Turkish Journal of Hematology Volume: 37 Issue: 1 / 2020

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Turkish Journal of Hematology Volume: 37 Issue: 1 / 2020

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

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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

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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.

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AIMS AND SCOPE

The Turkish Journal of Hematology is published quarterly (March, June,

September, and December) by the Turkish Society of Hematology. It is an

independent, non-profit peer-reviewed international English-language

periodical encompassing subjects relevant to hematology.

The Editorial Board of The Turkish Journal of Hematology adheres to

the principles of the World Association of Medical Editors (WAME),

International Council of Medical Journal Editors (ICMJE), Committee on

Publication Ethics (COPE), Consolidated Standards of Reporting Trials

(CONSORT) and Strengthening the Reporting of Observational Studies in

Epidemiology (STROBE).

The aim of The Turkish Journal of Hematology is to publish original

hematological research of the highest scientific quality and clinical

relevance. Additionally, educational material, reviews on basic

developments, editorial short notes, images in hematology, and letters

from hematology specialists and clinicians covering their experience and

comments on hematology and related medical fields as well as social

subjects are published. As of December 2015, The Turkish Journal of

Hematology does not accept case reports. Important new findings or data

about interesting hematological cases may be submitted as a brief report.

General practitioners interested in hematology and internal medicine

specialists are among our target audience, and The Turkish Journal of

Hematology aims to publish according to their needs. The Turkish Journal

of Hematology is indexed, as follows:

- PubMed Medline

- PubMed Central

- Science Citation Index Expanded

- EMBASE

- Scopus

- CINAHL

- Gale/Cengage Learning

- EBSCO

- DOAJ

- ProQuest

- Index Copernicus

- Tübitak/Ulakbim Turkish Medical Database

- Turk Medline

- 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.

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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).

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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

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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

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conducted with humans must state that the study protocol received

institutional review board approval and that the participants provided

informed consent.

Non-compliance with scientific accuracy is not in accord with scientific

ethics. Plagiarism: To re-publish, in whole or in part, the contents of

another author’s publication as one’s own without providing a reference.

Fabrication: To publish data and findings/results that do not exist.

Duplication: Use of data from another publication, which includes republishing

a manuscript in different languages. Salami slicing: To create

more than one publication by dividing the results of a study unnecessarily.

We disapprove of such unethical practices as plagiarism, fabrication,

duplication, and salami slicing, as well as efforts to influence the

review process with such practices as gifting authorship, inappropriate

acknowledgments, and references. Additionally, authors must respect

participants‘ right to privacy.

On the other hand, short abstracts published in congress books that do

not exceed 400 words and present data of preliminary research, and those

that are presented in an electronic environment, are not considered as

previously published work. Authors in such a situation must declare this

status on the first page of the manuscript and in the cover letter.

(The COPE flowchart is available at http://publicationethics.org.)

We use iThenticate to screen all submissions for plagiarism before

publication.

Conditions of Publication

All authors are required to affirm the following statements before their

manuscript is considered: 1. The manuscript is being submitted only

to The Turkish Journal of Hematology; 2. The manuscript will not be

submitted elsewhere while under consideration by The Turkish Journal

of Hematology; 3. The manuscript has not been published elsewhere,

and should it be published in The Turkish Journal of Hematology it will

not be published elsewhere without the permission of the editors (these

restrictions do not apply to abstracts or to press reports for presentations

at scientific meetings); 4. All authors are responsible for the manuscript’s

content; 5. All authors participated in the study concept and design,

analysis and interpretation of the data, and drafting or revising of the

manuscript and have approved the manuscript as submitted. In addition,

all authors are required to disclose any professional affiliation, financial

agreement, or other involvement with any company whose product

figures prominently in the submitted manuscript.

Authors of accepted manuscripts will receive electronic page proofs and

are responsible for proofreading and checking the entire article within

two days. Failure to return the proof in two days will delay publication. If

the authors cannot be reached by email or telephone within two weeks,

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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.

References

1. Jagasia M, Arora M, Flowers ME, Chao NJ, McCarthy PL, Cutler CS, Urbano-

Ispizua A, Pavletic SZ, Haagenson MD, Zhang MJ, Antin JH, Bolwell BJ,

Bredeson C, Cahn JY, Cairo M, Gale RP, Gupta V, Lee SJ, Litzow M, Weisdorf

DJ, Horowitz MM, Hahn T. Risk factors for acute GVHD and survival after

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Table 2. Summary of some of the current novel second-line

strategies.

Agent Comment Refs.

Mycophenolate

mofetil

Sirolimus

Cyclophosphamide

Antithymocyte

globulin (ATG)

Bortezomib

Infliximab,

etanercept

A potent inhibitor of lymphocyte

proliferation, and also frequently

used in reduced intensity allo-HSCT.

Sirolimus is an immunosuppressant

that binds to FKBP12 and inhibits

the mammalian target of the

rapamycin inhibitor to block

interleukin-2 (IL-2)-mediated signal

transduction. In phase II trials,

sirolimus was very effective in

combination with tacrolimus [21].

A phase 3 prospective, randomized

multicenter clinical trial showed

that tacrolimus/sirolimus prophylaxis

provided equivalent GvHD-free

survival when compared with

tacrolimus/methotrexate prophylaxis

after matched related donor

transplantation.

Cyclophosphamide alone or

in combination with other

immunosuppressive agents

was administered in two doses

for depletion of alloreactive

conventional T-cells, which were

highly induced immediately after

transplant, and was shown to induce

a relatively low incidence of GvHD

even in haploidentical transplant

recipients.

ATG has been associated with

reduced GvHD but has also been

discussed regarding effects on

infection risk, relapse, and survival.

While several randomized trials

showed a significant benefit of

ATG for the prophylaxis of GvHD,

especially cGvHD, a metaanalysis

from six randomized trials did not

report improved overall survival or

increased incidences of relapse and

nonrelapse mortality.

Bortezomib is a proteasome

inhibitor that has activity against

multiple myeloma. A phase I/II trial

demonstrated that it could be added

to tacrolimus and methotrexate as

aGvHD prophylaxis. This protocol

is promising as the 6-month

cumulative incidence of grade II-IV

aGvHD was 13%

Studies demonstrated elevated

levels of TNF-α and IL-1 in the

serum of patients with aGvHD.

Therapy of GvHD with humanized

anti-TNF-α (infliximab) or human

immunoglobulin G1 (etanercept) and

IL-1 receptor antagonist (IL-1Ra) has

shown some promise.

[20]

[21,22]

[23,24]

[25,26]

[27]

[28,29]

TNF: Tumor necrosis factor, IL: Interleukin, aGvHD: Acute graft‐versus‐host disease.

3


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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.

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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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Turk J Hematol 2020;37:20-29

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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Turk J Hematol 2020;37:20-29

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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Turk J Hematol 2020;37:20-29

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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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.

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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

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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

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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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6. Jegalian AG, Buxbaum NP, Facchetti F, Raffeld M, Pittaluga S, Wayne

AS, Jaffe ES. Blastic plasmacytoid dendritic cell neoplasm in children :

diagnostic features and clinical implications. Haematologica 2010;95:1873-

1879.

7. 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.

9. Murphy N, Owens D, Hinds E, Nelson N. Blastic plasmacytoid dendritic cell

neoplasm (BPDCN) in leukaemic phase without skin lesions: a diagnostic

and management challenge. Pathology 2019;51:439-441.

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+

hematodermic neoplasms): a review. Am J Clin Pathol 2005;123:662-675.

11. Jegalian AG, Facchetti F, Jaffe ES. Plasmacytoid dendritic cells. Adv Anat

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12. Feuillard J. Clinical and biologic features of CD4+CD56+ malignancies.

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13. Shi Y, Wang E. Blastic plasmacytoid dendritic cell neoplasm: A

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14. Petrella T, Comeau MR, Maynadié M, Couillault G, De Muret A, Maliszewski

CR, Dalac S, Durlach A, Galibert L. “Agranular CD4+ CD56+ hematodermic

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Mitsui T, Koiso H, Takizawa M, Uchiumi H, Saitoh T, Handa H, Murakami H,

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Puches R, Wiesner T, Cerroni L. Cutaneous manifestations of blastic

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K, Dictor M, Hansmann ML, Pileri SA, Dyer MJ, Sozzani S, Dikic I, Shaw

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2008;111:3778-3792

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23. Pagano L, Valentini CG, Grammatico S, Pulsoni A. Blastic plasmacytoid

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Suzumiya J. Long-term survival following autologous and allogeneic stem

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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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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.

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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

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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.

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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

71


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

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