
chemotherapy agent, doxorubicin, in order to see whether they
have synergistic effects. Doxorubicin had 2 times more antiproliferative
effect together with an IC 20
value of methylstat
on HL-60 cells.
Conclusion
According to our results, methylstat has potent apoptotic
effects on HL-60 cells. It might have therapeutic potential
to treat AML through the induction of apoptosis and antiproliferative
effects. However, it would be more effective to
show the chemotherapy response of methylstat with more cell
lines and on mouse models of human AML. That could be more
helpful for a better understanding of the clinical implications
of methylstat.
Chemotherapy targets both healthy, normal cells and cancer
cells. Therefore, it is important for an inhibitor to be specific
for cancer cells in order to minimize the side effects. For further
investigations, it might be better to apply methylstat to human
leukocyte cultures, expecting a higher IC 50
value compared to
AML cell lines. Furthermore, mice xenografts might be helpful
to comprehend the possible potential of methylstat for clinical
implications. In addition, there are a number of JmjC type
subfamilies and it would help to understand which of them
are overexpressed specifically for AML. Depending on that, new
specific therapeutic inhibitors for these JmjC demethylases
might be designed and applied in vivo and in vitro.
Acknowledgments
The National Scholarship Program granted this project for
undergraduate scientists supported by the Scientific and
Technological Research Council of Turkey (BİDEB 2209-A). We
are grateful to Prof. Udo Oppermann for supplying us with
methylstat from the University of Oxford (United Kingdom). The
authors acknowledge the assistance of the Bioengineering and
Biotechnology Application and Research Center staff of İzmir
Institute of Technology. We are deeply thankful to Miray Ünlü,
PhD student, for her assistance with the apoptotic assays.
Ethics
Ethics Committee Approval: There is no need for ethical
committee approval and informed consent since there were no
animal and human samples used in this study.
Informed Consent: There is no need for ethical committee
approval and informed consent since there were no animal and
human samples used in this study.
Authorship Contributions
Consept: D.K., N.H.; Design: Y.B.; Data Collection or Processing:
Y.B., S.E., Y.K., G.T.U., D.Ç.; Experiments: D.K., N.H.; Writing: Y.B.
Conflict of Interest: The authors declare no conflict of interest.
Financial Disclosure: The authors declare that this study
received no financial support.
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12
RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2019.2019.0128
Turk J Hematol 2020;37:13-19
Effects of the Proteasome Inhibitor Bortezomib in Combination
with Chemotherapy for the Treatment of Mantle Cell Lymphoma:
A Meta-analysis
Proteazom İnhibitörü Bortezomib’in Mantle Hücreli Lenfoma Tedavisinde Kemoterapi ile
Kombinasyonundaki Etkileri: Bir Meta-analiz
Shi-Jun Li 1 , Jian Hao 1 , Yu Mao 2 , Yu-Ling Si 1
1Tianjin 4 th Central Hospital, Clinic of Hematology, Tianjin, China
2Renmin Hospital, Clinic of Oncology, Tianjin, China
Abstract
Objective: The efficacy and the safety of bortezomib-based
chemotherapy were characterized in mantle cell lymphoma (MCL)
patients.
Materials and Methods: The PubMed, Cochrane Library, Clinical
Key, Science Direct, Oxford Journals, and China National Knowledge
Internet databases were searched up to 1 May 2019. The selected trials
needed to match the inclusion criteria and be carried out to evaluate
quality appraisal and the synthesis of efficacy and safety. The enrolled
MCL patients using bortezomib-based chemotherapy or chemotherapy
alone needed to have been compared. The overall response rate
(ORR), progression-free survival (PFS), and overall survival (OS) were
combined to evaluate the efficacy while serious adverse events (SAEs)
(grade III-IV peripheral neuropathy, neutropenia, and infection) were
used to evaluate the safety. The heterogeneity of the results were
analyzed simultaneously.
Results: A total of 620 patients were enrolled across four studies in
our meta-analysis, and the pooled results showed that the PFS [hazard
ratio (HR)=0.66, 95% confidence interval (CI)=0.54-0.82; p=0.0001)]
and OS (HR=0.73, 95% CI=0.55-0.96; p=0.03) of patients with
bortezomib-based chemotherapy were better than those of patients
with chemotherapy alone, unlike ORR (risk ratio=1.46, 95% CI=0.85-
2.49; p=0.17), while SAEs were prominent in the combination group.
Conclusion: MCL patients who are ineligible for transplant or
high-dose chemotherapy could benefit from bortezomib-based
chemotherapy.
Keywords: Bortezomib, Chemotherapy, Mantle cell lymphoma, Metaanalysis
Öz
Amaç: Mantle hücreli lenfoma (MCL) hastalarında Bortezomib bazlı
kemoterapinin etkinliği ve güvenilirliğinin belirlenmesi.
Gereç ve Yöntemler: 1 Mayıs 2019 tarihine kadar PubMed, Cochrane
Kütüphanesi, Klinik Anahtar, Doğrudan Bilim, Oxford Dergileri ve
Çin Ulusal Bilgi İnternet veritabanları araştırıldı. Seçilen çalışmaların
dahil edilme kriterlerini karşılaması ve kalite, ve etkinlik ve güvenlik
sentezi yapmış olması gerekiyordu. Çalışmaların bortezomib bazlı
kemoterapi ile sadece kemoterapi kullanan MCL hastalarını karşılıyor
olması gerekiyordu. Etkinliği değerlendirmek için genel yanıt oranı
(ORR), ilerlemesiz sağkalım (PFS) ve genel sağkalım (OS) birleştirilirken,
ciddi advers olaylar (SAE’ler) (3-4. düzey periferik nöropati, nötropeni
ve enfeksiyon) güvenliği değerlendirmek için kullanıldı. Sonuçların
heterojenliği aynı anda analiz edildi.
Bulgular: Meta-analizimizde dört çalışmaya toplam 620 hasta dahil
edilmişti ve toplu sonuçlarda PFS [tehlike oranı (HR)=0,66, %95 güven
aralığı (CI)=0,54-0,82; p=0,0001)] ve OS (HR=0,73, %95 CI=0,55-
0,96; p=0,03) ORR’den (risk oranı=1,46, %95 CI=0,85-2,49; p=0,17)
farklı olarak bortezomib bazlı kemoterapi alan hastalarda, tek başına
kemoterapi alan hastalardan daha iyi idi, SAE’ler ise kombinasyon
grubunda daha belirgindi.
Sonuç: Nakil veya yüksek doz kemoterapi için uygun olmayan MCL
hastaları bortezomib bazlı kemoterapiden yarar görebilir.
Anahtar Sözcükler: Bortezomib, Kemoterapi, Mantle hücreli lenfoma,
Meta-analiz
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Yu-Ling Si, M.D., Tianjin 4 th Central Hospital, Clinic of Hematology,
Tianjin, China
E-mail : skylovelu@aliyun.com ORCID: orcid.org/0000-0002-2728-1610
Received/Geliş tarihi: March 25, 2019
Accepted/Kabul tarihi: August 28, 2019
13
Li SJ, et al: The Efficacy and Safety of Bortezomib in MCL Turk J Hematol 2020;37:13-19
Introduction
Generally, mantle cell lymphoma (MCL) is an aggressive,
incurable subtype of non-Hodgkin B cell lymphoma [1,2,3],
with cyclin D1 overexpression resulting from t(11;14)
(q13;q32) translocation [4,5]. High-dose chemotherapy
with or without consolidation followed by autologous
hematopoietic stem cell transplantation (ASCT) is the firstline
treatment for MCL patient [2]. For patients not suitable
for high-dose chemotherapy or transplant, reduced-dose
chemotherapy is recommended [1,2,4]. However, there are
no generally accepted therapeutic approaches to date.
Combined chemotherapy regimens like cyclophosphamide,
doxorubicin, vincristine, prednisone, and rituximab (R-CHOP)
or rituximab, hyperfractionated cyclophosphamide, vincristine,
doxorubicin, and dexamethasone (hyper-CVAD), and/or highdose
consolidation therapies, are frequently used. However,
the median failure-free survival for standard therapy is only 8
to 20 months and the median survival of patients with highintensity
chemotherapy is 3-4 years [6]. A number of novel
agents were later approved for MCL, including bortezomib,
lenalidomide, and ibrutinib. Among them, ibrutinib obtained
the most significant effects with over 60% overall response
rate (ORR) and almost 20% complete remission (CR) in
relapsed/refractory (R/R) MCL [7], but it is not widely available
for patients in developing countries with expensive costs.
Lenalidomide did not benefit MCL patients with the minimum
ORR and CR in R/R MCL [8].
Bortezomib was confirmed to have a durable response and a
favorable rate of progression-free survival (PFS) in single-agent
data for R/R MCL in a multicenter phase II study [9], which
contributed to it being approved by the FDA for the treatment
of MCL patients in relapse after prior therapy. The SWOG S0601
trial further showed that the combination of bortezomib with
R-CHOP followed by bortezomib maintenance obtained a
doubled 2-year PFS rate compared with the R-CHOP regimen
alone (62% vs. 30%) in previously untreated MCL patients [10].
However, a randomized phase II study assessed the efficacy of
bortezomib plus CHOP versus CHOP in relapsed MCL patients
and showed that bortezomib-based chemotherapy had a nonsignificant
improvement on PFS (16.5 months vs. 8.1 months;
p=0.12) [11]. To obtain a better understanding of bortezomib
combination therapy in MCL patients, we performed a metaanalysis
of clinical trials to compare the efficacy and safety of
bortezomib-based chemotherapy in MCL patients.
Materials and Methods
Literature Sources
A literature review was performed by two reviewers
independently on the efficacy and safety of bortezomib-based
chemotherapy for MCL patients in the PubMed, Cochrane
Library, Clinical Key, Science Direct, Oxford Journals, and China
National Knowledge Infrastructure databases in both English
and Chinese. All relevant studies reported up to 1 May 2019
were searched and the search terms included “mantle-cell
lymphoma” or “MCL” and “bortezomib” or “Velcade” alone or
together. In addition, the published reference lists of those
articles were also checked for further eligible publications.
Inclusion Criteria
The eligible studies needed to conform to the following inclusion
criteria: (1) the trials enrolled MCL patients who were newly
diagnosed, previously untreated, in first CR, or relapsed; (2) the
trials included randomized controlled trials (RCTs) or prospective
cohort trials with a coincident or historical control group;
(3) the trials provided sufficient data on bortezomib-based
chemotherapy for MCL patients, including the hazard ratio (HR)
of the overall survival (OS) and the PFS or the odds ratio (OR)
of the clinical-pathological factors, which could be calculated
along with the corresponding 95% confidence interval (CI); (4)
if data were presented in more than one article, the most recent
or the most elaborate study would be selected; (5) reviews, case
reports, editorial comments, or letters to the editor without
original data were not included.
Data Collection and Quality Assessment
All titles and abstracts were screened by two reviewers
independently. Disagreements between the two reviewers
were settled by discussions to reevaluate the methodological
quality of original studies. The Jadad scale was used to evaluate
the methodological quality of the included RCTs, ranging from
0 to 7 points [12]. A high-quality study would have a score of
4 or greater. The Newcastle-Ottawa Quality Assessment Scale
(NOS) was used to evaluate the quality of the cohort trials
with a coincident or historical control group, ranging from
0 to 9 points. More than 5 points could be regarded as high
quality.
Outcome Calculation
Full extraction was performed on the comparative studies,
including RCTs and cohort trials with a coincident or historical
control group. The ORR, PFS, and OS were evaluated for
efficacy. Serious adverse events [(SAEs); grade III/IV peripheral
neuropathy, neutropenia, and infection)] were evaluated for
safety. Adverse events were classified in terms of each individual
clinical trial.
Statistical Analysis
RevMan version 5.2 was used to perform all calculations related
to the meta-analysis. Dichotomous data (ORR, peripheral
neuropathy, neutropenia, and infection) were calculated in
terms of a fixed or random effect model and expressed by
the risk ratio (RR) or OR with 95% CI. Time-to-event results
14
Turk J Hematol 2020;37:13-19
Li SJ, et al: The Efficacy and Safety of Bortezomib in MCL
were expressed by HR and 95% CI and pooled
with an inverse variance method through a
fixed effect model. Because ORR is not a minor
probability event, it was usually expressed as RR.
Adverse events were generally expressed as OR.
The inconsistency index (I 2 ) and the χ 2 -based
Cochran Q statistic were applied for heterogeneity
detection between clinical trials. In terms of
the values of the heterogeneity test, different
analysis models were chosen: if I 2 >50%, a random
effect model would be needed; in contrast, when
I 2 ≤50%, a fixed effect model would be selected.
When assessing the difference in outcome,
heterogeneity involving all trials was examined.
A value of p<0.05 was considered statistically
significant.
Results
Clinical Trials
We identified 2201 records in a primary literature
search. After removing 1719 studies that included
review articles, case reports, commentaries,
single-arm trials, and phase I trials, 482 articles
were identified for review. Then, after excluding
duplicate or redundant studies and those
lacking original data, only 4 eligible studies
met the inclusion criteria of this meta-analysis
[11,13,14,15]. All included clinical trials were
presented as full publications; the characteristics
of these trials are summarized in Table 1, including
the name of the first author, year of publication,
country, study design, detailed information on
patients, therapy regimens, median follow-up
time, PFS, OS, and quality score. As labeled in Table
1, Furtado et al. [11], Robak et al. [13], and Wu et
al. [14] were RCTs, and William et al. [15] was a
prospective cohort trial. All included clinical trials
were determined to be of high quality.
Overall Response Rate, Progression-free Survival,
and Overall Survival
The efficacy of bortezomib-based therapy could be
confirmed by ORR and survival analysis in the above
clinical trials (Table 1). The pooled RR for ORR was
1.46 (95% CI=0.85-2.49; p=0.17). There was no
significant difference between bortezomib-based
chemotherapy and chemotherapy alone in terms of
ORR. Bortezomib-based chemotherapy had distinctly
longer PFS (HR=0.66, 95% CI=0.54-0.82; p=0.0001)
and OS (HR=0.73, 95% CI=0.55-0.96; p=0.03) (Figure
1) than chemotherapy alone in MCL patients.
Table 1. Comparison among 4 trials for baseline characteristics, PFS, and OS.
Quality
score
Study
design
Overall
survival
(p-value)
[T/C]
Progressionfree
survival
(p-value)
[T/C]
Median
followup
time
[T/C]
Previous
therapy
[T/C]
Stage [T/C] Elevated
I II III IV
LDH
[T/C]
Male
(%)
[T/C]
Median
age
(range
[T/C])
Information
regarding
patients
Country Patients
and
therapy
regimens
(T/C)
First
author
(year)
4
(Jadad)
Randomized,
phase II trial
35.6/11.8
(34
months
OS)
(p=0.01)
16.5/8.1
(median
months)
(p=0.12)
65/91 0/2 4/1 7/5 12/15 N/A 10/4 34/34
(months)
69 (48-
73)
/71 (50-
83)
Relapse or
progression
MCL
UK B-CHOP
(n=23)/
CHOP
(n=23)
Furtado
et al.
[11]
5
(Jadad)
Randomized,
phase III trial
NR/56.3
(median
months)
(p=0.173)
24.7/14.4
(median
months)
(p<0.001)
73/75 N/A 12/16 49/42 182/186 88/86 N/A 40/40
(months)
65 (26-
88)
/66 (34-
82)
Newly
diagnosed
MCL
VR-CAP
(n=243)/
R-CHOP
(n=244)
28
countries
Robak
et al.
[13]
4
(Jadad)
Randomized,
phase III trial
N/A 56/29
(60-month
OS)
(p<0.05)
67/69 73.6/63.2 N/A 2/3 3/5 14/11 N/A 19/19 38/38
(months)
Relapsed
elderly MCL
China B-CHOP
(n=19)/
CHOP
(n=19)
Wu et
al.
[14]
7 (NOS)
Cohort,
phase II trial
72%/50%
(5-year)
OS)
(p=0.74)
57%/43%
(5-year PFS)
(p=0.37)
74/88 2/1 1/1 3/3 17/21 5/9 23/26 2.79/1.97
(years)
58 (36-
71)
/57 (36-
74)
CR1 MCL
followed by
ASCT
USA V-BEAM
(n=23)/
BEAM
(n=26)
William
et al.
[15]
The baseline characteristics, PFS, and OS of clinical trials as shown in the above table had no significant differences between treatment groups and control groups. T/C:Treatment group/control group; B-CHOP: Bortezomib, cyclophosphamide,
doxorubicin, vincristine, prednisone; CHOP: Cyclophosphamide, doxorubicin, vincristine, prednisone; VR-CAP: Bortezomib, rituximab, cyclophosphamide, doxorubicin, prednisone; R-CHOP: Rituximab, cyclophosphamide, doxorubicin, vincristine,
prednisone; V-BEAM: Bortezomib, carmustine, etoposide, cytarabine, melphalan; BEAM: Carmustine, etoposide, cytarabine, melphalan; ASCT: Autologous stem cell transplantation; LDH: Lactate dehydrogenase; PFS: Progression-free survival; OS:
Overall survival; Jadad, Jadad composite scale; NOS: Newcastle-Ottawa Quality Assessment Scale.
15
Li SJ, et al: The Efficacy and Safety of Bortezomib in MCL Turk J Hematol 2020;37:13-19
Figure 1. Pooled analyses of progression-free survival and overall survival.
CI: Confidence interval.
Serious Adverse Events
Three studies reported SAEs, including grade III/IV peripheral
neuropathy, grade III/IV neutropenia, and grade III/IV infection
[11,13,14]. The pooled OR for grade III/IV peripheral neuropathy,
grade III/IV neutropenia, and grade III/IV infection was 2.44 (95%
CI=1.02-5.83; p=0.04), 2.73 (95% CI=1.80-4.13; p<0.00001),
and 1.83 (95% CI=1.15-2.92; p=0.01) respectively. SAEs were
increased significantly in combination therapy compared with
chemotherapy alone.
Heterogeneity and Sensitivity Analysis
The heterogeneity of ORR was significantly different among
the 4 pooled trials (χ 2 =12.72; df=2; I 2 =84%; p=0.002). The
heterogeneity of grade III/IV peripheral neuropathy (χ 2 =0.74;
df=2; I 2 =0%; p=0.69), grade III/IV neutropenia (χ 2 =0.66; df=2;
I 2 =0%; p=0.72), and grade III/IV infection (χ 2 =1.11; df=2; I 2 =0%;
p=0.57) exhibited a non-significant difference among the four
pooled trials.
Discussion
MCL is an incurable aggressive B-cell lymphoma with poor
prognosis. The better treatment choice for MCL patients is
high-dose chemotherapy containing cytarabine, followed
by ASCT [16,17]. For patients who are either ineligible or not
considered for intensive chemotherapy and ASCT, the standard
R-CHOP regimen followed by rituximab maintenance is most
commonly used [18], which could improve response duration
compared with currently available therapies [19], but relapse
is inevitable. A number of novel agents have been approved
in the treatment of MCL, including bortezomib, lenalidomide,
and ibrutinib. Among them, ibrutinib, a first-generation BTK
inhibitor, obtained the most significant effects with over 60%
ORR and almost 20% CR in R/R MCL [7]. On the contrary,
lenalidomide obtained the minimum ORR and CR in R/R MCL
[8]. Although ibrutinib has changed the landscape of therapy
for MCL, it needs continuous administration until disease
progression or unacceptable drug-related toxicity. That will
be expensive and it is not widely available for patients in
developing countries, especially in China. In addition, most of
the patients receiving ibrutinib experienced common adverse
events, including diarrhea (54%), fatigue (50%), bleeding
(50%), nausea (33%), cytopenias (20%), atrial fibrillation (11%),
dyspnea (32%), and pneumonitis (8%) [7,20], inevitably leading
to the discontinuation of therapy. More recently, acalabrutinib,
a second-generation BTK inhibitor, has demonstrated promising
efficacy with 81% ORR and 40% CR for R/R MCL in a phase
II study along with lower rate of toxicities [21]. However, it
still needs continuous administration until disease progression
or unacceptable drug-related toxicity, which would be
unacceptable for most patients.
16
Turk J Hematol 2020;37:13-19
Li SJ, et al: The Efficacy and Safety of Bortezomib in MCL
Bortezomib, the first proteasome inhibitor, regulates multiple
cell signaling pathways related to the progress of MCL. It can
reversibly depress the 26S proteasome for inhibition of nuclear
factor-κB and TP53, and it can induce cell cycle arrest and
apoptosis [22]. Bortezomib was approved by the FDA for the
treatment of MCL patients in relapse after it was confirmed to
have 31% OS and median response duration of 9.3 months in
single-agent activity for R/R MCL [9]. Afterwards, bortezomib
obtained significant prolongation of PFS and OS in newly
diagnosed MCL patients when combined with standard
chemotherapy in a phase 3 clinical trial [13]. Based on these
studies, bortezomib was approved in the USA and Europe for the
treatment of MCL patients in both relapsed and upfront settings
[23,24]. Furthermore, bortezomib is low-cost and easy to obtain.
However, in a randomized phase II study, there was a nonsignificant
improvement in PFS (16.5 months vs. 8.1 months;
p=0.12) when assessing the efficacy of bortezomib-CHOP
(cyclophosphamide, doxorubicin, vincristine, and prednisone)
versus CHOP in relapsed MCL patients [11]. We thus collected
bortezomib-based clinical trials to explain the efficacy and
safety of bortezomib-based regimens.
In this meta-analysis, four studies were included [11,13,14,15].
There were no significant differences in baseline characteristics
between the treatment group and control group in each
clinical trial. Among them, three studies proved the benefits of
bortezomib regimens in MCL patients. The Furtado et al. [11]
and Wu et al. [14] studies showed the efficacy and toxicity of
a CHOP-bortezomib regimen compared with the CHOP regimen
in MCL patients at first relapse. In Furtado et al. [11], a marked
improvement in the quality of responses was achieved when
bortezomib was added to CHOP chemotherapy, with 82.6%
vs. 47.8% of patients obtaining an objective response (CHOPbortezomib
vs. CHOP, respectively). The OS in the CHOPbortezomib
arm was 35.6 months compared with 11.8 months
in the CHOP arm, but there was no difference in PFS between
the CHOP-bortezomib arm (16.5 months) and CHOP arm
(8.1 months), although 30.4% of patients progressed during
treatment in the CHOP arm compared to 8.7% in the CHOPbortezomib
arm [11]. Moreover, there were slightly more patients
experiencing additional toxicities attributed to the inclusion of
bortezomib. Wu et al. [14] also proved the benefits of a CHOPbortezomib
regimen for ORR and OS without increasing adverse
events. The ORR of CHOP-bortezomib was higher than that of
the CHOP arm (84.2% vs. 42.1%), and the median OS of the
CHOP-bortezomib arm was 56.0 months, which was longer
than the 29.0 months of the CHOP arm. Robak et al. [13]
compared the efficacy and toxicity between newly diagnosed
MCL patients who received R-CHOP and VR-CAP (bortezomib,
rituximab, cyclophosphamide, doxorubicin, and prednisone).
The results showed that the VR-CAP group had a significant
improvement in PFS but no significant improvement in OS and
ORR. Because the median OS was not reached in the VR-CAP
arm at the time of the study, there was only a non-significant
improvement in OS with an improved 4-year OS rate compared
with the R-CHOP arm (64% vs. 54%). Compared with the
R-CHOP group, the patients receiving VR-CAP treatment also
had more adverse events, which were mainly neutropenia and
thrombocytopenia. In addition, William et al. [15] compared the
OS and PFS between a BEAM (carmustine, etoposide, cytarabine,
and melphalan)-bortezomib regimen and a BEAM regimen in
MCL patients who were evaluated as CR1. The results showed
that patients receiving the BEAM-bortezomib regimen had
non-significant improvement in OS and PFS compared with
the BEAM regimen. In that study, the patients enrolled were
in the first CR after receiving upfront therapy with a rituximab
and hyper-CVAD regimen, and they received ASCT after a
BEAM-bortezomib/BEAM regimen treatment. Although there
were no significant improvements in OS and PFS, the authors
considered the benefit of bortezomib to have not been revealed
in the presence of ASCT; in other words, bortezomib could offer
benefits for MCL patients who are ineligible for ASCT. After all,
ASCT benefits only about 60% of MCL patients.
The reported ORR, OS, PFS, and SAE values were pooled from
the above four trials. The meta-analysis showed that patients
who received bortezomib-based therapy had longer PFS and OS
compared with those receiving chemotherapy alone, but there
was no significant difference in ORR. The reasons might be as
follows: first, the pooled data on ORR were extracted from three
studies (Furtado et al. [11], Robak et al. [13], and Wu et al. [14]),
among which Robak et al. [13] enrolled the most patients and
held the highest weight; however, Robak et al. [13] showed that
the bortezomib-based group had no significant improvement
in ORR, which might have influenced the heterogeneity of
ORR. Thus, more research should be conducted to reevaluate
the pooled ORR. Second, Robak et al. [13] showed that the
VR-CAP arm had a non-significant improvement in OS with
an improved 4-year OS rate compared with the R-CHOP arm
(64% vs. 54%); meanwhile, 132 patients (54%) in the R-CHOP
arm and 82 patients (34%) in the VR-CAP group received
subsequent anti-lymphoma therapy, and the type of subsequent
therapy was generally similar in the two groups. However, the
other three studies did not mention subsequent anti-lymphoma
therapy after disease progression, and subsequent antilymphoma
therapy might influence OS. Despite the advantages,
our results suggest that a bortezomib-based regimen might
cause significant increases in SAEs (i.e. grade III/IV peripheral
neuropathy, infection, and neutropenia).
In addition, the post hoc sub-analysis of Robak et al. [13]
assessed the efficacy and safety of VR-CAP and R-CHOP in 80
MCL patients aged <60 years who did not receive stem cell
transplantation despite medical eligibility [25]: the median
PFS and the median OS in the two groups were 42.6 vs. 20.6
17
Li SJ, et al: The Efficacy and Safety of Bortezomib in MCL Turk J Hematol 2020;37:13-19
months (HR=0.54; p=0.057) and not reached vs. 47.3 months
(HR=0.81; p=0.634), which suggested that VR-CAP had superior
efficacy to R-CHOP in suitable young MCL patients. Another
sub-group analysis of Robak et al. [13] investigated whether
VR-CAP compared with R-CHOP could improve outcomes in
East Asian patients with newly diagnosed MCL [26]: the results
supported the benefits of VR-CAP in East Asian patients with
MCL who are ineligible for transplant, as the median PFS was
27.7 months (VR-CAP) vs. 16.1 months (R-CHOP) (HR=0.58;
p=0.03), and the median OS was not reached (VR-CAP) vs. 56.3
months (R-CHOP).
This meta-analysis has some limitations. First of all, because a
great mass of phase 3 RCTs on bortezomib are not yet finished,
only three RCTs were included in our study, and this was the main
limitation of the meta-analysis. Second, some articles were short
of data on CR and the other common SAEs such as thrombotic
events, so we could not compare the outcomes of the two
regimens. Third, the clinical stage of the patients and the selection
of combined chemotherapies were disparate among all trials,
which would bring about heterogeneity. In the future, more phase
3 RCTs concerning bortezomib regimens with or without other
new medicines could help to formulate a conclusion regarding
MCL treatment; they may offer better efficacy and fewer adverse
events. Despite the limitations mentioned above, we have affirmed
that bortezomib-based regimens make a valuable contribution to
the treatment of MCL patients who are either ineligible or not
considered for intensive chemotherapy and ASCT.
Conclusion
In summary, bortezomib-based regimens for MCL patients were
more effective than chemotherapy alone in our analysis, but
with more grade III/IV adverse events. Bortezomib-based therapy
is more suitable for MCL patients who are either ineligible or
not considered for intensive chemotherapy and ASCT.
Ethics
Ethics Committee Approval: This article does not contain any
studies with human participants or animals performed by any
of the authors.
Informed Consent: The article presents a meta-analysis.
Therefore, informed consent is not required.
Authorship Contributions
Concept: S-J.L., J.H., Y.M., Y-L.S.; Design: S-J.L., J.H., Y.M., Y-L.S.;
Data Collection or Processing: S-J.L., J.H., Y.M., Y-L.S.; Writing: S-J.L.
Conflict of Interest: All authors declared that they have no
conflict of interest.
Financial Disclosure: There was no funding or financial support
for this article.
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19
RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2019.2019.0139
Turk J Hematol 2020;37:20-29
Predictive Values of Early Suppression of Tumorigenicity 2 for
Acute GVHD and Transplant-related Complications after Allogeneic
Stem Cell Transplantation: Prospective Observational Study
Allojenik Kök Hücre Transplantasyonu Sonrası Akut GVHD ve Transplantasyonla İlişkili
Komplikasyonlar İçin Tümörjenisite 2’nin Erken Baskılanmasının Prediktif Değeri: Prospektif
Gözlemsel Çalışma
Ayako Matsumura 1 , Takuya Miyazaki 1 , Takayoshi Tachibana 1 , Taiki Ando 2 , Megumi Koyama 2 , Satoshi Koyama 2 ,
Yoshimi Ishii 2 , Hiroyuki Takahashi 1 , Yuki Nakajima 1 , Ayumi Numata 2 , Wataru Yamamoto 2 , Kenji Motohashi 2 ,
Maki Hagihara 1 , Kenji Matsumoto 1 , Shin Fujisawa 2 , Hideaki Nakajima 1
1Yokohama City University Graduate School of Medicine, Department of Stem Cell and Immune Regulation, Kanagawa, Japan
2Yokohama City University Medical Center, Department of Hematology, Kanagawa, Japan
Abstract
Objective: A soluble form of suppression of tumorigenicity 2
(sST2) has emerged as a biomarker for acute graft-versus-host
disease (GVHD) and non-relapse mortality (NRM). We prospectively
monitored sST2 levels during the early phase of hematopoietic stem
cell transplantation (HSCT) and evaluated the clinical association with
transplant-related complications including acute GVHD.
Materials and Methods: Thirty-two adult Japanese patients who
received a first allogeneic HSCT were enrolled in this study. Levels of
sST2 were measured at fixed time points (pre-conditioning, day 0, day
14, day 21, and day 28).
Results: The median age was 50.5 years (range=16-66). With a
median follow-up of 21.5 months (range=0.9-35.4), 9 patients
developed grade II-IV acute GVHD. Median sST2 levels on the day
of HSCT were higher than baseline and reached the maximum value
(92.7 ng/mL; range=0-419.7) on day 21 after HSCT. The optimal cutoff
value of sST2 on day 14 for predicting grade II-IV acute GVHD was
determined as 100 ng/mL by ROC analysis. The cumulative incidence
of acute GVHD was 56.7% and 16.5% in the high- and low-sST2
groups, respectively (p<0.01). Multivariate analyses showed that
high sST2 levels at day 14 were associated with a higher incidence of
acute GVHD (hazard ratio=9.35, 95% confidence interval=2.92-30.0,
p<0.01). The cumulative incidence of NRM was increased in the highsST2
group (33% vs 0%, p<0.01), but all the patients died of non-GVHD
complications. Among 6 patients in the high-sST2 group without
grade II-IV GVHD, 5 patients developed veno-occlusive disease (VOD)
and one also had thrombotic microangiopathy (TMA).
Conclusion: The early assessment of sST2 after HSCT yielded predictive
values for the onset of acute GVHD and other transplant-related
complications including VOD and TMA.
Keywords: Suppression of tumorigenicity 2, Graft-versus-host disease,
Biomarker, Hematopoietic stem cell transplantation
Öz
Amaç: Akut graft-konakçı hastalığı (GVHD) ve relaps dışı mortalite
(NRM) için bir biyobelirteç olarak, tümör oluşumu 2’nin baskılanmasının
(sST2) çözünür bir formu ortaya konulmuştur. Hematopoietik kök
hücre transplantasyonunun (HSCT) erken safhasında sST2 düzeylerini
prospektif olarak izledik ve akut GVHD dahil transplantla ilişkili
komplikasyonlarla klinik ilişkisini değerlendirdik.
Gereç ve Yöntemler: Bu çalışmaya ilk allojenik HSCT’si olan 32
yetişkin Japon hasta alındı. sST2 seviyeleri sabit zaman noktalarında
(hazırlık rejimi öncesi, 0. gün, 14. gün, 21. gün ve 28. gün) ölçüldü.
Bulgular: Ortanca yaş 50,5 idi (aralık=16-66). Ortalama 21,5 aylık
takip süresi (aralık= 0,9-35,4) ile 9 hastada Grade II-IV akut GVHD
gelişti. Nakil gününde ortanca sST2 seviyeleri bazal değerlerden daha
yüksekti ve HSCT’den sonraki 21. günde maksimum değere (92,7 ng/mL;
aralık=0-419,7) ulaştı. Grade II-IV akut GVHD’yi tahmin etmek için 14.
günde sST2’nin optimal eşik değeri, ROC analizi ile 100 ng/mL olarak
belirlenmiştir. Akut GVHD’nin kümülatif insidansı yüksek ve düşük
sST2 gruplarında sırasıyla %56,7 ve %16,5 idi (p<0,01). Çok değişkenli
analizler 14. günde yüksek sST2 düzeylerinin daha yüksek akut GVHD
insidansı ile ilişkili olduğunu göstermiştir (tehlike oranı=9, 35, %95
güven aralığı=2,92-30,0, p<0,01). NRM’nin kümülatif insidansı yüksek
sST2 grubunda artmıştır (%33’e karşı %0, p<0,01), ancak tüm hastalar
GVHD dışı komplikasyonlardan öldü. Yüksek sST2 grubunda grade II-IV
GVHD olmayan 6 hastadan 5’inde veno-oklüzif hastalık (VOD) gelişti
ve birinde trombotik mikroanjiyopati (TMA) vardı.
Sonuç: HSCT sonrası sST2’nin erken değerlendirmesi, akut
GVHD’nin başlangıcı ve VOD ve TMA dahil transplantla ilişkili diğer
komplikasyonlar için prediktif değerler vermiştir.
Anahtar Sözcükler: Tümör oluşumu 2’nin baskılanması, Akut
graft-konakçı hastalığı, Biyomarker, Hematopoietik kök hücre
transplantasyonu
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Takuya Miyazaki, M.D., Yokohama City University Graduate School of
Medicine, Department of Stem Cell and Immune Regulation, Kanagawa, Japan
Phone : +81 45 787 2800
E-mail : takuya_m@yokohama-cu.ac.jp ORCID: orcid.org/0000-0003-1884-4757
Received/Geliş tarihi: April 2, 2019
Accepted/Kabul tarihi: August 28, 2019
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Turk J Hematol 2020;37:20-29
Matsumura A, et al: ST2 for Transplant-related Complications
Introduction
Acute graft-versus-host disease (GVHD) is one of the major
complications after allogeneic hematopoietic stem cell
transplantation (HSCT) and remains the leading cause of nonrelapse
mortality (NRM) [1,2]. Identifying useful biomarkers for
predicting onset or severity of acute GVHD at the early phase
of HSCT may help in the development of a more individualized
treatment strategy for GVHD. Recent studies show several
plasma biomarkers that correlate with acute GVHD: suppression
of tumorigenicity 2 (ST2) [3,4], interleukin (IL)-2 receptor α
[5], tumor necrosis factor receptor 1 (TNFR1) [5], hepatocyte
growth factor [5], IL-8 [5], and IL-6 [6] for systemic GVHD;
elafin for skin GVHD [7]; and regenerating islet-derived 3-α
[8] and T-cell immunoglobulin mucin-3 for gastrointestinal
GVHD [8,9,10,11,12]. Among these biomarkers, ST2 has emerged
as a promising biomarker for onset or steroid-resistant acute
GVHD and NRM [3,4,12,13,14]. In addition, recent studies have
demonstrated that high ST2 levels are associated with the
development of thrombotic microangiopathy (TMA) and venoocclusive
disease (VOD) after HSCT [15,16].
ST2 is a membrane receptor expressed on several immune cell
types that belong to the IL-1 receptor family. ST2 induces T
helper type 2 (Th2) and Treg immune responses, which play
important roles in GVHD [17]. A soluble form of ST2 (sST2)
is a decoy receptor for IL-33, which blocks the IL-33/ST2
pathway and drives Th2 cells toward a Th1 cell phenotype, and
it is thought to be important in the pathophysiology of GVHD
[18,19]. In murine models, the ST2/IL-33 axis has been reported
as a potential therapeutic target for GVHD [20,21].
Despite the accumulating evidence that higher sST2 levels are
associated with increased GVHD risk and subsequent mortality,
a clinical role of sST2 during the early phase of transplantation
has not been fully elucidated. We performed a multicenter,
prospective, observational study monitoring the serial changes
in sST2 levels before and early after HSCT with the aim of
determining the diagnostic and prognostic values for acute
GVHD, other transplant-related complications, and mortality.
Materials and Methods
Patients and Transplant Procedure
Patients who received first allogeneic HSCT for hematological
diseases were enrolled consecutively between February 2014 and
July 2015 at Yokohama City University Hospital or Yokohama
City University Medical Center. The selection of donor source
and conditioning regimen was based on patients’ hematological
diagnosis, donor availability, and patients’ clinical status.
Conditioning regimens were classified into myeloablative
conditioning (MAC) and reduced-intensity conditioning (RIC)
according to the definitions previously reported [22]. The
pre-transplant risk category was defined as standard or high
according to the diagnosis and the disease stage at the time of
transplantation, as described previously [23].
Written informed consent was obtained from all the patients
enrolled in this study before the start of the trial. This study was
approved by the institutional review boards of our university
hospital and medical center and was conducted in accordance
with the Declaration of Helsinki. All authors vouched for the
accuracy and completeness of the reported data, analyses, and
adherence to the study protocol.
Peripheral Blood Samples
Samples were collected prospectively before conditioning, on
the day of HSCT (day 0), and on days 14, 21, and 28 after HSCT.
Serum sST2 and IL-33 levels were measured by ELISA (Human
ST2/IL-1 R4 ELISA Kit and Human IL-33 ELISA Kit; R&D Systems,
Minneapolis, MN, USA). Absorbance was measured using a
microplate reader (Powerscan HT, DS Pharma Biomedical, Osaka,
Japan).
Transplant-related Complications
Acute GVHD was diagnosed clinically with histological
confirmation when available. The classification of acute GVHD
was based on the diagnostic criteria of the 1994 Consensus
Conference on Acute GVHD Grading [24]. GVHD prophylaxis
consisted of cyclosporine or tacrolimus with short-term
methotrexate. Anti-thymocyte globulin was administered
for HLA-serological mismatched transplant based on each
institution’s criteria.
Other transplant-related complications were diagnosed as
follows: TMA was diagnosed according to the Blood and Marrow
Clinical Trials Network and European Group for Blood and
Marrow Transplantation guidelines [25,26]. VOD was diagnosed
based on the Baltimore and Seattle criteria [27,28].
Statistical Analysis
Fisher’s exact test and the Mann-Whitney U test were used to
assess the categorical and continuous variables, respectively.
The receiver operating characteristics (ROC) curve from
logistic regression models with the area under the curve
(AUC) was used to present the correlation between sST2 and
other biomarkers. The Pearson test was used to determine the
correlation between sST2 and other biomarkers. The Kaplan-
Meier method was used to assess overall survival (OS) using the
log-rank test. For multivariate analysis, the Cox proportional
hazards method was used to assess the OS. The Gray test and
Fine-Gray test were used to assess the cumulative incidence
of GVHD and NRM. The competing risks were GVHD and death
prior to GVHD. Values of p<0.05 were considered statistically
significant. All analyses were performed using EZR version 1.36
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Matsumura A, et al: ST2 for Transplant-related Complications Turk J Hematol 2020;37:20-29
statistical software, which is a graphical user interface for R
version 3.4.1 [29].
Results
Patient Characteristics
The clinical characteristics of 32 patients are summarized in
Table 1. The transplant procedure was deemed heterogeneous
based on the donor source, conditioning regimen, and GVHD
prophylaxis.
Expression Patterns of sST2 Following Transplantation
First, we evaluated the expression patterns of sST2 by serial
sampling from individuals at a fixed time point before and early
after HSCT. Different sST2 expression patterns were observed
in each individual after the conditioning therapy (Figure
1A). Compared with median sST2 levels before conditioning
(median=25.9 ng/mL; range=0-42.7 ng/mL), sST2 levels on day 0
were remarkably elevated in most of the patients (median=51.4
ng/mL; range=0-227.9 ng/mL). The sST2 levels reached the
maximum value on day 21 after HSCT (median=92.7 ng/mL;
range=0-419.7 ng/mL) (Figure 1B).
skin involvement (stage 1 in one patient, stage 2 in five, and
stage 3 in four). None developed liver GVHD.
When comparing the sST2 levels in patients with and without
GVHD, median sST2 levels on day 14 after HSCT were relatively
higher in patients with GVHD, but with no significant difference
(Figure 4). Based on ROC curve analyses for predicting the
onset of acute GVHD, the sST2 levels on day 14 showed the
best AUC (0.66), with 66.7% sensitivity and 73.9% specificity
(Figure S1, A). The optimal cut-off point of sST2 was 100 ng/
mL based on the ROC analysis. We focused on day 14 sST2 levels
as the earliest time point for predicting acute GVHD. There was
no significant difference in patient characteristics between the
high-sST2 group (>100 ng/mL) and the low-sST2 group (≤100
ng/mL) (Table 1). The cumulative incidence of grades II-IV
GVHD was significantly higher in the high-sST2 group (56.7%)
than that in the low-sST2 group (16.5%) (p<0.01) (Figure 5A).
Effects of Conditioning Damages and Inflammatory Conditions
on sST2 Levels
As sST2 expression levels may depend on conditioning intensity
[4], the sST2 levels in patients who underwent MAC and RIC
were compared. The sST2 levels in patients who underwent MAC
were higher than those in patients who underwent RIC on days
0, 14, 21, and 28 without statistical difference between the two
groups (Figure 2). To determine whether sST2 was affected by
various inflammatory conditions after HSCT, the correlations
between sST2 and representative inflammatory markers, serum
ferritin and C-reactive protein (CRP), were estimated (Table 2).
sST2 levels were strongly correlated with ferritin and CRP levels
in all samples (r=0.456 and 0.615, respectively). Specifically,
sST2 was well correlated with CRP at days 0, 14, and 21 (r=0.717,
0.630, and 0.628, respectively). Furthermore, levels of serum IL-
33, the ligand of ST2, were under the detection limits in most of
the patients, resulting in no correlation of IL-33 with sST2 levels
(data not shown).
Association of sST2 Levels with Onset of Acute GVHD
The clinical courses of all patients are summarized in Figure 3.
With a median follow-up of 21.5 months (range=0.9-35.4) after
HSCT, 14 patients (43%) developed some grade of acute GVHD
[median days to onset=39 days (range=9-84); median days to
maximum grade=44 (range=15-94)]. The maximum grade of
GVHD was grade I in five patients, grade II in three, and grade III
in six; none developed grade IV. Nine patients had involvement
of the gastrointestinal tract (stage 1 in two patients, stage 2 in
two, stage 3 in four, and stage 4 in one), and ten patients had
Figure 1. Expression patterns of sST2 following transplantation.
A) Expression patterns of sST2 before conditioning (Pre) and on
days 0, 14, 21, and 28 after transplantation in individual patients.
B) Expression patterns of median sST2 values before conditioning
(Pre) and on days 0, 14, 21, and 28 after transplantation. Box plots
indicate medians, interquartiles, and ranges of sST2 levels.
sST2: Suppression of tumorigenicity 2.
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Matsumura A, et al: ST2 for Transplant-related Complications
Table 1. Patient characteristics.
Characteristics Total, n sST2 low, n sST2 high, n p
Age, median (range), years 50.5 (16-66) 49 (16-64) 51.5 (26-66) 0.60
Sex, male/female 20/12 11/9 9/3 0.45
Performance status 0.59
0 16 11 5
1 13 8 5
2 2 1 1
Unknown 1 0 1
Diagnosis 0.39
Acute myeloid leukemia 18 13 5
Acute lymphoid leukemia 4 2 2
Myelodysplastic syndrome 4 1 3
Malignant lymphoma 4 2 2
Multiple myeloma 1 1 0
Aplastic anemia 1 1 0
Disease risk † 0.70
Standard 23 15 8
High 9 5 4
Donor type 0.33
Unrelated donor 24 14 10
Related donor 8 6 2
Donor source 0.47
Bone marrow 24 16 8
Peripheral blood 4 3 1
Cord blood 4 1 3
HLA ‡ 0.70
Matched 21 14 7
Mismatched 11 6 5
Conditioning 1
MAC 21 13 8
RIC 11 7 4
HCT-CI 0.42
0 18 11 7
1-2 8 5 3
≥3 6 4 2
ATG used 0.34
Yes 7 3 4
No 25 17 8
GVHD prophylaxis 0.37
FK506 + sMTX 26 15 11
CyA + sMTX 6 5 1
Engraftment, median (range), days 17 (10-30) 17 (10-30) 18 (14-30) 0.61
sST2: Suppression of tumorigenicity, HLA: Human leukocyte antigen, MAC: Myeloablative conditioning, RIC: Reduced-intensity conditioning, HCT-CI: Hematopoietic cell transplantation
comorbidity index, ATG: Anti-thymocyte globulin, GVHD: Graft-versus-host disease, FK506: Tacrolimus, sMTX: Short-term methotrexate, CyA: Cyclosporine. † Disease risk: “standard risk”
indicates complete remission, partial response, untreated MDS, and aplastic anemia. “High risk” indicates all others [23].
‡ HLA serological match or mismatch (6-loci).
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Matsumura A, et al: ST2 for Transplant-related Complications
Turk J Hematol 2020;37:20-29
Multivariate analyses showed that high sST2 was associated
with higher incidence of acute GVHD (hazard ratio=9.35, 95%
confidence interval=2.92-30.0, p<0.01) (Table 3). sST2 levels at
other time points of days 0, 21, and 28 were not associated with
acute GVHD (data not shown). Although post-transplant CRP
and ferritin levels were well correlated with sST2 (Table 2), these
markers did not influence the cumulative incidence of acute
GVHD (cut-off values were calculated by ROC analysis=CRP 6.0
mg/dL and ferritin 1700 mg/dL, respectively) (Figure 5B and
Figure S1, B). These data suggest that the day 14 sST2 levels
are supportive findings for predicting the onset of acute GVHD.
Among the nine patients who had grade II-IV GVHD, six patients
were part of the high-sST2 group (Figure 6). The patients in the
high-sST2 group developed acute GVHD relatively early after
HSCT (median onset of GVHD=22 days; range=9-50), and sST2
levels were elevated several days before the onset of acute
GVHD. Four patients (cases 6, 7, 11, and 12) developed acute
GVHD before day 28, and the sST2 levels were the highest just
before the development of GVHD. On the other hand, lowsST2
patients did not develop GVHD in the early phase of HSCT
(median onset of GVHD=48 days; range=42-55).
Association of sST2 Levels with GVHD Severity and Target Organ
Involvement
The association of day 14 sST2 levels with GVHD severity was
examined, but the sST2 levels were not associated with the
grade of GVHD (data not shown). The cumulative incidence of
gastrointestinal GVHD was significantly increased in the highsST2
group (50% vs. 15%, p=0.03). However, there was no
significant association between sST2 levels and skin GVHD.
Association of sST2 Levels with Other Transplant-related
Complications
Figure 2. Comparison of sST2 expression patterns based on
conditioning intensity. Median sST2 levels in patients who
received MAC and RIC following transplantation. Box plots
indicate medians, interquartiles, and ranges of sST2 levels.
RIC: Reduced-intensity conditioning, MAC: Myeloablative conditioning,
sST2: Suppression of tumorigenicity 2.
Finally, we evaluated the association of sST2 levels with other
transplant-related complications and mortality (Figure 3).
The 1-year NRM and OS after HSCT was 12.5% and 65.6%,
respectively. The cumulative incidence of 1-year NRM was
significantly increased in the high-sST2 group on day 14 (33%
vs. 0%, p<0.01), while there was no significant difference in
1-year OS by univariate analysis (high sST2=50.0% vs. low
sST2=84.2%; p=0.11). Severe non-GVHD complications were
observed in 10 patients, including TMA (n=2), VOD (n=5), graft
failure (n=1), engraftment syndrome (n=4), sepsis (n=3), and
Figure 3. Clinical courses following HSCT in individual patients.
aGVHD: Acute graft-versus-host disease, Pt. No.: Patient number,
VOD: Veno-occlusive disease, TMA: Thrombotic microangiopathy,
ST2: Suppression of tumorigenicity 2, HSCT: Hematopoietic stem cell
transplantation.
Figure 4. Comparison of sST2 expression patterns in patients
with and without acute GVHD. Mean sST2 levels in patients
who developed grade II-IV acute GVHD or not following
transplantation. Bars indicate the mean ± SEM of sST2 levels.
aGVHD: Acute graft-versus-host disease, sST2: Suppression of
tumorigenicity 2, GVHD: Graft-versus-host disease.
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Matsumura A, et al: ST2 for Transplant-related Complications
Table 2. Correlation between suppression of tumorigenicity 2 and other inflammatory markers.
Pre Day 0 Day 14 Day 21 Day 28 Total
sST2 Median, ng/mL (range) 25.9 (0-42.7) 51.4 (0-227.9) 53.7 (0-419.7) 92.7 (0-419.7) 35.9 (0-419.3)
CRP Median, mg/dL (range) 0.16 (0-10.5) 1.5 (0-11.8) 4.8 (0.1-24.7) 1.7 (0.1-25.7) 0.6 (0-16.5)
r 0.303 0.717 0.630 0.628 0.476 0.615
p 0.092 <0.001 <0.001 <0.001 0.011 <0.001
Ferritin Median, ng/dL (range) 986 (100-2675) 1384 (257-7287) 1943 (432-5231) 2733 (859-9086) 2652 (896-9795)
r 0.463 0.304 0.322 0.467 0.284 0.456
p 0.009 0.116 0.078 0.009 0.135 <0.001
sST2: Suppression of tumorigenicity 2, CRP: C-reactive protein.
Table 3. Univariate and multivariate analyses for acute graft-versus-host disease.
Univariate
Variable n Day 100 aGVHD
% (95% CI)
Age
p
Multivariate
Hazard ratio
(95% CI)
<55 years 23 22.8 (7.9-42.9) Reference
≥55 years 9 49.2 (11.6-79.2) 0.29 3.48 (0.50-24.5) 0.21
Performance status
0 16 31.2 (0.1-0.5)
1 13 27.0 (0.1-0.6)
2 2 50.0 (0.0-1.0)
Unknown 1 NA 0.85
Disease risk
Standard 23 30.4 (0.1-0.5)
High 9 30.6 (0.0-0.7) 0.71
HLA
Matched 21 31.4 (12.2-52.9)
Mismatched 11 30.1 (5.8-60.3) 0.98
Conditioning
MAC 21 26.0 (8.8-47.3) Reference
RIC 11 39.4 (10.4-68.1) 0.53 1.94 (0.19-19.7) 0.57
HCT-CI
0 18 29.8 (0.1-0.5)
1-2 8 37.5 (0.1-0.7)
≥3 6 16.7 (0.0-0.6) 0.79
Donor source
Bone marrow 24 30.3 (13-49.6) Reference
Peripheral blood 4 25.0 (0.3-71.4) 0.37 (0.05-2.74) 0.33
Cord blood 4 NA 0.98 0.13 (0.02-0.81) 0.03
sST2, day 14
Low (≤100 ng/mL) 20 16.5 (3.8-37.1) Reference
High (>100 ng/mL) 12 56.7 (19.2-82.2) <0.01 9.35 (2.92-30.0) <0.01
GVHD: Graft-versus-host disease, MAC: Myeloablative conditioning, RIC: Reduced-intensity conditioning, HLA: Human leukocyte antigen, HCT-CI: Hematopoietic cell transplantationcomorbidity
index, aGVHD: Acute graft-versus-host disease, CI: Confidence interval, sST2: Suppression of tumorigenicity 2.
p
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Matsumura A, et al: ST2 for Transplant-related Complications
Turk J Hematol 2020;37:20-29
pneumonia (n=1), which often overlapped. Four patients in
the high-sST2 group died of transplant-related complications
including sepsis, graft failure, TMA, and pneumonia (cases 1, 3,
4, and 5) without developing grade II-IV GVHD. The two patients
who had TMA (cases 3 and 9) and five who had VOD (cases 1-5)
were included in the high-ST2 group. All patients who died had
severe overlapping complications including VOD.
Discussion
Previous studies have shown that ST2 may predict the onset
of acute GVHD, but no detailed analysis regarding the serial
monitoring of sST2 following HSCT has been reported thus far.
In this study, we performed a prospective evaluation of sST2
expression patterns by serial monitoring before and early after
HSCT and identified that early assessment of sST2 after HSCT
can yield predictive values for the onset of acute GVHD in
addition to other severe transplant-related complications, such
as TMA and VOD.
Although high sST2 levels on day 28 are associated with an
increased risk of acute GVHD in cord blood transplantation
[2], the optimal timing to measure sST2 remains unclear. We
conducted serial sST2 measurements at fixed time points during
HSCT and found that high sST2 levels around day 14 had the
most significant association for predicting GVHD development.
The expression patterns of sST2 in individual patients showed
that sST2 is relatively higher during exacerbation of acute
GVHD. As shown in Figure 6, the sST2 levels in patients with
early-phase GVHD were higher than those with late-phase
GVHD, suggesting that sST2 is clinically useful in predicting the
early phase of GVHD. As acute GVHD often develops in the early
Figure 5. High sST2 on day 14 is correlated with subsequent
GVHD development. A) Cumulative incidence of grade II-IV acute
GVHD by day 100 in patients with high sST2 (>100 ng/mL) and
low sST2 (≤100 ng/mL) levels on day 14 after transplantation. B)
Cumulative incidence of grade II-IV acute GVHD by day 100 in
patients with high-CRP (>6.0 mg/dL) and low-CRP (≤6.0 mg/dL)
levels on day 14 after transplantation. C) Cumulative incidence of
grade II-IV acute GVHD by day 100 in patients with high ferritin
(>1700 mg/dL) and low ferritin (≤1700 mg/dL) levels on day 14
after transplantation. The CRP and ferritin cut-off values were
calculated by ROC curve analysis for predicting the onset of acute
GVHD (ROC curves are available in Figure S1).
sST2: Suppression of tumorigenicity 2, CRP: C-reactive protein, GVHD:
Graft-versus-host disease.
Figure 6. Association of early-phase sST2 levels with subsequent
development of acute GVHD. Patient numbers correspond with
those in Figure 3. Clinical course of 14 patients who developed
grade I-IV acute GVHD. Dotted bars (grade I GVHD) or striped bars
(grade II-IV GVHD) indicate the duration of acute GVHD clinical
symptoms. Cut-off point of sST2 is 100 ng/mL.
aGVHD: Acute graft-versus-host disease, Pt. No.: Patient number, GVHD:
Graft-versus-host disease, sST2: Suppression of tumorigenicity 2.
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Matsumura A, et al: ST2 for Transplant-related Complications
phase of HSCT, it is advantageous to establish biomarkers that
can predict the onset of GVHD at earlier time points.
A soluble form of ST2 is released from endothelial cells,
epithelial cells, and fibroblasts in response to inflammatory
stimuli [30,31]. A previous study has shown that sST2 levels
are associated with conditioning intensity; sST2 levels were
two to four times higher after the MAC regimen than the RIC
regimen [4]. The present study showed that sST2 levels after
MAC were relatively higher than those after RIC. To reduce
the effect of conditioning intensity on sST2 levels, we also
examined the predictive values of the day 14/day 0 sST2 ratio
for acute GVHD, but we did not detect any relationship with
the development of GVHD (data not shown). Furthermore,
we examined the association between sST2 and targets of
GVHD involvement (skin, gastrointestinal tract, and liver)
and found that most of the patients in the high-sST2 group
developed gastrointestinal GVHD, which is thought to occur
due to endothelial damage. These findings imply that the
release of sST2 during GVHD exacerbation partly depends on
the degree of endothelial injury occurring after conditioning
therapy.
On the other hand, examining the association of sST2 with
other complications and mortality is also important. Vander
Lugt et al. [4] showed that high sST2 levels were associated
with NRM within 6 months after HSCT. Moreover, other
studies showed that high sST2 levels had a similar association
with increased risk of NRM [3,12,13]. In this study, although
it was difficult to evaluate this issue since only four patients
developed NRM, they all showed high sST2 levels on day
14. Notably, they died of sepsis, graft failure, TMA, and
pneumonia, without any GVHD, and all of them developed
VOD. Recent studies demonstrated that high sST2 levels
were significantly associated with TMA [15] and VOD [16],
both of which are characterized by endothelial cell injury.
In accordance with these observations, two patients who
developed TMA and five who developed VOD showed high
sST2 levels on day 14, even in our small cohort. In addition,
the correlation of sST2 with CRP and ferritin suggests that
sST2 is released under a variety of inflammatory conditions.
Several studies have shown that pro-inflammatory cytokines
and their receptors are potential GVHD biomarkers, but some
factors other than GVHD contribute to these cytokines’
release (e.g., TNF-α, TNFR1, IL-6) [5,6,32]. Therefore, caution
should be taken when diagnosing patients who have high
sST2 levels as sST2 is not a specific biomarker for acute GVHD.
Study Limitations
The limitations of this study include the small number of patients
and the heterogeneous patient populations and transplant
procedures, such as conditioning regimen, donor source, and
disease status. Even though we conducted detailed analyses of
sST2 expression patterns in individual patients, it was difficult
to exclude the multiple factors that can cause an increase in
sST2 (e.g., cardiac overload or infection).
Conclusion
We revealed that sST2 levels increased not only in patients
with acute GVHD but also in those with other life-threatening
complications, such as TMA, VOD, and severe infection, as identified
during patient monitoring. Although these complications
often overlap with each other in the clinical settings for HSCT,
gastrointestinal GVHD, TMA, and VOD, all of which are linked
to endothelial injury, may be key complications related to high
sST2 release. The early assessment of sST2 after HSCT may be a
predictive indicator for acute GVHD and other transplant-related
complications. Further studies with larger sample sizes and serial
monitoring are needed to clarify the clinical value of sST2.
Acknowledgments
We would like to thank all the members of the Yokohama
Cooperative Study Group for Hematology (YACHT) for
cooperating with this study and Dr. Etsuko Yamazaki for helpful
advice related to this work. We also thank Tom Kiper for his
review of the manuscript.
Ethics
Ethics Committee Approval: This study was approved by the
Institutional Review Board of Yokohama City University.
Informed Consent: It was obtained.
Authorship Contributions
Study Design: T.M., T.T.; Data Collection: A.M., T.M., T.T., T.A., M.K.,
S.K., Y.I., H.T., Y.N., A.N., W.Y., K. Mo., M.H., K. Ma., S.F.; Analysis:
A.M.; Writing: A.M., T.M.; Approval: H.N.
Conflict of Interest: The authors declare no conflicts of interest.
Financial Disclosure: The authors declare that this study
received no financial support.
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28
Turk J Hematol 2020;37:20-29
Matsumura A, et al: ST2 for Transplant-related Complications
Figure S1. ROC curves of sST2, CRP, and ferritin for predicting the development of acute GVHD. A) ROC curves for sST2 with area under
the curve (AUC) before conditioning and on days 0, 14, 21, and 28 after transplantation. B) ROC curves for CRP and ferritin with AUC
on day 14.
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characteristics.
29
RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2019.2019.0166
Turk J Hematol 2020;37:30-35
First-time Blood Donors Are Double-edged Swords for Blood
Transfusion Centers: A Retrospective Study in Southwest Iran
İlk Kez Kan Vericisi Olanlar Kan Transfüzyon Merkezleri için İki Ucu Keskin Bıçaktır:
Güneybatı İran’dan Retrospektif Bir Çalışma
Hamid Reza Niazkar 1 , Akbar Dorgalaleh 2 , Fariba Rad 3,4
1Gonabad University of Medical Sciences, Student Research Committee, Gonabad, Iran
2School of Allied Medical Science, Department of Hematology and Blood Transfusion, Tehran, Iran
3Yasuj University of Medical Sciences, Cellular and Molecular Research Center, Yasuj, Iran
4Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
Abstract
Objective: First-time blood donors are the most common group
of blood donors. They usually have different motivations for blood
donation, some of which provoke the donors to hide risk factors of
transfusion-transmissible infections (TTIs). Therefore, detection of TTIs
among first-time donors is crucial and can decrease the rate of TTIs
among blood recipients. This study aimed to evaluate the prevalence of
TTIs among first-time donors in the transfusion center of Kohgiluyeh
and Boyer-Ahmad Province (KBTC), Iran.
Materials and Methods: This retrospective study was conducted
with volunteer blood donors in 2004-2014 in the KBTC. Various data,
including sex, confidential unit exclusion (CUE), previous donation
history, and the laboratory findings of confirmatory tests, were
extracted from blood donor software. Data were analyzed by SPSS
using the chi-square test.
Results: Among 198,501 blood donors, 52,527 (26.46%) were firsttime
donors, while 145,974 donors (73.54%) were repeat and regular
donors. Most of the donors (94.5%) were male, while a minority (5.5%)
were female. The CUE option was chosen by 2,237 (1.13%) donors.
The incidence of hepatitis B surface antigen (HBsAg) and hepatitis C
virus (HCV) was 247 (0.13%) and 134 (0.07%) among the entire study
population, respectively. Three donors (0.002%) had confirmed human
immunodeficiency virus (HIV), while none of the blood donors were
positive for syphilis. Most of the donors with positive HBsAg (95.8%),
HCV (86.6%), and HIV (100%) infection were first-time donors.
Conclusion: Since TTIs are more common among first-time blood
donors than regular and repeat donors, special considerations should
be taken into account for this common group of blood donors.
Keywords: Transfusion-transmissible infections, Blood donor, Blood
donation
Öz
Amaç: Kan donörlerinin büyük çoğunluğunu ilk kez kan veren
donörler oluşturmaktadır. Bu kişilerin genellikle kan vermek için
farklı motivasyonları vardır ve bu da vericilerin transfüzyon ilişkili
enfeksiyonları (TTI) saklaması açısından risk oluşturur. Bu nedenle ilk
kez verici olanlarda TTI’ları tespit etmek çok önemlidir ve böylece kan
alıcılarında TTI’lar azaltılabilir. Bu çalışmada amaç İran’da Kohgiluyeh
ve Boyer-Ahmad Bölgesi (KBTC) transfüzyon merkezindeki ilk kez
verici olan kan donörlerinde TTI prevelansının araştırılmasıdır.
Gereç ve Yöntemler: Bu çalışma, KBTC’de 2004-2014 yılları arasında
gönüllü kan vericilerinin dahil edildiği retrospektif bir çalışmadır.
Cinsiyet, gizli ünite dışlaması (CUE), daha önceki kan bağışları, ve
doğrulama testlerinin laboratuvar bulguları gibi veriler, kan vericileri
yazılımından elde edilmiştir. Elde edilen veriler ki-kare testi kullanılarak
SPSS ile değerlendirilmiştir.
Bulgular: 198,501 kan vericisinden 52,527’i (%26,46) ilk kez kan
veren donörler, 145,974’i ise düzenli olarak kan verenler veya daha
önce kan vermis olanlardı (%73,54). Vericilerin çoğu erkekti (%94,5),
kadın verciler azınlıktaydı (5,5%). CUE seçeneği 2,237 (%1,13) verici
tarafından kullanılmıştı. Bütün çalışma grubunda hepatit B yüzey
antijeni (HBsAg) ve hepatit C virüsü (HCV) sıklığı sırasıyla 247 (%0,13)
ve 134 (%0,07) idi. Üç vericide doğrulanmış insan bağışıklık yetmezliği
virüsü (HIV) bulundu (%0,002), hiç bir hastada sifilis tespit edilmedi.
HBsAg (%95,8), HCV (86,6%), ve HIV (100%) enfeksiyonu olan
vericilerin çoğunluğu ilk kez kan vericisi olan gruptaydı.
Sonuç: TTI’lar tekrarlayan kez verici olanlar veya düzenli verici
olanlarla karşılaştırıldığında ilk kez verici olanlarda daha sık
görülmektedir ve bu vericilerin kullanılması durumunda özel
önlemlerin alınması gereklidir.
Anahtar Sözcükler: Transfüzyon ilişkili enfeksiyonlar, Kan vericisi,
Kan bağışı
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Fariba Rad, M.D., Yasuj University of Medical Sciences, Cellular
and Molecular Research Center, Yasuj, Iran and Blood Transfusion Research Center, High Institute for Research
and Education in Transfusion Medicine, Tehran, Iran
Phone : +98 9196028093
E-mail : fariba.rad89@gmail.com ORCID: orcid.org/0000-0002-6143-9979
Received/Geliş tarihi: April 26, 2019
Accepted/Kabul tarihi: September 2, 2019
30
Turk J Hematol 2020;37:30-35
Niazkar HR, et al: First-time Blood Donors in Southwest Iran
Introduction
Blood transfusion has a fundamental role in medical services.
Although blood donation can improve the quality of patients’
lives, it is one of the main causes of the transmission of viral
infections, including hepatitis B virus (HBV), hepatitis C virus
(HCV), and human immunodeficiency virus (HIV). Several studies
have shown that the chance of transmission of these infections
is about 1% in each blood unit transfusion, while a 6.2% chance
to transmit hepatitis was also reported [1,2,3,4].
Since blood can only be provided by human resources,
transfusion-transmissible infections (TTIs) via these products are
important issues. Therefore, blood safety is the most important
concern of blood transfusion worldwide [5]. In the process
of ensuring blood safety, donors have a crucial role [6]. As a
result, standard and relatively strict blood donation conditions
are used by blood transfusion centers to improve blood safety.
This includes a voluntary blood donation system, a confidential
unit exclusion (CUE) system, the exclusion of high-risk donors,
and standard and sensitive blood-borne disease detection
systems. High-risk donors are those with risky behaviors, such
as multiple sexual partners or intravenous drug injection, which
can increase the risk of TTIs [7,8,9].
According to the standards of blood transfusion organization,
volunteer donors are classified into two groups: first-time
donors and repeated and regular donors [10]. Several studies
have shown that first-time blood donors are the most common
group of blood donors [10]. These donors usually have different
motivations for blood donation. Some of these motivations
provoke donors not only to hide risk factors during interviews
but also to increase the risks of the blood donation system. At
the same time, studies in different communities have shown
that the prevalence of TTIs in first-time donors is higher than
that of other donors [10]. Therefore, evaluation and detection
of TTIs in first-time donors are crucial and can decrease the rate
of TTIs among blood recipients.
Despite various strategies and significant advances in the
detection and diagnosis of these diseases and the reduction
of the window period by new-generation diagnostic kits, TTIs
remain the most important challenge in blood transfusion. This
study aimed to evaluate the prevalence of TTIs among first-time
donors at the transfusion center of Kohgiluyeh and Boyer-
Ahmad Province, Iran (KBTC).
Materials and Methods
This retrospective study was performed with 198,501 volunteer
blood donors from 2004 to 2014 at the KBTC. Demographic
data including sex and CUE, as well as donation history and
laboratory findings of confirmatory tests, were extracted from
blood donation software. Blood donors were classified into two
groups as first-time donors or repeated and regular donors. A
first-time donor is a donor who is donating blood for the first
time. A repeated and regular donor is a donor who has donated
blood at least once in the past [10].
The results of HIV, HBV, HCV, and syphilis tests were
also extracted. The screening tests were performed with
commercially available enzyme-linked immunosorbent assay
kits.
Hepatitis B surface antigen (HBsAg) was checked with Behring
(Marburg, Germany), Siemens (Marburg, Germany), and Bio-Rad
(Hercules, CA, USA) kits; anti-HCV was checked with BioMerieux
(Marcy l’Etoile, France) and Avicenna (Avicenna Medical Center,
Moscow, Russia) kits; and HIV-Ab was tested with BioMerieux
(Marcy l’Etoile, France), Bio-Rad (Hercules, CA, USA), and
Adaltis (Montreal, Canada) kits. All repeatedly reactive results
were confirmed by a neutralization test (Behring, Marburg,
Germany) for HBV, a recombinant immunoblot assay for HCV
(Inonogenetic, Ghent, Belgium), or a western blot assay for HIV
(Inonogenetic, Ghent, Belgium).
According to Iranian Blood Transfusion Organization (IBTO)
policies, each blood unit with positive results confirmed for
blood-borne diseases was excluded and the corresponding
donor was recalled for counseling and appropriate treatment.
The rate of confirmed positive HBV, HCV, and HIV tests was
compared between the studied groups. The frequencies of
HBsAg, HIV, and HCV and 95% confidence intervals (CIs)
were calculated using SPSS software. The prevalence rate of
infections was calculated for each group and compared using
the chi-square test, and differences were considered significant
at p<0.05. This study was approved by the ethic committee of
Yasuj University of Medical Sciences (IR.Yuma.rec.1396.22).
Results
A total of 198,501 blood donations were performed from 2004
to 2014 at the KBTC. These included 52,527 (26.46%) first-time
donors and 145,974 (73.5%) repeated and regular donors. Out
of the total of 198,501, 187,691 donors (94.5%) were male and
10,810 donors (5.5%) were female (17.1:1). The CUE option was
chosen by 2,237 (1.13%) donors, while 196,264 (98.87%) of the
volunteer donors did not choose the CUE option and were thus
considered as the CUE-negative group. According to the history
of donation, the distribution of blood donors in CUE groups is
presented in Table 1.
Among the 198,501 blood donors, 247 (0.13%) and 134 (0.07%)
donors were positive for HBsAg and HCV, respectively. HBsAg
had higher prevalence compared to HCV infection in the
entire study population [0.13% and 0.07%, respectively; odds
ratio (OR)=1.84; 95% CI=1.49-2.27; p<0.0001]. Three donors
31
Niazkar HR, et al: First-time Blood Donors in Southwest Iran Turk J Hematol 2020;37:30-35
(0.002%) had confirmed HIV and none of the blood donors were
positive for syphilis. In addition, concomitant infections were
not detected in any donor.
Among the 52,527 first-time blood donors, 231 (0.44%), 116
(0.2%), and 3 (0.005%) donors were positive for HBsAg, HCV,
and HIV, respectively. HBsAg had a higher prevalence than HCV
infection among first-time donors (OR=1.98; 95% CI=1.59-
2.48; p<0.0001).
In the population studied in this investigation, 1.08% (24/2,237)
of CUE-positive and 0.18% (357/196,264) of CUE-negative
donors were positive for disease markers. Table 2 shows a
significantly higher prevalence of HBsAg and HCV in the
CUE-positive than CUE-negative donors (1.08% and 0.18%,
respectively; OR=5.84; 95% CI=3.875-8.820; p<0.0001).
The prevalence of confirmed HBsAg was 1.32% (14/1,060)
and 0.42% (217/51,467) among the first-time CUE-positive
and first-time CUE-negative donors, respectively (OR=3.1;
95% CI=1.814-5.312; p<0.001). The prevalence of confirmed
HCV was 0.85% (9/1,060) and 0.21% (107/51,467) among the
first-time CUE-positive and first-time CUE-negative donors,
respectively (OR=4.05; 95% CI=2.060-7.990; p<0.0001) (Table
3). There were 3 HIV-positive donors among the CUE-negative
first-time donors.
A significantly higher prevalence of HBsAg and HCV infection
was observed in male donors than females among the first-time
donors (p<0.001) (Table 4).
Discussion
Blood and its components are among the most important
causes of TTI transmission. The possibility of TTI transmission
in the transfusion of every blood unit is about 1% [6]. This is
a relatively high rate for transmission of blood-borne diseases
because some of these infections are severe, life-endangering
ones that are incurable or have a difficult treatment process
[6,7]. Thus, TTIs are a significant challenge for blood transfusion
services worldwide and require precise precautions. Different
factors such as vaccination programs, high-risk behaviors, and
the socioeconomic status of people can affect the risk of TTIs
in any community. In recent decades vaccination against HBV
significantly decreased the rate of TTIs in different countries.
In Iran, vaccination against HBV significantly decreased the
rate of HBV infections in comparison with countries without
this program or those with late establishment. The incidence of
HBV, HCV, and HIV is also higher in low-income countries than
in middle- and high-income countries. These data show that
there is a direct correlation between the economic condition of
countries and TTI incidence. Those countries with higher income
Table 1. Distribution of blood donors in confidential unit exclusion -positive and confidential unit exclusion-negative groups.
Group
Total donations,
n (%)
First-time,
n (%)
CUE-positive 2,237 (1.13) 1,060 (2.01)
p
Repeated and regular,
n (%)
1,177 (0.81)
CUE-negative 196,264 (98.87) 51,467 (97.99) 144,797 (99.15)
Total 198,501 (100) 52,527 (100) <0.001 145,974 (100)
CUE: Confidential unit exclusion.
p
<0.001
Table 2. The prevalence of confirmed hepatitis B surface antigen and hepatitis C virus among confidential unit exclusion-positive
and confidential unit exclusion-negative groups.
Group
Study population,
n (%)
HBsAg,
n (%)
p
HCV,
n (%)
p
Total,
n (%)
CUE-positive 2,237 (1.13) 14 (0.63) <0.0001 10 (0.45) <0.0001 24 (1.08) <0.0001
CUE-negative 196,264 (98.87) 233 (0.12) 124 (0.06) 357 (0.18)
Total 198,501 (100) 247 (0.13) 134 (0.07) 381 (0.2)
HCV: Hepatitis C virus, HBsAg: Hepatitis B surface antigen, CUE: Confidential unit exclusion.
p
Table 3. Comparison of hepatitis B surface antigen and hepatitis C virus prevalence among the confidential unit exclusionpositive
and confidential unit exclusion-negative first-time donors.
TTI Group of donations Number of donations Number positive, n (%) p
HBsAg First-time, CUE-positive 1,060 14 (1.32) <0.0001
First-time, CUE-negative 51,467 217 (0.42)
HCV First-time, CUE group 1,060 9 (0.85) <0.0001
First-time, non-CUE group 51,467 107 (0.21)
HCV: Hepatitis C virus, HBsAg: Hepatitis B surface antigen, CUE: Confidential unit exclusion.
32
Turk J Hematol 2020;37:30-35
Niazkar HR, et al: First-time Blood Donors in Southwest Iran
Table 4. Sex distribution of first-time reactive donors in whole study population.
Year
Total
donations
First time donors
2004 13,063 33 25 8 2.2
(1.114.4)
2005 13,270 18 14 4 2.4
(0.91-6.3)
2006 13,725 20 16 4 2.7
(1.01-7)
2007 13,687 22 19 3 3.9
(1.27-11.7)
Total
HbsAgpositive
Sex
OR Significance Total Sex
Male Female
(95% CI) level HCVpositive
Male Female
OR
(95% CI)
0.02 8 7 1 4.2
(0.61-28.8)
0.07 4 3 1 2.1
(0.30-15)
0.04 8 7 1 4.2
(0.61-28.8)
0.02 9 9 0 - -
2008 13,849 14 14 0 - - 4 4 0 - -
2009 17,110 19 16 3 3.35
(1.1-1.2)
2010 22,607 31 28 3 5.4
(1.8-16.4)
2011 25,557 30 28 2 7.7
(1.97-30)
0.03 17 16 1 8.7
(1.25-60)
0.003 24 23 1 12.2
(1.75-85)
0.003 14 14 0 - -
2012 23,902 20 20 0 - - 10 10 0 - -
2013 23,750 18 16 2 4.7
(1.2-18.4)
2014 17,981 6 5 1 3.2
(0.5-21.9)
HCV: Hepatitis C virus, HBsAg: Hepatitis B surface antigen, OR: Odds ratio, CI: Confidence interval.
0.02 10 10 0 - -
0.2 9 9 0 - -
Significance
level
0.1
0.4
0.1
0.02
0.01
can more easily provide preventive and vaccination programs
for their people than countries with lower income [11,12].
Education is another important factor that can significantly
decrease the rate of TTIs among blood donors, mostly by
reducing risky behaviors [13].
Due to the crucial role of blood safety in blood transfusion
services, huge efforts are made to improve the safety of
blood and its components [1,2,6,10,14]. These efforts are
performed in different stages of blood transfusion processes,
from blood donor selection to blood release [3,10,15]. Donor
selection is an important step in blood safety and different
studies have revealed that suitable and appropriate donor
selection can significantly improve blood safety [15,16]. It
was shown that blood components of repeated and regular
blood donors have a lower risk of TTI transmission than those
of first-time donors [10,17,18,19,20]. On the other hand, it
has been shown that first-time donors are the most common
donors in blood transfusion centers [10,18]. These two issues
highlight the importance of the donor selection process in
blood transfusion centers, which can significantly improve
the safety of blood and blood components. In our study,
about one-third of the donors were first-time donors. Similar
results were observed in several other studies in Iran and
other countries [15,17,18].
In our study, similar to many others, it was revealed that TTIs
are more common among first-time donors than repeated
and regular donors [6,15]. The CUE system is a commonly
used system in most blood transfusion centers. However, its
usefulness is questionable based on a considerable number of
studies [21,22,23]. Despite this issue, the IBTO has used this
system to improve the safety of blood and its components
[15,24]. Several studies confirmed that CUE is a relatively costbeneficial
system that can significantly improve the safety of
blood products [15,25]. In our study, this issue was observed and
the CUE option was more commonly used by first-time donors.
In our study, about 2% of the first-time donors used the CUE
option (CUE-positive), while only 0.8% of repeated and regular
donors did. This rate of CUE positivity among the first-time
donors in our study is lower than that in the study of Vogler
et al. [26], who reported about 5% CUE positivity among their
first-time donors.
Moreover, a significantly higher prevalence of TTIs was observed
among first-time donors with positive CUE in comparison with
the CUE-negative first-time donors. The higher prevalence of
TTIs among the first-time donors and the CUE-positive firsttime
donors is in agreement with other studies conducted
in Iran, Australia, the United Kingdom, and the Netherlands
[4,15,17,24,25].
33
Niazkar HR, et al: First-time Blood Donors in Southwest Iran
Turk J Hematol 2020;37:30-35
In our study, the prevalence of HBsAg and HCV was 0.13% and
0.07% among blood donors, respectively, and 0.44% and 0.2%
in the first-time blood donors, respectively. This rate of infection
in the KBTC is lower than those of other studies [1,2,5,27,28,29].
This discrepancy in the prevalence of TTIs in different populations
around the world reflects a variety of high-risk behaviors,
population risks, health statuses, and selection procedures in
those regions.
In our study, the prevalence of HBsAg and HCV infection had
increased during 2005-2007 and 2009-2011 among voluntary
first-time donors, while Farshadpour et al. [4], Amini Kafi‐Abad
et al. [30], and Khodabandehloo et al. [31] reported a decreased
trend in the prevalence of HBV and HCV between 2004 and 2012.
The reason for this increase in our study could be related to the
significant increase in the number of first-time blood donors,
which increased from 4,139 in 2005 to 7,031 in 2011. Also, this
increase could be related to a combination of other factors
including vaccination against HBV; low public knowledge
about blood-borne infections and routes of transmission in the
past such as traditional tattoos, traditional circumcision, and
cupping therapy; and the effectiveness of prospective donor
screening measures.
It seems that a higher rate of TTIs among first-time donors is
a relatively significant challenge for blood transfusion centers
and special policies such as CUE should be considered for these
donors to improve the safety of blood and its components.
Similar to other studies, our results showed a higher
prevalence of HBsAg compared to HCV in both total and firsttime
donors [30,32]. This high prevalence may be due to the
higher rate of HBV in the general population of this province,
whereas this issue was not determined in any other study
and consequently further studies are required to confirm this
issue [33,34].
Conclusion
Due to the high rate of TTIs among first-time donors, it is crucial
to implement some preventive programs among this common
type of blood donors to reduce the overall incidence of TTIs
among blood recipients.
Acknowledgment
The authors would like to thank all staff members of the KBTC.
Ethics
Ethics Committee Approval: This study was approved by the
ethics committee of Yasuj University of Medical Sciences (IR.
Yuma.rec.1396.22).
Informed Consent: Informed consent is not applicable to this
study.
Authorship Contributions
Analysis or Interpretation: H.R.N., A.D., F.R.; Literature Search:
H.R.N., A.D., F.R.; Writing: H.R.N., A.D., F.R.
Conflict of Interest: The authors declare no conflict of interest.
Financial Disclosure: The authors declare that this study
received no financial support.
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35
RESEARCH ARTICLE
DOI: 10.4274/tjh.galenos.2019.2019.0256
Turk J Hematol 2020;37:36-41
Percentages of CD4+CD8+ Double-positive T Lymphocytes in the
Peripheral Blood of Adults from a Blood Bank in Bogotá, Colombia
Kolombiya Bogota Kan Bankası Yetişkin Periferik Kan CD4+CD8+ Çift Pozitif T-Lenfosit
Yüzdeleri
Miguel S. Gonzalez-Mancera 1 , Natalia I. Bolaños 1 , Manuel Salamanca 1 †, Guillermo A. Orjuela 2 , Ayda N. Rodriguez 2 ,
John M. Gonzalez 1
1University of los Andes, School of Medicine, Grupo de Ciencias Básicas Médicas, Bogotá, Colombia
2National Blood Bank, Colombian Red Cross, Bogotá, Colombia
Abstract
Objective: CD4+CD8+ double-positive T-cells (DPTs) have been
classified as a separate T-cell subpopulation, with two main
phenotypes: CD4 high CD8 low and CD4 low CD8 high . In recent years,
the relevance of DPTs in the pathogenesis of infections, tumors, and
autoimmune diseases has been recognized. Reference values among
healthy individuals remain unknown. Therefore, the aim of this study
is to provide a reference value for DPTs in peripheral blood from
healthy donors in a blood bank in Bogotá, Colombia, and to determine
the activation status using a surface marker.
Materials and Methods: One hundred healthy donors were enrolled
in the study. Peripheral blood cells were stained for CD3, CD4,
CD8, and CD154 (CD40L), and cellular viability was assessed with
7-aminoactinomycin D and analyzed by flow cytometry.
Results: The median value for DPTs was 2.6% (interquartile
range=1.70%-3.67%). Women had higher percentages of DPTs than
men (3.3% vs. 2.1%). The subpopulation of CD4 low CD8 high showed
higher expression of CD154 than the other T-cell subpopulations.
Conclusion: DPT reference values were obtained from blood
bank donors. A sex difference was found, and the CD4 low CD8 high
subpopulation had the highest activation marker expression.
Keywords: Flow cytometry, Lymphocyte, Lymphocyte subpopulation,
T lymphocytes
Öz
Amaç: CD4+CD8+ çift pozitif T hücreleri (ÇPT) ayrı bir T hücre alt
popülasyonu olarak iki temel fenotip ile sınıflandırılmaktadır:
CD4 yüksek CD8 düşük ve CD4 düşük CD8 yüksek . Son yıllarda, ÇPT’lerin
enfeksiyonlar, tümörler ve otoimmün hastalıkların patogenezi ile
ilişkisi tanımlanmıştır. Sağlıklı bireyler arasında referans değerleri
bilinmemektedir. Bu nedenle, bu çalışmanın amacı, Kolombiya
Bogota’daki bir kan bankasındaki sağlıklı vericilerden alınan periferik
kandaki ÇPT’ler için bir referans değeri sağlamak ve bir yüzey belirteci
kullanarak aktivasyon durumunu belirlemektir.
Gereç ve Yöntemler: Çalışmaya yüz sağlıklı verici dahil edilmiştir.
Periferik kan hücreleri CD3, CD4, CD8 ve CD154 (CD40L) için işaretlendi
ve hücresel canlılık 7-aminoactinomycin D kullanarak akan hücre
ölçer ile analiz edildi.
Bulgular: ÇPT’ler için ortanca değer %2,6 (çeyrekler arası
aralık=%1,70-%3,67) olarak saptandı. Kadınlarda ÇPT yüzdesi
erkeklere göre daha yüksek bulundu (%3,3 karşı %2,1). CD4 düşük
CD8 yüksek alt popülasyonu diğer T hücre alt popülasyonlarından daha
yüksek CD154 ekspresyonu gösterdi.
Sonuç: ÇPT referans değerleri kan bankası vericilerinden elde edilmiştir.
Cinsiyetler arası fark ve CD4 düşük CD8 yüksek alt popülasyonunda da en
yüksek aktivasyon belirteci ekspresyonu saptanmıştır.
Anahtar Sözcükler: Hücre ölçer, Lenfosit, Lenfosit alt popülasyonu,
T lenfositleri
Introduction
Classically, T-cells have been classified according to the cell
surface markers CD4 and CD8. The expression of these proteins
is considered to be a mutually exclusive event reflecting the
specific functions of each major T-cell population in peripheral
blood: CD4+ or helper T-cells and CD8+ or cytotoxic T-cells.
However, with the use of multiparametric cellular analysis
methods, a variety of minor T-cell subpopulations have been
described [1], such as mature CD4+CD8+ or double-positive
T-cells (DPTs) [2,3]. This T-cell phenotype was initially described
in the thymus, where more than 80% of thymocytes expressed
both CD4+CD8+, which later commit to one cell lineage (CD8+
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: John M. González, M.D., University of los Andes,
School of Medicine, Grupo de Ciencias Básicas Médicas, Bogotá, Colombia
E-mail : johgonza@uniandes.edu.co ORCID: orcid.org/0000-0001-7251-5984
Received/Geliş tarihi: July 8, 2019
Accepted/Kabul tarihi: October 15, 2019
36
Turk J Hematol 2020;37:36-41
Gonzalez-Mancera MS, et al: Double-positive T-Cells in Blood Donors
or CD4+) after interaction with human leukocyte antigen (HLA)
class I or II molecules, respectively [4]. The origin of DPTs in the
peripheral blood of healthy individuals has been attributed to
the premature release of CD4+CD8+ T-cells from the thymus
to the periphery [5,6,7]. However, additional studies have
suggested that DPTs could originate from the acquisition of the
second marker by single-positive (either CD4+ or CD8+) T-cells
in the periphery [6,8]. Although several investigations support
that mature CD4+ T-cells are the source of DPTs, there is also
evidence that CD8+ T-cells could be the primary cellular type
[6]. Unlike immature thymic DPTs, peripheral DPTs exhibit the
functional properties of mature T-cells, including antigendependent
cytokine production, cytolytic activity, and expression
of markers associated with the memory phenotype [9,10]. DPTs
are divided into two main populations based on the differential
expression of each marker (CD4 high CD8 low and CD4 low CD8 high )
[1,2,3]. In healthy donors, CD4 high CD8 low cells have an effector or
memory phenotype (T EM
), whereas CD4low CD8 high cells display a
central memory phenotype (T CM
), which can switch to an effector
phenotype during viral infections such as hepatitis C virus (HCV)
and human immunodeficiency virus (HIV) [9,10]. Little is known
about the functionality of DPTs, though their function seems to
be disease-specific. DPTs exhibit cytotoxic potential in chronic
viral infections, such as lymphocytic choriomeningitis virus [11]
and HIV [12], and in certain types of cancer [13,14,15]. DPTs can
have a regulatory role in malignancies [13,14], systemic sclerosis
[16], and inflammatory bowel disease [17]. In autoimmune
diseases, DPTs can be found in different compartments; they
increase in peripheral blood among patients with myasthenia
gravis [18] but are found infiltrating the affected tissues in
autoimmune thyroid disease and rheumatoid arthritis [19,20]. In
systemic sclerosis and rheumatoid arthritis, DPTs secrete mainly
interleukin-4 [16,19], whereas in tumors, such as melanoma and
cutaneous lymphoma, the primary cytokine produced is tumor
necrosis factor (TNF)-α [13,14]. In chronic parasitic infections
such as Chagas disease, DPTs are not only increased in peripheral
blood [21] but are also found infiltrating the cardiac tissue in
patients with advanced chagasic cardiomyopathy [22,23].
Due to the growing interest in the study of DPT subpopulations
and their potential roles in specific diseases, it seems essential
to determine reference values among healthy individuals.
Therefore, the main goal of this study is to establish standard
values of DPTs and to evaluate their functional profile by
determining the presence of one specific activation marker in
suitable donors from a blood bank in Bogotá, Colombia.
Materials and Methods
Study and Donors
This is a descriptive and cross-sectional study of suitable donors
who volunteered for blood donation in 2017 at the National
Blood Bank of the Colombian Red Cross in Bogotá, Colombia.
The protocol and informed consent was approved by the Ethical
Committee of the University of los Andes (Act 209 of 2013).
One hundred and three donors were enrolled in this study and
provided informed consent. The demographic characteristics of
our study population are shown in Table 1. Three individuals
were excluded due to reactive serological tests for syphilis.
The study population included 55 men and 45 women who
fulfilled the donation requirements and had negative screening
tests (HIV, syphilis, hepatitis C virus, hepatitis B virus, Chagas
disease, and human T-cell lymphotropic virus). They ranged
from 19 to 61 years of age. Samples were obtained from citrate
phosphate dextrose anticoagulated blood bags and transported
refrigerated from the blood bank to the biomedical sciences
laboratory where the cellular analyses were conducted.
Cell Labeling and Cytometry Acquisition
Blood samples of 100 µL were used for labeling. Antibodies
included anti-CD3 APC (clone UCHT1), anti-CD4 PerCP (SK3),
anti-CD8 FITC (SK1), and anti-CD154 PE (TRAP1). All antibodies
were purchased from BD Pharmingen (BD, San Diego, CA, USA).
Blood was stained in darkness for 30 min at 4°C and then
incubated with a cell lysis buffer (BD FACS Lysing Solution)
for 15 min at room temperature. Subsequently, cells were
washed twice in phosphate-buffered saline (PBS) (Sigma-
Aldrich, St. Louis, MO, USA) (0.01 M, pH 7.4, PBS 1X) and gently
resuspended. Viability was assessed with 7-aminoactinomycin D
staining (7-AAD, BD). Samples were acquired and analyzed with
a FACSCanto II flow cytometer with FACSDiva 6.1 software (BD
Bioscience, San Jose, CA, USA). At least 5x10 4 cells were acquired
in the CD3+ T lymphocyte population gate according to their
forward scatter (FSC) and side scatter (SSC) features. The gating
strategy for CD3+ T-cells and DPTs is shown in Figure 1.
Statistical Analysis
Information about the donor characteristics is given in
percentages. The Shapiro-Wilk normality test was conducted for
all data obtained from the cellular analysis. A nonparametric
statistical analysis was performed in the study. The Mann-
Whitney U test was used to compare between two groups. The
Kruskal-Wallis test was used to compare among multiple groups.
The results are shown as medians and interquartile ranges
Table 1. Characteristics of the population studied.
Age groups
Ages 18-29 30-49 50-65 Total
Women 11 18 16 45
Men 6 33 16 55
No. 17 51 32 100
Age average (± SD) 24.4 (3.5) 40.9 (6.4) 55.7 (3.0) 42.9 (11.8)
Age in years, SD: Standard deviation.
37
Gonzalez-Mancera MS, et al: Double-positive T-Cells in Blood Donors Turk J Hematol 2020;37:36-41
(IQRs). Statistical analysis was performed using GraphPad Prism
7 software (San Diego, CA, USA). Significance was established
at p<0.05.
Results
T lymphocytes are a highly heterogeneous group of immune
cells that have been of great interest in clinical and biomedical
research studies. In an effort to establish values between the
different subpopulations of DPTs, lymphocytes were analyzed
from peripheral blood mononuclear cells from each blood donor
included in the study. Lymphocytes were gated according to FSC
vs. SSC features, as shown in Figure 1A. The median cell viability
was 99.15% (IQR=98.8%-99.4%) in all samples, as shown in
Figure 1B. CD3+ T-cells were subsequently identified according
to cell surface expression of CD4 or CD8, as shown in Figures
1C and 1D, respectively. DPTs were classified into two main
subpopulations, CD4 high CD8 low and CD4 low CD8 high , as shown in
Figure 1D. The median total percentage of DPTs among CD3+
T-cells in all samples studied was 2.6% (IQR=1.7%-3.67%), and
the CD4 high CD8 low subpopulation showed a median content
of 1.15% (IQR=0.8%-2.0%), whereas in the CD4 low CD8 high
subpopulation, it was 0.9% (IQR=0.5%-1.67%), as shown in
Figure 2A. CD4 high CD8 low accounted for 57.97% of DPTs, as
shown in Figure 2B. Total DPTs were analyzed according to sex.
Women showed a higher percentage of DPTs (median=3.3%;
IQR=2.2%-4.15%) than men (median=2.1%; IQR=1.6%-3.3%),
p=0.007, as shown in Figure 3. The activation status of DPTs
was assessed by using the surface marker expression of CD154,
also called CD40L, as shown in Figure 1E. The subpopulation
of CD4 low CD8 high showed higher expression of CD154 than the
other T-cell populations (p≤0.0001), as shown in Figure 4.
Discussion
In recent decades, there has been growing interest in CD4+CD8+
double-positive T lymphocytes, which are considered a separate
subpopulation of T-cells associated with different pathologic
conditions. In this study, a reference percentage value was
established among DPT subpopulations. An activation marker
was also studied in the blood samples of volunteer blood bank
donors. An increased frequency of DPTs was found in women
compared to men. Sex variance has been found in other blood
cell subpopulations, such as natural killer lymphocytes [24,25],
and it would be of particular interest to evaluate DPTs during
pregnancy and in placental tissue due to the sex difference
found.
The frequency of DPTs in peripheral blood does not increase in
HIV, HCV, or melanoma; however, this subpopulation exhibited
a higher expression of surface activation markers (i.e. HLA-DR
and CD38) and greater cytokine production (i.e. interferon-γ
and TNF-α) in individuals with these diseases when compared to
controls [7,9,10,14]. Nonetheless, in one study assessing HIV, an
increased frequency of DPTs expressing CD38 and HLA-DR was
associated with advanced disease in patients with CD4+ counts
Figure 1. Flow cytometry gating strategy. A) Peripheral blood leukocytes cells distributed in dot plot by flow cytometry according to FSC
versus SSC. B) Cell viability using 7-aminoactinomycin D. C) T-cells identified by the expression of CD3. D) Dot plot distribution showing
the expression of CD4 and CD8, and the gate on DPTs: CD4 high CD8 low and CD4 low CD8 high . E) Density plot showing CD154 expression
in each DPT subpopulation.
SSC: Side scatter, FSC: Forward scatter, DPTs: Double-positive T-cells, DPT: Double-positive T-cell.
38
Turk J Hematol 2020;37:36-41
Gonzalez-Mancera MS, et al: Double-positive T-Cells in Blood Donors
Figure 4. Expression of CD154 in single-positive and doublepositive
T-cell subpopulations. The subpopulation CD4 low CD8 high
had higher CD154 expression than other subpopulations of T-cells.
Data are displayed as medians with minimums and maximums.
patients with melanoma, there was an increased frequency
of DPTs in draining lymphoid nodes and tumor-infiltrating
lymphocytes [14].
Figure 2. A) Percentage of subpopulations of DPTs from the
total T lymphocytes. B) Percentage of subpopulations among the
total DPTs. Data are displayed as medians with minimums and
maximums.
DPTs: Double-positive T-cells.
Figure 3. Percentage of the total DPTs according to sex. Women
had higher percentages of DPT cells than men. Mann-Whitney,
p=0.007. Data are displayed as medians with minimums and
maximums.
DPT: Double-positive T-cell.
of <200 cells/µL [7], which are markers that have been widely
used to define T-cell activation by antigens [26]. Additionally,
an increased frequency of DPTs in peripheral blood was found
in chronic chagasic patients [22,23] and among individuals with
myasthenia gravis [18], and the percentage of DPTs interestingly
decreased after treatment in both diseases [18,23]. Among
In this study, a higher expression of CD154 (CD40L) was found
in CD4 low CD8 high cells. This activation marker has been used as an
indicator for antigen-specific T-cell activation in CD4+ T-cells
[27] and in CD8+ T-cells [28]. Remarkably, this DPT subpopulation
has an effector memory phenotype [9,10]. CD154 is the ligand
of CD40, and this axis has been found to be of particular
interest in the therapy of autoimmune diseases [29]. It would
be very important to elucidate the functional role of CD154 in
DPTs. Other markers have been studied on DPT cells, including
activation, homing, and differentiation markers [10,22]. Due
to the role of DPTs in the pathogenesis of several diseases, it
seems promising to study the expression of inhibitory molecules
such as PD-1 or CTLA-4. These molecules are currently targets
of immunotherapy for different tumor conditions [30,31,32]. In
previous reports, no other activation markers, such as CD38 or
HLA-DR, were found in DPTs from healthy donors [7,10,22].
In this study, a median DPT rate of 2.6% was found, which was a
higher result than that of the control donors in previous studies.
For instance, in controls used to study DPTs in HIV patients,
the median was 0.8% (IQR=0.1%-1.2%) [7]; in melanoma, the
mean was 0.9% [standard deviation (SD) ±0.6] [14]; in HCV
infection, the mean was 1% (SD ±0.6) [10]; and in chronic
Chagas disease, the mean was 1.1% (±0.5) [22]. However,
the control donors analyzed in these studies were from small
cohorts, and demographic information about blood donor
characteristics was lacking. Our study sample was significantly
larger than those included in prior investigations, which could
explain the increments evidenced in the results. Indeed, in one
study, the frequency of DPTs ranged from 0% to 5% in control
donors [18].
39
Gonzalez-Mancera MS, et al: Double-positive T-Cells in Blood Donors
Turk J Hematol 2020;37:36-41
Conclusion
These findings and the differences found between the sexes
can be used for future reference in specific populations and
diseases. The limitations of this study include the age restriction
of our sample and the limited screening tests performed for
each donor. To the best of our knowledge, this is the first study
to assess the frequency of DPTs in a large cohort of blood bank
donors.
Acknowledgments
We would like to thank all the donors who volunteered to be
a part of this study; the personnel of the National Blood Bank
of the Colombian Red Cross who aided in the collection of the
samples; and Juan Guillermo Ripoll, M.D., of the Mayo Clinic
for revising the manuscript. This project was performed and
finalized in memory of Manuel Salamanca, who conceived,
wrote, and standardized the protocols.
Ethics
Ethics Committee Approval: The protocol and informed consent
was approved by the Ethical Committee of the University of los
Andes (Act 209 of 2013).
Informed Consent: Blood sample acquisition, obtaining of
informed consent.
Authorship Contributions
Concept: G.A.O., A.N.R.; Design: M.S.; Data Collection or
Processing: M.S.G.M., N.I.B., J.M.G.; Analysis or Interpretation:
M.S.G.M., N.I.B., J.M.G.; Writing: M.S.G.M., N.I.B., J.M.G.
Conflict of Interest: The authors declare that they have no
conflict of interest.
Financial Disclosure: The authors declare that this study
received no financial support.
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41
PERSPECTIVES IN HEMATOLOGY
DOI: 10.4274/tjh.galenos.2019.2019.0241
Turk J Hematol 2020;37:42-47
WHO 2016 Definition of Chronic Myeloid Leukemia and Tyrosine
Kinase Inhibitors
Kronik Myeloid Lösemi WHO 2016 Tanımlaması ve Tirozin Kinaz İnhibitörleri
İbrahim C. Haznedaroğlu 1 , Işınsu Kuzu 2 , Osman İlhan 3
1Hacettepe University Faculty of Medicine, Department of Hematology, Ankara, Turkey
2Ankara University Faculty of Medicine, Department of Pathology, Ankara, Turkey
3Ankara University Faculty of Medicine, Therapeutic Apheresis Unit, Department of Hematology, Ankara, Turkey
Abstract
Philadelphia (Ph*)/BCR-ABL1-positive chronic myeloid leukemia
(CML) is considered as a chronic life-long disease, which could be
manageable with tyrosine kinase inhibitor (TKI) drugs. The aim of TKI
drug treatment is to provide age- and sex-matched duration of life
in a given patient with CML. Personalized CML treatment with TKI
drugs is the key strategy. Individual treatment approach includes the
harmonization of CML disease characteristics, clinical experience, and
best available clinical evidence. Specific CML disease characteristics
in a given patient include; CML disease risk, comorbidities, molecular
profile, compliance, lifestyle, and drug off-target risk profile. CML
research evidence includes; randomized clinical trials indicating
the data on the efficacy, safety, tolerability, toxicity, possible longterm
adverse events, and pharmacoeconomy of TKIs. Clinical and
physician experience includes TKI availability, TKI reimbursability, drug
experience, adherence, and BCR-ABL1 monitorization facilities. The
key decision of choosing a TKI of choosing TKIs for CML should be
made via the consideration of these variables. The aim of this paper
is to outline the latest 2016 World Health Organization definition of
CML and its proper management with TKI-class drugs.
Keywords: Chronic myeloid leukemia, CML, Tyrosine kinase inhibitor,
TKI
Öz
Philadelphia (Ph*)/BCR-ABL1 (+) kronik myeloid lösemi (KML), tirozin
kinaz inhibitörleri (TKİ) grubundan ilaçlarla yaşam boyu yönetilebilecek
kronik bir hastalıktır. TKİ ilaç tedavisinin hedefi, herhangi bir KML
hastasında aynı yaş ve cinsiyette sağlıklı bireylerde beklenen yaşam
süresi idamesini sağlamak olmalıdır. KML tedavisinde bireyselleştirilmiş
TKİ ilaç kullanımı anahtar stratejidir. Bireysel tedavi yaklaşımı; KML
hastalık özelliklerini, klinik deneyimi ve mevcut en iyi kanıtı uygunca
birleştirme esaslıdır. Herhangi bir KML hastasında özgül hastalık
özellikleri; KML hastalık riski, komorbiditeler, moleküler profil, hasta
uyumu, yaşam tarzı, ve ilaç temelli yan etki profilleridir. KML’de kritik
araştırma kanıtları; TKİ etkinlik, güvenilirlik, tolerabilite, toksisite,
uzun-dönem ilaç yan etkileri ve farmakoekonomi parametreleri için
karar verdirici nitelikte olan randomize klinik çalışmalardır. Klinik ve
hekim deneyimi; TKİ mevcudiyeti, TKİ geriödenebilirliği, ilaç deneyimi,
ilaca uyum ve izleyen klinikte BCR-ABL1 izlem olanakları olarak
özetlenebilir. KML seyrinde ana kritik TKİ kararına esas olarak sayılan bu
değişkenlerin dikkate alınması sonrasında ulaşılır. Bu makalenin amacı,
KML tanımlamasında en son kullanılan Dünya Sağlık Örgütü-2016
kriterleri eşliğinde TKİ grubu ilaçlar ile uygun KML yönetimi ilkelerini
tartışmaktır.
Anahtar Sözcükler: Kronik myeloid lösemi, KML, Tirozin kinaz
inhibitör, TKİ
Introduction
Philadelphia (Ph*)/BCR-ABL1-positive chronic myeloid leukemia
(CML) is a chronic neoplastic disease, which can be functionally
cured via the administration of tyrosine kinase inhibitor
(TKI) drugs [1]. The overall aim of TKI therapy in CML is to
provide normal life duration and quality to the patient. The
harmonization of CML disease characteristics, physician/clinic
facilities, and best clinical evidence is vital to reach this ultimate
aim [2,3]. The disease characteristics of a given patient include
CML disease risk, comorbidities, molecular profile, compliance,
lifestyle, and drug off-target risk profile. CML research evidence
includes randomized clinical trials indicating data on the safety,
efficacy, tolerability, toxicity, possible long-term adverse events,
and pharmacoeconomy of TKIs. Clinical experience involves TKI
availability, TKI reimbursability, drug experience, adherence, and
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: İbrahim C. Haznedaroğlu, M.D., Hacettepe University Faculty of
Medicine, Department of Hematology, Ankara, Turkey
Phone : +90 312 305 15 43
E-mail : haznedar@yahoo.com ORCID: orcid.org/0000-0001-8028-9462
Received/Geliş tarihi: June 27, 2019
Accepted/Kabul tarihi: October 15, 2019
42
Turk J Hematol 2020;37:42-47
Haznedaroğlu İC, et al: Tyrosine Kinase Inhibitors
monitorization facilities. The critical decision regarding TKIs for
CML should be done via the optimization of those variables for
every single CML patient (Figure 1) [3]. The aim of this paper
is to outline the proper TKI treatment for the management of
CML, as described in the 2016 World Health Organization (WHO)
classification [3].
2016 WHO Definition of Chronic Myeloid Leukemia
The essential clinicopathological characteristics of Ph*(+) CML
in the 2016 WHO classification are defined as follows [4];
Chronic Phase CML
This is a myeloproliferative neoplasm characterized by the
chromosomal translocation t(9;22) (q34.1;q11.2), resulting in
the BCR-ABL1 fusion gene and formation of the Philadelphia
chromosome (Ph*), which causes an increase in blood
granulocytes and bone marrow myeloid precursors as the
major proliferative component. Cryptic and variant forms of
the Philadelphia chromosome as well as additional cytogenetic
abnormalities may complicate the disease pathobiology.
Therefore, interphase fluorescence in situ hybridization (FISH),
chromosome banding analysis, and PCR should be integrated for
the diagnosis and follow-up of CML [5,6].
The disease is described in three main clinical phases, which
were significantly prognostic before the TKI treatment era. The
chronic phase is the initial phase. Disease progression is then
described in two phases as the accelerated phase (AP) and
blastic phase (BP). AP disease is characterized by 10%-19%
blasts in the bone marrow or peripheral blood. The criterion for
transformed BP is more than 20% blasts either in the blood or
in the bone marrow, or at extramedullary sites [4].
Typical peripheral blood findings in CP-CML are characterized
by increased neutrophils with various early-stage granulocytic
precursors. The diagnosis needs to be proven by demonstrating
the molecular abnormality of BCR-ABL1 fusion. Typical bone
marrow (BM) histopathology is demonstrated in Figures 2A-2D.
The presence of t(9;22) (q34.1;q11.2) or BCR-ABL1 abnormality
could be demonstrated by karyotype analysis, FISH, or PCRbased
methods. The most reliable and sensitive method is realtime
PCR. This method is important and should be preferred
especially for routine monitoring for the evaluation of the
response to TKI treatment [7].
Complete responders to TKI treatment are defined by <10x109/L
blood cell count and <450x109/L platelet count without any
immature granulocytes in differentiation and nonpalpable
spleen [4]. The bone marrow features and cellular compositions
are normal with the appearance of erythrocytic precursors. Such
a case is demonstrated in Figures 2E-2H.
Accelerated Phase CML
The typical BM histopathology for AP was described before the
TKI treatment era. Following the start of the TKI era, the criteria
were modified considering the therapy. Cases responding to TKI
treatment are characterized by normalization of the cellular
composition of the bone marrow as demonstrated in Figures 2I-
2M. Abnormal megakaryocytes associated with marked reticulin
or collagen fibrosis in accordance with typical AP-CML could be
present (Figure 2K). The AP criteria are listed below [4].
• The presence of t(9;22)(q34.1;q11.2) or BCR-ABL1 (via
molecular biology or karyotype analyses) together with genomic
cytogenetic evolution and/or TKI resistance.
• Genomic evolution may include second Ph*, trisomy 8,
isochromosome 17q, trisomy 19, complex karyotype, or 3q26.2
abnormalities.
• Persistent or increasing abnormal blood counts despite
TKI treatment (leukocytosis (>10x109/L), thrombocytosis
(>1000x109/L), or thrombocytopenia (<100x109/L) unrelated to
therapy, 20% or more basophils, 10%-19% blasts)
• Persistent or increasing splenomegaly.
Figure 1. The harmonization of individual disease characteristics,
the experience of physician/clinical facilities, and best clinical
evidence is essential for clinical decision-making in chronic
myeloid leukemia (CML).
CML: Chronic myeloid leukemia, TKI: Tyrosine kinase inhibitor.
• Occurrence of clinically significant driver mutations in BCR-
ABL1 during TKI therapy (particularly T315I).
• Additional clonal chromosomal abnormalities such as trisomy
8, isochromosome 17q, trisomy 19, or any new entity of complex
karyotype and 3q26.2 abnormalities or any new chromosomal
43
Haznedaroğlu İC, et al: Tyrosine Kinase Inhibitors Turk J Hematol 2020;37:42-47
abnormality in BCR-ABL fusion-positive cells occurring during
TKI treatment are the accepted criteria
There are also provisional response criteria to TKI treatment
as described in the 2016 WHO classification. These are: 1-
Failure to achieve complete response to the TKI treatment or
hematological resistance; 2- Any hematological, cytogenetic,
or molecular indications of resistance to TKI treatment; 3-
Occurrence of two or more mutations in the BCR-ABL fusion
gene during TKI therapy [4].
Blastic Phase CML
Typical BM histopathology is presented in Figures 2N-2S with
increased blastic infiltration in accordance with the typical BP-
CML clinical presentation. The relevant criteria follow [4].
• The presence of t(9;22)(q34.1;q11.2) or BCR-ABL1 (via
molecular biology or karyotype analyses) together with genomic
cytogenetic evolution and/or TKI resistance.
• Genomic evolution may include second Ph* chromosome,
trisomy 8, isochromosome 17q, trisomy 19, complex karyotype,
or 3q26.2 abnormalities.
• The presence of at least 20% blasts in the peripheral blood
and/or BM or the presence of extramedullary blastic infiltration
in any organ or tissue
• Persistent or increasing splenomegaly.
Figure 2. Bone marrow biopsy in chronic phase (CP) CML is
usually hypercellular with 100% cellularity (A). The bone marrow
cells are almost all composed of mature granulocytes and their
precursors (B). Reticulin could be seen, especially in the cases
with increased megakaryocytes, but usually does not increase
(C). Bone marrow aspirate is hypercellular, composed of maturing
granulocytic precursors with striking decrease in other precursors
(D). Cellularity decreases in the bone marrow of responders to
TKI treatment (E, F). The islands of erythroid precursors and
megakaryocytes as well as the granulocytic series reflect the
normal composition (G). Aspirate smears can also reflect the
normal cellular composition with erythroid precursors (H; green
arrows). Accelerated phase (AP) CML is characterized by increased
blasts of <10%-19% and/or megakaryocytes (I, J). Increase
in megakaryocyte population promotes reticulin fibrosis (K).
Immunohistochemistry is helpful, especially for demonstrating
blasts by CD34 staining (L). Blasts on bone marrow aspirates are
scattered between myeloid precursors (M; yellow arrows). The
blasts are the dominant cellular component in the bone marrow
of blastic phase (BP) CML (N, P). Presence of strikingly increased
blasts could be demonstrated by CD34 immunohistochemistry (R).
On bone marrow aspirate smears, blastic cells are also dominant
(S; red arrows).
Frontline Strategies for CML Patients
TKI drug treatment should be initiated as soon as possible in
patients newly diagnosed with CML. The aim of chronic TKI therapy
in CML is the restoration of normal hematopoiesis instead of the
neoplastic BCR-ABL1-induced myeloid neoplastic proliferation
and the prevention of BCR-ABL1-associated genomic instability
[8]. Distinct TKI frontline strategy pathways may be chosen to
obtain long-term treatment end-points in the personalized
treatment of de novo CML. Patient age, CML risk (based on Sokal,
Euro/Hasford, EUTOS, and ELTS scoring systems), comorbidities,
and the long-term aim of the TKI treatment (mainly prevention
of disease progression with life-long TKI drug administration or
treatment-free remission) are the main cornerstones for choosing
the frontline TKI strategy in CML [2,9].
Pathway 1 (Imatinib as the Frontline TKI for CML): Treatment
with oral generic imatinib mesylate at 400 mg daily can be
prescribed for any patient with CML as the initial therapy.
Switching to a second-generation TKI may be considered in
the case of resistance or intolerance during the CML followup
period. The rational reasons for choosing this path are
pharmacoeconomy, better tolerability, and less toxicity of
imatinib with regard to second-generation TKIs. Furthermore,
there is no difference of frontline dasatinib/nilotinib/bosutinib
compared to imatinib in terms of survival [2].
Pathway 2 (Second-Generation TKI as the Frontline Drug for
CML): Second-generation TKIs (nilotinib, dasatinib, bosutinib)
may be administered to patients at high Sokal disease risk
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Turk J Hematol 2020;37:42-47
Haznedaroğlu İC, et al: Tyrosine Kinase Inhibitors
of CML for the prevention of disease progression and blastic
crisis. The determination of disease risk may be defined using
the Sokal, Euro/Hasford, EUTOS, or ELTS scoring systems
[10]. The rationale for this path is the prevention of disease
progression, accelerated disease, and blastic crisis in highrisk
patients. CML patients with higher percentages of blasts,
basophils, and eosinophils and those with thrombocytosis, BM
fibrosis, and massive splenomegaly are candidates for frontline
second-generation TKI therapy [11]. Relatively young CML
patients representing the target subpopulation for treatmentfree
remission (TFR) should also be selected for the frontline
nilotinib or dasatinib approach. However, there is no overall
survival advantage between frontline imatinib and secondgeneration
TKI approaches [12,13,14]. Therefore, imatinib 400
mg treatment shall be chosen for patients with drug/diseaseassociated
comorbidities, for whom TFR is not a target [2].
In clinical practice, any TKI (imatinib, nilotinib, bosutinib,
or dasatinib) as frontline therapy can be chosen with the
optimization of the dosage with regard to the individual
disease/patient characteristics, life expectancy, lifestyle, and
comorbidities. TKI dosages (for example, imatinib 300 vs. 400
vs. 600 mg; dasatinib 50 vs. 100 vs. 140 mg; nilotinib 600 vs.
800 mg; ponatinib 15 mg vs. 30 mg; bosutinib 300 mg vs. 500
mg) could be tailored based on the tolerability, side effects, and
BCR-ABL1 levels of the CML patients. The doses of TKIs shall be
adopted based on the phase of CML and the line of TKI therapy.
Lower starting TKI doses for the sake of tolerability should be
titrated up to the standard doses in order to get hematological,
cytogenetic, and molecular responses with the observation of
toxicity, compliance, and tolerability. The rationale for the TFR
path, i.e. frontline second-generation TKIs, is to obtain faster
and deeper molecular responses including MR4.5 for TKI drug
cessation [14,15]. The EURO-SKI trial was performed with
molecular responders of MR4 with TKI-free long-term remissions,
representing an advantage of survival without TKI toxicities,
which may be referred to as “functional cure” [16]. Although the
most significant literature experience with TKI discontinuation
is with imatinib, patients with two-year administration of
second-generation TKIs and a two-year duration of MR4.5 are
ideal candidates for TKI drug discontinuation [2,16,17].
The response to TKI drug treatment in a patient with CML must
be monitored to check for full hematological (CHR), complete
cytogenetic (CCyR), and major molecular (MMR) remissions
regardless of the path that has been chosen. The clinicobiological
signs of normal hematopoiesis replacing Ph*(+) myeloid neoplasia
should be investigated. Next-generation molecular analyses
[18] may be incorporated in the follow-up of CML patients
to search for genomic stability of the disease. Current disease
guidelines such as those of the ELN or NCCN require CHR within
the first month, CCyR within the first year, and MMR within 18
months of TKI therapy. BCR-ABL1 of less than 10% within the
first 3 months after TKI is a very good prognostic sign called
early molecular response (EMR). However, there is little evidence
that switching to a second-generation TKI in the absence of
EMR might produce better disease outcomes and prevention of
disease progression [19,20]. Preliminary results of the DASCERN
study implied that CML patients without EMR to imatinib at 3
months who switched to dasatinib had a significantly increased
rate of MMR at 12 months when compared to patients receiving
imatinib mesylate. Longer follow-up duration is certainly
required to assess the impact of early switching of dasatinib at
3 months on the overall survival of patients [21].
Long-term adverse events associated with the chronic usage
of TKI drugs described by the ELN [22] represent an important
emerging challenge in everyday clinical practice for CML. Side
effects of TKIs are generally mild to moderate and easy to
manage in the middle periods of CML therapy [22]. Provisional
discontinuation of the drug may be a choice in the case of
serious adverse events. Close attention should be paid to
drug-drug interactions [23]. Cardiovascular toxicity with
ponatinib and nilotinib, pulmonary toxicity with dasatinib, and
gastrointestinal/metabolic toxicities with bosutinib and nilotinib
may require specific follow-up strategies for early adverse event
detection and proper clinical management [24]. If properly
managed, TKI therapies are well tolerated with improvement
of the drug-related symptoms in due course with a few dose
reductions or short drug holidays [25].
Salvage Strategies in CML Patients
Salvage strategies in CML mainly depend on the alternative
unused TKIs and allografting if all of the TKIs were used with
a T315I mutation. Decision-making in multi-TKI-resistant
CML should rely on the type of first-line treatment, type
of resistance (TKI mutation, TKI failure, TKI intolerance, TKI
incompliance), phase of disease, and transplant risk score
of the patient. Before the consideration of TKI alteration
during life-time management of CML, drug dose adjustments,
such as TKI dose decrements in the event of adverse events
and increments in the presence of insufficient BCR-ABL1
control, shall be performed. The optimal salvage therapeutic
strategy for CML will avoid both over- and under-treatment.
CML over-treatment may be described as aggressive clinical
intervention. For instance, the early/inappropriate decision
to apply a very risky hematopoietic stem cell transplantation
(HSCT) in a CML patient receiving a given second-generation
TKI and exhibiting inadequate response, in which a thirdgeneration
TKI or dose increments would produce a better
outcome, requires careful consideration. On the other hand,
the inability to detect warning signs of relapse/resistance in
follow-up resulting in TKI failure and/or blastic crisis may also
be considered as inappropriate management. ABL mutations
of T315I, Y253H, E255K, E255V, F359V, F359C, and F359I are
45
Haznedaroğlu İC, et al: Tyrosine Kinase Inhibitors Turk J Hematol 2020;37:42-47
poorly sensitive to nilotinib and T315I, T315A, F317L, F317V,
F317I, F317C, and V299L are the mutations poorly sensitive
to dasatinib. Ponatinib is the only TKI for T315I before HSCT.
The most challenging situations in patients with CML are
resistance to all available TKIs in patients who inability to
undergo transplantation, or recurrence after HSCT, especially
into blastic crisis [26]. The fourth-generation drug asciminib,
a specific TKI targeting the BCR-ABL1 myristoyl-binding
site, an allosteric regulatory domain, and PF-114 mesylate
[27], have the potential to treat patients with resistance to
ATP-binding-site TKIs, including T315I [28,29]. CML leukemic
stem cells expressing IL-1RAP can be targeted by CAR-T cells
(chimeric antigen receptor-engineered T lymphocytes) [30].
Manipulations of CML stem cells [31], neoplastic bone marrow
niche trafficking control [32], and the CRISPR/Cas9 system
with nanocarriers [33] seem to be future research areas in the
field of CML therapy.
Ethics
Informed Consent: There is no patient presentation or patient
information in the article.
Authorship Contributions
Surgical and Medical Practices: O.İ.; Concept: O.İ.; Design: O.İ.;
Data Collection or Processing: I.K.; Analysis or Interpretation:
I.K.; Literature Search: İ.C.H.; Writing: İ.C.H.
Conflict of Interest: The authors declare that they have no
conflict of interest.
Financial Disclosure: The authors declared that this study
received no financial support.
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BRIEF REPORT
DOI: 10.4274/tjh.galenos.2019.2019.0195
Turk J Hematol 2020;37:48-52
Blastic Plasmacytoid Dendritic Cell Neoplasia: A Single Center
Experience
Blastik Plazmasitoid Dendritik Hücreli Neoplazi: Tek Merkez Deneyimi
Ahu Senem Demiröz 1 , Cuyan Demirkesen 1 , Ayşe Salihoğlu 2 , Nükhet Tüzüner 1
1İstanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Department of Pathology, İstanbul, Turkey
2İstanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Department of İnternal Medicine, Hematology, İstanbul, Turkey
Abstract
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare
malignancy with skin tropism. The entity was recently defined and the
diagnosis is generally made by skin biopsies. It is necessary to apply
appropriate immunohistochemistry to recognize this rare entity. There
is no consensus on therapy and the survival rates are low. The aim of
this study is to describe the clinical and histopathological features of
BPDCN. We retrospectively reviewed 8 BPDCN cases of the Cerrahpaşa
Medical Faculty diagnosed between 2005 and 2019. We documented
the clinical findings, histopathologic diagnoses, and outcomes. The
mean age of the patients was 58.7 years (range=11-86 years), and
7 patients were male. The patients presented with erythematous or
purple papules, plaques, and papulonodular or nodular cutaneous
lesions. Two had lymphadenomegaly at presentation. In microscopic
evaluations, tumor cells infiltrated the entire dermis with a clear-cut
subepidermal Grenz zone in all cases. CD4, CD56, and CD123 were the
most frequently expressed immunohistochemical markers. The median
follow-up of 7 cases was 14 months, ranging from 6 to 48 months.
Three patients died of the disease, while 4 patients were still alive. Out
of 7 patients, 5 received chemotherapy. We found that the outcomes
of some patients were different from others but we did not link any
distinct clinical or histopathological characteristics to these different
outcomes.
Keywords: Acute leukemia, Other leukemia, Neoplasia
Öz
Blastik plazmasitoid dendritic hücreli neoplazi (BPDCN) nadir görülen,
malign bir tümördür. Bu antite son yıllarda tanımlanmış olup, tanı
genellikle deri biyopsisinde uygun immunhistokimya panelinin
uygulanması ile konulur. Tedavi konusunda tam bir fikir birliği yoktur
ve beklenen yaşam süresi kısadır. Bu çalışmanın amacı BPDCN’nin klinik
ve histopatolojik özelliklerinin tanımlanmasına katkıda bulunmaktır.
Cerrahpaşa Tıp Fakültesi’nde 2005 ile 2014 yılları arası tanı almış olan
8 olgu geriye dönük olarak incelenmiştir. Kliniğe başvuru bulguları,
histopatolojik tanı ve klinik gidiş incelenmiştir. Olguların ortalama
yaşı 58,7 (11-86) olup, 7 olgu erkektir. Başvuru şikayeti eritematöz
ya da mor renkli papül, plak, papülonodül ya da nodüler deri
lezyonlarıdır. İki olguda başvuru sırasında lenfadenomegali mevcuttur.
Mikroskobik olarak tümör hücreleri subepidermal grenz zon bırakarak
tüm dermisi infiltre etmektedir. CD4, CD56 ve CD123 en sık eksprese
olan immünhistokimyasal işaretleyicilerdir. Yedi olguda median takip
süresi 14 ay (6-48 ay) olup 3 olgu hastalığa bağlı kaybedilmiş, 4 olgu
hayattadır. Yedi olgunun 5 tanesi kemoterapi almıştır. Olguların bir
kısmında diğerlerine göre farklı klinik gidiş gözlenmiştir, ancak bu durum
incelenen klinik ve histokimyasal bulgularla ilişkilendirilememiştir.
Anahtar Sözcükler: Akut lösemi, Diğer lösemi, Neoplazi
Introduction
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a
clinically aggressive tumor derived from the precursors of
plasmacytoid dendritic cells (PDCs) with a high frequency
of cutaneous and bone marrow involvement and leukemic
dissemination [1]. The tumor is considered among the “acute
myeloid leukemia and related neoplasms” since 2008 [2] and
recently it was reclassified as a separate entity in the latest
World Health Organization classification scheme. The tumor cells
typically express CD4, CD56, CD43, CD45RA, and plasmacytoid
dendritic cell antigens (CD123, CD303, TCL1A, CD2AP, SPIB, and
type 1 interferon-dependent molecule MX1) [3]. The molecular
profile showed that this entity is much more related to myeloid
neoplasms [4].
In this study, we present 8 patients with BPDCN from a single
center to emphasize the clinical and histopathological features
of this rare entity.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Ahu Senem Demiröz, M.D., İstanbul University-Cerrahpasa,
Cerrahpasa Faculty of Medicine, Department of Pathology, İstanbul, Turkey
Phone : +90 505 441 81 73
E-mail : ahusenem@yahoo.com ORCID: orcid.org/0000-0002-8370-1130
Received/Geliş tarihi: May 17, 2019
Accepted/Kabul tarihi: November 19, 2019
48
Turk J Hematol 2020;37:48-52
Demiröz AS, et al: Blastic Plasmacytoid Dendritic Cell Neoplasia: A Single Center Experience
Materials and Methods
Eight cases of BPDCN were retrieved from the archives of
the Department of Pathology of the Cerrahpaşa Medical
Faculty. Data regarding the clinical features and follow-up
were obtained from the patients’ records and their attending
physicians. We retrospectively reviewed hematoxylin and eosin
and immunohistochemical (IHC) stained slides of skin and bone
marrow biopsies. All IHC stainings were performed on a VENTANA
BenchMark automated staining system using 4-µm paraffin
tissue sections. The primary antibodies used in this study were
CD4 (ready to use, Novocastra), CD56 (1:250, Cell Marque), CD123
(1:50, Novocastra), MPO (1:600, DAKO), CD68 (1:400, Novocastra),
TdT (1:400, Thermo Scientific), CD7 (1:40, Thermo Scientific), CD20
(1:250, Thermo Scientific), and CD3 (1:300, Novocastra).
Results
The clinical features of the 8 cases are summarized in Table 1.
The median follow-up of 7 cases was 14 months (ranging from
2 to 48 months). Three patients died of the disease, while 4
were still alive. Of 7 patients, 5 received chemotherapy. Three
of them (patients 4, 6, and 7), given the hyper-CVAD regimen,
were in remission during the follow-up period. One (patient 5)
was given the hyper-CVAD regimen fortified with methotrexate
and cytarabine, but he died 8 months after chemotherapy due
to systemic involvement. One (the child, patient 8) was given
a BFM-ALL high-risk regimen; he was in remission 11 months
after the initial diagnosis.
Tumor cells infiltrated the entire dermis with a subepidermal
Grenz zone in all cases. In one case, the infiltration reached the
subcutaneous fat tissue. The infiltration pattern was diffuse in
5 of the cases, patchy in 1 case, and both diffuse and patchy
in 2 cases. Tumor cells were medium-sized with fine chromatin
resembling lymphoblasts. Cytoplasm was variably abundant
and lacked granules. Mitotic activity was scored as 0-3 in one
high-power field (HPF) in 7 cases. A patient with subcutaneous
infiltration (14.2%, patient 3) had 2-5 mitotic findings per HPF.
Necrosis, vascular invasion, and angiotropism were not detected
in any of the cases.
The IHC features are summarized in Table 2.
Discussion
BPDCN is a recently described entity with an aggressive course.
There are only a few series published in the literature. The
largest such series comprised 91 patients [5]. This disease is still
an obscure entity with many unknowns.
It is typically seen in middle-aged or elderly men, although
pediatric cases have also been rarely reported. The age range
in our series was 11-86 years with a mean age of 58.7 years. Of
8 patients, one was a child (14.2%). The male:female ratio was
2.5-3:1 [2,6,7,8].
Table 1. Patient characteristics, clinical data, and outcomes.
Case Age/Sex Cutaneous lesion Size
(cm)
1 86/M Papulonodular and tumoral
lesions on the trunk and scalp
2 73/F Purple erythematous papules
and plaques on the chest, back,
and extremities
3 71/M Multiple purplish or skincolored
papules/nodules
4 59/M Purple infiltrated nodules
on the abdomen and back,
palpable papules on the leg
5 73/M Multiple purple-red nodules
and plaques on the face, back,
and chest
6 57/M Maculopapular lesions on
chest, back, and shoulder
7 40/M Indurated nodular lesions on
the extremities and chest
8 11/M Red macular bruise-like lesion
on the leg
Extracutaneous
lesion
Bone
marrow
infiltration
Treatment
Follow
up
(months)
5 Absent Absent NAa 2 DOD
0.5-1.5 Absent NAb NA Lost to
follow
up
0.5-2 Absent Absent NAa 4 DOD
10 Absent Present Hyper-CVAD
regimen
2-15 Absent Absent Hyper-CVAD regimen
with methotrexate
and cytarabine
Outcome
NA
48 NED
8 DOD
0.5-1 Cervical LAM Absent Hyper-CVAD regimen 36 NED
3.5 Cervical and
submandibular LAM
DOD: Dead of disease; NA: Not applicable; NED: No evidence of disease; LAM: Lymphadenomegaly.
a These patients could not receive chemotherapy and died immediately after the initial diagnosis.
b The patient refused bone marrow biopsy.
Absent Hyper-CVAD regimen 12 NED
3.5 Absent Present BFM-ALL high-risk
regimen
11 NED
49
Demiröz AS, et al: Blastic Plasmacytoid Dendritic Cell Neoplasia: A Single Center Experience Turk J Hematol 2020;37:48-52
The disease tends to involve multiple sites with a predilection
for the skin, followed by bone marrow, peripheral blood, and
lymph nodes [2]. Approximately 85% of the reported patients
presented with cutaneous involvement. There are also a few
cases reported without skin lesions [9]. The disease was limited
to the skin in half of the cases [10]. The skin lesions may be
localized or widespread and the appearance of the lesions varies
from small bruise-like areas to violaceous patches, nodules,
and ulcerated masses. In our series, 6 patients did not have any
extracutaneous involvement; only 2 out of 7 had bone marrow
infiltration. The skin lesions in our series consisted of a bruiselike
patch in one case (patient 8, the child); macules, plaques,
or maculopapular lesions in 3 cases; and nodules in 5 cases
with diameters varying from 0.5 to 15 cm. None of them were
ulcerated (Figures 1 and 2).
oncogenic transformation. PDCs are not present extensively in
the skin, but BPDCN expresses CD56 and has skin tropism by
binding specifically to E-selectin on dermal endothelial cells and
cutaneous T-cells [11,14,15,16]. This may be the cause of the
skin predilection of this tumor.
CD4, CD56, and CD123 are the most frequently expressed IHC
markers in BPDCN. Neither CD4 nor CD56 negativity excludes
the diagnosis [5,11,17,18]. There are no double-negative cases
known to date. All of our cases were CD4+ and CD56+. CD123
analysis was performed in 5 cases, which were all positive. It
The histology of our series was consistent with the literature
[2,11,12]. The only conspicuous feature is that the mitotic rate
was higher in the case with extension into the subcutaneous
fat (2-5/HPF), and this patient died 4 months after the
initial diagnosis. This indicates that as the disease progresses,
involvement of the subcutaneous fat tissue takes place [13].
There is only one study showing that patients with high
proliferative indexes have significantly better survival [5].
A few tumoral cells in the bone marrow or peripheral blood
are likely in the early phases of the disease, as was seen in 2
patients in our series, but overt leukemia is more characteristic
for advanced cases or relapses after therapy [13]. Leukemic
variants without cutaneous involvement have also been
documented [8,12]. Although not verified by biopsy, 2 patients
had lymphadenomegaly, which was considered as lymph
node involvement (25%). Extracutaneous involvement other
than lymph nodes is also seen in the spleen, liver, and tonsils.
Cytopenia is the most frequent feature at presentation, whereas
B symptoms are not common [3,12].
Circulating normal PDCs and BPDCN both express CD123,
TCL1, and CD4. CD56 acquisition by PDCs is associated with
Figure 1. Case 5; A) Papulo-nodular lesion on the patient’s
face. B) Diffuse tumoral infiltration including entire dermis and
narrow subepidermal grenz zone (hematoxylin & eosin, 400 x ).
C) Tumor cell morphology (hematoxylin & eosin, 400 x ). D, E, F)
Immunhistochemistry with CD4, CD56, CD123 (400 x ).
Table 2. Immunophenotypic profiles.
Antibody Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Results
CD4 + + + + + + + + 8/8
CD56 + + + + + + + + 8/8
CD123 ND ND + + ND + + + 5/5
MPO - - - - - - - - 0/8
CD68 - +* - +* - - +* +* 4/8
TdT ND ND - - + - - - 1/6
CD7 ND ND ND - ND - - ND 0/3
CD3 - - - - - - - - 0/8
CD20 - - - - - - - - 0/8
ND: Not done, *Paranuclear dot-like positivity.
50
Turk J Hematol 2020;37:48-52
Demiröz AS, et al: Blastic Plasmacytoid Dendritic Cell Neoplasia: A Single Center Experience
BDCA-2 is a specific marker of normal PDCs and is seen in a
certain proportion of BPDCN cases [19,21]. CD2AP is a very
selective marker for the differentiation of BPDCN from c-AML,
but it is also expressed in cortical thymocytes.
Flow cytometry analysis and cytogenetic and clonality studies
may also help in diagnosis and in the exclusion of mimickers.
It is pointed out in the literature that the median survival is
about 12-14 months [3,11,13]. Advanced age and stage are
associated with poor prognostic factors; however, relatively
favorable prognosis is seen in children [6]. The only child in
our series showed complete remission after treatment. Four of
our patients were older than 70 years, and 3 of them died of
the disease within 8 months of diagnosis. These outcomes are
parallel to the literature and the results are compatible with the
expected prognosis of the disease. However, the other 4 patients
in our series showed no evidence of disease after the median
follow-up of 26.75 months, which is unusual compared to the
literature. This may be due to the shorter length of follow-up
time. Recently, a few case series with longer overall survival
rates were published [22,23,24].
Figure 2. Case 7; A) Endurated nodular lesion on the arm,
B) Nodule on the arm was healed after chemotherapy. C)
Tumor cell morphology (hematoxylin & eosin, 400 x ). D, E, F)
Immunhistochemistry with CD4, CD56, CD123 (400 x ).
should be kept in mind that CD123 expression is seen also in
AML.
Myeloid sarcoma (MS), T-cell lymphoblastic leukemia/
lymphoma, NK cell lymphoma/leukemia, and some mature T-cell
lymphoma/leukemias are the most frequent morphological
mimickers of BPDCN and show some overlapping features
[13,19]. The diagnosis depends on exclusion. For the
diagnosis of BPDCN, the tumor should be negative for other
myelomonocytic, NK, T, and B lineage markers (CD34, CD8, MPO,
lysozyme, PAX5, CD20, CD79a, EBV, and T-cell receptor protein).
However, the expression of CD33, CD2, CD3, CD7, S100, CD38,
and CD10 may be observed [11,12,13]. CD68 immunoreactivity
with a paranuclear dot-like pattern is detected in most BPDCN
cases, which is similar to our cases, while the staining pattern
in MS is diffuse and cytoplasmic [5,10,18,20]. TdT expression
is reported in approximately one-third of BPDCN cases, while
only one case in our series was positive.
Several IHC markers (TCL-1, BDCA-2, CD2AP) are defined for the
differentiation of BPDCN from its mimickers. TCL-1 is expressed
in 90% of BPDCN cases and only 17% of c-AML, and it is also
seen in a broad variety of B cell lymphoproliferative disorders
and some T-cell disorders but is absent in NK cell lineages [19].
There is no consensus on therapy. Several treatment regimens
including therapies for non-Hodgkin lymphoma, ALL, and
AML have been used as alternative therapies. Multiagent
chemotherapy regimens as in ALL are the most accepted
applications for these patients. The disease often relapses after
chemotherapy and becomes resistant to the previous drugs
[3,4,11,13,23,25].
Conclusion
In summary, BPDCN is a rare disease with poor prognosis. More
studies are necessary to have a better understanding of the
disease for proper management.
Ethics
Ethics Committee Approval: Ethics committee approval was
not required.
Informed Consent: Informed consent was obtained from
patients or from the relatives of deceased patients.
Authorship Contributions
Concept: A.S.D., C.D.; Design: A.S.D., C.D.; Data Collection or
Processing: A.S.D., A.S.; Analysis or Interpretation: A.S.D., C.D.,
N.T.; Literature Search: A.S.D.; Writing: A.S.D.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
51
Demiröz AS, et al: Blastic Plasmacytoid Dendritic Cell Neoplasia: A Single Center Experience Turk J Hematol 2020;37:48-52
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AS, Jaffe ES. Blastic plasmacytoid dendritic cell neoplasm in children :
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7. Nizza D, Simoneaux SF. Blastic plasmacytoid dendritic cell neoplasm
presenting as a subcutaneous mass in an 8-year-old boy. Pediatr Radiol
2010;40 Suppl 1:S40-42.
8. Julia F, Petrella T, Beylot-Barry M, Bagot M, Lipsker D, Machet L, Joly P,
Dereure O, Wetterwald M, d’Incan M, Grange F, Cornillon J, Tertian G,
Maubec E, Saiag P, Barete S, Templier I, Aubin F, Dalle S. Blastic plasmacytoid
dendritic cell neoplasm : clinical features in 90 patients. Br J Dermatol
2013;169:579-586.
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neoplasm (BPDCN) in leukaemic phase without skin lesions: a diagnostic
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10. Petrella T, Bagot M, Willemze R, Beylot-Barry M, Vergier B, Delaunay M,
Meijer CJ, Courville P, Joly P, Grange F, De Muret A, Machet L, Dompmartin
A, Bosq J, Durlach A, Bernard P, Dalac S, Dechelotte P, D’Incan M,
Wechsler J, Teitell MA. Blastic NK-cell lymphomas (agranular CD4+CD56+
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11. Jegalian AG, Facchetti F, Jaffe ES. Plasmacytoid dendritic cells. Adv Anat
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Blood 2002;99:1556-1563.
13. Shi Y, Wang E. Blastic plasmacytoid dendritic cell neoplasm: A
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CR, Dalac S, Durlach A, Galibert L. “Agranular CD4+ CD56+ hematodermic
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52
IMAGES IN HEMATOLOGY
DOI: 10.4274/tjh.galenos.2019.2019.0068
Turk J Hematol 2020;37:53-54
Kasabach-Merritt Syndrome in an Adult
Yetişkinde Kasabach Merritt Sendromu
Milan Pantelic 1 , Masa Pantelic 2 , Petar Djuric 3 , Katarina Markovic 4 , Tamara Vucinic 1 , Jovan Todor Juloski 5
1Zvezdara University Medical Center, Department of Radiology, Belgrade, Serbia
2Zvezdara University Medical Center, Department of Gastroenterology, Belgrade, Serbia
3Zvezdara University Medical Center, Department of Nephrology, Belgrade, Serbia
4Zvezdara University Medical Center, Department of Hematology, Belgrade, Serbia
5Zvezdara University Medical Center, Department of Surgery, Belgrade, Serbia
Figure 1. Multislice computed tomography showed a large
retroperitoneal tumor.
Figure 2. Multislice computed tomography showed a large
retroperitoneal tumor.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Milan Pantelic, M.D., Zvezdara University Medical Center,
Department of Radiology, Belgrade, Serbia
Phone : +903816113810407
E-mail : milan.pantelic.rs@gmail.com ORCID: orcid.org/0000-0001-7366-2425
Received/Geliş tarihi: February 17, 2019
Accepted/Kabul tarihi: May 20, 2019
53
Pantelic M, et al: Kasabach-Merritt Syndrome in an Adult Turk J Hematol 2020;37:53-54
Figure 3. An exploratory laparotomy with biopsy was performed.
Kasabach-Merritt syndrome (KMS) is a vascular disease
characterized by the presence of thrombocytopenia, anemia,
disseminated intravascular coagulation (DIC), and vascular
lesions. It was first described in 1940 by Kasabach-Merritt
[1,2,3]. KMS often occurs during infancy and the neonatal
period and rarely in adults [1,2,4]. KMS is commonly associated
with kaposiform hemangioendothelioma and tufted angioma,
which are rare vascular tumors produced by the lymph and
capillary endothelium with positive immunohistochemical
staining for vascular markers (CD31, CD34) and focal positivity
for lymphatic markers (LYVE1, PROKS1, D2-40), while they are
negative for GLUT1 and Lewis Y antigen (markers specific to
hemangiomas) [1,5].
A 22-year-old woman presented to the emergency department
with abdominal pain, fever, and vomiting. Laboratory
evaluation showed moderate anemia, DIC (hypofibrinogenemia,
thrombocytopenia, prolonged prothrombin, and activated
partial thromboplastin time), and elevated D-dimer. Multislice
computed tomography showed a large retroperitoneal tumor
(Figures 1, 2). An exploratory laparotomy with biopsy was
performed (Figure 3). Histopathological examination revealed
a vascular lesion (positive for CD31 and CD34 positive; focally
positive for D2-40). The patient was transferred to another
hospital where she was treated with corticosteroids (prednisone
at 40 mg daily), fresh frozen plasma, and cryoprecipitate, which
led to an improvement. After six months, magnetic resonance
imaging showed a regression in tumor size.
Keywords: Kasabach-Meritt syndrome, Vascular lesion,
Retroperitoneal tumor, Multislice computed tomography
Anahtar Sözcükler: Kasabach-Merritt sendromu, Vasküler
lezyon, Retroperitoneal tümör, Bilgisayarlı tomografi
Informed Consent: A consent form was completed by all
participants.
Authorship Contributions
Concept: Mi.P., Ma.P., P.D., K.M., T.V., J.T.J; Design: Mi.P., Ma.P.,
P.D., K.M., T.V., J.T.J; Data Collection or Processing: Mi.P., Ma.P.,
P.D., K.M., T.V., J.T.J; Analysis or Interpretation: Mi.P., Ma.P., P.D.,
K.M., T.V., J.T.J; Literature Search: Mi.P., Ma.P., P.D., K.M., T.V., J.T.J;
Writing: Mi.P., Ma.P., P.D., K.M., T.V., J.T.J.
Conflict of Interest: The authors of this paper have no conflict
of interest, including specific financial interests, relationships,
and/or affiliations relevant to the subject matter or materials
included.
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Mahajan P, Margolin J, Iacobas I. Kasabach-Merritt phenomenon: classic
presentation and management options. Clin Med Insights Blood Disord
2017;10:1-5.
2. Li K, Tai M, Qin Z, Ge C. Clinical observations in mesh suture treatment
for infants of Kasabach-Merritt phenomenon. J Paediatr Child Health
2015;51:529-533.
3. Kasabach HH, Meritt KK. Capillary hemangioma with extensive purpura
report of a case. Am J Dis Child 1940;59:1063-1070.
4. Oak CY, Jun CH, Cho EA, Lee DH, Cho SB, Park CH, Joo YE, Kim HS, Rew JS,
Choi SK. Hepatic hemangioma with Kasabach-Merritt syndrome in an adult
patient. Korean J Gastroenterol 2016;67:220-223.
5. O’Rafferty C, O’Regan GM, Irvine AD, Smith OP. Recent advances in the
pathobiology and management of Kasabach-Merritt phenomenon. Br J
Haematol 2015;171:38-51.
54
IMAGES IN HEMATOLOGY
DOI: 10.4274/tjh.galenos.2019.2019.0171
Turk J Hematol 2020;37:55-56
Platelet Satellitism
Trombosit Satellitizmi
Yasemin Ardıçoğlu Akışın 1 , Nejat Akar 2
1TOBB-ETU Faculty of Medicine, Department of Biochemistry, Ankara, Turkey
2TOBB-ETU Faculty of Medicine, Department of Pediatrics, Ankara, Turkey
Figures 1 and 2. Platelet satellitism in a 5-year-old child with an upper respiratory tract infection accompanying asthma.
Platelet satellitism is a rare in vitro phenomenon that occurs
when an immunoglobulinG antibody directed against the
glycoprotein IIb/IIIa complex on the platelet membrane
forms in ethylenediaminetetraacetic acid-treated peripheral
blood at room temperature [1,2]. As the antibody coats the
platelets, platelets adhering to polymorphonuclear leukocytes
show a rosette-like appearance [3]. There is no definite causal
association with any disease. Severe rosetting may lead to a
misdiagnosis of thrombocytopenia unless peripheral smears are
examined [4].
The images presented here were obtained from a 5-year-old
child with an upper respiratory tract infection accompanying
asthma. Thrombocytopenia was not detected in complete blood
count (Figures 1 and 2).
Keywords: Platelet, Polymorphonuclear leukocytes, Satellitism
Anahtar Sözcükler: Trombosit, Polimorfonükleer lökosit,
Satellitizm
Informed Consent: The patient’s family consented to the
publication of this study.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Yasemin Ardıçoğlu Akışın, M.D., TOBB-ETU Faculty of Medicine,
Department of Biochemistry, Ankara, Turkey
Phone : +90 533 336 74 32
E-mail : yardicoglu@gmail.com ORCID: orcid.org/0000-0002-4109-0220
Received/Geliş tarihi: April 30, 2019
Accepted/Kabul tarihi: June 11, 2019
55
Ardıçoğlu Akışın Y and Akar N: Platelet Satellitism Turk J Hematol 2020;37:55-56
Authorship Contributions
Drafting, reviewing: Y.A.A.; Reviewing: Y.A.A., N.A.
Conflict of Interest: The authors declare that there is no conflict
of interest.
Financial Disclosure: The authors have no sources of support for
this work.
References
1. Bizzaro N, Goldschmeding R, von dem Borne AE. Platelet satellitism is Fc
gamma RIII (CD16) receptor-mediated. Am J Clin Pathol 1995;103:740-744.
2. Bobba RK, Doll DC. Platelet satellitism as a cause of spurious
thrombocytopenia. Blood 2012;119:4100.
3. LabCE. Pseudo-thrombocytopenia: Platelet Satellitism and Platelet
Clumping. LabCE, 2019. Available online at https://www.labce.com/
spg280957_pseudo_thrombocytopenia_platelet_satellitism_and_p.aspx
4. Chakrabart I. Platelet satellitism: a rare, interesting, in vitro phenomenon.
Indian J Hematol Blood Transfus 2014;30:213-214.
56
LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
Assessment of Patients with von Willebrand Disease with ISTH/
BAT and PBQ Scores
Von Willebrand Hastalığı Olgularının ISTH/BAT ve PBQ Skorları ile Değerlendirilmesi
Fatma Burcu Belen Apak 1 , Elif Gülsüm Ümit 2 , Yağmur Zengin 3 , Melike Sezgin Evim 4 , Ekrem Ünal 5 , Hasan Mücahit Özbaş 6 ,
Can Acıpayam 7
1Başkent University Faculty of Medicine, Department of Pediatric Hematology Oncology, Ankara, Turkey
2Trakya University Faculty of Medicine, Department of Hematology, Edirne, Turkey
3Başkent University Faculty of Medicine, Department of Biostatistics, Ankara, Turkey
4Uludağ University Faculty of Medicine, Department of Pediatric Hematology Oncology, Bursa, Turkey
5Erciyes University Faculty of Medicine, Department of Pediatric Hematology Oncology, Kayseri, Turkey
6Giresun University Faculty of Medicine, Department of Hematology, Giresun, Turkey
7Kahramanmaraş Sütçü İmam University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Kahramanmaraş, Turkey
To the Editor,
The Turkish Society of Hematology initiated the Turkish
Hemophilia Masterclass Academy program in 2016 to encourage
young hematologists entering the field of hemophilia. The
program involved 6 months of training, supported by monthly
exams. We, as a group of mentees from the Hemophilia
Masterclass Academy, aimed to evaluate the bleeding
phenotype of patients with von Willebrand Disease (VWD)
using the International Society of Thrombosis and Haemostasis-
Bleeding Assessment Tool (ISTH-BAT) and the Pediatric Bleeding
Questionnaire (PBQ) scores and investigate the correlation of
von Willebrand factor antigen (VWF:Ag) levels and bleeding
scores of the patients, as well as present the initial output of
our Masterclass Academy.
The study included 62 patients (aged 3-61 years) with the
diagnosis of VWD (54 patients with VWD type 1 and 8 with
VWD type 3). The ISTH-BAT and PBQ were administered to
patients who were ≥18 years old and <18 years old, respectively.
Informed consent was obtained from all patients.
The VWF:Ag levels, ristocetin cofactor activity (VWF:Ricof), and
FVIII levels were retrospectively reviewed from the patient records.
The cut-off point for a positive score was accepted as ≥2 for the
PBQ and ≥3 for the ISTH-BAT. Statistical analysis was performed
using SPSS 17.0. Demographic findings, median bleeding scores,
and VWF levels are presented in Table 1. Epistaxis, superficial
bleedings, bleeding from minor wounds, oral bleeding, umbilical
bleeding, and muscle hematoma were found to be statistically
significant in showing dependence between the diagnostic
status and the bleeding symptoms (p<0.05). The study aimed to
investigate the correlation between the VWF:Ag levels and the
bleeding scores in our patients. Our study showed that VWF:Ag
levels were inversely correlated with the bleeding scores of the
patients (Table 2).
The evaluation of bleeding scores dates back to the Vicenza
score that was used in VWD patients [1,2,3]. In 2009, Bowman
et al. [4] published the PBQ, and it was investigated in our
population with VWD previously [5]. In 2010, the ISTH-BAT
score was established by the ISTH/SSC Joint Working Group
[6,7]. One of the limitations of our study was the use of the PBQ
in children and ISTH-BAT in adults, which might have caused
heterogeneity in the evaluation of the scores. Moreover, the
lack of a control group, consisting of patients who had bleeding
symptoms but were normal by hemostatic tests, left us unable
to compare the VWD patients and normal individuals. Although
the median total scores of the types 1 and 3 VWD groups were
compatible with previous studies, the positive scores reported
in each subgroup for epistaxis, oral cavity bleeding, cutaneous
bleeding, and bleeding after minor wounds were found to
Table 1. Characteristics of von Willebrand type 1 and type 3
patients.
VWD type 1
(n=54)
VWD type 3
(n=8)
Median age, years (minimum-maximum) 28 (3-61) 17 (6-30)
Pediatric/Adult 13/41 3/5
Female 46 6
Male 8 2
VWF:Ag (IU/dL) 22.23 (2-50) 2.5 (0-10)
VWF:Ricof (IU/dL) 23.3 (8.3-45) 7.5 (6-23)
FVIII (IU/dL) 45 (15-70) 3 (1-11.5)
Median total score (minimum-maximum) 3 (0-19) 16 (9-27)
VWF:Ag: von Willebrand factor antigen level, VWF:Ricoll: von Willebrand Factor
ristocetin cofactor level, VWD: von Willebrand disease.
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LETTERS TO THE EDITOR Turk J Hematol 2020;37:57-76
Table 2. Bleeding scores positivity regarding each symptom in
von Willebrand type 1 and type 3 patients.
Bleeding symptom
VWD
type 1§
VWD
type 3*
Epistaxis 22% 100% 0.000
Cutaneous bleeding 3.7% 62.5% 0.000
Minor bleeding 5.6% 75% 0.000
Oral cavity bleeding 5.6% 75% 0.000
Bleeding after tooth extraction 22.2% 37.5% 0.295
Intramuscular bleeding 3.7% 37.5% 0.013
Hemarthrosis 3.7% 25% 0.077
Menorrhagia** 41.3% 50% 0.506
Central nervous system bleeding 1.9% 12.5% 0.243
Umbilical bleeding 1.9% 25% 0.041
Postsurgical bleeding 1.7% 0% 0.757
Cephalhematoma 0% 12.5% 0.129
Macroscopic hematuria 3.7% 12.5% 0.344
§ von Willebrand type 1 group (n=54). * von Willebrand type 3 group (n=8).
Epistaxis, superficial bleedings, bleeding from minor wounds, oral bleeding, umbilical
bleeding, muscle hematoma, and hemarthrosis were found to be statistically
significant in showing dependence between diagnosis status (VWD types 1-3) and
bleeding symptoms (p<0.05).
**Menorrhagia symptom was compared among female patients in both groups.
VWD: von Willebrand disease
be lower compared to those in the previous literature [4,5,8].
This may be explained by the inclusion of “low VWF levels”
(intermediate levels of VWF:Ag, 30-50 IU/dL) in the group with
type 1 VWD. People with “low VWF levels” falsely labeled as
“VWD type 1 patients” may have lower reported bleeding scores
compared to true VWD patients, leading to low positive scores.
Our study shows that the ISTH/BAT and PBQ can be useful in
the evaluation of the bleeding symptoms of patients. Further
studies with larger patient and control groups are warranted
to show the usage of bleeding scores in daily outpatient
practice. We, as the mentees of the Hemophilia Masterclass, feel
much appreciation to our mentors and the Turkish Society of
Hematology for their contributions to our progress in the field
of hemophilia.
Keywords: Von Willebrand Disease, Pediatric Bleeding
Questionnaire, ISTH/BAT score
Anahtar Sözcükler: Von Willebrand Hastalığı, Pediatrik Kanama
Skoru, ISTH/BAT skoru
p
Informed Consent: Informed consent was obtained from all
patients or their legal guardians.
Authorship Contributions
Surgical and Medical Practices: F.B.B.A., E.G.Ü., Y.Z., M.S.E., E.Ü.,
H.M.Ö., C.A.; Concept: F.B.B.A.; Design: F.B.B.A., E.G.Ü; Data
Collection or Processing: F.B.B.A., Y.Z.; Analysis or Interpretation:
Y.Z., F.B.B.A., E.G.Ü; Literature Search: F.B.B.A., E.G.Ü; Writing:
F.B.B.A.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Tosetto A, Castaman G, Rodeghiero F. Bleeding scores in inherited bleeding
disorders: clinical or research tools? Haemophilia 2008:14:415-422.
2. Rodeghiero F, Castaman G, Tosetto A, Batlle J, Baudo F, Cappeletti A, Casana
P, De Bosch N, Eikenboom JC, Federici AB, Lethagen S, Linari S, Srivastava A.
The discriminant power of bleeding history for the diagnosis of type 1 von
Willebrand disease: results from a multicenter study. J Thromb Haemoast
2005;3:2619-2626.
3. Tosetto A, Rodeghiero F, Castaman G, Goodeve A, Federici AB, Batlle J, Meyer
D, Fressinaud E, Mazurier C, Goudemand J, Eikenboom J, Schneppenheim R,
Budde U, Ingerslev J, Vorlova Z, Habart D, Holmberg L, Lethagen S, Pasi J,
Hill F, Peake I. A quantitative analysis of bleeding symptoms in type 1 von
Willebrand disease: results from a multicenter European study (MCMDM-1
VWD). J Thromb Haemost 2006;4:766-773.
4. Bowman M, Riddel J, Rand ML, Tosetto A, Silva M, James PD. Evaluation
of the diagnostic utility for von Willebrand disease of a pediatric bleeding
questionnaire. J Thromb Haemost 2009;7:1418-1421.
5. Belen B, Kocak U, Isik M, Keskin EY, Oner N, Sal E, Kaya Z, Yenicesu I, Gursel
T. Evaluation of Pediatric Bleeding Questionnaire in Turkish children with
von Willebrand disease and Platelet function disorders. Clin Appl Thromb
Hemost 2015;21:565-569.
6. Rodeghiero F, Tosetto A, Abshire T, Arnold DM, Coller B, James P, Neunert
C, Lillicrap D; ISTH/SSC Joint VWF and Perinatal/Pediatric Hemostasis
Subcommittees Working Group. ISTH/SSC bleeding assessment tool: a
standardized questionnaire and a proposal for a new bleeding score for
inherited bleeding disorders. J Thromb Haemost 2010;8:2063-2065.
7. Bidlingmaier C, Grote V, Budde U, Olivieri M, Kurnik K. Prospective evaluation
of pediatric bleeding questionnaire and the ISTH bleeding assessment tool
in children and parents in clinical routine. J Thromb Haemost 2012;10:1335-
1341.
8. Pathare A, Omrani SA, Al Hajri F, Al Obaidani N, Al Balushi B, Al Falahi K.
Bleeding score in type 1 von Willebrand disease patients using the ISTH-BAT
questionnaire. Int J Lab Hematol 2018;40:175-180.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Fatma Burcu Belen Apak, M.D., Başkent University Faculty of
Medicine, Department of Pediatric Hematology Oncology, Ankara, Turkey
Phone : +90 532 581 45 51
E-mail : draidabb@gmail.com ORCID: orcid.org/0000-0002-9278-6703
Received/Geliş tarihi: July 25, 2019
Accepted/Kabul tarihi: November 12, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0446
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Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
Children with Iron Deficiency Anemia Have a Tendency to
Hypercoagulation: An Evaluation by Thromboelastography
Demir Eksikliği Anemisi Olan Çocukların Hiperkoagülasyona Eğilimleri Vardır:
Tromboelastograf ile Bir Değerlendirme
Ceren Kılcı 1 , Lale Olcay 2 , Beril Özdemir 3 , Ali Fettah 4 , Meriç Yavuz Çolak 5
1Başkent University Faculty of Medicine, Department of Pediatrics, Ankara, Turkey
2Başkent University Faculty of Medicine, Department of Pediatrics, Department of Pediatric Hematology-Oncology, Ankara, Turkey
3Başkent University Faculty of Medicine, Department of Pediatrics, Ankara, Turkey
4Sami Ulus Pediatrics Training and Research Hospital, Department of Pediatric Hematology-Oncology Ankara, Turkey
5Başkent University, Faculty of Health Sciences, Ankara, Turkey
To the Editor,
In the literature, there are many reports about patients who
developed thrombosis with coexistent iron deficiency (ID) or ID
anemia (IDA) [1,2,3,4,5,6,7,8].
Moreover, the frequency of severe anemia [1] and IDA [2] in
patients who developed cerebral venous thrombosis (CVT) or
deep venous thrombosis (including pulmonary embolism),
respectively, was shown to be higher than in controls. The
occluded vessels were cerebral vessels in 96.2% and 46.4% of
the affected children and adults, respectively [8].
The predilection to hypercoagulation in ID/IDA was predicted to
be due to reactive thrombocytosis, microcytosis, dehydration,
infections, alterations in laminar flow, formation of turbulence,
corruption of the oxidant/antioxidant balance, increases in
platelet aggregation, increased procoagulants, and hypoxia
[3,4,5,6,7,8]. However, laboratory investigations of this topic are
still rare.
Herein we aim to provide laboratory evidence of the propensity
to thrombosis in IDA using thromboelastography, which can
qualitatively determine the status of coagulation as hyper- or
hypocoagulation, and to state whether the abnormality stems
from any pathology in primary hemostasis, secondary hemostasis,
or the fibrinolytic system or any effects of anticoagulants
or inhibitors within 30 min. With thromboelastography, the
formation, strength, elasticity, and firmness of a clot can be
shown using parameters such as reaction (R) time, clot formation
(K) time, alpha (α) angle, maximum amplitude (MA), maximum
lysis (LY30), and coagulation index (CI). Their functions and
implications are presented in Supplemental Table 1.
Blood samples from 34 IDA patients between the ages of 3.5 and
191 months and from 39 healthy children of 12 to 191 months of
age were studied using the flat cup test in thromboelastography
(TEG ® 5000 Thromboelastograph® Hemostasis Analyzer).
Patients with chronic (including thalassemia) or infectious/
inflammatory diseases, high c-reactive protein (CRP) levels,
obesity, hypertension, smoking habit, hyperuricemia, liver
or renal function abnormalities, vitamin B12 or folic acid
deficiencies, and self or family history of thrombosis or bleeding
were excluded from the study, as were those on any drug
therapy.
The thromboelastographic measurements observed in the IDA
and control groups were as follows: K, 1.4±0.6 vs. 1.8±1.1 min
(p=0.03); MA, 70.6±4.9 mm vs. 66.9±8.3 mm (p=0.05); LY30,
3.8±4.4 vs. 2.0±3.2 (p=0.12); R, 3.9±1.4 vs. 4.0±1.4 min (p=0.78);
α, 53.0±8.9° vs. 53.0±9.6° (p=0.91); and CI, 1.0±1.4 vs. 0.3±2.1
(p=0.19) (Supplemental Table 2; Figures 1A and 1B).
Significant decrease in K and increase in MA with borderline
significance compared to the controls implied hypercoagulability,
which was possibly due to increased fibrinogen levels and/or to
a lesser extent increased thrombocyte functions (Supplemental
Table 1). Inflammation-related hyperfibrinogenemia was a
remote possibility since patients with infection/inflammation
and high CRP levels were excluded; however, we could not
establish fibrinogen levels and thrombocyte functions. Other
studies showed normal levels of fibrinogen [9] and increased
[10] or decreased thrombocyte aggregation [11] in IDA.
Our findings revealed a positive linear relationship between
serum iron levels and α (p=0.034; r=0.339) and between red
blood cell distribution width (RDW) and α (p=0.004; r=0.448),
and an inverse linear relationship between RDW and K (p=0.048;
r=-0.319) in the control group.
In the IDA group, there was a positive and weak linear relationship
between ferritin and α (p=0.049; r=0.341), a positive linear
relationship between mean corpuscular volume (MCV) and MA
(p=0.04; r=0.353), and an inverse linear relationship between
thrombocyte count and K (p=0.041; r=-0.353).
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LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
frequent in patients with CVT than normal subjects and “severe
anemia” was an independent determinant of CVT. ID was an
independent predictor of venous thromboembolism recurrence
[12]. In our study, due to the limited number of participants, we
could not compare cases in terms of “severe” and “mild” anemia.
Figure 1A. A normal thromboelastography result for an individual
from the control group.
Indeed, of the 54 patients with coexistent ID/IDA and thrombosis
in the literature [8], 18% had thrombotic risk factors and 75.9%
had associated diseases/disorders [8], and both of our two
patients with both IDA and thrombosis had thrombotic risk
factors (100%) and an associated condition (100%). Since not
all patients in the literature were evaluated for thrombophilia
factors, this rate may be increased. We could not evaluate these
patients for accompanying thrombophilic factors.
We furthermore did not have an opportunity to compare
thromboelastography values before and after iron therapy.
However, our findings showed a propensity to hypercoagulation
in patients with IDA and confirm the results of previous studies
[1,2,8,9].
Figure 1B. Thromboelastography result for a patient with iron
deficiency anemia showing hypercoagulation.
Although the positive linear relationship of ferritin with α
and of MCV with MA pointed at hypocoagulability, the inverse
linear relationship between thrombocyte count and K pointed
at hypercoagulation, being correlated with the severity of
thrombocytosis, the latter of which is a usual finding in IDA.
These conflicting results of the correlation studies may be due
to the limited number of patients.
In another study similar to ours that investigated the
effect of IDA by rotational thromboelastometry (ROTEM),
normal coagulation test results were also revealed, whereby
maximum clot firmness in ROTEM, equivalent to MA in
thromboelastography, was increased in the IDA group and clot
formation time, equivalent to K in thromboelastography, was
decreased in the IDA group, both implying hypercoagulability.
This study also revealed similar thrombocyte counts in
the IDA and control groups despite a negative correlation
between thrombocyte count and CFT [equivalent to K in
thromboelastography] [9], as in our study.
The fact that none of our patients in this report had developed
thrombosis suggests that additional determinants may be required
for the development of thrombosis. Moreover, the real incidence
of thrombosis in IDA may be too low to be established in a small
cohort of patients such as ours. The duration and the severity of
anemia may be other factors for the initiation of thrombosis. In
a review of 54 patients who developed thrombosis on the basis
of ID/IDA, the majority had “severe” IDA [8], while Stolz et al.
[1] reported “not mild” but rather “severe” anemia to be more
Although further laboratory evaluations are required with
larger numbers of patients and the exclusion of accompanying
thrombophilic factors, IDA seems to be a new candidate among
thrombotic factors.
Keywords: Thromboelastograph, TEG, Iron deficiency anemia,
coagulation, Thromboembolism, Fibrinogen, Platelet functions
Anahtar Sözcükler: Tromboelastograf, TEG, Demir eksikliği
anemisi, Koagülasyon, Tromboembolizm, Fibrinojen, Trombosit
fonksiyonları
Acknowledgments
This work was derived from the residency dissertation of Dr.
Azize Ceren Kılcı, carried out during 2015-2016, and was
presented at the 2. Selim Hematoloji Güncelleme Sempozyumu
on 24-26 February 2017, Antalya (EPS-46). We would like to
thank Prof. Dr. Fatma Gümrük and Prof. Dr. Esra Baskın for
fruitful suggestions in the study; our patients and their parents
for joining our study; our nurses for collecting the blood
samples; Dr. Gürses Şahin for his collaboration in our clinical
research; and Çağdaş Kızılemiş for his collaboration in the
thromboelastography.
Informed Consent: Informed consent was received from the
parents of all participitants.
Authorship Contributions
Analysis or Interpretation: C.K., L.O., B.Ö., A.F., M.Y.Ç.; Literature
Search: C.K., L.O., B.Ö., A.F., M.Y.Ç.; Writing: L.O.
Conflict of Interest: No conflict of interest was declared by the
authors.
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Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Stolz E, Valdueza JM, Grebe M, Schlachetzki F, Schmitt E, Madlener K,
Rahimi A, Kempkes-Matthes B, Blaes F, Gerriets T, Kaps M. Anemia as a risk
factor for cerebral venous thrombosis? An old hypothesis revisited. Results
of a prospective study. J Neurol 2007;254:729-734.
2. Hung S, Lin H, Chung S. Association between venous thromboembolism
and iron-deficiency anemia: a population-based study. Blood Coagul
Fibrinolysis 2015;26:368-372.
3. Hartfield DS, Lowry NJ, Keene DL, Yager JY. Iron deficiency: a cause of stroke
in infants and children. Pediatr Neurol 1997;16:50-53.
4. Franchini M, Targher G, Montagnana M, Lippi G. Iron and thrombosis. Ann
Hematol 2008;87:167-173.
5. Akins PT, Glenn S, Nemeth PM, Derdeyn CP. Karotid artery thrombus
associated with severe iron-deficiency anemia and thrombocytosis. Stroke
1996;27:1002-1005.
7. Kinoshita Y, Taniura S, Shishido H, Nojima T, Kamitani H, Watanebe T.
Cerebral venous sinus thrombosis associated with iron deficiency: two case
reports. Neurol Med Chir 2006;46:589-593.
8. Ceren K, Lale O, Taner S, Z Ecevit, Murat Ö, Varan B. Thrombosis in iron
deficiency and iron deficiency anemia: A review of our cases and the
relevant literature. Open Acc J Oncol Med 2018;2:157-172.
9. Özdemir ZC, Kar YC, Gündüz E, Turhan Bozkurt A, Bör O. Evaluation of
hypercoagulability with rotational thromboelastometry in children with
iron deficiency anemia. Hematology 2018;23:664-668.
10. Tekin D, Yavuzer S, Tekin M, Akar N, Cin Ş. Possible effects of antioxidant
status on increased trombosit aggregation in childhood iron-deficiency
anemia. Ped Int 2001;43:74-77.
11. Çalışkan U, Öner AF, Kabakuş N, Koç H. Diminished trombosit aggregation in
patients with iron deficiency anemia. Clin Appl Thromb Hemost 1999;5:161-
163.
12. Potaczek DP, Jankowska EA, Wypasek E, Undas A. Iron deficiency: a novel risk
factor of recurrence in patients after unprovoked venous thromboembolism.
Pol Arch Med Wewn 2016;126:159-165.
6. Sushil B, Khan A, Hussain N, Gosalakkal J. Severe anemia causing cerebral
venous sinus thrombosis in an infant. J Pediatr Neurosci 2012;7:30-32.
Supplemental Table 1. Definition of thromboelastographic parameters [1,2].
Definition of parameter Indicated by increased values Indicated by decreased values
R (min)
(reaction time)
K (min)
(clot kinetics)
Alpha angle
(degree)
(clot kinetics)
MA (mm)
(maximum
amplitude)
CI (coagulation
index)
Maximum lysis
(LY30)
(%)
CI: Coagulation index; LY: Maximum lysis.
The time elapsed between the placement
of first blood sample in the device and
formation of fibrin (2 mm); associated
with plasma coagulation and inhibitor
activity
The time elapsed between the R time and
the time in which clot reaches 20 mm
of amplitude; associated with kinetics of
clot formation and measures the speed
of clot to attain a certain strength
Measures the speed of clot
polymerization; associated with
strengthening of clot
A direct function representing
maximum dynamic property of fibrin
and thrombocyte binding; associated
with thrombocyte function rather than
fibrinogen; denotes strength of fibrin
clot
Found by calculating linear indexes of R,
K, MA, and α angle values
Denotes the decline of the 30th minute
of amplitude after MA, as percentage;
indicates the degree of fibrinolysis and is
associated with clot stability
Factor deficiency
Anticoagulant therapy
Low fibrinogen level
Low thrombocyte count
Factor XIII deficiency
Low fibrinogen level
Thrombocytopenia
Insufficiency in thrombocyte functions
Use of heparin as anticoagulant therapy
Hypercoagulation
Hypercoagulation
If hypercoagulable status originates from
plasma (enzymatic hypercoagulability), R time is
decreased more than K time; if hypercoagulable
status originates from thrombocytes, both R
and K times are decreased
Values greater than +3 indicate
hypercoagulation
Values greater than 7.5% indicate increased
fibrinolysis
Hypercoagulation
Hypercoagulation
Major increase in fibrinogen,
and increased thrombocyte
function to a lesser extent
Factor deficiency
Fibrinogen impairment
Thrombocytopenia
Increased fibrinolysis
Thrombocytopenia
Thrombocytopathia
Hypofibrinogenemia
Values less than -3 indicate
hypocoagulation
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LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
Supplemental Table 2. Thromboelastographic parameters in the iron deficiency anemia group in comparison with the healthy
control group.
Mean ± SD z p
Clot formation time (K) (min) Iron deficiency anemia 1.4±0.6
0.03
-2.2
Control 1.8±1.1
Maximum amplitude (MA) (mm) Iron deficiency anemia 70.6±4.9 -1.94 0.05
Control 66.9±8.3
Maximum lysis (LY30) (%) Iron deficiency anemia 3.8±4.4 -1.57 0.12
Control 2.0±3.2
Reaction time (R) (min) Iron deficiency anemia 3.9±1.4 -0.02 0.78
Control 4.0±1.4
Alpha angle (α) (º) Iron deficiency anemia 53.0±8.9 -0.1 0.91
Control 53±9.6
Coagulation index (CI) Control 1.0±1.4 -1.29 0.19
Control 0.3±2.1
CI: Coagulation index; LY: Maximum lysis, SD: Standard deviation, MA: Maximum amplitude.
Supplemental References
1. Traverso CI, Caprini JA, Arcelus JI. The normal thromboelastogram and its interpretation. Semin Thromb Hemost 1995; 21: 7-13.
2. Thakur M, Ahmed AB, Tanaka KA. A Review of thromboelastography. Int J Periop Ultrasound Appl Technol 2012;1: 25-29.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Lale Olcay, M.D., Başkent University Faculty of Medicine,
Department of Pediatrics, Department of Pediatric Hematology-Oncology, Ankara, Turkey
Phone : +90 312 221 00 03
E-mail : laleolcay@hotmail.com.tr ORCID: orcid.org/0000-0002-5684-0581
Received/Geliş tarihi: January 17, 2019
Accepted/Kabul tarihi: December 18, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0027
Successful Hemostasis with Extended Half-life Recombinant
Factor VIII in Circumcision
Uzun Etkili Rekombinan Faktör VIII ile Başarılı Sünnet Operasyonu
Başak Koç 1 , Metin İshak Öztürk 2 , Bülent Zülfikar 1
1İstanbul University Oncology Institute, Department of Pediatric Hematology-Oncology, İstanbul, Turkey
2Haydarpaşa Training and Research Hospital, Department of Urology, İstanbul, Turkey
To the Editor,
Intensified coagulation factor replacement is essential for
surgical procedures in people with hemophilia A (HA). It is
indicated during surgery and in the postoperative period [1,2].
The efficacy and the safety of PEGylated recombinant human
full-length coagulation factor VIII (BAX 855) in prophylaxis and
treatment of bleeding episodes have already been reported and
its half-life in the circulation was proven to be 1.5 times longer
compared to standard half-life FVIII (SHL-FVIII) [3]. Circumcision
is a common surgical intervention in approximately half of the
world [4,5]. In this report, we aimed to present our experience
with extended half-life recombinant FVIII (EHL-rFVIII)-BAX 855
treatment for circumcision in two severe cases of HA.
The first patient was diagnosed at the age of 3 months with
severe HA (factor VIII = 0.001 IU/mL=0.1%) with no family history.
He started primary prophylaxis twice a week at the age of 17
months; however, his prophylaxis regimen had to be changed to
3 times a week at the age of 5 years old due to frequent bleeding
of the elbows. He was enrolled in an EHL-rFVIII clinical trial at 5.5
years old, and the prophylaxis was continued twice a week for 3
years with no bleeding. The second patient was diagnosed at the
age of 8 months with severe HA (factor VIII=0.003 IU/mL=0.3%);
he had a family history. He began primary prophylaxis twice a
week at the age of 15 months. He was enrolled in an EHL-rFVIII
clinical trial at 5.5 years old, and prophylaxis was continued twice
a week for 3 years with no bleeding. Both patients had no adverse
events and no inhibitors during this period. The two patients
were circumcised at 8 years old in a pediatric urology clinic. Both
patients were hospitalized on the day of circumcision. One patient
had phimosis and was hospitalized for 3 days; the other patient
was hospitalized for 1 day. Both patients were circumcised under
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Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
local anesthesia using a diathermic knife. Hemostasis control was
achieved by tranexamic acid and EHL-rFVIII. Both patients were
under prophylaxis at a dose of 45 IU/kg/day twice a week. The
circumcisions were performed on the prophylaxis day, and 2 extra
EHL-rFVIII doses (50 IU/kg/dose) were used during the prophylaxis
regimen. Factor FVIII level was assessed by chromogenic assay
on the first day of the circumcision. Factor VIII level was under
0.030 IU/mL for both patients at the beginning and 1.252 IU/mL
(125.2%) and 2.180 IU/mL (218%) at 30 min, respectively. Both
patients had regular wound healing. No unexpected bleeding or
wound infections were recorded. They returned to their routine
lives within 7 days.
Circumcision is a cultural and traditional surgical intervention,
and many patients want to be circumcised around the world. In
previously published series, it was reported that circumcision could
be performed with minimal complication rates by using a diathermic
knife. In this routine clinical practice, tranexamic acid and SHL-
FVIII products have been used for hemostasis with decreasing doses
between 4 and 14 days until wound healing occurs, depending
on the severity of hemophilia [6,7]. In another protocol in which
circumcision was performed under general anesthesia, fibrin glue
application with 2-3 days of factor supplementation was found to
be sufficient [8]. As we report here, just two extra doses of EHLrFVIII
were needed on postoperative days 1 and 2 for our patients
who underwent circumcision. Our experiences with these two
patients demonstrate that PEGylated rFVIII is well tolerated and
efficacious for bleeding prophylaxis before circumcision.
There are limited data on EHL-rFVIII products in surgical
interventions in the literature [9,10,11]. The first such prospective
study reported 15 surgical interventions with PEGylated EHLrFVIII
and hemostatic efficacy was excellent for all subjects in
both the intraoperative and perioperative period. Additionally, all
interventions were scored as excellent postoperatively, except for
one dental procedure that was graded as good. In addition, no
related adverse events, including thrombosis and inhibitors, were
recorded [9]. To the best of our knowledge, this is the first report
to indicate two successful circumcision procedures performed
under perioperative and postoperative EHL-rFVIII prophylaxis.
Additionally, successful prophylaxis was achieved with lower
frequency of factor supplementation compared to other series. As
we mentioned above, no adverse events, no thrombotic events, and
no inhibitor development were observed following prophylaxis.
In conclusion, EHL-rFVIII was safe and effective for circumcision
management in cases of severe HA. Decreasing the amount and
frequency of factor support seems to be possible according to
this report.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Keywords: Hemophilia, Surgery, Circumcision, BAX 855
Anahtar Sözcükler: Hemofili, Cerrahi, Sünnet, BAX 855
Informed Consent: Informed constent has been received.
Authorship Contributions
Surgical and Medical Practices: B.Z., B.K., M.İ.Ö.; Concept: B.Z.,
B.K., M.İ.Ö.; Design: B.Z., B.K.; Data Collection or Processing:
B.Z., B.K., M.İ.Ö.; Analysis or Interpretation: B.Z., B.K.; Literature
Search: B.Z., B.K.; Writing: B.Z., B.K.
Conflict of Interest: The authors declare that they have no
conflicts of interest with regard to this research.
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Ingerslev J, Hvid I. Surgery in hemophilia. The general view: patient selection,
timing, and preoperative assessment. Semin Hematol 2006;43:23-26.
2. Srivastava A, Brewer AK, Mauser-Bunschoten EP, Key NS, Kitchen S, Llinas A,
Ludlam CA, Mahlangu JN, Mulder K, Poon MC, Street A; Treatment Guidelines
Working Group on Behalf of the World Federation of Hemophilia. Guidelines
for the management of hemophilia. Haemophilia 2013;19:e1-e47.
3. Konkle BA, Stasyshyn O, Chowdary P, Bevan DH, Mant T, Shima M, Engl W,
Dyck-Jones J, Fuerlinger M, Patrone L, Ewenstein B, Abbuehl B. Pegylated,
full-length, recombinant factor VIII for prophylactic and on-demand
treatment of severe hemophilia A. Blood 2015;126:1078-1085.
4. Rizvi SAH, Naqvi SAA, Hussain M, Hasan AS. Religious circumcision: a Muslim
view. BJU Int 1999;83(Suppl.1):13-16.
5. Kavakli K, Kurugol Z, Goksen D, Nisli G. Should hemophiliac patients be
circumcised? Pediatr Hematol Oncol 2000;17:149-153.
6. Zulfikar B, Karaman MI, Ovali F. Circumcision in Hemophilia. An Overview.
Treatment of Hemophilia. Montreal, World Federation of Hemophilia, 2003.
7. Karaman MI, Zulfikar B, Özturk MI, Koca O, Akyüz M, Bezgal F. Circumcision
in bleeding disorders: improvement of our cost effective method with
diathermic knife. Urol J 2014;11:1406-1410.
8. Avanoglu A, Celik A, Ulman I, Özcan C, Kavaklı K, Nişli G, Gökdemir A. Safer
circumcision in patients with hemophilia: the use of fibrin glue for local
hemostasis. BJU Int 1999;83:91-94.
9. Hampton K, Chowdary P, Dunkley S, Ehrenforth S, Jacobsen L, Neff A,
Santagostino E, Sathar J, Takedani H, Takemoto CM, Négrier C. First report
on the safety and efficacy of an extended half‐life glyco PEG ylated
recombinant FVIII for major surgery in severe haemophilia A. Haemophilia
2017;23:689-696.
10. Mahlangu JN, Ragni M, Gupta N, Rangarajan S, Klamroth R, Oldenburg
J, Nogami K, Young G, Cristiano LM, Dong Y, Allen G, Pierce GF, Robinson
B. Long-acting recombinant factor VIII Fc fusion protein (rFVIIIFc) for
perioperative haemostatic management in severe haemophilia A. Thromb
Haemost 2016;116:1-8.
11. Brand B, Gruppo R, Wynn TT, Griskevicius L, Lopez Fernandez MF, Chapman
M, Dvorak T, Pavlova BG, Abbuehl BE. Efficacy and safety of pegylated fulllength
recombinant factor VIII with extended half‐life for perioperative
haemostasis in haemophilia A patients. Haemophilia 2016;22:e251-e258.
Address for Correspondence/Yazışma Adresi: Basak Koç, M.D., İstanbul University Oncology Institute,
Department of Pediatric Hematology-Oncology, İstanbul, Turkey
Phone : +90 212 414 24 34
E-mail : s_basakkoc@hotmail.com ORCID: orcid.org/0000-0002-0978-7992
Received/Geliş tarihi: August 18, 2019
Accepted/Kabul tarihi: December 11, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0305
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LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
Accidental High-dose Intrathecal Treatment: Late Results of a
Patient
Yanlışlıkla Uygulanan Yüksek Doz Metotreksat: Bir Hastada Geç Sonuçlar
Tiraje Celkan 1 , Evrim Çifçi Sunamak 2
1İstanbul University-Cerrahpaşa Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology Oncology, İstanbul, Turkey
2Dr. Lütfi Kırdar Kartal Training and Research Hospital, Child Health and Diseases, İstanbul, Turkey
To the Editor,
The increase in reporting accidental and overdose use of
childhood treatments, starting from 2014, is promising.
Understanding the dynamics of overdose may help in the
development of more effective prevention and control
strategies [1]. Herein, we want to share one of our patients
with an overdose of intrathecal treatment.
The survival rates of patients with leukemia and lymphoma
have improved after prophylactic intrathecal methotrexate
treatments. There have also been some case reports about
the mortality or morbidity of intrathecal methotrexate or
folinic acid [2,3,4,5,6,7,8,9]. We experienced a nursing error in
which 35 mg of intrathecal methotrexate was administered
instead of the planned 12 mg to a 3-year-old boy with T-cell
non-Hodgkin lymphoma, stage IV. The patient was receiving
the first day of the regimen of the second cytarabine block
of protocol I, phase 2, BFM (Berlin-Frankfurt-Münster). The
error was recognized after about 60 minutes. We performed
neither CSF drainage nor exchange as Kazancı et al. [9]
had done for their two patients. We thought of intrathecal
folinic acid administration, but there were no data about its
intrathecal use. Carboxypeptidase G2 was not available in
our country. We administered 9 mg of folinic acid (15 mg/
m 2 ) intravenously, followed by 100 mg of folinic acid (150
mg/m 2 ) infused in 6 hours. The patient was monitored for
toxic signs and symptoms. He developed no clinical signs.
Cranial computed tomography (CT) performed 2 days after
the incident revealed no morphological changes. He was
followed after this accident for 15 years. Although his
neurological development was normal and his most recent
cranial CT and electroencephalography results revealed no
sequelae, he has had two unsuccessful suicide attempts.
If a readily available source of information regarding the
action needed to be taken after such an incident had
been available, we could have approached the child more
comfortably and confidently, and our colleagues who
used intrathecal folinic acid would not have done so [4].
As methotrexate doses of up to 500 mg have not been
associated with untoward events, deciding when to hold an
intervention is important.
Keywords: Overdose, Intrathecal methotrexate, Folinic acid
Anahtar Sözcükler: Yüksek doz, İntratekal metotreksat, Folinik
asit
Informed Consent: Written informed consent was obtained.
Authorship Contributions
Surgical and Medical Practices: T.C.; Concept: T.C.; Design:
E.Ç.S.; Data Collection or Processing: E.Ç.S., T.C.; Analysis or
Interpretation: T.C.; Literature Search: E.Ç.S.; Writing: T.C.
Conflict of Interest: The authors declare that there is no conflict
of interest.
Financial Disclosure: There is no financial conflict of interest
to declare.
References
1. Jalal H, Buchanich JM, Roberts MS, Balmert LC, Zhang K, Burke DS. Changing
dynamics of the drug overdose epidemic in the United States from 1979
through 2016. Science 2018;361. pii: eaau1184.
2. Addiego JE Jr, Ridgway D, Bleyer WA. The acute management of intrathecal
methotrexate overdose: pharmacologic rationale and guidelines. J Pediatr
1981;98:825-828.
3. Riva L, Conter V, Rizzari C, Jankovic M, Sala A, Milani M. Successful
treatment of intrathecal methotrexate overdose with folinic acid rescue: a
case report. Acta Paediatr 1999;88:780-782.
4. Jardine LF, Ingram LC, Bleyer WA. Intrathecal leucovorin after intrathecal
methotrexate overdose. J Pediatr Hematol Oncol 1996;18:302-304.
5. Widemann BC, Balis FM, Shalabi A, Boron M, O’Brien M, Cole DE,
Jayaprakash N, Ivy P, Castle V, Muraszko K, Moertel CL, Trueworthy
R, Hermann RC, Moussa A, Hinton S, Reaman G, Poplack D, Adamson
PC. Treatment of accidental intrathecal methotrexate overdose with
intrathecal carboxypeptidase G2. J Natl Cancer Inst 2004;96:1557-
1559.
6. Jakobson AM, Kreuger A, Mortimer O, Henningsson S, Seidel H, Moe PJ.
Cerebrospinal fluid exchange after intrathecal methotrexate overdose. A
report of two cases. Acta Paediatr 1992;81:359-361.
7. Spiegel RJ, Cooper PR, Blum RH, Speyer JL, McBride D, Mangiardi J. Treatment
of massive intrathecal methotrexate overdose by ventriculolumbar
perfusion. N Engl J Med 1984;311:386-368.
64
Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
8. Bradley AM, Buie LW, Kuykendal A, Vorhees PM. Successful use of intrathecal
carboxypeptidase G2 for intrathecal methotrexate overdose: a case study
and review of the literature. Clin Lymphoma Myeloma Leuk 2013;13:166-
170.
9. Kazancı E, Gülen H, Erbay A, Vergin C. Treatment of intrathecal methotrexate
overdose with folinic acid rescue and lumbar cerebrospinal fluid exchange:
a report of two cases. Turk J Hematol 2011;28:63-67.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Evrim Çifçi Sunamak, M.D., Dr. Lütfi Kırdar Kartal Training and
Research Hospital, Child Health and Diseases, İstanbul, Turkey
Phone : +90 216 458 30 00
E-mail : evrimcifci@gmail.com ORCID: orcid.org/0000-0003-2952-3094
Received/Geliş tarihi: July 25, 2019
Accepted/Kabul tarihi: September 16, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0283
CMV-specific T-Cells for Treatment of CMV Infection after
Hematopoietic Stem Cell Transplantation in a Pediatric Case: First
Application in Turkey
Pediatrik Bir Olguda HKHN Sonrası CMV Spesifik T Hücre Kullanımı: Türkiye’deki İlk
Uygulama
Sevil Celilova 1 , Ersin Toret 1 , Başak Aksoy Adaklı 1 , Ercüment Ovalı 2 , Ceyhun Bozkurt 3
1Altınbaş University Faculty of Medicine, Medicalpark Bahçelievler Hospital, Department of Pediatric Hematology-Oncology & Bone Marrow
Transplantation Unit, İstanbul, Turkey
2Acıbadem University Faculty of Medicine, Altunizade Hospital, Department of Hematology, İstanbul, Turkey
3İstinye University Faculty of Medicine, Medicalpark Bahcelievler Hospital, Department of Pediatric Hematology-Oncology & Bone Marrow
Transplantation Unit, İstanbul, Turkey
To the Editor,
Cytomegalovirus (CMV) infection is still a major complication after
allogeneic hematopoietic stem cell transplantation (HSCT) [1,2].
Unfortunately, prolonged antiviral treatment of CMV infection
causes a delayed CMV-specific immune reconstitution. At this
point, adoptive immunotherapy by CMV-specific T-cells can
control CMV infection or provide immune reconstruction [3,4,5].
A 17-year-old boy with high-risk T-cell acute lymphoblastic
leukemia underwent HSCT from one antigen-mismatched
unrelated donor. He was conditioned with treosulfan,
fludarabine, thiotepa, and rabbit anti-thymocyte globulin
at 15 g/m 2 for 3 consecutive days (days -2 to 0). The patient
also received cyclosporine A (CsA) divided into two doses: 3
mg/kg daily from day -1 to post-transplant days +20 and +30
intravenously then switched to approximately 6 mg/kg peroral
daily (targeted blood concentration: 200-250 ng/mL with
monitoring). CsA was tapered quickly and stopped in the third
month of transplant due to renal failure. Methotrexate was
administered on days +1 (10 mg/m 2 ), +3 (8 mg/m 2 ), and +6 (8
mg/m 2 ). He achieved neutrophil engraftment on day +17 and
thrombocyte engraftment on day +32. Full donor chimerism was
observed in the first and third months. Lymphoid engraftment
was achieved on day +75 but generally the absolute lymphocyte
count was under 1500/mm 3 . He was CMV immunoglobulin G
(IgG)-seropositive and CMV-DNA polymerase chain reaction
(PCR) was negative before transplantation. Unfortunately, his
donor was CMV IgG-seronegative. CMV infection (reactivation)
occurred on day +19. Ganciclovir was started at 10 mg/kg/day
and no response was obtained in 14 days. CMV drug resistance
mutation was detected in the UL54 polymerase gene. Foscarnet
was administered at 180 mg/kg/day on day +34. First, an
increase of CD3+ lymphocytes was seen in the lymphocyte
subtype analyses around the third month after the transplant.
As a comorbidity, in spite of the fact that fluoroquinolone was
administered until +30 day, BK virus infection developed in
the patient and cidofovir was used at 5 mg/kg/week on days
+52, +67, and +79. No response was achieved with the antiviral
treatment and renal failure developed in the patient on day +82.
All antivirals were stopped. According to the recent literature,
the transplant council decided to use CMV-specific T-cells for
the patient’s ongoing CMV infection. Informed consent was
received from his family and the application was approved
by the Ministry of Health’s Scientific Advisory Commission on
Stem Cell Transplantation. In accordance with cGMP standards,
peptide-specific T lymphocytes were isolated and amplified by a
interferon-γ cytokine capture system using the fully automated
CliniMACS Prodigy device at Acıbadem Labcell, İstanbul. The
65
LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
Figure 1. Treatment process according to the course of CMV DNA titer (copies/mL).
CMV: Cytomegalovirus.
infusion doses of third-party CMV-specific T-cells were 2x10 4
cell/kg and 1x10 4 cell/kg in the 20 th and 22 nd weeks after
transplantation, respectively. While the recommended dose of
T-cells was 2x10 6 /m 2 [6], we reduced the dose due to the risk of
graft-versus-host disease (GvHD). The CMV-DNA PCR level was
higher than 1x10 5 copies/mL before infusion and had decreased
to 8x10 4 copies/mL on the 15 th day after infusion. The patient
had no immunosuppression at the time of T-cell infusion and did
not develop GvHD after the infusion. In follow-up, CMV-DNA
PCR increased to more than 3.5x10 5 copies/mL in the first month
of the cell infusion and the sixth month after transplantation.
In this period, CD3-CD16+56+ (natural killer) and CD3+CD8+
(T cytotoxic) lymphocyte subtypes were increased. Nevertheless,
the patient developed respiratory distress and CMV infection
was detected from the bronchoalveolar lavage sample, and
the CMV DNA titer was 152,000 copies/mL. After losing partial
response to CMV-specific T-cells, CMV pneumonia was proved
and then leflunomide was tested, but there was no response.
Finally, CMV-specific IgG was administered once weekly three
times. This treatment managed to decrease the CMV DNA copies
to under 20,000 copies/mL. The treatment process according to
the course of CMV DNA titer is shown in Figure 1.
CMV reactivations/infections are life-threatening complications
in the transplant setting, especially if the recipient and donor
are CMV mismatches. From our experience with this case, CMVspecific
T-cells can control viral replication to a certain extent,
but not enough for permanent results. The answer may be CMVspecific
IgG, which controlled CMV reactivation best in our case,
and antivirals may be used in combination.
Keywords: Childhood, Hematopoietic stem cell transplant, CMV,
Specific T-cell, Therapy
Anahtar Sözcükler: Çocukluk çağı, Hematopoetik kök hücre
nakli, CMV, Spesifik T hücre, Tedavi
Informed Consent: Informed consent was received from the family.
Authorship Contributions
Surgical and Medical Practices: S.C., E.T, B.A.A., E.O., C.B.; Design:
E.T., C.B.; Data collection or Processing: S.C., E.T, B.A.A.; Analyses
or Interpretration: E.O., C.B.; Literature Search: E.T., B.A.A. ;
Writing: E.T.
Conflict of Interest: No conflict of interest was declared by the
authors.
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Einsele H, Roosnek E, Rufer N, Sinzger C, Riegler S, Löffler J, Grigoleit U, Moris
A, Rammensee HG, Kanz L, Kleihauer A, Frank F, Jahn G, Hebart H. Infusion
of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection
not responding to antiviral chemotherapy. Blood 2002;99:3916-3922.
2. Espigado I, de la Cruz-Vicente F, BenMarzouk-Hidalgo OJ, Gracia-Ahufinger
I, Garcia-Lozano JR, Aguilar-Guisado M, Cisneros JM, Urbano-Ispizua A,
Perez-Romero P. Timing of CMV-specific effector memory T cells predicts
viral replication and survival after allogeneic hematopoietic stem cell
transplantation. Transpl Int 2014;27:1253-1262.
3. Boeckh M, Nichols WG, Papanicolaou G, Rubin R, Wingard JR, Zaia J.
Cytomegalovirus in hematopoietic stem cell transplant recipients: current
status, known challenges, and future strategies. Biol Blood Marrow
Transplant 2003;9:543-558.
4. Scheinberg P, Melenhorst JJ, Brenchley JM, Hill BJ, Hensel NF, Chattopadhyay
PK, Roederer M, Picker LJ, Price DA, Barrett AJ, Douek DC. The transfer of
adaptive immunity to CMV during hematopoietic stem cell transplantation
is dependent on the specificity and phenotype of CMV-specific T cells in the
donor. Blood 2009;114:5071-5080.
66
Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
5. Poiret T, Axelsson-Robertson R, Remberger M, Luo XH, Rao M, Nagchowdhury
A, Von Landenberg A, Ernberg I, Ringden O, Maeurer M. Cytomegalovirusspecific
CD8+ T-cells with different T-cell receptor affinities segregate T-cell
phenotypes and correlate with chronic graft-versus-host disease in patients
post-hematopoietic stem cell transplantation. Front Immunol 2018;9:760.
HE, Leen AM, Omer B. Off-the-shelf virus-specific T cells to treat BK virus,
human herpesvirus 6, cytomegalovirus, Epstein-Barr virus, and adenovirus
infections after allogeneic hematopoietic stem-cell transplantation. J Clin
Oncol 2017;35:3547-3557.
6. Tzannou I, Papadopoulou A, Naik S, Leung K, Martinez CA, Ramos CA,
Carrum G, Sasa G, Lulla P, Watanabe A, Kuvalekar M, Gee AP, Wu MF, Liu
H, Grilley BJ, Krance RA, Gottschalk S, Brenner MK, Rooney CM, Heslop
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Ersin Töret, M.D., Altınbaş University Faculty of Medicine,
Medicalpark Bahçelievler Hospital, Department of Pediatric Hematology-Oncology & Bone Marrow
Transplantation Unit, İstanbul, Turkey
Phone : +90 505 799 42 34
E-mail : drersintoret@hotmail.com ORCID: orcid.org/0000-0002-6379-8326
Received/Geliş tarihi: August 6, 2019
Accepted/Kabul tarihi: November 12, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0293
Comparison of Different Culture Conditions for Mesenchymal
Stem Cells from Human Umbilical Cord Wharton’s Jelly for Stem
Cell Therapy
Kök Hücre Tedavisi için İnsan Kordon Kanı Wharton Jel’inden Üretilen Mezenkimal Kök
Hücreler için Farklı Kültür Ortamlarının Karşılaştırılması
Yu Bao 1 , Shumin Huang 2 , Zhengyan Zhao 2
1Zhejiang University Faculty of Medicine, Children’s Hospital, Department of Nephrology, Zhejiang, China
2Zhejiang University Faculty of Medicine, Children’s Hospital, Clinic of Division of Child Health Care, Zhejiang, China
To the Editor,
Many recent studies have demonstrated that the umbilical cord
is an excellent source of mesenchymal stem cells (MSCs) [1,2,3].
However, in order to use human umbilical cord Wharton’s jellyderived
mesenchymal stem cells (hUC-MSCs) in clinical therapy,
a suitable culture procedure for good manufacturing practicecompliant
production is mandatory. Nutritional deficiency
is the major pathophysiological situation in an ischemic
microenvironment in the clinic [4]. Thus, the development of
serum-free culture systems is needed [5]. Furthermore, hypoxia
is common in vivo in mammals [6]. The average oxygen tension
falls to 1% in some cases of pathological ischemia, including
fracture hematoma, and in cases of myocardial ischemia [7].
Hence, the investigation of biological characteristics of hUC-
MSCs exposed to hypoxic and/or serum-free conditions is of
great interest.
In our study, we conducted parallel assays by using four cell
groups. For the hypoxic controls, cells from group A (n=10)
and group B (n=10) were exposed to 5% CO 2
and 94% N 2
in
an airtight modular incubator chamber (Billups-Rothenberg
Inc., Del Mar, CA, USA). The final oxygen tension was 1%-3% as
measured by an oximeter (Oxybaby M+, Witt Technology, Solza,
Italy). For the normoxic controls, cells from group C (n=10) and
group D (n=10) were placed in an incubator at 37 °C, 5% CO 2
,
and 21% O 2
. Cells from group A and group C were expanded in
a mixture of Dulbecco’s modified Eagle’s medium and nutrient
mixture F-12 (GIBCO, USA) supplemented with 10% fetal bovine
serum (GIBCO, USA). Cells from group B and group D were
expanded in StemPRO MSC serum-free medium (StemRD, USA).
Flow cytometric analysis, differentiation potential, proliferative
activities, cell cycle analysis, and apoptosis analysis of these
four cell populations were evaluated. We repeated all these
experiments 3 times.
Flow cytometry analysis of MSC-specific surface marker
expression showed that hUC-MSCs cultured under four
experimental conditions for six passages were positive
for CD44, CD73, CD90, CD105, CD29, and HLA-ABC (BD
Pharmingen, USA) and negative for CD34, CD45, CD14, and
HLA-DR (BD Pharmingen, USA); no significant differences
were detected between the four cell populations (Figure
1). This finding indicates that culturing cells under hypoxic
and/or serum-free conditions did not induce significant
variations in the typical MSC marker expression profile. hUC-
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Turk J Hematol 2020;37:57-76
MSCs maintained their multilineage differentiation potential
in vitro after expansion under various conditions [8]. MSCs
from all groups demonstrated a similar osteogenic phenotype,
as evidenced by positive staining for alizarin red S (Sigma-
Aldrich, USA) and deposits of calcified matrix (Figure 2). In
the case of adipogenic differentiation, cells from all groups
formed lipid vacuoles detected by oil red O (Sigma-Aldrich,
USA). There were no significant quantitative changes among
the groups (Figure 3). Thus, the results presented in this
report indicate that hypoxic and/or serum-free conditions
do not affect the biological characteristics of hUC-MSCs.
Under hypoxic and serum-free conditions, hUC-MSCs have
higher proliferation according to their growth curves (BD
Pharmingen, USA) and MTT assays (BD Pharmingen, USA) than
cells grown under normoxic and serum-containing culture
conditions, but without more apoptosis (Figures 4 and 5).
Figure 1. Flow cytometry of hUC-MSC samples [mean percentage
± SD (%)]. 1 and 2: Passage 3; 3 and 4: Passage 6.
SD: Standard deviation, hUC-MSC: Human umbilical cord Wharton’s
jelly-derived mesenchymal stem cells.
Figure 3. Multilineage differentiation potential of hUC-MSCs.
Adipogenesis was confirmed by neutral oil droplet formation
stained with oil red O. 1: Group A; 2: Group B; 3: Group C; 4:
Group D (original magnification: 100x, bar: 50 µm).
hUC-MSC: Human umbilical cord Wharton’s jelly-derived mesenchymal
stem cells.
Figure 2. Multilineage differentiation potential of hUC-MSCs.
Formation of mineralized matrix was detected by alizarin red S
staining. 1: Group A; 2: Group B; 3: Group C; 4: Group D (original
magnification: 200 x , bar: 50 µm).
hUC-MSC: Human umbilical cord Wharton’s jelly-derived mesenchymal
stem cells.
Figure 4. Expansion ability of the four groups. 1: Growth curve at
passage 3. 2: MTT assay at passage 3. 3: Cell cycle distribution at
passage 3 [mean percentage ± SD (%)]. 4: Cell cycle distribution
at passage 6 [mean percentage ± SD (%)].
SD: Standard deviation.
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Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
Authorship Contributions
Data collection or Processing: Y.B., S.H., Z.Z.; Analyses or
Interpretration: Y.B., S.H., Z.Z.; Literature Search: Y.B., S.H., Z.Z.;
Writing: Y.B., S.H., Z.Z.
Conflict of Interest: The authors of this paper have no conflicts
of interest, including specific financial interests, relationships,
and/or affiliations relevant to the subject matter or materials
included.
Financial Disclosure: The authors declared that this study
received no financial support.
Figure 5. Proportion of apoptotic hUC-MSCs cultured under
different conditions (mean ± SD).
SD: Standard deviation, hUC-MSC: Human umbilical cord Wharton’s
jelly-derived mesenchymal stem cells.
Taken together, our data indicate that hypoxic and serum-free
culture conditions do not influence the major properties of
hUC-MSCs. Under hypoxic and serum-free conditions, hUC-
MSCs showed higher proliferation, while their apoptosis rate
did not increase. This finding is consistent with that of previous
reports, which demonstrated enhanced proliferation of bone
marrow-derived MSCs (BM-MSCs) under hypoxic or serumfree
conditions [9,10]. Therefore, the availability of optimized
in vitro conditions, including hypoxia and serum-free media,
for hUC-MSC manipulations may have a substantial scientific
and clinical impact.
Funding: This work was funded by the Zhejiang Provincial
Natural Science Foundation of China (Grant No. LY18H070001).
Ethics Committee Approval: All procedures performed in
studies involving human participants were in accordance with
the ethical standards of the Medical Ethics Committee of the
Children’s Hospital School of Medicine, Zhejiang University
(reference number 1712048) and with the 1964 Helsinki
Declaration and its later amendments or comparable ethical
standards.
Informed Consent: Informed consent was obtained from all
individual participants included in the study.
References
1. Jin HJ, Bae YK, Kim M, Kwon SJ, Jeon HB, Choi SJ, Kim SW, Yang YS, Oh W,
Chang JW. Comparative analysis of human mesenchymal stem cells from
bone marrow, adipose tissue, and umbilical cord blood as sources of cell
therapy. Int J Mol Sci 2013;14:17986-8001.
2. Mahla RS. Stem cells applications in regenerative medicine and disease
therapeutics. Int J Cell Biol 2016;2016:6940283.
3. Chen S, Zhang W, Wang JM, Duan HT, Kong JH, Wang YX, Dong M, Bi X,
Song J. Differentiation of isolated human umbilical cord mesenchymal stem
cells into neural stem cells. Int J Ophthalmol 2016;9:41-47.
4. Lu HH, Li YF, Sheng ZQ, Wang Y. Preconditioning of stem cells for the
treatment of myocardial infarction. Chin Med J (Engl) 2012;125:378-384.
5. Chang Z, Hou T, Xing J, Wu X, Jin H, Li Z, Deng M, Xie Z, Xu J. Umbilical
cord Wharton’s jelly repeated culture system: a new device and method for
obtaining abundant mesenchymal stem cells for bone tissue engineering.
PLoS One 2014;9:e110764.
6. Swamynathan P, Venugopal P, Kannan S, Thej C, Kolkundar U, Bhagwat S, Ta
M, Majumdar AS, Balasubramanian S. Are serum-free and xeno-free culture
conditions ideal for large scale clinical grade expansion of Wharton’s jelly
derived mesenchymal stem cells? A comparative study. Stem Cell Res Ther
2014;5:88.
7. Huang Y, Chen B, Zhang J. Oxygen tension variation in ischemic
gastrocnemius muscle, marrow, and different hypoxic conditions in vitro.
Med Sci Monit 2014;20:2171-2176.
8. Li HZ, Chen Z, Hou CL, Tang YX, Wang F, Fu QG. Uric acid promotes osteogenic
differentiation and inhibits adipogenic differentiation of human bone
mesenchymal stem cells. J Biochem Mol Toxicol 2015;29:382-387.
9. Mohd Ali N, Boo L, Yeap SK, Ky H, Satharasinghe DA, Liew WC, Ong HK,
Cheong SK, Kamarul T. Probable impact of age and hypoxia on proliferation
and microRNA expression profile of bone marrow-derived human
mesenchymal stem cells. Peer J 2016;4:e1536.
10. Gottipamula S, Ashwin KM, Muttigi MS, Kannan S, Kolkundkar U,
Seetharam RN. Isolation, expansion and characterization of bone marrowderived
mesenchymal stromal cells in serum-free conditions. Cell Tissue Res
2014;356:123-135.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Zhengyan Zhao, Ph. M.D., Zhejiang University Faculty of
Medicine, Children’s Hospital, Clinic of Division of Child Health Care, Zhejiang, China
Phone : +8613805722351
E-mail : zhaozy@zju.edu.cn ORCID: orcid.org/0000-0002-5741-2431
Received/Geliş tarihi: December 23, 2018
Accepted/Kabul tarihi: November 12, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0439
69
LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
Vacuolated Blasts in the Bone Marrow of a Child with
Rhabdomyosarcoma
Rabdomyosarkomlu Çocukta Kemik İliğinde Vakuollü Blastlar
Eda Ataseven 1 , Dilek Ece 2 , Nazan Özsan 3 , Mehmet Kantar 4
1Ege University Faculty of Medicine, Department of Pediatric Hematology and Oncology, İzmir, Turkey
2Ege University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology and Oncology, İzmir, Turkey
3Ege University Faculty of Medicine, Department of Pathology, İzmir, Turkey
4Ege University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Oncology, İzmir, Turkey
To the Editor,
Rhabdomyosarcoma (RMS) is the most common soft tissue
sarcoma in children. The most common locations are the head/
neck region and genitourinary tract. Leukemic presentation
of RMS with diffuse bone marrow involvement and unknown
primary mass is very rare [1]. Most of the time it can be
misdiagnosed as acute leukemia.
A 3-year-old female patient was admitted to the hospital with
right arm pain, a limp while walking, and abdominal pain. From
her medical history we learned that one month earlier she was
diagnosed with a humerus fracture in the orthopedics clinic,
and despite fixation her pain had gradually increased and
disseminated. Magnetic resonance imaging of the humerus had
revealed diffuse bone marrow edema and pathological lymph
node enlargement in the axillary region. She was referred to our
clinic with a suspicion of hematologic malignancy.
RMS rarely involves the bone marrow. Blastic cells in RMS
resemble lymphoid blasts and it can mimic acute leukemia or
Burkitt’s lymphoma with marrow involvement, especially if
there are prominent vacuolizations, as in our case [1,2]. In the
literature, all patients presenting with bone marrow involvement
were diagnosed with alveolar type RMS [3]. We could not
distinguish the type of RMS in our patient. RMS should be kept
in mind in the differential diagnosis of patients, especially if
there are clustering blastic cells, multinucleated giant cells with
deep blue cytoplasm, and prominent vacuolization [4].
Upon physical examination, she had local swelling and pain
in the right arm. There was no organomegaly or pathological
lymph node enlargement on palpation. Laboratory examination
revealed hemoglobin level of 10.3 g/dL, white blood cell count
of 8.8x10 9 /L, and platelet count of 218x10 9 /L. Examination
of the peripheral blood smear was normal. Biochemical test
results were normal, except elevated lactate dehydrogenase and
uric acid levels. Abdominal ultrasonography was normal. We
performed bone marrow aspiration and biopsy with a suspicion
of leukemia.
The bone marrow aspiration smear showed immature cells
with disseminated intranuclear/intracytoplasmic vacuolization
(Figure 1). In the bone marrow biopsy, diffuse blastic infiltration
was noticed. Blastic cells were positive for myogenin and
desmin staining. The diagnosis was RMS metastasis in the
bone marrow. Work-up of the primary site of the disease was
performed. Abdominal computed tomography showed a huge
mass in the left pararectal fossa and multiple bone metastases
(Figure 2).
Figure 1. Bone marrow aspiration smear showed immature cells
with disseminated intranuclear/intracytoplasmic vacuolization.
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Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
Informed Consent: Informed consent form was received from
the family/patient to use medical records of the patient.
Authorship Contributions
Data collection or Processing: E.A., D.E., N.Ö., M.K.; Analyses or
Interpretration: E.A., D.E., N.Ö., M.K.; Literature Search: E.A., D.E.,
N.Ö., M.K.
Conflict of Interest: No author of this paper has a conflict of
interest, including specific financial interests, relationships, or
affiliations relevant to the subject matter or materials included
in this manuscript.
Financial Disclosure: The authors declared that this study
received no financial support.
Figure 2. Abdominal CT showed a huge mass in pararectal fossa.
CT: Computed tomography.
Keywords: Rhabdomyosarcoma, Vacuolated blasts, Bone marrow
involvement
Anahtar Sözcükler: Rabdomyosarkom, Vakuollü blast, Kemik
iliği tutulumu
References
1. Imataki O, Uemura M, Uchida S, Yokokura S, Takeuchi A, Ishikawa R, Kondo A,
Seo K, Kadowaki N. Complete mimicry: a case of alveolar rhabdomyosarcoma
masquerading as acute leukemia. Diagn Pathol 2017;12:77:1-7.
2. Naithani R, Kumar R, Mahapatra M, Agrawal N, Saxena R, Sharma S. Pelvic
alveolar rhabdomyosarcoma with bone marrow involvement misdiagnosed
as acute myeloid leukemia. Pediatr Hematol Oncol 2007;24:153-155.
3. Stall JN, Bailey NG. Metastatic alveolar rhabdomyosarcoma to the bone
marrow mimicking acute leukemia. Blood 2012;120:3632.
4. Patiroglu T, Isik B, Unal E, Canoz O, Deniz K, Kosemehmetoglu K, Karakukcu
M, Ozdemir MA. Cranial metastatic alveolar rhabdomyosarcoma mimicking
hematological malignancy in an adolescent boy. Childs Nerv Syst
2014;30:1737-1741.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Eda Ataseven, M.D., Ege University Faculty of Medicine,
Department of Pediatric Hematology and Oncology, İzmir, Turkey
Phone : +90 505 687 96 25
E-mail : edataseven@yahoo.com ORCID: orcid.org/0000-0003-3419-5814
Received/Geliş tarihi: August 28, 2019
Accepted/Kabul tarihi: November 27, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0324
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LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
T-Cell Lymphoblastic Lymphoma Showing Aberrant Synaptophysin
Expression in a Child
Çocuk Hastada Aberan Sinaptofizin Ekspresyonu Gösteren T Hücreli Lenfoblastik Lenfoma
Nazım Emrah Koçer, Bermal Hasbay, Fazilet Kayaselçuk
Başkent University Faculty of Medicine, Adana Dr. Turgut Noyan Research and Application Center Department of Pathology, Adana, Turkey
To the Editor,
The term “malignant small round cell tumor” represents a highly
aggressive group of tumors comprising small, monotonous,
relatively undifferentiated cells with high nucleus/cytoplasmic
ratios [1]. Despite histopathologic similarities, the treatment
modalities of each tumor in this generic group are different
than the others and differential diagnosis is crucial.
Histopathologic examination generally needs to be supported
by ancillary techniques, most commonly and practically by
immunohistochemistry, for the correct differential diagnosis.
Immunohistochemically aberrant expression of an antigen in a
small round cell tumor may cause a diagnostic dilemma.
Herein we report a case of T lymphoblastic lymphoma in a
9-year-old child showing aberrant synaptophysin expression.
A 9-year-old female patient was admitted to the hospital
with cough, dyspnea, and a mass in the left thoracic region.
Computerized tomography revealed a mass in the anterior
mediastinum; right paratracheal, hilar, and subcarinal
lymphadenomegalies; and severe left pleural effusion. Scanning
results of other regions were within normal limits. A Tru-Cut
biopsy was taken from the mediastinal mass. Histopathological
examination revealed a diffuse infiltration composed of
small cells with hyperchromatic nuclei and scant cytoplasm,
showing monomorphic atypia. In immunohistochemical study
the tumor cells were positive for LCA, CD2, CD3, CD10, CD1a,
TdT, and synaptophysin (Figure 1), while CD20, pancytokeratin,
CK-19, chromogranin A, and CD56 were negative. The Ki-67
proliferation index was higher than 90%. There was no bone
marrow infiltration.
The patient was diagnosed with T-cell lymphoblastic lymphoma.
Following adequate treatment, the patient is doing well in her
third year after the diagnosis.
Small round cell tumors most commonly affect the pediatric age
group and represent a variety of tumors, including non-Hodgkin
lymphomas, Ewing/primitive neuroectodermal tumor (PNET),
neuroblastoma, small cell osteosarcoma, rhabdomyosarcoma,
synovial sarcoma, hepatoblastoma, nephroblastoma, and
retinoblastoma [1]. Although each tumor needs different
management strategies and therapeutic modalities, due to the
Figure 1. Microphotographs reveal: A) diffuse infiltration of small cells with hyperchromatic nuclei, scant cytoplasm, and monomorphic
atypia (H&E, 400 x ); B) strong membranous immunohistochemical CD2 staining (CD2, 400 x ), C) diffuse nuclear TdT reactivity (TdT, 400 x );
and D) immunohistochemical synaptophysin expression (synaptophysin, 400 x ).
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Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
histopathologic similarities and frequently overlapping clinical
presentations, differential diagnosis can be challenging.
Histopathologic differential diagnosis requires a careful
assessment of slides for specific morphological features of each
tumor and the evaluation of an adequate immunohistochemical
spectrum by using thoroughly optimized antibodies. Aberrant
expression of an antigen may lead to misdiagnosis in
cases in which well-established diagnostic flow charts for
immunohistochemical profiles are not followed.
Expression of a neuroendocrine marker in lymphoma is
extremely rare. To our knowledge, there are only two cases of
synaptophysin expression reported in lymphoblastic lymphoma.
The first case was reported by Alvaro et al. [2], in a 6-yearold
girl, and the second case was reported by Patel et al. [3],
in a 42-year-old male patient. Both of these patients also had
jaundice in common as a presentation symptom. In our patient
the disease was limited to the mediastinum and there was no
sign of jaundice. The favorable prognosis suggests that aberrant
expression of synaptophysin had no prognostic impact in our
patient.
The most common synaptophysin-positive small round cell
tumors are PNET and neuroblastoma in childhood [1]. A
proper immunohistochemical panel covering major entities
in differential diagnosis and enabling cross-checks between
antigen expressions, combined with careful histopathologic
assessment, can prevent a misdiagnosis.
Keywords: Pediatric leukemias, Pediatric lymphomas, Acute
lymphoblastic leukemias
Anahtar Sözcükler: Pediyatrik lösemiler, Pediyatrik lenfomalar,
Akut lenfoblastik lösemiler
Informed Consent: The case is archival material of the Başkent
University Adana Dr. Turgut Noyan Research and Medical Center
and is presented as archival material in a retrospective manner.
The manuscript does not contain any personal data (e.g.,
photograph, radiological image, name) revealing the patient’s
identity. Thus, the patient is completely anonymous and the
manuscript ethically and legally does not require the patient’s
consent.
Authorship Contributions
Surgical and Medical Practices: N.E.K., B.H., F.K.; Concept: N.E.K.,
B.H.; Design: N.E.K.; Data Collection or Processing: : N.E.K.,
B.H., F.K.; Analysis or Interpretation: N.E.K., B.H., F.K.; Literature
Search: N.E.K., B.H.; Writing: N.E.K., B.H., F.K.
Conflict of Interest: The authors declare no conflict of interest.
Financial Disclosure: No funding from any source was used. The
authors declare no financial interest or support.
References
1. D’cruze L, Dutta R, Rao S, R A, Varadarajan S, Kuruvilla S. The role of
immunohistochemistry in the analysis of the spectrum of small round cell
tumours at a tertiary care centre. J Clin Diagn Res 2013;7:1377-1382.
2. Alvaro F, Jain M, Morris LL, Rice MS. Childhood acute lymphoblastic
leukaemia presenting as jaundice. J Paediatr Child Health 1996;32:466-468.
3. Patel KJ, Latif SU, de Calaca WM. An unusual presentation of precursor
T cell lymphoblastic leukemia/lymphoma with cholestatic jaundice: case
report. J Hematol Oncol 2009;2:12.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Nazım Emrah Koçer, M.D., Başkent University Faculty of
Medicine, Adana Dr. Turgut Noyan Research and Application Center Department of Pathology, Adana, Turkey
Phone : +90 505 273 05 78
E-mail : nekocer@baskent.edu.tr ORCID: orcid.org/0000-0002-5943-9283
Received/Geliş tarihi: August 18, 2019
Accepted/Kabul tarihi: December 18, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0307
73
LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
Hydroxyurea-induced Tooth Discoloration
Hidroksiürenin Neden Olduğu Dişte Renk Değişikliği
Muhammed Okuyucu 1 , Memiş Hilmi Atay 2
1Ondokuz Mayıs University Faculty of Medicine, Department of Internal Medicine, Samsun, Turkey
2Ondokuz Mayıs University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Samsun, Turkey
To the Editor,
Hydroxyurea is an antineoplastic agent that inhibits DNA
synthesis by inhibiting the enzyme ribonucleotide reductase
that catalyzes the conversion of ribonucleotide diphosphate
to deoxyribonucleotide triphosphate, which is used for
DNA synthesis and repair. It is often used for the treatment
of myeloproliferative disorders [1]. Most commonly, fever,
pneumonitis, and dermatological (eczema, ulceration),
gastrointestinal (mucositis), and hematological adverse effects
are reported in patients receiving hydroxyurea. Oral lesions and
brown staining of the fingers and toes has been reported rarely
[2,3,4].
An 82-year-old male patient was referred to the hematology
clinic with an elevated platelet count. Laboratory investigations
revealed a white blood cell count of 17,000/mm 3 (reference
range: 3,900-10,900/mm 3 ), hemoglobin of 17.5 g/dL (13.5-
16.9 g/dL), hematocrit of 54% (40%-49%), and platelet
count of 1,200,000/µL (166,000-308,000/µL). Upon physical
examination, the spleen was palpable (3 cm below the costal
margin). A peripheral smear showed a tear drop cell and giant
platelets. Grade 3 reticulin fibrosis was observed upon bone
marrow examination. With the diagnosis of myelofibrosis and
hydroxyurea (1.5 g/day), therapy was initiated. Upon follow-up
after 3 months of treatment, black discoloration of the teeth
was noted in the patient (Figure 1). The black pigmentation
was absent on his fingernails and toenails and he did not have
a history of smoking, nor had he started a new medication.
The patient was examined by a dentist, who thought that
the discoloration might have been caused by the drug. Since
the patient had a high risk of thrombosis (older age, rising
white blood cell and platelet counts), hydroxyurea was not
stopped. Because the tooth discoloration was not a lifethreatening
situation, the hydroxyurea dose was not modified.
The patient was referred to the dentist for dental cleaning. We
concluded that the black discoloration of the teeth associated
with hydroxyurea was a rare and unprecedented clinical
presentation.
Drug-induced tooth discoloration is categorized into two types:
extrinsic and intrinsic. The extrinsic type involves staining of the
outer surface of the tooth. Extrinsic factors include smoking,
excessive consumption of tea and coffee, and medications.
Solutions and antimicrobial preparations (amoxicillin-clavulanic
acid, ciprofloxacin, linezolid) containing iron have been most
prevalently associated with extrinsic discoloration of the teeth.
In intrinsic discoloration, stains are deposited within the enamel
of dentin during the development of tooth (e.g., tetracycline
stains). In extrinsic discoloration, stains are removed by dental
scaling and polishing [5]. It has been known that cutaneous
toxicities generally develop after prolonged hydroxyurea usage.
However, some publications in the literature reported that
cutaneous toxicity can occur within 3 months [6]. In this case,
we thought that with increasing age, erosion-related thinning
of the outer layer of the tooth and poor oral hygiene could have
caused discoloration of the tooth earlier.
In conclusion, this is the first case of tooth discoloration
associated with chronic use of hydroxyurea to be reported in
the literature. Clinicians should be aware of this uncommon
adverse effect of hydroxyurea.
Keywords: Hydroxyurea, Tooth discoloration, Myeloproliferative
disorders
Anahtar Sözcükler: Hidroksiüre, Dişte renk değişikliği,
Myeloproliferatif hastalıklar
Informed Consent: A consent form was completed by all
participants.
Authorship Contributions
Figure 1. Hydroxyurea induced tooth discoloration in the patient.
Data Collection or Processing: M.O., M.H.A; Analysis or
Interpretation: M.O., M.H.A; Literature Search: M.O., M.H.A;
Writing: M.O.
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Turk J Hematol 2020;37:57-76
LETTERS TO THE EDITOR
Conflict of Interest: The authors of this paper have no conflicts
of interest, including specific financial interests, relationships,
and/or affiliations relevant to the subject matter or materials
included.
Financial Disclosure: The authors declared that this study
received no financial support.
References
1. Singh A, Yong-Jie X. The cell killing mechanisms of hydroxyurea. Genes
2016;7:99-102.
2. Aste N, Fumo G, Contu F, Aste N, Biggio P. Nail pigmentation caused by
hydroxyurea: report of 9 cases. J Am Acad Dermatol 2002;47:146-147.
3. Antonioli E, Guglielmelli P, Pieri L, Finazzi M, Rumi E, Matinelli V, Vianelli
V, Randi ML, Bertozzi I, Stefano N, Za T, Rossi E, Ruggeri M, Elli E, Cacciola
E, Pogliani E, Rodeghiero F, Baccarani M, Passamonti F, Finazzi G, Rambaldi
A, Bosi A, Cazzola M, Barbui T, Vannuchi A. Hydroxyurea-related toxicity in
3,411 patients with Ph’-negative MPN. Am J Hematol 2012;87:552-554.
4. Mendonça R, Gueiros LA, Capellaro K, Pinheiro VR, Lopes MA. Oral lesions
associated with hydroxyurea treatment. Indian J Dent Res 2011;22:869-870.
5. Kumar A, Kumar V, Singh J, Hooda A, Dutta S. Drug-induced discoloration
of teeth: an updated review. Clin Pediatr 2012;51:181-185.
6. Ozyurt K, Baykan H, Ozturk P, Silay E. Case of longitudinal melanonychia
secondary to hydroxyurea. UHOD 2013;23:53-54.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Muhammed Okuyucu, M.D., Ondokuz Mayıs University Faculty
of Medicine, Department of Internal Medicine, Samsun, Turkey
Phone : +90 362 312 19 19
E-mail : muhammedokuyucu55@gmail.com ORCID: orcid.org/0000-0002-6026-2024
Received/Geliş tarihi: July 18, 2019
Accepted/Kabul tarihi: November 8, 2019
DOI: 10.4274/tjh.galenos.2019.2019.0275
Rare Presentation of Herpes Virus Lesions in a Case of Acute
Pre-B Lymphoblastic Leukemia
Akut Pre-B Lenfoblastik Lösemili Bir Olguda Herpes Virüs Lezyonlarının Nadir
Presentasyonu
Eylem Kaymaz, Zeliha Güzelküçük, Melek Işık, Neşe Yaralı
University of Health Sciences, Ankara Child Health and Diseases Hematology Oncology Training and Research Hospital, Clinic of Pediatrics,
Ankara, Turkey
To the Editor,
A 6-year-old girl with the diagnosis of acute pre-B lymphoblastic
leukemia had febrile neutropenia and pneumonia after induction
chemotherapy. Though wide-spectrum antibiotics were started
and then antifungal treatment was added, the fever could not
be controlled. During this period, a small vesicle resembling
herpes labialis developed at the edge of her lip and acyclovir
was added. The patient’s respiratory distress improved with
combined antibacterial and antifungal therapy and saturation
increased to normal levels after 1 week. During this period when
the patient was afebrile, 3-5 vesicles were noted on her palm
(Figure 1).
Herpes simplex virus (HSV) has two types, HSV-1 and HSV-2,
and these viruses are members of the herpesviruses family.
HSV can usually have lesions on different areas of body.
Clinical presentations range from asymptomatic infection to
mucocutaneous infections such as orolabial, ocular, genital
herpes, herpetic whitlow, herpes gladiatorum, and eczema
herpeticum as well as neonatal herpes, herpetic encephalitis,
and fatal dissemination [1,2]. The diagnosis of HSV infection
Figure 1. Vesicular lesions located on the hyperemic skin of the
palm.
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LETTERS TO THE EDITOR
Turk J Hematol 2020;37:57-76
can mostly be done with the clinical appearance of the lesions
and the history of the patient. It mostly produces oral and
perioral lesions but it may disseminate systematically and
cause secondary bacterial and fungal infections [3]. In children,
HSV infections on the hand most commonly occur on the
fingers and thumb, called herpetic whitlow. This infection can
be secondary to autoinoculation of the virus from a primary
oral HSV infection such as gingivostomatitis or inoculation by
a different person who bites or sucks on the finger [4,5]. The
palmar area is involved less commonly and can be transmitted
to others through contact with skin vesicles and also in patient
skin-to-skin contact. In our patient, the palmar lesion was
transmitted from her labial herpes.
Keywords: Herpes virus, Leukemia, Child
Anahtar Sözcükler: Herpes virüs, Lösemi, Çocuk
Informed Consent: It was obtained from parents or legal
guardians before the patient’s enrollment in the study.
Conflict of Interest: The authors declare no potential conflicts
of interest with respect to the research, authorship, and/or
publication of this article.
Financial Disclosure: The authors declare that this study
received no financial support.
References
1. Widener RW, Whitley RJ. Handbook of Clinical Neurology. Amsterdam, Alex
Tselis & John Booss, 2014.
2. Fatahzadeh M, Schwartz R. Human herpes simplex virus infections:
epidemiology, pathogenesis, symptomatology, diagnosis, and management.
J Am Acad Dermatol 2007;57:737-763.
3. Epstein J, Scully C. Herpes simplex virus in immunocompromised patients:
growing evidence of drug resistance. Oral Surg Oral Med Oral Pathol
1991;72:47-50.
4. Walker LG, Simmons BP, Lovallo JL. Pediatric herpetic hand infections. J
Hand Surg Am 1990;15:176-180.
5. Gill M, Arlette J, Buchan K. Herpes simplex virus infection of the hand. J Am
Acad Dermatol 1990;22:111-116.
Authorship Contributions
Concept: N.Y.; Design: Z.G., M.I.; Data Collection or Processing:
N.Y., Z.G.; Literature Search: Z.G., E.K.; Writing: E.K., Z.G.
©Copyright 2020 by Turkish Society of Hematology
Turkish Journal of Hematology, Published by Galenos Publishing House
Address for Correspondence/Yazışma Adresi: Zeliha Güzelküçük, M.D., University of Health Sciences,
Ankara Child Health and Diseases Hematology Oncology Training and Research Hospital, Clinic of Pediatrics,
Ankara, Turkey
Phone : +312 596 96 00
E-mail : drguzelkucuk@hotmail.com ORCID: orcid.org/0000-0003-1462-6867
Received/Geliş tarihi: October 24, 2018
Accepted/Kabul tarihi: November 23, 2018
DOI: 10.4274/tjh.galenos.2018.2018.0372
76