TJH-2018-2-baski

Volume 35 Issue 2 June 2018 80 TL

ISSN 1300-7777

Research Articles

The Role of CD200 and CD43 Expression in Differential Diagnosis Between Chronic Lymphocytic Leukemia and

Mantle Cell Lymphoma

Mesude Falay et al.; Ankara, Turkey

Association of Interleukin-2-330T/G and Interleukin-10-1082A/G Genetic Polymorphisms with

B-Cell Non-Hodgkin Lymphoma in a Cohort of Egyptians

Hala Aly Abdel Rahman, et al.; Cairo, Egypt

Myelodysplastic Syndrome in Pakistan: Clinicohematological Characteristics, Cytogenetic Profile, and Risk

Stratification

Rafia Mahmood et al.; Rawalpindi, Pakistan

Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated

Peptide in Plasma Cell Dyscrasias

Tamar Tadmor, et al.; Haifa, Israel

Acute Traumatic Coagulopathy: The Value of Histone in Pediatric Trauma Patients

Emel Ulusoy, et al.; İzmir, Turkey

Cover Picture:

Shivangi Harankhedkar et al.

Pleomorphic Multinucleated Plasma

Cells Simulating Megakaryocytes in

an Anaplastic Variant of Myeloma

2

Editor-in-Chief

Reyhan Küçükkaya

İstanbul, Turkey

rkucukkaya@hotmail.com

Associate Editors

Ayşegül Ünüvar

İstanbul, Turkey

aysegulu@hotmail.com

Cengiz Beyan

Ufuk University, Ankara, Turkey

cengizbeyan@hotmail.com

Hale Ören

Dokuz Eylül University, İzmir, Turkey

hale.oren@deu.edu.tr

İbrahim C. Haznedaroğlu

Hacettepe University, Ankara, Turkey

haznedar@yahoo.com

M. Cem Ar

İstanbul University Cerrahpaşa Faculty of

Medicine, İstanbul, Turkey

mcemar68@yahoo.com

Selami Koçak Toprak

Ankara University, Ankara, Turkey

sktoprak@yahoo.com

Semra Paydaş

Çukurova University, Adana, Turkey

sepay@cu.edu.tr

Assistant Editors

A. Emre Eşkazan

İstanbul University Cerrahpaşa Faculty of

Medicine, İstanbul, Turkey

Ali İrfan Emre Tekgündüz

Dr. A. Yurtaslan Ankara Oncology Training and

Research Hospital, Ankara, Turkey

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

Şule Ünal


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 Economy Technical University Hospital, Ankara, Turkey

Maryland School of Medicine, Baltimore, USA

Cedars-Sinai Medical Center, USA

Ankara, Turkey

Chicago Medical Center University, Chicago, USA

Ankara, Turkey

Karolinska University, Stockholm, Sweden

Mayo Clinic Saint Marys Hospital, USA

Heidelberg University, Heidelberg, Germany

Lille University, Lille, France

Loyola University, Maywood, USA

Karolinska University Hospital, Stockholm, Sweden

Frankfurt University, Frankfurt, Germany

Colorado Health Sciences University, USA

University Hospital Cologne, Cologne, Germany


Albert Ludwigs University, Germany

University of North Carolina School of Medicine, NC, USA

Southwestern Medical Center, Texas, USA

Georgia Health Sciences University, Augusta, USA

Pavia Medical School University, Pavia, Italy

Kings College Hospital, London, UK

Pierre et Marie Curie University, Paris, France

King’s Hospital, London, UK

Athens University, Athens, Greece

Torino University, Torino, Italy

Wayne State University School of Medicine, Detroit, USA

Rambam Medical Center, Haifa, Israel

University of Köln, Germany

AK St Georg, Hamburg, Germany

Memorial Şişli Hospital, İstanbul, Turkey

Santa Creu i Sant Pau Hospital, Barcelona, Spain

Mayo Clinic, Rochester, Minnesota, USA

İstanbul Cerrahpaşa University, İstanbul, Turkey

University of Minnesota, Minnesota, USA

The University of Texas MD Anderson Cancer Center, Houston, USA

San Martino University, Genoa, Italy

Language Editor

Leslie Demir

Statistic Editor

Hülya Ellidokuz

Editorial Office

İpek Durusu

Bengü Timoçin

A-I

Publishing

Services

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

ISSN: 1300-7777

Publishing Manager

Sorumlu Yazı İşleri Müdürü

Muhlis Cem Ar

Management Address

Yayın İdare Adresi

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

Türk Hematoloji Derneği adına yayın sahibi

Güner Hayri Özsan

Publishing House / Yayınevi

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

Print: Özgün Ofset Ticaret Ltd. Şti.

Yeşilce Mah. Aytekin Sok. No: 21 34418 4. Levent, İstanbul-Turkey

Phone: +90 212 280 0009

Printing Date / Basım Tarihi

25.05.2018

Cover Picture

Shivangi Harankhedkar et al.,

Pleomorphic Multinucleated Plasma Cells Simulating Megakaryocytes in an

Anaplastic Variant of Myeloma

Panel of photomicrographs: A) May-Grünwald Giemsa stained bone marrow

aspirate smear (100 x ) showing pleomorphic cells, with multilobation and

multinuclearity, with prominent inclusions (red arrows) and abundant

basophilic cytoplasm, and absence of perinuclear hof.

International scientific journal published quarterly.

Üç ayda bir yayımlanan İngilizce süreli yayındır.

Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile Turkish

Journal of Hematology’nin Türk Hematoloji Derneği İktisadi İşletmesi

tarafından yayınlanmasına karar vermiştir.

A-II

AIMS AND SCOPE

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

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

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

periodical encompassing subjects relevant to hematology.

The Editorial Board of The Turkish Journal of Hematology adheres to

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

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

Publication Ethics (COPE), Consolidated Standards of Reporting Trials

(CONSORT) and Strengthening the Reporting of Observational Studies in

Epidemiology (STROBE).

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

hematological research of the highest scientific quality and clinical

relevance. Additionally, educational material, reviews on basic

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

from hematology specialists and clinicians covering their experience and

comments on hematology and related medical fields as well as social

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

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

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

General practitioners interested in hematology and internal medicine

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

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

of Hematology is indexed, as follows:

- PubMed Medline

- PubMed Central

- Science Citation Index Expanded

- EMBASE

- Scopus

- CINAHL

- Gale/Cengage Learning

- EBSCO

- DOAJ

- ProQuest

- Index Copernicus

- Tübitak/Ulakbim Turkish Medical Database

- Turk Medline

Impact Factor: 0.686

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 sent free-of-charge to members

of Turkish Society of Hematology and libraries in Turkey and abroad.

Hematologists, other medical specialists that are interested in hematology,

and academicians could subscribe for only 40 $ per printed issue. All

published volumes are available in full text free-of-charge online at www.

tjh.com.tr.

Address: İlkbahar Mah., Turan Güneş Bulvarı, 613 Sok., No: 8, Çankaya,

Ankara, Turkey

Telephone: +90 312 490 98 97

Fax: +90 312 490 98 68

Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh

Web page: www.tjh.com.tr

E-mail: info@tjh.com.tr

Permissions

Requests for permission to reproduce published material should be sent to

the editorial office.

Editor: Professor Dr. Reyhan Küçükkaya

Adress: İlkbahar Mah, Turan Günes Bulvarı, 613 Sok., No: 8, Çankaya,

Ankara, Turkey

Telephone: +90 312 490 98 97

Fax: +90 312 490 98 68

Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh

Web page: www.tjh.com.tr

E-mail: info@tjh.com.tr

Publisher

Galenos 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.

The journal is printed on acid-free paper.

Editorial Policy

Following receipt of each manuscript, a checklist is completed by the

Editorial Assistant. The Editorial Assistant checks that each manuscript

contains all required components and adheres to the author guidelines,

after which time it will be forwarded to the Editor in Chief. Following the

Editor in Chief’s evaluation, each manuscript is forwarded to the Associate

Editor, who in turn assigns reviewers. Generally, all manuscripts will be

reviewed by at least three reviewers selected by the Associate Editor, based

on their relevant expertise. Associate editor could be assigned as a reviewer

along with the reviewers. After the reviewing process, all manuscripts are

evaluated in the Editorial Board Meeting.

Turkish Journal of Hematology’s editor and Editorial Board members are

active researchers. It is possible that they would desire to submit their

manuscript to the Turkish Journal of Hematology. This may be creating

a conflict of interest. These manuscripts will not be evaluated by the

submitting editor(s). The review process will be managed and decisions

made by editor-in-chief who will act independently. In some situation, this

process will be overseen by an outside independent expert in reviewing

submissions from editors.

A-III

TURKISH JOURNAL OF HEMATOLOGY

INSTRUCTIONS FOR AUTHORS

The Turkish Journal of Hematology accepts invited review articles, research

articles, brief reports, letters to the editor, and hematological images that

are relevant to the scope of hematology, on the condition that they have

not been previously published elsewhere. Basic science manuscripts,

such as randomized, cohort, cross-sectional, and case-control studies,

are given preference. All manuscripts are subject to editorial revision

to ensure they conform to the style adopted by the journal. There is a

double-blind reviewing system. Review articles are solicited by the Editorin-Chief.

Authors wishing to submit an unsolicited review article should

contact the Editor-in-Chief prior to submission in order to screen the

proposed topic for relevance and priority.

The Turkish Journal of Hematology does not charge any article submission

or processing charges.

Manuscripts should be prepared according to ICMJE guidelines (http://

www.icmje.org/). Original manuscripts require a structured abstract. Label

each section of the structured abstract with the appropriate subheading

(Objective, Materials and Methods, Results, and Conclusion). Letters to

the editor do not require an abstract. Research or project support should

be acknowledged as a footnote on the title page. Technical and other

assistance should be provided on the title page.

Original Manuscripts

Title Page

Title: The title should provide important information regarding the

manuscript’s content. The title must specify that the study is a cohort

study, cross-sectional study, case-control study, or randomized study (i.e.

Cao GY, Li KX, Jin PF, Yue XY, Yang C, Hu X. Comparative bioavailability

of ferrous succinate tablet formulations without correction for baseline

circadian changes in iron concentration in healthy Chinese male

subjects: A single-dose, randomized, 2-period crossover study. Clin Ther

2011;33:2054-2059).

The title page should include the authors’ names, degrees, and institutional/

professional affiliations and a short title, abbreviations, keywords, financial

disclosure statement, and conflict of interest statement. If a manuscript

includes authors from more than one institution, each author’s name

should be followed by a superscript number that corresponds to their

institution, which is listed separately. Please provide contact information

for the corresponding author, including name, e-mail address, and

telephone and fax numbers.

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).

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

A-IV

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



Review articles should not include more than 100 references. Reviews

should include a conclusion, in which a new hypothesis or study about the

subject may be posited. Do not publish methods for literature search or level

of evidence. Authors who will prepare review articles should already have

published research articles on the relevant subject. The study’s new and

important findings should be highlighted and interpreted in the Conclusion

section. There should be a maximum of two authors for review articles.

A-V

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.



References: Should not include more than 50 references

Images in Hematology


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


Letters can include no more than 500 words of text, 5-10 references, and

1 figure or table. No abstract is required, but please include a brief title.

Tables

Supply each table 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

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

A-VI

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

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

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

a manuscript in different languages. Salami slicing: To create

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

We disapprove of such unethical practices as plagiarism, fabrication,

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

review process with such practices as gifting authorship, inappropriate

acknowledgments, and references. Additionally, authors must respect

participants‘ right to privacy.

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

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

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

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

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

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

We use iThenticate to screen all submissions for plagiarism before

publication.

Conditions of Publication

All authors are required to affirm the following statements before their

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

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

submitted elsewhere while under consideration by The Turkish Journal

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

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

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

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

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

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

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

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

all authors are required to disclose any professional affiliation, financial

agreement, or other involvement with any company whose product

figures prominently in the submitted manuscript.

Authors of accepted manuscripts will receive electronic page proofs and

are responsible for proofreading and checking the entire article within

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

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

the manuscript will be rejected and will not be published in the journal.

Copyright

At the time of submission all authors will receive instructions for

submitting an online copyright form. No manuscript will be considered

for review until all authors have completed their copyright form. Please

note, it is our practice not to accept copyright forms via fax, e-mail, or

postal service unless there is a problem with the online author accounts

that cannot be resolved. Every effort should be made to use the online

copyright system. Corresponding authors can log in to the submission

system at any time to check the status of any co-author’s copyright form.

All accepted manuscripts become the permanent property of The Turkish

Journal of Hematology and may not be published elsewhere, in whole or

in part, without written permission.

Note: We cannot accept any copyright form that has been altered,

revised, amended, or otherwise changed. Our original copyright form

must be used as is.

Units of Measurement

Measurements should be reported using the metric system, according

to the International System of Units (SI). Consult the SI Unit Conversion

Guide, New England Journal of Medicine Books, 1992.

An extensive list of conversion factors can be found at https://www.

nist.gov/sites/default/files/documents/pml/wmd/metric/SP1038.pdf. For

more details, see http://www.amamanualofstyle.com/oso/public/jama/

si_conversion_table.html.

Abbreviations and Symbols

Use only standard abbreviations. Avoid abbreviations in the title and

abstract. The full term for an abbreviation should precede its first use

in the text, unless it is a standard abbreviation. All acronyms used in the

text should be expanded at first mention, followed by the abbreviation

in parentheses; thereafter the acronym only should appear in the text.

Acronyms may be used in the abstract if they occur 3 or more times

therein, but must be reintroduced in the body of the text. Generally,

abbreviations should be limited to those defined in the AMA Manual of

Style, current edition. A list of each abbreviation (and the corresponding

full term) used in the manuscript must be provided on the title page.

Online Manuscript Submission Process

The Turkish Journal of Hematology uses submission software powered

by ScholarOne Manuscripts. The website for submissions to The Turkish

Journal of Hematology is http://mc.manuscriptcentral.com/tjh. This

system is quick and convenient, both for authors and reviewers.

Setting Up an Account

New users to the submission site will need to register and enter their

account details before they can submit a manuscript. Log in, or click

the “Create Account” button if you are a first-time user. To create a

new account: After clicking the “Create Account” button, enter your

name and e-mail address, and then click the “Next” button. Your e-mail

address is very important. Enter your institution and address information,

as appropriate, and then click the “Next” Button. Enter a user ID and

password of your choice, select your area of expertise, and then click the

“Finish” button.

If you have an account, but have forgotten your log-in details, go to

“Password Help” on the journal’s online submission system and enter your

e-mail address. The system will send you an automatic user ID and a new

temporary password.

Full instructions and support are available on the site, and a user ID

and password can be obtained during your first visit. Full support for

authors is provided. Each page has a “Get Help Now” icon that connects

A-VII

directly to the online support system. Contact the journal administrator

with any questions about submitting your manuscript to the journal

(info@tjh.com.tr). For ScholarOne Manuscripts customer support, click

on the “Get Help Now” link on the top right-hand corner of every page

on the site.

The Electronic Submission Process

Log in to your author center. Once you have logged in, click the “Submit a

Manuscript” link in the menu bar. Enter the appropriate data and answer

the questions. You may copy and paste directly from your manuscript.

Click the “Next” button on each screen to save your work and advance

to the next screen.

Upload Files

Click on the “Browse” button and locate the file on your computer. Select

the appropriate designation for each file in the drop-down menu next to

the “Browse” button. When you have selected all the files you want to

upload, click the “Upload Files” button. Review your submission before

sending to the journal. Click the “Submit” button when you are finished

reviewing. You can use ScholarOne Manuscripts at any time to check

the status of your submission. The journal’s editorial office will inform

you by e-mail once a decision has been made. After your manuscript

has been submitted, a checklist will then be completed by the Editorial

Assistant. The Editorial Assistant will check that the manuscript contains

all required components and adheres to the author guidelines. Once the

Editorial Assistant is satisfied with the manuscript it will be forwarded to

the Senior Editor, who will assign an editor and reviewers.

The Review Processs

Each manuscript submitted to The Turkish Journal of Hematology is

subject to an initial review by the editorial office in order to determine

if it is aligned with the journal’s aims and scope and complies with

essential requirements. Manuscripts sent for peer review will be assigned

to one of the journal’s associate editors that has expertise relevant to the

manuscript’s content. All accepted manuscripts are sent to a statistical

and English language editor before publishing. Once papers have been

reviewed, the reviewers’ comments are sent to the Editor, who will then

make a preliminary decision on the paper. At this stage, based on the

feedback from reviewers, manuscripts can be accepted or rejected, or

revisions can be recommended. Following initial peer-review, articles

judged worthy of further consideration often require revision. Revised

manuscripts generally must be received within 3 months of the date of

the initial decision. Extensions must be requested from the Associate

Editor at least 2 weeks before the 3-month revision deadline expires;

The Turkish Journal of Hematology will reject manuscripts that are not

received within the 3-month revision deadline. Manuscripts with extensive

revision recommendations will be sent for further review (usually by the

same reviewers) upon their re-submission. When a manuscript is finally

accepted for publication, the Technical Editor undertakes a final edit

and a marked-up copy will be e-mailed to the corresponding author for

review and any final adjustments.

Submission of Revised Papers

When revising a manuscript based on the reviewers’ and Editor’s feedback,

please insert all changed text in red. Please do not use track changes, as

this feature can make reading difficult. To submit revised manuscripts,

please log in to your author center at ScholarOne Manuscripts. Your

manuscript will be stored under “Manuscripts with Decisions”. Please click

on the “Create a Revision” link located to the right of the manuscript title.

A revised manuscript number will be created for you; you will then need

to click on the “Continue Submission” button. You will then be guided

through a submission process very similar to that for new manuscripts.

You will be able to amend any details you wish. At stage 6 (“File Upload”),

please delete the file for your original manuscript and upload the revised

version. Additionally, please upload an anonymous cover letter, preferably

in table format, including a point-by-point response to the reviews’

revision recommendations. You will then need to review your paper as

a PDF and click the “Submit” button. Your revised manuscript will have

the same ID number as the original version, but with the addition of an R

and a number at the end, for example, TJH-2011-0001 for an original and

TJH-2011-0001.R1, indicating a first revision; subsequent revisions will

end with R2, R3, and so on. Please do not submit a revised manuscript

as a new paper, as revised manuscripts are processed differently. If you

click on the “Create a Revision” button and receive a message stating that

the revision option has expired, please contact the Editorial Assistant at

info@tjh.com.tr to reactivate the option.

English Language and Statistical Editing

All manuscripts are professionally edited by an English language editor

prior to publication. After papers have been accepted for publication,

manuscript files are forwarded to the statistical and English language

editors before publishing. Editors will make changes to the manuscript to

ensure it adheres to TJH requirements. Significant changes or concerns

are referred to corresponding authors for editing.

Online Early

The Turkish Journal of Hematology publishes abstracts of accepted

manuscripts online in advance of their publication in print. Once an

accepted manuscript has been edited, the authors have submitted any

final corrections, and all changes have been incorporated, the manuscript

will be published online. At that time the manuscript will receive a Digital

Object Identifier (DOI) number. Both forms can be found at www.tjh.

com.tr. Authors of accepted manuscripts will receive electronic page

proofs directly from the printer and are responsible for proofreading

and checking the entire manuscript, including tables, figures, and

references. Page proofs must be returned within 48 hours to avoid delays

in publication.

A-VIII

CONTENTS

Research Articles

94 The Role of CD200 and CD43 Expression in Differential Diagnosis Between Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Mesude Falay, Berna Afacan Öztürk, Kürsad Güneş, Yasin Kalpakçı, Simten Dağdaş, Funda Ceran, Gülsüm Özet; Ankara, Turkey

99 Association of Interleukin-2-330T/G and Interleukin-10-1082A/G Genetic Polymorphisms with B-Cell Non-Hodgkin

Lymphoma in a Cohort of Egyptians

Hala Aly Abdel Rahman, Mervat Mamdooh Khorshied, Ola Mohamed Reda Khorshid, Heba Mahmoud Mourad; Cairo, Egypt

109 Myelodysplastic Syndrome in Pakistan: Clinicohematological Characteristics, Cytogenetic Profile, and Risk Stratification

Rafia Mahmood, Chaudry Altaf, Parvez Ahmed, Saleem Ahmed Khan, Hamid Saeed Malik; Rawalpindi, Pakistan

116 Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated Peptide in

Plasma Cell Dyscrasias

Tamar Tadmor, Ilana Levy, Zahava Vadasz; Haifa, Israel

122 Acute Traumatic Coagulopathy: The Value of Histone in Pediatric Trauma Patients

Emel Ulusoy, Murat Duman, Aykut Çağlar, Tuncay Küme, Anıl Er, Fatma Akgül, Hale Çitlenbik, Durgül Yılmaz, Hale Ören; İzmir, Turkey

Brief Report

129 Use of a High-Purity Factor X Concentrate in Turkish Subjects with Hereditary Factor X Deficiency: Post Hoc Cohort

Subanalysis of a Phase 3 Study

Ahmet F. Öner, Tiraje Celkan, Çetin Timur, Miranda Norton, Kaan Kavaklı; Van, İstanbul, İzmir, Turkey; Hertfordshire, United Kingdom

Images in Hematology

134 Flaming Plasma Cell Leukemia

Reza Ranjbaran, Habibollah Golafshan; Shiraz, Iran

135 Improvement of Cutaneous Anaplastic Large Cell Lymphoma by Brentuximab Vedotin Monotherapy

Takashi Onaka, Tomoya Kitagawa, Chika Kawakami, Akihito Yonezawa; Fukuoka, Japan

Letters to the Editor

137 Glomerular and Tubular Functions in Transfusion-Dependent Thalassemia

Pathum Sookaromdee, Viroj Wiwanitkit; Bangkok, Thailand; Hainan, China

138 Use of Plerixafor to Mobilize a Healthy Donor Infected with Influenza A

Mahmut Yeral, Pelin Aytan, Can Boğa; Adana, Turkey

A-IX

139 Influenza A Infection and Stem Cell Mobilization

Sora Yasri, Viroj Wiwanitkit; Bangkok, Thailand; Hainan, China

141 Primary Mediastinal Large B-Cell Lymphoma As an Incidental Finding: Report of a Case

İpek Yönal-Hindilerden, Fehmi Hindilerden, Serkan Arslan, İbrahim Öner Doğan, Meliha Nalçacı; İstanbul, Turkey

142 A Rare Late Complication of Port Catheter Implantation: Embolization of the Catheter

Işık Odaman Al, Cengiz Bayram, Gizem Ersoy, Kazım Öztarhan, Alper Güzeltaş, Taner Kasar, Ezgi Uysalol, Başak Koç,

Ali Ayçiçek, Nihal Özdemir; İstanbul, Turkey

144 Nuclear Projections in Neutrophils for Supporting the Diagnosis of Trisomy 13

Şebnem Kader, Mehmet Mutlu, Filiz Aktürk Acar, Yakup Aslan, Erol Erduran; Trabzon, Turkey

145 Intravascular Large B-Cell Lymphoma of the Gallbladder

Bülent Çetin, Nalan Akyürek, Yavuz Metin, Feryal Karaca, İrem Bilgetekin, Ahmet Özet; Rize, Ankara, Adana, Turkey

147 Successful Treatment of Chronic Lymphocytic Leukemia Multifocal Central Nervous System Involvement with Ibrutinib

Anna Christoforidou, Georgios Kapsas, Zoe Bezirgiannidou, Spyros Papamichos, Ioannis Kotsianidis; Alexandroupolis, Greece

150 Pleomorphic Multinucleated Plasma Cells Simulating Megakaryocytes in an Anaplastic Variant of Myeloma

Shivangi Harankhedkar, Ruchi Gupta, Khaliqur Rahman; Uttar Pradesh, India

A-X

RESEARCH ARTICLE

DOI: 10.4274/tjh.2017.0085

Turk J Hematol 2018;35:94-98

The Role of CD200 and CD43 Expression in Differential

Diagnosis between Chronic Lymphocytic Leukemia and Mantle

Cell Lymphoma

Kronik Lenfositik Lösemi ve Mantle Hücreli Lenfoma Ayırıcı Tanısında CD200 ve CD43

Ekspresyonunun Rolü

Mesude Falay, Berna Afacan Öztürk, Kürşad Güneş, Yasin Kalpakçı, Simten Dağdaş, Funda Ceran, Gülsüm Özet

University Ministry of Health, Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey

Abstract

Objective: Atypical chronic lymphocytic leukemia (CLL) is most

frequently confused with mantle cell lymphoma (MCL). Several

markers may contribute to the diagnosis of CLL. However, there is

no consensus on which markers are needed to be used in flow

cytometry for the diagnosis of CLL. The aim of the present study was

to investigate the role of CD43 and CD200 markers in the differential

diagnosis between CLL and MCL.

Materials and Methods: To address this issue, 339 consecutive

patients with CLL and MCL were included in the flow cytometry

lymphoproliferative disease panel for evaluation of CD43 and CD200

expressions, but not in the Matutes scoring system.

Results: CD200 was expressed in 97.3% of atypical CLL cases, whereas

it was dimly expressed in only 6.1% of MCL cases. CD43 expression

was 95.7% in atypical CLL cases. In the MCL cases, its expression rate

was 39.4%.

Conclusion: CD43 and CD200 were found to be more valuable

markers than CD22, CD79b, and FMC7. CD43 and CD200 could also

be considered as definitive markers in atypical CLL patients, for whom

the Matutes scoring system remains ineffective.

Keywords: Chronic lymphocytic leukemia, Mantle cell lymphoma,

Immunophenotyping, CD200, CD43

Öz

Amaç: İmmünfenotip olarak atipik kronik lenfositik lösemi (KLL) ile

mantle cell lenfoma (MCL) sıklıkla karışabilmektedir. KLL tanısı için

birçok marker kullanılmaktadır, ancak akım sitometride KLL tanısı

için tam bir konsensüs oluşmamıştır. Bu çalışmada KLL ve MCL ayırıcı

tanısında CD43 ve CD200 ifadeleri araştırılmıştır.

Gereç ve Yöntemler: Matutes skorlama sisteminde olmayan CD43

ve CD200’ü akım sitometri lenfoproliferatif hastalık paneline dahil

ederek 339 KLL ve MCL olgusunda incelenmiştir.

Bulgular: Atipik KLL olgularının %97,3’ünde CD200 pozitifken MCL

olgularının ise sadece %6,1’inde düşük oranda ifade ediliyordu.

CD43’te atipik KLL olgularının %95,7’sinde ifade edilirken MCL

olgularının %39,4’ünde donuk ifade ediliyordu.

Sonuç: CD43 ve CD200; CD22, CD79b ve FMC7’ye göre daha anlamlı

bulundu. CD43 ve CD200 Matutes skorlama sistemi skorunun

yetersiz kaldığı KLL olgularının tanısında tamamlayıcı marker olarak

kullanılabilir.

Anahtar Sözcükler: Kronik lenfositik lösemi, Mantle cell lenfoma,

İmmünfenotiplendirme, CD200, CD43

Introduction

The World Health Organization (WHO) classification of

hematolymphoid system neoplasms is based on clinical,

morphological, immunophenotypic, and genetic features.

Mature B-cell lymphoproliferative diseases (LPDs) account for

more than 80% of hematolymphoid neoplasms [1]. Chronic

lymphocytic leukemia (CLL) is the most frequent type of LPD

[1,2]. Genetics has no role in the diagnosis of CLL, although there

are numerous genetic abnormalities. The presence of persistent

clonal B lymphocytosis (>5x10 9 /L lymphocytes) for more than 3

months is needed to make a diagnosis of CLL. It has characteristic

morphological features, as well as immunophenotypic features

in flow cytometry [1,2,3,4]. These include CD5+CD19+, CD23+,

©Copyright 2018 by Turkish Society of Hematology

Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Mesude FALAY, M.D.,

University Ministry of Health, Ankara Numune Training and Research Hospital, Clinic of Hematology, Ankara, Turkey

Phone : +90 545 408 90 08

E-mail : mesudey@gmail.com ORCID-ID: orcid.org/0000-0001-7846-3476

Received/Geliş tarihi: March 01, 2017

Accepted/Kabul tarihi: July 07, 2017

94


Falay M, et al: CLL and MCL Immunophenotyping

weak surface membrane immunoglobulins (sIg), and absent or

low expression of CD79b and FMC7 [3,4]. Immunophenotyping

has a major role in the diagnosis of CLL. However, CLL is a

quite heterogeneous disease; for this reason, it can be difficult

to diagnose [3,4,5,6,7]. Accordingly, a scoring system for the

diagnosis of CLL was first defined in 1994 by Matutes et al.

[8]. This scoring system consists of five parameters: CD5, CD22,

CD23, FMC7, and sIg. In 1997, Moreau et al. [9] replaced CD22 by

CD79b in the scoring system. According to this scoring system,

a score of 4-5 indicates typical CLL and a score of 3 indicates

atypical CLL, whereas a score of 0-2 excludes CLL [8,9]. Atypical

CLL is most frequently confused with mantle cell lymphoma

(MCL), which co-expresses CD5 and CD19 similarly to CLL

[4,10,11,12,13,14,15,16]. Generally, MCL is more aggressive and

requires a different therapeutic approach; therefore, differential

diagnosis between these two diseases should be performed

precisely. Histochemical or molecular tests [cyclin D1, SOX11,

t(11;14)] can be used for differential diagnosis [4,12]. Molecular

tests are not easily available, and they are time-consuming

and more expensive. For this reason, reliable additional new

markers have been investigated in cases in which the Matutes

score is inadequate. Several markers such as CD200 and CD43

may contribute to the diagnosis of CLL. However, there is no

consensus on which markers are needed to be used in flow

cytometry for the diagnosis of CLL. In the present study, we

aimed to investigate the role of markers that were included in

our LPD panel in flow cytometry but not in the Matutes scoring

system in the differential diagnosis between CLL and MCL.

Materials and Methods

Patients and Samples

The present study retrospectively evaluated the medical records

of 339 patients diagnosed with CLL (n=306) and MCL (n=33)

according to the WHO criteria [1]. For all patients, data on

complete blood count and peripheral blood (PB) and/or bone

marrow (BM) smear performed for morphological assessments

were obtained. All atypical CLL patients were evaluated for

cyclin D1 and/or t(11;14). Diagnosis of MCL was confirmed by

immunohistochemical detection of cyclin D1 in BM biopsies

or detection of t(11;14) by fluorescence in situ hybridization.

SOX11 expression was not evaluated.

Flow Cytometry Immunophenotyping

For flow cytometric study, fresh PB/BM samples were drawn

into 4-mL K3-EDTA tubes (BD Vacutainer, USA) and studied

immediately. Cells in suspension (2x10 6 cells in 50-100 µL per

tube) from the PB and BM samples were stained with monoclonal

antibodies (MoAbs) directed against cell surface markers via a

stain-lyse-and-then-wash direct immunofluorescence method

[17]. The MoAbs used for labeling in flow cytometry were obtained

from Beckman Coulter (BC, USA). A five-color staining was applied

for all samples using the following fluorochrome-conjugated

antibodies. MoAbs including fluorescein isothiocyanate (FITC),

phycoerythrin (PE), phycoerythrin-Texas red (ECD), phycoerythrin

cyanine 5 (PC5), and allophycocyanin (APC) were used for all

patients: CD45/CD5/CD10/CD19/CD23, CD19/CD103/CD22/

CD11c/CD25, CD5/CD20/sIgk/sIgλ CD45, CD19/CD3/CD79b/

CD22, and CD19/CD43/CD200/CD38. A tube containing Ig isotype

controls for FITC/PE/ECD/PC5/APC was used for all patients. Data

were immediately obtained at the end of sample staining using

a flow cytometer (Navios, BC, USA) and Kaluza Flow Cytometry

Analysis Software (BC, USA). For each sample, data from at least

10x10 4 events per tube were obtained. Instrument alignment was

confirmed daily using an alignment control bead (Flow-Check,

BC, USA). The accuracy and precision of cell counts were tested

using international quality controls purchased from the United

Kingdom National External Quality Assessment Scheme (UK

NEQAS LI, Sheffield, UK) (z-score range of -2.0 to 2.0). Briefly,

CD19+ B cells were selected (at least 2000 events according to

the threshold of the isotype control) from the data file using

conventional gating strategies (forward and side scatter and

the pattern of CD19 expression). As recommended by the British

Committee for Standards in Haematology guideline [2], a cutoff

value of 30% of lymphoid cells was accepted to indicate a

positive result with a given antibody using the Kaluza software.

The Matutes scoring was defined as ≥30% cell surface expression.

In all patients, the same fluorescent-labeled MoAbs were used

to ensure that the Matutes scoring was accurate. Diagnosis of

LPD was established according to the WHO classification based on

clinical data and morphologic, immunophenotypic, and genetic

criteria. The revised Matutes scoring system [9], based on the

immunophenotypic analysis of five membrane markers (CD5,

CD23, FMC7, sIg, CD79b), was used to classify all patients. This

scoring system assigns 1 point each for expression of CD5, CD23,

and sIg and for lack of expression of CD79b and FMC7. A score of

≥4 indicates typical CLL patients and a score of 3 or a lack of CD23

indicates atypical CLL patients. In all patients, cyclin D1 and/or

t(11;14) was used for the differential diagnosis. Diagnosis of MCL

was confirmed by cyclin D1 and/or t(11;14).

Statistical Analysis

Data analysis was performed using SPSS 15 for Windows (SPSS

Inc., Chicago, IL, USA). Descriptive statistics were expressed as

numbers and percentages. Categorical data were analyzed by

multivariate forward stepwise regression analysis, Pearson’s chisquare

test, or Fisher’s exact test as appropriate. A p-value of

less than 0.05 was considered statistically significant.

Results

The evaluation of 339 patients (100 females, 239 males) with

mean age of 68±10.4 years (range: 31-87 years) revealed

95



that median PB lymphocyte count at diagnosis was 19.8x10 9

lymphocytes/L (range: 0.8-274x10 9 lymphocytes/L). Of the

patients, 306 (90.26%) had CLL and 33 (9.74%) had MCL (Table

1). According to the Matutes scoring of CLL patients, 121 (40%)

patients had a score of ≥4 (of whom 105 (34.3%) had a score

of 4 and 16 (5.2%) had a score of 5), 178 (58.2%) patients had

a score of 3, 6 (2%) patients had a score of 2, and 1 patient

(0.3%) had a score of 1 (Table 2). There was no significant

difference between the typical and atypical CLL patients in

terms of morphological evaluation. In all atypical CLL cases,

cyclin D1 and/or t(11;14) were negative. The Matutes scores of

the MCL patients with positive cyclin D1 and/or t(11;14) were

3 in 7 (21.2%) patients, 2 in 11 (33.3%) patients, and 1 in 15

(45.5%) patients. There were no MCL patients with a score of

≥4. Regarding CD22, CD79b, FMC7, and CD23 expressions in the

Matutes score, CD23 expression was negative in 11 (3.5%) CLL

patients (3 had typical CLL and 8 had atypical CLL), whereas it

was positive in 6 (21.2%) MCL patients CD23 expression was

not diagnostic for CLL but it was significantly more expressed

in CLL patients (p<0.001). CD22, CD79b, and FMC7 expressions

were highly positive in atypical CLL patients (96.2%, 81.6%, and

97.3%, respectively) (Table 3); however, the difference was not

significant in the differential diagnosis between CLL and MCL

(p=1.000, p=0.431, and p=1.000, respectively). CD79b expression

was also positive in 38.8% of the CLL patients. No significant

difference was found between the CLL and MCL patients

regarding sIg expression intensity (p=0.385). Evaluations of

CD38, CD43, and CD200 expressions were included in the LPD

panel but not in the Matutes scoring system (Table 2). While

CD38 expression was moderate to strong in 93.9% of the MCL

patients, it was dimly expressed in 24% of both atypical and

typical CLL patients (p<0.001). When CD43 expression was

evaluated, 95.7% of the patients with atypical CLL and 98.3% of

the patients with typical CLL had moderate to strong expression.

Among the MCL patients, CD43 expression was dim to moderate

in 39.4% (p<0.001). When CD200 expression was evaluated, it

was moderate to strong in 95.8% of the CLL patients (3.6% had

Table 1. Demographic features.

Variables n=339

Age, years (range) 68.0±10.4 (45-89)

Sex

Female, n (%) 100 (29.4%)

Male, n (%) 239 (70.6%)

CLL, n (%) 306 (90.26%)

MCL, n (%) 33 (9.74%)

White blood cells, x10 3 19.8 (8-274)

Lymphocytes, x10 3 17.3 (6.8-240)

Hemoglobin, g/dL 12.7±2.27

Platelets, x10 3 202 (19-403)

CLL: Chronic lymphocytic leukemia, MCL: mantle cell lymphoma.

negative expression), whereas it was dimly expressed only in 2

MCL patients (6.1%) (p<0.001; Table 2). There was no significant

difference in CD200 expressions between the atypical and

typical CLL patients. In the differential diagnosis of MCL and

atypical CLL patients, multivariate forward stepwise regression

analysis revealed the most determinant marker to be CD200

(p<0.001, 95% CI; Table 4).

Table 2. Mantle cell lymphoma and chronic lymphocytic

leukemia patients’ Matutes scores.

Matutes score MCL (n) % CLL (n) %

1 (15) 45.5 (1) 0.3

2 (11) 33.3 (6) 2.0

3 (7) 21.2 (178) 58.2

4 (-) 0 (105) 34.3

5 (-) 0 (16) 5.2

CLL: Chronic lymphocytic leukemia, MCL: mantle cell lymphoma.

Table 3. Distribution of cases by marker positivity in the

differential diagnosis of mantle cell lymphoma and atypical

chronic lymphocytic leukemia score of ≤3, chronic lymphocytic

leukemia score of ≥4.

MCL

(n=33)

Atypical

CLL score

of ≤3

(n=185)

60%

p

p

(n=121)

40%

CD20 31 (93.9%) 184 (99.5%) 0.061 113 (93.4%) 1.000

CD22 32 (97.0%) 177 (96.2%) 1.000 90 (74.4%) 1.000

CD23 6 (21.2%) 177 (97.0%) <0.001 118 (96.7%) <0.001

CD79b 25 (75.8%) 151 (81.6%) 0.431 47 (38.8%) <0.001

CD25 10 (30.3%) 111 (60.0%) 0.002 77 (63.6%) 0.002

CD38 31 (93.9%) 185 (24.9%) <0.001 29 (24.0%) <0.001

CD200 2 (6.1%) 180 (97.3%) <0.001 117 (96.7%) <0.001

sIg 33 (100%) 74 (40%) 0.385 97 (80%) 0.410

CD43 13 (39.4%) 177 (95.7%) <0.001 119 (98.3%) <0.001

CD11C 11(33.3%) 116 (62.7%) 0.002 87 (71.9%) <0.001

FMC7 32 (97%) 180 (97.3%) 1.000 51 (42.1%) <0.001

MCL: Mantle cell lymphoma, CLL: chronic lymphocytic leukemia, sIg: surface membrane

immunoglobulins.

Table 4. Multivariate analysis for mantle cell lymphoma

and atypical chronic lymphocytic leukemia discrimination.

Odds ratio

95% Confidence

interval

Lower

Upper

Wald

CD200 1317.886 79.380 21879.729 25.115 <0.001

CD38 31.909 2.446 416.220 6.984 0.008

CD43 17.632 1.766 176.091 5.974 0.015

p

96



Discussion

The diagnosis of CLL is easy in the presence of characteristic

immunophenotypic features (CD5+CD19+ dual-positive,

CD23+, CD22-/low, CD79b-/low, sIg low, FMC7-, and CD20 low).

However, it is difficult to make a differential diagnosis of CLL

from MCL when immunophenotypic features are not typical.

In the present study, CD43 and CD200 expressions, which were

included in the LPD panel but not in the Matutes scoring system,

were found significant in the differential diagnosis between CLL

and MCL.

Immunophenotyping by flow cytometry, which is a frequently

used method, is beneficial in the distinction of CLL from

MCL [3,4,15]. However, there may be a problem for atypical

immunophenotypes in which the Matutes score is ≤3. Therefore,

it may be particularly difficult to distinguish some MCL cases

from atypical CLL cases. CD23 positivity is the most characteristic

feature of CLL [10,11]. Earlier studies have reported that CD23

negativity is a reliable marker in the distinction between CLL

and MCL [15]. In the present study, while 2.1% of the typical CLL

patients were CD23-negative, 21.2% of the MCL patients with

positive t(11;14) were CD23-positive. However, according to our

findings, CD23 alone was not efficient to make a differential

diagnosis between CLL and MCL [12,13,16]. On the other hand,

FMC7, which is an epitope of CD20, was expressed in 42.1% of

the typical CLL patients and 97.3% of the atypical CLL patients.

Similarly, the level of CD22 expression was closely correlated

with CD20. CD79b expression was also positive in 38.8% of

the CLL patients, which was considered in normal ranges. The

percent positivity and intensity of CD79b expression in MCL,

atypical CLL, and typical CLL is still controversial. CD22 and

FMC7 expressions are generally higher in MCL patients, whereas

in the present study, they were higher in both the CLL and MCL

patients. For this reason, the majority of the patients (58.2%)

were classified as having atypical CLL when Matutes scoring was

used. Earlier studies stated that FMC7, CD79b, and CD22 are not

efficient in making a differential diagnosis [1,17,18,19]. Every

manufacturer produces MoAbs in different clones and different

stains. There is a need for validation and standardization studies

on these MoAbs. At this point, the present study had a limitation

because the results were not checked with the use of different

MoAbs of different clones from different manufacturers.

With regard to CD38, CD43, and CD200, which were not included

in the Matutes scoring system, the different results obtained

in the present study between the CLL and MCL patients could

be partially explained by the individual differences among

the patients as well as the absence of specific techniques and

procedures in the flow cytometry. In the present study, CD38

expression was higher in the MCL patients than in the CLL

patients (p<0.001) but heterogeneous in the CLL patients; thus,

it was difficult to standardize. In addition, the LPD may have

a fluctuating course [20,21,22,23]. All of these factors need to

be taken into account while making a differential diagnosis

between CLL and MCL.

CD43 expression was first defined in 1999 by Harris et al. [24]

for the classification of malignant lymphomas. In the present

study, CD43 expression was higher in the CLL patients compared

with that in the MCL patients and it was quite effective in

accurate classification of the patients having Matutes scores of

≤3 according to the classical classification (p<0.001) [25,26,27].

In the present study, while 95.8% of the CLL patients showed

moderate to strong CD200 expression (3.6% had negative

expression), 6.1% of the MCL patients showed positive CD200

expression (p<0.001). There was no significant difference

in CD200 expressions between the atypical and typical CLL

patients. Moreover, CD200 was constantly expressed in the

typical CLL patients and was an excellent marker for its

differential diagnosis from MCL, as previously shown in other

studies [14,15,16,17,18,19].

Study Limitation

The limitation of the present study was to not evaluate CD200

and CD43 expressions in other LPD groups. If these expressions

were evaluated in other LPD groups, other diseases besides CLL

and MCL would have also been evaluated with regard to CD200

and CD43 expressions. However, as the number of patients

with other diseases was low in the present study, they were not

included.

Conclusion

In conclusion, there has not been a single marker identified

yet to make a definite diagnosis of CLL by flow cytometry.

Therefore, new markers for the differential diagnosis of CLL are

under investigation. The results of the present study revealed

that CD43 and CD200 in particular were more valuable markers

than CD22, CD79b, and FMC7, which are within the scope of

the Matutes scoring system. CD43 and CD200 could also be

considered as definitive markers in atypical CLL patients for

whom the Matutes scoring system remains ineffective. However,

as with the other markers, their heterogeneous distribution

and different rates of expression may still be in question. For

this reason, large-scale harmonization studies are needed

for patients with various diseases by defining standardized

sample preparation and staining, as well as specific techniques.

Identification of a new scoring system following these studies

would also be beneficial.

Ethics

Ethics Committee Approval: Ankara Numune Education

and Training Hospital Ethics Committee (decision number:

12.06.2015-1028).

97

Falay M, et al: CLL and MCL Immunophenotyping Turk J Hematol 2018;35:94-98

Informed Consent: Retrospective study.

Authorship Contributions

Surgical and Medical Practices: G.Ö., B.A.Ö., K.G.; Concept: M.F.;

Design: M.F.; Data Collection or Processing: M.F.; Analysis

or Interpretation: M.F., Y.K., S.D, F.C; Literature Search: M.F.;

Writing: M.F.

Conflict of Interest: The authors of this paper have no conflicts

of interest, including specific financial interests, relationships,

and/or affiliations relevant to the subject matter or materials

included.

References

1. Campo E, Swerdlow SH, Harris NL, Pileri S, Stein H, Jaffe ES. The 2008 WHO

classification of lymphoid neoplasms and beyond: evolving concepts and

practical applications. Blood 2011;117:5019-5032.

2. Oscier D, Dearden C, Eren E, Fegan C, Follows G, Hillmen P, Illidge T,

Matutes E, Milligan DW, Pettitt A, Schuh A, Wimperis J; British Committee

for Standards in Haematology. Guidelines on the diagnosis, investigation

and management of chronic lymphocytic leukaemia. Br J Haematol

2012;159:541-564.

3. Costa ES, Pedreira CE, Barrena S, Lecrevisse Q, Flores J, Quijano S,

Almeida J, del Carmen García-Macias M, Bottcher S, Van Dongen JJ,

Orfao A. Automated pattern-guided principal component analysis

vs expert-based immunophenotypic classification of B-cell chronic

lymphoproliferative disorders: a step forward in the standardization of

clinical immunophenotyping. Leukemia 2010;24:1927-1933.

4. Braylan RC. Impact of flow cytometry on the diagnosis and characterization

of lymphomas, chronic lymphoproliferative disorders and plasma cell

neoplasias. Cytometry A 2004;58:57-61.

5. Dreyling M, Hiddemann W; European MCL Network. Current treatment

standards and emerging strategies in mantle cell lymphoma. Hematology

Am Soc Hematol Educ Program 2009:542-551.

6. Sánchez ML, Almeida J, Vidriales B, López-Berges MC, García-Marcos MA,

Moro MJ, Corrales A, Calmuntia MJ, San Miguel JF, Orfao A. Incidence

of phenotypic aberrations in a series of 467 patients with B chronic

lymphoproliferative disorders: basis for the design of specific four-color

stainings to be used for minimal residual disease investigation. Leukemia

2002;16:1460-1469.

7. Dronca RS, Jevremovic D, Hanson CA, Rabe KG, Shanafelt TD, Morice WG,

Call TG, Kay NE, Collins CS, Schwager SM, Slager SL, Zent CS. CD5-positive

chronic B-cell lymphoproliferative disorders: diagnosis and prognosis of

a heterogeneous disease entity. Cytometry B Clin Cytom 2010;78(Suppl

1):35-41.

8. Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A, Que

TH, Catovsky D. The immunological profile of B-cell disorders and proposal

of a scoring system for the diagnosis of CLL. Leukemia 1994;8:1640-1645.

9. Moreau EJ, Matutes E, A’Hern RP, Morilla AM, Morilla RM, Owusu-Ankomah

KA, Seon BK, Catovsky D. Improvement of the chronic lymphocytic leukemia

scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin

Pathol 1997;108:378-382.

10. Medd PG, Clark N, Leyden K, Turner S, Strefford JA, Butler C, Collins GP,

Roberts DJ, Atoyebi W, Hatton CS. A novel scoring system combining

expression of CD23, CD20, and CD38 with platelet count predicts for the

presence of the t(11;14) translocation of mantle cell lymphoma. Cytometry

B Clin Cytom 2011;80:230-237.

11. Kroft SH. Uncovering clinically relevant phenotypic variations in

malignancies: CD23 in mantle cell lymphoma. Am J Clin Pathol

2008;130:159-161.

12. Schlette E, Fu K, Medeiros LJ. CD23 expression in mantle cell lymphoma:

clinicopathologic features of 18 cases. Am J Clin Pathol 2003;120:760-766.

13. Gao J, Peterson L, Nelson B, Goolsby C, Chen YH. Immunophenotypic

variations in mantle cell lymphoma. Am J Clin Pathol 2009;132:699-706.

14. Kilo MN, Dorfman DM. The utility of flow cytometric immunophenotypic

analysis in the distinction of small lymphocytic lymphoma/chronic

lymphocytic leukemia from mantle cell lymphoma. Am J Clin Pathol

1996;105:451-457.

15. Kaleem Z. Flow cytometric analysis of lymphomas: current status and

usefulness. Arch Pathol Lab Med 2006;130:1850-1858.

16. Gong JZ, Lagoo AS, Peters D, Horvatinovich J, Benz P, Buckley PJ. Value

of CD23 determination by flow cytometry in differentiating mantle

cell lymphoma from chronic lymphocytic leukemia/small lymphocytic

lymphoma. Am J Clin Pathol 2001;116:893-897.

17. Stewart CC, Stewart SJ. Immunophenotyping. Immunophenotyping. Curr

Protoc Cytom 2001;6:6.2.

18. Kraus TS, Sillings CN, Saxe DF, Li S, Jaye DL. The role of CD11c expression in

the diagnosis of mantle cell lymphoma. Am J Clin Pathol 2010;134:271-277.

19. Angelopoulou MK, Kontopidou FN, Pangalis GA. Adhesion molecules in

B-chronic lymphoproliferative disorders. Semin Hematol 1999;36:178-197.

20. Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW, Stevenson FK,

Oscier DG. CD38 expression and immunoglobulin variable region mutations

are independent prognostic variables in chronic lymphocytic leukemia,

but CD38 expression may vary during the course of the disease. Blood

2002;99:1023-1029.

21. Matrai Z. CD38 as a prognostic marker in CLL. Hematology 2005;10:39-46.

22. Kröber A, Seiler T, Benner A, Bullinger L, Brückle E, Lichter P, Döhner H,

Stilgenbauer S. V(H) mutation status, CD38 expression level, genomic

aberrations, and survival in chronic lymphocytic leukemia. Blood

2002;100:1410-1416.

23. Thompson PA, Tam CS. CD38 expression in CLL: a dynamic marker of

prognosis. Leuk Lymphoma 2014;55:1-2.

24. Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman

J, Lister TA, Bloomfield CD. World Health Organization classification of

neoplastic diseases of the hematopoietic and lymphoid tissues: report of

the Clinical Advisory Committee meeting-Airlie House, Virginia, November

1997. J Clin Oncol 1999;17:3835-3849.

25. Durrieu F, Geneviève F, Arnoulet C, Brumpt C, Capiod JC, Degenne M, Feuillard

J, Garand R, Kara-Terki A, Kulhein E, Maynadié M, Ochoa-Noguera ME, Plesa

A, Roussel M, Eghbali H, Truchan-Graczyk M, de Carvalho Bittencourt M,

Feugier P, Béné MC. Normal levels of peripheral CD19 + CD5 + CLL-like cells:

toward a defined threshold for CLL follow-up-a GEIL-GOELAMS study.

Cytometry B Clin Cytom 2011;80:346-353.

26. Deneys V, Michaux L, Leveugle P, Mazzon AM, Gillis E, Ferrant A, Scheiff JM,

De Bruyère M. Atypical lymphocytic leukemia and mantle cell lymphoma

immunologically very close: flow cytometric distinction by the use of CD20

and CD54 expression. Leukemia 2001;15:1458-1465.

27. Jung G, Eisenmann JC, Thiébault S, Hénon P. Cell surface CD43 determination

improves diagnostic precision in late B-cell diseases. Br J Haematol

2003;120:496-499.

98

RESEARCH ARTICLE

DOI: 10.4274/tjh.2017.0106


Association of Interleukin-2-330T/G and Interleukin-10-1082A/G

Genetic Polymorphisms with B-Cell Non-Hodgkin Lymphoma in a

Cohort of Egyptians

Bir Mısırlı Hasta Kohortunda İnterlökin-2-330T/G ve İnterlökin-10-1082A/G Genetik

Polimorfizmlerinin B-Hücreli Hodgkin Dışı Lenfoma ile İlişkisi

Hala Aly Abdel Rahman 1 , Mervat Mamdooh Khorshied 1 , Ola Mohamed Reda Khorshid 2 , Heba Mahmoud Mourad 1

1

Cairo University Kasr Alainy Faculty of Medicine, Department of Clinical and Chemical Pathology, Cairo, Egypt

2

Cairo University National Cancer Institute, Department of Medical Oncology, Cairo, Egypt

Abstract

Objective: Polymorphisms in the interleukin (IL)-2 and IL-10 genes

are known to be associated with susceptibility to different immunedysregulated

disorders and cancers such as non-Hodgkin lymphoma

(NHL). To explore the possible association between IL-2-330T/G and IL-

10-1082A/G single-nucleotide polymorphisms and the susceptibility

to B-cell NHL (B-NHL) in Egyptians, we conducted a case-control

study.

Materials and Methods: Genotyping of the studied genetic variations

was done for 100 B-NHL patients as well as 100 age- and sex-matched

healthy controls.

Results: The IL-2 variant allele occurred at a significantly higher rate

in patients than controls and was associated with susceptibility to

B-NHL [odds ratio (OR): 1.91, 95% confidence interval (CI): 1.28-2.85].

It was also associated with advanced performance status score. IL-2

polymorphism conferred an almost threefold increased risk of diffuse

large B-cell lymphoma (OR: 2.64, 95% CI: 1.35-5.15) and a fourfold

increased risk of indolent subtypes (OR: 4.34, 95% CI: 1.20-15.7). The

distribution of IL-10-1082A/G genotypes in our patients was close

to that of the controls. Co-inheritance of the variant genotypes of

IL-2 and the common genotype of IL-10 conferred an almost sixfold

increased risk (OR: 5.75, 95% CI: 1.39-23.72), while co-inheritance of

the variant genotypes of IL-2 and IL-10 conferred fivefold increased

risk of B-NHL (OR: 5.43, 95% CI: 1.44-20.45). The variant genotypes

of IL-2-330T/G and IL-10-1082A/G had no effect on the disease-free

survival of B-NHL patients.

Conclusion: The present study highlights the possible involvement of

the IL-2-330T/G genetic polymorphism in the susceptibility to B-NHL

in Egypt, especially indolent subtypes. Moreover, IL-10-1082A/G is not

a molecular susceptibility marker for B-NHL in Egyptians.

Keywords: Interleukin-2-330T/G, rs2069762, Interleukin-10-1082A/G,

rs1800896, B-cell non-Hodgkin lymphoma, Egypt

Öz

Amaç: İnterlökin (IL)-2 ve IL-10 genlerindeki polimorfizmlerin değişik

immün bozukluklar ve Hodgkin dışı lenfoma (HDL) gibi kanserlere

duyarlılık ile ilişkili olduğu bilinmektedir. Mısırlılardaki IL-2-330T/G

ve IL-10-1082A/G tek nükleotid polimorfizmleri ile B hücreli HDL’ye

(B-HDL) duyarlılık arasındaki olası ilişkinin araştırılması için bir olgukontrol

çalışması yapılmıştır.

Gereç ve Yöntemler: Bahsedilen genetik varyasyonlar için, 100 B-HDL

hastası ve yaş ile cinsiyet uyumlu 100 sağlıklı kontrole genotipleme

yapıldı.

Bulgular: IL-2 varyant alleli hastalarda kontrollere göre anlamlı olarak

daha yüksekti ve B-HDL duyarlılığı ile ilişkili bulundu [olasılık oranı

(OO): 1,91, %95 güven aralığı (GA): 1,28-2,85). Ayrıca bunun ileri

performans skoru ile de ilişkili olduğu görüldü. IL-2 polimorfizminin

diffüz büyük B hücreli lenfoma için yaklaşık üç kat (OO: 2,64; %95

GA: 1,35-5,15) ve yavaş seyirli (indolan) alt tiplerde dört kat artış

doğurmaktaydı (OO: 4,34, %95 GA: 1,20-15,7). IL-10-1082A/G

genotiplerinin dağılımı hastalar ve kontrollerde benzerdi. IL-2 varyant

genotipleri ile IL-10’un sık rastlanan genotiplerinin eş kalıtımı yaklaşık

altı kat artmış risk (OO: 5,75, %95 GA: 1,39-23,72) yaratmaktayken,

IL-2 ve IL-10 varyant genotiplerinin eş kalıtımı B-HDL riskinde beş

kat artışa (OO: 5,43, %95 GA: 1,44-20,45) neden olmaktaydı. IL-2-

330T/G ve IL-10-1082A/G variant genotiplerinin B-HDL hastalarında

hastalıksız sağkalım üzerine etkisi yoktu.

Sonuç: Bu çalışma Mısır’da, IL-2-330T/G genetik polimorfizmlerinin

özellikle yavaş seyirli B-HDL’ye yatkınlık ile olası ilişkisini

vurgulamaktadır. Ayrıca Mısırlılarda IL-10-1082A/G, B-HDL için

duyarlı bir moleküler belirteç değildir.

Anahtar Sözcükler: İnterlökin-2-330T/G, rs2069762, İnterlökin-10-

1082A/G, rs1800896, B-hücreli Hodgkin dışı lenfoma, Mısır



Address for Correspondence/Yazışma Adresi: Mervat MAMDOOH KHORSHIED, M.D.,

Cairo University Kasr Alainy Faculty of Medicine, Department of Clinical and Chemical Pathology, Cairo, Egypt

Phone : +202 235 644 80

E-mail : mervatkhorshied@hotmail.com ORCID-ID: orcid.org/0000-0003-2052-3768



99

Rahman HAA., et al: IL-2 and -10 Polymorphisms and B-NHL


Introduction

Despite the fact that there are some proven non-Hodgkin

lymphoma (NHL) risk factors, the etiology of NHL still warrants

extensive investigations [1]. Interleukin-2 (IL-2) has multiple

opposing functions in the immune system. It plays a master

role in T-cell growth and activation and in natural killer cellmediated

immune responses [2]. It has been reported to have

antitumor effects through its contribution in the development

of regulatory T cells, as well as expansion and apoptosis among

activated T cells [3]. It is postulated that low production of

IL-2 can suppress the antitumor response via the antibodydependent

cellular cytotoxicity (ADCC) seen in NHL patients,

thus increasing the susceptibility to develop NHL [4].

IL-10 has both immunosuppressive and antiangiogenic functions.

It thus has tumor-promoting as well as tumor-suppressing

properties [5]. It may protect malignant cells through the

inhibition of cytotoxic T lymphocyte-mediated tumor-specific

cell lysis. Thus, IL-10 has an important role in carcinogenesis and

it is postulated that it affects cancer risk, specifically for NHL

[6]. The IL-10 promotor region may influence its expression and

consequently alter susceptibility to NHL and disease outcome.

It has been hypothesized that decreased production of IL-10

may increase the risk of NHL by less effectively downregulating

the production of proinflammatory cytokines [7]. Accordingly,

genetic factors that downregulate IL-10 production may provide

a proinflammatory medium that favors lymphomagenesis [8].

However, other studies have hypothesized that IL-10, which is a

B-cell stimulatory cytokine, could promote lymphomagenesis [9].

Therefore, these conflicting findings suggest that dysregulation

in IL-10 in general could be a pivotal factor in NHL development.

The aim of the current work was to study the possible role of IL-

2-330T/G (rs2069762) and IL-10-1082A/G (rs1800896) singlenucleotide

polymorphisms (SNPs) as genetic risk factors for

B-cell NHL (B-NHL) in a group of Egyptian patients.


Study Population

This case-control study included 100 adult Egyptian B-NHL

patients recruited from the Department of Medical Oncology,

National Cancer Institute (NCI), Cairo University. These

comprised either de novo cases or patients attending the NCI

for follow-up. There were 54 males and 46 females. Their ages

ranged between 20 and 83 years with a mean age of 52.7 years.

One hundred unrelated age- and sex-matched volunteers were

included in the study as a control group. The research protocol

was approved by the Research Ethics Committee of the Kasr Al

Ainy Faculty of Medicine, Cairo University. From all participants,

informed consent was obtained in writing, and all procedures

were in accordance with the 1964 Helsinki Declaration.

Diagnosis and subtyping of B-NHL was performed according to

the World Health Organization classification of 2008. Patients

were subjected to thorough clinical examinations, as well as

laboratory investigations and radiological work-up for proper

clinical assessment. The demographic and clinical features of

the B-NHL patients are presented in Table 1.

Genotyping of IL-2-330T/G (rs2069762) and IL-10-1082A/G

(rs1800896)

DNA extraction from peripheral blood leukocytes was done

with the GeneJET Whole Blood Genomic DNA Purification

Mini Kit (Fermentas Life Sciences, Canada) according to the

manufacturer’s instructions. Samples were stored in the elution

buffer at -20 °C until use.

Detection of the IL-2-330T/G (rs2069762) SNP was performed

with the polymerase chain reaction-restriction fragment length

polymorphism (PCR-RFLP) technique according to Cavet et al.

[10]. The primer set used was as follows: forward, 5’-TAT TCA

CAT GTT CAG TGT AGT TCT-3’; and reverse, 5’-AGA CTG ACT

GAA TGG ATG TAG GTG-3’. Amplification was performed in a

thermocycler (PerkinElmer 9700; PerkinElmer, USA) using the

following program: 94 °C (5 min); then 30 cycles of 94 °C (1

min), 48° C (1 min), and 72 °C (1 min); and a final extension step

for 8 min at 72 °C. This SNP abolishes the restriction site that

can be recognized by the MaeI restriction enzyme; accordingly,

the T allele was restricted into two bands of 124 and 64 bp,

while the G allele remained a 188-bp band.

Genotyping of the IL-10-1082A/G (rs1800896) SNP was

performed by allele-specific PCR (ARMS) technique [11]. The

following primers were used: F-5’-AGCAACACTCCTCGTCGCAAC,

with either B1-5’-CCTATCCCTACTTCCCCC (G allele) or B2-5’-

CCTATCCCTACTTCCCCT (A allele). The thermocycler program

applied was 95 °C (10 min); then 30 cycles of 94 °C (30 s), 60 °C

(1 min), and 72 °C (1 min); and a final extension step for 7 min

at 72 °C. The AA genotype was identified by a single 153-bp

band in tube B2, while the homozygous variant (GG) showed

a 153-bp band in tube B1. The heterozygous variant (AG) was

identified by a 153-bp band in both tubes. To validate our results,

re-genotyping of 40 samples with respect to case-control status

was performed. The results were interpreted blindly and found

to be 100% concordant.

Treatment Regimen and Response to Therapy

All patients received the standard protocol treatment for NHL

at the NCI of Cairo University. Diffuse large B-cell lymphoma

(DLBCL) patients were treated according to stage and bulkiness.

Non-bulky (<10 cm) stage I-II cases including extranodal

presentations received 4 cycles of R-CHOP/21 days [rituximab at

100



Table 1. Demographic and clinical data of B-cell non-Hodgkin

lymphoma patients at presentation and their response to

therapy.

Item

B-NHL patients

(n=100)

Male 54/100

Sex

Female 46/100

B-symptoms: Fever, night sweats, weight

loss

25/100

Lymphadenopathy 81/100

Groups of lymph

nodes involved

Extranodal involvement

<2

≥2

Cervical 61/100

Axillary 42/100

Inguinal 38/100

Abdominal 33/100

Para-aortic 24/100

Submandibular 20/100

Mesenteric 9/100

79/100

21/100

Splenomegaly 40/100

Hepatomegaly 38/100

Clinical stage

I & II

III & IV

PS

Score <2

Score ≥2

IPI risk group

Low

Intermediate/High

IPI risk groups for DLBCL subtype (n=78)

Low/Intermediate low (1, 2)

Intermediate high/High (3, 4)

Histological aggressiveness

Indolent

Aggressive

Regimen of treatment

Chemotherapy

Chemotherapy & radiotherapy

No treatment

Response to treatment

CR

Non-CR

PR

PD

SD

Unavailable

23/100

77/100

61/100

39/100

29/100

71/100

44/78

34/78

21/100

79/100

78/100

17/100

5/100

59/100

27/100

17/100

4/100

6/100

14/100

PS: Performance status, IPI: International Prognostic Index, CR: complete remission,

PR: partial remission, PD: progressive disease, SD: stable disease, B-NHL: B-cell non-

Hodgkin lymphoma.

375 mg/m 2 , cyclophosphamide at 750 mg/m 2 , doxorubicin at 50

mg/m 2 , vincristine at 2 mg total dose, and prednisone at 100 mg

for 5 days, followed by involved field radiotherapy (IFRT)]. Stage

III or IV patients received 6-8 cycles of R-CHOP guided by the

patient’s response by positron emission tomography–computed

tomography, which was done after 4 cycles. Patients with initial

bulky disease received IFRT after their chemotherapy cycles.

Follicular lymphoma of stage I and II was treated with IFRT only,

while stages III and IV were treated if patients met the Groupe

d’Etude des Lymphomes Folliculaires criteria for initiation of

treatment. Mantle cell lymphoma patients were treated with

R-CHOP alternated with R-DHAP. Therapeutic responses were

assessed according to Oken et al. [12].


Data management and analysis were performed using SPSS

21. Data were explored for normality using the Kolmogorov-

Smirnov test and the Shapiro-Wilk test. Comparisons between

groups for parametric numeric variables were done using the

Student t-test, while for non-parametric numeric variables,

comparisons were done by the Mann-Whitney U test. Chi-square

or Fisher exact tests were used for comparing categorical data.

For risk estimation, the odds ratio (OR) and 95% confidence

interval (CI) were calculated. The Kaplan-Meier method was

used to assess disease-free survival (DFS). Differences between

survival curves were evaluated for statistical significance with

the log-rank test. All p-values are two-sided and p<0.05 was

considered significant.

Results

The genotypic and allelic frequencies of the IL-2-330T/G and IL-

10-1082A/G SNPs in B-NHL patients and controls are presented

in Tables 2 and 3. The genotypic distribution of the studied SNPs

was in agreement with Hardy-Weinberg equilibrium (p>0.05).

The IL-2-330T/G variant genotypes (TG and GG) are associated

with B-NHL risk, and the risk was higher for the indolent subtypes.

Statistical comparison revealed that a performance status score

of ≥2 was more common in patients harboring the variant

genotypes (Supplementary Tables 1 and 2). The distribution

of the variant genotypes of IL-10-1082A/G (AG and GG) did

not differ between B-NHL patients and controls. Extranodal

involvement of ≥2 sites was statistically more common in

patients having the common genotype (Supplementary Tables

3 and 4). Combined genotype analysis showed that B-NHL risk

increased almost sixfold in those having the variant genotypes

of IL-2-330T/G and the common genotype of IL-10-1082A/G

(AA), while co-inheritance of the variant genotypes of both

SNPs was associated with fivefold increased risk of B-NHL (OR:

5.43, 95% CI: 1.44-20.45).

101



Table 2. Distribution of interleukin-2-330T/G and interleukin-10-1082A/G genotypes in B-cell non-Hodgkin lymphoma patients

and controls.

Genotypes Controls, n (%) B-NHL patients, n (%) OR 95% CI p-value

TT 42 (42%) 20 (20%) (1) Ref.

IL-2-330T/G

TG 26 (26%) 38 (38%) 3.07 1.48-6.37 0.003

GG 32 (32%) 42 (42%) 2.76 1.36-5.57 0.005

TG & GG 58 (58%) 80 (80%) 2.90 1.54-5.44 0.001

T allele 0.55 0.39

G allele 0.45 0.61

1.91 1.28-2.85 <0.001

AA 28 (28%) 26 (26%) (1) Ref.

IL-10-1082A/G

Combined

genotypes

analysis,

IL-2/IL-10

GA 59 (59%) 51 (51%) 1.91 0.80-4.52 0.144

GG 13 (13%) 23 (23%) 0.93 0.49-1.79 0.830

GA & GG 72 (72%) 74 (74%) 1.11 0.59-2.07 0.750

A allele 0.575 0.515

G allele 0.425 0.485

0.79 0.53-1.16 0.228

TT/AA 3 (3%) 12 (12%) (1) Ref.

TT/GA &/or GG 17 (17%) 30 (30%) 2.27 0.56-9.17 0.251

TG &/or GG / AA 23 (23%) 16 (16%) 5.75 1.39-23.72 0.016

TG &/or GG / GA &/or GG 57 (57%) 42 (42%) 5.43 1.44-20.45 0.012

OR: Odds ratio, 95% CI: 95% confidence interval, Ref.: reference, B-NHL: B-cell non-Hodgkin lymphoma, IL: interleukin.

Table 3. Distribution of interleukin-2-330T/G and interleukin-10-1082A/G genotypes in indolent and aggressive subtypes of

B-cell non-Hodgkin lymphoma patients and controls.

Genotypes

Controls

(n=100)

Indolent B-NHL

(n=21)

OR

(95% CI)

p-value

Aggressive

B-NHL (n=79)

OR (95% CI)

p-value

IL-2-330

T/G

TT 42 (42%) 3 (14.3%) 4.34

17 (21.5%) 2.64

0.017

TG & GG 58 (58%) 18 (85.7%) (1.2-15.71)

62 (78.5%) (1.35-5.15)

0.004

IL-10-1082

A/G

AA 28 (28%) 3 (14.3%) 2.33

23 (29.1%) 0.95

0.191

(0.64-8.54)

(0.49-1.82)

GA & GG 72 (72%) 18 (85.7%) 56 (70.9%)

OR: Odds ratio, 95% CI: 95% confidence interval, B-NHL: B-cell non-Hodgkin lymphoma, IL: interleukin.

0.870

Regarding the potential role of these SNPs as molecular prognostic

markers, the 3-year and 5-year DFS rates were estimated. The

3-year DFS rate for the variant genotypes (GG or TG) of IL-2-

330T/G was 65.4% versus 69.2% for the common genotype (TT),

while the 5-year DFS rate for the variant genotypes (GG or TG)

was 45.3% versus 69.2% for the common genotype (TT) with

no statistically significant difference (p=0.211). The 3-year DFS

rate for the variant genotypes (GG or AG) of IL-10-1082A/G

was 60.7% versus 79.5% for the common genotype (AA), while

the 5-year DFS rate for the variant genotypes (GG or AG) was

49.1% versus 39.8% for the common genotype (AA), which was

statistically insignificant (p=0.205). Other potential prognostic

factors, such as the patients’ age at diagnosis, sex, clinical

stage, performance status, International Prognostic Index score,

extranodal involvement, and histopathological subtypes, did not

affect the DFS of our B-NHL patients (Supplementary Table 5).

Discussion

The relationship between the IL-2-330T/G SNP and NHL remains

ambiguous. Some studies showed that the variant (G) allele

correlates with decreased IL-2 production in vivo [13]. It has

been suggested that reduced IL-2 levels may downregulate the

antitumor response through ADCC and thus increase the risk of

NHL [4]. In the present study, 38% of B-NHL patients had the

102



heterozygous genotype (TG), while 42% had the homozygous

genotype (GG). These frequencies differed from those reported

by Song et al. [4], being 56.2% and 12.7% for the TG and

GG genotypes in Chinese NHL patients. This might be due to

ethnicity. In the study presented here, the frequency of the

variant genotypes was significantly higher in patients than

controls and was associated with increased risk of B-NHL among

Egyptians. This is in agreement with the study of Song et al. [4]

involving Chinese patients.

The IL-2-330T/G polymorphism was associated with advanced

performance status score. Otherwise, there was no association

between the IL-2-330T/G SNP and sex, presenting symptoms,

or other clinical and laboratory features, as well as response to

therapy. Song et al. [4] could not find any association between

the IL-2-330T/G SNP and clinical features in the Chinese patients

in their study. Based on the clinical behavior of the disease, our

patients were stratified into cases of indolent and aggressive

lymphomas. IL-2-330T/G polymorphic genotypes were found to

confer threefold increased risk of DLBCL, and the increase in risk

for indolent B-NHL was fourfold.

Being an anti-inflammatory cytokine, the main functions of IL-

10 are suppression of cytokine synthesis in Th1 cells as well as

downregulation of cytotoxic and cell-mediated inflammatory

responses [14]. It acts as an autocrine growth factor that

upregulates BCL-2 expression in some cases of B-cell neoplasms

[15]. High IL-10 levels were shown to be associated with poor

outcomes and shorter survival in B-NHL patients [16,17].

Genetic polymorphisms in the promotor area of the IL-10

gene have been reported to influence IL-10 levels. IL-10-1082

common (A) and variant (G) alleles respectively correlate with

low and high IL-10 expression levels [18]. Several studies have

investigated the association of IL-10 gene polymorphisms and

NHL susceptibility, reporting conflicting results. In the current

study, 74% of B-NHL patients harbored this genetic variation,

with 51% being heterozygous (AG) and 23% homozygous

(GG). These frequencies agree with those previously reported in

Australian patients, being 51% and 29% for AG and GG variant

genotypes, respectively [19]. Similarly, Lan et al. [20] found the

AG and GG genotypes in 52% and 23% of their female American

B-NHL patients, and these frequencies were close to those of

their controls. Extranodal involvement (i.e. the involvement of

≥2 extranodal sites) was more prominent in patients having

the common genotype. Otherwise, there were no statistical

differences between patients harboring the common or the

variant genotypes. Lech-Maranda et al. [24] found that DLBCL

patients harboring the variant genotypes had slightly higher

complete remission (CR) rates. They stated that patients with

elevated cytokine levels had significantly lower CR rates.

IL-10-1082A/G variant genotypes (AG and GG) were not

associated with susceptibility to either indolent or aggressive

B-NHL subtypes. Similar results were reported by Talaat et

al. [21], who concluded that IL-10-1082A/G polymorphic

genotypes could not be considered as a genetic risk factor for

DLBCL in Egyptians. Moreover, the studies of Kube et al. [22]

and Berglund et al. [23] revealed that the IL-10-1082A/G SNP

was not associated with susceptibility to aggressive B-NHL in

German or Swedish populations, respectively. Contrary to our

results, Purdue et al. [19] found that the frequency of the

variant genotypes conferred increased risk of DLBCL. Lech-

Maranda et al. [24] reported a similar frequency of the variant

genotypes in France, which was statistically significant when

compared to controls. They considered the IL-10-1082A/G SNP

as a genetic risk factor for DLBCL in the French population. Lan

et al. [20] stated that the GG homozygous variant genotype

was significantly associated with an increased risk for DLBCL

in female Americans. However, Cunningham et al. [25]

reported that the low-producing IL-10-1082 AA genotype

was significantly higher in patients with aggressive lymphoma

compared to controls.

Combined genotype analysis showed that B-NHL risk was

increased when IL-2-330T/G variant genotypes were coinherited

with either common or variant genotypes of IL-

10-1082A/G. Accordingly, we assume that B-NHL risk can be

attributed to the IL-2 rather than the IL-10 SNP.

Regarding DFS, none of the potentially known prognostic

factors affected the DFS of B-NHL patients. Furthermore, the

polymorphic genotypes of either IL-2-330T/G or IL-10-1082A/G

had no effect on the 3- and 5-year DFS rates of these patients.

Study Limitations

The relatively small sample size of this study is a limitation of

the present work. Larger sample size is recommended to validate

our results regarding the role of the studied SNPs as molecular

risk factors for B-NHL and to clarify their impact on therapeutic

response and disease course. Furthermore, IL-2 and IL-10

levels should have been examined to conclude the association

between the examined variations and NHL.

Conclusion

The current study highlights the possible involvement of the

IL-2-330T/G SNP in susceptibility to B-NHL. Moreover, IL-10-

1082A/G is not a molecular susceptibility marker for B-NHL in

Egyptians.

103



Ethics

Ethics Committee Approval: The research protocol was

approved by the Research Ethics Committee of the Departments

of Clinical Pathology and Medical Oncology, Cairo University,

and all procedures were performed in accordance with the 1964

Helsinki Declaration.

Informed Consent: Informed written consent was obtained

from all participants prior to enrollment in the study.

Supplementary Table 1. Comparison between B-cell non-Hodgkin lymphoma patients having wild genotype and polymorphic

genotypes of interleukin-2-330T/G regarding their clinical data.

Item

No (%)

IL-2 wild genotype

(n=20)

No (%)

IL-2 polymorphic genotypes

(n=80)

p-value

Sex

Male

Female

12/20 (60%)

8/20 (40%)

42/80 (52.5%)

38/80 (47.5%)

0.547

B-symptoms 6/20 (30%) 19/80 (23.8%) 0.564

Lymphadenopathy 17/20 (85%) 64/80 (80%) 0.610

Groups

of lymph

nodes

involved


<2

≥2

Cervical 14/20 (70%) 47/80 (58.8%) 0.356

Axillary 8/20 (40%) 34/80 (42.5%) 0.939

Inguinal 8/20 (40%) 30/80 (37.5%) 0.937

Submandibular 6/20 (30%) 14/80 (17.5%) 0.211

Abdominal 8/20 (40%) 25/80 (31.3%) 0.457

Mesenteric 4/20 (20%) 5/80 (6.3%) 0.055

Para-aortic 8/20 (40%) 16/80 (20%) 0.061

18/20 (90%)

2/20 (10%)

61/80 (76.3%)

19/80 (23.7%)

Splenomegaly 8/20 (40%) 32/80 (40%) 1.0

Hepatomegaly 6/20 (30%) 32/80 (40%) 0.453

Clinical stage

I & II

III & IV

PS

Score <2

Score ≥2

IPI risk group

Low

Intermediate/high

IPI risk groups for DLBCL subtype


Intermediate high/high (3, 4)

Treatment outcome

CR

Non-CR (PR, PD, SD)

Unavailable

Pathology

Indolent

Aggressive

8/20 (40%)

12/20 (60%)

16/20 (80%)

4/20 (20%)

9/20 (45%)

11/20 (55%)

12/17 (70.6%)

5/17 (29.4%)

12/20 (60%)

5/20 (25%)

3/20 (15%)

3/20 (15%)

17/20 (85%)

15/80 (18.8%)

65/80 (81.2%)

45/80 (56.3%)

35/80 (43.7%)

20/80 (25%)

60/80 (75%)

32/61 (52.5%)

29/61 (47.5%)

47/80 (58.8%)

22/80 (27.5%)

11/80 (13.7%)

18/80 (22.5%)

62/80 (77.5%)

*p-value <0.05 = significant, PS: Performance status, IPI: International Prognostic Index, CR: complete remission, PR: partial remission, PD: progressive disease, SD: stable disease,

IL: interleukin, DLBCL: Diffuse large B-cell lymphoma.

0.230

0.071

0.05*

0.078

0.183

0.971

0.461

104



Supplementary Table 2. Comparison between B-cell non-Hodgkin lymphoma patients with wild genotype and those with

polymorphic genotypes of interleukin-2-330T/G regarding their hematological data.

Item IL-2 wild genotype IL-2 polymorphic genotypes

Range Median Mean ± SD Range Median Mean ± SD

Hb, g/dL 9.60-16.70 12.80 12.59±1.83 5-17 11.85 11.68±2.34 0.092

p-value

Hemogram

TLC, x10 3 /cm 3 1.50-50 8.55 10.64±9.87 1.90-80 7.55 8.89±8.73 0.274

Plts, x10 3 /cm 3 133-491 300.50 281.25±113.37 14-675 278.50 295.40±138.87 0.829

LDH, IU/L 130-3531 239 499.45±751.66 135-3664 359 521.09±626.45 0.196

Hb: Hemoglobin, TLC: total leukocyte count, LDH: lactate dehydrogenase, Plts: platelets, IL: interleukin.


polymorphic genotypes of interleukin-10-1082A/G regarding their clinical data.

Item

IL-10 wild genotype

(n=26)


(n=74)

p-value

No. (%) No. (%)

Sex

Male

Female

11/26 (42.3%)

15/26 (57.7%)

43/74 (58.1%)

31/74 (41.9%)

0.164

B-symptoms 7/26 (26.9%) 18/74 (24.3%) 0.792

Lymphadenopathy 22/26 (84.6%) 59/74 (79.7%) 0.585

Cervical 15/26 (57.7%) 46/74 (62.2%) 0.688

Axillary 13/26 (50%) 29/74 (39.2%) 0.337

Inguinal 13/26 (50%) 25/74 (33.8%) 0.143

Groups of lymph

nodes involved

Submandibular 3/26 (11.5%) 17/74 (23%) 0.210

Abdominal 10/26 (38.5%) 23/74 (31.1%) 0.491

Mesenteric 1/26 (3.9%) 8/74 (10.8%) 0.439

Para-aortic 4/26 (15.4%) 20/74 (27%) 0.232


<2

16/26 (61.5%)

63/74 (85.1%)

≥2

10/26 (38.5%)

11/74 (14.9%)

0.011*

Splenomegaly 10/26 (38.5%) 30/74 (40.5%) 0.852

Hepatomegaly 10/26 (38.5%) 28/74 (37.8%) 0.955

Clinical stage

I & II

III & IV

PS

Score <2

Score ≥2

IPI risk group

Low

Intermediate/High

IPI risk groups for DLBCL subtype


Intermediate high/High (3, 4)

Treatment outcome

CR

Non-CR (PR, PD, SD)

Unavailable

Pathology

Indolent

Aggressive

*p-value <0.05=significant.

4/26 (15.4%)

22/26 (84.6%)

14/26 (53.9%)

12/26 (46.1%)

4/26 (15.4%)

22/26 (84.6%)

12/23 (52.2%)

11/23 (47.8%)

14/26 (53.8%)

8/26 (30.8%)

4/26 (15.4%)

3/26 (11.5%)

23/26 (88.7%)

19/74 (25.7%)

55/74 (74.3%)

47/74 (63.5%)

27/74 (36.5%)

25/74 (33.8%)

49/74 (66.2%)

32/55 (58.2%)

23/55 (41.8%)

45/74 (60.8%)

19/74 (25.7%)

10/74 (13.5%)

18/74 (24.3%)

56/74 (75.7%)

PS: Performance status, IPI: International Prognostic Index, CR: complete remission, PR: partial remission, PD: progressive disease, SD: stable disease, DLBCL: Diffuse large B-cell, IL: interleukin.

0.283

0.385

0.075

0.626

0.822

0.169

105




polymorphic genotypes of interleukin-10-1082A/G regarding their hematological data.

Item

IL-10 wild genotype


Range Median Mean ± SD Range Median Mean ± SD

p-value

Hb, g/dL 5-16.70 12 11.47±2.36 6.50-17 12 12±2.24 0.366

Hemogram

TLC, x10 3 /cm 3 3.20-80 7.30 10.18±14.46 1.50-50 8 8.91±6.04 0.368

Plts, x10 3 /cm 3 23-675 294 287.96±160.88 14-644 278.50 294.19±124.04 0.771

LDH, IU/L 155-2922 350 539.42±582.06 130-3664 297.50 508.79±674.97 0.346

Hb: Hemoglobin, TLC: total leukocyte count, LDH: lactate dehydrogenase, Plts: platelets, IL: interleukin, SD: standard deviation.

Supplementary Table 5. Disease-free survival of B-cell non-Hodgkin lymphoma patients.

Factors

Number of

cases

Number of

relapses

3 years 5 years Median p-value

All 100 22 61.8 49.6 56.5

Age

<60 71 16 68.5 56.2 130.6

≥60 29 6 57.1 28.6 56.5

Sex

Male 54 12 61.6 35.2 130.6

Female 46 10 67.2 67.2 47.4

Stage

I & II 23 4 71.1 71.1 -

III & IV 77 18 65.1 46.8 56.5

IPI

Intermediate/High 71 17 63.3 49.7 56.5

Low 29 5 76.7 38.4 47.4

IPI for DLBCL

Interm. high/High 34 8 49.8 33.2 31.8

Low/Interm/low 44 6 77.0 57.7 -

B-symp.

No 75 16 68.7 61.8 130.6

Yes 25 6 60.9 22.9 36.8

PS

<2 61 11 78.2 57.9 230.0

≥2 39 11 48.7 36.5 31.8

Spleen

No 60 12 71.9 46.2 56.5

Yes 40 10 57.4 47.8 36.8

Liver

No 62 12 70.7 49.5 56.5

Yes 38 10 59.3 49.4 36.8

Extranodal

<2 79 18 66.0 46.4 56.5

≥2 21 4 64.9 64.9 -

Hb

Abnormal 57 15 56.1 48.1 36.8

Normal 43 7 76.3 66.8 -

TLC

<11,000 79 16 66.9 50.2 -

≥11,000 21 6 84.4 36.8 36.8

Plts

≤150 17 3 85.7 64.3 130.6

>150 83 19 61.9 45.8 56.5

0.318

0.989

0.868

0.903

0.156

0.689

0.153

0.840

0.541

0.932

0.178

0.433

0.333

106



Supplementary Table 5. Continue.

LDH

Elevated 61 16 64.6 60.0 130.6

Normal 39 6 77.0 38.5 47.4

Pathology

Aggressive 79 15 64.6 48.4 56.5

Indolent 21 7 68.0 56.7 230.8

IL-2

TT 20 2 69.2 69.2 -

TG & GG 80 20 65.4 45.3 56.5

IL-10

AA 26 4 79.5 39.8 -

AG & GG 74 18 60.7 49.1 47.4

Hb: Hemoglobin, TLC: total leukocyte count, LDH: lactate dehydrogenase, Plts: platelets, IL: interleukin, IPI: International Prognostic Index.

0.616

0.889

0.211

0.205


Surgical and Medical Practices: O.M.R.K.; Concept: H.A.R.,

O.M.R.K.; Design: M.M.K., H.A.A.R. O.M.R.K.; Data Collection or

Processing: H.M.M.; Analysis or Interpretation: M.M.K., H.A.R.;

Literature Search: M.M.K., H.M.M.; Writing: M.M.K., H.M.M.,

H.A.R., O.M.R.K.

Conflict of Interest: The authors of this paper have no conflicts of

interest, including specific financial interests, relationships, and/or

affiliations relevant to the subject matter or materials included.

References

1. Wróbel T, Mazur G, Dzietczenia J, Gebura K, Kuliczkowski K, Bogunia-Kubik

K. VEGF and bFGF gene polymorphisms in patients with non-Hodgkin’s

lymphoma. Biomed Res Int 2013;2013:159813.

2. Gerber SA, Sorensen EW, Sedlacek AL, Lim JY, Skrombolas D, Frelinger JG,

Lord EM. Local expression of interleukin-2 by B16 melanoma cells results in

decreased tumour growth and long-term tumour dormancy. Immunology

2013;138:280-292.

3. D’Souza WN, Lefrançois L. IL-2 is not required for the initiation of CD8 T cell

cycling but sustains expansion. J Immunol 2003;171:5727-5735.

4. Song H, Chen L, Cha Z, Bai J. Interleukin 2 gene polymorphisms are

associated with non-Hodgkin lymphoma. DNA Cell Biol 2012; 31:1279-

1284.

5. Howell WM, Rose-Zerilli MJ. Cytokine gene polymorphisms, cancer

susceptibility, and prognosis. J Nutr 2007;137(1 Suppl):194-199.

6. Lech-Maranda E, Bienvenu J, Michallet AS, Houot R, Robak T, Coiffier B,

Salles G. Elevated IL-10 plasma levels correlate with poor prognosis in

diffuse large B-cell lymphoma. Eur Cytokine Netw 2006;17:60-66.

7. Rothman N, Skibola CF, Wang SS, Morgan G, Lan Q, Smith MT, Spinelli JJ,

Willett E, De Sanjose S, Cocco P, Berndt SI, Brennan P, Brooks-Wilson A,

Wacholder S, Becker N, Hartge P, Zheng T, Roman E, Holly EA, Boffetta P,

Armstrong B, Cozen W, Linet M, Bosch FX, Ennas MG, Holford TR, Gallagher

RP, Rollinson S, Bracci PM, Cerhan JR, Whitby D, Moore PS, Leaderer B,

Lai A, Spink C, Davis S, Bosch R, Scarpa A, Zhang Y, Severson RK, Yeager

M, Chanock S, Nieters A. Genetic vvariation in TNF and IL10 and risk of

non-Hodgkin lymphoma: a report from the InterLymph Consortium. Lancet

Oncol 2006;7:27-38.

8. Spink CF, Keen LJ, Mensah FK, Law GR, Bidwell JL, Morgan GJ. Association

between non-Hodgkin lymphoma and haplotypes in the TNF region. Br J

Haematol 2006;133:293-300.

9. Mittal RD, Manchanda PK. Association of interleukin (IL)-4 intron-3 and

IL-6 -174 G/C gene polymorphism with susceptibility to end-stage renal

disease. Immunogenetics 2007;59:159-165.

10. Cavet J, Middleton PG, Segall M, Noreen H, Davies SM, Dickinson

AM. Recipient tumor necrosis factor-alpha and interleukin-10 gene

polymorphisms associate with early mortality and acute graft-versus-host

disease severity in HLA-matched sibling bone marrow transplants. Blood

1991;94:3941-3946.

11. Breen EC, Boscardin WJ, Detels R, Jacobson LP, Smith MW, O’Brien SJ, Chmiel

JS, Rinaldo CR, Lai S, Martínez-Maza O. Non-Hodgkin’s B cell lymphoma

in persons with acquired immunodeficiency syndrome is associated with

increased serum levels of IL10, or the IL10 promoter -592 C/C genotype. Clin

Immunol 2003;109:119-129.

12. Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, Carbone

PP. Toxicity and response criteria of the Eastern Cooperative Oncology

Group. Am J Clin Oncol 1982;5:649-655.

13. Matesanz F, Fedetz M, Leyva L, Delgado C, Fernández O, Alcina A. Effects of

the multiple sclerosis associated -330 promoter polymorphism in IL2 allelic

expression. J Neuroimmunol 2004;148:212-217.

14. Wu MS, Huang SP, Chang YT, Shun CT, Chang MC, Lin MT, Wang HP, Lin JT.

Tumor necrosis factor-alpha and interleukin-10 promoter polymorphisms in

Epstein-Barr virus-associated gastric carcinoma. J Infect Dis 2002;185:106-

109.

15. Rousset F, Garcia E, Defrance T, Péronne C, Vezzio N, Hsu DH, Kastelein

R, Moore KW, Banchereau J. Interleukin 10 is a potent growth and

differentiation factor for activated human B lymphocytes. Proc Natl Acad

Sci U S A 1992;89:1890-1893.

16. Blay JY, Burdin N, Rousset F, Lenoir G, Biron P, Philip T, Banchereau J, Favrot

MC. Serum interleukin-10 in non-Hodgkin’s lymphoma: a prognostic factor.

Blood 1993;82:2169-2174.

17. Fayad L, Keating MJ, Reuben JM, O’Brien S, Lee BN, Lerner S, Kurzrock R.

Interleukin-6 and interleukin-10 levels in chronic lymphocytic leukemia:

correlation with phenotypic characteristics and outcome. Blood

2001;97:256-263.

18. Hulkkonen J, Pertovaara M, Antonen J, Lahdenpohja N, Pasternack A,

Hurme M. Genetic association between interleukin-10 promoter region

polymorphisms and primary Sjögren’s syndrome. Arthritis Rheum

2001;44:176-179.

19. Purdue MP, Lan Q, Kricker A, Grulich AE, Vajdic CM, Turner J, Whitby D,

Chanock S, Rothman N, Armstrong BK. Polymorphisms in immune function

genes and risk of non-Hodgkin lymphoma: findings from the New South

Wales non-Hodgkin Lymphoma Study. Carcinogenesis 2007;28:704-712.

20. Lan Q, Zheng T, Rothman N, Zhang Y, Wang SS, Shen M, Berndt SI, Zahm

SH, Holford TR, Leaderer B, Yeager M, Welch R, Boyle P, Zhang B, Zou K,

Zhu Y, Chanock S. Cytokine polymorphisms in the Th1/Th2 pathway and

susceptibility to non-Hodgkin lymphoma. Blood 2006;107:4101-4108.

21. Talaat RM, Abdel-Aziza AM, El-Maadawya EA, Abdel-Baryb N. Interleukin

10 gene promoter polymorphism and risk of diffuse large B cell lymphoma

(DLBCL). Egyptian Journal of Medical Human Genetics 2014;15:7-13.

22. Kube D, Hua TD, von Bonin F, Schoof N, Zeynalova S, Klöss M, Gocht D,

Potthoff B, Tzvetkov M, Brockmöller J, Löffler M, Pfreundschuh M, Trümper

107

Rahman HAA., et al: IL-2 and -10 Polymorphisms and B-NHL Turk J Hematol 2018;35:99-108

L. Effect of interleukin-10 gene polymorphisms on clinical outcome of

patients with aggressive non-Hodgkin’s lymphoma: an exploratory study.

Clin Cancer Res 2008;14:3777-3784.

23. Berglund M, Thunberg U, Roos G, Rosenquist R, Enblad G. The interleukin-10

gene promoter polymorphism (-1082) does not correlate with clinical

outcome in diffuse large B-cell lymphoma. Blood 2005;105:4894-4895.

24. Lech-Maranda E, Baseggio L, Bienvenu J, Charlot C, Berger F, Rigal

D, Warzocha K, Coiffier B, Salles G. Interleukin-10 gene promoter

polymorphisms influence the clinical outcome of diffuse large B-cell

lymphoma. Blood 2004;103:3529-3534.

25. Cunningham LM, Chapman C, Dunstan R, Bell MC, Joske DJ. Polymorphisms

in the interleukin 10 gene promoter are associated with susceptibility to

aggressive non-Hodgkin’s lymphoma. Leuk Lymphoma 2003;44:251-255.

108

RESEARCH ARTICLE

DOI: 10.4274/tjh.2017.0130


Myelodysplastic Syndrome in Pakistan: Clinicohematological

Characteristics, Cytogenetic Profile, and Risk Stratification

Pakistan’da Myelodisplastik Sendrom: Klinikohematolojik Özellikler, Sitogenetik Profil ve

Risk Stratifikasyonu

Rafia Mahmood, Chaudry Altaf, Parvez Ahmed, Saleem Ahmed Khan, Hamid Saeed Malik

Armed Forces Institute of Pathology, Department of Hematology, Rawalpindi, Pakistan

Abstract

Objective: Myelodysplastic syndrome (MDS) is a group of bone

marrow diseases that not only have variable morphological

presentation and heterogeneous clinical courses but also have a wide

range of cytogenetic abnormalities. Clinicohematological parameters

have a significant role in diagnosis and along with identification of

cytogenetic abnormalities are important for prognostic scoring and

risk stratification of patients to plan management and make treatment

decisions. This study aimed to determine the clinicohematological

characteristics, cytogenetic abnormalities, and risk stratification of

newly diagnosed de novo MDS patients.

Materials and Methods: This cross-sectional study was conducted in

the Department of Hematology, Armed Forces Institute of Pathology,

Rawalpindi, from January 2013 to January 2017. Patients were

diagnosed on the basis of World Health Organization criteria for

MDS, clinicohematological parameters were noted, and cytogenetic

analysis was performed. Risk stratification was done using the Revised

International Prognostic Scoring System.

Results: A total of 178 cases of MDS were analyzed, including 119

males (66.9%) and 59 females (33.1%). The median age was 58 years.

The most common presenting feature was anemia in 162 (91%) of

the patients. MDS with multilineage dysplasia was the most common

diagnosis, seen in 103 (57.9%) patients. A normal karyotype was seen in

95 (53.4%), while 83 (46.6%) showed clonal karyotypic abnormalities

at diagnosis. Of these, the common abnormalities found were trisomy

8, complex karyotype, and del 5q. Risk stratification revealed low-risk

disease in 73 (41%) patients.

Conclusion: Cytogenetic analysis showed the normal karyotype to be

the most common while risk stratification revealed a predominance of

low-risk disease at the time of presentation.

Keywords: Myelodysplastic syndrome, Cytogenetics, Revised

International Prognostic Scoring System

Öz

Amaç: Myelodisplastik sendrom (MDS) sadece değişken morfolojik

prezentasyona ve heterojen klinik seyre değil geniş sitogenetik

anormallikler yelpazesine de sahip olan bir grup kemik iliği hastalığıdır.

Klinikohematolojik parametreler tanıda önemli role sahiptir ve

sitogenetik anormalliklerin tanımlanması ile birlikte prognostik

skorlamada ve yönetimi planlamak ve tedavi kararlarını vermek için

risk stratifikasyonunda önemlidir. Bu çalışma, yeni tanı de novo MDS

hastalarında klinikohematolojik özellikler, sitogenetik anormallikler ve

risk stratifikasyonunu belirlemeyi amaçlamıştır.

Gereç ve Yöntemler: Bu kesitsel çalışma Rawalpindi Silahlı Kuvvetler

Patoloji Enstitüsü Hematoloji Departmanı’nda Ocak 2013’ten Ocak

2017 tarihine kadar sürdürülmüştür. Hastalar Dünya Sağlık Örgütü

MDS kriterlerine göre teşhis edildi, klinikohematolojik parametreler

not edildi ve sitogenetik analiz yapıldı. Risk stratifikasyonu Revize

Uluslararası Prognostik Skorlama Sistemi kullanılarak yapıldı.

Bulgular: Toplam 178 MDS olgusu, 119 erkek (%66,9) ve 59 kadın

(%33,1) analiz edildi. Medyan yaş 58 idi. Başvuruda en sık görülen

belirti olguların 162’sinde (%91) anemi idi. En sık tanı MDS çoklu seride

displazi olup 103 (%57,9) hastada görüldü. Teşhiste normal karyotip

95 (%53,4) olguda görülürken 83 (%46,6) olgu klonal karyotipik

anormallikler gösterdi. Bunlar arasında, en sık görülenler trizomi sekiz,

kompleks karyotip ve del5q idi. Risk stratifikasyonu 73 (%41) hastada

düşük-risk hastalık ortaya koydu.

Sonuç: Sitogenetik analiz en sık normal karyotipi gösterirken risk

stratifikasyonu tanı sırasında düşük-risk hastalığın çoğunlukta

olduğunu ortaya koymuştur.

Anahtar Sözcükler: Myelodisplastik sendrom, Sitogenetik, Revize

Uluslararası Prognostik Skorlama Sistemi



Address for Correspondence/Yazışma Adresi: Rafia MAHMOOD, M.D.,

Armed Forces Institute of Pathology, Department of Hematology, Rawalpindi, Pakistan

Phone : 923 365 182 270

E-mail : rafiamahmood@hotmail.com ORCID-ID: orcid.org/0000-0002-5394-9290


Accepted/Kabul tarihi: May 31, 2017

109

Mahmood R, et al: Myelodysplastic Syndrome in Pakistan


Introduction

Myelodysplastic syndrome (MDS) is a heterogeneous group of

clonal stem cell disorders characterized by peripheral blood

cytopenias, dysplasia, and ineffective hematopoiesis [1].

Patients have a variable clinical course and there is an increased

risk of myeloid leukemic transformation [2]. It is a disease of

the elderly; its incidence increases with age. It is slightly more

common in males with a male:female ratio of 1.4:1 [3]. While

a few patients may be detected incidentally when a routine

blood count reveals unexpected cytopenia, most present with

symptoms and signs of bone marrow failure. Notable findings

include fatigue due to anemia, infections, and bleeding [4].

Morphologic dysplasia is the hallmark of the disease [5]. Dysplasia

may be seen in any or all of the three lineages [6]. The World

Health Organization (WHO) has classified the myelodysplastic

syndromes based on the number of cytopenias, dysplasia in a

single lineage or in multiple lineages, cytogenetics, the number

of blast cells, and the presence or absence of ring sideroblasts

[5].

In addition to morphologic heterogeneity, MDS cases show

profound heterogeneity in their genetic presentation [7]. More

than half the patients show clonal chromosomal abnormalities

with a predominance of unbalanced abnormalities [8].

Cytogenetic analysis not only has an important role in diagnosis

where certain chromosomal abnormalities are considered

presumptive evidence of MDS, but also has important prognostic

implications. These cytogenetic abnormalities can be detected

by conventional metaphase karyotyping. However, fluorescent

in situ hybridization (FISH) has been seen to have a much

higher sensitivity for detection of del 5q [9]. These cytogenetic

findings serve as a basis for the characterization of cytogenetic

subgroups [10].

Over time, a better understanding of the biology of disease has

shown cytogenetics to be an important prognostic parameter

[11]. The Revised International Prognostic Scoring System

(R-IPSS) refines risk group definitions, aiming for better

prediction of individual prognosis. The parameters included are

the degree of cytopenias, the number of blast cells, and the

cytogenetic subgroup [12]. The prognostication of patients

based on individualized risk assessment not only predicts disease

progression but also provides an important tool in planning

management and making treatment decisions [13].

Most studies regarding MDS are from Western populations.

Disease biology, clinical presentations, and cytogenetic findings

are different and distinctive for population groups and can

show noticeable differences in geographic prevalence around

the world. The present study was designed with an aim to see

the clinicohematological features, cytogenetic profile, and risk

stratification of the patients of Pakistan (an Asian population)

as so far there is a lack of data on MDS in our region. This will

help to determine treatment protocols and prognosis.


Patients

This study was a cross-sectional analysis conducted in the

Department of Hematology of the Armed Forces Institute of

Pathology, Rawalpindi, from January 2013 to January 2017.

All patients were Pakistanis, of Asian origin, belonging to

different ethnic groups including Punjabis, Pashtuns, Sindhis,

Balochis, Kashmiris, and those from Gilgit-Baltistan. Patients

were between the ages of 30 and 85 years. These patients were

newly diagnosed with MDS and had no previous history of any

treatment. Patients who had failed culture (did not yield at

least 20 metaphases) in cytogenetic analysis were excluded

from the study. All subjects were thoroughly informed about

the study and written informed consent was obtained.

Clinicohematological Parameters

Detailed history was recorded and complete physical examination

was done. Symptoms and signs were noted. Complete blood

count, peripheral blood film, and bone marrow examination

were done and patients were diagnosed as having MDS based

on the WHO criteria.

Cytogenetics and FISH

Cytogenetic analysis was performed by using the conventional

G banding technique. A bone marrow specimen of 3 mL was

collected in sodium heparin. Metaphase chromosomes were

banded using the conventional Giemsa trypsin banding

technique and karyotyped according to the International System

for Human Cytogenetic Nomenclature criteria. At least twenty

metaphases were analyzed with the CytoVision semiautomated

image analysis and capture system.

Interphase FISH studies were performed on blood or bone

marrow specimens processed by standard methods for cultured

samples. The MetaSystems XL 5q31/5q33 probe (10 µL) was

applied to the target on the slide. A total of 500 nuclei were

analyzed per probe set by using a fluorescent microscope with

an orange green spectrum filter.

Risk Stratification

The patients were risk-stratified according to the R-IPSS.


Collected data were entered and analyzed using SPSS 20

(IBM Corp., Armonk, NY, USA). Quantitative variables, i.e. age,

hemoglobin (Hb), platelet count, and absolute neutrophil count

(ANC), have been presented as mean ± standard deviation.

110



Qualitative variables, i.e. sex, cytogenetics, and risk category,

have been presented as frequency and percentage.

Ethical Approval

This study was approved by the Ethical Review Committee of

the Armed Forces Institute of Pathology, Rawalpindi. Informed

written consent was received from the patients.

Results

A total of 178 patients were diagnosed as having de novo MDS.

The median age of the patients was 58 years. Out of 178 patients,

119 (66.9%) were male, while the remaining 59 (33.1%) patients

were female.

The most common presenting clinical feature was pallor,

followed by symptoms of fatigue, recurrent infections, and

bruising/bleeding; 118 (66%) of the patients were transfusiondependent

at the time of presentation. Mean Hb was 6.4 g/dL

and mean platelet count was 97x10 9 /L, while the mean ANC was

2.1x10 9 /L. Table 1 shows the clinicohematological parameters of

our patients. We classified our patients according to the 2016

revised WHO classification: 103 (57.9%) of the patients were in

the MDS-MLD (MDS with multilineage dysplasia) category while

36 (20.2%) cases were classified as MDS-SLD (MDS with single

lineage dysplasia), 16 (8.9%) as MDS-EB1 (MDS with excess

blasts-1), 12 (6.7%) as MDS-EB2 (MDS with excess blasts-2),

6 (3.4%) as MDS with isolated del (5q), 3 (1.7%) as MDS-RS-

SLD (MDS with ring sideroblasts with single lineage dysplasia),

and 2 (1.1%) as MDS-RS-MLD (MDS with ring sideroblasts with

multilineage dysplasia).

A normal karyotype was seen in 95 (53.4%) cases, while 83

(46.6%) patients showed clonal karyotypic abnormalities at

diagnosis (Figure 1 and 2). Of these, 56 (31.4%) had single and 8

Table 1. Clinicohematological parameters of the patients.

Parameters n=178 %

Hb <10 g/dL 174 97.8

Platelets <100x10 9 /L 99 55.6

ANC <1.5x10 9 /L 69 38.8

Cytopenia

Dysplasia

Blasts

Unicytopenia 45 25.3

Bicytopenia 69 38.8

Pancytopenia 64 35.9

Single lineage 49 27.5

Multilineage 129 72.5

PB <1%, BM <5% 150 84.8

PB 2%-4%, BM 5%-9% 16 8.9

PB 5%-19%, BM 10%-19% 12 6.2

Ring sideroblasts >15% 5 2.7

Hb: Hemoglobin, ANC: absolute neutrophil count, PB: peripheral blood, BM: bone

marrow.

(4.5%) had double cytogenetic abnormalities while 19 (10.7%)

had a complex karyotype. Of the cytogenetic abnormalities seen,

the most commonly found was trisomy 8 in 23 (12.9%) cases,

followed by del 5q in 13 (7.3%), monosomy 7 in 10 (5.6%), loss

of Y in 5 (2.8%), del 11q in 5 (2.8%), del 20q in 4 (2.2%), del 7q

in 3 (1.7%) and i(17q) in 1 (0.6%) patient. Other abnormalities,

including translocations, hyperdiploidy, hypodiploidy, deletions,

and monosomies, were seen in 8 (4.5%) of the patients. del 5q

was detected in 8 patients based on conventional cytogenetics

while in 5 patients it was missed by conventional cytogenetics

and detected by FISH.

Each parameter was assessed and scored according to the

R-IPSS. Based on the score, the patients were stratified into

five distinct risk groups. In the very-low-risk group, there were

17 (9.6%) patients, while there were 73 (41%) patients in the

low-risk group, 48 (27.1%) patients in the intermediate-risk

group, 24 (13.5%) patients in the high-risk group, and 16 (9.1%)

patients in the very-high-risk group.

Figure 1. Cytogenetics of the patients.

Figure 2. Common karyotypic abnormalities of the patients.

111



Discussion

Myelodysplastic syndromes show not only clinical heterogeneity

and genetic diversity but also a highly variable clinical course

[14]. Over the last decade a better understanding of the biology

of MDS has led to the identification of genetic molecular factors

that have diagnostic value as well as roles in determining the

disease course and prognosis [15]. Conventional cytogenetics

of all newly diagnosed MDS patients is thus of paramount

significance as it is an important component in risk-stratifying

the patients [16]. FISH has a much higher sensitivity and

has improved the detection of genomic aberrations in MDS,

especially del 5q [9].

To our knowledge, there are no comprehensive data available on

the clinicohematological features, cytogenetic profiles, and risk

stratification of MDS patients from our part of the country. The

Armed Forces Institute of Pathology is a tertiary care institute

and a referral center in the north of Pakistan. It caters to a

large number of patients from all over the country from very

different ethnic backgrounds. Our study aims to help clinicians

in structuring treatment decisions in light of cytogenetically

based risk stratification.

In our study, the median age of the patients was 58 years.

Similar findings have been reported in local studies. However,

a much higher age, 71 years, was reported by Greenberg et al.

[11] in a Western population. There is a major difference in the

age of presentation of our patients and the Western population.

These differences may be attributable to racial and geographic

differences and differences in disease biology in different

populations. Among our patients, males were more common as

compared to females (66.9% vs. 33.1%). The male-to-female

ratio was 2:1. Our observation coincides with the findings of

Sultan and Irfan [17], who reported a sex ratio of 1.6:1. Deeg et

al. [18] also reported a male predominance.

The most common presenting clinical findings of pallor

followed by fatigue observed in our study are consistent with

those reported by Narayanan [19] in the Indian population. Of

our patients, 66% were transfusion-dependent at the time of

presentation. A similar frequency of 58% was reported in the

Italian population, while Greenberg et al. [11] reported 32% of

the patients to be transfusion-dependent based on data from

eleven countries. Mean Hb was 6.4 g/dL. Chaubey et al. [20]

demonstrated mean Hb of 6.8 g/dL, which is in accordance with

our findings. In another study, Voso et al. [12] reported mean

Hb of 9.9 g/dL in Italian patients. There is a striking difference

in presenting Hb levels and transfusion dependency in our

population as compared to the Western populations studied by

Greenberg et al. [11]. This may be due to the fact that Pakistan

is a developing country and patients present late as they do not

have early access to tertiary care medical facilities. However,

these differences in presentation, with more than two-thirds

of our patients being transfusion-dependent, may affect the

overall treatment plan. These patients need further stratification

by evaluation of their erythropoietin levels, which will guide

further management. In patients with low erythropoietin levels

(less than 200 IU/L), early institution of erythropoietin therapy

predicts the response. Erythropoietin therapy not only improves

Hb levels but also enhances the quality of life without the risks

associated with blood transfusions. Iron chelation will also be an

Table 2. Comparison of clinicohematological characteristics with national and international studies.

Parameters

Our

study

Rashid

et al.

[21]

Sultan

and

Irfan

[17]

Ehsan and

Aziz

[22]

Chaubey

et al.

[20]

Narayanan

[19]

Avgerinou

et al.

[23]

Median age (years) 58 60 64 - 42 67 74 71 71

M:F ratio 2:1 1.4:1 1.7:1 1.6:1 - 2.3:1 2.4:1 1.1:1 1.5:1

Fatigue (%) 91 - 60 92.5 - 90 55 - -

Bleeding/bruising (%) 13.5 - 20 42.5 - 33.3 8 - -

Fever/infection (%) 25.3 - 33.3 55 - 31.7 15 - -

Pallor (%) 92 - 37.7 - - 75 - - -

Mean Hb (g/dL) 6.4 - 7.7 6.5 6.8 5.5 9.5 9.9 -

Mean platelet (x10 9 /l) 97 - 82.7 59.6 84.5 - 158 152 -

Mean ANC (x10 9 /l) 2.1 - 3.0 - - - 3.94 1.9 -

Transfusion dependent % 66 - - - - - - 58 32

Blasts

Voso

et al.

[12]

BM <5% 84.8 69 - - - - - 68 65

BM 5-10% 8.9 18.3 - - - - - 23 19

BM >10% 6.2 12.7 - - - - - 9 16

Median LDH (IU/L) 381 - - - - - - 317 -

Hb: Hemoglobin, ANC: absolute neutrophil count, BM: bone marrow, LDH: lactate dehydrogenase.

Greenberg

et al.

[11]

112



Table 3. Cytogenetic profile in comparison with national and international studies.

Parameters

Our

study

Rashid et

al. [21]

Chaubey et

al. [20]

Narayanan

[19]

Chen et

al. [25]

Lee et

al. [24]

Avgerinou et

al. [23]

Voso et

al. [12]

Normal karyotype 53.4 57.7 52.5 65.4 62.9 56 61.6 61 49

Abnormal karyotype 46.6 42.3 47.5 34.6 37.1 44 38.4 39 51

Complex karyotype 10.7 15.5 - - - 15.1 7.6 6 -

Trisomy 8 12.9 9.9 7.5 - 9.5 5.9 8.3 5 8.4

Del 5q 7.3 2.8 10 21.1 4.6 1.7 2.7 10.5 15.1

Monosomy 7 5.6 - 15 - 1.6 1.7 3 2 8

Loss of Y 2.8 2.8 - 5.8 - 2.5 5.8 - 2.8

del 11q 2.8 1.4 - - - - - - 1.1

del 20q 2.2 1.4 - - 5.4 - 2.2 5 3.6

del 7q 1.1 4.2 - 7.7 - - - - 3.1

Table 4. Comparison of cytogenetic subgroups and risk stratification.

Cytogenetic subgroup

AFIP

n=178

%

Narayanan [19]

n=52

%

Greenberg et al. [11]

n=7012

%

Very good 5.6 5.8 4 3

Good 62.9 86.5 72 77

Intermediate 15.2 7.7 13 13

Poor 12.9 - 4 4

Very poor 3.4 - 7 3

Risk stratification

Very low 9.6 9.6 19 38

Low 41.0 34.6 38 33

Intermediate 27.1 36.5 20 18

High 13.5 19.2 13 7

Very high 9.1 - 10 4

AFIP: Armed Forces Institute of Pathology.

Voso et al. [12]

n=380

%

Haase et

al. [8]

important consideration in patients who have received multiple

transfusions.

In our study, the mean platelet count was 97x10 9 /L, while a

mean platelet count of 100.5x10 9 /L was reported in Indians [20]

and 152x10 9 /L [12] in the Italian population. The mean ANC in

our study population was (2.1±1.8)x10 9 /L, which correlates with

the median ANC of 1.9x10 9 /L reported by Voso et al. [12]. The

clinicohematological characteristics of our study population are

compared with those of national and international studies in

Table 2.

On cytogenetic analysis, a normal karyotype was seen in

95 patients (53.4%), while 83 (46.6%) patients showed

clonal karyotypic abnormalities at diagnosis. Chromosomal

abnormalities were detected in 34.6% of cases by Narayanan

[19], 39% by Voso et al. [12], 42.3% by Rashid et al. [21], 47.5%

by Chaubey et al. [20], and 48% by Cao et al. [9]. A complex

karyotype, which carries poor overall survival, was seen in 10.7%

of our patients, while Rashid et al. [21] reported a frequency of

15.5%.

Table 3 shows a comparison of the cytogenetic profile with

national and international data. In our study, the most common

cytogenetic abnormality was trisomy 8 in 12.9% followed

by del 5q in 7.3% and monosomy 7 in 5.6% of the patients.

Rashid et al. [21] reported trisomy 8 to be the most common

cytogenetic abnormality with a frequency of 9.9%. However,

they reported a much lower frequency of del 5q in 2.8% of

the patients. This difference may be due to the difference in

the cytogenetic methodology adopted, as we used FISH for

detection of del 5q in addition to conventional cytogenetics,

as FISH has higher sensitivity. Chaubey et al. [20] reported

monosomy 7 as the most frequent cytogenetic abnormality

detected in 15%, followed by del 5q in 10% and trisomy 8 in

7.5% of Indian patients. In the Italian population [12], the most

common karyotypic abnormality reported is del 5q in 10.5%,

while much lower frequencies of 5% for trisomy 8 and 2% for

113



monosomy 7 have been reported. Identification of patients with

del 5q is particularly important as these patients are candidates

for treatment with the immunomodulatory drug lenalidomide.

Early initiation of treatment with lenalidomide not only leads

to transfusion independence but also induces cytogenetic

remission in this subgroup of patients.

The R-IPSS score is particularly useful in clinical decision-making

and selection of appropriate treatment options while at the same

time providing prognostic information and predicting outcome in

response to disease-modifying therapies. Upon risk stratification

by the R-IPSS, as shown in Table 4, most of our patients (41%)

were in the low-risk category, followed by 27.1% of the patients

in the intermediate-risk category. These findings are in accordance

with the findings of Greenberg et al. [11], who reported 38% of

their patients in the low-risk followed by 20% in the intermediaterisk

and 19% in the very-low-risk category. However, in an Italian

study, Voso et al. [12] reported 38% in the very-low-risk category,

followed by 33% in the low-risk and 18% in the intermediate-risk

category. Those patients in the high-risk and very-high-risk groups

need stringent regular monitoring as they have poor prognosis

and are potentially more likely to have disease progression and

transformation into acute myeloid leukemia.

Conclusion

Cytogenetic analysis showed the normal karyotype to be the

most common while among the cytogenetic abnormalities

detected trisomy 8 was the most common. Risk stratification

revealed a predominance of low-risk disease at the time of

presentation. The results of our study are in accordance with

other local studies with a few differences, which may be due

to differences in the method of detection of chromosomal

abnormalities. However, there are differences with studies in

other parts of the world. These differences may be attributable

to geographical and ethnic differences in disease biology and

genetics. As MDS has a heterogeneous clinical course, genetic

characterization of all newly diagnosed MDS patients is

important not only for diagnosis but also for risk stratification

so that individualized treatment can be instituted to improve

survival and for predicting outcome.

Acknowledgment

We are grateful for the technical support provided by Parvez

Iqbal.

Ethics

Ethics Committee Approval: Research Ethics and Academics

Department, Armed Forces Institute of Pathology, Rawalpindi,

Pakistan.

Informed Consent: Informed written consent was received

from the patients.


Surgical and Medical Practices: R.M., C.A.; Concept: R.M.;

Design: R.M., H.S.M.; Data Collection or Processing: R.M.;

Analysis or Interpretation: R.M.; Literature Search: R.M., C.A.

P.A., S.A.K.; Writing: R.M., C.A.




included.

References

1. de Swart L, Smith A, Johnston TW, Haase D, Droste J, Fenaux P, Symeonidis

A, Sanz G, Hellström-Lindberg E, Cermák J, Germing U, Stauder R, Georgescu

O, MacKenzie M, Malcovati L, Holm MS, Almeida AM, Madry K, Slama B,

Guerci-Bresler A, Sanhes L, Beyne-Rauzy O, Luño E, Bowen D, de Witte T.

Validation of the revised international prognostic scoring system (IPSS-R)

in patients with lower-risk myelodysplastic syndromes: a report from the

prospective European LeukaemiaNet MDS (EUMDS) registry. Br J Haematol

2015;170:372-383.

2. Fenaux P. Myelodysplastic syndromes: from pathogenesis and prognosis to

treatment. Semin Hematol 2004;41:6-12.

3. Ma X. Epidemiology of myelodysplastic syndromes. Am J Med 2012;125(7

Suppl):2-5.

4. Chatterjee T, Dixit A, Mohapatra M, Tyagi S, Gupta PK, Mishra P, Bhattacharya

M, Karan AS, Pati HP, Saxena R, Choudhry VP. Clinical, haematological and

histomorphological profile of adult myelodysplastic syndrome. Study of 96

cases in a single institute. Eur J Haematol 2004;73:93-97.

5. Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM,

Bloomfield CD, Cazzola M, Vardiman JW. The 2016 revision to the World

Health Organization classification of myeloid neoplasms and acute

leukemia. Blood 2016;127:2391-2405.

6. Brunning RD, Orazi A, Germing U, Le Beau MM, Porwit A, Baumann I,

Vardiman JW, Hellstrom-Lindberg E. Myelodysplastic syndromes. In:

Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J,

Vardiman JW, (eds). WHO Classification of Tumours of Haemopoietic and

Lymphoid Tissues. 4th ed. Lyon, IARC Press, 2008.

7. Haase D. Cytogenetic features in myelodysplastic syndromes. Ann Hematol

2008;87:515-526.

8. Haase D, Germing U, Schanz J, Pfeilstöcker M, Nösslinger T, Hildebrandt

B, Kundgen A, Lübbert M, Kunzmann R, Giagounidis AA, Aul C, Trümper

L, Krieger O, Stauder R, Müller TH, Wimazal F, Valent P, Fonatsch C, Steidl

C. New insights into the prognostic impact of the karyotype in MDS and

correlation with subtypes: evidence from a core dataset of 2124 patients.

Blood 2007;110:4385-4395.

9. Cao P, Li Y, Li X, Zhang G, Chen F. Detecting chromosomal aberrations in

myelodysplastic syndrome with fluorescence in situ hybridization and

conventional cytogenetic analysis. Zhong Nan Da Xue Xue Bao Yi Xue Ban

2014;39:605-611.

10. Jonas BA, Greenberg PL. MDS prognostic scoring systems-past, present and

future. Best Prac Res Clin Haematol 2015;28:3-13.

11. Greenberg PL, Tuechler H, Schanz J, Sanz G, Garcia-Manero G, Solé F,

Bennett JM, Bowen D, Fenaux P, Dreyfus F, Kantarjian H, Kuendgen A, Levis

A, Malcovati L, Cazzola M, Cermak J, Fonatsch C, Le Beau MM, Slovak ML,

Krieger O, Luebbert M, Maciejewski J, Magalhaes SM, Miyazaki Y, Pfeilstöcker

M, Sekeres M, Sperr WR, Stauder R, Tauro S, Valent P, Vallespi T, van de

Loosdrecht AA, Germing U, Haase D. Revised International Prognostic Scoring

System for myelodysplastic syndromes. Blood 2012;120:2454-2465.

12. Voso MT, Fenu S, Latagliata R, Buccisano F, Piciocchi A, Aloe-Spiriti MA,

Breccia M, Criscuolo M, Andriani A, Mancini S, Niscola P, Naso V, Nobile

114



C, Piccioni AL, D’Andrea M, D’Addosio A, Leone G, Venditti A. Revised

International Prognostic Scoring System (IPSS) predicts survival and

leukaemic evolution of MDS significantly better than the IPSS and WHO

prognostic scoring system: validation by the Gruppo Romano Mielodisplasie

Italian Regional Database. J Clin Oncol 2013;31:2671-2677.

13. Tefferi A, Vardiman JW. Myelodysplastic syndromes. N Engl J Med

2009;361:1872-1885.

14. Orazi A, Czader MB. Myelodysplastic syndromes. Am J Clin Pathol

2009;132:290-305.

15. Steensma DP, Bennett JM. The myelodysplastic syndromes: diagnosis and

treatment. Mayo Clin Proc 2006;81:104-130.

16. Look AT. Molecular pathogenesis of MDS. Hematology Am Soc Hematol

Educ Program 2005:156-160.

17. Sultan S, Irfan SM. Adult primary myelodysplastic syndrome: experience

from a tertiary care center in Pakistan. Asian Pac J Cancer Prev

2016:17:1535-1537.

18. Deeg HJ, Scott BL, Fang M, Shulman HM, Gyurkocza B, Myerson D, Pagel

JM, Platzbecker U, Ramakrishnan A, Radich JP, Sandmaier BM, Sorror M,

Stirewalt DL, Wilson WA, Storb R, Appelbaum FR, Gooley T. Five-group

cytogenetic risk classification, monosomal karyotype, and outcome after

hematopoietic cell transplantation for MDS or acute leukemia evolving

from MDS. Blood 2012;120:1398-1408.

19. Narayanan S. Clinical, hematological, and cytogenetic profile of adult

myelodysplastic syndrome in a tertiary care center. J Blood Med 2017;8:21-27.

20. Chaubey R, Sazawal S, Dada R, Mahapatra M, Saxena R. Cytogenetic profile

of Indian patients with de novo myelodysplastic syndromes. Indian J Med

Res 2011;134:452-457.

21. Rashid A, Khurshid M, Shaikh U, Adil S. Chromosomal abnormalities in primary

myelodysplastic syndrome. J Coll Physicians Surg Pak 2014;24:632-635.

22. Ehsan A, Aziz M. Clinico-haematological characteristics in Pakistani patients

of primary MDS according to WHO classification. J Coll Physicians Surg Pak

2010;20:232-236.

23. Avgerinou C, Alamanos Y, Zikos P, Lampropoulou P, Melachrinou M,

Labropoulou V, Tavernarakis I, Aktypi A, Kaiafas P, Raptis C, Kouraklis A,

Karakantza M, Symeonidis A. The incidence of myelodysplastic syndromes

in Western Greece is increasing. Ann Hematol 2013;92:877-887.

24. Lee JH, Lee JH, Shin YR, Lee JS, Kim WK, Chi HS, Park CJ, Seo EJ, Lee KH.

Application of different prognostic scoring systems and comparison

of FAB and WHO classification in Korean patients with MDS. Leukemia

2003;17:305-313.

25. Chen B, Zhao WL, Jin J, Xue YQ, Cheng X, Chen XT, Cui J, Chen ZM, Cao Q,

Yang G, Yao Y, Xia HL, Tong JH, Li JM, Chen J, Xiong SM, Shen ZX, Waxman S,

Chen Z, Chen SJ. Clinical and cytogenetic features of 508 Chinese patients

with MDS and comparison with those in Western countries. Leukemia

2005;19:767-775.

115

RESEARCH ARTICLE

DOI: 10.4274/tjh.2018.0022


Hierarchical Involvement of Myeloid-Derived Suppressor Cells

and Monocytes Expressing Latency-Associated Peptide in Plasma

Cell Dyscrasias

Plazma Hücreli Diskraziye Myeloid Kökenli Baskılayıcı Hücreler ve Latent Asosiye Peptit

Ekprese Eden Monositlerin Hiyerarşik Katılımı

Tamar Tadmor 1,2 Ilana Levy 3 , Zahava Vadasz 2,4

1

Bnai-Zion Medical Center, Clinic of Hematology, Haifa, Israel

2

The Ruth and Bruce Rappaport Faculty of Medicine, Clinic of Hematology, Haifa, Israel

3

Bnai-Zion Medical Center, Clinic of Internal Medicine B, Haifa, Israel

4

Bnai-Zion Medical Center, Clinic of Allergy and Clinical Immunology, Haifa, Israel

Abstract

Objective: Plasma cell dyscrasias (PCDs) are disorders of plasma

cells having in common the production of a monoclonal M-protein.

They include a spectrum of conditions that may represent a natural

progression of the same disease from monoclonal gammopathy

of unknown significance to asymptomatic and symptomatic

multiple myeloma, plasma cell leukemia, and Waldenström’s

macroglobulinemia. In PCDs, the immune system is actively suppressed

through the secretion of suppressive factors and the recruitment of

immune suppressive subpopulations. In this study, we examined the

expression of two subpopulations of cells with immunosuppressive

activity, monocytic myeloid-derived suppressor cells (MDSCs) and

monocytes expressing latency-associated peptide (LAP), in patients

with different PCDs and in healthy volunteers.

Materials and Methods: A total of 27 consecutive patients with

PCDs were included in this study. Nineteen healthy volunteers served

as controls.

Results: We observed a hierarchical correlation between disease

activity and the presence of monocytes with immunosuppressive

activity.

Conclusion: These results suggest that MDSCs and monocytes

expressing LAP have diverging roles in PCDs and may perhaps serve as

biomarkers of tumor activity and bulk.

Keywords: Multiple myeloma, Monoclonal gammopathy of unknown

significance, Myeloid-derived suppressor cells, Latency-associated

peptide

Öz

Amaç: Plazma hücreli diskrazi (PHD), monoklonal M-proteinin üretimine

sahip olan plazma hücrelerinin bozukluklarıdır. Aynı hastalığın,

önemi bilinmeyen monoklonal gammopatiden, asemptomatik ve

semptomatik multipl myeloma, plazma hücreli lösemi ve Waldenström

makroglobulinemiye doğru doğal ilerlemesini temsil edebilen bir dizi

spektrum içerir. PHD’lerde, baskılayıcı faktörlerin salgılanması ve

bağışıklık baskılayıcı alt popülasyonların katılımı ile bağışıklık sistemi

aktif olarak baskılanır. Bu çalışmada, PHD’lerin ve sağlıklı gönüllülerin,

immün baskılayıcı aktiviteye sahip iki alt popülasyonundaki; monositik

myeloid kökenli baskılayıcı hücreler (MKBH) ve latent asosiye peptit

(LAP) eksprese eden monositlerin, ekspresyonunu incelenmiştir.

Gereç ve Yöntemler: Bu çalışmaya PHD’li toplam 27 hasta dahil

edildi. On dokuz sağlıklı gönüllü, kontrol olarak kullanılmıştır.

Bulgular: Hastalık aktivitesi ile immünosüpresif aktivitesi olan

monositler arasında hiyerarşik bir ilişki gözlenmiştir.

Sonuç: Bu sonuçlar LAP anlatımı gösteren MKBH’lerin ve monositlerin,

PHD’lerde farklı rollere sahip olduğunu ve tümör aktivitesi ve kitle

biyobelirteçleri olarak kullanılabileceğini düşündürmektedir.

Anahtar Sözcükler: Multiple myelom, Önemi bilinmeyen monoklonal

gammopati, Myeloid kökenli baskılayıcı hücreler, Latent asosiye peptit



Address for Correspondence/Yazışma Adresi: Tamar TADMOR, M.D.,

Bnai-Zion Medical Center, Clinic of Hematology, Haifa, Israel

Phone : +972 48359407

E-mail : tamar.tadmor@b-zion.org.il ORCID-ID: orcid.org/0000-0002-3435-8612

Received/Geliş tarihi: January 13, 2018

Accepted/Kabul tarihi: March 23, 2018

116


Tadmor T, et al: Hierarchical Involvement of MDS Cells and Monocytes Expressing LAP in PCD

Introduction

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous

population of immature cells of granulocytic or monocytic

origin, which accumulate in a number of disorders including

solid tumors and hematological malignancies in particular

[1,2]. MDSCs inhibit T-cell proliferation and cytokine secretion,

favoring the recruitment of regulatory T cells (Tregs), and

are part of the immune regulatory subpopulations of cells

responsible for inhibition of the immune response, thereby

facilitating tumor escape [1,2].

Latency-associated peptide (LAP) is the N-terminal propeptide

of the transforming growth factor beta (TGF-β) precursor, which

binds noncovalently to TGF-β, forming a latent TGF-β complex.

When released into the extracellular milieu, LAP forms small

latent complexes with TGF-β1 [3,4,5]. TGF-β-LAP complexes

are present on the surface of various immune cells and have

been shown to play a role in immune regulation, promoting

the conversion of naive to activated Tregs, which induce Tregassociated

immunosuppression [3,4,5].

Bolzoni et al. [6] studied the function of CD14/CD16+ monocyte

subpopulations sorted from the bone marrow of patients with

monoclonal gammopathies at different stages of disease. In

this report, monocytes isolated from patients with multiple

myeloma (MM) showed activity that contributed to enhanced

osteoclast activation.

MM is the second most common hematological malignancy

in the United States and is invariably preceded by monoclonal

gammopathy of unknown significance (MGUS). Myeloma cells

are critically dependent on the tumor microenvironment for

their survival, progression, and proliferation, and a number of

recent studies have concentrated on targeted therapy of tumor

niche pathways [7,8,9].

MM is also associated with immune dysfunction, and several

reports have demonstrated increased numbers of MDSCs in

the bone marrow microenvironment, which contributes to

immunosuppression and tumor invasion [10,11,12,13,14,15,16].

Recently, we studied two immune subpopulations, monocytic

MDSCs and LAP-expressing monocytes, in the peripheral blood

of patients with different plasma cell dyscrasias (PCDs) and in

healthy volunteers and compared their frequencies.


A total of 27 consecutive patients with PCDs, classified according

to the International Myeloma Working Group as published in 2009

and updated in 2014-2015 [14,15] and seen in the Hematology

Unit of the Bnai Zion Medical Center in Haifa, Israel, between 2013

and 2015 were included in this study. For patients with plasma

cell leukemia, diagnosis was based on the percentage (≥20%)

and absolute number (≥2x10 9 /L) of plasma cells in the peripheral

blood, while Waldenström’s macroglobulinemia (WM) was defined

on the basis of the presence of immunoglobulin M monoclonal

gammopathy and ≥10% bone marrow lymphoplasmacytic

infiltration [17,18,19,20].

The cohort included 8 patients with MGUS, 14 with symptomatic

MM, 2 with plasma cell leukemia, and 3 with WM. Nineteen

healthy volunteers served as controls.

All samples were taken from treatment-naive patients, before

starting any therapy.

Written informed consent was obtained from all patients and

the study was approved by the hospital’s ethics committee.

Materials

Mononuclear cells were enriched from whole blood using

the Ficoll-Hypaque gradient (Lymphoprep, Oslo, Norway).

Fluorescence-activated cell sorting analysis was performed on

these mononuclear cells using the following antibodies: anti-

CD45 PC-5 (PE-Cy5), anti-CD14 PE (phycoerythrin), and anti-

HLA-DR FITC (fluorescein) (BD Biosciences, San Jose, CA, USA).

For staining, 0.5-1x10 6 mononuclear cells were stained and

incubated at room temperature for 30 min in the dark with the

above antibodies according to the manufacturer’s instructions

in 100 µL of PBS followed by red blood cell lysis (VersaLyse,

Beckman Coulter, Inc., Marseille, France). In addition, MDSCs

were characterized using antibodies to CD124 [interleukin

(IL)-4Ra], which is the common receptor for interleukin-4 (IL-

4). CD14+/HLA-DR neg/low cells were also gated for expression of

LAP using anti-LAP (clone 27232), obtained from R&D Systems

(Minneapolis, MN, USA).

Data were acquired with a Beckman Coulter Cytomics FC 500

flow cytometer and analyzed with CXP Software, version 2.2.

(Beckman Coulter, Brea, CA, USA).


All values were expressed as mean ± standard error of the mean.

For flow-cytometry data, values between groups of data were

tested for statistical significance.

The chi-square test was performed to determine whether data

were normally distributed and a two-tailed t-test was then applied

to the results. Significant p-values were those less than 0.05.

Results

The patient cohort included 11 males (41%) and 16 females

(59%); median age at diagnosis was 61 years (range: 45-86).

All patients were diagnosed and followed at the same medical

center. Patients’ characteristics are presented in Table 1.

117



Monocytic MDSC Expression

The mean number of circulating monocytic MDSCs in the

peripheral blood was defined by coexpression of positive CD14+

and dim expression of HLA-DR. The average expression was

5.9% (3.7%-8.1%) for the MGUS cohort, 12.5% (6.7%-27.2%)

for MM patients, 18.4% (14.6%-22%) in plasma cell leukemia

cases, 17.8% (16.5%-19%) in WM cases, and 5.5% (2.4%-7.9%)

in healthy controls.

No significant difference was observed between MGUS patients

and healthy volunteers (p=0.39), but the comparison with cases

of PCD was significant (p=0.002) (Figure 1a). Next, we analyzed

the monocyte subpopulation coexpressing CD124+, another

marker of MDSCs. Results obtained using mean numbers for

healthy controls and patients with MGUS, MM, plasma cell

leukemia, and WM were 8.1% (6.1%-11%), 4.4% (1.6%-7.1%),

15.7% (2.5%-17.5%), 18.4% (14.5%-22.3%), and 19.7%

(18.5%-20.9%), respectively (Figure 1b).

Results were statistically significant for all PCDs when compared

to healthy controls (p=0.03).

LAP Expression

The mean number of circulating monocyte/LAP+ cells in the

peripheral blood was defined by coexpression of positive CD14+

and LAP. The average expression was 6.5% (3.7%-9.1%) for

the MGUS cohort, 15.1% (12.1%-44%) for MM patients, 19%

(13.5%-23.2%) in plasma cell leukemia cases, 19.7% (16.9%-

23%) in WM cases, and 7.2% (5.9%-9.5%) in healthy controls.

No significant difference was observed between MGUS patients

and healthy volunteers (p=0.8), but results were significant for

other PCDs (p=0.018) (Figures 2a and 2b).

Discussion

Substantial advances in understanding the biology of PCD

progression have been achieved through the study of the bone

marrow microenvironment [8]. The bone marrow niche appears

to play an important role in the differentiation, proliferation,

migration, and survival of plasma cells. It is composed of a

heterogeneous cellular compartment that includes stromal cells,

osteoblasts, osteoclasts, endothelial cells, and immune cells [13].

Intercellular interaction appears to induce immune dysfunction,

Table 1. Patients’ demographic, clinical, and laboratory characteristics.

Characteristics MM WM PCL MGUS Healthy controls

Sex

Male

Female

5 (36%)

9 (64%)

1 (33%)

2 (67%)

1 (50%)

1 (50%)

4 (50%)

4 (50%)

6 (32%)

13 (68%)

Age 67.3±13.4 74.3±6.7 75.5±4.9 67.9±15.5 48.1±18.8

Hemoglobin (g/dL) 10.8±1.7 11.4±3.6 11.4±3.6 12.1±2.7 13.2±1.4

Creatinine (mg/dL) 1.4±1.3 0.9±0.3 0.9±0.1 1.4±1.3 0.8±0.2

Calcium (mg/dL) 9.5±1.9 10.4±1.3 9.8±0.4 8.9±1.5 9.5±0.2

Albumin (g/dL) 3.7±0.7 3.9±0.9 4.1±0.5 3.8±0.8 4.3±0.4

Beta-2-microglobulin (mg/L) 8.1±7.3 2.9±1.1 2.3±0 3.2±1.6 Unknown

M spike

IgG kappa

g/dL

IgG lambda

g/dL

IgA kappa

g/dL

IgA lambda

g/dL

IgM kappa

g/dL

IgM lambda

g/dL

FLC kappa

Kappa/lambda ratio

FLC lambda

Kappa/lambda ratio

5 (36%)

2.9±3.3

2 (14%)

2.7±3.1

-

-

1 (7%)

0.1±0

-

-

-

-

4 (28%)

57.3±66.0

2 (14%)

0.007±0.009

-

-

-

-

-

-

-

-

2 (67%)

0.45±0.07

1 (33%)

1.0±0

-

-

-

-

-

-

1 (50%)

0.1±0

-

-

-

-

-

-

-

-

1 (50%)

Unknown

-

-

8 (100%)

1.1±0.98

-

-

-

-

-

-

-

-

-

-

-

-

-

-

MM: Multiple myeloma, WM: Waldenström’s macroglobulinemia, PCL: plasma cell leukemia, MGUS: monoclonal gammopathy of unknown significance.

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

118



which is also an important feature of MGUS and MM and may

promote progression from a premalignant state to malignancy

[8,10,21,22,23].

Monocytes, macrophages, and mesenchymal stromal cells play

a role in MM pathogenesis, where they support the survival and

proliferation of neoplastic myeloma cells [25,26,27].

MDSCs are a heterogeneous population of immature myeloid

cells at different stages of maturation; they play a role in

cancer tolerance and function as an immunosuppressive cell

subpopulation [2].

Several studies have analyzed the frequency and function of

MDSCs in MM, indicating that they promote both myeloma

growth and osteoclast activity and are involved in cross-talk

with Treg cells, resulting in their expansion in the bone marrow

microenvironment [28,29,30,31].

We hypothesize that the enhanced activity of a monocyte

subpopulation with immunosuppressive activity may play a role

in patients with PCDs. We were able to demonstrate that, in

parallel to disease progression from MGUS to MM and plasma

cell leukemia, the number of monocytic MDSCs appears to

increase and they may express more IL-4R, which is critical

for suppression of MDSC function through the L4Ra-STAT6

pathway and thereby indicative of greater immune-related

activity [32].

The preliminary results that we report here are in keeping

with those of a recent study that also demonstrated increased

activity of CD14/CD16+ monocytes in different monoclonal

gammopathies in a hierarchical pattern. Indeed, these CD14/

C16+ monocytes isolated from MM patients appear to

contribute to bone disease and osteoclastogenesis via IL-21

overexpression [6].

Recently, a novel regulatory cell subset population has also

been described: Tregs and immature dendritic cells that express

human LAP (LAP+) [3,4,5,33,34,35]. To date, LAP+ expression

on monocytes or monocytic MDSCs has not yet been studied

extensively, but based on our lab’s preliminary results, showing

high expression of LAP on the surface of CD14+ mononuclear

cells isolated from patients with ankylosing spondylitis [35], we

decided to examine this phenomenon in patients with PCDs. Here

we indeed show that monocytes isolated from these patients

have higher positive expression of LAP and that the frequency

of its expression was correlated with disease progression.

Our results may have additional significance for biomarkers of

disease activity and we are currently initiating a study analyzing

these two subpopulations after therapy in symptomatic patients

with PCDs.

Figure 1. Flow-cytometry analysis of peripheral blood from

patients with different plasma cell dyscrasias in comparison to

healthy controls. a) Coexpression of CD14+/HLA-DR+dim. b)

Coexpression of CD14+/CD124+, both representing the average of

myeloid-derived suppressor cell (MDSC) percentage identified in

the peripheral blood of each cohort. c) An example of fluorescence

activated cell scanning analysis presenting peripheral blood

infiltrated by MDSCs in monoclonal gammopathy of unknown

significance, multiple myeloma, and plasma cell leukemia patients.

MM: Multiple myeloma, MGUS: monoclonal gammopathy of unknown

significance, MDSC: Myeloid-derived suppressor cell, LAP: latencyassociated

peptide, WM: Waldenström’s macroglobulinemia.

Figure 2. Flow-cytometry analysis of peripheral blood from

patients with different plasma cell dyscrasias in comparison to

healthy controls for the expression of latency-associated peptide

(LAP) on monocytes. a) Coexpression of CD14+/ LAP+. Results

represent the average percentage identified in the blood of each

cohort. b) An example of fluorescence activated cell scanning

analysis presenting peripheral blood infiltrated by monocytes/

LAP+ cells in a healthy control and a multiple myeloma patient.

LAP: Latency-associated peptide.

119



In addition, it has been reported that when effective therapy

for PCD is given, as with immunoregulatory lenalidomide

[36,37,38,39] and more recently treatment with daratumumab

[40], immunosuppressive MDSCs, Tregs, and Bregs are

reduced while the expression of CD4+ T-helper cells and

CD8+ cytotoxic T cells is increased, supporting a numerical

correlation between their frequency and disease activity.

Our study obviously has several limitations, including the limited

size of the cohort, the fact that these immunosuppressive

populations were isolated from peripheral blood and not bone

marrow, and the lack of functional assays.

Conclusion

In conclusion, we observed a hierarchical correlation between

the subtypes of PCD categories and the recruitment of two

subpopulations of monocytes, monocytic MDSCs and monocytes

expressing LAP, with immunosuppressive activity. These results

suggest that MDSCs and LAP play diverging roles in PCDs and

may have potential roles as markers of tumor activity. Our

results require further validation and we are now performing

a subsequent study to validate them and analyze the effect of

therapy on these two subpopulations.

Ethics

Ethics Committee Approval: The study was approved by the

hospital’s ethics committee.

Informed Consent: Written informed consent was obtained

from all patients.


Medical Practices: T.T., I.L., Z.V.; Concept: T.T.; Design: T.T., Z.V.; Data

Collection or Processing: Z.V., I.L.; Analysis or Interpretation: T.T.,

Z.V.; Literature Search: T.T.; Writing: T.T., I.L., Z.V.




included.

References

1. Tadmor T, Attias D, Polliack A. Myeloid-derived suppressor cells - their

role in haemato-oncological malignancies and other cancers and possible

implications for therapy. Br J Haematol 2011;153:557-567.

2. Gabrilovich DI, Nagaraj S. Myeloid-derived-supressor cells as regulators of

the immune system. Nat Rev Immunol 2009;9:162-174.

3. Gandhi R, Anderson DE, Weiner HL. Cutting edge: immature human dendritic

cells express latency-associated peptide and inhibit T cell activation in a

TGF-β-dependent manner. J Immunol 2007;178:4017-4021.

4. Gandhi R, Farez MF, Wang Y, Kozoriz D, Quintana FJ, Weiner HL. Cutting

edge: human latency-associated peptide + T cells: a novel regulatory T cell

subset. J Immunol 2010;184:4620-4624.

5. Slobodin G, Kaly L, Peri R, Kessel A, Rosner I, Toubi E, Rimar D, Boulman N,

Rozenbaum M, Odeh M. Higher expression of latency-associated peptide

on the surface of peripheral blood monocytes in patients with rheumatoid

arthritis may be protective against articular erosions. Inflammation

2013;36:1075-1078.

6. Bolzoni M, Ronchetti D, Storti P, Donofrio G, Marchica V, Costa F, Agnelli

L, Toscani D, Vescovini R, Todoerti K, Bonomini S, Sammarelli G, Vecchi A,

Guasco D, Accardi F, Palma BD, Gamberi B, Ferrari C, Neri A, Aversa F, Giuliani

N. IL21R expressing CD14+CD16+ monocytes expand in multiple myeloma

patients leading to increased osteoclasts. Haematologica 2017;102:773-

784.

7. Hu J, Van Valckenborgh E, Menu E, De Bruyne E, Vanderkerken K.

Understanding the hypoxic niche of multiple myeloma: therapeutic

implications and contributions of mouse models. Dis Model Mech

2012;5:763-771.

8. Jakubikova J, Cholujova D, Hideshima T, Gronesova P, Soltysova A, Harada

T, Joo J, Kong SY, Szalat RE, Richardson PG, Munshi NC, Dorfman DM,

Anderson KC. A novel 3D mesenchymal stem cell model of the multiple

myeloma bone marrow niche: biologic and clinical applications. Oncotarget

2016;7:77326-77341.

9. Xu Y, Zhang X, Liu H, Zhao P, Chen Y, Luo Y, Zhang Z, Wang X. Mesenchymal

stromal cells enhance the suppressive effects of myeloid-derived suppressor

cells of multiple myeloma. Leuk Lymphoma 2017;58:2668-2676.

10. Bianchi G, Ghobrial IM. Molecular mechanisms of effectiveness of novel

therapies in multiple myeloma. Leuk Lymphoma 2013;54:229-241.

11. Tadmor T. The growing link between multiple myeloma and myeloid derived

suppressor cells. Leuk Lymphoma 2014;55:2681-2682.

12. Brimnes MK, Vangsted AJ, Knudsen LM, Gimsing P, Gang AO, Johnsen HE,

Svane IM. Increased level of both CD4+FOXP3+ regulatory T cells and

CD14+HLA-DR–/low myeloid-derived suppressor cells and decreased level

of dendritic cells in patients with multiple myeloma. Scand J Immunol

2010;72:540-547.

13. De Veirman K, Van Ginderachter JA, Lub S, De Beule N, Thielemans K,

Bautmans I, Oyajobi BO, De Bruyne E, Menu E, Lemaire M, Van Riet I,

Vanderkerken K, Van Valckenborgh E. Multiple myeloma induces Mcl-1

expression and survival of myeloid-derived suppressor cells. Oncotarget

2015;6:10532-10547.

14. Favaloro J, Liyadipitiya T, Brown R, Yang S, Suen H, Woodland N, Nassif N,

Hart D, Fromm P, Weatherburn C, Gibson J, Ho PJ, Joshua D. Myeloid derived

suppressor cells are numerically, functionally and phenotypically different

in patients with multiple myeloma. Leuk Lymphoma 2014;55:2893-2900.

15. Wang Z, Zhang L, Wang H, Xiong S, Li Y, Tao Q, Xiao W, Qin H, Wang Y,

Zhai Z. Tumor-induced CD14 + HLA-DR –/low myeloid-derived suppressor cells

correlate with tumor progression and outcome of therapy in multiple

myeloma patients. Cancer Immunol Immunother 2015;64:389-399.

16. Malek E, de Lima M, Letterio JJ, Kim BG, Finke JH, Driscoll JJ, Giralt SA.

Myeloid-derived suppressor cells: the green light for myeloma immune

escape. Blood Rev 2016;30:341-348.

17. Kyle RA, Rajkumar SV. Criteria for diagnosis, staging, risk stratification and

response assessment of multiple myeloma. Leukemia 2009;23:3-9.

18. Palumbo A, Avet-Loiseau H, Oliva S, Lokhorst HM, Goldschmidt H, Rosinol

L, Richardson P, Caltagirone S, Lahuerta JJ, Facon T, Bringhen S, Gay F,

Attal M, Passera R, Spencer A, Offidani M, Kumar S, Musto P, Lonial S,

Petrucci MT, Orlowski RZ, Zamagni E, Morgan G, Dimopoulos MA, Durie BG,

Anderson KC, Sonneveld P, San Miguel J, Cavo M, Rajkumar SV, Moreau P.

Revised international staging system for multiple myeloma: a report from

International Myeloma Working Group. J Clin Oncol 2015;33:2863-2869.

19. Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, Mateos MV,

Kumar S, Hillengass J, Kastritis E, Richardson P, Landgren O, Paiva B, Dispenzieri

A, Weiss B, LeLeu X, Zweegman S, Lonial S, Rosinol L, Zamagni E, Jagannath

S, Sezer O, Kristinsson SY, Caers J, Usmani SZ, Lahuerta JJ, Johnsen HE, Beksac

M, Cavo M, Goldschmidt H, Terpos E, Kyle RA, Anderson KC, Durie BG, Miguel

JF. International Myeloma Working Group updated criteria for the diagnosis

of multiple myeloma. Lancet Oncol 2014;15:538-548.

120



20. Fernández de Larrea C, Kyle RA, Durie BG, Ludwig H, Usmani S, Vesole

DH, Hajek R, San Miguel JF, Sezer O, Sonneveld P, Kumar SK, Mahindra A,

Comenzo R, Palumbo A, Mazumber A, Anderson KC, Richardson PG, Badros

AZ, Caers J, Cavo M, LeLeu X, Dimopoulos MA, Chim CS, Schots R, Noeul A,

Fantl D, Mellqvist UH, Landgren O, Chanan-Khan A, Moreau P, Fonseca R,

Merlini G, Lahuerta JJ, Bladé J, Orlowski RZ, Shah JJ; International Myeloma

Working Group. Plasma cell leukemia: consensus statement on diagnostic

requirements, response criteria and treatment recommendations by the

International Myeloma Working Group. Leukemia 2013;27:780-791.

21. Manier S, Sacco A, Leleu X, Ghobrial IM, Roccaro AM. Bone marrow

microenvironment in multiple myeloma progression. J Biomed Biotechnol

2012;2012:157496.

22. Landgren O, Kyle RA, Pfeiffer RM, Katzmann JA, Caporaso NE, Hayes RB,

Dispenzieri A, Kumar S, Clark RJ, Baris D, Hoover R, Rajkumar SV. Monoclonal

gammopathy of undetermined significance (MGUS) consistently precedes

multiple myeloma: a prospective study. Blood 2009;113:5412-5417.

23. Pratt G, Goodyear O, Moss P. Immunodeficiency and immunotherapy in

multiple myeloma. Br J Haematol 2007;138:563-579.

24. Kim J, Denu RA, Dollar BA, Escalante LE, Kuether JP, Callander NS,

Asimakopoulos F, Hematti P. Macrophages and mesenchymal stromal cells

support survival and proliferation of multiple myeloma cells. Br J Haematol

2012;158:336-346.

25. Zheng Y, Cai Z, Wang S, Zhang X, Qian J, Hong S, Li H, Wang M, Yang J, Yi Q.

Macrophages are an abundant component of myeloma microenvironment

and protect myeloma cells from chemotherapy drug-induced apoptosis.

Blood 2009;114:3625-3628.

26. Ribatti D, Vacca A. The role of monocytes-macrophages in vasculogenesis in

multiple myeloma. Leukemia 2009;23:1535-1536.

27. Asimakopoulos F, Kim J, Denu RA, Hope C, Jensen JL, Ollar SJ, Hebron E,

Flanagan C, Callander N, Hematti P. Macrophages in multiple myeloma:

emerging concepts and therapeutic implications. Leuk Lymphoma

2013;54:2112-2121.

28. Görgün GT, Whitehill G, Anderson JL, Hideshima T, Maguire C, Laubach

J, Raje N, Munshi NC, Richardson PG, Anderson KC. Tumor-promoting

immune-suppressive myeloid-derived suppressor cells in the multiple

myeloma microenvironment in humans. Blood 2013;121:2975-2987.

29. Ramachandran IR, Martner A, Pisklakova A, Condamine T, Chase T, Vogl T,

Roth J, Gabrilovich D, Nefedova Y. Myeloid derived suppressor cells regulate

growth of multiple myeloma by inhibiting T cells in bone marrow. J Immunol

2013;190:3815-3823.

30. Van Valckenborgh E, Schouppe E, Movahedi K, De Bruyne E, Menu E, De

Baetselier P, Vanderkerken K, Van Ginderachter JA. Multiple myeloma induces

the immunosuppressive capacity of distinct myeloid-derived suppressor cell

subpopulations in the bone marrow. Leukemia 2012;26:2424-2428.

31. Zhuang J, Zhang J, Lwin ST, Edwards JR, Edwards CM, Mundy GR, Yang X.

Osteoclasts in multiple myeloma are derived from Gr-1+CD11b+myeloidderived

suppressor cells. PLoS One 2012;7:e48871. Roth F, De La Fuente AC,

Vella JL, Zoso A, Inverardi L, Serafini P.

32. Aptamer-mediated blockade of IL4Rα triggers apoptosis of MDSCs and

limits tumor progression. Cancer Res 2012;72:1373-1383.

33. Ali NA, Gaughan AA, Orosz CG, Baran CP, McMaken S, Wang Y, Eubank

TD, Hunter M, Lichtenberger FJ, Flavahan NA, Lawler J, Marsh CB. Latency

associated peptide has in vitro and in vivo immune effects independent of

TGF-β1. PLoS One 2008;3:e1914.

34. Zhang Y, Morgan R, Chen C, Cai Y, Clark E, Khan WN, Shin SU, Cho HM,

Al Bayati A, Pimentel A, Rosenblatt JD. Mammary-tumor-educated B cells

acquire LAP/TGF-β and PD-L1 expression and suppress antitumor immune

responses. Int Immunol 2016;28:423-433.

35. Slobodin G, Kessel A, Kuznets I, Peri R, Haj T, Rosner I, Toubi E, Odeh M.

CD14 bright LAP + mononuclear cells in peripheral blood positively correlate

with BASRI scores in patients with ankylosing spondylitis: a pilot study.

Joint Bone Spine 2012;79:633-634.

36. Handa H, Saitoh T, Murakami H. Immunomodulatory effects of lenalidomide.

Nihon Rinsho 2015;73:156-161.

37. Görgün G, Samur MK, Cowens KB, Paula S, Bianchi G, Anderson JE, White

RE, Singh A, Ohguchi H, Suzuki R, Kikuchi S, Harada T, Hideshima T, Tai YT,

Laubach JP, Raje N, Magrangeas F, Minvielle S, Avet-Loiseau H, Munshi NC,

Dorfman DM, Richardson PG, Anderson KC. Lenalidomide enhances immune

checkpoint blockade induced immune response in multiple myeloma. Clin

Cancer Res 2015;21:4607-4618.

38. Busch A, Zeh D, Janzen V, Mügge LO, Wolf D, Fingerhut L, Hahn-Ast C,

Maurer O, Brossart P, von Lilienfeld-Toal M. Treatment with lenalidomide

induces immunoactivating and counter-regulatory immunosuppressive

changes in myeloma patients. Clin Exp Immunol 2014;177:439-453.

39. Costa F, Vescovini R, Bolzoni M, Marchica V, Storti P, Toscani D, Accardi F,

Notarfranchi L, Dalla Palma B, Manferdini C, Manni S, Todaro G, Lisignoli

G, Piazza F, Aversa F, Giuliani N. Lenalidomide increases human dendritic

cell maturation in multiple myeloma patients targeting monocyte

differentiation and modulating mesenchymal stromal cell inhibitory

properties. Oncotarget 2017;8:53053-53067.

40. Krejcik J, Casneuf T, Nijhof IS, Verbist B, Bald J, Plesner T, Syed K, Liu

K, van de Donk NW, Weiss BM, Ahmadi T, Lokhorst HM, Mutis T, Sasser

AK. Daratumumab depletes CD38 + immune regulatory cells, promotes

T-cell expansion, and skews T-cell repertoire in multiple myeloma. Blood

2016;21;128:384-394.

121

RESEARCH ARTICLE

DOI: 10.4274/tjh.2017.0444


Acute Traumatic Coagulopathy: The Value of Histone in Pediatric

Trauma Patients

Akut Travma İlişkili Koagülopati: Pediatrik Travma Hastalarında Histonun Yeri

Emel Ulusoy 1 , Murat Duman 1 , Aykut Çağlar 1 , Tuncay Küme 2 , Anıl Er 1 , Fatma Akgül 1 , Hale Çitlenbik 1 ,

Durgül Yılmaz 1 , Hale Ören 3

1

Dokuz Eylül University Faculty of Medicine, Department of Pediatric Emergency Care, İzmir, Turkey

2

Dokuz Eylül University Faculty of Medicine, Department of Biochemistry, İzmir, Turkey

3

Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey

Abstract

Objective: Acute traumatic coagulopathy occurs after trauma

with impairment of hemostasis and activation of fibrinolysis. Some

endogenous substances may play roles in this failure of the coagulation

system. Extracellular histone is one such molecule that has recently

attracted attention. This study investigated the association between

plasma histone-complexed DNA (hcDNA) fragments and coagulation

abnormalities in pediatric trauma patients.

Materials and Methods: This prospective case-control study was

conducted in pediatric patients with trauma. Fifty trauma patients and

30 healthy controls were enrolled. Demographic data, anatomic injury

characteristics, coagulation parameters, computerized tomography

findings, trauma, and International Society on Thrombosis and

Haemostasis disseminated intravascular coagulation (ISTH DIC) scores

were recorded. Blood samples for hcDNA were collected and assessed

by enzyme-linked immunosorbent assay.

Results: Thirty-two patients had multiple trauma, while 18 patients

had isolated brain injury. hcDNA levels were significantly higher

in trauma patients than healthy controls (0.474 AU and 0.145 AU,

respectively). There was an association between plasma hcDNA levels

and trauma severity. Thirteen patients had acute coagulopathy of

trauma shock (ACoTS). ACoTS patients had higher plasma histone

levels than those without ACoTS (0.703 AU and 0.398 AU, respectively).

Plasma hcDNA levels were positively correlated with the ISTH DIC

score and length of stay in the intensive care unit and were negatively

correlated with fibrinogen level.

Conclusion: This study indicated that hcDNA levels were increased

in pediatric trauma patients and associated with the early phase of

coagulopathy. Further studies are needed to clarify the role of hcDNA

levels in mortality and disseminated intravascular coagulation.

Keywords: Acute traumatic coagulopathy, Children, Histone, Trauma

Öz

Amaç: Akut travma ilişkili koagülopati; travma sonrası ortaya çıkan,

hemostazda bozulma ve fibrinoliz aktivasyonudur. Koagülasyon

sistemindeki bu bozuklukta bazı endojen moleküller rol oynamaktadır.

Histon bu moleküllerden bir tanesi olup son dönemlerde dikkat

çekmeye başlamıştır. Bu çalışmada, pediatrik travma olgularında

histon-kompleks DNA (hcDNA) fragmanları ile koagülasyon

anormallikleri arasındaki ilişkinin incelenmesi amaçlanmıştır.

Gereç ve Yöntemler: Bu çalışma pediatrik travma olgularında yapılmış

prospektif olgu-kontrol çalışmasıdır. Çalışmaya 50 hasta ve 30 kontrol

olgusu dahil edildi. Tüm hastaların demografik verileri, travmanın

özellikleri, koagülasyon parametreleri, bilgisayarlı tomografi sonuçları,

travma skorları ve Dissemine İntravasküler Koagülasyon skoru (DİKS)

kaydedildi. hcDNA düzeyi için kan örnekleri alınarak enzim ilintili

immün test ile değerlendirildi.

Bulgular: Hastaların 32’sinde çoklu travma, 18’inde izole kafa travması

mevcuttu. hcDNA düzeyi travma olgularında sağlıklı kontrollere göre

istatistiksel olarak anlamlı yüksek bulundu (0,474 AU and 0,145 AU,

sırasıyla). Plazma hcDNA düzeyi ile travma ciddiyeti arasında anlamlı

ilişki saptandı. On üç hastada akut travma ilişkili koagülopati saptanmış

olup, bu hastaların akut travma ilişkili koagülopati olmayanlara göre

daha yüksek plazma histon düzeyine sahip oldukları görüldü (0,703

AU and 0,398 AU, sırasıyla). Plazma hcDNA düzeyinin, DİKS ve yoğun

bakımda kalış süresi ile pozitif; fibrinojen düzeyi ile negatif korelasyon

gösterdiği bulundu.

Sonuç: Bu çalışmada, pediatrik travma olgularında hcDNA düzeyinin

arttığı ve koagülopatinin erken fazıyla ilişkili olduğu gösterilmiştir.

hcDNA’nın dissemine intravasküler koagülasyon ve mortalite oranını

belirlemedeki yerini ortaya koymak için ileri çalışmalara ihtiyaç vardır.

Anahtar Sözcükler: Akut travma ilişkili koagülopati, Çocuk, Histon,

Travma



Address for Correspondence/Yazışma Adresi: Hale ÖREN, M.D.,

Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey

Phone : +90 232 412 60 01

E-mail : hale.oren@deu.edu.tr ORCID-ID: orcid.org/0000-0001-5760-8007

Received/Geliş tarihi: December 11, 2017


122


Ulusoy E, et al: Acute Traumatic Coagulopathy and Histone

Introduction

Trauma is the leading cause of visits to pediatric emergency

departments [1]. The majority of pediatric trauma is minor,

but it remains an important cause of morbidity and mortality

in childhood [2]. While massive bleeding is less common in

the pediatric trauma cohort, coagulation abnormalities have

been described in 10% to 77% of patients [3]. In this regard,

identification of coagulopathy and early intervention are

important in severely injured trauma patients [4,5].

Acute traumatic coagulopathy (ATC) is an endogenous process

that occurs after trauma with the impairment of hemostasis

and activation of fibrinolysis [6]. Patients with ATC frequently

meet the criteria for disseminated intravascular coagulation

(DIC). Currently available data suggest that ATC reflects the

early phase of DIC in trauma patients [7,8]. Three major factors

that are associated with subsequent development of ATC are

hemodilution, hypothermia, and acidosis, and its complex

nature is exacerbated by shock and tissue injury [9,10].

Accumulating evidence supports an important role of different

interactions between coagulation and inflammation in ATC.

Damage-associated molecules such as histone-complexed DNA

(hcDNA) fragments released after trauma play a significant role

in the balance of the coagulation system [11,12]. There are five

types of histones; all have alkaline structures. Histones form an

organized pattern with DNA in the cell nucleus by neutralizing

the acidic residues of the DNA [13]. Complex structures of

DNA, histones, and cell-specific granular proteins, known

as neutrophil extracellular traps (NETs), can be released into

circulation after stimulation by inflammatory cytokines [14,15].

During NET osis, which is a pathogen-induced cell death causing

NET release or tissue damage, nuclear and plasma membranes

dissolve or rupture and nuclear materials are released into the

circulation [12,16].

DIC can be subdivided into two different phenotypes:

fibrinolytic (hemorrhagic) and antifibrinolytic (thrombotic).

The antifibrinolytic phenotype is associated with plasminogen

activator inhibitor-1 and is seen in sepsis or the late phase of

trauma, while the fibrinolytic phenotype leads to coagulopathy

including primary and secondary fibrin(ogen)olysis in the early

phase of trauma [7]. NETs, contributing factors to coagulopathy

in the early stage of trauma, have various effects on the vascular

endothelium, platelets, erythrocytes, and coagulation proteins

[14,15]. Although NETs contain different components like

neutrophil granule enzymes and bactericidal molecules, the main

structure consists of DNA and histones [16]. hcDNA plays a role

in coagulopathy by increasing thrombin formation, activating

platelets, stimulating endothelial activation, inhibiting tissue

factor pathway inhibitor, causing thrombocytopenia, decreasing

fibrinogen, inhibiting anticoagulant protein C activation,

and stimulating factor XII-mediated thrombin generation

[15,17,18,19,20,21,22,23,24]. In addition, hcDNA complexes,

having an integrated linkage between inflammation and

coagulation, augment thrombin generation to a greater extent

than histones alone [15].

To date, few studies have been done assessing the extracellular

hcDNA fragment levels in trauma patients [12,25], while

there are no studies investigating this in the pediatric trauma

population. The aim of this study was to investigate the

relationship of histone with coagulopathy in pediatric trauma

patients and also to analyze coagulopathy frequency and its

relationship with clinical findings.


Study Population

This is a prospective case-control study conducted among

pediatric patients (1-16 years old) with multiple trauma or

isolated brain injury in a pediatric emergency department

between August 2014 and August 2015. Multiple trauma was

defined as injury to more than 1 body system, or at least 2

serious injuries to 1 body system [26]. Fifty trauma patients

were enrolled in the study. Patients with bleeding diathesis,

liver disease, arrival to the trauma center >2 h after injury

and/or >40 mL/kg intravenous fluid given before arrival to the

hospital, and usage of any drugs including antiplatelet drugs or

anticoagulants were all excluded.

Demographic data, patient characteristics, vital signs,

and anatomic injury characteristics were recorded.

The control group consisted of 30 children who were evaluated

in the outpatient clinic of our hospital for routine well-child

visits. None of the children had a history of drug usage, chronic

systemic disease, or any major trauma in the last 6 months.

Scoring Systems

Four scoring systems were used to assess patients upon

admission: the Glasgow Coma scale (GCS) [27], the Pediatric

Trauma score (PTS) [28], the Injury Severity score (ISS) [29], and

the International Society on Thrombosis and Haemostasis

(ISTH) DIC score [30]. GCS scores were classified as mild (14-

15), moderate (9-13), or severe (3-8) to describe the level

of consciousness. The PTS score was determined with six

parameters; the minimum score is -6 and the maximum score

is +12. Trauma severity is inversely correlated with PTS score

and a score of 8 or less indicates the need for trauma services.

The ISS consists of six body regions and produces values from 0

to 75. Major trauma is signified by an ISS score of greater than

15. If an injury is assigned an Abbreviated Injury Scale score of

6 (unsurvivable injury), the ISS score is automatically assigned

as 75. According to the ISTH DIC scale, overt DIC was diagnosed

if the total score was ≥5. The ISTH DIC score includes platelet

123



count, fibrinogen level, prothrombin time (PT), and fibrin

degradation products. Age-appropriate reference ranges within

our trauma center were used to determine prolonged PT (11.2-

14.4 s) and activated partial thromboplastin time (aPTT) (age 1-3

years: 30.6-39.9 s, age 4-7 years: 28.8-38.9 s, age 8-14 years:

28.1-39.1 s, age 14-18 years: 26.0-36.6 s) levels. The presence

of acute coagulopathy of trauma shock (ACoTS) was defined

as prolonged PT and/or aPTT according to the age-appropriate

references ranges [31,32,33,34,35].

The threshold for defining anemia is hemoglobin at or below the

2.5 th percentile for age, race, and sex [36]. Hypothermia is graded

as mild (36-34 °C), moderate (34-32 °C), or severe (<32 °C) [37].

Biochemical Analysis

Biochemical parameters of the study population were assessed

on admission. Venous blood gas, routine biochemistry, complete

blood count, PT, aPTT, fibrinogen, and D-dimer were evaluated

based on normal laboratory reference ranges in the hospital.

Peripheral venous blood samples were collected in blood tubes

with EDTA for hcDNA. Tubes were centrifuged at 1200 x g for

10 min and plasma samples were stored at -80 °C until analysis.

Plasma nucleosome levels were measured with the Cell Death

Detection ELISA PLUS commercial kit based on the principle of

sandwich enzyme immunoassay (Catalog No: 1774425, Roche

Diagnostics, Mannheim, Germany). Results were reported as

absorbance units (AU).

Ethical Approval

The study protocol was designed in compliance with

the Declaration of Helsinki. Informed consent was obtained

from parents or legal guardians before enrollment in the study.

The study was begun after receiving the approval of the Ethics

Committee of the Dokuz Eylül University Faculty of Medicine.


Statistical analysis was performed using SPSS 22.0 (IBM Corp.,

Armonk, NY, USA). Power hoc analysis was performed to

evaluate the sample size. Data are presented as medians with

interquartile ranges (IQRs) and 25 th -75 th percentiles. Histograms

were used to assess the normality of sample distributions. The

Kruskal-Wallis test was used for analyzing plasma hcDNA levels

among different groups. The Mann-Whitney U test was used

for comparing two groups. The chi-square test was used for

comparing group ratios. Correlations between parameters were

computed through Pearson correlation analysis. All t-tests were

two-tailed and group differences or correlations with p<0.05

were considered to be statistically significant. Receiver operating

curve (ROC) analysis was used to detect the optimal cut-off

points for separating the ACoTS group from the healthy control

group. Bonferroni correction was used for multiple comparisons.

Results

Fifty trauma patients and 30 healthy controls were enrolled in

the study. The study group and control children were comparable

in terms of age and sex distribution (Table 1).

Falls were the most frequent cause of injury; the second most

common was motor vehicle accidents (Table 2).

Eighteen (36%) patients had isolated brain injury while 32

(64%) patients had multiple trauma. Three patients had liver

laceration, 3 patients had spleen laceration, and 1 patient had

renal and spleen laceration. Twenty-one patients had anemia

and none of the patients had thrombocytopenia. Although no

patient had overt DIC, 13 patients had ACoTS. Ten patients had

only prolonged PT, 1 patient had only prolonged aPTT, and 2

patients had both. The median level of PT was 13.0 s (12.3-

14.2 s) and the median level of aPTT was 27.6 s (23.7-30.4 s)

in trauma patients. There were significant differences between

patients with ACoTS [PT: 15.6 s (14.9-16.9 s), aPTT: 31.8 s (27.6-

40.0 s)] and those without ACoTS [PT: 12.6 s (12.1-13.4 s),

aPTT: 25.9 s (22.3-29.1 s)] according to hemostasis parameters

(p=0.000 and p=0.001, respectively). Nineteen patients (38%)

were admitted to the intensive care unit (ICU). Emergency

endotracheal intubation was performed for 15 patients. The

overall mortality rate was 6%. Clinical characteristics of the

patients are shown in Table 3.

When we evaluated patients according to the ISS, we determined

that 21 patients had scores over 16 and only three patients had

75 points. Those three patients died.

Table 1. Demographic variables of patients and controls.

Patients

(n=50)

Controls

(n=30)

p-value

Age (IQR) 6.5 (3.0-11.5) 6.5 (3.8-9.5) 0.913*

Males, n (%) 33 (66.0) 20 (66.7) 0.951**

All data presented as median (IQR); *Mann-Whitney U test was used; **chi-square

test was used.

IQR: Interquartile range.

Table 2. Trauma mechanisms in the whole patient group.

Falls 20 (40)

Motor vehicle accidents 11 (22)

Pedestrians struck by a motor vehicle 9 (18)

Bicycle crashes 7 (14)

Others 3 (6)

Trauma mechanism,

n (%)

124



Table 3. Clinical and laboratory characteristics of the patients.

Multiple trauma

Isolated brain injury

n=50 (%)

32 (64)

18 (36)

Bone fracture 26 (52)

Open wounds 25 (50)

Hypotension 4 (8)

Blood transfusion 2 (4)

Pathological findings on CT*

Cranial

Abdominal

Thorax

31 (62)

7 (14)

28 (56)

Endotracheal intubation 15 (30)

Prolonged PT 12 (24)

Prolonged aPTT 3 (6)

Increased D-dimer 47 (94)

Decreased fibrinogen 2 (4)

Metabolic acidosis 30 (60)

Hypothermia 2 (4)

ICU stay 19 (38)

Mortality 3 (6)

*Pathological findings: Cranial CT: Facial-calvarial fracture, subarachnoid hemorrhage,

epidural hemorrhage, subdural hemorrhage, intraparenchymal hemorrhage, contusion,

pneumocephalus. Thorax CT: Pneumothorax, pulmonary contusion, fracture.

Abdominal CT: Spleen/renal/liver laceration, intraabdominal bleeding, intraabdominal

fluid collection, fracture.

CT: Computerized tomography, PT: prothrombin time, aPTT: activated partial

thromboplastin time, ICU: intensive care unit.

Plasma hcDNA levels were significantly higher in trauma patients

[0.474 AU (0.184-0.841 AU)] than in healthy controls [0.145 AU

(0.086-0.361 AU)] (p=0.008). ACoTS patients [0.703 AU (0.301-

0.897 AU)] had higher plasma histone levels than those without

ACoTS [0.398 AU (0.130-0.802 AU)]. We found significant

differences between hcDNA levels and groups according to the

GCS, PTS, ISS, and D-dimer (Table 4), but we did not find any

differences of hcDNA levels in terms of trauma type (p=0.338). ROC

curve analyses of hcDNA were performed. ROC analysis revealed

an optimal cut-off point at 0.186 AU for separating the ACoTS

patients from the control group. The sensitivity and specificity

were 76.0% and 66.4%, respectively. The area under the curve for

hcDNA was 0.679 (p=0.008) (Figure 1).

Plasma hcDNA levels were significantly correlated with ISTH

DIC score (r=0.433, p=0.002) and length of stay in the ICU

(r=0.314, p=0.026) in the whole study group. There was a

negative correlation between hcDNA levels and PTS score (r=-

0.464, p=0.001). When we investigated coagulation parameters,

we found a positive correlation between hcDNA levels and

D-dimer levels (r=0.597, p≤0.001) and a negative correlation

with fibrinogen (r=-0.342, p=0.015).

While most of the patients with organ laceration had higher

Table 4. Histone-complexed DNA levels of patients and controls.

Patients

Controls

Patients

GCS

PTS

ISS

ACoTS

No ACoTS

D-dimer

PT

APTT

Fibrinogen

hcDNA levels

(AU)

(n=50) 0.474 (0.184-0.841)

(n=30) 0.145 (0.086-0.361)

Male (n=17) 0.463 (0.094-0.853)

Female (n=33) 0.485 (0.243-0.802)

14-15 (n=27) 0.246 (0.095-0.709)

9-13 (n=11) 0.785 (0.666-0.932)

3-8 (n=12) 0.527 (0.252-0.871)

9-12 (n=21) 0.246 (0.098-0.747)

0-8 (n=29) 0.688 (0.404-0.864)

16-75 (n=26) 0.695 (0.379-0.865)

0-15 (n=24) 0.243 (0.094-0.652)

(n=13) 0.703 (0.301-0.897)

(n=37) 0.398 (0.130-0.802)

>0.5 (n=47) 0.503 (0.231-0.843)

≤0.5 (n=3) 0.094 (0.038-0.101)

>14.4 s (n=12) 0.767 (0.368-0.902)

≤14.4 s (n=38) 0.359 (0.119-0.793)

>Age-appropriate

references range

(n=3)

0.324 (0.094-0.324)

Normal (n=47) 0.485 (0.196-0.841)

<1.8 g/L (n=2) 0.892 (0.785-0.892)

≥1.8 g/L (n=48) 0.442 (0.160-0.838)

p-value

0.008*

0.759*

0.009**

0.007*

0.004*

0.044*

0.008*

0.013*

0.854

0.097

All data are presented as median (IQR); *Mann-Whitney U test was used; **Kruskal-

Wallis test was used.

hcDNA: Histone-complexed DNA; GCS: Glasgow Coma Scale; PTS: Pediatric Trauma Score; ISS: Injury

Severity Score; ACoTS: acute coagulopathy of trauma shock; PT: prothrombin time; aPTT: activated

partial thromboplastin time; IQR: interquartile range.

Figure 1. Receiver operating characteristic curve analyses of

histone-complexed DNA.

125



hcDNA levels than the median level in the trauma group, we

did not find any differences in hcDNA levels according to

pathological findings in computerized tomography (CT) of the

brain, thorax, or abdomen (p=0.342, p=0.229, and p=0.071,

respectively).

Discussion

In the present study, blood levels of hcDNA fragments and ATC

were assessed in pediatric trauma patients. The findings showed

that extracellular histone correlates with ATC and also trauma

severity.

In this study, ACoTS was determined in 13 (26%) patients. In the

literature there is a wide range, varying from 10% to 71%, for

the incidence of coagulopathy on admission after severe trauma

in the pediatric population [3]. Routine coagulation tests such as

PT, aPTT, international normalized ratio, fibrinogen, or fibrinogen

degradation product have been used to determine the presence

of coagulopathy, but Mann et al. [38] showed that these tests do

not indicate whole coagulation system abnormalities because

of reflecting only 4% of thrombin production. Different studies

have been performed to clarify mechanisms of coagulopathy

after trauma. In a large retrospective study it was found that

large-volume resuscitation with fluid during the management

of shock causes dilution of plasma proteins and coagulation

factors [39], but Brohi et al. [35] also showed that coagulopathy

could occur before excessive fluid resuscitation. Acidosis and

hypothermia can be easily observed in patients with especially

severe trauma, which cause clotting and platelet dysfunction.

Dirkmann et al. [40] showed that if both of them existed, a

synergistic effect occurred on coagulopathy and mortality was

increased. These factors increase the coagulopathy risks after

trauma, but the exact nature of this process is still not clear and

correction of acidosis and hypothermia does not always correct

the associated coagulopathy. This has led researchers to continue

investigating the additional underlying mechanisms. Damageassociated

molecules play a significant role in the balance of

the coagulation system in critically ill children [10,14]. In the

current study, we demonstrated the increase of hcDNA in the

early phase of coagulopathy without existing DIC in pediatric

trauma patients.

This study showed that plasma hcDNA levels were higher in the

trauma group than in healthy controls. This increase occurs because

of nuclear proteins being released out of the cell membrane with

cells dying in critically ill patients and in cases of trauma [41]. As

is well known, nuclear and plasma membranes must be damaged

for the release of intranuclear substances like histone or DNA to

occur after mechanical trauma [42]. In this regard, extracellular

hcDNA levels must be higher as trauma becomes more serious with

growing tissue damage. Kutcher et al. [25] showed that critically

injured adult trauma patients with high hcDNA levels had higher

ISS and lower GCS scores. In another study of adults, Johansson

et al. [12] found a correlation between the circulating hcDNA

levels and ISS values. In accordance with the literature, this study

shows a relationship between plasma hcDNA levels and trauma

severity according to GCS, PTS, and ISS scores. According to the

GCS, the highest hcDNA level was seen in the moderate GCS group.

Multiple organ injuries were mainly found in the moderate GCS

group. Among these trauma patients, only 2 patients had serious

organ injuries other than head trauma in the severe GCS group. The

amount of tissue damage was highest in the moderate GCS group

and lowest in the mild GCS group, consistent with histone levels.

The main finding of our study was that plasma hcDNA levels

were significantly correlated with coagulation parameters that

indicate coagulopathy in the pediatric trauma population.

After tissue injury, histone moves out of the cell membrane

and increases activated protein C (aPC). In turn, aPC inhibits

FV, FVIII, and PAI-1, thereby creating hypocoagulation and

hyperfibrinolysis [43]. The late phase of trauma can be

complicated with hypercoagulability and thromboembolic

events like prothrombotic states after depletion of aPC stores

as reported in septic patients [44]. In addition, extracellular

histone activates platelets by TLR2 and TLR4 to cause platelet

aggregation [17]. In experimental models with mice, histone

injection caused coagulopathy and bleeding with prolonged PT,

decreased fibrinogen, and fibrin deposition [45]. In this respect,

it seems that histone plays roles in both pro- and anticoagulant

processes. Two human studies examined blood histone levels

and coagulopathy in adult trauma patients [12,25]. The present

study demonstrated a hypocoagulopathic phase at the early

stage of trauma having an association between histone levels

and increased PT and aPTT and decreased fibrinogen.

Another result presented here is plasma hcDNA levels being

correlated with length of stay in the ICU in the whole study

group. A relationship between elevated histone levels and days

of mechanical ventilation was found in trauma patients by

Kutcher et al. [25]. This result is not surprising considering that

patients with high histone levels had high trauma severity and

coagulopathy. The effects of histone on lung tissue were also

shown in human and animal models. High histone levels caused

1.8-fold higher incidence of acute lung injury [25] along with

pulmonary edema, hemorrhage, and microvascular thrombosis

after injection of histone in animal models [46]. Nakahara

et al. [45] showed that extracellular histones caused platelet

aggregation, thrombotic occlusion of pulmonary capillaries,

and right-sided heart failure. We could not show a relationship

between histone levels and thorax, cranial, or abdominal CT

findings. Because these patients had multiple trauma, we could

not isolate any organ systems from the other tissues. When

these patients had higher ISS values, we attributed it to the

release of histone from dying cells that could not be shown

126



by imaging methods. We are unable to discuss whether a high

histone level is a marker for mortality due to the small size of

our patient population.

Treatment approaches have undergone more investigation

since the understanding of the important role of NETs in

coagulopathy. NETs may represent an attractive target for

antithrombotic therapy. In the literature, prevention of histone

toxicity on platelets and protection against histone-induced

thrombocytopenia by heparin were demonstrated [17,18].

Nakahara et al. [45] also demonstrated that recombinant

thrombomodulin protects mice against histone-induced lethal

thromboembolism. New therapeutic approaches have caused

excitement, with the better understanding of NETs including

hcDNA contributing to the pathophysiology of coagulopathy.

Conclusion

In conclusion, this study indicated that hcDNA levels increase in

pediatric trauma patients associated with coagulopathy. There

was an association between plasma hcDNA levels and trauma

severity according to GCS, PTS, and ISS scores. There was also

a significant correlation between hcDNA levels and length of

stay in the ICU. Further studies are needed to clarify the role of

high hcDNA levels in determining the functional significance of

these changes in therapy, DIC, and prediction of mortality.

Ethics

Ethics Committee Approval: The study was begun after

receiving the approval of the Ethics Committee of the Dokuz

Eylül University Faculty of Medicine.

Informed Consent: Informed consent was obtained from

parents or legal guardians before enrollment in the study.


Concept: E.U., H.Ö.; Design: E.U., F.A., H.Ç.; Data Collection or

Processing: E.U., A.Ç., A.E.; Analysis or Interpretation: E.U., M.D.,

D.Y., A.Ç., T.K.; Literature Search: H.Ö., M.D.; Writing: E.U., H.Ö.,

M.D., T.K.

Conflicts of Interest: The authors of this paper have no conflicts



included.

References

1. Wier LM, Hao Y, Owens P, Washington R. Overview of Children in the

Emergency Department, 2010. CUP Statistical Brief #157. Rockville, Agency

for Healthcare Research and Quality, 2013.

2. Avarello JT, Cantor RM. Pediatric major trauma: an approach to evaluation

and management. Emerg Med Clin North Am 2007;25:803-836.

3. Christians SC, Duhachek-Stapelman AL, Russell RT, Lisco SJ, Kerby JD, Pittet

JF. Coagulopathy after severe pediatric trauma. Shock 2014;41:476-490.

4. Rourke C, Curry N, Khan S, Taylor R, Raza I, Davenport R, Stanworth S, Brohi

K. Fibrinogen levels during trauma hemorrhage, response to replacement

therapy, and association with patient outcomes. J Thromb Haemost

2012;10:1342-1351.

5. Epstein DS, Mitra B, Cameron PA, Filtzgerald M, Rosenfeld JV. Normalization

of coagulopathy is associated with improved outcome after isolated

traumatic brain injury. J Clin Neurosci 2016;29:64-69.

6. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie RC, Pittet

JF. Acute coagulopathy of trauma: hypoperfusion induces systemic

anticoagulation and hyperfibrinolysis. J Trauma 2008;64:1211-1217.

7. Gando S, Otomo Y. Local hemostasis, immunothrombosis, and systemic

disseminated intravascular coagulation in trauma and traumatic shock. Crit

Care 2015;19:72.

8. Gando S. Acute coagulopathy of trauma shock and coagulopathy of

trauma: a rebuttal. You are now going down the wrong path. J Trauma

2009;67:381-383.

9. Frith D, Goslings JC, Gaarder C, Maegele M, Cohen MJ, Allard S, Johansson

PI, Stanworth S, Thiemermann C, Brohi K. Definition and drivers of acute

traumatic coagulopathy: clinical and experimental investigations. J Thromb

Haemostasis 2010;8:1919-1925.

10. Davenport R. Pathogenesis of acute traumatic coagulopathy. Transfusion

2013;53(Suppl 1):23-27.

11. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K,

Hauser CJ. Circulating mitochondrial DAMPs cause inflammatory responses

to injury. Nature 2010;464:104-107.

12. Johansson PI, Windelov NA, Rasmussen LS, Sørensen AM, Ostrowski SR.

Blood levels of histone-complexed DNA fragments are associated with

coagulopathy, inflammation and endothelial damage early after trauma. J

Emerg Trauma Shock 2013;6:171-175.

13. Felsenfeld G, Groudine M. Controlling the double helix. Nature

2003;421:448-453.

14. Kim JE, Lee N, Gu JY, Yoo HJ, Kim HK. Circulating levels of DNA histone

complex and dsDNA are independent prognostic factors of disseminated

intravascular coagulation. Thromb Res 2015;135:1064-1069.

15. Wisher JW, Becker RC. Antithrombotic therapy: new areas to understand

efficacy and bleeding. Expert Opin Ther Targets 2014;18:1427-1434.

16. Gould TJ, Lysov Z, Liaw PC. Extracellular DNA and histones: double-edged

swords in immunothrombosis. J Thromb Haemost 2015;13(Suppl 1):82-91.

17. Semeraro F, Ammollo CT, Morrissey JH, Dale GL, Friese P, Esmon NL, Esmon

CT. Extracellular histones promote thrombin generation through plateletdependent

mechanisms: involvement of platelet TLR2 and TLR4. Blood

2011;118:1952-1961.

18. Fuchs TA, Bhandari AA, Wagner DD. Histones induce rapid and profound

thrombocytopenia in mice. Blood 2011;118:3708-3714.

19. Martinod K, Wagner DD. Thrombosis: tangled up in NETs. Blood

2014;123:2768-2776.

20. Ammollo CT, Semeraro F, Xu J, Esmon NL, Esmon CT. Extracellular histones

increase plasma thrombin generation by impairing thrombomodulindependent

protein C activation. J Thromb Haemost 2011;9:1795-1803.

21. Komissarov AA, Florova G, Idell S. Effects of extracellular DNA on

plasminogen activation and fibrinolysis. J Biol Chem 2011;286:41949-

41962.

22. Barranco-Medina S, Pozzi N, Vogt AD, Di Cera E. Histone H4 promotes

prothrombin autoactivation. J Biol Chem 2013;288:35749-35757.

23. Xu J, Zhang X, Pelayo R, Monestier M, Ammollo CT, Semeraro F, Taylor FB,

Esmon NL, Lupu F, Esmon CT. Extracellular histones are major mediators of

death in sepsis. Nat Med 2009;15:1318-1321.

24. Altincicek B, Stotzel S, Wygrecka M, Preissner KT, Vilcinskas A. Host-derived

extracellular nucleic acids enhance innate immune responses, induce

coagulation, and prolong survival upon infection in insects. J Immunol

2008;181:2705-2712.

127



25. Kutcher ME, Xu J, Vilardi RF, Ho C, Esmon CT, Cohen MJ. Extracellular histone

release in response to traumatic injury: implications for a compensatory

role of activated protein C. J Trauma Acute Care Surg 2012;73:1389-1394.

26. Letts M, Davidson D, Lapner P. Multiple trauma in children: predicting

outcome and long-term results. Can J Surg 2002;45:126-131.

27. Teasdale G, Jennett B. Assessment of coma and impaired consciousness. A

practical scale. Lancet 1974;2:81-84.

28. Furnival RA, Schunk JE. ABCs of scoring systems for pediatric trauma.

Pediatr Emerg Care 1999;15:215-223.

29. Trauma.org. Injury Severity Score. www.trauma.org/archive/scores/iss.html

(accessed on 21 August 2013).

30. Taylor FB Jr, Toh CH, Hoots WK, Wada H, Levi M; Scientific Subcommittee on

Disseminated Intravascular Coagulation (DIC) of the International Society

on Thrombosis and Haemostasis (ISTH). Towards definition, clinical and

laboratory criteria, and a scoring system for disseminated intravascular

coagulation on behalf of the Scientific Subcommittee on Disseminated

Intravascular Coagulation of the International Society on Thrombosis and

Haemostasis. Thromb Haemost 2001;86:1327-1330.

31. Macleod JB, Lynn M, McKenney MG, Cohn SM, Murtha M. Early coagulopathy

predicts mortality in trauma. J Trauma 2003;55:39-44.

32. Johansson PI, Sørensen AM, Perner A, Welling KL, Wanscher M, Larsen

CF, Ostrowski SR. Disseminated intravascular coagulation or acute

coagulopathy of trauma shock early after trauma? An observational study.

Crit Care 2011;15:R272.

33. Johansson PI, Stensballe J, Rasmussen LS, Ostrowski SR. High circulating

adrenaline levels at admission predict increased mortality after trauma. J

Trauma Acute Care Surg 2012;72:428-436.

34. Curry NS, Davenport RA, Hunt BJ, Stanworth SJ. Transfusion strategies for

traumatic coagulopathy. Blood Rev 2012;26:223-232.

35. Brohi K, Singh J, Heron M, Coats T. Acute traumatic coagulopathy. J Trauma

2003;54:1127-1130.

36. Brugnara C, Oski FA, Nathan DG. Diagnostic approach to the anemic patient.

In: Orkin SH, Nathan DG, Ginsburg D, Look AT, Fisher DE, Lux S (eds). Nathan

and Oski’s Hematology and Oncology of Infancy and Childhood, 8th ed.

Philadelphia, WB Saunders, 2015.

37. Tsuei BJ, Kearney PA. Hypothermia in the trauma patient. Injury 2004;35:7-

15.

38. Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation.

Arterioscler Thromb Vasc Biol 2003;23:17-25.

39. Maegele M, Lefering R, Yucel N, Tjardes T, Rixen D, Paffrath T, Simanski C,

Neugebauer E, Bouillon B; AG Polytrauma of the German Trauma Society

(DGU). Early coagulopathy in multiple injury: an analysis from the German

Trauma Registry on 8724 patients. Injury 2007;38:298-304.

40. Dirkmann D, Hanke AA, Görlinger K, Peters J. Hypothermia and acidosis

synergistically impair coagulation in human whole blood. Anesth Analg

2008;106:1627-1632.

41. Allam R, Kumar SV, Darisipudi MN, Anders HJ. Extracellular histones in

tissue injury and inflammation. J Mol Med (Berl) 2014;92:465-472.

42. Hotchkiss RS, Strasser A, McDunn JE, Swanson PE. Cell death. N Engl J Med

2009;361:1570-1583.

43. Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation

of human factor VIII procoagulant protein by activated human protein C

and its analogy with factor V. Blood 1984;63:486-489.

44. Esmon CT. Protein C pathway in sepsis. Ann Med 2002;34:598-605.

45. Nakahara M, Ito T, Kawahara K, Yamamoto M, Nagasato T, Shrestha B, Yamada

S, Miyauchi T, Higuchi K, Takenaka T, Yasuda T, Matsunaga A, Kakihana Y,

Hashiguchi T, Kanmura Y, Maruyama I. Recombinant thrombomodulin

protects mice against histone-induced lethal thromboembolism. PLoS One

2013;8:e75961.

46. Abrams ST, Zhang N, Manson J, Liu T, Dart C, Baluwa F, Wang SS, Brohi

K, Kipar A, Yu W, Wang G, Toh CH. Circulating histones are mediators of

trauma associated lung injury. Am J Respir Crit Care Med 2013;187:160-

169.

128

BRIEF REPORT

DOI: 10.4274/tjh.2017.0446


Use of a High-Purity Factor X Concentrate in Turkish Subjects

with Hereditary Factor X Deficiency: Post Hoc Cohort Subanalysis

of a Phase 3 Study

Kalıtsal Faktör X Eksikliği Olan Türk Hastalarda Yüksek Saflıkta Faktör X Konsantresi

Kullanımı: Faz 3 Çalışmasının Post Hoc Kohort Alt Analizi

Ahmet F. Öner 1 , Tiraje Celkan 2 , Çetin Timur 3 , Miranda Norton 4 , Kaan Kavaklı 5

1

Yüzüncü Yıl University Faculty of Medicine, Department of Pediatric Hematology, Van, Turkey

2

İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology and Oncology, İstanbul, Turkey

3

İstanbul Medeniyet University, Göztepe Training and Research Hospital, Clinic of Pediatric Hematology, İstanbul, Turkey

4

Bio Products Laboratory Ltd., Elstree, Hertfordshire, United Kingdom

5

Ege University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey

Abstract

Hereditary factor X (FX) deficiency is a rare bleeding disorder more

prevalent in countries with high rates of consanguineous marriage. In

a prospective, open-label, multicenter phase 3 study, 25 IU/kg plasmaderived

factor X (pdFX) was administered as on-demand treatment or

short-term prophylaxis for 6 months to 2 years. In Turkish subjects

(n=6), 60.7% of bleeds were minor. A mean of 1.03 infusions were

used to treat each bleed, and mean total dose per bleed was 25.38

IU/kg. Turkish subjects rated pdFX efficacy as excellent or good for

all 84 assessable bleeds; investigators judged overall pdFX efficacy to

be excellent or good for all subjects. Turkish subjects had 51 adverse

events; 96% with known severity were mild/moderate, and 1 (infusionsite

pain) was possibly pdFX-related. These results demonstrate

that 25 IU/kg pdFX is safe and effective in this Turkish cohort

(ClinicalTrials.gov identifier: NCT00930176).

Keywords: Clinical trial, Clotting factor concentrate, Efficacy, Factor

X deficiency, Orphan drug, Safety

Öz

Kalıtsal faktör X (FX) eksikliği, akraba evliliklerinin yüksek oranda

görüldüğü ülkelerde daha sık olan nadir bir kanama bozukluğudur.

Prospektif, açık etiketli, çok merkezli bir faz 3 çalışmada 6 ay ila 2 yıl

boyunca gerektiği zaman veya kısa dönemli profilaktik olarak 25 IU/

kg plazma kaynaklı FX (pdFX) uygulanmıştır. Türk hastalarda (n=6)

kanamaların %60,7’si hafiftir. Her kanamayı tedavi etmek için ortalama

1,03 infüzyon gerekmiş ve kanama başına ortalama toplam doz 25,38

IU/kg olmuştur. Türk hastalar değerlendirilebilir 84 kanamanın tümü

için pdFX etkililiğini mükemmel veya iyi olarak derecelendirmiştir;

araştırmacılar genel pdFX etkililiğinin tüm hastalarda mükemmel veya

iyi olduğu kararına varmıştır. Türk hastalarda 51 advers olay gözlenmiştir;

şiddeti bilinenlerin %96’sı hafif/orta derecededir ve 1’i (infüzyon bölgesi

ağrısı) muhtemelen pdFX ile ilişkili olmuştur. Bu sonuçlar 25 IU/kg pdFX

kullanımının bu Türk kohortunda güvenli ve etkili olduğunu ortaya

koymaktadır (ClinicalTrials.gov tanımlayıcısı: NCT00930176).

Anahtar Sözcükler: Klinik çalışma, Pıhtılaşma faktörü konsantresi,

Etkililik, Faktör X eksikliği, Yetim ilaç, Güvenlilik

Introduction

Hereditary factor X (FX) deficiency (FXD) is a rare, autosomal

recessive coagulation disorder most prevalent in countries with

high rates of consanguineous marriage [1,2,3,4,5,6,7,8]. Patients

with severe FXD commonly present with bleeding into joints,

muscles, or mucous membranes [1,3]. Hereditary FXD is often

treated with fresh-frozen plasma (FFP) or prothrombin complex

concentrates (PCCs) [9,10], but single-factor concentrates, when

available, are recommended for treatment of rare bleeding

disorders [11].

A high-purity, high-potency, plasma-derived FX concentrate

(pdFX; Bio Products Laboratory Ltd., Elstree, UK) is approved in



Address for Correspondence/Yazışma Adresi: Miranda NORTON, PhD.,

Bio Products Laboratory Ltd., Elstree, Hertfordshire, United Kingdom

Phone : +44 20 8957 2661

E-mail : miranda.norton@bpl.co.uk ORCID-ID: orcid.org/0000-0003-4011-9877



129

Öner AF, et al: Use of pdFX in Turkish Subjects


the USA and the EU for on-demand treatment and bleeding

episode control in subjects aged ≥12 years with hereditary

FXD [12]. pdFX efficacy and safety were demonstrated in 5

subjects with hereditary FXD undergoing surgery [13] and in 16

subjects with hereditary FXD in a phase 3 trial conducted in the

USA, the UK, Spain, Germany, and Turkey [14].

This analysis evaluated pdFX use in the Turkish cohort (a

homogeneous subgroup in terms of the F10 mutation) from the

phase 3 trial [14].


This was a post hoc analysis of 6 Turkish subjects enrolled in

a prospective, open-label, multicenter, nonrandomized phase

3 study (ClinicalTrials.gov identifier, NCT00930176; EudraCT

identifier, 2009 0111145-18) [14] with independent ethics

committee approval for each study center, conducted in

accordance with good clinical practice guidelines [15]. All

subjects provided written informed consent.

As reported previously [14], enrolled subjects were aged ≥12

years with moderate or severe hereditary FXD (FX activity

[FX:C] <5 IU/dL) with ≥1 spontaneous/menorrhagic bleed in

the previous 12 months treated with FFP, PCCs, or a factor IX/X

concentrate. Subjects received on-demand pdFX at 25 IU/kg for

6 months to 2 years until ≥1 bleed had been treated; pdFX was

also used as short-term preventative therapy and presurgical

prophylaxis [13].

Assessments

pdFX efficacy, pharmacokinetics (PK), and safety were assessed

for the Turkish cohort as previously described for the overall

cohort [14,16]; optional F10 genotyping was also performed [17].

Subjects evaluated treatment efficacy for each bleed, and

investigators evaluated treatment efficacy for each subject.

Bleeds were categorized as menorrhagic, covert, or overt, and

pdFX efficacy for each bleed was categorized as “excellent,”

“good,” “poor,” or “unassessable” [14]. An independent data

review committee evaluated each bleed for assessability and

severity.

PK assessments were performed at baseline and 6 months

or after ≥1 bleed had been treated with pdFX as described

previously [16]. Plasma FX:C levels were measured via a onestage

clotting assay, and incremental recovery and half-life

were calculated.

Safety and tolerability assessments included adverse events

(AEs), infusion-site reactions, thrombogenicity markers, and

viral serology. FX inhibitor development was analyzed using

activated partial thromboplastin time-based inhibitor screens

and the Nijmegen-Bethesda assay.

Results

The Turkish cohort (Table 1) had a history of severe bleeds

treated using FFP or PCCs; one subject (17%) and 3 subjects

(50%, including the only subject with moderate FXD) had

received >150 days of exposure to FFP and PCCs, respectively.

All 6 subjects had the same homozygous missense mutation

in the F10 gene (p.Gly262Asp), including 3 who were known

relatives.

Hemostatic Efficacy

Of 92 pdFX-treated bleeds (range, 12-19; Figure 1), 84 were

eligible for primary efficacy analysis (Table 2). The median

number of bleeds was 1.05 per subject per month overall (range,

0.8-1.2), and 1.1 bleeds per month for the subject with moderate

FXD. The majority of bleeds (60.7%) were minor. Major bleeds

(39.3% of all episodes) included spontaneous bleeding, injury,

and menorrhagia.

Table 1. Subjects’ demographics and clinical characteristics (Turkish cohort; n=6).

Subject number Age Sex Basal FX:C (IU/dL)*

Severe FX deficiency (plasma FX:C <1 IU/dL)

Bleeding history †

Joint Muscle Menorrhagia Other ‡

1 20 M <1 N Y NA Y

2 19 F <1 N N Y Y

3 14 F <1 Y Y Y § Y

4 17 F <1 N N Y Y

5 17 F <1 N N Y N

Moderate FX deficiency (plasma FX:C ≥1 but <5 IU/dL)

6 12 M 1 Y N NA Y

*Lowest level recorded in subject’s lifetime (including during the study), † Includes all bleeds within the year prior to study entry and all significant bleeds

in the subject’s lifetime, ‡ Includes gastrointestinal, mucosal (not menorrhagia), pelvic, and unknown, § This subject was documented as having a history of

heavy menstrual bleeding; this had previously been reported as “no” due to lack of specific bleed details within the past year or in the subject’s lifetime.

FX: Factor X, FX:C: factor X activity, N: no; Y: yes, NA: not applicable.

130



Table 2. Characteristics of assessable* bleeding episodes (n=84) treated with plasma-derived FX and analyzed (Turkish cohort).

Number (%) of bleeds

All subjects Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6

Total bleeds 84 18 14 11 16 11 14

Bleed type

Menorrhagic 48 (57.1) 0 13 11 13 11 0

Covert 26 (31.0) 9 0 0 3 0 14

Overt 10 (11.9) 9 1 0 0 0 0

Bleed location

Mucosal 58 (69.0) 9 14 11 13 11 0

Joint 13 (15.5) 4 0 0 2 0 7

Muscle 11 (13.1) 5 0 0 1 0 5

Kidney 2 (2.4) 0 0 0 0 0 2

Bleed cause

Menorrhagia 48 (57.1) 0 13 11 13 11 0

Spontaneous 21 (25.0) 10 1 0 2 0 8

Injury 15 (17.9) 8 0 0 1 0 6

Bleed severity*

Major 33 (39.3) 6 0 5 7 1 14

Minor 51 (60.7) 12 14 6 9 10 0

*As assessed by the data review committee.

Subject-rated efficacy was “excellent” or “good” for each of

the 84 pdFX-treated assessable bleeds. Investigators rated pdFX

efficacy (on-demand, preventative, or surgical) as “excellent” in

4 subjects (67%) and “good” for 2 subjects (33%).

Figure 1. Summary of bleeding episodes treated with plasmaderived

FX (Turkish cohort).

pdFX: plasma-derived FX.

A total of 95 pdFX infusions (94 exposure days) were administered

(mean total dose, 22,596 IU or 389 IU/kg) to treat a bleed

(n=94) or for short-term preventative use (n=1) (Table 3). A mean

of 1.03 infusions were used to treat each bleed, and mean total

dose per bleed was 25.38 IU/kg. All 6 Turkish subjects completed

the study and then received on-demand pdFX compassionate

use for 1 year. During this time, 1 subject experienced a subdural

hematoma successfully treated with pdFX, followed by weekly

pdFX prophylaxis (2000 IU; ~30.8 IU/kg).

FX:C PK parameters following single intravenous pdFX doses

did not differ significantly between baseline and repeat PK

assessment visits. Mean pdFX incremental recovery was slightly

lower in the Turkish cohort than the overall cohort (1.77 vs. 2.00

IU/dL per IU/kg, respectively), while the mean terminal half-life

was similar (29.7 vs. 29.4 h, respectively).

Safety and Tolerability

Of 51 AEs reported by the Turkish subjects, 44 of 46 (96%) with

known severity were mild or moderate. The most frequently

reported AE was upper respiratory tract infection (9 events in 4

subjects, none of which were considered by the investigators to

be related to pdFX). Of the 51 AEs, 1 event in 1 subject (mild

infusion-site pain) was considered possibly pdFX-related; no AEs

were considered probably or very likely pdFX-related, and no AEs

resulted in death.

There were no inhibitors to FX, viral seroconversions, or

hypersensitivity reactions to pdFX. No evidence of thrombotic

events or clinical signs of thrombogenicity were observed.

131



Table 3. Summary of plasma-derived FX infusions (Turkish cohort).

Total use

Infusions (n) Total dose (IU) Total dose (IU/kg)

Mean 15.8 22,596 388.94

Median (range) 16.0 (12-19) 25,457 (12,312-31,308) 401.97 (284.6-484.9)

Use per month

Mean 0.95 NC 23.27

Median (range) 0.98 (0.7-1.1) NC (NC) 22.59 (18.7-30.4)

Treatments of bleeds

Mean 15.7 22,444 386.45

Median (range) 16 (12-19) 25,001 (12,312-31,308) 394.5 (284.6-484.9)

Use per month

Mean 0.94 NC 23.12

Median (range) 0.95 (0.7-1.1) NC (NC) 22.15 (18.7-30.4)

Preventative use*

Mean 1.0 912 14.95

Use per month

Mean 0.06 NC 0.87

*Data refer to a single infusion; therefore, medians and ranges are not presented. The single preventative dose was given following an injury to the subject’s leg, prior to the appearance

of swelling.

NC: Not calculated.

During the year of compassionate use, no product-related AEs

were reported. One pdFX infusion was given to treat bleeding

due to a urinary tract infection during pregnancy, with no

adverse effect on the baby.

Discussion

This post hoc analysis demonstrated the efficacy, PK, and safety

of pdFX in Turkish subjects with moderate or severe hereditary

FXD. One subject with moderate FXD (FX:C 1 IU/dL) nonetheless

had severe bleeding diathesis based on his bleeding and

treatment history.

The Turkish cohort required fewer infusions to treat each bleed

than the overall study cohort [14] (mean, 1.03 vs. 1.21 doses) and

consequently a lower total dose per bleed (mean, 25.38 vs.

31.00 IU/kg). The percentage of minor bleeds was higher in the

Turkish cohort than in the overall study population (60.7% vs.

47.1%), and preventative use was much lower (mean, 0.06 vs. 1.64

infusions per month). The slightly lower mean pdFX incremental

recovery among Turkish subjects versus the overall study

population [16] may derive from the small sample size. Across

94 exposure days, only 1 AE in 1 subject was considered by the

investigators to be possibly treatment-related.

All Turkish subjects had a homozygous F10 mutation

(p.Gly262Asp) resulting in an identical amino acid substitution. A

recent study of 12 Turkish patients with severe FXD identified

p.Gly262Asp in 11 of 12 patients (92%), this mutation being

associated with severe bleeding symptoms, suggesting the

potential value of mutational screening analysis in Turkey

and certain areas of Iran [18]. Other regional studies have

also suggested a correlation between genotype and clinical

manifestations of hereditary FXD [9,19]; additional studies are

needed, however, to confirm these findings.

Conclusion

In conclusion, pdFX is the first highly purified FX concentrate

developed for patients with hereditary FXD. The treatment

success rate observed in Turkish subjects (100%) was comparable

with that in the overall study population (98.4%) [14]. As

hereditary FXD is a rare disorder, this post hoc analysis is limited

by a small sample size. Nevertheless, these results demonstrate

that 25 IU/kg pdFX was safe and effective in Turkish patients

with moderate or severe hereditary FXD for on-demand

treatment of bleeding episodes.

Acknowledgments

Fiona Fernando, PhD, and Alexandra W. Davis (Ashfield Healthcare

Communications, Middletown, CT, USA) drafted and revised the

manuscript based on input from authors, and Dena McWain

(Ashfield Healthcare Communications) copyedited and styled the

manuscript per journal requirements. The authors would like to

thank the data review committee (Drs. Jørgen Ingerslev [Aarhus

University Hospital, Shejby, Denmark], Carol Kasper [University of

Southern California School of Medicine, Los Angeles, CA, USA],

and John Hanley [Newcastle Hospitals NHS Foundation Trust,

Newcastle upon Tyne, UK]) for their role in the study.

132



Ethics

Ethics Committee Approval: Ege University Medical Faculty

Clinical Trials Ethics Committee (approval number: 10-11.1/14).

Informed Consent: All subjects provided written informed

consent.


Surgical and Medical Practices: A.F.Ö., T.C., Ç.T., K.K.;

Concept: M.N.; Design: M.N.; Data Collection or

Processing: A.F.Ö., T.C., Ç.T., M.N., K.K.; Analysis or

Interpretation: M.N., K.K.; Literature Search: M.N.;

Writing: M.N.

Conflict of Interest: M.N. is an employee of Bio Products

Laboratory Ltd. K.K. has received investigational support from

Bio Products Laboratory Ltd. Other 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: Bio Products Laboratory (Elstree, UK)

provided support for this study and funding for medical

writing and editorial support in the development of this

manuscript. A.F.Ö.: Received educational support from Pfizer.

M.N.: Employee of Bio Products Laboratory. K.K.: Advisory

board member for Bayer, Novo Nordisk, Pfizer, and Shire;

received educational and investigational support from

Bayer, Bio Products Laboratory, CSL Behring, Novo Nordisk,

Octapharma, Pfizer, and Shire.

References

1. Brown DL, Kouides PA. Diagnosis and treatment of inherited factor X

deficiency. Haemophilia 2008;14:1176-1182.

2. Khair K, Kumar P, Mathias M, Efford J, Liesner R. Successful use of BPL

factor X concentrate in a child with severe factor X deficiency. J Haem Pract

2014;1:8-10.

3. Peyvandi F, Mannucci PM, Lak M, Abdoullahi M, Zeinali S, Sharifian R, Perry

D. Congenital factor X deficiency: spectrum of bleeding symptoms in 32

Iranian patients. Br J Haematol 1998;102:626-628.

4. Tuncbilek E, Koc I. Consanguineous marriage in Turkey and its impact on

fertility and mortality. Ann Hum Genet 1994;58:321-329.

5. Güz K, Dedeoğlu N, Lüleci G. The frequency and medical effects of

consanguineous marriages in Antalya, Turkey. Hereditas 1989;111:79-83.

6. Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation

disorders. Blood 2004;104:1243-1252.

7. Fışgın T, Balkan C, Celkan T, Kılınç Y, Türker M, Timur Ç, Gürsel T, Kürekçi E,

Duru F, Küpesiz A, Olcay L, Yılmaz Ş, Özgen Ü, Ünüvar A, Ören H, Kavaklı

K. Rare coagulation disorders: a retrospective analysis of 156 patients in

Turkey. Turk J Hematol 2012;29:48-54.

8. Menegatti M, Peyvandi F. Factor X deficiency. Semin Thromb Hemost

2009;35:407-415.

9. Karimi M, Vafafar A, Haghpanah S, Payandeh M, Eshghi P, Hoofar H,

Afrasiabi A, Gerdabi J, Ardeshiri R, Menegatti M, Peyvandi F. Efficacy of

prophylaxis and genotype-phenotype correlation in patients with severe

Factor X deficiency in Iran. Haemophilia 2012;18:211-215.

10. Mumford AD, Ackroyd S, Alikhan R, Bowles L, Chowdary P, Grainger J,

Mainwaring J, Mathias M, O’Connell N; BCSH Committee. Guideline for

the diagnosis and management of the rare coagulation disorders: a United

Kingdom Haemophilia Centre Doctors’ Organization guideline on behalf

of the British Committee for Standards in Haematology. Br J Haematol

2014;167:304-326.

11. Giangrande P, Seitz R, Behr-Gross ME, Berger K, Hilger A, Klein H, Schramm

W, Mannucci PM. Kreuth III: European consensus proposals for treatment

of haemophilia with coagulation factor concentrates. Haemophilia

2014;20:322-325.

12. Bio Products Laboratory. Coagadex® Prescribing Information. Available

online at http://www.coagadex.com/download/Coagadex_PI_10-2015.pdf.

Accessed 17 October 2017.

13. Escobar MA, Auerswald G, Austin S, Huang JN, Norton M, Millar CM.

Experience of a new high-purity factor X concentrate in subjects

with hereditary factor X deficiency undergoing surgery. Haemophilia

2016;22:713-720.

14. Austin SK, Kavakli K, Norton M, Peyvandi F, Shapiro A; FX Investigators

Group. Efficacy, safety, and pharmacokinetics of a new high-purity factor

X concentrate in subjects with hereditary factor X deficiency. Haemophilia

2016;22:419-425.

15. Dixon JR Jr. The International Conference on Harmonization Good Clinical

Practice Guideline. Qual Assur 1998;6:65-74.

16. Austin SK, Brindley C, Kavakli K, Norton M, Shapiro A; FX Investigators

Group. Pharmacokinetics of a high-purity plasma-derived factor X

concentrate in subjects with moderate or severe hereditary factor X

deficiency. Haemophilia 2016;22:426-432.

17. Mitchell M, Kavakli K, Norton M, Austin S. Genotype analysis of patients

with hereditary factor X deficiency enrolled in two phase 3 studies of pdFX,

a new high-purity factor X concentrate [abstract]. Blood 2015;126:3511.

18. Epcacan S, Menegatti M, Akbayram S, Cairo A, Peyvandi F, Oner AF.

Frequency of the p.Gly262Asp mutation in congenital Factor X deficiency.

Eur J Clin Invest 2015;45:1087-1091.

19. Herrmann FH, Auerswald G, Ruiz-Saez A, Navarrete M, Pollmann H, Lopaciuk

S, Batorova A, Wulff K; Greifswald Factor X Deficiency Study Group. Factor

X deficiency: clinical manifestation of 102 subjects from Europe and Latin

America with mutations in the factor 10 gene. Haemophilia 2006;12:479-489.

133

IMAGES IN HEMATOLOGY

DOI: 10.4274/tjh.2016.0388

Turk J Hematol 2018;35:134

Flaming Plasma Cell Leukemia

Alevsi Plazma Hücreli Lösemi

Reza Ranjbaran,

Habibollah Golafshan

Shiraz University of Medical Sciences, School of Paramedical Sciences, Diagnostic Laboratory Sciences and Technology Research Center, Shiraz, Iran

A 58-year-old man presented with anemia and splenomegaly.

Peripheral blood smear indicated rouleaux formation along

with 28% mononuclear cells with reddish-purple peripheral

cytoplasm suspicious for plasma cells (PCs). Flow cytometric

immunophenotyping of the peripheral blood revealed a large

mononuclear population positive for CD38, CD138, and CD20

and negative for CD45, CD19, and CD56. Intracytoplasmic

staining of kappa and lambda light-chains demonstrated lambda

restriction. The serum protein electrophoresis pattern illustrated

normal density in the γ-globulin region but an increase of about

threefold in the β-globulin fraction.

Regarding these findings, the patient was more likely to be

diagnosed with IgA monoclonal gammopathy [1]. However,

a definitive diagnosis was made by immunonephelometric

evaluation of serum immunoglobulins.This assay revealed 810

mg/dL IgG, 1595 mg/dL IgA, and 22 mg/dL IgM with a free

kappa/lambda ratio of 0.14.

PCs have particular morphological features including ovalshaped

structure and eccentric nuclei. IgA-secreting PCs have

cytoplasm with a pinkish tinge associated with the presence of

abundant glycoprotein and ribosomes and are totally known as

flame cells (Figure 1). PC leukemia, in particular the IgA variant,

is a rare and aggressive type of PC dyscrasia [2]. A well-prepared

peripheral blood smear can be very helpful in diagnosing and

determining the next diagnostic approach.

Keywords: Plasma cell leukemia, Flame cell, Immunoglobin A

Anahtar Sözcükler: Plazma hücre lösemi, Alevsi hücre,

İmmunoglobulin A

Figure 1. Flame cells.

Informed Consent: It was received.




included.

References

1. Attaelmannan M, Levinson SS. Understanding and identifying monoclonal

gammopathies. Clin Chem 2000; 46:1230-1238.

2. Singh T, Premalata C, Sajeevan K, Jain A, Batra U, Saini K, Satheesh C, Babu

KG, Lokanatha D. IgA plasma cell leukemia. J Lab Physicians 2009;1:19-21.



Address for Correspondence/Yazışma Adresi: Reza RANJBARAN, M.D.,

Shiraz University of Medical Sciences, Faculty of Paramedical Sciences, Diagnostic Laboratory

Sciences and Technology Research Center, Shiraz, Iran

Phone : +98 917 074 9518

E-mail : reza_ranjbaran2009@yahoo.com ORCID-ID: orcid.org/0000-0002-8890-0999

Received/Geliş tarihi: September 26, 2016

Accepted/Kabul tarihi: January 24, 2017

134

IMAGES IN HEMATOLOGY

DOI: 10.4274/tjh.2017.0448


Improvement of Cutaneous Anaplastic Large Cell Lymphoma by

Brentuximab Vedotin Monotherapy

Kutanöz Anaplastik Büyük Hücreli Lenfomada Brentuksimab Vedotin Monoterapisi ile Düzelme

Takashi Onaka 1 , Tomoya Kitagawa 1 , Chika Kawakami 2 , Akihito Yonezawa 1

1

Kokura Memorial Hospital, Clinic of Hematology, Kitakyushu, Fukuoka, Japan

2

University of Occupational and Environmental, Department of Dermatology, Fukuoka, Japan

Brentuximab vedotin (BV) is an antibody-drug conjugate

composed of a CD30-directed monoclonal antibody and

monomethyl auristatin E [1]. BV monotherapy showed good

response rates for cases of refractory and relapsed anaplastic large

cell lymphoma (ALCL), but only a few case reportsare available

for cutaneous localized ALCL (cALCL). We herein report the

treatment with BV of relapsed cALCL with an excellent response.

An 82-year-old female with relapsed cALCL had generalized

erythema accompanied by desquamation and could not extend

her fingers enough (Figure 1), with no lymph node lesions. Due

to the previous treatment with radiation, steroid ointment, and

systemic chemotherapy, we chose BV monotherapy for her,

dosing at 1.8 mg/kg every 21 days. After the third infusion, her

generalized erythemaand her finger movement were improved

(Figure 2). She did not have any severe adverse effects or infusion

reaction except for hematologic toxicity (leukocytopenia). She

has finished 6 courses of BV infusion and maintained remission

of skin lesions. There are several reports that showed the

effectiveness of BV treatment for cALCL [2,3], but the optimal

treatment interval and cycles, and the necessity of maintenance

therapy by using BV, are unclear. Further studies are needed to

evaluate BV treatment in cases of cALCL.

Figure 1. Generalized erythema accompanied by desquamation

before treatment with brentuximab vedotin.

Figure 2. Improvement of skin erythema accompanied by

desquamation after 4 cycles of brentuximab vedotin.



Address for Correspondence/Yazışma Adresi: Takashi ONAKA, M.D.,

Kokura Memorial Hospital, Clinic of Hematology,

Kitakyushu, Fukuoka, Japan T: 81-093-511-2000

E-mail : takashionaka3@gmail.com ORCID-ID: orcid.org/0000-0002-3149-2584



135

Onaka T, et al: Improvement of Cutaneous ALCL by Brentuximab Vedotin Monotherapy


Keywords: Brentuximab vedotin, Cutaneous ALCL

Anahtar Sözcükler: Brentuksimab vetodin, Kutanöz ABHL

Informed Consent: Informed consent was obtained from the

patient.

Conflict of Interest: The authors of this paper have no

conflicts of interest, including specific financial interests,

relationships,and/or affiliations relevant to the subject matter

or materials included.

References

1. Katz J, Janik JE, Younes A. Brentuximab vedotin (SGN-35). Clin Cancer Res

2011;17:6428-6436.

2. Desai A, Telang GH, Olszewski AJ. Remission of primary cutaneous anaplastic

large cell lymphoma after a brief course of brentuximab vedotin. Ann

Hematol 2013;92:567-568.

3. Kaffenberger BH, Kartono Winardi F, Frederickson J, Porcu P, Wong HK.

Periocular cutaneous anaplastic large cell lymphoma clearance with

brentuximab vedotin. J Clin Aesthet Dermatol 2013;6:29-31.

136

LETTERS TO THE EDITOR


Glomerular and Tubular Functions in Transfusion-Dependent

Thalassemia

Transfüzyona Bağımlı Talasemide Glomerüler ve Tübüler Fonksiyonlar

Pathum Sookaromdee 1 , Viroj Wiwanitkit 2

1

TWS Primary Care Center, Bangkok, Thailand

2

Hainan Medical University, Department of Tropical Medicine, Haikou, Hainan, China

To the Editor,

Annayev et al. [1] reported their interesting observations in

the publication entitled “Glomerular and Tubular Functions in

Children and Adults with Transfusion-Dependent Thalassemia”

(TDT). They concluded that “subclinical renal injury may be

present in TDT patients” [1]. We would like to share ideas

and experiences from our setting in Southeast Asia where

transfusion-dependent beta-thalassemia is very common. Renal

dysfunction is not uncommon in our thalassemic patients and

the degree of dysfunction varies [2]. In fact, the varying degree

of renal dysfunction in thalassemia patients is well known [3,4].

Patients with different variants of thalassemia have different

degrees of renal dysfunction [3,4,5]. Ong-ajyooth et al. [5]

noted that “The mechanism leading to the damage is not known

but it might be related to increased oxidative stress secondary

to tissue deposition of iron, as indicated by the raised levels of

serum and urine MDA”. Improved renal function is also observed

after stem cell transplantation therapy [6].

Keywords: Glomerular, Tubular, Thalassemia

Anahtar Sözcükler: Glomerüler, Tübüler, Talasemi




included.

References

1. Annayev A, Karakaş Z, Karaman S, Yalçıner A, Yılmaz A, Emre S. Glomerular

and tubular functions in children and adults with transfusion-dependent

thalassemia. Turk J Hematol 2018;35:66-70.

2. Sumboonnanonda A, Malasit P, Tanphaichitr VS, Ong-ajyooth S,

Sunthornchart S, Pattanakitsakul S, Petrarat S, Assateerawatt A, Vongjirad A.

Renal tubular function in beta-thalassemia. Pediatr Nephrol 1998;12:280-

283.

3. Nickavar A, Qmarsi A, Ansari S, Zarei E. Kidney function in patients with

different variants of beta-thalassemia. Iran J Kidney Dis 2017;11:132-137.

4. Uzun E, Balcı YI, Yüksel S, Aral YZ, Aybek H, Akdağ B. Glomerular and tubular

functions in children with different forms of beta thalassemia. Ren Fail

2015;37:1414-1418.

5. Ong-ajyooth L, Malasit P, Ong-ajyooth S, Fucharoen S, Pootrakul P,

Vasuvattakul S, Siritanaratkul N, Nilwarangkur S. Renal function in adult

beta-thalassemia/Hb E disease. Nephron 1998;78:156-161.

6. Sumboonnanonda A, Sanpakit K, Piyaphanee N. Renal tubule function in

beta-thalassemia after hematopoietic stem cell transplantation. Pediatr

Nephrol 2009;24:183-187.

Address for Correspondence/Yazışma Adresi: Pathum SOOKAROMDEE, M.D.,

TWS Primary Care Center, Bangkok, Thailand

Phone : 662 448 7892

E-mail : pathumsook@gmail.com ORCID-ID: orcid.org/0000-0002-8859-5322



DOI: 10.4274/tjh.2018.0083

137



Reply to the Authors:

To the Editor,

We thank Drs. Sookaromdee and Wiwanitkit for their interest and contribution to our article. There is a growing evidence of renal

dysfunction in patients with thalassemia. Although the process is multifactorial (the disease itself with regular transfusion, iron

accumulation in the parenchyma and toxicity of chelators), oxidative stress seems to be the main mechanism of renal damage.

Several studies have shown the beneficial effects of antioxidants (curcumin, glutamine) in prevention of chemotherapy-induced

nephrotoxicity by decreasing oxidative damage. Considering the significantly increased life expectancy of thalassemia patients with

long-term complications, we think the role and effects of antioxidant treatments in routine follow-up of the thalassemia patients

should be investigated in prospective studies.

Best Regards

Zeynep Karakaş, Serap Karaman

Use of Plerixafor to Mobilize a Healthy Donor Infected with

Influenza A

İnfluenza A ile Enfekte Olan Sağlıklı Bir Vericinin Plerixafor ile Mobilizasyonu

Mahmut Yeral, Pelin Aytan, Can Boğa

Başkent University Adana Practice and Research Center, Adult Bone Marrow Transplantation Center, Adana, Turkey

To the Editor,

The combined use of plerixafor and granulocyte-colony

stimulating factor (G-CSF) improves mobilization in poor

mobilizers. However, there are limited data available on the use

of plerixafor in healthy donors [1,2]. The effects of influenza A

infection on stem cell mobilization are not known.

A 46-year-old male was selected as an HLA-matched donor for

a patient diagnosed with acute myeloid leukemia (AML). Donor

assessment was performed in accordance with the standard

operating procedure prepared for JACIE (SOP: BMT-CU-006,

Donor Assessment and Safety). The donor was given 10 mg/kg/

day G-CSF. He developed a dry persistent cough, chills, fever

of 39 °C, fatigue, and flu-like symptoms on day 3 of G-CSF

administration. The donor was considered to have an upper

respiratory tract infection, which could not be attributed to

only G-CSF administration. The family members of the donor

were found to have similar symptoms. Thus, blood and urine

cultures were obtained and he was started on levofloxacin in

addition to paracetamol; G-CSF was continued. A respiratory

tract virus panel was performed on a nasal smear using a PCRbased

technique. The peripheral blood leukocyte count was

22,000/µL but CD34+ cells represented just 0.07% of all cells

(11/µL) on day 5 of G-CSF administration; this was considered

to reflect “poor mobilization”. Therefore, 0.24 mg/kg plerixafor

was administered “just in time,” in addition to G-CSF, on night

5, after the donor had been given all necessary information and

informed consent had been obtained. Two hours after the 11 th

dose of G-CSF, the leukocyte count was 45,000/µL, of which

0.33% (148/µL) were CD34+ cells. Peripheral stem cell apheresis

was performed using the Donor Spectra Optia Apheresis System

(Terumo BCT, Lakewood, CO, USA). A total of 15.20x10 8 nuclear

cells/kg were collected. The product contained 3.92x10 6 CD34+

cells/kg, 14.91x10 7 CD3+ cells/kg, 17.36x10 7 CD19+ cells/kg,

and 7.17x10 7 CD56+ cells/kg. G-CSF was discontinued after an

adequate number of stem cells had been collected, but the fever

persisted. Oseltamivir at 150 mg twice daily was then prescribed

for the donor because the respiratory tract virus panel

examination revealed influenza A infection. The fever became

controlled 24 h after oseltamivir administration. The plerixafor

procedure was considered to have permitted “sufficient

mobilization” in a healthy donor who could not be mobilized

with G-CSF probably because of his influenza infection.

Many factors including age, sex, body mass index, baseline

leukocyte count, and comorbid conditions may compromise

mobilization [3]. Although certain viral infections may cause

poor mobilization, data on the influence of influenza in this

context are rather limited [4]. Cytokine production or cytokine

storm developing during influenza infection may be presumed

to impair stem cell mobilization [5]. A combination of G-CSF and

138



plerixafor can be used to treat mobilization failure and is usually

well tolerated [6,7]. The only option upon stem cell mobilization

failure with G-CSF is bone marrow harvesting. Our donor was

given plerixafor “just in time”; he had an active infection and

did not consent to bone marrow harvesting. While plerixafor is

usually used for mobilization in lymphoma or myeloma patients,

literature data are available about its use in allogeneic settings

[8]. Stem cells in numbers adequate for safe transplantation

were collected in a single procedure.

This report indicates that influenza A may suppress the

hematopoietic system, negatively affecting stem cell

mobilization. The problem may be overcome by plerixafor

administration.

Keywords: Plerixafor, Influenza A, Healthy donor

Anahtar Sözcükler: Plerixafor, İnfluenza A, Sağlıklı verici




included.

References

1. Eyre TA, King AJ, Peniket A, Rocha V, Collins GP, Pawson R. Partial engraftment

following plerixafor rescue after failed sibling donor peripheral blood stem

cell harvest. Transfusion 2014;54:1231-1234.

2. Gattillo S, Marktel S, Rizzo L, Malato S, Malabarba L, Coppola M, Assanelli A,

Milani R, De Freitas T, Corti C, Bellio L, Ciceri F. Plerixafor on demand in ten

healthy family donors as a rescue strategy to achieve an adequate graft for

stem cell transplantation. Transfusion 2015;55:1993-2000.

3. Hölig K. G-CSF in healthy allogeneic stem cell donors. Transfus Med

Hemother 2013;40:225-235.

4. Rohn A, Kessler HH, Valentin T, Linkesch W, Neumeister P. Prophylactic

oseltamivir treatment for prevention of donor-recipient influenza A

H1N1 virus transmission does not compromise stem cell mobilization or

engraftment. Bone Marrow Transplant 2011;46:312-313.

5. Teijaro JR, Walsh KB, Rice S, Rosen H, Oldstone MB. Mapping the innate

signaling cascade essential for cytokine storm during influenza virus

infection. Proc Natl Acad Sci U S A 2014;111:3799-3804.

6. Hauge AW, Haastrup EK, Sengeløv H, Minulescu L, Dickmeiss E, Fischer-

Nielsen A. Addition of plerixafor for CD34+ cell mobilization in six healthy

stem cell donors ensured satisfactory grafts for transplantation. Transfusion

2014;54:1055-1058.

7. Flomenberg N, Devine SM, Dipersio JF, Liesveld JL, McCarty JM, Rowley

SD, Vesole DH, Badel K, Calandra G. The use of AMD3100 plus G-CSF for

autologous hematopoietic progenitor cell mobilization is superior to G-CSF

alone. Blood 2005;106:1867-1874.

8. Namdaroglu S, Korkmaz S, Altuntas F. Management of mobilization failure

in 2017. Transfus Apher Sci 2017;56:836-844.

Address for Correspondence/Yazışma Adresi: Mahmut YERAL, M.D.,

Başkent University Adana Practice and Research Center, Adult Bone Marrow Transplantation Center, Adana, Turkey

Phone : +90 322 327 27 27-2023

E-mail : drmyeral@gmail.com ORCID-ID: orcid.org/0000-0002-9580-628X

Received/Geliş tarihi: August 14, 2017


DOI: 10.4274/tjh.2017.0304

Influenza A Infection and Stem Cell Mobilization

İnfluenza A Enfeksiyonu ve Kök Hücre Mobilizasyonu

Sora Yasri 1 , Viroj Wiwanitkit 2

1

KMT Primary Care Center, Bangkok, Thailand

2

Hainan Medical University, Department of Tropical Medicine, Haikou, Hainan, China

To the Editor,

We read the publication entitled “Use of Plerixafor to Mobilize

a Healthy Donor Infected with Influenza A” and found it to

be very interesting [1]. Yeral et al. [1] mentioned that “The

effects of influenza A infection on stem cell mobilization are

not known” and concluded that “This report indicates that

influenza A may suppress the hematopoietic system, negatively

affecting stem cell mobilization. The problem may be overcome

by plerixafor administration” [1]. This article may provide a

new observation and confirm the usefulness of plerixafor in

achieving stem cell mobilization. Nevertheless, it should be

noted that this is not the first case of stem cell transplantation

in which the donor has influenza A infection. Lee et al. [2]

reported stem cell transplantation from a related donor infected

with influenza H1N1 2009 and in that case the transplantation

was completely done without noting any problem of stem cell

mobilization due to the influenza virus. Regardless of using

plerixafor, however, stem cell transplantation in cases in which

the donor has influenza infection is a considerable challenge

and it is questionable whether the procedure should be done

then or not.

139



Keywords: Influenza, Infection, Stem cell

Anahtar Sözcükler: İnfluenza, Enfeksiyon, Kök hücre




References

1. Yeral M, Aytan P, Boğa C. Use of plerixafor to mobilize a healthy donor

infected with influenza A. Turk J Hematol 2018;35:139-140.

2. Lee SH, Cheuh H, Yoo KH, Kim YJ, Sung KW, Koo HH, Kim DH, Kim SJ, Kim K,

Jang JH, Jung CW. Hematopoietic stem cell transplantation from a related

donor infected with influenza H1N1 2009. Transpl Infect Dis 2011;13:548-

550.

Address for Correspondence/Yazışma Adresi: Sora YASRI, M.D.,

KMT Primary Care Center, Bangkok, Thailand

Phone : 662 257 89 63

E-mail : sorayasri@outlook.co.th ORCID-ID: orcid.org/0000-0001-8292-6656


Accepted/Kabul tarihi: August 12, 2014

DOI: 10.4274/tjh.2018.0089

Reply to the Authors:

To the Editor,

We read the recent letter by Yasri and Wiwanitkit [1] regarding our manuscript with great interest. We are pleased with their

contributions and comments. The literature data with regard to the effect of influenza A on hematopoietic cell mobilization is limited

to only several case reports [2,3].

However it would not be incorrect to relate mobilization failure to Influenza in a donor who has no prior diseases, who is not using

any kind of medication or substance, and who is considered to be healthy in clinical and laboratory evaluations before mobilization.

Mobilization failure may be associated with cytokine increase, presence of viremia and viral titers. Lee et al. [2] reported three donors

who were infected with influenza. Mobilization was postponed for a short period of time in one donor due to poor mobilization risk.

Two donors could be mobilized with granulocyte-colony stimulating factor. Nevertheless one of the donors could not be regarded as

good mobilized. Because despite two days of apheresis procedure, the collected CD34+ cells from the healthy donor were ≤2×10 6/kg.

With our case we aimed to point out that influenza A may affect mobilization negatively and this condition may be overcome with

plerixafor. It should be known that mortality is inevitable in a patient who received myeloablative conditioning regimen without

stem cells.

References

1. Yasri S, Wiwanitkit V. Influenza A Infection and Stem Cell Mobilization, Turk J Hematol 2018;35:137-153.

Best Regards

Mahmut Yeral, Pelin Aytan, Can Boğa

2. Lee SH, Cheuh H, Yoo KH, Kim YJ, Sung KW, Koo HH, Kim DH, Kim SJ, Kim K, Jang JH, Jung CW. Hematopoietic stem cell transplantation from a related donor

infected with influenza H1N1 2009. Transpl Infect Dis 2011;13:548-550.

3. Rohn A, Kessler HH, Valentin T, Linkesch W, Neumeister P. Prophylactic oseltamivir treatment for prevention of donor-recipient influenza A H1N1 virus transmission

does not compromise stem cell mobilization or engraftment. Bone Marrow Transplant. 2011;46:312-313.

140



Primary Mediastinal Large B-Cell Lymphoma As an Incidental

Finding: Report of a Case

Tesadüfen Tanı Konulan Primer Mediastinal Büyük B Hücreli Lenfoma Olgusu

İpek Yönal-Hindilerden 1 , Fehmi Hindilerden 2 , Serkan Arslan 3 , İbrahim Öner Doğan 4 , Meliha Nalçacı 1

¹İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

2

Dr. Sadi Konuk Training and Research Hospital, Clinic of Hematology, İstanbul, Turkey

3

Dr. Sadi Konuk Training and Research Hospital, Clinic of Radiology, İstanbul, Turkey

4

İstanbul University İstanbul Faculty of Medicine, Department of Pathology, İstanbul, Turkey

To the Editor,

A 21-year-old female was examined for an incidentally detected

left parahilar mass on chest radiograph which was taken at

the time of job application (Figure 1a). Thoracic computed

tomography revealed a mass of 10x9x5 cm with irregular

lobulated borders in the anterior mediastinum invading the

pericardium (Figure 1b). Histopathological examination of the

anterior mediastinotomy material revealed large neoplastic

B cells staining positive for CD20 and MUM-1, negative for

CD10, and with a high Ki-67 proliferation index (80%-90%)

(Figure 2). On positron-emission tomography scan, only the

mediastinal mass showed increased fludeoxyglucose uptake

(SUV max

: 18) (Figure 1c). Final diagnosis was stage 1A primary

mediastinal large B-cell lymphoma (PMBCL). After 6 cycles of

R-CHOP, PET scan showed partial anatomical and metabolic

response. R-CHOP was completed to 8 cycles followed by

mediastinal radiation. She has now been disease-free for 2 years.

PMBCL, accounting for 2%-4% of all non-Hodgkin lymphomas,

often presents as a bulky anterior mediastinal mass and often

Figure 2. Histopathological examination of the mass. a) Diffuse

neoplastic infiltration on a partially sclerotic background

(hematoxylin and eosin stain, 40 x ). b) The clear-cell appearance

of the tumor cells (hematoxylin and eosin stain, 100 x ). c) The

appearance of round nuclei (centroblast-like) and clear cytoplasm

(hematoxylin and eosin stain, 400 x ). d) Infiltrated cells with CD20

expression (hematoxylin and eosin stain, 400 x ).

Figure 1. Radiological findings of primary mediastinal B-cell lymphoma. a) Appearance of the left parahilar mass on chest plain film. b)

Thorax computed tomography depicts a mass of 10x9x5 cm in the anterior mediastinum with irregular lobulated borders invading the

pericardium. c) Positron-emission tomography scan shows increased fludeoxyglucose uptake in the tumor.

141


invades surrounding structures such as the heart, lungs, pleura,

and superior vena cava [1,2]. Patients often present with cough,

dyspnea, chest pain, and superior vena cava syndrome [3].

R-CHOP plus consolidative mediastinal radiation is often an

option [4]. Herein, we report a rare case of asymptomatic PMBCL

with bulky mediastinal mass in which the patient achieved

complete remission after R-CHOP and mediastinal radiation.

Keywords: Mediastinal neoplasm, B-cell lymphoma, PMBCL

Anahtar Sözcükler: Mediastinal kitle, B hücreli lenfoma, PMBCL




References


1. Savage KJ. Primary mediastinal large B-cell lymphoma. Oncologist

2006;11:488-495.

2. Bhatt VR, Mourya R, Shrestha R, Armitage JO. Primary mediastinal large

B-cell lymphoma. Cancer Treat Rev 2015;41:476-485.

3. Abou-Elella AA, Weisenburger DD, Vose JM, Kollath JP, Lynch JC, Bast

MA, Bierman PJ, Greiner TC, Chan WC, Armitage JO. Primary mediastinal

large B-cell lymphoma: a clinicopathologic study of 43 patients from the

Nebraska Lymphoma Study Group. J Clin Oncol 1999;17:784-790.

4. Giri S, Bhatt VR, Pathak R, Bociek RG, Vose JM, Armitage JO. Role of radiation

therapy in primary mediastinal large B-cell lymphoma in rituximab era: a

US population-based analysis. Am J Hematol 2015;90:1052-1054.



Address for Correspondence/Yazışma Adresi: İpek YÖNAL-HİNDİLERDEN, M.D.,

İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine,

Division of Hematology, İstanbul, Turkey

E-mail : ipekyonal@hotmail.com ORCID-ID: orcid.org/0000-0003-3020-850X

Received/Geliş tarihi: February 08, 2016


DOI: 10.4274/tjh.2016.0057

A Rare Late Complication of Port Catheter Implantation:

Embolization of the Catheter

Nadir Görülen Bir Port Kateter Geç Komplikasyonu: Kateter Embolizasyonu

Işık Odaman Al 1 , Cengiz Bayram 1 , Gizem Ersoy 1 , Kazım Öztarhan 2 , Alper Güzeltaş 3 , Taner Kasar 3 , Ezgi Uysalol 1 ,

Başak Koç 1 , Ali Ayçiçek 1 , Nihal Özdemir 1

1University of Health Sciences, İstanbul Kanuni Sultan Süleyman Training and Research Hospital, Clinic of Pediatric Hematology and Oncology,

İstanbul, Turkey

2University of Health Sciences, İstanbul Kanuni Sultan Süleyman Training and Research Hospital, Clinic of Pediatric Cardiology, İstanbul, Turkey

3University of Health Sciences, İstanbul Mehmet Akif Ersoy Thoracic and Cardiovascular Surgery Training and Research Hospital, Clinic Pediatric

Cardiology, İstanbul, Turkey

To the Editor,

Children with cancer need long-term venous access due to the

long duration of therapy. Long-term totally implantable port

devices (TIPDs) are widely used in these patients for administration

of chemotherapeutic agents, parenteral nutrition, fluids, and

blood products [1,2]. Fracture and embolism of TIPDs are rare

complications but may cause serious results and mortality,

including pulmonary artery embolism, sepsis, arrhythmias, and

perforation of the caval vein [3,4,5]. Herein, we present a 9-yearold

male patient with pre-B acute lymphoblastic leukemia who was

admitted to the outpatient pediatric hematology and oncology

clinic at the 13 th month of maintenance therapy due to new onset

of non-flushing catheter. The patient had no other complaints.

On posterior anterior chest X-ray, the catheter was found to

be disconnected from its reservoir (Figure 1). Echocardiography

142

Figure 1. Chest X-ray showing disconnection of the catheter from

its reservoir.



and thorax computed tomography angiography of the patient

revealed the embolization of the catheter to the left pulmonary

artery (Figure 2). The embolized catheter was removed using an

interventional endovascular procedure under general anesthesia

through the femoral vein by an interventional cardiologist (Figure

3). Our case report highlights a rarely encountered complication

of TIPDs, which may be undiagnosed due to its rarity and lack of

symptoms in some patients, leading to serious complications.

Figure 2. Thorax computed tomography angiography of the

patient showing the embolization of the catheter to the left

pulmonary artery.

Keywords: Acute lymphoblastic leukemia, Catheter,

Complication

Anahtar Sözcükler: Akut lenfoblastik lösemi, Kateter,

Komplikasyon




References

1. Kurul S, Saip P, Aydin T. Totally implantable venous-access ports: local

problems and extravasation injury. Lancet Oncol 2002;3:684-692.

2. Intagliata E, Basile F, Vecchio R. Totally implantable catheter migration and

its percutaneous retrieval: case report and review of the literature. G Chir

2017;37:211-215.

3. Kassar O, Hammemi R, Ben Dhaou M, Kammoun S, Elloumi M. Spontaneous

fracture and migration of a totally implanted port device to pulmonary

artery in acute leukemia child. J Pediatr Hematol Oncol 2017;39:103-105.

4. Surov A, Buerke M, John E, Kösling S, Spielmann RP, Behrmann C.

Intravenous port catheter embolization: mechanisms, clinical features, and

management. Angiology 2008;59:90-97.

5. Ribeiro RC, Monteiro AC, Menezes QC, Schettini ST, Vianna SM. Totally

implantable catheter embolism: two related cases. Sao Paulo Med J

2008;126:347-349.

Figure 3. Removal of the catheter with an interventional

endovascular procedure from pulmonary artery.



Address for Correspondence/Yazışma Adresi: Cengiz BAYRAM M.D.,

University of Health Sciences, İstanbul Kanuni Sultan Süleyman Training and Research Hospital,

Clinic of Pediatric Hematology and Oncology, İstanbul, Turkey

Phone : +90 505 839 60 92

E-mail : cengizbayram2013@gmail.com ORCID-ID: orcid.org/0000-0003-2153-0628


Accepted/Kabul tarihi: April 20, 2017

DOI: 10.4274/tjh.2017.0134

143



Nuclear Projections in Neutrophils for Supporting the Diagnosis of

Trisomy 13

Trisomi 13 Tanısını Desteklemede Nötrofillerdeki Nükleer Çıkıntılar

Şebnem Kader 1 , Mehmet Mutlu 1 , Filiz Aktürk Acar 1 , Yakup Aslan 1 , Erol Erduran 2

1Karadeniz Technical University Faculty of Medicine, Division of Neonatology, Trabzon, Turkey

2Karadeniz Technical University Faculty of Medicine, Division Pediatric Hematology, Trabzon, Turkey

To the Editor,

Trisomy 13 is a rare genetic disorder characterized by severe

multiple congenital anomalies. Structural anomalies of

neutrophils may be supportive for the diagnosis of trisomy 13.

A newborn was born by vaginal delivery after 29 weeks of

pregnancy. Physical examination revealed symmetric growth

restriction, low-set hypoplastic ears, aplasia cutis congenita

areata on the vertex, postaxial polydactyly of the foot, bilateral

microphthalmia, an umbilical cord cyst, and heart murmurs.

Echocardiography showed truncus arteriosus type I. Review of

the peripheral blood smear revealed two or more small threadlike

pedunculated projections attached to the surface of the nuclei

in more than 60% of the neutrophils (Figure 1). The diagnosis of

trisomy 13 was made by chromosomal analysis. The infant died

at 2 days of life because of massive pulmonary hemorrhage.

The presence of threadlike pedunculated projections attached to

the surface of the nuclei of neutrophils was described in trisomy

of the D group of chromosomes (13, 14, and 15) and also in

trisomy 18 [1,2]. Two or more nuclear projections detected in

more than 15% of neutrophils may be highly suggestive of these

trisomies [3]. We suggest that identification of characteristic

structural anomalies of neutrophils on a blood smear may be

used for supporting the diagnosis of these trisomies.

Keywords: Trisomy 13, Blood smear, Neutrophilic nuclear

projections

Anahtar Sözcükler: Trisomi 13, Periferik yayma, Nötrofilik

nükleer projeksiyon

Figure 1. Peripheral blood smear showing threadlike

pedunculated projections attached to the surface of the nuclei

of neutrophils.

Informed Consent: Our patient’s parent gave consent.




included.

References

1. Huehns ER, Lutzner M, Hecht F. Nuclear abnormalities of the neutrophils in

D1 (13-15)-trisomy syndrome. Lancet 1964;1:589-590.

2. Kahwash BM, Nowacki NB, Kahwash SB. Aberrant (barbed-wire) nuclear

projections of neutrophils in trisomy 18 (Edwards syndrome). Case Rep

Hematol 2015;2015:163857.

3. Lakovschek IC, Streubel B, Ulm B. Natural outcome of trisomy 13, trisomy 18,

and triploidy after prenatal diagnosis. Am J Med Genet A 2011;155:2626-

2633.



Address for Correspondence/Yazışma Adresi: Mehmet MUTLU, M.D.,

Karadeniz Technical University Faculty of Medicine, Division of Neonatology, Trabzon, Turkey

Phone : +90 532 633 27 49

E-mail : drmehmetmutlu38@hotmail.com ORCID-ID: orcid.org/0000-0003-3666-3159

Received/Geliş tarihi: June 06, 2017


DOI: 10.4274/tjh.2017.0227

144



Intravascular Large B-Cell Lymphoma of the Gallbladder

Safra Kesesinin İntravasküler Diffüz Büyük Hücreli Lenfoması

Bülent Çetin 1 , Nalan Akyürek 2 , Yavuz Metin 3 , Feryal Karaca 4 , İrem Bilgetekin 5 , Ahmet Özet 6

1Recep Tayyip Erdoğan University Faculty of Medicine, Department of Internal Medicine, Division of Medical Oncology, Rize, Turkey

2Gazi University Faculty of Medicine, Department of Pathology, Ankara, Turkey

3Recep Tayyip Erdoğan University Faculty of Medicine, Department of Radiology, Rize, Turkey

4Adana Numune Training and Research Hospital, Clinic of Radiation Oncology, Adana, Turkey

5Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital, Clinic of Internal Medicine, Division of Medical Oncology,

Ankara, Turkey

6Gazi University Faculty of Medicine, Department of Internal Medicine, Division of Medical Oncology, Ankara, Turkey

To the Editor,

Intravascular large B-cell lymphoma (IVLBCL) is a rare type of

extranodal B-cell lymphoma characterized by the growth of

lymphoma cells within the lumina of small vessels. Two major

patterns of clinical presentation have been recognized: the first

is in European countries, with brain and skin involvement, and

the second in Asian countries, where patients typically present

with multiorgan failure, hepatosplenomegaly, pancytopenia,

and hemophagocytic syndrome [1,2,3,4,5]. Primary IVLBCL of

the gallbladder is exceedingly rare.

A 60-year-old male patient was admitted to the hospital with

fever, abdominal pain, and weight loss. Physical examination

showed an epigastric mass of approximately 4 cm in diameter

and the absence of hepatosplenomegaly and lymphadenopathy.

Laboratory tests revealed anemia (hemoglobin: 10 g/dL), with

normal leukocytes and platelets. Peripheral smear showed

normocytic-normochromic anemia without any abnormal cells.

Increases in liver function tests were positive laboratory findings

(aspartate aminotransferase: 240 U/L, alanine aminotransferase:

240 U/L, alkaline phosphatase: 740 U/L, gamma-glutamyl

transferase: 80 U/L, total bilirubin/direct bilirubin: 2.06/1.2 mg/

dL). Contrast-enhanced abdominal computerized tomography

(CT) for further evaluation revealed a greater curvature-based

mass of 8x5x5.5 cm in size, at the level of the distal gastric

corpus, significantly narrowing the gastric lumen (Figures 1A

and 1B). CT also showed hypodense areas in liver segments

5 and 8 and gallbladder stones, the largest being 1.5 cm in

diameter. Dynamic liver magnetic resonance imaging (MRI)

was performed to characterize the liver lesions. MRI revealed

calculous cholecystitis, choledocholithiasis, and a mass lesion of

6.5x3 cm in size, thought to be based on the greater curvature at

the corpus of the stomach. With no signs of distant metastasis,

the patient primarily underwent both cholecystectomy and

partial gastrectomy. Surgical biopsy of liver lesions revealed

nonspecific inflammatory changes and no evidence of a tumor,

while histologic examination confirmed a gastrointestinal

Figure 1. A, B) Axial computerized tomography image showing a

greater curvature-based intraluminal gastric mass (white arrows),

stones in the gallbladder (asterisk), and a vague hypodense area,

which was proven to be caused by cholangitis, in segment 5 of

the liver (black arrow). C) Intravascular B-cell lymphoma. The

numerous dilated blood vessels were filled with large, atypical,

centroblast-like lymphoid cells (hematoxylin and eosin, 400 x ). D)

CD20-positive atypical lymphoid cells (400 x ).

stromal tumor (GIST) of the stomach. Histological analysis

of the cholecystectomy material showed cells with irregular

nuclear contours and open chromatin confined to small vessels,

characteristic of the IVLBCL phenotype. These cells were strongly

positive for CD20 stain (Figures 1C and 1D). Since intravascular

infiltrations are easily missed on hematoxylin and eosin-stained

sections, bone marrow and liver biopsy slides were also stained

145

LETTERS TO THE EDITOR Turk J Hematol 2018;35:137-151

by CD20 and no evidence of intravascular lymphoma was

found. A whole-body integrated positron electron tomography-

CT scan for tumor staging showed diffusely increased uptake

of 18F-fludeoxyglucose in the liver (SUV max

: 7.0) and multiple

lymph node lesions including the submandibular, preauricular,

cervical, and jugular lymph nodes (SUV max

: 8.3). He was treated

with six cycles of an R-CHOP regimen. He did not show any

evidence of recurrence (normal gastroscopy and CT scan) at 36

months of follow-up.

IVLBCL usually occurs in adults in the sixth and seventh decades.

The tumor is often clinically unsuspected and can be easily

overlooked on biopsy. The diagnosis is most commonly made

at autopsy. The lymphoma cells are generally large with round

nuclei and prominent nucleoli. The malignant cells uniformly

express pan-B-cell antigens (CD20, CD79a) and variably express

other antigens such as CD5 (38%) and CD10 (13%) [2]. There

are no pathognomonic laboratory or radiologic abnormalities

associated with IVLBCL. Abdominal CT and MRI findings of our

patient with IVLBCL were nonspecific. What is the pathogenic

mechanism for simultaneous presentation of gallbladder

intravascular B-cell lymphoma with GIST? A unifying hypothesis

supports a single underlying genetic instability that could have

led to both diseases. The finding of two different neoplasms

in our patient seems to be coincidental rather than related

to the same pathogenic triggering. Central nervous system

symptoms, skin manifestations, bone marrow involvement,

and hemophagocytic syndrome are the most common clinical

and laboratory abnormalities, but these were not seen in our

case. Our patient presented with nonspecific symptoms and

laboratory abnormalities. The ability of IVLBCL to involve any

organ system further makes it very difficult to suspect this

condition in a patient with a rare presentation such as ours.

Keywords: Intravascular large B-cell lymphoma, Gallbladder,

Gastrointestinal stromal tumor

Anahtar Sözcükler: İntravasküler büyük hücreli lenfoma, Safra

kesesi, Gastrointestinal stromal tümör




included.

References

1. Ferreri AJ, Campo E, Seymour JF, Willemze R, Ilariucci F, Ambrosetti A, Zucca

E, Rossi G, López-Guillermo A, Pavlovsky MA, Geerts ML, Candoni A, Lestani

M, Asioli S, Milani M, Piris MA, Pileri S, Facchetti F, Cavalli F, Ponzoni M;

International Extranodal Lymphoma Study Group (IELSG). Intravascular

lymphoma: clinical presentation, natural history, management and

prognostic factors in a series of 38 cases, with special emphasis on the

“cutaneous variant.” Br J Haematol 2004;127:173-183.

2. Ferreri AJ, Dognini GP, Campo E, Willemze R, Seymour JF, Bairey O, Martelli

M, De Renz AO, Doglioni C, Montalbán C, Tedeschi A, Pavlovsky A, Morgan

S, Uziel L, Ferracci M, Ascani S, Gianelli U, Patriarca C, Facchetti F, Dalla

Libera A, Pertoldi B, Horváth B, Szomor A, Zucca E, Cavalli F, Ponzoni M;

International Extranodal Lymphoma Study Group (IELSG). Variations in

clinical presentation, frequency of hemophagocytosis and clinical behavior

of intravascular lymphoma diagnosed in different geographical regions.

Haematologica 2007;92:486-492.

3. Murase T, Nakamura S. An Asian variant of intravascular lymphomatosis:

an updated review of malignant histiocytosis-like B-cell lymphoma. Leuk

Lymphoma 1999;33:459-473.

4. Murase T, Nakamura S, Kawauchi K, Matsuzaki H, Sakai C, Inaba T, Nasu K,

Tashiro K, Suchi T, Saito H. An Asian variant of intravascular large B-cell

lymphoma: clinical, pathological and cytogenetic approaches to diffuse

large B-cell lymphoma associated with haemophagocytic syndrome. Br J

Haematol 2000;111:826-834.

5. Shimazaki C, Inaba T, Nakagawa M. B-cell lymphoma-associated

hemophagocytic syndrome. Leuk Lymphoma 2000;38:121-130.

Address for Correspondence/Yazışma Adresi: Bülent ÇETİN, M.D.,

Recep Tayyip Erdoğan University Faculty of Medicine, Department of Internal Medicine,

Division of Medical Oncology, Rize, Turkey

Phone : +90 505 884 26 94

E-mail : caretta06@hotmail.com ORCID-ID: orcid.org/0000-0001-8628-0864

Received/Geliş tarihi: July 24 , 2017


DOI: 10.4274/tjh.2017.0276

146



Successful Treatment of Chronic Lymphocytic Leukemia

Multifocal Central Nervous System Involvement with Ibrutinib

Kronik Lenfositik Löseminin Multifokal Santral Sinir Sistemi Tutulumunun İbrutinib ile

Başarılı Tedavisi

Anna Christoforidou 1 , Georgios Kapsas 2 , Zoe Bezirgiannidou 1 , Spyros Papamichos 1 , Ιoannis Kotsianidis 1

1Democritus University of Thrace, Department of Hematology, Alexandroupolis, Greece

2Democritus University of Thrace, Department of Radiology, Alexandroupolis, Greece

To the Editor,

Central nervous system involvement (CNSi) is rare in the course

of chronic lymphocytic leukemia (CLL). The frequency ranges

from 0.8% to 1% [1], and it is often underreported. Diagnosis is

challenging and there is no consensus on the optimal therapy

or survival. CNSi manifests as either leptomeningeal infiltration

or a focal parenchymal lesion, or both [1]. We describe the

case of a CLL patient who progressed with parenchymal CNS

involvement and was successfully treated with ibrutinib.

A 71-year-old woman was followed without treatment at

the hematology clinic for 12 years for asymptomatic CLL,

Binet stage I, exhibiting slowly progressive lymphocytosis and

mild hepatosplenomegaly. In March 2016 she presented with

expressive aphasia, memory problems, confusion, and headache,

but no B symptoms. Neurological examination confirmed the

mental and speech impairment but was otherwise unremarkable.

Thoracic and abdominal computed tomography scan showed no

lymphadenopathy or progression of visceromegaly. Her complete

blood count was unchanged compared to the previous year with

WBC lymphocytes at 14,652x10 9 /L, Htc at 45%, and platelets at

144x10 9 /L, with typical CLL morphology and immunophenotype

(CD19 83% with CD5+/CD23+/CD20+low/CD38-/sIglow) and

unmutated p53. IGH mutational analysis showed a mutated

clone with IGHV3-7/IGHD1-26/IGHJ4 rearrangement. Serum

chemistry was normal apart from elevated lactate dehydrogenase

at 303 U/L (upper normal limit: 248 U/L). Antinuclear antibody

and rheumatoid factor were negative; C-reactive protein,

C3, and C4 levels were within the normal limits. Magnetic

resonance imaging (MRI) showed a contrast-enhanced

irregularly shaped mass of 22x17x16 mm in the left frontal

lobe with intense edema and midline shift (Figure 1A). Lumbar

puncture showed 5/µL nucleated cell count, 5/µL erythrocytes,

0.4 g/L protein, and no monoclonal B lymphocytes (CD5/CD19)

by flow cytometry. Extensive investigations for infection with

cytomegalovirus, Epstein-Barr virus, human immunodeficiency

virus, herpes simplex virus, ortoxoplasma antibodies as well as

PCR for Cytomegalovirus DNA were negative in both serum and

cerebrospinal fluid. She was referred to a neurosurgeon but the

Figure 1. A) Initial presentation of the enhancing lesion in the

left frontal lobe (thick arrow), with considerable perilesional

edema. B, C, D) After one and four rituximab plus a high-dose

methylprednisolone cycles there was a reduction of the enhancing

lesion (thin arrow) and edema; however, new enhancing lesions

appeared in the left frontal operculum and the right middle

cerebellar peduncle (arrowheads). E) Brain magnetic resonance

imaging 5 months after ibrutinib therapy demonstrates complete

resolution of the cerebellar lesion and F) minimal enhancement

in the area of the lesion in the left frontal operculum (arrow). G)

Dynamic susceptibility contrast perfusion imaging. Comparison

between the enhancing lesion and the normal contralateral

side demonstrates an overshooting of the intensity curve of the

lesion above the baseline (arrow). This phenomenon is suggestive

of lymphoma [149x172 mm (72x72 DPI)].

patient was reluctant to undergo a core biopsy of the brain

lesion. However, dynamic susceptibility contrast MRI perfusion

imaging displayed a signal intensity curve overshooting above

the baseline that was suggestive of lymphoma (Figure 1G) [2].

Considering the above findings, the patient was treated

in an exploratory fashion with rituximab plus a high-dose

147

LETTERS TO THE EDITOR Turk J Hematol 2018;35:137-151

methylprednisolone (RHDM) regimen (rituximab at 500 mg/m 2

i.v. and methylprednisolone at 1g iv for 4 days). After 2 monthly

cycles, the neurological symptoms partially regressed, but her

MRI findings deteriorated with a new lesion on the left frontal

lobe, although the original lesion was impressively smaller

(Figures 1B and 1C). Continued RHDM resulted in a decrease of

lymphocytosis to 10.9x10 9 /L, but repeat MRI showed an atypical

pattern of older lesions receding coupled with the appearance

of new ones in multiple cerebral sites (Figure 1D). Since we did

not have proof of whether the infiltrating neoplastic cells were

identical to the original leukemic clone or a manifestation of

Richter’s syndrome (RS), second-line treatment was a challenge.

The patient was switched to ibrutinibat 420 mg per day, based

on the recent reports of ibrutinib’s CNS penetration and

effectiveness, even in high-grade lymphomas. Three months

later there was a partial improvement in the MRI findings and

no new lesions. Currently on the 15 th month of ibrutinib therapy,

she is completely symptom-free , shows partial response of CLL

and stable neuroimaging improvement, 21 months after initial

CNS involvement (Figure 1E, 1F).

Autopsy studies have found leukemic meningitis and

parenchymal brain involvement in up to 20% of CLL patients,

but clinical syndromes are very rarely reported [3], with the first

ever case published by Solal-Celigny et al. [4]. CNSi is diagnosed

by neuroimaging, cerebrospinal fluid evaluation, and core tissue

biopsy that differentiate between CLL, Richter’s transformation,

or another solid tumor. Strati et al. [1] reviewed 33 patients

with CLL CNSi and, among them, 11 out of 12 patients with CNS

RS had later developed systematic disease [1]. Our patient did

not at any point develop systematic Richter’s syndrome and has

an excellent clinical course during the 21 months of follow up

which is suggestive of a CLL rather than RS origin of the CNSi.

The treatment outcome of clinically apparent CNSi is unclear, as

most studies are retrospective. The management ranges from CLL

therapy alone [5] to CNS irradiation, intrathecal chemotherapy,

and intensive CNS-lymphoma modalities. Intrathecal rituximab

has been used in several case reports and in a small study for

high-grade CNS lymphomas but never in CLL [6]. In a recent

study the median overall survivalof patients with CLL or RS

brain involvement was 12 and 11 months, respectively [1]. On

the contrary, a cohort of 30 French patients had much better

overall survivalof 65% at 5 years [7]. Ibrutinib is an oral Bruton

tyrosine kinase inhibitor approved for B-CLL [8]. It is a small

molecule that crosses the blood-brain barrier with promising

results in CNS lymphoma, as shown in some cases of mantle

cell lymphoma [9,10,11], in Waldenström macroglobulinemia

patients [12,13,14], and, more importantly, in a phase I study

of 20 patients with relapsed/refractory CNS lymphoma showing

75% overall response rate, including 8 complete responses,

although responses were relatively short-lived [15]. Ibrutinib

has a convenient outpatient oral administration scheme with

minimal toxicity and is an attractive option for CNS lymphoma

Table 1. Characteristics of published cases of ibrutinib-treated chronic lymphocytic leukemia central nervous system involvement.

Time since CLL

diagnosis

Binet stage at

CNSi

CLL progression at

CNSi diagnosis

CNSi presentation

Patient 1 [16]

Median of 106

months*

Patient 2

[16]

Median of

106 months*

Patient 3

[16]

Median

of 106

months*

Patient 4 [16]

Median of 106

months*

Patient

5 [7]

Patient 6

[7]

Patient 7

[17]

Patient 8

(present

case)

N/A* N/A* N/A* 12 years

C B C A N/A N/A C A

Yes No Yes No N/A N/A N/A No

Nodular

enhancement

of left

parietal lobe

with nonspecific

periventricular

T2

hyperintensities

Leukemic

meningitis

Leukemic

meningitis

Thickening of

optic nerves and

chiasma; FLAIR

hyperintensities

with nodular

lesion of internal

occipitotemporal

region

N/A

N/A

Cervical

myelopathy

with

expansion of

the spinal

cord from C2

to C7

Del17p Yes Yes No Yes N/A N/A N/A No

CNS response to

ibrutinib

Duration of

response to

ibrutinib (months)

MRI

normalization

CR

CR

MRI near

normalization

N/A

N/A

MRI

normalization

9 14 8 9 N/A N/A 18 15

*Patients 1-6 were mentioned in the French cohort study [7], but only patients 1-4 had a detailed description in a separate publication [16].

MRI: Magnetic resonance imaging, CNSi: central nervous system involvement, CLL: chronic lymphocytic leukemia.

Multifocal

parenchymal

masses, with

biggest one

at 22x1x16

mm in the

left frontal

lobe

MRI near

normalization

148



compared to traditional intensive chemotherapy and/or

intrathecal therapy.

So far, there are seven published cases of CLL with CNSi treated

with ibrutinib monotherapy (Table 1): two with nodular masses

[7,16], four with leptomeningeal disease [7,16], and one with

cervical myelopathy [17]. None of these patients underwent

brain biopsy. All patients received the standard dose of 420

mg/day and all of them responded with sustained complete

responseor partial response, with a median follow-up of 8 to

18 months. Our patient had multiple brain masses and shows

an ongoing response to second line ibrutinib monotherapy for a

total of 21 months as per December 2017, when the latest brain

MRI was performed.

In conclusion, this case further supports the efficacy of ibrutinib

in CLL with CNSi, suggesting a potential future change in

the frontline management and also the outcome of this rare

condition.

Keywords: Chronic lymphocytic leukemia, Central nervous

system, CNS, Ibrutinib

Anahtar Sözcükler: Kronik lenfositik lösemi, Santral sinir

sistemi, SSS, İbrutinib




included.

References

1. Strati P, Uhm JH, Kaufmann TJ, Nabhan C, Parikh SA, Hanson CA, Chaffee

KG, Call TG, Shanafelt TD. Prevalence and characteristics of central nervous

system involvement by chronic lymphocytic leukemia. Haematologica

2016;101:458-465.

2. Mangla R, Kolar B, Zhu T, Zhong J, Almast J, Ekholm S. Percentage signal

recovery derived from MR dynamic susceptibility contrast imaging is useful

to differentiate common enhancing malignant lesions of the brain. AJNR

Am J Neuroradiol 2011;32:1004-1010.

3. Barcos M, Lane W, Gomez GA, Han T, Freeman A, Preisler H, Henderson E. An

autopsy study of 1206 acute and chronic leukemias (1958 to 1982). Cancer

1987;60:827-837.

4. Solal-Celigny P, Schuller E, Courouble Y, Gislon J, Elghozi D, Boivin

P. Cerebromeningeal location of chronic lymphoid leukemia. Rapid

immunochemical diagnosis and complete remission by intrathecal

chemotherapy. Presse Med 1983;12:2323-2325.

5. Benjamini O, Jain P, Schlette E, Sciffman JS, Estrov Z, Keating M. Chronic

lymphocytic leukemia with central nervous system involvement: a high-risk

disease? Clin Lymphoma Myeloma Leuk 2013;13:338-341.

6. Rubenstein JL, Fridlyand J, Abrey L, Shen A, Karch J, Wang E, Issa S, Damon

L, Prados M, McDermott M, O’Brien J, Haqq C, Shuman M. Phase I study of

intraventricular administration of rituximab in patients with recurrent CNS

and intraocular lymphoma. J Clin Oncol 2007;25:1350-1356.

7. Wanquet A, Birsen R, Bonnet C, Boubaya M, Choquet S, Dupuis J, Lepretre

S, Re D, Fahri J, Michallet AS, Ysebaert L, Lemal R, Lamy T, Delarue R,

Troussard X, Cymbalista F, Levy V, Dietrich PY, Leblond V, Aurran-Schleinitz

T. Management of central nervous system involvement in chronic

lymphocytic leukaemia: a retrospective cohort of 30 patients. Br J Haematol

2017;176:37-49.

8. Byrd JC, Furman RR, Coutre SE, Flinn IW, Burger JA, Blum KA, Grant B,

Sharman JP, Coleman M, Wierda WG, Jones JA, Zhao W, Heerema NA,

Johnson AJ, Sukbuntherng J, Chang BY, Clow F, Hedrick E, Buggy JJ, James

DF, O’Brien S. Targeting BTK with ibrutinib in relapsed chronic lymphocytic

leukemia. N Engl J Med 2013;369:32-42.

9. Tucker DL, Naylor G, Kruger A, Hamilton MS, Follows G, Rule SA. Ibrutinib

is a safe and effective therapy for systemic mantle cell lymphoma with

central nervous system involvement - a multi-centre case series from the

United Kingdom. Br J Haematol 2017;178:327-329.

10. Bernard S, Goldwirt L, Amorim S, Brice P, Briere J, de Kerviler E, Mourah S,

Sauvageon H, Thieblemont C. Activity of ibrutinib in mantle cell lymphoma

patients with central nervous system relapse. Blood 2015;126:1695-1698.

11. Gonzalez-Bonet LG, Garcia-Boyero R, Gaona-Morales J. Mantle cell

lymphoma with central nervous system involvement simulating bilateral

subdural hematomas. World Neurosurg 2017;99:808.

12. Cabannes-Hamy A, Lemal R, Goldwirt L, Poulain S, Amorim S, Perignon R,

Berger J, Brice P, De Kerviler E, Bay JO, Sauvageon H, Beldjord K, Mourah S,

Tournilhac O, Thieblemont C. Efficacy of ibrutinib in the treatment of Bing-

Neel syndrome. Am J Hematol 2016;91:17-19.

13. Castillo JJ, D’Sa S, Lunn MP, Minnema MC, Tedeschi A, Lansigan F, Palomba

ML, Varettoni M, Garcia-Sanz R, Nayak L, Lee EQ, Rinne ML, Norden AD,

Ghobrial IM, Treon SP. Central nervous system involvement by Waldenström

macroglobulinaemia (Bing-Neel syndrome): a multi-institutional

retrospective study. Br J Haematol 2016;172:709-715.

14. Mason C, Savona S, Rini JN, Castillo JJ, Xu L, Hunter ZR, Treon SP, Allen

SL. Ibrutinib penetrates the blood brain barrier and shows efficacy in the

therapy of Bing Neel syndrome. Br J Haematol 2017;179:339-341.

15. Grommes C, Pastore A, Gavrilovic I, Kaley T, Nolan C, Omuro AM, Wolfe

J, Pentsova E, Hatzoglou V, Mellinghoff I, DeAngelis L. Single-agent

ibrutinib in recurrent/refractory central nervous system lymphoma. Blood

2016;128:783.

16. Wanquet A, Birsen R, Lemal R, Hunault M, Leblond V, Aurran-Schleinitz

T. Ibrutinib responsive central nervous system involvement in chronic

lymphocytic leukemia. Blood 2016;127:2356-2358.

17. Tam CS, Kimber T, Seymour JF. Ibrutinib monotherapy as effective treatment

of central nervous system involvement by chronic lymphocytic leukaemia.

Br J Haematol 2017;176:829-831.

Address for Correspondence/Yazışma Adresi: Anna CHRISTOFORIDOU, M.D.,

Democritus University of Thrace, Department of Hematology, Alexandroupolis, Greece

Phone : +30 255 135 15 11

E-mail : annachristof@yahoo.gr ORCID-ID: orcid.org/0000-0002-3979-8318

Received/Geliş tarihi: August 20, 2017


DOI: 10.4274/tjh.2017.0313

149



Pleomorphic Multinucleated Plasma Cells Simulating

Megakaryocytes in an Anaplastic Variant of Myeloma

Anaplastik Variant Myelomda Megakaryositi Taklit Eden Pleomorfik Multinükleer Plazma

Hücreleri

Shivangi Harankhedkar, Ruchi Gupta, Khaliqur Rahman

Sanjay Gandhi Post Graduate Institute of Medical Sciences, Department of Hematology, Lucknow, Uttar Pradesh, India

To the Editor,

Myeloma cells are notorious for their morphological variations,

which range from mature-appearing plasma cells to other

poorly differentiated forms. The pleomorphic or anaplastic

variants are its uncommon rare variants, which may pose a

diagnostic dilemma in unprecedented cases. These anaplastic

variants may mimic high-grade lymphomas, leukemia, or even

metastatic carcinomas [1,2]. Anaplastic plasma cells may be

seen at diagnosis or evolve during the terminal phase of the

disease [3]. The correlation of this morphological variant with

treatment outcome is controversial, but it is believed to be

a harbinger of aggressive disease [4,5]. Herein we report the

case of an unsuspected multiple myeloma, where bone marrow

examination revealed the presence of bizarre plasma cells

simulating megakaryocytes.

An asymptomatic 65-year-old diabetic male presented

with bicytopenia. Complete blood count analysis showed

hemoglobin of 7 g/dL, total leukocyte count of 6.3x10 9 /L, and

51x10 9 /L platelets. The peripheral smear showed the presence

of occasional circulating plasma cells with minimal rouleaux

formation. Bone marrow examination revealed proliferation

of highly pleomorphic cells with multinucleation, simulating

megakaryocytes. Cells had moderate to abundant basophilic

cytoplasm, while nuclei were multilobulated, with open

chromatin and prominent nucleoli, along with a few intranuclear

basophilic inclusions (Figure 1A). Serum protein electrophoresis

revealed monoclonal protein of 0.19 g/dL, which was confirmed

to be IgA kappa on immunofixation (Figure 1B). The kappa/

lambda ratio was 427.6 and the β2 microglobulin level was 21.9

mg/L. Immunophenotypically, the cells expressed CD38, CD138,

CD56, and CD200 (Figures 1C-1E). FISH analysis, performed

after magnetic bead enrichment of plasma cells, showed the

presence of del(13q14.3). The patient was unfortunately lost to

follow-up.

Anaplastic multiple myeloma (AMM) is a rare morphological

variant of multiple myeloma, the true incidence of which is

largely unknown [1,2,6,7]. In the early 1990s, Allen and Coleman

[3] reviewed 108 cases of anaplastic myeloma, 68 of which

150

showed the presence of extramedullary disease. Other salient

characteristics of AMM, which have been observed by other

authors, too, include a younger age atpresentation, cytopenias,

predilection for IgA myelomas, and aggressive clinical course

[4,7,8,9]. Bahmanyar et al. [10] reviewed the genetic features

of 11 cases of AMM for the presence of myeloma-associated

genetic abnormalities and compared them with 188 newly

diagnosed non-anaplastic variants of MM. They observed

significantly higher frequencies of 1q21 amplification, 17p(p53)

deletion, and t(4,14). Additionally, the presence of complex

karyotype, del(13q14.3), t(1;19), and near tetraploidy has also

been reported [8,9,10]. The treatment outcome of this variant

is considered poor as per the older literature; however, patients

treated with triple-drug chemotherapeutic regimens in the

modern era have shown sustained responses [1,5,9].

To conclude, awareness of these variants in myeloma is

important for an accurate diagnosis. In cases where myeloma

cells show extreme “de-differentiation”, a multidisciplinary

Figure 1. Panel of photomicrographs: A) May-Grünwald Giemsa

stained bone marrow aspirate smear (100 x ) showing pleomorphic

cells, with multilobation and multinuclearity, with prominent

inclusions (red arrows) and abundant basophilic cytoplasm,

and absence of perinuclear hof; B) serum immunofixation

highlighting presence of IgA kappa monoclonal protein; C, D,

E) panel of dot plots documenting these atypical plasma cells

to be positive for CD38, CD138, CD200, and CD56 and negative

for CD45.



approach with the addition of immunophenotyping in the

diagnostic armamentarium is recommended. With the advent of

triple-drug regimens in myeloma therapy and autologous bone

marrow transplantation, the outcome of this variant needs to

be re-addressed inlarger studies.

Keywords: Myeloma, Anaplastic, Megakaryocytes

Anahtar Sözcükler: Myeloma, Anaplastik, Megakaryosit




References

1. Beljan Perak R, Karaman I, Sundov D, Jakelic Pitesa J, Novak A, Pavlovic

A. Anaplastic variant of plasma cell myeloma: a pitfall of morphlogical

identification. Acta Cytol 2016;60:275-276.

2. Rao S, Kar R, Pati HP. Anaplastic myeloma: a morphologic diagnostic

dilemma. Indian J Hematol Blood Transfus 2008;24:188-189.

3. Allen SL, Coleman M. Aggressive phase multiple myeloma: a terminal

anaplastic transformation resembling high-grade lymphoma. Cancer Invest

1990;8:417-424.

4. Zervas K, Constantinou N, Karakantza M, Tsigalidou-Balla V. Anaplastic

myeloma. Leuk Lymphoma 1995;16:515-518.

5. Agrawal M, Kanakry J, Arnold CA, Suzman DL, Mathieu L, Kasamon YL,

Gladstone DE, Ambinder RF, Ghosh N. Sustained remission and reversal of

end-organ dysfunction in a patient with anaplastic myeloma. Ann Hematol

2014;93:1245-1246.

6. Suchman AL, Coleman M, Mouradian JA, Wolf DJ, Saletan S. Aggressive

plasma cell myeloma: a terminal phase. Arch Intern Med 1981;141:1315-

1320.

7. Butler RC, Thomas SM, Thompson JM, Keat AC. Anaplastic myeloma in

systemic lupus erythematosus. Ann Rheum Dis 1984;43:653-655.

8. Sethi S, Miller I. Plasma cell myeloma with anaplastic transformation. Blood

2016;128:2106.

9. Chang H, Kajal B. Anaplastic variant of plasma cell myeloma with Dutcher

bodies. Blood 2016;127:3291.

10. Bahmanyar M, Qi X, Chang H. Genomic aberrations in anaplastic multiple

myeloma: high frequency of 1q21(CKS1B) amplifications. Leuk Res

2013;37:1726-1728.



Address for Correspondence/Yazışma Adresi: Ruchi GUPTA, M.D.,

Sanjay Gandhi Post Graduate Institute of Medical Sciences, Department of Hematology, Lucknow,

Uttar Pradesh, India

Phone : 800 490 4799

E-mail : ruchipgi@yahoo.co.in ORCID-ID: orcid.org/0000-0003-3427-9188

Received/Geliş tarihi: September 04, 2017

Accepted/Kabul tarihi: February 06, 2018

DOI: 10.4274/tjh.2017.0329

151

Advisory Board of This Issue (June 2018)

Ahmet Emre Eşkazan, Turkey

Antonio Medina Almeida, Portugal

Arbil Açıkalın, Turkey

Argiris Symeonidis, Greece

Canan Albayrak, Turkey

Claudio Cerchione, Italy

Donato Mannina, Italy

Emanuele Angelucci, Italy

Erdal Kurtoğlu, Turkey

Figen Atalay, Turkey

Güldane Cengiz Seval, Turkey

Hüseyin Gülen, Turkey

Jayadev Manikkam Umakanthan, USA

Melek Ergin, Turkey

Meltem Kurt Yüksel, Turkey

Mine Hekimgil, Turkey

Muhit Özcan, Turkey

Muhlis Cem Ar, Turkey

Mustafa Pehlivan, Turkey

Mustafa Yıldırım, Turkey

Müge Sayitoğlu, Turkey

Mükerrem Safalı, Turkey

Münci Yağcı, Turkey

Namık Özbek, Turkey

Nükhet Tüzüner, Turkey

Özgür Mehtap, Turkey

Özgür Rosti, Turkey

Pervin Topçuoğlu, Turkey

Prajwal Dhakal, USA

Rauf Haznedar, Turkey

Reyhan Küçükkaya, Turkey

Robert F. Cornell, USA

Tayfur Toptaş, Turkey

Teoman Soysal, Turkey

Ufuk Demirci, Turkey

Yavuz Bilgin, Turkey

Yookarin Khonglah, India

Zehra Çoban, Turkey

Zühre Kaya, Turkey

TJH-2018-2-baski

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?